WO2016161534A1 - Composición veterinaria de extracto de algas marinas y andrographis sp., útil para tratar infección en peces - Google Patents
Composición veterinaria de extracto de algas marinas y andrographis sp., útil para tratar infección en peces Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/53—Lamiaceae or Labiatae (Mint family), e.g. thyme, rosemary or lavender
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/163—Sugars; Polysaccharides
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/80—Feeding-stuffs specially adapted for particular animals for aquatic animals, e.g. fish, crustaceans or molluscs
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/02—Algae
- A61K36/03—Phaeophycota or phaeophyta (brown algae), e.g. Fucus
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/19—Acanthaceae (Acanthus family)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
Definitions
- the present invention relates to a veterinary composition
- a veterinary composition comprising an extract of seaweeds with a content of at least 5% of fucoidians and an extract of Andrographis sp plant with a content of at least 5% of andrographolides, which is useful in the control and prevention of infections caused by intracellular microorganisms in fish.
- cytokine macrophages such as IL-12 and IFN
- the diseases cause in the fish, symptoms such as erosions, warts, rashes or spots on the skin, fraying of fins, inflammation of the abdomen, erratic swimming, hemorrhages, lesions and ulcers in the pancreas, esophagus and stomach, muscle spasms and ascites, between others.
- piscirickettsia viruses such as viral hemorrhagic septicemia virus (HSV), infectious pancreatic necrosis virus (IPNv); Infectious hematopoietic necrosis virus (IHNV), Salmon alphavirus (SAV), Infectious salmon anemia (ISAv) and bacteria such as Francicella sp and Renibacterium salmoninarum.
- HSV viral hemorrhagic septicemia virus
- IPNv infectious pancreatic necrosis virus
- IHNV Infectious hematopoietic necrosis virus
- SAV Salmon alphavirus
- ISAv Infectious salmon anemia
- bacteria such as Francicella sp and Renibacterium salmoninarum.
- the ethlological agent corresponded to the first rlckettsla isolated and characterized from aquatic animals and was called Piscirickettsia salmonis (P. salmonis).
- This pathogen is an obligate, cytopathic intracellular parasite for different salmon cell lines and some warm-water fish. It is Gram negative, pleomorphic, usually cocoid, in pairs or in the form of a ring and a size between 0.5 - 1, 5 ⁇ in diameter (Fryer JL, Lannan CN, Garcés HL, Larenas JJ, Smith PA (1990) Isolation of a rickettsiales-like organism from diseased coho salmon ⁇ Oncorhynchus kisutch) in Chile. Fish Pathol; 25: 107-1 14).
- the clinical signs of piscirickettsiosis are characterized by swimming on the surface, slowly, erratically and sometimes in sliver.
- lethargy, anorexia, shock against the walls of the cage raft, bordering and darkening have been described.
- the most relevant external macroscopic lesions described include: peeling, gill pallor, ecchyotic and petechial hemorrhages at the base of the fins, nodules and skin ulcers up to 2 cm in diameter (Bravo, S .; Campos, M. (1989) Coho Salmon Syndrome.
- Piscirickettsiosis Atlantic salmon lesions (Psalm sa / af) naturally infected with Piscirickettsia salmonis, Av Cie ⁇ e Vet; 10: 53-58.). Hematocrit levels reflect severe anemia (Branson EJ, Nieto D ⁇ az-Mu ⁇ oz D. (1991). Description of a new disease condition oceurring in farmed coho salmon, Oncorhynchus kisutch (Walbaum), in South America. Journal Fish Disease; 14: 147-156.).
- the main lesions corresponded to necrosis in various organs and tissues, the most affected being the kidney, liver, spleen and intestine. It was also common to observe vascular lesions similar to those described for rickettsia in mammals, such as endothelial necrosis and thrombus formation.
- macrophages containing organisms within the cytoplasm, intravascular coagulation, perivascular inflammation, pericarditis, endocarditis, chronic inflammatory lesion in the lamina of the intestine increase in the number of granular cells of the intestinal granular stratum and hyperplasia and lamellar fusion (Cubillos V) were found.
- IPPN infectious pancreatic necrosis
- OIE World Organization for Animal Health
- RNA double stranded ribonucleic acid
- the VP2 protein stimulates the production of specific type neutralizing monoclonal antibodies (Nicholson BL. Use of monoclonal antibodies in Identification and characterization of fish viruses. Annu Rev Fish Dis 1993; 3: 241-257) and is thought to contain all recognized epitopes for these antibodies (Caswell-Reno P, Reno PW, Nicholson BL. Monoclonal antibodies to infectious pancreatic virus: analysis of viral epitopes and comparison of different isolates. J Gen Virol 1986; 67: 2193-2205).
- Infectlous pancreatlc necrosis ⁇ n Atlantic salmon, Salmo salar L J Fish Dis28, 383-389 In young that have completed the first feeding in a normal way, the outbreak is usually less explosive, being able to reach losses of 70% or more in a period of two months. Losses in larger animals can be between 10 and 20% (Roberts RJ, MD Pearson. 2005. Infectlous pancreatlc necrosis ⁇ n Atlantic salmon, Salmo salar L. J Fish Dis 28, 383-389).
- the affected fish show anorexla and swim irregularly (swim in corkscrews with ataxia lapses). These fish change to a dark color (hyperplgmentaclone) and have moderate exoftalmla and abdominal distension. Pale gills and hemorrhages in the ventral area, including the fins, are also observed. The fish are observed thin and with "hanging stools" of whitish color (Wolf K. Fish viruses and fish virus diseases. New York: Cornell Unlverslty Press, Ithaca, 1988).
- the main lesions found in the hlstopathological study include foci of coagulatlva necrosis in the pancreas, kidney and intestine.
- Pancreatic tissue looks degenerated, even in aclnares areas, with release of zymogen granules.
- the nuclei of the acinar cells are observed picnotic and of varying sizes. In many cases there is no infiltration of inflammatory cells. (McKnight IJ, Roberts RJ. The pathology of infectious pancreatic necrosis. I. The sequential histopathology of the naturally occurring condition. Br Vet J 1976; 132: 76-85).
- Virulence which is the relative ability of the pathogen to cause disease, is a manifestation of the interaction between the adverse effects produced by virus components and the defense mechanisms developed by the cells to try to eliminate the infection; however, the result of such interaction is always determined by the virus through its virulence factors, a function that can be exercised by any component of the viral particle (Lyles D. 2000. Cytopathogenesis and inhibition of host gene expression by RNA viruses. Microbiol and Mol Biol Rev 64, 709-724). The differences in the level of virulence observed between different strains of IPNV have been attributed to their genetic variation (Dobos P. 1995a. The molecular biology of infectious pancreatic necrosis virus.
- viral proteins considered as the main virulence factors of IPNV are VP2, a component of the outer shell of the viral capsid that participates in the recognition of the virus to cells; VP5 protein of inconclusive function, since it has been observed not to be necessary to establish infection and viral multiplication, but with antiapoptotic activity that apparently has no relation in the establishment of the carrier state.
- VP3 protein has been reported to induce apoptosis in culture cells, but is difficult to detect in an infection with complete viral particles.
- VP4 and VP1 have not been associated with adverse effects at the cellular level, but proteins of similar activity in other viruses have been observed with implication in the pathogenicity of the strains (Lyles D. 2000.
- RNA viruses Cytopathogenesis and inhibition of host gene expression by RNA viruses, Microbiol and Mol Biol Rev QA, 709-724; Liu M, VN Vakharia. 2004.
- VP1 protein of infectious bursal disease virus modulates the virulence in vivo.
- the host expresses a varied response aimed at preventing the infection or dissemination of the agent.
- the nonspecific defense system is the most important as a protection measure for fish and, within this, the interferon system (IFN) is one of the first lines of defense against viral infection through inducing synthesis of proteins that have antiviral activity;
- IFN interferon system
- Mxl protein and double stranded RNA-dependent klnasa protein (PKR) have been shown to have antivlral activity (Roberts RJ, MD Pearson. 2005. Infectious pancreatic necrosis in Atlantic salmon, Salmo salar L. J Fish Dis28 , 383-389).
- IFN interferon system
- IPNV has high antigenic and genotypic variability, characteristics that influence virus-cell interaction, virulence and the development of the carrier state; however, the mechanisms involved in these processes are not fully determined (Rodr ⁇ guez S, JJ Borrego, SI Pérez-Prieto. 2003. Infectious pancreatic necrosis virus: blology, pathogenesls, and diagnostic methods. Adv Vir Res 62, 1 13-165) .
- the procedure for the Identification of the NPI, recommended by the OIE, is based on the isolation of the VNPI in cell culture, followed by the immunological identification of the isolates by means of immunofluorescent tests (World Organization for Animal Health. Dlagnostic Manual for Aquatlc Animal Dlseases. France: OIE. 2003), seroneutrallzaconstru (Llentz JC, Springer JE. Neutralized test of Infectious pancreatic necrosis virus with polyvalent antlserum. J Wildl Dls 1973; 9: 120-124) and ELISA (Davis FJ, Laldler LA, Perry PW, Rosslngton D. Alcock R.
- VNPI VNPI-like protein
- the detection of VNPI in cell lines is consistent and simple, mainly in lines from homologous species. This is because 1) the virus is present in high titres in the tissues; 2) isolation can be done from non-sick animals; 3) there is no phase in which the virus cannot be isolated; 4) the time required for the isolation and identification of the agent is two to three weeks, which is not critical for the presentation of an epizootic, and 5) high sensitivity and easily observable cytopathic effect.
- Cell lines used for the isolation of VNPI include RTG-2 (rainbow trout gonad), CHSE-214 (chinook salmon embryo) and BF-2 (bluegill fry) (Kelly RK, Souter BW, Miller HR. Fish cells lines: comparisons of CHSE-214, FHM, and RTG-2 in assaying IHN and IPN viruses. J Fish Res Board Can 1978; 35: 1009-101 1).
- RT-PCR reverse transcription-polymerase chain reaction
- Rodr ⁇ guez S-JS Borrego JJ , Perez-Prieto SI. Comparative evaluation of five serological methods and RT-PCR assay for the detection of IPNV in fi sh. J Virol Methods 2001; 97: 23-31).
- the sensitivity of this technique has not been greater than that of cell culture, so the Virus isolation and serological confirmation are the processes of choice for the identification of VNPI.
- Andrographis paniculata in the treatment of a wide range of pathologies is known (Abu-Ghefreh, AA, Canatan, H. and Ezeamuzie, C. l. (2008) In vitro and in vivo anti-inflammatory effects of andrographolide International Immunopharmacology, 9: 313-318); including diseases such as the common cold, dysentery, fever, tonsillitis, diarrhea, liver pathologies, herpes, among others (Patarapanich, C, Laungcholatan, S., Mahaverawat, N., Chaichantipayuth, C, Pummangura, S.
- Bioorganic & Medicinal Chem istry 15: A2A7-A255), antithrombotic (Zhao, HY and Fang, WY (1 991) Antithrombotic effects of Andrographis paniculata nees in preventing myocardial infraction.Chin. Med. J., 104 (9): 770-5) hepatoprotectors (Siripong, P., Kongkathip, B., Preechanukool, K., Picha, P., Tunsuwan, K. and Taylor, W. (1 992) Cytotoxic diterpenoid constituents from Andrographis paniculata Nees leaves. J. Sci.
- Ther., 5 (3): 244 -51 is the aerial portions of Andrographis paniculata that are used to extract the phytochemical active substances.
- the extract contains diterpenes, flavonoids and stigmasteroles (Koteswara, R., Vimalamma, G., Venkata, R. and Yew, T. (2004) Flavonoids and andrographolides from Andrographis paniculata. Phyto., Volume 65, Issue 16, Pages 2317- 2321).
- Soclety 1: 1-5) and anti-inflammatory and antipyretic properties (Zhang, CY and Tan, BKH (1998) Vasorelaxatlon of rat thoraclc aorta caused by 14- deoxyandrographollde. Clin. Exp. Pharmacol. Physlol., 25: 424 - 429).
- the mechanism of antiviral action of sulfated polysaccharides is mainly by inhibiting the entry of enveloped viruses, such as Herpesviruses (HSV), into the host cell, by competition for cell surface receptors (Luscher-Mattli M. 2000. Polyanions a lost chance in the fight against HIV and other virus diseases. Antiviral chemistry; Schaeffer DJ, Krylov VS. 2000. Anti-HIV activity of extracts and compounds from algal and cyanobacteria. Ecotoxicology and environmental safety 45: 208-227; Witvrouw M, Pannecouque C, De Clerq E. 1997.
- HSV Herpesviruses
- Fucoidan was tested against several DNA and RNA-enveloped viruses, such as HSV, HCMV, VSV and HIV, and proved to be a potent and selective inhibitor of these viruses, regardless of their type of nucleic acid (Baba M, Snoeck R, Pauwels R , De Clercq E. 1988.
- Antimicrob sulfated polysaccharides are potent and selective inhibitors of various enveloped viruses, including herpes simplex virus, cytomegalovirus, vesicular stomatitis virus, and human immunodeficiency virus.
- Fucoidans from the brown seaweed Adenocystis utricularis extraction methods, antiviral activity and structural studies. Carbohydrate research 338: 153-65).
- various immunostimulatory compositions containing andrographolides or their derivatives or fucoidians are disclosed, for application in humans, see by way of example, the following patent literature: WO2005087223, WO9617605, WO20060081 15, WO20131 17149, CN102399245, RU2381807, CN1939413, CN1939420, JP2005082806, JP2002265370, WO0185709, WO0185710, US2002032229, JPH1 1228602, JPH01313433.
- immunostimulant pharmaceutical compositions of fucoidians and chitosan, and fucoidians and lactobacillus are known, see by way of example, JP2005060327 and JP2010235528.
- compositions comprising terpenes or their derivatives, and flavonoids and their derivatives, to restore the color of farmed or pigmentary fish, promote their growth or foods that enhance their immunity, see by way of example, the following literature Patent: JPH4166040, WO2006123939, JP2010124768 and CN103355491.
- antiviral or anti-sporozoal compositions comprising terpenes or their derivatives and flavonoids and their derivatives, to treat fish diseases, see by way of example, the following patent literature: CN 103989865 and KR201201 18805.
- the present invention relates to an immunostimulant synergistic composition for fish comprising fucoidians and andrographolides.
- the fucal order includes the union of Fucose units that varies according to the species analyzed but mainly shows glycosidic bonds of type (1 ⁇ 3) or (1 ⁇ 4) and sulfated groups can be located in positions C -2, C-3 or C-4.
- Examples of algae from which fucoidians of the fucal order can be obtained are: Fucus vesiculosus, Fucus evanescens, Fucus distichus, Fucus serratus, Pelvetia wrightii, Ascophyllum nodosum, Himanthalia Lorea, Bifurcar ⁇ a bifurcata, Sargassum stenophyllum, Hizikia fusiformearicaica Duriformearica.
- Examples of algae from which fucoidians of the order laminar and other brown algae can be obtained are: Lessonia nigrescens, Lessonia trabeculata, Lessonia vadosa, Macrocystis pyrifera, lindar ⁇ a pinnatifida, Padina pavonia, Laminaria angustata, Laminaria japan, Ecklonia kurome, Adenoistty utric menstrualis, Spatoglossum schroederi and Chordaria flagelliformis.
- the present invention relates to an immunostimulant synergistic composition for fish comprising fucoidians and andrographolides. More preferably, the present invention relates to an immunostimulant synergistic composition for fish comprising fucoidians and andrographolids, and which allows for effective control and prevention of infections caused by intracellular microorganisms. Even more particularly, the present invention relates more to a synergistic composition comprising fucoidians and andrographolides, and which allows effective control and prevention of piscirickettsiosis and infectious pancreatic necrosis (IPN), in fish.
- IPN pancreatic necrosis
- FIGURES Figure 1 Illustrates the conformation of distribution in fish ponds before challenge with P. salmonis, where DE Indicates diet with the composition of the present invention and C means diet without the composition of the present invention.
- Figure 2 Illustrates the conformation of distribution in the ponds during the challenge with P. salmonis, where DE indicates diet with the composition of the present invention, C means diet without the composition of the present invention and T means trojans.
- FIG. 3 Illustrates the analysis of IL-12 expression in SHK-1 cells treated with algae extract (FUTERPENOL®, a composition of botanical extracts and seaweed derivatives with bloactlvas molecules that promote Immunity against intracellular pathogens) , at different concentrations (0.01 -1 ⁇ / ⁇ ) and re-suspended in ethanol and water.
- the analysis of the effect of IL-12 expression was performed at 4 hours post-slow treatment in SHK-1 cells treated with the extract 0.001 ⁇ g / mL, 0.01 ⁇ g / mL and 0.1 ⁇ g / mL.
- the results show the means ⁇ standard error of triplicate samples.
- the * indicate significant differences with respect to the control without stimulus, analyzed by t-student, p ⁇ 0.05, ns, not significant.
- FIGS 4A and 4B Illustrates the analysis of the expression kinetics of IL-12 (4A) and IFN-I (4B) in SHK-1 cells treated with 0.5 nM Andrographólldo (AP), algae extract (BC) 1 ⁇ g / ml, the composition of the Invention (AP + BC) and diet alone as a control (C).
- AP Andrographólldo
- BC algae extract
- C diet alone as a control
- Figure 5 Illustrates the detection of P. salmonis in cells surviving the infection.
- the times of change of the bacterium in SHK-1 cells treated with 0.5 nM Andrographólldo (AP), algae extract (BlendC) 1 ⁇ g / ml, the composition of the Invention (AP + BlendC) and diet alone were determined as control (C).
- IL-12 (6A) and IFN-I (6B) Illustrates the analysis of the expression of IL-12 (6A) and IFN-I (6B) in cells surviving the infection with IPNv:
- the expression of IL-12 and IFN-I was performed in SHK-1 cells treated with the extract of algae (BlendC) 1 Mg / ml, the composition of the invention (AP + BlendC) and diet alone as a control (C) and infected with the virus.
- Figure 7 Illustrates the detection of IPNv in cells surviving the infection.
- Figure 8 Illustrates the distribution of the daily mortality of fish from the group fed only on a diet without the composition of the present invention, of the Trojans and those treated, during the 60 days of study by SRS challenge.
- Figure 9 Illustrates the cumulative percentage distribution of the daily mortality of fish from the group fed only on a diet without the composition of the present invention, of the Trojans and of the treated, during the 60 days of study by SRS challenge.
- the present invention relates to an immunostimulant synergistic composition for fish comprising fucoidians and andrographolides, where fucoidians are obtained from extracts prepared from Fucus vesiculosus, Fucus evanescens, Fucus distichus, Fucus serratus, Pelvetia wrightii, Ascophyllum nodosum, Himanthalia Lorea, Bifurcar ⁇ a bifurcata, Sargassum stenophyllum, Hizikia fusiforme, Durvillaea antarctica, Lessonia nigrescens, Lessonia trabeculata, Lessonia vadosa, Macrocystis pyrifera, Undaria pinnatifida, Padina pavonia, Laminaria angustata, Laminaria japónica, Ecklorisisis, Chloetrisatis, Chloetromatis, Ecklorisis, Chloetromatis, Echlorumis
- the andrographolids are obtained from extracts prepared from Andrographis affinis Nees, Andrographis beddomei, Andrographis echioides Nees, Andrographis elongata, Andrographis humifusa, Andrographis lineata Nees, Andrographis macrobotrys Nees, Andrographis nallamalayana, Andrographis neesiana, Andrographis ovata, Andrographis paniculata Nees, Andrographis rothii, Andrographis serpyllifolia, Andrographis Nees viscosula, Andrographis viscosula var.
- the fucoidane to andrographolide ratio is in the range of 5:95 to 20:80.
- the fucoidane to anrographolide ratio is 10:90.
- the present invention relates mainly to an immunostimulant synergistic composition for fish comprising fucoidians and andrographolids, and which allows for effective control and prevention of infections caused by intracellular microorganisms.
- the present invention relates more particularly to a synergistic composition
- a synergistic composition comprising fucoidians and andrographolides, and which allows effective control and prevention of piscirickettsiosis, viral hemorrhagic septicemia, infectious pancreatic necrosis, infectious hematopoietic necrosis, pancreatic disease and sleeping disease, infectious anemia of the Salmon, francicellosis and renibacteriosis.
- the present composition comprises synergistically comprising fucoidians and andrographolides, and which allows effective control and prevention of piscirickettsiosis and infectious pancreatic necrosis (IPN), in fish.
- IPN infectious pancreatic necrosis
- Example 1 Obtaining the aqueous extract of flour containing fucoidiano
- the dried brown algae were first pulverized by freezing them in liquid N 2 , and using a porcelain mortar, until obtaining an algae powder of 300 microns.
- Example 2 Obtaining Andrografolido and Preparation of Extract.
- Example 3 Preparation composition Andrografolido and Fucoidán.
- a mechanical mixture of both dry extracts was performed in a 90/10 proportion of algae extract and Andrograph ⁇ s sp. Extract, respectively.
- a KitchenAid Heavy Duty mixer (Model KS5SS, USA) with stainless steel container, adjustable speed and capacity> 1.5 L was used.
- the selected mixing speed was, according to the equipment, level 2 equivalent to ⁇ 70 rpm of the upper shaft.
- the mixing time was 10 minutes, the mixture was prepared according to the proportions of ingredients and the densities of these were determined through a gravimetric method with results of 0.77 grs / cm 3 .
- the SHK-1 cell line derived from the anterior kidney of Salmo salar, was used. The test was initiated when the cells presented 90% confluence, at which time they were stimulated with the algae extract (5% Fucoidans).
- the treatment was applied independently to SHK-1 cells, in L15 medium supplemented with 10% fetal bovine serum at a dose of 1 ig / m ⁇ , the cells were incubated at 20 ⁇ for 24 hours of stimulation time.
- RNA extraction was performed from the cells, using the RNA extraction kit (Omega-bio-tek), according to the manufacturer's protocol. Once the total RNA was obtained, the m RNAs were transformed to cDNA by means of the reverse transcription reaction, which was performed in a total volume of 20 ⁇ of solution, divided into two parts.
- the first reaction was carried out in a mixture containing 1.6 ⁇ of oligo-dT (1.25 ⁇ / ⁇ ) for gene expression analysis of 1.0 ⁇ markers of dNTPs (10 mM); 8.0 ⁇ of total RNA (5 ⁇ g) and 0.1 ⁇ of nuclease-free water and incubated for 10 min at 60 ⁇ to eliminate secondary mRNA structures.
- a second mixture comprising 1 ⁇ 1 of M-MLV reverse transcriptase (200 U), 4 ⁇ of 5X enzyme buffer and 0.5 ⁇ of recombinant RNAsaOUT ribonuclease inhibitor (40 U) was added to this solution.
- Each amplification reaction was performed using 2 ⁇ cDNA as temper, 0.2 ⁇ splitters (Table 1), 0.8 ⁇ MgCI 2 (25 mM), 1 ⁇ Lightcycler® Fast Start DNA Master SYBR Green amplification mixture in a volume of 10 ⁇ .
- the reaction was carried out in a LightCycler®1.5 thermal cycler.
- the program consisted of following steps: initial denaturation at 95 S C for 10 min, followed by a PCR reaction of 35 cycles each composed of denaturation at 95 S C for 10 s, pairing 58 S C for 10 s and extension at 70 S C for 10 s . Subsequently a cycle for obtaining the melting curve for 20 s at 95 S C, and finally a cooling cycle at 40 S C for 30 s.
- the SHK-1 cell line derived from the anterior kidney of Salmo salar, was used. The trial was started when the cells presented a 90% confluence, where they were stimulated with an extract of Andrographis sp (10% Total andrographolids).
- the treatment was applied independently to SHK-1 cells, in L15 medium supplemented with 10% fetal bovine serum in a dose of 5 nM of the extract of Andrographis sp, the cells were incubated at 20 for 24 hours of stimulation time.
- RNA extraction was performed from the cells, using the RNA Extraction Kit (Omega-bio-tek), according to the manufacturer's protocol. Once the total RNA was obtained, the m RNAs were transformed to cDNA by means of the reverse transcription reaction, which was performed in a total volume of 20 ⁇ of solution, divided into two parts.
- the first reaction was carried out in a mixture containing 1.6 ⁇ of oligo-dT (1.25 ⁇ / ⁇ ) for gene expression analysis of 1.0 ⁇ markers of dNTPs (10 mM); 8.0 ⁇ of total RNA (5 ⁇ g) and 0.1 ⁇ of nuclease-free water and incubated for 10 min at 60 ⁇ to eliminate secondary mRNA structures.
- Each amplification reaction was performed using 2 ⁇ cDNA as temper, 0.2 ⁇ splitters (Table 2), 0.8 ⁇ MgCI 2 (25 mM), 1 ⁇ Lightcycler® Fast Start DNA Master SYBR Green amplification mixture in a volume of 10 ⁇ .
- the reaction was carried out in a LightCycler®1.5 thermal cycler.
- the program consisted of following steps: initial denaturation at 95 S C for 10 min, followed by a PCR reaction of 35 cycles each composed of denaturation at 95 S C for 10 s, pairing 58 S C for 10 s and extension at 70 S C for 10 s . Subsequently a cycle for obtaining the melting curve for 20 s at 95 S C, and finally a cooling cycle at 40 S C for 30 s.
- Example 6 result of immune effect in fucoidian cell lines plus And rograf olidos
- the SHK-1 cell line derived from the anterior kidney of Salmo salar, was used. The test was started when the cells presented a 90% confluence, where they were stimulated with an extract of Andrographis s and an extract of brown algae.
- the treatment was applied jointly to SHK-1 cells, in L15 medium supplemented with 10% fetal bovine serum in doses of 1 ig / m ⁇ of brown algae extract (5% total fucoidians) and 5 nM of Andrographis extract sp (10% total andrographolids), the cells were incubated at 20 for 24 hours of stimulation time.
- RNA extraction from cells using the klt RNA extraction (Omega-blo-tek), according to the manufacturer's protocol.
- the m RNAs were transformed to cDNA by means of the reverse transcription reaction, which was performed in a total volume of 20 ⁇ of solution, divided into two parts.
- the first reaction was carried out in a mixture containing 1.6 ⁇ of ollgo-dT (1.25 ⁇ / ⁇ ) for analysis of gene expression of markers 1.0 ⁇ of dNTPs (10 mM); 8.0 ⁇ of total RNA (5 xg) and 0.1 ⁇ of nuclease-free water and incubated for 10 mln at 60 ⁇ to undermine secondary structures of mRNAs.
- a second mixture comprising 1 ⁇ of M-MLV reverse transcrlptase (200 U), 4 ⁇ of 5X enzyme buffer and 0.5 ⁇ of RNAsaOUT recombinant rlbonuclease inhibitor (40 U) was added to this solution. in a total volume of 5.5 ⁇ and will be incubated for 1 h at 370. Finally for the inactivation of the reverse transcriptase, the reaction mixture was incubated at 72 S C for 10 mln. The synthesized cDNA was stored at -20 S C for subsequent amplification by PCR or quantification by real time PCR (qPCR) for IFN-I and IL-12 genes related to the expression of EF-1 using the starting points indicated in the following table 3:
- Each amplification reaction was performed using 2 ⁇ of cDNA as temper, 0.2 ⁇ splitters (Table 3), 0.8 ⁇ MgCl 2 (25 mM), 1 ⁇ of amplification mixture Lightcycler® Fast Start DNA Master SYBR Green in a volume of 10 ⁇ .
- the reaction was carried out in a LlghtCycler®1.5 thermocoupler.
- the program consisted of the following steps: Initial denaturation at 95 S C for 10 mln, followed by a PCR reaction of 35 cycles each composed of denaturation at 95 S C for 10 s, pairing 58 S C for 10 s and extension at 70 S C for 10 s.
- Example 7 result of immune effect by P. salmonis challenge
- the SHK-1 cell line derived from the anterior kidney of Salmo salar, was used. The test was given ice when the cells presented a 90% confluence, where they were stimulated with a combination that will ensure a concentration of brown seaweed extract of 1 ⁇ g / ml and 5 nM of the extract of Andrographis sp, as well as stimulation Individual with a concentration of 1 ⁇ g / ml of brown seaweed extract, and Individual stimulation with a concentration of 5 nM of the extract of Andrographis sp. The treatments were applied independently to the SHK-1 cells, in L15 medium supplemented with 10% fetal bovine serum at the doses indicated above, the cells were incubated at 20 ⁇ for 24 hrs of Incubation.
- the duration of the challenge test was 9 days, the time required to obtain a 50% cytopathic effect in the control monolayers.
- the supernatants were harvested and the surviving cells were lysed with 200 ⁇ of TRK lysis buffer and stored in LS ml tubes at -eOO.
- RNA extraction was performed from the cells, using the RNA extraction kit (Omega-bio-tek), according to manufacturer's protocol
- the m RNAs were transformed to cDNA by means of the reverse transcription reaction, which was performed in a total volume of 20 ⁇ of solution, divided into two parts.
- the first reaction was carried out in a mixture containing 1.6 ⁇ of oligo-dT (1.25 ⁇ / ⁇ ) for gene expression analysis of 1.0 ⁇ markers of dNTPs (10 mM); 8.0 ⁇ of total RNA (5 ⁇ g) and 0.1 ⁇ of nuclease-free water and incubated for 10 min at ⁇ to eliminate secondary mRNA structures.
- Each amplification reaction was performed using 2 ⁇ of cDNA as temper, 0.2 ⁇ splitters (Table I), 0.8 ⁇ MgCI 2 (25 mM), 1 ⁇ of Lightcycler® Fast Start DNA Master SYBR Green amplification mixture in a volume of 10 ⁇ .
- the reaction was carried out in a LightCycler®1.5 thermal cycler.
- the program consisted of the following steps: initial denaturation at 95 S C for 10 min, followed by a PCR reaction of 35 cycles each composed of denaturation at 95 S C for 10 s, pairing 58 S C for 10 s and extension at 70 S C for 10 s.
- Example 8 Immunostimulation against intracellular microorganisms (/ ⁇ / ⁇ ) with the composition of the present invention in salmonid fish cell lines.
- a study was conducted on cell lines of salmonid fish, where the composition of the present invention was evaluated. Cell lines exposed to the combination of fucoidians + Andrografolides, cell lines exposed only to fucoidians, and subsequently, relevant molecular markers in the differentiation of ThO to Th1 such as IL-12 and IFN-1 were evaluated. The fucoidianos separately and the combination of these + Andrographolidos are significantly different in the surviving cells after a challenge with IPNV.
- composition of the present invention was an aqueous extract of seaweed with a percentage of 5% Fucoidian referential obtained from Macrocystes Pyrifera in combination with an extract of the Andrographis sp plant with a 1 0% of total andrographolides in a proportion 10% and 90% respectively, was incorporated into the fish food or diet, in a dose in the range of 0.5 to 2.5 Kg per ton of food. Preferably, in a dose of 1 Kg per ton of food.
- Samples of 30 fish were taken to be analyzed in the laboratory using real-time RT-PCR technique, to rule out the presence of IPNv, BKD and SRS.
- An exploratory sampling was carried out to determine the average population weight and to select the fish destined for the trial. Only animals with the required weight, good condition of adaptation to the saline environment and sanitary condition approved by the veterinarian (free of IPNv, SRS and BKD) were included. With the selection data, when making the marking, all animals that have a weight outside the selection range were excluded, as well as those with desquamation or that their condition was not adequate for the present study.
- the ponds were fed with a diet without the present composition, manually at 2-2.5% CP, with 100% ration in the morning. Daily, the unconsumed food from each pond was recovered, to subsequently estimate the actual feeding rate of each group.
- the fish were fed 2-2.5% bw / day, during acclimatization, treatment administration and pathogen challenge stage.
- the food was administered manually, delivering 100% of the ration during the morning. It should be noted that during the acclimatization and challenge a commercial diet without additives was administered.
- the amount of food supplied was adjusted regularly according to the expected growth for the species and mortality. Daily, the collection of food not consumed in the ponds was carried out, thus obtaining the actual consumption of food for the estimation of subsequent production indices.
- tissue samples were taken, considering the anterior kidney, the proximal intestine and blood samples to obtain plasma. The quantity of samples and sampling time are indicated in table 3 below.
- Anterior kidney samples were taken in two 0.5 cm 3 pieces, which were immersed in 2 ml eppendorf tubes (individually for each fish) containing 800 ⁇ of later RNA (Ambion). These samples were labeled and refrigerated (4-6 ⁇ ) for 24 h, then proceed to refrigeration of - ⁇ .
- the proximal intestine was destined for histological analysis, so they were placed in falcon tubes with buffered formalin. In this case, the number of samples per pond (3) was deposited in the same tube, labeled with date, pond number and treatment.
- Blood samples were deposited immediately after taking, in eppendorf tubes prepared with heparin (25UI). They were then centrifuged to remove the plasma, which was deposited in a new tube (previously labeled) and kept in the ultra freezer (- ⁇ ) until it was removed.
- the food with the composition of the present invention was delivered to three ponds (triplicate), as indicated in Table 4, for 30 consecutive days.
- the remaining ponds (controls) continued their feeding with a standard diet for the entire evaluation period.
- the delivery of the treatment was according to the above.
- Control corresponds to the commercial diet.
- Immunostimulant corresponds to the composition of the present invention. Then, a challenge was made with P. salmomis. Table 3 details the specifications of the P. salmonis isolate that was used in the inoculation of Trojan fish.
- the inoculum was delivered with TCID50 / ml determined through the Karber-Spearman method by the ADL Diagnostic Chile Ltda laboratory. In addition, the purity of the inoculum was evaluated, considering RT-PCR analysis ISAv, IPNv, BKD, F. psycrophilum and bacteriological cultures between TSA and TSA / s at 18 and 35 ° incubation.
- the fish were redistributed to perform the challenge. 3 ponds of 1 m 3 were formed , considering the mixture of treated and untreated fish at random in the new ponds, as indicated in table 6. At the time of the new distribution the pittag was read, assigning each chip the group and pond, see Figure 2.
- Treated means that a diet comprising the composition of the present invention has been provided.
- Control means that only a commercial diet has been provided.
- the challenge was carried out through cohabitation, which consisted of introducing fish infected with P. salmonis, Trojans, in healthy fish ponds (treated and controlled), as indicated in table 7, considering an infection pressure of 33%.
- the inoculum was administered to the group of trojans intratraltoneally at a rate of 0.2 ml / fish. Inoculation was performed according to the following procedure:
- the needle was inserted at an angle of approximately 45 s in the ventral midline, between the pectoral and pelvic fins, injecting 0.2 ml per fish.
- the plttag was read by assigning the group "Trojans", pond number and date of Inoculation to the chlp code.
- Tetai 120 Treated means that you have received the diet with the composition of the present invention.
- Control means that you have only received the commercial diet.
- the challenge lasted 60 days, during which time, it was expected to reach cumulative mortality of the control group, 40-60%, thus ending the trial.
- the mortality recorded during the challenge days was sent to the diagnostic laboratory to be analyzed by pathological observations.
- molecular analyzes were performed by the real-time RT-PCR technique, for IPN and SRS viruses, at 20% of the total, considering 15 Trojans, 30 from the treated group and 30 from the control group, to confirm the presence of the pathogen.
- Weight and length were measured at day 0 (acclimatization onset), 30 days after treatment administration and at the end of the challenge with P. salmonis, over 100% of the fish in each group. From the data, the Condition Factor (K), Feeding Rate (SFR), Specific Growth Rate (SGR),% Growth, Thermal Growth Rate (GF3) and Food Conversion Rate (FCRb) were calculated. .
- K Condition Factor
- SFR Feeding Rate
- SGR Specific Growth Rate
- GF3 Thermal Growth Rate
- FCRb Food Conversion Rate
- Table 9 Body weight, condition factor, coefficient of variation and weight gain at the end of the treatment administration stage.
- Treated means that the diet has been supplied with the composition of the invention.
- Control means that only the commercial diet has been supplied.
- Table 10 Food supplied,% SFR and feed conversion factor.
- Treated means that the diet has been supplied with the composition of the invention.
- Control means that only the commercial diet has been supplied.
- Table 10 summarizes the productive parameters obtained during the period of treatment administration (Diet with the present composition of the present invention). From the table it can be seen that the growth indicators (% growth, SGR and SFR) were similar between the treated group and the control group. The data of each group do not show significant differences in weight, obtained at the end of the administration (p> 0.05), in the specific growth rate SGR (p> 0.05) and in the thermal growth rate GF3 (p> 0.05).
- Table 1 1 Summary productive variables by treatment group
- Control means that only commercial diet has been provided.
- Treatment means that the commercial diet has been provided with the composition of the present invention.
- Table 1 1 shows the increase in biomass and accumulated growth (%) post administration of the commercial diet with the composition of the present invention.
- the biomass increase fluctuated from 6.35 to 7.28 kg and the accumulated growth of 135.7 to 162.2% between the different test ponds.
- SGR specific growth rate
- GF3 growth rate
- Control means that only the commercial diet has been supplied.
- control means that only the commercial diet has been supplied
- Table 14 shows the average weight, condition factor (K) and percentage of growth obtained at the end of the challenge for the control group and the treated group. As can be seen, the group treated with the composition of the present invention obtained greater average weight, condition factor and accumulated% growth at the end of the challenge.
- control means that only the commercial diet has been supplied
- control means that only the commercial diet has been supplied
- Treated means that the commercial diet plus the composition of the present invention has been supplied. As shown in the tables, the RPS at day 60 was 62.3% and then decreased to day 80 post challenge with 57.14%.
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MX2017012895A MX2017012895A (es) | 2015-04-08 | 2016-04-06 | Composicion veterinaria de extracto de algas marinas y andrographis sp, util para tratar infeccion en peces. |
BR112017021442-3A BR112017021442A2 (pt) | 2015-04-08 | 2016-04-06 | composição veterinária de extrato de algas marinhas útil para tratar infecção em peixes |
DKPA201770757A DK179845B1 (da) | 2015-04-08 | 2016-04-06 | Veterinary composition of marine algae and andrographis sp extracts, which can be used to treat infections in fish |
EP16776000.8A EP3281633A4 (en) | 2015-04-08 | 2016-04-06 | Veterinary composition of marine algae and andrographis sp extracts, which can be used to treat infections in fish |
CA2981989A CA2981989A1 (en) | 2015-04-08 | 2016-04-06 | Veterinary composition of marine algae and andrographis sp extracts, which can be used to treat infections in fish |
CR20170457A CR20170457A (es) | 2015-04-08 | 2016-04-06 | Composición veterinaria de extracto de algas marinas y andrographis, útil para tratar infección de peces |
US15/565,091 US11439680B2 (en) | 2015-04-08 | 2016-04-06 | Veterinary composition of marine algae and Andrographis sp extracts, which can be used to treat infections in fish |
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CL2015000876A CL2015000876A1 (es) | 2015-04-08 | 2015-04-08 | Composición veterinaria de extracto de algas marinas con al menos 5% de fucoidianos y extracto de planta andrographis sp con al menos 5% de andrografolidos, útil en el control y prevención de infecciones producidas por microorganismos intracelulares en peces. |
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CL2018003878A1 (es) * | 2018-12-28 | 2019-05-17 | Univ De Santiago De Chile 50% | Formulación que comprende extracto de crynodendron patagua y aceite esencial de satureja montana para la prevención y tratamiento de enfermedades causadas por piscirickettsia salmonis en individuos del género salmo. |
US11534469B2 (en) * | 2020-08-25 | 2022-12-27 | Vita Motus AG | Method for the stimulation of human immune cells |
CN114392284A (zh) * | 2022-01-26 | 2022-04-26 | 中国水产科学研究院黑龙江水产研究所 | 抗ihnv海带提取物的制备与应用 |
CN114392283A (zh) * | 2022-01-26 | 2022-04-26 | 中国水产科学研究院黑龙江水产研究所 | 抗ipnv的海带提取物及其应用 |
CN114392285A (zh) * | 2022-01-26 | 2022-04-26 | 中国水产科学研究院黑龙江水产研究所 | 抗ihnv和ipnv共感染海带提取物的制备与应用 |
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WO2006008115A1 (en) * | 2004-07-16 | 2006-01-26 | Universidad Austral De Chile | Diterpenic labdans as immunostimulants for treating infectious diseases |
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CN103989852A (zh) * | 2014-05-26 | 2014-08-20 | 湖州天健兽药有限公司 | 一种防治鱼病的中兽药 |
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WO2006008115A1 (en) * | 2004-07-16 | 2006-01-26 | Universidad Austral De Chile | Diterpenic labdans as immunostimulants for treating infectious diseases |
US20120189706A1 (en) * | 2009-10-06 | 2012-07-26 | Knocean Sciences, Inc. | Macrocystis Pyrifera Derived Health and Wellness Products and Methods of Using the Same |
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