WO2022173246A2 - Composition for preventing or treating novel coronavirus infection, comprising psidiuma guajava extract, pheophytin fraction thereof, or meroterpenoid-based compound isolated therefrom as active ingredient - Google Patents
Composition for preventing or treating novel coronavirus infection, comprising psidiuma guajava extract, pheophytin fraction thereof, or meroterpenoid-based compound isolated therefrom as active ingredient Download PDFInfo
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- WO2022173246A2 WO2022173246A2 PCT/KR2022/002045 KR2022002045W WO2022173246A2 WO 2022173246 A2 WO2022173246 A2 WO 2022173246A2 KR 2022002045 W KR2022002045 W KR 2022002045W WO 2022173246 A2 WO2022173246 A2 WO 2022173246A2
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- guava
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- extract
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- jeju
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- A61K36/18—Magnoliophyta (angiosperms)
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- A61K36/61—Myrtaceae (Myrtle family), e.g. teatree or eucalyptus
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- A—HUMAN NECESSITIES
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- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
- A01N65/08—Magnoliopsida [dicotyledons]
- A01N65/28—Myrtaceae [Myrtle family], e.g. teatree or clove
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- A—HUMAN NECESSITIES
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- A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A—HUMAN NECESSITIES
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- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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- A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline
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- A—HUMAN NECESSITIES
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
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- A—HUMAN NECESSITIES
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- A23V2250/00—Food ingredients
- A23V2250/30—Other Organic compounds
Definitions
- the present invention is a novel corona virus (SARS-CoV-2) containing as an active ingredient a guava ( Psidium guajava ) extract or a pheophytinated fraction thereof, or a meroterpenoid-based compound isolated therefrom, showing recovery ability against infection. It relates to a composition for the prevention, improvement or treatment of corona, and more specifically, by using a chemical method to increase the content of the active ingredient of the extract and mix it to show a synergistic effect between the constituent compounds to prevent and improve novel coronavirus infection Or it relates to a herbal composition having a synergistic effect in treatment.
- SARS-CoV-2 novel corona virus
- Coronavirus is an RNA virus belonging to the coronaviridae, and when viewed under an electron microscope, the shape of spikes on the surface protrude, reminiscent of a crown or the corona of the sun, hence its name (Englund, JA, et al. ., 2019).
- Coronavirinae and Torovirinae in the coronaviridae, and coronaviruses belong to the former.
- the coronavirus subfamily is further divided into four genus: Alphacoronavirus , Betacoronavirus , Gammacoronavirus and Deltacoronavirus .
- SARS Secute Respiratory Syndrome
- MERS Middle East Respiratory Syndrome
- Gene vaccine is a method to generate antibodies against the spike protein (S protein) forming gene by injecting mRNA into the human body.
- S protein spike protein
- Vaccines manufactured by Pfizer and Moderna reported 90% and 95% effectiveness, respectively.
- the storage method is -70°C and -20°C, respectively, so it is not easy to secure the stability of the vaccine.
- the viral vector vaccine is a method to form antibodies to the spike protein (S protein) using a recombinant viral vector by inserting the COVID-19 gene into a virus vector that has lost activity.
- AstraZeneca reported an effect of more than 62%.
- the vaccine works effectively, since the coronavirus undergoes mutations and frequent gene recombination in the process of propagation, there is a high possibility that the efficacy of the vaccine will not last long.
- guava Pumicutica, Psidium guajava
- guava Psidium guajava
- the leaves are consumed as tea and the fruit is consumed raw, in juices and jams.
- Guava has traditionally been used as a medicinal plant.
- guava was used as a folk remedy for diarrhea, dysentery, and acute gastroenteritis; in Peru, it was used for diarrhea, abdominal pain, vomiting, cough, and menstrual pain.
- guava was used for sore throat. It was used to rinse the mouth with juice.
- guava is known as a plant used for metabolic diseases such as obesity or diabetes, and there are studies reporting the antidiabetic effect of guava even in modern times (Huang, CS, et al., 2011; Oh., WK, et al. ., 2005; Shen, SC, et al., 2014).
- Chemical components reported from guava include flavonoids, benzophenones, tannins, and psidium meroterpenoids (Okuda, T., et al., 1987; Prabu, G.R., et al., 2006; Ukwueze, S.E., et al., 2015).
- guava contains a phthydium meroterpenoid characterized by a dihydropyran-ring junction between 3,5-diformyl-benzyl phloroglucinol and a terpenoid, and these meroterpenoids has been reported to have antidiabetic, anticancer and PDE4 inhibitor activity (Hou, J.Q., et al., 2019; Qin, X.-J., 2017; Tang, G.-H., et al., 2017) ).
- Korean Patent No. 102169476 discloses a composition for the prevention or treatment of type 2 severe acute respiratory syndrome coronavirus infection, and includes Zafirlukast, an asthma treatment, and Sulfinpyrazone, a gout treatment. showed inhibitory ability against SARS-CoV-2.
- Korean Patent Application Laid-Open No. 1020200053057 discloses a preventive and therapeutic agent for coronavirus, and a mixture of 14 substances such as calcium, phosphoric acid, folic acid, and quercetin is presented as a preventive and therapeutic agent for novel coronavirus.
- Korean Patent Application Laid-Open No. 1020200131784 discloses a composition for the prevention or treatment of type 2 severe acute respiratory syndrome coronavirus infection disease.
- Candidate drugs including sulfinpyrazone and allopurinol were derived.
- the anti-inflammatory effect of a composition containing guava in Korean Patent No. 101491493 the anti-inflammatory effect of a composition containing guava in Korean Patent No. 101072905, and guava in Korean Patent No. 101320946
- the above techniques are the composition of the present invention showing the prevention, improvement or treatment effect of novel coronavirus infection of the guava extract and the pheophytination fraction or the meroterpenoid-based compound isolated therefrom.
- It is an object of the present invention to provide a composition for preventing, improving or treating novel coronavirus infection comprising, as an active ingredient, a guava ( Psidium guajava ) extract or a pheophytinated fraction thereof or a meroterpenoid-based compound isolated therefrom.
- a guava Psidium guajava extract or a pheophytinated fraction thereof or a meroterpenoid-based compound isolated therefrom.
- Another object of the present invention is to provide a health functional food for preventing or improving symptoms of new corona virus infection, comprising as an active ingredient a guava extract or a pheophytinated fraction thereof or a meroterpenoid-based compound isolated therefrom. .
- Another object of the present invention is to provide a herbal composition having an excellent synergistic effect in preventing, improving or treating novel coronavirus infection, comprising a mixture according to the components of the guava ( Psidium guajava ) extract and a combination thereof.
- Another object of the present invention is to provide a disinfectant for preventing new corona virus infection comprising, as an active ingredient, a guava extract or a pheophytinated fraction thereof or a meroterpenoid-based compound isolated therefrom.
- Another object of the present invention is to provide a composition for animal feed for preventing or improving novel coronavirus infection, comprising as an active ingredient a guava extract or a pheophytinated fraction thereof or a meroterpenoid-based compound isolated therefrom.
- Another object of the present invention is to provide an animal drug for preventing or treating new corona virus infection, comprising a mixture according to the components of the guava ( Psidium guajava ) extract and a combination thereof as an active ingredient.
- the present invention provides a composition for preventing or treating novel coronavirus infection, comprising as an active ingredient a guava ( Psidium guajava ) extract or a pheophytinated fraction thereof or a meroterpenoid-based compound isolated therefrom.
- a guava Psidium guajava
- the guava ( Psidium guajava ) extract is porphyrin chlorophyll a (chlorophyll a), chlorophyll a ' (chlorophyll a '), chlorophyll b (chlorophyll b), chlorophyll b ' (chlorophyll b '), pheophytin It may include one or more selected from the group consisting of a (pheophytin a), pheophytin a' (pheophytin a'), pheophytin b (pheophytin b), and pheophytin b' (pheophytin b'), Preferably, at least one selected from the group consisting of pheophytin a, pheophytin a', pheophytin b, and pheophytin b' may include.
- the guava ( Psidium guajava ) extract may be obtained by extracting guava leaves or fruits with any one solvent selected from the group consisting of water, C1 to C4 lower alcohols, hexane, acetone, methylene chloride, ethyl acetate, and mixed solvents thereof.
- the guava leaf or fruit extract is an extract obtained by extracting guava leaves or fruits with 70-100% C1 to C4 aqueous solution of lower alcohol or lower alcohol or acetone, or hexane fractions and extracts obtained by pheophytinizing these hexane fractions and extracts It may be a fraction.
- the guava leaf or fruit extract was extracted three times by stirring for 2 hours using 95% ethanol on the guava leaf or fruit, and then concentrated under reduced pressure at 40° C. w/v] It can be prepared by concentration under reduced pressure of the hexane fraction obtained by solvent extraction with hexane of the suspension in citric acid solution, or the hexane fraction obtained by suspending the extract in water and storing it at 60° C. for at least one day It can be prepared by concentration under reduced pressure.
- Chlorophyll a (chlorophyll a), chlorophyll a ', chlorophyll b (chlorophyll b), chlorophyll b' (chlorophyll b '), pheophytin a (pheophytin a) of the porphyrins Pheophytin a', pheophytin b, and pheophytin b' can be prepared under similar conditions in all chlorophyll-producing plants.
- the polphyrin-based compound contained in the guava leaf or fruit is a hexane fraction of the guava leaf or fruit extracted with 70-100% C1 to C4 aqueous solution of lower alcohol or lower alcohol or acetone, and a hexane fraction obtained by pheophytinizing the extract. It can be obtained by separation, and preferably an extract extracted with 95% ethanol or a hexane fraction obtained by pheophytinizing the extract can be obtained by separating by chromatography.
- the chromatography is normal phase column chromatography (normal phase column chromatography), reverse phase column chromatography (reverse phase column chromatography), Diaion HP-20 column chromatography (Diaion HP-20 column chromatography), RP-18 column chromatography ( RP-18 column chromatography), LH-20 column chromatography (LH-20 column chromatography), preparative reversed-phase high performance chromatography, medium pressure liquid chromatography, High-performance liquid chromatography (HPLC) or the like may be used.
- normal phase column chromatography normal phase column chromatography
- reverse phase column chromatography reverse phase column chromatography
- Diaion HP-20 column chromatography Diaion HP-20 column chromatography
- RP-18 column chromatography RP-18 column chromatography
- LH-20 column chromatography LH-20 column chromatography
- HPLC High-performance liquid chromatography
- the guava extract is a guava leaf or fruit extracted with one or more solvents selected from the group consisting of water, C1 to C4 lower alcohols, hexane, acetone, methylene chloride, ethyl acetate, and mixed solvents thereof with distilled water. After suspending, it may be a fraction obtained by fractionation with an organic solvent.
- the organic solvent may be a C1-4 lower alcohol, ethyl acetate, hexane or acetone.
- the C1-4 lower alcohol may be methanol, ethanol, propanol, isopropanol, butanol, or the like.
- they are hexane and ethyl acetate, More preferably, they are hexane.
- the present invention provides at least one selected from Jeju guar capital A (compound 1 ), Jeju guar capital D (compound 2 ), Jeju guar capital E (compound 3 ) and Jeju guar capital H (compound 4 ) of the following formula 1 compounds may be included.
- the compound may be derived from guava.
- the present invention is selected from the group consisting of Jeju guar capital A (compound 1), Jeju guar capital D (compound 2), Jeju guar capital E (compound 3) and Jeju guar capital H (compound 4) of Formula 1 above It may include one or more compounds as an active ingredient, preferably comprising one or more compounds selected from the group consisting of Jeju guar capital A (compound 1) and Jeju guar capital D (compound 2) It relates to a composition for preventing or treating novel coronavirus infection, and animal medicine, a health functional food composition for improvement, and a composition for animal feed.
- the present invention also provides a guava extract obtained by extracting guava leaves or fruits using at least one selected from the group consisting of water, C1 to C4 lower alcohols, hexane, acetone, methylene chloride, ethyl acetate, and mixed solvents thereof as a solvent.
- the fractions from the second step were subjected to column chromatography, and Jeju guar capital A (compound 1), Jeju guar capital D (compound 2), Jeju guar capital E (compound 3) and Jeju guar capital H (compound 4) of Formula 1 above
- the present invention relates to a guava extract and a pheophytinated fraction or Jeju guar capital A (compound 1), Jeju guar capital D (compound 2), Jeju guar capital E (compound 3) and Jeju guar capital H of Formula 1 isolated therefrom (Compound 4) may be a composition for preventing or treating novel coronavirus infection, characterized in that it contains one or more compounds selected from the group consisting of as an active ingredient.
- the guava extract is porphyrin-type chlorophyll a (chlorophyll a), chlorophyll a' (chlorophyll a'), chlorophyll b (chlorophyll b), chlorophyll b' (chlorophyll b'), pheophytin a (pheophytin a) ), pheophytin a' (pheophytin a'), pheophytin b (pheophytin b), may include one or more selected from the group consisting of pheophytin b' (pheophytin b'), preferably pe It may include at least one selected from the group consisting of ophytin a (pheophytin a), pheophytin a' (pheophytin a'), pheophytin b (pheophytin b), and pheophytin b'
- the guava extract may be obtained by extracting guava leaves or fruits with any one solvent selected from the group consisting of water, C1 to C4 lower alcohols, hexane, acetone, methylene chloride, ethyl acetate, and mixed solvents thereof.
- the guava leaf or fruit extract is an extract obtained by extracting guava leaves or fruits with 70-100% C1 to C4 aqueous solution of lower alcohol or lower alcohol or acetone, or hexane fractions and extracts obtained by pheophytinizing these hexane fractions and extracts It may be a fraction.
- the guava leaf or fruit extract was extracted three times by stirring for 2 hours using 95% ethanol on the guava leaf or fruit, and then suspended in water, 0.5 N hydrochloric acid solution or 1% [w/v] citric acid aqueous solution. It can be prepared by concentrating the hexane fraction obtained by solvent extraction with hexane under reduced pressure, or by suspending the extract in water and storing it at 60° C. for at least one day, and then concentrating the solvent-extracted hexane fraction with hexane under reduced pressure.
- Chlorophyll a (chlorophyll a), chlorophyll a ', chlorophyll b (chlorophyll b), chlorophyll b' (chlorophyll b '), pheophytin a (pheophytin a) of the porphyrins Pheophytin a', pheophytin b, and pheophytin b' can be prepared under similar conditions in all chlorophyll-producing plants.
- the polphyrin-based compound contained in the guava leaf or fruit is a hexane fraction of the guava leaf or fruit extracted with 70-100% C1 to C4 aqueous solution of lower alcohol or lower alcohol or acetone, and a hexane fraction obtained by pheophytinizing the extract. It can be obtained by separation, and preferably, an extract extracted with 100% methanol or 95% ethanol or a hexane fraction obtained by pheophytinizing the extract can be obtained by separating by chromatography.
- the chromatography is normal phase column chromatography (normal phase column chromatography), reverse phase column chromatography (reverse phase column chromatography), Diaion HP-20 column chromatography (Diaion HP-20 column chromatography), RP-18 column chromatography ( RP-18 column chromatography), LH-20 column chromatography (LH-20 column chromatography), preparative reversed-phase high performance chromatography, medium pressure liquid chromatography, High-performance liquid chromatography (HPLC) or the like may be used.
- normal phase column chromatography normal phase column chromatography
- reverse phase column chromatography reverse phase column chromatography
- Diaion HP-20 column chromatography Diaion HP-20 column chromatography
- RP-18 column chromatography RP-18 column chromatography
- LH-20 column chromatography LH-20 column chromatography
- HPLC High-performance liquid chromatography
- the guava extract is a guava leaf or fruit extracted with one or more solvents selected from the group consisting of water, C1 to C4 lower alcohols, hexane, acetone, methylene chloride, ethyl acetate, and mixed solvents thereof with distilled water. After suspending, it may be a fraction obtained by fractionation with an organic solvent.
- the organic solvent may be a C1-4 lower alcohol, ethyl acetate, hexane or acetone.
- the C1-4 lower alcohol may be methanol, ethanol, propanol, isopropanol, butanol, or the like.
- they are hexane and ethyl acetate, More preferably, they are hexane.
- Compounds 1 to 4 of Formula 1 may be isolated from guava extract.
- Compounds 1 to 4 of Formula 1 isolated from the guava extract can be obtained by fractionation of the guava extract by chromatography, and the chromatography is performed by normal phase column chromatography, reverse phase column chromatography ), Diaion HP-20 column chromatography, RP-18 column chromatography, LH-20 column chromatography, Reversed phase preparation - It can be used by selecting from preparative reversed-phase high performance chromatography, medium pressure liquid chromatography, and high-performance liquid chromatography (HPLC).
- HPLC high-performance liquid chromatography
- the compound of the present invention may be synthesized according to a conventional method in the art, and may be prepared as a pharmaceutically acceptable salt.
- the plant is at least one selected from the group consisting of leaves, branches, stems, roots and fruits.
- the composition may be a composition for preventing or treating novel coronavirus infection.
- the pharmaceutical composition includes at least one compound selected from the group consisting of guava extract and pheophytination fraction or a meroterpenoid-based compound of Formula 1, which is a meroterpenoid compound isolated therefrom, and a pharmaceutically acceptable carrier. can do.
- the pharmaceutical composition comprising the guava extract and the pheophytinated hexane fraction or the meroterpenoid-based compound separated therefrom can be prepared as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc. according to a conventional method, respectively. It can be used in the form of oral dosage forms, external preparations, suppositories, and sterile injection solutions.
- Carriers, excipients and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose , methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
- Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and these solid preparations are the guava extract and pheophytinated hexane fraction of the present invention or the meroterpenoid compound isolated therefrom. It is prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc. with one or more compounds selected from the group consisting of compounds 1 to 4 of Formula 1.
- Liquid formulations for oral use include suspensions, solutions, emulsions, syrups, etc.
- various excipients such as wetting agents, sweeteners, fragrances, preservatives, etc. may be included.
- Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories.
- Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.
- injectable esters such as ethyl oleate.
- base of the suppository witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like can be used.
- the dosage of the pharmaceutical composition will vary depending on the age, sex, and weight of the subject to be treated, the specific disease or pathological condition to be treated, the severity of the disease or pathological condition, the route of administration, and the judgment of the prescriber. Dosage determination based on these factors is within the level of one of ordinary skill in the art, and generally the dosage is in the range of 0.1-1000 mg/kg/day. A more preferred dosage is 0.5-500 mg/kg/day. Administration may be administered once a day, or may be administered in several divided doses. The above dosage does not limit the scope of the present invention in any way.
- the pharmaceutical composition may be administered to mammals such as mice, livestock, companion animals, and humans by various routes. Any mode of administration can be envisaged, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine mucosal or intracerebrovascular injection.
- composition may be a food composition for preventing or treating novel coronavirus infection.
- the health functional food composition includes at least one compound selected from the group consisting of compounds 1 to 4 of Formula 1, which is a guava extract or a pheophytinated fraction thereof, or a meroterpenoid-based compound isolated therefrom, and a food pharmaceutically acceptable It may contain food supplement additives.
- At least one compound selected from the group consisting of compounds 1 to 4 of Formula 1, which is the guava extract or a pheophytinated fraction thereof, or a meroterpenoid-based compound isolated therefrom, comprises the total weight of the health functional food composition of the present invention. It may be included as 0.001 to 100% by weight as a basis.
- the food composition of the present invention includes the form of tablets, capsules, pills or liquids, and the guava extract of the present invention or a pheophytinated fraction thereof or a meroterpenoid-based compound isolated therefrom,
- Compound 1 of Formula 1 Foods to which one or more compounds selected from the group consisting of 4 can be added include, for example, various foods, beverages, gums, tea, vitamin complexes, health functional foods, and the like.
- Another aspect of the present invention is one selected from the group consisting of compounds 1 to 4 of Formula 1, which is the guava extract or the pheophytinated fraction thereof of the present invention or a meroterpenoid-based compound isolated therefrom.
- the composition comprising the above compound provides a composition for animal feed for preventing or improving novel coronavirus infection.
- the composition for animal feed may include a feed additive.
- the feed additive of the present invention corresponds to an auxiliary feed under the Feed Management Act.
- the type of animal feed is not particularly limited, and feeds commonly used in the art may be used.
- the feed include plant feeds such as grains, root fruits, food processing by-products, algae, fibers, pharmaceutical by-products, oils and fats, starches, gourds or grain by-products; and animal feeds such as proteins, inorganic materials, oils and fats, minerals, oils and fats, single cell proteins, zooplankton, or food. These may be used alone or in combination of two or more.
- the present invention also relates to a method for isolating compounds 1 to 4 of Formula 1, wherein guava leaves or fruits are mixed with water, C1 to C4 lower alcohols, hexane, acetone, methylene chloride, ethyl acetate, and mixed solvents thereof.
- the fractions from the second step were subjected to column chromatography, and Jeju guar capital A (compound 1), Jeju guar capital D (compound 2), Jeju guar capital E (compound 3) and Jeju guar capital H (compound 4) of the following formula 1 were added to column chromatography.
- the organic solvent of the second step may be a C1-4 lower alcohol, ethyl acetate, hexane, acetone, or the like.
- the C1-4 lower alcohol may be methanol, ethanol, propanol, isopropanol, butanol, or the like.
- they are hexane and ethyl acetate, More preferably, they are hexane.
- the fraction of step 2 is most preferably a hexane fraction obtained by suspending the concentrated extract of step 1 in distilled water and then fractionating with hexane.
- the column chromatography of the above three steps is normal phase column chromatography, reversed phase column chromatography, Diaion HP-20 column chromatography, RP-18 column chromatography, LH-20 column chromatography, preparative reverse phase-high performance liquid chromatography, It can be used by selecting from medium pressure liquid chromatography, high performance liquid chromatography, and the like.
- the present invention relates to a composition for the prevention, improvement or treatment of novel coronavirus infection comprising, as an active ingredient, a guava extract or a pheophytinated fraction thereof or a meroterpenoid-based compound isolated therefrom, a guava extract and a pheophytinated It was confirmed that the fraction or the meroterpenoid-based compound isolated therefrom exhibited cell lesion recovery ability against novel coronavirus infection.
- the guava extract of the present invention or its pheophytinated fraction or a meroterpenoid-based compound isolated therefrom can be used for the prevention, improvement or treatment of new corona infection, food, medical supplies, and composition for animal feed. It is expected that it will be possible
- FIG. 1 is a diagram showing the chemical structure of a compound isolated from guava leaves having an inhibitory effect on novel coronavirus (SARS-CoV-2).
- FIG. 2 is a chromatogram showing the presence or absence of meroterpenoid compounds in a leaf extract of guava, a hexane fraction thereof, and a pheophytination fraction using an inorganic acid.
- FIG. 3 is a chromatogram of a pheophytin-based compound using a leaf extract of guava, a hexane fraction of a dried leaf extract, and an inorganic acid.
- 5 is a chromatogram of a pheophytinated fraction through heat treatment of a guava leaf extract for 1 hour to 48 hours.
- FIG. 6 is a graph showing the cell lesion recovery ability for novel coronavirus (SARS-CoV-2) infection of each fraction including a leaf extract of guava and a pheophytination reaction.
- SARS-CoV-2 novel coronavirus
- FIG. 7 is a graph showing the cell lesion recovery ability against SARS-CoV-2 infection of compounds 1 to 4 isolated from leaf extracts of guava.
- Jeju Island located in the South Sea of Korea, is gradually approaching a subtropical climate due to global warming, and many farms located on Jeju Island are cultivating guava.
- the present inventors in the process of studying guava extracts showing recovery ability against novel coronavirus (SARS-CoV-2) infection, guava extracts and fractions or constituent compounds having phosidium meroterpenoid as a basic skeleton, and these constituent compounds It was discovered that the combination of liver is effective in preventing and improving the novel corona, and by using a chemical method, the content of the active ingredient of the extract is increased, and the present invention is completed by mixing it to show a synergistic effect between the constituent compounds.
- SARS-CoV-2 novel coronavirus
- the raw leaves and hot air-dried dried leaves of guava of the present invention were purchased from Jungeom Farm located in Jungeom-ri, Aewol-eup, Jeju-si, Korea. After 10 kg of dried guava leaves were crushed, 50 L of 95% [v/v] ethanol was added, and the mixture was extracted three times with an ultrasonic extractor for 99 minutes, and concentrated under reduced pressure to obtain 1.45 kg of a 95% ethanol extract.
- Fractions were sequentially obtained through chromatography of the extract obtained in Example 1.1, and antiviral activity of each fraction was evaluated to separate four new compounds from the fraction with the highest antiviral activity.
- Example 1.1 the extract obtained in Example 1.1 was always suspended in 2.0 L of distilled water, followed by sequential solvent fractionation with n -hexane and ethyl acetate to obtain an n -hexane fraction (240.3 g). Then, the n -hexane fraction (240.3 g) was subjected to normal phase column chromatography under gradient elution conditions of n -hexane:ethyl acetate:methanol (1:0:0 ⁇ 0:0:1[v:v:v]) (normal phase column chromatography) phase column chromatography) was performed to obtain 12 fractions (F.1 - 12).
- F.4 fraction (33.7 g) was subjected to normal phase column chromatography under gradient conditions of n -hexane:ethyl acetate (1:0 ⁇ 9:1 [v:v]) to obtain 7 small fractions (F. .4-1 - 4-7).
- F.4-7 reverse-phase MPLC was performed under a gradient condition of methanol: distilled water (0.1% formic acid) (9:1 ⁇ 0:1 [v:v]) to obtain 4 small fractions again (F.4- 7-1 - 4-7-4).
- Pheophytination and preparation of fractions of guava leaf extract are polphyrin-based compounds, which are demetallized, dephytylated, and decarbomethoxylated through a cooking process or digestion process. It is known that derivatives such as pheophytin, pyropheophytin, chlorophyllide, and pheophorbide are produced (Hayes & Ferruzzi, 2020).
- the present inventors confirmed the antiviral activity against SARS-CoV-2 of the guava extract, particularly the pheophytinated fraction, through a basic experiment, and the four compounds obtained in Example 2 were also abundant in the pheophytinated fraction. It was confirmed that it was included. Accordingly, a method for preparing a pheophytinated fraction capable of significantly increasing the content of pheophytin from guava leaf extract was established.
- the dried guava extract was suspended using a 0.5 N hydrochloric acid solution, and the solvent was fractionated with n -hexane for about 5 minutes, followed by solvent fractionation 2-3 times with the same volume of water. By lowering the acidity, a pheophytinated n- hexane fraction was finally obtained. Finally, after drying through a lyophilizer, it was dissolved in DMSO to make 10 mg/mL and used in the antiviral activity evaluation experiment.
- the experiment was performed by varying the concentration using citric acid, which is a safe organic acid present in plants. After drying the fresh guava leaves in the shade, they were extracted three times by stirring with 95% ethanol for 2 hours, and concentrated under reduced pressure to obtain the extract and used in the experiment. After preparing a 10% [w/v] citric acid aqueous solution, serial dilutions were made to prepare 10%, 5%, 1%, 0.5%, 0.25%, 0.1% citric acid aqueous solution.
- the guava extract prepared in Example 3.2 was suspended in distilled water and then left in a dry oven at 60° C. for 1 hour, 3 hours, 6 hours, 12 hours, 24 hours and 48 hours, followed by solvent fractionation with n -hexane and pheophytinylation An n- hexane fraction was obtained. Finally, fractions for each condition were prepared at 1 mg/mL for HPLC analysis.
- Example 3.1 95% ethanol extract of dried guava leaves, n -hexane fraction, and pheophytination fraction using 0.5N aqueous hydrochloric acid solution obtained in Example 3.1 were analyzed at a concentration of 10 mg/ml for the extract and 5 for the fraction. It was prepared using methanol or aqueous methanol solution at a concentration of mg/ml. Compounds 1 to 4 isolated in Example 1.2 were prepared at a concentration of 1 mg/ml.
- HPLC analysis was performed using a Thermo Fisher Scientific UltiMate 3000 system equipped with a DAD-Diode Array Detector 3000, and a YMC-Triart C18 (4.6 ⁇ 250.0 mm, 5 ⁇ m ) column was used.
- Analytical conditions the mobile phase of liquid chromatography was flowed at a flow rate of 1.0 ml/min using distilled water (mobile phase A) and acetonitrile (mobile phase B) containing 0.1% [v/v] formic acid, and a column temperature of 25 ° C., Analysis was performed at a sample temperature of 10°C, and 20 ⁇ l of each sample was injected.
- the chromatogram was observed at 290 nm, and the conditions of the mobile phase were as follows. It flowed with a gradient of 70-100% mobile phase B for 0-35.0 minutes, 100% mobile phase B for 35.0-55.0 minutes, and 70% mobile phase B for 55.1-60.0 minutes. Through the retention time and ultraviolet absorption pattern of compounds 1 to 4 isolated in Example 1-2, The presence or absence of the compounds included in each sample was checked, and the results are shown in FIG. 2 .
- n -hexane fraction and pheophytination fraction of the dried guava leaf extract obtained in Example 3.1 and the n -hexane fraction of the guava leaf extract obtained in Example 3.2 in the shade were mixed with methanol at a concentration of 1 mg/ml for HPLC analysis.
- methanol at a concentration of 1 mg/ml for HPLC analysis.
- polphyrin-based compounds were identified in all samples.
- chlorophyll a, b and pheophytin a, b and their derivatives were all confirmed, whereas the n -hexane fraction of the dried guava leaf was the same as the pheophytinated fraction of the same sample.
- Only pheophytin a, b and its derivatives were identified. This shows that as the purchased dried guava leaves are hot air-dried samples, pheophytinization by heat has progressed. In Example 5.3 below, pheophytination through heat treatment will be introduced to support these results.
- Example 3.2 After securing a pheophytination fraction and n -hexane fraction using citric acid for each concentration of the guava leaf extract obtained in Example 3.2, methanol was prepared at a concentration of 1 mg/ml. HPLC analysis was performed using the same system, column and analysis conditions as described in Example 5.1, and through the retention time and UV absorption pattern of the polphyrin-based compounds. The relative content of the substances included in the fractions for each concentration and the degree of pheophytinylation were compared through the intensity of the peak, and the results are shown in FIG. 4 .
- Example 3.3 After obtaining a pheophytinated fraction and an n -hexane fraction through heat treatment of the guava leaves obtained in Example 3.3, methanol was prepared at a concentration of 1 mg/ml. HPLC analysis was performed using the same system, column and analysis conditions as described in Example 5.1, and through the retention time and UV absorption pattern of the polphyrin-based compounds. The presence or absence of substances contained in each concentration fraction and the degree of pheophytinylation were compared with the relative content through the intensity of the peak, and the results are shown in FIG. 5 .
- Example 5.1 After concentrating the guava leaf extract, it was suspended in distilled water and left in a dry oven at 60 ° C for 1 hour, 3 hours, 6 hours, 12 hours, 24 hours and 48 hours to perform pheophytinization. It was confirmed that all chlorophyll a, b and derivatives thereof were completely pheophytinized even in samples left only for a period of time.
- Example 5.1 in the n -hexane fraction of the guava dried leaf extract dried by hot air, chlorophyll a, b and its derivatives were not identified, but only pheophytin a, b and its derivatives were confirmed, which also showed pheophytinylation by heat. to be.
- the pheophytination reaction is also possible through a method of heating at 60° C. to 100° C. (blanching) or processing the guava leaf itself with heat.
- SARS-CoV-2 severe acute respiratory syndrome coronavirus 2, NMC-CoV-02 strain
- SARS-CoV-2 virus was inoculated with mono-layered VERO cells, and after 3 days, the virus culture supernatant was collected, and the virus was inoculated into VERO cells cultured in DMEM medium containing 10% [v/v] FBS. It was used to measure antiviral activity.
- VERO cells After infecting VERO cells with the SARS-CoV-2 virus prepared in Example 6.1, VERO cells were dispensed at a density of 2 ⁇ 10 4 cells/well in this 96-well plate to 10% [v/v ] FBS (fetal bovine serum), 1% [v/v] PS (penicillin + streptomycin) and L-glutamine (L-glutamine, added 2ml based on 500ml medium) for one day in DMEM culture medium, and then the culture medium Washed with PBS solution.
- FBS fetal bovine serum
- PS penicillin + streptomycin
- L-glutamine L-glutamine
- infection medium infection media: DMEM
- SARS-CoV-2 virus at a concentration of 100 TCID 50
- PBS solution 50 ⁇ l of infection medium (infection media: DMEM) containing SARS-CoV-2 virus at a concentration of 100 TCID 50
- DMEM infection media
- the culture medium was washed with PBS solution.
- the compound of the present invention was treated and cultured for 3 days in DMEM culture medium containing 0.2 ⁇ g/ml trypsin, and the CPE caused by the virus was measured.
- FIGS. 6 and 7 The results of antiviral activity according to the dilution factor of each compound are shown in FIGS. 6 and 7 below.
- both the guava leaf extract and the fraction showed antiviral effect at a concentration of 20 ⁇ g /ml or less.
- the n -hexane fraction and the pheophytinated fraction showed antiviral activity even at 5 ⁇ g /ml and showed the best effect.
- antiviral activity was confirmed at a concentration of 40 ⁇ M or less for all compounds, which is shown in FIG. 7 .
- Compounds 1 and 2 showed activity at a concentration of 3.75 ⁇ M and showed the best antiviral effect.
- the meroterpenoid-based compound and the pheophytin-based compound exhibited antiviral effects, and at the same time, as confirmed through HPLC analysis in Examples 4 and 5.1, meloterpene-based compounds It was confirmed that the n -hexane fraction and the pheophytinated fraction containing the most abundant nooid compound and pheophytin compound exhibited the best antiviral effect.
- the guava extract, fraction, or pheophytinated fraction thereof of the present invention, or a meroterpenoid-based compound isolated therefrom is effective in preventing, improving or treating novel coronavirus infection.
- guava extract or pheophytinated fraction of the present invention or Jeju guar capital A (compound 1), Jeju guar capital D (compound 2), Jeju guar capital E (compound 3) and Jeju guar capital H (compound 4) 200 mg of at least one selected from the group consisting of 175.9 g of lactose, 180 g of potato starch and 32 g of colloidal silicic acid were mixed with each. After adding a 10% gelatin solution to this mixture, it was ground and passed through a 14 mesh sieve. This was dried, and 160 g of potato starch, 50 g of talc and 5 g of magnesium stearate were added thereto, and the resulting mixture was made into tablets.
- guava extract or pheophytinated fraction of the present invention or Jeju guar capital A (compound 1), Jeju guar capital D (compound 2), Jeju guar capital E (compound 3) and Jeju guar capital H (compound 4)
- At least one selected from the group consisting of flour was added in an amount of 0.1% by weight, and the mixture was used to prepare bread, cake, cookies, crackers and noodles to prepare health-promoting food.
- guava extract or pheophytinated fraction of the present invention or Jeju guar capital A (compound 1), Jeju guar capital D (compound 2), Jeju guar capital E (compound 3) and Jeju guar capital H (compound 4) At least one selected from the group consisting of tomato juice or carrot juice was added to 1,000 ml of each to prepare vegetable juice for health promotion.
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Abstract
The present invention relates to a composition for preventing or treating novel coronavirus infection, comprising, as an active ingredient, a Psidiuma guajava extract, a pheophytin fraction thereof, or a meroterpenoid-based compound isolated therefrom. By increasing, through a chemical method, the content of a porphyrin-related compound, which is an active compound, while containing the meroterpenoid-based compound, the composition may be advantageously used in the treatment and prevention of novel coronavirus infection through synergistic interactions of antiviral activity.
Description
본 발명은 신종코로나 바이러스(SARS-CoV-2) 감염에 대하여 회복능을 보이는 구아바(Psidium guajava) 추출물 또는 이의 페오피틴화 분획물 또는 이로부터 분리한 메로테르페노이드계 화합물을 유효성분으로 함유하는 신종코로나의 예방, 개선 또는 치료용 조성물에 관한 것으로, 더욱 상세하게는 화학적 방법을 사용하여 추출물의 활성성분의 함량을 높이고 구성화합물 간의 상승효과를 나타내도록 혼합하여 제조함으로써 신종 코로나 바이러스 감염의 예방, 개선 또는 치료에 상승효과를 갖는 생약조성물에 관한 것이다.The present invention is a novel corona virus (SARS-CoV-2) containing as an active ingredient a guava ( Psidium guajava ) extract or a pheophytinated fraction thereof, or a meroterpenoid-based compound isolated therefrom, showing recovery ability against infection. It relates to a composition for the prevention, improvement or treatment of corona, and more specifically, by using a chemical method to increase the content of the active ingredient of the extract and mix it to show a synergistic effect between the constituent compounds to prevent and improve novel coronavirus infection Or it relates to a herbal composition having a synergistic effect in treatment.
코로나바이러스는 코로나바이러스과(Coronaviridae)에 속하는 RNA 바이러스로서 전자현미경으로 보았을 때 표면의 스파이크(spikes) 모양이 튀어나와 있어 왕관 또는 태양의 코로나를 연상시킴으로 인하여 그 이름이 붙여졌다(Englund, J.A., et al., 2019). 코로나바이러스과에는 2개의 아과인 코로나바이러스 아과(Coronavirinae)와 토로바이러스 아과(Torovirinae)가 있으며 코로나바이러스는 전자에 속해있다. 코로나바이러스 아과는 다시 4개의 속(genus)인 알파-코로나바이러스(Alphacoronavirus), 베타-코로나바이러스(Betacoronavirus), 감마-코로나바이러스(Gammacoronavirus) 및 델타-코로나바이러스(Deltacoronavirus)로 나뉘어진다. 이 중 전자의 두 속인 알파와 베타-코로나바이러스는 사람과 동물에게 감염을 일으키고, 감마와 델타는 동물에게 감염을 일으킨다고 알려져 있다. 인간 코로나바이러스는 1960년대 중반에 처음 발견되었고, 현재까지 사람을 감염시키는 것으로 7종이 확인되었으며 이중 HCoV-229E, HCoV-NL63는 알파-코로나바이러스에, HCoV-OC43, HCoV-HKU1, SARS-CoV, SARS-CoV-2, MERS-CoV는 베타-코로나바이러스에 속해있다. 이외에도 돼지, 개, 고양이, 박쥐, 말 등 동물을 감염시켜 병원성을 나타내고 있다. Coronavirus is an RNA virus belonging to the coronaviridae, and when viewed under an electron microscope, the shape of spikes on the surface protrude, reminiscent of a crown or the corona of the sun, hence its name (Englund, JA, et al. ., 2019). There are two subfamilies, Coronavirinae and Torovirinae , in the coronaviridae, and coronaviruses belong to the former. The coronavirus subfamily is further divided into four genus: Alphacoronavirus , Betacoronavirus , Gammacoronavirus and Deltacoronavirus . Of these, the former two genera, alpha and beta-coronaviruses, infect humans and animals, and gamma and delta are known to infect animals. Human coronaviruses were first discovered in the mid-1960s, and seven species have been identified that infect humans so far, of which HCoV-229E and HCoV-NL63 are alpha-coronaviruses, HCoV-OC43, HCoV-HKU1, SARS-CoV SARS-CoV-2 and MERS-CoV belong to beta-coronaviruses. In addition, it shows pathogenicity by infecting animals such as pigs, dogs, cats, bats, and horses.
전 세계는 지난 2000년 이후 20년 동안 두 번의 코로나바이러스인 사스(SARS, 급성호흡기증후군)와 메르스(MERS, 중동호흡기증후군) 감염위기를 겪었고, 현재는 2019년 12월 중국 우한에서 발원한 코로나19(COVID-19)의 대유행 감염위기를 겪고 있다. 이들 코로나 바이러스는 모두 베타-코로나바이러스 속에 속해있지만 각기 다른 전파력과 치명률을 나타내었다. 사스의 경우, SARS-CoV를 병원체로 하여 2003년 2월에 중국에서 발생하였으며, 2020년 03월 03일 발표된 WHO의 통계자료(WHO, Coronavirus disease 2019 (COVID-19) Situation Report - 43, 2020)에 따르면 전 세계적으로 26개 국가가 감염되었으며 8,096명의 확진자, 774명의 사망자를 발생시켜 9.6%의 치명률을 보였다. 메르스의 경우, MERS-CoV를 병원체로 하여 2012년 중동지역 아라비아에서 발생하였으며, 동일 통계자료에 의하면 전 세계적으로 27개 국가가 감염되었으며 2,494명의 확진자, 858명의 사망자를 발생시켜 전파력은 낮지만 34-35%의 높은 치명률을 보였다. 2019년 12월에 중국에서 발생하여 현재까지도 지속되고 있는 COVID-19의 경우, 만 1년이 되는 2020년 12월 27일 기준 219개 국가가 감염되었으며, 8080만명의 확진자, 177만명의 사망자를 발생시켜 높은 전파력과 함께 상대적으로 다른 코로나 바이러스 질환인 사스나 메르스에 비하여 낮은 2.2%의 치명률을 보이고 있다. 그러나, 다른 바이러스에 비하여 증상이 발현되기 전에도 바이러스를 전파할 수 있는 가능성으로 인하여 COVID-19에 대한 국가적인 대응력을 어렵게 하고 있다. 전 세계 모든 국가들은 현 상황을 타개하기 위하여 다국적 제약회사를 중심으로 백신의 개발과 승인이 이루어지고 있는 상황이다(Park, S.E., 2020).The world has suffered from two coronavirus infection crises, SARS (Acute Respiratory Syndrome) and MERS (Middle East Respiratory Syndrome), in 20 years since 2000. 19 (COVID-19) is experiencing a pandemic infection crisis. All of these coronaviruses belong to the genus beta-coronaviruses, but they exhibited different transmission and lethality rates. In the case of SARS, it occurred in China in February 2003 with SARS-CoV as a pathogen, and the WHO, Coronavirus disease 2019 (COVID-19) Situation Report - 43, 2020 released on March 03, 2020 ), 26 countries were infected worldwide, 8,096 confirmed cases and 774 deaths, showing a fatality rate of 9.6%. In the case of MERS, it occurred in Arabia in the Middle East in 2012 with MERS-CoV as a pathogen. It showed a high fatality rate of 34-35%. In the case of COVID-19, which originated in China in December 2019 and continues to this day, as of December 27, 2020, which is one year old, 219 countries have been infected, 80.8 million confirmed cases, and 1.77 million deaths. It has a relatively low fatality rate of 2.2% compared to SARS and MERS, which are other corona virus diseases, along with high transmission power. However, compared to other viruses, the possibility of spreading the virus even before symptoms appear makes it difficult for the national response to COVID-19. In all countries around the world, vaccines are being developed and approved by multinational pharmaceutical companies to overcome the current situation (Park, S.E., 2020).
현재까지 COVID-19를 해결하기 위해 개발된 치료제는 없는 상황이며 각국의 다국적 제약회사를 중심으로 백신을 개발하여 대응에 나서고 있다. 임상을 받았거나 또는 앞두고 있는 미국과 유럽의 제약사인 화이자, 모더나, 아스트라제네카는 각기 유전자, 바이러스 벡터 타입의 백신 개발에 성공하였다. 유전자 백신은 스파이크 단백질(S protein) 형성 유전자인 mRNA를 인체내에 주입하여 이에 대항하는 항체가 생성되도록 하는 방법으로, 화이자와 모더나에서 제조된 백신은 각기 90%, 95%의 효과를 보고하였다. 그러나 저장방법이 각기 -70℃, -20℃로서 백신의 안정성 확보가 쉽지 않은 상태이다. 한편 바이러스 벡터 백신은 활동성을 없앤 바이러스 벡터에 COVID-19 유전자를 넣어 재조합 바이러스 벡터를 사용하여 스파이크 단백질(S protein)에 대한 항체를 형성하도록 하는 방법으로 아스트라제네카는 62% 이상의 효과를 보고하였다. 그러나, 비록 백신이 효과적으로 작용한다 하더라도 코로나바이러스는 증식과정에서 돌연변이 및 빈번한 유전자 재조합현상이 일어나기 때문에, 백신의 효용성이 오래가지 못할 가능성이 높다. 또한, 기존에 개발된 백신에 듣지 않는 새로운 변이 코로나 바이러스가 발생할 확률도 항상 존재한다.There is no treatment developed to solve COVID-19 so far, and multinational pharmaceutical companies in each country are developing vaccines to respond. Pfizer, Moderna, and AstraZeneca, which are US and European pharmaceutical companies that have undergone or are about to undergo clinical trials, have succeeded in developing vaccines of gene and viral vector types, respectively. Gene vaccine is a method to generate antibodies against the spike protein (S protein) forming gene by injecting mRNA into the human body. Vaccines manufactured by Pfizer and Moderna reported 90% and 95% effectiveness, respectively. However, the storage method is -70℃ and -20℃, respectively, so it is not easy to secure the stability of the vaccine. On the other hand, the viral vector vaccine is a method to form antibodies to the spike protein (S protein) using a recombinant viral vector by inserting the COVID-19 gene into a virus vector that has lost activity. AstraZeneca reported an effect of more than 62%. However, even if the vaccine works effectively, since the coronavirus undergoes mutations and frequent gene recombination in the process of propagation, there is a high possibility that the efficacy of the vaccine will not last long. In addition, there is always a possibility that a new mutant coronavirus that does not respond to previously developed vaccines will occur.
한편, 구아바(Guava, Psidium guajava)는 도금양과(Myrtaceae)의 상록 관목 또는 교목으로 아프리카, 남미 및 동남아시아의 열대 또는 아열대 지역에 널리 분포한다. 구아바는 전세계적으로 중요한 식량 작물로, 잎은 차로 음용하며 열매는 생식, 주스, 잼 등으로 섭취된다. 구아바는 전통적으로 약용식물로 사용되었는데, 중국은 민간요법으로 설사, 이질, 급성 위장염증에 구아바를 사용하였고, 페루에서는 설사와 복통, 구토, 기침, 생리통 등에 사용하였으며, 브라질에서는 목이 따끔거릴 때 구아바 주스로 입안을 씻어내는 데에 사용하였다. 또한 구아바는 비만 또는 당뇨와 같은 대사성 질환에 사용되는 식물로 알려져 있고, 현대에도 구아바의 항당뇨 효능을 보고한 연구결과가 있다(Huang, C.S., et al., 2011; Oh., W.K., et al., 2005; Shen, S.C., et al., 2014).On the other hand, guava (Guava, Psidium guajava ) is an evergreen shrub or tree of the Myrtaceae family, widely distributed in tropical or subtropical regions of Africa, South America and Southeast Asia. Guava is an important food crop worldwide. The leaves are consumed as tea and the fruit is consumed raw, in juices and jams. Guava has traditionally been used as a medicinal plant. In China, guava was used as a folk remedy for diarrhea, dysentery, and acute gastroenteritis; in Peru, it was used for diarrhea, abdominal pain, vomiting, cough, and menstrual pain. In Brazil, guava was used for sore throat. It was used to rinse the mouth with juice. In addition, guava is known as a plant used for metabolic diseases such as obesity or diabetes, and there are studies reporting the antidiabetic effect of guava even in modern times (Huang, CS, et al., 2011; Oh., WK, et al. ., 2005; Shen, SC, et al., 2014).
구아바로부터 보고된 화학성분으로는 플라보노이드(flavonoid), 벤조페논(benzophenone), 탄닌(tannin) 및 프지디움 메로테르페노이드(psidium meroterpenoid)이 보고되고 있다(Okuda, T., et al., 1987; Prabu, G.R., et al., 2006; Ukwueze, S.E., et al., 2015). 또한, 구아바에는 3,5-디포밀-벤질 플로로글루시놀과 테르페노이드 사이에 디하이드로 피란-고리 접합을 특징으로 하는 프지디움 메로테르페노이드가 존재하는 것으로 알려져 있으며, 이러한 메로테르페노이드는 항당뇨, 항암 및 PDE4 저해제 등의 활성이 보고된 바가 있다(Hou, J.Q., et al., 2019; Qin, X.-J., 2017; Tang, G.-H., et al., 2017).Chemical components reported from guava include flavonoids, benzophenones, tannins, and psidium meroterpenoids (Okuda, T., et al., 1987; Prabu, G.R., et al., 2006; Ukwueze, S.E., et al., 2015). In addition, it is known that guava contains a phthydium meroterpenoid characterized by a dihydropyran-ring junction between 3,5-diformyl-benzyl phloroglucinol and a terpenoid, and these meroterpenoids has been reported to have antidiabetic, anticancer and PDE4 inhibitor activity (Hou, J.Q., et al., 2019; Qin, X.-J., 2017; Tang, G.-H., et al., 2017) ).
한국등록특허 제102169476호는 제2형 중증급성호흡기증후군 코로나바이러스 감염 질환의 예방 또는 치료용 조성물을 개시하고 있으며, 천식치료제인 자피를루카스트(Zafirlukast)와 통풍치료제인 설핀파이라존(Sulfinpyrazone)의 SARS-CoV-2에 대한 억제능을 나타내었다. 한국공개특허 제1020200053057호는 코로나바이러스 예방 및 치료제를 개시하고 있으며, 칼슘, 인산, 엽산, 퀘세틴 등 14종 물질의 혼합물을 신종코로나 바이러스의 예방 및 치료제로 제시하고 있다. 한국공개특허 제1020200131784호는 제2형 중증급성호흡기증후군 코로나바이러스 감염 질환의 예방 또는 치료용 조성물을 개시하고 있으며, AI 딥러닝 알고리즘을 통해 2,700여개의 FDA 승인약물로부터 자피를루카스트(Zafirlukast)와 설핀파이라존(Sulfinpyrazone), 알로푸리놀(Allopurinol) 등을 포함한 후보약물을 도출하였다.Korean Patent No. 102169476 discloses a composition for the prevention or treatment of type 2 severe acute respiratory syndrome coronavirus infection, and includes Zafirlukast, an asthma treatment, and Sulfinpyrazone, a gout treatment. showed inhibitory ability against SARS-CoV-2. Korean Patent Application Laid-Open No. 1020200053057 discloses a preventive and therapeutic agent for coronavirus, and a mixture of 14 substances such as calcium, phosphoric acid, folic acid, and quercetin is presented as a preventive and therapeutic agent for novel coronavirus. Korean Patent Application Laid-Open No. 1020200131784 discloses a composition for the prevention or treatment of type 2 severe acute respiratory syndrome coronavirus infection disease. Candidate drugs including sulfinpyrazone and allopurinol were derived.
구아바를 이용한 특허로는 한국등록특허 제101491493호에 구아바를 포함하는 조성물의 항염증 효과가, 한국등록특허 제101072905호에 구아바를 포함하는 조성물의 항당뇨 효과가, 한국등록특허 제101320946호에 구아바의 전립선암 치료 효과가 기재되어 있으나, 상기 기술들은 구아바 추출물 및 페오피틴화 분획물 또는 이로부터 분리된 메로테르페노이드계 화합물의 신종코로나 바이러스 감염의 예방, 개선 또는 치료 효과를 나타내는 본 발명의 구성과 상이하다.As for the patents using guava, the anti-inflammatory effect of a composition containing guava in Korean Patent No. 101491493, the antidiabetic effect of a composition containing guava in Korean Patent No. 101072905, and guava in Korean Patent No. 101320946 Although the treatment effect of prostate cancer is described, the above techniques are the composition of the present invention showing the prevention, improvement or treatment effect of novel coronavirus infection of the guava extract and the pheophytination fraction or the meroterpenoid-based compound isolated therefrom. different
본 발명의 목적은 구아바(Psidium guajava) 추출물 또는 이의 페오피틴화 분획물 또는 이로부터 분리한 메로테르페노이드계 화합물을 유효성분으로 포함하는 신종코로나 바이러스 감염의 예방, 개선 또는 치료용 조성물을 제공하는 데 있다.It is an object of the present invention to provide a composition for preventing, improving or treating novel coronavirus infection comprising, as an active ingredient, a guava ( Psidium guajava ) extract or a pheophytinated fraction thereof or a meroterpenoid-based compound isolated therefrom. have.
본 발명의 또 다른 목적은 구아바 추출물 또는 이의 페오피틴화 분획물 또는 이로부터 분리한 메로테르페노이드계 화합물을 유효성분으로 포함하는 신종코로나 바이러스 감염의 예방 또는 증상 개선용 건강기능식품을 제공하는 데 있다.Another object of the present invention is to provide a health functional food for preventing or improving symptoms of new corona virus infection, comprising as an active ingredient a guava extract or a pheophytinated fraction thereof or a meroterpenoid-based compound isolated therefrom. .
본 발명의 또 다른 목적은 구아바(Psidium guajava) 추출물의 구성성분 및 이들 간의 조합에 따른 혼합물을 포함하는 신종코로나 바이러스 감염의 예방, 개선 또는 치료에 뛰어난 상승효과를 갖는 생약조성물을 제공하는 데 있다.Another object of the present invention is to provide a herbal composition having an excellent synergistic effect in preventing, improving or treating novel coronavirus infection, comprising a mixture according to the components of the guava ( Psidium guajava ) extract and a combination thereof.
본 발명의 또 다른 목적은 구아바 추출물 또는 이의 페오피틴화 분획물 또는 이로부터 분리한 메로테르페노이드계 화합물을 유효성분으로 포함하는 신종코로나 바이러스 감염 예방용 소독제를 제공하는 데 있다.Another object of the present invention is to provide a disinfectant for preventing new corona virus infection comprising, as an active ingredient, a guava extract or a pheophytinated fraction thereof or a meroterpenoid-based compound isolated therefrom.
본 발명의 또 다른 목적은 구아바 추출물 또는 이의 페오피틴화 분획물 또는 이로부터 분리한 메로테르페노이드계 화합물을 유효성분으로 포함하는 신종코로나 바이러스 감염의 예방 또는 개선용 동물 사료용 조성물을 제공하는 데 있다.Another object of the present invention is to provide a composition for animal feed for preventing or improving novel coronavirus infection, comprising as an active ingredient a guava extract or a pheophytinated fraction thereof or a meroterpenoid-based compound isolated therefrom.
본 발명의 또 다른 목적은 구아바(Psidium guajava) 추출물의 구성성분 및 이들 간의 조합에 따른 혼합물을 유효성분으로 포함하는 신종코로나 바이러스 감염의 예방 또는 치료용 동물 약품을 제공하는 데 있다.Another object of the present invention is to provide an animal drug for preventing or treating new corona virus infection, comprising a mixture according to the components of the guava ( Psidium guajava ) extract and a combination thereof as an active ingredient.
본 발명의 목적은 구아바(Psidium guajava) 추출물의 구성성분 및 이들 간의 조합에 따른 혼합물을 유효성분으로 포함하는 신종코로나 바이러스 감염의 예방 또는 개선용 동물 사료 첨가제를 제공하는 데 있다.It is an object of the present invention to provide an animal feed additive for preventing or improving novel coronavirus infection, comprising a mixture according to the components of the guava ( Psidium guajava ) extract and a combination thereof as an active ingredient.
본 발명의 목적은 구아바(Psidium guajava) 추출물로부터 분리한 신규 화합물을 제공하는 데 있다.It is an object of the present invention to provide a novel compound isolated from guava ( Psidium guajava ) extract.
본 발명은 구아바(Psidium guajava) 추출물 또는 이의 페오피틴화 분획물 또는 이로부터 분리한 메로테르페노이드계 화합물을 유효성분으로 포함하는 것을 특징으로 하는 신종코로나 바이러스 감염의 예방 또는 치료용 조성물을 제공한다.The present invention provides a composition for preventing or treating novel coronavirus infection, comprising as an active ingredient a guava ( Psidium guajava ) extract or a pheophytinated fraction thereof or a meroterpenoid-based compound isolated therefrom.
상기 구아바(Psidium guajava) 추출물은 폴피린(porphyrin)류의 클로로필 a(chlorophyll a), 클로로필 a’(chlorophyll a’), 클로로필 b(chlorophyll b), 클로로필 b’(chlorophyll b’), 페오피틴 a(pheophytin a), 페오피틴 a’(pheophytin a’), 페오피틴 b(pheophytin b), 페오피틴 b’(pheophytin b’) 으로 이루어진 군으로부터 선택된 1종 이상을 포함할 수 있으며, 바람직하게는 페오피틴 a(pheophytin a), 페오피틴 a’(pheophytin a’), 페오피틴 b(pheophytin b), 페오피틴 b’(pheophytin b’)으로 이루어진 군으로부터 선택된 1종 이상을 포함할 수 있다.The guava ( Psidium guajava ) extract is porphyrin chlorophyll a (chlorophyll a), chlorophyll a ' (chlorophyll a '), chlorophyll b (chlorophyll b), chlorophyll b ' (chlorophyll b '), pheophytin It may include one or more selected from the group consisting of a (pheophytin a), pheophytin a' (pheophytin a'), pheophytin b (pheophytin b), and pheophytin b' (pheophytin b'), Preferably, at least one selected from the group consisting of pheophytin a, pheophytin a', pheophytin b, and pheophytin b' may include.
상기 구아바(Psidium guajava) 추출물은 구아바 잎이나 과일을 물, C1 내지 C4의 저급 알코올, 헥산, 아세톤, 메틸렌 클로라이드, 에틸아세테이트 및 이들의 혼합용매로 구성된 군으로부터 선택된 어느 하나의 용매로 추출한 것일 수 있다. 바람직하게는 상기 구아바 잎이나 과일 추출물은 구아바 잎이나 과일을 70~100% C1 내지 C4의 저급 알코올 수용액 또는 저급 알코올 또는 아세톤으로 추출한 추출물이거나, 또는, 이들의 헥산 분획물 및 추출물을 페오피틴화한 헥산 분획물일 수 있다. 상기 구아바 잎이나 과일 추출물은 구아바 잎이나 과일에 95% 에탄올을 사용하여 2시간 동안 교반하여 3회에 걸쳐 추출한 뒤 40℃에서 감압 농축한 다음, 그 추출물을 물, 0.5 N 염산수용액 또는 1%[w/v] 구연산수용액으로 현탁시킨 것을 헥산으로 용매 추출하여 획득한 헥산 분획물을 감압 농축하여 제조할 수 있거나, 추출물을 물에 현탁한 후 60℃에서 하루 이상 보관한 후 헥산으로 용매 추출한 헥산 분획물을 감압 농축하여 제조할 수 있다.The guava ( Psidium guajava ) extract may be obtained by extracting guava leaves or fruits with any one solvent selected from the group consisting of water, C1 to C4 lower alcohols, hexane, acetone, methylene chloride, ethyl acetate, and mixed solvents thereof. . Preferably, the guava leaf or fruit extract is an extract obtained by extracting guava leaves or fruits with 70-100% C1 to C4 aqueous solution of lower alcohol or lower alcohol or acetone, or hexane fractions and extracts obtained by pheophytinizing these hexane fractions and extracts It may be a fraction. The guava leaf or fruit extract was extracted three times by stirring for 2 hours using 95% ethanol on the guava leaf or fruit, and then concentrated under reduced pressure at 40° C. w/v] It can be prepared by concentration under reduced pressure of the hexane fraction obtained by solvent extraction with hexane of the suspension in citric acid solution, or the hexane fraction obtained by suspending the extract in water and storing it at 60° C. for at least one day It can be prepared by concentration under reduced pressure.
상기 폴피린(porphyrin)류의 클로로필 a(chlorophyll a), 클로로필 a’(chlorophyll a’), 클로로필 b(chlorophyll b), 클로로필 b’(chlorophyll b’), 페오피틴 a(pheophytin a), 페오피틴 a’(pheophytin a’), 페오피틴 b(pheophytin b), 페오피틴 b’(pheophytin b’)는 엽록소를 생산하는 모든 식물에서 비슷한 조건으로 제조될 수 있다.Chlorophyll a (chlorophyll a), chlorophyll a ', chlorophyll b (chlorophyll b), chlorophyll b' (chlorophyll b '), pheophytin a (pheophytin a) of the porphyrins Pheophytin a', pheophytin b, and pheophytin b' can be prepared under similar conditions in all chlorophyll-producing plants.
상기 구아바 잎이나 과일에 포함되는 폴피린계 화합물은 구아바 잎이나 과일을 70~100% C1 내지 C4의 저급 알코올 수용액 또는 저급 알코올 또는 아세톤으로 추출한 추출물의 헥산 분획물 및 추출물을 페오피틴화한 헥산 분획물을 분리하여 얻을 수 있으며, 바람직하게는 95% 에탄올로 추출한 추출물 또는 추출물을 페오피틴화한 헥산 분획물을, 크로마토그래피로 분리하여 얻을 수 있다. 상기 크로마토그래피는 순상 컬럼 크로마토그래피(normal phase column chromatography), 역상 컬럼 크로마토그래피(reverse phase column chromatography), 디아이온 HP-20 컬럼 크로마토그래피(Diaion HP-20 column chromatography), RP-18 컬럼 크로마토그래피(RP-18 column chromatography), LH-20 컬럼 크로마토그래피(LH-20 column chromatography), 조제용 역상-고성능 액체 크로마토그래피(preparative reversed-phase high performance chromatography), 중압 액체 크로마토그래피(medium pressure liquid chromatography), 고성능 액체 크로마토그래피(high-performance liquid chromatography, HPLC) 등이 이용될 수 있다.The polphyrin-based compound contained in the guava leaf or fruit is a hexane fraction of the guava leaf or fruit extracted with 70-100% C1 to C4 aqueous solution of lower alcohol or lower alcohol or acetone, and a hexane fraction obtained by pheophytinizing the extract. It can be obtained by separation, and preferably an extract extracted with 95% ethanol or a hexane fraction obtained by pheophytinizing the extract can be obtained by separating by chromatography. The chromatography is normal phase column chromatography (normal phase column chromatography), reverse phase column chromatography (reverse phase column chromatography), Diaion HP-20 column chromatography (Diaion HP-20 column chromatography), RP-18 column chromatography ( RP-18 column chromatography), LH-20 column chromatography (LH-20 column chromatography), preparative reversed-phase high performance chromatography, medium pressure liquid chromatography, High-performance liquid chromatography (HPLC) or the like may be used.
또한, 상기 구아바 추출물은 구아바 잎 또는 과일을 물, C1 내지 C4의 저급 알코올, 헥산, 아세톤, 메틸렌 클로라이드, 에틸아세테이트 및 이들의 혼합용매로 구성된 군으로부터 선택되는 1종 이상의 용매로 추출한 추출물을 증류수로 현탁한 후, 유기용매로 분획하여 얻은 분획물일 수 있다.In addition, the guava extract is a guava leaf or fruit extracted with one or more solvents selected from the group consisting of water, C1 to C4 lower alcohols, hexane, acetone, methylene chloride, ethyl acetate, and mixed solvents thereof with distilled water. After suspending, it may be a fraction obtained by fractionation with an organic solvent.
상기 유기용매는 C1~4의 저급 알코올, 에틸아세테이트, 헥산 및 아세톤 등일 수 있다. 상기 C1~4의 저급 알코올은 메탄올, 에탄올, 프로판올, 이소프로판올, 부탄올 등일 수 있다. 바람직하게는 헥산, 에틸아세테이트이고, 더 바람직하게는 헥산이다.The organic solvent may be a C1-4 lower alcohol, ethyl acetate, hexane or acetone. The C1-4 lower alcohol may be methanol, ethanol, propanol, isopropanol, butanol, or the like. Preferably they are hexane and ethyl acetate, More preferably, they are hexane.
또한, 본 발명은 하기 화학식 1의 제주구아자본 A(화합물 1), 제주구아자본 D(화합물 2), 제주구아자본 E(화합물 3) 및 제주구아자본 H(화합물 4) 중에서 선택되는 1종 이상의 화합물을 포함할 수 있다.In addition, the present invention provides at least one selected from Jeju guar capital A (compound 1 ), Jeju guar capital D (compound 2 ), Jeju guar capital E (compound 3 ) and Jeju guar capital H (compound 4 ) of the following formula 1 compounds may be included.
[화학식 1] [Formula 1]
상기 화합물은 구아바로부터 유래된 것일 수 있다.The compound may be derived from guava.
또한, 본 발명은 상기 화학식 1의 제주구아자본 A(화합물 1), 제주구아자본 D(화합물 2), 제주구아자본 E(화합물 3) 및 제주구아자본 H(화합물 4)로 이루어진 군에서 선택되는 1종 이상의 화합물을 유효성분으로 포함할 수 있으며, 바람직하게는 제주구아자본 A(화합물 1) 및 제주구아자본 D(화합물 2)로 이루어진 군에서 선택되는 1종 이상의 화합물을 포함하는 것을 특징으로 하는 신종코로나 바이러스 감염의 예방 또는 치료용 조성물 및 동물약품, 개선용 건강기능식품 조성물 및 동물 사료용 조성물에 관한 것이다.In addition, the present invention is selected from the group consisting of Jeju guar capital A (compound 1), Jeju guar capital D (compound 2), Jeju guar capital E (compound 3) and Jeju guar capital H (compound 4) of Formula 1 above It may include one or more compounds as an active ingredient, preferably comprising one or more compounds selected from the group consisting of Jeju guar capital A (compound 1) and Jeju guar capital D (compound 2) It relates to a composition for preventing or treating novel coronavirus infection, and animal medicine, a health functional food composition for improvement, and a composition for animal feed.
본 발명은 또한, 구아바 잎 또는 과일을 물, C1 내지 C4의 저급 알코올, 헥산, 아세톤, 메틸렌 클로라이드, 에틸아세테이트 및 이들의 혼합용매로 구성된 군에서 선택되는 1종 이상을 용매로 하여 추출한 구아바 추출물을 수득하는 1단계; 상기 1단계의 구아바 추출물을 유기용매로 분획하여 분획물을 확보하는 2단계; 상기 2단계의 분획물을 컬럼 크로마토그래피에 가하여 상기 화학식 1의 제주구아자본 A(화합물 1), 제주구아자본 D(화합물 2), 제주구아자본 E(화합물 3) 및 제주구아자본 H(화합물 4)로 이루어진 군에서 선택되는 1종 이상의 화합물을 분리하는 3단계; 로 이루어진 상기 화학식 1의 화합물 1~4를 분리하는 방법에 관한 것이다.The present invention also provides a guava extract obtained by extracting guava leaves or fruits using at least one selected from the group consisting of water, C1 to C4 lower alcohols, hexane, acetone, methylene chloride, ethyl acetate, and mixed solvents thereof as a solvent. Step 1 to obtain; a second step of obtaining a fraction by fractionating the guava extract of step 1 with an organic solvent; The fractions from the second step were subjected to column chromatography, and Jeju guar capital A (compound 1), Jeju guar capital D (compound 2), Jeju guar capital E (compound 3) and Jeju guar capital H (compound 4) of Formula 1 above Step 3 of isolating one or more compounds selected from the group consisting of; It relates to a method for isolating compounds 1 to 4 of Formula 1 consisting of.
이하 본 발명을 상세하게 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 구아바 추출물 및 페오피틴화 분획물 또는 이로부터 분리한 상기 화학식 1의 제주구아자본 A(화합물 1), 제주구아자본 D(화합물 2), 제주구아자본 E(화합물 3) 및 제주구아자본 H(화합물 4)로 이루어진 군에서 선택되는 1종 이상의 화합물을 유효성분으로 포함하는 것을 특징으로 하는 신종코로나 바이러스 감염의 예방 또는 치료용 조성물일 수 있다.The present invention relates to a guava extract and a pheophytinated fraction or Jeju guar capital A (compound 1), Jeju guar capital D (compound 2), Jeju guar capital E (compound 3) and Jeju guar capital H of Formula 1 isolated therefrom (Compound 4) may be a composition for preventing or treating novel coronavirus infection, characterized in that it contains one or more compounds selected from the group consisting of as an active ingredient.
상기 구아바 추출물은 폴피린(porphyrin)류의 클로로필 a(chlorophyll a), 클로로필 a’(chlorophyll a’), 클로로필 b(chlorophyll b), 클로로필 b’(chlorophyll b’), 페오피틴 a(pheophytin a), 페오피틴 a’(pheophytin a’), 페오피틴 b(pheophytin b), 페오피틴 b’(pheophytin b’) 으로 이루어진 군으로부터 선택된 1종 이상을 포함할 수 있으며, 바람직하게는 페오피틴 a(pheophytin a), 페오피틴 a’(pheophytin a’), 페오피틴 b(pheophytin b), 페오피틴 b’(pheophytin b’)으로 이루어진 군으로부터 선택된 1종 이상을 포함할 수 있다.The guava extract is porphyrin-type chlorophyll a (chlorophyll a), chlorophyll a' (chlorophyll a'), chlorophyll b (chlorophyll b), chlorophyll b' (chlorophyll b'), pheophytin a (pheophytin a) ), pheophytin a' (pheophytin a'), pheophytin b (pheophytin b), may include one or more selected from the group consisting of pheophytin b' (pheophytin b'), preferably pe It may include at least one selected from the group consisting of ophytin a (pheophytin a), pheophytin a' (pheophytin a'), pheophytin b (pheophytin b), and pheophytin b' have.
상기 구아바 추출물은 구아바 잎이나 과일을 물, C1 내지 C4의 저급 알코올, 헥산, 아세톤, 메틸렌 클로라이드, 에틸아세테이트 및 이들의 혼합용매로 구성된 군으로부터 선택된 어느 하나의 용매로 추출한 것일 수 있다. 바람직하게는 상기 구아바 잎이나 과일 추출물은 구아바 잎이나 과일을 70~100% C1 내지 C4의 저급 알코올 수용액 또는 저급 알코올 또는 아세톤으로 추출한 추출물이거나, 또는, 이들의 헥산 분획물 및 추출물을 페오피틴화한 헥산 분획물일 수 있다. 상기 구아바 잎이나 과일 추출물은 구아바 잎이나 과일에 95% 에탄올을 사용하여 2시간 동안 교반하여 3회에 걸쳐 추출한 후, 물, 0.5 N 염산수용액 또는 1%[w/v] 구연산수용액으로 현탁시킨 것을 헥산으로 용매 추출하여 획득한 헥산 분획물을 감압 농축하여 제조할 수 있거나, 추출물을 물에 현탁한 후 60℃에서 하루 이상 보관한 후 헥산으로 용매 추출한 헥산 분획물을 감압 농축하여 제조할 수 있다.The guava extract may be obtained by extracting guava leaves or fruits with any one solvent selected from the group consisting of water, C1 to C4 lower alcohols, hexane, acetone, methylene chloride, ethyl acetate, and mixed solvents thereof. Preferably, the guava leaf or fruit extract is an extract obtained by extracting guava leaves or fruits with 70-100% C1 to C4 aqueous solution of lower alcohol or lower alcohol or acetone, or hexane fractions and extracts obtained by pheophytinizing these hexane fractions and extracts It may be a fraction. The guava leaf or fruit extract was extracted three times by stirring for 2 hours using 95% ethanol on the guava leaf or fruit, and then suspended in water, 0.5 N hydrochloric acid solution or 1% [w/v] citric acid aqueous solution. It can be prepared by concentrating the hexane fraction obtained by solvent extraction with hexane under reduced pressure, or by suspending the extract in water and storing it at 60° C. for at least one day, and then concentrating the solvent-extracted hexane fraction with hexane under reduced pressure.
상기 폴피린(porphyrin)류의 클로로필 a(chlorophyll a), 클로로필 a’(chlorophyll a’), 클로로필 b(chlorophyll b), 클로로필 b’(chlorophyll b’), 페오피틴 a(pheophytin a), 페오피틴 a’(pheophytin a’), 페오피틴 b(pheophytin b), 페오피틴 b’(pheophytin b’)는 엽록소를 생산하는 모든 식물에서 비슷한 조건으로 제조될 수 있다.Chlorophyll a (chlorophyll a), chlorophyll a ', chlorophyll b (chlorophyll b), chlorophyll b' (chlorophyll b '), pheophytin a (pheophytin a) of the porphyrins Pheophytin a', pheophytin b, and pheophytin b' can be prepared under similar conditions in all chlorophyll-producing plants.
상기 구아바 잎이나 과일에 포함되는 폴피린계 화합물은 구아바 잎이나 과일을 70~100% C1 내지 C4의 저급 알코올 수용액 또는 저급 알코올 또는 아세톤으로 추출한 추출물의 헥산 분획물 및 추출물을 페오피틴화한 헥산 분획물을 분리하여 얻을 수 있으며, 바람직하게는 100% 메탄올 또는 95% 에탄올로 추출한 추출물 또는 추출물을 페오피틴화한 헥산 분획물을 크로마토그래피로 분리하여 얻을 수 있다. 상기 크로마토그래피는 순상 컬럼 크로마토그래피(normal phase column chromatography), 역상 컬럼 크로마토그래피(reverse phase column chromatography), 디아이온 HP-20 컬럼 크로마토그래피(Diaion HP-20 column chromatography), RP-18 컬럼 크로마토그래피(RP-18 column chromatography), LH-20 컬럼 크로마토그래피(LH-20 column chromatography), 조제용 역상-고성능 액체 크로마토그래피(preparative reversed-phase high performance chromatography), 중압 액체 크로마토그래피(medium pressure liquid chromatography), 고성능 액체 크로마토그래피(high-performance liquid chromatography, HPLC) 등이 이용될 수 있다.The polphyrin-based compound contained in the guava leaf or fruit is a hexane fraction of the guava leaf or fruit extracted with 70-100% C1 to C4 aqueous solution of lower alcohol or lower alcohol or acetone, and a hexane fraction obtained by pheophytinizing the extract. It can be obtained by separation, and preferably, an extract extracted with 100% methanol or 95% ethanol or a hexane fraction obtained by pheophytinizing the extract can be obtained by separating by chromatography. The chromatography is normal phase column chromatography (normal phase column chromatography), reverse phase column chromatography (reverse phase column chromatography), Diaion HP-20 column chromatography (Diaion HP-20 column chromatography), RP-18 column chromatography ( RP-18 column chromatography), LH-20 column chromatography (LH-20 column chromatography), preparative reversed-phase high performance chromatography, medium pressure liquid chromatography, High-performance liquid chromatography (HPLC) or the like may be used.
또한, 상기 구아바 추출물은 구아바 잎 또는 과일을 물, C1 내지 C4의 저급 알코올, 헥산, 아세톤, 메틸렌 클로라이드, 에틸아세테이트 및 이들의 혼합용매로 구성된 군으로부터 선택되는 1종 이상의 용매로 추출한 추출물을 증류수로 현탁한 후, 유기용매로 분획하여 얻은 분획물일 수 있다.In addition, the guava extract is a guava leaf or fruit extracted with one or more solvents selected from the group consisting of water, C1 to C4 lower alcohols, hexane, acetone, methylene chloride, ethyl acetate, and mixed solvents thereof with distilled water. After suspending, it may be a fraction obtained by fractionation with an organic solvent.
상기 유기용매는 C1~4의 저급 알코올, 에틸아세테이트, 헥산 및 아세톤 등일 수 있다. 상기 C1~4의 저급 알코올은 메탄올, 에탄올, 프로판올, 이소프로판올, 부탄올 등일 수 있다. 바람직하게는 헥산, 에틸아세테이트이고, 더 바람직하게는 헥산이다.The organic solvent may be a C1-4 lower alcohol, ethyl acetate, hexane or acetone. The C1-4 lower alcohol may be methanol, ethanol, propanol, isopropanol, butanol, or the like. Preferably they are hexane and ethyl acetate, More preferably, they are hexane.
상기 화학식 1의 화합물 1~4은 구아바 추출물로부터 분리할 수 있다. Compounds 1 to 4 of Formula 1 may be isolated from guava extract.
상기 구아바 추출물로부터 분리된 화학식 1의 화합물 1~4는 구아바 추출물을 크로마토그래피로 분획하여 얻을 수 있으며, 상기 크로마토그래피는 순상 컬럼 크로마토그래피(normal phase column chromatography), 역상 컬럼 크로마토그래피(reverse phase column chromatography), 디아이온 HP-20 컬럼 크로마토그래피(Diaion HP-20 column chromatography), RP-18 컬럼 크로마토그래피(RP-18 column chromatography), LH-20 컬럼 크로마토그래피(LH-20 column chromatography), 조제용 역상-고성능 액체 크로마토그래피(preparative reversed-phase high performance chromatography), 중압 액체 크로마토그래피(medium pressure liquid chromatography), 고성능 액체 크로마토그래피(high-performance liquid chromatography, HPLC) 등에서 선택하여 사용할 수 있다. Compounds 1 to 4 of Formula 1 isolated from the guava extract can be obtained by fractionation of the guava extract by chromatography, and the chromatography is performed by normal phase column chromatography, reverse phase column chromatography ), Diaion HP-20 column chromatography, RP-18 column chromatography, LH-20 column chromatography, Reversed phase preparation - It can be used by selecting from preparative reversed-phase high performance chromatography, medium pressure liquid chromatography, and high-performance liquid chromatography (HPLC).
한편, 본 발명의 화합물은 당해 기술 분야에서 통상적인 방법에 따라 합성될 수 있으며, 약학적으로 허용 가능한 염으로 제조될 수 있다.Meanwhile, the compound of the present invention may be synthesized according to a conventional method in the art, and may be prepared as a pharmaceutically acceptable salt.
상기 식물은 잎, 가지, 줄기, 뿌리 및 과일로 이루어진 군에서 선택되는 1종 이상이다.The plant is at least one selected from the group consisting of leaves, branches, stems, roots and fruits.
상기 조성물은 신종코로나 바이러스 감염의 예방 또는 치료용 조성물일 수 있다.The composition may be a composition for preventing or treating novel coronavirus infection.
상기 약학 조성물은 구아바 추출물 및 페오피틴화 분획물 또는 이로부터 분리된 메로테르페노이드계 화합물인 상기 화학식 1의 화합물 1~4로 이루어진 군에서 선택되는 1종 이상의 화합물 및 약학적으로 허용 가능한 담체를 포함할 수 있다.The pharmaceutical composition includes at least one compound selected from the group consisting of guava extract and pheophytination fraction or a meroterpenoid-based compound of Formula 1, which is a meroterpenoid compound isolated therefrom, and a pharmaceutically acceptable carrier. can do.
상기 구아바 추출물 및 페오피틴화 헥산 분획물 또는 이로부터 분리된 메로테르페노이드계 화합물을 포함하는 약학 조성물은 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 및 멸균 주사용액의 형태로 제형화하여 사용될 수 있다. 상기 약학 조성물에 포함될 수 있는 담체, 부형제 및 희석제로는 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로스, 메틸 셀룰로스, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있다. 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 본 발명의 구아바 추출물 및 페오피틴화 헥산 분획물 또는 이로부터 분리된 메로테르페노이드계 화합물인 상기 화학식 1의 화합물 1~4로 이루어진 군에서 선택되는 1종 이상의 화합물에 적어도 하나 이상의 부형제, 예를 들면, 전분, 탄산칼슘, 수크로스 또는 락토즈, 젤라틴 등을 섞어 조제된다. 또한 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크 같은 윤활제들도 사용된다. 경구를 위한 액상 제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조제제, 좌제가 포함된다. 비수성용제, 현탁제로는 프로필렌글리콜, 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세로젤라틴 등이 사용될 수 있다.The pharmaceutical composition comprising the guava extract and the pheophytinated hexane fraction or the meroterpenoid-based compound separated therefrom can be prepared as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc. according to a conventional method, respectively. It can be used in the form of oral dosage forms, external preparations, suppositories, and sterile injection solutions. Carriers, excipients and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose , methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and these solid preparations are the guava extract and pheophytinated hexane fraction of the present invention or the meroterpenoid compound isolated therefrom. It is prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc. with one or more compounds selected from the group consisting of compounds 1 to 4 of Formula 1. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid formulations for oral use include suspensions, solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, preservatives, etc. may be included. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like can be used.
상기 약학 조성물의 투여량은 치료받을 대상의 연령, 성별, 체중과, 치료할 특정 질환 또는 병리 상태, 질환 또는 병리 상태의 심각도, 투여경로 및 처방자의 판단에 따라 달라질 것이다. 이러한 인자에 기초한 투여량 결정은 당업자의 수준 내에 있으며, 일반적으로 투여량은 0.1~1000㎎/㎏/일의 범위이다. 더 바람직한 투여량은 0.5~500㎎/㎏/일이다. 투여는 하루에 한 번 투여할 수도 있고, 수 회 나누어 투여할 수도 있다. 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다.The dosage of the pharmaceutical composition will vary depending on the age, sex, and weight of the subject to be treated, the specific disease or pathological condition to be treated, the severity of the disease or pathological condition, the route of administration, and the judgment of the prescriber. Dosage determination based on these factors is within the level of one of ordinary skill in the art, and generally the dosage is in the range of 0.1-1000 mg/kg/day. A more preferred dosage is 0.5-500 mg/kg/day. Administration may be administered once a day, or may be administered in several divided doses. The above dosage does not limit the scope of the present invention in any way.
상기 약학 조성물은 쥐, 가축, 반려동물, 인간 등의 포유동물에 다양한 경로로 투여될 수 있다. 투여의 모든 방식은 예상될 수 있는데, 예를 들면, 경구, 직장 또는 정맥, 근육, 피하, 자궁 내 점막 또는 뇌혈관 내 주사에 의해 투여될 수 있다.The pharmaceutical composition may be administered to mammals such as mice, livestock, companion animals, and humans by various routes. Any mode of administration can be envisaged, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine mucosal or intracerebrovascular injection.
또한, 상기 조성물은 신종코로나 바이러스 감염의 예방 또는 치료용 식품 조성물일 수 있다.In addition, the composition may be a food composition for preventing or treating novel coronavirus infection.
상기 건강기능식품 조성물은 구아바 추출물 또는 이의 페오피틴화 분획물 또는 이로부터 분리한 메로테르페노이드계 화합물인 상기 화학식 1의 화합물 1~4로 이루어진 군에서 선택되는 1종 이상의 화합물 및 식품학적으로 허용 가능한 식품보조 첨가제를 포함할 수 있다.The health functional food composition includes at least one compound selected from the group consisting of compounds 1 to 4 of Formula 1, which is a guava extract or a pheophytinated fraction thereof, or a meroterpenoid-based compound isolated therefrom, and a food pharmaceutically acceptable It may contain food supplement additives.
상기 구아바 추출물 또는 이의 페오피틴화 분획물 또는 이로부터 분리한 메로테르페노이드계 화합물인 상기 화학식 1의 화합물 1~4로 이루어진 군에서 선택되는 1종 이상의 화합물은 본 발명의 건강기능식품 조성물 총 중량을 기준으로 0.001~100중량%로 하여 포함될 수 있다.At least one compound selected from the group consisting of compounds 1 to 4 of Formula 1, which is the guava extract or a pheophytinated fraction thereof, or a meroterpenoid-based compound isolated therefrom, comprises the total weight of the health functional food composition of the present invention. It may be included as 0.001 to 100% by weight as a basis.
본 발명의 식품 조성물은 정제, 캡슐제, 환제 또는 액제 등의 형태를 포함하며, 본 발명의 구아바 추출물 또는 이의 페오피틴화 분획물 또는 이로부터 분리한 메로테르페노이드계 화합물인 상기 화학식 1의 화합물 1~4로 이루어진 군에서 선택되는 1종 이상의 화합물을 첨가할 수 있는 식품으로는, 예를 들어, 각종 식품류, 음료, 껌, 차, 비타민 복합제, 건강기능성식품류 등이 있다.The food composition of the present invention includes the form of tablets, capsules, pills or liquids, and the guava extract of the present invention or a pheophytinated fraction thereof or a meroterpenoid-based compound isolated therefrom, Compound 1 of Formula 1 Foods to which one or more compounds selected from the group consisting of 4 can be added include, for example, various foods, beverages, gums, tea, vitamin complexes, health functional foods, and the like.
본 발명의 또 다른 하나의 양태는, 본 발명의 상기 구아바 추출물 또는 이의 페오피틴화 분획물 또는 이로부터 분리한 메로테르페노이드계 화합물인 상기 화학식 1의 화합물 1~4로 이루어진 군에서 선택되는 1종 이상의 화합물을 포함하는 조성물은 신종코로나 바이러스 감염의 예방 또는 개선용 동물 사료용 조성물을 제공한다.Another aspect of the present invention is one selected from the group consisting of compounds 1 to 4 of Formula 1, which is the guava extract or the pheophytinated fraction thereof of the present invention or a meroterpenoid-based compound isolated therefrom. The composition comprising the above compound provides a composition for animal feed for preventing or improving novel coronavirus infection.
상기 동물 사료용 조성물은 사료 첨가제를 포함할 수 있다. 본 발명의 사료첨가제는 사료 관리법상의 보조 사료에 해당한다.The composition for animal feed may include a feed additive. The feed additive of the present invention corresponds to an auxiliary feed under the Feed Management Act.
상기 동물 사료의 종류는 특별히 제한되지 아니하며, 당해 기술 분야에서 통상적으로 사용되는 사료를 사용할 수 있다. 상기 사료의 비제한적인 예로는, 곡물류, 근과류, 식품 가공 부산물류, 조류, 섬유질류, 제약 부산물류, 유지류, 전분류, 박류 또는 곡물 부산물류 등과 같은 식물성 사료; 단백질류, 무기물류, 유지류, 광물성류, 유지류, 단세포 단백질류, 동물성 플랑크톤류 또는 음식물 등과 같은 동물성 사료를 들 수 있다. 이들은 단독으로 사용되거나 2종 이상을 혼합하여 사용될 수 있다.The type of animal feed is not particularly limited, and feeds commonly used in the art may be used. Non-limiting examples of the feed include plant feeds such as grains, root fruits, food processing by-products, algae, fibers, pharmaceutical by-products, oils and fats, starches, gourds or grain by-products; and animal feeds such as proteins, inorganic materials, oils and fats, minerals, oils and fats, single cell proteins, zooplankton, or food. These may be used alone or in combination of two or more.
본 발명은 또한, 상기 화학식 1의 화합물 1~4을 분리하는 방법에 관한 것으로, 구아바를 잎 또는 과일을 물, C1 내지 C4의 저급 알코올, 헥산, 아세톤, 메틸렌 클로라이드, 에틸아세테이트 및 이들의 혼합용매로 구성된 군에서 선택되는 1종 이상을 용매로 하여 추출한 구아바 추출물을 수득하는 1단계; 상기 1단계의 구아바 추출물을 유기용매로 분획하여 분획물을 확보하는 2단계; 상기 2단계의 분획물을 컬럼 크로마토그래피에 가하여 하기 화학식 1의 제주구아자본 A(화합물 1), 제주구아자본 D(화합물 2), 제주구아자본 E(화합물 3) 및 제주구아자본 H(화합물 4)로 이루어진 군에서 선택되는 1종 이상의 화합물을 분리하는 3단계;로 이루어질 수 있다.The present invention also relates to a method for isolating compounds 1 to 4 of Formula 1, wherein guava leaves or fruits are mixed with water, C1 to C4 lower alcohols, hexane, acetone, methylene chloride, ethyl acetate, and mixed solvents thereof. Step 1 to obtain a guava extract extracted by using at least one selected from the group consisting of as a solvent; a second step of obtaining a fraction by fractionating the guava extract of step 1 with an organic solvent; The fractions from the second step were subjected to column chromatography, and Jeju guar capital A (compound 1), Jeju guar capital D (compound 2), Jeju guar capital E (compound 3) and Jeju guar capital H (compound 4) of the following formula 1 were added to column chromatography. 3 steps of isolating one or more compounds selected from the group consisting of;
상기 2단계의 유기용매는 C1~4의 저급 알코올, 에틸아세테이트, 헥산 및 아세톤 등일 수 있다. 상기 C1~4의 저급 알코올은 메탄올, 에탄올, 프로판올, 이소프로판올, 부탄올 등일 수 있다. 바람직하게는 헥산, 에틸아세테이트이고, 더 바람직하게는 헥산이다.The organic solvent of the second step may be a C1-4 lower alcohol, ethyl acetate, hexane, acetone, or the like. The C1-4 lower alcohol may be methanol, ethanol, propanol, isopropanol, butanol, or the like. Preferably they are hexane and ethyl acetate, More preferably, they are hexane.
상기 2단계의 분획물은 상기 1단계의 농축한 추출물을 증류수로 현탁한 후, 헥산으로 분획한 헥산 분획물이 가장 바람직하다.The fraction of step 2 is most preferably a hexane fraction obtained by suspending the concentrated extract of step 1 in distilled water and then fractionating with hexane.
상기 3단계의 컬럼 크로마토그래피는 순상 컬럼 크로마토그래피, 역상 컬럼 크로마토그래피, 디아이온 HP-20 컬럼 크로마토그래피, RP-18 컬럼 크로마토그래피, LH-20 컬럼 크로마토그래피, 조제용 역상-고성능 액체 크로마토그래피, 중압 액체 크로마토그래피, 고성능 액체 크로마토그래피 등에서 선택하여 사용할 수 있다.The column chromatography of the above three steps is normal phase column chromatography, reversed phase column chromatography, Diaion HP-20 column chromatography, RP-18 column chromatography, LH-20 column chromatography, preparative reverse phase-high performance liquid chromatography, It can be used by selecting from medium pressure liquid chromatography, high performance liquid chromatography, and the like.
본 발명은 구아바 추출물 또는 이의 페오피틴화 분획물 또는 이로부터 분리한 메로테르페노이드계 화합물을 유효성분으로 포함하는 신종코로나 바이러스 감염의 예방, 개선 또는 치료용 조성물에 관한 것으로, 구아바 추출물 및 페오피틴화 분획물 또는 이로부터 분리한 메로테르페노이드계 화합물이 신종코로나 바이러스 감염에 대하여 세포병변 회복능을 나타냄을 확인하였다.The present invention relates to a composition for the prevention, improvement or treatment of novel coronavirus infection comprising, as an active ingredient, a guava extract or a pheophytinated fraction thereof or a meroterpenoid-based compound isolated therefrom, a guava extract and a pheophytinated It was confirmed that the fraction or the meroterpenoid-based compound isolated therefrom exhibited cell lesion recovery ability against novel coronavirus infection.
이를 통해, 본 발명의 구아바 추출물 또는 이의 페오피틴화 분획물 또는 이로부터 분리한 메로테르페노이드계 화합물을 신종코로나의 감염의 예방, 개선 또는 치료용 의약품, 식품, 의료용품, 동물 사료용 조성물 개발에 이용할 수 있을 것으로 기대된다.Through this, the guava extract of the present invention or its pheophytinated fraction or a meroterpenoid-based compound isolated therefrom can be used for the prevention, improvement or treatment of new corona infection, food, medical supplies, and composition for animal feed. it is expected that it will be possible
도 1은 신종 코로나 바이러스(SARS-CoV-2)에 저해효과를 가지는 구아바 잎에서 분리한 화합물의 화학구조를 보여준 그림이다.1 is a diagram showing the chemical structure of a compound isolated from guava leaves having an inhibitory effect on novel coronavirus (SARS-CoV-2).
도 2은 구아바의 잎 추출물, 이의 헥산 분획물 및 무기산을 이용한 페오피틴화 분획물에서 메로테르페노이드계 화합물의 존재유무를 나타내주는 크로마토그램이다.2 is a chromatogram showing the presence or absence of meroterpenoid compounds in a leaf extract of guava, a hexane fraction thereof, and a pheophytination fraction using an inorganic acid.
도 3는 구아바의 잎 추출물, 건잎 추출물의 헥산 분획물 및 무기산을 이용한 페오피틴계 화합물의 크로마토그램이다.3 is a chromatogram of a pheophytin-based compound using a leaf extract of guava, a hexane fraction of a dried leaf extract, and an inorganic acid.
도 4는 구아바의 잎 추출물을 각 조건의 유기산처리하여 얻은 페오피틴화 분획물의 크로마토그램이다.4 is a chromatogram of a pheophytinated fraction obtained by treating a leaf extract of guava with an organic acid under each condition.
도 5는 구아바 잎 추출물을 1시간~48시간 열처리를 통한 페오피틴화 분획물의 크로마토그램이다.5 is a chromatogram of a pheophytinated fraction through heat treatment of a guava leaf extract for 1 hour to 48 hours.
도 6은 구아바의 잎 추출물 및 페오피틴화 반응물을 포함한 각 분획물의 신종 코로나 바이러스(SARS-CoV-2) 감염에 대한 세포병변 회복능을 나타낸 그래프이다.6 is a graph showing the cell lesion recovery ability for novel coronavirus (SARS-CoV-2) infection of each fraction including a leaf extract of guava and a pheophytination reaction.
도 7은 구아바의 잎 추출물로부터 분리한 화합물 1~4의 SARS-CoV-2 감염에 대한 세포병변 회복능을 나타낸 그래프이다.7 is a graph showing the cell lesion recovery ability against SARS-CoV-2 infection of compounds 1 to 4 isolated from leaf extracts of guava.
대한민국의 남해 상에 위치하는 제주도는 지구 온난화로 인해 점진적으로 아열대 기후에 가까워짐에 따라 제주도에 위치한 많은 농가에서 구아바를 재배하고 있다. 본 발명자들은 신종코로나 바이러스(SARS-CoV-2) 감염에 대하여 회복능을 보이는 구아바 추출물을 연구하는 과정에서, 구아바 추출물과 분획물 또는 프지디움 메로테르페노이드를 기본 골격으로하는 구성화합물 및 이들 구성화합물 간의 조합이 신종코로나의 예방 및 개선에 효과가 있음을 발견하여 화학적 방법을 사용함으로 추출물의 활성성분의 함량을 높이고 구성화합물 간의 상승효과를 나타내도록 혼합하여 제조함으로써 본 발명을 완성하였다.Jeju Island, located in the South Sea of Korea, is gradually approaching a subtropical climate due to global warming, and many farms located on Jeju Island are cultivating guava. The present inventors, in the process of studying guava extracts showing recovery ability against novel coronavirus (SARS-CoV-2) infection, guava extracts and fractions or constituent compounds having phosidium meroterpenoid as a basic skeleton, and these constituent compounds It was discovered that the combination of liver is effective in preventing and improving the novel corona, and by using a chemical method, the content of the active ingredient of the extract is increased, and the present invention is completed by mixing it to show a synergistic effect between the constituent compounds.
이하, 본 발명의 바람직한 실시예들을 상세히 설명하기로 한다. 그러나, 본 발명은 여기서 설명되는 실시예에 한정 되지 않고 다른 형태로 구체화될 수도 있다. 오히려, 여기서 소개되는 내용이 철저하고 완전해지고, 그리고 당업자에게 본 발명의 사상을 충분히 전달하기 위해 제공하는 것이다.Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, it is provided so that this disclosure will be thorough and complete, and will fully convey the spirit of the invention to those skilled in the art.
<실시예 1 : 구아바 추출물 제조 및 추출물로부터 화합물 분리><Example 1: Preparation of Guava Extract and Compound Separation from Extract>
1.1 구아바 추출물 및 분획물의 제조1.1 Preparation of Guava Extracts and Fractions
본 발명의 구아바의 생잎 및 열풍건조된 건잎은 대한민국 제주시 애월읍 중엄리에 위치한 중엄농장에서 구입한 것을 이용하였다. 구아바 건잎 10 kg을 잘게 부순 후 95 % [v/v] 에탄올 50ℓ를 가한 후 99분 동안 초음파 추출기로 3회에 걸쳐 추출하고, 이를 감압 농축하여, 95% 에탄올 추출물 1.45 kg을 수득하였다.The raw leaves and hot air-dried dried leaves of guava of the present invention were purchased from Jungeom Farm located in Jungeom-ri, Aewol-eup, Jeju-si, Korea. After 10 kg of dried guava leaves were crushed, 50 L of 95% [v/v] ethanol was added, and the mixture was extracted three times with an ultrasonic extractor for 99 minutes, and concentrated under reduced pressure to obtain 1.45 kg of a 95% ethanol extract.
이와 동시에, 표준화된 추출법을 정립하기 위하여 동일한 시료 5 g을 50 mL의 95% 에탄올로 교반 추출하였다(40°C, 1.5 시간/회, 총 3회). 동결건조한 후, 물에 현탁하여 n-헥산, 에틸아세테이트, n-부탄올의 순서대로 용매분획을 실시하였다. 최종적으로 추출물을 비롯한 각 분획물을 동결건조기를 통하여 말린 후, DMSO에 녹여 10 mg/mL로 만들어 항바이러스 활성 평가 실험에 사용하였다.At the same time, in order to establish a standardized extraction method, 5 g of the same sample was stirred and extracted with 50 mL of 95% ethanol (40°C, 1.5 hours/time, 3 times in total). After freeze-drying, it was suspended in water to perform solvent fractionation in the order of n -hexane, ethyl acetate, and n -butanol. Finally, each fraction including the extract was dried through a lyophilizer, dissolved in DMSO to make 10 mg/mL, and used in an antiviral activity evaluation experiment.
1.2 구아바 추출물로부터 화합물 분리1.2 Compound Isolation from Guava Extract
상기 실시예 1.1에서 수득한 추출물을 크로마토그래피를 통하여 순차적으로 분획물을 얻고, 각 분획물의 항바이러스 활성 평가를 실시하여 가장 항바이러스 활성이 뛰어난 분획으로부터 4종의 신규화합물을 분리하였다.Fractions were sequentially obtained through chromatography of the extract obtained in Example 1.1, and antiviral activity of each fraction was evaluated to separate four new compounds from the fraction with the highest antiviral activity.
먼저, 상시 실시예 1.1에서 수득한 추출물을 증류수 2.0ℓ에 현탁한 후 n-헥산, 에틸아세테이트로 순차적 용매분획하여 n-헥산 분획물(240.3 g)을 얻었다. 이후 n-헥산 분획 (240.3 g)을 n-헥산:에틸아세테이트:메탄올 (1:0:0 → 0:0:1[v:v:v])의 농도구배 용출 조건으로 순상 컬럼 크로마토그래피(normal phase column chromatography)를 실시하여 12개의 분획을 얻었다 (F.1 - 12). F.4 분획물(33.7 g)에 대하여 n-헥산:에틸아세테이트 (1:0 → 9:1[v:v])의 농도 구배 조건으로 순상 컬럼 크로마토그래피를 실시하여 7개의 소분획을 얻었다 (F.4-1 - 4-7). F.4-7에 대하여 메탄올:증류수 (0.1% 폼산) (9:1 → 0:1[v:v]) 농도 구배 조건으로 역상 MPLC를 실시하여 다시 4개의 소분획을 얻었다 (F.4-7-1 - 4-7-4). F. 4-7-3 분획에 대하여 아세토니트릴(acetonitrile):증류수 (91:9 → 93:7[v:v])을 이동상으로 HPLC[컬럼:YMC Triart C18 column 10 × 250 mm; 입자 크기:5 μm; 유속:3.6 ml/min; UV detection: 254 nm]를 실시하여 화합물 1 (8.4 mg, t
R = 47.2 min)을 얻었다. F.5 (18.0 g)에 대하여 n-헥산:에틸아세테이트 (99:1 → 9:1[v/v])의 농도 구배 조건으로 순상 컬럼 크로마토그래피를 실시하여 8개의 소분획을 얻었다 (F.5-1 - 5-8). F.5-6 에 대하여 메탄올:증류수 (0.1% 폼산) (9:1 → 0:1[v/v]) 농도 구배 조건으로 역상 MPLC를 실시하여 다시 6개의 소분획을 얻었다 (F.5-6-1 - 5-6-6). F.5-6-3에 대하여 아세토니트릴:증류수(0.1% 폼산) (87:13 → 90:10[v/v])을 이동상으로 HPLC [컬럼:YMC Triart C18 column 10 × 250 mm; 입자크기:5 μm; 유속:3.0 ml/min; UV detection: 254 nm]를 실시하여 화합물 2 (21.0 mg, t
R = 46.0 min), 화합물 3 (4.0 mg, t
R = 46.5 min), 그리고 화합물 4 (8.1 mg, t
R = 31.3 min)을 얻었다. First, the extract obtained in Example 1.1 was always suspended in 2.0 L of distilled water, followed by sequential solvent fractionation with n -hexane and ethyl acetate to obtain an n -hexane fraction (240.3 g). Then, the n -hexane fraction (240.3 g) was subjected to normal phase column chromatography under gradient elution conditions of n -hexane:ethyl acetate:methanol (1:0:0 → 0:0:1[v:v:v]) (normal phase column chromatography) phase column chromatography) was performed to obtain 12 fractions (F.1 - 12). F.4 fraction (33.7 g) was subjected to normal phase column chromatography under gradient conditions of n -hexane:ethyl acetate (1:0 → 9:1 [v:v]) to obtain 7 small fractions (F. .4-1 - 4-7). For F.4-7, reverse-phase MPLC was performed under a gradient condition of methanol: distilled water (0.1% formic acid) (9:1 → 0:1 [v:v]) to obtain 4 small fractions again (F.4- 7-1 - 4-7-4). F. For fractions 4-7-3, HPLC [column: YMC Triart C18 column 10×250 mm; Particle size: 5 μm; flow rate: 3.6 ml/min; UV detection: 254 nm] to obtain compound 1 (8.4 mg, t R = 47.2 min). F.5 (18.0 g) was subjected to normal phase column chromatography under gradient conditions of n -hexane:ethyl acetate (99:1 → 9:1 [v/v]) to obtain 8 small fractions (F. 5-1 - 5-8). For F.5-6, reverse-phase MPLC was performed under a gradient condition of methanol: distilled water (0.1% formic acid) (9:1 → 0:1 [v/v]) to obtain 6 small fractions again (F.5- 6-1 - 5-6-6). For F.5-6-3, HPLC [column: YMC Triart C18 column 10×250 mm; Particle size: 5 μm; flow rate: 3.0 ml/min; UV detection: 254 nm] to obtain compound 2 (21.0 mg, t R = 46.0 min), compound 3 (4.0 mg, t R = 46.5 min), and compound 4 (8.1 mg, t R = 31.3 min) .
<실시예 2 : 화합물의 물리화학적 구조 확인><Example 2: Confirmation of the physicochemical structure of the compound>
실시예 2.1 제주구아자본 A(화합물 1).Example 2.1 Jeju Guar Capital A (Compound 1).
Jejuguajavone A;Jejuguajavone A;
노란색을 띤 점성 물질;yellowish viscous substance;
UV (메탄올) : λ
max (log ε) 205 (4.52), 287 (4.69) ㎚;UV (methanol): λ max (log ε) 205 (4.52), 287 (4.69) nm;
IR (KBr) ν
max : 2954, 2927, 2870, 1634, 1442, 1383, 1306, 1178, 856㎝-1;IR (KBr) ν max: 2954, 2927, 2870, 1634, 1442, 1383, 1306, 1178, 856 cm -1 ;
HRESIMS m/z 439.2476 [M - H]- (calcd for C27H35O5, 439.2484);HRESIMS m / z 439.2476 [M - H] - (calcd for C 27 H 35 O 5 , 439.2484);
1H NMR 및 13C NMR 데이터는 하기 표 1 및 표 2 참조. 1 H NMR and 13 C NMR data see Tables 1 and 2 below.
2.2 제주구아자본 D(화합물 2).2.2 Jeju Guar Capital D (Compound 2).
Jejuguajavone D;Jejuguajavone D;
노란색을 띤 점성 물질;yellowish viscous substance;
UV (메탄올) λ
max (log ε) : 296㎚ (4.72);UV (methanol) λ max (log ε): 296 nm (4.72);
IR (KBr) ν
max : 3410, 2953, 2928, 2867, 1614, 1420, 1369, 1206, 1165, 1135, 895㎝-1;IR (KBr) ν max : 3410, 2953, 2928, 2867, 1614, 1420, 1369, 1206, 1165, 1135, 895 cm -1 ;
HRESIMS m/z 439.2849 [M - H]- (calcd for C28H39O4, 439.2848);HRESIMS m / z 439.2849 [M - H] - (calcd for C 28 H 39 O 4 , 439.2848);
1H NMR 및 13C NMR 데이터는 하기 표 1 및 표 2 참조. 1 H NMR and 13 C NMR data see Tables 1 and 2 below.
2.3 제주구아자본 E(화합물 3).2.3 Jeju Guar Capital E (Compound 3).
Jejuguajavone E;Jejuguajavone E;
노란색을 띤 점성 물질;yellowish viscous substance;
UV (메탄올) λ
max (log ε) : 296㎚ (4.93);UV (methanol) λ max (log ε): 296 nm (4.93);
IR (KBr) ν
max : 3415, 2952, 2928, 2861, 1614, 1461, 1421, 1377, 1162, 1136, 889㎝-1;IR (KBr) ν max : 3415, 2952, 2928, 2861, 1614, 1461, 1421, 1377, 1162, 1136, 889 cm -1 ;
HRESIMS m/z 453.3013 [M - H]- (calcd for C29H41O4, 453.3005);HRESIMS m / z 453.3013 [M - H] - (calcd for C 29 H 41 O 4 , 453.3005);
1H NMR 및 13C NMR 데이터는 하기 표 1 및 표 2 참조. 1 H NMR and 13 C NMR data see Tables 1 and 2 below.
2.4 제주구아자본 H(화합물 4).2.4 Jeju Guar Capital H (Compound 4).
Jejuguajavone H;Jejuguajavone H;
무색 점성 물질;colorless viscous substance;
[α] -40° [(-)-4] (c 0.10, 메탄올); [α] +37° [(+)-4] (c 0.10, 메탄올)[α] -40° [(-)- 4 ] ( c 0.10, methanol); [α] +37° [(+)- 4 ] ( c 0.10, methanol)
UV (메탄올) λ
max (log ε) : 297㎚ (4.31);UV (methanol) λ max (log ε): 297 nm (4.31);
IR (KBr) ν
max : 3421, 2954, 2930, 2868, 1611, 1420, 1368, 1295, 1245, 1207, 1164, 1132, 1083, 1045, 984㎝-1;IR (KBr) ν max : 3421, 2954, 2930, 2868, 1611, 1420, 1368, 1295, 1245, 1207, 1164, 1132, 1083, 1045, 984 cm -1 ;
HRESIMS m/z 439.2844 [M - H]- (calcd for C28H39O4, 439.2848);HRESIMS m / z 439.2844 [M - H] - (calcd for C 28 H 39 O 4 , 439.2848);
1H NMR 및 13C NMR 데이터는 하기 표 1 및 표 2 참조. 1 H NMR and 13 C NMR data see Tables 1 and 2 below.
1H-NMR Spectroscopic Data (in CDCl3) 1 H-NMR Spectroscopic Data (in CDCl 3 ) | ||||
NoNo |
화합물 1
a
|
화합물 2
b
|
화합물 3
a
|
화합물 4
a
|
1One | 2.14, m2.14, m | 2.12, m2.12, m | 2.09, m2.09, m |
1.85, m1.85, |
22 |
α 1.71, m β 1.57, mα 1.71, m β 1.57, m |
α 1.55, m β 1.69, mα 1.55, m β 1.69, m |
α 1.57, m β 1.46, mα 1.57, m β 1.46, m |
α 1.34, m β 1.16, mα 1.34, m β 1.16, |
33 |
α 2.50, m β 2.15, mα 2.50, m β 2.15, m |
α 2.45, m β 2.16, mα 2.45, m β 2.16, m |
α 2.46, ddd (13.5, 10.0, 2.5) β 2.11, mα 2.46, ddd (13.5, 10.0, 2.5) β 2.11, m |
α 2.11, m β 1.85, mα 2.11, m β 1.85, |
55 | 2.40, m2.40, m | 2.43, m2.43, m | 2.63, q (9.0)2.63, q (9.0) | 5.02, dd (12.0, 4.2)5.02, dd (12.0, 4.2) |
66 |
α 1.70, m β 1.62, mα 1.70, m β 1.62, m |
α 1.67, m β 1.61, mα 1.67, m β 1.61, m |
α 1.66, m β 1.76, dd (10.5, 8.0)α 1.66, m β 1.76, dd (10.5, 8.0) |
α 2.18, t (12.6, 4.2) β 1.76, dd (12.6, 4.2)α 2.18, t (12.6, 4.2) β 1.76, dd (12.6, 4.2) |
88 | 1.97, m1.97, m | 1.95, m1.95, m | 1.63, m1.63, m |
5.15, m5.15, |
99 |
α 1.83, dddd (15.6, 12.6, 5.4, 3.0) β 1,44, mα 1.83, dddd (15.6, 12.6, 5.4, 3.0) |
α 1.87, dddd (15.0, 12.5, 5.5, 3.0) β 1,45, dddd (15.0, 11.0, 5.5, 3.0)α 1.87, dddd (15.0, 12.5, 5.5, 3.0) |
α 1.54, m β 1,51, mα 1.54, m |
5.15, m5.15, |
1010 |
α 2.09, m β 1.94, mα 2.09, m β 1.94, m |
α 2.16, m β 1.93, mα 2.16, m β 1.93, m |
α 1.84, m β 2.14, mα 1.84, m β 2.14, m |
α 2.36, m β 2.60, d (14.4)α 2.36, m β 2.60, d (14.4) |
1212 | 0.99, s0.99, s | 0.98, s0.98, s | 0.94, s0.94, s | 1.01, s1.01, s |
1313 | 1.00, s1.00, s | 0.97, s0.97, s | 0.98, s0.98, s | 1.05, s1.05, s |
1414 | 1.11, s1.11, s | 1.14, s1.14, s | 1.08, s1.08, s | 1.13, s1.13, s |
1515 |
4.94, br s 4.89, br s4.94, br s 4.89, br s |
4.87, br s 4.84, br s4.87, br s 4.84, br s |
4.81, br s 4.72, br s4.81, br s 4.72, br s |
1.62, s1.62, s |
1‘One' | 2.45, m2.45, m |
2.55, m 2.08, m2.55, m 2.08, m |
2.04, m 2.78, m2.04, m 2.78, m |
α 2.05, m β 2.91, dd (15.6, 6.0)α 2.05, m β 2.91, dd (15.6, 6.0) |
8‘8' |
2.18, m 1.43, m2.18, m 1.43, m |
-- | -- | -- |
9‘9' |
1.02, m 0.83, m1.02, m 0.83, m |
3.06, dd (16.0, 6.0) 2.79, dd (16.0, 7.5)3.06, dd (16.0, 6.0) 2.79, dd (16.0, 7.5) |
2.97, td (7.5, 5.5)2.97, td (7.5, 5.5) |
2.95, dd (14.4, 6.6) 2.79, dd (14.4, 6.0)2.95, dd (14.4, 6.6) 2.79, dd (14.4, 6.0) |
10‘10' | 0.81, m0.81, m | 2.22, m2.22, m | 1.63, m1.63, m | 2.21, m2.21, m |
11‘11' | 10.00, s10.00, s | 0.91, d (6.5)0.91, d (6.5) | 1.32, m1.32, m | 0.95, d (6.6)0.95, d (6.6) |
12‘12' | 10.15, s10.15, s | 0.96, d (6.5)0.96, d (6.5) | 1.34, m1.34, m | 0.93, d (6.6)0.93, d (6.6) |
13‘13' | -- | 2.03, s2.03, s | 0.89, m0.89, m | 2.06, s2.06, s |
14‘14' | -- | -- | 2.04, s2.04, s | -- |
5‘-OH5'-OH | 13.31, s13.31, s | 14.14, s14.14, s | 14.18, s14.18, s | 14.17, s14.17, s |
7’-OH7'-OH | 13.40, s13.40, s | 5.19, s5.19, s | 5.20, s5.20, s | 5.23, br s5.23, br s |
acquired at a600 MHz, b500MHz. acquired at a 600 MHz, b 500 MHz. |
13C-NMR Spectroscopic Data (in CDCl3) 13 C-NMR Spectroscopic Data (in CDCl 3 ) | ||||
NoNo |
화합물 1
a
|
화합물 2
b
|
화합물 3
a
|
화합물 4
a
|
1One | 39.0 39.0 | 33.6 33.6 | 33.6 33.6 | 34.8 34.8 |
22 | 33.1 33.1 | 34.1 34.1 | 34.3 34.3 | 30.5 30.5 |
33 | 35.7 35.7 | 35.3 35.3 | 35.3 35.3 | 37.9 37.9 |
44 | 152.3152.3 | 152.2 152.2 | 152.4 152.4 | 136.6 136.6 |
55 | 42.7 42.7 | 42.1 42.1 | 42.1 42.1 | 123.2 123.2 |
66 | 36.4 36.4 | 36.5 36.5 | 36.6 36.6 | 41.5 41.5 |
77 | 33.8 33.8 | 33.7 33.7 | 33.8 33.8 | 38.3 38.3 |
88 | 53.6 53.6 | 52.7 52.7 | 52.8 52.8 | 142.9 142.9 |
99 | 22.6 22.6 | 23.0 23.0 | 23.0 23.0 | 120.2 120.2 |
1010 | 38.0 38.0 | 37.9 37.9 | 38.0 38.0 | 42.8 42.8 |
1111 | 84.0 84.0 | 82.1 82.1 | 82.1 82.1 | 82.2 82.2 |
1212 | 22.2 22.2 | 22.1 22.1 | 22.2 22.2 | 24.3 24.3 |
1313 | 30.6 30.6 | 30.3 30.3 | 30.3 30.3 | 30.3 30.3 |
1414 | 21.4 21.4 | 20.8 20.8 | 20.9 20.9 | 20.0 20.0 |
1515 | 110.4 110.4 | 110.4 110.4 | 110.2 110.2 | 17.2 17.2 |
1‘One' | 35.1 35.1 | 26.1 26.1 | 26.2 26.2 | 23.8 23.8 |
2‘2' | 106.1106.1 | 99.6 99.6 | 99.7 99.7 | 99.4 99.4 |
3‘3' | 164.5164.5 | 154.5 154.5 | 154.7 154.7 | 154.5 154.5 |
4‘4' | 105.1105.1 | 106.0 106.0 | 105.8 105.8 | 106.0 106.0 |
5‘5' | 167.9167.9 | 162.6 162.6 | 162.6 162.6 | 162.8 162.8 |
6‘6' | 104.1104.1 | 101.1 101.1 | 101.5 101.5 | 101.4 101.4 |
7‘7' | 169.1169.1 | 157.3 157.3 | 157.4 157.4 | 157.4 157.4 |
8‘8' | 31.6 31.6 | 205.8 205.8 | 206.4 206.4 | 206.1 206.1 |
9‘9' | 18.3 18.3 | 53.3 53.3 | 44.6 44.6 | 53.4 53.4 |
10‘10' | 14.5 14.5 | 25.0 25.0 | 24.8 24.8 | 25.5 25.5 |
11‘11' | 192.3192.3 | 23.0 23.0 | 31.8 31.8 | 23.0 23.0 |
12‘12' | 192.0 192.0 | 22.6 22.6 | 22.7 22.7 | 22.8 22.8 |
13‘13' | 7.0 7.0 | 14.1 14.1 | 7.17.1 | |
14‘14' | 7.1 7.1 | |||
acquired at a150 MHz, b125MHz. acquired at a 150 MHz, b 125 MHz. |
<실시예 3. 구아바 잎 추출물의 페오피틴화 및 분획물의 제조>페오피틴 A 및 B는 폴피린계 화합물로, 조리과정 또는 소화과정을 통하여 탈메탈화, 탈피틸화, 탈카르보메톡실화가 일어나 페오피틴류(pheophytin), 파이로페오피틴류(pyropheophytin), 클로로필라이드류(chlorophyllide), 페오폴바이드류(pheophorbide) 등의 유도체가 생성되는 것으로 알려져 있다(Hayes & Ferruzzi, 2020). 본 발명자들은 기초실험을 통해 구아바 추출물, 특히 페오피틴화 분획물의 SARS-CoV-2에 대한 항바이러스 활성을 확인하였으며, 또한 상기 실시예 2에서 수득한 4종의 화합물이 페오피틴화 분획물에도 풍부하게 포함되어 있음을 확인하였다. 이에 따라, 구아바 잎 추출물로부터 페오피틴의 함량을 현저히 높일 수 있는 페오피틴화 분획물을 제조하는 방법을 확립하였다. <Example 3. Pheophytination and preparation of fractions of guava leaf extract> Pheophytins A and B are polphyrin-based compounds, which are demetallized, dephytylated, and decarbomethoxylated through a cooking process or digestion process. It is known that derivatives such as pheophytin, pyropheophytin, chlorophyllide, and pheophorbide are produced (Hayes & Ferruzzi, 2020). The present inventors confirmed the antiviral activity against SARS-CoV-2 of the guava extract, particularly the pheophytinated fraction, through a basic experiment, and the four compounds obtained in Example 2 were also abundant in the pheophytinated fraction. It was confirmed that it was included. Accordingly, a method for preparing a pheophytinated fraction capable of significantly increasing the content of pheophytin from guava leaf extract was established.
3.1 무기산을 이용한 구아바 잎 추출물의 페오피틴화 및 분획물의 제조3.1 Pheophytination of Guava Leaf Extract Using Inorganic Acids and Preparation of Fractions
무기산을 이용한 페오피틴화 반응을 확립하기 위하여 구아바 건잎 추출물을 0.5 N의 염산수용액을 사용하여 현탁시키고 n-헥산과 5분 가량 용매분획을 한 후, 동일 부피의 물로 2-3회 용매분획을 실시함으로 산도를 낮추어 최종적으로 페오피틴화 n-헥산 분획물을 수득하였다. 최종적으로 동결건조기를 통하여 말린 후, DMSO에 녹여 10 mg/mL로 만들어 항바이러스 활성 평가 실험에 사용하였다.In order to establish a pheophytinylation reaction using an inorganic acid, the dried guava extract was suspended using a 0.5 N hydrochloric acid solution, and the solvent was fractionated with n -hexane for about 5 minutes, followed by solvent fractionation 2-3 times with the same volume of water. By lowering the acidity, a pheophytinated n- hexane fraction was finally obtained. Finally, after drying through a lyophilizer, it was dissolved in DMSO to make 10 mg/mL and used in the antiviral activity evaluation experiment.
3.2 유기산을 이용한 구아바 잎 추출물의 페오피틴화 및 분획물의 제조3.2 Pheophytination of Guava Leaf Extract Using Organic Acids and Preparation of Fractions
페오피틴화 조건 설정에 있어서 식물에 존재하면서 안전한 유기산인 구연산을 사용하여 농도를 달리함으로 실험을 수행하였다. 구아바 생잎을 음건한 후, 95% 에탄올로 2시간 동안 교반하여 3회에 걸쳐 추출하고 감압농축하여 그 추출물을 얻어 실험에 사용하였다. 10%[w/v] 구연산 수용액을 제조한 후 연속적으로 희석하여 10%, 5%, 1%, 0.5%, 0.25%, 0.1% 구연산 수용액을 준비하였다. 각 농도별 구연산수용액을 사용하여 추출물을 현탁시키고 n-헥산과 5분 가량 용매분획을 한 후, 동일 부피의 물로 2-3회 용매분획을 실시함으로 산도를 낮추어 최종적으로 페오피틴화 n-헥산 분획물을 수득하였다. 최종적으로 HPLC 분석을 위해 각 농도별 분획물을 1 mg/mL로 준비하였다.In setting the pheophytination conditions, the experiment was performed by varying the concentration using citric acid, which is a safe organic acid present in plants. After drying the fresh guava leaves in the shade, they were extracted three times by stirring with 95% ethanol for 2 hours, and concentrated under reduced pressure to obtain the extract and used in the experiment. After preparing a 10% [w/v] citric acid aqueous solution, serial dilutions were made to prepare 10%, 5%, 1%, 0.5%, 0.25%, 0.1% citric acid aqueous solution. After suspending the extract using an aqueous citric acid solution for each concentration, solvent fractionation with n -hexane for about 5 minutes, and solvent fractionation 2-3 times with the same volume of water to lower the acidity and finally pheophytinated n- hexane fraction was obtained. Finally, fractions for each concentration were prepared at 1 mg/mL for HPLC analysis.
3.3 열처리 방법을 이용한 구아바 잎 추출물의 페오피틴화 및 분획물의 제조3.3 Pheophytination of guava leaf extract using heat treatment method and preparation of fractions
상기 실시예 3.2 에서 준비한 구아바 추출물을 증류수에 현탁한 후 60℃ 드라이오븐에 1시간, 3시간, 6시간, 12시간, 24시간 및 48시간 동안 방치한 후 n-헥산과 용매분획하여 페오피틴화 n-헥산 분획물을 수득하였다. 최종적으로 HPLC 분석을 위해 각 조건별 분획물을 1 mg/mL로 준비하였다.The guava extract prepared in Example 3.2 was suspended in distilled water and then left in a dry oven at 60° C. for 1 hour, 3 hours, 6 hours, 12 hours, 24 hours and 48 hours, followed by solvent fractionation with n -hexane and pheophytinylation An n- hexane fraction was obtained. Finally, fractions for each condition were prepared at 1 mg/mL for HPLC analysis.
<실시예 4. HPLC를 통한 구아바 추출물, <Example 4. Guava extract via HPLC,
nn
-헥산 분획물과 페오피틴화 분획물에서 화합물 1-4의 확인>-Identification of compound 1-4 in hexane fraction and pheophytination fraction>
구아바 건잎의 95% 에탄올 추출물, n-헥산 분획물 및 상기 실시예 3.1에서 수득한 0.5N 염산수용액을 이용한 페오피틴화 분획물을 HPLC 분석을 위해 추출물의 경우 10 mg/ml의 농도로, 분획물의 경우 5 mg/ml의 농도로 메탄올 또는 메탄올 수용액을 이용하여 준비하였다. 상기 실시예 1.2에서 분리한 화합물 1~4는 1 mg/ml의 농도로 준비하였다.For HPLC analysis, 95% ethanol extract of dried guava leaves, n -hexane fraction, and pheophytination fraction using 0.5N aqueous hydrochloric acid solution obtained in Example 3.1 were analyzed at a concentration of 10 mg/ml for the extract and 5 for the fraction. It was prepared using methanol or aqueous methanol solution at a concentration of mg/ml. Compounds 1 to 4 isolated in Example 1.2 were prepared at a concentration of 1 mg/ml.
HPLC 분석은 DAD-Diode Array Detector 3000이 장착된 Thermo Fisher Scientific UltiMate 3000 시스템을 통해 실시하였고, 컬럼은 YMC-Triart C18 (4.6×250.0 ㎜, 5μm)을 사용하였다. 분석 조건은, 액체크로마토그래피의 이동상은 0.1%[v/v] 포름산을 포함하는 증류수(이동상 A)와 아세토니트릴(이동상 B)을 사용하여 유속 1.0㎖/분으로 흘려주었고, 컬럼 온도 25℃, 시료 온도 10℃에서 분석을 시행하였으며 각 시료는 20㎕씩 주입하였다. 크로마토그램은 290 nm에서 관찰하였으며, 이동상의 조건은 다음과 같다. 0~35.0분 70~100% 이동상 B 농도구배로, 35.0~55.0분 100% 이동상 B, 55.1~60.0분 70% 이동상 B로 흘려주었다. 상기 실시예 1-2에서 분리한 화합물 1~4의 머무름 시간 및 자외선 흡광 패턴을 통하여 각 시료에 포함된 화합물들의 존재유무를 확인하였고, 그 결과를 도 2에 나타내었다.HPLC analysis was performed using a Thermo Fisher Scientific UltiMate 3000 system equipped with a DAD-Diode Array Detector 3000, and a YMC-Triart C18 (4.6×250.0 mm, 5 μm ) column was used. Analytical conditions, the mobile phase of liquid chromatography was flowed at a flow rate of 1.0 ml/min using distilled water (mobile phase A) and acetonitrile (mobile phase B) containing 0.1% [v/v] formic acid, and a column temperature of 25 ° C., Analysis was performed at a sample temperature of 10°C, and 20 μl of each sample was injected. The chromatogram was observed at 290 nm, and the conditions of the mobile phase were as follows. It flowed with a gradient of 70-100% mobile phase B for 0-35.0 minutes, 100% mobile phase B for 35.0-55.0 minutes, and 70% mobile phase B for 55.1-60.0 minutes. Through the retention time and ultraviolet absorption pattern of compounds 1 to 4 isolated in Example 1-2, The presence or absence of the compounds included in each sample was checked, and the results are shown in FIG. 2 .
도 2에서 확인할 수 있듯, 구아바 잎 95% 에탄올 추출물의 n-헥산 분획물 및 페오피틴화 분획물에서 화합물 1~4가 뚜렷하게 확인되었다. 또한 이 결과로부터 페오피틴화 분획물에도 화합물 1~4가 사라지거나 변하지 않고 그대로 존재하고 있음을 확인하였다.As can be seen in FIG. 2 , compounds 1 to 4 were clearly identified in the n -hexane fraction and the pheophytination fraction of the 95% ethanol extract of guava leaf. In addition, from this result, it was confirmed that compounds 1 to 4 were present without disappearing or changing even in the pheophytinated fraction.
<실시예 5. HPLC를 통한 구아바 잎 추출물의 페오피틴화 변화도 측정><Example 5. Measurement of pheophytinylation gradient of guava leaf extract through HPLC>
5.1 무기산을 이용한 구아바 잎 추출물의 페오피틴화 변화도 확인5.1 Confirmation of Pheophytination Change of Guava Leaf Extract Using Inorganic Acid
상기 실시예 3.1에서 수득한 구아바 건잎 추출물의 n-헥산 분획물과 페오피틴화 분획물 및 상기 실시예 3.2에서 수득한 음건한 구아바 잎 추출물의 n-헥산 분획물을 HPLC 분석을 위해 1mg/ml의 농도로 메탄올을 이용하여 준비하였다. HPLC 분석은 상기 실시예 4에서 기재한 것과 동일한 시스템 및 칼럼을 사용하였으며, 분석 조건으로 액체크로마토그래피의 이동상은 에틸아세테이트:메탄올:증류수 (15:65:20, [v:v:v]) 혼합물 (이동상 A)과 에틸아세테이트:메탄올:증류수 (60:30:10, [v:v:v]) 혼합물 (이동상 B)을 사용하여 흘려주었고, 컬럼 온도 25℃, 시료 온도 10℃에서 분석을 시행하였으며 각 시료는 20㎕씩 주입하였다. 크로마토그램은 654 nm에서 관찰하였으며, 이동상 및 유속 조건은 다음과 같다. 0~6.0분 0% 이동상 B로 유속 1.3 ml/분, 6.0~7.0분 0~30% 이동상 B로 유속 1.3 ml/분~1.5ml/분, 7.0~10.0분 30% 이동상 B로 유속 1.5 ml/분, 10.0~11.0분 30~40% 이동상 B로 유속 1.5 ml/분, 11.0~12.0분 40~50% 이동상 B로 유속 1.5ml/분, 12.0~13.0분 50~100% 이동상 B로 유속 1.5ml/분, 13.0-24.0분 100% 이동상 B로 유속 1.5 ml/분, 24.0~26.1분 100~0% 이동상 B로 유속 1.5ml/분~1.3ml/분, 26.1~33.0분 0% 이동상 B로 유속 1.3ml/분으로 흘려주었다. 폴피린계 화합물들의 머무름 시간 및 자외선 흡광 패턴을 통하여 각 분획에 포함되어있는 물질들의 존재유무와 페오피틴화 정도를 피크의 강도를 통하여 상대적인 함량을 비교하였고, 그 결과를 도 3에 나타내었다.The n -hexane fraction and pheophytination fraction of the dried guava leaf extract obtained in Example 3.1 and the n -hexane fraction of the guava leaf extract obtained in Example 3.2 in the shade were mixed with methanol at a concentration of 1 mg/ml for HPLC analysis. was prepared using For HPLC analysis, the same system and column as those described in Example 4 were used, and the mobile phase of liquid chromatography was an ethyl acetate:methanol:distilled water (15:65:20, [v:v:v]) mixture as an analysis condition. (mobile phase A) and ethyl acetate:methanol:distilled water (60:30:10, [v:v:v]) were flowed using a mixture (mobile phase B), and the analysis was performed at a column temperature of 25°C and a sample temperature of 10°C. 20 μl of each sample was injected. The chromatogram was observed at 654 nm, and the mobile phase and flow rate conditions were as follows. 0 to 6.0 min 0% mobile phase B 1.3 ml/min, 6.0 to 7.0 min 0 to 30% mobile phase B 1.3 ml/min to 1.5 ml/min, 7.0 to 10.0 min 30% mobile phase B 1.5 ml/min min, 10.0-11.0 min 30-40% flow rate 1.5 ml/min to mobile phase B, 11.0 to 12.0 min 40-50% flow rate 1.5 ml/min to mobile phase B, 12.0 to 13.0 min 50-100% flow rate 1.5 ml to mobile phase B /min, 13.0-24.0 min Flow rate to 100% mobile phase B 1.5 ml/min, 24.0 to 26.1 min Flow rate to 100-0% mobile phase B 1.5 ml/min to 1.3 ml/min, 26.1 to 33.0 min Flow rate to 0% mobile phase B It flowed at 1.3ml/min. Through the retention time and ultraviolet absorption pattern of polphyrin-based compounds The relative content of the substances contained in each fraction and the degree of pheophytinylation were compared through the intensity of the peak, and the results are shown in FIG. 3 .
도 3에서 보듯이, 모든 시료에서 폴피린계 화합물이 확인되었다. 음건한 구아바 생잎 추출물의 n-헥산 분획물의 경우, 클로로필 a, b와 페오피틴 a, b 및 그 유도체가 모두 확인된 반면, 구아바 건잎의 n-헥산 분획물은 동일시료의 페오피틴화 분획물과 같이 페오피틴 a, b 및 그 유도체만 확인되었다. 이는 구입한 구아바 건잎이 열풍건조된 시료임에 따라, 열에 의한 페오피틴화가 진행된 것임을 보여준다. 하기 실시예 5.3에서 이러한 결과를 뒷받침할 열처리를 통한 페오피틴화를 소개할 것이다.As shown in FIG. 3 , polphyrin-based compounds were identified in all samples. In the case of the n -hexane fraction of the dry guava leaf extract, chlorophyll a, b and pheophytin a, b and their derivatives were all confirmed, whereas the n -hexane fraction of the dried guava leaf was the same as the pheophytinated fraction of the same sample. Only pheophytin a, b and its derivatives were identified. This shows that as the purchased dried guava leaves are hot air-dried samples, pheophytinization by heat has progressed. In Example 5.3 below, pheophytination through heat treatment will be introduced to support these results.
5.2 유기산을 이용한 구아바 잎 추출물의 페오피틴화 변화도 확인5.2 Confirmation of Pheophytination Change of Guava Leaf Extract Using Organic Acid
상기 실시예 3.2에서 수득한 구아바 잎 추출물의 농도별 구연산을 이용한 페오피틴화 분획물과 n-헥산 분획물을 확보한 후, 1 mg/ml의 농도로 메탄올을 이용하여 준비하였다. HPLC 분석은 상기 실시예 5.1에서 기재한 것과 동일한 시스템, 칼럼 및 분석조건을 이용하였으며, 폴피린계 화합물들의 머무름 시간 및 자외선 흡광 패턴을 통하여 각 농도별 분획에 포함되어있는 물질들의 존재유무와 페오피틴화 정도를 피크의 강도를 통하여 상대적인 함량을 비교하였고, 그 결과를 도 4에 나타내었다. 10%, 5.0%, 1.0%, 0.5%, 0.25%, 0.1%의 서로 다른 농도로 준비한 구연산수용액으로 페오피틴화를 실시한 결과, 도 4에서 보듯이 저농도의 0.1% 구연산수용액에서도 모든 클로로필 a, b 및 그 유도체가 전부 페오피틴화 되는 것을 확인하였다.After securing a pheophytination fraction and n -hexane fraction using citric acid for each concentration of the guava leaf extract obtained in Example 3.2, methanol was prepared at a concentration of 1 mg/ml. HPLC analysis was performed using the same system, column and analysis conditions as described in Example 5.1, and through the retention time and UV absorption pattern of the polphyrin-based compounds. The relative content of the substances included in the fractions for each concentration and the degree of pheophytinylation were compared through the intensity of the peak, and the results are shown in FIG. 4 . As a result of pheophytinization with citric acid aqueous solutions prepared at different concentrations of 10%, 5.0%, 1.0%, 0.5%, 0.25%, and 0.1%, as shown in FIG. 4, all chlorophyll a and b And it was confirmed that all of the derivatives were pheophytinized.
5.3 열처리를 이용한 구아바 잎 추출물의 페오피틴화 변화도 확인5.3 Confirmation of Pheophytination Change of Guava Leaf Extract Using Heat Treatment
상기 실시예 3.3에서 수득한 구아바 잎의 열처리를 통한 페오피틴화 분획물과 n-헥산 분획물을 확보한 후, 1 mg/ml의 농도로 메탄올을 이용하여 준비하였다. HPLC 분석은 상기 실시예 5.1에서 기재한 것과 동일한 시스템, 칼럼 및 분석조건을 이용하였으며, 폴피린계 화합물들의 머무름 시간 및 자외선 흡광 패턴을 통하여 각 농도별 분획에 포함되어있는 물질들의 존재유무와 페오피틴화 정도를 피크의 강도를 통하여 상대적인 함량을 비교하였고, 그 결과를 도 5에 나타내었다. 구아바 잎 추출물을 농축한 후 증류수에 현탁시켜 60℃ 드라이오븐에 1시간, 3시간, 6시간, 12시간, 24시간 및 48시간 단위로 방치하여 페오피틴화를 실시한 결과, 도 5에서 보듯이 1시간 동안만 방치한 시료에서도 모든 클로로필 a, b 및 그 유도체가 완전히 페오피틴화 되는 것을 확인하였다. 상기 실시예 5.1에서 열풍건조된 구아바 건잎 추출물의 n-헥산 분획물에서 클로로필 a, b 및 그 유도체는 확인되지 않고 페오피틴 a, b 및 그 유도체만 확인된 것 또한 열에 의한 페오피틴화를 나타내는 결과이다. 열에 의한 클로로필 a, b의 페오피틴화가 확인됨에 따라, 60℃에서 100℃ (데치기, blanching)를 하거나 구아바 잎 자체를 열로써 가공하는 방법을 통하여 페오피틴화 반응 또한 가능하다.After obtaining a pheophytinated fraction and an n -hexane fraction through heat treatment of the guava leaves obtained in Example 3.3, methanol was prepared at a concentration of 1 mg/ml. HPLC analysis was performed using the same system, column and analysis conditions as described in Example 5.1, and through the retention time and UV absorption pattern of the polphyrin-based compounds. The presence or absence of substances contained in each concentration fraction and the degree of pheophytinylation were compared with the relative content through the intensity of the peak, and the results are shown in FIG. 5 . After concentrating the guava leaf extract, it was suspended in distilled water and left in a dry oven at 60 ° C for 1 hour, 3 hours, 6 hours, 12 hours, 24 hours and 48 hours to perform pheophytinization. It was confirmed that all chlorophyll a, b and derivatives thereof were completely pheophytinized even in samples left only for a period of time. In Example 5.1, in the n -hexane fraction of the guava dried leaf extract dried by hot air, chlorophyll a, b and its derivatives were not identified, but only pheophytin a, b and its derivatives were confirmed, which also showed pheophytinylation by heat. to be. As the pheophytination of chlorophyll a and b by heat has been confirmed, the pheophytination reaction is also possible through a method of heating at 60° C. to 100° C. (blanching) or processing the guava leaf itself with heat.
<실시예 6. 신종코로나 바이러스에 대한 구아바 잎 추출물 및 분획물 또는 분리한 화합물의 억제능 평가><Example 6. Evaluation of inhibitory ability of guava leaf extracts and fractions or isolated compounds against novel coronavirus>
6.1 코로나 바이러스의 준비6.1 Preparation of coronavirus
SARS-CoV-2(Severe acute respiratory syndrome coronavirus 2, NMC-CoV-02 strain)는 충북대학교(청주, 한국)에서 VERO 세포를 호스트(host)로 하여 배양한 후 제공된 것을 사용하였다. SARS-CoV-2 바이러스를 mono-layered VERO 세포 접종하고 3일 후 바이러스 배양 상층액 분액을 채취하고, 바이러스를 10%[v/v] FBS가 포함된 DMEM 배지에서 배양 유지된 VERO 세포에 접종하여 항바이러스 활성 측정에 사용하였다.SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2, NMC-CoV-02 strain) was provided after culturing VERO cells as a host at Chungbuk National University (Cheongju, Korea). SARS-CoV-2 virus was inoculated with mono-layered VERO cells, and after 3 days, the virus culture supernatant was collected, and the virus was inoculated into VERO cells cultured in DMEM medium containing 10% [v/v] FBS. It was used to measure antiviral activity.
6.2 CPE 어세이6.2 CPE Assay
상기 실시예 6.1에서 준비된 SARS-CoV-2 바이러스를 VERO 세포에 감염시킨 후, 본 96-웰(well) 플레이트에 2×104세포/웰의 밀도로 VERO 세포를 분주하여 10%[v/v] FBS(fetal bovine serum), 1%[v/v] PS(penicillin+streptomycin) 및 L-글루타민(L-glutamine, 500㎖ 배지 기준 2㎖ 첨가)을 포함한 DMEM 배양액에서 하루 동안 증식시킨 뒤 배양액을 PBS 용액으로 세척하였다. 상기 세척된 VERO 세포에 SARS-CoV-2 바이러스가 100 TCID50의 농도로 포함된 감염 배지(infection media : DMEM) 50㎕를 넣어, 2시간 동안 감염시킨 후 배양액을 PBS 용액으로 세척하였다. 이후, 본 발명의 화합물를 처리하여, 0.2㎍/㎖ trypsin을 포함한 DMEM 배양액에서 3일 동안 배양하고, 바이러스에 의한 CPE를 측정하였다. 각 화합물의 희석배수에 따른 항바이러스 활성 결과를 하기 도6 및 도7에 나타내었다.After infecting VERO cells with the SARS-CoV-2 virus prepared in Example 6.1, VERO cells were dispensed at a density of 2×10 4 cells/well in this 96-well plate to 10% [v/v ] FBS (fetal bovine serum), 1% [v/v] PS (penicillin + streptomycin) and L-glutamine (L-glutamine, added 2㎖ based on 500㎖ medium) for one day in DMEM culture medium, and then the culture medium Washed with PBS solution. 50 μl of infection medium (infection media: DMEM) containing SARS-CoV-2 virus at a concentration of 100 TCID 50 was added to the washed VERO cells, and after infection for 2 hours, the culture medium was washed with PBS solution. Thereafter, the compound of the present invention was treated and cultured for 3 days in DMEM culture medium containing 0.2 μg/ml trypsin, and the CPE caused by the virus was measured. The results of antiviral activity according to the dilution factor of each compound are shown in FIGS. 6 and 7 below.
6.3 구아바 잎 추출물 및 분획물 또는 분리한 화합물의 신종코로나 바이러스에 대한 항바이러스 활성 확인6.3 Confirmation of antiviral activity against novel coronavirus of guava leaf extracts and fractions or isolated compounds
구아바 잎 추출물 및 분획물이 VERO 세포에서 SARS-CoV-2 바이러스에 대한 항바이러스 활성이 있는 결과를 도 6에 나타내었다.The results of guava leaf extracts and fractions having antiviral activity against SARS-CoV-2 virus in VERO cells are shown in FIG. 6 .
도 6에서 보는 바와 같이, 구아바 잎 추출물 및 분획물 모두 20 μg/ml 농도 이하에서 항바이러스 효과가 나타나는 것을 확인하였다. 특히 n-헥산 분획물 및 페오피틴화 분획물이 5 μg/ml 에서도 항바이러스 활성을 나타내며 가장 우수한 효과를 보였다. 또한, 상기 실시예 2의 화합물 1~4를 처리한 결과, 모든 화합물에 대하여 40 μM 농도 이하에서 항바이러스 활성이 확인되었으며 이는 도 7에 나타내었다. 화합물 1 및 2의 경우 3.75 μM 농도에서 활성을 나타내며 가장 우수한 항바이러스 효과를 보였다.As shown in FIG. 6 , it was confirmed that both the guava leaf extract and the fraction showed antiviral effect at a concentration of 20 μg /ml or less. In particular, the n -hexane fraction and the pheophytinated fraction showed antiviral activity even at 5 μg /ml and showed the best effect. In addition, as a result of treatment with compounds 1 to 4 of Example 2, antiviral activity was confirmed at a concentration of 40 μM or less for all compounds, which is shown in FIG. 7 . Compounds 1 and 2 showed activity at a concentration of 3.75 μM and showed the best antiviral effect.
본 발명에서 실시한 활성평가에 따르면 메로테르페노이드계 화합물 및 페오피틴계 화합물이 항바이러스 효과를 나타낸 것을 확인함과 동시에, 상기 실시예 4 및 실시예 5.1에서 HPLC 분석을 통해 확인한 바에 따라 메로테르페노이드계 화합물 및 페오피틴계 화합물이 가장 풍부하게 함유된 n-헥산 분획물 및 페오피틴화 분획물이 가장 우수한 항바이러스 효과를 나타내는 것을 확인하였다.According to the activity evaluation conducted in the present invention, it was confirmed that the meroterpenoid-based compound and the pheophytin-based compound exhibited antiviral effects, and at the same time, as confirmed through HPLC analysis in Examples 4 and 5.1, meloterpene-based compounds It was confirmed that the n -hexane fraction and the pheophytinated fraction containing the most abundant nooid compound and pheophytin compound exhibited the best antiviral effect.
이를 통해, 본 발명의 구아바 추출물, 분획물 또는 이의 페오피틴화 분획물 또는 이로부터 분리한 메로테르페노이드계 화합물이 신종코로나 바이러스 감염의 예방, 개선 또는 치료에 효과를 나타낸다는 것을 알 수 있다.Through this, it can be seen that the guava extract, fraction, or pheophytinated fraction thereof of the present invention, or a meroterpenoid-based compound isolated therefrom, is effective in preventing, improving or treating novel coronavirus infection.
<제제예 1. 약학적 제제><Formulation Example 1. Pharmaceutical formulation>
제제예 1.1 정제의 제조Formulation Example 1.1 Preparation of tablets
본 발명의 구아바 추출물(또는 페오피틴화 분획물) 20g 또는 제주구아자본 A(화합물 1), 제주구아자본 D(화합물 2), 제주구아자본 E(화합물 3) 및 제주구아자본 H(화합물 4)로 이루어진 군에서 선택되는 1종 이상 200 mg을 각각 락토오스 175.9 g, 감자전분 180 g 및 콜로이드성 규산 32 g과 혼합하였다. 이 혼합물에 10% 젤라틴 용액을 첨가시킨 후, 분쇄해서 14 메쉬체를 통과시켰다. 이것을 건조시키고 여기에 감자전분 160 g, 활석 50 g 및 스테아린산 마그네슘 5 g을 첨가해서 얻은 혼합물을 정제로 만들었다.20 g of the guava extract (or pheophytinated fraction) of the present invention or Jeju guar capital A (compound 1), Jeju guar capital D (compound 2), Jeju guar capital E (compound 3) and Jeju guar capital H (compound 4) 200 mg of at least one selected from the group consisting of 175.9 g of lactose, 180 g of potato starch and 32 g of colloidal silicic acid were mixed with each. After adding a 10% gelatin solution to this mixture, it was ground and passed through a 14 mesh sieve. This was dried, and 160 g of potato starch, 50 g of talc and 5 g of magnesium stearate were added thereto, and the resulting mixture was made into tablets.
제제예 1.2 주사액제의 제조Formulation Example 1.2 Preparation of injection solution
본 발명의 구아바 추출물(또는 페오피틴화 분획물) 20g 또는 제주구아자본 A(화합물 1), 제주구아자본 D(화합물 2), 제주구아자본 E(화합물 3) 및 제주구아자본 H(화합물 4)로 이루어진 군에서 선택되는 1종 이상 100mg, 염화나트륨 0.6 g 및 아스코르브산 0.1 g을 증류수에 용해시켜서 100 ㎖를 만들었다. 이 용액을 병에 넣고 20 ℃에서 30분간 가열하여 멸균시켰다.20 g of the guava extract (or pheophytinated fraction) of the present invention or Jeju guar capital A (compound 1), Jeju guar capital D (compound 2), Jeju guar capital E (compound 3) and Jeju guar capital H (compound 4) 100 mg of one or more selected from the group consisting of, 0.6 g of sodium chloride and 0.1 g of ascorbic acid were dissolved in distilled water to make 100 ml. This solution was placed in a bottle and sterilized by heating at 20 °C for 30 minutes.
<제제예 2. 식품 제조><Formulation Example 2. Food Manufacturing>
제제예 2.1 조리용 양념의 제조Formulation Example 2.1 Preparation of cooking seasoning
본 발명의 구아바 추출물(또는 페오피틴화 분획물) 20g 또는 제주구아자본 A(화합물 1), 제주구아자본 D(화합물 2), 제주구아자본 E(화합물 3) 및 제주구아자본 H(화합물 4)로 이루어진 군에서 선택되는 1종 이상을 각각 조리용 양념에 1중량%로 첨가하여 건강 증진용 조리용 양념을 제조하였다.20 g of the guava extract (or pheophytinated fraction) of the present invention or Jeju guar capital A (compound 1), Jeju guar capital D (compound 2), Jeju guar capital E (compound 3) and Jeju guar capital H (compound 4) At least one kind selected from the group consisting of each was added to the cooking seasoning in an amount of 1% by weight to prepare a cooking seasoning for health promotion.
제제예 2.2 밀가루 식품의 제조Formulation Example 2.2 Preparation of wheat flour food
본 발명의 구아바 추출물(또는 페오피틴화 분획물) 20g 또는 제주구아자본 A(화합물 1), 제주구아자본 D(화합물 2), 제주구아자본 E(화합물 3) 및 제주구아자본 H(화합물 4)로 이루어진 군에서 선택되는 1종 이상을 각각 밀가루에 0.1중량% 로 첨가하고, 이 혼합물을 이용하여 빵, 케이크, 쿠키, 크래커 및 면류를 제조하여 건강 증진용 식품을 제조하였다.20 g of the guava extract (or pheophytinated fraction) of the present invention or Jeju guar capital A (compound 1), Jeju guar capital D (compound 2), Jeju guar capital E (compound 3) and Jeju guar capital H (compound 4) At least one selected from the group consisting of flour was added in an amount of 0.1% by weight, and the mixture was used to prepare bread, cake, cookies, crackers and noodles to prepare health-promoting food.
제제예 2.3 스프 및 육즙 (gravies)의 제조Formulation Example 2.3 Preparation of soups and gravies
본 발명의 구아바 추출물(또는 페오피틴화 분획물) 20g 또는 제주구아자본 A(화합물 1), 제주구아자본 D(화합물 2), 제주구아자본 E(화합물 3) 및 제주구아자본 H(화합물 4)로 이루어진 군에서 선택되는 1종 이상을 각각 육즙에 0.1중량%로 첨가하여 건강 증진용 수프 및 육즙을 제조하였다.20 g of the guava extract (or pheophytinated fraction) of the present invention or Jeju guar capital A (compound 1), Jeju guar capital D (compound 2), Jeju guar capital E (compound 3) and Jeju guar capital H (compound 4) At least one selected from the group consisting of each was added to the broth in an amount of 0.1% by weight to prepare a health-improving soup and broth.
제제예 2.4 유제품 (dairy products)의 제조Formulation Example 2.4 Preparation of dairy products
본 발명의 구아바 추출물(또는 페오피틴화 분획물) 20g 또는 제주구아자본 A(화합물 1), 제주구아자본 D(화합물 2), 제주구아자본 E(화합물 3) 및 제주구아자본 H(화합물 4)로 이루어진 군에서 선택되는 1종 이상을 각각 우유에 0.1중량%로 첨가하고, 상기 우유를 이용하여 버터 및 아이스크림과 같은 다양한 유제품을 제조하였다.20 g of the guava extract (or pheophytinated fraction) of the present invention or Jeju guar capital A (compound 1), Jeju guar capital D (compound 2), Jeju guar capital E (compound 3) and Jeju guar capital H (compound 4) At least one selected from the group consisting of milk was added in an amount of 0.1 wt%, respectively, and various dairy products such as butter and ice cream were prepared using the milk.
제제예 2.5 야채주스 제조Formulation Example 2.5 Preparation of vegetable juice
본 발명의 구아바 추출물(또는 페오피틴화 분획물) 20g 또는 제주구아자본 A(화합물 1), 제주구아자본 D(화합물 2), 제주구아자본 E(화합물 3) 및 제주구아자본 H(화합물 4)로 이루어진 군에서 선택되는 1종 이상을 각각 토마토주스 또는 당근주스 1,000 ㎖에 가하여 건강 증진용 야채주스를 제조하였다.20 g of the guava extract (or pheophytinated fraction) of the present invention or Jeju guar capital A (compound 1), Jeju guar capital D (compound 2), Jeju guar capital E (compound 3) and Jeju guar capital H (compound 4) At least one selected from the group consisting of tomato juice or carrot juice was added to 1,000 ml of each to prepare vegetable juice for health promotion.
제제예 2.6 과일주스 제조Formulation Example 2.6 Fruit Juice Preparation
본 발명의 구아바 추출물(또는 페오피틴화 분획물) 20g 또는 제주구아자본 A(화합물 1), 제주구아자본 D(화합물 2), 제주구아자본 E(화합물 3) 및 제주구아자본 H(화합물 4)로 이루어진 군에서 선택되는 1종 이상의 0.1 g을 사과주스 또는 포도주스 1,000 ㎖에 가하여 건강 증진용 과일주스를 제조하였다.20 g of the guava extract (or pheophytinated fraction) of the present invention or Jeju guar capital A (compound 1), Jeju guar capital D (compound 2), Jeju guar capital E (compound 3) and Jeju guar capital H (compound 4) Fruit juice for health promotion was prepared by adding 0.1 g of at least one selected from the group consisting of apple juice or grape juice to 1,000 ml.
Claims (19)
- 구아바(Psidiuma guajava) 추출물 또는 이의 페오피틴화 분획물 또는 이로부터 분리한 메로테르페노이드계 화합물을 유효성분으로 포함하는 신종코로나 바이러스 감염의 예방 또는 치료용 조성물.A composition for preventing or treating novel coronavirus infection comprising, as an active ingredient, a guava ( Psidiuma guajava ) extract or a pheophytinated fraction thereof or a meroterpenoid-based compound isolated therefrom.
- 제1항에 있어서,According to claim 1,상기 구아바 추출물은 구아바 잎 또는 과일을 각각 물, C1 내지 C4의 저급 알코올, 헥산, 아세톤, 메틸렌 클로라이드, 에틸아세테이트 및 이들의 혼합용매로 구성된 군으로부터 선택된 어느 하나의 용매로 추출한 것을 특징으로 하는 신종코로나 바이러스 감염의 예방 또는 치료용 조성물.The guava extract is a new corona, characterized in that the guava leaf or fruit is extracted with any one solvent selected from the group consisting of water, C1 to C4 lower alcohol, hexane, acetone, methylene chloride, ethyl acetate, and mixed solvents thereof, respectively A composition for preventing or treating a viral infection.
- 제1항에 있어서,According to claim 1,상기 구아바 추출물의 페오피틴화 분획물은 구아바 잎 또는 과일 추출물을 0.5 ~ 1.0N의 염산수용액에 현탁시킨 후, 헥산, 메틸렌 클로라이드, 에틸아세테이트 및 이들의 혼합용매로 구성된 군으로부터 선택된 어느 하나의 용매로 추출한 것을 특징으로 하는 신종코로나 바이러스 감염의 예방 또는 치료용 조성물.The pheophytinated fraction of the guava extract is obtained by suspending a guava leaf or fruit extract in a 0.5 to 1.0N hydrochloric acid solution, followed by extraction with any one solvent selected from the group consisting of hexane, methylene chloride, ethyl acetate, and a mixed solvent thereof. A composition for preventing or treating novel coronavirus infection, characterized in that.
- 제1항에 있어서,According to claim 1,상기 구아바 추출물의 페오피틴화 분획물은 구아바 잎 또는 과일 추출물을 0.1 ~ 10 %(w/v) 의 구연산수용액에 현탁시킨 후, 헥산, 메틸렌 클로라이드, 에틸아세테이트 및 이들의 혼합용매로 구성된 군으로부터 선택된 어느 하나의 용매로 추출한 것을 특징으로 하는 신종코로나 바이러스 감염의 예방 또는 치료용 조성물.The pheophytination fraction of the guava extract is obtained by suspending the guava leaf or fruit extract in 0.1 to 10% (w/v) citric acid aqueous solution, A composition for preventing or treating novel coronavirus infection, characterized in that it is extracted with a single solvent.
- 제1항에 있어서,According to claim 1,상기 구아바 추출물의 페오피틴화 분획물은 구아바 잎 또는 과일 추출물을 물에 현탁시킨 후, 60 ℃에서 1시간 ~ 48시간 방치한 다음, 헥산, 메틸렌 클로라이드, 에틸아세테이트 및 이들의 혼합용매로 구성된 군으로부터 선택된 어느 하나의 용매로 추출한 것을 특징으로 하는 신종코로나 바이러스 감염의 예방 또는 치료용 조성물.The pheophytinated fraction of the guava extract is selected from the group consisting of guava leaf or fruit extract suspended in water, left at 60° C. for 1 hour to 48 hours, and then hexane, methylene chloride, ethyl acetate, and a mixed solvent thereof. A composition for preventing or treating novel coronavirus infection, characterized in that it is extracted with any one solvent.
- 제1항 내지 제5항 중 어느 한 항에 있어서,6. The method according to any one of claims 1 to 5,상기 신종코로나 바이러스 감염의 예방 또는 치료용 조성물은 하기 화학식 1의 제주구아자본 A(화합물1), 제주구아자본 D(화합물 2), 제주구아자본 E(화합물 3) 및 제주구아자본 H(화합물 4)로 이루어진 군에서 선택되는 1종 이상의 화합물을 유효성분으로 포함하는 것을 특징으로 하는 신종코로나 바이러스 감염의 예방 또는 치료용 약학 조성물.The composition for preventing or treating novel coronavirus infection is Jeju guar capital A (compound 1), Jeju guar capital D (compound 2), Jeju guar capital E (compound 3) and Jeju guar capital H (compound 4) of the following formula 1 ) A pharmaceutical composition for preventing or treating novel coronavirus infection, characterized in that it contains one or more compounds selected from the group consisting of as an active ingredient.[화학식 1][Formula 1]
- 구아바 추출물 또는 이의 페오피틴화 분획물 또는 이로부터 분리한 메로테르페노이드계 화합물을 유효성분으로 포함하는 신종코로나 바이러스 감염의 예방 또는 증상 개선용 건강기능식품.A health functional food for preventing or improving symptoms of novel coronavirus infection, comprising as an active ingredient a guava extract or a pheophytinated fraction thereof or a meroterpenoid-based compound isolated therefrom.
- 제7항에 있어서,8. The method of claim 7,상기 구아바 추출물은 구아바 잎 또는 과일을 각각 물, C1 내지 C4의 저급 알코올, 헥산, 아세톤, 메틸렌 클로라이드, 에틸아세테이트 및 이들의 혼합용매로 구성된 군으로부터 선택된 어느 하나의 용매로 추출한 것을 특징으로 하는 신종코로나 바이러스 감염의 예방 또는 증상 개선용 건강기능식품.The guava extract is a new corona, characterized in that the guava leaf or fruit is extracted with any one solvent selected from the group consisting of water, C1 to C4 lower alcohol, hexane, acetone, methylene chloride, ethyl acetate, and mixed solvents thereof, respectively Health functional food for preventing or improving symptoms of viral infection.
- 제7항에 있어서,8. The method of claim 7,상기 구아바 추출물의 페오피틴화 분획물은 구아바 잎 또는 과일 추출물을 0.5 ~ 1.0 N의 염산수용액에 현탁시킨 후, 헥산, 메틸렌 클로라이드, 에틸아세테이트 및 이들의 혼합용매로 구성된 군으로부터 선택된 어느 하나의 용매로 추출한 것을 특징으로 하는 신종코로나 바이러스 감염의 예방 또는 증상 개선용 건강기능식품.The pheophytinated fraction of the guava extract is obtained by suspending a guava leaf or fruit extract in a 0.5 to 1.0 N aqueous hydrochloric acid solution, followed by extraction with any one solvent selected from the group consisting of hexane, methylene chloride, ethyl acetate, and a mixed solvent thereof. Health functional food for preventing or improving symptoms of new coronavirus infection, characterized in that.
- 제7항에 있어서,8. The method of claim 7,상기 구아바 추출물의 페오피틴화 분획물은 구아바 잎 또는 과일 추출물을 0.05 ~ 10 %(w/v) 의 구연산수용액에 현탁시킨 후, 헥산, 메틸렌 클로라이드, 에틸아세테이트 및 이들의 혼합용매로 구성된 군으로부터 선택된 어느 하나의 용매로 추출한 것을 특징으로 하는 신종코로나 바이러스 감염의 예방 또는 증상 개선용 건강기능식품.The pheophytination fraction of the guava extract is obtained by suspending the guava leaf or fruit extract in 0.05 to 10% (w/v) citric acid aqueous solution, A health functional food for preventing or improving symptoms of novel coronavirus infection, characterized in that it is extracted with a single solvent.
- 제7항에 있어서,8. The method of claim 7,상기 구아바 추출물의 페오피틴화 분획물은 구아바 잎 또는 과일 추출물을 물에 현탁시킨 후, 60 ℃에서 1시간 ~ 48시간 방치한 다음, 헥산, 메틸렌 클로라이드, 에틸아세테이트 및 이들의 혼합용매로 구성된 군으로부터 선택된 어느 하나의 용매로 추출한 것을 특징으로 하는 신종코로나 바이러스 감염의 예방 또는 증상 개선용 건강기능식품.The pheophytinated fraction of the guava extract is selected from the group consisting of guava leaf or fruit extract suspended in water, left at 60° C. for 1 hour to 48 hours, and then hexane, methylene chloride, ethyl acetate, and a mixed solvent thereof. A health functional food for preventing or improving symptoms of novel coronavirus infection, characterized in that it is extracted with any one solvent.
- 제7항 내지 제12항 중 어느 한 항에 있어서,13. The method according to any one of claims 7 to 12,상기 신종코로나 바이러스 감염의 증상 개선용 건강기능식품은 하기 화학식 1의 제주구아자본 A(화합물 1), 제주구아자본 D(화합물 2), 제주구아자본 E(화합물 3) 및 제주구아자본 H(화합물 4)로 이루어진 군에서 선택되는 1종 이상의 화합물을 유효성분으로 포함하는 것을 특징으로 하는 신종코로나 바이러스 감염의 예방 또는 증상 개선용 건강기능식품.The health functional food for improving symptoms of novel coronavirus infection is Jeju guar capital A (compound 1), Jeju guar capital D (compound 2), Jeju guar capital E (compound 3) and Jeju guar capital H (compound) 4) Health functional food for preventing or improving symptoms of new coronavirus infection, characterized in that it contains one or more compounds selected from the group consisting of as an active ingredient.[화학식 1][Formula 1]
- 구아바 추출물 또는 이의 페오피틴화 분획물 또는 이로부터 분리한 메로테르페노이드계 화합물을 유효성분으로 포함하는 신종코로나 바이러스 감염 예방용 소독제.A disinfectant for preventing new corona virus infection comprising, as an active ingredient, a guava extract or a pheophytinated fraction thereof, or a meroterpenoid-based compound isolated therefrom.
- 제13항에 있어서,14. The method of claim 13,상기 구아바 추출물의 페오피틴화 분획물은 구아바 잎 또는 과일 추출물을 0.5 ~ 1.0 N의 염산수용액에 현탁시킨 후, 헥산, 메틸렌 클로라이드, 에틸아세테이트 및 이들의 혼합용매로 구성된 군으로부터 선택된 어느 하나의 용매로 추출한 것을 특징으로 하는 신종코로나 바이러스 감염 예방용 소독제.The pheophytinated fraction of the guava extract is obtained by suspending a guava leaf or fruit extract in a 0.5 to 1.0 N aqueous hydrochloric acid solution, followed by extraction with any one solvent selected from the group consisting of hexane, methylene chloride, ethyl acetate, and a mixed solvent thereof. Disinfectant for prevention of new corona virus infection, characterized in that.
- 제13항에 있어서,14. The method of claim 13,상기 구아바 추출물의 페오피틴화 분획물은 구아바 잎 또는 과일 추출물을 0.1 ~ 10 %(w/v) 의 구연산수용액에 현탁시킨 후, 헥산, 메틸렌 클로라이드, 에틸아세테이트 및 이들의 혼합용매로 구성된 군으로부터 선택된 어느 하나의 용매로 추출한 것을 특징으로 하는 신종코로나 바이러스 감염 예방용 소독제.The pheophytinized fraction of the guava extract is obtained by suspending the guava leaf or fruit extract in 0.1 to 10% (w/v) citric acid aqueous solution, A disinfectant for preventing new corona virus infection, characterized in that it is extracted with a single solvent.
- 제13항에 있어서,14. The method of claim 13,상기 구아바 추출물의 페오피틴화 분획물은 구아바 잎 또는 과일 추출물을 물에 현탁시킨 후, 60 ℃에서 1시간 ~ 48시간 방치한 다음, 헥산, 메틸렌 클로라이드, 에틸아세테이트 및 이들의 혼합용매로 구성된 군으로부터 선택된 어느 하나의 용매로 추출한 것을 특징으로 하는 신종코로나 바이러스 감염 예방용 소독제.The pheophytinated fraction of the guava extract is selected from the group consisting of guava leaf or fruit extract suspended in water, left at 60° C. for 1 hour to 48 hours, and then hexane, methylene chloride, ethyl acetate, and a mixed solvent thereof. A disinfectant for preventing new corona virus infection, characterized in that it is extracted with any one solvent.
- 제13항 내지 제16항 중 어느 한 항에서 있어서,17. The method according to any one of claims 13 to 16,상기 신종코로나 바이러스 감염 예방용 소독제은 하기 화학식 1의 제주구아자본 A(화합물 1), 제주구아자본 D(화합물 2), 제주구아자본 E(화합물 3) 및 제주구아자본 H(화합물 4) 로 이루어진 군으로부터 하나 이상을 포함하는 것을 특징으로 하는 신종코로나 바이러스 감염 예방용 소독제.The disinfectant for preventing new corona virus infection is a group consisting of Jeju guar capital A (compound 1), Jeju guar capital D (compound 2), Jeju guar capital E (compound 3) and Jeju guar capital H (compound 4) of the following Chemical Formula 1 A disinfectant for preventing new corona virus infection, characterized in that it contains one or more from.[화학식 1][Formula 1]
- 구아바 추출물 또는 이의 페오피틴화 분획물 또는 이로부터 분리한 메로테르페노이드계 화합물을 유효성분으로 포함하는 신종코로나 바이러스 감염의 예방 또는 개선용 동물 사료용 조성물.A composition for animal feed for preventing or improving new corona virus infection, comprising as an active ingredient a guava extract or a pheophytinated fraction thereof or a meroterpenoid-based compound isolated therefrom.
- 하기 화학식 1의 제주구아자본 A(화합물 1), 제주구아자본 D(화합물 2), 제주구아자본 E(화합물 3) 및 제주구아자본 H(화합물 4)로 이루어진 군에서 선택되는 1종의 신규 화합물.One new compound selected from the group consisting of Jeju guar capital A (compound 1), Jeju guar capital D (compound 2), Jeju guar capital E (compound 3) and Jeju guar capital H (compound 4) of the following formula 1 .[화학식 1][Formula 1]
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PCT/KR2022/002045 WO2022173246A2 (en) | 2021-02-10 | 2022-02-10 | Composition for preventing or treating novel coronavirus infection, comprising psidiuma guajava extract, pheophytin fraction thereof, or meroterpenoid-based compound isolated therefrom as active ingredient |
Country Status (2)
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KR (1) | KR20220115432A (en) |
WO (1) | WO2022173246A2 (en) |
Family Cites Families (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR101072905B1 (en) | 2010-02-04 | 2011-10-17 | 노상근 | The anti-diabetic composition containing extracts of Psidium guajava and Acanthopanax senticosus |
KR101320946B1 (en) | 2011-04-01 | 2013-10-23 | 경희대학교 산학협력단 | Composition for Treating Prostate Cancer Comprising Hexane Fraction from Guava Leaf Extract As Active Ingredient |
KR101491493B1 (en) | 2013-04-04 | 2015-02-09 | 한국식품연구원 | Anti-inflammatory pharmaceutical composition comprising tangerine pericarp and Psidium guajava extract |
KR20190053490A (en) * | 2017-11-10 | 2019-05-20 | 동성제약주식회사 | Process for preparing high purity pheophytin, Chlorin e6, and Chlorin e6 and PVP complex from natural products containing chlorophyll |
KR102176525B1 (en) | 2018-11-07 | 2020-11-10 | 한국과학기술원 | Method of Preparing Hierarchical Wrinkle Structure by Using Sacrificial Layer and Hierarchical Wrinkle Structure Prepared Thereby |
KR102169476B1 (en) | 2020-05-20 | 2020-10-23 | (주)신테카바이오 | Composition for preventing or treating sars coronavirus 2 infection disease |
KR20200131784A (en) | 2020-11-04 | 2020-11-24 | (주)신테카바이오 | Composition for preventing or treating sars coronavirus 2 infection disease |
-
2021
- 2021-02-10 KR KR1020210019447A patent/KR20220115432A/en not_active IP Right Cessation
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2022
- 2022-02-10 WO PCT/KR2022/002045 patent/WO2022173246A2/en active Application Filing
Also Published As
Publication number | Publication date |
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KR20220115432A (en) | 2022-08-17 |
WO2022173246A3 (en) | 2022-10-06 |
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