CN105111267A - Preparation method of ganoderol B - Google Patents

Abstract

The invention discloses a preparation method of ganoderol B. The method comprises steps as follows: (1), extracting and enriching ganoderol B; (2), separating ganoderol B with a high-speed countercurrent chromatography method. The preparation method of ganoderol B is simple to operate, low-pollution and low in cost, and the yield and the purity of ganoderol are high.

Description

A kind of preparation method of ganoderol B

Technical field

The invention belongs to field of natural medicinal chemistry, relate to a kind of preparation method of ganoderol B specifically.

Background technology

Ganoderol B is triterpene compound, CAS 104700-96-1, molecular formula C ₃₀h ₄₈o ₂, molecular weight 440.71, molecular structural formula is as follows:

Ganoderol B is the secondary metabolite of glossy ganoderma, there is one of multiple bioactive triterpene substance, the relevant pharmacology document display reported, ganoderol B energy check melanin generates, reduce freckle, as the inhibitor of cholesterol biosynthesis, alpha-glucosidase inhibitor, suppresses prostate cancer cell to increase.

The preparation method of existing ganoderol B is confined to column chromatography and chromatography, but these methods have consuming time, and preparation amount is little, and there is the problem of dead absorption, exploitation is applicable to industrialization, and the preparation method of scale operation ganoderol B accords with the demands of the market, and has realistic meaning.

Summary of the invention

The object of the present invention is to provide a kind of preparation method of ganoderol B, the method comprises the steps:

(1) extraction and enrichment ganoderol B: Ganoderma mycelium is added 90-99% alcohol solution dipping and extract, extracting solution concentrates; Concentrated solution extracts by sherwood oil, chloroform, ethyl acetate successively respectively; Acetic acid ethyl acetate extract is carried out concentrating under reduced pressure, through purification on normal-phase silica gel (100-200 order) column chromatography, chloroform: methyl alcohol=98:2-9:1 wash-out successively, wherein chloroform: methyl alcohol=98:2 wash-out obtains the component containing ganoderol B;

(2) high-speed countercurrent chromatography is separated ganoderol B: be separated the component containing ganoderol B with high-speed counter-current chromatograph; Then detect with high-efficient liquid phase chromatogram technique analysis, collect, concentrated, dry, be separated and obtain ganoderol B.

Specifically, the preparation method of a kind of ganoderol B of the present invention, comprises the steps:

(1) extraction and enrichment ganoderol B: 85-99% ethanolic soln soak at room temperature Ganoderma mycelium being added 5-10 times of volume extracts 3-5 time, and each 40-60 hour, then concentrates extracting solution; Concentrated solution carries out concussion extraction 3-5 time by the sherwood oil of 1-5 times of volume, chloroform, ethyl acetate successively respectively, each standing 2-8 hour, acetic acid ethyl acetate extract is merged, concentrating under reduced pressure, through purification on normal-phase silica gel (100-200 order) column chromatography, with chloroform: methyl alcohol=98:2-9:1 wash-out successively, detect through thin layer chromatography, merge similar components, after ganoderol B standard control, find chloroform: methyl alcohol=98:2 wash-out obtains the component containing ganoderol B;

(2) high-speed countercurrent chromatography is separated ganoderol B: solvent system is made up of normal hexane, ethyl acetate, methyl alcohol, water, mix by 11-15:22-26:16-22:8-11 volume ratio, get the phase that fixes mutually, lower is moving phase mutually, extract is with lower phased soln and sample introduction, rotating speed 700-900rmp, flow velocity is 1-3ml/min, and every 10 ~ 20mL is collected as a cut; Then detect each test tube with high-efficient liquid phase chromatogram technique analysis and collect liquid composition, and compare with ganoderol B standard substance, collect by heterogeneity, concentrated, dry, be separated and obtain ganoderol B.

High-speed countercurrent chromatography is a kind of continuous liquid luquid partition chromatography technology of Solid Free carrier compared with traditional chromatographic technique, utilize this kind of technology to have a lot of advantage, do not need solid support to make carrier, there is not irreversible adsorption, sample can all reclaim, disengaging time is short and separation efficiency is high.In addition, this technological operation simple and flexible, at room temperature carries out, and also can have good separating effect to the sample of instability, solvent for use also can reclaim, and is a kind of method that economical and effective obtains triterpene compound in glossy ganoderma.

Adopt method of the present invention to prepare ganoderol B, have that the time is short, simple to operate, cost is low and yield is high, purity advantages of higher.

Accompanying drawing illustrates:

Fig. 1 is that the high-speed counter-current of embodiment 1 is separated spectrogram

Fig. 2 is the high performance liquid phase spectrogram of the ganoderol B obtained in embodiment 1

Fig. 3 is that the high-speed counter-current of embodiment 2 is separated spectrogram

Fig. 4 is the high performance liquid phase spectrogram of the ganoderol B obtained in embodiment 2

Embodiment

Strains tested: Shanghai agriculture glossy ganoderma No. 1 Ganodermalucidum is provided by China Committee for Culture Collection of Microorganisms's agricultural edible mushrooms branch center, Shanghai, microorganism center, strain number: GanodermalucidumG0119.

High-speed counter-current chromatograph: purchased from Shanghai Tauto Biotechnology Co., Ltd., INSTRUMENT MODEL is: TBE-300B.

High performance liquid chromatograph: Waters Products, INSTRUMENT MODEL is 2695-717-600E.

Ganoderol B standard substance: purchased from national standard standard of physical sample message center.

Purification on normal-phase silica gel (100-200 order): Haiyang Chemical Plant, Qingdao;

Thin-layer chromatography (TLC) plate (HSGF254): Yantai Hui You silica gel development corporation, Ltd.;

All the other reagent are common commercially available prod.

Embodiment 1:

The Ganoderma mycelium be incubated on PDA flat board is cut into pea size, be inoculated in and be equipped with in the 500mL triangular flask of 200mLBD substratum, (150r/min cultivated by shaking table, 26 DEG C) 7d, obtained primary seed solution, then primary seed solution is received by inoculum size 20% and be equipped with in the 1000mL triangular flask of 400mLBD substratum, (150r/min cultivated by shaking table, 26 DEG C) 3d obtains secondary seed solution, is used for the two benches fermentation done after this.Be inoculated in the 1000mL triangular flask of 400mlBD substratum by secondary seed solution by inoculum size 10%, cultivate (150r/min, 26 DEG C) 3d at shaking table, this is the concussion cultivation of first stage.Taken off by the fermented liquid that concussion is cultivated, rest on 26 DEG C, cultivate 14d under dark condition, this is the quiescent culture of subordinate phase.

Get the Ganoderma mycelium 8.9kg of above-mentioned two benches fermentation, the 95% aqueous ethanolic solution soak at room temperature adding 7 times of volumes extracts 3 times, each 48 hours, then by extracting solution concentrating under reduced pressure; Concentrated solution adds the sherwood oil of 2 times of volumes, chloroform, each 3 times of ethyl acetate concussion extraction successively respectively, leaves standstill 2 hours at every turn.Acetic acid ethyl acetate extract is merged, concentrating under reduced pressure obtains solid phase extraction thing 50g, cross silicagel column (100-200 order), with chloroform: methyl alcohol=98:2-9:1 wash-out successively, detect through thin layer chromatography, merge similar components, after ganoderol B standard control, find chloroform: methyl alcohol=98:2 wash-out obtains the component containing ganoderol B, concentrate and be weighed as 4g.Get normal hexane, ethyl acetate, methyl alcohol, water, mix by the volume ratio of 14:24:22:10, after abundant layering, get and fill high speed adverse current chromatogram pipe mutually and to fix phase, open and turn main frame, rotating speed 850rpm/min, pump into simultaneously and do moving phase mutually down, flow velocity is 2ml/min, with moving phase sample dissolution 330mg, by sampling valve sample introduction, UV-detector on-line monitoring, every 10ml is collected as a cut.HPLC analyzing and testing separation case, and compare with ganoderol B standard substance, collect the cut merging ganoderol B, concentrate drying, records by high performance liquid phase area normalization method the ganoderol B 15.2mg that purity is 94%.

Embodiment 2:

Get Ganoderma mycelium (fermentation process is with the embodiment 1) 8.9kg of two benches fermentation, the 95% aqueous ethanolic solution soak at room temperature adding 7 times of volumes extracts 3 times, each 48 hours, then by extracting solution concentrating under reduced pressure; Concentrated solution adds the sherwood oil of 2 times of volumes, chloroform, each 3 times of ethyl acetate concussion extraction successively respectively, leaves standstill 2 hours at every turn.Acetic acid ethyl acetate extract is merged, concentrating under reduced pressure obtains solid phase extraction thing 50g, cross silicagel column (100-200 order), with chloroform: methyl alcohol=98:2-9:1 wash-out successively, detect through thin layer chromatography, merge similar components, after ganoderol B standard control, find chloroform: methyl alcohol=98:2 wash-out obtains the component containing ganoderol B, concentrate and be weighed as 4g.Get normal hexane, ethyl acetate, methyl alcohol, water, mix by the volume ratio of 12:24:20:10, after abundant layering, get and fill high speed adverse current chromatogram pipe mutually and to fix phase, open and turn main frame, rotating speed 850rpm/min, pump into simultaneously and do moving phase mutually down, flow velocity is 2ml/min, with moving phase sample dissolution 150mg, by sampling valve sample introduction, UV-detector on-line monitoring, every 10ml is collected as a cut.HPLC analyzing and testing separation case, and compare with ganoderol B standard substance, collect the cut merging ganoderol B, concentrate drying, records by high performance liquid phase area normalization method the ganoderol B 7.3mg that purity is 94%.

Claims (2)

1. a preparation method for ganoderol B, is characterized in that the method comprises the steps:

(1) extraction and enrichment ganoderol B: Ganoderma mycelium is added 90-99% alcohol solution dipping and extract, extracting solution concentrates; Concentrated solution extracts by sherwood oil, chloroform, ethyl acetate successively respectively; Acetic acid ethyl acetate extract is carried out concentrating under reduced pressure, through purification on normal-phase silica gel 100-200 order column chromatography, chloroform: methyl alcohol=98:2-9:1 wash-out successively, wherein chloroform: methyl alcohol=98:2 wash-out obtains the component containing ganoderol B;

(2) high-speed countercurrent chromatography is separated ganoderol B: be separated the component containing ganoderol B with high-speed counter-current chromatograph; Then detect with high-efficient liquid phase chromatogram technique analysis, collect, concentrated, dry, be separated and obtain ganoderol B.

2. the preparation method of ganoderic acid T according to claim 1, is characterized in that the method comprises the steps:

(1) extraction and enrichment ganoderol B: 85-99% ethanolic soln soak at room temperature Ganoderma mycelium being added 5-10 times of volume extracts 3-5 time, and each 40-60 hour, then concentrates extracting solution; Concentrated solution carries out concussion extraction 3-5 time by the sherwood oil of 1-5 times of volume, chloroform, ethyl acetate successively respectively, each standing 2-8 hour, acetic acid ethyl acetate extract is merged, concentrating under reduced pressure, through purification on normal-phase silica gel 100-200 order column chromatography, with chloroform: methyl alcohol=98:2-9:1 wash-out successively, detect through thin layer chromatography, merge similar components, after ganoderol B standard control, find chloroform: methyl alcohol=98:2 wash-out obtains the component containing ganoderol B;

(2) high-speed countercurrent chromatography is separated ganoderol B: solvent system is made up of normal hexane, ethyl acetate, methyl alcohol, water, mix by 11-15:22-26:16-22:8-11 volume ratio, get the phase that fixes mutually, lower is moving phase mutually, extract is with lower phased soln and sample introduction, rotating speed 700-900rmp, flow velocity is 1-3ml/min, and every 10 ~ 20mL is collected as a cut; Then detect each test tube with high-efficient liquid phase chromatogram technique analysis and collect liquid composition, and compare with ganoderol B standard substance, collect by heterogeneity, concentrated, dry, be separated and obtain ganoderol B.