Enzymatic production of phosphorylated equol

Abstract

To provide novel phosphorylated equol with improved solubility in water.SOLUTION: The present invention provides a phosphorylated equol represented by formula (1) or a pharmacologically acceptable salt thereof (where, R1 is a phosphate group, R2 is a hydroxy group).SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a novel phosphorylated equol and a method for producing said phosphorylated equol using an enzyme. [Background technology]

[0002] In soybeans, for example, isoflavones exist as glycosides covalently bound to sugars, such as daidzin, glycitin, and genistin, with only small amounts of aglycones present. These glycosides are also malonylated and acetylated. Once ingested, these glycosides are converted into daidzein, glycitein, and genistein, respectively, by digestive enzymes or the enzyme β-glucosidase produced by intestinal bacteria. Furthermore, daidzein is known to be enzymatically converted by intestinal bacteria to dihydrodaidzein, and then to O-desmethylangolensin (O-DMA) or equol.

[0003] Of these metabolites, equol is known to have the highest estrogenic activity (Non-Patent Documents 1 and 2). However, in humans, there are individual differences in isoflavone metabolism, and as mentioned above, few people possess intestinal bacteria capable of fermenting daidzein to produce equol. It has been revealed that the prevalence of such intestinal bacteria is approximately 50% in Japanese and approximately 30% in Westerners (Non-Patent Documents 3 and 4). Therefore, there has been a problem in that people who do not possess equol-producing bacteria are unable to produce equol in their bodies even if they ingest legume foods such as soybeans.

[0004] To overcome these challenges, attempts have been made in recent years to produce equol exogenously using anaerobic microorganisms such as lactic acid bacteria (Patent Documents 1 to 4). However, equol has the problem of low solubility in water. Increasing its solubility in water would increase the efficiency with which equol is absorbed into the body, making it effective when incorporated into foods and other foods.

[0005] Equol glycoside is known as an equol compound with increased water solubility (Patent Document 5). It is also known that dehydroequol can be phosphorylated by chemical synthesis (Patent Document 6), and that daidzein and genistein can be phosphorylated using a microbial phosphorylating enzyme (Patent Document 7). There are also reports of equol phosphorylated at the 7th carbon and at two carbons, the 7th and 4' carbons (Patent Document 6). However, Patent Document 6 does not disclose spectral data for the 7th carbon-phosphorylated equol, and no evidence has been obtained that such a compound has actually been synthesized. Furthermore, when phosphorylated equol is chemically synthesized, there are safety concerns regarding the catalysts used, etc. Therefore, there has been a need to establish a method for producing phosphorylated equol using microbiological techniques. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Patent Publication No. 2006-204296 [Patent Document 2] Special Publication 2006-504409 [Patent Document 3] Patent Publication No. 2008-61584 [Patent Document 4] Patent Publication No. 2010-104241 [Patent Document 5] WO2008 / 126752 [Patent Document 6] Special Publication 2007-511542 [Patent Document 7] Patent Publication No. 2002-238594 [Non-patent literature]

[0007] [Non-Patent Document 1] Schmitt, E. et al., Toxicol. In Vitro, 15, 433-439 (2001) [Non-patent document 2] Sathyamoorthy, N. and Wang, TT, Eur. J. Cancer, 33, 2384-2389 (1997) [Non-patent document 3] Arai, Y. et al., J. Epidemiol., 10, 127-135 (2000) [Non-patent document 4] Setchell, KD et al., J. Nutr., 133, 1027-1035 (2003) Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention was made in light of these circumstances, and its purpose is to provide a novel phosphorylated equol in which only the 4' carbon or the 7' carbon is phosphorylated, and a method for producing said phosphorylated equol using an enzyme. [Means for solving the problem]

[0009] The present inventors have conducted extensive research to solve the above-mentioned problems. Specifically, they searched for enzymes capable of phosphorylating only the 4' carbon or only the 7 carbon of equol from among various kinases. As a result, they discovered that the 4' carbon or the 7 carbon of equol can be specifically phosphorylated by using kinase T, kinase Q, kinase S, kinase N, and kinase L obtained from Thermostable Enzyme Research Institute Co., Ltd. [These enzymes are available as Phosphorylation Enzymes (Thermostable Enzyme Research Institute) T, Q, S, N, and L, respectively.]. That is, the present invention provides equol phosphorylated only at the 4' carbon or the 7 carbon, and methods for producing such phosphorylated equol. Specifically, the present invention provides the following [1] to

[12] .

[0010] [1] Phosphorylated equol having one phosphate group or a pharmaceutically acceptable salt thereof. [2] Phosphorylated equol represented by the following chemical formula (1) or a pharmaceutically acceptable salt thereof: [ka] In the formula, one of R1 and R2 is a hydroxyl group, and the other is a phosphate group. [3] The phosphorylated equol or a pharmaceutically acceptable salt thereof according to [2], wherein R1 is a phosphate group and R2 is a hydroxyl group. [4] The phosphorylated equol or a pharmaceutically acceptable salt thereof according to [2], wherein R1 is a hydroxyl group and R2 is a phosphate group. [5] A method for producing phosphorylated equol having one phosphate group, comprising the step of contacting equol with a kinase in the presence of a phosphate donor. [6] The method according to [5], wherein the kinase is a kinase that specifically phosphorylates only the 4' carbon or only the 7' carbon of equol, and the phosphorylated equol having one phosphate group is phosphorylated equol represented by the following chemical formula (1): [ka] In the formula, one of R1 and R2 is a hydroxyl group, and the other is a phosphate group. [7] The method according to [5] or [6], wherein the kinase is any of the enzymes available from Thermostable Enzyme Research Institute under the names of kinase T, kinase Q, kinase S, kinase N, or kinase L. [8] The method according to any one of [5] to [7], wherein the phosphate donor is polyphosphate, ATP, phosphoenolpyruvate, or creatine phosphate. [9] The method according to any one of [6] to [8], wherein R1 is a phosphate group and R2 is a hydroxyl group.

[10] The method according to any one of [6] to [8], wherein R1 is a hydroxyl group and R2 is a phosphate group.

[11] Phosphorylated equol obtained by the method according to any one of [5] to

[10] .

[12] A composition comprising the phosphorylated equol according to any one of [1] to [3] or a pharmaceutically acceptable salt thereof, or the phosphorylated equol according to

[11] . [Effects of the Invention]

[0011] The present invention provides a novel phosphorylated equol in which only the 4' carbon or the 7 carbon is phosphorylated, and a method for producing the same. The phosphorylated equol provided by the present invention is expected to have improved water solubility, resulting in improved absorption efficiency into the body and improved bioavailability when orally ingested. It can also be used to reduce the bitterness of foods. Therefore, the phosphorylated equol of the present invention is useful in the food industry, etc. The phosphorylated equol of the present invention is also believed to have the female hormone and antioxidant effects of equol, and is therefore useful for preventing and / or ameliorating diseases and symptoms such as menopausal disorders (menopausal complaints, osteoporosis, hyperlipidemia), osteoporosis, prostatic hyperplasia, and metabolic syndrome. [Brief explanation of the drawings]

[0012] [Figure 1] 1 is a graph showing the amount of phosphorylated equol produced by kinases A to G, kinase I, kinase J, and kinases L to U. The vertical axis shows the amount produced, and the horizontal axis shows the enzymes used in the experiment. [Figure 2] FIG. 2 shows a chromatogram of the reaction mixture obtained using kinase T. [Figure 3] FIG. 3 shows a total ion chromatogram (ESI-neg) in LC / MS analysis. [Figure 4] FIG. 4 shows a total ion chromatogram (ESI-pos) in LC / MS analysis. [Figure 5] FIG. 5 shows the LC / MS mass spectrum (ESI-neg). [Figure 6] FIG. 6 shows the LC / MS mass spectrum (ESI-pos). [Figure 7] FIG. 7 is a graph showing the progress of the phosphorylated equol production reaction. [Figure 8] FIG. 8 shows a total ion chromatogram (ESI-neg) in the LC / MS / MS analysis. [Figure 9] FIG. 9 shows a total ion chromatogram (ESI-pos) in the LC / MS / MS analysis. [Figure 10] FIG. 10 shows the LC / MS / MS mass spectrum (ESI-neg, P-1). [Figure 11] FIG. 11 shows the mass spectrum (ESI-pos, P-1) of LC / MS / MS. [Figure 12] FIG. 12 shows the LC / MS / MS mass spectrum (ESI-neg, P-2). [Figure 13] FIG. 13 shows the mass spectrum (ESI-pos, P-2) of LC / MS / MS. [Figure 14] FIG. 14 shows the LC / MS / MS fragment analysis pattern (P-1). [Figure 15]FIG. 15 shows the LC / MS / MS fragment analysis pattern (P-2). [Figure 16] FIG. 16 shows phosphorylated equol that can be inferred from the fragment analysis pattern. DETAILED DESCRIPTION OF THE INVENTION

[0013] The present invention relates to phosphorylated equol having one phosphate group. Specifically, the present invention relates to phosphorylated equol represented by the following chemical formula (1) or a pharmaceutically acceptable salt thereof. [ka] In chemical formula (1), one of R1 and R2 is a hydroxyl group, and the other is a phosphate group.

[0014] The present invention also relates to phosphorylated equol in which R1 is a phosphate group and R2 is a hydroxyl group in the above chemical formula (1). Alternatively, the present invention relates to phosphorylated equol in which R1 is a hydroxyl group and R2 is a phosphate group. The former phosphorylated equol can be described as equol phosphorylated at the 4' carbon, or 4'-O-phosphoequol. Meanwhile, the latter phosphorylated equol can be described as equol phosphorylated at the 7 carbon, or 7-O-phosphoequol.

[0015] Equol, where the 4' carbon is phosphorylated, or 4'-O-phosphoequol, is represented by the following chemical formula (2): [ka]

[0016] Equol with the 7th carbon phosphorylated, or 7-O-phosphoequol, is represented by the following chemical formula (3). [ka]

[0017] In the present invention, pharmaceutically acceptable salts include, for example, acid addition salts, metal salts, and organic base addition salts. Examples of acid addition salts include, but are not limited to, inorganic acid salts such as hydrochloride, sulfate, and phosphate, and organic acid salts such as acetate, maleate, fumarate, tartrate, and citrate. Examples of metal salts include, but are not limited to, alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as magnesium salt and calcium salt, aluminum salt, and zinc salt. Examples of organic base addition salts include, but are not limited to, salts formed with primary amines such as methylamine, ethylamine, and aniline; secondary amines such as dimethylamine, diethylamine, pyrrolidine, piperidine, morpholine, and piperazine; and tertiary amines such as trimethylamine, triethylamine, N,N-dimethylaniline, and pyridine; and ammonium salts.

[0018] The phosphorylated equol of the present invention can be produced by a method comprising the step of contacting equol with a kinase in the presence of a phosphate donor. In the present invention, equol may be either R-equol or S-equol. Equol can be produced by methods known to those skilled in the art (for example, one method for producing equol using anaerobic microorganisms capable of producing equol involves fermenting daidzein with the anaerobic microorganisms in a gas phase consisting of one or more gases, including hydrogen, and then recovering equol (Patent Document 7 / JP 2002-238594 A). Alternatively, commercially available equol from suppliers such as Daicel Chemical Industries (China) Co., Ltd. can also be used.

[0019] Furthermore, the kinase of the present invention is preferably an enzyme capable of specifically phosphorylating only the 4' carbon or only the 7 carbon of equol. Examples of such enzymes include, but are not limited to, kinase T, kinase Q, kinase S, kinase N, and kinase L, all of which are available from Thermostable Enzyme Research Institute, Inc. Thermostable Enzyme Research Institute (Address: 5-5-2 Minatojima Minamimachi 5-chome, Chuo-ku, Kobe; Website: http: / / www.tainetsu.com / ) manages and stores these enzymes in association with their names. Therefore, kinases T, Q, S, N, and L of the present invention can be obtained by informing Thermostable Enzyme Research Institute of the name of the desired enzyme.

[0020] The kinases of the present invention also include homologs of these kinases. In the present invention, homologs of kinase T, kinase Q, kinase S, kinase N, and kinase L refer to proteins that contain amino acid sequences derived from these enzymes by deletion, substitution, insertion, and / or addition of one or more amino acids, and that have the activity of specifically phosphorylating only the 4' carbon or only the 7' carbon of equol. The number of amino acids to be modified is not particularly limited, as long as the modified protein retains the aforementioned physicochemical properties. However, modifications are generally within 50 amino acids, preferably within 30 amino acids, and more preferably within 10 amino acids (e.g., within 5 amino acids, within 3 amino acids). Alternatively, modifications of, for example, 20% or less, specifically 10% or less (e.g., within 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%) of the amino acid sequence are acceptable. In other words, proteins containing amino acid sequences that are preferably 80% or more, more preferably 90% or more (e.g., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more) homologous to the amino acid sequences of kinase T, kinase Q, kinase S, kinase N, and kinase L are also included in the kinases of the present invention.

[0021] Generally, to maintain protein function, it is preferable that the amino acid to be substituted be an amino acid with properties similar to the amino acid before substitution. Such substitutions of amino acid residues are called conservative substitutions. For example, Ala, Val, Leu, Ile, Pro, Met, Phe, and Trp are all classified as nonpolar amino acids and therefore have similar properties. Examples of uncharged amino acids include Gly, Ser, Thr, Cys, Tyr, Asn, and Gln. Examples of acidic amino acids include Asp and Glu. Examples of basic amino acids include Lys, Arg, and His. Amino acid substitutions within each of these groups are permitted.

[0022] In the present invention, polyphosphate, ATP, phosphoenolpyruvate, creatine phosphate, etc. can be used as the phosphate donor, but are not limited to these. In the present invention, polyphosphate refers to a polymer whose basic unit is phosphate (PO4). The polyphosphate of the present invention includes polyphosphate with a linear structure and polyphosphate with a cyclic structure.

[0023] In the present invention, the reaction conditions for equol and kinase may be, but are not limited to, the following conditions. Reaction temperature: preferably 4 to 60°C, more preferably 15 to 40°C, and even more preferably 36 to 38°C. pH: preferably 2 to 12, more preferably 4 to 6, and even more preferably 4.4 to 4.6. Equol concentration: preferably 1 to 20 mM, more preferably 4 to 10 mM, and even more preferably 4.5 to 6.5 mM. · Phosphatase concentration: preferably 10 to 1000 μg / mL, more preferably 100 to 700 μg / mL, and even more preferably 300 to 500 μg / mL. Amount of phosphate donor: preferably 1 to 20%, more preferably 5 to 15%, and even more preferably 8 to 12%. In the present invention, "contact" can also be replaced with "mixing" or "adding".

[0024] The method for producing phosphorylated equol of the present invention can additionally include a step of recovering the produced equol phosphate. Phosphorylated equol can be recovered, for example, by the following method. The liquid containing enzymatically converted equol is heated, for example, at 70°C for 30 minutes, then returned to room temperature and centrifuged to remove insoluble matter, yielding a supernatant. This supernatant is concentrated under reduced pressure using a rotary evaporator, the pH adjusted to approximately 3, and an equal volume of methanol is added and mixed thoroughly. This liquid is then passed through a silica gel column, followed by a 2 / 8 mixture of water and methanol, and fractions are collected. Fractions containing phosphorylated equol can be collected based on UV absorption at 280 nm and HPLC analysis results. The collected liquid is then concentrated under reduced pressure using a rotary evaporator and dried to obtain a solid.

[0025] Furthermore, those skilled in the art can identify equol phosphorylated at only the 4' carbon or the 7' carbon using analytical chemistry techniques well known to those skilled in the art, such as LC / MS, LC / MS / MS, and NMR.

[0026] The present invention further relates to phosphorylated equol obtained by the above-mentioned method for producing phosphorylated equol. Specifically, the present invention relates to phosphorylated equol obtained by a method including a step of contacting equol with a kinase. As described above, the phosphorylated equol obtained by this method is a novel phosphorylated equol in which only the 4' carbon or only the 7 carbon is specifically phosphorylated.

[0027] The present invention also relates to a composition containing phosphorylated equol or a pharmaceutically acceptable salt thereof (hereinafter referred to as a phosphorylated equol-containing composition). The composition of the present invention can be provided as an ingredient for pharmaceuticals, foods, beverages, cosmetics, etc. The phosphorylated equol-containing composition of the present invention is expected to be more efficiently taken up into the body than a non-phosphorylated equol-containing composition. Examples of phosphorylated equol include, but are not limited to, equol phosphorylated at the 4' carbon (4'-O-phosphoequol) and equol phosphorylated at the 7 carbon (7-O-phosphoequol).

[0028] When the phosphorylated equol-containing composition of the present invention is provided as a pharmaceutical, its dosage form can be selected depending on the disease to be prevented or treated, the pharmaceutical's usage pattern, administration route, etc. Examples include tablets, coated tablets, pills, capsules, granules, powders, liquids, suspensions, emulsions, syrups, injections, suppositories, infusions, decoctions, tinctures, etc. These various preparations can be formulated in accordance with conventional methods using known adjuvants commonly used in the pharmaceutical formulation field, such as fillers, extenders, excipients, binders, humectants, disintegrants, surfactants, lubricants, colorants, flavorings, solubilizers, suspending agents, and coating agents, in addition to the main drug, as needed. Furthermore, these pharmaceutical preparations may contain colorants, preservatives, fragrances, flavors, sweeteners, and other pharmaceuticals. The phosphorylated equol-containing composition of the present invention can be used for the prevention and treatment of breast cancer, prostate cancer, osteoporosis, heart disease, and menopausal disorders.

[0029] When the phosphorylated equol-containing composition of the present invention is provided as a food or drink, it can be used as a general food, a food for specified health uses, a nutritional supplement, a functional food, a food for the sick, a food additive, a supplement, etc. Examples of food forms that may be used include soft drinks, milk, pudding, jelly, candy, gum, gummy candy, yogurt, chocolate, soup, cookies, snacks, ice cream, popsicles, bread, cake, cream puffs, ham, meat sauce, curry, stew, cheese, butter, dressing, etc.

[0030] The phosphorylated equol-containing composition of the present invention can contain water, protein, carbohydrates, lipids, vitamins, minerals, organic acids, organic bases, fruit juice, flavors, etc. as its main ingredients. Proteins include animal and vegetable proteins such as whole milk powder, skim milk powder, partially skim milk powder, casein, soy protein, egg protein, and meat protein, as well as their hydrolysates and butter. Carbohydrates include sugars, modified starches (textlin, soluble starch, British starch, oxidized starch, starch esters, starch ethers, etc.), and dietary fiber. Lipids include animal fats and oils such as lard, fish oil, and their fractionated, hydrogenated, and interesterified oils; and vegetable fats and oils such as palm oil, safflower oil, corn oil, rapeseed oil, coconut oil, and their fractionated, hydrogenated, and interesterified oils. Examples of vitamins include vitamin A, carotenes, B vitamins, vitamin C, D vitamins, vitamin E, K vitamins, vitamin P, vitamin Q, niacin, nicotinic acid, pantothenic acid, biotin, inositol, choline, and folic acid. Examples of minerals include calcium, potassium, magnesium, sodium, copper, iron, manganese, zinc, selenium, and whey minerals. Examples of organic acids include malic acid, citric acid, lactic acid, and tartaric acid. Two or more of these ingredients can be used in combination, and synthetic products and / or foods containing large amounts of these ingredients can also be used.

[0031] The proportion of the phosphorylated equol-containing composition in these foods can be appropriately determined depending on the type of food, the content of phosphorylated equol, the age and sex of the person taking it, the expected effect, etc. An example is, but is not limited to, a proportion of 0.01-100g, preferably 0.1-10g, and more preferably 0.5-5g per 100g of food. The daily intake of foods containing a phosphorylated equol-containing composition varies depending on the content of phosphorylated equol in the composition, the age and weight of the person taking it, the frequency of intake, etc., but can be, for example, an amount of composition equivalent to 0.1-10g per adult per day.

[0032] Furthermore, when the phosphorylated equol-containing composition of the present invention is provided as a cosmetic, the composition can be prepared in various desired forms, such as liquids such as aqueous solutions, lotions, sprays, suspensions, and emulsions, solids such as powders, granules, and blocks, semi-solids such as creams and pastes, and gels. Such cosmetics are useful as various cosmetic materials, including face washes, emulsions, creams, gels, essences (beauty serums), packs, masks, and other basic cosmetics, foundations, lipsticks, and other makeup cosmetics, oral cosmetics, fragrance cosmetics, hair cosmetics, and body cosmetics. The phosphorylated equol-containing composition of the present invention can be enclosed in an appropriate container such as a bottle, bag, can, spray can, spray container, box, or pack, as needed.

[0033] The proportion of the phosphorylated equol-containing composition in cosmetics of the present invention is not particularly limited and can be determined appropriately depending on the type of cosmetic, the content of phosphorylated equol, etc. One example is, but is not limited to, a proportion of 0.01-10 g, preferably 0.1-5 g, of the composition (in dry weight terms) per 100 g of cosmetic product. All prior art documents cited in this specification are hereby incorporated by reference. [Example]

[0034] The present invention will be described in detail below with reference to examples, but the present invention is not limited thereto. Example 1 <Screening> 5 mL of water was added to a polyphosphoric acid solution (ACROS Organics) while cooling, followed by 5 mL of triethylamine (Wako Pure Chemical Industries, Ltd.) while cooling (Solution A). Equol (Daicel Chiral Technologies (China) Co., Ltd., trade name "(S)-EQUOL") was dissolved in 5 mL of triethylamine (Wako Pure Chemical Industries, Ltd.) and mixed with Solution A. The pH was adjusted to approximately 4.5 while cooling using 35% NaOH solution, and the total volume was adjusted to 47.5 mL with water (Solution B). To 0.95 mL of solution B, 0.05 mL of each kinase solution (Kinase A to Kinase U, manufactured by Thermostable Enzyme Laboratory Co., Ltd., website address: http: / / www.tainetsu.com / document.php?pid=1) was added, and the mixture was allowed to react for 24 hours at 37°C. 0.45 mL of HPLC mobile phase was added to 0.05 mL of the reacted solution and mixed to prepare an HPLC sample. The reaction composition and HPLC analysis conditions are shown below.

[0035] Reaction solution composition Equol concentration: 1.2 mg / mL (5 mM) Polyphosphate concentration: 3.75% Triethylamine: 20% Enzyme concentration: 50μg / mL

[0036] ·HPLC analysis conditions Column: YMC-Pack ODS-A (150 x 4.6 mm ID) Mobile phase: 25 mM formic acid (pH 2.5):methanol = 55:45 Flow rate: 0.5mL / min Detection: UV (280 nm) Oven temperature: 40℃ Injection volume: 10μL

[0037] As a result, it was found that phosphorylase T had the highest reactivity, followed by phosphorylase Q, phosphorylase S, phosphorylase N, and phosphorylase L in that order (Figure 1). Further, as a result of HPLC analysis of the reaction solution by phosphorylase T under the HPLC analysis conditions described below, a peak thought to be phosphorylated equol was confirmed at a retention time of 6.91 minutes (Figure 2). Since the peak at a retention time of 6.91 minutes appeared only after the reaction, it was estimated to be phosphorylated equol.

[0038] Example 2 <Confirmation of equol phosphate> Using the reaction solution obtained in Example 1, it was confirmed that the peak at a retention time of 6.91 minutes was phosphorylated equol. As a result of analysis by LC / MS, it was suggested by the molecular weight of the compound that it was phosphorylated equol with one phosphate group attached to equol. In the mass spectrum, peaks were observed at 321.2 in ESI-neg (Figure 5) and at 323.0 in ESI-pos (Figure 6), indicating that a compound with a molecular weight of 322 was present. This molecular weight corresponded to phosphorylated equol with one phosphate group attached to equol. The LC / MS analysis conditions are shown below.

[0039] ·LC / MS analysis conditions <HPLC analysis conditions> Column: InertSustain C18 (manufactured by GL Sciences) 2.1φ×150mm (C-50) Mobile phase: (A) 0.1% formic acid aqueous solution (B) HPLC grade MeOH Gradient conditions: (A) / (B) = 80 / 20 (5 min) → 15 min → 0 / 100 (10 min) Flow rate: 0.2 mL / min Oven temperature: 40°C Detection: PDA (signal 280 nm, bandwidth 10 nm, reference off, Peak width > 0.05 min, slit 4 nm) Injection volume: 5 μL

[0040] Mass spectrometer: Quadrupole triple stage LC / MS / MS device (manufactured by Micro Mass) Ion source: ESI-pos,neg Measurement mode: Scan analysis (m / z 50-1050) Capillary: 3.00kV (pos,neg) Cone: 50(pos), 80(neg) RF Lens1:20.0(pos), 50.0(neg) Desolvency temperature: 200℃ Source Temp.: 80℃ Cone Gas Flow: 66L / hr Desolvation Gas Flow: 690L / hr

[0041] Example 3 <Production of equol phosphate (1 L scale)> 200 mL of water was added to a polyphosphate solution (Wako Pure Chemical Industries, Ltd.) while cooling, followed by 150 mL of triethylamine (Wako Pure Chemical Industries, Ltd.) while cooling. The pH was adjusted to approximately 3.0 with 35% NaOH solution while cooling (Solution A). 1.5 g of equol (Daicel Chiral Chemicals (China) Co., Ltd., trade name "(S)-EQUOL") was dissolved in 50 mL of triethylamine (Wako Pure Chemical Industries, Ltd.), mixed with Solution A while cooling, and then the pH was adjusted to approximately 4.5 with 35% NaOH solution while cooling. 3.9 mL of 1286 U / mL phosphorylase T was added, and the solution was diluted to 1000 mL with water and incubated at 37°C for 26 hours.

[0042] Reaction solution composition Equol concentration: 1.5g / L (6.2mM) Polyphosphate concentration: 10% Triethylamine: 20% Kinase T: 5U / mL

[0043] As a result of HPLC analysis by UV detection, it was confirmed that 1.2 mM of phosphorylated equol was produced after the reaction (Figure 7).

[0044] Example 4 <Reconfirmation of equol phosphate> Using the reaction solution obtained in Example 3, phosphorylation of equol was confirmed by LC / MS / MS. Fragment analysis was performed from the mass spectrum of LC / MS / MS, and an attempt was made to identify the reaction product of Example 3 from its cleavage pattern. First, in the total ion chromatogram, two peaks appeared prominently after the reaction (Figures 8 and 9). They were designated as P-1 and P-2, respectively, and mass spectrum analysis was performed. From the fragment analysis pattern of LC / MS / MS, characteristic peaks of P-1 were 123.0, 187.0, and 213.0 (Figure 14). Considering the patterns shown in Figures 14 and 16 comprehensively, it was confirmed that P-1 corresponded to the peak of 4'-O-phosphorylated equol. Similarly, from the fragment analysis pattern shown in Figure 15, characteristic peaks of P-2 were 107.0, 186.7, and 202.9. Considering the patterns shown in Figures 15 and 16 comprehensively, it was confirmed that P-2 corresponded to the peak of 7-O-phosphorylated equol. From the above, it was confirmed that the reaction product of Example 3 was 4'-O-phosphorylated equol and 7-O-phosphorylated equol. The analysis conditions by LC / MS / MS are as follows.

[0045] ·LC / MS / MS analysis conditions <HPLC conditions> Column: SunShell PFP (manufactured by ChromaNik Technologies) 2.1φ×150mm (C-54) Eluent: (A) 0.1% formic acid aqueous solution (B) HPLC-grade MeOH Gradient conditions: (A) / (B) = 80 / 20 (5 min) → 15 min → 0 / 100 (10 min) Flow rate: 0.2 mL / min Oven Temperature: 40°C Detection: PDA (Signal 280 nm, Bandwidth 10 nm, Reference off, Peak width > 0.05 min, Slit 4 nm) Injection volume: 5 μL

[0046] Mass spectrometer: Quadrupole triple-stage LC / MS / MS instrument (manufactured by Micro Mass) Ion source: ESI-pos, neg Measurement mode: Scan analysis (m / z 50 - 1050) Capillary: 3.00 kV (pos, neg) Cone: 50 (pos), 80 (neg) RF Lens1: 20.0 (pos), 50.0 (neg) Desolvation Temp: 200°C Source Temp.: 80°C Cone Gas Flow: 75 L / hr Desolvation Gas Flow: 680 L / hr

[0047] <MS / MS Conditions (ESI-pos)> LM1 Resolution: 15.0 HM1 Resolution: 15.0 Ion Energy1: 0.5 Entrance: -2 Collision: 20 Exit: 0.1 LM2 Resolution: 15.0 HM2 Resolution: 15.0 Ion Energy2: 2.0 Multiplier: 650 V

Industrial Applicability

[0048] The present invention provides a novel phosphorylated equol and a method for producing the phosphorylated equol. The phosphorylated equol of the present invention has improved water solubility and improved absorption efficiency into the body compared to non-phosphorylated equol. It also has reduced bitterness. Therefore, the phosphorylated equol of the present invention is particularly useful in the food industry. Furthermore, the phosphorylated equol of the present invention is believed to have the same female hormone and antioxidant effects as equol, and is therefore useful for preventing and / or ameliorating diseases and symptoms such as menopausal disorders (menopausal complaints, osteoporosis, hyperlipidemia), osteoporosis, prostatic hyperplasia, and metabolic syndrome.

Claims

1. A composition comprising 4'-O-phosphoequol or a pharmaceutically acceptable salt thereof and 7-O-phosphoequol or a pharmaceutically acceptable salt thereof.

2. A pharmaceutical comprising the composition described in claim 1.

3. A food or beverage comprising the composition described in claim 1.

4. A food or beverage as described in claim 3, containing 0.01-100g, 0.1-10g, or 0.5-5g of the composition per 100g of food or beverage.

5. A food or beverage as described in claim 3 or 4, which is a food for specified health uses, a nutritional supplement, a functional food, a food for the sick, a food additive, or a supplement.

6. A cosmetic comprising the composition described in claim 1.

7. The cosmetic product according to claim 6, comprising 0.01-10g or 0.1-5g of the composition per 100g of the cosmetic product.

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