JP2000290188A - Xanthine oxidase inhibitor - Google Patents

Abstract

(57) [Problem] To provide a xanthine oxidase inhibitor comprising an extract from a highly safe plant as an active ingredient. SOLUTION: As a result of comparing the xanthine oxidase inhibitory activities of ten kinds of plant materials, it was found that the activity of Pleurotus serrata (banaba) was the strongest. "Crude extract" obtained by extracting banaba with hot water, etc., "resin adsorption component" obtained by adsorbing this crude extract on styrene-divinylbenzene synthetic resin, etc. "Organic solvent-soluble component" obtained by a partitioning operation with a solvent, and "Elagic acid""Ellagic acid derivative""Ellagic acid-like compound""" obtained by purifying this organic solvent-soluble component by high-performance liquid chromatography All of the "lignans" were confirmed to have a xanthine oxidase inhibitory action, and a xanthine oxidase inhibitor containing these extracted components as active ingredients was provided.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a xanthine oxidase inhibitor containing a plant-derived component as an active ingredient.

[0002]

BACKGROUND OF THE INVENTION Xanthine oxidase, which is abundant in mammalian liver, small intestinal mucosa and milk, produces uric acid from hypoxanthine via xanthine in the final stage of the purine compound degradation pathway.

[0003] An increase in the uric acid concentration in the blood causes various diseases such as gout as hyperuricemia. Therefore, in order to correct hyperuricemia, administration of a uric acid production inhibitor, ie, a xanthine oxidase inhibitor, is clinically performed. The development of a physiologically active substance capable of suppressing the activity of xanthine oxidase has been watched.

By the way, a number of compounds having a xanthine oxidase inhibitory action have been reported to date, but most of them have been synthesized by chemical methods and have never been said to be highly safe for the human body. . Recently, a xanthine oxidase inhibitor derived from a natural product has been disclosed from the viewpoint of safety to the human body (Japanese Patent Application Laid-Open No. H5-244963).
And Japanese Patent Application Laid-Open No. 9-202733), the effects of inhibiting xanthine oxidase were not sufficiently satisfactory.

[0005] Therefore, the present invention is to provide a xanthine oxidase inhibitor having a highly safe natural product-derived component as an active ingredient and having an excellent activity of inhibiting xanthine oxidase activity.

[0006]

Means for Solving the Problems In order to solve the above-mentioned problems, the present inventors have studied the use of xanthine oxidase inhibitors for plants which have been used for food for many years and whose safety to the human body has been confirmed. I considered something. As a result, they have found that an extract of Lagerstroemia speciosa, which is a plant belonging to the genus Lagerstroemia, belonging to the genus Larsaceae, has a strong xanthine oxidase inhibitory effect, and has led to the present invention.

[0007] That is, the xanthine oxidase inhibitor of the present invention is characterized by containing, as an active ingredient, a component extracted from a plant belonging to the genus Lagerstroemia.

In the present invention, any plant belonging to the genus Lagerstroemia can be used, but "Lagerstroemia speciosa" is particularly preferred. This "Lagers Lagers
troemia speciosa) grows in the tropics, including the Philippines, and is called banana. Since this extract is used as everyday tea in the Philippines, its safety for the human body is guaranteed. It can be said that there is.

In the present invention, the parts of the plant that can be used for extraction include leaves, stems, flowers, woody parts, bark parts, roots, and the like. In the extraction, these plant parts are dried and pulverized. It is preferable to use them.

[0010] As the "extracted component" in the present invention, a "crude extract" obtained by extracting the above plant with water (including hot water), an organic solvent or a mixed solution thereof is used. Can be. The extraction temperature at this time is 40 to 10
It is preferable to set the temperature to 0 ° C, especially to 95 to 100 ° C. As the organic solvent, methanol, ethanol, it is preferable to use a water-soluble organic solvent such as acetone,
If necessary, a combination of water and an organic solvent can be used. The amount of the hot water, the organic solvent, and the mixed solution of the organic solvent and the hot water for extracting the plant is about 1:10 to 1: 100, especially about 1:25, based on the weight of the dried plant. Is preferred.

The "resin adsorbed component" obtained by separating and purifying the above "crude extract" by a chromatography technique can also be used as the "extracted component" in the present invention.
That is, a styrene-divinylbenzene-based synthetic resin (Diaion HP-10, 20, 30, 40, 50 manufactured by Mitsubishi Kasei Kogyo Co., Ltd., Amberlite XAD-2,4 manufactured by Organo Co., Ltd., or Duolite S manufactured by Sumitomo Chemical Co., Ltd.) Series) or dextran-based synthetic resin (Sephadex LH manufactured by Pharmacia)
-20) and the above “crude extract” is adsorbed, and the “resin adsorbed component” is eluted with an organic solvent and, if necessary, a mixed solution of water and an organic solvent. For example, Diai
The "crude extract" is supplied to a column packed with on HP-20, the non-adsorbed components are washed with water, and then the "resin adsorbed components" are eluted and recovered with methanol.

Further, the "resin-adsorbed component" is obtained by distributing water and an organic solvent such as chloroform, hexane, ethyl acetate and butanol into "organic solvent-soluble component".
Can also be preferably used as the "extracted component" in the present invention.

Further, a "high-performance liquid chromatography fractionation component" obtained by purifying or isolating the "organic solvent-soluble component" using high performance liquid chromatography.
Can also be used as the “extracted component” in the present invention. High performance liquid chromatography can be performed on a column using a separation mode such as reverse phase, normal phase, gel filtration, ion exchange, and hydrophobicity, but is preferably performed in reverse phase mode from the economical viewpoint. A preferred example is Diaion HP-20
The resin-adsorbed fraction was partitioned with ethyl acetate and butanol to obtain a fraction soluble in ethyl acetate and a fraction soluble in butanol. The fraction soluble in ethyl acetate was subjected to 50% methanol (1% ) Can be used to separate the main components.

"High performance liquid chromatography preparative components"
Contains ellagic acid, an ellagic acid derivative, a compound similar to ellagic acid, and lignans as main components, and it has been confirmed that each of these fractions has an excellent xanthine oxidase inhibitory action. Therefore, any of the thus obtained ellagic acid, ellagic acid derivative, ellagic acid-like compound, lignans,
The above mixture can also be used very effectively as the "extracted component" in the present invention.

The method for purifying the above-mentioned ellagic acid, ellagic acid derivative, ellagic acid-like compound and lignans is not limited to the above-mentioned method, and if they are obtained from other plants, they may be similarly inhibited by xanthine oxidase. It can be used as an active ingredient of an agent. Furthermore, the extracted components obtained in each of the above steps can be used in combination. As the ellagic acid derivative, valoneic aci described later
Other than d dilactone, those having an ellagic acid structure as a basic skeleton and a sugar group, a lipid, an amino acid, a polyphenol, or the like bonded to the hydroxyl group, or those having the hydroxyl group substituted or conjugated to a methyl group, an acetyl group, or the like. In these cases, it is considered that the same effect as valoneic acid dilactone is exerted. Ellagic acid analogs include 3,4,8,9,10-pentahydroxy-dibenzo [bd] pyra
Compounds similar in structure to ellagic acid, such as n-6-one, such as ellagitannin, which produces ellagic acid when hydrolyzed, are 3,4,8,9,10-pentane
It is thought that it exerts the same effect as hydroxy-dibenzo [bd] pyran-6-one.

The banaba extract according to the present invention has an effect of inhibiting the activity of xanthine oxidase and can suppress the production of uric acid, as is apparent from the examples described later. Therefore, it can be used for the prevention and treatment of various diseases caused by hyperuricemia such as gout, and further, various diseases caused by active oxygen such as inflammation, aging, carcinogenesis, arteriosclerosis, and cerebral disorder. It is also effective in the prevention and treatment of various diseases.

The xanthine oxidase inhibitor of the present invention can be variously provided as pharmaceuticals, quasi-drugs, cosmetics, foods, beverages (cans, plastic bottles, bottles, etc.). For example, it can be provided as a banaba tea drink having a xanthine oxidase inhibitory action. The dosage form may be dried by freeze-drying or spray-drying and provided as a dry powder, or may be a liquid, tablet,
Powders, granules, dragees, capsules, suspensions, solutions, emulsions,
Ampules, injections and the like can also be used.

The above-mentioned "extracted component" alone can be used alone as an active ingredient of a xanthine oxidase inhibitor, but is also known as a food having an xanthine oxidase inhibitory action or an antihyperuricemia agent. The effect can be further enhanced by using allobrinol, alloxanthine, blobeneside known as uric diuretic, benzbromarone, or the like in combination.

[0019]

BEST MODE FOR CARRYING OUT THE INVENTION (Schooling from Plant Materials) Ten kinds of plant materials known as so-called healthy tea were collected, and their xanthine oxidase enzyme activity inhibitory effects were compared and examined. After crushing each plant material with a mill,
0.5 g of the mixture is placed in a screwed Erlenmeyer flask.
ml was added and extracted for 30 minutes. The obtained extract was filtered with a filter, and the effect of inhibiting xanthine oxidase enzyme activity was examined by the measurement method described below. The result is shown in FIG.

From the results shown in FIG. 1, it was found that the component (crude extract) obtained by extracting banaba leaves was superior to other materials. Therefore, a hot water extract (crude extract) of banaba leaves was prepared, fractionated by HP-20 column chromatography and organic solvent extraction as follows, and the xanthine oxidase inhibitory activity was measured for each fraction. I decided that.

(Extraction and Purification of Banaba Leaf) Banana leaves were extracted and purified in the steps shown in FIG. 2, and the xanthine oxidase inhibitory activity of each fraction was measured by the measurement method described later.

That is, fresh banaba leaves produced in the Philippines were forcibly and sufficiently dried in an appropriate amount, and crushed and thoroughly mixed, and 1 kg of banaba leaves were extracted with 50 liters of hot water at 95 to 100 ° C. for 30 minutes. At this time, the brix of the extraction solution was about 1.5%. The obtained extract was filtered and centrifuged, and further concentrated by an evaporator,
This was spray-dried to obtain a “hot water extract”, that is, a crude extract.

Next, a styrene-divinylbenzene synthetic resin (Diaion HP-20 manufactured by Mitsubishi Kasei Kogyo Co., Ltd.) was placed in a glass column having a diameter of 10 cm and a length of 100 cm.
After filling 5 liters, the filler was washed with methanol and further washed with distilled water, and then the above “hot water extract” (total amount)
Was dissolved in 1 liter of distilled water, and the solution was passed through a glass column. Then, about 6 times the amount of distilled water was passed through the column, and the fraction not adsorbed to the styrene-divinylbenzene-based synthetic resin (hereinafter referred to as "the fraction") was used. HP-20 non-adsorbed fraction), and then flowed about 5 times the amount of methanol with respect to the column capacity to flow the fraction adsorbed on the styrene-divinylbenzene synthetic resin (hereinafter referred to as "HP-20 adsorbed fraction"). Fractionation "). The “HP” thus obtained
The “-20 non-adsorbed fraction” and the “HP-20 adsorbed fraction” were respectively concentrated and freeze-dried to obtain dried products.

Next, the obtained “HP-20 adsorption fraction” 2
Dissolve 00 g in 1.5 liters of distilled water, add 2 liters of ethyl acetate, shake well with a separating funnel,
The “ethyl acetate-extracted fraction” was collected as the ethyl acetate-soluble component, and the above-mentioned ethyl acetate-extracting operation was repeated twice to obtain an “ethyl acetate-extracted fraction”, which was concentrated and freeze-dried. To obtain a dried product.

On the other hand, 1.5 liters of butanol was added to the remaining distilled water remaining in the separating funnel and shaken sufficiently, and then a butanol extraction fraction was collected. This was repeated twice to obtain a "butanol-extracted fraction", which was concentrated and freeze-dried to obtain a dried product.

Regarding the above “ethyl acetate fraction”,
Purification was performed using preparative high performance liquid chromatography. The column is a YMC column ODS 120A (20 mm ID x
250mm), 50% methanol (containing 1% acetic acid)
Was flowed at a flow rate of 6 ml / min at room temperature. The eluted components were monitored by ultraviolet absorption at 254 nm. At this time, five large peaks are detected (FIG. 3). Each peak was collected using a fraction collector, concentrated, and then lyophilized to obtain a dried product (“high-performance liquid chromatography fractionation component”).

The "hot water extract", "HP-20 non-adsorbed fraction", "HP-20 adsorbed fraction", "ethyl acetate extracted fraction", and "butanol extracted fraction" obtained in each of the above-described steps.
The "high-performance liquid chromatography preparative component" was measured for xanthine oxidase inhibitory activity by the measurement method described later, and was compared.

FIG. 4 shows the xanthine oxidase inhibitory activity at the fractionation stage. Thus, the activity can be increased by adsorbing the crude extract to HP-20, and the activity can be further increased by extracting the HP-20 adsorbed fraction with ethyl acetate and butanol. It was found that the ethyl acetate and butanol extract components exhibited higher activities.

FIG. 5 shows the xanthine oxidase inhibitory activity of the peak obtained by purifying the ethyl acetate fraction by high performance liquid chromatography. From this, peak No. 1
Other than the above, the inhibitory activity was higher than that of the ethyl acetate fraction itself. Instrumental analysis using NMR and mass spectrum for peak No. 2 which had particularly strong inhibitory activity showed that lignans, 2,5-bis- (4-hydroxy-3,5-dimethoxyphenyl)-
3,4-bis- (hydroxymethyl)-(2,3,4,5) -tetrahydrofuran
(Formula 1) and valoneic acid dilact, an ellagic acid derivative
One was identified as a mixture. further,
About peak No. 3, it is 3,4,8,
9,10-pentahydroxy-dibenzo [bd] pyran-6-one
The peak No. 4 was identified as ellagic acid (Chem. 4).

[0030]

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[0031]

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[0032]

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[0033]

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(Measurement of Xanthine Oxidase Inhibitory Activity) Using xanthine as a substrate, the amount of uric acid produced by xanthine oxidase, which is an oxidase, was measured.
All reagents used were products of Wako Pure Chemical Industries. 0.1 M phosphate buffer (pH 7.4) in the cell of the spectrophotometer in advance
0.3 ml, sample solution 0.02 ml or 0.1 ml.
1 ml, distilled water 0.18 ml or 0.1 ml, 0.12
0.1 ml of U / mL xanthine oxidase was mixed, and the reaction was started by adding 0.1 mM xanthine. The generated uric acid was monitored for absorbance at 295 nm. A phosphate buffer was added to the reference cell instead of xanthine oxidase. The xanthine oxidase inhibitory activity was evaluated by suppressing the increase in absorbance due to the addition of a sample solution, with the case where no sample was added as a control taken as 100%.

[Brief description of the drawings]

FIG. 1 is a graph comparing 10 kinds of plant materials with hot water and comparing the resulting extracts with xanthine oxidase inhibitory activities.

FIG. 2 is a diagram showing an example of steps of extraction and purification of P. persicae (banaba).

FIG. 3 is a graph showing an elution pattern obtained by preparative high performance liquid chromatography.

FIG. 4: Hot water extract of P. persica (Banaba)
4 is a graph comparing the xanthine oxidase inhibitory activities of HP20-adsorbed, non-HP20-adsorbed, and HP20-adsorbed fractions partitioned with ethyl acetate and butanol, and the water residue.

FIG. 5 is a graph comparing the xanthine oxidase inhibitory activities of the peaks obtained by preparative high performance liquid chromatography.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61K 31/35 A61K 31/35 31/366 31/365 601 (72) Inventor Takami Tsunoda Sagara, Haibara-gun, Shizuoka Town Goddess 21 F-term in ITO EN Central Research Laboratories Co., Ltd.

Claims (5)

[Claims] 1. A xanthine oxidase inhibitor comprising, as an active ingredient, an ingredient extracted from a plant belonging to the genus Lagerstroemia. 2. A "crude extract" obtained by extracting Lagerstroemia speciosa belonging to the genus Lagerstroemia using water, hot water, an organic solvent, or a mixed solution comprising at least two of these, as an active ingredient. A xanthine oxidase inhibitor. 3. Lagerstroemia speciosa belonging to the genus Lagerstroemia is extracted using water, hot water, an organic solvent or a mixed solution comprising at least two of these, and the resulting “crude extract” is styrene. A xanthine oxidase inhibitor containing, as an active ingredient, a “resin-adsorbed component” obtained by adsorbing to a divinylbenzene-based synthetic resin or a dextran-based synthetic resin. 4. Lagerstroemia speciosa belonging to the genus Lagerstroemia is extracted using water, hot water, an organic solvent or a mixed solution comprising at least two of these, and the obtained “crude extract” is styrene. -Adsorbed on a divinylbenzene-based synthetic resin or dextran-based synthetic resin, and the obtained "resin-adsorbed component" is obtained by a partitioning operation between water and an organic solvent such as chloroform, hexane, ethyl acetate, and butanol. A xanthine oxidase inhibitor comprising a "soluble component" as an active ingredient. 5. A Lagerstroemia speciosa belonging to the genus Lagerstroemia is extracted using water, hot water, an organic solvent or a mixed solution comprising at least two of these, and the obtained “crude extract” is styrene. -Adsorbed on a divinylbenzene-based synthetic resin or dextran-based synthetic resin, and the obtained "resin-adsorbed component" is subjected to a partitioning operation between water and an organic solvent such as chloroform, hexane, ethyl acetate, butanol, etc. , And the "organic solvent-soluble component" is purified by high-performance liquid chromatography to obtain ellagic acid, an ellagic acid derivative, an ellagic acid-like compound, or a lignan, or a mixture of two or more of these. A xanthine oxidase inhibitor contained as a component.