WO2022052394A1 - 一种飞燕草素酰基化花色苷的制备方法 - Google Patents

一种飞燕草素酰基化花色苷的制备方法 Download PDF

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WO2022052394A1
WO2022052394A1 PCT/CN2021/070637 CN2021070637W WO2022052394A1 WO 2022052394 A1 WO2022052394 A1 WO 2022052394A1 CN 2021070637 W CN2021070637 W CN 2021070637W WO 2022052394 A1 WO2022052394 A1 WO 2022052394A1
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phase
anthocyanin
acid
volume
concentration
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French (fr)
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陈卫
谢佳宏
徐阳
崔昊昕
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浙江大学
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Priority to JP2023516730A priority Critical patent/JP2023542661A/ja
Priority to US17/674,860 priority patent/US11981697B2/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
    • 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
    • B01D11/0261—Solvent extraction of solids comprising vibrating mechanisms, e.g. mechanical, acoustical
    • B01D11/0265—Applying ultrasound
    • 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
    • B01D15/42—Selective adsorption, e.g. chromatography characterised by the development mode, e.g. by displacement or by elution
    • B01D15/424—Elution mode
    • B01D15/426—Specific type of solvent
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
    • C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
    • C07H17/06—Benzopyran radicals
    • C07H17/065—Benzo[b]pyrans

Definitions

  • the invention relates to the technical field of separation and purification of natural products, in particular to a method for separating and preparing delphinidin-3-O-(6-O-p-coumaroyl) glucoside.
  • Grapes are the fruit of the Vitis genus plant and are cultivated all over the world. Grapes have high nutritional value.
  • Anthocyanins are one of the important polyphenols in grapes. Studies have shown that grapes contain delphinidin, malvain, paeoniflorin and petunidin and other anthocyanin aglycones combined with glucose to form anthocyanins.
  • delphinidin, malvain, paeoniflorin and petunidin and other anthocyanin aglycones combined with glucose to form anthocyanins.
  • due to the sensitivity of anthocyanins to light, temperature and pH their chemical properties are relatively unstable and their bioavailability is low, which greatly limit the practical application of anthocyanins.
  • acylated anthocyanins also have biological activity, and have more stable chemical properties and higher bioavailability.
  • the grape contains a large amount of anthocyanin, which indicates that grape anthocyanin has a better market prospect.
  • High performance liquid chromatography is a chromatography technology based on the principle of solid-liquid adsorption.
  • adsorbents such as silica gel as the stationary phase
  • separation is performed according to the difference in the binding ability of different compound molecules to the stationary phase.
  • the separation effect mainly depends on the properties of the stationary phase filler. (eg composition, particle size), and whether the LC method is good.
  • High performance liquid chromatography has the advantages of stability, reliability and good repeatability.
  • Countercurrent chromatography is a liquid-liquid chromatography technique in which both the stationary phase and the mobile phase are liquid. The principle is to separate the molecules of different compounds according to the difference in the distribution coefficients between the stationary phase and the mobile phase.
  • the separation effect of countercurrent chromatography mainly depends on whether the two-phase solvent system used is suitable.
  • Countercurrent chromatography has the advantages of simple sample pretreatment, wide application range, less sample loss, and large processing capacity. At present, in the preparation of grape anthocyanins, extraction, macroporous resin and single column chromatography or chromatographic technology are mainly used for separation and purification.
  • CN104177460A discloses a method for preparing 3,5-disaccharide anthocyanins.
  • the method uses ultrasonic-assisted extraction, extraction, purification and other steps, but the obtained product is an anthocyanin mixture containing three different disaccharides.
  • Anthocyanins, and the product does not involve acylated anthocyanins.
  • CN102229633A discloses a method for separating and preparing five high-purity anthocyanin monomers from grape skins. This method uses leaching, macroporous resin purification, and liquid-phase purification to obtain five anthocyanins. However, due to the use of two-step preparation liquid-phase purification, the sample is lost and the purification yield is reduced. The purity of the anthocyanins (malvain acetyl glucoside, malvain trans-coumaroyl glucoside) was relatively low, only 91.7% and 95.5%, respectively.
  • CN108976268A discloses a method for preparing two main anthocyanin standard products of thorn grape.
  • the method adopts macroporous resin to adsorb and enrich the turbid juice of thorn grape, and then elute and freeze-dry to obtain crude anthocyanins, and then use high-speed Countercurrent chromatography, using water-n-butanol-methyl tert-butyl ether-acetonitrile-trifluoroacetic acid (volume ratio 5:4:1:2:0.001 or 5:3:1:1:0.001) as a two-phase solvent system After separation, two anthocyanins were obtained with purities of 95.8% and 92.2%, respectively.
  • the invention discloses a method based on preparative liquid chromatography and high-speed countercurrent chromatography.
  • the combined method because the present invention can realize the preparation of high-purity delphinidin-3-O-(6-O-p-coumaroyl) glucoside monomers in large quantities from grapes with complex anthocyanin components, It provides new ideas for the development and utilization of grape resources in my country.
  • a method for separating and preparing delphinidin-3-O-(6-O-p-coumaroyl) glucoside comprising the steps:
  • phase A is pure acetonitrile
  • phase B is formic acid aqueous solution with a volume percent concentration of formic acid of 1% to 2%;
  • the gradient elution program is: 0-4min, 5%-20% phase A; 4-18min, 20%-25% phase A; 18-21min, 25%-35% phase A; 21-24min, 35%-60 %A phase; 24-27min, 60%-5%A phase; 27-30min, 5%A phase;
  • the flow rate is 8-10mL/min, the column temperature is 30°C, and the detection wavelength is 520nm;
  • the detection wavelength is 280 nm, the fractions with retention time of 105-115 min are collected, concentrated under reduced pressure, and freeze-dried to obtain the target compound delphinidin-3-O-(6-O-p-coumaroyl)glucoside.
  • the percentages of raw materials in the present invention all refer to the volume percentage concentration, and the various solutions in the present invention, unless otherwise specified, use water as the solvent.
  • step (1) the acidic alcohol solution is extracted and concentrated to obtain the crude extract of grape skin anthocyanins. Specifically: washing the grapes and taking the skins, mixing with the acidic ethanol solution, and beating after mixing, and the temperature is below 50 ° C (preferably room temperature) Ultrasonic extraction, filtration, the filtrate is concentrated under reduced pressure at 40-50°C to remove ethanol to obtain a crude extract of grape skin anthocyanins;
  • the material-to-liquid ratio of grape skin and acidic ethanol solution is 1g:4-8mL;
  • the volume concentration of ethanol is 50%-80%, preferably 60%-70%, and the volume concentration of acid is 0.1%-1%;
  • the ultrasonic extraction time is 40-120min.
  • the acid is selected from at least one of hydrochloric acid, formic acid and acetic acid.
  • step (2) the macroporous resin purification method is specifically:
  • the crude extract of grape skin anthocyanins was injected into the macroporous resin, and then eluted with 0, 5%, 20%, and 40% acidic ethanol solutions of 4 times the column volume (4 BV) of ethanol by volume, and the volume of ethanol was collected.
  • the acidic ethanol eluent with a concentration of 40% is evaporated under reduced pressure at 40-50°C to remove the ethanol to obtain the anthocyanin eluent;
  • the macroporous resin is selected from AB-8, D101, XAD-7, HPD-100 or DM-130, its specific surface area is 450-550m 2 /g, the average pore size is 10-50nm, and the particle size is in the range of 0.3-1.25 mm;
  • the acidic ethanol solution is selected from an ethanol solution with an acid volume percentage concentration of 0.1% to 1.5%, wherein the acid is selected from at least one of hydrochloric acid, formic acid and acetic acid.
  • step (3) the organic solvent is ethyl acetate.
  • step (3) it is preferable to extract at a ratio of the organic solvent to the anthocyanin eluent volume ratio of 1:1, and extract more than 2 times.
  • step (4) the lyophilized anthocyanin powder can be dissolved in phase B or water.
  • the liquid chromatographic column used by the preparative liquid chromatography system is a C18 column, and the single injection volume is 10-40 mg in terms of anthocyanin freeze-dried powder, and the volume after the decompression concentration is before the concentration. 40%-70% of the volume.
  • step (5) the temperature of the high-speed countercurrent chromatographic instrument is stabilized at 20-30 ° C, the positive connection is forward, and the stationary phase is pumped, and then the rotating speed is adjusted to 800-950 r/min, and the mobile phase is passed into the mobile phase at a flow rate of 2 mL/min. Equilibrium, the amount of each injection is 30-200mg based on the crude anthocyanin monomer.
  • the main advantages of the present invention include:
  • a method for separating delphinidin-3-O-(6-O-p-coumaroyl) glucoside (molecular structure shown in Figure 1) from grape skin was established for the first time, and the yield was not less than 8 mg/ kg of grape skins, with a purity of not less than 98%.
  • delphidin-3-O-(6-O-p-coumaroyl can be prepared in large quantities in grape raw materials with complex polyphenol components ) glucoside, has the advantages of large processing capacity and good repeatability, which is convenient for industrial production.
  • Fig. 1 is the molecular structure diagram of delphinidin-3-O-(6-O-p-coumaroyl) glucoside;
  • Fig. 2 is in embodiment 1, the high performance liquid chromatogram of grape skin anthocyanin crude extract
  • Example 3 is a high-performance liquid chromatogram that contains delphinidin-3-O-(6-O-p-coumaroyl) glucoside moiety after separation and purification by macroporous resin in Example 1;
  • Fig. 4 is the high-speed countercurrent chromatogram in embodiment 1;
  • Fig. 5 is in embodiment 1, the high performance liquid chromatogram of final product delphinidin-3-O-(6-O-p-coumaroyl) glucoside;
  • Fig. 6 is in embodiment 1, the primary mass spectrogram and secondary mass spectrogram of delphinidin-3-O-(6-O-p-coumaroyl) glucoside;
  • Fig. 7 is the high performance liquid chromatogram of final product in Comparative Example 1;
  • FIG. 8 is a high-performance liquid chromatogram of the final product in Comparative Example 2.
  • the AB-8 macroporous resin was loaded into the chromatographic column, and ethanol, 0.5mol/L hydrochloric acid solution, 0.5mol/L sodium hydroxide solution were successively used, and after washing with water, the anthocyanin crude extract was subjected to 0.2BV/L.
  • the flow rate of h is injected into the chromatography column.
  • the mixture was extracted twice with ethyl acetate, and the aqueous phase was taken, concentrated under appropriate reduced pressure, and lyophilized to obtain lyophilized anthocyanin powder.
  • the high-performance liquid chromatogram of the fraction containing delphinidin-3-O-(6-O-p-coumaroyl) glucoside is shown in FIG. 3 .
  • Liquid-phase separation was performed using an Ultimate XB-C18 (7 ⁇ m, 21.2 ⁇ 250 mm) preparative chromatographic column.
  • the mobile phase consisted of pure acetonitrile (phase A) and 1.5% formic acid in water (phase B).
  • the gradient elution method is as follows: 0-4min, 5%-20% phase A; 4-18min, 20%-25% phase A; 18-21min, 25%-35% phase A; 21-24min, 35%-60 %A phase; 24-27min, 60%-5%A phase; 27-30min, 5%A phase, flow rate is 10mL/min, column temperature is 30°C, detection wavelength is 520nm.
  • the anthocyanin freeze-dried powder is dissolved and then injected, and the injection volume is 4mL, and the 16.5-18.0min components are collected, concentrated under reduced pressure, and then freeze-dried to obtain delphinidin-3-O-(6-O-pteroflora Bean acyl) glucoside monomer crude product.
  • Methyl tert-butyl ether methanol: water: trifluoroacetic acid was placed in a separatory funnel in a volume ratio of 2:2:3:0.001, shaken well, and after standing for 30 min, the upper and lower phases were separated, and ultrasonically degassed respectively. 30min.
  • the instrument temperature of the high-speed countercurrent chromatography system was stabilized at 20°C, the stationary phase was pumped, and then the rotational speed was adjusted to 850r/min.
  • the crude lyophilized powder was dissolved in 1 mL of mobile phase, filtered with a microporous membrane, and injected into the sample. A single injection of 10 mL was performed and detected under an ultraviolet detector with a detection wavelength of 280 nm.
  • the prepared anthocyanin sample was injected into the mass spectrometer, and the sample was analyzed according to the mass spectrum (Fig. 6), and it was confirmed that the mass number of the separated anthocyanin was normal.
  • the grapes were washed and peeled to obtain 2kg of grape skins, and 80% ethanol solution containing 0.5% (v/v) hydrochloric acid was added according to the ratio of material to liquid ratio of 1g: 6mL to fully mix, and ultrasonically extracted for 60min, (control temperature below 50°C) , protected from light), filtered with gauze, the filtrate was centrifuged at 4000 rpm for 10 min, and the supernatant was taken. The filter residue was extracted once more in the same way. The filtrates were combined and filtered again using a Buchner funnel. The filtrate was evaporated under reduced pressure at 45°C to remove ethanol and concentrated to obtain a crude extract of grape skin anthocyanins.
  • the AB-8 macroporous resin was loaded into the chromatographic column, and ethanol, 0.5mol/L hydrochloric acid solution, 0.5mol/L sodium hydroxide solution were successively used, and after washing with water, the anthocyanin crude extract was subjected to 0.2BV/L.
  • the flow rate of h is injected into the chromatography column.
  • use acid water (containing 0.5% hydrochloric acid), 5%, 20%, and 40% acidic ethanol (containing 0.5% hydrochloric acid) for 4 column volumes in sequence to elute collect the 40% acidic ethanol eluent, reduce The ethanol was removed by evaporation under pressure. Then, in a ratio of 1:1, the mixture was extracted twice with ethyl acetate, and the aqueous phase was taken, concentrated under appropriate reduced pressure, and freeze-dried to obtain freeze-dried anthocyanin powder.
  • Liquid-phase separation was performed using an Ultimate XB-C18 (7 ⁇ m, 21.2 ⁇ 250 mm) preparative chromatographic column.
  • the mobile phase consisted of pure acetonitrile (phase A) and 1.5% formic acid in water (phase B).
  • the gradient elution method is as follows: 0-4min, 5%-20% phase A; 4-18min, 20%-25% phase A; 18-21min, 25%-35% phase A; 21-24min, 35%-60 %A phase; 24-27min, 60%-5%A phase; 27-30min, 5%A phase, flow rate is 10mL/min, column temperature is 30°C, detection wavelength is 520nm.
  • the anthocyanin freeze-dried powder is dissolved and then injected, and the injection volume is 4mL, and the 16.5-18.0min components are collected, concentrated under reduced pressure, and then freeze-dried to obtain delphinidin-3-O-(6-O-pteroflora Bean acyl) glucoside monomer crude product.
  • Methyl tert-butyl ether methanol: water: trifluoroacetic acid was placed in a separatory funnel in a volume ratio of 2:2:3:0.001, shaken well, and after standing for 30 min, the upper and lower phases were separated, and ultrasonically degassed respectively. 30min.
  • the instrument temperature of the high-speed countercurrent chromatography system was stabilized at 25°C, the stationary phase was pumped, and then the rotational speed was adjusted to 850r/min.
  • the crude monomer lyophilized powder was dissolved in a proportion of 1 mL of mobile phase, filtered with a microporous membrane, and injected into the sample.
  • a single injection of 10 mL was performed and detected under an ultraviolet detector with a detection wavelength of 280 nm.
  • the target peak fractions of 105-115min were collected, concentrated under reduced pressure, and lyophilized to obtain 19 mg of delphinidin-3-O-(6-O-p-coumaroyl) glucoside with HPLC purity of 98.2%.
  • the AB-8 macroporous resin was loaded into the chromatographic column, and ethanol, 0.5mol/L hydrochloric acid solution, 0.5mol/L sodium hydroxide solution were successively used, and after washing with water, the anthocyanin crude extract was subjected to 0.2BV/L.
  • the flow rate of h is injected into the chromatography column.
  • use acid water (containing 0.5% hydrochloric acid), 5%, 20%, and 40% acidic ethanol (containing 0.5% hydrochloric acid) for 2 column volumes in sequence to elute collect the 40% acidic ethanol eluent, reduce The ethanol was removed by evaporation under pressure. Then, in a ratio of 1:1, the mixture was extracted twice with ethyl acetate, and the aqueous phase was taken, concentrated under appropriate reduced pressure and lyophilized to obtain anthocyanin lyophilized powder.
  • Liquid-phase separation was performed using an Ultimate XB-C18 (7 ⁇ m, 21.2 ⁇ 250 mm) preparative chromatographic column.
  • the mobile phase consisted of pure acetonitrile (phase A) and 1.5% formic acid in water (phase B).
  • the gradient elution method is as follows: 0-4min, 5%-20%A; 4-18min, 20%-25%A; 18-21min, 25%-35%A; 21-24min, 35%-60%A; 24-27min, 60%-5%A; 27-30min, 5%A, the flow rate is 10mL/min, the column temperature is 30°C, and the detection wavelength is 520nm.
  • the lyophilized anthocyanin powder is dissolved and then injected, and the injection volume is 3 mL, and the 16.5-18.0min components are collected, concentrated under reduced pressure, and then freeze-dried to obtain delphinidin-3-O-(6-O-pteroflora Bean acyl) glucoside monomer crude product.
  • Methyl tert-butyl ether methanol: water: trifluoroacetic acid was placed in a separatory funnel in a volume ratio of 2:2:3:0.001, shaken well, and after standing for 30 min, the upper and lower phases were separated, and ultrasonically degassed respectively. 30min. Stabilize the instrument temperature of the high-speed countercurrent chromatography system at 25 °C, pump the stationary phase, then adjust the speed to 850 r/min, connect the forward rotation, pass the mobile phase at a flow rate of 2 mL/min until equilibrium, and dissolve the lyophilized powder at a rate of 10 mg per 10 mg.
  • the crude monomer lyophilized powder was dissolved in a proportion of 1 mL of mobile phase, filtered with a microporous membrane, and injected into the sample. A single injection of 15 mL was performed and detected under an ultraviolet detector with a detection wavelength of 280 nm.
  • the target peak fractions of 105-115 min were collected and concentrated under reduced pressure, and 163 mg of delphinidin-3-O-(6-O-p-coumaroyl) glucoside was obtained by lyophilization, and the HPLC purity was 98.4%.
  • the preparation process is the same as that of Example 1, the difference is only that the step of high-speed countercurrent chromatographic purification is removed, and other steps remain unchanged.
  • the mixture of delphinidin-3-O-(6-O-p-coumaroyl) glucoside could not obtain delphinidin-3-O-(6-O-p-coumaroyl) glucoside monomer.
  • the preparation process is the same as that of Example 1, except that the flow rate in the high-speed countercurrent chromatography purification is set to 5 mL/min. After testing, although the target component delphinidin-3-O-(6-O-p-coumaroyl) glucoside can be collected, but because the flow rate is too fast, the target component is not completely compatible with other anthocyanins and other impurities. After separation, the purity of the obtained target component was only 90% (as shown in Figure 8).
  • the preparation process is the same as that of Example 1, except that the solvent system separated by high-speed countercurrent chromatography is replaced by n-butanol: methyl tert-butyl ether: methanol: water: trifluoroacetic acid at 2:2:1:5:0.001 system.
  • the target compound delphinidin-3-O-(6-O-p-coumaroyl) glucoside was mainly retained in the upper phase, and the target compound could not be collected.
  • the preparation process is the same as that of Example 1, except that the solvent system separated by high-speed countercurrent chromatography is replaced by a system of methyl tert-butyl ether: methanol: water: trifluoroacetic acid in a ratio of 2:1:3:0.001. After testing, the target delphinidin-3-O-(6-O-p-coumaroyl) glucoside could not be obtained.
  • the preparation process is the same as that of Example 1, and the difference is only to change the component collection time in the preparative liquid chromatography purification process. If the component collection time is not in the range of 16.5-18.0min, then the delphinidin-3-O containing delphinidin-3-O cannot be obtained. - Components of (6-O-p-coumaroyl)glucoside. If the fraction collection time is included and wider than the range of 16.5-18.0 min, it will affect the purity of the final isolated delphinidin-3-O-(6-O-p-coumaroyl) glucoside monomer.
  • the preparation process is the same as that of Example 1, except that the extraction process is changed instead of using an acid ethanol solution for extraction, and an acid-free ethanol solution is used instead.
  • the other steps remain unchanged, then the final target product delphinidin-3-O-(6-O-p-coumaroyl) glucoside monomer yield is 2mg/kg grape skin, much lower than 8mg/kg grape skin .

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Abstract

本发明公开了一种分离制备飞燕草素-3-O-(6-O-对香豆酰)葡萄糖苷的方法，通过提取，大孔树脂纯化，萃取，制备液相色谱和高速逆流色谱等步骤，从葡萄中分离纯化得到高纯度的飞燕草素-3-O-(6-O-对香豆酰)葡萄糖苷单体。通过该方法可以从10kg的葡萄皮中获得至少80mg的飞燕草素-3-O-(6-O-对香豆酰)葡萄糖苷，纯度可达到不低于98％。本方法具有操作简单，处理量大，重复性好等优点，为我国葡萄资源的开发利用提供了新的思路。

Description

一种飞燕草素酰基化花色苷的制备方法 技术领域
本发明涉及天然产物的分离纯化技术领域，具体涉及一种分离制备飞燕草素-3-O-(6-O-对香豆酰)葡萄糖苷的方法。
背景技术
葡萄是葡萄科葡萄属植物的果实，在世界各地均有栽培。葡萄具有很高的营养价值。花色苷是葡萄中重要的多酚类物质之一，研究表明，葡萄中含有飞燕草素、锦葵素、芍药素和矮牵牛素等花青素苷元与葡萄糖结合形成的花色苷。但由于花色苷对光、温度和pH敏感，导致其化学性质相对不稳定，而且生物利用度较低，这些性质极大地限制了花色苷的实际应用。已有研究表明，相对于非酰化花色苷，酰化花色苷同样具有生物活性，并且有更稳定的化学性质以及更高的生物利用度。而葡萄中含有大量对香豆酰化的花色苷，这说明葡萄花色苷具有更好的市场前景。
近年来，固相萃取技术(SPE)、制备型高效液相色谱技术(preparative-HPLC)和高速逆流色谱技术(HSCCC)等新型纯化技术开始得到发展和应用。高效液相色谱是基于固液吸附原理的色谱技术，通过使用硅胶等吸附剂作为固定相，根据不同化合物分子与固定相的结合能力的差异性而分离，分离效果主要取决于固定相填料的性质(如组成，粒径大小)，以及液相色谱方法是否良好。高效液相色谱技术具有稳定、可靠、重复性好等优点。逆流色谱是一种液液色谱技术，其固定相和流动相都是液态的，原理是根据不同化合物分子在固定相和流动相的分配系数的差异而分离。逆流色谱法的分离效果，主要取决于使用的两相溶剂体系是否合适。逆流色谱技术具有样品前处理简单、适用范围广、样品损失少、处理量大等优点。目前在葡萄花色苷的制备中，主要采用萃取、大孔树脂和单一的柱层析或色谱技术进行分离纯化。
如CN104177460A中公开了一种3,5-二糖类花色苷的制备方法，该方法使用超声辅助提取、萃取、纯化等步骤，但获得的产物为花色苷混合物， 含有三种不同的二糖类花色苷，并且产物不涉及酰化花色苷。
又如CN102229633A中公开了一种从葡萄皮中分离制备五种高纯度花色苷单体的方法。该方法使用浸提，大孔树脂纯化，制备液相的方法纯化得到了五种花色苷，然而由于使用了两步制备液相纯化，导致样品损耗，降低了纯化得率，且其中两种酰化花色苷(锦葵素乙酰葡萄糖苷，锦葵素反式香豆酰葡萄糖苷)的纯度相对较低，分别仅为91.7％和95.5％。
CN108976268A中公开了一种制备刺葡萄两个主要花色苷标准品的方法，该方法采用大孔树脂对刺葡萄浊汁进行吸附富集，随后经过洗脱，冷冻干燥得到花色苷粗品，再使用高速逆流色谱，以水-正丁醇-甲基叔丁基醚-乙腈-三氟乙酸(体积比5：4：1：2：0.001或5：3：1：1：0.001)为两相溶剂体系分离，得到两种花色苷，纯度分别为95.8％和92.2％。但从刺葡萄浊汁HPLC图中，可以看到其花色苷组成简单，由高速逆流色谱的局限性可推测出，当分离的样品花色苷组分复杂时，使用该方法可能难以得到目标花色苷。
由于酰化花色苷的分离纯化困难，导致目前市面上没有商业化的酰化花色苷标准品。因此，研究开发一种从复杂花色苷原料如葡萄中纯化酰化花色苷单体的工艺，对推动花色苷标准品市场及开发葡萄深加工产品具有重要意义。
发明内容
针对本领域存在的不足之处，根据酰化花色苷的特性，考虑到高效液相色谱技术和高速逆流色谱技术具有不同的分离原理，本发明公开了一种基于制备液相色谱与高速逆流色谱联用的方法，由于，本发明可以实现从花色苷组分复杂的葡萄中，大量制备高纯度的飞燕草素-3-O-(6-O-对香豆酰)葡萄糖苷单体，为我国葡萄资源的开发利用提供新的思路。
一种分离制备飞燕草素-3-O-(6-O-对香豆酰)葡萄糖苷的方法，包括步骤：
(1)醇提浓缩：以葡萄为原料，经酸性醇溶液提取，浓缩得到葡萄皮花色苷粗提液；
(2)大孔树脂纯化：将所述葡萄皮花色苷粗提液注入大孔树脂，经洗脱，浓缩得到花色苷洗脱液；
(3)萃取：先使用有机溶剂萃取所述花色苷洗脱液，再经减压浓缩，冻干后得到花色苷冻干粉；
(4)制备液相色谱纯化：将花色苷冻干粉溶解，注入制备型液相色谱系统中进行纯化，并用紫外检测器下检测，具体参数条件如下：
流动相：A相为纯乙腈，B相为甲酸体积百分浓度为1％～2％的甲酸水溶液；
梯度洗脱程序为：0-4min，5％-20％A相；4-18min，20％-25％A相；18-21min，25％-35％A相；21-24min，35％-60％A相；24-27min，60％-5％A相；27-30min，5％A相；
流速为8-10mL/min，柱温为30℃，检测波长为520nm；
根据液相色谱图收集保留时间为16.5-18.0min的组分，之后减压浓缩，冻干，得到花色苷单体粗品；
(5)高速逆流色谱分离：将甲基叔丁基醚、甲醇、水和三氟乙酸以2:2:3:0.001的体积比混合作为两相溶剂体系，且以上相为固定相，下相为流动相，依次将固定相和流动相泵入高速逆流色谱仪器中，两相在管路中达到平衡后，将花色苷单体粗品用流动相溶解，进样并在紫外检测器下检测，检测波长为280nm，收集保留时间为105-115min的组分并减压浓缩、冻干，得到目标化合物飞燕草素-3-O-(6-O-对香豆酰)葡萄糖苷。
如无特殊说明，本发明中出现的原料百分比均指体积百分浓度，本发明中出现的各种溶液，如无特别说明，均以水作为溶剂。
步骤(1)中，所述酸性醇溶液提取，浓缩得到葡萄皮花色苷粗提液具体为：将葡萄洗净取皮，与酸性乙醇溶液混合，混匀后打浆，50℃以下(优选室温)超声提取，过滤，滤液在40-50℃减压浓缩除去乙醇，得到葡萄皮花色苷粗提液；
葡萄皮与酸性乙醇溶液的料液比为1g:4-8mL；
所述酸性乙醇溶液中，乙醇体积浓度为50％-80％，优选60％-70％，酸体积浓度为0.1％-1％；
所述超声提取时间为40-120min。
步骤(1)中，所述酸性乙醇溶液中，酸选自盐酸、甲酸、乙酸中的至少一种。
步骤(2)中，所述大孔树脂纯化方法具体为：
将葡萄皮花色苷粗提液注入大孔树脂中，之后依次用乙醇体积浓度为0，5％，20％，40％的酸性乙醇溶液各4倍柱体积(4 BV)洗脱，收集乙醇体积浓度为40％的酸性乙醇洗脱液，40-50℃减压蒸发除去乙醇，得到花色苷洗脱液；
所述大孔树脂选自AB-8，D101，XAD-7，HPD-100或DM-130，其比表面积为450-550m 2/g，平均孔径为10-50nm，粒径范围在0.3-1.25mm；
步骤(2)中，所述酸性乙醇溶液选自酸的体积百分浓度为0.1％～1.5％的乙醇溶液，其中酸选自盐酸、甲酸、乙酸中的至少一种。
步骤(3)中，所述有机溶剂为乙酸乙酯。
步骤(3)中，优选以所述有机溶剂与花色苷洗脱液体积比1:1的比例萃取，并萃取2次以上。
步骤(4)中，所述花色苷冻干粉可以用B相或水溶解。
步骤(4)中，制备型液相色谱系统使用的液相色谱柱为C18柱，单次进样量以花色苷冻干粉计为10-40mg，所述减压浓缩后的体积为浓缩前体积的40％-70％。
步骤(5)中，高速逆流色谱仪器温度稳定在20-30℃，正接正转，泵入固定相，之后调节转速至800-950r/min，以2mL/min的流速通入流动相并使之平衡，每次进样量以花色苷单体粗品计为30-200mg。
本发明与现有技术相比，主要优点包括：
1、首次建立了从葡萄皮中分离飞燕草素-3-O-(6-O-对香豆酰)葡萄糖苷(分子结构如图1所示)的方法，产率不低于8mg/kg葡萄皮，纯度不低于98％。
2、通过将制备液相色谱和高速逆流色谱联用，能够在多酚组分复杂的葡萄原料中，大批量地制备得到飞燕草素-3-O-(6-O-对香豆酰)葡萄糖苷，有处理量大、重复性好等优点，便于实现工业化生产。
附图说明
图1为飞燕草素-3-O-(6-O-对香豆酰)葡萄糖苷的分子结构图；
图2为实施例1中，葡萄皮花色苷粗提液的高效液相色谱图；
图3为实施例1中，经大孔树脂分离纯化后，含有飞燕草素-3-O-(6-O-对香豆酰)葡萄糖苷部分的高效液相色谱图；
图4为实施例1中的高速逆流色谱图；
图5为实施例1中，最终产物飞燕草素-3-O-(6-O-对香豆酰)葡萄糖苷的高效液相色谱图；
图6为实施例1中，飞燕草素-3-O-(6-O-对香豆酰)葡萄糖苷的一级质谱图和二级质谱图；
图7为对比例1中最终产物的高效液相色谱图；
图8为对比例2中最终产物的高效液相色谱图。
具体实施方式
下面结合附图及具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的操作方法，通常按照常规条件，或按照制造厂商所建议的条件。
实施例1
将葡萄洗净剥皮，得到8kg葡萄皮，按照料液比1g：4mL的比例加入含0.5％(v/v)盐酸的70％的乙醇溶液充分混合，超声提取120min，(控制温度在50℃以下，避光)，纱布过滤，滤液在4000rpm下离心10min，取上清液。滤渣按相同的方法再提取1次。将滤液合并，使用布氏漏斗再过滤一次。滤液于45℃下减压蒸发除去乙醇并浓缩，得到葡萄皮花色苷粗提液。葡萄皮花色苷粗提液的高效液相色谱图如图2所示。
将AB-8大孔树脂装入层析柱，并依次使用乙醇，0.5mol/L的盐酸溶液，0.5mol/L的氢氧化钠溶液，水清洗后，将花色苷粗提液以0.2BV/h的流速注入层析柱中。上样后，依次使用酸水(含0.5％盐酸)，5％、20％、40％的酸性乙醇(含0.5％盐酸)各4倍柱体积洗脱，收集40％酸性乙醇洗脱液，减压蒸发除去乙醇。然后以1：1的比例，使用乙酸乙酯萃取2遍，取水相，适当减压浓缩，冻干后，得到花色苷冻干粉。经大孔树脂分离纯化后，含有飞燕草素-3-O-(6-O-对香豆酰)葡萄糖苷部分的高效液相色谱图如图3所示。
液相分离使用Ultimate XB-C18(7μm,21.2×250mm)制备色谱柱。流动相用纯乙腈(A相)和1.5％的甲酸水溶液(B相)组成。梯度洗脱方法如下：0-4min，5％-20％A相；4-18min，20％-25％A相；18-21min，25％-35％ A相；21-24min，35％-60％A相；24-27min，60％-5％A相；27-30min，5％A相，流速为10mL/min，柱温为30℃，检测波长为520nm。将花色苷冻干粉溶解后进样，进样量为4mL，收集16.5-18.0min组分，减压浓缩，然后冷冻干燥，得到飞燕草素-3-O-(6-O-对香豆酰)葡萄糖苷单体粗品。
甲基叔丁基醚：甲醇：水：三氟乙酸按2：2：3：0.001的体积比置于分液漏斗中，充分摇匀，静置30min后，将上下相分离，分别超声脱气30min。使高速逆流色谱系统的仪器温度稳定在20℃，泵入固定相，之后调节转速至850r/min，正接正转，以2mL/min的流速通入流动相直到平衡，以每5mg冻干粉溶于1mL流动相中的比例溶解粗品冻干粉，使用微孔滤膜过滤后进样，单次进样10mL，并在紫外检测器下检测，检测波长280nm。收集105-115min的目标峰组分(如图4)并减压浓缩，冻干即得到70mg飞燕草素-3-O-(6-O-对香豆酰)葡萄糖苷，高效液相色谱图如图5所示，HPLC纯度为98.7％。
将制得花色苷样品在质谱仪中进样，根据质谱图对样品进行分析(图6)，确认分离得到的花色苷质量数正常。
实施例2
将葡萄洗净剥皮，得到2kg葡萄皮，按照料液比1g：6mL的比例加入含0.5％(v/v)盐酸的80％的乙醇溶液充分混合，超声提取60min，(控制温度在50℃以下，避光)，纱布过滤，滤液在4000rpm下离心10min，取上清液。滤渣按相同的方法再提取1次。将滤液合并，使用布氏漏斗再过滤一次。滤液于45℃下减压蒸发除去乙醇并浓缩，得到葡萄皮花色苷粗提液。
将AB-8大孔树脂装入层析柱，并依次使用乙醇，0.5mol/L的盐酸溶液，0.5mol/L的氢氧化钠溶液，水清洗后，将花色苷粗提液以0.2BV/h的流速注入层析柱中。上样后，依次使用酸水(含0.5％盐酸)，5％、20％、40％的酸性乙醇(含0.5％盐酸)各4倍柱体积洗脱，收集40％酸性乙醇洗脱液，减压蒸发除去乙醇。然后以1：1的比例，使用乙酸乙酯萃取2遍，取水相，经适当减压浓缩，冻干后，得到花色苷冻干粉。
液相分离使用Ultimate XB-C18(7μm,21.2×250mm)制备色谱柱。流动相用纯乙腈(A相)和1.5％的甲酸水溶液(B相)组成。梯度洗脱方法 如下：0-4min，5％-20％A相；4-18min，20％-25％A相；18-21min，25％-35％A相；21-24min，35％-60％A相；24-27min，60％-5％A相；27-30min，5％A相，流速为10mL/min，柱温为30℃，检测波长为520nm。将花色苷冻干粉溶解后进样，进样量为4mL，收集16.5-18.0min组分，减压浓缩，然后冷冻干燥，得到飞燕草素-3-O-(6-O-对香豆酰)葡萄糖苷单体粗品。
甲基叔丁基醚：甲醇：水：三氟乙酸按2：2：3：0.001的体积比置于分液漏斗中，充分摇匀，静置30min后，将上下相分离，分别超声脱气30min。使高速逆流色谱系统的仪器温度稳定在25℃，泵入固定相，之后调节转速至850r/min，正接正转，以2mL/min的流速通入流动相直到平衡，以每5mg冻干粉溶于1mL流动相中的比例溶解单体粗品冻干粉，使用微孔滤膜过滤后进样，单次进样10mL，并在紫外检测器下检测，检测波长280nm。收集105-115min的目标峰组分并减压浓缩，冻干即得到19mg飞燕草素-3-O-(6-O-对香豆酰)葡萄糖苷，HPLC纯度为98.2％。
实施例3
将葡萄洗净剥皮，得到20kg葡萄皮，按照料液比1g：4mL的比例加入含0.5％(v/v)盐酸的70％的乙醇溶液充分混合，超声提取90min，(控制温度在50℃以下，避光)，纱布过滤，滤液在4000rpm下离心10min，取上清液。滤渣按相同的方法再提取1次。将滤液合并，使用布氏漏斗再过滤一次。滤液于45℃下减压蒸发除去乙醇并浓缩，得到葡萄皮花色苷粗提液。
将AB-8大孔树脂装入层析柱，并依次使用乙醇，0.5mol/L的盐酸溶液，0.5mol/L的氢氧化钠溶液，水清洗后，将花色苷粗提液以0.2BV/h的流速注入层析柱中。上样后，依次使用酸水(含0.5％盐酸)，5％、20％、40％的酸性乙醇(含0.5％盐酸)各2倍柱体积洗脱，收集40％酸性乙醇洗脱液，减压蒸发除去乙醇。然后以1：1的比例，使用乙酸乙酯萃取2遍，取水相，适当减压浓缩冻干后，得到花色苷冻干粉。
液相分离使用Ultimate XB-C18(7μm,21.2×250mm)制备色谱柱。流动相用纯乙腈(A相)和1.5％的甲酸水溶液(B相)组成。梯度洗脱方法如下：0-4min，5％-20％A；4-18min，20％-25％A；18-21min，25％-35％A；21-24min，35％-60％A；24-27min，60％-5％A；27-30min，5％A， 流速为10mL/min，柱温为30℃，检测波长为520nm。将花色苷冻干粉溶解后进样，进样量为3mL，收集16.5-18.0min组分，减压浓缩，然后冷冻干燥，得到飞燕草素-3-O-(6-O-对香豆酰)葡萄糖苷单体粗品。
甲基叔丁基醚：甲醇：水：三氟乙酸按2：2：3：0.001的体积比置于分液漏斗中，充分摇匀，静置30min后，将上下相分离，分别超声脱气30min。使高速逆流色谱系统的仪器温度稳定在25℃，泵入固定相，之后调节转速至850r/min，正接正转，以2mL/min的流速通入流动相直到平衡，以每10mg冻干粉溶于1mL流动相中的比例溶解单体粗品冻干粉，使用微孔滤膜过滤后进样，单次进样15mL，并在紫外检测器下检测，检测波长280nm。收集105-115min的目标峰组分并减压浓缩，冻干即得到163mg飞燕草素-3-O-(6-O-对香豆酰)葡萄糖苷，HPLC纯度为98.4％。
对比例1
制备工艺与实施例1的相同，区别仅在于去掉高速逆流色谱纯化的步骤，其他步骤不变，所得最终产物的高效液相色谱图如图7所示，可知，该对比例只能得到含飞燕草素-3-O-(6-O-对香豆酰)葡萄糖苷的混合物，无法得到飞燕草素-3-O-(6-O-对香豆酰)葡萄糖苷单体。
对比例2
制备工艺与实施例1的相同，区别仅在于将高速逆流色谱纯化中的流速设置为5mL/min。经测试，虽然能收集到目标组分飞燕草素-3-O-(6-O-对香豆酰)葡萄糖苷，但是由于流速太快，目标组分没有完全与其他花色苷与其他杂质分离，得到的目标组分纯度仅为90％(如图8)。
对比例3
制备工艺与实施例1的相同，区别仅在于将高速逆流色谱分离的溶剂体系替换为正丁醇：甲基叔丁基醚：甲醇：水：三氟乙酸按2：2：1：5：0.001的体系。经测试，目标化合物飞燕草素-3-O-(6-O-对香豆酰)葡萄糖苷主要保留在上相中，无法收集得到目标物。
对比例4
制备工艺与实施例1的相同，区别仅在于将高速逆流色谱分离的溶剂体系替换为甲基叔丁基醚：甲醇：水：三氟乙酸按2：1：3：0.001的体系。经测试，无法得到目标物飞燕草素-3-O-(6-O-对香豆酰)葡萄糖苷。
对比例5
制备工艺与实施例1的相同，区别仅在于改变制备液相色谱纯化过程中组分收集时间，若组分收集时间不在16.5-18.0min的范围，则无法得到含飞燕草素-3-O-(6-O-对香豆酰)葡萄糖苷的组分。若组分收集时间包含且宽于16.5-18.0min的范围，则会影响最后分离得到的飞燕草素-3-O-(6-O-对香豆酰)葡萄糖苷单体的纯度。
对比例6
制备工艺与实施例1的相同，区别仅在于改变提取过程不使用酸性乙醇溶液提取，改用不含酸的乙醇溶液提取。其他步骤不变，则最后目标产物飞燕草素-3-O-(6-O-对香豆酰)葡萄糖苷单体的产率为2mg/kg葡萄皮，远低于8mg/kg葡萄皮。
此外应理解，在阅读了本发明的上述描述内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (7)

  1. 一种分离制备飞燕草素-3-O-(6-O-对香豆酰)葡萄糖苷的方法，其特征在于，包括步骤：
    (1)醇提浓缩：以葡萄为原料，经酸性醇溶液提取，浓缩得到葡萄皮花色苷粗提液；
    (2)大孔树脂纯化：将所述葡萄皮花色苷粗提液注入大孔树脂，经洗脱，浓缩得到花色苷洗脱液；
    (3)萃取：先使用有机溶剂萃取所述花色苷洗脱液，再经减压浓缩，冻干后得到花色苷冻干粉；
    (4)制备液相色谱纯化：将花色苷冻干粉溶解，注入制备型液相色谱系统中进行纯化，并用紫外检测器下检测，具体参数条件如下：
    流动相：A相为纯乙腈，B相为甲酸体积百分浓度为1％～2％的甲酸水溶液；
    梯度洗脱程序为：0-4min，5％-20％A相；4-18min，20％-25％A相；18-21min，25％-35％A相；21-24min，35％-60％A相；24-27min，60％-5％A相；27-30min，5％A相；
    流速为8-10mL/min，柱温为30℃，检测波长为520nm；
    根据液相色谱图收集保留时间为16.5-18.0min的组分，之后减压浓缩，冻干，得到花色苷单体粗品；
    (5)高速逆流色谱分离：将甲基叔丁基醚、甲醇、水和三氟乙酸以2:2:3:0.001的体积比混合作为两相溶剂体系，且以上相为固定相，下相为流动相，依次将固定相和流动相泵入高速逆流色谱仪器中，两相在管路中达到平衡后，将花色苷单体粗品用流动相溶解，进样并在紫外检测器下检测，检测波长为280nm，收集保留时间为105-115min的组分并减压浓缩、冻干，得到目标化合物飞燕草素-3-O-(6-O-对香豆酰)葡萄糖苷。
  2. 根据权利要求1所述的方法，其特征在于，步骤(1)中，所述酸性醇溶液提取，浓缩得到葡萄皮花色苷粗提液，具体为：将葡萄洗净取皮，与酸性乙醇溶液混合，混匀后打浆，50℃以下超声提取，过滤，滤液在40-50℃减压浓缩除去乙醇，得到葡萄皮花色苷粗提液；
    葡萄皮与酸性乙醇溶液的料液比为1g:4-8mL；
    所述酸性乙醇溶液中，乙醇体积浓度为50％-80％，酸体积浓度为0.1％-1％；
    所述超声提取时间为40-120min。
  3. 根据权利要求2所述的方法，其特征在于，所述酸性乙醇溶液中，酸选自盐酸、甲酸、乙酸中的至少一种。
  4. 根据权利要求1所述的方法，其特征在于，步骤(2)中，所述大孔树脂纯化方法具体为：
    将葡萄皮花色苷粗提液注入大孔树脂中，之后依次用乙醇体积浓度为0，5％，20％，40％的酸性乙醇溶液各4倍柱体积洗脱，收集乙醇体积浓度为40％的酸性乙醇洗脱液，40-50℃减压蒸发除去乙醇，得到花色苷洗脱液；
    所述大孔树脂选自AB-8，D101，XAD-7，HPD-100或DM-130，其比表面积为450-550m 2/g，平均孔径为10-50nm，粒径范围在0.3-1.25mm；
    所述酸性乙醇溶液选自酸的体积百分浓度为0.1％～1.5％的乙醇溶液，其中酸选自盐酸、甲酸、乙酸中的至少一种。
  5. 根据权利要求1所述的方法，其特征在于，步骤(3)中，所述有机溶剂为乙酸乙酯。
  6. 根据权利要求1所述的方法，其特征在于，步骤(4)中，制备型液相色谱系统使用的液相色谱柱为C18柱，单次进样量以花色苷冻干粉计为10-40mg，所述减压浓缩后的体积为浓缩前体积的40％-70％。
  7. 根据权利要求1所述的方法，其特征在于，步骤(5)中，高速逆流色谱仪器温度稳定在20-30℃，正接正转，泵入固定相，之后调节转速至800-950r/min，以2mL/min的流速通入流动相并使之平衡，每次进样量以花色苷单体粗品计为30-200mg。
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