阅读说明：本技术 采用大孔树脂串联动态轴向压缩柱分离纯化大蓟总苷的方法 (Method for separating and purifying Japanese thistle herb total glycosides by adopting macroporous resin series dynamic axial compression column ) 是由 李坤平 郭姣 于 2021-07-15 设计创作，主要内容包括：本发明公开了一种采用大孔树脂串联动态轴向压缩柱分离纯化大蓟总苷的方法,包括药材净选、热水提取、絮凝沉降、过滤、大孔树脂富集、洗脱、浓缩、动态轴向压缩柱层析纯化、干燥处理等步骤。本发明方法避免了传统溶剂萃取,提取分离纯化过程繁琐、有机溶剂用量大、能耗高等缺点,提高了大蓟总苷加工技术的环境友好性,且在不改变目标产品特征成份的前提下,通过实时检测,流份收集切换操作,可以提高大蓟总苷的纯度、工艺指向性明确,操作简单、分离效率高。(The invention discloses a method for separating and purifying Japanese thistle herb total glycosides by adopting a macroporous resin series dynamic axial compression column, which comprises the steps of medicinal material cleaning, hot water extraction, flocculation and sedimentation, filtration, macroporous resin enrichment, elution, concentration, dynamic axial compression column chromatography purification, drying treatment and the like. The method provided by the invention overcomes the defects of complicated extraction, separation and purification processes, large organic solvent consumption, high energy consumption and the like of the traditional solvent, improves the environmental friendliness of the thistle total glycoside processing technology, and can improve the purity of the thistle total glycoside through real-time detection and stream collection switching operation on the premise of not changing the characteristic components of a target product, and has the advantages of definite process directivity, simplicity in operation and high separation efficiency.)

1. A method for separating and purifying Japanese thistle herb total glycosides by adopting a macroporous resin series dynamic axial compression column is characterized by comprising the following steps:

(1) heating and extracting herba seu radix Cirsii Japonici with pure water, coarse filtering to obtain crude extractive solution, and flocculating

Settling, taking the supernatant, and filtering to obtain clear supernatant;

(2) enriching the clear supernatant by macroporous resin, eluting, concentrating, and filtering with membrane to obtain membrane filtrate;

(3) purifying the membrane filtrate by dynamic axial compression column chromatography, collecting fluid containing pectolinarin and linarin by LC-qTOF-MS method for rapid identification of precise molecular weight and locking retention time of reference substance, concentrating, and drying to obtain herba seu radix Cirsii Japonici total glycosides.

2. The method for separating and purifying the cynara scolymus total glycosides according to claim 1, wherein the pure water extraction time in step (1) is 1-4 hours, the extraction temperature is 90-95 ℃, the extraction times are 1-2, and the ratio of the material to the liquid is 1: 10-30.

3. The method for separating and purifying the Japanese thistle total glycosides by adopting the macroporous resin series dynamic axial compression column according to claim 1, wherein in the step (1), the rough filtration of the extracting solution is to filter the extracting solution by adopting a sintered metal filter head of 90-100 mu m and connecting the filter head with a vacuum pump.

4. The method for separating and purifying the cynara scolymus total glycosides by adopting the macroporous resin series dynamic axial compression column according to claim 1, wherein in the step (1), the supernatant is filtered by adopting a sintered metal filter head of 50-60 mu m connected with a vacuum pump.

5. The method for separating and purifying herba seu radix Cirsii Japonici total glycosides according to claim 1, wherein in step (2), the macroporous resin is AB-8 or D-101, and the eluting solvent is 60-95% methanol or ethanol.

6. A method according to claim 1, comprisingThe method for separating and purifying the Japanese thistle herb total glycosides by using the macroporous resin series dynamic axial compression column is characterized in that in the step (3), the dynamic axial compression column uses C₁₈The reverse phase silica gel bonded phase is filler; methanol is taken as a mobile phase A and 0.05 percent formic acid aqueous solution is taken as a mobile phase B; gradient elution, wherein the elution procedure is 0-17 min, and 95% B → 44% B; 17-21 min, 44% B → 38% B; 21-32 min, 38% B → 5% B; 32-35 min, 5% B → 5% B; 35-37 min, 5% → 95% B; 37-45 min, 95% B → 95% B; flow rate 70 mL/min^-1The detection wavelengths are 254nm and 330 nm, the column temperature is 25 ℃, and the sample injection amount is 10 mL.

7. The method for separating and purifying herba seu radix Cirsii Japonici total glycosides according to claim 1, wherein the drying in step (3) is freeze drying or vacuum heating drying.

8. The method for separating and purifying herba seu radix Cirsii Japonici total glycosides of claim 1, wherein in step (3), the qTOF-MS M/z ([ M-H ] M/z) of pectolinarioside and linarin is determined by using macroporous resin series dynamic axial compression column]^-) 621.1755 + -0.05 Da and 591.1654 + -0.05 Da respectively, and the dynamic axial compression column chromatography retention time is about 24.45 + -0.2 min.

9. The circium japonicum total glycosides prepared by the method of any one of claims 1-8, which consists essentially of 2 components: the total weight content of pectolinarin and linarin is not less than 90%.

10. The circium japonicum total glycosides of claim 9, wherein the weight content of pectolinarin is 75-85% and the weight content of linarin is 5-15%.

Technical Field

The invention belongs to the technical field of separation and purification, and particularly relates to a method for separating and purifying Japanese thistle herb total glycosides by adopting a macroporous resin series dynamic axial compression column.

Background

Herba seu radix Cirsii Japonici (also called Carex Dactylicapni, radix Raphani, etc.), and its base plant is herba seu radix Cirsii Japonici of Cirsium of CompositaeCirsium japonicumDC. The dried aerial parts of Japanese thistle herb are used as the medicine and collected in the Chinese pharmacopoeia part I (2020 edition). Circium japonicum is cool in nature, sweet and bitter in taste; it enters heart and liver meridians. Herba seu radix Cirsii Japonici is rich in flavonoids, pectolinarin, linarin, hesperidin, and Salix purpureaChuanyuanhuang and the like have been reported.

The pectolinarigenin and the linarin are flavonoid oxygen glycoside compounds, have good physiological activities of resisting inflammation, protecting liver, reducing blood sugar, resisting tumor and the like, and have great development and utilization values. Through previous researches of the applicant, the content of the pectolinarin, the linarin and the like in the circium japonicum is low (the content of the pectolinarin in the cirium japonicum is not lower than 0.2 percent according to the regulation of pharmacopoeia). However, the Japanese thistle herb is perennial herb, is wild in hillsides, roadside and the like, is distributed in south and north provinces of China, has rich resources and low cost, is convenient to artificially cultivate, is an excellent source of the pectolinarin and the linarin, and the mixture of the pectolinarin and the linarin has the same biological activity as the two, so the Japanese thistle herb and the linarin are prepared by mixing the two, and the mixture of the two is named as Japanese thistle total glycoside (TFC)₉₀）。

At present, the extraction, separation and purification of the components of the circium japonicum usually adopt several steps or the whole process of the following processes: coarse crushing raw materials, soaking in water or solvent, heating reflux extraction or ultrasonic extraction, filtering and centrifuging, precipitating, concentrating, separating by macroporous resin adsorption, extracting with solvent, performing column chromatography, concentrating by evaporation under reduced pressure, recovering solvent, spray drying or freeze drying, and recrystallizing. The traditional extraction and separation process has the defects of long time consumption, high energy consumption, large solvent consumption, poor environmental compatibility, low product purity and the like.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention mainly aims to provide the method for separating and purifying the cirsium japonicum total glycosides by adopting the macroporous resin series dynamic axial compression column, the method avoids the defects of complicated extraction, separation and purification process, large organic solvent consumption, high energy consumption and the like of the traditional solvent extraction, improves the environmental friendliness of the cirsium japonicum total glycoside processing technology, and can improve the purity of the cirsium japonicum total glycosides, and has definite process directivity, simple operation and high separation efficiency by real-time detection and fraction collection switching operation on the premise of not changing the characteristic components of target products.

The invention is realized by the following technical scheme:

a method for separating and purifying Japanese thistle herb total glycosides by adopting a macroporous resin series dynamic axial compression column comprises the following steps:

(1) heating and extracting herba seu radix Cirsii Japonici with pure water, coarse filtering to obtain crude extractive solution, and flocculating

Settling, taking the supernatant, and filtering to obtain clear supernatant;

(2) enriching the clear supernatant by macroporous resin, eluting, concentrating, and filtering with membrane to obtain membrane filtrate;

(3) purifying the membrane filtrate by dynamic axial compression column chromatography, collecting fluid containing pectolinarin and linarin by LC-qTOF-MS method for rapid identification of precise molecular weight and locking retention time of reference substance, concentrating, and drying to obtain herba seu radix Cirsii Japonici total glycosides.

Preferably, in the step (1), the extraction time of the pure water is 1-4 hours, the extraction temperature is 90-95 ℃, the extraction times are 1-2 times, and the ratio of material to liquid is 1: 10-30.

Preferably, in the step (1), the rough filtration of the extracting solution is to filter the extracting solution by connecting a 90-100 mu m sintered metal filter head with a vacuum pump.

Preferably, in the step (1), the supernatant is filtered by connecting a 50-60 mu m sintered metal filter head with a vacuum pump.

Preferably, in the step (2), the macroporous resin is AB-8 or D-101, and the elution solvent is 70-95% methanol or ethanol.

Preferably, in step (3), the dynamic axial compression column is compressed by C₁₈The reverse phase silica gel bonded phase is filler; methanol is taken as a mobile phase A and 0.05 percent formic acid aqueous solution is taken as a mobile phase B; gradient elution, wherein the elution procedure is 0-17 min, and 95% B → 44% B; 17-21 min, 44% B → 38% B; 21-32 min, 38% B → 5% B; 32-35 min, 5% B → 5% B; 35-37 min, 5% → 95% B; 37-45 min, 95% B → 95% B; flow rate 70 mL/min^-1The detection wavelengths are 254nm and 330 nm, the column temperature is 25 ℃, and the sample injection amount is 10 mL.

Preferably, in the step (3), the drying is freeze drying or vacuum heating drying.

In the step (3), the step (c),qTOF-MS M/z ([ M-H) of pectolinaride and linarin]^-) 621.1755 + -0.05 Da and 591.1654 + -0.05 Da respectively, and the dynamic axial compression column chromatography retention time is about 24.45 + -0.2 min.

The invention also provides the circium japonicum total glycosides prepared by the method, which mainly comprise 2 components: the total weight content of pectolinarin and linarin is not less than 90%.

Preferably, the content of pectolinarin in the composition is 75-85 wt%, and the content of linarin in the composition is 5-15 wt%.

Compared with the prior art, the invention has the following beneficial effects:

the invention provides a novel method for separating and purifying the setose thistle total glycosides by a macroporous resin enrichment series dynamic axial compression column, which overcomes the defects of complicated extraction, separation and purification process, large organic solvent consumption and high energy consumption in the prior art, and improves the purification efficiency and the purity of the setose thistle total glycosides by adopting a method of LC-qTOF-MS accurate molecular weight rapid identification and contrast retention time locking. Meanwhile, the method adopts a dynamic axial compression column chromatography technology to purify the cirsium japonicum total glycosides, and the experimental and industrial amplification reliability is high; the macroporous resin enrichment series dynamic axial compression column chromatography technology adopted by the invention has compact process and reduces the pollution risk link.

Drawings

FIG. 1 is a liquid chromatogram of the separated and prepared herba seu radix Cirsii Japonici total glycosides; wherein A is a dynamic axial compression column chromatogram, and B is an HPLC analysis chromatogram.

FIG. 2 is a high resolution mass spectrum of linarin prepared by separation according to the present invention;

FIG. 3 is the high resolution mass spectrum of pectolinarin prepared by separation according to the present invention.

Detailed Description

The present invention is further illustrated by the following specific embodiments, which are not intended to limit the scope of the invention.

Example 1:

a method for separating and purifying Japanese thistle herb total glycosides by adopting a macroporous resin series dynamic axial compression column comprises the following steps:

(1) taking 1kg of the overground part of Japanese thistle as a raw material, cleaning, removing impurities, adding pure water, heating and extracting for 2 times at 90-95 ℃, wherein the ratio of materials to liquids in the first extraction is 1: 20, the extraction time is 2 hours, and the ratio of the material to the liquid in the second extraction is 1: 15, the extraction time is 1.5h,

coarse filtering the two extracting solutions by connecting a 90-100 mu m sintered metal filter head with a vacuum pump, combining to obtain a coarse extracting solution, adding a flocculating agent (0.005% polyacrylamide) into the coarse extracting solution for flocculation and sedimentation, taking supernatant, and filtering the supernatant by connecting a 50-60 mu m sintered metal filter head with a vacuum pump to obtain clarified supernatant;

(2) pouring the clear supernatant into pretreated D101 macroporous adsorption resin, and statically adsorbing for 12 h; then, filtering the resin after adsorption, rinsing with 2 times of pure water, eluting with 95% ethanol for 3 times, each time for 30 minutes, collecting the eluate, dissolving with 70% ethanol after concentration under reduced pressure, and filtering with a 0.45 μm membrane;

(3) preparing a chromatographic system by adopting Jiangsu Hanbang science and technology Limited, configuring an NP7000 infusion pump and an NU3000 UV/VIS detector, connecting a DAC-HB dynamic axial compression column (50 multiplied by 650 mm) in series, taking octadecylsilane chemically bonded silica (C18, Hua Spectrum New science and technology Limited, ZK 2016080701) as a filler, taking methanol (A) -0.05% formic acid aqueous solution (B) as a mobile phase, and performing gradient elution, wherein the elution procedure is 0-17 min, and 95% B → 44% B; 17-21 min, 44% B → 38% B; 21-32 min, 38% B → 5% B; 32-35 min, 5% B → 5% B; 35-37 min, 5% → 95% B; 37-45 min, 95% B → 95% B; flow rate 70 mL/min^-1The detection wavelengths are 254nm and 330 nm, the column temperature is 25 ℃, and the sample injection amount is 10 mL; the chromatogram is shown in FIG. 1.

A retention time index is established with pectolinarin control (Chengdu Egyo method Biotech limited, AF 21021105), and the retention time is about 24.45 min. Performing molecular weight measurement on the target fraction by LC-qTOF-MS, and collecting fractions containing pectolinarin and linarin, and their qTOF-MS M/z ([ M-H ]]^-) 621.1755Da and 591.1654Da respectively (as in FIGS. 2-3);

concentrating the target fraction under reduced pressure, and lyophilizing with vacuum freeze-dryer (Christ Alpha 1-2LDplus type lyophilizer, Germany) to obtain pale yellow powder 1.812 g; the cold trap temperature of the freeze dryer is set at-55 deg.C, freezing temperature is-36 deg.C, and vacuum degree is 0.20 mbar.

About 10mg of the obtained pale yellow powder was accurately weighed and dissolved in 70% ethanol and made to 10mL volume, and analyzed by HPLC (Waters, model 2695, DAD detector) (chromatogram shown in FIG. 1), wherein the contents of pectolinarin and linarin were 84.17% and 7.96%, respectively, by external standard method.

Wherein, chromatographic conditions of HPLC are as follows: waters 2695 high performance liquid chromatograph (PDA 2998 detector, Empower 2 chromatography workstation), Kromasil 100-5C18 (4.6X 250mm, 5 μm) as chromatographic column, gradient elution (0-15 min, 10% B → 14% B, 15-20 min, 14% B → 24% B, 20-33 min, 24% B → 26% B, 33-54 min, 26% B → 57% B, 54-59 min, 57% → 68% B, 59-65 min, 68% B → 90% B, flow rate 1.0 mL. min) with 0.05% aqueous formic acid solution (A) -acetonitrile (B) as mobile phase^-1The detection wavelength is 330 nm, the column temperature is 30 ℃, and the sample injection amount is 10 mu L.

Similarly, according to the method specified in the setose thistle item of the 2020 version of Chinese pharmacopoeia, the content of the pectolinarin is determined to be 0.23 percent, and the content of the linarin is determined to be 0.05 percent.

Example 2:

a method for separating and purifying Japanese thistle herb total glycosides by adopting a macroporous resin series dynamic axial compression column comprises the following steps:

(1) taking 1kg of the overground part of Japanese thistle as a raw material, cleaning, removing impurities, adding pure water, heating and extracting for 2 times at 90-95 ℃, wherein the ratio of materials to liquids in the first extraction is 1: 15, the extraction time is 2 hours, and the ratio of the material to the liquid in the second extraction is 1: 10, the extraction time is 1.5h,

coarse filtration is carried out on the two extracting solutions by connecting a sintered metal filter head of 90-100 mu m with a vacuum pump, crude extracting solutions are obtained by combination, a flocculating agent (0.0075% polyacrylamide) is added into the crude extracting solutions for flocculation and sedimentation, and supernatant liquid is obtained and filtered by connecting a sintered metal filter head of 50-60 mu m with a vacuum pump, so that clear supernatant liquid is obtained;

(2) pouring the clear supernatant into pretreated AB-8 macroporous adsorption resin, and statically adsorbing for 12 h; then, filtering out the resin after adsorption, rinsing with 2 times of pure water, eluting with methanol for 3 times, each time for 20 minutes, collecting the eluent, dissolving with methanol after decompression and concentration, and filtering with a 0.45 mu m membrane;

(3) preparing a chromatographic system by adopting Jiangsu Hanbang science and technology Limited, configuring an NP7000 infusion pump and an NU3000 UV/VIS detector, connecting a DAC-HB dynamic axial compression column (50 multiplied by 650 mm) in series, taking octadecylsilane chemically bonded silica (C18, Hua Spectrum New science and technology Limited, ZK 2016080701) as a filler, taking methanol (A) -0.05% formic acid aqueous solution (B) as a mobile phase, and performing gradient elution, wherein the elution procedure is 0-17 min, and 95% B → 44% B; 17-21 min, 44% B → 38% B; 21-32 min, 38% B → 5% B; 32-35 min, 5% B → 5% B; 35-37 min, 5% → 95% B; 37-45 min, 95% B → 95% B; flow rate 70 mL/min^-1The detection wavelengths are 254nm and 330 nm, the column temperature is 25 ℃, and the sample injection amount is 10 mL;

concentrating the target fraction under reduced pressure, drying in vacuum heating drying oven (DZF type vacuum drying oven), and grinding to obtain light yellow TFC₉₀Powder 1.502 g. The vacuum degree of the drying oven is-0.085 MPa, and the temperature of the drying oven is 65 +/-1 ℃.

The resulting pale yellow powder, about 10mg, was accurately weighed and dissolved in 70% ethanol and made up to 10mL for HPLC analysis (Waters, model 2695, DAD detector) and calculated by the external standard method, where the contents of pectolinarin and linarin were 84.76% and 8.27%, respectively.

Example 3:

a method for separating and purifying Japanese thistle herb total glycosides by adopting a macroporous resin series dynamic axial compression column comprises the following steps:

(1) taking 5kg of the overground part of Japanese thistle as a raw material, cleaning, removing impurities, adding pure water, heating and extracting for 2 times at 90-95 ℃, wherein the ratio of materials to liquids in the first extraction is 1: 25, the extraction time is 2 hours, the operation is repeated for 1 time for the second time, the two extracting solutions are roughly filtered by connecting a sintered metal filter head of 90-100 mu m with a vacuum pump, crude extracting solutions are obtained through combination, a flocculating agent (0.01% polyacrylamide) is added into the crude extracting solutions for flocculation and sedimentation, supernatant liquid is obtained through connecting a sintered metal filter head of 50-60 mu m with a vacuum pump, and clear supernatant liquid is obtained;

(2) pouring the clear supernatant into pretreated D101 macroporous adsorption resin, and statically adsorbing for 12 h; then, filtering out the adsorbed resin, rinsing with 2 times of pure water, eluting with methanol for 3 times, each time for 20 minutes, collecting the eluent, rinsing the macroporous resin eluted with the last methanol for 4 times with the pure water, each time for 2 times of the volume of the resin, adding the filtered macroporous resin into the residual liquid adsorbed before, repeating the adsorption and elution operations, combining the eluents obtained in two times, dissolving with 70% ethanol after decompression and concentration, and filtering with a 0.45 mu m membrane;

(3) preparing a chromatographic system by adopting Jiangsu Hanbang science and technology Limited, configuring an NP7000 infusion pump and an NU3000 UV/VIS detector, connecting a DAC-HB dynamic axial compression column (50 multiplied by 650 mm) in series, taking octadecylsilane chemically bonded silica (C18, Hua Spectrum New science and technology Limited, ZK 2016080701) as a filler, taking methanol (A) -0.05% formic acid aqueous solution (B) as a mobile phase, and performing gradient elution, wherein the elution procedure is 0-17 min, and 95% B → 44% B; 17-21 min, 44% B → 38% B; 21-32 min, 38% B → 5% B; 32-35 min, 5% B → 5% B; 35-37 min, 5% → 95% B; 37-45 min, 95% B → 95% B; flow rate 70 mL/min^-1The detection wavelengths are 254nm and 330 nm, the column temperature is 25 ℃, and the sample injection amount is 10 mL;

concentrating the target fraction under reduced pressure, placing in a vacuum heating drying oven, drying, and grinding to obtain light yellow TFC₉₀7.724g of powder. The vacuum degree of the drying box is-0.085 to-0.090 MPa, and the temperature of the drying box is 65 +/-1 ℃.

The resulting pale yellow powder, about 10mg, was accurately weighed and dissolved in 70% ethanol and made up to 10mL for HPLC analysis (Waters, model 2695, DAD detector) and calculated by the external standard method, where the contents of pectolinarin and linarin were 83.74% and 7.25%, respectively.