CN111551656B - A kind of preparation method of high-purity corilagin - Google Patents
A kind of preparation method of high-purity corilagin Download PDFInfo
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- TUSDEZXZIZRFGC-UHFFFAOYSA-N 1-O-galloyl-3,6-(R)-HHDP-beta-D-glucose Natural products OC1C(O2)COC(=O)C3=CC(O)=C(O)C(O)=C3C3=C(O)C(O)=C(O)C=C3C(=O)OC1C(O)C2OC(=O)C1=CC(O)=C(O)C(O)=C1 TUSDEZXZIZRFGC-UHFFFAOYSA-N 0.000 title claims abstract description 112
- 229920002786 Corilagin Polymers 0.000 title claims abstract description 112
- TUSDEZXZIZRFGC-XIGLUPEJSA-N corilagin Chemical compound O([C@H]1[C@H](O)[C@H]2OC(=O)C3=CC(O)=C(O)C(O)=C3C3=C(O)C(O)=C(O)C=C3C(=O)OC[C@@H](O1)[C@H]2O)C(=O)C1=CC(O)=C(O)C(O)=C1 TUSDEZXZIZRFGC-XIGLUPEJSA-N 0.000 title claims abstract description 112
- CPWYQGWOJMNXGJ-UHFFFAOYSA-N corilagin Natural products OC1C2COC(=O)c3c(O)c(O)c(O)c(O)c3c4c(O)c(O)c(O)c(O)c4C(=O)OC1C(O)C(OC(=O)c5cc(O)c(O)c(O)c5)O2 CPWYQGWOJMNXGJ-UHFFFAOYSA-N 0.000 title claims abstract description 112
- 238000002360 preparation method Methods 0.000 title claims abstract description 11
- 241000219991 Lythraceae Species 0.000 claims abstract description 38
- 235000014360 Punica granatum Nutrition 0.000 claims abstract description 38
- 238000004587 chromatography analysis Methods 0.000 claims abstract description 10
- 238000000034 method Methods 0.000 claims abstract description 9
- 238000000746 purification Methods 0.000 claims abstract description 5
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 156
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 150
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 104
- 239000012141 concentrate Substances 0.000 claims description 53
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 52
- 235000019253 formic acid Nutrition 0.000 claims description 52
- 239000007864 aqueous solution Substances 0.000 claims description 48
- 239000007788 liquid Substances 0.000 claims description 48
- 239000000843 powder Substances 0.000 claims description 46
- 238000000926 separation method Methods 0.000 claims description 33
- 239000000203 mixture Substances 0.000 claims description 26
- 238000010828 elution Methods 0.000 claims description 25
- 239000003153 chemical reaction reagent Substances 0.000 claims description 24
- XELZGAJCZANUQH-UHFFFAOYSA-N methyl 1-acetylthieno[3,2-c]pyrazole-5-carboxylate Chemical compound CC(=O)N1N=CC2=C1C=C(C(=O)OC)S2 XELZGAJCZANUQH-UHFFFAOYSA-N 0.000 claims description 24
- 239000007787 solid Substances 0.000 claims description 24
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 24
- HQVFCQRVQFYGRJ-UHFFFAOYSA-N formic acid;hydrate Chemical compound O.OC=O HQVFCQRVQFYGRJ-UHFFFAOYSA-N 0.000 claims description 2
- 238000011084 recovery Methods 0.000 abstract description 23
- 239000000463 material Substances 0.000 abstract description 7
- 239000004480 active ingredient Substances 0.000 abstract description 5
- 238000003908 quality control method Methods 0.000 abstract description 4
- 239000013558 reference substance Substances 0.000 abstract description 4
- 238000005227 gel permeation chromatography Methods 0.000 abstract description 3
- 239000002994 raw material Substances 0.000 abstract description 3
- 238000004366 reverse phase liquid chromatography Methods 0.000 abstract description 3
- 239000012071 phase Substances 0.000 description 69
- 238000001514 detection method Methods 0.000 description 43
- 239000007791 liquid phase Substances 0.000 description 23
- 239000000945 filler Substances 0.000 description 22
- 239000012074 organic phase Substances 0.000 description 22
- 239000002245 particle Substances 0.000 description 22
- 238000011067 equilibration Methods 0.000 description 10
- 238000010586 diagram Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 1
- 208000034507 Haematemesis Diseases 0.000 description 1
- 206010033078 Otitis media Diseases 0.000 description 1
- 206010053476 Traumatic haemorrhage Diseases 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 208000001780 epistaxis Diseases 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- -1 polyphenol compounds Chemical class 0.000 description 1
- 235000013824 polyphenols Nutrition 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
Description
技术领域technical field
本发明涉及一种高纯度柯里拉京的制备方法。The invention relates to a preparation method of high-purity corilagin.
背景技术Background technique
色谱作为一种分离手段在现代社会被广泛用于制药、化工等领域。利用吸附力、分配系数、溶解度等原理上的差异,目标化合物在色谱柱上可取得连续的分布,形成可分开的“色带”(无颜色的物质在特定检测器下会显示不同的吸收峰)。色谱技术的应用领域很广,不同需求下对色谱技术的要求大不相同。As a means of separation, chromatography is widely used in pharmaceutical, chemical and other fields in modern society. Utilizing the differences in principles such as adsorption force, distribution coefficient, solubility, etc., the target compound can obtain continuous distribution on the chromatographic column, forming separable "color bands" (colorless substances will show different absorption peaks under specific detectors) ). Chromatographic technology has a wide range of applications, and the requirements for chromatographic technology are quite different under different needs.
石榴,又称安石榴,是一种在我国尤其是新疆地区(古称“西域”)有着广泛种植的水果。石榴花作为药用,有止鼻衄、吐血及外伤出血的作用,亦可治白带过多。外用则可治中耳炎。石榴花中含有丰富的植物多酚类化合物,具有多种多样的抗菌抗病毒活性。其中,柯里拉京是石榴花中的一种主要活性成分。纯化获得高纯度的柯里拉京对照品,可用于国家标准品证书的申报,为石榴花或其他以柯里拉京为有效成分的药材质量控制做出贡献。Pomegranate, also known as pomegranate, is a fruit widely grown in my country, especially in Xinjiang (called "Western Regions" in ancient times). As a medicine, pomegranate flower can stop epistaxis, hematemesis and traumatic bleeding, and can also treat excessive leucorrhea. External use can cure otitis media. Pomegranate flowers are rich in plant polyphenol compounds, which have various antibacterial and antiviral activities. Among them, Corilagin is a main active ingredient in pomegranate flower. The high-purity corilagin reference substance obtained by purification can be used for the declaration of the national standard product certificate, and contributes to the quality control of pomegranate flower or other medicinal materials with corilagin as the active ingredient.
发明内容Contents of the invention
本发明的目的在于,提供一种高纯度柯里拉京的制备方法,该方法通过正交色谱及非线性色谱原理相结合,先后用反相色谱法和凝胶色谱法,以石榴花提取物为原料,从中获得了高纯度的柯里拉京对照品。该方法获得的柯里拉京纯度高,产率大,回收率高,满足柯里拉京国家标准品的申报要求,可为石榴花或其他以柯里拉京为有效成分的药材质量控制做出贡献。这对含有柯里拉京的药材的质量标准研究具有十分重要的意义。与传统的柯里拉京纯化工艺相比,该方法简单易行,量大且纯度高,因而颇具产业价值。The object of the present invention is to provide a kind of preparation method of high-purity corilagin, which combines the principles of orthogonal chromatography and nonlinear chromatography, successively uses reverse phase chromatography and gel chromatography, and uses pomegranate flower extract as Raw material, from which a high-purity Corylatin reference substance was obtained. The corilagin obtained by the method has high purity, high yield and high recovery rate, meets the declaration requirements of the national standard product of corilagin, and can contribute to the quality control of pomegranate flower or other medicinal materials containing corilagin as active ingredients. This is of great significance to the research on the quality standard of medicinal materials containing corilagin. Compared with the traditional Corilagin purification process, the method is simple and easy to implement, with large quantity and high purity, and thus has considerable industrial value.
本发明所述的一种高纯度柯里拉京的制备方法,按下列步骤进行:A kind of preparation method of high-purity corilagin of the present invention, carry out according to the following steps:
a、通过配以二极管阵列检测器的高效液相色谱仪进行分离纯化,调整高效液相色谱仪波长为254-280nm和320-380nm,将甲醇和甲酸含量为0.05-2.0%甲酸水混合,其中甲醇含量控制在20%-40%,作为流动相,平衡色谱柱时间5-20min,称取石榴花提取物0.2-20g,溶于1-20mL水中,进样至色谱柱分离,接取包含有柯里拉京的组分,用旋转蒸发仪在35-55℃下浓缩至10mL,待用;a. Carry out separation and purification by a high-performance liquid chromatograph equipped with a diode array detector, adjust the wavelength of the high-performance liquid chromatograph to 254-280nm and 320-380nm, and mix methanol and formic acid with 0.05-2.0% formic acid water, wherein The methanol content is controlled at 20%-40%. As the mobile phase, equilibrate the chromatographic column for 5-20min, weigh 0.2-20g of pomegranate flower extract, dissolve it in 1-20mL water, inject the sample into the chromatographic column for separation, and take the Concentrate the components of Corilagin at 35-55°C with a rotary evaporator to 10 mL, and set aside;
b、保持步骤a中的色谱条件,将乙腈和甲酸含量为0.05%-2.0%水溶液混合,其中乙腈含量控制在7%-25%,作为流动相,平衡色谱柱时间5-20min,将步骤a中所得组分进样至色谱柱分离,接取包含柯里拉京的组分,用旋转蒸发仪在35-55℃下浓缩至0.2-2mL,待用;b. Keep the chromatographic conditions in step a, mix acetonitrile and formic acid with 0.05%-2.0% aqueous solution, wherein the acetonitrile content is controlled at 7%-25%, as mobile phase, equilibrate the chromatographic column for 5-20min, and step a Inject the components obtained in the sample into the chromatographic column for separation, take the components containing Corylatin, and use a rotary evaporator to concentrate to 0.2-2mL at 35-55°C for use;
c、将步骤b中浓缩获取组分样品上至葡聚糖凝胶LH-20层析柱上,长度控制在5-200cm,柱内径在0.5-3.0cm,以甲醇为洗脱试剂进行层析柱洗脱,接取洗脱液,合并包含柯里拉京的组分,用旋转蒸发仪在35-55℃下浓缩至2mL,真空冷冻干燥,得到高纯度柯里拉京白色固体粉末。c. Put the concentrated and obtained component samples in step b onto the Sephadex LH-20 chromatography column, the length is controlled at 5-200cm, the inner diameter of the column is 0.5-3.0cm, and methanol is used as the eluting reagent for chromatography Column elution, take the eluate, combine the fractions containing corilagin, concentrate to 2 mL at 35-55°C with a rotary evaporator, and freeze-dry in vacuum to obtain a white solid powder of high-purity corilagin.
步骤a、步骤b、步骤c中浓缩温度范围35-40℃。The concentration temperature range in step a, step b and step c is 35-40°C.
步骤a中甲醇含量控制在25-30%。Methanol content is controlled at 25-30% in the step a.
步骤b中乙腈含量控制在7-15%。Acetonitrile content is controlled at 7-15% in step b.
步骤b中包含柯里拉京组分浓缩至0.2mL-0.5mL。The fraction containing corilagin in step b was concentrated to 0.2mL-0.5mL.
步骤a和步骤b中甲酸水溶液中甲酸含量范围在0.1-0.5%。The formic acid content in the formic acid aqueous solution in step a and step b is in the range of 0.1-0.5%.
步骤a和步骤b中作为流动相,平衡色谱柱时间15-20min。In step a and step b, it is used as the mobile phase, and the time for equilibrating the chromatographic column is 15-20min.
本发明所述的一种高纯度柯里拉京的制备方法,主要是基于正交色谱原理及非线性色谱容量相结合,先后用反相色谱法和凝胶色谱法,以石榴花提取物为原料,从石榴花提取物中获得了高纯度的柯里拉京对照品。该方法获得的柯里拉京纯度高,产率大,回收率高,满足柯里拉京国家标准品的申报要求,可为石榴花或其他以柯里拉京为有效成分的药材质量控制做出贡献。这对含有柯里拉京的药材的质量标准研究具有十分重要的意义The preparation method of a kind of high-purity corilagin of the present invention is mainly based on the combination of orthogonal chromatographic principle and nonlinear chromatographic capacity, using reverse phase chromatography and gel chromatography successively, using pomegranate flower extract as raw material , from the pomegranate flower extract obtained a high-purity corilagin reference substance. The corilagin obtained by the method has high purity, high yield and high recovery rate, meets the declaration requirements of the national standard product of corilagin, and can contribute to the quality control of pomegranate flower or other medicinal materials containing corilagin as active ingredients. This is of great significance to the quality standard research of medicinal materials containing corilagin
附图说明Description of drawings
图1为本发明中石榴花提取物在步骤a中的分离色谱示意图。Figure 1 is a schematic diagram of the separation chromatogram of the pomegranate flower extract in step a of the present invention.
图2为本发明中步骤a中所得组分在步骤b的色谱条件下分离示意图。Fig. 2 is a schematic diagram of separation of components obtained in step a under the chromatographic conditions of step b in the present invention.
图3为本发明中过完凝胶柱后所得柯里拉京的纯度测定图。Fig. 3 is the purity determination figure of the obtained corilagin after passing through the gel column in the present invention.
具体实施方式Detailed ways
下面结合具体实施例,进一步详细阐述本发明。Below in conjunction with specific embodiment, further elaborate the present invention in detail.
实施例1Example 1
a、将一根反相C18色谱柱(规格为长250毫米,直径80毫米,填料粒径10微米)连接在一台配有双通道紫外检测器的高效液相色谱仪(又称DAD检测器),高效液相色谱仪的由双泵组成,泵A连有甲醇为有机相,泵B连有甲酸含量为0.1%的水溶液,通过色谱工作站控制泵A和泵B的流速(总流速为180mL/min),其中A相占20%,平衡色谱柱时间15分钟,柱温控制在25℃,调整DAD检测器波长分别为254nm和320nm,称取0.2g石榴花提取物粉末,溶于1mL水中,用0.45μm的有机滤头过滤,至于液相小瓶中待测,色谱柱平衡以后通过进样器进样,接取包含有柯里拉京的组分,用旋转蒸发仪在温度35℃下浓缩至10mL,待用;a, a reversed-phase C18 chromatographic column (the specification is 250 millimeters in length, 80 millimeters in diameter, 10 microns of filler particle diameter) is connected to a high-performance liquid chromatograph (also known as DAD detector) equipped with a dual-channel ultraviolet detector ), the high-performance liquid chromatograph is made up of double pumps, and the pump A is connected with methanol as the organic phase, and the pump B is connected with an aqueous solution with a formic acid content of 0.1%. /min), wherein phase A accounts for 20%, the equilibration time of the chromatographic column is 15 minutes, the column temperature is controlled at 25°C, the wavelength of the DAD detector is adjusted to 254nm and 320nm respectively, and 0.2g of pomegranate flower extract powder is weighed and dissolved in 1mL of water , filter with a 0.45μm organic filter head, and put it in the liquid phase vial to be tested. After the chromatographic column is equilibrated, the sample is injected through the injector, and the component containing Corylatin is collected and concentrated at a temperature of 35°C with a rotary evaporator. to 10mL, set aside;
b、保持步骤a中的色谱条件,将乙腈和甲酸含量为0.1%的水溶液混合,其中乙腈含量控制在7%,作为流动相,平衡色谱柱时间15min,柱温控制在25℃,DAD检测器检测波长分别为254nm和320nm,将步骤a中所得组分进样至色谱柱分离,接取包含柯里拉京的组分,用旋转蒸发仪在温度35℃下浓缩至0.2mL,待用;b. Maintain the chromatographic conditions in step a, mix acetonitrile and an aqueous solution with a formic acid content of 0.1%, wherein the acetonitrile content is controlled at 7%, as the mobile phase, the time for equilibrating the chromatographic column is 15min, the column temperature is controlled at 25°C, and the DAD detector The detection wavelengths are 254nm and 320nm, respectively. Inject the components obtained in step a into the chromatographic column for separation, take the components containing Corylatin, and concentrate them to 0.2mL with a rotary evaporator at a temperature of 35°C for use;
c、将步骤b中浓缩获取组分样品上至葡聚糖凝胶LH-20层析柱上,长度控制在15cm,柱内径在0.5cm,以甲醇为洗脱试剂进行层析柱洗脱,接取洗脱液,合并包含柯里拉京的组分,用旋转蒸发仪在35℃下浓缩至2mL,真空冷冻干燥,得到高纯度柯里拉京白色固体粉末2.0mg,纯度98.5%,柯里拉京回收率58.8%。c. Put the concentrated and obtained component samples in step b onto the Sephadex LH-20 chromatographic column, the length is controlled at 15 cm, the inner diameter of the column is 0.5 cm, and the chromatographic column is eluted with methanol as the elution reagent. Take the eluate, combine the components containing Corilagin, concentrate to 2 mL at 35°C with a rotary evaporator, and freeze-dry in vacuum to obtain 2.0 mg of high-purity Corilagin white solid powder with a purity of 98.5%. Corilagin The recovery rate was 58.8%.
实施例2Example 2
a、将一根反相C18色谱柱(规格为长250毫米,直径80毫米,填料粒径10微米)连接在一台配有双通道紫外检测器的高效液相色谱仪(又称DAD检测器),高效液相色谱仪的由双泵组成,泵A连有甲醇为有机相,泵B连有甲酸含量为0.1%的水溶液,通过色谱工作站控制泵A和泵B的流速(总流速为180mL/min),其中A相占20%,平衡色谱柱时间15分钟,柱温控制在25℃,DAD检测器检测波长分别为254nm和320nm,称取20g石榴花提取物粉末,溶于20mL水中,用0.45μm的有机滤头过滤,至于液相小瓶中待测,色谱柱平衡以后通过进样器进样,接取包含有柯里拉京的组分,用旋转蒸发仪在温度35℃下浓缩至10mL,待用;a, a reversed-phase C18 chromatographic column (the specification is 250 millimeters in length, 80 millimeters in diameter, 10 microns of filler particle diameter) is connected to a high-performance liquid chromatograph (also known as DAD detector) equipped with a dual-channel ultraviolet detector ), the high-performance liquid chromatograph is made up of double pumps, and the pump A is connected with methanol as the organic phase, and the pump B is connected with an aqueous solution with a formic acid content of 0.1%. /min), wherein phase A accounts for 20%, the time for equilibrating the chromatographic column is 15 minutes, the column temperature is controlled at 25°C, the detection wavelength of the DAD detector is 254nm and 320nm respectively, and 20g of pomegranate flower extract powder is weighed, dissolved in 20mL of water, Filter with a 0.45 μm organic filter head, and put it in a liquid phase vial to be tested. After the chromatographic column is equilibrated, the sample is injected through the injector, and the components containing corilagin are collected and concentrated at a temperature of 35°C with a rotary evaporator. 10mL, ready for use;
b、保持步骤a中的色谱条件,将乙腈和甲酸含量为0.1%的水溶液混合,其中乙腈含量控制在7%,作为流动相,平衡色谱柱时间15min,柱温控制在25℃,DAD检测器检测波长分别为254nm和320nm,将步骤a中所得组分进样至色谱柱分离,接取包含柯里拉京的组分,用旋转蒸发仪在35℃下浓缩至0.2mL,待用;b. Maintain the chromatographic conditions in step a, mix acetonitrile and an aqueous solution with a formic acid content of 0.1%, wherein the acetonitrile content is controlled at 7%, as the mobile phase, the time for equilibrating the chromatographic column is 15min, the column temperature is controlled at 25°C, and the DAD detector The detection wavelengths are 254nm and 320nm, respectively. Inject the components obtained in step a into the chromatographic column for separation, take the components containing Corylatin, concentrate them to 0.2mL at 35°C with a rotary evaporator, and set aside;
c、将步骤b中浓缩获取组分样品上至葡聚糖凝胶LH-20层析柱上,长度控制在15cm,柱内径在0.5cm,以甲醇为洗脱试剂进行层析柱洗脱,接取洗脱液,合并包含柯里拉京的组分,用旋转蒸发仪在温度35℃下浓缩至2mL,真空冷冻干燥,得到高纯度柯里拉京白色固体粉末235mg,纯度97.8%,柯里拉京回收率69.1%。c. Put the concentrated and obtained component samples in step b onto the Sephadex LH-20 chromatographic column, the length is controlled at 15 cm, the inner diameter of the column is 0.5 cm, and the chromatographic column is eluted with methanol as the elution reagent. Take the eluate, combine the components containing Corilagin, concentrate to 2mL with a rotary evaporator at a temperature of 35°C, and freeze-dry in vacuum to obtain 235mg of high-purity Corilagin white solid powder with a purity of 97.8%. Corilagin The recovery rate was 69.1%.
实施例3Example 3
a、将一根反相C18色谱柱(规格为长250毫米,直径80毫米,填料粒径10微米)连接在一台配有双通道紫外检测器的高效液相色谱仪(又称DAD检测器),高效液相色谱仪的由双泵组成,泵A连有甲醇为有机相,泵B连有甲酸含量为0.1%的水溶液,通过色谱工作站控制泵A和泵B的流速(总流速为180mL/min),其中A相占40%,平衡色谱柱时间15分钟,柱温控制在25℃,DAD检测器检测波长分别为254nm和320nm,称取20g石榴花提取物粉末,溶于20mL水中,用0.45μm的有机滤头过滤,至于液相小瓶中待测,色谱柱平衡以后通过进样器进样,接取包含有柯里拉京的组分,用旋转蒸发仪在35℃下浓缩至10mL,待用;a, a reversed-phase C18 chromatographic column (the specification is 250 millimeters in length, 80 millimeters in diameter, 10 microns of filler particle diameter) is connected to a high-performance liquid chromatograph (also known as DAD detector) equipped with a dual-channel ultraviolet detector ), the high-performance liquid chromatograph is made up of double pumps, and the pump A is connected with methanol as the organic phase, and the pump B is connected with an aqueous solution with a formic acid content of 0.1%. /min), wherein phase A accounts for 40%, the time for equilibrating the chromatographic column is 15 minutes, the column temperature is controlled at 25°C, the detection wavelength of the DAD detector is 254nm and 320nm respectively, and 20g of pomegranate flower extract powder is weighed, dissolved in 20mL of water, Filter with a 0.45 μm organic filter head, and put it in a liquid phase vial to be tested. After the chromatographic column is equilibrated, inject the sample through the injector, take the component containing corilagin, and concentrate it to 10 mL at 35 °C with a rotary evaporator ,stand-by;
b、保持步骤a中的色谱条件,将乙腈和甲酸含量为0.1%的水溶液混合,其中乙腈含量控制在7%,作为流动相,平衡色谱柱时间10min,柱温控制在25℃,DAD检测器检测波长分别为254nm和320nm,将步骤a中所得组分进样至色谱柱分离,接取包含柯里拉京的组分,用旋转蒸发仪在温度35℃下浓缩至0.2mL,待用;b. Maintain the chromatographic conditions in step a, mix acetonitrile and an aqueous solution with a formic acid content of 0.1%, wherein the acetonitrile content is controlled at 7%, as the mobile phase, equilibrate the chromatographic column for 10 minutes, and the column temperature is controlled at 25 ° C, DAD detector The detection wavelengths are 254nm and 320nm, respectively. Inject the components obtained in step a into the chromatographic column for separation, take the components containing Corylatin, and concentrate them to 0.2mL with a rotary evaporator at a temperature of 35°C for use;
c、将步骤b中浓缩获取组分样品上至葡聚糖凝胶LH-20层析柱上,长度控制在15cm,柱内径在0.5cm,以甲醇为洗脱试剂进行层析柱洗脱,接取洗脱液,合并包含柯里拉京的组分,用旋转蒸发仪在35℃下浓缩至2mL,真空冷冻干燥,得到高纯度柯里拉京白色固体粉末302mg,纯度95.4%,柯里拉京回收率88.8%。c. Put the concentrated and obtained component samples in step b onto the Sephadex LH-20 chromatographic column, the length is controlled at 15 cm, the inner diameter of the column is 0.5 cm, and the chromatographic column is eluted with methanol as the elution reagent. Take the eluate, combine the components containing Corilagin, concentrate to 2mL with a rotary evaporator at 35°C, and freeze-dry in vacuo to obtain 302 mg of high-purity Corilagin white solid powder with a purity of 95.4%, which was recovered by Corilagin The rate is 88.8%.
实施例4Example 4
a、将一根反相C18色谱柱(规格为长250毫米,直径80毫米,填料粒径10微米)连接在一台配有双通道紫外检测器的高效液相色谱仪(又称DAD检测器),高效液相色谱仪的由双泵组成,泵A连有甲醇为有机相,泵B连有甲酸含量为0.1%的水溶液,通过色谱工作站控制泵A和泵B的流速(总流速为180mL/min),其中A相占25%,平衡色谱柱时间15分钟,柱温控制在25℃,DAD检测器检测波长分别为260nm和360nm,称取20g石榴花提取物粉末,溶于20mL水中,用0.45μm的有机滤头过滤,至于液相小瓶中待测,色谱柱平衡以后通过进样器进样,接取包含有柯里拉京的组分,用旋转蒸发仪在35℃下浓缩至10mL,待用;a, a reversed-phase C18 chromatographic column (the specification is 250 millimeters in length, 80 millimeters in diameter, 10 microns of filler particle diameter) is connected to a high-performance liquid chromatograph (also known as DAD detector) equipped with a dual-channel ultraviolet detector ), the high-performance liquid chromatograph is made up of double pumps, and the pump A is connected with methanol as the organic phase, and the pump B is connected with an aqueous solution with a formic acid content of 0.1%. /min), wherein phase A accounts for 25%, the equilibration time of the chromatographic column is 15 minutes, the column temperature is controlled at 25°C, and the detection wavelength of the DAD detector is 260nm and 360nm respectively, and 20g pomegranate flower extract powder is weighed, dissolved in 20mL water, Filter with a 0.45 μm organic filter head, and put it in a liquid phase vial to be tested. After the chromatographic column is equilibrated, inject the sample through the injector, take the component containing corilagin, and concentrate it to 10 mL at 35 °C with a rotary evaporator ,stand-by;
b、保持步骤a中的色谱条件,将乙腈和甲酸含量为0.1%的水溶液混合,其中乙腈含量控制在10%,作为流动相,平衡色谱柱时间15min,柱温控制在25℃,DAD检测器检测波长分别为260nm和360nm,将步骤a中所得组分进样至色谱柱分离,接取包含柯里拉京的组分,用旋转蒸发仪在温度35℃下浓缩至0.2mL,待用;b. Maintain the chromatographic conditions in step a, mix acetonitrile and an aqueous solution with a formic acid content of 0.1%, wherein the acetonitrile content is controlled at 10%, as the mobile phase, equilibrate the chromatographic column for 15 minutes, and the column temperature is controlled at 25 ° C. DAD detector The detection wavelengths are 260nm and 360nm respectively. Inject the components obtained in step a into the chromatographic column for separation, take the components containing Corylatin, concentrate them to 0.2mL with a rotary evaporator at a temperature of 35°C, and set aside;
c、将步骤b中浓缩获取组分样品上至葡聚糖凝胶LH-20层析柱上,长度控制在15cm,柱内径在0.5cm,以甲醇为洗脱试剂进行层析柱洗脱,接取洗脱液,合并包含柯里拉京的组分,用旋转蒸发仪在温度35℃下浓缩至2mL,真空冷冻干燥,得到高纯度柯里拉京白色固体粉末230mg,纯度98.9%,柯里拉京回收率67.6%。c. Put the concentrated and obtained component samples in step b onto the Sephadex LH-20 chromatographic column, the length is controlled at 15 cm, the inner diameter of the column is 0.5 cm, and the chromatographic column is eluted with methanol as the elution reagent. Take the eluate, combine the components containing Corilagin, concentrate to 2mL with a rotary evaporator at a temperature of 35°C, and freeze-dry in vacuum to obtain 230mg of high-purity Corilagin white solid powder with a purity of 98.9%. Corilagin The recovery rate was 67.6%.
实施例5Example 5
a、将一根反相C18色谱柱(规格为长250毫米,直径80毫米,填料粒径10微米)连接在一台配有双通道紫外检测器的高效液相色谱仪(又称DAD检测器),高效液相色谱仪的由双泵组成,泵A连有甲醇为有机相,泵B连有甲酸含量为0.1%的水溶液,通过色谱工作站控制泵A和泵B的流速(总流速为180mL/min),其中A相占30%,平衡色谱柱时间15分钟,柱温控制在25℃,DAD检测器检测波长分别为254nm和320nm,称取20g石榴花提取物粉末,溶于20mL水中,用0.45μm的有机滤头过滤,至于液相小瓶中待测,色谱柱平衡以后通过进样器进样,接取包含有柯里拉京的组分,用旋转蒸发仪在35℃下浓缩至10mL,待用;a, a reversed-phase C18 chromatographic column (the specification is 250 millimeters in length, 80 millimeters in diameter, 10 microns of filler particle diameter) is connected to a high-performance liquid chromatograph (also known as DAD detector) equipped with a dual-channel ultraviolet detector ), the high-performance liquid chromatograph is made up of double pumps, and the pump A is connected with methanol as the organic phase, and the pump B is connected with an aqueous solution with a formic acid content of 0.1%. /min), wherein phase A accounts for 30%, the time for equilibrating the chromatographic column is 15 minutes, the column temperature is controlled at 25°C, the detection wavelength of the DAD detector is 254nm and 320nm respectively, and 20g of pomegranate flower extract powder is weighed, dissolved in 20mL of water, Filter with a 0.45 μm organic filter head, and put it in a liquid phase vial to be tested. After the chromatographic column is equilibrated, inject the sample through the injector, take the component containing corilagin, and concentrate it to 10 mL at 35 °C with a rotary evaporator ,stand-by;
b、保持步骤a中的色谱条件,将乙腈和甲酸含量为0.1%的水溶液混合,其中乙腈含量控制在7%,作为流动相,平衡色谱柱时间20min,柱温控制在25℃,DAD检测器检测波长分别为254nm和320nm,将步骤a中所得组分进样至色谱柱分离,接取包含柯里拉京的组分,用旋转蒸发仪在温度35℃下浓缩至0.2mL,待用;b. Maintain the chromatographic conditions in step a, mix acetonitrile and an aqueous solution with a formic acid content of 0.1%, wherein the acetonitrile content is controlled at 7%, as the mobile phase, equilibrate the chromatographic column for 20 minutes, and the column temperature is controlled at 25 ° C, DAD detector The detection wavelengths are 254nm and 320nm, respectively. Inject the components obtained in step a into the chromatographic column for separation, take the components containing Corylatin, and concentrate them to 0.2mL with a rotary evaporator at a temperature of 35°C for use;
c、将步骤b中浓缩获取组分样品上至葡聚糖凝胶LH-20层析柱上,长度控制在15cm,柱内径在1.0cm,以甲醇为洗脱试剂进行层析柱洗脱,接取洗脱液,合并包含柯里拉京的组分,用旋转蒸发仪在温度38℃下浓缩至2mL,真空冷冻干燥,得到高纯度柯里拉京白色固体粉末234mg,纯度98.8%,柯里拉京回收率68.8%。c. Put the concentrated and obtained component sample in step b onto the Sephadex LH-20 chromatographic column, the length is controlled at 15 cm, the inner diameter of the column is 1.0 cm, and the chromatographic column is eluted with methanol as the elution reagent. Take the eluate, combine the components containing Corilagin, concentrate to 2mL with a rotary evaporator at a temperature of 38°C, and freeze-dry in vacuum to obtain 234mg of high-purity Corilagin white solid powder with a purity of 98.8%. Corilagin The recovery rate was 68.8%.
实施例6Example 6
a、将一根反相C18色谱柱(规格为长250毫米,直径80毫米,填料粒径10微米)连接在一台配有双通道紫外检测器的高效液相色谱仪(又称DAD检测器),高效液相色谱仪的由双泵组成,泵A连有甲醇为有机相,泵B连有甲酸含量为0.1%的水溶液,通过色谱工作站控制泵A和泵B的流速(总流速为180mL/min),其中A相占30%,平衡色谱柱时间15分钟,柱温控制在25℃,DAD检测器检测波长分别为254nm和320nm,称取20g石榴花提取物粉末,溶于20mL水中,用0.45μm的有机滤头过滤,至于液相小瓶中待测,色谱柱平衡以后通过进样器进样,接取包含有柯里拉京的组分,用旋转蒸发仪在温度35℃下浓缩至10mL,待用;a, a reversed-phase C18 chromatographic column (the specification is 250 millimeters in length, 80 millimeters in diameter, 10 microns of filler particle diameter) is connected to a high-performance liquid chromatograph (also known as DAD detector) equipped with a dual-channel ultraviolet detector ), the high-performance liquid chromatograph is made up of double pumps, and the pump A is connected with methanol as the organic phase, and the pump B is connected with an aqueous solution with a formic acid content of 0.1%. /min), wherein phase A accounts for 30%, the time for equilibrating the chromatographic column is 15 minutes, the column temperature is controlled at 25°C, the detection wavelength of the DAD detector is 254nm and 320nm respectively, and 20g of pomegranate flower extract powder is weighed, dissolved in 20mL of water, Filter with a 0.45 μm organic filter head, and put it in a liquid phase vial to be tested. After the chromatographic column is equilibrated, the sample is injected through the injector, and the components containing corilagin are collected and concentrated at a temperature of 35°C with a rotary evaporator. 10mL, ready for use;
b、保持步骤a中的色谱条件,将乙腈和甲酸含量为0.1%的水溶液混合,其中乙腈含量控制在25%,作为流动相,平衡色谱柱时间15min,柱温控制在25℃,DAD检测器检测波长分别为254nm和320nm,将步骤a中所得组分进样至色谱柱分离,接取包含柯里拉京的组分,用旋转蒸发仪在温度35℃下浓缩至0.2mL,待用;b. Maintain the chromatographic conditions in step a, mix acetonitrile and an aqueous solution with a formic acid content of 0.1%, wherein the acetonitrile content is controlled at 25%, as the mobile phase, equilibrate the chromatographic column for 15 minutes, the column temperature is controlled at 25 ° C, and the DAD detector The detection wavelengths are 254nm and 320nm, respectively. Inject the components obtained in step a into the chromatographic column for separation, take the components containing Corylatin, and concentrate them to 0.2mL with a rotary evaporator at a temperature of 35°C for use;
c、将步骤b中浓缩获取组分样品上至葡聚糖凝胶LH-20层析柱上,长度控制在15cm,柱内径在1.5cm,以甲醇为洗脱试剂进行层析柱洗脱,接取洗脱液,合并包含柯里拉京的组分,用旋转蒸发仪在温度40℃下浓缩至2mL,真空冷冻干燥,得到高纯度柯里拉京白色固体粉末312mg,纯度93.2%,柯里拉京回收率91.8%。c. Put the concentrated and obtained component samples in step b onto the Sephadex LH-20 chromatographic column, the length is controlled at 15 cm, the inner diameter of the column is 1.5 cm, and the chromatographic column is eluted with methanol as the elution reagent. Take the eluate, combine the components containing Corilagin, concentrate to 2mL with a rotary evaporator at a temperature of 40°C, and freeze-dry in vacuum to obtain 312 mg of high-purity Corilagin white solid powder with a purity of 93.2%. Corilagin The recovery rate is 91.8%.
实施例7Example 7
a、将一根反相C18色谱柱(规格为长250毫米,直径80毫米,填料粒径10微米)连接在一台配有双通道紫外检测器的高效液相色谱仪(又称DAD检测器),高效液相色谱仪的由双泵组成,泵A连有甲醇为有机相,泵B连有甲酸含量为0.1%的水溶液,通过色谱工作站控制泵A和泵B的流速(总流速为180mL/min),其中A相占30%,平衡色谱柱时间15分钟,柱温控制在25℃,DAD检测器检测波长分别为254nm和320nm,称取20g石榴花提取物粉末,溶于20mL水中,用0.45μm的有机滤头过滤,至于液相小瓶中待测,色谱柱平衡以后通过进样器进样,接取包含有柯里拉京的组分,用旋转蒸发仪在温度35℃下浓缩至10mL,待用;a, a reversed-phase C18 chromatographic column (the specification is 250 millimeters in length, 80 millimeters in diameter, 10 microns of filler particle diameter) is connected to a high-performance liquid chromatograph (also known as DAD detector) equipped with a dual-channel ultraviolet detector ), the high-performance liquid chromatograph is made up of double pumps, and the pump A is connected with methanol as the organic phase, and the pump B is connected with an aqueous solution with a formic acid content of 0.1%. /min), wherein phase A accounts for 30%, the time for equilibrating the chromatographic column is 15 minutes, the column temperature is controlled at 25°C, the detection wavelength of the DAD detector is 254nm and 320nm respectively, and 20g of pomegranate flower extract powder is weighed, dissolved in 20mL of water, Filter with a 0.45 μm organic filter head, and put it in a liquid phase vial to be tested. After the chromatographic column is equilibrated, the sample is injected through the injector, and the components containing corilagin are collected and concentrated at a temperature of 35°C with a rotary evaporator. 10mL, ready for use;
b、保持步骤a中的色谱条件,将乙腈和甲酸含量为0.1%的水溶液混合,其中乙腈含量控制在15%,作为流动相,平衡色谱柱时间20min,柱温控制在25℃,DAD检测器检测波长分别为254nm和320nm,将步骤a中所得组分进样至色谱柱分离,接取包含柯里拉京的组分,用旋转蒸发仪在温度40℃下浓缩至0.5mL,待用;b. Maintain the chromatographic conditions in step a, mix acetonitrile and an aqueous solution with a formic acid content of 0.1%, wherein the acetonitrile content is controlled at 15%, as the mobile phase, equilibrate the chromatographic column for 20 minutes, and the column temperature is controlled at 25 ° C. DAD detector The detection wavelengths are 254nm and 320nm, respectively. Inject the components obtained in step a into the chromatographic column for separation, take the components containing Corylatin, concentrate them to 0.5mL at a temperature of 40°C with a rotary evaporator, and set aside;
c、将步骤b中浓缩获取组分样品上至葡聚糖凝胶LH-20层析柱上,长度控制在15cm,柱内径在2.5cm,以甲醇为洗脱试剂进行层析柱洗脱,接取洗脱液,合并包含柯里拉京的组分,用旋转蒸发仪在温度45℃下浓缩至2mL,真空冷冻干燥,得到高纯度柯里拉京白色固体粉末288mg,纯度98.4%,柯里拉京回收率84.7%。c. Put the concentrated and obtained component samples in step b on the Sephadex LH-20 chromatographic column, the length is controlled at 15 cm, the inner diameter of the column is 2.5 cm, and the chromatographic column is eluted with methanol as the elution reagent. Take the eluate, combine the components containing Corilagin, concentrate to 2mL with a rotary evaporator at a temperature of 45°C, and freeze-dry in vacuum to obtain 288 mg of high-purity Corilagin white solid powder with a purity of 98.4%. Corilagin The recovery rate was 84.7%.
实施例8Example 8
a、将一根反相C18色谱柱(规格为长250毫米,直径80毫米,填料粒径10微米)连接在一台配有双通道紫外检测器的高效液相色谱仪(又称DAD检测器),高效液相色谱仪的由双泵组成,泵A连有甲醇为有机相,泵B连有甲酸含量为0.05%的水溶液,通过色谱工作站控制泵A和泵B的流速(总流速为180mL/min),其中A相占30%,平衡色谱柱时间15分钟,柱温控制在25℃,高效液相色谱仪检测波长分别为254nm和320nm,称取20g石榴花提取物粉末,溶于20mL水中,用0.45μm的有机滤头过滤,至于液相小瓶中待测,色谱柱平衡以后通过进样器进样,接取包含有柯里拉京的组分,用旋转蒸发仪在温度35℃下浓缩至10mL,待用;a, a reversed-phase C18 chromatographic column (the specification is 250 millimeters in length, 80 millimeters in diameter, 10 microns of filler particle diameter) is connected to a high-performance liquid chromatograph (also known as DAD detector) equipped with a dual-channel ultraviolet detector ), the high performance liquid chromatograph is made up of double pumps, and the pump A is connected with methanol as an organic phase, and the pump B is connected with an aqueous solution with a formic acid content of 0.05%. /min), wherein phase A accounts for 30%, the equilibration time of the chromatographic column is 15 minutes, the column temperature is controlled at 25°C, the detection wavelength of the high performance liquid chromatography is 254nm and 320nm respectively, and 20g of pomegranate flower extract powder is weighed, dissolved in 20mL Filter the water with a 0.45μm organic filter head, and put it in the liquid phase vial to be tested. After the chromatographic column is equilibrated, the sample is injected through the injector, and the components containing Corylatin are collected, and the temperature is 35°C with a rotary evaporator. Concentrate to 10mL, set aside;
b、保持步骤a中的色谱条件,将乙腈和甲酸含量为0.05%的水溶液混合,其中乙腈含量控制在15%,作为流动相,平衡色谱柱时间10min,柱温控制在25℃,高效液相色谱仪检测波长分别为254nm和320nm,将步骤a中所得组分进样至色谱柱分离,接取包含柯里拉京的组分,用旋转蒸发仪在温度50℃下浓缩至1mL,待用;b. Maintain the chromatographic conditions in step a, mix acetonitrile and an aqueous solution with a formic acid content of 0.05%, wherein the acetonitrile content is controlled at 15%, as the mobile phase, the time for equilibrating the chromatographic column is 10min, the column temperature is controlled at 25°C, and the high performance liquid phase The detection wavelengths of the chromatograph are 254nm and 320nm, respectively. The components obtained in step a are injected into the chromatographic column for separation, and the components containing Corylatin are collected and concentrated to 1mL at a temperature of 50°C with a rotary evaporator for use;
c、将步骤b中浓缩获取组分样品上至葡聚糖凝胶LH-20层析柱上,长度控制在15cm,柱内径在2.5cm,以甲醇为洗脱试剂进行层析柱洗脱,接取洗脱液,合并包含柯里拉京的组分,用旋转蒸发仪在温度50℃下浓缩至2mL,真空冷冻干燥,得到高纯度柯里拉京白色固体粉末270mg,纯度96.4%,柯里拉京回收率79.4%。c. Put the concentrated and obtained component samples in step b on the Sephadex LH-20 chromatographic column, the length is controlled at 15 cm, the inner diameter of the column is 2.5 cm, and the chromatographic column is eluted with methanol as the elution reagent. Take the eluate, combine the components containing Corilagin, concentrate to 2mL with a rotary evaporator at a temperature of 50°C, and freeze-dry in vacuum to obtain 270 mg of high-purity Corilagin white solid powder with a purity of 96.4%. Corilagin The recovery rate was 79.4%.
实施例9Example 9
a、将一根反相C18色谱柱(规格为长250毫米,直径80毫米,填料粒径10微米)连接在一台配有双通道紫外检测器的高效液相色谱仪(又称DAD检测器),高效液相色谱仪的由双泵组成,泵A连有甲醇为有机相,泵B连有甲酸含量为0.5%的水溶液,通过色谱工作站控制泵A和泵B的流速(总流速为180mL/min),其中A相占30%,平衡色谱柱时间15分钟,柱温控制在25℃,DAD检测器检测波长分别为254nm和320nm,称取20g石榴花提取物粉末,溶于20mL水中,用0.45μm的有机滤头过滤,至于液相小瓶中待测,色谱柱平衡以后通过进样器进样,接取包含有柯里拉京的组分,用旋转蒸发仪在温度35℃下浓缩至10mL,待用;a, a reversed-phase C18 chromatographic column (the specification is 250 millimeters in length, 80 millimeters in diameter, 10 microns of filler particle diameter) is connected to a high-performance liquid chromatograph (also known as DAD detector) equipped with a dual-channel ultraviolet detector ), the high performance liquid chromatograph is made up of double pumps, and the pump A is connected with methanol as the organic phase, and the pump B is connected with the aqueous solution with a formic acid content of 0.5%, and the flow rate of the pump A and the pump B is controlled by the chromatography workstation (the total flow rate is 180mL /min), wherein phase A accounts for 30%, the time for equilibrating the chromatographic column is 15 minutes, the column temperature is controlled at 25°C, the detection wavelength of the DAD detector is 254nm and 320nm respectively, and 20g of pomegranate flower extract powder is weighed, dissolved in 20mL of water, Filter with a 0.45 μm organic filter head, and put it in a liquid phase vial to be tested. After the chromatographic column is equilibrated, the sample is injected through the injector, and the components containing corilagin are collected and concentrated at a temperature of 35°C with a rotary evaporator. 10mL, ready for use;
b、保持步骤a中的色谱条件,将乙腈和甲酸含量为0.5%的水溶液混合,其中乙腈含量控制在15%,作为流动相,平衡色谱柱时间20min,柱温控制在25℃,DAD检测器检测波长分别为254nm和320nm,将步骤a中所得组分进样至色谱柱分离,接取包含柯里拉京的组分,用旋转蒸发仪在温度45℃下浓缩至0.5mL,待用;b. Maintain the chromatographic conditions in step a, mix acetonitrile and an aqueous solution with a formic acid content of 0.5%, wherein the acetonitrile content is controlled at 15%, as the mobile phase, the time for equilibrating the chromatographic column is 20min, the column temperature is controlled at 25°C, and the DAD detector The detection wavelengths are 254nm and 320nm respectively. Inject the components obtained in step a into the chromatographic column for separation, take the components containing Corylatin, and concentrate them to 0.5mL with a rotary evaporator at a temperature of 45°C for use;
c、将步骤b中浓缩获取组分样品上至葡聚糖凝胶LH-20层析柱上,长度控制在15cm,柱内径在2.5cm,以甲醇为洗脱试剂进行层析柱洗脱,接取洗脱液,合并包含柯里拉京的组分,用旋转蒸发仪在温度55℃下浓缩至2mL,真空冷冻干燥,得到高纯度柯里拉京白色固体粉末201mg,纯度97.4%,柯里拉京回收率59.1%。c. Put the concentrated and obtained component samples in step b on the Sephadex LH-20 chromatographic column, the length is controlled at 15 cm, the inner diameter of the column is 2.5 cm, and the chromatographic column is eluted with methanol as the elution reagent. Take the eluate, combine the components containing Corilagin, concentrate to 2mL with a rotary evaporator at a temperature of 55°C, and freeze-dry in vacuum to obtain 201mg of high-purity Corilagin white solid powder with a purity of 97.4%. The recovery rate was 59.1%.
实施例10Example 10
a、将一根反相C18色谱柱(规格为长250毫米,直径80毫米,填料粒径10微米)连接在一台配有双通道紫外检测器的高效液相色谱仪(又称DAD检测器),高效液相色谱仪的由双泵组成,泵A连有甲醇为有机相,泵B连有甲酸含量为2.0%的水溶液,通过色谱工作站控制泵A和泵B的流速(总流速为180mL/min),其中A相占30%,平衡色谱柱时间15分钟,柱温控制在25℃,DAD检测器检测波长分别为254nm和320nm,称取20g石榴花提取物粉末,溶于20mL水中,用0.45μm的有机滤头过滤,至于液相小瓶中待测,色谱柱平衡以后通过进样器进样,接取包含有柯里拉京的组分,用旋转蒸发仪在温度35℃下浓缩至10mL,待用;a, a reversed-phase C18 chromatographic column (the specification is 250 millimeters in length, 80 millimeters in diameter, 10 microns of filler particle diameter) is connected to a high-performance liquid chromatograph (also known as DAD detector) equipped with a dual-channel ultraviolet detector ), the high performance liquid chromatograph is made up of double pumps, and the pump A is connected with methanol as the organic phase, and the pump B is connected with the aqueous solution with a formic acid content of 2.0%. /min), wherein phase A accounts for 30%, the time for equilibrating the chromatographic column is 15 minutes, the column temperature is controlled at 25°C, the detection wavelength of the DAD detector is 254nm and 320nm respectively, and 20g of pomegranate flower extract powder is weighed, dissolved in 20mL of water, Filter with a 0.45 μm organic filter head, and put it in a liquid phase vial to be tested. After the chromatographic column is equilibrated, the sample is injected through the injector, and the components containing corilagin are collected and concentrated at a temperature of 35°C with a rotary evaporator. 10mL, ready for use;
b、保持步骤a中的色谱条件,将乙腈和甲酸含量为2.0%的水溶液混合,其中乙腈含量控制在25%,作为流动相,平衡色谱柱时间20min,柱温控制在25℃,DAD检测器检测波长分别为254nm和320nm,将步骤a中所得组分进样至色谱柱分离,接取包含柯里拉京的组分,用旋转蒸发仪在温度35℃下浓缩至2mL,待用;b. Maintain the chromatographic conditions in step a, mix acetonitrile and an aqueous solution with a formic acid content of 2.0%, wherein the acetonitrile content is controlled at 25%, as the mobile phase, equilibrate the chromatographic column for 20 minutes, and the column temperature is controlled at 25 ° C. DAD detector The detection wavelengths are 254nm and 320nm, respectively. Inject the components obtained in step a into the chromatographic column for separation, take the component containing Corylatin, and concentrate it to 2mL at a temperature of 35°C with a rotary evaporator for use;
c、将步骤b中浓缩获取组分样品上至葡聚糖凝胶LH-20层析柱上,长度控制在15cm,柱内径在3.0cm,以甲醇为洗脱试剂进行层析柱洗脱,接取洗脱液,合并包含柯里拉京的组分,用旋转蒸发仪在温度55℃下浓缩至2mL,真空冷冻干燥,得到高纯度柯里拉京白色固体粉末223mg,纯度91.6%,柯里拉京回收率65.6%。c. Put the concentrated and obtained component samples in step b onto the Sephadex LH-20 chromatographic column, the length is controlled at 15 cm, the inner diameter of the column is 3.0 cm, and the chromatographic column is eluted with methanol as the elution reagent. Take the eluate, combine the components containing Corilagin, concentrate to 2mL with a rotary evaporator at a temperature of 55°C, and freeze-dry in vacuum to obtain 223mg of high-purity Corilagin white solid powder with a purity of 91.6%. Corilagin The recovery rate was 65.6%.
实施例11Example 11
a、将一根反相C18色谱柱(规格为长250毫米,直径80毫米,填料粒径10微米)连接在一台配有双通道紫外检测器的高效液相色谱仪(又称DAD检测器),高效液相色谱仪的由双泵组成,泵A连有甲醇为有机相,泵B连有甲酸含量为0.1%的水溶液,通过色谱工作站控制泵A和泵B的流速(总流速为180mL/min),其中A相占30%,平衡色谱柱时间15分钟,柱温控制在25℃,DAD检测器检测波长分别为254nm和320nm,称取20g石榴花提取物粉末,溶于20mL水中,用0.45μm的有机滤头过滤,至于液相小瓶中待测,色谱柱平衡以后通过进样器进样,接取包含有柯里拉京的组分,用旋转蒸发仪在温度55℃下浓缩至10mL,待用;a, a reversed-phase C18 chromatographic column (the specification is 250 millimeters in length, 80 millimeters in diameter, 10 microns of filler particle diameter) is connected to a high-performance liquid chromatograph (also known as DAD detector) equipped with a dual-channel ultraviolet detector ), the high-performance liquid chromatograph is made up of double pumps, and the pump A is connected with methanol as the organic phase, and the pump B is connected with an aqueous solution with a formic acid content of 0.1%. /min), wherein phase A accounts for 30%, the time for equilibrating the chromatographic column is 15 minutes, the column temperature is controlled at 25°C, the detection wavelength of the DAD detector is 254nm and 320nm respectively, and 20g of pomegranate flower extract powder is weighed, dissolved in 20mL of water, Filter with a 0.45 μm organic filter head, and put it in a liquid phase vial to be tested. After the chromatographic column is equilibrated, the sample is injected through the injector, and the components containing Corylatin are collected and concentrated at a temperature of 55 °C with a rotary evaporator to 10mL, ready for use;
b、保持步骤a中的色谱条件,将乙腈和甲酸含量为0.1%的水溶液混合,其中乙腈含量控制在15%,作为流动相,平衡色谱柱时间5min,柱温控制在25℃,DAD检测器检测波长分别为254nm和320nm,将步骤a中所得组分进样至色谱柱分离,接取包含柯里拉京的组分,用旋转蒸发仪在温度55℃下浓缩至0.2mL,待用;b. Keep the chromatographic conditions in step a, mix acetonitrile and an aqueous solution with a formic acid content of 0.1%, wherein the acetonitrile content is controlled at 15%, as the mobile phase, equilibrate the chromatographic column for 5 minutes, and the column temperature is controlled at 25 ° C. DAD detector The detection wavelengths are 254nm and 320nm respectively. Inject the components obtained in step a into the chromatographic column for separation, take the components containing Corylatin, concentrate them to 0.2mL with a rotary evaporator at a temperature of 55°C, and set aside;
c、将步骤b中浓缩获取组分样品上至葡聚糖凝胶LH-20层析柱上,长度控制在15cm,柱内径在0.5cm,以甲醇为洗脱试剂进行层析柱洗脱,接取洗脱液,合并包含柯里拉京的组分,用旋转蒸发仪在55℃下浓缩至2mL,真空冷冻干燥,得到高纯度柯里拉京白色固体粉末275mg,纯度92.4%,柯里拉京回收率80.9%。c. Put the concentrated and obtained component samples in step b onto the Sephadex LH-20 chromatographic column, the length is controlled at 15 cm, the inner diameter of the column is 0.5 cm, and the chromatographic column is eluted with methanol as the elution reagent. Take the eluate, combine the components containing Corilagin, concentrate to 2mL with a rotary evaporator at 55°C, and freeze-dry in vacuo to obtain 275mg of high-purity Corilagin white solid powder with a purity of 92.4%, which was recovered by Corilagin The rate is 80.9%.
实施例12Example 12
a、将一根反相C18色谱柱(规格为长250毫米,直径80毫米,填料粒径10微米)连接在一台配有双通道紫外检测器的高效液相色谱仪(又称DAD检测器),高效液相色谱仪的由双泵组成,泵A连有甲醇为有机相,泵B连有甲酸含量为0.1%的水溶液,通过色谱工作站控制泵A和泵B的流速(总流速为180mL/min),其中A相占30%,平衡色谱柱时间15分钟,柱温控制在25℃,DAD检测器检测波长分别为254nm和320nm,称取20g石榴花提取物粉末,溶于20mL水中,用0.45μm的有机滤头过滤,至于液相小瓶中待测,色谱柱平衡以后通过进样器进样,接取包含有柯里拉京的组分,用旋转蒸发仪在温度40℃下浓缩至10mL,待用;a, a reversed-phase C18 chromatographic column (the specification is 250 millimeters in length, 80 millimeters in diameter, 10 microns of filler particle diameter) is connected to a high-performance liquid chromatograph (also known as DAD detector) equipped with a dual-channel ultraviolet detector ), the high-performance liquid chromatograph is made up of double pumps, and the pump A is connected with methanol as the organic phase, and the pump B is connected with an aqueous solution with a formic acid content of 0.1%. /min), wherein phase A accounts for 30%, the equilibration column time is 15 minutes, the column temperature is controlled at 25°C, the detection wavelength of the DAD detector is 254nm and 320nm respectively, and 20g pomegranate flower extract powder is weighed, dissolved in 20mL water, Filter with a 0.45 μm organic filter head, and put it in a liquid phase vial to be tested. After the chromatographic column is equilibrated, the sample is injected through the injector, and the components containing Corylatin are collected and concentrated at a temperature of 40 °C with a rotary evaporator to 10mL, ready for use;
b、保持步骤a中的色谱条件,将乙腈和甲酸含量为0.1%的水溶液混合,其中乙腈含量控制在18%,作为流动相,平衡色谱柱时间8min,柱温控制在25℃,DAD检测器检测波长分别为254nm和320nm,将步骤a中所得组分进样至色谱柱分离,接取包含柯里拉京的组分,用旋转蒸发仪在温度40℃下浓缩至0.5mL,待用;b. Keep the chromatographic conditions in step a, mix acetonitrile and an aqueous solution with a formic acid content of 0.1%, wherein the acetonitrile content is controlled at 18%, as the mobile phase, the equilibration time of the chromatographic column is 8min, the column temperature is controlled at 25°C, and the DAD detector The detection wavelengths are 254nm and 320nm, respectively. Inject the components obtained in step a into the chromatographic column for separation, take the components containing Corylatin, concentrate them to 0.5mL at a temperature of 40°C with a rotary evaporator, and set aside;
c、将步骤b中浓缩获取组分样品上至葡聚糖凝胶LH-20层析柱上,长度控制在15cm,柱内径在2.5cm,以甲醇为洗脱试剂进行层析柱洗脱,接取洗脱液,合并包含柯里拉京的组分,用旋转蒸发仪在温度50℃下浓缩至2mL,真空冷冻干燥,得到高纯度柯里拉京白色固体粉末280mg,纯度98.6%,柯里拉京回收率82.4%。c. Put the concentrated and obtained component samples in step b on the Sephadex LH-20 chromatographic column, the length is controlled at 15 cm, the inner diameter of the column is 2.5 cm, and the chromatographic column is eluted with methanol as the elution reagent. Take the eluate, combine the components containing Corilagin, concentrate to 2mL with a rotary evaporator at a temperature of 50°C, and freeze-dry in vacuum to obtain 280mg of high-purity Corilagin white solid powder with a purity of 98.6%. The recovery rate was 82.4%.
实施例13Example 13
a、将一根反相C18色谱柱(规格为长250毫米,直径80毫米,填料粒径10微米)连接在一台配有双通道紫外检测器的高效液相色谱仪(又称DAD检测器),高效液相色谱仪的由双泵组成,泵A连有甲醇为有机相,泵B连有甲酸含量为0.1%的水溶液,通过色谱工作站控制泵A和泵B的流速(总流速为180mL/min),其中A相占30%,平衡色谱柱时间15分钟,柱温控制在25℃,DAD检测器检测波长分别为254nm和320nm,称取20g石榴花提取物粉末,溶于20mL水中,用0.45μm的有机滤头过滤,至于液相小瓶中待测,色谱柱平衡以后通过进样器进样,接取包含有柯里拉京的组分,用旋转蒸发仪在温度40℃下浓缩至10mL,待用;a, a reversed-phase C18 chromatographic column (the specification is 250 millimeters in length, 80 millimeters in diameter, 10 microns of filler particle diameter) is connected to a high-performance liquid chromatograph (also known as DAD detector) equipped with a dual-channel ultraviolet detector ), the high-performance liquid chromatograph is made up of double pumps, and the pump A is connected with methanol as the organic phase, and the pump B is connected with an aqueous solution with a formic acid content of 0.1%. /min), wherein phase A accounts for 30%, the time for equilibrating the chromatographic column is 15 minutes, the column temperature is controlled at 25°C, the detection wavelength of the DAD detector is 254nm and 320nm respectively, and 20g of pomegranate flower extract powder is weighed, dissolved in 20mL of water, Filter with a 0.45 μm organic filter head, and put it in a liquid phase vial to be tested. After the chromatographic column is equilibrated, the sample is injected through the injector, and the components containing Corylatin are collected and concentrated at a temperature of 40 °C with a rotary evaporator to 10mL, ready for use;
b、保持步骤a中的色谱条件,将乙腈和甲酸含量为0.1%的水溶液混合,其中乙腈含量控制在20%,作为流动相,平衡色谱柱时间18min,柱温控制在25℃,DAD检测器检测波长分别为254nm和320nm,将步骤a中所得组分进样至色谱柱分离,接取包含柯里拉京的组分,用旋转蒸发仪在温度40℃下浓缩至0.2mL,待用;b. Maintain the chromatographic conditions in step a, mix acetonitrile and an aqueous solution with a formic acid content of 0.1%, wherein the acetonitrile content is controlled at 20%, as the mobile phase, the equilibration time of the chromatographic column is 18min, the column temperature is controlled at 25 ° C, and the DAD detector The detection wavelengths are 254nm and 320nm respectively. Inject the components obtained in step a into the chromatographic column for separation, take the components containing Corylatin, concentrate them to 0.2mL with a rotary evaporator at a temperature of 40°C, and set aside;
c、将步骤b中浓缩获取组分样品上至葡聚糖凝胶LH-20层析柱上,长度控制在5cm,柱内径在1.5cm,以甲醇为洗脱试剂进行层析柱洗脱,接取洗脱液,合并包含柯里拉京的组分,用旋转蒸发仪在温度53℃下浓缩至2mL,真空冷冻干燥,得到高纯度柯里拉京白色固体粉末296mg,纯度89.8%,柯里拉京回收率87.0%。c. Put the concentrated and obtained component samples in step b on the Sephadex LH-20 chromatographic column, the length is controlled at 5 cm, the inner diameter of the column is 1.5 cm, and the chromatographic column is eluted with methanol as the elution reagent. Take the eluate, combine the components containing Corilagin, concentrate to 2mL with a rotary evaporator at a temperature of 53°C, and freeze-dry in vacuum to obtain 296mg of high-purity Corilagin white solid powder with a purity of 89.8%. Corilagin The recovery rate was 87.0%.
实施例14Example 14
a、将一根反相C18色谱柱(规格为长250毫米,直径80毫米,填料粒径10微米)连接在一台配有双通道紫外检测器的高效液相色谱仪(又称DAD检测器),高效液相色谱仪的由双泵组成,泵A连有甲醇为有机相,泵B连有甲酸含量为0.1%的水溶液,通过色谱工作站控制泵A和泵B的流速(总流速为180mL/min),其中A相占30%,平衡色谱柱时间15分钟,柱温控制在25℃,DAD检测器检测波长分别为254nm和320nm,称取20g石榴花提取物粉末,溶于20mL水中,用0.45μm的有机滤头过滤,至于液相小瓶中待测,色谱柱平衡以后通过进样器进样,接取包含有柯里拉京的组分,用旋转蒸发仪在温度40℃下浓缩至10mL,待用;a, a reversed-phase C18 chromatographic column (the specification is 250 millimeters in length, 80 millimeters in diameter, 10 microns of filler particle diameter) is connected to a high-performance liquid chromatograph (also known as DAD detector) equipped with a dual-channel ultraviolet detector ), the high-performance liquid chromatograph is made up of double pumps, and the pump A is connected with methanol as the organic phase, and the pump B is connected with an aqueous solution with a formic acid content of 0.1%. /min), wherein phase A accounts for 30%, the time for equilibrating the chromatographic column is 15 minutes, the column temperature is controlled at 25°C, the detection wavelength of the DAD detector is 254nm and 320nm respectively, and 20g of pomegranate flower extract powder is weighed, dissolved in 20mL of water, Filter with a 0.45 μm organic filter head, and put it in a liquid phase vial to be tested. After the chromatographic column is equilibrated, the sample is injected through the injector, and the components containing Corylatin are collected and concentrated at a temperature of 40 °C with a rotary evaporator to 10mL, ready for use;
b、保持步骤a中的色谱条件,将乙腈和甲酸含量为0.1%的水溶液混合,其中乙腈含量控制在15%,作为流动相,平衡色谱柱时间5min,柱温控制在25℃,DAD检测器检测波长分别为254nm和320nm,将步骤a中所得组分进样至色谱柱分离,接取包含柯里拉京的组分,用旋转蒸发仪在温度40℃下浓缩至2mL,待用;b. Keep the chromatographic conditions in step a, mix acetonitrile and an aqueous solution with a formic acid content of 0.1%, wherein the acetonitrile content is controlled at 15%, as the mobile phase, equilibrate the chromatographic column for 5 minutes, and the column temperature is controlled at 25 ° C. DAD detector The detection wavelengths are 254nm and 320nm respectively. Inject the components obtained in step a into the chromatographic column for separation, take the components containing Corylatin, concentrate them to 2mL at a temperature of 40°C with a rotary evaporator, and set aside;
c、将步骤b中浓缩获取组分样品上至葡聚糖凝胶LH-20层析柱上,长度控制在200cm,柱内径在3.0cm,以甲醇为洗脱试剂进行层析柱洗脱,接取洗脱液,合并包含柯里拉京的组分,用旋转蒸发仪在温度55℃下浓缩至2mL,真空冷冻干燥,得到高纯度柯里拉京白色固体粉末262mg,纯度98.1%,柯里拉京回收率77.0%。c. Put the concentrated and obtained component samples in step b on the Sephadex LH-20 chromatographic column, the length is controlled at 200 cm, the inner diameter of the column is 3.0 cm, and the chromatographic column is eluted with methanol as the elution reagent. Take the eluate, combine the components containing Corilagin, concentrate to 2mL with a rotary evaporator at a temperature of 55°C, and freeze-dry in vacuum to obtain 262mg of high-purity Corilagin white solid powder with a purity of 98.1%. Corilagin The recovery rate was 77.0%.
实施例15Example 15
a、将一根反相C18色谱柱(规格为长250毫米,直径80毫米,填料粒径10微米)连接在一台配有双通道紫外检测器的高效液相色谱仪(又称DAD检测器),高效液相色谱仪的由双泵组成,泵A连有甲醇为有机相,泵B连有甲酸含量为0.1%的水溶液,通过色谱工作站控制泵A和泵B的流速(总流速为180mL/min),其中A相占30%,平衡色谱柱时间15分钟,柱温控制在25℃,DAD检测器检测波长分别为254nm和320nm,称取20g石榴花提取物粉末,溶于20mL水中,用0.45μm的有机滤头过滤,至于液相小瓶中待测,色谱柱平衡以后通过进样器进样,接取包含有柯里拉京的组分,用旋转蒸发仪在温度40℃下浓缩至10mL,待用;a, a reversed-phase C18 chromatographic column (the specification is 250 millimeters in length, 80 millimeters in diameter, 10 microns of filler particle diameter) is connected to a high-performance liquid chromatograph (also known as DAD detector) equipped with a dual-channel ultraviolet detector ), the high-performance liquid chromatograph is made up of double pumps, and the pump A is connected with methanol as the organic phase, and the pump B is connected with an aqueous solution with a formic acid content of 0.1%. /min), wherein phase A accounts for 30%, the time for equilibrating the chromatographic column is 15 minutes, the column temperature is controlled at 25°C, the detection wavelength of the DAD detector is 254nm and 320nm respectively, and 20g of pomegranate flower extract powder is weighed, dissolved in 20mL of water, Filter with a 0.45 μm organic filter head, and put it in a liquid phase vial to be tested. After the chromatographic column is equilibrated, the sample is injected through the injector, and the components containing Corylatin are collected and concentrated at a temperature of 40 °C with a rotary evaporator to 10mL, ready for use;
b、保持步骤a中的色谱条件,将乙腈和甲酸含量为0.1%的水溶液混合,其中乙腈含量控制在10%,作为流动相,平衡色谱柱时间15min,柱温控制在25℃,DAD检测器检测波长分别为254nm和320nm,将步骤a中所得组分进样至色谱柱分离,接取包含柯里拉京的组分,用旋转蒸发仪在温度40℃下浓缩至0.2mL,待用;b. Maintain the chromatographic conditions in step a, mix acetonitrile and an aqueous solution with a formic acid content of 0.1%, wherein the acetonitrile content is controlled at 10%, as the mobile phase, equilibrate the chromatographic column for 15 minutes, and the column temperature is controlled at 25 ° C. DAD detector The detection wavelengths are 254nm and 320nm respectively. Inject the components obtained in step a into the chromatographic column for separation, take the components containing Corylatin, concentrate them to 0.2mL with a rotary evaporator at a temperature of 40°C, and set aside;
c、将步骤b中浓缩获取组分样品上至葡聚糖凝胶LH-20层析柱上,长度控制在15cm,柱内径在3.0cm,以甲醇为洗脱试剂进行层析柱洗脱,接取洗脱液,合并包含柯里拉京的组分,用旋转蒸发仪在温度55℃下浓缩至2mL,真空冷冻干燥,得到高纯度柯里拉京白色固体粉末267mg,纯度98.69%,柯里拉京回收率78.8%。c. Put the concentrated and obtained component samples in step b onto the Sephadex LH-20 chromatographic column, the length is controlled at 15 cm, the inner diameter of the column is 3.0 cm, and the chromatographic column is eluted with methanol as the elution reagent. Take the eluate, combine the components containing Corilagin, concentrate to 2mL with a rotary evaporator at a temperature of 55°C, and freeze-dry in vacuum to obtain 267mg of high-purity Corilagin white solid powder with a purity of 98.69%. Corilagin The recovery rate was 78.8%.
实施例16Example 16
a、将一根反相C18色谱柱(规格为长250毫米,直径80毫米,填料粒径10微米)连接在一台配有双通道紫外检测器的高效液相色谱仪(又称DAD检测器),高效液相色谱仪的由双泵组成,泵A连有甲醇为有机相,泵B连有甲酸含量为0.1%的水溶液,通过色谱工作站控制泵A和泵B的流速(总流速为180mL/min),其中A相占30%,平衡色谱柱时间15分钟,柱温控制在25℃,DAD检测器检测波长分别为254nm和320nm,称取20g石榴花提取物粉末,溶于20mL水中,用0.45μm的有机滤头过滤,至于液相小瓶中待测,色谱柱平衡以后通过进样器进样,接取包含有柯里拉京的组分,用旋转蒸发仪在温度40℃下浓缩至10mL,待用;a, a reversed-phase C18 chromatographic column (the specification is 250 millimeters in length, 80 millimeters in diameter, 10 microns of filler particle diameter) is connected to a high-performance liquid chromatograph (also known as DAD detector) equipped with a dual-channel ultraviolet detector ), the high-performance liquid chromatograph is made up of double pumps, and the pump A is connected with methanol as the organic phase, and the pump B is connected with an aqueous solution with a formic acid content of 0.1%. /min), wherein phase A accounts for 30%, the time for equilibrating the chromatographic column is 15 minutes, the column temperature is controlled at 25°C, the detection wavelength of the DAD detector is 254nm and 320nm respectively, and 20g of pomegranate flower extract powder is weighed, dissolved in 20mL of water, Filter with a 0.45 μm organic filter head, and put it in a liquid phase vial to be tested. After the chromatographic column is equilibrated, the sample is injected through the injector, and the components containing Corylatin are collected and concentrated at a temperature of 40 °C with a rotary evaporator to 10mL, ready for use;
b、保持步骤a中的色谱条件,将乙腈和甲酸含量为0.1%的水溶液混合,其中乙腈含量控制在7%,作为流动相,平衡色谱柱时间15min,柱温控制在25℃,DAD检测器检测波长分别为254nm和320nm,将步骤a中所得组分进样至色谱柱分离,接取包含柯里拉京的组分,用旋转蒸发仪在温度40℃下浓缩至2.0mL,待用;b. Maintain the chromatographic conditions in step a, mix acetonitrile and an aqueous solution with a formic acid content of 0.1%, wherein the acetonitrile content is controlled at 7%, as the mobile phase, the time for equilibrating the chromatographic column is 15min, the column temperature is controlled at 25°C, and the DAD detector The detection wavelengths are 254nm and 320nm respectively. Inject the components obtained in step a into the chromatographic column for separation, take the components containing Corylatin, concentrate them to 2.0mL with a rotary evaporator at a temperature of 40°C, and set aside;
c、将步骤b中浓缩获取组分样品上至葡聚糖凝胶LH-20层析柱上,长度控制在15cm,柱内径在3.0cm,以甲醇为洗脱试剂进行层析柱洗脱,接取洗脱液,合并包含柯里拉京的组分,用旋转蒸发仪在温度55℃下浓缩至2mL,真空冷冻干燥,得到高纯度柯里拉京白色固体粉末255mg,纯度98.2%,柯里拉京回收率75.0%。c. Put the concentrated and obtained component samples in step b onto the Sephadex LH-20 chromatographic column, the length is controlled at 15 cm, the inner diameter of the column is 3.0 cm, and the chromatographic column is eluted with methanol as the elution reagent. Take the eluate, combine the components containing Corilagin, concentrate to 2mL with a rotary evaporator at a temperature of 55°C, and freeze-dry in vacuum to obtain 255 mg of high-purity Corilagin white solid powder with a purity of 98.2%. Corilagin The recovery rate was 75.0%.
实施例17Example 17
a、将一根反相C18色谱柱(规格为长250毫米,直径80毫米,填料粒径10微米)连接在一台配有双通道紫外检测器的高效液相色谱仪(又称DAD检测器),高效液相色谱仪的由双泵组成,泵A连有甲醇为有机相,泵B连有甲酸含量为0.1%的水溶液,通过色谱工作站控制泵A和泵B的流速(总流速为180mL/min),其中A相占30%,平衡色谱柱时间15分钟,柱温控制在25℃,DAD检测器检测波长分别为254nm和320nm,称取20g石榴花提取物粉末,溶于20mL水中,用0.45μm的有机滤头过滤,至于液相小瓶中待测,色谱柱平衡以后通过进样器进样,接取包含有柯里拉京的组分,用旋转蒸发仪在温度40℃下浓缩至10mL,待用;a, a reversed-phase C18 chromatographic column (the specification is 250 millimeters in length, 80 millimeters in diameter, 10 microns of filler particle diameter) is connected to a high-performance liquid chromatograph (also known as DAD detector) equipped with a dual-channel ultraviolet detector ), the high-performance liquid chromatograph is made up of double pumps, and the pump A is connected with methanol as the organic phase, and the pump B is connected with an aqueous solution with a formic acid content of 0.1%. /min), wherein phase A accounts for 30%, the time for equilibrating the chromatographic column is 15 minutes, the column temperature is controlled at 25°C, the detection wavelength of the DAD detector is 254nm and 320nm respectively, and 20g of pomegranate flower extract powder is weighed, dissolved in 20mL of water, Filter with a 0.45 μm organic filter head, and put it in a liquid phase vial to be tested. After the chromatographic column is equilibrated, the sample is injected through the injector, and the components containing Corylatin are collected and concentrated at a temperature of 40 °C with a rotary evaporator to 10mL, ready for use;
b、保持步骤a中的色谱条件,将乙腈和甲酸含量为0.1%的水溶液混合,其中乙腈含量控制在20%,作为流动相,平衡色谱柱时间15min,柱温控制在25℃,DAD检测器检测波长分别为254nm和320nm,将步骤a中所得组分进样至色谱柱分离,接取包含柯里拉京的组分,用旋转蒸发仪在温度40℃下浓缩至2.0mL,待用;b. Maintain the chromatographic conditions in step a, mix acetonitrile and an aqueous solution with a formic acid content of 0.1%, wherein the acetonitrile content is controlled at 20%, as the mobile phase, the equilibration time of the chromatographic column is 15min, the column temperature is controlled at 25 ° C, and the DAD detector The detection wavelengths are 254nm and 320nm respectively. Inject the components obtained in step a into the chromatographic column for separation, take the components containing Corylatin, concentrate them to 2.0mL with a rotary evaporator at a temperature of 40°C, and set aside;
c、将步骤b中浓缩获取组分样品上至葡聚糖凝胶LH-20层析柱上,长度控制在15cm,柱内径在3.0cm,以甲醇为洗脱试剂进行层析柱洗脱,接取洗脱液,合并包含柯里拉京的组分,用旋转蒸发仪在温度55℃下浓缩至2mL,真空冷冻干燥,得到高纯度柯里拉京白色固体粉末268mg,纯度98.7%,柯里拉京回收率78.8%。c. Put the concentrated and obtained component samples in step b onto the Sephadex LH-20 chromatographic column, the length is controlled at 15 cm, the inner diameter of the column is 3.0 cm, and the chromatographic column is eluted with methanol as the elution reagent. Take the eluate, combine the components containing Corilagin, concentrate to 2mL with a rotary evaporator at a temperature of 55°C, and freeze-dry in vacuum to obtain 268mg of high-purity Corilagin white solid powder with a purity of 98.7%. Corilagin The recovery rate was 78.8%.
实施例18Example 18
a、将一根反相C18色谱柱(规格为长250毫米,直径80毫米,填料粒径10微米)连接在一台配有双通道紫外检测器的高效液相色谱仪(又称DAD检测器),高效液相色谱仪的由双泵组成,泵A连有甲醇为有机相,泵B连有甲酸含量为0.1%的水溶液,通过色谱工作站控制泵A和泵B的流速(总流速为180mL/min),其中A相占30%,平衡色谱柱时间20分钟,柱温控制在25℃,DAD检测器检测波长分别为254nm和320nm,称取20g石榴花提取物粉末,溶于20mL水中,用0.45μm的有机滤头过滤,至于液相小瓶中待测,色谱柱平衡以后通过进样器进样,接取包含有柯里拉京的组分,用旋转蒸发仪在温度40℃下浓缩至10mL,待用;a, a reversed-phase C18 chromatographic column (the specification is 250 millimeters in length, 80 millimeters in diameter, 10 microns of filler particle diameter) is connected to a high-performance liquid chromatograph (also known as DAD detector) equipped with a dual-channel ultraviolet detector ), the high-performance liquid chromatograph is made up of double pumps, and the pump A is connected with methanol as the organic phase, and the pump B is connected with an aqueous solution with a formic acid content of 0.1%. /min), wherein phase A accounts for 30%, the equilibration column time is 20 minutes, the column temperature is controlled at 25°C, the detection wavelength of the DAD detector is 254nm and 320nm respectively, and 20g pomegranate flower extract powder is weighed, dissolved in 20mL water, Filter with a 0.45 μm organic filter head, and put it in a liquid phase vial to be tested. After the chromatographic column is equilibrated, the sample is injected through the injector, and the components containing Corylatin are collected and concentrated at a temperature of 40 °C with a rotary evaporator to 10mL, ready for use;
b、保持步骤a中的色谱条件,将乙腈和甲酸含量为0.1%的水溶液混合,其中乙腈含量控制在15%,作为流动相,平衡色谱柱时间20min,柱温控制在25℃,DAD检测器检测波长分别为254nm和320nm,将步骤a中所得组分进样至色谱柱分离,接取包含柯里拉京的组分,用旋转蒸发仪在温度40℃下浓缩至2.0mL,待用;b. Maintain the chromatographic conditions in step a, mix acetonitrile and an aqueous solution with a formic acid content of 0.1%, wherein the acetonitrile content is controlled at 15%, as the mobile phase, equilibrate the chromatographic column for 20 minutes, and the column temperature is controlled at 25 ° C. DAD detector The detection wavelengths are 254nm and 320nm respectively. Inject the components obtained in step a into the chromatographic column for separation, take the components containing Corylatin, concentrate them to 2.0mL with a rotary evaporator at a temperature of 40°C, and set aside;
c、将步骤b中浓缩获取组分样品上至葡聚糖凝胶LH-20层析柱上,长度控制在15cm,柱内径在3.0cm,以甲醇为洗脱试剂进行层析柱洗脱,接取洗脱液,合并包含柯里拉京的组分,用旋转蒸发仪在温度55℃下浓缩至2mL,真空冷冻干燥,得到高纯度柯里拉京白色固体粉末269mg,纯度98.9%,柯里拉京回收率79.1%。c. Put the concentrated and obtained component samples in step b onto the Sephadex LH-20 chromatographic column, the length is controlled at 15 cm, the inner diameter of the column is 3.0 cm, and the chromatographic column is eluted with methanol as the elution reagent. Take the eluate, combine the components containing Corilagin, concentrate to 2mL with a rotary evaporator at a temperature of 55°C, and freeze-dry in vacuum to obtain 269mg of high-purity Corilagin white solid powder with a purity of 98.9%. The recovery rate was 79.1%.
实施例19Example 19
a、将一根反相C18色谱柱(规格为长250毫米,直径80毫米,填料粒径10微米)连接在一台配有双通道紫外检测器的高效液相色谱仪(又称DAD检测器),高效液相色谱仪的由双泵组成,泵A连有甲醇为有机相,泵B连有甲酸含量为0.1%的水溶液,通过色谱工作站控制泵A和泵B的流速(总流速为180mL/min),其中A相占30%,平衡色谱柱时间5分钟,柱温控制在25℃,DAD检测器检测波长分别为254nm和320nm,称取20g石榴花提取物粉末,溶于20mL水中,用0.45μm的有机滤头过滤,至于液相小瓶中待测,色谱柱平衡以后通过进样器进样,接取包含有柯里拉京的组分,用旋转蒸发仪在温度40℃下浓缩至10mL,待用;a, a reversed-phase C18 chromatographic column (the specification is 250 millimeters in length, 80 millimeters in diameter, 10 microns of filler particle diameter) is connected to a high-performance liquid chromatograph (also known as DAD detector) equipped with a dual-channel ultraviolet detector ), the high-performance liquid chromatograph is made up of double pumps, and the pump A is connected with methanol as the organic phase, and the pump B is connected with an aqueous solution with a formic acid content of 0.1%. /min), wherein phase A accounts for 30%, the equilibration column time is 5 minutes, the column temperature is controlled at 25°C, the detection wavelength of the DAD detector is 254nm and 320nm respectively, and 20g pomegranate flower extract powder is weighed, dissolved in 20mL water, Filter with a 0.45 μm organic filter head, and put it in a liquid phase vial to be tested. After the chromatographic column is equilibrated, the sample is injected through the injector, and the components containing Corylatin are collected and concentrated at a temperature of 40 °C with a rotary evaporator to 10mL, ready for use;
b、保持步骤a中的色谱条件,将乙腈和甲酸含量为0.1%的水溶液混合,其中乙腈含量控制在15%,作为流动相,平衡色谱柱时间5min,柱温控制在25℃,DAD检测器检测波长分别为254nm和320nm,将步骤a中所得组分进样至色谱柱分离,接取包含柯里拉京的组分,用旋转蒸发仪在温度40℃下浓缩至2.0mL,待用;b. Keep the chromatographic conditions in step a, mix acetonitrile and an aqueous solution with a formic acid content of 0.1%, wherein the acetonitrile content is controlled at 15%, as the mobile phase, equilibrate the chromatographic column for 5 minutes, and the column temperature is controlled at 25 ° C. DAD detector The detection wavelengths are 254nm and 320nm respectively. Inject the components obtained in step a into the chromatographic column for separation, take the components containing Corylatin, concentrate them to 2.0mL with a rotary evaporator at a temperature of 40°C, and set aside;
c、将步骤b中浓缩获取组分样品上至葡聚糖凝胶LH-20层析柱上,长度控制在15cm,柱内径在3.0cm,以甲醇为洗脱试剂进行层析柱洗脱,接取洗脱液,合并包含柯里拉京的组分,用旋转蒸发仪在温度55℃下浓缩至2mL,真空冷冻干燥,得到高纯度柯里拉京白色固体粉末213mg,纯度94.6%,柯里拉京回收率62.6%。c. Put the concentrated and obtained component samples in step b onto the Sephadex LH-20 chromatographic column, the length is controlled at 15 cm, the inner diameter of the column is 3.0 cm, and the chromatographic column is eluted with methanol as the elution reagent. Take the eluate, combine the components containing Corilagin, concentrate to 2mL with a rotary evaporator at a temperature of 55°C, and freeze-dry in vacuum to obtain 213mg of high-purity Corilagin white solid powder with a purity of 94.6%. Corilagin The recovery rate was 62.6%.
实施例20Example 20
a、将一根反相C18色谱柱(规格为长250毫米,直径80毫米,填料粒径10微米)连接在一台配有双通道紫外检测器的高效液相色谱仪(又称DAD检测器),高效液相色谱仪的由双泵组成,泵A连有甲醇为有机相,泵B连有甲酸含量为0.1%的水溶液,通过色谱工作站控制泵A和泵B的流速(总流速为180mL/min),其中A相占30%,平衡色谱柱时间15分钟,柱温控制在25℃,DAD检测器检测波长分别为280nm和380nm,称取20g石榴花提取物粉末,溶于20mL水中,用0.45μm的有机滤头过滤,至于液相小瓶中待测,色谱柱平衡以后通过进样器进样,接取包含有柯里拉京的组分,用旋转蒸发仪在温度40℃下浓缩至10mL,待用;a, a reversed-phase C18 chromatographic column (the specification is 250 millimeters in length, 80 millimeters in diameter, 10 microns of filler particle diameter) is connected to a high-performance liquid chromatograph (also known as DAD detector) equipped with a dual-channel ultraviolet detector ), the high-performance liquid chromatograph is made up of double pumps, and the pump A is connected with methanol as the organic phase, and the pump B is connected with an aqueous solution with a formic acid content of 0.1%. /min), wherein phase A accounts for 30%, the time for equilibrating the chromatographic column is 15 minutes, the column temperature is controlled at 25°C, the detection wavelength of the DAD detector is 280nm and 380nm respectively, and 20g of pomegranate flower extract powder is weighed, dissolved in 20mL of water, Filter with a 0.45 μm organic filter head, and put it in a liquid phase vial to be tested. After the chromatographic column is equilibrated, the sample is injected through the injector, and the components containing Corylatin are collected and concentrated at a temperature of 40 °C with a rotary evaporator to 10mL, ready for use;
b、保持步骤a中的色谱条件,将乙腈和甲酸含量为0.1%的水溶液混合,其中乙腈含量控制在15%,作为流动相,平衡色谱柱时间15min,柱温控制在25℃,DAD检测器检测波长分别为280nm和380nm,将步骤a中所得组分进样至色谱柱分离,接取包含柯里拉京的组分,用旋转蒸发仪在温度40℃下浓缩至1.5mL,待用;b. Maintain the chromatographic conditions in step a, mix acetonitrile and an aqueous solution with a formic acid content of 0.1%, wherein the acetonitrile content is controlled at 15%, as the mobile phase, equilibrate the chromatographic column for 15 minutes, and the column temperature is controlled at 25 ° C. DAD detector The detection wavelengths are 280nm and 380nm, respectively. Inject the components obtained in step a into the chromatographic column for separation, take the components containing corilagin, concentrate them to 1.5mL with a rotary evaporator at a temperature of 40°C, and set aside;
c、将步骤b中浓缩获取组分样品上至葡聚糖凝胶LH-20层析柱上,长度控制在15cm,柱内径在0.5cm,以甲醇为洗脱试剂进行层析柱洗脱,接取洗脱液,合并包含柯里拉京的组分,用旋转蒸发仪在温度55℃下浓缩至2mL,真空冷冻干燥,得到高纯度柯里拉京白色固体粉末278mg,纯度98.6%,柯里拉京回收率81.7%。c. Put the concentrated and obtained component samples in step b onto the Sephadex LH-20 chromatographic column, the length is controlled at 15 cm, the inner diameter of the column is 0.5 cm, and the chromatographic column is eluted with methanol as the elution reagent. Take the eluate, combine the components containing Corilagin, concentrate to 2mL with a rotary evaporator at a temperature of 55°C, and freeze-dry in vacuum to obtain 278 mg of high-purity Corilagin white solid powder with a purity of 98.6%. The recovery rate was 81.7%.
实施例21Example 21
a、将一根反相C18色谱柱(规格为长250毫米,直径80毫米,填料粒径10微米)连接在一台配有双通道紫外检测器的高效液相色谱仪(又称DAD检测器),高效液相色谱仪的由双泵组成,泵A连有甲醇为有机相,泵B连有甲酸含量为0.2%的水溶液,通过色谱工作站控制泵A和泵B的流速(总流速为180mL/min),其中A相占27%,平衡色谱柱时间17分钟,柱温控制在25℃,DAD检测器检测波长分别为254nm和366nm,称取16.8g石榴花提取物粉末,溶于20mL水中,用0.45μm的有机滤头过滤,至于液相小瓶中待测,色谱柱平衡以后通过进样器进样,接取包含有柯里拉京的组分,用旋转蒸发仪在温度37℃下浓缩至10mL,待用;a, a reversed-phase C18 chromatographic column (the specification is 250 millimeters in length, 80 millimeters in diameter, 10 microns of filler particle diameter) is connected to a high-performance liquid chromatograph (also known as DAD detector) equipped with a dual-channel ultraviolet detector ), the high performance liquid chromatograph is made up of double pumps, and the pump A is connected with methanol as an organic phase, and the pump B is connected with an aqueous solution with a formic acid content of 0.2%. /min), wherein phase A accounts for 27%, the time for equilibrating the chromatographic column is 17 minutes, the column temperature is controlled at 25°C, the detection wavelength of the DAD detector is 254nm and 366nm respectively, and 16.8g of pomegranate flower extract powder is weighed and dissolved in 20mL of water , filter with a 0.45μm organic filter head, and put it in the liquid phase vial to be tested. After the chromatographic column is equilibrated, the sample is injected through the injector, and the component containing Corylatin is collected and concentrated at a temperature of 37°C with a rotary evaporator. to 10mL, set aside;
b、保持步骤a中的色谱条件,将乙腈和甲酸含量为0.1%的水溶液混合,其中乙腈含量控制在10%,作为流动相,平衡色谱柱时间17min,柱温控制在25℃,DAD检测器检测波长分别为254nm和366nm,将步骤a中所得组分进样至色谱柱分离,接取包含柯里拉京的组分,用旋转蒸发仪在温度37℃下浓缩至0.3mL,待用;b. Keep the chromatographic conditions in step a, mix acetonitrile and an aqueous solution with a formic acid content of 0.1%, wherein the acetonitrile content is controlled at 10%, as the mobile phase, the equilibration time of the chromatographic column is 17min, the column temperature is controlled at 25 ° C, and the DAD detector The detection wavelengths are 254nm and 366nm, respectively. Inject the components obtained in step a into the chromatographic column for separation, take the components containing corilagin, concentrate them to 0.3mL with a rotary evaporator at a temperature of 37°C, and set aside;
c、将步骤b中浓缩获取组分样品上至葡聚糖凝胶LH-20层析柱上,长度控制在100cm,柱内径在1.0cm,以甲醇为洗脱试剂进行层析柱洗脱,接取洗脱液,合并包含柯里拉京的组分,用旋转蒸发仪在温度55℃下浓缩至2mL,真空冷冻干燥,得到高纯度柯里拉京白色固体粉末216mg,纯度98.7%,柯里拉京回收率75.8%。c. Put the concentrated and obtained component samples in step b onto the Sephadex LH-20 chromatographic column, the length is controlled at 100 cm, the inner diameter of the column is 1.0 cm, and the chromatographic column is eluted with methanol as the elution reagent. Take the eluate, combine the components containing Corilagin, concentrate to 2mL with a rotary evaporator at a temperature of 55°C, and freeze-dry in vacuum to obtain 216mg of high-purity Corilagin white solid powder with a purity of 98.7%. Corilagin The recovery rate was 75.8%.
实施例22Example 22
a、将一根反相C18色谱柱(规格为长250毫米,直径80毫米,填料粒径10微米)连接在一台配有双通道紫外检测器的高效液相色谱仪(又称DAD检测器),高效液相色谱仪的由双泵组成,泵A连有甲醇为有机相,泵B连有甲酸含量为0.1%的水溶液,通过色谱工作站控制泵A和泵B的流速(总流速为180mL/min),其中A相占27.4%,平衡色谱柱时间15分钟,柱温控制在25℃,DAD检测器检测波长分别为254nm和366nm,称取16.8g石榴花提取物粉末,溶于20mL水中,用0.45μm的有机滤头过滤,至于液相小瓶中待测,色谱柱平衡以后通过进样器进样,其分离色谱图如图1所示,接取包含有柯里拉京的组分,用旋转蒸发仪在温度40℃下浓缩至10mL,待用;a, a reversed-phase C18 chromatographic column (the specification is 250 millimeters in length, 80 millimeters in diameter, 10 microns of filler particle diameter) is connected to a high-performance liquid chromatograph (also known as DAD detector) equipped with a dual-channel ultraviolet detector ), the high-performance liquid chromatograph is made up of double pumps, and the pump A is connected with methanol as the organic phase, and the pump B is connected with an aqueous solution with a formic acid content of 0.1%. /min), wherein phase A accounts for 27.4%, equilibrates the
b、保持步骤a中的色谱条件,将乙腈和甲酸含量为0.1%的水溶液混合,其中乙腈含量控制在10.8%,作为流动相,平衡色谱柱时间15min,柱温控制在25℃,DAD检测器检测波长分别为254nm和366nm,将步骤a中所得组分进样至色谱柱分离,其分离色谱图如图2所示,接取包含柯里拉京的组分,用旋转蒸发仪在温度40℃下浓缩至0.5mL,待用;b. Maintain the chromatographic conditions in step a, mix acetonitrile and an aqueous solution with a formic acid content of 0.1%, wherein the acetonitrile content is controlled at 10.8%, as the mobile phase, equilibrate the chromatographic column for 15 minutes, and the column temperature is controlled at 25 ° C. DAD detector The detection wavelengths are 254nm and 366nm, respectively, and the components obtained in step a are injected into the chromatographic column for separation. The separation chromatogram is shown in Figure 2. The components containing Corylatin are taken, and the temperature is 40°C with a rotary evaporator. Concentrate to 0.5mL, set aside;
c、将步骤b中浓缩获取组分样品上至葡聚糖凝胶LH-20层析柱上,长度控制在13.5cm,柱内径在1.0cm,以甲醇为洗脱试剂进行层析柱洗脱,接取洗脱液,合并包含柯里拉京的组分,用旋转蒸发仪在温度55℃下浓缩至2mL,真空冷冻干燥,得到高纯度柯里拉京白色固体粉末228mg,纯度98.4%,柯里拉京回收率80.0%,其纯度鉴定图如图3所示。c. Put the concentrated and obtained component samples in step b onto the Sephadex LH-20 chromatographic column, the length is controlled at 13.5cm, the inner diameter of the column is 1.0cm, and methanol is used as the elution reagent for chromatographic column elution , take the eluate, combine the components containing Corilagin, concentrate to 2mL with a rotary evaporator at a temperature of 55°C, and freeze-dry in vacuo to obtain 228 mg of high-purity Corilagin white solid powder with a purity of 98.4%. The recovery rate of Beijing was 80.0%, and its purity identification diagram is shown in Figure 3.
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