Method for producing phosphorylated equol using enzymes

Abstract

This invention provides a novel phosphorylated equol with enhanced solubility in water. [Solution] Phosphorylated equol represented by formula (1) or a pharmaceutically acceptable salt thereof. TIFF2026063030000006.tif34170 (wherein R1 is a phosphate group and R2 is a hydroxyl group)

Description

Technical Field

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

Background Art

[0002] Isoflavones exist in soybeans, for example, in the form of glycosides covalently bonded to sugars, such as daidzin, glycitin, and genistin, and only a very small amount exists in the form of aglycones. Some of these glycosides are further malonylated or acetylated. When these glycosides enter the human or animal body, they are converted into daidzein, glycitein, and genistein, respectively, by the action of digestive enzymes or enzymes produced by intestinal bacteria, such as β-glucosidase. Furthermore, it is known that daidzein is enzymatically converted to O-desmethylangolensin (O-DMA) or equol via dihydrodaidzein by the action of intestinal bacteria.

[0003] Equol is known to have the highest estrogenic activity among these metabolites (Non-Patent Documents 1 and 2). However, in humans, there are individual differences in the metabolism of isoflavones, and few people possess intestinal bacteria capable of fermenting daidzein to produce equol as described above. It has been revealed that the prevalence rate of such intestinal bacteria is about 50% in Japanese and about 30% in Westerners (Non-Patent Documents 3 and 4). Therefore, there has been a problem that people who do not possess equol-producing bacteria cannot produce equol in the body even when they ingest leguminous foods such as soybeans.

[0004] To overcome these challenges, attempts have recently been made to produce equol extracellularly using anaerobic microorganisms such as lactic acid bacteria (Patent Documents 1-4). However, equol has the problem of low solubility in water. If its solubility in water can be increased, the efficiency of equol absorption into the body can be improved, making it effective when included in foods and other products.

[0005] Equol glycosides are known as equol compounds 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 phosphorylation enzymes derived from microorganisms (Patent Document 7). There are also reports on equol with phosphorylation at position 7 and equol with phosphorylation at two positions, 7 and 4' (Patent Document 6). However, regarding equol with phosphorylation at position 7, Patent Document 6 does not disclose spectral data of the compound, and there is no evidence that such a compound has actually been synthesized. Furthermore, when phosphorylated equol is chemically synthesized, there are safety concerns regarding the catalysts and other materials used. 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] Japanese Patent Publication No. 2006-204296 [Patent Document 2] Special Publication 2006-504409 [Patent Document 3] Japanese Patent Publication No. 2008-61584 [Patent Document 4] Japanese Patent Publication No. 2010-104241 [Patent Document 5] WO2008 / 126752 [Patent Document 6] Special Publication 2007-511542 [Patent Document 7] Japanese 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) [Overview of the project] [Problems that the invention aims to solve]

[0008] This invention has been made in view of the above circumstances, and its purpose is to provide a novel phosphorylated equol in which only the carbon at the 4' position or the carbon at the 7th position is phosphorylated, and a method for producing said phosphorylated equol using an enzyme. [Means for solving the problem]

[0009] The inventors diligently conducted research to solve the above problems. Specifically, the inventors attempted to search for an enzyme that can phosphorylate only the 4' carbon or only the 7' carbon of equol from among various phosphorylation enzymes. As a result, the inventors found that by using phosphorylation enzymes T, Q, S, N, and L obtained from the Heat-Resistant Enzyme Research Institute Co., Ltd. [These enzymes can be obtained as Phosphorylation Enzyme (Heat-Resistant Enzyme Research Institute) T, Q, S, N, L respectively], it is possible to specifically phosphorylate the 4' carbon or the 7' carbon of equol. That is, the present invention provides equol in which only the 4' carbon or the 7' carbon is phosphorylated, and a method for producing said phosphorylated equol, and specifically 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] However, in the formula, either R1 or R2 is a hydroxyl group, and the other is a phosphate group. [3] A phosphorylated equol as described in [2] or a pharmaceutically acceptable salt thereof, wherein R1 is a phosphate group and R2 is a hydroxyl group. [4] Phosphorylated equol as described in [2] or a pharmaceutically acceptable salt thereof, 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 phosphorylation enzyme in the presence of a phosphate donor. [6] The method according to [5], wherein the phosphorylation enzyme is a phosphorylation enzyme that specifically phosphorylates only the carbon at the 4' position or only the carbon at the 7th position of equol, and the phosphorylated equol having one phosphate group is the phosphorylated equol represented by the following chemical formula (1). [ka] However, in the formula, either 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 phosphorylating enzyme is any one of the enzymes available under the names of phosphorylating enzyme T, phosphorylating enzyme Q, phosphorylating enzyme S, phosphorylating enzyme N or phosphorylating enzyme L from the Institute of Thermostable Enzymes. 〔8〕The method according to any one of 〔5〕to〔7〕, wherein the phosphate donor is polyphosphoric acid, 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〕, a pharmaceutically acceptable salt thereof, or the phosphorylated equol according to 〔11〕.

Advantages of the Invention

[0011] According to the present invention, a novel phosphorylated equol in which only the carbon at the 4'-position or the carbon at the 7-position is phosphorylated and a method for producing the same are provided. The phosphorylated equol provided by the present invention is expected to have an improved uptake efficiency into the body due to an improvement in water solubility and an improved bioavailability in oral ingestion. It can also be used for the purpose of reducing the bitterness of foods. Therefore, the phosphorylated equol of the present invention is useful in the food field and the like. Further, the phosphorylated equol of the present invention is considered to have the female hormone action and antioxidant action possessed by equol. Therefore, the phosphorylated equol of the present invention is also useful for the prevention and / or improvement of diseases and symptoms such as menopause disorder (menopausal syndrome, osteoporosis, hyperlipidemia), osteoporosis, prostatic hypertrophy, metabolic syndrome and the like.

Brief Description of the Drawings

[0012] [Figure 1] Figure 1 is a graph showing the amount of phosphorylated equol produced by phosphorylation enzymes A-G, I, J, and L-U. The vertical axis represents the amount produced, and the horizontal axis represents the enzyme used in the experiment. [Figure 2] Figure 2 shows the chromatogram of the reaction solution obtained using phosphorylation enzyme T. [Figure 3] Figure 3 shows the total ion chromatogram (ESI-neg) obtained from LC / MS analysis. [Figure 4] Figure 4 shows the total ion chromatogram (ESI-pos) obtained from LC / MS analysis. [Figure 5] Figure 5 shows the LC / MS mass spectrum (ESI-neg). [Figure 6] Figure 6 shows the mass spectrum (ESI-pos) of the LC / MS. [Figure 7] Figure 7 is a graph showing the progression of the reaction to produce phosphorylated equol. [Figure 8] Figure 8 shows the total ion chromatogram (ESI-neg) obtained from LC / MS / MS analysis. [Figure 9] Figure 9 shows the total ion chromatogram (ESI-pos) obtained from LC / MS / MS analysis. [Figure 10] Figure 10 shows the LC / MS / MS mass spectrum (ESI-neg, P-1). [Figure 11] Figure 11 shows the LC / MS / MS mass spectrum (ESI-pos, P-1). [Figure 12] Figure 12 shows the LC / MS / MS mass spectrum (ESI-neg, P-2). [Figure 13] Figure 13 shows the LC / MS / MS mass spectrum (ESI-pos, P-2). [Figure 14] Figure 14 shows the fragment analysis pattern (P-1) from LC / MS / MS. [Figure 15]Figure 15 shows the fragment analysis pattern (P-2) of LC / MS / MS. [Figure 16] Figure 16 shows the phosphorylated equol that can be inferred from the fragment analysis pattern. [Modes for carrying out 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), either R1 or R2 is a hydroxyl group, and the other is a phosphate group.

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

[0015] Equol, or 4'-O-phosphoequol, in which the carbon at the 4' position is phosphorylated, is represented by the following chemical formula (2). [ka]

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

[0017] In the present invention, examples of pharmaceutically acceptable salts include 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; as well as ammonium salts.

[0018] The phosphorylated equol of the present invention can be produced by a method comprising the step of contacting equol with a phosphorylation enzyme 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, as a method for producing equol using anaerobic microorganisms capable of producing equol, daidzein is fermented by anaerobic microorganisms in a gas phase consisting of one or more gases including hydrogen, and equol is recovered (Patent Document 7 / JP 2002-238594)). Alternatively, commercially available equol from suppliers such as DAIEL CHIRAL THCHNOLOGIES (CHINA) Co., Ltd. can be used.

[0019] Furthermore, the phosphorylation enzymes of the present invention are preferably enzymes that can specifically phosphorylate only the carbon at the 4' position or only the carbon at the 7th position of equol. Examples of such enzymes include, but are not limited to, phosphorylation enzymes T, Q, S, N, and L, which are available from the Heat-Resistant Enzyme Research Institute Co., Ltd. The Heat-Resistant Enzyme Research Institute Co., Ltd. (address: 5-2, Minamimachi 5-chome, Chuo-ku, Kobe City, website address: http: / / www.tainetsu.com / ) manages and stores these enzymes in association with their names. Therefore, phosphorylation enzymes T, Q, S, N, and L of the present invention can be obtained by informing the Heat-Resistant Enzyme Research Institute of the name of the enzyme you wish to obtain.

[0020] The phosphorylation enzymes of the present invention also include homologs of these phosphorylation enzymes. In the present invention, homologs of phosphorylation enzyme T, phosphorylation enzyme Q, phosphorylation enzyme S, phosphorylation enzyme N, and phosphorylation enzyme L mean proteins that include 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 that have the activity to specifically phosphorylate 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 generally it is within 50 amino acids, preferably within 30 amino acids, more preferably within 10 amino acids (e.g., within 5 amino acids, within 3 amino acids). 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 the entire amino acid sequence is permitted. In other words, proteins containing amino acid sequences that have homology to the amino acid sequences of phosphorylation enzyme T, phosphorylation enzyme Q, phosphorylation enzyme S, phosphorylation enzyme N, and phosphorylation enzyme L, preferably 80% or more, more preferably 90% or more (for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more), are also included in the phosphorylation enzymes of the present invention.

[0021] Generally, to maintain protein function, it is preferable that the substituted amino acid has properties similar to the original amino acid. Such amino acid substitutions 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 permissible.

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

[0023] In the present invention, the following conditions can be used as reaction conditions for equol and phosphorylation enzymes, but are not limited to these. Reaction temperature: preferably 4-60°C, more preferably 15-40°C, and more preferably 36-38°C. pH: Preferably 2 to 12, more preferably pH 4 to 6, and more preferably pH 4.4 to 4.6. • Equol concentration: preferably 1 to 20 mM, more preferably 4 to 10 mM, and more preferably 4.5 to 6.5 mM. • Concentration of phosphorylation enzyme: preferably 10 to 1000 μg / mL, more preferably 100 to 700 μg / mL, and more preferably 300 to 500 μg / mL. • Amount of phosphate donor: preferably 1-20%, more preferably 5-15%, more preferably 8-12%. In this invention, "contact" can be replaced with terms such as "mixing" or "addition."

[0024] The present invention provides a method for producing phosphorylated equol, which may include an additional step of recovering the generated phosphate equol. Phosphorylated equol can be recovered, for example, by the following method: The solution from which equol has been enzymatically decontaminated is heated at 70 degrees Celsius for 30 minutes, for example, according to a conventional method, then returned to room temperature, and insoluble matter is removed by centrifugation. The supernatant can then be recovered. This supernatant is concentrated under reduced pressure using a rotary evaporator, the pH is adjusted to around 3, and an equal volume of methanol is added and mixed well. After passing this solution through a silica gel column, a water / methanol = 2 / 8 solution is passed through to recover the fraction. The recovery of the fraction containing phosphorylated equol can be performed by referring to the UV absorption at 280 nm and the results of HPLC analysis. Furthermore, the recovered solution can be concentrated under reduced pressure using a rotary evaporator and dried to obtain a solidified product.

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

[0026] Furthermore, the present invention relates to phosphorylated equol obtained by the above-described method for producing phosphorylated equol. Specifically, it relates to phosphorylated equol obtained by a method that includes a step of contacting equol with a phosphorylation enzyme. The phosphorylated equol obtained by this method is a novel phosphorylated equol in which only the carbon at the 4' position or only the carbon at the 7th position is specifically phosphorylated, as described above.

[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 a material for pharmaceuticals, food and beverages, cosmetics, etc. Compared with an unphosphorylated equol-containing composition, the phosphorylated equol-containing composition of the present invention is expected to have improved uptake efficiency into the body. Examples of phosphorylated equol include, but are not limited to, equol in which the carbon at the 4' position is phosphorylated (4'-O-phosphoequol) and equol in which the carbon at the 7' position is phosphorylated (7-O-phosphoequol).

[0028] When providing the phosphorylated equol-containing composition of the present invention as a pharmaceutical product, the 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 formulations can be formulated according to conventional methods using, as necessary, fillers, bulking agents, excipients, binders, humectants, disintegrants, surfactants, lubricants, colorants, flavoring agents, solubilizers, suspensions, coatings, and other known auxiliary agents that are commonly used in the pharmaceutical formulation technology. Furthermore, these pharmaceutical formulations may contain colorants, preservatives, fragrances, flavoring agents, 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 food or beverage, it can be used not only as a general food, but also as 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 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., containing the phosphorylated equol-containing composition of the present invention.

[0030] The phosphorylated equol-containing composition of the present invention can be used as a main component of water, protein, carbohydrates, lipids, vitamins, minerals, organic acids, organic bases, fruit juice, flavors, etc. Examples of proteins include whole milk powder, skim milk powder, partially skim milk powder, casein, soy protein, chicken egg protein, meat protein, and other animal and plant proteins, as well as their hydrolysates, butter, etc. Examples of carbohydrates include sugars, modified starch (including dextrin, soluble starch, British starch, oxidized starch, starch esters, starch ethers, etc.), and dietary fiber. Examples of lipids include animal fats and oils such as lard, fish oil, fractionated oils therefrom, hydrogenated oils, and transesterified oils, and vegetable fats and oils such as palm oil, safflower oil, corn oil, rapeseed oil, coconut oil, fractionated oils therefrom, hydrogenated oils, and transesterified oils. Examples of vitamins include vitamin A, carotenes, B vitamins, vitamin C, vitamin D, vitamin E, vitamin K, 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 large amounts of these components may also be used.

[0031] The proportion of phosphorylated equol-containing compositions 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 consuming it, and the expected effects. As an example, a proportion of 0.01-100g, preferably 0.1-10g, and more preferably 0.5-5g per 100g of food can be given, but is not limited to these. The daily intake of food containing a phosphorylated equol-containing composition will vary depending on the content of phosphorylated equol in the composition, the age and weight of the person consuming it, the number of times it is consumed, etc., but for example, an amount equivalent to 0.1-10g of the composition per day for an adult can be given.

[0032] Furthermore, when providing the phosphorylated equol-containing composition of the present invention as a cosmetic, the composition can be prepared into various desired forms of cosmetics, such as liquids like aqueous solutions, lotions, sprays, suspensions, and emulsifiers; solids like powders, granules, and blocks; semi-solids like creams and pastes; and gels. Such cosmetics are useful as various cosmetic materials such as facial cleansers, lotions, creams, gels, essences (serums), packs and masks, makeup cosmetics like foundations and lipsticks, oral cosmetics, fragrance cosmetics, hair cosmetics, and body cosmetics. The phosphorylated equol-containing composition of the present invention can be sealed in a suitable container such as a bottle, bag, can, spray can, atomizer, box, or pack, as needed.

[0033] In cosmetics containing the phosphorylated equol-containing composition of the present invention, the proportion of the composition in the cosmetic is not particularly limited. It can be appropriately set depending on the type of cosmetic, the content of phosphorylated equol, etc., but as an example, the proportion of the composition (on a dry weight basis) is 0.01-10g, preferably 0.1-5g, per 100g of cosmetic, but is not limited to these. All prior art documents cited herein are incorporated herein 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> To a polyphosphate solution (ACROS Organics), 5 mL of water was added 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., trade name "(S)-EQUOL") was dissolved in 5 mL of triethylamine (Wako Pure Chemical Industries, Ltd.), mixed with Solution A, and the pH was adjusted to around 4.5 using a 35% NaOH solution while cooling, 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 phosphorylation enzyme solution (phosphorylation enzymes A to U, manufactured by Tainetetsu Co., Ltd., website address: http: / / www.tainetsu.com / document.php?pid=1) was added, and the mixture was reacted at 37°C for 24 hours. After the reaction, 0.05 mL of the solution was mixed with 0.45 mL of HPLC mobile phase to obtain the HPLC sample. The reaction mixture and HPLC analysis conditions are shown below.

[0035] • Composition of reaction solution 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 (280nm) 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 considered 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 that it was phosphorylated equol with one phosphate group attached to equol based on the molecular weight of the compound. 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 for 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) Desolution Temp: 200℃ Source Temp: 80℃ Cone Gas Flow: 66 L / hr Desolvation Gas Flow: 690 L / hr

[0041] Example 3: Production of equol phosphorus oxide (1L scale) To a polyphosphate solution (Wako Pure Chemical Industries, Ltd.), 200 mL of water was added while cooling, then 150 mL of triethylamine (Wako Pure Chemical Industries, Ltd.) was added while cooling, and the pH was adjusted to around 3.0 using a 35% NaOH solution while cooling (Solution A). 1.5 g of equol (DAICEL CHIRAL THCHNOLOGIES (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 the pH was adjusted to around 4.5 using a 35% NaOH solution while cooling. 3.9 mL of phosphorylation enzyme T1286 U / mL was added, the volume was made up to 1000 mL with water, and the mixture was reacted at 37°C for 26 hours.

[0042] • Composition of reaction solution Equol concentration: 1.5g / L (6.2mM) Polyphosphate concentration: 10% Triethylamine: 20% Phosphorylation enzyme T: 5 U / 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 123.0, 187.0, and 213.0 were obtained as the 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, characteristic peaks of 107.0, 186.7, and 202.9 were obtained as the 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 × 150 mm (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] This invention provides a novel phosphorylated equol and a method for producing said phosphorylated equol. Compared to unphosphorylated equol, the phosphorylated equol of this invention has improved solubility in water and improved absorption efficiency into the body. Furthermore, its bitterness is reduced. Therefore, the phosphorylated equol of this invention is particularly useful in the food industry. Furthermore, the phosphorylated equol of the present invention is thought to possess the estrogen-like and antioxidant effects of equol. Therefore, the phosphorylated equol of the present invention is useful for the prevention and / or improvement of diseases and symptoms such as menopausal disorders (menopausal symptoms, osteoporosis, hyperlipidemia), osteoporosis, benign 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 product comprising the composition described in claim 1.

3. Food and beverages comprising the composition described in claim 1.

4. The food or beverage according to claim 3, comprising 0.01-100g, 0.1-10g, or 0.5-5g of the composition per 100g of food or beverage.

5. Foods and beverages according to claim 3 or 4, which are Foods for Specified Health Uses, nutritional supplements, functional foods, foods for sick people, food additives, or supplements.

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

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

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