CN104846026A - Method for extracting taxol from Hainan Pestalotiopsis sp. F metabolite - Google Patents
Method for extracting taxol from Hainan Pestalotiopsis sp. F metabolite Download PDFInfo
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- CN104846026A CN104846026A CN201510161710.7A CN201510161710A CN104846026A CN 104846026 A CN104846026 A CN 104846026A CN 201510161710 A CN201510161710 A CN 201510161710A CN 104846026 A CN104846026 A CN 104846026A
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Abstract
The invention discloses a method for extracting taxol from a Hainan Pestalotiopsis sp. F metabolite. The method comprises the steps of thallus culturing and fermentation; secondary metabolite extraction; and column chromatography purification. Taxol produced by the extracted Hainan Pestalotiopsis sp. F has a same structure with standard taxol, and the output is 1466.87[mu]g/L. The taxol output of the above fungus in panda scurf is substantially higher than that of the fungus from other animals. Production of taxol from the fungus may be a potential candidate method for industrial large-scale production of taxol.
Description
Technical field
The present invention relates to the extracting method of taxol, specifically, relate to a kind of method intending extracting pestalotia bacteria meta-bolites taxol from Hainan.
Background technology
Taxol (taxol) is separated from yewtree (Taxus brevifolia Nutt) by Wani etc. the earliest, is that a kind of diterpene-kind compound with antitumour activity of complexity is used for the treatment of ovarian cancer, mammary cancer, nonsmall-cell lung cancer, esophagus cancer etc.Schiff confirm the taxol mechanism of action be by with tubulin binding, promote that it is polymerized, anticancer mitotic division, stop the infinite multiplication of cancer cells.
At present, mainly from the bark of Ramulus et folium taxi cuspidatae, taxol is extracted.Due to Ramulus et folium taxi cuspidatae system rare plant, and taxol in bark content is very low, and extracting taxol by bark cannot meet the need of market, and seriously destroys natural resources, jeopardizes ecological flat.1993, Stierle etc. isolated a kind of Taxol-producing fungi from the phloem of yewtree, had opened up a new way by Taxol Producion by Microbe Fermentation.1996, Strobel etc. were separated to the paclitaxel produced endogenetic fungus of a strain from Xizang Taxus chinensis, and output reaches 50ug/L; 2000, Wang etc. isolated a strain taxol-producing endophytic fungi from southerm yew, and output reaches 185.4ug/L; Zhou Dongpo etc. are from taxus chinensis in northeast one strain endogenetic fungus, and be the genus new to China-treelike menu of China, output is 51.06-125.70ug/L.In recent years, Chinese scholars were studied by the screening to taxol Producing Strain and the optimization aspect to fermentation condition.Govindarajan Kathiravan etc. ferments to undercoat plan dish stey, detects that content of taxol is 640ug/L; Upright grade isolates strain green muscardine fungus output after fermentation from Qin_Ba mountain areas China's Ramulus et folium taxi cuspidatae stem and root can reach 846.1ug/L; Zhang Yuan waits the novel species filtering out a strain Alternaria from southerm yew phloem, and in its nutrient solution, content of taxol is 1003ug/L, substantially reaches demand of industrial production; Upright Jin Rui etc. screen the initial optimization that the paclitaxel produced endogenetic fungus XC1-07 of a strain carries out fermentation condition, make the output of taxol be 1124.34ug/L.We isolate a strain Taxol-producing fungi, through being accredited as plan Pestalotia from the dermopathic giant panda site of disease of trouble.Confirm its paclitaxel produced ability by TLC, UV, HPLC, NMR many kinds of detection meanss simultaneously.
2011, from the giant panda scurf of the Giant Panda in China protection and research centre that are arranged in Yaan, isolate strain Hainan plan dish stey.Hainan plan dish stey is generally a kind of endogenetic fungus separated from the plants such as Podocarpus macrophyllus.As far as we know, this is the first time report finding Hainan plan dish stey in animal, and every other plan dish stey species, be all separated from Chinese yew.Although the amount isolating taxol from non-animal is still very low, whether the amount that isolated Hainan plan dish stey is paclitaxel produced from animal-origin is still unknown higher than the level of non-animal resource.Non-animal fungi Taxol Yield is low, and Hainan, giant panda source plan dish stey Taxol Yield seldom has report.
Summary of the invention
The object of the invention is to overcome the defect existed in prior art, there is provided a kind of method intending extracting pestalotia bacteria meta-bolites taxol from Hainan, the method has been investigated isolated Hainan plan dish stey from giant panda scurf and has been produced the output of taxol.Its concrete technical scheme is:
Intend from Hainan the method extracting taxol pestalotia bacteria meta-bolites, comprise the following steps:
Step 1: the cultivation of thalline and fermentation
Separation being intended pestalotia bacteria strain from Hainan of giant panda body surface is inoculated on PDA solid medium, 28 DEG C of lucifuges are cultivated, after microscopy produces spore, rinse bacterium colony by 20ml stroke-physiological saline solution and make suspension, suspension is moved into a sterile test tube, airtightly put into whirlpool 15s, bacteria suspension is fully mixed;
Then with pipettor, the bacteria suspension prepared is proceeded to CPDA fermented liquid, with concussion incubator, 28 DEG C, 200r/min isothermal vibration cultivate 21 ~ 28 days;
Step 2: the extraction of secondary metabolite
CPDA fermented liquid 4 DEG C, 10000r/min, centrifugal 10min, collect supernatant liquor and mycelium respectively.Add 0.5gNaCO
3to in supernatant liquor, NaCl can be added simultaneously, use isopyknic ethyl acetate: acetone extract three times, each 4h, adds methyl alcohol to mycelium and stirs, with 24h freezing after Ultrasonic Cell Disruptor smudge cells, obtain organic phase and mycelium through centrifugal, mycelium carries out surname extraction 8h again, and solvent is methylene dichloride, then merge above-mentioned three part organic phases 35 DEG C of spin concentration on Rotary Evaporators and become medicinal extract shape, medicinal extract is dissolved in 10ml methyl alcohol for column chromatography;
Step 3: column chromatography purification
After AB-8 macroporous resin carries out pre-treatment, wet method dress post.With dehydrated alcohol with the flow velocity of 1.0ml/min rush post stable after, wash-out is carried out with the ethanolic soln that concentration is 30%, 40%, 50%, 60%, 70%, flow velocity is 1.5ml/min, collects elutriant, carries out TLC detection, exhibition coating systems, under 254nm ultraviolet lamp, observe fluorescence, then develop the color with the vitriol oil spraying of 1% Vanillin, observe and compare with or without Japanese yew, collect the above-mentioned elutriant with characteristic locus coeruleus, repeated again after concentrated post obtain purifying for twice after sample.
Further, the volume ratio of bacteria suspension described in step 1 and fermented liquid is 1: 100.
Further, the weight of mycelia described in step 2: methyl alcohol volume=1g: 10ml.
Further, opening up coating systems in step 3 is methylene dichloride and methyl alcohol, and its volume ratio is 20: 1.
Compared with prior art, beneficial effect of the present invention is:
The taxol of Hainan plan dish stey fungi that the inventive method is extracted, and standard taxol has identical structure, and output is 1466.87 μ g/L.This fungi in giant panda scurf, paclitaxel produced amount is significantly higher than the fungi from non-animal.It may be a potential alternative approach of producing for taxol industrial scale that fungi produces taxol.
Embodiment
Below in conjunction with specific embodiment, technical scheme of the present invention is described in more detail.
Bacterial strain in the inventive method
Be separated and intend pestalotia bacteria strain from Hainan of giant panda body surface.Hainan is intended pestalotia bacteria (giant panda source) and is collected in Bi Fengxia town, Sichuan Province China province Ya'an China protection giant panda study base.
Substratum and fermented liquid
PDA solid medium; CPDA fermented liquid: 200g potato, 20g glucose, 3g KH2PO4,1.5gMgSO4,0.15g trisodium citrate (replacing citric acid with trisodium citrate in experiment), 0.01g VB1,1000mL tap water, pH6.5.
Embodiment 1
Intend from Hainan the method extracting taxol pestalotia bacteria meta-bolites, comprise the following steps:
Step 1: the cultivation of thalline and fermentation
Separation being intended pestalotia bacteria strain from Hainan of giant panda body surface is inoculated on PDA solid medium, 28 DEG C of lucifuges are cultivated, after microscopy produces spore, rinse bacterium colony by 20ml stroke-physiological saline solution and make suspension, suspension is moved into a sterile test tube, airtightly put into whirlpool 15s, bacteria suspension is fully mixed;
Then with pipettor the bacteria suspension prepared proceeded to CPDA fermented liquid (by bacteria suspension: fermented liquid=1: 100), with concussion incubator, 28 DEG C, 200r/min isothermal vibration cultivates 21 days;
Step 2: the extraction of secondary metabolite
CPDA fermented liquid 4 DEG C, 10000r/min, centrifugal 10min, collect supernatant liquor and mycelium respectively.Add 0.5gNaCO
3(amount of 1000ml) is in supernatant liquor, NaCl can be added simultaneously, use isopyknic ethyl acetate: acetone extract three times, each 4h, add methyl alcohol (by mycelia weight: methyl alcohol volume=1: 10g:ml) to mycelium to stir, with 24h freezing after Ultrasonic Cell Disruptor smudge cells, organic phase and mycelium is obtained through centrifugal, mycelium carries out surname extraction 8h again, solvent is methylene dichloride, then merge above-mentioned three part organic phases 35 DEG C of spin concentration on Rotary Evaporators and become medicinal extract shape, medicinal extract is dissolved in 10ml methyl alcohol for column chromatography;
Step 3: column chromatography purification
After AB-8 macroporous resin carries out pre-treatment, wet method dress post.With dehydrated alcohol with the flow velocity of 1.0ml/min rush post stable after, wash-out is carried out with the ethanolic soln that concentration is 30%, flow velocity is 1.5ml/min, collects elutriant, carries out TLC detection, exhibition coating systems (methylene dichloride: methyl alcohol=20: 1), under 254nm ultraviolet lamp, observe fluorescence, then develop the color with the vitriol oil spraying of 1% Vanillin, observe and compare with or without Japanese yew, collect the above-mentioned elutriant with characteristic locus coeruleus, repeated again after concentrated post obtain purifying for twice after sample.
Embodiment 2
Intend from Hainan the method extracting taxol pestalotia bacteria meta-bolites, comprise the following steps:
Step 1: the cultivation of thalline and fermentation
Separation being intended pestalotia bacteria strain from Hainan of giant panda body surface is inoculated on PDA solid medium, 28 DEG C of lucifuges are cultivated, after microscopy produces spore, rinse bacterium colony by 20ml stroke-physiological saline solution and make suspension, suspension is moved into a sterile test tube, airtightly put into whirlpool 15s, bacteria suspension is fully mixed;
Then with pipettor the bacteria suspension prepared proceeded to CPDA fermented liquid (by bacteria suspension: fermented liquid=1: 100), with concussion incubator, 28 DEG C, 200r/min isothermal vibration cultivates 25 days;
Step 2: the extraction of secondary metabolite
CPDA fermented liquid 4 DEG C, 10000r/min, centrifugal 10min, collect supernatant liquor and mycelium respectively.Add 0.5gNaCO
3(amount of 1000ml) is in supernatant liquor, NaCl can be added simultaneously, use isopyknic ethyl acetate: acetone extract three times, each 4h, add methyl alcohol (by mycelia weight: methyl alcohol volume=1: 10g:ml) to mycelium to stir, with 24h freezing after Ultrasonic Cell Disruptor smudge cells, organic phase and mycelium is obtained through centrifugal, mycelium carries out surname extraction 8h again, solvent is methylene dichloride, then merge above-mentioned three part organic phases 35 DEG C of spin concentration on Rotary Evaporators and become medicinal extract shape, medicinal extract is dissolved in 10ml methyl alcohol for column chromatography;
Step 3: column chromatography purification
After AB-8 macroporous resin carries out pre-treatment, wet method dress post.With dehydrated alcohol with the flow velocity of 1.0ml/min rush post stable after, wash-out is carried out with the ethanolic soln that concentration is 40%, flow velocity is 1.5ml/min, collects elutriant, carries out TLC detection, exhibition coating systems (methylene dichloride: methyl alcohol=20: 1), under 254nm ultraviolet lamp, observe fluorescence, then develop the color with the vitriol oil spraying of 1% Vanillin, observe and compare with or without Japanese yew, collect the above-mentioned elutriant with characteristic locus coeruleus, repeated again after concentrated post obtain purifying for twice after sample.
Embodiment 3
Intend from Hainan the method extracting taxol pestalotia bacteria meta-bolites, comprise the following steps:
Step 1: the cultivation of thalline and fermentation
Separation being intended pestalotia bacteria strain from Hainan of giant panda body surface is inoculated on PDA solid medium, 28 DEG C of lucifuges are cultivated, after microscopy produces spore, rinse bacterium colony by 20ml stroke-physiological saline solution and make suspension, suspension is moved into a sterile test tube, airtightly put into whirlpool 15s, bacteria suspension is fully mixed;
Then with pipettor the bacteria suspension prepared proceeded to CPDA fermented liquid (by bacteria suspension: fermented liquid=1: 100), with concussion incubator, 28 DEG C, 200r/min isothermal vibration cultivates 28 days;
Step 2: the extraction of secondary metabolite
CPDA fermented liquid 4 DEG C, 10000r/min, centrifugal 10min, collect supernatant liquor and mycelium respectively.Add 0.5gNaCO
3(amount of 1000ml) is in supernatant liquor, NaCl can be added simultaneously, use isopyknic ethyl acetate: acetone extract three times, each 4h, add methyl alcohol (by mycelia weight: methyl alcohol volume=1: 10g:ml) to mycelium to stir, with 24h freezing after Ultrasonic Cell Disruptor smudge cells, organic phase and mycelium is obtained through centrifugal, mycelium carries out surname extraction 8h again, solvent is methylene dichloride, then merge above-mentioned three part organic phases 35 DEG C of spin concentration on Rotary Evaporators and become medicinal extract shape, medicinal extract is dissolved in 10ml methyl alcohol for column chromatography;
Step 3: column chromatography purification
After AB-8 macroporous resin carries out pre-treatment, wet method dress post.With dehydrated alcohol with the flow velocity of 1.0ml/min rush post stable after, wash-out is carried out with the ethanolic soln that concentration is 50%, flow velocity is 1.5ml/min, collects elutriant, carries out TLC detection, exhibition coating systems (methylene dichloride: methyl alcohol=20: 1), under 254nm ultraviolet lamp, observe fluorescence, then develop the color with the vitriol oil spraying of 1% Vanillin, observe and compare with or without Japanese yew, collect the above-mentioned elutriant with characteristic locus coeruleus, repeated again after concentrated post obtain purifying for twice after sample.
Embodiment 4
Intend from Hainan the method extracting taxol pestalotia bacteria meta-bolites, comprise the following steps:
Step 1: the cultivation of thalline and fermentation
Separation being intended pestalotia bacteria strain from Hainan of giant panda body surface is inoculated on PDA solid medium, 28 DEG C of lucifuges are cultivated, after microscopy produces spore, rinse bacterium colony by 20ml stroke-physiological saline solution and make suspension, suspension is moved into a sterile test tube, airtightly put into whirlpool 15s, bacteria suspension is fully mixed;
Then with pipettor the bacteria suspension prepared proceeded to CPDA fermented liquid (by bacteria suspension: fermented liquid=1: 100), with concussion incubator, 28 DEG C, 200r/min isothermal vibration cultivates 23 days;
Step 2: the extraction of secondary metabolite
CPDA fermented liquid 4 DEG C, 10000r/min, centrifugal 10min, collect supernatant liquor and mycelium respectively.Add 0.5gNaCO
3(amount of 1000ml) is in supernatant liquor, NaCl can be added simultaneously, use isopyknic ethyl acetate: acetone extract three times, each 4h, add methyl alcohol (by mycelia weight: methyl alcohol volume=1: 10g:ml) to mycelium to stir, with 24h freezing after Ultrasonic Cell Disruptor smudge cells, organic phase and mycelium is obtained through centrifugal, mycelium carries out surname extraction 8h again, solvent is methylene dichloride, then merge above-mentioned three part organic phases 35 DEG C of spin concentration on Rotary Evaporators and become medicinal extract shape, medicinal extract is dissolved in 10ml methyl alcohol for column chromatography;
Step 3: column chromatography purification
After AB-8 macroporous resin carries out pre-treatment, wet method dress post.With dehydrated alcohol with the flow velocity of 1.0ml/min rush post stable after, wash-out is carried out with the ethanolic soln that concentration is 60%, flow velocity is 1.5ml/min, collects elutriant, carries out TLC detection, exhibition coating systems (methylene dichloride: methyl alcohol=20: 1), under 254nm ultraviolet lamp, observe fluorescence, then develop the color with the vitriol oil spraying of 1% Vanillin, observe and compare with or without Japanese yew, collect the above-mentioned elutriant with characteristic locus coeruleus, repeated again after concentrated post obtain purifying for twice after sample.
Embodiment 5
Intend from Hainan the method extracting taxol pestalotia bacteria meta-bolites, comprise the following steps:
Step 1: the cultivation of thalline and fermentation
Separation being intended pestalotia bacteria strain from Hainan of giant panda body surface is inoculated on PDA solid medium, 28 DEG C of lucifuges are cultivated, after microscopy produces spore, rinse bacterium colony by 20ml stroke-physiological saline solution and make suspension, suspension is moved into a sterile test tube, airtightly put into whirlpool 15s, bacteria suspension is fully mixed;
Then with pipettor the bacteria suspension prepared proceeded to CPDA fermented liquid (by bacteria suspension: fermented liquid=1: 100), with concussion incubator, 28 DEG C, 200r/min isothermal vibration cultivates 25 days;
Step 2: the extraction of secondary metabolite
CPDA fermented liquid 4 DEG C, 10000r/min, centrifugal 10min, collect supernatant liquor and mycelium respectively.Add 0.5gNaCO
3(amount of 1000ml) is in supernatant liquor, NaCl can be added simultaneously, use isopyknic ethyl acetate: acetone extract three times, each 4h, add methyl alcohol (by mycelia weight: methyl alcohol volume=1: 10g:ml) to mycelium to stir, with 24h freezing after Ultrasonic Cell Disruptor smudge cells, organic phase and mycelium is obtained through centrifugal, mycelium carries out surname extraction 8h again, solvent is methylene dichloride, then merge above-mentioned three part organic phases 35 DEG C of spin concentration on Rotary Evaporators and become medicinal extract shape, medicinal extract is dissolved in 10ml methyl alcohol for column chromatography;
Step 3: column chromatography purification
After AB-8 macroporous resin carries out pre-treatment, wet method dress post.With dehydrated alcohol with the flow velocity of 1.0ml/min rush post stable after, wash-out is carried out with the ethanolic soln that concentration is 70%, flow velocity is 1.5ml/min, collects elutriant, carries out TLC detection, exhibition coating systems (methylene dichloride: methyl alcohol=20: 1), under 254nm ultraviolet lamp, observe fluorescence, then develop the color with the vitriol oil spraying of 1% Vanillin, observe and compare with or without Japanese yew, collect the above-mentioned elutriant with characteristic locus coeruleus, repeated again after concentrated post obtain purifying for twice after sample.
The above; be only the present invention's preferably embodiment; protection scope of the present invention is not limited thereto; anyly be familiar with those skilled in the art in the technical scope that the present invention discloses, the simple change of the technical scheme that can obtain apparently or equivalence are replaced and are all fallen within the scope of protection of the present invention.
Claims (4)
1. intend from Hainan the method extracting taxol pestalotia bacteria meta-bolites, it is characterized in that, comprise the following steps:
Step 1: the cultivation of thalline and fermentation
Separation being intended pestalotia bacteria strain from Hainan of giant panda body surface is inoculated on PDA solid medium, 28 DEG C of lucifuges are cultivated, after microscopy produces spore, rinse bacterium colony by 20ml stroke-physiological saline solution and make suspension, suspension is moved into a sterile test tube, airtightly put into whirlpool 15s, bacteria suspension is fully mixed;
Then with pipettor, the bacteria suspension prepared is proceeded to CPDA fermented liquid, with concussion incubator, 28 DEG C, 200r/min isothermal vibration cultivate 21 ~ 28 days;
Step 2: the extraction of secondary metabolite
CPDA fermented liquid 4 DEG C, 10000r/min, centrifugal 10min, collect supernatant liquor and mycelium respectively; Add 0.5gNaCO
3to in supernatant liquor, add NaCl simultaneously, use isopyknic ethyl acetate: acetone extract three times, each 4h, adds methyl alcohol to mycelium and stirs, with 24h freezing after Ultrasonic Cell Disruptor smudge cells, obtain organic phase and mycelium through centrifugal, mycelium carries out surname extraction 8h again, and solvent is methylene dichloride, then merge above-mentioned three part organic phases 35 DEG C of spin concentration on Rotary Evaporators and become medicinal extract shape, medicinal extract is dissolved in 10ml methyl alcohol for column chromatography;
Step 3: column chromatography purification
After AB-8 macroporous resin carries out pre-treatment, wet method dress post; With dehydrated alcohol with the flow velocity of 1.0ml/min rush post stable after, wash-out is carried out with the ethanolic soln that concentration is 30%, 40%, 50%, 60%, 70%, flow velocity is 1.5ml/min, collects elutriant, carries out TLC detection, exhibition coating systems, under 254nm ultraviolet lamp, observe fluorescence, then develop the color with the vitriol oil spraying of 1% Vanillin, observe and compare with or without Japanese yew, collect the above-mentioned elutriant with characteristic locus coeruleus, repeated again after concentrated post obtain purifying for twice after sample.
2. the method extracting taxol from the plan pestalotia bacteria meta-bolites of Hainan according to claim 1, it is characterized in that, the volume ratio of bacteria suspension described in step 1 and fermented liquid is 1: 100.
3. the method extracting taxol from the plan pestalotia bacteria meta-bolites of Hainan according to claim 1, is characterized in that, the weight of mycelia described in step 2: methyl alcohol volume=1g: 10ml.
4. the method extracting taxol from the plan pestalotia bacteria meta-bolites of Hainan according to claim 1, it is characterized in that, opening up coating systems in step 3 is methylene dichloride and methyl alcohol, and its volume ratio is 20: 1.
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Cited By (1)
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| CN112592327A (en) * | 2020-12-15 | 2021-04-02 | 大连大学 | Polyketide separated from microbial pestalotiopsis, separation method and application thereof |
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| CN112592327A (en) * | 2020-12-15 | 2021-04-02 | 大连大学 | Polyketide separated from microbial pestalotiopsis, separation method and application thereof |
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