KR20220115432A - Composition for prevention or treatment of severe acute respiratory syndrome corona virus infection comprising extract of leaves or fruits of Psidium guajava or pheophytinized fraction or meroterpenoids isolated from therefrom as active ingredients - Google Patents

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 can be advantageously used in the treatment and prevention of novel coronavirus infection through synergistic interactions of antiviral activity.

Description

Guava (Psidiuma guajava) extract or a pheophytinated fraction thereof or a composition for preventing or treating novel coronavirus infection comprising a meroterpenoid-based compound isolated therefrom as an active ingredient {Composition for prevention or treatment of severe acute respiratory syndrome corona virus infection comprising extract of leaves or fruits of Psidium guajava or pheophytinized fraction or meroterpenoids isolated from therefrom as active ingredients}

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.

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 is causing pathogenicity by infecting animals such as pigs, dogs, cats, bats, and horses.

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).

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.

On the other hand, guava ( Psidium guajava ) is an evergreen shrub or tree of the family Myrtaceae, 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).

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) ).

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.

As for 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 the novel coronavirus infection of the guava extract and the pheophytination fraction or the meroterpenoid-based compound isolated therefrom. different

Korean Patent No. 102169476, a composition for prevention or treatment of type 2 severe acute respiratory syndrome coronavirus-infected disease, registered on October 19, 2020. Korean Patent Publication No. 1020200053057. Coronavirus prevention and treatment, 2020. 05. 02. applied. Korean Patent Application Laid-Open No. 1020200131784, a composition for preventing or treating a type 2 severe acute respiratory syndrome coronavirus infection disease, filed on November 04, 2020. Korean Patent Registration No. 101491493, anti-inflammatory pharmaceutical composition comprising citrus peel and guava extract, registered on 02.09.2015. Korean Patent Registration No. 101072905, anti-diabetic composition containing extracts of guava and spiny hornwort as main components, registered on October 06, 2011. Korean Patent No. 101320946, a composition for treating prostate cancer comprising a hexane fraction of guava leaf extract as an active ingredient, registered on October 16, 2013.

Belouzard, S., et al., Mechanisms of coronavirus cell entry mediated by the viral spike protein. Viruses, 4, 1011-1033, 2012. Brian, D.A., Baric R.C., Coronavirus genome structure and replication, Coronavirus replication and reverse genetics. Berlin: Springer, 1-30, 2005. Englund, J.A., et al., Human coronaviruses, including Middle East respiratory syndrome coronavirus. Feigin and Cherry's textbook of pediatric infectious disease. 8th ed. Philadelphia: Elsevier Inc., 1846-1854, 2019. Hou, J.Q., et al., Psiguadiols A-J, rearranged meroterpenoids as potent PTP1B inhibitors from Psidium guajava. J. Nat. Prod., 82(12), 3267-3278, 2019. Huang, C.S., et al., Antihyperglycemic and antioxidative potential of Psidium guajava fruit in streptozotocin-induced diabetic rats. Food Chem. Toxicol., 49(9), 2189-2195, 2011. Letko, M., et al., Functional assessment of cell entry and receptor usage for SARS-CoV-2 and other lineage B betacoronaviruses. Nat. Microbiol., 5, 562-569, 2020. Oh, W.K., et al., Antidiabetic effects of extracts from Psidium guajava. J. Ethnopharmacol., 96(3), 411-415, 2005. Okuda, T., et al., Guavins A, C and D, complex tannins from Psidium guajava. Chem. Pharm. Bull., 35(1), 443-446, 1987. Park, S.E., Epidemiology, virology, and clinical features of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; coronavirus disease-19). Pediatr. Infec. Vaccine, 27, 1-10, 2020. Prabu, G.R., et al., Guaijaverin - a plant flavonoid as potential antiplaque agent against Streptococcus mutans. J. Appl. Microbiol., 101(2), 487-495, 2006. Shen, S.C., et al., Effect of guava (Psidium guajava Linn.) leaf soluble solids on glucose metabolism in type 2 diabetic rats. Phytother. Res., 22(11), 1458-1464, 2008. Ukwueze, S.E., et al., A new antibacterial benzophenone glycoside from Psidium guajava (Linn.) leaves. Nat. Prod. Res., 29(18), 1728-1734, 2015. World Health Organization, Coronavirus disease 2019 (COVID-19): situation report, 43. World Health Organization, 1-7, 2020. Qin, X.-J., et al., Meroterpenoids with antitumor activities from guava (Psidium guajava). J. Agric. Food Chem., 65(24), 4993-4999, 2017. Tang, G.-H., et al. Psiguajadials A-K: unusual psidium meroterpenoids as phosphodiesterase-4 inhibitors from the leaves of Psidium guajava. Sci. Rep. 7(1), 1047-1061, 2017. Zhang, H., et al., Angiotensin-converting enzyme 2 (ACE2) as a SARS-CoV-2 receptor: molecular mechanisms and potential therapeutic target. Intensive Care Med., 46, 586-590, 2020.

An object of the present invention is guava ( Psidium guajava ) extract or its pheophytinated fraction or to provide a composition for preventing, improving, or treating novel coronavirus infection comprising a meroterpenoid-based compound isolated therefrom as an active ingredient.

Another object of the present invention is to provide 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. .

Another object of the present invention is guava ( Psidium guajava ) to provide a herbal composition having an excellent synergistic effect in the prevention, improvement or treatment of novel coronavirus infection, including a mixture according to the components of the extract and combinations 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 preventing or improving new corona virus infection for animal feed comprising a guava extract or a pheophytinated fraction thereof or a meroterpenoid-based compound isolated therefrom as an active ingredient.

Another object of the present invention is guava ( Psidium guajava ) to provide an animal drug for the prevention or treatment of new coronavirus infection comprising a mixture according to the components of the extract and a combination thereof as an active ingredient.

An object of the present invention is guava ( Psidium guajava ) It is to provide an animal feed additive for the prevention or improvement of new coronavirus infection comprising a mixture according to the components of the extract and a combination thereof as an active ingredient.

An object of the present invention is guava ( Psidium guajava ) to provide a novel compound isolated from an extract.

The present invention is guava ( Psidium guajava ) extract or its pheophytinated fraction, or a meroterpenoid-based compound isolated therefrom, as an active ingredient, provides a composition for preventing or treating novel coronavirus infection.

The guava ( Psidium guajava ) extracts of porphyrins are chlorophyll a (chlorophyll a), chlorophyll a' (chlorophyll a'), chlorophyll b (chlorophyll b), chlorophyll b' (chlorophyll b'), 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 pheophytin a, pheophytin a', pheophytin b, and pheophytin b'. have.

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 leaf or fruit with 70-100% C1 to C4 aqueous solution of lower alcohol or lower alcohol or acetone, or a hexane fraction and pheophytinized extract thereof 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.

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.

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.

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.

[Formula 1]

The compound may be derived from guava.

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.

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.

Hereinafter, the present invention will be described in detail.

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' have.

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.

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.

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.

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.

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).

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.

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. 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.

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.

In addition, the composition may be a food composition for preventing or treating novel coronavirus infection.

The health functional food composition is one or more compounds 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. 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.

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;

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.

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.

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

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).
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.
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.
4 is a chromatogram of a pheophytinated fraction obtained by treating a leaf extract of guava with an organic acid under each condition.
5 is a chromatogram of a pheophytinated fraction through heat treatment of a guava leaf extract for 1 hour to 48 hours.
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.
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.

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.

<Example 1: guava Preparation of extracts and isolation of compounds from extracts>

1.1 guava extract and fraction of Produce

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.

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 guava Separation of compounds from extracts

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.

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) .

<Example 2: Confirmation of the physicochemical structure of the compound>

Example 2.1 Jeju Gua Capital A (Compound 1).

Jejuguajavone A;

yellowish viscous substance;

+96° (c 0.10, 메탄올);[α]

+96° ( c 0.10, methanol);

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 cm ^-1 ;

HRESIMS m / z 439.2476 [M - H] ^- (calcd for C ₂₇ H ₃₅ O ₅ , 439.2484);

¹ H NMR and ¹³ C NMR data see Tables 1 and 2 below.

2.2 Jeju Gua Capital D (Compound 2).

Jejuguajavone D;

yellowish viscous substance;

+11° (c 0.10, 메탄올);[α]

+11° ( c 0.10, methanol);

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 cm ^-1 ;

HRESIMS m / z 439.2849 [M - H] ^- (calcd for C ₂₈ H ₃₉ O ₄ , 439.2848);

¹ H NMR and ¹³ C NMR data see Tables 1 and 2 below.

2.3 Jeju Gua Capital E (Compound 3).

Jejuguajavone E;

yellowish viscous substance;

+9° (c 0.10, 메탄올);[α]

+9° ( c 0.10, methanol);

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 cm ^-1 ;

HRESIMS m / z 453.3013 [M - H] ^- (calcd for C ₂₉ H ₄₁ O ₄ , 453.3005);

¹ H NMR and ¹³ C NMR data see Tables 1 and 2 below.

2.4 Jeju Gua Capital H (compound 4).

Jejuguajavone H;

colorless viscous substance;

-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 (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 cm ^-1 ;

HRESIMS m / z 439.2844 [M - H] ^- (calcd for C ₂₈ H ₃₉ O ₄ , 439.2848);

¹ H NMR and ¹³ C NMR data see Tables 1 and 2 below.

¹ H-NMR Spectroscopic Data (in CDCl ₃ ) No compound 1 ^a compound 2 ^b compound 3 ^a compound 4 ^a One 2.14, m 2.12, m 2.09, m 1.85, m 2 α 1.71, m
β 1.57, m α 1.55, m
β 1.69, m α 1.57, m
β 1.46, m α 1.34, m
β 1.16, m 3 α 2.50, m
β 2.15, m α 2.45, m
β 2.16, m α 2.46, ddd (13.5, 10.0, 2.5)
β 2.11, m α 2.11, m
β 1.85, m 5 2.40, m 2.43, m 2.63, q (9.0) 5.02, dd (12.0, 4.2) 6 α 1.70, m
β 1.62, m α 1.67, m
β 1.61, m 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) 8 1.97, m 1.95, m 1.63, m 5.15, m 9 α 1.83, dddd (15.6, 12.6, 5.4, 3.0)
β 1,44, m 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 1.54, m
β 1,51, m 5.15, m 10 α 2.09, m
β 1.94, m α 2.16, m
β 1.93, m α 1.84, m
β 2.14, m α 2.36, m
β 2.60, d (14.4) 12 0.99, s 0.98, s 0.94, s 1.01, s 13 1.00, s 0.97, s 0.98, s 1.05, s 14 1.11, s 1.14, s 1.08, s 1.13, s 15 4.94, br s
4.89, br s 4.87, br s
4.84, br s 4.81, br s
4.72, br s 1.62, s One' 2.45, m 2.55, m
2.08, m 2.04, m
2.78, m α 2.05, m
β 2.91, dd (15.6, 6.0) 8' 2.18, m
1.43, m - - - 9' 1.02, m
0.83, m 3.06, dd (16.0, 6.0)
2.79, dd (16.0, 7.5) 2.97, td (7.5, 5.5) 2.95, dd (14.4, 6.6)
2.79, dd (14.4, 6.0) 10' 0.81, m 2.22, m 1.63, m 2.21, m 11' 10.00, s 0.91, d (6.5) 1.32, m 0.95, d (6.6) 12' 10.15, s 0.96, d (6.5) 1.34, m 0.93, d (6.6) 13' - 2.03, s 0.89, m 2.06, s 14' - - 2.04, s - 5'-OH 13.31, s 14.14, s 14.18, s 14.17, s 7'-OH 13.40, s 5.19, s 5.20, s 5.23, br s acquired at ^a 600 MHz, ^b 500 MHz.

¹³ C-NMR Spectroscopic Data (in CDCl ₃ ) No compound 1 ^a compound 2 ^b compound 3 ^a compound 4 ^a One 39.0 33.6 33.6 34.8 2 33.1 34.1 34.3 30.5 3 35.7 35.3 35.3 37.9 4 152.3 152.2 152.4 136.6 5 42.7 42.1 42.1 123.2 6 36.4 36.5 36.6 41.5 7 33.8 33.7 33.8 38.3 8 53.6 52.7 52.8 142.9 9 22.6 23.0 23.0 120.2 10 38.0 37.9 38.0 42.8 11 84.0 82.1 82.1 82.2 12 22.2 22.1 22.2 24.3 13 30.6 30.3 30.3 30.3 14 21.4 20.8 20.9 20.0 15 110.4 110.4 110.2 17.2 One' 35.1 26.1 26.2 23.8 2' 106.1 99.6 99.7 99.4 3' 164.5 154.5 154.7 154.5 4' 105.1 106.0 105.8 106.0 5' 167.9 162.6 162.6 162.8 6' 104.1 101.1 101.5 101.4 7' 169.1 157.3 157.4 157.4 8' 31.6 205.8 206.4 206.1 9' 18.3 53.3 44.6 53.4 10' 14.5 25.0 24.8 25.5 11' 192.3 23.0 31.8 23.0 12' 192.0 22.6 22.7 22.8 13' 7.0 14.1 7.1 14' 7.1 acquired at ^a 150 MHz, ^b 125 MHz.

<Example 3. guava of leaf extract Pheophytination and fraction of Manufacturing>

Pheophytins A and B are polphyrin-based compounds, and demetallation, depitylation, and decarbomethoxylation occur through the cooking process or digestion process, so that pheophytin, pyropheophytin, It is known that derivatives such as 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 Using inorganic acids guava of leaf extract Pheophytination and fraction of Produce

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 Using organic acids guava of leaf extract Pheophytination and fraction of Produce

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 Using the heat treatment method guava of leaf extract Pheophytination and fraction of Produce

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 4. HPLC through guava extract, n - hexane fraction and Pheophytination in fractions Identification of compound 1-4>

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 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 .

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.

<Example 5. HPLC through guava of leaf extract Pheophytination Gradient Measurement>

5.1 Using inorganic acids guava of leaf extract Pheophytination check the gradient

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 .

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 Using organic acids guava of leaf extract Pheophytination check the gradient

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 contained in each concentration fraction 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 pheophytination 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 Using heat treatment guava of leaf extract Pheophytination check the gradient

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.

< Example 6. About Novel Corona Virus guava leaf extract and fraction or the isolated compound. deterrent Rating>

6.1 Preparation of coronavirus

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 assay

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 guava leaf extract and fraction Or check the antiviral activity of the isolated compound against the novel coronavirus

Figure 6 shows the results of guava leaf extracts and fractions having antiviral activity against SARS-CoV-2 virus in VERO cells.

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.

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, meroterpene 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.

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.

< Formulation example 1. Pharmaceutical preparations>

Formulation example 1.1 Preparation of tablets

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 one or more 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.

Formulation example 1.2 Preparation of injection solution

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.

<Formulation Example 2. Food Manufacturing>

Formulation example 2.1 Preparation of cooking seasoning

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 cooking seasoning in an amount of 1% by weight to prepare a cooking seasoning for health promotion.

Formulation example 2.2 Manufacture of wheat flour food

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.

Formulation example 2.3 soup and preparation of gravies

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.

Formulation example 2.4 Manufacture of dairy products

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 milk in an amount of 0.1% by weight, and various dairy products such as butter and ice cream were prepared using the milk.

Formulation example 2.5 Vegetable Juice Manufacturing

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.

Formulation example 2.6 Fruit Juice Manufacturing

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)

Guava ( Psidiuma) guajava ) extract or a pheophytinated fraction thereof, or a composition for preventing or treating novel coronavirus infection comprising a meroterpenoid-based compound isolated therefrom as an active ingredient. The method of claim 1,
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. The method of claim 1,
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. The method of claim 1,
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 composition for preventing or treating novel coronavirus infection, characterized in that it is extracted with a single solvent. According to claim 1,
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. 6. The method according to any one of claims 1 to 5,
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.
[Formula 1]

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. 8. The method of claim 7,
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. 8. The method of claim 7,
The pheophytination fraction of the guava extract is obtained by suspending the 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 novel coronavirus infection, characterized in that. 8. The method of claim 7,
The pheophytinated fraction of the guava extract is obtained by suspending guava leaf or fruit extract in 0.05 to 10% (w/v) aqueous citric acid solution, A health functional food for preventing or improving symptoms of novel coronavirus infection, characterized in that it is extracted with a single solvent. 8. The method of claim 7,
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 new coronavirus infection, characterized in that it is extracted with any one solvent. 13. The method according to any one of claims 7 to 12,
The health functional food for improving symptoms of the 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.
[Formula 1]

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. 14. The method of claim 13,
The pheophytination fraction of the guava extract is obtained by suspending the 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. 14. The method of claim 13,
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. 14. The method of claim 13,
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. 17. The method according to any one of claims 13 to 16,
The disinfectant for preventing new coronavirus 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 formula 1 A disinfectant for preventing new corona virus infection, characterized in that it contains one or more from.
[Formula 1]

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. 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 .
[Formula 1]

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