KR101457621B1 - Methods for Preparing Rg3 or Rg2 Group Ginsenosides and Compositions for Preventing or Treating Metabolic Diseases - Google Patents

Abstract

The present invention relates to a method for producing Rg3 group or Rg2 group ginsenoside and a composition for preventing or treating metabolic diseases. The present invention provides a method for preparing Rg3 group and Rg2 group ginsenoside, which is most optimized in terms of simplicity and efficiency. According to the present invention, at least 80% by weight of Rg3 group jinnenoside and Rg2 group jinnenoside can be obtained. The Rg3 group ginsenoside produced by the present invention is highly suitable for the prevention or treatment of metabolic diseases, particularly obesity, diabetes and diabetic complications, and exerts excellent in vivo efficacy. The Rg3 group ginsenoside produced by the present invention exerts excellent in vivo efficacy against various diabetic complications and is very effective in relieving the symptoms of diabetic patients in the early, middle, and late stages of diabetes.

Description

[0001] The present invention relates to a method for preparing ginsenoside Rg3 or Rg2 group, and a method for preventing or treating metabolic diseases,

The present invention relates to a process for producing Rg3 or Rg2 group ginsenosides and a composition for the prevention or treatment of metabolic diseases.

Diabetes is one of the typical metabolic diseases and chronic adult diseases. Diabetes patients in Korea account for about 5% of the total population, at least 2.5 million people.

Diabetes is a disease in which sugar in the blood is not used as energy because of lack of insulin secretion, insulin action and insufficiency and remains in the blood. The causes of diabetes include westernization of diet, obesity, lack of exercise, and stress. In developed countries such as the United States, Europe and Japan, socioeconomic fluctuations due to the increase in obesity and diabetes are great, and in Korea, too, diabetes is rapidly increasing due to economic development, and more than 300 million patients will develop by 2025 (Kim, Y., Diabetes Care , 1995, 18: 545-548).

Diabetes is largely divided into insulin-dependent type 1 diabetes and non-dependent type 2 diabetes. Type 1 diabetes patients have little or no secretion of insulin due to genetic or viral infection, and the amount of insulin secreted by type 2 diabetics is normal. In many cases, type 2 diabetes, which is accompanied by obesity, is a disease in which insulin resistance is induced and an abnormality occurs in glucose metabolism due to an increase in fatty acids in blood, resulting in increase in blood glucose level in the blood. In addition, Abnormalities of secretion and abnormal signal transduction are also thought to be the cause of type 2 diabetes. Insulin resistance is a key factor in the pathogenesis of type 2 diabetes, type 2 appear in more than 90% of diabetic patients, it is believed that before the onset of hyperglycemia preceding years (Haffner SM, et al JAMA, 1990, 263:. 2893- 2898). Insulin resistance and the resulting aggregation of metabolic abnormalities and vascular system abnormalities are referred to as insulin resistance syndrome or metabolic syndrome or metabolic syndrome. Insulin resistance and the metabolic syndrome are associated with an increased risk of cardiovascular disease, especially coronary heart disease (Isomaa B, et al. Diabetes Care , 2001, 24: 683-9), has been reported to increase the risk of coronary artery disease and mortality by a factor of 3 due to metabolic syndrome (Lakka H, et al. JAMA , 2002, 288: 2709-2716).

Metabolic syndrome refers to a disorder in which multiple diseases occur simultaneously, such as diabetes, hypertension, hyperlipidemia, obesity, coronary or arteriosclerosis, caused by chronic metabolic disorders. Reaven (Reaven GM, Diabetes , 1988, 37: 1595 -1607). Metabolic syndrome is characterized by insulin resistance, hypertension, and dyslipidemia, most of which are accompanied by overweight or obesity. Metabolic syndrome is also a risk factor for cardiovascular disease and has been reported to be associated with death from all causes. Unlike type 1 diabetes, the prevalence of metabolic syndrome is higher in type 2 diabetic patients and it is known that type 2 diabetes patients have increased mortality when accompanied by metabolic syndrome (Bonora E, et al. Diabet Med ., 2004, 21: 52-8; Ford ES, Diabetes Care ., 2005, 28: 1769-78; Alexander CM, et al. Diabetes , 2003, 52: 1210-1214). In addition, studies on the association of macrovascular and microvascular complications of type 2 diabetes with the components of metabolic syndrome such as hypertension and dyslipidemia have been published (Mykkanen L, et al. Diabetologia , 1993, 36 : 553-559; Haffner SM, et al. Diabetes , 1992, 41: 715-722).

The most serious problems of metabolic syndrome are the occurrence of chronic complications such as diabetic retinopathy, nephropathy, hyperlipidemia, cardiovascular diseases (stroke, angina, myocardial infarction, peripheral vascular disease) (Wolf SP, Br Med Bull ., 1993, 49: 642-652). Most of these chronic complications are irreversible after they have been developed. Until now, there is no way to completely block this process. If the proper treatment is not performed, serious symptoms will be caused and the patient will die. Therefore, for the effective management or treatment of metabolic syndrome with complex symptoms, it is ideal to have a blood glucose lowering effect for maintenance of normal blood sugar and at the same time to treat nephropathy, hepatopathy and hyperlipemia. However, And they are separately taking their hypoglycemic agents, hypotensive agents, and cholesterol drugs separately.

Conventional herbal composition compositions for the prevention and treatment of diabetes or diabetic complications include obesity and metabolism including the extract of Evodiae Fructus, Imperatae Rhizoma and Citrus Unshiu Markovich as active ingredients, A composition for preventing or treating Metabolic Syndrome or Syndrome X is disclosed in Korean Patent Publication No. 2009-0114093 and Korean Patent Publication No. 2010-0956278 discloses a composition for preventing or treating Metabolic Syndrome or Syndrome X, A composition for treating or preventing diabetes or diabetic complications comprising an extract of a herbal composition comprising a herbal extract of Hwangjeong, Baekjoo, mokmundong, corn oil and ginseng as an active ingredient. Korean Patent Laid-Open Publication No. 2006-0025990 also discloses a method for producing a compound of formula (I), which is a compound of formula A composition for preventing diabetes mellitus containing a complex herbal medicine extract comprising at least one herbal composition, at least one herb, at least one herb, at least one herb, at least one herb, at least one herb, at least one herbaceous plant, at least one herbaceous plant, Furthermore, Korean Patent Laid-Open Publication No. 2003-0059952 discloses a composition comprising an extract of a perennial excipient and an oriental herbal medicine complex, that is, an extract made of rhubarb, chaebol, algae, persimmon, windshield,

However, there is still a demand for the development of a substance that can more effectively prevent or treat diabetes or diabetic complications.

Examples of representative species of ginseng include Panax ginseng CAMeyer, Panax quinquefolium L., which is native to Korea and has excellent efficacy in Korea, Panax ginseng CAMeyer, which is native to North America, (Panax notoginseng FHChen), and Panax japonicus CAMeyer which is native to southwestern China and Japan. So far, many pharmacological tests have shown that ginseng strengthens the nonspecific resistance to stress in the body and has an antioxidant action. It also improves hypertension, enhances insulin action, strengthens blood glucose level in allogenic diabetic mice, promotes glucose and lipid metabolism, And so on.

Since the pharmacological effect of ginseng has been proved to be ginsenoside, studies on the ginsenosides of ginseng or red ginseng have been continuously carried out. Ginsenosides are classified into PPT (protopanaxatriol) ginsenoside, PPD (protopanaxadiol) ginsenoside and oleene ginsenoside according to their structural characteristics, and they are known to have different pharmacological actions.

Rd, Ra2, Rb1, Rb2, Rb3, Rc, Rd, Rg3, Rg5, Rh2, Rh3, F2 and CK and the PPT class ginsenosides include Re, Rf, Rg1, Rg2, Rg4, Rh1, Rh4, and the like.

The ginsenosides Rb1, Rb2, Rb3, Rc, and Rd have the highest contents of ginsenosides, while the triols have Re, Rf, and Rg1. Rg2 and other highly active ginsenosides and highly active ginsenosides contain only very small amounts (the content of Rg3 is about three-tenths of the total ginsenoside).

The known pharmacological actions of the Rg3 group (Rg3, Rk1 and Rg5) are antidiabetic, anti-thrombotic, anticancer, and memory-improving actions and particularly effective for diabetes and diabetic complications (containing insulin- .

Rarely effective ginsenosides of the Rg3 group (Rg3, Rk1 and Rg5) have been actively studied as health functional foods, functional cosmetic materials and new drugs.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have sought to develop a process by which Rg3 group ginsenosides and Rg2 group ginsenosides can be prepared with improved yields by a simpler process. As a result, the present inventors have applied a ginseng extract to a suitable resin and developed a process including a saccharide decomposition process, thereby confirming that Rg3 group ginsenoside and Rg2 group ginsenoside can be provided at a high yield. The present inventors also completed the present invention by confirming that the Rg3 group ginsenoside prepared by the method of the present invention is very effective for the treatment of metabolic diseases.

It is therefore an object of the present invention to provide a process for the preparation of Rg3 group ginsenosides or Rg2 group ginsenosides.

It is another object of the present invention to provide a composition for preventing, improving or treating metabolic diseases.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention provides a process for preparing an Rg3 group ginsenoside or Rg2 group ginsenoside comprising the steps of:

(a) obtaining ginseng or red ginseng extract;

(b) applying the ginseng or red ginseng extract to an adsorption chromatography using a resin composed of a copolymer of styrene and benzene to adsorb the ginsenosides in the extract to the resin;

(c) eluting the adsorbed ginsenoside to obtain protopanaxatriol (PPT) ginsenoside and PPD (protopanaxadiol) ginsenoside; And

(d) sugar degrading the PPD family ginsenosides or PPT family ginsenosides to obtain Rg2 group ginsenosides comprising Rg3 group ginsenosides or Rg2 comprising Rg3.

The present inventors have sought to develop a process by which Rg3 group ginsenosides and Rg2 group ginsenosides can be prepared with improved yields by a simpler process. As a result, the present inventors have applied a ginseng extract to a suitable resin and developed a process including a saccharide decomposition process, thereby confirming that Rg3 group ginsenoside and Rg2 group ginsenoside can be provided at a high yield. In addition, the present inventors have found that the Rg3 group ginsenoside prepared by the method of the present invention is very effective for the treatment of metabolic diseases.

The method of the present invention will be described in detail in each step as follows:

Step (a): Obtain ginseng or red ginseng extract

First, ginseng or red ginseng extract is obtained.

The term " ginseng " in the present specification encompasses fresh ginseng, ginseng, white ginseng, direct ginseng, ginseng, taegeuk ginseng, Ginseng used in the present invention, it is possible to use the various well-known ginseng, goryeosam (Panax ginseng , goat ginseng, camellia ginseng, western ginseng , P. quiquefolius , P. notoginseng , P. japonicus , P. trifolium , and P. hippocampus . pseudoginseng , and P. vietnamensis . In addition, the term " ginseng " in the present specification is a term encompassing not only the underground part of ginseng but also a tissue or organ (for example, fruit, leaf or stem)

The term " red ginseng " in the present specification means that ginseng is produced by conventional methods.

Ginseng or red ginseng extract means extracted from various organs or parts of ginseng (eg, roots, leaves, flowers, stems, etc.). Preferably, the ginseng extract in the present invention comprises (a) water, (b) an anhydrous or hydrated lower alcohol having 1 to 4 carbon atoms (methanol, ethanol (alcohol), propanol, butanol, etc.) (D) acetone, (e) ethyl acetate, (f) chloroform, (g) butyl acetate or (h) 1,3-butylene glycol as extraction solvents and more preferably Is obtained from ginseng using water, methanol or ethanol (alcohol). It is obvious to those skilled in the art that the extract of the present invention can be used to obtain ginseng or red ginseng extract which exhibits substantially the same effect not only by the above-mentioned extraction solvent but also by other extraction solvent.

In addition, the extract of the present invention includes not only the extract obtained by the above-mentioned extraction solvent, but also an extract obtained through a conventional purification process. For example, by separation using an ultrafiltration membrane having a constant molecular weight cut-off value, separation by various chromatographies (made for separation by size, charge, hydrophobicity or affinity), and the like, The fraction is also included in the ginseng extract of the present invention. The ginseng extract of the present invention can be prepared in powder form by an additional process such as vacuum distillation and freeze drying or spray drying.

Step (b): Adsorption chromatography

Next, the ginseng or red ginseng extract is applied to adsorption chromatography using a resin composed of a copolymer of styrene and benzene to adsorb ginsenosides in ginseng or red ginseng extract to the resin.

One of the features of the present invention is to apply a ginseng or red ginseng extract to an adsorption chromatography resin using a resin composed of a copolymer of styrene and benzene to separate the PPT series ginsenosides and the PPD series ginsenosides contained in the ginseng extract .

According to a preferred embodiment of the present invention, in the resin made of the copolymer of styrene and benzene, the vinyl group is bonded to the benzene, and more preferably, the benzene is divinylbenzene.

According to a preferred embodiment of the present invention, the resin used in the present invention has a ratio of 300-1000 m ² / g, more preferably 400-8000 m ² / g, even more preferably 500-700 m ² / g Surface area. This specific surface area is an important factor for the resin used in the present invention to efficiently separate the PPT and PPD series ginsenosides.

For the efficiency of PPT and PPD series ginsenosides to be separated, the resins used in the present invention need to have a suitable pore volume. According to a preferred embodiment of the present invention, the resin used in the present invention is preferably 1.0 to 2.0 ml / g, more preferably 1.0 to 1.8 ml / g, even more preferably 1.1 to 1.7 ml / g, Lt; RTI ID = 0.0 > ml / g. ≪ / RTI >

In addition, for the efficiency of separation of PPT and PPD series ginenoside, the styrene and benzene copolymer resins used in the present invention need to have a suitable pore radius. According to a preferred embodiment of the present invention, the resin used in the present invention has a thickness of 50-1000 A, more preferably 100-800 A, even more preferably 100-500 A, even more preferably 200-400 A Lt; / RTI >

According to a preferred embodiment of the present invention, ginseng or red ginseng extract is applied to a styrene and a benzene copolymer resin, and then water (for example, distilled water (distilled water) is added to remove the hydrophilic non-saponin component (glucose, sucrose, sorbitol, ) ≪ / RTI >

Step (c): to give PPD and PPT in series ginsenoside

The adsorbed ginsenosides are eluted to obtain PPT series ginsenosides and PPD series ginsenosides.

The elution may be carried out using various elution solvents, preferably using alcohol, more preferably methanol or ethanol (alcohol), most preferably ethanol (alcohol). The alcohol as an eluting solvent is preferably 10-95 v / v%, more preferably 20-90 v / v%, even more preferably 40-85 v / v%, even more preferably 50-80 v / v%, most preferably 70-80 v / v% alcohol.

The amount of the eluting solvent used is preferably 1-10 times, more preferably 1-6 times the resin volume. For example, when 70-80 v / v% of ethanol is used, when 1-3-fold ethanol is applied, the PPT series ginsenosides are first eluted, and then 4-6 times ethanol is applied to the PPD series ginsenoside .

According to a preferred embodiment of the present invention, the PPT family ginsenosides obtained in this elution process include Rg1, Re and Rf. According to a preferred embodiment of the present invention, the PPD series ginsenosides obtained in this elution process include Rb1, Rc, Rb2 and Rd.

Step (d): yield of Rg3 group ginsenoside Rg2 and ginsenoside group by decomposing sugar

Finally, sugar degradation of the PPD family ginsenosides or PPT family ginsenosides yields Rg2 group ginsenosides comprising Rg3 or Rg2 group ginsenosides comprising Rg2.

One of the features of the present invention is to degrade the sugar groups of the PPD and PPT family ginsenosides separated by the adsorption chromatography process described above to obtain the desired Rg3 group ginsenosides and Rg2 group ginsenosides.

The saccharide decomposition in the present invention can be carried out through various methods known in the art, but it is preferably carried out using a saccharide-hydrolyzing enzyme.

The sugar hydrolase used in the present invention is preferably a sugar hydrolytic enzyme derived from ginseng (or red ginseng). The ginseng-derived saccharide hydrolase used in the present invention can be prepared, for example, as follows. Ginseng can be obtained from ginseng itself by repeating the step of increasing and drying at 50-100 ° C.

By the conversion reaction, the PPD family ginsenosides form Rg3 group ginsenosides containing Rg3, and the PPT family ginsenosides form Rg2 group ginsenosides including Rg2.

According to a preferred embodiment of the present invention, step (d) is carried out in the presence of the extract of step (a) (preferably, the concentrate of the extract of step (a)). In this case, saponin hydrolase, i.e., glycosidase, contained in the extract of step (a) is involved in bioconversion of this step.

According to a preferred embodiment of the present invention, the saccharide decomposition process is performed, and the reaction product is applied to the resin used in step (b) and washed with water to remove the hydrophilic component (sugar). The Rg3 group jinnenoside bound to the resin or the Rg2 group jinnenoside is then eluted with a suitable eluting solvent (e. G., Ethanol) to finally obtain Rg3 group jinnenoside or Rg2 group jinnenoside.

According to a preferred embodiment of the present invention, the method further comprises a step of discoloring the saccharide-decomposed product or the hydrophilic component-removed product by applying the product to the anion exchange resin. In this case, the anion exchange resin used includes a variety of anion exchange resins known in the art, and is preferably a strongly basic anion exchange resin. In the anion exchange resin used in the present invention, a positively charged functional group is covalently bonded. Preferably, the functional group charged on the anion exchange resin is an amine or an amino group, more preferably a primary amine, a secondary amine, a tertiary amine or a quaternary amine, most preferably a quaternary amine.

According to a preferred embodiment of the present invention, before carrying out step (d), the PPT series ginsenosides or PPD series ginsenosides obtained in step (c) are pulverized and the PPT series ginsenoside powder is adjusted to pH 2.5 -5.0 acetic acid and PPD ginsenoside powder are dissolved in citric acid at pH 3.0-6.0 to prepare a solution. To the thus-prepared solution, a sugar hydrolytic enzyme (for example, a concentrated solution of the extract of step (a)) is added to conduct a bioconversion reaction.

The term " Rg3 group ginsenoside " as used herein means ginsenoside comprising Rg3 [20 (S) -Rg3 and 20 (R) -Rg3], Rk1 and Rg5. The term " Rg2 group ginsenoside " means a ginsenoside comprising Rg2 [20 (S) -Rg2 and 20 (R) -Rg2], Rg4 and Rg6.

According to a preferred embodiment of the present invention, the Rg3 group ginsenoside finally obtained by the process of the present invention is at least 80% by weight based on the final product.

According to a preferred embodiment of the present invention, the Rg3 group ginsenosides finally obtained by the process of the present invention comprise the total weight of Rg3 [20 (S) -Rg3 and 20 (R) -Rg3], Rk1 and Rg5 30 to 55 parts by weight (more preferably 30 to 50 parts by weight, still more preferably 30 to 45 parts by weight) of Rg3 [including 20 (S) -Rg3 and 20 (R) (More preferably 15-40 parts by weight, still more preferably 20-30 parts by weight) of Rk1 and 20-40 parts by weight (more preferably 25-40 parts by weight, even more preferably, 30-40 parts by weight).

According to a preferred embodiment of the present invention, the Rg2 group ginsenoside finally obtained by the process of the present invention is at least 80% by weight based on the final product, more preferably Rg2 [20 (S) -Rg2 and 25 to 50 parts by weight of Rg2 [20 (S) -Rg2 and 20 (R) -Rg2] based on 100 parts by weight of the total weight of Rg4 and Rg6, 20-45 (Even more preferably from 30 to 45 parts by weight, still more preferably from 35 to 45 parts by weight) and Rg6 from 10 to 25 parts by weight (even more preferably from 13 to 25 parts by weight, 15-20 parts by weight).

According to another aspect of the present invention, the present invention provides a method for producing a resin composition comprising 30 to 55 parts by weight of Rg3 [including 20 (S) -Rg3 and 20 (R) -Rg3] based on 100 parts by weight of the total weight of Rg3, Rk1 and Rg5, 10 to 30 parts by weight and Rg5 of 20 to 40 parts by weight as an active ingredient for preventing, ameliorating or treating metabolic diseases.

According to a preferred embodiment of the present invention, the active ingredient in the composition of the present invention contains Rg3 [including 20 (S) -Rg3 and 20 (R) -Rg3], Rg3 and Rg5, 20 (S) -Rg3 and 20 (R) -Rg3), 15-40 parts by weight of Rk1 and 25-40 parts by weight of Rg5, more preferably Rg3 [20 20 (R) -Rg3], 20-30 parts by weight of Rk1 and 30-40 parts by weight of Rg5.

Rg3 used in the composition of the present invention comprises 20 (S) -Rg3 and 20 (R) -Rg3, preferably a weight ratio of 20 (S) -Rg3: 20 (R) : 1, more preferably from 1: 0.8 to 0.8: 1, even more preferably from 1: 0.9 to 0.9: 1, even more preferably from 1: 0.95 to 0.95: Weight ratio.

The metabolic diseases to be prevented, ameliorated or treated by the present invention are preferably metabolites selected from the group consisting of diabetes, diabetic complications, obesity, hyperlipidemia, fatty liver, arteriosclerosis, nephropathy, hepatopathy, hypertension, stroke, heart failure and renal failure Disease.

The diabetic complications to be prevented, ameliorated or treated by the present invention are those selected from the group consisting of diabetic nephropathy, diabetic hepatopathy, hypertension, arteriosclerosis, stroke and heart failure.

According to a preferred embodiment of the present invention, Rg3 [including 20 (S) -Rg3 and 20 (R) -Rg3 as active ingredients], Rk1 and Rg5 are prepared by the method of the present invention described above.

Specifically, the effects of the composition according to the present invention on body weight, changes in ovarian fat mass, changes in ovarian adipocyte diameter and changes in abdominal fat cell diameter were examined. As a result, weight loss, (Fig. 1). As shown in Fig. 1, the ovarian adipocyte size reduction and abdominal adipocyte size reduction effect are excellent.

 In addition, the composition according to the present invention exhibits hypoglycemic activity, and thus can be useful for preventing or treating complications induced by diabetes or diabetes. Specifically, an experiment on the effect of the composition according to the present invention on blood glucose has shown that the blood glucose lowering effect is excellent, so that it can be effectively used for preventing or treating diabetes or diabetic complications (see Example 2) .

The composition according to the present invention exhibits antihyperlipidemic activity and thus can be usefully used for the prevention or treatment of complications caused by antihyperlipidemia, diabetes or diabetes. Specifically, the present inventors conducted an experiment to measure changes in blood total cholesterol and triglyceride content of blood, changes in low density lipoprotein (LDL) and high density lipoprotein (HDL) content in blood, and found that total cholesterol (Eg, Example 3), which is effective for preventing or treating anti-hyperlipemia, diabetes, or diabetic complications because it has a low content, a low triglyceride content in blood, a low LDL content in blood, and an HDL content increase effect.

Furthermore, the composition according to the present invention exhibits liver-protecting activity and thus can be effectively used for preventing or treating complications arising from hepatopathy, diabetes or diabetes. Specifically, the present inventors conducted an experiment to measure changes in liver weight, blood aspartame aminotransferase (AST) and alanine aminotransferase (ALT) content of the composition according to the present invention. As a result, liver weight gain, blood AST And the effect of decreasing the ALT content of the blood is excellent, so that it can be usefully used for preventing or treating hepatopathy, diabetes or diabetic complication (see Example 4).

In addition, the composition according to the present invention exhibits a kidney-protective activity and thus can be useful for preventing or treating complications arising from nephropathy, diabetes or diabetes. Specifically, the present inventors conducted experiments to measure changes in blood urea nitrogen (BUN) and creatinine content and changes in the number of denatured tubular cells of the composition according to the present invention. As a result, the blood BUN content, the blood creatinine content, And thus can be effectively used for preventing or treating nephropathy, diabetes or diabetic complication (see Example 5).

The compositions of the present invention may be prepared with pharmaceutical compositions and food compositions.

When the composition of the present invention is manufactured from a pharmaceutical composition, the composition of the present invention includes a pharmaceutically acceptable carrier. Such pharmaceutically acceptable carriers are those conventionally used in the field of manufacture and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, But are not limited to, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations include, but are not limited to, Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention can be administered orally or parenterally. In the case of parenteral administration, the composition can be administered by intravenous injection, subcutaneous injection, muscle injection, intraperitoneal injection, transdermal administration, etc., .

The appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate and responsiveness of the patient, Usually, a skilled physician can readily determine and prescribe dosages effective for the desired treatment or prophylaxis. According to a preferred embodiment of the present invention, the daily dosage of the pharmaceutical composition of the present invention is 0.001-10000 mg / kg.

The pharmaceutical composition of the present invention may be formulated into a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of excipients, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

The composition of the present invention can also be manufactured into a food composition. The food composition of the present invention may contain, in addition to the active ingredient, components that are ordinarily added in the manufacture of food, for example, proteins, carbohydrates, fats, nutrients, seasonings and flavors. Examples of the above-mentioned carbohydrates are monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, oligosaccharides and the like; And polysaccharides such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. Natural flavorings such as tau martin and stevia extract (e.g., rebaudioside A and glycyrrhizin) and synthetic flavorings (saccharine, aspartame, etc.) can be used as flavorings. For example, when the food composition of the present invention is prepared as a drink, it may further contain citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, juice, mulberry extract, jujube extract, licorice extract, have.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a method for preparing Rg3 and Rg2 group ginsenosides, which are most optimized in terms of simplicity and efficiency.

(b) According to the present invention, at least 80% by weight of Rg3 group jinnenoside and Rg2 group jinnenoside can be obtained.

(c) The process of the present invention is well suited for mass production of industrial scale.

(d) The Rg3 group ginsenosides prepared by the present invention are highly suitable for the prevention or treatment of metabolic diseases, particularly obesity, diabetes and diabetic complications, and exert excellent in vivo efficacy.

(e) The Rg3 group ginsenoside produced by the present invention exerts excellent in vivo efficacy against various diabetic complications, and is very effective in alleviating the symptoms of diabetic patients in the early, middle, and late stages of diabetes.

Figure 1 shows the HPLC (high performance liquid chromatography) analysis results for the Rg3 group ginsenosides prepared by the present invention.
Figure 2 shows the HPLC analysis of Rg2 group ginsenosides prepared by the present invention.
Figure 3 shows the results of body weight change of obese mice orally administered with Rg3 group or CLA prepared by the present invention for 28 days.
Fig. 4 shows the results of serum leptin levels in obese mice orally administered with Rg3 group or CLA prepared according to the present invention for 28 days.
Figure 5 shows the results of serum adiponectin levels in obese mice orally administered for 28 days with Rg3 group or CLA produced by the present invention.
6A is a representative photograph of H & E staining for observing histologic changes in the epididymal fat layer of an obese mouse administered orally for 28 days with Rg3 group or CLA prepared by the present invention.
FIG. 6B is a graph showing the average diameter of adipocytes in the epididymal fat layer of an obesity mouse administered orally for 28 days with Rg3 group or CLA prepared by the present invention. FIG.
FIG. 7A is a representative photograph of H & E staining for observing histological changes in abdominal wall fat layer of obese mice administered orally for 28 days with Rg3 group or CLA prepared according to the present invention.
FIG. 7B is a graph showing the thickness of the abdominal fat layer of an obese mouse administered orally for 28 days with Rg3 group or CLA prepared by the present invention. FIG.
8A is a representative photograph of H & E staining for observation of histological changes in the pancreas of an obese mouse administered orally for 28 days with Rg3 group or CLA prepared according to the present invention.
FIG. 8B is a graph showing the ratio of the pancreatic enzyme raw granules to the Rg3 group or CLA produced by the present invention in obese mice orally administered for 28 days.
FIG. 8C is a graph showing the average number and percentage of pancreatic islets of obese mice administered orally for 28 days with the Rg3 group or CLA prepared according to the present invention. FIG.
9 is a graph showing insulin levels in plasma of obese mice orally administered with Rg3 group or CLA prepared by the present invention for 28 days.
FIG. 10A is a representative photograph of H & E staining for observation of insulin-immunoreactive cells (IR) histologic changes in the pancreas of an obese mouse administered orally for 28 days with Rg3 group or CLA prepared according to the present invention.
FIG. 10B is a graph showing the average number of insulin-immunoreactive cells (IR) in the pancreas of an obese mouse administered orally for 28 days with Rg3 group or CLA prepared by the present invention.
FIG. 11A is a representative photograph of H & E staining for observation of glucagon-immunoreactive cells (IR) histologic changes in the pancreas of an obese mouse administered orally for 28 days with Rg3 group or CLA prepared by the present invention.
FIG. 11B is a graph showing the average number of glucagon-immunoreactive cells (IRs) in the pancreas of obese mice orally administered with Rg3 group or CLA produced by the present invention for 28 days.
FIG. 12 is a graph showing the ratio of insulin / glucagon-immunoreactive cells (IR) of the pancreas of an obese mouse administered orally for 28 days to Rg3 group or CLA produced by the present invention.
FIG. 13A is a representative photograph of H & E staining for observation of histological changes in obese mice in which Rg3 group or CLA prepared according to the present invention was orally administered for 28 days.
FIG. 13B is a graph showing the proportion of hepatic hepatitis in liver of obese mice orally administered with Rg3 group or CLA orally for 28 days and the mean diameter of hepatocytes prepared by the present invention.
14A is a representative photograph of H & E staining for observation of histological changes in kidney of an obese mouse administered orally for 28 days with Rg3 group or CLA prepared according to the present invention.
FIG. 14B is a graph showing the number of abnormal glomeruli and the percentage of epilepsy in the kidney of an obese mouse administered orally for 28 days with Rg3 group or CLA prepared by the present invention. FIG.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Throughout this specification, "%" used to denote the concentration of a particular substance is intended to include solids / solids (wt / wt), solid / liquid (wt / The liquid / liquid is (vol / vol)%.

Preparation Example: Preparation of Rg3 group and Rg2 group

After washing ginseng, the ginseng saponin-decomposing enzyme contained in ginseng roots was used to control the temperature and time of roasting in the roasted roast, and then red ginseng was produced in a dryer (moisture content less than 15%) to produce red ginseng Respectively. Subsequently, 75% ethanol (6 times by weight) was added to the weight of red ginseng raw material, and the mixture was heated and circulated at 60 ° C for 8 hours (primary extract). The above primary extract was further fed with 70% 5 times the amount of the alcohol, followed by heating and circulating extraction at 70 ° C for 8 hours to obtain a secondary extract. The primary extract and the secondary extract were combined, filtered at 5 탆 and concentrated to 60 Brix at 45 ° C or lower. Next, for the third extraction, 6 times of distilled water was added to the concentrate, followed by extraction at 85 ° C for 8 hours, filtration, and concentration at 60 Brix.

The concentrate obtained by combining the primary extract and the secondary extract was diluted to 5-10 Brix, and then a mixture of styrene and divinyl benzene (DVB) having a specific surface area of 600 m ² / g, a pore volume of 1.3 ml / g and a pore radius of 200-300 Å Saponin was adsorbed on a resin made of a copolymer (Sigma-Aldrich) and then washed with 6 times of distilled water to remove non-saponin components as hydrophilic substances (glucose, sucrose, sorbitol, minerals, vitamins, organic acids and proteins) Respectively. Then, as the elution solvent, the ratio of the solvent (95%): distilled water volume (v / v) 18:82, 22:78, 26:74, 32:68, 37:63, 41:59 and 100: (PPT) series of ginsenosides were separated and the ginsenosides of the paraxanthiol (PPD) series were sequentially separated (see Table 1).

The results of ginsenoside separation by the above adsorption chromatography are shown in the following Table 1:

division Volume ratio (vol / vol) of ethanol: distilled water used - Solvent condition Gin Senocide 18:82 22:78 26:74 32:68 37:63 41:59 Rg1 2.4 20.1 12.5 1.9 0 0 Re 1.5 32.4 20 1.1 0 0 Rf 0 0 2.8 3.5 3 2.5 Total tree 3.9 52.5 35.3 6.5 3 2.5 Total triol weight% 99.9 93.7 39.8 7.7 4.5 3.2 Rb1 0 3.5 26.1 29.3 19.4 9.6 Rc 0 0 12.5 20.1 20.5 14.7 Rb2 0 0 11.2 17.5 18.2 13.4 Rd 0 0 3.6 9.5 18.3 36.9 Total Dior 0 3.5 53.4 76.4 76.4 74.6 Total diol weight% 0 6.3 60.2 92.2 95.4 96.8 Sum 3.9 56 88.7 82.9 80.1 77.1

The separated PPD solution and PPT solution were concentrated under reduced pressure and vacuum dried to be powdered.

To prepare the Rg3 group, a 5 wt% solution of the powdered raw PPD was prepared using citric acid (pH 5.0), and the same amount of the third extract (including ginseng saponin degrading enzyme) was added and the mixture was heated at 50 ° C for 10 hours Lt; / RTI > Then, the digestion enzyme was inactivated by heating at 100 DEG C for 1 hour and then cooled.

In order to prepare the Rg2 group, 5 wt% solution of the powdered raw PPT was prepared using acetic acid (pH 4.0), and the same amount of the third extract (including ginsenoside decomposing enzyme) was added thereto, And reacted for 10 hours. Then, the digestion enzyme was inactivated by heating at 100 DEG C for 1 hour, followed by cooling, and the protein precipitate was separated.

The reaction product was adsorbed on a resin composed of a copolymer of styrene and DVB (divinylbenzene), and water-soluble substance (sugar) and non-saponin component were removed with 6-8 times volume of distilled water. Then, ginsenoside was extracted from the resin by passing 5-6 times 75% ethanol of the volume through the resin, and then decolorized by passing through an anion exchange decoloring resin (Autran Biotech) with a quaternary amine group. Then, concentrated, dried and pulverized to, Rg3; a product comprising _{(C 42 H 72 O 13 785.01} ) to about 35% by weight including at least and Rg3 group (Rg3, Rk1 and Rg5) to about 90% by weight or more . The Rg3 group is derived from the PPD powder. Also, Rg2; for _{(C 42 H 72 O 13 784.94} ) a, Rg2 group (Rg2, Rg4 and Rg6) and containing at least 45% by weight to prepare a product containing at least 85% by weight. The Rg2 group is derived from the PPT powder.

The results of HPLC (high performance liquid chromatography, Waters 2695 Separation Module with Waters 2996 Photodiode Array Detector) analysis for the Rg3 group prepared in the present invention are shown in FIG. 1 and Table 2.

Gin Seno side name Retention time (minutes) area % area 20 (S) -Rg3 83.441 4715269 20.91 20 (R) -Rg3 83.871 4928972 21.86 Rk1 87.948 4907312 21.76 Rg5 88.381 7998208 35.47

As can be seen from FIG. 1 and Table 2, the present invention provides an Rg3 group (Rg3 group) having about 43 weight% of Rg3 [including 20 (S) -Rg3 and 20 (R) -Rg3], about 22 weight% of Rk1 and about 35 weight% of Rg5 Jinsei laborers were able to get these.

The results of HPLC analysis of the Rg2 group prepared in the present invention are shown in FIG. As can be seen in FIG. 2, the present invention provides the Rg2 group of the present invention comprising 20 ( S ) -Rg2 and about 45% by weight of 20 ( R ) -Rg2, about 17% by weight of Rg6 and about 38% by weight of Rg4. there was.

Experimental Example

Materials and Experiments

Rg3  group

Samples containing 90% by weight of the Rg3 group prepared in the above Preparation Example and containing 43% by weight of Rg3, 22% by weight of Rk1 and 35% by weight of Rg5 were used for the efficacy demonstration.

Experimental animals and breeding

Five 5-week old C57BL / 6JJms mice (SLC, Japan) and 5 male genetically obese 5-week-old C57BL / 6JHam-ob / ob mice (C57BL / 6J strain) (SLC, Japan) Were used for the experiment for 28 days. The animals were divided into five polycarbonate cages and kept in a room maintained at a temperature of 20-25 ° C and a humidity of 40-45%. Light and dark cycles were provided for 12 hours, and rodents' general diets (Samyang, Korea) and water were unlimited.

Experimental group (5 per group)

1. Normal control group: normal mice (C57BL / 6JJms), medium administration

2. obesity control (ob / ob): Obese mice (C57BL / 6JHamSlc-ob / ob)

3. CLA: Obesity mouse (C57BL / 6JHamSlc-ob / ob), conjugated linoleic acid 750 mg / kg / day

4. Rg3 group-10: obesity mouse (C57BL / 6JHamSlc-ob / ob), Rg3 group 10 mg / kg / day

5. Rg3 group-50: Obesity mouse (C57BL / 6JHamSlc-ob / ob), Rg3 group 50 mg / kg / day

6. Rg3 group-100: Obesity mouse (C57BL / 6JHamSlc-ob / ob), Rg3 group 100 mg / kg /

Test material preparation and administration

The Rg3 group was provided in Arimed Therapeutics (Arimed Therapeutics, Korea) in the form of a pale yellow powder, and CLA was provided in RexGene Biotech Co., Korea in the form of a capsule containing a pale pink solution. The Rg3 group was stored at 4 ℃ in the refrigerator. The CLA was kept in the dryer until use to protect it from light and moisture. After 4 weeks of adaptation, all test materials were dissolved or suspended in distilled water for 28 days and then orally injected once daily into the oral cavity using a sonde in a 1 ml syringe. Rg3 group 10, 50, 100 mg / kg and CLA 750 mg / kg were administered at a dose of 10 ml / kg using distilled water as the medium. In the normal control and obesity control groups, only the same amount of distilled water was administered orally instead of the test substance (Table 3).

Experimental composition group animal Dose (mg / kg / day) Treated drug Control group Normal C57BL / 6JJms 10 ml / kg Medium (sterile distilled water) Obesity C57BL / 6JHam-ob / ob 10 ml / kg Medium (sterile distilled water) Reference group Obesity C57BL / 6JHam-ob / ob 750 ml / kg CLA Working group Obesity C57BL / 6JHam-ob / ob 10 ml / kg Rg3 Group Obesity C57BL / 6JHam-ob / ob 50 ml / kg Rg3 Group Obesity C57BL / 6JHam-ob / ob 100 ml / kg Rg3 Group

Rg3 Group: Ginsenoside Rg3 Group

CLA: conjugated linoleic acid, reference group

The test substances were orally administered once a day at a dose of 10 ml / kg to distilled water for 28 days after 4 weeks of compliance.

Test substance

Material name: Ginsenoside Rg3 group (Rg3 group prepared in the above production example)

Arrival day: July 4, 2011

Approved test substance Dose: 10, 50, 100 mg / kg

Formulation: Light yellow powder

Solubility: Dissolve distilled water in medium at 80 ° C to 10 mg / ml.

Storage: Cold storage at 4 ℃

Reference substance

Material name: Conjugated Linoleic Acid (CLA)

Arrival day: July 4, 2011

Approved test substance dose: 750 mg / kg based on previous studies (Hue et al., 2009)

Source: RexGene Biotech Co. Ltd. (Chungbuk, Korea)

Formulation: Pale pink solution

Solubility: Suspended in white to 75 mg / ml with distilled water as a medium.

Storage: Store at room temperature until use

Weight change

Changes in body weight were measured using an automatic electric balance (Precisa Instrument, Switzland) once daily for 28 days from the day before the start of the administration. All animals fasted overnight (except for water, about 12 hours) to reduce the differences that could be caused by the feed at the beginning and end of the dose. The weight gain was calculated as follows:

Formula 1. Weight gain (g):

= Weight at the end of the dose - weight at the beginning of the dose (test substance administered from 0 to 28 days)

Average Daily Feed Intake Measurements

Each cage was fed with 150 g of feed, and the amount of feed that was provided to the automatic electrical balance after 24 hours was measured. The difference between the feed amount and the measured feed amount was considered as the average daily feed intake (g / day / day) of each mouse as follows:

Formula 2. Average Daily Feed Intake (g / day / grains):

= {Amount of feed provided (150 g) - feed amount measured after 24 hours}

These measurements were performed on days 1, 7, 14, 21, 25, and 27, respectively.

Long-term weighing

To determine the weight (g) of the liver, pancreas, kidney, and epididymal fat layer after the end of the experiment and to reduce the difference from individual tea, the relative weight (% body weight) was calculated using weight and absolute weight at the end of the experiment as follows:

Formula 3. Relative Organ Weight (%)

= {(Absolute long-term weight / weight at the end of the experiment) × 100}

Blood glucose level measurement

On the 28th day after the administration, blood is collected from the vena cava and stored in a NAF glucose vesicle tube (Becton Dickinson, USA) and separated from the plasma. Blood glucose levels were measured using an automated blood analyzer (Toshiba 200 FR, Japan).

Plasma Insulin Level Measurement

To measure plasma insulin levels, blood is drawn from the vena cava at the end of the experiment and placed in a heparin-pristled vial tube (Becton Dickinson, New York, NJ, USA) for plasmapheresis. Plasma insulin levels were analyzed by enzyme-linked immunosorbent assay (ELISA, Boerhringer Mannheim, Mannheim, Germany) as in previous studies (Kim et al., 2007).

Measurement of serum biochemical indicators

At the end of the experiment, approximately 1 ml of venous blood is collected from the vena cava under anesthesia. All blood samples are centrifuged at 15,000 rpm for 10 minutes at room temperature using a clotted activated serum tube. Then, the values of AST, ALT, BUN, creatinine, TC, FFA and triglyceride in the serum are measured using an automatic blood analyzer (AU400, Olympus, Japan).

Serum Leptin and Adiponectin  Numerical measurement

Serum was isolated from the collected blood by the usual method for the measurement of serum adiponectin levels. Levels of serum leptin (Linco Research, USA) and adiponectin (Otsuka Pharm., Japan) were measured using a commercially available ELISA kit as in the previous study (Sahai et al., 2004).

Histopathology

After long-term weighing, the dorsal abdominal fat layer in the left lobe of the liver, the left kidney, the splenic lobe of the pancreas, the epididymal fat layer and the muscularis quadratus lumborum were collected. Samples taken are fixed in 10% neutral buffered formalin. After paraffin embedding, successive sections are made to 3-4 μm thickness and representative sections are stained with hematoxylin-eosin (H & E) for optical microscopy. Histopathologic features of each organs were then observed.

Measurement of tissue morphology

Based on previous studies analyzing the morphometry of metabolic disease mice (Jung et al., 2011) or diabetic rats (Kim et al., 2006, 2007), the prevalence of hepatitis, hepatocellular hypertrophy, vasodilatory glomeruli, Edema, adipocytosis and hyperplasia, percentage of the area occupied by pancreatic enzyme source granules, number of pancreatic islands and percent occupation were measured as follows.

Percentage of fatty acid changes in the fatty liver of the liver (percentage of the fatty acid changes in the fatty tissues of the liver ): Using the automatic image analysis process (DMI; Daegu, KOREA) ² ).

Hepatocyte hypertrophy (mean diameter of hepatocytes): Using an automated image analysis process, the mean diameter of the hepatocytes was calculated in micrometers within a defined compartment visible on a computer monitor; At least 10 hepatocytes per liver tissue were analyzed.

Number of atrophied glomeruli (average number of capillary dilated atrophic glomeruli): Using an automated image analysis process, the average number of vasodilated glomeruli among 100 glomeruli (number / 100 glomeruli; one region per sample) was calculated.

Adipocyte hyperplasia (mean diameter of adipocytes): Using an automated image analysis process, the mean diameter of the epididymal tissue and dorsal abdominal white adipocytes was calculated as μm in a defined compartment visible on a computer monitor; At least 10 fat cells per fat layer were analyzed.

Thickness of the accumulated dorsal fat layer : The thickness of the accumulated dorsal fat layer was calculated in mm using an automatic image analysis process.

Percentage of the area occupied by the pancreatic enzyme source granules : Using an automated image analysis process, the average of the area occupied by the enzyme source granules was calculated as the percentage between one compartment of the pancreas (% pancreatic tissue / mm ² ).

Pancreatic islet number: using the automated image analysis process, and calculates the average number of pancreatic islands in the appropriate portion of the pancreas (the number of pancreatic parenchyma / 10 mm ^2).

Percentage of pancreatic islands occupied : Using an automated image analysis process, the average of the area occupied by the pancreatic islets was calculated as the percentage between the appropriate compartment of the pancreas (% of pancreatic tissue / 10 mm ² ).

In this analysis, histopathologists analyzed the distribution of blind treated groups.

Immunohistochemistry

Portions of tissue from other serial sections were stained with the immunohistochemical avidin-biotin-peroxidase (ABC) method (Edwin and Leigh, 1999). (DiaSorin, MN, USA, dilution: 1: 2,000) or rabbit polyclonal glucagon (DiaSorin, MN, USA, dilution: 1: 2,000) antiserum was used at this time and the steps were as follows:

1. The sections are incubated in methanol containing 0.3% H ₂ O ₂ at room temperature for 30 minutes to block the activity of endogenous peroxidase.

2. Rinse 3 times with 0.01 M phosphate buffered saline (PBS; pH 7.2).

3. The sections are treated with a normal HoleSerum blotting solution (Vector Lab. Inc., CA, USA, Dilution 1: 100) and blocked for 1 hour at room temperature in a hygrostat to block non-specific binding of immunoglobulin.

4. Wash three times with 0.01M PBS.

5. Treat the sections with primary insulin and glucagon antisera and place them overnight at 4 ° C in a humidifier.

6. Wash three times with 0.01 M PBS.

7. Treat the sections with biotinylated universal secondary anti-bodies (Vector Lab. Inc., CA, USA, Dilution 1:50) and incubate for 1 hour at room temperature.

8. Wash three times with 0.01 M PBS.

9. The sections are treated with Vectastain Elite ABC Kit (Vector Lab. Inc., CA, USA, Dilution 1:50) and incubated for 1 hour at room temperature.

10. Wash three times with 0.01M PBS.

11. The sections are treated with peroxidase substrate kit (Vector Lab. Inc., CA) and incubated at room temperature for 5 minutes.

12. Wash three times with 0.01M PBS.

13. Stain contrast with Meyer hematoxylin solution.

Measurement of tissue morphology

Based on previous studies analyzing histological morphology of endocrine cells in normal ddN mice (Lee et al., 2010) or diabetic rats (Kim et al., 2006, 2007), the immunoreactivity of insulin and glucagon, Of the cells were considered as positive immunoreactive (IR) cells. And the number of insulin- and glucagon-immunoreactive cells (IRs) in the ratio of these (insulin / glucagon-IR cells) were measured as follows.

Number of Insulin- and Glucagon IR Cells : Using an automated image analysis process, the average number of insulin- and glucagon-IR cells was measured in a defined compartment (mm ² of pancreatic tissue) visible on a computer monitor.

Insulin / glucagon- IR cells : Using the average number of insulin- and glucagon-IR cells measured in the same area of serial sections, their ratios were calculated as follows.

Formula 4. Insulin / glucagon-IR cells (ratio)

= (Mean number of insulin-IR cells / average number of glucagon-IR cells)

In this analysis, histopathologists analyzed the distribution of blind treated groups.

Statistical analysis

All values are expressed as mean ± standard deviation (SD) for 5 dogs. Multiple comparative assays were performed for different dose groups. Leven (1981) was used for variance homogeneity. If the Levine test results show a deviation without any validity from the homogeneity of the change, the result is a one-way ANOVA test to determine which pair of group comparisons have changed significantly and to determine the least significant difference (LSD) Multiple-comparison tests were conducted and analyzed. When valid deviations from variance homogeneity were found in the Levin test, a non-parametric comparison test and a Kruskal-Wallis test were performed. When valid differences were observed in the Kruskal-Wallis test, the Whitney U-Wilcoxon Rank is W test was performed only to determine the specific pair of group comparisons. Statistical analysis was performed using SPSS for Windows (Release 14.0K, SPSS Inc., USA) (Ludbrook, 1997). And the percent change compared to the percentage of obesity control was calculated to help understand the efficacy of the test substance and the percentage change between obesity control and normal control was calculated as follows to observe induction status:

Formula 5. Percent change compared to normal control (%)

= [((Obesity control data - normal control data) / normal control data) 100]

Formula 6. Percent change compared to obesity control (%) (%)

= [((Data of administration groups - data of obesity control group) / data of obesity control group] x 100]

Experiment result

Impact on obesity

Effect on weight change

Before the start of the experiment, significant increase (p <0.01) in body weight was observed in all obese mouse experimental groups compared to the normal control group. In the CLA, Rg3 group 10 and 50 mg / kg administration groups, Body weight decreased significantly (p <0.01 or p <0.05) compared to obesity control group and 28 days body weight gain was also significantly decreased (p <0.01) compared to obese control group. On the other hand, the 100 mg / kg Rg3 group did not show any significant difference compared to the obesity control group. However, the significant decrease in body weight began to appear from the 16th day of administration, and the 28-day body weight gain was also significantly decreased (p <0.01) (Table 4 and Figure 3).

Weight gain in obese mice orally administered with Rg3 group or CLA for 28 days group Weight (g) Weight gain
[B] - [A] Start dosing End of dose Control group Normal group 21.02 + - 0.54 23.62 ± 0.81 2.60 ± 0.40 Obesity (ob / ob) 39.76 + 4.26 ^a 48.26 ± 3.80 ^b 8.50 ± 1.48 ^b Reference group (CLA) 39.76 ± 3.02 ^a 39.88 ± 3.10 ^bc 0.12 ± 1.71 ^bc Rg3 group treated group 10 mg / kg 36.88 + 4.26 ^a 39.82 ± 5.84 ^bd 2.94 ± 2.05 ^c 50 mg / kg 39.52 ± 3.74 ^a 40.72 ± 3.96 ^bd 1.20 ± 0.91 ^c 100 mg / kg 40.12 + - 6.25 ^a 41.66 + 4.98 ^b 1.54 + - 2.81 ^c

Values are mean ± SD of experiments for 5 animals.

Rg3 Group: Ginsenoside Rg3 Group

CLA: conjugated linoleic acid, reference group

All animals were fasted overnight at the start and end of treatment.

The statistical significance of ^a p was p <0.01 and compared with the normal group value by LDS test.

^b p was statistically significant at p <0.01 and compared with normal group by MW test.

The statistical significance level of ^c p was p <0.01 and the statistical significance level of ^d p was p <0.05 and compared with obesity group (ob / ob) by MW test.

The body weight gain was 226.92% during the 28 days of the obesity control group compared with that of the normal control group, and the obesity group treated with CLA, Rg3 group 10, 50 and 100 mg / kg showed -98.59, -65.41, -85.88 and -81.88%, respectively.

Changes in feed intake

There was a significant (p <0.01) increase in feed intake in all obese mouse experimental groups compared to the normal control group. However, in addition to the CLA group, there was also a meaningless change in the feed intake incidence as compared to the obesity control group, with a decrease in mean daily feed intake (p <0.05) compared to the obese control group on days 14, 21, 25 and 27 (Table 5).

The average daily feed intake of obese mice administered orally with Rg3 group or CLA for 28 days group The measured feed intakes (g / day / day) 1 day 7 days 14 days 21st 25th 27th Control group Normal group 5.10 + - 0.67 4.42 0.38 4.42 0.38 4.64 0.11 4.54 ± 0.32 4.40 + - 0.48 Obesity group
(ob / ob) 7.16 ± 0.30 ^a 6.64 + - 0.43 ^a 6.74 + 0.13 ^a 6.98 ± 0.39 ^a 6.68 + 0.33 ^a 6.46 + 0.43 ^a Reference group
(CLA) 7.12 + - 0.36 ^a 6.42 ± 0.36 ^a 5.00 ± 1.17 ^b 5.44 ± 0.50 ^b 4.26 ± 0.53 ^b 4.12 ± 0.93 ^b Rg3 group treated group 10 mg / kg 6.66 ± 0.76 ^a 6.72 ± 0.69 ^a 6.66 ± 0.69 ^a 6.90 ± 0.86 ^a 6.60 + - 0.42 ^a 6.28 ± 0.48 ^a 50 mg / kg 6.96 ± 0.58 ^a 6.84 ± 0.22 ^a 6.78 ± 0.48 ^a 6.84 ± 0.68 ^a 6.58 ± 0.38 ^a 6.52 + 0.33 ^a 100 mg / kg 7.22 + 0.40 ^a 6.68 ± 0.52 ^a 6.62 ± 0.58 ^a 6.84 ± 0.80 ^a 6.56 ± 0.30 ^a 6.48 + 0.70 ^a

Values are mean ± SD of experiments for 5 animals.

Rg3 Group: Ginsenoside Rg3 Group

CLA: conjugated linoleic acid, reference group

The statistical significance of ^a p was p <0.01 and compared with the normal group value by LDS test.

^b p was statistically significant (p <0.01) and compared with obesity group (ob / ob) by LDS test.

After 14 days of feeding, the feed intake was 52.49% higher in the obese control group than in the normal control group. In the CLA, Rg3 group 10, 50 and 100 mg / kg group, the feed intake was -25.82, -1.19 and 0.59 And -1.78%, respectively.

Feed intake after 27 days of treatment was 46.82% higher in the obese control group compared to the control group, and -36.22, -2.71, and 0.93, respectively, in the CLA, Rg3 group 10, 50 and 100 mg / And 0.31%, respectively.

Changes in epididymal fat mass

In the obese control group, relative and absolute weight of the epididymal fat was significantly increased (p <0.01) compared to the normal control group, but the decrease in fat weight was significantly (p <0.01) (Table 6 and Table 7).

Absolute organ weights of obese mice administered orally with Rg3 group or CLA for 28 days group Absolute long-term weight (g) liver kidney Epididymis Pancreas Control group Normal group 0.915 0.028 0.133 ± 0.009 0.080 0.023 0.116 + 0.013 Obesity (ob / ob) 3.605 ± 0.475 ^a 0.223 ± 0.008 ^c 1.117 ± 0.292 ^a 0.136 + 0.040 Reference group (CLA) 3.163 + 0.565 ^a 0.171 ± 0.023 ^de 0.624 ± 0.133 ^ab 0.111 + 0.027 Rg3 group treated group 10 mg / kg 2.193 ± 0.728 ^ab 0.195 + 0.012 ^ce 0.581 ± 0.105 ^ab 0.139 + 0.013 50 mg / kg 2.092 ± 0.480 ^ab 0.178 ± 0.016 ^ce 0.552 + 0.078 ^ab 0.125 + 0.023 100 mg / kg 2.156 ± 0.224 ^ab 0.187 ± 0.012 ^ce 0.572 ± 0.107 ^ab 0.143 + 0.029

Values are mean ± SD of experiments for 5 animals.

Rg3 Group: Ginsenoside Rg3 Group

CLA: conjugated linoleic acid, reference group

The statistical significance of ^a p was p <0.01 and compared with the normal group value by LDS test.

^b p was statistically significant (p <0.01) and compared with obesity group (ob / ob) by LDS test.

The statistical significance level of ^c p was p <0.01 and the statistical significance level of ^d p was p <0.05 and compared with the normal group value by MW test.

The statistical significance of ^e p was p <0.01 and compared with the obesity group (ob / ob) by MW test.

Relative organ weights of obese mice administered orally with Rg3 group or CLA for 28 days group Relative organ weight (% body weight) liver kidney Epididymis Pancreas Control group Normal group 3.873 + 0.030 0.564 + 0.030 0.335 + 0.084 0.489 + 0.048 Obesity (ob / ob) 7.458 ± 0.593 ^a 0.463 ± 0.034 ^d 2.307 ± 0.504 ^a 0.284 + 0.094 ^a Reference group (CLA) 7.956 ± 1.407 ^a 0.428 + 0.048 ^d 1.555 + - 0.235 ^ac 0.275 ± 0.056 ^a Rg3 group treated group 10 mg / kg 5.428 ± 1.099 ^bc 0.501 + - 0.100 1.465 + 0.231 ^ac 0.359 + 0.082 ^b 50 mg / kg 5.119 ± 0.952 ^bc 0.441 0.061 ^e 1.355 + 0.135 ^ac 0.309 + 0.064 ^a 100 mg / kg 5.217 ± 0.689 ^bc 0.455 ± 0.058 ^e 1.401 ± 0.360 ^ac 0.351 + 0.101 ^a

Values are mean ± SD of experiments for 5 animals.

Rg3 Group: Ginsenoside Rg3 Group

CLA: conjugated linoleic acid, reference group

statistical significance of p is ^a statistical significance of p <0.01 and p ^b is p <0.05, and were compared with control group value by the LSD test.

The statistical significance of ^c p was p <0.01 and compared with obesity group (ob / ob) value by LSD test.

The statistical significance level of ^d p was p <0.01 and the statistical significance level of ^e p was p <0.05 and compared with the normal group value by MW test.

The absolute weight of epididymal fat was 1303.52% in the obese control group, and -44.15, -47.99 and -50.57 in the CLA, Rg3 group, 10, 50 and 100 mg / And -48.80%, respectively.

The relative weight of epididymal fat was 589.34% in the obese control group and -32.63, -36.50 and -41.26% in the CLA, Rg3 group, 10, 50 and 100 mg / And -39.29%, respectively.

Serum Leptin  Change in content

In the obesity control group, serum leptin levels were significantly decreased (p <0.01) as compared with the normal control group, but the blood leptin levels were similar to those of the obesity control group in CLA and all three dose groups of Rg3 group ).

Serum leptin levels were -85.10% in the obese control group and 9.86, 2.70, -3.50 and 2.86% in the CLA, Rg3 group, 10, 50 and 100 mg / Change.

Serum Adiponectin  Change in content

In the obesity control group, adiponectin content was significantly decreased (p <0.01) compared to the normal control group. However, in all the Rg3 group treated group, there was a significant (p <0.01) increase in the serum adiponectin content . On the other hand, the CLA-treated group showed a similar change in serum adiponectin content as in the obesity control (Fig. 5).

The levels of adiponectin in the serum were -59.84% in the obese control group and -3.14, 35.18, 73.14 and 52.84% in the CLA, Rg3 group, 10, 50 and 100 mg / Change.

Fat cell Diameter  change

In the obesity control group, significant adipocyte hypertrophy was observed, and the increase in the diameter of the epididymal and abdominal fat cells was significant (p <0.01) as compared with the normal control group. However, in all of the Rg3 group and the CLA group, (P < 0.01) epididymal and abdominal fat adipocyte diameters (Figs. 6 and 7), respectively.

The lipid peroxidase-induced adipocyte diameter was 262.37% in the obese control group, but -32.49, -37.67, -49.17, and -38.0 in the CLA, Rg3 group 10, 50 and 100 mg / And 43.04%, respectively.

In the CLA, Rg3 group, 10, 50 and 100 mg / kg group, the abdominal fat cell diameter was -22.25, -46.10, -51.19 and - 47.65%, respectively.

Changes in abdominal fat thickness

In the obesity control group, accumulation of the adipose tissue was recognized and the accumulation of abdominal fat thickness was significantly increased (p <0.01) compared to the normal control group. However, the accumulation of abdominal fat thickness was significantly increased (p <0.01) And a decrease in fat thickness was respectively recognized (Fig. 7).

The cumulative abdominal fat thickness was 385.09% in the obese control group and -24.39, -36.99, -42.97, and -36.99 in the CLA, Rg3 group, 10, 50 and 100 mg / -41.26%, respectively.

Enzyme source granule  Change in content

In the obese control group, the secretion of the enzyme granules from the exocrine glands was increased, and the ratio of the enzyme granules in the exocrine pancreas was significantly decreased (p <0.01) (P < 0.01 or p < 0.05) in the Rg3 group treated group compared to the obese control group (Fig. 8).

The percentage of pancreatic exocrine granules in the obese control group was -52.28% higher than that of the normal control group. In the CLA, Rg3 group 10, 50 and 100 mg / kg group, the ratio of the enzyme granules was 33.11, 27.57, 86.36 And 56.55%, respectively.

Anti-diabetic  effect

Changes in blood glucose

In the obese control group, there was a significant increase in blood glucose (p <0.01) compared to the normal control group, but a decrease in blood glucose was recognized in all the treatment groups compared to the obesity control group (p <0.01) 8).

Biochemical markers in serum of obese mice administered orally with Rg3 group or CLA for 28 days Biochemical indicators of serum glucose
(mg / dl) AST
(IU / l) ALT
(IU / l) BUN
(mg / dl) Creatinine
(mg / dl) TC
(mg / dl) FFA
(mmol / l) Triglyceride
(mg / dl) Control group Normal group 140.20
± 12.66 68.20
± 11.10 24.00
± 4.47 31.20
± 4.15 0.34
± 0.04 104.20
± 5.45 0.60
± 0.09 85.60
± 12.56 Obesity group
(ob / ob) 618.00
± 74.49 ^a 369.40
± 44.52 ^a 362.20
± 54.85 ^a 46.80
± 5.45 ^a 0.42
± 0.02 ^a 285.00
± 10.58 ^a 1.75
± 0.18 ^a 129.40
± 5.77 ^a Reference group
(CLA) 464.80
± 48.00 ^ac 302.60
± 24.87 ^ac 259.60
± 51.03 ^ac 38.00
± 3.54 ^bc 0.36
± 0.03 ^d 249.60
± 11.08 ^ac 1.40
± 0.10 ^ac 104.80
± 7.12 ^ac Rg3 group treated group 10 mg / kg 427.20
± 66.94 ^ac 272.80
± 36.32 ^ac 239.00
± 44.03 ^ac 37.60
± 3.36 ^bc 0.35
0.04 ^c 224.00
± 16.29 ^ac 1.32
± 0.14 ^ac 101.80
± 5.54 ^ac 50 mg / kg 316.40
± 68.79 ^ac 211.40
± 29.20 ^ac 199.60
± 21.02 ^ac 34.00
± 3.16 ^c 0.34
± 0.03 ^c 203.00
± 7.31 ^ac 1.15
± 0.14 ^ac 96.00
± 9.67 ^c 100 mg / kg 347.40
± 64.10 ^ac 227.20
± 35.72 ^ac 217.20
± 28.67 ^ac 35.20
± 2.77 ^c 0.34
± 0.02 ^c 208.80
± 11.41 ^ac 1.23
± 0.12 ^ac 97.60
± 7.40 ^bc

Values are mean ± SD of experiments for 5 animals.

Rg3 Group: Ginsenoside Rg3 Group

CLA: conjugated linoleic acid, reference group

statistical significance of p is ^a statistical significance of p <0.01 and p ^b is p <0.05, and were compared with control group value by the LSD test.

^The significance level of ^c p was p <0.01 and the significance level of ^d p was p <0.05 and compared with obesity group (ob / ob) by LSD test.

In the obese control group, blood glucose was 340.80% higher than that of the normal control group. In the CLA, Rg3 group, 10, 50 and 100 mg / kg group, -24.79, -30.87, -48.80 and -43.79 %, Respectively.

Changes in Plasma Insulin Content

In the obese control group, plasma insulin content was significantly increased (p <0.01) as compared to the normal control group, but the plasma insulin content was significantly decreased in all the treatment groups compared to the obesity control group (p <0.01) (Fig. 9).

Plasma insulin content was 1353.97% in the obese control group compared with the control group, and -27.51, -49.73, -77.52, and -77.52 in the CLA, Rg3 group 10, 50 and 100 mg / 71.43%, respectively.

Pancreas weight  change

(P < 0.01 or p < 0.05) relative to the normal control group was observed in all obesity test groups, but in addition to the increase in relative pancreatic relative weight in all Rg3 group treated groups compared with the obesity control group, Significant changes in pancreatic weight compared to the control group were not observed in all treatment groups (Table 6 and Table 7).

In the obese control group, the absolute pancreas weight was 17.30%, and in the CLA and Rg3 group 10, 50 and 100 mg / kg groups, the -18.44, 2.80, -7.96 and 5.16% Change.

The relative weight of the pancreas was -41.84% in the obese control group compared with the control group. In the CLA, Rg3 group 10, 50 and 100 mg / kg administration groups, -3.17, 26.30, 8.62 and 23.37% Change.

Pancreatic islet  Changes in abnormal growth and expansion

In the obese control group, prominent pancreatic islet proliferation was recognized, and the number of pancreatic islets and pancreatic islets to total pancreatic islets was significantly increased (p <0.01) compared to the normal control group. However, CLA and all three doses of Rg3 In the group treated group, the number and ratio of pancreatic islets were significantly decreased (p <0.01) compared to the obese control group (Fig. 8).

The mean number of pancreatic islets was 264.86% in the obese control group compared with the control group. In the CLA, Rg3 group 10, 50 and 100 mg / kg group, the number of pancreatic islets was -28.15, -43.70, -51.85 And -50.37%, respectively.

The ratio of pancreatic islets in the obese control group was 521.27% compared to the normal control group. In the CLA, Rg3 group 10, 50 and 100 mg / kg group, the ratio of -20.57, -32.26, -45.26 And -41.14%, respectively.

Pancreatic islet  Insulin and glucagon IR  Changes in cells

In the obese control group, a significant increase in endocrine cells was observed, and a significant increase (p <0.01) in insulin and glucagon-immunoreactive cells (IR) was observed compared with the normal control group. However, CLA and all three doses of Rg3 In the group treated group, the number of insulin and glucagon IR decreased significantly (p <0.01) compared to the obese control group, respectively. In addition, the increase in insulin and glucagon-immunoreactive cells (IR) was recognized simultaneously in all obese mouse experimental groups, and the ratio of insulin / glucagon IR cells was recognized similar to the normal control group (Fig. 10-12).

The mean insulin IR number was 213.87% in the obese control group compared to the control group, and -35.99, -42.80, -53.91% in the CLA, Rg3 group 10, 50 and 100 mg / And -52.29%, respectively.

The mean number of glucagon IRs was 228.44% in the obese control group compared to the control group, and -37.62, -43.80, -54.52 in the CLA, Rg3 group 10, 50 and 100 mg / And -52.13%, respectively.

The ratio of insulin / glucagon IR was -5.84% in the obese control group, and 3.36, 1.60 and 4.68 in the CLA, Rg3 group, 10, 50 and 100 mg / 1.57%, respectively.

Effect on hyperlipemia

Serum TC  Change in content

In the obese control group, there was a significant increase (p <0.01) in the TC content in the serum compared to the normal control group. However, in the CLA, Rg3 group 10, 50 and 100 mg / ) TC content were recognized (Table 8).

The TC content of serum was 173.51% in the obese control group compared with the control group, and -12.42, -21.40, -28.77 and -12.47% in the CLA, Rg3 group 10, 50 and 100 mg / And 26.74%, respectively.

FFA  Change in content

In the obese control group, the FFA content was significantly increased (p <0.01) compared to the normal control group, but the FFA content was significantly decreased in all the treatment groups (p <0.01) compared to the obesity control group 8).

Serum FFA content was 193.96% in the obese control group, and -20.21, -24.89, -34.25 and -24.25% in the CLA, Rg3 group, 10, 50 and 100 mg / And 29.57%, respectively.

Serum Triglyceride  Change in content

In the obese control group, serum triglyceride content was significantly increased (p <0.01) compared with the normal control group, but the decrease in the triglyceride content was significantly (p <0.01) Respectively (Table 8).

Serum triglyceride content was 51.17% in the obese control group, and -19.01, -21.33, -25.81, and -25.81 in the CLA, Rg3 group, 10, 50 and 100 mg / -24.57%, respectively.

Effect on liver damage

Changes in liver weight

In the obesity control group, significant (p < 0.01) relative and absolute weight gains were observed compared to the normal control group, but all of the Rg3 group treated group showed a significant decrease in liver weight (p < 0.01) compared to the obesity control group . On the other hand, changes in liver weight similar to those in the obesity control group were recognized in the CLA-treated group (Table 6 and Table 7).

The mean absolute liver weight of the obese control group was 294.12% compared with that of the normal control group. In the CLA, Rg3 group 10, 50 and 100 mg / kg group, the -12.28, -39.18, -41.98 and -40.21% Respectively.

The relative weight of liver was 92.55% in the obese control group and 6.67, -27.23, -31.37 and -30.05% in the CLA, Rg3 group, 10, 50 and 100 mg / Change.

Serum AST Change in content

In the obese control group, serum AST content was significantly increased (p <0.01) compared to the normal control group, but in the CLA, Rg3 group 10, 50 and 100 mg / A decrease in serum AST levels was observed (Table 8).

Serum AST levels were 441.64% in the obese control group and -18.08, -26.15, -42.77 and -38.49% in the CLA, Rg3 group, 10, 50 and 100 mg / Respectively.

Serum ALT Change in content

In the obese control group, serum ALT level was significantly increased (p <0.01) compared to the normal control group. However, in all CLA and Rg3 group treated groups, the decrease in serum ALT level was significant (p <0.01) (Table 8).

Serum ALT levels were 1409.17% in the obese control group, and -28.33, -34.01, -44.89 and -40.03 in the CLA, Rg3 group, 10, 50 and 100 mg / %, Respectively.

Fatty hepatitis  change

Liver hepatitis (ratio of fat area changed in liver tissue), which is characterized by severe hepatocellular hypertrophy due to fat accumulation in liver cells, was significantly increased (p <0.01) in liver fat ratio compared to normal control in obese control group (P < 0.01 or p < 0.05), respectively, as compared with the obesity control group (Fig. 13).

In the obese control group, fat change rate was 1193.99% in the obese control group, and -11.26, -49.14, -61.54 and -11.57% in the CLA, Rg3 group 10, 50 and 100 mg / -54.79%, respectively.

Changes in hepatocellular hypertrophy

In the obese control group, there was a significant (p <0.01) increase in the diameter of the hepatocyte (hypertrophy) compared to the normal control group, but the decrease in the hepatocyte diameter was significantly (p <0.01 or p <0.05) Respectively (Fig. 13).

The diameter of the hepatocytes was 65.00% in the obese control group and -1.61, -28.39, -21.71 and -22.19% in the CLA, Rg3 group, 10, 50 and 100 mg / Respectively.

Influence on kidney damage

Height-weighted  change

In the obesity control group, the increase in the absolute absolute weight and the relative weight decrease were significant (p <0.01) as compared with the normal control group. However, in all CLA and Rg3 group treated groups, Weight reduction, respectively. On the other hand, significant changes in relative weight relative to the obesity control group were not observed in all treatment groups (Table 6 and Table 7).

The absolute height of kidneys was 67.12% higher in the obese control group than in the obese control group. In the CLA, Rg3 group 10, 50 and 100 mg / kg group, -23.27, -12.40, -20.04 and -15.81% Respectively.

The relative weight of the kidneys was -17.85% in the obese control group and -7.75, 8.23, -4.87 and -1.84% in the CLA, Rg3 group, 10, 50 and 100 mg / Respectively.

Serum BUN Change in content

In the obese control group, serum BUN level was significantly increased (p <0.01) compared to the normal control group. However, CLA, Rg3 group 10, 50 and 100 mg / ), Respectively (Table 8).

Serum BUN content was 50.00% in the obese control group and -18.18, -19.66, -27.35 and -24.79 in the CLA, Rg3 group, 10, 50 and 100 mg / %, Respectively.

Changes in serum creatinine content

In the obese control group, serum creatinine was significantly increased (p <0.01) compared to the normal control group, but in the CLA, Rg3 group, 10, 50 and 100 mg / And a decrease in the serum creatinine level, respectively (Table 8).

Serum creatinine levels were 23.81% in the obese control group, -12.98, -14.90, -18.75 and -17.79 in the CLA, Rg3 group, 10, 50 and 100 mg / %, Respectively.

Renal histopathological change

In the obesity control group, renal degeneration characterized by marked capillary-obstructed glomerular atrophy and swelling was recognized, and the number of glomeruli and renal epilepsy showing capillary dilatational atrophy (p <0.01) (P < 0.01), respectively, as compared with the obesity control group (Fig. 14).

The number of metabolic glomeruli indicating capillary vasoconstrictive atrophy was 1326.32% in the obese control group compared to the normal control group. In the CLA, Rg3 group, 10, 50 and 100 mg / 69.20, -71.96 and -75.25%, respectively.

In the obese control group, 332.26% change was observed in the obesity control group. In the CLA, Rg3 group 10, 50 and 100 mg / kg administration groups, the -33.52, -61.78 and -51.18 %, Respectively.

Review

In this study, the anti-obesity and antidiabetic effects of the Rg3 group were compared with those of obesity mice [Lindstr, 2007; Kurundkar et al., 2011]. In this study, blood glucose lowering and antidiabetic effects were evaluated based on histopathological changes of blood glucose, pancreatic weight, and endocrine pancreas. Liver protective effect was evaluated by liver weight, histopathological changes in liver, changes of serum AST and ALT contents And the effects on hyperlipidemia were based on changes in plasma TC, FFA and triglyceride content and the kidney protective effect was evaluated based on histopathological changes in kidney weight, serum creatinine, BUN and kidney. The anti-obesity effect was also evaluated based on the histopathological changes of feed intake, body weight, fat around the epididymis, serum leptin and adiponectin content, abdominal and epididymal fat, and pancreatic exocrine portion. Experimental results were compared with the CLA 750 mg / kg group [Hue et al., 2009], which is known to have a significant anti-obesity and antidiabetic effect.

As a result of this experiment, obese mice were reported in previous reports [Haluzik et al., 2004; Margalit et al., 2006; Wang et al., 2011; Xu et al., 2011; Yoon and Kim, 2011], and the common complications related to diabetes such as hyperlipidemia, liver and kidney damage were observed, but the obesity and diabetes findings in obese mice were associated with three dose of Rg3 group 10, 50 and 100 mg / kg). In particular, the Rg3 group at 10 mg / kg showed better anti-obesity and antidiabetic effects than the CLA at 750 mg / kg, and the dose-dependent anti-obesity and anti-diabetic effect of Rg3 group In the group of 100 mg / kg of Rg3 group, the increase of diabetes mellitus was observed, but the similar or relatively low effect was observed in 50 mg / kg group, suggesting that absorption saturation of Rg3 group occurred between 50-100 mg / kg.

Obesity mouse is an obesity model in which an appetite control protein, leptin, is genetically modified [Haluzik et al., 2004], which is the most commonly used animal model for developing obesity and diabetes treatment with obesity and type 2 diabetes One [Haluzik et al., 2004; Margalit et al., 2006; Wang et al., 2011; Xu et al., 2011; Yoon and Kim, 2011]. The obese mice used in this experiment also showed a significant increase in body weight from the 5th week of intake. However, CLA, Rg3 group 10 and 50 mg / kg administration group showed significant increase from 20, 26 and 17 days Weight gain was recognized, and the 28-day body weight gain was also significantly (p <0.01) lower than the obese control group. In addition, the 100 mg / kg Rg3 group did not show any significant difference compared to the obesity control group, but the significant decrease in body weight started to be recognized from the 16th day of administration, and the 28 day gain was also significantly decreased (p <0.01) Respectively. Therefore, 10 mg / kg of Rg3 group showed better weight gain inhibitory effect than CLA 750 mg / kg.

And fat accumulation is the most common phenomenon resulting from obesity, which is known to result in hypertrophy of adipocytes during histopathological examination [Morange et al., 2000]. Adipocyte secretion changes in obesity and insulin resistance, as well as adipocyte secretion, are known to play a role in both endocrine and secretory functions, as well as role as a simple energy store [Fujita et al., 2005] (Fujita et al., 2005; Mitchell et al., 2005]. It is known that leptin is a kind of adipocaine secreted mainly by adipocytes and plays a role of appetite suppression [Bjorbaek and Kahn 2004]. Adiponectin is also a relatively new adipokine, [Maeda et al., 1996], which is known to be released into the blood [Arita et al., 1999], is significantly reduced in obesity in humans and animals, and directly linked to insulin resistance and obesity [Mitchell et al., 2005 ]. As a result of this experiment, the Rg3 group significantly inhibited the fat accumulation, fat cell hypertrophy and blood adipocine content of obese mice, which are animal model of leptin gene mutation, similarly to CLA, It had no other effect.

The enzyme-derived granules of the pancreatic exocrine pancreas contain mainly enzymes involved in the digestion of fat and protein [Gartner and Hiatt, 2007], and it is known that the pancreas in obesity leads to a marked decrease of these enzyme granules with fat accumulation [Tasso et al., 1971; Wilson et al., 1988]. In the present study, the obesity control group showed a marked decrease of the accumulation enzyme granules compared with the normal control group. However, the decrease of the granules of the enzyme source was remarkably suppressed by the administration of the CLA and Rg3 groups, The effect of reducing fat absorption is thought to be effective for obesity, but the exact mechanism is unknown.

The increase in blood sugar is the most basic phenomenon of diabetes [Sathishsekar and Subramanian, 2005]. Obese mice used in this experiment also showed typical hyperglycemia. On the other hand, the increase in blood glucose level was significantly reduced by 28 days of continuous CLA or Rg3 group administration, and these CLA and Rg3 groups are considered to be highly effective for the treatment of diabetes. In addition, as type 2 diabetes progresses, abnormal proliferation of the pancreatic islets as a part of insulin resistance is induced [Terauchi et al., 2007], more insulin secretion is required to control increased blood sugar, and consequently, Insulin-producing cells are accompanied by abnormal proliferation, and glucagon-producing cells are also increased at a similar rate to control [Han et al., 2007; Noriega-Let al., 2007; Terauchi et al., 2007]. The results of this experiment showed that the abnormal proliferation of the pancreatic islets and the increase in the number of insulin and glucagon immunoreactive cells per unit area were observed in the obese control group as compared with the normal control group. However, the change of the pancreatic endocrine part was observed by administration of CLA and Rg3 Respectively. The hyperglycemic status in obese mice leads to hyperlipemia in the form of complications [Hue et al., 2009; Chen et al., 2010], and in general, the increase of FFA, triglyceride and TC in hyperlipidemia is problematic [Kamada et al., 2005]. As a result of this experiment, a decrease in serum FFA, triglyceride, and TC levels exceeding CLA 750 mg / kg was observed in the 10 mg / kg group of the Rg3 group, and it was observed that the Rg3 group significantly inhibited hyperlipemia caused by obesity or diabetic complications.

As diabetes progresses, hepatocyte damage due to fibrosis and abnormal actions results in an increase in liver weight and consequently an increase in blood AST and ALT levels [Ceriello et al., 1992; Sathishsekar and Subramanian, 2005]. These liver damage is referred to as diabetic hepatopathy, and these symptoms have been used as direct findings in diabetic hepatopathy [Quine and Raghu, 2005]. AST, commonly known as SGOT, and ALT, also known as SGPT, are the most common blood chemistries to measure liver damage [Sodikoff, 1995]. As a result of this experiment, it was observed that the Rg3 group significantly inhibited the increase of liver weight and the increase of blood AST and ALT content in obese mice, and especially the histopathological examination showed significant inhibition of fat change and hepatocyte hypertrophy , The Rg3 group was observed to effectively block diabetic hepatopathy.

As the diabetic state becomes chronic, renal lesions are caused by glomerular atrophy, fibrosis, inflammatory cell infiltration, tubular necrosis and concomitant increase in blood BUN and creatinine content, which is called diabetic nephropathy [Trachtman et al. , 2002; Clark et al., 2004]. Serum BUN and creatinine content are the most representative blood chemistries for renal status [Sodikoff, 1995]. Obesity mice are also known to progress to renal diabetic nephropathy as part of a typical type 2 diabetes complication [Hudkins et al., 2010; Alpers and Hudkins, 2011]. As a result of this experiment, capillary glomerular atrophy and an increase in the proportion of epileptic seizure, that is, epileptic edema, with increase in renal weight, blood BUN and creatinine level were recognized histopathologically, It was observed that the progression of diabetic nephropathy in obese mice was markedly suppressed.

Significant increases in feed intake were observed in all obese mice compared to the normal control group, but in the CLA-treated group, significant reductions in feed intake were observed compared with the obesity control group, similar to previous reports [Hue et al., 2009] The antiobesity and antidiabetic effects of CLA were observed to be mainly caused by a decrease in feed intake. On the other hand, all of the three doses of the Rg3 group showed similar changes in feed intake to those of the obesity control group at all measurement days, and the anti-obesity and antidiabetic effects of the Rg3 group are thought to be different from the feed intake reductions. Peroxisome Proliferator Activated Receptor-γ (PPAR-γ) [Chawla et al., 1994], which is present in high concentrations in adipose tissue, has been used for the treatment of obesity and type 2 diabetes And it has been reported that the activity of PPAR-γ is increased in a variety of obese and diabetic animal models [Burant et al., 1997; Memon et al., 2000; Hue et al., 2009]. In addition, AMP activated protein kinase (AMPK) also plays an important role in intracellular energy homeostasis [Sakoda et al., 2002; Motoshima et al., 2006], AMP activity has been shown to inhibit lipid accumulation and antidiabetic effects [Bae et al., 2007; Hwahng et al., 2009]. Rg3 group also showed anti-obesity effect through AMPK activity and PPAR-y inhibition by in vitro method through 3T3-L1 cells [Hwang et al., 2009], the anti-obesity and anti-obesity of Rg3 group recognized in the present invention The diabetic effect is also thought to be due to AMPK activity and PPAR-γ inhibition.

conclusion

The present study demonstrated that Rg3 group was orally administered to obese mice for 28 days, and that the Rg3 group effectively inhibited obesity, diabetes and related complications such as hyperlipidemia, liver damage, and nephropathy. Thus, the Rg3 group will be a potent new therapeutic for obesity and its associated type 2 diabetes (for example, the AMPK activity, also known as the mechanism of action, and the metabolic syndrome mediated by PPAR-gamma inhibition). In this study, the anti-obesity and anti-diabetic effects of Rg3 group at 10 mg / kg were superior to those of CLA 750 mg / kg.

Sasa ( Acknowledgment )

This research was supported by the Korean Research Foundation as the fund of the Ministry of Education, Science and Technology in 2011 (No.2011-0030124) (This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government [MEST] (No.2011-0030124)).

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Claims (21)

A process for preparing Rg2 group ginsenosides comprising Rg3 group ginsenosides or Rg2, Rg4 and Rg6 comprising Rg3, Rk1 and Rg5 comprising the steps of:
(a) obtaining ginseng or red ginseng extract;
(b) applying the ginseng or red ginseng extract to an adsorption chromatography using a resin composed of a copolymer of styrene and benzene to adsorb the ginsenosides in the extract to the resin;
(c) eluting the adsorbed ginsenoside to obtain protopanaxatriol (PPT) ginsenoside and PPD (protopanaxadiol) ginsenoside; And
(d) sugar degrading the PPD family ginsenoside or PPT family ginsenoside to obtain an Rg2 group ginsenoside comprising Rg3, Rk1 and Rg5 or Rg2 group ginsenosides comprising Rg2, Rg4 and Rg6 .
The method according to claim 1, wherein the benzene of the resin is bonded to a vinyl group.
The method according to claim 2, wherein the benzene of the resin is divinylbenzene.
The method according to claim 1, wherein the resin has a specific surface area of 300 to 1000 m ² / g.
The method of claim 1, wherein the resin has a pore volume of 1.0-2.0 ml / g.
The method of claim 1, wherein the resin has a pore radius of 50-1000 A.
The method according to claim 1, wherein the elution of step (c) is carried out using alcohol.
2. The method of claim 1, wherein the PPT family ginsenosides obtained in step (c) comprise Rg1, Re and Rf.
2. The method of claim 1, wherein the PPD family ginsenoside obtained in step (c) comprises Rb1, Rc, Rb2 and Rd.
The method of claim 1, wherein the method further comprises washing the resin with water between steps (b) and (c) to remove the hydrophilic material.
The method according to claim 1, wherein the saccharification is carried out using a glycosidase.
12. The method according to claim 11, wherein the saccharide hydrolase is a saccharide-hydrolyzing enzyme derived from ginseng or red ginseng.
The method according to claim 1, wherein the method further comprises a step of applying decolorization results to the anion exchange resin after decolorization to decolorize the anion exchange resin.
The method according to claim 1, wherein step (d) is carried out in the presence of the extract of step (a).
[Claim 3] The composition according to claim 1, wherein the Rg3 group ginsenoside comprises Rg3, Rk1 and Rg5, wherein 30 to 55 parts by weight of Rg3 and 10 to 30 parts by weight of Rk1, based on 100 parts by weight of Rg3, Rk1 and Rg5, And Rg5 is 20 to 40 parts by weight.
The composition of claim 1, wherein the Rg2 group ginsenoside comprises Rg2, Rg4, and Rg6, wherein the total weight of Rg2, Rg4, and Rg6 is 25-50 parts by weight based on 100 parts by weight of Rg2, 20-45 parts by weight of Rg4 And Rg6 is 10-25 parts by weight.
Rg3, Rk1 and Rg5 as the active ingredient, 30 to 55 parts by weight of Rg3, 10 to 30 parts by weight of Rk1 and 20 to 40 parts by weight of Rg5 based on 100 parts by weight of the total weight of Rg3, Rk1 and Rg5 Is a metabolic disease selected from the group consisting of diabetes, obesity, hyperlipidemia, fatty liver, atherosclerosis, diabetic nephropathy and diabetic hepatopathy, which is produced by the method of any one of claims 1 to 16 A pharmaceutical composition for preventing, ameliorating or treating a disease.
delete delete delete Rg3, Rk1 and Rg5 as the active ingredient, 30 to 55 parts by weight of Rg3, 10 to 30 parts by weight of Rk1 and 20 to 40 parts by weight of Rg5 based on 100 parts by weight of the total weight of Rg3, Rk1 and Rg5 Is a metabolic disease selected from the group consisting of diabetes, obesity, hyperlipidemia, fatty liver, atherosclerosis, diabetic nephropathy and diabetic hepatopathy, which is produced by the method of any one of claims 1 to 16 &Lt; / RTI &gt;

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