KR102491460B1 - Method for increasing bioconversion of glycoside to aglycone using Scutellaria baicalensis extract and composition obtained thereby - Google Patents

Abstract

The present invention relates to a method for increasing aglycone by bioconversion of glycosides of gold extract and a composition containing the gold extract by the method as an active ingredient, and gold ( Scutellaria baicalensis ) in 70% ethanol It is characterized in that it comprises the step of treating a hydrolase to the golden extract extracted in.
The method of bioconverting the glycosides of gold extract to increase the non-glycosides is simple, low-cost and quick time compared to the conventional manufacturing method, and the glycosides Baicalin or Wogonoside are converted to the non-glycosides baicalin ( baicalein) or Wogonin has an effect that can be increased. In addition, the golden extract with increased non-glycoside has excellent anti-viral efficacy against porcine epidemic diarrhea (PED) virus, and is effective in preventing and treating porcine epidemic diarrhea caused by the virus, so related pharmaceuticals It can be used in industry as well as feed and feed additive.

Description

A method for increasing non-glycosides by bioconversion of glycosides of gold extract and a composition comprising the gold extract by the method as an active ingredient {Method for increasing bioconversion of glycoside to aglycone using Scutellaria baicalensis extract and composition obtained thereby}

The present invention relates to a method for increasing glycosides of gold extract to non-glycosides and a composition containing the gold extract by the method as an active ingredient.

Secondary metabolites such as alkaloids, flavonoids, phenolic compounds, and quinones present in plants have antibiotic efficacy [Choi et al., J Kor Plant Res 19(4): 491-496, 2006.], and has fewer side effects compared to artificially synthesized compounds.

Most of the various physiologically active substances derived from natural products exist in the form of glycosides, which are sugar-attached forms, and thus have a higher molecular weight than non-glycoside forms and have relatively low bioavailability due to low absorption into cells. On the other hand, the non-glycoside structure has high solubility in the lipids of the intestinal cell membrane, and thus it is easy to penetrate the intestinal cell membrane [Kim et al., 2010, Korean J Food Cookery Sci 17:214-219]. Examples include genistein and ginsenosides. Although these substances are effective physiological substances, they take a long time to move to the large intestine and convert to non-glycosides by intestinal microorganisms, so the absorption rate in the body is low, so they are practically in the body. Studies on non-glycosides, which are metabolite forms that exhibit effective physiological activity through absorption, are in progress [(Kim et al., 2006, J Life Sci 16:136-140), (Quan et al., 2008, J Ginseng Res 32 :226-231), (Lim et al., 2009, J Korean Soc Food Sci Nutr 38:1167-1173.)].

Golden ( Scutellaria baicalensis ) is a dicotyledonous perennial plant of the currency plant order Lamiaceae and is used as a herbal medicine. The active medicinal ingredients known so far include Baicalein, Baicalin, Wogonin, Wogonoside, and the like. In China, gold was used for high fever, dry cough, hematemesis, constipation, etc. [Kim et al., 2006, J Korean Soc Food Sci Nutr 35(5): 543-548)], and as a pharmacological action, it is antibacterial [(Bae et al., Bae et al. al., 2005, J Kor Micribiol Biotechnol 33(1): 35-40: 2005), (Leach, 2011, Bioscience Horizons, 4: 119-127.)], antifungal [(Wang et al., 2015, Microb Pathog, 87:21-9)], antiviral (Nayak et al., 2014, J Antimicrob Chemother, 69:1298-1310.)] and hepatoprotective [(Pen et al., 2012, J Ethnopharmacol, 139:829 -837.)]. In particular, gold is known to have various antibacterial effects against pork Salmonella [(Kim et al., 2008, Korean J. Oriental Physiology & Pathology 22:619-623.), (Kim et al., 2015, J. Int. Korean Med. 36:165-179.)].

In recent years, the use of antibiotics in the livestock industry has been restricted worldwide, but when a disease occurs, rapid transmission occurs due to dense breeding, etc., resulting in a rapid increase in mortality, resulting in economic loss to farms. In particular, salmonellosis is a general term for diseases caused by bacteria of the genus Salmonella, and causes great damage to the livestock industry. In poultry, fowl typhoid is caused by Salmonella gallinarum , and in swine, salmonellosis is caused by Salmonella choleraesius and Salmonella typhimurium infection. In addition, in pigs, not only salmonellosis, but also a high frequency of infection with porcine epidemic diarrhea (PED) virus, which causes vomiting and watery diarrhea, causes serious economic losses. In particular, when Salmonella bacteria and viruses are mixed infection, negative effects such as weight loss and gain occur than each infection alone, and higher infections can occur with synergy [(Yang et al., 2017, Korean J Vet Res, 57 , 235-243.), (Wills et al., 2000, Vet Microbiol. 71(3-4):177-192.)].

Therefore, in the present invention, the non-glycoside content of the active substances was increased through bioconversion of the golden extract, and the antiviral activity of the PED virus of the bioconverted golden extract was revealed, thereby completing the present invention.

An object of the present invention is to provide a method for increasing non-glycone by bioconversion of glycosides of golden extract and a composition containing the golden extract by the method as an active ingredient.

In addition, an object of the present invention is a pharmaceutical composition for the prevention or treatment of PEDv infection comprising a gold extract as an active ingredient by a method of increasing non-glycone by bioconverting glycoside of gold extract, prevention of PEDv infection Or to provide a feed composition for improvement, and a feed additive composition for preventing or improving PEDv infection.

In order to achieve the above object, a method of increasing aglycone by bioconverting the glycoside of the gold extract according to a preferred embodiment of the present invention is a golden extract obtained by extracting gold ( Scutellaria baicalensis ) in ethanol. It may include the step of treating a hydrolase to.

In one embodiment, the hydrolase is pectinase, cellulase, glucan 1,4-alpha-glucosidase, beta-glucosidase ( β-glucosidase), glucanase, and arabinanase, characterized in that at least one selected from the group consisting of.

In one embodiment, the glycoside is baicalin, wogonoside, daidzin, genistin, glycitin, saponin, procyanidin , naringenin, rutinose, hesperidin, Mogroside V, amygdalin and 3-phenol coumarin selected from the group consisting of It is characterized by one or more types.

In addition, the aglycosome is baicalein, wogonin, genistein, daidzein, glycitein, sapogenin, 3,4-hydroxyphenyl Acetic acid (3,4-Hydroxyphenylaetic acid), 4-HPA, m-coumaric acid, p-coumaric acid, O-beta-D-glucuronides, stilbenoids , rutin, hesparetin, mogroside IIIE, mendelonitrile, and benzaldehyde.

In one embodiment, it is characterized in that the hydrolase is treated with 5-20% (w / w) of the total weight of the golden extract.

In another embodiment, it is characterized in that the treatment is performed under a temperature condition of 30 to 60 degrees at pH 4 to 9.

In one embodiment, gold ( Scutellaria baicalensis ) Provides a method for producing a pharmaceutical composition for preventing or treating PEDv infection, comprising the step of treating a golden extract obtained by extracting it in ethanol with a hydrolase.

In one embodiment, gold ( Scutellaria baicalensis ) Provides a method for producing a feed composition for preventing or improving PEDv infection, comprising the step of treating a golden extract extracted with 70% ethanol with a hydrolase.

In one embodiment, gold ( Scutellaria baicalensis ) Provides a method for producing a feed additive composition for preventing or improving PEDv infection, comprising the step of treating a hydrolase in a golden extract extracted in ethanol.

According to one embodiment of the present invention, a composition comprising a golden extract treated with a hydrolase by a bioconversion method as an active ingredient is provided.

According to one embodiment of the present invention, there is provided a pharmaceutical composition for preventing or treating PEDv infection comprising, as an active ingredient, a golden extract treated with a hydrolase by a biotransformation method.

According to one embodiment of the present invention, a feed composition for preventing or improving PEDv infection comprising a golden extract treated with a hydrolase by a bioconversion method as an active ingredient is provided.

According to one embodiment of the present invention, a feed additive composition for preventing or improving PEDv infection comprising a golden extract treated with a hydrolase by a bioconversion method as an active ingredient is provided.

The method of increasing the non-glycosides by bioconversion of the glycosides of the gold extract of the present invention is simpler, cheaper, and faster than conventional manufacturing methods, and converts glycosides such as Baicalin or Wogonoside into non-glycosides. There is an effect that can be increased with baicalein or Wogonin. In addition, the golden extract with increased non-glycoside has excellent anti-viral efficacy against porcine epidemic diarrhea (PED) virus, and is effective in preventing and treating porcine epidemic diarrhea caused by the virus, so related pharmaceuticals It can be usefully used in industry as well as feed and feed additives.

1 is a diagram showing the results of analyzing the content of active substances (baicalin, baicalein, wogonoside, wogonin) for three kinds of golden raw material and one kind of golden extract (Gianon).
Figure 2 is a diagram showing the results of analyzing the content of baicalein and wogonin according to each pH condition and temperature condition in order to confirm the optimization conditions of the bioconversion golden extract.
Figure 3a is a result of confirming the cell viability (%) by time (24 hours, 48 hours) after treating VERO-76 cells with dilutions of golden extract (1000-fold, 500-fold, 250-fold, 100-fold, 10-fold) for each concentration is a diagram showing
Figure 3b shows VERO-76 cells treated with dilutions of golden extract (1000-fold, 500-fold, 250-fold, 100-fold, 10-fold) for each concentration and inoculated with PEDv by date (0, 1, 2, 3, 4 days) ) It is a diagram showing the results of confirming antiviral efficacy.
Figure 4a shows the cell viability (%) by time (24 hours, 48 hours) after treating VERO-76 cells with dilutions of bioconverted golden extract (1000-fold, 500-fold, 250-fold, 100-fold, 10-fold) at each concentration. It is a diagram showing the confirmed result.
Figure 4b shows VERO-76 cells treated with dilutions of bioconverted golden extract (1000-fold, 500-fold, 250-fold, 100-fold, 10-fold) at each concentration and inoculated with PEDv by date (0, 1, 2, 3, 4 days) is a diagram showing the results of confirming antiviral efficacy.

The present invention provides a bioconversion method for increasing glycosides of golden extracts to non-glycones, including the step of treating golden extracts with hydrolase ( Scutellaria baicalensis extract ). .

In the present invention, the term "bioconversion" refers to a process of converting an added substance into a chemically modified form using the physiological function of an organism. means to increase

The hydrolase is pectinase, cellulase, Viscozyme® L (β-glucanase), AMG (glucan 1,4-α-glucosidase), Plantase UF (pectinase), Pluszyme (β-glucosidase) , At least one selected from the group consisting of Peclyve UF cleaner (glucanase, arabinanase) and Peclyve PR (pectinase), preferably pectinase (Pectinase) or in order to increase the glycoside of the gold extract of the present invention to a non-glycoside , but not limited thereto.

The glycosides are baicalin, wogonoside, daidzin, genistin, glycitin, saponin, procyanidin, naringenin At least one selected from the group consisting of rutinose, hesperidin, Mogroside V, amygdalin, and 3-Phenyl coumarin, preferably Preferably Baicalin or Wogonoside, but is not limited thereto.

The non-glycosides are baicalein, wogonin, genistein, daidzein, glycitein, sapogenin, 3,4-hydroxyphenylacetic acid ( 3,4-Hydroxyphenylaetic acid), 4-HPA, m-coumaric acid, p-coumaric acid, O-beta-D-glucuronides, stilbenoids, rutin At least one selected from the group consisting of (rutin), hesparetin, Mogroside IIIE, mendelonitrile and benzaldehyde, preferably baicalein or Wogonin, but is not limited thereto.

The treatment may be performed with 5-20% (w/w) of hydrolase based on the total weight of the golden extract, preferably 7-15% (w/w), more preferably 10 It can be processed as % (w/w). In addition, the treatment may be a temperature condition of 30 to 60 degrees at pH 4 to 9, preferably 30 to 50 degrees at pH 4 to 7, but to achieve the purpose of bioconverting the glycoside of the gold extract of the present invention to a non-glycoside. If it is for, it is not limited to this.

In one embodiment of the present invention, as a result of comparing the active substance content of the conventional golden raw material or golden extract, the bioconversion including Baicalin and Wogonoside having a high content of glycosides Samples were selected for Thereafter, as a result of treating the selected samples with 10 types of hydrolases, it was confirmed that pectinase is the most excellent as a bioconversion enzyme. In order to optimize bioconversion, as a result of enzymatic treatment at different temperatures and pHs, it was confirmed that optimal bioconversion conditions appeared at 50 ° C. and pH 5 conditions.

In the present invention, for biotransformation, enzymes can be treated with gold extract (substrate) to perform an enzymatic reaction under optimal temperature and pH conditions, and the "enzymatic reaction" is a substrate and an enzyme under appropriately artificially controlled environmental conditions. means to react.

In addition, the present invention provides a pharmaceutical composition for preventing or treating porcine epidemic diarrhea (PED) virus infection comprising the golden extract prepared by the bioconversion method as an active ingredient.

The pharmaceutical composition of the present invention may further include an adjuvant in addition to the gold extract prepared by the bioconversion method. Any adjuvant known in the art may be used without limitation, but, for example, Freund's complete adjuvant or incomplete adjuvant may be further included to increase the effect.

The pharmaceutical composition according to the present invention may be prepared in the form of incorporating the active ingredient into a pharmaceutically acceptable carrier. Here, the pharmaceutically acceptable carrier includes carriers, excipients and diluents commonly used in the pharmaceutical field. Pharmaceutically acceptable carriers usable in the pharmaceutical composition of the present invention include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The pharmaceutical composition of the present invention may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories or sterile injection solutions according to conventional methods, respectively. .

When formulated, it may be 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 contain at least one or more excipients such as starch, calcium carbonate, sucrose, lactose, and gelatin in addition to active ingredients. It can be prepared by mixing etc. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, solutions for oral administration, emulsions, syrups, etc. In addition to commonly used diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. can Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base for suppositories, witepsol, tween 61, cacao paper, laurin paper, glycerogelatin, and the like may be used.

The pharmaceutical composition according to the present invention can be administered to a subject by various routes. All modes of administration are contemplated, eg oral, intravenous, intramuscular, subcutaneous, intraperitoneal injection.

The dosage of the pharmaceutical composition according to the present invention is selected in consideration of the age, weight, sex, and physical condition of the subject. It is obvious that the concentration of the single domain antibody included in the pharmaceutical composition can be variously selected depending on the subject, and is preferably included in the pharmaceutical composition at a concentration of 0.01 to 5,000 ug/ml. If the concentration is less than 0.01 ug / ml, pharmaceutical activity may not appear, and if it exceeds 5,000 ug / ml, it may be toxic to the human body.

In one embodiment of the present invention, in order to confirm the PED antiviral efficacy of the bioconversion golden extract of the present invention, as a result of performing the virus content and cytotoxicity test, the bioconversion of the present invention compared to the conventional golden extract It was confirmed that the gold extract had low viral content and low cytotoxicity, and it was confirmed that the antiviral effect of the PED of the biotransformed golden extract of the present invention was excellent.

In addition, the present invention provides a feed composition for preventing or improving PEDv infection comprising a golden extract prepared by a bioconversion method as an active ingredient.

In addition, the present invention provides a feed additive composition for preventing or improving PEDv infection comprising a golden extract prepared by a bioconversion method as an active ingredient.

The feed composition of the present invention replaces conventional antibiotics and inhibits the growth of harmful PEDv, improving the health of animals, improving the weight gain and quality of livestock, and increasing milk production and immunity. . The feed composition of the present invention may be prepared in the form of fermented feed, formulated feed, pellet form and silage. The fermented feed can be prepared by fermenting organic matter by adding various microorganisms or enzymes other than the active ingredient of the present invention, and the formulated feed can be prepared by mixing several types of general feed with the active ingredient of the present invention. . Feed in the form of pellets can be prepared by applying heat and pressure to the compound feed, etc. in a pellet machine, and silage can be prepared by fermenting green feed. Wet fermented feed collects and transports organic matter such as food waste, mixes excipients for sterilization and moisture control in a certain ratio, and ferments for more than 24 hours at a temperature suitable for fermentation, so that the moisture content is about 70%. It can be made by adjusting. The fermented dry feed can be prepared by further subjecting the wet fermented feed to a drying process to adjust the moisture content to about 30% to 40%.

The feed composition of the present invention may further include components added to conventional feed. Examples of ingredients added to such feeds may include grain powder, meat powder, and pulses. In the above, the grain powder may use at least one selected from rice flour, wheat flour, barley flour, and corn flour. In the above meat powder, any one or more powdered meat powder selected from among chicken, beef, pork, and ostrich meat may be used. In the above, at least one selected from soybeans, kidney beans, peas, and black beans may be used as the pulse.

In the feed composition of the present invention, in addition to grain powder, meat powder, and pulses, which are ingredients added to conventional feeds mentioned above, at least one selected from nutrients and minerals may be added to increase the nutritional properties of the feed, and the feed quality In order to prevent the degradation of antifungal agents, antioxidants, anticoagulants, emulsifiers, and at least one selected from a binder may be included.

In addition, the present invention provides a method for preparing a pharmaceutical composition for preventing or treating PEDv infection, comprising treating the golden extract with a hydrolase.

In addition, the present invention provides a method for producing a feed composition for preventing or improving PEDv infection.

In addition, the present invention provides a method for preparing a feed additive composition for preventing or improving PEDv infection.

In addition, the present invention provides a composition for bioconverting glycosides of golden extracts to non-glycosides, including golden extract treated with hydrolase as an active ingredient.

The present invention will be described in more detail through the following examples. However, the following examples are only for specifying the content of the present invention, and the present invention is not limited thereto.

<Example 1> Selection of golden raw material

In order to analyze the contents of baicalin, baicalein, wogonoside, and wogonin, the active ingredients of 3 types of gold source and 1 type of gold extract (Gianon), 1 g of gold was 70% After mixing with ethanol at a volume ratio of 1/200, extraction was performed at 85° C. for 90 minutes in a heating mantle. The extracted sample was filtered through a 0.2 um filter, and the four active ingredients were analyzed using high-performance liquid chromatography (HPLC) [Yu et al., 2013, Oncol Rep. 2013 Nov;30(5):2411-2418]. Standards include 95% Baicalin (572667, sigma), 98% Baicalein (465119, sigma), 95% Wogonoside (SMB00081, sigma), 98% Wogonin (W0769, sigma) was used, and as an analytical column, an RStech column (C18-T51002546) with a particle size of about 5 μm, a column length of about 25 cm, and a column diameter of about 4.6 mm was used. The column temperature was 30 ° C. and the detector wavelength was 275 nm, injecting 10 ul of each of the above samples, and a mobile phase solvent (18% methanol, 25% acetonitrile, 57% water, 0.2% acetic acid) at a rate of 0.8 ml / min for 80 minutes. The sample was developed using

As shown in Figure 1, as a result of comparing the content of the active substance of the golden extract and the golden extract, it was confirmed that the golden extract had high baicalin and ugonoside content, and the golden extract (Gianon) was a sample for future bioconversion. was used as

<Example 2> Selection of bioconversion enzyme

Bioconversion was confirmed by treating the selected gold extract with 10 hydrolases, respectively. Enzymes used include β-glucanase (Yinong, China), Pectinase (Yinong, China), Cellulase (Yinong, China), Viscozyme L (Novozymes, Denmark), AMG (Novozymes, Denmark), Plantase UF (Vision, Korea) , Pluszyme (Vision, Korea), β-glucuronidase (Sigma, USA), Peclyve UF cleaner (Lyven, France), and Peclyve PR (Lyven, France). After simply mixing the gold extract in 0.1 M acetate buffer at pH 4.5 at a volume ratio of 1/10, the gold extract was treated with 10% of a hydrolase by weight and reacted for 8 hours at an appropriate activity temperature for each enzyme.

As shown in Table 1, it was confirmed that urgonine among active substances increased about 1.7 times during bioconversion with Yinong's pectinase (EC number: 3.2.1.15, 30,000 U/g).

<Example 3> Optimization of bioconversion of gold extract

In order to optimize the bioconversion process of the golden extract using the enzyme pectinase selected according to Example 2, enzyme treatment was performed under different temperature and pH conditions. Specifically, the enzyme was treated under the conditions of 37 degrees at pH5, 60 degrees at pH5, 50 degrees at pH5, 50 degrees at pH6, 50 degrees at pH7, and 50 degrees at pH8 to confirm bioconversion optimization conditions.

As shown in Figure 2, it was confirmed that the content of baicalein and urgonin, which are substances after bioconversion at 50 ° C. and pH5 conditions, increased by 2.88 times and 2.06 times, respectively, compared to the gold extract.

<Example 4> Confirmation of antiviral efficacy

4-1. Preparation of bioconverted golden extract of the present invention for antiviral efficacy

In order to confirm the antiviral efficacy of the golden extract and the bioconverted golden extract of the present invention against PEDv, each was mixed with DMSO (Dimethyl sulfoxide, Bio basic) at a volume ratio of 1/20 and diluted 20 times. The content of the indicator substance of the extracted sample is shown in Table 2 below. The 20-fold diluted sample was further diluted 10-fold, 100-fold, 250-fold, 500-fold, and 1000-fold, respectively, and used for cytotoxicity and antiviral experiments. The virus used was PEDv (Porcine Epidemic Diarrhea Virus, ISU46065), and in order to evaluate the antiviral effect of the evaluation substance against the virus, the Agriculture, Forestry and Livestock Quarantine Headquarters Notification No. 2016-91, Regulations on Safety and Effectiveness Review of Animal Drugs, etc. The test was conducted according to.

4-2. Antiviral efficacy test of the bioconverted golden extract of the present invention against PED virus

In order to test the antiviral efficacy of the bioconverted golden extract of the present invention against PED virus, the antiviral effect against the virus was determined to be effective when the killing of 10 ⁴ TCID ₅₀ /mL or more was confirmed compared to the pathogen control group. The PED virus culture was subcultured by inoculating the blank virus strain into the VERO-76 cell line, using DMEM medium supplemented with 5% fetal bovine serum, and testing the content of the blank virus in advance to test that it is 10 ⁶ TCID ₅₀ / mL or more used in The gold extract, or the treatment of the biotransformed golden extract of the present invention, is inoculated into a 12-well tissue culture plate in which VERO-76 cells form a monolayer, 1 ml of each reaction solution for each dilution of the material, and incubated for 2 hours in a 5% CO ₂ , 37 ℃ incubator treated during. Thereafter, all of the reaction solution was removed, and 300 ul of the virus was dispensed and adsorbed for 1 hour in a 5% CO ₂ , 37°C incubator. After 1 hour, the virus was removed, replaced with 2 ml of the reaction solution for each dilution of the material, and then cultured for 5 days in a 5%, CO _{2 ,} 37°C incubator. 330 ul of each culture medium was collected every 24 hours from the moment of treatment to test the antiviral activity of the substance, and VERO-76 cells formed a monolayer on a 96-well tissue culture plate with more than 2 repetitions per dilution, 100 reaction solution per dilution Inoculated by ul, and then adsorbed for 1 hour in a 5% CO ₂ 37°C incubator. After that, all the diluted solution was removed, replaced with cell culture medium, and cultured for 5 days in an incubator at 5% CO _{2 and} 37°C. The presence or absence of the virus was finally determined by confirming the cytopathic effect (CPE). Virus content was calculated using the Kaerber method (FIGS. 3b and 4b).

In addition, in the cytotoxicity test, VERO-76 cells were used in DMEM medium supplemented with 5% fetal serum. In a 96-well tissue culture plate in which VERO-76 cells formed a monolayer, 100 ul of the reaction solution for each dilution of the materials was dispensed three times or more, and treated in a 5% CO ₂ , 37 ° C incubator for 24 hours and 48 hours, respectively. After 24 hours and 48 hours, the reaction solution was discarded, and 100 ul of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) (2.5 mg/ml) was dispensed for 140 minutes. adsorbed. After 140 minutes, it was dried for more than 16 hours in a 65° C. dryer. To determine cytotoxicity, 100 ul of DMSO was dispensed and absorbance was measured at 540 nm. If cell viability of 80% or less was observed within 48 hours of substance treatment, significant cytotoxicity was determined (FIGS. 3a and 4a).

As shown in Figures 3a and 3b, virus growth was not observed when Vero-76 cells were treated with 10-fold and 100-fold diluted golden extract, but cytotoxicity of 20% or more when treated with 10-fold and 100-fold diluted golden extract , it was confirmed that the antiviral effect on PED was not effective. On the other hand, as shown in Figures 4a and 4b, when the golden extract diluted 100 times and 250 times compared to the pathogen control group, the cytotoxicity was not significant compared to the control group. % viral growth inhibition was observed, confirming that the antiviral effect against PEDv was effective.

Claims (13)

Gold ( Scutellaria baicalensis ) Including the step of treating a hydrolytic enzyme to a gold extract extracted in ethanol,
The hydrolase is pectinase, cellulase, glucan 1,4-alpha-glucosidase, beta-glucosidase, glucan A method for increasing non-glycosides by bioconversion of glycosides of gold extract, characterized in that at least one selected from the group consisting of glucanase and arabinanase. delete The method of claim 1, wherein the glycoside is baicalin, wogonoside, daidzin, genistin, glycitin, saponin, procyanidin , naringenin, rutinose, hesperidin, Mogroside V, amygdalin and 3-phenol coumarin selected from the group consisting of A method for increasing non-glycosides by bioconversion of glycosides of gold extract, characterized in that at least one kind. According to claim 1,
The non-glycosides are baicalein, Wogonin, genistein, daidzein, glycitein, sapogenin, 3,4-hydroxyphenylacetic acid ( 3,4-Hydroxyphenylaetic acid), 4-HPA, m-coumaric acid, p-coumaric acid, O-beta-D-glucuronides, stilbenoids, rutin (rutin), hesparetin, mogroside IIIE, mendelonitrile and benzaldehyde, characterized in that at least one selected from the group consisting of A method of converting and increasing non-glycosides. According to claim 1,
The treatment is a method of increasing non-glycosides by bioconverting the glycosides of the golden extract, characterized in that the hydrolase is treated with 5-20% (w / w) of the total weight of the golden extract. According to claim 1,
The treatment is a method of increasing non-glycosides by bioconverting the glycosides of the gold extract, characterized in that the treatment at pH 4 to 9 at a temperature condition of 30 to 60 degrees. Gold ( Scutellaria baicalensis ) Including the step of treating the golden extract extracted with 70% ethanol with a hydrolase,
The hydrolase is pectinase, cellulase, glucan 1,4-alpha-glucosidase, beta-glucosidase, glucan Method for producing a pharmaceutical composition for preventing or treating PEDv infection, characterized in that at least one selected from the group consisting of glucanase and arabinanase. Gold ( Scutellaria baicalensis ) Including the step of treating the golden extract extracted with 70% ethanol with a hydrolase,
The hydrolase is pectinase, cellulase, glucan 1,4-alpha-glucosidase, beta-glucosidase, glucan Method for producing a feed composition for preventing or improving PEDv infection, characterized in that at least one selected from the group consisting of glucanase and arabinanase. Gold ( Scutellaria baicalensis ) Including the step of treating the golden extract extracted with 70% ethanol with a hydrolase,
The hydrolase is pectinase, cellulase, glucan 1,4-alpha-glucosidase, beta-glucosidase, glucan Method for producing a feed additive composition for preventing or improving PEDv infection, characterized in that at least one selected from the group consisting of glucanase and arabinanase. A composition comprising, as an active ingredient, a golden extract treated with a hydrolase by the bioconversion method of claim 1. A pharmaceutical composition for the prevention or treatment of PEDv infections comprising, as an active ingredient, a golden extract treated with a hydrolase by the bioconversion method of claim 1. A feed composition for preventing or improving PEDv infection comprising, as an active ingredient, a golden extract treated with a hydrolase by the bioconversion method of claim 1. A feed additive composition for preventing or improving PEDv infection comprising, as an active ingredient, a golden extract treated with a hydrolase by the bioconversion method of claim 1.

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