KR100496146B1 - Composition comprising the extract of Glycyrrhiza uralensis FISCH., Curcuma longa L. and Perilla frutescens BRITT. for the prevention and treatment of gastritis and gastric ulcer - Google Patents

Abstract

The invention licorice (Glycyrrhiza uralensis FISCH.), Turmeric (Curcuma longa L.) And lobular by using the extract and the combined crude drugs including those of (Perilla frutescens BRITT.) Inhibiting the histamine receptor activity and to reduce gastric acid secretion of histamine To provide a safe and effective medicine and dietary supplement for the prevention and treatment of gastritis and gastric ulcer disease associated with the activity of the receptor.

Description

Composition comprising the extract of Glycyrrhiza uralensis FISCH., Curcuma longa L. and Perilla frutescens BRITT. for the prevention and treatment of gastritis and gastric ulcer}

The present invention is a licorice, turmeric, leaflet extract that has been widely used as a herbal medicine since ancient times An antagonistic effect on histamine receptors is used to treat gastritis and gastric ulcers or as a dietary supplement.

Gastritis and gastric ulcers are caused by infections of Helicobacter pylori , which are parasitic in the stomach, and the effects of gastric acid and pepsin, which are factors that cause the gastrointestinal tract to break down, and mucosa, a protective factor that protects the stomach wall from This is caused by a break in equilibrium between functions. In addition, many drugs, including non steroidal anti-inflammatory drugs, are associated with side effects such as gastrointestinal bleeding, which is considered to be a major cause of gastritis and gastric ulcers. Therefore, in order to treat gastritis and gastric ulcers, antibiotics that suppress Helicobacter pylori, or drugs that inhibit gastric acid secretion, should be administered, depending on the cause. Most treatments for gastritis and gastric ulcers developed to date have a wide variety of mechanisms by removing or lowering acid secreted from the stomach (Wollin A., Clin. Invest. Med., 10 , pp209-214, 1987).

In order to eliminate the bacterium Helicobacter pylori infected with the double digestive organs, various drugs mainly on antibacterial agents have been tried. For example, bismuth preparations such as colloidal bismuth citrate and bismuth salicylic acid, antibacterial agents such as amoxicillin, ampicillin, clarithromycin, oploxacin and tetracycline, hydrogenation agents such as tinidazole and metronidazole Attempts have been made to administer proton pump inhibitors alone or in combination with two or three agents.

On the other hand, the drug that has long been used for the treatment of gastrointestinal disorders is antacid. The effect on symptoms is the fastest because it directly neutralizes the stomach acid secreted in the stomach, protecting the stomach and eliminating heartburn. The most representative drug is a suspension made of a mixture of aluminum hydroxide and magnesium hydroxide, and drinks such as sodium bicarbonate are on the market. However, antacids do not prevent gastric acid from coming out, and they act to neutralize gastric acid already secreted for a short time. It can also cause side effects such as urolithiasis. Therefore, the drugs that prevent the secretion of gastric acid more fundamentally was searched, and then acetylcholine receptor inhibitors and prostaglandin receptor active drugs were developed. However, they are not widely used because of the side effects that occur in the nerve cells other than the gastrointestinal tract because they can be present and work throughout the blood and peripheral nervous system.

On the other hand, quinoline derivatives are known to exhibit the effect of inhibiting gastric acid secretion in mammals, and many attempts have now been made to develop them as reversible gastric acid secretion inhibitors (European Patent Application No. 87-307824.0; US Patent No. 5,362,743; PCT Application KR94-29274; European Patent Application No. 89-301801.0; European Patent Application No. 89-301805.1; European Patent Application No. 89-301802.8; European Patent Application No. 88-306583.1; PCT Application KR-97-00074; Korea Patent Application No. 96-38314; Korean Patent Application No. 97-30692; Korean Patent Application No. 97-30693).

Currently, the most popular drugs are gastric hydrogen ion carrier inhibitors and histamine receptor antagonists. Hydrogen ion carriers exist only in the stomach wall and are involved in the synthesis of gastric acid by discharging hydrogen ions into the gastrointestinal tract (Sachs G, Spenney JG, et al; Physiol. Rev., 58 , pp106-173, 1978), omeprazole In case of using a hydrogen ion carrier inhibitor such as), it is possible to lower the amount of gastric acid synthesized, and benzimidazole derivatives including pyridine represented by omeprazole as gastric acid secretion inhibitors have been widely used. Benzimidazole derivatives have an excellent effect of inhibiting gastric acid secretion, but they have an irreversible mechanism of action, and when administered for a long time, the drug accumulates in the body and the drug persists even when the medication is stopped, or the stomach wall becomes thick due to the dose. Problems such as losing side effects have been reported.

Histamine receptors, on the other hand, have a more pronounced effect on the gastrointestinal tract than other organs and promote gastric acid secretion. Thus histamine receptor inhibitors can be applied to inhibit gastric acid secretion.

Although histamine receptor inhibitors, which are currently developed and used, have relatively low toxicity, they have side effects when administered in the long term in clinical practice (about 1-3% of cimetidine), and the central nervous system such as headache, dizziness and vomiting. Related diseases and diarrhea and skin redness have been reported. However, the most lethal ones are impotence, loss of libido, and gynecomastia in men. These drugs are the hydroxylation of estradiol. ) To increase estradiol in plasma.

In particular, the histamine receptor of interest in the present invention plays an important role in gastric acid secretion. Histamine receptors, when activated in combination with histamine, result in the activation of adenylyl cyclase to increase the concentration of cyclic AMPs that induce intracellular signaling and eventually protein kinase A A) (Gespach C, et al; Mol. Pharmacol., 22 , pp547-553, 1982 .: Sawutz DG, Kalinyak K, et al; J. Pharmacol. Exp. Ther., 231, pp 1-7, 1984).

Although the treatment of peptic ulcers has been developed into various classes of drugs and various treatment strategies using different drugs can be suggested, they do not fundamentally show a direct healing effect to heal wounds of peptic ulcers. These drugs only play a role in suppressing the attack from gastric acid to keep the wounds from worsening, and the disappearance of the ulcer is inevitably dependent on the body's natural healing power. This limitation makes the treatment of peptic ulcers require very long drug administration. Therefore, not only the short-term acute toxicity of the drug but also chronic toxicity and its side effects can be considered as a good treatment.

Therefore, the present inventors analyzed the components of various traditional herbal medicines that have already been proved to overcome the limitations of conventional gastritis and gastric ulcer treatments, and searched for the effects of treating gastritis and gastric ulcers, and the fraction mixture of licorice, turmeric and leaflet extracts and these Gastric inflammation and gastric ulcer therapeutic components were obtained from a combined herbal medicine.

The turmeric (Curcuma longa L.) Used in the present invention is a ginger and a yearly plant is tropical Asian origin of (Zingiberaceae), grown in the country, except for mountainous areas of southern China and elsewhere and domestic northern and rhizomes (根莖) The medicinal herbs Known ingredients include turmerone, zingerene, phellandrene, 1,8-cineole, bornol and dehydroturmerone. , Arabinose, fructose, glucose, starch, organic acid, etc.The root is yellow crystalline diketone compound curcumin and its derivative p-hydroxycinnamoylferuloylmethane And about 0.3% of yellow pigment of p, p'-dihydroxydicinnamoylmethane, other essential oils 1-5%, nonvolatile oil 2.4%, starch 50%, crude fiber 5%, ash 4%, water 16% There. It promotes blood circulation and is used for the treatment of bruises and fish blood as well as shoulder joint pain. It shows blood cholesterol lowering effect and inhibitory effect on liver virus (Information and Shin, Min-Kyo; Herbal Dictionary), Daesaeng, Yeonglimsa, pp874 ~ 875, 1998).

Licorice ( Glycyrrhiza uralensis FISCH.) Is a perennial herb that is a perennial herb, and licorice is not only used in oriental medicine to increase the efficacy, but also to alleviate various pain and urgency as an alleviation and analgesic medicine. It is used for external use and has its own weakness such as neutralization of poison. For example, licorice has the effect of protecting the liver and smoothing the liver and regulating fatigue, and taking licorice reduces the burden on the liver by its detoxification. Triterpene-based saponins and glycyrrhizin are contained in the roots and roots of plants and have all detoxification effects on bacterial toxins as well as drug addiction, food poisoning, and metabolism in the body. There is a function and regulation of thyroid sebum hormone. Licorice has anti-inflammatory and anti-allergic reactions, antagonistic action against acetylcholine, and has the effect of enhancing the adrenal-induced cardiovascular effect (Jung, Ji-Sup and Shin, Min-Kyo; Ilbo Herbal Medicine, Yeonglimsa, pp684-687 , 1998).

In addition, perilla frutescens BRITT (perilla frutes), an annual herb of Labiatae, is called leaflet and seed in medicinal plants, and essential oil of the entire leaflet. ), About 55% of perylaldehyde, 20-30% of l-limonene, and a small amount of α-pinene, and arginine, cumic acid, and cyanidin-3- (6-p). -Coumaryl-β-D-glucoside) -5-β-D-glucoside, and the refined fat and oil component of the leaf contains isoeegoma ketone and the like. Used as a sweat, antitussive, dry, diuretic, soothing and analgesic. Small stems are old stems of the lobules and the roots of the lobules, which are effective in combating gout, sweating and expectoration, lowering the qi, dizziness, body pain and nasal congestion, It is effective in treating runny nose (Information and Shin, Min-Kyo; Doha Herbal Medicine Dictionary, Younglimsa, pp855-856, 1998).

Turmeric and lobules have been shown to benefit the stomach through folk remedies, but in practice the above-mentioned literature and research reports have reported no therapeutic effects on gastritis and gastric ulcer disease. Accordingly, the present inventors have attempted to provide gastritis and gastric ulcer therapeutic agents and gastroprotective foods using extracts, fractions, and complex herbal medicines comprising licorice, turmeric and leaflets.

As a result, the present invention was completed by confirming that licorice, turmeric and leaflet extracts according to the present invention and the complex herbal containing the same have an inhibitory effect on gastritis and gastric ulcer.

It is an object of the present invention to provide a pharmaceutical composition and dietary supplement containing complex herbal extracts and fractions including licorice, turmeric and leaflets effective for the treatment and prevention of gastritis and gastric ulcer disease.

In accordance with the above object, the present invention provides a composition for the prevention and treatment of gastritis and gastric ulcer containing a non-polar solvent soluble extract of a complex herbal medicine comprising licorice, turmeric and leaflets.

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The non-polar solvent includes ethyl acetate, chloroform, hexane solvent, and preferably ethyl acetate. The complex herbal medicine additionally includes Paeonia lactiflora , small leaves ( Cardamomi Fructus ), Herbal medicines such as Bokbunja ( Rubus coreanus ), Phlomis umbrosa , Curcuma zedoaria, Cnidium monnieri , Cnidium officinale , etc. may be included.

Method for separating the extracts and fractions from the herbal medicine of the present invention is as follows.

Hereinafter, the present invention will be described in detail.

The herbal extract of the present invention is about 5 to 20 times, preferably about 10 to 15 times, water, lower alcohol or about 1: 0.1 to 1:10, preferably 1, the weight of the dried herbal weight (kg). These mixed solvents having a mixing ratio (kg / L) of: 0.2 to 1: 5 are used at an extraction temperature of 20 to 50 ° C, preferably 20 to 30 ° C, for about 1 hour to 2 days, preferably about 2 hours to 1 The crude extract which is a soluble extract according to water, a lower alcohol, or a mixed solvent thereof may be obtained by an extraction method such as ultrasonic extraction, reflux cooling extraction, and the like.

The present invention provides a composition for the prevention and treatment of gastritis and gastric ulcer comprising crude extracts obtained in the extraction process.

In addition, the non-polar solvent soluble extract of the present invention, after suspending the crude extract in water, the suspension is added 1 to 10 times by adding about 1 to 10 times, preferably about 1 to 5 times the volume of a polar or nonpolar solvent, Preferably it can be obtained by fractionation 2 to 5 times. It is also possible to further carry out conventional fractionation processes (Harborne JB Phytochemical methods: A guide to modern techniques of plant analysis. 3rd Ed. Pp 6-7, 1998).

In addition, purified purified fraction of the active ingredient of the present invention can be separated by fractionating and concentrating the components dissolved using the non-polar solvent soluble extract, and then separated by silica gel column chromatography and thin layer column chromatography (TLC).

Hereinafter, the separation process of the complex herbal extracts and fractions including licorice, turmeric, leaflets,

For example, the first step of grinding licorice, turmeric, leaflets and extracted with a polar solvent such as water, methanol or ethanol and concentrated under reduced pressure to obtain a crude extract soluble in a polar solvent;

A second step of dissolving the crude extract in a nonpolar solvent such as ethyl acetate and suspending it, followed by filtration to separate the nonpolar solvent soluble extract layer and to concentrate to obtain a nonpolar solvent soluble extract available in the nonpolar solvent;

The two-phase nonpolar solvent soluble extract is then subjected to column chromatography and TLC, wherein the silica gel column is developed with a hexane: ethyl acetate 10: 1 to 1: 1 (w / w) ratio, preferably a 3: 1 ratio. Solvent was developed on a column, and after fractionation, fractions having an R _f value of 0.25 to 0.30 (developing solvent conditions, hexane: ethyl acetate = 3: 1) were collected on a TLC to form a single aliquot, and dried to obtain a purified fraction in a purified form. It consists of a third step.

In another aspect, the present invention provides a purified fraction obtained by the production method of the three steps effective for the prevention and treatment of gastritis, gastric ulcer.

In addition, the present invention provides a pharmaceutical composition for preventing and treating gastritis and gastric ulcer comprising the purified fraction.

The composition for preventing and treating gastritis and gastric ulcer of the present invention comprises 0.5 to 50% by weight of the extract or fraction based on the total weight of the composition.

Compositions comprising extracts or fractions of the invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

Compositions comprising extracts or fractions of the invention may further comprise suitable carriers, excipients and diluents according to conventional methods.

Carriers, excipients and diluents that may be included in the compositions comprising extracts or fractions of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin , Calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

Compositions comprising extracts or fractions according to the invention are in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterile injectable solutions, respectively, according to conventional methods. Can be formulated and used.

The amount of the extract or fraction of the present invention may vary depending on the age, sex, and weight of the patient, but may be administered once to several times in an amount of 0.1 to 500 mg / kg. In addition, the dosage of the extract or fraction may be increased or decreased depending on the route of administration, degree of disease, sex, weight, age, and the like. Therefore, the above dosage does not limit the scope of the present invention in any aspect.

The composition comprising the extract or fraction of the present invention can be used in a variety of drugs, food and beverages for the prevention and treatment of gastritis and gastric ulcer. Examples of the food to which the licorice, turmeric and leaflet extracts can be added include various foods, beverages, gums, teas, vitamin complexes, and health supplements.

Licorice, turmeric and leaflet extracts or fractions of the present invention have little toxicity and side effects, and thus are drugs that can be used with confidence even for prolonged administration.

The extract or fraction of the present invention may be added to food or beverage for the purpose of preventing gastritis and gastric ulcer. At this time, the amount of the extract or fraction in the food or beverage is generally added to the health food composition of the present invention 0.01 to 30% by weight of the total food weight, the health beverage composition is 0.02 to 30 g based on 100 ml, Preferably it can be added in the ratio of 0.3-1 g.

The health beverage composition of the present invention is not particularly limited in the liquid component except for containing the extract as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates, etc. as additional ingredients, as in general beverages. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of said natural carbohydrates is generally about 1-20 g, preferably about 5-12 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, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and salts thereof. , Organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. The compositions of the present invention may also contain pulp for the production of natural fruit juices and fruit juice beverages and vegetable beverages. These components can be used independently or in combination. The proportion of such additives is not so critical but is 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.

Hereinafter, the present invention will be described in more detail based on the following examples.

However, the content of the present invention is not limited by the following examples.

 Example 1. Preparation of crude extracts of licorice, turmeric and lobule

3 kg each of dried licorice, turmeric and leaflets (Hoangbang per Pohang Rejuvenation) were pulverized with a grinder, and 20 liters of 80% ethanol (water: ethanol = 20:80) was added for 2 hours to extract the active substance in the medicinal herb. The supernatant was filtered through filter paper (Whatman) to remove debris from each sample. In order to increase the extraction rate as much as possible, 80% ethanol of the same volume was poured again into the sample and extracted for 18 hours or more, and the supernatant was filtered again with a filter paper and the ethanol extract twice was concentrated in a large concentrator (EYELA, model name N-11, Japan) was used to remove the 80% ethanol solvent and concentrated crude extracts of licorice, turmeric and leaflet to obtain 400 g of licorice, 100g of turmeric and 175g of leaflet.

Example 2. Preparation of Non-polar Solvent Soluble Extracts of Licorice, Turmeric and Leaflets

Each crude extract concentrate of licorice, turmeric and leaflet of Example 1 was mixed with 1.5 liters of ethyl acetate, and then shaken, fractionated into an ethyl acetate insoluble layer (lower layer) and an ethyl acetate soluble layer (upper layer), and then ethyl acetate soluble layer. Was collected. The same volume of ethyl acetate solvent as the lower layer (water layer) was repeated five times in the same manner as described above to obtain a substance that is soluble in the ethyl acetate layer as much as possible until the color of the solution became light, and then the collected ethyl acetate soluble layer. Magnesium sulfate (MgSO ₄ ) was used to remove the water-soluble components as much as possible, and the ethyl acetate solvent was removed using a large condenser (EYELA, model N-11, Japan). 56.3 g of solvent-soluble extract licorice, 49.5 g of turmeric, and 49.8 g of lobule were obtained, respectively.

Example 3 Preparation of Purified Fractions of Licorice, Turmeric, and Lobules

Example 2 of the development solvent (hexane (Hex): ethyl acetate (EA)) of the thin layer chromatography (TLC) using various ratios of 4: 1, 3: 1, 2: 1 and 1: 1 The nonpolar solvent soluble extract of each sample was continuously developed, and silica gel column chromatography was performed by selecting a mixed developing solvent (Hex: EA = 3: 1) having a development rate of 0.25. 56.3 g of licorice, 49.5 g of turmeric, and 49.8 g of lobules of Example 2 were dissolved in 500 ml of ethyl acetate, and then mixed evenly with silica gel 60 (MERCK) in the same volume (250 ml), and a small concentrator (EYELA, N- 1N, Japan), the ethyl acetate solvent was removed, and then the non-polar solvent-soluble extract and silica gel mixture of each sample collected were ground into a powder form using a mortar, and the mixture was determined by using a TLC fraction. 3: 1), followed by developing on a column to fractionate. Fractions of 100 licorice, 69 turmeric and 140 lobules were obtained by dropping the solution at a rate that yielded a volume of 50 ml at 20 minutes, and each fraction was used for TLC (developing solvent Hex: EA = 3). : 1) was performed again, and fractions having similar TLC R _f values of 0.25-0.30 were collected and mixed into a single fraction with reference to the fractional ratio. The developing solvent was then removed using a lyophilizer (Ilshin Science, FD5508, Korea), and the purified fractions with similar TLC patterns were collected and 2.5 g (R _f 0.23-0.42) and 5.79 g (R _f 0.23) turmeric, respectively. -0.57) and 2.9 g (R _f 0.14-0.46) leaflets were purified (see Figure 1).

Example 4 Preparation of a Complex Herb Containing Licorice, Turmeric, and Lobules

The purified fractions of licorice, turmeric and leaflet prepared in Example 3 were dissolved in a solvent and mixed in 1: 1: 1 (6mg: 6mg: 6mg), respectively, and other herbal ingredients (peony, soybean, bokbunja, fast, sesame, filamentous, 80% ethanol extract of Cheongung was dissolved in a solvent 7mg) was prepared at a concentration of 25mg / ㎖ was used in the experiment.

Experimental Example 1. Effect of gastric acid secretion on cell signaling system of licorice, turmeric and leaflet extract

Cancer cells (Human Leukemia cell line) U937 cells (Korea Cell Line Bank, 21593) was treated with licorice, turmeric, leaflet extract and fractions of Examples 1 to 3, and then increased due to the activation of histamine receptors. By observing the inhibition of cAMP, the effect of licorice, turmeric and leaflet extracts on gastric acid secretion-related cell signaling system was confirmed.

U937 cells were treated with 10 μM of histamine, 10 μM of ranitidine (SIGMA, R-101), and 0.3 μg / μl of licorice, turmeric, and lobular crude extract of Example 1, and soluble licorice, turmeric, and lobular ethyl acetate soluble extract of Example 2, respectively. 0.3 μg / μL was treated for 20 minutes to determine the amount of cAMP production (see FIGS. 2 and 3). In addition, the licorice, turmeric and leaflet fractions (F9, F7 and F11, respectively) with the lowest cytotoxicity were treated by concentrations to observe cAMP production (see FIGS. 4A, 4B and 4C).

Ranitidine is an antagonist of histamine 2 receptors that inhibits gastric acid secretion and is used to treat peptic ulcers. Inhibition of acid secretion, such as during stimulation, has been reported, and was used as a positive control for comparing the gastric ulcer inhibitory effect of licorice, turmeric, leaflet extract.

Figure 2 was observed the effect of the production of cAMP in U937 cells using licorice, turmeric, leaflet crude extract, in the experimental group showing histamine receptor activity by treatment with histamine 10μM was produced more than 6pmole cAMP licorice, turmeric, leaflet Crude extract showed a similar or reduced effect of cAMP concentration compared to the control group (1 pmole) and ranitidine treated group (1.5 pmole) untreated group (see Fig. 2). Ethyl acetate extracts of licorice, turmeric and leaflets were also similar in production to cranitidine-treated group and cAMP production, especially in the case of turmeric ethyl acetate extract, which significantly reduced cAMP production (see FIG. 3).

When the concentration of purified fraction (F9) of licorice was changed from 1 pg / μl to 3 μg / μl and the concentration of cAMP produced was examined, the maximum amount of cAMP produced when the concentration of licorice purified fraction (F9) was 1 μg / μl or more It was shown to have a high effect of inhibiting gastric acid secretion due to antagonistic action on the histamine H ₂ receptor (see FIG. 4A), and the turmeric purified fraction (F7) had a concentration of 1 ng / μl to 2 μg / μl. When the concentration of cAMP was examined, the concentration of purified fraction of turmeric (F7) was 1 μg / μl, indicating that the highest amount of cAMP production was found to have a large effect of inhibiting gastric acid secretion (see FIG. 4B). In the case of leaflets, when the concentration of purified fraction (F11) was varied from 30 ng / µl to 3 µg / µl, the concentration of purified fraction (F11) was 3 µg / µl. The lowest amount of cAMP production was shown to have a large effect of inhibiting gastric acid secretion by inhibiting histamine H ₂ receptor activity (see FIG. 4C).

In conclusion, licorice, turmeric, lobular crude extracts, nonpolar soluble extracts, and purified fractions were found to inhibit gastric acid secretion by inhibiting cAMP production, and reaffirmed that they could be effectively used for the prevention and treatment of gastritis and gastric ulcers. Could.

Experimental Example 2. Cytotoxicity Test of Licorice, Turmeric and Leaflet Extracts

Cancer cells (Human Leukemia cell line) U937 cells were treated with licorice, turmeric, and lobular extracts of Examples 1 to 3, and then the toxicity to the cells was determined by MTT (3- (4,5-dimethylthiazole-). 2-yl) -2,5-diphenyltetrazolium bromide thiazolyl blue) test method was confirmed that the cytotoxicity is not large.

About 10 ⁵ U937 cells were put into a 96-well plate, 100 mg / μl of licorice, turmeric, lobular crude extract and ethyl acetate extract obtained in Example 1 were incubated for 24 hours, and then 2 mg / ml. 25 μl of MTT solution was added to each well and incubated for 2 hours. Then add 100µl of SS solution (Solubilizing solution: SDS is the main raw material) and react for 16 hours. Optical Density (OD) was measured in a 96-well plate as it was at 570 nm wavelength in an ELISA reader. Using the resulting absorbance values, the control absorbance values of only the cells without the sample extract were subtracted from the control group absorbed values without subtracting the absorbance of the group containing only the culture solution, MTT solution, and SS solution as the default value (100%). The absorbance of each group of sample additives was compared.

9 shows the cytotoxic effects of MTT assay in U937 cells using crude extracts of licorice, turmeric and leaflets, and ethyl acetate extract, showing overall higher survival rate than the control group, and more in crude extracts than ethyl acetate extract. High cell viability is shown (see FIG. 5).

Similarly, when the cytotoxicity test with licorice, turmeric and leaflet purified fractions, the highest cell viability was shown in the licorice purified fraction (F8), turmeric purified fraction (F7), leaflet purified fraction (F11), respectively (Fig. 6a, 6b and 6c).

Experimental Example 3. Gastric ulcer inhibitory effect of licorice, turmeric, lobular nonpolar solvent soluble extract

In order to confirm the therapeutic effect of gastritis and gastric ulcer of licorice, turmeric, lobular non-polar solvent soluble extract of Example 2, animal experiments were performed using the extract of the three herbs and ranitidine.

Five-week-old Spraugue-Dawley (SD) -based male rats (Hyochang Science) were fasted for 24 hours and water was supplied in sufficient amounts. After 24 hours, the water supply was stopped and only 1 ml of methyl cellulose (50 mg Methylcellulose / 10 ml physiological saline), a solvent, was used orally in the control group and ranitidine (SIGMA, which is used as a therapeutic agent for peptic ulcer in other experimental groups). 5 mg / kg of R-101) and 20 mg / kg of licorice, turmeric and leaflet ethyl acetate extracts were orally administered to another experimental group, respectively. After an hour, the abdomen was opened by about 1 cm to seal the pyloric region. After 8 hours, the cervical spine was dissected, the esophagus and the gastric inlet were closed, and the stomach was extracted. The gastric juice was collected through the pyloric region using a syringe. Using a new syringe, inject 15 ml of a 4% formaldehyde solution into the stomach, immerse the tissue in 4% formaldehyde and fix it, and after a day, spread the tissue along the upper part of the stomach, X length = 1 mm x 1 mm) to compare the size of the gastric ulcer lesion.

Gastric acid secretion was measured to compare the gastric juice collected in the stomach process, the gastric juice was precipitated at 1000 rpm for 5 minutes, centrifuged at 1000 rpm for 5 minutes using only 1 ml of the supernatant, and the supernatant was again obtained to obtain 0.01 N hydroxide. Titration experiments were performed using sodium (NaOH), and mEq / 8 hour values were calculated by multiplying the amount of gastric acid secretion with 0.01 N sodium hydroxide.

As a result, Figure 7a and 7b is a picture of gastric ulcer in the stomach of the rat by basal acid secretion, a control group using only methyl cellulose (see Figure 7a) and licorice, turmeric, leaflet ethyl acetate extract 20mg / kg administration group ( 7b), it can be seen that there is a significant effect on gastric ulcer lesion size, and comparing the actual lesion area, a control group using only methyl cellulose solvent, a positive control group administered with ranitidine 5mg / kg and licorice, turmeric, leaflets When comparing the experimental groups administered with the nonpolar solvent-soluble extract 20 mg / kg, respectively, it was confirmed that the administration of licorice, turmeric, leaflet extract reduced the area of gastric ulcer lesions produced by the basal acid in the stomach to suppress the induction (Fig. 8).

9 is a comparison of basal acid secretion in the control group, ranitidine administration group and licorice, turmeric, leaflet ethyl acetate extract administration group, licorice and turmeric ethyl acetate extracts significantly inhibited gastric acid secretion effect than ranitidine, a positive control group that inhibits gastric acid secretion (See FIG. 9).

As a result of the same result, the amount of basal acid secreted from the stomach of the rat was 0.01 N sodium hydroxide, and the acid-base was shown in FIG. 10. The experimental group using licorice and turmeric ethyl acetate extract showed an appropriate amount less than that of the positive control ranitidine. It showed an effect on inhibition (see FIG. 10).

Looking at the above results, it was found that when the licorice, turmeric, leaflet ethyl acetate extract was used, gastric acid secretion was suppressed, and a significant inhibitory effect of gastric ulcer was also seen.

 Experimental Example 4. Gastric ulcer inhibitory effect of licorice, turmeric, leaflet purified fractions and complex herbal medicines containing them

In the same manner as in Experiment 3, licorice, turmeric, leaflet purified fraction of Example 3 and the combined herbal medicine of Example 4 was confirmed the effect of inhibiting gastric ulcer induced by indomethacin.

1 hour after administration of licorice, turmeric, lobular tablet fraction and combined herbal medicine, rats were anesthetized with ether, and then opened for 1 cm to seal the pyloric region of the stomach and duodenum, and 40 mg / kg of indomethacin was injected into the duodenum. The vessels were sealed and quarantined for 8 hours.

Indomethacin, which is used as a control group, is a nonsteroidal anti-inflammatory drug (NSAID). The analgesic and anti-inflammatory effect is shown by inhibiting the production of prostaglandin from arachidonic acid. The most common side effect of NSAIDs is gastrointestinal ulcer, which is sometimes accompanied by bleeding anemia. Patients chronically using NSAIDs have two to three times more gastrointestinal side effects than non-users. The mechanism of side effects is due to the inhibition of the biosynthesis of prostaglandins such as prostacycline (PGI ₂ ; 5-HT-5-hydroxytryptamine) and prostaglandin E2 (PGE ₂ ), which play a protective role in the gastric mucosa. Eicosanoids such as PGI ₂ and PGE ₂ inhibit acid secretion by the stomach, increase mucosal blood flow and promote secretion of cytoprotective mucus in the intestine.

In the case of the control group, 40 mg / kg of indomethacin (SIGMA, I-7378) was administered to induce gastric ulcer, and the experimental group of indomethacin and 100 mg / kg of purified fractions of licorice, turmeric and leaflet, respectively, and the composite of Example 4 The herbal medicine was treated to determine the effect of suppressing gastric ulcer.

11a and 11b are the control group administered only a solvent after the administration of indomethacin 40mg / kg, licorice, turmeric, leaflet fraction 100mg / kg and the combined herbal drug after the administration of indomethacin 40mg / kg, respectively The results showed that gastric ulcer was inhibited by licorice, turmeric, and leaflet purified fractions, and the best effect on the suppression of gastric ulcer was administered when the combined herbal medicine containing licorice, turmeric, and leaflet purified fractions (Fig. 11b).

Comparing the actual gastric ulcer lesion area, the indomethacin 40 mg / kg administration group, the solvent-treated control group, the experimental group administered 100 mg / kg of licorice, turmeric and leaflet fractions, respectively, and the licorice, turmeric and leaflet fractions were included. Comparing the combination drug group, the area of gastric ulcer lesions produced by indomethacin in the stomach was significantly reduced by 1/10 or more compared to the control group, and also in the licorice, turmeric, and leaflet fractions. The lesion area was reduced by 1/5 to 1/10 (see FIG. 12).

The results of the above study showed that crude extracts, non-polar solvent soluble extracts and purified fractions of complex herbal medicines including licorice, turmeric, and lobule showed the effect of inhibiting cellular signaling of histamine receptors related to gastric acid secretion, and reduced gastric acid secretion and administered to rats. By showing a significant inhibitory effect of gastric ulcers, it was confirmed that it can be effectively used for the prevention and treatment of gastritis and gastric ulcers.

Complex herbal crude extracts including licorice, turmeric, lobular, non-polar solvent soluble extracts and purified fractions have excellent efficacy in suppressing gastritis and gastric ulcers, and thus can be used as new therapeutic agents effective in preventing and treating gastrointestinal inflammation, ulcers.

1 is a TLC result of licorice, turmeric, leaflet purified fractions and mixtures thereof,

2 is a graph showing the effect of cAMP production in U937 cells treated with 10 μM of histamine for 20 minutes using 300 ng / μl of crude extracts of licorice, turmeric and lobule,

3 is a graph showing the effect of cAMP production in U937 cells treated with 10 μM of histamine for 20 minutes using 300 ng / μl of a non-polar solvent (EA) soluble extract of licorice, turmeric and leaflets.

Figure 4a is a graph showing the effect of the production of cAMP in U937 cells when the concentration was treated for 20 minutes with histamine 10μM using the fraction (F9) was a low toxicity among the licorice purified fraction, Figure 4b is a purified turmeric fraction When the concentration was treated for 10 minutes with histamine 10μM using the fraction (F7) that was low toxicity, it is a graph showing the effect of cAMP production in U937 cells, Figure 4c is a fraction of low leaf toxicity fraction (F11) When the concentration was treated for 20 minutes with histamine 10μM using a graph showing the effect of cAMP production in U937 cells,

5 is a graph showing the cytotoxic effect through MTT assay using 100ng / μl crude extract of licorice, turmeric, leaflet and 100ng / μl non-polar solvent (EA) soluble extract,

Figure 6a is a graph showing the cytotoxic effect of the fraction by MTT assay using 50ng / μl purified fraction of licorice, Figure 6b is a cytotoxic effect of the fraction by MTT assay using 50ng / μl purified fraction of turmeric Figure 6c is a graph showing the cytotoxic effect of each fraction through the MTT assay using 50ng / μl purified fraction of lobule,

Figure 7a is a picture of gastric ulcer by basal acid secretion of the control group administered with methyl cellulose and no medicinal extract, Figure 7b is from the stomach of rats administered 20 mg / kg of licorice, turmeric and leaflet non-polar solvent (EA) soluble extract This picture shows the effect of suppressing gastric ulcer,

FIG. 8 is a graph comparing the area of gastric ulcer lesions produced by basal acid in the stomach of rats to which the control group was administered 20 mg / kg of lanitidine administration group, licorice, turmeric and lobular nonpolar solvent (EA) soluble extract,

9 is a graph comparing the percentage of basal acid secretion in the stomach of rats treated with a control group and a lanitidine-treated group, licorice, turmeric and lobular non-polar solvent (EA) soluble extracts, in percentage;

FIG. 10 is a graph of the amount converted from the amount of basal acid secreted from the control group and 20 mg / kg of the lanitidine-administered group, 20 mg / kg of licorice, turmeric and leaflet non-polar solvent (EA) soluble extracts, and titration amount of 0.01 N sodium hydroxide,

Figure 11a is a picture of gastric ulcer of the control group administered with 40 mg / kg of methylcellulose and indomethacin, Figure 11b is a combined herbal 100mg / kg including licorice, turmeric, leaflet purified fraction and each licorice, turmeric, leaflet purified fraction 100 It is a photograph showing the gastric ulcer inhibitory effect of the rat in which the mg / kg with indomethacin 40mg / kg was administered.

12 is a control group treated with methylcellulose and only indomethacin 40mg / kg and indomethacin and each licorice purified fraction (F9) 100mg / kg, turmeric purified fraction (F7) 100mg / kg, leaflet purified fraction (F11) This is a graph comparing the area of gastric ulcer lesions in rats treated with 100 mg / kg and 100 mg / kg of the combined herbal medicine.

Claims (14)

delete delete delete delete delete A pharmaceutical composition for the prevention and treatment of gastritis and gastric ulcer containing ethyl acetate soluble extract of a compound herbal medicine comprising licorice, turmeric and leaflets. delete delete 7. The composition of claim 6, wherein the complex herbal additionally comprises peony, soybean paste, bokbunja, fast, extract, casualty and uterus. After grinding dried licorice, turmeric and leaflets, extracted with polar solvent such as water, methanol or ethanol, concentrated under reduced pressure to obtain crude extracts soluble in polar solvents, and dissolved the crude extracts with non-polar solvents such as ethyl acetate. The non-polar solvent soluble extract layer obtained by filtration of this suspension was separated and concentrated to obtain a non-polar solvent soluble extract soluble in the non-polar solvent, and the R _f value on the TLC obtained by performing column chromatography and TLC on the non-polar solvent soluble extract. A pharmaceutical composition for the prevention and treatment of gastritis and gastric ulcers containing a purified fraction of purified form having 0.25 to 0.30 (developing solvent conditions, hexane: ethylacetate = 3: 1). delete 11. A composition according to claim 6 or 10 comprising 0.5 to 50% of said extract relative to the total weight of the composition. delete delete