JP2003171283A - Xanthine oxidase inhibitory agent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a food or medicine effective for the prevention and treatment of gout and hyperuricemia, having no fear of side effects even on using it continuously and having an effective and highly safe improving activity for the gout and hyperuricemia. <P>SOLUTION: This xanthine oxidase inhibitory agent contains a compound expressed by the following formula (1) (wherein, R<SB>1</SB>is H or rhamnose residue of which hydroxyl group may be blocked; and R<SB>2</SB>is H, or glucose, maltose, maltotriose or maltotetraose residue of which hydroxyl group may be blocked). The compound and the food or medicine containing the same are useful for the prevention and treatment of the gout and its risk factor, hyperuricemia. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD [0001] The present invention relates to foods and pharmaceuticals having an improved activity of xanthine oxidase and improving gout and hyperuricemia containing a compound which inhibits the synthesis of uric acid as an active ingredient.

[0002]

BACKGROUND OF THE INVENTION Gout is based on hyperuricemia due to abnormal metabolism of purines, and also has gouty nodules, joint dysfunction, and joint deformity due to the deposition of urate crystals in or around the joints. It is a disease that occurs and is a troublesome disease that causes many complications such as renal disorders and vascular disorders.

Conventionally, there are not so many patients with gout and hyperuricemia in Japan, and therefore, there has been little interest in preventing gout and hyperuricemia. However, in recent years, lifestyles have changed rapidly, and the number of cases of gout is increasing due to an increase in the number of people who eat high-calorie, high-protein and high-fat diets, or an increase in stress. There is a growing interest in the prevention and treatment of the risk factor hyperuricemia.

Gout and hyperuricemia are diseases caused by an increase in uric acid in blood, and the mechanism thereof is caused by the change of xanthine in the body into uric acid by the action of xanthine oxidase ( Masashi Ogasawara et al .; Purine and pyrimidine metabolism 18
(1) 1-10 1994). That is, the prevention and treatment of gout and hyperuricemia are basically controlled so that the blood uric acid level does not reach an unfavorable level. By reducing blood uric acid level to an appropriate level, patients with severe gout can also reduce acute attacks, stop chronic progression, prevent renal-vascular complications, and prevent deterioration (Tatsuo Hosoya). Mebio 17 (6) 24-29 2000).

Conventionally, as the drug for adjusting the blood uric acid level, a uric acid excretion agent "proveneside" and a uric acid production inhibitor "alopurinol" have been provided, but their effects are transient, gastrointestinal disorders and rashes. However, there are problems such as side effects such as leukopenia, thrombocytopenia, liver damage and renal damage. It has also been pointed out that allopurinol has a poor solubility. Further, as a plant-derived xanthine oxidase inhibitor, 2'-O-glucosyl-isovitexin (JP-A-5-238943) and kaempferol-3-O-α-arabinopyranoside-2 "-garate (JP-A-6- 172184), 4-amino-1H-pyrazolo [3,4-d] pyrimidin-3-one (JP-A-5-31
, Etc. have been isolated and reported, but there are problems that the activity is weak and side effects are accompanied to some extent.

[0006]

The object of the present invention is to effectively prevent and treat gout and hyperuricemia, and to prevent effective side effects even when used continuously, and to provide highly safe gout and It is intended to provide a food or medicine having a hyperuricemia-improving action.

[Means for Solving the Problems]

The present inventor is a perennial plant belonging to the family Rosaceae, and has long been an agent for antipyretics of rhizomes and flowers, edema and rheumatism as diuretics, and flu and measles as sweating, tonic and analgesic agents, and diarrhea of leaves. The common snow cane that has been used as a stop (generic name: spirea, Fili
pendula ulmaria), quercetin-4'-glycoside and quercetin-3-glycoside were isolated, and it was found that this substance has a strong xanthine oxidase inhibitory activity, and a patent application has been filed (Japanese Patent Application 2000- 336993). As a result of further study by the present inventors, quercetin-4′-glycoside and quercetin
The present inventors have completed the present invention by finding that among the 3-glycosides, the compound represented by the following formula (1) has a strong xanthine oxidase inhibitory activity. That is, the present invention is a xanthine oxidase inhibitor containing a compound represented by the following formula (1). The present invention is also an agent for gout or hyperuricemia, which comprises the compound represented by the formula (1). Furthermore, the present invention is a food or medicine containing the above-mentioned xanthine oxidase inhibitor or the agent for gout or hyperuricemia.

[0008]

[Chemical 2] (In the formula, R ₁ represents a rhamnose residue in which a hydrogen atom or a hydroxyl group may be protected, and R ₂ represents glucose, maltose, maltotriose, maltotetraose in which a hydrogen atom or a hydroxyl group may be protected. Represents a residue.)

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION Examples of the compound represented by the formula (1) of the present invention include rutin, α-monoglycosyl rutin, isoquercitrin and the like. α-Monoglycosyl rutin and isoquercitrin can be produced as a mixture by the following method. That is, rutin (quercetin-3
-Rutinose) and dextrin dissolved in a solution of cyclomaltodextrin glucanotransferase to transfer sugar from dextrin to rutin, which can be purified for production. Further, as these compounds, commercially available products can be used. As a commercially available product, for example, αG rutin P or αG rutin PS manufactured by Toyo Pure Sugar Co., Ltd. can be mentioned as a mixture of α-monoglycosyl rutin and isoquercitrin.

The mixture of α-monoglycosyl rutin and isoquercitrin of the present invention has a very high solubility in water (about 120 g / 100 g of water) and about 500 of rutin (about 0.01 g / 100 g of water).
It has 0 times the water solubility. In addition, it has the feature of being dissolved in 50% alcohol at a high concentration. Furthermore, it has high stability on the acidic side, and is stable in boiling water for 1 hour or more at pH 3 to pH 5. Good for long-term storage, 0.02% aqueous solution
No changes in the components were observed even when left at room temperature and outdoor direct sunlight at pH 3 for 54 days. In addition, no change in composition was observed even after storage at room temperature for 2 years. In addition, it has an ultraviolet absorption characteristic peculiar to flavonols, and UV-A (320 nm to 400 nm
nm) has strong absorption. In addition, it is a powder that has a white or yellow color and has a faint bitterness.

The fact that the compound of the present invention has a xanthine oxidase inhibitory activity has never been known so far, and is a new finding obtained by the present invention. The compound according to the present invention has an excellent xanthine oxidase inhibitory activity and can be used as a food or a drug for the purpose of preventing and improving gout and hyperuricemia.

The gout or hyperuricemia improving agent of the present invention can be administered either orally or parenterally. Upon administration, the active ingredient can be mixed with a solid or liquid non-toxic pharmaceutical carrier suitable for administration methods such as oral administration, rectal administration and injection, and then administered in the form of a conventional pharmaceutical preparation. Examples of such preparations include solid preparations such as tablets, granules, powders and capsules, liquid preparations such as solutions, suspensions and emulsions, and freeze-dried preparations. It can be prepared by conventional means. Examples of the above-mentioned non-toxic pharmaceutical carrier include glucose, lactose, sucrose, starch, mannitol, dextrin, fatty acid glyceride, polyethylene glycol, hydroxyethyl starch, ethylene glycol, polyoxyethylene sorbitan fatty acid ester,
Amino acids, gelatin, albumin, water, physiological saline and the like can be mentioned. Further, if necessary, conventional additives such as a stabilizer, a wetting agent, an emulsifier, a binder and an isotonicity agent can be appropriately added.

The food of the present invention means, as it is, or by adding various nutritional components, or by including it in foods and drinks.
It is eaten as a health food or food material useful for the treatment and prevention of gout or hyperuricemia. For example, after adding the above-mentioned suitable auxiliaries, using a conventional means, a form suitable for food, such as granules, granules, tablets, capsules, paste, etc. may be provided for food, In addition, various foods, for example, processed meat products such as ham and sausage,
It may be used by adding it to processed fish products such as kamaboko and chikuwa, bread, confectionery, butter, milk powder, fermented dairy products, or added to water, fruit juice, milk, soft drinks and other beverages.

The effective dose of the compound of the present invention depends on the patient's age, body weight, symptom, patient's degree, administration route, administration schedule, formulation form, strength of inhibitory activity of the compound, etc.
For example, in the case of oral administration, it is generally 0.01 to 1000 mg / kg body weight per day, preferably
The dose may be about 10 to 600 mg / kg body weight and may be administered in several divided doses per day. The compounds of the invention have low toxicity, for example:
Even when the rats were orally administered at a dose of 500 mg / kg daily for 100 days, no deaths were observed and no change in body weight was observed.

[0015]

EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited thereto. Example 1 [Production of a mixture of isoquercitrin and α-monoglucosylrutin] Using reagent rutin (manufactured by Wako Pure Chemical Industries, Ltd.), isoquercitrin and α-monoglucosylrutin were prepared based on Patent Nos. 2926411 and 3155466. A mixture was produced. 1 part by weight of rutin and 10 parts by weight of dextrin were mixed with 100 parts by weight of hot water at 80 ° C to prepare a suspension-like solution containing a high amount of rutin, in which cyclomaltodextrin glucanotransferase derived from Bacillus stearothermophilus (stock (Hayashibara Biochemical Laboratory Co., Ltd.) was added to 20 units per dextrin glam, and the mixture was stirred at pH 6.0 and 75 ° C for 24 hours.
The reaction was carried out for a time to generate α-glucosyl rutin.

The reaction solution is heated to inactivate the enzyme, and the pH is adjusted to about
Adjusted to 5.0 and 0.5% per α-glucosyl rutin
Glucoamylase (manufactured by Nagase & Co., Ltd.) and 0.5%
Hesperidinase (manufactured by Tanabe Seiyaku Co., Ltd.) was added,
The mixture was reacted at 55 ° C. for 20 hours with stirring to generate monoglucosyl rutin and isoquercitrin. The reaction solution was heated to deactivate the enzyme, and after filtration, the filtrate was passed through a column filled with an intermediate polar resin (trade name: Amberlite XAD-7). After washing with water to remove impurities such as glucose, the mixture was desorbed with 50 v / v% ethanol, concentrated and powdered to obtain a mixture of isoquercitrin and α-monoglucosylrutin. The obtained isoquercitrin and α-
When the monoglucosyl rutin mixture was analyzed using high performance liquid chromatography (JASCO Corporation: AS-950 type), the weight ratio of α-monoglucosyl rutin and isoquercitrin was about 5: 1. there were. Hereinafter, this mixture is referred to as αG rutin.

<HPLC conditions> Sample concentration: 1 mg / ml Column: YMC-Pack ODS-AQ (250 mm × 4.6) (manufactured by YMC Co., Ltd.) Flow rate: 0.5 ml / min. Eluent: water / methanol / acetic acid = 65 / 30/5 detection: UV 254nm

Example 2 [Measurement of xanthine oxidase inhibitory activity] Xanthine oxidase (XOD) inhibitory activity measuring method (WEN-SHING CHA
XOD inhibitory activity was measured according to N., et al., ANTICANCER RESEARCH 15 703-708 1995). That is, in a test tube
40 μl of xanthine oxidase (0.04 units / 40 μl, manufactured by Oriental Yeast Co., Ltd.) dissolved in 0.1 M phosphate buffer (pH 7.5) and 350 μl of 0.1 M phosphate buffer (pH 7.5)
Then, 10 μl of a sample (1 mg / ml) in which the sample compound was dissolved in a 10% dimethylsulfoxide aqueous solution was added, and preincubation was performed at 37 ° C. for 10 minutes. Then, 600 μl of 0.1 mM xanthine aqueous solution (manufactured by Wako Pure Chemical Industries, Ltd.) dissolved in 0.1 M phosphate buffer (pH 7.5) was added, and the mixture was reacted at 37 ° C. for 10 minutes. The reaction was stopped by heating in hot water of 100 ° C. for 3 minutes, and after sufficiently stirring, the amount of uric acid produced was measured by HPLC (hereinafter, the amount of uric acid produced is A).

As sample compounds, commercially available rutin (manufactured by Wako Pure Chemical Industries, Ltd.) and isoquercitrin (EXTR
ASYNTHESE), α-monoglucosyl rutin (manufactured by Toyo Seika Co., Ltd.) and αG rutin obtained in Example 1 were used. As a control experiment, a reaction solution containing no sample (hereinafter, the amount of uric acid produced in the reaction solution containing no sample in the control experiment is referred to as B), a reaction solution containing no enzyme (hereinafter, a reaction solution containing no enzyme) The amount of uric acid produced in the sample is C), and the amount of uric acid produced in the reaction liquid containing no sample and enzyme (hereinafter, the amount of uric acid produced in the sample and the reaction liquid not containing enzyme is D) Was measured by HPLC, and the xanthine oxidase inhibitory activity was calculated according to the following formula, and shown as an IC ₅₀ value (concentration that inhibits 50%) (μg / ml). Inhibition rate (%) = [1- (AC) / (BD)] × 10
0

The results are shown in Table 1. As a comparison, 2'-O-glucosyl-isovitexin, which has already been reported to have xanthine oxidase inhibitory activity,
Kaempferol-3-O-α-arabinopyranoside-2 "-garrate, 4-amino-1H-pyrazolo [3,4-d] pyrimidin-3-one, and allopurinol, a commercially available xanthine oxidase inhibitor The same test as described above was performed on the above, and the results are also shown in Table 1.

[0021]

[Table 1]

It can be seen from Table 1 that the compounds of the present invention, α-monoglucosylrutin, rutin, isoquercitrin and αG rutin obtained in Example 1 have high xanthine oxidase inhibitory activity.

Example 3 [Evaluation of efficacy using hyperuricemia model rat] Y. Osada was used to evaluate efficacy using hyperuricemia model rat.
Et al. (European Journal of Pharmacology, 2411)
83-188 1993) with a partial modification. In other words, 4-week-old male SD rats (Shimizu Experimental Materials Co., Ltd., Kyoto City) were purchased, and after sufficient quarantine training, general conditions were observed and body weights were measured, and animals with good health conditions were selected. Use at age, stratify by body weight and make up each group by random sampling. Group composition was performed on the day before the administration day. After fasting the animals for 24 hours, first, a sample prepared by adjusting αG rutin obtained in Example 1 to a 125 mg / kg body weight with 0.5% CMC (carboxymethylcellulose) aqueous solution was orally administered. At this time, the 0.5% CMC aqueous solution alone was administered to the untreated group and the hyperuricemia group (control group). Then, 2 hours later, 125 mg / kg oxonic acid (Aldrich Ja
Pan) 0.5% CMC aqueous solution was orally administered to induce hyperuricemia (only 0.5% CMC aqueous solution was administered to the untreated group). Blood was collected 1 hour before the administration of the solvent or the test substance (3 hours before the administration of oxonic acid), 0.5 hours after the administration of the test substance, and oxonic aci.
d Perform 1, 2, 3, 4, 5, 6 hours after administration from the fundus to prepare plasma for uric acid measurement. In measuring uric acid, the results of measurement using DRI-CHEM3030 (manufactured by FUJIFILM) are shown in FIG. As can be seen from FIG. 1, the compound of the present invention (αG rutin) has a blood uric acid level lowering effect (oxonic ac).
2 to 6 hours after id administration).

Example 4 [Production of tablet] Using the αG rutin obtained in Example 1, a tablet having the following composition was produced according to a conventional method. (Composition) (Composition: wt%) αG Rutin 24 Lactose 63 Corn Starch 12 Guar Gum 1

Example 5 [Manufacture of juice] Using the αG rutin obtained in Example 1, a juice having the following composition was manufactured according to a conventional method. (Composition) (Composition:% by weight) Frozen concentrated Unshu mandarin orange juice 5.0 Fructose glucose liquid sugar 11.0 Citric acid 0.2 L-ascorbic acid 0.02 Fragrance 0.2 Pigment 0.1 αG rutin 0.2 Water 83.28

[0026]

EFFECTS OF THE INVENTION Since the compounds of the present invention have xanthine oxidase inhibitory activity, these compounds and foods or pharmaceuticals containing them are useful for the prevention and treatment of gout and its risk factor, hyperuricemia. is there.

[Brief description of drawings]

FIG. 1 is a diagram showing the results of a blood uric acid level lowering test using a hyperuricemia model rat.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12N 9/99 C12N 9/99 (72) Inventor Takashi Naito 12-13 Kamishinano, Totsuka-ku, Yokohama-shi, Kanagawa Incorporated company FANCL Central Research Institute (72) Inventor Toshimitsu Kambara 1089-8 Ai, Shinichi-machi, Ashina-gun, Hiroshima Prefecture Maruzen Pharmaceutical Co., Ltd. Research Institute (72) Nobuaki Odo Aikata-cho, Shinichi-machi, Hiroshima Prefecture 1089- 8 Maruzen Pharmaceutical Co., Ltd. Research Institute (72) Inventor Takashi Yumoto 1-6-41 Iwasaki Nishi, Ichihara City, Chiba Prefecture Toyo Seika Co., Ltd. Chiba Factory F-term (reference) 4B018 LB08 LE05 MD42 MD48 ME14 MF12 4C057 AA17 BB02 BB03 DD01 KK08 4C086 AA01 AA02 EA04 MA01 MA04 NA14 ZA96 ZC20

Claims (4)

[Claims] 1. The following formula (1): (In the formula, R ₁ represents a rhamnose residue in which a hydrogen atom or a hydroxyl group may be protected, and R ₂ represents hydrogen atom, a glucose in which a hydroxyl group may be protected, maltose, maltotriose or maltotetraose. A xanthine oxidase inhibitor containing a compound represented by a residue. 2. An agent for gout or hyperuricemia containing the compound represented by the formula (1). 3. A food containing the agent according to claim 1 or 2. 4. A medicine containing the agent according to claim 1 or 2.