WO2021022819A1 - 一种基于异构化特征的安石榴苷纯化方法 - Google Patents

一种基于异构化特征的安石榴苷纯化方法 Download PDF

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WO2021022819A1
WO2021022819A1 PCT/CN2020/082035 CN2020082035W WO2021022819A1 WO 2021022819 A1 WO2021022819 A1 WO 2021022819A1 CN 2020082035 W CN2020082035 W CN 2020082035W WO 2021022819 A1 WO2021022819 A1 WO 2021022819A1
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punicalagin
components
puunicalagin
temperature
cut
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French (fr)
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艾萨·阿吉艾克拜尔
孙光映
赵永昕
阿布都艾尼·木尼热
阿地力·古丽契热
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中国科学院新疆理化技术研究所
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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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
    • B01D15/18—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to flow patterns
    • B01D15/1814—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to flow patterns recycling of the fraction to be distributed
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
    • B01D15/24—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to the treatment of the fractions to be distributed
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/32—Bonded phase chromatography
    • B01D15/325—Reversed phase
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/01—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing oxygen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H9/00—Compounds containing a hetero ring sharing at least two hetero atoms with a saccharide radical
    • C07H9/02—Compounds containing a hetero ring sharing at least two hetero atoms with a saccharide radical the hetero ring containing only oxygen as ring hetero atoms

Definitions

  • the invention relates to a method for purifying punicalagin based on isomerization characteristics.
  • Isomers are compounds with the same molecular weight but different structures and are widely found in natural products. There are many types of isomers, among which there are several types of special structures, such as certain epimers and cis-trans isomers, which can be transformed into each other under certain conditions. At present, the research on this isomerization reaction has been more mature and in-depth. However, the introduction of this isomerization feature into the field of separation and purification to solve the problem of the matrix effect of the purification of isomerizable compounds is a completely new concept. There is currently no report in the literature. Structured purification method.
  • isomerizable compounds usually show two or more peaks on the chromatogram. Connect one of the chromatographic peak components (which may contain impurities that are very similar to its polarity), and under certain conditions, accelerate the conversion into components with all isomers coexisting; at this time, the components are still on the chromatogram Can show all isomer peaks. Selectively cut off all the isomer components except the original chromatographic peak components, which can effectively shield the impurities embedded in the original chromatographic peak, and then under certain conditions can be transformed into each isomer uniformly existing without any impurities The monomer compound.
  • Punicarin is a kind of plant polyphenol with ellagic acid as the mother nucleus and rich in pomegranate peel medicinal materials. It has good anti-inflammatory, antibacterial, antiviral, tissue cancer cell proliferation and other biological activities. A compound with great potential for medicine.
  • punicalagin there is a glucoside with one end carbon, which can produce anomeric isomers, namely, two isomers of ⁇ -pusmoside and ⁇ -pusmoside, which appear on the chromatogram. Separate isomer peak. The two isomers can be converted into each other.
  • the main purification methods of punicalagin are mainly column chromatography, liquid chromatography and countercurrent chromatography, as well as the coordinated use of various methods.
  • the corresponding isomerization purification method was developed, combined with pilot-scale chromatography technology, to obtain high-purity punicalagin monomer compounds in large quantities.
  • the key point of the method of the present invention is the establishment of isomerization purification, and it is used for the batch preparation of high-purity punicalagin monomer compounds, which has very important significance for the research and development of new drugs targeting punicalagin.
  • the established isomerization purification method is suitable for the purification of isomerizable compounds, is innovative and has a certain demonstration.
  • the purpose of the present invention is to provide a method for purifying punicalagin based on the characteristics of isomerization.
  • the method uses pomegranate peel extract as a raw material, based on the fact that the punicalagin in the pomegranate peel extract has two mutually convertible isomers.
  • the structural characteristics are used to avoid the impurities contained in the punicalagin, and the pilot-scale preparative liquid chromatography is used to obtain a large amount of punicalarin with a purity higher than 98% from the complex extract of pomegranate peel.
  • the method is very simple, and the obtained punicalagin has high purity and large preparation quantity, and has a strong reference value for the purification and preparation of compounds with isomerization characteristics.
  • the mobile phase is methanol with a volume ratio of 1 ⁇ 5:9 ⁇ 5 and 0.01% ⁇ 5% (v/v) formic acid water, the flow rate is controlled at 50 ⁇ 300mL/min, and the UV detection wavelength is 220 ⁇ 280nm and 350 ⁇ 380nm, balance for 3 ⁇ 30min;
  • step c Take the components at the position of ⁇ -puunicarin after concentration in step b, and re-inject the sample to reversed-phase chromatography under the same conditions according to step a, and cut out the two chromatographic peaks of ⁇ -puunicalain and ⁇ -puunicalain respectively.
  • the components cut from the position of ⁇ -pusmoginine are qualified products; the qualified products are concentrated at a temperature of 30-50 °C, freeze-dried to obtain powder, and the purity is higher than 98% by high performance liquid chromatography; ⁇ -The components cut from the punicalagin position are concentrated at a temperature of 30 to 50°C, and cyclic injection according to the chromatographic conditions of step a to prepare high-purity punicalagin;
  • step b take the components at the position of ⁇ -puunicalagin after concentration in step b, and re-inject them into reversed-phase chromatography under the same conditions according to step a, and cut out two chromatograms of ⁇ -puunicalagin and ⁇ -puunicalagin respectively Peaks, where the components cut from the position of ⁇ -puunicalagin are qualified products; the components cut from the position of ⁇ -puunicalagin are concentrated at a temperature of 30-50°C, and then injected into step a reversed-phase chromatography, Separate the two chromatographic peaks of ⁇ -puunicarin and ⁇ -puunicalain, among which the components cut from the position of ⁇ -puunicalagin are qualified products; combine the two components obtained from the position of ⁇ -pusmoside, and step c The components cut from the position of ⁇ -pusmoside are combined, concentrated at a temperature of 30 ⁇ 50°C, freeze-dried to obtain a light yellow powder, and the
  • step b 0.1-10 g of pomegranate peel extract is weighed and dissolved in 10-50 mL of water.
  • the centrifugal rotation speed is 5000-10000 revolutions/minute, and the time is 3-5 minutes.
  • the method for purifying punicalagin according to the present invention is characterized in that the concentration is concentrated to 10-50 mL. Further preferably, the concentration is performed using a rotary evaporator.
  • the method of purifying punicalagin based on isomerization characteristics of the present invention is carried out according to the following steps:
  • the mobile phase is methanol and 0.1% formic acid water with a volume ratio of 12:88, the flow rate is controlled at 180mL/min, the temperature is room temperature, the UV detection wavelength is 254nm and 366nm, equilibrate for 15min, and set aside;
  • step c Take the components at the position of ⁇ -puunicalain after concentration in step b, and re-inject the sample to the pilot reversed phase chromatography under the same conditions according to step a, and cut the two chromatograms of ⁇ -puunidinin and ⁇ -puunicalain respectively
  • the peak where the components cut from the position of ⁇ -puunicalagin are qualified products, they are concentrated with a rotary evaporator at a temperature of 39°C, freeze-dried to obtain a pale yellow powder, weighed, a total of 137 mg, and detected by high performance liquid chromatography Obtain 98% of its purity, and the components cut from the position of ⁇ -puunicalagin were concentrated to 20 mL with a rotary evaporator at a temperature of 50°C, and then cyclic injection was performed according to the chromatographic conditions of step a to prepare high-purity punicalagin;
  • step b take the components at the position of ⁇ -puunicarin after concentration in step b, and re-inject the sample to the pilot reversed phase chromatogram of the same conditions according to step a, and cut out the two chromatographic peaks of ⁇ -pusmoside and ⁇ -pusmoside respectively ,
  • the components cut off from the position of ⁇ -puunicalagin are qualified products; the components cut off from the position of ⁇ -puunicalagin are concentrated to 20 mL with a rotary evaporator at a temperature of 50 °C, and then injected into step a Test reversed-phase chromatography, cut out the two chromatographic peaks of ⁇ -puunicalain and ⁇ -puunicalain respectively, and the components cut from the position of ⁇ -puunicalagin are qualified products.
  • the chromatographic column used in the method is a reversed-phase C18 chromatographic column, wherein the volume of the quantitative loop for injection is 30 mL, which is greater than the sample volume by 20 mL .
  • the isomerization feature-based purification method of punicalagin develops an isomerization purification method using the isomerization characteristics of punicalagin and is used for the batch purification of high-purity punicalagin. It is simple and practical, has a large preparation amount, and the purity of the obtained punicalagin is higher than 98%.
  • the established isomerization purification method is suitable for the purification of isomerizable compounds, is innovative and has a certain demonstration.
  • Figure 1 is a chromatogram of the pomegranate peel extract involved in the present invention on a pilot-scale reversed phase chromatography
  • Figure 2 is a preparative chromatogram of the ⁇ -puunicalagin component 1 involved in the present invention after being concentrated and reloaded onto the pilot reverse phase chromatogram;
  • Fig. 3 is a liquid chromatographic identification diagram of ⁇ -puunicalagin component 3 obtained in the present invention.
  • Figure 4 is a preparative chromatogram of the ⁇ -puunicalagin component 4 involved in the present invention after being concentrated and reloaded onto the pilot reverse phase chromatogram;
  • Figure 5 is a liquid chromatographic identification diagram of the combined ⁇ -puunicalagin component 5 and ⁇ -puunicalagin component 7 obtained in the present invention.
  • ⁇ -pusmoside component 2 with a time range of 12.5 to 20 minutes and the ⁇ -pusmoside component 3 with a time range of 25 to 35 minutes are collected into different component tanks.
  • ⁇ -Punica granatum component 3 is a qualified product, concentrated with a rotary evaporator at a temperature of 39°C, freeze-dried to obtain a light yellow powder, weighed, a total of 137 mg, and its purity is higher than 98 by high performance liquid chromatography.
  • the ⁇ -puunicalagin component 2 is concentrated to 20 mL with a rotary evaporator at a temperature of 50°C, and cyclic injection according to the chromatographic conditions of step a can be used to prepare high-purity punicalagin.
  • ⁇ -puunicalagin component 4 Re-inject the ⁇ -puunicalagin component 4 into the balanced pilot-phase reverse phase chromatographic column according to step a.
  • the chromatogram is shown in Figure 4.
  • the ⁇ -puunicalagin component 5 with a time range of 12.5 min to 20 min and the ⁇ -puunicalagin component 6 with a time range of 25 min to 35 min were collected into different component tanks.
  • ⁇ -Punica granatum component 5 is a qualified product.
  • the ⁇ -puunicalagin component 6 was concentrated to 20 mL with a rotary evaporator at a temperature of 50°C, and the sample was re-injected into the balanced pilot reverse phase chromatographic column according to step a.
  • the chromatogram is still shown in Figure 4.
  • the ⁇ -pusmoside component 7 with a time range of 12.5 to 20 minutes and the ⁇ -pusmoside component 8 with a time range of 25 to 35 minutes are collected into different component tanks.
  • ⁇ -Punica granatum component 7 is a qualified product, combined with ⁇ -Punica granatum component 5, concentrated with a rotary evaporator at a temperature of 39°C, freeze-dried to obtain a light yellow powder, weighed, a total of 280 mg, and passed the high efficiency Liquid chromatography detected that its purity was higher than 98%, as shown in Figure 5.
  • the ⁇ -puunicalagin component 8 is concentrated to 20 mL with a rotary evaporator at a temperature of 50° C., and cyclic injection according to the chromatographic conditions of step a can be used to prepare high-purity punicalagin.
  • reversed-phase chromatographic columns involved in the examples in addition to reversed-phase C18 chromatographic columns, can be used reversed-phase chromatographic columns including reversed-phase C8 column, reversed-phase C4 column, reversed-phase C30 column, reversed-phase C2 column, reversed-phase cyano column , Reversed-phase polystyrene column.
  • the column length can be used between 50mm and 1000mm, the inner diameter is between 2.1mm and 2000mm, and the particle size of the filler can be Use between 1.2 ⁇ m and 500 ⁇ m.

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Abstract

本发明涉及一种基于异构化特征的安石榴苷纯化制备方法，该方法以石榴皮提取物为原料，基于石榴皮提取物中安石榴苷具有两个可相互转化的异构体的结构特点来规避包含在安石榴苷中的杂质，利用中试级制备液相色谱实现了从复杂的石榴皮提取物中获得了大量纯度高于98％的安石榴苷。该方法十分简便，所得安石榴苷的纯度高、制备量大，且对具有异构化特征化合物的纯化制备具有很强的借鉴价值。

Description

一种基于异构化特征的安石榴苷纯化方法
相关申请的交叉参考
该申请要求2019年08月02日提交的中国专利申请号为201910711349.9的专利申请的优先权，该专利申请在此被完全引入作为参考。
技术领域
本发明涉及一种基于异构化特征的安石榴苷纯化方法。
背景技术
从复杂的天然产物药材或提取物中获得大量高纯度的单体化合物，一直是一个热点难点问题。天然产物的成分多，结构组成非常复杂，在单一化合物的周围往往分布着结构高度相似的化合物，在分离过程中对目标化合物造成极其严重的干扰，这种干扰往往被称为基质效应。在传统的柱色谱、液相色谱、逆流色谱中，这种基质效应往往给纯化合物的分离带来了很大的困扰，甚至得不到纯度合格的产品。因此，发明一种可以有效屏蔽基质效应的分离方法，且能应用于简单的色谱分离手段，提高从天然产物中单体化合物的纯化效率，具有十分重要的意义。
同分异构体是分子量相同，而结构不相同的化合物，在天然产物中广泛存在。同分异构体的种类繁多，其中有几类特殊的结构，如某些差向异构体和顺反异构体，在一定条件下可以相互转化。目前，对这种异构化反应的研究已经较为成熟深入。然而，将这种异构化的特点引入到分离纯化领域中来解决可异构化化合物的纯化的基质效应问题，则是一个全新的概念，目前尚无任何文献进行报道，可称之为异构化纯化法。
异构化纯化法基本原理为：可异构化的化合物，通常在色谱上显示两个或更多的峰。将其中的一个色谱峰组分接下(其中可包含与其极性非常相似的杂质)，在一定的条件下加速转化成所有异构体共存的组分；此时的组分在色谱图上仍可展现所有的异构体峰。选择性切下除原色谱峰组分之外的所有异构体组分，即可有效屏蔽原色谱峰包埋的杂质，再在一定条件下转化成各异构体均匀存在、且无任何杂质的单体化合物。
安石榴苷是一种以鞣花酸为母核的，在石榴皮药材中含量丰富的植物多酚，具有很好的抗炎、抗菌、抗病毒、组织癌细胞增殖等生物活性，是一种很有成药潜力的化合物。安石榴苷的结构中，存在一个葡萄糖苷，具有一个端头碳，可产 生端基异构体，即α-安石榴苷和β-安石榴苷两个异构体，在色谱上呈现两个单独的异构体峰。两个异构体间可以相互转化。目前主要的安石榴苷的主要纯化手段主要为柱色谱、液相色谱法和逆流色谱法，以及各手段的配合使用。由于天然产物的基质效应十分复杂，纯化难度很大，因而造成合格的高纯样品的极低，甚至不能达到极高的纯度。利用安石榴苷的可异构化特征，开发相应的异构化纯化方法，结合中试色谱技术，大批量得到高纯度的安石榴苷单体化合物。本发明所述方法的关键点就是异构化纯化的建立，并用于高纯度安石榴苷单体化合物的批量制备，这对以安石榴苷为目标的新药研发具有十分重要的意义。所建立的异构化纯化方法适用于可异构化化合物的纯化，创新性强，具有一定的示范性。
发明内容
本发明的目的在于，提供一种基于异构化特征的安石榴苷纯化方法，该方法以石榴皮提取物为原料，基于石榴皮提取物中安石榴苷具有两个可相互转化的异构体的结构特点来规避包含在安石榴苷中的杂质，利用中试级制备液相色谱实现了从复杂的石榴皮提取物中获得了大量纯度高于98％的安石榴苷。该方法十分简便，所得安石榴苷的纯度高、制备量大，且对具有异构化特征化合物的纯化制备具有很强的借鉴价值。
本发明的基于异构化特征的安石榴苷纯化方法，包括以下步骤：
a、取反相色谱柱，流动相为体积比1～5：9～5的甲醇和0.01％～5％(v/v)甲酸水，流速控制在50～300mL/min，紫外检测波长为220～280nm和350～380nm，平衡3～30min；
b、称取石榴皮提取物，溶于水中，离心，取上清液，进样至已平衡的反相色谱柱中，分别切取α-安石榴苷和β-安石榴苷两个色谱峰，并分别温度30～50℃下浓缩；
c、取步骤b浓缩后的α-安石榴苷位置的组分，按照步骤a重新进样至同条件的反相色谱，分别切取α-安石榴苷和β-安石榴苷两个色谱峰，其中β-安石榴苷位置切下来的组分为合格产品；将合格产品在温度30～50℃下浓缩，冷冻干燥得到粉末，并通过高效液相色谱法检测得其纯度高于98％；α-安石榴苷位置切下来的组分，在温度30～50℃下浓缩，按步骤a色谱条件循环进样再制备高纯度的安石榴苷；
和/或，取步骤b浓缩后的β-安石榴苷位置的组分，按照步骤a重新进样至同条件的反相色谱，分别切取α-安石榴苷和β-安石榴苷两个色谱峰，其中α-安 石榴苷位置切下来的组分为合格产品；将β-安石榴苷位置切下来的组分，在温度30～50℃下浓缩，再进样至步骤a反相色谱，分别切取α-安石榴苷和β-安石榴苷两个色谱峰，其中α-安石榴苷位置切下来的组分为合格产品；合并两部分α-安石榴苷位置所得组分，与步骤c中β-安石榴苷位置切下的组分合并，在温度30～50℃下浓缩，冷冻干燥得到淡黄色粉末，并通过高效液相色谱法检测得其纯度高于98％，β-安石榴苷位置切下来的组分在温度30～50℃下浓缩，按步骤a色谱条件循环进样再制备高纯度的安石榴苷。
根据本发明所述的安石榴苷纯化方法，其中优选的，步骤b称取0.1～10g石榴皮提取物，溶于10～50mL水中。
根据本发明所述的安石榴苷纯化方法，其中优选的，步骤b中离心转速5000～10000转/分钟，时间3～5分钟。
根据本发明所述的安石榴苷纯化方法，其特征在于，所述浓缩均浓缩至10～50mL。进一步地优选的，所述浓缩使用旋转蒸发仪进行。
根据本发明前述任一所述的安石榴苷纯化方法，作为一种优选方式，本发明所述的一种基于异构化特征的安石榴苷纯化方法，按下列步骤进行：
a、取中试反相色谱柱，流动相为体积比12:88的甲醇和0.1％甲酸水，流速控制在180mL/min，温度室温，紫外检测波长为254nm和366nm，平衡15min，待用；
b、称取6g石榴皮提取物，溶于20mL水中，离心，离心转速10000转/分钟，时间5分钟，取上清液，通过六通阀进样至已平衡的中试反相色谱柱中，分别切取α-安石榴苷和β-安石榴苷两个色谱峰，并分别在温度50℃下用旋转蒸发仪浓缩至20mL；
c、取步骤b浓缩后的α-安石榴苷位置的组分，按照步骤a重新进样至同条件的中试反相色谱，分别切取α-安石榴苷和β-安石榴苷两个色谱峰，其中β-安石榴苷位置切下来的组分为合格产品，用旋转蒸发仪在温度39℃下浓缩，冷冻干燥得到淡黄色粉末，称重，共计137mg，并通过高效液相色谱法检测得其纯度98％，α-安石榴苷位置切下来的组分，在温度50℃下用旋转蒸发仪浓缩至20mL，按步骤a色谱条件循环进样再制备高纯度的安石榴苷；
或取步骤b浓缩后的β-安石榴苷位置的组分，按照步骤a重新进样至同条件的中试反相色谱，分别切取α-安石榴苷和β-安石榴苷两个色谱峰，其中α-安石榴苷位置切下来的组分为合格产品；β-安石榴苷位置切下来的组分，用旋蒸蒸发 仪在温度50℃下浓缩至20mL，再进样至步骤a中试反相色谱，分别切取α-安石榴苷和β-安石榴苷两个色谱峰，其中α-安石榴苷位置切下来的组分为合格产品。合并两部分α-安石榴苷位置所得组分，用旋转蒸发仪在温度39℃下浓缩，冷冻干燥得到淡黄色粉末，称重，共计280mg，并通过高效液相色谱法检测得其纯度高于98％，β-安石榴苷位置切下来的组分在温度50℃下用旋转蒸发仪浓缩至20mL，按步骤a色谱条件循环进样再制备高纯度的安石榴苷。
本发明所述的一种基于异构化特征的安石榴苷纯化方法，该方法中所用的色谱柱，为反相C18色谱柱，其中进样用的定量环体积为30mL，大于上样体积20mL。
本发明所述的一种基于异构化特征的安石榴苷纯化方法，利用安石榴苷可异构化的特征开发出异构化纯化方法，并用于高纯度安石榴苷的批量纯化，该方法简单实用，制备量大，所得安石榴苷纯度高于98％，所建立的异构化纯化方法适用于可异构化化合物的纯化，创新性强，具有一定的示范性。
附图说明
图1为本发明所涉及的石榴皮提取物在中试反相色谱上的色谱图；
图2为本发明所涉及的α-安石榴苷组分1浓缩后重新上样至中试反相色谱上的制备色谱图；
图3为本发明得到的β-安石榴苷组分3的液相色谱鉴定图；
图4为本发明所涉及的β-安石榴苷组分4浓缩后重新上样至中试反相色谱上的制备色谱图；
图5为本发明得到的α-安石榴苷组分5和α-安石榴苷组分7合并后的液相色谱鉴定图。
具体实施方式
下面结合具体实施例，进一步详细阐述本发明。
实施例1
a、取中试反相色谱柱，规格柱长250mm×内径80mm，填料粒径10μm，流动相为体积比12:88的甲醇和0.1％甲酸水，流速控制在180mL/min，温度室温，紫外检测波长为254nm和366nm，平衡15min，待用；
b、称取6g石榴皮提取物，其中安石榴苷含量约为32％，溶于20mL水中，离心，离心转速10000转/分钟，时间5分钟，取上清液，通过六通阀进样至已 平衡的中试反相色谱柱中如图1所示，将时间范围为10min至15min间的组分α-安石榴苷组分1收集到组分罐中，并分别在温度50℃下用旋转蒸发仪浓缩至20mL；
c、将组分1按照步骤a重新进样至平衡好的中试反相色谱柱中，其色谱图如图2所示。将时间范围为12.5min至20min间的α-安石榴苷组分2和时间范围为25min至35min间的β-安石榴苷组分3分别收集到不同的组分罐中。β-安石榴苷组分3为合格产品，用旋转蒸发仪在温度39℃下浓缩，冷冻干燥得到淡黄色粉末，称重，共计137mg，并通过高效液相色谱法检测得其纯度高于98％，如图3所示。α-安石榴苷组分2在温度50℃下用旋转蒸发仪浓缩至20mL，按步骤a色谱条件循环进样可用来再制备高纯度的安石榴苷。
实施例2
a、取中试反相色谱柱，规格柱长250mm×内径80mm，填料粒径10μm，流动相为体积比12:88的甲醇和0.1％甲酸水，流速控制在180mL/min，温度室温，紫外检测波长为254nm和366nm，平衡15min，待用；
b、称取6g石榴皮提取物，其中安石榴苷含量约为32％，溶于20mL水中，离心，离心转速10000转/分钟，时间5分钟，取上清液，通过六通阀进样至已平衡的中试反相色谱柱中如图1所示，将时间范围为17min至30min间的组分β-安石榴苷组分4收集到组分罐中，并分别在温度50℃下用旋转蒸发仪浓缩至20mL；
c、将β-安石榴苷组分4按照步骤a重新进样至平衡好的中试反相色谱柱中，其色谱图如图4所示。将时间范围为12.5min至20min间的α-安石榴苷组分5和时间范围为25min至35min间的β-安石榴苷组分6分别收集到不同的组分罐中。α-安石榴苷组分5为合格产品。
将β-安石榴苷组分6在温度50℃下用旋转蒸发仪浓缩至20mL，按照步骤a重新进样至平衡好的中试反相色谱柱中，其色谱图仍如图4所示。将时间范围为12.5min至20min间的α-安石榴苷组分7和时间范围为25min至35min间的β-安石榴苷组分8分别收集到不同的组分罐中。α-安石榴苷组分7为合格产品，与α-安石榴苷组分5合并，用旋转蒸发仪在温度39℃下浓缩，冷冻干燥得到淡黄色粉末，称重，共计280mg，并通过高效液相色谱法检测得其纯度高于98％，如图5所示。β-安石榴苷组分8在温度50℃下用旋转蒸发仪浓缩至20mL，按步骤a色谱条件循环进样可用来再制备高纯度的安石榴苷。
实施例中所涉及反相色谱流动相，除甲醇-0.1％甲酸水混合物外，可使用乙腈-0.1％甲酸水混合物、四氢呋喃-0.1％甲酸水混合物，甲醇-乙腈-0.1％甲酸水混合，甲醇-四氢呋喃-0.1％甲酸水混合物，乙腈-四氢呋喃-0.1％甲酸水混合物，甲醇-乙腈-四氢呋喃-0.1％甲酸水混合物。其流动相中0.1％甲酸水含量的比例应控制在50％至95％之间。
实施例中所涉及反相色谱柱，除反相C18色谱柱外，可使用反相色谱柱包括反相C8柱、反相C4柱，反相C30柱，反相C2柱，反相氰基柱，反相聚苯乙烯柱。
实施例中所涉及的反相色谱柱规格，除柱长250mm、内径80mm、填料粒径10μm外，可使用柱长在50mm至1000mm之间，内径在2.1mm至2000mm之间，填料粒径可使用1.2μm至500μm之间。

Claims (6)

  1. 一种基于异构化特征的安石榴苷纯化方法，包括以下步骤：
    a、取反相色谱柱，流动相为体积比1～5：9～5的甲醇和0.01％～5％甲酸水，流速控制在50～300mL/min，紫外检测波长为220～280nm和350～380nm，平衡3～30min；
    b、称取石榴皮提取物，溶于水中，离心，取上清液，进样至已平衡的反相色谱柱中，分别切取α-安石榴苷和β-安石榴苷两个色谱峰，并分别温度30℃～50℃下浓缩；
    c、取步骤b浓缩后的α-安石榴苷位置的组分，按照步骤a重新进样至同条件的反相色谱，分别切取α-安石榴苷和β-安石榴苷两个色谱峰，其中β-安石榴苷位置切下来的组分为合格产品；将合格产品在温度30℃～50℃下浓缩，冷冻干燥得到粉末，并通过高效液相色谱法检测得其纯度高于98％；α-安石榴苷位置切下来的组分，在温度30℃～50℃下浓缩，按步骤a色谱条件循环进样再制备高纯度的安石榴苷；
    和/或，取步骤b浓缩后的β-安石榴苷位置的组分，按照步骤a重新进样至同条件的反相色谱，分别切取α-安石榴苷和β-安石榴苷两个色谱峰，其中α-安石榴苷位置切下来的组分为合格产品；将β-安石榴苷位置切下来的组分，在温度30～50℃下浓缩，再进样至步骤a反相色谱，分别切取α-安石榴苷和β-安石榴苷两个色谱峰，其中α-安石榴苷位置切下来的组分为合格产品；合并两部分α-安石榴苷位置所得组分，与步骤c中β-安石榴苷位置切下的组分合并，在温度30℃～50℃下浓缩，冷冻干燥得到淡黄色粉末，并通过高效液相色谱法检测得其纯度高于98％，β-安石榴苷位置切下来的组分在温度30～50℃下浓缩，按步骤a色谱条件循环进样再制备高纯度的安石榴苷。
  2. 根据权利要求1所述的安石榴苷纯化方法，其特征在于，步骤b称取0.1～10g石榴皮提取物，溶于10～50mL水中。
  3. 根据权利要求1所述的安石榴苷纯化方法，其特征在于，步骤b中离心转速5000～10000转/分钟，时间3～10分钟。
  4. 根据权利要求1所述的安石榴苷纯化方法，其特征在于，所述浓缩均浓缩至10～50mL。
  5. 根据权利要求1或4所述的安石榴苷纯化方法，其特征在于，所述浓缩使用旋转蒸发仪进行。
  6. 根据权利要求1-5任一所述的安石榴苷纯化方法，其特征在于按下列步骤进行：
    a、取中试反相色谱柱，流动相为体积比12:88的甲醇和0.1％甲酸水，流速控制在180mL/min，温度室温，紫外检测波长为254nm和366nm，平衡15min，待用；
    b、称取6g石榴皮提取物，溶于20mL水中，离心，离心转速10000转/分钟，时间5分钟，取上清液，通过六通阀进样至已平衡的中试反相色谱柱中，分别切取α-安石榴苷和β-安石榴苷两个色谱峰，并分别在温度50℃下用旋转蒸发仪浓缩至20mL；
    c、取步骤b浓缩后的α-安石榴苷位置的组分，按照步骤a重新进样至同条件的中试反相色谱，分别切取α-安石榴苷和β-安石榴苷两个色谱峰，其中β-安石榴苷位置切下来的组分为合格产品；用旋转蒸发仪在温度39℃下浓缩，冷冻干燥得到淡黄色粉末，称重，共计137mg，并通过高效液相色谱法检测得其纯度高于98％；α-安石榴苷位置切下来的组分，在温度50℃下用旋转蒸发仪浓缩至20mL，按步骤a色谱条件循环进样再制备高纯度的安石榴苷；
    或取步骤b浓缩后的β-安石榴苷位置的组分，按照步骤a重新进样至同条件的中试反相色谱，分别切取α-安石榴苷和β-安石榴苷两个色谱峰，其中α-安石榴苷位置切下来的组分为合格产品；β-安石榴苷位置切下来的组分，用旋蒸蒸发仪在温度50℃下浓缩至20mL，再进样至步骤a中试反相色谱，分别切取α-安石榴苷和β-安石榴苷两个色谱峰，其中α-安石榴苷位置切下来的组分为合格产品。合并两部分α-安石榴苷位置所得组分，与步骤c中β-安石榴苷位置切下的组分合并，用旋转蒸发仪在温度39℃下浓缩，冷冻干燥得到淡黄色粉末，称重，共计280mg，并通过高效液相色谱法检测得其纯度高于98％，β-安石榴苷位置切下来的组分在温度50℃下用旋转蒸发仪浓缩至20mL，按步骤a色谱条件循环进样再制备高纯度的安石榴苷。
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