KR20130060435A

KR20130060435A - Neutraceutical composition comprising the extract of processed panax ginseng for hepato-protective activity

Info

Publication number: KR20130060435A
Application number: KR1020110126486A
Authority: KR
Inventors: 김복득; 이혜진; 김진희; 황귀서; 박정일; 이양범; 김성용; 박용성; 박인순
Original assignee: 주식회사 진생사이언스; 가천대학교 산학협력단
Priority date: 2011-11-30
Filing date: 2011-11-30
Publication date: 2013-06-10

Abstract

The present invention provides liver protection and liver disease improvement, comprising Panax genus plant extract as an active ingredient, the sum of the contents of ginsenosides Rg3, Rg5, Rk1 is greater than the sum of the contents of ginsenosides Rb1, Rb2, Rc, Rd, and The present invention relates to a preventive dietary supplement, in particular, a composition containing the processed Panax genus plant extract of the present invention as an active ingredient, the effect on apoptosis by treating t-BHP on HepG2 cells, which are human liver cell lines. Experiments and their effects on NO production and oxidative factors, lipid peroxidation, and LDH, GOT, and GPT release levels caused by cell membrane destruction were measured.

Description

Nutraceutical composition comprising the extract of processed Panax Ginseng for hepato-protective activity}

It relates to a health functional food showing a hepatoprotective effect containing the processed ginseng extract of the present invention.

[Document 1] Brenneisen, P., Wenk, J., Klotz. LO, Wlaschek, M., Briviba, K., Krieg, T., Sies, H., Scharffetter-Kochanek, K., Central role of ferrous / ferric iron in the ultraviolet B-mediated signaling pathway leading to increased interstitial collagenase (MMP-1) and stromelysin-1 (MMP-3) mRNA levels in cultured human dermal fibroblasts. J. Biol. Chem. 273, 5279-5287, 1998.

[Literature 2] Masaki, H., Atsumi, T., Sakurai, H. Detection of hydrogen peroxide and hydroxyl radicals in murine skin fibroblasts under UVB irradiation. Biochem . Biophys . Res . Commun . 206, pp. 474-479, 1995.

[Literature 3] Understanding of Korean ginseng, Korean Ginseng Society, p 9, 1995; Advances in Ginseng Research, Korea Ginseng Society, p 127, 1998)

[Reference 4] Understanding Korean Ginseng, Korean Ginseng Society, pp 9-18, 2008).

[Reference 5] Kim W.Y. et al., Journal of Natural Products, vol. 63, pp 1702-1704, 2000;

6 Kwon S W, et al., J. Chromatogr. A, 921 (2), pp 335-339, 2001;

Document 7 Park I.H. et. al., Archives of Pharmacal Research, 25, pp. 428-432 and 837-841, 2002;

Document 8 Yong-Sam Keum, Kwang-Kyun Park, Jong-Min Lee, Kyung-soo Chun., Antioxidant and anti-tumor promoting activities of the methanol extract of heat-processed ginseng. J. CancerLetters . 150, pp. 41-48,2000.

9 C. Kim, So R. Kim, George J. Markelonis, Tae H. Oh., Ginsenosides Rb1 and Rg3 Protect Cultured Rat Cortical Cells From Glutamate-Induced Neurodegeneration. J. Neuroscience Res . 53, pp. 426-432, 1998.

Keun Young Jung, Dong seon Kim, Platelet Activating Factor Antagonist Activity of Ginsenosides. Biol . Pharm . Bull . 21.PP.79-80, 1998.

11 W Kim, et al., J. Nat . Prod . , 63 (12) , pp 1702-1704, 2001;

12 Kwon SW, et al., J. Chromatogr . A. , 921 (2) , pp 335-339, 2001

[Document 13] Korean Patent No. 10-0192678 Processed Ginseng Product with Enhanced Drug Effect, 1999

The present invention relates to a health functional food for improving or preventing hepatocellular protection and liver disease, which contains the processed Panax genus plant extract as an active ingredient.

Cell damage is known to cause various diseases by inhibiting the function of cells. Lipid peroxidation in vivo causes chemical chemotaxis of multinucleated cells such as macrophages and neutrophils, and stimulates the secretion of leukotriene, collagenase, elastase, oxidase, etc. Cause abnormalities. Lipid peroxide also acts as a major cause of hepatocellular necrosis, which is formed mainly by the reaction of unsaturated fatty acids in the cell membrane with free radicals activated oxygen. Causes of lipid peroxide production in vivo include UV, X-rays, as well as environmental substances such as carbon tetrachloride and tert-butyl hydroperoxide (t-BHP) alcohols, hazardous food additives, and nonsteroidal anti-inflammatory agents such as acetaminophen. The same physical factors and other heavy metals are known in various ways (1-2). Oxidative stress, which is involved in lipid peroxidation, destroys cells and inhibits function through DNA mutations, changes in enzyme activity, disruption of signaling systems through receptor oxidation, and changes in the osmotic pressure of cells through changes in ion channels. Therefore, hepatocytes involved in metabolic processes of various chemicals can be easily destroyed lipid peroxide not only by alcohol consumption but also various drugs. Therefore, various drug developments have been developed to protect liver cells, which have the function of removing various oxidative free radicals including lipid peroxide produced from hepatocytes.

Panax is a perennial herbaceous plant belonging to the genus Araliaceae in plant taxonomy. Representative species include Korean ginseng ( Panax ginseng ), flower ginseng ( Panax quinquefolia ), ginseng (samchi, Panax notoginseng ), Vietnamese ginseng ( Panax vietnamensis ) and Panax japonicus (People's Understanding, Korean Ginseng Society, p 9, 1995; Advances in Ginseng Research, Korean Ginseng Society, p 127, 1998). Thior ( Panax elegatior ), Panax wanzianus ( Panax wangianus , Panax bipinnatifidus ), Panax pseudoinsins ( Panax pseudoginseng ).

Ginseng, one of the most famous Panax plants, is one of the most widely used herbal medicines in Asia. It has been widely used in Asia for many years. It has been widely used for many purposes such as fatigue, nourishment, improvement of cognitive function, memory, blood circulation improvement, And the inhibition of platelet aggregation are known. (Understanding of Korean Ginseng, Korean Ginseng Society, pp 9-18, 2008).

On the other hand, heat-processing or treating a genus of Panax plants with a weak acid removes some sugars and alcohol groups from the ginsenoside structure, and the new structure of ginsenosides having stronger potency, ie, ginsenosides Rg3, Rg5, Rk1, Rk2 , Rk3, Rs1, Rs2, Rs3, F4, Rh2, Rh3, Rh4 and the like are known to be produced (Kim WY et al, Journal of Natural Products, vol. 63, pp1702-1704, 2000; Kwon SW, et al. , J. Chromatogr.A, 921 (2), pp 335-339, 2001; Park IH et. Al., Archives of Pharmacal Research, 25, pp.428-432 and 837-841, 2002).

Republic of Korea Patent No. 192678 and U.S. Patent No. 5,76,460 include processed panax plants in which the sum of the contents of ginsenosides (Rg3 + Rg5 + Rk1) is greater than the sum of the contents of (Rb1 + Rb2 + Rc + Rd). Hereinafter, the ginseng) is described, wherein the ginseng is a processed ginseng by dramatically increasing the content of Rg ₃ , Rg ₅ , Rk ₁ , which is highly effective by treating ginseng ( Panaxginseng ) at high temperature and high pressure. The efficacy of sun ginseng has been reported anti-cancer action (7), brain function improving action (8), platelet aggregation ability blocking action (9) and the like.

However, none of the above documents have yet described or taught the therapeutic effect of the processed Panax genus plant extracts on hepatoprotective and hepatic diseases.

The present inventors attempted to evaluate the action of inhibiting the injury of hepatocytes by tert-butyl hydroperoxide (t-BHP) using extract of ginseng, a processed ginseng. To this end, hepG2 cells, human hepatocytes, were treated with t-BHP to measure apoptosis, and their effects on NO production and lipid peroxidation, oxidative factors, and LDH, GOT, and GPT glass induced by cell membrane destruction were measured. By measuring the liver protection and liver disease improvement and prevention effect was completed the present invention.

In order to solve the above object, the present invention is processed Panax genus plant extract in which the sum of the ginsenoside Rg3, Rk1, Rg5 content is higher than the sum of the content of ginsenoside Rb1, Rb2, Rc, Rd It provides a dietary supplement for liver protection and liver disease improvement and prevention containing as an active ingredient.

Processed Panax genus plant as defined herein, preferably a ginseng (Panax ginseng), hwagisam (Panax quinquefolia ), Whole Chilsam (samchi, Panax notoginseng , Panax vietnamensis , Panax japonicus , Panax elegatior , Panax wangianus , Panax bipinnatifidus , or Panax pseudosin ( Panax vipinnatifidus ) Panax pseudoginseng ) artificially processed plants, and more preferably relates to processed ginseng with enhanced efficacy by heat treatment at high temperatures. More specifically, the ginseng is heated at a high temperature of 120 to 180 ° C. for 0.5 to 20 hours to increase the active ingredient so that the ratio of ginsenosides (Rg 3 + Rg 5) / (Rc + Rd + RbR + Rb₂) is 1.0 or more. Contains processed ginseng.

The extract is a lower alcohol such as water, methanol, ethanol, butanol, or a mixed solvent thereof, preferably water or 10 to 100% water and ethanol mixed solvent, more preferably, about 50 to 90% water and ethanol mixed Extracts soluble in the solvent.

The liver disease is characterized by including acute hepatitis, chronic hepatitis, fatty liver disease, cirrhosis of the liver, liver fibrosis, and liver cancer, preferably fatty liver disease.

Hereinafter, the method of obtaining the extract of the present invention will be described in more detail.

For example, after washing and drying the fruits, stems or roots of the Panax plants, respectively, the Panax plants are heat treated at a high temperature of 120 to 180 ° C. for 0.5 to 20 hours and then about 1 times the weight of the individual samples. To 30 times, preferably about 5 to 15 times (w / v) volume of water, C ₁ to C ₄ lower alcohols or mixed solvents thereof, preferably water or ethanol, more preferably water or Heat extraction method, ultrasonic extraction method, reflux extraction method for about 1 to 6 hours, preferably 3 to 5 hours at a reaction temperature of about 70 to 120 ℃, preferably 80 to 110 ℃ using 50 to 90% ethanol as the extraction solvent A second step of repeated extraction 1 to 10 times, preferably 2 to 7 times by a conventional extraction method such as a reflux extraction method; The processed Extract of Phanax spp. Of the present invention can be obtained through the third step of filtration and concentration under reduced pressure of the extract obtained in the above step.

In another aspect, the present invention provides a dietary supplement and food additives for the improvement and prevention of liver protection and liver disease containing the above-described manufacturing method and processed Panax genus plant extract prepared by the production method as an active ingredient.

The extracts prepared above were measured for apoptosis by treatment of HepG2 cells, human hepatocytes, with t-BHP, and their effects on NO production and lipid peroxidation, oxidative factors, and LDH and GOT induced by cell membrane destruction. As a result of measuring the degree of GPT release, it was confirmed that it is useful for the protection of hepatocytes and the treatment and prevention of liver disease.

Extract of the present invention comprises from 0.01 to 99% by weight of the extract relative to the total weight of the composition.

&Quot; Health functional food "as defined herein means food prepared and processed using raw materials or ingredients having functionality useful to the human body in accordance with Law No. 6727 on Health Functional Foods." Functional " Structure and function of the nutrient to control or physiological effects, such as to obtain a beneficial effect for health is intended to eat.

The dietary supplement of the present invention comprises the extract in an amount of 0.01 to 95%, preferably 1 to 80% by weight, based on the total weight of the composition.

In addition, the health functional food of the present invention can be manufactured and processed as a health functional food in the form of tablets, capsules, powders, granules, liquids, pills and the like for the purpose of preventing the disease.

Health functional foods containing the extract of the present invention can be used in a variety of drugs, foods and beverages for the prevention and improvement of the target disease. Examples of the foods to which the extract of the present invention can be added include various foods, beverages, gums, tea, vitamin complexes, health supplements and the like, and they can be used as powders, granules, tablets, capsules or beverages have.

Foods to which the extract of the present invention can be added include, for example, various foods, beverages, gums, tea, vitamin complexes, and health functional foods.

In addition, the present invention may be added to food or beverage for the purpose of preventing and improving the disease. At this time, the amount of the extract in the food or drink may be 0.01 to 15% by weight of the total food, and the health beverage composition may be added in a proportion of 0.02 to 5 g, preferably 0.3 to 1 g, based on 100 ml .

The health functional beverage composition of the present invention has no particular limitation on the other ingredients other than the above-mentioned extract as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. Natural flavors (tau martin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

In addition to the above, the extract of the present invention can also be used as a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and aging agents (cheese, chocolate, etc.), pectic acid and its salts, Salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages and the like. In addition, the sample of the present invention may contain natural fruit juice and fruit flesh for the production of fruit juice drinks and vegetable drinks. These components can be used independently or in combination. The proportion of such additives is not so critical but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the sample of the present invention.

As described above, the composition containing the processed Panax genus plant extract of the present invention as an active ingredient treatment effect t-BHP to HepG2 cells, a human liver cell line, the effect on cell death and NO production and lipid peroxidation of oxidative factors The results of the experiment and the measurement of the degree of LDH, GOT and GPT release caused by cell membrane destruction can be useful for the protection of liver cells and for the improvement and prevention of liver disease.

1 is a diagram showing the inhibitory effect of hepatic cell death of the processed ginseng extract (SG) of the present invention (where * P> 0.01, t-BHP, SG not compared with all treated; ** P> 0.05, t-BHP Compared with only treatment, #P> 0.01, showing comparison with only treatment with t-BHP);
Figure 2 is a diagram showing the inhibitory effect of hepatic cell membrane destruction of the processed ginseng extract (SG) of the present invention (where * P> 0.01, compared with only t-BHP; ** P> 0.05, t-BHP only treatment Comparison with one);
Figure 3 is a diagram showing the effect of inhibiting GOT free from hepatocytes of the processed ginseng extract (SG) of the present invention (where * P> 0.01, t-BHP, SG all compared with not treated; ** P> 0.05 , a comparison with the treatment with only t-BHP);
Figure 4 is a diagram showing the effect of inhibiting GPT free from hepatocytes of SG of the processed ginseng extract (SG) of the present invention (where * P> 0.05, compared with the treatment with t-BHP; ** P> 0.01, t A comparison with the treatment with only BHP);
Figure 5 is a diagram showing the inhibitory effect of hepatic cell peroxidation production of the processed ginseng extract (SG) of the present invention (where * P> 0.05, compared with only t-BHP; ** P> 0.01, t-BHP Only comparisons with treatments).

Hereinafter, the present invention will be described in detail by the following Examples and Experimental Examples.

However, it should be understood that the present invention is not limited thereto.

Example 1 Preparation of Processed Ginseng Extract

Methods, such as steam (Kim WY, et al, J. Nat Prod, 63 (12), pp 1702-1704, 2001;..... Kwon SW, et al, J. Chromatogr A., 921 (2), pp 335-339, 2001).

That is, dried ginseng (Gumsan ginseng market) was finely chopped, and heated using steam at 120 ° C. and 15 atm for 4 hours. 2 L of 80% ethanol was added to 1 kg of processed ginseng (hereinafter referred to as ginseng), and the extract obtained by reflux extraction for 4 hours or more in a water bath was concentrated under reduced pressure and freeze-dried to obtain about 300 g of an extract powder. In the experiment, the ginseng extract (hereinafter referred to as SG) was dissolved in a medium using DMSO (Sigma D-2650), and used after passing a filter paper having a pore size of 0.45 μm. (Rg3 + Rg5 + Rk1) / (Rb1 + Rb2 + Rc + Rd) = 6.1 of the ginseng extract used in the experiment.

Example 2. Analysis of Saponin Content of Sea Ginseng Extract

The ginseng saponin component contained in the processed ginseng prepared in Example 1 according to the present invention was analyzed by the following method.

Four 40 ml stainless steel vessels were added with 5 g of white ginseng and 5 ml of water, respectively, and then sealed and heated at 110 ° C. for 2 hours, 120 ° C. for 2 hours and 3 hours, and 130 ° C. for 2 hours. 5 g of each of the processed ginseng and the commercially produced white ginseng and red ginseng were taken, extracted three times with 100 ml of methanol, concentrated, suspended in water, and extracted three times with 100 ml of ether. The remaining aqueous layer was extracted three times with 100 mL of butanol, and then the butanol fraction was concentrated. → 80/20/15, detector: analyzed by ELSD (Evaporative light scattering detector). The measured results are shown in Table 1 below.

Saponin
Sample Rb ₁ Rb ₂ Rc Rd Rg ₃ Rg ₅ (Rg ₃ + Rg ₅₎ /
_{(Rc + Rd + Rb 1 +} Rb 2) 120/3 hour 10.82 6.91 8.52 7.64 28.01 14.01 1.24 120/2 hours 10.46 7.74 10.66 5.58 24.12 11.35 1.03 130/2 hours 4.06 3.44 3.98 3.82 21.00 16.17 2.43 110/2 hours 19.02 11.55 10.68 8.37 7.02 4.39 0.23 White ginseng 22.15 3.25 5.86 2.47 0.00 0.00 0.00 Red ginseng 30.11 9.69 12.45 1.76 1.05 1.05 0.01

As can be seen from the results shown in the above Table 1, the processed ginseng obtained by the heat treatment according to the present invention significantly increases the contents of Rg and Rg components, which are ginseng saponins which are almost or completely absent from fresh ginseng, white ginseng and red ginseng And exhibits excellent drug efficacy.

According to the results as described above, in order to more specifically confirm the change in the content of saponin components, in particular ginsenoside Rg and Rg according to the change in the heating temperature for ginseng, heating is 100 ℃, 110 ℃, 120 ℃, 130 ℃, Rg and Rg content of ginseng obtained by 2 hours at 150 ° C., 160 ° C., 180 ° C. and 200 ° C. was measured and compared with the content in ginseng (salt) that was not heated. The results are shown in Table 2 below.

Heating temperature
(time)
Kinds Untreated
(Fresh ginseng) 100
2 hours 110
2 hours 120
2 hours 130
2 hours 150
2 hours 160
2 hours 180
2 hours 200
2 hours Rg ₃ 0.00 0.02 0.08 0.17 0.86 0.44 0.45 0.35 0.23 Rg ₅ 0.00 0.02 0.05 0.08 0.44 0.53 0.56 0.48 0.38

Note) Each ingredient content is expressed as% of the amount of fresh ginseng used.

As seen above, the content of ginsenosides such as Rg and Rg is significantly increased in the case of processed ginseng heated at 120 to 180 ° C as in the present invention compared to the case of untreated ginseng or red ginseng (100 ° C heating). It can be seen that. (Rg3 + Rg5 + Rk1) / (Rb1 + Rb2 + Rc + Rd) = 6.1 of the ginseng extract used in the experiment.

Reference Example 1. Experimental preparation

1-1. reagent

MTT, 1,3,3,3-tetraethoxypropane and tert-butylhydroxyperoxide (t-BHP) were purchased from Sigma (St. Louis, MO) and measured for lactic acid dehydrogenase activity. Kits, geoti, and phyti measurement kits were purchased from Bio Clinical System. 2-thiobarbituric acid (TBA) was purchased from TKOYO KASEI KOGYO (Toshima, kita-Ku, Tokyo, JAPAN).

1-2. Cell culture

The cells used in the experiment were HepG2, a human liver cell line, purchased from Korea Cell Line Bank. Cells were cultured in DMEM medium containing 10% FBS (Lonza, Walkersville, MD USA) (WelGENE, Daegu, KOREA). The temperature of the incubator is 37 ° C and maintained at 5% carbon dioxide concentration. On the second day, when the culture dish was about 80% full, the cells were transferred to a new culture dish.

Reference Example 2. Statistical Processing

All experimental results were statistically analyzed using one-way ANOVA, and significance was assessed at p <0.05 level using Student-Newman-Keuls Test.

Experimental Example 1. Hepatocellular Survival Measurement Experiment

In order to test the effect on the viability of hepatic ginseng extract obtained in the above example was performed by applying the method disclosed in the literature as follows (Sohn JH, Han KL, Lee SH, Hwang JK. Protective effects of panduratin A against oxidative damage of tert-butylhydroperoxide in human HepG2 cells.Biol Pharm Bull. 2005; 28 (6): 1083-60)

Hepatocytes obtained in the reference example were dispensed about 5 X 10 ⁵ in a 96-well plate and cultured for 12 hours, and then treated with SG samples so as to have respective concentrations in DMEM medium without serum. After 30 minutes, incubated for 3 hours under or without tert-butyl hydroperoxide. After the treatment, the cells were washed once with saline, 100 ml of 0.5 mg / mL MTT solution was added thereto, and the reaction was performed for 3 hours in a 37 ° C incubator by blocking the light. After the reaction, the cells were washed once with saline, 100 ml of DMSO was added to extract the reaction solution, and the absorbance was measured at 530 nm using a microplate reader (GENios A5082, TECAN, Maennedort, Switzerland).

After SG was added to DMEM medium without serum to be 0, 5, 10, and 20 ㎍ / mL, respectively, and pretreated for 30 minutes, 1.25 mM tert-butyl hydroperoxide was added and incubated in a 5% carbon dioxide incubator for 3 hours. Treatment of tertiary-butyl hydroperoxide at a concentration of 1.25 mM resulted in about 50% cell damage compared to that without treatment (FIG. 1). However, when the concentration of SG was added to 0, 5, 10 and 20 ㎍ / mL, the higher the concentration of SG was found to reduce the cell damage. 50% reduction in cell viability after treatment with t-BHP was observed to recover to about 78.6% when 20 μ / mL of SG was added. SG was found to increase the survival rate of hepatocytes damaged by tert-butyl hydroperoxide in proportion to concentration.

Experimental Example 2. Hepatocytes LDH Glass impact test

In order to examine the effect of hepatocellular LDH release of the ginseng extract obtained in the above example, the experiment was performed by applying the method disclosed in the literature (Lima CF, Fernandes-Ferreira M, Pereira-Wilson C. Phenolic compounds protect HepG2). cells from oxidative damage: relevance of glutathione levels.Life Sci. 2006; 79 (21): 2056-68)

In order to measure the activity of LDH enzyme, hepatocytes were divided into ⁵ × 10 ⁵ cells in a 96-well plate and cultured for 12 hours, and then treated with SG and cultured to each concentration in a medium without serum. After 30 minutes, tert-butyl hydroperoxide was added and incubated for 3 hours. After the treatment, 50 ml of a colorant reagent containing lithium lactate substrate, NAD, nitrotetrazolium blue, and 1-methoxy PMS was added to 20 ml of the culture solution and reacted at 37 ° C for 10 minutes. After the reaction was completed, 100 ml of hydrochloric acid solution was added to stop the reaction, and the absorbance was measured at 570 nm using a microplate reader within 60 minutes. The activity value of the LDH enzyme was expressed in WU (Wroblewski units) using the absorbance of the LDH enzyme standard solution.

LDH (lactate dehydrogenase) enzyme is an enzyme present in the cytoplasm that normally does not pass through the cell membrane, but when the cell membrane is damaged, it is released extracellularly, that is, into the culture medium. The lactic acid dehydrogenase in the culture solution dehydrogenates the lactic acid in the substrate solution in the kit to produce pyruvic acid and NAD, and the produced NAD reduces nitrotezozolium blue in the reaction solution to produce deformazan. After the reaction is completed, the chromaticity of the reactant deformazan is measured using a microplate reader. Based on the data obtained from the MTT measurement, hepatic cells were treated with 1.25 mM tert-butyl hydroperoxide, which caused about 50% cell death, to observe the activity of lactate dehydrogenase released extracellularly (see FIG. 2). About 5 times (108.5 WU) of lactate dehydrogenase activity was increased in the culture compared to the tri-butyl hydroperoxide-treated cell population (26.3 WU), and the concentration of SG was increased to 5, 10 and 20 g / mL. Treatment reduced to 74.5 (significance less than 0.01), 70.5 (significance less than 0.01), and 41.3 WU (significance less than 0.05; relative to cells treated with 1.25 mM t-BHP only) relative to concentration. That is, oxidative damage of HepG2 cells by tert-butyl hydroperoxide was inhibited by SG.

Experimental Example 3. from liver cells GOT And GPT Glass impact test

In order to examine the effect on the GOT and GPT release from the hepatocytes of the ginseng extract obtained in the above examples, the experiment was performed as follows (Lima CF, Fernandes-Ferreira M, Pereira-Wilson C.). Phenolic compounds protect HepG2 cells from oxidative damage: relevance of glutathione levels.Life Sci. 2006; 79 (21): 2056-68)

GOT and GPT were measured in the medium to measure cell membrane destruction. Hepatocytes were divided into ⁵ × 10 ⁵ cells in a 96-well plate and cultured for 12 hours, and then treated with SG and cultured in a serum-free medium. After 30 minutes, tert-butyl hydroperoxide was added and incubated for 3 hours. In 50 ml of the culture solution, add 30 ml of geotid substrate solution (alpha-ketoglutarate and L-aspartic acid) and gefiti substrate solution (alpha-ketoglutarate and L-alanine), respectively, at 37 ° C. Reaction was continued for a while. 30 ml of 2,4-dinitrophenylhydrazine color reagent (Sigma-aldrich) was added to the reaction product and allowed to stand at room temperature. After 20 minutes, 100 ml of sodium hydroxide solution was added and left to stand at room temperature for 10 minutes for color development. Within 60 minutes absorbance was measured at a wavelength of 505 nm using a microplate reader (iGNOS, TECAN). GOT and GPT activities were expressed in Karmen / mL using standard calibration curves.

GOT is an enzyme in hepatocytes, which is released into culture when hepatic cells are damaged by tert-butyl hydroperoxide. Therefore, the increase in the activity of geothyroidism in the culture medium implies the damage of the hepatocytes of the cells. When the reaction solution is added to the culture solution for the geoty substrate solution (alpha-ketoglutarate acid and L-aspartic acid) in the kit, the substrate generates oxaloacetic acid and pyruvic acid by geoty. The resulting oxaloacetic acid and pyruvic acid change the 2,4-dinitrophenylhydrazine in the color reagent (young reagent) into the hydrazine in the kit, and sodium hydroxide solution is added thereto to develop the reaction solution to light brown color. Was measured using an instrument (iGNOS, TECAN). 1.25 mM tert-butyl hydroperoxide was treated to liver cells to observe the activity of the extracellularly released geotyse enzymes (Fig. 3), compared with the cell group not treated with tert-butyl hydroperoxide (3 Karmen / mL). About 10-fold (33 Karmen / mL) geotypic activity was increased in the culture, where SG was treated at concentrations of 5, 10 and 20 g / mL, depending on the concentration of 17.5 (probability below 0.05) and 12 ( Significant probability less than 0.05) and 7.5 Karmen / mL (significant probability less than 0.05; compared with cell groups treated only with 1.25 mM tert-butyl hydroperoxide). As a result, it was confirmed that the extracellular release of geoty by tertiary-butyl hydroperoxide was inhibited by SG in liver cells.

GPT is an enzyme in hepatocytes and, like geotin and lactic acid dehydrogenase, when liver cells are damaged by tert-butyl hydroperoxide, they are released into the culture medium. Therefore, the increase in the activity of pipi activity in the culture medium means damage of the hepatocytes of the cells. When the substrate solution for gephyti (alpha-ketoglutarate acid and L-alanine) in the kit was added to the culture solution and reacted, the substrate produced oxaloacetic acid and pyruvic acid by zipi. You can determine the extent of hepatocellular damage by measuring the chromaticity according to the same principle as the geotye measurement. Treatment of 1.25 mM tert-butyl hydroperoxide to liver cells observed the activity of the zipi enzyme released extracellularly (FIG. 4), compared to the cell group not treated with tert-butyl hydroperoxide (3 Karmen / mL). Eighty times (24 Karmen / mL) zipi activity was increased in the culture, where SG was treated at concentrations of 5, 10 and 20 g / mL, depending on the concentration of 20 (below 0.05 probability) and 17.5 ( Significant probability less than 0.05) and 11 Karmen / mL (significance less than 0.01; compared with cell groups treated only with 1.25 mM tert-butyl hydroperoxide). In other words, SG reduced the cellular damage of HepG2 by tert-butyl hydroperoxide.

Experimental Example 4. Measurement of lipid peroxide production in hepatocytes

In order to examine the effect of the ginseng extract obtained in the above example on the amount of lipid peroxide production of hepatocytes, the experiment was performed by applying the method disclosed in the literature (Noh et al. 2010. Antioxidant effects of the chestnut (Castanea crenata) ) inner shell extract in t -BHP-treated HepG2 cells, and CCl ₄ -and high-fat diet-treated mice.Food and Chemical Toxicology 48,3177-83).

Hepatocytes were divided into ⁵ × 10 ⁵ cells in a 96-well plate and cultured for 12 hours, and then treated with SG in a serum-free medium at different concentrations and incubated for 30 minutes. After tertiary-butyl hydroperoxide was added and incubated for 3 hours, the culture solution was removed, and 150 ml of 6% Triton X-100 was treated for 5 minutes. It was mixed with 20 ml of trichloroacetic acid solution and left for 15 minutes on ice, followed by centrifugation at low temperature for 15 minutes at 1,500 rpm. An equal amount of 0.67% thiobarbituric acid solution was added to 120 ml of the supernatant obtained by centrifugation, boiled for 10 minutes, cooled at room temperature, and chromaticity was measured at 535 nm using a microplate reader. The calibration curve was prepared using standard 1,1,3,3-tetraethoxypropane, and the degree of lipid peroxidation was expressed by the molar concentration of thiobarbituric acid reactant (TBARS).

Lipid peroxidation is a well-known cell damage mechanism in animals and plants that increases when cells or tissues undergo oxidative stress. In particular, the unsaturated lipids of cell membranes are known to be the most easily peroxidized, and as a result, the naturally occurring product is malondialdehyde (MDA). The thiobarbituric acid reaction product (TBARS) assay is a representative measurement method of lipid peroxidation, in which malondialdehyde reacts with thiobarbituric acid to form an adduct and measures the degree of lipid peroxidation by measuring its chromaticity. Lipid peroxidation increases with increasing chromaticity, and it can be expected that cell and tissue damage caused by oxidative stress has occurred. After pretreatment of SG at 0, 5, 10, and 20 ㎍ / mL concentrations to liver cells, and the addition of 1.25 mM tert-butyl hydroperoxide, the cell lysates were reacted with thiobarbituric acid solution. Observation of the concentration of the acid reactant (FIG. 5) showed a significant increase (0.39 mM) compared to the cell group without treatment with tert-butyl hydroperoxide (0.09 mM), where the SG concentration was 5, 10 μg / mL. Treatment reduced to 0.37 mM (below 0.05 probability) and 0.3 mM (below 0.01 probability) depending on concentration, and 0.17 mM (below 0.01 probability; 1.25 mM tert-butyl hydroperoxide with 20 μg / mL SG treatment). Compared to the cell group treated only) by about 69%. That is, it was confirmed that lipid peroxidation in liver cells caused by tert-butyl hydroperoxide can be suppressed by treating SG.

Experimental Example 5. Single dose toxicity exam

A single dose toxicity test was performed using the processed ginseng extract prepared in Example using a mouse. As a result of the single dose toxicity test, no mortality was observed for 2 weeks at 2 g / kg, which is the dose prescribed by ICH, and no significant abnormality was found in weight gain and feed intake. . Therefore, each extract of the present invention was found to be a safe drug.

Hereinafter, formulation examples of the composition containing the extract of the present invention will be described, but the present invention is not intended to be limited thereto but is specifically described.

Formulation example One. Sanje Produce

SG Extract 200 mg

Lactose 100 mg

Talc 10 mg

The above components are mixed and filled in airtight bags to prepare powders.

Formulation example 2. Preparation of tablets

SG Extract 200 mg

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.

Formulation example 3. Preparation of capsules

SG Extract 200 mg

Crystalline cellulose 3 mg

Lactose 14.8 mg

Magnesium stearate 0.2 mg

The above components are mixed in accordance with a conventional method for producing a capsule, and filled in a gelatin capsule to prepare a capsule.

Formulation example 4. Liquid Produce

SG Extract 200 mg

10 g per isomer

5 g mannitol

Purified water

Each component was added and dissolved in purified water according to the usual liquid preparation method, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed, and purified water was added thereto. The whole was added with purified water to adjust the total volume to 100 ml, And sterilized to prepare a liquid preparation.

The above components were mixed according to a conventional health drink manufacturing method, and the mixture was heated at 85 DEG C for about 1 hour with stirring, and the solution thus prepared was filtered to obtain a sterilized 2-liter container, which was sealed and sterilized, &Lt; / RTI >

Although the composition ratio is a mixture of the components suitable for the preferred beverage as a preferred embodiment, the blending ratio may be arbitrarily varied according to the regional and national preferences such as the demand level, the demanding country, and the intended use.

Claims

Hepatoprotective, containing as an active ingredient, a processed Panax genus plant extract with an increased active ingredient such that the sum of the ginsenosides Rg3, Rk1, and Rg5 is higher than the sum of the contents of the ginsenosides Rb1, Rb2, Rc, and Rd; Health functional foods for improving and preventing liver disease.

The method of claim 1,
The Panax genus plants Ginseng (Panax ginseng ), Panax ginseng ( Panax quinquefolia ), Whole Chisam (Samchi, Panax notoginseng ), Vietnamese Ginseng ( Panax vietnamensis ), Panax ginseng ( Panax japonicus ), Panax elegatior , Panax wanzianus ( Panax wangianus , Panax bipinnatifidus ), or Panax health functional food characterized by plants artificially processed pseudoginseng ).

The method of claim 1,
The Panax genus plants are treated with ginseng at a high temperature of 120 to 180 ° C. for 0.5 to 20 hours so that the ratio of ginsenosides (Rg 3 + Rg 5) / (Rc + Rd + Rb ₁ + Rb ₂ ) is 1.0 or more. Health functional food characterized by processed ginseng with increased ingredients.

The method of claim 1,
The extract is a health functional food which is a lower alcohol such as water, methanol, ethanol, butanol, or a mixed solvent thereof.

The method of claim 1,
The liver disease is acute hepatitis, chronic hepatitis, fatty liver disease, liver cirrhosis, liver fibrosis, and liver cancer health functional food.

The method of claim 1,
The health functional food form is a health functional food is a powder, granules, tablets, capsules or beverages.