WO2020252800A1 - 一种增强酪氨酸酶抑制活性的方法 - Google Patents

一种增强酪氨酸酶抑制活性的方法 Download PDF

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WO2020252800A1
WO2020252800A1 PCT/CN2019/092511 CN2019092511W WO2020252800A1 WO 2020252800 A1 WO2020252800 A1 WO 2020252800A1 CN 2019092511 W CN2019092511 W CN 2019092511W WO 2020252800 A1 WO2020252800 A1 WO 2020252800A1
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acid
tyrosinase
quercetin
tyrosinase inhibitor
isorhamnetin
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French (fr)
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范柳萍
于群
李进伟
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Jiangnan University
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Jiangnan University
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G13/00—Protection of plants
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23B—PRESERVATION OF FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES; CHEMICAL RIPENING OF FRUIT OR VEGETABLES
    • A23B2/00—Preservation of foods or foodstuffs, in general
    • A23B2/70—Preservation of foods or foodstuffs, in general by treatment with chemicals
    • A23B2/725—Preservation of foods or foodstuffs, in general by treatment with chemicals in the form of liquids or solids
    • A23B2/729—Organic compounds; Microorganisms; Enzymes
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/192—Carboxylic acids, e.g. valproic acid having aromatic groups, e.g. sulindac, 2-aryl-propionic acids, ethacrynic acid 
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
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    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
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    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
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    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • 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
    • A61K8/36—Carboxylic acids; Salts or anhydrides thereof
    • 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
    • A61K8/36—Carboxylic acids; Salts or anhydrides thereof
    • A61K8/365—Hydroxycarboxylic acids; Ketocarboxylic acids
    • 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
    • A61K8/36—Carboxylic acids; Salts or anhydrides thereof
    • A61K8/368—Carboxylic acids; Salts or anhydrides thereof with carboxyl groups directly bound to carbon atoms of aromatic rings
    • 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
    • A61K8/37—Esters of carboxylic acids
    • A61K8/375—Esters of carboxylic acids the alcohol moiety containing more than one hydroxy group
    • 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
    • A61K8/49—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds
    • A61K8/4973—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with oxygen as the only hetero atom
    • A61K8/498—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with oxygen as the only hetero atom having 6-membered rings or their condensed derivatives, e.g. coumarin
    • 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
    • A61K8/63—Steroids; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
    • A61K2800/59—Mixtures
    • A61K2800/592—Mixtures of compounds complementing their respective functions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
    • A61K2800/59—Mixtures
    • A61K2800/592—Mixtures of compounds complementing their respective functions
    • A61K2800/5922—At least two compounds being classified in the same subclass of A61K8/18
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/74—Biological properties of particular ingredients
    • A61K2800/78—Enzyme modulators, e.g. Enzyme agonists
    • A61K2800/782—Enzyme inhibitors; Enzyme antagonists

Definitions

  • the invention relates to a method for enhancing the inhibitory activity of tyrosinase and belongs to the technical field of intensive and deep processing of fruits and vegetables.
  • Tyrosinase is the key rate-limiting enzyme for melanin formation in bacteria, fungi, plants, mammals and humans. It is one of the reasons for the browning of fresh fruits and vegetables, beverages and the production of melanin in human skin. Tyrosinase inhibitors can effectively reduce the production of melanin and browning in food systems by inhibiting the activity of tyrosinase, thereby reducing the adverse effects caused by tyrosinase. Therefore, in recent years, with tyrosinase Enzyme inhibitors are constantly being discovered, and their research and development have also attracted increasing attention.
  • Phenolic substances (quercetin, ferulic acid, cinnamic acid), saponins (dioscin, proto-dioscin) are common active ingredients in fruit and vegetable products, which are anti-anxiety, diuretic, improve sleep quality, lower blood sugar, anti-tumor, Improve memory, anti-aging, reduce irritability and fatigue and other functions, and they also have significant effects on whitening.
  • tyrosinase inhibitors are mainly divided into animal sources (silk gum, collagen peptides, etc.), plant sources (platycodin, myricetin, catechin, aloin, etc.) according to their sources, and microbial sources (botrytis , Kojic acid, etc.), chemical compounds (vitamin C, superoxide dismutase, hydrogen peroxide, etc.).
  • animal sources sik gum, collagen peptides, etc.
  • plant sources platycodin, myricetin, catechin, aloin, etc.
  • microbial sources botrytis , Kojic acid, etc.
  • chemical compounds vitamin C, superoxide dismutase, hydrogen peroxide, etc.
  • the present invention uses quercetin, cinnamic acid and ferulic acid as representative polyphenols, and original diosgenin and diosgenin as representative saponins.
  • the method of heating treatment improves its inhibitory effect on tyrosinase.
  • the first object of the present invention is to provide a method for enhancing the activity of tyrosinase inhibitors, by heating treatment to increase the inhibitory effect of tyrosinase inhibitors on tyrosinase; the tyrosinase inhibition
  • the agent is composed of phenolic acid with a concentration of 0.6-1.8 mg/mL and saponin with a concentration of 0.5-1.5 mg/mL; the heating treatment conditions are: the heating temperature is 130-170° C., and the time is 30-120 min.
  • the second object of the present invention is to provide a tyrosinase inhibitor composed of phenolic acid at a concentration of 0.6-1.8 mg/mL and saponin at a concentration of 0.5-1.5 mg/mL.
  • the phenolic acid is three of quercetin, cinnamic acid, ferulic acid, gallic acid, and isorhamnetin, including but not limited to the following combinations:
  • the dosage ratio of any three components of phenolic acid is 1: (1-10): (1-10).
  • the saponins include one of original diosgenin, diosgenin, and smilagein.
  • the third objective of the present invention is to provide the application of the tyrosinase inhibitor in the fields of beauty and health care, treatment of pigmented skin diseases, pest control, and food preservation.
  • the fourth object of the present invention is to provide a composition containing the above-mentioned tyrosinase inhibitor.
  • the composition further includes a compound represented by formula (I):
  • the fifth object of the present invention is to provide a compound that inhibits tyrosinase activity, the structural formula of the compound is shown in formula (I):
  • polyphenols and saponins are compounded to form a tyrosinase inhibitor, the relative activity of tyrosinase can be as low as 5% after treatment, and the three components of polyphenols can produce a synergistic effect;
  • Figure 1 is the liquid quality diagram of the mixture of quercetin, cinnamic acid and ferulic acid before heating;
  • Figure 2 shows the liquid quality diagram of the mixture of quercetin, ferulic acid, and cinnamic acid after heating at 150°C for 30 minutes;
  • Figure 3 shows the structure of newly formed substances.
  • Determination method of tyrosinase activity using spectrophotometry, using 2.8mL 2.5mM L-dopa solution as substrate, taking 100 ⁇ L of the treated inhibitor solution, and then adding 100 ⁇ L 0.2mg/mL tyrosine After the enzyme solution was incubated at 30°C for 15 minutes, its absorbance was measured at 475nm, and the tyrosinase activity without inhibitor was recorded as 100%.
  • the calculation formula is as follows:
  • a 1 and A 2 represent the absorbance values of the blank group at 0 min and 15 min
  • B 1 and B 2 represent the absorbance values of the sample at 0 min and 15 min.
  • the evaluation method of the interaction refer to the literature such as Fan et al. (2017, Food & Function) and Cai et al. (2012, Journal of Agricultural and Food Chemistry), the specific method is: use 2.8 mL 2.5 mM L-dopa solution as the substrate, Take 100 ⁇ L of inhibitor solution with different treatment methods and different ratios, and then add 100 ⁇ L 0.2mg/mL tyrosinase solution, incubate at 30°C for 15min, and measure the change in absorbance at 475nm.
  • Vab (or Vabc, Vabcd)-V* ⁇ -0.1 is synergistic effect (SY); when -0.1 ⁇ Vab (or Vabc, Vabcd)-V* ⁇ +0.1 is additive effect (AD); when Vab (or Vabc) ,Vabcd)-V*>+0.10 superposition effect (SU).
  • the technical scheme of the present invention includes the following steps:
  • the technical scheme of the present invention includes the following steps:
  • the technical scheme of the present invention includes the following steps:
  • Saponins and polyphenols are mixed: the polyphenols in step 1) are mixed with smilaxogenin at a concentration of 1.5 mg/mL;
  • the technical scheme of the present invention includes the following steps:
  • the technical scheme of the present invention includes the following steps:
  • quercetin, cinnamic acid and isorhamnetin are mixed according to the ratio of 5mM quercetin+5mM cinnamic acid+0.5mM isorhamnetin; in an implementation method of the present invention, its characteristics
  • the dosage of the quercetin, ferulic acid and isorhamnetin is 5mM quercetin+5mM ferulic acid+0.5mM isorhamnetin.
  • the technical scheme of the present invention includes the following steps:
  • Tyrosinase inhibitor Relative activity of tyrosinase (%) Vabc V* 2 (Va ⁇ Vb ⁇ Vc) Vabc-V* 2
  • Example 1 33.62 0.7870 0.9819 -0.195SY
  • Example 2 48.71 0.7739 0.9039 -0.130SY
  • Example 3 40.30 0.8196 0.9605 -0.141SY
  • Example 4 44.23 0.7344 0.8724 -0.138SY
  • Example 6 35.33 0.7644 0.9467 -0.182SY
  • any three phenolic acid combinations can produce synergistic effects.
  • the tyrosinase inhibitor prepared by the method of the present invention is used to treat tyrosinase, the relative activity of tyrosinase can be as low as 33.62%, and it has a good inhibitory effect on tyrosinase.
  • Comparative Example 1 The effect of different components of tyrosinase inhibitor on the inhibitory activity of tyrosinase
  • step 1) Mix the polyphenols according to the polyphenol formulas in Tables 2 to 4, and the other parameters are the same as in Example 1.
  • the p-tyrosine is determined by spectrophotometry.
  • the inhibitory effect of the enzyme the results are shown in Tables 2 to 4.
  • Example 2 According to the method of Example 1, the difference is that a single phenolic acid is used, namely 5mM quercetin, 5mM cinnamic acid, 5mM ferulic acid, 5mM gallic acid, 5mM isorhamnetin, and other parameters are the same as in Example 1.
  • the measured relative activities (%) of tyrosinase were 80.69%, 102.32%, 107.56%, 97.25%, 82.52%, respectively.
  • SY is a synergistic effect
  • AD is a superimposed effect
  • SU is a secondary superimposed effect.
  • SY is a synergistic effect
  • AD is a superimposed effect
  • SU is a secondary superimposed effect.
  • SY is a synergistic effect
  • AD is a superimposed effect
  • SU is a secondary superimposed effect.
  • Comparative Example 2 The effect of heat treatment on the inhibition of tyrosinase activity by tyrosinase inhibitors
  • Example 1 According to the method of Example 1, the difference is that the heat treatment is not performed, and other parameters are the same as in Example 1.
  • the measured relative activity of tyrosinase is 78.82%, and the relative activity of tyrosinase after heat treatment is 33.62 % (Example 1), indicating that heat treatment is beneficial to the inhibitory effect of tyrosinase inhibitor on tyrosinase.
  • Example 2 According to the method of Example 1, the difference is that the heating temperature is set to 100°C, and other parameters are the same as in Example 1.
  • the measured relative activity of tyrosinase is 70.56%.
  • the relative activity of oxidase is 33.62% (Example 1), indicating that too low temperature will affect the effect of tyrosinase inhibitors.
  • Example 3 According to the method of Example 1, the difference is that the heating temperature is set to 200°C, and the other parameters are the same as in Example 1.
  • the measured relative activity of tyrosinase is 65.78%, and the tyrosinase is heated at 150°C.
  • the relative activity of oxidase is 33.62% (Example 1), indicating that too high temperature will affect the effect of tyrosinase inhibitors.

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Abstract

本发明公开了一种增强酪氨酸酶抑制活性的方法，属于果蔬精深加工技术领域。本发明以0.6-1.8mg/mL浓度的酚酸和0.5-1.5mg/mL浓度的皂苷组成酪氨酸酶抑制剂；所述酚酸是槲皮素、肉桂酸、阿魏酸、没食子酸、异鼠李素中的三种；酚酸任意三种组分用量比为1：(1-10)：(1-10)；所述皂苷包括原薯蓣皂苷、薯蓣皂苷、菝葜皂苷元中的一种。本发明采用多酚和皂苷复配成酪氨酸酶抑制剂，处理后酪氨酸酶相对活性最低可达5％，且三种组分的多酚可产生协同效应；高温会使得皂苷去糖基化，生成多酚聚合物等高酪氨酸酶抑制作用的物质，因此大大提高了酪氨酸酶抑制剂对酪氨酸酶的抑制作用。

Description

一种增强酪氨酸酶抑制活性的方法 技术领域
本发明涉及一种增强酪氨酸酶抑制活性的方法，属于果蔬精深加工技术领域。
背景技术
酪氨酸酶是细菌、真菌、植物、哺乳动物以及人体中黑色素形成的关键限速酶，是导致新鲜果蔬、饮料产生褐变、人体皮肤黑色素产生的原因之一。而酪氨酸酶抑制剂可以通过抑制酪氨酸酶的活性，有效的减少黑色素和食品体系褐变的产生，从而降低因酪氨酸酶产生的不利影响，因此，近年来随着酪氨酸酶抑制剂不断发现，对它的研究开发也日益受到人们重视。酚类物质(槲皮素、阿魏酸、肉桂酸)、皂苷(薯蓣皂苷、原薯蓣皂苷)是果蔬产品中常见的活性成分，具有抗焦虑、利尿、改善睡眠质量、降血糖、抗肿瘤、改善记忆、抗衰老、降低烦躁和疲劳感等功能，同时它们对美白也具有显著效果。
现有的酪氨酸酶抑制剂根据其来源主要分为动物来源(蚕丝胶、胶原肽等)，植物来源(桔梗皂苷、杨梅素、儿茶素、芦荟苷等)，微生物来源(葡萄孢菌、曲酸等)，化学合成物(维生素C、超氧化物歧化酶、过氧化氢等)。目前有关酪氨酸酶抑制剂的研究主要集中在单一活性成分对酪氨酸酶活性的影响，其中人工合成化合物副作用较强，而天然活性成分则存在抑制效果不显著的特点。
发明内容
针对上述问题，本发明以槲皮素、肉桂酸和阿魏酸为代表多酚，以原薯蓣皂苷和薯蓣皂苷为代表皂苷，经过加热处理的方法提高其对酪氨酸酶的抑制作用。
本发明的第一个目的是提供一种增强酪氨酸酶抑制剂活性的方法，通过加热处理的方式提高酪氨酸酶抑制剂对酪氨酸酶的抑制作用；所述酪氨酸酶抑制剂由0.6-1.8mg/mL浓度的酚酸和0.5-1.5mg/mL浓度的皂苷组成；所述加热处理条件为：加热温度为130-170℃，时间为30-120min。
本发明的第二个目的是提供一种酪氨酸酶抑制剂，该酪氨酸酶抑制剂由0.6-1.8mg/mL浓度的酚酸和0.5-1.5mg/mL浓度的皂苷组成。
在本发明一种实施方法中，所述酚酸是槲皮素、肉桂酸、阿魏酸、没食子酸、异鼠李素中的三种，包括但不限于以下几种组合：
(a)槲皮素，肉桂酸和阿魏酸；
(b)槲皮素，没食子酸和阿魏酸；
(c)槲皮素，没食子酸和异鼠李素；
(d)槲皮素，肉桂酸和异鼠李素；
(e)槲皮素，阿魏酸和异鼠李素。
在本发明一种实施方法中，酚酸任意三种组分用量比为1：(1-10)：(1-10)。
在本发明一种实施方法中，所述皂苷包括原薯蓣皂苷、薯蓣皂苷、菝葜皂苷元中的一种。
本发明的第三个目的是提供所述酪氨酸酶抑制剂在美容保健、色素型皮肤病的治疗、病虫害防治以及食品保鲜等方面的应用。
本发明的第四个目的是提供一种含有上述酪氨酸酶抑制剂的组合物。
在本发明一种实施方式中，所述组合物还包括式(Ⅰ)所示的化合物：
Figure PCTCN2019092511-appb-000001
本发明的第五个目的是提供一种具有抑制酪氨酸酶活性的化合物，所述化合物的结构式如式(Ⅰ)所示：
Figure PCTCN2019092511-appb-000002
本发明的有益效果：
(1)本发明采用多酚和皂苷复配成酪氨酸酶抑制剂，处理后酪氨酸酶相对活性最低可达5％，且三种组分的多酚可产生协同效应；
(2)高温会使得皂苷去糖基化，生成多酚聚合物等高酪氨酸酶抑制作用的物质，因此大大提高了酪氨酸酶抑制剂对酪氨酸酶的抑制作用。
附图说明
图1为槲皮素、肉桂酸、阿魏酸混合物加热前液质图；
图2为槲皮素、阿魏酸、肉桂酸经150℃加热30min后混合物的液质图；
图3为新生成物质结构图。
具体实施方式
1、酪氨酸酶活性的测定方法：采用分光光度法，用2.8mL 2.5mM L-多巴溶液作为底物， 取处理好的抑制剂溶液100μL，然后加入100μL 0.2mg/mL的酪氨酸酶溶液在30℃下孵育15min后在475nm下测量其吸光值变化，以未加抑制剂的酪氨酸酶活性记为100％，计算公式如下：
酪氨酸酶相对活性/％＝(B 2-B 1)/(A 2-A 1)×100
A 1和A 2表示空白组在0min和15min时的吸光值，B 1和B 2表示样品在0min和15min时的吸光值。
2、相互作用的评价方法：参考Fan等(2017，Food&Function)和Cai等(2012，Journal of Agricultural and Food Chemistry)等文献，具体方法为：用2.8mL 2.5mM L-多巴溶液作为底物，取不同处理方法和不同配比的抑制剂溶液100μL，然后加入100μL 0.2mg/mL的酪氨酸酶溶液在30℃下孵育15min后在475nm下测量其吸光值变化。
当Vab(或Vabc,Vabcd)-V*<-0.1为协同效应(SY)；当-0.1<Vab(或Vabc,Vabcd)-V*<+0.1为叠加效应(AD)；当Vab(或Vabc,Vabcd)-V*>+0.10次叠加效应(SU)。
实施例1：
本发明的技术方案包括以下步骤：
1)多酚混匀：将槲皮素，肉桂酸和阿魏酸按照5mM槲皮素+5mM肉桂酸+5mM阿魏酸配比混合；
2)皂苷与多酚混匀：将步骤1)中多酚与浓度为1.0mg/mL的原薯蓣皂苷混合；
3)加热：将步骤2)中的样品溶液在150℃下进行加热处理，时间为30min；
4)测定其对酪氨酸酶的抑制作用，将上述不同处理后的溶液稀释成不同浓度，通过分光光度法测定其对酪氨酸酶的抑制作用，结果见表1。
由图1和2可知，在150℃、30min的加热条件下，新生成了保留时间为10.11min的物质，且根据浓度推断，此物质可能是由于阿魏酸经过酯化反应生成的新物质。由图3可知，可推测出生成的新物质的结构式如式(Ⅰ)所示：
Figure PCTCN2019092511-appb-000003
实施例2：
本发明的技术方案包括以下步骤：
1)多酚混匀：将槲皮素，肉桂酸和阿魏酸按照5mM槲皮素+0.5mM肉桂酸+5mM阿魏酸 配比混合；
2)皂苷与多酚混匀：将步骤1)中多酚与浓度为0.5mg/mL的原薯蓣皂苷混合；
3)加热：将步骤2)中的样品溶液在150℃下进行加热处理，时间为30min；
4)测定其对酪氨酸酶的抑制作用，将上述不同处理后的溶液稀释成不同浓度，通过分光光度法测定其对酪氨酸酶的抑制作用，结果见表1。
实施例3：
本发明的技术方案包括以下步骤：
1)多酚混匀：将槲皮素，没食子酸和阿魏酸按照5mM槲皮素+0.5mM没食子酸+5mM阿魏酸配比混合；
2)皂苷与多酚混匀：将步骤1)中多酚与浓度为1.5mg/mL的菝葜皂苷元混合；
3)加热：将步骤2)中的样品溶液在130℃下进行加热处理，时间为30min；
4)测定其对酪氨酸酶的抑制作用，将上述不同处理后的溶液稀释成不同浓度，通过分光光度法测定其对酪氨酸酶的抑制作用，结果见表1。
实施例4：
本发明的技术方案包括以下步骤：
1)多酚混匀：将槲皮素，没食子酸和异鼠李素按照5mM槲皮素+0.5mM没食子酸+0.5mM异鼠李素配比混合；
2)皂苷与多酚混匀：将步骤1)中多酚与浓度为1.0mg/mL的原薯蓣皂苷混合；
3)加热：将步骤2)中的样品溶液在170℃下进行加热处理，时间为30min；
4)测定其对酪氨酸酶的抑制作用，将上述不同处理后的溶液稀释成不同浓度，通过分光光度法测定其对酪氨酸酶的抑制作用，结果见表1。
实施例5：
本发明的技术方案包括以下步骤：
1)多酚混匀：将槲皮素，肉桂酸和异鼠李素按照5mM槲皮素+5mM肉桂酸+0.5mM异鼠李素配比混合；在本发明一种实施方法中，其特征在于，所述槲皮素，阿魏酸和异鼠李素的用量为5mM槲皮素+5mM阿魏酸+0.5mM异鼠李素。
2)皂苷与多酚混匀：将步骤1)中多酚与浓度为1.0mg/mL的原薯蓣皂苷混合；
3)加热：将步骤2)中的样品溶液在150℃下进行加热处理，时间为30min；
4)测定其对酪氨酸酶的抑制作用，将上述不同处理后的溶液稀释成不同浓度，通过分光光度法测定其对酪氨酸酶的抑制作用，结果见表1。
实施例6：
本发明的技术方案包括以下步骤：
1)多酚混匀：将槲皮素，阿魏酸和异鼠李素按照5mM槲皮素+5mM阿魏酸+0.5mM异鼠李素配比混合；
2)皂苷与多酚混匀：将步骤1)中多酚与浓度为1.0mg/mL的原薯蓣皂苷混合；
3)加热：将步骤2)中的样品溶液在150℃下进行加热处理，时间为30min；
4)测定其对酪氨酸酶的抑制作用，将上述不同处理后的溶液稀释成不同浓度，通过分光光度法测定其对酪氨酸酶的抑制作用，结果见表1。
表1酪氨酸酶抑制剂对酪氨酸酶的作用效果
酪氨酸酶抑制剂 酪氨酸酶相对活性(％) Vabc V* 2(Va×Vb×Vc) Vabc-V* 2
实施例1 33.62 0.7870 0.9819 -0.195SY
实施例2 48.71 0.7739 0.9039 -0.130SY
实施例3 40.30 0.8196 0.9605 -0.141SY
实施例4 44.23 0.7344 0.8724 -0.138SY
实施例5 35.24 0.7252 0.8918 -0.167SY
实施例6 35.33 0.7644 0.9467 -0.182SY
由表1可知，任意三种酚酸复配都能产生协同作用。另外，采用本发明方法制备的酪氨酸酶抑制剂处理酪氨酸酶，酪氨酸酶相对活性最低可达33.62％，对酪氨酸酶具有较好的抑制效果。
对比例1：不同组分的酪氨酸酶抑制剂对酪氨酸酶的抑制活性的影响
1、采用实施例1的方法，区别在于，步骤1)分别按照表2～4中的多酚配方将多酚混匀，其它参数条件同实施例1，通过分光光度法测定其对酪氨酸酶的抑制作用，结果见表2～4。
由表2～4可知，单一酚酸对酪氨酸酶呈现激活的作用效果，但当与槲皮素复配后呈现了明显的抑制作用。双组分多酚和四组分多酚组合的抑制剂只能达到叠加效应，不能实现协同效应。
2、按照实施例1的方法，区别在于，采用单一酚酸，即5mM槲皮素、5mM肉桂酸、5mM阿魏酸、5mM没食子酸、5mM异鼠李素，其它参数条件同实施例1，测得的酪氨酸酶相对活性(％)分别为80.69％、102.32％、107.56％、97.25％、82.52％。
表2双组分多酚组成的酪氨酸酶抑制剂对酪氨酸酶的作用效果
Figure PCTCN2019092511-appb-000004
注：SY为协同效应；AD为叠加效应；SU为次叠加效应。
表3三组分多酚组成的酪氨酸酶抑制剂对酪氨酸酶的作用效果
Figure PCTCN2019092511-appb-000005
注：SY为协同效应；AD为叠加效应；SU为次叠加效应。
表4四组分多酚组成的酪氨酸酶抑制剂对酪氨酸酶的作用效果
Figure PCTCN2019092511-appb-000006
注：SY为协同效应；AD为叠加效应；SU为次叠加效应。
对比例2：加热处理对酪氨酸酶抑制剂抑制酪氨酸酶活性的影响
1、按照实施例1的方法，区别在于，不进行加热处理，其它参数条件同实施例1，测得的酪氨酸酶相对活性为78.82％，而加热处理后酪氨酸酶相对活性为33.62％(实施例1)，说明加热处理有利于酪氨酸酶抑制剂对酪氨酸酶的抑制作用。
2、按照实施例1的方法，区别在于，将加热温度设置为100℃，其它参数条件同实施例1，测得的酪氨酸酶相对活性为70.56％，而采用150℃加热处理后酪氨酸酶相对活性为33.62％(实施例1)，说明温度过低会影响酪氨酸酶抑制剂的作用。
3、按照实施例1的方法，区别在于，将加热温度设置为200℃，其它参数条件同实施例1，测得的酪氨酸酶相对活性为65.78％，而采用150℃加热处理后酪氨酸酶相对活性为33.62％(实施例1)，说明温度过高会影响酪氨酸酶抑制剂的作用。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。

Claims (16)

  1. 一种增强酪氨酸酶抑制剂活性的方法，其特征在于，通过加热处理的方式提高酪氨酸酶抑制剂对酪氨酸酶的抑制作用；所述酪氨酸酶抑制剂由0.6-1.8mg/mL浓度的酚酸和0.5-1.5mg/mL浓度的皂苷组成；所述加热处理条件为：加热温度为130-170℃，时间为30-120min。
  2. 一种酪氨酸酶抑制剂，其特征在于，该酪氨酸酶抑制剂由0.6-1.8mg/mL浓度的酚酸和0.5-1.5mg/mL浓度的皂苷组成。
  3. 根据权利要求2所述的酪氨酸酶抑制剂，其特征在于，所述酚酸由槲皮素、肉桂酸、阿魏酸、异鼠李素、没食子酸中的任意三种组成。
  4. 根据权利要求3所述的酪氨酸酶抑制剂，其特征在于，所述酚酸包括但不限于以下几种组合：
    (a)槲皮素，肉桂酸和阿魏酸；
    (b)槲皮素，没食子酸和阿魏酸；
    (c)槲皮素，没食子酸和异鼠李素；
    (d)槲皮素，肉桂酸和异鼠李素；
    (e)槲皮素，阿魏酸和异鼠李素。
  5. 根据权利要求3所述的酪氨酸酶抑制剂，其特征在于，酚酸任意三种组分用量比为1：(1-10)：(1-10)。
  6. 根据权利要求3所述的酪氨酸酶抑制剂，其特征在于，酚酸由5mM槲皮素、5mM肉桂酸和5mM阿魏酸组成。
  7. 根据权利要求3所述的酪氨酸酶抑制剂，其特征在于，酚酸由5mM槲皮素、0.5mM肉桂酸和5mM阿魏酸组成。
  8. 根据权利要求3所述的酪氨酸酶抑制剂，其特征在于，酚酸由5mM槲皮素、0.5mM没食子酸和5mM阿魏酸组成。
  9. 根据权利要求3所述的酪氨酸酶抑制剂，其特征在于，酚酸由5mM槲皮素、0.5mM没食子酸和0.5mM异鼠李素组成。
  10. 根据权利要求3所述的酪氨酸酶抑制剂，其特征在于，酚酸由5mM槲皮素、5mM肉桂酸和0.5mM异鼠李素组成。
  11. 根据权利要求3所述的酪氨酸酶抑制剂，其特征在于，酚酸由5mM槲皮素、5mM阿魏酸和0.5mM异鼠李素组成。
  12. 根据权利要求2所述的酪氨酸酶抑制剂，其特征在于，所述皂苷是原薯蓣皂苷、薯蓣皂苷、菝葜皂苷元中的一种。
  13. 权利要求2所述的酪氨酸酶抑制剂在美容保健、色素型皮肤病的治疗、病虫害防治 以及食品保鲜等方面的应用。
  14. 含有权利要求2所述的酪氨酸酶抑制剂的组合物。
  15. 根据权利要求14所述的组合物，其特征在于，所述组合物还包括式1所示的化合物：
    Figure PCTCN2019092511-appb-100001
  16. 一种具有抑制酪氨酸酶活性的化合物，其特征在于，所述化合物的结构式如式(Ⅰ)所示：
    Figure PCTCN2019092511-appb-100002
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CHUN LIANG; JU-HWAN LIM; SEUNG-HYUNG KIM; DONG-SEON KIM;: "Dioscin: A synergistic tyrosinase inhibitor from the roots of", FOOD CHEMISTRY, ELSEVIER LTD., NL, vol. 134, no. 2, 1 March 2012 (2012-03-01), NL, pages 1146 - 1148, XP028417813, ISSN: 0308-8146, DOI: 10.1016/j.foodchem.2012.03.003 *
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