KR102583001B1 - Composition for preventing or treating severe acute respiratory syndrome- corona virus infection comprising Chlorella sp. extract or pheophytinized fraction or pyphyrins or carotenones isolated therefrom as active ingredients - Google Patents

Abstract

The present invention relates to a composition for the prevention or treatment of novel coronavirus infection comprising Chlorella ( Chlorella sp.) extract and pheophytinylated fraction, or a polphyrin-based or carotenone-based compound isolated therefrom, as an active ingredient, and a chemical method. Through this, the content of pheophytin A (compound 1) and pheophytin B (compound 2), which are active compounds, are polphyrine compounds, and by further including carotenone compounds, the antiviral activity is synergized, especially in humans. It can be useful in the treatment and prevention of new coronavirus infection.

Description

Composition for preventing or treating novel coronavirus infection comprising Chlorella (Chlorella sp.) extract and its pheophytinylated fraction, or a polphyrin-based or carotenone-based compound obtained therefrom as an active ingredient {Composition for preventing or treating severe acute respiratory syndrome- corona virus infection comprising Chlorella sp. extract or pheophytinized fraction or pyphyrins or carotenones isolated therefrom as active ingredients}

The present invention relates to Chlorella , which shows recovery ability against novel coronavirus (SARS-CoV-2) infection. sp.) It relates to extracts and fractions or constituent compounds and compositions for the prevention, improvement or treatment of new coronaviruses containing them as active ingredients. More specifically, using physical and chemical methods to increase the content of active ingredients in the extract and the constituent compounds. It relates to a herbal composition that has a synergistic effect in preventing, improving, or treating new coronavirus infection by manufacturing it to show a synergistic effect between the liver.

Coronavirus is an RNA virus belonging to the coronavirus family ( Coronaviridae ) and was given its name because when viewed under an electron microscope, the spikes on the surface protrude, reminiscent of a crown or solar corona (Englund et al., 2019 ). The coronavirus family has two subfamilies, coronavirus subfamily ( Coronavirinae ) and torovirus subfamily ( Torovirinae ), and coronavirus belongs to the former. The coronavirus subfamily is further divided into four genera: Alphacoronavirus , Betacoronavirus , Gammacoronavirus , and Deltacoronavirus . Among these, the former two genera, alpha and beta-coronaviruses, are known to cause infection in humans and animals, while gamma and delta are known to cause infection in animals. Human coronaviruses were first discovered in the mid-1960s, and to date, seven types have been confirmed to infect humans, 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 pathogenic by infecting animals such as pigs, dogs, cats, bats, and horses.

The world has experienced two coronavirus infection crises, SARS (Acute Respiratory Syndrome) and MERS (Middle East Respiratory Syndrome), over the past 20 years since 2000, and the current coronavirus outbreak originated in Wuhan, China in December 2019. We are experiencing a pandemic infection crisis due to COVID-19. Although these coronaviruses all belong to the beta-coronavirus genus, they have different transmissibility and fatality rates. In the case of SARS, it occurred in China in February 2003 with SARS-CoV as the pathogen, and according to WHO's statistical data published on March 3, 2020 (WHO, Coronavirus disease 2019 (COVID-19) Situation Report - 43, 2019 ), 26 countries were infected worldwide, resulting in 8,096 confirmed cases and 774 deaths, resulting in a fatality rate of 9.6%. In the case of MERS, with MERS-CoV as the pathogen, it occurred in Arabia in the Middle East in 2012. According to the same statistical data, 27 countries around the world were infected, resulting in 2,494 confirmed cases and 858 deaths, so the transmissibility is low. It showed a high fatality rate of 34-35%. In the case of COVID-19, which broke out in China in December 2019 and continues to this day, 218 countries have been infected as of December 23, 2020, a full year ago, with 78.4 million confirmed cases and 1.72 million people. It has a high transmissibility and a fatality rate of 2.2%, which is relatively low compared to other coronavirus diseases such as SARS or MERS. However, compared to other viruses, the possibility of spreading the virus even before symptoms appear makes national response to COVID-19 difficult. All countries around the world are developing and approving vaccines led by multinational pharmaceutical companies to overcome the current situation (Park, 2020).

To date, there has been no treatment developed to solve COVID-19, and multinational pharmaceutical companies in each country are responding by developing vaccines. However, even if the vaccine works effectively, the effectiveness of the vaccine is highly unlikely to last long because mutations and frequent genetic recombination occur during the coronavirus's proliferation process. In addition, there is always a possibility that new mutant strains of the coronavirus will arise that do not respond to previously developed vaccines.

Because it is difficult to develop effective treatments in a short period of time, multinational pharmaceutical companies around the world are using drug repositioning to target already approved drugs or drugs that are not approved but for which clinical trial data already exists. 19 Efforts are being made to find a cure and it is being approved for emergency use. Although drug development through drug repurposing has great advantages in terms of cost and time, there are several problems related to patents and approvals. Also, in essence, the effectiveness of these single substances may not last long due to the emergence of resistant viruses. For this reason, it is important not only to develop vaccines and new drugs, but also to scientifically clearly prove the protective effect against COVID-19 of plants that are close to us and can be easily used and consumed as food, and to enable people to utilize them. It can be said to be a necessary strategy to overcome.

Meanwhile, chlorella is a single-celled organism belonging to green algae that grows in fresh water, a type of plankton, and is rich in various nutrients such as protein, chlorophyll, vitamins, minerals, and amino acids. It became famous after being studied as a food for astronauts by the National Aeronautics and Space Administration (NASA). In addition, 73% of the population over 50 years of age in Japan, one of the world's major longevity countries, is recognized as a health food to the extent that 73% of people take chlorella products. Chlorella's known effects so far include supplying protein, promoting metabolism, and improving immunity. In particular, CGF (Chlorella growth factor), a chlorella extract, is known to promote growth and development in children and promote hematopoiesis.

Chlorophyll is the main component of chlorella. Chlorophyll includes polphyrin-based compounds, chlorophyll A and chlorophyll B, and is demetalized, dephytylated, and decarboxylated through cooking or digestion processes. Bomethoxylation may occur to produce derivatives such as pheophytin, pyropheophytin, chlorophyllide, and pheophorbide (Hayes & Ferruzzi, 2020) . These substances are major components commonly contained in green plants, and have been reported to have clinical effects on leukopenia, aflatoxin, and cancer, and have also been confirmed to have an inhibitory effect on HIV and herpes virus (Ulbricht et al., 2014). There has been no confirmation regarding the treatment or prevention of the new coronavirus.

As a prior art, Korean Patent No. 102169476 discloses a composition for preventing or treating type 2 severe acute respiratory syndrome coronavirus infection disease, including Zafirlukast, an asthma treatment, and sulfinpyrazone, a gout treatment. (Sulfinpyrazone) showed inhibitory ability against SARS-CoV-2. Korean Patent Application No. 1020200053057 discloses coronavirus prevention and treatment, and suggests a mixture of 14 substances, including calcium, phosphoric acid, folic acid, and quercetin, as a prevention and treatment for the novel coronavirus. Korean Patent Application No. 1020200131784 discloses a composition for preventing or treating type 2 severe acute respiratory syndrome coronavirus infection disease, and Zafirlukast and Zafirlukast are selected from over 2,700 FDA-approved drugs through an AI deep learning algorithm. Candidate drugs including Sulfinpyrazone and Allopurinol were derived. However, they all have differences in the configuration of the present invention.

Korean Patent No. 102169476, Composition for preventing or treating type 2 severe acute respiratory syndrome coronavirus infectious disease, registered on October 19, 2020. Korean Patent Application No. 1020200053057. Coronavirus prevention and treatment, filed on May 2, 2020. Korean Patent Application No. 1020200131784, Composition for preventing or treating type 2 severe acute respiratory syndrome coronavirus infection disease, applied on November 04, 2020.

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. Egner P.A., et al., Identification and characterization of chlorin e4 ethyl ester in sera of individuals participating in the chlorophyllin chemoprevention trial. Chem. Res. Toxicol., 13, 900-906, 2000. 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. Hayes M., Ferruzzi M.G., Update on the bioavailability and chemopreventative mechanisms of dietary chlorophyll derivatives. Nutr. Res., 81, 19-37, 2020. Hsu C.-Y., et al., Organ-specific distribution of chlorophyll-related compounds from dietary spinachi in rabbits. Indian J. Biochem. Biophys., 51, 388-395, 2014. 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. McQuistan, T.J., et al., Cancer chemoprevention by dietary chlorophylls: a 12,000-animal dose-dose matrix biomarker and tumor study. Food Chem. Toxicol., 50, 341-352, 2012. 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. Ulbricht C., et al., An evidence-based systematic review of chlorophyll by the natural standard research collaboration. J Diet. Suppl., 11, 198-239, 2014. World Health Organization, Coronavirus disease 2019 (COVID-19): situation report, 43. World Health Organization, 1-7, 2020. 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.

The object of the present invention is to provide a composition for preventing or treating novel coronavirus infection containing Chlorella ( Chlorella sp.) extract or a pheophytinylated fraction thereof, or a polphyrine-based or carotenone-based compound isolated therefrom as an active ingredient. It is there.

Another object of the present invention is to prevent or improve symptoms of new coronavirus infection, comprising Chlorella ( Chlorella sp.) extract or pheophytinylated fraction or polphyrin-based or carotenone-based compound isolated therefrom as an active ingredient. The goal is to provide functional foods.

Another object of the present invention is to provide a disinfectant for preventing novel coronavirus infection containing Chlorella ( Chlorella sp.) extract or a pheophytinylated fraction thereof, or a polphyrine-based or carotenone-based compound isolated therefrom as an active ingredient. there is.

Another object of the present invention is to provide an animal drug for preventing or treating new coronavirus infection, which contains as an active ingredient the components of Chlorella sp. extract and a mixture thereof.

The purpose of the present invention is to provide an animal feed additive for preventing or improving new coronavirus infection, which contains the components of Chlorella sp. extract and a mixture thereof as an active ingredient.

The present invention provides a composition for preventing or treating novel coronavirus infection, comprising as an active ingredient a Chlorella ( Chlorella sp.) extract, a pheophytinylated fraction thereof, or a polphyrine-based or carotenone-based compound isolated therefrom.

The chlorella extract is porphyrin-type chlorophyll a, chlorophyll a', chlorophyll b, chlorophyll b', and pheophytin a. ), pheophytin a', pheophytin b, pheophytin b', carotenone compound α -apo-13-carotenone or β -apo- It may contain one or more selected from the group consisting of 13-carotenone, preferably pheophytin a, pheophytin a', pheophytin b, pheophytin It may include one or more selected from the group consisting of pheophytin b', α -apo-13-carotenone, or β -apo-13-carotenone.

The Chlorella extract may be obtained by extracting Chlorella sp. powder 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 Chlorella extract may be an extract obtained by extracting Chlorella with a 70-100% C1 to C4 lower alcohol aqueous solution, lower alcohol, or acetone, or may be a hexane fraction thereof or a hexane fraction obtained by pheophytinating the extract. The Chlorella extract is prepared by extracting Chlorella ( Chlorella sp.) three times by stirring for 2 hours using 95% ethanol, and then concentrating the hexane fraction obtained by solvent extraction with water or 0.5 N aqueous hydrochloric acid and hexane under reduced pressure. can do.

Of the porphyrins, chlorophyll a, chlorophyll a', chlorophyll b, chlorophyll b', pheophytin a, Pheophytin a', pheophytin b, and pheophytin b' can be produced under similar conditions from all plants that produce chlorophyll.

The compounds contained in the Chlorella extract can be obtained by separating the hexane fraction of the extract obtained by extracting Chlorella with a 70-100% C1 to C4 lower alcohol aqueous solution or lower alcohol or acetone and the hexane fraction obtained by pheophytinizing the extract, preferably Can be obtained by separating the extract extracted with 100% methanol or 95% ethanol or the hexane fraction obtained by pheophytinylating the extract by chromatography. The chromatography includes silica gel column chromatography, LH-20 column chromatography, RP-18 column chromatography, and thin layer chromatography. ), high performance liquid chromatography, etc. can be used.

The Chlorella ( Chlorella sp.) extract of the present invention is obtained by extracting Chlorella powder 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. You can.

The pheophytinated fraction of the Chlorella ( Chlorella sp.) extract of the present invention contains 0.05 to 10% (w/v) of the Chlorella extract. It may be suspended in an aqueous citric acid solution and extracted with any solvent selected from the group consisting of hexane, methylene chloride, ethyl acetate, and mixed solvents thereof.

In addition, the pheophytinated fraction of the Chlorella ( Chlorella sp.) extract of the present invention is prepared by suspending the Chlorella extract in water, leaving it at 60°C for 30 minutes to 48 hours, and then adding hexane, methylene chloride, ethyl acetate, and a mixed solvent thereof. It may be extracted with any one solvent selected from the group consisting of.

The composition for preventing or treating novel coronavirus infection of the present invention includes pheophytin A (compound 1), pheophytin B (compound 2), alpha-apo-13-carotenone (compound 3), and beta of the following formula 1: It may include one or more from the group consisting of -apo-13-carotenone (Compound 4).

[Formula 1]

Meanwhile, the compounds of the present invention can be synthesized according to methods common in the art, and can also be prepared as pharmaceutically acceptable salts.

The pharmaceutical composition containing the chlorella extract and the pheophytinylated hexane fraction or the polphyrin-based or carotenone-based compound isolated therefrom is prepared as powder, granule, tablet, capsule, suspension, emulsion, syrup, respectively, according to conventional methods. It can be formulated and used in the form of oral dosage forms such as aerosols, external preparations, suppositories, and sterile injectable solutions. Carriers, excipients, and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, and cellulose. , methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations include the chlorella extract and pheophytinylated hexane fraction of the present invention or the polphyrine or carotenone series isolated therefrom. It is prepared by mixing the compound with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, and gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, oral solutions, emulsions, syrups, etc. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. . Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao, laurel, glycerogelatin, etc. can be used.

The dosage of the composition will vary depending on the age, gender, and weight of the subject to be treated, the specific disease or pathological state to be treated, the severity of the disease or pathological state, the route of administration, and the judgment of the prescriber. Dosage determinations based on these factors are within the level of one skilled in the art, and dosages generally range from 0.01 mg/kg/day to approximately 2000 mg/kg/day. A more preferred dosage is 0.1 mg/kg/day to 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 can be administered to mammals such as rats, livestock, and humans through various routes. All modes of administration are contemplated, for example, oral, rectal or by intravenous, intramuscular, subcutaneous, intrauterine mucosal or intracerebrovascular injection. The Chlorella ( Chlorella sp.) extract and pheophytinized fraction of the present invention, or the polphyrine-based or carotenone-based compounds isolated therefrom, have almost no toxicity and side effects, and are therefore drugs that can be safely used even when taken for a long period of time for preventive purposes.

The present invention provides a health functional food for preventing or improving new coronavirus infection containing Chlorella ( Chlorella sp.) extract or a pheophytinylated fraction thereof, or a polphyrine-based or carotenone-based compound isolated therefrom as an active ingredient. .

The Chlorella ( Chlorella sp.) extract may be obtained by extracting Chlorella powder 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. . In addition, the pheophytinated fraction of the Chlorella ( Chlorella sp.) extract is obtained by suspending the Chlorella extract in a 0.5 to 1.0 N aqueous hydrochloric acid solution, suspending it in a 0.05 to 10% (w/v) aqueous citric acid solution, or suspending it in water. After doing so, it may be left at 60°C for 30 minutes to 48 hours and then extracted with any one solvent selected from the group consisting of hexane, methylene chloride, ethyl acetate, and mixed solvents thereof.

The health functional foods for preventing or improving new coronavirus infection include pheophytin A (compound 1), pheophytin B (compound 2), alpha-apo-13-carotenone (compound 3), and beta-apo-13. -It may contain one or more from the group consisting of carotenone (compound 4).

The present invention provides a disinfectant for preventing novel coronavirus infection containing Chlorella ( Chlorella sp.) extract or a pheophytinylated fraction thereof, or a polphyrine-based or carotenone-based compound isolated therefrom as an active ingredient.

The Chlorella ( Chlorella sp.) extract may be obtained by extracting Chlorella powder 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. . In addition, the pheophytinated fraction of the Chlorella ( Chlorella sp.) extract is obtained by suspending the Chlorella extract in a 0.5 to 1.0 N aqueous hydrochloric acid solution, or 0.05 to 10% (w/v) Suspended in an aqueous citric acid solution or in water, left at 60°C for 30 minutes to 48 hours, and then extracted with any solvent selected from the group consisting of hexane, methylene chloride, ethyl acetate, and mixed solvents thereof. You can.

The disinfectant for preventing new coronavirus infection includes pheophytin A (Compound 1), pheophytin B (Compound 2), alpha-apo-13-carotenone (Compound 3), and beta-apo-13-carotenone (Compound 4). ) may include one or more from the group consisting of.

The present invention provides an animal drug for preventing or treating new coronavirus infection or an animal feed additive for preventing or improving new coronavirus infection, which contains the components of Chlorella sp. extract and mixtures thereof as active ingredients. provides.

By producing Chlorella ( Chlorella sp.) extract and pheophytinylated fraction, or polphyrine-based or carotenone-based compounds isolated therefrom, which show cell lesion recovery ability against new coronavirus infection according to the present invention, new coronavirus can be prevented in animals and humans. It is expected that it can be useful as a medicine for preventing or treating infections. In addition, the pheophytinylated fraction or the polphyrin-based compound isolated therefrom can be manufactured from chlorophyll, a green plant, and can be usefully used as a medicine for preventing or treating new coronavirus infections in animals and humans.

Figure 1 is a chromatogram showing the presence or absence and relative content of polphyrine or carotenone compounds in each fraction including Chlorella sp. extract.
Figure 2 is a graph showing the cell lesion recovery ability of Chlorella ( Chlorella sp.) extract, each fraction, and a single compound derived from Chlorella against novel coronavirus (SARS-CoV-2) infection.
Figure 3 is a graph showing the increased cell lesion recovery ability against novel coronavirus (SARS-CoV-2) infection through mixing of Chlorella ( Chlorella sp.) extract fractions.
Figure 4 is a chromatogram showing the chemical composition of Chlorella ( Chlorella sp.) active subfraction (C1.1-C1.8).
Figure 5 is a graph showing the increased cell lesion recovery ability against novel coronavirus (SARS-CoV-2) infection through mixing of active small fractions of Chlorella ( Chlorella sp.).
Figure 6 shows a chromatogram showing the relative content changes of pheophytin A and B by (A) pheophytinizing Chlorella ( Chlorella sp.) extract by varying the concentration of inorganic acid, and (B) the extract containing the pheophytinylated fraction and This is a chromatogram showing the presence or absence of apo-13-carotenone compounds, which are active substances, in the fraction.
Figure 7 is a chromatogram showing the relative content changes of pheophytin A and B by pheophytinizing Chlorella ( Chlorella sp.) extract by varying the concentration of organic acid.
Figure 8 is a chromatogram showing the relative content change of pheophytin A and B by pheophytinizing Chlorella ( Chlorella sp.) extract by varying the heating treatment time.
Figure 9 shows spinach ( Spinacia This is a chromatogram showing the relative content changes of pheophytin A and B by converting the oleracea extract into pheophytin by varying the concentration of inorganic acid.
Figure 10 shows the inhibitory ability of Chlorella ( Chlorella sp.) extract, pheophytinylated fraction containing polphyrin-based and carotenone-based compounds, and pheophytin A and B mixture against novel coronavirus (SARS-CoV-2) infection. This result was confirmed through animal testing.

The present inventor, while conducting research to discover candidate natural products with pharmacological activity against the novel coronavirus targeting natural product extracts from the Bioactive Substance Resource Bank, began by confirming that the extract of Chlorella has an excellent antiviral effect. The present invention was completed by identifying active substances and their synergistic combinations through bioactivity-guided fractionation and isolation and maximization of the content of active substances.

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, the content introduced herein is provided to be thorough and complete, and to fully convey the spirit of the present invention to those skilled in the art.

< Example 1. Chlorella extract and of fractions Manufacturing>

Chlorella ( Chlorella sp.) powder was purchased from a commercial company and extracted three times with 100 g of 95% ethanol by stirring to minimize chlorophyll destruction. After drying the extract, 2.1 g of crude extract was obtained. Afterwards, in order to identify the active substance by activity-guided fractionation and isolation, medium pressure liquid chromatography (MPLC) was used under the following conditions (solvent: EA/MeOH/H). ₂ O; Column: Reverleris C18, 120 g; Flow rate: 15 ml/mL; Detection wavelength: 365, 400 nm; Gradient elution: 0-40 min 0/70/30 → 0/100/0 (EA/MeOH/H ₂ O), 40-110 min 0/100/0 → 50/50/0, 110-130 min 50/50/0 → 100/0/0) and divided into 9 fractions (C1-C9), each fraction The HPLC chromatogram for the fractions was confirmed, and the main compounds contained in each fraction are shown in Figure 1 by referring to HRMS (high resolution mass spectrometry), UV patterns, and previously reported spectroscopic data. In addition to the nine small fractions including the obtained extract, in subsequent experiments, chlorophyll a, chlorophyll b, sodium copper chlorophyll, and beta-carotene were purchased from Sigma-Aldrich as single substances derived from chlorella, and their inhibitory ability against SARS-CoV-2 was tested. Measured together.

< Example 2. Chlorella extract and of fractions inhibitory ability Confirm>

Cytopathic effect (CPE) assay was performed to confirm the effect of the Chlorella extract and fraction of the present invention on the proliferation of coronavirus.

2-1. Corona Virus Preparation

SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2, NMC-CoV-02 strain) was used after culturing VERO cells as a host at Chungbuk National University (Cheongju, Korea). SARS-CoV-2 virus was inoculated into mono-layered VERO cells, an aliquot of the virus culture supernatant was collected 3 days later, and the virus was inoculated into VERO cells cultured in DMEM medium containing 10% [v/v] FBS. It was used to measure antiviral activity.

Example 2-2. CPE Assay

After infecting VERO cells with the SARS-CoV-2 virus prepared in Example 2-1, the extract and fraction prepared in Example 1 were processed and CPE assay was performed. VERO cells were distributed in a 96-well plate at a density of 2 × 10 ⁴ cells/well, supplemented with 10% [v/v] FBS (fetal bovine serum) and 1% [v/v] PS (penicillin + streptomycin). and L-glutamine (L-glutamine, 2 ml added per 500 ml medium) for one day, and then the culture was washed with a PBS solution. The washed VERO cells were added with 50㎕ of infection media (DMEM + 1㎍/㎖ trypsin) containing the SARS-CoV-2 virus at a concentration of 100 TCID ₅₀ , infected for 2 hours, and then cultured in a PBS solution. Washed with . Afterwards, the extracts and fractions of the present invention were treated at different concentrations, cultured in DMEM culture medium containing (infection media: DMEM + 0.2 ㎍/ml trypsin) for 3 days, and the effect on viruses was measured. After 3 days of culture, the CPE effect was confirmed depending on the concentration of the extract and fraction to measure the survival of VERO cells. The results of virus titer measurement according to the concentration of extracts and fractions are shown in Figure 2.

As shown in Figure 2, among the fractions obtained from Chlorella, fractions 1 (C1), 7 (C7), and 8 (C8) showed an inhibitory effect on the growth of SARS-CoV-2. When comparing the physical and chemical properties of each fraction with Figure 1, it was confirmed that C1 is a complex and C7 and C8 are parts containing pheophytin B and A as the main compounds, respectively. In each Chlorella fraction, fractions C2 and C3, which contain carotenoid compounds as main compounds, did not show inhibitory activity, and fractions C5 and C4, which contain chlorophyll A and B as main compounds, respectively, did not show inhibitory activity. These results showed that single substances (chlorophyll A, chlorophyll B, sodium copper chlorophyll, beta-carotene) purchased from Sigma-Aldrich were evaluated for their inhibitory ability against SARS-CoV-2 in the same medium experiment, and no inhibitory activity was shown. It matched. Therefore, it was confirmed that the substances showing inhibitory activity in Chlorella are those contained in fraction C1 and pheophytin B and A, the main compounds of fractions C7 and C8, which show inhibitory activity against SARS-CoV-2.

< Example 3. About the new coronavirus of active fraction (C1-C9) Check the synergy effect according to combination>

In order to confirm the synergistic effect of the combination of fractions C1, C7, and C8, which showed the strongest inhibitory ability in Example 2, a cell lesion inhibition effect assay was performed in the same manner as in Example 2, and the results are shown in Figure 3 indicated. Referring to Figure 3, it was confirmed that the combination of fractions C1, C7, and C8, which showed the strongest activity in a single fraction, showed a stronger synergy effect than when each single fraction was treated. In particular, the combinations C1 + C7 and C1 + C8 containing fraction C1 showed a stronger inhibitory effect than C7 + C8, which does not contain C1, indicating that the compound contained in C1 has a synergistic effect with the effect of C7 and C8. Confirmed. In addition, fraction C1 + C7 + C8 showed stronger inhibitory ability than C1 + C7 and C1 + C8, so the constituents of fraction C1 were confirmed.

< Example 4. active fraction C1's small fraction Manufacturing>

The C1 fraction that showed the strongest activity in Example 3 was subjected to the following conditions (solvent: MeOH/H ₂ O (0.1% FA); column: Reverleris C18, 120 g; flow rate: 15 ml/mL; detection wavelength: 365 , 400 nm; gradient elution: 0-80 min 10/0 → 100/0, 80-120 min 100/0) and divided into eight fractions (C1.1-C1.8), and HPLC chromatography for each subfraction. Grams were confirmed and shown in Figure 4.

< Example 5. About the new coronavirus of the active fraction (C1.1-C1.8) Check the synergy effect according to combination>

Based on the results of Example 3, in order to identify substances showing a synergistic effect from C1, the combination variable was fixed to C7, which showed the second strongest inhibitory ability, excluding C1, and small fractions of C1 (C1.1 - C1. The synergy effect with 8) was confirmed. The results are shown in Figure 5. Referring to Figure 5, C1.6 and C1.7 showed the strongest effect among single fractions. Through this, it can be expected that C1.6 and C1.7 contain substances involved in activity, and when referring to Figure 4 of Example 4, C1.6 and C1.7 contain substances of the same series. It was possible to predict this through the UV pattern. Meanwhile, in combination with C7, C1.6 showed the strongest synergistic effect. However, since it was confirmed through additional experiments that C1.6 was particularly unstable in heat and transformed into other forms, experiments were started to separate the active material from the relatively stable C1.7.

< Example 6. From active fraction Separation of compounds>

An experiment was conducted to isolate the active substance from fractions C1.6, C7, and C8, which showed the strongest activity in Examples 2 to 5. First, to obtain a single substance from C7 (37.7 mg) and C8 (13.7 mg), high performance liquid chromatography (HPLC) was used under the following conditions (solvent: A-MeOH/H ₂ O, B- EA; Column: YMC triart C18 10x250; Flow rate: 3 ml/mL; Detection wavelength: 450, 654 nm; Gradient elution: 0-25 min 50/50 (A/B) (C7), 0-26 min 54/46 (A/B) (C8)), and as a result, pheophytin B (0.3) (compound 2) and pheophytin A (1.1 mg) (compound 1) were obtained, respectively.

Additionally, the small fraction C1.7 High Performance Liquid Chromatography (HPLC) was used under the following conditions (solvent: A-MeCN, BH ₂ O (0.1% FA); column: Optimapak C18 10x250; flow rate: 4 ml/mL; detection wavelength: β -apo-13-carotenone (1.0 mg) (compound 3) was separated using gradient elution: 330, 450 nm; gradient elution: 0-25 min 55/45 (A/B)).

< Example 7. Confirmation of physical and chemical structures of compounds isolated from Chlorella>

Example 7-1. Pheophytin A (Compound 1)

Pheophytin A;

Dark blue powder;

ESIMS m/z 871.6 [M + H] ⁺ ;

¹ H-NMR (CDCl ₃ , 800 MHz): δ _H 3.22 (1H, s, H-2a), 7.98 (1H, dd, J = 17.6, 11.2 Hz, H-3a), 6.16 (1H, d, J = 11.2 Hz, H-3b), 6.27 (1H, d, J = 18.4 Hz, H-3b), 9.37 (1H, s, H-5), 3.38 (3H, s, H-7a), 3.67 (2H) , m, H-8a), 1.68 (3H, t, J = 8.0 Hz, H-8b), 9.50 (1H, s, H-10), 3.67 (3H, s, H-12a), 6.24 (1H, s, H-13b), 3.86 (3H, s, H-13d), 4.44 (1H, m, H-17), 4.47 (1H, m, H-18), 1.78 (3H, d, J = 7.2, H-18a), 8.54 (1H, s, H-20).

¹³ C NMR (CDCl ₃ , 200 MHz): δ _C 142.0 (C-1), 131.8 (C-2), 12.1 (C-2a), 129.1 (C-3a), 136.3 (C-3b), 136.2 ( C-4), 97.5 (C-5), 155.7 (C-6), 136.2 (C-7), 11.3 (C-7a), 145.2 (C-8), 19.5 (C-8a), 17.4 (C) -8b), 151.0 (C-9), 104.4 (C-10), 137.9 (C-11), 129.1 (C-12), 12.1 (C-12a), 129.0 (C-13), 189.6 (C- 13a), 64.7 (C-13b), 172.2 (C-13c), 51.1 (C-13d), 149.6 (C-14), 105.2 (C-15), 161.2 (C-16), 52.8 (C-17) ), 29.8 (C-17a), 172.9 (C-17c), 50.1 (C-18), 23.1 (C-18a), 169.6 (C-19), 93.1 (C-20).

Example 7-2. Pheophytin B (Compound 2)

Pheophytin B;

brown powder;

ESIMS m/z 885.5 [M + H] ⁺ ;

¹ H-NMR (CDCl ₃ , 800 MHz): δ _H 3.38 (1H, s, H-2a), 7.99 (1H, dd, J = 17.7, 10.4 Hz, H-3a), 6.23 (1H, dd, J = 12.6, 1.7 Hz, H-3b), 6.37 (1H, br d, J = 18.0 Hz, H-3b), 10.35 (1H, s, H-5), 11.13 (3H, s, H-7a), 9.63 (1H, s, H-10), 3.68 (3H, s, H-12a), 6.24 (1H, s, H-13b), 3.91 (3H, s, H-13d), 4.20 (1H, br d) , J = 8.8 Hz, H-17), 4.40-4.60 (1H, m, H-18), 8.54 (1H, s, H-20).

Example 7-3. Alpha- Apo -13- Carotenone (Compound 3)

α -apo-13-carotenone;

yellow resin;

ESIMS m/z 259.2 [M + H] ⁺ ;

¹ H-NMR (CDCl ₃ , 800 MHz): δ _H 7.54 (1H, dd, J = 15.2, 11.2 Hz), 6.16 (1H, d, J = 15.2 Hz), 6.15 (1H, d, J = 15.6 Hz) ), 6.14 (1H, d, J = 11.4 Hz), 5.79 (1H, dd, J = 16.0, 9.6 Hz), 5.44 (1H, br s), 2.29 (3H, s), 2.22 (1H, d, J = 9.1 Hz), 2.00 (3H, s), 1.58 (3H, s), 1.44 (1H, m), 1.20 (1H, m), 0.91 (3H, s), 0.83 (3H, s).

¹³ C NMR (CDCl ₃ , 200 MHz): δ _C 198.7, 145.3, 139.5, 136.0, 135.5, 133.8, 129.6, 127.4, 121.6, 55.1, 32.8, 31.7, 27.9, 27.8, 27.1 , 23.2, 23.2, 13.7.

Example 7-4. Beta- Apo -13- Carotenone (Compound 4)

β -apo-13-carotenone;

yellow resin;

ESIMS m/z 259.2 [M + H] ⁺ ;

¹ H-NMR (CDCl ₃ , 800 MHz): δ _H 7.57 (1H, dd, J = 15.2, 12.0 Hz), 6.42 (1H, d, J = 16.0 Hz), 6.16-6.19 (3H, m), 2.30 (3H, s), 2.06 (3H, s), 2.02-2.06 (2H, m), 1.72 (3H, s), 1.59-1.63 (2H, m), 1.47-1.48 (2H, m), 1.03 (6H) , s).

¹³ C NMR (CDCl ₃ , 200 MHz): δ _C 198.5, 145.6, 139.3, 137.5, 136.7, 131.3, 129.3, 127.7, 39.6, 34.3, 33.1, 28.9, 27.6, 21.7, 19.1, 13.1.

According to the above results, pheophytin A, pheophytin B, and apo-13-carotenone-based compounds alpha-apo-13-carotenone (Compound 3) or beta-apo-13-carotenone (Compound 4) in Chlorella extract ) showed inhibitory activity against SARS-CoV-2, and their mixture was confirmed to exhibit synergistically excellent antiviral activity against SARS-CoV-2.

< Example 8. Pheophytinylation fraction Recipe>

Based on the results of Examples 2 to 5, the present inventors established a method of preparing a pheophytinylated fraction from a chlorophyll extract that can significantly increase the content of pheophytin with antiviral activity.

Example 8-1. using inorganic acid Pheophytinylation fraction manufacturing

In order to establish a pheophytinylation reaction using an inorganic acid, pheophytinylation was performed using an extract obtained by stirring 95% ethanol from Chlorella powder and varying the conditions of the hydrochloric acid solution at 0.1 N, 0.5 N, and 1 N. To confirm the increase/decrease in pheophytin A and pheophytin B in the extract without acid treatment and in the pheophytin fraction under different conditions, the extract and pheophytinized fraction were dissolved in 100% acetone and dissolved in 1 mg/mL. Adjust the concentration to the following analysis conditions (solvent: A-EA/MeOH/H ₂ O (15/65/20), B-EA/MeOH/H ₂ O (60/30/10); column: YMC triart C18 4.6x250; flow rate: 1.5 ml/min; detection wavelength: 210, 254, 450, 654 nm; gradient elution (A/B): 0-6 min (flow rate: 1.3 ml/min) 100/0, 6-7 min (Flow rate: 1.3 → 1.5 ml/min) 100/0 → 70/30, 7-10 min 70/30, 10-11 min 70/30 → 60/40, 11-12 min 60/40 → 50/50, 12-13 min 50/50 → 0/100, 13-24 min 0/100, 24-26 min 0/100 → 100/0, 26-26.1 min 100/0) and the results were analyzed using UHPLC. It is shown in Figure 6. Through comparison of the information on the molecular weight of each substance and the UV-Vis absorption spectrum unique to each substance on the right with existing literature, pheophytin A, pheophytin A, and Pheophytin B, chlorophyll A, and chlorophyll B were confirmed. In 0.1 N HCl, the areas of the peaks corresponding to chlorophyll A and chlorophyll B decreased, while the areas of the peaks corresponding to pheophytin A and pheophytin B increased. It was found that there was an increase. At 0.5 N, peaks corresponding to chlorophyll A and chlorophyll B were not identified, and the peak areas of pheophytin A and pheophytin B increased, and at 1.0 N, the same pattern was observed. Through this, it was confirmed that when a 0.5 N aqueous hydrochloric acid solution was used, sufficient demetalization of chlorophyll A and chlorophyll B occurred and the entire amount was converted to pheophytin A and pheophytin B, respectively.

Example 8-2. using organic acids Pheophytinylation fraction manufacturing

To set the conditions for pheophytinization, an experiment was performed using citric acid, a safe organic acid present in plants, at different concentrations. 0.05 to 10% (w/v) of the chlorella extract After suspending in an aqueous citric acid solution, the subsequent experiment was conducted in the same manner as in Example 8-1, and the results are shown in a chromatogram in Figure 7. When referring to the results, it can be seen that the entire amount of chlorophyll A and B was converted to pheophytin A and B in citric acid of 0.5% or more. Therefore, pheophytin A and B can be easily made by using safe organic acids, and citric acid and When foods containing the same organic acid are consumed together with chlorella, the content of pheophytin A and B increases, so an increased protective effect against SARS-CoV-2 can be expected.

Example 8-3. using heat treatment method Pheophytinylation fraction manufacturing

In order to set the conditions for pheophytinization according to heat treatment, the Chlorella extract was suspended in water in the same manner as in Example 8-1 above, and then the heat treatment time was varied from 30 minutes to 48 hours at 60 ° C. The experiment was conducted, and the results are shown in a chromatogram in Figure 8. Referring to the results, it was found that most of the chlorophyll A and B was converted to pheophytin A and B when treated for 24 hours, and when treated for 48 hours, chlorophyll A and B were not confirmed. Through this, it can be seen that as the content of pheophytin A and B increases through heat processing of food, increased inhibitory ability against SARS-CoV-2 can be expected. In addition, the results of a preliminary experiment measuring the degree of change in pheophytin A and B after leaving the plant leaves, including chloroplasts, in a dryer at 60°C showed that pheophytinization occurs even when the chlorella powder itself is left in a dryer. I found out that it was possible. These results can also be applied to pheophytinization through the blanching method, which involves heat treatment in boiling water for a short time in the general food processing process.

< Example 9. Spinach extract Pheophytinylation of fractions Manufacturing>

In order to confirm that the pheophytination of Example 8 is not limited to chlorella and can be applied to plants with green leaves, a pheophytination fraction was obtained from spinach, which is known to have a high content of chlorophyll A and B. A pheophytinated n- hexane fraction was obtained in the same manner as in Example 8-1. In addition, it is also possible to obtain a pheophytinated fraction by citric acid and heat treatment as in Examples 8-2 and 8-3. The results are shown in Figure 9. As can be seen in the UV-Vis absorption spectrum on the right side of Figure 9, the substance was confirmed through the specific UV patterns of chlorophyll A, chlorophyll B, pheophytin A, and pheophytin B, which are comparison objects.

< Example 10. For animal testing Pheophytin Mass separation of A and B>

Based on the results of Example 5, an experiment was conducted for mass separation of pheophytin A and B to confirm efficacy in an animal model. In order to separate a large amount of substances, a single substance was isolated from spinach, which is reported to contain a lot of chlorophyll A and B. 5.0 kg of spinach was extracted three times by stirring with acetone to minimize loss of chlorophyll A and B. Afterwards, 73 g of the pheophytinylated fraction was obtained using the method of Example 8-1, and it was confirmed through a chromatogram that it contained pheophytin A and B as well as carotenone derivatives. In addition, 1.95 g and 0.76 g of pheophytin A and B, respectively, were separated from the pheophytin fraction under the following conditions (normal phase column chromatography, particle size: 43-60 ㎍; n -hexane: Acetone = 3:1). did.

< Example 11. New coronavirus based on a combination of active compounds isolated from chlorella inhibitory Animal efficacy evaluation>

In order to conduct this experiment, 9 golden hamsters (syrian gloden hamsters) weighing 100 g were prepared per group, and after an adaptation period of 1 week, they were provided during the day at 12-hour intervals (7:00 a.m. to 17:00 p.m.). It was managed according to the night cycle. The groups were 1) control group (DMSO:PEG400:DW = 1:8:16 solution), 2) pheophytin A + B administration group (25 mg/head/day), 3) pheophytin fraction administration group (25 mg/head) /day) and 4) Chlorella extract (25 mg/head/day).

Hamsters in all groups were challenged intranasally with the SARS-CoV-2 (NMC-CoV-02) virus at a dose of 10 ^4.0 TCID ₅₀ , and then administered 0.5 compounds at the same time every day (every 24 hours) starting from the day of challenge. It was administered orally in ml each. On the 3rd and 7th days after challenge, virus titers were measured in the nasal and lung tissues of hamsters. The virus titer results in each tissue are shown in Figure 10.

As shown in Figure 10, when compared to the nasal tissue results control group (5dpi: 4.9log ₁₀ TCID ₅₀ /g, 7dpi: 3.0log ₁₀ TCID ₅₀ /g), pheophytin A + B (5dpi: 3.8log ₁₀ TCID ₅₀ /g, 7dpi: 1.9log ₁₀ TCID ₅₀ /g), pheophytin fraction (5dpi: 2.1log ₁₀ TCID ₅₀ /g, 7dpi: 1.8log ₁₀ TCID ₅₀ /g), chlorella extract (5dpi: 2.6log ₁₀ TCID ₅₀ /g, 7dpi: 1.9log ₁₀ TCID ₅₀ /g) It was confirmed that the virus titer was significantly lowered on the 5th and 7th days after challenge vaccination in both groups. In particular, the pheophytinylated fraction showed the strongest activity at 5 dpi, showing that the rate of antiviral activity was faster than that of the chlorella extract or pheophytin A + B mixture.

In addition, when comparing the lung tissue results with the control group (7dpi: 3.2log ₁₀ TCID ₅₀ /g), pheophytin A + B (7dpi: 2.2log ₁₀ TCID ₅₀ /g), pheophytin fraction (7dpi: 1.8log ₁₀ TCID ₅₀ /g) and Chlorella extract (7dpi: 2.0log ₁₀ TCID ₅₀ /g) groups showed a significant decrease in virus titer at 7 days after challenge.

The above results show that chlorella extract or a mixture of pheophytin A and B has strong antiviral activity against SARS-CoV-2, and that the pheophytin fraction has a higher antiviral activity than chlorella extract or a mixture of pheophytin A and B. It was found that it showed viral activity. As can be seen in Figures 6A and 6B, substances other than pheophytin A and B contained in the pheophytin fraction, for example, carotenone derivatives such as apo-13-carotenone compounds, are included, thereby forming carotenone derivatives. It can be inferred that it has strong antiviral activity by producing a synergistic effect with pheophytin A and B.

< example 1. Pharmaceutical preparations>

example 1-1. manufacture of tablets

20 g of the chlorella extract (or pheophytinylated fraction) of the present invention, or pheophytin A, pheophytin B, 200 mg of at least one selected from the group consisting of alpha-apo-13-carotenone and beta-apo-13-carotenone were mixed with 175.9 g of lactose, 180 g of potato starch, and 32 g of colloidal silicic acid, respectively. A 10% gelatin solution was added to this mixture, then pulverized 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.

example 1-2. Manufacturing of injectable solutions

Pheophytin A, pheophytin B of the present invention, 100 mg of at least one selected from the group consisting of alpha-apo-13-carotenone and beta-apo-13-carotenone, 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.

< example 2. Food manufacturing>

example 2-1. Manufacturing of cooking seasoning

Chlorella extract (or pheophytinylated fraction) of the present invention or pheophytin A, pheophytin B, A cooking seasoning for health promotion was prepared by adding 1% by weight of one or more selected from the group consisting of alpha-apo-13-carotenone and beta-apo-13-carotenone to the cooking seasoning.

example 2-2. Manufacturing of flour foods

Chlorella extract (or pheophytinylated fraction) of the present invention or pheophytin A, pheophytin B, At least one selected from the group consisting of alpha-apo-13-carotenone and beta-apo-13-carotenone is added to flour in an amount of 0.1% by weight, and this mixture is used to make bread, cakes, cookies, crackers and Noodles were manufactured to produce food for health promotion.

example 2-3. soup and preparation of gravies

Chlorella extract (or pheophytinylated fraction) of the present invention or pheophytin A, pheophytin B, Health-promoting soup and broth were prepared by adding 0.1% by weight of one or more 2 selected from the group consisting of alpha-apo-13-carotenone and beta-apo-13-carotenone to the broth, respectively.

example 2-4. Manufacturing of dairy products

Chlorella extract (or pheophytinylated fraction) of the present invention or pheophytin A, pheophytin B, At least one selected from the group consisting of alpha-apo-13-carotenone and beta-apo-13-carotenone is added to milk at 0.1% by weight, and the milk is used to make various dairy products such as butter and ice cream. Manufactured.

example 2-5. Vegetable juice manufacturing

20 g of the chlorella extract (or pheophytinylated fraction) of the present invention, or pheophytin A, pheophytin B, Vegetable juice for health promotion was prepared by adding 200 mg of at least one selected from the group consisting of alpha-apo-13-carotenone and beta-apo-13-carotenone to 1,000 ml of tomato juice or sugar root juice, respectively.

example 2-6. Fruit juice manufacturing

20 g of the chlorella extract (or pheophytinylated fraction) of the present invention, or pheophytin A, pheophytin B, Fruit juice for health promotion was prepared by adding 200 mg of at least one selected from the group consisting of alpha-apo-13-carotenone and beta-apo-13-carotenone to 1,000 ml of apple juice or grape juice.

Claims (17)

In the composition for preventing or treating new coronavirus infection containing Chlorella (Chlorella sp.) extract or a pheophytinated fraction thereof as an active ingredient,
The composition for preventing or treating novel coronavirus infection includes pheophytin A (compound 1), pheophytin B (compound 2), alpha-apo-13-carotenone (compound 3), and beta-apo of the following formula 1: A composition for preventing or treating novel coronavirus infection, characterized in that it contains at least one selected from the group consisting of -13-carotenone (Compound 4).
[Formula 1]
According to paragraph 1,
The Chlorella ( Chlorella sp.) extract is used for the prevention or treatment of new coronavirus infection, characterized in that Chlorella powder is extracted with any one solvent selected from the group consisting of water, C1 to C4 lower alcohol, and mixed solvents thereof. Composition. According to paragraph 1,
The pheophytinized fraction of the Chlorella ( Chlorella sp.) extract is obtained by suspending the Chlorella extract in a 0.5 to 1.0 N aqueous hydrochloric acid solution, followed by adding any one solvent selected from the group consisting of hexane, methylene chloride, ethyl acetate, and mixed solvents thereof. A composition for preventing or treating new coronavirus infection, characterized in that it is extracted with . According to paragraph 1,
The pheophytinated fraction of the Chlorella ( Chlorella sp.) extract is obtained by suspending the Chlorella extract in 0.05 to 10% (w/v) citric acid aqueous solution and then using hexane, methylene chloride, ethyl acetate, and a mixed solvent thereof. A composition for preventing or treating new coronavirus infection, characterized in that it is extracted with any selected solvent. According to paragraph 1,
The pheophytinized fraction of the Chlorella ( Chlorella sp.) extract is prepared by suspending the Chlorella extract in water, leaving it at 60°C for 30 minutes to 48 hours, and then mixing it with hexane, methylene chloride, ethyl acetate, and a mixed solvent thereof. A composition for preventing or treating new coronavirus infection, characterized in that it is extracted with any one solvent selected from. delete In the health functional food for preventing or improving symptoms of new coronavirus infection containing Chlorella (Chlorella sp.) extract or its pheophytinylated fraction as an active ingredient,
The health functional food for preventing or improving the symptoms of novel coronavirus infection includes pheophytin A (compound 1), pheophytin B (compound 2), alpha-apo-13-carotenone (compound 3), and A health functional food for preventing or improving symptoms of new coronavirus infection, comprising at least one selected from the group consisting of beta-apo-13-carotenone (compound 4).
[Formula 1]
. In clause 7,
The Chlorella ( Chlorella sp.) extract prevents or improves symptoms of new coronavirus infection, characterized in that chlorella powder is extracted with any one solvent selected from the group consisting of water, C1 to C4 lower alcohol, and mixed solvents thereof. Health functional food. In clause 7,
The pheophytinized fraction of the Chlorella ( Chlorella sp.) extract is obtained by suspending the Chlorella extract in a 0.5 to 1.0 N aqueous hydrochloric acid solution, followed by adding any one solvent selected from the group consisting of hexane, methylene chloride, ethyl acetate, and mixed solvents thereof. A health functional food for preventing or improving symptoms of new coronavirus infection, characterized by being extracted from . In clause 7,
The pheophytinated fraction of the Chlorella ( Chlorella sp.) extract is obtained by suspending the Chlorella extract in 0.05 to 10% (w/v) citric acid aqueous solution and then using hexane, methylene chloride, ethyl acetate, and a mixed solvent thereof. A health functional food for preventing or improving symptoms of new coronavirus infection, characterized by extraction with a selected solvent. In clause 7,
The pheophytinized fraction of the Chlorella ( Chlorella sp.) extract is prepared by suspending the Chlorella extract in water, leaving it at 60°C for 30 minutes to 48 hours, and then mixing it with 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 selected from. delete In a disinfectant for preventing novel coronavirus infection containing Chlorella (Chlorella sp.) extract or a pheophytinylated fraction thereof as an active ingredient,
The disinfectant for preventing new coronavirus infection is pheophytin A (compound 1), pheophytin B (compound 2), alpha-apo-13-carotenone (compound 3), and beta-apo-13-caro of the following formula 1: A disinfectant for preventing novel coronavirus infection, characterized in that it contains at least one selected from the group consisting of Tenon (Compound 4).
[Formula 1]
According to clause 13,
The pheophytinized fraction of the Chlorella ( Chlorella sp.) extract is obtained by suspending the Chlorella extract in a 0.5 to 1.0 N aqueous hydrochloric acid solution, followed by adding any one solvent selected from the group consisting of hexane, methylene chloride, ethyl acetate, and mixed solvents thereof. A disinfectant for preventing new coronavirus infection, characterized by being extracted from . According to clause 13,
The pheophytinized fraction of the Chlorella ( Chlorella sp.) extract contains 0.05 to 10% (w/v) of the Chlorella extract. A disinfectant for preventing novel coronavirus infection, characterized in that it is suspended in an aqueous citric acid solution and extracted with any solvent selected from the group consisting of hexane, methylene chloride, ethyl acetate, and mixed solvents thereof. According to clause 13,
The pheophytinized fraction of the Chlorella ( Chlorella sp.) extract is prepared by suspending the Chlorella extract in water, leaving it at 60°C for 30 minutes to 48 hours, and then mixing it with hexane, methylene chloride, ethyl acetate, and a mixed solvent thereof. A disinfectant for preventing new coronavirus infection, characterized in that it is extracted with any one solvent selected from. delete