KR101461589B1 - Pharmaceutical composition for prevention and treatment of liver toxicity disease comprising Socheongryong-Tang or lactic acid bacteria fermented Socheongryong-Tang - Google Patents

Abstract

The present invention relates to a composition comprising a fermented product of lactic acid bacteria in Socheongryongtang and Socheongryongtang. More specifically, the present invention relates to a composition comprising a fermented product of Socheongryongtang produced by inoculating, culturing and fermenting lactic acid bacteria in Socheongryongtang and Socheongryongtang with hepatocyte- To a pharmaceutical composition for the prevention and treatment of hepatotoxic diseases.

Description

[0001] The present invention relates to a pharmaceutical composition for the prevention and treatment of hepatotoxic diseases including lactic acid bacteria fermented by Socheongryongtang or Sochi-ryongtang (Socheongryong-tang or lactic acid bacteria fermented Socheongryong-Tang)

The present invention relates to the use of fermented lactic acid bacteria of Socheongryongtang and Sochungryongtang, and more specifically to a pharmaceutical composition for preventing or treating a hepatotoxic disease comprising at least one of fermented lactic acid bacteria of Socheongryongtang or Sochungryongtang as an effective ingredient. The present invention also relates to a health functional food for preventing or ameliorating a hepatotoxic disease comprising one or more of lactic acid bacteria fermented by Soo-ryongtang and Sochi-ryongtang.

The liver is known to be closely related to mental activities, such as blood storage and circulation, blood circulation control and defense detoxification in the human body. Since our body is always exposed to various pollutants and toxic substances by industrialization, the liver is constantly suffering from detoxification. Moreover, liver damage due to mental stress is a serious problem. If mental restoration is performed, damaged liver cells are restored, but in a busy modern society, they can not afford to take a mental rest, so mental stress, And the human body can not defend and detoxify the immune system by causing abnormalities and cause other diseases. Liver disease is classified into viral liver disease, alcoholic liver disease, drug toxic liver disease, fatty liver, autoimmune liver disease, metabolic liver disease and other liver diseases depending on the cause of the disease. Since liver disease is not recognized until it has progressed considerably since it has no subjective symptoms at the beginning, it is at the top of the cause of death not only in Korea but also in the world. However, since there is no effective therapeutic agent, development of drugs that can treat and prevent liver damage is urgently needed to be.

Studies on liver protective activity were mainly focused on two pathways. One is to evaluate the detoxification reaction of toxic liver toxins such as carbon tetrachloride, tBHP, acetaminophen and hydrogen peroxide, and the other is serum glutamate pyruvate transaminase (GPT), alanine amino transferase ALT) and serum glutamate oxaloacetate transaminase (GOT, aspartate alanine aminotransferase (AST)).

Among them, hydrogen peroxide, which is a main component of reactive oxygen species, is known to induce apoptosis of various cells among various types of physiological oxidative stress inducers that cause apoptosis. In addition, tBHP, which is a typical substance causing lipid peroxidation, is decomposed into alkoxy or peroxy which can oxidize cell components by cytochrome P450 enzyme in hepatocytes, and these products result in DNA damage and eventually kill cells (Rush et al., Toxicol, Appl. Pharmanol. 78, p. 473, 1985). In addition, tBHP has been reported to induce ALT, AST, LDH leakage, MDA (malondialdehyde) formation and GSH deficiency in hepatocytes (Altman et al., Mutat Res. 306, pp35, 1994).

Meanwhile, Socheongryongtang is a well-known respiratory disease drug in Korea, Japan and China. It is composed of eight kinds of herbal medicines such as mahwang, peony, sesin, health, licorice, Socheongryongtang is mainly used for allergic rhinitis, allergic asthma, cold, etc. It is also known to be used for conjunctivitis, eczema, blister and nephritis. Recently, it has been found that Sohyeongryongtang acts antihistamines, and it has also been shown in other articles that it exerts an action of bronchial smooth muscle. It has also been reported that IL-4 and IgE levels in asthmatic patients are reduced and inflammatory cell infiltration into the airways is prevented to help asthma treatment. However, there is no research literature or other data on the composition for the prevention and treatment of hepatotoxic diseases containing Lactobacillus fermented product of Soo-ryong-tang or Soo-ryong-tang.

Thus, the present inventors confirmed that the fermented product of Sohwa-ryongtang lactic acid fermented by the inoculation of Lactobacillus acidophilus with Sochi-Ryongtang and Sochungryongtang has a protective function for preventing and treating hepatocyte damage against hepatotoxicity caused by oxidative stress such as hydrogen peroxide or tBHP, .

The present invention is to provide a pharmaceutical composition comprising one or more of Sochyungryongtang or Sohryungrytang lactic acid fermentation product having the preventive and therapeutic effect of hepatotoxic diseases.

In order to solve the above problems, the present invention provides a pharmaceutical composition for the prevention and treatment of hepatotoxic diseases comprising one or more of lactic acid bacteria fermented by Soojungryongtang and Sochungryongtang.

As used herein, the term " Socheongryongtang " is a prescription used for the treatment of coughing, pertussis and bronchitis caused by a cold. It is a prescription contained in the " upper limit " it means.

In clinical applications, this prescription is a typical recipe for seawater and dyspnea, which is a symptom of cold sputum that also serves as a symptom. In recent years, it has been shown that Sohyeongryongtang has antihistamine action, and other studies have shown that it exerts an action of bronchial smooth muscle. It is also known that IL-4 and IgE levels in asthmatic patients are reduced and inflammatory cell infiltration into the airways is blocked to aid asthma treatment. As an example of the prescription, it contains 5.62 g of each of mahwang, vinegar, omija, and vanha, and 3,75 g of seshin, health, licorice and licorice root.

The term " fermented product of lactic acid bacteria " used in the present invention means the result of culture obtained from a culture obtained by inoculating lactic acid bacteria into a culture medium on which lactic acid bacteria can grow. The term " culture supernatant " or " culture supernatant solution " obtained from a culture obtained by inoculating lactic acid bacteria with Socheongryongtang.

As a specific example, the lactic acid bacteria used in the present invention include Bifidobacterium, Lactobacillus, Streptococcus, Leuconostoc, Pediococcus and Lactococcus. Microorganisms belonging to the genus Lactobacillus can be used. Preferably, microorganisms belonging to the genus Lactobacillus can be used, but the present invention is not limited thereto.

The term " hepatotoxic disease " as used herein refers to a disease caused by cell damage caused by toxicity of hepatocyte, and specifically refers to damage to the liver, viral liver damage, hepatitis, liver cirrhosis, liver cancer, hepatic coma, Do not.

The term " preventive " used in the present invention means all actions that inhibit or delay the incidence of hepatotoxicity by using the fermented product of Lactobacillus of Socheongryongtang and Socheongryongtang according to the present invention.

The term " treatment " used in the present invention means all the actions of improving or improving the symptom of hepatotoxicity by using the fermented product of Lactobacillus of Sowongryongtang and Socheongryongtang according to the present invention.

The Lactobacillus fermented product of Sohyeongryongtang and Soojungryongtang of the present invention has a hepatocyte protective effect against hepatotoxicity by preventing oxidative stress induced by excess of active oxygen or cell damage induced by tBHP.

According to a specific example, Socheongryongtang significantly increased cell survival rate with respect to hepatocyte toxicity induced by hydrogen peroxide, decreased DNA fragmentation, decreased the activity of ALT, AST and LDH and decreased the amount of active oxygen (Figs. 3 and 4).

According to another specific embodiment, the fermented product of Lactobacillus acidophilus of Socheongryongtang remarkably increases the cell survival rate of hepatotoxicity induced by hydrogen peroxide and tBHP (tert-butyl hydroperoxide), and at the same time, Respectively.

The appropriate concentration of the Sowongryongtang or lactic acid bacteria fermented product for preventing and treating the hepatotoxic disease of the present invention is not particularly limited, but it may be preferably 10 μg / ml to 1,000 μg / ml. The pharmaceutical composition for preventing and treating the hepatotoxic disease according to the present invention may further comprise a pharmaceutically acceptable carrier, excipient or diluent.

Examples of pharmaceutically acceptable carriers include, but are not limited to, saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome, And if necessary, may further contain other commonly used additives such as antioxidants and buffers. In addition, a target organ or tissue-specific antibody or other ligand can be used in combination with the carrier so as to specifically act on the target organ. The carrier, excipient, or additive may be any conventional formulation, and the carrier, excipient, or additive is not limited by the above examples.

The composition according to the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols or the like, oral preparations, suppositories or sterilized injection solutions according to a conventional method. In detail, when formulating, it can be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants and the like which are usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient, such as starch, calcium carbonate, sucrose, lactose, gelatin, Can be mixed and prepared. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Examples of liquid formulations for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations and suppositories. Examples of the non-aqueous solution and suspension include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like. Examples of the suppository base include withexol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

As a concrete example, the fermented product of Lactobacillus thunbergii of the present invention can be prepared by the following steps: mixing herbal medicine including horse mackerel, peanut, sesquin, health, licorice, (Step 1); A step (step 2) of preparing a culture supernatant of Sowongyangyotang by inoculating mycelium of lactic acid bacteria into the thus prepared Sowongryongtang; And fermenting the supernatant liquid (step 3).

The step 1 is a step of preparing Sohyeongryongtang to produce a fermented product of Lactobacillus japonica. The composition can be prepared by a bath extraction method after preparing the pharmacological preparations such as mahwang, peony, sesin, health, licorice, licorice, bamboo, and omija. Specifically, the herbal medicine materials can be dipped in the living water and extracted with hot water to prepare a rice wine. The thus-prepared Syo-ryong-tang can be prepared into a powder form by freeze-drying.

Step 2 is a step of inoculating lactic acid bacteria into Socheongryongtang to prepare a fermented product of Sukyangryongtang lactic acid bacteria. The lactic acid bacteria may be microorganisms such as Bifidobacterium, Lactobacillus, Streptococcus, Leuconostoc, Pediococcus and Lactococcus. , Preferably microorganisms belonging to the genus Lactobacillus.

The step 3 is a step of fermenting a culture solution of Sowongyangyotang inoculated with lactic acid bacteria to produce a fermented product of Soychonryangtang lactic acid bacteria, and the fermentation is preferably carried out at 35 to 45 DEG C for 1 to 3 days under an aerobic atmosphere. More preferably, the lactic acid bacteria are inoculated into Socheonglong-tang and fermented in an aerobic atmosphere at 37 DEG C for 48 hours.

According to a specific embodiment, the pH of Socheongryongtang was adjusted to pH 8.0 using 1M sodium hydroxide to prepare a fermented product of Lactobacillus thunbergii. The fermented product was sterilized at 121 ° C for 15 minutes under high pressure, cooled at room temperature, The sterilized herbal formulations were inoculated at a concentration of 1% (v / v), fermented at 37 ° C for 24 hours and 48 hours, and then lyophilized to produce fermented lactic acid bacteria.

The present invention also provides a health functional food for preventing or ameliorating a hepatotoxic disease comprising one or more of the lactic acid fermented products of Socheongryongtang and Sochungryongtang.

As a specific example, the lactic acid bacteria used for the fermented product of Sohryungryongtang in health functional foods for the prevention or amelioration of hepatotoxic diseases include Bifidobacterium, Lactobacillus, Streptococcus, Leuconostoc ), Pediococcus, and Lactococcus. Preferably, microorganisms belonging to the genus Lactobacillus can be used, but the present invention is not limited thereto.

As a specific example, the fermented product of Soychonryongtang of the health functional food for preventing or ameliorating the hepatotoxic diseases can be prepared by the following steps: Mixing the herbal medicine including Maqin, peony, sesin, health, licorice, (Step 1); A step (step 2) of preparing a culture supernatant of Sowongyangyotang by inoculating mycelium of lactic acid bacteria into the thus prepared Sowongryongtang; And fermenting the supernatant liquid (step 3).

The step 1 is a step of preparing Sohyeongryongtang to produce a fermented product of Lactobacillus japonica. The composition can be prepared by a bath extraction method after preparing the pharmacological preparations such as mahwang, peony, sesin, health, licorice, licorice, bamboo, and omija. Specifically, the herbal medicine materials can be dipped in the living water and extracted with hot water to prepare a rice wine. The thus-prepared Syo-ryong-tang can be prepared into a powder form by freeze-drying.

Step 2 is a step of inoculating lactic acid bacteria into Socheongryongtang to prepare a fermented product of Sukyangryongtang lactic acid bacteria. The lactic acid bacteria may be microorganisms such as Bifidobacterium, Lactobacillus, Streptococcus, Leuconostoc, Pediococcus and Lactococcus. , Preferably microorganisms belonging to the genus Lactobacillus.

The step 3 is a step of fermenting a culture solution of Sowongyangyotang inoculated with lactic acid bacteria to produce a fermented product of Soychonryangtang lactic acid bacteria, and the fermentation is preferably carried out at 35 to 45 DEG C for 1 to 3 days under an aerobic atmosphere. More preferably, the lactic acid bacteria are inoculated into Socheonglong-tang and fermented in an aerobic atmosphere at 37 DEG C for 48 hours.

Preferably, the fermented soybean lactic acid bacteria according to the present invention may be added to foods or beverages for the purpose of preventing or ameliorating a hepatotoxic disease. The type of food is not particularly limited, and examples thereof include confectionery, bakery products, Drinks such as various foods, water, soft drinks and fruit drinks, gum, tea, vitamin complex, seasoning, and health functional foods.

The present invention relates to a pharmaceutical composition for prevention and treatment of hepatotoxic diseases or a pharmaceutical composition for preventing or treating hepatotoxic diseases or a pharmaceutical composition for preventing or treating hepatotoxic diseases since the composition comprising the fermented product of Lactobacillus acidus of Socheongryongtang and Sochungryongtang has hepatocyte protective activity against oxidative stress induced by hydrogen peroxide and cytotoxicity caused by tBHP There is an effect that it can be effectively used as a health functional food for prevention or improvement.

FIG. 1 is a graph of HPLC analysis of Lactobacillus amylophilus (B) obtained from Sochungryongtang (A) and Sochungryongtang.
FIG. 2 shows the results of MTT test on the protective effect of Sowoungryongtang on hydrogen peroxide-induced hepatocellular toxicity.
FIG. 3 shows the results of DNA electrophoresis on the protective effect of Sueongryongtang against hydrogen peroxide-induced hepatocellular toxicity.
FIG. 4 shows the results of measurement of the decrease in ALT, AST and LDH activity of hepatocytes pretreated with Socheongryongtang.
FIG. 5 shows the results of measurement of the reduction effect of Sowongryong-tang on the production of hydrogen peroxide-induced hepatocyte reactive oxygen (Cont: control group, H ₂ O ₂ : hydrogen peroxide treated group, H ₂ O ₂ + SCRT: hydrogen peroxide and treated rice bran.
FIGS. 6A and 6B show the results of measurement of the reduction effect of sueuroyongtang on tBHP-induced hepatocyte activation oxygen production (Cont: control group, tBHP: tBHP treatment group, tBHP + CY: tBHP and shitungryogang treatment group).
FIG. 7 shows the result of MTT test for the protective effect of Lactobacillus thunbergii on hydrogen peroxide-induced hepatocellular toxicity (CY: Sohyang Ryang Tang, CYC: The pH of Sochi Ryongtang was adjusted to pH 8.0 with 1M sodium hydroxide, (A) Lactobacillus casei (1), (B) Lactobacillus acidophilus, (B) Lactobacillus acidophilus, (B) Lactobacillus acidophilus, (C) (C) Lactobacillus casei (2), (D) Lactobacillus plantarum, (E) Lactobacillus amylophilus, (L) Lactobacillus curvatus, (G) Lactobacillus delbrueckus subsp. Lactis, , (J) Bifidobacterium thermophilum).
FIG. 8 shows the result of MTT test for the tBHP-induced hepatocellular toxicity (CY: Sohyang Ryongtang, CYC: the pH of Sochyangryongtang was adjusted to pH 8.0 with 1M sodium hydroxide, (B) Lactobacillus acidophilus, (C) Lactobacillus acidophilus, (C) Lactobacillus acidophilus, (C) Lactobacillus acidophilus, (C) Lactobacillus casei, (D) Lactobacillus plantarum, (E) Lactobacillus amylophillus, (L) Lactobacillus curvatus, (G) Lactobacillus delbrueckus subsp. Lactis, (L) Lactobacillus casei, (B) Bifidobacterium brevis, (J) Bifidobacterium thermophilum).
FIGS. 9A and 9B show the results of measurement of the reduction effect of lactic acid bacteria of Soychonryangtang to tBHP-induced hepatocyte reactive oxygen production (Cont: Control group, tBHP: tBHP treated group, tBHP + CY (E): tBHP and Lactobacillus Lactobacillus amylophilus fermentation).

Hereinafter, the constitution and effects of the present invention will be described in more detail through examples. These embodiments are only for illustrating the present invention, and the scope of the present invention is not limited by these embodiments.

Manufacturing example : Socheongryongtang  Produce

(Daejeon Metropolitan City, 23-4 Middle East), while Daejeon, Gyeonggi Province was purchased from Poongsan Pharmaceutical Co., Ltd. (311-1, Suhhadong, Gyeongbuk and Gyeongbuk) , Licorice, and omija were purchased from Meryung (377-26, Jinno-dong, Yeongcheon, Gyeongbuk). After that, test substances were prepared by the method of hot water extraction (Korea, Gyeongseo Extractor cosmos-660) in the order of mahwang, peony, sesin, health, licorice, Deokgyori 800) for 1 hour, followed by hot water extraction for 180 minutes, and Sowongryongtang was prepared in powder form using a freeze dryer (Ilshin FD5512, Korea).

Example  One. Sochyong-tang  Lactobacillus Fermented  Produce

1-1. Lactic acid bacteria  culture

45 Lactobacillus spp. And 2 Bifidobacterium spp. Were purchased from the Food Microbiology Gene Bank of Korea Food Research Institute and used for the experiment. The strains were cultivated in a standard medium (aerobic bacteria: MRS medium, anaerobic bacteria: Reinforced clostridial medium), and initial bacterial counts were adjusted to 10 ⁵ -10 ⁶ .

Lactobacillus strain broth for growth are MRS broth was used ^{(Difco TM Lactobacilli MRS Broth, Becton} Dickinson, Franklin Lakes, NJ, USA), For-based substitute slope medium and viable cell count measuring medium MRS agar medium (Bacto ^TM Lactobacilli MRS AGAR, Becton Dickinson). The composition of the MRS medium for the growth of Lactobacillus strains was as follows: 10 g / l peptone, 10 g / l juice, 5 g / l yeast extract, 20 g / l glucose, 1 ml / K ₂ HPO ₄ , 5 g / l sodium acetate, 2 g / l triammonium citrate, 0.2 g / l MgSO ₄ 7H ₂ O, 0.2 g / l MnSO ₄ 4H ₂ O, pH 6.2-6.6.

Reinforced Clostridial medium (Becton Dickinson) was used as a liquid medium for the growth of Bifidobacterium strains. The composition of Reinforced Clostridial medium for Bifidobacterium strain propagation is as follows: 10 g / L bacto-tryptose, 10 g / L bacterium-juice, 3 g / l bacto-yeast extract, 5 g / Strose, 5 g / l sodium chloride, 1 g / l water-soluble starch, 0.5 g / l L-cystine hydrochloric acid, 3 g / l sodium acetate, 0.5 g / l bacto-agar medium pH 7.6.

1-2. Using lactic acid bacteria Sochyong-tang  Lactobacillus Fermented  Produce

The pH of Socheongryong tang prepared in Preparation Example 1 was adjusted to pH 8.0 using 1M sodium hydroxide, sterilized at 121 ° C for 15 minutes, and cooled at room temperature. After that, 47 kinds of prevalent strains were inoculated at 1% (v / v) concentration in the sterilized herbal formulations and the pH change was observed after fermentation at 37 ° C for 24 hours and 48 hours. As a result, 10 strains of the following Table 1 were selected as the strain having a high pH-lowering ability and used in the production of fermented herbal medicine.

Bacteria Species A Lactobacillus casei (1) B Lactobacillus acidophillus C Lactobacillus casei (2) D Lactobacillus plantarum E Lactobacillus amylophillus F Lactobacillus curvatus G Lactobacillus delbrusal subsp. lactis H Lactobacillus casei (3) I Bifidobacterium brevis J Bifidobacterium thermophilum

To prepare the fermented herbal composition, 10 kinds of the lactic acid bacteria selected above were inoculated into the prepared medium at a concentration of 1% (v / v), cultured at 37 DEG C for 48 hours, and centrifuged to obtain a culture supernatant , And the obtained culture supernatant was lyophilized to prepare a fermented product of Sohryungryangtang lactic acid bacteria.

1-3. Announcement Metabolite  analysis

As medicinal herb indicators, there are Puerarin, daidzin, genistin, daidzein, genistein, hesperidin, hesperetin, The naringin was purchased from Sigma Chemical Co. (St. Louis, Mo., USA) and purchased as paeoniflorin, glycyrrhizin,? -Glycyrrhetinic acid,? -Glycyrrhetinic acid acid, baicalin, baicalein, loganin and aucubin were purchased from Wako pure chemical (Osaka, Japan).

2 ml of the fermented product of Sowongryongtang lactic acid bacterium prepared in Example 1-2 was taken and lyophilized. 4 ml of ethanol was added to the lyophilized sample, and the mixture was shaken at 60 ° C for 25 minutes in a shaking incubator, centrifuged (1,500 g × 1,000 sec at 10 ° C) to remove the residues, and then filtered through a 0.2 μm syringe filter (Millipore Co., Bedford, Mass., USA) and subjected to HPLC analysis. The results are shown in FIG.

As a result, as can be seen from Fig. 1, it was possible to observe an increase or decrease of the peak compared to the case of Syoheungryotang (Fig. 1 (A)) which was not fermented in the case of Socheongryongtang fermented by lactic acid bacteria (Fig. 1 (B) And it was confirmed that the composition of Sochyong -

Example  2: Socheongryongtang Hepatotoxicity  Prevention and treatment effectiveness evaluation

2-1. Primary culture of hepatocytes

Primary hepatocyte culture was performed using a two - stage collagenase perfusion method modified from Berry 's and Friend' s methods. The rats were anesthetized with intravenous injection of Zoletil and rumpun, followed by laparotomy. Twenty-four gauze catheters were intubated through the portal vein, perfused with Krebs-Ringer-HEPES buffer solution and circulated in a digestive solution consisting of collagenase (Sigma, USA) . After the hepatocytes were digested, hepatocytes were liberated by stripping the hepatic membrane and filtered using a nylon mesh. The filtrate was centrifuged at 400 rpm for 3 minutes, the supernatant was discarded, and the precipitated hepatocytes were suspended in a buffer solution containing BSA (Sigma, USA) and centrifuged again. After the precipitated hepatocytes were suspended in the culture solution to obtain a cell suspension, the hepatocyte concentration was adjusted to be 2 x 10 < ⁶ > cells / ml and transplanted into a culture container previously coated with gelatin. The culture medium contained 10% FBS (Hyclone), 500 U / L insulin (Roche, Switzerland), 2 mM L-glutamine (Lonza), 15 mM HEPES (Lonza, Switzerland), 10 ⁵ U / l penicillin, 100 mg / William's E media (Gibco) containing mycine (Gibco, USA) was used. In a 37 ° C incubator maintained at constant temperature and humidity, CO ₂ 5% mixed gas was supplied and cultured.

2-2. Socheongryongtang  Cell viability test

In order to confirm the effect of protecting the hepatocytes of Socheongryongtang prepared in the above Preparation Example, MTT test was performed on the cytotoxicity induced by hydrogen peroxide.

The cells were pretreated with 10, 30, 100, 300, 1000 ㎍ / ml for 1 hour, treated with 1 mM of hydrogen peroxide and incubated for 24 hours. MTT reagent was added to MTT Reagents were added. After addition of MTT reagent and incubation for 1 hour, the culture solution was removed, and 150 μl of DMSO was added to each well of the culture dish. After shaking for 30 minutes, absorbance was measured at 570 nm using an ELISA reader. The relative cell viability of the control cells treated with Socheongryongtang and hydrogen peroxide was determined as 100%. The results are shown in Fig.

As shown in FIG. 2, the cell survival rate of the cell group (-, +) treated with hydrogen peroxide for the control group (-, -) without any treatment decreased to 50% or less. However, the cell survival rate was remarkably increased with addition of Socheongryongtang. In particular, when 100. And 300 .mu.g / ml of Socheongryongtang added, the cell survival rate was restored to 100% by completely blocking the cytotoxicity by hydrogen peroxide there was.

2-3. Socheongryongtang DNA  Fragmentation test

DNA fragmentation test was performed in order to confirm the cell protection effect by preprocessing of Sowongkyung-tang prepared in the above-mentioned preparation example in the cells in which cell death was induced by hydrogen peroxide.

As a control group, genomic DNA was extracted from primary hepatocytes of mouse and electrophoresis was performed, and the fragmentation pattern was observed. In addition, pre-treatment of Sowongyangryotang was performed on the primary hepatocytes of the same mouse for 1 hour, 1 mM of hydrogen peroxide was treated for 24 hours, and genomic DNA was extracted and subjected to electrophoresis. The results are shown in Fig.

In FIG. 3, DNA fragmentation was not observed in the control group (-, -) and Sowongryongtang treated group (-, +) without any treatment, but DNA fragmentation was observed in the hydrogen peroxide alone treatment group (+, -) , And the cells were killed by hydrogen peroxide. On the other hand, in the group (+, +) pretreated with Socheongryongtang, DNA was observed to the same degree as the control group and Socheongryongtang alone, and it was confirmed that DNA fragmentation was suppressed to a good extent by pretreatment of Socheongryongtang.

2-4. Socheongryongtang  Cytotoxicity test

The amount of ALT, AST, and LDH was measured in order to confirm the reduction of hydrogen peroxide - induced hepatocellular toxicity when pretreated with.

The primary hepatocytes of mouse were pretreated for 1 hour and treated with 1 mM of hydrogen peroxide for 6 hours. Cell culture medium was collected to investigate the amount of LDH and the levels of ALT and AST, which are indicators of cytotoxicity, The results are shown in Fig.

As shown in FIG. 4, the activity of ALT, AST and LDH was increased when hydrogen peroxide alone was treated. However, it was confirmed that the activity of ALT, AST and LDH by hydrogen peroxide was significantly decreased at 100 ㎍ / ml and 1000 ㎍ / ml of Socheongri - tang when pretreated with Socheongryongtang. In particular, at a concentration of 1000 μg / ml of Sociedryongtang, the activity was lower than that of ALT, AST and LDH of the control group, which was not treated.

2-5. Socheongryongtang H ₂ O ₂  Inductive On hepatotoxicity  Active oxygen Degree of formation  exam

To confirm the inhibitory effect of Socheongryongtang on the production of active oxygen, the amount of reactive oxygen species in the cells was measured by pretreatment of Socheongryongtang in mouse primary hepatocytes for 1 hour and treatment of 1 mM of hydrogen peroxide for 4 hours using FACs. Respectively. As a negative control, the amount of active oxygen in the mouse primary hepatocytes without any addition was measured, and the amount of active oxygen generated in the mouse primary hepatocytes treated with only hydrogen peroxide was measured as a positive control.

As shown in FIG. 5, it was confirmed that the amount of active oxygen was increased when hydrogen peroxide was added to the amount of active oxygen in the control group (cont) in which nothing was added. On the other hand, it was confirmed that Sochi - ryong - tang reduced active oxygen induced by hydrogen peroxide when pretreatment of Socheongryong - tang produced less active oxygen than the control group treated with hydrogen peroxide alone.

2-6. Socheongryongtang tBHP  Inductive On hepatotoxicity  Active oxygen Degree of formation  exam

In order to confirm the inhibitory effect of Socheongryongtang on the production of active oxygen, the amount of reactive oxygen species in the cells was measured by FACs after 1 hour pretreatment of Sukyangryongtang with mouse primary hepatocytes and treatment with 0.1 mM or 0.2 mM of tBHP for 4 hours. The results are shown in Figs. 6A and 6B. The amount of active oxygen in the primary hepatocytes without any addition of the negative control group (cont) was measured in FIG. 6A, and the amount of reactive oxygen generated in the primary hepatocytes treated with only 0.1 mM or 0.2 mM of tBHP as a positive control group was measured and compared.

As can be seen from FIG. 6A, it was confirmed that the amount of active oxygen increased with addition of tBHP to the amount of active oxygen in the control (cont) in which nothing was added. On the other hand, as shown in FIG. 6B, when pre-treated with Socheongryongtang, less active oxygen was produced than in the positive control group treated with tBHP alone, confirming that Socheongryongtang decreased reactive oxygen induced by tBHP.

Example  3: Sochyong-tang  Lactobacillus Fermented Hepatotoxicity  Prevention and treatment effectiveness evaluation

3-1. Sochyong-tang  Lactobacillus Fermented  Cell viability test (hydrogen peroxide induced MTT assay )

To confirm the protective effect of hepatocyte on the fermented product of Sohryungryongtang lactic acid bacteria prepared in the above example, MTT test was performed on the cytotoxicity induced by hydrogen peroxide.

The hepatocytes cultured in Example 2-1 were pretreated with 10 kinds of the fermented lactic acid bacteria of Sochyongryotang prepared in Example 1 at the concentrations of 100, 200, 500, and 1000 / / ml for 1 hour and treated with 1 mM of hydrogen peroxide And cultured for 24 hours. MTT reagent was added so that the culture solution and MTT reagent had a ratio of 10: 1. MTT reagent was added and incubated for 1 hour. After removing the culture medium, 150 μl of DMSO was added to each well of the culture dish and shaken for 30 minutes. Absorbance was measured at 570 nm using an ELISA reader. The relative cell viability of the control group (con) without any treatment was determined to be 100%. As a positive control, the pH of Sowongryongtang (CY) prepared in Preparation Example and the pH of the Sowongryongtang were adjusted to pH 8.0 using 1M sodium hydroxide , And the cell viability of Sojung Ryong Tang (CYC) treated at 121 ° C for 15 minutes under high pressure sterilization was determined. The results are shown in FIG.

As can be seen in FIG. 7, the cells treated with hydrogen peroxide for the negative control group treated with nothing showed a cell survival rate of less than 40%. However, it was confirmed that the cell survival rate was remarkably increased by adding the fermented lactic acid bacterium of Sohryungryungtang.

3-2. Sochyong-tang  Lactobacillus Fermented  Cell viability test ( tBHP  By induction MTT assay )

To confirm the protective effect of hepatocyte on the fermented product of Sohryungryangtang lactic acid bacteria prepared in the above example, MTT test was performed on tBHP-induced cytotoxicity.

The same test as in Example 3-1 was carried out except that tBHP was used instead of hydrogen peroxide. The results are shown in FIG.

As can be seen from FIG. 8, cells treated with tBHP for the control group (con), which had not been treated with anything, had a cell survival rate of 50% or less. However, it was confirmed that the cell survival rate was remarkably increased by pretreating the fermented product of Lactobacillus thunbergii.

3-3. Sochyong-tang  Lactobacillus Fermented  Active oxygen Generative  exam

To confirm the effect of fermentation of lactic acid bacteria fermented by Sochyongryuntang, the primary hepatocytes of mouse were pretreated with 200 ㎍ / ml fermented soybean milk fermented product (CY (E)) for 1 hour and treated with 1 mM tBHP 0.1 mM or 0.2 mM for 4 hours After that, the amount of reactive oxygen generated in the cells was measured using FACs. The results are shown in FIGS. 9A and 9B. As a negative control, the amount of active oxygen of primary mouse hepatocytes without any addition was measured, and the amount of active oxygen generated in mouse primary hepatocytes treated with tBHP alone as a positive control was measured and compared.

As can be seen from FIG. 9A, it was confirmed that the amount of active oxygen was increased when tBHP was added to the amount of active oxygen in the control group to which nothing was added. On the other hand, as shown in FIG. 9B, pretreatment of Lactobacillus amylophilus fermented product (CY (E)) produced less active oxygen than that of the tBHP-treated positive control, so that fermented lactic acid bacteria of Sochungryongtang were induced by tBHP Of the total amount of active oxygen.

Claims (8)

A pharmaceutical composition for the prevention and treatment of tBHP induced hepatotoxic diseases, which comprises Bifidobacterium brevis or Bifidobacterium sulphurum thermophilum fermented product of Socheongryongtang.
delete The pharmaceutical composition for the prevention and treatment of hepatotoxic diseases according to claim 1, wherein the fermented product of Socheongryongtang is prepared by the following steps:
Preparing a sorghum-yang-tang by mixing and extracting hot-water extracts of herbal medicines including horse mackerel, peanut, sesquin, health, licorice,
Preparing a culture solution of Sikyeongryongtang by inoculating the prepared Sikyeongryongtang with Bifidobacterium brevis or Bifidobacterium sulfamoyl mycelium; And
The step of fermenting the supernatant of the supernatant.
4. The pharmaceutical composition according to claim 3, wherein the fermentation is carried out in an aerobic atmosphere at a temperature of 35 DEG C to 45 DEG C for 1 to 3 days.
A health functional food for prevention and improvement of tBHP induced hepatotoxic diseases including Bifidobacterium brevis or Bifidobacterium sulfamoil fermented product of Socheongryongtang.
delete [Claim 6] The method according to claim 5, wherein the fermented product of Sowok-ryongtang is prepared by the following steps:
Preparing a sorghum-yang-tang by mixing and extracting hot-water extracts of herbal medicines including horse mackerel, peanut, sesquin, health, licorice,
Preparing a culture solution of Sikyeongryongtang by inoculating the prepared Sikyeongryongtang with Bifidobacterium brevis or Bifidobacterium sulfamoyl mycelium; And
The step of fermenting the supernatant of the supernatant.
The health functional food for preventing and improving tBHP induced hepatotoxic diseases according to claim 7, wherein the fermentation is carried out at a temperature of 35 ° C to 45 ° C under an aerobic atmosphere for 1 to 3 days.

Images