WO2012064158A2 - Composition comprising fermented medicinal herbs for treating irritable bowel syndrome - Google Patents

Abstract

The present invention relates to a pharmaceutical composition and functional health food for treating irritable bowel syndrome, comprising a mixture of fermented medicinal herbs, wherein the mixture contains fermented Atractylodis Rhizoma Alba, Massa Medicata Fermentata, and Semen Dolichoris. More particularly, the present invention relates to a pharmaceutical composition and functional health food for treating irritable bowel syndrome, comprising a mixture of fermented medicinal herbs, wherein the mixture contains Atractylodis Rhizoma Alba, Massa Medicata Fermentata, and Semen Dolichoris which are fermented under optimum fermentation conditions for maximizing the effects thereof in treating functional gastrointestinal disorders.

Description

Formal composition using fermented herbal medicine

The present invention relates to a formal pharmaceutical composition and health functional food comprising a mixture of herbal medicines containing fermented Baekchul, Singok and Baekpok. More specifically, the present invention relates to a formal pharmaceutical composition and a dietary supplement comprising a mixture of herbal medicines containing fermented baekchul, singok and baekryeom as optimal fermentation conditions for maximizing the efficacy of functional bowel disease.

Irritable bowel syndrome (IBS) is a disease characterized by abdominal pain and changes in bowel habits (diarrhea, constipation, or repeated diarrhea and constipation). Typical symptoms are intermittent but sometimes persistent and last for at least three months. It is a chronic disease that should be present. Although it is estimated that about 42.4 million people suffer from constipation, diarrhea or both symptoms of irritable bowel syndrome in the United States, only 30% of people are treated because IBS is not life-threatening. Of these, only 8.8% of them take prescription drugs.

IBS disease is more common in people who are nervous or stressed, have a weak stomach, have a lack of exercise, and are meticulous and timid. For example, it is necessary to develop treatment and prevention techniques for various diseases such as office workers, examinees, young housewives, job drivers, postpartum or postoperative weakness, irregular smokers and drinkers.

Irritable bowel syndrome has many causes, but it is closely related to the imbalance of the intestinal microorganisms, that is, the lack of beneficial bacteria and the excessive number of harmful bacteria that adversely affect metabolism, so in the West, many probiotic agents called probiotics It is utilized.

At present, the market for formal medicine is increasing in connection with lifestyle diseases of the modern people.The development of new products that can enhance the functioning of the digestive system and 'strengthen' the bowel is not the 'formal medicine' which is eaten only when the stomach hurts or diarrhea. It is desperately required. As Korea is gaining global recognition in fermenting foods such as kimchi and soybean paste, it is thought that it will be able to enhance its competitiveness by developing a composition combining herbal medicine and fermentation technology. Fermented herbal medicines with controlled microorganisms and living strains are expected to increase added value.

People with chronic diseases that are likely to lack intestinal microorganisms can enhance the effectiveness of Chinese medicine by administering fermented herbs that have been fermented outside the body, which can be a cornerstone for the role of microorganisms, pharmacological action of natural products, and development of natural medicines. have. In addition, because the Chinese herbal medicine is late absorption and late expression, it is necessary to develop a herbal medicine that is expressed quickly and quickly.

Thus, the present inventors produce a component having an improved yield yield and improved toxin reduction efficacy of the natural-derived formal component in the mixed fermentation tablet by using the fermented natural product resources, and has a formal effect through the mixed fermentation of beneficial bacteria of the formal intestinal efficacy verified. The present invention has been completed by maximizing the development of a high functional material having an effect on chronic irritable bowel disease and stomach ailment, food intolerance, and developing a composition that can be utilized for health promotion of all the people based on the material.

It is an object of the present invention to provide a pharmaceutical composition for dietary supplements and health functional food comprising a mixture of herbal medicines containing fermented baekchul, singok and baekryeom. More specifically, it is to provide a formal pharmaceutical composition and health functional food comprising a mixture of herbal medicines containing fermented Baekchul, Singok and Baekdu as fermentation conditions to maximize the efficacy of functional bowel disease.

In order to achieve the object of the present invention as described above, the present invention provides a formal composition comprising a mixture of fermented Baekchul, Singok and Baekdudu.

In the present invention, the fermentation of the baekchul is preferably fermented with Bacillus licheniformis (Bacillus licheniformis) as a fermentation strain of 1 to 3% glucose. In addition, the fermentation of the new song is preferably fermented with 1% to 3% glucose as a fermentation strain of Leuconostoc mesenteroides. In addition, the fermentation of the white migraine is preferably fermented with 1% to 3% LB broth as Bacillus rickeniformis as a fermentation strain.

The ratio of fermented baekchul, new song, and white bean contained in the mixture is preferably in the range of 0.5 to 2 parts by weight of fermented new song and 0.5 to 2 parts by weight of fermented white bean, based on 1 part by weight of fermented baekchul. More preferably, the weight ratio of the white out, the new song, and the white bean is 1: 1: 1. In addition, the mixture may be administered to the subject in an amount of 50 to 200 mg / kg, preferably administered to the subject in an amount of 50 to 100 mg / kg.

In addition, the present invention provides a dietary supplement for dietary supplement comprising a mixture of fermented Baekchul, Singok and Baekpok. The ratio of fermented baekchul, new song, and white bean contained in the mixture is preferably in the range of 0.5 to 2 parts by weight of fermented new song and 0.5 to 2 parts by weight of fermented white bean, based on 1 part by weight of fermented baekchul. More preferably, the weight ratio of the white out, the new song, and the white bean is 1: 1: 1.

The composition of the present invention can be used as a medicine and health food by using it because it has excellent suitability effect, has no absorption rate and no toxicity or side effects in vivo, and excellent stability.

1 is a graph showing the effect of various concentrations of fermented or non-fermented herbal medicines on RAW264.7 cells on cell viability. C represents a control group.

Figure 2 is a graph related to the protective action of fermented and non-fermented herbal medicine against LPS-induced cytotoxicity. C represents the control group and a, b represent significant differences from the control and LPS treated groups, respectively (p <0.05).

Figure 3 is a graph showing the effect of the herbal medicine on the gene expression of iNOS expressed in RAW264.7 cells. C represents the control group and a represents the significant difference from the LPS treated group (p <0.05).

Figure 4 is a graph showing the effect of the herbal medicine on COX-2 expression in LPS induced RAW264.7 cells. C represents the control group and a represents the significant difference from the LPS treated group (p <0.05).

5 is a graph showing the inhibitory effect of fermented herbal and non-fermented herbal medicines on NO produced from RAW264.7 cells by LPS. C represents the control group and a represents the significant difference from the LPS treated group (p <0.05).

FIG. 6 is a graph showing the effect on LPS-induced endotoxin levels of mixed fermented and unfermented or colostrum. a shows a significant difference from the group treated with LPS (p <0.05).

7 is a graph showing the effects of mixed fermented and non-fermented herbal medicines on serum CRP levels induced by LPS.

FIG. 8 is a graph showing the lactulose / mannitol ratio following the injection of mixed fermented or unfermented or colostrum in rats treated with LPS. a shows a significant difference from the group treated with LPS (p <0.05).

9 is a graph of the water content of feces following injection of mixed fermented or unfermented or probiotics in rats treated with neomycin. a shows significant difference from the group treated with neomycin (p <0.05).

FIG. 10 is a graph of CRP levels following infusion of fermented or unfermented or probiotics in rats treated with neomycin. a shows significant difference from the group treated with neomycin (p <0.05).

FIG. 11 is a graph of interferon-γ following injection of mixed fermented or unfermented or probiotics in rats treated with neomycin. a shows significant difference from the group treated with neomycin (p <0.05).

FIG. 12 is a micrograph of colon tissue following administration of mixed fermented or unfermented or probiotics in rats treated with neomycin. In antibiotic-induced diarrhea, intestinal membrane damage, infiltration of inflammatory cells, edema and discontinuity of lower tissues were observed, but pathologic findings were clearly improved in the intestinal mucosa of fermented herbs.

FIG. 13 is a graph showing Lactobacillus proliferation following injection of mixed fermented or unfermented or colostrum in rats treated with LPS. It can be seen that the microorganisms of the genus Lactobacillus significantly increased in the fermented herbal medicine group compared to the control group.

14 is a graph showing the intestinal beneficial bacteria (Bifidobacterium) proliferation following injection of mixed fermented or unfermented or colostrum in rats treated with LPS. Compared to the control group, the fermented Chinese herbal medicine group showed significantly increased Bifidobacterium genus.

The present invention provides a formal pharmaceutical composition comprising a mixture of fermented baekchul, singok and baekryeom.

The pharmaceutical composition of the present invention includes fermented medicinal herb containing fermented Baekchul, Singok and Baekdudu at optimum fermentation conditions for maximizing the efficacy of functional bowel disease, and Baekchul, Singok contained in the mixture of the herbal medicine , The ratio of Baekdudu is preferably in the range of 0.5-2 parts by weight of fermented new song and 0.5-2 parts by weight of fermented Baengdu with respect to 1 part by weight of fermented Baekchul. It is more preferable that it is a weight ratio of: 1.

"Atractylodis Rhizoma alba" in the present invention belongs to the Asteraceae, and its components are known as Atractylone, Atractylenolide, Beta-Eudesmol and Heynesol. Exudation acts as a diuretic and sweating effect on the abnormality of water metabolism in the digestive tract and subcutaneous tissue, and it is effective in pain and gastroenteritis.

In addition, "new song" in the present invention is a medicinal herb made by mixing fermented flour, bran, spear juice, fermented juice, cheongho juice, hanginni, red bean, etc., is used as a drying agent and a fire extinguishing agent.

In the present invention, "white flakes (Latin name Dolichos Semen, scientific name Dolichoris lablab L., scientific name Leguminosae)" is a white, dried seed, round egg shape. It strengthens the stomach, harmonizes the middle and lowers the heat, has the effect of reducing moisture, and cures the loss of appetite due to vomiting and diarrhea. The taste is sweet and the properties are slightly warm. It contains 0.227% protein, 0.018% fat, 0.57% carbohydrate, calcium, phosphorus, zinc and iron, and has trypsin inhibitor, amylase inhibitor, hemagglutinin A and B Contains.

Preferred fermentation conditions for maximizing the efficacy of functional intestinal diseases of the respective herbs are as follows.

First, in case of baekchul, it can be dissolved in autoclaved Milli-Q water and fermented. It is preferred to inject 1-3% LB broth or 1-3% glucose as the medium during fermentation, more preferably 2% glucose. After the mixture is cooled by autoclaving, bacterial inoculum can be injected and fermented. The bacterial inoculum is preferably Bacillus rickeniformis, Lactobacillus acidophilus or Leukonostock mesenteroides, and more preferably Bacillus rickeniformis as a fermentation strain. .

For white miso, it can be dissolved in autoclaved Milli-Q water and fermented. During fermentation, it is preferred to inject 1-3% LB broth or 1-3% glucose as the medium, more preferably inject 2.5% LB broth. After the mixture is cooled by autoclaving, bacterial inoculum can be injected and fermented. The bacterial inoculum is preferably Bacillus rickeniformis, Lactobacillus ashidophilus or Leukonostock mesenteroides, and more preferably Bacillus rickeniformis as a fermentation strain.

The new song can be fermented by dissolving in autoclaved Milli-Q water. It is preferred to inject 1-3% LB broth or 1-3% glucose as the medium during fermentation, more preferably 2% glucose. The mixture may be cooled by autoclaving and then fermented by injecting bacterial inoculum. The bacterial inoculum is preferably Bacillus rickeniformis, Lactobacillus ashidophyllus or Leukonostock mesenteroides as the fermentation strain, and more preferably using Leukonostock mesenteroides.

In the above fermentation strain, fermentation at 30-32 ° C. for Bacillus rickeniformis and 33-36 ° C. for leukonostock mesenteroides is preferable as the optimum growth temperature of the strain.

In addition, the formal pharmaceutical composition of the present invention may further include a pharmaceutically acceptable additive. The additives may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

Pharmaceutical dosage forms of the compositions of the present invention may be used in the form of their pharmaceutically acceptable salts, and may be used alone or in combination with other pharmaceutically active compounds, as well as in any suitable collection.

The pharmaceutical preparations comprising the compositions according to the invention can each be in the form of tablets, pills, capsules, powders, suspensions, granules or extracts in the form of oral formulations, external preparations, suppositories and sterile injectable solutions according to conventional methods. Can be formulated and used. Carriers, excipients and diluents that may be included in the pharmaceutical formulation comprising the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium Phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxy benzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, which may comprise at least one excipient in the composition, for example starch, calcium carbonate, sucrose or lactose, gelatin The mixture is prepared. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, utopsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

Preferred dosages of the compositions of the present invention vary depending on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. In one embodiment of the invention, the fermentation mixture may be administered to the subject in an amount of 50 to 200 mg / kg, preferably administered to the subject in an amount of 50 to 100 mg / kg. Administration may be administered once a day or may be divided several times.

The composition of the present invention can be administered to mammals such as rats, mice, livestock, humans, etc. by various routes. All modes of administration can be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

In addition, the present invention provides a dietary supplement for dietary supplement comprising a mixture of fermented Baekchul, Singok and Baekpok.

Formal health functional food of the present invention is characterized in that it comprises the fermentation mixture as an active ingredient showing the efficacy of the prevention and treatment of functional bowel disease. Examples of foods to which the fermented products of the herb may be added include, but are not limited to, various foods, powders, granules, tablets, capsules, syrups, beverages, gums, teas, vitamin complexes, and health functional foods. no.

In addition, the fermentation mixture may be added to food or beverage for the purpose of preventing the functional bowel disease. At this time, the amount of the fermented product in the food or beverage may be added to 0.01 to 15% by weight of the total food weight, the health beverage composition may be added in a ratio of 0.02 to 5g, preferably 0.3 to 1g based on 100ml. .

The health functional beverage composition of the present invention is not particularly limited to other ingredients except the fermented product as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. . Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (tauumatin, stevia extract (e.g., Rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of natural carbohydrates 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 fermented products of the present invention include various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavoring agents, colorants and neutralizing agents (such as cheese, chocolate), pectic acid and salts thereof, alginic acid and Salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. In addition, the fermented products of the present invention may contain fruit flesh for the production of natural fruit juices and fruit juice beverages and vegetable beverages. These components can be used independently or in combination. The proportion of such additives is not so critical but is usually chosen in the range of 0 to about 20 parts by weight per 100 parts by weight of the fermentation of the invention.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are only for illustrating the present invention, and the contents of the present invention are not limited by the following examples.

Example 1 Preparation of Fermented Composition

20 g of each of baekchul, new song and white migraine were put into 200 ml of autoclaved Milli-Q water (Millipore Corp., Milford, Mass., USA), and then each medicine was extracted by ultrasonication at 70 ° C., followed by 70 for 3 hours. It was left at 占 폚. Each medicinal extract was subjected to autoclaving at 121 ° C. for 20 minutes. Thereafter, LB bacterial medium or glucose in powder form was injected during their fermentation. After cooling the sterilized mixture, 2% (v / v) of bacterial inoculum was injected into the medium, followed by 24 at 31 ° C. (Bacillus sp.) Or 35.4 ° C. (Leuconostoc sp.). Fermented for hours.

The non-fermented preparation of each herb was carried out in a similar manner as above, but 2% (v / v) sterile bacterial medium was used in place of the bacterial inoculum. In animal experiments, non-fermented products of individual herbals were sterilized (sterilized non-fermented products) to kill bacteria, and the experimental group was used without sterilization.

Example 2 In Vitro Efficacy of Composition

<2-1> Survival Rate Analysis

The rodent macrophage cell line, RAW264.7 cells, was cultured in DMEM using 10% heat inactivated fetal calf serum, 2 mM L-glutamine, 100 U / ml penicillin, and 100 mcg / ml streptomycin. Incubated at 37 ° C., 5% CO ₂ incubator. RAW264.7 cells were dispensed in 24-well plates at 2 × 10 ⁵ cells / well and then incubated overnight in incubator. Thereafter, 10 mcg / ml of LPS (Pseudomonas aeruginosa 10, Sigma, St. Louis, 10%) in RAW264.7 cells with or without varying concentrations (0.5% to 10% v / v) of fermented and non-fermented herbs. MO, USA) and incubated for 24 hours. Media and fermentation strains for each herbal medicine are shown in Table 1 below.

Table 1

number	Herbal Medicine	badge	Fermentation strain
1-1	new song	2% glucose	Lactobacillus ashdophyllus
1-2	new song	2% glucose	Leukonostock Mecentteroides
2-1	Whiteness	none	none
2-2	Whiteness	2.5% LB Broth	Bacillus rickenformis
2-3	Whiteness	2% glucose	Bacillus rickenformis
3-1	White migraine	2.5% LB Broth	Bacillus rickenformis
3-2	White migraine	2% glucose	Bacillus rickenformis

Reduction of reaction was induced by incubating for 3 hours after adding MTT, and medium was carefully removed from each well and DMSO solution was added to completely dissolve formazan crystal byproduct. The absorbance of each well was measured at 570 nm using a microplate reader (Spectramax Plus, Molecular Devices, Sunnyvale, Calif., USA) and cell viability was expressed as OD value.

<2-1-1> Effect of Medicinal Herbs on Cell Viability and Protective Effects against LPS-induced Cytotoxicity

1, in the MTT cytotoxicity assay, the experimental group treated with the fermented herbal and non-fermented herbal did not cause cytotoxicity at any dose compared to the control group.

In addition, looking at Figure 2, when treated with 10mcg / ㎖ LPS showed a cytotoxic effect causing a 58% loss of cell viability compared to the control. All of the fermented and non-fermented herbal treatments except the experimental group treated with 1-1 and 3-2 samples at 0.5% concentration maintained higher cell viability than the LPS-only treatment. Of the 2-3 and 3-1 experimental groups, the 10% treatment group showed the highest protective effect against LPS-induced cytotoxicity, with cell survival rates up to 102% and 103%, respectively.

<2-1-2> Effect of Herbal Medicine on Gene Expression of iNOS Expressed in RAW264.7 Cells

RAW264.7 cells were dispensed into 6-well plates at 8 × 10 ⁵ cells / well and incubated overnight in incubator. Thereafter, 5% and 10% fermented herbal and non-fermented Chinese herbs were administered or not treated with 10 mcg / ㎖ LPS was incubated for 24 hours. RNA was then extracted from the cells using Trizol® reagent (Invitrogen, Carsbad, CA, USA) and 2 μg RNA was extracted from the cDNA synthesis kit (SprintTM RT Complete Oligo- (dT) 18, Clontech, Mountain View, CA, USA ) Was reverse transcribed to obtain the first chain cDNA. The final volume of 20 mcL samples were analyzed by RT-PCR on a LightCycler instrument (Roche Applied Science, Indianapolis, Idaho, USA) using LightCycler-FastStart DNA Master SYBR Green (Roche Diagnostic, Roche Applied Science).

RT-PCR was performed to investigate the effect of the drug on gene expression of iNOS and COX-2 expressed in RAW264.7 cells. For this purpose, RAW264.7 cells were dispensed in 6-well plates at 8 × 10 ⁵ cells / well and incubated overnight in incubator. Since 5% and 10% fermented herbal or non-fermented Chinese medicine was administered or not treated with 10mcg / ㎖ LPS was incubated for 24 hours. RNA was then extracted from the cells using Trizol® (Invitrogen, Carsbad, CA, USA), reverse transcription of 2mcg RNA, followed by cDNA synthesis kit (SprintTM RT Complete Oligo- (dT) 18, Clontech, Mountain View, CA , USA) to obtain the first-chain cDNA. Samples of final volume of 20 mcL were subjected to RT-PCR analysis on a LightCycler instrument (Roche Applied Science, Indianapolis, Idaho, USA) using a LightCycler FastStart DNA Master SYBR Green kit (Roche Diagnostic, Roche Applied Science). At this time, PCR was performed using a primer pair (Bioneer, Daejeon, Korea) described in SEQ ID NO: 1 to SEQ ID NO: 6 (Table 2).

TABLE 2 Primer Sequences for iNOS, COX-2 and GAPDH

primer	Sequence	SEQ ID NO:
iNOS-forward	5'-AGCCCAACAATACAAGATGACCCTA-3 '	One
iNOS-reverse	5'-TTCCTGTTGTTTCTATTTCCTTTGT-3 '	2
COX-2-forward	5'-AGAAGGAAATGGCTGCAGAA-3 '	3
COX-2-reverse	5'-GCTCGGCTTCCAGTATTGAG-3 '	4
GAPDH-forward	5'-TGATGACATCAAGAAGGTGGTGAAG-3 '	5
GAPDH-reverse	5'-TCCTTGGAGGCCATGTAGGCCAT-3 '	6

PCR amplification was performed as follows: COX-2 (40 amplifications after 95 ° C. 10 min incubation step, 95 ° C. 10 sec denaturation, 53 ° C. 10 sec annealing, and 72 ° C. 15 sec extension): GAPDH (55 amplifications) , 94 ° C. 3 min incubation, 94 ° C. 30 sec denaturation, 56 ° C. 30 sec annealing, 72 ° C. 90 sec extension): and PCR parameters for iNOS were identical to the PCR parameters of GAPDH except for 55 times amplification of GAPDH. . In order to confirm the specificity of the reaction, melting curve analysis was performed immediately after amplification, and data was analyzed using LightCycler Software provided by Roche Diagnostic. In each assay, water was used instead of template cDNA to check the incidence of primer-dimer formation as a negative control. Transcript levels of COX-2 and iNOS were normalized using GAPDH of international standards.

As a result, iNOS expression was significantly increased after LPS treatment (17,750 fold over control, FIG. 3). Except for the 3-2 experimental group treated with 10%, iNOS expression was significantly suppressed in the fermented or non-fermented herbal groups (p <0.05). In the 1-2 experimental group, iNOS expression was more suppressed than in the 1-1 experimental group. In the 1-2 experimental group, 70% of the iNOS expression was suppressed compared to the LPS-induced iNOS expression. Appeared to inhibit. In addition, the 5% treatment group of the 3-2 sample was inhibited by 48% compared to LPS-induced iNOS expression.

In addition, the expression of COX-2 was increased by 275-fold in LPS-induced RAW264.7 cells. LPS-induced COX-2 gene expression was significantly inhibited in all fermented and non-fermented herbal treatment groups except for groups treated with 5% and 10% treated 1-1 samples and 5% treated 3-3 samples. (P <0.05). In addition, it was shown that 2-1 to 2-3 experimental groups and 3-1 experimental groups most inhibit the expression of COX-2 gene induced by LPS (FIG. 4).

<2-1-3> Inhibitory Effects of Fermented and Non-Fermented Medicinal Herbs on NO Produced from RAW264.7 Cells by LPS

RAW264.7 cells were dispensed into 6-well plates at 8 × 10 ⁵ cells / well and incubated overnight in incubator. Thereafter, 5% and 10% fermented herbal and non-fermented Chinese herbs were administered or not treated with 10 mcg / ㎖ LPS was incubated for 24 hours. NO generated from the cell line was measured using colorimetric analysis using Griess reagent (Promega, Madison, WI, USA). 100mcL of culture medium and 100mcL of Griess reagent were mixed, incubated at room temperature for 10 minutes, and the absorbance was measured at 540 nm. The concentration of NO was estimated using the prepared sodium nitrite standard curve.

As a result, the concentration of NO in the cell culture by LPS was increased by about 16 times than when only the cells were cultured. LPS-induced NO production was significantly inhibited in all fermented and non-fermented herbs (p <0.05). In particular, the 2-1 experimental group and the 2-3 experimental group were found to most inhibit LPS-induced NO production (FIG. 5).

Example 3. In vivo endotoxin reduction and increased enteric permeability improvement experiment

<3-1> Anti-inflammatory Activity of Fermented Herbal Medicine Mix and Non-Fermented Herbal Medicine

In the in vitro experiment of Example 2, the animal experiment was performed by selecting a pair showing the most significant anti-inflammatory effect in each herbal-microbial combination. In other words, Ryukonostoke Mesenteroidesu of the new song Bacillus rickenformis in fermented products, leachate and white migraine The fermented products were mixed and used for the experiment. The weight ratio of the mixed new song, Baekchul, Baekdu fermentation was 1: 1: 1.

Male Sprague Dawley rats weighing 200 ± 20g at 8 weeks of age can be fed freely of solid feed and water in the nursery, which is composed of constant temperature (20 ± 2 ℃), humidity, light for 12 hours, and dark for 12 hours. It was made. The experimental procedure was carried out according to the animal care guidelines of the NIH, and with the prior consent of the Animal Ethics Committee of Dongguk University. Rats were randomly assigned to 6 groups after environmental compliance. (1) control, (2) LPS-treated, (3) LPS-treated mixed fermented herbal medicine (300 mg / kg), (4) LPS-treated mixed fermented herbal medicine (600 mg / kg), (5 ) Mixed non-fermented and overpressure sterilized herbal medicine (600 mg / kg), (6) LPS-treated colostrum.

LPS from Escherichia coli 055: B5, Sigma, St. Louis, MO, USA was dissolved in physiological saline and administered in an amount of 1 mg / kg from group 2 to group 6, and group 1 was physiological without LPS. Saline was administered. A mixture of 1-2, 2-3, and 3-1 fermented or not fermented to groups 3, 4, and 5 rats, 300 mg / kg and 600 mg / kg, respectively, for 5 days prior to LPS administration. Administered. On the other hand, rats belonging to groups 1 and 6 were administered water and colostrum (Diatech Korea, Seoul, Korea; dose: 1ml / rat using 10% v / v solution) instead of the herbal medicine according to the plan. After 12 hours of LPS administration, rats were transferred to their respective metabolic cages, after which no food was consumed except for 12 hours. Twenty four hours after LPS administration, 1 ml aqueous test solution containing 66 mg / ml lactulose and 50 mg / ml mannitol was administered orally to each rat by intubation. In this situation, rats were maintained for 24 hours with only water allowed, followed by urine and blood tests for further analysis.

<3-2> Inhibitory Effects of Fermented and Non-Fermented Medicinal Herbs on Endotoxin Levels of Serum Induced by LPS

Serum endotoxin levels were measured using a Limulus Ameobocyte Lyasate (LAL) -based kit (Diatech Korea, Seoul, Korea) with an Endo-Check TM analyzer (Diatech Korea, Seoul, Korea). Serum CRP was analyzed by ELISA using rat-specific kits available from BD Biosciences (San Diego, CA, USA) and Thermo Scientific (Rockford, IL, USA). LPS is known to cause endotoxin shock as an extracellular membrane component of Gram-negative bacteria.

As a result, the endotoxin level was increased by 300 times compared to the control by LPS administration (300 fold over control, Figure 6). LPS-induced endotoxin levels in the group administered LPS and oral colostrum for 5 days after oral administration of mixed fermented herbal medicines (300 mg / kg, 600 mg / kg) for 5 days Significantly decreased (p <0.05). The group receiving mixed fermented herbal medicine (600 mg / kg) reduced LPS-induced endotoxin levels by 65%, and the group receiving mixed fermented herbal medicine (300 mg / kg) reduced 58%. The colostrum group was reduced by 62%. Mixed non-fermented herbal and sterile fermented herbal groups did not significantly reduce LPS-induced endotoxin levels (p> 0.05) (FIG. 6).

<3-3> Effect of Mixed Fermented and Non Fermented Medicinal Herbs on LPS-Induced Serum CRP Levels

To assess systemic inflammation, CRP was measured using a kit for rats from BD Biosciences (San Diego, CA, USA).

As a result, LPS-induced serum CRP levels increased by 64% compared to the control. Serum CRP levels in the non-fermented and sterile fermented and mixed colostrum groups and the colostrum-treated group were lower than those in the LPS group, but not statistically significant. p> 0.05) (FIG. 7).

<3-4> Effect of Mixed Fermented and Non Fermented Medicinal Herbs on the L / M Ratio of LPS Treated Rats

Increased gut permeability is also known as leaky gut syndrome, and microbial toxins such as endotoxins, food toxins, etc. may be systemic if the integrity of the intestinal mucosa decreases due to stress, drugs, and alcohol. Entering the bloodstream circulatory system causes systemic inflammation and is known to cause various chronic diseases. Lactulose, a large molecule, should not normally be excreted from urine, while mannitol, a small molecule, is detected in urine. This ratio can be used to measure the integrity and intestinal permeability of the intestinal mucosa.

Urine lactulose and mannitol levels were measured using the K-Lactul and K-Manol kit purchased from Megazyme (Bray, Co. Wicklow, Ireland). Assays were performed according to the kit manufacturer's instructions. Urine lactulose and mannitol levels were determined by the percentage recovered through ingestion and calculated the L / M ratio.

As a result, the mixed fermented Chinese herbal medicine significantly reduced the ratio of lactulose / mannitol (p <0.05) depending on the dose, and the group containing the mixed non-fermented Chinese medicine and the steamed Chinese herbal medicine and the colostrum-treated group Did not significantly reduce the ratio of lactose / mannitol (p> 0.05) (FIG. 8).

Example 4. In vivo antibiotic-induced diarrhea inhibitory effect experiment

Male sprag-dolly (200 ± 20 g, 6 weeks) rats were maintained at controlled temperature and humidity. The rats were kept in a 12-hour day / night cycle environment with randomly ingested food and water, and the experiment was conducted under the prior permission of Dongguk University Animal Ethics Committee in accordance with the Animal Breeding Guidelines of the National Institutes of Health.

Neomycin was used to cause diarrhea in rats after taking antibiotics. Animals adapted to this were randomly divided into six groups as follows: (1) control group, (2) neomycin injection group, (3) mixed fermentation herbal and neomycin injection group, (4) mixed non-fermentation Herbal medicine and neomycin injection group, (5) mixed non-fermented sterilized herbal medicine and neomycin injection group, (6) probiotics and neomycin injection group. Neomycin (product of La Jolla Calbiochem / EMD Biosciences, Calif.) Was dissolved in sterile purified water, and animals in groups 2-6 were injected 800 mg / kg orally once a day for 7 consecutive days. In contrast, only sterile purified water was injected into group 1. Rats in groups 3, 4, and 5 correspond to 600 mg / kg of the fermented mixture, the non-fermented mixture, and the non-fermented autoclaved materials 1-2, 2-3 and 3-1, respectively, for the herbal medicine. The dose was given once a day for 8 consecutive days (from the day before initiating neomycin). On the other hand, instead of herbal medicine, animals belonging to Group 1 and Group 6 were treated with water and probiotics (Lactobacillus ashdophyllus, 1.0 × 10 ¹¹ CFU / g, manufactured by CellBiotech, respectively. / Kg). After this diet, blood and feces were collected for further analysis. Animals were sacrificed to remove the cecum, and the extracted cecum was washed in phosphate buffered saline and fixed in formalin solution to prevent degeneration of the villi. Plasma was obtained by separating the blood of the test subject in a centrifuge at 4 ℃, 1,000 × g for 15 minutes. All samples were stored at −70 ° C. until analysis.

<4-1> Effects of Mixed Fermented and Non-Fermented Medicinal Herbs on Body Weight and Fecal Characteristics of Neomycin-treated Rats-Measurement of Water Content in Feces

The feces of each rat collected were weighed and recorded immediately. It was then centrifuged for 2 hours and weighed again in the dry state. The water content of the feces was calculated through the following formula.

Moisture Content (%) = ((Original Moisture Weight (g)-Dry Weight (g)) / Original Moisture Weight (g)) × 100

As a result, the stool began to appear semisolid between days 2 and 3 of neomycin injection. Injection of the herbal medicines or probiotics mentioned above did not change the amount of stool compared to neomycin. However, a marked increase in stool moisture content is evidence of neomycin infusion by indicating the onset of diarrhea symptoms. However, the water content of the stool did not show a clear change according to the herbal medicine and probiotics injection mentioned above (Fig. 9).

<4-2> Effect of Mixed Fermented and Non-Fermented Medicinal Herbs on CRP and Interferon-γ Levels in Neomycin Treated Rats-Determination of CRP, Interferon γ Concentrations

Endotoxin levels in serum were measured using an Endo-Check ™ analyzer (Diatech, South Korea) provided by Diatech using a kit based on the Mycobacterium toxin test (Limulus Ameobocyte Lyasate). Followed by manufacturer's instructions. Serum CRP and interferon-γ levels were measured by ELISA (enzyme-linked immunosorbent assay) using BD commercial kits from BD Biosciences (San Diego, Calif.) And Thermo Scientific (Rockford, Ill., USA), respectively. Next, we analyzed the changes in CRP and interferon-γ following neomycin injection to determine whether the expression of diarrhea-induced symptoms caused by antibiotics was related to the process of inflammation.

As a result, the CRP levels of the animals exposed to neomycin show the induction of an acute immune response to neomycin. Infusion of probiotics resulted in a significant decrease in CRP in neomycin dosed rats. This suggests the anti-inflammatory action of the drug. Regular treatment with fermented / non-fermented sterile herbal mixtures significantly reduced neomycin-induced CRP levels, demonstrating the anti-inflammatory (anti-inflammatory) action of the two herbs. However, for reasons not yet clear, a mixture of non-fermented non-sterile herbal medicines did not significantly affect serum CRP levels in neomycin-dosed rats (FIG. 10). In addition, treatment with neomycin significantly increased serum IFN- [gamma] in the animals tested, which is further empowering the expression of immune responses to neomycin. Interestingly, treatment with all herbal preparations resulted in a significant decrease in serum IFN-γ levels in neomycin dosed rats (FIG. 11). These results indicate that the herbal medicines have anti-inflammatory effects and thus have the ability to protect the intestinal mucosa from the immune response following antibiotic administration that can cause diarrhea.

<4-3> Histological Change

The cecum was fully fixed in formalin and then transferred to 70% ethanol solution and stored at 4 ° C. until analysis. Tissue paraffin processing was as follows: 1 hour in 70% methanol, 1 hour in 90% methanol, 1 hour in 95% methanol, 1 hour in 100% methanol, 1 hour in 100% ethanol, 1 in xylene Time (2 Changes), 30 minutes in paraffin (65 ° C.). Next, paraffin blocks of tissues were prepared. Each block was made using a thin slicer to make 4 μM tissue sections, soaked in a water bath, and fixed on a clean glass slide. The glass slide was heated in a 65 ° C. oven for 20 minutes to secure the tissue to the glass slide. The glass slides were rehydrated with deparaffins in the following steps: 2 minutes in xylene (3 changes); 100% ethanol (2 changes); 95% ethanol; 80% ethanol; water. Hematoxylin & Eosin (H & E) tissue staining was performed as follows: 2 minutes in hematoxylin; Rinsing with running water; Hydrochloric acid alcohol (76.6% ethanol and 1/300 (v / v) concentrated HCl); water; Ammonia solution (0.084% (w / v) NH 4 OH); Rinsing with running water for 5 minutes; 80% ethanol; Eosin 15 seconds; 95% ethanol (2 changes); 100% ethanol (2 changes). The prepared slides were observed under a microscope and each image was captured.

As a result, in the untreated group, the intestinal mucosa was cleanly preserved, and normal villi growth and lower tissue were well preserved. In the antibiotic-induced diarrhea group, damage to the intestinal mucosa, infiltration of inflammatory cells, edema and discontinuity of the lower tissue were observed, but pathological findings were clearly improved in the intestinal mucosa of the group administered with fermented herbal medicine (FIG. 12).

An analysis of the cecum tissues was conducted to evaluate whether neomycin causes lesions in the digestive tract and whether herbal medicines can cure such damage. Neomycin-administered rats were observed with cecal tissue lesions such as loss of epithelial integrity, inflammatory symptoms including edema and loss of crypts. The cecal tissue profile in the neomycin dose group exposed to fermented non-sterile herbal medicine material was similar to that of the control group, suggesting that the herbal material is protective against neomycin dosed tissue damage and gut inflammation. In contrast, treatment of non-fermented (sterile / non-sterile) herbal mixtures showed only modest effects on neomycin administered tissue lesions in the cecum of rats. Exposure to probiotics has shown a stronger protective effect than neo-fermented medicinal substances on cecal tissue damage in neomycin-treated rats, which is an important role of inflammation in neomycin-treated tissue lesions in the digestive tract. From the above results, it was confirmed that the fermented herbal mixture has a strong anti-inflammatory (anti-inflammatory) effect and can be used for the treatment of tissue damage caused by neomycin.

Example 5 Intestinal Microbiota Improvement Effect in In vivo LPS Treated Enteritis Model Animals

Animal feces were collected before sacrifice and stored at -80 ° C for analysis. Fecal DNA was extracted using the kit and the purity was verified through an A260 / A280 ratio and the approximate content was measured. Primers for each genus were searched for in the literature, ordered, and reacted by real-time PCR to determine specificity. The procedure of obtaining optimal reaction conditions of Lactobacillus, Bifidobacterium, Clostridium, Salmonella, Universal bacteria, etc. and confirming by melting curve was repeated. Finally, relative abundance was measured by subtracting the number of each genus from universal bacteria. The harmful bacteria Salmonella and Clostridium were rarely detected because they were less exposed to the external environment, and significant differences were observed between the genus Lactobacillus and Bifidobacterium (Fig. 13, 14).

Although embodiments of the present invention have been described above with reference to the accompanying drawings, those skilled in the art to which the present invention pertains may be embodied in other specific forms without changing the technical spirit or essential features of the present invention. I can understand that. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

Claims (10)

1. Formal pharmaceutical composition comprising a mixture of fermented baekchul, new song and baek head.
2. The method according to claim 1,

   Fermentation of the leachate is a formal pharmaceutical composition, characterized in that the fermentation strain Bacillus rickeniformis as a fermentation strain 1 to 3% glucose.
3. The method according to claim 1,

   Fermentation of the new song is a formal pharmaceutical composition, characterized in that the fermentation of 1 ~ 3% glucose as a medium as a fermentation strain of leukonostock mesenteroides.
4. The method according to claim 1,

   Fermentation of the white migraine is a formal pharmaceutical composition, characterized in that the fermentation strain Bacillus rickeniformis as a fermentation strain 1 to 3% LB broth.
5. The method according to claim 1,

   The ratio of fermented Baekchul, Singok, Baekdudu contained in the mixture is 0.5 to 2 parts by weight of fermented Baekgok and 0.5 to 2 parts by weight of fermented Baekdu with respect to 1 part by weight of fermented Baekchul. Composition.
6. The method according to claim 5,

   The ratio of fermented Baekchul, Singok, Baekmul contained in the mixture is a weight ratio of 1: 1: 1, characterized in that the formal pharmaceutical composition.
7. The method according to claim 1,

   The amount of the mixture is for formal pharmaceutical composition, characterized in that administered to the subject in an amount of 50 to 100mg / kg.
8. Formal dietary supplement containing a mixture of fermented baekchul, new song and baek head.
9. The method according to claim 1,

   Formal health, characterized in that the ratio of fermented baekchul, new song, white migraine contained in the mixture is in the range of 0.5 to 2 parts by weight of fermented new song and 0.5 to 2 parts by weight of fermented white bean, based on 1 part by weight of fermented baekchul. Nutraceutical.
10. The method according to claim 9,

    Fermented Baekchul, Singok, Baekdudu ratio contained in the mixture is characterized in that the weight ratio of 1: 1: 1.

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