Method for producing phosphorylated equol using enzyme

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] For example, in soybeans, isoflavones exist as glycosides covalently bound to sugars, such as daidzin, glycitin, and genistin, and only small amounts of aglycones exist. Some of these glycosides are further malonylated or acetylated. When these glycosides enter the human or animal body, they are converted to daidzein, glycitein, and genistein, respectively, by the action of digestive enzymes or β-glucosidase, an enzyme produced by intestinal bacteria. Furthermore, it is known that daidzein is enzymatically converted by the action of intestinal bacteria to dihydrodaidzein, and then to O-desmethylangolensin (O-DMA) or equol.

[0003] Of these metabolic products, 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, only a few people possess intestinal bacteria that have the ability to ferment daidzein and 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] In order to overcome these problems, in recent years, attempts have been made to produce equol externally using anaerobic microorganisms such as lactic acid bacteria (Patent Documents 1 to 4). However, equol has a problem of low solubility in water. If the solubility in water could be increased, the efficiency of equol intake into the body could be increased, which would be effective when it is added to foods, etc.

[0005] Equol glycoside is known as an equol compound with increased solubility in water (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 phosphorylating enzyme derived from a microorganism (Patent Document 7). There are also reports on equol phosphorylated at the 7th carbon and equol phosphorylated at two carbons, the 7th and 4' positions (Patent Document 6). However, Patent Document 6 does not disclose any spectral data for equol phosphorylated at the 7th carbon, 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 demand for the establishment of 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 table 2006-504409 [Patent Document 3] Patent Publication No. 2008-61584 [Patent Document 4] Patent Publication 2010-104241 [Patent Document 5] WO2008 / 126752 [Patent Document 6] Special table 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 has been made in consideration of these circumstances, and its object is to provide a novel phosphorylated equol in which only the 4' carbon or the 7th 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 problems. Specifically, the present inventors have attempted to search for an enzyme that can phosphorylate only the 4' carbon or only the 7' carbon of equol from among various kinases. As a result, the present inventors have found 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 Heat-Stable Enzyme Laboratory Co., Ltd. [these enzymes are available as Phosphorylation Enzyme (Heat-Stable Enzyme Laboratory) T, Q, S, N, and L, respectively.]. That is, the present invention provides equol in which only the 4' carbon or the 7 carbon is phosphorylated, and a method for producing the phosphorylated equol, and specifically provides the following [1] to

[12] .

[0010] [1] Phosphorylated equol having one phosphate group or a pharma- ceutically acceptable salt thereof. [2] Phosphorylated equol represented by the following chemical formula (1) or a pharma- ceutically 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 pharma- ceutically acceptable salt thereof according to [2], wherein R1 is a phosphate group and R2 is a hydroxyl group. [4] The phosphorylated equol or a pharma- ceutically 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 Institute under the name 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 pharma- ceutically acceptable salt thereof, or the phosphorylated equol according to

[11] . Effect 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, which improves its efficiency of absorption into the body, and to improve its bioavailability when orally ingested. It can also be used to reduce the bitterness of foods. Thus, the phosphorylated equol of the present invention is useful in the food industry and the like. The phosphorylated equol of the present invention is also believed to have the female hormone and antioxidant effects that equol has, and is therefore useful for preventing and / or improving diseases and symptoms such as menopausal disorders (menopausal indefinite symptoms, osteoporosis, hyperlipidemia), osteoporosis, prostatic hyperplasia, and metabolic syndrome. [Brief description 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. [Diagram 2] FIG. 2 shows a chromatogram of the reaction solution obtained using kinase T. [Diagram 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 the LC / MS analysis. [Diagram 5] FIG. 5 shows the LC / MS mass spectrum (ESI-neg). [Figure 6] FIG. 6 shows the mass spectrum (ESI-pos) of LC / MS. [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 mass spectrum (ESI-neg, P-1) of LC / MS / MS. [Figure 11] FIG. 11 shows the mass spectrum (ESI-pos, P-1) of LC / MS / MS. [Figure 12] FIG. 12 shows the mass spectrum (ESI-neg, P-2) of LC / MS / MS. [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 is suspected from the fragment analysis pattern. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[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 pharma- ceutically 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 referred to as equol phosphorylated at the 4' carbon or 4'-O-phosphoequol. Meanwhile, the latter phosphorylated equol can be referred to as equol phosphorylated at the 7 carbon or 7-O-phosphoequol.

[0015] Equol with the 4´ carbon 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, examples of pharma- ceutically 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 of 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 recovering equol (Patent Document 7 / JP 2002-238594 A)). Alternatively, equol commercially available from suppliers such as DAICEL CHIRAL THCHNOLOGIES (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 available from Heat-Stable Enzyme Laboratory Co., Ltd. Heat-Stable Enzyme Laboratory Co., Ltd. (Address: 5-5-2 Minatojima Minamimachi 5-chome, Chuo-ku, Kobe City, Website address: http: / / www.tainetsu.com / ) manages and stores these enzymes in association with their names. Therefore, the kinases T, Q, S, N, and L of the present invention can be obtained by informing Heat-Stable Enzyme Laboratory of the name of the enzyme desired to be obtained.

[0020] The kinase of the present invention also includes homologues of these kinases. In the present invention, the homologues of kinase T, kinase Q, kinase S, kinase N, and kinase L refer to proteins that contain an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, and / or added to the amino acid sequence of these enzymes, and have the activity of specifically phosphorylating only the 4' carbon or only the 7th carbon of equol. The number of amino acids to be modified is not particularly limited as long as the modified protein has the above-mentioned physicochemical properties, but is generally 50 amino acids or less, preferably 30 amino acids or less, and more preferably 10 amino acids or less (e.g., 5 amino acids or less, 3 amino acids or less). Alternatively, modification of, for example, 20% or less, specifically 10% or less (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% or less) of amino acid residues in the entire amino acid sequence is permitted. In other words, proteins containing an amino acid sequence that has a homology of preferably 80% or more, more preferably 90% or more (e.g., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more) 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] In general, in order to maintain the function of a protein, it is preferable that the amino acid to be substituted is an amino acid having similar properties to the amino acid before substitution. Such substitution of amino acid residues is called conservative substitution. For example, Ala, Val, Leu, Ile, Pro, Met, Phe, and Trp are all classified as non-polar amino acids, and therefore have similar properties to each other. In addition, examples of uncharged amino acids include Gly, Ser, Thr, Cys, Tyr, Asn, and Gln. In addition, examples of acidic amino acids include Asp and Glu. In addition, 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, the phosphate donor may be, but is not limited to, polyphosphate, ATP, phosphoenolpyruvate, creatine phosphate, etc. In the present invention, polyphosphate refers to phosphate (PO 4 The polyphosphate of the present invention includes polyphosphates having a linear structure and polyphosphates having a cyclic structure.

[0023] In the present invention, the reaction conditions for equol and kinase can 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 pH 4 to 6, and even more preferably pH 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 1-20%, more preferably 5-15%, even more preferably 8-12%. In the present invention, "contact" can also be expressed as "mixing" or "adding".

[0024] The method for producing phosphorylated equol of the present invention may additionally include a step of recovering the produced equol phosphate. Phosphorylated equol can be recovered, for example, by the following method. The liquid in which equol has been enzyme-reduced is heated, for example, in a conventional manner, at 70°C for 30 minutes, then cooled to room temperature, and insoluble matter is removed by centrifugation. This allows the recovery of a supernatant. This supernatant is concentrated under reduced pressure using a rotary evaporator, the pH is adjusted to about 3, and an equal amount of methanol is added and mixed well. This liquid is passed through a silica gel column, and then water / methanol=2 / 8 is passed through to recover the fraction. The recovery of the fraction containing phosphorylated equol can be performed with reference to UV absorption at 280 nm and the results of HPLC analysis. The recovered liquid is then concentrated under reduced pressure using a rotary evaporator and dried to obtain a solidified product.

[0025] Furthermore, a person skilled in the art can identify equol in which only the 4' carbon or the 7th carbon is phosphorylated 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 pharma- ceutically acceptable salt thereof (hereinafter referred to as phosphorylated equol-containing composition). The composition of the present invention can be provided as a material for medicines, foods and 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 product, its dosage form can be selected according to the disease to be prevented or treated, the form of use of the pharmaceutical product, the route of administration, 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 according to conventional methods using known auxiliary agents that can be commonly used in the pharmaceutical formulation technical field, such as fillers, extenders, excipients, binders, moisturizing agents, disintegrants, surfactants, lubricants, colorants, flavoring agents, dissolution aids, suspensions, coating agents, etc., in addition to the main drug as necessary. In addition, colorants, preservatives, fragrances, flavoring agents, sweeteners, and other pharmaceutical products may be contained in the pharmaceutical preparations. 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 food or drink, it can be used as general food, as well as specific health foods, nutritional supplements, functional foods, foods for the sick, food additives, supplements, etc. Examples of the form of food that contains the phosphorylated equol-containing composition of the present invention 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 use water, protein, carbohydrate, lipid, vitamins, minerals, organic acids, organic bases, fruit juice, flavors, etc. as main components. Examples of 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. Examples of carbohydrates include sugars, processed starch (textlin, soluble starch, British starch, oxidized starch, starch ester, starch ether, etc.), and dietary fiber. Examples of lipids include animal fats and oils such as lard, fish oil, and their fractionated oils, hydrogenated oils, and transesterified oils, and vegetable fats and oils such as palm oil, safflower oil, corn oil, rapeseed oil, coconut oil, and their fractionated oils, hydrogenated oils, and transesterified oils. Examples of vitamins include vitamin A, carotenes, vitamin B group, vitamin C, vitamin D group, vitamin E, vitamin K group, 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 components can be used in combination, and synthetic products and / or foods containing a large amount of these may be used.

[0031] The blending ratio of the phosphorylated equol-containing composition in these foods can be appropriately set depending on the type of food, the content of phosphorylated equol, the age and sex of the person taking it, the expected effect, etc. One example is a ratio of 0.01-100g, preferably 0.1-10g, and more preferably 0.5-5g per 100g of food, but is not limited to these. The daily intake amount of food containing a composition containing phosphorylated equol varies depending on the content of phosphorylated equol in the composition, the age and weight of the person taking it, the number of times it is taken, etc., but an example is an amount of the 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 product, the composition can be prepared into various desired cosmetic 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, gels, etc. Such cosmetics are useful as various types of cosmetics, such as face washes, milky lotions, creams, gels, essences (beauty serums), packs and masks, makeup cosmetics such as foundations and lipsticks, oral cosmetics, fragrance cosmetics, hair cosmetics, body cosmetics, etc. 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 necessary.

[0033] In cosmetics containing the phosphorylated equol-containing composition of the present invention, the blending ratio of the composition in the cosmetics is not particularly limited. It can be appropriately set depending on the type of the cosmetic, the content of phosphorylated equol, etc., but an example is a ratio in which the total amount of the composition (calculated as dry weight) is 0.01-10 g, preferably 0.1-5 g, per 100 g of cosmetic, but is not limited thereto. All prior art documents cited in this specification are hereby incorporated by reference. EXAMPLES

[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 the polyphosphoric acid solution (ACROS Organics) while cooling, and then 5 mL of triethylamine (Wako Pure Chemical Industries, Ltd.) was added while cooling (Solution A). Equol (DAICEL CHIRAL TECHNOLOGIES (CHINA) CO., LTD., product name "(S)-EQUOL") was dissolved in 5 mL of triethylamine (Wako Pure Chemical Industries, Ltd.) and mixed with Solution A. The pH was then adjusted to around 4.5 while cooling using 35% NaOH solution, and the total volume was made up to 47.5 mL with water (Solution B). To 0.95 mL of solution B, 0.05 mL of each of the kinase solutions (Kinase A to Kinase U, manufactured by Heat-Stable Enzyme Laboratory Co., Ltd., website address: http: / / www.tainetsu.com / document.php?pid=1) was added, and the mixture was reacted for 24 hours at 37° C. After the reaction, 0.05 mL of the solution was mixed with 0.45 mL of HPLC mobile phase to prepare an HPLC sample. The reaction composition and HPLC analysis conditions are shown below.

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

[0036] ·HPLC analysis conditions Column: YMC-Pack ODS-A (150×4.6mm 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 the 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, a peak was 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 was consistent with phosphorylated equol with one phosphate group attached to equol. The analysis conditions of LC / MS 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) Decomposition Temp.: 200℃ Source Temp.: 80℃ Cone Gas Flow: 66L / hr Desolvation Gas Flow: 690L / hr

[0041] Example 3 <Production of Equol Phosphate (1 L Scale)> 200mL of water was added to the polyphosphoric acid solution (Wako Pure Chemical Industries, Ltd.) while cooling, then 150mL of triethylamine (Wako Pure Chemical Industries, Ltd.) was added while cooling, and the pH was adjusted to about 3.0 with 35% NaOH solution while cooling (Solution A). 1.5g of equol (DAICEL CHIRAL THCHNOLOGIES (CHINA) co., LTD., product name "(S)-EQUOL") was dissolved in 50mL of triethylamine (Wako Pure Chemical Industries, Ltd.), mixed with Solution A while cooling, and the pH was adjusted to about 4.5 with 35% NaOH solution while cooling. 3.9mL of phosphorylation enzyme T1286U / mL was added, and the solution was made up to 1000mL with water, and reacted at 37℃ 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 was completed (Figure 7).

[0044] Example 4 <Reconfirmation of Equolic Acid> The reaction solution obtained in Example 3 was used to confirm phosphorylated equol 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, 123.0, 187.0, and 213.0 were obtained as characteristic peaks of P-1 (Figure 14). Considering the patterns shown in Figures 14 and 16 comprehensively, it was confirmed that P-1 was the peak corresponding to 4'-O-phosphorylated equol. Similarly, from the fragment analysis pattern shown in Figure 15, 107.0, 186.7, and 202.9 were obtained as characteristic peaks of P-2. Considering the patterns shown in Figures 15 and 16 comprehensively, it was confirmed that P-2 was the peak corresponding to 7-O-phosphorylated equol. From the above, it was confirmed that the reaction products of Example 3 were 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 said 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. In addition, it has reduced bitterness. Therefore, the phosphorylated equol of the present invention is particularly useful in the food industry. The phosphorylated equol of the present invention is also believed to have the female hormone and antioxidant effects that equol has, and is therefore useful for preventing and / or improving diseases and symptoms such as menopausal disorders (menopausal indefinite symptoms, osteoporosis, hyperlipidemia), osteoporosis, prostatic hyperplasia, and metabolic syndrome.

Claims

1. 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, the method, wherein the kinase is a kinase that specifically phosphorylates only the 7-carbon atom of equol, and the phosphorylated equol having one phosphate group is represented by the following chemical formula (1): 【Chemistry 1】 In the formula, R1 is a hydroxyl group and R2 is a phosphate group.

2. 2. The method of claim 1, wherein the phosphate donor is polyphosphate, ATP, phosphoenolpyruvate, or creatine phosphate.

3. A method for screening a kinase, comprising the steps of: (a) contacting a test kinase with a solution containing equol in the presence of a phosphate donor; and (b) selecting a kinase that produces phosphorylated equol having one phosphate group from among the test kinases; And, the method, wherein the phosphorylated equol having one phosphate group is phosphorylated equol represented by the following chemical formula (1): 【Chemistry 2】 In the formula, R1 is a hydroxyl group and R2 is a phosphate group.

4. 4. The method of claim 3, wherein the phosphate donor is polyphosphate, ATP, phosphoenolpyruvate, or creatine phosphate.

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