CN108822178B - Preparation method of low-polarity rare ginsenoside Rg5/Rk1 and Rh3/Rk2 - Google Patents

Abstract

The invention provides a preparation method of rare ginsenoside quasi-monomer compound Rg5/Rk1 (containing Rg5 and an isomer Rk1) and rare ginsenoside quasi-monomer compound Rh3/Rk2 (containing Rh3 and an isomer Rk 2). compared with the existing preparation method of rare ginsenoside, the preparation method is simple and low in cost, and meanwhile, on the basis, the invention also provides methods for preparing rare ginsenoside monomer compounds Rg5 and Rk1 and rare ginsenoside monomer compounds Rh3 and Rk 2; the method can separate and prepare rare ginsenoside quasi-monomer compound Rg5/Rk1 and monomer components thereof and rare ginsenoside quasi-monomer compound Rh3/Rk2 and monomer components thereof at low cost, high efficiency and large scale by simple column chromatography separation technology.

Description

Preparation method of low-polarity rare ginsenoside Rg5/Rk1 and Rh3/Rk2

Technical Field

The invention relates to the field of medicines, in particular to a preparation method of low-polarity rare ginsenoside.

Background

Cancer is the second largest disease in the world that impairs human life, and the mortality rate is second only to cardiovascular and cerebrovascular diseases, which is one of the most important factors in human death. The world cancer report, the latest version of which is responsible for the international cancer research center (IARC), the official cancer institution of the world health organization, has predicted that global cancer cases will exhibit a rapid proliferation situation, ranging from 1400 million people in 2012 to 1900 million people in 2025 year by year, and up to 2400 million people in 2035 year. The report also showed that nearly half of the globally new cancer cases appeared in asia in 2012, most of which were in china, which was the top of new cancer cases. In 2012, 307 million cancer patients are newly added in China and cause about 220 million deaths, which account for 21.9% and 26.8% of the global total amount, respectively. The data of the world health is slightly lower than the statistics of China. 2012's of data published by national tumor registration centers show that about 350 million new cancer cases and about 250 million people die each year in china.

There are three major modes of treatment commonly used today for cancer: surgery, radiation therapy and medication, and the choice of which therapy depends on the location of the tumor, the degree of malignancy, the degree of progression and the physical state of the patient. Of the three modes, surgical treatments are often limited in effectiveness due to cancer cell invasion and spread to adjacent tissues or distant metastasis; the therapeutic methods of radiotherapy are limited by the damage to other normal tissues in the body; the medicine treatment method is the most basic treatment method for advanced diffuse and metastatic malignant tumors. Although chemotherapy for directly killing tumor cells has been developed and advanced obviously in the past decades and becomes the medium of tumor drug therapy, the defects of poor effect, small drug selectivity, more toxic and side effects and serious side effects on slow-growing solid tumors become important limiting factors in clinical treatment. The fourth mode following surgery, radiation therapy and chemotherapy is biological treatment of tumors, which primarily regulates the body's own biological response through the action of tumor host defense mechanisms or biological agents, thereby inhibiting or eliminating tumors; although biological treatment has no great toxic and side effects, the price is high due to strict technical requirements and complex process, and a plurality of cancer patients and family members are difficult to bear, so that the popularization of the biological treatment in the field of cancer treatment is influenced.

Due to the above limitations, research and development of natural antitumor drugs are receiving more and more attention. The natural anticancer medicine has important functions in inhibiting or killing tumor cell, regulating body's immunity, improving symptom and characteristic, reducing toxic side effect of radiotherapy and chemotherapy, and regulating tumor after disease. Thus, natural plant neotherapy will become the fifth mode following surgery, radiation therapy, chemotherapy, and biological therapy.

Araliaceae (araliaceae) panax (panax) plants, such as ginseng (p.ginseng), American ginseng (p.quinquefolius), panax notoginseng (p.notoginseng), panax japonicus (p.uaponicus), cucurbitaceae Gynostemma pentaphylla (Gynostemma orientalis thunb Mak) and the like are Chinese famous traditional medicinal plants, and the main effective components of the araliaceae (araliaceae) panax ginseng (panax x) plant are dammarane type tetracyclic triterpene ginsenoside series compounds. More than 60 prototype ginsenosides in araliaceae plants have been found, and can be divided into two main classes: 1) panaxadiol saponins (Ra, Rb1, Rb2, Rb3, Rc, Rd, etc.); 2) the panaxatriol ginsenoside (Re, Rf, Rg1, Rg2, Rh1, etc.) is composed of aglycone and sugar, is usually easily dissolved in water, and has the curative effect mainly comprising: immune regulation function, improving microcirculation function, regulating digestive function, enhancing memory and learning ability, anti-aging, tranquilization and the like, but does not show obvious anti-tumor activity.

The low-polarity rare ginsenoside is present only in wild ginseng, or processed ginseng and panax notoginseng products such as red ginseng, black ginseng and cooked panax notoginseng, is present only in ten-thousandth, is hardly soluble in water, is usually dissolved only in low-polarity organic solvents such as ethanol or ethyl acetate, is called low-polarity rare ginsenoside, and mainly comprises secondary metabolic derivatives (C-k, Rg3, Rh2, Rh1, aPPD, aPPT and the like) generated by degrading the original ginsenoside through glycosidic bonds, and converted derivatives (Rg5, Rk1, 3, Rk2, Delta (20-21) PPD, RK1, Rh4, Rk3, F4, F6, Delta (20-21) PPT, Delta (20-22) PPT and the like) with a multi-double bond structure formed by simultaneous desugarization and dehydration of side chains. The low-polarity rare ginsenoside not only has the original biological activity of the common prototype ginsenoside, but also shows brand new pharmaceutical activities such as anti-tumor, anti-virus and the like which the prototype saponin does not have, and has extremely high medicinal value and application prospect.

So far, although the rare metabolic ginsenosides Rg3 and Rh2 are produced in large scale in markets at home and abroad, no enterprise can simultaneously produce high-content (> 30-95%) rare metabolic ginsenosides and rare transformed ginsenosides in large scale, especially Rh3, Rk2, Rg5 and RK1 with high anticancer activity. Therefore, the research on the preparation methods of the two low-polarity rare ginsenosides has important significance for finding ingredients with stronger activity and developing new antitumor drugs.

Disclosure of Invention

The invention aims to solve the technical problem of providing a preparation method of rare ginsenoside quasi-monomer compound Rg5/Rk1 and quasi-monomer compound Rh3/Rk2, and on the basis, the invention also provides a purification preparation method of rare ginsenoside monomer quasi-monomer compound Rg5/Rk1 and quasi-monomer compound Rh3/Rk2 respectively.

The first aspect of the present invention provides a method for preparing the rare ginsenoside, which comprises: pulverizing the stem and leaf of the pyrolytic araliaceae plant into powder, putting the stem and leaf powder into a steam pressure cooker for cooking, then carrying out vacuum drying on the cooked stem and leaf powder to obtain dry cooked stem and leaf powder, soaking the dry cooked stem and leaf powder in 95% ethanol, then filtering, and concentrating the filtrate to obtain the extract of the stem and leaf powder of the pyrolytic araliaceae plant containing the low-polarity rare ginsenoside; sequentially adding ethanol, sodium hydroxide, benzoyl peroxide, and oxygen into stem and leaf extract of pyrolytic Araliaceae plant containing low-polarity rare ginsenoside, and heating reaction solution to obtain pyrolytic Araliaceae plant selected from Ginseng radix, radix Panacis Quinquefolii and Notoginseng radix;

in a preferred embodiment, the stem leaves are pulverized into powder by a pulverizer,

in a preferred embodiment, the steaming is carried out in a steam pressure cooker for 20 hours at the temperature of 120 ℃ and the pressure of 0.15MPa,

in a preferred embodiment, the steamed stem and leaf powder is dried in vacuum at 80 ℃ for 8 hours in an oven,

in a preferred embodiment, the step of concentrating the filtrate to obtain an ethanol extract of stem and leaf powder further comprises: recovering ethanol for reuse, soaking the residue for 5 times, mixing to obtain extract of stem and leaf,

in a preferred embodiment, the heating reaction is carried out in a four-mouth bottle, the four-mouth bottle is provided with a thermometer, an oxygen inlet pipe, a sealing rubber pipe connected to the top of the four-mouth bottle, a condensation pipe, the other end of the rubber pipe is connected with a funnel, the funnel is immersed in a beaker filled with liquid paraffin,

in a preferred embodiment, ethanol is added and then stirred for 10 minutes,

in a preferred embodiment, the heating reaction is heated until the ethanol is in a reflux state, the reaction is carried out for 36 hours, in a preferred embodiment, after the heating reaction is finished, the reaction liquid is concentrated to be in a paste shape by using a rotary evaporator, distilled water is added until solid precipitates are released, the solid precipitates are collected by filtering and washed by water, then the solid precipitates are dried and dehydrated, n-hexane is firstly used for extracting to remove low-polarity substances, then absolute ethyl alcohol is used for extracting, and the solvent is evaporated to dryness to obtain the rare ginsenoside.

In a preferable example, the prepared rare ginsenoside is added into an ethyl acetate solution for ultrasonic treatment, then n-hexane is added for shaking up and standing, and then filtration is carried out to obtain filter residue;

in a preferred embodiment, the ethyl acetate solution is added for 30min of ultrasonic treatment.

The second aspect of the invention also provides a method for purifying rare ginsenoside quasi-monomer compounds Rg5/Rk1 and Rh3/Rk2, which comprises the following steps: adding silica gel into first ethyl acetate and n-hexane solvent, stirring uniformly, loading into a column, wherein the volume ratio of ethyl acetate to n-hexane in the first ethyl acetate and n-hexane solvent is 1:1, mixing a concentrated solution obtained by combining filtrate with the silica gel, grinding the silica gel after sample mixing, loading into the column, flushing 3 retention volumes by using the first ethyl acetate and n-hexane solution, collecting Rh3 and Rk2 in the 2 nd and 3 rd retention volumes, flushing 3 retention volumes by using the second ethyl acetate and n-hexane solution in a ratio of 5:1, flushing 6 retention volumes altogether, and collecting Rg5 and Rk1 in the volume ratio of ethyl acetate to n-hexane in the second ethyl acetate and n-hexane solvent in the 5 th and 6 th retention volumes;

in a preferred embodiment, the silica gel is 200-300 mesh,

in a preferred embodiment, the specification of the silica gel column after column packing is 20cm long and 4cm wide,

in a preferred embodiment, the retention volume of the packed chromatographic column is 200mL,

in a preferred embodiment, silica gel is selected and added into a first ethyl acetate and n-hexane solvent, the mixture is stirred uniformly and loaded into a column, the volume ratio of ethyl acetate to n-hexane in the first ethyl acetate and n-hexane solvent is 1:1, the concentrated solution obtained by combining the filtrates and the silica gel are mixed, the silica gel after sample mixing is ground into fine powder and loaded into the column, the first ethyl acetate and n-hexane solution is firstly used for flushing 3 retention volumes, the 2 nd and 3 rd retention volumes are collected Rh3 and Rk2, the ethyl acetate to n-hexane solution is used for flushing 3 retention volumes in the middle, the ethyl acetate and n-hexane solution used in the middle is used for flushing 2:1, the second ethyl acetate and n-hexane solution are used for flushing 3 retention volumes in the middle, the volume ratio of ethyl acetate to n-hexane in the n-hexane solvent is 5:1, 9 retention volumes are flushed together, the 5 th and 6 th retention volumes are collected, delta (20-21) PPT/delta (20-22) PPT is collected, the 8 th and 9 th retention volumes collect Rg5 and Rk 1.

The terms used in the present invention have the following definitions, unless otherwise described:

the term "rare ginsenoside" as used herein means a compound containing predominantly one or more rare ginsenoside monomeric compounds Rg5, Rk1 and Rh3, Rk2 or rare ginsenoside quasi-monomeric compounds Rg5/Rk1 and Rh3/Rk 2.

The term "rare ginsenoside quasimonoid compound Rg5/Rk 1" as used herein means a rare ginsenoside monomer Rg5 having the following structural formula:

and a complex of one isomer of rare ginsenoside Rk1 having the following structural formula:

the term "rare ginsenoside quasimonoid compound Rh3/Rk 2" as used herein means rare ginsenoside monomer Rh3 having the following structural formula:

and a complex of one isomer of rare ginsenoside Rk2 having the following structural formula:

the invention provides a preparation method of a composition containing rare ginsenoside quasi-monomer compound Rg5/Rk1 and rare ginsenoside quasi-monomer compound Rh3/Rk2, compared with the existing method for preparing rare ginsenoside, the preparation method is simple and low in cost, meanwhile, on the basis, the invention also provides a method for preparing rare ginsenoside monomer Rg5/Rk1 and rare ginsenoside monomer Rh3/Rk2, the method can separate and prepare rare ginsenoside quasi-monomer compound Rg5/Rk1 and rare ginsenoside quasi-monomer compound Rh3/Rk2 in a large scale with low cost and high efficiency through a simple column chromatography separation technology, and lays a foundation for further developing related medicaments.

Drawings

Fig. 1 is a molecular structure schematic diagram of rare ginsenoside monomer Rg 5.

Fig. 2 is a molecular structure diagram of rare ginsenoside monomer Rk 1.

Fig. 3 is a molecular structure diagram of rare ginsenoside monomer Rh 3.

Fig. 4 is a schematic molecular structure diagram of rare ginsenoside monomer Rk 2.

Detailed Description

The main component of diol group ginsenoside in the araliaceae plant extract can generate new rare saponin components such as Rs4, Rs5, Rg5, Rk1 and the like after the glycosidic bond cleavage reaction and the side chain dehydration reaction in the steaming process, 2-4 glycosidic bonds are connected on the 3-position carbon (C) of the tetracyclic triterpene skeleton, and if the glycosidic bonds in the diol group ginsenoside components are further broken to form the rare saponins with stronger activity: it is difficult to use components such as Rg5/Rk1 (containing two glycosidic bonds), Rk2/Rh3 (containing only one glycosidic bond), and Δ (20-21) PPD/Δ (20-22) PPD (containing no glycosidic bond). Although the remaining glycosidic bond may be further cleaved by reaction under strong or weak acid (high temperature) conditions after steaming, many side reactions may occur, such as: the ring-closing reaction of the side chain, the addition reaction of the double bonds of the side chain and the like can only separate a small amount of rare saponins Rg5/Rk1, Rk2/Rh3 and delta (20-21) PPD/delta (20-22) PPD components, and the large-scale production preparation and the real application can not be realized; if the double oxidation reaction is carried out under the alkaline condition after the steaming, the side chain side reactions can be avoided, and the large-scale preparation of the rare saponin components can be realized. Meanwhile, the invention discovers that the oxidation degradation degree of the glycosidic bond can be effectively controlled by using alkalis with different alkaline strengths, such as sodium alkoxide, sodium hydroxide and the like, so that products Rg5/Rk1, Rk2/Rh3 containing only 1-2 glycosidic bonds or products delta (20-21) PPD/delta (20-22) PPD without glycosidic bonds are formed, and the invention is particularly beneficial to industrial production.

The invention provides a comprehensive preparation method of the rare ginsenoside composition, which comprises the following steps: sequentially adding ethanol, sodium hydroxide, benzoyl peroxide, and oxygen into the stem and leaf extract of the pyrolytic Araliaceae plant containing low-polarity rare ginsenoside, and heating the reaction solution to react to obtain the final product, wherein the pyrolytic Araliaceae plant containing low-polarity rare ginsenoside is preferably selected from wild ginseng, red ginseng, black ginseng, American ginseng and pseudo-ginseng;

in a preferred embodiment, the heating reaction is carried out in a four-mouth bottle, the four-mouth bottle is provided with a thermometer, an oxygen inlet pipe, a sealing rubber pipe connected to the top of the four-mouth bottle, a condensation pipe, the other end of the rubber pipe is connected with a funnel, the funnel is immersed in a beaker filled with liquid paraffin,

in a preferred embodiment, ethanol is added and then stirred for 10 minutes,

in a preferred embodiment, the heating reaction is heated until the ethanol is in a reflux state, the reaction is carried out for 36 hours, in a preferred embodiment, after the heating reaction is finished, the reaction liquid is concentrated to be in a paste shape by using a rotary evaporator, distilled water is added until solid precipitates are released, the solid precipitates are collected by filtering and washed by water, then the solid precipitates are dried and dehydrated, n-hexane is firstly used for extracting to remove low-polarity substances, then absolute ethyl alcohol is used for extracting, and the solvent is evaporated to dryness to obtain the rare ginsenoside.

In a preferable example, the prepared rare ginsenoside is added into an ethyl acetate solution for ultrasonic treatment, then n-hexane is added for shaking up and standing, and then filtration is carried out to obtain filter residue;

in a preferred embodiment, the ethyl acetate solution is added for 30min of ultrasonic treatment.

The second aspect of the invention also provides a method for purifying rare ginsenoside quasi-monomer compounds Rg5/Rk1 and Rh3/Rk2, which comprises the following steps: adding silica gel into first ethyl acetate and n-hexane solvent, stirring uniformly, loading into a column, wherein the volume ratio of ethyl acetate to n-hexane in the first ethyl acetate and n-hexane solvent is 1:1, mixing the concentrated solution obtained by combining the filtrates with the silica gel, grinding the silica gel after sample mixing into fine powder, loading into the column, flushing 3 retention volumes by using the first ethyl acetate and n-hexane solution, collecting Rh3 and Rk2 by using the 2 nd and 3 rd retention volumes, flushing 3 retention volumes by using the second ethyl acetate and n-hexane solution at the ratio of 5:1, flushing 6 retention volumes altogether, and collecting Rg5 and Rk1 by using the second ethyl acetate and n-hexane solvent at the volume ratio of 5:1 and the 5 th and 6 th retention volumes;

in a preferred embodiment, the silica gel is 200-300 mesh,

in a preferred embodiment, the specification of the silica gel column after column packing is 20cm long and 4cm wide,

in a preferred embodiment, the retention volume of the packed chromatographic column is 200mL,

in a preferred embodiment, silica gel is selected and added into a first ethyl acetate and n-hexane solvent, the mixture is stirred uniformly and loaded into a column, the volume ratio of ethyl acetate to n-hexane in the first ethyl acetate and n-hexane solvent is 1:1, the concentrated solution obtained by combining the filtrates and the silica gel are mixed, the silica gel after sample mixing is ground into fine powder and loaded into the column, the first ethyl acetate and n-hexane solution is firstly used for flushing 3 retention volumes, the 2 nd and 3 rd retention volumes are collected Rh3 and Rk2, the ethyl acetate to n-hexane solution is used for flushing 3 retention volumes in the middle, the ethyl acetate and n-hexane solution used in the middle is used for flushing 2:1, the second ethyl acetate and n-hexane solution are used for flushing 3 retention volumes in the middle, the volume ratio of ethyl acetate to n-hexane in the n-hexane solvent is 5:1, 9 retention volumes are flushed together, the 5 th and 6 th retention volumes are collected, delta (20-21) PPT/delta (20-22) PPT is collected, the 8 th and 9 th retention volumes collect Rg5 and Rk 1.

In a preferred embodiment of the first and second aspects of the present invention, the extract of the stem and leaf of the pyrolyzed plant containing rare ginsenoside with low polarity is prepared by the following method: pulverizing the pyrolyzed stem leaves of the plant of the Araliaceae family into powder, putting the stem leaf powder into a steam pressure cooker for cooking, then carrying out vacuum drying on the cooked stem leaf powder to obtain dry cooked stem leaf powder, soaking the dry cooked stem leaf powder in 95% ethanol, then filtering, and concentrating the filtrate to obtain the stem leaf powder extract;

in a preferred embodiment, the stem leaves are pulverized into powder by a pulverizer,

in a preferred embodiment, the cooking condition in the steam pressure cooker is 120 ℃ and 0.15MPa for 20 hours,

in a preferred embodiment, the steam cooked stem and leaf powder is put into an oven for 8 hours at 80 ℃ under the vacuum drying condition,

in a preferred embodiment, the step of concentrating the filtrate to obtain the ethanol extract of stem leaf powder further comprises: recovering ethanol for reuse, soaking the residue for 5 times, and mixing to obtain the extract of the stem and leaf.

Unless otherwise defined, the terms used herein have the ordinary meanings as commonly understood in the art to which this invention belongs.

The present invention is described below with reference to specific examples, which are intended to be illustrative only and are not to be construed as limiting the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Example 1 methods for preparing Low-polarity rare ginsenoside compositions and Low-polarity rare ginsenoside monomers Rh3, Rk2, Rg5 and Rk1 from stems and leaves of Panax notoginseng

This example provides a method for preparing a low-polarity rare ginsenoside monomer Rh3 having the following structural formula:

and a preparation method of a low-polarity rare ginsenoside monomer Rk2 with the following structural formula:

meanwhile, the embodiment also provides a low-polarity rare ginsenoside composition and a preparation method of a low-polarity rare ginsenoside monomer Rg5 with the following structural formula:

and a preparation method of a low-polarity rare ginsenoside monomer Rk1 with the following structural formula:

(1) extraction and preparation:

pulverizing stem and leaf of Notoginseng radix (10kg) from Yunnan province purchased from Chinese medicinal material wholesale market into powder by pulverizer, placing in medical steam pressure cooker, steaming at 120 deg.C under 0.15MPa for 20h, mixing Notoginseng radix stem and leaf powder with water to the greatest extent, vacuum drying in oven at 80 deg.C for 8h to obtain dried steamed stem and leaf powder, soaking with 5kg of 95% ethanol for two days, filtering, concentrating filtrate to obtain ethanol extract of steamed stem and leaf powder, recovering ethanol, soaking residue for 5 times, and mixing to obtain 650g of steamed stem and leaf extract of Notoginseng radix.

Adding 200 g of cooked notoginseng stem and leaf extract and 1500mL of ethanol into a 2L four-mouth bottle provided with a thermometer, an oxygen inlet pipe and a condenser pipe connected to the top of the bottle (the other end of the rubber pipe is connected with a funnel and is immersed into a beaker filled with liquid paraffin), stirring for 10 minutes, adding 200 g of sodium hydroxide, stirring until NaOH is completely dissolved, adding 10g of benzoyl peroxide, starting introducing oxygen, heating the reaction solution until the ethanol is in a reflux state, and reacting for 36 hours. After the reaction is finished, concentrating the reaction solution to be paste by using a rotary evaporator, adding 1500mL of distilled water, releasing solid precipitate, filtering and collecting solid, washing the solid with water until the pH value is about 7, drying the solid in a drying oven at 110 ℃ to remove water, extracting the solid by using n-hexane to remove low-polarity substances, extracting the solid by using 3x100mL absolute ethyl alcohol for three times, combining the ethanol solution, evaporating the solvent to dryness to obtain 90g of a rare ginsenoside mixture crude product, and refining the rare ginsenoside monomer by using the following silicon column chromatography technology for the crude product.

(2) The separation process flow comprises the following steps:

(3) operating process and process conditions

Extraction of rare ginsenoside crude product

The purpose is as follows: the effective substances are extracted by removing pigments by using solubility difference.

The method comprises the following steps: adding 20g of the rare ginsenoside crude product into 200mL of water-saturated ethyl acetate solution, performing ultrasonic treatment for 30min, filtering, adding 100mL of water-saturated ethyl acetate into filter residue, performing ultrasonic treatment for 30min, filtering, combining the water-saturated ethyl acetate solutions, adding 300mL of n-hexane, shaking uniformly, standing, filtering, combining the filtrates, concentrating and evaporating to dryness.

Silica gel column separation of Rh3/Rk2, Rg5/Rk1

The purpose is as follows: separating Rh3/Rk2 and Rg5/Rk1 with silica gel column and removing most of pigment

The method comprises the following steps: 90g of silica gel (200 meshes and 300 meshes) is added into 300mL of solution (ethyl acetate: n-hexane 1: 1) and stirred uniformly and filled into a column, and the specification of the silica gel column is 20cm long and 4cm wide. The concentrated solution was mixed with 15g of silica gel, and the mixed silica gel was finely ground and packed. The retention volume was approximately 200mL, and 3 retention volumes were first flushed with ethyl acetate/n-hexane 1:1 and then with ethyl acetate/n-hexane 5:1 for a total of 6 retention volumes. Collecting the 2 nd and 3 nd retention volumes and evaporating the solvent to dryness to obtain a crude Rh3/Rk2 product, and collecting the 5 th and 6 th retention volumes and evaporating the solvent to dryness to obtain a crude Rg5/Rk1 product.

The high pressure fast preparation of purified Rk1/Rg5 and Rk2/Rh 3.

(1) Experimental reagent: chromatographically pure acetonitrile (manufactured by concider), ultrapure water: Milli-Q homemade

(2) The instrument equipment comprises: a) full-automatic preparation of chromatograph: GILSON model: GX-281; b) HITACHI high performance liquid chromatograph (configuration: quaternary pump, UV detector, online degassing system, autosampler, column oven, AgilentOpenLab data processing system).

(3) The preparation conditions are as follows:

a chromatographic column: waters Symmetry C187 um 19 250

Mobile phase: 80% acetonitrile in water at an equal temperature of 15min

Wavelength: 203 nm; sample introduction amount: 500 mul; flow rate: 10 mL/min; column temperature: and (4) room temperature.

(4) Content determination liquid phase conditions

A chromatographic column: waters Symmetry C185 um 4.6.6 mm 150mm (or similar column)

Wavelength: 203 nm; sample introduction amount: 20 mu l of the mixture; flow rate: 1 mL/min; column temperature: at 30 ℃.

Gradient: phase A: water; phase B: acetonitrile

t/min	0	10	20	30	35	45	46	55
									B％	45	45	65	85	95	95	45	45

(5) Preparing a test solution:

rk1/Rg5 test solution: the crude product was dissolved in about 30mg of 3mL of methanol.

Rk2/Rh3 test solution: the crude product was dissolved in about 20mg of methanol in 2 mL.

1. Collecting: the peak emergence time of Rk1/Rg5 is about 6-8 min; the peak emergence time of Rk2/Rh3 is about 11-13 min.

2. And (3) drying: the collected liquid was prepared, distilled under reduced pressure to an organic phase free, and the sample was lyophilized.

3. And (3) determination: and (4) detecting the content of the freeze-dried sample by an area normalization method.

Example 2 preparation of Low-polarity rare ginsenoside composition and Low-polarity rare ginsenoside monomers Rh3, Rk2, Delta (20-21) PPT, Delta (20-22) PPT, Rg5 and Rk1 from Ginseng radix

Extraction and preparation:

pulverizing stem and leaf of Ginseng radix (1.0kg) from Jilin purchased from Chinese medicinal material wholesale market into powder with pulverizer, placing in medical steam pressure cooker, steaming at 120 deg.C under 0.15MPa for 20 hr (mixing stem and leaf powder of Ginseng radix with water is avoided as much as possible); then placing into oven, vacuum drying at 80 deg.C for 8 hr to obtain dried and steamed ginseng stem and leaf powder, soaking with 5kg 95% ethanol for two days, filtering, and concentrating the filtrate to obtain ethanol extract of steamed ginseng stem and leaf powder. And recovering ethanol, repeatedly using, soaking filter residue for 5 times, and mixing to obtain 650g of steamed ginseng stem leaf extract.

The separation, purification and crystallization method is slightly improved based on the embodiment 1, and specifically comprises the following steps:

the method comprises the following steps: 90g of silica gel (200 meshes and 300 meshes) is added into 300mL of solution (ethyl acetate: n-hexane 1: 1) and stirred uniformly and filled into a column, and the specification of the silica gel column is 20cm long and 4cm wide. The concentrated solution was mixed with 15g of silica gel, and the mixed silica gel was finely ground and packed. The retention volume is about 200ml, firstly, ethyl acetate and n-hexane are used for flushing 3 retention volumes 1:1, then ethyl acetate and n-hexane are used for flushing 3 retention volumes 2:1, and then ethyl acetate and n-hexane are used for flushing 3 retention volumes 5:1 to obtain 9 retention volumes. Collecting the 2 nd and 3 nd retention volumes and evaporating the solvent to dryness to obtain a crude Rh3/Rk2 product, collecting the 5 th and 6 th retention volumes and evaporating the solvent to dryness to obtain a crude Delta (20-21) PPT/Delta (20-22) PPT product, collecting the 8 th and 9 th retention volumes and evaporating the solvent to dryness to obtain a crude Rg5/Rk1 product.

Comparative example 1: in the heated or slightly acidic environment, the total ginsenoside part in Ginseng radix or Notoginseng radix is subjected to sugar-reducing hydrolysis to generate secondary ginsenoside

(1) Pretreatment of raw materials: washing radix Panacis Quinquefolii with water to remove impurities such as silt, pulverizing, and sieving with 40 mesh sieve;

(2) steam explosion treatment: wetting ginseng powder with 10% acetic acid to make the water content of the ginseng powder be 35%, placing the ginseng powder in a steam explosion tank, introducing steam until the pressure in the steam explosion tank is 1.5MPa, and performing steam explosion treatment for 60 minutes;

(3) a step of biological complex enzyme treatment, which is to adjust the water content of the steam-exploded raw materials to 40% by using water, add a biological complex enzyme preparation (containing 25% of cellulase, 25% of α -amylase, 10% of xylanase, 5% of laccase, 20% of protease, 10% of pectinase and 5% of mannase) according to the amount of 0.5% of the material ratio, uniformly mix, and hydrolyze for 5 hours at the conditions of pH5.5 and 30 ℃;

(4) drying and sterilizing: carrying out reduced pressure drying and microwave sterilization on the reaction system obtained by the treatment in the step (3);

(5) and (3) detection: sampling ginseng powder raw materials, carrying out batch inspection, and detecting the contents of ginsenoside (including monomer ginsenoside Rg3 and Rh2), panaxan and ginseng protein in a sample;

(6) packaging: packaging the ginseng powder qualified by inspection according to 25kg per barrel, and sealing and storing.

However, these compounds need to react under higher temperature and stronger acidic condition, and the side chain cyclization of the product is easy to occur to form panaxadiol and panaxatriol type secondary ginsenoside, and other side reactions also occur, so that it is difficult to prepare the ginseng secondary saponins with stronger pharmacological activity on a large scale (Rk1/Rg5, Rk3/Rh4 and Rk2/Rh 3).

On the other hand, the preparation of the secondary ginsenoside (Rg3, Rh1 and Rh2) can also be carried out by an alkaline hydrolysis method, and the panaxatriol ginsenoside can obtain Rh1 and aPPT; the diol group of ginsenoside can obtain Rg3, Rh2 and aPPD.

Comparative example 2

Dissolving 10g of NaOH in 100mL of glycol solution, adding 10g of total saponins of American ginseng stems and leaves, heating and hydrolyzing for 1 hour under stirring, controlling the temperature at 190 ℃, cooling to room temperature, adding 50 times of water for dilution, extracting with ethyl acetate, recovering ethyl acetate to obtain 7g of product, and verifying by TLC that the product contains ginsenoside Rg3, Rh2, Rh1 and Rg2, protopanaxadiol and protopanaxatriol.

Comparative example 3

18kg of pseudo-ginseng (specification: countless heads, purchased from Yunnan) is crushed into powder (100 meshes and 200 meshes), is soaked in 30kg of 95 percent ethanol for two days, is filtered, is concentrated into a pseudo-ginseng ethanol extract, is recycled and repeatedly used for soaking filter residue for six times, is finally accumulated to obtain 3.37kg of the pseudo-ginseng ethanol extract, is dissolved in water, is extracted for three times by petroleum ether, is extracted from a water phase for four times by n-butyl alcohol, and is concentrated by the n-butyl alcohol layer to obtain 1.78kg of the n-butyl alcohol extract of the total saponins of pseudo-ginseng.

Dissolving 100g of total saponin extract in 1300mL of n-butanol, heating, stirring, adding 132.6g (1.56mol, concentration: 1.2mol/L) of sodium ethoxide (chemical purity: 80%), introducing oxygen, reacting at 90 deg.C for 65 hr, and finishing the reaction. Cooling the reaction solution to room temperature, washing with n-butanol saturated water, concentrating the n-butanol layer, dissolving with water, extracting with ethyl acetate, washing the ethyl acetate layer with water, and drying. Concentrating, purifying by silica gel column chromatography [ gradient elution with 1-5% methanol/chloroform solution ] to obtain 6g of protopanaxadiol (A2), wherein the purity is 97.93% by HPLC; protopanaxatriol (A3)11g, purity 99.95% by HPLC. The physicochemical data determined for compound (a3) correspond to literature values: chen Yingjie et al, Journal of Shenyang College of pharmacy, 1987, 4(4), 282-289.

The physicochemical data determined for compound (a3) are as follows:

1H NMR(300MHz，CDCl3)：δ5.18(d，1H)，4.14(m，1H)，3.6(m，1H)，3.2(s，1H)，2.15-1.61(m，20H)，1.58-1.17(m，12H)，1.05(s，5H)，1.01(s，3H)，0.95(d，8H)。

13C NMR(300MHz，CDCl3)：132.1，125.2，78.8，74.7，71.0，68.9，61.4，53.7，51.7，49.8，47.7，47.2，41.2，39.6，39.4，39.1，34.8，31.4，31.3，31.2，27.3，27.2，26.7，26.0，22.6，18.0，17.5，17.4，17.1，15.8。

the above methods using alkaline hydrolysis have the common disadvantages of low yield of the target product and high reaction temperature, and particularly, the steric configuration of C-20 position is not changed when ginsenoside is subjected to alkaline hydrolysis, and-OH at C-20 position and-H at C-21 or C-22 position are not dehydrated to form a double bond between C-20 and C-21 or C-22, so that novel secondary ginsenosides (Rk1/Rg5, Rk3/Rh4, Rk2/Rh3, etc.) cannot be directly prepared by alkaline hydrolysis.

Claims (4)

1. A method for preparing rare ginsenoside, which comprises the following steps: pulverizing stems and leaves of Araliaceae plants into powder, steaming the stem and leaf powder in a steam pressure cooker, vacuum drying the steamed stem and leaf powder to obtain dried steamed stem and leaf powder, soaking the dried steamed stem and leaf powder in 95% ethanol, filtering, and concentrating the filtrate to obtain the extract of the stem and leaf powder of the pyrolyzed Araliaceae plants containing the low-polarity rare ginsenoside; sequentially adding ethanol, sodium hydroxide and benzoyl peroxide into a stem and leaf extract of a pyrolyzed araliaceae plant containing low-polarity rare ginsenoside, introducing oxygen, heating a reaction solution for reaction, concentrating the reaction solution into a paste by using a rotary evaporator after the heating reaction is finished, adding distilled water until solid precipitate is released, filtering, collecting the solid precipitate, washing with water, drying to remove water, extracting by using n-hexane to remove low-polarity substance impurities, extracting by using absolute ethyl alcohol, and evaporating a solvent to obtain a crude product of the rare ginsenoside; wherein the plant of Araliaceae is selected from Ginseng radix, radix Panacis Quinquefolii and Notoginseng radix;

optionally, the stem leaves are pulverized into powder by a pulverizer,

optionally, the steaming is carried out for 20 hours in a steam pressure cooker at the temperature of 120 ℃ and the pressure of 0.15MPa,

optionally, vacuum drying the steamed stem and leaf powder at 80 deg.C for 8 hr,

optionally, the step of concentrating the filtrate to obtain the stem leaf powder extract further comprises: recovering ethanol for reuse, soaking the residue for 5 times, mixing to obtain the extract of stem and leaf powder,

optionally, the heating reaction is carried out in a four-mouth bottle, the four-mouth bottle is provided with a thermometer, an oxygen inlet pipe, a sealing rubber pipe and a condensation pipe, the top of the four-mouth bottle is connected with a funnel, the other end of the rubber pipe is connected with a funnel, the funnel is immersed in a beaker filled with liquid paraffin,

optionally, stirring for 10 minutes after adding ethanol,

optionally, the heating reaction is heated until the ethanol is in a reflux state, and the reaction is carried out for 36 hours.

2. The preparation method according to claim 1, characterized in that the prepared crude rare ginsenoside product is added into an ethyl acetate solution for ultrasonic treatment, then n-hexane is added for shaking up and standing, then filtration is carried out to obtain filter residue and filtrate, then the filtrate is combined and concentrated and evaporated to dryness to obtain a concentrated solution;

optionally, the addition of the ethyl acetate solution is sonicated for 30 min.

3. The preparation method according to claim 2, characterized in that silica gel is added with a first ethyl acetate and a normal hexane solvent, stirred uniformly and loaded into a column, the volume ratio of ethyl acetate to normal hexane in the first ethyl acetate and normal hexane solvent is 1:1, a concentrated solution obtained by combining filtrates is mixed with silica gel, the silica gel after sample mixing is ground and loaded into the column, the first ethyl acetate and normal hexane solution is firstly used for flushing 3 retention volumes, the 2 nd and 3 rd retention volumes are collected with Rh3 and Rk2, the second ethyl acetate and normal hexane solution is then used for flushing 3 retention volumes with 5:1, and the total flushing is carried out for 6 retention volumes, the volume ratio of ethyl acetate to ethyl hexane in the normal hexane solvent is 5:1, and the 5 th and 6 th retention volumes are collected with 5 and Rk 1;

optionally, the silica gel is 200-300 meshes,

optionally, the specification of the silica gel column after column filling is 20cm long and 4cm wide,

optionally, the retention volume of the packed column is 200 mL.

4. The preparation method according to claim 3, characterized in that silica gel is added with a first ethyl acetate and n-hexane solvent, stirred uniformly and loaded into a column, the volume ratio of ethyl acetate to n-hexane in the first ethyl acetate and n-hexane solvent is 1:1, the concentrated solution obtained by combining the filtrates is mixed with silica gel, the silica gel after sample mixing is ground and loaded into the column, the first ethyl acetate and n-hexane solution is used for washing 3 retention volumes, the 2 nd and 3 rd retention volumes are used for collecting Rh3 and Rk2, and ethyl acetate is used for the middle: flushing 3 retention volumes by using n-hexane at a ratio of 2:1, wherein the volume ratio of ethyl acetate to the n-hexane in the n-hexane solvent used in the middle is 2:1, flushing 3 retention volumes by using a second ethyl acetate and an n-hexane solution at a ratio of 5:1, wherein the volume ratio of the second ethyl acetate to the n-hexane in the n-hexane solvent is 5:1, flushing 9 retention volumes altogether, and collecting Rg5 and Rk1 at the 8 th retention volume and the 9 th retention volume;

optionally, the silica gel is 200-300 meshes,

optionally, the specification of the silica gel column after column filling is 20cm long and 4cm wide,

optionally, the retention volume of the packed column is 200 mL.

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