KR20110014401A - Composition for curing nsaids-induced gastric antral ulceration comprising the extract of rubus coreanus - Google Patents

Abstract

PURPOSE: A composition containing Rubus coreanus Miquel extract for preventing and treating gastritis and gastric ulcer is provided to enhance antioxidant enzyme and to suppress lipid peroxidation. CONSTITUTION: A composition for preventing and treating gastric ulcer and gastritis caused by non-steroidal anti-inflammatory drug(NSAIDs) contains Rubus coreanus Miquel extract as an active ingredient, having cyaniding 3-glucoside and cyaniding 3-rutinoside. The Rubus coreanus Miquel extract is obtained by extracting with alcohol solvent, filtering the extract, drying, and filling in a column chromatography. The NSAIDs is naproxen.

Description

Composition for curing NSAIDs-induced gastric antral ulceration comprising the extract of Rubus coreanus}

The present invention relates to a composition for preventing and treating gastritis and gastric ulcer caused by nonsteroidal anti-inflammatory drugs (NSAIDs) containing bokbunja extract, and more specifically, cyanidin 3-glucoside and cyanidin 3 It relates to a composition for preventing and treating gastritis and gastric ulcer caused by nonsteroidal anti-inflammatory drugs (NSAIDs) comprising bokbunja extract containing cyanidin 3-rutinoside as an active ingredient.

Many nonsteroidal anti-inflammatory drugs (NSAIDs) have been widely used clinically as anti-inflammatory and analgesics, but gastrointestinal disease is the most serious side effect in patients taking NSAIDs. It is known to cause severe gastric mucosal damage, including bleeding, ulceration, perforation, and the like. The mechanism of action of NSAIDs is to inhibit the synthesis of prostaglandins from arachidonic acid by inhibiting cyclooxygenase (COX). Prostaglandins increase the chemotaxis and vascular permeability for fever, edema, erythema and leukocytes. NSAIDs exert an anti-inflammatory effect by inhibiting these effects of prostaglandins. Prostaglandins play a physiological role as well as inflammation. It suppresses acid secretion of the gastric mucosa, promotes mucus secretion, maintains tension of the gastroesophageal sphincter, dilates renal vessels, and maintains renal blood flow when fluids decrease. Physiological functions of prostaglandins are inhibited by NSAIDs, resulting in side effects such as gastrointestinal side effects and nephrotoxicity.

Naproxen, a noncorticosteroid drug belonging to NSAIDs, causes gastritis and gastric ulcers in the antrum area of the stomach when orally administered to rats. The generation of gastric antral ulceration plays a crucial role in the generation of oxygen free radicals and lipid peroxidation. Gastrointestinal ulcers include attack factors (gastric acid, pepsin, vagus nerve stimulation, gastrin secretion, increased gastric wall cells and damage to gastric mucosa) and protective factors (bicarbonate secretion, mucus production, mucosal production, local mucosal blood flow and prostaglandins) Occurs when the balance between) is disrupted, as ulcers caused by NSAIDs such as naproxen are caused by the inhibition of prostaglandin synthesis. Treatment agents for such ulcers include antacids that inhibit attack factors, anti-pepsin agents, mucoprotective agents that enhance defense factors, mucosal blood flow enhancers, and H2-receptor antagonists that suppress gastric secretion.

On the other hand, anthocyanins (anthocyanin) is a pigment of the flavonoid system widely distributed in nature, and prevents the formation of deposits in the arteries to clear blood and reduce the risk of heart disease and stroke. It is also known to have strong antioxidant activity. It is very effective in effectively neutralizing free radicals, which have been identified as a cause of disease and aging. Free radicals are compounds produced by the process of oxygen and nutrients to make energy. When an appropriate amount is present, it attacks and eliminates bacteria or foreign substances.However, if the amount is increased, it circulates in the body to block blood vessels, damage cells, and damage the hormone system. The damage is serious, including confusion and cancer or diabetes. Among the substances having an antioxidant effect, anthocyanin has a 5 to 7 times stronger effect than tocopherol, which is known as an antioxidant. The antioxidant ability of these anthocyanins is believed to represent a defense mechanism against many diseases resulting from the attack of reactive oxygen species (ROS).

Accordingly, the present inventors have made a thorough research to overcome the problems of the prior art, it was confirmed that the extract extracted from the bokbunja is effective in inhibiting lipid peroxidation, increased SOD activity, increased catalase activity, increased glutathione peroxidase activity By doing so, the present invention was confirmed to be effective in treating gastritis and gastric ulcer caused by nonsteroidal anti-inflammatory drugs, and completed the present invention.

Therefore, the main object of the present invention is bokbunja-derived cyanidin 3-glucoside and cyanidin 3-rutinoside against gastritis and gastric ulcer caused by taking nonsteroidal anti-inflammatory drugs. To determine the therapeutic applicability of, and to provide a composition for treating gastritis and gastric ulcer using the same.

According to an aspect of the present invention, the present invention is a nonsteroidal composition comprising bokbunja extract containing cyanidin 3-glucoside and cyanidin 3-rutinoside as an active ingredient. Provided is a composition for preventing and treating gastritis and gastric ulcer caused by sex anti-inflammatory agents (NSAIDs).

In the composition for preventing and treating gastritis and gastric ulcer of the present invention, the bokbunja extract is extracted with a hydrophilic organic solvent such as water, alcohol, ethyl acetate, ethyl ether, or a mixed solvent thereof, and is generally used for filtration and purification of natural extracts. Although it may be prepared by various methods used, preferably, the bokbunja fruit is extracted with an alcohol containing hydrochloric acid (HCl), the obtained extract is obtained by filtering and drying, and then the dried product is dissolved in distilled water and then column chromatography ( It is characterized by obtaining a pigment fraction by filling in column chromatography).

In the composition for preventing and treating gastritis and gastric ulcer of the present invention, the NSAIDs are naproxen (Naproxen). The naproxen of the present invention is a noncorticosteroid drug belonging to NSAIDs, and when orally administered to rats, induces gastritis and gastric ulcerations in the gastric antrum area, caused by naproxen The generation of gastric antral ulceration plays a crucial role in the generation of oxygen free radicals and lipid peroxidation. Therefore, the composition of the present invention contains an extract of Bokbunja as an active ingredient in order to induce antioxidant activity for the purpose of inhibiting the production of oxygen free radicals and preventing lipid peroxidation.

In the composition for preventing and treating gastritis and gastric ulcer of the present invention, the composition is characterized by inhibiting lipid peroxidation and activating antioxidant enzymes in vivo. Gastritis and gastric ulcer caused by NSAIDs of the present invention can be prevented or treated by activating antioxidant enzymes that inhibit lipid peroxidation in vivo and induce antioxidant actions such as catalase and glutathione peroxidase, as mentioned above. have. In the embodiment of the present invention it was confirmed that the inhibitory effect of lipid peroxidation and the activity of antioxidant enzymes in rats administered the bokbunja extract significantly increased.

The bokbunja extract of the present invention may be prepared by a method known in the pharmaceutical art in order to be used as such a prophylactic and therapeutic composition, and may be mixed with itself or a pharmaceutically acceptable carrier, excipient, diluent, etc. It may be prepared and used in formulations such as tablets, capsules or injections. They can also be administered orally or parenterally.

The dosage for administering the bokbunja extract according to the present invention as an active ingredient may be appropriately selected according to the age, sex and symptoms of the disease of the patient, and preferably, the bokbunja extract of 0.1 to 100 mg / kg per day may be administered. have.

In the present invention, by using the gastric ulcer mouse model induced by NSAIDs to confirm the prevention and treatment effect of the bokbunja extract in vivo, to provide a composition for treating gastritis and gastric ulcer by NSAIDs containing the bokbunja extract as an active ingredient It became. NSAIDs of the present invention cause gastric ulcer more than duodenal ulcer and do not pathologically well. NSAIDs cause gastric mucosal damage by inhibiting the synthesis of prostaglandin, which acts as an important defense factor in the gastric mucosa in vivo. Gastric ulcer bleeding is the most common side effect of NSAIDs and is caused by the inhibition of cyclooxygenase (COX), a rate-limiting enzyme. Cox-1 is constantly expressed in gastric mucosa, platelets, and kidneys and plays an important role in maintaining homeostasis of tissues. Cox-2 is induced by cytokines and mainly expressed in inflammatory sites. In addition, NSAIDs are biologically active substances that increase the production of leukotriene, which causes an allergic or inflammatory response, induces increased intercelluar adhension molecule (IMC) expression, increased neutrophils and increased oxygen free radicals, and inhibits platelet function. Induces chronic bleeding.

Unlike gastric ulcers caused by the NSAIDs of the present invention, there are also ulcers caused by pathological causes such as H. pylori. The treatment of ulcers by H. pylori is to prevent several signaling systems from operating by blocking cytotoxic substances such as cagA produced by anti-H. Pylori function into cells, and treating them by the NSAIDs of the present invention. Since the treatment is induced by selective inhibition of COX, which can block the synthesis of prostaglandins, and not a pathological cause, the treatment mechanisms are different from each other.

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for illustrating the present invention, and thus the scope of the present invention is not construed as being limited by these embodiments.

Example 1. Isolation and Analysis of Bokbunja Extract.

Bokbunja used in the present invention was obtained in 2006 grown in Gochang, Jeonbuk. Separation process of the bokbunja extract from the bokbunja is as follows. 10 g of the bokbunja fruit was precipitated in methanol containing 0.3% HCl and stirred for 24 hours to obtain a methanol extract. The methanol extract was filtered using Whatman NO.4 filter paper, and then completely dried using a rotary evaporator (EYELA, Japan), and then the resulting dried product was dissolved in 100 ml of distilled water and column chromatograph using Amberlite XAD7 resin. Filling was performed by column chromatography to obtain a pigment fraction. The obtained pigment fraction was analyzed for pigment using UPLC MS / MS (Waters, Milford MA, US).

1 shows the results of UPLC analysis of the extract. As shown in Figure 1, the extract extracted from the bimodal molecule consists of a single component (Fig. 1A), it was confirmed that the single component is anthocyanin (Fig. 1B, Fig. 1C). The extract was separated using an open column, and analyzed using a total ion chromatogram. As a result, peaks were identified at 449.1 and 595.1. To determine the molecular weight of this fraction, the molecular weight was confirmed by UPLC / Q-TOF (Ultra-high Performance Liquid Chromatography / Quadrupole-Time of Flight) analysis, and 449.1 (m / z) was obtained from cyanidin 3-glucoside, 519 (m / z) was confirmed to be cyanidin 3-rutinoside.

FIG. 1B shows cyanidin 3-glucoside, and FIG. 1C shows cyanidin 3-rutinoside, and MS to determine whether the two types of cyanidins are single. This graph shows 449 (m / z) and 519 (m / z), respectively, using the / MS spectra.

Example 2 Treatment of Experimental Animals and Methods of Making Naproxen-Induced Gastric Ulcer Model

 250-250 g of 7-week-old female rats (Sprage-Dawley rats) were purchased from Samtaco. The rats were placed in rat cages and reared in a controlled room with a temperature of 22-24 ° C., a humidity of 70-75%, 12 hours light and 12 hours dark, and fed a normal laboratory diet. The mice were fasted for 18 hours before the experiment and provided feed and tap water for the duration of the experiment after the first naproxen administration.

The mice used in the experiment were divided into three groups.

1.Normal group: group that did not receive anything

2. Control group: naproxen (110 mg / kg) group only

3. Experimental group: After administration of naproxen (110 mg / kg), bokbunja extract was administered in doses of 20, 50 and 80 mg / kg, respectively.

The naproxen (Sigma, US) was dissolved in distilled water and used twice a day (09:00 am, 09:00 pm) orally administered to experimental mice by orogastric gavage. The bokbunja extract was orally administered for 3 days. Experimental rats were sacrificed by ether anesthesia 1 to 5 days after naproxen administration, and then the stomach of the mice was excised along greater curvature and washed with 0.9% saline.

Example 3 Measurement of Changes in Lipid Peroxidation by Administration of Bokbunja Extract.

To determine the degree of lipid peroxidation, levels of thiobarbiturin acid (TBA) reactants were measured in gastric mucosa of experimental rats. To homogenate the gastric mucosa (stomach homogenate) was added 8.1% sodium dodecyl sulfate (SDS), 20% acetic acid (acetic acid, pH 3.5), 0.8% TBA and reacted at 95 ℃ for 1 hour. Thereafter, the mixture was cooled with cooling water, mixed in a reactant (reactants) in a ratio of 15: 1, n-butanol and pyridine were added, shaken strongly for 1 minute, and centrifuged at 4,000 rpm for 10 minutes. Absorbance was measured at 532 nm, and the measurement results were expressed as MDA (malondialdehyde) (nmole) per g of tissue weight. In addition, the degree of lipid peroxidation was calculated from a standard curve using malondialdehyde tetrabutylammonium salt.

2 is a graph showing the results of lipid peroxidation. As shown in Figure 2, the control group was administered only naproxen 110 mg / kg MDA concentration was 146.54 nmol / g / g tissue weight, the MDA concentration of the normal group without treatment was tissue weight (g) It was found to be 66.27 nmol / g, thereby confirming that the concentration of MDA was increased by naproxen. In order to confirm the lipid peroxidation inhibitory ability of the bokbunja extract after pre-treatment of the extract by concentration (20, 50, 80 mg / kg), in the oral administration of naproxen group, the concentration of MDA in the administration concentration of bokbunja extract The tendency to decrease is confirmed. In particular, it was confirmed that the concentration of MDA was most reduced in the group administered 80 mg / kg bokbunja extract.

Example 4 Determination of Changes in SOD Activity by Administration of Bokbunja Extract.

It was performed in 50 mM sodium phosphate buffer (pH 7.8) containing 3 ml of 0.1 mM EDTA in a 25 ° C. cuvette. The reaction mixture uses 0.1 mM ferricytochrome C and 0.1 mM xanthine to produce sufficient xanthine xanthine to produce a reduction of 0.025 Absorbance Unit of Pericytochrome C per minute at 550 nm. oxidase). Tissue homogenate was mixed with the reaction mixture (50 mM calcium phosphate buffer (pH 7.8) with 0.1 mM EDTA, 0.1 mM pericytochrome C, 0.1 mM xanthine). Kinetic spectrophotometric analysis was started at 550 nm as soon as xanthine oxidase was added, and the amount of SOD required to inhibit 50% reduction of cytochrome C under defined conditions was defined as 1 unit. Absorbance was measured at 550 nm, and the results are expressed in units per mg of protein.

3 is a graph showing the results of SOD activity measurement. As shown in FIG. 3, the SOD concentration of the control group administered only naproxen 110 mg / kg was 980.82 U / mg per mg protein, and the SOD concentration of the normal group without treatment was 1266.02 U / mg mg protein. As shown, it was confirmed that naproxen reduced the SOD concentration. After pre-treatment of bokbunja extract by concentration (20, 50, 80 mg / kg), the concentration of SOD increased in proportion to the dose of the extract in the oral administration of naproxen. In particular, it was confirmed that the concentration of SOD was most increased in the group administered 80 mg / kg bokbunja extract.

Example 5 Measurement of Catalase Activity Change by Administration of Bokbunja Extract.

Catalase activity in the gastric mucosa of experimental mice was measured according to the method of Aebi (1974). The reaction mixture consisted of 50 mM calcium phosphate buffer and 10 mM H ₂ O ₂ , pH 7.0, and the experiment was performed by combining 1.9 ml of the calcium phosphate buffer, 1 ml of the H ₂ O _2, and 100 μm of homogenized tissue. Under defined conditions, the amount of catalase required to degrade 1.0 μmole of H ₂ O ₂ per minute at pH 7.0 was defined as 1 unit. Absorbance was measured at 240 nm for 2 minutes and the results are expressed in units per mg of protein.

4 is a graph showing the results of catalase activity measurement. As shown in FIG. 4, the CAT concentration of the control group administered only naproxen 110 mg / kg was 22.83 U / mg per mg protein, and the CAT concentration of the normal group without any treatment was 28.35 U / mg mg protein. Appeared. Accordingly, it was confirmed that the CAT concentration was reduced by naproxen. After pre-treatment of the bokbunja extract by concentration (20, 50, 80 mg / kg), the concentration of CAT in the oral administration of naproxen was confirmed to increase in proportion to the dose of the extract. In particular, it was confirmed that the concentration of CAT was most increased in the group administered 80 mg / kg bokbunja extract.

Example 6 Determination of Glutathione peroxidation Activity Change by Administration of Bokbunja Extract.

Glutathione peroxidase (GSH-px) activity in gastric mucosa of experimental mice was measured according to a modified method of Lawrence and burk (1976) method. The reaction mixture consisted of 50 mM calcium phosphate buffer with pH 8.0 and glutathione peroxidase assay buffer containing 0.5 mM EDTA, and NADHP assay reagent containing 5 mM NADHP, 42 mM GSH and 10 unit / ml glutathione reductase. A homogenate solution and a sample of supernatant fluid with 50 mM calcium phosphate buffer were prepared by centrifuging 1,000 g at 4 ° C. for 10 minutes. To the cuvette, 900 μl of glutathione peroxidase assay buffer, 50 μl of NADHP assay reagent, and 50 μl of sample were continuously added and mixed well (the total amount of cuvette was 1.00 ml). The reaction was started by adding 10 μl of 30 mM tert-butyl hydroperoxide (tBHP) or cumene hydroperoxide (CHP) (80%). Absorbance was recorded and measured by Wavelength: 340 nm / Initial delay: 15 seconds / Interval: 10 seconds / Number of readings: 6. The total amount of glutathione peroxidase was determined as the sum of the results of each experiment using 30 mM t-BHP and CHP (80%). The level of GSH-px was expressed in terms of μmole / min / mg of protein.

5 is a graph showing the results of measuring glutathione peroxidase (GSH-px) activity. As shown in FIG. 5, the concentration of GSH-px in the control group administered only naproxen 110 mg / kg was 4.44 nmol / min / mg of protein, and the concentration of GSH-px in the normal group without treatment was 15.77. nmol / min / mg of protein. As a result, it was confirmed that the concentration of GSH-px by naproxen was reduced. After pre-treatment of the bokbunja extract at different concentrations (20, 50, 80 mg / kg), the concentration of GSH-px increased in proportion to the concentration of the extract in the oral administration of naproxen. In particular, it was confirmed that the concentration of GSH-px most increased in the group administered 80 mg / kg bokbunja extract.

As described above, according to the present invention, the bokbunja extract containing cyanidin 3-glucoside and cyanidin 3-rutinoside has a SOD with lipid peroxidation in vivo. Because it increases the activity of antioxidant enzymes such as catalase and glutathione peroxidase, it is useful for the development of therapeutics and nutraceuticals that have excellent therapeutic effect against gastritis and gastric ulcer caused by NSAIDs. It is expected to be able.

Figure 1 shows the UPLC analysis data of the extract extracted from bokbunja.

Figure 2 is a measure of the degree of lipid peroxidation according to the concentration of bokbunja extract.

Figure 3 measures the SOD activity according to the concentration of bokbunja extract.

Figure 4 is a measure of the catalase activity according to the concentration of bokbunja extract.

Figure 5 is a measure of glutathione peroxidase activity according to the concentration of bokbunja extract.

Claims (4)

Gastritis induced by nonsteroidal anti-inflammatory drugs (NSAIDs) comprising bokbunja extract containing cyanidin 3-glucoside and cyanidin 3-rutinoside as active ingredients, and Composition for preventing and treating gastric ulcer. The method of claim 1, wherein the bokbunja extract is extracted with an alcohol solvent, the obtained extract is obtained by filtering and drying the extract, and then packed into column chromatography to obtain a pigment fraction. Therapeutic composition. According to claim 1, wherein the NSAIDs are naproxen (Naproxen) composition for preventing and treating gastritis and gastric ulcer. The composition for preventing and treating gastritis and gastric ulcer according to claim 1, wherein the composition inhibits lipid peroxidation in vivo and activates antioxidant enzymes.

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