WO2020041851A2 - Composition pharmaceutique, procédé d'obtention et utilisation de métabolites secondaires produits par le champignon exserohilum rostratum dans la régénération cellulaire - Google Patents

Composition pharmaceutique, procédé d'obtention et utilisation de métabolites secondaires produits par le champignon exserohilum rostratum dans la régénération cellulaire 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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WO2020041851A3 (fr
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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/pt
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Publication of WO2020041851A2 publication Critical patent/WO2020041851A2/fr
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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

La présente invention concerne une composition pharmaceutique comprenant des métabolites secondaires produits par le champignon Exserohilum rostratum dans la régénération cellulaire. Plus particulièrement, la présente invention concerne une composition pharmaceutique comprenant des métabolites secondaires monocérine et anularine I produits à partir de la culture du champignon Exserohilum rostratum pour la régénération cellulaire. Selon un autre aspect, la présente invention concerne le procédé d'obtention et d'utilisation desdits métabolites secondaires produits par le champignon Exserohilum rostratum dans la régénération cellulaire.
PCT/BR2019/050360 2018-08-31 2019-09-02 Composition pharmaceutique, procédé d'obtention et utilisation de métabolites secondaires produits par le champignon exserohilum rostratum dans la régénération cellulaire WO2020041851A2 (fr)

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BR102018067418-8A BR102018067418A2 (pt) 2018-08-31 2018-08-31 Composição farmacêutica, processo de obtenção e uso de metabólitos secundários produzido pelo fungo exserohilum rostratum na regeneração celular e tecidual
BRBR1020180674188 2018-08-31
BR102019018195-8A BR102019018195A2 (pt) 2018-08-31 2019-08-30 Composição farmacêutica, processo de obtenção e uso de metabólitos secundários produzido pelo fungo exserohilum rostratum na regeneração celular
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