WO2021042922A1 - 一种四没食子酰葡萄糖的制备方法 - Google Patents

一种四没食子酰葡萄糖的制备方法 Download PDF

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Publication number
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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Prior art keywords
preparation
leaf tea
flow rate
hairy
extraction
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PCT/CN2020/106263
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English (en)
French (fr)
Chinese (zh)
Inventor
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/ja
Publication of WO2021042922A1 publication Critical patent/WO2021042922A1/zh

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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

Definitions

  • 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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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicines Containing Plant Substances (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
  • Saccharide Compounds (AREA)
PCT/CN2020/106263 2019-09-02 2020-07-31 一种四没食子酰葡萄糖的制备方法 WO2021042922A1 (zh)

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JP2022506663A JP7305870B2 (ja) 2019-09-02 2020-07-31 テトラガロイルグルコースの製造方法

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113376271A (zh) * 2021-05-26 2021-09-10 华东理工大学 一种基于lc-ms的生物样品中壳寡糖的高灵敏度检测分析方法
CN114891052A (zh) * 2022-05-10 2022-08-12 合肥工业大学 一种从油茶蒲中制备1,2,3,6-四-O-没食子酰-β-D-吡喃葡萄糖苷的方法

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110437289B (zh) * 2019-09-02 2020-07-07 广东省农业科学院茶叶研究所 一种四没食子酰葡萄糖的制备方法

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CN105669631A (zh) * 2015-12-30 2016-06-15 中国科学院西北高原生物研究所 一种委陵菜属植物提取物及从中分离四种单宁类化合物的方法
CN105732250A (zh) * 2016-02-06 2016-07-06 福建农林大学 一种高纯度灰树花多酚组分的制备方法
CN105859803A (zh) * 2016-05-09 2016-08-17 中国农业科学院特产研究所 一种没食子酰基葡萄糖的制备方法
CN107029051A (zh) * 2016-12-09 2017-08-11 昭平县科学技术指导站 一种茶叶中茶多酚的提取工艺
CN110437289A (zh) * 2019-09-02 2019-11-12 广东省农业科学院茶叶研究所 一种四没食子酰葡萄糖的制备方法

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CN101525357A (zh) * 2008-03-07 2009-09-09 首都医科大学 一种从中药中分离制备五没食子酰基葡萄糖的方法
CN108976268B (zh) * 2018-06-21 2021-12-24 湖南农业大学 一种制备刺葡萄两个主要花色苷标准品的方法

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CN105669631A (zh) * 2015-12-30 2016-06-15 中国科学院西北高原生物研究所 一种委陵菜属植物提取物及从中分离四种单宁类化合物的方法
CN105732250A (zh) * 2016-02-06 2016-07-06 福建农林大学 一种高纯度灰树花多酚组分的制备方法
CN105859803A (zh) * 2016-05-09 2016-08-17 中国农业科学院特产研究所 一种没食子酰基葡萄糖的制备方法
CN107029051A (zh) * 2016-12-09 2017-08-11 昭平县科学技术指导站 一种茶叶中茶多酚的提取工艺
CN110437289A (zh) * 2019-09-02 2019-11-12 广东省农业科学院茶叶研究所 一种四没食子酰葡萄糖的制备方法

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113376271A (zh) * 2021-05-26 2021-09-10 华东理工大学 一种基于lc-ms的生物样品中壳寡糖的高灵敏度检测分析方法
CN114891052A (zh) * 2022-05-10 2022-08-12 合肥工业大学 一种从油茶蒲中制备1,2,3,6-四-O-没食子酰-β-D-吡喃葡萄糖苷的方法
CN114891052B (zh) * 2022-05-10 2024-04-12 合肥工业大学 一种从油茶蒲中制备1,2,3,6-四-O-没食子酰-β-D-吡喃葡萄糖苷的方法

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