CN102126941B - Preparation method of euphorbia peplus plant extract for preparing antitumor medicament - Google Patents
Preparation method of euphorbia peplus plant extract for preparing antitumor medicament Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title description 5
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- GNFTZDOKVXKIBK-UHFFFAOYSA-N 3-(2-methoxyethoxy)benzohydrazide Chemical compound COCCOC1=CC=CC(C(=O)NN)=C1 GNFTZDOKVXKIBK-UHFFFAOYSA-N 0.000 claims description 6
- 239000012535 impurity Substances 0.000 claims description 6
- 241000701408 Euphorbia kansui Species 0.000 claims description 5
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- QDRKDTQENPPHOJ-UHFFFAOYSA-N sodium ethoxide Chemical compound [Na+].CC[O-] QDRKDTQENPPHOJ-UHFFFAOYSA-N 0.000 claims description 3
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- XDTMQSROBMDMFD-UHFFFAOYSA-N Cyclohexane Chemical compound C1CCCCC1 XDTMQSROBMDMFD-UHFFFAOYSA-N 0.000 description 2
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- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 description 1
- CHWRSCGUEQEHOH-UHFFFAOYSA-N potassium oxide Chemical compound [O-2].[K+].[K+] CHWRSCGUEQEHOH-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention relates to a method for extracting a euphorbia peplus plant extract for preparing an antitumor medicament from a euphorbia peplus plant. The method comprises the following process steps of: smashing raw materials; performing reflux extraction; concentrating under reduced pressure; extracting; performing back-extraction; performing alkaline hydrolysis; adjusting PH value to 7; concentrating under reduced pressure; performing silica gel column chromatography; performing gradient elution with petroleum ether and ethyl acetate; concentrating; crystallizing; and drying. The method has the advantages of capability of realizing industrial production scale because industrially readily-available separating materials and reagents are adopted, high product quality, content of over 99.9percent and strong international market competitiveness.
Description
Technical Field
The invention relates to a traditional Chinese medicine extraction method, in particular to a preparation method of an extract of a plant of euphorbia of euphorbiaceae for preparing an anti-tumor medicine, and particularly relates to a preparation method of high-purity euphorbia diterpene alcohol.
Background
Tumor (tumor) is a new organism formed by the loss of normal regulation and control of local tissue cells on gene level under the action of various tumorigenic factors, resulting in clonal abnormal hyperplasia. Since the 70 s of the 20 th century, the incidence and mortality of malignant tumors in China have been on the rising trend. In recent years, however, the incidence and mortality of some major foreign tumors has begun to decline, mainly due to the ongoing research and development of medicine.
The natural compound ingenol-3-angelate separated and screened from Euphorbia plants (Euphorbia peplus) in the family euphorbiaceae by combination of australian Peplin Biotech Ltd and Queensland Institute of Medical Research has significant anti-tumor activity, the action mechanism is to promote the activity of Protein Kinase (PKC), the clinical cure rate of the phase II on skin cancer is more than 90%, the 3 months in 2010, and the clinical result of the phase III published by the denmark LEO Pharma also shows that the expected good effect is achieved.
The domestic natural product of the same kernel also has anti-tumor activity of extracts of plants of the Euphorbiaceae Euphorbia, especially Euphorbia diterpenol derivatives. Euphol (ingenol), a novel and complex structure, belongs to tetracyclic diterpenes. The serial derivatives with ingenol as mother nucleus have obvious anticancer and antiviral activity. Medical workers at home and abroad do a great deal of intensive work on the series of derivatives and obtain a lot of achievements.
In 2003, 4 ingenol derivatives were isolated from roots of Euphorbia kansui (Euphorbia kansui) by yao et al, Shenyang pharmaceutical university, and activity tests showed that all of them had significant antitumor activity. In 2008, shanghai pharmaceutical research institute, dodean et al, classified 16 ingenol derivatives from the whole plant of euphorbia lactiflora (eupolyphbia esula) all have anti-tumor activity.
Since the discovery of significant activity of ingenol derivatives, the series of compounds with ingenol and its parent nucleus continues to be highly appreciated by researchers in world medicine. They have conducted extensive and intensive research around this class of compounds and published a number of papers 120 in international well-known journals. Although the synthesis of ingenol by a chemical method is reported, the total synthesis cost of the ingenol cannot be reduced due to a novel and complicated structure, so that the ingenol is an expensive compound all the time, and the research and the application of derivatives of the ingenol are seriously influenced.
Euphorbiae Lathyridis semen is mature seed of Euphorbiae Lathyridis semen (Euphorbiae Lathyridis) of Euphorbiae of Euphorbiaceae, is a toxic Chinese medicine, and is recorded in Chinese pharmacopoeia. The semen Euphorbiae is rich in 40-50% of fatty oil, and has been studied as energy plant in China. Before ingenol can not be completely synthesized, caper euphorbia seed is used as a raw material for preparing the ingenol in order to meet the requirement of synthesizing derivatives of the ingenol. Although Giovanni appendix is equal to the process for preparing ingenol reported in 1999 (J.Nat.Prod.1999.62, 76-79), it has severely limited its application due to the use of expensive cyclohexane and the use of potassium cyanide, a highly toxic chemical.
Disclosure of Invention
The invention is carried out on the premise that the process of ingenol total synthesis is not mature and the cost is high, and aims to overcome the defects in the prior art and provide the process for extracting the euphorbia diterpenoid alcohol from the euphorbia plant in the euphorbiaceae, which saves the cost, reduces the price of the medicine and improves the yield.
The invention is realized by the following steps:
a) crushing raw materials: pulverizing Euphorbiaceae Euphorbia into granules;
b) reflux extraction: taking 1 weight part of the crushed raw materials, and carrying out reflux extraction by using a solvent of 80-100% by volume ratio, wherein the solvent is methanol, ethanol or a mixture thereof;
c) and (3) concentrating under reduced pressure: mixing the extractive solutions, pumping into a concentrating tank, concentrating under reduced pressure to obtain extract, and cooling;
d) and (3) extraction: adding petroleum ether into the extract for extraction, and separating the upper layer petroleum ether from the lower layer water and insoluble solid;
e) back extraction: collecting petroleum ether liquid, placing the petroleum ether liquid in an extraction tank, adding acetonitrile, stirring for 5 minutes, standing for layering, discharging lower acetonitrile extract, performing back extraction for 2 times according to the same operation, and combining the acetonitrile extracted for 3 times;
f) and (3) concentrating under reduced pressure: recovering acetonitrile extract under reduced pressure to obtain soft extract;
g) alkali hydrolysis: dissolving the thick paste in methanol to obtain a sample solution, and adding alkali for hydrolysis for 0-48 hours;
h) neutralizing: adding hydrochloric acid into the alkali hydrolysate to adjust the pH value to 7;
i) and (3) concentrating under reduced pressure: recovering the neutralized solution under reduced pressure to obtain extract;
j) silica gel chromatography: mixing the extract and silica gel at a weight ratio of 1: 1, oven drying, performing silica gel column chromatography, eluting with petroleum ether and ethyl acetate at a ratio of 1: 1 to remove impurities, and eluting with ethyl acetate to obtain ethyl acetate eluate;
k) and (3) crystallization and purification: concentrating the ethyl acetate eluate under reduced pressure, placing in refrigerator at-20-5 deg.C for 0-48 hr, and crystallizing to obtain extract of Euphorbia plant of Euphorbiaceae.
Wherein,
the plant of Euphorbiaceae Euphorbia in step a) is preferably Euphorbiae Lathyridis semen, radix kansui;
the solvent for reflux extraction in the step b) is preferably methanol or 95% ethanol, the reflux extraction frequency is 3 times, 5 parts by weight of the solvent is added for reflux extraction for 2 hours for the first time, 4 parts by weight of the solvent is added for reflux extraction for 1.5 hours for the second time, and 4 parts by weight of the solvent is added for reflux extraction for 1.5 hours for the third time;
0.5 part by weight of petroleum ether is added in the step d);
the volume of the acetonitrile added in the step e) is 1/3 of the volume of the petroleum ether;
the concentration of the sample solution in the step g) is 0.1 g/ml;
the alkali added in the step g) is sodium hydroxide, strong potassium oxide, sodium methoxide and sodium ethoxide, preferably sodium hydroxide and sodium methoxide, and the concentration of the alkali is 0.1-0.5M after the alkali is added;
step g) alkaline hydrolysis to 0.25M sodium hydroxide at 30 ℃ for 24 hours or 0.2M sodium methoxide at 25 ℃ for 24 hours;
the crystallization in step k) is carried out under conditions of-15 ℃ for 24 hours.
By HPLC, Infrared (IR), charcoal (C13C-NMR spectrum and hydrogen (C-NMR)1H-NMR) spectrum detection and identification, the extract of the plant of euphorbia of euphorbiaceae prepared by the above method is high-purity euphorbia diterpenoid alcohol (ingenol).
The content is determined according to the high performance liquid chromatography (appendix VID) of Chinese pharmacopoeia.
The chromatographic condition takes octadecylsilane chemically bonded silica as a filler; methanol-water (53: 47) as mobile phase; detection by an evaporative light scattering detector. The number of theoretical plates should not be less than 1000 calculated from the ingenol peak.
The invention preferably adopts two plants of the stephania japonica and the euphorbia kansui as extraction sources, and has the advantages that: the two plants are medicinal plants which are widely planted in China, and the two plants are both varieties recorded in Chinese pharmacopoeia, which is beneficial to continuously, stably and massively obtaining raw materials, and ensures the continuous, stable and massive supply of ingenol from the source, so that the natural ecological environment is not damaged due to the predatory collection of resources caused by the massive demand of the ingenol.
The invention has the advantages and innovation points that:
(1) the invention uses petroleum ether with low price and easy obtaining to replace cyclohexane with high price in the prior art, and greatly reduces the production cost while achieving good extraction and separation effects.
(2) The invention uses sodium hydroxide and sodium methoxide with low price and easy obtaining to replace potassium cyanide which is a highly toxic chemical product hardly obtained in industry, and can achieve better hydrolysis effect, simultaneously enable industrial production to become possible, and greatly reduce production cost.
(3) The time of alkaline hydrolysis in the prior art is 7 days, and the invention adopts sodium hydroxide and sodium methoxide, so that the time of alkaline hydrolysis is shortened to be within 1 day, and the production period of ingenol is greatly shortened.
(4) The invention adopts ethyl acetate for recrystallization, can remove the fat-soluble impurities such as alkene alcohol and the like still remained after the silica gel column chromatography by means of a crystallization method, and the purity of the product can reach more than 99.9 percent.
Drawings
FIG. 1 shows the structural formula of euphorbia diterpene alcohols;
FIG. 2 is an HPLC chromatogram of euphorbia diterpenoid alcohol obtained by the present invention;
FIG. 3 is an IR spectrum of the euphorbia diterpenoid alcohol obtained by the present invention;
FIG. 4 shows the preparation of ingenol obtained by the present invention1H-NMR spectrum;
FIG. 5 shows the preparation of ingenol obtained by the present invention13A C-NMR spectrum;
Detailed Description
The invention is further illustrated by the following specific embodiments:
example 1
a) Selecting 100kg of semen Euphorbiae, and pulverizing into granules.
b) Reflux extraction: extracting the pulverized plant granules with methanol under reflux for 3 times. Adding 600L methanol for reflux extraction for 2 hr for the first time, adding 500L methanol for reflux extraction for 1.5 hr for the second time, adding 500L methanol for reflux extraction for 1.5 hr for the third time,
c) and (3) concentrating under reduced pressure: mixing the above three extractive solutions, pumping into a concentrating tank, concentrating under reduced pressure to obtain a thin paste, and cooling.
d) And (3) extraction: adding 60L petroleum ether to extract the above thin paste. The upper petroleum ether layer was separated from the lower water and insoluble solids.
e) Back extraction: collecting the petroleum ether liquid, placing the petroleum ether liquid in an extraction tank, adding 20L of acetonitrile, stirring for 5 minutes, standing for layering, discharging the lower acetonitrile extract, performing back extraction for 2 times according to the same operation, and combining the acetonitrile extracted for 3 times.
f) And (3) concentrating under reduced pressure: and decompressing and recycling the acetonitrile extract liquid to be extractum.
g) Alkali hydrolysis: the sample was dissolved in 40L of methanol, and 400g of sodium hydroxide was added. The temperature is 30 ℃ for 24 hours.
h) Neutralizing: adding hydrochloric acid about 550ml into the above alkaline hydrolysis solution to adjust pH to 7
i) And (3) concentrating under reduced pressure: and decompressing and recycling the methanol solution with the pH value adjusted to 7 to be extractum.
j) Silica gel chromatography: mixing the above extract with 600g silica gel, oven drying, separating by chromatography in a chromatographic column with 3kg silica gel in the lower layer, eluting with petroleum ether and ethyl acetate at ratio of 1: 1 to remove impurities, and eluting with ethyl acetate to obtain the desired compound euphorbia diterpene alcohol.
k) And (3) crystallization: concentrating the ethyl acetate part under reduced pressure, standing at-5 deg.C for 24 hr, crystallizing to separate out Euphorbia diterpene alcohol 18g, and detecting with high performance liquid chromatography to obtain a content of above 99.9%.
Example 2
a) Selecting 100kg of euphorbia kansui, and crushing into granules.
b) Reflux extraction: extracting the pulverized plant granules with methanol under reflux for 3 times. Adding 500L of the mixture for reflux extraction for 2 hours for the first time, adding 400L of the mixture for reflux extraction for 1.5 hours for the second time, adding 400L of the mixture for reflux extraction for 1.5 hours for the third time,
c) and (3) concentrating under reduced pressure: mixing the above three extractive solutions, pumping into a concentrating tank, concentrating under reduced pressure to obtain a thin paste, and cooling.
d) And (3) extraction: adding 50L petroleum ether to extract the above thin paste. The upper petroleum ether layer was separated from the lower water and insoluble solids.
e) Back extraction: collecting the petroleum ether liquid, placing the petroleum ether liquid in an extraction tank, adding 16L of acetonitrile, stirring for 5 minutes, standing for layering, discharging the lower acetonitrile extract, performing back extraction for 2 times according to the same operation, and combining the acetonitrile extracted for 3 times.
f) And (3) concentrating under reduced pressure: and decompressing and recycling the acetonitrile extract liquid to be extractum.
g) Alkali hydrolysis: the sample was dissolved in 40L of methanol, and 216g of sodium methoxide was added. The temperature is 30 ℃ for 24 hours.
h) Neutralizing: adding hydrochloric acid about 30ml into the above alkaline hydrolysis liquid to adjust pH to 7
i) And (3) concentrating under reduced pressure: and decompressing and recycling the methanol solution with the pH value adjusted to 7 to be extractum.
j) Silica gel chromatography: mixing the above extract with 500g silica gel, oven drying, separating by chromatography in a chromatographic column with lower layer containing 2.5kg silica gel, eluting with petroleum ether and ethyl acetate at ratio of 1: 1 to remove impurities, and eluting with ethyl acetate to obtain the desired compound Euphorbia diterpene alcohol.
k) And (3) crystallization: concentrating the ethyl acetate part under reduced pressure, standing at-5 deg.C for 24 hr, crystallizing to separate out 15g of euphorbia diterpene alcohol, and detecting with high performance liquid chromatography to obtain a content of above 99.9%.
Example 3
a) Selecting 100kg of semen Euphorbiae, and pulverizing into granules.
b) Reflux extraction: extracting the pulverized plant granules with 95% ethanol under reflux for 3 times. Adding 600L for reflux extraction for 2 hr for the first time, adding 500L for reflux extraction for 1.5 hr for the second time, adding 500L for reflux extraction for 1.5 hr for the third time,
c) and (3) concentrating under reduced pressure: mixing the above three extractive solutions, pumping into a concentrating tank, concentrating under reduced pressure to obtain a thin paste, and cooling.
d) And (3) extraction: adding 60L petroleum ether to extract the above thin paste. The upper petroleum ether layer was separated from the lower water and insoluble solids.
e) Back extraction: collecting the petroleum ether liquid, placing the petroleum ether liquid in an extraction tank, adding 20L of acetonitrile, stirring for 5 minutes, standing for layering, discharging the lower acetonitrile extract, performing back extraction for 2 times according to the same operation, and combining the acetonitrile extracted for 3 times.
f) And (3) concentrating under reduced pressure: and decompressing and recycling the acetonitrile extract liquid to be extractum.
g) Alkali hydrolysis: the sample was dissolved in 50L of methanol, and 350g of sodium ethoxide was added. The temperature is 30 ℃ for 24 hours.
h) Neutralizing: adding hydrochloric acid 38ml to the above alkaline hydrolysate to adjust pH to 7
i) And (3) concentrating under reduced pressure: and decompressing and recycling the methanol solution with the pH value adjusted to 7 to be extractum.
j) Silica gel chromatography: mixing the above extract with 450g silica gel, oven drying, separating by chromatography in a chromatographic column with lower layer containing 2.5kg silica gel, eluting with petroleum ether and ethyl acetate at ratio of 1: 1 to remove impurities, and eluting with ethyl acetate to obtain the desired compound euphorbia diterpene alcohol.
k) And (3) crystallization: concentrating the ethyl acetate part under reduced pressure, standing at-20 deg.C for 24 hr, crystallizing to separate out 17g of euphorbia diterpene alcohol, and detecting with high performance liquid chromatography to obtain content of above 99.9%.
Claims (5)
1. A method for preparing extract of Euphorbia plant of Euphorbiaceae for preparing antitumor drug comprises the following steps:
a) crushing raw materials: pulverizing Euphorbiaceae Euphorbia into granules;
b) reflux extraction: taking 1 weight part of the crushed raw materials, and carrying out reflux extraction by using a solvent of 80-100% by volume ratio, wherein the solvent is methanol, ethanol or a mixture thereof;
c) and (3) concentrating under reduced pressure: mixing the extractive solutions, pumping into a concentrating tank, concentrating under reduced pressure to obtain extract, and cooling;
d) and (3) extraction: adding petroleum ether into the extract for extraction, and separating the upper layer petroleum ether from the lower layer water and insoluble solid;
e) back extraction: collecting petroleum ether liquid, placing the petroleum ether liquid in an extraction tank, adding acetonitrile, stirring for 5 minutes, standing for layering, discharging lower acetonitrile extract, performing back extraction for 2 times according to the same operation, and combining the acetonitrile extracted for 3 times;
f) and (3) concentrating under reduced pressure: recovering acetonitrile extract under reduced pressure to obtain soft extract;
g) alkali hydrolysis: dissolving the thick paste in methanol to obtain a sample solution, and adding alkali for hydrolysis for 0-48 hours;
h) neutralizing: adding hydrochloric acid into the alkali hydrolysate to adjust the pH value to 7;
i) and (3) concentrating under reduced pressure: recovering the neutralized solution under reduced pressure to obtain extract;
j) silica gel chromatography: mixing the extract and silica gel at a weight ratio of 1: 1, oven drying, performing silica gel column chromatography, eluting with petroleum ether and ethyl acetate at a ratio of 1: 1 to remove impurities, and eluting with ethyl acetate to obtain ethyl acetate eluate;
k) and (3) crystallization and purification: concentrating the ethyl acetate eluate under reduced pressure, and placing in refrigerator at-20-5 deg.C for 0-48 hr to precipitate crystals to obtain extract of Euphorbia plant of Euphorbiaceae;
in the step, the plant of Euphorbiaceae Euphorbia is Euphorbia lathyris or Euphorbia kansui, and the extract of Euphorbia of Euphorbiaceae is Euphorbia diterpene alcohol;
0.5 part by weight of petroleum ether is added in the step d);
the volume of the acetonitrile added in the step e) is 1/3 of the volume of the petroleum ether;
the alkali added in the step g) is sodium hydroxide, potassium hydroxide, sodium methoxide and sodium ethoxide, and the concentration of the alkali is 0.1-0.5M after the alkali is added.
2. The method according to claim 1, wherein the solvent for reflux extraction in step b) is methanol or 95% ethanol, the number of times of reflux extraction is 3, 5 parts by weight of the solvent is added for reflux extraction for 2 hours for the first time, 4 parts by weight of the solvent is added for reflux extraction for 1.5 hours for the second time, and 4 parts by weight of the solvent is added for reflux extraction for 1.5 hours for the third time.
3. The method according to claim 1, wherein the concentration of the sample solution in the step g) is 0.1 g/mL.
4. The process according to claim 1, wherein the step g) of alkaline hydrolysis is carried out by adding 0.25M sodium hydroxide at 30 ℃ for 24 hours or 0.2M sodium methoxide at 25 ℃ for 24 hours.
5. The method according to claim 1, wherein the crystallization in step k) is carried out at-15 ℃ for 24 hours.
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| CN103655674A (en) * | 2012-09-11 | 2014-03-26 | 清华大学深圳研究生院 | Euphorbia esula extract as well as preparation method and application thereof |
| CN105272838B (en) * | 2014-06-11 | 2017-12-01 | 成都普瑞法科技开发有限公司 | The isolation and purification method of ingenol extract |
| CN104447633A (en) * | 2014-11-28 | 2015-03-25 | 天津耀宇生物技术有限公司 | Preparation method of terpenoid |
| CN105982945A (en) * | 2015-02-15 | 2016-10-05 | 沈阳药科大学 | Preparation method and medicinal application of euphorbium antitumor extract and composition thereof |
| DK3270686T3 (en) * | 2015-03-20 | 2020-03-09 | Phyton Holdings Llc | Preparation of ingenol, ingenol esters and / or tiglian-3-one derivatives by Euphorbiaceae plant cell suspension cultures |
| CN105061169B (en) * | 2015-08-03 | 2017-08-18 | 江苏红豆杉药业有限公司 | A kind of ingenol and preparation method thereof |
| CN105152889A (en) * | 2015-08-19 | 2015-12-16 | 西安岳达植物科技有限公司 | Extraction method of ingenol |
| CN106083555A (en) * | 2016-06-14 | 2016-11-09 | 宁波绿之健药业有限公司 | The extracting method of ingenol |
| CN107778159B (en) * | 2016-08-24 | 2021-04-06 | 天津中新药业研究院有限公司 | Preparation method of euphorbia lathyris diterpene alcohol |
| CN107714755A (en) * | 2017-08-24 | 2018-02-23 | 吉林大学 | A kind of Lathyrol ester active component and preparation method thereof |
| CN107693568A (en) * | 2017-11-03 | 2018-02-16 | 九州天润（武汉）中药研究院有限公司 | A kind of preparation method of defatted MOLEPLANT SEED |
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