CN103276041B - Method for extracting hopene from moelleriellaochracea - Google Patents
Method for extracting hopene from moelleriellaochracea Download PDFInfo
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- CN103276041B CN103276041B CN201310229308.9A CN201310229308A CN103276041B CN 103276041 B CN103276041 B CN 103276041B CN 201310229308 A CN201310229308 A CN 201310229308A CN 103276041 B CN103276041 B CN 103276041B
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- hopene
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Abstract
The invention discloses a method for extracting hopene from moelleriellaochracea. The method comprises the steps as follows: 1), activating bacterial strains; 2), performing primary culture; 3), performing secondary culture; 4), performing pressure reduction and suction filtration to obtain mycelium which is dried at the constant temperature; 5), grinding and sieving; 6), soaking ground mycelium by ethyl acetate; and 7), subjecting a leaching agent to rotating concentration, sample stirring, 200-300 meshes of silica-gel column chromatography, rotational steaming and concentration, eluting by trichloromethane, natural drying and recrystallization by methanol, and HREIMS (high resolution electron impact mass spectrometry) and NMR (nuclear magnetic resonance) spectrum analyses prove thatthe obtained material is hopene.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind ofly strangle from reddish brown little Mo the method for extracting hopene bacterium.
Background technology
Reddish brown little Mo strangle bacterium (
moelleriellaochracea) be the important member of entomogenous fungi, its metabolite has all represented huge potentiality to be exploited in biological control and the aspect such as medical: 1. not strangling bacterium can cause aleyrodid and the epiphytotics generation of shell class insect group, and this makes it can become effective biocontrol agent; 2. its meta-bolites have preferably antibacterial, kill plasmodium activity, and insect cell and tumour cell are had to stronger toxicity, almost non-toxic to mammalian cell.
In society now, people pay attention to curative effect further, particularly for the treatment of the major diseases such as cancer.Thereby the effective antitumor medicine of finding different sources receives much concern already.In the metabolite of entomogenous fungi, contain the multiple desinsections such as polypeptide, alkaloid, terpenoid, sterol, antitumor, anti-inflammatory, antibacterial material.Therefore,, to the abundant exploitation of entomogenous fungi, the development of mankind's medical level will be conducive to.
Summary of the invention
The object of the present invention is to provide and a kind ofly strangle from reddish brown little Mo the method for extracting hopene bacterium, to promote its pharmacological research and the development and application as lead compound.
For achieving the above object, the present invention adopts following technical scheme:
Strangling from reddish brown little Mo the method for extracting hopene bacterium comprises the following steps:
1) reddish brown little Mo is strangled to bacterium (Qiu Junzhi etc., reddish brown little Mo strangles bacterium in Chinese discovery. fungus journal, 2009.28 (1): 148-150) after activation, get in bacterium piece inoculation PDB substratum, under 160r/min, 25 DEG C of conditions, cultured continuously 8 days is elementary cultivation, again be transferred in PDB substratum by 10% inoculum size, continuing cultured continuously under 160r/min, 25 DEG C of conditions 28 days is secondary cultivation.
2) decompress filter obtains mycelium, and mycelium grinds to form powdery after freeze-day with constant temperature at 40 DEG C, sieves, with ethyl acetate immersion, ultrasonic, repeats 3 times.Merge soak solution, revolve and steam to obtain mycelium medicinal extract.
3), by 200 ~ 300 order silicagel columns on mycelium medicinal extract, with sherwood oil and acetone gradient elution, collect sherwood oil: acetone volume ratio=20:1 part elutriant, concentrated by rotary evaporation, chloroform washes out, and naturally volatilizes, by recrystallizing methanol, obtain hopene through HREIMS, NMR spectrum analysis again.
4) use mtt assay to carry out the mensuration of cytoactive.
Being prepared as of described PDB substratum: peeling potatoes, stripping and slicing, takes 200g, boiling water boiling 30min, 6 layers of filtered through gauze, take 20g glucose, and adding distil water is settled to 1000mL, pH nature.121 DEG C of autoclavings, 20min.
The size of inoculating bacterium piece in described step 1) is 0.5 × 0.5cm, and inoculum size is 4.
Described step 2) in soak 24h, ultrasonic 1h with 3 times of volumes of acetic acid ethyl esters.
In described step 3), recrystallization concrete operations are:, naturally volatilize, after crystal is separated out the hopene that the contains a small amount of impurity dissolving obtaining with chloroform, wash away impurity with methyl alcohol again, separate out and discard, remaining crystal is again with recrystallization after dissolve with methanol, until obtain single hopene.
Remarkable advantage of the present invention is: the present invention fungi fast to breed, that the cycle is short is as material, and cost is low, simple to operate, reproducible, can make purer material by simple mode.The data that obtain according to cytoactive detection can illustrate that this kind of compound has excellent anti-tumor activity.
Brief description of the drawings
Fig. 1 is the thin-layer chromatography TLC of hopene.Vitriol oil colour developing, for red, illustrates that it is triterpene substance.
Fig. 2 is hopene
1h NMR spectrum.In figure, show to exist 7 methyl signals, wherein 6 are connected on quaternary carbon, and another one is connected on the two keys of quaternary carbon.
Fig. 3 is hopene
13c NMR spectrum.Wherein have as seen from the figure 30 carbon signals, and wherein 2 be double-linked carbon, illustrate and have vinyl group.
Fig. 4 is hopene
13c NMR spectrum (under) and DEPT 135 compose (on).Two figure are contrasted to rear discovery, wherein contain 6 quaternary carbons, 12 secondary carbon.
Fig. 5 is the EI-MS spectrum of hopene.Be 410 according to m/z given in figure, can infer that its relative molecular mass is 410.
Fig. 6 is the chemical structure of hopene.
Embodiment
embodiment 1
The preparation of hopene
1) reddish brown little Mo is strangled to bacterium (this laboratory field acquisition, separation and purification preservation strain) activation after, with 0.5 × 0.5cm(4 piece) be inoculated in PDB substratum (peeling potatoes, stripping and slicing, takes 200g, boiling water boiling 30min, 6 layers of filtered through gauze, take 20g glucose, adding distil water is settled to 1000mL, pH nature.Divide and install in 250ml triangular flask, every bottled 100ml, 121 DEG C of autoclavings, 20min, under 160r/min, 25 DEG C of conditions, cultured continuously 8 days is elementary cultivation, again be transferred in PDB substratum by 10% inoculum size, continuing cultured continuously under 160r/min, 25 DEG C of conditions 28 days is secondary cultivation;
2) decompress filter obtains mycelium, and mycelium grinds to form powdery after freeze-day with constant temperature at 40 DEG C, sieves, with 3 times of ethyl acetate immersions, ultrasonic, repeats 3 times.Merge soak solution, revolve and steam to obtain mycelium medicinal extract.
3) mycelium medicinal extract and bacterium immersion cream are merged, upper 200 ~ 300 order silicagel columns, with sherwood oil and acetone gradient elution, collect sherwood oil: acetone volume ratio=20:1 part elutriant, concentrated by rotary evaporation, chloroform washes out, and naturally volatilizes, by recrystallizing methanol, obtain hopene through HREIMS, NMR spectrum analysis again.
embodiment 2
The experiment of anti-tumor activity
The compound that adopts embodiment 1 to obtain is tested as follows:
Clone and culture condition thereof: the present invention's clone used is human liver cancer cell strain SMMC-7721.Cell, with containing the DMEM culture medium culturing of 10% foetal calf serum, wherein contains every milliliter of 100 unit penicillin and 100 microgram Streptomycin sulphates, and it is after the culture dish of 10 centimetres that cell is inoculated in diameter, 37 DEG C, 5%CO
2in environment, cultivate, when cell covers with, go down to posterity by tryptic digestion method.
Cytotoxicity test: cytotoxicity adopts mtt assay to measure.Cell is become to single cell suspension with 0.25% tryptic digestion, adopt blood cell to calculate version and carry out viable count, adjusting viable cell concentrations is 5 × 10
4every milliliter is inoculated in 96 well culture plates, every hole 160 microlitres.Culture plate is put into CO
2in incubator, at 37 DEG C, 5%CO
2and under saturated humidity condition, cultivate 24 hours.After cell attachment, nutrient solution is inhaled and abandoned, taking the cell without hopene processing as control group, taking the cell of different concns hopene processing as experimental group, each concentration arranges respectively 3 multiple holes.When cultivation finishes first 4 hours, every hole adds 20 microlitre MTT.In incubator, continue to hatch 4 hours, abandon supernatant, every hole adds 100 microlitre DMSO, shakes 10 minutes, puts microplate reader and measures OD value, and wavelength is set to 490nm.Calculate survival rate by following formula, map simultaneously and try to achieve half degree of killing and wounding (IC
50), the cytotoxicity of evaluation medicine.
Average OD value × 100% of the average OD value/control wells of survival rate %=medicine feeding hole.
Calculating hopene through mapping is 12 μ g/mL to the IC50 of human liver cancer cell strain SMMC-7721.
The new compound that above-described embodiment makes has excellent anti-tumor activity to human liver cancer cell strain SMMC-7721.
The foregoing is only preferred embodiment of the present invention, all equalizations of doing according to the present patent application the scope of the claims change and modify, and all should belong to covering scope of the present invention.
Claims (3)
1. strangle from reddish brown little Mo a method of extracting hopene bacterium, it is characterized in that: comprise the following steps:
1) reddish brown little Mo is strangled after bacterium activation, get in bacterium piece inoculation PDB substratum, under 160r/min, 25 DEG C of conditions, cultured continuously 8 days is elementary cultivation, is again transferred in PDB substratum by 10% inoculum size, and continuing cultured continuously under 160r/min, 25 DEG C of conditions 28 days is secondary cultivation;
2) decompress filter obtains mycelium, and mycelium grinds to form powdery after freeze-day with constant temperature at 40 DEG C, sieves, with ethyl acetate immersion, ultrasonic, repeats 3 times, merges soak solution, revolves and steams to obtain mycelium medicinal extract;
3), by 200 ~ 300 order silicagel columns on mycelium medicinal extract, with sherwood oil and acetone gradient elution, collect sherwood oil: acetone volume ratio=20:1 part elutriant, concentrated by rotary evaporation, chloroform washes out, and naturally volatilizes, then obtains hopene by recrystallizing methanol.
2. according to claim 1ly strangle from reddish brown little Mo the method for extracting hopene bacterium, it is characterized in that: the size of inoculating bacterium piece in described step 1) is 0.5 × 0.5cm, inoculum size is 4.
3. according to claim 1ly strangle from reddish brown little Mo the method for extracting hopene bacterium, it is characterized in that: described step 2) in soak 24h, ultrasonic 1h with 3 times of volumes of acetic acid ethyl esters.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201310229308.9A CN103276041B (en) | 2013-06-09 | 2013-06-09 | Method for extracting hopene from moelleriellaochracea |
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| CN102080049B (en) * | 2010-12-03 | 2013-01-09 | 大连民族学院 | Ginseng endogenesis zygorhynchus moelleri mildew as well as method for preparing ginsenoside Rd by utilizing same |
| CN102838650B (en) * | 2012-08-15 | 2014-04-09 | 福建农林大学 | Method of extracting dustanin from Moelleriella ochracea |
| CN103014090B (en) * | 2012-12-14 | 2014-05-21 | 福建农林大学 | Method for extracting bis-benzene oxepin-11(6H) keto-1, 10-dihydroxy, 3-methyl-7, 8-dimethoxy from Moller bacteria |
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