JP2019156780A - Xanthine oxidase inhibitor - Google Patents

Abstract

To provide xanthine oxidase inhibitors that are derived from coffee, to provide medicines and functional foods containing the xanthine oxidase inhibitors as an active ingredient, and to provide methods for recovering the fractions containing a compound with xanthine oxidase inhibitory activity from coffee roasted beans as a composition with xanthine oxidase inhibitory activity.SOLUTION: A quinic acid lactone derivative represented by any one of the following general formulas (1) to (3) is used as an active ingredient [in the formulas (1) to (3), R, R, and Rrepresent each independently a hydrogen atom, a caffeoyl group, a feruloyl group, or a coumaroyl group, however, the case where two or more of Rto Rare hydrogen atoms is excluded] .SELECTED DRAWING: None

Description

  The present invention relates to a xanthine oxidase inhibitor derived from food and drink.

  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. Patent Document 5 reports that ferruoylquinic acid lactones or coumaroylquinic acid lactones have xanthine oxidase inhibitory activity. 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.

Japanese Patent No. 4988166 Japanese Patent No. 5044643 Japanese Patent No. 5437672 Japanese Patent No. 5498067 JP 2017-048185 A

Honda, et al., Bioscience, Biotechnology, and Biochemistry, 2014, vol. 78 (12), p.2110-2116.

  The xanthine oxidase inhibitory active ingredients described in Patent Documents 1 to 5 are difficult to purify and isolate from natural products as raw materials and are not sufficiently active. 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.

  The present invention is a xanthine oxidase inhibitor derived from coffee, which is a taste beverage most used in the world and is expected to have a gout prevention effect, a pharmaceutical and functional food containing the xanthine oxidase inhibitor as an active ingredient, and It is an object of the present invention to provide a method for recovering a fraction containing a compound having xanthine oxidase inhibitory activity from roasted coffee beans as a composition having xanthine oxidase inhibitory activity.

  As a result of diligent research to solve the above-mentioned problems, the present inventors have found that quinic acid is a quinic acid lactone in which one or more compounds selected from the group consisting of caffeic acid, ferulic acid, and coumaric acid are ester-bonded. The lactone derivative was found to have a strong xanthine oxidase inhibitory activity and completed the present invention.

[1] The xanthine oxidase inhibitor according to the first aspect of the present invention includes the following general formulas (1) to (3).

[In the formulas (1) to (3), R ¹ , R ² , and R ³ each independently represent a hydrogen atom, a caffeoyl group, a feruloyl group, or a coumaroyl group. However, the case where two or more of R ^{1 to} R ³ are hydrogen atoms is excluded. ]
A xanthine oxidase inhibitor comprising a quinic acid lactone derivative represented by any of the above:
[2] As the xanthine oxidase inhibitor of [1], any ^{two of} R ¹ , R ² , and R ³ are each independently a caffeoyl group, a feruloyl group, or a coumaroyl group, The remaining one is preferably a hydrogen atom.
The xanthine oxidase inhibitor of [3] [1], wherein of R ^1, R ^2, and R ^3, 2 two or but a caffeoyl group, is preferably one of the hydrogen atoms remain.
[4] The xanthine oxidase inhibitor of [1] is represented by the general formula (2) or (3), and R ¹ , R ² are independent of each other among R ¹ , R ² , and R ^3. Thus, it is preferable to use a quinic acid lactone derivative, which is a caffeoyl group, a feruloyl group, or a coumaroyl group, and R ³ is a hydrogen atom as an active ingredient.
[5] The xanthine oxidase inhibitor according to the second embodiment of the present invention is a fat extracted with a fat-soluble organic solvent from a hot water extract of roasted coffee beans whose L * value of ground beans is 30 or more and 45 or less. A soluble organic solvent extract is used as an active ingredient.
[6] The method for producing a composition having xanthine oxidase inhibitory activity according to the third aspect of the present invention comprises a hot water extract of roasted coffee beans having a L * value of 30 to 45 and fat-soluble. After mixing an organic solvent, it has the fat-soluble organic-solvent layer collection | recovery process which collect | recovers only a fat-soluble organic-solvent layer as a composition which has a xanthine oxidase inhibitory activity.
[7] As a method for producing the composition having xanthine oxidase inhibitory activity of [6], 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
[8] As a method for producing the composition having xanthine oxidase inhibitory activity of [6] or [7], after the fat-soluble organic solvent layer recovery step, from the recovered fat-soluble organic solvent layer, the following general formula: (1)-(3)

[In the formulas (1) to (3), R ¹ , R ² , and R ³ each independently represent a hydrogen atom, a caffeoyl group, a feruloyl group, or a coumaroyl group. However, the case where two or more of R ^{1 to} R ³ are hydrogen atoms is excluded. ]
It is preferable to have a fractionation process which collect | recovers the fraction containing the quinic acid lactone derivative represented by either.
[9] A pharmaceutical product according to the fourth aspect of the present invention is characterized by containing the xanthine oxidase inhibitor according to any one of [1] to [5].
[10] The pharmaceutical product of [9] is preferably used for the treatment or prevention of recurrence of gout.
[11] The functional food according to the fifth aspect of the present invention contains the xanthine oxidase inhibitor of any one of [1] to [5], and reduces the uric acid level in serum.

  The xanthine oxidase inhibitor according to the present invention contains, as an active ingredient, a quinic acid lactone derivative that is a component that is naturally contained in coffee in a relatively large amount. For this reason, since the xanthine oxidase inhibitor has a high uric acid level reducing effect and can be administered relatively safely, it is suitable as an active ingredient for functional foods such as supplements and an active ingredient for pharmaceuticals. In particular, it is suitable as an active ingredient of pharmaceuticals for the prevention and treatment of gout and hyperuricemia and as an active ingredient of functional foods for reducing serum uric acid levels.

It is the figure which showed the relationship between the content (per 1g of green beans) of the various components in roasted coffee beans, and the roasting degree in the reference example 1. FIG. It is the figure which showed the relationship between the color tone (L * value) of roasted coffee beans, and the content of diCQLs (per 1 g of green beans) in Reference Example 1. 2 is a preparative HPLC chromatogram of a heated product (ultra shallow roast) of 5-caffeoylquinic acid in Example 1. FIG. It is the figure which showed the measurement result of the xanthine oxidase inhibition rate (%) of the solution which prepared each fraction shown in FIG. 3 to solid content concentration 0.3 mg / mL or 0.1 mg / mL. In Example 1, the inhibition rate of xanthine oxidase of 3,4-diCQL, 4,5-diCQL, 3,5-diCQL, 5-CQL, 3-CQL, and 4-CQL was measured, and the IC ₅₀ was examined. It is the figure which showed the result.

  In the present invention and the present specification, xanthine oxidase inhibitory action means an action to suppress various physiological functions caused by an increase in blood uric acid level, and xanthine oxidase inhibitor is an agent having a uric acid level reducing action. is there.

The xanthine oxidase inhibitor according to the present invention contains a quinic acid lactone derivative represented by any one of the following general formulas (1) to (3) as an active ingredient. In the following general formulas (1) to (3), R ¹ , R ² , and R ³ each independently represent a hydrogen atom, a caffeoyl group, a feruloyl group, or a coumaroyl group. However, the case where two or more of R ^{1 to} R ³ are hydrogen atoms is excluded.

  The caffeoyl group is represented by the following formula (a), the feruloyl group is represented by the following formula (b), and the coumaroyl group is represented by the following formula (c). In formulas (a) to (c), a bond marked with “*” represents a bond with an oxygen atom bonded to the cyclohexane ring in general formulas (1) to (3).

As the quinic acid lactone derivatives represented by the general formulas (1) to (3), any one of R ¹ , R ² and R ³ is a hydrogen atom, and the remaining two are caffeoyl groups and feruloyl. Or a compound that is a coumaroyl group, and all of R ¹ , R ² , and R ³ may be each independently a caffeoyl group, a feruloyl group, or a coumaroyl group.

When any one of R ¹ , R ² , and R ³ is a hydrogen atom, the remaining two groups may be the same group or different groups. Similarly, when all of R ¹ , R ² , and R ³ are a caffeoyl group, a feruloyl group, or a coumaroyl group, these three groups may all be the same group, or two or more groups may be combined. It may be done.

Table 1 shows combinations of R ¹ , R ² , and R ³ groups of the quinic acid lactone derivatives represented by the general formulas (1) to (3). In Table 1, “H” represents a hydrogen atom, “(a)” represents a caffeoyl group, “(b)” represents a feruloyl group, and “(c)” represents a coumaroyl group.

The active ingredient of the xanthine oxidase inhibitor according to the present invention is a quinic acid lactone derivative represented by the general formulas (1) to (3), wherein R ¹ and R ² are each independently a caffeoyl group, A quinic acid lactone derivative which is a feruloyl group or a coumaroyl group and R ³ is a hydrogen atom is preferred, and is a quinic acid lactone derivative represented by the general formulas (1) to (3), which is a combination 1, The quinic acid lactone derivative represented by 2 or 3 is more preferred, and is the quinic acid lactone derivative represented by the general formula (2) or (3), which is represented by the combination 1, 2, or 3 in Table 1. More preferred is a quinic acid lactone derivative represented by the general formula (2) or (3), and a quinic acid lactone derivative represented by the combination 1 in Table 1 is particularly preferred.

  The xanthine oxidase inhibitor according to the present invention may contain only one kind of quinic acid lactone derivative represented by any one of the general formulas (1) to (3), and contains two or more kinds in combination. It may be. For example, the xanthine oxidase inhibitor according to the present invention may contain only dicaffeoylquinic acid lactone represented by the general formula (1) of the combination 1 in Table 1 as an active ingredient. The quinic acid lactone derivative represented by the general formula (1) of the combination 1 in Table 1 and the quinic acid lactone derivative represented by the general formula (2) in the combination 1 of Table 1 and the general formula (3) of the combination of Table 1 It may contain a quinic acid lactone derivative.

The quinic acid lactone derivatives represented by the general formulas (1) to (3) are, for example, quinic acid lactones (general formulas (1) to (3), in which all of R ¹ , R ² , and R ³ are The compound can be synthesized by dehydration condensation of a hydroxyl group of a compound that is a hydrogen atom and a carboxyl group of caffeic acid, ferulic acid, or p-coumaric acid. The dehydration condensation reaction can be performed by a conventional method.

  The quinic acid lactone derivatives represented by the general formula (1) include, for example, 1-caffeoylquinic acid (CAS ID: 1241-87-8), 3-caffeoylquinic acid (CAS ID: 327-97-). 9), 5-Caffeoylquinic acid, 1,3-dicaffeoylquinic acid (CAS ID: 19870-46-3), 3,4-dicaffeoylquinic acid (CAS ID: 14534-61-3), 4,5-dicaffeoylquinic acid (CAS ID: 57378-72-0), 1,3-dicaffeoylquinic acid (CAS ID: 30964-13-7), 3,5-dicaffeoylquinic acid (CAS CAS ID: 2450-53-5), 4,5-Diferloylquinic acid, 3,5-Diferloylquinic acid, 3-Ferloylquinic acid (CAS ID: 1899-29-2), 4-Ferro Roylquinic acid (CAS ID: 2613-86-7), 5-feruloylquinic acid, 4,5-diferloylquinic acid, 3,5-diferloylquinic acid, 3,4-diferloylquinic acid , 3-p-Cumaloy Quinic acid (CAS ID: 1899-30-5), 4-p-coumaroylquinic acid (CAS ID: 93451-44-6), 5-p-coumaroylquinic acid (CAS ID: 32451-86-8), 1-p Coumaroyl quinic acid, 3-caffeoyl-4-feruloyl quinic acid, 3-caffeoyl-5-feruloyl quinic acid, 4-caffeoyl-5-feruloyl quinic acid, 1-caffeoyl-5-feruloyl quina Carboxyl group bonded to cyclohexane ring by heat treatment of chlorogenic acid such as acid, 3-feruloyl-4-caffeoylquinic acid, 3-feruloyl-5-caffeoylquinic acid, 4-feruloyl-5-caffeoylquinic acid And a hydroxyl group can be synthesized by dehydration reaction. Lactonization by heat treatment of chlorogenic acid can be performed by a conventional method.

  The quinic acid lactone derivatives represented by the general formulas (1) to (3) are relatively abundantly contained in roasted coffee beans having a low roasting degree, and the content decreases rather as the roasting degree increases. The quinic acid lactone derivative can be extracted from a hot water extract of roasted coffee beans with a fat-soluble organic solvent. That is, the quinic acid lactone derivatives represented by the general formulas (1) to (3) can be extracted and recovered from a hot water extract of roasted coffee beans with a low roasting degree by extraction with a fat-soluble organic solvent. it can. 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 a composition according to the present invention”) is obtained from roasted coffee beans having a low roasting degree. By extracting the quinic acid lactone derivative represented by the general formulas (1) to (3), a composition having xanthine oxidase inhibitory activity (hereinafter sometimes referred to as “xanthine oxidase inhibitory composition”) is obtained. is there. Specifically, after mixing a hot water extract of roasted coffee beans with a low roasting degree and a fat-soluble organic solvent, only the fat-soluble organic solvent layer is recovered as a composition having xanthine oxidase inhibitory activity. An organic solvent layer recovery step;

  The roasting degree of roasted coffee beans can be generally expressed 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 the ground beans are analyzed with a color difference meter has an L * value of about 21 for deep roasted beans, an L * value of about 25 for medium roasted beans, L * value is about 30 for green beans.

  In the present invention and the present specification, the L * value of roasted coffee beans is the L * value of ground beans obtained by grinding roasted coffee beans. The L * value of pulverized beans can be measured, for example, by placing the pulverized beans in an ultraviolet-visible spectrophotometer with an integrating sphere, measuring the reflected light spectrum, and performing color calculation from the obtained reflected light spectrum.

  Since the content of the quinic acid lactone derivative represented by the general formulas (1) to (3) is higher, the roasted coffee beans used as a raw material in the method for producing the composition according to the present invention include L * A value of 30 or more and 45 or less is preferable, a value of 30 or more and 40 or less is more preferable, a value of 32 or more and 40 or less is more preferable, and a value of 32 or more and 38 or less is more preferable. In addition, roasted coffee beans with such a large L * value of ground beans are insufficiently roasted, have almost no coffee-like flavor, have a very strong acidity, and are generally not suitable for coffee beverages. is there.

  The type and production area of roasted coffee beans used as a raw material are not particularly limited, and may be Arabica, Robusta, Revelica, or a blend of these. Good. 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.

  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 with a fat-soluble organic solvent, the quinic acid lactone derivatives represented by the general formulas (1) to (3) in the hot water extract are extracted into the fat-soluble organic solvent. Is done. 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 may be an organic solvent that can be phase-separated from water and can dissolve relatively low-polarity components such as quinic acid lactone derivatives represented by the general formulas (1) to (3). Although not particularly limited, an organic solvent having high volatility is preferable 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 the extraction efficiency of the quinic acid lactone derivative represented by the general formulas (1) to (3) is high.

  The hot water extract of roasted coffee beans may be directly subjected to extraction with a fat-soluble organic solvent, and the quinic acid lactone derivatives represented by the general formulas (1) to (3) are recovered. You may use for the extraction by a fat-soluble organic solvent, after performing the process which removes a compound with large molecular weight. Since the hot water extract of roasted coffee beans may contain high molecular compounds having xanthine oxidase promoting activity, the high molecular weight fraction should be removed from the hot water extract of roasted coffee beans in advance. Thus, a fat-soluble organic solvent extract having a higher concentration of the quinic acid lactone derivative represented by the general formulas (1) to (3) can be 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 represented by the general formulas (1) to (3). And a method of recovering as a fraction containing a quinic acid lactone derivative.

  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 inhibitory 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 inhibition composition containing the quinic acid lactone derivative represented by general formula (1)-(3) 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 components other than the quinic acid lactone derivatives represented by the general formulas (1) to (3). Then, the manufacturing method of the composition which concerns on this invention is a quinic acid lactone derivative represented by General formula (1)-(3) further from this fat-soluble organic-solvent extract after a fat-soluble organic-solvent layer collection | recovery process. The process of refine | purifying may be included. That is, the manufacturing method of the composition which concerns on this invention is general formula (1)-(3) from the collect | recovered fat-soluble organic solvent layer (fat-soluble organic solvent extract) after the said fat-soluble organic solvent layer collection | recovery process. And a fractionation step of recovering the quinic acid lactone derivative represented by In the fractionation step, the quinic acid lactone derivatives represented by the general formulas (1) to (3) contained in two or more types may be collected together and separated for each type of quinic acid lactone derivative. May be collected. 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 the quinic acid lactone derivative represented by the general formulas (1) to (3) is not particularly limited, and the target is generally obtained from a composition containing a plurality of compounds. The method can be appropriately selected from methods used for purifying a compound. 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, a general formula (1) is obtained from a fat-soluble organic solvent extract by 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). The quinic acid lactone derivatives represented by) to (3) can be purified together, or each can be isolated and purified.

  The xanthine oxidase inhibitor according to the present invention may be composed only of a quinic acid lactone derivative represented by any one of the general formulas (1) to (3), which is an active ingredient, and contains other components. It may be a thing. As said other component, what is necessary is just a thing which does not impair the xanthine oxidase inhibitory effect by the quinic acid lactone derivative represented by either of general formula (1)-(3), for example, an excipient | filler, a binder, Fluidity improver (anti-caking agent), stabilizer, preservative, pH adjuster, solubilizer, suspending agent, emulsifier, thickener, flavoring agent, sweetener, acidulant, flavor, colorant, etc. Various substances used as the above may be appropriately contained depending on the 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. Examples of the dosage form include tablets, capsules, granules, powders, syrups, sprays, injections, suppositories, eye drops, and nasal drops. Since the dosage is easy, the dosage form of the xanthine oxidase inhibitor according to the present invention is preferably a tablet, capsule, granule, powder, syrup, or the like suitable for oral administration.

  The quinic acid lactone derivative, which is an active ingredient of the xanthine oxidase inhibitor according to the present invention, is a substance that is relatively contained in roasted coffee beans and can be taken relatively safely. Therefore, these are suitable as raw materials for foods, drinks, feeds, cosmetics, pharmaceuticals and the like, and in particular, foods, drinks, feeds, cosmetics, pharmaceuticals that are ingested in order to suppress the effects of uric acid production or accumulation in the body. It is suitable as an active ingredient. Specifically, the xanthine oxidase inhibitor according to the present invention is useful as an active ingredient of functional foods such as pharmaceuticals and supplements used for the prevention or treatment of various diseases caused by the production or accumulation of uric acid.

  Further, for example, the xanthine oxidase inhibitor according to the present invention can be used as it is added to liquid coffee, instant coffee and the like. 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]

<Roasting method of coffee beans>
In the following examples and the like, unless otherwise specified, coffee beans were roasted as follows.
First, green coffee beans were put into a test tube that had been adjusted to a heating temperature (210 ° C.). Next, this test tube is placed in a heating device “METAL BATH MB-1H-U II” (product number: 123240, manufactured by Koike Seimitsu Seisakusho) and heated at 210 ° C. for a predetermined time to obtain roasted coffee beans. It was. The temperature control of the heating device was performed using a waterproof digital thermometer “Safety Thermo SN3000” (product number: NO123736, manufactured by Thermal Engineering). Further, during heating, water generated at the upper part of the test tube was absorbed by a tissue and removed.

  The content of various components in the roasted coffee beans was determined as the content equivalent to green beans by measuring the weight of the coffee beans before and after heating and calculating the weight reduction rate.

<Measurement of L * value of coffee beans>
In the following examples and the like, L * values of coffee beans were measured as follows unless otherwise specified.
First, roasted coffee beans were pulverized using a coffee mill (MJ516, manufactured by Melita Japan).
The green coffee beans were pulverized using a pulverizer “Blender WB-1” (manufactured by Osaka Chemical Co., Ltd.).

  Next, the obtained pulverized product was put into a powder cell (PSH-002, manufactured by JASCO Corporation), and an ultraviolet-visible spectrophotometer “V-750 spectrophotometer equipped with an integrating sphere (ISV-922, manufactured by JASCO Corporation) was installed. ”(Manufactured by JASCO Corporation), and the reflected light spectrum was measured under the following conditions.

(Spectral conditions)
Measurement mode:% T
Band width: 5nm
Scanning speed: 400 nm / min Data interval: 1 nm
Wavelength: 380 to 800 nm

  Based on the spectrum pattern obtained for each coffee bean, color calculation is performed from the spectrum under the following conditions using a color evaluation (color diagnosis) program (v. 2.1.1., Manufactured by JASCO Corporation). Therefore, the color tone of the coffee beans was evaluated by L * a * b *.

(Color calculation conditions)
Color system: L * a * b *
Field of view: 2 degrees Light source: D65
Data interval: 5nm
Color matching function: JIS Z8701-1999.

  A white plate (X = 95.04, Y = 99.99, Z = 108.87) and a Roast Color Classification System coffee color plate “SCAA (Specialty Coffee Association of America)” (manufactured by Agtron) were used for calibration. The coffee color plate was cut to a size that fits into the powder cell, and the reflected light spectrum was measured in the same manner as the crushed roasted beans to calculate the color from the spectrum.

Coffee color plates L ^* values (mean ± SE, n = 3 ): [Plate No.95 (very light): 32.38 ± 0.10, Plate No.85 (light): 30.55 ± 0.08, Plate No.75 (moderately light ): 28.70 ± 0.12, Plate No.65 (light medium): 27.45 ± 0.15, Plate No.55 (medium): 24.93 ± 0.09, Plate No.45 (moderately dark) 22.69 ± 0.12, Plate No.35 (dark) : 21.24 ± 0.09, Plate No.25 (very dark): 20.43 ± 0.09]

[Reference Example 1]
Various components were separated and purified from roasted coffee beans, and the xanthine oxidase inhibitory activity of each component was examined.

<Roasted coffee beans>
2.3 g of green coffee beans (Brazil Santos N02 (Code: BS · G)) were heated and roasted at 210 ° C. for 10, 20, 30, and 40 minutes.
It was as Table 2 when the brightness of each roasted coffee bean was measured.

<Hot water extraction of roasted coffee beans>
First, 10 mL of hot water was added to 1.00 g of crushed coffee beans or roasted coffee beans, and then the mixture was allowed to stand at room temperature for 30 minutes while being stirred once every 15 minutes to perform hot water extraction. Subsequently, the whole supernatant was collected by centrifugation (2000 rpm, 5 minutes). 10 mL of hot water was newly added to the coffee bean residue from which the supernatant was removed, and hot water extraction was performed in the same manner. Centrifugation was performed to collect the entire amount of the supernatant. The remaining coffee bean residue was extracted with hot water in the same manner, extracted with hot water in the same manner, and centrifuged to collect the entire supernatant. The total amount of hot water extract (supernatant) obtained by hot water extraction three times in total was made up to 30 mL.

<Various component analysis>
Pyrogallol (Pyr) and dicaffeoylquinic acid lactones (diCQLs) from the ethyl acetate fraction, chlorogenic acids (CAs), chlorogenic acid lactones (CLs) and dicaffeoylquinic acids (diCQAs) from the hot water extract, Each was analyzed and quantified under the following conditions.

(Pyr quantitative) [calibration curve: y = 26,747x-1812.7 (range of x = 0.25-5 nmol)]
Column: J-Pak Symphonia C18 (4.6 × 250 mm)
Solvent: (A) 0.1% phosphoric acid _{/ H 2 O, (B)} CH 3 CN
Gradient conditions: (B) 0% (10 minutes), 10% (20 minutes), 100% (25-35 minutes), 0% (40-55 minutes)
Flow rate: 1.0 mL / min Detection wavelength: 268 nm
Injection volume: 20μL

(Quantification of CAs or CLs) [calibration curve: y = 905, 995x + 135,060 (range of x = 0.5-5 nmol)]
Column: COSMOSIL 5C18-AR-II (4.6 × 250 mm)
Solvent: (A) 0.1% phosphoric acid / H ₂ O, (B) CH ₃ CN
Gradient conditions: (B) 5% (0 minutes), 45% (40 minutes), 100% (50-55 minutes), 5% (55.1-65 minutes)
Flow rate: 1.0 mL / min Detection wavelength: 320 nm
Injection volume: 20μL

(Quantification of diCQAs or diCQLs) [calibration curve: y = 1,094,612x + 55,117 (range of x = 0.1-5 nmol)]
Column: COSMOSIL PBr (3.0 x 150 mm)
Solvent: (A) 0.1% phosphoric acid / H ₂ O: (B) CH ₃ CN = 70: 30 (volume ratio)
Flow rate: 0.7 mL / min Detection wavelength: 320 nm
Injection volume: 20μL

  FIG. 1 is a diagram in which the results (mean ± SE, n = 3) obtained by converting the content of each component per gram of green beans are plotted for each roasting time of green coffee beans. The numbers in parentheses below the heating time on the horizontal axis indicate the L * values for each coffee bean. FIG. 2 is a graph in which the horizontal axis is plotted as the color tone (L * value) of coffee beans, and the vertical axis is expressed as the content of diCQLs (per 1 g of green beans). As a result, the longer the heating time, the lower the content of chlorogenic acid and the higher the pyrogallol content. The content of diCQLs was maximized with ultra-shallow roasted beans with a roasting time of 10 minutes. In particular, it was confirmed that roasted coffee beans having an L * value of 30 to 45 have a high content of diCQLs. In addition, the hot water extract of roasted coffee beans having an L * value of 30 to 45 for ground beans exhibited a strong acidity and astringency (eggumi) and was not suitable as a coffee beverage.

[Example 1]
The following three types of dicaffeoylquinic acid lactone [3,4-O-dicaffeoyl-γ-quinide (3,4-diCQL), 4,5-O-dicaffeoyl-muco-γ-quinide (4,5-diCQL) And 3,5-O-dicaffeoyl-epi-δ-quinide (3,5-diCQL)] were isolated and purified, and each xanthine oxidase activity was examined.

<Purification of 4,5-diCQL and 3,5-diCQL>
4,5-diCQL and 3,5-diCQL were isolated and purified from the heated product obtained by heating 5-caffeoylquinic acid (5-CQA), respectively.
Specifically, first, 2 g of 5-CQA (refined product) was subjected to heat treatment (ultra shallow roasting) at 210 ° C. for 10 minutes in the same manner as roasted coffee beans.
The obtained heated product was subjected to HPLC using an ODS column under the following conditions, and each fraction was collected.

(HPLC preparative conditions)
Column: ODS column “COSMOSIL 5C18-AR-II” (20 × 250 mm)
Solvent: (A) 1% AcOH / H ₂ O, (B) CH ₃ CN
Gradient conditions: (B) 0-20 minutes (20%), 20.1-40 minutes (25%), 40.1-60 minutes (30%), 60.1-80 minutes (100%)
Flow rate: 9.6 mL / min Detection wavelength: 320 nm

  FIG. 3 shows a chromatogram of preparative HPLC. In the chromatogram, “1” to “14” indicate each fraction collected. Table 3 shows the results of measurement of the concentrate weight (mg) of each fraction. In the table, “Ex” is an insoluble substance at the time of chromatographic sample preparation and is a portion that could not be chromatographed.

  A xanthine oxidase inhibitory activity was examined for a solution in which each fraction was adjusted to a solid content concentration of 0.3 mg / mL or 0.1 mg / mL. The results are shown in FIG. In the figure, “FC: ca. 0.3 mg / mL” is the result of the solution prepared with each solid fraction concentration of 0.3 mg / mL. “FC: ca. 0.1 mg / mL” indicates that each fraction is solid. It is the result of the solution prepared to the partial concentration of 0.1 mg / mL. Fraction 2 is a fraction containing chlorogenic acid known to have xanthine oxidase inhibitory activity. Fraction 12 having the highest xanthine oxidase inhibitory activity had higher xanthine oxidase inhibitory activity than fraction 2.

40 mg of the fraction 12 having the strongest activity was used as a solvent (A) 1% AcOH / H ₂ O and (B) CH ₃ CN mixed solvent [(A) :( B) = 70: 30 (volume Except for the ratio, the recycle preparative HPLC was performed 5 times under the same conditions as the HPLC conditions for the purification of diCQL in this example, and further purified. As a result, 14 mg of 4,5-diCQL and 8 mg of 3,5-diCQL were isolated and purified.

<Isolation and purification of 3,4-diCQL>
3,4-diCQL was hardly seen in the 5-CQA heated product. Therefore, it was isolated and purified from a heated product obtained by heating 3,4-diCQA isolated and purified from 100 g of Indonesian coffee green beans (Indonesia WIB-1, Code: IN · G).
Specifically, first, a mixture of 30 mg of 3,4-diCQA and 3 g of sea sand was subjected to heat treatment (ultra shallow roasting) at 210 ° C. for 10 minutes, similarly to roasting of coffee beans.
The obtained heated product was used except that the solvent was (A) a mixed solvent of 1% AcOH / H ₂ O and (B) CH ₃ CN [(A) :( B) = 70: 30 (volume ratio)]. Recycle preparative HPLC was performed 3 times under the same conditions as the HPLC conditions for purifying diCQL of this Example, and 10 mg of 3,4-diCQL was isolated and purified.

  The structural analysis data of each diCQLs is shown below.

<3,4-diCQL>
DART-MS m / z 497 [M + H] ⁺ ; ¹ H NMR (400 MHz in CD ₃ OD) δppm 2.10-2.35 (2H, m, H2 _{ax, eq} ), 2.40-2.70 (2H, m, H6 _{ax , eq} ), 4.95 (1H, dd, J = 6.6, 5.0 Hz, H5 _eq ), 5.18 (1H, ddd, J = 11.5, 6.6, 4.7 Hz, H3 _ax ), 5.62 (1H, dd, J = 5.0, 4.7 Hz, H4 _eq ), 6.15 (1H, d, J = 16.0 Hz, H5 _' ), 6.38 (1H, d, J = 16.0 Hz, H5''), 6.68 (1H, d, J = 8.0 Hz, H3 '), 6.78 (1H, d, J = 8.0 Hz, H3 ”), 6.81 (1H, d, J = 8.0, 2.0 Hz, H2'), 6.96 (1H, dd, J = 8.0, 2.0 Hz, H2” ), 6.99 (1H, d, J = 2.0 Hz, H1 '), 7.09 (1H, d, J = 2.0 Hz, H1''), 7.48 (1H, d, J = 16.0 Hz, H4'), 7.63 (1H , d, J = 16.0 Hz, H4 ”); NOEs were observed 8.8% between the peaks at 5.18 and 5.62 ppm, 1.7% between the peaks at 5.18 and 2.10 ppm, 3.2% between the peaks at 5.62 and 4.95 ppm.

<4,5-diCQL>
DART-MS m / z 497 [M + H] ⁺ ; ¹ H NMR (400 MHz in CD ₃ OD) δppm 2.16 (1H, dd, J = 14.0, 1.3 Hz, H6 _ax ), 2.40-2.47 (3H, m , H2 _{ax, eq} , H6 _eq ), 4.95 (1H, ddd, J = 6.0, 5.0, 1.3 Hz, H3 _eq ), 5.12 (1H, brd, J = 5.0 Hz, H4 _eq ), 5.29 (1H, dd, J = 5.0, 1.3 Hz, H5 _eq ), 6.20 (1H, d, J = 16.0 Hz, H5 _' ), 6.35 (1H, d, J = 16.0 Hz, H5``), 6.78 (1H, d, J = 8.0 Hz, H3 '), 6.80 (1H, d, J = 8.0 Hz, H3 ”), 6.96 (1H, dd, J = 8.0, 2.0 Hz, H2'), 7.00 (1H, dd, J = 8.0, 2.0 Hz, H2 ”), 7.05 (1H, d, J = 2.0 Hz, H1 '), 7.08 (1H, d, J = 2.0 Hz, H1”), 7.57 (1H, d, J = 16.0 Hz, H4') , 7.65 (1H, d, J = 16.0 Hz, H4 ”); NOEs were observed 2.7% between the peaks at 5.29 and 2.47 ppm, 1.3% between the peaks at 5.29 and 2.16 ppm, 5.0% between the peaks at 5.29 and 5.12 ppm.

<3,5-diCQL>
DART-MS m / z 497 [ M + H] +; 1 H NMR (400 MHz in CD 3 OD) δppm 1.87-1.94 (2H, m, H2 eq, H6 eq), 2.45 (1H, dd, J = 15.2 , 10.0 Hz, H6 _ax ), 2.68 (1H, dd, J = 15.2, 9.2 Hz, H2 _ax ), 4.90 (1H, dd, J = 4.4, 1.6 Hz, H4 _eq ), 5.27 (1H, ddd, J = 10.0, 5.0, 4.4 Hz, H5 _ax ), 5.54 (1H, dd, J = 9.2, 2.8, 1.6 Hz, H3 _eq ), 6.29 (1H, d, J = 16.0 Hz, H5 _' ), 6.32 (1H, d , J = 16.0 Hz, H5``), 6.76 (1H, d, J = 8.0 Hz, H3 '), 6.78 (1H, d, J = 8.0 Hz, H3''), 6.96 (1H, dd, J = 8.0 , 2.0 Hz, H2 '), 6.99 (1H, dd, J = 8.0, 2.0 Hz, H2 ”), 7.05 (1H, d, J = 2.0 Hz, H1'), 7.07 (1H, d, J = 2.0 Hz , H1 ”), 7.58 (1H, d, J = 16.0 Hz, H4 '), 7.64 (1H, d, J = 16.0 Hz, H4”); NOEs were observed 3.5% between the peaks at 5.54 and 2.68 ppm, 2.8 % between the peaks at 5.27 and 2.45 ppm.

<Measurement of xanthine oxidase inhibition rate>
Xanthine oxidase inhibition rate of purified 3,4-diCQL, 4,5-diCQL, 3,5-diCQL, 5-CQL, 3-CQL, and 4-CQL was measured, and xanthine oxidase inhibition strength (IC ₅₀ ) I investigated. The measurement results are shown in FIG. The IC ₅₀ of 3,4-diCQL, 4,5-diCQL and 3,5-diCQL are very low, 350 μM, 21.5 μM and 47.8 μM, respectively, which have strong xanthine oxidase inhibitory activity It was confirmed that In particular, 4,5-diCQL and 3,5-diCQL had stronger xanthine oxidase inhibitory activity than CQLs known to have xanthine oxidase inhibitory activity.

Claims (11)

The following general formulas (1) to (3)

[In the formulas (1) to (3), R ¹ , R ² , and R ³ each independently represent a hydrogen atom, a caffeoyl group, a feruloyl group, or a coumaroyl group. However, the case where two or more of R ^{1 to} R ³ are hydrogen atoms is excluded. ]
A xanthine oxidase inhibitor comprising a quinic acid lactone derivative represented by any of the above: ^2. The xanthine according to claim ¹ , wherein any ^{two of} R ¹ , R ² , and R ³ are each independently a caffeoyl group, a feruloyl group, or a coumaroyl group, and the remaining one is a hydrogen atom. Oxidase inhibitor. The xanthine oxidase inhibitor according to claim ¹ , wherein any ^{two of} R ¹ , R ² , and R ³ are caffeoyl groups, and the remaining one is a hydrogen atom. It is represented by the general formula (2) or (3), and among R ¹ , R ² , and R ³ , R ¹ and R ² are each independently a caffeoyl group, a feruloyl group, or a coumaroyl group. The xanthine oxidase inhibitor according to claim 1, wherein a quinic acid lactone derivative in which R ³ is a hydrogen atom is an active ingredient.   A xanthine oxidase inhibitor comprising, as an active ingredient, a fat-soluble organic solvent extract extracted from a hot water extract of roasted coffee beans having a L * value of pulverized beans of 30 to 45, with a fat-soluble organic solvent.   After mixing the hot water extract of roasted coffee beans with a L * value of pulverized beans of 30 to 45 and a fat-soluble organic solvent, only the fat-soluble organic solvent layer is recovered as a composition having xanthine oxidase inhibitory activity. A method for producing a composition having xanthine oxidase inhibitory activity, comprising a step of collecting a fat-soluble organic solvent layer.   The method for producing a composition having xanthine oxidase inhibitory activity according to claim 6, wherein the fat-soluble organic solvent is ethyl acetate, hexane, chloroform, dichloromethane, diethyl ether, or a mixed solvent containing any of these. After the fat-soluble organic solvent layer recovery step, from the recovered fat-soluble organic solvent layer, the following general formulas (1) to (3)

[In the formulas (1) to (3), R ¹ , R ² , and R ³ each independently represent a hydrogen atom, a caffeoyl group, a feruloyl group, or a coumaroyl group. However, the case where two or more of R ^{1 to} R ³ are hydrogen atoms is excluded. ]
The manufacturing method of the composition which has the fractionation process which collect | recovers the fraction containing the quinic acid lactone derivative represented by either of Claim 6 or 7 which has a xanthine oxidase inhibitory activity.   A pharmaceutical comprising the xanthine oxidase inhibitor according to any one of claims 1 to 5.   The pharmaceutical product according to claim 9, which is used for treatment of gout or prevention of recurrence.   The functional food which contains the xanthine oxidase inhibitor as described in any one of Claims 1-5, and reduces the uric acid level in serum.

Images