JP2006182777A - Method for synthesizing inclusion compounds of hesperetin and naringenin - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide inclusion compounds of hesperetin and naringenin having improved absorbability in vivo as a method for improving the absorbability in vivo of hesperidin and naringin which are flavanone present in the nature; and to provide a method for synthesizing them. <P>SOLUTION: The method for synthesizing the inclusion compound of the flavanone which is the inclusion compound of the hesperetin or the naringenin is provided. In one example of the method, the method comprises a step for obtaining the inclusion compound of the hesperidin or the naringin by including the hesperidin or the naringin with a β-cyclodextrin, a branched β-cyclodextrin or a modified product thereof, and a step for obtaining the inclusion compound of the hesperetin or the naringenin by hydrolyzing the inclusion compound of the hesperidin or the naringin. In another aspect, the hesperetin or the naringenin can directly be included. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for producing a flavanone clathrate compound, and a food and a medicine containing the flavanone clathrate compound.

  Polyphenol is a general term for plant components having a plurality of phenolic hydroxyl groups in the same molecule. Polyphenol is contained in various plants and is a substance excellent in antioxidant ability. Polyphenols have various physiological activities and are used in various applications such as pharmaceuticals and foods. Among polyphenols, research on flavonoids is in progress.

  For flavonoids, for example, the following physiological activities are known. Hesperidin and rutin have long been known as vitamin P, and vitamin P is known to have a blood pressure lowering effect (Non-patent Document 1: Shintaro Kamiya, New Vitaminology (Japan Vitamin Society) 1969, p439). Hesperidin further has an antiallergic action (Non-patent Document 2: Hideaki Matsuda et al., Pharmaceutical Journal, 111, 193-198 (1991), Non-patent Document 3: JA Da Silva Emim et al., J. Pharm. Pharmacol., 46, 118-712 (1994)), an action to decrease LDL-cholesterol and improve blood cholesterol level (Non-patent Document 4: MT Monoforte et al., Il Farmaco, 50, 595-599 (1995)), anti Cancer action (Non-patent document 5: T. Tanaka et al., Cancer Research, 54, 4653-4659 (1994), Non-patent document 6: T. Tanaka et al., Cancer Research, 57, 246-252 (1997), Non-patent document 7: T. Tanaka et al., Carcinogen esis, 18, 761-769 (1997), non-patent document 8: T. Tanaka et al., Carcinogenesis, 18, 957-965 (1997)) have been reported. Non-Patent Document 9 (A. Garg, S. Garg, LJD Zaneveld and AK Singla, Chemistry and pharmacology of the citrus bioflavonoid hesperidin, PHYTOTHERAPY 1565). The chemistry and pharmacological actions of are outlined. Non-patent document 9 describes that hesperidin has various effects such as an action of reducing the permeability and vulnerability of capillary walls, an action of suppressing colitis, an action of suppressing arthritis, and an action of suppressing aggregation of platelets and cells, in addition to the above-mentioned actions. It describes having a physiological effect.

  Furthermore, in recent studies, hesperidin is expected to promote the differentiation of preadipocytes and improve symptoms such as diabetes. Hesperidin also has an effect of improving blood circulation, an effect of improving blood circulation by improving shoulder stiffness, coldness and the like (Patent Document 1: JP-A-11-171778), an effect of improving skin condition (Patent Document 2: JP-A 2003-325135). And the osteoporosis improving effect (Patent Document 3: JP 2002-234844 A).

  Naringin is known as a citrus bitter substance and is used in foods and beverages for the purpose of imparting a bitter taste. Naringenin, an aglycone thereof, is antimutagenic (Non-Patent Document 10: WL Bear and RW Teel. Effects of citrus flavonoids on the mutagenicity of Acetycetic acid. : 3609-3614 (2000)), cell growth inhibitory action (Non-Patent Document 11: T. Takahashi et al., Structure-activity relations of the flavonoids and the induction of granity in HL60 human myeloid leukemia cells.Bioscience, Biotechnology and Biochemistry 62: 2199-2204 (1998)), Non-Patent Document 12: M. D. Herrera and c. noradrenaline in rat vas deferens-I. Evidence for postoperative alpha-2 adrenergic receptor.Gen.Pharmac.24: 739-742 (1993)), non-patent document 13: G. D. Carlo et al., Inhibition of intensional mobility and section by flavonoids in mice and rats: structure-activity relationship 105. Journal of Pharm. Summanen et al., Effects of simple aromatic compounds and flavonoids on Ca2 + fluxes in rat piloty GH4C1 cells. It has been reported to have various physiological activities such as European Journal of Pharmacology 414: 125-133 (2001)).

  Flavonoids are sparingly soluble in the pH range of 3 to 10 and tend to form precipitates. For example, baicalein, a type of flavonoid, absorbs only about 1/3 million of the dose. Thus, flavonoids are known to have extremely poor absorption efficiency into the body (Non-patent Document 15: W. Wakui et al., J. Chromat., 575, 131-136 (1992)). Unless it is absorbed into the body, the physiological activity of the flavonoid is not sufficiently exhibited in vivo. Therefore, it has been eagerly desired to increase the absorption efficiency.

  Naturally occurring flavonoids generally exist in the form of glycosides in which a sugar is bound to an aglycone. Flavonoids in the form of glycosides are considered to be taken into the body from the large intestine after sugar is hydrolyzed by enzymes produced by bacteria present in the intestinal tract after ingestion by animals. . Various attempts to increase the amount of flavonoids absorbed into the body have been studied. As one of them, a method for improving the solubility of flavonoids has been studied.

For example, Non-Patent Document 16 (Kayoko Shimoi et al., Journal of Agricultural and Food Chemistry, Vol. 51, No. 9 (2003)) is aglycone quercetin, quercetin diglycoside tritin, and quercetin triglyceride. The absorption of 4 ^G- α-D-glucopyranosylrutin (αG-rutin), which is a glycosylated product, into the rat body is compared. According to Non-Patent Document 16, the absorbability increases in the order of quercetin <rutin <αG-rutin.

  The absorbability and water solubility of quercetin, rutin and αG-rutin are summarized in Table 1 below and FIG. Absorptivity is based on AUC0 → 24h in Table 1 of Non-Patent Document 16. Here, for AUC0 → 24h, the value of the sum of the unmodified product and the methylated product (for example, in the case of quercetin, the sum of quercetin, tamalizetin and isorhamnetin) was used. As a relative ratio, the ratio when αUC-rutin AUC0 → 24h is 1.0 is shown. Solubility shows known values for rutin and αG-rutin. Quercetin is almost insoluble in water. In FIG. 5, the solubility of quercetin was plotted as 0.01 in order to place it on a logarithmic graph.

  It can be seen from Table 1 and FIG. 5 that the absorbability increases as the solubility increases. From these facts, it is considered that the absorbability increases if the solubility of flavonoids is increased.

  In general, glycosylation of flavonoids increases solubility. For this reason, various attempts have been made to increase the solubility by glycosylating flavonoids.

  For example, Patent Document 4 (Japanese Patent Application Laid-Open No. 2000-78956) discloses that a sugar is bound to a flavonoid (for example, hesperidin, which is a glycoside), so that the glycosylated compound has a higher rate than the non-glycosylated compound in the body. To be absorbed.

  Patent Document 5 (Japanese Patent Application Laid-Open No. 2000-78955) discloses that a flavonoid (for example, hesperidin that is a glycoside) and a flavonoid are both administered to a living body by administering a sugar transfer compound and a flavonoid to the living body. It is described that the absorption efficiency of is greatly improved.

  As another method for improving the solubility of flavonoids in water, a method of including flavonoids with cyclodextrin is known. For example, Patent Document 6 (Japanese Patent Laid-Open No. 6-54664) describes that the solubility in water can be significantly improved by inclusion of rutin, which is a glycoside of quercetin, with cyclodextrin.

  However, on the other hand, even if rutin is included in β-cyclodextrin, there is almost no influence on the absorbency (Non-patent Document 17: Kouzou Miyake et al. There are also β-cyclodextrins, Pharmaceutical development and technology, 5 (3), 399-407 (2000)).

  Thus, conventionally, attempts have been made to improve the solubility of flavonoids as one of the methods for improving the absorbability of flavonoids. In order to improve the solubility of flavonoids, attempts have been mainly made to further glycosylate flavonoids, which are glycosides, or to include them with cyclodextrins.

  Non-Patent Document 18 (R. Ficarra et al., Study of flavonids / β-cyclodextrins inclusion compounds by NMR, FT-IR, DSC, X-ray investigation 2-, 200 of biopharmaceuticals. Hesperetin, hesperidin, naringenin and naringin are included in β-cyclodextrin to check whether they are included. Non-Patent Document 18 describes that inclusion compound formation increases solubility in water and suggests that it may improve drug dissolution and absorption in therapeutic formulations. . However, Non-Patent Document 18 does not investigate the absorbability itself of these complexes. Moreover, it is not described which has higher bioavailability between a glycoside of hesperetin and a hesperetin inclusion product. In the method described in Non-Patent Document 18, an inclusion compound is prepared using an organic solvent such as methanol. Since methanol is harmful to the human body, the inclusion compound prepared using this method cannot be used as a food.

  Furthermore, as another method for improving the absorbability, a method of cleaving a flavonoid glycoside sugar and taking it as a flavonoid aglycone has been tried.

  In the case of hesperidin, hesperetin, an aglycon, is more easily absorbed than hesperidin, a glycoside, as described in Non-Patent Document 19 (Booth AN et al., Metabolic fat of hesperidin, eroditictyol, homeochemicaltyol and diosmin, J. Biol. J. Biol. 230: 661-668 (1958)).

  For rutin, the method of increasing the solubility by inclusion with β-cyclodextrin has little effect on improving the absorbability. The method of transferring a sugar to rutin to form a glycoside has a higher effect of increasing the solubility than the inclusion of rutin with cyclodextrin, and also has a high effect of further increasing the absorbability.

  For hesperidin, the method of cleaving the sugar of hesperidin to form the aglycone with the lowest solubility is better than the method of increasing the solubility by transferring the sugar to hesperidin to form a glycoside. The effect of raising the sex is high.

  Thus, it is considered that the method for increasing the absorbency differs depending on the type of flavonoid, and it is difficult to predict the method for increasing the absorbability most.

A method for efficiently including a hesperetin inclusion product and a naringenin inclusion product is also not known.
JP-A-11-171778 (first page) JP 2003-325135 A (first page) JP 2002-234844 A (first page) JP 2000-78956 A (summary of the first page) Japanese Unexamined Patent Publication No. 2000-79955 (2nd page) JP-A-6-54664 (first page) Shintaro Kamiya, New Vitaminology (Japan Vitamin Society) 1969, p439 Hideaki Matsuda et al., Pharmaceutical Journal, 111, 193-198 (1991) J. et al. A. Da Silva Emim et al. Pharm. Pharmacol. 46, 118-712 (1994) M.M. T. T. et al. Monforte et al., Il Farmaco, 50, 595-599 (1995). T. T. et al. Tanaka et al., Cancer Research, 54, 4653-4659 (1994). T. T. et al. Tanaka et al., Cancer Research, 57, 246-252 (1997). T. T. et al. Tanaka et al., Carcinogenesis, 18, 761-769 (1997). T. T. et al. Tanaka et al., Carcinogenesis, 18, 957-965 (1997). A. Garg, S .; Garg, L.G. J. et al. D. Zaneveld and A.M. K. Singla, Chemistry and pharmacology of the citrus bioflavonoid hesperidin, PHYTOOTHERAP REARCH 15, 655-669 (2001) W. L. Bear and R.M. W. Tele. Effects of citrus flavonoids on the mutagenicity of heterocyclic amines and on cytochrome P450 1A2 activity. Anticancer Research 20: 3609-3614 (2000) T. T. et al. Takahashi et al., Structure-activity relations of flavonoids and the induction of granularity-or monocytic-differentiation in HL60 humani. Bioscience, Biotechnology and Biochemistry 62: 2199-2204 (1998) M.M. D. Herrera and E.H. Marhuenda Effect of Naringin and Naringenin on Contractions Induced By Noradrenalin in rat vas deferens-I. Evidence for postsynthetic alpha-2 adrenergic receptor. Gen. Pharmac. 24: 739-742 (1993) G. D. Carlo et al., Inhibition of intensional mobility and section by flavonoids in mice and rats: structure-activity relations. Journal of Pharmacology and Pharmacology 45: 1054-1059 (1993) J. et al. Summanen et al., Effects of simple aromatic compounds and flavonoids on Ca2 + fluxes in rat piloty GH4C1 cells. European Journal of Pharmacology 414: 125-133 (2001) W. Wakui et al. Chromatog. , 575, 131-136 (1992) Kayoko Shimoi et al., Journal of Agricultural and Food Chemistry, Vol. 51, No. 9 (2003) Kouzou Miyake et al., Improvement of solidity and oral bioavailability of routine by complex3 with 2-hydropropyline-β-cyclodextrin R. Ficarra et al., Study of flavonids / β-cyclodextrins inclusion complexes by NMR, FT-IR, DSC, X-ray incubation. , Journal of pharmaceutical and biomedical analysis 29 (2002) 1005-1014. Boot AN et al., Metabolic fate of hesperidin, erodidictol, homoerodytyol and diosmin, J. et al. Biol. Chem. 230: 661-668 (1958)

  The present invention is intended to solve the above problems, and as a method for enhancing the bioabsorbability of hesperidin or naringin, which is a naturally occurring glycoside form, hesperetin inclusion compound and naringenin inclusion compound are synthesized. It aims to provide a way to do.

  As a result of intensive studies to solve the above problems, the present inventors have removed hesperidin or naringin, which is a naturally occurring glycoside form, by hydrolyzing it to form hesperetin or naringenin which are aglycones. These are further included by β-cyclodextrin, branched β-cyclodextrin or a modified product thereof, or hesperidin or naringin is first included by β-cyclodextrin, branched β-cyclodextrin or a modified product thereof. After that, it was found that the method of changing the inclusion of flavonoid aglycone by removing sugar from these by hydrolysis was the most effective method for increasing the bioabsorbability of hesperidin or naringin. The present invention has been completed.

The method of the present invention is a method for synthesizing a flavanone inclusion compound which is a hesperetin inclusion compound or a naringenin inclusion compound,
Inclusion of hesperidin or naringin with β-cyclodextrin, branched β-cyclodextrin or a modification thereof to obtain a hesperidin inclusion compound or naringin inclusion compound; and the hesperidin inclusion compound or the naringin inclusion compound Is hydrolyzed to obtain a hesperetin inclusion compound or a naringenin inclusion compound.

  In one embodiment, the hydrolyzing step can be performed by allowing rhamnosidase and glucosidase to act on the hesperidin inclusion compound or the naringin inclusion compound.

  In one embodiment, the β-cyclodextrin, branched β-cyclodextrin, or a modified product thereof is a β-cyclodextrin, branched β-cyclodextrin having 1 to 6 glucose residues linked as side chains. It can be selected from the group consisting of cyclodextrins, and their chemically modified products.

  In one embodiment, the hesperidin or naringin can be included by branched β-cyclodextrin.

  In one embodiment, the branched β-cyclodextrin may be a compound in which two glucose residues are linked as side chains to β-cyclodextrin.

  In one embodiment, the flavanone inclusion compound can be a hesperetin inclusion compound.

Another method of the present invention is a method of synthesizing a flavanone inclusion compound which is a hesperetin inclusion compound or a naringenin inclusion compound,
By mixing hesperetin or naringenin with a basic suspension or aqueous solution containing hesperetin or naringenin and an acidic aqueous solution containing β-cyclodextrin, branched β-cyclodextrin or a modification thereof, β-cyclodextrin , Inclusion by a branched β-cyclodextrin or a modified product thereof to obtain a hesperetin inclusion compound or a naringenin inclusion compound.

  In one embodiment, the β-cyclodextrin, branched β-cyclodextrin or a modified product thereof is β-cyclodextrin, branched β-cyclodextrin having 1 to 6 glucose residues linked as side chains. It can be selected from the group consisting of cyclodextrins, and their chemically modified products.

  In one embodiment, the hesperetin or naringenin may be included by branched β-cyclodextrin.

  In one embodiment, the branched β-cyclodextrin may be a compound in which two glucose residues are linked as side chains to β-cyclodextrin.

  In one embodiment, the flavanone inclusion compound can be a hesperetin inclusion compound.

Yet another method of the present invention is a method of synthesizing a flavanone inclusion compound which is a hesperetin inclusion compound or a naringenin inclusion compound,
By mixing an organic solvent solution containing hesperetin or naringenin and an aqueous solution containing β-cyclodextrin, branched β-cyclodextrin or a modification thereof, hesperetin or naringenin is mixed with β-cyclodextrin, branched β-cyclodextrin or Including the step of inclusion by these modifications to obtain a hesperetin inclusion compound or a naringenin inclusion compound, wherein the organic solvent is ethanol, isopropanol or tetrahydrofuran.

  The food of the present invention is a food containing a flavanone inclusion compound, wherein the flavanone is hesperetin or naringenin, and the flavanone is included by β-cyclodextrin, branched β-cyclodextrin or a modified product thereof. ing.

  In one embodiment, the food of the present invention can be a health food.

  In one embodiment, the health food of the present invention may be for improving blood flow and improving coldness.

  In one embodiment, the health food of the present invention may be for improving skin condition.

  In one embodiment, the health food of the present invention may be for lowering cholesterol.

  In one embodiment, the health food of the present invention may be for improving allergic symptoms.

  In one embodiment, the health food of the present invention may be anti-inflammatory.

  The pharmaceutical of the present invention is a pharmaceutical comprising a flavanone inclusion compound, wherein the flavanone is hesperetin or naringenin, and the flavanone is included by β-cyclodextrin, branched β-cyclodextrin or a modified product thereof. ing.

  The skin external preparation of the present invention is a skin external preparation containing a flavanone inclusion compound, wherein the flavanone is hesperetin or naringenin, and the flavanone is β-cyclodextrin, branched β-cyclodextrin or a modified product thereof. Is included.

  The inclusion compound of the present invention is a flavanone inclusion compound, and the flavanone is hesperetin or naringenin, and the flavanone is included by branched β-cyclodextrin or chemically modified β-cyclodextrin.

  Hesperidin or naringin is hydrolyzed to hesperetin or naringenin, and then these are included by β-cyclodextrin, branched β-cyclodextrin or a modification thereof, or hesperidin or naringin is converted to β-cyclodextrin, branched β-cyclodine. Inclusion with dextrin or a modified product thereof, and further, by removing the sugar by hydrolysis and converting it to an inclusion product of flavonoid aglycone, hesperetin inclusion compound and naringenin inclusion compound with extremely high bioabsorbability are obtained. Provided with high efficiency. Since these inclusions are highly bioabsorbable, they can be absorbed into the body quickly with high efficiency. Therefore, the physiological effects similar to those of taking hesperidin or naringin can be exerted only by taking a small amount.

  Hereinafter, the present invention will be described in detail.

  According to the present invention, a method for producing a flavanone inclusion compound is provided. In the flavanone inclusion compound, flavanone is hesperetin or naringenin, and the flavanone is included by β-cyclodextrin, branched β-cyclodextrin or a modified product thereof.

  In the present specification, an “inclusion compound” is a molecular complex, in which one chemical species forms a 1 to 3 dimensional molecular space, and the other chemical species is incorporated into the space (that is, an inclusion compound). A compound produced by contact). A compound that provides space is called a host, and a chemical species that is included is called a guest. In the flavanone inclusion compound synthesized in the present invention, β-cyclodextrin, branched β-cyclodextrin or a modified product thereof is a host, and flavanone is a guest.

(1. Flavanone)
The flavanone used as a guest in the inclusion compound synthesized in the present invention is hesperetin or naringenin. The flavanone is preferably hesperetin.

Hesperetin is also called 3 ', 5,7-trihydroxy-4'-methoxyflavanone. Hesperetin has the following structure:

  Hesperetin is an aglycon of hesperidin. Hesperetin can be obtained by degrading hesperidin with hesperidinase or naringinase. Hesperidin is a kind of flavonoid and is contained in citrus fruits (for example, Wenzhou orange, Summer orange, Iyo orange, Navel orange, Valencia orange), and especially in Wenzhou orange. Hesperidin is contained in a large amount in tangerines. Hesperidin is also most abundant in the white mesocarp, which is between the yellow rind and the flesh, even in the mandarin orange region. Hesperidin can be extracted from citrus peel, fruit juice or seed. For example, when extracting hesperidin from a squeezed citrus juice of Unshu mandarin orange, calcium hydroxide is added to adjust the pH to 11.5 to 12.5, and the mixture is stirred and squeezed, and then centrifuged. A method has been shown in which an acid is added to this solution to adjust the pH to 5 to 5.5, followed by heating, and then recovering the precipitated hesperidin by centrifugation (Japanese Patent Laid-Open No. 8-188593). Subsequently, hesperetin can be obtained by preparing an aqueous hesperidin solution and degrading the sugar moiety (eg, by the action of naringinase or hydrolysis under acidic conditions). Details of methods for obtaining hesperidin from citrus fruits are described, for example, in King FE and Robertson A. et al. , Natural glucosides, Part3. J. et al. Chem. soc. (II): 1704-1709 (1931). Alternatively, commercially available hesperidin may be used as a raw material. Hesperidin is sold, for example, by Hamari Sangyo Co., Ltd. Hesperetin is an aglycon of hesperidin. Details of the method for obtaining hesperetin from hesperidin by acid degradation are described in, for example, Asahina Y. et al. Flavanone glucosides (V): Reduction of flavone and flavonol derivatives, J. et al. Pharm. Soc-Japan 50: 217-223 (1931). Hesperetin can also be obtained by enzymatic degradation of hesperidin with rhamnosidase and glucosidase.

Naringenin is also called 4 ', 5,7-trihydroxyflavanone. Naringenin has the following structure:

  Naringenin exists naturally as its glycosylated product. That is, naringenin is an aglycone of naringin (also called naringin). Naringenin can be obtained by breaking down naringin with naringinase. Naringin is a kind of flavonoid and is contained in a large amount in citrus fruits, particularly grapefruit (Citrus paradisi MACF.), Pomelo (Citrus grandis) and the like. Naringin is a citrus bitter substance. Naringin is generally contained in the grapefruit fruit by 0.03-0.1%. Naringin is also most abundant in white mesocarp, which is between grape skin and flesh, even in grapefruit areas. Naringin can be isolated from grapefruit, pomelo and other fruit skins, juices or seeds by extraction with water or an organic solvent (eg, ethanol or methanol) at room temperature. The solubility of naringin is 0.1% by weight with respect to water, but dissolves 1.0% by weight or more in an alkaline aqueous solution. Therefore, naringin in fruit skin such as grapefruit and pomelo, fruit juice or seeds may be first crystallized by dissolving in an alkaline aqueous solution and neutralizing with an acid. Thereafter, naringinin can be obtained by preparing an aqueous naringin solution and degrading the sugar moiety by the action of naringinase. Details of the method of obtaining naringin from grapefruit are described in, for example, Determination of bioflavonoids in grapefruit juice. (Orii Y et al., 1997 Clinical Pharmacology). Alternatively, commercially available naringin may be used as a raw material. Naringin is sold, for example, by Saneigen FFI Co., Ltd. Details of the method for obtaining naringenin from naringin are described in, for example, the enzyme handbook Michio Kosaki, published by Jijinshokan.

(2. β-cyclodextrin, branched β-cyclodextrin or a modified product thereof)
In the inclusion compound synthesized in the present invention, β-cyclodextrin, branched β-cyclodextrin or a modified product thereof acts as a host. These hosts are already known to take various organic compounds inside their cavities and form complexes. Since the interior of the cavity of cyclodextrin is in a relatively hydrophobic environment, it tends to selectively incorporate lipophilic substances or functional groups that are relatively lipophilic in the hydrophilic molecular structure in an environment using water. There is.

  As used herein, “β-cyclodextrin” refers to a maltooligosaccharide composed of seven cyclic α- (1 → 4) linked D-glucopyranose units. In β-cyclodextrin, three-dimensionally, glucose residues are coordinated so as to surround the bottom wall of the bucket. β-cyclodextrin can be prepared by allowing cyclodextrin glucanotransferase (CGTase) to act on a polysaccharide such as starch. The production method of β-cyclodextrin is described in, for example, cyclodextrin—Basics and Applications— (published by Fujio Toda, Sangyo Tosho). β-cyclodextrin is also commercially available from, for example, Yokohama International Bio-Institute.

  As used herein, “branched β-cyclodextrin” refers to what is commonly referred to as “branched β-cyclodextrin” in which one or more glucose residues are linked to β-cyclodextrin as a side chain, and β- Galactosyl-branched β cyclodextrin in which a galactosyl group is bonded to the C4 hydroxyl group or C6 hydroxyl group of a glucose residue bonded to cyclodextrin by β-bonding, and galactosyl branched β in which a galactosyl group is directly bonded to β-cyclodextrin -Cyclodextrin, 6-O-α-D-mannosyl-β-cyclodextrin with β-cyclodextrin bound to mannose residue, C4-hydroxyl group or C6-hydroxyl group of glucose residue bound to β-cyclodextrin A mannose-branched β cyclode, which has a mannose residue bonded to it by an α-bond Commonly referred to as “heterobranched β-cyclodextrin” such as 6-O-β-D-N-acetylglucosaminyl-β-cyclodextrin in which N-acetylglucosamine residue is bound to xistrin and β-cyclodextrin Say what you are. As long as the inclusion ability is maintained and structurally possible, the binding residue can be linked to any position of the β-cyclodextrin, but is preferably linked to the position on the bottom side of the bucket. As long as the inclusion ability is maintained and structurally possible, the residues to be bound can be linked to one or more of β-cyclodextrin, but are preferably linked to one site. The number of residues linked to β-cyclodextrin is preferably 1-6, more preferably 1-5, even more preferably 1-4, and even more preferably 1-3. More preferably, it is 1 or 2, and most preferably 2. The chain composed of a plurality of residues linked to β-cyclodextrin may be linear or branched. Branched cyclodextrins exhibit a higher water solubility than cyclodextrins.

  The branched β-cyclodextrin, for example, does not first decompose cyclodextrins such as α-amylase, β-amylase, and glucoamylase into a β-cyclodextrin-containing starch degradation product obtained by allowing CGTase to act on starch? Alternatively, an enzyme that is difficult to decompose is added to decompose the acyclic dextrin remaining in the starch decomposition product to generate an oligosaccharide. Next, the starch degradation product containing β-cyclodextrin and oligosaccharide can be produced by allowing a debranching enzyme such as pullulanase or isoamylase to act to link the oligosaccharide to cyclodextrin. Alternatively, it can also be produced by adding maltose, maltotriose or the like to an aqueous solution containing pure β-cyclodextrin and allowing a debranching enzyme to act on this solution. Alternatively, when a cyclodextrin-forming enzyme is allowed to act on starch, branched β-cyclodextrin is formed in the solution (Japanese Patent Laid-Open No. 6-54664), and these may be used. A method for producing a branched β-cyclodextrin is described, for example, in JP-A-6-14789. Branched β-cyclodextrin is also commercially available from, for example, Yokohama International Bio-Institute.

  In the present specification, the “modified product thereof” refers to a modified product of β-cyclodextrin or a modified product of branched β-cyclodextrin. For convenience, branched β-cyclodextrin is not included in the modified β-cyclodextrin. β-cyclodextrin and branched β-cyclodextrin can be optionally modified as long as they maintain the inclusion ability and are structurally possible. The modification is preferably a chemical modification. Examples of chemical modifications include methylation, propylation, monoacetylation, triacetylation, monochlorotriazinylation, hydroxyethylation, 2-hydroxypropylation, 2,3-dihydroxypropylation, 2-hydroxybutylation , 2-hydroxyisobutylation, diethylaminoethylation, trimethylammoniopropylation and the like. Specific examples of commercially available chemically modified β-cyclodextrin include: CAVASOL W7 M, CAVASOLW7 M Pharma, and CAVASOL W7 M TL (all manufactured by Wacker Chemicals East Asia Co., Ltd.) β-cyclodextrin; hydroxypropyl-β-cyclodextrin commercially available as CAVASOLW7 HP and CAVASOL W7 HP Pharma (both manufactured by Wacker Chemicals East Asia Co., Ltd.); CAVASOL W7 A (all manufactured by Wacker Chemicals East Asia Co., Ltd.) ) Monoacetyl-β-cyclodextrin commercially available as CAVASOL W7 TA (both manufactured by Wacker Chemicals East Asia Co., Ltd.) Triacetyl -β- cyclodextrin are commercially available as, and Cavasol W7 MCT (both Wacker Chemicals East Ltd. ASIA, Ltd.) monochlorotriazinyl sulfonyl -β- cyclodextrin marketed as. Methods for chemically modifying β-cyclodextrin or branched β-cyclodextrin are known in the art, and are described, for example, in cyclodextrin—Basics and Applications— (Fujio Toda, published by Sangyo Tosho).

  In the method for synthesizing the clathrate compound of the present invention, one type of β-cyclodextrin, branched β-cyclodextrin or a modified product thereof may be used alone, or two or more types may be mixed and used. Also good. One type is preferably used alone. In the synthesis method of the present invention, β-cyclodextrin, branched β-cyclodextrin or a modified product thereof can be purified and used, but these cyclodextrins are prepared and a solution not purified is used. Also good. It is preferable to use a purified product.

または

であり、βＣＤ−Ｒは、（Ｃ_６Ｈ_１０Ｏ_５）_６（Ｃ_６Ｈ_９Ｏ_５）−Ｒ^１を示し、Ｒ^１は、Ｈ、−（Ｃ_６Ｈ_１１Ｏ_５）（Ｃ_６Ｈ_１０Ｏ_５）_ｓ、低級アルキル、置換アルキル、アシルおよびアジニルからなる群より選択され、ｍ、ｎおよびｓはそれぞれ１以上の任意の整数である。周知のとおり、βＣＤは、グルコース残基が７つ連なった環状構造をとる。 (3. Flavanone inclusion compound)
The flavanone inclusion compound synthesized in the present invention is represented by the general formula [F] _m · [βCD-R] _n . Where F is

Or

And βCD-R represents (C ₆ H ₁₀ O ₅ ) ₆ (C ₆ H ₉ O ₅ ) —R ¹ , wherein R ¹ represents H, — (C ₆ H ₁₁ O ₅ ) (C ₆ H ₁₀ O ₅ ) _s , lower alkyl, substituted alkyl, acyl and azinyl, where m, n and s are each any integer of 1 or greater. As is well known, βCD has a cyclic structure in which seven glucose residues are linked.

  As used herein, “lower alkyl” refers to a group that may be straight or branched, having from 1 to 6 carbon atoms in the chain. Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, n-pentyl, heptyl, nonyl, decyl, fluoromethyl, trifluoromethyl and cyclopropylmethyl.

As used herein, “substituted alkyl” means that an alkyl group is substituted with one or more substituents, and these substituents may be the same or different. Each substituent is halo, alkyl, aryl, cycloalkyl, cyano, hydroxy, alkoxy, alkylthio, amino, —NH (alkyl), —NH (cycloalkyl), —N (alkyl) ₂ , carboxy and —C (O ) Independently selected from the group consisting of O-alkyl.

  As used herein, “acyl” refers to an HC (O) — group, an alkyl-C (O) — group, an alkenyl-C (O) — group, an alkynyl-C (O) — group, a cycloalkyl— Means a C (O)-group, a cycloalkenyl-C (O)-group or a cycloalkynyl-C (O)-group; Non-limiting examples of suitable acyl groups include formyl, acetyl, propanoyl, 2-methylpropanoyl and cyclohexanoyl.

  In the flavanone inclusion compound synthesized in the present invention, flavanone is hesperetin or naringenin, and this flavanone is included by β-cyclodextrin, branched β-cyclodextrin or a modified product thereof. The flavanone is preferably encapsulated by branched β-cyclodextrin.

  m: n is preferably 1: 1, m is preferably 1, and n is preferably 1.

R ¹ is preferably-(C ₆ H ₁₁ O ₅ ) (C ₆ H ₁₀ O ₅ ) _s . _{- (C 6 H 11 O 5} ) (C 6 H 10 O 5) s may be also branched be linear. It is preferably linear.

  s is preferably 0-5, more preferably 0-4, even more preferably 0-3, even more preferably 0-2, even more preferably 0-1 and most preferably Is 1.

  Formation of the inclusion compound from the host and guest can be confirmed by methods known in the art. For example, it can be confirmed by X-ray diffraction, nuclear magnetic resonance (NMR), differential scanning calorimetry (DSC), Fourier transform infrared spectrometer (FT-IR) and the like.

  In the flavanone clathrate compound synthesized in the present invention, the host and the guest are clathrated at a molecular ratio of 1: 1, which can be confirmed by, for example, NMR.

(4. Method for synthesizing flavanone inclusion compound)
A flavanone inclusion compound is synthesized by the method of the present invention. The method of the present invention is any of the following methods.

(4.1 Method of Obtaining Aglycone Inclusion Compound by Encapsulating Glycoside and then Hydrolyzing Sugar Part)
A first method of the present invention is a method for synthesizing a flavanone inclusion compound, wherein hesperidin or naringin is included by β-cyclodextrin, branched β-cyclodextrin or a modified product thereof, and the hesperidin inclusion compound Or a step of obtaining a naringin clathrate compound; and a step of hydrolyzing the hesperidin clathrate compound or the naringin clathrate compound to obtain a hesperetin clathrate compound or a naringenin clathrate compound.

  As a general method for synthesizing an inclusion compound, a saturated aqueous solution method for producing an inclusion compound in a saturated aqueous solution of cyclodextrin, a kneading method for obtaining an inclusion compound by kneading cyclodextrin with water and a guest substance, There is a spray drying method in which a polysaccharide aqueous solution containing dextrin or cyclodextrin and a guest substance are spray dried to obtain a powder containing an inclusion compound. Details of these methods are known in the art. Details of the kneading method for kneading cyclodextrin, water and a guest substance to obtain an inclusion compound are described in, for example, preparation of CD inclusion flavor powder and its release properties (Hidefumi Yoshii, 2004, Japan Food Engineering Association) . For details of the spray-drying method of obtaining powder containing inclusion compounds by spray-drying cyclodextrin or an aqueous polysaccharide solution containing cyclodextrin and the guest substance, for example, research on preparation and properties of functional food powder (Hidefumi Yoshii) (Abstracts of Annual Meeting of the Japan Food Engineering Society 2003)

  In the synthesis method of the present invention, first, hesperidin or naringin, which is a glycoside, is clathrated with β-cyclodextrin, branched β-cyclodextrin or a modified product thereof to obtain a hesperidin inclusion compound or naringin inclusion compound. . The synthetic method of the present invention has the advantage that hesperidin and naringin are natural products and can be obtained very easily and inexpensively compared to hesperetin and naringenin.

  The hesperidin clathrate compound or naringin clathrate compound can be prepared by the following procedure A1 or B1.

(A1. Procedure using basic aqueous solution)
When a basic aqueous solution of hesperidin or naringin and an acidic aqueous solution of β-cyclodextrin, branched β-cyclodextrin, or a modified product thereof are mixed and neutralized, an inclusion compound is formed in this neutralized solution. Is done. Since the solubility of hesperidin or naringin increases when it is made basic, it is preferable to make the aqueous solution of hesperidin or naringin basic, more preferably about 10 to about 12, and about 10.5 to about 11 .5 is most preferable. To make the solution basic, any base known in the art can be used. Examples of bases that can be used to make the solution basic include: sodium hydroxide, sodium carbonate, sodium bicarbonate, sodium acetate, sodium citrate, potassium hydroxide, potassium carbonate, potassium phosphate, Calcium hydroxide, calcium carbonate and citrus. When the obtained flavanone inclusion compound is used for food applications, the base is preferably a base that can be used as a food additive. One type of base may be used alone, or two or more types of bases may be mixed and used.

  When synthesizing without using an organic solvent, there is no safety concern, so that the obtained inclusion compound can be used for food. In addition, when an organic solvent is not used, it is not necessary to confirm that no harmful substances remain, so that there is an advantage that it is simple and costs are low.

(B1. Procedure using organic solvent)
An inclusion compound may be formed by stirring an aqueous solution of β-cyclodextrin, branched β-cyclodextrin or a modified product thereof in a solution obtained by dissolving hesperidin or naringin in an organic solvent. . As an organic solvent, a water-soluble thing is good, Preferably methanol, ethanol, isopropanol, acetone, tetrahydrofuran etc. are mentioned.

  In any of the procedures of A1 and B1, the molar concentration ratio of hesperidin or naringin to β-cyclodextrin, branched β-cyclodextrin or a modified product thereof during mixing can be arbitrarily set. , Preferably 1: 2 to 1: 6, more preferably 1: 3 to 1: 4.

  Next, the hesperidin clathrate compound or the naringin clathrate compound is hydrolyzed to obtain a hesperetin clathrate compound or a naringenin clathrate compound. The hydrolysis of the hesperidin inclusion compound or the naringin inclusion compound may be carried out enzymatically or chemically.

  Rhamnosidase and glucosidase are used when the hydrolysis is carried out enzymatically. Rhamnosidase hydrolyzes the rhamnose residue in hesperidin and naringin. Glucosidase hydrolyzes glucose residues in hesperidin and naringin. Rhamnosidase and glucosidase are readily available in the art.

  For example, an enzyme commonly referred to as naringinase is a mixture comprising rhamnosidase and β-glucosidase. Naringinase is extracted from a culture solution of Aspergillus sp. (For example, Aspergillus usamii and Aspergillus niger) or Penicillium sp. (Penicillium decumbens) or Coniothyrium sp. Can be obtained.

  The extraction is preferably performed at 0 ° C to 15 ° C. Concentration is preferably performed at 0 ° C to 30 ° C. The ethanol treatment is preferably performed at 0 ° C to 15 ° C. Naringinase is marketed by Tanabe Seiyaku Co., Ltd. as naringinase.

  Enzymes commonly referred to as hesperidinases include rhamnosidase and β-glucosidase. Hesperidinase can be obtained by extracting with water from a culture solution of filamentous fungi (Aspergillus sp. (For example, Aspergillus niger) or Penicillium sp. (For example, Penicillium decumbens)), and then treating with ethanol.

  The extraction is preferably performed at 0 ° C to 15 ° C. Concentration is preferably performed at 0 ° C to 30 ° C. The ethanol treatment is preferably performed at 0 ° C to 15 ° C. Hesperidinase is sold by Tanabe Seiyaku Co., Ltd. as soluble hesperidinase <Tanabe> No.2.

  In addition to these, any other glucosidase and rhamnosidase may be used.

  The hesperidin inclusion compound or naringin inclusion compound may be purified before the glucosidase and rhamnosidase are allowed to act, or the next step may be performed without purifying the formed hesperidin inclusion compound or naringin inclusion compound. Also good. The pH of the solution containing the hesperidin clathrate compound or naringin clathrate compound is arbitrarily set, but is preferably adjusted to be acidic in order to optimize the enzyme reaction, more preferably the pH is about 4-5. It is adjusted to become. In order to stably maintain the state of inclusion and dissolution in an acidic solution and increase the yield of hesperetin inclusion or naringenin inclusion after the reaction, the concentration of hesperidin or naringin in the reaction solution is increased. 0.1 mg / ml to 15 mg / ml, preferably 4 mg / ml to 12 mg / ml, and the molar ratio of hesperidin or naringin to cyclodextrin is 1: 2 to 1: 6, preferably 1: 3 to 1: 4. To do.

  The amount of glucosidase and rhamnosidase added to the hesperidin inclusion compound or the naringin inclusion compound can be arbitrarily set. The amount of glucosidase is preferably from about 0.001% to about 10% by weight, more preferably from about 0.01% to about 5% by weight, and even more preferably from about 0.05% to about 5% by weight. 1% by weight. The amount of rhamnosidase is preferably from about 0.001% to about 10%, more preferably from about 0.01% to about 5%, and even more preferably from about 0.05% to about 10%. 1% by weight. For example, when naringinase is used as glucosidase and rhamnosidase, the amount of naringinase is preferably about 0.01 wt% to about 10 wt%, more preferably about 0.05 wt% to about 5 wt%, More preferably, it is about 0.1% by weight to about 1% by weight.

  The temperature of the solution in reacting the hesperidin clathrate compound or naringin clathrate compound with the enzyme can be arbitrarily set, but is preferably about 10 ° C to about 70 ° C, more preferably about 20 ° C to about 65 ° C. ° C, more preferably about 30 ° C to about 60 ° C, and most preferably about 40 ° C to about 55 ° C. Of course, the reaction temperature is set in consideration of the optimum reaction temperature and reaction efficiency of the enzyme used. The reaction time can be arbitrarily set, but is preferably about 30 minutes to about 100 hours, more preferably about 1 hour to about 80 hours, further preferably about 5 hours to about 60 hours, Preferably, it is about 10 hours to about 50 hours. After the enzyme reaction, the reaction solution may be heated at a temperature higher than that of the enzyme reaction (for example, about 50 ° C. to about 90 ° C., preferably about 60 ° C. to about 80 ° C.) for an arbitrary time (for example, about 1 hour to about 6 hours, preferably about 2 hours to about 4 hours).

  When rhamnosidase and glucosidase thus act on the hesperidin inclusion compound or naringin inclusion compound, the sugar moiety of these flavanones is hydrolyzed to form a hesperetin inclusion compound or naringenin inclusion compound.

  When the hydrolysis is performed chemically, the hesperidin clathrate compound or naringin clathrate compound can be acid hydrolyzed. Any acid can be used for acid hydrolysis as long as it does not degrade the β-cyclodextrin and aglycone moieties. Examples of acids that can be used for acid hydrolysis include hydrochloric acid, sulfuric acid, nitric acid. When the obtained flavanone inclusion compound is used for food applications, the acid is preferably an acid that can be used as a food additive. It is preferable to use hydrochloric acid. One kind of acid may be used alone, or two or more kinds of acids may be mixed and used. The acid concentration at the start of hydrolysis is preferably about 0.1N to about 10N, more preferably about 1N to about 5N. If the acid concentration is too low, the residual ratio of undegraded hesperidin may increase. If the acid concentration is too high, inclusion by β-cyclodextrin may be removed, or the β-cyclodextrin part and the aglycon part may be decomposed.

  The reaction temperature during the acid hydrolysis is not particularly limited, but it is preferable to heat the solution to near the boiling temperature. The reaction time for acid hydrolysis is not particularly limited, but is preferably about 1 minute to about 100 hours, more preferably about 10 minutes to about 50 hours, and even more preferably about 1 hour to about 40 hours. And most preferably from about 10 hours to about 30 hours. If the reaction time is too short, acid hydrolysis may be insufficient. If the reaction time is too long, inclusion by β-cyclodextrin may be removed, or the β-cyclodextrin part and the aglycon part may be decomposed.

(4.2 Method of directly including β-cyclodextrin in aglycone)
Hesperetin inclusion compound or naringenin inclusion compound can be synthesized by directly including β-cyclodextrin, branched β-cyclodextrin or a modified product thereof in aglycone. This method of the present invention comprises (A2) mixing a basic suspension or basic aqueous solution containing hesperetin or naringenin and an acidic aqueous solution containing β-cyclodextrin, branched β-cyclodextrin or a modified product thereof. A step of obtaining hesperetin inclusion compound or naringenin inclusion compound by inclusion of hesperetin or naringenin with β-cyclodextrin, branched β-cyclodextrin or a modification thereof, or (B2) an organic containing hesperetin or naringenin By mixing the solvent solution with an aqueous solution containing β-cyclodextrin, branched β-cyclodextrin or a modified product thereof, hesperetin or naringenin is added to the β-cyclodextrin, branched β-cyclodextrin or a modified product thereof. To obtain a hesperetin inclusion compound or a naringenin inclusion compound. Even when β-cyclodextrin and the like are directly included in the aglycon, the same inclusion method as that described for the case of including hesperidin or naringin is used.

  The hesperetin inclusion compound or naringenin inclusion compound can be prepared by the following procedure A2 or B2.

(A2. Procedure using basic aqueous solution)
For example, when neutralizing by mixing a basic suspension or aqueous solution of hesperetin or naringenin and an acidic aqueous solution of β-cyclodextrin, branched β-cyclodextrin or a modified product thereof, An inclusion compound is formed in the solution. Since the solubility of hesperetin or naringenin increases when it is made basic, it is preferable to make the suspension or aqueous solution of hesperidin or naringenin basic, more preferably about 10 to about 14, and about 11. Most preferred is 5 to about 13.5. To make the suspension or aqueous solution basic, any base known in the art can be used. Examples of bases that can be used to make a suspension or aqueous solution basic include: sodium hydroxide, sodium carbonate, sodium bicarbonate, sodium acetate, sodium citrate, potassium hydroxide, potassium carbonate, Potassium phosphate, calcium hydroxide, calcium carbonate and citrus. When the obtained flavanone inclusion compound is used for food applications, the base is preferably a base that can be used as a food additive. One type of base may be used alone, or two or more types of bases may be mixed and used.

  When synthesizing without using an organic solvent, there is no safety concern, so that the obtained inclusion compound can be used for food. In addition, when an organic solvent is not used, it is not necessary to confirm that no harmful substances remain, so that there is an advantage that it is simple and costs are low.

(B2. Procedure using organic solvent)
Even if hesperetin or naringenin is dissolved in an organic solvent, an inclusion compound can be formed by stirring an aqueous solution of β-cyclodextrin, branched β-cyclodextrin or a modification thereof little by little while stirring. Good. As an organic solvent, a water-soluble thing is good, Preferably ethanol, isopropanol, tetrahydrofuran, etc. are mentioned.

  In any of the procedures of A2 and B2, the molar concentration ratio of hesperetin or naringenin and β-cyclodextrin during mixing can be arbitrarily set, but is preferably 1: 1 to 1: 6. Yes, more preferably 1: 2 to 1: 4.

  Mixing can take place at any temperature. After mixing, the mixed solution may be heated at a higher temperature (for example, about 50 ° C. to about 90 ° C., preferably about 60 ° C. to about 80 ° C.) for any time (for example, about 1 ° C.) as necessary. Hours to about 6 hours, preferably about 2 hours to about 4 hours).

(5. Method for confirming the solubility of the flavanone inclusion compound)
The solubility of the flavanone inclusion compound can be confirmed by methods known in the art. For example, 1.7 parts by volume of β-cyclodextrin solution in 0.1N HCl is added to 2.2 parts by volume of flavanone basic solution of various concentrations in 0.1N NaOH while stirring little by little. The solubility can be easily confirmed by forming a flavanone inclusion compound while changing the pH to neutral and confirming whether precipitation occurs. Alternatively, for example, the solubility can be confirmed by adding a flavanone clathrate compound to 20 ° C. water and confirming whether or not it dissolves when stirred for 30 minutes.

(6. Method for confirming absorption of flavanone inclusion compound)
Absorbency of the flavanone inclusion compound can be confirmed using methods known in the art. The absorbability of the flavanone inclusion compound can be measured, for example, as follows. Pre-bred (eg, about 3 days to 1 week) animals (eg, 6 week old male ddY mice) are fasted (eg, about 14 hours), and a flavanone inclusion compound solution (eg, 200 μl) at a predetermined concentration is used. Is administered orally. It may be administered directly to the stomach using a sonde. After administration, blood is collected over time (eg, from the heart) and serum is separated. Next, a solution containing sulfatase and glucuronidase is added to the serum for reaction, then acetonitrile is added for centrifugation, and the supernatant is dried by concentration centrifugation. This was dissolved in acetonitrile / methanol / water (50:20:30, v / v / v), stirred, sonicated, subjected to high performance liquid chromatography (HPLC) and detected with an electrochemical detector, The amount of flavanone derivative in the obtained serum is quantified.

Alternatively, the resulting blood is A. Boutin et al. (The American)
After pretreatment by society for Pharmacology and Experimental Therapeutics, 21, 1157-1166 (1993), the amount of flavanone inclusion compound contained therein may be quantified using HPLC. J. et al. A. The method of Boutin et al. Is as follows. Add 1000 μl of acetonitrile to 500 μl of collected blood, shake well, and let stand for 15 minutes. After centrifugation at 5500 rpm for 20 minutes, the supernatant is taken and lyophilized. Lyophilized samples are dissolved in 100 μl of acetonitrile / distilled water (20/80; V / V, 0.01 M NaOH) solution and analyzed by HPLC. The conditions for HPLC are slightly different depending on the flavanone inclusion compound to be measured, but the basic conditions are as follows. Column: ODS, column temperature: 40 ° C., eluent: acetonitrile / distilled water (20/80; V / V), flow rate: 0.5 ml / min, detection: UV 280 nm. The flavanone inclusion compound concentration in the blood is determined by preparing a calibration curve using flavanone compounds with known concentrations. In addition to mice, rats can also be used as experimental animals.

(7. Food containing flavanone inclusion compound)
The food of the present invention contains the flavanone inclusion compound synthesized by the method of the present invention. The food product can be any food product. The food may be solid, semi-solid, or liquid, but is preferably liquid. The food is preferably a health food, more preferably a health drink, but is not limited thereto. The health food can be used for the same normal use as the flavanone (ie, hesperetin or naringenin) in the flavanone inclusion compound contained in the health food. Examples of uses and effects of health foods include blood flow improvement and cooling improvement, skin condition improvement, cholesterol lowering, allergy symptom improvement, and anti-inflammatory use. In addition to the flavanone inclusion compound, the food product of the present invention may contain strawberry tea extract. By including the strawberry tea extract, a synergistic effect between the physiological activity of the flavanone inclusion compound and the physiological activity of the strawberry tea extract can be obtained. In particular, when the food of the present invention is for allergic symptom improvement, it is preferable that the food of the present invention contains strawberry tea extract in addition to the flavanone inclusion compound.

  Foods include, for example, frozen desserts (ice cream, ice milk, ice confections, etc.), palatable beverages (eg, soft drinks, carbonated beverages (cider, ramune, etc.), condiment beverages, alcoholic beverages, powdered juices, etc.) Milk, yogurt, ice cream, butter, margarine, cheese, whipped cream, etc., confectionery (Western confectionery, Japanese confectionery, snack confectionery, etc.), such as anko, yokan, bun, chocolate, gum, jelly, agar, apricot tofu, cake, castella , Cookies, rice crackers, tablet confectionery, etc.), bread, rice cakes, marine products (rice cakes, chikuwa, etc.), processed meat products (sausage, ham, etc.), processed fruit products (jam, marmalade, fruit sauce, etc.), seasonings ( Dressing, mayonnaise, miso, etc.), noodles (udon, soba, etc.), pickles, and meat, fish, fruit bottling, canned food, etc. That.

  The clathrate compound synthesized by the method of the present invention does not require any special process to be added to the food, and is added with the raw material at the initial stage of the food production process, added during the production process, or the production process. Add at the end of. As an addition method, a normal method such as mixing, kneading, dissolution, dipping, spraying, spraying, and application is selected according to the type and properties of the food. The food of the present invention can be prepared according to methods known to those skilled in the art.

(8. Drugs containing flavanone inclusion compounds)
The pharmaceutical agent of the present invention contains the flavanone inclusion compound synthesized by the method of the present invention. The medicament can be any medicament. The pharmaceutical may be solid, semi-solid, or liquid, but is preferably liquid.

  The clathrate compound synthesized by the method of the present invention does not require any special process to be added to the drug, and is added with the raw material at the beginning of the drug manufacturing process, added during the manufacturing process, or the manufacturing process. Add at the end of. As the addition method, a normal method such as mixing, kneading, dissolution, dipping, spraying, spraying, and application is selected according to the type and properties of the pharmaceutical. The medicament of the present invention can be prepared according to methods known to those skilled in the art.

(9. Other uses of flavanone inclusion compounds)
The flavanone inclusion compound synthesized by the method of the present invention can be used in various other applications. An example of such a use is a skin external preparation. Examples of skin external preparations include, for example, lotion, milky lotion, cream, cosmetic liquid, hair nourishing agent, hair restorer, pack, lipstick, lip balm, makeup base lotion, makeup base cream, foundation, eye color, cheek color, Shampoo, rinse, hair liquid, hair nick, permanent wave agent, hair color, treatment, bath preparation, hand cream, leg cream, neck cream, body lotion, etc. The skin external preparation containing the flavanone inclusion compound synthesized by the method of the present invention can be prepared according to methods known to those skilled in the art.

  In the present specification, a garment manufactured from a fiber or fabric by binding the flavanone inclusion compound to the fiber, mixing it with a fiber material, impregnating the fiber, or applying it to the surface of the fabric. Clothes containing flavanone inclusion compounds in a method of use in which the flavanone inclusion compound is percutaneously absorbed when the skin (for example, underwear) comes into contact with the skin are also included in the concept of an external preparation for skin. Bonding the flavanone inclusion compound to the fiber can be performed, for example, by crosslinking. A method of bonding a compound to a fiber, a method of mixing with a fiber material, a method of impregnating a fiber, a method of applying to a fabric surface, and the like are known in the art.

  The clathrate compound synthesized by the method of the present invention does not require any special process to be added to the external preparation for skin. Whether it is added with the raw material at the beginning of the manufacturing process of the external preparation for skin or during the manufacturing process. Or added at the end of the manufacturing process. As the addition method, a normal method such as mixing, kneading, dissolution, dipping, spraying, spraying, and application is selected according to the type and properties of the external preparation for skin. The topical skin preparation synthesized by the method of the present invention can be prepared according to methods known to those skilled in the art.

<Production Example 1: Production of inclusion hesperetin via inclusion hesperidin>
1 part by weight of hesperidin (purchased from Hamari Sangyo Co., Ltd.) was added to 100 parts by weight of 0.1N NaOH and dissolved to prepare a hesperidin solution (pH = 12.5). 9.772 parts by weight of branched β-cyclodextrin (G2-β-CD (purchased from Yokohama International Bio-Laboratory Co., Ltd.)) was added to 76.8 parts by weight of 0.1N HCl and dissolved, and branched β-cyclodone. A dextrin solution (pH = 1.2) was prepared. A hesperidin clathrate solution was prepared by adding the branched β-cyclodextrin solution to the hesperidin solution little by little while stirring to prepare a hesperidin clathrate solution (hesperidin concentration 5.65 mg / g, molar ratio of hesperidin: branched β-cyclodextrin). = 1: 4). It was confirmed by NMR that hesperidin inclusions were formed in this solution.

  To this solution was added 5N HCl (about 5 parts by weight) to adjust the pH to 4.5, and then 0.88 parts by weight of naringinase (purchased from Tanabe Seiyaku Co., Ltd.) was added and dissolved. This solution was kept at 50 ° C. for 48 hours with shaking to allow nanringinase to act on the inclusion hesperidin. The reaction solution was kept at 75 ° C. for 4 hours to obtain a hesperetin clathrate solution. The reaction solution is centrifuged and the supernatant is removed. The mobile phase is acetonitrile / water (25:75, v / v), the flow rate is 1 ml / min, and the detector is UV (wavelength 280 nm). HPLC) analysis revealed that most of the inclusions of hesperetin were present in the solution, and more than 95% of the original hesperidin inclusions had been changed to hesperetin inclusions. It was. It was confirmed by NMR that hesperetin was included in the branched β-cyclodextrin.

  As a result, it was found that the hesperetin inclusion product was obtained by cleaving the rhamnose and glucose bound to hesperetin while the majority of hesperidin was included in the branched cyclodextrin.

  In order to remove cleaved glucose, rhamnose, unreacted branched β-cyclodextrin, salts and the like from this hesperetin clathrate solution using the difference in adsorptivity by the porous synthetic adsorbent, The product was passed through a column packed with trade name Amberlite XAD 16HP manufactured by KK). Only the inclusion hesperetin was adsorbed to the column, and the other substances flowed out. The column on which the inclusion hesperetin was adsorbed was washed with water, and then a solution of ethanol 50 v / v% was passed through it to allow inclusion hesperetin to flow out of the column. After the ethanol in the effluent was distilled off, the remaining solution was spray-dried to obtain a powder. This powder showed good solubility in water, and the hesperetin contained in the powder was included by almost 100% branched β-cyclodextrin, indicating that no unencapsulated hesperetin remained. And confirmed by analysis by FT-IR.

<Production Example 2: Production of inclusion hesperetin by directly inclusion of hesperetin>
A suspension was prepared by adding 1 part by weight of hesperidin (purchased from Hamari Sangyo Co., Ltd.) to 100 parts by weight of distilled water, and adjusted to pH = 2 with 1N HCl. This solution was kept for 24 hours while maintaining at 100 ° C. to hydrolyze hesperidin. The precipitate after the reaction contained hesperetin, hesperetin-7-glucoside and unreacted hesperidin.

  These products are completely dissolved in 0.1N sodium hydroxide solution (pH = about 11), and then separated into hesperetin and other substances using the difference in adsorptivity by the hydrophobic synthetic adsorbent. The solution was passed through a column packed with Amersham Sephadex LH-20 manufactured by Amersham Biosciences. The discharged 100 ml solution contained hesperetin-7-glucoside and unreacted hesperidin. When 100 ml of water was passed through this column, the first 70 ml contained impurities, hesperetin-7-glucoside and unreacted hesperidin, and the remaining 30 ml contained hesperetin. Further, 200 ml of a 50% ethanol / v)% solution was passed through to drain the entire amount of hesperetin from this column. The precipitate after the ethanol in the effluent containing hesperetin was distilled off was dried to obtain a powder of hesperetin.

  A hesperetin solution (pH = 12.3) was prepared by dissolving hesperetin powder in 0.1N NaOH to a concentration of 9 mg / ml. Branched β-cyclodextrin (G2-β-CD (purchased from Yokohama International Bio-Laboratory Co., Ltd.)) is dissolved in 0.1N HCl to a concentration of 118 mg / ml, and a branched β-cyclodextrin solution (pH = 1.2) was prepared. A hesperetin clathrate solution was prepared by adding 1.7 parts by volume of the branched β-cyclodextrin solution to 2.2 parts by volume of the hesperetin solution while stirring little by little to change the pH of the solution to the neutral region. This reaction solution was held at 85 ° C. for 6 hours.

  In order to remove unreacted branched β-cyclodextrin, salts and the like from this hesperetin clathrate solution by utilizing the difference in adsorptivity by the porous synthetic adsorbent, trade name Amberlite XAD 16HP manufactured by Organo Corp. The solution was passed through a column packed with. Only the inclusion hesperetin was adsorbed to the column, and the other substances flowed out. The column on which the inclusion hesperetin was adsorbed was washed with water, and then a solution of ethanol 50 v / v% was passed through it to allow inclusion hesperetin to flow out of the column. After the ethanol in the effluent was distilled off, the remaining solution was spray dried and spray dried to obtain a powder. This powder shows good solubility in water, and the hesperetin contained in the powder is included by almost 100% branched β-cyclodextrin, and there is no unencapsulated hesperetin remaining. This was confirmed by analysis by DSC and FT-IR.

<Solubility test 1: Inclusion test with β-cyclodextrin>
Β-cyclodextrin (purchased from Yokohama International Bio-Laboratory Co., Ltd.) was dissolved in 0.1N HCl to a concentration of 15 mg / ml to prepare a β-cyclodextrin solution. Various flavonoid aglycones were dissolved in 0.1 N NaOH to a concentration of 2 mg / ml to prepare aglycone solutions. As the flavonoid aglycone, hesperetin, naringenin, luteolin, apigenin, daidzein (all manufactured by Sigma-Aldrich Japan) and quercetin (manufactured by Wako Pure Chemical Industries, Ltd.) were used. By adding 1.7 parts by volume of β-cyclodextrin solution into 2.2 parts by volume of aglycon solution while stirring little by little to change the pH of the solution to the neutral region, β-cyclodextrin inclusion product of flavonoid aglycone A solution was prepared.

  After completion of mixing the β-cyclodextrin solution and the aglycone solution, the mixture was allowed to stand for 30 minutes, and it was visually judged whether or not precipitation occurred. When any flavonoid aglycone was used, no precipitation occurred and the solution was in a solution state. This indicates that β-cyclodextrin is suitable as an inclusion host for flavanone aglycone.

<Solubility test 2: Inclusion test with branched β-cyclodextrin>
Branched β-cyclodextrin to 0.1N HCl (maltose residue linked to one of glucose constituting β-cyclodextrin by α1-6 bond; G2-β-CD (Yokohama International Bio-Research Co., Ltd.) To obtain a concentration of 117.9 mg / ml, and a branched β-cyclodextrin solution was prepared so that various flavonoid aglycones in 0.1N NaOH had a concentration of 9.09 mg / ml. A helicetin, naringenin, quercetin, luteolin, apigenin and daidzein were used as flavonoid aglycones, and 1.7 parts by volume of the branched β-cyclodextrin solution was added to 2.2 parts by volume of the aglycon solution. By adding a little at a time with stirring, change the pH of the solution to the neutral range. Thus, a branched β-cyclodextrin clathrate solution of flavonoid aglycone was prepared.

  After completion of mixing the branched β-cyclodextrin solution and the aglycone solution, the mixture was allowed to stand for 30 minutes, and whether or not precipitation occurred was visually determined. As a result, when hesperetin and naringenin, which are aglycones of flavanone, were used, no precipitation occurred in the solution state. However, precipitation occurred when flavonoid aglycones of other flavones, flavanols, and isoflavones were used. From this, it was found that branched β-cyclodextrin is suitable as an inclusion host for flavanone aglycone.

<Solubility Comparison Test 1: Inclusion Test with α-Cyclodextrin>
Α-Cyclodextrin was dissolved in 0.1N HCl to a concentration of 45 mg / ml to prepare an α-cyclodextrin solution. Various flavonoid aglycones were dissolved in 0.1 N NaOH to a concentration of 6 mg / ml to prepare aglycone solutions. Hesperetin, naringenin, quercetin, luteolin, apigenin and daidzein were used as flavonoid aglycones. By adding 1.7 parts by volume of α-cyclodextrin solution to 2.2 parts by volume of aglycon solution while stirring little by little, and changing the pH to neutral range, α-cyclodextrin inclusion solution of flavonoid aglycone Was prepared.

  After the mixing of the α-cyclodextrin solution and the aglycone solution was completed, the mixture was left for 30 minutes, and whether or not precipitation occurred was visually determined. As a result, precipitation occurred even when any flavonoid aglycone was used. This indicates that α-cyclodextrin is not suitable as an inclusion host for flavonoid aglycones.

<Solubility Comparison Test 2: Inclusion Test with Branched α-Cyclodextrin>
117.9 mg / ml of 0.1-N HCl in branched-α-cyclodextrin (one of glucose constituting α-cyclodextrin having a maltose residue linked by α1-6 bond; G2-α-CD) Was dissolved to give a branched α-cyclodextrin solution. Various flavonoid aglycones were dissolved in 0.1N NaOH to a concentration of 9.09 mg / ml to prepare aglycone solutions. Hesperetin, naringenin, quercetin, luteolin, apigenin and daidzein were used as flavonoid aglycones. By adding 1.7 parts by volume of the branched α-cyclodextrin solution to 2.2 parts by volume of the aglycon solution while stirring little by little to change the pH of the solution to the neutral region, the branched α-cyclodextrin envelope of the flavonoid aglycone is added. A inclusion solution was prepared.

  After completion of mixing the branched α-cyclodextrin solution and the aglycone solution, the mixture was left for 30 minutes, and it was visually judged whether or not precipitation occurred. As a result, precipitation occurred even when any flavonoid aglycone was used. This indicates that branched α-cyclodextrin is not suitable as an inclusion host for flavonoid aglycones.

<Solubility Comparison Test 3: Inclusion Test with γ-Cyclodextrin>
A γ-cyclodextrin solution was prepared by dissolving γ-cyclodextrin in 0.1 N HCl to a concentration of 90 mg / ml. Various flavonoid aglycones were dissolved in 0.1N NaOH to a concentration of 9.09 mg / ml to prepare aglycone solutions. Hesperetin, naringenin, quercetin, luteolin, apigenin and daidzein were used as flavonoid aglycones. By adding 1.7 parts by volume of γ-cyclodextrin solution to 2.2 parts by volume of aglycon solution while stirring little by little, and changing the pH of the solution to a neutral region, γ-cyclodextrin inclusion product of flavonoid aglycone A solution was prepared.

  After mixing of the γ-cyclodextrin solution and the aglycone solution, the mixture was allowed to stand for 30 minutes, and whether or not precipitation occurred was visually determined. As a result, precipitation occurred even when any flavonoid aglycone was used. This indicates that γ-cyclodextrin is not suitable as an inclusion host for flavonoid aglycones.

<Solubility Test 3: Solubility of β-Cyclodextrin Inclusion Solution>
A β-cyclodextrin solution was prepared by dissolving β-cyclodextrin in 0.1 N HCl to a concentration of 15 mg / ml. Various flavonoid aglycones were dissolved in 0.1N NaOH at different concentrations to prepare aglycone solutions. As flavonoids, hesperetin, naringenin, luteolin, quercetin, apigenin and daidzein were used. By adding 1.7 parts by volume of β-cyclodextrin solution into 2.2 parts by volume of aglycon solution while stirring little by little to change the pH of the solution to the neutral region, β-cyclodextrin inclusion product of flavonoid aglycone A solution was prepared.

  After completion of mixing the β-cyclodextrin and the aglycone solution, the mixture was allowed to stand for 30 minutes, and whether or not precipitation occurred was visually determined. Thus, the presence or absence of precipitation due to the difference in the aglycone concentration was confirmed. The results are shown in Table 2.

  The maximum final aglycone concentration at which no precipitation occurred was considered solubility. The solubility of hesperetin βCD inclusion complex is 2.8 mg / ml, the solubility of naringenin βCD inclusion complex is 2.8 mg / ml, the solubility of quercetin βCD inclusion complex is 1.4 mg / ml, and luteolin The solubility of βCD inclusion product was 1.7 mg / ml, the solubility of apigenin βCD inclusion product was 1.1 mg / ml, and the solubility of daidzein βCD inclusion product was 1.1 mg / ml. These solubilities are shown graphically in FIG. As a result, it was found that the inclusion solution of hesperetin and naringenin flavanones was more soluble than the inclusion solution of other flavonoids.

<Solubility Test 4: Solubility of Branched β-Cyclodextrin Inclusion Solution>
Branched β-cyclodextrin to 0.1N HCl (maltose residue linked to one of glucose constituting β-cyclodextrin by α1-6 bond; G2-β-CD (Yokohama International Bio-Laboratory Co., Ltd.) Purchased) was dissolved to a concentration of 150 mg / ml to prepare a branched β-cyclodextrin solution (pH = 1.2). Various flavonoid aglycones were dissolved in 0.1N NaOH at different concentrations to prepare aglycone solutions (pH = about 12 to 13). Hesperetin and quercetin were used as flavonoid aglycones. By adding 1.7 parts by volume of the branched β-cyclodextrin solution to 2.2 parts by volume of the aglycon solution while stirring little by little to change the pH of the solution to the neutral region, the branched β-cyclodextrin envelope of the flavonoid aglycone is added. A inclusion solution was prepared.

  After completion of mixing the branched β-cyclodextrin solution and the aglycone solution, the mixture was allowed to stand for 30 minutes, and whether or not precipitation occurred was visually determined. Thus, the presence or absence of precipitation due to the difference in the aglycone concentration was confirmed. The results are shown in Table 3.

  The maximum final aglycone concentration at which no precipitation occurred was considered solubility. The solubility of the hesperetin G2-βCD inclusion product was 7.3 mg / ml, and the solubility of the quercetin G2-βCD inclusion product was 3.4 mg / ml. These solubilities are shown graphically in FIG. As a result, it was found that the inclusion solution of hesperetin was significantly more soluble than the inclusion solution of quercetin.

<Evaluation Example 1: Absorption test of hesperetin inclusion product (G2-β-CD)>
Branched β-cyclodextrin with 8.47 ml of 0.1N HCl (maltose residue linked to one of glucose constituting β-cyclodextrin by α1-6 bond; G2-β-CD (Yokohama International Bio Co., Ltd.) 1 g) was dissolved to obtain a branched β-cyclodextrin solution. 100 mg of hesperetin was dissolved in 11 ml of 0.1N NaOH to prepare a hesperetin solution. While sonicating, the hesperetin solution was added to the branched β-cyclodextrin solution to obtain a G2-β-CD inclusion hesperetin solution (hesperetin concentration 5 mg / ml).

  Forty-seven ddY mice (♂), 6 weeks old, were fed MF (produced by Oriental Yeast Co., Ltd.) as a feed and preliminarily raised for 3 days and then fasted for 14 hours before test substance administration. Thereafter, 200 μl of a G2-β-CD inclusion hesperetin solution (hesperetin concentration: 5 mg / ml) was orally administered. Blood was collected from the hearts of 5 to 6 mice at 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, and 9 hours after administration, Serum was separated by a conventional method.

  In order to quantify the amount of hesperidin derivative in the collected serum sample, sulfatase H-2 (manufactured by Sigma-Aldrich Japan Co., Ltd., sulfatase 4,080 units / ml and glucuronidase 141,000 units / ml) was added to each serum sample 50 μl. Was diluted 10-fold with acetate buffer (0.1 M, pH 5.0), and 50 μl was added and reacted at 37 ° C. for 2 hours. Next, 750 μl of acetonitrile was added to the reaction solution, and the mixture was sufficiently stirred, followed by centrifugation. The supernatant was solidified by concentration centrifugation to obtain a solid. This solid was dissolved in 100 to 400 μl of acetonitrile / methanol / water (50:20:30, v / v / v), stirred for about 15 minutes, subjected to high performance liquid chromatography, and subjected to electrochemical detection (ESA). Analysis was performed using Coulochem III). The concentration of hesperetin in each serum sample was determined by converting hesperetin as a standard. The results are shown in FIG. In addition, the area under the curve (AUC) of 0 to 9 hours of this graph was calculated. The results are shown in FIG.

<Evaluation Example 2: Absorbency test of dissolved hesperetin inclusion product (β-CD)>
1 g of β-cyclodextrin (β-CD, purchased from Yokohama International Bio-Laboratory Co., Ltd.) was dissolved in 8.47 ml of 0.1N HCl, and 10 ml of distilled water was added thereto to obtain a β-cyclodextrin solution. 100 mg of hesperetin (manufactured by Sigma-Aldrich Japan Co., Ltd.) was dissolved in 11 ml of 0.1N NaOH to obtain a hesperetin solution. While sonicating, the hesperetin solution was added to the β-cyclodextrin solution to obtain a β-CD inclusion hesperetin solution (hesperetin concentration 3.33 mg / ml). 300 μl of this solution was orally administered to 47 ddY mice (♂) under the same conditions as in Evaluation Example 1, and the absorbability was measured in the same manner as in Example 1. In this evaluation example, in order to administer the same amount of hesperetin as in evaluation example 1 while keeping the inclusion in a dissolved state, the hesperetin concentration is lowered and the administration volume is increased as compared with evaluation example 1. ing. The results are shown in FIG. In addition, the area under the curve (AUC) of 0 to 9 hours of this graph was calculated. The results are shown in FIG.

<Evaluation Example 3: Absorptivity test of suspended hesperetin inclusion product (β-CD)>
1 g of β-cyclodextrin (β-CD, purchased from Yokohama International Bio-Laboratory Co., Ltd.) was dissolved in 8.47 ml of 0.1N HCl to obtain a β-cyclodextrin solution. 100 mg of hesperetin (manufactured by Sigma-Aldrich Japan Co., Ltd.) was dissolved in 11 ml of 0.1N NaOH to obtain a hesperetin solution. While sonicating, the hesperetin solution was added to the β-cyclodextrin solution to obtain a β-CD inclusion hesperetin solution (hesperetin concentration 5 mg / ml). 200 μl of this solution was orally administered to 47 ddY mice (♂) under the same conditions as in Example 1, and the absorbability was measured in the same manner as in Example 1. In this evaluation example, in order to administer hesperetin having the same concentration, the same administration volume and the same weight as those of evaluation example 1, administration was performed in a suspended state. The results are shown in FIG. In addition, the area under the curve (AUC) of 0 to 9 hours of this graph was calculated. The results are shown in FIG.

<Evaluation Comparative Example 1: Hesperetin absorption test>
A hesperetin suspension was prepared by adding 50 mg of hesperetin (purchased from Sigma-Aldrich Japan Co., Ltd.) to distilled water (hesperetin concentration 5 mg / ml). This solution was stirred well and 200 μl was quickly taken and orally administered to 47 ddY mice (♂) under the same conditions as in Example 1. Absorbency was measured in the same manner as in Example 1. The results are shown in FIG. In addition, the area under the curve (AUC) of 0 to 9 hours of this graph was calculated. The results are shown in FIG.

<Evaluation Comparative Example 2: Hesperidin Absorption Test>
Hesperidin suspension (prepared from Sigma-Aldrich Japan Co., Ltd .; purity 80%) 126 mg (101 mg in terms of hesperidin) was added to distilled water to prepare a hesperidin suspension (hesperetin concentration 5 mg / ml). This solution was stirred well and 200 μl was quickly taken and orally administered to 47 ddY mice (♂) under the same conditions as in Example 1. Absorbency was measured in the same manner as in Example 1. The results are shown in FIG. In addition, the area under the curve (AUC) of 0 to 9 hours of this graph was calculated. The results are shown in FIG.

<Evaluation Comparative Example 3: Absorbency test of enzyme-treated hesperidin>
Enzyme-treated hesperidin (has one glucose residue added to hesperidin; purchased from Ezaki Glico Co., Ltd.) 128 mg was added to distilled water to prepare an enzyme-treated hesperidin solution (hesperetin concentration 5 mg / ml). 200 μl of this solution was taken and orally administered to 47 ddY mice (♂) under the same conditions as in Example 1. Absorbability was measured in the same manner as in Example 1. The results are shown in FIG. In addition, the area under the curve (AUC) of 0 to 9 hours of this graph was calculated. The results are shown in FIG.

<Evaluation Comparative Example 4: Absorbency test of quercetin (G2-β-CD) clathrated with branched β-cyclodextrin>
1.2 g of G2-β-cyclodextrin (G2-β-CD, purchased from Yokohama International Bio-Laboratory Co., Ltd.) was dissolved in 8.47 ml of 0.1N HCl to obtain a branched β-cyclodextrin solution. 60 mg of quercetin (purchased from Wako Pure Chemical Industries, Ltd.) was dissolved in 10.8 ml of 0.1N NaOH to obtain a quercetin solution. While sonicating, the quercetin solution was added to the G2-β-cyclodextrin solution to obtain a G2-β-CD inclusion quercetin solution (quercetin concentration 3 mg / ml). 200 μl of this solution was orally administered to 47 ddY mice (マ ウ ス) under the same conditions as in Example 1, and the absorption amounts of quercetin and its metabolites, tamalizetin and isorhamnetin were measured in the same manner as in Example 1. The sum of them was evaluated as absorbency. The results are shown in FIG. In addition, the area under the curve (AUC) of 0 to 9 hours of this graph was calculated. The results are shown in FIG.

<Evaluation Comparative Example 5: Absorption test of quercetin>
Quercetin suspension was prepared by adding 30 mg of quercetin (purchased from Wako Pure Chemical Industries, Ltd.) to distilled water (quercetin concentration 3 mg / ml). This solution was stirred well and immediately 200 μl was taken and orally administered to 47 ddY mice (♂) under the same conditions as in Example 1. The absorbability of quercetin was measured in the same manner as in Example 1. The results are shown in FIG. In addition, the area under the curve (AUC) of 0 to 9 hours of this graph was calculated. The results are shown in FIG.

<Summary of Evaluation Examples 1 to 3 and Evaluation Comparative Examples 1 to 5>

  In Table 5, the relative ratio indicates the ratio when the AUC of the enzyme treatment (PA-T) is 1.0.

  In Table 7, the relative ratio indicates the ratio when the AUC of quercetin is 0.06. This is because the relative ratio of AUC of quercetin in Table 1 was 0.06, and this was adjusted.

  As a result of Evaluation Examples 1 to 3 and Evaluation Comparative Examples 1 to 3, the serum hesperetin concentration 15 minutes after administration of the dissolved hesperetin-β-cyclodextrin inclusion product was determined by comparing hesperetin, hesperidin and enzyme-treated hesperidin. It was shown to be about 6 times, about 700 times, and about 180 times higher than the serum hesperetin concentration when administered. Even when hesperetin-β-cyclodextrin inclusion product was deposited, the serum hesperetin concentration 15 minutes after administration was higher than the serum hesperetin concentration when hesperetin, hesperidin and enzyme-treated hesperidin were administered. High concentrations of about 9 times, about 970 times and about 240 times were shown, respectively. Furthermore, the serum hesperetin concentration 15 minutes after administration of the hesperetin-G2-β-cyclodextrin inclusion product is about 9 times as compared with the serum hesperetin concentration when hesperetin, hesperidin and enzyme-treated hesperidin are administered. , About 1060 times and about 270 times higher concentrations.

  The AUC (area under blood concentration) of 0 to 9 hours after administration of the hesperetin-G2-β-cyclodextrin inclusion product was compared with the AUC when hesperetin, hesperidin and enzyme-treated hesperidin were administered, respectively. Remarkably high values were obtained, such as about 4.4 times, about 64 times and about 27 times. From these results, it was found that inclusion efficiency of hesperetin with G2-β-cyclodextrin or β-CD increases the absorption efficiency into the body.

  As a result, the absorbability was βCD inclusion> G2-β-CD inclusion >> hesperetin >> enzyme treatment> hesperidin.

  As can be seen from Table 5 and FIG. 4, when the aglycon was hesperetin, there was no relationship between the increase in solubility and absorbability. This was completely unexpected from the conventional knowledge that when the aglycone is quercetin as shown in FIG. It was also found that the absorbability increased even if the solubility of the clathrate compound clathrated with β-cyclodextrin did not increase so much.

  According to the results of the experiments by the present inventors, there is little difference in the effect of improving the absorption, although there is a difference in solubility between the hesperetin inclusion product of branched β-cyclodextrin and the hesperetin inclusion product of β-cyclodextrin. I found that there was no. Therefore, the increase in solubility in water due to inclusion is not the only reason for the improvement in absorption, but some changes such as change in polarity due to inclusion of hesperetin in cyclodextrin affect the improvement in absorption. It is thought that there is a possibility.

(Absorption test in mice)
Naringenin G2-βCD inclusion product, naringenin or naringin is administered to mice in the same manner as in Example 1, and the absorbability is tested. In the case of naringin, as in the case of hesperidin, naringenin is included. It can be seen that the absorbability is remarkably improved. Further, heparidin G2-βCD inclusion complex or methyl hesperidin was administered to mice in the same manner as in Evaluation Example 1, and the absorbability was tested, so that the absorbability was significantly higher than that obtained when hesperetin inclusion complex was given. It turns out that it does not improve.

<Evaluation Example 4A-1: Human absorbability test of hesperetin inclusion product (G2-β-CD)>
270 g of enzyme-treated hesperidin (manufactured by Ezaki Glico Co., Ltd.) was dissolved in 4.5 L of tap water, and about 40 ml of 5N HCl was added to adjust the pH to 5.5. To this solution, 9 g of naringinase and 9 g of glucoamylase were added and dissolved, and the reaction was carried out for 48 hours while maintaining the temperature at 55 ° C. to recover the precipitate. To purify the precipitate, 4.5 L of water was added, and about 40 ml of 5N NaOH was added to adjust the pH to 11.0 to dissolve all the precipitate once. Thereafter, about 60 ml of 5N HCl was added to lower the pH to 2.0 to precipitate hesperetin, and the enzyme-treated hesperidin, hesperidin and hesperetin-7-glucoside remaining in the supernatant were removed. This operation was repeated once, and the collected precipitate was washed twice with water, and then the precipitate was freeze-dried to obtain about 30 g of powder. When this powder was passed through high performance liquid chromatography and the peak area of hesperetin was examined, it was about 98%. 1800 mg of this purified hesperetin was dissolved in 300 ml of 0.1N NaOH to prepare a hesperetin solution.

  17.6 g of branched β-cyclodextrin (G2-β-CD (purchased from Yokohama International Bio-Laboratory Co., Ltd.)) is dissolved in 240 ml of 0.1N HCl, and this is added little by little to the hesperetin solution while stirring. A hesperetin clathrate solution was prepared while summing (pH = 7.5), and water was added to make the total volume 600 ml (converted to hesperetin: 3 mg / ml).

  Five healthy 27-44 year old adult male subjects were fasted from 9:30 pm the previous day, and 100 ml of hesperetin inclusion product solution (hesperetin content 300 mg) was ingested at 9:30 am. Before ingestion, 30 minutes and 1 hour later, about 10 ml of blood was collected over time from the brachial vein, and serum was separated by centrifugation.

  In order to quantify the amount of hesperidin derivative in the collected serum sample, 500 μl of acetate buffer (0.1 M, pH 4.5) was added to 500 μl of each serum sample. Furthermore, sulfatase H-2 (manufactured by Sigma-Aldrich Japan Co., Ltd., consisting of sulfatase 4080 units / ml and glucuronidase 141,000 units / ml) diluted 10 times with acetate buffer (0.1 M, pH 5.0) 100 μl was added and reacted at 37 ° C. for 2 hours. Next, 500 μl of oxalic acid (0.01 M) was added, and after sufficient stirring, the mixture was centrifuged at 8000 rpm for 5 minutes, and the supernatant was loaded on Oasis HLB 30 mg / 1 cc (manufactured by Nippon Waters Co., Ltd.) that had been equilibrated. After washing with 1 ml of oxalic acid (0.01M) and 1 ml of water, elution was recovered with 1 ml of methanol. This was solidified by concentration centrifugation. 200 μl of acetonitrile was added here, and after stirring, sonicated for about 15 minutes, dissolved, centrifuged, and the supernatant was subjected to high performance liquid chromatography and analyzed using an electrochemical detector (Colochem III manufactured by ESA). . The concentration of hesperetin in each serum sample was determined by converting hesperetin as a standard. The results are shown in FIG.

<Evaluation Example 4A-2: Human Absorption Test of Hesperetin Inclusion Product (β-CD)>
1-3.5 g of β-cyclodextrin (β-CD (purchased from Yokohama International Bio-Laboratory Co., Ltd.)) was dissolved in 240 ml of 0.1N HCl, and this was added to the hesperetin solution little by little with stirring to neutralize (pH = 7.5) A hesperetin clathrate solution was prepared, and water was added to make the total volume 600 ml (converted to hesperetin: 3 mg / ml).

  For the same subject as Evaluation Example 4A-1, the absorbency was measured in the same manner as in Evaluation Example 4A-1. The results are shown in FIG.

<Evaluation Comparative Example 6A: Human absorbability test of hesperetin>
A hesperetin suspension was prepared by adding 300 mg of hesperetin prepared in the same manner as in Evaluation Example 4A-1 to 100 ml of tap water (hesperetin concentration: 3 mg / ml). The same subject as Evaluation Example 4A-1 was ingested after thoroughly stirring this solution, and the absorbency was measured in the same manner as in Evaluation Example 4A-1. The results are shown in FIG.

<Summary of Evaluation Example 4A-1, Evaluation Example 4A-2, and Evaluation Comparative Example 6A>

  As shown in FIG. 8 and Table 8, the amount of hesperetin absorbed when the G2-βCD inclusion product was ingested was significantly higher at 30 minutes and 1 hour after ingestion than when hesperetin was ingested. It was. Similarly, when the β-CD inclusion product was ingested, the amount of hesperetin absorbed was significantly higher 30 minutes and 1 hour after ingestion than when hesperetin was ingested. Therefore, as a result of Evaluation Example 4A-1, Evaluation Example 4A-2 and Evaluation Comparative Example 6A, in the same way as the results of the mouse in FIG. 2, by inclusion of hesperetin with G2-βCD or β-CD, It was found that the absorbability was remarkably improved.

(Absorption test in humans)
Regarding hesperetin βCD inclusion product, hesperidin, and enzyme-treated hesperidin, the absorbability is remarkably improved by including hesperetin by administering to humans and testing the absorbability in the same manner as in Evaluation Example 4A-1. I understand that.

<Evaluation Example 4B and Evaluation Comparative Example 6B: Human absorbability test of hesperetin inclusion product (G2-β-CD) and enzyme-treated hesperidin>
A healthy 27-year-old adult male and a 25-year-old adult female were used as subjects, fasted after 9:30 pm the previous day, and prepared in the same manner as in Evaluation Example 4A-1, except that the hesperetin content was different. 100 ml of a hesperetin inclusion product (G2-β-CD inclusion product) solution (hesperetin content: 100 mg; evaluation example 4B) or an enzyme-treated hesperidin solution prepared in the same manner as in evaluation comparative example 3 except that the hesperetin content is different (Hesperetin content 100 mg; Evaluation Comparative Example 6B) was ingested at 9:30 in the morning. Before ingestion, 15 minutes, 30 minutes, 45 minutes, and 60 minutes, about 10 ml of blood was collected over time from the brachial vein, and serum was separated by centrifugation. The amount of hesperidin derivative in the collected serum sample was measured by the same method as in Evaluation Example 4A-1. The results are shown in FIG.

<Summary of Evaluation Example 4B and Evaluation Comparative Example 6B>

  As shown in FIG. 9 and Table 9, the amount of hesperetin absorbed when the G2-βCD inclusion product was ingested showed a high value throughout the period from 15 minutes to 60 minutes after ingestion of the enzyme-treated hesperidin. It was. As a result, it was found that inclusion of hesperetin with G2-βCD significantly improved the absorbability compared to enzyme-treated hesperidin.

<Evaluation Comparative Example 6C-1 and Evaluation Comparative Example 6C-2: Human Absorption Test of Hesperidin and Enzyme-treated Hesperidin>
4 healthy adult males and 3 adult females as subjects, fasting after 9:30 pm the previous day, adding 100 ml of a suspension of hesperidin (manufactured by Hamari Pharmaceutical Co., Ltd.) and stirring in distilled water ( Hesperetin content 1500 mg; Evaluation Comparative Example 6C-1), or 100 ml of an enzyme-treated hesperidin solution prepared in the same manner as in Evaluation Comparative Example 3 except that the Hesperetin content is different (Hesperetin content 1500 mg; Evaluation Comparative Example 6C-2) Ingested at 9:30. Before ingestion, 30 minutes later, 1 hour later, and 2 hours later, approximately 10 ml of blood was collected over time from the brachial vein, and serum was separated by centrifugation. The amount of hesperidin derivative in the collected serum sample was measured by the same method as in Evaluation Example 4A-1. The results are shown in FIG.

<Summary of Evaluation Comparative Example 6C-1 and Evaluation Comparative Example 6C-2>

  As shown in FIG. 10 and Table 10, the amount of hesperetin absorbed when the enzyme-treated hesperidin was collected showed a higher value at 30 minutes and 1 hour after the intake of hesperidin. As a result, it was found that enzyme-treated hesperidin has higher absorbability than hesperidin.

  Considering evaluation example 4A-1, evaluation example 4A-2, evaluation example 4B, evaluation comparative example 6A, evaluation comparative example 6B, evaluation comparative example 6C-1 and evaluation comparative example 6C-2, hesperetin G2 It was found that the -β-CD inclusion product and the hesperetin β-CD inclusion product all had extremely higher absorbability than any of the enzyme-treated hesperidin, hesperidin and hesperetin.

(Various effects of hesperetin)
Administration of hesperetin-branched βCD inclusion product to humans, mice, rats, etc. confirms the allergic reaction inhibitory effect.

<Evaluation Example 5: Hesperetin branch (G2) βCD inclusion compound allergic reaction inhibitory effect verification test>
After preliminary breeding of 42 5-week-old female BALB / c mice, 7 mice were administered so that the body weight would be equal, control group (Group A), tea extract extract group (Group B), enzyme-treated hesperidin administration Group (group C1, 600 mg / kg; group C2, 120 mg / kg), hesperetin inclusion product (hesperetin branched (G2) βCD inclusion product produced in the same manner as in Evaluation Example 1) administration group (groups D1, D2) ), 甜茶 and hesperetin inclusion product administration group (group E). Mice were orally administered 200 μL of the test substance solution with a gastric sonde for 5 consecutive days. At this time, the test substance solution was adjusted to the dosage shown in Table 11. On the third day after oral administration, 5 μl of DNP-specific IgE antibody 1375 ng / ml (manufactured by Sigma) was intradermally injected into one pinna for sensitization. One hour after oral administration on the fifth day, 0.05 mg of DNP-conjugated bovine serum albumin dissolved in 0.2 ml of 0.25% Evans blue-containing physiological saline was administered via the tail vein to induce an allergic reaction. 30 minutes after the inducing, both auricles were excised, and the central part was punched out using a punch for punching leather with a diameter of 7 mm. For each pinna, exuded Evans blue was extracted with formamide, and its concentration was measured by absorbance at 620 nm. For group A, the average of the difference between the amount of pigment exudation on the sensitized side pinna and the amount of dye exudation on the non-sensitized side pinna was defined as 1, and the allergic reaction inhibition rate of each group was calculated. The result is shown in FIG.

  As shown in FIG. 11, the group B to which the strawberry tea extract was administered alone had a tendency to suppress the allergic reaction compared to the group A to which distilled water was administered, but the effect was weak. This result suggests that the tea extract alone has an allergic reaction inhibitory effect.

  No allergy-suppressing effect was observed in groups C1 and C2 to which enzyme-treated hesperidin was administered alone.

  In group D1 to which hesperetin inclusion was administered alone, a high allergic reaction inhibitory effect exceeding 60% was observed compared to group A to which distilled water was administered, but in group D2 with a reduced dose, the effect was low. . From these results, it was shown that the hesperetin inclusion compound alone has an allergic reaction inhibitory effect. The enzyme-treated hesperidin dose in group C and the hesperetin inclusion dose in group D1 are equivalent in terms of hesperetin. Hesperetin inclusions are thought to release hesperetin in the body. Hesperidin is known to have an allergic reaction inhibitory action. Hesperidin is decomposed and absorbed in the body by hesperetin, which is an aglycone, and hesperetin is considered to exert an antiallergic effect. From this, it is considered that the activity of suppressing the allergic reaction of the hesperetin inclusion product was exerted strongly due to the improvement of hesperetin absorbability. Therefore, by administering as a hesperetin inclusion compound, a significantly higher allergic reaction inhibitory effect than when hesperidin is administered can be exerted by a small amount of ingestion.

  In group E, which was administered in combination with B group and equal dose of tea, and D2 group and hesperetin inclusion compound, about 60% of allergic reaction inhibitory effect was observed compared to group A administered with distilled water. This effect is significantly higher than the total allergic reaction inhibition rate (about 17%) when each dose is given alone. This result suggests that the allergic reaction inhibitory effect was synergistically enhanced by combining the tea extract and the hesperetin inclusion product even at a dose alone having a low allergic reaction inhibitory effect.

  The blood cholesterol lowering effect is confirmed by administering the hesperetin-branched βCD inclusion product to humans, mice, rats and the like.

<Evaluation Example 6: Cholesterol elevation inhibitory effect verification test of hesperetin G2βCD inclusion product>
Thirty 6-week-old male BALB / c mice were preliminarily reared, and then 10 mice each, so that the body weight was equal, control group (distilled water administration), hesperetin administration group, and hesperetin G2βCD inclusion product (production implementation) The hesperetin branch (G2) βCD inclusion product produced in the same manner as in Example 1 was divided into three groups. After being divided into three groups, mice were fed a 1% cholesterol supplemented diet for 4 weeks. This 1% cholesterol-added diet is a high-cholesterol diet, and its composition is 10 g of cholesterol (Wako Pure Chemical), 200 g of casein (Oriental yeast), 541 g of corn starch (Oriental yeast), soybean oil (Wako Pure). The drug was 100 g, DL-methionine (Wako Pure Chemical) 2 g, mineral mix (oriental yeast) 40 g, vitamin mix (oriental yeast) 22 g, and cellulose (oriental yeast) 85 g. During this 4-week feeding period, mice were orally administered 200 μL of the test substance solution 5 times a week with a stomach tube. The test substance solution was distilled water in the control group, the hesperetin administration group was a 0.5% hesperetin solution, and the hesperetin G2βCD inclusion product administration group was adjusted so that the hesperetin administration amount was equivalent to the hesperetin administration group A solution of the hesperetin G2βCD inclusion product was obtained. Since the 0.5% hesperetin solution does not completely dissolve, administration was performed in a sufficiently suspended state.

  After 4 weeks, the mice were sacrificed by cervical dislocation and dissected, and body weight, organ weight (weight of liver, heart, spleen, peri-testicular fat and peri-kidney fat), blood and liver cholesterol, HDL cholesterol And neutral fat was measured. Specifically, after measuring body weight, the mouse was dislocated from the cervical vertebrae at the time of dissection, and blood was collected from the heart. Then, the liver, heart, spleen, peri-testicular fat and peri-renal fat were removed, and the weight was measured and removed. The liver was immediately frozen in liquid nitrogen and stored frozen at -80 ° C. Regarding blood test items (cholesterol, HDL cholesterol and triglyceride), at the time of dissection, the collected blood was separated into serum with a centrifuge, and then a commercially available lipid measurement kit (cholesterol E test Wako, HDL-cholesterol E). Test Wako, Triglyceride E Test Wako, all manufactured by Wako Pure Chemical Industries, Ltd.). For test items in the liver (cholesterol, HDL cholesterol and triglycerides), 0.5 g of the frozen and stored liver was weighed, followed by the Folch method (J. Folch et al., J. Biol. Chem.,). 266, 497, 1957), the lipid was extracted with chloroform, concentrated to dryness under reduced pressure, dissolved in ethanol, and further dissolved in Triton X-100 in distilled water (5 g / L, sodium cholate 3 mmol / L). A solution obtained by adding 100 μL) was measured with a commercially available kit in the same manner as serum.

  The average of the results is shown in Table 12. As shown in Table 12, compared with the control group, the hesperetin clathrate administration group was about 15%, the hesperetin group was about 7%, and the increase in liver cholesterol was suppressed. Peri-testicular fat and perirenal fat weight are indicators of visceral fat and were measured at the time of dissection. As for the weight of peripheral fat in the testis, compared to the control group, the hesperetin inclusion group administration group showed a value about 16% lower, and the hesperetin administration group showed a value about 14% lower. Regarding the weight of peripheral kidney fat, the hesperetin inclusion group administration group showed a value about 36% lower and the hesperetin group about 29% lower than the control group. There was no difference in body weight, liver, heart, or spleen weight between the groups (not shown in the table). All of these groups showed that each index of lipid decreased when hesperetin was administered. It was found that the lipid administration effect was higher in the inclusion administration group than in the hesperetin administration group. These results are considered to be due to the improvement in the absorption of hesperetin in the hesperetin inclusion group administration group as seen in the results of FIGS.

  By administering the hesperetin-branched βCD inclusion product to humans, mice, rats, etc., the blood flow improving effect and the cooling property improving effect are confirmed.

<Evaluation Example 7: Blood flow improvement effect verification test of hesperetin G2βCD inclusion product>
It has been reported that enzyme-treated hesperidin has an effect of improving blood flow and is effective for cold disease (58th and 59th Japan Society of Nutrition and Food, Yoshitani et al.). Hesperetin inclusion (corresponding to hesperetin equivalent of 40 mg) in a 27-year-old woman diagnosed with cold syndrome according to criteria for cold syndrome (Yoshitoshi Terasawa: Biopharmaceutical Journal, 41 (2), 1987) (the same method as Evaluation Example 4A-1) The effect of improving blood flow when hesperidin (G2 βCD inclusion product) or enzyme-treated hesperidin prepared in 1) was dissolved in 100 ml of water at 37 ° C. was compared by the cooling load method (reference: Masahiko Kano, “ Peripheral Circulation Disorder and Thermography ", BIOMEDICAL THERMOLOGY, 17 (2), 111-113, 1998; Research, "Bulletin of Niigata University College of Medical Technology, 7 (2), 215-226, 2000). The blood flow improvement value when only 100 ml of water at 37 ° C. was ingested was also measured as a control. After taking a test beverage for 2 hours or more after eating and resting for 30 minutes, the left hand was immersed in a thermostatic bath with a water flow set at 15 ° C. for 1 minute, and a cooling load was applied. Thereafter, the body surface temperature of the left fingertip was measured by thermography (NEC Sanei TH3100MR) for 30 minutes and every 5 minutes, and the blood flow rate was measured by a laser blood flow meter (Omega Wave FLO-N1).

  The results are shown in Table 13, Table 14, FIG. 12 and FIG. FIG. 12 shows the surface temperature recovery rate when the temperature immediately before cooling is 100% and the temperature immediately after cooling is 0%. FIG. 13 is represented by the rate of change when the value immediately before cooling is 100%. As shown in FIG. 12, a high temperature recovery rate was recognized more rapidly by ingesting hesperetin inclusions than when ingesting enzyme-treated hesperidin. Further, as shown in FIG. 13, by ingesting the hesperetin inclusion product, a faster recovery of blood flow was observed than with the enzyme-treated hesperidin. As seen in the results of FIGS. 8 and 9, the above results are considered to be due to the rapid absorption of hesperetin in the body and the increase in the absorption amount of hesperetin inclusion.

  By administering the hesperetin-branched βCD inclusion product to humans, mice, rats, etc., the skin condition improving effect is confirmed.

  The anti-inflammatory effect is confirmed by administering hesperetin branched βCD inclusion compound to humans, mice, rats and the like.

<Evaluation Example 8: Anti-inflammatory effect verification test of hesperetin branched βCD inclusion product>
After preliminary breeding of 10 5-week-old female BALB / c mice, 5 mice each, so that the body weight becomes equal, control group (group A), hesperetin inclusion (in the same manner as in Evaluation Example 1) The hesperetin branch (G2) βCD inclusion product produced) was divided into two groups (group B). Mice were orally administered 200 μL of the test substance solution with a gastric sonde for 4 consecutive days. The test substance solution was adjusted to the dosage shown in Table 15. On the fourth day after oral administration, 10 μL / ear of 0.06% croton oil (Sigma) was applied to the center of the left auricle of the mouse. Three hours later, 0.2 ml of 1% Evans blue-containing physiological saline was administered from the tail vein, and one hour later, the auricle was excised. The center part was punched out using a punch for punching leather with a diameter of 7 mm, and Evans blue exuding on the auricle was extracted with formamide, and its concentration was measured by absorbance at 620 nm. The difference in inflammation between the right auricle pigment and the left auricle pigment exudation amount of the A group was set to 1, and the inflammation suppression rate of the B group was evaluated. The results are shown in Table 15.

  As shown in Table 15, in group B administered with hesperetin inclusion product, inflammation was suppressed 41% compared to group A administered with distilled water. From these results, it was shown that the hesperetin inclusion product has an anti-inflammatory effect.

  As mentioned above, although this invention has been illustrated using preferable embodiment of this invention, this invention should not be limited and limited to this embodiment. It is understood that the scope of the present invention should be construed only by the claims. It is understood that those skilled in the art can implement an equivalent range based on the description of the present invention and the common general technical knowledge from the description of specific preferred embodiments of the present invention. Patents, patent applications, and documents cited herein should be incorporated by reference in their entirety, as if the contents themselves were specifically described herein. Understood.

  According to the present invention, a hesperetin inclusion compound and a naringenin inclusion compound having extremely high bioabsorbability are provided.

FIG. 1 is a graph showing the solubility of inclusion compounds obtained by inclusion of various flavonoid aglycones with β-cyclodextrin (βCD) or branched β-cyclodextrin (G2βCD). FIG. 2 shows hesperetin clathrated with branched β-cyclodextrin (G2βCD inclusion; evaluation example 1), hesperetin clathrated with β-cyclodextrin in a dissolved state (βCD inclusion 200 μl; evaluation example 2), Hesperetin (βCD inclusion 300 μl; evaluation example 3), hesperetin (hesperetin; evaluation comparative example 1), hesperidin (hesperidin; evaluation comparative example 2), and enzyme treatment in the precipitated state, clathrated with β-cyclodextrin 2 is a graph showing the time course of hesperetin concentration in mouse heart blood after oral administration of hesperidin (enzyme treatment (PA-T); Evaluation Comparative Example 3) to which a sugar chain was added by the method described above. FIG. 3 shows hesperetin clathrated with branched β-cyclodextrin (G2βCD inclusion; evaluation example 1), hesperetin clathrated with β-cyclodextrin in the dissolved state (βCD inclusion 200 μl; evaluation example 2), Hesperetin (βCD inclusion 300 μl; evaluation example 3), hesperetin (hesperetin; evaluation comparative example 1), hesperidin (hesperidin; evaluation comparative example 2), and enzyme treatment in the precipitated state, clathrated with β-cyclodextrin Shows the area under the curve (AUC) of 0 to 9 hours of hesperetin concentration in mouse heart blood after oral administration of hesperidin (enzyme treatment (PA-T); Evaluation Comparative Example 3) to which the sugar chain was added by It is a graph. FIG. 4 shows hesperetin clathrated with branched β-cyclodextrin (G2βCD inclusion; evaluation example 1), hesperetin clathrated with β-cyclodextrin in a dissolved state (βCD inclusion 200 μl; evaluation example 2), It is the graph which took the relative ratio of the absorptivity of the hesperetin ((beta) CD inclusion inclusion 300microliter; evaluation example 3) of the inclusion state of (beta) -cyclodextrin in the precipitated state, and took the solubility on the Y-axis. Fig. 5 shows the relative absorption ratio of rutin (enzyme-treated (αG rutin)), rutin (rutin), and quercetin (quercetin) absorbed by enzyme treatment on the X-axis and solubility on the Y-axis. It is a graph. FIG. 6 shows the concentration of quercetin in the heart blood of mice after oral administration of quercetin (G2βCD inclusion quercetin; Evaluation Comparative Example 4) or quercetin (Quercetin; Evaluation Comparative Example 5) encapsulated with branched β-cyclodextrin. It is a graph which shows the sum total of a tamalizetin density | concentration and an isorhamnetin density | concentration with time. FIG. 7 shows the concentration of quercetin in mouse heart blood after oral administration of quercetin (G2βCD inclusion quercetin; evaluation comparative example 4) or quercetin (quercetin; evaluation comparative example 5) encapsulated with branched β-cyclodextrin. It is a graph which shows the area under a curve (AUC) of 0 to 9 hours of the sum total of tamalizetin concentration and isorhamnetin concentration. FIG. 8 shows hesperetin clathrated with branched β-cyclodextrin (G2βCD inclusion product; evaluation example 4A-1), hesperetin clathrated with β-cyclodextrin (βCD inclusion product; evaluation conducted) It is a graph which shows the hesperetin density | concentration in a human serum sample after ingesting the human ingestion of Example 4A-2) or hesperetin (Hesperetin; evaluation comparative example 6A-1). FIG. 9 shows humans after oral intake of hesperetin (G2βCD inclusion product; evaluation example 4B) or enzyme-treated hesperidin (enzyme-treated hesperidin; evaluation comparative example 6B) encapsulated with branched β-cyclodextrin. It is a graph which shows the hesperetin density | concentration in a serum sample with time. FIG. 10 shows the time course of hesperetin concentration in a human serum sample after orally ingesting hesperidin (hesperidin; evaluation comparative example 6C-1) or enzyme-treated hesperidin (enzyme-treated hesperidin; evaluation comparative example 6C-2). FIG. FIG. 11 shows distilled water, strawberry tea extract (80 mg / kg), enzyme-treated hesperidin (600 mg / kg or 120 mg / kg), hesperetin clathrated with branched β-cyclodextrin (hesperetin inclusion; 550 mg / kg or 55 mg) / Kg) or an allergic reaction inhibition rate (%) compared to the allergic reaction of group A when a mixture of strawberry tea extract and hesperetin inclusion product is orally administered to mice. FIG. 12 is a graph showing the change over time in the recovery rate of the surface temperature when a control (water), enzyme-treated hesperidin or hesperetin inclusion product is orally ingested by humans. The vertical axis represents the recovery rate (%), and the horizontal axis represents the elapsed time (minutes). FIG. 13 is a graph showing the change over time in the recovery rate of blood flow when a control (water), enzyme-treated hesperidin or hesperetin inclusion product is orally ingested by humans. The vertical axis represents the recovery rate (%), and the horizontal axis represents the elapsed time (minutes).

Claims (22)

A method of synthesizing a flavanone inclusion compound which is a hesperetin inclusion compound or a naringenin inclusion compound,
Inclusion of hesperidin or naringin with β-cyclodextrin, branched β-cyclodextrin or a modification thereof to obtain a hesperidin inclusion compound or naringin inclusion compound; and the hesperidin inclusion compound or the naringin inclusion compound Hydrolyzing to obtain a hesperetin inclusion compound or a naringenin inclusion compound.   The method according to claim 1, wherein the hydrolyzing step is carried out by allowing rhamnosidase and glucosidase to act on the hesperidin inclusion compound or the naringin inclusion compound.   The β-cyclodextrin, the branched β-cyclodextrin or a modified product thereof is a β-cyclodextrin, a branched β-cyclodextrin in which 1 to 6 glucose residues are linked as side chains to the β-cyclodextrin, and their The method of claim 1, wherein the method is selected from the group consisting of chemical modifications.   The method according to claim 1, wherein the hesperidin or naringin is included by branched β-cyclodextrin.   The method according to claim 4, wherein the branched β-cyclodextrin is a compound in which two glucose residues are linked to the β-cyclodextrin as side chains.   The method according to claim 1, wherein the flavanone inclusion compound is a hesperetin inclusion compound. A method of synthesizing a flavanone inclusion compound which is a hesperetin inclusion compound or a naringenin inclusion compound,
By mixing hesperetin or naringenin with a basic suspension or aqueous solution containing hesperetin or naringenin and an acidic aqueous solution containing β-cyclodextrin, branched β-cyclodextrin or a modification thereof, β-cyclodextrin , Including a step of inclusion by a branched β-cyclodextrin or a modified product thereof to obtain a hesperetin inclusion compound or a naringenin inclusion compound.   The β-cyclodextrin, the branched β-cyclodextrin or a modified product thereof is a β-cyclodextrin, a branched β-cyclodextrin in which 1 to 6 glucose residues are linked as side chains to the β-cyclodextrin, and their 8. The method of claim 7, wherein the method is selected from the group consisting of chemical modifications.   The method according to claim 7, wherein the hesperetin or naringenin is included by branched β-cyclodextrin.   The method according to claim 9, wherein the branched β-cyclodextrin is a compound in which two glucose residues are linked as side chains to β-cyclodextrin.   The method according to claim 7, wherein the flavanone inclusion compound is a hesperetin inclusion compound. A method of synthesizing a flavanone inclusion compound which is a hesperetin inclusion compound or a naringenin inclusion compound,
By mixing an organic solvent solution containing hesperetin or naringenin and an aqueous solution containing β-cyclodextrin, branched β-cyclodextrin or a modification thereof, hesperetin or naringenin is mixed with β-cyclodextrin, branched β-cyclodextrin or A method comprising inclusion by a modification thereof to obtain a hesperetin inclusion compound or a naringenin inclusion compound, wherein the organic solvent is ethanol, isopropanol or tetrahydrofuran;   A food comprising a flavanone inclusion compound, wherein the flavanone is hesperetin or naringenin, and the flavanone is included by β-cyclodextrin, branched β-cyclodextrin or a modified product thereof.   The food according to claim 13, which is a health food.   The food according to claim 14, which is used for improving blood flow and cooling.   The food according to claim 14, which is for improving skin condition.   The food according to claim 14, which is used for lowering cholesterol.   The food according to claim 14, which is used for improving allergic symptoms.   The food according to claim 14, which is used for anti-inflammation.   A pharmaceutical comprising a flavanone inclusion compound, wherein the flavanone is hesperetin or naringenin, and the flavanone is included by β-cyclodextrin, branched β-cyclodextrin or a modification thereof.   A skin external preparation containing a flavanone inclusion compound, wherein the flavanone is hesperetin or naringenin, and the flavanone is included by β-cyclodextrin, branched β-cyclodextrin or a modification thereof Topical agent.   A clathrate inclusion compound, wherein the flavanone is hesperetin or naringenin, and the flavanone is clathrated by branched β-cyclodextrin or chemically modified β-cyclodextrin.

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