WO2022241749A1 - 一种原飞燕草素b9没食子酸酯的化学合成方法 - Google Patents

一种原飞燕草素b9没食子酸酯的化学合成方法 Download PDF

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WO2022241749A1
WO2022241749A1 PCT/CN2021/095046 CN2021095046W WO2022241749A1 WO 2022241749 A1 WO2022241749 A1 WO 2022241749A1 CN 2021095046 W CN2021095046 W CN 2021095046W WO 2022241749 A1 WO2022241749 A1 WO 2022241749A1
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gallate
mobile phase
prodelphinidin
hydrochloric acid
purification
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PCT/CN2021/095046
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French (fr)
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陈士国
徐新雷
潘海波
叶兴乾
王艺
程焕
陈健乐
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浙江大学
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Priority to PCT/CN2021/095046 priority Critical patent/WO2022241749A1/zh
Priority to US17/779,274 priority patent/US20240182435A1/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/58—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4
    • C07D311/64—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4 with oxygen atoms directly attached in position 8
    • 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
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D11/00—Solvent extraction
    • B01D11/02—Solvent extraction of solids
    • B01D11/0288—Applications, solvents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/58—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4
    • C07D311/60—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4 with aryl radicals attached in position 2
    • C07D311/62—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4 with aryl radicals attached in position 2 with oxygen atoms directly attached in position 3, e.g. anthocyanidins

Definitions

  • the invention relates to the field of plant functional components, in particular to a preparation method of prodelphinidin B9 gallate.
  • Proanthocyanidins are a kind of polymerized polyphenols with flavan-3-ol as the structural unit. They are widely found in plants and are the second largest group of dietary polyphenols after lignin. Its main structural units are (epi)catechin, (epi)afotoin, (epi)gallocatechin (E)GC and its gallate. A large number of studies have shown that proanthocyanidins have various beneficial biological activities such as anti-oxidation, hypoglycemic, and weight loss. And these functions are closely related to its structure, especially the type of structural unit and degree of aggregation. First, the degree of polymerization of proanthocyanidins determines its bioavailability.
  • proanthocyanidins with a degree of polymerization greater than 4 are not absorbed at all.
  • the density of phenolic hydroxyl groups in proanthocyanidins is closely related to its biological activity. Studies have shown that compared with proanthocyanidins without galloyl groups, proanthocyanidins containing galloyl groups in their structural units exhibit stronger biological activity due to their higher density of phenolic hydroxyl groups. Among them, procyanidin with (E)GC and its gallate (E)GCG as structural units is a proanthocyanidin with strong activity.
  • proanthocyanidins which have been studied more, use (epi)catechin, (epi)alfocin and their gallate as structural units. Due to their rich sources, the structure-activity relationship research using their dimers as materials is relatively clear. ; while the studies on procyanidin are based on the proanthocyanidin mixture.
  • the proanthocyanidins derived from bayberry leaves have a typical prodelphinidin structure, the main structural units are EGC and EGCG, and have strong antioxidant activity in vitro, but due to their high degree of polymerization, most of them are distributed between 9.5 and 26.7 However, the bioavailability is extremely low, the in vivo activity is poor, and the mechanism of action is unclear. The content of prodelphinidin with low degree of polymerization is extremely low, and the separation and purification are difficult, which leads to the stagnation of its structure-activity relationship research.
  • the object of the present invention is to provide a kind of chemical synthesis method of protodelphinidin B9 gallate
  • the present invention uses bayberry leaf proanthocyanidin as raw material, takes EGCG as nucleophilic substrate, synthesizes two kinds of bis-gallates under acid catalysis Polyprotodelphinidin gallate (protodelphinidin B-3'-gallate and protodelphinidin B-3,3'-bis-gallate), increasing the content of its components, reducing further Purification difficulty.
  • the obtained dimerized procyanidin gallate is a B-type proanthocyanidin dimer with EGCG as the structural unit, which not only improves the bioavailability of procyanidin-type proanthocyanidins, but also provides a basis for the study of its structure-activity relationship. High purity material.
  • a chemical synthesis method of prodelphinidin B9 gallate specifically:
  • protodelphinidin B-3'-gallate and protodelphinidin B-3,3'-digallate have the following structural formula:
  • bayberry leaf proanthocyanidins The extraction and purification methods of bayberry leaf proanthocyanidins refer to the patent "A Method for Separating and Preparing Red bayberry leaf proanthocyanidins", and the purity of the obtained bayberry leaf proanthocyanidins is 86.4%; 98%.
  • the drying method is as follows: the reaction solution after reacting for 40 minutes is rotatably evaporated at 40°C to remove methanol and a small amount of water to obtain dry powder of the crude reaction product;
  • Shimadzu LC-20 semi-preparative liquid chromatography is used to separate and purify the obtained reaction crude product to obtain prodelphinidin B9 gallate.
  • the separation and purification also includes the second purification:
  • Shim-pack GIST C18 (20mmx250mm, 5 ⁇ m) was used for the second purification, mobile phase A was ultrapure water, and mobile phase B was 98vol% acetonitrile (containing 0.05vol% phosphoric acid). Dissolve the purified prodelphinidin B9 gallate in the mobile phase A and B mixture with a volume ratio of 80:20 to prepare a 50 mg/mL solution. After injecting 2 mL, the mobile phase was eluted and purified. The flow rate of the mobile phase was 15 mL/min, and the elution gradient was: 0-5 min, 20-40 vol% B; 5-15 min, 40 vol % B.
  • the invention uses bayberry leaf proanthocyanidin as a raw material, uses EGCG as a nucleophile, attacks the structural unit EGCG of bayberry leaf proanthocyanidin and the C4 site of EGC under the catalysis of hydrochloric acid, and synthesizes prodelphinidin B9 gallate .
  • EGCG EGCG
  • the purity and yield of prodelphinidin B9 gallate prepared by the method provided by the invention are both higher. It can be directly applied in the food field as a nutritional enhancer and natural antioxidant.
  • Fig. 1 is the synthetic HPLC analytical spectrum of former delphinidin B9 gallate in embodiment 1;
  • Fig. 2 is the synthetic HPLC analysis spectrum of former delphinidin B9 gallate in embodiment 2;
  • Fig. 3 is the synthetic HPLC analytical spectrum of former delphinidin B9 gallate in embodiment 3;
  • Fig. 4 is the synthetic HPLC analysis spectrum of former delphinidin B9 gallate in embodiment 4;
  • Fig. 5 is the synthetic HPLC analysis spectrum of former delphinidin B9 gallate in embodiment 5;
  • Fig. 6 is the synthetic HPLC analysis spectrum of former delphinidin B9 gallate in embodiment 6;
  • Figure 7 is the HPLC analysis spectrum of prodelphinidin B-3'-gallate prepared in Example 1, with a purity of 93.2%.
  • Fig. 8 is the HPLC analysis spectrum of prodelphinidin B-3,3'-digallate prepared in Example 1, the purity is 96.6%.
  • Fig. 9 is the mass spectrum of prodelphinidin B-3'-gallate prepared in Example 1, with molecular weights of 762.
  • Figure 10 is the mass spectrum of prodelphinidin B-3,3'-digallate prepared in Example 1, with a molecular weight of 914.
  • Figure 11 is the 13 C NMR chart of prodelphinidin B-3'-gallate prepared in Example 1;
  • Figure 12 is the 13 C NMR chart of prodelphinidin B-3,3'-digallate prepared in Example 1;
  • the peak eluting time of prodelphinidin B-3'-gallate 1 and prodelphinidin B-3,3'-bis-gallate 2 are respectively 9.9min and 11.7min min.
  • reaction condition reaction condition, and its drying, separation and purification (twice purification), specifically as follows:
  • Shim-pack GIST C18 (20mmx250mm, 5 ⁇ m) was used for the second purification, mobile phase A was ultrapure water, and mobile phase B was 98vol% acetonitrile (containing 0.05vol% phosphoric acid). Dissolve the purified prodelphinidin B9 gallate in the mobile phase A and B mixture with a volume ratio of 80:20 to prepare a 50 mg/mL solution. After injecting 2 mL, the mobile phase was eluted and purified. The flow rate of the mobile phase was 15 mL/min, and the elution gradient was: 0-5 min, 20-40 vol% B; 5-15 min, 40 vol % B.
  • Adopt high-performance liquid chromatography to analyze former delphinidin B9 gallate
  • specific analysis condition is: high-performance liquid chromatography is Waters 2695, and detector is Waters 2489 ultraviolet-visible light detector, and detection wavelength is 280nm, chromatographic column
  • the model is Luna hilic (4.6mmx250mm, 5 ⁇ m, Phenomemex Inc.); mobile phase A is acetonitrile (containing 0.5vol% acetic acid), mobile phase B is ultrapure water; gradient elution: 0-30min, 12vol%-27vol%B , 30-45min, 27vol%-80vol%B, 45-46min, 80vol%-12vol%B, 46-55min, 12vol%B; flow rate: 0.8mL/min; injection volume: 10 ⁇ L; column temperature: 30°C.
  • the purity and structure detection method of the prepared prodeldelnidin B9 gallate is as follows:
  • Liquid chromatography-mass spectrometry analysis Fast liquid phase-triple quadrupole mass spectrometer Agilent 1290/6460 Triple Quad was used for analysis, and the chromatographic conditions were the same as above.
  • the mass spectrometry conditions are: ionization mode is ESI negative ion mode; mass-to-charge ratio scanning range is 50-1500m/z; desolvation temperature is 400°C; capillary voltage is 3.5kV; collision energy is 16eV.
  • Figures 7 and 8 are the HPLC analysis charts of the prepared prodelphinidin B-3'-gallate and prodelphinidin B-3,3'-digallate, the purity of which is 93.2% and 96.6% respectively.
  • Figures 9 and 10 are mass spectrograms of prodeldelnidin B-3'-gallate and prodeldelnidin B-3,3'-bis-gallate prepared by the present invention. It can be seen that the molecular weights are respectively 762 and 914.
  • Figures 11 and 12 are 13 C NMR figures of prodelphinidin B-3'-gallate and prodelphinidin B-3,3'-bisgallate prepared in the present invention; the carbonyl group of gallic acid ( 166ppm) and galloyl ring carbon signals (138ppm and 110ppm) are the characteristics of galloesterified flavan-3-ol, indicating that its structural unit contains EGCG, and usually A-type proanthocyanidin has a broad peak at 102-104ppm signal, which was not detected, indicating the absence of a bond of type A in the structure, prodelphinidin B-3'-gallate and prodelphinidin B-3,3'-digallate.
  • Embodiment 6 is a diagrammatic representation of Embodiment 6

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Abstract

本发明的目的是提供一种原飞燕草素B9没食子酸酯的化学合成方法，以杨梅叶原花色素为原料，利用EGCG作为亲核试剂，在盐酸催化作用下攻击杨梅叶原花色素的结构单元EGCG和EGC的C4位点，合成原飞燕草素B9没食子酸酯。相较于从茶叶、杨梅叶等材料中提取、分离原飞燕草素B9没食子酸酯，本发明提供的方法所制得的原飞燕草素B9没食子酸酯纯度和得率均较高，可直接应用于食品领域中作为营养增强剂和天然抗氧化剂。

Description

一种原飞燕草素B9没食子酸酯的化学合成方法 技术领域
本发明涉及植物功能组分领域，具体涉及一种原飞燕草素B9没食子酸酯的制备方法。
背景技术
原花色素是一类以黄烷-3-醇为结构单元的聚合型多酚，广泛存在植物中，是继木质素后的第二大类膳食多酚。其主要结构单元是(表)儿茶素、(表)阿福豆素、(表)没食子儿茶素(E)GC及其没食子酸酯。大量研究表明原花色素具有抗氧化、降血糖、减肥等多种有益生物活性。而这些功能与其结构密切相关，尤其是结构单元种类和聚合度。首先，原花色素聚合度决定了其生物利用率，随着聚合度增加，生物利用率降低，聚合度大于4的原花色素完全不被吸收。原花色素结构单元酚羟基密度与其生物活性密切相关。已有研究表明，相比没有棓酰基的原花色素，结构单元中含有棓酰基的原花色素因其更高的酚羟基密度而表现更强的生物活性。其中，以(E)GC及其没食子酸酯(E)GCG为结构单元的原飞燕草素是活性较强的原花色素。
目前研究较多的原花色素以(表)儿茶素、(表)阿福豆素及其没食子酸酯为结构单元，由于其来源丰富，以其二聚体为材料的构效关系研究较为清晰；而关于原飞燕草素的研究均基于原花色素混合物。来源于杨梅叶的原花色素具有典型的原飞燕草素结构，主要结构单元为EGC和EGCG，体外抗氧化活性极强，但是由于其聚合度高，大部分聚合度分布在9.5到26.7之间，生物利用率极低，体内活性差，且作用机制模糊。低聚合度原飞燕草素的含量极低，分离纯化困难大，导致其构效关系研究停滞。
发明内容
本发明的目的是提供一种原飞燕草素B9没食子酸酯的化学合成方法，本发明以杨梅叶原花色素为原料，以EGCG为亲核底物，在酸催化作用下合成两种二聚原飞燕草素没食子酸酯(原飞燕草素B-3'-没食子酸酯和原飞燕草素B-3,3'-双没食子酸酯)，提高其组分含量，降低进一步纯化难度。所得二聚原飞燕草素没食子酸酯是以EGCG为结构单元的B型原花色素二聚体，不仅提高原飞燕草素型原花色素生物利用率，还为其构效关系研究提供高纯度材料。
本发明采用的技术方案具体为：
一种原飞燕草素B9没食子酸酯的化学合成方法，具体为：
分别配置浓度为10-400mg/mL杨梅叶原花色素的含盐酸溶液和10-400mg/mL表没食子儿茶素没食子酸酯的含盐酸溶液；其中，两种含盐酸溶液的盐酸浓度均为0.1-1.0mol/L；将盐酸浓度相同的杨梅叶原花色素和EGCG的含盐酸溶液按体积比1:2-2:1混合后，在20-60℃条件下反应40min，经干燥、分离纯化后获得原飞燕草素B9没食子酸酯，所述原飞燕草素B9没食子酸酯包括原飞燕草素B-3'-没食子酸酯和原飞燕草素B-3,3'-双没食子酸酯。
其中，包括原飞燕草素B-3'-没食子酸酯和原飞燕草素B-3,3'-双没食子酸酯，具有如下结构式：
Figure PCTCN2021095046-appb-000001
所述杨梅叶原花色素提取、纯化方法参照专利《一种分离制备杨梅叶原花色素的方法》，制得的杨梅叶原花色素纯度为86.4％；EGCG为市售HPLC级试剂，纯度≥98％。
作为一优选方案，干燥的方法具体为：反应40min后的反应液在40℃下旋转蒸发，去除甲醇和少量水，获得反应粗产物干粉；
作为一优选方案，利用岛津LC-20型半制备液相色谱对获得的反应粗产物进行分离纯化，获得原飞燕草素B9没食子酸酯。
纯化采用S系列X5H制备柱(10mmx250mm，5μm)，流动相A为含0.5vol％乙酸的乙腈，流动相B为超纯水。将反应粗产物溶解于体积比为88：12的流动相A、B混合液，配制成20mg/mL的溶液。进样2mL后，进行流动相洗脱纯化，流动相流速为5mL/min，洗脱 梯度为：0-10min，12-27vol％B，10-20min，60vol％B，20-26min 12vol％B。设置检测波长为280nm，根据液相图谱，收集5.5-6.0min的流出液，将流出液于40℃条件下旋转蒸发去除乙腈，冷冻干燥后获得原飞燕草素B9没食子酸酯纯化物。
进一步地，所述分离纯化还包括第二次纯化：
第二次纯化采用Shim-pack GIST C18(20mmx250mm，5μm)，流动相A为超纯水，流动相B为98vol％乙腈(含0.05vol％磷酸)。将原飞燕草素B9没食子酸酯纯化物溶解于体积比为80：20的流动相A、B混合液，配制成50mg/mL的溶液。进样2mL后，进行流动相洗脱纯化，流动相流速为15mL/min，洗脱梯度为：0-5min，20-40vol％B；5-15min，40vol％B。设置检测波长为280nm，根据液相图谱，在不同出峰时间分别收集流出液，将流出液于40℃条件下旋转蒸发去除乙腈，冷冻干燥后分别获得原飞燕草素B-3'-没食子酸酯和原飞燕草素B-3,3'-双没食子酸酯。
本发明以杨梅叶原花色素为原料，利用EGCG作为亲核试剂，在盐酸催化作用下攻击杨梅叶原花色素的结构单元EGCG和EGC的C4位点，合成原飞燕草素B9没食子酸酯。相较于从茶叶、杨梅叶等材料中提取、分离原飞燕草素B9没食子酸酯，本发明提供的方法所制得的原飞燕草素B9没食子酸酯纯度和得率均较高。可直接应用于食品领域中作为营养增强剂和天然抗氧化剂。
附图说明
图1为实施例1中原飞燕草素B9没食子酸酯合成HPLC分析图谱；
图2为实施例2中原飞燕草素B9没食子酸酯合成HPLC分析图谱；
图3为实施例3中原飞燕草素B9没食子酸酯合成HPLC分析图谱；
图4为实施例4中原飞燕草素B9没食子酸酯合成HPLC分析图谱；
图5为实施例5中原飞燕草素B9没食子酸酯合成HPLC分析图谱；
图6为实施例6中原飞燕草素B9没食子酸酯合成HPLC分析图谱；
图7为实施例1制备的原飞燕草素B-3'-没食子酸酯HPLC分析图谱，纯度为93.2％
图8为实施例1制备的原飞燕草素B-3,3'-双没食子酸酯HPLC分析图谱，纯度为96.6％。
图9为实施例1制备的原飞燕草素B-3'-没食子酸酯的质谱图，分子量分别为762。
图10为实施例1制备的原飞燕草素B-3,3'-双没食子酸酯的质谱图，分子量为914。
图11为实施例1制备的原飞燕草素B-3'-没食子酸酯的 13C NMR图；
图12为实施例1制备的原飞燕草素B-3,3'-双没食子酸酯的 13C NMR图；
其中，图1-6中，原飞燕草素B-3'-没食子酸酯①和原飞燕草素B-3,3'-双没食子酸酯②的出峰时间分别在9.9min和11.7min。
具体实施方式
为使本发明的目的、技术方案更加清楚，下面对本发明的具体实施方式做进一步详细说明，但并不以此来限制本发明。各实施例的差异在于反应条件，其干燥、分离纯化(两次纯化)，具体如下：
干燥：将反应40min后的反应液在40℃下旋转蒸发，去除甲醇和少量水，获得反应粗产物干粉；
分离纯化：
第一次纯化采用S系列X5H制备柱(10mmx250mm，5μm)，流动相A为含0.5vol％乙酸的乙腈，流动相B为超纯水。将反应粗产物溶解于体积比为88：12的流动相A、B混合液，配制成20mg/mL的溶液。进样2mL后，进行流动相洗脱纯化，流动相流速为5mL/min，洗脱梯度为：0-10min，12-27vol％B，10-20min，60vol％B，20-26min 12vol％B。设置检测波长为280nm，根据液相图谱，收集5.5-6.0min的流出液，将流出液于40℃条件下旋转蒸发去除乙腈，冷冻干燥后获得原飞燕草素B9没食子酸酯纯化物。
第二次纯化采用Shim-pack GIST C18(20mmx250mm，5μm)，流动相A为超纯水，流动相B为98vol％乙腈(含0.05vol％磷酸)。将原飞燕草素B9没食子酸酯纯化物溶解于体积比为80：20的流动相A、B混合液，配制成50mg/mL的溶液。进样2mL后，进行流动相洗脱纯化，流动相流速为15mL/min，洗脱梯度为：0-5min，20-40vol％B；5-15min，40vol％B。设置检测波长为280nm，根据液相图谱，在不同出峰时间分别收集流出液，将流出液于40℃条件下旋转蒸发去除乙腈，冷冻干燥后分别获得原飞燕草素B-3'-没食子酸酯和原飞燕草素B-3,3'-双没食子酸酯。
另外，制得的原飞燕草素B9没食子酸酯合成检测方法为：
采用高效液相色谱(HPLC)分析原飞燕草素B9没食子酸酯，具体分析条件为：高效液相色谱为Waters 2695，检测器为Waters 2489紫外-可见光检测器，检测波长为280nm，色谱柱型号为Luna hilic(4.6mmx250mm，5μm，Phenomemex Inc.)；流动相A为乙腈(含0.5vol％乙酸)，流动相B为超纯水；梯度洗脱：0-30min，12vol％-27vol％B，30-45min，27vol％-80vol％B，45-46min，80vol％-12vol％B，46-55min，12vol％B；流速：0.8mL/min；进样量：10μL；柱温：30℃。
制得的原飞燕草素B9没食子酸酯纯度和结构检测方法为：
液质联用分析：采用快速液相-三重串联四极杆质谱联用仪Agilent 1290/6460 Triple Quad进行分析，色谱条件同上。质谱条件为：电离模式为ESI负离子模式；质荷比扫描范围为50-1500m/z；去溶剂化温度为400℃；毛细管电压为3.5kV；碰撞能量为16eV。
13C NMR分析：采用Agilent DD2-600超导核磁共振波谱仪进行分析，以氘代甲醇为溶剂。
实施例1：
称取杨梅叶原花色素和EGCG各1g，分别溶解于50mL 0.1mol/L HCl甲醇溶液，搅拌均匀至澄清，将50mL杨梅叶原花色素溶液和50mLEGCG溶液在具塞锥形瓶中混合，在40℃下水浴加热40min，经干燥、分离纯化后经检测，如图1所示，原飞燕草素B-3'-没食子酸酯产率为8.5％，原飞燕草素B-3,3'-双没食子酸酯产率20.0％。
图7和8为制备的原飞燕草素B-3'-没食子酸酯和原飞燕草素B-3,3'-双没食子酸酯HPLC分析图谱，纯度分别为93.2％和96.6％。
图9和10为本发明制备的原飞燕草素B-3'-没食子酸酯和原飞燕草素B-3,3'-双没食子酸酯的质谱图，可以看出，分子量分别为762和914。
图11和12为本发明制备的原飞燕草素B-3'-没食子酸酯和原飞燕草素B-3,3'-双没食子酸酯的 13C NMR图；棓酸的羰基(166ppm)及棓酰环碳信号(138ppm和110ppm)是棓酸酯化的黄烷-3-醇的特征，说明其结构单元含EGCG，同时通常A型原花色素在102-104ppm处有一宽峰信号，未检测到该信号，表明结构中不存在A型键，为原飞燕草素B-3'-没食子酸酯和原飞燕草素B-3,3'-双没食子酸酯。
实施例2：
称取杨梅叶原花色素和EGCG各1g，分别溶解于100mL 0.1mol/L HCl甲醇溶液，搅拌均匀至澄清，将50mL杨梅叶原花色素溶液和25mL EGCG溶液在具塞锥形瓶中混合，在40℃下水浴加热40min，经干燥、分离纯化后经检测，如图2所示，原飞燕草素B-3'-没食子酸酯产率为6.2％，原飞燕草素B-3,3'-双没食子酸酯产率11.6％。
实施例3：
称取杨梅叶原花色素和EGCG各1g，分别溶解于50mL 0.1mol/L HCl甲醇溶液，搅拌均匀至澄清，将50mL杨梅叶原花色素溶液和50mL EGCG溶液在具塞锥形瓶中混合，在60℃下水浴加热40min，经干燥、分离纯化后经检测，如图3所示，原飞燕草素B-3'-没食子酸酯产率为4.0％，原飞燕草素B-3,3'-双没食子酸酯产率18.0％。
实施例4：
称取杨梅叶原花色素和EGCG各1g，分别溶解于5.0mL 0.1mol/L HCl甲醇溶液，搅拌均匀至澄清，将2.5mL杨梅叶原花色素溶液和5.0mL EGCG溶液在具塞锥形瓶中混合，在20℃下水浴加热40min，经干燥、分离纯化后经检测，如图4所示，原飞燕草素B-3'-没食子酸酯产率为16.6％，原飞燕草素B-3,3'-双没食子酸酯产率19.5％。
实施例5：
称取杨梅叶原花色素和EGCG各2g，分别溶解于5.0mL 0.1mol/L HCl甲醇溶液，搅拌均匀至澄清，将2.5mL杨梅叶原花色素溶液和5.0mLEGCG溶液在具塞锥形瓶中混合，在40℃下水浴加热40min，经干燥、分离纯化后经检测，如图5所示，原飞燕草素B-3'-没食子酸酯产率为27.9％，原飞燕草素B-3,3'-双没食子酸酯产率21.2％。
实施例6：
称取杨梅叶原花色素和EGCG各1g，分别溶解于50.0mL 1mol/L HCl甲醇溶液，搅拌均匀至澄清，将50mL杨梅叶原花色素溶液和50mLEGCG溶液在具塞锥形瓶中混合，在40℃下水浴加热40min，经干燥、分离纯化后经检测，如图4所示，原飞燕草素B-3'-没食子酸酯产率为6.8％，原飞燕草素B-3,3'-双没食子酸酯产率18.0％。
以上所述仅为本发明的优选实施例，所述实施例并非用于限制本发明的专利保护范围，因此凡是运用本发明的说明书内容所作的等同结构变化，同理均应包含在本发明所附权利要求的保护范围内。

Claims (6)

  1. 一种原飞燕草素B9没食子酸酯的化学合成方法，其特征在于，具体为：
    分别配置浓度为10-400mg/mL杨梅叶原花色素的含盐酸溶液和10-400mg/mL表没食子儿茶素没食子酸酯的含盐酸溶液；其中，两种含盐酸溶液的盐酸浓度均为0.1-1.0mol/L；将盐酸浓度相同的杨梅叶原花色素和EGCG的含盐酸溶液按体积比1:2-2:1混合后，在20-60℃条件下反应40min，经干燥、分离纯化后获得原飞燕草素B9没食子酸酯，所述原飞燕草素B9没食子酸酯包括原飞燕草素B-3'-没食子酸酯和原飞燕草素B-3,3'-双没食子酸酯。
  2. 根据权利要求1所述的化学合成方法，其特征在于，所述配置浓度为10-400mg/mL杨梅叶原花色素的含盐酸溶液和10-400mg/mL表没食子儿茶素没食子酸酯的含盐酸溶液采用的溶剂为甲醇或乙醇。
  3. 根据权利要求1所述的化学合成方法，其特征在于，所述杨梅叶原花色素纯度为86.4％；EGCG纯度≥98％。
  4. 根据权利要求1所述的化学合成方法，其特征在于，干燥的方法具体为：
    将反应40min后的反应液在40℃下旋转蒸发，去除甲醇和少量水，获得反应粗产物干粉。
  5. 根据权利要求1所述的化学合成方法，其特征在于，所述分离纯化的方法具体为：
    采用S系列X5H制备柱，流动相A为含0.5vol％乙酸的乙腈，流动相B为超纯水；将反应粗产物溶解于体积比为88：12的流动相A、B混合液，配制成20mg/mL的溶液；进样2mL后，进行流动相洗脱纯化，流动相流速为5mL/min，洗脱梯度为：0-10min，12-27vol％B，10-20min，60vol％B，20-26min 12vol％B；设置检测波长为280nm，根据液相图谱，收集5.5-6.0min的流出液，将流出液于40℃条件下旋转蒸发去除乙腈，冷冻干燥后获得原飞燕草素B9没食子酸酯纯化物。
  6. 根据权利要求5所述的化学合成方法，其特征在于，所述分离纯化还包括第二次纯化：
    第二次纯化采用Shim-pack GIST C18，流动相A为超纯水，流动相B为含0.05vol％磷酸的98vol％乙腈；将原飞燕草素B9没食子酸酯纯化物溶解于体积比为80：20的流动相A、B混合液，配制成50mg/mL的溶液；进样2mL后，进行流动相洗脱纯化，流动相流速为15mL/min，洗脱梯度为：0-5min，20-40vol％B；5-15min，40vol％B；设置检测波长为280 nm，根据液相图谱，在不同出峰时间分别收集流出液，将流出液于40℃条件下旋转蒸发去除乙腈，冷冻干燥后分别获得原飞燕草素B-3'-没食子酸酯和原飞燕草素B-3,3'-双没食子酸酯。
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