CN103290088A - 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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- CN103290088A CN103290088A CN2013102356764A CN201310235676A CN103290088A CN 103290088 A CN103290088 A CN 103290088A CN 2013102356764 A CN2013102356764 A CN 2013102356764A CN 201310235676 A CN201310235676 A CN 201310235676A CN 103290088 A CN103290088 A CN 103290088A
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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 of method of reining in extraction triterpene substance zeorin the bacterium from reddish brown Xiao Mo.
Background technology
Reddish brown Xiao Mo rein in bacterium (
Moelleriella ochracea) be under the jurisdiction of Ascomycota, caprophyl guiding principle, Hypocreales, Clavicipitaceae, do not rein in Pseudomonas.Not reining in bacterium, is the important pathogenic fungi of aleyrodid, has obtained unusual effect aspect biological control, its meta-bolites also present preferably antibiotic, kill the plasmodium activity, and insect and tumour cell are had stronger toxicity.People separate from its meta-bolites, are purified into some activated materials, 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.As seen, do not rein in bacterium and medically have potential using value.If want large-scale production and application, at first to consider extracting method, the extracted amount optimization of target compound.
Summary of the invention
The objective of the invention is to disclose a kind of method of reining in extraction triterpene substance zeorin the bacterium from reddish brown Xiao Mo, to promote it in the development and application of medical field.
The technical scheme that the present invention takes is as follows:
A kind of method of reining in extraction triterpene substance zeorin the bacterium from reddish brown Xiao Mo may further comprise the steps:
1) after reddish brown Xiao Mo reins in the bacterium activation, getting the bacterium piece is inoculated in the PDB substratum, cultured continuously 7 ~ 10 days was elementary cultivation under 100 ~ 160r/min, 24 ~ 28 ℃ of conditions, be transferred to again in the PDB substratum by 8% ~ 10% inoculum size, static cultivation 35 ~ 40 days was secondary cultivation under 24 ℃ ~ 28 ℃ conditions;
2) bacterium liquid and mycelium filtering separation, mycelium soaked 48～100 hours in 3 ℃～15 ℃, organic solvent, filter soak solution, revolve steam temperature control 35 ℃～40 ℃ 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 the separating funnel, the ethyl acetate that adds 1～2 times is shaken the bottle extraction, shake a bottle rotating speed 150r/min～280r/min, shake the static 150min of 20～60min, revolve steaming ethyl acetate partial concentration and get crude extract, reclaim ethyl acetate;
3) the acetic acid ethyl dissolution crude extract of 1 times of crude extract volume, last 200-300 order silicagel column, sherwood oil: ethyl acetate volume ratio 60:1 begins gradient elution, collects sherwood oil: ethyl acetate volume ratio 30:1～10:1 part elutriant, revolve inspissation contract extract A;
4) the acetic acid ethyl dissolution extract A of 1 times of crude extract volume, last 200-300 order silicagel column, sherwood oil: ethyl acetate volume ratio 30:1 begins gradient elution, collect sherwood oil: ethyl acetate volume ratio 15:1 part elutriant, revolve inspissation contract medicinal extract, chloroform dissolves, and is placed in 4 ℃ of refrigerators to volatilize, and getting the lenticular material is zeorin.
Wherein, the PDB substratum is in the described step 1): the 4g potato powder, and 20g glucose, boiling water boils 30min, adds single water that steams and be settled to 1L after 4 layers of filtered through gauze.
Described step 2) organic solvent can select acetone, ethanol, methyl alcohol in.
Remarkable advantage of the present invention:
1) raw material is drawn materials simply;
2) equipment is simple, operation easily, and cost is low;
3) the compound purity height that makes.
Description of drawings
Fig. 1: the zeorin of extraction is the 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
Below be several specific embodiment of the present invention, further specify the present invention, but the present invention be not limited only to this.
1) reddish brown Xiao Mo reins in bacterium (Qiu Junzhi etc., reddish brown Xiao Mo reins in bacterium in the discovery of China. the fungus journal, 2009.28 (1): 148-150) after the activation, getting the bacterium piece is inoculated into the PDB substratum (boiling water boils 30min for 4g potato powder, 20g glucose, after 4 layers of filtered through gauze, add single water that steams and be settled to 1L) in, cultured continuously 8 d are elementary cultivation under 150 r/min, 25 ℃ of conditions, are transferred to again in the PDB substratum by 9% inoculum size, and static cultivation 38 d are secondary cultivation under 25 ℃ of conditions;
2) bacterium liquid and mycelium filtering separation, mycelium soaked 70 hours in 4 ℃, ethanol, filter soak solution, revolve steam temperature control 38 ℃ medicinal extract, reclaim ethanol.The medicinal extract that is dissolved in water, water: medicinal extract weight ratio=70:1, pour the water solubles in the separating funnel into, the ethyl acetate that adds 1.5 times is shaken the bottle extraction, shakes bottle rotating speed 200 r/min, shakes static 150 min of 40 min, revolve steaming ethyl acetate partial concentration and get crude extract, reclaim ethyl acetate;
3) the acetic acid ethyl dissolution crude extract of 1 times of crude extract volume, last 200-300 order silicagel column, sherwood oil: ethyl acetate volume ratio 60:1 begins gradient elution, collects sherwood oil: ethyl acetate volume ratio 25:1 part elutriant, revolve inspissation contract extract A;
4) the acetic acid ethyl dissolution extract A of 1 times of crude extract volume, last 200-300 order silicagel column, sherwood oil: ethyl acetate volume ratio 30:1 begins gradient elution, collect sherwood oil: ethyl acetate volume ratio 15:1 part elutriant, revolve inspissation contract medicinal extract, chloroform dissolves, and is placed in 4 ℃ of refrigerators to volatilize, and getting the 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 that obtains 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 8 angular methyl(group)s that are connected on the quaternary carbon are arranged, infer that it is triterpene compound.(1H m) is shown as 1 hydrogen signal on company's oxygen methyne to δ 3.965.Fig. 3 (
13C NMR) there are not phenyl ring conjugated double bond, ethylene linkage and three bond structure in the prompting compound.δ 73.938,69.326 prompting have 2 even the 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 unanimity of H NMR.Pass through compound
13C NMR spectrum is found with the contrast of DEPT spectrum, is contained 6 quaternary carbons in the compound, 10 secondary carbon.Data and document (Isaka M by 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 of 3.13%(extraction yield %=/mycelium medicinal extract total amount * 100%).
Embodiment 2
1) after reddish brown Xiao Mo reins in bacterium activation, get the bacterium piece and be inoculated in the PDB substratum, cultured continuously 7 days was elementary cultivation under 100r/min, 24 ℃ of conditions, was transferred to again in the PDB substratum by 8% inoculum size, and static cultivation 35 days was secondary cultivation under 24 ℃ of conditions;
2) bacterium liquid and mycelium filtering separation, mycelium soaked 48 hours in 3 ℃, acetone, filter soak solution, revolve steam temperature control 35 ℃ medicinal extract, reclaim acetone; The medicinal extract that is dissolved in water, water: medicinal extract weight ratio=50:1, pour the water solubles in the separating funnel into, the ethyl acetate that adds 2 times is shaken the bottle extraction, shakes a bottle rotating speed 150r/min, shakes the static 150min of 20min, revolve steaming ethyl acetate partial concentration and get crude extract, reclaim ethyl acetate;
3) the acetic acid ethyl dissolution crude extract of 1 times of crude extract volume, last 200-300 order silicagel column, sherwood oil: ethyl acetate volume ratio 60:1 begins gradient elution, collects sherwood oil: ethyl acetate volume ratio 30:1 part elutriant, revolve inspissation contract extract A;
4) the acetic acid ethyl dissolution extract A of 1 times of crude extract volume, last 200-300 order silicagel column, sherwood oil: ethyl acetate volume ratio 30:1 begins gradient elution, collect sherwood oil: ethyl acetate volume ratio 15:1 part elutriant, revolve the inspissation contracting, chloroform dissolves, and is placed in 4 ℃ of refrigerators to volatilize, and getting the lenticular material verified is zeorin.
5) empirical tests obtains purity and is 99% zeorin.
Extraction yield: the pure zeorin weight of 2.58%(extraction yield %=/mycelium medicinal extract total amount * 100%).
Embodiment 3
1) after reddish brown Xiao Mo reins in the bacterium activation, getting the bacterium piece is inoculated in the PDB substratum, cultured continuously 10 days was elementary cultivation under 160r/min, 28 ℃ of conditions, was transferred to again in the PDB substratum by 10% inoculum size, and static cultivation 40 days was secondary cultivation under 28 ℃ of conditions;
2) bacterium liquid and mycelium filtering separation, mycelium soaked 100 hours in 15 ℃, methyl alcohol, filter soak solution, revolve steam temperature control 40 ℃ medicinal extract, reclaim methyl alcohol; The medicinal extract that is dissolved in water, water: medicinal extract weight ratio=100:1, pour the water solubles in the separating funnel into, the ethyl acetate that adds 1 times is shaken the bottle extraction, shakes a bottle rotating speed 280r/min, shakes the static 150min of 60min, revolve steaming ethyl acetate partial concentration and get crude extract, reclaim ethyl acetate;
3) the acetic acid ethyl dissolution crude extract of 1 times of crude extract volume, last 200-300 order silicagel column, sherwood oil: ethyl acetate volume ratio 60:1 begins gradient elution, collects sherwood oil: ethyl acetate volume ratio 10:1 part elutriant, revolve inspissation contract extract A;
4) the acetic acid ethyl dissolution extract A of 1 times of crude extract volume, last 200-300 order silicagel column, sherwood oil: ethyl acetate volume ratio 30:1 begins gradient elution, collect sherwood oil: ethyl acetate volume ratio 15:1 part elutriant, revolve inspissation contract medicinal extract, chloroform dissolves, and is placed in 4 ℃ of refrigerators to volatilize, and gets lenticular material zeorin.
5) empirical tests obtains purity and is 99% zeorin.
Extraction yield: the pure zeorin weight of 2.67%(extraction yield %=/mycelium medicinal extract total amount * 100%).
Claims (3)
1. rein in the bacterium method of extracting the triterpene substance zeorin from reddish brown Xiao Mo for one kind, it is characterized in that this method may further comprise the steps:
1) after reddish brown Xiao Mo reins in the bacterium activation, getting the bacterium piece is inoculated in the PDB substratum, cultured continuously 7 ~ 10 days was elementary cultivation under 100 ~ 160r/min, 24 ~ 28 ℃ of conditions, be transferred to again in the PDB substratum by 8% ~ 10% inoculum size, static cultivation 35 ~ 40 days was secondary cultivation under 24 ℃ ~ 28 ℃ conditions;
2) bacterium liquid and mycelium filtering separation, mycelium soaked 48～100 hours in 3 ℃～15 ℃, organic solvent, filter soak solution, revolve steam temperature control 35 ℃～40 ℃ 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 the separating funnel, the ethyl acetate that adds 1～2 times is shaken the bottle extraction, shake a bottle rotating speed 150r/min～280r/min, shake 20～60min, static 150min, revolve steaming ethyl acetate partial concentration and get crude extract, reclaim ethyl acetate;
3) the acetic acid ethyl dissolution crude extract of 1 times of crude extract volume, last 200-300 order silicagel column, sherwood oil: ethyl acetate volume ratio 60:1 begins gradient elution, collects sherwood oil: ethyl acetate volume ratio 30:1～10:1 part elutriant, revolve inspissation contract extract A;
4) the acetic acid ethyl dissolution extract A of 1 times of crude extract volume, last 200-300 order silicagel column, sherwood oil: ethyl acetate volume ratio 30:1 begins gradient elution, collect sherwood oil: ethyl acetate volume ratio 15:1 part elutriant, revolve inspissation contract medicinal extract, chloroform dissolves, and is placed in 4 ℃ of refrigerators to volatilize, and getting the lenticular material is zeorin.
2. according to claims 1 described method of reining in extraction triterpene substance zeorin the bacterium from reddish brown Xiao Mo, it is characterized in that: the PDB substratum is in the described step 1): the 4g potato powder, 20g glucose, boiling water boils 30min, adds single water that steams and be settled to 1L after 4 layers of filtered through gauze.
3. according to claims 1 described method of reining in extraction triterpene substance zeorin the bacterium from reddish brown Xiao Mo, it is characterized in that: organic solvent can select acetone, ethanol, methyl alcohol described step 2).
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107955819A (en) * | 2017-12-23 | 2018-04-24 | 福建农林大学 | A kind of culture medium and method for not strangling bacterium fermenting and producing triterpene |
| CN115385979A (en) * | 2022-09-07 | 2022-11-25 | 福建农林大学 | Method for extracting hopane triterpenoids from Raffaelea lauricola |
Citations (3)
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|---|---|---|---|---|
| CN102080049A (en) * | 2010-12-03 | 2011-06-01 | 大连民族学院 | Ginseng endogenesis zygorhynchus moelleri mildew as well as method for preparing ginsenoside Rd by utilizing same |
| CN102838650A (en) * | 2012-08-15 | 2012-12-26 | 福建农林大学 | Method of extracting dustanin from Moelleriella ochracea |
| CN103014090A (en) * | 2012-12-14 | 2013-04-03 | 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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2013
- 2013-06-15 CN CN201310235676.4A patent/CN103290088B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102080049A (en) * | 2010-12-03 | 2011-06-01 | 大连民族学院 | Ginseng endogenesis zygorhynchus moelleri mildew as well as method for preparing ginsenoside Rd by utilizing same |
| CN102838650A (en) * | 2012-08-15 | 2012-12-26 | 福建农林大学 | Method of extracting dustanin from Moelleriella ochracea |
| CN103014090A (en) * | 2012-12-14 | 2013-04-03 | 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 |
Non-Patent Citations (3)
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| MASAHIKO ISAKA等: "Hopane triterpenes as chemotaxonomic markers for the scale insect pathogens Hypocrella s. lat. and Aschersonia", 《MYCOLOGICAL RESEARCH》 * |
| QIU JUN-ZHI等: "The finding of Moelleriella ochracea(Ascomycota, Clavicipitaceae) in China", 《菌物学报》 * |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107955819A (en) * | 2017-12-23 | 2018-04-24 | 福建农林大学 | A kind of culture medium and method for not strangling bacterium fermenting and producing triterpene |
| CN115385979A (en) * | 2022-09-07 | 2022-11-25 | 福建农林大学 | Method for extracting hopane triterpenoids from Raffaelea lauricola |
| CN115385979B (en) * | 2022-09-07 | 2024-01-30 | 福建农林大学 | Method for extracting hopane type triterpene compound from Raffaelea lauricola |
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