KR20230114999A - Composition for preventing, alleviating or treating colitis containing extract of Amomum villosum Loureiro as an active ingredient - Google Patents

Abstract

The present invention relates to a composition for preventing, alleviating or treating colitis containing an Amomum villosum extract as an active ingredient. The present invention is characterized by the efficacy of the Amomum villosum extract in inhibiting the expression of IL-8, an inflammatory cytokine, and the expression of COX-2 protein in HT-29 colon cell line.

Description

Composition for preventing, alleviating or treating colitis containing extract of Amomum villosum Loureiro as an active ingredient}

The present invention relates to a composition for preventing, improving or treating colitis containing an extract of Yangchunsa as an active ingredient. The present invention is characterized in that it is based on the efficacy of inhibiting the expression of IL-8, an inflammatory cytokine, and the expression of COX-2 protein in the HT-29 colon cell line of Yangchunsa extract.

Colitis is a broad term for an inflammatory disease of unknown cause occurring in the digestive tract, and there are various types of it, and representative colitis includes ulcerative colitis.

Ulcerative colitis mainly occurs in the sigmoid colon and rectum on the left side of the colon. It often also occurs in the ileum, the end of the small intestine that connects to the large intestine. It is characterized by inflammation and ulceration of the mucous membrane inside the large intestine, which consists of the colon and rectum. The main symptoms are bloody diarrhea, abdominal pain, and stool urgency. They have bowel movements several times a day, complain of dehydration, anemia, fever, and weight loss.

Although the etiology of ulcerative colitis is not precisely known, it is presumed to be caused by psychological factors, environmental factors such as smoking/diet, or physical and genetic factors of each patient. It has been recognized as a disease with high incidence in Western countries, such as Europe and the United States, where the incidence is predominantly meat-eating, and has been recognized as a disease with low incidence in Korea.

On the other hand, since all the patho-physiological findings of colitis have not been revealed, effective and appropriate treatments are very scarce. Currently used treatment regimens for ulcerative colitis include pharmacological treatment and surgical treatment. Drugs used for pharmacological treatment include aminosalicylate agents such as sulfasalazine and mesalazine, steroid agents, and infliximab. In this case, immunosuppressants such as azathioprine, 6-mercaptopurine, and cyclosporin, corticosteroids such as prednisone, and the like are used.

However, long-term administration of these drugs may cause serious side effects such as nausea, vomiting, indigestion, anorexia, and headache, as well as skin rashes due to hypersensitivity reactions, fever, pancreatitis, hepatitis, hemolytic anemia, and bone marrow suppression. For example, mesalazine, which is most widely used for ulcerative colitis, is highly absorbed through the placenta into the fetus even at low concentrations, so its use in pregnant women is limited, and systemic hypersensitivity and interstitial nephritis have been reported as side effects. Corticosteroids are useful for short-term palliative therapy, but are used only for short-term therapy due to severe systemic side effects when used long-term. In addition, the above drugs are not drugs targeted at the pathogenesis of colitis, but are used as temporary measures, and thus cannot cure colitis. A surgical treatment technique includes colon resection, but side effects such as appendicitis, night fecal incontinence, and decreased fertility in women have been reported.

Therefore, there is an urgent need to discover new substances for the treatment of colitis with high efficacy and safety.

Looking at prior art literature, regarding Yangchunsa, Korean Registered Patent Publication No. 10-1952644 describes a composition for preventing, improving or treating obesity containing Yangchunsa extract, and Korean Registered Patent Publication No. 10-1952648 discloses Yangchunsa extract. A composition for preventing and improving the treatment of fatty liver containing is described.

In addition, regarding colitis, Korean Patent Registration No. 10-2084973 discloses a composition for preventing or treating colitis containing Enterococcus faecalis as an active ingredient. Experiments on efficacy are described. In addition, Korean Registered Patent Publication No. 10-2150548 discloses a composition for preventing or treating colitis containing a mixed herbal medicine extract and sulfasalazine as active ingredients. Efficacy for inhibition has been described.

Republic of Korea Registered Patent Publication No. 10-1952644 (registered on February 21, 2019) Republic of Korea Registered Patent Publication No. 10-1952648 (registered on February 21, 2019) Republic of Korea Registered Patent Publication No. 10-2084973 (registered on February 28, 2020) Republic of Korea Registered Patent Publication No. 10-2150548 (registered on August 26, 2020)

The composition according to the present invention has the efficacy of preventing, improving or treating colitis. In particular, Yangchunsa extract, an active ingredient of the composition according to the present invention, has an inhibitory effect on the expression of IL-8, an inflammatory cytokine, and an expression of COX-2 protein in the HT-29 colon cell line.

1 is a graph showing the results of LPS survival rate measurement when Yangchunsa extract was administered to HT-29 colon cell line.
Figure 2 is a graph showing the results of measuring the amount of IL-8, an inflammatory cytokine, when the Yangchunsa extract was administered to the HT-29 colon cell line.
Figure 3 is a photograph showing the result of measuring the expression level of COX-2 protein when the Yangchunsa extract was administered to the HT-29 colon cell line.

Terms or words used in this specification and claims should not be construed as being limited to their usual or dictionary meanings, and the inventors may appropriately define the concept of terms in order to best explain their invention. It should be interpreted as a meaning and concept consistent with the technical idea of the present invention based on the principle that there is.

Therefore, the examples, reference examples, experimental examples, formulation examples and matters shown in the drawings described in this specification are only the most preferred examples of the present invention and do not represent all of the technical ideas of the present invention. It should be understood that there may be various equivalents and variations that can be substituted for them.

Sain (砂仁) refers to a cluster of ripe fruits or seeds of Amomum villosum Loureiro var. xanthioides TLWu et Senjen or Amomum villosum Loureiro of the Zingiberaceae family. So, in the present invention, the fruit of Yangchunsa was used as a material.

For reference, Yangchunsa is mainly produced in Fujian, Guangdong, Guangxi, and Yunnan, and deer hornsa is mainly produced in Indochina Peninsula regions such as Cambodia, India, Laos, Myanmar, Thailand, and Vietnam, and some regions in Guangxi and Yunnan in China. .(Flora of China, v24:347-356.2000). In ancient times, Nokgaksa Temple was referred to separately under the name of ‘chuksain’ because the production area was different.

On the other hand, the fruit of Haenamsa (海南砂Amomum longiligulare TLWu), which is recognized as genuine in China but treated as fake in Korea, is also being distributed in large quantities. However, Shen et al. (SHEN Li WANG Yang JIANG Ku et al. Comparison of HPLC Fingerprints of Amomum villosum Lour. ，Amomurn villosum Lour. var. xanthioides TLWu et Senjen and Amomum longiligulare TLWu. Chinese Pharmaceutical Journal. 2016: 51(12); 1039 -1043.) showed a high degree of similarity in the composition of the three types of deaths as a result of the RP-HPLC study. However, while Haenamsa and Yangchunsa had similar chemical components, Noggaksa had a clear difference from the previous two, so the pharmacological and clinical efficacy of these It was said that comparisons should be made. In the end, it would be said that the cause of death of Nokgaksa and that of Yangchunsa are obviously different.

Example 1. Preparation of Yangchunsa Extract

Yangchunsa ( Amomum villosum Lour.) was purchased from Kwangmyeongdang Pharmaceutical Co., Ltd. (Ulsan, Korea) and verified for authenticity in the herbal medicine department of Wonkwang University College of Oriental Medicine. Then, the filtrate was concentrated and dried under reduced pressure to obtain an extract.

Experimental Example 1. Cell culture and cytotoxicity test

HT-29 human colon epithelial cells used in the experiment were purchased from the Korea Cell Line Bank (Seoul, Republic of Korea). Cells were cultured in DMEM (Hyclone, USA) containing 10% FBS, 100 IU/ml penicillin, and 100 μg/ml streptomycin. In the experiment, HT-29 cells were pretreated with 100, 200, and 500 μg/ml Yangchunsa extract (AVE) for 1 hour, followed by 1 μg/ml LPS (E. coli strain B8:0127; Sigma Chemical) for 24 hours. Exposed. CCK-8 assay (Dojindo, Japan) for cytotoxicity was performed according to the manufacturer's instructions.

Figure 1 is a graph showing the results of measuring LPS viability when Yangchunsa extract was administered to HT-29 colon cell line. (Effects of AVE on the cell viability of HT-29 colon epithelial cells. The cells were preincubated in the presence of the Indicated concentrations of AVE for 1 h and then treated with LPS (1 μg/mL) for 24 h. Cell viability was assessed using the CCK-8 kit. The results are presented as the mean ± SD of three individual experiments.)

Looking at it, it can be seen that the survival rate was 100% or close to 100% for 100, 200, and 500 μg / ml Yangchunsa extract.

Experimental Example 2. IL-8 measurement

Monoclonal anti-IL-8 (R&D Systems) suspended in coating buffer (14.2 mM Na2CO3, 34.9 mM NaHCO3, 3.1 mM NaN3, pH 9.6) was added to a microtiter plate (Costa, Corning, NY, USA) overnight at 4 Incubated at °C. Plates were washed three times with phosphate buffered saline containing 0.05% Tween-20 (PBST) and blocked with PBST containing 1% bovine serum albumin for 1 hour at 37 °C. 100 microliters of sample or diluted standard solution was added to the plate, and the plate was incubated for 2 hours at room temperature (RT). After washing with PBST, 100 μl of biotinylated goat anti-mouse IgG (R&D Systems) was added, and the plate was incubated at 37 °C for 1 hour. The color was developed by adding streptavidin-horseradish hyperosidase (HRP) conjugate (Amersham, Buckinghamshire, UK), adding 100 μl of tetramethylbenzidine and incubating for 30 min at RT. Reactions were stopped with 4M H2SO4 and OD values were measured at 450 nm using an ELISA reader.

Figure 2 is a graph showing the results of measuring the amount of IL-8, an inflammatory cytokine, when Yangchunsa extract was administered to HT-29 colon cell line. (Effects of AVE on IL-8 production in LPS-stimulated HT-29 colon epithelial cells.The cells were preincubated for 1h with AVE, then incubated with LPS for 12 h.IL-8 levels were then determined by ELISA (mean±SD, n = 3).** p < 0.01, compared with untreated control ; ^# p < 0.05 and ^## p < 0.01, compared with LPS-treated control.)

Looking at it, compared to about 78 pg/mL when Yangchunsa extract was not administered, when 100, 200, and 500 μg/ml Yangchunsa extract were administered, IL-8 increased in a concentration-dependent manner at about 61, 50, and 40 pg/mL, respectively. expression was suppressed. In particular, when administered at 500 ug/ml, an IL-8 expression inhibitory effect of about 50% was shown.

Experimental Example 3. iNOS protein expression level measurement

iNOS protein expression was determined by Western blotting. HT-29 cell reset was performed in ice-cold lysis buffer (50 mM Tris, pH 7.4, 1 mM EDTA, 0.1% Triton X-100, 1 mM PMSF, 25 μg/ml leupeptin and 20 μg/ml pepstatin). Equivalent protein samples were resolved on 10% SDS-polyacryglamide gels and blocked with TBST [10 mM Tris (pH 7.4), 100 mM NaCl, 0.5% Tween 20] containing 5% non-fat milk for 30 min at RT. was transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA). For immunodetection, membranes were incubated overnight with anti-COX-2 antibody (1:1000, Cell Signaling, USA) in TBST containing 1% milk powder. Then incubated with HRP conjugated anti-mouse IgG (1:1000, Santa Cruz, CA, USA). Bands were visualized by enhanced chemiluminescence (ECL kit, Thermo, USA).

Figure 3 is a photograph showing the result of measuring the expression level of COX-2 protein when Yangchunsa extract was administered to HT-29 colon cell line. (Effects of AVE on the expression of LPS-induced iNOS protein in HT-29 colon Epithelial cells. Cells were pretreated with AVE at the indicated concentrations for 1 h and then treated with LPS (1 μg/mL) for 24 h. Cell lysates were prepared and the COX-2 and actin protein levels were determined by western blotting.)

Looking at it, it can be seen that the COX-2 band appears distinctly when there is only LPS without administration of Yangchunsa extract, but the brightness of the band decreases rapidly when 100, 200, and 500 μg/ml Yangchunsa extract is administered. It can be confirmed that iNOS protein expression was suppressed by the administration of the extract.

Example 2. Composition for preventing, improving or treating colitis containing Yangchunsa extract as an active ingredient

Yangchunsa extract contained in the composition according to the present invention has the effect of inhibiting the expression of IL-8, an inflammatory cytokine of the HT-29 colon cell line, and the expression of COX-2 protein. A composition containing the Yangchunsa extract according to Example 1 as an active ingredient was prepared and used. In Example 1, Yangchunsa was extracted with distilled water, but an extract extracted with alcohol or an organic solvent may also be used.

Based on the results of Experimental Examples 1 to 3, the Yangchunsa extract in the composition according to the present invention is included in an amount of 100 to 500 μg/ml based on HT-29 cells.

The present invention can provide a colitis prevention agent formulated in a pharmaceutical unit dosage form by adding the Yangchunsa extract as an active ingredient and a pharmaceutically acceptable carrier, excipient or diluent. Here, the carrier, excipient, and diluent include toze, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, undecided quality cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

In addition, the pharmaceutical dosage form may be used in the form of a pharmaceutically acceptable salt, and may be used alone or in combination with other pharmaceutically active compounds as well as in a suitable set. In addition, when formulating the active ingredient, it may be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. In addition, the pharmaceutical dosage form is formulated according to conventional methods into oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterile injection solutions. can

The solid preparation for oral administration may be prepared by mixing the extract with at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used.

Preparations for parenteral administration may include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used as the non-aqueous solvent or suspending agent.

As a base for the suppository, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogeratin and the like may be used.

A preferred dose of the extract of the present invention varies depending on the condition and weight of the patient, the severity of the disease, age, sex, drug type, administration route and period, but can be appropriately selected by those skilled in the art. However, for a desirable effect, the extract of the present invention is preferably administered at 0.001 to 300 mg/kg, and administration may be administered once a day or divided several times. The dosage is not intended to limit the scope of the present invention in any way.

The extract of the present invention can be administered to mammals such as rats, mice, livestock, and humans through various routes. All modes of administration are contemplated, eg oral, rectal or intravenous, intramuscular or subcutaneous administration.

A health functional food composition for preventing or improving colitis can be provided by adding a food additive to the active ingredient of the present invention.

Foods to which the active ingredient can be added include, for example, various foods, beverages, chewing gum, tea, vitamin complexes, health functional foods, and the like.

The amount of the active ingredient in the food or beverage may be added in an amount of 0.01 to 20% by weight of the total weight of the food or beverage, and the health drink composition may be added in an amount of 0.02 to 5 g, preferably 0.3 to 1 g, based on 100 ml. there is.

The health functional beverage composition of the present invention is not particularly limited in other ingredients other than containing the extract, and may contain various flavoring agents or natural carbohydrates as additional ingredients like conventional beverages. Examples of the aforementioned natural carbohydrates include monosaccharides such as glucose, fructose; disaccharides such as maltose, sucrose and the like and polysaccharides such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. In addition to the above, natural flavors (thaumatin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavoring agents. 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 is various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and enhancers (cheese, chocolate, etc.), pectic acid and its salts, organic acids, protection It may contain a colloidal thickener, a pH adjusting agent, a stabilizer, a preservative, glycerin, alcohol, a carbonating agent used in carbonated beverages, and the like.

In addition, the composition 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 extract of the present invention.

So far, the present invention has been mainly looked at with respect to preferred embodiments.

The embodiments described in this specification and the configurations shown in the drawings relate to one of the most preferred embodiments of the present invention, and do not represent all of the technical spirit of the present invention, so various equivalents and modifications that can replace them It should be understood that there may be examples.

Therefore, the present invention is not limited to the presented embodiments, and within the equivalent scope of the technical idea of the present invention and the technical idea described in the claims to be described below by those skilled in the art to which the present invention belongs There may be embodiments in which various modifications and changes are possible.

Claims (4)

Yangchunsa (Amomum villosum Lour.) A food composition for preventing or improving colitis containing an extract as an active ingredient. The method of claim 1,
The Yangchunsa extract is a food composition for preventing or improving colitis, characterized in that extracted with distilled water, alcohol or an organic solvent. According to claim 1 or 2,
The Yangchunsa extract is a food composition for the prevention or improvement of colitis, characterized in that it possesses the inflammatory cytokine IL-8 expression inhibitory effect and COX-2 protein expression inhibitory effect in the HT-29 colon cell line. Yangchunsa (Amomum villosum Lour.) A pharmaceutical composition for preventing or treating colitis containing an extract as an active ingredient.