WO2021042922A1 - Preparation method for tetragalloylglucose - Google Patents

Preparation method for tetragalloylglucose Download PDF

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WO2021042922A1
WO2021042922A1 PCT/CN2020/106263 CN2020106263W WO2021042922A1 WO 2021042922 A1 WO2021042922 A1 WO 2021042922A1 CN 2020106263 W CN2020106263 W CN 2020106263W WO 2021042922 A1 WO2021042922 A1 WO 2021042922A1
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preparation
leaf tea
flow rate
hairy
extraction
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PCT/CN2020/106263
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Chinese (zh)
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乔小燕
马成英
陈维
胡蝶
苗爱清
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广东省农业科学院茶叶研究所
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Priority to JP2022506663A priority Critical patent/JP7305870B2/en
Publication of WO2021042922A1 publication Critical patent/WO2021042922A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C07H1/06—Separation; Purification
    • C07H1/08—Separation; Purification from natural products
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H13/00—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids
    • C07H13/02—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids
    • C07H13/08—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids having the esterifying carboxyl radicals directly attached to carbocyclic rings

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  • the invention relates to the technical field of separation and purification of active ingredients of natural products, in particular to a preparation method of tetragalloyl glucoside.
  • Galloyl glucose is a polyphenol compound, which is widely present in many kinds of medicinal materials.
  • 1,2,3,4,6-pentagalloyl glucose has antioxidant activity, which can activate insulin-mediated glucose transduction signals. It can prevent diabetes and protect the liver; it can also bind to toxins in the human body and play an anti-endotoxin effect.
  • 1,2,3,4,6-pentagalloyl glucose has antiviral functions, such as inhibiting varicella-zoster virus replication; hepatitis B virus (HBV), hepatitis C virus (HCV) , Human immunodeficiency virus (HIV) and herpes simplex virus (HSV).
  • Hairy leaf tea (Camellia ptilophylla Chang) is a tea tree resource unique to Guangdong. It is world-renowned because it does not contain caffeine. However, due to the poor palatability of processed teas from Maoye tea, although Maoye tea is well known, its development and utilization are relatively backward. In recent years, many studies have shown that galloyl glucose has a variety of biological activities such as anti-tumor, antibacterial, and antioxidant activities. The applicant found that the hairy leaf tea contains higher tetragalloyl glucose, which is of great significance to the development and utilization of the hairy leaf tea. In view of the literature on the anti-oxidation and anti-viral effects of galloyl glucose, the establishment of a purification method for galloyl glucose in the tea and the identification of its structure are of great significance to the further development and utilization of hairy leaf tea.
  • the purpose of the present invention is to provide a method for preparing hairy leaf tea tetragalloyl glucose, by which high-purity tetragalloyl glucose can be prepared in large quantities.
  • a preparation method of tetragalloyl glucose includes the following steps,
  • the dosage is 0.2% trifluoroacetic acid aqueous solution, n-butanol , Ethyl acetate, methyl tert-butyl ether, acetonitrile and n-hexane, as a two-phase solvent system, fully mixed and let stand overnight, the two phases are separated and ultrasonically degassed, the upper phase is used as the stationary phase, and the lower phase is used as the mobile phase; Under the conditions of flow rate 1.6 ⁇ 2.0mL/min and detection wavelength of 280nm, the hair leaf tea extract prepared above was subjected to high-speed countercurrent chromatography (HSCCC) separation, the effluent was collected for 119 ⁇ 130min according to the peak shape, and the collected effluent was concentrated After drying, the compound tetragalloyl glucose is obtained.
  • HSC high-speed countercurrent chromatography
  • the extraction temperature is 50°C-60°C.
  • the amount of the distilled water is 9-11 times that of the hairy leaf tea, more preferably 10 times.
  • the extraction is performed twice, each time is 25 minutes to 60 minutes, more preferably 30 minutes to 40 minutes.
  • the pH value is adjusted to 3 with hydrochloric acid.
  • adsorption is performed through a chromatography column equipped with a polar or weakly polar macroporous adsorption resin at a flow rate of 3-4 BV/h, preferably AB-8 macroporous adsorption resin.
  • the rotation speed of the host during the high-speed countercurrent chromatography (HSCCC) separation is 850 ⁇ 50 rpm, and the separation temperature is 20 ⁇ 1°C.
  • the concentration of ethanol is 50 ⁇ 5%, preferably 50%.
  • the hairy leaf tea is Nankunshan hairy leaf tea.
  • the volume ratio of 0.2% trifluoroacetic acid aqueous solution, n-butanol, ethyl acetate, methyl tert-butyl ether, acetonitrile, and n-hexane is 4:1:1:1.5:1:1 .
  • the flow rate is 1.95-2.0 mL/min, and/or the sample amount is 300 ⁇ 50 mg of the hairy leaf tea extract.
  • the preparation method of the tetragalloyl glucose of the present invention uses the hairy leaf tea as a raw material for extraction. After the hairy leaf tea extract is purified by a macroporous adsorption resin, the tetragalloyl glucose is separated by optimized high-speed countercurrent chromatography.
  • the preparation method of the present invention has the advantages of simplicity, good reproducibility, high purity of tetragalloyl glucose and high yield, can be used for mass preparation of compounds, and lays a material foundation for further activity research.
  • Figure 1 shows the HPLC chromatogram of the raw material extract of Maoye Tea.
  • Figure 2 is a separation chromatogram of HSCCC of system 4 in Example 1.
  • Figure 3 is the HPLC purity detection diagram of the compound separated in System 4 in Example 1.
  • Figure 4 is a separation diagram of HSCCC of System 2 in Example 2.
  • FIG. 5 is an HPLC chart of the separation product of system 2 in Example 2.
  • HCCC high-speed countercurrent
  • HPLC conditions Column: Agilent ZORBAX SB-C18 (5 ⁇ m, 4.6 ⁇ 250mm), mobile phase A is 0.2% phosphoric acid aqueous solution, and B is acetonitrile. The elution gradient is: 0 ⁇ 25min: B 5% ⁇ 72%; 25 ⁇ 30min: B 28% ⁇ 32%, flow rate is 1.0mL/min, DAD detector, injection volume is 20 ⁇ L, column temperature: 28°C.
  • the different solvents in the system were prepared in the proportions shown in Table 1 and then allowed to stand for stratification after shaking.
  • Remove 3 mL of the lower phase solvent add a small amount of the above-prepared sample of the hairy leaf tea extract, and measure the area of each target peak in the lower phase solvent (before S extraction) by HPLC. Then take an equal volume of the upper phase for extraction, determine the target peak area in the lower phase after extraction (after S extraction), and calculate the partition coefficient K of each component according to the following formula (1).
  • K value select a suitable solvent system as the stationary phase and mobile phase of HSCCC.
  • System 1 is a medium solvent system. Due to the strong water solubility of the target component, the value of the partition coefficient (K) in this system is too small.
  • System 2 adds acid on the basis of system 1, and changes the system ratio. It is found that the K value of the target component is more suitable. However, after the computer operation, the result shows that the purity of the target component is very low, and many of them have low hydrophilicity. The content impurities flow out together with the target component, and the separation effect is completely unattainable.
  • System 3 is a commonly used system for separation of components with strong hydrophilicity, but the K value for the target component is too large; therefore, the experiment combines the components of systems 1 and 2 and adjusts the solvent ratio to find that systems 4 and 5
  • the K value is better, that is, 0.2% trifluoroacetic acid aqueous solution, n-butanol, ethyl acetate, methyl tert-butyl ether, acetonitrile, n-hexane are used as the solvent system, and the sample loading operation obtains a satisfactory separation effect, preferably
  • High-speed counter-current chromatography column TBE-300A high-speed counter-current chromatograph (Shanghai Tongtian Biotechnology Co., Ltd.): equipped with PTFE column, inner diameter 1.6mm, column volume 280mL, rotation speed 0-1000r/min, TBP-50A pump, TBD-2000UV detector, LX-300 thermostat.
  • Example 1 The structure of the compound described in Example 1 was identified and separated by mass spectrometry and nuclear magnetic resonance.

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Abstract

Disclosed is a preparation method for tetragalloylglucose. The method comprises: using Camellia ptilophylla Chang as a raw material for extraction, purifying the Camellia ptilophylla Chang extract by using a macroporous adsorption resin, and performing separation by means of high-speed countercurrent chromatography to obtain tetragalloylglucose. The preparation method of the present invention has the advantages of a simple process, good reproducibility, high purity and yield of tetragalloylglucose, can be used for the mass preparation of compounds, and lays a material foundation for further study on activity.

Description

一种四没食子酰葡萄糖的制备方法A kind of preparation method of tetragalloyl glucose 技术领域Technical field
本发明涉及天然产物活性成分的分离纯化技术领域，具体为一种四没食子酰葡萄糖苷的制备方法。The invention relates to the technical field of separation and purification of active ingredients of natural products, in particular to a preparation method of tetragalloyl glucoside.
背景技术Background technique
没食子酰葡萄糖是一种多酚化合物，广泛存在于多种药材中。现研究较多的是1,2,3,4,6-五没食子酰葡萄糖，1,2,3,4,6-五没食子酰葡萄糖具有抗氧化活性，通过激活胰岛素介导的葡萄糖转导信号途径，起到预防糖尿病和保护肝脏的功能；也能与人体内毒素结合，起到抗内毒素作用。除此之外，1,2,3,4,6-五没食子酰葡萄糖有抗病毒的功能，如抑制水痘-带状疱疹病毒复制；乙型肝炎病毒(HBV)、丙型肝炎病毒(HCV)、人体免疫机能丧失病毒(HIV)和单纯疱疹病毒(HSV)。Galloyl glucose is a polyphenol compound, which is widely present in many kinds of medicinal materials. Currently, more researches are on 1,2,3,4,6-pentagalloyl glucose. 1,2,3,4,6-pentagalloyl glucose has antioxidant activity, which can activate insulin-mediated glucose transduction signals. It can prevent diabetes and protect the liver; it can also bind to toxins in the human body and play an anti-endotoxin effect. In addition, 1,2,3,4,6-pentagalloyl glucose has antiviral functions, such as inhibiting varicella-zoster virus replication; hepatitis B virus (HBV), hepatitis C virus (HCV) , Human immunodeficiency virus (HIV) and herpes simplex virus (HSV).
茶是我国传统的重要经济作物，其保健功效为世人所知。毛叶茶(Camellia ptilophylla Chang)是广东特有的茶树资源，因不含咖啡碱而闻名于世。但由于毛叶茶加工茶类适口性差的问题，使得毛叶茶虽然被人所知，开发利用却较为落后。近年来，很多研究表明，没食子酰葡萄糖具有抗肿瘤、抗菌、抗氧化活性等多种生物活性。申请人发现毛叶茶中含有较高的四没食子酰葡萄糖，对毛叶茶的开发利用具有重要意义。鉴于文献对于没食子酰葡萄糖抗氧化和抗病毒功效的相关研究，建立该茶中的没食子酰葡萄糖的纯化方法并鉴定其结构对毛叶茶的进一步开发利用具有重要意义。Tea is an important traditional economic crop in my country, and its health benefits are well known to the world. Hairy leaf tea (Camellia ptilophylla Chang) is a tea tree resource unique to Guangdong. It is world-renowned because it does not contain caffeine. However, due to the poor palatability of processed teas from Maoye tea, although Maoye tea is well known, its development and utilization are relatively backward. In recent years, many studies have shown that galloyl glucose has a variety of biological activities such as anti-tumor, antibacterial, and antioxidant activities. The applicant found that the hairy leaf tea contains higher tetragalloyl glucose, which is of great significance to the development and utilization of the hairy leaf tea. In view of the literature on the anti-oxidation and anti-viral effects of galloyl glucose, the establishment of a purification method for galloyl glucose in the tea and the identification of its structure are of great significance to the further development and utilization of hairy leaf tea.
发明内容Summary of the invention
本发明的目的是提供一种毛叶茶四没食子酰葡萄糖的制备方法，通过该方法，可以大量制备得到高纯度的四没食子酰葡萄糖。The purpose of the present invention is to provide a method for preparing hairy leaf tea tetragalloyl glucose, by which high-purity tetragalloyl glucose can be prepared in large quantities.
一种四没食子酰葡萄糖的制备方法，包括以下步骤，A preparation method of tetragalloyl glucose includes the following steps,
A.毛叶茶提取液的制备A. Preparation of hair leaf tea extract
取毛叶茶，粉碎过筛，加入蒸馏水超声提取和过滤，1-3次收集滤液，调节pH值3±0.1，以3-4BV/h的流速通过层析柱进行吸附，吸附结束后静置30min-60min(目标组分被紧密吸附在色谱柱上，避免被水洗去)；先用3-4BV的水以4-6BV/h的流速淋洗，然后用4-6BV质量浓度为30～70％％的乙醇水溶液以2-4BV/h的流速洗脱至洗脱液无色，收集乙醇洗脱液，浓缩后冷冻干燥，得毛叶茶提取物；Take the hairy leaf tea, smash and sieve, add distilled water for ultrasonic extraction and filtration, collect the filtrate 1-3 times, adjust the pH value to 3 ± 0.1, and pass through the chromatography column at a flow rate of 3-4 BV/h for adsorption, and then stand still after the adsorption is completed 30min-60min (the target component is tightly adsorbed on the chromatographic column to avoid being washed away by water); first rinse with 3-4BV water at a flow rate of 4-6BV/h, and then use 4-6BV with a mass concentration of 30～70 The %% ethanol aqueous solution is eluted at a flow rate of 2-4BV/h until the eluate is colorless, the ethanol eluate is collected, concentrated and freeze-dried to obtain the hairy leaf tea extract;
B.高速逆流色谱法分离：B. High-speed countercurrent chromatography separation:
按体积比(4±0.1)：(1±0.1)：(1±0.1)：(1.5±0.1)：(1±0.1)：(1±0.1)用量取0.2％三氟乙酸水溶液、正丁醇、乙酸乙酯、甲基叔丁基醚、乙腈和正已烷，作为两相溶剂体系，充分混合后静置过夜，两相分离后超声脱气，上相作固定相，下相作流动相；在流速1.6～2.0mL/min、检测波长280nm条件下，将上述制得的毛叶茶提取物进行高速逆流色谱(HSCCC)分离，根据峰形收集119～130min流出液，将收集的流出液浓缩后干燥，得化合物四没食子酰基葡萄糖。According to the volume ratio (4±0.1): (1±0.1): (1±0.1): (1.5±0.1): (1±0.1): (1±0.1) the dosage is 0.2% trifluoroacetic acid aqueous solution, n-butanol , Ethyl acetate, methyl tert-butyl ether, acetonitrile and n-hexane, as a two-phase solvent system, fully mixed and let stand overnight, the two phases are separated and ultrasonically degassed, the upper phase is used as the stationary phase, and the lower phase is used as the mobile phase; Under the conditions of flow rate 1.6～2.0mL/min and detection wavelength of 280nm, the hair leaf tea extract prepared above was subjected to high-speed countercurrent chromatography (HSCCC) separation, the effluent was collected for 119～130min according to the peak shape, and the collected effluent was concentrated After drying, the compound tetragalloyl glucose is obtained.
在其中一个实施例中，所述提取温度为50℃～60℃。In one of the embodiments, the extraction temperature is 50°C-60°C.
在其中一个实施例中，所述蒸馏水的用量为毛叶茶的9～11倍，更优选为10倍。In one of the embodiments, the amount of the distilled water is 9-11 times that of the hairy leaf tea, more preferably 10 times.
在其中一个实施例中，提取2次，每次25min～60min，更优选为30min～40min。In one of the embodiments, the extraction is performed twice, each time is 25 minutes to 60 minutes, more preferably 30 minutes to 40 minutes.
在其中一个实施例中，用盐酸调节pH值为3。In one of the embodiments, the pH value is adjusted to 3 with hydrochloric acid.
在其中一个实施例中，通过装有极性或弱极性大孔吸附树脂的层析柱以3-4BV/h流速进行吸附，优选AB-8大孔吸附树脂。In one of the embodiments, adsorption is performed through a chromatography column equipped with a polar or weakly polar macroporous adsorption resin at a flow rate of 3-4 BV/h, preferably AB-8 macroporous adsorption resin.
在其中一个实施例中，高速逆流色谱(HSCCC)分离时的主机转速850±50rpm，分离温度20±1℃。In one of the embodiments, the rotation speed of the host during the high-speed countercurrent chromatography (HSCCC) separation is 850±50 rpm, and the separation temperature is 20±1°C.
在其中一个实施例中，乙醇的浓度为50±5％，优选50％。In one of the embodiments, the concentration of ethanol is 50±5%, preferably 50%.
在其中一个实施例中，粉碎后过10目-30目筛。In one of the embodiments, after crushing, it is passed through a 10-30 mesh sieve.
在其中一个实施例中，所述毛叶茶为南昆山毛叶茶。In one of the embodiments, the hairy leaf tea is Nankunshan hairy leaf tea.
在其中一个实施例中，0.2％三氟乙酸水溶液，正丁醇，乙酸乙酯，甲基叔丁基醚，乙腈，正已烷的体积比为：4:1:1:1.5:1:1。In one of the embodiments, the volume ratio of 0.2% trifluoroacetic acid aqueous solution, n-butanol, ethyl acetate, methyl tert-butyl ether, acetonitrile, and n-hexane is 4:1:1:1.5:1:1 .
在其中一个实施例中，流速1.95～2.0mL/min，和/或上样量为300±50mg的毛叶茶提取物。In one of the embodiments, the flow rate is 1.95-2.0 mL/min, and/or the sample amount is 300±50 mg of the hairy leaf tea extract.
通过以上的工艺参数的选择和优化，可以高效地得到有效的提取物。Through the selection and optimization of the above process parameters, effective extracts can be efficiently obtained.
本发明所述四没食子酰葡萄糖的制备方法，以毛叶茶为原料进行提取，毛叶茶提取物经大孔吸附树脂纯化后，采用优化后的高速逆流色谱法分离得到四没食子酰基葡萄糖。本发明所述制备方法具有简便，重现性好，四没食子酰基葡萄糖的纯度高，收率高的优点，可用于化合物大量制备，并为进一步活性研究奠定了物质基础。The preparation method of the tetragalloyl glucose of the present invention uses the hairy leaf tea as a raw material for extraction. After the hairy leaf tea extract is purified by a macroporous adsorption resin, the tetragalloyl glucose is separated by optimized high-speed countercurrent chromatography. The preparation method of the present invention has the advantages of simplicity, good reproducibility, high purity of tetragalloyl glucose and high yield, can be used for mass preparation of compounds, and lays a material foundation for further activity research.
附图说明Description of the drawings
图1为毛叶茶原料提取物HPLC色谱图。Figure 1 shows the HPLC chromatogram of the raw material extract of Maoye Tea.
图2为实施例1中体系4的HSCCC分离色谱图。Figure 2 is a separation chromatogram of HSCCC of system 4 in Example 1.
图3为实施例1中体系4分离化合物的HPLC纯度检测图。Figure 3 is the HPLC purity detection diagram of the compound separated in System 4 in Example 1.
图4为实施例2中体系2的HSCCC分离图。Figure 4 is a separation diagram of HSCCC of System 2 in Example 2.
图5为实施例2中体系2分离物HPLC图。FIG. 5 is an HPLC chart of the separation product of system 2 in Example 2. FIG.
图6实施例3中体系5的HSCCC分离图。Fig. 6 HSCCC separation diagram of System 5 in Example 3.
图7实施例3中体系5的分离物HPLC图。Fig. 7 HPLC chart of the separated product of system 5 in Example 3.
具体实施方式detailed description
除非另有定义，本发明所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本发明的说明书中所使用的术语只是为了描述具体的实施例的目的，不用于限制本发明。本发明所使用的术语“和/或”包括一个或多个相关的所列项目的任意的和所有的组合。Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the technical field of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments, and are not used to limit the present invention. The term "and/or" used in the present invention includes any and all combinations of one or more related listed items.
为了便于理解本发明，下面将对本发明进行更全面的描述。本发明可以以许多不同的形式来实现，并不限于本文所描述的实施例。相反地，提供这些实施例的目的是使对本发明公开内容的理解更加透彻全面。In order to facilitate the understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.
实施例1毛叶茶中四没食子酸葡萄糖的制备Example 1 Preparation of Tetragallic Acid Glucose in Hairy Leaf Tea
1、高速逆流(HSCCC)上样原料(毛叶茶提取物)的制备1. Preparation of high-speed countercurrent (HSCCC) loading material (hair leaf tea extract)
取粉碎过10目筛的毛叶茶粉末200g，用为毛叶茶粉末10倍重量的蒸馏水在50～60℃下提取2次，第一次60min，后一次30min，收集提取液，合并，用盐酸调节pH值3，通过装有800mlAB-8大孔吸附树脂的层析柱以4BV/h的流速进行吸附，吸附后静置30min，先用1800ml水以6BV/h的流速淋洗，然后用50％质量浓度的乙醇水溶液洗脱1800ml，流速4BV/h，收集洗脱液，浓缩后冷冻干燥，得毛叶茶提取物，作为高速逆流的上样原料。Take 200g of the hairy leaf tea powder crushed through a 10-mesh sieve, extract 2 times at 50～60℃ with distilled water 10 times the weight of the hairy leaf tea powder, the first time is 60 minutes, the next time is 30 minutes, the extracts are collected, combined, and used Adjust pH to 3 with hydrochloric acid, and pass through a chromatography column equipped with 800ml AB-8 macroporous adsorption resin at a flow rate of 4BV/h. After adsorption, let it stand for 30min. First rinse with 1800ml water at a flow rate of 6BV/h, and then use The 50% mass concentration of ethanol aqueous solution was eluted with 1800ml at a flow rate of 4BV/h. The eluate was collected, concentrated and freeze-dried to obtain the hairy leaf tea extract, which was used as a high-speed countercurrent sample material.
2、HPLC检测方法2. HPLC detection method
高效液相色谱条件：色谱柱：Agilent ZORBAX SB-C18(5μm,4.6×250mm)，流动相A为0.2％的磷酸水溶液，B为乙腈。洗脱梯度为：0→25min:B 5％→72％；25→30min:B 28％→32％，流速为1.0mL/min，DAD检测器，进样量为20μL，柱温：28℃。HPLC conditions: Column: Agilent ZORBAX SB-C18 (5μm, 4.6×250mm), mobile phase A is 0.2% phosphoric acid aqueous solution, and B is acetonitrile. The elution gradient is: 0→25min: B 5%→72%; 25→30min: B 28%→32%, flow rate is 1.0mL/min, DAD detector, injection volume is 20μL, column temperature: 28°C.
取少量的上述制备的毛叶茶提取物用水溶解，过膜进样，HPLC谱图见图1，由图1可见，毛叶茶提取物中含有多个化学成分，保留时间为23.4min的化合物为目标组分。Take a small amount of the above-prepared hair leaf tea extract to dissolve in water, and inject the sample through the membrane. The HPLC spectrum is shown in Figure 1. It can be seen from Figure 1 that the hair leaf tea extract contains multiple chemical components with a retention time of 23.4 min. For the target component.
3、高速逆流色谱(HSCCC)分离3. High-speed countercurrent chromatography (HSCCC) separation
3.1溶剂***的选择3.1 Choice of solvent system
通过对多个体系进行筛选，将该体系中不同溶剂按下表1中的比例配制，震摇后静置分层。移取3mL下相溶剂，加入少量上述制备的毛叶茶提取物样品中，用HPLC测定样品在下相溶剂中各目标峰的面积(S萃前)。再取等体积上相进行萃取，测定萃后下相中目标峰面积(S萃后)，按下式(1)计算出各组分的分配系数K。根据K值选择适合的溶剂体系作为HSCCC的固定相与流动相。By screening multiple systems, the different solvents in the system were prepared in the proportions shown in Table 1 and then allowed to stand for stratification after shaking. Remove 3 mL of the lower phase solvent, add a small amount of the above-prepared sample of the hairy leaf tea extract, and measure the area of each target peak in the lower phase solvent (before S extraction) by HPLC. Then take an equal volume of the upper phase for extraction, determine the target peak area in the lower phase after extraction (after S extraction), and calculate the partition coefficient K of each component according to the following formula (1). According to the K value, select a suitable solvent system as the stationary phase and mobile phase of HSCCC.
式(1)：K＝(S 萃前-S 萃后)/S 萃前 Formula (1): K = (S before extraction-after S extraction ) / before S extraction
体系1为中等溶剂体系，由于目标组分的水溶性强，在该体系中的分配系数(K)值太小。System 1 is a medium solvent system. Due to the strong water solubility of the target component, the value of the partition coefficient (K) in this system is too small.
体系2是在体系1的基础上加入酸，并改变体系的配比，发现目标组分的K值较为合适，但经上机操作后结果显示，目标组分纯度很低，很多亲水性低含量杂质与目标组分一起流出，完全达不到分离效果。 System 2 adds acid on the basis of system 1, and changes the system ratio. It is found that the K value of the target component is more suitable. However, after the computer operation, the result shows that the purity of the target component is very low, and many of them have low hydrophilicity. The content impurities flow out together with the target component, and the separation effect is completely unattainable.
体系3为亲水性强的成分分离常用体系，但对目标组分的K值又太大；因此，实验将体系1、2组成相结合，并通过调整溶剂的配比，发现体系4、5的K值较好，即以0.2％三氟乙酸水溶液、正丁醇、乙酸乙酯、甲基叔丁基醚、乙 腈、正已烷作为溶剂体系，上样操作获得较满意的分离效果，优选体系为体系4，即体积百分比0.2％的三氟乙酸水溶液:正丁醇:乙酸乙酯:甲基叔丁基醚:乙腈:正已烷＝4:1:1:1.5:1:1(V/V)。System 3 is a commonly used system for separation of components with strong hydrophilicity, but the K value for the target component is too large; therefore, the experiment combines the components of systems 1 and 2 and adjusts the solvent ratio to find that systems 4 and 5 The K value is better, that is, 0.2% trifluoroacetic acid aqueous solution, n-butanol, ethyl acetate, methyl tert-butyl ether, acetonitrile, n-hexane are used as the solvent system, and the sample loading operation obtains a satisfactory separation effect, preferably The system is system 4, which is a 0.2% aqueous solution of trifluoroacetic acid: n-butanol: ethyl acetate: methyl tert-butyl ether: acetonitrile: n-hexane=4:1:1:1.5:1:1 (V /V).
表1溶剂体系K值筛选表Table 1 K value screening table of solvent system
Figure PCTCN2020106263-appb-000001
Figure PCTCN2020106263-appb-000001
3.2 HSCCC操作条件3.2 HSCCC operating conditions
高速逆流色谱柱：TBE-300A高速逆流色谱仪(上海同田生物技术有限公司)：配置有聚四氟乙烯柱，内径1.6mm，柱容积280mL，转速0-1000r/min，TBP-50A泵，TBD-2000UV检测器，LX-300恒温器。High-speed counter-current chromatography column: TBE-300A high-speed counter-current chromatograph (Shanghai Tongtian Biotechnology Co., Ltd.): equipped with PTFE column, inner diameter 1.6mm, column volume 280mL, rotation speed 0-1000r/min, TBP-50A pump, TBD-2000UV detector, LX-300 thermostat.
按上述体系4配置溶剂体系，充分混合，静置过夜，两相分离后超声脱气30min。两相分离后超声脱气，上相作固定相，下相作流动相；下相流速2.0mL/min、主机转速850rpm，柱温箱20℃，波长280nm，在该条件下固定相的保留率62％(保留率越高，即上相在仪器管道中的体积就越大，有利于萃取分离)，上样量为300mg，HSCCC分离图谱见图2，收集119～130min的流出液， 55℃旋转蒸发浓缩后冷冻干燥得3.17mg目标化合物。Configure the solvent system according to the above system 4, mix well, let it stand overnight, and ultrasonically degas for 30 minutes after the two phases are separated. After the two phases are separated, ultrasonic degassing, the upper phase is used as the stationary phase, and the lower phase is used as the mobile phase; the flow rate of the lower phase is 2.0mL/min, the host speed is 850rpm, the column oven is 20℃, and the wavelength is 280nm. The retention rate of the stationary phase under this condition 62% (the higher the retention rate, the larger the volume of the upper phase in the instrument pipeline, which is conducive to extraction and separation), the sample amount is 300 mg, and the HSCCC separation chart is shown in Figure 2. The effluent of 119 to 130 minutes is collected at 55°C. It was concentrated by rotary evaporation and freeze-dried to obtain 3.17 mg of the target compound.
根据HPLC(分析条件同上)在254nm下的色谱图，见图3，采用峰面积归一法计算目标化合物纯度，目标化合物纯度为92.3％。According to the chromatogram of HPLC (analysis conditions as above) at 254 nm, as shown in Figure 3, the purity of the target compound was calculated by the peak area normalization method, and the purity of the target compound was 92.3%.
采用质谱和核磁共振鉴定分离实施例1所述化合物的结构。The structure of the compound described in Example 1 was identified and separated by mass spectrometry and nuclear magnetic resonance.
化合物：棕色粉末，ESI-MS m/z:787.1012[M－H] –，分量式为C 34H 28O 22； 1H-NMR(500MHz,DMSO-d6)δ:6.99(2H,s,H-2””,6””),6.97(2H,s,H-2”’,6”’),6.95(2H,s,H-2’,6’),6.92(2H,s,H-2”,6”),6.10(1H,d,J＝8.5Hz,H-1),5.89(H,m,H-3),5.20(1H,dd,J＝8.5Hz,7.5Hz,H-2),4.27(2H,m,H-6),4.16(H,m,H-5),3.88(1H,s,H-4)； Compound: brown powder, ESI-MS m/z: 787.1012[M-H] – , the component formula is C 34 H 28 O 22 ; 1 H-NMR (500MHz, DMSO-d6) δ: 6.99 (2H, s, H -2””,6””),6.97(2H,s,H-2”',6”'),6.95(2H,s,H-2',6'),6.92(2H,s,H- 2”,6”),6.10(1H,d,J=8.5Hz,H-1),5.89(H,m,H-3), 5.20(1H,dd,J=8.5Hz,7.5Hz,H- 2), 4.27(2H,m,H-6), 4.16(H,m,H-5), 3.88(1H,s,H-4);
13C-NMR(125MHz,DMSO-d6):δ62.3(C-6),66.22(C-4),70.58C-2),71.65(C-5),72.87(C-3),92.43(C-1),119.38(C-1’),109.48(C-2’,6’),146.10(C-3’,5’),139.94(C-4’),164.97(C-7’),165.33(C-7”),166.00(C-7”’),109.36(C-2”,6”),109.23(C-2”’,6”’),109.23(C-2””,6””),118.03(C-1”),119.31(C-1”’),119.49(C-1””),139.24(C-4”),139.14(C-4””),143.02(C-3”,5”),145.98(C-3”’,5”’),145.95(C-3””,5””)。 13 C-NMR(125MHz, DMSO-d6): δ62.3(C-6), 66.22(C-4), 70.58C-2), 71.65(C-5), 72.87(C-3), 92.43( C-1),119.38(C-1'),109.48(C-2',6'),146.10(C-3',5'),139.94(C-4'),164.97(C-7') ,165.33(C-7”),166.00(C-7”'),109.36(C-2”,6”),109.23(C-2”',6”'),109.23(C-2””, 6””),118.03(C-1”),119.31(C-1”'),119.49(C-1””),139.24(C-4”),139.14(C-4””),143.02( C-3”, 5”), 145.98 (C-3”', 5”'), 145.95 (C-3””, 5””).
根据1H和13C NMR数据和目前公开的资料(例如：肖世基,郭大乐,何达海,等.猕猴桃藤山柳化学成分研究[J].中草药,2016,(03):383-387；舒希凯，芍药花抗氧化活性成分的分离和鉴定[M]，山东师范大学)，确定化合物为：1,2,3,6--四-O-没食子酰基-葡萄糖，其化学结构式如下。According to 1H and 13C NMR data and currently public information (eg: Xiao Shiji, Guo Dale, He Dahai, etc.. Study on the chemical constituents of kiwifruit, vine and willow[J].Chinese Herbal Medicine,2016,(03):383-387; Shu Xikai, Paeonia lactiflora antioxidant Isolation and identification of active ingredients [M], Shandong Normal University), determined that the compound is: 1,2,3,6-tetra-O-galloyl-glucose, and its chemical structure is as follows.
Figure PCTCN2020106263-appb-000002
Figure PCTCN2020106263-appb-000002
实施例2体系2的分离及分离效果Example 2 Separation and separation effect of system 2
按上述体系2配置溶剂体系，充分混合，静置过夜，两相分离后超声脱气30min。下相流速2.0mL/min、主机转速850rpm，柱温箱20℃，波长280nm，在该条件下固定相的保留率为51％，上样量300mg，HSCCC分离图谱见图4，收集195～210min的流出液，HPLC检测结果见图5，结果表明，在该段流出液中将原料中低含量的两个组分也富集并与目标组分完全不能分离。Configure the solvent system according to system 2 above, mix well, let it stand overnight, and ultrasonically degas for 30 minutes after the two phases are separated. The flow rate of the lower phase is 2.0mL/min, the speed of the host is 850rpm, the column oven is 20℃, and the wavelength is 280nm. Under these conditions, the retention rate of the stationary phase is 51%, and the loading amount is 300mg. The HSCCC separation chart is shown in Figure 4, and the collection is 195～210min. The effluent of HPLC is shown in Figure 5. The results show that the two low-content components in the raw material are also enriched in the effluent of this section and cannot be separated from the target component at all.
实施例3体系5的分离及分离效果Example 3 Separation and separation effect of system 5
按上述体系5配置溶剂体系，充分混合，静置过夜，两相分离后超声脱气30min。两相分离后超声脱气，上相作固定相，下相作流动相；下相流速2.0mL/min、主机转速850rpm，柱温箱20℃，波长280nm，在该条件下固定相的保留率53％，上样量300mg，HSCCC分离图谱见图6，收集141～147min的流出液，55℃旋转蒸发浓缩后冷冻干燥得约1.8mg目标化合物。HPLC(分析条件 同上)在254nm下的色谱图，见图7，采用峰面积归一法计算化合物纯度，化合物纯度为90.6％。结果表明，该方法的固定相保留率、目标化合物得率和纯度均低于体系4的配比。Configure the solvent system according to the above system 5, mix well, let it stand overnight, and ultrasonically degas for 30 minutes after the two phases are separated. After the two phases are separated, ultrasonic degassing, the upper phase is used as the stationary phase, and the lower phase is used as the mobile phase; the flow rate of the lower phase is 2.0mL/min, the host speed is 850rpm, the column oven is 20℃, and the wavelength is 280nm. The retention rate of the stationary phase under this condition 53%, the loading amount is 300 mg, and the HSCCC separation chart is shown in Figure 6. The effluent from 141 to 147 min is collected, concentrated by rotary evaporation at 55°C and freeze-dried to obtain about 1.8 mg of the target compound. The chromatogram of HPLC (analysis conditions as above) at 254 nm is shown in Fig. 7. The compound purity was calculated by the peak area normalization method, and the compound purity was 90.6%. The results show that the retention rate of the stationary phase, the yield of the target compound and the purity of this method are all lower than the ratio of System 4.
本文中所举例说明的发明可适当地在不存在本文中未具体公开的任何要素、限制的情况下实施。因此，例如术语“包含/包括”等应理解为开放式的且没有限制。另外，本文中采用的术语和表达用作描述而非限制的术语，并且并非旨在使用排除所示出且描述的特征或其部分的任何等同特征的此类术语和表达，但是应认识到，在本发明所要求保护的范围可进行多种修改。因此，应理解，尽管已通过优选实施方案和任选特征具体公开了本发明，但是本领域技术人员可采用本文中公开的其中体现发明的修改方案和变化方案，并且这样的修改方案和变化方案被认为在本发明的范围之内。The invention exemplified herein can be suitably implemented without any elements or limitations not specifically disclosed herein. Therefore, for example, the terms "include/include" and the like should be understood as open-ended and not restrictive. In addition, the terms and expressions used herein are used as descriptive rather than restrictive terms, and are not intended to use such terms and expressions that exclude the features shown and described or any equivalent features of parts thereof, but it should be recognized that Various modifications can be made within the scope of protection claimed by the present invention. Therefore, it should be understood that although the present invention has been specifically disclosed through preferred embodiments and optional features, those skilled in the art can adopt the modifications and variations disclosed herein in which the invention is embodied, and such modifications and variations It is considered to be within the scope of the present invention.
已在本文中对本发明进行了广泛且一般性的描述。落入一般性公开内容内的较窄类别和亚属分组各自也形成本发明的一部分。这包括具有从该属中除去任何主题的条件或负面限制的本发明的一般性描述，不管所删除内容是否在本文中具体详述。The present invention has been described broadly and generically herein. Each of the narrower categories and subgenus groupings that fall within the general disclosure also forms part of the present invention. This includes a general description of the present invention with conditions or negative limitations to remove any subject matter from the genus, regardless of whether the deleted content is specifically detailed herein.

Claims (12)

  1. 一种四没食子酰葡萄糖的制备方法，包括以下步骤，A preparation method of tetragalloyl glucose includes the following steps,
    A.毛叶茶提取液的制备：A. Preparation of hair leaf tea extract:
    取毛叶茶，粉碎过筛，加入蒸馏水超声提取和过滤1-3次，收集滤液，调节pH值3.0±0.1，以3-4BV/h流速通过层析柱进行吸附，吸附后静置30min-60min，先用3-4BV的水以4-6BV/h的流速淋洗，然后用4-6BV质量浓度为50±5％的乙醇水溶液以2-4BV/h的流速洗脱至洗脱液无色，收集乙醇洗脱液，浓缩后冷冻干燥，得毛叶茶提取物；Take the hairy leaf tea, smash through a sieve, add distilled water for ultrasonic extraction and filter 1-3 times, collect the filtrate, adjust the pH value to 3.0±0.1, and pass through the chromatography column at a flow rate of 3-4BV/h for adsorption, and then stand for 30min after adsorption. For 60 minutes, first rinse with 3-4BV water at a flow rate of 4-6BV/h, and then use 4-6BV with a mass concentration of 50±5% ethanol aqueous solution at a flow rate of 2-4BV/h to elute until the eluent has no Color, collect the ethanol eluate, concentrate and freeze-dry to obtain the hairy leaf tea extract;
    B.高速逆流色谱法分离：B. High-speed countercurrent chromatography separation:
    按体积比(4±0.1):(1±0.1):(1±0.1):(1.5±0.1):(1±0.1):(1±0.1)用量取0.2％三氟乙酸水溶液、正丁醇、乙酸乙酯、甲基叔丁基醚、乙腈和正已烷，作为两相溶剂体系，充分混合后静置过夜；According to the volume ratio (4±0.1): (1±0.1): (1±0.1): (1.5±0.1): (1±0.1): (1±0.1), take 0.2% trifluoroacetic acid aqueous solution and n-butanol , Ethyl acetate, methyl tert-butyl ether, acetonitrile and n-hexane, as a two-phase solvent system, fully mixed and allowed to stand overnight;
    两相分离后超声脱气，上相作固定相，下相作流动相，在流速1.6～2.0mL/min、检测波长280nm条件下，将上述制得的毛叶茶提取物进行高速逆流色谱分离，根据峰形收集119～130min流出液，将收集的流出液浓缩后干燥，得四没食子酰基葡萄糖，所述四没食子酰基葡萄糖为1,2,3,6--四-O-没食子酰基-葡萄糖。After the two phases are separated, ultrasonically degassed, the upper phase is used as the stationary phase, and the lower phase is used as the mobile phase. Under the conditions of a flow rate of 1.6～2.0mL/min and a detection wavelength of 280nm, the above-prepared hair leaf tea extract is subjected to high-speed countercurrent chromatographic separation , Collect the effluent for 119～130min according to the peak shape, concentrate the collected effluent and dry to obtain tetragalloyl glucose, the tetragalloyl glucose is 1,2,3,6-tetra-O-galloyl-glucose .
  2. 根据权利要求1所述的制备方法，其特征是，所述蒸馏水的用量为毛叶茶的9-11倍。The preparation method according to claim 1, wherein the amount of the distilled water is 9-11 times that of the hairy leaf tea.
  3. 根据权利要求2所述的制备方法，其特征是，所述蒸馏水的用量为毛叶茶的10倍。The preparation method according to claim 2, wherein the amount of the distilled water is 10 times that of the hairy leaf tea.
  4. 根据权利要求1所述的制备方法，其特征是，所述提取温度为50℃～60℃；和/或提取2次，每次25min～60min。The preparation method according to claim 1, wherein the extraction temperature is 50°C-60°C; and/or the extraction is performed twice, 25min-60min each time.
  5. 根据权利要求1所述的制备方法，其特征是，提取2次，每次为30min～40min。The preparation method according to claim 1, characterized in that the extraction is performed twice, and each time is 30 minutes to 40 minutes.
  6. 根据权利要求1所述的制备方法，其特征是，用盐酸调节pH值为3。The preparation method according to claim 1, wherein the pH value is adjusted to 3 with hydrochloric acid.
  7. 根据权利要求1所述的制备方法，其特征是，通过装有AB-8大孔吸附树脂的层析柱以3-4BV/h流速进行吸附；和或高速逆流色谱分离时，主机转速850±50rpm，分离温度25±5℃。The preparation method according to claim 1, characterized in that the adsorption is carried out at a flow rate of 3-4 BV/h through a chromatography column equipped with AB-8 macroporous adsorption resin; and or during high-speed countercurrent chromatographic separation, the host rotation speed is 850± 50rpm, separation temperature 25±5℃.
  8. 根据权利要求1-5任一项所述的制备方法，其特征是，所述乙醇水溶液的浓度为50％。The preparation method according to any one of claims 1 to 5, wherein the concentration of the ethanol aqueous solution is 50%.
  9. 根据权利要求1-5任一项所述的制备方法，其特征是，粉碎后过10目-30目筛。The preparation method according to any one of claims 1 to 5, characterized in that, after crushing, it is passed through a 10 mesh to 30 mesh sieve.
  10. 根据权利要求1-5任一项所述的制备方法，其特征是，所述毛叶茶为南昆山毛叶茶。The preparation method according to any one of claims 1 to 5, wherein the hairy leaf tea is Nankunshan hairy leaf tea.
  11. 根据权利要求1-5任一项所述的制备方法，其特征是，所述0.2％三氟乙酸水溶液、正丁醇、乙酸乙酯、甲基叔丁基醚、乙腈、正已烷的体积比为：4:1:1:1.5:1:1。The preparation method according to any one of claims 1 to 5, wherein the volume of the 0.2% trifluoroacetic acid aqueous solution, n-butanol, ethyl acetate, methyl tert-butyl ether, acetonitrile and n-hexane The ratio is 4:1:1:1.5:1:1.
  12. 根据权利要求1-5任一项所述的制备方法，其特征是，上样量为300±50mg的毛叶茶提取物。The preparation method according to any one of claims 1 to 5, wherein the loading amount is 300±50 mg of the hairy leaf tea extract.
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CN114891052A (en) * 2022-05-10 2022-08-12 合肥工业大学 Method for preparing 1,2,3, 6-tetra-O-galloyl-beta-D-glucopyranoside from camellia oleifera abel

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