CN103290088B - Method for extracting triterpenoid type substance, namely zeorin from moelleriella ochracea - Google Patents
Method for extracting triterpenoid type substance, namely zeorin from moelleriella ochracea Download PDFInfo
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- CN103290088B CN103290088B CN201310235676.4A CN201310235676A CN103290088B CN 103290088 B CN103290088 B CN 103290088B CN 201310235676 A CN201310235676 A CN 201310235676A CN 103290088 B CN103290088 B CN 103290088B
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- ethyl acetate
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Abstract
The invention discloses a method for extracting a triterpenoid type substance, namely zeorin from moelleriella ochracea. The method comprises the following steps: activating a strain, performing primary culture and secondary culture, performing suction filtration on a fermentation solution, performing cold soaking extraction on mycelia, performing rotary evaporation on a soaking solution, adding water for dissolution, performing shake flask extraction with an organic solvent to obtain a crude extract, performing chromatographic purification on the crude extract through a silica gel column, collecting eluate, performing rotary evaporation and concentration, dissolving by using chloroform and placing into a refrigerator at the temperature of 4 DEG C for evaporation to dryness so as to get a zeorin crystal. The method disclosed by the invention has the advantages of available raw materials, simple equipment and low cost and is easy to operate, and the purity of a prepared compound is high.
Description
Technical field
The present invention relates to a kind ofly strangle from reddish brown little Mo the method for extracting triterpene substance zeorin bacterium.
Background technology
Reddish brown little Mo strangle bacterium (
moelleriella ochracea) be under the jurisdiction of Ascomycota, caprophyl guiding principle, Hypocreales, Clavicipitaceae, do not strangle Pseudomonas.Not strangling bacterium, is the important pathogen fungi of aleyrodid, aspect biological control, has obtained unusual effect, its meta-bolites also present preferably antibacterial, kill plasmodium activity, and insect and tumour cell are had to stronger toxicity.People separate, are purified into some activated materials from its meta-bolites, as zeorin, and its molecular formula C
30h
52o
2, molecular weight 444, is five yuan of carbocyclic ring materials of triterpenes, has the effect of anti-mycobacterium tuberculosis, IC
50=12.5 μ g/ml.Visible, do not strangle bacterium and medically there is potential using value.If want large-scale production and application, first to consider extracting method, the extracted amount optimization of target compound.
Summary of the invention
The object of the invention is openly a kind ofly to strangle from reddish brown little Mo the method for extracting triterpene substance zeorin bacterium, to promote its development and application at medical field.
The technical scheme that the present invention takes is as follows:
Strangle from reddish brown little Mo a method of extracting triterpene substance zeorin bacterium, comprise the following steps:
1) reddish brown little Mo strangles after bacterium activation, getting bacterium piece is inoculated in PDB substratum, under 100 ~ 160r/min, 24 ~ 28 DEG C of conditions, cultured continuously 7 ~ 10 days is elementary cultivation, again be transferred in PDB substratum by 8% ~ 10% inoculum size, under 24 DEG C ~ 28 DEG C conditions, static cultivation 35 ~ 40 days is secondary cultivation;
2) bacterium liquid and mycelium filtering separation, mycelium soaks 48～100 hours in 3 DEG C～15 DEG C, organic solvent, filters to obtain soak solution, revolve steam temperature be controlled at 35 DEG C～40 DEG C medicinal extract, reclaim organic solvent;
Medicinal extract is dissolved in water, water: medicinal extract weight ratio=50:1～100:1, the water solubles is poured in separating funnel, add the ethyl acetate of 1～2 times to carry out shaking flask extraction, shaking flask rotating speed 150r/min～280r/min, shake the static 150min of 20～60min, revolve steaming ethyl acetate partial concentration and obtain crude extract, reclaim ethyl acetate;
3) the acetic acid ethyl dissolution crude extract of 1 times of crude extract volume, upper 200-300 order silicagel column, sherwood oil: ethyl acetate volume ratio 60:1 starts gradient elution, collect sherwood oil: ethyl acetate volume ratio 30:1～10:1 part elutriant, concentrated by rotary evaporation obtains extract A;
4) the acetic acid ethyl dissolution extract A of 1 times of crude extract volume, upper 200-300 order silicagel column, sherwood oil: ethyl acetate volume ratio 30:1 starts gradient elution, collect sherwood oil: ethyl acetate volume ratio 15:1 part elutriant, concentrated by rotary evaporation obtains medicinal extract, chloroform dissolves, and is placed in 4 DEG C of refrigerators and volatilizes, and obtaining lenticular material is zeorin.
Wherein, described step 1) in PDB substratum be: 4g potato powder, 20g glucose, boiling water boiling 30min adds single water that steams and is settled to 1L after 4 layers of filtered through gauze.
Described step 2) in organic solvent can select acetone, ethanol, methyl alcohol.
Remarkable advantage of the present invention:
1) raw material is drawn materials simply;
2) equipment is simple, easily operation, and cost is low;
3) compound purity making is high.
Brief description of the drawings
Fig. 1: the zeorin of extraction is purity proof diagram on silica-gel plate
Fig. 2: the zeorin of extraction
1h NMR spectrum
Fig. 3: the zeorin of extraction
13c NMR spectrum
Fig. 4: the chemical structure of zeorin.
Embodiment
Be below several specific embodiment of the present invention, further illustrate the present invention, but the present invention be not limited only to this.
embodiment 1
1) reddish brown little Mo strangles bacterium (Qiu Junzhi etc., reddish brown little Mo strangles bacterium in Chinese discovery. fungus journal, 2009.28 (1): 148-150) after activation, get bacterium piece and be inoculated into PDB substratum (4g potato powder, 20g glucose, boiling water boiling 30min, after 4 layers of filtered through gauze, add single water that steams and be settled to 1L) in, under 150 r/min, 25 DEG C of conditions, cultured continuously 8 d are elementary cultivation, again be transferred in PDB substratum by 9% inoculum size, under 25 DEG C of conditions, static cultivation 38 d are secondary cultivation;
2) bacterium liquid and mycelium filtering separation, mycelium soaks 70 hours in 4 DEG C, ethanol, filters to obtain soak solution, revolve steam temperature be controlled at 38 DEG C medicinal extract, reclaim ethanol.The medicinal extract that is dissolved in water, water: medicinal extract weight ratio=70:1, pours the water solubles in separating funnel into, add the ethyl acetate of 1.5 times to carry out shaking flask extraction, shaking flask rotating speed 200 r/min, shake static 150 min of 40 min, revolve steaming ethyl acetate partial concentration and obtain crude extract, reclaim ethyl acetate;
3) the acetic acid ethyl dissolution crude extract of 1 times of crude extract volume, upper 200-300 order silicagel column, sherwood oil: ethyl acetate volume ratio 60:1 starts gradient elution, collect sherwood oil: ethyl acetate volume ratio 25:1 part elutriant, concentrated by rotary evaporation obtains extract A;
4) the acetic acid ethyl dissolution extract A of 1 times of crude extract volume, upper 200-300 order silicagel column, sherwood oil: ethyl acetate volume ratio 30:1 starts gradient elution, collect sherwood oil: ethyl acetate volume ratio 15:1 part elutriant, concentrated by rotary evaporation obtains medicinal extract, chloroform dissolves, and is placed in 4 DEG C of refrigerators and volatilizes, and obtaining lenticular material is zeorin.
5) purity of extract checking: as shown in Figure 1, developping agent is methyl alcohol: methylene chloride volume ratio=1:20, R
f=0.43, can determine that the zeorin purity obtaining is 99%.
6) structure verification of extract: from Fig. 2 (
1h NMR
3) can see δ 1.208 (3H, s), 1.183 (3H, s), 1.159 (3H, s), 1.043 (3H, s), 1.016 (3H, s), 0.978 (3H, s), 0.870 (3H, s), 0.764 (3H, s) prompting has 8 angular methyl(group)s that are connected on quaternary carbon, infers that it is triterpene compound.δ 3.965 (1H, m) is shown as 1 hydrogen signal on company's oxygen methyne.Fig. 3 (
13c NMR) there is not phenyl ring conjugated double bond, ethylene linkage and three bond structure in compound in prompting.δ 73.938,69.326 prompting have 2 even oxygen carbon exist, δ 22.121,21.918,21.052,18.526,18.277,17.129,17.078,16.089 promptings have 8 angular methyl(group)s to exist, this with
1the inferred results of H NMR is consistent.Pass through compound
13c NMR spectrum is found with the contrast of DEPT spectrum, is contained 6 quaternary carbons, 10 secondary carbon in compound.By data and document (the Isaka M of Fig. 2, Fig. 3, Hywel-Jones NL, Sappan M, Mongkolsamrit S, Saidaengkham S. Hopane triterpenes as chemotaxonomic markers for the scale insect pathogens
hypocrellas. lat. and
aschersonia. Mycological Research, 2009,113 (4): 491-497) contrast, provable this material is zeorin, chemical structure is as shown in Figure 4.
7) extraction yield: the pure zeorin weight/mycelium of 3.13%(extraction yield %=medicinal extract total amount × 100%).
embodiment 2
1) reddish brown little Mo strangles after bacterium activation, getting bacterium piece is inoculated in PDB substratum, under 100r/min, 24 DEG C of conditions, cultured continuously 7 days is elementary cultivation, is again transferred in PDB substratum by 8% inoculum size, and under 24 DEG C of conditions, static cultivation 35 days is secondary cultivation;
2) bacterium liquid and mycelium filtering separation, mycelium soaks 48 hours in 3 DEG C, acetone, filters to obtain soak solution, revolve steam temperature be controlled at 35 DEG C medicinal extract, reclaim acetone; The medicinal extract that is dissolved in water, water: medicinal extract weight ratio=50:1, pours the water solubles in separating funnel into, add the ethyl acetate of 2 times to carry out shaking flask extraction, shaking flask rotating speed 150r/min, shakes the static 150min of 20min, revolve steaming ethyl acetate partial concentration and obtain crude extract, reclaim ethyl acetate;
3) the acetic acid ethyl dissolution crude extract of 1 times of crude extract volume, upper 200-300 order silicagel column, sherwood oil: ethyl acetate volume ratio 60:1 starts gradient elution, collect sherwood oil: ethyl acetate volume ratio 30:1 part elutriant, concentrated by rotary evaporation obtains extract A;
4) the acetic acid ethyl dissolution extract A of 1 times of crude extract volume, upper 200-300 order silicagel column, sherwood oil: ethyl acetate volume ratio 30:1 starts gradient elution, collect sherwood oil: ethyl acetate volume ratio 15:1 part elutriant, concentrated by rotary evaporation, chloroform dissolves, and is placed in 4 DEG C of refrigerators and volatilizes, and obtaining lenticular material verified is zeorin.
5) empirical tests, obtains purity and is 99% zeorin.
Extraction yield: the pure zeorin weight/mycelium of 2.58%(extraction yield %=medicinal extract total amount × 100%).
embodiment 3
1) reddish brown little Mo strangles after bacterium activation, getting bacterium piece is inoculated in PDB substratum, under 160r/min, 28 DEG C of conditions, cultured continuously 10 days is elementary cultivation, is again transferred in PDB substratum by 10% inoculum size, and under 28 DEG C of conditions, static cultivation 40 days is secondary cultivation;
2) bacterium liquid and mycelium filtering separation, mycelium soaks 100 hours in 15 DEG C, methyl alcohol, filters to obtain soak solution, revolve steam temperature be controlled at 40 DEG C medicinal extract, reclaim methyl alcohol; The medicinal extract that is dissolved in water, water: medicinal extract weight ratio=100:1, pours the water solubles in separating funnel into, add the ethyl acetate of 1 times to carry out shaking flask extraction, shaking flask rotating speed 280r/min, shakes the static 150min of 60min, revolve steaming ethyl acetate partial concentration and obtain crude extract, reclaim ethyl acetate;
3) the acetic acid ethyl dissolution crude extract of 1 times of crude extract volume, upper 200-300 order silicagel column, sherwood oil: ethyl acetate volume ratio 60:1 starts gradient elution, collect sherwood oil: ethyl acetate volume ratio 10:1 part elutriant, concentrated by rotary evaporation obtains extract A;
4) the acetic acid ethyl dissolution extract A of 1 times of crude extract volume, upper 200-300 order silicagel column, sherwood oil: ethyl acetate volume ratio 30:1 starts gradient elution, collect sherwood oil: ethyl acetate volume ratio 15:1 part elutriant, concentrated by rotary evaporation obtains medicinal extract, chloroform dissolves, and is placed in 4 DEG C of refrigerators and volatilizes, and obtains lenticular material zeorin.
5) empirical tests, obtains purity and is 99% zeorin.
Extraction yield: the pure zeorin weight/mycelium of 2.67%(extraction yield %=medicinal extract total amount × 100%).
Claims (1)
1. strangle from reddish brown little Mo a method of extracting triterpene substance zeorin bacterium, it is characterized in that: the method comprises the following steps:
1) reddish brown little Mo strangles after bacterium activation, getting bacterium piece is inoculated in PDB substratum, under 100 ~ 160r/min, 24 ~ 28 DEG C of conditions, cultured continuously 7 ~ 10 days is elementary cultivation, again be transferred in PDB substratum by 8% ~ 10% inoculum size, under 24 DEG C ~ 28 DEG C conditions, static cultivation 35 ~ 40 days is secondary cultivation;
Described PDB substratum is: 4g potato powder, and 20g glucose, boiling water boiling 30min adds single water that steams and is settled to 1L after 4 layers of filtered through gauze;
2) bacterium liquid and mycelium filtering separation, mycelium soaks 48～100 hours in 3 DEG C～15 DEG C, organic solvent, filters to obtain soak solution, revolve steam temperature be controlled at 35 DEG C～40 DEG C medicinal extract, reclaim organic solvent; Medicinal extract is dissolved in water, water: medicinal extract weight ratio=50:1～100:1, the water solubles is poured in separating funnel, add the ethyl acetate of 1～2 times to carry out shaking flask extraction, shaking flask rotating speed 150r/min～280r/min, shakes 20～60min, static 150min, revolve steaming ethyl acetate partial concentration and obtain crude extract, reclaim ethyl acetate;
Described organic solvent can select acetone, ethanol, methyl alcohol;
3) the acetic acid ethyl dissolution crude extract of 1 times of crude extract volume, upper 200-300 order silicagel column, sherwood oil: ethyl acetate volume ratio 60:1 starts gradient elution, collect sherwood oil: ethyl acetate volume ratio 30:1～10:1 part elutriant, concentrated by rotary evaporation obtains extract A;
4) the acetic acid ethyl dissolution extract A of 1 times of crude extract volume, upper 200-300 order silicagel column, sherwood oil: ethyl acetate volume ratio 30:1 starts gradient elution, collect sherwood oil: ethyl acetate volume ratio 15:1 part elutriant, concentrated by rotary evaporation obtains medicinal extract, chloroform dissolves, and is placed in 4 DEG C of refrigerators and volatilizes, and obtaining lenticular material is zeorin.
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