WO2019178980A1

WO2019178980A1 - Method for preparing gynostemma pentaphylla makino saponin lvi, xlvi, l, li, damulin a or damulin b

Info

Publication number: WO2019178980A1
Application number: PCT/CN2018/093947
Authority: WO
Inventors: 娄志春
Original assignee: 娄志春
Priority date: 2018-03-23
Filing date: 2018-07-01
Publication date: 2019-09-26

Abstract

A method for preparing gynostemma pentaphylla makino saponin LVI, XLVI, L, LI, Damulin A or Damulin B. In said method, Gynostemma cardiospermum Cogn.ex Oliv is used as a raw material to prepare a high content of target product by means of extraction and purification. The raw material is easy to obtain, and the cost is low. In addition, using cunninghamella blakesleeana AS3.153 strain to respectively convert gynostemma pentaphylla makino saponin LI and L to Damulin A and Damulin B by means of a microbial conversion method is also disclosed, and said method has a high conversion rate.

Description

Method for preparing Gynostemma saponin LVI, XLVI, L, LI, Da Mulin A or Da Mulin B

Technical field

The invention belongs to the field of biomedicine, and particularly relates to a method for preparing Gynostemma saponins LVI, XLVI, L, LI, Damulin A or Damulin B.

Background technique

Gynostemma pentaphyllum (Thunb.) Makino, also known as nectar, genus, and roots, is a perennial herbaceous vine of the genus Gynostemma. It is mainly grown in the Qinling Mountains and south of the Yangtze River in China. It was first published in the Ming Dynasty's "Rescue Materia Medica" for wild vegetables. In the "Compendium of Materia Medica", it began to be used as the "blackberry". "China Zhuang Pharmacy": "Tongfu three ways and two ways, clearing away heat and detoxification, cough and phlegm. For chronic bronchitis, viral hepatitis, pyelonephritis, gastroenteritis, diarrhea, hypertension, arteriosclerosis, hyperlipidemia , hemorrhoids swollen poison, snake bites." The main chemical components of Gynostemma pentaphyllum are saponins and polysaccharides, and also contain a small amount of flavonoids, terpenoids, organic acids, proteins, vitamins, fats, fibers and other components as well as trace elements such as zinc, copper, magnesium, iron, manganese and selenium. Gynostemma pentaphyllum has anti-tumor, hypoglycemic, anti-aging, anti-fibrosis and other pharmacological effects. Its main active ingredient, Gynostemma saponin, has no side effects, so it has great application prospects in the development of health foods and new drugs. It is a kind of New dual-use plant resources.

There are many saponins in Gynostemma pentaphyllum. The more studied and active ones are Gynostemma saponins LVI, XLVI, L, LI, Damulin A and Damulin B. Among them, Gynostemma saponins LVI, XLVI content is higher, the other four are very low content of rare saponins (the conversion products of Gynostemma saponins LVI, XLVI).

At present, Gynostemma saponins LVI and XLVI are extracted and purified from Gynostemma pentaphyllum. Gynostemma saponins L, LI, Damulin A and Damulin B are extracted and purified by heat-treated Gynostemma pentaphyllum. (Reference: Distiller by UPLC-MS of Four dammaranetype saponins from heat-processed Gynostemma pentaphyllum; Bioscience, Biotechnology, and Biochemistry, 2014; Vol. 78, No. 2, 311-316). There are two shortcomings in the preparation of Gynostemma pentaphyllum LVI, XLVI, L, LI, Damulin A and Damulin B from Gynostemma pentaphyllum:

First, the price of Gynostemma pentaphyllum is relatively high and the cost is high. Since the National Science and Technology Commission listed Gynostemma in the "Spark Program" in 1986 as the first place in the "precious Chinese herbal medicines" to be developed, the market price of Gynostemma pentaphyllum has increased significantly.

Secondly, the unheated Gynostemma pentaphyllum L. saponins L, LI, Da Mulin A and Da Mulin B are very low, which is difficult to separate and purify; heating is complicated, and after heating, Gynostemma pentaphyllum L, LI, Damulin A and Damulin B The content is not increased synchronously, and the heating temperature and time are difficult to control (this phenomenon can also be reflected in the above cited documents).

There are 13 species and 2 varieties of 2 subgenera in the genus Gynostemma. The 2 subgenera are the genus Gynostemma and the subgenus Glycyrrhiza. The above-mentioned Gynostemma pentaphyllum (Thunb.) Makino is one of the genus Gynostemma (Document: Gynostemma) Classification systems and distribution, Journal of Plant Taxonomy, 1995). In the research invention, Gynostemma basically contains Gynostemma saponins, and the species are basically the same, but the content of specific Gynostemma saponins in different plants is quite different. Gynostemma cardiospermum Cogn.ex Oliv, which is produced in western Hubei, southwestern Shaanxi, and Sichuan, belongs to the genus Amaranthus. Its main feature is that the fruit is a capsule. When mature, the tip is cracked along the abdominal suture, with three surviving ridges. Style, other characteristics are basically consistent with Gynostemma pentaphyllum (Thunb.) Makino. It has not been reported that the content of public Gynostemma saponins LVI, XLVI, L, LI, Damulin A, B in Gynostemma cardiospermum Cogn.ex Oliv is significantly higher than that of Gynostemma pentaphyllum (Thunb.) Makino.

Summary of the invention

The present invention is directed to a method of preparing Gynostemma saponin LVI, XLVI, L, LI, Damulin A or Damulin B.

The above objectives are achieved by the following technical solutions:

The preparation method of Gynostemma saponins LVI, XLVI, L, LI, Da Mulin A and Da Mulin B includes the following steps:

In step S1, the leaves of Gynostemma cardiospermum Cogn.ex Oliv are collected, dried in the air, extracted with an aqueous solution of ethanol and refluxed, filtered through a gauze, and the filtrate is concentrated to an alcohol-free taste;

In step S2, the extract concentrated to the non-alcoholic taste is applied to the D101 macroporous adsorption resin column, the resin diameter ratio is 1:10, and the mixed resin accounts for 1/10 of the total amount of the resin, and the wet packing is performed, and the resin is mixed. The sample was firstly eluted with 10BV of 30% ethanol at a flow rate of 10BV/h, and then eluted with 10BV of 85% ethanol at a flow rate of 10BV/h. The 3-10BV eluate was collected and concentrated to an alcohol-free atmosphere. Obtaining an aqueous solution of total sapon

Step S3, adjusting the total saponin aqueous solution to pH value 6.0, adding chitosan/attapulgite clay composite, stirring at room temperature, standing, filtering, collecting filtrate, adjusting pH to neutral, and lyophilizing to obtain refined total saponin powder ;

In step S4, the purified total saponin powder is applied to a normal phase silica gel column, the diameter ratio of the silica gel is 1:10, and the mixed silica gel accounts for 1/10 of the total amount of the silica gel. The dry method is packed in a column, and the silica gel is loaded; The ratio of 20:1:2, 10:1:2, 5:1:2, 2:1:2 of dichloromethane/methanol/acetone mixed solvent was eluted at a rate of 12 BV/h, and eluted 10 BV, 6BV, 5BV, 3BV;

Step S5, respectively, collecting 20:1:2 dichloromethane/methanol/acetone eluting 7-8BV, 9-10BV eluent and concentrating and drying to obtain Damulin B and Damulin A; respectively collecting 10:1:2 dichloride Methane/methanol/acetone elution 3-4BV, 5-6BV eluate was concentrated and dried to obtain Gynostemma saponin L, Gynostemma saponin LI; 5:1:2 dichloromethane/methanol/acetone elution 4-5BV elution The solution was concentrated and dried to obtain Gynostemma saponin XLVI; the 2:1:2 dichloromethane/methanol/acetone eluted 2-3 BV eluate was concentrated and dried to obtain Gynostemma saponin LVI.

Preferably, the chitosan/attapulgite clay composite is prepared by dissolving 1 g of chitosan in 100 mL of a 1% aqueous solution of acetic acid to obtain a chitosan sol, and adding 8 g of attapulgite clay to the sol. After stirring at room temperature for 48 hours, the upper layer solution was poured out after standing for 2 hours, and the residue was dried at 50 ° C, ground and dried.

Preferably, the concentration of the aqueous ethanol solution in step S1 is 75% by volume.

Preferably, the solid to liquid ratio extracted in step S1 is 1:20.

Preferably, 1 g of the total saponin aqueous solution is added to 50 g of the chitosan/attapulgite clay composite in step S3.

Preferably, the chitosan/attapulgite clay composite is added in step S3, stirred for 24 hours, allowed to stand for 2 hours, and then filtered.

Preferably, step S3 adjusts the pH with hydrochloric acid and aqueous ammonia.

A method for preparing Damulin A by microbial transformation method comprises the following steps:

Step S1, preparation of spores and seeds: inoculation of S. glabrata AS3.153 strain on slant medium, and incubating at 28 ° C for 7 days at constant temperature to obtain a culture with vigorous mycelium growth and spore-rich; taking appropriate amount of spores The seed bottle was shaken and cultured at 28 ° C for 24 h to obtain a seed culture;

Step S2, microbial transformation: the seed culture was transferred to a conversion flask at a seeding rate of 5%, and pre-cultured at 28 ° C for 24 h, and then a sterile aqueous solution of Gynostemma pentaphyllum LI was added to make the final concentration of Gynostemma pentaphyllum LI 0.2 g/L, 28 ° C. Shake culture for 96h;

Step S3, the conversion product is collected: the supernatant of the transformation culture is taken, extracted with ethyl acetate, and the ethyl acetate extract is collected and concentrated to obtain a crude conversion product;

Step S4, the crude product of the conversion product is applied to a normal phase silica gel column, the diameter ratio of the silica gel is 1:10, and the sampled silica gel accounts for 1/10 of the total amount of the silica gel. The dry method is packed in a column, and the silica gel is loaded; For 40:1:2, 20:1:2 dichloromethane/methanol/acetone mixed solvent, elute at a rate of 12 BV/h, elute 8BV, 10BV, respectively; collect 20:1:2 dichloromethane/methanol /acetic acid eluted 9-10 BV eluate was concentrated and dried to obtain Da Mulin A.

A method for preparing Damulin B by microbial transformation method comprises the following steps:

Step S2, microbial transformation: the seed culture was transferred to a conversion flask at a seeding rate of 5%, and pre-cultured at 28 ° C for 24 h, and then a sterile aqueous solution of Gynostemma pentaphyllum L was added to make the final concentration of Gynostemma pentaphyllum L 0.2 g/L, 28 ° C. Shake culture for 96h;

Step S3, the conversion product was collected: the supernatant of the transformation culture was taken, extracted with ethyl acetate, and the ethyl acetate extract was collected and concentrated to obtain a crude product.

Step S4, the crude product of the conversion product is applied to a normal phase silica gel column, the diameter ratio of the silica gel is 1:10, and the sampled silica gel accounts for 1/10 of the total amount of the silica gel. The dry method is packed in a column, and the silica gel is loaded; The 40:1:2, 20:1:2 dichloromethane/methanol/acetone mixed solvent was eluted at a rate of 12 BV/h, eluting 8BV, 8BV, respectively; collecting 20:1:2 dichloromethane/methanol /acetic acid eluted 7-8BV eluate was concentrated and dried to obtain Da Mulin B.

Technical advantages of the invention:

1. The present invention uses Gynostemma cardiospermum Cogn.ex Oliv, which belongs to the same genus but different subgenus, to replace Gynostemma pentaphyllum (Thunb.) Makino as a raw material, and can extract and purify and obtain a large amount of Gynostemma saponins LVI, XLVI, L, LI, and up to The contents of Mulin A and Da Mulin B, Gynostemma saponins LVI, XLVI, L, LI, Da Mulin A and Da Mulin B in Gynostemma cardiospermum Cogn.ex Oliv leaves are in Gynostemma pentaphyllum (Thunb.) Makino leaves, respectively. The content is 10-50 times; and the Gynostemma cardiospermum Cogn.ex Oliv is not commercialized, and the cost is negligible.

2. The step S3 of the present invention uses the chitosan/attapulgite clay composite to refine the total saponin aqueous solution, thereby effectively reducing the impurities in the total saponin, greatly simplifying the subsequent normal phase silica gel column chromatography process, and the product purity is high.

3. The present invention finds that when the transformation raw material is Gynostemma pentaphyllum LI, the strain A. sinensis AS3.153 will be converted into Damulin A; when the transformation material is Gynostemma saponin L, the short thorn The .153 strain will convert it to Da Mulin B. The configuration of the C20 hydroxyl group of Gynostemma pentaphyllum LI and Gynostemma saponin L determines the dehydration selectivity of the strain A. sinensis AS3.153, and the strain of C. sinensis AS3.153 is selective for dehydration at this position. It is possible to selectively prepare Damulin A or Damulin B, and the utilization rate of raw materials is high.

DRAWINGS

Figure 1 is a high performance liquid chromatographic spectrum of the aqueous solution of total saponin in step S2;

2 is a high performance liquid chromatographic spectrum of the refined total saponin powder in step S3;

3 is a high performance liquid chromatographic spectrum of Da Mulin B purified by silica gel column chromatography;

4 is a high performance liquid chromatographic spectrum of Damulin A purified by silica gel column chromatography;

Figure 5 is a high performance liquid chromatographic spectrum of Gynostemma saponin L purified by silica gel column chromatography;

6 is a high performance liquid chromatographic spectrum of Gynostemma pentaphyllum LI purified by silica gel column chromatography;

Figure 7 is a high performance liquid chromatographic spectrum of Gynostemma saponin XLVI purified by silica gel column chromatography;

Figure 8 is a high performance liquid chromatographic spectrum of Gynostemma saponin LVI purified by silica gel column chromatography;

Figure 9 is the HPLC analysis results of the crude product of the conversion product of Example 2. The upper figure shows the chromatogram of the reference solution (the synthesis of Damulin A and B monomers, the concentration is 0.1 mg/mL), and the figure below shows the chromatogram of the test solution. Figure (preparation of crude product 0.5mg dissolved in an appropriate amount of methanol);

Figure 10 is the HPLC analysis results of the crude product of the conversion product of Example 3. The upper figure shows the chromatogram of the reference solution (the synthesis of Damulin A and B monomers, the concentration is 0.1 mg/mL), and the figure below shows the chromatogram of the test solution. Figure (prepared from the conversion product 0.5 mg in an appropriate amount of methanol).

detailed description

The substantial content of the present invention will be specifically described below with reference to the embodiments, but those skilled in the art should understand that the scope of the present invention should not be limited to the specific embodiments.

Example 1: Preparation of Gynostemma saponins LVI, XLVI, L, LI, Damulin A and Damulin B

First, the experimental materials

The leaves of Gynostemma cardiospermum Cogn.ex Oliv were collected from Fangxian County, Shiyan City, Hubei Province. The leaves of Gynostemma pentaphyllum (Thunb.) Makino were collected from Suining, Shaoyang, Hunan Province.

The chitosan/attapulgite clay composite preparation method comprises the following steps: 1 g of chitosan is dissolved in 100 mL of a 1% aqueous solution of acetic acid to obtain a chitosan sol, and 8 g of attapulgite clay is added to the sol, and stirred at room temperature for 48 hours. After standing for 2 h, the upper layer solution was poured out, and the residue was dried at 50 ° C, ground and dried.

Different concentrations of ethanol refer to aqueous solutions of different volume percentages of ethanol.

Second, the experimental method

1. Preparation of Gynostemma saponins LVI, XLVI, L, LI, Damulin A and Damulin B

Including the following steps:

In step S1, the leaves of Gynostemma cardiospermum Cogn.ex Oliv are collected, dried in the air, and extracted with a 75% by volume aqueous solution of ethanol, and the solid-liquid ratio is 1:20, and extracted 3 times, each time for 2 hours, gauze. Filtration, and the combined filtrates were concentrated to an alcohol-free taste;

In step S3, the total saponin aqueous solution is adjusted to pH 6.0 with hydrochloric acid, chitosan/attapulgite clay composite is added, and 1 g of total saponin aqueous solution is added to 50 g of chitosan/attapulgite clay composite, stirred for 24 hours, and allowed to stand for 2 h. After filtration, the filtrate is collected, the pH is adjusted to neutral by ammonia water, and lyophilized to obtain a refined total saponin powder;

In step S4, the purified total saponin powder is applied to a normal phase silica gel column, the diameter ratio of the silica gel is 1:10, and the mixed silica gel accounts for 1/10 of the total amount of the silica gel. The dry method is packed in a column, and the silica gel is loaded (refined total saponin). After the powder is dissolved in methanol, it is uniformly mixed with the mixed silica gel, and the mass ratio of the total saponin powder to the sampled silica gel is 1:1); the volume ratio is 20:1:2, 10:1:2, 5:1 in sequence. : 2, 2:1:2 of dichloromethane / methanol / acetone mixed solvent eluted at a rate of 12BV / h, eluting 10BV, 6BV, 5BV, 3BV;

2, high performance liquid chromatography analysis conditions

Chromatograph: LC-20ADXR high pressure pump; SPD-M20A diode array UV visible light detector; CTO-20AC column oven; CBM-20A system controller; SIL-20ACXR autosampler;

Column: Agilent ZORBAX Extend-C18 (250 mm x 4.6 mm, 5 μm);

Mobile phase A phase: water (including pentadyltetrahydrofuran);

Mobile phase B phase: acetonitrile (including pentadyltetrahydrofuran);

Elution procedure: 0-3 min, 20% phase B; 3-15 min, 20%→40% phase B; 15-30 min, 40%→80% phase B;

Flow rate: 1.0 mL/min;

Column temperature: 30 ° C;

Detection wavelength: 203 nm;

Injection volume: 10 μL.

Third, the experimental results

The aqueous solution of the total saponin in step S2 is diluted 5 times as a high-performance liquid chromatography injection solution, and the total saponin powder is purified in step S3 to dissolve into a 0.5 mg/mL solution as a high-performance liquid chromatography injection solution. 1 is a high performance liquid chromatographic spectrum of the aqueous solution of total saponin in step S2, and FIG. 2 is a high performance liquid chromatographic spectrum of the purified total saponin powder in step S3. It can be seen from Fig. 1 and Fig. 2 that the total saponin aqueous solution obtained in step S2 has a complicated composition, and the impurity component in the refined total saponin is greatly reduced in step S3, indicating that the chitosan/attapulgite clay composite can effectively adsorb and remove total saponins at pH 6.0. The impurity component in the aqueous solution. After removing impurities, it helps to reduce the difficulty of subsequent silica gel column chromatography.

The purified total saponins are separated and purified by silica gel column chromatography in steps S4 and S5 to obtain Gynostemma saponins LVI, XLVI, L, LI, Damulin A and Damulin B monomers. Fig. 3 is a high performance liquid chromatographic spectrum of Damulin B purified by silica gel column chromatography, and Fig. 4 is a high performance liquid chromatographic spectrum of Damulin A purified by silica gel column chromatography, and Fig. 5 is purified by silica gel column chromatography. High performance liquid chromatographic analysis of Gynostemma pentaphyllum L, Figure 6 is a high performance liquid chromatographic analysis of Gynostemma pentaphyllum LI purified by silica gel column chromatography, and Figure 7 is a high performance liquid phase of Gynostemma saponin XLVI purified by silica gel column chromatography. Chromatographic analysis of the spectrum, Figure 8 is a high performance liquid chromatographic analysis of Gynostemma saponin LVI purified by silica gel column chromatography. As can be seen from Fig. 3-8, the purity of Gynostemma saponins LVI, XLVI, L, LI, Da Mulin A and Da Mulin B is very high, above 95%. Such high purity Gynostemma saponins LVI, XLVI, L, LI, Damulin A and Damulin B can be prepared by one silica gel column chromatography, mainly benefiting from the purification of chitosan/attapulgite clay complex in step S3. Remove impurities.

Example 2: Preparation of Da Mulin A by Gynostemma saponin LI

Step S1, spore and seed preparation: the strain of C. sinensis AS3.153 preserved at 4 ° C was inoculated on a slant medium, and cultured at 28 ° C for 7 days at constant temperature to obtain a culture with vigorous hyphae growth and spore-rich; A spore-containing mycelium was taken by a sterile inoculating loop, and only the spores were placed in a seed bottle (50 mL medium in a 250 mL flask), and cultured at 28 ° C for 24 hours to obtain a seed culture;

Step S2, microbial transformation: the seed culture is transferred to a conversion flask (100 mL triangular flask containing 20 mL medium) at a 5% inoculation amount, and pre-cultured at 28 ° C for 24 h, and then a sterile aqueous solution of Gynostemma pentaphyllum LI is added to make Gynostemma saponin LI end. The concentration was 0.2 g/L, and the culture was shaken at 28 ° C for 96 h;

Step S3, the conversion product is collected: the supernatant of the transformation culture is taken, extracted three times with an equal volume of ethyl acetate, and the ethyl acetate extract is collected, and concentrated to dryness to obtain a crude conversion product;

In step S4, the crude product of the conversion product is applied to a normal phase silica gel column, the diameter ratio of the silica gel is 1:10, and the sampled silica gel accounts for 1/10 of the total amount of the silica gel. The dry column is packed and the silica gel is loaded (the crude product for conversion is used) After the methanol is dissolved, it is uniformly mixed with the mixed silica gel, and the mass ratio of the crude product to the sampled silica gel is 1:1); the volume ratio is 40:1:2, 20:1:2 in dichloromethane/methanol/ The acetone mixed solvent was eluted at a rate of 12 BV/h, and 8 BV and 10 BV were respectively eluted; the eluate of 9:10 BV was collected by 20:1:2 dichloromethane/methanol/acetone and concentrated to obtain Da Mulin A;

The slant medium is potato sucrose agar medium, the composition is: sucrose 1.5g, agar 2.0g, potato 20g and distilled water 100ml; seed bottle and transformation bottle medium composition: glucose 2.0g, yeast extract 0.5g, peptone 0.5 g, sodium chloride 0.5g, dipotassium hydrogen phosphate 0.5g and distilled water 100ml, the pH was adjusted to 8.3. After sterilization at 115 ° C for 30 min, use.

After conversion, 1 mole of Gynostemma saponin LI can be transformed, isolated and purified to obtain 0.92 mol of Da Mulin A, the conversion rate is above 90%, and the purity of Da Mulin A is 98.2%.

Example 3: Preparation of Da Mulin B by Gynostemma

Step S1, spore and seed preparation: the strain of C. sinensis AS3.153 stored at 4 ° C was inoculated on the slant medium, and cultured at 28 ° C for 7 days at constant temperature to obtain a culture with strong mycelium growth and spore rich; A spore-containing mycelium was taken by a sterile inoculating loop, and only the spores were placed in a seed bottle (50 mL medium in a 250 mL flask), and cultured at 28 ° C for 24 hours to obtain a seed culture;

Step S2, microbial transformation: the seed culture is transferred to a conversion flask (100 mL triangular flask containing 20 mL medium) at a 5% inoculation amount, and pre-cultured at 28 ° C for 24 h, and then a sterile aqueous solution of Gynostemma pentaphyllum L is added to make Gynostemma saponin L The concentration was 0.2 g/L, and the culture was shaken at 28 ° C for 96 h;

In step S4, the crude product of the conversion product is applied to a normal phase silica gel column, the diameter ratio of the silica gel is 1:10, and the sampled silica gel accounts for 1/10 of the total amount of the silica gel. The dry column is packed and the silica gel is loaded (the crude product for conversion is used) After the methanol is dissolved, it is uniformly mixed with the mixed silica gel, and the mass ratio of the crude product to the sampled silica gel is 1:1); the volume ratio is 40:1:2, 20:1:2 in dichloromethane/methanol/ The acetone mixed solvent was eluted at a rate of 12 BV/h, and 8 BV and 8 BV were respectively eluted; the eluate of 7-8 BV was eluted by collecting 20:1:2 dichloromethane/methanol/acetone and concentrated to obtain Da Mulin B;

After conversion, 1 mole of Gynostemma saponin L can be converted, isolated and purified to obtain 0.91 mol of Da Mulin B, the conversion rate is above 90%, and the purity of Da Mulin B is 98.5%.

Example 4: Comparison of the contents of A and B in Damulin in the crude products of Examples 2 and 3 by high performance liquid chromatography

High performance liquid chromatography analysis conditions:

Column: Agilent ZORBAX Extend-C18 (250 mm x 4.6 mm, 5 μm);

Mobile phase A phase: water (including pentadyltetrahydrofuran);

Mobile phase B phase: acetonitrile (including pentadyltetrahydrofuran);

Flow rate: 1.0 mL/min;

Column temperature: 30 ° C;

Detection wavelength: 203 nm;

Injection volume: 10 μL.

Figure 9 is the HPLC analysis results of the crude product of the conversion product of Example 2. The upper figure shows the chromatogram of the reference solution (the synthesis of Damulin A and B monomers, the concentration is 0.1 mg/mL), and the figure below shows the chromatogram of the test solution. Figure (prepared from the conversion product 0.5 mg in an appropriate amount of methanol). Figure 10 is the HPLC analysis results of the crude product of the conversion product of Example 3. The upper figure shows the chromatogram of the reference solution (the synthesis of Damulin A and B monomers, the concentration is 0.1 mg/mL), and the figure below shows the chromatogram of the test solution. Figure (prepared from the conversion product 0.5 mg in an appropriate amount of methanol).

It can be seen from Fig. 9 and Fig. 10 that when the transformation raw material is Gynostemma saponin LI, the strain A. sinensis AS3.153 will be basically converted into Damulin A; when the transformation raw material is Gynostemma saponin L, the short thorn is small. The C. sinensis AS3.153 strain will convert it to Damulin B. The configuration of the C20 hydroxyl group of Gynostemma pentaphyllum LI and Gynostemma saponin L determines the dehydration selectivity of the strain A. sinensis AS3.153, and the strain of C. sinensis AS3.153 is selective for dehydration at this position. It is possible to selectively prepare Damulin A or Damulin B, and the utilization rate of raw materials is high.

Claims

A method for preparing Gynostemma saponin LVI, XLVI, L, LI, Damulin A or Damulin B, characterized in that it comprises the following steps:

In step S1, the leaves of Gynostemma cardiospermum Cogn.ex Oliv are collected, dried in the air, extracted with an aqueous solution of ethanol and refluxed, filtered through a gauze, and the filtrate is concentrated to an alcohol-free taste;

In step S2, the extract concentrated to the non-alcoholic taste is applied to the D101 macroporous adsorption resin column, the resin diameter ratio is 1:10, and the mixed resin accounts for 1/10 of the total amount of the resin, and the wet packing is performed, and the resin is mixed. The sample was firstly eluted with 10BV of 30% ethanol at a flow rate of 10BV/h, and then eluted with 10BV of 85% ethanol at a flow rate of 10BV/h. The 3-10BV eluate was collected and concentrated to an alcohol-free atmosphere. Obtaining an aqueous solution of total sapon

Step S3, adjusting the total saponin aqueous solution to pH value 6.0, adding chitosan/attapulgite clay composite, stirring at room temperature, standing, filtering, collecting filtrate, adjusting pH to neutral, and lyophilizing to obtain refined total saponin powder ;

In step S4, the purified total saponin powder is applied to a normal phase silica gel column, the diameter ratio of the silica gel is 1:10, and the mixed silica gel accounts for 1/10 of the total amount of the silica gel. The dry method is packed in a column, and the silica gel is loaded; The ratio of 20:1:2, 10:1:2, 5:1:2, 2:1:2 of dichloromethane/methanol/acetone mixed solvent was eluted at a rate of 12 BV/h, and eluted 10 BV, 6BV, 5BV, 3BV;

Step S5, respectively, collecting 20:1:2 dichloromethane/methanol/acetone eluting 7-8BV, 9-10BV eluent and concentrating and drying to obtain Damulin B and Damulin A; respectively collecting 10:1:2 dichloride Methane/methanol/acetone elution 3-4BV, 5-6BV eluate was concentrated and dried to obtain Gynostemma saponin L, Gynostemma saponin LI; 5:1:2 dichloromethane/methanol/acetone elution 4-5BV elution The solution was concentrated and dried to obtain Gynostemma saponin XLVI; the 2:1:2 dichloromethane/methanol/acetone eluted 2-3 BV eluate was concentrated and dried to obtain Gynostemma saponin LVI.
The method according to claim 1, wherein the chitosan/attapulgite clay composite is prepared by dissolving 1 g of chitosan in 100 mL of a 1% aqueous solution of acetic acid to obtain a chitosan sol. 8 g of attapulgite clay was added to the sol, and the mixture was stirred at room temperature for 48 hours. After standing for 2 hours, the upper layer solution was poured out, and the residue was dried at 50 ° C, ground and dried.
The method according to claim 1, wherein the concentration of the aqueous ethanol solution in step S1 is 75% by volume.
The method according to claim 3, wherein the solid-liquid ratio extracted in step S1 is 1:20.
The method according to claim 1, characterized in that in step S3, 1 L of total saponin aqueous solution is added to 50 g of chitosan/attapulgite clay composite.
The method according to claim 5, wherein the chitosan/attapulgite clay composite is added in step S3, stirred for 24 hours, allowed to stand for 2 hours, and then filtered.
The method of claim 1 wherein step S3 adjusts the pH with hydrochloric acid and aqueous ammonia.
A method for preparing Damulin A by microbial transformation method, comprising the steps of:

Step S1, preparation of spores and seeds: inoculation of S. glabrata AS3.153 strain on slant medium, and incubating at 28 ° C for 7 days at constant temperature to obtain a culture with vigorous mycelium growth and spore-rich; taking appropriate amount of spores The seed bottle was shaken and cultured at 28 ° C for 24 h to obtain a seed culture;

Step S2, microbial transformation: the seed culture was transferred to a conversion flask at a seeding rate of 5%, and pre-cultured at 28 ° C for 24 h, and then a sterile aqueous solution of Gynostemma pentaphyllum LI was added to make the final concentration of Gynostemma pentaphyllum LI 0.2 g/L, 28 ° C. Shake culture for 96h;

Step S3, the conversion product was collected: the supernatant of the transformation culture was taken, extracted with ethyl acetate, and the ethyl acetate extract was collected and concentrated to obtain a crude product.

Step S4, the crude product of the conversion product is applied to a normal phase silica gel column, the diameter ratio of the silica gel is 1:10, and the sampled silica gel accounts for 1/10 of the total amount of the silica gel. The dry method is packed in a column, and the silica gel is loaded; For 40:1:2, 20:1:2 dichloromethane/methanol/acetone mixed solvent, elute at a rate of 12 BV/h, elute 8BV, 10BV, respectively; collect 20:1:2 dichloromethane/methanol /acetic acid eluted 9-10 BV eluate was concentrated and dried to obtain Da Mulin A.
A method for preparing Damulin B by a microbial transformation method, comprising the steps of:

Step S1, preparation of spores and seeds: inoculation of S. glabrata AS3.153 strain on slant medium, and incubating at 28 ° C for 7 days at constant temperature to obtain a culture with vigorous mycelium growth and spore-rich; taking appropriate amount of spores The seed bottle was shaken and cultured at 28 ° C for 24 h to obtain a seed culture;

Step S2, microbial transformation: the seed culture was transferred to a conversion flask at a seeding rate of 5%, and pre-cultured at 28 ° C for 24 h, and then a sterile aqueous solution of Gynostemma pentaphyllum L was added to make the final concentration of Gynostemma pentaphyllum L 0.2 g/L, 28 ° C. Shake culture for 96h;

Step S3, the conversion product was collected: the supernatant of the transformation culture was taken, extracted with ethyl acetate, and the ethyl acetate extract was collected and concentrated to obtain a crude product.

Step S4, the crude product of the conversion product is applied to a normal phase silica gel column, the diameter ratio of the silica gel is 1:10, and the sampled silica gel accounts for 1/10 of the total amount of the silica gel. The dry method is packed in a column, and the silica gel is loaded; The 40:1:2, 20:1:2 dichloromethane/methanol/acetone mixed solvent was eluted at a rate of 12 BV/h, eluting 8BV, 8BV, respectively; collecting 20:1:2 dichloromethane/methanol /acetic acid eluted 7-8BV eluate was concentrated and dried to obtain Da Mulin B.