Process for the production of flavonoid inclusion compounds

Abstract

A method for producing a flavone inclusion compound, comprising a detachment step of treating a poorly soluble flavone having a rhamnose glycoside structure with an enzyme having rhamnodiastase activity in the presence of a cyclodextrin to detach rhamnose. According to the production method of the present invention, a flavone inclusion compound, a flavone glycoside composition, which is excellent in solubility in water, can be efficiently produced, and can be suitably utilized in the fields of pharmaceuticals, food and drink, health foods, foods for specified health uses, and cosmetics, etc.

Description

[0001] This application is a divisional application of PCT application with an international filing date of January 31, 2018, international application number PCT / JP2018 / 003177, Chinese national phase application number 201880023105.5, and invention title "Method for manufacturing flavonoid inclusion compounds". Technical Field

[0002] This invention relates to methods for manufacturing flavonoid inclusion compounds, methods for manufacturing flavonoid glycoside compositions, flavonoid inclusion compounds, compositions containing flavonoid inclusion compounds, isoquercitrin glycoside compositions, hesperidin-7-glucoside compositions, naringenin-7-glucoside compositions, food and beverage products, medical products, or cosmetics containing these compounds or compositions, and methods for improving the solubility of poorly soluble flavonoids having a rhamnoside structure. Background Technology

[0003] Flavonoids possess antioxidant properties, thus they are used to prevent the deterioration of food flavor and the fading of pigments. In Japan's lists of food additives, existing additives, and antioxidants, numerous catechins, enzyme-treated rutin, rutin extracts, tea extracts, and bayberry extracts, all containing flavonoids as active ingredients, are reported. Furthermore, as physiological agents, flavonoids have been reported to have anti-tumor effects, lower cholesterol, reduce blood pressure, lower blood sugar, and reduce body fat. They are also widely used in pharmaceuticals, food products, health foods, foods for specific health purposes, and cosmetics.

[0004] Flavonoids are found in vegetables, fruits, tea, and other foods, with over 3,000 known species. However, because most are poorly soluble in water, they are difficult to use in soft drinks, liquids, and other foods, beverages, pharmaceuticals, and cosmetics that require high water solubility. For example, typical flavonoids such as hesperidin and rutin have a water solubility of less than 0.01%, making them unsuitable for use in soft drinks and lotions.

[0005] Poorly soluble flavonoids can be divided into those with and without rhamnoside structures. It has been reported that compared with rutin, hesperidin, and naringin, which have rhamnoside structures, isoquercitrin, hesperidin-7-glucoside, naringenin, and naringenin-7-glucoside, which are desorbed from rhamnoside, have higher in vivo absorption in rats (Non-Patent Literature 1-3).

[0006] Furthermore, it is known that by encapsulating poorly soluble flavonoids with cyclodextrin or glycosylating poorly soluble flavonoids, their in vivo absorption can be improved, effectively demonstrating physiological activity. For example, the inclusion compound of isoquercitrin (1M)·γ-cyclodextrin (5M) has been reported to have a higher in vivo absorption rate in humans compared to isoquercitrin (Patent Document 1); in mouse experiments, the hesperidin·β-cyclodextrin inclusion compound, etc., showed higher in vivo absorption (AUC0-9 hours) compared to hesperidin, and greater effects in inhibiting allergic reactions, improving blood flow, and improving cold intolerance (Patent Document 2); the naringenin·hydroxypropylβ-cyclodextrin inclusion compound, compared to naringenin, showed increased in vivo absorption (in rats), reduced VLDL (very low-density lipoprotein), and increased glucose clearance (Non-Patent Document 4). Regarding glycosylation, it has been reported, for example, that the anti-allergic effect in mice follows the order of "enzyme-treated rutin < isoquercitrin < enzyme-treated isoquercitrin," with the removal of rhamnose and water-soluble enzyme-treated isoquercitrin showing the most effective effect (Non-Patent Literature 5).

[0007] In addition to the aforementioned documents, methods for removing rhamnose from insoluble flavonoids having a rhamnoside structure are disclosed, for example, in Patent Documents 3-6. Methods for encapsulating insoluble flavonoids with cyclodextrin are disclosed, for example, in Patent Documents 2, 7, and 8. Methods for glycosylation of insoluble flavonoids are disclosed, for example, in Patent Documents 9 and 10. Furthermore, Patent Document 11 discloses a method for making insoluble flavonoids water-soluble by coexisting the insoluble flavonoid with a composition of soybean saponins and / or malonyl isoflavone glycosides in an aqueous medium to increase their solubility.

[0008] In addition, as a method for improving the solubility of poorly soluble flavonoids, a method for improving water solubility characterized by combining poorly soluble flavonoids with water-soluble flavonoid glycosides has been disclosed (Patent Documents 3-4, Patent Document 12); characterized by water-soluble flavonoids containing poorly soluble flavonoid-β-cyclodextrin and glycosylhesperidin (Patent Document 8).

[0009] Existing technical documents Patent documents Patent Document 1: Japanese Patent No. 5002072 Patent Document 2: Japanese Patent No. 5000884 Patent Document 3: Japanese Patent No. 4902151 Patent Document 4: Japanese Patent No. 3833775 Patent Document 5: Japanese Patent No. 4498277 Patent Document 6: Japanese Patent No. 5985229 Patent Document 7: Japanese Patent No. 3135912 Patent Document 8: Japanese Patent No. 5000373 Patent Document 9: Japanese Patent No. 4202439 Patent Document 10: Japanese Patent No. 3989561 Patent Document 11: Japanese Patent Application Publication No. 2011-225586 Patent Document 12: Japanese Patent Application Publication No. 7-10898 Non-patent literature Non-patent literature 1: British Journal of Nutrition, 102, 976-984, 2009 Non-patent literature 2: Biological & Pharmaceutical Bulletin, 32(12), 2034-2040, 2009 Non-patent literature 3: American Journal of Physiology: Gastrointestinal and Liver Physiology, 279, 1148-1154, 2000 Non-patent literature 4: PLOS ONE (4), e18033, 2011 Non-patent literature 5: Journal of Natural Medicines, Oct, 67(4), 881-6, 2013. Summary of the Invention

[0010] The technical problem that the invention aims to solve However, the manufacturing methods disclosed in the aforementioned prior art documents cannot be said to have good production efficiency, and they do not fully meet the requirements for the solubility of the obtained flavonoids in water, so further improvements are expected.

[0011] The objective of this invention is to provide a simple and efficient method for manufacturing flavonoid inclusion compounds and flavonoid glycoside compositions with excellent water solubility. Furthermore, it provides flavonoid inclusion compounds with excellent water solubility, compositions containing flavonoid inclusion compounds, isoquercitrin glycoside compositions, hesperidin-7-glucoside compositions, naringenin-7-glucoside compositions, food and beverage products, medical products, or cosmetics containing these compounds or compositions, and methods for improving the solubility of poorly soluble flavonoids having a rhamnoside structure.

[0012] Means for solving technical problems This invention relates to: [1] A method for manufacturing flavonoid inclusion compounds, comprising: a removal step of removing rhamnose by treating an insoluble flavonoid having a rhamnoside structure with an enzyme having rhamnosidease activity in the presence of cyclodextrin. [2] A method for manufacturing a flavonoid glycoside composition, comprising: a glycosylation step of treating a flavonoid inclusion compound obtained by the manufacturing method described in [1] with a glycosyltransferase to glycosylate it; [3] A method for manufacturing a flavonoid glycoside composition, comprising: a desorption step of removing rhamnose by treating an insoluble flavonoid having a rhamnose glycoside structure with an enzyme having rhamnosidease activity in the presence of cyclodextrin; and a glycosylation step of treating the flavonoid inclusion compound obtained by the aforementioned desorption step with a glycosyltransferase to glycosylate it. [4] Flavonoid inclusion compounds, which are flavonoid inclusion compounds formed by isoquercitrin encapsulating γ-cyclodextrin, wherein the molar ratio of the aforementioned isoquercitrin to the aforementioned γ-cyclodextrin (γ-cyclodextrin / isoquercitrin) is 0.9 to 1.8, and the solubility of the aforementioned isoquercitrin in water is more than 2%. [5] Flavonoid inclusion compounds, which are flavonoid inclusion compounds formed by isoquercitrin encapsulating γ-cyclodextrin, wherein the molar ratio of the aforementioned isoquercitrin to the aforementioned γ-cyclodextrin (γ-cyclodextrin / isoquercitrin) is 0.9 to 4.0, and the solubility of the aforementioned isoquercitrin in water is more than 2.5%; [6] Flavonoid inclusion compounds, which are flavonoid inclusion compounds formed by isoquercitrin encapsulating β-cyclodextrin, wherein the molar ratio of the aforementioned isoquercitrin to the aforementioned β-cyclodextrin (β-cyclodextrin / isoquercitrin) is 1.0 to 3.0, and the solubility of the aforementioned isoquercitrin in water is greater than 0.1%; [7] Flavonoid inclusion compound, which is a flavonoid inclusion compound formed by hesperidin-7-glucoside encapsulating cyclodextrin, wherein the molar ratio of the aforementioned hesperidin-7-glucoside to the aforementioned cyclodextrin (cyclodextrin / hesperidin-7-glucoside) is 1.0 to 3.0, and the solubility of the aforementioned hesperidin-7-glucoside in water is greater than 0.01%; [8] Flavonoid inclusion compound, which is a flavonoid inclusion compound formed by naringenin-7-glucoside encapsulating β-cyclodextrin, wherein the molar ratio of the aforementioned naringenin-7-glucoside to the aforementioned β-cyclodextrin (β-cyclodextrin / naringenin-7-glucoside) is 1.0 to 3.0, and the solubility of the aforementioned naringenin-7-glucoside in water is greater than 0.01%; [9] A composition containing a flavonoid inclusion compound, comprising any one of [4] to [8] a flavonoid inclusion compound and rhamnose, wherein the molar ratio of the flavonoid inclusion compound to the rhamnose (rhamnose / flavonoid inclusion compound) is 0.8 to 1.2;

[10] A composition containing a flavonoid inclusion compound, comprising any one of [4] to [8] a flavonoid inclusion compound and a poorly soluble flavonoid having a rhamnoside structure, wherein the molar ratio of the flavonoid in the aforementioned flavonoid inclusion compound to the aforementioned poorly soluble flavonoid (poorly soluble flavonoid / flavonoid in the inclusion compound) is 0.001 to 0.1;

[11] An isoquercitrin glycoside composition, which is an isoquercitrin glycoside composition containing a compound represented by the following general formula (1), wherein the content of the glycoside with n=0 is 10 mol% or more and 30 mol% or less, the content of the glycoside with n=1 to 3 is 50 mol% or less, and the content of the glycoside with n=4 or more is 30 mol% or more. [Chemistry 1] In general formula (1), Glc means glucose residue and n means an integer greater than or equal to 0 or 1;

[12] A hesperidin-7-glucoside glycoside composition, which is a hesperidin-7-glucoside glycoside composition containing a compound represented by the following general formula (2), wherein the content of the glycoside with n=0 is 10 mol% or more and 30 mol% or less, the content of the glycoside with n=1 to 3 is 50 mol% or less, and the content of the glycoside with n=4 or more is 30 mol% or more. [Chemistry 2] In general formula (2), Glc means glucose residue and n means an integer greater than or equal to 0 or 1;

[13] A naringenin-7-glucoside composition, which is a naringenin-7-glucoside composition containing a compound represented by the following general formula (3), wherein the content of the glycoside with n=0 is 10 mol% or more and 30 mol% or less, the content of the glycoside with n=1 to 3 is 50 mol% or less, and the content of the glycoside with n=4 or more is 30 mol% or more. [Chemistry 3] In general formula (3), Glc means glucose residue and n means an integer greater than or equal to 0 or 1;

[14] A food or beverage comprising one or more compounds or compositions selected from the following: a flavonoid inclusion compound obtained by the manufacturing method described in [1]; a flavonoid glycoside composition obtained by the manufacturing method described in [2] or [3]; a flavonoid inclusion compound described in any one of [4] to [8]; a composition containing a flavonoid inclusion compound described in [9] or

[10] ; an isoquercitrin glycoside composition described in

[11] ; a hesperidin-7-glucoside glycoside composition described in

[12] ; and a naringin-7-glucoside glycoside composition described in

[13] .

[15] A medicine comprising one or more compounds or compositions selected from the manufacturing method described in [1], the manufacturing method described in [2] or [3], the flavonoid inclusion compound described in any one of [4] to [8], the composition containing the flavonoid inclusion compound described in [9] or

[10] , the isoquercitrin glycoside composition described in

[11] , the hesperidin-7-glucoside composition described in

[12] , and the naringerin-7-glucoside composition described in

[13] .

[16] A cosmetic comprising one or more compounds or compositions selected from the manufacturing method described in [1], a flavonoid inclusion compound obtained by the manufacturing method described in [2] or [3], a flavonoid inclusion compound described in any one of [4] to [8], a composition containing a flavonoid inclusion compound described in [9] or

[10] , an isoquercitrin glycoside composition described in

[11] , a hesperidin-7-glucoside composition described in

[12] , and a naringenin-7-glucoside composition described in

[13] ; and

[17] A method for improving the solubility of a poorly soluble flavonoid having a rhamnoside structure, wherein the poorly soluble flavonoid having a rhamnoside structure, and a flavonoid inclusion compound obtained by the manufacturing method described in [1] or any one of [4] to [8], are mixed in a medium such that the molar ratio of flavonoid in the aforementioned flavonoid inclusion compound to the aforementioned poorly soluble flavonoid (flavonoid in the inclusion compound / poorly soluble flavonoid) is 0.1 to 0.9.

[0013] Invention Effects According to the present invention, a simple and efficient method for manufacturing flavonoid inclusion compounds and flavonoid glycoside compositions with excellent water solubility is provided. Furthermore, methods for improving the solubility of poorly soluble flavonoid inclusion compounds, compositions containing flavonoid inclusion compounds, isoquercitrin glycoside compositions, hesperidin-7-glucoside compositions, naringenin-7-glucoside compositions, food and beverage products, medical products or cosmetics containing these compounds or compositions, and methods for improving the solubility of poorly soluble flavonoids having a rhamnoside structure are also provided. Attached Figure Description

[0014] Figure 1 The HPLC chromatogram of Example 39 is shown.

[0015] Figure 2 : This shows the HPLC chromatogram of Example 40. Detailed Implementation

[0016] The inventors conducted research on the aforementioned issues and discovered that by desquamating the rhamnose in poorly soluble flavonoids with a rhamnoside structure in the presence of cyclodextrin, flavonoid inclusion compounds can be produced simultaneously with rhamnose desquamation, and this method is more efficient than conventional methods that involve separate desquamation and inclusion steps. Furthermore, it was unexpectedly discovered that the flavonoid inclusion compounds obtained by this method exhibit superior water solubility compared to those produced by conventional methods. In this invention, various cyclic oligosaccharides can be used in addition to cyclodextrin. Here, cyclic oligosaccharides refer to compounds in which monosaccharides are linked in a cyclic structure, and more specifically, examples include cyclodextrin, cycloglucan, cyclofructan, cycloalternan, and cluster dextrin. The following description uses cyclodextrin as an example, but the invention is not limited to this, and other cyclic oligosaccharides can also be used.

[0017] The method for manufacturing the flavonoid inclusion compound of the present invention includes a descrambling step of treating an insoluble flavonoid having a rhamnoside structure with an enzyme having rhamnosidease activity in the presence of cyclodextrin to remove rhamnosine.

[0018] The separation step is the process of separating rhamnose from insoluble flavonoids with a rhamnoside structure to obtain inclusion compounds of flavonoids without a rhamnoside structure and cyclodextrin (also known as "flavonoid inclusion compounds"). The separation step can be carried out in a solvent such as water, either by standing or with stirring. To prevent oxidation or browning during the reaction, the air in the headspace of the reaction system can be replaced with an inert gas such as nitrogen. Furthermore, antioxidants such as ascorbic acid can be added to the reaction system. The separation step can be terminated by known methods such as heating the reaction solution to inactivate the enzyme.

[0019] Examples of poorly soluble flavonoids having a rhamnoside structure include those selected from flavonols, flavanones, flavones, and isoflavones. Those having a structure in which one or more, preferably two or more hydroxyl groups are bonded to the benzene ring of the flavonoid skeleton, and which contains rhamnose. Here, "poorly soluble" means a solubility in water at 25°C of 1.0% by mass or less, preferably 0.1% or less, more preferably 0.01% by mass or less. Specific examples include: rutin, hesperidin, naringin, geraniol, sennain, myricetin, neohesperidin, luteolin-7-rutinoside, delphinidin-3-rutinoside, cyanidin-3-rutinoside, isorhamnetin-3-rutinoside, kaempferol-3-rutinoside, apigenin-7-rutinoside, robinin-7-rutinoside, and their derivatives. Examples of derivatives include acetylation, malonylation, and methylation.

[0020] There is no particular limitation on the amount of insoluble flavonoids with rhamnoside structures used in the reaction system. Preferably, it can be set to 0.1-20% by mass, more preferably 1-15% by mass, and even more preferably 2-14% by mass. When two or more insoluble flavonoids with rhamnoside structures are used, the amount used refers to their total amount.

[0021] The raw materials containing the insoluble flavonoids with a rhamnoside structure used in the manufacturing method of the present invention do not require special purification, but purification is preferred. The content of the insoluble flavonoids with a rhamnoside structure in the aforementioned raw materials is not particularly limited; preferably 5% or more, more preferably 20% or more, further preferably 50% or more, further preferably 80% or more, and further preferably 90% or more can be used.

[0022] There are no particular limitations on the cyclodextrin (CD) present in the separation step, but it is more preferable to use one or more selected from β-cyclodextrin (β-CD), branched β-cyclodextrin (branched β-CD), and γ-cyclodextrin (γ-CD). Cyclodextrin is a type of cyclic oligosaccharide in which D-glucose is linked by α-1,4 glycosidic bonds to form a cyclic structure; β-cyclodextrin has 7 glycosidic residues linked together, and γ-cyclodextrin has 8. Branched β-CD is a substance with one or more glucose residues, galactosyl groups, or hydroxypropyl groups attached to the β-CD as side chains, such as maltose-β-CD (G2-β-CD) and hydroxypropyl-β-CD (HP-β-CD). It should be noted that "in the presence of cyclodextrin" refers to the state in which cyclodextrin is present in the separation reaction system.

[0023] The amount of cyclodextrin present is not particularly limited, but in the reaction system it can be preferably 0.01 to 60% by mass, more preferably 1 to 50% by mass, and even more preferably 3 to 40% by mass. When two or more types of cyclodextrin are used, the amount refers to their total amount.

[0024] From an efficiency point of view, the molar ratio of cyclodextrin to insoluble flavonoids with rhamnoside structure (cyclodextrin / flavonoid) is preferably 0.01 or more, more preferably 0.1 or more, even more preferably 0.9 or more, even more preferably 1.0 or more. From an economic point of view, it is preferably 10.0 or less, more preferably 6.0 or less, even more preferably 4.0 or less, even more preferably 3.0 or less.

[0025] As an enzyme with rhamnosidase activity, its source is not limited; enzymes with rhamnosidase activity from all sources, including animal, plant, and microbial sources, can be used. Furthermore, it can also be a recombinant enzyme. In addition, the enzyme's form is not particularly limited.

[0026] Specific examples of enzymes with rhamnosidase activity include hesperidinase, naringinase, β-glucosidase, and pectinase.

[0027] The amount of enzyme with rhamnosidase activity used varies depending on the type of enzyme used, the reaction conditions, and the type of poorly soluble flavonoids with a rhamnoside structure in the raw material. In the case of, for example, hesperidinase, naringinase, and β-glucosidase, the amount is preferably 0.01 to 1000 U per 1 g of poorly soluble flavonoids with a rhamnoside structure. The reaction conditions can be selected according to the characteristics of the enzyme used, with the reaction temperature and pH of the reaction solution preferably set to pH 3 to 7, and more preferably to pH 3.5 to 6.5. In addition, the poorly soluble flavonoids with a rhamnoside structure can be dissolved in an alkaline region and then the enzyme reaction can be carried out under conditions below pH 7. As a solvent used in the reaction system, an aqueous medium can be used. In this specification, an aqueous medium refers to an aqueous solution of water or an organic solvent. Examples of water include tap water, distilled water, ion-exchanged water, and purified water. As an organic solvent, there is no particular limitation as long as it is an organic solvent that is uniformly mixed with water. From the viewpoint of being suitable for food, ethanol is preferred as an organic solvent. Furthermore, the reaction temperature is preferably 10–80°C, more preferably 40–75°C. Additionally, the reaction time varies depending on the type of enzyme, and can be set, for example, 1–100 hours, preferably 2–24 hours.

[0028] Enzymes with rhamnosidase activity may also have glucosidase activity. It is not limited to using glucosidase activity to obtain aglycone inclusion compounds (quercetin inclusion compounds, hesperidin inclusion compounds, naringin inclusion compounds, myricetin inclusion compounds, etc.) from insoluble flavonoids (hesperidin, rutin, naringin, myricetin, etc.) with rhamnoside structures. These are also included in the flavonoid inclusion compounds described in this invention.

[0029] As described above, the resulting flavonoid inclusion compounds are inclusion compounds of flavonoids and cyclodextrins that do not possess a rhamnoside structure. Here, inclusion compound refers to a compound formed by one chemical species forming a molecular-scale space and including another chemical species due to its shape and size being suitable for that space.

[0030] Examples of flavonoids that do not possess a rhamnoside structure include isoquercitrin, quercetin, hesperidin-7-glucoside, hesperidin, naringenin-7-glucoside (cherry glycoside), naringenin, luteolin-7-glucoside, geraniol-7-glucoside, myricetin, sennaol-7-glucoside, delphinidin-3-glucoside, cyanidin-3-glucoside, isorhamnetin-3-glucoside, kaempferol-3-glucoside, apigenin-7-rutin glycoside, and robinin-7-glucoside.

[0031] Specific examples of the structural formulas of insoluble flavonoids with and without rhamnoside structures are shown below. The structural formulas of rutin (RTN), hesperidin (HSP), and naringin (NRG) with rhamnoside structures, and isoquercitrin (IQC), quercetin (QCT), hesperidin-7-glucoside (HPT-7G), hesperidin (HPT), naringin-7-glucoside (prunin) (NGN-7G), and naringin (NGN) without rhamnoside structures are as follows.

[0032] [Chemistry 4] From an efficiency point of view, the molar ratio of cyclodextrin to flavonoid inclusion complexes without rhamnoside structure (cyclodextrin / flavonoid) is preferably 0.01 or more, more preferably 0.1 or more, even more preferably 0.9 or more, even more preferably 1.0 or more. From an economic point of view, it is preferably 10.0 or less, more preferably 6.0 or less, even more preferably 4.0 or less, even more preferably 3.0 or less.

[0033] The yield of the generated flavonoid inclusion compound is preferably 10-100%, more preferably 40-100%, more preferably 70-100%, and even more preferably 90-100%. The yield is the conversion rate from insoluble flavonoids with rhamnoside structures to flavonoids without rhamnoside structures, which can be calculated by the method described in the examples below. It should be noted that the generated flavonoid inclusion compound is not limited in proportion to the flavonoids with rhamnoside structures (e.g., rutin, hesperidin, naringin, etc.) used as raw materials, or to other flavonoids that may be present in the raw materials (e.g., quercetin, kaempferol-3-rutinoside, kaempferol-3-glucoside, hesperidin, naringin, etc.), depending on the flavonoid content and conversion rate of the raw materials used.

[0034] In the generated flavonoid inclusion compound or the composition containing flavonoid inclusion compounds described later (sometimes both are referred to as "flavonoid inclusion compounds, etc."), the solubility of the flavonoid moiety in water depends on the type and amount of the poorly soluble flavonoid with a rhamnoside structure and the cyclodextrin used, and is preferably 0.01% or more, more preferably 0.015% or more, further preferably 0.02% or more, further preferably 1.0% or more, further preferably 2.0% or more, further preferably 2.5% or more, and further preferably 3% or more. There is no particular upper limit, and it can be set to, for example, 20% or less. In this specification, the solubility of the flavonoid moiety in water is a mass percentage concentration at 25°C, which can be determined by the method described in the examples described later.

[0035] The specific method is as follows.

[0036] Method 1-1 The isoquercitrin is a flavonoid inclusion compound containing γ-cyclodextrin. From the viewpoint of suppressing production costs, the molar ratio of the aforementioned isoquercitrin to the aforementioned γ-cyclodextrin in the inclusion compound (γ-cyclodextrin / isoquercitrin) is preferably 0.9 to 4.0, more preferably 0.9 to 1.8, and the solubility of the aforementioned isoquercitrin in water is preferably 0.01% or more, more preferably 2% or more, further preferably 2.5% or more, and even more preferably 3% or more.

[0037] Method 1-2 The isoquercitrin is a flavonoid inclusion compound containing β-cyclodextrin, wherein the molar ratio of the aforementioned isoquercitrin to the aforementioned β-cyclodextrin in the inclusion compound (β-cyclodextrin / isoquercitrin) is 1.0 to 3.0, and the solubility of the aforementioned isoquercitrin in water is preferably 0.01% or more, more preferably 0.02% or more, even more preferably 0.03% or more, and even more preferably 0.05% or more.

[0038] Methods 1-3 The flavonoid inclusion compound containing hesperidin-7-glucoside in cyclodextrin, wherein the molar ratio of hesperidin-7-glucoside to cyclodextrin in the inclusion compound (cyclodextrin / hesperidin-7-glucoside) is 1.0 to 3.0, and the solubility of hesperidin-7-glucoside in water is preferably 0.01% or more, more preferably 0.02% or more, and even more preferably 0.03% or more.

[0039] Methods 1-4 The flavonoid inclusion compound containing naringenin-7-glucoside in β-cyclodextrin, wherein the molar ratio of naringenin-7-glucoside to β-cyclodextrin in the inclusion compound (cyclodextrin / naringenin-7-glucoside) is 1.0 to 3.0, and the solubility of naringenin-7-glucoside in water is preferably 0.01% or more, more preferably 0.02% or more, and even more preferably 0.03% or more.

[0040] According to the method for manufacturing the flavonoid inclusion compound of the present invention, without purification, a composition containing the flavonoid inclusion compound and rhamnose can be obtained. In this case, the molar ratio (rhamnose / flavonoid) of flavonoid inclusion compound to desorbed rhamnose in the aforementioned flavonoid inclusion compound is 0.8 to 1.2.

[0041] According to the method for manufacturing the flavonoid inclusion compound of the present invention, when the aforementioned yield is not 100%, a poorly soluble flavonoid having a rhamnoside structure is contained as an unreacted substance. From the viewpoint of long-term stability, the molar ratio (poorly soluble flavonoid / flavonoid in the inclusion compound) of the flavonoid inclusion compound containing such an unreacted substance to the aforementioned poorly soluble flavonoid (poorly soluble flavonoid / flavonoid in the inclusion compound) is preferably 0.1 or less, more preferably 0.08 or less, and even more preferably 0.05 or less. The lower limit is not particularly limited and can be 0.001 or more, 0.003 or more, 0.004 or more, or 0.01 or more.

[0042] Furthermore, it has been unexpectedly discovered that the flavonoid inclusion compound obtained by the manufacturing method of the present invention can improve the solubility of insoluble flavonoids with a rhamnoside structure. More specifically, by mixing insoluble flavonoids with a rhamnoside structure and the flavonoid inclusion compound obtained by the manufacturing method of the present invention in a medium, with the molar ratio of flavonoids in the inclusion compound to the insoluble flavonoids (flavonoids in the inclusion compound / insoluble flavonoids) preferably 0.1 to 0.9, more preferably 0.1 to 0.7, and even more preferably 0.1 to 0.3, the solubility of insoluble flavonoids with a rhamnoside structure can be improved. Here, "medium" refers to an aqueous medium, or an aqueous solution containing food additives such as sugars, salts, acidulants, sweeteners, flavorings, glycerin, propylene glycol, lemon extract, or traditional Chinese medicine extracts. This method for improving solubility can be implemented by directly using a composition containing a flavonoid inclusion compound with unreacted material, or by adding a flavonoid inclusion compound to a poorly soluble flavonoid having a rhamnoside structure. Here, for poorly soluble flavonoids having a rhamnoside structure, the flavonoid inclusion compound obtained by the manufacturing method of the present invention, as mentioned above, examples of combinations such as the following can be cited: rutin and isoquercitrin-γ-cyclodextrin inclusion compound, hesperidin and hesperidin-7-glucoside-β-cyclodextrin inclusion compound, naringin and naringin-7-glucoside-β-cyclodextrin inclusion compound, and rutin and naringin-7-glucoside-β-cyclodextrin inclusion compound.

[0043] The method for manufacturing the flavonoid inclusion compound of the present invention is not particularly limited in terms of purification as needed, except for the separation step. Purification can be performed through resin treatment steps (adsorption, ion exchange, etc.), membrane treatment steps (ultrafiltration, reverse osmosis, zeta potential membrane treatment, etc.), electrodialysis, salting out, acid precipitation, recrystallization, solvent fractionation, etc. For example, the composition containing the flavonoid inclusion compound obtained in the separation step can be adsorbed with a porous synthetic adsorbent, washed with water to remove rhamnose, etc., and then subjected to alcohol dissolution and spray drying to obtain a purified powder. Alternatively, after alcohol dissolution, a diluent or other additives other than the composition can be included. It should be noted that rhamnose, etc., can also be fractionated for use in the food, pharmaceutical, quasi-pharmaceutical, and cosmetic industries. Furthermore, only flavonoids can be purified from the manufactured flavonoid inclusion compound.

[0044] As a diluent, there are no particular limitations as long as it does not impair the effect of the present invention. Examples include sugars such as sucrose, glucose, dextrin, starches, trehalose, lactose, maltose, syrup, and liquid sugars; alcohols such as ethanol, propylene glycol, and glycerol; sugar alcohols such as sorbitol, mannitol, xylitol, erythritol, maltitol, reducing syrup, and mannitol; or water. Furthermore, as additives, examples include phosphates, organic acids, chelating agents, and antioxidants such as ascorbic acid.

[0045] Next, the method for manufacturing the flavonoid glycoside composition of the present invention will be described.

[0046] The method for manufacturing the flavonoid glycoside composition of the present invention includes a glycosylation step of treating the flavonoid inclusion compound obtained by the method for manufacturing the flavonoid inclusion compound of the present invention with a glycosyltransferase to glycosylate it. Specifically, it includes a descaling step of treating an insoluble flavonoid having a rhamnoside structure with an enzyme having rhamnosidase activity in the presence of cyclodextrin to descalcify the rhamnose, and a glycosylation step of treating the flavonoid inclusion compound obtained via the aforementioned descaling step with a glycosyltransferase to glycosylate it.

[0047] The separation step and the flavonoid inclusion compounds obtained via the separation step are as described above. It should be noted that obtaining via the separation step does not exclude methods that include steps other than the separation step, but also includes methods that optionally include obtaining via purification steps, etc.

[0048] The glycosylation step involves reacting the flavonoid inclusion compound obtained in the desorption step with a glycosyltransferase to glycosylate it, thereby yielding a flavonoid glycoside composition. Furthermore, similar to the desorption step, the glycosylation step can be carried out in a solvent such as water, either by standing or by stirring. To prevent oxidation or browning during the reaction, the air in the headspace of the reaction system can be replaced with an inert gas such as nitrogen. Additionally, antioxidants such as ascorbic acid can be added to the reaction system. The glycosylation step can be terminated by known methods such as heating the reaction solution to inactivate the enzyme.

[0049] In the glycosylation step, the cyclodextrin of the flavonoid inclusion compound becomes a sugar donor, and flavonoid glycoside compositions can be produced. There are no restrictions on the additional supply of sugar donors. Specific examples of additional sugar donors include starch, dextrin, starch partial hydrolysates such as maltodextrin, xylooligosaccharides, and products containing them.

[0050] As a glycosyltransferase, there are no particular restrictions as long as the enzyme possesses glycotransfer activity against the flavonoid inclusion compound obtained via the cleavage step. The source of the glycosyltransferase is not limited; glycosyltransferases from all sources, including animal, plant, and microbial sources, can be used. Furthermore, artificial enzymes obtained using recombination technology, partial hydrolysis, etc., can also be used. In addition, the form of the glycosyltransferase is not particularly limited; dried enzyme proteins, enzymes immobilized with an insoluble carrier, and liquids containing enzyme proteins can be used.

[0051] Specific examples of glycosyltransferases include cyclodextrin glucosyltransferase, glucosyltransferase, α-glucosidase, β-glucosidase, α-galactosidase, β-galactosidase, α-amylase, xylanase, pullulanase, arabinofuranase, etc.

[0052] The amount of glycosyltransferase used varies depending on the type of enzyme used, the conditions of the glycosyltransfer reaction, and the type of sugar. For example, in the case of cyclodextrin glucosyltransferase, it is preferably 1 to 10,000 U relative to 1 g of the flavonoid inclusion compound. When glycosylating poorly soluble flavonoids, the enzymatic reaction is usually carried out in an alkaline region to solubilize the flavonoids. However, in alkaline regions above pH 7, the stability of the flavonoids deteriorates, the solvent decomposes, and browns, requiring a browning step and a desalting step due to alkali neutralization. However, for the flavonoid inclusion compound obtained by the manufacturing method of the present invention, since the poorly soluble flavonoids are solubilized at high concentrations even at pH 7, the enzymatic reaction proceeds efficiently with glycosylation even at pH 7. Therefore, from the viewpoint of production efficiency and quality, a pH of 3 to 7 is preferred, and more preferably pH 6 to 6.8. Glycosyltransfer can be carried out in an alkaline region, or it can be carried out under conditions of adjusting to an alkaline region and then adjusting to pH 7 or below. Aqueous medium can be used as a solvent in the reaction system. Furthermore, the reaction temperature is preferably 40–70°C, more preferably 50–65°C. The reaction time varies depending on the type of enzyme, and can be set, for example, 0.5–120 hours, preferably 1–30 hours. Moreover, from the viewpoint of production efficiency, after the separation step, it is preferable to continuously change the temperature and pH to optimal levels and add glycosyltransferase to carry out the glycosylation step.

[0053] The sugars bound to flavonoids can be either α-bonds or β-bonds. The type of sugar is not particularly limited, but preferably at least one type selected from five- or six-carbon sugars such as glucose, galactose, and fructose. Furthermore, the number of sugars bound is preferably 1 to 30, more preferably 1 to 25, even more preferably 1 to 20, even more preferably 1 to 15, and even more preferably 1 to 10. Flavonoid glycoside compositions refer to mixtures containing glycosides to which the aforementioned sugars are bound on flavonoids. The ratio of the number of each glycoside bound is not limited, but from the viewpoint of not impairing the aroma of food and beverages, the following configuration is preferred.

[0054] Method 2-1 An isoquercitrin glycoside composition comprising a compound of the following general formula (1), wherein the content of the glycoside with n=0 is 10 mol% or more and 30 mol% or less, the content of the glycoside with n=1 to 3 is 50 mol% or less, and the content of the glycoside with n=4 or more is 30 mol% or more. Preferably, the content of the glycoside with n=0 is 10 mol% or more and 30 mol% or less, the content of the glycoside with n=1 to 3 is 35 mol% or more and 45 mol% or less, and the content of the glycoside with n=4 or more is 30 mol% or more and 50 mol% or less. [Chemistry 5] In general formula (1), Glc means glucose residue and n means an integer greater than or equal to 0 or 1.

[0055] Method 2-2 A hesperidin-7-glucoside glycoside composition, comprising a compound of the following general formula (2), wherein the content of the glycoside with n=0 is 10 mol% or more and 30 mol% or less, the content of the glycoside with n=1 to 3 is 50 mol% or less, and the content of the glycoside with n=4 or more is 30 mol% or more. Preferably, the content of the glycoside with n=0 is 10 mol% or more and 25 mol% or less, the content of the glycoside with n=1 to 3 is 35 mol% or more and 50 mol% or less, and the content of the glycoside with n=4 or more is 30 mol% or more and 50 mol% or less. [Chemistry 6] In general formula (2), Glc means glucose residue and n means an integer greater than or equal to 0 or 1.

[0056] Method 2-3 The naringenin-7-glucoside composition is a naringenin-7-glucoside composition containing a compound represented by the following general formula (3), wherein the content of the glycoside with n=0 is 10 mol% or more and 30 mol% or less, the content of the glycoside with n=1 to 3 is 50 mol% or less, and the content of the glycoside with n=4 or more is 30 mol% or more. [Chemistry 7] In general formula (3), Glc means glucose residue and n means an integer greater than or equal to 0 or 1.

[0057] It should be noted that the number of glucose groups (n) can be arbitrarily adjusted. For example, after the flavonoid glycoside composition is generated, by treating it with various amylases (α-amylase, β-amylase, glucoamylase, α-glucosidase, etc.) alone or in combination, the number of glucose sugar chains in the flavonoid glycoside composition molecule can be reduced, resulting in a flavonoid glycoside composition with arbitrary glucose sugar chain length.

[0058] The method for manufacturing the flavonoid glycoside composition of the present invention, apart from the separation step and the glycosylation step, is not particularly limited in terms of purification as needed. Purification can be performed through resin treatment steps (adsorption, ion exchange, etc.), membrane treatment steps (ultrafiltration, reverse osmosis, zeta potential membrane treatment, etc.), electrodialysis, salting out, acid precipitation, recrystallization, solvent fractionation, etc. For example, the flavonoid glycoside composition obtained in the glycosylation step can be adsorbed onto a porous synthetic adsorbent, washed with water, dissolved in alcohol, and then spray-dried to obtain a purified powder. Furthermore, after alcohol dissolution, a diluent or other additives, other than the composition, can be included.

[0059] As specific examples of diluents, they are the same as those described in the methods for manufacturing flavonoid inclusion compounds.

[0060] The solubility of the flavonoid glycoside composition obtained by the manufacturing method of the present invention in water, in terms of flavonoid conversion value, is preferably 0.01% or more, more preferably 0.015% or more, even more preferably 0.02% or more, even more preferably 0.1% or more, and even more preferably 0.5% or more. There is no particular upper limit, and it can be set, for example, below 20%.

[0061] The flavonoid inclusion compounds and flavonoid glycoside compositions obtained by the manufacturing method of the present invention can be combined with insoluble flavonoids having a rhamnoside structure that are said to have slow absorption in vivo, or with flavonoid glycoside compositions having a rhamnoside structure, to form a food with continuously improved absorption rate in vivo. Examples include combinations of isoquercitrin inclusion compounds and rutin, combinations of isoquercitrin glycoside compositions and rutin glycoside compositions (e.g., αG rutin, Toyo Seisakusho Co., Ltd.), combinations of hesperidin-7-glucoside inclusion compounds and hesperidin glycoside compositions (e.g., αG hesperidin, Toyo Seisakusho Co., Ltd.), and combinations of hesperidin-7-glucoside glycoside compositions and hesperidin glycoside compositions (e.g., monoglucosyl hesperidin, Hayashihara Co., Ltd.).

[0062] Furthermore, the flavonoid glycoside composition obtained by the manufacturing method of the present invention can improve the solubility of other poorly soluble flavonoids by combining them with other poorly soluble flavonoids. Examples of such combinations include isoquercitrin glycoside composition with rutin, hesperidin-7-glucoside glycoside composition with hesperidin, and hesperidin-7-glucoside glycoside composition with myricetin. Moreover, the molar ratio (glycoside composition / other poorly soluble flavonoid) is preferably 0.1 to 0.5, more preferably 0.1 to 0.3, and even more preferably 0.1 to 0.15.

[0063] As described above, the flavonoid inclusion compounds and / or flavonoid glycoside compositions obtained by the manufacturing method of the present invention exhibit excellent in vivo absorption rates. Furthermore, they also demonstrate excellent performance in preventing fading, preventing flavor degradation, and maintaining shelf stability. Therefore, they are suitable for use as food compositions, pharmaceutical compositions, cosmetic compositions, and food additive compositions. More specifically, they can be used as raw materials for anti-allergy, anti-oxidation, anti-cancer, anti-inflammatory, intestinal flora improvement, deodorization, inhibition of plasma cholesterol rise, inhibition of blood pressure rise, inhibition of blood sugar rise, inhibition of platelet aggregation, prevention of dementia, body fat burning, inhibition of body fat accumulation, improvement of endurance, anti-fatigue, improvement of cold intolerance, improvement of skin condition, hair growth, inhibition of muscle atrophy, and sleep aid. In addition, they can be used as antioxidants, anti-fading agents, and flavor degradation preventers in food additives. Food additive compositions can be added to prevent the deterioration of sweeteners, colorants, preservatives, thickeners and stabilizers, color developers, bleaching agents, mold inhibitors, gum bases, bittering agents, gloss agents, acidulants, flavoring agents, emulsifiers, reinforcing agents, manufacturing agents, and fragrances, and to prepare mixed formulations. That is, the present invention can provide food products, pharmaceuticals, cosmetics, etc., containing flavonoid inclusion compounds and / or flavonoid glycoside compositions obtained by the manufacturing method of the present invention.

[0064] Food and beverage products include food and drinks, such as nutritional supplements, health foods, foods for specific health purposes, functional foods, foods for therapeutic purposes, comprehensive health foods, supplements, tea drinks, coffee drinks, fruit juices, soft drinks, and beverages.

[0065] The medicine includes medicines or quasi-medicines, preferably oral preparations or topical preparations for skin use, and can be formulated as liquids, tablets, granules, pills, syrups, lotions, sprays, or ointments.

[0066] As a cosmetic, it can be formulated as cream, liquid lotion, emulsion lotion, or spray.

[0067] The amount of flavonoid inclusion compounds and / or flavonoid glycoside compositions in the food, pharmaceutical, or cosmetic products of the present invention is not particularly limited, and can be appropriately designed with reference to the preferred daily intake of flavonoids and taking into account solubility, flavor, etc. For example, the amount of the flavonoid portion of the flavonoid inclusion compounds and / or flavonoid glycoside compositions obtained by the manufacturing method of the present invention in the food composition can be set to preferably 0.001 to 30% by mass, more preferably 0.01 to 20% by mass, and even more preferably 0.02 to 10% by mass. The amount in the food composition can also be determined in a way that allows the flavonoid inclusion compounds and / or flavonoid glycoside compositions to be ingested in one to several doses (e.g., three times) per day, preferably 10 mg to 20 g, more preferably 30 mg to 10 g, and even more preferably 100 mg to 5 g. Furthermore, as a measure of the effect of flavonoids, the amount of flavonoid inclusion compounds and / or flavonoid glycoside compositions in the food additive formulation can be used in amounts preferably 0.001 to 50% by mass, more preferably 0.01 to 40% by mass, and even more preferably 0.1 to 30% by mass. Example

[0068] The present invention will now be described in more detail by way of examples, but the invention is not limited to these examples in any way. It should be noted that, unless otherwise specified, "%" means "mass %".

[0069] Preparation of compositions containing flavonoid inclusion compounds Examples 1-31 In a 1000ml beaker, insoluble flavonoids (rutin or hesperidin) with rhamnoside structure and cyclodextrin were added as shown in Tables 1 and 2, and water was added to a volume of 1000g. The temperature was adjusted to 70℃ and pH 4.5. Then, while stirring, 3-30g of naringinase (Amano Engineering Co., Ltd. 155u / g) was added. After reacting for 24 hours, the mixture was returned to room temperature and filtered through filter paper to obtain a composition of flavonoid inclusion compounds (isoquercetin or hesperidin-7-glucoside) without rhamnoside structure and cyclodextrin, and a composition of flavonoid inclusion compounds containing desorbed rhamnose.

[0070] Comparative Examples 1-3 The compositions of Comparative Examples 1 and 3 were prepared in the same manner as in Examples 16 and 17, except that cyclodextrin was not added. Furthermore, the composition of Comparative Example 2 was prepared by adding dextrin instead of cyclodextrin, except that dextrin was added, and the process was repeated in the same manner as in Example 16.

[0071] Comparative Example 101 In a 100 ml beaker, isoquercitrin and γ-cyclodextrin prepared as shown in Table 1-2 were added, and water was added to a final volume of 100 g. The mixture was stirred at 70 °C and pH 4.5 for 24 hours, then returned to room temperature and filtered through filter paper to prepare a composition containing an inclusion compound of isoquercitrin and γ-cyclodextrin.

[0072] Preparation of isoquercitrin 10g of rutin used in Table 1 was added to 100L of aqueous solution and adjusted to 70℃ and pH 4.5. Then, while stirring, 1g of naringinase (Amano Enzaim Co., Ltd. 155u / g) was added, and the mixture was recovered and dried to obtain 7.2g of isoquercitrin with a purity of over 96%. The isoquercitrin was confirmed to be identical using HPLC with the reagent Wako.

[0073] Examples 101-109 Using the raw materials shown in Table 2-2, except as otherwise described in Examples 1-31, compositions containing inclusion compounds of naringenin-7-glucoside and β-cyclodextrin were prepared.

[0074] Comparative Example 102 The composition of Comparative Example 102 was prepared in the same manner as in Example 104, without the addition of cyclodextrin.

[0075] The details used in Tables 1, 1-2, 2, and 2-2 are as follows: RTN: Rutin prepared as follows 50 kg of buds from the legume *Sophora japonica* was soaked in 500 L of hot water for 3 hours to obtain a filtrate. The filtrate was then cooled to room temperature and filtered off. The precipitate was washed with water, recrystallized, and dried to obtain 3190 g of rutin with a purity of over 96%. The rutin (Wako) peak was identified as identical using HPLC. HSP: Hesperidin (content ≥ 97%, manufactured by Hamari Pharmaceutical Co., Ltd.) NRG: Naringin (95% or higher, manufactured by SIGMA) β-CD: β-cyclodextrin (manufactured by Biotech Co., Ltd.) γ-CD: γ-Cyclodextrin (manufactured by パールエース Co., Ltd.) Dextrin: Sandec #70 (manufactured by Sanwa Starch Industry Co., Ltd.)

[0076] Conversion rate of rutin to isoquercitrin The unfiltered reaction mixtures from Examples 1-16 and Comparative Examples 1-2 were used as the assay samples. The conversion rate (%) was calculated as follows: HPLC area ratio (peak area of ​​isoquercitrin / peak area of ​​rutin) < HPLC conditions: column: CAPCELLPAK C18 SIZE 4.6 mm × 250 mm (SHISEIDO), eluent: 20% (v / v) acetonitrile / 0.1% phosphoric acid aqueous solution, detection: 351 nm, flow rate: 0.4 ml / min, column temperature: 70 °C > . The conversion rate (%) was calculated as: peak area of ​​isoquercitrin × 100 / (peak area of ​​rutin + peak area of ​​isoquercitrin). The peaks of isoquercitrin were confirmed by HPLC using the reagent isoquercitrin (Wako). The conversion rates in Examples 1-16 were all above 96%. On the other hand, compared with Example 16 using the same amount of enzyme, the conversion rate in Comparative Example 1 was lower at 56%, and the conversion rate in Comparative Example 2 was lower at 57%.

[0077] Conversion rate of hesperidin to hesperidin-7-glucoside The unfiltered reaction mixtures from Examples 17-31 and Comparative Example 3 were used as the test samples. The conversion rate (%) was calculated using the following HPLC (SHIMADZU) conditions: column: CAPCELL PAK C18 SIZE 4.6 mm × 250 mm (SHISEIDO), eluent: 40% (v / v) acetonitrile / 0.1% phosphoric acid aqueous solution, detection: 280 nm, flow rate: 0.4 ml / min, column temperature: 70 °C > , calculated as: peak area of ​​hesperidin-7-glucoside × 100 / (peak area of ​​rutin + peak area of ​​hesperidin-7-glucoside). Hesperidin-7-glucoside was confirmed as the same peak by HPLC using a dried product identified by NMR as hesperidin-7-glucoside. The conversion rates in Examples 17-31 were all above 96%. On the other hand, the conversion rate in Comparative Example 3 was lower at 57%.

[0078] Conversion rate of naringin to naringenin-7-glucoside The unfiltered reaction mixtures from Examples 101-109 and Comparative Example 102 were used as the test samples. The conversion rate was calculated based on the HPLC (SHIMADZU) area ratio (peak area of ​​naringenin-7-glucoside / peak area of ​​naringin) < HPLC conditions: column: CAPCELL PAK C18 SIZE 4.6 mm × 250 mm (SHISEIDO), eluent: 25% (v / v) acetonitrile / 0.1% phosphoric acid aqueous solution, detection: 280 nm, flow rate: 0.4 ml / min, column temperature: 70 °C >. That is, the conversion rate (%) was calculated as: peak area of ​​naringenin-7-glucoside × 100 / (peak area of ​​naringin + peak area of ​​naringenin-7-glucoside). Naringenin-7-glucoside was confirmed as the same peak using the reagent naringenin-7-glucoside (Wako) by HPLC. The conversion rates of Examples 101-109 and Comparative Example 102 were above 95%.

[0079] Isoquercetin (IQC) concentration (absorbent assay) After the reaction-complete solutions of Examples 1-16 and Comparative Examples 1, 2, and 101 were allowed to stand at room temperature, 1 ml of the supernatant was filtered through a filter and used as the test sample. A calibration curve was constructed using rutin (Wako) at an absorbance of 351 nm (0.1% phosphoric acid solution). The rutin concentration was calculated from the absorbance of the test sample, corrected for conversion, and multiplied by 0.761 (the molecular weight ratio of isoquercitrin / rutin (464.38 / 610.52 = 0.761)). The resulting value was then used to calculate the isoquercitrin concentration. The results are shown in Tables 1 and 1-2. It should be noted that the conversion rate used for concentration calculation was obtained by HPLC determination of the same sample as the one used for concentration analysis.

[0080] Hesperidin-7-glucoside (HPT-7G) concentration (absorbent assay) After the reaction solutions of Examples 17-31 and Comparative Example 3 were allowed to stand at room temperature, 1 ml of the supernatant was filtered and used as the test sample. Using the reagent hesperidin (Wako), a calibration curve was constructed at an absorbance of 280 nm (0.1% phosphoric acid solution). The hesperidin concentration was calculated from the absorbance of the test sample, corrected by the conversion rate from HPLC analysis, and then multiplied by 0.761 (the molecular weight ratio of hesperidin-7-glucoside / hesperidin (464.42 / 610.56 = 0.761)). The resulting value was then calculated as the hesperidin-7-glucoside concentration. The results are shown in Table 2. It should be noted that the conversion rate used for concentration calculation was calculated by performing HPLC analysis on the same sample as the concentration analysis.

[0081] Concentration of naringenin-7-glucoside (NGN-7G) (absorbent assay) After the reaction end solutions of Examples 101-109 and Comparative Example 102 were allowed to stand at room temperature, 1 ml of the supernatant was filtered through a filter and used as the test sample. A calibration curve was prepared using naringin (manufactured by SIGMA, hereinafter NRG) at an absorbance of 280 nm (0.1% phosphoric acid solution). The naringin concentration was calculated from the absorbance of the test sample, corrected by the conversion rate from HPLC analysis, and multiplied by 0.748 (the molecular weight ratio of naringin / naringenin-7-glucoside (434.39 / 580.54 = 0.748)). The resulting value was then used to calculate the naringenin-7-glucoside concentration. The results are shown in Table 2-2. It should be noted that the conversion rate used in the concentration calculation was calculated by performing HPLC analysis on the same sample as the concentration analysis.

[0082] Molar ratios (CD / IQC (molar ratio), CD / HPT-7G (molar ratio), CD / NGN-7G (molar ratio)) (HPLC sugar analysis) After the reaction-complete solutions of Examples 1-31, 101-109, and Comparative Example 101 were allowed to stand at room temperature, 1 ml of the supernatant was filtered through a filter and used as the assay sample. HPLC (SHIMADZU) analysis <HPLC conditions: Column: Inertsil NH2 (4.6×150 mm), eluent: 65% acetonitrile / water (v / v), detection: differential refractometer RID-10A (SHIMADZU), flow rate: 1 ml / min, column temperature: 40℃. After preparing calibration curves for β-cyclodextrin (Wako) and γ-cyclodextrin (Wako), the molar concentration of cyclodextrin in the sample was calculated. The molar ratios of cyclodextrin / isoquercetin, cyclodextrin / hesperidin-7-glucoside, and cyclodextrin / naringenin-7-glucoside were calculated based on the molar concentrations of isoquercitrin, hesperidin-7-glucoside, and naringenin-7-glucoside. The results are shown in Tables 1, 1-2, 2, and 2-2. It should be noted that the molar ratio of the filtrate after the reaction is the same in the case of the freeze-dried product.

[0083] Solubility (IQC solubility, HPT-7G solubility, NGN-7G solubility) The reaction-completed solutions of Examples 1-31, 101-109 and Comparative Examples 1-3, 101, and 102 were allowed to stand at room temperature, filtered through filter paper, and then freeze-dried to obtain dried products. The prepared dried products were added to a 100ml beaker containing 50ml of water at 50°C with stirring until they precipitated out and could not be completely dissolved. After standing at room temperature (25°C), 1ml of the supernatant was filtered, and the concentrations of isoquercitrin, hesperidin-7-glucoside, and naringenin-7-glucoside were calculated by absorbance analysis as solubility. In cases where the amount of dried product was insufficient during solubility determination, the required amount was obtained by repeating the experiment of the same examples, and the solubility was measured. Furthermore, in Examples 1-31, 101-109, and Comparative Example 101, the inclusion of flavonoids in cyclodextrins was confirmed by differential scanning calorimetry (DSC), nuclear magnetic resonance (NMR), and Fourier transform infrared spectrophotometry (FT-IR). The solubility results are shown in Tables 1, 1-2, 2, and 2-2. It should be noted that after the reactions in Examples 1-31 and 101-109 were completed, the room-temperature filtrate was dialyzed to remove rhamnose, and the freeze-dried flavonoid inclusion compounds obtained showed substantially the same solubility.

[0084] Notes in Table 1 (1) Rutin concentration (mass%) at the start of the reaction (2) β-cyclodextrin concentration at the start of the reaction (mass%) (3) The initial concentration of γ-cyclodextrin (mass%) at the start of the reaction (4) Dextrin concentration at the start of the reaction (mass%) (5) Cyclodextrin / rutin molar ratio at the start of the reaction (6) The concentration of isoquercitrin in the filtrate after the reaction (mass%) (7) Cyclodextrin / isoquercetin (molar ratio) of the filtrate after the reaction. (8) Solubility of isoquercitrin in the freeze-dried filtrate after the reaction (mass %).

[0085] Notes to Table 1-2 (11) Concentration of isoquercitrin (mass%) at the start of heating and stirring (12) Concentration of γ-cyclodextrin at the start of heating and stirring (mass%) (13) Cyclodextrin / isoquercetin (molar ratio) at the start of heating and stirring (14) The concentration of isoquercitrin in the filtrate after heating and stirring (mass%) (15) Cyclodextrin / isoquercetin (molar ratio) of the filtrate after heating and stirring (16) Solubility of isoquercitrin in the freeze-dried filtrate after heating and stirring (mass %).

[0086] As clearly shown in Table 1, according to the manufacturing method of the present invention, the inclusion complex of isoquercitrin and cyclodextrin can be obtained efficiently while rutin undergoes a rhamnose desorption reaction. On the other hand, in Comparative Examples 1 and 2, the conversion rate and solubility were low. It should be noted that the reaction solutions and reaction end solutions of Examples 7, 8 and Comparative Examples 1-3 were in suspension. However, although the reaction solutions of Examples 1-6 and Examples 9-16 were in suspension at the beginning and middle of the reaction, they dissolved at the end of the reaction and after standing at room temperature thereafter. The inclusion rate of the isoquercitrin-cyclodextrin inclusion compound (isoquercitrin concentration in the inclusion compound (concentration of the filtrate after standing at room temperature after the reaction) × 100 / isoquercitrin concentration in the reaction end solution (unfiltered mixture)) was approximately 100%. However, as in Comparative Example 101 in Table 1-2, when only isoquercitrin and γ-cyclodextrin with the same composition as in Example 10 were mixed and heated, the mixture remained in a suspended state. The inclusion rate (isoquercitrin concentration in the inclusion compound (concentration of the filtrate after standing at room temperature after the reaction) × 100 / isoquercitrin concentration in the unfiltered mixture after the reaction) was also low at 12%, and the solubility of the freeze-dried filtrate was also low.

[0087] Notes in Table 2 (21) Hesperidin concentration at the start of the reaction (mass%) (22) β-cyclodextrin concentration at the start of the reaction (mass%) (23) Initial concentration of γ-cyclodextrin (mass%) at the start of the reaction (24) Cyclodextrin / hesperidin molar ratio at the start of the reaction (25) Concentration (mass%) of hesperidin-7-glucoside in the filtrate after the reaction. (26) Cyclodextrin / hesperidin-7-glucoside (molar ratio) in the filtrate after the reaction. (27) Solubility (mass%) of hesperidin-7-glucoside in the freeze-dried filtrate after the reaction.

[0088] As clearly shown in Table 2, according to the manufacturing method of the present invention, the inclusion complex of hesperidin-7-glucoside and cyclodextrin can be obtained efficiently while the rhamnose desorption reaction is carried out by hesperidin. On the other hand, in Comparative Example 3, the conversion rate was low and the solubility was also low. It should be noted that in Examples 17, 26 and Comparative Example 3, the reaction solution and the reaction end solution were in suspension. However, although the reaction solution in Examples 18-25 and Examples 27-31 was in suspension at the beginning and middle of the reaction, it dissolved at the end of the reaction and when left to stand at room temperature. The inclusion rate of the hesperidin-7-glucoside-cyclodextrin inclusion compound (the concentration of hesperidin-7-glucoside in the inclusion compound (the concentration of the filtrate after standing at room temperature after the reaction) × 100 / the concentration of hesperidin-7-glucoside in the reaction end solution (unfiltered mixture)) was approximately 100%.

[0089] Notes to Table 2-2 (31) Naringin concentration at the start of the reaction (mass%) (32) β-cyclodextrin concentration at the start of the reaction (mass%) (33) Cyclodextrin / naringin molar ratio at the start of the reaction (34) Concentration (mass%) of naringenin-7-glucoside in the filtrate after the reaction. (35) Cyclodextrin / naringenin-7-glucoside (molar ratio) of the filtrate after the reaction. (36) Solubility (mass%) of naringenin-7-glucoside in the freeze-dried filtrate after the reaction.

[0090] As clearly shown in Table 2-2, according to the manufacturing method of the present invention, the inclusion complex of naringenin-7-glucoside and β-cyclodextrin can be obtained efficiently while the rhamnose desorption reaction of naringin is being carried out, and the solubility is also improved. The conversion rate of Comparative Example 102 is over 95%, but precipitation occurs immediately upon standing at room temperature after the reaction, resulting in low solubility. However, after the reaction of Examples 101 to 109, the filtrate after standing at room temperature is dissolved, and the inclusion rate of the naringenin-7-glucoside-cyclodextrin inclusion compound (concentration of naringenin-7-glucoside in the inclusion compound (concentration of the filtrate after standing at room temperature after the reaction) × 100 / concentration of naringenin-7-glucoside in the reaction end solution (unfiltered mixture)) is approximately 100%.

[0091] The molar ratio (rhamnose / flavonoid) in compositions containing flavonoid inclusion compounds. In addition, after determining the rhamnose content of the filtrate after the reaction in Examples 18–25, 27–31, and 101–109 (under the same conditions as HPLC sugar analysis, with a calibration curve made using rhamnose (Wako), the molar concentration of rhamnose was calculated, and the molar ratio (rhamnose / flavonoid) to the flavonoids of the inclusion compound was 0.8–1.2.

[0092] Flavor evaluation of flavonoid inclusion compounds 100 ml of each of the reaction-complete solutions from Examples 10-15 were added to a dialysis membrane (Spectra / Por CE dialysis tube, MWCO 500-1000, manufactured by funakoshi) and dialyzed in 10 L of water (5 water exchanges, 10°C) to remove rhamnose. Each solution was then freeze-dried to obtain 10 g to 30 g of dried product. The dried product was added to commercially available carbonated beverages (sugar-free) ("Minami-Alps Tennensui Sparkling", manufactured by Suntory), coffee beverages (sugar-free) ("WONDAGOLD BLACK", manufactured by Asahi Beverages), and green tea ("Oi Ocha", manufactured by Itoen) at a concentration of 0.1% by mass (equivalent to isoquercitrin). Five judges performed sensory evaluations (off-taste, sweetness) using the unadded products as a control. The average scores were calculated according to the evaluation criteria described below. The results are shown in Table 3.

[0093] Odor evaluation criteria 1: I have a strong smell 2: I can smell a slightly strong odor. 3: I noticed an unusual odor 4: A slight odor was detected. 5: No unusual odor was detected.

[0094] Sweetness evaluation criteria 1: I strongly felt the sweetness. 2: A slightly strong sweetness can be detected. 3: You can taste the sweetness 4: Slightly sweet taste 5: I did not taste any sweetness.

[0095] As clearly shown in Table 3, a molar ratio (γCD / IQC) of 1.0–1.8 results in less off-flavor and sweetness compared to a molar ratio of 2.0–4.0, making it a preferred choice from the perspective of minimizing the impact on the flavor of beverages and other foods. Furthermore, although not shown in the table, for hesperidin-7-glucoside inclusion compounds, a molar ratio (CD / HPT-7G) of 1.0–1.9 results in less off-flavor (flavor different from the unadded) and sweetness compared to a molar ratio of 2.0–3.0, making it suitable for use in food and beverage products.

[0096] Preparation of flavonoid glycoside compositions Examples 32-39 A small amount of alkali was added to the reaction solution prepared in Example 4 (70°C, pH 4.5, isoquercitrin concentration 2.3% by mass) to adjust the temperature to 60°C and pH 6.5. Then, 20g of cyclodextrin glucosyltransferase (CGTase: Amano Engineering Co., Ltd., trade name "Composite", 600U / ml) was added to start the reaction and maintained for 24 hours. The resulting reaction solution was heated to sterilize, filtered, and then freeze-dried to obtain 158g of isoquercitrin glycoside composition (sample 1) containing the compound shown in general formula (1). The solubility of the obtained isoquercitrin glycoside composition (sample 1) in water, calculated as isoquercitrin, was 2.7%. The obtained isoquercitrin glycoside composition (sample 1) was dissolved in water and passed through a column packed with Diaion HP-20 (porous synthetic adsorption resin, manufactured by Mitsubishi Kemica Co., Ltd.) to adsorb the isoquercitrin glycoside composition. The column was washed with twice the resin capacity of water to remove sugars such as rhamnose. Then, the adsorbed components were eluted with twice the resin capacity of 65% (v / v) ethanol. The eluent was concentrated and freeze-dried to prepare the glycoside composition of Example 39. The HPLC chromatogram of Example 39 is shown below. Figure 1 The result was the same as the HPLC chromatogram of Sample 1. Furthermore, following the same method as in Example 39, the isoquercitrin glycoside composition (Sample 1) was adsorbed and washed with water, and then dissolved in ethanol at a concentration of 10–60% (v / v). These dissolves (10, 20, 30, 40, 50, and 60% (v / v) dissolves) were combined and the molar ratio adjusted, concentrated, and freeze-dried to prepare the glycoside compositions of Examples 32–38. The solubility of the glycoside compositions of Examples 32–39 in water, based on the isoquercitrin conversion value, was ≥10%. It should be noted that when preparing reaction solutions at pH 7.5 and pH 8.5 with the same amount of enzyme during the glycotransfer reaction, approximately the same amount of isoquercitrin glycoside composition was produced; however, due to partial decomposition of flavonoids, the solution turned brownish-black. Therefore, the product obtained from the reaction at pH 6.5 was used. It should be noted that the reaction solutions prepared in Examples 1-3 and 10-16 also produced the same HPLC chromatograms as Sample 1 under the same conditions. Figure 1 The isoquercitrin glycoside composition, [Chemistry 8] In general formula (1), Glc means glucose residue and n means an integer greater than or equal to 0 or 1.

[0097] Examples 40-46 A small amount of alkali was added to the reaction solution prepared in Example 22 (70°C, pH 4.5, hesperidin-7-glucoside concentration 2.9% by mass) to adjust the temperature to 60°C and pH 6.5. Then, 5g of cyclodextrin glucosyltransferase (CGTase: Amano Engineering Co., Ltd., trade name "Composite", 600U / ml) was added to start the reaction and maintained for 24 hours. The resulting reaction solution was heated to sterilize, filtered, and spray-dried to obtain 136g of a hesperidin-7-glucoside glycoside composition containing the compound shown in general formula (2) (sample 2). The solubility of the obtained hesperidin-7-glucoside glycoside composition (sample 2) in water, converted to hesperidin-7-glucoside, was 5.1%. The obtained hesperidin-7-glucoside glycoside composition (sample 2) was dissolved in water and passed through a column packed with Diaion HP-20 (porous synthetic adsorption resin, manufactured by Mitsubishi Kemica Co., Ltd.) to adsorb the hesperidin-7-glucoside glycoside composition. The column was washed with twice the resin capacity of water to remove sugars such as rhamnose. Then, the adsorbed components were eluted with twice the resin capacity of 65% (v / v) ethanol. The eluent was concentrated and freeze-dried to prepare the glycoside composition of Example 40. The HPLC chromatogram of Example 40 is shown below. Figure 2 The result was the same as the HPLC chromatogram of Sample 2. Furthermore, following the same method as in Example 40, the hesperidin-7-glucoside glycoside composition (Sample 2) was adsorbed and washed with water, followed by dissolution with ethanol at a concentration of 10–60% (v / v). These dissolution solutions (10, 20, 30, 40, 50, and 60% (v / v) dissolution solutions) were combined and their molar ratios adjusted, concentrated, and freeze-dried to prepare the glycoside compositions of Examples 41–46. The solubility of the glycoside compositions of Examples 40–46 in water, converted to hesperidin-7-glucoside, was greater than 10%. It should be noted that when preparing reaction solutions at pH 7.5 and pH 8.5 with the same amount of enzyme during the glycotransfer reaction, approximately the same amount of hesperidin-7-glucoside glycoside composition was produced; however, due to partial decomposition of the flavonoids, the solution turned brownish-black. Therefore, the product obtained from the reaction at pH 6.5 was used. It should be noted that the reaction solutions prepared in Examples 21, 23, and 27–31 also produced the same HPLC chromatograms as Sample 2 under the same conditions. Figure 2 The isoquercitrin glycoside composition, [Chemical Formula 9] In General Formula (2), Glc means a glucose residue, and n means an integer of 0 or more than 1.

[0098] Solubility (IQC conversion value, HPT-7G conversion value) The solubility of the flavone glycoside compositions of Samples 1 and 2 and Examples 32 to 46 was calculated for the isoquercetin concentration and the hesperetin-7-glucoside concentration by the same method as the aforementioned IQC solubility and HPT-7G solubility, and set as the isoquercetin conversion value and the hesperetin-7-glucoside conversion value. It should be noted that if each conversion value is 10% or more and no precipitation is observed, the solubility is described as 10% or more.

[0099] The molar ratio (%) in the flavone glycoside compositions of Examples 32 to 46 was calculated by the following formula based on the analysis results under the following HPLC (SIMADZU) conditions. The results are shown in Table 4. It should be noted that Figure 1 , Figure 2 Each peak from n = 0 to n = 7 of Molar ratio (%) = area of each peak of the flavone glycoside composition × 100 / total peak area of the flavone glycoside composition.

[0100] <HPLC conditions: Examples 32 to 39> Column: CAPCELL PAK C18 SIZE 4.6 mm × 250 mm (SHISEIDO) Eluent: water / acetonitrile / phosphoric acid = 799 / 200 / 1 (volume ratio) Detection: absorbance measurement at a wavelength of 351 nm Flow rate: 0.4 ml / min Column temperature: 70 °C <HPLC conditions: Examples 40 to 46> Column: CAPCELL PAK C18 SIZE 4.6 mm × 250 mm (SHISEIDO) Eluent: water / acetonitrile / phosphoric acid = 849 / 150 / 1 (volume ratio) Detection: absorbance measurement at a wavelength of 280 nm Flow rate: 0.4 ml / min Column temperature: 70 °C.

[0101] Flavor evaluation of the flavone glycoside composition The freeze-dried flavonoid glycoside compositions of Examples 32-46 were added to acidic sugar solutions (pH 3.1, Brix 10°) to achieve an isoquercitrin equivalent concentration of 0.05% (Examples 32-39) or a hesperidin-7-glucoside equivalent concentration of 0.05% (Examples 40-46). Functional evaluations (bitterness, spiciness, astringency) were performed by seven judges. The average scores were calculated according to the evaluation criteria described below. It should be noted that the evaluation scores for bitterness, spiciness, and astringency in the acidic sugar solution (freshly prepared and dissolved) containing 0.05% isoquercitrin prepared in Comparative Example 101 were set to the highest score of 1 for comparison. Furthermore, since no precipitation was observed in the acidic sugar solution containing 0.05% isoquercitrin at room temperature for 30 minutes after preparation, the functional evaluation was performed during this period. The results are shown in Table 4.

[0102] Evaluation criteria for bitterness 1: I strongly felt the bitterness. 2: A slightly strong bitter taste was detected. 3: You can taste bitterness 4: Slightly bitter taste 5: I did not taste bitter.

[0103] Evaluation criteria for spiciness 1: I strongly felt the spiciness. 2: A slightly strong spiciness can be detected. 3: You can taste the spiciness. 4: Slightly spicy taste 5: I did not taste any spiciness.

[0104] Astringency evaluation criteria 1: I have a strong astringent taste. 2: A slightly strong astringent taste can be detected. 3: You can taste the astringency. 4: Slightly astringent taste 5: No astringent taste was detected.

[0105] As shown in Table 4, in the glycoside compositions of Examples 36, 38, 39, 40, and 41, the content of glycosides with n=0 in general formulas (1) and (2) is 10 mol% or more and 30 mol% or less, the content of glycosides with n=1 to 3 is 50 mol% or less, and the content of glycosides with n=4 or more is 30 mol% or more. In the functional evaluation using acidic sugar solutions, bitterness, spiciness, and astringency are significantly reduced, making them suitable for food and beverage applications. It should be noted that the glycoside compositions of Examples 32 to 46 all exhibit excellent solubility and are suitable for applications unrelated to taste, such as cosmetics. Furthermore, although not shown in the table, for the naringenin-7-glucoside glycoside compositions obtained using the reaction solutions prepared in Examples 104 to 106, when the content of glycosides with n=0 is 10 mol% or more and 30 mol% or less, the content of glycosides with n=1 to 3 is 50 mol% or less, and the content of glycosides with n=4 or more is 30 mol% or more, under acidic sugar solution, in a functional evaluation based on a naringenin-7-glucoside equivalent concentration of 0.05%, bitterness, spiciness, and astringency are significantly reduced.

[0106] Molar ratio of flavonoid glycosides (rhamnose / flavonoids) The molar ratio (rhamnose / flavonoid) of the molar concentration calculated after determining the rhamnose content of samples 1 and 2 (under the same conditions as HPLC sugar analysis, with a calibration curve made using rhamnose (Wako)) and the molar concentration calculated based on the conversion values ​​of isoquercitrin and hesperidin-7-glucoside is 0.8 to 1.2.

[0107] Evaluation of colorfastness The composition containing flavonoid inclusion compounds from Example 16 and the flavonoid glycoside composition from Example 39 were added to an acidic sugar solution (pH 3.0) containing 0.05% red cabbage pigment preparation to make the isoquercitrin equivalent concentration 0.005%. The pigment residue rate after 4 hours of UV fading meter treatment was compared, and an anti-fading effect was observed compared with the additive-free product. The results are shown in Table 5.

[0108] [Table 5] Table 5 Pigment residue rate (%) Example 16 96 Example 39 95 Additive-free 56

[0109] As clearly shown in Table 5, an anti-fading effect on red cabbage pigment was observed in compositions containing isoquercitrin-γ-cyclodextrin and compositions containing isoquercitrin glycosides.

[0110] Evaluation of the effectiveness in preventing aroma degradation (milk) 100 ml of the reaction solution from Example 16 was added to a dialysis membrane (Spectra / Por CE dialysis tube, MWCO 500-1000, manufactured by Funakoshi Co., Ltd.) and dialyzed in 10 L of water (5 water exchanges, 10°C) to remove rhamnose. The solution was then freeze-dried to obtain 22 g of dried product. The obtained dried product and the flavonoid glycoside composition from Example 39 were added to a 100 ml transparent glass bottle to achieve an isoquercitrin concentration of 0.005% in commercially available milk (3.5% milk fat, "Meiji Emulsion", manufactured by Meiji Co., Ltd.). The aroma after irradiation with a fluorescent lamp (20,000 lux, 5 hours, 10°C) was compared with the average score of 10 judges using the following evaluation criteria. The results showed an effect in preventing aroma degradation. The results are shown in Table 6.

[0111] <Evaluation Criteria> 1: Significant changes compared to unirradiated samples 2: Slightly significant changes compared to the unirradiated sample 3: Slightly different from the unirradiated sample 4: Very slightly different from the unirradiated sample 5: No change compared to the unirradiated sample.

[0112] [Table 6] Table 6 Effect of preventing aroma deterioration (milk) Example 16 3.8 Example 39 3.9 Additive-free 2.2

[0113] As clearly shown in Table 6, the combination of isoquercitrin-γ-cyclodextrin inclusion compounds and isoquercitrin glycosides showed an effect in preventing the deterioration of flavor in milk.

[0114] Evaluation of the effect of preventing aroma degradation (jelly) Grapefruit jelly without additives was prepared using grapefruit juice (1 / 6) 0.5%, gelatin 3%, grapefruit juice capsules 1%, maltitol 6%, and safflower yellow preparation 0.025%. 100 ml of the reaction end solution from Examples 22 and 28 was added to a dialysis membrane (Spectra / Por CE dialysis tube, MWCO 500-1000, manufactured by Funakoshi Co., Ltd.) and dialyzed in 10 L of water (5 water exchanges, 10°C) to remove rhamnose. The solution was then freeze-dried, yielding 12 g of dried product in Example 22 and 16 g in Example 28. The obtained dried products and the flavonoid glycoside composition from Example 40 were added to the additive-free grapefruit jelly at a hesperidin-7-glucoside concentration of 0.005% to prepare grapefruit jelly with additives. They were then heated to 93°C, sealed in clear glass bottles, and cooled. The aromas after one month of storage at room temperature in a room exposed to normal fluorescent light were compared using the average scores of 10 judges according to the following evaluation criteria. The results showed that each additive had an effect in preventing aroma degradation. The results are shown in Table 7.

[0115] <Evaluation Criteria> 1: Significant changes compared to unirradiated samples 2: Slightly significant changes compared to the unirradiated sample 3: Slightly different from the unirradiated sample 4: Very slightly different from the unirradiated sample 5: No change compared to the unirradiated sample.

[0116] [Table 7] Table 7 Fragrance degradation prevention effect (jelly) Example 22 4.1 Example 28 4.0 Example 40 4.2 Additive-free 1.9

[0117] As clearly shown in Table 7, the aroma degradation prevention effect in grapefruit jelly was observed for hesperidin-7-glucoside-β-cyclodextrin inclusion compounds, hesperidin-7-glucoside-γ-cyclodextrin inclusion compounds, and hesperidin-7-glucoside glycoside compositions.

[0118] Evaluation of storage stability (acidic sugar solution) The isoquercitrin prepared in Comparative Example 101, the composition containing flavonoid inclusion compounds in Example 16, and the flavonoid glycoside composition in Example 39 were dissolved in an acidic sugar solution with a pH of 3 containing 0.03% isoquercitrin. The solutions were then heat-packed (93°C) into 100ml glass vials. After cooling, the solutions were allowed to stand for 4 months at 4°C, 25°C, and 40°C, respectively, and the presence or absence of precipitation was observed visually. Clear solutions without observed precipitation were marked as ○, and solutions with observed precipitation were marked as ×. The results are shown in Table 8.

[0119] Acidic sugar solution formula (quality%) 1. Sugar 10 2. Citric acid (crystals) 0.08 3. Adjust pH using sodium citrate (pH 3) 4. Water balance.

[0120] [Table 8] Table 8 4℃ 25℃ 40℃ Isoquercetin × × × Example 16 ○ ○ ○ Example 39 ○ ○ ○

[0121] As shown in Table 8, when isoquercitrin was added to the acidic sugar solution, precipitation was observed immediately after storage under any of the following conditions: refrigeration (4°C), room temperature (25°C), and 40°C. However, when isoquercitrin-γ-cyclodextrin inclusion compound and isoquercitrin glycoside combination were added, no precipitation was observed even after standing for 4 months, and no precipitation was observed even after long-term storage for 5 months.

[0122] Evaluation of storage stability (green tea) Hesperidin (manufactured by Hamari Pharmaceutical Co., Ltd.), hesperidin-7-glucoside (the product prepared below), the flavonoid inclusion compound of Example 22, and the flavonoid glycoside composition of Example 40 were added to commercially available green tea ("Oiocha", manufactured by Itoen Co., Ltd.) at a concentration equivalent to 0.03% of hesperidin-7-glucoside. After standing at 4°C and 25°C for 7 days, the presence or absence of precipitation was observed visually. Clear tea with no observed precipitation was marked as ○, and tea with observed precipitation was marked as ×. The results are shown in Table 9.

[0123] 7g of hesperidin (manufactured by Hamari Pharmaceutical Co., Ltd.) was added to 100L of aqueous solution, and the temperature was adjusted to 70°C and pH 4.5. Then, while stirring, 0.5g of naringinase (Amano Engineering Co., Ltd., 155u / g) was added, and the mixture was recovered and dried to obtain 4.2g of hesperidin-7-glucoside with a purity of over 96%. As described above, it was analyzed by NMR and HPLC to confirm its hesperidin-7-glucoside composition and its content.

[0124] [Table 9] Table 9 4℃ 25℃ hesperidin × × Hesperidin-7-glucoside × × Example 22 ○ ○ Example 40 ○ ○

[0125] As shown in Table 9, when hesperidin and hesperidin-7-glucoside were added to green tea, precipitation was observed immediately after storage under either refrigeration (4°C) or room temperature (25°C). However, when compositions containing hesperidin-7-glucoside inclusion compounds and hesperidin-7-glucoside glycosides were added, no precipitation was observed after only 7 days of standing.

[0126] Evaluation of shelf-life stability (lemon beverage) The composition of naringin (manufactured by SIGMA), naringenin-7-glucoside (the product prepared below), and the flavonoid inclusion compound of Example 109 was added to a commercially available lemon beverage (C1000 Lemon Wouter, manufactured by Hauswälness Foods) at a concentration of 0.3% (converted from naringenin-7-glucoside). After standing at 4°C and 25°C for one month, the presence or absence of precipitation was observed visually. Clear samples without observed precipitation were marked as ○, and samples with observed precipitation were marked as ×. The results are shown in Table 9-2.

[0127] After the reaction in Comparative Example 102 was completed, the precipitate, after standing at room temperature, was recovered, then washed with water, recrystallized, and dried to obtain 13 g of naringenin-7-glucoside with a purity of over 95%. The precipitate and its content were analyzed by HPLC using the reagent naringenin-7-glucoside (Wako).

[0128] [Table 9-2] Table 9-2 4℃ 25℃ Naringin × × Naringenin-7-glucoside × × Example 109 ○ ○

[0129] As shown in Table 9-2, when naringin and naringenin-7-glucoside were added to lemon beverages, precipitation was observed under either refrigeration (4°C) or room temperature (25°C). However, when a composition containing naringenin-7-glucoside inclusion compounds was added, no precipitation was observed even after standing for 1 month.

[0130] Evaluation of in vivo absorption Nine-week-old male Wister rats were fed MF (Oryzanol yeast strain) as feed for 7 days, and then fasted one day before administration of the test substance. Then, the following substances were administered orally once (catheter-forced oral administration, n=2): 100 μmol / kg (IQC equivalent) of the dried product of Example 16 prepared in the evaluation of aroma deterioration prevention, rutin suspension (Alps Pharmaceuticals, 100 μmol / kg (IQC equivalent)), 1000 μmol / kg (HPT-7G equivalent) of the dried product of Example 22 prepared in the evaluation of aroma deterioration prevention, and hesperidin suspension (Hamari Pharmaceuticals, 1000 μmol / kg (HPT-7G equivalent)). Blood was collected from the rat's tail vein at 30 minutes, 1 hour, and 3 hours, and plasma was obtained by centrifugation. The amount of quercetin derivatives in the collected serum samples was determined according to the method of Makino et al. ( Biol.pharm.Bull. 32(12) 2034,2009), the amount of hesperidin derivatives was determined according to the method of Yamada et al. ( Biosci.Biotechnol.Biochem,70(6),1386,2006), high performance liquid chromatography (SHIMADZU) was used, and analysis was performed using a photodiode array detector (SPD-M30A, SHIMADZU). The results are shown in Tables 10 and 11. Table 10 shows the concentrations (μM) of quercetin and quercetin derivatives (isorhamnoside, tamarindin) and their summation area under the plasma concentration-time curve (AUC) (μM·h) from 0 to 3 hours. In addition, since hesperidin derivatives were not detected, Table 11 shows the concentrations (μM) of hesperidin and its area under the plasma concentration-time curve (AUC) (μM·h) from 0 to 3 hours.

[0131] As shown in Tables 10 and 11, it can be seen that in the comparison of AUC 1–3 hours, isoquercitrin-γ-cyclodextrin inclusion compounds and hesperidin-7-glucoside-β-cyclodextrin inclusion compounds were absorbed by rats more efficiently than rutin and hesperidin. Furthermore, although not recorded in the tables, the in vivo absorption rate of the isoquercitrin glycoside composition of Example 39 at 100 μmol / kg (IQC conversion value) was approximately the same as that of Example 16, and the in vivo absorption rate of the hesperidin-7-glucoside glycoside composition of Example 40 at 1000 μmol / kg (HPT-7G conversion value) was approximately the same as that of Example 22.

[0132] Improved solubility of poorly soluble flavonoids Examples 110-113 Using the amounts of ingredients shown in Table 12, rutin (RTN) and the inclusion compound of isoquercitrin and γ-cyclodextrin (IQC-rCD) were dissolved in acidic sugar solution (pH 3.1, Brix 10°) and hot-packed into 100 ml glass bottles. The prepared solutions were cooled and stored under refrigeration (4°C for 6 months), and the presence or absence of precipitation was observed visually. The results are shown in Table 12.

[0133] Comparative Examples 103-106, Reference Examples Using the amounts of ingredients shown in Table 13, rutin (RTN) and isoquercitrin (IQC) were dissolved in acidic sugar solution, prepared, cooled, and refrigerated in the same manner as in Examples 110-113, and the presence or absence of precipitation was observed visually. The results are shown in Table 13.

[0134] In Tables 12 and 13, IQC / RTN (molar ratio) is the area ratio (peak area of ​​isoquercitrin / peak area of ​​rutin) expressed as a molar ratio after HPLC analysis (SHIMADZU, under the same conditions as conversion) of 1 ml of acidic sugar solution after dissolving the prepared components as the test sample.

[0135] Notes to Tables 12 and 13 (41) Rutin concentration (mass%) in acidic sugar solution (42) Concentration (mass%) of isoquercitrin-γ-cyclodextrin inclusion compound in acidic sugar solution (43) Concentration of isoquercitrin in acidic sugar solution (mass%) (44) Concentration of γ-cyclodextrin in acidic sugar solution (mass%) (45) Solubility: No precipitation was observed: Small amount of precipitate: + The amount of precipitate is slightly higher: ++ Large amount of precipitate: +++ (46) Isoquercetin / rutin (molar ratio) in acidic sugar solution.

[0136] As shown in Tables 12 and 13, in Examples 110–113 with the addition of isoquercitrin-γ-cyclodextrin inclusion compounds, the solubility of rutin was improved, particularly when the molar ratio of isoquercitrin to rutin (isoquercitrin / rutin) was in the range of 0.1–0.7, and no precipitation was observed. Similar results were also observed in the freeze-dried flavonoid inclusion compounds of Examples 10, 13, and 14 (products from which rhamnose was removed by dialysis), and in the freeze-dried compositions of flavonoid inclusion compounds containing rhamnose. On the other hand, as shown in Table 13, precipitation was observed in those with only isoquercitrin added.

[0137] Furthermore, in Tables 12 and 13, the same results were obtained with isoquercitrin and isoquercitrin-β-cyclodextrin (freeze-dried products of Examples 1-7), or with hesperidin and hesperidin-7-glucoside-β-cyclodextrin instead of rutin (freeze-dried products of Examples 18-23), or hesperidin-7-glucoside-γ-cyclodextrin (freeze-dried products of Examples 27-31).

[0138] Evaluation of the long-term stability and astringency of isoquercitrin-γ-cyclodextrin inclusion compounds Examples 114-117 Except for the amounts of components shown in Table 14, solutions were prepared in the same manner as in Examples 110-113, and the astringency was functionally evaluated immediately after cooling. Furthermore, solutions with the same amounts of components were prepared separately, refrigerated (4°C, 12 months), and the presence or absence of precipitation was observed visually. The results are shown in Table 14.

[0139] Comparative Examples 107-110 Except for the amounts of components shown in Table 15, solutions were prepared in the same manner as in Comparative Examples 103-106, and the astringency was evaluated functionally immediately after cooling. Furthermore, solutions with the same amounts of components were prepared separately, refrigerated (4°C, 12 months), and the presence or absence of precipitation was observed visually. The results are shown in Table 15.

[0140] In Tables 14 and 15, RTN / IQC (molar ratio) is the area ratio (peak area of ​​rutin / peak area of ​​isoquercitrin) expressed as a molar ratio after HPLC analysis (SHIMADZU, under the same conditions as conversion) of 1 ml of acidic sugar solution after dissolving the prepared components as the test sample.

[0141] Notes to Tables 14 and 15 (51) Concentration (mass%) of isoquercitrin-γ-cyclodextrin inclusion compound in acidic sugar solution (52) Concentration of isoquercitrin in acidic sugar solution (mass%) (53) Concentration of γ-cyclodextrin in acidic sugar solution (mass%) (54) Rutin concentration (mass%) in acidic sugar solution (55) Solubility: No precipitation was observed: Small amount of precipitate: + The amount of precipitate is slightly higher: ++ Large amount of precipitate: +++ (56) Rutin / isoquercetin ratio in acidic sugar solution (57) After the acidic sugar solution was prepared and cooled, it was compared by 10 judges with the acidic sugar solution without additives. The sample with the strongest astringent taste was scored as 3 points, followed by 2 points and 1 point. The average value is shown.

[0142] It should be noted that no precipitation was observed in Comparative Examples 107–110 after preparation and cooling, and at room temperature for 30 minutes. Therefore, functional evaluation was performed during this period.

[0143] As shown in Table 14, it was confirmed that even when a specific amount of rutin is present in the composition containing isoquercitrin and γ-cyclodextrin obtained by the manufacturing method of the present invention, the long-term stability of the inclusion compound is not a problem. Furthermore, it was confirmed that the astringency is weak, and its impact on the flavor when added to food and beverages is minimal. The astringency is particularly weak when the RTN / IQC (molar ratio) is 0.08 or less (Examples 114-117). These results were also observed in the freeze-dried flavonoid inclusion compounds of Examples 10, 13, and 14 (products from which rhamnose was removed by dialysis) and the freeze-dried composition containing rhamnose and flavonoid inclusion compounds. On the other hand, as shown in Table 15, precipitation was observed in the compositions containing isoquercitrin when rutin was present, and the astringency was also strong.

[0144] Furthermore, in Tables 14 and 15, the same results were obtained with isoquercitrin and isoquercitrin-β-cyclodextrin (freeze-dried products of Examples 1-7), or with hesperidin and hesperidin-7-glucoside-β-cyclodextrin instead of rutin (freeze-dried products of Examples 18-23), or hesperidin-7-glucoside-γ-cyclodextrin (freeze-dried products of Examples 27-31).

[0145] The specific contents of the ingredients used in Examples 110-117, Comparative Examples 103-110, and Reference Examples are as follows. RTN: A solution of 90g of 99.5% ethanol (by volume) and 10g of rutin (prepared product: isoquercitrin / rutin molar ratio of 0.3 / 99.7) dissolved by heating. IQC-rCD: Freeze-dried product of Example 16 (product with rhamnose removed by dialysis, (rutin / isoquercetin molar ratio of 0.3 / 99.7)). IQC: A product obtained by heating and dissolving 18g of 99.5% ethanol (by volume) and 2g of isoquercitrin (prepared product: molar ratio of rutin / isoquercitrin is 0.3 / 99.7).

[0146] Formulation examples of compositions containing flavonoid inclusion compounds and flavonoid glycoside compositions. Formula Example 1: Grapefruit Beverage To prevent flavor degradation, a beverage containing the dried product of the composition of Example 16 containing isoquercitrin-γ-cyclodextrin inclusion compounds was prepared. This product can be suitably used as a beverage. Ingredient weight % Grapefruit concentrate juice 5.0 0.9g of a mixed liquid sugar of glucose and fructose. Maltitol 2.0 Acidulant 0.3 Vitamin C 0.02 Spices 0.1 0.02g of dried product from Example 16 Water balance Total: 100.

[0147] Recipe Example 2: Jelly To prevent flavor degradation, a dried product containing the composition of Example 22, comprising hesperidin-7-glucoside-β-cyclodextrin inclusion compound, was prepared as a jelly. This product can be suitably used as a food (jelly). Ingredient weight % Sugar 10.0 Lemon concentrate juice 8.5g Gardenia yellow preparation 0.004 Acidulant 1.0 Gelation agent 1.5 Vitamin C 0.02 Spices 0.2 0.04g of dried product from Example 22 Water balance Total: 100.

[0148] Formula Example 3: Cosmetics To improve dull, puffy skin, a cosmetic containing a dried product of the hesperidin-7-glucoside composition of Example 40 is prepared. This product can be suitably used as a skin care cosmetic. Ingredient weight % Glycerin 5.0 Propylene glycol 4.0 0.1% oleyl alcohol Surfactant 2.0 10.0g of ethanol Spices 0.1 0.26g of dried product from Example 40 Pure water balance Total: 100.

[0149] Formulation Example 4: Tablets To alleviate body temperature, tablets containing the dried product of the composition of Example 22, which contains hesperidin-7-glucoside-β-cyclodextrin inclusion compound, are prepared. This product can be suitably used as a health food. Ingredient weight % Maltitol 69.0 Trehalose 12.9 Acidulant 2.5 Stearic acid (Ca 0.5) Vitamin C 0.02 Spices 0.08 15.0g of dried material from Example 22 Total: 100.

[0150] Recipe Example 5: Coffee Beverage To reduce body fat, a coffee beverage containing a dried product of the isoquercitrin glycoside composition of Example 39 was prepared. This product can be suitably used as a food for specific health purposes. Ingredient weight % Coffee extract 32.6 Sugar 6.0 Spices 0.06 0.06g of dried product from Example 39 Water balance Total: 100.

[0151] Formula Example 6: Black Tea Beverage To reduce neutral fats, a black tea beverage containing the dried product of the hesperidin-7-glucoside composition of Example 40 was prepared. This product can be suitably used as a functional food. Ingredient weight % Black tea extract 18.6 Sodium bicarbonate 0.002 0.003% sucralose Vitamin C 0.03 Spices 0.1 0.06g of dried product from Example 40 Water balance Total: 100.

[0152] Formula Example 7: Hair Growth Agent To improve scalp health, a hair growth agent was prepared containing a dried product of the composition of Example 22 containing hesperidin-7-glucoside-β-cyclodextrin inclusion compound. Ingredient weight % Ethanol 60.0 Japanese Swertia Extract 5.0 Acetate tocopherol 0.2 Panthenol ethyl ether 0.2 Propylene glycol 5.0 Preservative 0.1 Spices 0.2 0.03g of dried product from Example 22 Pure water balance Total: 100.

[0153] Formula Example 8: Shampoo To prevent inflammation, a shampoo was prepared containing the dried product of the composition of Example 27 containing hesperidin-7-glucoside-γ-cyclodextrin inclusion compound. Ingredient weight % Polyoxyethylene (2) lauryl ether Sodium sulfate 9.0 Sodium lauryl sulfate 4.0 Coconut oil fatty acid amamidopropyl betaine 3.0 Highly polymeric methylpolysiloxane 2.0 Methyl polysiloxane 1.0 Coconut oil fatty acid monoethanolamide 1.0 Propylene glycol 2.0 2.0g of ethylene distearate Preservative 0.1 Spices 0.1 0.03g of dried product from Example 27 Water balance Total: 100.

[0154] Formula Example 9: Weight Loss Tablets For weight loss, tablets are prepared containing the dried product of the composition of Example 109 containing naringenin-7-glucoside-β-cyclodextrin inclusion compound. This product can be suitably used as a health food. Ingredient weight % Maltitol 64.0 Trehalose 12.9 Acidulant 2.5 Stearic acid (Ca 0.5) Vitamin C 0.02 Spices 0.08 20.0g of dried material from Example 109 Total: 100.

[0155] Industrial practicality According to the manufacturing method of the present invention, flavonoid inclusion compounds and flavonoid glycoside compositions with excellent water solubility can be efficiently produced and can be appropriately used in the fields of pharmaceuticals, food and beverages, health foods, foods for specific health purposes, and cosmetics.

Claims

1. A method for producing flavonoid inclusion compounds, comprising a separation step, the separation step including: Rhamnose is removed from insoluble flavonoids with rhamnoside structures by treatment with an enzyme with rhamnosidase activity in the presence of cyclodextrin. Among them, poor solubility refers to a solubility of less than 1.0% by mass in water at 25°C.

2. The manufacturing method according to claim 1, wherein, The insoluble flavonoid having a rhamnoside structure is selected from one or more of the following: rutin, hesperidin, naringin, geraniin, sennain, myricetin, neohesperidin, luteolin-7-rutinoside, delphinidin-3-rutinoside, cyanidin-3-rutinoside, isorhamnetin-3-rutinoside, kaempferol-3-rutinoside, apigenin-7-rutinoside, and robinin-7-rutinoside.

3. The manufacturing method according to claim 1 or 2, wherein, The cyclodextrin is present in a proportion of 0.01 moles or more relative to 1 mole of the insoluble flavonoid having a rhamnoside structure.

4. The manufacturing method according to any one of claims 1 to 3, wherein, The flavonoid inclusion compound is a flavonoid inclusion compound formed by the inclusion of the poorly soluble flavonoid without rhamnoside structure into cyclodextrin, wherein the molar ratio (cyclodextrin / flavonoid) of the cyclodextrin to the poorly soluble flavonoid without rhamnoside structure is 1.0 to 3.

0.

5. The manufacturing method according to any one of claims 1 to 4, wherein, The flavonoid inclusion compounds are selected from: (i) A flavonoid inclusion compound formed by isoquercitrin encapsulating γ-cyclodextrin, wherein the molar ratio of γ-cyclodextrin to isoquercitrin (γ-cyclodextrin / isoquercitrin) is 0.9 to 4.0; (ii) A flavonoid inclusion compound formed by isoquercitrin encapsulating β-cyclodextrin, wherein the molar ratio of β-cyclodextrin to isoquercitrin (β-cyclodextrin / isoquercitrin) is 1.0 to 3.0; (iii) A flavonoid inclusion compound formed by encapsulating hesperidin-7-glucoside in cyclodextrin, wherein the molar ratio of said cyclodextrin to said hesperidin-7-glucoside (cyclodextrin / hesperidin-7-glucoside) is 1.0 to 3.0; and (iv) A flavonoid inclusion compound formed by encapsulating naringenin-7-glucoside in β-cyclodextrin, wherein the molar ratio of β-cyclodextrin to naringenin-7-glucoside (β-cyclodextrin / naringenin-7-glucoside) is 1.0 to 3.

0.

6. The manufacturing method according to claim 1, further comprising: The glycosylation step involves treating the flavonoid inclusion compound obtained from the detachment step with a glycosyltransferase to glycosylate the flavonoid inclusion compound.

7. Selected from the following flavonoid inclusion compounds, (i) Flavonoid inclusion compounds formed by isoquercitrin encapsulating γ-cyclodextrin, wherein, The molar ratio of γ-cyclodextrin to isoquercitrin (γ-cyclodextrin / isoquercitrin) is 0.9 to 1.8, and the solubility of isoquercitrin in water at 25°C is more than 2%. (ii) A flavonoid inclusion compound formed by isoquercitrin encapsulating γ-cyclodextrin, wherein the molar ratio of γ-cyclodextrin to isoquercitrin (γ-cyclodextrin / isoquercitrin) is 0.9 to 4.0, and the solubility of isoquercitrin in water at 25°C is more than 2.5%. (iii) A flavonoid inclusion compound formed by isoquercitrin encapsulating β-cyclodextrin, wherein the molar ratio of β-cyclodextrin to isoquercitrin (β-cyclodextrin / isoquercitrin) is 1.0 to 3.0, and the solubility of isoquercitrin in water at 25°C is more than 0.1%. (iv) A flavonoid inclusion compound formed by encapsulating hesperidin-7-glucoside in cyclodextrin, wherein the molar ratio of the cyclodextrin to the hesperidin-7-glucoside (cyclodextrin / hesperidin-7-glucoside) is 1.0 to 3.0, and the hesperidin-7-glucoside has a solubility of more than 0.01% in water at 25°C; and (v) A flavonoid inclusion compound formed by encapsulating naringenin-7-glucoside in β-cyclodextrin, wherein the molar ratio of β-cyclodextrin to naringenin-7-glucoside (β-cyclodextrin / naringenin-7-glucoside) is 1.0 to 3.0, and the solubility of naringenin-7-glucoside in water at 25°C is greater than 0.01%.

8. A composition containing flavonoid inclusion compounds, comprising: (i) the flavonoid inclusion compound of claim 7, and rhamnose, wherein the molar ratio (rhamnose / flavonoid) of rhamnose to flavonoids in the flavonoid inclusion compound is 0.8 to 1.2; or (ii) The flavonoid inclusion compound of claim 7, and the insoluble flavonoid having a rhamnoside structure, wherein the molar ratio of the insoluble flavonoid to the flavonoid in the flavonoid inclusion compound (insoluble flavonoid / flavonoid in the inclusion compound) is 0.001 to 0.

1.

9. A product that is a food, beverage, pharmaceutical or cosmetic product, comprising one or more compounds or compositions selected from the following: a flavonoid inclusion compound obtained by the manufacturing method of any one of claims 1 to 5; a flavonoid glycoside composition obtained by the manufacturing method of claim 6; a flavonoid inclusion compound of claim 7; and a composition containing a flavonoid inclusion compound of claim 8.

10. A method for improving the solubility of poorly soluble flavonoids with rhamnoside structures, wherein, The insoluble flavonoid having a rhamnoside structure, and the flavonoid inclusion compound obtained by the manufacturing method according to any one of claims 1 to 5 or the flavonoid inclusion compound according to claim 7, are mixed in a medium such that the molar ratio of flavonoid in the inclusion compound to the insoluble flavonoid (flavonoid in the inclusion compound / insoluble flavonoid) is 0.1 to 0.9.

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