KR100905747B1 - Compositions for curing NSAIDs-induced gastric antral ulceration comprising anthocyanin - Google Patents

Abstract

The present invention relates to a composition for treating gastritis and gastric ulcer caused by nonsteroidal anti-inflammatory drugs (NSAIDs) containing anthocyanins, and more particularly, characterized in that it contains anthocyanins extracted from black rice as an active ingredient. It relates to a composition for preventing and treating gastritis, gastric ulcer caused by anti-inflammatory agents (NSAIDs).

According to the present invention, whether anthocyanin extracted from black rice is effective in gastritis and gastric ulcer induced by NSAIDs was confirmed through inhibition of lipid peroxidation, increased SOD activity, increased catalase activity, and increased glutathione peroxidase activity. Therefore, the therapeutic effect on gastritis and gastric ulcer induced by nonsteroidal anti-inflammatory agent from the black rice-derived anthocyanin can be expected.

Anthocyanin, black rice, gastritis, gastric ulcer, naproxen

Description

Composition for the treatment of gastritis and gastric ulcer induced by nonsteroidal anti-inflammatory agent containing anthocyanin {Compositions for curing NSAIDs-induced gastric antral ulceration comprising anthocyanin}

The present invention relates to a composition for treating gastritis and gastric ulcer caused by a nonsteroidal anti-inflammatory agent containing anthocyanin, and more particularly, an anthocyanin extracted from black rice as an active ingredient, a nonsteroidal anti-inflammatory agent (NSAIDs) It relates to a composition for preventing and treating gastritis and gastric ulcer caused by a).

Although many nonsteroidal anti-inflammatory drugs (NSAIDs) have been widely used clinically as anti-inflammatory and analgesics, the most serious side effects of patients taking NSAIDs are gastrointestinal disorders. Administration is known to cause severe gastric mucosal damage, including bleeding, ulceration, perforation, and the like. Naproxen is a noncorticosteroid drug belonging to NSAIDs that causes gastritis and gastric ulcerations in the antrum of the stomach when orally administered to rats, caused by naproxen 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 the like, and H2-receptor antagonists that suppress gastric secretion. Recently, as Helicobacter pyrroli has been recognized as an important causative agent of peptic ulcer, antimicrobial agents have been used to treat gastrointestinal ulcers.

On the other hand, anthocyanins are flavonoid pigments widely distributed in nature and are known to have strong antioxidant activity. Anthocyanins have the ability to remove free radicals and free radicals. This ability 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 intensive studies to overcome the problems of the prior art, and confirmed that anthocyanin extracted from black rice is effective in inhibiting lipid peroxidation, increasing SOD activity, increasing catalase activity, and increasing glutathione peroxidase activity. By doing so, it was confirmed that it is effective in the treatment of gastritis and gastric ulcer caused by nonsteroidal anti-inflammatory drugs, thereby completing the present invention.

Therefore, the main object of the present invention is to identify the therapeutic applicability of anthocyanin to gastritis and gastric ulcer caused by taking nonsteroidal anti-inflammatory and to provide its novel use.

According to one aspect of the invention, the present invention provides a composition for the prevention and treatment of gastritis, gastric ulcer caused by nonsteroidal anti-inflammatory agents (NSAIDs) containing anthocyanin as an active ingredient.

In the composition of the present invention, the anthocyanin (anthocyanin) is usually extracted from the plant as a natural pigment is used to contain anthocyanin extracted from purple sweet potato, black beans, black sesame, bokbunja, red grapes, blueberries, udi as an active ingredient However, it is preferable to contain anthocyanin extracted from black rice as an active ingredient. In the embodiment of the present invention, in order to extract the anthocyanin derived from black rice, the bran of black rice, which is a portion having a particularly high content of physiologically active ingredients, was used.

In the present invention, the anthocyanin may be prepared by various methods generally used for filtration and purification of natural extracts by extracting with a hydrophilic organic solvent such as water, alcohol, or a mixed solvent thereof, preferably black rice The bran was extracted with ethanol, filtered through a filter paper, and separated using a column filled with an ion exchange resin. In the embodiment of the present invention, Whatman No. 1 filter paper was used as the filter paper, and Amberlite XAD7 resin was used as the ion exchange resin.

In the composition of the present invention, the nonsteroidal anti-inflammatory agent is characterized in that naproxen (Naproxen). Naproxen of the present invention is a noncorticosteroid drug belonging to NSAIDs, which causes gastritis and gastric ulcerations in the antrum of the stomach when orally administered to rats. The generation of gastric antral ulceration caused by the production of oxygen free radicals (lipid peroxidation) plays a decisive role. Therefore, the composition of the present invention contains black rice-derived anthocyanin 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 of the present invention, the composition is characterized by inhibiting lipid peroxidation and activating antioxidant enzymes in vivo. Gastritis and gastric ulcer caused by the NSAIDs of the present invention, as mentioned above, can be prevented or treated by activating antioxidant enzymes that inhibit lipid peroxidation in vivo and induce antioxidant activities such as catalase and glutathione peroxidase. have. In the embodiment of the present invention, it was confirmed that lipid peroxidation inhibitory activity and antioxidant enzyme activity were markedly increased in the rats administered with black rice-derived anthocyanin.

The black rice-derived anthocyanin of the present invention may be prepared by a method known in the pharmaceutical art in order to be used as a composition for preventing and treating the above, and may be mixed with itself or a pharmaceutically acceptable carrier, excipient, diluent, and the like. It can be prepared and used in the form of powders, granules, tablets, capsules or injections. They can also be administered orally or parenterally.

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

In the present invention, by using the gastric ulcer mouse model induced by NSAIDs to confirm the prevention and treatment effect of anthocyanin in vivo, it was to provide a composition for treating gastritis and gastric ulcer by NSAIDs containing anthocyanin as an active ingredient. In particular, naproxen is more commonly used than other NSAIDs in the treatment of arthritis patients, and gastric ulcers caused by naproxen are mainly induced in the gastric antrum. Conforms. Therefore, in the present invention, naproxen was selected as an NSAID for inducing ulceration in rats, and an excellent anti-ulcer effect of anthocyanin in the naproxen-induced gastric antral ulcer model induced by such naproxen This has led to improved use of gastritis and gastric ulcer use.

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

As described above, according to the present invention, the anthocyanin extracted from black rice increases the activity of antioxidant enzymes such as SOD, catalase, and glutathione peroxidase in combination with lipid peroxidation in vivo, and therefore, gastritis induced by NSAIDs And it is expected to be useful in the development of therapeutics and nutraceuticals that exhibit excellent therapeutic effects against gastric ulcers.

Example 1 Extraction and Analysis of Anthocyanins from Black Rice

Black rice (black jinju rice) used for anthocyanin extraction was obtained from RDA. The process of extracting anthocyanin from the black rice is as follows. 0.5 g of bran of black rice is precipitated in ethanol containing 0.% TFA (Trifluoroacetic acid) and left for 24 hours. The ethanol extract is then filtered using Whatman NO.1 filter paper and then completely dried using a rotary evaporator. The obtained dried material was dissolved in 100 ml of distilled water and then packed into column chromatography using Amberlite XAD7 resin. Anthocyanins obtained by performing ion exchange chromatography as described above were analyzed by HPLC (FIG. 1).

As shown in Figure 1, the extract extracted from the black rice consists of a single component (A of Figure 1), it was confirmed that the single component is anthocyanin (B of Figure 1).

Example 2 Treatment of Experimental Animal Group and Method of Making Naproxen-Induced Gastric Ulcer Model

230-250 g of 7-week old male rats (Sprage-Dawley rats) were purchased from Biolink. Rats were housed in cages and kept in a room controlled at 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 rats were fasted for 18 hours prior to the experiment. After the first naproxen dose, the rats were fed feed and tap water for the duration of the experiment.

Naproxen (sodium salt) is Sigma Chemical Co. It was purchased from (St Louis, MO) and used in dissolved distilled water and continuously orally administered using a feeding needle. Anthocyanins were immediately dissolved in distilled water immediately before use and orally administered to mice at doses of 5, 25 and 50 ml / kg, respectively. Naproxen (120 mg / kg dose range) was administered to rats by orogastric gavage twice a day (09 and 21 o'clock). Mice were killed via ether anesthesia 1-5 days after naproxen. Stomach of rats was cut according to greater curvature and washed with 0.9% saline.

The mice were specifically divided into 3 groups (8 animals per group). The normal group orally administered distilled water twice a day at 9 and 21 o'clock in the same dose as the other groups. Control group was orally administered naproxen (120 mg / kg) twice a day at 9 and 21 o'clock. The experimental group (Anthocyanin-administered group) was orally administered naproxen (120 mg / kg) twice a day at 9 and 21 o'clock, and then anthocyanin at 9, 9 and 21 hours twice a day at 5, 25 and 50 mg / kg doses, respectively. Administration via orogastric gavage.

In addition, gastric ulcers were identified by routine histological evaluation of the stomach of several mice. Gastric damage by naproxen was mainly confirmed in the form of ulcers. Ulcers were visually judged by the depth penetrating the gastric mucosal surface. Gastric ulcers were identified mainly in the gastric antrum and not in gastric corpus.

Example 3 Measurement of Changes in Lipid Peroxidation by Anthocyanin Administration

To determine the degree of lipid peroxidation, the level of TBA (thiobarbituric acid) reactants in the gastric mucosa of rats was measured. To homogenate the gastric mucosa (stomach homogenate) was added 8.1% sodium dodecyl sulfate, 20% acetic acid (pH 3.5), 0.8% TBA and reacted at 95 ℃ for 1 hour. Then, the mixture was cooled with tap water and n-butanol and pyridine (15: 1 v / v) mixed at a ratio of 15: 1 was added to the reactants, followed by strong shaking for 1 minute. And centrifuged for 10 minutes at 4000rpm. Absorbance was measured at 532 nm and the results were expressed in MDA (nmole) per g of tissue weight. The degree of lipid peroxidation was calculated from a standard curve using malondialdehyde tetrabutylammonium salt.

As a result, as shown in Figure 2, the concentration of MDA (Malondialdehyde) in the gastric mucosa, which is an indicator of lipid peroxidation, was 156.92 ± 6.03 nmol / g of tissue in the normal group. In the control group, 266.94 ± 8.0 nmol / g of tissue was nearly twice as high as the normal group. On the other hand, 5, 25, and 50 mg / kg of anthocyanin showed a significant dose-dependent decrease in lipid peroxidation with 232.43 ± 6.78, 185.59 ± 5.28 and 167.08 ± 9.91 nmol / g of tissue, respectively. All results were expressed as mean ± standard deviation, and when comparing the significance (P <0.05) between the control group, normal group and experimental group, Tukey's studentized range test was used.

Example 4 Measurement of Changes in SOD Activity by Administration of Anthocyanin

SOD activity in gastric mucosa of mice after oral administration of anthocyanin was measured. Experiments were performed in 50 mM potassium phosphate buffer (pH 7.8) containing 3 mL of 0.1 mM EDTA in a 25 ° C. cuvette. The reaction mixture produces a reduction rate of 0.1 mM ferricytochrome C, 0.1 mM xanthine, and ferricytochrome C of 0.025 absorbance units per minute at 550 nm. Sufficient xanthine oxidase was included. Tissue homogenate was mixed with the reaction mixture (50 mM potassium phosphate buffer (pH 7.8) with 0.1 mM EDTA, 0.1 mM pericytochrome C, 0.1 mM xanthine). Kinetic spectrophotometric analysis started at 550 nm as soon as xanthine oxidase was added. Under defined conditions, the amount of SOD required to inhibit the reduction of cytochrome C by 50% is defined as 1 unit. Absorbance was measured at 550 nm and the results expressed in units per mg of protein.

As a result, as shown in Figure 3, the SOD activity of the control group was 4.30 ± 1.43 units / mg of protein significantly decreased compared to the normal group (10.03 ± 1.43 units / mg of protein). However, anthocyanin 5, 25, 50 mg / kg group showed 5.77 ± 0.75, 8.75 ± 1.38 and 9.73 ± 1.60 units / mg of protein, respectively, and significantly increased SOD activity. In particular, SOD activity at anthocyanin doses of 25 mg / kg and 50 mg / kg showed a much higher increase compared to the control.

Example 5 Measurement of Catalase Activity Change by Anthocyanin Administration

Catalase activity in the gastric mucosa of rats after oral administration of anthocyanin was determined according to the method of Aebi (1974). The reaction mixture consisted of 50 mM potassium phosphate buffer at pH 7.0 and 10 mM H ₂ O ₂ . The experiment was performed at 3 mL by combining 1.9 mL of 50 mM potassium phosphate buffer at pH 7.0, 1 mL of 10 mM H ₂ O _2, and 100 μl of tissue homogenate. Under defined conditions, the amount of catalase required to degrade 1.0 μmole H ₂ O ₂ per minute at 25 ° C., pH 7.0 was defined as 1 unit. Absorbance was measured for 2 minutes at 240 nm and the results are expressed in units per mg of protein.

As a result, the catalase activity was 5.2867 ± 1.6702 units / mg of protein in the control group administered naproxen, as shown in Figure 4 significantly compared to the normal group (36.0850 ± 2.7979 units / mg of protein). On the other hand, in the experimental group administered anthocyanin 5, 25, 50 mg / kg 6.6600 ± 1.7739, 14.7325 ± 2.3110 and 34.6300 ± 2.4914 units / mg of protein, respectively, significantly increased compared to the control group. In particular, catalase activity at anthocyanin doses of 25 mg / kg and 50 mg / kg showed a much higher increase compared to the control.

Example 6 Measurement of Changes in Glutathione Peroxidase Activity by Anthocyanin Administration

Glutathione peroxidase (GSH-px) activity in the gastric mucosa of rats after oral administration of anthocyanin was determined according to a modified method of Lawrence and Burk [1976]. The reaction mixture consisted of glutathione peroxidase assay buffer (50 mM potassium phosphate buffer pH 8.0, 0.5 mM EDTA) and NADPH assay reagent (5 mM NADPH, 42 mM GSH, 10 unit / mL glutathione reductase). Samples of homonatate solution and supernatant fluid with 50 mM potassium phosphate buffer were prepared by centrifugation at 1,000 g for 10 minutes at 4 ° C. 900 μl of glutathioneperoxidase assay buffer, 50 μl of NADPH assay reagent and 50 μl of sample were continuously added to the cuvette and mixed well (the total amount of cuvette was 1.00 mL). The reaction was initiated by the addition of 10 [mu] l of 30 mM tert-butyl hydroperoxide or Cumene hydroperoxide (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 enzyme (glutathione peroxidase) was determined by the sum of the results of each experiment using 30 mM tert-butyl hydroperoxide and Cumene hydroperoxide (80%). The level of GSH is expressed in terms of μmole / min / mg of protein.

As a result, the GSH-px activity in the control group administered naproxen as shown in Figure 5 significantly reduced 5.62 ± 0.91 μmole / min / mg of protein compared to the normal group (9.94 ± 0.24 μmole / min / mg of protein) It was. On the other hand, in the experimental group administered anthocyanin 5, 25 and 50 mg / kg, it was significantly increased compared with the control group with 6.15 ± 0.62, 7.99 ± 0.72 and 9.34 ± 0.38 μmole / min / mg of protein, respectively.

Example 7 Observation of Changes in Gastric Tissue by Anthocyanin Administration

Murine stomach tissues were fixed with 10% neutral formalin, foamed with paraffin, and cut into 4-μcm thick. Then, it was observed by staining with hematoxylin and eosin according to the general procedure.

Referring to FIG. 6, the gastric mucosal images of the untreated rats, that is, the normal rats treated with nothing were shown. In comparison, the Naproxen-treated rats: In gastric ulcer-induced model (NSAIDs), oral administration of naproxen, the gastric mucosal part was observed to be damaged. In the case of anthocyanin-pretreated rats (oral administration of naproxen after administration of anti-ulcer substance anthocyanin from black rice), the gastric mucosal portion of the gastric mucosal area was less damaged by the anti-ulcer substance anthocyanin than the experimental group treated with naproxen only. Was observed.

Figure 1 shows the HPLC analysis data of the anthocyanin extracted from black rice.

2 is a measure of the degree of lipid peroxidation according to the concentration of anthocyanin.

Figure 3 measures the SOD activity according to the concentration of anthocyanin administration.

Figure 4 measures the catalase activity according to the concentration of anthocyanin administration.

5 is a measure of glutathione peroxidase activity according to the concentration of anthocyanin.

Claims (5)

delete delete Cyanidin 3-O-glucoside and peonidine 3-0-glucoside (Cheondin 3-O-glucoside) isolated from a black rice bran extracted with ethanol, filtered through a filter paper and packed with an ion exchange resin. Gastric inflammation, gastric ulcer prevention and treatment composition caused by nonsteroidal anti-inflammatory drugs (NSAIDs) containing anthocyanin consisting of 3-O-glucoside) as an active ingredient. 4. The composition of claim 3, wherein the nonsteroidal anti-inflammatory agent is naproxen. 4. The composition of claim 3, wherein the composition inhibits lipid peroxidation and activates antioxidant enzymes in vivo.

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