WO2020041851A2 - Pharmaceutical composition, process for obtaining and using secondary metabolites produced by the fungus exserohilum rostratum in cell regeneration - Google Patents

Pharmaceutical composition, process for obtaining and using secondary metabolites produced by the fungus exserohilum rostratum in cell regeneration Download PDF

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WO2020041851A2
WO2020041851A2 PCT/BR2019/050360 BR2019050360W WO2020041851A2 WO 2020041851 A2 WO2020041851 A2 WO 2020041851A2 BR 2019050360 W BR2019050360 W BR 2019050360W WO 2020041851 A2 WO2020041851 A2 WO 2020041851A2
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cells
annularin
monocerine
cell
fact
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PCT/BR2019/050360
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French (fr)
Portuguese (pt)
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WO2020041851A3 (en
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Ana Olívia DE SOUZA
Janice ONUKI
Durvanei Augusto Maria
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Instituto Butantan
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Priority claimed from BR102019018195-8A external-priority patent/BR102019018195A2/en
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Publication of WO2020041851A2 publication Critical patent/WO2020041851A2/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A61K31/366—Lactones having six-membered rings, e.g. delta-lactones
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungal isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/02—Oxygen as only ring hetero atoms
    • C12P17/06—Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
    • C12P17/181—Heterocyclic compounds containing oxygen atoms as the only ring heteroatoms in the condensed system, e.g. Salinomycin, Septamycin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/62—Carboxylic acid esters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi

Definitions

  • the present invention relates to the use of a secondary metabolite produced by the fungus Exserohilum rostratum in cell and tissue regeneration.
  • the present invention relates to the use of secondary metabolites monocerin and annularin I, organic compounds obtained from the culture of the fungus Exserohilum rostratum for cell regeneration in HUVEC endothelial cells and normal FN1 fibroblasts.
  • Endophytic fungi are fungi that live inside plants, generally inhabiting their aerial parts, such as leaves and stems, without apparently causing any damage to their hosts. Therefore, they differ from phytopathogenic microorganisms, which are harmful to plants, causing disease.
  • Endophyte microorganisms were first mentioned in the early 19th century, but it was Bary (1866) who first outlined a possible distinction between endophytes and phytopathogens. In the late 1970s, it was found that endophytes provide the host plant with protection against insects and pests, against other pathogenic microorganisms and even against herbivores. Currently, it is known that endophytic fungi can produce molecules that act as toxins, antibiotics, growth factors and other drugs, and many other products of biotechnological interest (AZEVEDO, 2004). Endophytic fungi attach to plants through natural openings and wounds that serve as entry doors sometimes in the secondary side roots. Other entry points are natural openings such as stomata and hydatodes, openings caused by insects and structures of pathogenic fungi, such as appressoriums.
  • MS primary and secondary metabolites
  • Primary metabolites are the small molecules produced during vegetative growth. They are used in food and feed industries including, for example: alcohols (ethanol), amino acids (monosodium glumate, lysine, threonine, phenylalanine, tryptophan), flavoring nucleotides (5-guanyl acid, 5-isosinic acid), organic acids (acetic , propionic, fumaric, lactic), polyols (glycerol, mannitol, xylitol), polysaccharides (xanthan), sugars (fructose, ribose) and vitamins (riboflavin, cyanocobalamin, biotin) (DEMAIN, 2000; RAJASEKARAN et al., 2008).
  • alcohols ethanol
  • amino acids monosodium glumate, lysine, threonine, phenylalanine, tryptophan
  • flavoring nucleotides (5-guanyl acid, 5-isos
  • Secondary metabolites are synthesized when microbial growth is in the stationary phase; they are often bioactive and of low molecular weight. They are of great importance to civilization due to the antibiotic activity of pharmaceutical importance, as well as immunosuppressive and toxic activity. They are not normally derived from the substrate used for cell growth, but are synthesized from a primary metabolite. In general, these metabolites are formed when large amounts of primary metabolite precursors, such as amino acids, acetate, pyruvate and others, are accumulated. They present some characteristics such as: restricted taxonomic distribution, that is, not all strains of the same species are capable of producing a certain metabolite and are not essential for the growth and reproduction of the organism.
  • Cultivation conditions especially the composition of the medium, control the formation of these metabolites which are produced as a group of interrelated structures. They can be overexpressed and are encoded by sets of expendable genes (JAY, 2005; KELLER et al., 2005; MARTIN et al., 2005; YU & KELLER, 2005; NIGAM, 2009).
  • MS are important for the organisms that produce them, functioning as sex hormones, ionophores, defense against bacteria, fungi, amoebae, insects and plants, symbiosis agent, differentiation effectors and unknown activities (DEMAIN & ADRIO, 2008). MS are usually separated into five groups: amino acid derivatives, non-ribosomal peptides, polyketides, fatty acid derivatives and polyketide-peptide hybrids (KEMPKEN & ROHLFS, 2010; ROZE et al., 2011). [007] The genes for the biosynthesis of secondary metabolites are usually organized in "clusters" in the producing strains.
  • clusters include, in addition to the genes encoding biosynthetic enzymes and regulatory proteins, genes for resistance to the toxic action of secondary metabolites (to prevent the suicide of the producing species) and genes for the secretion of these metabolites (MARTIN et al. , 2005). It is usual for different species of fungi to have one or more secondary metabolites in common, many of which are produced by phylogenetically very different fungi (FRISVAD et al., 2008).
  • annularins A-H ( Figure 1) and classified as polyketide metabolites (Li et al., 2003).
  • AF annularins are 3,4,5-tri-substituted a-pyrones and GH annularins are 3,4-disubstituted a-b-unsaturated g-lactones.
  • Annularins A, B, C and F inhibited the growth of Bacillus subtilus (ATCC 6051) by inducing an 8-10 mm halo with 200 pg / disc tested and at the same concentration, annularin C inhibited the growth of Staphylococcus aureus (ATCC 29213) with 14 mm halo. No compound showed action against C. albicans (ATCC 90029) and with the exception of compounds D and E (not tested) the others were not effective against A. flavus (NRRL 6541).
  • Annularins A-F are -pyrones, members of a general class commonly found in fungi.
  • a study for the synthesis of annularins was proposed by Motodate et al., (2010) and the total synthesis of annularin F was reported for the first time in 2006 (Kurdyumov et al., 2006).
  • the annularins A-F are presumably derived from the polyketide pathway and there is a hypothesis that the annularins G and H originate from an analogous biosynthetic pathway, but with a different stage of ring closure.
  • monocerine originates as heptacetide, which was demonstrated in a biosynthesis study of this molecule with the incorporation of 2 H, 13 C and 18 0 acetates in D. ravenelii (Scott et al., 1984).
  • the canopy plant is a small shrub endemic to the Caatinga, found in sandy soil with the presence of intense sun, absence of water nearby and is characterized by the presence of latex in the stem interior and intense aroma.
  • This plant was collected by our group, and a desiccant was deposited at the Herbarium of the State University of Feira de Santana (UEFS) under the number Tombo 161257.
  • the desiccant was identified as Croton blanchet ⁇ anus belonging to the Euphorb ⁇ aceae family by Botany Daniela Carneiro Santos Torres, from UEFS, specialist in the Euphorb ⁇ aceae Family.
  • the Euphorb ⁇ aceae family is one of the largest of the Angiospermae, with about 300 genera and approximately 7,500 species distributed worldwide. In Brazil this family of plants is widely distributed in different types of vegetation, but it is considered one of the most representative of the Caatinga. According to Albuquerque et al., (2002) the Euphorb ⁇ aceae family is used by the inhabitants of the Caatinga for medicinal purposes, such as, for example, the species of the genus Croton, such as C. argirophylloides (popular name: quince or sacatinga), C. rham ⁇ n ⁇ fol ⁇ us (popular name: canopy) and C. sonder ⁇ anus (popular name: quince). These plants are also used as insect repellent and as a source of wood.
  • C. argirophylloides popular name: quince or sacatinga
  • C. rham ⁇ n ⁇ fol ⁇ us popular name: canopy
  • C. sonder ⁇ anus popular name: quince
  • the endophytic fungus encoded as FV3 was isolated from the plant leaf and taxonomically identified by Molecular Biology techniques (Raeder and Broda 1985; Gonzáles-Mendoza et al., 2010) by our collaborator Prof. Wellington L. de Ara ⁇ jo of the Department of Microbiology at ICB / USP, as referring to the species Exserohilum rostratum.
  • This species has terrestrial and marine origin and can be found as a pathogen in marine invertebrates or as a phytopathogen causing diseases in fruits and plant roots (Sappapan et al., 2008). In Thailand this species has already been isolated as an endophytic from leaves of Stemona sp.
  • the present invention discloses the use of the secondary metabolites monocerin and annularin I in cell regeneration in HUVEC endothelial cells and normal FN1 fibroblasts. In this sense, the use of the secondary metabolites monocerine and annularin I in cell regeneration in HUVEC endothelial cells and normal FN1 fibroblasts is quite different from that proposed by any prior art document.
  • the present invention will provide significant advantages in relation to the use of the secondary metabolites monocerin and annularin I obtained from the culture of the fungus Exserohilum rostratum for use in cell regeneration, such as for example endothelial cells HUVEC, macrophage fibroblasts, etc., enabling an increase in their performance and presenting a more favorable cost / benefit ratio.
  • Figure 1 shows the structures of the annular compounds AH (1-8) described in the literature and obtained from the culture of the aquatic fungus Annulatascus triseptatus, monocerine (Fl) and anularin I (F2) obtained from the culture the fungus Exserohilum rostratum;
  • FIG. 2 shows the HUVEC endothelial cells in the process of cell multiplication.
  • Red arrows indicate cells in the process of cell multiplication and black cells in the process of apoptosis;
  • the graph indicates the chromatographic profile of the extract obtained from the culture of the fungus in potato dextrose (BD) medium of the compounds of the present invention.
  • the conditions of the analysis were in column C18 (250 x 4, 6 mm), isocratic elution system with 56.0% B in 30 minutes (solution A: H 2 0 / TFA, 99.9 / 0.1%; solution B: Me0H / H 2 0 / TFA, 90: 9.9: 0.1%) at a flow rate of 1 ml / min.
  • Figure 4 shows a graph of the chromatographic profile of the extract obtained from the culture of the fungus in Czapek medium of the compounds of the present invention.
  • the conditions of the analysis were in column C18 (250 x 4, 6 mm), isocratic elution system 56.0% B in 30 minutes (solution A: H 2 0 / TFA, 99.9 / 0.1%; solution B: MeOH / H 2 O / TFA, 90: 9.9: 0.1%), and with a flow rate of 1 mL / min.
  • Fl monocerine.
  • Obs. absence of annularin I.
  • Figure 5 shows a graph of the chromatographic profile of the extract obtained from the culture of the fungus in malt extract medium (Synth brand) of the compounds of the present invention.
  • the conditions of the analysis were in column C18 (250 x 4.6 mm), isocratic elution system 56.0% B in 35 minutes (solution A: H 2 0 / TFA, 99.9 / 0.1%; solution B: MeOH / fbO / TFA, 90: 9.9: 0.1%), and with a flow rate of 1 ml / min.
  • Figure 6 shows a graph of the chromatographic profile of the extract obtained from the fungus culture in a malt extract medium (Himedia brand).
  • the conditions of the analysis were in column C18 (250 x 4, 6 mm), isocratic elution system 56.0% B in 30 minutes (solution A: H 2 0 / TFA, 99.9 / 0.1%; solution B: MeOH / fbO / TFA, 90: 9.9: 0.1%), and with a flow rate of 1 ml / min.
  • Figure 7 shows a graph of the chromatographic profile of the extract obtained from the culture of the fungus in Minimum Medium of the compounds of the present invention.
  • the conditions of the analysis were in column C18 (250 x 4, 6 mm), in an isocratic elution system 56.0% of B in 35 minutes (solution A: H2O / TFA, 99.9 / 0.1%; solution B : Me0H / H 2 0 / TFA, 90: 9.9: 0.1%) at a flow rate of 1 ml / min.
  • Fl monocerine
  • F2 annularin I.
  • Figure 8 shows a graph of the chromatographic profile of the YPSS sample obtained from the fungus culture.
  • Figure 9 shows a graph of the chromatographic profile of the YESD sample.
  • Column C18 250 x 4.6 mm
  • isocratic elution system 56 0% B in 30 minutes
  • solution A H 2 0 / TFA, 99.9 / 0.1%
  • solution B Me0H / H 20 / TFA, 90: 9.9: 0.1%)
  • flow rate 1 mL / min.
  • Figure 10 shows a graph of the chromatographic profile of the PYG1 sample, on the HPLC in column C18 (250 x 4.6 mm), in an isocratic elution system 56.0% B in 30 minutes (solution A: H 2 0 / TFA 99.9 / 0.1%, solution B: Me0H / H 2 0 / TFA, 90: 9.9: 0.1%) at a flow rate of 1 ml / min.
  • Figure 11 shows a standard curve for monocerine (Fl fraction) at wavelengths of 214, 254 and 280 nm according to the concentrations of the injected monocerine.
  • Figure 12 shows a standard curve for annularin I (fraction F2) at wavelengths of 214, 254 and 280 nm, according to the concentrations of annularin I that was injected.
  • Figure 13 shows the cell viability of HUVEC endothelial cells after treatment with monocerine and annularin I at concentrations of 0.02 to 10 mM, at 24, 48 and 72 h. Zero equal to the RPMI 1640 control corresponding to 100% cell viability.
  • Figure 14 shows the cell viability of normal FN1 fibroblasts after treatment with monocerine and annularin I at concentrations of 0.02 to 10 mM, at 24, 48 and 72 h. Zero equal to the RPMI 1640 control corresponding to 100% cell viability.
  • Figure 15 shows the distribution of HUVEC endothelial cells in the phases of the cell cycle after treatment with annularin I in concentrations of 0.02; 0.15; 0.625 and 2.5 mM, at 6, 24, 48 and 72 h. Zero equal to the control treatment with RPMI 1640 culture medium;
  • Figure 16 shows a cell cycle analysis of HUVECs and FN1 fibroblasts treated with annularin I from 0.02 to 0.625 mM at 6, 24, 48 and 72 h.
  • the bars represent the proportions of G2 / M proliferative cells; in the synthesis of the S phase; Quiescent cell G0 / G1 and debris in sub-Gl (fragmented DNA).
  • the data represent mean ⁇ SD of three independent experiments. * Significantly different from the untreated control * p ⁇ 0.05; ** p ⁇ 0.01 and *** p ⁇ 0.001.
  • Figure 17 shows the distribution of HUVEC endothelial cells and normal FN1 fibroblasts in the cell cycle phases after treatment with the monocerine compound in concentrations of 0.02; 0.15 and 0.625 at 24, 48 and 72 h. Zero equal to the control treatment with RPMI 1640 culture medium.
  • Figure 18 shows the proliferative rate of normal human FN1 fibroblasts and endothelial cells (EC).
  • the treatment with annularin I was unable to alter the cell proliferation of fibroblast cells, but for EC the treatment changed the cell proliferative capacity at all treatments and times.
  • Figure 19 shows Western blotting representative of VEGF-R1 expression in HUVEC incubations with different concentrations of anularin I (1 and 2: RPMI; 3 and 4: 0.15 mM, 5 and 6: 0, 625 mM ) for 24 hours.
  • Glyceraldehyde triphosphate dehydrogenase (GAPDH) was used as loading control.
  • Figure 20 shows the relative expression of proteins in HUVEC cells after treatment with 0.15 and 0.625 mM of annularin I for 6 and 24 h. Results expressed by the proportion of proteins in relation to the controls (GADPH or b- Actin (mean ⁇ SEM) relativized with a value of 1.
  • Figure 21 shows senescent HUVEC endothelial cells after treatment with 0.02 annularin I; 0.15 and 0.625 mM. Assay performed by b-galactosidases. Mean ⁇ SD for 24 and 48 h. Statistical analysis using the One-way ANOVA / Dunnett multiple comparison test (between concentrations). significant index ** ** ** p ⁇ 0.01 for comparisons of treatment at 6 and 24 h and the control group.
  • Figure 22 shows the percentage of HUVEC endothelial cells and normal FN1 senescent and non-senescent fibroblasts after treatment with annularin I or monocerine in concentrations of 0.02 to 1.25 mM. Assay with b-galactosidase.
  • Figure 23 shows representative photomicrographs of endothelial cells treated with 0.02 (1B) annularin I 0.15 (1C), 0.625 mM (1D) for 24 h and 0.02 (2B) 0.15 ( 2C), 0.625 mM (2D) for 48 h.
  • 24 h and 48 h controls are represented by AI and 2A, respectively.
  • c 1.3 S non-senescent detected by the b-galactosidase assay.
  • Figure 25 shows the determination of apoptosis in HUVECs cells by double staining with annexin V-FITC / propidium iodide. Distribution of cells in viable cells, in the process of necrosis-like cell death, early or late apoptosis after treatment with 0.02 to 1.25 mM monocerine. The control corresponds to treatment with RPMI 1640 only.
  • Figure 26A shows the chromatographic profile of purification of the SPE100 monocerine fraction by the first step (A): Method: column C18 (250 x 10 mm), isocratic elution system 48.0% B in 60 minutes and flow rate 2.5 mL / min.
  • Figure 26B shows the purified monocerine after the second purification step; Method: column C18 (250 x 4.6 mm), isocratic elution system 56.0% B in 30 minutes and flow rate 1.0 mL / min.
  • Solution A H20 / TFA (99.9 / 0.1%)
  • solution B MeOH / H20 / TFA (90: 9.9: 0.1%).
  • Figure 27 shows a graphical representation of the wound size measured by the Pachymeter.
  • Wound size represented as mean ⁇ SD for groups of animals treated for 14 days.
  • n 80, data were analyzed by unidirectional ANOVA followed by multiple Tukey-Kramer comparisons using Graph pad Prism 5.1. The treatments were compared with the untreated group and there was no statistically significant difference.
  • Figure 28 shows a graphical representation of the wound size measured by the In Vivo X-ray image (MS-FX Pro).
  • the wound size is represented as mean ⁇ SEM for groups of animals treated for 14 days.
  • N 4 animals in each group.
  • Two animals from each group were used to measure the size of the wound by MS-Fx Pro.
  • the data were calculated using Graph pad Prism 5.1 and the treatments were compared with the untreated group.
  • Figure 29 shows an in vivo X-ray image for wounds performed by MS FX PRO. This figure represents the size of the wounds of two animals per group treated for 24 h, 48 h and 72 h. By comparison with the control groups (G1 and G5), it is possible to observe that the wounds of animals treated with 0.005% monocerine started to be well reduced after 72 h;
  • Figure 30 shows an in vivo X-ray image for wounds performed by MS FX PRO. This figure represents the size of the wounds of two animals per treated group for 7, 10 and 14 days. By comparison with the control groups (G1 and G5), it is possible to observe that the wounds of animals treated with 0.005% monocerine were almost completely healed after 10 days;
  • Figure 31 shows photomicrographs obtained by Scanning Electron Microscopy of the dermis of mice treated for 24 h, 72 h, 7 and 14 days.
  • Untreated control group Gl Maintenance of the extracellular matrix of the scar fragment from 24 hours to 14 days (red arrows); Collagenase group (G2): presence of numerous dead cells and erythrocytes identified from 24 hours to 14 days (blue arrows); Monocerine 0.0006% (G3): strong formation of collagen fibers; Monokerin 0, 005% (G4): Collagen fibers (yellow arrow) - fibronectins observed from 24 to 14 days (yellow arrow); Vehicle (G5): microfibrils, red blood cells, inflammatory cells (green arrow) until the 14th day;
  • Figure 32 shows staining of animal tissues from 24 hours of experiments. Magnitude of 10x. A) Hemotoxilin and Eosin (H&E); B) Pricosirius Red (PR) and C) Masson Trichome (MT) staining. Control Group (Gl), Collagenase (G2), monocerine 0.0006% (G3), monocerine 0.005% (G4) and vehicle (G5).
  • H&E Hemotoxilin and Eosin
  • PR Pricosirius Red
  • MT Masson Trichome
  • Figure 33 shows the staining of animal tissues after 72 h of experiments. Magnitude of 10x.
  • Figure 34 shows the staining of animal tissues from 7-day experiments. Magnitude of 10x. A) Hemotoxilin and Eosin (H&E); B) Pricosirius Red (PR) and C) Masson Trichome (MT) staining. Control Group (Gl), Collagenase (G2), monocerine 0.0006% (G3), monocerine 0.005% (G4) and vehicle (G5);
  • Figure 35 shows the staining of animal tissues from 7-day experiments. Magnitude of 10x. A) Hemotoxilin and Eosin (H&E); B) Pricosirius Red (PR) and C) Masson Trichome (MT) staining. Control Group (Gl), Collagenase (G2), monocerine 0.0006% (G3), monocerine 0.005% (G4) and vehicle (G5);
  • Figure 37 shows photomicrographs of Balb / C mouse skin stained with Picrosirius red and evaluated by polarized microscopy using a standardized program (microimaging software) Zen Blue 2.6 for image capture. The images were captured for 24 h, 72 h, 7 and 14 days treatment, allowing the observation of all skin constituents. Birefringence of hair (shades of green) and collagen (x10). Ep: epidermis; C: collagens; D: dermis; Gs: sebaceous glands; Gp: sweat glands; *: hair; arrow: pili arrector muscle.
  • Figure 38 shows the hematological parameters for individual types of blood cells.
  • the indicators related to red blood cells and immune cells belonging to the class of leukocytes were analyzed in peripheral blood from different time intervals 24h. 7d and 14 d.
  • the following groups are illustrated by different column colors. Control group (white); Collagenase (gray); Monocerin 0.0006% (red); Monocerin 0.005% (blue); Vehicle (black).
  • Figure 39 shows the hematological analysis for individual types of blood cells.
  • the indicators related to red blood cells and immune cells belonging to the leukocyte class were analyzed in peripheral blood at different time intervals 24h, 7d and 14d.
  • the following groups illustrate the different colors.
  • Control group (white); Collagenase (gray); Monocerin 0.0006% (red); Monocerin 0.005% (blue); Vehicle (black).
  • Figure 40 shows a photomicrograph of tissues immunostained with antibody against VEGF in the control and treated groups for 24 h. For all groups, it is possible to observe immunoreactivity against VEGF (arrows), while for negative controls there was no immunoreactivity as expected; The exception is for the negative control reaction for the Gl group, which was also immunoreactive, but of low intensity, as expected.
  • Scale bar 40 x 100 pm. The data were analyzed by measuring the pixels of the 1024 x 2048 pixel scale bar.
  • Figure 41 shows a graphical representation of the area% of histointensity immune to VEGF calculated by Image j and obtained data calculated by Origin 9.1.
  • Figure 43 shows a graphical representation of the wound size measured by the In Vivo X-ray image (MS-FX Pro).
  • the wound size is represented as mean ⁇ SEM for groups of animals treated for 14 days.
  • N 4 animals in each group.
  • Two animals from each group were used to measure the size of the wound by MS-Fx Pro.
  • the data were calculated using Graph pad Prism 5.1 and the treatments were compared with the untreated group.
  • Figure 44 shows an in vivo X-ray image for wounds performed by MS FX PRO. This figure represents the size of the wounds of one animal per group treated for 24 h, 72 h, 7, 10 and 14 days. By comparison with the control groups, it can be seen that the wounds of animals treated with 0.003% and 0.0005% anularin I were almost completely healed after 10 days.
  • Figure 45 shows hematological parameters for individual types of blood cells.
  • the indicators related to red blood cells and immune cells belonging to the class of leukocytes were analyzed in peripheral blood from different time intervals of 24 h, 7 and 14 days. The following groups are illustrated by different column colors. Control group (white); Collagenase (gray); Annularin I 0.003% (red); Annularin I 0.0005% (blue); Vehicle (black).
  • Figure 46 shows hematological parameters for individual types of blood cells.
  • the indicators related to red blood cells and immune cells belonging to the class of leukocytes were analyzed in peripheral blood from different time intervals of 24 h, 7 and 14 days. The following groups are illustrated by different column colors. Control group (white); Collagenase (gray); Annularin I 0.003% (red); Annularin I 0.0005% (blue); Vehicle (black).
  • Figure 47 shows photomicrographs obtained by SEM of the dermis of mice treated for 24 h, 72 h, 7 and 14 days.
  • Untreated control group Gl maintenance of the extracellular matrix of the scar fragment from 24 hours to 14 days (red arrows);
  • Collagenase group (G2) presence of numerous dead cells and erythrocytes identified from 24 hours to 14 days (blue arrows);
  • 0.003% annularine (G3) strong formation of collagen fibers;
  • Annularin 0, 0005% G4: Collagen fibers (yellow arrow) - fibronectins observed from 24 hours to 14 days (yellow arrow);
  • Vehicle (G5) microfibrils, red blood cells, inflammatory cells (green arrow) until the 14th day.
  • Figure 48 shows the staining of animal tissues after 24 hours of experiments. Magnitude of 10x. A) Hemotoxilin and Eosin (H&E); B) Picrosirius Red (PR) and C) Masson Trichome (MT) staining. Control Group (Gl), Collagenase (G2), Annularine 0.003% (G3), Annularine 0.0005% (G4) and vehicle (G5).
  • H&E Hemotoxilin and Eosin
  • PR Picrosirius Red
  • MT Masson Trichome
  • Figure 49 shows the staining of animal tissues after 72 h of experiments. Magnitude of 10x.
  • H&E Hemotoxilin and Eosin
  • PR Picrosirius Red coloring
  • MT Masson Trichome
  • Control Group Gl
  • Collagenase G2
  • Annularine 0.003%
  • G4 Annularine 0.0005%
  • Vehicle G5
  • Figure 50 shows the staining of animal tissues from 7-day experiments. Magnitude of 10x. A) Hemotoxilin and Eosin (H&E); B) Picrosirius Red (PR) and C) Masson Trichome (MT) staining. Control Group (Gl), Collagenase (G2), Annularine 0.003% (G3), Annularine 0.0005% (G4) and Vehicle (G5).
  • H&E Hemotoxilin and Eosin
  • PR Picrosirius Red
  • MT Masson Trichome
  • Figure 51 shows the staining of animal tissues from 14-day experiments. Magnitude of 10x. A) Hemotoxilin and Eosin (H&E); B) Picrosirius Red (PR) and C) Masson Trichome (MT) staining. Control Group (Gl), Collagenase (G2), Annularine 0.003% (G3), Annularine 0.0005% (G4) and Vehicle (G5).
  • H&E Hemotoxilin and Eosin
  • PR Picrosirius Red
  • MT Masson Trichome
  • Figure 52 shows a 3D graphic illustration of fabrics with Picrosirius red coloring from 24h to 14 days.
  • Ep epidermis
  • C collagens
  • D dermis
  • Gs sebaceous glands
  • Gp sweat glands
  • * hair
  • arrow pectoral arrector muscle
  • Figure 53 shows the percentage of collagen formation induced by treatments with annularin I up to 14 days. Analysis using the J 5.1 image and data calculated using a 5.1 graphic prism.
  • Figure 54 shows the percentage of collagen formation induced by treatments with monocerine up to 14 days. Analysis using the J 5.1 image and data calculated using a 5.1 graphic prism.
  • Figure 55 shows a 3D graphic representation of tissues using a polarized microscope. Quantification of collagen formation by 0.0006% and 0.005% monocerin and 0.0005% and 0.003% anularin I treatments. The area occupied by collagen fibers was quantified using Image J and Graph Pad Prism 5.1 software. Collagen fibers exhibited polarizing colors in red-orange, while for controls, collagen fibers were sparse and immature. The control of the untreated group showed intense green - yellow birefringence, suggesting that the collagen content was reduced and its fibers were very loosely compacted. Green fibers - typical of type III collagen were most frequently located in the upper dermis and epidermis in the groups treated with anularin.
  • control groups control, collagenase and vehicle
  • had thick red and yellow fibers typically of type I collagen located in the lower deep dermis.
  • the groups treated with monocerine and annularin I showed an arrangement of visible and organized collagen fibers from the groups treated with annularin.
  • the present invention relates to the use of secondary metabolite produced by the fungus Exserohilum rostratum in cell regeneration.
  • the present invention relates to the use of the secondary metabolites monocerin and annularin I obtained from the culture of the fungus Exserohilum rostratum for cell regeneration having as reference the effect on HUVEC endothelial cells and normal FN1 fibroblasts.
  • the present invention refers to the use of secondary metabolites, such as monocerine and annularin I produced by the fungus Exserohilum rostratum, as an agent that induces cell proliferation, a process of fundamental importance in tissue repair, as, for example, in wound healing.
  • secondary metabolites such as monocerine and annularin I produced by the fungus Exserohilum rostratum
  • the fungus E. rostratum was grown in potato dextrose culture medium for 15 days at 28 ° C and 150 rpm, and the culture broth / supernatant was subjected to solid phase extraction (Solid Phase Extraction - SPE) with C18 cartridge (Spe-ed SPE Cartridges - Octadecyl C18 / 18%), and the retained material was extracted with 100% methanol resulting in the crude extract.
  • This extract showed antifungal action against Cryptococcus neoformans ATCC 90112 r Trichophytum rubrum IOC 4527, Candida albicans ATCC 36802 and Aspergillus fumigatus IOC 4526 (CLSI, 2002a, b) with minimum inhibitory concentrations of 31.25 to 500 pg / mL (Table 1) .
  • the crude extract was subjected to high performance liquid chromatography (HPLC) in a C8 column (250 x 10 mm) and an isocratic elution system ranging from 44 to 60% with a flow of 2.5 to 4 mL / min (solution A: H 2 0 / TFA, 99.9 / 0.1%; solution B: Me0H / H 2 0 / TFA, 90: 9.9: 0.1%) and two main fractions were obtained and refurified using phenyl column (250 x 4.6 mm) and 44 to 60% isocratic elution.
  • HPLC high performance liquid chromatography
  • the pure F1 and F2 fractions were evaluated for antifungal action, and physicochemically characterized by 1 H and 13 C nuclear magnetic resonance and mass spectrometry as the monocerine compounds (2S, 3aR, 9bR) - 6-hydroxy -7,8-dimethoxy-2-propyl-2,3,3a, 9b-tetrahydro-5H-bore [3, 2-c] isochromen-5-one, and 4-methoxy-5-methyl- 6 annularin -butil-2H-piran-2-one, respectively, according to the structures represented in Figure 1.
  • the characterization was carried out with the collaboration of Prof. Roberto Berlinck from IQSC / USP.
  • annularin I This annularin was also obtained by other researchers and named as annularin I (Pinheiro et al, 2016). Following the nomenclature of the group of annularins previously described (AH), in the sequence this would be annularin I. This molecule differs structurally from the annularins AH previously described and has an additional CH 2 compared to annularin D.
  • Figure 1 shows the structures of the annular compounds AH (1-8) described in the literature and obtained from the culture of the aquatic fungus Annulatascus triseptatus, monocerine (Fl) and annularin I (F2) obtained from the culture of the fungus Exserohilum rostratum.
  • Annularins A, B, C and F inhibited the growth of Bacillus subtilus (ATCC 6051) by inducing an 8-10 mm halo with 200 pg / disc tested and at the same concentration, annularin C inhibited the growth of Staphylococcus aureus (ATCC 29213) with 14 mm halo. none compound showed action against C. albicans (ATCC 90029) and with the exception of compounds D and E (not tested) the others were not effective against A. flavus (NRRL 6541).
  • Annularins A-F are ⁇ -pyrones, members of a general class commonly found in fungi.
  • a study for the synthesis of annularins was proposed by Motodate et al. , (2010) and the total synthesis of annularin F was first reported in 2006 (Kurdyumov et al., 2006).
  • the annularins A-F are presumably derived from the polyketide pathway and there is a hypothesis that the annularins G and H originate from an analogous biosynthetic pathway, but with a different stage of ring closure.
  • Monocerine inhibited the growth of the dermatophyte fungus T. rubrum, the yeast Cand ⁇ da albicans and the filamentous fungus Aspergillus fumigatus with MICs of 15 to 500 gg / mL.
  • Annularin I inhibited the growth of the dermatophyte T. rubrum only at 250 gg / mL, and was not effective on C. albicans and A. fumigatus until the concentration of 500 gg / mL (see Table 1).
  • cytotoxic activity was evaluated in cells of the tumor line K562 (chronic erythrocytic leukemia) and B16F10 (murine melanoma) by the MTT assay that assesses cell viability by the action of the mitochondrial succinate dehydrogenase enzyme (Mosmann, 1983).
  • Figure 2 shows the HUVEC endothelial cells in the process of cell multiplication. Control (A) and cell treatments with annularin I in mM, B (0.07), C (0.14), D (0.28), E (0.56), and F (1.12) . Red arrows indicate cells in the process of cell multiplication and black cells in the process of apoptosis.
  • annularin I as an agent that induces cell proliferation
  • a process of fundamental importance in tissue repair such as, for example, in wound healing, new tests were performed in HUVEC endothelial cells and in normal FN1 fibroblasts.
  • the fungus E. rostratum was grown in a plate containing potato dextrose agar (BDA) culture medium for 7 days, and in an oven at 28 ° C. Subsequently, with the help of disposable and sterile Pasteur loops, the colony was fragmented and carefully transferred to a 250 mL Erlenmeyer flask containing 50 mL of liquid culture media: Dextrose Potato (BD), Czapek Dox CD Medium), Minimal Medium (MM), Malt Extract 2% brand Synth (EMS), Malt Extract 2% brand Himedia (EMH), YPSS, YESD and PYG. The culture was performed in duplicate for 15 days at 28 ° C, and at 150 rpm.
  • BDA potato dextrose agar
  • the fungal suspension was filtered through a polyester or cotton membrane (gauze) (filtration 1) and later on glass wool (filtration 2) to remove the highest proportion of biomass, and the pH of the supernatant was measured with a pH indicator strip.
  • the culture supernatant (filtrate 2) was transferred to 500 ml plastic bottles and frozen at -20 ° C for at least 48 hours.
  • the freezing stage is very important for the precipitation of small particles such as spore residues and / or cells not removed in filtration 2.
  • filtrate 2 was subjected to a new filtration in a glass fiber membrane with 1 mpi porosity (filtration 3) and then subjected to solid phase extraction (EFS).
  • EFS solid phase extraction
  • C18 cartridges (Octadecyl C18 / 18% - 50 mg / 6mL) were conditioned with 100% methanol PA, washed with distilled water and filtrate 3 was added to the cartridge with the aid of a vacuum pump .
  • the product retained in the cartridge was extracted with 100% P.A. methanol resulting in the crude extract.
  • the extract solvent was completely removed by rotary evaporation under vacuum in a water bath at a maximum temperature of 45 ° C.
  • Extraction can also be by liquid-liquid extraction (ELL) using solvents such as acetate ethyl, hexane, dichloromethane, etc. as well as the extraction can also be in solid phase using Octadecyl C18 / 18% discs.
  • Octadecyl C18 / 18% extraction would be efficient using cartridges or discs with any of the commercially available sorbents based on organic groups, such as C18, C8, C4, C2, cyclohexyl, phenyl, cyanopropyl, aminopropyl (NH2), chemically linked to silica.
  • Figure 4 represents the chromatographic profile of the extracts obtained from the cultures in Czapek Dox (MC) medium. It can be seen that the extract did not contain MS beyond those expected, however, it did not induce the production of the compound annularin I.
  • Figure 5 shows the chromatographic profile of the extract obtained from the culture in malt extract medium (brand Synth). Column C18 (250 x 4, 6 mm), isocratic elution system 56.0% B in 35 minutes (solution A: H 2 0 / TFA,
  • Figure 7 represents the chromatographic profile of the extract obtained from the minimal culture medium (MM).
  • MM culture medium in addition to the monocerine and annularin I compounds, other compounds were detected, but annularin I was detected with low absorbance, indicating low concentration.
  • Figures 8, 9 and 10 represent the chromatographic profile of extracts obtained from cultures in YPSS, YESD and PYG media, respectively. For these extracts, the fractions were detected, but with very low absorbances indicating the low concentration of the compounds present in the extract. In addition, other compounds not identified by our group were also detected. Quantification of monocerine and annularin I compounds produced in different culture media
  • mAU milli absorbance
  • the wavelength of 214 nm was the one with the highest resolution and, therefore, was used for the quantification of these compounds in the extracts, respectively.
  • Table 13A Calculation of the average percentage of monocerine produced by the culture of the fungus Exserohilum rostratum in different culture media. Calculation considering all data in Table 16.
  • Table 15A Calculation of the average percentage of annularin I produced by the culture of the fungus Exserohilum rostratum in different culture media. Calculation considering all positive data in Table 17.
  • the flasks were removed from the culture and the biomass removed by filtration on a polyester membrane (filtration 1), and then on a 50 mm glass fiber membrane and a porosity of 1 mpi (filtration 2) with vacuum pump aid.
  • the culture supernatants were applied in 500 mg / 6 mL solid-phase extraction cartridges (SPE) previously conditioned (with three applications of pure methanol and distilled water), respectively, and the extraction of the retained material was carried out with methanol. at 25, 50, 75 and 100%, without TFA. For 25, 50 and 75% a volume of column for extraction. For 100%, three column volumes were used, leaving the silica to dry at the end of the procedure.
  • SPE solid-phase extraction cartridges
  • the monocerine and annularin I compounds present in the SPE fractions were purified by HPLC on a C18 or phenyl column (250x46 mm) in an isocratic method of 48-50% mobile phase B in 50 or 55 minutes with detection at 214, 254 and 280 nm.
  • the pool resulting from the collections was submitted to a rotary evaporation to eliminate the solvent and obtain the compounds completely pure and dry.
  • the compounds monocerine and annularin I were not extracted by extraction with 25% methanol, and therefore this percentage of methanol can be used to remove residues of culture medium that may have been retained in the stationary phase of the cartridge.
  • the compounds obtained from the SPE75 and SPE100 fractions of each culture time were purified and the mass quantified for analysis of the culture time with higher yield in the production of the compounds.
  • the most suitable culture time is 15 days because it favors the production of monocerine and annularin I compounds.
  • the set of results indicates that monocerin induces greater cytotoxic effects compared to annularin I in endothelial cells, being able to modify the proportion of cells in G0 / G1 and cells with fragmented DNA.
  • Annularin I was shown to be a compound with the potential to induce proliferation in endothelial cells, maintaining the proportion of cells in G0 / G1, decreasing cells with fragmented DNA and inducing senescence which may mean the maintenance of proliferating cells. These effects were accompanied by the differential expression of proteins that regulate the control points and progression of the cell cycle.
  • the endothelial cells used in the study are of the ATCC CRL-1730 / HUVEC lineage, and the cells of normal human fibroblasts FN1 were in a previous study isolated from eyelid obtained from blepharoplasty surgery at the Faculty of Medicine of the University of S ⁇ o Paulo, and the procedures approved by the Ethics Council (Ethics Committee for Analysis of Research Projects at HCFMUSP - CAPPesq HCFMUSP No. 921/06). These cells were established in culture, frozen and stored for use indefinitely after reactivation in culture.
  • the cells were cultured at 37 ° C in RPMI-1640 medium containing supplements (SFB 5-10%, penicillin and streptomycin), and in a humid atmosphere and 5% CO2. After subconfluence, the cells were processed and resuspended in RPMI-1640 culture medium with SFB and antibiotics. For each assay, cell concentration was adjusted according to the necessary recommendation.
  • the monocerine and annularin I compounds were dissolved in dimethyl sulfoxide, filtered through a 0.22 pm regenerated cellulose membrane and stored in aliquots at -20 ° C. At the time of use, the compounds were diluted in 10 mM sodium phosphate buffer (PBS), and then in RPMI 1640 culture medium (without SFB and with antibiotics) in the desired concentrations for each assay.
  • PBS sodium phosphate buffer
  • RPMI 1640 culture medium without SFB and with antibiotics
  • the HUVEC and FN1 cells in RPMI-1640 culture medium were plated in 96 well plates and flat bottom, in the concentration of 1x10 4 cells / well. After adhesion, the culture medium was removed and the cells were treated with monocerine or annularin I in concentrations of 0.02 to 10 mM. After 24, 48 and 72 h cell viability was assessed by absorbance reading at 540 nm according to the protocol previously described (Mosmann, 1983) and figures 13 and 14 represent the values of the percentages of viable cells after treatments.
  • HUVEC cells become more sensitive to treatment with annularin I only within 72 h and for the concentration of and / or above 1.25 mM. For 0.625 mM, around 88% of the cells remained viable at all times of treatment. For 24 h, cell viability was 71.34; 65.74 and 55.97 for treatments with 1.25; 2.5 and 5 mM, respectively, as shown in Table 17.
  • Table 17 Representation of the percentage of viable HUVEC cells by the MTT method (mean ⁇ SD), after treatment with annularin I and monocerine in concentrations of 0.02 to 10 mM.
  • HUVEC endothelial cells were little sensitive to the action of monocerin. In 24 h, cell viability was different from the control treatment only for 10 mM and for 48 h, from 2.5 mM.
  • cell viability shows close values for both 24 h and 48 h. From 0.625 mM, cell viability is statistically different from that of the control group at 24 h, and for 48 h only at 10 mM. For the treatment of 72 h cell viability is reduced from 0.08 mM of annularin. For 2.5 mM of annularin there was 58.13 ⁇ 9.94%; 69.19 ⁇ 10.54% and 68.5318.31% of viable cells, with treatments for 24, 48 and 72 h, respectively as shown in Table 18.
  • Table 18 Representation of the percentage of viable FN1 fibroblasts by the MTT method (mean ⁇ SD), after treatment with annularin I and monocerine in concentrations of 0.02 to 10 mM.
  • Figure 13 shows cell viability data of HUVEC endothelial cells and normal FN1 fibroblasts after treatment with monocerine in concentrations of 0.02 to 10 mM, at 24, 48 and 72 h. Zero equal to the RPMI 1640 control corresponding to 100% cell viability.
  • Figure 14 shows cell viability data of HUVEC endothelial cells and normal FN1 fibroblasts after treatment with annularin I at concentrations of 0.02 to 10 mM, at 24, 48 and 72 h. Zero equal to the RPMI 1640 control corresponding to 100% cell viability.
  • Statistical analysis by One-way ANOVA / Dunnett ' s multiple comparison test (between concentrations).
  • HUVEC and FN1 cells (2x10 5 cells / well, 6-well plate) were treated with 0.02 annularin I; 0.15; 0, 625 and 2.5 mM, and with 0.02 monocerine;
  • the cells were centrifuged and the pellet was homogenized with buffer containing Triton X-100, RNAse and propidium iodide, and analyzed in a flow cytometer FACsCalibur (BD).
  • BD flow cytometer
  • the acquisition of the cell population, on average of 10,000 events was performed by the program "Cell Quest” (BD) and the DNA content measured by the fluorescence intensity was analyzed using the ModFit software version 4.0.
  • the results are expressed as an average percentage of cells in the different phases of the cell cycle: Quiescent phase G0 / G1, Synthesis phase - S and Phase G2 / M (mitosis).
  • Figure 15 shows the distribution of HUVEC and FN1 endothelial cells in the phases of the cell cycle after treatment with annularin I in concentrations of 0.02; 0.15; 0.625 and 2.5 mM, at 6, 24, 48 and 72 h. Zero equal to the control treatment with RPMI 1640 culture medium.
  • CFSE-DA is an intracellular fluorescent marker that is divided equally between daughter cells. In this system, cell division can be evaluated in multiple generations by flow cytometry, allowing the identification of up to 10 generations in vitro and in vivo.
  • the entry of the CFSE-DA dye into the cells is due to the deacetylated form that makes it permeable and allows rapid flow through the plasma membrane. Esterases present in the cells cleave the acetate resulting in the CFSE form which is much less permeable and which is concentrated inside the cell.
  • the CFSE-DA and CFSE forms have the amine-reactive succinimidyl side chain, but only the CFSE form is fluorescent.
  • the high intracellular concentration of CFSE facilitates rapid, and high level of intracellular protein labeling. Cell labeling must occur quickly to obtain a homogeneously labeled cell population, which is critical in differentiating cells that have passed through various cell divisions (Quah and Parish, 2010; Lyons et al., 2013.).
  • the marker diffuses into the cell and binds covalently to intracellular amines, resulting in a very stable fluorescent label that can be fixed with aldehydes.
  • the excess of the unconjugated reagent diffuses passively into the extracellular medium and is removed with the supernatant by centrifugation.
  • Emission and excitation peaks after hydrolysis are at 492 and 517 nm, respectively.
  • the labeled cells can be analyzed by flow cytometry on equipment with an excitation source at 488 nm.
  • cells were labeled with CFSE-DA (1 pL of 5 mM CFSE-DA to 1 mL of suspension containing 1x10 s cells resulting in the final concentration of 5 nM of the marker) and transferred to 6-well plates in the concentration of 1 x 10 5 cells / well. After cell adhesion, cells were treated for 24, 48 and 72 h with 0.02 to 2.5 mM annularin I and monocerin diluted in RPMI-1640 culture medium without SFB.
  • the cells were trypsinized, centrifuged and the supernatant discarded.
  • the cell pellet was resuspended in 1 mL of buffer for flow cytometry and stored in a refrigerator for further analysis.
  • the analyzes were performed using the percentage of responsive cells and the number of divisions of each cell analyzed, broken down according to the content of CFSE-DA by flow cytometry in FACSCalibur® cytometer (Becton-Dickinson Immunocytometry Systems, San Jose , CA, USA) and analyzed by acquisition and analysis in the CellQuest Pro Modfit Becton-Dickson program (Modfit-BD).
  • Figure 17 shows the cell proliferation index by mean ⁇ SD for HUVEC endothelial cells and FN1 fibroblasts treated with annularin I and monocerine in concentrations of 0.02 mM to 2.5 mM at times of 6, 24, 48 and 72 h.
  • CFSE-DA is an intracellular fluorescent marker that is divided equally between daughter cells. In this system, cell division can be evaluated in multiple generations by flow cytometry, allowing the identification of up to 10 generations in vitro and in vivo.
  • the entry of the CFSE-DA dye into the cells is due to the deacetylated form that makes it permeable and allows rapid flow through the plasma membrane. Esterases present in the cells cleave the acetate resulting in the CFSE form which is much less permeable and which is concentrated inside the cell.
  • the CFSE-DA and CFSE forms have the amine-reactive succinimidyl side chain, but only the CFSE form is fluorescent.
  • the high intracellular concentration of CFSE facilitates rapid, and high level of intracellular protein labeling. Cell labeling must occur quickly to obtain a homogeneously labeled cell population, which is critical in differentiating cells that have passed through various cell divisions (Quah and Parish, 2010; Lyons et al., 2013).
  • the marker diffuses into the cell and binds covalently to intracellular amines, resulting in a very stable fluorescent label that can be fixed with aldehydes.
  • the excess of the unconjugated reagent diffuses passively into the extracellular medium and is removed with the supernatant by centrifugation.
  • the emission and excitation peaks after hydrolysis are at 492 and 517 nm, respectively.
  • the labeled cells can be analyzed by flow cytometry on equipment with an excitation source at 488 nm.
  • the cells were labeled with CFSE-DA (1 pL of 5 mM CFSE-DA to 1 mL of suspension containing 1 x 106 cells resulting in the final concentration of 5 nM of the marker) and transferred to 6-well plates in the concentration of 1 x 10 5 cells / well. After cell adhesion, cells were treated for 24, 48 and 72 h with 0.02 to 2.5 mM annularin I and monocerin diluted in RPMI-1640 culture medium without SFB.
  • the cells were trypsinized, centrifuged and the supernatant discarded.
  • the cell pellet was resuspended in 1 mL of buffer for flow cytometry and stored in a refrigerator for further analysis.
  • the analyzes were performed using the percentage of responsive cells and the number of divisions of each cell analyzed, broken down according to the content of CFSE-DA by flow cytometry in FACSCalibur® cytometer (Becton-Dickinson Immunocytometry Systems, San Jose , CA, USA) and analyzed by acquisition and analysis in the CellQuest Pro Modfit Becton-Dickson program (Modfit-BD).
  • Figure 17 shows the cell proliferation index by mean ⁇ SD for HUVEC endothelial cells and FN1 fibroblasts treated with anularin I at concentrations of 0.02; 0.15 and 0.625 mM at 24, 48 and 72 h.
  • Figure 18 shows the cell proliferation index by mean ⁇ SD for HUVEC endothelial cells and FN1 fibroblasts treated with monocerine at concentrations of 0.02 and 0.15 mM at 24, 48 and 72 h.
  • HUVEC endothelial cells (8x10 10 cells / well, 6-well plate) were treated with 0.15 and 0.625 mM annularin I for 6 and 24 h. After treatment, the cells were washed with PBS and lysed with 400 pL of RIPA buffer / well (containing protease and phosphatase inhibitors and EDTA) in an ice bath. After centrifugation, the protein concentration was determined in the protein lysate and stored at -80 ° C until use.
  • proteins were transferred to a polyvinylidene fluoride (PVDF) membrane for 30-45 min with a voltage of 20 V in a semi-dry system using transfer buffer (12 mM Tris, 96 mM Glycine, 20% methanol , pH 8.3). After transfer, the membrane was blocked with blocking buffer (1% BSA in TBS-T) at 4 ° C for about 12 hours (overnight) and washed 3 times with TBS-T, 5 min each under constant agitation of 60 rpm. The first antibody (Abcam) was diluted in 1% BSA in TBS-T.
  • blocking buffer 1% BSA in TBS-T
  • the membrane was placed in contact over its entire surface with an adequate amount of the antibody solution and incubated at room temperature for 1 h or at 4 ° C for about 12 hours (overnight) under constant agitation, depending on the antibody. Then the membrane was washed 3 times with TBS-T and incubated with the second anti-rabbit or anti-mouse antibody (Thermo brand) as before, for 1 hour at room temperature. The membrane was washed again with TBS-T for 3 times and the markings revealed using the SuperS ⁇ gnal® West Dura Extended Duration Substrate reagent (Thermo Scientific) and the digital imaging equipment ChemiDoc MP (Bio-Rad Lab) with chemiluminescence detection. The densitometry of the bands corresponding to the labeling of the proteins with the antibodies were performed using the Image Lab software (Bio-Rad Lab).
  • HUVEC endothelial cells were treated with annularin I at 0.15 and 0.625 mM for 6 and 24 h and protein expression was evaluated for all proteins listed in Table 21 using GADPH or b-actin proteins as loading control. .
  • Figure 19 shows a Western blotting representative of VEGF-R1 expression in HUVEC incubations with different concentrations of anularin I (1 and 2: RPMI; 3 and 4: 0.15 mM, 5 and 6: 0, 625 mM) for 24 hours.
  • Glyceraldehyde triphosphate dehydrogenase (GAPDH) was used as loading control.
  • Figure 22 shows the expression proteins by the mean ⁇ SEM.
  • senescence-associated b-galactosidase (SA-Bgal) (Dimri et al., 1995) is a marker commonly used for cells in senescence. This activity derives from the overexpression of lysosomal acid ⁇ -galactosidase.
  • HUVEC endothelial cells and normal FN1 fibroblasts (1x10 5 cells / well, 12-well plate), after adhesion, were treated with annularin I or monocerine in concentrations of 0.02 to 1.25 mM in triplicate, and treatments maintained for 6, 24, 48 or 72 h.
  • Control cells received only culture medium (without SFB and with antibiotics). The treatments were removed, the cells carefully "washed” with PBS and fixed with fixation buffer (Sigma Code F1797) for 6-7 min at room temperature (0.6 mL / well).
  • the cells were marked with 0.5 ml of the b-galactosidase solution / well (Kit Sigma code CS0030), kept in an oven at 37 ° C without CO2 until they stained blue (time of 2 -10 h).
  • the b-galactosidase solution was removed, the cells were covered with 70% glycerol and kept in the refrigerator until observation under an optical microscope. For each well, images from three fields were photographed, and cells stained blue (senescent) or without staining (non senescent) were counted.
  • results were expressed as a percentage of senescent and non-senescent cells, for each concentration tested in the two cell lines.
  • HUVEC endothelial cells and normal FN1 fibroblasts treated with annularin I or monocerin in concentrations of 0.02 to 1.25 mM were evaluated with respect to induction of senescence.
  • annularin I induced greater senescence in HUVECs cells, mainly to 0, 625 mM, and consequently there is a decrease in the percentage of non-senescent cells.
  • Table 21 Percentage of senescent and non-senescent HUVEC endothelial cells after treatment with anularin I in concentrations of 0.02; 0.15 and 0.625 mM.
  • Table 23 Percentage of senescent and non-senescent HUVEC endothelial cells after treatment with 0.02 to 1.25 mM monocerin (mean ⁇ SD).
  • NS not senescent
  • S senescent
  • SD standard deviation
  • Table 24 Percentage of normal senescent and non-senescent FN1 fibroblasts after treatment with 0.02 to 1.25 mM monocerin (mean ⁇ SD).
  • FN1 normal FN1 fibroblasts
  • NS not senescent
  • S senescent
  • SD standard deviation
  • Figure 22 shows the percentage of HUVEC endothelial cells and normal senescent and non-senescent FN1 fibroblasts after treatment with monocerine in concentrations of 0.02 to 1.25 mM. Assay with b-galactosidase.
  • Figure 23 shows representative photomicrographs of endothelial gutters treated with annularin
  • Annexin V is a phospholipid with vascular anticoagulant activity that is found in greater proportion in the cytosolic face of cell membranes.
  • the utility of annexin V in flow cytometry applications is derived from its selective affinity for negatively charged phospholipids.
  • Annexin V / FITC is usually used in association with a vital cell marker, such as propidium iodide (PI).
  • PI propidium iodide
  • Viable cells have an intact plasma membrane, and thus they are able to exclude the vital dye PI, while the membranes of dead cells are permeable to the dye, which is incorporated into the DNA.
  • HUVECs cells were plated at 4x10 5 cells / well (in a 6-well plate), and after adhesion they were treated with 0.02 to 1.25 mM monocerin (in RPMI 1640 without SFB and antibiotics), in sextuplicate and for 24 h. After trypsinization, the cells were transferred to 2 mL tubes and processed for labeling with annexin V and propidium iodide according to the manufacturer's protocol (Appendix-V-FITC Molecular Probes) and in the absence of light direct. The cells were resuspended in 250 pL of FACs buffer with 4% paraformaldehyde and the reading was performed in a flow cytometer.
  • untreated and unmarked HUVEC cells untreated cells up to the annexin V labeling step, untreated cells up to the propidium iodide labeling stage and untreated and annexed V labeled cells and propidium iodide were used.
  • Figure 24 illustrates the classification of cells according to the annexin V / propidium iodide labeling, and the results show that even at the concentration of 1.25 mM the cells are viable, in a state of necrosis, apoptosis initial or late in the proportion of 91.53%, 2.28%, 3.65% and 0.53%, respectively (Table 27, figure 25). This data is in agreement with the results found in the cell viability assay by MTT and senescence after treatment of cells with monocerine at 1.25 for 24 h, indicating the absence of cytotoxicity of this compound in HUVEC cells in the period of 24 h.
  • Figure 25 shows determination of apoptosis in HUVECs cells by double staining with annexin V-FITC / propidium iodide. Distribution of cells in viable cells, in the process of necrosis-like cell death, early or late apoptosis after treatment with 0.02 to 1.25 mM monocerine. The control corresponds to treatment with RPMI 1640 only.
  • HUVECs cells were treated with monocerine and after staining with acridine Orange or rhodamine 123 were analyzed by confocal laser scanning microscopy. The observations regarding each concentration of the monocerine and treatments were described and images were recorded.
  • Acridine-orange is a fluorescence dye used to stain acid vacuoles (lysosomes, endosomes and autophagosomes), RNA and DNA in living cells.
  • the dye is interleaved in double-stranded nucleic acids (DsDNA) being detected as green fluorescence at 530 nm. It also binds electrostatically to phosphate groups in single-stranded nucleic acids (ssDNA), RNA or vacuoles, and in this case, detection is at 640 nm (McMaster and Carmichael, 1977).
  • Rhodamine 123 is a fluorescent marker, known to inhibit the function of the electrical potential of mitochondria. Rhodamine 123 binds to mitochondrial membranes and inhibits transport processes, especially electron transport, delaying internal cell respiration.
  • SEM Scanning Electron Microscopy
  • the cascade of the apoptotic process can be initiated by two main routes: the extrinsic, which is mediated by death receptors present on the cell surface, and the intrinsic, which involves changes in the mitochondria (Burz et al., 2009; Portt et al., 2011). These pathways are regulated by several proteins, such as p53, members of the Bcl-2 family (B-celllymphomaprotein2), IAPs (apoptosis inhibiting proteins) and MAPKs (mitogen-activated protein kinases) (Liu et al., 2011; Sankari et al., 2012).
  • the granules adhered to the membrane of the HUVECs cells of the control group (which received only culture medium), changed significantly after treatment with monocerine depending on the time and concentration, which favored the maintenance of cytotoxic activity, as demonstrated in the cell viability (MTT), amount of DNA fragmented in the cell cycle stages, and the different proportions of senescent cells found after treatment.
  • the factors that are possibly responsible for these cytological changes are histone deacetylase inhibitors, which relax chromatin without physically damaging DNA, repairable DNA damage, ATM activation (ataxia telangiectasia) and p53 suppression, which directly influence the dynamics of distribution and progression of the cell cycle phases.
  • the activity of the monocerine and annularin I compounds on the proliferation of normal human cells was evaluated using the cell cycle phase analysis to compare the results obtained between the cells treated with different concentrations of the compounds and the untreated group.
  • the transition from the G1 to S cycle phase requires the assembly and activation of a DNA replication complex to initiate DNA synthesis.
  • HUVECs endothelial cells undergo contact inhibition, inhibit their proliferation after confluence and become quiescent.
  • the cell cycle in eukaryotic cells controls the progression, between and within the phases, through control points that coordinate the proliferation of the cells with the surrounding environment, ensuring precisely replication and division.
  • Fibroblasts are the main cellular components of connective tissues found in most organs of the body. Maintaining the balance between proliferation and differentiation is crucial for homeostasis. After activation by injury, DNA damage, oxidative stress or mechanical injury, fibroblasts activate the migration, adhere to the provisional extracellular matrix and proliferate (Eckes et al., 2014).
  • This phenomenon can occur through several mechanisms involved in the control of cell proliferation and differentiation, among them, the mechanisms that control the progression of the cell cycle.
  • G0 Gl
  • Gl Gl
  • Gl Gl
  • mitosis cells of continuous replication
  • the cells that entered Gl progress in the cycle and reach a critical phase in the Gl / S transition, restriction point, a limiting step for replication. Passing this point of restriction, normal cells become irreversibly compromised with DNA replication.
  • CDKs cyclin-dependent kinases
  • VEGF vascular endothelial growth factor
  • hypoxia-inducing factor HIF-la
  • VEGF-R1 hypoxia-inducing factor receptor 1
  • FGF-2 FGF-2
  • fibroblasts Several factors contribute to the increase in VEGF expression, among them, the low concentration of intracellular oxygen blocks the degradation of the hypoxia-inducing factor (HIF-la), which increases the levels of expression of its receptor (VEGF-R1) and determines intracellular hypoxia, which, in turn, determines the increase of its activity by activating the transcription of the VEGF gene or factors related to its expression such as FGF-2, expressed in fibroblasts (Boudreau and Myers, 2003).
  • HIF-la hypoxia-inducing factor
  • VEGF and FGF-2 are important factors in inducing the formation of new blood vessels.
  • VEGF-R1 expression was evaluated in HUVECs endothelial cells, treated for 6 and 24 h with the compound annularin I, compared to the untreated control group.
  • the results showed increased expression of VEGF-R1 receptors after 6 h of treatment, and for FGF-2 expression this increase was observed mainly after 24 h which clearly demonstrates the effects of regulation of proliferation and endothelial differentiation. This phenomenon is characterized by the induction of the vascular sprout growth receptor and endothelial expansion, and the induction of the fibroblast growth factor responsible for the maintenance of cell proliferation.
  • FGF-2 is a member of a family of 13 growth factors structurally linked and related to heparin. It is ubiquitously expressed in cells of mesodermal and neuroectodermal origin, and in a variety of cells. In vitro, FGF-2 is a potent mitogen for different types of cells, including vascular endothelial cells and fibroblasts. When endothelial cells are cultured, FGF-2 induces an antigenic phenotype that consists of increased proliferation, migration, production and expression of specific integrins. In vivo, in turn, is a potent inducer of angiogenesis and has pleiotropic effects on the development and differentiation in various organs.
  • Cell proliferation can be influenced by physiological and pathological conditions, being largely controlled by signals, soluble or contact-dependent factors, of microenvironments that act by stimulating or inhibiting it.
  • the most important mechanism of cell proliferation is the conversion of quiescent cells into proliferative cells.
  • the recruitment of quiescent cells and the progression of the cell cycle require stimulating signals to overcome the physiological inhibition of cell proliferation.
  • Cell cycle activities are controlled by cyclins, cyclin-dependent kinases (CDK-s) and their inhibitors.
  • CDK-s cyclin-dependent kinases
  • Cip / Kip There are two main classes of inhibitors: the Cip / Kip and INK-4 / ARF families. Within the Cip / Kip family, p21, p27 and p53 stand out, belonging to the INK-4 / ARF family, p6 and p4, which function as suppressors of cell cycle progression.
  • the transcriptional activity of p21 is under the control of the p53 protein. P21 will also compete with cyclin D with the same goal of causing the cell cycle to stop. P27 responds to growth suppressors and will compete with the cyclin E / CDK-2 complex, also causing a cell cycle to stop at the Gl / S restriction point, changing the proportion of standing and quiescent / senescent cells.
  • the tumor suppressor protein p53 is a key protein in apoptosis of HUVEC cells and plays an important role in apoptosis signal transduction pathways in several cell types, including fibroblasts and vascular endothelial cells.
  • the function of p53 is of great importance in genotoxic stress where it modulates and integrates several types of responses that control apoptosis, cell cycle arrest, senescence and other physiological processes (Speidel, 2010).
  • p27 levels are increased in quiescent cells and fall rapidly after stimulation by mitogens.
  • the increase in p27 expression in HUVECs cells with the compound annularin I possibly represents the effects of cell proliferation caused by the compound in the periods of 6 and 24 h.
  • the p21 protein is a critical effector of the p53-dependent pathway and causes a stop in the cell cycle by inhibiting cyclin-dependent kinase. Therefore, p21 plays a central role in apoptosis, and is also related to maintaining the differentiating state of endothelial cells.
  • Annularin I proved to be a compound with the potential to induce proliferation in endothelial cells, maintaining the proportion of cells in G0 / G1, decreasing cells with fragmented DNA and inducing senescence which may mean the maintenance of proliferating cells. These effects were accompanied by the differential expression of proteins that regulate the control points and progression of the cell cycle.
  • annularin I tests for morphological analysis by scanning electron microscopy (SEM), mitochondrial analysis by laser confocal microscopy and gene expression assay will be performed soon.
  • SEM scanning electron microscopy
  • mitochondrial analysis by laser confocal microscopy and gene expression assay will be performed soon.
  • G1 Control group - untreated
  • G2 Control group - treatment with collagenase (commercial product - Kollagenase Cristalia® - containing 0.6 U / g of collagenase plus 1% chloramphenicol;
  • G3 Group treated with 0.0006% monocerine
  • G4 Group treated with 0.005% monocerine
  • G7 Group treated with vehicle - cream without the active ingredient.
  • Balb / C mice (4 animals / group) were treated daily with the formulations for a period of 24 h, 72 h, 7 and 14 days.
  • the E. rostratum fungus was grown on potato dextrose agar (PDA) for seven days at 28 ° C, and after growth some colonies were transferred to an Erlenmeyer flask containing 800 mL of PDB and incubated in a orbital shaker (Marconi®, MA-830) at 28 ° C, at 150 rpm for 15 days. Each culture was carried out with six Erlenmeyers in a total of 6.4 liters per lot. Several batches were grown to obtain the necessary monocerine for the tests.
  • the SPE-100 fraction was purified by high performance liquid chromatography (HPLC) by an isocratic method (40-54% B, LC / MS ACE ⁇ preparative dimensions column C18 250x10x10 mm; mobile phases: A: H20 / TFA (99, 9: 0.1%) and B: MeOH / H20 / TFA (90: 9.9: 0.1%) and the fraction obtained from the SPE-100 subsequently dried using a Speed-vac system (CHRIST RVC 2-18 CD plus).
  • the fraction obtained F1 containing impure monocerine which was purified again in a second step in the same condition using a smaller column of preparative dimensions LC / MS ACE ⁇ (C18 250x4.6 mm). Pure monocerine was analyzed by mass spectrometry by gas chromatography (GC-MS Agilent 7890 / 5975C) to guarantee the degree of purity PREPARATIONS OF FORMULATIONS CONTAINING MONOCERINE
  • a nonionic modified base cream was prepared according to the National Formulary of the Brazilian Pharmacopoeia 5th edition (BRAZIL, 2012). Briefly, a emulsion was prepared with an aqueous phase containing propylene glycol, methyl paraben and purified water, which after heating was added to an oil phase containing a nonionic self-emulsifying base, mineral oil and propyl paraben.
  • mice Male Balb / C mice, 6 to 8 weeks old, weighing 20 to 22 g, were obtained from the Central Animal Center of the Butantan Institute and kept in boxes (four animals per box) with clippings pine, water and food ad libitum. The animals were kept in the experimental clinical facilities of the Molecular Biology Laboratory of the Butantan Institute under controlled temperature and light cycle for at least one week before the tests.
  • the animals received general anesthesia with ketamine (10 mg / kg) and xylazine (100 mg / kg) for intraperitoneal administration and the dorsal region was scraped and cleaned with 70% ethanol. The dorsal area of the mouse was marked with a 10 mm circular stamp and then the excision wound was carefully induced using scissors.
  • G2 control group - treatment with collagenase (commercial product Kollagenase Cristalia® - containing 0.6 U / g of collagenase plus 1% chloramphenicol);
  • the mouse received isoflurane inhalation anesthesia with an overdose of the recommended general dose and the blood sample was collected by cardiac puncture and added to a tube containing anticoagulant coated with EDTA, for additional biochemical analyzes.
  • the wound surface on the skin was collected after euthanizing each animal with the push to separate the animal's neck.
  • the granulation tissue was formed in the area of the lesion, which was carefully excised, leaving a 5 mm margin of normal skin for histological and morphological evaluation and determination of hydroxyproline.
  • the X-ray also monitored the wound size of the animals in the 14-day treatment. For this, two animals from each group (Gl, G2, G3, G4 and G5) received isoflurane anesthesia, and the wound image was recorded by an in vivo X-ray image (MS FX PRO Bruker®) at 24 h, 72 h, 7, 10 and 14 days (SATO et al., 2016). The analysis of the wound size was performed with Bruker MI Software Version 7.2 and calculated using GraphPad Prism 5.0. The data were expressed by comparison with the control group.
  • Serum was stored for cytokine analysis and whole blood was analyzed for white blood cells, platelets, neutrophils and monocytes. The results were transformed into graphs using the Graph Pad Prism software
  • tissue fragments collected were previously fixed in 10% formaldehyde, gradually dehydrated in a sequence of alcohols (70, 80, 95, 100 and 100%, 1 hour each), diaphanized in xylene for (2 hours) and followed by immersion in paraffin (Histosec-MERCK®), 5 pm sections were obtained in an automatic microtome (Leica, RM2165®) to prepare slides for staining (TOLOSA et al. 2003). The slides were analyzed under an optical microscope (Nikon 80i®) in a 40x objective.
  • tissue sections were stained with Mayers Hematoxylin and Eosin Y (HE) to estimate the degrees of wound healing and epidermal regeneration and granulation tissue formation, as previously described (KIM et al., 2003).
  • HE Mayers Hematoxylin and Eosin Y
  • the red sections of picrosirius were stained with the Commercial Kit (Sigma Aldrich) (Cat # P6744-1GA). For the wax picrosirius red and paraffin hydrate sections, then stain the cores with Mayers hematoxylin for 5 minutes and then wash the slides for 10 minutes under running water. After that, stain the fabric slides in red picrosirius for 45 minutes and place in the incubator at 30 ° C. Then, washed in two changes of acidified water it also physically removed the water from the slides by vigorous stirring and then dehydrates the tissue slides in three exchanges of 100% ethanol and , in the end, cleaned in Xylene for 5 minutes each (2x) and mounted the glide in resinous medium. The intensity of the red color referring to the collagen distribution was calculated by the area that adjusts the field using Image Pro Plus 4.5.1 (Media Cybernetics, Silver Spring, MD). The percentage of collagen was calculated for each group treated for 14 days.
  • the spots were analyzed microscopically using a 20x and 40x objective lens from an optical microscope connected to a digital camera (Coolpix 990; Nikon Eclipse80i). Representative areas of the effects of the treatments were photographed to record tissue regeneration in the dermis and epidermis and the formation of tissue granulation (KIM et al., 2012).
  • Picrosirius' red color was also analyzed by a polarized microscope.
  • the special coloring of Picrosirius red highlights the natural birefringence of collagen fibers when exposed to polarized light microscopy (Olympus BX-model®, with U-POT and U-ANT filter).
  • results of birefringence also allow to evaluate the organization of collagen fibers in the tissues, as possible artifacts that may occur.
  • the filters and the condenser have been adapted to allow a good image capture.
  • the program for image capture was Zeiss Lite (version 2.6, Zen blue, Germany
  • Antigenic recovery was performed with citrate buffer (pH 6.0, 4x 5x) in a water bath at 95 ° C, followed by blocking the endogenous activity of peroxidase for 15 min at room temperature and protected from light with 3% of hydrogen peroxide (H2O2) diluted in distilled water and blocking nonspecific binding with goat serum diluted 2% in 1 x PBS for 30 minutes.
  • citrate buffer pH 6.0, 4x 5x
  • H2O2O2 hydrogen peroxide
  • the antibodies were compared to the negative control, which used 0.2% BSA instead of the primary antibody.
  • Seven fields from an immunostained section (VEGF) were selected and captured for each sample. Quantification was evaluated on high quality captured images (2048 and 1536 pixel buffer) using Image Pro Plus 4.5.1 (Media Cybernetics, Silver Spring, MD) and processed using Origin 9.1 and GraphPad Prism 5.1. The staining result was expressed as mean ⁇ standard deviation using the Image J software. This analysis was performed only for the 24-hour treatment group. RESULTS
  • Figure 26A shows the HPLC profile of the first purification step, when the F1 fraction containing unclean monocerine was collected. After evaporation of methanol, the F1 fraction was fully purified in the second step, as shown in Figure 26B.
  • Monocerine was characterized as previously described by 1H and 13C nuclear magnetic resonance (NMR) (NMR) and mass spectrometry (MS) and used for the cream formulation at 0.0006% and 0.005%.
  • the wound size was measured daily by caliper and the sizes are represented, from the first 24 hours to the 14th day, for all treatment groups.
  • the wound size on the seventh day of the groups treated with 0.0006% (Group G3) and 0.005% (Group G4) monocerine was 4.75 ⁇ 0.80 and 3.72 ⁇ 0.42 mm, respectively, while for 10 days it was 2.20 ⁇ 0.62 mm and 1.26 ⁇ 0.29 mm, respectively.
  • the size of collagenase commercial cream (group G2) was higher, and 5.62 ⁇ 0.39 and 2.6 ⁇ 0.69 mm, respectively (Table 27).
  • the size of the wound is also shown in Figure 27 for the period of 24 hours, 72 hours and 7 and 14 days.
  • Table 26 Intensity of clinical evaluation for edema, hyperemia, bleeding, granulation and crusting.
  • Table 27 Daily measurement of wound sizes by caliper and represented by means ⁇ SD for four animals / group in tests (mm).
  • the size of the wounds of the animals treated for 14 days was also monitored by in vivo X-ray imaging (MS Fx Pro) at time zero 24 h, 48 h, 72 h, 7 d, 10 of 14 days and the data are represented in figure 28 and table 28. The images were recorded and are represented in figures 29 and 30. The analysis allows to observe the healing and wound closure in the group treated with 0.0006% and 0.005% monocerin.
  • the granulation tissue appeared fragile with monokin in the 0.005% ointment, possibly due to the increase in the ultrastructural images of the dermis captured by SEM showing the granulation tissues of the animals in the group treated with 0.0006% monocerine (G3) with the presence of numerous dead cells and abundant blood cells; it is also possible to observe fibronectin and red blood in tissues infiltrated by capillaries.
  • G3 monocerine
  • fibronectin and red blood red blood in tissues infiltrated by capillaries.
  • the distribution of the collagen well (red arrow).
  • the wound tissues for 7 and 14 days histological experiment are shown in Figures 33 and 34.
  • the untreated control (Gl): (H&E) visible fibroblast cells of the scar tissue and blood vessels (yellow arrow); (PR) collagens are dense and organized (orange arrow); A denser layer of epidermis (MT) (red arrows) was observed at 7 and 14 days of treatment.
  • (PR) adipose tissues were observed and inflammation was also observed until the 14th day (orange arrow);
  • MT The large number of inflammatory cells was also present in the treatment of 7 and 14 days (red arrow).
  • the 0.0006% monocerine group (G3): (H&E) registered good distribution of epithelial cells and organized skin tissue (red arrow); (PR) collagen and epithelial layer were dense and better than the control groups (orange arrow); (MT).
  • the special red dye of picrosirius has the ability to increase the natural birefringence of collagen when exposed to polarized light.
  • Type I collagen would show a yellow-red color, while type III would be green, as shown in Figure 37.
  • red blood cells red blood cells
  • white blood cells white blood cells
  • platelets 0.0006% monocerine and 0.005%
  • Groups G3 and G4 had the highest levels of red blood cells in 24 h, compared to other control groups Gl, G2-collagenase and vehicle G5. Later, after 7 days of proliferation. Red blood cells maintained their balance and almost similar results were observed for all groups, except for the vehicle in the G5 group, which showed a low and significant number of red blood cells compared to the groups treated with monocerine (G3 and G4).
  • the groups treated with 0.0006% and 0.005% monocerin showed higher levels of leukocytes when compared to the other groups (Gl, G2 and G5). Slightly higher levels of leukocytes were observed after 7 days in the untreated Gl control group. Whereas, after 14 days, the groups treated with monocerine (G3 and G4) were taller than other control groups (Gl, G2 and G5). In the case of platelets within 24 h, the groups treated with monocerine in the inflammation phase (G3 and G4) had higher levels of platelets than other control groups (Gl, G2 and G5), which leads to skin regeneration and immune response after application of groups treated with monocerine. Interestingly, platelet levels were lower in the G3 group treated with 0.0006% monocerine than in the G4 group treated with 0.005% monocerine.
  • the G2 group showed monocytes level slightly higher than the monocerine G3 and G4 in 0.0006% and 0.005%. At that time, the level of monocytes in groups Gl and G5 was significantly lower than groups G2, G3 and G4 ( Figure 38).
  • Immunohistochemical analyzes were performed for VEGF. This analysis will be repeated including the analysis of TIMP-2 antibodies. MMP-3 and MMP-9 are also involved in the wound process.
  • Figure 40 shows a photomicrograph of tissues immunostained with antibody against VEGF in the control and treated groups for 24 h. For all groups it is possible observe the immunoreactivity against VEGF (arrows), while for the negative controls there was no immunoreactivity as expected; The exception is for the negative control reaction for the Gl group, which was also immunoreactive, but of low intensity, as expected.
  • Scale bar 40 x 100 pm. The data were analyzed by measuring the pixels of the 1024 x 2048 pixel scale bar.
  • monocerine was incorporated into a cream formulation at 0.0006% and 0.005% and its effectiveness was evaluated in an in vivo cutaneous wound surgical model.
  • the treatment of the lesions started after about 3 h after induction in the animals.
  • the wound healing process is characterized by dynamic and interactive events involving soluble mediators, blood cells, ECM and parenchymal cells, which results in the permanent restoration of anatomical and functional integrity.
  • ordered refers to the sequence of phases of wound healing that include inflammation, formation and remodeling of tissues (SINGER; CLARK, 1999).
  • the secondary metabolites of the fungus have been associated with a strong anti-inflammatory, antimicrobial and antioxidant activity, essential for wound healing.
  • the specific activities of E. rostratum secondary metabolite extracts and their beneficial constituents for wound healing have been reported to include inhibitory effects on bleeding, increased wound contraction, increased levels of basic fibroblast growth factor (FGF) and platelet-derived growth factor and stimulation of hematological parameters such as white and red blood cells (AGYARE C; AKINDELE; STEENKAMP, 2019).
  • Hematological parameters are important markers of the disease in human and veterinary medicine.
  • the immune system and neuroendocrine systems are the two main components that maintain body homeostasis.
  • Peripheral blood samples can indicate abnormalities in the body, which generally cause several threats to human health, including devastating autoimmune or metabolic diseases.
  • the data indicate that, by the two methods used to measure the size of the wound (X-ray and caliper) for the groups treated with 0.0006% and 0.005% monocerin (G3 and G4), the reduction and closure of the wound were more efficient than in the control groups.
  • Blood clotting is a host defense mechanism that, in addition to inflammatory responses, not only helps to protect the integrity of the vascular system, but also promotes repair after tissue damage.
  • the formation of a blood clot serves not only to close the edges of the wound, but also to pass through the fibronectins, which provides a temporary matrix in which the fibroblasts endothelial cells and keratinocytes can enter the wound (MONROE DM; MAUREANE, 2012).
  • Fibroblasts were involved in the synthesis of collagen, glycosaminoglycans (GAGs). proteoglycans and adhesive glycoproteins and recognized as of essential importance in the healing process of wounds treated for 72 h. predominantly in monocerine in the groups treated with 0.0006% and 0.005% (G3 and G4). However, the group treated with G2 collagenase showed collagen degradation that clearly elaborates the sign of inflammation.
  • the last stage of skin wound healing is the remodeling phase in which the provisional extracellular matrix is remodeled.
  • the injured area is completely re-epithelized and a contractile response mediated by myofibroblast occurs. Due to their multiple collagen binding sites, myofibroblasts bind to collagen fibers and contract the reduction of the wound area, as shown in the results of Figures 31, 32, 33, 34 and 35.
  • the wounds treated with annularin I showed contraction, coagulation, granulation and sutured or stapled wound edges contracting sharply with time and the application of annularin in wounds. Therefore, the wounds of the control group were also observed and showed an effective healing on the 13th and 14th day, which were also observed and photographed.
  • the size of the wound was measured daily by a caliper and the sizes are represented, from the first 24 hours to the 14th day, for all treatment groups.
  • the wound size on the seventh day of the groups treated with 0.005% (Group G3) and 0.0006% (Group G4) anularin was 4.48 ⁇ 0.18 and 4.39 ⁇ 0.10, respectively, while 10 days was 1.81 ⁇ 0.86 and 1.62 ⁇ 0.30 mm, respectively.
  • the size of collagenase commercial cream (group G2) was higher, and 5.62 ⁇ 0.39 and 2.6 ⁇ 0.69 mm, respectively ( Figure 42, Table 30).
  • the size of the wound is also represented in the Figure 1 for a period of 24 hours, 72 hours and 7 and 14 days.
  • FIG. 42 The graphic illustration of figure 42 shows that the groups treated with 0.003% and 0.0005% anularin healed less than 14 days compared to the untreated groups. Rapid skin remodeling of the treated anularin I groups was observed.
  • Figure 43, Table 31 the results show that the groups treated with anularin and the untreated groups healed within 14 days.
  • the wound size for treated groups decreased anularina I better than the untreated group;
  • the skin was more organized and compact and no scarring was observed.
  • the wounds of the untreated groups were healed, but the scar was small.
  • Table 30 Measurement of wound size daily by caliper and represented by means ⁇ SD for four animals / group in millimeters (mm).
  • Table 31 Measurement of wound size by in vivo radiological image (MS Fx pro) in millimeters (mm) represented by means ⁇ SD for 2 animals / group
  • red blood cells red blood cells
  • white blood cells white blood cells
  • platelets white blood cells
  • annularin I the group treated with annularin I mainly with a concentration of 0.003%, with a greater number of leukocytes and PLT until the 14th day, compared to the untreated groups.
  • the data allow us to conclude that the group treated with annularin I also proliferates immune cells, which leads to better skin remodeling.
  • Picrosirius red dye has the ability to increase the natural birefringence of collagen when exposed to polarized light.
  • Type I collagen would show a yellow-red color, while type III would be green, as shown in Figure 12.
  • the groups treated with monocerine and annularin I showed a dense and collagen compact compared to control groups (control, collagenase and vehicle).
  • the untreated control group showed significantly low collagen expression.
  • the groups treated with monocerine and annularin I had more yellow and red fibers, which was seen possibly indicating the presence of type I collagen.
  • the green fibers typically of type III collagen
  • the control groups control, collagenase and vehicle
  • had thick red and yellow fibers typically of type I collagen located in the lower deep dermis.
  • the groups treated with monocerine and annularin I showed an arrangement of visible and organized collagen fibers from the groups treated with annularin.
  • compositions comprising the compound (2S, 3aR, 9bR) - 6-hydroxy-7, 8-dimethoxy- 2-propyl-2, 3, 3a, 9b-tetrahydro-5H-bore [3, 2-c] isochromen-5-one (monocerin), and at least one pharmaceutically acceptable carrier.
  • compositions comprising the compound 4-methoxy-5-methyl-6-butyl-2H-pyran-2-one (anularin I), and at least one pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier includes, but is not limited to, any carrier that does not interfere with the effectiveness of the biological activity of the active ingredients and is non-toxic to the patient to whom it is administered.
  • suitable pharmaceutical carriers include phosphate buffered saline solutions, water, emulsions, such as oil / water emulsions, various types of wetting agents, sterile solutions, etc.
  • Such vehicles can be formulated by conventional methods and can be administered to the individual in the dose and in therapeutic regimens most appropriate to each case.
  • the compositions are sterile, but can also be prepared under aseptic conditions.
  • compositions can also contain adjuvants, preservatives, emulsifying agents and dispersing agents.
  • Pharmaceutical formulations can be for human, and / or veterinary / animal use.
  • the formulations can be prepared in the form of particulate systems such as, for example, microparticles, nanoparticles, microspheres, nanospheres, liposomes, in carrier complexes as in cyclodextrins (alpha, beta and gamma); still in the form of controlled release.
  • the formulations can be prepared by the combination of the monocerine and annularin I compounds in the most appropriate proportions and doses / concentrations.
  • At formulations can be in the form of creams, ointments, gels, adhesives, sprays, including nanotechnology-based formulations, or even the association with other active principles with the same biological effect, or even association with antimicrobials.
  • compositions of the present invention can be carried out in different ways, for example by intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal, particularly, the main ones are cutaneous, subcutaneous, topical, intradermal, rectal, intraocular, nasal and auricular.
  • the route of administration depends on the type of treatment and the type of compound contained in the pharmaceutical composition.
  • the dosage regimen will be determined by the doctor and other clinical factors.
  • dosages for any patient depend on many factors, including the patient's weight, body surface area, age, sex, pharmaceutical composition carrying the particular compound to be administered, time and route of administration, the type of therapy, general health status and other factors to be considered and administered concurrently.
  • the present invention relates to the use of secondary metabolite produced by the fungus Exserohilum rostratum in cell regeneration.
  • the present invention relates to the use of the secondary metabolites monocerine and annularin I obtained from the culture of the fungus Exserohilum rostratum for the preparation of drugs for cell regeneration, preferably cell regeneration in HUVEC endothelial cells and normal FN1 fibroblasts.
  • compositions of the present invention can be used as an agent that induces cell proliferation, preferably in tissue repair.
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Abstract

The present invention relates to a pharmaceutical composition comprising secondary metabolites produced by the fungus Exserohilum rostratum for cell regeneration. Particularly, the present invention relates to a pharmaceutical composition comprising the secondary metabolites monocerin and annularin I produced from the culture of the fungus Exserohilum rostratum for cell regeneration. In another aspect, the present invention relates to the process for obtaining and using said secondary metabolites produced by the fungus Exserohilum rostratum in cell regeneration.

Description

"COMPOSIÇÃO FARMACÊUTICA, PROCESSO DE OBTENÇÃO E USO DE METABÓLITOS SECUNDÁRIOS PRODUZIDO PELO FUNGO EXSEROHILUM ROSTRATUM NA REGENERAÇÃO CELULAR E TEClDUAL" .  "PHARMACEUTICAL COMPOSITION, PROCESS OF OBTAINING AND USE OF SECONDARY METABOLITES PRODUCED BY THE FUNGUS EXSEROHILUM ROSTRATUM IN CELL AND TECHNICAL REGENERATION".
Campo da Invenção  Field of the Invention
A presente invenção se refere ao uso de metabólito secundário produzido pelo fungo Exserohilum rostratum na regeneração celular e tecidual . Particularmente, a presente invenção se refere ao uso dos metabólitos secundários monocerina e anularina I, compostos orgânicos obtidos a partir da cultura do fungo Exserohilum rostratum para regeneração celular em células endoteliais HUVEC e fibroblastos normais FN1.  The present invention relates to the use of a secondary metabolite produced by the fungus Exserohilum rostratum in cell and tissue regeneration. In particular, the present invention relates to the use of secondary metabolites monocerin and annularin I, organic compounds obtained from the culture of the fungus Exserohilum rostratum for cell regeneration in HUVEC endothelial cells and normal FN1 fibroblasts.
Antecedentes da Invenção  Background of the Invention
[001] Fungos endofiticos são fungos que vivem no interior das plantas, habitando, de modo geral, suas partes aéreas, como folhas e caules, sem causar aparentemente nenhum dano a seus hospedeiros. Portanto, eles se diferenciam dos microrganismos fitopatogênicos , que são prejudiciais às plantas, causando-lhes doenças.  [001] Endophytic fungi are fungi that live inside plants, generally inhabiting their aerial parts, such as leaves and stems, without apparently causing any damage to their hosts. Therefore, they differ from phytopathogenic microorganisms, which are harmful to plants, causing disease.
[002] Microrganismos endófitos foram mencionados pela primeira vez no inicio do século XIX, mas foi Bary (1866) quem primeiro delineou uma possível distinção entre endófitos e fitopatógenos . No final dos anos 70, verificou- se que os endófitos propiciam à planta hospedeira proteção contra insetos e pragas, contra outros microrganismos patogênicos e inclusive contra herbívoros. Atualmente, sabem-se que os fungos endófitos podem produzir moléculas que atuam como toxinas, antibióticos, fatores de crescimento e outros fármacos, e ainda muitos outros produtos de interesse biotecnológicos (AZEVEDO, 2004) . Os fungos endofiticos fixam nas plantas por aberturas naturais e feridas que servem como portas de entrada vezes nas raízes secundárias laterais. Outras portas de entrada são as aberturas naturais como estômatos e hidatódios, aberturas causadas por insetos e por estruturas de fungos patogênicos, como os apressórios. [002] Endophyte microorganisms were first mentioned in the early 19th century, but it was Bary (1866) who first outlined a possible distinction between endophytes and phytopathogens. In the late 1970s, it was found that endophytes provide the host plant with protection against insects and pests, against other pathogenic microorganisms and even against herbivores. Currently, it is known that endophytic fungi can produce molecules that act as toxins, antibiotics, growth factors and other drugs, and many other products of biotechnological interest (AZEVEDO, 2004). Endophytic fungi attach to plants through natural openings and wounds that serve as entry doors sometimes in the secondary side roots. Other entry points are natural openings such as stomata and hydatodes, openings caused by insects and structures of pathogenic fungi, such as appressoriums.
Metabólitos primários e secundários produzidos por microrganismos  Primary and secondary metabolites produced by microorganisms
[003] Uma grande vantagem da prospecção química de metabólitos primários e secundários (MS) produzidos por microrganismos, em relação às demais fontes, é o fato de que microrganismos podem ser cultivados em larga escala em fermentadores . Também não existe prejuízo ao ecossistema, como pode ocorrer com a retirada de plantas e outros organismos de áreas naturais, nem problemas éticos como os que podem advir da prospecção de metabólitos bioativos a partir de insetos, anfíbios e outras espécies animais (TAKAHASHI & LUCAS, 2007) .  [003] A great advantage of the chemical prospecting of primary and secondary metabolites (MS) produced by microorganisms, in relation to other sources, is the fact that microorganisms can be cultivated on a large scale in fermenters. There is also no damage to the ecosystem, as can occur with the removal of plants and other organisms from natural areas, nor ethical problems such as those that may arise from the prospect of bioactive metabolites from insects, amphibians and other animal species (TAKAHASHI & LUCAS, 2007).
Metabólitos Primários  Primary Metabolites
[004] Metabólitos primários são as pequenas moléculas produzidas ao longo do crescimento vegetativo. São usados em indústrias alimentícias e de ração incluindo, por exemplo: álcoois (etanol), aminoácidos (glumato monossódico, lisina, treonina, fenilalanina, triptofano) , nucleotídeos flavorizantes (ácido 5-guanílico, ácido 5-isosínico) , ácidos orgânicos (acético, propiônico, fumárico, láctico) , polióis (glicerol, manitol, xilitol), polissacarídeos (xantana) , açúcares (frutose, ribose) e vitaminas ( riboflavina, cianocobalamina, biotina) (DEMAIN, 2000; RAJASEKARAN et al . ,2008) .  [004] Primary metabolites are the small molecules produced during vegetative growth. They are used in food and feed industries including, for example: alcohols (ethanol), amino acids (monosodium glumate, lysine, threonine, phenylalanine, tryptophan), flavoring nucleotides (5-guanyl acid, 5-isosinic acid), organic acids (acetic , propionic, fumaric, lactic), polyols (glycerol, mannitol, xylitol), polysaccharides (xanthan), sugars (fructose, ribose) and vitamins (riboflavin, cyanocobalamin, biotin) (DEMAIN, 2000; RAJASEKARAN et al., 2008).
Metabólitos Secundários (MS)  Secondary Metabolites (MS)
[005] Metabólitos secundários são sintetizados quando o crescimento microbiano está na fase estacionária; são frequentemente bioativos e de baixa massa molecular. Apresentam grande importância à humanidade devido à atividade antibiótica de importância farmacêutica, bem como atividade imunossupressora e tóxica. Normalmente não são derivados do substrato utilizado para o crescimento celular, e sim sendo sintetizados a partir de um metabólito primário. Em geral, esses metabólitos são formados quando grandes quantidades de precursores de metabólitos primários, tais como aminoácidos, acetato, piruvato e outros, são acumulados. Apresentam algumas caracteristicas como: distribuição taxonômica restrita, ou seja, nem todas as linhagens de uma mesma espécie são capazes de produzir determinado metabólito e não são essenciais para o crescimento e reprodução do organismo. As condições de cultivo como especialmente a composição do meio controlam a formação destes metabólitos que são produzidos como um grupo de estruturas inter-relacionadas. Podem ser super expressos e são codificados por conjuntos de genes dispensáveis (JAY, 2005; KELLER et al . ,2005; MARTIN et al . , 2005; YU & KELLER, 2005; NIGAM, 2009) . [005] Secondary metabolites are synthesized when microbial growth is in the stationary phase; they are often bioactive and of low molecular weight. They are of great importance to humanity due to the antibiotic activity of pharmaceutical importance, as well as immunosuppressive and toxic activity. They are not normally derived from the substrate used for cell growth, but are synthesized from a primary metabolite. In general, these metabolites are formed when large amounts of primary metabolite precursors, such as amino acids, acetate, pyruvate and others, are accumulated. They present some characteristics such as: restricted taxonomic distribution, that is, not all strains of the same species are capable of producing a certain metabolite and are not essential for the growth and reproduction of the organism. Cultivation conditions, especially the composition of the medium, control the formation of these metabolites which are produced as a group of interrelated structures. They can be overexpressed and are encoded by sets of expendable genes (JAY, 2005; KELLER et al., 2005; MARTIN et al., 2005; YU & KELLER, 2005; NIGAM, 2009).
[006] Na natureza, MS são importantes para os organismos que os produzem, funcionando como hormônios sexuais, ionóforos, defesa contra bactérias, fungos, amebas, insetos e plantas, agente de simbiose, efetores de diferenciação e atividades desconhecidas (DEMAIN & ADRIO, 2008) . Os MS são usualmente separados em cinco grupos: derivados de aminoácidos, peptideos não ribossomais, policetideos , derivados de ácidos graxos e híbridos de policetídeos-peptídeos (KEMPKEN & ROHLFS , 2010; ROZE et al . , 2011) . [007] Os genes para a biossintese de metabólitos secundários são usualmente organizados em " clusters" nas linhagens produtoras. Estes " clusters " incluem, além dos genes que codificam as enzimas biossintéticas e as proteínas regulatórias , genes para a resistência à ação tóxica dos metabólitos secundários (para evitar o suicídio das espécies produtoras) e genes para a secreção desses metabólitos (MARTIN et al . , 2005) . É usual que diferentes espécies de fungos tenham um ou mais metabólitos secundários em comum, sendo muitos deles produzidos por fungos filogeneticamente muito diferentes (FRISVAD et al . , 2008) . [006] In nature, MS are important for the organisms that produce them, functioning as sex hormones, ionophores, defense against bacteria, fungi, amoebae, insects and plants, symbiosis agent, differentiation effectors and unknown activities (DEMAIN & ADRIO, 2008). MS are usually separated into five groups: amino acid derivatives, non-ribosomal peptides, polyketides, fatty acid derivatives and polyketide-peptide hybrids (KEMPKEN & ROHLFS, 2010; ROZE et al., 2011). [007] The genes for the biosynthesis of secondary metabolites are usually organized in "clusters" in the producing strains. These "clusters" include, in addition to the genes encoding biosynthetic enzymes and regulatory proteins, genes for resistance to the toxic action of secondary metabolites (to prevent the suicide of the producing species) and genes for the secretion of these metabolites (MARTIN et al. , 2005). It is usual for different species of fungi to have one or more secondary metabolites in common, many of which are produced by phylogenetically very different fungi (FRISVAD et al., 2008).
[008] A síntese de MS pode garantir ao fungo, vantagem em habitat no qual existe competição com outros microrganismos. Portanto, muitos desses metabólitos apresentam efeitos tóxicos ou inibitórios em outros organismos (KHALDI et al . , 2010) . Devido a essas propriedades bioativas, muitos metabólitos secundários têm sido utilizados para uso farmacêutico como antibióticos, agentes hipocolesterolemiantes , antitumorais e imunossupressores , sendo que, poucos MS de sucesso não apresentam atividade antibiótica. Uma desvantagem é que micotoxinas e compostos produzidos por fungos patogênicos que induzem aumento da virulência tem impacto negativo na sociedade (DEMAIN, 1999; SHWAB & KELLELR, 2008) .  [008] The synthesis of DM can guarantee the fungus an advantage in a habitat in which there is competition with other microorganisms. Therefore, many of these metabolites have toxic or inhibitory effects on other organisms (KHALDI et al., 2010). Due to these bioactive properties, many secondary metabolites have been used for pharmaceutical use, such as antibiotics, hypocholesterolemic agents, antitumor agents and immunosuppressants, and few successful MS do not have antibiotic activity. A disadvantage is that mycotoxins and compounds produced by pathogenic fungi that induce increased virulence have a negative impact on society (DEMAIN, 1999; SHWAB & KELLELR, 2008).
[009] Devido à ampla distribuição destes metabólitos por todo o Reino Fungi, esta se torna uma característica importante para a classificação e a identificação de fungos. A Quimiotaxonomia em fungos, baseada em MS tem sido utilizada com sucesso em diversos gêneros de Ascomicetos como Alternaria Aspergillus , Fusarium, Hypoxylon, Penicillium, Stachybotrys , Xylaria e em poucos gêneros de Basidiomicetos , mas não em Zigomicetos e Quitridiomicetos (FRISVAD et al . 2008) . [009] Due to the wide distribution of these metabolites throughout the Fungi Kingdom, this becomes an important feature for the classification and identification of fungi. Fungal chemotaxonomy, based on MS, has been used successfully in several genera of Ascomycetes such as Alternaria Aspergillus, Fusarium, Hypoxylon, Penicillium, Stachybotrys, Xylaria and in few genera of Basidiomycetes, but not in Zygomycetes and Quitridiomycetes (FRISVAD et al. 2008).
Metabólitos secundários Anularina e Monocerina  Secondary metabolites Annularin and Monocerin
[0010] No estado da técnica, são poucos os relatos sobre o isolamento e síntese de monocerinas, assim como, as atividades biológicas já descritas para este composto. Para as anularinas os relatos são ainda mais escassos.  [0010] In the state of the art, there are few reports on the isolation and synthesis of monokines, as well as the biological activities already described for this compound. For annularins, reports are even more scarce.
[0011] As monocerinas ( 12S ) -12-hidroximonocerina e ( 12R) -12-hidroximonocerina já foram isoladas do fungo endofítico Microdochium bolleyí e E. rostratum junto com a monocerina ( 2S , 3aR, 9bR) - 6-hidroxi-7 , 8-dimetoxi-2-propil- 2, 3, 3a, 9b-tetra-hidro-5H-furo [3, 2-c] isocromen-5-ona (Sappapan et al . , 2008; Zhang et al . , 2008) que também já foi isolada de outras fontes, como da cultura de E. turcum (Robeson e Strobel, 1982; Cuq et al . , 1993), Helmínthosporíum monoceras (Claydon et al . , 1979), Fusarium larvarium (Claydon et al . , 1979; Grove e Pople, 1979), e de Dreschlera ravenelli (Scott et al . , 1984) .  [0011] Monocerins (12S) -12-hydroxymonocerine and (12R) -12-hydroxymonocerine have already been isolated from the endophytic fungus Microdochium bolleyí and E. rostratum together with monocerine (2S, 3aR, 9bR) - 6-hydroxy-7, 8-dimethoxy-2-propyl-2, 3, 3a, 9b-tetrahydro-5H-hole [3,2-c] isochromen-5-one (Sappapan et al., 2008; Zhang et al., 2008) which has also been isolated from other sources, such as E. turcum (Robeson and Strobel, 1982; Cuq et al., 1993), Helminthosporíum monoceras (Claydon et al., 1979), Fusarium larvarium (Claydon et al., 1979; Grove and Pople, 1979), and Dreschlera ravenelli (Scott et al., 1984).
[0012] O estudo do metabolismo secundário do fungo E. rostratum levou a identificação de fitotoxinas com ação herbicida (Tan et al . , 2004), e do extrato bruto da extração com acetato de etila, o derivado de isocumarina 11- hidroximonocerina foi isolado junto com 12-hidroximonocerina e monocerina cuja potencial ação antimalárica (IC50 de 0,68 mM) e de seus análogos foi pela primeira vez descrita (Sappapan et al . , 2008) . As monocerinas tem ainda ação antifúngica, inseticida e fitotóxica (Sappapan et al . , 2008) .  [0012] The study of the secondary metabolism of the fungus E. rostratum led to the identification of phytotoxins with herbicidal action (Tan et al., 2004), and of the crude extract from the extraction with ethyl acetate, the derivative of isocoumarine 11-hydroxymonocerine was isolated together with 12-hydroxymonocerine and monocerine whose potential antimalarial action (IC50 of 0.68 mM) and its analogs was first described (Sappapan et al., 2008). Monocerines also have antifungal, insecticidal and phytotoxic effects (Sappapan et al., 2008).
[0013] Da cultura do fungo aquático A. triseptatus, o extrato obtido da extração com acetato de etila apresentou ação antifúngica e antibacteriana e o estudo químico resultou no isolamento de 8 compostos, denominados como anularinas A- H (Figura 1) e classificados como metabólitos policetideos (Li et ai., 2003) . As anularinas A-F são a-pironas 3,4,5- trisubstituídas e as anularinas G-H são g-lactonas 3,4- disubstituídas a, b-insaturadas . As anularinas A, B, C e F inibiram o crescimento de Bacíllus subtílus (ATCC 6051) induzindo halo de 8-10 mm com 200 pg/disco testado e nesta mesma concentração, a anularina C inibiu o crescimento de Staphylococcus aureus (ATCC 29213) com halo de 14 mm. Nenhum composto apresentou ação contra C. albicans (ATCC 90029) e com exceção dos compostos D e E (não testados) os demais não foram efetivos contra A. flavus (NRRL 6541) . [0013] From the culture of the aquatic fungus A. triseptatus, the extract obtained from extraction with ethyl acetate showed antifungal and antibacterial action and the chemical study resulted in the isolation of 8 compounds, named as annularins A-H (Figure 1) and classified as polyketide metabolites (Li et al., 2003). AF annularins are 3,4,5-tri-substituted a-pyrones and GH annularins are 3,4-disubstituted a-b-unsaturated g-lactones. Annularins A, B, C and F inhibited the growth of Bacillus subtilus (ATCC 6051) by inducing an 8-10 mm halo with 200 pg / disc tested and at the same concentration, annularin C inhibited the growth of Staphylococcus aureus (ATCC 29213) with 14 mm halo. No compound showed action against C. albicans (ATCC 90029) and with the exception of compounds D and E (not tested) the others were not effective against A. flavus (NRRL 6541).
[0014] Anularinas A-F são -pironas, membros de uma classe geral comumente encontrada em fungos. Um estudo para a síntese de anularinas foi proposto por Motodate et ai., (2010) e a síntese total de anularina F foi relatada pela primeira vez em 2006 (Kurdyumov et ai., 2006) . Biogeneticamente, as anularinas A-F são presumivelmente derivadas da via de policetídeo e existe a hipótese de que as anularinas G e H tenham origem em uma via biossintética análoga, mas com uma diferente etapa de fechamento do anel.  [0014] Annularins A-F are -pyrones, members of a general class commonly found in fungi. A study for the synthesis of annularins was proposed by Motodate et al., (2010) and the total synthesis of annularin F was reported for the first time in 2006 (Kurdyumov et al., 2006). Biogenetically, the annularins A-F are presumably derived from the polyketide pathway and there is a hypothesis that the annularins G and H originate from an analogous biosynthetic pathway, but with a different stage of ring closure.
[0015] De forma semelhante, a monocerina tem origem como heptacetídeo, o que foi demonstrado em um estudo de biossíntese dessa molécula com a incorporação de acetatos marcados com 2H, 13C e 180 em D. ravenelii (Scott et ai., 1984) . [0015] Similarly, monocerine originates as heptacetide, which was demonstrated in a biosynthesis study of this molecule with the incorporation of 2 H, 13 C and 18 0 acetates in D. ravenelii (Scott et al., 1984).
Origem do fungo Exserohilum rostratum  Origin of the fungus Exserohilum rostratum
[0016] A planta velame é um arbusto endémico da Caatinga, de pequeno porte, encontrado em terreno arenoso com presença de sol intenso, ausência de água nas proximidades e se caracteriza pela presença de látex no interior do caule e intenso aroma. Esta planta foi coletada por nosso grupo, e uma exsicata foi depositada no Herbário da Universidade Estadual de Feira de Santana (UEFS) sob o número de Tombo 161257. A exsicata foi identificada como Croton blanchetíanus pertencente à família Euphorbíaceae pela Botânica Daniela Carneiro Santos Torres, da UEFS, especialista na Família Euphorbíaceae. [0016] The canopy plant is a small shrub endemic to the Caatinga, found in sandy soil with the presence of intense sun, absence of water nearby and is characterized by the presence of latex in the stem interior and intense aroma. This plant was collected by our group, and a desiccant was deposited at the Herbarium of the State University of Feira de Santana (UEFS) under the number Tombo 161257. The desiccant was identified as Croton blanchetíanus belonging to the Euphorbíaceae family by Botany Daniela Carneiro Santos Torres, from UEFS, specialist in the Euphorbíaceae Family.
[0017] A família Euphorbíaceae é uma das maiores das Angíospermae, com cerca de 300 gêneros e aproximadamente 7.500 espécies distribuídas em todo o mundo. No Brasil esta família de plantas está amplamente distribuída nos diferentes tipos de vegetação, mas é considerada uma das mais representativas da Caatinga. Segundo Albuquerque et ai., (2002) a família Euphorbíaceae é utilizada pelos habitantes da Caatinga com finalidades medicinais, como por exemplo, as espécies do gênero Croton, como C. argirophylloides (nome popular: marmeleiro-branco ou sacatinga) , C. rhamínífolíus (nome popular: velame) e C. sonderíanus (nome popular: marmeleiro) . Estas plantas também são usadas como repelente de insetos e como fonte de madeira.  [0017] The Euphorbíaceae family is one of the largest of the Angiospermae, with about 300 genera and approximately 7,500 species distributed worldwide. In Brazil this family of plants is widely distributed in different types of vegetation, but it is considered one of the most representative of the Caatinga. According to Albuquerque et al., (2002) the Euphorbíaceae family is used by the inhabitants of the Caatinga for medicinal purposes, such as, for example, the species of the genus Croton, such as C. argirophylloides (popular name: quince or sacatinga), C. rhamínífolíus (popular name: canopy) and C. sonderíanus (popular name: quince). These plants are also used as insect repellent and as a source of wood.
[0018] Caules e folhas da planta velame (C. blanchetíanus) foram processados em nosso laboratório para isolamento de fungos endofíticos de acordo com o procedimento descrito por Araújo et ai., (2001) . Deste processo, 70 fungos endofíticos foram isolados e os extratos orgânicos, obtidos após cultura, foram avaliados em relação à ação antifúngica contra espécies de fungos patogênicos para o homem.  [0018] Stems and leaves of the canopy plant (C. blanchetíanus) were processed in our laboratory to isolate endophytic fungi according to the procedure described by Araújo et al., (2001). From this process, 70 endophytic fungi were isolated and the organic extracts, obtained after culture, were evaluated in relation to the antifungal action against species of fungi that are pathogenic to man.
[0019] Dentre os isolados, o fungo endofítico codificado como FV3 foi isolado da folha da planta e taxonomicamente identificado por técnicas de Biologia Molecular (Raeder e Broda 1985; Gonzáles-Mendoza et ai., 2010) pelo grupo do nosso colaborador Prof. Wellington L. de Araújo do Departamento de Microbiologia do ICB/USP, como referente a espécie Exserohilum rostratum. [0019] Among the isolates, the endophytic fungus encoded as FV3 was isolated from the plant leaf and taxonomically identified by Molecular Biology techniques (Raeder and Broda 1985; Gonzáles-Mendoza et al., 2010) by our collaborator Prof. Wellington L. de Araújo of the Department of Microbiology at ICB / USP, as referring to the species Exserohilum rostratum.
[0020] Esta espécie tem origem terrestre e marinha e pode ser encontrada como patógeno de invertebrados marinhos ou como um fitopatógeno causando doenças em frutos e raízes de plantas (Sappapan et al . , 2008) . Na Thailandia esta espécie já foi isolada como endofítico de folhas de Stemona sp .  [0020] This species has terrestrial and marine origin and can be found as a pathogen in marine invertebrates or as a phytopathogen causing diseases in fruits and plant roots (Sappapan et al., 2008). In Thailand this species has already been isolated as an endophytic from leaves of Stemona sp.
[0021] O artigo de autoria de Pina, J.R.S. (UFPA); Pinheiro, E.A.A. (UFPA); Leite, E.A. (UFPA); Marinho, A.M.R. [0021] The article by Pina, J.R.S. (UFPA); Pinheiro, E.A.A. (UFPA); Leite, E.A. (UFPA); Marinho, A.M.R.
(UFPA) e intitulado "Novos policetideos com atividade antibacteriana isolados do fungo Exserohilum rostratum endófito de Bauhinia guianensis" apresentado no 56° Congresso Brasileiro de Química de 07 a 11 de novembro de 2016 em Belém do Pará revela a partir de uma matriz vegetal ( Bauhinia guianensis) , onde isolou-se a espécie fúngica Exserohilum rostratum 1.11 Er, ao qual foi obtida a biomassa e, assim, foram isolados, os esteroides ergosterol (1) e peróxido de ergosterol (2); a isocumarina, monocerina (3) e os policetideos de ocorrência inédita na literatura, denominados de anularina I (4) e anularina J (5) . Concomitante foram realizados ensaios antimicrobianos . As annularinas I (4) e J (5) apresentaram boas atividades bacteriostática, frente à B. subtilis e E. coli, destacando- se 4 que apresentou atividade bacteriostática na mesma concentração do antibiótico de referência. Quanto ao ensaio de citotoxidade as substâncias mostraram-se inativas. (UFPA) and entitled "New polyketides with antibacterial activity isolated from the endophytic fungus Exserohilum rostratum from Bauhinia guianensis" presented at the 56th Brazilian Congress of Chemistry from November 7 to 11, 2016 in Belém do Pará reveals from a plant matrix (Bauhinia guianensis), where the fungal species Exserohilum rostratum 1.11 Er was isolated, from which the biomass was obtained and thus the steroids ergosterol (1) and ergosterol peroxide (2) were isolated; isocoumarin, monocerine (3) and polyketides of unprecedented occurrence in the literature, called annularin I (4) and annularin J (5). Concomitantly, antimicrobial assays were performed. Annularins I (4) and J (5) showed good bacteriostatic activities, compared to B. subtilis and E. coli, with 4 standing out with bacteriostatic activity at the same concentration as the reference antibiotic. As for the cytotoxicity test, the substances were inactive.
[0022] O referido artigo de autoria de Pina, J.R.S. revela que as anularinas I (4) e J (5) apresentaram boas atividades bacteriostática, frente à B. subtilis e E. coli , destacando-se (4) que apresentou atividade bacteriostática na mesma concentração do antibiótico de referência. Quanto ao ensaio de citotoxidade as substâncias mostraram-se inativas . [0022] The referred article by Pina, JRS reveals that annularins I (4) and J (5) showed good bacteriostatic activities, compared to B. subtilis and E. coli, standing out (4) that showed bacteriostatic activity at the same concentration as the reference antibiotic. As for the cytotoxicity test, the substances were inactive.
[0023] A presente invenção revela o uso dos metabólitos secundários monocerina e anularina I na regeneração celular em células endoteliais HUVEC e fibroblastos normais FN1. Neste sentido, o uso dos metabólitos secundários monocerina e anularina I na regeneração celular em células endoteliais HUVEC e fibroblastos normais FN1 é bastante diferente daquele proposto por qualquer documento do estado da técnica.  [0023] The present invention discloses the use of the secondary metabolites monocerin and annularin I in cell regeneration in HUVEC endothelial cells and normal FN1 fibroblasts. In this sense, the use of the secondary metabolites monocerine and annularin I in cell regeneration in HUVEC endothelial cells and normal FN1 fibroblasts is quite different from that proposed by any prior art document.
[0024] Como pode ser observado nenhum documento do estado da técnica descreve ou muito menos sugere o uso dos metabólitos secundários monocerina e anularina I a partir da cultura do fungo Exserohilum rostratum para regeneração celular, baseando-se em dados experimentais em células endoteliais HUVEC e fibroblastos normais FN1.  [0024] As can be seen, no prior art document describes or much less suggests the use of secondary metabolites monocerin and annularin I from the culture of the fungus Exserohilum rostratum for cell regeneration, based on experimental data in HUVEC and endothelial cells normal FN1 fibroblasts.
Sumário da Invenção  Summary of the Invention
[0025] Para solucionar os problemas acima mencionados, a presente invenção propiciará vantagens significativas em relação ao uso dos metabólitos secundários monocerina e anularina I obtidos a partir da cultura do fungo Exserohilum rostratum para uso na regeneração celular, como por exemplo de células endoteliais HUVEC, fibroblastos macrófagos, etc., possibilitando um aumento do seu desempenho e apresentando uma relação custo/beneficio mais favorável .  [0025] To solve the problems mentioned above, the present invention will provide significant advantages in relation to the use of the secondary metabolites monocerin and annularin I obtained from the culture of the fungus Exserohilum rostratum for use in cell regeneration, such as for example endothelial cells HUVEC, macrophage fibroblasts, etc., enabling an increase in their performance and presenting a more favorable cost / benefit ratio.
[0026] Considerando os dados da literatura, a monocerina e a anularina I obtidas a partir do fungo E. rostratum podem ter sido biossintetizadas pela via de policetideos . Diversos compostos policetídeos são descritos como potentes antibióticos, antitumorais , antifúngicos, agentes imunossupressores e antivirais (Yadav et al . , 2003) . Este contexto reforça a relevância da investigação dos efeitos biológicos desses dois compostos e constitui um desafio, principalmente com relação a anularina I ainda pouco estudada . [0026] Considering the literature data, monocerine and anularin I obtained from the fungus E. rostratum may have been biosynthesized by polyketides. Several polyketide compounds are described as potent antibiotics, antitumor, antifungal, immunosuppressive and antiviral agents (Yadav et al., 2003). This context reinforces the relevance of investigating the biological effects of these two compounds and constitutes a challenge, especially with regard to annularin I, which has not yet been studied.
Breve Descrição dos Desenhos  Brief Description of Drawings
[0027] A estrutura e operação da presente invenção, juntamente com vantagens adicionais da mesma podem ser mais bem entendidas mediante referência aos desenhos em anexo e a seguinte descrição:  [0027] The structure and operation of the present invention, together with additional advantages of the same can be better understood by reference to the attached drawings and the following description:
[0028] A Figura 1 mostra as estruturas dos compostos anularinas A-H (1-8) descritas na literatura e obtidas a partir da cultura do fungo aquático Annulatascus triseptatus, da monocerina (Fl) e da anularina I (F2) obtidas a partir da cultura do fungo Exserohilum rostratum;  [0028] Figure 1 shows the structures of the annular compounds AH (1-8) described in the literature and obtained from the culture of the aquatic fungus Annulatascus triseptatus, monocerine (Fl) and anularin I (F2) obtained from the culture the fungus Exserohilum rostratum;
[0029] A Figura 2 mostra as células endoteliais HUVEC em processo de multiplicação celular. Controle (A) e tratamentos das células com o composto anularina I da presente invenção em mM, B (0,07), C (0,14), D (0,28), E (0,56), e F (1,12) . Setas na cor vermelha indicam células em processo de multiplicação celular e na cor preta células em processo de apoptose;  [0029] Figure 2 shows the HUVEC endothelial cells in the process of cell multiplication. Control (A) and cell treatments with the annularin I compound of the present invention in mM, B (0.07), C (0.14), D (0.28), E (0.56), and F ( 1.12). Red arrows indicate cells in the process of cell multiplication and black cells in the process of apoptosis;
[0030] Na Figura 3, o gráfico indica o perfil cromatográfico do extrato obtido da cultura do fungo em meio batata dextrose (BD) dos compostos da presente invenção. As condições da análise foram em coluna C18 (250 x 4, 6 mm), sistema de eluição isocrática com 56, 0 % de B em 30 minutos (solução A:H20/TFA, 99,9/0,1%; solução B: Me0H/H20/TFA, 90:9,9:0,1%), e com fluxo de 1 mL/min. Fl=monocerina e F2=anularina I . [0030] In Figure 3, the graph indicates the chromatographic profile of the extract obtained from the culture of the fungus in potato dextrose (BD) medium of the compounds of the present invention. The conditions of the analysis were in column C18 (250 x 4, 6 mm), isocratic elution system with 56.0% B in 30 minutes (solution A: H 2 0 / TFA, 99.9 / 0.1%; solution B: Me0H / H 2 0 / TFA, 90: 9.9: 0.1%) at a flow rate of 1 ml / min. Fl = monocerine and F2 = annularin I.
[0031] A figura 4 mostra um gráfico do perfil cromatográfico do extrato obtido da cultura do fungo em meio Czapek dos compostos da presente invenção. As condições da análise foram em coluna C18 (250 x 4, 6 mm), sistema de eluição isocrática 56,0 % de B em 30 minutos (solução A: H20/TFA, 99,9/0,1%; solução B: Me0H/H20/TFA, 90:9,9:0,1%), e com fluxo de 1 mL/min. Fl=monocerina . Obs . : ausência de anularina I . [0031] Figure 4 shows a graph of the chromatographic profile of the extract obtained from the culture of the fungus in Czapek medium of the compounds of the present invention. The conditions of the analysis were in column C18 (250 x 4, 6 mm), isocratic elution system 56.0% B in 30 minutes (solution A: H 2 0 / TFA, 99.9 / 0.1%; solution B: MeOH / H 2 O / TFA, 90: 9.9: 0.1%), and with a flow rate of 1 mL / min. Fl = monocerine. Obs. : absence of annularin I.
[0032] A Figura 5 mostra um gráfico do perfil cromatográfico do extrato obtido da cultura do fungo em meio extrato de malte (marca Synth) dos compostos da presente invenção. As condições da análise foram em coluna C18 (250 x 4,6 mm), sistema de eluição isocrática 56,0 % de B em 35 minutos (solução A:H20/TFA, 99,9/0,1%; solução B: MeOH/fbO/TFA, 90:9,9:0,1%), e com fluxo de 1 mL/min. Fl=monocerina e F2=anularina I . [0032] Figure 5 shows a graph of the chromatographic profile of the extract obtained from the culture of the fungus in malt extract medium (Synth brand) of the compounds of the present invention. The conditions of the analysis were in column C18 (250 x 4.6 mm), isocratic elution system 56.0% B in 35 minutes (solution A: H 2 0 / TFA, 99.9 / 0.1%; solution B: MeOH / fbO / TFA, 90: 9.9: 0.1%), and with a flow rate of 1 ml / min. Fl = monocerine and F2 = annularin I.
[0033] A Figura 6 mostra um gráfico do perfil cromatográfico do extrato obtido da cultura do fungo em meio extrato de malte (marca Himedia) . As condições da análise foram em coluna C18 (250 x 4, 6 mm), sistema de eluição isocrática 56,0 % de B em 30 minutos (solução A:H20/TFA, 99,9/0,1%; solução B: MeOH/fbO/TFA, 90:9,9:0,1%), e com fluxo de 1 mL/min. Fl=monocerina e F2=anularina I. [0033] Figure 6 shows a graph of the chromatographic profile of the extract obtained from the fungus culture in a malt extract medium (Himedia brand). The conditions of the analysis were in column C18 (250 x 4, 6 mm), isocratic elution system 56.0% B in 30 minutes (solution A: H 2 0 / TFA, 99.9 / 0.1%; solution B: MeOH / fbO / TFA, 90: 9.9: 0.1%), and with a flow rate of 1 ml / min. Fl = monocerine and F2 = annularin I.
[0034] A Figura 7 mostra um gráfico do perfil cromatográfico do extrato obtido da cultura do fungo em Meio Mínimo dos compostos da presente invenção. As condições da análise foram em coluna C18 (250 x 4, 6 mm), em sistema de eluição isocrática 56,0 % de B em 35 minutos (solução A: H2O/TFA, 99,9/0,1%; solução B: Me0H/H20/TFA, 90:9,9:0,1%), e com fluxo de 1 mL/min. Fl=monocerina e F2=anularina I. [0035] A Figura 8 mostra um gráfico do perfil cromatográfico da amostra YPSS obtida da cultura do fungo. As condições da análise foram em coluna C18 (250 x 4,6 mm), em sistema de eluição isocrática 56, 0 % de B em 35 minutos (solução A:H20/TFA, 99,9/0,1%; solução B: Me0H/H20/TFA, 90:9,9:0,1%), e com fluxo de 1 mL/min. Fl=monocerina e F2=anularina I . [0034] Figure 7 shows a graph of the chromatographic profile of the extract obtained from the culture of the fungus in Minimum Medium of the compounds of the present invention. The conditions of the analysis were in column C18 (250 x 4, 6 mm), in an isocratic elution system 56.0% of B in 35 minutes (solution A: H2O / TFA, 99.9 / 0.1%; solution B : Me0H / H 2 0 / TFA, 90: 9.9: 0.1%) at a flow rate of 1 ml / min. Fl = monocerine and F2 = annularin I. [0035] Figure 8 shows a graph of the chromatographic profile of the YPSS sample obtained from the fungus culture. The conditions of the analysis were in column C18 (250 x 4.6 mm), in an isocratic elution system 56, 0% B in 35 minutes (solution A: H 2 0 / TFA, 99.9 / 0.1%; solution B: Me0H / H 2 0 / TFA, 90: 9.9: 0.1%) at a flow rate of 1 ml / min. Fl = monocerine and F2 = annularin I.
[0036] A Figura 9 mostra um gráfico do perfil cromatográfico da amostra YESD. Coluna C18 (250 x 4,6 mm), em sistema de eluição isocrática 56, 0 % de B em 30 minutos (solução A:H20/TFA, 99,9/0,1%; solução B: Me0H/H20/TFA, 90:9,9:0,1%), e com fluxo de 1 mL/min. [0036] Figure 9 shows a graph of the chromatographic profile of the YESD sample. Column C18 (250 x 4.6 mm), in an isocratic elution system 56, 0% B in 30 minutes (solution A: H 2 0 / TFA, 99.9 / 0.1%; solution B: Me0H / H 20 / TFA, 90: 9.9: 0.1%), and with a flow rate of 1 mL / min.
[0037] A Figura 10 mostra um gráfico do perfil cromatográfico da amostra PYG1, no HPLC em coluna C18 (250 x 4,6 mm), em sistema de eluição isocrática 56,0 % de B em 30 minutos (solução A:H20/TFA, 99,9/0,1%; solução B: Me0H/H20/TFA, 90:9,9:0,1%), e com fluxo de 1 mL/min. [0037] Figure 10 shows a graph of the chromatographic profile of the PYG1 sample, on the HPLC in column C18 (250 x 4.6 mm), in an isocratic elution system 56.0% B in 30 minutes (solution A: H 2 0 / TFA 99.9 / 0.1%, solution B: Me0H / H 2 0 / TFA, 90: 9.9: 0.1%) at a flow rate of 1 ml / min.
[0038] A Figura 11 mostra uma Curva padrão para monocerina (fração Fl) nos comprimentos de onda de 214, 254 e 280 nm de acordo com as concentrações de monocerina que foi injetada.  [0038] Figure 11 shows a standard curve for monocerine (Fl fraction) at wavelengths of 214, 254 and 280 nm according to the concentrations of the injected monocerine.
[0039] A Figura 12 mostra uma curva padrão para anularina I (fração F2 ) nos comprimentos de onda de 214, 254 e 280 nm, de acordo com as concentrações de anularina I que foi injetada.  [0039] Figure 12 shows a standard curve for annularin I (fraction F2) at wavelengths of 214, 254 and 280 nm, according to the concentrations of annularin I that was injected.
[0040] A Figura 13 mostra a viabilidade celular de células endoteliais HUVEC após o tratamento com monocerina e anularina I nas concentrações de 0,02 a 10 mM, nos tempos de 24, 48 e 72 h. Zero igual ao controle RPMI 1640 correspondente a 100% de viabilidade celular. Análise estatística por One-way ANOVA / Dunnett's multiple comparison test (entre concentrações) . índice de significância de * r<0,1; ** p<0,05; *** p<0,01 para comparações entre os tratamentos em 24 e 48 h e o grupo controle não tratado (0) . índice de significância de # p< 0,01%; ## p < 0,05%, ### p< 0,01% e #### p< 0,001% para comparações entre o controle e o tempo de 72 h. [0040] Figure 13 shows the cell viability of HUVEC endothelial cells after treatment with monocerine and annularin I at concentrations of 0.02 to 10 mM, at 24, 48 and 72 h. Zero equal to the RPMI 1640 control corresponding to 100% cell viability. Statistical analysis by One-way ANOVA / Dunnett ' s multiple comparison test (between concentrations). significance index of * r <0.1; ** p <0.05; *** p <0.01 for comparisons between treatments at 24 and 48 h and the untreated control group (0). significance index of # p <0.01%;## p <0.05%, ### p <0.01% and #### p <0.001% for comparisons between the control and the 72 h time.
[0041] A Figura 14 mostra a viabilidade celular de fibroblastos normais FN1 após o tratamento com monocerina e anularina I nas concentrações de 0,02 a 10 mM, nos tempos de 24, 48 e 72 h. Zero igual ao controle RPMI 1640 correspondente a 100% de viabilidade celular. Análise estatística por One-way ANOVA / Dunnett's multiple comparison test (entre concentrações) . índice de significância de * r<0,1; ** p<0,05; *** p<0,01 para comparações entre os tratamentos em 24 e 48 h e o grupo controle não tratado (0) . índice de significância de # p< 0,01%; ## p < 0,05%, ### p< 0,01% e #### p< 0,001% para comparações entre o controle e o tempo de 72 h. [0041] Figure 14 shows the cell viability of normal FN1 fibroblasts after treatment with monocerine and annularin I at concentrations of 0.02 to 10 mM, at 24, 48 and 72 h. Zero equal to the RPMI 1640 control corresponding to 100% cell viability. Statistical analysis by One-way ANOVA / Dunnett ' s multiple comparison test (between concentrations). significance index of * r <0.1; ** p <0.05; *** p <0.01 for comparisons between treatments at 24 and 48 h and the untreated control group (0). significance index of # p <0.01%;## p <0.05%, ### p <0.01% and #### p <0.001% for comparisons between the control and the 72 h time.
[0042] A Figura 15 mostra a distribuição de células endoteliais HUVEC nas fases do ciclo celular após o tratamento com anularina I nas concentrações de 0,02; 0,15; 0,625 e 2,5 mM, nos tempos de 6, 24, 48 e 72 h. Zero igual ao tratamento controle com meio de cultura RPMI 1640;  [0042] Figure 15 shows the distribution of HUVEC endothelial cells in the phases of the cell cycle after treatment with annularin I in concentrations of 0.02; 0.15; 0.625 and 2.5 mM, at 6, 24, 48 and 72 h. Zero equal to the control treatment with RPMI 1640 culture medium;
[0043] A Figura 16 mostra uma análise de ciclo celular de HUVECs e fibroblastos FN1 tratados com anularina I de 0,02 a 0, 625 mM a 6, 24, 48 e 72 h. As barras representam as proporções de células proliferativas G2 / M; na síntese da fase S; Célula quiescente G0 / G1 e detritos em sub-Gl (DNA fragmentado) . Os dados representam média ± DP de três experimentos independentes. * Significativamente diferente do controle não tratado * p <0,05; ** p <0,01 e *** p <0,001. [0044] A Figura 17 mostra a distribuição de células endoteliais HUVEC e fibroblastos normais FN1 nas fases do ciclo celular após o tratamento com o composto monocerina nas concentrações de 0,02; 0,15 e 0,625 nos tempos de 24, 48 e 72 h. Zero igual ao tratamento controle com meio de cultura RPMI 1640. [0043] Figure 16 shows a cell cycle analysis of HUVECs and FN1 fibroblasts treated with annularin I from 0.02 to 0.625 mM at 6, 24, 48 and 72 h. The bars represent the proportions of G2 / M proliferative cells; in the synthesis of the S phase; Quiescent cell G0 / G1 and debris in sub-Gl (fragmented DNA). The data represent mean ± SD of three independent experiments. * Significantly different from the untreated control * p <0.05; ** p <0.01 and *** p <0.001. [0044] Figure 17 shows the distribution of HUVEC endothelial cells and normal FN1 fibroblasts in the cell cycle phases after treatment with the monocerine compound in concentrations of 0.02; 0.15 and 0.625 at 24, 48 and 72 h. Zero equal to the control treatment with RPMI 1640 culture medium.
[0045] A Figura 18 mostra a taxa proliferativa de fibroblastos humanos normais FN1 e células endoteliais (EC) . O tratamento com anularina I foi incapaz de alterar a proliferação celular de células de fibroblastos, mas para EC o tratamento modificou a capacidade proliferativa celular em todos os tratamentos e tempos. Média ± DP. Análise estatística por teste de comparação múltipla One-way ANOVA / Dunnett (entre concentrações) .  [0045] Figure 18 shows the proliferative rate of normal human FN1 fibroblasts and endothelial cells (EC). The treatment with annularin I was unable to alter the cell proliferation of fibroblast cells, but for EC the treatment changed the cell proliferative capacity at all treatments and times. Mean ± SD. Statistical analysis by multiple comparison test One-way ANOVA / Dunnett (between concentrations).
[0046] A Figura 19 mostra Western blotting representativo da expressão de VEGF-R1 nas incubações de HUVEC com diferentes concentrações de anularina I (1 e 2: RPMI; 3 e 4: 0,15 mM, 5 e 6: 0, 625 mM) por 24h. Gliceraldeído trifosfato desidrogenase (GAPDH) foi utilizado como controle de carregamento.  [0046] Figure 19 shows Western blotting representative of VEGF-R1 expression in HUVEC incubations with different concentrations of anularin I (1 and 2: RPMI; 3 and 4: 0.15 mM, 5 and 6: 0, 625 mM ) for 24 hours. Glyceraldehyde triphosphate dehydrogenase (GAPDH) was used as loading control.
[0047] A Figura 20 mostra a expressão relativa de proteínas em células HUVEC após tratamento com 0,15 and 0,625 mM de anularina I por 6 e 24 h. Resultados expressos pela proporção de proteínas em relação aos controles (GADPH ou b- Actina (media ± SEM) relativizados com valor de 1.  [0047] Figure 20 shows the relative expression of proteins in HUVEC cells after treatment with 0.15 and 0.625 mM of annularin I for 6 and 24 h. Results expressed by the proportion of proteins in relation to the controls (GADPH or b- Actin (mean ± SEM) relativized with a value of 1.
[0048] A Figura 21 mostra células endoteliais HUVEC senescentes após tratamento com anularina I em 0,02; 0,15 e 0,625 mM. Ensaio realizado por b-galactosidases . Média ± DP por 24 e 48 h. Análise estatística pelo teste de comparação múltipla One-way ANOVA / Dunnett (entre as concentrações) . índice significante ** ** ** p <0,01 para comparações de tratamento às 6 e 24 heo grupo controle. [0048] Figure 21 shows senescent HUVEC endothelial cells after treatment with 0.02 annularin I; 0.15 and 0.625 mM. Assay performed by b-galactosidases. Mean ± SD for 24 and 48 h. Statistical analysis using the One-way ANOVA / Dunnett multiple comparison test (between concentrations). significant index ** ** ** p <0.01 for comparisons of treatment at 6 and 24 h and the control group.
[0049] A Figura 22 mostra a porcentagem de células endoteliais HUVEC e fibroblastos normais FN1 senescentes e não senescentes após tratamento com anularina I ou monocerina nas concentrações de 0,02 a 1,25 mM. Ensaio com b- galactosidase .  [0049] Figure 22 shows the percentage of HUVEC endothelial cells and normal FN1 senescent and non-senescent fibroblasts after treatment with annularin I or monocerine in concentrations of 0.02 to 1.25 mM. Assay with b-galactosidase.
[0050] A Figura 23 mostra fotomicrografias representativas de células endoteliais trat:adas com anularina I a 0,02 (1B) 0,15 (1C), 0,625 mM (1D) durante 24 h e 0,02 (2B) 0, 15 (2C) , 0, 625 mM (2D) durante 48 h. Controles de 24 h e 48 h são representados por IA e 2A, respec ti amente .
Figure imgf000017_0001
;ao c 1.3 S não senescentes detectadas pelo ensaio da b-galactosidase . [0050] Figure 23 shows representative photomicrographs of endothelial cells treated with 0.02 (1B) annularin I 0.15 (1C), 0.625 mM (1D) for 24 h and 0.02 (2B) 0.15 ( 2C), 0.625 mM (2D) for 48 h. 24 h and 48 h controls are represented by AI and 2A, respectively.
Figure imgf000017_0001
c 1.3 S non-senescent detected by the b-galactosidase assay.
[0051] A Figura 24 mostra um histograma da análise de apoptose em células HUVECs tratadas com monocerina de 0,02 a 1,25 mM. Análise pela marcação dupla com anexina V- FITC/Iodeto de propideo e por citometria de fluxo. Gráficos representativos do tipo dot plot para cada uma das concentrações testadas. Quadrantes: inferior esquerdo= células viáveis; inferior direito= células em apoptose (anexina-V +) ; superior direito= células em apoptose tardia (anexina-V + e iodeto de propideo +; superior esquerdo= células em necrose (iodeto de propideo +) .  [0051] Figure 24 shows a histogram of apoptosis analysis in HUVECs cells treated with 0.02 to 1.25 mM monocerin. Analysis by double labeling with annexin V-FITC / propidium iodide and by flow cytometry. Representative dot plot graphics for each of the tested concentrations. Quadrants: lower left = viable cells; lower right = apoptosis cells (annexin-V +); upper right = cells in late apoptosis (annexin-V + and propidium iodide +; upper left = cells in necrosis (propidium iodide +).
[0052] A Figura 25 mostra a determinação de apoptose em células HUVECs pela dupla marcação com anexina V- FITC/Iodeto de propideo. Distribuição das células em viáveis, em processo de morte celular do tipo necrose, apoptose inicial ou tardia após tratamento com monocerina de 0,02 a 1,25 mM. O controle corresponde a tratamento somente com RPMI 1640.  [0052] Figure 25 shows the determination of apoptosis in HUVECs cells by double staining with annexin V-FITC / propidium iodide. Distribution of cells in viable cells, in the process of necrosis-like cell death, early or late apoptosis after treatment with 0.02 to 1.25 mM monocerine. The control corresponds to treatment with RPMI 1640 only.
[0053] A Figura 26A mostra o perfil cromatográfico da purificação da fração monocerina SPE100 pela primeira etapa (A) : Método: coluna C18 (250 x 10 mm), sistema de eluição isocrática 48,0% B em 60 minutos e vazão 2,5 mL / min . [0053] Figure 26A shows the chromatographic profile of purification of the SPE100 monocerine fraction by the first step (A): Method: column C18 (250 x 10 mm), isocratic elution system 48.0% B in 60 minutes and flow rate 2.5 mL / min.
[0054] A Figura 26B mostra a monocerina purificada após a segunda etapa de purificação; Método: coluna C18 (250 x 4,6 mm), sistema de eluição isocrática 56,0% B em 30 minutos e taxa de fluxo 1,0 mL / min. Solução A: H20 / TFA (99,9 / 0,1%), solução B: MeOH / H20 / TFA (90: 9,9: 0,1%) .  [0054] Figure 26B shows the purified monocerine after the second purification step; Method: column C18 (250 x 4.6 mm), isocratic elution system 56.0% B in 30 minutes and flow rate 1.0 mL / min. Solution A: H20 / TFA (99.9 / 0.1%), solution B: MeOH / H20 / TFA (90: 9.9: 0.1%).
[0055] A Figura 27 mostra uma representação gráfica do tamanho da ferida medida pelo Paquímetro. Tamanho da ferida representado como média ± DP para grupos de animais tratados por 14 dias. n = 80, os dados foram analisados por ANOVA unidirecional seguida de comparações múltiplas de Tukey-Kramer usando o Graph pad Prism 5.1. Os tratamentos foram comparados com o grupo não tratado e não houve diferença estatística significante .  [0055] Figure 27 shows a graphical representation of the wound size measured by the Pachymeter. Wound size represented as mean ± SD for groups of animals treated for 14 days. n = 80, data were analyzed by unidirectional ANOVA followed by multiple Tukey-Kramer comparisons using Graph pad Prism 5.1. The treatments were compared with the untreated group and there was no statistically significant difference.
[0056] A figura 28 mostra uma representação gráfica do tamanho da ferida medida pela imagem In Vivo de raios-X (MS-FX Pro) . O tamanho da ferida é representado como média ± SEM para grupos de animais tratados por 14 dias. N = 4 animais em cada grupo. Foram utilizados dois animais de cada grupo para medir o tamanho da ferida por MS-Fx Pro. Os dados foram calculados usando o Graph pad Prism 5.1 e os tratamentos foram comparados com o grupo não tratado.  [0056] Figure 28 shows a graphical representation of the wound size measured by the In Vivo X-ray image (MS-FX Pro). The wound size is represented as mean ± SEM for groups of animals treated for 14 days. N = 4 animals in each group. Two animals from each group were used to measure the size of the wound by MS-Fx Pro. The data were calculated using Graph pad Prism 5.1 and the treatments were compared with the untreated group.
[0057] A Figura 29 mostra uma imagem de raios X ín vivo para feridas realizadas por MS FX PRO. Esta figura representa o tamanho das feridas de dois animais por grupo tratado por 24 h, 48 he 72 h. Por comparação com os grupos controle (G1 e G5) , é possível observar que as feridas de animais tratados com monocerina a 0,005% começaram a ser bem reduzidas após 72 h; [0057] Figure 29 shows an in vivo X-ray image for wounds performed by MS FX PRO. This figure represents the size of the wounds of two animals per group treated for 24 h, 48 h and 72 h. By comparison with the control groups (G1 and G5), it is possible to observe that the wounds of animals treated with 0.005% monocerine started to be well reduced after 72 h;
[0058] A figura 30 mostra uma imagem de raios X ín vivo para feridas realizadas por MS FX PRO. Esta figura representa o tamanho das feridas de dois animais por grupo tratado por 7, 10 e 14 dias. Por comparação com os grupos controle (G1 e G5) , é possível observar que as feridas de animais tratados com monocerina a 0,005% foram quase totalmente cicatrizadas após 10 dias;  [0058] Figure 30 shows an in vivo X-ray image for wounds performed by MS FX PRO. This figure represents the size of the wounds of two animals per treated group for 7, 10 and 14 days. By comparison with the control groups (G1 and G5), it is possible to observe that the wounds of animals treated with 0.005% monocerine were almost completely healed after 10 days;
[0059] A Figura 31 mostra fotomicrografias obtidas por Microscopia Eletrónica de Varredura da derme de camundongos tratados por 24 h, 72 h, 7 e 14 dias. Grupo controle não tratado Gl) : Manutenção da matriz extracelular do fragmento de cicatriz de 24 horas a 14 dias (setas vermelhas); Grupo colagenase (G2) : presença de numerosas células mortas e hemácias identificadas de 24 horas a 14 dias (setas azuis); Monocerina 0,0006% (G3) : forte formação de fibras colágenas; Monocerina 0, 005% (G4) : Fibras de colágeno (seta amarela) - fibronectinas observadas de 24 a 14 dias (seta amarela); Veículo (G5) : microfibrilas , glóbulos vermelhos, células inflamatórias (seta verde) até o 14° dia;  [0059] Figure 31 shows photomicrographs obtained by Scanning Electron Microscopy of the dermis of mice treated for 24 h, 72 h, 7 and 14 days. Untreated control group Gl): Maintenance of the extracellular matrix of the scar fragment from 24 hours to 14 days (red arrows); Collagenase group (G2): presence of numerous dead cells and erythrocytes identified from 24 hours to 14 days (blue arrows); Monocerine 0.0006% (G3): strong formation of collagen fibers; Monokerin 0, 005% (G4): Collagen fibers (yellow arrow) - fibronectins observed from 24 to 14 days (yellow arrow); Vehicle (G5): microfibrils, red blood cells, inflammatory cells (green arrow) until the 14th day;
[0060] A Figura 32 mostra coloração de tecidos de animais a partir de 24 horas de experiências. Magnitude de lOx. A) Hemotoxilina e Eosina (H&E); B) Coloração Pricosirius Red (PR) e C) Masson Trichome (MT) . Grupo Controle (Gl), Colagenase (G2), monocerina 0,0006% (G3), monocerina 0,005% (G4) e veículo (G5) .  [0060] Figure 32 shows staining of animal tissues from 24 hours of experiments. Magnitude of 10x. A) Hemotoxilin and Eosin (H&E); B) Pricosirius Red (PR) and C) Masson Trichome (MT) staining. Control Group (Gl), Collagenase (G2), monocerine 0.0006% (G3), monocerine 0.005% (G4) and vehicle (G5).
[0061] A Figura 33 mostra a coloração de tecidos de animais a partir de 72 h de experiências. Magnitude de lOx. A) Hemotoxilina e Eosina (H&E) ; B) Coloração Pricosirius Red (PR) e C) Masson Trichome (MT) . Grupo Controle (Gl), Colagenase (G2), monocerina 0,0006% (G3), monocerina 0,005% (G4) e veiculo (G5) . [0061] Figure 33 shows the staining of animal tissues after 72 h of experiments. Magnitude of 10x. A) Hemotoxilin and Eosin (H&E); B) Pricosirius Red (PR) and C) Masson Trichome (MT) staining. Control Group (Gl), Collagenase (G2), 0.0006% monocerine (G3), 0.005% monocerine (G4) and vehicle (G5).
[0062] A Figura 34 mostra a coloração de tecidos de animais a partir de experiências de 7 dias. Magnitude de lOx. A) Hemotoxilina e Eosina (H&E); B) Coloração Pricosirius Red (PR) e C) Masson Trichome (MT) . Grupo Controle (Gl), Colagenase (G2), monocerina 0,0006% (G3), monocerina 0,005% (G4) e veiculo (G5) ;  [0062] Figure 34 shows the staining of animal tissues from 7-day experiments. Magnitude of 10x. A) Hemotoxilin and Eosin (H&E); B) Pricosirius Red (PR) and C) Masson Trichome (MT) staining. Control Group (Gl), Collagenase (G2), monocerine 0.0006% (G3), monocerine 0.005% (G4) and vehicle (G5);
[0063] A Figura 35 mostra a coloração de tecidos de animais a partir de experiências de 7 dias. Magnitude de lOx. A) Hemotoxilina e Eosina (H&E); B) Coloração Pricosirius Red (PR) e C) Masson Trichome (MT) . Grupo Controle (Gl), Colagenase (G2), monocerina 0,0006% (G3), monocerina 0,005% (G4) e veiculo (G5) ;  [0063] Figure 35 shows the staining of animal tissues from 7-day experiments. Magnitude of 10x. A) Hemotoxilin and Eosin (H&E); B) Pricosirius Red (PR) and C) Masson Trichome (MT) staining. Control Group (Gl), Collagenase (G2), monocerine 0.0006% (G3), monocerine 0.005% (G4) and vehicle (G5);
[0064] A figura 36 mostra o percentual de colágeno presente nos tratamentos em grupo de 24 horas a 14 dias. Dados representados pela média ± SEM para animais em cada grupo (N = 4) . Dados analisados por ANOVA unidirecional seguida de comparações múltiplas de Tukey-Kramer . Os tratamentos com monocerina foram comparados com o grupo não tratado e quando estatisticamente significante são representados por * P <0,005;  [0064] Figure 36 shows the percentage of collagen present in group treatments from 24 hours to 14 days. Data represented by the mean ± SEM for animals in each group (N = 4). Data analyzed by unidirectional ANOVA followed by multiple Tukey-Kramer comparisons. The treatments with monocerine were compared with the untreated group and when statistically significant they are represented by * P <0.005;
[0065] A Figura 37 mostra fotomicrografias da pele de camundongo Balb / C coradas com vermelho de Picrosirius e avaliadas por microscopia polarizada usando um programa padronizado (software de microimagem) Zen Blue 2.6 para captura de imagens . As imagens foram capturadas para tratamento de 24 h, 72 h, 7 e 14 dias, permitindo a observação de todos os constituintes da pele. Birrefringência de cabelos (tons de verde) e colágeno (xlO) . Ep : epiderme; C: colágenos; D: derme; Gs : glândulas sebáceas; Gp : glândulas sudoríparas; *: cabelos; seta: músculo arrector pili. [0065] Figure 37 shows photomicrographs of Balb / C mouse skin stained with Picrosirius red and evaluated by polarized microscopy using a standardized program (microimaging software) Zen Blue 2.6 for image capture. The images were captured for 24 h, 72 h, 7 and 14 days treatment, allowing the observation of all skin constituents. Birefringence of hair (shades of green) and collagen (x10). Ep: epidermis; C: collagens; D: dermis; Gs: sebaceous glands; Gp: sweat glands; *: hair; arrow: pili arrector muscle.
[0066] A figura 38 mostra os parâmetros hematológicos para tipos individuais de células sanguíneas. Os indicadores relacionados às hemácias e as células imunes pertencentes à classe de leucócitos foram analisados no sangue periférico a partir de diferentes intervalos de tempo 24h. 7d e 14 d. Os seguintes grupos são ilustrados por diferentes cores de coluna. Grupo controle (branco); Colagenase (cinza); Monocerina 0,0006% (vermelho); Monocerina 0,005% (azul); Veículo (preto) .  [0066] Figure 38 shows the hematological parameters for individual types of blood cells. The indicators related to red blood cells and immune cells belonging to the class of leukocytes were analyzed in peripheral blood from different time intervals 24h. 7d and 14 d. The following groups are illustrated by different column colors. Control group (white); Collagenase (gray); Monocerin 0.0006% (red); Monocerin 0.005% (blue); Vehicle (black).
[0067] A Figura 39 mostra a análise hematológica para tipos individuais de células sanguíneas. Os indicadores relacionados às hemácias e as células imunes pertencentes à classe de leucócitos foram analisados no sangue periférico em diferentes intervalos de tempo 24h, 7d e 14d. Os seguintes grupos ilustram as cores diferentes. Grupo controle (branco); Colagenase (cinza); Monocerina 0,0006% (vermelho); Monocerina 0,005% (azul); Veículo (preto) .  [0067] Figure 39 shows the hematological analysis for individual types of blood cells. The indicators related to red blood cells and immune cells belonging to the leukocyte class were analyzed in peripheral blood at different time intervals 24h, 7d and 14d. The following groups illustrate the different colors. Control group (white); Collagenase (gray); Monocerin 0.0006% (red); Monocerin 0.005% (blue); Vehicle (black).
[0068] A Figura 40 mostra uma fotomicrografia de tecidos imunocorados com anticorpo contra VEGF nos grupos controle e tratado por 24 h. Para todos os grupos é possível observar a imunorreatividade contra o VEGF (setas), enquanto para os controles negativos não houve imunorreatividade conforme o esperado; A exceção é para a reação de controle negativo para o grupo Gl, que também foi imunorreativo, mas de baixa intensidade, conforme o esperado. Barra de escala 40 x 100 pm. Os dados foram analisados medindo os pixels da barra de escala de 1024 x 2048 pixels.  [0068] Figure 40 shows a photomicrograph of tissues immunostained with antibody against VEGF in the control and treated groups for 24 h. For all groups, it is possible to observe immunoreactivity against VEGF (arrows), while for negative controls there was no immunoreactivity as expected; The exception is for the negative control reaction for the Gl group, which was also immunoreactive, but of low intensity, as expected. Scale bar 40 x 100 pm. The data were analyzed by measuring the pixels of the 1024 x 2048 pixel scale bar.
[0069] A Figura 41 mostra uma representação gráfica da área % de histointensidade imune ao VEGF calculada pela Imagem j e dados obtidos calculados pela Origem 9.1. [0070] A figura 42 mostra uma representação gráfica do tamanho da ferida medida pelo Paquimeter. Tamanho da ferida representado como média ± DP para grupos de animais tratados por 14 dias. n = 80, os dados foram analisados por ANOVA unidirecional seguida de comparações múltiplas de Tukey-Kramer usando o Graph pad Prism 5.1. Os tratamentos foram comparados com o grupo não tratado e não houve diferença estatística significante . [0069] Figure 41 shows a graphical representation of the area% of histointensity immune to VEGF calculated by Image j and obtained data calculated by Origin 9.1. [0070] Figure 42 shows a graphical representation of the size of the wound measured by the Paquimeter. Wound size represented as mean ± SD for groups of animals treated for 14 days. n = 80, data were analyzed by unidirectional ANOVA followed by multiple Tukey-Kramer comparisons using Graph pad Prism 5.1. The treatments were compared with the untreated group and there was no statistically significant difference.
[0071] A figura 43 mostra uma representação gráfica do tamanho da ferida medida pela imagem In Vivo de raios-X (MS-FX Pro) . O tamanho da ferida é representado como média ± SEM para grupos de animais tratados por 14 dias. N = 4 animais em cada grupo. Foram utilizados dois animais de cada grupo para medir o tamanho da ferida por MS-Fx Pro. Os dados foram calculados usando o Graph pad Prism 5.1 e os tratamentos foram comparados com o grupo não tratado.  [0071] Figure 43 shows a graphical representation of the wound size measured by the In Vivo X-ray image (MS-FX Pro). The wound size is represented as mean ± SEM for groups of animals treated for 14 days. N = 4 animals in each group. Two animals from each group were used to measure the size of the wound by MS-Fx Pro. The data were calculated using Graph pad Prism 5.1 and the treatments were compared with the untreated group.
[0072] A figura 44 mostra imagem de raios X ín vivo para feridas realizadas por MS FX PRO. Esta figura representa o tamanho das feridas de um animal por grupo tratado por 24 h, 72 h, 7, 10 e 14 dias. Por comparação com os grupos controles, é possível observar que as feridas de animais tratados com anularina I a 0,003% e 0,0005% foram quase totalmente cicatrizadas após 10 dias.  [0072] Figure 44 shows an in vivo X-ray image for wounds performed by MS FX PRO. This figure represents the size of the wounds of one animal per group treated for 24 h, 72 h, 7, 10 and 14 days. By comparison with the control groups, it can be seen that the wounds of animals treated with 0.003% and 0.0005% anularin I were almost completely healed after 10 days.
[0073] A Figura 45 mostra parâmetros hematológicos para tipos individuais de células sanguíneas. Os indicadores relacionados às hemácias e as células imunes pertencentes à classe de leucócitos foram analisados no sangue periférico a partir de diferentes intervalos de tempo de 24 h, 7 e 14 dias. Os seguintes grupos são ilustrados por diferentes cores de coluna. Grupo controle (branco); Colagenase (cinza); Anularina I 0,003% (vermelho); Anularina I 0,0005% (azul); Veiculo (preto) . [0073] Figure 45 shows hematological parameters for individual types of blood cells. The indicators related to red blood cells and immune cells belonging to the class of leukocytes were analyzed in peripheral blood from different time intervals of 24 h, 7 and 14 days. The following groups are illustrated by different column colors. Control group (white); Collagenase (gray); Annularin I 0.003% (red); Annularin I 0.0005% (blue); Vehicle (black).
[0074] A Figura 46 mostra Parâmetros hematológicos para tipos individuais de células sanguíneas. Os indicadores relacionados às hemácias e as células imunes pertencentes à classe de leucócitos foram analisados no sangue periférico a partir de diferentes intervalos de tempo de 24 h, 7 e 14 dias. Os seguintes grupos são ilustrados por diferentes cores de coluna. Grupo controle (branco); Colagenase (cinza); Anularina I 0,003% (vermelho); Anularina I 0,0005% (azul); Veículo (preto) .  [0074] Figure 46 shows hematological parameters for individual types of blood cells. The indicators related to red blood cells and immune cells belonging to the class of leukocytes were analyzed in peripheral blood from different time intervals of 24 h, 7 and 14 days. The following groups are illustrated by different column colors. Control group (white); Collagenase (gray); Annularin I 0.003% (red); Annularin I 0.0005% (blue); Vehicle (black).
[0075] A figura 47 mostra fotomicrografias obtidas por MEV da derme de camundongos tratados por 24 h, 72 h, 7 e 14 dias. Grupo controle não tratado Gl) : manutenção da matriz extracelular do fragmento de cicatriz de 24 horas a 14 dias (setas vermelhas); Grupo colagenase (G2) : presença de numerosas células mortas e hemácias identificadas de 24 horas a 14 dias (setas azuis); Anularina 0,003% (G3) : forte formação de fibras de colágeno; Anularina 0, 0005% (G4) : Fibras de colágeno (seta amarela) - fibronectinas observadas de 24 horas a 14 dias (seta amarela) ; Veículo (G5) : microfibrilas , glóbulos vermelhos, células inflamatórias (seta verde) até o 14° dia.  [0075] Figure 47 shows photomicrographs obtained by SEM of the dermis of mice treated for 24 h, 72 h, 7 and 14 days. Untreated control group Gl): maintenance of the extracellular matrix of the scar fragment from 24 hours to 14 days (red arrows); Collagenase group (G2): presence of numerous dead cells and erythrocytes identified from 24 hours to 14 days (blue arrows); 0.003% annularine (G3): strong formation of collagen fibers; Annularin 0, 0005% (G4): Collagen fibers (yellow arrow) - fibronectins observed from 24 hours to 14 days (yellow arrow); Vehicle (G5): microfibrils, red blood cells, inflammatory cells (green arrow) until the 14th day.
[0076] A figura 48 mostra a coloração de tecidos de animais a partir de 24 horas de experiências. Magnitude de lOx. A) Hemotoxilina e Eosina (H&E); B) Coloração Picrosirius Red (PR) e C) Masson Trichome (MT) . Grupo Controle (Gl), Colagenase (G2), Anularina 0,003% (G3), Anularina 0,0005% (G4) e veículo (G5) .  [0076] Figure 48 shows the staining of animal tissues after 24 hours of experiments. Magnitude of 10x. A) Hemotoxilin and Eosin (H&E); B) Picrosirius Red (PR) and C) Masson Trichome (MT) staining. Control Group (Gl), Collagenase (G2), Annularine 0.003% (G3), Annularine 0.0005% (G4) and vehicle (G5).
[0077] A Figura 49 mostra a coloração de tecidos de animais a partir de 72 h de experiências. Magnitude de lOx. A) Hemotoxilina e Eosina (H&E) ; B) Coloração Picrosirius Red (PR) e C) Masson Trichome (MT) . Grupo Controle (Gl), Colagenase (G2), Anularina 0,003% (G3), Anularina 0,0005% (G4) e Veiculo (G5) . [0077] Figure 49 shows the staining of animal tissues after 72 h of experiments. Magnitude of 10x. A) Hemotoxilin and Eosin (H&E); B) Picrosirius Red coloring (PR) and C) Masson Trichome (MT). Control Group (Gl), Collagenase (G2), Annularine 0.003% (G3), Annularine 0.0005% (G4) and Vehicle (G5).
[0078] A Figura 50 mostra a coloração de tecidos de animais a partir de experiências de 7 dias. Magnitude de lOx. A) Hemotoxilina e Eosina (H&E); B) Coloração Picrosirius Red (PR) e C) Masson Trichome (MT) . Grupo Controle (Gl), Colagenase (G2), Anularina 0,003% (G3), Anularina 0,0005% (G4) e Veiculo (G5) .  [0078] Figure 50 shows the staining of animal tissues from 7-day experiments. Magnitude of 10x. A) Hemotoxilin and Eosin (H&E); B) Picrosirius Red (PR) and C) Masson Trichome (MT) staining. Control Group (Gl), Collagenase (G2), Annularine 0.003% (G3), Annularine 0.0005% (G4) and Vehicle (G5).
[0079] A Figura 51 mostra a coloração de tecidos de animais de experiências de 14 dias. Magnitude de lOx. A) Hemotoxilina e Eosina (H&E) ; B) Coloração Picrosirius Red (PR) e C) Masson Trichome (MT) . Grupo Controle (Gl), Colagenase (G2), Anularina 0,003% (G3), Anularina 0,0005% (G4) e Veiculo (G5) .  [0079] Figure 51 shows the staining of animal tissues from 14-day experiments. Magnitude of 10x. A) Hemotoxilin and Eosin (H&E); B) Picrosirius Red (PR) and C) Masson Trichome (MT) staining. Control Group (Gl), Collagenase (G2), Annularine 0.003% (G3), Annularine 0.0005% (G4) and Vehicle (G5).
[0080] A figura 52 mostra uma ilustração gráfica em 3D de tecidos com coloração vermelha Picrosirius de 24h a 14 dias. Fotomicrografias da pele de camundongo Balb / C coradas com vermelho Picrosirius e avaliadas por microscopia polarizada usando um programa padronizado (software de microimagem) Zen Blue 2.6 para captura de imagem. As imagens foram capturadas para tratamento de 24 h, 72 h, 7 e 14 dias, permitindo a observação de todos os constituintes da pele. Birrefringência de cabelos (tons de verde) e colágeno (xlO) . Ep : epiderme; C: colágenos; D: derme; Gs : glândulas sebáceas; Gp : glândulas sudoríparas; *: cabelos; seta: músculo arrector pílí  [0080] Figure 52 shows a 3D graphic illustration of fabrics with Picrosirius red coloring from 24h to 14 days. Photomicrographs of the Balb / C mouse skin stained with Picrosirius red and evaluated by polarized microscopy using a standardized program (microimaging software) Zen Blue 2.6 for image capture. The images were captured for 24 h, 72 h, 7 and 14 days treatment, allowing the observation of all skin constituents. Birefringence of hair (shades of green) and collagen (x10). Ep: epidermis; C: collagens; D: dermis; Gs: sebaceous glands; Gp: sweat glands; *: hair; arrow: pectoral arrector muscle
[0081] A Figura 53 mostra o percentual de formação de colágeno induzido por tratamentos com anularina I até 14 dias. Análise pela imagem J 5.1 e dados calculados por prisma gráfico 5.1. [0082] A Figura 54 mostra a porcentagem de formação de colágeno induzida por tratamentos com monocerina até 14 dias. Análise pela imagem J 5.1 e dados calculados por prisma gráfico 5.1. [0081] Figure 53 shows the percentage of collagen formation induced by treatments with annularin I up to 14 days. Analysis using the J 5.1 image and data calculated using a 5.1 graphic prism. [0082] Figure 54 shows the percentage of collagen formation induced by treatments with monocerine up to 14 days. Analysis using the J 5.1 image and data calculated using a 5.1 graphic prism.
[0083] A Figura 55 mostra uma representação gráfica 3D de tecidos usando microscópio polarizado. Quantificação da formação de colágeno por monocerina a 0,0006% e 0,005% e tratamentos de anularina I a 0,0005% e 0,003%. A área ocupada por fibras de colágeno foi quantificada usando o Image J e o software Graph Pad Prism 5.1. As fibras de colágeno exibiram cores de polarização no vermelho-laranja, enquanto para os controles as fibras de colágeno eram esparsas e imaturas. O controle do grupo não tratado exibiu intensa birrefringência verde - amarela, sugerindo que o conteúdo de colágeno foi reduzido e suas fibras estavam muito frouxamente compactadas. Fibras verdes - típicas do colágeno tipo III foram mais frequentemente localizadas na derme superior e na epiderme nos grupos tratados com anularina. Os grupos controle (controle, colagenase e veículo) apresentaram fibras grossas vermelhas e amarelas (típicas do colágeno tipo I) localizadas na derme profunda inferior. No geral, os grupos tratados com monocerina e anularina I mostraram arranjo de fibras colágenas visíveis e organizadas dos grupos tratados com anularina.  [0083] Figure 55 shows a 3D graphic representation of tissues using a polarized microscope. Quantification of collagen formation by 0.0006% and 0.005% monocerin and 0.0005% and 0.003% anularin I treatments. The area occupied by collagen fibers was quantified using Image J and Graph Pad Prism 5.1 software. Collagen fibers exhibited polarizing colors in red-orange, while for controls, collagen fibers were sparse and immature. The control of the untreated group showed intense green - yellow birefringence, suggesting that the collagen content was reduced and its fibers were very loosely compacted. Green fibers - typical of type III collagen were most frequently located in the upper dermis and epidermis in the groups treated with anularin. The control groups (control, collagenase and vehicle) had thick red and yellow fibers (typical of type I collagen) located in the lower deep dermis. In general, the groups treated with monocerine and annularin I showed an arrangement of visible and organized collagen fibers from the groups treated with annularin.
Descrição Detalhada da Invenção  Detailed Description of the Invention
[0084] Embora a presente invenção possa ser suscetível a diferentes concretizações, é mostrada nos desenhos e na seguinte discussão detalhada, uma concretização preferida com o entendimento de que a presente concretização deve ser considerada uma exemplificação dos princípios da invenção e não pretende limitar a presente invenção ao que foi ilustrado e descrito neste relatório. [0084] Although the present invention may be susceptible to different embodiments, it is shown in the drawings and in the following detailed discussion, a preferred embodiment with the understanding that the present embodiment should be considered an example of the principles of the invention and is not intended to limit the present invention to what was illustrated and described in this report.
[0085] A presente invenção se refere ao uso de metabólito secundário produzido pelo fungo Exserohilum rostratum na regeneração celular. Particularmente, a presente invenção se refere ao uso dos metabólitos secundários monocerina e anularina I obtidos a partir da cultura do fungo Exserohilum rostratum para regeneração celular tendo como referência o efeito em células endoteliais HUVEC e fibroblastos normais FN1.  [0085] The present invention relates to the use of secondary metabolite produced by the fungus Exserohilum rostratum in cell regeneration. In particular, the present invention relates to the use of the secondary metabolites monocerin and annularin I obtained from the culture of the fungus Exserohilum rostratum for cell regeneration having as reference the effect on HUVEC endothelial cells and normal FN1 fibroblasts.
[0086] Basicamente, a presente invenção se refere ao uso de metabólito secundário, tais como monocerina e anularina I produzido pelo fungo Exserohilum rostratum, como agente indutor de proliferação celular, processo de fundamental importância no reparo de tecidos, como, por exemplo, na cicatrização de feridas.  [0086] Basically, the present invention refers to the use of secondary metabolites, such as monocerine and annularin I produced by the fungus Exserohilum rostratum, as an agent that induces cell proliferation, a process of fundamental importance in tissue repair, as, for example, in wound healing.
Produção e purificação dos metabólitos secundários, monocerina e anularina I a partir da cultura do fungo Exserohilum rostratum  Production and purification of secondary metabolites, monocerine and annularin I from the culture of the fungus Exserohilum rostratum
[0087] Para a produção de metabólitos secundários, o fungo E. rostratum foi cultivado em meio de cultura batata dextrose por 15 dias a 28°C e 150 rpm, e o caldo/sobrenadante da cultura foi submetido a extração em fase sólida (Solid Phase Extraction - SPE) com cartucho C18 (Spe-ed SPE Cartridges - Octadecyl C18/18%), e o material retido foi extraído com metanol a 100% resultando o extrato bruto.  [0087] For the production of secondary metabolites, the fungus E. rostratum was grown in potato dextrose culture medium for 15 days at 28 ° C and 150 rpm, and the culture broth / supernatant was subjected to solid phase extraction (Solid Phase Extraction - SPE) with C18 cartridge (Spe-ed SPE Cartridges - Octadecyl C18 / 18%), and the retained material was extracted with 100% methanol resulting in the crude extract.
[0088] Este extrato apresentou ação antifúngica contra Cryptococcus neoformans ATCC 90112r Trichophytum rubrum IOC 4527, Candida albicans ATCC 36802 e Aspergillus fumigatus IOC 4526 (CLSI, 2002a ,b) com concentrações inibitórias mínimas de 31,25 a 500 pg/mL (Tabela 1) . [0088] This extract showed antifungal action against Cryptococcus neoformans ATCC 90112 r Trichophytum rubrum IOC 4527, Candida albicans ATCC 36802 and Aspergillus fumigatus IOC 4526 (CLSI, 2002a, b) with minimum inhibitory concentrations of 31.25 to 500 pg / mL (Table 1) .
Tabela 1. Ação antifúngica do extrato bruto e dos compostos monocerina e anularina I produzidos pelo fungo E. rostratum (CIM em gg/mL) Table 1. Antifungal action of crude extract and compounds monocerine and annularin I produced by the fungus E. rostratum (MIC in gg / mL)
Figure imgf000027_0001
Figure imgf000027_0001
[0089] Para fracionamento, o extrato bruto foi submetido à cromatografia liquida de alta eficiência (CLAE) em coluna C8 (250 x 10 mm) e sistema de eluição isocrática variando de 44 a 60% com fluxo de 2,5 a 4 mL/min (solução A: H20/TFA, 99,9/0,1%; solução B: Me0H/H20/TFA, 90:9,9:0,1%) e duas frações principais foram obtidas e repurifiçadas usando coluna fenil (250 x 4,6 mm) e eluição isocrática de 44 a 60%. As frações F1 e F2 obtidas puras foram avaliadas em relação à ação antifúngica, e fisico-quimicamente caracterizadas por ressonância magnética nuclear de 1H e 13C e espectrometria de massas como sendo os compostos monocerina (2S, 3aR, 9bR) - 6-hidroxi-7 , 8-dimetoxi-2-propil-2 , 3, 3a, 9b- tetra-hidro-5H-furo [3, 2-c] isocromen-5-ona, e anularina 4- metoxi-5-metil- 6-butil-2H-piran-2-one, respectivamente, conforme estruturas representadas na Figura 1. A caracterização foi realizada com a colaboração do Prof. Roberto Berlinck do IQSC/USP. [0089] For fractionation, the crude extract was subjected to high performance liquid chromatography (HPLC) in a C8 column (250 x 10 mm) and an isocratic elution system ranging from 44 to 60% with a flow of 2.5 to 4 mL / min (solution A: H 2 0 / TFA, 99.9 / 0.1%; solution B: Me0H / H 2 0 / TFA, 90: 9.9: 0.1%) and two main fractions were obtained and refurified using phenyl column (250 x 4.6 mm) and 44 to 60% isocratic elution. The pure F1 and F2 fractions were evaluated for antifungal action, and physicochemically characterized by 1 H and 13 C nuclear magnetic resonance and mass spectrometry as the monocerine compounds (2S, 3aR, 9bR) - 6-hydroxy -7,8-dimethoxy-2-propyl-2,3,3a, 9b-tetrahydro-5H-bore [3, 2-c] isochromen-5-one, and 4-methoxy-5-methyl- 6 annularin -butil-2H-piran-2-one, respectively, according to the structures represented in Figure 1. The characterization was carried out with the collaboration of Prof. Roberto Berlinck from IQSC / USP.
[0090] Esta anularina também foi obtida por outros pesquisadores e denominada como anularina I (Pinheiro et ai, 2016) . Seguindo a nomenclatura do grupo de anularinas até então descritas (A-H) , na sequência esta seria anularina I. Esta molécula difere estruturalmente das anularinas A-H anteriormente descritas e tem um CH2 a mais em relação a anularina D. [0090] This annularin was also obtained by other researchers and named as annularin I (Pinheiro et al, 2016). Following the nomenclature of the group of annularins previously described (AH), in the sequence this would be annularin I. This molecule differs structurally from the annularins AH previously described and has an additional CH 2 compared to annularin D.
Caracterização físico-química da monocerina e anularina I: Monocerlna (nome IUPAC) : (2S, 3aR, 9bR) -6-hldroxl-7, 8- dlmetoxl-2-propll-2, 3, 3a , 9b-tetra-hldro-5H-fu.ro [3,2-c] lsocromen-5-ona  Physico-chemical characterization of monocerine and annularin I: Monocerlna (IUPAC name): (2S, 3aR, 9bR) -6-hldroxl-7, 8-dlmethoxy-2-propll-2, 3, 3a, 9b-tetra-hldro- 5H-fu.ro [3,2-c] lsocromen-5-one
[0091] ¾ RMN (499, 8 MHz , CDCI3) : d 11,17 (s, OH); [0091] ¾ NMR (499, 8 MHz, CDCl3): d 11.17 (s, OH);
6,85 (s, 1H) ; 5,20 ( dd, 6,0 e 3,2Hz, 1H) ; 4,6 (d, 3,2 Hz, 1H) ; 4,00 (m, 1H) ; 3,89 (s, OCH3); 3,71 (s, OCH3); 2,60 (ddd, 14,5; 8,4 e 6,0 Hz, 2H) ; 1,90 (dd, 8,4 e 6,0 Hz, 2H) ; 1,47 (m, 2H) ; 1,26 (m, 2H) ; 0,84 (t, 7,3 Hz, 3H) . 13C NMR (125,76.85 (s, 1H); 5.20 (dd, 6.0 and 3.2 Hz, 1H); 4.6 (d, 3.2 Hz, 1H); 4.00 (m, 1H); 3.89 (s, OCH3); 3.71 (s, OCH3); 2.60 (ddd, 14.5; 8.4 and 6.0 Hz, 2H); 1.90 (dd, 8.4 and 6.0 Hz, 2H); 1.47 (m, 2H); 1.26 (m, 2H); 0.84 (t, 7.3 Hz, 3H). 13 C NMR (125.7
MHz, CDCI3) : d 168,1; 158,8; 155,3; 136,7; 132,4; 105,7;MHz, CDCI3): d 168.1; 158.8; 155.3; 136.7; 132.4; 105.7;
102,0; 82,0; 77,9; 73,9; 60,4; 56,8; 38,8; 38,3; 19,1; 14,4. EM: M+ = 308,1; [M-C3H7 ] +=265 , 1 102.0; 82.0; 77.9; 73.9; 60.4; 56.8; 38.8; 38.3; 19.1; 14.4. MS: M + = 308.1; [M-C3H7] + = 265, 1
Anularina I (nome IUPAC) : 4-metoxl-5-metil-6-butil-2H-píran- 2-ona  Annularin I (IUPAC name): 4-methoxy-5-methyl-6-butyl-2H-píran-2-one
[0092] ¾ RMN (499, 8 MHz, CDCI3) : d 5,53 (s, 1H) , [0092] ¾ NMR (499, 8 MHz, CDCl3): d 5.53 (s, 1H),
2,27 (t, 6,4 Hz, 2H) , 1,29 (m, 4H) , 0,86 (t, 7,7 Hz, 3H) ,2.27 (t, 6.4 Hz, 2H), 1.29 (m, 4H), 0.86 (t, 7.7 Hz, 3H),
3,81 (s, 3H) , 2,16 (s, 3H) · 13C NMR (125,7 MHz, CDCI3 ) d 170,8;3.81 (s, 3H), 2.16 (s, 3H) · 13 C NMR (125.7 MHz, CDCl3) d 170.8;
163,4; 158,3; 111,1; 87,8; 57,0; 31,4; 23,6; 22,2; 17,2;163.4; 158.3; 111.1; 87.8; 57.0; 31.4; 23.6; 22.2; 17.2;
14,1. EM: M+ = 196,1; [M-C2H3O] +=153, 0. 14.1. MS: M + = 196.1; [M-C2H3O] + = 153.0.
[0093] A Figura 1 mostra as estruturas dos compostos anularinas A-H (1-8) descritas na literatura e obtidas a partir da cultura do fungo aquático Annulatascus triseptatus, da monocerina (Fl) e da anularina I (F2) obtidas a partir da cultura do fungo Exserohilum rostratum.  [0093] Figure 1 shows the structures of the annular compounds AH (1-8) described in the literature and obtained from the culture of the aquatic fungus Annulatascus triseptatus, monocerine (Fl) and annularin I (F2) obtained from the culture of the fungus Exserohilum rostratum.
Anularina e monocerlna — origem e atividade biológica Annularin and monocerlna - origin and biological activity
[0094] Na literatura, são poucos os relatos sobre o isolamento e síntese de monocerinas, assim como as atividades biológicas já descritas para este composto. Para as anularinas os relatos são ainda mais escassos. As monocerinas ( 12S ) -12-hidroximonocerina e ( 12R) -12-hidroximonocerina já foram isoladas do fungo endofitico Microdochium bolleyí e E. rostratum junto com a monocerina (2S, 3aR, 9bR) - 6-hidroxi-7 , 8- dimetoxi-2-propil-2 , 3, 3a, 9b-tetra-hidro-5H-furo [3, 2-c] isocromen-5-ona (Sappapan et al . , 2008; Zhang et al . , 2008) que também já foi isolada de outras fontes, como da cultura de E. turcum (Robeson e Strobel, 1982; Cuq et al . , 1993),[0094] In the literature, there are few reports on the isolation and synthesis of monokines, as well as the biological activities already described for this compound. For annularins, reports are even more scarce. Monocerines (12S) -12-hydroxymonocerine and (12R) -12-hydroxymonocerine have already been isolated from the endophytic fungus Microdochium bolleyí and E. rostratum together with monocerine (2S, 3aR, 9bR) - 6-hydroxy-7, 8-dimethoxy-2 -propyl-2, 3, 3a, 9b-tetrahydro-5H-bore [3, 2-c] isochromen-5-one (Sappapan et al., 2008; Zhang et al., 2008) which has also been isolated from other sources, such as E. turcum culture (Robeson and Strobel, 1982; Cuq et al., 1993),
Helmínthosporíum monoceras (Claydon et al . , 1979), Fusarium larvarium (Claydon et al . , 1979; Grove e Pople, 1979), e de Dreschlera ravenelli (Scott et al . , 1984) .0 estudo do metabolismo secundário do fungo E. rostratum levou a identificação de fitotoxinas com ação herbicida (Tan et al . , 2004), e do extrato bruto da extração com acetato de etila, o derivado de isocumarina 11-hidroximonocerina foi isolado junto com 12-hidroximonocerina e monocerina cuja potencial ação antimalárica (IC50 de 0,68 mM) e de seus análogos foi pela primeira vez descrita (Sappapan et al . , 2008) . As monocerinas tem ainda ação antifúngica, inseticida e fitotóxica (Sappapan et al . , 2008) . Helminthosporíum monoceras (Claydon et al., 1979), Fusarium larvarium (Claydon et al., 1979; Grove and Pople, 1979), and Dreschlera ravenelli (Scott et al., 1984) .0 study of the secondary metabolism of the fungus E. rostratum led to the identification of phytotoxins with herbicidal action (Tan et al., 2004), and of the crude extract from the extraction with ethyl acetate, the 11-hydroxymonocerine isocoumarin derivative was isolated together with 12-hydroxymonocerine and monocerine whose potential antimalarial action ( IC50 of 0.68 mM) and its analogs was first described (Sappapan et al., 2008). Monocerines also have antifungal, insecticidal and phytotoxic effects (Sappapan et al., 2008).
[0095] Da cultura do fungo aquático A. triseptatus, o extrato obtido da extração com acetato de etila apresentou ação antifúngica e antibacteriana e o estudo químico resultou no isolamento de 8 compostos, denominados como anularinas A- H (Figura 1) e classificados como metabólitos policetídeos (Li et al . , 2003) . As anularinas A-F são a-pironas 3,4,5- trisubstituídas e as anularinas G-H são y-lactonas 3,4- disubstituídas a, b-insaturadas . As anularinas A, B, C e F inibiram o crescimento de Bacíllus subtílus (ATCC 6051) induzindo halo de 8-10 mm com 200 pg/disco testado e nesta mesma concentração, a anularina C inibiu o crescimento de Staphylococcus aureus (ATCC 29213) com halo de 14 mm. Nenhum composto apresentou ação contra C. albicans (ATCC 90029) e com exceção dos compostos D e E (não testados) os demais não foram efetivos contra A. flavus (NRRL 6541) . [0095] From the culture of the aquatic fungus A. triseptatus, the extract obtained from the extraction with ethyl acetate showed antifungal and antibacterial action and the chemical study resulted in the isolation of 8 compounds, called anularins A-H (Figure 1) and classified as polyketide metabolites (Li et al., 2003). AF annularins are 3,4,5-tri-substituted α-pyronines and GH annularins are 3,4-disubstituted α-pyrones, b-unsaturated. Annularins A, B, C and F inhibited the growth of Bacillus subtilus (ATCC 6051) by inducing an 8-10 mm halo with 200 pg / disc tested and at the same concentration, annularin C inhibited the growth of Staphylococcus aureus (ATCC 29213) with 14 mm halo. none compound showed action against C. albicans (ATCC 90029) and with the exception of compounds D and E (not tested) the others were not effective against A. flavus (NRRL 6541).
[0096] Anularinas A-F são a-pironas, membros de uma classe geral comumente encontrada em fungos. Um estudo para a síntese de anularinas foi proposto por Motodate et al . , (2010) e a síntese total de anularina F foi relatada pela primeira vez em 2006 (Kurdyumov et al . , 2006) . Biogeneticamente, as anularinas A-F são presumivelmente derivadas da via de policetídeo e existe a hipótese de que as anularinas G e H tenham origem em uma via biossintética análoga, mas com uma diferente etapa de fechamento do anel.  [0096] Annularins A-F are α-pyrones, members of a general class commonly found in fungi. A study for the synthesis of annularins was proposed by Motodate et al. , (2010) and the total synthesis of annularin F was first reported in 2006 (Kurdyumov et al., 2006). Biogenetically, the annularins A-F are presumably derived from the polyketide pathway and there is a hypothesis that the annularins G and H originate from an analogous biosynthetic pathway, but with a different stage of ring closure.
[0097] De forma semelhante, a monocerina tem origem como heptacetídeo, o que foi demonstrado em um estudo de biossíntese dessa molécula com a incorporação de acetatos marcados com 2H, 13C e 180 em D. ravenelii (Scott et al . ,[0097] Similarly, monocerine originates as heptacetide, which was demonstrated in a biosynthesis study of this molecule with the incorporation of 2 H, 13 C and 18 0 acetates in D. ravenelii (Scott et al.,
1984) . 1984).
[0098] Considerando os dados da literatura, a monocerina e a anularina I obtidas na presente invenção a partir da cultura do fungo E. rostratum podem ter sido biossintetizadas pela via de policetídeos . Diversos compostos policetídeos são descritos como potentes antibióticos, antitumorais , antifúngicos, agentes imunossupressores e antivirais (Yadav et al . , 2003) . Este contexto reforça a relevância da investigação dos efeitos biológicos desses dois compostos e constitui um desafio, principalmente para a anularina I ainda pouco estudada.  [0098] Considering the literature data, monocerine and annularin I obtained in the present invention from the culture of the fungus E. rostratum may have been biosynthesized by the polyketide route. Several polyketide compounds are described as potent antibiotics, antitumor, antifungal, immunosuppressive and antiviral agents (Yadav et al., 2003). This context reinforces the relevance of investigating the biological effects of these two compounds and constitutes a challenge, especially for annularin I, which has not yet been studied.
Estudos preliminares da monocerina e anularina I produzidas pelo fungo E. rostratum. Preliminary studies of monocerine and annularin I produced by the fungus E. rostratum.
[0099] A monocerina inibiu o crescimento do fungo dermatófito T. rubrum, da levedura Candída albicans e do fungo filamentoso Aspergillus fumigatus com CIMs de 15 a 500 gg/mL. A anularina I inibiu o crescimento do dermatófito T. rubrum somente a 250 gg/mL, e não foi efetiva sobre C. albicans e A. fumigatus até a concentração de 500 gg/mL (ver Tabela 1 ) . [0099] Monocerine inhibited the growth of the dermatophyte fungus T. rubrum, the yeast Candída albicans and the filamentous fungus Aspergillus fumigatus with MICs of 15 to 500 gg / mL. Annularin I inhibited the growth of the dermatophyte T. rubrum only at 250 gg / mL, and was not effective on C. albicans and A. fumigatus until the concentration of 500 gg / mL (see Table 1).
[00100] A ação antifúngica para a monocerina e anularina I não é significativa quando comparada com compostos antifúngicos de uso na clinica. Como alternativa de aplicação, a atividade citotóxica foi avaliada em células da linhagem tumoral K562 ( leucemia eritrocitica crónica) e B16F10 (melanoma murino) pelo ensaio do MTT que avalia a viabilidade celular pela ação da enzima mitocondrial succinato desidrogenase (Mosmann, 1983) .  [00100] The antifungal action for monocerine and annularin I is not significant when compared with antifungal compounds used in the clinic. As an alternative of application, cytotoxic activity was evaluated in cells of the tumor line K562 (chronic erythrocytic leukemia) and B16F10 (murine melanoma) by the MTT assay that assesses cell viability by the action of the mitochondrial succinate dehydrogenase enzyme (Mosmann, 1983).
[00101] A monocerina e a anularina I, até a concentração de 2 mM, não diminuíram a viabilidade celular de forma significativa nas células K-562 (leucemia humana) e B16F10 (melanoma murino), e em algumas concentrações promoveram proliferação celular. Este efeito de indução de proliferação celular foi um resultado não esperado; porém, muito relevante e interessante do ponto de vista de aplicação terapêutica .  [00101] Monocerine and annularin I, up to a concentration of 2 mM, did not significantly decrease cell viability in K-562 (human leukemia) and B16F10 (murine melanoma) cells, and in some concentrations promoted cell proliferation. This cell proliferation-inducing effect was an unexpected result; however, very relevant and interesting from the point of view of therapeutic application.
[00102] Para confirmação deste efeito, ensaios para análise da capacidade proliferativa da monocerina e da anularina I foram realizados em células endoteliais da veia do cordão umbilical humano (HUVEC) , também pelo método do MTT, e por observação da morfologia por microscopia. O resultado anterior foi confirmado, e nestas células, a monocerina e a anularina I induziram proliferação na proporção de duas e cinco vezes, respectivamente, em relação ao controle. Pela análise morfológica foi possível observar células em multiplicação celular, como representado na Figura 2 para o composto anularina I. A viabilidade celular também foi avaliada para estes dois compostos em macrófagos J774 até a concentração de 1 mM. A monocerina induziu perda da viabilidade celular com somente 50% das células viáveis na concentração de 45 mM (IC50) e a anularina I não foi tóxica para as células que permaneceram totalmente viáveis até a máxima concentração testada de 1 mM. [00102] To confirm this effect, assays to analyze the proliferative capacity of monocerine and annularin I were performed on human umbilical cord vein endothelial cells (HUVEC), also by the MTT method, and by observing the morphology by microscopy. The previous result was confirmed, and in these cells, monocerine and annularin I induced proliferation two and five times, respectively, in relation to the control. Through the morphological analysis it was possible to observe cells in cell multiplication, as represented in Figure 2 for the compound annularin I. Cell viability was also evaluated for these two compounds in J774 macrophages up to a concentration of 1 mM. Monokerin induced loss of cell viability with only 50% of viable cells at a concentration of 45 mM (IC50) and annularin I was not toxic for cells that remained fully viable until the maximum tested concentration of 1 mM.
[00103] A Figura 2 mostra as células endoteliais HUVEC em processo de multiplicação celular. Controle (A) e tratamentos das células com a anularina I em mM, B (0,07), C (0,14), D (0,28), E (0,56), e F (1,12). Setas na cor vermelha indicam células em processo de multiplicação celular e na cor preta células em processo de apoptose.  [00103] Figure 2 shows the HUVEC endothelial cells in the process of cell multiplication. Control (A) and cell treatments with annularin I in mM, B (0.07), C (0.14), D (0.28), E (0.56), and F (1.12) . Red arrows indicate cells in the process of cell multiplication and black cells in the process of apoptosis.
[00104] Considerando estes dados, e a perspectiva para um estudo mais aprofundado da aplicação da anularina I como agente indutor de proliferação celular, processo de fundamental importância no reparo de tecidos, como, por exemplo, na cicatrização de feridas, novos ensaios foram realizados em células endoteliais HUVEC e em fibroblastos normais FN1.  [00104] Considering these data, and the perspective for a more in-depth study of the application of annularin I as an agent that induces cell proliferation, a process of fundamental importance in tissue repair, such as, for example, in wound healing, new tests were performed in HUVEC endothelial cells and in normal FN1 fibroblasts.
[00105] Em estudo paralelo foi realizado a cultura do fungo E. rostratum em diferentes condições para otimização da produção dos metabólitos secundários monocerina e anularina I .  [00105] In a parallel study, the culture of the fungus E. rostratum was carried out under different conditions to optimize the production of the secondary metabolites monocerine and annularin I.
Procedimento experimental para otimização da produção dos compostos monocerina e anularina I  Experimental procedure to optimize the production of monocerine and annularin I compounds
Cultura do fungo e processo de extração dos Metabólitos Secundários (MS)  Fungus culture and extraction process of Secondary Metabolites (MS)
[00106] O fungo E. rostratum foi cultivado em placa contendo meio de cultura batata dextrose ágar (BDA) por 7 dias, e em estufa a 28°C. Posteriormente, com o auxilio de alças de Pasteur descartáveis e estéreis, a colónia foi fragmentada e transferida cuidadosamente para frasco erlenmeyer de 250 mL contendo 50 mL dos meios de cultura líquidos: Batata Dextrose (BD) , Meio Czapek Dox CD), Meio Mínimo (MM), Extrato de Malte 2% marca Synth (EMS), Extrato de Malte 2% marca Himedia (EMH) , YPSS, YESD e PYG . A cultura foi realizada em duplicata por 15 dias a 28°C, e a 150 rpm. [00106] The fungus E. rostratum was grown in a plate containing potato dextrose agar (BDA) culture medium for 7 days, and in an oven at 28 ° C. Subsequently, with the help of disposable and sterile Pasteur loops, the colony was fragmented and carefully transferred to a 250 mL Erlenmeyer flask containing 50 mL of liquid culture media: Dextrose Potato (BD), Czapek Dox CD Medium), Minimal Medium (MM), Malt Extract 2% brand Synth (EMS), Malt Extract 2% brand Himedia (EMH), YPSS, YESD and PYG. The culture was performed in duplicate for 15 days at 28 ° C, and at 150 rpm.
[00107] Após a cultura, em cabine de biossegurança nível II, a suspensão fúngica foi filtrada em membrana de poliéster ou algodão (gaze) (filtração 1) e posteriormente em lã de vidro (filtração 2) para remoção da maior proporção de biomassa, e o pH do sobrenadante foi medido com fita indicadora de pH.  [00107] After the culture, in a level II biosafety cabin, the fungal suspension was filtered through a polyester or cotton membrane (gauze) (filtration 1) and later on glass wool (filtration 2) to remove the highest proportion of biomass, and the pH of the supernatant was measured with a pH indicator strip.
[00108] O sobrenadante da cultura (filtrado 2) foi transferido para garrafas plásticas de 500 mL e congelado a -20°C por pelo menos 48 horas. A etapa de congelamento é importantíssima para a precipitação de pequenas partículas como resíduos de esporos e/ou células não removidos na filtração 2. Em seguida, o filtrado 2 foi submetido à nova filtração em membrana de fibra de vidro com porosidade de 1 mpi (filtração 3) e então submetido a extração em fase sólida (EFS ) . Para a EFS (ou SPE) , cartuchos C18 (Octadecyl C18/18% - 50 mg/6mL) foram condicionados com metanol P.A. a 100%, lavados com água destilada e o filtrado 3 foi sendo adicionado ao cartucho com auxílio de bomba de vácuo. O produto retido no cartucho foi extraído com metanol P.A. 100% resultando no extrato bruto. Após a extração, o solvente do extrato foi totalmente removido por rotaevaporação sob vácuo em banho-maria na temperatura máxima de 45°C.  [00108] The culture supernatant (filtrate 2) was transferred to 500 ml plastic bottles and frozen at -20 ° C for at least 48 hours. The freezing stage is very important for the precipitation of small particles such as spore residues and / or cells not removed in filtration 2. Then, filtrate 2 was subjected to a new filtration in a glass fiber membrane with 1 mpi porosity (filtration 3) and then subjected to solid phase extraction (EFS). For EFS (or SPE), C18 cartridges (Octadecyl C18 / 18% - 50 mg / 6mL) were conditioned with 100% methanol PA, washed with distilled water and filtrate 3 was added to the cartridge with the aid of a vacuum pump . The product retained in the cartridge was extracted with 100% P.A. methanol resulting in the crude extract. After extraction, the extract solvent was completely removed by rotary evaporation under vacuum in a water bath at a maximum temperature of 45 ° C.
[00109] A extração pode ser também por extração líquido-líquido (ELL) usando solventes como acetato de etila, hexano, diclorometano, etc. assim como a extração pode ser também em fase sólida usando discos de Octadecil C18/18%. Além de Octadecyl C18/18%, a extração seria eficiente usando cartuchos ou discos com algum dos sorventes comercialmente disponíveis e baseados em grupos orgânicos, como C18, C8, C4, C2, cicloexil, fenil, cianopropil, aminopropil (NH2), ligados quimicamente à sílica. [00109] Extraction can also be by liquid-liquid extraction (ELL) using solvents such as acetate ethyl, hexane, dichloromethane, etc. as well as the extraction can also be in solid phase using Octadecyl C18 / 18% discs. In addition to Octadecyl C18 / 18%, extraction would be efficient using cartridges or discs with any of the commercially available sorbents based on organic groups, such as C18, C8, C4, C2, cyclohexyl, phenyl, cyanopropyl, aminopropyl (NH2), chemically linked to silica.
[00110] As vantagens apresentadas pela EFS em comparação com a extração líquido-líquido clássica são: menor consumo de solvente orgânico, não formação de emulsões, facilidade de automação, altas porcentagens de recuperação do analito, volumes reduzidos de resíduos tóxicos, capacidade de aumentar seletivamente a concentração do analito e disponibilidade comercial de muitos equipamentos e sorventes para EFS,  [00110] The advantages presented by EFS in comparison with the classic liquid-liquid extraction are: lower consumption of organic solvent, no formation of emulsions, ease of automation, high percentages of analyte recovery, reduced volumes of toxic residues, ability to increase selectively the analyte concentration and commercial availability of many equipment and sorbents for EFS,
Análise Instrumental  Instrumental analysis
Os extratos obtidos de cada cultura nos diferentes meios foram pesados e dissolvidos em metanol na concentração de 5 mg/mL para serem analisados por cromatografia líquida de alta eficiência (CLAE/HPLC) de fase reversa, em coluna C18 (250 x 4,6 mm) e sistema de eluição isocrática com 56,0 % de fase móvel B em 30-35 minutos (solução A:H20/TFA, 99,9/0,1%; solução B: MeOH/ThO/TFA, 90:9,9:0,1%) . Por CLAE duas frações principais F1 (monocerina) e F2 (anularina I) foram detectadas e o perfil cromatográfico foi analisado para quantificação da porcentagem dos compostos. A quantificação dos compostos monocerina e anularina I presentes nos extratos foi determinada por comparação a uma curva padrão previamente estabelecida . The extracts obtained from each culture in the different media were weighed and dissolved in methanol at a concentration of 5 mg / mL to be analyzed by reverse phase high performance liquid chromatography (HPLC / HPLC), in a C18 column (250 x 4.6 mm ) and isocratic elution system with 56.0% mobile phase B in 30-35 minutes (solution A: H 2 0 / TFA, 99.9 / 0.1%; solution B: MeOH / ThO / TFA, 90: 9.9: 0.1%). By HPLC, two main fractions F1 (monocerine) and F2 (annularin I) were detected and the chromatographic profile was analyzed to quantify the percentage of compounds. The quantification of the monocerine and annularin I compounds present in the extracts was determined by comparison to a previously established standard curve.
[00111] Conforme apresentado na Tabela 2, as massas brutas dos extratos obtidos foram quantificadas e os valores estão apresentados pela média da cultura em duplicata ± o desvio padrão. [00111] As shown in Table 2, the gross masses of the extracts obtained were quantified and the values are presented by the culture mean in duplicate ± the standard deviation.
[00112] Embora o inoculo seja estimado, de forma a conter o mesmo número de unidades formadoras de colónias por inoculo, pode se observar, que para o meio Czapeck houve grande diferença nos valores da massa obtida resultando em um alto valor do desvio padrão. Até o momento não temos explicação para este fato.  [00112] Although the inoculum is estimated, in order to contain the same number of colony forming units per inoculum, it can be observed that for the Czapeck medium there was a large difference in the values of the mass obtained resulting in a high value of the standard deviation. So far we have no explanation for this fact.
[00113] Após a cultura do fungo E. rostratum nos diferentes meios de cultura, os valores de pH dos filtrados 2 das culturas foram medidos com fita indicadora de pH e os valores estão apresentados na Tabela 2.  [00113] After the culture of the E. rostratum fungus in the different culture media, the pH values of the filtrates 2 of the cultures were measured with a pH indicator tape and the values are shown in Table 2.
Tabela 2. Massa bruta em miligramas dos extratos obtidos para os diferentes meios de cultura usados no cultivo do fungo E. rostratum e valores de pH.  Table 2. Gross mass in milligrams of the extracts obtained for the different culture media used in the cultivation of the fungus E. rostratum and pH values.
Figure imgf000035_0001
Figure imgf000035_0001
*Valor médio das duplicatas, SD=desvio padrão.  * Average value of duplicates, SD = standard deviation.
[00114] Comparando com a massa dos compostos monocerina e anularina I obtidos de diferentes meios de cultura pode-se observar a relação do pH com a produção desses metabólitos secundários. O meio BD que apresentou maior rendimento possui o pH na faixa de 5,5. Os meios em extrato de malte, YPSS, YESD e PYG possuem o pH na faixa 7, 0-9,0 e além dos compostos monocerina e anularina I também induziram a produção de outros metabólitos detectados nas condições da corrida cromatográfica e presentes nos respectivos cromatogramas . Portanto, pode-se concluir que o pH ácido na faixa de 5,5 é um fator importante para a produção dos compostos de interesse e pode ser estudado mais detalhadamente . [00114] Comparing with the mass of the monocerine and annularin I compounds obtained from different culture media, one can observe the relationship of pH with the production of these secondary metabolites. The BD medium with the highest yield has a pH in the range of 5.5. The malt extract, YPSS, YESD and PYG media have pH in the range 7, 0-9.0 and in addition to the monocerine and annularin I compounds also induced the production of other metabolites detected in the chromatographic run conditions and present in the respective chromatograms. Therefore, it can be concluded that the acid pH in the range of 5.5 is an important factor for the production of the compounds of interest and can be studied in more detail.
[00115] Os extratos obtidos de cada cultura foram analisados por Cromatografia Liquida de Alta Eficiência (CLAE/HPLC) e os perfis cromatográficos estão representados nas figuras 3 a 10.  [00115] The extracts obtained from each culture were analyzed by High Performance Liquid Chromatography (HPLC / HPLC) and the chromatographic profiles are represented in figures 3 to 10.
[00116] De acordo com o perfil cromatográfico do extrato das culturas em meio liquido BD apresentado na figura 3, os dois compostos de interesse, monocerina e anularina I foram produzidos, e nas condições cromatográficas usadas no estudo a presença de outros compostos não foi detectada.  [00116] According to the chromatographic profile of the cultures extract in BD liquid medium presented in figure 3, the two compounds of interest, monocerine and annularin I were produced, and in the chromatographic conditions used in the study the presence of other compounds was not detected .
[00117] O perfil cromatográfico das amostras obtidas do meio de cultura Extrato de Malte 2% (marca Synth - EMS) é apresentado na figura 5. Para este meio de cultura, os dois compostos monocerina e anularina I foram detectados, entretanto, a anularina I apresentou baixa absorbância indicando a baixa concentração na amostra. Para a cultura neste meio, pelo método de análise outros MS interferentes não foram detectados.  [00117] The chromatographic profile of the samples obtained from the 2% Malt Extract culture medium (Synth - EMS brand) is shown in figure 5. For this culture medium, the two monocerine and annularin I compounds were detected, however, the annularin I showed low absorbance indicating low concentration in the sample. For culture in this medium, by the method of analysis other interfering MS were not detected.
[00118] A figura 4 representa o perfil cromatográfico dos extratos obtidos das culturas em meio Czapek Dox (MC) . Pode-se observar que o extrato não continha MS além daqueles esperados, porém, o mesmo não induziu a produção do composto anularina I .  [00118] Figure 4 represents the chromatographic profile of the extracts obtained from the cultures in Czapek Dox (MC) medium. It can be seen that the extract did not contain MS beyond those expected, however, it did not induce the production of the compound annularin I.
[00119] A Figura 5 mostra o perfil cromatográfico do extrato obtido da cultura em meio extrato de malte (marca Synth) . Coluna C18 (250 x 4, 6 mm) , sistema de eluição isocrática 56,0 % de B em 35 minutos (solução A:H20/TFA,[00119] Figure 5 shows the chromatographic profile of the extract obtained from the culture in malt extract medium (brand Synth). Column C18 (250 x 4, 6 mm), isocratic elution system 56.0% B in 35 minutes (solution A: H 2 0 / TFA,
99,9/0,1%; solução B: Me0H/H20/TFA, 90:9,9:0,1%), e com fluxo de 1 mL/min. Fl=monocerina e F2=anularina I. 99.9 / 0.1%; Solution B: Me0H / H 2 0 / TFA, 90: 9.9: 0.1%) at a flow rate of 1 ml / min. Fl = monocerine and F2 = annularin I.
[00120] Os compostos monocerina e anularina I foram detectados nos extratos produzidos no meio de cultura em extrato de malte (marca Himedia - EMH) (figura 6), entretanto, apresentou outros compostos.  [00120] The monocerine and annularin I compounds were detected in the extracts produced in the culture medium in malt extract (Himedia brand - EMH) (figure 6), however, presented other compounds.
[00121] A figura 7 representa o perfil cromatográfico do extrato obtido do meio de cultura mínimo (MM) . No meio de cultura MM além dos compostos monocerina e anularina I outros compostos foram detectados, porém a anularina I foi detectada com baixa absorbância, indicando baixa concentração.  [00121] Figure 7 represents the chromatographic profile of the extract obtained from the minimal culture medium (MM). In the MM culture medium, in addition to the monocerine and annularin I compounds, other compounds were detected, but annularin I was detected with low absorbance, indicating low concentration.
[00122] As figuras 8, 9 e 10 representam o perfil cromatográfico dos extratos obtidos das culturas em meios YPSS, YESD e PYG, respectivamente . Para estes extratos, as frações foram detectadas, porém com absorbâncias muito baixas indicando a baixa concentração dos compostos presentes no extrato. Além disso, outros compostos não identificados pelo nosso grupo também foram detectados. Quantificação dos compostos monocerina e anularina I produzidos nos diferentes meios de cultura  [00122] Figures 8, 9 and 10 represent the chromatographic profile of extracts obtained from cultures in YPSS, YESD and PYG media, respectively. For these extracts, the fractions were detected, but with very low absorbances indicating the low concentration of the compounds present in the extract. In addition, other compounds not identified by our group were also detected. Quantification of monocerine and annularin I compounds produced in different culture media
[00123] Para a quantificação dos compostos monocerina e anularina I presentes nos extratos orgânicos, uma curva padrão com estes compostos, previamente obtidos, foi preparada utilizando cromatográfia liquida de alta eficiência (CLAE/HPLC) em coluna C18 250 x 4, 6 mm, em sistema de eluição isocrática 56, 0 % de fase móvel B em 35 minutos (solução A:H20/TFA, 99,9/0,1%; solução B: Me0H/H20/TFA, 90:9,9:0,1%) e detecção a 214 nm, 254 nm e 280 nm. [00123] For the quantification of monocerine and annularin I compounds present in organic extracts, a standard curve with these compounds, previously obtained, was prepared using high performance liquid chromatography (HPLC / HPLC) in a C18 column 250 x 4, 6 mm, in an isocratic elution system 56, 0% of mobile phase B in 35 minutes (solution A: H 2 0 / TFA, 99.9 / 0.1%; solution B: MeOH / H 2 0 / TFA, 90: 9, 9: 0.1%) and detection at 214 nm, 254 nm and 280 nm.
[00124] Inicialmente, os compostos monocerina e anularina I foram pesados, dissolvidos em metanol puro grau espectroscópico na concentração de 5 mg/mL, e em seguida as soluções foram diluídas para concentrações de 1 a 12,5 pg/10 pL para se estabelecer uma curva linear (ver Tabela 5) . [00124] Initially, the monocerine and annularin I were weighed, dissolved in pure spectroscopic grade methanol at a concentration of 5 mg / mL, and then the solutions were diluted to concentrations of 1 to 12.5 pg / 10 pL to establish a linear curve (see Table 5).
[00125] Os compostos monocerina e anularina I nas diferentes concentrações foram injetados no HPLC, em volume de 10 pL, em duplicata, e eluídos em coluna C18 250 x 4,6 mm, em sistema de eluição isocrática 56, 0 % de fase móvel B em 35 minutos (solução AitUO/TFA, 99,9/0,1%; solução B: MeOH/TUO/TFA, 90:9,9:0,1%) e detecção a 214 nm, 254 nm e 280 nm.  [00125] The monocerine and annularin I compounds in different concentrations were injected into the HPLC, in a volume of 10 pL, in duplicate, and eluted in a C18 column 250 x 4.6 mm, in an isocratic elution system 56, 0% mobile phase B in 35 minutes (AitUO / TFA solution, 99.9 / 0.1%; solution B: MeOH / TUO / TFA, 90: 9.9: 0.1%) and detection at 214 nm, 254 nm and 280 nm .
[00126] Os valores da área sob a curva para cada composto, nos comprimentos de onda de 214 nm, 254 nm e 280 nm foram calculados e usados para a elaboração da curva padrão .  [00126] The values of the area under the curve for each compound, at the wavelengths of 214 nm, 254 nm and 280 nm were calculated and used for the elaboration of the standard curve.
Tabela 5. Dados utilizados para elaboração das curvas padrão para 214, 254 e 280 nm para a monocerina (Fl) . Table 5. Data used for the elaboration of the standard curves for 214, 254 and 280 nm for monocerine (Fl).
Figure imgf000038_0001
Figure imgf000038_0001
Monocerina Area Area Area (pg injetados] (mAU) 214 (mAU) 254 (mAU) 280 Monocerina Area Area Area (injected pg) (mAU) 214 (mAU) 254 (mAU) 280
1 48,44 11, 15 24,231 48.44 11, 15 24.23
2 91,33 21, 08 45, 082 91.33 21, 08 45, 08
3 133, 15 3 133, 15
5 57, 68 125,26 5 57, 68 125.26
6 69, 61 151,366 69, 61 151.36
7.5 353,38 7.5 353.38
10 486,71 114,49 243, 99 10 486.71 114.49 243, 99
12.5 610, 83 149, 95 303,33 mAU= mili absorbância 12.5 610, 83 149, 95 303.33 mAU = milli absorbance
Tabela 6. Dados da regressão linear para a fração Fl Table 6. Linear regression data for the fraction Fr
(monocerina) nos comprimentos de onda de 214, 254 e 280 nm. Intensidade da área. Comprimento (monocerine) at wavelengths of 214, 254 and 280 nm. Intensity of the area. Length
214 nm 254 nm 280 nm de onda  214 nm 254 nm 280 nm wave
Interceptação -8,09524 ± 4,63767 -2,16267 ± 1,54243 0,35482 ± 2,60569 Declive 49,23365 ± 0,62869 11,9706 ± 0,21047 24,41392 ± 0,35555 Pearson ' s r 0, 99967 0, 99938 0, 99958 R2 0, 99935 0,99877 0, 99915 Intercept -8.09524 ± 4.63767 -2.16267 ± 1.54243 0.35482 ± 2.60569 Slope 49.23365 ± 0.62869 11.9706 ± 0.21047 24.41392 ± 0.355555 Pearson 'sr 0 , 99967 0, 99938 0, 99958 R 2 0, 99935 0.99877 0, 99915
[00127] Equação reta para monocerina (214 nm) : y  [00127] Straight equation for monocerine (214 nm): y
49, 234x - 8,0952, ou x= y + 8,0952/49,234.  49, 234x - 8.0952, or x = y + 8.0952 / 49.234.
Tabela 7. Dados utilizados para elaboração das curvas padrão para 214, 254 e 280 nm para a anularina I (F2) . Intensidade da área sob a curva em mAU.  Table 7. Data used to prepare the standard curves for 214, 254 and 280 nm for annularin I (F2). Intensity of the area under the curve in mAU.
Anularina I Área Área Área  Annularine I Area Area Area
(pg injetados) (mAU) 214 nm (mAU) 254 nm (mAU) 280 nm  (injected pg) (mAU) 214 nm (mAU) 254 nm (mAU) 280 nm
2 134, 62 23,70 92, 18  2,134, 62 23,70 92, 18
3 174, 08 29, 33 116, 91  3 174, 08 29, 33 116, 91
5 278,31 50, 89 200,26  5 278.31 50, 89 200.26
7.5 388,47 76, 83 298, 88  7.5 388.47 76, 83 298, 88
10 509, 94 98,53 388,49  10 509, 94 98.53 388.49
12.5 601, 18 129, 22 501,74  12.5 601, 18 129, 22 501.74
mAU= mili absorbância mAU = milli absorbance
Tabela 8. Dados da regressão linear para anularina I (fração F2 ) . Intensidade da Área sob a curva em mAU.  Table 8. Linear regression data for annularin I (fraction F2). Area intensity under the mAU curve.
Comprimento  Length
214 nm 254 nm 280 nm  214 nm 254 nm 280 nm
de onda  wave
Interceptação 45,57738 ± 7,78852 1,01994 ± 2,12705 5,71057 ± 6,29037 Declive 45,32822 ± 1,01903 10,05948 ± 0,2783 39,10473 ± 0,82302 Pearson ' s r 0,99899 0,99847 0,99912  Intercept 45.57738 ± 7.78852 1.01994 ± 2.12705 5.71057 ± 6.29037 Slope 45.32822 ± 1.01903 10.05948 ± 0.2783 39.10473 ± 0.82302 Pearson 'sr 0.99899 0.99847 0.999912
R2 0,99798 0,99695 0,99823 R 2 0.99798 0.99695 0.99823
mAU= mili absorbância; Equação da reta para anularina I (214 nm) : y = 45,328x + 45,577, ou x= y - 45,577/45,328; mAU = milli absorbance; Line equation for annularin I (214 nm): y = 45.328x + 45.577, or x = y - 45.577 / 45.328;
[00128] Tanto para monocerina como para anularina I, as curvas padrão apresentaram coeficiente de correlação (R2) > 0,99 atendendo o requisito da resolução de conformidade da Anvisa RE 899 de 2003. [00128] For both monocerine and annularin I, the standard curves showed a correlation coefficient (R 2 ) > 0.99 meeting the requirement of Anvisa RE 899 2003 compliance resolution.
[00129] Para a monocerina e anularina I com base no coeficiente de correlação, o comprimento de onda de 214 nm foi o que apresentou maior resolução e, portanto, foi utilizado para a quantificação desses compostos nos extratos, respectivamente .  [00129] For monocerine and annularin I based on the correlation coefficient, the wavelength of 214 nm was the one with the highest resolution and, therefore, was used for the quantification of these compounds in the extracts, respectively.
[00130] Após serem efetuadas as análises dos extratos das culturas em diferentes meios, por CLAE/HPLC, a concentração dos compostos monocerina e anularina I produzidos em cada cultura foi calculada considerando a área (mAU) de acordo com cada intensidade de absorbância apresentada (altura por mAU) para a monocerina (Fl) e anularina I (F2) em 214 nm, de acordo com as tabelas 13, 14, e 15 e com as equações das retas (abaixo) e calculadas para cada curva padrão dos compostos.  [00130] After analyzing the extracts of the cultures in different media, by HPLC / HPLC, the concentration of the monocerine and annularin I compounds produced in each culture was calculated considering the area (mAU) according to each absorbance intensity presented ( height per mAU) for monocerine (Fl) and annularin I (F2) at 214 nm, according to tables 13, 14, and 15 and with the equations of the lines (below) and calculated for each standard curve of the compounds.
[00131] Equação para monocerina (214 nm) : y = 49,234x - 8,0952, ou x= y + 8,0952/49,234  [00131] Equation for monocerine (214 nm): y = 49.234x - 8.0952, or x = y + 8.0952 / 49.234
[00132] Equação para anularina I (214 nm) : y = 45, 328x + 45,577, ou x= y - 45,577/45,328  [00132] Equation for annularin I (214 nm): y = 45, 328x + 45.577, or x = y - 45.577 / 45.328
Tabela 9. Determinação da área sob a curva (mAU) e da absorbância (mAU) das frações Fl (monocerina) e F2 (anularina I) em 214 nm, presentes nos extratos da fração SPE100.  Table 9. Determination of the area under the curve (mAU) and absorbance (mAU) of the fractions Fl (monocerine) and F2 (annularin I) at 214 nm, present in the extracts of the SPE100 fraction.
Extratos Massa Fl - monocerina F2 - anularina I Mass Fl extracts - F2 monocerine - annularine I
(SPE100) injetada (mr) Area Abs Área Abs . (SPE100) injected (mr) Area Abs Area Abs.
Figure imgf000040_0001
Figure imgf000040_0001
Figure imgf000041_0001
Figure imgf000041_0001
*absorbância indetectável  * undetectable absorbance
Tabela 10. Determinação da área sob a curva (mAU) e da absorbância (mAU) das frações F1 (monocerina) e F2 (anularina I) em 254 nm, presentes nos extratos da fração SPE100. Table 10. Determination of the area under the curve (mAU) and absorbance (mAU) of the F1 (monocerine) and F2 fractions (annularin I) at 254 nm, present in the extracts of the SPE100 fraction.
Extratos Massa F1 - monocerina F2 - anularina I Mass F1 extracts - F2 monocerine - annularine I
(SPE100) inj etada ( mg) Área Abs . Área Abs . (SPE100) inj eted (mg) Area Abs. Abs area.
Figure imgf000042_0001
Figure imgf000043_0001
Figure imgf000042_0001
Figure imgf000043_0001
Tabela 11. Determinação da área sob a curva (mAU*mL) e da absorbância (mAU) das frações F1 (monocerina) e F2 (anularina Table 11. Determination of the area under the curve (mAU * mL) and absorbance (mAU) of the F1 (monocerine) and F2 (annularin) fractions
I) em 280 nm, presentes nos extratos da fração SPE100. I) at 280 nm, present in the extracts of the SPE100 fraction.
Extratos Massa F1 - monocerina F2 - anularina I Mass F1 extracts - F2 monocerine - annularine I
(SPE100) injetada (mV) Área Abs . Área Abs . (SPE100) injected (mV) Abs area. Abs area.
Figure imgf000043_0002
YESD1 25 64, 97 10, 53 0, 50 2,06
Figure imgf000043_0002
YESD1 25 64, 97 10, 53 0, 50 2.06
YESD1 50 82,55 14, 62 44,93 6, 07YESD1 50 82.55 14, 62 44.93 6, 07
YESDl 100 16,40 27,89 10, 90 11,54 YESDl 100 16.40 27.89 10, 90 11.54
!!lll 111111 !! lll 111111
YPSS1 100 12, 60 25, 44 11,17 18,15YPSS1 100 12, 60 25, 44 11.17 18.15
YPSS2 25 11, 80 17, 88 6, 22 8,14YPSS2 25 11, 80 17, 88 6, 22 8.14
YPSS2 50 7, 64 16,78 6, 52 11,08YPSS2 50 7, 64 16.78 6, 52 11.08
Comparação da massa de monocerina e anularina I produzidas, em porcentagem, nos diferentes meios de cultura. Comparison of the mass of monocerine and annularin I produced, in percentage, in the different culture media.
[00133] Para cada meio de cultura foi calculado a porcentagem de monocerina e de anularina I produzida e o resultado final (Tabelas 12-14) mostra que a ordem de rendimento para a monocerina a partir do extrato bruto foi BD > EM > MM > YESD > PYG > YPSS e para anularina I foi BD > EM >>>> demais meios.  [00133] For each culture medium, the percentage of monocerine and annularin I produced was calculated and the final result (Tables 12-14) shows that the order of yield for monocerine from the crude extract was BD> EM> MM> YESD> PYG> YPSS and for annularin I was BD> EM >>>> other means.
[00134] Alguns resultados apresentaram valores negativos para os MS monocerina e anularina I devido à baixa concentração presente no extrato.  [00134] Some results showed negative values for MS monocerine and annularin I due to the low concentration present in the extract.
Tabela 12. Valores das concentrações de monocerina (área sob a curva em 214 nm) de acordo com a equação da reta y = 49, 234x - 8,0952, ou x= y + 8,0952/49,234.  Table 12. Values of monocerine concentrations (area under the curve at 214 nm) according to the line equation y = 49, 234x - 8.0952, or x = y + 8.0952 / 49.234.
Figure imgf000044_0001
Figure imgf000044_0001
Figure imgf000045_0001
Figure imgf000045_0001
Tabela 13A. Cálculo da porcentagem média de monocerina produzida pela cultura do fungo Exserohilum rostratum nos diferentes meios de culturas. Cálculo considerando todos os dados da Tabela 16. Table 13A. Calculation of the average percentage of monocerine produced by the culture of the fungus Exserohilum rostratum in different culture media. Calculation considering all data in Table 16.
Figure imgf000045_0002
Figure imgf000045_0002
Figure imgf000046_0001
Figure imgf000046_0001
Tabela 13B. Cálculo da porcentagem de monocerina nos extratos brutos excluindo alguns dados Table 13B. Calculation of the percentage of monocerine in the crude extracts excluding some data
Figure imgf000046_0002
Figure imgf000046_0002
Tabela 14. Valores das concentrações de anularina I (área sob a curva em 214 nm) de acordo com a equação da reta y = 45, 328x + 45, 577, ou x= y - 45, 577/45, 328. Table 14. Values of concentrations of annularin I (area under the curve at 214 nm) according to the equation of the line y = 45, 328x + 45, 577, or x = y - 45, 577/45, 328.
Figure imgf000046_0003
Figure imgf000046_0003
Figure imgf000047_0001
Figure imgf000047_0001
*absorbância não detectável , portanto não existe pico e nem área sob a curva, e nem massa detectada.  * Absorbance undetectable, so there is no peak or area under the curve, and no detected mass.
Tabela 15A. Cálculo da porcentagem média de anularina I produzida pela cultura do fungo Exserohilum rostratum nos diferentes meios de culturas. Cálculo considerando todos os dados positivos da Tabela 17. Table 15A. Calculation of the average percentage of annularin I produced by the culture of the fungus Exserohilum rostratum in different culture media. Calculation considering all positive data in Table 17.
Figure imgf000048_0001
Figure imgf000048_0001
*Nos meios MM, MC, PYG e YPSS o fungo Exserohilum rostratum não produziu anularina I.  * In MM, MC, PYG and YPSS, the fungus Exserohilum rostratum did not produce annularin I.
Avaliação do tempo de cultura mais adequado para a produção dos compostos monocerina e anularina I .  Evaluation of the most appropriate culture time for the production of monocerine and annularin I compounds.
[00135] Colónias do fungo E. rostratum foram adicionadas em tubo de ensaio de 50 mL contendo 10 mL de solução salina e a suspensão homogeneizada. Para os inóculos, 1 mL da suspensão foi adicionado a erlenmeyers de 250 mL contendo 50 mL de meio batata dextrose (BD) , que foram incubados em shaker a 28°C e 150 rpm. Os frascos foram mantidos em cultura por 2, 3, 7, 10, 15, [00135] Colonies of the E. rostratum fungus were added in a 50 ml test tube containing 10 ml of saline and the suspension homogenized. For the inoculants, 1 mL of the suspension was added to 250 mL conical flasks containing 50 mL of potato dextrose (BD) medium, which were incubated in a shaker at 28 ° C and 150 rpm. The flasks were kept in culture for 2, 3, 7, 10, 15,
21 e 28 dias. 21 and 28 days.
[00136] Nos tempos específicos, os frascos foram retirados da cultura e a biomassa removida por filtração em membrana de poliéster (filtração 1), e em seguida em membrana de fibra de vidro de 50 mm e porosidade de 1 mpi (filtração 2) com auxílio de bomba de vácuo.  [00136] At the specified times, the flasks were removed from the culture and the biomass removed by filtration on a polyester membrane (filtration 1), and then on a 50 mm glass fiber membrane and a porosity of 1 mpi (filtration 2) with vacuum pump aid.
[00137] Os sobrenadantes das culturas foram aplicados em cartuchos de extração em fase sólida (SPE) de 500 mg/ 6 mL previamente condicionados (com três aplicações de metanol puro e água destilada) , respectivamente e a extração do material retido foi realizada com metanol a 25, 50, 75 e 100%, sem TFA. Para 25, 50 e 75% foram usados um volume de coluna para a extração. Para 100% usou-se três volumes de coluna deixando a sílica secar no final do procedimento. [00137] The culture supernatants were applied in 500 mg / 6 mL solid-phase extraction cartridges (SPE) previously conditioned (with three applications of pure methanol and distilled water), respectively, and the extraction of the retained material was carried out with methanol. at 25, 50, 75 and 100%, without TFA. For 25, 50 and 75% a volume of column for extraction. For 100%, three column volumes were used, leaving the silica to dry at the end of the procedure.
[00138] O material extraído foi totalmente seco em rotaevaporador e as frações SPE25, SPE50, SPE75 e SPE100 foram obtidas.  [00138] The extracted material was totally dried in a rotary evaporator and the fractions SPE25, SPE50, SPE75 and SPE100 were obtained.
[00139] As frações foram ressuspendidas em 400 pL de metanol com pureza analítica (grau espectroscópico - HPLC) e 5 pL de cada amostra foram injetados em cromatógrafo líquido (HPLC) . A eluição foi realizada com método gradiente de 0 a 100 % de B em 30 minutos. O perfil analítico permitiu detectar a presença ou não dos compostos monocerina e anularina I, e comparar as suas proporções em cada fração.  [00139] The fractions were resuspended in 400 pL of methanol with analytical purity (spectroscopic grade - HPLC) and 5 pL of each sample were injected in a liquid chromatograph (HPLC). Elution was performed using a 0 to 100% B gradient method in 30 minutes. The analytical profile made it possible to detect the presence or absence of monocerine and annularin I compounds, and to compare their proportions in each fraction.
[00140] Os compostos monocerina e anularina I presentes nas frações SPE foram purificados por HPLC em coluna C18 ou fenil (250x46 mm) em método isocrático de 48- 50% de fase móvel B em 50 ou 55 minutos com detecção a 214, 254 e 280 nm. O pool resultante das coletas foi submetido a rotaevaporação para eliminação do solvente e obtenção dos compostos totalmente puros e secos.  [00140] The monocerine and annularin I compounds present in the SPE fractions were purified by HPLC on a C18 or phenyl column (250x46 mm) in an isocratic method of 48-50% mobile phase B in 50 or 55 minutes with detection at 214, 254 and 280 nm. The pool resulting from the collections was submitted to a rotary evaporation to eliminate the solvent and obtain the compounds completely pure and dry.
[00141] O rendimento de cada cultura, nos diferentes tempos, e dos respectivos compostos monocerina e anularina I estão representados na tabela 16.  [00141] The yield of each culture, at different times, and of the respective monocerine and annularin I compounds are shown in table 16.
[00142] Pode-se concluir que nos tempos de 2 e 3 dias não ocorreu a produção dos compostos monocerina e anularina I que não foram obtidos pelo método de extração empregado e consequentemente não detectados por HPLC.  [00142] It can be concluded that in the times of 2 and 3 days there was no production of the monocerine and annularin I compounds that were not obtained by the extraction method employed and consequently not detected by HPLC.
[00143] Os compostos monocerina e anularina I não foram extraídos por extração com 25% de metanol, e, portanto, esta porcentagem de metanol pode ser usada para remoção de resíduos de meio de cultura que possa ter sido retido na fase estacionária do cartucho. [00144] Os compostos obtidos das frações SPE75 e SPE100 de cada tempo de cultura foram purificados e a massa quantificada para análise do tempo de cultura com maior rendimento na produção dos compostos. [00143] The compounds monocerine and annularin I were not extracted by extraction with 25% methanol, and therefore this percentage of methanol can be used to remove residues of culture medium that may have been retained in the stationary phase of the cartridge. [00144] The compounds obtained from the SPE75 and SPE100 fractions of each culture time were purified and the mass quantified for analysis of the culture time with higher yield in the production of the compounds.
[00145] Pela análise dos dados pode-se concluir que o tempo de 15 dias é o mais efetivo para a produção do extrato bruto e também para os compostos monocerina e anularina I .  [00145] By analyzing the data it can be concluded that the time of 15 days is the most effective for the production of the crude extract and also for the mono-keratin and annularin I compounds.
[00146] Como esperado, o tempo de 21 e de 28 dias não são adequados porque provavelmente a cultura entra em fase de declínio e morte das células fúngicas, e consequentemente ocorre baixo rendimento dos compostos de interesse que podem estar sendo de alguma forma degradados no meio.  [00146] As expected, the time of 21 and 28 days is not adequate because the culture probably enters the phase of decline and death of fungal cells, and consequently there is a low yield of the compounds of interest that may be being somehow degraded in the middle.
[00147] Para cada 1,824 mg de monocerina obteve-se 1 mg de anularina I confirmando a proporção de maior produção de monocerina durante a cultura.  [00147] For each 1.824 mg of monocerine, 1 mg of annularin I was obtained, confirming the proportion of greater production of monocerine during culture.
Tabela 16. Rendimento das culturas do fungo E. rostratum em meio de cultura batata dextrose nos tempos de 2 a 28 dias, a 150 rpm e 28°C.  Table 16. Yield of cultures of the fungus E. rostratum in potato dextrose culture medium in the periods of 2 to 28 days, at 150 rpm and 28 ° C.
Figure imgf000050_0001
Figure imgf000050_0001
ND - não determinado; SPE=extração em fase sólida [00148] De acordo com os resultados obtidos, o meio de cultura batata dextrose (BD) foi considerado o mais adequado para a cultura do fungo Exserohilum rostratum e maior rendimento na obtenção dos metabólitos secundários monocerina e anularina. ND - not determined; SPE = solid phase extraction [00148] According to the results obtained, the potato dextrose (BD) culture medium was considered the most suitable for the cultivation of the fungus Exserohilum rostratum and the highest yield in obtaining the secondary metabolites monocerine and annularin.
[00149] Também se pode observar melhor efetividade da cultura em pH na faixa de 5, 5-6,0.  [00149] It is also possible to observe better culture effectiveness at pH in the range of 5, 5-6.0.
[00150] O tempo de cultura mais adequado é de 15 dias porque favorece a produção dos compostos monocerina e anularina I .  [00150] The most suitable culture time is 15 days because it favors the production of monocerine and annularin I compounds.
EFEITO BIOLÓGICO  BIOLOGICAL EFFECT
Estudo da capacidade regenerativa endotelial do metabólito secundário anularina I produzida pelo fungo Exserohilum rostratum  Study of the endothelial regenerative capacity of the secondary metabolite annularin I produced by the fungus Exserohilum rostratum
[00151] O conjunto de resultados indica que a monocerina induz maiores efeitos citotóxicos em relação à anularina I em células endoteliais, sendo capaz de modificar a proporção de células em G0/G1 e células com DNA fragmentado .  [00151] The set of results indicates that monocerin induces greater cytotoxic effects compared to annularin I in endothelial cells, being able to modify the proportion of cells in G0 / G1 and cells with fragmented DNA.
[00152] A anularina I se mostrou um composto com potencial na indução de proliferação em células endoteliais, manutenção da proporção de células em G0/G1, diminuição das células com DNA fragmentado e indução de senescência que pode significar a manutenção de células em proliferação. Esses efeitos foram acompanhados pela expressão diferencial de proteínas que regulam os pontos de controle e progressão do ciclo celular.  [00152] Annularin I was shown to be a compound with the potential to induce proliferation in endothelial cells, maintaining the proportion of cells in G0 / G1, decreasing cells with fragmented DNA and inducing senescence which may mean the maintenance of proliferating cells. These effects were accompanied by the differential expression of proteins that regulate the control points and progression of the cell cycle.
Cultura das células endoteliais e fibroblastos  Culture of endothelial cells and fibroblasts
[00153] As células endoteliais usadas no estudo são da linhagem ATCC CRL-1730/HUVEC, e as células de fibroblastos normais humanos FN1 foram em estudo anterior isoladas da pálpebra de olho obtida de cirurgia de blefaroplastia na Faculdade de Medicina da Universidade de São Paulo, e os procedimentos aprovados pelo Conselho de Ética ( Comissão de Ética para Análise de Projetos de Pesquisa do HCFMUSP - CAPPesq HCFMUSP No . 921/06) . Essas células foram estabelecidas em cultura, congeladas e armazenadas para uso por tempo indefinido após a reativação em cultura. [00153] The endothelial cells used in the study are of the ATCC CRL-1730 / HUVEC lineage, and the cells of normal human fibroblasts FN1 were in a previous study isolated from eyelid obtained from blepharoplasty surgery at the Faculty of Medicine of the University of São Paulo, and the procedures approved by the Ethics Council (Ethics Committee for Analysis of Research Projects at HCFMUSP - CAPPesq HCFMUSP No. 921/06). These cells were established in culture, frozen and stored for use indefinitely after reactivation in culture.
[00154] As células foram cultivadas a 37°C em meio RPMI-1640 contendo suplementos (SFB 5-10%, penicilina e estreptomicina) , e em atmosfera úmida e 5% de CO2. Após a subconfluência, as células foram processadas e ressuspensas em meio de cultura RPMI-1640 com SFB e antibióticos. Para cada ensaio, a concentração celular foi ajustada de acordo com a recomendação necessária.  [00154] The cells were cultured at 37 ° C in RPMI-1640 medium containing supplements (SFB 5-10%, penicillin and streptomycin), and in a humid atmosphere and 5% CO2. After subconfluence, the cells were processed and resuspended in RPMI-1640 culture medium with SFB and antibiotics. For each assay, cell concentration was adjusted according to the necessary recommendation.
Preparação dos compostos anularina I e monocerina  Preparation of the compounds annularin I and monocerine
[00155] Para uso nos ensaios em cultura celular, os compostos monocerina e anularina I foram dissolvidos em dimetilsulfóxido, filtrados em membrana de celulose regenerada de 0,22 pm e armazenados em aliquotas, a -20 °C. No momento do uso, os compostos foram diluídos em tampão fosfato de sódio (PBS) a 10 mM, e em seguida em meio de cultura RPMI 1640 (sem SFB e com antibióticos) nas concentrações desejadas para cada ensaio.  [00155] For use in cell culture assays, the monocerine and annularin I compounds were dissolved in dimethyl sulfoxide, filtered through a 0.22 pm regenerated cellulose membrane and stored in aliquots at -20 ° C. At the time of use, the compounds were diluted in 10 mM sodium phosphate buffer (PBS), and then in RPMI 1640 culture medium (without SFB and with antibiotics) in the desired concentrations for each assay.
Avaliação da viabilidade celular pelo ensaio do MTT  Assessment of cell viability by the MTT assay
[00156] Para o ensaio de viabilidade celular as células HUVEC e FN1 em meio de cultura RPMI-1640 foram plaqueadas em placas de 96 cavidades e fundo chato, na concentração de lxlO4 células/cavidade. Após adesão, o meio de cultura foi removido e as células foram tratadas com monocerina ou anularina I nas concentrações de 0,02 a 10 mM. Após 24, 48 e 72 h a viabilidade celular foi avaliada por leitura da absorbância a 540 nm de acordo com protocolo anteriormente descrito (Mosmann, 1983) e as figuras 13 e 14 representam os valores das porcentagens de células viáveis após os tratamentos. [00156] For the cell viability assay the HUVEC and FN1 cells in RPMI-1640 culture medium were plated in 96 well plates and flat bottom, in the concentration of 1x10 4 cells / well. After adhesion, the culture medium was removed and the cells were treated with monocerine or annularin I in concentrations of 0.02 to 10 mM. After 24, 48 and 72 h cell viability was assessed by absorbance reading at 540 nm according to the protocol previously described (Mosmann, 1983) and figures 13 and 14 represent the values of the percentages of viable cells after treatments.
[00157] A análise estatística mostra que até 5 mM o efeito da anularina I sobre as células HUVEC não é estatisticamente diferente do controle até o tempo de 48 h, com exceção do tratamento a 0,04 mM que induziu proliferação celular .  [00157] Statistical analysis shows that up to 5 mM the effect of annularin I on HUVEC cells is not statistically different from the control until the time of 48 h, with the exception of the 0.04 mM treatment that induced cell proliferation.
[00158] As células HUVEC tornam-se mais sensíveis ao tratamento com anularina I somente no tempo de 72 h e para a concentração de e/ou acima de 1,25 mM. Para 0,625 mM, em torno de 88% das células permaneceram viáveis em todos os tempos de tratamento. Para o tempo de 24 h a viabilidade celular foi de 71,34; 65,74 e 55,97 para tratamentos com 1,25; 2,5 e 5 mM, respectivamente conforme apresentado na Tabela 17.  [00158] HUVEC cells become more sensitive to treatment with annularin I only within 72 h and for the concentration of and / or above 1.25 mM. For 0.625 mM, around 88% of the cells remained viable at all times of treatment. For 24 h, cell viability was 71.34; 65.74 and 55.97 for treatments with 1.25; 2.5 and 5 mM, respectively, as shown in Table 17.
Tabela 17. Representação da porcentagem de células HUVEC viáveis pelo método do MTT (média ± SD) , após tratamento com anularina I e monocerina nas concentrações de 0,02 a 10 mM.  Table 17. Representation of the percentage of viable HUVEC cells by the MTT method (mean ± SD), after treatment with annularin I and monocerine in concentrations of 0.02 to 10 mM.
Figure imgf000053_0001
Figure imgf000054_0001
Figure imgf000053_0001
Figure imgf000054_0001
[00159] Para os tratamentos em 24 e 48 h, as células endoteliais HUVEC foram pouco sensíveis à ação da monocerina. Em 24 h, a viabilidade celular foi diferente do tratamento controle somente para 10 mM e para 48 h, a partir de 2,5 mM.  [00159] For treatments at 24 and 48 h, HUVEC endothelial cells were little sensitive to the action of monocerin. In 24 h, cell viability was different from the control treatment only for 10 mM and for 48 h, from 2.5 mM.
[00160] Com relação ao tempo de 72 h a viabilidade celular foi diferente do controle para todas as concentrações de monocerina testadas. Para as concentrações de 0,02; 0,04; 0,15; 0,32; 0, 625 e 1,25 mM para o tratamento de 24 h, 99, 04; 99,55; 100,02; 95,61; 95,84 e 83,73% das células estavam viáveis, respectivamente . Para 0,08 mM houve proliferação celular com maior número de células que no grupo controle, porém este valor não é estatisticamente significante .  [00160] Regarding the 72 h time, cell viability was different from the control for all tested monocerine concentrations. For concentrations of 0.02; 0.04; 0.15; 0.32; 0.625 and 1.25 mM for the 24 h, 99, 04 treatment; 99.55; 100.02; 95.61; 95.84 and 83.73% of the cells were viable, respectively. For 0.08 mM there was cell proliferation with a greater number of cells than in the control group, however this value is not statistically significant.
[00161] Em fibroblastos FN1 tratados com anularina I a viabilidade celular apresenta valores próximos tanto para 24 h, como para 48 h. A partir de 0, 625 mM a viabilidade celular é estatisticamente diferente da do grupo controle no tempo de 24 h, e para 48 h somente a 10 mM. Para o tratamento de 72 h a viabilidade celular é reduzida a partir de 0,08 mM de anularina. Para 2,5 mM de anularina haviam 58,13 ± 9,94%; 69,19 ± 10,54% e 68,5318,31% de células viáveis, com os tratamentos por 24, 48 e 72 h, respectivamente conforme mostrado na Tabela 18. Para 5 mM, nos tempos de 24 e 48 h de tratamento ainda haviam 62,63 ± 16,20 e 67,68 ± 7,86% de células viáveis, respectivamente. Para 10 mM, em 24 h, 53,25 ± 11,07 % das células estavam viáveis. [00161] In FN1 fibroblasts treated with annularin I, cell viability shows close values for both 24 h and 48 h. From 0.625 mM, cell viability is statistically different from that of the control group at 24 h, and for 48 h only at 10 mM. For the treatment of 72 h cell viability is reduced from 0.08 mM of annularin. For 2.5 mM of annularin there was 58.13 ± 9.94%; 69.19 ± 10.54% and 68.5318.31% of viable cells, with treatments for 24, 48 and 72 h, respectively as shown in Table 18. For 5 mM, in the 24 and 48 h treatment times there were still 62.63 ± 16.20 and 67.68 ± 7.86% of viable cells, respectively. For 10 mM, in 24 h, 53.25 ± 11.07% of the cells were viable.
Tabela 18. Representação da porcentagem de fibroblastos FN1 viáveis pelo método do MTT (média ± SD) , após tratamento com anularina I e monocerina nas concentrações de 0,02 a 10 mM.  Table 18. Representation of the percentage of viable FN1 fibroblasts by the MTT method (mean ± SD), after treatment with annularin I and monocerine in concentrations of 0.02 to 10 mM.
Figure imgf000055_0001
Figure imgf000056_0001
Figure imgf000055_0001
Figure imgf000056_0001
[00162] Para monocerina, em fibroblastos FN1 a viabilidade celular é diferente da do controle a partir de 0,15 mM nos tempos de 48 h e 72 h. Em 24 h somente a partir de 2,5 mM. A partir de 2,5 mM, em 24 h, a viabilidade celular de fibroblastos era inferior a 50%. Para os tempos de 48 e 72 h, com 0, 625 mM de monocerina havia em torno de 40% de viabilidade celular.  [00162] For monocerine, in FN1 fibroblasts the cell viability is different from that of the control from 0.15 mM in the 48 h and 72 h times. In 24 hours only from 2.5 mM. After 2.5 mM, in 24 h, the cell viability of fibroblasts was less than 50%. For the 48 and 72 h times, with 0, 625 mM of monocerine, there was around 40% cell viability.
[00163] Os dados mostram que em fibroblastos FN1, a monocerina é menos citotóxica que a anularina I . Porém, em células endoteliais HUVEC ocorre efeito inverso. Pode-se observar baixa citotoxidade induzida pela anularina, e até 0,32 mM o índice de viabilidade celular está próximo a 100%.  [00163] The data show that in FN1 fibroblasts, monocerine is less cytotoxic than annularin I. However, in HUVEC endothelial cells, the opposite effect occurs. Low annularin-induced cytotoxicity can be observed, and up to 0.32 mM the cell viability index is close to 100%.
[00164] Como esperado, a maior citotoxicidade foi observada com 10 mM de anularina I e de monocerina em todos os tempos de tratamento.  [00164] As expected, the highest cytotoxicity was observed with 10 mM annularin I and monocerine at all times of treatment.
[00165] A Figura 13 apresenta dados de viabilidade celular de células endoteliais HUVEC e fibroblastos normais FN1 após o tratamento com monocerina nas concentrações de 0,02 a 10 mM, nos tempos de 24, 48 e 72 h. Zero igual ao controle RPMI 1640 correspondente a 100% de viabilidade celular. Análise estatística por One-way ANOVA / Dunnett's multiple comparison test (entre concentrações) . índice de significância de * r<0,1; ** p<0,05; *** p<0,01 para comparações entre os tratamentos em 24 e 48 h e o grupo controle não tratado (0) . índice de significância de # p< 0,01%; ## p < 0,05%, ### p< 0,01% e #### p< 0,001% para comparações entre o controle e o tempo de 72 h. [00166] A Figura 14 mostra dados de viabilidade celular de células endoteliais HUVEC e fibroblastos normais FN1 após o tratamento com anularina I nas concentrações de 0,02 a 10 mM, nos tempos de 24, 48 e 72 h. Zero igual ao controle RPMI 1640 correspondente a 100% de viabilidade celular. Análise estatística por One-way ANOVA / Dunnett's multiple comparison test (entre concentrações) . índice de significância de * r<0,1; ** p<0,05; *** p<0,01 para comparações entre os tratamentos em 24 e 48 h e o grupo controle não tratado (0) . índice de significância de # p< 0,01%; ## p < 0,05%, ### p< 0,01% e #### p< 0,001% para comparações entre o controle e o tempo de 72 h. [00165] Figure 13 shows cell viability data of HUVEC endothelial cells and normal FN1 fibroblasts after treatment with monocerine in concentrations of 0.02 to 10 mM, at 24, 48 and 72 h. Zero equal to the RPMI 1640 control corresponding to 100% cell viability. Statistical analysis by One-way ANOVA / Dunnett ' s multiple comparison test (between concentrations). significance index of * r <0.1; ** p <0.05; *** p <0.01 for comparisons between treatments at 24 and 48 h and the untreated control group (0). significance index of # p <0.01%;## p <0.05%, ### p <0.01% and #### p <0.001% for comparisons between the control and the 72 h time. [00166] Figure 14 shows cell viability data of HUVEC endothelial cells and normal FN1 fibroblasts after treatment with annularin I at concentrations of 0.02 to 10 mM, at 24, 48 and 72 h. Zero equal to the RPMI 1640 control corresponding to 100% cell viability. Statistical analysis by One-way ANOVA / Dunnett ' s multiple comparison test (between concentrations). significance index of * r <0.1; ** p <0.05; *** p <0.01 for comparisons between treatments at 24 and 48 h and the untreated control group (0). significance index of # p <0.01%;## p <0.05%, ### p <0.01% and #### p <0.001% for comparisons between the control and the 72 h time.
Análise das fases do ciclo celular de células endoteliais HUVEC e fibroblastos humanos normais FN1 tratados com anularina I e monocerina - Análise por Cítometría de Fluxo  Analysis of the cell cycle phases of HUVEC endothelial cells and normal human FN1 fibroblasts treated with annularin I and monocerine - Flow Cytometric Analysis
[00167] Para análise do efeito da anularina I e da monocerina no ciclo celular, as células HUVEC e FN1 (2xl05 células/poço, placa de 6 poços) foram tratadas com anularina I a 0,02; 0,15; 0, 625 e 2,5 mM, e com monocerina a 0,02;[00167] For analysis of the effect of annularin I and monocerine on the cell cycle, HUVEC and FN1 cells (2x10 5 cells / well, 6-well plate) were treated with 0.02 annularin I; 0.15; 0, 625 and 2.5 mM, and with 0.02 monocerine;
0,15 e 0,625 mM por 6, 24, 48 e 72 h. Após o tratamento, as células foram tripsinizadas, ressuspensas em PBS, fixadas com álcool RNase, e armazenadas a -20°C para posterior análise . 0.15 and 0.625 mM for 6, 24, 48 and 72 h. After treatment, the cells were trypsinized, resuspended in PBS, fixed with RNase alcohol, and stored at -20 ° C for further analysis.
[00168] As células foram centrifugadas e o pellet foi homogeneizado com tampão contendo Triton X-100, RNAse e iodeto de propídeo, e analisadas em citômetro de fluxo FACsCalibur (BD) . No citômetro de fluxo, a aquisição da população celular, em média de 10.000 eventos foi realizada pelo programa "Cell Quest" (BD) e o conteúdo de DNA medido pela intensidade de fluorescência foi analisado usando o software ModFit versão 4.0. Os resultados estão expressos em porcentagem média de células nas diferentes fases do ciclo celular: Fase quiescente G0/G1, Fase de síntese - S e Fase G2/M (mitose) . [00168] The cells were centrifuged and the pellet was homogenized with buffer containing Triton X-100, RNAse and propidium iodide, and analyzed in a flow cytometer FACsCalibur (BD). In the flow cytometer, the acquisition of the cell population, on average of 10,000 events was performed by the program "Cell Quest" (BD) and the DNA content measured by the fluorescence intensity was analyzed using the ModFit software version 4.0. The results are expressed as an average percentage of cells in the different phases of the cell cycle: Quiescent phase G0 / G1, Synthesis phase - S and Phase G2 / M (mitosis).
[00169] A análise dos dados foi realizada usando o software ModFit versão 4.0, e os resultados estão expressos nas figuras 15 e 16, em porcentagem média de células nas diferentes fases do ciclo celular: Fase quiescente G0/G1, Fase de síntese - S e Fase G2/M (mitose) .  [00169] Data analysis was performed using ModFit software version 4.0, and the results are expressed in figures 15 and 16, in average percentage of cells in the different phases of the cell cycle: Quiescent phase G0 / G1, Synthesis phase - S and Phase G2 / M (mitosis).
[00170] A figura 15 mostra a distribuição de células endoteliais HUVEC e FN1 nas fases do ciclo celular após o tratamento com anularina I nas concentrações de 0,02; 0,15; 0,625 e 2,5 mM, nos tempos de 6, 24, 48 e 72 h. Zero igual ao tratamento controle com meio de cultura RPMI 1640.  [00170] Figure 15 shows the distribution of HUVEC and FN1 endothelial cells in the phases of the cell cycle after treatment with annularin I in concentrations of 0.02; 0.15; 0.625 and 2.5 mM, at 6, 24, 48 and 72 h. Zero equal to the control treatment with RPMI 1640 culture medium.
[00171] Em fibroblastos tratados por 24 e 48 h com 0,02 e 0,15 mM de anularina I há um aumento das células na fase S. Para 72 h, o aumento ocorre somente para 0,02 mM. O aumento ocorre de acordo com a proporção de células com DNA fragmentado (Figura 15) .  [00171] In fibroblasts treated for 24 and 48 h with 0.02 and 0.15 mM of annularin I, there is an increase in cells in the S phase. For 72 h, the increase occurs only to 0.02 mM. The increase occurs according to the proportion of cells with fragmented DNA (Figure 15).
[00172] Nas células HUVECs, por sua vez o tratamento com anularina I após 24 e 48 horas aumentou a presença de células na fase de proliferação G2M e após 72 h induziu aumento na fase S (Figura 15) . Para células HUVECs e fibroblastos humanos tratadas com monocerina ocorre parada do ciclo celular em G0/G1 em todos os tempos do tratamento. Nas duas linhagens celulares a porcentagem de células com DNA fragmentado é similar ao grupo controle. Para fibroblastos ocorre aumento de células na fase S, e G2M não difere do controle. Para HUVECs as células em fase S são também similares ao controle.  [00172] In HUVECs cells, in turn, treatment with annularin I after 24 and 48 hours increased the presence of cells in the G2M proliferation phase and after 72 h induced an increase in the S phase (Figure 15). For HUVECs cells and human fibroblasts treated with monocerine, the cell cycle stops in G0 / G1 at all times of the treatment. In both cell lines, the percentage of cells with fragmented DNA is similar to the control group. For fibroblasts there is an increase in cells in the S phase, and G2M does not differ from the control. For HUVECs, S-phase cells are also similar to the control.
Determinação da proliferação celular [00173] O estudo da indução de proliferação celular por ação da anularina I e monocerina em células HUVEC e FN1 foi realizado por marcação das células com CFSE-DA (CFSE - Carboxifluoresceina diacetato succinimidil ester) (Milovanova et al . , 2004) . Determination of cell proliferation [00173] The study of cell proliferation induction by the action of annularin I and monocerin in HUVEC and FN1 cells was performed by labeling the cells with CFSE-DA (CFSE - Carboxyfluorescein diacetate succinimidil ester) (Milovanova et al., 2004).
[00174] O CFSE-DA é um marcador fluorescente intracelular que é dividido igualmente entre células filhas. Neste sistema, a divisão celular pode ser avaliada em múltiplas gerações por citometria de fluxo, permitindo a identificação de até 10 gerações ín vitro e ín vivo.  [00174] CFSE-DA is an intracellular fluorescent marker that is divided equally between daughter cells. In this system, cell division can be evaluated in multiple generations by flow cytometry, allowing the identification of up to 10 generations in vitro and in vivo.
[00175] A entrada do corante CFSE-DA nas células é pela forma deacetilada que o torna permeável e permite o rápido fluxo através da membrana plasmática. Esterases presentes nas células clivam o acetato resultando na forma CFSE muito menos permeável e que se concentra no interior da célula. As formas CFSE-DA e CFSE possuem a cadeia lateral succinimidil reativa a aminas, mas somente a forma CFSE é fluorescente. A alta concentração intracelular de CFSE facilita a rápida, e o alto nível de marcação de proteínas intracelulares. A marcação das células deve ocorrer de forma rápida para se obter população de células homogeneamente marcadas, o que é crítico na diferenciação das células que passaram por várias divisões celulares (Quah e Parish, 2010; Lyons et al . , 2013.) .  [00175] The entry of the CFSE-DA dye into the cells is due to the deacetylated form that makes it permeable and allows rapid flow through the plasma membrane. Esterases present in the cells cleave the acetate resulting in the CFSE form which is much less permeable and which is concentrated inside the cell. The CFSE-DA and CFSE forms have the amine-reactive succinimidyl side chain, but only the CFSE form is fluorescent. The high intracellular concentration of CFSE facilitates rapid, and high level of intracellular protein labeling. Cell labeling must occur quickly to obtain a homogeneously labeled cell population, which is critical in differentiating cells that have passed through various cell divisions (Quah and Parish, 2010; Lyons et al., 2013.).
[00176] O marcador difunde-se para o interior da célula e se liga covalentemente a aminas intracelulares, resultando em marcação fluorescente bastante estável que pode ser fixada com aldeídos. O excesso do reagente não conjugado difunde-se passivamente para o meio extracelular e é removido junto com o sobrenadante por centrifugação.  [00176] The marker diffuses into the cell and binds covalently to intracellular amines, resulting in a very stable fluorescent label that can be fixed with aldehydes. The excess of the unconjugated reagent diffuses passively into the extracellular medium and is removed with the supernatant by centrifugation.
[00177] Os picos de emissão e excitação após hidrólise são a 492 e 517 nm, respectivamente . As células marcadas podem ser analisadas por citometria de fluxo em equipamento com fonte de excitação a 488 nm. [00177] Emission and excitation peaks after hydrolysis are at 492 and 517 nm, respectively. The labeled cells can be analyzed by flow cytometry on equipment with an excitation source at 488 nm.
[00178] Neste estudo, as células foram marcadas com CFSE-DA (1 pL de CFSE-DA a 5 mM para 1 mL de suspensão contendo 1x10s células resultando na concentração final de 5 nM do marcador) e transferidas para placas de 6 poços na concentração de lxlO5 células/poço. Após a adesão celular as células foram tratadas por 24, 48 e 72 h com anularina I e monocerina de 0,02 a 2,5 mM diluídas em meio de cultura RPMI- 1640 sem SFB . [00178] In this study, cells were labeled with CFSE-DA (1 pL of 5 mM CFSE-DA to 1 mL of suspension containing 1x10 s cells resulting in the final concentration of 5 nM of the marker) and transferred to 6-well plates in the concentration of 1 x 10 5 cells / well. After cell adhesion, cells were treated for 24, 48 and 72 h with 0.02 to 2.5 mM annularin I and monocerin diluted in RPMI-1640 culture medium without SFB.
[00179] Após o tratamento, as células foram tripsinizadas, centrifugadas e o sobrenadante descartado. O pellet celular foi ressuspenso em 1 mL de tampão para citometria de fuxo e armazenado em geladeira para posterior análise .  [00179] After the treatment, the cells were trypsinized, centrifuged and the supernatant discarded. The cell pellet was resuspended in 1 mL of buffer for flow cytometry and stored in a refrigerator for further analysis.
[00180] As análises foram realizadas utilizando a percentagem de células responsivas e o número de divisões de cada célula analisada, discriminadas de acordo com o conteúdo de CFSE-DA por citometria de fluxo em citometro FACSCalibur® (Becton-Dickinson Immunocytometry Systems, San José, CA, EUA) e analisados pela aquisição e análise no programa CellQuest Pro Modfit Becton-Dickson (Modfit-BD) .  [00180] The analyzes were performed using the percentage of responsive cells and the number of divisions of each cell analyzed, broken down according to the content of CFSE-DA by flow cytometry in FACSCalibur® cytometer (Becton-Dickinson Immunocytometry Systems, San José , CA, USA) and analyzed by acquisition and analysis in the CellQuest Pro Modfit Becton-Dickson program (Modfit-BD).
[00181] A análise estatística dos resultados de proliferação celular não indicou diferença significativa entre o tratamento com anularina I e monocerina nas células HUVECs e em fibroblastos FN1.  [00181] Statistical analysis of cell proliferation results did not indicate a significant difference between treatment with annularin I and monocerin in HUVECs cells and in FN1 fibroblasts.
[00182] A Figura 17 mostra o índice de proliferação celular pela média ± SD para células endoteliais HUVEC e fibroblastos FN1 tratados com anularina I e monocerina em concentrações de 0,02 mM a 2,5 mM nos tempos de 6, 24, 48 e 72 h. Pela análise estatística por One-way ANOVA / Dunnett's multiple comparison test (entre concentrações) não houve significância estatística entre o controle e os tratamentos em nenhum dos tratamentos nos diferentes tempos. [00182] Figure 17 shows the cell proliferation index by mean ± SD for HUVEC endothelial cells and FN1 fibroblasts treated with annularin I and monocerine in concentrations of 0.02 mM to 2.5 mM at times of 6, 24, 48 and 72 h. The statistical analysis by one-way ANOVA / Dunnett's multiple comparison test (between concentrations) there was no statistical significance between control and treatments in any of the treatments at different times.
Determinação da proliferação celular Determination of cell proliferation
[00183] O estudo da indução de proliferação celular por ação da anularina I e monocerina em células HUVEC e FN1 foi realizado por marcação das células com CFSE-DA (CFSE - Carboxifluoresceína diacetato succinimidil ester) (Milovanova et al . , 2004) .  [00183] The study of cell proliferation induction by action of annularin I and monocerin in HUVEC and FN1 cells was performed by labeling the cells with CFSE-DA (CFSE - Carboxyfluorescein diacetate succinimidil ester) (Milovanova et al., 2004).
[00184] O CFSE-DA é um marcador fluorescente intracelular que é dividido igualmente entre células filhas. Neste sistema, a divisão celular pode ser avaliada em múltiplas gerações por citometria de fluxo, permitindo a identificação de até 10 gerações ín vitro e ín vivo.  [00184] CFSE-DA is an intracellular fluorescent marker that is divided equally between daughter cells. In this system, cell division can be evaluated in multiple generations by flow cytometry, allowing the identification of up to 10 generations in vitro and in vivo.
[00185] A entrada do corante CFSE-DA nas células é pela forma deacetilada que o torna permeável e permite o rápido fluxo através da membrana plasmática. Esterases presentes nas células clivam o acetato resultando na forma CFSE muito menos permeável e que se concentra no interior da célula. As formas CFSE-DA e CFSE possuem a cadeia lateral succinimidil reativa a aminas, mas somente a forma CFSE é fluorescente. A alta concentração intracelular de CFSE facilita a rápida, e o alto nível de marcação de proteínas intracelulares. A marcação das células deve ocorrer de forma rápida para se obter população de células homogeneamente marcadas, o que é crítico na diferenciação das células que passaram por várias divisões celulares (Quah e Parish, 2010; Lyons et al . , 2013) .  [00185] The entry of the CFSE-DA dye into the cells is due to the deacetylated form that makes it permeable and allows rapid flow through the plasma membrane. Esterases present in the cells cleave the acetate resulting in the CFSE form which is much less permeable and which is concentrated inside the cell. The CFSE-DA and CFSE forms have the amine-reactive succinimidyl side chain, but only the CFSE form is fluorescent. The high intracellular concentration of CFSE facilitates rapid, and high level of intracellular protein labeling. Cell labeling must occur quickly to obtain a homogeneously labeled cell population, which is critical in differentiating cells that have passed through various cell divisions (Quah and Parish, 2010; Lyons et al., 2013).
[00186] O marcador difunde-se para o interior da célula e se liga covalentemente a aminas intracelulares, resultando em marcação fluorescente bastante estável que pode ser fixada com aldeídos. O excesso do reagente não conjugado difunde-se passivamente para o meio extracelular e é removido junto com o sobrenadante por centrifugação. [00186] The marker diffuses into the cell and binds covalently to intracellular amines, resulting in a very stable fluorescent label that can be fixed with aldehydes. The excess of the unconjugated reagent diffuses passively into the extracellular medium and is removed with the supernatant by centrifugation.
[00187] Os picos de emissão e excitação após hidrólise são a 492 e 517 nm, respectivamente . As células marcadas podem ser analisadas por citometria de fluxo em equipamento com fonte de excitação a 488 nm.  [00187] The emission and excitation peaks after hydrolysis are at 492 and 517 nm, respectively. The labeled cells can be analyzed by flow cytometry on equipment with an excitation source at 488 nm.
[00188] Neste estudo, as células foram marcadas com CFSE-DA (1 pL de CFSE-DA a 5 mM para 1 mL de suspensão contendo lxlO6 células resultando na concentração final de 5 nM do marcador) e transferidas para placas de 6 poços na concentração de lxlO5 células/poço. Após a adesão celular as células foram tratadas por 24, 48 e 72 h com anularina I e monocerina de 0,02 a 2,5 mM diluídas em meio de cultura RPMI- 1640 sem SFB . [00188] In this study, the cells were labeled with CFSE-DA (1 pL of 5 mM CFSE-DA to 1 mL of suspension containing 1 x 106 cells resulting in the final concentration of 5 nM of the marker) and transferred to 6-well plates in the concentration of 1 x 10 5 cells / well. After cell adhesion, cells were treated for 24, 48 and 72 h with 0.02 to 2.5 mM annularin I and monocerin diluted in RPMI-1640 culture medium without SFB.
[00189] Após o tratamento, as células foram tripsinizadas, centrifugadas e o sobrenadante descartado. O pellet celular foi ressuspenso em 1 mL de tampão para citometria de fuxo e armazenado em geladeira para posterior análise .  [00189] After the treatment, the cells were trypsinized, centrifuged and the supernatant discarded. The cell pellet was resuspended in 1 mL of buffer for flow cytometry and stored in a refrigerator for further analysis.
[00190] As análises foram realizadas utilizando a percentagem de células responsivas e o número de divisões de cada célula analisada, discriminadas de acordo com o conteúdo de CFSE-DA por citometria de fluxo em citometro FACSCalibur® (Becton-Dickinson Immunocytometry Systems, San José, CA, EUA) e analisados pela aquisição e análise no programa CellQuest Pro Modfit Becton-Dickson (Modfit-BD) .  [00190] The analyzes were performed using the percentage of responsive cells and the number of divisions of each cell analyzed, broken down according to the content of CFSE-DA by flow cytometry in FACSCalibur® cytometer (Becton-Dickinson Immunocytometry Systems, San José , CA, USA) and analyzed by acquisition and analysis in the CellQuest Pro Modfit Becton-Dickson program (Modfit-BD).
[00191] A análise estatística dos resultados de proliferação celular não indicou diferença significativa entre o tratamento com anularina I e monocerina nas células HUVECs e em fibroblastos FN1. [00191] Statistical analysis of cell proliferation results did not indicate significant difference between treatment with annularin I and monocerine in cells HUVECs and FN1 fibroblasts.
[00192] A Figura 17 mostra o índice de proliferação celular pela média ± SD para células endoteliais HUVEC e fibroblastos FN1 tratados com anularina I nas concentrações de 0,02; 0,15 e 0,625 mM nos tempos de 24, 48 e 72 h. Pela análise estatística por One-way ANOVA / Dunnett's multiple comparison test (entre concentrações) não houve significância estatística entre o controle e os tratamentos em nenhum dos tratamentos nos diferentes tempos. [00192] Figure 17 shows the cell proliferation index by mean ± SD for HUVEC endothelial cells and FN1 fibroblasts treated with anularin I at concentrations of 0.02; 0.15 and 0.625 mM at 24, 48 and 72 h. The statistical analysis by one-way ANOVA / Dunnett's multiple comparison test (between concentrations) there was no statistical significance between control and treatments in any of the treatments at different times.
[00193] Os resultados estão expressos na figura 17 e pode-se observar que a anularina I não induziu proliferação celular em fibroblastos nas concentrações testadas nos tempos de 24 h, 48 h e 72 h, porém para células HUVECs há uma tendência de aumento da proliferação para as concentrações de 0,02 e 0,15 mM nos três tempos de tratamento. Para 48 e 72 h a proliferação celular é menor do que no controle e este dado está em consonância com os dados de viabilidade celular que mostraram redução da viabilidade celular para esta concentração e tempo de tratamento.  [00193] The results are shown in figure 17 and it can be seen that annularin I did not induce cell proliferation in fibroblasts at the concentrations tested at 24 h, 48 h and 72 h, however for HUVECs there is a tendency for increased proliferation for concentrations of 0.02 and 0.15 mM in the three treatment times. For 48 and 72 h the cell proliferation is less than in the control and this data is in line with the cell viability data that showed a reduction in cell viability for this concentration and treatment time.
[00194] A Figura 18 mostra o índice de proliferação celular pela média ± SD para células endoteliais HUVEC e fibroblastos FN1 tratados com monocerina nas concentrações de 0,02 e 0,15 mM nos tempos de 24, 48 e 72 h. Pela análise estatística por One-way ANOVA / Dunnett's multiple comparison test (entre concentrações) não houve significância estatística entre o controle e os trataentos em nenhum dos tratamentos nos diferentes tempos. [00194] Figure 18 shows the cell proliferation index by mean ± SD for HUVEC endothelial cells and FN1 fibroblasts treated with monocerine at concentrations of 0.02 and 0.15 mM at 24, 48 and 72 h. The statistical analysis by one-way ANOVA / Dunnett's multiple comparison test (between concentrations) there was no statistical significance between control and trataentos in the treatments at different times.
[00195] Em fibroblastos, em 24 h a monocerina a 0,15 mM induziu pequeno aumento na proliferação celular. Para 48 h e 72 h, nos grupos tratados o índice foi inferior ao dos grupos controles, resultado também observado no ensaio de viabilidade celular. Para células HUVECs em 24 h houve pequeno aumento do índice de proliferação, porém em 48 e 72 h o índice é similar ao dos grupos controles. [00195] In fibroblasts, in 24 ha 0.15 mM monocerine induced a small increase in cell proliferation. For 48 h and 72 h, in the treated groups the index was lower than in the control groups, a result also observed in the cell viability. For HUVECs cells at 24 h there was a small increase in the proliferation index, however at 48 and 72 h the index is similar to that of the control groups.
Expressão proteínas - análise do efeito da anularina sobre células endoteliais HUVEC por Western Blotting  Protein expression - analysis of the effect of anularin on HUVEC endothelial cells by Western Blotting
Obtenção do Usado celular eletroforese transferência e Western Blotting  Obtaining Used Cell Electrophoresis Transfer and Western Blotting
[00196] As células endoteliais HUVEC (8xl05 células/poço, placa de 6 poços) foram tratadas com anularina I a 0,15 e 0, 625 mM por 6 e 24 h. Após o tratamento, as células foram lavadas com PBS e lisadas com 400 pL de tampão RIPA/poço (contendo inibidores de proteases e fosfatases e EDTA) em banho de gelo. Após centrifugação, a concentração de proteínas foi determinada no lisado proteico e este armazenado a -80°C até a utilização. [00196] HUVEC endothelial cells (8x10 10 cells / well, 6-well plate) were treated with 0.15 and 0.625 mM annularin I for 6 and 24 h. After treatment, the cells were washed with PBS and lysed with 400 pL of RIPA buffer / well (containing protease and phosphatase inhibitors and EDTA) in an ice bath. After centrifugation, the protein concentration was determined in the protein lysate and stored at -80 ° C until use.
[00197] A análise da expressão de proteínas específicas foi feita no lisado total por eletroforese em gel de 12,5 % ou 4-20 % de poliacrilamida contendo dodecil sulfato de sódio (SDS-PAGE- Mini PROTEAN TGX pre-cast gel, Bio-Rad Lab) a 130 V por 10 min e 100 V por 2 horas em tampão SDS-PAGE (25 mM Tris, 192 mM Glicina, 0,1 % SDS, pH 8,3), utilizando 20 ou 40 pg do lisado proteico e 10 pL de marcador de peso molecular de proteínas PageRuler™ (Plus) Prestaíned Protein Ladder 10-250 kDa ou Spectra™ Multicolor Hígh Range Protein Ladder 40-300 kDa (Thermo Scientific) . Após a eletroforese, as proteínas foram transferidas para uma membrana de fluoreto de polivinilideno (PVDF) por 30-45 min com voltagem de 20 V em um sistema semi-seco utilizando tampão de transferência (12 mM Tris, 96 mM Glicina, 20 % metanol, pH 8,3) . Após a transferência, a membrana foi bloqueada com tampão de bloqueio (BSA 1 % em TBS-T) a 4 °C por mais ou menos 12 horas (overnight) e lavada 3 vezes com TBS-T, 5 min cada sob agitação constante de 60 rpm. O primeiro anticorpo (Abcam) foi diluído em BSA 1 % em TBS-T. A membrana foi colocada em contato em toda a sua superfície com uma quantidade adequada da solução de anticorpo e incubada a temperatura ambiente por 1 h ou a 4°C por mais ou menos 12 horas (overnight) sob agitação constante, dependendo do anticorpo. A seguir a membrana foi lavada 3 vezes com TBS-T e incubada com o segundo anticorpo anti- coelho ou anti-camundongo (marca Thermo) como anteriormente, por l h a temperatura ambiente. A membrana foi novamente lavada com TBS-T por 3 vezes e as marcações reveladas utilizando o reagente SuperSígnal® West Dura Extended Duration Substrate (Thermo Scientific) e o equipamento de imagem digital ChemiDoc MP (Bio-Rad Lab) com detecção de quimioluminescência . As densitometrias das bandas correspondentes à marcação das proteínas com os anticorpos foram realizadas utilizando o software Image Lab (Bio-Rad Lab) . [00197] The analysis of the expression of specific proteins was made in the total lysate by 12.5% or 4-20% polyacrylamide gel electrophoresis containing sodium dodecyl sulfate (SDS-PAGE-Mini PROTEAN TGX pre-cast gel, Bio -Rad Lab) at 130 V for 10 min and 100 V for 2 hours in SDS-PAGE buffer (25 mM Tris, 192 mM Glycine, 0.1% SDS, pH 8.3), using 20 or 40 pg of protein lysate and 10 pL of PageRuler ™ Protein Molecular Weight Marker (Plus) Prestaíned Protein Ladder 10-250 kDa or Spectra ™ Multicolor Hi Range Protein Ladder 40-300 kDa (Thermo Scientific). After electrophoresis, proteins were transferred to a polyvinylidene fluoride (PVDF) membrane for 30-45 min with a voltage of 20 V in a semi-dry system using transfer buffer (12 mM Tris, 96 mM Glycine, 20% methanol , pH 8.3). After transfer, the membrane was blocked with blocking buffer (1% BSA in TBS-T) at 4 ° C for about 12 hours (overnight) and washed 3 times with TBS-T, 5 min each under constant agitation of 60 rpm. The first antibody (Abcam) was diluted in 1% BSA in TBS-T. The membrane was placed in contact over its entire surface with an adequate amount of the antibody solution and incubated at room temperature for 1 h or at 4 ° C for about 12 hours (overnight) under constant agitation, depending on the antibody. Then the membrane was washed 3 times with TBS-T and incubated with the second anti-rabbit or anti-mouse antibody (Thermo brand) as before, for 1 hour at room temperature. The membrane was washed again with TBS-T for 3 times and the markings revealed using the SuperSígnal® West Dura Extended Duration Substrate reagent (Thermo Scientific) and the digital imaging equipment ChemiDoc MP (Bio-Rad Lab) with chemiluminescence detection. The densitometry of the bands corresponding to the labeling of the proteins with the antibodies were performed using the Image Lab software (Bio-Rad Lab).
[00198] Para a análise da expressão proteica as condições do gel, transferência e diluições foram padronizadas para os seguintes anticorpos: ciclina Dl, p53, p21 , FGF-2, Ki-67, p27, VEGF-1, TGF-b, b-actina e GAPDH . As condições padronizadas estão descritas na Tabela 19, e após os testes, 40 pg do lisado proteico das amostras foram usados em gel de 12,5 % ou 4-20 % seguindo as seguintes diluições dos anticorpos primários e secundários.  [00198] For protein expression analysis, gel conditions, transfer and dilutions were standardized for the following antibodies: cyclin D1, p53, p21, FGF-2, Ki-67, p27, VEGF-1, TGF-b, b -actin and GAPDH. The standardized conditions are described in Table 19, and after the tests, 40 pg of the protein lysate from the samples were used in 12.5% or 4-20% gel following the following dilutions of the primary and secondary antibodies.
Tabela 19. Condições para diluição dos anticorpos nos ensaios de expressão proteica.
Figure imgf000065_0001
Table 19. Conditions for dilution of antibodies in protein expression assays.
Figure imgf000065_0001
Figure imgf000066_0001
Figure imgf000066_0001
PM= peso molecular  MW = molecular weight
[00199] As células endoteliais HUVEC foram tratadas com anularina I a 0,15 e 0, 625 mM por 6 e 24 h e a expressão proteica foi avaliada para todas as proteínas listadas na Tabela 21 utilizando como controle de carregamento as proteínas GADPH ou b-actina.  [00199] HUVEC endothelial cells were treated with annularin I at 0.15 and 0.625 mM for 6 and 24 h and protein expression was evaluated for all proteins listed in Table 21 using GADPH or b-actin proteins as loading control. .
[00200] A Figura 19 mostra um Western blotting representativo da expressão de VEGF-R1 nas incubações de HUVEC com diferentes concentrações de anularina I (1 e 2: RPMI; 3 e 4: 0,15 mM, 5 e 6: 0, 625 mM) por 24h. Gliceraldeído trifosfato desidrogenase (GAPDH) foi utilizado como controle de carregamento.  [00200] Figure 19 shows a Western blotting representative of VEGF-R1 expression in HUVEC incubations with different concentrations of anularin I (1 and 2: RPMI; 3 and 4: 0.15 mM, 5 and 6: 0, 625 mM) for 24 hours. Glyceraldehyde triphosphate dehydrogenase (GAPDH) was used as loading control.
[00201] Os resultados da expressão proteica estão expressos na Tabela 20 e nas figuras 19, 20 e 21. Pode-se observar que ocorreu redução da expressão de p27 para os grupos tratados com anularina I nos tempos de 6 h, porém de forma estatisticamente significante somente para 0, 625 mM. Em 24 h observa-se efeito inverso com expressivo aumento para as duas concentrações testadas, porém não significante. Tabela 20. Expressão proteica para células HUVECs tratadas com anularina I a 0,15 e 0,625 mM nos tempos de 6 h e 24 h.
Figure imgf000066_0002
Figure imgf000067_0001
[00201] The results of protein expression are expressed in Table 20 and in figures 19, 20 and 21. It can be seen that there was a reduction in the expression of p27 for the groups treated with annularin I within 6 h, but statistically significant only for 0, 625 mM. In 24 h there is an inverse effect with a significant increase for the two concentrations tested, but not significant. Table 20. Protein expression for HUVECs cells treated with 0.15 and 0.625 mM annularin I at 6 h and 24 h.
Figure imgf000066_0002
Figure imgf000067_0001
DP= desvio padrão. * nos gráficos os valores estão em erro padrão .  SD = standard deviation. * in the graphs the values are in standard error.
[00202] Para ciclina Dl também ocorreu redução da expressão em 6 h, mas não significante, e em 24 h ocorreu aumento da expressão, estatisticamente significante, principalmente para 0,15 mM com maior aumento.  [00202] For cyclin Dl there was also a reduction in expression in 6 h, but not significant, and in 24 h there was an increase in expression, statistically significant, mainly to 0.15 mM with greater increase.
[00203] Os níveis de p53 estão bem acima dos níveis do grupo controle e pode-se observar que este aumento inicia- se a partir do tempo de 6 h atingindo maior valor para o tratamento com 0,625 mM em 24 h.  [00203] The levels of p53 are well above the levels of the control group and it can be observed that this increase starts from the time of 6 h reaching a higher value for the treatment with 0.625 mM in 24 h.
[00204] Ocorre redução da expressão de p21 em 6 h para 0, 625 mM e expressivo aumento em 24 h; aumento de TGF-b em 6 h para 0,15 mM, e em 24 h nas duas concentrações. [00205] Com 6 h houve significante redução de FGF-2 para tratamento com 0,625 mM, mas para 0,15 mM em 6 h e em 24 h nas duas concentrações, a expressão foi muito superior ao controle, e estatisticamente significante nas duas concentrações testadas. [00204] There is a reduction in p21 expression in 6 h to 0.625 mM and a significant increase in 24 h; increase of TGF-b in 6 h to 0.15 mM, and in 24 h in both concentrations. [00205] At 6 h there was a significant reduction in FGF-2 for treatment with 0.625 mM, but to 0.15 mM in 6 h and in 24 h at both concentrations, the expression was much higher than the control, and statistically significant at both concentrations tested .
[00206] Para VEGF-R1 o aumento foi expressivo em 6 h, mas para 24 h foi reduzido em relação ao controle nas duas concentrações avaliadas.  [00206] For VEGF-R1 the increase was significant in 6 h, but for 24 h it was reduced in relation to the control in the two concentrations evaluated.
[00207] A Figura 22 mostra a expressão proteínas pela media ± SEM. Análise estatística por One-way ANOVA / Dunnett's multiple comparison test (entre concentrações) . índice de significância de * r<0,1; ** p<0,05; *** p<0,01 para comparações entre os tratamentos em 6 e 24 h e o grupo controle não tratado (0) . índice de significância de · p< 0,01%; ·· p < 0,05% e ··· p< 0,01% para comparações entre os tempos de 6 e 24 h para as concentrações de 0,15 e 0,625 mM de anularina. [00207] Figure 22 shows the expression proteins by the mean ± SEM. Statistical analysis by One-way ANOVA / Dunnett ' s multiple comparison test (between concentrations). significance index of * r <0.1; ** p <0.05; *** p <0.01 for comparisons between treatments at 6 and 24 h and the untreated control group (0). significance index of · p <0.01%; ·· p <0.05% and ··· p <0.01% for comparisons between 6 and 24 h for concentrations of 0.15 and 0.625 mM of annularin.
[00208] Para TGF-b houve aumento da expressão em 6 h para 0,15 e 0,625 mM, e em 24 h a expressão foi superior ao grupo controle somente para 0,15 mM; para 0,625 mM, embora de forma não significativa, a expressão diminuiu de 1,0 ± 0,174 para 0,791 ± 0,223 conforme pode ser observado na Tabela 23.  [00208] For TGF-b there was an increase in expression in 6 h to 0.15 and 0.625 mM, and in 24 h the expression was superior to the control group only to 0.15 mM; to 0.625 mM, although not significantly, the expression decreased from 1.0 ± 0.174 to 0.791 ± 0.223 as can be seen in Table 23.
Avaliação da senescência celular — Ensaio enzimático da b- galactosidase  Evaluation of cell senescence - Enzymatic assay of b-galactosidase
[00209] Uma das características da senescência é a paralização do crescimento celular. Além disso, as células senescentes geralmente aumentam em tamanho, muitas vezes duplicando em volume e, se aderentes, adquirem uma morfologia achatada. A coloração histoquímica para a b-galactosidase associada à senescência (SA-Bgal) (Dimri et al . , 1995) é um marcador comumente usado para células em senescência. Esta atividade deriva da super-expressão de b-galactosidase ácida lisossomal . [00209] One of the characteristics of senescence is the paralysis of cell growth. In addition, senescent cells generally increase in size, often doubling in volume and, if adherent, acquire a flattened morphology. Histochemical staining for senescence-associated b-galactosidase (SA-Bgal) (Dimri et al., 1995) is a marker commonly used for cells in senescence. This activity derives from the overexpression of lysosomal acid β-galactosidase.
[00210] As células endoteliais HUVEC e fibroblastos normais FN1 (lxlO5 células/poço, placa de 12 poços), após a adesão, foram tratadas com anularina I ou monocerina nas concentrações de 0,02 a 1,25 mM em triplicata, e os tratamentos mantidos por 6, 24, 48 ou 72 h. Células controles receberam somente meio de cultura (sem SFB e com antibióticos) . Os tratamentos foram removidos, as células cuidadosamente "lavadas" com PBS e fixadas com tampão de fixação (Sigma Código F1797) por 6-7 min em temperatura ambiente (0,6 mL/poço) . Após nova "lavagem" com PBS, as células foram marcadas com 0,5 mL da solução de b- galactosidase/poço (Kit Sigma código CS0030), mantidas em estufa a 37°C sem CO2 até se corarem de azul (tempo de 2-10 h) . [00210] HUVEC endothelial cells and normal FN1 fibroblasts (1x10 5 cells / well, 12-well plate), after adhesion, were treated with annularin I or monocerine in concentrations of 0.02 to 1.25 mM in triplicate, and treatments maintained for 6, 24, 48 or 72 h. Control cells received only culture medium (without SFB and with antibiotics). The treatments were removed, the cells carefully "washed" with PBS and fixed with fixation buffer (Sigma Code F1797) for 6-7 min at room temperature (0.6 mL / well). After a new "wash" with PBS, the cells were marked with 0.5 ml of the b-galactosidase solution / well (Kit Sigma code CS0030), kept in an oven at 37 ° C without CO2 until they stained blue (time of 2 -10 h).
[00211] A solução de b-galactosidase foi removida, as células foram cobertas com glicerol a 70% e mantidas em geladeira até a observação em microscópio óptico. Para cada poço, as imagens de três campos foram fotografadas, e as células coradas de azul ( senescentes ) ou sem coloração (não senescentes) foram contadas.  [00211] The b-galactosidase solution was removed, the cells were covered with 70% glycerol and kept in the refrigerator until observation under an optical microscope. For each well, images from three fields were photographed, and cells stained blue (senescent) or without staining (non senescent) were counted.
[00212] Os resultados foram expressos em porcentagem de células senescentes e não senescentes, para cada concentração testada nas duas linhagens celulares.  [00212] The results were expressed as a percentage of senescent and non-senescent cells, for each concentration tested in the two cell lines.
[00213] As células endoteliais HUVEC e fibroblastos normais FN1 tratadas com anularina I ou monocerina nas concentrações de 0,02 a 1,25 mM foram avaliadas com relação à indução de senescência.  [00213] HUVEC endothelial cells and normal FN1 fibroblasts treated with annularin I or monocerin in concentrations of 0.02 to 1.25 mM were evaluated with respect to induction of senescence.
[00214] Os resultados estão expressos nas figuras 22 e 23 e tabelas 22, 23 e 24, em porcentagem de células senescentes e não senescentes, para cada concentração testada nas duas linhagens celulares, e ilustrados na figura 25. [00214] The results are expressed in figures 22 and 23 and tables 22, 23 and 24, in percentage of senescent and non-senescent cells, for each concentration tested in the two cell lines, and illustrated in figure 25.
[00215] Pode-se observar que com 24 h e 48 h de tratamento, a anularina I induziu maior senescência nas células HUVECs, principalmente para 0, 625 mM, e consequentemente ocorre diminuição na porcentagem de células não senescentes.  [00215] It can be seen that with 24 h and 48 h of treatment, annularin I induced greater senescence in HUVECs cells, mainly to 0, 625 mM, and consequently there is a decrease in the percentage of non-senescent cells.
Tabela 21. Porcentagem de células endoteliais HUVEC senescentes e não senescentes após tratamento com anularina I nas concentrações de 0,02; 0,15 e 0,625 mM.  Table 21. Percentage of senescent and non-senescent HUVEC endothelial cells after treatment with anularin I in concentrations of 0.02; 0.15 and 0.625 mM.
Figure imgf000070_0001
Figure imgf000070_0001
S= senescentes; NS= não senescente; SD= desvio padrão.  S = senescent; NS = not senescent; SD = standard deviation.
[00216] A tabela 22 abaixo mostra a expressão relativa de proteínas representadas na Figura 20.  [00216] Table 22 below shows the relative expression of proteins represented in Figure 20.
Figure imgf000070_0002
Figure imgf000070_0002
Figure imgf000071_0001
Figure imgf000071_0001
endoteliais HUVEC senescentes e não senescentes após tratamento com anularina I nas concentrações de 0,02; 0,15 e 0,625 mM. Ensaio com b-galactosidase . senescent and non-senescent HUVEC endothelials after treatment with 0.02 annularin I; 0.15 and 0.625 mM. Assay with b-galactosidase.
[00218] Pela figura 22, pode-se verificar que para células HUVECs, a porcentagem de células senescentes com o tratamento com monocerina foi similar à de células controle que receberam somente meio de cultura em todos os tempos de tratamento. Pode-se concluir que em células HUVECs o índice de senescência induzido pela monocerina é muito baixo.  [00218] From figure 22, it can be seen that for HUVECs cells, the percentage of senescent cells with the treatment with monocerine was similar to that of control cells that received only culture medium at all times of treatment. It can be concluded that in HUVECs the senescence index induced by monocerine is very low.
[00219] Para fibroblastos FN1, em 24 h ocorreu alta porcentagem de senescência inclusive no grupo controle. A análise dos gráficos referentes aos tratamentos de 48 e 72 h (Figura 23) mostra que nesses tempos, com o aumento da concentração de monocerina ocorre também aumento da porcentagem de célula em senescência. Este resultado é incompatível com os de 24 h, tempo no qual já havia alta porcentagem de senescência. [00219] For FN1 fibroblasts, a high percentage of senescence occurred within 24 h, including in the control group. The analysis of the graphs referring to the treatments of 48 and 72 h (Figure 23) shows that in those times, with the increase of the concentration of monocerine, there is also an increase in the percentage of cells in senescence. This result is incompatible with 24-hour ones, in which time there was already a high percentage of senescence.
[00220] Para fibroblastos que são células humanas primárias, os efeitos de senescência, são comumente observados nessas condições de cultivo, pela cinética de crescimento celular já que não são linhagem imortalizada. [00220] For fibroblasts that are primary human cells, senescence effects are commonly observed in these culture conditions, due to cell growth kinetics since they are not immortalized lineage.
Tabela 23. Porcentagem de células endoteliais HUVEC senescentes e não senescentes após tratamento com monocerina a 0,02 a 1,25 mM (media ± SD) . Table 23. Percentage of senescent and non-senescent HUVEC endothelial cells after treatment with 0.02 to 1.25 mM monocerin (mean ± SD).
Figure imgf000072_0001
Figure imgf000072_0001
NS= não senescente; S= senescente; SD=desvio padrão.  NS = not senescent; S = senescent; SD = standard deviation.
Tabela 24. Porcentagem de fibroblastos normais FN1 senescentes e não senescentes após tratamento com monocerina a 0,02 a 1,25 mM (media ± SD) .  Table 24. Percentage of normal senescent and non-senescent FN1 fibroblasts after treatment with 0.02 to 1.25 mM monocerin (mean ± SD).
Figure imgf000072_0002
Figure imgf000072_0002
Figure imgf000073_0002
Figure imgf000073_0002
FN1= fibroblastos normais FN1; NS= não senescente; S= senescente; SD=desvio padrão.  FN1 = normal FN1 fibroblasts; NS = not senescent; S = senescent; SD = standard deviation.
[00221] Em 24 h as células estavam em senescência e este fenômeno foi observado na análise microscópica das células. Para os tratamentos de 48 e 72 h, pela análise microscópica das lâminas pode-se constatar baixíssima densidade celular indicando que as células senescentes haviam perdido a adesão e consequentemente foram eliminadas no processo de fixação das células. Em decorrência desse fato, as células senescentes e não senescentes representadas em porcentagem, nos gráficos para os tempos de 48 e 72 h são referentes somente àquelas que não perderam a adesão, e consequentemente o valor dos desvios padrão são bem altos.  [00221] In 24 h the cells were in senescence and this phenomenon was observed in the microscopic analysis of the cells. For treatments of 48 and 72 h, microscopic analysis of the slides showed a very low cell density indicating that the senescent cells had lost their adhesion and were consequently eliminated in the process of fixing the cells. As a result of this fact, the senescent and non-senescent cells represented in percentage, in the graphs for the times of 48 and 72 h, refer only to those that have not lost adherence, and consequently the value of the standard deviations is quite high.
[00222] A Figura 22 mostra a porcentagem de células endoteliais HUVEC e fibroblastos normais FN1 senescentes e não senescentes após tratamento com monocerina nas concentrações de 0,02 a 1,25 mM. Ensaio com b-galactosidase .  [00222] Figure 22 shows the percentage of HUVEC endothelial cells and normal senescent and non-senescent FN1 fibroblasts after treatment with monocerine in concentrations of 0.02 to 1.25 mM. Assay with b-galactosidase.
[00223] A Figura 23 mostra fotomicrografias representativas de cuias endoteliais tratadas com anularina [00223] Figure 23 shows representative photomicrographs of endothelial gutters treated with annularin
I
Figure imgf000073_0001
0, 02I
Figure imgf000073_0001
0.02
( 2B } 0,15 ( 2( 0,625 mM (2D) durante 48 h . Controles de 24 he 48 h são representados por IA e 2A, respectivamente . As células em azul são senescentes e incolores são células não senescentes detectadas pelo ensaio da b-galactosidase) . (2B} 0.15 (2 (0.625 mM (2D) for 48 h. Controls of 24 h and 48 h are represented by AI and 2A, respectively. The cells in blue are senescent and colorless are non-senescent cells detected by the b assay galactosidase).
Ensaio realizado com b-galactosidase . Determinação de apoptose em células HUVECs por marcação com anexina V-FITC/Iodeto de propídeo e leitura por citometria de fluxo Assay performed with b-galactosidase. Determination of apoptosis in HUVECs by annexin labeling V-FITC / propidium iodide and reading by flow cytometry
[00224] A possibilidade de indução de apoptose foi avaliada pela dupla coloração com anexina V-FITC e iodeto de propídeo .  [00224] The possibility of inducing apoptosis was assessed by double staining with annexin V-FITC and propidium iodide.
[00225] A anexina V é um fosfolipídio com atividade anticoagulante vascular que é encontrada em maior proporção na face citosólica das membranas celulares. A utilidade da anexina V em aplicações de citometria de fluxo é derivada de sua afinidade seletiva para fosfolipídios negativamente carregados. A anexina V/FITC é usualmente utilizada em associação com um marcador vital de células, como por exemplo, iodeto de propídeo (PI) . Células viáveis apresentam a membrana plasmática íntegra, e dessa forma, conseguem excluir o corante vital PI, enquanto as membranas de células mortas são permeáveis ao corante, que se incorpora no DNA. Com isso, a partir destes marcadores é possível identificar células viáveis (Anexina V/FITC e PI negativas), células em um estágio recente de apoptose (Anexina V/FITC positivas e PI negativas) e células que estão em um estágio tardio de apoptose ou mesmo mortas (Anexina V/FITC e PI positivas) (Koopman et ai., 1994) .  [00225] Annexin V is a phospholipid with vascular anticoagulant activity that is found in greater proportion in the cytosolic face of cell membranes. The utility of annexin V in flow cytometry applications is derived from its selective affinity for negatively charged phospholipids. Annexin V / FITC is usually used in association with a vital cell marker, such as propidium iodide (PI). Viable cells have an intact plasma membrane, and thus they are able to exclude the vital dye PI, while the membranes of dead cells are permeable to the dye, which is incorporated into the DNA. Thus, from these markers it is possible to identify viable cells (Annex V / FITC and PI negative), cells in a recent stage of apoptosis (Annex Annex V / FITC positive and PI negative) and cells that are in a late stage of apoptosis or even dead (Appendix V / FITC and PI positive) (Koopman et al., 1994).
[00226] Para o ensaio, as células HUVECs foram plaqueadas a 4xl05 células/poço (em placa de 6 poços), e após a adesão foram tratadas com monocerina de 0,02 a 1,25 mM (em RPMI 1640 sem SFB e com antibióticos), em sextuplicata e por 24 h. Após tripsinização, as células foram transferidas para tubos de 2 mL e processadas para marcação com anexina V e iodeto de propídeo de acordo com o protocolo do fabricante (Anexina-V-FITC Molecular Probes) e na ausência de luz direta. As células foram ressuspensas em 250 pL de tampão FACs com paraformaldeido 4% e a leitura realizada em citômetro de fluxo. Como controles foram usadas células HUVECs sem tratamento e sem marcação, células sem tratamento até a etapa da marcação da anexina V, células sem tratamento até a etapa da marcação com iodeto de propideo e células sem tratamento e marcadas com anexina V e iodeto de propideo. [00226] For the assay, HUVECs cells were plated at 4x10 5 cells / well (in a 6-well plate), and after adhesion they were treated with 0.02 to 1.25 mM monocerin (in RPMI 1640 without SFB and antibiotics), in sextuplicate and for 24 h. After trypsinization, the cells were transferred to 2 mL tubes and processed for labeling with annexin V and propidium iodide according to the manufacturer's protocol (Appendix-V-FITC Molecular Probes) and in the absence of light direct. The cells were resuspended in 250 pL of FACs buffer with 4% paraformaldehyde and the reading was performed in a flow cytometer. As controls, untreated and unmarked HUVEC cells, untreated cells up to the annexin V labeling step, untreated cells up to the propidium iodide labeling stage and untreated and annexed V labeled cells and propidium iodide were used.
[00227] A figura 24 ilustra a classificação das células de acordo com a marcação com anexina V/iodeto de propideo, e os resultados mostram que mesmo na concentração de 1,25 mM as células encontram-se viáveis, em estado de necrose, apoptose inicial ou tardia na proporção de 91,53%, 2,28%, 3,65% e 0,53%, respectivamente (Tabela 27, figura 25) . Este dado está de acordo com os resultados encontrados no ensaio de viabilidade celular por MTT e de senescência após tratamento das células com monocerina a 1,25 por 24 h, indicando ausência de citotoxidade desse composto em células HUVEC no período de 24 h.  [00227] Figure 24 illustrates the classification of cells according to the annexin V / propidium iodide labeling, and the results show that even at the concentration of 1.25 mM the cells are viable, in a state of necrosis, apoptosis initial or late in the proportion of 91.53%, 2.28%, 3.65% and 0.53%, respectively (Table 27, figure 25). This data is in agreement with the results found in the cell viability assay by MTT and senescence after treatment of cells with monocerine at 1.25 for 24 h, indicating the absence of cytotoxicity of this compound in HUVEC cells in the period of 24 h.
[00228] O ensaio foi em quadruplicata, mas a análise estatística não foi realizada por se tratar de resultado de somente um ensaio.  [00228] The trial was performed in quadruplicate, but the statistical analysis was not performed because it was the result of only one trial.
Tabela 25. Determinação de apoptose em células HUVECs tratadas com monocerina de 0,02 a 1,25 mM. Valores em porcentagem.  Table 25. Determination of apoptosis in HUVECs cells treated with 0.02 to 1.25 mM monocerin. Percentage values.
Figure imgf000075_0001
[00229] A Figura 25 mostra determinação de apoptose em células HUVECs pela dupla marcação com anexina V- FITC/Iodeto de propideo. Distribuição das células em viáveis, em processo de morte celular do tipo necrose, apoptose inicial ou tardia após tratamento com monocerina de 0,02 a 1,25 mM. O controle corresponde a tratamento somente com RPMI 1640.
Figure imgf000075_0001
[00229] Figure 25 shows determination of apoptosis in HUVECs cells by double staining with annexin V-FITC / propidium iodide. Distribution of cells in viable cells, in the process of necrosis-like cell death, early or late apoptosis after treatment with 0.02 to 1.25 mM monocerine. The control corresponds to treatment with RPMI 1640 only.
Análise por microscopia de varredura a laser confocal  Confocal laser scanning microscopy analysis
[00230] As células HUVECs foram tratadas com monocerina e após marcação com acridina Orange ou rodamina 123 foram analisadas por microscopia de varredura a laser confocal. As observações referentes a cada concentração da monocerina e tratamentos foram descritas e as imagens foram registradas .  [00230] HUVECs cells were treated with monocerine and after staining with acridine Orange or rhodamine 123 were analyzed by confocal laser scanning microscopy. The observations regarding each concentration of the monocerine and treatments were described and images were recorded.
[00231] Acridina-orange é um corante de fluorescência utilizado para corar vacúolos ácidos (lisossomas, endossomas e autofagossomas ) , RNA e DNA em células vivas.  [00231] Acridine-orange is a fluorescence dye used to stain acid vacuoles (lysosomes, endosomes and autophagosomes), RNA and DNA in living cells.
[00232] O corante se intercala nos ácidos nucléicos em dupla fita (DsDNA) sendo detectado como fluorescência verde a 530 nm. Também se liga eletrostaticamente a grupos fosfatos em ácidos nucléicos em fita simples (ssDNA), RNA ou vacúolos, e nesse caso, a detecção é em 640 nm (McMaster e Carmichael, 1977) .  [00232] The dye is interleaved in double-stranded nucleic acids (DsDNA) being detected as green fluorescence at 530 nm. It also binds electrostatically to phosphate groups in single-stranded nucleic acids (ssDNA), RNA or vacuoles, and in this case, detection is at 640 nm (McMaster and Carmichael, 1977).
[00233] A rodamina 123 é um marcador fluorescente, conhecido por inibir a função do potencial elétrico das mitocôndrias . Rodamina 123 liga-se às membranas da mitocôndria e inibi os processos de transporte, especialmente o transporte de elétrons, retardando a respiração celular interna.  [00233] Rhodamine 123 is a fluorescent marker, known to inhibit the function of the electrical potential of mitochondria. Rhodamine 123 binds to mitochondrial membranes and inhibits transport processes, especially electron transport, delaying internal cell respiration.
Microscopia Eletrónica de Varredura (MEV) [00234] As análises obtidas no MEV são formadas através de um feixe de elétrons que é usado para varrer a amostra, o qual emite os chamados elétrons secundários e ocorre interação de um feixe primário com a superfície de interesse. As imagens obtidas neste estudo forneceram informações analíticas das alterações e arranjos da ultraestrutura ou da morfologia das células nas diferentes condições de tratamento e período de análise. Scanning Electron Microscopy (SEM) [00234] The analyzes obtained in the SEM are formed through an electron beam that is used to scan the sample, which emits the so-called secondary electrons and there is interaction of a primary beam with the surface of interest. The images obtained in this study provided analytical information on the changes and arrangements of the ultrastructure or the morphology of the cells in the different treatment conditions and period of analysis.
[00235] A cascata do processo apoptótico pode ser iniciada por duas principais vias: a extrínseca, que é mediada por receptores de morte presentes na superfície celular, e a intrínseca, que envolve alterações na mitocôndria (Burz et al . , 2009; Portt et al . , 2011) . Estas vias são reguladas por diversas proteínas, como p53, os membros da família Bcl-2 (B-celllymphomaprotein2 ) , as IAPs (proteínas inibidoras da apoptose) e as MAPKs (proteínas quinases ativadas por mitógenos) (Liu et al . , 2011; Sankari et al . , 2012 ) .  [00235] The cascade of the apoptotic process can be initiated by two main routes: the extrinsic, which is mediated by death receptors present on the cell surface, and the intrinsic, which involves changes in the mitochondria (Burz et al., 2009; Portt et al., 2011). These pathways are regulated by several proteins, such as p53, members of the Bcl-2 family (B-celllymphomaprotein2), IAPs (apoptosis inhibiting proteins) and MAPKs (mitogen-activated protein kinases) (Liu et al., 2011; Sankari et al., 2012).
[00236] Diversos são os fatores que podem desencadear a apoptose, entre eles: ligação de moléculas a receptores de membrana, agentes quimioterápicos , radiação ionizante, danos no DNA, choque térmico, privação de fatores de crescimento, baixa quantidade de nutrientes e níveis aumentados de espécies reativas do oxigénio (Hengartner, 2000) .  [00236] Several are the factors that can trigger apoptosis, among them: binding of molecules to membrane receptors, chemotherapeutic agents, ionizing radiation, DNA damage, thermal shock, deprivation of growth factors, low amount of nutrients and increased levels reactive oxygen species (Hengartner, 2000).
[00237] A análise ultraestrutural das células endoteliais HUVECs por MEV demonstraram que as células morrem após a ativação de mecanismos pré-apoptóticos , dependentes da concentração, e do tempo de exposição ao composto monocerina .  [00237] The ultrastructural analysis of HUVECs endothelial cells by SEM demonstrated that the cells die after the activation of pre-apoptotic mechanisms, dependent on concentration, and the time of exposure to the monocerine compound.
[00238] Os resultados de MEV indicam que as alterações na membrana plasmática durante a apoptose são principalmente de natureza bioquímica, não envolvendo alterações morfológicas com exceção da perda das microvilosidades com consequente retração citoplasmática, que foi observada nas células tratadas dependendo do tempo de tratamento, sendo essas as principais características da apoptose, destacando- se pela formação de corpos apoptóticos e a degradação da membrana celular. Os grânulos aderidos à membrana das células HUVECs do grupo controle (que recebeu somente meio de cultura) , se modificaram significativamente após o tratamento com a monocerina dependendo do tempo e da concentração, o que favoreceu a manutenção da atividade citotóxica, como demonstrados nos ensaios de viabilidade celular (MTT) , quantidade de DNA fragmentado nas fases do ciclo celular, e das diferentes proporções de células senescentes encontradas após o tratamento. [00238] SEM results indicate that changes in the plasma membrane during apoptosis are mainly of biochemical nature, not involving morphological changes except for the loss of microvilli with consequent cytoplasmic retraction, which was observed in the treated cells depending on the treatment time, these being the main characteristics of apoptosis, standing out for the formation of apoptotic bodies and degradation of the cell membrane. The granules adhered to the membrane of the HUVECs cells of the control group (which received only culture medium), changed significantly after treatment with monocerine depending on the time and concentration, which favored the maintenance of cytotoxic activity, as demonstrated in the cell viability (MTT), amount of DNA fragmented in the cell cycle stages, and the different proportions of senescent cells found after treatment.
[00239] A senescência celular foi descrita pela primeira vez por Hayflick e Moorhead como a perda progressiva e irreversível do potencial proliferativo das células somáticas (Hayflick e Moorhead, 1961) . Este fenómeno caracteriza-se não apenas por uma perda de capacidade replicativa, mas também por uma série de alterações na morfologia celular, expressão gênica, metabolismo, epigenética, entre outros.  [00239] Cell senescence was first described by Hayflick and Moorhead as the progressive and irreversible loss of the proliferative potential of somatic cells (Hayflick and Moorhead, 1961). This phenomenon is characterized not only by a loss of replicative capacity, but also by a series of changes in cell morphology, gene expression, metabolism, epigenetics, among others.
[00240] A resistência à apoptose produz células senescentes .  [00240] Resistance to apoptosis produces senescent cells.
[00241] Nos experimentos com células HUVECs tratadas com anularina I observou-se diminuição da proporção de células nas diferentes fases do ciclo celular com aumento das células com DNA fragmentado. Isto provavelmente se deve a capacidade das células endoteliais produzirem substâncias, como por exemplo, fatores de crescimento e aminas vasoativas que alteram o ambiente tecidual e podem contribuir, não só para o processo de envelhecimento, para a génese de doenças como também em processo de reparo tecidual (Carneiro, 2007) . [00241] In experiments with HUVEC cells treated with annularin I, a decrease in the proportion of cells in the different phases of the cell cycle was observed, with an increase in cells with fragmented DNA. This is probably due to the ability of endothelial cells to produce substances, such as growth factors and vasoactive amines. which alter the tissue environment and can contribute, not only to the aging process, to the genesis of diseases but also to the tissue repair process (Carneiro, 2007).
[00242] Dimri et al . demostraram que a expressão da enzima b-galactosidase está aumentada em células senescentes num estado de senescência replicativa associada a atividade enzimática da b-galactosidase (SA - b- Gal), em pH 6,0 (Dimri et al . , 1995) .  [00242] Dimri et al. demonstrated that the expression of the enzyme b-galactosidase is increased in senescent cells in a state of replicative senescence associated with the enzymatic activity of b-galactosidase (SA - b-Gal), at pH 6.0 (Dimri et al., 1995).
[00243] Nesta etapa de senescência replicativa, normalmente as células atingem confluência e adquirem aumento em seu volume celular e expandem seus prolongamentos citoplasmáticos , semelhantes às células mesenquimais , e podem tornar-se multinucleadas e altamente granuladas. Estas caracteristicas foram encontradas nas células HUVECs após o tratamento com o composto monocerina nos diferentes períodos, o que foi observado no ensaio por MEV e microscopia confocal a laser.  [00243] In this stage of replicative senescence, cells normally reach confluence and acquire an increase in their cell volume and expand their cytoplasmic prolongations, similar to mesenchymal cells, and can become multinucleated and highly granulated. These characteristics were found in HUVECs cells after treatment with the monocerine compound in the different periods, which was observed in the test by SEM and confocal laser microscopy.
[00244] Os fatores que possivelmente são responsáveis por essas modificações citológicas são os inibidores da histona desacetilase, que relaxam a cromatina sem danificar fisicamente o DNA, danos reparáveis ao DNA, ativação da ATM (ataxia telangiectasia) e a supressão da p53, o que influenciam diretamente a dinâmica de distribuição e progressão das fases do ciclo celular.  [00244] The factors that are possibly responsible for these cytological changes are histone deacetylase inhibitors, which relax chromatin without physically damaging DNA, repairable DNA damage, ATM activation (ataxia telangiectasia) and p53 suppression, which directly influence the dynamics of distribution and progression of the cell cycle phases.
[00245] Dados recentes indicam que as células senescentes desempenham uma variedade de funções durante processos como desenvolvimento embrionário, supressão tumoral, cicatrização de feridas e reparação de tecidos (Krizhanovsky et al . , 2008; Munoz-Espín et al . , 2013; Demaria et al . , 2014; Ritschka et al . , 2017) . Fatos que corroboram com o possível papel da monocerina e anularina I no equilíbrio entre os fatores de proliferação e senescência como verificado nos ensaios da marcação da enzima b- galactosidase e na determinação dos índices proliferativos . [00245] Recent data indicate that senescent cells perform a variety of functions during processes such as embryonic development, tumor suppression, wound healing and tissue repair (Krizhanovsky et al., 2008; Munoz-Espín et al., 2013; Demaria et al., 2014; Ritschka et al., 2017). Facts that corroborate the possible role of monocerine and annularin I in the balance between proliferation and senescence factors as verified in the assays of the labeling of the enzyme b-galactosidase and in the determination of proliferative indices.
[00246] A atividade dos compostos monocerina e anularina I sobre a proliferação de células normais humanas foi avaliada utilizando a análise de fase do ciclo celular para comparar os resultados obtidos entre as células tratadas com diferentes concentrações dos compostos e o grupo não tratado. A transição da fase do ciclo G1 para S requer a montagem e ativação de um complexo de replicação de DNA para iniciar a síntese de DNA. As células endoteliais HUVECs sofrem inibição de contato, inibem a sua proliferação após a confluência e tornam-se quiescentes. O ciclo celular em células de eucariotas controla a progressão, entre e dentro das fases, através de pontos de controle que coordenam a proliferação das células com o ambiente circundante, assegurando com precisão a replicação e divisão.  [00246] The activity of the monocerine and annularin I compounds on the proliferation of normal human cells was evaluated using the cell cycle phase analysis to compare the results obtained between the cells treated with different concentrations of the compounds and the untreated group. The transition from the G1 to S cycle phase requires the assembly and activation of a DNA replication complex to initiate DNA synthesis. HUVECs endothelial cells undergo contact inhibition, inhibit their proliferation after confluence and become quiescent. The cell cycle in eukaryotic cells controls the progression, between and within the phases, through control points that coordinate the proliferation of the cells with the surrounding environment, ensuring precisely replication and division.
[00247] Em fibroblastos humanos tratados por 24 e 48 h com 0,02 e 0,15 mM do composto anularina I há um aumento das células na fase S. Para 72 h, o aumento ocorre somente para 0,02 mM. O aumento ocorre de acordo com a proporção de células com DNA fragmentado. Nas células HUVECs, por sua vez o tratamento com anularina I, aumentou após 24 e 48 horas a presença de células na fase de proliferação G2M e posteriormente, induziu a parada das células na fase G2 / M.  [00247] In human fibroblasts treated for 24 and 48 h with 0.02 and 0.15 mM of the compound annularin I there is an increase in the cells in the S phase. For 72 h, the increase occurs only to 0.02 mM. The increase occurs according to the proportion of cells with fragmented DNA. In HUVECs cells, in turn, treatment with annularin I increased the presence of cells in the G2M proliferation phase after 24 and 48 hours and subsequently induced the arrest of cells in the G2 / M phase.
[00248] Os fibroblastos são os principais componentes celulares dos tecidos conjuntivos encontrados na maioria dos órgãos do corpo. A manutenção do equilíbrio entre proliferação e diferenciação é crucial para a homeostase. Após a ativação por lesão, danos no DNA, estresse oxidativo ou injuria mecânica, os fibroblastos ativam processo de migração, aderem à matriz extracelular provisória e proliferam (Eckes et ai., 2014) . [00248] Fibroblasts are the main cellular components of connective tissues found in most organs of the body. Maintaining the balance between proliferation and differentiation is crucial for homeostasis. After activation by injury, DNA damage, oxidative stress or mechanical injury, fibroblasts activate the migration, adhere to the provisional extracellular matrix and proliferate (Eckes et al., 2014).
[00249] Este fenômeno pode ocorrer por diversos mecanismos envolvidos no controle de proliferação e diferenciação celular, dentre estes, os mecanismos que controlam a progressão do ciclo celular.  [00249] This phenomenon can occur through several mechanisms involved in the control of cell proliferation and differentiation, among them, the mechanisms that control the progression of the cell cycle.
[00250] Para entrar no ciclo, as células quiescentes devem passar pela transição de G0 para Gl, que funciona como uma porta de entrada para o ciclo celular. Esta transição envolve a transcrição de um grande conjunto de genes, incluindo vários proto-oncogenes e genes necessários para a síntese e a tradução de proteínas. As células podem se manter em G0 ou em Gl (Células quiescentes) ou em seguida completar a mitose (células de replicação contínua) . As células que entraram em Gl progridem no ciclo e atingem uma fase crítica na transição Gl/S, ponto de restrição, um passo limitante para a replicação. Passando este ponto de restrição, células normais se tornam irreversivelmente comprometidas com a replicação do DNA. A progressão através do ciclo celular, particularmente a transição Gl/S, é fortemente regulada por proteínas chamadas ciclinas e enzimas associadas chamadas quinases dependentes de ciclina (CDKs) . As CDKs adquirem atividade catalítica através da formação de complexos com as ciclinas. CDKs ativadas por esses complexos conduzem o ciclo celular e outras proteínas são fosforiladas que são críticas para a progressão do ciclo celular. Neste contexto, a expressão das proteínas ciclina Dl, p53, p21 e p27 demostraram os efeitos de controle dos pontos de restrição e controle da progressão do ciclo celular promovidos pelo composto anularina I . [00251] Outras proteínas avaliadas no estudo foram a expressão do VEGF-R1 e FGF-2 após o tratamento com o composto anularina I em células endoteliais HUVECs . [00250] To enter the cycle, quiescent cells must go through the transition from G0 to Gl, which acts as a gateway to the cell cycle. This transition involves the transcription of a large set of genes, including several proto-oncogenes and genes necessary for protein synthesis and translation. The cells can be maintained in G0 or Gl (quiescent cells) or then complete mitosis (cells of continuous replication). The cells that entered Gl progress in the cycle and reach a critical phase in the Gl / S transition, restriction point, a limiting step for replication. Passing this point of restriction, normal cells become irreversibly compromised with DNA replication. Progression through the cell cycle, particularly the Gl / S transition, is strongly regulated by proteins called cyclins and associated enzymes called cyclin-dependent kinases (CDKs). CDKs acquire catalytic activity through the formation of complexes with cyclins. CDKs activated by these complexes drive the cell cycle and other proteins are phosphorylated which are critical for cell cycle progression. In this context, the expression of the proteins cyclin Dl, p53, p21 and p27 demonstrated the effects of controlling the restriction points and controlling the progression of the cell cycle promoted by the compound annularin I. [00251] Other proteins evaluated in the study were the expression of VEGF-R1 and FGF-2 after treatment with the compound annularin I in HUVECs endothelial cells.
[00252] O fator de crescimento vascular endotelial (VEGF) é um potente e seletivo fator mitogênico para o endotélio. Ele produz uma rápida e completa resposta angiogênica e um aumento da permeabilidade capilar. Produzido e secretado por uma série de células normais, ele tem expressão marcada em células tumorais e endoteliais, ou o endotélio o ativa por fatores inflamatórios. O VEGF pertence a uma família das glicoproteínas , que inclui o VEGF- A, VEGF-B, VEGF-C, VEGF-D e o fator de crescimento placentário. O principal mediador da angiogênese e vasculogenese é o VEGF-A, usualmente referido como VEGF. Vários fatores contribuem para o aumento da expressão do VEGF, dentre eles, a baixa concentração de oxigénio intracelular bloqueia a degradação do fator indutor da hipóxia (HIF-la) , o que eleva os níveis de expressão do seu receptor (VEGF-R1) e determina a hipoxia intracelular, que, por sua vez, determina o aumento de sua atividade pela ativação da transcrição do gene VEGF ou dos fatores relacionados a sua expressão como o FGF-2, expresso em fibroblastos (Boudreau e Myers, 2003) .  [00252] The vascular endothelial growth factor (VEGF) is a potent and selective mitogenic factor for the endothelium. It produces a rapid and complete angiogenic response and an increase in capillary permeability. Produced and secreted by a series of normal cells, it has marked expression in tumor and endothelial cells, or the endothelium activates it by inflammatory factors. VEGF belongs to a family of glycoproteins, which includes VEGF-A, VEGF-B, VEGF-C, VEGF-D and the placental growth factor. The main mediator of angiogenesis and vasculogenesis is VEGF-A, usually referred to as VEGF. Several factors contribute to the increase in VEGF expression, among them, the low concentration of intracellular oxygen blocks the degradation of the hypoxia-inducing factor (HIF-la), which increases the levels of expression of its receptor (VEGF-R1) and determines intracellular hypoxia, which, in turn, determines the increase of its activity by activating the transcription of the VEGF gene or factors related to its expression such as FGF-2, expressed in fibroblasts (Boudreau and Myers, 2003).
[00253] O VEGF e o FGF-2 são fatores importantes na indução da formação de novos vasos sanguíneos.  [00253] VEGF and FGF-2 are important factors in inducing the formation of new blood vessels.
[00254] A expressão do fator VEGF facilita a migração e a proliferação das células endoteliais, resultando nos processos de vasculogênese ou angiogênese. Neste trabalho, a expressão do VEGF-R1 foi avaliada nas células endoteliais HUVECs, tratadas por 6 e 24 h com o composto anularina I, comparado ao grupo controle não tratado. Para o composto anularina I, os resultados mostraram aumento da expressão dos receptores de VEGF-R1 após 6 h de tratamento, e para a expressão do FGF-2 esse aumento foi observado principalmente após 24 h o que demonstra claramente os efeitos de regulação da proliferação e diferenciação endotelial. Esse fenômeno é caracterizado pela indução do receptor de crescimento de brotamento vascular e expansão endotelial, e indução do fator de crescimento de fibroblasto responsável pela manutenção da proliferação celular. [00254] The expression of the VEGF factor facilitates the migration and proliferation of endothelial cells, resulting in the processes of vasculogenesis or angiogenesis. In this work, VEGF-R1 expression was evaluated in HUVECs endothelial cells, treated for 6 and 24 h with the compound annularin I, compared to the untreated control group. For the compound annularin I, the results showed increased expression of VEGF-R1 receptors after 6 h of treatment, and for FGF-2 expression this increase was observed mainly after 24 h which clearly demonstrates the effects of regulation of proliferation and endothelial differentiation. This phenomenon is characterized by the induction of the vascular sprout growth receptor and endothelial expansion, and the induction of the fibroblast growth factor responsible for the maintenance of cell proliferation.
[00255] O FGF-2 é membro de uma família de 13 fatores de crescimento ligados estruturalmente e relacionados à heparina. É expresso ubiquamente em células de origem mesodermal e neuroectodérmica, e em uma variedade de células. In vitro, FGF-2 é um potente mitógeno para diferentes tipos de células, incluindo células endoteliais vasculares e fibroblastos . Quando as células endoteliais são cultivadas, o FGF-2 induz um fenótipo antigênico que consiste em aumento da proliferação, migração, produção e expressão de integrinas específicas. In vivo, por sua vez, é um potente indutor da angiogênese e tem efeitos pleiotrópicos no desenvolvimento e diferenciação em vários órgãos.  [00255] FGF-2 is a member of a family of 13 growth factors structurally linked and related to heparin. It is ubiquitously expressed in cells of mesodermal and neuroectodermal origin, and in a variety of cells. In vitro, FGF-2 is a potent mitogen for different types of cells, including vascular endothelial cells and fibroblasts. When endothelial cells are cultured, FGF-2 induces an antigenic phenotype that consists of increased proliferation, migration, production and expression of specific integrins. In vivo, in turn, is a potent inducer of angiogenesis and has pleiotropic effects on the development and differentiation in various organs.
[00256] A proliferação celular pode ser influenciada por condições fisiológicas e patológicas, sendo amplamente controlada por sinais, fatores solúveis ou contato- dependentes, de microambientes que agem estimulando-a ou inibindo-a. O mecanismo de proliferação celular mais importante é a conversão de células quiescentes em proliferativas . O recrutamento das células quiescentes e a progressão do ciclo celular requerem sinais estimulantes para sobrepujar a inibição fisiológica da proliferação celular. As atividades do ciclo celular são comandadas por ciclinas, cinases dependentes de ciclina (CDK-s) e seus inibidores. Durante todo o ciclo, a função das ciclinas é ativar as CDK-s, sendo que seus níveis caem após exercerem essa função. A atividade dos complexos ciclina-CDK é regulada por fatores inibidores de CDK-s. Existem duas classes principais de inibidores: as famílias Cip/Kip e INK-4/ARF. Dentro da família Cip/Kip destacam-se p21, p27 e p53 e, pertencentes à família INK-4/ARF, pl6 e pl4, que funcionam como supressores da progressão do ciclo celular. [00256] Cell proliferation can be influenced by physiological and pathological conditions, being largely controlled by signals, soluble or contact-dependent factors, of microenvironments that act by stimulating or inhibiting it. The most important mechanism of cell proliferation is the conversion of quiescent cells into proliferative cells. The recruitment of quiescent cells and the progression of the cell cycle require stimulating signals to overcome the physiological inhibition of cell proliferation. Cell cycle activities are controlled by cyclins, cyclin-dependent kinases (CDK-s) and their inhibitors. During the entire cycle, the function of the cyclins is to activate the CDK-s, and their levels fall after exercising this function. The activity of the cyclin-CDK complexes is regulated by CDK-s inhibitory factors. There are two main classes of inhibitors: the Cip / Kip and INK-4 / ARF families. Within the Cip / Kip family, p21, p27 and p53 stand out, belonging to the INK-4 / ARF family, p6 and p4, which function as suppressors of cell cycle progression.
[00257] A atividade transcricional de p21 está sob o controle da proteína p53. A p21 também irá competir com a ciclina D com o mesmo objetivo de provocar a parada no ciclo celular. A p27 responde aos supressores de crescimento e irá competir com o complexo ciclina E/CDK-2, também provocando uma parada do ciclo celular no ponto de restrição Gl/S, modificando a proporção de células paradas e quiescentes/senescentes .  [00257] The transcriptional activity of p21 is under the control of the p53 protein. P21 will also compete with cyclin D with the same goal of causing the cell cycle to stop. P27 responds to growth suppressors and will compete with the cyclin E / CDK-2 complex, also causing a cell cycle to stop at the Gl / S restriction point, changing the proportion of standing and quiescent / senescent cells.
[00258] A proteína supressora de tumor p53 é proteína chave na apoptose de células HUVEC e desempenha um papel importante nas vias de transdução de sinal de apoptose em diversos tipos de células, incluindo fibroblastos e células endoteliais vasculares. A função de p53 é de grande importância no estresse genotóxico onde modula e integra vários tipos de resposta que controlam a apoptose, a parada do ciclo celular, senescência e outros processos fisiológicos (Speidel, 2010) .  [00258] The tumor suppressor protein p53 is a key protein in apoptosis of HUVEC cells and plays an important role in apoptosis signal transduction pathways in several cell types, including fibroblasts and vascular endothelial cells. The function of p53 is of great importance in genotoxic stress where it modulates and integrates several types of responses that control apoptosis, cell cycle arrest, senescence and other physiological processes (Speidel, 2010).
[00259] Normalmente, os níveis de p27 apresentam-se aumentados em células quiescentes e caem rapidamente após estimulação por mitógenos. O aumento na expressão p27 em célula HUVECs com o composto anularina I, possivelmente representa os efeitos de proliferação celular provocados pelo composto nos períodos de 6 e 24 h. Por outro lado, a proteína p21 é um efetor crítico da via p53-dependente e provoca uma parada no ciclo celular pela inibição da cinase dependente de ciclina. Portanto, p21 exerce papel central na apoptose, e também está relacionada com a manutenção do estado de diferenciação das células endoteliais. Os resultados das expressões das proteínas inibidoras da progressão do ciclo celular p21, p27 e p53 por Western blot corroboram com os dados do teste de proliferação celular e da determinação da proporção de células em apoptose (ensaio de senescência, MEV e Microscopia de confocal) . Anularina I em HUVECs não induz efeitos de morte celular por apoptose dependente de p53, nos períodos de tempo estudados. [00259] Normally, p27 levels are increased in quiescent cells and fall rapidly after stimulation by mitogens. The increase in p27 expression in HUVECs cells with the compound annularin I, possibly represents the effects of cell proliferation caused by the compound in the periods of 6 and 24 h. On the other hand, the p21 protein is a critical effector of the p53-dependent pathway and causes a stop in the cell cycle by inhibiting cyclin-dependent kinase. Therefore, p21 plays a central role in apoptosis, and is also related to maintaining the differentiating state of endothelial cells. The results of the expression of proteins inhibiting the cell cycle progression p21, p27 and p53 by Western blot corroborate with the data from the cell proliferation test and the determination of the proportion of cells undergoing apoptosis (senescence test, SEM and confocal microscopy). Annularin I in HUVECs does not induce cell death effects due to p53-dependent apoptosis, in the studied periods of time.
[00260] O conjunto de resultados indica que a monocerina induz maiores efeitos citotóxicos em relação à anularina I em células endoteliais, sendo capaz de modificar a proporção de células em G0/G1 e células com DNA fragmentado .  [00260] The set of results indicates that monocerin induces greater cytotoxic effects compared to annularin I in endothelial cells, being able to modify the proportion of cells in G0 / G1 and cells with fragmented DNA.
[00261] A anularina I se mostrou um composto com potencial na indução de proliferação em células endoteliais, manutenção da proporção de células em G0/G1, diminuição das células com DNA fragmentado e indução de senescência que pode significar a manutenção de células em proliferação. Esses efeitos foram acompanhados pela expressão diferencial de proteínas que regulam os pontos de controle e progressão do ciclo celular.  [00261] Annularin I proved to be a compound with the potential to induce proliferation in endothelial cells, maintaining the proportion of cells in G0 / G1, decreasing cells with fragmented DNA and inducing senescence which may mean the maintenance of proliferating cells. These effects were accompanied by the differential expression of proteins that regulate the control points and progression of the cell cycle.
[00262] Para a monocerina, em decorrência do seu efeito de inibição da proliferação em fibroblastos , seria interessante avaliar a indução da expressão de proteínas relacionadas aos pontos de controle e progressão do ciclo celular, assim como realizado para a anularina I, na perspectiva de caracterizar os diferentes mecanismos de ação envolvidos neste efeito, como por exemplo, em cicatrização hiperplásica . [00262] For monocerine, due to its inhibition effect on proliferation in fibroblasts, it would be interesting to evaluate the induction of the expression of proteins related to the control points and progression of the cell cycle, as performed for annularin I, in perspective of characterizing the different mechanisms of action involved in this effect, such as, for example, in hyperplastic healing.
[00263] Para a anularina I, os ensaios de análise morfológica por microscopia eletrónica de varredura (MEV) , análise mitocondrial por microscopia confocal a laser e ensaio de expressão gênica serão realizados em breve. No entanto, considerando os ensaios já realizados e os efeitos da anularina I e seu potencial de ação em células endoteliais, os dados direcionam para a importância do estudo em modelos experimentais de remodelamento e reparo tecidual, e de cicatrização ín vivo.  [00263] For annularin I, tests for morphological analysis by scanning electron microscopy (SEM), mitochondrial analysis by laser confocal microscopy and gene expression assay will be performed soon. However, considering the tests already carried out and the effects of annularin I and its action potential on endothelial cells, the data point to the importance of studying experimental models of tissue remodeling and repair, and of healing in vivo.
Efeito dos compostos monocerina e anularina I avaliado in vivo, em um modelo de ferida experimental nos camundongos  Effect of monocerine and annularin I compounds evaluated in vivo, in an experimental wound model in mice
[00264] Devido ao seu potencial no remodelamento de tecidos, especialmente no controle de processos de cicatrização de feridas, o efeito dos compostos monocerina e anularina I foi avaliado ín vivo, em um modelo de ferida experimental nos camundongos Balb/C por 24 h, 72 h, 7 e 14 dias .  [00264] Due to its potential in tissue remodeling, especially in the control of wound healing processes, the effect of the monocerine and annularin I compounds was evaluated in vivo, in an experimental wound model in Balb / C mice for 24 h, 72 h, 7 and 14 days.
[00265] Os compostos puros foram dissolvidos em propilenoglicol e incorporados em uma formulação na forma de creme (formulação já conhecida e descrita no estado da técnica) nas concentrações de 0,0005 e 0,003% para anularina I e de 0,0006% e 0,005% para monocerina.  [00265] The pure compounds were dissolved in propylene glycol and incorporated into a cream formulation (formulation already known and described in the state of the art) at concentrations of 0.0005 and 0.003% for annularin I and 0.0006% and 0.005 % for monocerine.
[00266] As feridas foram induzidas no dorso dos animais no tamanho de 1 cm2, e os tratamentos dos animais foram da seguinte forma: [00266] The wounds were induced on the animals' back in the size of 1 cm 2 , and the treatments of the animals were as follows:
G1 : Grupo controle - não tratado; G2 : Grupo controle - tratamento com colagenase (produto comercial - Kollagenase Cristalia® - contendo 0.6 U/g de colagenase mais cloranfenicol 1%; G1: Control group - untreated; G2: Control group - treatment with collagenase (commercial product - Kollagenase Cristalia® - containing 0.6 U / g of collagenase plus 1% chloramphenicol;
G3 : Grupo tratado com monocerina a 0,0006%;  G3: Group treated with 0.0006% monocerine;
G4 : Grupo tratado com monocerina a 0,005%;  G4: Group treated with 0.005% monocerine;
G5 : Grupo tratado com anularina I a 0,0005%;  G5: Group treated with 0.0005% annularin I;
G6: Grupo tratado com anularina I a 0,003%;  G6: Group treated with 0.003% annularin I;
G7 : Grupo tratado com veiculo - creme sem o principio ativo .  G7: Group treated with vehicle - cream without the active ingredient.
[00267] Camundongos Balb / C (4 animais / grupo) foram tratados diariamente com as formulações pelo período de 24 h, 72 h, 7 e 14 dias.  [00267] Balb / C mice (4 animals / group) were treated daily with the formulations for a period of 24 h, 72 h, 7 and 14 days.
[00268] Após o tratamento, o sangue dos animais foi coletado para análises bioquímicas e hematológicas. O tamanho da ferida foi medido diariamente usando um paquímetro, bem como raio X MS FX-pro (Bruker) . A reepitelização tecidual foi analisada por microscopia óptica usando a coloração das células com hematoxilina e eosina, picrosirius red e tricrômico de Masson, para detectar alterações histopatológicas, quantificar o conteúdo de fibras colágenas e colágeno tipo I. Além disso, o tecido regenerativo também foi analisado por imunohistoquímica e microscopia eletrónica de varredura (MEV) . Os parâmetros bioquímicos, hematológicos e histológicos indicaram efeito proliferativo devido à presença de fibroblastos , queratinócitos , células epiteliais e fibras colágenas.  [00268] After treatment, the animals' blood was collected for biochemical and hematological analyzes. The wound size was measured daily using a caliper, as well as an MS FX-pro X-ray (Bruker). Tissue reepithelialization was analyzed by optical microscopy using cell staining with hematoxylin and eosin, Picrosirius red and Masson's trichrome, to detect histopathological changes, quantify the content of collagen fibers and type I collagen. In addition, the regenerative tissue was also analyzed by immunohistochemistry and scanning electron microscopy (SEM). The biochemical, hematological and histological parameters indicated a proliferative effect due to the presence of fibroblasts, keratinocytes, epithelial cells and collagen fibers.
[00269] O tratamento com as formulações contendo os compostos foi muito eficaz e reduziu o tamanho da ferida dos animais em 7 dias promovendo a cicatrização da ferida em 13 dias sem cicatrizes na pele regenerada. OBTENÇÃO E PURIFICAÇÃO DA MONOCERINA SECUNDÁRIA DE METABOLITE PRODUZIDA PELO FUNGUS E. ROSTRATUM [00269] The treatment with the formulations containing the compounds was very effective and reduced the wound size of the animals in 7 days promoting wound healing in 13 days without scarring on the regenerated skin. OBTAINING AND PURIFICATION OF SECONDARY METABOLITE MONOCERINE PRODUCED BY FUNGUS E. ROSTRATUM
[00270] O fungo E. rostratum foi cultivado em meio de agar dextrose de batata (PDA) por sete dias a 28 °C, e após o crescimento algumas colónias foram transferidas para um balão de Erlenmeyer contendo 800 mL de PDB e incubadas em um agitador orbital (Marconi® , MA-830) a 28 ° C, a 150 rpm por 15 dias. Cada cultura foi realizada com seis Erlenmeyers em um total de 6,4 litros por lote. Vários lotes foram cultivados para obter monocerina necessária para os ensaios. Após a cultura, a biomassa foi removida por filtração e o caldo de cultura foi submetido a extração em fase sólida (SPE) em cartucho C18 (Cartuchos Speed SPE - Octadecyl C18 / 18%, 2 gramas) . A fração de metanol SPE-100 foi seca por evaporação em rota (Bucchi © R-210) . A fração SPE-100 foi purificada por cromatografia liquida de alta eficiência (HPLC) por um método isocrático (40-54% B, LC / MS ACE © dimensões preparativas coluna C18 250x10x10 mm; fases móveis: A: H20 / TFA (99,9: 0,1 %) e B: MeOH / H20 / TFA (90: 9,9: 0,1%) e a fração obtida do SPE-100 posteriormente seca usando um sistema Speed-vac (CHRIST RVC 2-18 CD plus) . A fração obtida F1 contendo monocerina impura que foi purificada novamente em uma segunda etapa na mesma condição usando uma coluna menor de dimensões preparativas LC / MS ACE © ( C18 250x4,6 mm) . A monocerina pura foi analisada por espectrometria de massa por cromatografia em fase gasosa (GC-MS Agilent 7890 / 5975C) para garantir o grau de pureza. PREPARAÇÕES DE FORMULAÇÕES QUE CONTÊM MONOCERINA  [00270] The E. rostratum fungus was grown on potato dextrose agar (PDA) for seven days at 28 ° C, and after growth some colonies were transferred to an Erlenmeyer flask containing 800 mL of PDB and incubated in a orbital shaker (Marconi®, MA-830) at 28 ° C, at 150 rpm for 15 days. Each culture was carried out with six Erlenmeyers in a total of 6.4 liters per lot. Several batches were grown to obtain the necessary monocerine for the tests. After culture, the biomass was removed by filtration and the culture broth was subjected to extraction in solid phase (SPE) in C18 cartridge (Speed SPE Cartridges - Octadecyl C18 / 18%, 2 grams). The SPE-100 methanol fraction was dried by evaporation en route (Bucchi © R-210). The SPE-100 fraction was purified by high performance liquid chromatography (HPLC) by an isocratic method (40-54% B, LC / MS ACE © preparative dimensions column C18 250x10x10 mm; mobile phases: A: H20 / TFA (99, 9: 0.1%) and B: MeOH / H20 / TFA (90: 9.9: 0.1%) and the fraction obtained from the SPE-100 subsequently dried using a Speed-vac system (CHRIST RVC 2-18 CD plus). The fraction obtained F1 containing impure monocerine which was purified again in a second step in the same condition using a smaller column of preparative dimensions LC / MS ACE © (C18 250x4.6 mm). Pure monocerine was analyzed by mass spectrometry by gas chromatography (GC-MS Agilent 7890 / 5975C) to guarantee the degree of purity PREPARATIONS OF FORMULATIONS CONTAINING MONOCERINE
[00271] Um creme de base não iônico modificado foi preparado de acordo com o Formulário Nacional da Farmacopeia Brasileira, 5a edição (BRASIL, 2012) . Resumidamente, uma emulsão foi preparada com uma fase aquosa contendo propilenoglicol , metil parabeno e água purificada, que após aquecimento foi adicionada a uma fase oleosa contendo uma base auto-emulsificante não iônica, óleo mineral e propil parabeno . [00271] A nonionic modified base cream was prepared according to the National Formulary of the Brazilian Pharmacopoeia 5th edition (BRAZIL, 2012). Briefly, a emulsion was prepared with an aqueous phase containing propylene glycol, methyl paraben and purified water, which after heating was added to an oil phase containing a nonionic self-emulsifying base, mineral oil and propyl paraben.
[00272] A monocerina foi dissolvida em propilenoglicol e incorporada no creme resfriado, gota a gota, para obter as concentrações finais de 0,0006% e 0,005%. As formulações foram preparadas em cabine de fluxo laminar para garantir condições assépticas e mantidas na geladeira até o uso.  [00272] Monocerine was dissolved in propylene glycol and incorporated into the cooled cream, drop by drop, to obtain the final concentrations of 0.0006% and 0.005%. The formulations were prepared in a laminar flow cabinet to ensure aseptic conditions and kept in the refrigerator until use.
ENSAIO IN VIVO IN VIVO TEST
[00273] Para este experimento, camundongos machos Balb / C, de 6 a 8 semanas de idade, com peso de 20 a 22 g, foram obtidos do Centro Animal Central do Instituto Butantan e mantidos em caixas (quatro animais por caixa) com aparas de pinho, água e comida ad libitum. Os animais foram mantidos nas instalações clinicas experimentais do Laboratório de Biologia Molecular do Instituto Butantan sob temperatura controlada e ciclo de luz pelo menos por uma semana antes dos ensaios.  [00273] For this experiment, male Balb / C mice, 6 to 8 weeks old, weighing 20 to 22 g, were obtained from the Central Animal Center of the Butantan Institute and kept in boxes (four animals per box) with clippings pine, water and food ad libitum. The animals were kept in the experimental clinical facilities of the Molecular Biology Laboratory of the Butantan Institute under controlled temperature and light cycle for at least one week before the tests.
[00274] O protocolo experimental ín vivo foi aprovado pelo Comité de Ética em Uso de Animais do Instituto Butantan (CEUAIB # 3932260218) e pelo Comité de Ética em Uso de [00274] The in vivo experimental protocol was approved by the Butantan Institute's Animal Use Ethics Committee (CEUAIB # 3932260218) and by the Animal Use Ethics Committee
Animais da Faculdade de Medicina Veterinária e Zootecnia da Universidade de São Paulo (CEUA # 9897150818) . Animals from the Faculty of Veterinary Medicine and Zootechnics, University of São Paulo (CEUA # 9897150818).
INDUÇÃO E TRATAMENTO DE FERIDAS  WOUND INDUCTION AND TREATMENT
[00275] Os animais receberam anestesia geral com cetamina (10 mg / kg) e xilazina (100 mg / kg) por administração intraperitoneal e a região dorsal foi raspada e limpa com etanol a 70%. A área dorsal do mouse foi marcada com carimbo circular de 10 mm e, em seguida, a ferida de excisão foi cuidadosamente induzida usando uma tesoura. [00275] The animals received general anesthesia with ketamine (10 mg / kg) and xylazine (100 mg / kg) for intraperitoneal administration and the dorsal region was scraped and cleaned with 70% ethanol. The dorsal area of the mouse was marked with a 10 mm circular stamp and then the excision wound was carefully induced using scissors.
[00276] A ferida foi deixada sem roupa após a cirurgia e os animais foram colocados em colchões térmicos para melhor recuperação, a fim de evitar qualquer tipo de perda de animais .  [00276] The wound was left undressed after the surgery and the animals were placed on thermal mattresses for better recovery, in order to avoid any type of loss of animals.
[00277] Antes da indução da ferida e após o processo pós-operatório, o animal recebeu tramadol por via intraperitoneal a 20 mg / kg como analgésico.  [00277] Before the induction of the wound and after the postoperative process, the animal received tramadol intraperitoneally at 20 mg / kg as an analgesic.
[00278] Oitenta animais foram divididos aleatoriamente em cinco grupos com quatro animais cada, e foram tratados diariamente por 24 h, 72 h, 7 e 14 dias com 50 mg de seu respectivo tratamento, conforme descrito abaixo. Grupos Gl, G2, G3, G4 e G5.  [00278] Eighty animals were randomly divided into five groups with four animals each, and were treated daily for 24 h, 72 h, 7 and 14 days with 50 mg of their respective treatment, as described below. Groups G1, G2, G3, G4 and G5.
G1 : grupo controle - animais não tratados;  G1: control group - untreated animals;
G2 : grupo controle - tratamento com colagenase (produto comercial Kollagenase Cristalia® - contendo 0, 6 U / g de colagenase mais cloranfenicol 1%);  G2: control group - treatment with collagenase (commercial product Kollagenase Cristalia® - containing 0.6 U / g of collagenase plus 1% chloramphenicol);
G3 : tratamento com monocerina a 0,0006%;  G3: treatment with 0.0006% monocerine;
G4 : tratamento com monocerina a 0,005%;  G4: treatment with 0.005% monocerine;
G5 : tratamento com o veiculo (formulação sem monocerina)  G5: treatment with the vehicle (formulation without monocerine)
[00279] Após o final do teste, às 24 h, 72 h, 7 e 14 dias, o camundongo recebeu anestesia por inalação de isoflurano com overdose da dose geral recomendada e a amostra de sangue foi coletada por punção cardíaca e adicionada a um tubo contendo anticoagulante revestido com EDTA, para análises bioquímicas adicionais. A superfície da ferida na pele foi coletada após a eutanásia de cada animal com o empurrão para separar o pescoço do animal. O tecido de granulação se formou na área da lesão, que foi cuidadosamente excisada, deixando uma margem de 5 mm da pele normal para avaliação histológica e morfológica e determinação da hidroxiprolina . [00279] After the end of the test, at 24 h, 72 h, 7 and 14 days, the mouse received isoflurane inhalation anesthesia with an overdose of the recommended general dose and the blood sample was collected by cardiac puncture and added to a tube containing anticoagulant coated with EDTA, for additional biochemical analyzes. The wound surface on the skin was collected after euthanizing each animal with the push to separate the animal's neck. The granulation tissue was formed in the area of the lesion, which was carefully excised, leaving a 5 mm margin of normal skin for histological and morphological evaluation and determination of hydroxyproline.
[00280] Para análise histológica e morfológica, o tecido da ferida excisado de todos os grupos foi lavado com solução salina tamponada com fosfato (PBS) três vezes e fixado com formalina a 10% por 24 h, e mantido à temperatura ambiente até que a respectiva análise fosse realizada.  [00280] For histological and morphological analysis, the excised wound tissue from all groups was washed with phosphate buffered saline (PBS) three times and fixed with 10% formalin for 24 h, and kept at room temperature until the respective analysis was carried out.
AVALIAÇÃO CLÍNICA DO PROCESSO DE CICATRIZAÇÃO DA PELE CLINICAL EVALUATION OF THE SKIN HEALING PROCESS
[00281] A avaliação clinica do processo de cicatrização da pele foi realizada diariamente. Aspectos macroscópicos das áreas e bordas da ferida foram avaliados quanto a edema, hiperemia, sangramento, granulação e formação de crostas. A presença ou ausência desses processos foi atribuída aos seguintes escores: baixa (*), moderada (**) e alta intensidade (***) .  [00281] The clinical evaluation of the skin healing process was performed daily. Macroscopic aspects of the wound areas and borders were evaluated for edema, hyperemia, bleeding, granulation and crusting. The presence or absence of these processes was attributed to the following scores: low (*), moderate (**) and high intensity (***).
[00282] Diariamente, antes dos tratamentos, a medição de cada ferida era realizada com paquímetro. Para cada animal, a borda da ferida foi medida considerando quatro lados opostos e, em seguida, o meio foi calculado para o tamanho e o fechamento da ferida. Os dados foram calculados e expressos por comparação com o grupo controle.  [00282] Daily, before treatments, the measurement of each wound was performed with a caliper. For each animal, the wound edge was measured considering four opposite sides, and then the medium was calculated for the size and closure of the wound. The data were calculated and expressed by comparison with the control group.
[00283] O raio-X também monitorou o tamanho da ferida dos animais no tratamento de 14 dias. Para isso, dois animais de cada grupo (Gl, G2, G3, G4 e G5) receberam anestesia com isoflurano, e a imagem da ferida foi registrada por imagem de raios X In vivo (MS FX PRO Bruker®) às 24 h, 72 h , 7, 10 e 14 dias (SATO et ai., 2016) . A análise do tamanho da ferida foi realizada com o Bruker MI Software Versão 7.2 e calculada usando o GraphPad Prism 5.0. Os dados foram expressos por comparação com o grupo controle. [00283] The X-ray also monitored the wound size of the animals in the 14-day treatment. For this, two animals from each group (Gl, G2, G3, G4 and G5) received isoflurane anesthesia, and the wound image was recorded by an in vivo X-ray image (MS FX PRO Bruker®) at 24 h, 72 h, 7, 10 and 14 days (SATO et al., 2016). The analysis of the wound size was performed with Bruker MI Software Version 7.2 and calculated using GraphPad Prism 5.0. The data were expressed by comparison with the control group.
ANÁLISE DE HEMATOLOGIA  HEMATOLOGY ANALYSIS
[00284] As amostras de sangue coletadas dos animais em diferentes intervalos de tempo nas 24 h, 72 h, 7 e 14 dias foram centri fugadas por (Eppendorf ® Centrifuge 5424 R Mini Spin Plus) a 2.000 rpm por 10 minutos para separar o soro e o plasma da amostra de sangue, analisados hematologicamente usando o analisador de hematologia IDEXX ProCyte Dx®.  [00284] Blood samples collected from animals at different time intervals at 24 h, 72 h, 7 and 14 days were leaked by (Eppendorf ® Centrifuge 5424 R Mini Spin Plus) at 2,000 rpm for 10 minutes to separate the serum and the plasma of the blood sample, analyzed hematologically using the IDEXX ProCyte Dx® hematology analyzer.
[00285] O soro foi armazenado para análise de citocinas e o sangue total analisado para glóbulos brancos, plaquetas, neutrófilos e monócitos. Os resultados foram transformados em gráficos usando o software Graph Pad Prism [00285] Serum was stored for cytokine analysis and whole blood was analyzed for white blood cells, platelets, neutrophils and monocytes. The results were transformed into graphs using the Graph Pad Prism software
5.0. 5.0.
ANÁLISE MORFOLÓGICA E HISTOLÓGICA DO TECIDO FERIDO  MORPHOLOGICAL AND HISTOLOGICAL ANALYSIS OF WOUNDED TISSUE
[00286] Para a análise da amostra, os fragmentos de tecidos coletados, com intervalos de tempo determinados 24h, 72h, 7 e 14 dias, foram previamente fixados em formaldeido a 10%, desidratados gradualmente em uma sequência de álcoois (70, 80, 95, 100 e 100%, 1 hora em cada), diafanizados em xilol por (2 horas) e seguidos de imersão em parafina (Histosec-MERCK®) , seções de 5 pm foram obtidas em um micrótomo automático (Leica, RM2165®) para preparar as lâminas para coloração (TOLOSA et al . 2003) . As lâminas foram analisadas sob um microscópio óptico (Nikon 80i®) em uma objetiva de 40x.  [00286] For the sample analysis, the tissue fragments collected, with determined time intervals 24h, 72h, 7 and 14 days, were previously fixed in 10% formaldehyde, gradually dehydrated in a sequence of alcohols (70, 80, 95, 100 and 100%, 1 hour each), diaphanized in xylene for (2 hours) and followed by immersion in paraffin (Histosec-MERCK®), 5 pm sections were obtained in an automatic microtome (Leica, RM2165®) to prepare slides for staining (TOLOSA et al. 2003). The slides were analyzed under an optical microscope (Nikon 80i®) in a 40x objective.
MICROSCOPIA ELETRÓNICA DE VARREDURA (MEV)  ELECTRONIC SCAN MICROSCOPY (SEM)
[00287] a MEV foi realizado para avaliar a organização da matriz extracelular (MEC) e colágenos. Para cada grupo, os tecidos foram fixados em formol a 10%, pós-fixados com 1% de tetróxido de ósmio (pH 7,4) a 4 ° C por 1 hora. Posteriormente, as amostras foram lavadas primeiro com solução salina tamponada com fosfato a 1% (PBS) (3x por 5 minutos) e também com água destilada (3x por 3 minutos) usando um ultra-som (exclusivo®) . Em seguida, as amostras foram desidratadas gradualmente em etanol (50%, 70%, 90% e[00287] SEM was performed to assess the organization of the extracellular matrix (MEC) and collagens. For each group, the tissues were fixed in 10% formaldehyde, post-fixed with 1% osmium tetroxide (pH 7.4) at 4 ° C for 1 hour. Subsequently, the samples were washed first with 1% phosphate buffered saline (PBS) (3x for 5 minutes) and also with distilled water (3x for 3 minutes) using an ultrasound (exclusive®). Then, the samples were gradually dehydrated in ethanol (50%, 70%, 90% and
100%) em um aparelho de ponto critico seco (LEICA EM CPD300®) , e montadas em tocos de alumínio e cobertas com ouro, através do aparelho blasterer sputter com (EMITECH K550®) e analisados pelo microscópio SEM Zeiss (Leo 435 VP®) no Setor de Anatomia dos "Animais Domésticos e Silvestres" na "Faculdade de Medicina Veterinária e Zootecnia" (EMVZ) da Universidade de São Paulo, de acordo com estudo anterior. ANÁLISE HISTOLÓGICA PELO HEMATOXYLIN / EOSIN, E COLORAÇÃO POR VERMELHO DE PICROSIRIUS E MASSON TRICHOME 100%) in a dry critical point device (LEICA EM CPD300®), and mounted on aluminum stumps and covered with gold, using the blasterer sputter device (EMITECH K550®) and analyzed by the SEM Zeiss microscope (Leo 435 VP® ) in the Anatomy Sector of "Domestic and Wild Animals" at the "Faculty of Veterinary Medicine and Zootechnics" (EMVZ) of the University of São Paulo, according to a previous study. HISTOLOGICAL ANALYSIS BY HEMATOXYLIN / EOSIN, AND RED COLORING OF PICROSIRIUS AND MASSON TRICHOME
[00288] As seções de tecidos foram coradas com Mayers Hematoxilina e Eosina Y (HE) para estimar os graus de cicatrização de feridas e regeneração epidérmica e formação de tecido de granulação, conforme descrito anteriormente (KIM et al . , 2003) .  [00288] The tissue sections were stained with Mayers Hematoxylin and Eosin Y (HE) to estimate the degrees of wound healing and epidermal regeneration and granulation tissue formation, as previously described (KIM et al., 2003).
[00289] Seções adicionais foram coradas com vermelho Picrosirius para observação de fibras de colágeno, fibras musculares, citoplasma e glóbulos vermelhos, e com tricrômio de Masson para observar eritrócitos, citoplasma, fibrina, músculo e colágeno (KIM et al . , 2012)  [00289] Additional sections were stained with Picrosirius red to observe collagen fibers, muscle fibers, cytoplasm and red blood cells, and with Masson's trichrome to observe erythrocytes, cytoplasm, fibrin, muscle and collagen (KIM et al., 2012)
[00290] As seções vermelhas de picrosirius foram coradas com o Kit comercial (Sigma Aldrich) (Cat # P6744- 1GA) . Para as seções de picrosirius red de wax e hidrate parafina, depois manche os núcleos com Mayers hematoxilina por 5 minutos e depois lave as lâminas por 10 minutos em água corrente. Depois disso, manche as lâminas de tecido em vermelho picrosirius por 45 minutos e coloque na incubadora a 30° C. Em seguida, lavada em duas trocas de água acidificada também removeu a água fisicamente das lâminas por agitação vigorosa e depois desidrata as lâminas de tecido em três trocas de etanol a 100% e, no final, limpa em Xileno por 5 minutos cada (2x) e montou o desliza em meio resinoso. A intensidade da cor vermelha referente à distribuição do colágeno foi calculada pela área que ajusta o campo usando o Image Pro Plus 4.5.1 (Media Cybernetics, Silver Spring, MD) . A porcentagem de colágeno foi calculada para cada grupo tratado por 14 dias. [00290] The red sections of picrosirius were stained with the Commercial Kit (Sigma Aldrich) (Cat # P6744-1GA). For the wax picrosirius red and paraffin hydrate sections, then stain the cores with Mayers hematoxylin for 5 minutes and then wash the slides for 10 minutes under running water. After that, stain the fabric slides in red picrosirius for 45 minutes and place in the incubator at 30 ° C. Then, washed in two changes of acidified water it also physically removed the water from the slides by vigorous stirring and then dehydrates the tissue slides in three exchanges of 100% ethanol and , in the end, cleaned in Xylene for 5 minutes each (2x) and mounted the glide in resinous medium. The intensity of the red color referring to the collagen distribution was calculated by the area that adjusts the field using Image Pro Plus 4.5.1 (Media Cybernetics, Silver Spring, MD). The percentage of collagen was calculated for each group treated for 14 days.
[00291] As manchas foram analisadas microscopicamente usando uma lente objetiva de 20x e 40x de um microscópio óptico conectado a uma câmera digital (Coolpix 990; Nikon Eclipse80i) . Áreas representativas dos efeitos dos tratamentos foram fotografadas para registrar a regeneração tecidual na derme e epiderme e a formação de granulação tecidual (KIM et al . , 2012) .  [00291] The spots were analyzed microscopically using a 20x and 40x objective lens from an optical microscope connected to a digital camera (Coolpix 990; Nikon Eclipse80i). Representative areas of the effects of the treatments were photographed to record tissue regeneration in the dermis and epidermis and the formation of tissue granulation (KIM et al., 2012).
ANÁLISE DE TECIDOS DE VERMELHO DE PICROSIRIUS USANDO MICROSCÓPIO POLARIZADO  ANALYSIS OF PICROSIRIUS RED TISSUES USING POLARIZED MICROSCOPE
[00292] A coloração vermelha de Picrosirius também foi analisada por microscópio polarizado. A coloração especial do vermelho de Picrosirius destaca a birrefringência natural das fibras de colágeno quando expostas à microscopia de luz polarizada (Olympus BX-model®, com filtro U-POT e U-ANT) .  [00292] Picrosirius' red color was also analyzed by a polarized microscope. The special coloring of Picrosirius red highlights the natural birefringence of collagen fibers when exposed to polarized light microscopy (Olympus BX-model®, with U-POT and U-ANT filter).
[00293] Os resultados da birrefringência permitem avaliar também a organização das fibras de colágeno nos tecidos, como possíveis artefatos que possam ocorrer.  [00293] The results of birefringence also allow to evaluate the organization of collagen fibers in the tissues, as possible artifacts that may occur.
[00294] Os filtros e o condensador foram adaptados para permitir uma boa captura de imagem. O programa para captura de imagens foi Zeiss Lite (versão 2.6, Zen blue, Alemanha [00294] The filters and the condenser have been adapted to allow a good image capture. The program for image capture was Zeiss Lite (version 2.6, Zen blue, Germany
https : / /www. zeiss . com/microscopy/ int/products/microscope- software . html . https: / / www. zeiss. com / microscopy / int / products / microscope- software. html.
AVALIAÇÃO IMUNOHISTOQUÍMICA E MORFOMÉ TRI CA  IMMUNOHISTOCHEMICAL EVALUATION AND MORPHOMÉ TRI CA
[00295] A recuperação antigênica foi realizada com tampão citrato (pH 6,0, 4x 5x) em banho-maria a 95 ° C, seguido pelo bloqueio da atividade endógena da peroxidase por 15 min à temperatura ambiente e protegido da luz com 3% de peróxido de hidrogénio (H2O2) diluído em água destilada e bloqueando a ligação inespecífica com soro de cabra diluído a 2% em lx PBS por 30 minutos.  [00295] Antigenic recovery was performed with citrate buffer (pH 6.0, 4x 5x) in a water bath at 95 ° C, followed by blocking the endogenous activity of peroxidase for 15 min at room temperature and protected from light with 3% of hydrogen peroxide (H2O2) diluted in distilled water and blocking nonspecific binding with goat serum diluted 2% in 1 x PBS for 30 minutes.
[00296] Em seguida, as seções de tecido embebidas em parafina foram incubadas com os anticorpos primários: coelho policlonal anti-VEGF 1: 200 (anticorpo anti-VEGF MSDS) [00296] Then, the sections of tissue embedded in paraffin were incubated with the primary antibodies: polyclonal rabbit anti-VEGF 1: 200 (anti-VEGF MSDS antibody)
(Millipore Cat . # ABS82 Lote # ABS82 Lote # 2142671), durante a noite em uma câmara úmida a 4 0 C e depois lavado em tampão fosfato (PBS, NaCl 136,9 mM, KC1 4,8 mM, KH2P04 14,8 mM e NaOH 8,7 mM, pH 7,2) . A interação da proteína inespecífica será bloqueada com albumina sérica bovina a 2% por 30 minutos. A reação foi detectada por um kit Dako Advance HRP que continha o anticorpo secundário (# K6068, DakoCarpinteria, EUA) e a cor desenvolvida com diaminobenzidina DAB (# K3468, Dako) . (Millipore Cat. # ABS82 ABS82 Lot # Lot # 2142671) overnight in a moist chamber at 4 0 C and then washed in phosphate buffered saline (PBS, NaCl 136.9 mM, KC1 4.8 mM, KH2P04 14.8 mM and 8.7 mM NaOH, pH 7.2). The interaction of the non-specific protein will be blocked with 2% bovine serum albumin for 30 minutes. The reaction was detected by a Dako Advance HRP kit that contained the secondary antibody (# K6068, DakoCarpinteria, USA) and the color developed with diaminobenzidine DAB (# K3468, Dako).
[00297] As lâminas foram levemente coradas com hematoxilina . Entre cada passo após a incubação do anticorpo, as lâminas foram lavadas em PBS contendo BSA a 0,2%. Finalmente, as lâminas são montadas e visualizadas ao microscópio .  [00297] The slides were lightly stained with hematoxylin. Between each step after the antibody incubation, the slides were washed in PBS containing 0.2% BSA. Finally, the slides are assembled and viewed under a microscope.
[00298] Além disso, os anticorpos foram comparados ao controle negativo, que utilizava BSA a 0,2% em vez do anticorpo primário. Foram selecionados sete campos de uma seção imunocorada (VEGF) e capturados para cada amostra. A quantificação foi avaliada em imagens capturadas de alta qualidade (buffer de 2048 c 1536 pixels) usando o Image Pro Plus 4.5.1 (Media Cybernetics, Silver Spring, MD) e processada através do Origin 9.1 e GraphPad Prism 5.1. O resultado da coloração foi expresso como média ± desvio padrão através do software Image J. Esta análise foi realizada apenas para o grupo de tratamento de 24 horas. RESULTADOS[00298] In addition, the antibodies were compared to the negative control, which used 0.2% BSA instead of the primary antibody. Seven fields from an immunostained section (VEGF) were selected and captured for each sample. Quantification was evaluated on high quality captured images (2048 and 1536 pixel buffer) using Image Pro Plus 4.5.1 (Media Cybernetics, Silver Spring, MD) and processed using Origin 9.1 and GraphPad Prism 5.1. The staining result was expressed as mean ± standard deviation using the Image J software. This analysis was performed only for the 24-hour treatment group. RESULTS
OBTENÇÃO E PURIFICAÇÃO DE MONOCERINA OBTAINING AND PURIFYING MONOCERINE
[00299] Como esperado, a monocerina foi produzida com sucesso pela cultura do fungo E. rostratum e após o processo de purificação por HPLC, o composto puro foi obtido.  [00299] As expected, monocerin was successfully produced by culturing the fungus E. rostratum and after the HPLC purification process, the pure compound was obtained.
[00300] A Figura 26A mostra o perfil de HPLC da primeira etapa de purificação, quando a fração F1 contendo monocerina impura foi coletada. Após a evaporação do metanol, a fração F1 foi totalmente purificada no segundo passo, como mostra a Figura 26B.  [00300] Figure 26A shows the HPLC profile of the first purification step, when the F1 fraction containing unclean monocerine was collected. After evaporation of methanol, the F1 fraction was fully purified in the second step, as shown in Figure 26B.
[00301] A monocerina foi caracterizada como previamente descrito por ressonância magnética nuclear (RMN) 1H e 13C (RMN) e espectrometria de massa (MS) e usada para a formulação de creme a 0,0006% e 0,005%.  [00301] Monocerine was characterized as previously described by 1H and 13C nuclear magnetic resonance (NMR) (NMR) and mass spectrometry (MS) and used for the cream formulation at 0.0006% and 0.005%.
AVALIAÇÃO CLÍNICA E MEDIÇÃO DO TAMANHO DA FERIDA  CLINICAL EVALUATION AND MEASUREMENT OF WOUND SIZE
[00302] Os procedimentos cirúrgicos foram realizados com sucesso para todos os grupos de camundongos. Todos os dias, os animais passavam pelo processo de cicatrização de feridas e suas bordas avaliadas macroscopicamente quanto a edema, hiperemia, sangramento, granulação e formação de crostas. A presença ou ausência desses processos foi atribuída como intensidade baixa (*), moderada (**) ou alta (***) . Essas características macroscópicas estão representadas na Tabela 26. [00302] Surgical procedures were successfully performed for all groups of mice. Every day, the animals went through the wound healing process and their edges were evaluated macroscopically for edema, hyperemia, bleeding, granulation and crusting. The presence or absence of these processes was attributed as low (*), moderate (**) or high intensity (***). These macroscopic characteristics are shown in Table 26.
[00303] Pela análise clinica, foi possível observar a presença de sangramento e coagulação nos animais de todos os grupos. Entretanto, o mesmo evento esteve presente até o 5o dia no grupo tratado com colagenase (G2) e até o 3o dia no grupo tratado com monocerina a 0,0006% (G3) e 0,005% (G4) . Por fim, o grupo que recebeu apenas o veículo (G5) apresentou atraso na cicatrização e recuperação até o 10° dia. Um grau significativo de cicatrização foi observado no quinto e no décimo dia nos aspectos clínicos dos grupos tratados com g3 e g4 monocerina, mostrados na Tabela 26. [00303] By clinical analysis, it was possible to observe the presence of bleeding and coagulation in animals of all groups. However, the same event attended by the 5 day in the group treated with collagenase (G2) and up to 3 days in the group treated with the monocerina 0.0006% (G3) and 0.005% (G4). Finally, the group that received only the vehicle (G5) showed delayed healing and recovery until the 10th day. A significant degree of healing was observed on the fifth and tenth days in the clinical aspects of the groups treated with g3 and g4 monocerine, shown in Table 26.
[00304] O tamanho da ferida foi medido diariamente por paquímetro e os tamanhos estão representados, desde as primeiras 24 horas até o 14° dia, para todos os grupos de tratamento. O tamanho da ferida no sétimo dia dos grupos tratados com monocerina a 0,0006% (Grupo G3) e 0,005% (Grupo G4) foi de 4,75 ± 0,80 e 3,72 ± 0,42 mm, respectivamente, enquanto durante 10 dias foi de 2,20 ± 0,62 mm e 1,26 ± 0,29 mm, respectivamente. No 7o e 10° dia, o tamanho do creme comercial com colagenase (Grupo G2 ) foi maior, sendo 5,62 ± 0,39 e 2,6 ± 0,69 mm, respectivamente (Tabela 27) . O tamanho da ferida também é representado na figura 27 pelo período de 24 horas, 72 horas e 7o e 14° dia. [00304] The wound size was measured daily by caliper and the sizes are represented, from the first 24 hours to the 14th day, for all treatment groups. The wound size on the seventh day of the groups treated with 0.0006% (Group G3) and 0.005% (Group G4) monocerine was 4.75 ± 0.80 and 3.72 ± 0.42 mm, respectively, while for 10 days it was 2.20 ± 0.62 mm and 1.26 ± 0.29 mm, respectively. In the 7th and 10 th day, the size of collagenase commercial cream (group G2) was higher, and 5.62 ± 0.39 and 2.6 ± 0.69 mm, respectively (Table 27). The size of the wound is also shown in Figure 27 for the period of 24 hours, 72 hours and 7 and 14 days.
Tabela 26 - Intensidade da avaliação clínica para edema, hiperemia, sangramento, granulação e formação de crostas.  Table 26 - Intensity of clinical evaluation for edema, hyperemia, bleeding, granulation and crusting.
Figure imgf000097_0001
Figure imgf000097_0001
Figure imgf000098_0001
Figure imgf000098_0001
*** = alta intensidade; ** = media intensidade; * = baixa intesidade; ausência representada como (-) .  *** = high intensity; ** = medium intensity; * = low intensity; absence represented as (-).
Tabela 27 - Medição diária dos tamanhos das feridas por paquímetro e representada pelo meio ± DP para quatro animais / grupo em ensaios (mm) .  Table 27 - Daily measurement of wound sizes by caliper and represented by means ± SD for four animals / group in tests (mm).
Figure imgf000098_0002
[00305] O tamanho das feridas dos animais tratados por 14 dias também foi monitorado por imagem ín vivo de raios-X (MS Fx Pro) no tempo zero 24 h, 48 h, 72 h, 7 d, 10 d e 14 dias e os dados são representado na figura 28 e na tabela 28. As imagens foram registradas e estão representadas nas figuras 29 e 30. A análise permite observar a cicatrização e o fechamento da ferida no grupo tratado com monocerina a 0,0006% e 0,005%.
Figure imgf000098_0002
[00305] The size of the wounds of the animals treated for 14 days was also monitored by in vivo X-ray imaging (MS Fx Pro) at time zero 24 h, 48 h, 72 h, 7 d, 10 of 14 days and the data are represented in figure 28 and table 28. The images were recorded and are represented in figures 29 and 30. The analysis allows to observe the healing and wound closure in the group treated with 0.0006% and 0.005% monocerin.
Tabela 28- Medição do tamanho das feridas por imagem radiológica in vivo (MS Fx pro) em milímetros (mm) representada pelos meios ± DP para 2 animais / grupo.  Table 28- Measurement of wound size by in vivo radiological image (MS Fx pro) in millimeters (mm) represented by means ± SD for 2 animals / group.
Figure imgf000099_0001
Figure imgf000099_0001
ANALISE MORFOLOGI CA DA FERIDA POR MICROSCOPIA ELE TRÓNICA DE VARREDURA  MORPHOLOGICAL ANALYSIS OF THE WOUND BY MICROSCOPY HE TRONIC SCAN
[00306] Imagens de microscopia eletrónica de varredura (Figura 31 mostram que camundongos do grupo não tratado (Gl) e do grupo colagenase (G2) demonstraram integridade da matriz extracelular , mas uma baixa deposição de fibras de colágeno dimensionalmente imaturas quando comparadas ao grupo tratado com monocerina a 0,0006% (G3) e 0,005% (G4) .Os animais do grupo que recebeu apenas o veículo (G5) apresentaram processo inflamatório e presença de coagulação no 7o dia. [00306] Scanning electron microscopy images (Figure 31 show that mice from the untreated group (Gl) and the collagenase group (G2) demonstrated integrity of the extracellular matrix, but a low deposition of dimensionally immature collagen fibers when compared to the treated group with 0.0006% (G3) and 0.005% (G4) monocerine. The animals in the group that received only the vehicle (G5) showed inflammation and coagulation in the presence of 7 days.
[00307] Além disso, o tecido de granulação parecia frágil com monocerina na pomada a 0,005%, possivelmente devido ao aumento da imagens ultra-estruturais da derme capturada por MEV mostram os tecidos de granulação dos animais do grupo tratado com monocerina 0,0006% (G3) com presença de numerosas células mortas e abundantes células sanguíneas; também é possível observar fibronectina e sangue vermelho em tecidos infiltrados por capilares. Há evidências consistentes de que mecanismos complexos regulam a correlação entre inflamação e coagulação em. As feridas cirúrgicas tratadas com monocerina em ambas as concentrações (grupos G3 e G4) exibiram formações de coágulos e muitas fibronectinas foram observadas após 7 dias.  [00307] In addition, the granulation tissue appeared fragile with monokin in the 0.005% ointment, possibly due to the increase in the ultrastructural images of the dermis captured by SEM showing the granulation tissues of the animals in the group treated with 0.0006% monocerine (G3) with the presence of numerous dead cells and abundant blood cells; it is also possible to observe fibronectin and red blood in tissues infiltrated by capillaries. There is consistent evidence that complex mechanisms regulate the correlation between inflammation and coagulation in. Surgical wounds treated with monocerine in both concentrations (groups G3 and G4) exhibited clot formation and many fibronectins were observed after 7 days.
[00308] A pele ferida dos grupos tratados com monocerina (G3 e G4) revelou claramente que a ativação das células de reepitelização da pele era mais eficaz e mais rápida do que os grupos de controle de veículo e não tratados .  [00308] The injured skin of the groups treated with monocerine (G3 and G4) clearly revealed that the activation of the skin's re-epithelialization cells was more effective and faster than the vehicle control and untreated groups.
ANÁLISE HISTOLÓGICA DOS TECIDOS FERIDOS  HISTOLOGICAL ANALYSIS OF WOUNDED TISSUES
[00309] Pela avaliação histológica dos retalhos cutâneos revelados pela coloração HE, foi possível observar regressão das lesões com melhor epitelização nos grupos não tratado (Gl) e tratado com colagenase (G2)), mas esse processo foi mais eficaz, mostrando melhor reorganização dos derme nos dois grupos tratados com monocerina (G3 e G4) até o 14° dia. A indicação clara dos tecidos histológicos da ferida nas experiências de 24 e 72 h é mostrada nas Figuras 31 e 32. [00310] Para o grupo não tratado (Gl), é possível observar: (H&E) - tecido cicatricial visível grande número de células e vasos sanguíneos de fibroblastos (amarelo seta) ; (PR) redução de colágeno necrose densa e organizada (seta laranja); (MT) grande número de células inflamatórias cicatrizes visíveis (setas vermelhas) estavam presentes. No caso de tratamento com colagenase (G2) : cicatrizes de inflamação (H&E), células de necrose (seta amarela) (PR) inflamação dos tecidos adiposos (seta laranja); (MT) grande número de células inflamatórias resistiu claramente às células de resposta imune na cicatrização de feridas (seta vermelha) . Para o tratamento de monocerina a 0, 0006%, as evidências são: (H&E) - boa distribuição de células epiteliais e tecido organizado da pele (seta vermelha); (PR) colágeno e camada epitelial densa (seta laranja); (MT) grande formação de fibroblastos, células inflamatórias reduzidas foram marcadas nas figuras 31 e 32 dos tecidos corados com intervalos de 24 e 72 horas. [00309] By histological evaluation of the skin flaps revealed by HE staining, it was possible to observe regression of lesions with better epithelialization in the untreated (Gl) and treated with collagenase (G2) groups, but this process was more effective, showing a better reorganization of the dermis in the two groups treated with monocerine (G3 and G4) until the 14th day. The clear indication of the histological tissues of the wound in the 24 and 72 h experiments is shown in Figures 31 and 32. [00310] For the untreated group (Gl), it is possible to observe: (H&E) - scar tissue visible a large number of fibroblast cells and blood vessels (yellow arrow); (PR) reduction of dense and organized collagen necrosis (orange arrow); (MT) a large number of visible scar inflammatory cells (red arrows) were present. In the case of treatment with collagenase (G2): scars of inflammation (H&E), necrosis cells (yellow arrow) (PR) inflammation of adipose tissues (orange arrow); (MT) a large number of inflammatory cells clearly resisted immune response cells in wound healing (red arrow). For the treatment of monocerine at 0, 0006%, the evidence is: (H&E) - good distribution of epithelial cells and organized skin tissue (red arrow); (PR) collagen and dense epithelial layer (orange arrow); (MT) large fibroblast formation, reduced inflammatory cells were marked in figures 31 and 32 of the stained tissues at 24 and 72 hour intervals.
[00311] A distribuição do poço de colágenos (seta vermelha) . O grupo monocerina 0, 005% (G4) : (H&E) - Neste grupo as células epiteliais são mais organizadas e há alta distribuição de colágeno (seta amarela); (PR) grande número de células de fibroblastos indica sinais de cicatrização e os colágenos também estavam apoiando a regeneração da pele (seta laranja); (MT) abundantes células de fibroblastos e a camada de epiderme foram mais organizadas (seta vermelha) . Menos, mas não o último, no grupo tratado com veículo (G5) (H&E) - grande número de células inflamatórias (seta amarela); (PR) privação de colágenos (seta laranja); (MT) grande número de células inflamatórias adiposas (seta vermelha) . [00312] Os tecidos da ferida para experimento histológico de 7 e 14 dias são mostrados nas Figuras 33 e 34. O controle não tratado (Gl) : (H&E) células visíveis de fibroblastos do tecido cicatricial e vasos sanguíneos (seta amarela); (PR) os colágenos são densos e organizados (seta laranja); Camada de epiderme (MT) mais densa (setas vermelhas) foi observada nos 7 e 14 dias de tratamento. Tratamento com colagenase (G2) : foram observadas inflamação (H&E) e cicatrizes (seta amarela); (PR) tecidos adiposos foram observados e também inflamação foi observada até o 14° dia (seta laranja); (MT) O grande número de células inflamatórias também esteve presente no tratamento de 7 e 14 dias (seta vermelha) . O grupo monocerina 0,0006% (G3) : (H&E) registrou boa distribuição de células epiteliais e tecido cutâneo organizado (seta vermelha); (PR) colágeno e camada epitelial foram densos e melhores que os grupos controle (seta laranja) ; (MT) . [00311] The distribution of the collagen well (red arrow). The monocerine group 0, 005% (G4): (H&E) - In this group the epithelial cells are more organized and there is a high distribution of collagen (yellow arrow); (PR) a large number of fibroblast cells indicate signs of healing and collagens were also supporting skin regeneration (orange arrow); (MT) abundant fibroblast cells and the epidermis layer were more organized (red arrow). Less, but not the last, in the group treated with vehicle (G5) (H&E) - large number of inflammatory cells (yellow arrow); (PR) collagen deprivation (orange arrow); (MT) large number of adipose inflammatory cells (red arrow). [00312] The wound tissues for 7 and 14 days histological experiment are shown in Figures 33 and 34. The untreated control (Gl): (H&E) visible fibroblast cells of the scar tissue and blood vessels (yellow arrow); (PR) collagens are dense and organized (orange arrow); A denser layer of epidermis (MT) (red arrows) was observed at 7 and 14 days of treatment. Treatment with collagenase (G2): inflammation (H&E) and scarring (yellow arrow) were observed; (PR) adipose tissues were observed and inflammation was also observed until the 14th day (orange arrow); (MT) The large number of inflammatory cells was also present in the treatment of 7 and 14 days (red arrow). The 0.0006% monocerine group (G3): (H&E) registered good distribution of epithelial cells and organized skin tissue (red arrow); (PR) collagen and epithelial layer were dense and better than the control groups (orange arrow); (MT).
[00313] A grande formação de fibroblastos no intervalo de 7 dias. Além disso, as células inflamatórias foram reduzidas no tratamento de 7 e 14 dias (seta vermelha) . Grupo monocerina a 0, 005% (G4) : (H&E) A proliferação nas células epiteliais foi revelada (seta amarela); (PR) ocorre a queratinização e os colágenos estão bem organizados (seta laranja) mostrados nas figuras 33 e 34 em intervalos de 7 dias; (MT) células de fibroblastos abundantes e camada de epiderme organizada levam à recuperação e cicatrização em menos de 14 dias (seta vermelha); Por fim, no grupo veículo (G5) : (H&E) , foi observado pequeno número de células inflamatórias no 14° dia (seta amarela); Por MT, um grande número de células inflamatórias e tecidos adiposos é mostrado nas figuras 33 e 34 para o intervalo de 7 e 14 dias, o que significa que as feridas não foram totalmente recuperadas e confiável (seta vermelha) . [00313] The great formation of fibroblasts in the interval of 7 days. In addition, inflammatory cells were reduced in the treatment of 7 and 14 days (red arrow). 0.005% monocerine group (G4): (H&E) The proliferation in epithelial cells was revealed (yellow arrow); (PR) keratinization occurs and the collagens are well organized (orange arrow) shown in figures 33 and 34 at 7-day intervals; (MT) abundant fibroblast cells and an organized epidermis layer lead to recovery and healing in less than 14 days (red arrow); Finally, in the vehicle group (G5): (H&E), a small number of inflammatory cells was observed on the 14th day (yellow arrow); By TM, a large number of inflammatory cells and adipose tissues are shown in figures 33 and 34 for the interval of 7 and 14 days, which it means that the wounds have not been fully recovered and reliable (red arrow).
[00314] A avaliação pela coloração por Picrosirius permitiu reconhecer a densidade total de colágeno devido à presença de fibras de colágeno (amarelo avermelhado nas figuras 31, 32, 33, 34 e 35) . A área ocupada por fibras de colágeno foi quantificada usando a Imagem J e o software Graph Pad Prism 5.1, apresentado na figura 35. Os dados foram obtidos para quatro animais por cada grupo e representados pela porcentagem de meios ± DP mencionados na Tabela 28. O valor final foi representado comparando todos os grupos (Tabela 28) . Em comparação com os grupos controles (Gl, G2 e G5) . A porcentagem de colágeno foi mais intensa nos grupos G3 e G4 tratados com monocerina a 0,0006% e 0,005% para todo o tempo dos experimentos. A maior porcentagem de colágeno foi observada no experimento de 14 dias, sendo 60,44 ± 5,47% e 70,36 ± 13,31%, respectivamente .  [00314] Picrosirius staining allowed to recognize the total collagen density due to the presence of collagen fibers (reddish yellow in figures 31, 32, 33, 34 and 35). The area occupied by collagen fibers was quantified using Image J and the Graph Pad Prism 5.1 software, shown in figure 35. Data were obtained for four animals for each group and represented by the percentage of means ± SD mentioned in Table 28. The final value was represented by comparing all groups (Table 28). In comparison with the control groups (Gl, G2 and G5). The percentage of collagen was more intense in groups G3 and G4 treated with 0.0006% and 0.005% monocerine for the entire duration of the experiments. The highest percentage of collagen was observed in the 14-day experiment, with 60.44 ± 5.47% and 70.36 ± 13.31%, respectively.
ANÁLISE DE TECIDOS DE VERMELHO DE PICROSIRIUS USANDO MICROSCÓPIO POLARIZADO  ANALYSIS OF PICROSIRIUS RED TISSUES USING POLARIZED MICROSCOPE
[00315] O corante especial vermelho de picrosirius tem a capacidade de aumentar a birrefringência natural do colágeno quando exposto à luz polarizada. O colágeno tipo I mostraria uma cor amarelo-vermelho, enquanto o tipo III seria verde, como mostrado na Figura 37.  [00315] The special red dye of picrosirius has the ability to increase the natural birefringence of collagen when exposed to polarized light. Type I collagen would show a yellow-red color, while type III would be green, as shown in Figure 37.
[00316] Às 24 e 72 horas, os grupos tratados com monocerina G3 a 0,0006% e G4 a 0,005% mostraram um arranjo denso e compacto de colágenos em comparação aos grupos controle (Gl, G2 e G5) . Nas 24 e 72 horas, o grupo Gl não tratado mostrou uma expressão significativamente baixa de colágenos  [00316] At 24 and 72 hours, the groups treated with 0.0006% G3 monocerin and 0.005% G4 showed a dense and compact arrangement of collagens compared to the control groups (Gl, G2 and G5). At 24 and 72 hours, the untreated Gl group showed significantly low collagen expression
No tratamento por 7 dias, houve uma clara diferença observada entre os grupos controle (Gl. G2 e G5) e os grupos tratados com monocerina G3 e G4, onde a população de fibras colágenas exibiu cores de polarização no vermelho-laranja, enquanto para os controles o colágeno as fibras eram escassas e imaturas. O grupo Gl não tratado exibiu intensa birrefringência verde - amarela, sugerindo que o conteúdo de colágeno foi reduzido e suas fibras estavam muito frouxamente empacotadas . In the treatment for 7 days, there was a clear difference observed between the control groups (Gl. G2 and G5) and the groups treated with monocerine G3 and G4, where the population of collagen fibers showed polarization colors in red-orange, while for controls the collagen fibers were scarce and immature. The untreated Gl group exhibited intense green - yellow birefringence, suggesting that the collagen content was reduced and its fibers were very loosely packed.
[00317] No entanto, no 14° dia para os grupos tratados com monocerina a 0, 0006% e 0, 005% (G3. G4), mais fibras amarelas e vermelhas foram vistas, possivelmente indicando a presença de colágeno tipo I. As fibras verdes (típicas do colágeno tipo III) foram mais frequentemente localizadas na derme superior e na epiderme no grupo G3 tratado com monocerina a 0,005%. Os grupos controle (Gl, G2 e G5) apresentaram fibras grossas vermelhas e amarelas (típicas do colágeno tipo I ) localizada na derme profunda inferior. No geral, os grupos tratados com monocerina foram de 0,0006% e 0,005% (G3 e G4) . A figura 37 mostra a disposição das fibras colágenas visíveis e organizadas dos grupos tratados com monocerina .  [00317] However, on the 14th day for the groups treated with 0, 0006% and 0, 005% monocerin (G3. G4), more yellow and red fibers were seen, possibly indicating the presence of type I collagen. green fibers (typical of type III collagen) were more frequently located in the upper dermis and epidermis in the G3 group treated with 0.005% monocerin. The control groups (Gl, G2 and G5) had thick red and yellow fibers (typical of type I collagen) located in the lower deep dermis. In general, the groups treated with monocerine were 0.0006% and 0.005% (G3 and G4). Figure 37 shows the arrangement of the visible and organized collagen fibers of the groups treated with monocerine.
ANÁLISE DE HEMATOLOGIA  HEMATOLOGY ANALYSIS
[00318] Neste estudo, foi examinado o sangue periférico de camundongos Balb / C entre os grupos controle Gl (não tratado) , G2 (colagenase) e G5 (veículo) e tratados com monocerina a 0,0006% e 0,005% (G3 e G4) . Os dados estão representados nas figuras 38 e 39.  [00318] In this study, the peripheral blood of Balb / C mice was examined between the control groups Gl (untreated), G2 (collagenase) and G5 (vehicle) and treated with 0.0006% and 0.005% monocerin (G3 and G4). The data are represented in figures 38 and 39.
[00319] Foram analisadas as três principais categorias de células imunes, como glóbulos vermelhos (glóbulos vermelhos), glóbulos brancos (glóbulos brancos) e plaquetas. Como mostrado na Figura 38, a monocerina a 0,0006% e 0,005% (Grupos G3 e G4) apresentou os níveis mais altos de hemácias em 24 h, em comparação com outros grupos de controle Gl, G2-colagenase e veículo G5. Mais tarde, após 7 dias em fase de proliferação. As hemácias mantiveram seu equilíbrio e resultados quase semelhantes foram observados para todos os grupos, exceto o veículo do grupo G5, que mostrou um número baixo e significativo de glóbulos vermelhos em comparação aos grupos tratados com monocerina (G3 e G4) . [00319] The three main categories of immune cells were analyzed, such as red blood cells (red blood cells), white blood cells (white blood cells) and platelets. As shown in Figure 38, 0.0006% monocerine and 0.005% (Groups G3 and G4) had the highest levels of red blood cells in 24 h, compared to other control groups Gl, G2-collagenase and vehicle G5. Later, after 7 days of proliferation. Red blood cells maintained their balance and almost similar results were observed for all groups, except for the vehicle in the G5 group, which showed a low and significant number of red blood cells compared to the groups treated with monocerine (G3 and G4).
[00320] Em 24 horas, os grupos tratados com monocerina a 0,0006% e 0,005% (G3 e G4) apresentaram níveis mais elevados de leucócitos quando comparados aos demais grupos (Gl, G2 e G5) . Níveis levemente mais altos de leucócitos foram observados após 7 dias no grupo Gl controle não tratado. Enquanto, após 14 dias, os grupos tratados com monocerina (G3 e G4) eram mais altos do que outros grupos controle (Gl, G2 e G5) . No caso de plaquetas em 24 h, os grupos tratados com monocerina em fase de inflamação (G3 e G4) apresentaram níveis mais altos de plaquetas do que outros grupos controle (Gl, G2 e G5) , o que leva à regeneração da pele e à resposta imune após a aplicação de grupos tratados com monocerina. Curiosamente, os níveis de plaquetas foram mais baixos no grupo G3 tratado com monocerina a 0,0006% do que no grupo G4 tratado com monocerina a 0,005%.  [00320] In 24 hours, the groups treated with 0.0006% and 0.005% monocerin (G3 and G4) showed higher levels of leukocytes when compared to the other groups (Gl, G2 and G5). Slightly higher levels of leukocytes were observed after 7 days in the untreated Gl control group. Whereas, after 14 days, the groups treated with monocerine (G3 and G4) were taller than other control groups (Gl, G2 and G5). In the case of platelets within 24 h, the groups treated with monocerine in the inflammation phase (G3 and G4) had higher levels of platelets than other control groups (Gl, G2 and G5), which leads to skin regeneration and immune response after application of groups treated with monocerine. Interestingly, platelet levels were lower in the G3 group treated with 0.0006% monocerine than in the G4 group treated with 0.005% monocerine.
[00321] Adicionalmente, na fase de proliferação após 7 dias, o nível de plaquetas foi maior no grupo G2 de colagenase G2 em comparação com outros grupos, mas os grupos monocerina G3 e G4 foram maiores que o grupo não tratado Gl e o grupo tratado com veículo G5. Na fase de remodelação após 10 dias, as plaquetas do grupo G3, G4 tratadas com monocerina foram observadas mais altas do que os outros grupos controle Gl e G5, exceto o grupo G2 Collagenase. [00322] Parâmetros adicionais relacionados à RBC, como teste de hematócrito e concentração de hemoglobina, também foram analisados. Curiosamente, em 24 h grupos tratados com monocerina (G3 e G4) acentuadamente uma concentração mais alta de hemoglobina em comparação com outros grupos Gl, G2 e G5. Em 7 dias, os grupos tratados com monocerina (G3 e G4) apresentaram maior nível de concentração do que outros grupos controle, exceto o grupo não tratado (Gl) . No entanto, em 14 dias todas as leituras dos grupos foram mantidas com nível quase idêntico de HGB, também não houve distinção no percentual de volume de células de hematócrito entre todos os grupos, o mesmo comportamento do HCT foi registrado e mostrado no (Figura 39) . [00321] Additionally, in the proliferation phase after 7 days, the level of platelets was higher in the G2 group of collagenase G2 compared to other groups, but the monocerine groups G3 and G4 were higher than the untreated group Gl and the treated group with G5 vehicle. In the remodeling phase after 10 days, the platelets of the G3, G4 group treated with monocerine were observed higher than the other control groups Gl and G5, except for the group G2 Collagenase. [00322] Additional parameters related to RBC, such as hematocrit test and hemoglobin concentration, were also analyzed. Interestingly, in 24 h groups treated with monocerine (G3 and G4) markedly a higher concentration of hemoglobin compared to other groups Gl, G2 and G5. In 7 days, the groups treated with monocerine (G3 and G4) showed a higher concentration level than other control groups, except the untreated group (Gl). However, in 14 days all the readings of the groups were maintained with an almost identical level of HGB, there was also no distinction in the percentage of hematocrit cell volume between all groups, the same behavior of the HCT was recorded and shown in (Figure 39 ).
[00323] As subpopulações de leucócitos em grupos tratados com 24 horas de monocerina (G3 e G4) apresentaram os níveis mais baixos de linfócitos em comparação com outros grupos (Gl, G2 e G5) . Em 7 dias, o nível de linfócitos para o grupo monocerina em 0,0006% e 0,005% (G3 e G4) foi superior ao grupo não tratado controle Gl (Figura 37) . Além disso, em 14 dias a monocerina a 0,0006% (grupo G3) foi superior aos grupos Gl, G2 e G3 e o nível de monocerina a 0, 005% (grupo G4) foi superior aos grupos Gl e G2, mas inferior aos grupos G4 e G5  [00323] Leukocyte subpopulations in groups treated with 24 hours of monocerine (G3 and G4) showed the lowest levels of lymphocytes compared to other groups (Gl, G2 and G5). In 7 days, the lymphocyte level for the monocerine group by 0.0006% and 0.005% (G3 and G4) was higher than the untreated control group Gl (Figure 37). In addition, in 14 days the 0.0006% monocerine (group G3) was higher than the groups Gl, G2 and G3 and the level of 0.05% monocerine (group G4) was higher than the groups Gl and G2, but lower to groups G4 and G5
[00324] Nas 24h, o grupo G2 apresentou nível de monócitos levemente superior ao monocerina G3 e G4 em 0,0006% e 0,005%. Nesse momento, o nível de monócitos dos grupos Gl e G5 era significativamente menor que os grupos G2, G3 e G4 ( Figura 38 ) .  [00324] In the 24h, the G2 group showed monocytes level slightly higher than the monocerine G3 and G4 in 0.0006% and 0.005%. At that time, the level of monocytes in groups Gl and G5 was significantly lower than groups G2, G3 and G4 (Figure 38).
[00325] A porcentagem de monócitos para o grupo Gl controle - não tratado. A monocerina G3 e G4 a 0, 0006% e 0,005% foi maior que o grupo G2 colagenase e o grupo veículo G5 por 7 dias de tratamento. No final do experimento, aos 14 dias, a monocerina no grupo tratado com 0, 005% (G4) foi significativamente maior do que nos outros grupos G1 controle não tratado. Colagenase G2. G3 monocerina a 0,0006% e grupo G5 de veículos (Gráfico 5) . [00325] The percentage of monocytes for the control group GI - untreated. Monocerine G3 and G4 at 0.0006% and 0.005% was higher than the G2 collagenase group and the vehicle group G5 for 7 days of treatment. At the end of the experiment, at 14 days, the monocerine in the group treated with 0.005% (G4) was significantly higher than in the other G1 untreated control groups. Collagenase G2. 0.0006% monocerina G3 and vehicle G5 group (Graph 5).
[00326] Para os neutrófilos em 24 h, os grupos Gl, G2 e G5 apresentaram os níveis mais baixos em comparação aos grupos tratados com G3 e G4. Em 7 dias, o Gl apresentou nível ligeiramente superior aos demais grupos G2, G3, G4 e G5. Para G4 monocerina no nível de 0,005% de neutrófilos foi maior que G2 colagenase. Grupo de veículos G5 e para monocerina G3 a 0,0006%. Curiosamente, em 14 dias, a monocerina G4 a 0, 005% foi maior que nos grupos Gl, G2, G3 e G5 (Figura 39) .  [00326] For neutrophils in 24 h, groups Gl, G2 and G5 showed the lowest levels compared to groups treated with G3 and G4. In 7 days, the Gl showed a slightly higher level than the other groups G2, G3, G4 and G5. For monocerine G4, the level of 0.005% neutrophils was greater than G2 collagenase. Group of vehicles G5 and for single-grain G3 0.0006%. Interestingly, in 14 days, monocerin G4 at 0.005% was higher than in groups Gl, G2, G3 and G5 (Figure 39).
[00327] No geral, os grupos tratados com monocerina a 0,0006% e 0,005% (G3 e G4) mostraram presença significativa de linfócitos, neutrófilos e monócitos e populações maiores de granulócitos . Os resultados obtidos deixaram os sinais de entendimento sobre o tratamento com monocerina, que foram efetivamente contrários à resposta inflamatória e ajudam a ativar a resposta das células imunes no mecanismo de cura, em comparação com outros grupos de controle apresentados nas Figuras 38 e 39.  [00327] In general, the groups treated with 0.0006% and 0.005% monocerin (G3 and G4) showed a significant presence of lymphocytes, neutrophils and monocytes and larger populations of granulocytes. The results obtained left the signs of understanding about the treatment with monocerine, which were effectively contrary to the inflammatory response and help to activate the response of the immune cells in the healing mechanism, in comparison with other control groups presented in Figures 38 and 39.
ANÁLISES IMUNOHISTOQUÍMICAS  IMMUNOHISTOCHEMICAL ANALYSIS
[00328] As análises imunoistoquímicas foram realizadas para o VEGF. Esta análise será repetida incluindo a análise dos anticorpos TIMP-2. MMP-3 e MMP-9 também envolvidos no processo da ferida.  [00328] Immunohistochemical analyzes were performed for VEGF. This analysis will be repeated including the analysis of TIMP-2 antibodies. MMP-3 and MMP-9 are also involved in the wound process.
[00329] A Figura 40 mostra uma fotomicrografia de tecidos imunocorados com anticorpo contra VEGF nos grupos controle e tratado por 24 h. Para todos os grupos é possível observar a imunorreatividade contra o VEGF (setas), enquanto para os controles negativos não houve imunorreatividade conforme o esperado; A exceção é para a reação de controle negativo para o grupo Gl, que também foi imunorreativo, mas de baixa intensidade, conforme o esperado. Barra de escala 40 x 100 pm. Os dados foram analisados medindo os pixels da barra de escala de 1024 x 2048 pixels. [00329] Figure 40 shows a photomicrograph of tissues immunostained with antibody against VEGF in the control and treated groups for 24 h. For all groups it is possible observe the immunoreactivity against VEGF (arrows), while for the negative controls there was no immunoreactivity as expected; The exception is for the negative control reaction for the Gl group, which was also immunoreactive, but of low intensity, as expected. Scale bar 40 x 100 pm. The data were analyzed by measuring the pixels of the 1024 x 2048 pixel scale bar.
[00330] Os dados para imunocoloração contra VEGF mostraram que, em comparação com o grupo controle não tratado, o tratamento com grupo Collagenase (G2) apresentou maior imunorreatividade, com 87,2% de colágeno, e os grupos monocerina tratados com 0,005% e 0,0006%, respectivamente, 85,4 e 83,5%. Os dados são representados na Figura 41.  [00330] Data for immunostaining against VEGF showed that, compared to the untreated control group, treatment with the Collagenase group (G2) showed greater immunoreactivity, with 87.2% collagen, and the monocerine groups treated with 0.005% and 0.0006%, respectively, 85.4 and 83.5%. The data are represented in Figure 41.
[00331] O metabolito secundário dos fungos mostrou potencial e tem sido consistentemente uma importante fonte de agentes terapêuticos (VOLK et al . , 2013) .  [00331] The secondary metabolite of fungi has shown potential and has consistently been an important source of therapeutic agents (VOLK et al., 2013).
[00332] Portanto, neste estudo, a monocerina foi incorporada em uma formulação em creme a 0,0006% e 0,005% e sua eficácia foi avaliada em um modelo cirúrgico cutâneo in vivo de ferida. A fim de avaliar o efeito da monocerina, embora nos estágios iniciais do processo de reparação tecidual, o tratamento das lesões tenha começado após cerca de 3 h após a indução nos animais.  [00332] Therefore, in this study, monocerine was incorporated into a cream formulation at 0.0006% and 0.005% and its effectiveness was evaluated in an in vivo cutaneous wound surgical model. In order to assess the effect of monocerine, although in the early stages of the tissue repair process, the treatment of the lesions started after about 3 h after induction in the animals.
[00333] A ação dos metabólitos secundários no sistema imunológico não foi estudada extensivamente, mas a atividade anti-inflamatória dos flavonóides mostrou ser atribuída à sua capacidade de inibir a degranulação de neutrófilos; diminuindo a liberação de ácido araquidônico e outros mediadores das células imunes (NIJVELDT et al . , 2001) .  [00333] The action of secondary metabolites in the immune system has not been studied extensively, but the anti-inflammatory activity of flavonoids has been shown to be attributed to their ability to inhibit neutrophil degranulation; decreasing the release of arachidonic acid and other immune cell mediators (NIJVELDT et al., 2001).
[00334] Esse efeito provavelmente pode interferir no reparo da pele. O processo de cicatrização de feridas é caracterizado por eventos dinâmicos e interativos que envolvem mediadores solúveis, células sanguíneas, MEC e células parenquimatosas , o que resulta na restauração permanente da integridade anatômica e funcional. O termo "ordenado" refere-se à sequência de fases de cicatrização de feridas que incluem inflamação, formação e remodelação de tecidos (SINGER; CLARK, 1999) . [00334] This effect is likely to interfere with skin repair. The wound healing process is characterized by dynamic and interactive events involving soluble mediators, blood cells, ECM and parenchymal cells, which results in the permanent restoration of anatomical and functional integrity. The term "ordered" refers to the sequence of phases of wound healing that include inflammation, formation and remodeling of tissues (SINGER; CLARK, 1999).
[00335] Os metabólitos secundários do fungo foram associados a uma forte atividade anti-inflamatória, antimicrobiana e antioxidante, essenciais para a cicatrização de feridas. As atividades específicas dos extratos de metabólito secundário de E. rostratum e seus constituintes benéficos para a cicatrização de feridas foram relatadas como incluindo efeitos inibitórios no sangramento, aumento da contração da ferida, aumento dos níveis do fator básico de crescimento de fibroblastos (FGF) e fator de crescimento derivado de plaquetas e estimulação de parâmetros hematológicos como glóbulos brancos e vermelhos (AGYARE C; AKINDELE ; STEENKAMP, 2019) .  [00335] The secondary metabolites of the fungus have been associated with a strong anti-inflammatory, antimicrobial and antioxidant activity, essential for wound healing. The specific activities of E. rostratum secondary metabolite extracts and their beneficial constituents for wound healing have been reported to include inhibitory effects on bleeding, increased wound contraction, increased levels of basic fibroblast growth factor (FGF) and platelet-derived growth factor and stimulation of hematological parameters such as white and red blood cells (AGYARE C; AKINDELE; STEENKAMP, 2019).
[00336] Os parâmetros hematológicos são importantes marcadores da doença na medicina humana e veterinária. O sistema imunológico e os sistemas neuroendócrinos são os dois componentes principais que mantêm a homeostase corporal. Amostras de sangue periférico podem indicar anormalidades no corpo, que geralmente causam várias ameaças à saúde humana, incluindo doenças autoimunes ou metabólicas devastadoras .  [00336] Hematological parameters are important markers of the disease in human and veterinary medicine. The immune system and neuroendocrine systems are the two main components that maintain body homeostasis. Peripheral blood samples can indicate abnormalities in the body, which generally cause several threats to human health, including devastating autoimmune or metabolic diseases.
[00337] Na análise geral dos resultados referentes à proporção da área da ferida em relação ao tempo inicial de pós-operatório, houve um efeito do grupo, o tempo da análise e a interação entre esses fatores (teste ANOVA one-way de medidas repetitivas , p <0,005) . [00337] In the general analysis of the results regarding the proportion of the wound area in relation to the initial postoperative time, there was an effect of the group, the time of the analysis and the interaction between these factors (one-way ANOVA test of repetitive measures, p <0.005).
[00338] Na análise por grupos ao longo de diferentes tempos de cicatrização, foi observada uma redução significativa na proporção da área da ferida em relação ao tempo inicial (teste de comparação de Tukey, p <0,05) . Para os grupos G3 e G4 houve diferença significativa entre os 7 e 14 dias (p> 0,05), que apresentaram redução progressiva da área da ferida (p <0,05) em todos os momentos de cicatrização. Na comparação entre os grupos experimentais em cada momento de análise, foi evidenciado que a proporção da área da ferida para o grupo veiculo G5 e grupo G2 colagenase aos 7 e 14 dias foi menor em relação aos grupos G3 e G4 tratados com monocerina (pós -Teste de Tukey, p <0,005), e não houve diferença significativa entre os grupos nos 3 e 7 dias de cicatrização. Os dados indicam que, pelos dois métodos utilizados para medir o tamanho da ferida (raio X e paquímetro) para os grupos tratados com monocerina a 0,0006% e 0,005% (G3 e G4 ) , a redução e o fechamento da ferida foram mais eficientes do que em os grupos de controle.  [00338] In the analysis by groups over different healing times, a significant reduction was observed in the proportion of the wound area in relation to the initial time (Tukey comparison test, p <0.05). For groups G3 and G4 there was a significant difference between 7 and 14 days (p> 0.05), which showed a progressive reduction in the wound area (p <0.05) at all times of healing. In the comparison between the experimental groups at each moment of analysis, it was evidenced that the proportion of the wound area for the vehicle group G5 and the group G2 collagenase at 7 and 14 days was lower in relation to the groups G3 and G4 treated with monocerine (post - Tukey's test, p <0.005), and there was no significant difference between groups in the 3 and 7 days of healing. The data indicate that, by the two methods used to measure the size of the wound (X-ray and caliper) for the groups treated with 0.0006% and 0.005% monocerin (G3 and G4), the reduction and closure of the wound were more efficient than in the control groups.
[00339] A investigação hematológica mostrou que os grupos tratados com monocerina (G3 e G4) apresentaram alta porcentagem de hemácias e leucócitos em comparação com outros grupos controle (Gl, G2 e G5) . Em 14 dias, todos os grupos mantiveram o nível de distribuição das células, mas em 24 horas, 72 horas e 7 dias, a distribuição de leucócitos e hemácias nos grupos tratados com monocerina (G3 e G4) foi maior que em outros grupos controle.  [00339] The hematological investigation showed that the groups treated with monocerine (G3 and G4) had a high percentage of red blood cells and leukocytes in comparison with other control groups (Gl, G2 and G5). At 14 days, all groups maintained the level of cell distribution, but at 24 hours, 72 hours and 7 days, the distribution of leukocytes and red blood cells in the groups treated with monocerine (G3 and G4) was greater than in other control groups.
[00340] Os animais tratados com a formulação contendo monocerina em ambas as concentrações apresentaram redução estatisticamente significativa do tamanho da ferida e a pele foi mais compacta e densa. Nos animais tratados com a formulação, transportando monocerina, a formação de tecido e a reepitelização foram eficazes, com aumento da distribuição e deposição de colágeno, e angiogênese significativamente melhorada nas lesões cutâneas tratadas com monocerina. Durante o processo, observou-se uma formação clara de crosta granular e sangue, coagulação sem sintomas graves de sangramento e liquido mucoso ou com vazamento nas feridas tratadas com monocerina. [00340] Animals treated with the formulation containing monocerine in both concentrations showed a statistically significant reduction in wound size and skin it was more compact and dense. In animals treated with the formulation, carrying monocerine, tissue formation and reepithelization were effective, with increased distribution and deposition of collagen, and significantly improved angiogenesis in skin lesions treated with monocerine. During the process, a clear formation of granular crust and blood was observed, coagulation without severe symptoms of bleeding and mucous fluid or leakage in wounds treated with monocerine.
[00341] A coagulação sanguínea é um mecanismo de defesa do hospedeiro que, paralelamente às respostas inflamatórias, não apenas ajuda a proteger a integridade do sistema vascular, mas também promove o reparo após lesão tecidual . A formação de um coágulo sanguíneo serve não apenas para fechar as bordas da ferida, mas também para atravessar as fibronectinas , o que fornece uma matriz provisória na qual os fibroblastos células endoteliais e queratinócitos podem entrar na ferida (MONROE DM; MAUREANE, 2012) .  [00341] Blood clotting is a host defense mechanism that, in addition to inflammatory responses, not only helps to protect the integrity of the vascular system, but also promotes repair after tissue damage. The formation of a blood clot serves not only to close the edges of the wound, but also to pass through the fibronectins, which provides a temporary matrix in which the fibroblasts endothelial cells and keratinocytes can enter the wound (MONROE DM; MAUREANE, 2012).
[00342] Os fibroblastos estavam envolvidos na síntese de colágeno, glicosaminoglicanos (GAGs) . proteoglicanos e glicoproteínas adesivas e reconhecidos como de importância essencial no processo de cicatrização de feridas tratadas por 72 h. predominantemente em monocerina nos grupos tratados com 0,0006% e 0,005% (G3 e G4 ) . No entanto, o grupo tratado com G2 colagenase mostrou a degradação de colágenos que elabora claramente o sinal de inflamação.  [00342] Fibroblasts were involved in the synthesis of collagen, glycosaminoglycans (GAGs). proteoglycans and adhesive glycoproteins and recognized as of essential importance in the healing process of wounds treated for 72 h. predominantly in monocerine in the groups treated with 0.0006% and 0.005% (G3 and G4). However, the group treated with G2 collagenase showed collagen degradation that clearly elaborates the sign of inflammation.
[00343] Os dados observados na análise SEM na Figura 6 permitem concluir que os animais G3 e G4 tratados com monocerina apresentaram tendência à regeneração dos tecidos e melhor formação de fibronectinas colágenas e glóbulos vermelhos, o que foi indicação de cicatrização em animais. A análise histológica dos tecidos dos animais revelou que a organização da pele e a distribuição de colágeno dos animais tratados com monocerina foi melhor que os grupos G1 (não tratado e tratamento com veiculo G5) e mesmo que G2 (tratamento com colagenase) em 24 horas e 14 dias. [00343] The data observed in the SEM analysis in Figure 6 allow us to conclude that the animals G3 and G4 treated with monocerine showed a tendency for tissue regeneration and better formation of collagen fibronectins and red blood cells, which was an indication of healing in animals. Histological analysis of animal tissues revealed that the skin organization and collagen distribution of animals treated with monocerine was better than the G1 (untreated and G5 vehicle) groups and even G2 (collagenase treatment) in 24 hours. and 14 days.
[00344] TENGRUP et al . , (1988) mencionaram que uma reação inflamatória persistente com acúmulo de leucócitos polimorfo-nucleares pode ser parcialmente responsável pelo baixo conteúdo de colágeno no tecido de granulação. Uma vez que estas células contêm enzimas degradadoras de colagénio, p. colagenase. Como no grupo tratado com colagenase (G2) os animais feridos na pele mostraram exatamente a degradação dos colágenos e inflamação nos intervalos de 24, 72, 7 e 14 dias. A avaliação clinica mostrou que os grupos tratados com monocerina (G3 e G4) exibiram a organização do tecido de granulação. Este tipo de organização também foi observado pela luz polarizada do microscópio na pele da ferida corada com vermelho de Picrosirius dos grupos tratados com monocerina. Foi possível observar que a distribuição dos colágenos foi mais densa e superior à dos demais grupos controle (Gl, G2 e G5) , o que evidenciou que o tecido de granulação estava iniciando em seu nível macro molecular.  [00344] TENGRUP et al. , (1988) mentioned that a persistent inflammatory reaction with accumulation of polymorphonuclear leukocytes may be partially responsible for the low collagen content in the granulation tissue. Since these cells contain collagen-degrading enzymes, e.g. collagenase. As in the group treated with collagenase (G2), the animals injured in the skin showed exactly the collagen degradation and inflammation in the intervals of 24, 72, 7 and 14 days. The clinical evaluation showed that the groups treated with monocerine (G3 and G4) exhibited the organization of the granulation tissue. This type of organization was also observed by the polarized light of the microscope on the skin of the wound stained with Picrosirius red from the groups treated with monocerine. It was possible to observe that the distribution of collagens was more dense and higher than that of the other control groups (Gl, G2 and G5), which showed that the granulation tissue was starting at its macro molecular level.
[00345] Além disso, os grupos G3 e G4 tratados com monocerina apresentaram reepitelização da pele após 24 horas de lesão, também observam que a atividade mais evidente na fase proliferativa da cicatrização, terminando durante a remodelação da matriz extracelular . Os queratinócitos da monocerina nos grupos tratados com 0,005% (G4) estavam presentes na borda das feridas secretam fatores de crescimento que estimulam a proliferação e migração dessas células para a cobertura da área lesada. [00346] O colágeno, o principal constituinte dérmico, pode ser analisado ou quantificado através de diferentes técnicas, como imuno-histoquímica, reação em cadeia da polimerase por microscopia confocal (PCR) e múltiplas manchas (COELHO et al . , 2018) . Neste estudo, os cortes histológicos corados com vermelho Picrosirius foram analisados sob microscopia de luz polarizada na figura 37. [00345] In addition, groups G3 and G4 treated with monocerine showed re-epithelialization of the skin after 24 hours of injury, they also note that the most evident activity in the proliferative phase of healing, ending during remodeling of the extracellular matrix. Monocerine keratinocytes in the groups treated with 0.005% (G4) were present at the edge of the wounds that secrete growth factors that stimulate the proliferation and migration of these cells to cover the injured area. [00346] Collagen, the main dermal constituent, can be analyzed or quantified through different techniques, such as immunohistochemistry, polymerase chain reaction by confocal microscopy (PCR) and multiple spots (COELHO et al., 2018). In this study, histological sections stained with Picrosirius red were analyzed under polarized light microscopy in figure 37.
[00347] O último estágio da cicatrização da ferida cutânea é a fase de remodelação na qual a matriz extracelular provisória é remodelada. Após 14 dias de tratamento com monocerina a 0,005% (Grupo G3), a área lesada é completamente reepitelizada e ocorre uma resposta contrátil mediada por miofibroblasto . Devido aos seus múltiplos locais de ligação com colágeno, os miofibroblastos se ligam às fibras de colágeno e contraem a redução da área da ferida, como mostrado nos resultados das Figuras 31, 32, 33, 34 e 35.  [00347] The last stage of skin wound healing is the remodeling phase in which the provisional extracellular matrix is remodeled. After 14 days of treatment with 0.005% monocerine (Group G3), the injured area is completely re-epithelized and a contractile response mediated by myofibroblast occurs. Due to their multiple collagen binding sites, myofibroblasts bind to collagen fibers and contract the reduction of the wound area, as shown in the results of Figures 31, 32, 33, 34 and 35.
[00348] Os dados mostram que o metabólito secundário monocerina a 0,0006% e 0,005% realizado em uma formulação em creme estimula a ativação da proliferação de fibroblastos das células epiteliais, o aumento do colágeno tipo 1 e a diminuição do infiltrado inflamatório.  [00348] The data show that the 0.0006% and 0.005% monocerine secondary metabolite carried out in a cream formulation stimulates the activation of fibroblast proliferation of epithelial cells, an increase in type 1 collagen and a decrease in inflammatory infiltrate.
[00349] Durante toda a avaliação histológica por três processos de coloração, por MEV e por microscopia polarizada, é possível concluir que a monocerina transportada em uma formulação em creme foi um agente eficaz para a remodelação da pele, conforme ilustrado nos resultados, principalmente nas Figuras 31, 32, 33, 34, 35 e 36.  [00349] Throughout the histological evaluation by three staining processes, by SEM and by polarized microscopy, it is possible to conclude that the monokine transported in a cream formulation was an effective agent for skin remodeling, as illustrated in the results, mainly in Figures 31, 32, 33, 34, 35 and 36.
[00350] Os resultados aqui apresentados indicam que a monocerina é um composto muito promissor com potencial para ser aplicado em formulações para cicatrização de feridas. [00351] Os dados apresentados foram obtidos a partir da primeira etapa dos ensaios in vivo realizados apenas com quatro animais por grupo. Em uma próxima etapa, os ensaios In Vivo serão repetidos com mais quatro animais por grupo para obter dados suficientes para uma análise estatística confiável . [00350] The results presented here indicate that monocerine is a very promising compound with the potential to be applied in formulations for wound healing. [00351] The data presented were obtained from the first stage of in vivo tests performed with only four animals per group. In a next step, the In Vivo assays will be repeated with four more animals per group to obtain sufficient data for reliable statistical analysis.
[00352] É evidente que a formulação portadora da monocerina em sua maior concentração de 0,005% foi mais eficaz no processo de cicatrização de feridas. No entanto, a faixa preferencial com formulação contendo monocerina é de 0,0006%, a 0,05%. Esses medicamentos de fontes naturais podem levar a melhores formas de terapia para pacientes com feridas agudas, crónicas e cirúrgicas na pele.  [00352] It is evident that the formulation containing monoerine in its highest concentration of 0.005% was more effective in the process of wound healing. However, the preferred range with formulation containing monocerine is 0.0006%, at 0.05%. These drugs from natural sources can lead to better forms of therapy for patients with acute, chronic and surgical wounds on the skin.
EFE I TO DO COMPOSTO ANULARINA I AVALIADO IN VIVO, EM UM MODELO DE FERIDA EXPERIMENTAL NOS CAMUNDONGOS .  EFFECT OF THE ANULARINE COMPOUND I ASSESSED IN VIVO, IN AN EXPERIMENTAL WOUND MODEL IN THE MICE.
Avaliação clínica da ferida  Clinical wound assessment
[00353] Pela análise clínica (Tabela 29), foi possível observar a presença de sangramento e coagulação nos animais de todos os grupos. No entanto, o mesmo evento esteve presente até o 5o dia para o grupo tratado com colagenase (G2) e até o 3o dia para os grupos tratados com anularina a 0, 005% (G3) e 0, 0006% (G4) . Por fim, o grupo que recebeu apenas o veículo (G5) apresentou atraso na cicatrização e recuperação até o 10° dia. Observou-se uma diferença proeminente entre os grupos controle e o grau de cicatrização no quinto e décimo dia nos aspectos clínicos dos grupos tratados com anularina G3 e G4, mostrados na Tabela. A partir da avaliação clínica, as feridas tratadas com anularina I mostraram contração, coagulação, granulação e bordas suturadas ou grampeadas da ferida se contraindo acentuadamente com o tempo e a aplicação de compostos de anularina nas feridas. Portanto, as feridas do grupo controle também foram observadas e mostraram uma cura eficaz no 13° e no 14° dia, que também foram observadas e fotografadas. [00353] By clinical analysis (Table 29), it was possible to observe the presence of bleeding and coagulation in animals of all groups. However, the same event attended by the 5th day of the group treated with collagenase (G2) and to the third day for the groups treated with anularina 0, 005% (G3) and 0. 0006% (G4) . Finally, the group that received only the vehicle (G5) showed delayed healing and recovery until the 10th day. There was a prominent difference between the control groups and the degree of healing on the fifth and tenth days in the clinical aspects of the groups treated with annularin G3 and G4, shown in the Table. From the clinical evaluation, the wounds treated with annularin I showed contraction, coagulation, granulation and sutured or stapled wound edges contracting sharply with time and the application of annularin in wounds. Therefore, the wounds of the control group were also observed and showed an effective healing on the 13th and 14th day, which were also observed and photographed.
Tabela 29. Intensidade da avaliação clinica para edema, hiperemia, sangramento, granulação e formação de crostas  Table 29. Intensity of clinical evaluation for edema, hyperemia, bleeding, granulation and crusting
Figure imgf000115_0001
Figure imgf000115_0001
= alta intensidade; ** = Média intensidade; * = baixa intensidade; Ausência representada como (-) .  = high intensity; ** = Medium intensity; * = low intensity; Absence represented as (-).
Tamanho da ferida  Wound size
[00354] O tamanho da ferida foi medido diariamente por paquímetro e os tamanhos estão representados, desde as primeiras 24 horas até o 14° dia, para todos os grupos de tratamento. O tamanho da ferida no sétimo dia dos grupos tratados com anularina a 0,005% (Grupo G3 ) e 0,0006% (Grupo G4) foi de 4,48 ± 0,18 e 4,39 ± 0,10, respectivamente, enquanto durante 10 dias foi de 1,81 ± 0,86 e 1,62 ± 0,30 mm, respectivamente. No 7o e 10° dia, o tamanho do creme comercial com colagenase (Grupo G2 ) foi maior, sendo 5,62 ± 0,39 e 2,6 ± 0,69 mm, respectivamente (Figura 42, Tabela 30) . O tamanho da ferida também é representado no gráfico 1 pelo período de 24 horas, 72 horas e 7o e 14° dia. [00354] The size of the wound was measured daily by a caliper and the sizes are represented, from the first 24 hours to the 14th day, for all treatment groups. The wound size on the seventh day of the groups treated with 0.005% (Group G3) and 0.0006% (Group G4) anularin was 4.48 ± 0.18 and 4.39 ± 0.10, respectively, while 10 days was 1.81 ± 0.86 and 1.62 ± 0.30 mm, respectively. In the 7th and 10 th day, the size of collagenase commercial cream (group G2) was higher, and 5.62 ± 0.39 and 2.6 ± 0.69 mm, respectively (Figure 42, Table 30). The size of the wound is also represented in the Figure 1 for a period of 24 hours, 72 hours and 7 and 14 days.
[00355] A ilustração gráfica da figura 42 mostra que os grupos tratados com anularina a 0,003% e 0,0005% curaram menos de 14 dias em comparação com os grupos não tratados. Observou-se uma rápida remodelação da pele dos grupos tratados de anularina I. No caso de MsFx pro X ray (Figura 43, Tabela 31), os resultados mostram que os grupos tratados com anularina e os grupos não tratados curaram em 14 dias. Por representação gráfica, também observou que após 24 h até o 7o dia, o tamanho da ferida para os grupos tratados com anularina I diminuiu melhor do que o grupo não tratado; Para o grupo tratado com anularina I, a pele estava mais organizada e compacta e nenhuma cicatriz foi observada. No 14° dia, as feridas dos grupos não tratados foram curadas, mas a cicatriz foi pequena. [00355] The graphic illustration of figure 42 shows that the groups treated with 0.003% and 0.0005% anularin healed less than 14 days compared to the untreated groups. Rapid skin remodeling of the treated anularin I groups was observed. In the case of MsFx pro X ray (Figure 43, Table 31), the results show that the groups treated with anularin and the untreated groups healed within 14 days. For graphic representation, also observed that after 24 h until day 7, the wound size for treated groups decreased anularina I better than the untreated group; For the group treated with annularin I, the skin was more organized and compact and no scarring was observed. On the 14th day, the wounds of the untreated groups were healed, but the scar was small.
[00356] Os resultados obtidos pela Radiografia In Vivo Image indicam que, para os grupos tratados com anularina a 0,005% (Grupo G3) e 0,0003% (Grupo 4), as feridas foram cicatrizadas de forma tão eficaz e precoce, quando comparadas a outros grupos. No caso do grupo tratado com colagenase (G2), as feridas não foram cicatrizadas adequadamente. Para o grupo não tratado (Gl), é possível observar que a ferida não foi cicatrizada e houve cicatriz na pele. No caso do grupo que recebeu apenas o veículo (G5) no 14° dia do tratamento, o tamanho das feridas do animal foi maior do que o grupo tratado com anularina. Este efeito é representado na Figura 44.  [00356] The results obtained by Radiography In Vivo Image indicate that, for the groups treated with 0.005% (Group G3) and 0.0003% (Group 4) anularin, the wounds were healed so effectively and early, when compared other groups. In the case of the group treated with collagenase (G2), the wounds were not healed properly. For the untreated group (Gl), it is possible to observe that the wound was not healed and there was a scar on the skin. In the case of the group that received only the vehicle (G5) on the 14th day of treatment, the size of the animal's wounds was larger than the group treated with annularin. This effect is represented in Figure 44.
Tabela 30. Medição do tamanho das feridas diariamente por paquímetro e representada por meio ± DP para quatro animais / grupo em milímetros (mm) .
Figure imgf000116_0001
Figure imgf000117_0001
Table 30. Measurement of wound size daily by caliper and represented by means ± SD for four animals / group in millimeters (mm).
Figure imgf000116_0001
Figure imgf000117_0001
Tabela 31. Medição do tamanho das feridas por imagem radiológica ín vivo (MS Fx pro) em milímetros (mm) representada pelos meios ± DP para 2 animais / grupo  Table 31. Measurement of wound size by in vivo radiological image (MS Fx pro) in millimeters (mm) represented by means ± SD for 2 animals / group
Figure imgf000117_0002
Figure imgf000118_0001
Figure imgf000117_0002
Figure imgf000118_0001
ANÁLISE DE HEMATOLOGIA  HEMATOLOGY ANALYSIS
[00357] As amostras de sangue coletadas dos animais em diferentes intervalos de tempo nas 24 h, 72 h, 7 e 14 dias foram centrifugadas por (Eppendorf ® Centrifuge 5424 R Mini Spin Plus) a 2.000 rpm por 10 minutos para separar o soro e o plasma da amostra de sangue, analisados hematologicamente usando o analisador de hematologia IDEXX ProCyte Dx®.  [00357] Blood samples collected from the animals at different time intervals at 24 h, 72 h, 7 and 14 days were centrifuged by (Eppendorf ® Centrifuge 5424 R Mini Spin Plus) at 2,000 rpm for 10 minutes to separate the serum and the plasma of the blood sample, analyzed hematologically using the IDEXX ProCyte Dx® hematology analyzer.
[00358] Foram analisadas as três principais categorias de células imunes, como glóbulos vermelhos (glóbulos vermelhos), glóbulos brancos (glóbulos brancos) e plaquetas. Conforme mostrado nas Figuras 45 e 46 para a anularina I a 0,003% e 0,0005%, apresentaram os níveis mais altos de hemácias em 24 h, em comparação com outros grupos de controle Gl, G2-colagenase e veículo G5. Mais tarde, após 7 dias, o grupo tratado com anularina I principalmente com concentração de 0,003%, com maior número de leucócitos e PLT até o 14° dia, em comparação com os grupos não tratados. Os dados permitem concluir que o grupo tratado com anularina I também prolifera células imunes, o que leva a uma melhor remodelação da pele.  [00358] The three main categories of immune cells were analyzed, such as red blood cells (red blood cells), white blood cells (white blood cells) and platelets. As shown in Figures 45 and 46 for annularin I at 0.003% and 0.0005%, they had the highest levels of red blood cells within 24 h compared to other control groups Gl, G2-collagenase and vehicle G5. Later, after 7 days, the group treated with annularin I mainly with a concentration of 0.003%, with a greater number of leukocytes and PLT until the 14th day, compared to the untreated groups. The data allow us to conclude that the group treated with annularin I also proliferates immune cells, which leads to better skin remodeling.
ANÁLISE MORFOLÓGICA DA FERIDA POR MICROSCOPIA ELE TRÓNICA DE VARREDURA  MORPHOLOGICAL ANALYSIS OF THE WOUND BY MICROSCOPY HE TRONIC SCAN
[00359] Imagens de microscopia eletrónica de varredura (figura 47) mostram que camundongos do grupo não tratado (Gl) e do grupo colagenase (G2) demonstraram integridade da matriz extracelular , mas uma baixa deposição de fibras de colágeno dimensionalmente imaturas quando comparadas com o grupo tratado com anularina a 0,003% e 0,0005 % Os animais do grupo que recebeu apenas o veículo (G5) apresentaram processo inflamatório e presença de coagulação no 7o dia. Além disso, o tecido de granulação parecia frágil com anularina a 0,005% de pomada, possivelmente devido ao aumento da permeabilidade vascular. Imagens ultra-estruturais da derme capturada por MEV mostram os tecidos de granulação dos animais do grupo tratado com anularina a 0,0005% e 0,003% no 7o dia também com presença de numerosas células mortas e células sanguíneas abundantes; também é possível observar fibronectina e glóbulos vermelhos em tecidos infiltrados por capilares. Há evidências consistentes de que mecanismos complexos regulam a correlação entre inflamação e coagulação. As feridas cirúrgicas tratadas com anularina em ambas as concentrações exibiram formações de coágulos e muitas fibronectinas foram observadas após 24 horas e 14 dias. [00359] Scanning electron microscopy images (figure 47) show that mice from the untreated group (Gl) and the collagenase group (G2) demonstrated integrity of the extracellular matrix, but a low deposition of dimensionally immature collagen fibers when compared with group treated with 0.003% and 0.0005% anularin The animals in the group that received only the vehicle (G5) showed inflammation and coagulation in the presence of 7 days. In addition, the granulation tissue appeared fragile with 0.005% annularin ointment, possibly due to increased vascular permeability. Ultrastructural images captured from the dermis by SEM showing the granulation tissue of animals treated with anularina 0.0005% and 0.003% on day 7 also the presence of numerous dead cells and abundant blood cells; it is also possible to observe fibronectin and red blood cells in tissues infiltrated by capillaries. There is consistent evidence that complex mechanisms regulate the correlation between inflammation and coagulation. Surgical wounds treated with anularin in both concentrations exhibited clot formation and many fibronectins were observed after 24 hours and 14 days.
Avaliação Histológica  Histological Evaluation
[00360] Pela avaliação histológica dos retalhos cutâneos pela coloração da Hematoxilina Eosina (HE) , foi possível observar regressão das lesões com melhor epitelização nos grupos não tratado (Gl) e tratado com colagenase (G2), mas esse processo foi mais eficaz, mostrando melhor re -organização da derme em ambos os grupos tratados com anularina I até o 14° dia.  [00360] By histological evaluation of skin flaps by staining Hematoxylin Eosin (HE), it was possible to observe regression of lesions with better epithelialization in the untreated (Gl) and treated with collagenase (G2) groups, but this process was more effective, showing better reorganization of the dermis in both groups treated with annularin I until the 14th day.
[00361] A indicação clara dos tecidos histológicos da ferida nas experiências de 24 e 72 h, 7 e 14 h dias foi mostrada nas Figuras 47 a 51. Para o grupo não tratado (Gl), é possível observar: (H&E) - tecido cicatricial visível grande número de fibroblastos células e vasos sanguíneos (seta amarela); (PR) redução de colágeno necrose densa e organizada (seta laranja); (MT) grande número de células inflamatórias cicatrizes visíveis (setas vermelhas) estavam presentes. [00361] The clear indication of the histological tissues of the wound in the experiments of 24 and 72 h, 7 and 14 h days was shown in Figures 47 to 51. For the untreated group (Gl), it is possible to observe: (H&E) - tissue visible scarring large number of fibroblasts cells and blood vessels (yellow arrow); (PR) reduction of dense and organized collagen necrosis (orange arrow); (MT) large number of visible scar inflammatory cells (red arrows) were present.
[00362] No caso de tratamento com colagenase (G2) : cicatrizes de inflamação (H&E), inflamação dos tecidos adiposos das células necróticas (PR) ; (MT) grande número de células inflamatórias resistiu claramente às células de resposta imune na cicatrização de feridas. Para o tratamento com anularina I em 0,003% e 0,0005%, as evidências são: (H&E) - boa distribuição de células epiteliais e tecido cutâneo organizado; (PR) colágeno e camada epitelial densa; (MT) grande formação de fibroblastos , células inflamatórias reduzidas foram marcadas nas figuras s de 24 h e 72 h, intervalos de tempo de 7 e 14 dias tecidos manchados.  [00362] In the case of treatment with collagenase (G2): scars of inflammation (H&E), inflammation of the adipose tissues of necrotic cells (PR); (MT) a large number of inflammatory cells clearly resisted immune response cells in wound healing. For treatment with annularin I in 0.003% and 0.0005%, the evidence is: (H&E) - good distribution of epithelial cells and organized skin tissue; (PR) collagen and dense epithelial layer; (MT) large fibroblast formation, reduced inflammatory cells were marked in the figures at 24 h and 72 h, time intervals of 7 and 14 days stained tissues.
[00363] A distribuição do poço de colágenos nos grupos tratados com anularina e as forças mecânicas dos colágenos são mais fortes e organizadas em comparação aos grupos de controle. Os grupos tratados com anularina mostraram melhores camadas celulares epiteliais e há alta distribuição de colágeno (PR) ; um grande número de células de fibroblastos indica sinais de cura e os colágenos também estavam apoiando a regeneração da pele observada e marcada também em (MT) ; células de fibroblastos abundantes e a camada de epiderme foram mais organizadas em grupos tratados com anularina .  [00363] The distribution of the collagen well in the groups treated with annularin and the mechanical forces of the collagens are stronger and more organized compared to the control groups. The groups treated with anularin showed better epithelial cell layers and there is a high distribution of collagen (PR); a large number of fibroblast cells indicate signs of healing and collagens were also supporting the skin regeneration observed and also marked in (MT); abundant fibroblast cells and the epidermis layer were more organized in groups treated with anularin.
[00364] Portanto, mais fraca distribuição e intensidade de colágenos no grupo tratado com veiculo também em (H&E) - colorindo grande número de células inflamatórias; (PR) privação de colágenos; (MT) grande número de células adiposas inflamatórias foi observado até o 14° dia.  [00364] Therefore, weaker distribution and intensity of collagens in the group treated with vehicle also in (H&E) - staining a large number of inflammatory cells; (PR) collagen deprivation; (MT) a large number of inflammatory fat cells were observed until the 14th day.
ANÁLISE DE TECIDOS DE VERMELHO DE PICROSIRIUS USANDO MICROSCÓPIO POLARIZADO - DADOS PARA MONOCERINA E ANNULARINA ANALYSIS OF PICROSIRIUS RED TISSUES USING POLARIZED MICROSCOPE - DATA FOR MONOCERINE AND ANNULARIN
I [00365] O corante vermelho de Picrosirius tem a capacidade de aumentar a birrefringência natural do colágeno quando exposto à luz polarizada. I [00365] Picrosirius red dye has the ability to increase the natural birefringence of collagen when exposed to polarized light.
[00366] A intensidade da formação de colágeno induzida pelos grupos tratados com monocerina e anularina I foi maior ao longo dos 14 dias de tratamento, em comparação aos controles (Figuras 53 e 54) . Para ambos os compostos, a maior concentração mostrou mais intensidade de colágeno em comparação com as menores concentrações. Após os 7 dias, os grupos tratados com colagenase mostraram que a intensidade dos colágenos estava degradada e os resultados calculados mostraram a menor intensidade dos colágenos em 7 e 14 dias.  [00366] The intensity of collagen formation induced by the groups treated with monocerine and annularin I was higher over the 14 days of treatment, compared to controls (Figures 53 and 54). For both compounds, the highest concentration showed more collagen intensity compared to the lowest concentrations. After 7 days, the groups treated with collagenase showed that the collagen intensity was degraded and the calculated results showed the lowest collagen intensity in 7 and 14 days.
[00367] A avaliação por coloração com vermelho de Picrosirius permitiu reconhecer a densidade total de colágeno devido à presença de fibras de colágeno (amarelo avermelhado na figura 55) . A área ocupada pelas fibras de colágeno foi quantificada usando o Image J e o software Graph Pad Prism 5.1, apresentado nas Figuras 53 e 54. Os dados foram obtidos para quatro animais de cada grupo e representados pela porcentagem de média ± DP . O valor final foi representado comparando todos os grupos com os grupos controles (controle, colagenase e veiculo) . A porcentagem de colágeno foi mais intensa nos grupos tratados com monocerina e anularina I em todas as concentrações durante todo o tempo das experiências. A maior porcentagem de colágeno foi observada no experimento de 14 dias e mostrada no gráfico 6 e 7.  [00367] The evaluation by staining with Picrosirius red allowed to recognize the total collagen density due to the presence of collagen fibers (reddish yellow in figure 55). The area occupied by the collagen fibers was quantified using Image J and the Graph Pad Prism 5.1 software, shown in Figures 53 and 54. Data were obtained for four animals in each group and represented by the percentage of mean ± SD. The final value was represented by comparing all groups with the control groups (control, collagenase and vehicle). The percentage of collagen was more intense in the groups treated with monocerine and annularin I in all concentrations during the entire time of the experiments. The highest percentage of collagen was observed in the 14-day experiment and shown in graphs 6 and 7.
[00368] O colágeno tipo I mostraria uma cor amarelo-vermelho, enquanto o tipo III seria verde, como mostrado na Figura 12. Às 24 e 72 h, os grupos tratados com monocerina e anularina I mostraram um arranjo denso e compacto de colágeno em comparação aos grupos controle (controle , colagenase e veiculo) . Nas 24 e 72 horas, o grupo controle não tratado mostrou uma expressão significativamente baixa de colágenos. [00368] Type I collagen would show a yellow-red color, while type III would be green, as shown in Figure 12. At 24 and 72 h, the groups treated with monocerine and annularin I showed a dense and collagen compact compared to control groups (control, collagenase and vehicle). At 24 and 72 hours, the untreated control group showed significantly low collagen expression.
[00369] No tratamento por 7 dias, houve uma clara diferença observada entre os grupos controle e os grupos tratados com monocerina e anularina I, que mostraram uma quantidade mais densa e compacta de colágenos. A população de fibras de colágeno exibiu cores de polarização no vermelho-laranja, enquanto para os controles as fibras de colágeno eram esparsas e imaturas. O controle do grupo não tratado exibiu intensa birrefringência verde - amarela, sugerindo que o conteúdo de colágeno foi reduzido e suas fibras estavam muito frouxamente compactadas.  [00369] In the treatment for 7 days, there was a clear difference observed between the control groups and the groups treated with monocerine and annularin I, which showed a more dense and compact amount of collagens. The population of collagen fibers exhibited polarization colors in red-orange, while for controls the collagen fibers were sparse and immature. The control of the untreated group showed intense green - yellow birefringence, suggesting that the collagen content was reduced and its fibers were very loosely compacted.
[00370] No entanto, no 14° dia, os grupos tratados com monocerina e anularina I apresentaram mais fibras amarelas e vermelhas, o que foi visto possivelmente indicando a presença de colágeno tipo I. As fibras verdes (típicas do colágeno tipo III) foram mais frequentemente localizadas na derme superior e na epiderme nos grupos tratados com anularina. Os grupos controle (controle, colagenase e veículo) apresentaram fibras grossas vermelhas e amarelas (típicas do colágeno tipo I) localizadas na derme profunda inferior. No geral, os grupos tratados com monocerina e anularina I mostraram arranjo de fibras colágenas visíveis e organizadas dos grupos tratados com anularina .  [00370] However, on the 14th day, the groups treated with monocerine and annularin I had more yellow and red fibers, which was seen possibly indicating the presence of type I collagen. The green fibers (typical of type III collagen) were most often located in the upper dermis and epidermis in the groups treated with anularin. The control groups (control, collagenase and vehicle) had thick red and yellow fibers (typical of type I collagen) located in the lower deep dermis. In general, the groups treated with monocerine and annularin I showed an arrangement of visible and organized collagen fibers from the groups treated with annularin.
COMPOSIÇÃO FARMACÊUTICA  PHARMACEUTICAL COMPOSITION
[00371] Em outro aspecto da presente invenção, proporcionam-se aqui composições farmacêuticas que compreendem o composto ( 2S , 3aR, 9bR) - 6-hidroxi-7 , 8-dimetoxi- 2-propil-2, 3, 3a, 9b-tetra-hidro-5H-furo [3, 2-c] isocromen-5- ona (monocerina) , e pelo menos, um veiculo farmaceuticamente aceitável . [00371] In another aspect of the present invention, pharmaceutical compositions are provided herein comprising the compound (2S, 3aR, 9bR) - 6-hydroxy-7, 8-dimethoxy- 2-propyl-2, 3, 3a, 9b-tetrahydro-5H-bore [3, 2-c] isochromen-5-one (monocerin), and at least one pharmaceutically acceptable carrier.
[00372] Em outro aspecto, proporcionam-se aqui composições farmacêuticas que compreendem o composto 4- metoxi-5-metil- 6-butil-2H-piran-2-one (anularina I), e pelo menos, um veiculo farmaceuticamente aceitável.  [00372] In another aspect, pharmaceutical compositions are provided herein comprising the compound 4-methoxy-5-methyl-6-butyl-2H-pyran-2-one (anularin I), and at least one pharmaceutically acceptable carrier.
[00373] O termo "veiculo farmaceuticamente aceitável" inclui, mas não está limitado a qualquer veiculo que não interfira com a eficácia da atividade biológica dos dos princípios ativos e que não seja tóxico para o paciente a quem é administrado. Exemplos de veículos farmacêuticos adequados são bem conhecidos no estado da técnica e incluem soluções salinas tamponadas com fosfato, água, emulsões, tais como emulsões óleo / água, vários tipos de agentes molhantes, soluções estéreis, etc. Tais veículos podem ser formulados por métodos convencionais e podem ser administrados ao indivíduo na dose e em esquemas terapêuticos mais adequados a cada caso. De preferência, as composições são estéreis, mas também podem ser preparadas em condições assépticas. Estas composições podem também conter adjuvantes, conservantes, agentes emulsionantes e agentes dispersantes . As formulações farmacêuticas podem ser para uso humano, e/ou veterinário/animal. As formulações podem ser preparadas na forma de sistemas particulados como por exemplo, micropartícuias , nanopartícuias , microesferas, nanoesferas, lipossomas, em complexos carreadores como em ciclodextrinas (alfa, beta e gama) ; ainda na forma de liberação controlada. As formulações podem ser preparadas pela associação dos compostos monocerina e anularina I nas proporções e doses/concentrações mais adequadas. As formulações podem ser na forma de cremes, pomadas, géis, adesivos, sprays, incluindo ainda formulações de base nanotecnológica, ou ainda a associação com outros princípios ativos de mesmo efeito biológico, ou ainda associação com antimicrobianos . [00373] The term "pharmaceutically acceptable carrier" includes, but is not limited to, any carrier that does not interfere with the effectiveness of the biological activity of the active ingredients and is non-toxic to the patient to whom it is administered. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil / water emulsions, various types of wetting agents, sterile solutions, etc. Such vehicles can be formulated by conventional methods and can be administered to the individual in the dose and in therapeutic regimens most appropriate to each case. Preferably, the compositions are sterile, but can also be prepared under aseptic conditions. These compositions can also contain adjuvants, preservatives, emulsifying agents and dispersing agents. Pharmaceutical formulations can be for human, and / or veterinary / animal use. The formulations can be prepared in the form of particulate systems such as, for example, microparticles, nanoparticles, microspheres, nanospheres, liposomes, in carrier complexes as in cyclodextrins (alpha, beta and gamma); still in the form of controlled release. The formulations can be prepared by the combination of the monocerine and annularin I compounds in the most appropriate proportions and doses / concentrations. At formulations can be in the form of creams, ointments, gels, adhesives, sprays, including nanotechnology-based formulations, or even the association with other active principles with the same biological effect, or even association with antimicrobials.
[00374] A administração das composições da presente invenção pode ser efetuada por diferentes maneiras, por exemplo por via intravenosa, intraperitoneal , subcutânea, intramuscular, tópica ou intradérmica, particularmente, as principais são cutânea, subcutânea, tópica, intradérmica, retal, intraocular, nasal e auricular.  [00374] The administration of the compositions of the present invention can be carried out in different ways, for example by intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal, particularly, the main ones are cutaneous, subcutaneous, topical, intradermal, rectal, intraocular, nasal and auricular.
[00375] A via de administração, é claro, depende do tipo de tratamento e do tipo de composto contido na composição farmacêutica. O regime de dosagem será determinado pelo médico e de outros fatores clínicos. Como é bem conhecido nas técnicas médicas, as dosagens para qualquer paciente dependem de muitos fatores, incluindo o peso do paciente, a área de superfície corporal, a idade, o sexo, a composição farmacêutica carreando o composto em particular a ser administrado, o tempo e via de administração, o tipo de terapia, estado geral de saúde e outros fatores a serem considerados e administrados concomitantemente .  [00375] The route of administration, of course, depends on the type of treatment and the type of compound contained in the pharmaceutical composition. The dosage regimen will be determined by the doctor and other clinical factors. As is well known in medical techniques, dosages for any patient depend on many factors, including the patient's weight, body surface area, age, sex, pharmaceutical composition carrying the particular compound to be administered, time and route of administration, the type of therapy, general health status and other factors to be considered and administered concurrently.
USO DAS COMPOSIÇÕES FARMACÊUTICAS DA PRESENTE INVENÇÃO .  USE OF THE PHARMACEUTICAL COMPOSITIONS OF THE PRESENT INVENTION.
[00376] A presente invenção se refere ao uso de metabólito secundário produzido pelo fungo Exserohilum rostratum na regeneração celular. Particularmente, a presente invenção se refere ao uso dos metabólitos secundários monocerina e anularina I obtidos a partir da cultura do fungo Exserohilum rostratum para preparação de medicamentos para regeneração celular, preferencialmente, regeneração celular em células endoteliais HUVEC e fibroblastos normais FN1. [00376] The present invention relates to the use of secondary metabolite produced by the fungus Exserohilum rostratum in cell regeneration. In particular, the present invention relates to the use of the secondary metabolites monocerine and annularin I obtained from the culture of the fungus Exserohilum rostratum for the preparation of drugs for cell regeneration, preferably cell regeneration in HUVEC endothelial cells and normal FN1 fibroblasts.
[00377] Particularmente, as composições farmacêuticas da presente invenção podem ser utilizadas como agente indutor de proliferação celular, preferencialmente, no reparo de tecidos .  [00377] In particular, the pharmaceutical compositions of the present invention can be used as an agent that induces cell proliferation, preferably in tissue repair.
[00378] Assim, as concretizações apresentadas na presente invenção não limitam a totalidade das possibilidades, será entendido que várias omissões, substituições e alterações podem ser feitas por um técnico versado no assunto, sem se afastar do espirito e escopo da presente invenção.  [00378] Thus, the embodiments presented in the present invention do not limit the totality of possibilities, it will be understood that various omissions, substitutions and changes can be made by a technician versed in the subject, without departing from the spirit and scope of the present invention.
[00379] É expressamente previsto que todas as combinações dos elementos que desempenham a mesma função substancialmente da mesma forma para alcançar os mesmos resultados estão dentro do escopo da invenção. Substituições de elementos de uma concretização descrita para outra são também totalmente pretendidos e contemplados.  [00379] It is expressly provided that all combinations of elements that perform the same function in substantially the same way to achieve the same results are within the scope of the invention. Substitutions of elements from one embodiment described to another are also fully intended and contemplated.
[00380] Também é preciso entender que os desenhos não estão necessariamente em escala, mas que eles são apenas de natureza conceituai. A intenção é, portanto, ser limitada, tal como indicado pelo escopo das reivindicações anexas. REFERÊNCIAS  [00380] It is also necessary to understand that the drawings are not necessarily to scale, but that they are only of a conceptual nature. The intention is therefore to be limited, as indicated by the scope of the appended claims. REFERENCES
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Claims

REIVINDICAÇÕES
1. Composição farmacêutica para regeneração celular caracterizada pelo fato de que compreende o composto (2S, 3aR, 9bR) - 6-hidroxi-7 , 8-dimetoxi-2-propil-2 , 3, 3a, 9b- tetra-hidro-5H-furo [3, 2-c] isocromen-5-ona (monocerina) e um veiculo farmaceuticamente aceitável.  1. Pharmaceutical composition for cell regeneration characterized by the fact that it comprises the compound (2S, 3aR, 9bR) - 6-hydroxy-7, 8-dimethoxy-2-propyl-2, 3, 3a, 9b-tetrahydro-5H -puncture [3, 2-c] isochromen-5-one (monocerine) and a pharmaceutically acceptable carrier.
2. Composição farmacêutica, de acordo com a reivindicação 1, caracterizada pelo fato de que a monocerina está na composição em uma concentração entre 0,0006% a 0,005  2. Pharmaceutical composition, according to claim 1, characterized by the fact that the monocerine is in the composition in a concentration between 0.0006% to 0.005
3. Composição farmacêutica para regeneração celular caracterizada pelo fato de que compreende o composto 4- metoxi-5-metil- 6-butil-2H-piran-2-one (anularina I) e um veiculo farmaceuticamente aceitável. 3. Pharmaceutical composition for cell regeneration characterized by the fact that it comprises the compound 4-methoxy-5-methyl-6-butyl-2H-pyran-2-one (annularin I) and a pharmaceutically acceptable carrier.
4. Composição farmacêutica, de acordo com a reivindicação 3, caracterizada pelo fato de que a anularina I está na composição em uma concentração entre a 0,0006% a 0, 005% .  4. Pharmaceutical composition, according to claim 3, characterized by the fact that annularin I is in the composition in a concentration between 0.0006% to 0.005%.
5. Composição farmacêutica para regeneração celular caracterizada pelo fato de que compreende a associação dos compostos ( 2S , 3aR, 9bR) - 6-hidroxi-7 , 8-dimetoxi-2-propil- 5. Pharmaceutical composition for cell regeneration characterized by the fact that it comprises the combination of compounds (2S, 3aR, 9bR) - 6-hydroxy-7, 8-dimethoxy-2-propyl-
2, 3, 3a, 9b-tetra-hidro-5H-furo [3, 2-c] isocromen-5-ona2, 3, 3a, 9b-tetrahydro-5H-hole [3, 2-c] isochromen-5-one
(monocerina) e 4-metoxi-5-metil- 6-butil-2H-piran-2-one (anularina I) e um veiculo farmaceuticamente aceitável. (monocerine) and 4-methoxy-5-methyl-6-butyl-2H-pyran-2-one (annularin I) and a pharmaceutically acceptable carrier.
6. Processo de produção de metabólitos secundários a partir da cultura do fungo Exserohilum rostratum caracterizado pelo fato de que compreende as seguintes etapas :  6. Process of production of secondary metabolites from the culture of the fungus Exserohilum rostratum characterized by the fact that it comprises the following steps:
a) cultivar o fungo E. rostratum em um meio de cultura contendo uma fonte de carbono por 15 dias entre 25°C a 45° C, sem agitação, ou com agitação atingindo até 400 rpm; b) submeter o caldo da cultura a congelamento, entre zero a -20°C por pelo menos 48-72 horas e em seguida proceder nova filtração em membrana de vidro, para remoção de resíduos de cultura como esporos e/ou micelas fúngicas para posterior extração em fase sólida (Solid Phase Extraction - SPE) com cartucho C18 (Spe-ed SPE Cartridges - Octadecyl C18/18%), c) remover os resíduos do meio de cultura e impurezas com metanol a 20% no cartucho. a) cultivate the fungus E. rostratum in a culture medium containing a carbon source for 15 days between 25 ° C to 45 ° C, without agitation, or with agitation reaching up to 400 rpm; b) freeze the culture broth, between zero at -20 ° C for at least 48-72 hours and then proceed with new filtration on a glass membrane, to remove culture residues such as spores and / or fungal micelles for later solid phase extraction (Solid Phase Extraction - SPE) with C18 cartridge (Spe-ed SPE Cartridges - Octadecyl C18 / 18%), c) remove residues from the culture medium and impurities with 20% methanol in the cartridge.
d) extrair o material retido com um solvente orgânico e) obter a fração SPE100.  d) extract the retained material with an organic solvent e) obtain the SPE100 fraction.
7. Processo de produção de metabólitos secundários, de acordo com a reivindicação 6, caracterizado pelo fato de que na etapa a, o meio contendo uma fonte de carbono é, preferencialmente, um meio batata dextrose (BD) .  7. Secondary metabolite production process, according to claim 6, characterized by the fact that in step a, the medium containing a carbon source is preferably a potato dextrose (BD) medium.
8. Processo de produção de metabólitos secundários, de acordo com a reivindicação 6, caracterizado pelo fato de que na etapa b, a extração, opcionalmente pode ser também por extração líquido-líquido (ELL) usando solventes como acetato de etila, hexano, diclorometano, ou a extração pode ser feita também em fase sólida usando discos de Octadecil C18/18%.  8. Secondary metabolite production process, according to claim 6, characterized by the fact that in step b, the extraction optionally can also be by liquid-liquid extraction (ELL) using solvents such as ethyl acetate, hexane, dichloromethane , or extraction can also be done in solid phase using Octadecyl C18 / 18% discs.
9. Processo de produção de metabólitos secundários, de acordo com a reivindicação 6, caracterizado pelo fato de que na etapa e, o solvente orgânico pertence ao grupo selecionado entre metanol, acetonitrila, acetato de etila, preferencialmente, metanol a 100%.  9. Secondary metabolite production process, according to claim 6, characterized by the fact that in step e, the organic solvent belongs to the group selected from methanol, acetonitrile, ethyl acetate, preferably 100% methanol.
10. Processo de produção de metabólitos secundários, de acordo com a reivindicação 6, caracterizado pelo fato de que compreende ainda submeter a fração SPE100 a uma etapa de fracionamento .  10. Secondary metabolite production process, according to claim 6, characterized by the fact that it also comprises subjecting the SPE100 fraction to a fractionation step.
11. Processo de produção de metabólitos secundários, de acordo com a reivindicação 10, caracterizado pelo fato de que na etapa de fracionamento a fração SPE100 é submetida à cromatografia liquida de alta eficiência (CLAE) em coluna C18, C8 ou fenil (250 x 4,6 mm; 250 x 7,75 mm; ou 250 x 10 mm) e sistema de eluição isocrática variando de 44 a 60% com fluxo de 1,0 a 4 mL/min (solução A:H2<D/TFA, 99,9/0,1%; solução B: MeOH/PRO/TFA, 90:9,9:0,1%), e duas frações principais (F1 e F2 ) foram obtidas e repurifiçadas usando coluna C18, C8 ou fenil (250 x 4,6 mm; 250 x 7,75 mm; ou 250 x 10 mm) com eluição isocrática de 44 a 60%, ou sistema de eluição com gradiente de 0 a 50-56 % de B seguido de isocrático de 50-56 % B em 25-30 minutos e gradiente de 50- 56% B a 0 % de B em 10 minutos. 11. Secondary metabolite production process, according to claim 10, characterized by the fact that that in the fractionation stage the SPE100 fraction is subjected to high performance liquid chromatography (HPLC) in a C18, C8 or phenyl column (250 x 4.6 mm; 250 x 7.75 mm; or 250 x 10 mm) and isocratic elution ranging from 44 to 60% with a flow rate of 1.0 to 4 mL / min (solution A: H2 <D / TFA, 99.9 / 0.1%; solution B: MeOH / PRO / TFA, 90: 9 , 9: 0.1%), and two main fractions (F1 and F2) were obtained and refurified using column C18, C8 or phenyl (250 x 4.6 mm; 250 x 7.75 mm; or 250 x 10 mm) with 44 to 60% isocratic elution, or elution system with 0 to 50-56% B gradient followed by 50-56% B isocratic in 25-30 minutes and 50- 56% B to 0% gradient B in 10 minutes.
12. Processo de produção de metabólitos secundários, de acordo com a reivindicação 11, caracterizado pelo fato de que frações F1 e F2 obtidas são os compostos monocerina - (2S, 3aR, 9bR) - 6-hidroxi-7 , 8-dimetoxi-2-propil-2 , 3, 3a, 9b- tetra-hidro-5H-furo [3, 2-c] isocromen-5-ona, e anularina I - 4-metoxi-5-metil-6-butil-2H-piran-2-one, respectivamente .  12. Secondary metabolite production process, according to claim 11, characterized by the fact that fractions F1 and F2 obtained are the monocerine compounds - (2S, 3aR, 9bR) - 6-hydroxy-7, 8-dimethoxy-2 -propyl-2, 3, 3a, 9b-tetrahydro-5H-bore [3, 2-c] isochromen-5-one, and annularin I - 4-methoxy-5-methyl-6-butyl-2H-pyran -2-one, respectively.
13. Uso dos metabólitos secundários monocerina e anularina I produzidos a partir da cultura do fungo Exserohilum rostratum conforme definido pelas reivindicações 6 a 12 caracterizado pelo fato de que é para preparação de um medicamento para regeneração celular.  13. Use of the secondary metabolites monocerine and annularin I produced from the culture of the fungus Exserohilum rostratum as defined by claims 6 to 12 characterized by the fact that it is for the preparation of a drug for cell regeneration.
14. Uso dos metabólitos secundários monocerina e anularina I, de acordo com a reivindicação 13, caracterizado pelo fato de que é como agente indutor de proliferação celular, preferencialmente, no reparo de tecidos.  14. Use of secondary metabolites monocerine and annularin I, according to claim 13, characterized by the fact that it is as an agent that induces cell proliferation, preferably in tissue repair.
15. Uso dos metabólitos secundários monocerina e anularina I, de acordo com a reivindicação 14, caracterizado pelo fato de que o reparo de tecidos é, preferencialmente, na cicatrização de feridas. 15. Use of the secondary metabolites monocerin and annularin I, according to claim 14, characterized by the fact that tissue repair is preferably in wound healing.
16. Uso dos metabólitos secundários monocerina e anularina I produzidos a partir da cultura do fungo Exserohilum rostratum conforme definido pelas reivindicações 6 a 13 caracterizado pelo fato de que é para preparação de um medicamento para ação antifúngica contra Cryptococcus neoformans, Tríchophytum rubrum, Candida albicans e Aspergillus fumígatus com concentrações inibitórias mínimas de 31,25 a 500 pg/mL. 16. Use of secondary metabolites monocerin and annularin I produced from the culture of the fungus Exserohilum rostratum as defined by claims 6 to 13 characterized by the fact that it is for the preparation of a drug for antifungal action against Cryptococcus neoformans, Tríchophytum rubrum, Candida albicans and Aspergillus fumígatus with minimum inhibitory concentrations of 31.25 to 500 pg / mL.
PCT/BR2019/050360 2018-08-31 2019-09-02 Pharmaceutical composition, process for obtaining and using secondary metabolites produced by the fungus exserohilum rostratum in cell regeneration WO2020041851A2 (en)

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