Background Art As Korean intakes the hot and salty food and the alcoholic beverage in excess, gastritis diseases have been occurred frequently. However, there has not been yet developed therapeutically effective treatment to protect gastritis diseases in Korea.

Gastric acid has been widely known as the cause of disease such as gastritis diseases and reflux esophagitis. Recently, various scientific studies on the gastritis diseases and pharmacological treatments of the disease have been reported so that the secret pharmacological effects are paid attention to modern scientific approaches (Nakamura K, et al; Jpn. J Pharmacol. 32 (3), pp445-56,1982 ; Okabe S, et al; Jpn. J. Pharmacol.

69 (4), pp317-23, 1995).

Rumex aqaticus herba belonging to polygonaceae is a perennial plant and grows naturally in far-eastern Asia region, Japan, Europe, northern America region, etc.

It has been reported that Rumex species plant contains various compounds, for example, 3-acetyl-2-methyl-1, 5-dihydroxy-2,3-epoxynaphthoquinol isolated from Rumex japonicus (Zee O. P., et al ; Arch Pharm. Res., Aug 21 (4), pp385-6,1998), 1-o- P-D-glucopyranosyl emodin isolated from Rumex gemlini Radix (Kang Y, et al; Chung Kuo Chung Yao Tsa Chih, Dec 21 (12), pp741-2,1996), quercetin-3-rhamnoside, champerol-3-rhamnopyranosyl (lof3-6) galactoside, quercetin-3-glycosyl (lu-4) galactoside and emodin isolated from Rumex chalepensis (Hasan A, et al; Phytochemistry, Jul. 39 (5), ppl211-3, 1995) and chrysophanic acid, emodin and nepodin (nepodin; 2-acetyl-1, 8-dihydroxy-3-methyl naphthalene) isolated from Rumex luminastrum (Abe el-Fattah H. , et al ; Acta Pharm Hung, May 64 (3), pp83-5,1994).

It has been known to be effective as insecticide and has been used to treat disease such as diarrhea, antifebrile, edema, jaundice and constipation.

Especially, the extract of its radix is effective to treat the symptoms of the womb bleeding, epistaxis, emesis, diarrhea, chronic enteritis, constipation, tonsillitis, inflammation, cancer, lucosis, cold, phthisis, cough, bronchitis, nephritis and etc.

Also the extract is efficient in alleviation of fever, regulation of body temperature, promotion of bile secretion, reduction of blood coagulation time by lowering capillary permeability, urination operation and protection of liver function.

However, there have been no disclosure or suggestion about therapeutic activity of quercetin-3-0-D-D-glucuronide against gastritis diseases and reflux esophagitis.

Disclosure of the invention Accordingly, it is an object of the present invention to provide a process for preparing quercetin-3-O-P-D-glucuronide (QGC) of the following Chemical Formula 1 isolated from the leaves of Rumex aquaticus herba.

[Chemical Formula 1] OH , AOH HOX O-Glucuronic acid OH O It is another object of the present invention to provide a pharmaceutical composition comprising a pharmaceutically effective amount of quercetin-3-0-P-D- glucuronide (QGC) with pharmaceutically acceptable carriers, excipients and diluents for prevention and treatment of gastritis diseases and reflux esophagitis.

It is another object of the present invention to provide use of QGC compound for preparation of medicament for preventing or treating gastritis diseases and reflux esophagitis.

It is another object of the present invention to provide a health food comprising above quercetin-3-0-D-D-glucuronide for the prevention of gastritis diseases and reflux esophagitis and sitologically acceptable additive.

In preferred embodiment of the present invention, there provided the process of preparing QGC compound from the leaves of Rumex aquaticus herba comprising the steps of; obtaining the crude extract of the leaves of Rumex aquaticus herba ; defatting the crude extract by using non-polar solvent; removing sugar and mineral salts from above defatted extract and obtaining flavonoid fraction by using non-ionic exchange column chromatography; and purifying the fraction by recrystallizing to obtain above compound.

Above described QGC compound can be prepared by following steps: 1. 1St step : the preparation of crude extract Specially, at the 1St step, Rumex aquaticus herba leaves is sliced and dried in the shade. Then, dried plant is mixed with one to thirty times, preferably, two to eight times volumes of water or Cl-C5 lower alcohol, or the mixture thereof; and is extracted at a temperature ranging from 40~60°C, preferably 50°C, for a period <BR> <BR> ranging from 1 to 24 hrs, preferably 3hrs, with 1 to 10 times, , preferably 6 times, to obtain an aqueous crude extract. The extract is centrifuged, filtered, concentrated under a reduced pressure and the crude extract is sealed and kept in refrigerator until use. Additionally, above concentrate can be lyopliilized to obtain an extract powder.

2. 2 step : defatting step The crude extract obtained from 1st step, is suspended in distilled water, separated to water-soluble layer and water-insoluble layer; mixed with non-polar organic solvent, such as chloroform, ether, methylene chloride, hexane, ethyl-acetate, and acetone to remove fatty component therefrom.

3. 3rd step: obtaining step of flavonoid containing fraction The defatted extract prepared in 2nd step, is subjected to removement of nonpolar organic solvent, absorption to column chromatography non-ionic resin, and elution

with water in order to eliminate sugar and mineral components, and consecutively, elution with methanol, 10 to 60% ethyl alcohol, or Cl-C5 lower alcohol or obtain flavonoid containing fraction.

4. 4th step : separation of QGC The flavonoid containing fraction obtained in 3rd step, is subjected to column chromatography and recrystallized to separate and obtain pure inventive QGC.

The present invention also provides a pharmaceutical composition comprising quercetin-3-0-P-D-glucuronide (QGC) prepared by above-mentioned process as an active ingredient for the prevention and treatment of gastritis diseases and reflux esophagitis.

The pharmaceutical composition of the present invention for the treatment of gastritis diseases and reflux esophagitis contains between 0.5 to 50% by weight of QGC compound based on the total weight of the composition.

It is still another object of the present invention to provide a method of treating a mammal or human afflicted with gastritis and reflux esophagitis comprising administering to a mammal or human an effective amount of said compound prepared by above process and pharmaceutically acceptable carrier thereof.

The inventive composition may additionally comprise conventional carrier, diluents in accordance with a using method. It is preferable that said carrier is used as appropriate substance according to the usage and application method, but it is not limited. Appropriate diluents are listed in the written text of Remington's Pharmaceutical Science (Mack Publishing co. , Easton PA).

Hereinafter, the following formulation methods and excipients are merely exemplary and in no way limit the invention.

The pharmaceutical composition of the present invention in pharmaceutically acceptable carrier, excipient and diluents may be contains lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, maltitol, starches, acacia rubber, alginate, gelatin, calcium

phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxy benzoate, propylhydroxybenzoate, magnesium stearate and mineral oil.

The formulations may additionally include fillers, anti-agglutinating agents, lubricating agents, wetting agents, flavoring agents, emulsifiers, preservatives and the like. The compositions of the invention may be formulated so as to provide quick, sustained or delayed release of the active ingredient after their administration to a patient by employing any of the procedures well known in the art.

The pharmaceutical composition of the present invention may be prepared in any form, such as oral dosage form (powder, tablet, capsule, aqueous medicine, syrup, granule, etc. ) or sterile injectible preparation (solution, suspension, emulsion).

The desirable dose of the inventive QGC compound varies depending on the condition and weigh of the subject, severity, drug form, route and period of administration, and may be chosen by those skilled in the art. However, in order to obtain desirable effects, it is generally recommended to administrate at the amount ranging 0.1 to 500mg/kg by weigh/day of the inventive extract or compound of the present invention. The dose may be administrated in single or divided into several times per day. However, it should be understood that the amount of the active ingredient actually is administrated ought to be determined in light of various relevant factors including the condition to be treated, the chosen route of administration, the age, sex and body weight of the individual patient, and the severity of the patient's symptom. And above administration is not limited to the present invention in any manner.

Examples of addable food comprising aforementioned compound of the present invention are various food, beverage, gum, vitamin complex, health improving food and the like.

Above described composition therein can be added to food, additive or beverage for prevention of gastritis diseases. For the purpose of preventing gastritis diseases, wherein, the amount of above described extract or compounds of the present invention in food or beverage may generally range from about 0.1 to 15 w/w %, preferably 1 to 10 w/w % of

total weight of food for the health food composition and 1 to 30 g, preferably 3 to 10 g per 100 mu the health beverage composition.

Providing that the health beverage composition of the present invention contains above described extract or compounds of the present invention as an essential component in the indicated ratio, there is no particular limitation on the other liquid component, wherein the other component can be various deodorant or natural carbohydrate, etc. such as conventional beverage. Examples of aforementioned natural carbohydrate are monosaccharide such as glucose, fructose, etc; disaccharide such as maltose, sucrose, etc.; conventional sugar such as dextrin and cyclodextrin ; and sugar alcohol such as xylitol, erythritol, etc. As the other deodorant than aforementioned ones, natural deodorant such as taumatin and stevia extract such as levaudioside A, glycyrrhizin <BR> <BR> et al. , and synthetic deodorant such as saccharin, aspartam et al. , may be useful favorably.

The amount of above described natural carbohydrate ratio is generally ranges from about 1 to 20 g, preferably 5 to 12 g per 100mE of present beverage composition.

To investigate the protective effect of inventive composition on the reflux esophagitis, we induced reflux esophagitis to the mouse model and confirmed that inventive compound significantly and dose-dependently prevented the development of reflux esophagitis and gastritis, and the lipid peroxidation of cellular membrane and furthermore, decreased the gastric volume and acid output.

The present invention is more specifically explained by the following examples.

However, it should be understood that the present invention is not limited to these examples in any manner.

Brief Description of the Drawings The above and other objects, features and other advantages of the present invention will more clearly understood from the following detailed description taken in conjunction with the accompanying drawing, in which; Fig. 1 shows the process for preparing QGC isolated from Rumex aquaticus herba

leaves, Fig. 2 shows the effects of quercetin and QGC on reflux esophagitis induced surgically in rats, Fig. 3 shows the effects of quercetin and QGC on gastritis diseases induced surgically in rats, Fig. 4 shows the administration effects of quercetin and QGC on gastric volume of gastric contents for reflux esophagitis, Fig. 5 shows the administration effects of quercetin and QGC on gastric acid output of gastric contents for reflux esophagitis, Fig. 6 shows the value of malondialdehyde transformed into lipid peroxidation level by using esophagitis mucosa obtained from reflux esophagitis group after administration of quercetin and QGC.

Best Mode for Carrying Out the Invention The following Example and Experimental Examples are intended to further illustrate the present invention without limiting its scope.

Example 1. Preparation of QGC (1) 4 kg leaves of Rumex aquaticus herba (medical herb garden, Chungang Univ., Korea) was mixed with 12 liter of 80 % methanol and was subjected to reflux extraction for 3 hrs at 40 ~ 60°C to obtain a crude extract thereof.

After evaporating the solvent with rotary vacuum evaporator (Eyela, FD-5N, Japan), the obtained 120g of the concentrated extract was dissolved, suspended with 500 mE of distilled water, filtered with filter paper and then divided into water-soluble and water insoluble layer.

And then the water insoluble layer was removed and water soluble layer was defatted with 3 liter of chloroform.

After water soluble layer was concentrated under reduced pressure to eliminate chloroform, it was subjected to adsorption process with non-ionic resin for column chromatography (XAD-2) and followed by eluting with 400ml of distilled water to remove sugar and mineral salts therefrom. It was eluted with increasing volume of

MeOH such as 20,40, 60,80% and 100% methanol successively.

Finally, 6 g of flavonoid-containing fraction isolated from 40% methanol elution fraction was obtained.

The fraction was recrystalized to obtain 2.1 g of pure QGC, which was identified and confirmed as quercetin-3-O-p-D-glucuronide having molecular weight of 654 by the result of instrumental analysis as. followings (Fig. 1); mp : 193-195°C (-) FAB MS (m/z) : 477 [M-H]-, 301 [M- (GIcUA=H)]- 'H-NMR (300MHz, DMSO-d6); 7.59 (1H, dd, J 2. 4,6. 3Hz, H-6'), 7.51 (1H, s, H-2'), 6. 82 (1H, d, J 2. 4,8. 4Hz, H-5'), 6.39 (1H, d, J=2. 1Hz, H-8), 6.19 (1H, d, J=2. 1Hz, H-6), 5.48 (1H, d, J=7.2Hz, GluUAH) 3C-NMR (75MHz, DMSO-d6); 156.4 (C-2), 133.3 (C-3), 177.4 (C-4), 161.4 (C-5), 98.9 (C-6), 164.5 (C-7), 93.7 (C-8), 156.5 (C-9), 104.0 (C-10), 121.0 (C-1'), 115.3 (C-2'), 145.1 (C-3'), 148.8 (C-4'), 116.2 (C-5'), 121.9 (C-6'), 101.2 (GluUA : C-1), 73.9 (GluUA : C-2), 76.1 (GluUA : C-3), 71.4 (GluUA : C-4), 75.9 (GluUA : C-5), 170.0 (GluUA : C-6) Example 2. Preparation of QGC (2) 4 kg the leaves of Rumex aquaticus herba leaves was mixed with 12 liter of 80% ethanol and the crude extract was prepared, processed and purified according to the identical method of Example 1 except using other non-ionic resin (Diaion HP-20) to separate inventive compound QGC.

Finally, quercetin-3-O-ß-D-glucuronide of molecular weight 654 was obtained and identified by instrumental analysis as shown in Example 1.

Experimental Example 1. Effect of QGC in reflux esophagitis in rat To confirm protective effect of quercetin-3-O-p-D-glucuropyranoside (QGC) on

the reflux esophagitis, we have adopted the evaluation methods to check the effect on reflux esophagitis disclosed in the literatures (Nakamura K, et al; Jpn. J. Pharmacol., 32 (3), pp445-56,1982 : Okabe S, et al; Jpn. J. Pharmacol., 69 (4), pp317-23, 1995).

Male Sprague-Dawley rats (Hanlim, Korea) weighing about 200 g of a body weight were used for the experiments. The rats were starved for 24 hrs before the experiments, but were freely accessed to drinking water. All animals were kept in raised mesh-bottom cages to prevent coprophagy. Under ether anesthesia, the abdomen was incised along the midline and then both the pylorus and limiting ridge (transitional region between the forestomach and corpus) were simultaneously ligated. A longitudinal cardiomyotomy of about 1 cm length across the gastroesophageal junction was performed to enhance reflux.

QGC prepared in Example 1 and quercetin (Q 0125, Sigma) dissolved in 0.01% <BR> <BR> dimethyl sulfoxide were administered intraduodenally (i. d. ) at the amount of lOmg/kg immediately after ligation of the pylorus and limiting ridge. The volume of the drug or vehicle was 1 ml/kg of body weight. The drugs were prepared freshly each time. Six hours later, the animals were killed by cervical dislocation and then the esophagus was harvested and collected gastric juice.

In the experiment of reflux esophagitis, severe ulcerations were observed in the esophagus of control group, while both QGC (lOmg/kg) and quercetin (lOmg/kg) treated groups decreased the development of reflux esophagitis significantly (Fig. 2).

The level of ulcer index shows that QGC has more potent efficacy than quercetin, 2.1 and 3.9, respectively. QGC and quercetin reduced a significant increase in index of gastric ulceration, 3.8 and 4.1, respectively, induced by exposure of the gastric mucosa to indomethacin (0, no visible lesions; 1, a few erosions; 2, total area of lesions 30 mm2 ; 3, total area of lesions 30 mm2 ; 4, perforation, Data are mean i SEM.

Significant differences from the corresponding control, respectively *P<0.05).

Experimental Example 2. Effect of QGC in gatiritis in rats Male Sprague-Dawley rats (Hanlim, Korea) weighing a body weight of about

200 g were used for the experiments. The rats were starved for 24 hrs before the experiments, but were freely allowed to drinking water. All animals were kept in raised mesh-bottom cages to prevent coprophagy. QGC and quercetin were administered per <BR> <BR> os (p. o. ) one hour before indomethacin (50 mg/kg, I7378, Sigma) administration. The volume of the drug or vehicle was 2 ml/kg of body weight. The drugs were prepared freshly each time. After five hour, the animals were sacrificed and then their stomachs were excised, fixed with 3% of formalin. The stomachs were opened along the greater curvature and spread out with pins on a cork board. The area (mm2) of mucosal erosive lesions was measured under a dissecting microscope with a squared grid (X10 ; Olympus, Tokyo, Japan).

As shown in Fig. 3, in gastritis, quercetin and QGC administered i. d. (0.1 mg/kg) groups showed the gastric lesion of ulceration, 4.8 cm2 and 0.9 cm2, respectively.

QGC significantly reduced an increase in gastric lesion of ulceration (6.0 cm2) induced by exposure of the gastric mucosa to indomethacin. (Data are mean i SEM.

Significant differences from the corresponding control, quercetin, *P<0.05, ***P<0. 001, respectively) Quercetin administered i. d. (3mg/kg) group decreased the gastric volume (1. 8mu), comparing with 2. 1mQ of that of control group (indometacin treated group (Fig. 4).

QGC administered i. d. (3mg/kg) group decreased the gastric volume (0. 6m) comparing with the normal group (1. 6mu).

QGC significantly decreased the gastric volume and this effect decrease the ulceration of gastric and esophageal mucosa (Data are mean SEM. Significant differences from the corresponding control denotes respectively *P<0.05).

Administration of quercetin (lmg/kg) and QGC (lmg/kg) in dose-dependent <BR> <BR> manner decreased acid output, i. e. , the levels of acidity were 0.97mEq/hr and 0. 45mEq/hr, respectively (Fig. 5). This result showed reducing effect of QGC comparing with that of control (1. lmEq/hr) administered with indomethacin.

From these results, it is suggested that the inhibitory effects of QGC on the reflux esophagitis and gastritis on rats (Data are mean SEM. Significant differences from

the corresponding control denotes respectively. *P<0.05) Experimental Example 3. Effect of QGC in lipid peroxidation of cellular membrane To detect lipid peroxidation, esophageal mucosa was harvested, sonicated in 1 ml of Tris-HCl buffer (pH 7.0). After centrifugation at 600g for 10 min at 4°C (Micro 17TR, Hanil, Korea), 0.9 ml of trichloroacetic acid (8%) was added to 0.3 ml of supernatant. After centrifugation at 10, 000g for 5 min at 4°C, 0.25 ml of thiobarbituric acid (TBA, 1%) was added to 1 ml of supernatant and the resulting solution was heated at 100°C for 20 min. The tubes were cooled, 2 ml of n-butanol was added and each tubes was vortexed for 90 sec. After centrifugation at 3, 000g for 5 min at 4°C, 1 ml of butanol phase was utilized for TBARS assay at 532 nm (UV- 160A, Shimadzu, Japan) against malonaldehydebis (dimethyl acetal) standards.

Results were expressed as ng/mg protein. Protein assay was determined according to the Bradford method.

Lipid peroxidation of cellular membrane is the result of cell damage by free radicals. Malondialdehyde (MDA), the end product of lipid peroxidation, was reacted with TBA to show fluorescence and that is a sensitive marker of oxidative stress (Buege JA, Aust SD. Microsomal lipid peroxidation. Methods in Enzymol. 52, pp302- 310,1978).

As to reflux esophagitis, quercetin and QGC administered i. d. (0. 1 mg/kg) groups showed 3.4 mol/kg and 2. 7mol/kg of MDA level, respectively, comparing with that of control group (3. 1mol/mg). QGC administered group significantly decreased the amounts of MDA formation, which shows the inhibition of reflux esophagitis- associated lipid peroxidations comparing with quercetin administration group (Fig. 6).

This result suggests that QGC has an antioxidative effect.

Finally, this study demonstrates that QGC showed potent efficacy on the development of reflux esophagitis and indomethacin-induced gastritis by the inhibition of gastric secretion and the prevention of oxidative stress. The QGC is more potent

than quercetin, one of anti-inflammatory drug.

Preparation of powder Dried powder of Example 1 50mg Lactose 100mg Talc lOmg Powder preparation was prepared by mixing above components and filling sealed package.

Preparation of tablet Dried powder of Example 1 50mg Corn Starch 100mg Lactose 100mg Magnesium Stearate 2mg Tablet preparation was prepared by mixing above components and entabletting.

Preparation of capsule Dried powder of Example 1 50mg Corn Starch 100mg Lactose 100mg Magnesium Stearate 2mg Tablet preparation was prepared by mixing above components and filling gelatin capsule by conventional gelatin preparation method.

Preparation of injection Dried powder of Example 1 50mg Distilled water for injection optimum amount PH controller optimum amount Injection preparation was prepared by dissolving active component, controlling pH to about 7.5 and then filling all the components in 2mQ ample and sterilizing by conventional injection preparation method.

Preparation of liquid Dried powder of Example 1 0. 1-8 Og Sugar 5~10g Citric acid 0. 05-0. 3% Caramel 0. 005-0. 02%

Vitamin C 0. 1-1% Distilled water 79-94% C02 gas 0. 5-0. 82% Liquid preparation was prepared by dissolving active component, filling all the components and sterilizing by conventional liquid preparation method.

The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the present invention, and all such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.

Industrial Applicability The pharmaceutical composition comprising inventive quercetin-3-0-P-D- glucuronide (QGC) isolated from Rumex aquaticus herba leaves may be promising drug for the prevention and treatment of gastritis diseases and reflux esophagitis.