KR20030091404A - Process for Preparing Quercetin-7-O-β-D-glucuronide (QGC) isolated from Rumex Aquaticus Herba and Composition comprising the compound for the prevention and treatment of gastritis diseases and reversal esophagitis - Google Patents

Abstract

PURPOSE: Provided are a process for preparing Quercetin-7-O-β-D-glucuronide (QGC) isolated from Rumex aquaticus herba and a composition comprising the same for the prevention and treatment of gastritis diseases and reversal esophagitis. The separated QGC has excellent therapeutic effect on gastritis and reversal esophagitis. CONSTITUTION: A process for separating Quercetin-7-O-β-D-glucuronide(QAC) from Rumex aquaticus herba is characterized by the steps of: defatting a water or lower alcohol extract of Rumex aquaticus using a nonpolar solvent, such as ether or methylenechloride; removing saccharide and inorganic salt using nonionic exchange resin; and performing recrystallization of 40% fractions.

Description

Method for isolating quercetin-7-o-beta-di-glucuronide isolated from rhubarb and composition for the prevention and treatment of gastritis and reflux esophagitis diseases containing the compound {Process for Preparing Quercetin-7-O- β-D-glucuronide (QGC) isolated from Rumex Aquaticus Herba and Composition comprising the compound for the prevention and treatment of gastritis diseases and reversal esophagitis}

The present invention relates to pharmaceutical compositions and dietary supplements effective for diseases of the gastrointestinal tract.

Koreans develop gastritis caused by excessive consumption of spicy and salty foods and alcoholic beverages, and the high incidence of vomiting due to the excessive intake of irritating foods and alcohols is also likely to cause reflux esophagitis. In Korea, however, basic research and development of therapeutic drugs are poor. Gastric acid has long been recognized as a major attacking factor for various gastric acid-related diseases such as peptic ulcer and reflux esophagitis, and therapeutic drugs have been developed with gastric acid eradication as the main target for treating ulcers. This has been required. (Nakamura K, et al; Jpn. J. Pharmacol. 32 (3), pp 445-56, 1982; Okabe S, et al; Jpn. J. Pharmacol. 69 (4), pp 317-23, 1995)

Ground rhubarb (Rumex aquaticusHerba) is a perennial herb of the genusaceae, and it is distributed in Korea (Gyeongbuk, Hamnam), Japan, Europe, and North America. Known constituents of the Rumex species A bullshit(Rumex japonicus)3-acetyl-2-methyl-1, 5-dihydroxy-2, 3-epoxynaphthoquinol (Zee OP, et al;Arch. Pharm. Res. Aug21(4), pp 385-6, 1998)Rumex gmelini)1-O-β-D-glucopyranosyl emodine (Kang Y, et al;Chung Kuo Chung Yao Tsa Chih, Dec21(12) pp741-2, 1996), the same plant (Rumex chalepensis)Quercetin-3-lamnoside, camphorol-3-lamnopyranosyl (1β6) galactosid, quercetin-3-glycosyl (1β4) galactosid, emodine (Hasan A, et al;Phytochemistry, Jul.39(5) pp1211-3, 1995), the same plant (Rumex luminastrumChrysophan acid, emodin, nepodin (2-acetyl-1, 8-dihydroxy-3-methyl nahthalene) (Abe el-Fattah H., et al;Acta pharm hung, May64(3), pp83-5, 1994), and also contains a strong blood sugar component. It is used as a medicine for insecticides, diarrhea, fever, edema, jaundice, constipation, and postpartum pain in oriental medicine, especially as a substitute for rhubarb. Rooted water also helps to stop bleeding of the uterus and nosebleeds, and it is also effective for vomiting, diarrhea, acute enteritis, constipation, lymphadenitis, tonsillitis, etc. It can also have a significant therapeutic effect on myeloid or lymphoid leukemia. In addition, the effect of lowering the fever and sweating, cold, pulmonary tuberculosis, cough, bronchitis, nephritis is also effective. It has a lowering action, antipyretic and body temperature lowering action, promotes the secretion of bile, and decreases permeability of capillaries, thereby shortening blood coagulation time. Extensive antibacterial action, staphylococcus aureus, gonococcus, diphtheria bacteria, typhoid bacteria, inhibits the development of dysentery. In addition, diuretic and hepatic function-protecting effects are shown.

However, none of these documents discloses that quercetin-7-O-β-D-glucuronide (QGC) is effective for peptic ulcer and reflux esophagitis.

It is an object of the present invention to separate quercetin-7-O-β-D-glucuronide from isolated rhubarb leaves effective for the treatment and prevention of gastritis and reflux esophagitis diseases. It is to provide a pharmaceutical composition and dietary supplement containing glucuronide.

1 is a separation process for separating QGC from rhubarb,

2 is a graph showing the inhibitory effect on reflux esophagitis of rats of quercetin and QGC,

3 is a graph showing the inhibitory effect on quercetin and QGC in gastritis of rats,

4 is a graph showing changes in gastric juice amount for reflux esophagitis of quercetin and QGC,

5 is a graph showing the change in acid output of gastric juice for reflux esophagitis of quercetin and QGC,

6 is a graph showing the degree of lipid peroxidation as malondialdehyde value using esophageal mucosa obtained from reflux esophagitis group after administration of quercetin and QGC.

In accordance with the above object, the present invention provides a method for separating quercetin-7-O-β-D-glucuronide (QGC) of the general formula (1) from the leaves of Rumex Aquaticus Herba.

The QGC compound of the present invention is degreasing the water or the lower alcohol soluble extract of the rhubarb leaf by using a nonpolar solvent such as chloroform, ether or methylene chloride, and removing sugar and inorganic salt by using a nonionic exchange resin and recrystallization. It can be separated purely.

Hereinafter, specifically describing the separation process of the QGC compound,

A first step of extracting the ground rhubarb leaves by hydrothermal extraction with a lower alcohol solvent such as water, methanol or ethanol, and a mixed solvent thereof and concentrating under reduced pressure to obtain X;

A second step of dissolving the X in distilled water, suspending and dividing the filtrate into an insoluble part and a soluble part, and then degreasing the water soluble part with a nonpolar organic solvent such as chloroform, ether or methylene chloride;

A third step of removing the non-polar organic solvent such as chloroform, ether or methylene chloride from the water-soluble part degreased above and then adsorbing the non-ionic resin for column chromatography;

A fourth step of removing the sugar and inorganic salts eluted with distilled water and eluting with 10-60% ethyl alcohol or methyl alcohol to obtain a flavonoid-containing fraction;

This flavonoid containing fraction can be obtained through a series of separation steps that recrystallize to separate pure QGC.

The present invention also provides a pharmaceutical composition for the treatment of gastritis and reflux esophagitis comprising quercetin-7-O-β-D-glucuronide as an active ingredient.

The composition for the treatment of gastritis and reflux esophagitis of the present invention comprises the QGC compound in an amount of about 0.5 to 50% by weight based on the total weight of the composition.

Compositions comprising the QGCs of the invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

Carriers, excipients and diluents that may be included in the composition comprising the QGC of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium Phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The composition comprising the QGC according to the present invention may be formulated in the form of powders, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories, and sterile injectable solutions, respectively, according to a conventional method. Can be used.

The amount of the QGC compound may vary depending on the age, sex, and weight of the patient, but may be administered once or in several divided doses of 0.1 to 500 mg / kg body weight per day. The dosage of QGC may be increased or decreased depending on the route of administration, degree of disease, sex, weight, age, and the like. Therefore, the above dosage does not limit the scope of the present invention in any aspect.

The composition containing the QGC compound according to the present invention can be used in various forms such as pharmaceuticals, foods and beverages for the prevention or treatment of peptic ulcer and reflux esophagitis. Examples of the food to which the QGC compound can be added include various foods, beverages, gums, teas, vitamin complexes, and health supplements.

In addition, the QGC compounds according to the present invention can be added to food or beverages for the purpose of preventing peptic ulcer and reflux esophagitis. At this time, the amount of the compound in the food or beverage, in the case of the general health food composition of the present invention can be added to 0.1 to 15% by weight, preferably 1 to 10% by weight of the total food weight, It can be added in the ratio of 1-30 g, Preferably it is 3-10 g based on 100 ml.

The health beverage composition of the present invention has no particular limitation on the liquid component except for containing the above-mentioned QGC compound as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates, etc. as additional ingredients, as in general beverages. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.), and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The ratio of said natural carbohydrate is generally about 1-20 g, preferably about 5-12 g per 100 ml of the composition of the present invention.

In addition to the above, the composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and its Salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. The compositions of the present invention may also contain pulp for the production of natural fruit juices and fruit juice beverages and vegetable beverages. These components can be used independently or in combination. The proportion of such additives is not so critical but is generally selected from the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

The effects of the composition of the present invention on the prevention and treatment of peptic ulcer and reflux esophagitis were tested by applying a model in which the reflux esophagitis was surgically induced in rats to evaluate the effect in the reflux esophagitis model. The inhibitory effect on the lipid peroxide of the cell membrane was verified.

The present invention is described in more detail based on the following examples, although the present invention is not limited thereto.

Example 1. Preparation of QGC

Extract the extract obtained by adding 80% methanol of three times the volume to 4 kg of fresh rhubarb collected from Chung-Ang University herbal medicine and reflux heating at a temperature of 40-60 ° C. with an evaporator (Eyela, FD-5N Japan). Concentrated under reduced pressure to obtain 120 g of concentrated X, the obtained X was dissolved in 500 ml of distilled water, suspended, filtered with filter paper, divided into water insoluble and water soluble layers, and then the water insoluble layer was removed and the water soluble layer was 30OO. It was degreased with ml of chloroform.

After removing the chloroform solvent by depressurizing condenser with decompression concentrator, it was adsorbed with nonionic resin for column chromatography (XAD-2), eluted with 4000 ml of distilled water to remove sugars and inorganic salts. Elution was carried out sequentially with 40% methanol, 60% methanol, 80% methanol and 100% methanol to obtain 6 g of flavonoid-containing fractions eluted with 40% methanol. (See Figure 1)

6 g of the flavonoid-containing fractions were recrystallized to isolate 2.1 g of pure QGC. Confirmed.

mp: 193-195 ° C

(-) FAB MS ( m / z ): 477 [M−H] ⁻ , 301 [M− (GlcUA = H)] ⁻

¹ H-NMR (300 MHz, DMSO- d ₆ );

7.59 (1H, dd, J = 2.4, 6.3 Hz, H-6 '), 7.51 (1H, s, H-2'),

6.82 (1H, d, J = 2.4, 8.4 Hz, H-5 '), 6.39 (1H, d, J = 2.1 Hz, H-8),

6.19 (1H, d, J = 2.1 μs, H-6), 5.48 (1H, d, J = 7.2 μs, GluUA H)

¹³ C-NMR (75 MHz, DMSO- d ₆ );

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

To 4 kg of rhubarb, three times of 80% ethanol was added, concentrated and suspended under reduced pressure and filtered in the same manner as in Example 1, and then divided into a water insoluble layer and a water soluble layer, and then the water insoluble layer was removed. The soluble layer was degreased with 30OO ml of chloroform.

After removing the chloroform solvent by depressurizing condenser with decompression condenser, it was adsorbed with nonionic resin (Diaion HP-20) for column chromatography, eluted with 4000 ml of distilled water to remove sugar and inorganic salt. Elution was carried out sequentially with ethanol, 40% ethanol, 60% ethanol, 80% ethanol, 100% ethanol to obtain 6 g of flavonoid-containing fractions eluted with 40% ethanol.

The same instrumental analysis experiment as in Example 1 was carried out to identify and confirm that 6 g of the flavonoid-containing fraction, which was recrystallized, was quercetin-7-O-β-D-glucuronide (QGC) having a molecular weight of 654.

Experimental Example 1. Drug efficacy test for gastritis and reflux esophagitis of QGC

To confirm the therapeutic effect of QGC on reflux esophagitis, Nakamura K, et al; Jpn. J. Pharmacol., 32 (3), pp 445-56, 1982: Okabe S, et al; Jpn. J. Pharmacol., 69 (4), pp 317-23, 1995) was used in this experiment by measuring the treatment effect for reflux esophagitis.

As a method for the treatment of reflux esophagitis for this substance, 5 week old Spraugue-Dawley (SD) male rats (Harlims) were fasted for 24 hours, and water was supplied in a sufficient amount. After measuring the weight, the abdomen was ligated, and the upper part of the stomach was ligated, and an injured sphincter exercise was abnormally caused by giving a 1 cm length of wound to the contact portion forming the boundary between the esophagus and the stomach.

The pyloric region, which borders the stomach and duodenum, was also ligated so that the stomach acid flowed back into the esophagus without moving to the duodenum. Quercetin (Q0125, Sigma) and the QGC obtained in Example 1 were injected in the duodenum in the gastric direction and sutured after 6 hours. The drug was injected into the stomach in the duodenum and sutured, and after 6 hours, the cervical spine was degenerated, the esophagus was extracted, and the gastric juice was collected to observe the degree of esophagitis and gastric juice was measured.

Five-week-old Spraugue-Dawley (SD) male rats were fasted for 24 hours and water was supplied in sufficient amounts. After measuring the body weight, the test substance was orally administered first, and 1 hour later, oral administration of indomethacin (I7378, Sigma) at a concentration of 50 mg / kg, and after 5 hours, the cervical spinal bone was removed and the stomach was extracted and 3% formalin ( After fixation with formalin, the length of gastritis was measured.

Esophageal mucosa is taken to determine the degree of peroxidation, and then ultrasonically pulverized in 1 ml of Tris-HCl buffer solution (pH 7.0). After centrifugation at 600 x g and 4 DEG C for 5 minutes, 0.9 ml of trichloroacetic acid (8%) is added to 0.3 ml of the supernatant. After centrifugation at 10,000 × g and 5 ° C. for 5 minutes, 0.25 ml of TBA (1%) was added to 1 ml of the supernatant, the tube was cooled, 2 ml of n -butanol was added and 3,000 × g at 5 ° C. for 5 minutes. After centrifugation, the absorbance was measured at 532 nm with 1 ml of a butanol separation layer. The standard uses malonaldehyde bis-dimethyl acetal. The results were expressed in pmol / mg protein and protein quantitative analysis was carried out by the Bradford method.

As shown in FIG. 2, quercetin (10 mg / kg) showed about 3.9 and QGC (10 mg / kg) showed about 2.1, indicating that quercetin causes gastritis for reflux esophagitis. There was a slight decrease in ulceration index (4.1) from the untreated group containing only the kidneys, and QGC showed a significantly lower ulceration index than that of the normal group (3.8). (Ulcer index 0 indicates a state without ulcers, 1 indicates a state with a slight ulcer size, 2 indicates a state with an ulcer size of 30 mm 2 or less, 3 indicates a state with an ulcer size of 30 mm 2 or more, 4 means complete ulceration of the mucosa and * P <0.05 for untreated group)

In addition, as shown in FIG. 3, in gastritis, quercetin and QGC showed gastric mucosal ulcer areas of 4.8 and 0.9 cm 2 at constant dose (0.1 mg / kg), which is indomethaced to gastric mucosal ulcers. Quercetin was slightly decreased compared to the gastric mucosal ulcer area (approximately 6.0) of the untreated group, and the QGC had a significantly reduced gastric mucosal protection (* P <0.05, for the untreated group). *** P <0.001 for quercetin

In addition, as shown in FIG. 4, in gastritis, quercetin showed 1.8 ml in gastric acid secretion at a constant concentration (3 mg / kg) compared to the amount of gastric juice (2.1) of the untreated group containing only indomethacin. , QGC was 0.6ml compared with normal gastric juice (1.6) at constant dose (3mg / kg). It was confirmed that QGC had a strong gastric acid inhibitory activity (* P <0.05 for untreated group, respectively).

In addition, as shown in FIG. 5, in the total acid discharge (mEq / hour) in the stomach, quercetin (1 mg / kg) and QGC (1 mg / kg) were 0.97 mEq / hour and 0.45 mEq / hour, respectively. It was found that the quercetin had a lesser effect than the total acid emissions of the untreated group (approximately 1.1 mEq / hour) and that QGC had a much reduced acid emission (* P <0.05 for normal groups, respectively).

Figure 6 shows the degree of peroxidation of lipids in terms of malondialdehyde, membrane lipid peroxidation occurs as free radicals cause cell damage. Malondialdehyde, the final product of lipid peroxidation, specifically reacts with TBA reagent to fluoresce. And become a sensitive marker of the degree of peroxidation (Buege JA, Aust SD. Microsomal lipid peroxidation.Methods in Enzymol. 52, pp302-310, 1978). For reflux esophagitis, quercetin (3 mg / kg) and QGC (mg / kg) showed 3.4 mol / mg and 2.7 mol / mg, respectively, compared to the malondialdehyde value (3.1 mol / mg) of the normal group. It was confirmed that QGC showed much lower concentration than malondialdehyde (MDA) level in reflux esophagitis.

In conclusion, QGC has been shown to be effective in treating gastric ulcer and reflux esophagitis, and it can be reconfirmed that QGC compound combined with glucuronic acid shows excellent gastric mucosal protection and reflux esophagitis. there was.

Quercetin-7-0-β-D-glucuronide (QGC) isolated from Rumex Aquaticus Herba has been shown to be effective as a new therapeutic drug for ulcers and reflux esophagitis of gastritis. It can be effectively used for inflammation, ulcers and reflux esophagitis of the gastrointestinal tract.

Claims (6)

Water or lower alcohol soluble extracts of the leaves of Rumex Aquaticus Herba were degreased using non-polar solvents such as chloroform, ether or methylene chloride, and sugars and inorganic salts were removed using nonionic exchange resins. Separation method of quercetin-7-O-β-D-glucuronide compound (QGC), characterized in that the recrystallization of the% fraction. The method of claim 1, further comprising: a first step of extracting the ground rhubarb leaf by hot water extraction with a lower alcohol solvent such as water, methanol or ethanol, and a mixed solvent thereof, followed by concentration under reduced pressure; A second step of dissolving the X in distilled water and suspending it, filtering and dividing the mixture into a water insoluble layer and a water soluble layer, and then degreasing the water soluble layer with a nonpolar organic solvent such as chloroform, ether or methylene chloride; A third step of removing the non-polar organic solvent such as chloroform, ether or methylene chloride from the solubilized part of the degreased water and then adsorbing the non-ionic resin for column chromatography; A fourth step of removing the sugar and inorganic salt eluted with distilled water and eluting with 10 to 60% ethyl alcohol or methyl alcohol to obtain a flavonoid-containing fraction; Separation method of QGC which recrystallizes this flavonoid containing fraction. Pharmaceutical composition for the treatment of gastrointestinal inflammation, ulcer and reflux esophagitis comprising quercetin-7-O-β-D-glucuronide as an active ingredient. The composition of claim 3, wherein the quercetin-7-O-β-D-glucuronide compound is present in an amount of about 0.5 to 50 wt% based on the total weight of the composition. A dietary supplement comprising quercetin-7-O-β-D-glucuronide and a foodstuff acceptable food supplement, which exhibit a prophylactic effect of gastrointestinal inflammation, ulcers and reflux esophagitis. The dietary supplement according to claim 5, which is a health beverage.