CN102603758A - Extraction method of camptothecin and 9-hydroxymethyl camptothecin from tender leaves and twigs of Nothapodytes nimmoniana - Google Patents
Extraction method of camptothecin and 9-hydroxymethyl camptothecin from tender leaves and twigs of Nothapodytes nimmoniana Download PDFInfo
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Abstract
The invention relates to the field of drug extraction, in particular to an extraction method of two plant anticancer drugs, namely camptothecin and 9-hydroxymethyl camptothecin, from tender leaves and twigs of Nothapodytes nimmoniana. The invention provides a method for extracting camptothecin and 9-hydroxymethyl camptothecin by microbial fermentation and high purity extraction. The method can greatly increase the content of camptothecin in tender leaves and twigs of Nothapodytes nimmoniana so as to increase the yield of camptothecin, overcome the defect of low extraction amount of camptothecin in prior arts, decrease drug cost and save land source.
Description
Technical field
The present invention relates to the extract drugs field, especially a kind of method that adopts abstraction technique from the tender leaf of natural medicinal plant form nothapodytes and spray, to extract NSC 94600 and 9-methylol NSC 94600 two kind of plant cancer therapy drugs.
Background technology
NSC 94600 is a kind of botanical anticancer medicine, and intestines and stomach and Head and Neck cancer etc. is had short term effect preferably.Traditional NSC 94600 is from fruit of Central-South, the southwestern camplotheca acuminata of China and root skin, to extract to obtain.The content of NSC 94600 in camplotheca acuminata is extremely low then, depends merely on and from camplotheca acuminata, extracts the application demand that obviously can't satisfy NSC 94600.1976, Chinese chemist is high, and happy living the grade successfully synthesized the racemization NSC 94600, opened new era of domestic synthetic NSC 94600.Afterwards, there is scientists to improve the method for NSC 94600 synthetic again successively.Yet the NSC 94600 of synthetic is not natural after all, and production technique and cost are also higher, makes artificial synthesis can't promote at all.
Form nothapodytes is a kind of medicinal plant that uses high-tech means domestic artificial growth in Guangdong Province, have document (phytochemry, 1972) to report in this plant abroad and contain NSC 94600, and its content is up to 2/10000ths. five.This manual work for NSC 94600 is extracted and has been brought good news.2008, publication number was that CN101289452A discloses a kind of method of from form nothapodytes, extracting NSC 94600, and this method adopts abstraction technique, from the branches and leaves of form nothapodytes, has extracted the high purity NSC 94600.But this method adopts traditional extraction means, and its output still has much room for improvement.
Summary of the invention
For overcoming the low defective of traditional NSC 94600 extracted amount, the present invention provides a kind of method of utilizing microbial fermentation to carry out high purity NSC 94600 and the extraction of 9-methylol NSC 94600 more earlier.This method can greatly improve the camptothecine content in Root-bark of Pittosporumlike Nothapodytes tender leaf and the spray, thereby improves the yield of NSC 94600.
The present invention provides the method for extracting NSC 94600 a kind of tender leaf and spray from form nothapodytes, and its step comprises:
(1) pre-treatment: choose fresh form nothapodytes tender leaf and spray, dry back grind into powder;
(2) fermentation: press the enzyme material and add fermented liquid, fermented 1 ~ 3 day than 1.5:1 (w/w); Leavening temperature is 20 ~ 37 ℃;
Said fermented liquid is the high sugared dry yeast in Angel or sends out dry yeast sharp that its concentration is 1 ~ 11 ‰ (w/w);
(3) extract: the ratio in 0.5 ~ 3:1 (w/w) in tunning adds extracting solution, heating and extracting; Said extracting solution is that concentration is 10 ~ 90% ethanolic soln; The temperature of said heating and extracting is controlled between 40 ~ 75 ℃.
(4) filter: the leaching supernatant promptly gets extracting solution;
(5) concentrate: in said extracted liquid, add the sulfurous gas and the ferrous sulfate (inhibitor) of 0.5 ~ 10% (W/V), concentrate, get enriched material;
(6) extraction: in enriched material, add extraction agent enriched material is extracted, and reclaim extraction agent, obtain extracting enriched material, said extraction agent is sherwood oil or methylene dichloride;
(7), purifying: add 10% ~ 90% acetone soln in the enriched material in extraction, adopt silica gel column chromatography earlier, preliminary percent crystallinity reaches about 70%; Carry out lower pressure column layer post again, crystallization purity reaches about 80%; Adopt the FLASH medium pressure column chromatography again, collect elutriant, its crystallization purity reaches about 95%; Carry out recrystallization at last, obtaining purity is 99% finished product.
Described recrystallization is that ratio adding methyl alcohol, acetone, the N (w/w) in 3:2:6 carries out recrystallization in elutriant, collects the xln airing, promptly obtains purity and reaches 99% NSC 94600.
The present invention provides the method for extracting 9-methylol NSC 94600 a kind of tender leaf and spray from form nothapodytes again, and its step comprises:
(1) pre-treatment: choose fresh form nothapodytes tender leaf and spray, dry back grind into powder;
(2) fermentation: add fermented liquid by the enzyme material than 1.5:1, fermented 1 ~ 3 day; Leavening temperature is 20 ~ 37 ℃, and said fermented liquid is the high sugared dry yeast in Angel or sends out dry yeast sharp that its concentration is 1 ~ 11 ‰ (w/w);
(3) extract: in tunning, add extracting solution, heating and extracting; Said extracting solution is an ethanolic soln, and its concentration is: 10 ~ 90%, and said heating and extracting, temperature are controlled between 40 ~ 75 ℃;
(4) concentrate: in said extracted liquid, add 0.5 ~ 10% sulfurous gas and ferrous sulfate (inhibitor), concentrate, must concentrated extract;
(5) extraction: in enriched material, add extraction agent enriched material is extracted, and reclaim extraction agent, obtain extracting enriched material.Said extraction agent is sherwood oil or methylene dichloride;
(6) separate: in the extraction enriched material, add acetone soln, left standstill after stirring 12 ~ 48 hours, suction filtration keeps filtrating and filter cake; With filter cake cruel or methyl chloride dissolving with acetone or acetate second, stir behind the companion suction filtration once more, reclaim filtrating, twice filtrating is mixed, and the filtrating that obtains being contains the bullion of 9-hydroxyl first hydrogen base NSC 94600;
(7) purifying: the bullion of 9-hydroxyl first hydrogen base NSC 94600 is carried out column chromatography, and the Fractional Collections elutriant carries out HPLC and detects, and purity is pulverized greater than the direct concentrate drying of 98% component, obtains finished product; Purity repeats column chromatography less than 90%; Purity is carried out recrystallization at 90% ~ 98% the acetone that adds, and more than or equal to 98%, the crystal drying and crushing obtains finished product up to the purity of 9-hydroxyl first hydrogen base NSC 94600.Said elutriant is an acetone, or Fatty Alcohol(C12-C14 and C12-C18), and its concentration is 20 ~ 90% (w/w).
Embodiment
Following case study on implementation is to describe in further detail of the present invention, but it does not mean that any restriction of the present invention.
Embodiment 1:
Win fresh form nothapodytes tender leaf 500g, put into baking oven dry 12 hours, be poured in the fermentation vat after being ground to powder, add the aqueous solution 750g of dry yeast, holding temperature was fermented 3 days at 28 ℃ ~ 30 ℃;
Tunning is put into the 5L flask, add 50% ethanolic soln 2L, put into 60 ℃ of thermostat water bath insulations 12 hours, the filtering filter residue.
In supernatant, add sulfurous gas 30g, vacuum concentration is 300mL to concentrating liquid measure;
Add sherwood oil 300mL, leave standstill after stirring after 24 hours and remove supernatant layer, lower floor then is the extraction enriched material.
Ratio in 1:1 (w/w) adds 90% acetone soln in the extraction enriched material, slowly be poured into and carry out chromatography in the silicagel column; Carry out low pressure column chromatography again after collecting elutriant; The elutriant of collecting joins that compression leg carries out chromatography among the FLASH again, collects elutriant.
Ratio adding methyl alcohol, acetone, N (w/w) in 3:2:6 in elutriant carry out recrystallization, collect the xln airing, promptly obtain purity and reach 99% NSC 94600.
The invention provides a kind of method of utilizing microbial fermentation to carry out high purity NSC 94600 and the extraction of 9-methylol NSC 94600 more earlier.This method can greatly improve the camptothecine content in Root-bark of Pittosporumlike Nothapodytes tender leaf and the spray, thereby improves the yield of NSC 94600.Overcome the low defective of traditional NSC 94600 extracted amount, reduced the medicine cost, saved land resources.
Embodiment 2:
Choose fresh form nothapodytes spray and tender leaf 1Kg, insolation is 5 days under burning sun, is poured in the fermentation vat after pulverizing, and adds the aqueous solution 750g of dry yeast, and holding temperature was fermented 2 days at 35 ℃ ~ 37 ℃;
Tunning is put into the 2L flask, add 99% ethanolic soln 1L, put into 70 ℃ of thermostat water bath insulations 24 hours, the filtering filter residue.
In supernatant, add ferrous sulfate 50g, vacuum concentration is 150mL to concentrating liquid measure;
Add methylene dichloride 200mL, leave standstill after stirring after 10 hours and remove supernatant layer, lower floor then is the extraction enriched material.
Ratio in 1:2 (w/w) adds 50% acetone soln in the extraction enriched material, slowly be poured into and carry out chromatography in the silicagel column; Carry out low pressure column chromatography again after collecting elutriant; The elutriant of collecting joins that compression leg carries out chromatography among the FLASH again, collects elutriant.
Ratio adding methyl alcohol, acetone, N (w/w) in 3:2:6 in elutriant carry out recrystallization, collect the crystal airing, promptly obtain purity and reach 99% NSC 94600.
Embodiment 3:
Win fresh form nothapodytes tender leaf 500g, put into baking oven dry 12 hours, be poured in the fermentation vat after being ground to powder, add the aqueous solution 1Kg of dry yeast?, holding temperature was fermented 2 days at 20 ℃ ~ 23 ℃;
Tunning is put into the 10L flask, add and carry 50% ethanolic soln 5L, put into 50 ℃ of thermostat water bath insulations 48 hours, the filtering filter residue.
In supernatant, add sulfurous gas 80g, vacuum concentration is 500mL to concentrating liquid measure;
Add sherwood oil 500mL, leave standstill after stirring after 24 hours and pour out supernatant layer, lower floor then is the extraction enriched material.Supernatant adds petroleum ether solution in the 1:1 ratio again, leaves standstill after stirring after 24 hours and pours out supernatant layer, obtains lower floor's extraction enriched material and extraction enriched material before and merges.
In the extraction enriched material, add 70% acetone soln in 1:1 (w/w) ratio; Left standstill after stirring 12 ~ 24 hours, suction filtration is collected an amount of 50% acetone of filtrating adding and is carried out column chromatography; The Fractional Collections elutriant carries out HPLC and detects; Purity is pulverized greater than the direct concentrate drying of 98% component, obtains 9-hydroxyl first hydrogen base NSC 94600 finished product; Purity repeats column chromatography less than 90%; Purity is carried out recrystallization at 90% ~ 98 the acetone that adds, and more than or equal to 98%, the crystal drying and crushing obtains finished product up to the purity of 9-hydroxyl first hydrogen base NSC 94600.
Embodiment 4:
Get fresh form nothapodytes spray and tender leaf 10Kg, insolation is 5 days under burning sun, is poured in the fermentation vat after pulverizing, and adds the aqueous solution 15Kg of dry yeast, and holding temperature was fermented 1 day at 32 ℃ ~ 35 ℃;
Tunning is put into the 10L extractor, adds 99% ethanolic soln 5L, be heated to 70 ℃ after insulation 24 hours, filter, collect filtrating.
In filtrating, add ferrous sulfate 500g, vacuum concentration is 500mL to concentrating liquid measure;
Add 50% dichloromethane solution 1L, leave standstill after stirring after 48 hours and remove supernatant layer, lower floor then is the extraction enriched material.Supernatant adds 70% dichloromethane solution in 1:1 (w/w) ratio again, leaves standstill after stirring after 24 hours and pours out supernatant layer, obtains lower floor's extraction enriched material and extraction enriched material before and merges.
In the extraction enriched material, add 75% acetone soln in 1:2 (w/w) ratio; Left standstill after stirring 12 ~ 24 hours, suction filtration is collected an amount of 75% Fatty Alcohol(C12-C14 and C12-C18) of filtrating adding and is carried out column chromatography; The Fractional Collections elutriant carries out HPLC and detects; Purity is pulverized greater than the direct concentrate drying of 98% component, obtains 9-hydroxyl first hydrogen base NSC 94600 finished product; Purity repeats column chromatography less than 90%; Purity is carried out recrystallization at 90% ~ 98% 90% Fatty Alcohol(C12-C14 and C12-C18) that adds, and more than or equal to 98%, the crystal drying and crushing obtains finished product up to the purity of 9-hydroxyl first hydrogen base NSC 94600.
Claims (5)
1. method of from the tender leaf of form nothapodytes and spray, extracting NSC 94600, its step comprises:
Pre-treatment: choose fresh form nothapodytes tender leaf and spray, dry back grind into powder;
Fermentation: press the enzyme material and add fermented liquid, fermented 1 ~ 3 day than 1.5:1 (w/w); Leavening temperature is 20 ~ 37 ℃, and said fermented liquid is the high sugared dry yeast in Angel or sends out dry yeast sharp that its concentration is 1 ~ 11 ‰ (w/w);
Extract: the ratio in 0.5 ~ 3:1 (w/w) in tunning adds extracting solution, heating and extracting; Said extracting solution is that concentration is 10 ~ 90% ethanolic soln; The temperature of said heating and extracting is controlled between 40 ~ 75 ℃;
Filter: the leaching supernatant promptly gets extracting solution;
(5), concentrate: in said extracted liquid, add the sulfurous gas and the ferrous sulfate of 0.5 ~ 10% (W/V), concentrate, get enriched material;
(6), extraction: in enriched material, add extraction agent enriched material is extracted, and reclaim extraction agent, obtain extracting enriched material, said extraction agent is sherwood oil or methylene dichloride;
(7), purifying: add 10% ~ 90% acetone soln in the enriched material in extraction, adopt silica gel column chromatography earlier, preliminary percent crystallinity reaches about 70%; Carry out lower pressure column layer post again, crystallization purity reaches about 80%; Adopt the FLASH medium pressure column chromatography again, collect elutriant, its crystallization purity reaches about 95%; Carry out recrystallization at last, obtaining purity is 99% finished product.
2. extract the method for NSC 94600 a kind of tender leaf and spray from form nothapodytes according to claim 1, its step: (1) described fermented liquid is the high sugared dry yeast in Angel or sends out dry yeast sharp that its concentration is 1 ~ 11 ‰ (w/w).
3. extract the method for NSC 94600 a kind of tender leaf and spray from form nothapodytes according to claim 1 and 2, the described extraction agent of its step (6) is sherwood oil or methylene dichloride.
4. extract the method for NSC 94600 a kind of tender leaf and spray from form nothapodytes according to claim 1 and 2, the described recrystallization of its step (7) is in elutriant, to add methyl alcohol, acetone, N (w/w) in the ratio of 3:2:6 to carry out recrystallization.
5. method of from the tender leaf of form nothapodytes and spray, extracting 9-methylol NSC 94600, its step comprises:
(1) pre-treatment: choose fresh form nothapodytes tender leaf and spray, dry back grind into powder;
(2) fermentation: add fermented liquid by the enzyme material than 1.5:1, fermented 1 ~ 3 day; Leavening temperature is 20 ~ 37 ℃, and said fermented liquid is the high sugared dry yeast in Angel or sends out dry yeast sharp that its concentration is 1 ~ 11 ‰ (w/w);
(3) extract: in tunning, add extracting solution, heating and extracting; Said extracting solution is an ethanolic soln, and its concentration is: 10 ~ 90%, and said heating and extracting, temperature are controlled between 40 ~ 75 ℃;
(4) concentrate: in said extracted liquid, add 0.5 ~ 10% sulfurous gas and ferrous sulfate (inhibitor), concentrate, must concentrated extract;
(5) extraction: in enriched material, add extraction agent enriched material is extracted, and reclaim extraction agent, obtain extracting enriched material,
Said extraction agent is sherwood oil or methylene dichloride;
(6) separate: in the extraction enriched material, add acetone soln, left standstill after stirring 12 ~ 48 hours, suction filtration keeps filtrating and filter cake; With filter cake cruel or methyl chloride dissolving with acetone or acetate second, stir behind the companion suction filtration once more, reclaim filtrating, twice filtrating is mixed, and the filtrating that obtains being contains the bullion of 9-hydroxyl first hydrogen base NSC 94600;
(7) purifying: the bullion of 9-hydroxyl first hydrogen base NSC 94600 is carried out column chromatography, and the Fractional Collections elutriant carries out HPLC and detects, and purity is pulverized greater than the direct concentrate drying of 98% component, obtains finished product; Purity repeats column chromatography less than 90%; Purity is carried out recrystallization at 90% ~ 98% the acetone that adds, and more than or equal to 98%, the crystal drying and crushing obtains finished product up to the purity of 9-hydroxyl first hydrogen base NSC 94600.
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| CN201210030118XA CN102603758A (en) | 2012-02-12 | 2012-02-12 | Extraction method of camptothecin and 9-hydroxymethyl camptothecin from tender leaves and twigs of Nothapodytes nimmoniana |
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| CN201210030118XA CN102603758A (en) | 2012-02-12 | 2012-02-12 | Extraction method of camptothecin and 9-hydroxymethyl camptothecin from tender leaves and twigs of Nothapodytes nimmoniana |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015010293A1 (en) * | 2013-07-24 | 2015-01-29 | Zhang Yuanjin | Anticancer pharmaceutical composition, preparation method for same, and use thereof |
| CN105053041A (en) * | 2015-08-11 | 2015-11-18 | 长沙秋点兵信息科技有限公司 | Pesticide for crops |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US6111108A (en) * | 1997-07-01 | 2000-08-29 | Midwest Research Institute | Extraction of biologically active components from Camptotheca acuminata with supercritical fluids |
| US20040192917A1 (en) * | 2003-03-31 | 2004-09-30 | Srivastava Santosh Kumar | Process for isolation of anticancer agent camptothecin from nothapodytes foetida |
| CN101289452A (en) * | 2008-06-12 | 2008-10-22 | 张苑金 | Process for abstracting camptothecine form nothapodytes |
| CN102051393A (en) * | 2009-11-10 | 2011-05-11 | 恩施市锦华生物工程有限责任公司 | Method for extracting camptothecin and 9-methoxyl camptothecin from root-bark of pittosporumlike nothapodytes |
| CN102093374A (en) * | 2010-11-30 | 2011-06-15 | 东北林业大学 | Method for efficiently extracting camptothecin derivative |
| CN102127089A (en) * | 2010-11-03 | 2011-07-20 | 张守力 | Method for extracting camptothecin |
-
2012
- 2012-02-12 CN CN201210030118XA patent/CN102603758A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6111108A (en) * | 1997-07-01 | 2000-08-29 | Midwest Research Institute | Extraction of biologically active components from Camptotheca acuminata with supercritical fluids |
| US20040192917A1 (en) * | 2003-03-31 | 2004-09-30 | Srivastava Santosh Kumar | Process for isolation of anticancer agent camptothecin from nothapodytes foetida |
| CN101289452A (en) * | 2008-06-12 | 2008-10-22 | 张苑金 | Process for abstracting camptothecine form nothapodytes |
| CN102051393A (en) * | 2009-11-10 | 2011-05-11 | 恩施市锦华生物工程有限责任公司 | Method for extracting camptothecin and 9-methoxyl camptothecin from root-bark of pittosporumlike nothapodytes |
| CN102127089A (en) * | 2010-11-03 | 2011-07-20 | 张守力 | Method for extracting camptothecin |
| CN102093374A (en) * | 2010-11-30 | 2011-06-15 | 东北林业大学 | Method for efficiently extracting camptothecin derivative |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015010293A1 (en) * | 2013-07-24 | 2015-01-29 | Zhang Yuanjin | Anticancer pharmaceutical composition, preparation method for same, and use thereof |
| CN105053041A (en) * | 2015-08-11 | 2015-11-18 | 长沙秋点兵信息科技有限公司 | Pesticide for crops |
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Application publication date: 20120725 |