CN108623649B

CN108623649B - Method for separating and purifying notoginsenoside Fc from total saponins of panax notoginseng leaves

Info

Publication number: CN108623649B
Application number: CN201810612165.2A
Authority: CN
Inventors: 高月; 刘星; 李海舟
Original assignee: Kunming University of Science and Technology
Current assignee: Kunming University of Science and Technology
Priority date: 2018-06-14
Filing date: 2018-06-14
Publication date: 2021-05-14
Anticipated expiration: 2038-06-14
Also published as: CN108623649A

Abstract

The invention discloses a method for separating and purifying notoginsenoside Fc from notoginsenoside leaf total saponins, which is characterized in that notoginsenoside leaf total saponins are used as raw materials, a crude notoginsenoside Fc product is prepared by medium-pressure column chromatography, and the crude notoginsenoside Fc product is purified by reverse-phase high-performance liquid chromatography and normal-phase high-performance liquid chromatography. The pure product with the content of more than 98 percent can be obtained, and the grade of the reference substance is achieved.

Description

Method for separating and purifying notoginsenoside Fc from total saponins of panax notoginseng leaves

Technical Field

The invention relates to the technical field of extraction, separation and purification of effective components of traditional Chinese medicines, in particular to a method for separating and purifying notoginsenoside Fc in panax notoginseng leaf total saponins.

Background

Notoginseng radix, also known as Notoginseng radix, radix Stephaniae Sinicae, is Araliaceae plant Notoginseng radixPanax notoginsengDried roots and rhizomes of (Burk.) f.h. Chen. Mainly distributed in Yunnan, Guangxi, Jiangxi, Sichuan and other places. The traditional effects are used for stopping bleeding, promoting blood circulation, removing blood stasis, relieving swelling, relieving pain and the like. Modern researches have found that the medicine has various physiological effects on cardiovascular and cerebrovascular systems, nervous systems, immune systems and the like. Due to the diverse biological activities and the increased use amount of panax notoginseng, the supply amount of panax notoginseng roots is difficult to meet the increasing market demand. Based on comprehensive utilization and optimized utilization of pseudo-ginseng resources, development of new resources and utilization of other plant part resources are urgently needed. Annual leaf recovery may be another source compared to the long periods required for root systems. Different parts of pseudo-ginseng are rich in different types of saponins, and the significant difference of leaves and roots of pseudo-ginseng is that the leaves contain rich 20(S) -protopanaxadiol type saponins. At present, according to literature reports, the notoginsenoside Fc has obvious effects of inhibiting platelet aggregation, improving myocardial ischemia, protecting cardiac muscle and the like, and can be used for preventing and treating various thrombotic diseases such as myocardial ischemia, myocardial infarction and the like. Zhengliyang and the like also prove that the notoginsenoside Fc can reduce the urine protein of db/db mice with type 2 diabetes, delay the progress of nephropathy and has the kidney protection effect. From the results, the research on the effective components of the saponin in the panax notoginseng leaf is necessary, and the study can optimize the resource utilization and has good medicinal value. Wherein the notoginsenoside Fc is 20(S) -protopanaxadiol saponin, and is the main effective active component in folium Notoginseng total saponin. Therefore, the extraction and purification of the active component notoginsenoside Fc in the total saponins of panax notoginseng leaves has great significanceAnd (5) defining. Until now, no patent report on the separation and purification of high-content notoginsenoside Fc is found.

Disclosure of Invention

The invention provides a method for separating and purifying notoginsenoside Fc from notoginsenoside, which takes notoginsenoside as a raw material, concentrates a part containing notoginsenoside Fc by column chromatography separation, and purifies the concentrated part to finally obtain high-purity notoginsenoside Fc, thereby providing important basis for comprehensive development and utilization of notoginsenoside Fc and large-scale preparation of notoginsenoside Fc.

The technical scheme adopted by the invention is as follows:

a method for separating and purifying notoginsenoside Fc from folium Notoginseng total saponin comprises the following steps:

(1) dissolving folium Notoginseng total saponin in methanol water solution, performing intermediate pressure column chromatography with LK-20 resin, isocratic eluting with methanol-water system, collecting eluate, and concentrating to obtain Notoginseng radix saponin Fc crude product;

(2) dissolving the crude product of notoginsenoside Fc with methanol, filtering, preparing by reversed phase high performance liquid chromatography, collecting eluate containing notoginsenoside Fc, concentrating, dissolving with methanol, and standing to precipitate white crystal;

(3) dissolving the white crystal with chromatographic methanol, purifying with forward high performance liquid chromatography, and collecting eluate containing notoginsenoside Fc to obtain purified notoginsenoside Fc.

Dissolving the panax notoginseng leaf total saponins in a methanol aqueous solution with the volume ratio of 20-60% in the step (1); isocratic elution is carried out by using a methanol-water system with the volume ratio of 71-74%, and the flow rate of medium-pressure column chromatography is 15-30 mL/min.

In the step (2), the reversed-phase high performance liquid chromatography adopts a C18 column and an acetonitrile-water system with the volume ratio of 28-31% to perform isocratic elution, and the flow rate is 15-20 mL/min.

And (3) performing isocratic elution on the forward high performance liquid chromatography in the step (3) by using a HILIC column and an acetonitrile-water system with the volume ratio of 30-33%, wherein the flow rate is 15-20 mL/min.

The invention has the advantages that:

1. the LK-20 resin is adopted for separation and preparation of the notoginsenoside Fc for the first time, the notoginsenoside Fc crude product can be obtained quickly and efficiently, the operation process is simplified, and the resin can be repeatedly used, so that the method is economic and environment-friendly;

2. the medium-pressure preparation is adopted in the separation process, so that the operation is simple and the time is saved; in the process of separating the medium-pressure resin column, no toxic and expensive organic solvent is used, so that the cost is saved and the environment is protected;

3. the HILIC chromatographic column is a neutral amide bonding phase with a unique structure, has outstanding hydrophilicity, and avoids acidity and heterogeneity of silanol groups on the surface of pure silica gel. The high-proportion acetonitrile-water is used as a mobile phase, so that a strong polar compound which is difficult to be reserved on a traditional reversed phase chromatographic column can be reserved;

4. the notoginsenoside Fc with the purity of more than 98 percent can be obtained, the grade of a reference substance is reached, and the method can be used for preparing a large amount of notoginsenoside Fc.

Detailed Description

In order to make the technical details of the invention known to those skilled in the art, the technical solutions of the invention are further described below with reference to specific embodiments.

In the following examples, the total saponins of panax notoginseng leaves were purchased from yuxi city and pharmaceutical limited, Yunnan province; the column C18 (YMC-Pack ODS-A, 250 mm. times.20 mm, 5 μm) was A product of YMC Co. of Tokyo, Japan, and the HILIC column (XAmide, 150 mm. times.4.6 mm, 5 μm) was A product of science and technology Co., Ltd, of Wako, Beijing.

Example 1

Dissolving 100g of folium Notoginseng total saponin with 20% methanol water solution, and filtering. Performing intermediate pressure column chromatography with LK-20 resin, isocratic eluting with 71% methanol-water system at flow rate of 20mL/min, collecting eluate, and concentrating to obtain notoginsenoside Fc crude product. Dissolving the crude product of notoginsenoside Fc with methanol, filtering, performing isocratic elution with C18 column via 28% acetonitrile-water system at flow rate of 18mL/min, collecting eluate containing notoginsenoside Fc, and detecting notoginsenoside Fc at 203nm with a peak area of 91.7%. Concentrating the collected eluent containing notoginsenoside Fc, dissolving with methanol, placing until white crystals are separated out, dissolving the white crystals with chromatographic methanol, performing isocratic elution with a HILIC chromatographic column through an acetonitrile-water system with the volume ratio of 30% at the flow rate of 16mL/min, collecting the eluent containing notoginsenoside Fc to obtain purified notoginsenoside Fc, wherein the detection content is 98.2%, the drying weight is 330mg, and the yield is 0.33%.

Example 2

Dissolving 100g of folium Notoginseng total saponin in 30% methanol water solution, and filtering. Performing intermediate pressure column chromatography with LK-20 resin, isocratic eluting with 72% methanol-water system at flow rate of 15mL/min, collecting eluate, and concentrating to obtain notoginsenoside Fc crude product. Dissolving the crude product of notoginsenoside Fc with methanol, filtering, performing isocratic elution with C18 column via 29% acetonitrile-water system at flow rate of 19mL/min, collecting eluate containing notoginsenoside Fc, and detecting notoginsenoside Fc at 203nm with a peak area of 91.5%. Concentrating the collected eluent containing notoginsenoside Fc, dissolving with methanol, placing until white crystals are separated out, dissolving the white crystals with chromatographic methanol, performing isocratic elution with a HILIC chromatographic column through an acetonitrile-water system with the volume ratio of 31% at the flow rate of 18mL/min, collecting the eluent containing notoginsenoside Fc to obtain purified notoginsenoside Fc, wherein the detection content is 98.3%, the drying weight is 320mg, and the yield is 0.32%.

Example 3

Dissolving 100g of folium Notoginseng total saponin in 45% methanol water solution, and filtering. Performing intermediate pressure column chromatography with LK-20 resin, isocratic eluting with 73% methanol-water system at flow rate of 25mL/min, collecting eluate, and concentrating to obtain notoginsenoside Fc crude product. Dissolving the crude product of notoginsenoside Fc with methanol, filtering, performing isocratic elution with C18 column via 30% acetonitrile-water system at flow rate of 15mL/min, collecting eluate containing notoginsenoside Fc, and detecting notoginsenoside Fc at 203nm with a peak area of 91.5%. Concentrating the collected eluent containing notoginsenoside Fc, dissolving with methanol, placing until white crystals are separated out, dissolving the white crystals with chromatographic methanol, performing isocratic elution with a HILIC chromatographic column through an acetonitrile-water system with the volume ratio of 32% at the flow rate of 15mL/min, collecting the eluent containing notoginsenoside Fc to obtain purified notoginsenoside Fc, wherein the detection content is 98.3%, the drying weight is 360mg, and the yield is 0.36%.

Example 4

Dissolving 100g of folium Notoginseng total saponin in 60% methanol water solution, and filtering. Performing intermediate pressure column chromatography with LK-20 resin, isocratic eluting with 74% methanol-water system at flow rate of 30mL/min, collecting eluate, and concentrating to obtain notoginsenoside Fc crude product. Dissolving the crude product of notoginsenoside Fc with methanol, filtering, performing isocratic elution with C18 column via 31% acetonitrile-water system at flow rate of 16mL/min, collecting eluate containing notoginsenoside Fc, and detecting notoginsenoside Fc at 203nm with a peak area of 91.4%. Concentrating the collected eluent containing notoginsenoside Fc, dissolving with methanol, placing until white crystals are separated out, dissolving the white crystals with chromatographic methanol, performing isocratic elution with a HILIC chromatographic column through an acetonitrile-water system with the volume ratio of 33% at the flow rate of 20mL/min, collecting the eluent containing notoginsenoside Fc to obtain purified notoginsenoside Fc, wherein the detection content is 98.1%, the drying weight is 370mg, and the yield is 0.37%.

Claims

1. A method for separating and purifying notoginsenoside Fc from folium Notoginseng total saponin comprises the following steps:

(1) dissolving folium Notoginseng total saponin in methanol water solution, performing intermediate pressure column chromatography with LK-20 resin, performing isocratic elution with 71-74% methanol-water system, collecting eluate, and concentrating to obtain Notoginseng radix saponin Fc crude product;

(2) dissolving the crude product of notoginsenoside Fc with methanol, filtering, preparing by reversed phase high performance liquid chromatography, isocratic eluting with 28-31% acetonitrile-water system, collecting eluate containing notoginsenoside Fc, concentrating, dissolving with methanol, and standing to precipitate white crystal;

(3) dissolving white crystal with chromatographic methanol, purifying with forward high performance liquid chromatography, isocratic eluting with 30-33% acetonitrile-water system, and collecting eluate containing notoginsenoside Fc to obtain purified notoginsenoside Fc.

2. The method according to claim 1, wherein the total saponins of panax notoginseng leaves in step (1) are dissolved in 20-60% by volume of methanol aqueous solution.

3. The method according to claim 1, wherein the flow rate of the medium-pressure column chromatography in the step (1) is 15 to 30 mL/min.

4. The method of claim 1, wherein the reversed-phase high performance liquid chromatography in step (2) is performed using a C18 column at a flow rate of 15-20 mL/min.

5. The method as claimed in claim 1, wherein the forward high performance liquid chromatography in step (3) uses HILIC column at a flow rate of 15-20 mL/min.