WO2024007901A1 - Method for preparing oxyresveratrol - Google Patents

Method for preparing oxyresveratrol Download PDF

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WO2024007901A1
WO2024007901A1 PCT/CN2023/102920 CN2023102920W WO2024007901A1 WO 2024007901 A1 WO2024007901 A1 WO 2024007901A1 CN 2023102920 W CN2023102920 W CN 2023102920W WO 2024007901 A1 WO2024007901 A1 WO 2024007901A1
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Prior art keywords
enzymatic hydrolysis
morin
oxidized resveratrol
resveratrol
oxidized
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PCT/CN2023/102920
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French (fr)
Chinese (zh)
Inventor
张誉荠
周戟
李从严
周雄武
黄灿
罗慧
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云南英格生物技术有限公司
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Publication of WO2024007901A1 publication Critical patent/WO2024007901A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/22—Preparation of oxygen-containing organic compounds containing a hydroxy group aromatic
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/33—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
    • A61K8/34—Alcohols
    • A61K8/347—Phenols
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C37/00—Preparation of compounds having hydroxy or O-metal groups bound to a carbon atom of a six-membered aromatic ring
    • C07C37/68—Purification; separation; Use of additives, e.g. for stabilisation
    • C07C37/70—Purification; separation; Use of additives, e.g. for stabilisation by physical treatment
    • C07C37/82—Purification; separation; Use of additives, e.g. for stabilisation by physical treatment by solid-liquid treatment; by chemisorption
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

  • the invention belongs to the technical field of medicinal chemistry, and specifically relates to a preparation method of oxidized resveratrol.
  • oxidized resveratrol in the existing technology is mainly carried out by traditional extraction and purification methods.
  • content of oxidized resveratrol in plants is low and the source range is narrow, it is difficult to effectively obtain oxidized resveratrol using traditional extraction methods. alcohol.
  • CN103194493A describes a method for preparing oxidized resveratrol by using mulberry branch powder as the fermentation substrate and Aspergillus niger as the fermentation strain to carry out fermentation. This method has a long time period from strain activation to the end of fermentation, and the operation It is complex, and other impurities are easily produced during microbial fermentation, making separation and purification difficult.
  • CN104803830A describes a process for preparing high-purity oxidized resveratrol by repeatedly extracting, extracting, and decolorizing organic reagents such as ethanol and ethyl acetate.
  • the process uses organic reagents such as ethyl acetate and petroleum ether. It is more harmful to the environment and operators, and the operation is more cumbersome.
  • the object of the present invention is to provide a safe, low-cost, simple and efficient method for preparing oxidized resveratrol.
  • a first aspect of the invention provides a method for preparing oxidized resveratrol, comprising the steps:
  • the enzymatic hydrolysis rate of morin A is ⁇ 60%, preferably ⁇ 70%, more preferably ⁇ 80%, and most preferably ⁇ 90%.
  • step (S2) the enzymatic hydrolysis rate of morin A is 60-97%, preferably 70-96%, 80-95%.
  • step (S2) enzymatic hydrolysis treatment is performed in an aqueous phase system.
  • the solvent of the aqueous phase system is water, or a water/ethanol mixed solvent with an ethanol content of ⁇ 10% (preferably ⁇ 5wt%, more preferably ⁇ 1wt%).
  • step (S2) compared with the raw material containing morin A before enzymatic hydrolysis, the relative increase of oxidized resveratrol in the enzymatic hydrolysis product is ⁇ 300%, preferably Ground ⁇ 400%, more preferably ⁇ 500%, most preferably ⁇ 600%.
  • step (S2) the relative increase amplitude of oxidized resveratrol is 50-2500%, preferably 100-2000%, more preferably 500-2000%.
  • step (S2) in the enzymatic hydrolysis product, the concentration of oxidized resveratrol is ⁇ 0.15 mg/g, preferably ⁇ 0.30 mg/g, preferably ⁇ 0.50 mg/g.
  • the concentration of oxidized resveratrol in the enzymatic hydrolysis product is 0.10-0.80 mg/g, preferably 0.2-0.7 mg/g, preferably 0.3-0.6 mg/g.
  • step (S2) in the enzymatic hydrolysis product, the concentration of morin A is ⁇ 0.20 mg/g, preferably ⁇ 0.15 mg/g, preferably ⁇ 0.10 mg/g.
  • the concentration of morin A in the enzymatic hydrolysis product is 0.05-0.20 mg/g, preferably 0.06-0.15 mg/g, preferably 0.08-0.10 mg /g.
  • the concentration of morrin A is 0.30-2.00 mg/g, preferably 0.40-1.80 mg/g. .
  • the concentration of oxidized resveratrol in the raw material containing morin A is ⁇ 0.10 mg/g, preferably ⁇ 0.08 mg/g, Preferably ⁇ 0.06mg/g.
  • the raw material containing morin A is a water extract, ethanol extract, or ethanol/water extract of mulberry branches or mulberry bark medicinal materials.
  • the raw material containing morin A is a plant extract, preferably an extract of a Moraceae plant.
  • the extract includes: extracts of roots, stems, leaves, and fruits.
  • the plant extract includes: mulberry branch extract, mulberry white bark extract, or a combination thereof.
  • the raw material containing morin A is an extract of mulberry branches or mulberry white bark medicinal materials.
  • step (S2) the ratio of the dosage E1 of ⁇ -glucanase to the weight W1 of morin A (E1/W1) is 02-25, preferably 6-20, More preferably 6-15, such as about 10.
  • step (S2) the enzymatic hydrolysis time is 0.5-3 hours, preferably 1-2 hours.
  • the method further includes the steps of:
  • step (S3) Separate oxidized resveratrol from the enzymatic hydrolysis product subjected to enzymatic hydrolysis treatment in step (S2) to obtain separated crude oxidized resveratrol.
  • the method further includes the steps of:
  • the separation uses macroporous adsorption resin to separate and oxidize resveratrol.
  • the macroporous resin resin model is: AB-8, D101, DM301, or a combination thereof.
  • the conditions for purification of the macroporous adsorption resin include: sample loading, water washing, ethanol with a mass percentage concentration of 30% to 85%, etc.
  • step (S2) ⁇ -glucanase is used for enzymatic hydrolysis to obtain an enzymatic hydrolysis solution (or enzymatic hydrolysis product) containing oxidized resveratrol.
  • step (S3) the oxidized resveratrol is separated from the enzymatic hydrolysis solution.
  • the method further includes:
  • the prepared oxidized resveratrol is added to the final product as an antioxidant or whitening additive.
  • a second aspect of the present invention provides an oxidized resveratrol prepared by the method of claim 1.
  • a third aspect of the present invention provides the use of a ⁇ -glucanase, which is used to enzymatically hydrolyze morrin A, thereby converting morrin A into oxidized white sugar. veratrol, or
  • the ⁇ -glucanase is used to prepare an enzyme preparation, which is used to enzymatically hydrolyze morin A, thereby converting morin A into oxidized resveratrol.
  • Figure 1 shows the chemical structural formula of morin A.
  • Figure 2 shows the chemical structural formula of oxidized resveratrol.
  • Figure 3 shows a process flow diagram of the present invention.
  • Figure 4 shows a schematic diagram of the enzymatic hydrolysis of the present invention.
  • Figure 5 shows the effects of different types of enzymes on the enzymatic hydrolysis rate of morin A and the increase in oxidized resveratrol.
  • Figure 6 shows the HPLC detection chart of the content of enzymatically hydrolyzed components of ⁇ -glucanase of the present invention.
  • Figure 7 shows the effects of different enzymatic hydrolysis times on the enzymatic hydrolysis rate of morin A and the increase in oxidized resveratrol.
  • Figure 8 shows the effects of different enzyme addition amounts on the enzymatic hydrolysis rate of morin A and the increase in oxidized resveratrol.
  • Figure 9 shows the effect of the extract prepared in the present invention on inhibiting tyrosinase.
  • Figure 10 shows that the extract prepared by the present invention can effectively scavenge DPPH (1,1-diphenyl-2-picrylhydrazyl) free radicals.
  • the present invention unexpectedly discovered an enzymatic bioconversion method that can significantly increase the production content of oxidized resveratrol.
  • the present invention also unexpectedly discovered that the highest conversion rate of oxidized resveratrol can be obtained by using ⁇ -glucanase to enzymatically hydrolyze morin A. On this basis, the present invention was completed.
  • Mulberroside A is a dihydroxystilbene compound, also known as oxidized resveratrol glycoside. It mainly exists in Moraceae and Morus plants. Its chemical structure formula (see Figure 1) is as follows:
  • morin A Compared with oxidized resveratrol (see Figure 2), morin A has two more sugar groups connected through glycosidic bonds.
  • Morrin A is worse than oxidized resveratrol in terms of antioxidant and inhibitory tyrosinase activity, especially in inhibiting tyrosinase activity.
  • the IC50 value of the inhibitory rate of tyrosinase by morin A is 53.6 ⁇ M, while the IC50 value of oxidized resveratrol is only 0.49 ⁇ M. The difference between the two is more than 100 times.
  • Oxidized resveratrol chemically named 2,4,3',5'-tetrahydroxystilbene, is a stilbene derivative of stilbene and is mainly found in plants of the genus Morus and Jackfruit. Its chemical structural formula is as follows:
  • oxidized resveratrol has a strong inhibitory effect on tyrosinase activity, thereby inhibiting melanin production.
  • oxidized resveratrol is the most promising dietary functional compound with anti-browning, whitening and other effects. Its oral drugs or related foods are safe and low-toxic.
  • oxidized resveratrol has few toxic side effects, large market demand, low content in plants, complex extraction processes, and narrow sources, it is expensive and severely restricts its use in cosmetics, natural medicines, health foods, and Development and utilization of functional drinks.
  • a method for preparing a product with high content of oxidized resveratrol is provided.
  • the method of the present invention includes the steps:
  • the present invention provides a method for preparing a product containing high content of oxidized resveratrol, which includes the following steps:
  • Enzymatic hydrolysis Use HPLC to measure the content of morin A in the above ethanol extract, and then according to the detection results, add 50% to 2000% ( ⁇ -glucan) according to the total mass of morin A in the above ethanol extract.
  • the ratio of the amount of carbohydrase E1 to the weight W1 of morin A (E1/W1) is 2-20, preferably 6-15, more preferably 8-12) ⁇ -glucanase, xylan Carbohydrate, pectinase or cellulase, or a combination thereof, is used for enzymatic hydrolysis; the enzymatic hydrolysis time is usually 0.5h to 3h, and filtered to obtain the enzymatic hydrolysis filtrate;
  • (iv) Drying Concentrate and/or dry the analytical solution to obtain a product containing oxidized resveratrol. Typically, the concentration is concentrated under reduced pressure at a temperature of 40°C to 60°C, with a vacuum degree of less than 20 Pa, and the drying is freeze-drying at a freezing temperature of -70°C to -50°C.
  • step (S2) or the enzymatic hydrolysis step ⁇ -glucanase is added for enzymatic hydrolysis.
  • the ratio of the dosage E1 of ⁇ -glucanase to the weight W1 of morin A is 0.5-20, preferably about 2-20 or 6-15, more preferably Land 6-12, as about 10.
  • the enzymatic hydrolysis time is not particularly limited, usually 0.5-3 hours, such as 1-2h or 1-3h, preferably about 2h.
  • compositions and uses of oxidized resveratrol Compositions and uses of oxidized resveratrol
  • Oxidized resveratrol has strong tyrosinase inhibition and antioxidant activity, so it can be widely used in cosmetics, natural medicines and health products.
  • the present invention also provides the use of oxidized resveratrol prepared by the method of the present invention.
  • Representative applications include (but are not limited to): cosmetic compositions or products, pharmaceutical compositions or medicines, dietary supplements, etc.
  • the cosmetic composition of the present invention includes the oxidized resveratrol of the present invention; and a cosmetically acceptable carrier.
  • the oxidized resveratrol of the present invention can be prepared into a cosmetic composition using conventional methods.
  • Representative cosmetics include (but are not limited to): solid dosage forms, semi-solid dosage forms, or liquid dosage forms, such as solutions, gels, Cream, lotion, spray, ointment, cream, paste, cake, powder, patch, etc.
  • the oxidized resveratrol of the present invention can also be used to prepare pharmaceutical compositions.
  • the pharmaceutical composition of the present invention includes the oxidized resveratrol of the present invention; and a pharmaceutically acceptable carrier.
  • the dosage form of the pharmaceutical composition of the present invention is not particularly limited, and representative dosage forms include (but are not limited to): ointments, creams, gels, pastes, patches, etc.
  • the medicament can be prepared by generally known preparation techniques, and suitable pharmaceutical additives can be added to the medicament.
  • Examples of pharmaceutical additives include excipients, binders, decomposers, lubricants, flow aids, suspending agents, emulsifiers, stabilizers, heat preservation (wetting) agents, preservatives, solvents, solubilizers, preservatives, Flavoring agents, sweeteners, dyes, spices, propellants, etc., these pharmaceutical additives can be selected and added in appropriate amounts within a range that does not affect the effects of the present invention.
  • ingredients commonly used in cosmetics or pharmaceuticals may be added to the cosmetics or pharmaceuticals of the present invention, such as film-forming agents, oil-soluble gelling agents, organically modified clay minerals, and resins. , moisturizers, preservatives, antibacterial agents, flavors, salts, antioxidants, pH adjusters, chelating agents, cooling agents, anti-inflammatory agents, skin beautifying ingredients (whitening agents, cell active agents, skin roughness improving agents, blood Circulation accelerators, skin astringents, anti-fat leakage agents, etc.), vitamins, amino acids, nucleic acids, hormones, inclusion compounds, etc.
  • Suitable oil-soluble gelling agents include (but are not limited to): metal soaps such as aluminum stearate, magnesium stearate, zinc myristate; N-lauroyl-L-glutamic acid, ⁇ , ⁇ -di -Amino acid derivatives such as n-butylamine; cyclodextrin fatty acid esters such as cyclodextrin palmitate, cyclodextrin stearate, cyclodextrin 2-ethylhexanoate palmitate; sucrose palmitate, Sucrose fatty acid esters such as sucrose stearate; benzylidene derivatives of sorbitol such as monobenzylidene sorbitol and dibenzylidene sorbitol; dimethylbenzyldodecyl ammonium montmorillonite clay, dibenzylidene sorbitol and other sucrose fatty acid esters; As a gelling agent for organically modified clay minerals such as
  • Suitable humectants include (but are not limited to): glycerin, sorbitol, propylene glycol, dipropylene glycol, 1,3-butanediol, glucose, xylitol, maltitol, polyethylene glycol, hyaluronic acid, Chondroitin sulfate, pyrrolidone carboxylate, polyoxyethylene methyl glucoside, polyoxypropylene methyl glucoside, etc.
  • Suitable antibacterial preservatives include (but are not limited to): alkyl parabens, benzoic acid, sodium benzoate, sorbic acid, potassium sorbate, phenoxyethanol, etc.
  • Antibacterial agents include: benzoic acid, salicylic acid , carbolic acid, sorbic acid, alkyl parahydroxybenzoate, p-chlorom-cresol, hexachlorophenol, benzalkonium chloride, chlorhexidine chloride, trichloro-N-anilide, triclosan, photosensitizer , phenoxyethanol, etc.
  • Suitable antioxidants include (but are not limited to): tocopherol, butylated hydroxyanisole, dibutylated hydroxytoluene, phytic acid, etc.; pH adjusters include: lactic acid, citric acid, glycolic acid, succinic acid, tartaric acid, dl -Malic acid, potassium carbonate, sodium bicarbonate, ammonium bicarbonate, etc.; chelating agents include alanine, ethylenediaminetetraacetic acid sodium salt, sodium polyphosphate, sodium metaphosphate, phosphoric acid, etc.; cooling agents include: L-menthol , camphor, etc. Anti-inflammatory agents include: allantoin, glycyrrhetinic acid, glycyrrhizic acid, tranexamic acid, Azulene, etc.
  • Representative skin beautifying ingredients include (but are not limited to): placenta extract, arbutin, glutathione, saxifrage extract and other whitening agents; royal jelly, photosensitizer, cholesterol derivatives, calf blood Cell active agents such as extracts; skin roughness improving agents; valeramide nonanoate, benzyl nicotinate, ⁇ -butoxyethyl nicotinate, capsaicin, Gingerone, cantharidin tincture, fish oil, caffeine, tannic acid, ⁇ -borneol, nicotinic acid tocopherol, hexanicotinic acid inositol ester, cyclomandelyl, cinnarizine, tolazoline, acetylcholine, verapamil , stephanatine, ⁇ -oryzanol and other blood circulation promoters; zinc oxide, tannic acid and other skin astringents; vitamins include: vitamin A oil, rosin oil,
  • Suitable amino acids include (but are not limited to): glycine, valine, leucine, isoleucine, serine, threonine, phenylalanine, arginine, lysine, aspartic acid , glutamic acid, cystine, cysteine, methionine, tryptophan, etc.
  • nucleic acids include deoxyribonucleic acid, etc.
  • hormones include estradiol, vinyl estradiol, etc.
  • cosmetics include skin care cosmetics, hair care cosmetics, color cosmetics, and ultraviolet protection cosmetics.
  • hair care products such as shampoo, conditioner, hair care essence, and hair mask
  • basic cosmetics such as lotions, creams, lotions, sunscreens, facial mask materials, facial cleansers, essences, etc.
  • makeup cosmetics such as foundation, white powder, blush, etc. wait.
  • the form of the cosmetic product is not particularly limited, and may be liquid, emulsion, cream, solid, paste, gel, powder, multi-layered, mousse, or spray. wait.
  • the present invention is the first enzymatic method for biotransformation, which efficiently transforms morin A, which is relatively high in content and widely distributed in plants, into an oxidative whitening agent with low content but strong tyrosinase inhibitory activity.
  • the enzymatic hydrolysis rate of veratrol and morin A reaches over 50%, up to 93.56%.
  • the extraction and elution solvents of the present invention are ethanol, which is safe, non-toxic, low-cost and recyclable.
  • the extract containing morin A is used for enzymatic hydrolysis.
  • the raw material can be extracted by heating and refluxing.
  • the preparation method is simple, the equipment requirements are low, the cost is low, and it is suitable for industrial production.
  • the present invention uses macroporous resin for purification, the resin cost is relatively low, and the resin regeneration is simple.
  • the high-purity oxidized resveratrol product of the present invention has a stronger effect of inhibiting tyrosinase and can be used as an antioxidant additive (antioxidant ) is added to many different products including cosmetics.
  • ⁇ -Glucanase microbial source, enzyme activity 700EGU/g.
  • Cellulase microbial source, enzyme activity 700EGU/g.
  • Xylanase microbial source, enzyme activity 500FXUs/g.
  • Pectinase microbial source, enzyme activity 8600PGNU/g.
  • the present invention utilizes a preparation method of biological transformation to increase the content of oxidized resveratrol, which includes the following steps:
  • Example 2 Repeat Example 1, with the only difference being that ⁇ -glucanase is replaced with cellulase (Example 2), xylanase (Example 3), and pectinase (Example 4) (enzyme dosage Same as Example 1), thereby preparing sample No. 2, 3 and 4 respectively.
  • ⁇ -glucanase has extremely excellent ability to convert morrin A. Not only the enzymatic hydrolysis rate of morrin A is as high as about 80%, which is about 20.6 times the conversion ability of cellulase, and the fruit The conversion capacity of glue enzyme is about 31.0 times.
  • Example 7 In order to increase the amount of oxidized resveratrol, raw materials with higher content of morin A were selected to carry out Examples 5 to 7. Sample Nos. 5, 6 and 7 were thus prepared respectively.
  • enzymatic hydrolysis for 3 hours there is not much difference between enzymatic hydrolysis for 3 hours and enzymatic hydrolysis for 2 hours. Considering the cost issue, enzymatic hydrolysis for 2 hours can be selected as the appropriate enzymatic hydrolysis time.
  • Example 8 Repeat Example 1, with the only difference being that the enzyme addition amount is replaced by 50% (Example 8), 100% (Example 9), and 150% (Example 8) of the total mass of morin A in the ethanol extract. 10), 200% (Example 11), 600% (Example 12), 1000% (Example 13) or 2000% (Example 14), thereby preparing samples No. 8, 9, 10, 11, 12, 13 or 14.
  • the E1/W1 ratio is 6 (i.e. 600%), the amount of oxidized resveratrol is significantly increased; when the amount of ⁇ -glucanase E1 and the weight of mulberry glycoside A W1
  • the ratio (E1/W1) is 6 to 10
  • the production of oxidized resveratrol increases slowly.
  • the ratio is 10
  • the maximum production amount is reached; when the ratio of the dosage E1 of ⁇ -glucanase to the weight W1 of morrin A (E1/W1) is 11 to 20, although the enzymatic hydrolysis rate of morrin A gradually increased, reaching a maximum of 94.22% when the ratio was 20, but the production of oxidized resveratrol slowly decreased.
  • the appropriate dosage of ⁇ -glucanase E1 and the weight W1 of morin A can be selected to be 8-12 (such as 10). That is, the enzyme addition amount is selected to be 800-1200% (such as about 1000%) of the total mass of morin A in the ethanol extract.
  • the tyrosinase inhibitory efficacy test was performed on the Morus alba bark extract (sample No. 13) prepared in Example 13.
  • the specific implementation method is as follows:
  • Reagent A Weigh 7.16g Na 2 HPO 4 ⁇ 12H 2 O and dilute to 100mL with distilled water;
  • Reagent B Weigh 3.12g NaH 2 PO 4 ⁇ 2H 2 O and dilute to 100mL with distilled water; or weigh 2.72g of KH 2 PO 4 and dilute to 100mL;
  • Control group Take 250 ⁇ L of PBS buffer, add 750 ⁇ L of L-tyrosine solution, and incubate at 37°C for 30 minutes; add 50 ⁇ L of tyrosinase solution, react at 37°C for 30 minutes, and measure the absorbance value at 450 nm.
  • Experimental group Take 25 ⁇ L, 50 ⁇ L, 125 ⁇ L and 250 ⁇ L of the test solution respectively, make up to 250 ⁇ L with PBS buffer, add 750 ⁇ L L-tyrosine solution, and incubate at 37°C for 30 min; add 50 ⁇ L of tyrosinase solution, and incubate at 37 React at °C for 30 minutes, and measure the absorbance value at 450nm.
  • Blank group Take 25 ⁇ L, 50 ⁇ L, 125 ⁇ L and 250 ⁇ L of the test solution respectively, make up to 250 ⁇ L with PBS buffer, add 750 ⁇ L L-tyrosine solution, and incubate at 37°C for 30 min; add 50 ⁇ L of PBS buffer, and incubate at 37°C. React for 30 minutes and measure the absorbance value at 450nm.
  • Positive control group Take 25 ⁇ L, 50 ⁇ L, 125 ⁇ L and 250 ⁇ L of arbutinol solution respectively, make up to 250 ⁇ L with PBS buffer, add 750 ⁇ L L-tyrosine solution, and incubate at 37°C for 30 min; add 50 ⁇ L of tyrosinase solution , react at 37°C for 30 minutes, and measure the absorbance value at 450nm.
  • Tyrosinase inhibition rate (%) (A 0 -A 1 +A 2 )/A 0 *100%
  • A0 is the absorbance value of the control group
  • A1 is the absorbance value of the experimental group
  • A2 is the absorbance value of the blank group
  • A2 of the positive control group is 0.
  • Example No. 13 A DPPH free radical scavenging test was performed on the Morus alba bark extract (sample No. 13) prepared in Example 13.
  • the specific implementation method is as follows:
  • Control group Take 0.025mL, 0.05mL, 0.1mL, 0.2mL, 0.4mL, and 0.5mL samples into deep well plates respectively, make up to 0.5mL with distilled water, add 2.0mL DPPH methanol solution, mix well, and place in the dark for 30 minutes Then, take 0.2 mL and put it into a 96-well plate, and use a microplate reader to measure the absorbance at 520 nm.
  • Blank group Take 0.025mL, 0.05mL, 0.1mL, 0.2mL, 0.4mL, and 0.5mL samples into the deep well plate respectively, make up to 0.5mL with distilled water, add 2.0mL methanol solution, mix well, and place in the dark for 30 minutes. , take 0.2mL and put it into a 96-well plate, and measure the absorbance at 520nm with a microplate reader.
  • Positive control group Take 0.025mL, 0.05mL, 0.1mL, 0.2mL, 0.4mL, 0.5mL Vc methanol solution and deep well plate respectively, make up to 0.5mL with distilled water, add 2.0mL DPPH methanol solution, mix well, and darken After leaving for 30 minutes, take 0.2 mL and place it in a 96-well plate, and measure the absorbance at 520 nm with a microplate reader.
  • Control group Add 0.5 mL distilled water and 2.0 mL DPPH methanol solution, mix well, place in a dark place for 30 minutes, take 0.2 mL and put it into a 96-well plate, and use a microplate reader to measure the absorbance at 520 nm.
  • DPPH radical scavenging rate (%) (A 0 -A 1 +A 2 )/A 0 *100%
  • A0 is the absorbance value of the control group
  • A1 is the absorbance value of the experimental group
  • A2 is the absorbance value of the blank group
  • A2 of the positive control group is 0.

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Abstract

Provided is a method for preparing oxyresveratrol, comprising the steps of: (S1) providing a raw material containing mulberroside A; and (S2) using β-glucanase to perform enzymatic hydrolysis treatment on the raw material containing mulberroside A, and converting mulberroside A into oxyresveratrol so as to obtain an enzymatic hydrolysate. The method can remarkably increase the yield of oxyresveratrol, the enzymatic hydrolysis rate of mulberroside A can be as high as about 90%, and the yield of oxyresveratrol is greatly increased. The method has mild reaction conditions, simple post-treatment, and no need for special reagents, and is suitable for industrial production.

Description

氧化白藜芦醇的制备方法Preparation method of oxidized resveratrol 技术领域Technical field
本发明属于药物化学技术领域，具体地涉及氧化白藜芦醇的制备方法。The invention belongs to the technical field of medicinal chemistry, and specifically relates to a preparation method of oxidized resveratrol.
背景技术Background technique
现有技术制备氧化白藜芦醇主要以传统的提取纯化方法进行，但是因为氧化白藜芦醇在植物中含量较低，来源范围较窄，故传统的提取方法较难有效获得氧化白藜芦醇。The preparation of oxidized resveratrol in the existing technology is mainly carried out by traditional extraction and purification methods. However, because the content of oxidized resveratrol in plants is low and the source range is narrow, it is difficult to effectively obtain oxidized resveratrol using traditional extraction methods. alcohol.
CN103194493A中描述了一种以桑枝粉为发酵底物，利用黑曲霉为发酵菌种进行发酵从而制备氧化白藜芦醇的方法，该方法存在从菌种活化到发酵结束时间周期较长，操作复杂，且微生物发酵中易产生其他杂质导致分离纯化较为困难的问题。CN103194493A describes a method for preparing oxidized resveratrol by using mulberry branch powder as the fermentation substrate and Aspergillus niger as the fermentation strain to carry out fermentation. This method has a long time period from strain activation to the end of fermentation, and the operation It is complex, and other impurities are easily produced during microbial fermentation, making separation and purification difficult.
CN104803830A中描述了一种利用乙醇及乙酸乙酯等有机试剂进行反复提取、萃取、脱色从而制备高纯的氧化白藜芦醇的工艺，该工艺使用到的有机试剂例如乙酸乙酯、石油醚，对环境及操作人员危害较大，且操作较为繁琐。CN104803830A describes a process for preparing high-purity oxidized resveratrol by repeatedly extracting, extracting, and decolorizing organic reagents such as ethanol and ethyl acetate. The process uses organic reagents such as ethyl acetate and petroleum ether. It is more harmful to the environment and operators, and the operation is more cumbersome.
因此，本领域迫切需要开发一种可以安全、低成本、简单、高效地制备氧化白藜芦醇的方法。Therefore, there is an urgent need in this field to develop a method for preparing oxidized resveratrol safely, at low cost, simply and efficiently.
发明内容Contents of the invention
本发明的目的在于提供一种安全、低成本、简单、高效地制备氧化白藜芦醇的方法。The object of the present invention is to provide a safe, low-cost, simple and efficient method for preparing oxidized resveratrol.
本发明的第一方面，提供了一种氧化白藜芦醇的制备方法，包括步骤：A first aspect of the invention provides a method for preparing oxidized resveratrol, comprising the steps:
(S1)提供含桑皮苷A的原料；(S1) Provide raw materials containing morin A;
(S2)将所述含桑皮苷A的原料，用β-葡聚糖酶进行酶解处理，将桑皮苷A转化为氧化白藜芦醇，从而获得酶解产物：
(S2) Enzymatically hydrolyze the raw material containing moryside A with β-glucanase to convert moryside A into oxidized resveratrol, thereby obtaining an enzymatic hydrolysis product:
在另一优选例中，在步骤(S2)中，桑皮苷A的酶解率≥60％，较佳地≥70％，更佳地≥80％，最佳地≥90％。In another preferred example, in step (S2), the enzymatic hydrolysis rate of morin A is ≥60%, preferably ≥70%, more preferably ≥80%, and most preferably ≥90%.
在另一优选例中，在步骤(S2)中，桑皮苷A的酶解率为60-97％，较佳地70-96％， 80-95％。In another preferred example, in step (S2), the enzymatic hydrolysis rate of morin A is 60-97%, preferably 70-96%, 80-95%.
在另一优选例中，在步骤(S2)中，在水相体系中进行酶解处理。In another preferred embodiment, in step (S2), enzymatic hydrolysis treatment is performed in an aqueous phase system.
在另一优选例中，所述水相体系的溶剂为水，或乙醇含量≤10％(较佳地≤5wt％，更佳地≤1wt％)的水/乙醇混合溶剂。In another preferred embodiment, the solvent of the aqueous phase system is water, or a water/ethanol mixed solvent with an ethanol content of ≤10% (preferably ≤5wt%, more preferably ≤1wt%).
在另一优选例中，在步骤(S2)中，与酶解前的含桑皮苷A的原料相比，所述酶解产物中氧化白藜芦醇的相对增加幅度≥300％，较佳地≥400％，更佳地≥500％，最佳地≥600％。In another preferred example, in step (S2), compared with the raw material containing morin A before enzymatic hydrolysis, the relative increase of oxidized resveratrol in the enzymatic hydrolysis product is ≥300%, preferably Ground ≥ 400%, more preferably ≥ 500%, most preferably ≥ 600%.
在另一优选例中，在步骤(S2)中，氧化白藜芦醇的相对增加幅度为50-2500％，较佳地100-2000％，更佳地500-2000％。In another preferred example, in step (S2), the relative increase amplitude of oxidized resveratrol is 50-2500%, preferably 100-2000%, more preferably 500-2000%.
在另一优选例中，在步骤(S2)中，酶解产物中，氧化白藜芦醇的浓度≥0.15mg/g，较佳地≥0.30mg/g，较佳地≥0.50mg/g。In another preferred example, in step (S2), in the enzymatic hydrolysis product, the concentration of oxidized resveratrol is ≥0.15 mg/g, preferably ≥0.30 mg/g, preferably ≥0.50 mg/g.
在另一优选例中，在步骤(S2)中，酶解产物中，氧化白藜芦醇的浓度为0.10-0.80mg/g，较佳地0.2-0.7mg/g，较佳地0.3-0.6mg/g。In another preferred example, in step (S2), the concentration of oxidized resveratrol in the enzymatic hydrolysis product is 0.10-0.80 mg/g, preferably 0.2-0.7 mg/g, preferably 0.3-0.6 mg/g.
在另一优选例中，在步骤(S2)中，酶解产物中，桑皮苷A的浓度≤0.20mg/g，较佳地≤0.15mg/g，较佳地≤0.10mg/g。In another preferred example, in step (S2), in the enzymatic hydrolysis product, the concentration of morin A is ≤0.20 mg/g, preferably ≤0.15 mg/g, preferably ≤0.10 mg/g.
在另一优选例中，在步骤(S2)中，酶解产物中，桑皮苷A的浓度为0.05-0.20mg/g，较佳地0.06-0.15mg/g，较佳地0.08-0.10mg/g。In another preferred example, in step (S2), the concentration of morin A in the enzymatic hydrolysis product is 0.05-0.20 mg/g, preferably 0.06-0.15 mg/g, preferably 0.08-0.10 mg /g.
在另一优选例中，在所述酶解处理之前，所述的含桑皮苷A的原料中，桑皮苷A的浓度为0.30-2.00mg/g，较佳地0.40-1.80mg/g。In another preferred example, before the enzymatic hydrolysis treatment, in the raw material containing morrin A, the concentration of morrin A is 0.30-2.00 mg/g, preferably 0.40-1.80 mg/g. .
在另一优选例中，在所述酶解处理之前，所述的含桑皮苷A的原料中，氧化白藜芦醇的浓度为≤0.10mg/g，较佳地≤0.08mg/g，较佳地≤0.06mg/g。In another preferred example, before the enzymatic hydrolysis treatment, the concentration of oxidized resveratrol in the raw material containing morin A is ≤0.10 mg/g, preferably ≤0.08 mg/g, Preferably ≤0.06mg/g.
在另一优选例中，所述的含桑皮苷A的原料为桑枝或桑白皮药材的水提取物、乙醇提取物、或乙醇/水提取物。In another preferred embodiment, the raw material containing morin A is a water extract, ethanol extract, or ethanol/water extract of mulberry branches or mulberry bark medicinal materials.
在另一优选例中，所述的含桑皮苷A的原料为植物提取物，优选地桑科植物的提取物。In another preferred example, the raw material containing morin A is a plant extract, preferably an extract of a Moraceae plant.
在另一优选例中，所述的提取物包括：根、茎、叶、果的提取物。In another preferred embodiment, the extract includes: extracts of roots, stems, leaves, and fruits.
在另一优选例中，所述的植物提取物包括：桑枝提取物、桑白皮提取物、或其组合。In another preferred embodiment, the plant extract includes: mulberry branch extract, mulberry white bark extract, or a combination thereof.
在另一优选例中，所述的含桑皮苷A的原料为桑枝或桑白皮药材的提取物。In another preferred embodiment, the raw material containing morin A is an extract of mulberry branches or mulberry white bark medicinal materials.
在另一优选例中，在步骤(S2)中，β-葡聚糖酶的用量E1与桑皮苷A重量W1之比(E1/W1)为02-25,较佳地为6-20，更佳地为6-15，如约10。In another preferred example, in step (S2), the ratio of the dosage E1 of β-glucanase to the weight W1 of morin A (E1/W1) is 02-25, preferably 6-20, More preferably 6-15, such as about 10.
在另一优选例中，在步骤(S2)中，酶解时间为0.5-3小时，优选地为1-2h。 In another preferred example, in step (S2), the enzymatic hydrolysis time is 0.5-3 hours, preferably 1-2 hours.
在另一优选例中，所述方法还包括步骤：In another preferred embodiment, the method further includes the steps of:
(S3)从(S2)步骤中经酶解处理的酶解产物中，分离氧化白藜芦醇，获得分离的氧化白藜芦醇粗品。(S3) Separate oxidized resveratrol from the enzymatic hydrolysis product subjected to enzymatic hydrolysis treatment in step (S2) to obtain separated crude oxidized resveratrol.
在另一优选例中，所述方法还包括步骤：In another preferred embodiment, the method further includes the steps of:
(S4)对分离的氧化白藜芦醇粗品进行精制和/或干燥。(S4) Refining and/or drying the separated crude oxidized resveratrol.
在另一优选例中，所述的分离采用大孔吸附树脂进行分离氧化白藜芦醇。In another preferred embodiment, the separation uses macroporous adsorption resin to separate and oxidize resveratrol.
在另一优选例中，所述大孔树脂树脂型号为：AB-8、D101、DM301、或其组合。In another preferred example, the macroporous resin resin model is: AB-8, D101, DM301, or a combination thereof.
在另一优选例中，所述大孔吸附树脂纯化的条件包括：上样，水洗，质量百分比浓度为30％～85％的乙醇等。在另一优选例中，在步骤(S2)中，用β-葡聚糖酶进行酶解，获得含氧化白藜芦醇的酶解液(或酶解产物)。In another preferred example, the conditions for purification of the macroporous adsorption resin include: sample loading, water washing, ethanol with a mass percentage concentration of 30% to 85%, etc. In another preferred embodiment, in step (S2), β-glucanase is used for enzymatic hydrolysis to obtain an enzymatic hydrolysis solution (or enzymatic hydrolysis product) containing oxidized resveratrol.
在另一优选例中，在步骤(S3)中，从所述酶解液中分离出所述的氧化白藜芦醇。In another preferred example, in step (S3), the oxidized resveratrol is separated from the enzymatic hydrolysis solution.
在另一优选例中，所述方法还包括：In another preferred embodiment, the method further includes:
将制备的氧化白藜芦醇，作为抗氧化或美白的添加剂添加入终产品。The prepared oxidized resveratrol is added to the final product as an antioxidant or whitening additive.
本发明的第二方面，提供了一种氧化白藜芦醇，所述的氧化白藜芦醇是用权利要求1所述的方法制备的。A second aspect of the present invention provides an oxidized resveratrol prepared by the method of claim 1.
本发明的第三方面，提供了一种β-葡聚糖酶的用途，所述β-葡聚糖酶被用于对桑皮苷A进行酶解，从而将桑皮苷A转化为氧化白藜芦醇，或A third aspect of the present invention provides the use of a β-glucanase, which is used to enzymatically hydrolyze morrin A, thereby converting morrin A into oxidized white sugar. veratrol, or
所述β-葡聚糖酶被用于制备酶制剂，所述酶制剂用于对桑皮苷A进行酶解，从而将桑皮苷A转化为氧化白藜芦醇。The β-glucanase is used to prepare an enzyme preparation, which is used to enzymatically hydrolyze morin A, thereby converting morin A into oxidized resveratrol.
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described one by one here.
附图说明Description of the drawings
图1显示了桑皮苷A的化学结构式。Figure 1 shows the chemical structural formula of morin A.
图2显示了氧化白藜芦醇的化学结构式。Figure 2 shows the chemical structural formula of oxidized resveratrol.
图3显示了本发明的工艺流程图。Figure 3 shows a process flow diagram of the present invention.
图4显示了本发明的酶解示意图。 Figure 4 shows a schematic diagram of the enzymatic hydrolysis of the present invention.
图5显示了不同种类酶对桑皮苷A酶解率及氧化白藜芦醇增加量的影响。Figure 5 shows the effects of different types of enzymes on the enzymatic hydrolysis rate of morin A and the increase in oxidized resveratrol.
图6显示了本发明β-葡聚糖酶酶解成分含量HPLC检测图。Figure 6 shows the HPLC detection chart of the content of enzymatically hydrolyzed components of β-glucanase of the present invention.
图7显示了不同酶解时间对桑皮苷A酶解率及氧化白藜芦醇增加量的影响。Figure 7 shows the effects of different enzymatic hydrolysis times on the enzymatic hydrolysis rate of morin A and the increase in oxidized resveratrol.
图8显示了不同酶添加量对桑皮苷A酶解率及氧化白藜芦醇增加量的影响。Figure 8 shows the effects of different enzyme addition amounts on the enzymatic hydrolysis rate of morin A and the increase in oxidized resveratrol.
图9显示了本发明制备的提取物抑制酪氨酸酶的效果图。Figure 9 shows the effect of the extract prepared in the present invention on inhibiting tyrosinase.
图10显示了本发明制备的提取物可有效清除DPPH(1,1-二苯基-2-苦基肼)自由基。Figure 10 shows that the extract prepared by the present invention can effectively scavenge DPPH (1,1-diphenyl-2-picrylhydrazyl) free radicals.
具体实施方式Detailed ways
本发明人经过广泛而深入的研究，通过大量筛选和测试，提供了一种提高氧化白藜芦醇含量的制备方法和应用。本发明意外地发现了一种酶解的生物转化方法，可以显著提高氧化白藜芦醇的制备含量。本发明还意外地发现使用β-葡聚糖酶酶解桑皮苷A的方法，可获得最高的氧化白藜芦醇的转化率。在此基础上完成了本发明。After extensive and in-depth research and extensive screening and testing, the inventor provided a preparation method and application for increasing the content of oxidized resveratrol. The present invention unexpectedly discovered an enzymatic bioconversion method that can significantly increase the production content of oxidized resveratrol. The present invention also unexpectedly discovered that the highest conversion rate of oxidized resveratrol can be obtained by using β-glucanase to enzymatically hydrolyze morin A. On this basis, the present invention was completed.
实验表明，本发明方法能极其有效地将桑枝或桑白皮等原料中桑皮苷A转为氧化白藜芦醇，使氧化白藜芦醇增加量达2000％以上。Experiments show that the method of the present invention can extremely effectively convert morin A in raw materials such as mulberry branches or mulberry bark into oxidized resveratrol, increasing the amount of oxidized resveratrol by more than 2000%.
桑皮苷AMorrin A
桑皮苷A(mulberroside A)为二羟基芪类化合物，又称氧化白藜醇苷，主要存在于桑科和桑属植物中，其化学结构式(见图1)如下：
Mulberroside A is a dihydroxystilbene compound, also known as oxidized resveratrol glycoside. It mainly exists in Moraceae and Morus plants. Its chemical structure formula (see Figure 1) is as follows:
与氧化白藜芦醇(见图2)相比，桑皮苷A多了两个通过糖苷键连接的糖基。Compared with oxidized resveratrol (see Figure 2), morin A has two more sugar groups connected through glycosidic bonds.
在抗氧化及抑制酪氨酸酶活性方面，桑皮苷A都比氧化白藜芦醇差，特别是在抑制酪氨酸酶活性方面。据文献报道桑皮苷A对酪氨酸酶的抑制率IC50值为53.6μM，而氧化白藜芦醇IC50值仅为0.49μM，两者比较相差100多倍。Morrin A is worse than oxidized resveratrol in terms of antioxidant and inhibitory tyrosinase activity, especially in inhibiting tyrosinase activity. According to literature reports, the IC50 value of the inhibitory rate of tyrosinase by morin A is 53.6 μM, while the IC50 value of oxidized resveratrol is only 0.49 μM. The difference between the two is more than 100 times.
氧化白藜芦醇 Oxidized resveratrol
氧化白藜芦醇，化学名为2，4，3'，5'-四羟基芪，是二苯乙烯衍生物的芪类物质，主要存在于桑属、波罗蜜属等植物。其化学结构式如下：
Oxidized resveratrol, chemically named 2,4,3',5'-tetrahydroxystilbene, is a stilbene derivative of stilbene and is mainly found in plants of the genus Morus and Jackfruit. Its chemical structural formula is as follows:
药理研究表明，氧化白藜芦醇对酪氨酸酶活性具有强烈的抑制作用，从而抑制黑色素的生成。近年来，更有大量研究显示氧化白藜芦醇是最有前景研制出抗褐变、美白等作用的膳食功能性化合物，其口服药物或者相关食品安全且低毒。Pharmacological studies have shown that oxidized resveratrol has a strong inhibitory effect on tyrosinase activity, thereby inhibiting melanin production. In recent years, a large number of studies have shown that oxidized resveratrol is the most promising dietary functional compound with anti-browning, whitening and other effects. Its oral drugs or related foods are safe and low-toxic.
由于氧化白藜芦醇具有毒副作用小，市场需求大，在植物中含量较低，且提取工艺过程复杂，来源窄等特点，从而导致价格昂贵，严重制约了在化妆品、天然药物、保健食品及功能饮料的开发及利用。Because oxidized resveratrol has few toxic side effects, large market demand, low content in plants, complex extraction processes, and narrow sources, it is expensive and severely restricts its use in cosmetics, natural medicines, health foods, and Development and utilization of functional drinks.
制备方法Preparation
本发明中，提供了一种制备高含量氧化白藜芦醇的产品的方法。典型地，本发明方法包括步骤：In the present invention, a method for preparing a product with high content of oxidized resveratrol is provided. Typically, the method of the present invention includes the steps:
(S1)提供含桑皮苷A的原料；(S1) Provide raw materials containing morin A;
(S2)将所述含桑皮苷A的原料，用β-葡聚糖酶进行酶解，从而将桑皮苷A转化为氧化白藜芦醇；(S2) Enzymatically hydrolyze the raw material containing morin A with β-glucanase, thereby converting morin A into oxidized resveratrol;
(S3)从(S2)步骤中经酶解处理的酶解产物中，分离氧化白藜芦醇，获得分离的氧化白藜芦醇粗品；(S3) Separate oxidized resveratrol from the enzymatic hydrolysis product treated in step (S2) to obtain separated crude oxidized resveratrol;
(S4)对分离的氧化白藜芦醇粗品进行精制和/或干燥，从而获得含高含量氧化白藜芦醇的产品。(S4) Refining and/or drying the separated crude oxidized resveratrol to obtain a product containing high content of oxidized resveratrol.
在一个优选例中，本发明提供一种制备含高含量氧化白藜芦醇的产品的制备方法,包括如下步骤：In a preferred embodiment, the present invention provides a method for preparing a product containing high content of oxidized resveratrol, which includes the following steps:
(i)提取：取桑枝或桑白皮药材，将其打至粗粉，而后加入醇/水混合溶剂(如质量百分比浓度为60％～80％乙醇水溶液)，然后进行提取(例如，在常压回流提取1～3次(每次0.5-2小时，或约1h))，合并提取液，过滤，浓缩，得乙醇提取物；(i) Extraction: Take mulberry branches or mulberry white bark medicinal materials, beat them into coarse powder, then add an alcohol/water mixed solvent (such as a mass percentage concentration of 60% to 80% ethanol aqueous solution), and then extract (for example, in Extract under normal pressure reflux 1 to 3 times (0.5-2 hours each time, or about 1 hour)), combine the extracts, filter and concentrate to obtain ethanol extract;
(ii)酶解：利用HPLC测定上述乙醇提取物中桑皮苷A的含量，而后根据检测结果，按照上述乙醇提取物中桑皮苷A的总质量加入50％～2000％(β-葡聚糖酶的用量E1与桑皮苷A重量W1之比(E1/W1)为2-20,较佳地为6-15，更佳地为8-12)的β-葡聚糖酶、木聚糖酶、果胶酶或纤维素酶、或其组合，进行酶解；酶解时间通常为0.5h～3h，过滤，得酶解滤液；(ii) Enzymatic hydrolysis: Use HPLC to measure the content of morin A in the above ethanol extract, and then according to the detection results, add 50% to 2000% (β-glucan) according to the total mass of morin A in the above ethanol extract. The ratio of the amount of carbohydrase E1 to the weight W1 of morin A (E1/W1) is 2-20, preferably 6-15, more preferably 8-12) β-glucanase, xylan Carbohydrate, pectinase or cellulase, or a combination thereof, is used for enzymatic hydrolysis; the enzymatic hydrolysis time is usually 0.5h to 3h, and filtered to obtain the enzymatic hydrolysis filtrate;
(iii)大孔吸附树脂纯化：将上述酶解滤液上样于大孔吸附树脂，代表性的树脂型号包括：AB-8、D101或DM301或类似的树脂，然后用洗脱液进行洗脱，从而获得含氧化白藜芦醇的解析液；在该步骤中，代表性的洗脱液包括(但并不限于)：质量百分比浓度为30％～85％的乙醇的水溶液；此外，在该步骤中，可以任选地在上样后用水进行洗涤；(iii) Purification of macroporous adsorption resin: Load the above enzymatic hydrolysis filtrate onto macroporous adsorption resin. Representative resin models include: AB-8, D101 or DM301 or similar resins, and then use eluent to elute. Thus, an analytical solution containing oxidized resveratrol is obtained; in this step, representative eluents include (but are not limited to): an aqueous solution of ethanol with a mass percentage concentration of 30% to 85%; in addition, in this step , you can optionally wash with water after loading the sample;
(iv)干燥：将所述解析液进行浓缩和/或干燥，从而获得含氧化白藜芦醇的产品。典型地，所述浓缩为在40℃～60℃的条件下进行减压浓缩，真空度小于20Pa，并且所述干燥为在-70℃～-50℃的冻结温度下进行冷冻干燥。(iv) Drying: Concentrate and/or dry the analytical solution to obtain a product containing oxidized resveratrol. Typically, the concentration is concentrated under reduced pressure at a temperature of 40°C to 60°C, with a vacuum degree of less than 20 Pa, and the drying is freeze-drying at a freezing temperature of -70°C to -50°C.
在另一优选例中，在步骤(S2)或酶解步骤中，加入β-葡聚糖酶进行酶解。In another preferred embodiment, in step (S2) or the enzymatic hydrolysis step, β-glucanase is added for enzymatic hydrolysis.
在另一优选例中，β-葡聚糖酶的用量E1与桑皮苷A重量W1之比(E1/W1)为0.5-20,较佳地为约2-20或6-15，更佳地6-12，如约10。In another preferred example, the ratio of the dosage E1 of β-glucanase to the weight W1 of morin A (E1/W1) is 0.5-20, preferably about 2-20 or 6-15, more preferably Land 6-12, as about 10.
在另一优选例中，在本发明中，酶解时间没有特别限制，通常为0.5-3小时，例如为1h-2h或1-3h,优选地为约2h。In another preferred example, in the present invention, the enzymatic hydrolysis time is not particularly limited, usually 0.5-3 hours, such as 1-2h or 1-3h, preferably about 2h.
含氧化白藜芦醇组合物及用途Compositions and uses of oxidized resveratrol
氧化白藜芦醇具有较强的抑制酪氨酸酶及抗氧化活性，因此可以广泛应用于化妆品、天然药物和保健品等中。Oxidized resveratrol has strong tyrosinase inhibition and antioxidant activity, so it can be widely used in cosmetics, natural medicines and health products.
本发明还提供了用本发明方法制备的氧化白藜芦醇的应用。代表性的应用包括(但并不限于)：化妆品组合物或产品、药物组合物或药品、膳食补充剂等。The present invention also provides the use of oxidized resveratrol prepared by the method of the present invention. Representative applications include (but are not limited to): cosmetic compositions or products, pharmaceutical compositions or medicines, dietary supplements, etc.
以化妆品组合物为例，本发明的化妆品组合物包括本发明所述的氧化白藜芦醇；和化妆品可接受的载体。典型地，可用常规方法将本发明的氧化白藜芦醇制备成化妆品组合物，代表性的化妆品包括(但并不限于)：固体剂型、半固体剂型、或液体剂型，如溶液、凝胶、膏霜、乳液、喷雾、膏剂、霜剂、糊剂、饼、粉剂、贴剂等。Taking a cosmetic composition as an example, the cosmetic composition of the present invention includes the oxidized resveratrol of the present invention; and a cosmetically acceptable carrier. Typically, the oxidized resveratrol of the present invention can be prepared into a cosmetic composition using conventional methods. Representative cosmetics include (but are not limited to): solid dosage forms, semi-solid dosage forms, or liquid dosage forms, such as solutions, gels, Cream, lotion, spray, ointment, cream, paste, cake, powder, patch, etc.
本发明的氧化白藜芦醇还可用于制成药物组合物。本发明的药物组合物包括本发明所述的氧化白藜芦醇；和药学上可接受的载体。本发明药物组合物的剂型没有特别限制，代表性的剂型包括(但并不限于)：如膏剂、霜剂、凝胶剂、糊剂、贴剂等。药物能够由通常已知的制备技术来制备，并且合适的药物添加剂能够被添加到该药物中。 The oxidized resveratrol of the present invention can also be used to prepare pharmaceutical compositions. The pharmaceutical composition of the present invention includes the oxidized resveratrol of the present invention; and a pharmaceutically acceptable carrier. The dosage form of the pharmaceutical composition of the present invention is not particularly limited, and representative dosage forms include (but are not limited to): ointments, creams, gels, pastes, patches, etc. The medicament can be prepared by generally known preparation techniques, and suitable pharmaceutical additives can be added to the medicament.
药物添加剂的例子包括赋形剂、粘结剂、分解剂、润滑剂、流动助剂、悬浮剂、乳化剂、稳定剂、保温(润湿)剂、防腐剂、溶剂、增溶剂、防腐剂、调味剂、增甜剂、染料、香料、推进剂等，这些药物添加剂可以进行选择并以在不影响本发明的效果的范围内的合适量添加。Examples of pharmaceutical additives include excipients, binders, decomposers, lubricants, flow aids, suspending agents, emulsifiers, stabilizers, heat preservation (wetting) agents, preservatives, solvents, solubilizers, preservatives, Flavoring agents, sweeteners, dyes, spices, propellants, etc., these pharmaceutical additives can be selected and added in appropriate amounts within a range that does not affect the effects of the present invention.
在不防碍本发明的效果的范围内，本发明的化妆品或药品中可以添加在化妆品或药品中常用的其它成分，例如成膜剂、油溶性凝胶化剂、有机改性粘土矿物、树脂、保湿剂、防腐剂、抗菌剂、香精、盐类、抗氧化剂、pH调节剂、螯合剂、清凉剂、抗炎剂、皮肤美化用成分(美白剂、细胞活性剂、皮肤粗糙改善剂、血液循环促进剂、皮肤收敛剂、抗脂漏剂等)、维生素类、氨基酸类、核酸、激素、包合化合物等。Within the scope that does not hinder the effects of the present invention, other ingredients commonly used in cosmetics or pharmaceuticals may be added to the cosmetics or pharmaceuticals of the present invention, such as film-forming agents, oil-soluble gelling agents, organically modified clay minerals, and resins. , moisturizers, preservatives, antibacterial agents, flavors, salts, antioxidants, pH adjusters, chelating agents, cooling agents, anti-inflammatory agents, skin beautifying ingredients (whitening agents, cell active agents, skin roughness improving agents, blood Circulation accelerators, skin astringents, anti-fat leakage agents, etc.), vitamins, amino acids, nucleic acids, hormones, inclusion compounds, etc.
合适的油溶性凝胶化剂包括(但并不限于)：硬脂酸铝、硬脂酸镁、肉豆蔻酸锌等金属皂；N-月桂酰基-L-谷氨酸、α，γ-二-正丁基胺等氨基酸衍生物；环糊精棕榈酸酯、环糊精硬脂酸酯、环糊精2-乙基己酸棕榈酸酯等环糊精脂肪酸酯；蔗糖棕榈酸酯、蔗糖硬脂酸酯等蔗糖脂肪酸酯；一亚苄基山梨醇、二亚苄基山梨醇等山梨醇的亚苄基衍生物；二甲基苄基十二烷基铵蒙脱石粘土、二甲基二十八烷基铵蒙脱石粘土等有机改性粘土矿物等的凝胶化剂，可以根据需要使用一种，或者使用二种或以上。Suitable oil-soluble gelling agents include (but are not limited to): metal soaps such as aluminum stearate, magnesium stearate, zinc myristate; N-lauroyl-L-glutamic acid, α,γ-di -Amino acid derivatives such as n-butylamine; cyclodextrin fatty acid esters such as cyclodextrin palmitate, cyclodextrin stearate, cyclodextrin 2-ethylhexanoate palmitate; sucrose palmitate, Sucrose fatty acid esters such as sucrose stearate; benzylidene derivatives of sorbitol such as monobenzylidene sorbitol and dibenzylidene sorbitol; dimethylbenzyldodecyl ammonium montmorillonite clay, dibenzylidene sorbitol and other sucrose fatty acid esters; As a gelling agent for organically modified clay minerals such as methyloctadecyl ammonium montmorillonite clay, one type, or two or more types may be used as needed.
合适的保湿剂包括(但并不限于)：甘油、山梨醇、丙二醇、双丙甘醇、1，3-丁二醇、葡萄糖、木糖醇、麦芽糖醇、聚乙二醇、透明质酸、硫酸软骨素、吡咯烷酮羧酸盐、聚氧乙烯甲基葡糖苷、聚氧丙烯甲基葡糖苷等。Suitable humectants include (but are not limited to): glycerin, sorbitol, propylene glycol, dipropylene glycol, 1,3-butanediol, glucose, xylitol, maltitol, polyethylene glycol, hyaluronic acid, Chondroitin sulfate, pyrrolidone carboxylate, polyoxyethylene methyl glucoside, polyoxypropylene methyl glucoside, etc.
合适的抗菌防腐剂包括(但并不限于)：对羟基苯甲酸烷基酯、苯甲酸、苯甲酸钠、山梨酸、山梨酸钾、苯氧基乙醇等，抗菌剂有：苯甲酸、水杨酸、石炭酸、山梨酸、对羟基苯甲酸烷基酯、对氯间甲酚、六氯酚、苯扎氯铵、氯化洗必泰、三氯-N-碳酰苯胺、三氯生、感光素、苯氧基乙醇等。Suitable antibacterial preservatives include (but are not limited to): alkyl parabens, benzoic acid, sodium benzoate, sorbic acid, potassium sorbate, phenoxyethanol, etc. Antibacterial agents include: benzoic acid, salicylic acid , carbolic acid, sorbic acid, alkyl parahydroxybenzoate, p-chlorom-cresol, hexachlorophenol, benzalkonium chloride, chlorhexidine chloride, trichloro-N-anilide, triclosan, photosensitizer , phenoxyethanol, etc.
合适的抗氧化剂包括(但并不限于)：生育酚、丁基羟基茴香醚、二丁基羟基甲苯、植酸等，pH调节剂有：乳酸、柠檬酸、乙醇酸、琥珀酸、酒石酸、dl-苹果酸、碳酸钾、碳酸氢钠、碳酸氢铵等，螯合剂有丙氨酸、乙二胺四乙酸钠盐、多磷酸钠、偏磷酸钠、磷酸等，清凉剂有：L-薄荷醇、樟脑等，抗炎剂有：尿囊素、甘草亭酸、甘草酸、凝血酸、甘葡环烃(Azulene)等。Suitable antioxidants include (but are not limited to): tocopherol, butylated hydroxyanisole, dibutylated hydroxytoluene, phytic acid, etc.; pH adjusters include: lactic acid, citric acid, glycolic acid, succinic acid, tartaric acid, dl -Malic acid, potassium carbonate, sodium bicarbonate, ammonium bicarbonate, etc.; chelating agents include alanine, ethylenediaminetetraacetic acid sodium salt, sodium polyphosphate, sodium metaphosphate, phosphoric acid, etc.; cooling agents include: L-menthol , camphor, etc. Anti-inflammatory agents include: allantoin, glycyrrhetinic acid, glycyrrhizic acid, tranexamic acid, Azulene, etc.
代表性的皮肤美化用成分包括(但并不限于)：胎盘提取液、熊果苷、谷胱甘肽、虎耳草提取物等美白剂；蜂王浆、感光素、胆甾醇衍生物、小牛血液提取液等细胞活性剂；皮肤粗糙改善剂；壬酸缬草酰胺、烟酸苄酯、烟酸β-丁氧基乙酯、辣椒素、 姜油酮、斑蝥酊、鱼石脂、咖啡因、鞣酸、α-冰片、烟酸生育酚、六烟酸肌醇脂、环扁桃酯、桂利嗪、妥拉唑啉、乙酰胆碱、维拉帕米、千金藤素、γ-谷维醇等血液循环促进剂；氧化锌、鞣酸等皮肤收敛剂；维生素类有：维生素A油、松香油、乙酸松香油、棕榈酸松香油等维生素A类；核黄素、丁酸核黄素、黄素腺嘌呤核苷酸等维生素B2类；吡多辛盐酸盐、吡多辛二辛酸酯、吡多辛三棕榈酸酯等维生素B6类；L-抗坏血酸、L-抗坏血酸二棕榈酸酯等维生素C类；麦角钙化醇、胆钙化醇等维生素D类；α-生育酚、β-生育酚、γ-生育酚、乙酸dl-α-生育酚、烟酸dl-α-生育酚、琥珀酸dl-α-生育酚等维生素E类；维生素H；维生素P；烟酸、烟酸苄酯、烟酰胺等烟酸类；泛酸钙、D-泛醇、泛酰基乙基醚、乙酰基泛酰基乙基醚等泛酸类；生物素等。Representative skin beautifying ingredients include (but are not limited to): placenta extract, arbutin, glutathione, saxifrage extract and other whitening agents; royal jelly, photosensitizer, cholesterol derivatives, calf blood Cell active agents such as extracts; skin roughness improving agents; valeramide nonanoate, benzyl nicotinate, β-butoxyethyl nicotinate, capsaicin, Gingerone, cantharidin tincture, fish oil, caffeine, tannic acid, α-borneol, nicotinic acid tocopherol, hexanicotinic acid inositol ester, cyclomandelyl, cinnarizine, tolazoline, acetylcholine, verapamil , stephanatine, γ-oryzanol and other blood circulation promoters; zinc oxide, tannic acid and other skin astringents; vitamins include: vitamin A oil, rosin oil, acetate rosin oil, palmitic acid rosin oil and other vitamin A; Riboflavin, riboflavin butyrate, flavin adenine nucleotide and other vitamin B2 types; vitamin B6 types such as pyridoxine hydrochloride, pyridoxine dicaprylate, pyridoxine tripalmitate and other vitamins; L - Vitamin C such as ascorbic acid, L-ascorbyl dipalmitate; Vitamin D such as ergocalciferol, cholecalciferol; α-tocopherol, β-tocopherol, γ-tocopherol, dl-α-tocopherol acetate, Niacin dl-α-tocopherol, dl-α-tocopherol succinate and other vitamin E; vitamin H; vitamin P; niacin, niacin benzyl ester, nicotinamide and other niacin; calcium pantothenate, D-panthenol , pantothenic acid ethyl ether, acetyl pantothenic acid ethyl ether, etc.; biotin, etc.
合适的氨基酸类包括(但并不限于)：甘氨酸、缬氨酸、亮氨酸、异亮氨酸、丝氨酸、苏氨酸、苯丙氨酸、精氨酸、赖氨酸、天冬氨酸、谷氨酸、胱氨酸、半胱氨酸、甲硫氨酸、色氨酸等，核酸有脱氧核糖核酸等，激素有雌二醇、乙烯基雌二醇等。Suitable amino acids include (but are not limited to): glycine, valine, leucine, isoleucine, serine, threonine, phenylalanine, arginine, lysine, aspartic acid , glutamic acid, cystine, cysteine, methionine, tryptophan, etc., nucleic acids include deoxyribonucleic acid, etc., and hormones include estradiol, vinyl estradiol, etc.
在本发明中，化妆品的优选例子包括：皮肤护理化妆品、毛发护理化妆品、彩妆化妆品、防紫外线化妆品。例如有洗发水、护发素、护发精华、发膜等护发产品；乳液、霜、露、防晒霜、面膜材料、洗面奶、精华液等基础化妆品；粉底、白粉、腮红等彩妆化妆品等。In the present invention, preferred examples of cosmetics include skin care cosmetics, hair care cosmetics, color cosmetics, and ultraviolet protection cosmetics. For example, there are hair care products such as shampoo, conditioner, hair care essence, and hair mask; basic cosmetics such as lotions, creams, lotions, sunscreens, facial mask materials, facial cleansers, essences, etc.; makeup cosmetics such as foundation, white powder, blush, etc. wait.
在本发明中，对于化妆品产品的形态没有特别限定，可以是液状、乳液状、霜状、固体状、糊状、凝胶状、粉末状、多层状、摩丝状(mousse)、喷雾状等。In the present invention, the form of the cosmetic product is not particularly limited, and may be liquid, emulsion, cream, solid, paste, gel, powder, multi-layered, mousse, or spray. wait.
本发明的主要优点包括：The main advantages of the present invention include:
(1)本发明首次酶法进行生物转化，将植物中含量较高、分布较广的桑皮苷A进行高效转化，使之成为含量较低，但抑制酪氨酸酶活性极强的氧化白藜芦醇；桑皮苷A酶解率达到50％以上，最高可达93.56％。(1) The present invention is the first enzymatic method for biotransformation, which efficiently transforms morin A, which is relatively high in content and widely distributed in plants, into an oxidative whitening agent with low content but strong tyrosinase inhibitory activity. The enzymatic hydrolysis rate of veratrol and morin A reaches over 50%, up to 93.56%.
(2)本发明提取及洗脱溶剂均为乙醇，溶剂安全无毒，成本较低，可回收。(2) The extraction and elution solvents of the present invention are ethanol, which is safe, non-toxic, low-cost and recyclable.
(3)本发明中，将含桑皮苷A的提取物用于酶解，所述原料可通过加热回流提取，制法方法简便、设备要求低、成本低廉，适合工业化生产。(3) In the present invention, the extract containing morin A is used for enzymatic hydrolysis. The raw material can be extracted by heating and refluxing. The preparation method is simple, the equipment requirements are low, the cost is low, and it is suitable for industrial production.
(4)本发明使用大孔树脂进行纯化，树脂成本较为低廉，树脂再生简便。(4) The present invention uses macroporous resin for purification, the resin cost is relatively low, and the resin regeneration is simple.
(5)与酶解前的桑枝或桑白皮提取物相比，本发明高纯度的氧化白藜芦醇产品具有更强的抑制酪氨酸酶的功效，可作为抗氧化添加剂(抗氧化剂)添加到化妆品等多种不同产品中。 (5) Compared with the mulberry branch or mulberry bark extract before enzymatic hydrolysis, the high-purity oxidized resveratrol product of the present invention has a stronger effect of inhibiting tyrosinase and can be used as an antioxidant additive (antioxidant ) is added to many different products including cosmetics.
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the invention and are not intended to limit the scope of the invention. Experimental methods without specifying specific conditions in the following examples usually follow conventional conditions or conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.
材料Material
β-葡聚糖酶：微生物来源，酶活力700EGU/g。β-Glucanase: microbial source, enzyme activity 700EGU/g.
纤维素酶：微生物来源，酶活力700EGU/g。Cellulase: microbial source, enzyme activity 700EGU/g.
木聚糖酶：微生物来源，酶活力500FXUs/g。Xylanase: microbial source, enzyme activity 500FXUs/g.
果胶酶：微生物来源，酶活力8600PGNU/g。Pectinase: microbial source, enzyme activity 8600PGNU/g.
上述酶均为市售产品。The above-mentioned enzymes are all commercially available products.
实施例1Example 1
氧化白藜芦醇的制备(样品No.1)Preparation of oxidized resveratrol (sample No. 1)
本发明利用生物转化的制备方法，提高氧化白藜芦醇含量，包括以下步骤：The present invention utilizes a preparation method of biological transformation to increase the content of oxidized resveratrol, which includes the following steps:
(1)提取：取桑白皮药材，将其打至粗粉，按首次料液比1：10(g：mL)，第二次料液比1：10(g：mL)分别加入60％(v/v)乙醇，回流提取两次，每次1h，减压抽滤，浓缩，得乙醇提取物；(1) Extraction: Take the mulberry bark medicinal material, beat it into coarse powder, add 60% respectively according to the first material-liquid ratio 1:10 (g: mL) and the second time material-liquid ratio 1:10 (g: mL). (v/v) ethanol, reflux and extract twice, 1 hour each time, filter under reduced pressure, and concentrate to obtain ethanol extract;
(2)酶解：HPLC测定上述乙醇提取物中桑皮苷A的含量，按照桑皮苷A总质量的400％(w/w)添加酶，在水溶液中进行酶解，酶解时间2h，减压抽滤，得酶解滤液；利用HPLC测定上述乙醇提取物中酶解前后桑皮苷A及氧化白藜芦醇含量；(2) Enzymatic hydrolysis: HPLC determines the content of morin A in the above ethanol extract, adds enzyme according to 400% (w/w) of the total mass of morin A, and performs enzymatic hydrolysis in an aqueous solution. The enzymatic hydrolysis time is 2 hours. Filter under reduced pressure to obtain the enzymatic hydrolysis filtrate; use HPLC to measure the contents of morin A and oxidized resveratrol in the above ethanol extract before and after enzymatic hydrolysis;
(3)大孔吸附树脂纯化：将上述酶解滤液上样于DM301大孔吸附树脂进行纯化，收集解析液；(3) Purification of macroporous adsorption resin: Load the above enzymatic hydrolysis filtrate onto DM301 macroporous adsorption resin for purification, and collect the analytical solution;
(4)干燥：将所述解析液进行减压浓缩、冷冻干燥，得到样品No.1。(4) Drying: The analysis liquid was concentrated under reduced pressure and freeze-dried to obtain sample No. 1.
实施例2～4Examples 2 to 4
氧化白藜芦醇的制备(样品No.2、3和4)Preparation of oxidized resveratrol (Sample No. 2, 3 and 4)
重复实施例1，不同点仅在于：将β-葡聚糖酶分别替换为纤维素酶(实施例2)、木聚糖酶(实施例3)、果胶酶(实施例4)(酶用量同实施例1)，从而分别制得样品No.2、3和4。Repeat Example 1, with the only difference being that β-glucanase is replaced with cellulase (Example 2), xylanase (Example 3), and pectinase (Example 4) (enzyme dosage Same as Example 1), thereby preparing sample No. 2, 3 and 4 respectively.
实施例1～4的实验结果如表1和图5-6所示： The experimental results of Examples 1 to 4 are shown in Table 1 and Figures 5-6:
表1不同酶对桑皮苷A转化的影响
Table 1 Effects of different enzymes on the conversion of morrin A
如表1及图5所示，大多数酶无法有效地将桑皮苷A转化为氧化白藜芦醇，有的甚至导致氧化白藜芦醇含量下降(如实施例3的木聚糖酶)。这提示，在采用多种不同的酶(纤维素酶、木聚糖酶、果胶酶)进行处理时，虽然桑皮苷A的含量有所下降，下降百分比(酶解率)为20-30％不等，但是酶解产物中，所需的氧化白藜芦醇的含量却基本上未增加，增加幅度为-6％至3％不等。As shown in Table 1 and Figure 5, most enzymes cannot effectively convert morin A into oxidized resveratrol, and some even cause the content of oxidized resveratrol to decrease (such as the xylanase in Example 3) . This suggests that when using a variety of different enzymes (cellulase, xylanase, pectinase) for treatment, although the content of morin A has decreased, the decrease percentage (enzymatic hydrolysis rate) is 20-30 % varies, but the required content of oxidized resveratrol in the enzymatic hydrolysis product has basically not increased, with an increase ranging from -6% to 3%.
出乎意料的是，β-葡聚糖酶具有极其优异的转化桑皮苷A能力，不仅桑皮苷A酶解率高达约80％，为纤维素酶转化能力的约20.6倍，和为果胶酶转化能力的约31.0倍。Unexpectedly, β-glucanase has extremely excellent ability to convert morrin A. Not only the enzymatic hydrolysis rate of morrin A is as high as about 80%, which is about 20.6 times the conversion ability of cellulase, and the fruit The conversion capacity of glue enzyme is about 31.0 times.
此外，出乎意料的是，采用β-葡聚糖酶时，在酶解产物中，氧化白藜芦醇的含量显著增加，增加幅度为约64％。In addition, unexpectedly, when β-glucanase was used, the content of oxidized resveratrol in the enzymatic hydrolyzate increased significantly, with an increase of approximately 64%.
实施例5～7Examples 5 to 7
氧化白藜芦醇的制备(样品No.5、6和7)Preparation of oxidized resveratrol (Sample No. 5, 6 and 7)
重复实施例1，不同点仅在于：将酶添加量替换为乙醇提取物中桑皮苷A的总质量的1000％，将酶解时间分别替换为1h(实施例5)、2h(实施例6)、3h(实施例7)；为提高氧化白藜芦醇的增加量，选用桑皮苷A含量更高的原料进行实施例5～7。从而分别制得样品No.5、6和7。Repeat Example 1, with the only difference being that the enzyme addition amount is replaced by 1000% of the total mass of morin A in the ethanol extract, and the enzymatic hydrolysis time is replaced by 1h (Example 5) and 2h (Example 6) respectively. ), 3h (Example 7); In order to increase the amount of oxidized resveratrol, raw materials with higher content of morin A were selected to carry out Examples 5 to 7. Sample Nos. 5, 6 and 7 were thus prepared respectively.
实施例5～7的实验结果如表2和图7所示：The experimental results of Examples 5 to 7 are shown in Table 2 and Figure 7:
表2酶解时间对桑皮苷A转化的影响

Table 2 Effect of enzymatic hydrolysis time on the conversion of morrin A

从表2及图7可以看出，酶解时间3h时具有最优的桑皮苷A转化效果，酶解率可达95.74％，氧化白藜芦醇增加量达1896.55％。It can be seen from Table 2 and Figure 7 that the enzymatic hydrolysis time of 3 hours has the optimal conversion effect of morin A, the enzymatic hydrolysis rate can reach 95.74%, and the increase in oxidized resveratrol can reach 1896.55%.
然而，酶解3h与酶解2h相比差别不大，考虑到成本问题，可以选择酶解2h作为合适的酶解时间。However, there is not much difference between enzymatic hydrolysis for 3 hours and enzymatic hydrolysis for 2 hours. Considering the cost issue, enzymatic hydrolysis for 2 hours can be selected as the appropriate enzymatic hydrolysis time.
实施例8～14Examples 8 to 14
氧化白藜芦醇的制备(样品No.8至14)Preparation of oxidized resveratrol (Sample No. 8 to 14)
重复实施例1，不同点仅在于：将酶添加量分别替换为乙醇提取物中桑皮苷A的总质量的50％(实施例8)、100％(实施例9)、150％(实施例10)、200％(实施例11)、600％(实施例12)、1000％(实施例13)或2000％(实施例14)中的一种，从而分别制得样品No.8、9、10、11、12、13或14。Repeat Example 1, with the only difference being that the enzyme addition amount is replaced by 50% (Example 8), 100% (Example 9), and 150% (Example 8) of the total mass of morin A in the ethanol extract. 10), 200% (Example 11), 600% (Example 12), 1000% (Example 13) or 2000% (Example 14), thereby preparing samples No. 8, 9, 10, 11, 12, 13 or 14.
实施例8～14的实验结果如表3和图8所示：The experimental results of Examples 8 to 14 are shown in Table 3 and Figure 8:
表3酶添加量对桑皮苷A转化的影响
Table 3 Effect of enzyme addition on the conversion of morin A
从表3及图8可以看出，β-葡聚糖酶的用量E1与桑皮苷A重量W1之比(E1/W1)为0.5-2时，生成氧化白藜芦醇的量增加不显著(增加幅度为16-23％不等)。It can be seen from Table 3 and Figure 8 that when the ratio of the dosage E1 of β-glucanase to the weight W1 of morin A (E1/W1) is 0.5-2, the amount of oxidized resveratrol produced does not increase significantly. (The increase ranges from 16-23%).
然而，出乎意料的是，当E1/W1比值为6(即600％)时，生成氧化白藜芦醇的量显著增加；当β-葡聚糖酶的用量E1与桑皮苷A重量W1之比(E1/W1)为6～10时，氧化白藜芦醇的生成量增加缓慢。当比值为10时，达到最大生成量；当β-葡聚糖酶的用量E1与桑皮苷A重量W1之比(E1/W1)为11～20时，虽然桑皮苷A酶解率逐渐增高，在比值为20时达到最高至94.22％，但氧化白藜芦醇的生成量反而有缓慢减少。However, unexpectedly, when the E1/W1 ratio is 6 (i.e. 600%), the amount of oxidized resveratrol is significantly increased; when the amount of β-glucanase E1 and the weight of mulberry glycoside A W1 When the ratio (E1/W1) is 6 to 10, the production of oxidized resveratrol increases slowly. When the ratio is 10, the maximum production amount is reached; when the ratio of the dosage E1 of β-glucanase to the weight W1 of morrin A (E1/W1) is 11 to 20, although the enzymatic hydrolysis rate of morrin A gradually increased, reaching a maximum of 94.22% when the ratio was 20, but the production of oxidized resveratrol slowly decreased.
基于氧化白藜芦醇生成量和材料成本的考虑，故可选择合适的β-葡聚糖酶的用量E1与桑皮苷A重量W1比值(E1/W1)为8-12(如10)，即选择酶添加量为乙醇提取物中桑皮苷A的总质量800-1200％(如约1000％)。Based on the consideration of the production amount of oxidized resveratrol and material cost, the appropriate dosage of β-glucanase E1 and the weight W1 of morin A (E1/W1) can be selected to be 8-12 (such as 10). That is, the enzyme addition amount is selected to be 800-1200% (such as about 1000%) of the total mass of morin A in the ethanol extract.
实施例15Example 15
抑制酪氨酸酶功效Inhibit tyrosinase effect
针对实施例13制得的桑白皮提取物(样品No.13)，进行抑制酪氨酸酶功效测试，具体实施方式如下：The tyrosinase inhibitory efficacy test was performed on the Morus alba bark extract (sample No. 13) prepared in Example 13. The specific implementation method is as follows:
15.1试验试剂及配制方法15.1 Test reagents and preparation methods
15.1.1 PBS缓冲液(0.1M，pH 6.8)15.1.1 PBS buffer (0.1M, pH 6.8)
试剂A：称取7.16g Na2HPO4·12H2O用蒸馏水定容到100mL；Reagent A: Weigh 7.16g Na 2 HPO 4 ·12H 2 O and dilute to 100mL with distilled water;
试剂B：称取3.12g NaH2PO4·2H2O用蒸馏水定容到100mL；或者称取KH2PO4 2.72g，定容到100mL；Reagent B: Weigh 3.12g NaH 2 PO 4 ·2H 2 O and dilute to 100mL with distilled water; or weigh 2.72g of KH 2 PO 4 and dilute to 100mL;
取49mL试剂A与51mL试剂B混合均匀，稀释一倍(加入100mL蒸馏水)，即得。Take 49mL of reagent A and 51mL of reagent B, mix them evenly, and dilute it twice (add 100mL of distilled water) to get it.
15.1.2 L-酪氨酸溶液15.1.2 L-tyrosine solution
准确称取L-酪氨酸4.5mg，用PBS缓冲液(1.1)定容到50mL，即得。Accurately weigh 4.5 mg of L-tyrosine and dilute to 50 mL with PBS buffer (1.1).
15.1.3酪氨酸酶溶液15.1.3 Tyrosinase solution
准确称量5mg酪氨酸酶，用移液枪加入10mL PBS缓冲液(1.1)，即得。Accurately weigh 5 mg of tyrosinase and add 10 mL of PBS buffer (1.1) with a pipette.
15.1.4 1％β-熊果苷醇溶液(阳性对照)15.1.4 1% β-arbutinol solution (positive control)
称取β-熊果苷0.1g，用10％乙醇溶液定容到10mL，即得。Weigh 0.1g of β-arbutin and dilute to 10 mL with 10% ethanol solution.
15.2试验方法15.2 Test methods
15.2.1检测样品配制15.2.1 Test sample preparation
用PBS缓冲液(1.1)将其配制为适宜浓度的溶液，即可进行检测。 Use PBS buffer (1.1) to prepare a solution of appropriate concentration, and then the test can be carried out.
15.2.2检测流程15.2.2 Testing process
对照组：取250μL PBS缓冲液，加入750μL L-酪氨酸溶液，于37℃下保温30min；加入酪氨酸酶溶液50μL，于37℃下反应30min，于450nm下测定其吸光度值。Control group: Take 250 μL of PBS buffer, add 750 μL of L-tyrosine solution, and incubate at 37°C for 30 minutes; add 50 μL of tyrosinase solution, react at 37°C for 30 minutes, and measure the absorbance value at 450 nm.
实验组：分别取待测液25μL、50μL、125μL及250μL，用PBS缓冲液补足到250μL，加入750μL L-酪氨酸溶液，于37℃下保温30min；加入酪氨酸酶溶液50μL，于37℃下反应30min，于450nm下测定其吸光度值。Experimental group: Take 25 μL, 50 μL, 125 μL and 250 μL of the test solution respectively, make up to 250 μL with PBS buffer, add 750 μL L-tyrosine solution, and incubate at 37°C for 30 min; add 50 μL of tyrosinase solution, and incubate at 37 React at ℃ for 30 minutes, and measure the absorbance value at 450nm.
空白组：分别取待测液25μL、50μL、125μL及250μL，用PBS缓冲液补足到250μL，加入750μL L-酪氨酸溶液，于37℃下保温30min；加入PBS缓冲液50μL，于37℃下反应30min，于450nm下测定其吸光度值。Blank group: Take 25 μL, 50 μL, 125 μL and 250 μL of the test solution respectively, make up to 250 μL with PBS buffer, add 750 μL L-tyrosine solution, and incubate at 37°C for 30 min; add 50 μL of PBS buffer, and incubate at 37°C. React for 30 minutes and measure the absorbance value at 450nm.
阳性对照组：分别取熊果苷醇溶液25μL、50μL、125μL及250μL，用PBS缓冲液补足到250μL，加入750μL L-酪氨酸溶液，于37℃下保温30min；加入酪氨酸酶溶液50μL，于37℃下反应30min，于450nm下测定其吸光度值。Positive control group: Take 25 μL, 50 μL, 125 μL and 250 μL of arbutinol solution respectively, make up to 250 μL with PBS buffer, add 750 μL L-tyrosine solution, and incubate at 37°C for 30 min; add 50 μL of tyrosinase solution , react at 37°C for 30 minutes, and measure the absorbance value at 450nm.
15.2.3试验结果15.2.3 Test results
试验结果如图9所示，在该试验条件下，阳性对照β-熊果苷的IC50＝4.893mg/mL，桑白皮提取物(样品No.13)IC50＝1.365μg/mL，是阳性对照的3585倍，具有较强的酪氨酸酶抑制效果。The test results are shown in Figure 9. Under the test conditions, the IC50 of the positive control β-arbutin = 4.893 mg/mL, and the IC50 of the mulberry bark extract (sample No. 13) = 1.365 μg/mL, which is the positive control. 3585 times of that, with strong tyrosinase inhibitory effect.
酪氨酸酶抑制率(％)＝(A0-A1+A2)/A0*100％Tyrosinase inhibition rate (%) = (A 0 -A 1 +A 2 )/A 0 *100%
其中A0为对照组吸光度值；A1为实验组吸光度值；A2为空白组吸光度值；阳性对照组A2为0。Among them, A0 is the absorbance value of the control group; A1 is the absorbance value of the experimental group; A2 is the absorbance value of the blank group; A2 of the positive control group is 0.
实施例16Example 16
DPPH自由基清除活性DPPH free radical scavenging activity
针对实施例13制得的桑白皮提取物(样品No.13)，进行DPPH自由基清除测试，具体实施方式如下：A DPPH free radical scavenging test was performed on the Morus alba bark extract (sample No. 13) prepared in Example 13. The specific implementation method is as follows:
16.1试验试剂及配制方法16.1 Test reagents and preparation methods
16.1.1 DPPH甲醇溶液16.1.1 DPPH methanol solution
称取7.88gDPPH，用甲醇定容至100mL，暗处存放。Weigh 7.88gDPPH, dilute to 100mL with methanol, and store in a dark place.
16.1.2 Vc甲醇溶液(阳性对照) 16.1.2 Vc methanol solution (positive control)
称取10mg抗坏血酸，用甲醇定容至100mL。Weigh 10 mg of ascorbic acid and dilute to 100 mL with methanol.
16.2试验方法16.2 Test methods
16.2.1检测样品配制16.2.1 Test sample preparation
用50％甲醇溶液将其配制为适宜浓度的溶液，即可进行检测。Use 50% methanol solution to prepare a solution of appropriate concentration, and then the test can be carried out.
16.2.2检测流程16.2.2 Testing process
对照组：分别取0.025mL、0.05mL、0.1mL、0.2mL、0.4mL、0.5mL样品与深孔板中，用蒸馏水补足至0.5mL，加入2.0mL DPPH甲醇溶液，混匀，暗处放置30min后，取0.2mL与96孔板中，用酶标仪测定520nm处吸光度。Control group: Take 0.025mL, 0.05mL, 0.1mL, 0.2mL, 0.4mL, and 0.5mL samples into deep well plates respectively, make up to 0.5mL with distilled water, add 2.0mL DPPH methanol solution, mix well, and place in the dark for 30 minutes Then, take 0.2 mL and put it into a 96-well plate, and use a microplate reader to measure the absorbance at 520 nm.
空白组：分别取0.025mL、0.05mL、0.1mL、0.2mL、0.4mL、0.5mL样品与深孔板中，用蒸馏水补足至0.5mL，加入2.0mL甲醇溶液，混匀，暗处放置30min后，取0.2mL与96孔板中，用酶标仪测定520nm处吸光度。Blank group: Take 0.025mL, 0.05mL, 0.1mL, 0.2mL, 0.4mL, and 0.5mL samples into the deep well plate respectively, make up to 0.5mL with distilled water, add 2.0mL methanol solution, mix well, and place in the dark for 30 minutes. , take 0.2mL and put it into a 96-well plate, and measure the absorbance at 520nm with a microplate reader.
阳性对照组：分别取0.025mL、0.05mL、0.1mL、0.2mL、0.4mL、0.5mL Vc甲醇溶液与深孔板中，用蒸馏水补足至0.5mL，加入2.0mL DPPH甲醇溶液，混匀，暗处放置30min后，取0.2mL与96孔板中，用酶标仪测定520nm处吸光度。Positive control group: Take 0.025mL, 0.05mL, 0.1mL, 0.2mL, 0.4mL, 0.5mL Vc methanol solution and deep well plate respectively, make up to 0.5mL with distilled water, add 2.0mL DPPH methanol solution, mix well, and darken After leaving for 30 minutes, take 0.2 mL and place it in a 96-well plate, and measure the absorbance at 520 nm with a microplate reader.
对照组：加入0.5mL蒸馏水及2.0mL DPPH甲醇溶液，混匀，暗处放置30min后，取0.2mL与96孔板中，用酶标仪测定520nm处吸光度。Control group: Add 0.5 mL distilled water and 2.0 mL DPPH methanol solution, mix well, place in a dark place for 30 minutes, take 0.2 mL and put it into a 96-well plate, and use a microplate reader to measure the absorbance at 520 nm.
16.2.3试验结果16.2.3 Test results
试验结果如图10所示，在该试验条件下，桑白皮提取物(样品No.13)对DPPH清除率最高达到95％以上，其IC50＝138.86μg/mL；整体来看，氧化白藜芦醇含量为2.8％的提取物DPPH清除率虽然低于同等浓度的抗坏血酸，但其浓度与DPPH清除率之间具有较好的线性关系，具有一定的抗氧化作用。The test results are shown in Figure 10. Under the test conditions, the DPPH clearance rate of Morus alba extract (sample No. 13) reached a maximum of more than 95%, and its IC50 = 138.86 μg/mL; overall, the oxidized white quinoa Although the DPPH clearance rate of the extract with a retinol content of 2.8% is lower than that of ascorbic acid with the same concentration, there is a good linear relationship between its concentration and DPPH clearance rate, and it has a certain antioxidant effect.
DPPH自由基清除率(％)＝(A0-A1+A2)/A0*100％DPPH radical scavenging rate (%) = (A 0 -A 1 +A 2 )/A 0 *100%
其中A0为对照组吸光度值；A1为实验组吸光度值；A2为空白组吸光度值；阳性对照组A2为0。Among them, A0 is the absorbance value of the control group; A1 is the absorbance value of the experimental group; A2 is the absorbance value of the blank group; A2 of the positive control group is 0.
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。 All documents mentioned in this application are incorporated by reference in this application to the same extent as if each individual document was individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims (10)

  1. 一种氧化白藜芦醇的制备方法，其特征在于，包括步骤：A method for preparing oxidized resveratrol, which is characterized by comprising the steps:
    (S1)提供含桑皮苷A的原料；(S1) Provide raw materials containing morin A;
    (S2)将所述含桑皮苷A的原料，用β-葡聚糖酶进行酶解处理，将桑皮苷A转化为氧化白藜芦醇，从而获得酶解产物：
    (S2) Enzymatically hydrolyze the raw material containing moryside A with β-glucanase to convert moryside A into oxidized resveratrol, thereby obtaining an enzymatic hydrolysis product:
    其中，在步骤(S2)中，按重量计的β-葡聚糖酶的用量E1与桑皮苷A重量W1之比为6-20。Wherein, in step (S2), the ratio by weight of the amount of β-glucanase E1 to the weight W1 of morin A is 6-20.
  2. 如权利要求1所述的方法，其特征在于，在步骤(S2)中，桑皮苷A的酶解率≥90％。The method of claim 1, wherein in step (S2), the enzymatic hydrolysis rate of morin A is ≥90%.
  3. 如权利要求1所述的方法，其特征在于，在步骤(S2)中，与酶解前的含桑皮苷A的原料相比，所述酶解产物中氧化白藜芦醇的相对增加幅度≥1000％。The method according to claim 1, characterized in that, in step (S2), compared with the raw material containing morin A before enzymatic hydrolysis, the relative increase of oxidized resveratrol in the enzymatic hydrolysis product is ≥1000%.
  4. 如权利要求1所述的方法，其特征在于，所述的含桑皮苷A的原料为桑枝或桑白皮药材的水提取物、乙醇提取物、或乙醇/水提取物。The method according to claim 1, characterized in that the raw material containing mulberry glycoside A is a water extract, an ethanol extract, or an ethanol/water extract of mulberry branches or mulberry bark medicinal materials.
  5. 如权利要求1所述的方法，其特征在于，在步骤(S2)中，β-葡聚糖酶的用量E1与桑皮苷A重量W1之比(E1/W1)为6-15。The method according to claim 1, characterized in that, in step (S2), the ratio of the dosage E1 of β-glucanase to the weight W1 of morin A (E1/W1) is 6-15.
  6. 如权利要求1所述的方法，其特征在于，在酶解处理之前，所述的含桑皮苷A的原料中，桑皮苷A的浓度为0.30-2.00mg/g。The method according to claim 1, characterized in that, before the enzymatic hydrolysis treatment, the concentration of morrin A in the raw material containing morrin A is 0.30-2.00 mg/g.
  7. 如权利要求1所述的方法，其特征在于，在步骤(S2)中，在所述酶解产物中，桑皮苷A的浓度≤0.10mg/g；和/或，在所述酶解产物中，氧化白藜芦醇的浓度≥0.30mg/g。The method of claim 1, wherein in step (S2), in the enzymatic hydrolysis product, the concentration of morin A is ≤0.10 mg/g; and/or, in the enzymatic hydrolysis product Among them, the concentration of oxidized resveratrol is ≥0.30mg/g.
  8. 如权利要求1所述的方法，其特征在于，所述方法还包括步骤：The method of claim 1, further comprising the steps of:
    (S3)从(S2)步骤中经酶解处理的酶解产物中，分离氧化白藜芦醇，获得分离的氧化白藜芦醇粗品。(S3) Separate oxidized resveratrol from the enzymatic hydrolysis product subjected to enzymatic hydrolysis treatment in step (S2) to obtain separated crude oxidized resveratrol.
  9. 如权利要求8所述的方法，其特征在于，所述的分离采用大孔吸附树脂进行分离氧化白藜芦醇。The method of claim 8, characterized in that the separation uses macroporous adsorption resin to separate and oxidize resveratrol.
  10. 如权利要求1-9中任一项所述的方法，其特征在于，所述方法还包括：The method according to any one of claims 1-9, characterized in that the method further includes:
    将制备的氧化白藜芦醇，作为抗氧化或美白的添加剂添加入终产品。 The prepared oxidized resveratrol is added to the final product as an antioxidant or whitening additive.
PCT/CN2023/102920 2022-07-04 2023-06-27 Method for preparing oxyresveratrol WO2024007901A1 (en)

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