CN105111267B - A kind of preparation method of ganoderol B - Google Patents
A kind of preparation method of ganoderol B Download PDFInfo
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- CN105111267B CN105111267B CN201510650324.4A CN201510650324A CN105111267B CN 105111267 B CN105111267 B CN 105111267B CN 201510650324 A CN201510650324 A CN 201510650324A CN 105111267 B CN105111267 B CN 105111267B
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Abstract
The invention discloses a kind of preparation method of ganoderol B, the method comprising the steps of；(1) extract and be enriched with ganoderol B；(2) high-speed countercurrent chromatography separation ganoderol B.A kind of preparation method of ganoderol B of the present invention is simple to operate, pollutes small, cost is low, and yield, the purity of ganoderol B are high.
Description
Technical field
The invention belongs to field of natural medicinal chemistry, more particularly to a kind of preparation method of ganoderol B.
Background technology
Ganoderol B is triterpene compound, CAS 104700-96-1, molecular formula C30H48O2, molecular weight 440.71, molecule
Structural formula is as follows：
Ganoderol B is one of the secondary metabolite of ganoderma lucidum, the triterpene substance with multiple biological activities, has been reported
Related pharmacology document shows that ganoderol B can suppress melanin generation, reduce freckle, be used as the inhibitor of cholesterol biosynthesis, α-Portugal
Polyglycoside enzyme inhibitor, suppresses prostate gland cancer cell and increases.
The preparation method of existing ganoderol B is confined to column chromatography and chromatography, but these methods have time-consuming, preparation
Small, the problem of there is dead absorption is measured, exploitation is adapted to industrialization, and the preparation method of large-scale production ganoderol B accords with the demands of the market,
There is realistic meaning.
The content of the invention
It is an object of the invention to provide a kind of preparation method of ganoderol B, this method comprises the following steps：
(1) extract and be enriched with ganoderol B：Ganoderma lucidum mycelium is added into 90-99% alcohol solution dippings to extract, extract solution is dense
Contracting；Concentrate is extracted with petroleum ether, chloroform, ethyl acetate respectively successively；Acetic acid ethyl acetate extract is concentrated under reduced pressure,
Through purification on normal-phase silica gel (100-200 mesh) column chromatography, chloroform:Methanol=98:2-9:1 elutes successively, wherein chloroform:Methanol=98:2 wash
It is de- to obtain the component containing ganoderol B；
(2) high-speed countercurrent chromatography separation ganoderol B：The component containing ganoderol B is carried out with high-speed counter-current chromatograph
Separation；Then detected with high-efficient liquid phase chromatogram technique analysis, collect, concentrate, drying, isolated ganoderol B.
Specifically, a kind of preparation method of ganoderol B of the invention, comprises the following steps：
(1) extract and be enriched with ganoderol B：Ganoderma lucidum mycelium is added to the 85-99% ethanol solution normal temperature of 5-10 times of volume
Soak extraction 3-5 times, it is each 40-60 hours, then extract solution is concentrated；Concentrate is successively respectively with the oil of 1-5 times of volume
Ether, chloroform, ethyl acetate carry out concussion extraction 3-5 times, stand 2-8 hours every time, acetic acid ethyl acetate extract is merged, and depressurize dense
Contracting, through purification on normal-phase silica gel (100-200 mesh) column chromatography, uses chloroform:Methanol=98:2-9:1 elutes successively, is examined through thin layer chromatography
Survey, merge after similar components, with ganoderol B standard control, find chloroform:Methanol=98:2 afford containing ganoderol B
Component；
(2) high-speed countercurrent chromatography separation ganoderol B：Dicyandiamide solution is made up of n-hexane, ethyl acetate, methanol, water, is pressed
11-15：22-26：16-22：8-11 volume ratios are mixed, and take phase to fix phase, and lower phase is mobile phase, the lower phased soln of extract
And sample introduction, rotating speed 700-900rmp, flow velocity is 1-3ml/min, and every 10~20mL is collected as a cut；Then efficient liquid phase is used
Chromatography analysis detect each test tube collection liquid composition, and are compared with ganoderol B standard items, collect, concentrate, do by heterogeneity
It is dry, isolated ganoderol B.
High-speed countercurrent chromatography is a kind of continuous liquid-liquid distribution color of Solid Free carrier compared with traditional chromatographic technique
Spectral technology, has many advantages using this kind of technology, be not required to solid support make carrier, can in the absence of Irreversible Adsorption, sample
With whole recovery, disengaging time is short and separative efficiency is high.In addition, the technical operation simple and flexible, is carried out, to shakiness at room temperature
Fixed sample can also have good separating effect, and solvent for use can also be reclaimed, and be that a kind of economical and effective obtains triterpene in ganoderma lucidum
The method of class compound.
Ganoderol B is prepared using the method for the present invention, with the time is short, simple to operate, cost is low and yield is high, purity is high
The advantages of.
Brief description of the drawings：
Fig. 1 separates spectrogram for the high-speed counter-current of embodiment 1
Fig. 2 is the efficient liquid phase spectrogram of the ganoderol B obtained in embodiment 1
Fig. 3 separates spectrogram for the high-speed counter-current of embodiment 2
Fig. 4 is the efficient liquid phase spectrogram of the ganoderol B obtained in embodiment 2
Embodiment
Strains tested：No. 1 Ganoderma lucidum of Shanghai agriculture ganoderma lucidum is by China Committee for Culture Collection of Microorganisms's agriculture
Industry microorganism center Shanghai edible mushroom branch center is provided, strain number：Ganoderma lucidum G0119.
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 items：Purchased from national standard standard of physical sample message center.
Purification on normal-phase silica gel (100-200 mesh)：Haiyang Chemical Plant, Qingdao；
Thin-layer chromatography (TLC) plate (HSGF254)：Yantai Hui You silica gel development corporation, Ltd.；
Remaining reagent is ordinary commercial products.
Embodiment 1：
The ganoderma lucidum mycelium being incubated on PDA plate is cut into pea size, is inoculated in equipped with 200mL BD culture mediums
In 500mL triangular flasks, primary seed solution is made in shaking table culture (150r/min, 26 DEG C) 7d, then primary seed solution by inoculation
Amount 20% is connected in the 1000mL triangular flasks equipped with 400mL BD culture mediums, and shaking table culture (150r/min, 26 DEG C) 3d is made two
Level seed liquor, for doing two benches fermentation hereafter.Secondary seed solution is inoculated into 400ml BD culture mediums by inoculum concentration 10%
1000mL triangular flasks in, in shaking table culture (150r/min, 26 DEG C) 3d, this is the concussion and cultivate of first stage.By concussion training
Foster zymotic fluid is removed, and is rested on 26 DEG C, is cultivated 14d under dark condition, and this is the quiescent culture of second stage.
The ganoderma lucidum mycelium 8.9kg of above-mentioned two benches fermentation is taken, 95% ethanol water soak at room temperature of 7 times of volumes is added
Extract 3 times, 48 hours every time, then extract solution is concentrated under reduced pressure；Concentrate is separately added into the petroleum ether of 2 times of volumes, chlorine successively
Imitative, ethyl acetate concussion is extracted each 3 times, stands 2 hours every time.Acetic acid ethyl acetate extract is merged, solid is concentrated under reduced pressure to give
Extract 50g, crosses silicagel column (100-200 mesh), uses chloroform:Methanol=98:2-9:1 elutes successively, is examined through thin layer chromatography
Survey, merge after similar components, with ganoderol B standard control, find chloroform:Methanol=98:2 afford containing ganoderol B
Component, concentration be weighed as 4g.N-hexane, ethyl acetate, methanol, water are taken, by 14:24:22:10 volume ratio mixing, fully
After layering, take phase to fill high speed adverse current chromatogram pipe and fix phase, open and turn main frame, rotating speed 850rpm/min, while being pumped into lower phase
Mobile phase is done, flow velocity is 2ml/min, uses mobile phase sample dissolution 330mg, by sample introduction valve injection, UV-detector is monitored on-line,
A cut is collected as per 10ml.HPLC analysis detection separation situations, and compared with ganoderol B standard items, collect and merge spirit
Sesame alcohol B cut, is concentrated and dried, and the ganoderol B 15.2mg that purity is 94% is measured with efficient liquid phase area normalization method.
Embodiment 2：
Ganoderma lucidum mycelium (the fermentation process be the same as Example 1) 8.9kg for taking two benches to ferment, adds 95% second of 7 times of volumes
Alcohol solution soak at room temperature is extracted 3 times, 48 hours every time, then extract solution is concentrated under reduced pressure；Concentrate is separately added into 2 times successively
The petroleum ether of volume, chloroform, ethyl acetate concussion are extracted each 3 times, stand 2 hours every time.Acetic acid ethyl acetate extract is merged, subtracted
Pressure is concentrated to give solid phase extraction thing 50g, crosses silicagel column (100-200 mesh), uses chloroform:Methanol=98:2-9:1 elutes successively, warp
Thin layer chromatography is detected, is merged after similar components, with ganoderol B standard control, finds chloroform:Methanol=98:2 elute
To the component containing ganoderol B, concentration is weighed as 4g.N-hexane, ethyl acetate, methanol, water are taken, by 12:24:20:10 body
Product is than mixing, fully after layering, takes phase to fill high speed adverse current chromatogram pipe and fixes phase, opens and turn main frame, rotating speed 850rpm/min,
Being pumped into lower phase does mobile phase simultaneously, and flow velocity is 2ml/min, uses mobile phase sample dissolution 150mg, by sample introduction valve injection, ultraviolet inspection
Device on-line monitoring is surveyed, a cut is collected as per 10ml.HPLC analysis detection separation situations, and opposed with ganoderol B standard items
Than collecting the cut for merging ganoderol B, being concentrated and dried, the ganoderma lucidum that purity is 94% is measured with efficient liquid phase area normalization method
Alcohol B 7.3mg.
Claims (1)
1. a kind of preparation method of ganoderol B, it is characterised in that this method comprises the following steps：
(1) extract and be enriched with ganoderol B：Ganoderma lucidum mycelium is added to the 90-99% ethanol solution soak at room temperature of 5-10 times of volume
Extract 3-5 times, it is each 40-60 hours, then extract solution is concentrated；Concentrate is successively respectively with the petroleum ether of 1-5 times of volume, chlorine
Imitative, ethyl acetate carries out concussion extraction 3-5 times, stands 2-8 hours every time, acetic acid ethyl acetate extract is merged, is concentrated under reduced pressure, and passes through
Purification on normal-phase silica gel 100-200 mesh column chromatographies, use chloroform:Methanol=98:2-9:1 elutes successively, is detected through thin layer chromatography, merges
After similar components, with ganoderol B standard control, chloroform is found:Methanol=98:2 afford the component containing ganoderol B；
(2) high-speed countercurrent chromatography separation ganoderol B：Dicyandiamide solution is made up of n-hexane, ethyl acetate, methanol, water, by 11-
15：22-26：16-22：8-11 volume ratios are mixed, and take phase to fix phase, and lower phase is mobile phase, extract with lower phased soln simultaneously
Sample introduction, rotating speed 700-900rmp, flow velocity is 1-3ml/min, and every 10~20mL is collected as a cut；Then high-efficient liquid phase color is used
Chromatography detects each test tube collection liquid composition, and is compared with ganoderol B standard items, collects, concentrates, does by heterogeneity
It is dry, isolated ganoderol B.
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