KR100963339B1 - Anti-peptic ulcer preparation comprising rice prolamin - Google Patents

Abstract

The present invention relates to an anti-extinguishing ulcer composition comprising rice prolamin (prolamin) as an active ingredient.

The anti-digestive ulcer composition of the present invention can be widely used because of high safety by including a protein of a commercial food as an active ingredient as well as having an excellent prophylactic or therapeutic effect in peptic ulcer.

Prolamin, Rice, Protein, Anti-Peptic Ulcer

Description

Antidigestive ulcer composition comprising rice prolamin {ANTI-PEPTIC ULCER PREPARATION COMPRISING RICE PROLAMIN}

The present invention provides an anti-digestive ulcer composition comprising rice prolamin as an active ingredient; And rice prolamin as an active ingredient, and further comprising at least one selected from the group consisting of tea extract, ginseng extract, barley extract, (-)-epicatechin (EC), tannic acid, and cysteine as activation promoters It relates to an antidigestive ulcer composition.

The main purpose of drug treatment for peptic ulcers is to relieve pain, heal the ulcer itself, and incidentally to prevent ulcer recurrences and complications. To this end, anti-ulcers currently used include antacids, H ₂ -receptor antagonists, hydrogen ion pump inhibitors, and anti-helicobacter pylori, but most of these drugs have various side effects.

On the other hand, the present inventors have reported that glutinous rice extract effectively suppresses the occurrence of gastric ulcers, has an excellent effect in healing the ulcers generated, as well as high safety for the living body as an extract of a commercial food (Ahn et al., Korea Patent Nos. 0208969, 1999; Ahn et al., Korean Patent Nos. 0253740, 2000; Ahn et al., Korean Patent No. 10-0627820, 2006). However, the above invention did not reveal the anti-ulcer active substance present in the glutinous rice extract.

The present inventors continued the study to identify the components having anti-digestive ulcer activity, and as a result, the present inventors completed the present invention by discovering that the prolamin protein contained in glutinous rice or non-glutinous rice is the main component showing such anti-digestive ulcer activity.

That is, an object of the present invention is to provide a composition having excellent anti-digestive ulcer activity.

In order to achieve the above object, the present invention provides an anti-extinguishing ulcer composition comprising rice prolamin as an active ingredient.

In addition, in the present invention, an anti-digestive ulcer composition containing such rice prolamin as an active ingredient tea extract, ginseng extract, barley extract, (-)-epicatechin (epicatechin), tannic acid (tannic acid) and cysteine (cysteine) It has been found that synergistic effects on anti-digestive ulcer activity can be obtained by further including one or more selected from the group consisting of The activity promoting auxiliary component is preferably included 1 to 80 parts by weight based on 1 part by weight of rice prolamin.

Rice prolamin, the active ingredient of the anti-digestive ulcer composition of the present invention, can be purified from glutinous or non-glutinous rice, and prolamin derivatives having a similar structure can be used.

The composition may be formulated in an oral dosage form and may also contain a pharmaceutically acceptable carrier.

The dose of the present composition is preferably administered 0.3 to 3mg of glutinous rice prolamin per Kg of body weight, preferably 1 to 10 times a day.

The composition comprising the rice prolamin of the present invention as an active ingredient has an excellent prophylactic or therapeutic effect in peptic ulcer.

Hereinafter, the present invention will be described in more detail with reference to the following examples and test examples. However, the following Examples and Test Examples are only for illustrating the present invention, but the scope of the present invention is not limited thereto.

Example  1: production of glutinous rice extract

Five times the weight of distilled water was added to 150 g of glutinous rice powder and shaken three times for 30 seconds using a blender. After shaking, the supernatant was obtained by centrifugation at 3,000 × g for 10 minutes, and the protein concentration of the obtained supernatant was measured using a BCA kit of Pierce, USA, and the result was 1.42 mg / ml. The supernatant was diluted with distilled water so that the protein concentration was 1 mg / ml, which was used as a glutinous rice extract.

Example  2: Preparation of Sedimentation Fraction and Soluble Fraction of Glutinous Rice Extract

500 ml of the glutinous rice extract prepared in Example 1 was left at room temperature for 15 hours to allow sufficient precipitation, followed by centrifugation at 2,000 × g for 10 minutes to separate the precipitate into a clear liquid portion, and the precipitate was suspended in 500 ml of distilled water. The glutinous rice extract was used as the precipitation fraction, and the clear liquid portion was used as the glutinous rice extract soluble fraction.

Example  3: Preparation of Non-rice Extract and its Soluble Fraction

Soluble fraction of non-rice extract was prepared in the same manner as in Examples 1 and 2. That is, 5 times weight of distilled water was added to 50 g of non-glutinous rice powder, and shaken three times for 30 seconds using a mixer, followed by centrifugation at 3,000 × g for 10 minutes to obtain a supernatant, which was diluted so that the protein concentration was 1 mg / ml. Used as an extract. After the rice extract was left at room temperature for 15 hours to sufficiently precipitate, centrifuged at 2,000 × g for 10 minutes, and the clear liquid portion of the upper layer was used as the soluble fraction of the rice extract.

Example  4: Preparation of Barley and Wheat Extracts and Their Soluble Fractions

Five times the weight of distilled water was added to 50 g of barley and wheat powder, respectively, and shaken three times for 30 seconds using a blender. After shaking, the supernatant was obtained by centrifugation at 3,000 × g for 10 minutes, and the protein concentration was diluted to 1 mg / ml, which was used as barley extract and wheat extract, respectively. A portion of the extract was centrifuged again at 15,000 × g for 20 minutes to separate the precipitate and the clear liquid portion, and the clear liquid portion was used as an soluble fraction of barley extract and wheat extract, respectively.

Example  5: soluble fraction of fresh ginseng extract

Distilled water of 10-fold weight was added to 25 g of fresh ginseng and shaken three times for 30 seconds using a blender. After shaking, centrifugation was carried out at 15,000 × g for 20 minutes to separate the precipitate and the clear liquid portion, and the clear liquid portion was used as a soluble fraction of fresh ginseng extract.

Example  6: Tea extract  Soluble fraction manufacturing

Green tea (Korean Pacific, trade name: Halla), black tea (made by Lipton, UK), and oolong tea (中國 福建 烏龍 茶) 10g each were put into 100-fold boiling distilled water, extracted for 10 minutes, and filtered. In subsequent experiments, the filtrate was used as it was, or freeze-dried to make a powder (about 2.2g of green tea, about 2.1g of black tea, about 2.0g of oolong tea).

Example  7: rice Prolamin  refine

100 g of glutinous rice and non-glutinous rice powder was extracted with 300 ml of distilled water for 4 hours, followed by centrifugation at 2,000 × g for 10 minutes to obtain a precipitate, and the distilled water extraction process was repeated once again. After distilled water extraction, the precipitate was extracted again with 300 ml of 0.5 M NaCl for 4 hours, and the same extraction process was repeated once more.

The precipitate obtained through the above procedure was extracted with 300 ml of 2-propanol for 1 hour, and the same extraction procedure was repeated once more. The 2-propanol extracts were combined, filtered and dried in the hood.

The dried material was sufficiently dissolved in 50 ml of 70% ethanol and filtered. Three times the volume of cold acetone was slowly added to the filtrate to allow sufficient protein precipitation and centrifugation at 2,000 × g for 20 minutes to obtain a precipitate. The precipitate was again dissolved in 30 ml of 70% ethanol and precipitated with three times the volume of cold acetone two more times, and the precipitate was dialyzed with distilled water for 24 hours. Through the above procedure, 62 mg and 85 mg of glutinous rice and non-glutinous prolamin were obtained, respectively.

Experimental Example  One: In rice extract  Present Antidigestibility  Identification of ulcer active substance 同 定 )

(One) Antidigestibility  How to Measure Ulcer Effects

Anti-inflammatory ulcer effects were measured using the method described by Yamasaki et al. (Eur. J. Pharmacol. 142, 23-29, 1987). That is, gastric ulcers were induced by oral administration of 1 ml of ethanol to a male rat (Sprague-Dawley) weighing 250 ± 20 g. To measure the activity of the antidigestive ulcer composition, 1 ml of the test composition was administered orally 30 minutes before ethanol administration. After 1.5 hours of ethanol administration, the stomach was excised under ether anesthesia, and 10 ml of 1% formalin was injected and fixed for 30 minutes. Distilled water was used as a control. After formalin fixation, the stomach was cut along the Taiwanese gland, the inner surface of the stomach was taken with a digital camera, and the area of the gastric mucosal injury was measured by planimetry on the enlarged photographic image. The degree of damage to the gastric mucosa was expressed by the gastric ulcer index, which is the percentage of the area of injury to the total area of the gastric mucosa. Gastric ulcer inhibition rate (%) of the anti-digestive ulcer composition was calculated by the following equation:

% Of gastric ulcer inhibition rate of antidigestive ulcer composition = [(gastric ulcer index of distilled water control group-gastric ulcer index of test group) / gastric ulcer index of distilled water control group] X 100%

Statistical significance verification of the measured results was performed according to one-way ANOVA and Scheffe test.

(2) Rice Extract  Other grain extracts Antidigestibility  Ulcer activity comparison

In order to compare the anti-digestive ulcer activity of the glutinous rice extract, non-rice extract, barley extract, and wheat extract prepared in Examples 1, 3 and 4, 1 ml of these extracts were orally administered to the rats of the experimental group, and after 30 minutes, ethanol 1 The amount of gastric ulcer was measured by oral administration to the rats, and the results are shown in FIG. 1. The number of rats in each experimental group were to 6, measured values were expressed as mean ± standard deviation, ^{** p <0.01} represents the significance of the difference for the control group distilled water.

As shown in FIG. 1, glutinous rice extract showed the strongest anti-digestive ulcer activity, and non-glutinous rice extract showed a pronounced anti-digestive ulcer activity, although lower than glutinous rice extract. However, barley extract and wheat extract did not show significant antidigestive ulcer activity.

(3) Precipitation and Soluble Fraction of Glutinous Rice Extract Antidigestibility  Ulcer activity distribution

The anti-inflammatory ulcer activity of glutinous rice extract was the most prominent, and in the following experiments, we tried to identify the anti-digestive ulcer active substances in the glutinous rice extract. First, in order to know whether the anti-digestive ulcer activity of the glutinous rice extract is present in the precipitate fraction and the soluble fraction, two portions of the glutinous rice extract prepared in Example 1 and the glutinous rice extract prepared in Example 2 were orally added to the rats of the experimental group. After 30 minutes, 1 ml of ethanol was orally administered to the rats, and the incidence of gastric ulcer was measured. Distilled water was used as a control. The results are shown in FIG. The number of rats in each experimental group was 6, and the mean value was expressed as the mean ± standard deviation. ^＊ p <0.05 and ^＊＊ p <0.01 represent the difference of the difference with respect to the distilled water control, respectively. ^†† p <0.01 is the glutinous rice. The significance of the difference with respect to the extract administration group is shown.

As shown in Figure 2, the average gastric ulcer index of the distilled water control group, glutinous rice extract group, sediment fraction fraction group, soluble fraction administration group is 66.5 ± 10.4, 16.0 ± 8.9, 46.3 ± 10.7 and 61.5 ± 11.7%, respectively, from three test groups Gastric ulcer inhibition rate was 75.9% 30.4% and 7.5%, respectively. In other words, the precipitate fraction showed about 40% anti-inflammatory ulcer activity of glutinous rice extract, whereas the soluble fraction did not show significant anti-digestive ulcer activity. The above results suggest that the main ingredient showing the anti-digestive ulcer activity of the glutinous rice extract is present in the sedimentation fraction, but does not show the maximum activity alone, and for that purpose, it is possible to require the assistance of the auxiliary ingredient present in the soluble fraction.

(4) Sedimentation fraction of glutinous rice extract Antidigestibility  Promotional Effects of Various Substances on Ulcer Activity

Non-rice, barley, ginseng and tea extracts prepared in Examples 3 to 6 to see if the soluble fraction of extract of other grains or natural foods can promote the anti-digestive ulcer activity of the glutinous rice precipitate fraction instead of the soluble fraction of glutinous rice extract. Soluble fraction of was added to sedimentation fraction of glutinous rice extract to measure anti-inflammatory ulcer activity. To this end, the glutinous rice extract precipitate prepared in Example 2 was centrifuged, the supernatant was discarded, the additive was added, and the precipitate was suspended and administered to rats.

As a result, as shown in Table 1, soluble fractions of grain extracts such as rice and barley, as well as soluble fractions of fresh ginseng extract and tea (green tea, black tea, oolong tea) extracts alone showed significant anti-inflammatory ulcer activity. Without promoting the anti-inflammatory ulcer activity of the sediment fraction of glutinous rice extract, the effect of tea extract was the most pronounced.

In order to determine which components in the tea extracts exhibited the above facilitating effect, anti-inflammatory ulcer activity was measured by adding a highly likely substance to the glutinous rice extract precipitation fraction.

As a result, as shown in Table 2, (-)-epicatechin (EC), tannic acid, and cysteine at concentrations of 10 mM each significantly promoted the anti-digestive ulcer activity of the glutinous rice extract precipitation fraction. On the other hand, ascorbic acid, reduced glutathione, (-)-epigallocatechin gallate (EGCG), phytic acid, gallic acid, caffeine, etc., including other amino acids, did not show a clear promoting effect at 10 mM concentration.

^{a The} displayed value is mean ± standard deviation

^＊ p <0.05, ＊＊ p <0.01: Significance of difference with distilled water control group

^† p <0.05, ^†† p <0.01: Significance of difference between glutinous rice extract and sediment fraction group

^{a The} displayed value is mean ± standard deviation

^＊ p <0.05, ＊＊ p <0.01: Significance of difference with distilled water control group

^† p <0.05: Significance of tea in glutinous rice extract sediment fraction administration group

(5) Sedimentation fraction of glutinous rice extract Antidigestibility  Effect of heat treatment and pepsin treatment on ulcer activity

The glutinous rice extract precipitate was heated in boiling water for 5 minutes, or reacted with 0.1 mg / ml pepsin for 1 hour at 37 ° C. in the presence of 1% lactic acid, and calibrated to pH 6.8 by adding a small amount of 5N NaOH to each sample. Green tea extract of ㎎ / ㎖ was added and administered to the rats to measure the anti-inflammatory ulcer activity, the results are shown in FIG. The number of rats in each experimental group was 6, and the measured value was expressed as mean ± standard deviation. ^＊＊ p <0.01 indicates the difference of the difference to the distilled water control group, and ^†† p <0.01 indicates the control group before heating or pepsin treatment. The significance of the difference for.

As shown in FIG. 3, as the glutinous rice extract precipitate fraction was heat-treated at high temperature or treated with pepsin, the anti-digestive ulcer activity of the precipitate fraction was completely lost. In the test composition in which the glutinous rice extract precipitate fraction was reacted with 1% lactic acid without pepsin for 1 hour and then the pH was neutralized, most of the anti-digestive ulcer activity was maintained. The above experimental results indicate that the anti-digestive ulcer active substance in the precipitate fraction is easily inactivated by heating and pepsin, which strongly suggests that the active substance is a protein.

(6) globulin in sediment fraction of glutinous rice extract, Glutelin  And Prolamin Antidigestibility  Ulcer activity comparison

In order to determine which of the globulin, glutelin and prolamin proteins in the glutinous rice extract precipitation fractions are the main constituents exhibiting anti-inflammatory ulcer activity, the precipitation fractions were respectively 0.5M NaCl, 1% lactic acid, 70 After extracting with 4% ethanol for 4 hours and centrifuged, the anti-inflammatory ulcer activity was measured in the extract and the precipitate fraction of the residue was carried out after dialysis in distilled water for 24 hours (Fig. 4). When measuring the anti-digestive ulcer activity, 2 mg / ml green tea extract was added to the test composition. The number of rats of each test group was 6, measured values were expressed as mean ± SD, ^{* p} <0.05 and ^{** p <0.01} represents the significance of the difference for each of the distilled water control group, ^†† p <0.01 was The difference between the precipitated fraction of glutinous rice extract and the green tea extract mixed group before extraction was significant.

As shown in FIG. 4, when extracted with 0.5 M NaCl or 1% lactic acid, most of the anti-extinguishing ulcer activity remains in the sediment fraction, while extracting with 70% ethanol elutes a large portion of the anti-digestive ulcer activity. Antidigestive ulcer activity did not remain apparent.

The above results strongly suggest the possibility that prolamin is an anti-digestive ulcer in the sediment fraction of glutinous rice extract.

(7) refined glutinous rice Prolamin Antidigestibility  Ulcer activity

In order to confirm the anti-digestive ulcer activity of glutinous rice prolamin, after purifying the prolamin from glutinous rice powder, the anti-digestive ulcer activity was measured. First, the protein purified from glutinous rice in Example 7 was subjected to electrophoresis (SDS-PAGE) according to the method of Laemmli (Nature 192, 680-682, 1970). FIG. 5 shows the results of electrophoresis performed by adding 10-30 µg of protein samples to 15% polyacrylamide gel. The purified sample showed a single polypeptide band with a molecular weight of about 13 KDa, which is in good agreement with the structural properties of rice prolamin. Therefore, it was confirmed that the sample prepared from glutinous rice in Example 7 was a highly purified prolamine protein.

The anti-digestive ulcer activity of the glutinous rice prolamin prepared in Example 7 was measured in a dose dependent manner (FIG. 6). At this time, 10mM EC was added as an activation promoter. The number of rats in each experimental group was 6, and the measured value was expressed as mean ± standard deviation, and ^* p <0.05 and ^** p <0.01 indicate the difference of difference with the distilled water control group.

As a result, the dose of prolamin showed significant anti-digestive ulcer activity when administered at 0.3 mg / kg body weight, and the anti-digestive ulcer activity increased with increasing dose, reaching a maximum of 3 mg / kg body weight. .

(8) Refined Rice Prolamin Antidigestibility  Ulcer activity

As shown in FIG. 1, since a considerable amount of anti-digestive ulcer activity was also detected in non-glutinous rice extract, we examined the activity by purifying prolamin from non-glutinous rice by the same method as glutinous rice prolamin. Electrophoresis (SDS-PAGE) of the purified prolamin sample from non-glutinous rice in Example 7 (Fig. 7) showed a major polypeptide band having a molecular weight of about 13 KDa, which produced a highly purified protein sample. Results indicate that it is a rollamine.

In addition, as a result of the dose-dependent measurement of the anti-digestive ulcer activity of the purified uncooked rice prolamin as shown in Fig. 6 (Fig. 8), it shows a significant activity from the dose of 3 mg per kg body weight of the animal and the dose is increased As a result, anti-inflammatory ulcer activity increased. However, as in Example 7, purified rice prolamin was much lower in specific activity than glutinous rice prolamin.

     1 is a result of comparing the anti-inflammatory ulcer activity of glutinous rice extract, non-rice extract, barley extract and wheat extract.

2 is a result of comparing the anti-inflammatory ulcer activity of the whole glutinous rice extract, the sediment fraction of the glutinous rice extract, and the soluble fraction of the glutinous rice extract.

Figure 3 shows the change of anti-digestive ulcer activity during the heat treatment of the precipitate fraction of glutinous rice extract at 100 ° C. for 5 minutes or pepsin treatment.

Figure 4 shows the anti-digestive ulcer activity distributed in the extract and the residue precipitate fraction when the precipitate fraction of glutinous rice extract was extracted with 0.5M NaCl, 1% lactic acid, and 70% ethanol, respectively.

5 is the result of electrophoresis (SDS-PAGE) of the purified prolamin from glutinous rice powder.

Figure 6 shows the dose-dependent anti-digestive ulcer activity of prolamine purified from glutinous rice powder.

Figure 7 is the result of electrophoresis (SDS-PAGE) of the purified prolamin from non-glutinous rice powder.

Figure 8 shows the dose-dependent anti-digestive ulcer activity of prolamine purified from non-rice powder.

Claims (5)

An anti-extinguishing ulcer composition comprising rice prolamin (prolamin) as an active ingredient. The method of claim 1, An anti-digestive ulcer composition further comprising at least one selected from the group consisting of tea extract, ginseng extract, barley extract, (-)-epicatechin (EC), tannic acid, and cysteine as an activation promoter. The method of claim 2, An anti-extinguishing ulcer composition comprising 1 to 80 parts by weight of an activation promoting ingredient per 1 part by weight of rice prolamin. The method of claim 1, An antidigestive ulcer composition wherein the rice is glutinous or non-glutinous. The method of claim 1, Anti-digestive ulcer composition, characterized in that it is formulated as a preparation for oral administration

Images