KR20100120614A - Composition comprising the platycoside saponins isolated from the root of platycodon grandiflorum and its platycodin d enriched-saponin fraction obtained by enzymatic conversion, intervening the lipid metabolic disorders - Google Patents

Abstract

PURPOSE: A composition containing Platycodon grandiflorum A. DC. saponin fraction and platycodin of high content is provided to suppress adipocyte differentiation and to prevent and treat adipose metabolism. CONSTITUTION: A novel Platycodon grandiflorum A. DC. saponin fraction with enhanced drug efficiency contains 50.0-98.0(w/w)% of platycodon D, 0-35.0(w/w)% of platycodon E and 3.0-20.0(w/w)% of platycodon D3. A method for preparing Platycodon grandiflorum A. DC. saponin fraction comprises: a step of isolating Platycodon grandiflorum A. DC. extract by reverse phase resin, polystyrenes or ion exchange resin column to obtain Platycodon grandiflorum A. DC. saponin fraction; and a step of bio-conversing the fraction by enzyme. A pharmaceutical composition for preventing and treating lipid metabolic disease contains Platycodon grandiflorum A. DC. saponin fraction or saponin compound as an active ingredient.

Description

Composition containing the platycoside saponins isolated from the root of the composition of the gilyeong saponin isolated from the gilgi extract and a lipid metabolic disease containing the high content of the platycodine D bioconverted from the fraction as an active ingredient. of Platycodon grandiflorum and its platycodin D enriched-saponin fraction obtained by enzymatic conversion, intervening the lipid metabolic disorders}

The present invention relates to a composition for the prophylaxis and treatment of lipid metabolic diseases, which contains a gilyeong saponin fraction isolated from the gilyeong extract and a high content of Platicodin D, which is bioconverted from the fraction as an active ingredient.

Document 1 arey VJ. et al., Body fat distribution and risk of non-insulin-dependent diabetes mellitus in women, The nurses' health study, Am J Epidemiol, 145 , pp. 614-619, 1997

Golay A. et al., Obesity and NIDDMP, the retrograde regulation concept, Diabetes Rev , 5 , pp.69-82, 1997

Document 3 Hotamisligil GS. et al., Tumor necrosis factor a, a key component of the obesity-diabetes link, Diabetes , 43 , pp.1271-1278, 1994

4 Biol. Pharm. Bull., 11 , p. 2114, 2008

Document 5 Eur. J. Pharmacol., 537 , p. 1, 2006

Document 6 Life Sci., 76 , p.2315, 2005

Document 7 Int. J. Cancer, 261 , p. 2674, 2008

8, Cancer Lett., 261 , p. 98, 2008

Document 9 Am. J. Chin. Med., 32 , p. 257, 2004

Reference Document 10 Planta Med., 68 , p. 794, 2002

Document 11 Arch. Pharm. Res., 27 , p. 1048, 2004

12. Eur. J. Pharmacol., 537 , p. 1, 2006

13 J. Food Sci., 73 , p. 195, 2008

[14] Chem Pharm Bull, (Tokyo) 54 , p. 557, 2006

Document 15 Chem. Pharm. Bull, (Tokyo) 54 , p. 1285, 2006

16. J. Chem. Soc. Perkin Trans., I , p.661, 1984

Reference 17 Planta Med., 71 , p.566, 2005

18 Akiyama T., Chem. Pharm. Bull., 20 , p. 1957, 1972

19 Akiyama T., Chem. Pharm. Bull., 24 , p. 1965, 1972

20. Kubota T., Chem. Commun ., P. 1313, 1969

[Document 21] Republic of Korea Registration No. 10-0564927

[Document 22] Korean Registration No. 10-0750333

[Reference 23] Republic of Korea Registration No. 10-0696647

24 Hahn J., Nutr., 130 , p. 2760, 2000

[Document 25] Korean Patent Application No. 10-2008-0081605

26 J. Choromatgr. 27 , p. A 1135, 2006

[27] Bradford MM., Et al., Ann Biochem, 72 , pp. 248-254, 1976

In general, obesity is a phenomenon in which excess energy in the body accumulates into fat, causing abnormal metabolism due to abnormal body fat, and obesity is the most common nutritional problem in the industrial society. 3, 20% of children are reported as obese patients, more than 70% of type 2 diabetic patients, obesity, and in recent years, the simple diet of obesity has increased significantly as the eating habits are westernized and the culture level is improved. Since obesity causes or worsens the occurrence of complications related to metabolic diseases such as hypertension, arteriosclerosis, hyperlipidemia, fatty liver and diabetes, obesity is recommended to be prevented in advance. It is very important for the prevention and health of various diseases to control the body metabolism through improvement.

Specifically, obesity is regulated by an abnormal mechanism of energy metabolism in the body, thereby storing the surplus energy in fat cells, hypertrophy obesity that increases fat size as fat cells increase, and fat scale by increasing the number of fat cells This can be divided into increased proliferative obesity. Among these, hypertrophic obesity is limited in the amount of fat that can accumulate in each fat cell, so severe obesity is not a lot of cases, but proliferative obesity increases the number of fat cells can store a large amount of fat It can be severe obesity.

In obesity, energy metabolism is mainly lipid, and both lipid breakdown and lipid synthesis are increased. Increased lipolysis and thus increased use of free fatty acids are a hallmark of obesity, especially abdominal obesity, and a high correlation between fat volume and lipid oxidation is observed, suggesting that increased fat volume may contribute to increased free fatty acid levels and lipid oxidation. I think. Lipid oxidation is observed in the early stages of obesity, but also on fasting or after glucose load.

Many prospective studies have also shown that obesity precedes glucose tolerance and insulin-independent diabetes (Carey VJ. Et al., Body fat distribution and risk of non-insulin-dependent diabetes mellitus in women, The nurses' health study, Am J Epidemiol, 145 , pp. 614-619, 1997). In the Framingham study, obesity was found to be more common in obese people, and in most studies, obese people with impaired glucose tolerance were at higher risk for developing insulin-independent diabetes.

The exact mechanism of progression from obesity to diabetes is unknown, but increased insulin resistance is believed to play an important role. Felber et al. Reported that obesity is associated with normal glucose tolerance, impaired glucose tolerance, diabetes with hyperinsulinemia and diabetes with hypoinsulinemia, according to epidemiological observations. It was classified into four groups and progressed from obesity to diabetes (Golay A. et al., Obesity and NIDDMP, the retrograde regulation concept, Diabetes Rev , 5 , pp.69-82, 1997).

Given the contextual evidence, it is thought that adipocytes contribute to the development of insulin resistance, that is, factors or metabolic messengers secreted from adipocytes are expected to inhibit the action of insulin in muscle and liver. The first and most widely recognized of these factors is the free fatty acids that are hydrolyzed from triglycerides stored in adipose tissue. In addition, TNF-α and leptin, which are released from adipocytes, have recently been suggested as a cause of insulin resistance in obesity (Hotamisligil GS. Et al., Tumor necrosis factor a, a key component of the obesity-diabetes link, Diabetes , 43 , pp. 1271-1278, 1994).

To evaluate the efficacy related to the obesity effect, 3T3-L1 fibroblasts have been studied using the method of consuming fat accumulated in the differentiated state of fat when it differentiates into adipocytes or inhibiting the newly synthesized fat. At the level, there is a drawback that the results cannot be reproduced identically in animal experiments. In addition, as the obesity treatment agents, the most commonly used is orlistat and reductyl (sibutramine), but in the small intestine, xenical which inhibits lipase is produced by fatty stool, gas production, etc. Side effects are well known, and in the case of reductil, side effects such as headache, dry mouth, anorexia and insomnia have been reported by increasing serotonin and noradrenaline levels in the sympathetic nervous system, requiring a safe and new mechanism of treatment. to be.

Gilkyung is the root of Platycodon grandiflorum A. DC., A plant of Campanulaceae , commonly called Gilkyoung in Korea, Jiegeng in China, and Kikyo in Japan. Has been. Gilkyung has been used mainly for the treatment of expectoration, antitussive action, cough and bronchitis, and anti-inflammatory effects through NF-kB and NO assay (Biol. Pharm. Bull., 11 , p. 2114, 2008; Eur. J. Pharmacol , 537 , p.1, 2006; Life Sci., 76 , p.2315, 2005) Anticancer effect (Int. J. Cancer, 261 , p.2674, 2008; Cancer Lett., 261 , p.98, 2008 ), Pain relief effect (Am. J. Chin. Med., 32 , p.257, 2004; Planta Med., 68 , p.794, 2002), inhibitory effect on digestive enzyme secretion (Arch. Pharm. Res., 27) , p.1048, 2004), and cholesterol metabolism improving effects (Eur. J. Pharmacol., 537 , p. 1, 2006; J. Food Sci., 73 , p. 195, 2008).

Deulyimyeo gilgyeong saponin as the main pharmacological components of gilgyeong, triterpene saponins of gilgyeong saponin of oleic Annan (oleanane) contained about 2% of the sequence (platycosides; platycoside A, platycodin D _2, D _3, D, polygalacin D, etc.) More than 30 species are known so far ( Chem Pharm Bull, (Tokyo) 54 , p.557, 2006; Chem. Pharm. Bull, (Tokyo) 54 , p.1285, 2006; J. Chem. Soc.Perkin Trans , I , p . 661, 1984; Planta Med., 71 , p.566, 2005). The main forms of saponenin are basically saponins with a glycoside of saponin and polygalacic acid as the glycosides of pladicodigenin, depending on the presence or absence of an alcohol group at the glycoside 4, and these saponins Several saponins have been reported until recently, depending on the type and number of sugars bound to 3 and 28 of Akiyama T., Chem. Pharm. Bull., 20 , p. 1957, 1972; Akiyama T., Chem Pharm.Bull., 24 , p. 1965, 1972; Kubota T., Chem. Commun ., P.1313, 1969), among which are Platicodin D, D ₃ and Platicoside E, Polygalacin D is the main component of Gilkyung in content.

In particular, the Platicodin D is a saponin having a major pharmacological component of Gildy ( Platycodon grandiflorum A. DC.), Anti-apoptosis (Korean Register No. 10-0564927), cholesterol lowering (cholestrol-lowering ), Republic of Korea Registration No. 10-0750333) anti-inflammation (Korea Registration No. 10-0696647) is known for its efficacy, a single component of high pharmacological value (Hahn J., Nutr., 130 , p.2760, 2000), but the disadvantage of the Platicodin D, which includes a large number of saponins with a high polarity and similar structure to compensate for the sharp drop in yield when separated by the present inventors By using a high-speed countercurrent chromatography that maximizes the separation efficiency of the material by rotating the column at high speed, a large amount of gilsaponin-containing fractions and purified purified Platicodin D from the fractions in a short time Li and production methods have been patented (Korean Patent Application No. 10-2008-0081605), but in spite of the various effects described above, many limitations are due to the very low content of Platicodin D contained in the original cilantro saponin. There is this.

No one in this document teaches or describes a composition for the prophylaxis and treatment of lipid metabolic diseases, which contains the gilyeong saponin fraction isolated from the gilyeong extract and a high content of platycodine D, which is bioconverted from the fraction, as an active ingredient. .

Accordingly, the present inventors measured the effect of inhibiting adipocyte differentiation by using the pathogenic saponin fraction isolated from the pathogenic extract and the high content of placodin D bioconverted by enzyme from the fraction, and as a result, the adipocyte differentiation improving ability was excellent. The present invention was completed by confirming that there is an excellent effect in the prevention and treatment of lipid metabolism diseases.

In order to achieve the above object, the present invention provides a novel gilyeong saponin fraction containing a large amount of Platicodin D isolated from the gilyeong extract.

More specifically, the present invention comprises the first step of obtaining the Gilyeong extract by extracting Gilyeong; A second step of obtaining the citric saponin fraction from the extract by at least one separation method selected from a reversed phase resin, a pure resin, a polystyrene-based or ion exchange resin column; 50.0∼98.0% (w / w)% of Platicodin D prepared by a third step process in which the fraction is separated into a single long-length saponin compound, in particular, a single Platicodin D component, which is bioconverted by an enzyme. A novel long-length saponin fraction with enhanced efficacy, containing 0-35.0 (w / w)% of Tycodine E, and 0-20.0 (w / w)% of Platicodine D ₃ , is provided.

In another aspect, the present invention provides a pharmaceutical composition for the prevention and treatment of lipid metabolic diseases, containing as an active ingredient the isolated gilyeong saponin compound, which is bioconverted from the gilyeong saponin fraction separated from the gilyeong extract or by enzyme.

In another aspect, the present invention provides a health functional food for the prevention and improvement of lipid metabolism diseases containing the gilyeong saponin fraction separated from the gilyeong extract or an isolated gilyeong saponin compound bioconverted by the enzyme from the fraction as an active ingredient.

Extracts as defined herein include water, including purified water, lower alcohols having 1 to 4 carbon atoms or mixed solvents thereof, preferably water, methanol or ethanol mixed solvents, more preferably extracts soluble in water or methanol.

In order to achieve the above object, the present invention is a first step of obtaining a Gilyeong extract by extracting Gilyeong; A second step of obtaining the citric saponin fraction from the extract by at least one separation method selected from a reversed phase resin, a pure resin, a polystyrene-based or ion exchange resin column; Separation and production method for separating a large amount of long saponin compound from a long length saponin compound, a third step process of separating only a single platycodine D component in a large amount by the enzymatic bioconversion of the fraction, in particular a single placodin D component to provide.

As defined herein, an “enzyme” is a common glycolytic enzyme, such as glucosidase, cellulase, lactase, mannosidase, glycosidase, galactosidase, naringinase, hesperididases, or glucronidase. It is preferred, _and more preferably, comprises glucosidase, cellulase, lactase, naringinase, hesperididase or glucronidase, even more preferably glucosidase or cellulase.

As defined herein, “longitudinal saponin compounds” include Platicoside A, Platicodine D ₂ , Platicodine D ₃ , Platicoside E, Diapio-Platinicoside E, Platicodone D, Diapio-Placicodine D, Platicodine D ₃ , 2-O-acetyl Platicodine D, 3-O-acetyl Platicodine D, 2-O-Acetyl Platicodine D ₂ , 3-O-Acetyl Platicodine D ₂ , 2-O-acetyl Platicodine D ₃ , 3-O-acetyl Platicodine D ₃ , Polygalacin D ₂ , polygalacin D _3, polygalacin D, or diapio-platicodine D ₃ and the like _, preferably placoside E, platicodine D ₃ or platicodine D, more preferably platicodine D is included.

The lipid metabolism diseases include obesity, diabetes, hypercholesterolemia, hyperlipidemia, hypolipidemia, lipoproteinosis or arteriosclerosis, preferably obesity or diabetes.

Hereinafter, the present invention will be described in detail.

More specifically, the step of obtaining the gilyeong extract of the first step of the manufacturing process, for example, about 1 to 30 times the volume of the dried gilyeong dry weight, preferably 5 to 15 times the volume (w / v%) water, methanol, ethanol, butanol and other solvents selected from lower alcohols having 1 to 4 carbon atoms or mixed solvents thereof are added, and about 0 to 100 ℃, preferably 1 to 20 hours at room temperature Preferably, the extraction method such as cold extraction, hot water extraction, ultrasonic extraction, reflux cooling extraction, or heat extraction method for 3 to 15 hours, preferably about 1 to 7 times, preferably 2 to 5 times by hot water extraction method After extraction, the mixture was filtered and concentrated under reduced pressure to obtain the Giltung extract of the present invention.

Obtaining the gilyeong saponin fraction of the second step, for example, the gilyeong extract obtained in step 1, such as inverse resin (C18), pure resin (silica gel), polystyrene (Diaion-based) and ion exchange resin column It is adsorbed on a polymer synthetic resin, preferably polystyrene (Diaion-based) to remove the polar substance obtained from the polar solvent by using a mixed solvent of water and alcohol, preferably a mixed solvent of water and methanol, and polarity than the solvent The step of distilling off these low constituents may yield the long-length saponin fraction of the present invention from a less polar solvent.

The step of obtaining a single long-length saponin compound of the third step, for example, the fraction of the long-length saponin obtained in the second step is glucosidase, cellulase, lactase, mannosidase, glycosidase, galactosidase, narin Ginase, hesperididase or glucoronidase and the like are preferred, _and more preferably, glucosidase, cellulase, lactase, naringinase, hesperididase or glucoronidase, even more preferably glucosidase Of the present invention through an enzymatic reaction process (bioconversion) by adding a sidase or cellulase enzyme to react at about 20 to 50 ° C, preferably 30 to 40 ° C, for 6 hours to 48 hours, preferably about 24 hours. It is possible to obtain a component having a high content of longitudinal saponin compounds, for example, a content of 50 to 99% of Platicodin D.

The present invention provides a pharmaceutical composition for the prophylaxis and treatment of lipid metabolic diseases, which comprises a gilyeong saponin fraction isolated from the gilyeong extract obtained by the above-mentioned manufacturing process and a high content of gilyeong saponin compound bioconverted from the fraction as an active ingredient. to provide.

Pharmaceutical compositions for the prevention and treatment of lipid metabolism diseases of the present invention, the total weight of the composition is 0.1 to 50% by weight of the gilyeong saponin fraction separated from the gilyeong extract and a high content of gilyeong saponin compound bioconverted from this fraction by enzyme Included in%.

The pharmaceutical composition comprising the gilyeong saponin fraction isolated from the gilgi extract of the present invention and a high content of gilgi saponin compound added with an enzyme from the fraction further comprises suitable carriers, excipients and diluents commonly used in the preparation of pharmaceutical compositions. can do.

The pharmaceutical composition comprising the gilyeong saponin fraction isolated from the gilyeong extract according to the present invention and a high content of gilyeong saponin compound bioconverted from the fraction by enzyme, respectively, is prepared in accordance with conventional methods, powders, granules, tablets, capsules, Oral dosage forms such as suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterile injectable solutions. Examples of carriers, excipients and diluents which may be included in the composition comprising the gilyeong saponin fraction isolated from the gilgin extract and the high content of gilgin saponin compound bioconverted from the fraction are lactose, dextrose, sucrose, sorbitol , Mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxy Benzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid preparations include at least one excipient such as starch, calcium carbonate, sucrose in the extract. ) Or lactose, gelatin and the like are mixed. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

Preferred dosages of the gilyeong saponin fraction isolated from the gilyeong extract of the present invention and the high content of gilyeong saponin compounds bioconverted from the fraction by enzymes are dependent on the condition and body weight of the patient, the extent of the disease, the form of the drug, the route and duration of the patient. It depends, but can be appropriately selected by those skilled in the art. However, for the desired effect, the ciliary saponin fraction isolated from the ciliaceae extract of the present invention and the high content of ciliform saponin compound bioconverted by the enzyme from the fraction are 0.0001 to 100 mg / kg per day, preferably 0.001 to 100 Administration at mg / kg is recommended. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.

The ciliary saponin fraction isolated from the ciliaceae extract of the present invention and the high content of ciliform saponin compound bioconverted from the fraction can be administered to mammals such as mice, mice, livestock, humans and the like by various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

In addition, the present invention is a health for the prevention and improvement of lipid metabolic diseases containing the gilyeong saponin fraction separated from the gilyeong extract obtained by the above manufacturing process and a high content of gilyeong saponin compound bioconverted by the enzyme from the fraction as an active ingredient Provide nutraceuticals.

As a food which can add the gilgi saponin fraction isolate | separated from the gilgi extract of this invention, and the high content gilgi saponin compound bioconverted from this fraction by enzyme, For example, various foods, drinks, gum, tea, vitamin Complex, health functional foods, and the like.

It may also be added to foods or beverages for the purpose of preventing and improving lipid metabolism diseases. At this time, the amount of the extract in the food or beverage may be added in 0.01 to 15% by weight of the total food weight, the health beverage composition may be added in a ratio of 0.02 to 5g, preferably 0.3 to 1g based on 100ml. .

Health functional food of the present invention includes the form of tablets, capsules, pills, liquids and the like.

The health functional beverage composition of the present invention is not particularly limited to other ingredients except for containing the forget-me-not extract as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. . Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (tauumatin, stevia extract (e.g., Rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of said natural carbohydrates is generally about 1-20 g, preferably about 5-12 g per 100 ml of the composition of the present invention.

In addition to the above, the gilyeong saponin fraction separated from the gilyeong extract of the present invention and the high content gilyeong saponin compound bioconverted by the enzyme from the fraction are various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, etc. Flavoring agents, coloring and neutralizing agents (cheese, chocolate, etc.), pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohols, carbonated beverages used in carbonated drinks A topical agent etc. can be contained. In addition, the gilyeong saponin fraction isolated from the gilyeong extract of the present invention and the high content of gilyeong saponin compound, which is bioconverted from the fraction by enzyme, may contain the flesh for the production of natural fruit juice and fruit juice beverage and vegetable beverage. These components can be used independently or in combination. The proportion of such additives is not so critical but is usually selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the extract of the present invention.

As described above, the gilyeong saponin fraction isolated from the gilyeong extract of the present invention and the high content of placodin D bioconverted by enzyme from this fraction have an excellent inhibitory effect on adipocyte differentiation, thereby preventing lipid metabolism disease. And it can be usefully used as a therapeutic pharmaceutical composition.

Hereinafter, the present invention will be described in detail. However, the following Examples, Reference Examples and Experimental Examples are merely illustrative of the present invention, but the content of the present invention is not limited thereto.

Reference Example 1.HPLC Experiment Preparation

All reagents used for HPLC analysis were purchased from HPLC solvents of JT Baker (USA), and HPLC (HITACHI L-6200) and autoinjector (Autoinjector, Shimadzu SIL-9A) were used for HPLC to check the composition of saponin. ), A vaporized light scattering detector (ELSD, Sedex 75) was used, and water (90%): acetonitrile (10%) in a gradient method with a retention time of 60 min and 1 ml / min. A single long-length saponin was separated using a reversed-phase C18 column (Zorbax SB-Aq c18, 150 × 4.6 mm ID, 5 μm particles) manufactured by Agilent, and confirmed by HPLC upon purity measurement ( J Chromatogr. 27 , p. A 1135, 2006).

Example 1.Preparation of Gilpyeong Extract

1-1. Preparation of Gilkyung 30% Methanol Extract

After purchasing 1kg of dried kilns as cut form manufactured and packaged by Ire Pharmaceutical Co., Ltd. sold in Gyeongdong market, put them in a 5L reactor and add 3L of 30% methanol to Soxhlet extract method. Extracted by heating 3 times at 100 ℃ for 3 hours, filtered using ADVANTEC paper filter, the filtrate was concentrated using a vacuum distillation (EYELA, JP / N-1000) and freeze-dried to form a powder Obtained 350g (hereinafter referred to as 'ME') of 30% methanol extract.

1-2. Preparation of Gilkyung Hydrothermal Extract

After purchasing 1kg of dried kilns as cut form manufactured and packaged by Ire Pharmaceutical Co., Ltd. sold in Gyeongdong market, put them in 5L reactor and add 3L of 100% distilled water to Soxhlet extract method. Extracted by heating 3 times at 100 ℃ for 3 hours, filtered using ADVANTEC paper filter, the filtrate was concentrated using a vacuum distillation (EYELA, JP / N-1000) and freeze-dried to form a powder Obtained 500 g of hot water extract (hereinafter, referred to as 'WE').

Example 2. Preparation of Gilt Saponin Fraction

In order to remove the polar component of the WE (gilyeong hot water extract) of Example 1-2, 200g of WE obtained in Example 1-2 was dissolved again in 15L of water (distilled water) to polymer synthetic resin (Diaion HP-20 gel) The column (100 × 10 cm) filled with 1.5 kg of water was chromatographed using water: methanol solvent system, washed with 15 L of water and 30% methanol each to remove polar substances including polysaccharides, and then flowed again with 15 L of 100% methanol. The obtained fractions were concentrated under reduced pressure using a vacuum distillation machine and a lyophilizer (Eyela, JP / N-1000) to obtain about 4 g of gildo irradiation ponin (hereinafter referred to as CS).

Example 3. Analysis of Gildo Irradiation Phonin

5 mg of the polar component-removed PS obtained from Example 2 was removed in a 1.5 mL Eppendorf tube (AXYGEN Scientific, USA), dissolved in 1 mL of distilled water, and the dissolved sample was injected into a 1 ml total plastic syringe (1 ml). Norm-ject syringe, Henke Sass Wolf, Germany) and a PVDF syringe filter (0.45㎛, PVDF syringe filter, Whatma n, USA) were used for sample pretreatment and 20 μl injection into HPLC for analysis.

As a result, the absolute amounts of Platicoside E, Platicodine D ₃ and Platicodine D contained in 5 mg PS were 0.26, 0.16 and 0.67 mg, respectively. The relative amounts were 23.8, 14.9 and 61.3%.

REFERENCE EXAMPLE 2 Changes in Components of Irradiated Phosphorin and Increased Effect of Platincodine D

2-1. Effect of Glucosidase Enzyme on Changes in Components of Platinum and Platincodin D

Beta glucosidase (G0395) derived from almonds was purchased from Sigma. The activity of the enzyme is expressed in units (U), 1 unit represents the degree of releasing 1.0 μmole of glucose per minute from salicin at pH 5.0 and temperature 37 ° C., and the enzyme purchased is 4.6 units per mg. Activity.

5 mg of PS of Example 2 having the polar material removed therefrom was dissolved in 1 mL of distilled water in a 1.5 mL Eppendorf tube (AXYGEN Scientific, USA), and then betaglucosidase solid type 5 units (1.09 mg; 4.6) was dissolved in the dissolved sample. u / 1mg) was added and reacted for 24 hours in a 37.5 ° C shake incubator. After the reaction, the sample was immersed in water at 100 ° C. for about 3 minutes, and the sample was pretreated using a 1 ml whole-plastic syringe (norm-ject syringe) and a PVDF syringe filter, and then injected into a 20 μl HPLC. The data obtained from the analysis were used to quantify each amount of Platicoside E, Platicodine D ₃ and Platicodine D, and the absolute amounts of the three substances in mg and the relative proportions in%.

Experimental results showed that the absolute amounts of Platicoside E, Platicodine D ₃ and Platicodine D were 0.17, 0.19 and 0.84 mg, respectively. Relative proportions were 14.2, 15.5, and 70.3%.

2-2. Effect of Glucosidase Enzyme on Changes in Components of Platinum and Platincodin D

Beta glucosidase (49290) from almonds was purchased from Fluka. The activity of the enzyme is expressed in units (U), 1 unit represents the degree of releasing 1.0 μmole of glucose per minute from salicin at pH 5.0 and temperature 37 ° C., and the enzyme purchased was 8.92 units per mg. Activity.

5 mg of PS of Example 2 having the polar material removed was dissolved in 1 mL of distilled water in a 1.5 mL Eppendorf tube (AXYGEN Scientific, USA), and then 5 units (0.56 mg; 8.92 mg) of betaglucosidase was dissolved in the dissolved sample. u / 1mg) was added and reacted for 24 hours in a 37.5 ° C. shaking incubator. After the reaction, the sample was immersed in water at 100 ° C. for about 3 minutes, and the sample was pretreated using a 1 ml whole-plastic syringe (norm-ject syringe) and a PVDF syringe filter (PVDF syringe filter). The data obtained from the analysis were used to quantify each amount of Platicoside E, Platicodine D ₃ and Platicodine D, and the absolute amounts of the three substances in mg and the relative proportions in%.

Experimental results showed that the absolute amounts of Platicoside E, Platicodine D ₃ and Platicodine D were 0.25, 0.17 and 0.76 mg, respectively. Relative proportions were 21.2, 14.4, and 64.3%.

2-3. Effect of Cellulase Enzyme on Changes of Composition and Increase of Platincodin D

Solid cellulase (C8546) derived from Trichoderma lizsey was purchased from Sigma. The activity of the enzyme is expressed in units (U). One unit represents the degree of releasing 1.0 μmole of glucose within one hour from cellulose at pH 5.0 and a temperature of 37 ° C. The enzyme purchased is 6 units per mg. Activity.

5 mg of PS of Example 2, from which the polar material was removed, was placed in a 1.5 mL Eppendorf tube (AXYGEN Scientific, USA), dissolved in 1 mL of distilled water, and 5 units (0.83 mg; 6 u / 1 mg) of cellulase were added to the dissolved sample. The reaction was carried out for 24 hours in a 37.5 ℃ shake incubator. After the reaction, the sample was immersed in water at 100 ° C. for about 3 minutes, and the sample was pretreated using a 1 ml whole-plastic syringe (norm-ject syringe) and a PVDF syringe filter (PVDF syringe filter). The data obtained from the analysis were used to quantify each amount of Platicoside E, Platicodine D ₃ and Platicodine D, and the absolute amounts of the three substances in mg and the relative proportions in%.

Experimental results showed that the absolute amounts of Platicoside E, Platicodine D ₃ and Platicodine D were 0.0, 0.0 and 1.58 mg, respectively. Relative proportions were 0.0, 0.0 and 100%.

2-4. Effect of Cellulase Enzyme on Changes of Composition and Increase of Platincodin D

It was purchased from Sigma as liquid cellulase (C2730) derived from Trichoderma liz. The activity of the enzyme is expressed in units (U) and 1 unit represents the degree of releasing 1.0 μmole of glucose within 1 hour from cellulose at pH 5.0 and 37 ° C. The purchased enzyme is 6 units per mg. Activity.

5 mg of PS of Example 2 having the polar material removed was dissolved in 1 mL of distilled water in a 1.5 mL Eppendorf tube (AXYGEN Scientific, USA), and then 5 units (5.96 μl) of cellulase liquid and 1 unit (1.19) were dissolved in the dissolved sample. 0.84 unit / μl) was added and reacted for 24 hours in a 37.5 ° C shake incubator. After the reaction, the sample was immersed in water at 100 ° C. for about 3 minutes, and the sample was pretreated using a 1 ml whole-plastic syringe (norm-ject syringe) and a PVDF syringe filter (PVDF syringe filter). The data obtained from the analysis were used to quantify each amount of Platicoside E, Platicodine D ₃ and Platicodine D, and the absolute amounts of the three substances in mg and the relative proportions in%.

The experimental results showed that the absolute amounts of Platicoside E, Platicodine D ₃ and Platicodine D were 0.0, 0.0, 1.49 mg and the relative ratios were 0.0, 0.0 and 100%, respectively. The relative proportions of Platicoside E, Platicodine D ₃ and Platicodine D were 0, 5.7, and 94.3%, respectively.

2-5. Effect of Galactosidase Enzyme on Changes in Composition of Platinum and Platincodin D

Galactosidase (G5160) from Aspergillus aurise was purchased from Sigma. The activity of the enzyme is expressed in units (U). One unit represents the degree to which 1.0 μmole of lactose is released per minute at pH 4.5 and a temperature of 30 ° C., and the purchased enzyme has an activity of 10 units per mg.

5 mg of PS of Example 2, in which the polar material was removed, was dissolved in 1 mL of distilled water in a 1.5 mL Eppendorf tube (AXYGEN Scientific, USA), and 0.5 mg (5 units; 10 u / 1 mg of galactosidase) was dissolved in the dissolved sample. ) Was added and reacted for 24 hours in a 37.5 ° C reciprocating shake incubator. After the reaction, the sample was immersed in water at 100 ° C. for about 3 minutes, and the sample was pretreated using a 1 ml whole-plastic syringe (norm-ject syringe) and a PVDF syringe filter (PVDF syringe filter). The data obtained from the analysis were used to quantify each amount of Platicoside E, Platicodine D ₃ and Platicodine D, and the absolute amounts of the three substances in mg and the relative proportions in%.

Experimental results showed that the absolute amounts of Platicoside E, Platicodine D ₃ and Platicodine D were 0.06, 0.15 and 1.24 mg, respectively. Relative proportions were 4.2, 10.6, 85.3%.

2-6. Effect of Gluronidase Enzyme on Changes in Components of Platinum and Platincodin D

It was purchased from Sigma with glucronidase (G7646) derived from Escherichia coli . The activity of the enzyme is expressed in units (U) and 1 unit represents 1.0 ug of phenolphthalein released per hour from phenolphthalein gluronide at pH 6.8 and temperature of 37 ° C. The purchased enzyme is 5,292,000 units per 1g protein. Activity.

5 mg of PS of Example 2 from which the polar material was removed was dissolved in 1 mL of distilled water in a 1.5 mL Eppendorf tube (AXYGEN Scientific, USA), and 5 units of glucronidase were added to the dissolved sample to reciprocate shaking at 37.5 ° C. The reaction was carried out for 24 hours in a shake incubator. After the reaction, the sample was immersed in water at 100 ° C. for 3 minutes, and the sample was pretreated by using a 1 ml whole-plastic syringe (norm-ject syringe) and a PVDF syringe filter (PVDF syringe filter). The data obtained from the analysis were used to quantify each amount of Platicoside E, Platicodine D ₃ and Platicodine D, and the absolute amounts of the three substances in mg and the relative proportions in%.

Experimental results showed that the absolute amounts of Platicoside E, Platicodine D ₃ and Platicodine D were 0.30, 0.18 and 0.77 mg, respectively. Relative proportions were 24.2, 14.2 and 61.6%.

2-7. Effect of Gluronidase Enzyme on Changes in Components of Platinum and Platincodin D

Gluronidadase (G7017) from Helix pomatia was purchased from Sigma. The activity of the enzyme is expressed in units (U), where 1 unit represents 1.0 ug of phenolphthalein released per hour from phenolphthalein gluronide at pH 6.8 and temperature of 37 ° C., and the purchased enzyme is 1,145,000 per 1g protein. The activity of the unit.

5 mg of PS of Example 2 from which the polar material was removed was dissolved in 1 mL of distilled water in a 1.5 mL Eppendorf tube (AXYGEN Scientific, USA), and 5 units of glucronidase were added to the dissolved sample to reciprocate shaking at 37.5 ° C. The reaction was carried out for 24 hours in a shake incubator. After the reaction, the sample was immersed in water at 100 ° C. for 3 minutes, and the sample was pretreated by using a 1 ml whole-plastic syringe (norm-ject syringe) and a PVDF syringe filter (PVDF syringe filter). The data obtained from the analysis were used to quantify each amount of Platicoside E, Platicodine D ₃ and Platicodine D, and the absolute amounts of the three substances in mg and the relative proportions in%.

As a result, the absolute amounts of Platicoside E, Platicodine D ₃ and Platicodine D were 0.24, 0.18 and 0.76 mg, respectively. Relative proportions were 20.4, 15.4, and 64.2%.

Example 4 Preparation of High Content Platincodin D Using Cellulase Enzyme

After removing the polar material PS 2 g of Example 2 in a 1 L container and dissolved in 400 mL of distilled water, 240 μl (201.6 unit) enzyme of cellulase liquid of Reference Example 2-4 was added to the dissolved sample. To 37.5 ° C. in a shake incubator for 24 hours. After the reaction, the solution was immersed in water at 100 ° C. for about 3 minutes, and the reaction product was poured into a column chromatography filled with Diaion-HP20 packing. The reaction product was started with water, and then prepared with 100% methanol (water, 30% methanol, 100% methanol). After collecting the% methanol fraction, the fraction was removed using a vacuum distillation apparatus, concentrated and lyophilized to obtain a high content of Platicodin D (hereinafter referred to as 'GPD') to which the enzyme was added.

The peaks of Platicoside E, Diapio-Platinicoside E, Platicodine D ₃ , Polygalacin D or Diapio-Platicicodine D ₃ decreased over time in the HPLC chromatogram and eventually disappeared. At the same time, an increase in Platicodin D and Diapio-Platinicodine D can be observed, which means that one or two more glucoses are attached to C-3 of Platicodin D. ₃ , and the glucose of Platicoside E is separated by cellulase, so the above compounds are converted to Platicodin D, and Diapio-Platinicoside E and Diapio-Platinicotin D _{3 are} also Diapio-Plati All are converted to ticodine D.

Experimental Example 1. Adipocyte differentiation inhibitory effect

DMEM (Dulbecco's Modified Eagle Medium, Gibco-BRL, Grand Island, NY) containing 10% bovine calf serum of pre-adipocytes and 3T3-L1 cells (American Tissue Culture.Collection, ATCC) , USA), and seeded by suspending 4 x 10 ⁴ cells per well in 2 ml in 6-well plates. After incubation for about 24 to 36 hours in the same medium, the cell density of 90% in the culture of fat precursor cells is 10μg / ml insulin, 0.5mM IBM (IBMX, 3-isobutyl-) for 3T3-L1 1-methylxanthine), 1 μM dexamethasone, and 10% fetal bovine serum (Fetal Bovine Serum) were added to DMEM medium and treated for 48 to 55 hours to induce differentiation into mature adipocytes. At this time, 4 μg / ml of WE (longitudinal hot water extract) obtained in the above example, 4 μg / ml of CS (irradiated ponin), 5 μM of PD (platicodine D), and 4 μg / ml of GPD (platicodine D with enzyme) were DMSO. After dissolving in the same concentration of DMSO was treated together to be used in each concentration treatment group, as a control (hereinafter referred to as 'DM') 10μg / ml insulin (insulin), 0.5mM IBM (IBMX, 3-isobutyl -1-methylxanthine) and 1 μM dexamethasone are added and dissolved in DMSO under the same conditions. After 48 hours of induction of differentiation, the drug was again treated with 10 μg / ml insulin (10% FBS / DMEM medium containing insulin) and again treated with 10% FBS / DMEM medium after 48 hours.

1-1. Western blotting

5-6 days after induction into adipocytes, using the Bradford method (Bradford MM., Et al., Ann Biochem, 72 , pp. 248-254, 1976) using the fetal bovine serum after protein extraction from the cell line Quantification by After 20 g of protein was denatured at 95 ° C. for 5 min, electrophoresis was performed on a 12% discontinuous sodium dodecylsulfate (SDS-PAGE) polyacrylamide gel, and the electrophoretic protein was poly- for 50 min at 20 volts. Transfer to vinylidene difluoride (PVDF, Amersham Bioscience, USA) membrane. 5% skim milk powder was dissolved in TTBS (Tris-Tween buffered saline) as blocking buffer and reacted for 30 minutes at room temperature, and c / EBP-α (Santacruz) and PPAR-γ2 (Santacruz) antibodies were reacted for 3 hours. After washing three times at 10 minute intervals. Secondary antibody anti-rabbit IgG-HRP, anti-mouse IgG-HRP, (1: 2,000 dilution, Santacruz, USA) were reacted for 1 hour at room temperature, and then washed three times for 10 minutes with TTBS once again through a washing step. , And reacted with ECL (enhanced chemiluminescence) solution for 2 minutes, and compared the thickness of the band (band) appeared on the film was confirmed the presence and absence of protein expression.

As a result, as shown in FIG. 2, c / EBP-α and PPAR-γ expression levels were inhibited in the order of PD> GPD> CS> WE. However, when the concentration of the single compound uM was converted into ug units. , PD (Platincodine D) is about 6.12 ug / ml, which is higher than GPD (Placocodin D with enzyme), especially GPD (Placocodin D with enzyme) is a representative fat. Synthetic protein cEBP-α and PPAR-γ expression level was found to show at least the same or superior effect than PD (platicodine D) (see Fig. 2).

1-2. Inhibitory Effect of Adipocyte Differentiation

After 8 to 10 days after the differentiation into adipocytes, the resulting fat is identified through Oil-red O staining. Remove the plate from the incubator to observe the cells, remove the cell medium and wash the cells 2 or 3 times with PBS. If too much PBS is added, the cells will fall off, and the well wall will be injected into the well wall. 0.5% Oil prepared in Reference Example 2 by adding 1 ml of 10% formaldehyde to each plate and fixing the cells at room temperature for 1 hour, removing 10% formalin and washing it with distilled water 2-3 times. 1 ml of -red O staining reagent is added to each plate and stained for about 2 hours at room temperature. After staining, the staining reagent is removed, washed with distilled water, opened by drying the lid, and then image scanned. After washing the stained reagent 2-3 times with 60% isopropanol on the stained cells, optical microscope After the dyeing was completed, 1 ml of 4% NPE-40 (IGEPAL) solution was added to each plate for 5 minutes to dissolve the dye, and the absorbance was measured and quantified at 490 nm.

As a result, as shown in FIG. 3, the control group had 3T3-L1 differentiation into adipocytes well and changed its shape to a rounded shape, and at the same time, fat began to accumulate and the accumulated fat appeared in several layers. It can be seen that this was observed in the oil red O staining results, while the stained fat, PD> GPD> CS> WE was found to have an inhibitory effect of adipocyte differentiation (see Figure 3).

In addition, as shown in Figure 4, when the degree of fat converted to 100% based on the control group, WE (gilpyeong hot water extract) 78%, CS (irradiationponin) 62%, PD (platicodine D) is 52%, GPD (platicodine D with enzyme) was 55% (see Figure 4).

Therefore, when the amount of GPD is measured in the same manner as the PD, the inhibitory effect of adipocyte differentiation may be at least the same or excellent.

Although a formulation example of a pharmaceutical composition comprising a gilyeong saponin fraction isolated from the gilyeong extract of the present invention and a high content of gilyeong saponin compound added with an enzyme from the fraction is described, the present invention is not intended to be limited thereto. To do so.

Formulation Example 1 Preparation of Powder

PD 20 mg

Lactose 100 mg

Talc 10 mg

The above ingredients are mixed and filled in an airtight cloth to prepare a powder.

Formulation Example 2 Preparation of Tablet

WE 10 mg

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, tablets are prepared by tableting according to a conventional method for preparing tablets.

Formulation Example 3 Preparation of Capsule

CS 10 mg

3 mg of crystalline cellulose

Lactose 14.8 mg

Magnesium Stearate 0.2 mg

According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare capsules.

 Formulation Example 4 Preparation of Injection

GPD 10 mg

Mannitol 180 mg

Sterile distilled water for injection 2974 mg

Na ₂ HPO ₄ 12H ₂ O 26 mg

According to the conventional method for preparing an injection, the amount of the above ingredient is prepared per ampoule (2 ml).

Formulation Example 5 Preparation of Liquid

PD 20 mg

10 g of isomerized sugar

5 g of mannitol

Purified water

According to the conventional method of preparing a liquid solution, each component is added to the purified water to dissolve it, the lemon flavor is added, the above components are mixed, the purified water is added, the whole is adjusted to 100 ml by adding purified water, and then filled in a brown bottle. The solution is prepared by sterilization.

 Formulation Example 6 Preparation of Healthy Drink

GPD 100 mg

15 g of vitamin C

100 g of vitamin E (powder)

Iron lactate 19.75 g

3.5 g of zinc oxide

Nicotinamide 3.5 g

0.2 g of vitamin A

Vitamin B ₁ 0.25 g

Vitamin B ₂ 0.3 g

Water quantification

After mixing the above components in accordance with a conventional healthy beverage production method, and stirred and heated at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed sterilization and then refrigerated and stored in the present invention For the preparation of healthy beverage compositions.

Although the composition ratio is a composition that is relatively suitable for the preferred beverage in a preferred embodiment, the compounding ratio may be arbitrarily modified according to regional and ethnic preferences such as demand hierarchy, demand country, and usage.

[National R & D project supporting this invention]

[Task unique number]

       A080974

[Name of Buddha]

       Ministry of Health and Welfare

[Name of research project]

       Health and Medical Technology Development Project

[Name of Research Project]

       Development of unannounced functional whitening material and product using Platicodin D

[Host]

        Biospectrum

[Research period]

        April 01, 2009-March 31, 2010

1 is a diagram showing the results of the component analysis of Gil-kyung (flower bellflower) irradiated with cellulase enzyme,

2 is a diagram showing the degree of c / EBP-α and PPAR-γ expression of WE, CS, PD and GPD,

Figure 3 is a diagram showing the inhibitory effect of adipocyte differentiation according to the oil red O staining results of WE, CS, PD and GPD,

Figure 4 is a diagram showing the numerical comparison of the inhibitory effect of adipocyte differentiation according to the result of oil red O staining of WE, CS, PD and GPD.

Claims (12)

A first step of extracting the tillering to obtain the tillering extract; A second step of obtaining the citric saponin fraction from the extract by at least one separation method selected from a reversed phase resin, a pure resin, a polystyrene-based or ion exchange resin column; Platicodine D 50.0-98.0 (w / w)%, Platicodine E 0-35.0 (w /) prepared through a third step process containing a large amount of a single long-length saponin compound, which is bioconverted by the fraction by enzyme. A novel medicinal saponin fraction with enhanced potency, containing w)%, and Platicodin D ₃ 0-20.0 (w / w)%. A pharmaceutical composition for the prophylaxis and treatment of lipid metabolism diseases, comprising as an active ingredient a gilyeong saponin fraction isolated from the gilyeong extract of claim 1 or a biocides isolated from the fraction by an enzyme. The pharmaceutical composition according to claim 2, wherein the extract is an extract available in water including purified water, a lower alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof. The pharmaceutical composition according to claim 2, wherein the enzyme is glucosidase, cellulase, lactase, mannosidase, glycosidase, galactosidase, naringinase, hesperididase or glucronidase. The method according to claim 2, wherein the long saponin compound is Platicoside A, Platicodine D ₂ , Platicodine D ₃ , Platicoside E, Diapio-Platinicoside E, Platicodone D, Diapio-Placicodine D, Platicodine D ₃ , 2-O-acetyl Platicodine D, 3-O-acetyl Platicodine D, 2-O-Acetyl Platicodine D ₂ , 3-O-Acetyl Platicodine D ₂ , 2-O-acetyl Platicodine D ₃ , 3-O-acetyl Platicodine D ₃ , Polygalacin D ₂ , a polygalacin D _3, a polygalacin D or a diapio-platicodine D ₃ pharmaceutical composition. The pharmaceutical composition of claim 2, wherein the lipid metabolism disease is obesity, diabetes mellitus, hypercholesterolemia, hyperlipidemia, hypolipidemia, lipoproteinosis or arteriosclerosis. A health functional food for the prevention and improvement of a lipid metabolism disease, comprising as an active ingredient the gilyeong saponin fraction separated from the gilyeong extract of claim 1 or the gilyeong saponin compound isolated from the fraction by enzyme. A dietary supplement according to claim 7 in the form of a powder, granule, tablet, capsule or beverage. A first step of extracting the tillering to obtain the tillering extract; A second step of obtaining the citric saponin fraction from the extract by at least one separation method selected from a reversed phase resin, a pure phase resin, a polystyrene-based or ion exchange resin column; Separation and production method for separating a large amount of the gilyeong saponin compound of the third step process containing a component containing a large amount of the gilyeong saponin Platicodine D compound that the fraction was bioconverted by the enzyme. The method of claim 9, The step of obtaining the gilyeong extract of the first step, the lower alcohol having 1 to 4 carbon atoms of water, methanol, ethanol, butanol containing purified water of 1 to 30 times the volume (w / v%) of the dried gilyeong dry weight Or by adding a solvent selected from these mixed solvents, and extracting 1 to 7 times by an extraction method of cold extraction, hot water extraction, ultrasonic extraction, reflux cooling extraction, or heat extraction for 1 to 20 hours at 0 to 100 ° C. and room temperature. Filtration and concentration under reduced pressure to obtain a Gilyeong extract. The method of claim 9, Obtaining the gilyeong saponin fraction of the second step, the gilyeong extract obtained in the step 1 to a polymer synthetic resin, such as inverse resin (C18), pure resin (silica gel), polystyrene (Diaion) and ion exchange resin column Adsorbing to remove the polar material obtained from the polar solvent by using a mixed solvent of water and alcohol as the effluent solvent, and distilling the components having a lower polarity than the effluent solvent to obtain a long-length saponin fraction from the lower polar solvent. The method of claim 9, The step of obtaining a single gilyeong saponin compound of the third step, glucosidase, cellulase, lactase, mannosidase, glycosidase, galactosidase, naringinase, hess A method for obtaining a high content of saponin saponin compound extract through the enzymatic reaction process (bioconversion) which is carried out at 20 to 50 ° C. for 6 hours to 48 hours by adding a ferrididase or gluronidase enzyme.

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