KR102567235B1 - Composition for the prevention and treatment of Inflammatory Bowl Disease - Google Patents

Abstract

The present invention relates to a composition for the prevention, treatment, or improvement of inflammatory bowel disease containing a mixed extract containing at least two of Mokhyang, Gaja, and Health, as an active ingredient, and a method for preparing the same, and more specifically, to a mixture of Mokhyang, Health, and Gaja Inhibition of mononuclear cell adhesion in intestinal epithelial cells, inhibition of inflammatory cytokines production, improvement of colitis in animal models of DSS (Dextran sodium sulfate)-induced colitis, and TNBS (2,4,6-trinitrobenzenesulfonic acid)-induced clones It relates to a pharmaceutical composition having an effect of improving Crohn's disease in an animal model of the disease.

Description

Composition for the prevention and treatment of inflammatory bowel disease {Composition for the prevention and treatment of Inflammatory Bowl Disease}

The present invention relates to the use of a mixed extract of Mokhyang, Gaja and Health, and more specifically, to a composition for preventing, treating or improving inflammatory bowel disease containing a mixed extract containing at least two of Mokhyang, Gaja and Health as an active ingredient , and its use.

Inflammatory bowel disease (IBD) is an incurable disease in which chronic inflammation or ulcers are caused in the mucous membranes of the large intestine and small intestine, and diarrhea and bloody stools continue for a long time and recur.

Inflammatory bowel disease is a more common disease in Westerners, but it has been rapidly increasing in Korea since the 1980s. Approximately 15% of patients with intestinal disease have a family history in the immediate family.

Inflammatory bowel disease can be caused by environmental, genetic and immune factors (Kitahora, T. (2012) Familial prevalence of inflammatory bowel disease, Nihon Rinsho, 70 Suppl 1, 44-47; Lowe, A. M., Roy, P. O., M, B. P., Michel, P., Bitton, A., St-Onge, L., & Brassard, P. (2009) Epidemiology of Crohn's disease in Quebec, Canada. Inflamm Bowel Dis, 15(3) , 429-435), and the exact etiology of inflammatory bowel disease remains unclear (Toumi, R., Abdelouhab, K., Rafa, H., Soufli, I., Raissi-Kerboua, D., Djeraba, Z., & Touil-Boukoffa, C. (2013) Beneficial role of the probiotic mixture Ultrabiotique on maintaining the integrity of intestinal mucosal barrier in DSS-induced experimental colitis.Immunopharmacol Immunotoxicol, 35(3), 403-409).

Although the cause of inflammatory bowel disease is still unclear, it is estimated that inflammatory mediators and activation of immune cells are important etiologies due to environmental or genetic factors and autoimmune diseases.

Inflammatory bowel disease is classified into two diseases: ulcerative colitis and Crohn's disease, which are clinically similar but differ in histological, endoscopic and immunological aspects. Activation of factors and immune cells is known to be an important disease.

Sustained or inappropriate activation of the intestinal immune system plays an important role in the pathophysiology of chronic mucositis inflammation, particularly by infiltration of neutrophils, macrophages, lymphocytes and mast cells, eventually resulting in mucosal destruction and ulceration.

In the inflammatory response that is mainly involved in the pathogenesis of inflammatory bowel disease, inflammation such as TNF-α (Tumor necrosis factor-α), interleukin-6 (IL-6) and interleukin-8 (IL-8) Response-promoting cytokines play a major role.

In particular, TNF-α is highly expressed in the colonic lumen and colonic epithelial cells of ulcerative colitis patients, and according to recent studies, TNF-α is known to play an important role in the pathogenesis of ulcerative colitis. Infliximab, an anti-TNF-α antibody, is known to be effective not only in the treatment of boils, but also in the treatment of previously untreated Crohn's disease. However, these treatments are expensive and cause side effects such as fluid reactions or infectious complications in some patients.

Current treatments for inflammatory bowel disease include 5-aminosalicylic acid (5-ASA) drugs that block the production of prostaglandins, such as sulfasalazine and mesalazine. or steroid-type immunosuppressants are used, and if the disease does not respond to steroid treatment, immunosuppressants such as azathioprine, 6-mercaptopurine, and cyclosporin are also used, but there is no drug that can be expected to cure the disease.

In addition, long-term administration of these drugs may cause side effects such as nausea, vomiting, indigestion, anorexia, and headache, as well as leukopenia due to hypersensitivity reactions, skin rash, fever, pancreatitis, hepatitis, hemolytic anemia, and bone marrow suppression. .

On the other hand, natural extracts and formulations using them have been used to alleviate and treat various diseases ranging from cough and cold to parasitic infection and inflammation. More than 60% of anticancer drugs and more than 75% of infectious diseases in the market today originate from natural products. This is because natural products are very diverse and provide high specific physiological activity.

However, there is no reliable treatment for inflammatory bowel disease yet, and the development of effective therapeutic agents for these diseases is required, and for this purpose, natural extracts useful for inflammatory bowel disease are needed.

Therefore, the present inventors have made diligent efforts to develop a composition derived from mixed extracts of natural plants that does not cause side effects by using natural plant materials and is effective for inflammatory bowel disease. Inhibiting the production of inflammatory cytokines TNF-α (tumor necrosis factor-α) and IL-6 (interleukin-6), inhibiting monocyte infiltration, and DSS (Dextran sodium sulfate)-induced colitis animal model and TNBS (2,4,6 -trinitrobenzenesulfonic acid) induced Crohn's disease animal model to recover the length of the large intestine, reduce weight, improve the condition of diarrhea and bloody stool, and also verify the effective dose through the effect of the administered dose of two or more mixed extracts among Mokhyang, Gaja and Health Thus, the present invention was completed.

1. Korea Patent No. 10-1337389 2. Korea Patent No. 10-1446396 3. Korean Patent Publication No. 10-2004-0018475 4. US registered patent US 9044502 B2

1. Kim So-yeon, Park Jae-woo, Ryu Bong-ha (2013) Effects of Tangmok-Hyang on DSS (Dextran Sulfate Sodium)-Induced Inflammatory Bowel Disease Animal Models. Korean J. Orient. Int. Med. 34(2), 134-146 2. Kitahora, T. (2012) Familial prevalence of inflammatory bowel disease, Nihon Rinsho, 70, 44-47 3. Lowe, A. M., Roy, P. O., M, B. P., Michel, P., Bitton, A., St-Onge, L., & Brassard, P. (2009) Epidemiology of Crohn's disease in Quebec, Canada. Inflamm Bowel Dis, 15(3), 429-435 4. Toumi, R., Abdelouhab, K., Rafa, H., Soufli, I., Raissi-Kerboua, D., Djeraba, Z., & Touil-Boukoffa, C. (2013) Beneficial role of the probiotic mixture Ultrabiotique on maintaining the integrity of intestinal mucosal barrier in DSS-induced experimental colitis. Immunopharmacol Immunotoxicol, 35(3), 403-409 5. Moreau J. (2014) Crohn's disease and ulcerative colitis. Rev Infirm. 199, 16-18 6. Shi D, Das J, Das G. (2006) Inflammatory bowel disease requires the interplay between innate and adaptive immune signals. Cell Res. 16(1), 70-74 7. Mashhadi NS, Ghiasvand R, Askari G, Hariri M, Darvishi L, Mofid MR. (2013) Anti-oxidative and anti-inflammatory effects of ginger in health and physical activity: review of current evidence. Int J Prev Med. 4, 36-42 8. Shen CL, Hong KJ, Kim SW. (2005) Comparative effects of ginger root (Zingiber officinale Rosc.) on the production of inflammatory mediators in normal and osteoarthrotic sow chondrocytes. J Med Food. 8(2), 149-153 9. Thomson M, Al-Qattan KK, Al-Sawan SM, Alnaqeeb MA, Khan I, Ali M. (2002) The use of ginger (Zingiber officinale Rosc.) as a potential anti-inflammatory and antithrombotic agent. Prostaglandins Leukot Essential Fatty Acids. 67(6), 475-478 10. Kim HR, Kim JM, Kim MS, Hwang JK, Park YJ, Yang SH, Kim HJ, Ryu DG, Lee DS, Oh H, Kim YC, Rhee YJ, Moon BS, Yun JM, Kwon KB, Lee YR. (2014) Saussurea lappa extract suppresses TPA-induced cell invasion via inhibition of NF-κB-dependent MMP-9 expression in MCF-7 breast cancer cells. BMC Complement Altern Med. 14, 170-178 11. Choi HG, Lee DS, Li B, Choi YH, Lee SH, Kim YC (2012) Santamarin, a sesquiterpene lactone isolated from Saussurea lappa, represses LPS-induced inflammatory responses via expression of heme oxygenase-1 in murine macrophage cells. Int Immunopharmacol. 13(3), 271-279 12. Zhao F, Xu H, He EQ, Jiang YT, Liu K. (2008) Inhibitory effects of sesquiterpenes from Saussurea lappa on the overproduction of nitric oxide and TNF-alpha release in LPS-activated macrophages. J Asian Nat Prod Res. 10(11-12), 1045-1053 13. Gokhale AB, Damre AS, Kulkami KR, Saraf MN. (2002) Preliminary evaluation of anti-inflammatory and anti-arthritic activity of S. lappa, A. speciosa and A. aspera. Phytomedicine. 9(5), 433-437

The main object of the present invention is to provide various uses of a mixed extract containing at least two of Ekomhyang, Gaja and Health.

One object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of inflammatory bowel disease containing a mixed extract containing at least two of Morocco, Gaza and Health as an active ingredient and a method for preparing the same.

Another object of the present invention is to provide a health functional food for the prevention or improvement of inflammatory bowel disease containing a mixed extract containing at least two of saffron, gaza and health as an active ingredient.

In addition, an object of the present invention is a pharmaceutical composition or health functional food for the prevention, treatment or improvement of inflammatory bowel disease containing a mixed extract containing at least two of cedar, gaza and health as an active ingredient, effective use of the mixed extract to provide capacity.

In order to solve the above problems,

In one embodiment, the present invention provides a pharmaceutical composition for the prevention or treatment of inflammatory bowel disease containing a mixed extract containing at least two of Morphaceae, Gaza and Health as an active ingredient.

At this time, the mixed extract may include, for example, in the case of a mixed extract of Mokhyang and Gaja, Mokhyang: Gaja at a weight ratio of 1: 1; In the case of a mixed extract of cedar and health, cedar: health may be included in a weight ratio of 1: 1; In the case of a mixed extract of health and gaja, health: gaja may be included in a weight ratio of 1: 1; In the case of the mixed extract of Mokhyang, Gaja, and Health, Mokhyang: Gaja: Health may be included in a weight ratio of 2: 2: 1.

The mixed extract may be a crude extract, a polar solvent-soluble extract, or a non-polar solvent-soluble extract of C. japonica, Gaza, and health, and the crude extract is soluble in a solvent selected from water including purified water, methanol, ethanol, butanol, or mixed solvents thereof The polar solvent soluble extract may be an extract soluble in a solvent selected from water, ethanol, butanol, or a mixed solvent thereof, and the non-polar solvent soluble extract may be an extract soluble in hexane, chloroform, dichloromethane or ethyl acetate. may be available extracts.

In a preferred embodiment, the mixed extract of the present invention may be extracted using water, C1-C4 lower alcohol or a mixture thereof as a solvent, and in particular, a hot water extract or an ethanol extract may be used, most preferably In particular, 50 to 100% of ethanol extract can be used.

The extract of the present invention is a mixed extract obtained by mixing the Mokhyang and the Gaja or the Health, and then extracting the mixture, or the mixed extract is the Mokhyang extract obtained from the Mokhyang and the Gaja extract obtained from the Gaja, or the above It may be an extract obtained by mixing health extracts obtained from health.

In addition, the extract of the present invention is a mixed extract obtained by mixing the Mokhyang, the Gaja, and the Health, and then extracting the mixture, or the mixed extract is the Mokhyang extract obtained from the Mokhyang and the Gaja extract obtained from the Gaja And it may be an extract obtained by mixing health extracts obtained from the health.

On the other hand, the mixture may further include extracts such as peony, rhubarb, geoman, humpback, broiler, horseback riding, Shiho, sage, baekchul, and uiyiin in addition to mokhyang, gaza and health.

At this time, the mixed extract containing at least two of cedar, gaja and health can be extracted at 20 to 120 ° C for 1 to 15 hours, for example, at 60 to 120 ° C for 1 to 6 hours. .

The mixed extract can prevent and treat inflammatory bowel disease by suppressing any one or more of the production of IL-6 or TNF-α, which are inflammatory mediators.

The inflammatory bowel disease includes Crohn's disease, ulcerative colitis, intestinal Behçet's disease, intestinal tuberculosis and enteritis, diarrhea, and the like. Ulcerative colitis is a chronic disease in which the large intestine becomes inflamed and ulcers form. It causes hemorrhagic diarrhea, severe abdominal pain, and seizures accompanied by fever. The exact cause is unknown, but it is caused by environmental and genetic factors and autoimmune Illness is believed to be the cause. Crohn's disease, also called localized enteritis or granulomatous ileitis or ileocolitis, is a chronic inflammation of the intestinal wall that occurs anywhere in the digestive tract.

In the present invention, the mixed extract is used to recover the length of the large intestine and improve the condition of weight loss, diarrhea, and bloody stool in an animal model of DSS (Dextran sodium sulfate)-induced colitis and a 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced Crohn's disease animal model. It can have more than one effect.

In addition, the present invention, as another embodiment, provides a health functional food for preventing or improving inflammatory bowel disease containing a mixed extract containing at least two of cedar, gaza and health as an active ingredient, and its use.

The health functional food may be prepared as, for example, powder, granule, tablet, capsule, syrup or beverage.

In another embodiment, the present invention is a pharmaceutical composition or health functional food for the prevention, treatment or improvement of inflammatory bowel disease containing a mixed extract containing at least two of cedar, gaza and health as an active ingredient, the mixed extract provides an effective dose of

An effective amount of the mixed extract having excellent anti-inflammatory efficacy may be 0.1 to 50% by weight of the extract based on the total weight of the composition. Preferably, the mixed extract may be at a concentration of 50 to 400 mg/kg, and most preferably at a concentration of 200 to 400 mg/kg.

As such, the present invention relates to the anti-inflammatory function of a mixed extract containing at least two of Eckhyang, Gaza and Health, more specifically, the inflammatory cytokines TNF-α (tumor necrosis factor-α), IL-6 (interleukin-α) 6) Restoration of colon length, weight loss, diarrhea, and bloody stool in animal models of DSS (Dextran sodium sulfate)-induced colitis and TNBS (2,4,6-trinitrobenzenesulfonic acid)-induced Crohn's disease animal models It includes all of the various uses using an effective amount showing an improvement effect and an excellent anti-inflammatory effect.

Since the composition containing the extracts of Mokhyang, Garza and Health mixed according to the present invention has excellent anti-inflammatory and colitis improving effects and has little cytotoxicity, it is suitable for preventing and treating inflammatory diseases, especially inflammatory bowel diseases, including pharmaceuticals, processed foods, It can be usefully used as an active ingredient in compositions such as functional foods, food additives, functional beverages, or beverage additives.

1 is a graph showing the effect of inhibiting mononuclear cell adhesion in intestinal epithelial cells of the extracts of Mokhyang and Health mixture.
Figure 2 is a graph showing the TNF-α production inhibitory ability of Mokhyang and health mixed extract.
Figure 3 is a graph showing the IL-6 production inhibitory ability of Mokhyang and health mixed extract.
Figure 4 is a graph showing the colon length recovery effect by Mokhyang and healthy mixed extract in DSS (Dextran sodium sulfate) induced colitis animal model.
Figure 5 is a graph showing the disease activity for weight loss, diarrhea, improvement of the state of bloody feces by Mokhyang and health mixed extract in DSS-induced colitis animal model.
Figure 6 is a photograph showing the intestinal mucosal recovery effect by Mokhyang and healthy mixed extract in DSS-induced colitis animal model.
Figure 7 shows the effect of MPO activity reduction by the extract of Mokhyang and health mixture.
8 is a result of cytotoxicity analysis (MTT analysis) according to the ethanol-containing concentration of the solvent used in the extract of Mokhyang and health mixture.
Figure 9 is a graph showing the ability to inhibit NO production according to the ethanol-containing concentration of the solvent used in the mixed extract of Mokhyang and health.
10 is a result of measuring cell viability according to the blending (mixing) ratio of Mokhyang and Health.
Figure 11 is the result of confirming the NO production inhibition ability according to the blending (mixing) ratio of Mokhyang and health.
12 is a graph showing the effect of inhibiting monocyte adhesion in intestinal epithelial cells according to the mixing ratio of Mokhyang and Health.
13 is a graph showing the inhibitory effect of monocyte adhesion ability in intestinal epithelial cells of the mixed extracts of Mokhyang and Gaja.
14 is a graph showing the inhibitory effect of mixed extracts of Mokchyang and Gajaja on the production of TNF-α, an inflammatory cytokine induced by LPS.
15 is a graph showing the inhibitory effect of mixed extracts of Mokchyang and Gajaja on the production of IL-6, an inflammatory cytokine, induced by LPS.
16 is a graph showing the effects of the mixed extracts of Mokhyang and Gaza in DSS-induced colitis animal models.
17 is a graph measuring the activity of MPO (Myeloperoxidase) for the inhibition of inflammatory cell infiltration in intestinal tissue by the mixed extracts of Mokhyang and Gaza in DSS-induced colitis animal model.
18 is a graph showing the effects of a mixed extract of Mokhyang and Gajaja in a 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced Crohn's disease animal model.
Figure 19 is a graph showing the monocyte adhesion inhibitory effect on intestinal epithelial cells of health and Gaja mixed extracts.
20 is a graph showing the inhibitory effect of the mixed extract of health and Gajaja on the production of IL-6, an inflammatory cytokine induced by LPS.
21 is a graph showing the health and effects of Gaza mixed extracts in DSS-induced colitis animal models.
22 is a graph showing the effects of health and Gaja mixture extracts in TNBS-induced Crohn's disease animal models.
23 is a graph showing the inhibitory effect on mononuclear cell adhesion ability in intestinal epithelial cells of the extracts of Mokhyang, Gaza and Health mixture.
24 is a graph showing the inhibitory effect of the extracts of Mokhyang, Gajaja and Health mixture on the production of IL-6, an inflammatory cytokine induced by LPS.
25 is a graph showing the effect according to the mixing ratio of the extracts of Mokhyang, Gaza, and Health mixture in DSS-induced colitis animal model.
26 is a graph showing the effect according to the mixing ratio of the extracts of Mokhyang, Gaza and Heunghyang mixed extracts in TNBS-induced Crohn's disease animal model.
27 is a graph showing the effect of inhibiting monocyte adhesion in intestinal epithelial cells according to the extraction solvents of the extracts of Mokhyang, Gaza and Health mixed extracts.
28 is a graph showing the ability to inhibit the production of IL-6, an inflammatory cytokine, according to the extraction solvent of the mixed extracts of Mokhyang, Gaja and Health.
29 is a graph showing the effects of the extraction solvents of the extracts of Mokhyang, Gaza, and Health mixed extracts in DSS-induced colitis animal models.
30 is a graph showing the effects of the extraction solvents of the extracts of Mokhyang, Gaza, and Health mixture in TNBS-induced Crohn's disease animal model.
31 is a graph showing the effect according to the administered dose of Mokhyang, Gaza and Heunghyang mixed extracts in DSS-induced colitis animal models.
32 is a graph showing the effect according to the dose of Mokhyang, Gaza and Heunghyang mixed extracts in TNBS-induced Crohn's disease animal model.

Definitions of terms used in the present invention are as follows.

“Extract” refers to a preparation obtained by squeezing a crude drug into an appropriate leachate and concentrating the leachate by evaporating the leachate, but is not limited thereto, such as, but not limited to, an extract obtained by extraction treatment, a dilution or concentrate of an extract, and a dried product obtained by drying the extract. , It may be a crude product or a purified product thereof. The extract can be prepared using a general extraction method, separation and purification method known in the art. The extraction method is not limited thereto, but preferably, methods such as hot water extraction, hot water extraction, cold needle extraction, reflux cooling extraction, or ultrasonic extraction may be used. In the specification of the present invention, fractions are also included in the extract.

"Crude extract" is a solvent selected from water including purified water, lower alcohols having 1 to 4 carbon atoms such as methanol, ethanol, butanol, or mixed solvents thereof, preferably water and ethanol mixed solvents, more preferably 50 to 100% Contains extracts that are soluble in ethanol.

The "polar solvent-soluble extract" includes an extract soluble in a solvent selected from water, methanol, ethanol, butanol, or a mixed solvent thereof, preferably water or ethanol.

"Non-polar solvent soluble extract" includes extracts soluble in hexane, chloroform, dichloromethane or ethyl acetate, preferably hexane, dichloromethane or ethyl acetate, more preferably hexane or ethyl acetate solvent.

A "carrier" is defined as a compound that facilitates the addition of a compound into a cell or tissue. For example, dimethylsulfoxide (DMSO) is a commonly used carrier that facilitates the introduction of many organic compounds into the cells or tissues of living organisms.

A "diluent" is defined as a compound that is diluted in water to dissolve the compound as well as to stabilize the biologically active form of the subject compound. Salts dissolved in buffer solutions are used as diluents in the art. A commonly used buffer solution is phosphate buffered saline because it mimics the salt state of human solutions. Because buffer salts can control the pH of a solution at low concentrations, buffer diluents rarely modify the biological activity of a compound.

"Subject" or "patient" means any single individual in need of treatment, including humans, cattle, dogs, guinea pigs, rabbits, chickens, insects, etc., and also clinical research trials that do not show any clinical manifestations of disease. Any subject who participated in an epidemiologic study or a subject used as a control group was included.

"Tissue or cell sample" means a collection of similar cells obtained from tissue of a subject or patient. The source of the tissue or cell sample may be solid tissue from a fresh, frozen and/or preserved organ or tissue sample or biopsy or aspirate; blood or any blood component; It may be a cell at any point in the subject's pregnancy or development. Tissue samples can also be primary or cultured cells or cell lines.

“Administering” means providing a composition to a subject by any suitable method.

An "effective amount" is an appropriate amount that produces beneficial or desirable clinical or biochemical results. An effective amount can be administered once or more. For purposes of this invention, an effective amount is an amount suitable for temporarily alleviating, ameliorating, stabilizing, reversing, slowing or delaying the progression of a disease state. A composition is said to be "pharmaceutically or physiologically acceptable" if the recipient animal is able to tolerate administration of the composition, or if administration of the composition to the animal is suitable. An agent can be said to be administered in a "therapeutically effective amount" when the amount administered is physiologically important. An agent is physiologically meaningful if the presence of the agent results in a physiologically detectable change in the recipient patient.

The term "treating", unless stated otherwise, means reversing, alleviating, inhibiting the progression of, or preventing the disease or disorder to which the term applies, or one or more symptoms of the disease or disorder. means that As used herein, the term 'treatment' refers to the act of treating when 'treating' is defined as above.

"Health functional food" means a food in which the functionality of a general food is improved by adding an extract of one embodiment of the present invention to a general food. Functionality can be roughly divided into physical properties and physiological functions. When the extract of the present invention is added to general foods, the physical properties and physiological functions of general foods will be improved, and the present invention comprehensively treats foods with such improved functions as 'health functions'. Define 'food'.

"About" means 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4 for a reference amount, level, value, number, frequency, percentage, measure, size, amount, weight or length means an amount, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by 3, 2 or 1%.

All technical terms used in the present invention, unless defined otherwise, are used with the same meaning as commonly understood by one of ordinary skill in the art related to the present invention. In addition, although preferred methods or samples are described in this specification, those similar or equivalent thereto are also included in the scope of the present invention. The contents of all publications incorporated by reference herein are incorporated herein by reference.

Hereinafter, the present invention will be described in detail.

The present invention relates to the use of a mixed extract containing at least two of Mokhyang, Gaja and Health, and to the display of specific physiological activities and functions contained in the mixed extract containing at least two of Mokhyang, Gaja and Health.

[Effective Substance]

Aucklandia lappa Decne is a perennial plant belonging to the Asteraceae/Compositae family. Its flowering period is July-September and its fruiting period is August-October. It grows in relatively high mountains, and its origin is India. In China, it is cultivated in Yunnan, Guangseong, and Sichuan. It is also called Nammokhyang, Gwangmokhyang, Unmokhyang, Wolseo Mokhyang, and Cheonmokhyang.

Because the scent of wood is warm, it shows good therapeutic effects on abdominal pain caused by cold stomach, flatulence, vomiting, diarrhea, etc., and is also effective for dysentery, enhancing the function of the large intestine, urinating well, and reducing pain. It is known to have a stopping effect.

Health (Zingiber officinale Rosc.) is a dried rhizome of ginger. It is a perennial herb native to tropical Asia belonging to the genus Ginger and Ginger. , About 50 species are distributed in Malaysia, and in Korea, both ginger and ginger are cultivated in ginger, and flower ginger is cultivated in ginger, and flower ginger grows wild under forests in southern islands. Health is also called health ginger, gyungang, baekgang, and raw health.

It is spicy in nature, warm in nature, and has no poison, so it acts on the lungs, stomach, and nasal passages, and also acts on the heart meridians, liver meridians, and gall meridians. It has the effect of dissipating dead skin, releasing cold energy, stopping vomiting and communicating phlegm, and treating cold, wind, vomiting, phlegm, asthma, seawater, bloating, and diarrhea. In addition, it is known to help energy, promote blood circulation, remove cold and wind, detoxify, anti-inflammatory, remove the smell of toothache, stop pain, and soothe wetness.

Gaza (Terminalia chebula Retzius) is a mature fruit of Gaza, which is a tropical tall tree native to India, Myanmar, Malaysia, and Sichuan, China. A yellow pigment called myrovalan is extracted from the fruit juice and used as furniture wood. is also used Various names of Gaza are called Gaza, such as Gari-wolf (訶黎勒), Gari-wolf (訶梨勒), Sufengja (隨風子), Gari (訶梨), and Gari (訶黎).

Gaja has a slight peculiar smell, and the medicinal properties are bitter, slightly sour, astringent and warm. It lowers the qi, removes old phlegm and cough, is effective in shortness of breath, old diarrhea, dysentery, college, and prolapse, helps digestion, stimulates the appetite, and comforts the fetus in the stomach. In addition, as pharmacological action, it has been reported that it inhibits bacteria against convergence, branching, and Pseudomonas aeruginosa.

In one aspect of the present invention, an extract or fraction combining two or more of the above-mentioned Mokhyang, Gaja, and Health is used.

The extract or fraction may be extracted or fractionated from various parts of the chamomile, Gaza, and health.

[0040] The extracts of the saffron, gaja, and health fractions can be obtained as each solvent fraction through a process of fractionating organic solvents from non-polar to polar. A suitable solvent for fractionation may be water, ethanol, methanol, hexane, chloroform, dichloromethane, ethyl acetate, butanol or a mixture thereof.

The present invention of extract of chamomile, Gajaja, health may be prepared and used by a method known in the art, a modified method thereof, or a method according to the present invention.

For example, it can be extracted by cold extraction, hot water extraction, ultrasonic extraction, reflux cooling extraction or heating extraction. According to one embodiment of the present invention, the extract of the present invention can be extracted through hot water extraction or reflux cooling extraction, Extraction may be repeated 1 to 10 times, 1 to 8 times, or 1 to 6 times.

In addition, the extracts or fractions of saffron, gaza, health can be prepared in a powder state by additional processes such as distillation under reduced pressure and freeze drying or spray drying. In addition, the extract or fraction is further purified into fractions using various chromatography methods such as silica gel column chromatography, thin layer chromatography, and high performance liquid chromatography. can also be obtained

Therefore, in the present invention, the extract of Alpine, Gaza, and Health is a concept that includes all extracts, fractions, and purified products obtained in each step of extraction, fractionation, or purification, and dilutions, concentrates, or dried products thereof.

As one specific example, it can be prepared by the following method.

i) extracting by adding an extraction solvent to two or more substances selected from the group consisting of Mokhyang, Gaja, and Health;

ii) filtering the extract of step i); and

iii) drying the filtered extract of step ii).

In i), any solvent acceptable in the art may be used as the extraction solvent, and water or an organic solvent may be used. The extraction solvent is preferably water, alcohol or a mixture thereof. It is preferable to use a C1 to C4 lower alcohol as the alcohol, and more preferably to use ethanol as the lower alcohol.

For example, alcohol having 1 to 4 carbon atoms including purified water, methanol, ethanol, propanol, isopropanol, butanol, acetone, and ether Various solvents such as benzene, chloroform, ethyl acetate, methylene chloride, hexane and cyclohexane may be used alone or in combination, but Not limited.

The extraction solvent is, for example, 50-100%, 50-95%, 50-90%, 50-85%, 50-80%, 50-75%, 50-70%, 50-65%, or 50 Can be used at ~60% concentration. Preferably, it can be used at a concentration of 50 to 60%.

As an extraction method, shaking extraction, Soxhlet extraction, or reflux extraction is preferably used, but is not limited thereto. It is preferable to extract by adding 1 to 10 times the extraction solvent to the amount of cedar and ghee, cedar and health, or health and ghee. The extraction temperature is preferably 20 to 120 ° C, but is not limited thereto. In addition, the extraction time may be 1 to 20 hours, preferably 1 to 15 hours, and more preferably 1 to 6 hours, without limitation.

In the above method, the drying method of step iii) includes vacuum drying, vacuum drying, boiling drying, spray drying or freeze drying.

As a specific example, in the case of preparing a mixed extract containing at least two of Mokhyang, Gaja and Health, for example, Mokhyang and Gaja mixed extract, Mokhyang and Health mixed extract, or Health and Gaza mixed extract, preferably Mokhyang: Gaza, woodhyang: health or health: gaja is mixed in a weight ratio of 1 to 3: 1 to 3, more preferably woodhyang: gaja, woodhyang: health or health: gaja 1: 1, mixed weight From about 1 to 30 times the volume, preferably 5 to 15 times the volume (w / v%) of water including purified water, lower alcohols having 1 to 4 carbon atoms such as methanol, ethanol, butanol, or mixed solvents thereof A selected solvent, preferably a mixed solvent of water and ethanol, for example, 50% to 100% ethanol is added, and the mixture is heated at about 20 to 120 ° C, preferably 60 to 100 ° C for 1 to 15 hours, preferably 1 to 6 After extracting by extraction method such as cold extraction, hot water extraction, ultrasonic extraction, reflux extraction, or heating extraction method, preferably by reflux extraction method, filtered and dried for a period of time, the wood scent of the present invention: Gaza, wood scent: health or health: Gaja A mixed crude extract containing

As another specific example, in the case of preparing a mixed extract containing all of Mokhyang, Gaja, and Health, Mokhyang: Gaja: Health may be mixed in a weight ratio of 1 to 3: 1 to 3: 1 to 3. As a specific example, Mokhyang: Gaja: Health may be mixed at a ratio of 1:1:1, 2:2:1, 2:1:1, 1:2:1, or 1:1:2. As a preferred embodiment, Mokhyang: Gaja: Health can be mixed in a ratio of 2: 2: 1 or 2: 1: 1, and as a more preferred example, Mokhyang: Gaja: Health can be mixed in a ratio of 2: 2: 1 can

As another example, the mixed extract containing two or more of the present invention, Gaza, and Health, is about 1 to 30 times the volume of each weight, preferably 5 to 15 times the volume (w / v%) A solvent selected from water including purified water, lower alcohols having 1 to 4 carbon atoms such as methanol, ethanol, and butanol, or mixed solvents thereof, preferably a mixed solvent of water and ethanol, preferably 50 to 100% ethanol, is added to about 20 to 120° C., preferably 60 to 100° C., for 1 to 15 hours, preferably 1 to 6 hours, by an extraction method such as cold extraction, hot water extraction, ultrasonic extraction, reflux extraction, or heating extraction, preferably under reflux. After each extraction by the extraction method, filtering, mixing of the respective extracts in a volume ratio of the above-described ratio, and then drying, a mixed crude extract containing two or more of the extracts of Mokhyang, Gajaja, and Health can be obtained.

In one embodiment of the present invention, a mixed extract containing the extract of cedar and gamhyang or cedar and health extract; And a mixed extract containing the extracts of chamomile, Gaza, and health was obtained.

Optionally, the extract of the present invention may further include an extract of one or more substances selected from the group consisting of white peony, Shiho, horseback riding, rhubarb, black man, black pepper, broiler, cine, baekchul, uiyiin, and the like.

Therefore, the present invention includes a method for preparing a mixed extract containing at least two of kokhyang, Gaza and health.

The above manufacturing method is only an exemplary method thereof, and may be appropriately modified and used by various methods based on the technology in the art. For example, non-exemplified extraction methods according to the present invention can be successfully performed with modifications obvious to those skilled in the art.

Those of ordinary skill in the field to which the present invention pertains, specific for the preparation of a mixed extract and a hop extract containing sage, sage, and health according to the present invention Since reaction conditions and the like can be confirmed through examples to be described later, a detailed description thereof will be omitted.

[Inflammatory Bowel Disease]

The present invention relates to the function of preventing, treating and/or improving inflammatory bowel disease of a mixed extract containing at least two of the above-prepared Mokhyang, Gaja and Health.

In the present invention, an inflammatory disease is any condition characterized by a local or systemic biological defense reaction caused by an external physicochemical stimulus or an external infectious agent infection such as bacteria, fungi, viruses, various allergens, or other causes. These reactions may be caused by activation of various inflammatory mediators and immune cells, and activation of related enzymes (eg, iNOS, COX-2, etc.), secretion of inflammatory mediators (eg, NO, TNF-α, IL-6, secretion of IL-1β and PGE2), body fluid infiltration, cell migration, tissue destruction, etc., accompanied by a series of complex physiological reactions, and symptoms such as erythema, pain, edema, fever, deterioration or loss of specific body functions, etc. may appear outwardly.

Since the inflammatory disease may have an acute, chronic, ulcerative, allergic or necrotic nature, as long as any disease is included in the inflammatory disease, whether it is acute, chronic, ulcerative, allergic or necrotic regardless of whether

In the present invention, the inflammatory disease may include inflammatory bowel disease.

"Inflammatory bowl disease (IBD)" is a chronic inflammatory disease of unknown cause that invades the digestive tract, and is an incurable disease in which diarrhea and bloody stools continue for a long time and recur.

As a general term for inflammatory bowel disease, it can be largely divided into ulcerative colitis (UC) and Crohn's disease (CD). Although the clinical features of the two diseases are different, both take a chronic course, and since the cause and pathophysiology are not known, it is called inflammatory bowel disease for convenience.

Inflammatory bowel disease is usually divided into ulcerative colitis and Crohn's disease, but intestinal Behcet's disease, which is relatively common in Korea, also belongs to this category. In addition to mouth ulcers, genital ulcers, and ocular symptoms, Behcet's disease can invade multiple organs, including the skin, blood vessels, gastrointestinal tract, central nervous system, heart, and lungs.

"Crohn's disease (CD)" and "ulcerative colitis (UC)" are chronic inflammatory bowel diseases of unknown etiology.

Unlike ulcerative colitis, Crohn's disease can affect any part of the intestine. The most prominent feature of Crohn's disease is the enlargement of granular, reddish-purple edema in the intestinal wall. As inflammation develops, these granulomas often lose their localized boundaries and integrate with the surrounding tissue. Diarrhea and intestinal obstruction are the main clinical features. Like ulcerative colitis, the course of Crohn's disease can be persistent or recurrent, mild or severe, but unlike ulcerative colitis, Crohn's disease cannot be cured by resection of the causative segment of intestine. Most patients who develop Crohn's disease require surgery at some point, but continued medical treatment is common because subsequent relapses are common.

"Ulcerative colitis (UC)" affects the large intestine. The disease course may be continuous or recurrent, mild or severe. The earliest lesion is an inflammatory infiltrate forming an abscess at the base of the Liberkun crypt. Adhesion of these distended and ruptured crypts tends to separate the overlying mucosa from its blood supply, resulting in ulceration. Symptoms of this disease include cramps, lower abdominal pain, rectal bleeding, and frequent, loose stools consisting mainly of blood, pus, and mucus with sparse fecal particles. Acute, severe or chronic, persistent ulcerative colitis may require total colectomy. The clinical picture of UC is highly variable, its onset may be insidious or sudden, and may include diarrhea, secondary episodes, and recurrent rectal bleeding. Fulminant involvement of the entire colon can result in toxic megacolon, a life-threatening emergency. Extraintestinal indications include arthritis, pyoderma gangrenosum, uveitis, and erythema nodosum.

Therefore, the inflammatory bowel disease that the mixed extract containing at least two of Eckhyang, Gaza and Health is intended to treat, prevent, and improve is, for example, Crohn's disease, ulcerative colitis, intestinal Behcet's disease, It includes at least one selected from the group consisting of intestinal tuberculosis enteritis, and diarrhea.

Monocyte adhesion inhibitory effect on intestinal epithelial cells of a mixed extract containing two or more of japonica, Gaza, and health through one embodiment of the present invention; LPS-induced inflammatory cytokine TNF-α, IL-6 production inhibitory effect; By observing the effects of recovery of colon length, weight loss, diarrhea, and bloody stool in animal models of DSS (Dextran sodium sulfate)-induced colitis and TNBS (Trinitrobenzene sulfonic acid)-induced Crohn's disease, the following specific functions were confirmed. .

(i) The mixed extract containing at least two of Mokhyang, Gaja and Health of the present invention has the ability to inhibit infiltration of mononuclear cells into the intestinal mucosa.

It is known that many monocytes infiltrate the intestinal mucosa of patients with inflammatory bowel disease (IBD). Monocytes, also called monocytes, are a type of phagocytes present in the blood that can differentiate into macrophages or dendritic cells, and play a role in regulating tissue homeostasis, immunity, and inflammatory responses. Infiltration of these monocytes into the intestinal mucosa eventually leads to mucosal destruction and ulceration, resulting in dysfunction and damage to the intestinal mucosa. Therefore, inhibition of monocyte infiltration in the intestinal mucosa can be said to be an important factor in preventing or treating intramucosal inflammation and ulceration.

In one embodiment of the present invention, it was confirmed that the effect of the mixed extract containing at least two of Mokhyang, Gaja, and Health was excellent in inhibiting the ability of intestinal epithelial cells to adhere to monocytes.

(ii) The mixed extract containing at least two of Moghyang, Gaja and Health of the present invention has an effect of inhibiting the production of inflammatory cytokines.

Monocytes and lymphocytes are thought to be involved in mucosal damage in the lesions of patients with inflammatory bowel disease (IBD), and inflammatory cytokines such as TNF-α and IL-6 produced from these cells cause intestinal inflammation or ulceration. , causing intestinal mucosal dysfunction and damage.

TNF-α is a factor that induces neutrophils to the relevant site at the beginning of the inflammatory response and causes and exacerbates the acute inflammatory response, and IL-6 is a representative inflammatory cytokine that is synthesized and secreted by various factors to cause acute or chronic inflammatory diseases. and play an important role in the process.

In one embodiment of the present invention, it was confirmed that the mixed extract containing two or more of the extracts of saffron, gaja, and health inhibited the production of inflammatory cytokines TNF- and IL-6 of macrophages induced by LPS.

(iii) The mixed extract containing two or more of the present invention, japonica, and health, has an improvement effect in the DSS inflammatory bowel disease animal model and the TNBS Crohn's disease animal model.

In one embodiment of the present invention, an animal model of DSS (dextran sodium sulfate)-induced inflammatory bowel disease and a 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced Crohn's disease animal model are made, and the animal model is used to And as a result of observing the effect of improving colitis or Crohn's disease of a mixed extract containing two or more of health, the length of the large intestine of the animal model was restored, and disease activity showing the degree of weight loss, diarrhea and bloody stool (Disease activity index, DAI) Also, the excellent improvement effect was confirmed. In addition, as a result of confirming the state of oxidative stress in immune diseases, when the mixed extract was treated, it was confirmed that the MPO activity was inhibited, so the excellent oxidative stress inhibitory effect was confirmed.

(iv) In one embodiment, when the mixed extract containing at least two of cedar, gaza and health is a 50% ethanol extract or at a specific mixing ratio of two or more substances, excellent cell proliferation and NO (nitric oxide) production inhibition had

Extraction solvents can be important for extracting components useful for treating inflammatory bowel disease from natural materials, etc., and depending on the extraction solvents, there may be differences in the amount of active ingredients extracted and the extraction of unnecessary components. It can be a meaningful technical feature.

In one embodiment of the present invention, as a result of confirming the cytotoxicity and NO production inhibitory effect of the mixed extract extracted by varying the concentration of ethanol in the extraction solvent, the 50% ethanol extract is not cytotoxic and promotes cell proliferation, and 76% It was confirmed that the NO production inhibitory effect was shown.

In addition, in the case of mixed extracts, differences in active ingredients or effects obtained may vary depending on the mixing ratio of each natural material or herbal medicine. Therefore, finding a blending ratio that can maximize efficacy while minimizing toxicity can also be a technically significant feature.

In one embodiment of the present invention, the effect of inhibiting the production of NO, which is a major factor in cytotoxicity and inflammation, and the inhibitory effect of monocyte infiltration that causes ulcers of the mixed extract according to the blending ratio (w / w) of Kochhyang and Gaja or health It was confirmed, and it was confirmed that Mokhyang: Gaja or Health showed an excellent effect when mixed in a ratio of about 1: 1.

In another embodiment of the present invention, the effect of inhibiting monocyte infiltration and the production of inflammatory cytokines that cause ulcers of the mixed extract according to the mixing ratio (w / w) of Mokhyang, Garza and Health, and DSS inflammatory bowel disease animal model and The colitis improvement effect was confirmed in the TNBS Crohn's disease animal model, and it was confirmed that the excellent effect was exhibited when Mokhyang: Gaja: Health was mixed in a ratio of about 2: 2: 1.

(v) In one embodiment of the present invention, the effect of improving colitis in the DSS inflammatory bowel disease animal model and the TNBS Crohn's disease animal model according to the effective dose of the mixed extract containing at least two of Mokhyang and Gaza or Health was confirmed. .

As a result of confirming the effect of the dose of the mixed extract, both the DSS inflammatory bowel disease animal model and the TNBS Crohn's disease animal model showed that as the dose increased, the intestinal length, disease activity (DAI), and intestinal weight per intestinal length were restored to normal. It was confirmed that, when administered at 400 mg/kg, the best colitis improvement effect was shown.

As such, the present invention includes all of the various uses of the inflammatory bowel disease prevention, treatment and / or improvement function of the mixed extract containing at least two of the above-mentioned Mokhyang, Gaja and Health.

[Pharmaceutical composition]

In one embodiment, the present invention relates to a pharmaceutical composition for the prevention or treatment of inflammatory bowel disease containing a mixed extract containing at least two of Morphaceae, Gaza and Health as an active ingredient.

The preventive and therapeutic effects of inflammatory bowel disease defined in the present invention inhibit mononuclear cell infiltration into the intestinal mucosa, inhibit the production of inflammatory cytokines TNF-α and IL-6, restore the length of the large intestine, and restore body weight according to colitis. It is characterized in that it works by reducing diarrhea, diarrhea and bloody stool.

In addition, the present invention provides a method for treating and preventing inflammatory diseases comprising administering the pharmaceutical composition to a subject suffering from inflammatory bowel disease.

The pharmaceutical composition for preventing and treating inflammatory bowel disease containing the mixed extract of the present invention preferably contains 0.1 to 50% by weight of the extract based on the total weight of the composition. More preferably, the mixed extract is used at a concentration of 50 to 400 mg/kg, most preferably 200 to 400 mg/kg.

The pharmaceutical composition containing the mixed extract of the present invention may further include suitable carriers, excipients and diluents commonly used in the preparation of pharmaceutical compositions.

In addition, the composition containing the mixed extract may be formulated or used in combination with drugs such as steroid drugs, antihistamines, anti-inflammatory drugs and antibiotics that are already used.

The pharmaceutical composition containing the mixed extract is formulated in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories and sterile injection solutions, respectively, according to conventional methods. can be used interchangeably. In the present invention, it can be preferably formulated as an oral dosage form.

Carriers, excipients and diluents that may be contained in the composition containing the extract of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum , alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. there is.

When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations contain at least one excipient such as starch, calcium carbonate, sucrose or lactose in the compound. , gelatin, etc. are mixed and prepared. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, solutions for oral use, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. .

Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base for the suppository, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogeratin and the like may be used.

In the administration method of the composition of the present invention, it can be used for oral administration and parenteral administration, and the administration method is not specifically limited, but it is preferably used as an oral administration agent.

The amount of the composition of the present invention may vary depending on the patient's age, sex, and weight, but may be administered at 0.0001 to 400 mg/kg. In addition, the dosage may be increased or decreased depending on the route of administration, severity of disease, sex, weight, age, and the like. It can be administered once a day or divided into several doses. Therefore, the dosage is not intended to limit the scope of the present invention in any way.

In one embodiment of the present invention, the colitis amelioration effect was confirmed in the DSS inflammatory bowel disease animal model and the TNBS Crohn's disease animal model according to the effective dose of the mixed extract containing at least two of Mokhyang and Gaja or Health, and that Results In both the DSS inflammatory bowel disease animal model and the TNBS Crohn's disease animal model, it was confirmed that the intestinal length, disease activity (DAI), and intestinal weight per intestinal length were restored to normal as the dose increased, and when administered at 400 mg/kg It showed the best colitis improvement effect.

The pharmaceutical composition may be administered to mammals such as rats, mice, livestock, and humans through various routes. All modes of administration are contemplated, including intravenous, intraperitoneal, intramuscular, intraarterial, oral, intracardiac, intramedullary, intrathecal, transdermal, enteral, subcutaneous, sublingual or topical administration, but Not limited.

The pharmaceutical dosage form of the composition of the present invention may be used in the form of a pharmaceutically acceptable salt thereof, and may also be used alone or in combination with other pharmaceutically active compounds as well as in a suitable set. In addition, the pharmaceutical composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy or biological response modifiers for the prevention or treatment of inflammatory bowel disease or related complications.

[Health functional food]

On the other hand, the present invention relates to a health functional food for preventing or improving inflammatory bowel disease containing, as an active ingredient, a mixed extract containing at least two of Mokhyang, Gaja and Health.

Functionality can be roughly divided into physical properties and physiological functions. When a mixed extract containing two or more of the present invention is added to general food, the physical properties and physiological functionality of the general food will be improved. For example, prevention and improvement of Chron's disease, ulcerative colitis, intestinal Behcet's disease, intestinal tuberculosis and enteritis, diarrhea, etc. Functional food for use can be prepared. In addition, it will be possible to manufacture functional enhanced foods using the same.

That is, the compound or pharmaceutically acceptable salt thereof containing a mixed extract comprising two or more of the present invention, saffron, japonica and health, can be used as a main component or an additive or adjuvant of food in the preparation of various functional foods and health functional foods. can

In the present invention, there is no particular limitation on the type of food, and examples of food to which a mixed extract containing two or more of the above-mentioned Mokhyang, Gaja, and Health can be added include various types of food, powders, granules, tablets, capsules, syrups, and beverages. , gums, chocolates, candies, snacks, teas, vitamin complexes, etc., and may include all health foods in a conventional sense.

The health beverage composition of the present invention has no particular limitations on liquid components other than containing the mixed extract as an essential component in the indicated ratio, and may contain various flavoring agents or natural carbohydrates as additional components like conventional beverages. . Examples of the aforementioned natural carbohydrates include monosaccharides such as glucose, disaccharides such as fructose, such as maltose, sucrose, etc., and polysaccharides such as dextrins, cyclodextrins, and the like. It is sugar and sugar alcohols, such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (thaumatin, stevia extract (e.g. rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can advantageously be used. The proportion 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 composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and enhancers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its It may contain salts, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, and the like. In addition, the compositions of the present invention may contain fruit flesh for preparing natural fruit juice, fruit juice beverages, and vegetable beverages. These components may be used independently or in combination. The proportion of these additives is not critical, but is generally selected from the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

In addition, in the present invention, since the mixed extract containing at least two of Alfalfa, Garza and Health is a herbal ingredient, it can be safely used even when taken for a long period of time for the purpose of treating, improving, and preventing diseases.

As such, the present invention is an effect of inhibiting monocyte adhesion in intestinal epithelial cells, an inhibitory effect on the production of TNF-α, an inflammatory cytokine induced by LPS, IL-6, and DSS (Dextran sodium sulfate)-induced colitis animal model and TNBS (2,4,6-trinitrobenzenesulfonic acid)-induced Crohn's disease animal model with recovery of colon length, weight loss, diarrhea, and improvement in bloody stools, and an effective amount showing excellent anti-inflammatory effects It includes all the various uses using .

Example

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

experimental material

1. Material and Sample Extraction

Two or more of 5 g of Mokhyang, 5 g of Health, and 5 g of Gaja were mixed, and 100 ml of 50% ethanol aqueous solution, which is 10 times the amount of the sample weight, was added, followed by reflux extraction at 95 ° C for 2 hours. The obtained extract was filtered and then freeze-dried. It was used as a sample for this experiment while being stored in a refrigerator in a powder state. The present experiment was conducted by preparing a mixed extract of Mokhyang and health, a mixed extract of Mokhyeong and Gaja, a mixed extract of health and Gaja, and a mixed extract of Mokhyang, health and Gaja.

2. Reagents and instruments

(1) Reagents and devices for measuring the effect of inhibiting monocyte adhesion in intestinal epithelial cells

Human colon epithelial cell line HT-29 cells (American Type Culture Collections, Rockville, MA, USA), human monocyte cell line U937 cells (ATCC, Rockville, MA, USA), fetal bovine serum (FBS), penicillin/streptomycin, 2' ,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF/AM), 5-aminosalicylic acid (5-ASA) and a microplate reader for measurement (TECAN, Gr, Austria) was used.

(2) Anti-inflammatory reagents and instruments

Macrophages (American Type Culture Collections, Rockville, MA, USA), 5-aminosalicylic acid (5-ASA), dexamethasone, TNF-α ELISA kit (R&D Systems, USA), IL-6 ELISA kit (R&D Systems, USA) and a microplate reader (TECAN, Gr, Austria) for measurement was used.

(3) DSS animal model production reagent

C57BL/6 female mice (17-19 g, 6 weeks old, Daehan Biolink (Eumseong, Korea)), 5-aminosalicylic acid (5-ASA), CMC (Carboxymethyl Cellulose), and DSS (Dextran sodium sulfate) were used.

(4) TNBS animal model production reagent

ICR female mice (17-19 g, 6 weeks old, Daehan Biolink (Eumseong, Korea)), 5-aminosalicylic acid (5-ASA), and 2,4,6-trinitro-benzene sulfonic acid (TNBS) were used.

Experiment method

1. Cell viability test (cytotoxicity test)

Cells were plated and cultured at a certain number according to the purpose of the test. After 24 hours, the medium was replaced with a medium that did not contain FBS, and then cultured for 24 hours, and the prepared extract was treated. After 24 hours of material treatment, the medium was removed, and 5 mg/ml MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl Tetrazolium Bromide) reagent was treated with 40 μl/well and further incubated for 4 hours. did After 4 hours, the medium was removed, and 1 ml of dimethylsulfoxide (DMSO, Amresco, 0231-500ML) was added and shaken for 10 minutes. Then, 200 μl of each was taken into 96 wells and the absorbance was measured at 540 nm. The degree of cytotoxicity was expressed as a percentage based on the absorbance intensity treated with the solvent in which the substance was dissolved as a control.

2. NO production inhibitory effect

Human colon epithelial cell line HT-29 cells (American Type Culture Collections, Rockville, MA, USA) were cultured in Dulbecco's Modified Eagle's Medium (DMEM), 10% Fatal bovine serum (FBS), and 1% Antibiotic-Antimycotic (GIBCO, Cat No. 15240). -062) in a 100 mm/60.1 cm² culture dish at 37°C and 5% CO ₂ conditions. When these HT-29 cells proliferate by more than 80%, 10 ⁵ cells/well are dispensed into a 6-well plate, cultured until they proliferate by more than 80% in cell culture conditions, then the medium is removed, and then a new sample containing the extract prepared at an appropriate concentration is obtained. After exchange with medium, it was additionally cultured under cell culture conditions for 48 hours. A culture solution of these cells was taken and used as a test sample. Test analysis was performed according to the manufacturer's manual using a Total NO/Nitrite/Nitrate ELISA kit (R&D systems, Cat.# KGE001). Briefly, in the test order, 50 μl of the cultured cell culture medium by processing the sample was put into a 96-well plate, treated with Griess Reagent, reacted at room temperature for 10 minutes, and absorbance was measured at 540 nm.

3. Monocytes in intestinal epithelial cells adhesion inhibitory effect

Human colon epithelial cell line HT-29 cells (American Type Culture Collections, Rockville, MA, USA) and human monocyte cell line U937 cells (ATCC, Rockville, MA, USA) were cultured in fetal bovine serum (FBS) and 1% penicillin/streptomycin. The cells were cultured in RPMI 1640 containing 95% air and 5% CO ₂ , and the cell line was maintained at 37°C.

Monocyte-colon epithelial cell adhesion was assessed with 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF/AM, 10 μg/ml) for 30 min at 37°C. It was measured using U937 cells labeled with . HT-29 cells (2X10 ⁶ cells/well) cultured for 24 hours in a 48 well plate were treated with TNF-α (100ng/ml) and simultaneously prepared extract (100μg/ml) and cultured for 24 hours.

At this time, as a positive control, 20 mM 5-aminosalicylic acid (5-ASA) was used. Then, HT-29 cells were co-cultured with BCECF/AM-labeled U937 cells (5X10 ⁵ cells/well) at 37°C for 30 minutes. Non-adherent U937 cells were removed and washed twice with PBS. HT-29 cells with attached U937 cells were lysed using 0.1% Triton X-100 dissolved in 0.1 mol/L Tris. Fluorescence of these cell lysates was measured using a microplate reader (TECAN, Gr, Austria) using luminescence at 485 nm and 520 nm.

4. TNF -α production inhibitory effect

Macrophages (American Type Culture Collections, Rockville, MA, USA) cultured in a 24-well plate were pretreated with LPS (1 μg/mL) for 1 hour, and then treated with 100 μg/ml extract, 20 mM 5-ASA, and 20 μM dexamethasone The cell culture medium cultured for 24 hours was used. First, the TNF-α capture antibody was placed in a 96-well ELISA plate and reacted overnight. The plate was washed 6 times with 1xPBS (PBS-T) containing 0.05% Tween-20 and then blocked with 1x assay diluent solution containing 2% BSA for 1 hour at room temperature. After washing the plate 6 times with 1x PBS-T, the cell culture medium or TNF-α standard protein was added and reacted at room temperature for 2 hours. It was again washed 6 times with 1x PBS-T and reacted at room temperature for 2 hours with a detection antibody. The plate was again washed 6 times with 1x PBS-T and reacted at room temperature for 30 minutes with an avidin-HRP solution. After washing with 1x PBS-T solution 6 times, TMB substrate solution was added thereto, and reaction was performed at room temperature for the last 30 minutes, and reaction stop solution (1M H ₃ PO ₄ ) was added to stop the reaction. The degree of reaction of the plate was measured by absorbance at 450 nm with a microplate reader (TECAN, Gr, Austria).

5. IL-6 production inhibitory effect

Macrophages (American Type Culture Collections, Rockville, MA, USA) cultured in a 24-well plate were pretreated with LPS (1 μg/mL) for 1 hour, and then treated with 50 μg/ml extract, 20 mM 5-ASA, and 20 μM dexamethasone The cell culture medium cultured for 24 hours was used. First, the capture antibody of IL-6 was put into a 96-well ELISA plate and reacted overnight. The plate was washed 6 times with 1xPBS (PBS-T) containing 0.05% Tween-20 and then blocked with 1x assay diluent solution containing 2% BSA for 1 hour at room temperature.

After washing the plate 6 times with 1x PBS-T, the cell culture medium or IL-6 standard protein was added and reacted at room temperature for 2 hours.

It was again washed 6 times with 1x PBS-T and reacted at room temperature for 2 hours with a detection antibody. The plate was again washed 6 times with 1x PBS-T and reacted at room temperature for 30 minutes by adding an avidin-HRP solution. After washing with 1x PBS-T solution 6 times, TMB substrate solution was added thereto, and reaction was performed at room temperature for the last 30 minutes, and reaction stop solution (1M H ₃ PO ₄ ) was added to stop the reaction. The degree of reaction of the plate was measured by absorbance at 450 nm with a microplate reader (TECAN, Gr, Austria).

6. Myeloperoxidase ( MPO ) via active measurement oxidative stress suppression effect

200 μl of lysis buffer was added to 100 mg of colon tissue collected during autopsy, homogenization was performed, and the supernatant obtained by operating the centrifuge at 10000 rpm for 20 minutes was used. First, MPO's capture antibody was put into a 96-well ELISA plate and reacted overnight. The plate was washed 6 times with 1X PBS (PBS-T) containing 0.05% Tween-20 and then blocked with 1x assay diluent solution containing 2% BSA for 1 hour at room temperature. After washing the plate 6 times with 1x PBS-T, the prepared supernatant or MPO standard protein was added and reacted at room temperature for 2 hours. It was again washed 6 times with 1x PBS-T and reacted at room temperature for 2 hours with a detection antibody. The plate was washed again with 1x PBS-T 6 times and reacted at room temperature for 30 minutes with Streptavidin-HRP solution. After washing with 1x PBS-T solution 6 times, TMB substrate solution was added thereto, and reaction was performed at room temperature for the last 30 minutes, and reaction stop solution (1M H ₃ PO ₄ ) was added to stop the reaction. The degree of reaction of the plate was measured by absorbance at 450 nm with a microplate reader (TECAN, Gr, Austria).

7. Colitis improvement effect in DSS animal model

To construct an experimental model of inflammatory bowel disease, C57BL/6 female mice (17-19 g), 6 weeks old, were purchased from Daehan Biolink (Eumseong, Korea) and allowed to consume solid food and water freely for one week at a constant humidity ( 50%), a constant temperature (22 ℃), and a 12-hour period in which the contrast was adjusted to the experimental environment for 1 week. After undergoing an adaptation period for one week, they were divided into a normal control group, a colitis-induced group, a control drug administration group (5-ASA 200 mg/kg), and an extract administration group. The control drug (5-ASA) and extract were dissolved in 0.5% CMC (Carboxymethyl Cellulose), and then orally administered once a day together with 3% DSS (Dextran sodium sulfate) intake for 7 days. Normal control group and colitis-induced group Silver 0.5% CMC was orally administered. The colitis-inducing group, the control drug-administered group, and the extract-administered group freely consumed 3% DSS for one week, and the normal control group consumed water freely. Then, on the 8th day, the mice were sacrificed, and the cecum was extracted from the cecum to the anus immediately before the anus. marked with a score.

8. TNBS Improvement effect of Crohn's disease in animal models

To construct an experimental model of inflammatory bowel disease, 7-week-old ICR female mice (25-27 g) were purchased from Daehan Biolink (Eumseong, Korea) and fed solid food and water freely for one week at a constant humidity (50± 10%), a constant temperature (22 ± 2 ℃), and a 12-hour cycle, the light and shade were adjusted to the experimental environment for 1 week. After undergoing an adaptation period for one week, they were divided into a normal control group, a colitis-induced group, a control drug administration group (5-ASA 200 mg/kg), and an extract administration group (200 mg/kg). They were fasted 24 hours before TNBS injection. Animals in the colitis-inducing group, control drug-administered group, and extract-administered group were anesthetized with ether, and then TNBS (0.5 mg) was dissolved in 50% ethanol and injected into the anus using a PE zone at an injection depth of 2-3 cm. . For about 1 minute after injection, the head was placed downward and the tail was raised to prevent the injected solution from flowing out again through the anus, so that the drug was absorbed into the intestine as much as possible. After dissolving the control drug (5-ASA) and extract in 0.5% CMC (Carboxymethyl Cellulose), oral administration was administered once a day for 5 days, and 0.5% CMC was orally administered to the normal control group and the colitis-induced group. After oral administration on the 5th day, fasting until the next day, mice were sacrificed, and the large intestine from the cecum to the anus just before the anus was removed. The length and appearance of the excised colon were observed, and the degree of weight loss, constipation, and intestinal edema was confirmed and marked as a score.

Example One : elecampane and efficacy confirmation for health blend extract

1-1: monocyte infiltration inhibition

The monocyte invasion inhibitory ability of the extracts of the present invention and health mixture was confirmed.

As a result, the fluorescence-labeled U937 cell adhesion to HT-29 colonic epithelial cells was significantly increased by TNF-α stimulation, and was inhibited by 18.93% by the control drug (5-ASA). In the case of the single extracts of Mokhyang (purified water) and Health, respectively, the inhibitory effect was 25.21 and 12.36%, respectively, but in the case of the mixed extract of the present invention, it was confirmed that the inhibitory effect was 44.15%, and compared to the control drug, it was confirmed that the efficacy was equal or higher (Table 1, Fig. 1). In addition, the mixed extract of the present invention showed excellent efficacy compared to the single health single extract and the single extract of Mokhyang (purified water). Through these results, it was confirmed that the extract inhibited the infiltration of monocytes more effectively than the control drug.

division density Adhesion inhibition ability (%) 5-ASA (positive control) 20 mM 18.93 Mokhyang (purified water) 100 μg/ml 25.21 health 100 μg/ml 12.36 wood scent + health 100 μg/ml 44.15

1-2: Inhibitory effect on inflammatory cytokine production

It was intended to confirm the ability to inhibit the production of inflammatory cytokines of the extracts of Mokhyang and Health of the present invention.

(1) TNF-α production inhibitory effect

As a result of confirming the concentration of TNF-α, it was found that the expression of TNF-α was significantly reduced in the sample and control group than in the group treated with LPS alone, and in the case of 5-ASA used as a control, the production of TNF-α In the case of dexamethasone, the production of TNF-α was suppressed by 38.46%.

In the case of single extracts of Mokhyang (purified water) and health, respectively, 8.76 and 18.87% of inhibitory effects were shown, but in the case of the extract of the present invention, the production of TNF-α was inhibited by 65.9% (Table 2, Figure 2).

division density TNF-α inhibition (%) 5ASA 20 mM 11.09 Dexamethasone 20 µM 38.46 Mokhyang (purified water) 100 μg/ml 8.76 health 100 μg/ml 18.87 wood scent + health 100 μg/ml 65.90

(2) IL-6 production inhibitory effect

In addition, as a result of checking the concentration of IL-6, it was found that the expression of IL-6 was significantly reduced in the sample and control group than in the LPS-only group, and in the case of 5-ASA used as a control, IL-6 The production of 6 was inhibited by 52.78%, and in the case of dexamethasone, the production of IL-6 was inhibited by 49.89%.

In the case of single extracts of Mokhyang (purified water) and Health, respectively, 5.40 and 19.53% of inhibitory effects were shown, but in the case of the extract of the present invention, the production of IL-6 was inhibited by 94.05% (Table 3, Figure 3).

division density IL-6 inhibition (%) 5ASA 20 mM 52.78 Dexamethasone 20 µM 49.89 Mokhyang (purified water) 100 μg/ml 5.40 health 100 μg/ml 19.53 wood scent + health 100 μg/ml 94.05

Through these results, it was confirmed that the inflammatory cytokine inhibitory effect in the extract of the present invention was greater than that of the control group, and also showed excellent efficacy in the mixed extract of the present invention compared to the single healthy and single extract of Mokhyang (purified water). Judging from this, it was found that the IBD-related inflammatory Cytokine production inhibited in vitro test results had a very good anti-inflammatory effect.

1-3: Colitis improvement effect in DSS animal model

In the DSS inflammatory bowel disease animal model, the colitis treatment and improvement effects of the extracts of Mokhyang and Health mixture of the present invention were confirmed.

As a result of checking the length of the intestine, it was confirmed that the length of the intestine in the colitis-induced group was about 1.7 cm shorter than that of the normal group. In the case of the control group, it was confirmed that the length increased by 3.6% compared to the colitis-inducing group. In the case of Mokhyang single extract, the length increased by 8.22% compared to the colitis-inducing group, whereas in the case of the mixed extract-administered group of the present invention, the length increased by 12.8% compared to the colitis-inducing group. It was confirmed that (Table 5, Figure 4).

In addition, as a result of observing the appearance and showing the disease activity (Table 4), diarrhea and bloody stool were found in the colitis-induced group, showing a high value of 8.25. In the case of the control group, an improvement effect of about 45% compared to the colitis-induced group . In the case of Mokhyang single extract, the effect was 57.57% compared to the colitis-inducing group, whereas the mixed extract administration group of the present invention showed an effect of 78% compared to the colitis-inducing group, showing an improvement effect equal to or greater than that of the control drug administration group and the single extract ( Table 5, Figure 5).

score weight loss (%) stool consistency degree of bleeding 0 doesn't exist commonly doesn't exist One 1-5 2 5-10 Thinness latent bleeding 3 10-20 4 >20 diarrhea heavy bleeding

division colon length DAI normal control 6.9 0.83 colitis inducing group 5.23 8.25 Control drug administration group 5.42 4.50 Mokhyang (purified water) 5.42 4.50 elecampane 5.66 3.50 wood scent + health 5.90 1.75

In addition, as a result of obtaining intestinal mucosal tissue and confirming the tissue state using immunostaining, it was confirmed that more empty spaces caused by necrosis of the intestinal mucosa were seen in DSS animals than in the intestinal mucosa of normal animals.

In the case of administration of the control group, it was confirmed that the intestinal mucosa was partially recovered, but when compared with the normal group, it was found that part of the intestinal mucosa was still not recovered. On the other hand, when the mixed extract of the present invention was administered, it was confirmed that the intestinal mucosa was recovered to a degree similar to that of the normal group, indicating that it had an excellent effect on the recovery of the damaged intestinal mucosa (FIG. 6).

In addition, as a result of measuring myeloperoxidase (MPO) activity to confirm the state of oxidative stress in immune diseases,

In the colitis-induced group, an MPO activity level of about 0.85 was confirmed, and in the case of the control drug administration group and the single extract of Mokhyang, the MPO activity levels were reduced by 17.6% and 23.5%, respectively.

The mixed extract-administered group of the present invention exhibited an MPO activity reduction effect of about 52.9% compared to the colitis-inducing group, and exhibited a better oxidative stress inhibitory effect than the control drug-administered group and the single extract of Mokhyang (FIG. 7).

Through these results, it was found that the extracts of the present invention can be usefully used in the prevention or treatment of inflammatory bowel disease because they show excellent anti-inflammatory and colitis-improving effects in the case of the mixed extract of Mokhyang and Health.

Furthermore, in order to find out the conditions of the mixed extract that exerts a more optimal effect, the effect was confirmed by varying the solvent concentration and mixing ratio.

1-4: according to solvent concentration elecampane and cytotoxicity test of health mixture extract

(1) MTT analysis

Cytotoxicity was compared according to the ethanol-containing concentration of the solvent used in the mixed extracts of Mokhyang and Health of the present invention.

As a result, it was confirmed that all of the 30%, 50%, and 95% ethanol extracts showed no cytotoxicity. In particular, in the case of 50% ethanol extract, cell viability was increased by about 330% compared to the control group, and cell proliferation was promoted (FIG. 8).

It was confirmed that there was no significant difference in cytotoxicity according to the concentration of ethanol, and among them, it was found that the 50% ethanol extract would be effective in cell proliferation in case of deficiency such as cell death due to inflammation by promoting cell proliferation.

(2) NO production inhibition ability

Furthermore, NO production inhibitory ability was compared according to the ethanol-containing concentration of the solvent used in the mixed extracts of Mokhyang and Health of the present invention.

As a result, the 30% ethanol extract showed 48% inhibitory activity, and the 50% ethanol extract and 95% ethanol extract showed 76% and 68% inhibition of NO production, respectively. This is a result showing a superior effect than the 37% inhibitory ability of 5-ASA, which is a positive control group (FIG. 9).

Through these results, it was confirmed that the NO production inhibitory effect according to the concentration of ethanol was superior to that of the positive control, and in particular, the 50% ethanol extract showed the best NO production inhibitory effect.

According to the results of Examples 1-4 above, the extracts of Mokhyang and Health mixed extracts extracted using 50% ethanol solvent, which promotes cell proliferation without cytotoxicity and has the best inhibitory effect on NO production related to inflammation, are prepared in the following examples. was used to confirm the effect.

1-5: elecampane and cytotoxicity test according to the mixing ratio of health

In order to confirm the blending ratio having the best efficacy of the mixed extract of Mokhyang and health of the present invention, the cytotoxicity of each mixed extract was confirmed with a difference in the blending ratio of Mokhyang and health.

As a result, it was confirmed that cytotoxicity appeared as the ratio of health increased in the mixed extract (FIG. 10). In particular, when the ratio of Mokhyang:Healthy was 3:7 to 1:9, the cell viability was only 60% or less, and when the ratio of Mokhyang:Healthy was 9:1 to 4:6, more than 70% of the cells It was confirmed that there was a survival rate.

In addition, the NO production inhibitory ability of each mixed extract was confirmed with a difference in the blending ratio of Mokhyang and Health.

As the ratio of health in the mixed extract increased, the NO production inhibitory effect was shown, and among them, it was confirmed that more than 70% of NO production was inhibited when Mokhyang: health was 6: 4 to 1: 9 (FIG. 11). In particular, it was found that, preferably, the production of NO, which is important for inflammation, was suppressed when the ratio of Mokhyang:Health was 6:4 or 5:5.

1-6: elecampane and monocytes in intestinal epithelial cells according to the mixing ratio of health adhesion inhibitory effect

In order to confirm the blending ratio having the best efficacy, the mononuclear cell invasion inhibitory ability of each mixed extract was confirmed with a difference in the blending ratio of Mokhyang and Health.

As shown in FIG. 12, in the case of mixed extracts with different blending ratios of Mokhyang and Health, it was confirmed that mononuclear cell infiltration was inhibited as the ratio of Health increased, and when Mokhyang: Health was 5: 5, the inhibition was 25.06%. , 6: 4, it was confirmed that the inhibitory effect was 32.5%.

The combination ratio showing the best NO production inhibition and mononuclear cell invasion inhibition without cytotoxicity was the case of Mokhyang: Health, 6: 4 or 5: 5 (i.e., 1: 1).

Example 2 : elecampane Confirmation of efficacy for Gaza mixed extract

On the other hand, the present inventors performed the following experiment using the mixed extract of Mokhyang and Gajaja.

2-1 Monocytes in intestinal epithelial cells adhesion inhibitory effect

The present invention confirmed the monocyte invasion inhibitory ability of the mixed extracts of Mokhyang and Gajaja.

The adhesion of fluorescently labeled U937 cells to HT-29 colonic epithelial cells was significantly increased by TNF-α stimulation, and was inhibited by 19.94% by the control drug (5-ASA). In the case of the single extract of Mokhyang (purified water), each showed an inhibitory effect of 15.21%, but in the case of the mixed extract of the present invention, it was confirmed that the inhibitory effect was 21.18%, and compared to the control drug, it was confirmed that the same or higher efficacy appeared (Table 6, FIG. 13) .

In addition, the mixed extract of the present invention showed excellent efficacy compared to the single extract of Mokhyang (purified water). Through these results, it was confirmed that the extract inhibited monocyte infiltration as effectively as the control drug.

division density Adhesion inhibition ability (%) 5-ASA (positive control) 20 mM 18.93 Mokhyang (purified water) 100 μg/ml 25.21 Mokhyang + Let's go 100 μg/ml 49.36

2-2 Production of inflammatory cytokines inhibition

(1) TNF-α production inhibitory effect

As a result of confirming the ability of the mixed extracts of the present invention to inhibit the production of TNF-α, it was found that the expression of TNF-α was significantly reduced in the sample and control group compared to the LPS-only group. In the case of 5-ASA, the production of TNF-α was suppressed by 11.09%, and in the case of dexamethasone, the production of TNF-α was suppressed by 38.46%.

In the case of Mokhyang (purified water) single extract, each showed an inhibitory effect of 8.76%, but in the case of the mixed extract of the present invention, the production of TNF-α was inhibited by 62.71% (Table 7, FIG. 14).

division density TNF-α inhibitory ability (%) 5-ASA (positive control) 20 mM 11.09 Dexamethasone (positive control) 20 µM 38.46 Mokhyang (purified water) 100 μg/ml 8.76 Mokhyang + Let's go 100 μg/ml 62.71

(2) IL-6 production inhibitory effect

As a result of additionally confirming the concentration of IL-6, it was found that the expression of IL-6 was significantly reduced in the sample and control group than in the LPS-only group, and in the case of 5-ASA used as a control, IL-6 production was inhibited by 67.68%, and in the case of dexamethasone, IL-6 production was inhibited by 49.89%.

In the case of a single extract of Mokhyang (purified water), each showed an inhibitory effect of 7.48%, but in the case of the extract of the present invention, the production of IL-6 was inhibited by 95.19% (Table 8, FIG. 15).

division density IL-6 inhibition (%) 5-ASA (positive control) 20 mM 52.78 Dexamethasone (positive control) 20 µM 49.89 Mokhyang (purified water) 100 μg/ml 5.40 Mokhyang + Let's go 100 μg/ml 95.08

2-3. Colitis improvement effect in DSS animal model

In the DSS inflammatory bowel disease animal model, the therapeutic and ameliorative effects of the mixed extract of Mokhyang and Gaza of the present invention on colitis were confirmed.

As a result of checking the intestinal length, it was confirmed that the intestinal length in the colitis-induced group was shortened by about 1.7cm compared to the normal group, and in the case of the control drug-treated group, it was confirmed that the intestinal length increased by 3.6% compared to the colitis-induced group. In the case of the extract, it was confirmed that the length increased by 8.22% compared to the colitis-inducing group, and in the case of the mixed extract administration group of the present invention, the length increased by 10.5% compared to the colitis-inducing group (Table 9, FIG. 16A).

division Large intestine length (cm) DAI normal control 6.9 67.68 colitis inducing group 5.23 49.89 Control drug administration group 5.42 7.48 Mokhyang (purified water) 5.42 95.19 Mokhyang + Let's go 5.78 4.5

In addition, as a result of observing the appearance state and showing the disease activity, diarrhea and bloody stool were found in the colitis-induced group, showing a high value of 8.25, and in the case of the control drug-administered group, it showed an improvement effect of about 45% compared to the colitis-induced group. The mixed extract administration group of the present invention showed an effect of 45% compared to the colitis-inducing group, showing an improvement effect equivalent to that of the control drug administration group (Table 9, Fig. 16 B).

As a result of measuring myeloperoxidase (MPO) activity to confirm the status of oxidative stress in immune diseases, the MPO activity level was about 0.9 in the colitis-induced group, and about 22% in the control drug-treated group. MPO activity values are shown. The mixed extract administration group of the present invention showed an effect of reducing MPO activity by about 50% compared to the colitis-inducing group, and showed a better oxidative stress inhibitory effect than the control drug administration group (FIG. 17).

2-4 TNBS Improvement effect of Crohn's disease in animal models

ICR female mice (17-19 g, 6 weeks old, Daehan Biolink (Eumseong, Korea)), 5-aminosalicylic acid (5-ASA), and 2,4,6-trinitro-benzene sulfonic acid (TNBS) were used.

In the TNBS Crohn's disease animal model, the treatment and improvement effects of the mixed extract of Mokhyang and Gaza of the present invention were confirmed.

As a result of checking the weight of the intestine per intestine length, it was confirmed that the Crohn's disease group was about 37mg/cm, which was increased by about 4 to 5mg/cm compared to the normal group. The control drug-administered group showed little difference when compared to the Crohn's disease-induced group, which indicates that there is no effect in reducing the thickened barrier.

On the other hand, in the case of the group administered with the mixed extract of Mokhyang and Gaza of the present invention, it was confirmed that the group reduced by about 16% compared to the Crohn's disease-induced group. confirmed (FIG. 18 A).

In addition, as a result of observing the appearance and showing the disease activity, diarrhea and bloody stool were found in the Crohn's disease-induced group, showing a high value of about 7. showed On the other hand, the mixed extract administration group of the present invention showed an effect of about 45% compared to the Crohn's disease-induced group, showing a superior improvement effect than the control drug administration group (FIG. 18 B).

As a result of measuring MPO activity to confirm the state of oxidative stress in immune diseases, the MPO activity level of about 0.38 was confirmed in the Crohn's disease-induced group, and about 15% of MPO decreased in the control drug-treated group. The activity values are shown. The mixed extract administration group of the present invention showed an effect of reducing MPO activity by about 21% compared to the Crohn's disease-induced group, and exhibited an oxidative stress inhibitory effect equal to or higher than that of the control drug administration group (FIG. 18 C).

From these results, it was found that the mixed extract of Mokhyang and Gajaja of the present invention can be usefully used for preventing or treating inflammatory bowel disease because it shows excellent anti-inflammatory and Crohn's disease improving effects.

Example 3: Confirmation of efficacy for health and Gaza mixed extract

On the other hand, the present inventors performed the following experiment using the mixed extract of Geonggung and Gajaja.

3-1 Monocytes in intestinal epithelial cells adhesion inhibitory effect

The present invention confirmed the monocyte invasion inhibition ability of the mixed extract of health and Gajaja.

The adhesion of fluorescently labeled U937 cells to HT-29 colonic epithelial cells was significantly increased by TNF-α stimulation, and was inhibited by 18.93% by the control drug (5-ASA). In the case of the health or Gaja single extract, 12.35 and 14.55% of the inhibitory effects were shown, respectively, but in the case of the mixed extract of the present invention, it was confirmed that the inhibitory effect was 31.76%, and it was confirmed that the efficacy was equal or higher than that of the control drug (Table 10, FIG. 19 ).

In addition, the mixed extract of the present invention showed excellent efficacy compared to the single extract of health or Gaza. Through these results, it was confirmed that the extract inhibited monocyte infiltration as effectively as the control drug.

division density Adhesion inhibition ability (%) 5-ASA (positive control) 20 mM 18.93 health 100 μg/ml 12.35 let's go 100 μg/ml 14.55 health + let's go 100 μg/ml 31.76

3-2 Production of inflammatory cytokines inhibition

As a result of confirming the concentration of IL-6 by the mixed extract of health and Gajaja of the present invention, it was found that the expression of IL-6 was significantly reduced in the sample and control group than in the LPS-only group. In the case of 5-ASA, the production of IL-6 was inhibited by 50.98%.

In the case of health or Gaja single extract, 18.87 and 19.88% of inhibitory effects were shown, respectively, but in the case of extract of the present invention, IL-6 production was inhibited by 76.55% (Table 11, FIG. 20).

division density IL-6 inhibition (%) 5-ASA (positive control) 20 mM 50.98 health 100 μg/ml 18.87 let's go 100 μg/ml 19.88 health + let's go 100 μg/ml 76.55

3-3. Colitis improvement effect in DSS animal model

In the DSS inflammatory bowel disease animal model, the therapeutic and ameliorative effects of the health and Gaza mixture extract of the present invention on colitis were confirmed.

As a result of checking the length of the intestine, it was confirmed that the length of the intestine in the colitis-induced group was shortened by about 2 cm compared to the normal group, and when the mixed extract of the present invention was administered, it was confirmed that it increased by about 9.2% compared to the colitis-induced group (Table 12, Fig. 21 A).

In addition, as a result of observing the appearance state and showing the disease activity, diarrhea and bloody stool were found in the colitis-induced group, showing a high value of 7.25, but in the case of the mixed extract administration group of the present invention, it showed an improvement effect of 4.75 (Table 12, Figure 21 B).

And, as a result of confirming the weight of the intestine per intestine length, it was confirmed that the colitis-inducing group was about 32 mg/cm, which increased by about 9 mg/cm compared to the normal group. In the case of the mixed extract administration group of the present invention, compared with the colitis-inducing group. It was confirmed that it was effective in improving the thickened barrier, as it could be confirmed that it slightly decreased when it was performed (Table 12, FIG. 21 C).

division Large intestine length (cm) DAI weight per area normal control 7.42±0.1 0 22.25±0.81 colitis inducing group 5.44±0.12 7.25±0.25 31.51±1.2 Control drug administration group 5.31±0.16 6.88±0.44 30.66±0.76 health + let's go 5.93±0.4 4.75±0.25 29.12±0.55

3-4 TNBS Improvement effect of Crohn's disease in animal models

ICR female mice (17-19 g, 6 weeks old, Daehan Biolink (Eumseong, Korea)), 5-aminosalicylic acid (5-ASA), and 2,4,6-trinitro-benzene sulfonic acid (TNBS) were used.

In the TNBS Crohn's disease animal model, the therapeutic and ameliorative effects of the mixed extract of the present invention on Crohn's disease were confirmed.

As a result of confirming the length of the intestine, it was confirmed that the long length of the Crohn's disease-induced group was increased compared to the Crohn's disease-induced group in the case of the mixed extract administration group of the present invention (Table 13, FIG. 22A).

In addition, as a result of checking the intestinal weight per intestinal length, it was confirmed that the Crohn's disease group was about 74mg/cm, which was about 49mg/cm higher than that of the normal group. The control drug-administered group showed a difference of about 6.5 mg/cm when compared to the Crohn's disease-induced group, which indicates that there is some effect in reducing the thickened barrier.

On the other hand, in the case of the group administered with the mixed extract of the health and Gaja of the present invention, it was confirmed that the group reduced by about 23% compared to the Crohn's disease-inducing group, and through this, the mixed extract of the present invention is effective in improving the barrier thickened due to Crohn's disease confirmed (Table 13, Figure 22 B).

In addition, as a result of observing the appearance and showing the disease activity, diarrhea and bloody stool were found in the Crohn's disease-induced group, showing a high value of about 11. showed On the other hand, the mixed extract administration group of the present invention showed an effect of about 27% compared to the Crohn's disease-induced group, showing a superior improvement effect than the control drug administration group (Table 13, Fig. 22C).

division Large intestine length (cm) DAI weight per area normal control 9.64±0.54 0 25.75±1.51 colitis inducing group 7.92±0.56 11.20±0.89 74.12±7.71 Control drug administration group 8.33±0.22 10.56±1.40 67.67±9.30 health + let's go 8.75±0.25 8.33±0.97 57.49±4.46

Through these results, it was found that the mixed extract of the present invention can be usefully used for the prevention or treatment of inflammatory bowel disease because it shows excellent anti-inflammatory effects and Crohn's disease improvement effects.

Example 4 : elecampane , Confirmation of efficacy for Gaza and health mixed extracts

In addition, the present inventors mixed three kinds of wood, gaja, and health according to the ratios of Nos. 1 to 5 in Table 10 below, added 50% ethanol aqueous solution in an amount of 7 times the sample weight, and extracted at 80 ° C. for 3 hours under reflux. , The obtained extract was filtered, freeze-dried, and used as a sample in this experiment while being stored in a refrigerator in a powder state.

number Go: Let's Go: Health Amount of 50% aqueous ethanol solution One 100g : 100g : 100g 2100ml 2 200g : 200g : 100g 3500ml 3 200g : 100g : 100g 2800ml 4 100g : 200g : 100g 2800ml 5 100g : 100g : 200g 2800ml

4-1 Monocytes in intestinal epithelial cells adhesion inhibitory effect

The adhesion of fluorescently labeled U937 cells to HT-29 colonic epithelial cells was significantly increased by TNF-α stimulation, and was inhibited by about 25% by the control drug (5-ASA).

In the case of No. 1 to 5 Mokhyang, Gaja, and Health mixed samples, all of them showed an excellent inhibitory effect of about 60 to 70% compared to the control group, especially in the case of a 2: 2: 1 mixing ratio of Mokhyang: Gaza: Health The adhesion inhibitory activity was excellent. (Table 15, Figure 23).

division density Adhesion inhibitory activity (%) control - TNF-a (negative control) 100 ng/ml - 5ASA (positive control) 20 mM 24.48 Gohyang: Go: Health = 1:1:1 100ug/ml 59.66 Gohyang: Go: Health = 2:2:1 100ug/ml 73.27 Gohyang: Go: Health = 2:1:1 100ug/ml 64.97 Gohyang: Go: Health = 1:2:1 100ug/ml 66.82 Gohyang: Go: Health = 1:1:2 100ug/ml 58.13

4-2 Production of inflammatory cytokine IL-6 inhibition

As a result of confirming the concentration of IL-6 in the mixed samples of Mokhyang, Gaza and Health No. 1 to 5, it was found that the expression of IL-6 was significantly reduced. Or, in the case of a mixing ratio of 2: 1: 1, the production of IL-6 was most effectively inhibited (Table 16, FIG. 24).

division density IL-6 production inhibitory ability (%) 5ASA 20 mM 49.89 Gohyang: Go: Health = 1:1:1 50ug/ml 54.30 Gohyang: Go: Health = 2:2:1 50ug/ml 94.92 Gohyang: Go: Health = 2:1:1 50ug/ml 91.15 Gohyang: Go: Health = 1:2:1 50ug/ml 42.94 Gohyang: Go: Health = 1:1:2 50ug/ml 53.56

4-3. Colitis improvement effect in DSS animal model

In the DSS inflammatory bowel disease animal model, the colitis treatment and amelioration effects of the extracts of the present invention, Gaza, and Health mixture were confirmed.

As a result of confirming the intestinal length, it was confirmed that the intestinal length was increased compared to the colitis-induced group in the case of the mixed extract administration group of the present invention compared to the intestinal length in the colitis-induced group. In particular, in the case of the mixing ratio of 2: 2: 1 of Mokhyang: Gaja: Health, the longest intestine length was found (Table 17, Fig. 25 A).

In addition, as a result of observing the appearance state and showing the disease activity, diarrhea and bloody stool were found in the colitis-induced group, showing a high value of 6.75, but all of the mixed extract administration groups of the present invention showed an improvement effect with DAI 5.13 or less. In particular, in the case of a mixing ratio of 2: 2: 1 of Mokhyang: Gaja: Health, the improvement effect was most remarkable at about DAI 2.25 (Table 17, Fig. 25 B).

And, as a result of confirming the weight of the intestine per intestine length, it was confirmed that the colitis-inducing group was about 32 mg/cm, which increased by about 9 mg/cm compared to the normal group, and in the case of the mixed extract administration group of the present invention, compared with the colitis-inducing group. It was confirmed that it was effective in improving the thickened barrier, as it could be confirmed that it slightly decreased when it was applied. In particular, in the case of a mixing ratio of 2: 2: 1 of Mokhyang: Gaja: Health, the thickness reduction effect was significant (Table 17, FIG. 25 C).

division colon length DAI weight per area normal control 7.8 0 23.87 colitis inducing group 5.44 6.75 32.36 Control drug administration group 5.77 5.75 32.52 Gohyang: Go: Health = 1:1:1 5.91 5.00 32.07 Gohyang: Go: Health = 2:2:1 6.48 2.25 28.27 Gohyang: Go: Health = 2:1:1 5.88 4.00 28.91 Gohyang: Go: Health = 1:2:1 5.88 3.75 32.41 Gohyang: Go: Health = 1:1:2 5.55 5.13 30.35

4-4 TNBS Improvement effect of Crohn's disease in animal models

Similar to the colitis model, the Crohn's disease treatment and improvement effects of the extract of the present invention in the TNBS Crohn's disease animal model were confirmed.

As a result of confirming the intestinal length, it was confirmed that the intestinal length increased compared to the Crohn's disease-induced group in the case of the mixed extract administration group of the present invention compared to the intestinal length in the Crohn's disease-induced group. In particular, in the case of the mixing ratio of 2: 2: 1 of Mokhyang: Gaja: Health, the longest intestine length was found (Table 18, Fig. 26 A).

In addition, even when the disease activity was observed by observing the appearance, all of the groups administered with the mixed extract of the present invention showed an improvement effect, especially in the case of a 2:2:1 mixing ratio of Mokhyang:Gaja:Health, an improvement effect of about DAI 3 appeared most prominently (Table 18, Fig. 26 B).

In addition, as a result of confirming the weight of the intestine per intestine length, it was confirmed that the mixed extract administration group of the present invention was effective in improving the thickened barrier. appeared large (Table 18, Figure 26 C).

division colon length DAI weight per area normal control 9.38 0 26.48 Crohn's disease-causing group 8.28 6.60 50.70 Control drug administration group 8.27 6.30 48.66 Gohyang: Go: Health = 1:1:1 8.26 5.43 39.66 Gohyang: Go: Health = 2:2:1 9.13 3.00 33.32 Gohyang: Go: Health = 2:1:1 9.11 6.00 48.56 Gohyang: Go: Health = 1:2:1 8.40 6.29 41.51 Gohyang: Go: Health = 1:1:2 8.13 6.67 55.64

From these results, it can be seen that the mixed extract of Mokhyang, Garza and Health of the present invention can be usefully used for the prevention or treatment of inflammatory bowel disease, since it shows excellent anti-inflammatory effects, colitis improving effects, and Crohn's disease improving effects. could

Example 5: elecampane , let's go and health blend of extracts in the extraction solvent Confirmation of efficacy according to

In addition, the present inventors mixed three types of wood, gaja, and health in a ratio of 200 g: 200 g: 100 g, and then extracted the extraction solvents in Nos. 1 to 3 of Table 15 in an amount 7 times the sample weight. The mixture was extracted under reflux at 80 ° C for 3 hours, and the obtained extract was filtered, freeze-dried, and used as a sample for this experiment while being stored in a refrigerator in a powder state.

number extraction solvent Amount of extraction solvent One 30% ethanol 3500ml 2 50% ethanol 3500ml 3 70% ethanol 3500ml

5-1 Monocytes in intestinal epithelial cells adhesion inhibitory effect

The adhesion of fluorescently labeled U937 cells to HT-29 colonic epithelial cells was significantly increased by TNF-α stimulation, and was inhibited by about 25% by the control drug (5-ASA).

In the case of 30% ethanol, 50% ethanol, and 70% ethanol-extracted Mokhyang, Gaja, and Health mixture samples, all of them showed an excellent inhibitory effect of about 60 to 80% compared to the control group, especially in the case of 50% ethanol solvent extraction, the most adhesion inhibition activity was excellent. (Table 20, Figure 27).

division density Adhesion inhibitory activity (%) 5ASA (positive control) 20 mM 24.48 30% ethanol 100ug/ml 55.22 50% ethanol 100ug/ml 79.24 70% ethanol 100ug/ml 59.19

5-2 Production of the inflammatory cytokine IL-6 inhibition

As a result of checking the concentration of IL-6 in the case of Mokhyang, Gaza and Heunghyang mixed samples extracted with 30% ethanol, 50% ethanol and 70% ethanol, it was found that the expression of IL-6 was significantly reduced. In particular, 50% ethanol In the case of solvent extraction, the production of IL-6 was most effectively inhibited (Table 21, FIG. 28).

division density IL-6 production inhibitory ability (%) 5ASA 20 mM 49.35 30% ethanol 50ug/ml 89.96 50% ethanol 50ug/ml 95.93 70% ethanol 50ug/ml 88.65

5-3. Colitis improvement effect in DSS animal model

In the DSS inflammatory bowel disease animal model, the effect of treating and improving colitis according to the extraction solvent of the extracts of the present invention, the mixed extract of Mokhyang, Gaza and Heunghyang was confirmed.

As a result of confirming the intestinal length, it was confirmed that the intestinal length was increased compared to the colitis-induced group in the case of the mixed extract administration group of the present invention compared to the intestinal length in the colitis-induced group. In particular, in the case of 50% ethanol solvent extraction, the longest length was found (Table 22, Fig. 29 A).

In addition, as a result of observing the external appearance and showing the disease activity, diarrhea and bloody stool were found in the colitis-induced group, showing a high value of 6.67, but in the case of the ethanol aqueous solution extract administration group of the present invention, all showed an improvement effect with DAI 4.5 or less. . In particular, in the case of 50% ethanol solvent extract, the improvement effect was most remarkable at about DAI 3.3 (Table 22, FIG. 29 B).

And as a result of checking the weight of the intestine per intestine length, it was confirmed that the colitis-inducing group was about 32 mg/cm, which increased by about 9 mg/cm compared to the normal group. As it can be confirmed that it is slightly reduced (about 27 mg / kg) when it is used, it was confirmed that it is effective in improving the thickened barrier (Table 22, FIG. 29 C).

division colon length DAI weight per area normal control 7.45 0 23.80 colitis inducing group 5.31 6.67 32.10 Control drug administration group 5.45 5.33 31.42 30% ethanol 5.51 4.50 32.35 50% ethanol 6.30 3.33 27.15 70% ethanol 5.60 4.33 32.69

5-4 TNBS Improvement effect of Crohn's disease in animal models

Similar to the colitis model, in the TNBS Crohn's disease animal model, the treatment and improvement of Crohn's disease according to the extraction solvent of the extract of the present invention was confirmed.

As a result of confirming the intestinal length, it was confirmed that the intestinal length increased compared to the Crohn's disease-induced group in the case of the mixed extract administration group of the present invention compared to the intestinal length in the Crohn's disease-induced group. In particular, in the case of 50% ethanol solvent extraction, the longest length was found (Table 23, Fig. 30 A).

In addition, even when examining the disease activity by observing the appearance state, all of the groups administered with the mixed extract of the present invention showed an improvement effect, especially in the case of 50% ethanol solvent extraction, the improvement effect was most remarkable at about DAI 3 (Table 23, Figure 30 B).

And as a result of checking the weight of the intestine per intestine length, it was confirmed that the Crohn's disease-induced group increased by about 34 mg/kg compared to the normal group at about 60 mg/kg. When compared, it was confirmed that it significantly decreased, and in particular, in the case of 50% ethanol solvent extraction, it significantly decreased to about 34 mg / kg, which confirmed that it was effective in improving the thickened barrier (Table 23, FIG. 30 C).

division colon length DAI weight per area normal control 9.50 0 26.48 Crohn's disease-causing group 7.63 12.00 60.58 Control drug administration group 8.28 8.20 46.55 30% ethanol 9.22 7.63 45.41 50% ethanol 9.44 3.88 34.68 70% ethanol 8.93 8.13 46.49

Through these results, the mixed extracts of two or more of Mokhyang, Gaja and Health of the present invention showed excellent anti-inflammatory, colitis and Crohn's disease improvement effects in most of the extraction solvents, especially in 50% ethanol extraction, the best anti-inflammatory effect and Since the effect of improving colitis and Crohn's disease was confirmed, it was found that it would be useful for preventing or treating inflammatory bowel disease.

Example 6: elecampane , Confirmation of the efficacy according to the administered dose of Gaza and health mixture extracts

In addition, the present inventors mixed 3 types of wood, gaja, and health in a ratio of 200g: 200g: 100g, and then added 50% ethanol in an amount 7 times the sample weight and refluxed at 80 ° C for 3 hours, respectively. Extraction was performed, and the obtained extract was filtered, freeze-dried, and used as a sample in this experiment while being stored in a refrigerator in a powder state.

6-1. Colitis improvement effect in DSS animal model

In the DSS inflammatory bowel disease animal model, the effect of the treatment and improvement of colitis according to the administered dose of the extracts of the present invention, Gaja, and Health mixture was confirmed.

The colitis treatment and improvement effect shown by administering the mixed extract at 50, 100, 200, and 400 mg/kg, respectively, was confirmed.

As a result of confirming the intestinal length, it was confirmed that the intestinal length was increased compared to the colitis-induced group in the case of the mixed extract administration group of the present invention compared to the intestinal length in the colitis-induced group. In particular, when administered at a dose of 400 mg/kg, the longest intestinal length was observed (Table 24, Fig. 31 A).

In addition, as a result of observing the external appearance and showing the disease activity, diarrhea and bloody stool were found in the colitis-induced group, showing a high value of 7.25, but all of the mixed extract administration groups of the present invention showed an improvement effect of DAI 5.5 or less. In particular, when administered at a dose of 400 mg/kg, the improvement effect was most remarkable, with a DAI of 2.88 (Table 24, FIG. 31 B).

And as a result of checking the weight of the intestine per intestine length, it was confirmed that the colitis-inducing group was about 31 mg/cm, which increased by about 9 mg/cm compared to the normal group. It was confirmed that it slightly decreased when administered, and in particular, when administered at a dose of 400 mg / kg, it was significantly reduced to 26 mg / cm, which confirmed that it was effective in improving the thickened barrier (Table 24, FIG. 31 C).

division colon length DAI weight per area normal control 7.42±0.1 0 22.25±0.81 colitis inducing group 5.44±0.12 7.25±0.25 31.51±1.2 Control drug administration group 5.31±0.16 6.88±0.44 30.66±0.76 50 mg/kg 5.63±0.2 5.5±0.57 28.56±0.88 100 mg/kg 5.89±0.15 5±0.27 29.42±1.25 200 mg/kg 6.2±0.08 3.38±0.6 27.82±0.43 400 mg/kg 6.46±0.28 2.88±0.48 26.52±0.94

6-2 TNBS Improvement effect of Crohn's disease in animal models

Similar to the colitis model, in the TNBS Crohn's disease animal model, the effect of treatment and improvement of Crohn's disease according to the administered dose of the extract of the present invention, Gaja, and Health mixed extract was confirmed.

The colitis treatment and improvement effect shown by administering the mixed extract at 50, 100, 200, and 400 mg/kg, respectively, was confirmed.

As a result of checking the intestinal length, it was confirmed that the intestinal length was increased compared to the Crohn's disease-induced group in the case of the mixed extract administration group of the present invention compared to the intestinal length in the Crohn's disease-induced group. In particular, when administered at a dose of 400 mg/kg, the longest intestinal length was observed (Table 25, Fig. 32 A).

In addition, even when examining the disease activity by observing the appearance state, the group administered with the mixed extract of the present invention showed an improvement effect compared to the Crohn's disease-induced group (DAI 11), especially when administered at a dose of 400 mg/kg DAI The improvement effect was most remarkable at about 6.5 (Table 25, Fig. 32 B).

In addition, as a result of confirming the weight of the intestine per intestine length, it was confirmed that the Crohn's disease-induced group increased by about 49 mg/cm compared to the normal group at about 74 mg/cm, and in the case of the mixed extract administration group of the present invention, the Crohn's disease-induced group It was confirmed that it decreased when compared to , and in particular, when administered at a dose of 400 mg / kg, it significantly decreased to 50 mg / cm, and it was confirmed that it was effective in improving the thickened barrier through this (Table 25, FIG. 32 C ).

division colon length DAI weight per area normal control 9.64±0.54 0 25.75±1.51 Crohn's disease-causing group 7.92±0.56 11.20±0.89 74.12±7.71 Control drug administration group 8.33±0.22 10.56±1.40 67.67±9.30 50 mg/kg 8.76±0.42 10.1±1.69 71.05±10.20 100 mg/kg 8.97±0.37 8.38±1.48 58.08±8.04 200 mg/kg 9.1±0.26 7±1.73 52.06±9.66 400 mg/kg 9.34±0.15 6.5±0.9 50.24±4.95

Through these results, the mixture of two or more extracts of Mokhyang, Gaza and Health of the present invention showed excellent anti-inflammatory, colitis and Crohn's disease improvement effects at most doses, especially when 400 mg/kg was administered. Since the inflammatory effect, the effect of improving colitis, and the effect of improving Crohn's disease were confirmed, it was found that it would be useful for preventing or treating inflammatory bowel disease.

Claims (22)

It contains a mixed extract containing at least two of Mokhyang, Gaja, and Health as an active ingredient, and the mixed extract is a 50% ethanol extract, and the blending ratio of Mokhyang, Gaja, and Health is 1 to 2: 1 to 2: 1, respectively. A pharmaceutical composition for preventing or treating inflammatory bowel disease, characterized in that it comprises a weight ratio of ~2
delete delete delete delete delete delete delete The pharmaceutical composition for the prevention or treatment of inflammatory bowel disease according to claim 1, wherein the pharmaceutical composition comprises 2:2:1 weight ratio of Mokhyang:Gaja:Healthy.
delete delete delete delete The pharmaceutical composition for preventing or treating inflammatory bowel disease according to claim 1, wherein the content of the mixed extract contained in the composition is in an amount of 100 to 400 mg/kg.
The pharmaceutical composition for preventing or treating inflammatory bowel disease according to claim 1, wherein the composition is for oral use.
The pharmaceutical composition for preventing or treating inflammatory bowel disease according to claim 1, wherein the mixed extract inhibits at least one of IL-6 and TNF-α production, which are inflammatory mediators.
The inflammatory bowel disease according to claim 1, wherein the inflammatory bowel disease is at least one selected from the group consisting of Crohn's disease, ulcerative colitis, enteric Behcet's disease, intestinal tuberculosis enteritis, and diarrhea. A pharmaceutical composition for preventing or treating a disease.
It contains a mixed extract containing at least two of Mokhyang, Gaja, and Health as an active ingredient, and the mixed extract is a 50% ethanol extract, and the blending ratio of Mokhyang, Gaja, and Health is 1 to 2: 1 to 2: 1, respectively. A health functional food for preventing or improving inflammatory bowel disease, characterized in that it comprises a weight ratio of ~2.
[Claim 19] The health functional food for preventing or improving inflammatory bowel disease according to claim 18, wherein the weight ratio of 2: 2: 1 is 2: 2: 1.
delete The functional food for preventing or improving inflammatory bowel disease according to claim 18, wherein the functional food is powder, granule, tablet, capsule, syrup or beverage.
The inflammatory bowel disease according to claim 18, wherein the inflammatory bowel disease is at least one selected from the group consisting of Crohn's disease, ulcerative colitis, enteric Behçet's disease, intestinal tuberculosis enteritis, and diarrhea. Health functional food for preventing or improving diseases.

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