KR102531825B1 - Method Preparing Ginsenosides F2 or Rg3 Using Fruits of Panax ginseng - Google Patents

Abstract

In the present invention, ginseng fruit extract is fermented with a Bacillus sp microorganism, and the fermented product is treated with Pectinex or Viscozyme enzyme, or the ginseng fruit extract is fermented with a Lactobacillus sp microorganism Disclosed is a method for producing ginsenoside F2 or Rg3 using ginseng fruit, characterized in that by treating with citric acid therein.

Description

Method for preparing ginsenosides F2 or Rg3 using ginseng fruit {Method Preparing Ginsenosides F2 or Rg3 Using Fruits of Panax ginseng}

The present invention relates to a method for preparing ginsenoside F2 or Rg3 using ginseng fruits.

Ginseng is a perennial herb, and 6-7 species are known worldwide. Currently, Korean ginseng ( Panax ginseng CA MEYER), American Guangdong ginseng ( Panax quinquefolium L.), Chinese ginseng ( Panax notoginseng ), and Japanese bamboo ginseng ( Panax japonicus) CA MEYER), etc. are used as medicinal materials. The root (Ginseng radix) is divided into white ginseng (Ginseng Radix alba) and red ginseng (Ginseng Radix Rubra) according to the processing method. It is made by steaming and drying the roots with steam, etc., and has a light yellowish brown or reddish brown color. The unique color of red ginseng, which is different from white ginseng, is due to the gelatinization of ginseng starch by heat treatment when red ginseng is steamed, and the change in tissue color by amino-carbonyl reaction.

Ginseng has been widely used in East Asian countries such as China, Korea, and Japan for about 2,000 years as a medicine or as a restorative agent for health promotion. Saponin (ginsenoside), the main active ingredient of ginseng, has immunity enhancement, anti-inflammatory, anti-allergic, anti-cancer, erectile dysfunction, blood pressure lowering, anti-cholesterol, anti-thrombotic, anti-diabetic, adult disease and It has been reported that it has preventive and therapeutic effects on aging (J. Immunol., 148:1519-25, 1992; Lee FC., Facts about ginseng, the elixir of life. Hollyn International. New Jersey, 1992; Huang KC ., The pharmacology of Chinese herbs.CRC Press.Florida, 1999;Chang HM., Pharmacology and application of Chinese material medica.Vol 1.World Scientific.Singapore, 1986;Biol.Parm.Bull.17:635-9, 1994; Biol. Pharm. Bull. 18:1197-1202, 1995).

As such, the main component showing physiological activity in ginseng is saponin. Ginseng saponin is a neutral substance of bisdesmoside, which is a combination of glucose, arabinose, xylose, and rhamnose, etc. It is a glycoside. These are named Ginsenoside Rx according to the order of polarity by column chromatography. More than 40 species of ginsenosides have been isolated and identified to date, and these ginsenosides are divided into PPD (Protopanaxadiol) series and PPT (Protopanaxatriol) series, and their basic chemical structure is shown in [Formula 1] below. As can be seen in [Table 1] below, PPD-type saponins have a basic structure in which R ₂ is hydrogen (H) and PPD, and ginsenosides Rb1, Rb2, Rg3, Rc, Rd, F2, compound Y, compound K, etc. are present here. belongs to, and PPT-type saponins have R ₁ as hydrogen (H) and PPT is the basic structure, and ginsenosides Re, Rf, Rg1, and Rh1 belong to this group.

[Formula 1]

In general, many glycoside compounds that exist in nature tend to increase their physiological activity when sugar is decomposed into non-saccharides rather than themselves (Med. Pharm. Soc. 1992, 9:1-13). In the case of ginseng saponin, ginsenosides Rg3, Rh1, Rh2, F2, compound Y, compound K, etc. produced by hydrolysis of some of the sugars rather than ginsenosides Rb1, Rb2, Rd, and Re in which three or more sugars are bonded. It is known to exhibit much superior effects in terms of absorption into the living body or physiological activity (Ginseng Res. 2003, 27:129-134). For example, general ginsenosides such as Rb1, Rb2, and Rc, which are PPD saponins, are not immediately absorbed into the human body, but are degraded by human intestinal bacteria or enzymes to form ginsenosides F1, F2, Rg3, and compound-K. It is absorbed only after the component is converted and its efficacy is expressed (Planta Medica 63:463-40, 1997; Drug Metab. Dispos. 31:1065-71, 2003). Ginsenoside F2, a precursor of compound-K, has been reported to have an activity to improve degenerative brain diseases or improve memory by inhibiting nerve cell damage (Korean Patent Registration No. 10-1509056), and ginsenoside Rg3 Immunity enhancement, blood flow improvement, fatigue recovery, anticancer effect, and liver function improvement effect have been reported (Korean J. Medic. Crop Sci., 21, 401-404, 2013).

The present invention discloses a method for preparing ginsenoside F2 or Rg3 using ginseng fruits.

An object of the present invention is to provide a method for preparing ginsenoside F2 or Rg3 using ginseng fruit.

Other objects or specific objects of the present invention will be presented below.

As confirmed in the Examples below, the present inventors fermented ginseng fruit extract with a Bacillus sp. microorganism and treated the fermented product with Pectinex or Viscozyme enzyme to obtain ginnoside F2 In addition, it was confirmed that the ginsenoside Rg3 content increased when the ginseng fruit extract was fermented with Lactobacillus sp. microorganisms and treated with citric acid.

The present invention is provided based on these experimental results, and in one aspect, the present invention relates to a method for preparing ginsenoside F2. The method for preparing ginsenoside F2 of the present invention includes (a) preparing a ginseng fruit extract. (b) inoculating and culturing a Bacillus sp microorganism in a ginseng fruit extract, (c) hydrolyzing the culture with any one enzyme of Pectinex and Viscozyme It consists of including the step of doing.

In the present invention, the ginseng fruit extract refers to a product obtained by leaching the flesh and skin of ginseng fruit, preferably ginseng fruit, with water, an organic solvent, or a mixture thereof. The organic solvent is a lower alcohol having 1 to 4 carbon atoms (methanol, ethanol, butanol, etc.), methylene chloride, ethylene, acetone, hexane, ether, chloroform, ethyl acetate, butyl acetate, N,N-dimethylformamide (DMF), dimethyl It refers to sulfoxide (DMSO), 1,3-butylene glycol, propylene glycol, etc., preferably ethanol. As the extraction method, any method such as cooling, reflux, warming, ultrasonic radiation, and supercritical extraction may be applied. After extraction, it may be desirable to remove extraction residues (solids thereafter) through filtration, centrifugation, or standing for a certain period of time. In particular, in the present invention, the ginseng fruit extract was extracted with hot water (60 to 90 ° C), extracted with hot water (60 to 90 ° C), removed from the extraction residue, added with ethanol to the supernatant from which the extraction residue was removed, and reacted for a certain period of time (20 to 240 minutes). It is preferably a supernatant obtained by centrifugation. When ethanol is added to react and centrifuged, sugar components other than ginsenoside present in the supernatant are removed, thereby increasing the concentration of ginsenoside in the supernatant after centrifugation.

In the present invention, the ginseng fruit extract may be adjusted to an appropriate pH for the smooth growth and proliferation of microorganisms of the genus Bacillus to be inoculated, for example, pH 6.0 to pH 7.5.

In addition, carbon sources, nitrogen sources, and/or trace elements known in the art may be added to the ginseng fruit extract to promote or assist the growth and proliferation of microorganisms of the genus Bacillus to be inoculated.

The carbon source is preferably a sugar such as a monosaccharide, disaccharide or polysaccharide. For example, glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch, starch hydrolyzate and the like can be used. Complex compounds, such as molasses or by-products of sugar refining processes, may also be used. In some cases, oil such as soybean oil or sunflower oil, organic acid such as acetic acid, or glycerol may be advantageous as a carbon source. These carbon sources may be included in the medium alone or in a mixture of two or more, and whether included in the medium alone or in a mixture of two or more, 0.1% (w / w) to 20% (w / w) It can be included in the medium as a range.

As the nitrogen source, an inorganic nitrogen compound, an organic nitrogen compound, or a complex compound containing these compounds may be used. Examples of inorganic nitrogen compounds include ammonia, ammonium salts (eg, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc.), nitrate, and the like, and examples of organic nitrogen compounds include urea, amino acids, and the like. Complex compounds include yeast extract, soytone, peptone, tryptone, malt extract, broth, soybean powder, soybean okara powder, cheonggukjang powder, soybean paste Natural nitrogen sources, such as powder, etc. are mentioned. One or more of these nitrogen sources may also be used in combination, and may be included in the medium in the range of 0.1% (w/w) to 8.0% (w/w). When using a natural nitrogen source, it is preferable to sterilize and use for the same reasons as described in relation to the case of using a natural carbon source.

Trace elements include calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper, iron, phosphorus, sulfur, etc. These trace elements may be added to the medium in the form of salt compounds, and the medium of these trace elements In order to increase solubility and maintain a solution state, a chelating agent such as catechol or citric acid may be added together. Examples of the salt compound form include sodium hydrogen phosphate, magnesium sulfate, iron chloride, calcium salt, manganese salt, cobalt salt, molybthenate salt, chelate metal salt and the like. These trace elements may be used in combination of one or more, and may be included in the medium in the range of 0.001% (w/w) to 3.0% (w/w).

In addition, the medium may optionally be prepared by including a growth promoter such as biotin, riboflavin, thiamine, folic acid, nicotinic acid, pantothenate, or pyridoxine.

Regarding the optimization of other medium formulations, there are many references in the art including Applied Microbiol. Physiology, A Practical Approach (Editors P.M. Rhodes, P.F. Stanbury, IRL Press (1997) pp. 53-73, ISBN 0 19 963577 3). Literature is known and reference may be made to these known documents.

Also, in the present invention, any Bacillus sp. microorganism may be used as the microorganism of the genus Bacillus. Specifically, as such microorganisms, Bacillus subtilis microorganisms that can be used in the method of the present invention include Bacillus subtilis ( B. subtilis ), Bacillus lichneformis ( B. lichneformis ), Bacillus megaterium ( B. megaterium ), Bacillus Amyloliquefaciens ( B. amyloliquefaciens ), Bacillus natto ( B. natto ) and the like are representative, in addition to Bacillus anthracis ( B.antharcis ), Bacillus lentus ( B.lentus ), Bacillus fermirus ( B. pumilus ), Bacillus thuringiensis ( B.thuringiensis ), Bacillus albei ( B.alvei ), Bacillus azotopixanseu ( B.azotofixans ), Bacillus macerans ( B.macerans ), Bacillus polymyxa ( B.polymyxa ), Bacillus papilliae ( B.popilliae ), Bacillus coagulans ( B.coagulans ), Bacillus stearothermophilus ( B.stearothermophilus ), Bacillus pasteurii ( B.pasteurii ), Bacillus paricus ( B.sphaericus ), Bacillus Fastidiosus ( B. fastidiosus ) and the like. Preferably it is the Bacillus sp. S9 strain (accession number KCTC 13945BP) used in the examples below. This strain is Stenotrophomonas sp. It is deposited with accession number KCTC 13945BP as a mixture with S7 strain, and when this mixed strain is cultured in R2A agar at a temperature of 35 ° C., only Bacillus sp. S9 strain can be isolated, cultured and used.

After the Bacillus genus microorganism is inoculated, it may be cultured with shaking at a temperature of 25 to 45 ° C. for one or more days, preferably for 1 to 10 days.

The Pectinex Ultra Passover enzyme used in the present invention is an enzyme having Polygalacturonase activity, and the Viscozyme enzyme has a complex activity of beta-glucanase, cellulase and xylanase. eggplant is an enzyme This enzymatic treatment is preferably performed at a pH of 4 to 6 and a temperature of 25 to 50 ° C., as confirmed in the following examples.

Such enzymatic treatment may be preferably performed for a supernatant having a high ginsenoside content by centrifuging the culture of the microorganisms of the genus Bacillus and recovering the supernatant, as in the following examples. Here, the enzymatic treatment may be performed on the concentrate obtained by concentration of the supernatant under reduced pressure, freeze-drying, or the like, or may be performed on the suspension after suspending the concentrate in distilled water.

In the present invention, the ginseng extract or the culture is preferably used after being sterilized to prevent unintended fermentation by other microorganisms. Sterilization may be performed using methods known in the art, such as high-temperature and high-pressure sterilization and ultraviolet irradiation.

In another aspect, the present invention relates to a method for preparing ginsenoside Rg3, which includes (a) preparing a ginseng fruit extract, (b) adding ginseng fruit extract to Lactobacillus Inoculating and culturing microorganisms of the genus Lactobacillus sp., and (c) treating the culture with citric acid.

In the method of the present invention, the step of preparing the ginseng fruit extract may refer to the above.

Also, in the present invention, any Lactobacillus microorganism may be used as the microorganism of the genus Lactobacillus, for example, Lactobacillus plantarum , Lactobacillus delbrueckii , Lactobacillus bulgaricus , Lactobacillus casei , Lactobacillus brevis , Lactobacillus acidophilus , etc. may be used, preferably Lactobacillus sp S11 strain (Accession No. KCTC13946BP) can be used.

In addition, in the present invention, citric acid is used for the purpose of increasing the Rg3 recovery rate, and is in the range of 0.5 to 10% (v / v), preferably in the range of 0.5 to 7% (v / v) in the culture by microorganisms of the genus Lactobacillus. It may be added to 80 ~ 140 ℃ range, preferably in the range of 100 ~ 130 ℃ 5 minutes to 2 hours, preferably 5 minutes to 1 hour. This citric acid treatment may be performed on the supernatant after centrifugation of the culture, and may be performed on the concentrate obtained by methods such as concentration by darkening or freeze-drying, or by suspending the concentrate in distilled water and performing the suspension. there is.

Meanwhile, since ginsenosides F2 and Rg3 obtained by the production method of the present invention have various physiological activities, they can be commercialized as pharmaceutical compositions, health functional foods, and functional cosmetics. Specifically, the pharmaceutical composition, health functional food, and functional cosmetic are antidiabetic, antihypertensive, antihyperlipidemia, cardiovascular disease treatment or prevention, atopic dermatitis improvement, anti-obesity, rejuvenation, and skin wrinkle improvement with these ginsenoside components. , skin whitening, etc. may have uses or functions known in the art.

When the ginsenosides F2 and Rg3 obtained by the production method of the present invention are commercialized and used as a pharmaceutical composition, the pharmaceutical composition includes a pharmaceutically acceptable carrier in addition to the ginsenoside, and is known in the art. Depending on the route of administration, it may be prepared as an oral dosage form or a parenteral dosage form. The route of administration herein may be any appropriate route including topical route, oral route, intravenous route, intramuscular route, and direct absorption through mucosal tissue, and two or more routes may be used in combination. An example of a combination of two or more routes is a case in which two or more formulations of drugs are combined according to the route of administration, for example, one drug is firstly administered intravenously and the other drug is secondly administered through a topical route.

Pharmaceutically acceptable carriers are well known in the art depending on the route of administration or dosage form, and specifically, reference may be made to the pharmacopoeia of each country including the "Korean Pharmacopoeia".

When the pharmaceutical composition of the present invention is prepared as an oral dosage form, powder, granule, tablet, pill, dragee, capsule, liquid, gel, syrup, suspension, wafer according to a method known in the art together with a suitable carrier. It can be prepared in formulations such as Examples of suitable carriers include sugars such as lactose, glucose, sucrose, dextrose, sorbitol, mannitol, and xylitol, starches such as corn starch, potato starch, and wheat starch, cellulose, methylcellulose, ethylcellulose, sodium carboxymethylcellulose, Celluloses such as hydroxypropylmethylcellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, mineral oil, malt, gelatin, talc, polyol, vegetable oil, ethanol, Serol etc. are mentioned. In the case of formulation, appropriate binders, lubricants, disintegrants, coloring agents, diluents, etc. may be included as needed. Suitable binders include starch, magnesium aluminum silicate, starch ferrite, gelatin, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone, glucose, corn sweetener, sodium alginate, polyethylene glycol, wax, and the like, and oleic acid as a lubricant Sodium, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, silica, talcum, stearic acid, magnesium salts and calcium salts thereof, polyethylene glycol, etc. may be mentioned. As disintegrants, starch, methyl cellulose , agar, bentonite, xanthan gum, starch, alginic acid or its sodium salt, and the like. Examples of the diluent include lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, and glycine.

When the pharmaceutical composition of the present invention is prepared as a parenteral formulation, it may be formulated in the form of an injection, transdermal administration, nasal inhalation, and suppository along with a suitable carrier according to a method known in the art. When formulated as an injection, an aqueous isotonic solution or suspension may be used as a suitable carrier, and specifically, an isotonic solution such as phosphate buffered saline (PBS) containing triethanolamine, sterile water for injection, or 5% dextrose may be used. . When formulated as a transdermal formulation, it may be formulated in the form of ointments, creams, lotions, gels, external solutions, pastas, liniments, air rolls, and the like. In the case of nasal inhalation, it can be formulated in the form of an aerosol spray using a suitable propellant such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, etc., and when formulated as a suppository, the carrier is Witepsol ( witepsol), tween 61, polyethylene glycols, cacao fat, laurin fat, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, sorbitan fatty acid esters, and the like can be used.

Regarding the specific formulation of the pharmaceutical composition, it is known in the art, and for example, reference may be made to Remington's Pharmaceutical Sciences (19th ed., 1995) and the like. These documents are considered as part of this specification.

The preferred dosage of the pharmaceutical composition of the present invention is in the range of 0.001 mg/kg to 10 g/kg per day, preferably 0.001 mg/kg to 1 g, depending on the patient's condition, weight, sex, age, severity of the patient, and route of administration. /kg can be in the range. Administration can be done once a day or divided into several times. These dosages should not be construed as limiting the scope of the present invention in any respect.

In another aspect, when the ginsenosides F2 and Rg3 obtained by the production method of the present invention are commercialized and used as a health functional food composition, the food composition can be manufactured in any form, such as tea, juice, carbonated beverages, ions Beverages such as beverages, processed oils such as milk and yogurt, foods such as chewing gum, rice cakes, Korean snacks, bread, snacks, and noodles, tablets, capsules, pills, granules, liquids, powders, flakes, pastes, syrups, gels, and jellies It can be manufactured into health functional food formulations such as , bars, etc.

In addition, the food composition of the present invention may take any product classification as long as it conforms to the enforcement regulations at the time of manufacture and distribution in legal and functional classification. For example, health functional food according to the Korean 「Health Functional Food Act」, or snacks, legumes, teas, beverages according to each food type according to the Food Code (MFDS notification 「Food Standards and Specifications」) of the Korea 「Food Sanitation Act」 , special purpose foods, etc.

The food composition of the present invention may include food additives in addition to the fermentation broth. Food additives may generally be understood as substances that are added to, mixed with, or infiltrated into food in manufacturing, processing, or preserving food. Since food is consumed daily and for a long period of time, its safety must be guaranteed. According to the Food Additives Code under the laws of each country governing the manufacture and distribution of food (in Korea, it is the 「Food Sanitation Act」), safety-guaranteed food additives are limitedly regulated in terms of ingredients or functions. In the Korean Food Additives Codex (MFDS notification 「Standards and Specifications for Food Additives」), food additives are classified into chemical synthetic products, natural additives and mixed preparations in terms of ingredients and are regulated. It is divided into additives, preservatives, emulsifiers, acidulants, and thickeners.

Sweeteners are used to impart appropriate sweetness to foods, and both natural and synthetic sweeteners can be used in the composition of the present invention. Preferably, a natural sweetener is used, and examples of the natural sweetener include sugar sweeteners such as corn syrup solids, honey, sucrose, fructose, lactose, and maltose.

Flavors may be used to improve taste or aroma, and both natural and synthetic flavors may be used. Preferably, it is the case of using a natural one. In case of using natural ones, in addition to flavor, the purpose of enhancing nutrition can also be combined. As a natural flavoring agent, it may be obtained from apples, lemons, tangerines, grapes, strawberries, peaches, etc., or obtained from green tea leaves, roundworms, bamboo leaves, cinnamon, chrysanthemum leaves, jasmine, and the like. In addition, those obtained from ginseng (red ginseng), bamboo shoots, aloe vera, ginkgo, etc. can be used. Natural flavors can be liquid concentrates or solid extracts. In some cases, synthetic flavors may be used, and synthetic flavors may include esters, alcohols, aldehydes, terpenes, and the like.

As preservatives, sodium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate, EDTA (ethylenediaminetetraacetic acid), etc. may be used, and as emulsifiers, acacia gum, carboxymethylcellulose, xanthan gum, pectin and the like, and examples of the acidulant include acid salt, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, phosphoric acid, and the like. Acidulants may be added to the food composition to have an appropriate acidity for the purpose of inhibiting the growth of microorganisms in addition to improving taste.

As the thickening agent, a suspending agent, a sedimentation agent, a gel forming agent, a swelling agent, and the like may be used.

In addition to the food additives described above, the food composition of the present invention may include physiologically active substances or minerals known in the art and whose stability is guaranteed as food additives for the purpose of supplementing or reinforcing functionality and nutrition.

Examples of such physiologically active substances include catechins contained in green tea, vitamins such as vitamin B1, vitamin C, vitamin E, and vitamin B12, tocopherol, dibenzoylthiamine, and the like. Examples of minerals include calcium preparations such as calcium citrate and magnesium stearate. magnesium preparations such as the like, iron preparations such as iron citrate, chromium chloride, potassium iodine, selenium, germanium, vanadium, zinc, and the like.

The food composition of the present invention may include the above-mentioned food additives in an appropriate amount to achieve the purpose of addition according to the type of product.

Regarding other food additives that may be included in the food composition of the present invention, reference may be made to national food codes or food additive codes.

In another aspect, when the ginsenosides F2 and Rg3 obtained by the manufacturing method of the present invention are commercialized and used as a functional cosmetic composition, the cosmetic composition includes ingredients commonly used in cosmetic compositions, such as stabilizers, in addition to the fermentation broth, conventional adjuvants such as solubilizers, surfactants, vitamins, colors and flavors, and carriers.

When the formulation of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as a carrier component. can

When the formulation of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and in particular, in the case of a spray, additional chlorofluorohydrocarbon, propane / May contain a propellant such as butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, solubilizing agent or emulsifying agent is used as a carrier component, specifically water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol aliphatic esters, polyethylene glycol, fatty acid esters of sorbitan, and the like may be used.

When the formulation of the present invention is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, or polyoxyethylene sorbitan ester, microcrystals Star cellulose, aluminum metahydroxide, bentonite, agar, and the like can be used.

When the formulation of the present invention is surfactant-containing cleansing, as carrier components, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid amide Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters and the like can be used.

The cosmetic composition of the present invention may be prepared according to a method for preparing a cosmetic composition conventionally performed in the art, except for including ginsenosides F2 and Rg3 obtained by the preparation method of the present invention.

As described above, according to the present invention, it is possible to provide a method capable of preparing ginsenosides F2 and Rg3 with high physiological activity from ginseng fruit in high yield. These ginsenosides F2 and Rg3 can be usefully used in health functional foods and pharmaceuticals.

1 is a chromatogram of ginsenoside F2.
2 is a chromatogram of ginsenoside Rg3.
Figure 3 is a schematic diagram of the manufacturing process of ginsenosides F2 and Rg3.

Hereinafter, the present invention will be described with reference to examples. However, the scope of the present invention is not limited to these examples.

<Example> Manufacturing method of ginsenoside F2 and ginsenoside Rg3

<Example 1> Manufacturing method of ginsenoside F2

After washing and drying the ginseng fruit (1kg), 1-fold distilled water was added, and then the ginseng seed was removed using a ginseng seed remover (model name: HP718, Jinsung Industry), and the flesh and skin of the ginseng fruit were recovered. The recovered pulp and skin suspension was heated at 80 ° C. for 6 hours for hot water extraction, and then left at 4 ° C. for 12 hours to precipitate solids. After taking the supernatant, an equal volume of ethanol (99.9%) was added thereto, heated at 60 ° C. for 30 minutes, centrifuged (8000 rpm, 30 minutes) to take the supernatant, concentrated under reduced pressure, and suspended the concentrate in distilled water. It was made to be 300 ml. Here, the acidity was adjusted to pH 6.5 with 1M NaOH solution, sterilized (15 minutes at 121 ° C), and inoculated with Bacillus sp S9 strain (Accession No. KCTC 13945BP) cultured on an R2A agar plate in the form of a colony. and cultured with shaking for 2 days at 30 ° C. and 180 rpm. After incubation, the supernatant was collected by centrifugation (8000 rpm, 30 minutes), adjusted to pH 5 with citric acid, sterilized by heating at 80 ° C, cooled to room temperature, and 10% (v / v) each enzyme under the conditions of Table 2 below. After addition, it was reacted at 80 rpm at 40 °C. The enzyme activity was stopped by treatment with high temperature (100° C.), and the ginsenoside content was measured using HPLC. Ginsenoside content measurement using HPLC was analyzed according to the method of Kim Keum-suk et al. (Korean J. Medicinal Crop Sci. 24(1):47-54, 2016). For HPLC analysis, 300 ul of the sample was mixed with 1.2 ml of methanol, ultrasonically extracted, centrifuged, and 1 ml of the supernatant was taken. The ginsenoside was partially purified using a solid phase extractor (SPE), and an Agilent 1100 series HPLC system (Agilent Technologies, USA) was used as the instrument, and ginsenoside standard was purchased from Embo Laboratories.

processing enzyme enzyme activity manufacturing company Treatment temperature and time Pectinex
(Ultra Passover) Polygalacturonase Novozyme 40℃, 24 hours, pH 5 Viscozymes β-glucanase, Cellulase, Xylanse Novozyme 40℃, 24 hours, pH 5

The HPLC analysis results are shown in Figure 1, where A is an HPLC chromatogram before enzyme treatment, B is a chromatogram of Pectinex enzyme-treated material, and C is a chromatogram of Viscozyme enzyme-treated material. Referring to chromatogram B, ginsenoside Rd has the highest content, and it is determined that this ginsenoside Rd has been converted to ginsenoside F2 by Pectinex and Viscozyme enzyme treatment. Ginsenoside F2 obtained by Pectinex and Viscozyme enzyme treatment was 68 ng/ul and 88 ng/ul, respectively.

<Example 2> Manufacturing method of ginsenoside Rg3

After washing and drying the ginseng fruit (1kg), 1-fold distilled water was added, and then the ginseng seed was removed using a ginseng seed remover (model name: HP718, Jinseong Industry), and the flesh and skin of the ginseng fruit were recovered. The recovered pulp and skin suspension was heated at 80 ° C. for 6 hours for hot water extraction, and then left at 4 ° C. for 12 hours to precipitate solids. After taking the supernatant, an equal volume of ethanol (99.9%) was added thereto, heated at 60 ° C. for 30 minutes, centrifuged (8000 rpm, 30 minutes) to take the supernatant, concentrated under reduced pressure, and suspended the concentrate in distilled water. It was made to 300 ml. cultured on R2A agar plates. Bacillus genus ( Bacillus sp ) S11 strain (Accession No. KCTC 13946BP) was inoculated in the form of a colony and cultured for 2 days at 30 ° C. and 180 rpm with shaking. After culturing, the supernatant was recovered by centrifugation (8000 rpm, 30 minutes).

After adding 5% (v/v) citric acid to the recovered supernatant and reacting by heating at 121° C. for 10 minutes, the ginsenoside Rg3 content was measured using HPLC in the same manner as in Example 1. The HPLC chromatogram is shown in FIG. 2 together with the standard chromatogram. When treated with citric acid, the ginsenoside Rg3 content was 54.5 ng/ul.

Figure 3 schematically shows the manufacturing process of ginsenosides F2 and Rg3 according to Examples 1 and 2.

Claims (9)

(a) preparing a ginseng fruit extract, (b) inoculating and culturing a Bacillus sp microorganism in the ginseng fruit extract, and (c) hydrolyzing the culture with Pectinex Including,
The ginseng fruit is a ginseng fruit from which seeds have been removed,
The ginseng fruit extract is obtained by adding ethanol to the supernatant of the hot water extract of ginseng fruit, centrifuging the supernatant, concentrating the supernatant under reduced pressure, and suspending the concentrate in distilled water,
The microorganism of the genus Bacillus is S9 strain of the genus Bacillus (accession number KCTC 13945BP),
The culture is a supernatant or a concentrate obtained by centrifugation after culture after step (b) and before step (c),
The method for producing ginsenoside F2, characterized in that the Pectinex enzyme is an enzyme having polygalacturonase activity.
According to claim 1,
Before the step (b), a step of adjusting the pH of the ginseng fruit extract to a range of 6.0 to 7.5 is added.
According to claim 1,
The method characterized in that it further comprises the step of adjusting the pH to 5 by adding citric acid to the culture after step (b) and before step (c).




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