CN117120068A - Radix Sanguisorbae extract composition for inhibiting SARS-CoV-2 3CL protease and RdRp activity - Google Patents
Radix Sanguisorbae extract composition for inhibiting SARS-CoV-2 3CL protease and RdRp activity Download PDFInfo
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Abstract
The present invention relates to novel use of radix Sanguisorbae extract for inhibiting activities of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2,Severe acute respiratory syndrome coronavirus 2) virus 3CL protease and RNA-dependent RNA polymerase (RdRp, RNA-dependent RNA polymerase), and composition comprising the radix Sanguisorbae extract of the present invention as an active ingredient, which inhibit activities of 3CL protease and RdRp, and have an effect of preventing or treating infection caused by SARS-CoV-2 and its variant viruses.
Description
Technical Field
The present invention relates to the function of sanguisorba officinalis (Sangusorba officinalis Linne) extract for inhibiting the activity of 3C-like protease (3C-like protease) (3 CL protease) and RNA-dependent RNA polymerase (RNA-dependent RNA Polymerase, rdRp) held by SARS-CoV-2 virus and its use.
Background
The radix Sanguisorbae refers to cucumber fragrance, which is perennial herb belonging to Rosaceae. Is distributed in korea, china, japan, russia and the like, and is a wild grass grown in mountains and wild lands. Since ancient times, sanguisorba officinalis is utilized as a detoxification, scald, diarrhea, respiratory and gastrointestinal disease relieving agent, and is reported to have various effects of antiallergic, anticancer, antioxidant and the like.
Conventionally, as a solvent for extracting plants, methanol (methanol), ethanol (ethanol) or water is used, and hot water extract of sanguisorba officinalis is reported to have useful effects, and it has been reported through previous studies that it has anticancer effects of inhibiting cell proliferation of oral cancer (oral cancer) and enhancing immune function (immunopotentiation). The hot water extraction method is easier to extract natural substances than other methods, and can obtain useful components in large amounts, and is low in toxicity because of the extraction method that does not use chemical components such as ethanol, and the extract has a strong efficacy and can be regarded as safe in food intake. In addition, the garden burnet is a herb family growing in mountain land, and has the advantages of easy cultivation, strong autogenous power and easy acquisition.
Although korean patent publication No. 10-17842540000 discloses "a composition for preventing or treating sepsis and a health functional food composition comprising sanguisorba officinalis extract as an active ingredient" and korean patent publication No. 2011-017540 discloses "a pharmaceutical composition for preventing or treating helicobacter pylori and a health food comprising sanguisorba officinalis extract as an active ingredient", no preventive, ameliorative or therapeutic effect of sanguisorba officinalis extract on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2,Severe acute respiratory syndrome coronavirus 2) infection has been reported in the literature so far.
The present inventors confirmed that the sanguisorba officinalis extract has an effect of preventing or treating infection caused by SARS-CoV-2, thereby completing the present invention.
Disclosure of Invention
Technical problem
The radix Sanguisorbae refers to cucumber fragrance, which is perennial herb belonging to Rosaceae. Is distributed in korea, china, japan, russia and the like, and is a wild grass grown in mountains and wild lands. Since ancient times, sanguisorba officinalis is utilized as a detoxification, scald, diarrhea, respiratory and gastrointestinal disease relieving agent, and is reported to have various effects of antiallergic, anticancer, antioxidant and the like.
Conventionally, as a solvent for extracting plants, methanol (methanol), ethanol (ethanol) or water is used, and hot water extract of sanguisorba officinalis is reported to have useful effects, and it has been reported through previous studies that it has anticancer effects of inhibiting cell proliferation of oral cancer (oral cancer) and enhancing immune function (immunopotentiation). The hot water extraction method is easier to extract natural substances than other methods, and can obtain useful components in large amounts, and is low in toxicity because of the extraction method that does not use chemical components such as ethanol, and the extract has a strong efficacy and can be regarded as safe in food intake. In addition, the garden burnet is a herb family growing in mountain land, and has the advantages of easy cultivation, strong autogenous power and easy acquisition.
Although korean patent publication No. 10-17842540000 discloses "a composition for preventing or treating sepsis and a health functional food composition comprising sanguisorba officinalis extract as an active ingredient" and korean patent publication No. 2011-017540 discloses "a pharmaceutical composition for preventing or treating helicobacter pylori and a health food comprising sanguisorba officinalis extract as an active ingredient", no preventive, ameliorative or therapeutic effect of sanguisorba officinalis extract on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2,Severe acute respiratory syndrome coronavirus 2) infection has been reported in the literature so far.
The present inventors confirmed that the sanguisorba officinalis extract has an effect of preventing or treating infection caused by SARS-CoV-2, thereby completing the present invention.
Technical proposal
In order to achieve the above object, the present invention provides a composition for treating and/or preventing COVID-19 induced by SARS-CoV-2 by inhibiting the activities of 3CL protease and RdRp, comprising an extract of Sanguisorbae radix as an active ingredient.
Also provided is a health functional food composition for preventing and/or ameliorating infection caused by SARS-CoV-2, which comprises a Sanguisorbae extract as an active ingredient.
Also, provided is a method for producing a pharmaceutical composition for preventing or treating infection caused by SARS-CoV-2 virus, comprising:
drying sanguisorba officinalis;
a step of extracting the dried sanguisorba officinalis as described above with a solvent at 30 to 120 ℃ to form a solution; and
and freeze-drying the extracted solution to prepare a powder.
Technical effects
The composition comprising the sanguisorba officinalis extract of the present invention as an active ingredient inhibits SARS-CoV-2 specific 3CL protease and RdRp activity, thereby having an effect of treating COVID-19 induced by SARS-CoV-2.
Drawings
FIG. 1 is a graph showing the results of inhibition of 3CL protease by a Sanguisorbae radix extract, a negative control group and a positive control group in an in vitro assay (in-vitro assay). * P <0.01; * P <0.001
FIG. 2 is a graph showing the results of inhibition of SARS-CoV-2 3CL protease activity by a Sanguisorbae radix extract, a negative control group and a positive control group in Cell-based assay (Cell-based). * P <0.05; * P <0.01; * P <0.001
FIG. 3 is a graph showing the results of inhibition of SARS-CoV-2RdRp activity by the Sanguisorbae radix extract, the negative control group and the positive control group in an in vitro assay (in-vitro assay). * P <0.01; * P <0.001
FIG. 4 is a graph showing the results of inhibition of SARS-CoV-2 proliferation by Sanguisorba officinalis extract, negative control, positive control 1 (GC 376) and positive control 2 (Remdesivir) at the cellular level.
FIG. 5 is a graph showing the results of inhibition of weight loss by sanguisorba officinalis extract, negative control group, positive control group in SARS-CoV-2 infected hamsters. * P <0.05; * P <0.01; * P <0.001
FIG. 6 is a graph showing the results of confirming that the virus proliferation was inhibited by the sanguisorba extract, the negative control group, and the positive control group in lung tissue of hamster infected with SARS-CoV-2 by RT-PCR. * P <0.05
FIG. 7 is a graph showing the results of confirming that the virus proliferation was inhibited by the sanguisorba officinalis extract, the negative control group, and the positive control group in lung tissue of hamsters infected with SARS-CoV-2 by plaque assay (plaque assay). M, average (mean value)
FIG. 8 is a graph showing the results of confirming that the virus proliferation was inhibited by the sanguisorba extract, the negative control group, and the positive control group in lung tissue of hamster infected with SARS-CoV-2 by tissue staining.
FIG. 9 is a graph showing the results of inhibition of inflammation by sanguisorba officinalis extract, negative control group, positive control group in lung tissue of hamster infected with SARS-CoV-2. * P <0.05
Best mode for carrying out the invention
The invention relates to the use of sanguisorba officinalis extract for inhibiting SARS-CoV-2 virus 3CL protease and RdRp activity.
Detailed Description
The present invention will be described in detail below.
The present invention provides a pharmaceutical composition for preventing or treating infection caused by SARS-CoV-2, comprising an extract of Sanguisorbae radix as an active ingredient.
SARS-CoV-2 is a virus that causes respiratory syndrome by infection, designated as the first stage of legal infection. At present, global epidemics (pandemic) are induced and very serious severe cases are induced, leading to mortality in the elderly over 80 years of age up to 25%. The intranasal penetration of SARS-CoV-2 is designated as a major cause of transmission by means of droplets in the air or contact with humans.
SARS-CoV-2, which has flowed into the human body, is bound to the receptor for Angiotensin converting enzyme 2 (ACE 2, angiotenin-Converting Enzyme 2) on the cell surface, and is impregnated into the cell, and a large amount of new virus is produced by division and proliferation (Jackson CB, et al, mechanics of SARS-CoV-2entry into cells.Nat Rev Mol Cell Biol.2021 5;1-18). The SARS-CoV-2 produced is proliferated by repeating the same process of re-secreting it out of the cell and re-penetrating it into other cells. Thus, the mechanisms associated with cellular infection, intracellular replication and proliferation of SARS-CoV-2 are major targets for therapeutic development.
SARS-CoV-2 produces multiple variants including delta-type variants derived primarily from genetic mutations within the spike domain (Harvey, W.T, et al SARS-CoV-2variants,spike mutations and immune escape.Nat Rev Microbiol19,2021 409-424) that saturate the host cell.
SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2) was the first known severe acute respiratory syndrome coronavirus 2in 2019 and was classified as positive-sense single-stranded RNA virus (stranded RNA virus). The disease infected by this virus is designated as coronavirus infectious disease-19 (Coronavirus disease 2019), abbreviated as covd-19. The SARS-CoV-2 disease is likely to be coronavirus infectious disease-19. The coronavirus infectious disease-19 may be a respiratory disease. The respiratory disease may be pneumonia. The symptoms of coronavirus infection-19 may be at least one of fever, tiredness, cough, dyspnea, phlegm, sore throat, headache, hemoptysis, nausea and diarrhea.
The SARS-CoV-2 virus described above can comprise variants thereof.
The present invention is characterized in that the mutation is generated in the raised protein (spike protein) of SARS-CoV-2 virus to form the above-mentioned variant.
On the other hand, it is known that, in addition to the mutation in the raised protein (spike protein) of SARS-CoV-2 virus, a virus having a gene mutation at a site related to the activities of 3CL protease and RdRp has not been reported, and thus, an inhibitor targeting the activities of 3CL protease and RdRp can also be expected to have the same therapeutic efficacy on the mutant virus.
The present invention is characterized in that a composition comprising the above-mentioned sanguisorba officinalis extract as an active ingredient can inhibit or inhibit the activity of 3C-like protease (3C-like protease).
The present invention is characterized in that a composition comprising the above-mentioned sanguisorba officinalis extract as an active ingredient can inhibit SARS-CoV-2 specific 3CL protease and RdRp activity.
The 3CL protease is a protein which is encoded in the SARS-CoV-2 gene together with papain-like protease and is essential for the production of functional proteins required for the in vivo proliferation of SARS-CoV-2 (Anirudhan V, et al, targeting SARS-CoV-2viral proteases as atherapeutic strategy to treat COVID-19.J Med Virol.2021 93 (5): 2722-2734). The functional protein required for the proliferation of SARS-CoV-2 is expressed as a polypeptide by the protein synthesis system of the host cell. Specifically, each protein covalently linked to the polypeptide does not exert its inherent function, but is cleaved by a protease (protease) to obtain its function when it is present alone (Anirudhan V, et al targeting SARS-CoV-2viral proteases as a therapeutic strategy to treat COVID-19.J Med Virol.2021 93 (5): 2722-2734). It is known that 3CL protease acts on 11 cleavage sites (cleavage sites) present in a polypeptide to produce a functional protein, whereas papain-like protease acts on 3 cleavage sites (cleavage sites) to produce a functional protein (Anirudhan V, et al, targeting SARS-CoV-2viral proteases as a therapeutic strategy to treat COVID-19.J Med Virol.2021 93 (5): 2722-2734). Thus, inhibiting 3CL protease can inhibit the production of a plurality of functional proteins, compared to papain-like protease, and this enzyme can be a major goal for the development of SARS-CoV-2 therapeutics.
The invention is characterized in that the composition can block or inhibit RdRp (RNA-dependent RNA polymerase) activity.
The RdRp is an enzyme responsible for replication and transcription of the RNA genome in most RNA viruses, including SARS-CoV-2 (Aftab, S.O., et al analysis of SARS-CoV-2RNA-dependent RNA polymerase as a potential therapeutic drug target using acomputational appreach.J Transl Med 2020, 18, 275).
The present inventors have found that the present invention is completed by screening substances inhibiting the enzymatic activities of SARS-CoV-2 specific 3CL protease and RdRp using hot water extracts of 223 crude drugs in order to develop a therapeutic and prophylactic agent for SARS-CoV-2.
In a specific embodiment of the present invention, sanguisorba officinalis extract is prepared using sanguisorba officinalis, and used in vitro, cell test and animal experiment using hamster. As a result, it was confirmed that the sanguisorba officinalis extract of the present invention can be usefully used as a pharmaceutical composition for preventing or treating infection caused by the SARS-CoV-2 virus by exhibiting a inhibitory effect on proliferation of SARS-CoV-2 (see fig. 1 to 9) by inhibiting 3CL protease and RdRp, compared to a negative control group in which the sanguisorba officinalis extract was not used.
Further, as shown in example 2 below, the 3CL glow assay kit (glow assay kit) (Meng Da, molecular (Montana Molecular), usa) used for this experiment was based on GFP fusion (fusion) proteins in which a domain (domain) cleaved by a 3CL protease was attached to the c-terminal end of a green fluorescent protein (GFP protein). GFP fusion (fusion) proteins do not appear fluorescent whereas pure GFP proteins, in which the c-terminal fusion domain (c-terminal fusion domain) is cleaved by the 3CL protease, appear fluorescent. That is, when 3CL protease is inhibited, fluorescence generated from GFP fusion (fusion) protein is also reduced. Therefore, by reducing the fluorescence generation, inhibition of SARS-CoV-2 3CL protease activity by the sanguisorba officinalis extract was confirmed.
As shown in example 5 below, as a positive control group, rdRp inhibitor Mo Nuola (molnupiravir) containing 100mg/kg to 300mg/kg, preferably 250mg/kg of methyl cellulose solvent (MC vehicle) can be orally administered to hamsters, but is not limited thereto. When the content of Mo Nuola is less than the above range, the inhibition effect of SARS-CoV-2 expressed in hamsters is very small, and when it exceeds the above range, the practical benefit in terms of cost is small. The reason for using high concentration (250 mg/kg) is that high concentration Mo Nuola of hamster (molupiravir) exerts inhibitory effect of SARS-CoV-2 (Rosenke K et al. Oraliy precipitated MK-4482 inhibitors SARS-CoV-2replication in the Syrian hamster model.Nat Commun.2021 12 (1): 2295) differently from ferret (ferret) (Cox RM et al. Therapeutic administered ribonucleoside analogue MK-4482/EIDD-2801blocks SARS-CoV-2transmission in ferrets.Nat Microbiol.2021 6 (1): 11-18) which uses low concentration of minora (15 mg/kg).
The present invention is characterized in that the above-mentioned sanguisorba officinalis extract can be extracted with water, ethanol or a solvent as a mixture thereof.
The solvent may be water, ethanol, a lower alcohol having 1 to 5 carbon atoms, or an aqueous solution of a lower alcohol having 1 to 5 carbon atoms, but is not limited thereto.
When extracted by the above solvent, the extraction temperature is 20 to 130 ℃, preferably 30 to 120 ℃, more preferably 90 to 110 ℃, but is not limited thereto.
The pharmaceutical compositions may comprise a pharmaceutically acceptable carrier, excipient or diluent. The term "pharmaceutically acceptable" of the invention refers to those properties that exhibit no toxicity to cells or humans exposed to the above-described compositions. Compositions comprising a pharmaceutically acceptable carrier are likely to be in a variety of dosage forms for oral or parenteral administration. In the preparation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant, which are generally used, may be used. The carrier, excipient and diluent may be one or more selected from the group consisting of lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, physiological saline, methylparaben, propylparaben, talc, magnesium stearate and mineral oil, dextrin, calcium carbonate, propylene glycol and liquid paraffin, but are not limited thereto, and conventional carriers, excipients or diluents may be used. The above components can be used alone or in combination with radix Sanguisorbae extract as effective component.
The pharmaceutical composition may be in any form selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, oral liquid preparations, emulsions, syrups, sterilized aqueous solutions, nonaqueous solvents, suspensions, emulsions, freeze-dried preparations and suppositories.
The base material of the suppository can be semisynthetic fatty acid ester (witepsol), polyethylene glycol (macrogol), tween (tween) 60, cocoa butter, trilaurin, glycerogelatin, etc.
The pharmaceutical composition may have any one of dosage forms selected from the group consisting of powder, granule, tablet, capsule and liquid form.
The pharmaceutical administration forms of the above extracts may be used alone or in combination with other pharmaceutically active compounds and can be suitably combined.
The pharmaceutical composition can be orally administered, and can be solid preparation or liquid preparation. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and these solid preparations are formulated by mixing one or more excipients such as starch, calcium carbonate (calcium carbonate), sucrose (sucrose), lactose (lactose), gelatin, and the like with the above extract. Besides the simple excipient, a lubricant such as magnesium stearate and talc is used. As liquid preparations for oral administration, there are corresponding suspending agents, oral liquid preparations, emulsions, syrups and the like, and besides water and liquid paraffin, which are often used as simple diluents, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be contained. Formulations for parenteral administration include sterile aqueous solutions, nonaqueous solvents, suspensions, emulsions, freeze-dried formulations, suppositories. As the nonaqueous solvent and suspending agent, propylene glycol (propylene glycol), vegetable oil such as polyethylene glycol and olive oil, injectable ester such as ethyl oleate, and the like can be used.
The pharmaceutical compositions of the present invention can be administered in a pharmaceutically effective amount. The administration amount is not particularly limited and may vary depending on the in vivo absorbability, body weight, age, sex, health state, diet, administration time, administration method, excretion rate, severity of disease, etc. The pharmaceutical composition of the present invention is prepared in consideration of an effective amount range, and thus the unit dosage form preparation in such a dosage form may be administered as needed using a method of administration specialized according to the judgment of an expert and personal need for monitoring or observing the administration of the pharmaceutical agent, or a plurality of administrations at prescribed time intervals. The above administration is preferably once daily, and may be divided into a plurality of administrations.
The invention is characterized in that the sanguisorba officinalis extract is extracted from the roots of cucumber (Sanguisorba officinalis L.), the roots of sanguisorba longifolia (Sanguisorba longifolia) or a mixture thereof.
Also, the present invention provides a food composition for preventing or ameliorating infection caused by SARS-CoV-2 or a disease caused thereby, comprising an extract of Sanguisorbae radix as an active ingredient.
The present invention is characterized in that the food may be one or more of health functional foods and beverages.
When the above-mentioned sanguisorba officinalis extract is used as a food additive, the above-mentioned sanguisorba officinalis extract may be directly added or may be used together with other foods or food ingredients, and may be suitably used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined depending on the purpose of use (prophylactic, healthy or therapeutic treatment). In general, when preparing a food or beverage, the extract of the present invention can be added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less, relative to the raw materials. However, the above amount may be less than the above range for long-term intake for health and hygiene purposes or for health conditioning purposes, and the active ingredient may be used in an amount not less than the above range because of no problem in safety.
The kind of the above health functional food is not particularly limited. Examples of foods to which the above-mentioned sanguisorba extract can be added include meats, sausages, breads, chocolates, candies, snacks, biscuits, pizzas, instant noodles, other noodles, chewing gums, dairy products including ice cream, various soups, drink water, tea, health drinks, alcoholic beverages, vitamin complexes, and the like, and can include health functional foods in the conventional sense.
The health functional beverage composition of the present invention may contain various flavors or natural carbohydrates, etc. as additional ingredients as in conventional beverages. The natural carbohydrate is a monosaccharide such as glucose and fructose, a disaccharide such as maltose and sucrose, a polysaccharide such as dextrin and cyclodextrin, a sugar alcohol such as xylitol, sorbitol and erythritol. As the sweetener, natural sweeteners such as thaumatin and stevioside extract, synthetic sweeteners such as saccharin and aspartame, etc. can be used.
In addition to the above-mentioned health functional foods, the health functional foods of the present invention may contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acids and salts thereof, alginic acids and salts thereof, organic acids, protective colloid tackifiers, pH adjusters, stabilizers, preservatives, glycerin, ethanol, carbonating agents for use in carbonated beverages, and the like. In addition, pulp for preparing natural juice, juice beverage and vegetable beverage may be included. Such components may be used alone or may be used in combination. The ratio of such additives is not critical, but is generally selected in the range of 0.01 to 2 parts by weight based on 100 parts by weight of the composition of the present invention.
The above sanguisorba officinalis extract is preferably prepared by a preparation method including the following steps, but is not limited thereto:
1) Drying sanguisorba officinalis;
2) A step of extracting the dried sanguisorba officinalis as described above with a solvent at 30 to 120 ℃ to form a solution; and
3) And freeze-drying the extracted solution to prepare a powder.
The present invention provides a process for preparing a pharmaceutical composition for preventing or treating infection caused by SARS-CoV-2, characterized by comprising:
1) Drying sanguisorba officinalis;
2) A step of extracting the dried sanguisorba officinalis as described above with a solvent at 30 to 120 ℃ to form a solution; and
3) And freeze-drying the extracted solution to prepare a powder.
The present invention also provides a method for producing a health functional food composition for preventing or ameliorating infection caused by SARS-CoV-2, comprising:
1) Drying sanguisorba officinalis;
2) A step of extracting the dried sanguisorba officinalis as described above with a solvent at 30 to 120 ℃ to form a solution; and
3) And freeze-drying the extracted solution to prepare a powder.
In the above method, the sanguisorba officinalis of step 1) is not limited to cultivated cucumber aroma or sanguisorba longifolia, or commercially available sanguisorba officinalis, etc., and any part such as leaf, root, stem and branch may be used, but is not limited thereto, but the root of cucumber aroma or the root of sanguisorba longifolia is most preferably used according to a preferred embodiment of the present invention.
In the above method, the extraction solvent of step 1) is preferably water, ethanol, alcohol or a mixture thereof and an organic solvent. As the above-mentioned ethanol, a lower alcohol having 1 to 5 carbon atoms is preferably used, and as the lower alcohol, ethanol or methanol is preferably used. As the extraction method, hot water extraction, oscillation extraction, soxhlet extraction, or reflux extraction is preferably used, but is not limited thereto. Preferably, the above extraction solvent is added to the total weight of dried sanguisorba officinalis in an amount of 5 to 30 times, more preferably 20 times. The extraction temperature is preferably 20 to 130 ℃, more preferably 90 to 110 ℃, but is not limited thereto. The extraction time is preferably 2 to 48 hours, more preferably 15 to 30 hours, and most preferably 24 hours, but is not limited thereto. Further, the number of extraction is preferably 1 to 5, more preferably 3 to 4, and most preferably 3, repeated extraction, but is not limited thereto.
The above-mentioned sanguisorba officinalis extract is preferably 30 to 95% alcoholic extract, more preferably 40 to 90% alcoholic extract, and most preferably 50 to 80% alcoholic extract, and for less than 50% of the above-mentioned alcoholic extract, the extraction efficiency is lowered, so that the intended effect for preventing or treating infection caused by SARS-CoV-2 virus cannot be exhibited, and for more than 80% alcoholic extract, the production cost of the sanguisorba officinalis extract is undesirably increased.
A composition comprising the sanguisorba officinalis extract prepared by the above preparation method as an active ingredient, characterized in that 3CL protease and RdRp activities are inhibited, thereby exhibiting an effect for preventing or treating infection caused by SARS-CoV-2.
The present invention will be described in detail with reference to examples and experimental examples.
However, the following examples and experimental examples are only for illustrating the present invention, and the present invention is not limited to the following examples and experimental examples.
EXAMPLE 1 preparation of sanguisorba officinalis extract
Distilled water 20 times the total weight of dried radix Sanguisorbae (root of Cucumis sativus (Sanguisorba officinalis Linne)) was added to the dried radix Sanguisorbae, and the mixture was cold-immersed for 1 hour and 30 minutes. Then, the solution was extracted with a large shaker under reflux (reflux) at 100℃for 1 hour 30 minutes. Filtering the extracted solution under reduced pressure, removing impurities, and freeze drying at-20deg.C to obtain powder. A solution (20 mg/mL) containing the Sanguisorbae radix extract was prepared by putting 0.2g of the above lyophilized Sanguisorbae radix powder into 10mL of distilled water, and used for in vitro (in-vitro) and cell test. For animal experiments using hamsters, a solution containing a sanguisorba officinalis extract was prepared by mixing 2g of a freeze-dried sanguisorba officinalis powder with 0.1g of an excipient and putting into 10mL of distilled water.
EXAMPLE 2 inhibition of SARS-CoV-2 specific 3CL protease Activity based on Sanguisorba extract
2-1 analysis of 3CL protease Activity Using in vitro experiments
The 3CL protease in vitro (in-vitro) activity assay was performed at the test tube level as described below. This experiment utilized the SARS-CoV-2 3CL protease (3C-like protease) inhibitor detection kit (inhibitor assay kit) (BPS bioscience, USA). After a substrate (substrate) that exhibits fluorescence when bound to the active site of 3CL protease is reacted with a test sample, fluorescence is measured by a known method, and the 3CL protease inhibitory effect of the sample is confirmed. As a negative control group, H was used in the same volume as the sample 2 O, as a positive control group for 3CL protease activity inhibition, GC376 of 0.1, 1 and 10. Mu.M was used, respectively. The reaction volume was 50. Mu.l, and the treated Sanguisorbae radix extract was 2, 20, 200. Mu.g/ml, respectively.
As a result, as shown in FIG. 1, the IC50 was 26.8. Mu.g/mL, and the sanguisorba officinalis extract was equal to or more than that of the positive control group, inhibiting the activity of SARS-CoV-2 3CL protease.
2-2 analysis of 3CL protease Activity Using cell-based analysis
Cell level detection (Cell-based assay) was performed on the 3CL protease activity inhibitory efficacy of sanguisorba officinalis extract using 293 cells. For this experiment, the 3CLglow assay kit (Montana Molecular, usa) was used.
Inoculation (seed) in 293 cells (1X 10 3 And) 24-well plate (24-well plate). The next day (after 18-24 hours), a baculovirus (1 x 10) containing 3CL protease and its matrix, i.e., GFP fusion protein (fusion protein) was used 3 Personal) (Montana Molecular, U.S) Infection in 293 cells. Then, the cells were treated with the Sanguisorbae radix extract at concentrations of 1, 10, 100. Mu.g/ml, respectively. As a negative control group, treatment H 2 O, as positive control group, GC376 of 0.1, 1, 10. Mu.M was used, respectively. After 24 hours, the inhibitory effect of the sanguisorba officinalis extract was observed by fluorescence microscopy (magnification 100X, scale bar=100 μm).
As a result, as shown in fig. 2, the number of fluorescence-expressing cells was significantly reduced in the sanguisorba extract-treated group similarly to GC376 as a positive control group. At the cellular level of the sanguisorba officinalis extract, the SARS-CoV-2 3CL protease activity inhibition IC50 was 25.98. Mu.g/ml, very similar to the results of the in-vitro assay described previously. Thus, it was confirmed that the sanguisorba officinalis extract has a remarkable effect of inhibiting the activity of SARS-CoV-2 3CL protease at a cellular level.
EXAMPLE 3 inhibition of SARS-CoV-2 specific RdRp Activity based on Sanguisorba extract
RdRp (RNA-dependent RNA polymerase) in vitro (in-vitro) activity assays were performed at the test tube level as described below. The product used in the experiment was purchased from RdRp inhibitor assay kit (Profoldin, USA). Efficacy of the test sample is determined if it is based on a matrix that exhibits fluorescence when bound to RNA amplified according to RdRp. As a negative control group concerning the inhibition of RdRp activity, H was used 2 O, as a positive control group, aurintricarboxylic acid (Aurintricarboxylic acid) (ATA) (Hung HC et al, inhibition of enterovirus 71replication and the viral 3Dpolymerase by aurintricarboxylic acid.J Antimicrob Chemother.2010 65 (4): 676-83) was used. The reaction volume was 50. Mu.l, and the treated Sanguisorbae radix extract was 2, 20, 200. Mu.g/ml, respectively.
As a result, as shown in FIG. 3, the IC50 was 66.7. Mu.g/ml, and the sanguisorba officinalis extract was equal to or more than that of the positive control group, inhibiting the activity of RdRp.
EXAMPLE 4 inhibition of proliferation of SARS-CoV-2 in VERO-E6 cells based on Sanguisorba extract
In a 96-well plate, 8×10 per well (well) is dispensed 3 cells/100. Mu.L of VERO-E6 cells at 37℃with 5% CO 2 Overnight (overright) culture in an incubator,the day of the test reached a state of about 80% full (confusing). VERO-E6 cells are cells derived from cardiac epithelial cells of African green monkeys and are used in virus infection experiments (Govorkova EA, et al African green monkey kidney (Vero) cells provide an alternative host cell system for influenza Aand B viruses J virol.1996;70 (8): 5519-5524). As positive control groups for this experiment, 3CL protease inhibitor (inhibitor) GC376 and ryanodevir (Remdesivir) as RdRp inhibitor (inhibitor) (Gilead Sciences, usa) were used. For the sample concentration, the concentration of 8.14. Mu.g/ml non-toxic to VERO-E6 cells in the sanguisorba officinalis extract was taken as the highest concentration, diluted 2-fold, and the lowest concentration was reached to 0.51. Mu.g/ml. The highest concentration of GC376 and Rede-Sivir was 12.5. Mu.g/ml, diluted 2-fold and the lowest concentration was 1.56. Mu.g/ml. The diluted sample was mixed with SARS-CoV-2-containing medium (200 TCID 50 ) After adjusting to 200. Mu.l volume, CO was used at 37 ℃ 2 The mixture was allowed to stand in the incubator for 1 hour. Then, after the medium is removed from the cells, it is replaced with a medium containing the sample and virus. After 3 days of culture, cytopathic effect (cytopathic effect) (CPE) of apoptosis induced by virus proliferation was observed with a microscope, and the degree of CPE inhibition by the sample inhibiting virus proliferation was observed. This experiment was repeated 3 times and the sample concentration for CPE inhibition was calculated as an average.
As a result, as shown in FIG. 4, it was confirmed that the average CPE inhibition by GC376 was 3.125. Mu.g/ml, the average CPE inhibition by Rede-Sivir was 8.333. Mu.g/ml, and the average CPE inhibition by Sanguisorba officinalis extract was 4.069. Mu.g/ml. Thus, it was confirmed that the sanguisorba officinalis extract of the present invention has a SARS-CoV-2 proliferation inhibitory effect at a cellular level, which is Wei Jiang times as high as that of the drug actually prescribed for patients suffering from SARS-CoV-2 infection.
EXAMPLE 5 efficacy of Sanguisorba officinalis extract in inhibiting SARS-CoV-2 proliferation in hamster animal test
5-1 construction of animal model of SARS-CoV-2 infection Using hamsters and oral administration of test substances
Subculturing the known virus SARS-CoV-2 (NCCP 43326) to obtain a strain of 10 4 TCID50/mL virus preparationFor phosphate buffered saline (PBS, phosphate buffered saline) solutions. Then, 100. Mu.L of SARS-CoV-2 virus solution was administered into the left nasal cavity of hamster. Immediately after virus infection, as a negative control group, an excipient Methyl Cellulose (MC) solvent (vehicle) was orally administered, and as a positive control group, MC vehicle containing RdRp inhibitor Mo Nuola (molnupirvir) (merck, usa) at a high concentration (250 mg/kg) was orally administered. For the Sanguisorbae radix extract, MC vehicle was used for oral administration at 100 mg/kg. Each test substance was administered 2 times per day. The significance between the test substance dosing groups was determined using student t-test, statistical analysis using Prism 7.04 (GraphPad Software inc., san Diego, CA, USA) and judged statistically significant when the p-value was less than 0.05.
Inhibiting weight loss in hamsters infected with 5-2SARS-CoV-2 by means of Sanguisorba officinalis extract
The body weight of each subject was measured by electronic weighing scale daily for 7 days from the current day by inoculating SARS-CoV-2 infected hamster with virus and administering test substance as described in example 5-1.
As a result, as shown in FIG. 5, when SARS-CoV-2 infected hamsters were administered only the negative control solvent (vehicle), the body weight was reduced between 2 and 4 days after infection. In contrast, the sanguisorba extract-administered group had an inhibitory effect on body weight loss similar to that of Mo Nuola-vir (molupiravir) as a positive control group. Therefore, the sanguisorba officinalis extract was found to strongly inhibit the division and proliferation of SARS-CoV-2 in hamster animal test and to restore biostable properties, thereby inducing the normalization of body weight.
5-3 confirmation of inhibition of SARS-CoV-2 proliferation in hamster lung by Sanguisorba officinalis extract Using Real time reverse transcription-polymerase chain reaction (Real time RT-PCR) technique
On day 3 of infection (3Day post infection,3DPI), a part of the lesion was excised from the hamster lung tissue, and a predetermined amount of Wizol was placed therein TM (Wizbiosolution, seongnam, korea) samples, after being crushed with beads for tissue crushing, were extracted from the supernatant as total RNA. Total (Total) RNA extracted was extracted using Nanodrop (Na)noDrop2000, sameiser technology (Thermo scientific)). Using WizScript TM cDNA synthesis kit (synthesis kit) (Wizbiosolution) cDNA was synthesized from 1. Mu.g total RNA. Then, the synthesized cDNA was used with a probeWizPure TM qPCR Master-UDG (WizbioSolution), and uses the E gene specific primer (primer) or RdRp gene specific primer of SARS-CoV2, is suitable for real-time reverse transcription polymerase chain reaction (real-time RT-PCR). The base sequences of the primers used in real-time RT-PCT are set forth in Table 1.
TABLE 1
The lung tissue-derived virus residual amount was quantitatively analyzed by using the number of copies (copy) of the virus gene corresponding to total RNA per ng or. Mu.g based on a standard curve (standard curve) prepared by using SARS-CoV-2RNA derived from a known copy number.
As a result, as shown in fig. 6, it was confirmed that the sanguisorba officinalis extract had an effect of significantly inhibiting viral proliferation in lung tissue of infected animals, as compared with Mo Nuola viral inhibiting proliferation of SARS-CoV-2 by inhibiting activity of RdRp.
5-4 confirmation of inhibition of SARS-CoV-2 proliferation in hamster lung by Sanguisorba officinalis extract using Plaque assay (Plaque assay)
On day 3 of infection (3 DPI), the lung tissue excised from hamster lung tissue was homogenized by adding 10 μl of PBS per 1mg (homogenization). After centrifugation at 5000rpm for 3 minutes, the supernatant was recovered, and after volume adjustment to 200. Mu.l using PBS serial dilution, african green monkey kidney cells (Vero E6) grown in 12-well plates were treated. After shaking the virus and allowing it to contact Vero E6 cells uniformly at 15 minute intervals for 1 hour, the supernatant was removed, and 1. Mu.ml of agarose overlapping (agarose overlay) medium (medium) containing 1% agarose (agaros) was added to each well (well). After confirming that agarose overlay (agaros overlay) medium had hardened, the 12-well plate was inverted and incubated for 96 hours to see if plaques (plaque) formed. Agarose overlay (agaros overlay) medium was removed and stained with crystal violet (crystal violet). The number of plaques (plaques) per g of tissue was calculated by visually comparing the dilution of the sample with the number of plaques (plaques) derived from the virus of known copy number.
As shown in fig. 7, it was found that the sanguisorba officinalis extract has an effect of significantly inhibiting the proliferation of SARS-CoV-2 in the lung tissue of hamsters compared to Mo Nuola as an RdRp inhibitor.
5-5 inhibition of SARS-CoV-2 proliferation in hamster lung by sanguisorba officinalis extract was confirmed by immunohistochemical staining (immunohistochemistry)
Day 3 (3 DPI) infection, hamster lung tissue was excised, paraffin blocks were prepared, and tissue sections were sectioned at 5 μm thickness. Thereafter, the antibody against SARS-CoV-2 specific nucleocapsid (nucleoapsid) (Cat# 40143-MM05; sino Biological Inc.)) was used and the antibody was subjected to a known tissue staining method.
As a result, as shown in fig. 8, a strong browning reaction (nucleocapsid reaction) was observed in the epithelial cells in the hamster lung tissue, in which infection with SARS-CoV-2 was observed in the solvent (vehicle) treatment group. In contrast, for the sanguisorba extract treated group, the nucleocapsid response was similar to Mo Nuola of the positive control group. Thus, it was confirmed by immunohistochemical staining that sanguisorba officinalis extract inhibited the division and proliferation of SARS-CoV-2 in hamster lung tissue.
5-6 radix Sanguisorbae extract for inhibiting hamster pneumonia caused by SARS-CoV-2 virus
On day 3 of infection (3 DPI), 3 hamster sections were removed from each group, and after right lung tissue was fixed in 10% neutral formalin fixed (NBF, neutralized buffered formalin) solution, histopathological examination was performed. After paraffin blocks were prepared using lung tissue fixed at 10% NBF, the tissue was sectioned at a thickness of 5 μm. Afterwards, inflammation scores were assessed by Hematoxylin-Eosin (H & E, hematoxylin & Eosin) staining and light microscopy. The score (scoring) is determined by scoring with 0-3 units according to the degree of inflammation based on the literature (see literature 9). The value 0 is a value without lesions, and the score is 1 for 10% or less, 2 for 10% -50% or more, and 3 for 50% or more, and when bleeding or edema is observed, 0.5 score is additionally given.
As shown in fig. 9, it was confirmed that the sanguisorba officinalis extract had the same or more effects as those of the positive control Mo Nuola strain in relieving inflammation in hamster lung tissue caused by infection with SARS-CoV-2.
Industrial applicability
The use of the sanguisorba officinalis extract of the present invention to inhibit SARS-CoV-2 virus 3CL protease and RdRp activity can be used for developing pharmaceuticals.
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Claims (20)
1. A pharmaceutical composition for preventing or treating infection caused by SARS-CoV-2, comprising a sanguisorba officinalis extract as an active ingredient.
2. The pharmaceutical composition for preventing or treating infection by SARS-CoV-2 according to claim 1, wherein said SARS-CoV-2 comprises a variant thereof.
3. The pharmaceutical composition for preventing or treating infection by SARS-CoV-2 according to claim 2, wherein the variant is formed by a mutation in the protuberant protein of SARS-CoV-2.
4. The pharmaceutical composition for preventing or treating infection by SARS-CoV-2 according to claim 1, wherein said pharmaceutical composition blocks 3C-like protease activity.
5. The pharmaceutical composition for preventing or treating infection by SARS-CoV-2 according to claim 1, wherein said pharmaceutical composition blocks RNA-dependent RNA polymerase activity.
6. The pharmaceutical composition for preventing or treating infection caused by SARS-CoV-2 according to claim 1, wherein said sanguisorba extract is extracted with water, ethanol or a solvent as a mixture thereof.
7. The pharmaceutical composition for preventing or treating infection by SARS-CoV-2 according to claim 1, wherein said pharmaceutical composition comprises a pharmaceutically acceptable carrier, excipient or diluent.
8. The pharmaceutical composition for preventing or treating infection caused by SARS-CoV-2 according to claim 1, wherein said pharmaceutical composition has any one dosage form selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, oral liquid preparations, emulsions, syrups, sterilized aqueous solutions, nonaqueous solvents, emulsions, freeze-dried preparations and suppositories.
9. The pharmaceutical composition for preventing or treating infection caused by SARS-CoV-2 according to claim 1, wherein said pharmaceutical composition has any one dosage form selected from the group consisting of powder, granule, tablet, capsule and liquid form.
10. The pharmaceutical composition for preventing or treating infection caused by SARS-CoV-2 according to claim 1, wherein said sanguisorba extract is extracted from roots of cucumber, roots of sanguisorba longifolia or a mixture thereof.
11. A health functional food composition for preventing or improving infection caused by severe acute respiratory syndrome coronavirus 2, characterized by comprising sanguisorba officinalis extract as an active ingredient.
12. The health-functional food composition for preventing or ameliorating infection caused by SARS-CoV-2 according to claim 11, wherein said SARS-CoV-2 virus comprises a variant thereof.
13. The health functional food composition for preventing or ameliorating infection caused by SARS-CoV-2 as claimed in claim 11, wherein said variant is formed by mutation of a raised protein of SARS-CoV-2 virus.
14. The health-functional food composition for preventing or ameliorating infection caused by SARS-CoV-2 according to claim 11, wherein said health-functional food composition blocks 3C-like protease activity.
15. The health-functional food composition for preventing or ameliorating infection caused by SARS-CoV-2 according to claim 11, wherein said health-functional food composition inhibits RNA-dependent RNA polymerase activity.
16. A method of preparing a pharmaceutical composition for preventing or treating infection caused by SARS-CoV-2, comprising:
drying sanguisorba officinalis;
a step of solvent-extracting the dried sanguisorba officinalis at 30 to 120 ℃ to form a solution; and
and a step of freeze-drying the extracted solution to prepare a powder.
17. The method for preparing a pharmaceutical composition for preventing or treating infection caused by SARS-CoV-2 according to claim 16, wherein the solvent is any one or more selected from the group consisting of water, ethanol, or a mixture thereof.
18. The method of preparing a pharmaceutical composition for preventing or treating infection by SARS-CoV-2 according to claim 16, wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier, excipient or diluent.
19. The method for preparing a pharmaceutical composition for preventing or treating infection caused by SARS-CoV-2 according to claim 16, wherein said pharmaceutical composition has any one dosage form selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, oral liquid preparations, emulsions, syrups, sterilized aqueous solutions, nonaqueous solvents, emulsions, freeze-dried preparations and suppositories.
20. The method for producing a pharmaceutical composition for preventing or treating infection caused by SARS-CoV-2 according to claim 16, wherein said pharmaceutical composition has any one of dosage forms selected from the group consisting of powder, granule, tablet, capsule and liquid form.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR10-2021-0045995 | 2021-04-08 | ||
| KR10-2021-0170899 | 2021-12-02 | ||
| KR1020210170899A KR102479180B1 (en) | 2021-04-08 | 2021-12-02 | Sanguisorba officinalis Linne extract having a suppressive effect against enzymatic activity of the SARS-CoV-2 3C-like protease and RNA-dependent RNA Polymerase |
| PCT/KR2021/018706 WO2022215827A1 (en) | 2021-04-08 | 2021-12-10 | Sanguisorba officinalis linne extract composition inhibiting 3cl protease and rdrp activity of sars-cov-2 |
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| CN117120068A true CN117120068A (en) | 2023-11-24 |
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| CN202180096830.7A Pending CN117120068A (en) | 2021-04-08 | 2021-12-10 | Radix Sanguisorbae extract composition for inhibiting SARS-CoV-2 3CL protease and RdRp activity |
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