JP2017048185A - Xanthine oxidase inhibitor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a xanthine oxidase inhibitor derived from food and drink, and a method for producing a composition containing the inhibitor.SOLUTION: A method for producing a composition having xanthine oxidase inhibitory activity comprises a liposoluble organic solvent layer recovery process in which, after hot water extract of roasted coffee beans and liposoluble organic solvent are mixed, only a liposoluble organic solvent layer is recovered as a composition having xanthine oxidase inhibitory activity. A xanthine oxidase inhibitor contains a liposoluble organic solvent extract extracted from the hot water extract of roasted coffee beans with the liposoluble organic solvent as an active ingredient.SELECTED DRAWING: None

Description

  The present invention relates to a xanthine oxidase inhibitor derived from food and drink and a method for producing a composition containing the inhibitor.

  Gout, which is known as a lifestyle-related disease, is a disease accompanied by severe pain in the joints such as the big toe due to hyperuricemia caused by abnormal metabolism of purines. Such pain occurs because increased uric acid in the liquid crystallizes and deposits in the joint. In recent years, dietary habits have changed rapidly, and an increasing number of people have high-calorie, high-protein, high-fat meals. Along with this change in diet, the number of gout patients is increasing year by year, and there is an increasing interest in the prevention and treatment of gout and hyperuricemia.

  Gout is a disease caused by an increase in uric acid in blood, and controlling uric acid in blood within a normal value is the basis for prevention and treatment of diseases such as gout and hyperuricemia. Xanthine oxidase is an enzyme that plays an important role in the synthesis of uric acid in vivo, and a drug that inhibits xanthine oxidase is useful as a prophylactic and therapeutic drug for gout and hyperuricemia.

  However, the uric acid synthesis inhibitor “allopurinol” or the like conventionally used as a drug for adjusting blood uric acid level has problems such as transient xanthine oxidase inhibitory activity and side effects. Therefore, there is a demand for a uric acid level reducing agent derived from a natural product that has no or little side effects and is derived from foods and drinks that are taken daily. For example, Patent Document 1 discloses a xanthine oxidase inhibitor containing, as an active ingredient, a component extracted from whiskey or a beech family Quercus plant used in the production thereof, and Patent Document 2 is derived from a fermented barley. Serum uric acid level-reducing agents containing these ingredients as active ingredients are described. Patent Document 3 describes that an ascofilum extract and a tamarind extract are effective in reducing serum uric acid levels, and Patent Document 4 describes xanthine oxidase inhibition using rose extract as an active ingredient. Agents are disclosed. Furthermore, Non-Patent Document 1 reports that the chlorogenic acid lactone contained in the hot water extract of roasted coffee beans has xanthine oxidase inhibitory activity. In addition, Non-Patent Document 2 reports that pyrogallol has a weak xanthine oxidase inhibitory activity, and Non-Patent Document 3 reports that purpurogallin has a xanthine oxidase inhibitory activity.

Japanese Patent No. 4988166 Japanese Patent No. 5044643 Japanese Patent No. 5437672 Japanese Patent No. 5498067

Honda, et al., Bioscience, Biotechnology, and Biochemistry, 2014, vol. 78 (12), p.2110-2116. Gray and Felsher, Experimental Biology and Medicine, 1945, vol.59 (2), p.287-289. Sheu, et al., Anticancer Research, 1998, vol.18, p.263-268.

  The xanthine oxidase inhibitory active ingredients described in Patent Documents 1 to 4 are difficult to purify and isolate from natural products as raw materials. On the other hand, Non-Patent Document 1 does not describe anything other than chlorogenic acid lactone as a substance having xanthine oxidase inhibitory activity contained in the hot water extract of roasted coffee beans. Further, the efficiency of extraction and purification from a hot water extract of roasted coffee beans of chlorogenic acid lactone is not sufficient. Non-Patent Document 2 does not describe pulprogalin, and Non-Patent Document 3 does not describe pyrogallol.

  The present invention relates to a xanthine oxidase inhibitor derived from roasted coffee beans, which is the most popular beverage in the world, and a fraction containing a compound having xanthine oxidase inhibitory activity from roasted coffee beans. It aims at providing the method of collect | recovering as a composition which has inhibitory activity.

  As a result of intensive studies to solve the above problems, the present inventors have found that a substance having xanthine oxidase inhibitory activity contained in roasted coffee beans is a fat-soluble organic solvent from a hot water extract of roasted coffee beans. It can be efficiently recovered by extraction, and the ferroylquinic acid lactones and coumaroylquinic acid lactones contained in the recovered fat-soluble organic solvent extract have a powerful xanthine oxidase inhibitory activity and have been recovered It was found that pyrogallol was contained in the fat-soluble organic solvent extract, and that pyrogallol and its alkali-treated product also had strong xanthine oxidase inhibitory activity, and the present invention was completed.

[1] A method for producing a composition having xanthine oxidase inhibitory activity according to the first aspect of the present invention comprises mixing a hot water extract of roasted coffee beans with a fat-soluble organic solvent, and then only the fat-soluble organic solvent layer. It has the fat-soluble organic-solvent layer collection | recovery process which collect | recovers as a composition which has xanthine oxidase inhibitory activity, It is characterized by the above-mentioned.
[2] As a method for producing the composition having xanthine oxidase inhibitory activity of [1], the fat-soluble organic solvent is ethyl acetate, hexane, chloroform, dichloromethane, diethyl ether, or a mixed solvent containing any of these. It is preferable that
[3] The method for producing the composition having xanthine oxidase inhibitory activity of [1] or [2] includes pyrogallol from the recovered fat-soluble organic solvent layer after the fat-soluble organic solvent layer recovery step. It is preferable to have a fractionation step of collecting the fraction.
[4] The method for producing the composition having xanthine oxidase inhibitory activity of [3] includes an alkali treatment step of adjusting the fraction containing pyrogallol to pH 6.5 to 8.0 after the fractionation step. It is also preferable to have it.
[5] As a method for producing the composition having xanthine oxidase inhibitory activity of [4], the fraction adjusted to pH 7 to 8 after the alkali treatment step is adjusted to pH 4.5 to 5.5. It is also preferable to have an acid treatment step.
[6] The xanthine oxidase inhibitor according to the second aspect of the present invention is characterized in that an extract extracted from a hot water extract of roasted coffee beans with a fat-soluble organic solvent is an active ingredient.
[7] The xanthine oxidase inhibitor according to the third aspect of the present invention is characterized by containing feroylquinic acid lactone as an active ingredient.
[8] The xanthine oxidase inhibitor according to the fourth aspect of the present invention is characterized by comprising coumaroylquinic acid lactones as active ingredients.
[9] A pharmaceutical product according to the fifth aspect of the present invention is characterized by containing the xanthine oxidase inhibitor according to any one of [6] to [8].

  By the method for producing a composition having xanthine oxidase inhibitory activity according to the present invention, a substance having xanthine oxidase inhibitory activity contained in roasted coffee beans is efficiently extracted, and a composition having very high xanthine oxidase inhibitory activity is obtained. Can be manufactured. In addition, the xanthine oxidase inhibitor according to the present invention is derived from roasted coffee beans in addition to high xanthine oxidase inhibitory activity, and can be administered relatively safely, and prevention and treatment of gout and hyperuricemia Suitable as an active ingredient of pharmaceuticals for

It is the chromatogram of preparative HPLC of the ethyl acetate extract prepared from the hot water extract of shallow roasted beans in Example 3. It is the chromatogram of preparative HPLC of the ethyl acetate extract prepared from the hot water extract of deep roasted beans in Example 3. It is a chromatogram of preparative HPLC fraction 2 of deep roasted beans in Example 3. It is a chromatogram of preparative HPLC fraction 3 of deep roasted beans in Example 3. It is the figure in Example 4 which showed the measurement result of the xanthine oxidase inhibition rate (%) of each fraction which fractionated the ethyl acetate soluble fraction by MPLC. Fr. obtained by fractionating the ethyl acetate-soluble fraction in Example 4 by MPLC. 2 is a chromatogram of 2 preparative HPLC. It is the figure in Example 5 which showed the time-dependent change of content of the pyrogallol and the pulprogalin in the solution which incubated pyrogallol on each pH conditions. In Example 5, it is the figure which showed the time-dependent change of content of the purpurogallin in the solution which incubated the purpurogallin at pH7.4. It is the figure in Example 6 which showed the time-dependent change of content of pyrogallol and purpurogallin, and a xanthine oxidase inhibition rate (%) of the solution which incubated pyrogallol at pH 7.4.

  The xanthine oxidase inhibitor according to the present invention contains a compound having xanthine oxidase inhibitory activity contained in roasted coffee beans as an active ingredient. In addition to the chlorogenic acid lactones disclosed in Non-Patent Document 1, ferroylquinic acid lactones, coumaroylquinic acid lactones, and pyrogallol as compounds having xanthine oxidase inhibitory activity contained in roasted coffee beans (1,2,3-Benzenetriol). In addition, compounds obtained by alkali treatment of pyrogallol such as purpurogallin (2,3,4,6-Tetrahydry-5H-benzocyclohexane-5-one) also have strong xanthine oxidase inhibitory activity. It can be an active ingredient of the xanthine oxidase inhibitor according to the invention.

  In the present specification, “chlorogenic acid lactones” refers to neochlorogenic acid lactone and cryptochlorogenic acid lactone. That is, “chlorogenic acid lactones” is a general term for 3-caffeoylquinic acid lactone and 4-caffeoylquinic acid lactone.

Ferroylquinic acid lactones are compounds of molecular formula C ₁₇ H ₁₈ O ₈ having a structure in which the carboxyl group of ferulic acid and the hydroxyl group of quinic acid lactone are dehydrated and condensed. For example, the following formula (1-1) to The compound represented by (1-5) is mentioned. Coumaroyl quinic acid lactones are compounds of molecular formula C ₁₇ H ₁₈ O ₈ having a structure in which the carboxyl group of p-coumaric acid and the hydroxyl group of quinic acid lactone are dehydrated and condensed. For example, the following formula (2-1) to The compound represented by (2-5) is mentioned.

  Ferroylquinic acid lactones, coumaroylquinic acid lactones, and pyrogallol can be recovered by extraction from a hot water extract of roasted coffee beans by fat-soluble organic solvent extraction. That is, the method for producing a composition having xanthine oxidase inhibitory activity according to the present invention (hereinafter sometimes referred to as “the method for producing the composition according to the present invention”) is obtained from roasted coffee beans and ferroylquinic acid lactone. , Coumaroylquinic acid lactones, and pyrogallol-containing compounds having xanthine oxidase inhibitory activity are extracted to obtain a composition having xanthine oxidase inhibitory activity (hereinafter sometimes referred to as “xanthine oxidase inhibitory composition”). It is. Specifically, after mixing the hot water extract of roasted coffee beans and a fat-soluble organic solvent, the method has a fat-soluble organic solvent layer recovery step of recovering only the fat-soluble organic solvent layer as a xanthine oxidase-inhibiting composition.

  The roasted coffee beans used as a raw material are not particularly limited as long as they are roasted, and the type and production area of the coffee beans are not particularly limited, and may be Arabica, Robusta, or Riberica. It may be a blend of these. Also, the roasting method is not particularly limited, and it is generally used for coffee beans such as direct fire roasting method, hot air roasting method, far infrared roasting method, charcoal fire roasting method, microwave roasting method and the like. Any method used for roasting may be used.

  Roasted coffee beans contain compounds having xanthine oxidase inhibitory activity such as ferroylquinic acid lactones, coumaroylquinic acid lactones, chlorogenic acid lactones, and pyrogallol, regardless of the roasting degree. . For this reason, the roasting degree of roasted coffee beans used as a raw material is not particularly limited, and may be an extremely shallow roast, a shallow roast, a medium roast, or a deep roast. It may be roasted. Increased content of pyrogallol, ferroylquinic acid lactones and coumaroylquinic acid lactones, and an unknown active ingredient different from chlorogenic acid lactones (active ingredient contained in higher polar fraction than chlorogenic acid lactones) Since the content is increased, the roasted coffee beans used as a raw material preferably have a higher roasting degree, more preferably a medium-roasted or deep-roasted roasted coffee bean, and particularly preferably a deep-roasted roasted coffee bean.

  In addition, the roasting degree of coffee beans can be represented by lightness (L * value). The lower the roasting degree, the higher the lightness, and the higher the roasting degree, the smaller the lightness. For example, the lightness when pulverized beans are analyzed with a color difference meter has an L value of about 18 for deep roasted beans, an L value of about 21 for medium roasted beans, and a roasted beans of shallow roast. The L value is about 24.

  Since extraction efficiency becomes high, it is preferable that the roasted coffee beans are pulverized before being extracted with hot water. The roasted coffee beans can be pulverized using a general pulverizer such as a roll mill. The degree of pulverization is not particularly limited, and roasted coffee beans of various shapes such as coarsely ground, mediumly ground, medium ground, medium finely ground, and finely ground can be used.

  The hot water extract of roasted coffee beans is obtained by bringing heated water into contact with the pulverized product of roasted coffee beans and extracting a water-soluble solid content. The extraction method can be performed by a method generally used when brewing coffee or a method used when extracting soluble solids from a pulverized product of roasted coffee beans when producing instant coffee. . Specifically, any of a drip type, an espresso type, a siphon type, a percolator type, a coffee press (French press) type and the like may be used.

  By mixing the hot water extract of roasted coffee beans and a fat-soluble organic solvent, compounds having xanthine oxidase inhibitory activity such as ferroylquinic acid lactones, coumaroylquinic acid lactones, and pyrogallol in the hot water extract are obtained. Extracted into a fat-soluble organic solvent. That is, the fat-soluble organic solvent layer (fat-soluble organic solvent extract) can be used as the xanthine oxidase-inhibiting composition in the present invention, and this is separated and recovered from the aqueous layer and insoluble matter. Extraction with the fat-soluble organic solvent and recovery of the fat-soluble organic solvent layer can be carried out in the same manner as the extraction method and the liquid separation method with a general organic solvent.

  The fat-soluble organic solvent is not particularly limited as long as it is phase-separable from water and can dissolve relatively low-polarity components such as ferroylquinic acid lactones, coumaroylquinic acid lactones, and pyrogallol. Although not intended, a highly volatile organic solvent is preferred because it can be easily removed from the recovered fat-soluble organic solvent extract. Specific examples of the fat-soluble organic solvent include ethyl acetate, hexane, chloroform, dichloromethane (methylene chloride), diethyl ether, or a mixed solvent containing at least one of these. In the method for producing the composition according to the present invention, ethyl acetate is particularly preferable because extraction efficiency of various cinnamic acid-bonded quinic acid lactones such as ferroylquinic acid lactones and pyrogallol is high.

  The hot water extract of roasted coffee beans may be directly subjected to extraction with a fat-soluble organic solvent, and ferruoylquinic acid lactones, coumaroylquinic acid lactones, and pyrogallol are recovered but have a higher molecular weight than this. After performing the process which removes a compound, you may use for the extraction by a fat-soluble organic solvent. Since the hot water extract of roasted coffee beans contains a polymer compound having xanthine oxidase accelerating activity, by removing the high molecular weight fraction from the hot water extract of roasted coffee beans in advance, A fat-soluble organic solvent extract having high xanthine oxidase inhibitory activity is obtained. As a method for removing the high molecular weight fraction, for example, an ultrafiltration membrane having a fractional molecular weight of 1000 to 3000 is used, and the fraction that has passed through the ultrafiltration membrane is used as a fraction containing ferroylquinic acid lactones and the like. The method of collecting is mentioned.

  When the manufacturing method of the composition which concerns on this invention consists only of a fat-soluble organic-solvent layer collection | recovery process, the said fat-soluble organic-solvent extract is a xanthine oxidase inhibition composition manufactured by the said manufacturing method. The fat-soluble organic solvent extract thus obtained can be concentrated after removing the fat-soluble organic solvent. Moreover, the solid xanthine oxidase inhibitory composition containing feroylquinic acid lactone etc. is obtained by removing a fat-soluble organic solvent completely from the said composition. For removing the fat-soluble organic solvent, a method generally used for removing the organic solvent such as an evaporator can be appropriately used.

  The obtained fat-soluble organic solvent extract contains ferroylquinic acid lactones, coumaroylquinic acid lactones, chlorogenic acid lactones, pyrogallol and the like. Therefore, the method for producing a composition according to the present invention further comprises a step of purifying a specific compound having xanthine oxidase inhibitory activity from the fat-soluble organic solvent extract after the step of collecting the fat-soluble organic solvent layer. May be. That is, the method for producing a composition according to the present invention includes a specific method having xanthine oxidase inhibitory activity from the collected fat-soluble organic solvent layer (fat-soluble organic solvent extract) after the fat-soluble organic solvent layer collecting step. You may have the fractionation process which collect | recovers a compound. As the compound recovered in the fractionation step, pyrogallol, ferroylquinic acid lactones or coumaroylquinic acid lactones are preferable. In addition, “recovery of a specific compound” in the fractionation step refers to a ratio (mass) of the target compound to be recovered in the total composition after recovery of the total fat-soluble organic solvent extract before recovery ( The method is not limited to a method for completely isolating and purifying the compound.

  In the fractionation step, the method for recovering pyrogallol, ferroylquinic acid lactones, coumaroylquinic acid lactones, etc. is not particularly limited, and the target compound is generally purified from a composition containing a plurality of compounds. It can be used by appropriately selecting from the methods used in this case. Examples of the method include column chromatography. The fraction containing the target compound can be collected using the retention time of the chemically synthesized product (standard product) of the compound as an index. For example, from a fat-soluble organic solvent extract, by using a high-performance liquid column chromatography (HPLC) method or a medium pressure liquid chromatography (MPLC) method using a reverse phase system column such as an ODS column (C18 column), feruloquinic acid Lactones, coumaroylquinic acid lactones, and pyrogallol can be isolated and purified.

  The stability of pyrogallol is affected by pH. Pyrogallol is stable under acidic conditions of about pH 4.0 to 5.5. However, it is unstable at higher pH, for example, pH 6.0 to 8.0, and pyrogallol disappears when pyrogallol reacts with each other and is converted into purpurogallin. Purpurogallin also has high xanthine oxidase inhibitory activity and can be used as an active ingredient of the xanthine oxidase inhibitor according to the present invention.

  For example, in the fractionation step, after collecting a fraction containing pyrogallol, an alkali treatment step of adjusting the fraction to pH 6.0 to 8.0 can be performed to obtain an alkali-treated product of pyrogallol. it can. In the conversion reaction of pyrogallol to other compounds, the higher the pH, the faster the reaction rate. For this reason, in an alkali treatment process, it is preferable to adjust the fraction containing pyrogallol to pH 6.5-8.0, and it is more preferable to adjust to pH 7.0-8.0. The alkali treatment time may be a time sufficient to convert pyrogallol to pulprogalin or the like, and can be appropriately adjusted in consideration of pH. For example, 10 to 300 minutes are preferred, 10 to 200 minutes are more preferred, 10 to 120 minutes are more preferred, and 10 to 90 minutes are even more preferred.

  Purpurogallin is not very stable under alkaline conditions and is degraded. Then, in the manufacturing method of the composition which concerns on this invention, the acid treatment which adjusts the said fraction further adjusted to pH 6.5-8.0 after the said alkali treatment process to pH 4.5-5.5. It is also preferable to have a process.

  The alkali treatment and acid treatment of the fraction containing pyrogallol can be performed by a general pH adjustment treatment. For example, the pH of the fraction can be adjusted by adding a buffer having a target pH or mixing an alkali compound or an acid. The buffer can be appropriately selected from those commonly used in the field such as phosphate buffer, Tris buffer, citrate buffer, acetate buffer, borate buffer and the like. The alkali treatment can also be performed by a treatment in which a fraction containing pyrogallol is contacted with an alkaline resin such as an anion exchange resin. The acid treatment can also be performed by a treatment in which an oxidizing agent such as oxidized vitamin C (dehydroascorbic acid) is mixed with the fraction containing pyrogallol.

  The xanthine oxidase inhibitory composition produced by the method for producing a composition according to the present invention, ferroylquinic acid lactones, coumaroylquinic acid lactones, pyrogallol and purpurogallin have xanthine oxidase inhibitory activity. Therefore, these can be used as active ingredients of xanthine oxidase inhibitors. These are all coffee-derived substances that have been widely taken as drinks since ancient times, and can be taken relatively safely. Therefore, these are suitable as raw materials for foods and drinks, feeds, cosmetics, pharmaceuticals and the like, and are particularly useful as raw materials for pharmaceuticals and supplements used for the prevention and treatment of gout and hyperuricemia.

  The xanthine oxidase inhibitor according to the present invention is selected from the group consisting of a xanthine oxidase inhibitor composition produced by the method for producing a composition according to the present invention, ferroylquinic acid lactones, coumaroylquinic acid lactones, pyrogallol, and purprogalin. One or more of these ingredients are active ingredients. Ferroylquinic acid lactones, coumaroylquinic acid lactones, pyrogallol, and pulprogalin as active ingredients may not be derived from a hot water extract of roasted coffee beans, and may be, for example, chemically synthesized products. . Among the xanthine oxidase-inhibiting compositions produced by the method for producing a composition according to the present invention, among others, the fat-soluble organic solvent extract can be obtained more easily without requiring an operation by the HPLC method. It contains ferroylquinic acid lactones, coumaroylquinic acid lactones, chlorogenic acid lactones, and pyrogallol, and is very useful as an active ingredient for xanthine oxidase inhibitors. On the other hand, since it has higher xanthine oxidase inhibitory activity, the xanthine oxidase inhibitor according to the present invention was obtained by fractionating a compound having xanthine oxidase inhibitory activity from the fat-soluble organic solvent extract. It is also preferable. Among them, those having at least one of ferroylquinic acid lactones and coumaroylquinic acid lactones as active ingredients and those having at least one of pyrogallol and purprogalin as active ingredients are preferred. Those having both of them as active ingredients and those having both pyrogallol and purpurogallin as active ingredients may be used.

  The xanthine oxidase inhibitor according to the present invention may contain only feroylquinic acid lactones, coumaroylquinic acid lactones, pyrogallol, and purpurogallin as active ingredients, or may contain other ingredients. Good. Examples of other active ingredients include compounds having xanthine oxidase inhibitory activity derived from other roasted coffee beans, such as chlorogenic acid lactones, and xanthine oxidase inhibitor other than the hot water extract of roasted coffee beans The extract which extracted the compound which has activity is mentioned.

  In addition, the xanthine oxidase inhibitor according to the present invention may be composed only of an active ingredient having xanthine oxidase inhibitory activity, or may contain other ingredients. The other components may be those that do not impair the xanthine oxidase inhibitory action by xanthine oxidase inhibitory composition, ferroylquinic acid lactones, coumaroylquinic acid lactones, pyrogallol, and purprogalin, such as excipients, binding Agents, fluidity improvers (anti-caking agents), stabilizers, preservatives, pH adjusters, solubilizers, suspending agents, emulsifiers, thickeners, flavoring agents, sweeteners, acidulants, flavorings, coloring Various substances used as a material and the like may be appropriately contained according to desired product quality.

  The dosage form of the xanthine oxidase inhibitor according to the present invention is not particularly limited, and various dosage forms can be applied. Since the xanthine oxidase inhibitor according to the present invention exhibits a xanthine oxidase inhibitory effect when taken orally, those suitable for oral administration are preferred. Examples of the dosage form include tablets, capsules, granules, powders, syrups and the like.

  For example, the xanthine oxidase inhibitor composition from which the solvent has been removed, and purified ferroylquinic acid lactones, coumaroylquinic acid lactones, pyrogallol, and purpurogallin can be used as they are added to liquid coffee, instant coffee, etc. . Here, as liquid coffee, the coffee drink (or what is called a drink containing coffee) put into a can or what is called a PET bottle container and marketed is mentioned. Moreover, as instant coffee, what is called soluble powder coffee which removed the water | moisture content by spraying or freeze-drying the extract which extracted the roasting ground coffee with hot water is mentioned. Examples of the coffee mix beverage include beverages obtained by adding and mixing soluble powdered coffee with sugar, creaming powder, and the like.

  EXAMPLES Next, although an Example is shown and this invention is demonstrated further in detail, this invention is not limited to a following example etc.

<Measurement of xanthine oxidase inhibitory activity>
In the following examples and the like, unless otherwise stated, the xanthine oxidase inhibitory activity of samples was measured as follows.
First, 10 μL of a 1 mmol / L xanthine aqueous solution, 10 μL of a sample dissolved in DMSO (dimethyl sulfoxide), and 160 μL of 12.5 mmol / L phosphate buffer (pH 7.8) were mixed at 37 ° C. Preincubation for minutes. A control reaction solution using DMSO instead of the sample was used. Next, 20 μL of XO buffer (xanthine oxidase dissolved in buffer to 0.027 unit / mL) was added to the reaction solution, incubated at 37 ° C. for 10 minutes, reacted, and then 3% HCLO _The reaction was stopped by adding 25 μL of ₄ aqueous solution. Thereafter, 20 μL of the reaction solution was injected into the HPLC system under the following conditions to determine the uric acid concentration.

HPLC conditions;
Column: Mightysil RP-18 GP Aqua (250 × 4.6 mm (inner diameter)) (manufactured by Kanto Chemical Co., Inc.)
_Solvent: CH 3 OH-0.1% phosphoric acid aqueous solution (2.5: 97.5 (v / v )),
Flow rate: 0.5 mL / min,
Temperature: 35 ° C
Detection wavelength: 290 nm and 270 nm.

The xanthine oxidase inhibition rate was calculated by the following formula.
[Inhibition rate (%)] = ([Control uric acid peak area] − [Sample uric acid peak area]) × 100 / [Control uric acid peak area]

[Reference Example 1]
The effects of coffee beans type and roasting degree on xanthine oxidase inhibitory activity of xanthine oxidase inhibitory composition obtained from hot water extract of coffee beans were investigated. The coffee beans used were a total of 8 kinds of Brazilian Arabica "Brazil Santos" and Vietnamese robusta seeds, shallow roasted beans, medium roasted beans, and deep roasted beans. Table 1 shows the results of measuring the color of each coffee bean using a color difference meter. In the table, “h ^* ” represents a hue angle, “C” represents saturation, and “L ^* ” and “L ^{* 2} ” represent lightness. Hue angle, saturation, and lightness “L ^* ” were measured on the beans before grinding, and lightness “L ^{* 2} ” was measured on the ground beans.

  10 g of each coffee bean powder was immersed in 50 mL of boiling water, heated for 1 hour, and extracted with hot water while stirring. After removing the coffee powder by filter filtration, a portion of the obtained hot water extract is taken to measure the solid content (mg), and based on the measurement results, each hot water extract is solidified. About the sample which adjusted final concentration of to 0.6 mg / mL, the xanthine oxidase inhibitory activity was measured and the inhibition rate (%) of each sample was calculated | required. The results are shown in Table 1. As a result, in any coffee beans, the green beans slightly promoted xanthine oxidase activity, and the roasted beans inhibited xanthine oxidase activity. There was almost no difference depending on the roasting degree of roasted beans, and all showed inhibition of about 20%.

  Moreover, when the content of chlorogenic acids (neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid) and chlorogenic acid lactones in each hot water extract was measured by the method described in Non-Patent Document 1, all were chlorogenic acid. The content of chlorogenic acids was higher than that of lactones, and chlorogenic acids were the main components. Moreover, the tendency for both chlorogenic acids and chlorogenic acid lactones to decrease as the roasting degree increased was observed.

[Reference Example 2]
The hot water extract of roasted coffee beans was fractionated by molecular weight, and the xanthine oxidase inhibitory activity of each fraction was examined.
First, a hot water extract was prepared in the same manner as in Reference Example 1, using a powder of medium roasted beans (brightness L ^* = 32.23) of Brazilian Arabica species “Brazil Santos”. Three hot water extract solutions prepared by dissolving 30.0 mg of the prepared hot water extract in 1 mL of water were prepared, and one was filtered through an ultrafiltration membrane (Nanosep 3K Omega, Pall) with a molecular weight cut off of 3000. The filtrate that passed through the ultrafiltration membrane was designated as “MW <3K fraction”, and the fraction retained on the ultrafiltration membrane was designated as “MW> 3K fraction”. Similarly, about one of three hot water extract solutions, it filtered with the ultrafiltration membrane (Vivacon 500, Sartorius) of 2000 molecular weight cut off, and the filtrate which passed the ultrafiltration membrane is "MW <2K drawing. The fraction retained on the ultrafiltration membrane was designated as “MW> 2K fraction”. One hot water extract solution remains, using the vitamin _{B 12} molecular weight of about 1000 as an index, open column chromatography fractionation (filler: TOYOPEAL HW-40F, TOSOH Co.) performed earlier than vitamin B12 The eluted fraction was designated as “MW> 1K fraction”, the fraction containing vitamin B12 and eluted after vitamin B12 was designated as “MW <1K fraction”.

  A fraction of each fraction was collected to measure the solid content (mg), and based on the measurement results, each sample fraction was adjusted to a final solid content concentration of 0.3 mg / mL. The oxidase inhibitory activity was measured, and the inhibition rate (%) of each sample was determined. The results of the inhibition rate (%) are shown in Table 2 together with the solid content (mg) of each fraction. As a result, xanthine oxidase inhibitory activity was observed in the fraction having a low molecular weight, but xanthine oxidase activity was promoted in the fraction having a high molecular weight.

[Reference Example 3]
The ethanol extract, acetone extract, and ethyl acetate extract of roasted coffee beans were examined for xanthine oxidase inhibitory activity.
In the case of using ethanol as an extraction solvent, first, 1 g of medium roasted bean powder of Brazilian Arabica species “Brazil Santos” is immersed in 5 mL of ethanol, extracted with heating and stirring at 60 ° C. for 1 hour. The filtrate (one-time ethanol extract) was recovered by filtration through a filter. The roasted coffee bean powder, which is the residue on the filter, was immersed in 5 mL of fresh ethanol, heated at 60 ° C. for 1 hour and extracted with stirring, and then the filtrate (twice ethanol extract) was collected by filtration. . The roasted coffee bean powder, which is the residue on the filter, was immersed in 5 mL of fresh ethanol, extracted with heating and stirring at 60 ° C. for 1 hour, and then the filtrate (3 times ethanol extract) was collected by filter filtration. . All recovered ethanol extracts were mixed. The same was performed when acetone and ethyl acetate were used as the extraction solvent.

  A part of each solvent extract was collected to measure the solid content (mg), and based on the measurement results, each solvent extract was subjected to a final solid content concentration of 0.3 mg / mL or 0.15 mg / About the sample adjusted to mL, xanthine oxidase inhibitory activity was measured and the inhibition rate (%) of each sample was calculated | required. The results of the inhibition rate (%) are shown in Table 3 together with the extracted solid content (mg) of each solvent extract and the content of chlorogenic acid lactone (nmol / 1.0 mg). The content of chlorogenic acid lactone in each solvent extract was measured by the method described in Non-Patent Document 1.

  As a result, although the inhibition rate of each solvent extract was correlated with the content of chlorogenic acid lactone, the inhibition activity was insufficient. In other words, a compound having xanthine oxidase inhibitory activity in roasted coffee beans is poor in extraction efficiency when an organic solvent extraction is directly performed from the roasted coffee bean powder, and an extract having sufficient inhibitory activity is obtained. Not confirmed.

[Example 1]
The xanthine oxidase inhibitory activity was investigated about the dichloromethane extract and ethyl acetate extract of the hot water extract of roasted coffee beans.
First, a hot water extract was obtained in the same manner as in Reference Example 1, using a powder of medium roasted beans from Brazilian Arabica species “Brazil Santos”.

<Extraction method 1>
Extraction was performed by adding 5 mL of dichloromethane to 5 mL hot water extract (solid content: 100.0 mg) and stirring. The generated micellar insoluble matter was removed by cotton plug filtration, and the filtration residue was 7.3 mg. Then, the filtrate was separated and recovered by separating into a dichloromethane layer (dichloromethane extract) and an aqueous layer. To the collected aqueous layer, 5 mL of ethyl acetate was added and stirred for extraction. The generated micellar insoluble matter was removed by cotton plug filtration, and the filtrate was separated, and separated into an ethyl acetate layer (one ethyl acetate extract) and an aqueous layer. To the collected aqueous layer, 5 mL of ethyl acetate was added and stirred for extraction. The generated micellar insoluble matter was removed by cotton plug filtration, and the filtrate was separated and recovered by separating into an ethyl acetate layer (extracted twice with ethyl acetate) and an aqueous layer. A mixture of the ethyl acetate extract and the ethyl acetate extract was used as the ethyl acetate extract. In addition, the total amount of micelle-like insoluble matter generated by mixing ethyl acetate (total amount of two filtration residues) was 3.3 mg.

<Extraction method 2>
Extraction was performed by adding 5 mL of ethyl acetate to 5 mL of hot water extract (solid content: 100.0 mg) and stirring. The generated micellar insoluble matter was removed by cotton plug filtration, and the filtrate was separated, and separated into an ethyl acetate layer (one ethyl acetate extract) and an aqueous layer. To the collected aqueous layer, 5 mL of ethyl acetate was added and stirred for extraction. The generated micellar insoluble matter was removed by cotton plug filtration, and the filtrate was separated and recovered by separating into an ethyl acetate layer (extracted twice with ethyl acetate) and an aqueous layer. A mixture of the ethyl acetate extract and the ethyl acetate extract was used as the ethyl acetate extract. In addition, the total amount of micellar insoluble matter generated by mixing ethyl acetate (total amount of two filtration residues) was 5.6 mg.

  Regarding the dichloromethane extract, ethyl acetate extract, and aqueous layer obtained in Extraction Method 1, and the ethyl acetate extract and aqueous layer obtained in Extraction Method 2, the solid content (mg) and xanthine oxidase inhibitory activity were determined. Examined. Xanthine oxidase inhibitory activity was measured using a sample in which the final concentration of solids was adjusted to 0.3 mg / mL. Table 4 shows the measurement results.

  As shown in Table 4, in an organic solvent extract of a hot water extract of roasted coffee beans, particularly an extract with ethyl acetate, a strong xanthine oxidase inhibitory activity exceeding 60% was confirmed at a low concentration of 300 ppm by mass. It was done. From these results, it was found that a powerful xanthine oxidase inhibitor derived from roasted coffee beans can be efficiently extracted from a hot water extract of roasted coffee beans by a simple technique of extraction with a fat-soluble organic solvent.

[Example 2]
The xanthine oxidase inhibitory activity was investigated about the extract extracted with the hot water extract of the roasted coffee beans from which a kind and the roasting degree differ with ethyl acetate. The coffee beans used were a total of nine types of shallow roast beans, medium roast beans, and deep roast beans of Brazilian Arabica “Brazil Santos”, Vietnamese Robusta, and Columbian Arabica “Excelso”.
2.00 g of each roasted coffee bean powder was immersed in 10 mL of boiling water, heated for 1 hour, and extracted with hot water while stirring. Next, 5 mL of ethyl acetate was added in two portions to the hot water extract from which the solid content was removed by filtration in the same manner as in Extraction Method 2 of Example 1 to obtain an ethyl acetate extract. A part of the obtained ethyl acetate extract was collected and the solid content (mg) was measured. Based on the measurement results, each ethyl acetate extract had a final solid content concentration of 0.03, 0.0. About the sample adjusted to 1 or 0.3 mg / mL, the xanthine oxidase inhibitory activity was measured and the inhibition rate (%) of each sample was calculated | required. For each ethyl acetate extract, the 50% inhibitory concentration (IC ₅₀ ) was determined from the inhibition rates (%) at final concentrations of 0.03, 0.1, and 0.3 mg / mL. Table 5 shows the solid content (mg), inhibition rate (%), and IC ₅₀ (mg / mL) of each ethyl acetate extract.

As a result, an ethyl acetate extract having a very high inhibition rate of about 60% was obtained from any roasted coffee beans. Thus, it was confirmed that an extract having a high xanthine oxidase inhibitory activity can be obtained by extracting the hot water extract of roasted coffee beans with ethyl acetate regardless of the production region and type of the coffee beans. Further, it was found that in any roasted coffee beans, deep roasted beans had the smallest IC ₅₀ , and deep roasted beans yielded an ethyl acetate extract having a stronger xanthine oxidase inhibitory activity.

  Non-patent document 1 describes the content of chlorogenic acid lactones for ethyl acetate extracts prepared from a total of 6 types of Brazilian Arabica and Vietnamese Robusta lightly roasted beans, medium roasted beans, and deeply roasted beans. It measured by the method of. Table 6 shows the measurement results. As a result, it was observed that the content of chlorogenic lactones tended to decrease as the roasting degree increased. From the results of Tables 5 and 6, in the ethyl acetate extract prepared from deep roasted beans, compounds having xanthine oxidase inhibitory activity other than chlorogenic acid lactones are present. It was suggested that inhibitory activity was achieved.

[Example 3]
A compound with xanthine oxidase inhibitory activity was isolated and identified in ethyl acetate extract prepared from hot water extract of Brazilian Arabica light and deep roast beans.
First, in the same manner as in Example 2, hot water extracts were prepared from powders of lightly roasted beans and deep roasted beans of Brazilian Arabica, and ethyl acetate extracts were prepared from the hot water extracts. This ethyl acetate extract was separated by HPLC using an ODS column, and fraction 1 eluted before chlorogenic acid lactones (fraction before chlorogenic acid lactones) and chlorogenic acid lactones were eluted. Fraction 2 (chlorogen acid lactones eluted fraction) and fraction 3 (fraction after chlorogenic acid lactones elution) eluted after chlorogenic acid lactones. Preparative HPLC conditions were as follows.

Preparative HPLC conditions;
Column: Cosmosil 5C18-AR-II (250 × 20 mm (inner diameter), manufactured by Nacalai Tesque),
Solvent A: 1% acetic acid-containing aqueous solution,
Solvent B: acetonitrile,
Elution conditions: Until elution of chlorogenic acid lactones [A: B = 85: 15 (v / v)], elution time of chlorogenic acid lactones [A: B = 50: 50 (v / v)], chlorogenic acid lactone [A: B = 0: 100 (v / v)] after elution
Flow rate: 9.6 mL / min,
Detection wavelength: 320 nm.

  Fig. 1 shows a preparative HPLC chromatogram of an ethyl acetate extract prepared from a hot roast bean extract. Fig. 2 shows an ethyl acetate extract prepared from a deep roast bean extract. Preparative HPLC chromatograms are shown respectively. In the figure, "1" is fraction 1 (fraction before chlorogenic acid lactones elution), "2" is fraction 2 (chlorogenic acid lactones elution fraction), and "3" is fraction 3 (chlorogenic acid lactones). The fraction after elution) is shown.

For each fraction, in the same manner as in Example 2, the final concentration 0.03, 0.1, and 0.3 mg / mL inhibition of the (%) was determined 50% inhibition concentration _{(IC 50).} Table 7 shows the inhibition rate (%) and IC ₅₀ (mg / mL) at a final concentration of 0.3 mg / mL for each fraction. As a result, xanthine oxidase inhibitory activity was observed in all of Fraction 1, Fraction 2, and Fraction 3 for both deeply roasted beans and lightly roasted beans. That is, it was found that all fractions contained substances having high xanthine oxidase inhibitory activity other than chlorogenic lactones with known activity.

  The total component analysis by HPLC-TOFMS was performed for the compounds contained in fractions 1 to 3 of deep roasted beans. The conditions for HPLC and TOFMS were as follows. The elemental composition of the peak compound was calculated using MassLynx software (V.4.1, manufactured by Waters) using high-resolution MS data from deprotonated molecule ions.

HPLC conditions;
Column: Cosmosil 5C18-AR-II (250 × 4.6 mm (inner diameter), manufactured by Nacalai Tesque),
Solvent A: 1% acetic acid-containing aqueous solution,
Solvent B: acetonitrile,
Linear gradient conditions: from 5% solvent B (0 minutes) to 45% solvent B (80 minutes), further to 100% solvent B (100 minutes),
Flow rate: 0.5 mL / min,
Detection: 280 nm and 320 nm.

TOFMS conditions;
ESI: negative,
Capillary voltage: 1.2 kV
Cone voltage: 23V
Ion source temperature: 120 ° C.
Desolvation temperature: 450 ° C
Cone gas flow rate: 50 L / h,
Desolvent gas flow rate: 800 L / h,
MSE low collision energy: 6V
MSE high collision energy: 20-30V.

  In the fraction 1 of deep roasted beans, the main compound peak and its molecular weight data were not obtained in the HPLC-TOFMS analysis, but in the HPLC-TOFMS analysis of the fraction 2, four main peaks and their MS data were confirmed. (FIG. 3). Numbers 1 to 4 are assigned in order from the peak with the short retention time, and Table 8 shows the MS data of these peaks.

  The HPLC-TOFMS chromatogram of fraction 3 of deep roasted beans confirmed six main peaks (FIG. 4). Numbers 5-10 are assigned in order from the peak with the shortest retention time, and Table 9 shows the MS data of these peaks.

  From the molecular weight related ions and fragment ions of each main peak of fractions 2 and 3 obtained by HPLC-TOFMS, peaks 1 to 4 of fraction 2 are lactones of chlorogenic acid (positional isomers of caffeic acid moiety), The main peak 7 of fraction 3 is feroylquinic acid lactone (positional isomer of ferulic acid moiety is unknown) together with 5, and peak 6 is coumaroylquinic acid lactone (position of coumaric acid moiety is unknown) It was confirmed. From these results, it is found that the fat-soluble organic solvent extract of the hot water extract of roasted coffee beans contains ferroylquinic acid lactones and coumaroylquinic acid lactones in addition to chlorogenic acid lactones. all right. Ferroylquinic acid lactones and coumaroylquinic acid lactones are also considered to have a strong xanthine oxidase inhibitory activity derived from deep roasted beans.

[Example 4]
For ethyl acetate extract of hot water extract of roasted coffee beans, compounds having xanthine oxidase inhibitory activity other than ferroylquinic acid lactones and coumaroylquinic acid lactones were examined.
First, 2 kg of roasted coffee bean powder (lightness L ^* value: 21.7) was immersed in 10 L of boiling water and heated for 2 hours with occasional stirring to perform hot water extraction. Next, the hot water extract from which the solid content was removed by filtration was extracted twice with 10 L of ethyl acetate in the same manner as in Extraction Method 2 of Example 1 to obtain an ethyl acetate extract. The obtained ethyl acetate extract was concentrated under reduced pressure to prepare 32.3 g of an ethyl acetate soluble fraction.

  Of the ethyl acetate soluble fraction, 0.35 g was separated into 10 fractions (Fr. 1 to Fr. 10) by MPLC using an ODS column. The MPLC conditions were as follows.

MPLC conditions;
Column: ULTRA PACK ODS-S-50 (300 × 50 mm (inner diameter), manufactured by Yamazensha),
Solvent: Mixed solvent of acetonitrile / 0.1% acetic acid-containing aqueous solution (12.5 / 87.5 (volume ratio)),
Flow rate: 9.6 mL / min.

  A part of each fraction was collected and the solid content (mg) was measured. 1 is 31.8 mg, Fr. 2 is 15.5 mg, Fr. 3 is 10.7 mg, Fr. 4 is 105.4 mg, Fr. 5 is 29.9 mg, Fr. 6 is 9.8 mg, Fr. 7 is 9.4 mg, Fr. 8 is 5.7 mg, Fr. 9 is 8.3 mg, Fr. 10 was 12.1 mg. Moreover, about the sample which adjusted the final concentration of each fraction to 0.15 mg / mL, the xanthine oxidase inhibitory activity was measured and the inhibition rate (%) of each sample was calculated | required. The measurement results are shown in FIG. 5 (n = 3). In FIG. 5, “*” indicates p <0.05 relative to the control by t-test test.

  Next, Fr. 2 were separated by HPLC using an ODS column, and 6 fractions were fractionated from the peak of the obtained chromatogram. The obtained chromatogram and the fractions collected (Fr. 2-1 to Fr. 2-6) are shown in FIG. The HPLC conditions were as follows.

HPLC conditions;
Column: Cosmosil 5C18-AR-II (250 × 4.6 mm (inner diameter), manufactured by Nacalai Tesque),
Solvent A: 1% acetic acid-containing aqueous solution,
Solvent B: acetonitrile,
Stepwise elution conditions: 5% solvent B (between 0 and 16 minutes), then 10% solvent B (between 16 and 28 minutes), then 100% solvent B (between 28 and 44 minutes),
Flow rate: 9.6 mL / min,
Detection: 280 nm.

  About each fraction (Fr.1-Fr.6), the xanthine oxidase inhibitory activity was measured and the inhibition rate (%) of each sample was calculated | required, Fr. 2-1 had the highest xanthine oxidase inhibitory activity, and it was suggested that the compound having a peak at a retention time of 6.5 minutes has a very strong xanthine oxidase inhibitory activity in the chromatogram of FIG. Therefore, the Fr. From 2 under the same HPLC conditions as described above, a fraction containing a compound having a peak at a retention time of 6.5 minutes was collected. The collected fraction had a solid content of 14.3 mg. This fraction was further purified by gel filtration chromatography (solvent: methanol) using a Sephadex LH-20 column (560 × 2.5 mm (inner diameter)) to isolate 4 mg of pure active substance. The obtained active substance was identified as pyrogallol from the following NMR and MS data.

_{^{1 H-NMR (CD 3 OD}} , 300MHz): δ6.31 (2H, AA 'B) and δ6.51 (1H, AA' B) .

DART-TOFMS: m / z 127.0383 [M + H] ⁺ (monoisotopic mass of C ₆ H ₅ O ₃ is 127.0395), m / z 125.0231 [M−H] ⁻ (C ₆ H ₃ O The monoisotopic mass of ₃ is 125.0239).

Xanthine oxidase inhibitory activity was measured for pyrogallol and its structurally similar compounds. As a result, pyrogallol the _{IC 50} is that 0.73μmol / L, gout agent allopurinol _(IC 50: _0.50μmol / L) showed activity intensity comparable to. On the other hand, when the xanthine oxidase inhibition rate at 200 μmol / L of pyrogallol was 100%, the relative inhibition rate (%) of each compound was 31.7 ± 1.5% for 1,2,4-trihydroxybenzene, and chlorolucinol, respectively. Is 5.92 ± 0.21%, hydroquinone is −8.94 ± 1.0%, catechol is −3.39 ± 2.4%, resorcinol is −0.41 ± 2.6%, Such a high activity was not observed.

[Example 5]
The stability of pyrogallol at pH 5.0, 6.0, 72, and 7.8 was investigated. Note that pH 5.0 corresponds to the pH of the hot water extract of shallow roasted coffee beans, pH 6.0 corresponds to the pH of the hot water extract of deep roasted coffee beans, and pH 7.4 corresponds to the pH in the living body. PH 7.8 is a measurement condition for xanthine oxidase inhibitory activity.

  Specifically, 10 μL of 4 mM pyrogallol (chemically synthesized product) and 190 μL of 12.5 mM phosphate buffer (pH 5.0, 6.0, 7.4, or 7.8) were mixed, and the resulting sample The solution was incubated for 290 minutes at 37 ° C. A part of each sample solution was sampled at 0, 10, 80, 150, 220, and 290 minutes after the start of incubation, and component analysis was performed by HPLC. The HPLC conditions were as follows.

HPLC conditions;
Column: Cosmosil 5C18-AR-II (250 × 4.6 mm (inner diameter), manufactured by Nacalai Tesque),
Solvent A: 0.1% phosphoric acid-containing aqueous solution,
Solvent B: acetonitrile,
Linear gradient conditions: 5% solvent B (0 minutes) to 45% solvent B (40 minutes), then 100% solvent B (50 minutes), 100% solvent B maintained up to 55 minutes, then 5% solvent B (55.1 minutes) and maintain 5% solvent B for up to 65 minutes,
Flow rate: 1.0 mL / min,
Detection: 254, 280, 320 nm,
Injection volume: 20 μL.

  The measurement results are shown in FIG. FIG. 7A shows the result of the pH 7.8 sample solution, FIG. 7B shows the result of the pH 7.4 sample solution, and FIG. 7C shows the result of the pH 6.0 sample solution. (D) shows the result of the sample solution of pH 5.0, respectively. In the figure, “Pyr” is the measurement result of pyrogallol. As a result, it was found that pyrogallol was stable and hardly decreased at pH 5.0, but disappeared as the incubation time increased at pH 6.0 to 7.8. Pyrogallol disappeared slowly over 5 hours at pH 6.0, but at pH 7.4 and pH 7.8, pyrogallol decreased to about half after 10 minutes from the start of incubation, and almost all of it disappeared in about 1 hour. .

In the sample of pH 5.0, no other peak was confirmed by HPLC, but in the sample of pH 6.0 to 7.8, another compound peak was confirmed to correlate with the disappearance of pyrogallol. The compound contained in the peak was identified as purpurogallin from the result of comparing the HPLC retention time and the characteristic UV-VIS spectrum (λmax) with the standard data. Purpurogallin used as a sample was prepared by pyrogallol NaIO ₃ oxidation (Evans and Dehn, Journal of the American Chemical Society, 1930, vol. 52, p. 3647-3649). The NMR and MS data of purpurogallin are shown below.

¹ H-NMR (CH ₃ OD): δ ppm 6.72 (1H, dd, J = 15.2 and 12.4 Hz), 6.86 (1H, s), 7.06 (1H, d, J = 15) .2 Hz), 7.29 (1H, d, J = 12.4 Hz).

HR-DART-MS: m / z 221.0469 [M + H] ⁺ (monoisotopic mass of C ₁₁ H ₉ O ₅ is 221.0450).

  Measurement results of the content of purpurogallin contained in each sample solution are shown in FIGS. In the figure, “Pur” is the measurement result of purpurogallin. As a result, purpurogallin was not detected at pH 5.0, but was slowly produced at pH 6.0, and at pH 7.4 and pH 7.8, it was rapidly produced by 10 minutes after the start of incubation, After that, it gradually decreased and almost disappeared in about 4 hours.

In addition, the stability of purpurogallin at pH 7.4 was examined.
Specifically, 50 μL of 0.4 mM or 4 mM purpurogallin (chemically synthesized product) and 950 μL of 12.5 mM phosphate buffer (pH 7.4) were mixed, and the resulting sample solution (purpurogallin concentration: 20 μM or 200 μM). Was incubated at 37 ° C. for 300 minutes. A part of each sample solution was sampled at 0, 30, 60, 120, 180, 240, and 300 minutes after the start of incubation, and component analysis was performed by HPLC. The HPLC conditions were the same as the pyrogallol conditions except that the detection wavelength was changed from 254 nm to 268 nm.

  The measurement results are shown in FIG. FIG. 8A shows the result of the sample solution having a purpurogallin concentration of 20 μM, and FIG. 7B shows the result of the sample solution having a purprogalin concentration of 200 μM. As a result, in all the sample solutions, purpurogallin gradually decreased and almost all disappeared in 5 hours from the start of incubation.

[Example 6]
When pyrogallol was incubated under the condition of pH 7.4, the changes over time in the contents of pyrogallol and purpurogallin and the santin oxidase inhibitory activity were examined.
Specifically, 50 μL of 400 μM pyrogallol (chemically synthesized product) and 950 μL of 12.5 mM phosphate buffer (pH 7.4) were mixed, and the obtained sample solution was incubated at 37 ° C. for 300 minutes. A part of each sample solution was sampled at 0, 30, 60, 120, 240, and 300 minutes after the start of incubation, and component analysis by HPLC and measurement of santin oxidase inhibitory activity were performed.

  Component analysis by HPLC was performed using 80 μL of sample solution with 8% of 3% perchloric acid added, and HPLC conditions were as follows: the injection amount was 22 μL, and the detection wavelength was changed from 254 nm to 268 nm. The same conditions as in Example 5 were used.

Xanthine oxidase inhibitory activity was measured by first preparing a solution in which 10 μL of 1 mmol / L xanthine aqueous solution and 160 μL of 12.5 mM phosphate buffer (pH 7.4) were mixed, and adding 10 μL of the sample solution to the solution. Immediately after stirring, 20 μL of XO buffer (xanthine oxidase dissolved in buffer to 0.011 unit / mL) was added, reacted at 37 ° C. for 10 minutes, and then reacted with 3% HCLO ₄ aqueous solution. The reaction was stopped by adding 25 μL. Thereafter, 20 μL of the reaction solution was injected into the HPLC system, the uric acid concentration was determined, and the xanthine oxidase inhibition rate (%) was determined. A control reaction solution was prepared by using 10 μL of 12.5 mM phosphate buffer (pH 7.4) instead of 10 μL of the sample solution. The HPLC conditions were the same as the HPLC conditions described in <Measurement of xanthine oxidase inhibitory activity> except that the flow rate was changed to 1.0 mL / min.

  The measurement results are shown in FIG. 9 (n = 2). In the figure, “Pyr” is the result of pyrogallol content (left vertical axis), “Pur” is the result of purpurogallin content (μM, left vertical axis), and “XOI” is the xanthine oxidase inhibition rate ( %, Right vertical axis). Pyrogallol was reduced to less than half after 30 minutes after addition of phosphate buffer at pH 7.4 (after the start of incubation), and the total amount disappeared after 2 hours. The amount of purpurogallin reached its maximum at 30 minutes after the start of incubation, and then gradually decreased, and almost all disappeared after 240 minutes. On the other hand, the inhibition rate of xanthine oxidase is 34.1% at the start of incubation (before purpurogallin production), and increases to about 60% at 30 to 60 minutes after the start of incubation. It gradually decreased, and almost no xanthine oxidase inhibitory activity was observed at 240 minutes after the start of incubation. As shown in FIG. 9, the increase / decrease in the xanthine oxidase inhibition rate was linked to the purpurogallin content. From these results, it was suggested that both pyrogallol and purprogalin have xanthine oxidase inhibitory activity, but that of purpurogallin is stronger than that of pyrogallol.

Claims (9)

  After mixing the hot water extract of roasted coffee beans and a fat-soluble organic solvent, it has a fat-soluble organic solvent layer recovery step of recovering only the fat-soluble organic solvent layer as a composition having xanthine oxidase inhibitory activity And a method for producing a composition having xanthine oxidase inhibitory activity.   The method for producing a composition having xanthine oxidase inhibitory activity according to claim 1, wherein the fat-soluble organic solvent is ethyl acetate, hexane, chloroform, dichloromethane, diethyl ether, or a mixed solvent containing any of these.   The composition which has a xanthine oxidase inhibitory activity of Claim 1 or 2 which has the fractionation process which collect | recovers the fraction containing a pyrogallol from the collect | recovered fat-soluble organic solvent layer after the said fat-soluble organic solvent layer collection | recovery process. Manufacturing method.   The manufacturing method of the composition which has the xanthine oxidase inhibitory activity of Claim 3 which has an alkali treatment process which adjusts the fraction containing the said pyrogallol to pH 6.5-8.0 after the said fractionation process.   It has a xanthine oxidase inhibitory activity of Claim 4 which has an acid treatment process which adjusts the said fraction adjusted to pH 6.5-8.0 to pH 4.5-5.5 after the said alkali treatment process. A method for producing the composition.   A xanthine oxidase inhibitor comprising a fat-soluble organic solvent extract extracted from a hot water extract of roasted coffee beans with a fat-soluble organic solvent as an active ingredient.   A xanthine oxidase inhibitor characterized by comprising feroylquinic acid lactone as an active ingredient.   A xanthine oxidase inhibitor comprising coumaroylquinic acid lactone as an active ingredient.   A pharmaceutical comprising the xanthine oxidase inhibitor according to any one of claims 6 to 8.