JP2011516467A - Anti-peptic ulcer composition containing rice prolamin - Google Patents

Abstract

  The present invention relates to an anti-peptic ulcer composition comprising rice prolamin as an active ingredient. The anti-peptic ulcer composition of the present invention has an excellent preventive or therapeutic effect in peptic ulcer, and since it contains a protein of general food as an active ingredient, it can be used in a wide range.

Description

  The present invention relates to an anti-peptic ulcer composition containing rice prolamin as an active ingredient, which comprises a tea extract, a water extract, a barley extract, (-)-epicatechin (EC), tannic acid and cysteine At least one selected from the above is further included as an auxiliary component for promoting the activity of rice prolamin.

The main purpose of drug treatment for peptic ulcer is to relieve pain, heal the ulcer itself, and prevent recurrence and complications of the ulcer. For this purpose, various kinds of drugs such as antacids, H ₂ -receptor antagonists, proton pump inhibitors, anti-Helicobacter pylori preparations are generally used. However, if these drugs are used regularly over a long period, various side effects may occur, so care must be taken.

  In addition, the inventors of the present invention not only have a glutinous rice extract that effectively suppresses the occurrence of gastric ulcer and has an excellent effect on healing of the generated ulcer, but also derived from general foods. It is reported that the safety is high (Korea patents 0208969, 0253740 and 10-0627820). However, the above patents did not reveal the main substance with anti-peptic ulcer activity present in glutinous rice extract.

Korean Patent No. 0208969 Korean Patent No. 0253740 Korean Patent No. 10-0627820

  As a result of repeated studies to clarify a substance having anti-peptic ulcer activity in glutinous rice extract, the present inventors have found that rice prolamin has such an activity, and completed the present invention. It was.

  The present invention provides an anti-peptic ulcer composition comprising rice prolamin as an active ingredient.

  In addition, the inventors of the present invention include a composition comprising rice prolamin as an active ingredient, from the group consisting of tea extract, water extract, barley extract, (−)-epicatechin, tannic acid and cysteine. It has been found that the effect is enhanced by further including at least one selected component as an auxiliary component. Such an auxiliary component is preferably contained in an amount of 1 to 80 parts by weight based on 1 part by weight of rice prolamin.

  Rice prolamin which is an active ingredient of the anti-peptic ulcer composition of the present invention can be purified from glutinous rice or glutinous rice, and prolamin derivatives having a structure similar to rice prolamin can also be used. The composition of the present invention can be formulated into preparations for oral administration and can contain a pharmaceutically acceptable carrier.

  The dosage of this composition is preferably 0.3 to 3 mg of glutinous rice prolamin per kg body weight, and 1 to 10 times a day is recommended.

  The composition of the present invention containing rice prolamin as an active ingredient has excellent preventive and therapeutic effects in peptic ulcer.

FIG. 1 shows the anti-peptic ulcer activity of glutinous rice extract, glutinous rice extract, barley extract and wheat extract. FIG. 2 shows the anti-peptic ulcer activity of the whole glutinous rice extract, the precipitated fraction of the glutinous rice extract, and the soluble fraction of the glutinous rice extract. FIG. 3 shows the change in anti-peptic ulcer activity when the precipitate fraction of glutinous rice extract is heat-treated at 100 ° C. for 5 minutes or treated with pepsin. Figure 4 shows the anti-digestion of the solubilized subfraction and the residual precipitate fraction when the precipitated fraction of glutinous rice extract was extracted with 0.5M NaCl, 1% lactic acid, and 70% ethanol, respectively. It shows the distribution of sexual ulcer activity. FIG. 5 shows an SDS-PAGE analysis of prolamin purified from glutinous rice. FIG. 6 shows the dose-dependent anti-peptic ulcer activity of prolamin purified from glutinous rice. FIG. 7 shows an SDS-PAGE analysis of prolamin purified from glutinous rice. FIG. 8 shows the dose-dependent anti-peptic ulcer activity of prolamin purified from glutinous rice.

  While various aspects of the present invention have been described above, specific embodiments of the present invention will be described in the following examples and experimental examples. However, the scope of the present invention is not limited by these.

[Example 1]: Preparation of glutinous rice extract 150 g of glutinous rice powder was added to 5 times weight of distilled water, and vigorously stirred three times for 30 seconds using a vortex mixer. After stirring, the mixture was centrifuged at 3,000 × g for 10 minutes to obtain a supernatant. The protein concentration of the supernatant measured using a Pierce BCA kit in the United States was 1.42 mg / ml. The supernatant was diluted with distilled water to a protein concentration of 1 mg / ml, and this was used as a glutinous rice extract.

[Example 2]: Preparation of precipitate fraction and soluble fraction of glutinous rice extract 500 ml of glutinous rice extract prepared in Example 1 was allowed to stand at room temperature for 15 hours to form a precipitate. Centrifuged for minutes. The obtained precipitate was suspended in 500 ml of distilled water and used as a precipitate fraction of the glutinous rice extract. The clear supernatant was used as the soluble fraction of glutinous rice extract.

[Example 3]: Preparation of glutinous rice extract and its soluble fraction A glutinous rice extract and its soluble fraction were prepared in the same manner as in Examples 1 and 2. 50 g of glutinous rice powder was added to 5 times the weight of distilled water and vigorously stirred three times for 30 seconds using a vortex mixer. After stirring, the mixture was centrifuged at 3,000 × g for 10 minutes to obtain a supernatant, which was diluted to a protein concentration of 1 mg / ml, and used as a glutinous rice extract. The glutinous rice extract was allowed to stand at room temperature for 15 hours to form a precipitate, and then centrifuged at 2,000 × g for 10 minutes to obtain a clear supernatant, which was used as a soluble fraction of the glutinous rice extract.

[Example 4]: Preparation of barley extract and wheat extract and their soluble fractions 50 g each of barley and wheat flour was added to 5 times weight of distilled water and vigorously stirred three times for 30 seconds using a vortex mixer did. After stirring, the mixture was centrifuged at 3,000 × g for 10 minutes to obtain a supernatant. These supernatants were diluted to a protein concentration of 1 mg / ml and used as barley extract and wheat extract, respectively. If necessary, these extracts were centrifuged at 15,000 × g for 20 minutes, and the resulting clear supernatant was used as a soluble fraction of barley and wheat extracts.

[Example 5]: Preparation of soluble fraction of water extract Extract 25 g of water extract was added to 10 times weight of distilled water, and vigorously stirred 3 times for 30 seconds using a vortex mixer. After stirring, the mixture was centrifuged at 15,000 × g for 20 minutes, and the resulting clear supernatant was used as the soluble fraction of the water extract.

[Example 6]: Preparation of soluble fraction of tea extract 10g each of green tea (Amore Pacific, Korea, trade name: Hanra), black tea (Lipton, UK), and oolong tea (Fujian, China) It was extracted for 10 minutes in boiling water with 100 times weight and filtered. In this examination, this filtrate was used as it was or freeze-dried.

[Example 7]: Purification of rice prolamin 100 g of glutinous rice or glutinous rice powder was extracted with 300 ml of distilled water for 4 hours, and centrifuged at 2,000 xg for 10 minutes to remove the supernatant. The above extraction process was repeated once. The resulting precipitate was then extracted twice with 300 ml of 0.5M NaCl for 4 hours. The precipitate thus obtained was extracted with 300 ml of 2-propanol for 1 hour, and the extraction was repeated once. Two propanol extracts were collected and dried in a hood.

  The dried product was dissolved in 50 ml of 70% ethanol and filtered. The filtrate was then slowly added with 3 volumes of ice-cold acetone to form a protein precipitate and centrifuged at 2,000 × g for 20 minutes. The precipitate thus obtained was dissolved in 30 ml of 70% ethanol, filtered and added to 3 volumes of ice-cold acetone to form a protein precipitate. This process was repeated once. The obtained protein precipitate was dialyzed against distilled water for 24 hours. Finally, 62 mg of glutinous rice prolamin and 85 mg of glutinous rice prolamin were obtained.

Experimental example 1: Identification of anti-peptic ulcer substances present in rice extract
(1) Measurement of anti-peptic ulcer activity
Measurements were made with slight modifications to the method described in Yamasaki et al. (Eur. J. Pharmacol. 142, 23-29, 1987). Male rats (Sprague-Dawley) weighing 250 ± 20 g were orally administered with 1 ml of ethanol to induce gastric ulcer. To measure the activity of the anti-peptic ulcer preparation, 1 ml of the test preparation was orally administered 30 minutes before ethanol administration. The same amount of distilled water was administered to the control group. Rats were sacrificed under ether anesthesia 1.5 hours after ethanol administration, and the stomach was removed. 10% 1% formalin was injected into the stomach and fixed for 30 minutes. After formalin fixation, the stomach was incised along the large vagina and the inner surface was photographed with a digital camera. Thereafter, the area of damage to the gastric mucosa was measured planimetrically on the photograph and expressed as an ulcer index. This index is the ratio of the damaged site to the total area of the gland. The ulcer formation inhibition rate (%) of the test preparation was calculated as follows.

  The statistical significance of all data was verified by one-way analysis of variance (one-way ANOVA) and Scheffe test.

(2) Comparison of anti-peptic ulcer activity between rice extract and other cereal extracts To compare the anti-peptic ulcer activity of glutinous rice extract, glutinous rice extract, barley extract and wheat extract, respectively 1 ml of extract was orally administered to rats. After 30 minutes, 1 ml of ethanol was orally administered to rats to evaluate the degree of damage to the gastric mucosa. The result is shown in FIG. The number of rats in each group was 6, and the measured values were expressed as mean ± standard deviation. ^** p <0.01 is when compared with the control group.

  As shown in FIG. 1, the glutinous rice extract showed the strongest anti-peptic ulcer activity, and the glutinous rice extract also showed a significant anti-peptic ulcer activity although it was lower than the glutinous rice extract. However, neither the barley extract nor the wheat extract showed significant anti-peptic ulcer activity. Therefore, in the following experimental examples, glutinous rice extract was mainly used to identify anti-peptic ulcer substances.

(3) Distribution of anti-peptic ulcer activity in the precipitated fraction and soluble fraction of glutinous rice extract The first step to determine in which fraction the anti-peptic ulcer substance of glutinous rice extract is present As described above, the precipitate and soluble fraction of the glutinous rice extract prepared in Examples 1 and 2 were orally administered to rats (1 ml / rat). After 30 minutes, ethanol was orally administered (1 ml / rat), and the degree of damage to the gastric mucosa was evaluated. The result is shown in figure 2. The number of rats in each experimental group was 6, and the measured values were expressed as mean ± standard deviation. ^* P <0.05 and ^** p <0.01 are when compared with the distilled water group. ^†† p <0.01 is when compared to the glutinous rice extract group.

  As shown in FIG. 2, the average ulcer index of the control group administered with distilled water, the glutinous rice extract administration group, the glutinous rice extract precipitation fraction administration group and the glutinous rice extract soluble fraction administration group was 66.5 ± 10.4, respectively. 16.0 ± 8.9, 46.3 ± 10.7 and 61.5 ± 11.7%, and the ulcer formation inhibition rates of the latter three were 75.9%, 30.4% and 7.5%, respectively. From this result, it can be seen that the precipitate fraction showed considerable anti-peptic ulcer activity (about 40% of the glutinous rice extract), whereas the soluble fraction did not show significant anti-peptic ulcer activity. This result indicates that the main anti-peptic ulcerous substance is present in the precipitated fraction rather than the soluble fraction of glutinous rice extract. Furthermore, the main substance suggests that a soluble activating component is required for maximum activity.

(4) Enhancement of the anti-peptic ulcer activity of the glutinous rice extract fraction by various substances The soluble fraction of other cereals and natural foods also has the anti-peptic ulcer activity of the glutinous rice extract fraction. In order to investigate whether it can be enhanced, each of the soluble fractions of glutinous rice extract, barley extract, mizusane extract and tea extract prepared in Examples 3 to 6 was added to the glutinous rice extract precipitation fraction. Anti-peptic ulcer activity was measured before and after. For this experimental example, the glutinous rice extract precipitate fraction prepared in Example 2 was centrifuged to remove the supernatant. The precipitate was then mixed with each of the soluble fractions of the above extract and the mixture was administered to experimental rats.

  As shown in Table 1, not only cereal extracts such as glutinous rice and barley extract, but also all soluble fractions of sangu extract and tea (green tea, black tea and oolong tea) extract are significantly anti-digested by themselves. The anti-peptic ulcer activity of the glutinous rice extract precipitate fraction could be enhanced. Of these tested soluble fractions, tea extract was found to show the strongest enhancement effect.

  In order to identify the compounds contributing to the enhancing effects shown in Table 1, several candidate compounds were added to the glutinous rice extract precipitate fraction to measure changes in anti-peptic ulcer activity.

  As shown in Table 2, 10 mM (-)-epicatechin (EC), tannic acid and cysteine were found to significantly enhance the anti-peptic ulcer activity of the glutinous rice extract precipitate fraction. On the other hand, other amino acids, ascorbic acid, reduced glutathione, (−)-epigallocatechin gallate, phytic acid, gallic acid and caffeine did not show a significant enhancing effect at 10 mM.

(5) Effect of heat treatment and pepsin treatment on the anti-peptic ulcer activity of the glutinous rice extract fraction Fraction of the glutinous rice extract fraction with boiling water for 5 minutes or 0.1% in the presence of 1% lactic acid The mixture was reacted with mg / ml pepsin at 37 ° C for 1 hour. Thereafter, each treated product was mixed with 2 mg / ml green tea extract and administered to rats to measure anti-peptic ulcer activity. The result is shown in FIG. The number of rats in each group was 6, and the measured values were expressed as mean ± standard deviation. ^** p <0.01 is when compared with the distilled water group. ^†† p <0.01 is compared to the untreated group.

  As shown in FIG. 3, when the glutinous rice extract precipitate fraction was heat-treated or treated with pepsin, the anti-peptic ulcer activity of the fraction was completely lost. The activity was maintained only when the glutinous rice extract fraction was reacted with 1% lactic acid without pepsin. These results suggest that the substance exhibiting anti-peptic ulcer activity in the glutinous rice extract precipitate fraction may be a protein.

(6) Comparison of anti-digestible ulcer activity between globulin, glutelin and prolamin protein in glutinous rice extract precipitate fraction Any of globulin, glutelin and prolamin protein present in glutinous rice extract fraction is anti-digestible In order to examine whether ulcer activity was exhibited, the precipitate fractions were extracted with 0.5 M NaCl, 1% lactic acid and 70% ethanol for 4 hours, respectively, and centrifuged at 3,000 × g for 10 minutes. Thereafter, the anti-peptic ulcer activity was measured after dialyzing with distilled water for 24 hours for the solubilized subfraction, and the activity was measured by suspending the precipitate with distilled water for the residual sediment subfraction (FIG. 4). . To measure anti-peptic ulcer activity, 2 mg / ml green tea extract was added as an activator to the subfraction. The number of rats in each group was 6, and the measured values were expressed as mean ± standard deviation. ^* P <0.05 and ^** p <0.01 are when compared with the distilled water group. ^†† p <0.01 is when compared with the untreated precipitate fraction group.

  As shown in FIG. 4, most of the anti-peptic ulcer activity remained in the precipitate fraction after extraction with 0.5M NaCl or 1% lactic acid. However, extraction with 70% ethanol lost all anti-peptic ulcer activity. Instead, the solubilized subfraction of 70% ethanol extract showed strong anti-peptic ulcer activity.

  The above results strongly suggest that prolamin exhibits the anti-peptic ulcer activity of the glutinous rice extract precipitate fraction.

(7) Anti-peptic ulcer activity of purified glutinous rice prolamin To confirm the anti-peptic ulcer activity of glutinous rice prolamin, prolamin was purified from glutinous rice powder as in Example 7. Thereafter, the purified protein was first analyzed by SDS-PAGE according to the method of Laemmli (Nature 192, 680-682, 1970) to evaluate the purity. FIG. 5 shows the results of SDS-PAGE analysis performed by adding 10-30 μg of protein to a 15% polyacrylamide gel. In the analysis, a single polypeptide band having a molecular weight of about 13 KDa was detected. This result is consistent with previous reports on the characteristics of rice prolamin. Therefore, it is shown that the protein prepared from glutinous rice in Example 7 is a highly purified prolamin protein.

Next, the anti-peptic ulcer activity of glutinous rice prolamin was measured in a dose-dependent manner (FIG. 6). In this experiment, 10 mM epicatechin (EC) was added as an activator. The number of rats in each group was 6, and the measured values were expressed as mean ± standard deviation. ^* P <0.05 and ^** p <0.01 are compared with the control group administered with distilled water.

  As shown in FIG. 6, the anti-peptic ulcer activity continuously increased as the prolamin dose increased. At a dose of 0.3 mg / kg body weight, prolamin began to show significant anti-peptic ulcer activity, reaching a maximum when the dose was 3 mg / kg body weight.

(8) Anti-peptic ulcer activity of purified glutinous rice prolamin Since glutinous rice extract also showed significant anti-peptic ulcer activity (Fig. 1), the anti-digestibility of prolamin purified from glutinous rice in the same manner as (7) The ulcer activity was examined. SDS-PAGE analysis of a prolamin preparation obtained from glutinous rice detected a single polypeptide band of 13 KDa (FIG. 7), indicating that the prepared protein is highly purified prolamin.

  Purified glutinous rice prolamin exhibited a dose-dependent anti-peptic ulcer activity (FIG. 8). However, its specific activity was much lower than glutinous rice prolamin.

Claims (5)

  An anti-peptic ulcer composition comprising rice prolamin as an active ingredient.   At least one selected from the group consisting of tea extract, water extract, barley extract, (-)-epicatechin (EC), tannic acid and cysteine as an auxiliary component for promoting anti-peptic ulcer activity 2. The anti-peptic ulcer composition according to claim 1, further comprising:   3. The anti-peptic ulcer composition according to claim 2, comprising 1 to 80 parts by weight of the auxiliary component with respect to 1 part by weight of the rice prolamin.   2. The anti-peptic ulcer composition according to claim 1, wherein the rice is glutinous rice or glutinous rice.   2. The anti-peptic ulcer composition according to claim 1, which is formulated into a preparation for oral administration.