CN114292279B - Extraction and separation method of cyclopamine as anti-cancer active ingredient of veratrum nigrum - Google Patents
Extraction and separation method of cyclopamine as anti-cancer active ingredient of veratrum nigrum Download PDFInfo
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Abstract
The invention relates to the technical field of medicines, in particular to a method for extracting and separating cyclopamine which is an anti-cancer active ingredient of veratrum. The method mainly aims at the problems of low extraction amount of cyclopamine, complex extraction steps, high extraction difficulty and large input amount, and provides the following technical scheme: step one: selecting freeze-dried veratrum as a raw material, adding ethanol into a centrifuge tube, and uniformly mixing to obtain an extracting solution; step two: adjusting the pH value and then adding petroleum ether; step three: adding dichloromethane extract into the product, evaporating to dryness, and marking as part A; step four: adding dichloromethane into the lower layer liquid, evaporating to dryness, and marking as part B; step five: evaporating the ethanol layer to dryness, and marking as part C; step six: A. the B and C extracts were dissolved in HPLC assay solution to obtain cyclopamine. The extraction and separation method of cyclopamine provided by the invention can efficiently extract and separate cyclopamine which is an anticancer active ingredient of veratrum, has low use equipment cost, is simple and convenient and easy to operate, is suitable for popularization and use, and is mainly applied to extraction of cyclopamine.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a method for extracting and separating cyclopamine which is an anti-cancer active ingredient of veratrum.
Background
Medically, cancer refers to malignant tumors originating in epithelial tissue, the most common of which; cancer has biological characteristics such as abnormal cell differentiation and proliferation, growth loss control, infiltration, metastasis and the like, the occurrence of the cancer is a multi-factor and multi-step complex process, which is divided into three processes of cancer causing, cancer promoting and evolution, and the cure rate of the cancer is lower, so that the expression of "talk about the color change of the cancer" is only known, along with the continuous development of the medical industry, the medical field finds that cyclopamine has a certain effect on cancer treatment through continuous experimental research, cyclopamine realizes the treatment effect by inducing the differentiation of tumor cells and the apoptosis of the tumor cells, and has no influence on normal tissue cells including normal basal epidermis and undifferentiated cells of hair follicles; cyclopamine causes apoptosis by a non-genotoxic mechanism and thus is unlike radiotherapy and most of the currently used chemotherapeutics which act by damaging DNA. These novel effects not achieved by previous cancer chemotherapeutics render cyclopamine suitable for cancer treatment, for the treatment of BCC' S other tumors; the patent CN1525859B proposes the use of cyclopamine in the preparation of medicaments for treating basal cell carcinoma and other tumors, in which the differentiation and apoptosis of basal cell carcinoma cells induced by local treatment of cyclopamine is proposed, thus it can be known that cyclopamine has a certain effect in the treatment of cancer; the veratrum is a traditional Chinese medicinal material, and cyclopamine can be separated, but the prior art does not provide a high-efficiency and simple separation means, so that the problems of low extraction amount of cyclopamine in the veratrum and the like occur.
Disclosure of Invention
The invention aims at solving the problems of low extraction quantity of cyclopamine in veratrum, complex extraction steps, high extraction difficulty and input quantity and the like in the existing means in the background technology, and provides an extraction and separation method of cyclopamine serving as an anticancer active ingredient of veratrum.
The technical scheme of the invention is as follows: an extraction and separation method of cyclopamine which is an anticancer active ingredient of veratrum nigrum comprises the following steps:
step one: selecting the processed freeze-dried veratrum as a raw material, placing the freeze-dried veratrum in a centrifuge tube, adding 10-40ml of ethanol, uniformly mixing, and centrifuging for 10min by a centrifugal device to obtain an extracting solution;
step two: concentrating the extract obtained in the step one to 10ml by a cloth evaporator, regulating the PH value of the concentrated extract to 4 by adopting 5% acetic acid, adding 10ml of petroleum ether at 50 ℃, uniformly mixing, and discarding a petroleum ether layer;
step three: adding 10ml of dichloromethane extract into the product obtained in the second step, uniformly mixing, standing, sucking the extract, placing the extract into a new glass tube, repeatedly extracting with 10ml of dichloromethane, combining the extracts, evaporating to dryness, and marking as a part A;
step four: adding 10ml of dichloromethane into the residual lower layer liquid in the third step, uniformly mixing, standing, sucking the extracting solution, placing the extracting solution in a new glass tube, repeatedly extracting with 10ml of dichloromethane, combining the extracting solutions, evaporating to dryness, and marking as a part B;
step five: evaporating the residual ethanol layers in the third step and the fourth step to dryness, and marking the dried ethanol layers as a C part;
step six: the extracts of parts A, B and C are dissolved in HPLC determination solution, and the extracts are separated and assayed to obtain the final product cyclopamine.
Preferably, in the first step, the processed freeze-dried veratrum is selected as a raw material, the freeze-dried veratrum is placed in a centrifuge tube, 20ml of ethanol is added for uniform mixing, and the mixture is centrifuged for 10min by a centrifugal device to obtain an extract.
Preferably, in the first step, the processed freeze-dried veratrum is selected as a raw material, the freeze-dried veratrum is placed in a centrifuge tube, 30ml of ethanol is added for uniform mixing, and the extract is obtained by centrifugation for 10min through a centrifugal device.
Preferably, in the first step, the processed freeze-dried veratrum is selected as a raw material, the freeze-dried veratrum is placed in a centrifuge tube, 40ml of ethanol is added for uniform mixing, and the mixture is centrifuged for 10min by a centrifugal device to obtain an extract.
Preferably, in the first step, the processed freeze-dried veratrum is selected as a raw material, the freeze-dried veratrum is placed in a centrifuge tube, 10ml of ethanol is added for uniform mixing, and the extract is obtained by centrifugation for 10min through a centrifugal device.
Preferably, the ethanol is grade A, and the rotating speed of the centrifugal equipment is 7000 revolutions.
Preferably, the HPLC adopts isocratic elution, the isocratic eluent adopts a mixed composition of ammonium cerium nitrate, water and tetrahydrofuran, and the mass ratio of the ammonium cerium nitrate, the water and the tetrahydrofuran is 42:57.9:0.1.
Compared with the prior art, the invention has the following beneficial technical effects:
according to the invention, cyclopamine is obtained by taking veratrum as a raw material, the obtaining cost of cyclopamine is effectively reduced, the extracting solution is obtained by ethanol, the extracting solution is concentrated, the PH value is adjusted by acetic acid, the extracting solution is concentrated by petroleum ether uniformly mixing, different layers of the treated extracting solution are treated by combining dichloromethane, the A part, the B part and the C part are obtained, and finally, the cyclopamine which is qualified in final measurement is obtained by HPLC, wherein the input amount of the used extraction and separation equipment is small, the extraction difficulty is low, and the extraction method is simple and efficient;
in conclusion, the cyclopamine extraction and separation method provided by the invention can efficiently extract and separate the cyclopamine which is an anticancer active ingredient of veratrum, has low equipment cost, is simple and convenient to operate, and is suitable for popularization and use.
Detailed Description
The technical scheme of the invention is further described below with reference to specific embodiments.
Example 1
The invention provides a method for extracting and separating cyclopamine which is an anticancer active ingredient of veratrum, comprising the following steps:
step one: selecting the processed freeze-dried veratrum as a raw material, placing the freeze-dried veratrum into a centrifuge tube, adding 20ml of ethanol, uniformly mixing, centrifuging for 10min by using a centrifugal device to obtain an extracting solution, wherein the ethanol is of grade A, and the rotating speed of the centrifugal device is 7000;
step two: concentrating the extract obtained in the step one to 10ml by a cloth evaporator, regulating the PH value of the concentrated extract to 4 by adopting 5% acetic acid, adding 10ml of petroleum ether at 50 ℃, uniformly mixing, and discarding a petroleum ether layer;
step three: adding 10ml of dichloromethane extract into the product obtained in the second step, uniformly mixing, standing, sucking the extract, placing the extract into a new glass tube, repeatedly extracting with 10ml of dichloromethane, combining the extracts, evaporating to dryness, and marking as a part A;
step four: adding 10ml of dichloromethane into the residual lower layer liquid in the third step, uniformly mixing, standing, sucking the extracting solution, placing the extracting solution in a new glass tube, repeatedly extracting with 10ml of dichloromethane, combining the extracting solutions, evaporating to dryness, and marking as a part B;
step five: evaporating the residual ethanol layers in the third step and the fourth step to dryness, and marking the dried ethanol layers as a C part;
step six: dissolving the extracts of parts A, B and C in HPLC determination solution, separating and determining the extract to obtain cyclopamine as final product; HPLC adopts isocratic elution, isocratic eluent adopts a mixed composition of ceric ammonium nitrate, water and tetrahydrofuran, and the mass ratio of the ceric ammonium nitrate, the water and the tetrahydrofuran is 42:57.9:0.1.
Example two
The invention provides a method for extracting and separating cyclopamine which is an anticancer active ingredient of veratrum, and the embodiment comprises the following steps:
step one: selecting the processed freeze-dried veratrum as a raw material, placing the freeze-dried veratrum into a centrifuge tube, adding 30ml of ethanol, uniformly mixing, centrifuging for 10min by using a centrifugal device to obtain an extracting solution, wherein the ethanol is of grade A, and the rotating speed of the centrifugal device is 7000;
step two: concentrating the extract obtained in the step one to 10ml by a cloth evaporator, regulating the PH value of the concentrated extract to 4 by adopting 5% acetic acid, adding 10ml of petroleum ether at 50 ℃, uniformly mixing, and discarding a petroleum ether layer;
step three: adding 10ml of dichloromethane extract into the product obtained in the second step, uniformly mixing, standing, sucking the extract, placing the extract into a new glass tube, repeatedly extracting with 10ml of dichloromethane, combining the extracts, evaporating to dryness, and marking as a part A;
step four: adding 10ml of dichloromethane into the residual lower layer liquid in the third step, uniformly mixing, standing, sucking the extracting solution, placing the extracting solution in a new glass tube, repeatedly extracting with 10ml of dichloromethane, combining the extracting solutions, evaporating to dryness, and marking as a part B;
step five: evaporating the residual ethanol layers in the third step and the fourth step to dryness, and marking the dried ethanol layers as a C part;
step six: dissolving the extracts of parts A, B and C in HPLC determination solution, separating and determining the extract to obtain cyclopamine as final product; HPLC adopts isocratic elution, isocratic eluent adopts a mixed composition of ceric ammonium nitrate, water and tetrahydrofuran, and the mass ratio of the ceric ammonium nitrate, the water and the tetrahydrofuran is 42:57.9:0.1.
Example III
The invention provides a method for extracting and separating cyclopamine which is an anticancer active ingredient of veratrum, and the embodiment comprises the following steps:
step one: selecting the processed freeze-dried veratrum as a raw material, placing the freeze-dried veratrum in a centrifuge tube, adding 40ml of ethanol, uniformly mixing, centrifuging for 10min by using a centrifugal device to obtain an extracting solution, wherein the ethanol is of grade A, and the rotating speed of the centrifugal device is 7000;
step two: concentrating the extract obtained in the step one to 10ml by a cloth evaporator, regulating the PH value of the concentrated extract to 4 by adopting 5% acetic acid, adding 10ml of petroleum ether at 50 ℃, uniformly mixing, and discarding a petroleum ether layer;
step three: adding 10ml of dichloromethane extract into the product obtained in the second step, uniformly mixing, standing, sucking the extract, placing the extract into a new glass tube, repeatedly extracting with 10ml of dichloromethane, combining the extracts, evaporating to dryness, and marking as a part A;
step four: adding 10ml of dichloromethane into the residual lower layer liquid in the third step, uniformly mixing, standing, sucking the extracting solution, placing the extracting solution in a new glass tube, repeatedly extracting with 10ml of dichloromethane, combining the extracting solutions, evaporating to dryness, and marking as a part B;
step five: evaporating the residual ethanol layers in the third step and the fourth step to dryness, and marking the dried ethanol layers as a C part;
step six: dissolving the extracts of parts A, B and C in HPLC determination solution, separating and determining the extract to obtain cyclopamine as final product; HPLC adopts isocratic elution, isocratic eluent adopts a mixed composition of ceric ammonium nitrate, water and tetrahydrofuran, and the mass ratio of the ceric ammonium nitrate, the water and the tetrahydrofuran is 42:57.9:0.1.
Example IV
The invention provides a method for extracting and separating cyclopamine which is an anticancer active ingredient of veratrum, comprising the following steps:
step one: selecting the processed freeze-dried veratrum as a raw material, placing the freeze-dried veratrum into a centrifuge tube, adding 10ml of ethanol, uniformly mixing, centrifuging for 10min by using a centrifugal device to obtain an extracting solution, wherein the ethanol is of grade A, and the rotating speed of the centrifugal device is 7000;
step two: concentrating the extract obtained in the step one to 10ml by a cloth evaporator, regulating the PH value of the concentrated extract to 4 by adopting 5% acetic acid, adding 10ml of petroleum ether at 50 ℃, uniformly mixing, and discarding a petroleum ether layer;
step three: adding 10ml of dichloromethane extract into the product obtained in the second step, uniformly mixing, standing, sucking the extract, placing the extract into a new glass tube, repeatedly extracting with 10ml of dichloromethane, combining the extracts, evaporating to dryness, and marking as a part A;
step four: adding 10ml of dichloromethane into the residual lower layer liquid in the third step, uniformly mixing, standing, sucking the extracting solution, placing the extracting solution in a new glass tube, repeatedly extracting with 10ml of dichloromethane, combining the extracting solutions, evaporating to dryness, and marking as a part B;
step five: evaporating the residual ethanol layers in the third step and the fourth step to dryness, and marking the dried ethanol layers as a C part;
step six: dissolving the extracts of parts A, B and C in HPLC determination solution, separating and determining the extract to obtain cyclopamine as final product; HPLC adopts isocratic elution, isocratic eluent adopts a mixed composition of ceric ammonium nitrate, water and tetrahydrofuran, and the mass ratio of the ceric ammonium nitrate, the water and the tetrahydrofuran is 42:57.9:0.1.
Cyclopamine was extracted and isolated by the preparation method of examples one to four using 100g of freeze-dried veratrum, the quality and purity of the extracted cyclopamine are shown in the following table:
| example 1 | Example two | Example III | Example IV | |
| Extraction quality (g) | 12.36 | 18.21 | 14.55 | 13.56 |
| Purity (%) | 80.65 | 88.23 | 82.04 | 83.54 |
The highest extraction quality, high yield, and highest purity of cyclopamine were obtained from the extraction of cyclopamine in example two by combining the above tables.
The above-described embodiments are merely a few preferred embodiments of the present invention, and many alternative modifications and combinations of the above-described embodiments will be apparent to those skilled in the art based on the technical solutions of the present invention and the related teachings of the above-described embodiments.
Claims (6)
1. A method for extracting and separating cyclopamine which is an anticancer active ingredient of veratrum, which is characterized by comprising the following steps:
step one: selecting the processed freeze-dried veratrum as a raw material, placing the freeze-dried veratrum in a centrifuge tube, adding 10-40ml of ethanol, uniformly mixing, and centrifuging for 10min by a centrifugal device to obtain an extracting solution;
step two: concentrating the extract obtained in the first step to 10ml by a cloth evaporator, adjusting the pH value of the concentrated extract to 4 by 5% acetic acid, adding 10ml petroleum ether at 40-60 ℃, uniformly mixing, and discarding a petroleum ether layer;
step three: adding 10ml of dichloromethane into the product obtained in the second step, uniformly mixing, standing, sucking the extracting solution, placing the extracting solution in a new glass tube, repeatedly extracting with 10ml of dichloromethane, combining the extracting solutions, evaporating to dryness, and marking as a part A;
step four: adding 10ml of dichloromethane into the residual lower layer liquid in the third step, uniformly mixing, standing, sucking the extracting solution, placing the extracting solution in a new glass tube, repeatedly extracting with 10ml of dichloromethane, combining the extracting solutions, evaporating to dryness, and marking as a part B;
step five: evaporating the residual ethanol layers in the third step and the fourth step to dryness, and marking the dried ethanol layers as a C part;
step six: dissolving the extracts of parts A, B and C in HPLC determination solution, separating and determining the extract to obtain cyclopamine as final product; the HPLC adopts isocratic elution, isocratic eluent adopts a mixed composition of ceric ammonium nitrate, water and tetrahydrofuran, and the mass ratio of the ceric ammonium nitrate to the water to the tetrahydrofuran is 42:57.9:0.1.
2. The method for extracting and separating cyclopamine as an anticancer active ingredient from veratrum nigrum of claim 1, wherein the step one is to use the processed freeze-dried veratrum nigrum as a raw material, place the freeze-dried veratrum nigrum in a centrifuge tube, add 20ml of ethanol, mix uniformly, centrifuge for 10min by a centrifuge device, and obtain an extract.
3. The method for extracting and separating cyclopamine as an anticancer active ingredient from veratrum nigrum of claim 1, wherein the step one is to use the processed freeze-dried veratrum nigrum as a raw material, place the freeze-dried veratrum nigrum in a centrifuge tube, add 30ml of ethanol, mix uniformly, centrifuge for 10min by a centrifuge device, and obtain an extract.
4. The method for extracting and separating cyclopamine as an anticancer active ingredient from veratrum nigrum of claim 1, wherein the step one is to use the processed freeze-dried veratrum nigrum as a raw material, place the freeze-dried veratrum nigrum in a centrifuge tube, add 40ml of ethanol, mix uniformly, centrifuge for 10min by a centrifuge device, and obtain an extract.
5. The method for extracting and separating cyclopamine as an anticancer active ingredient from veratrum nigrum of claim 1, wherein the step one is to use the processed freeze-dried veratrum nigrum as a raw material, place the freeze-dried veratrum nigrum in a centrifuge tube, add 10ml of ethanol, mix uniformly, centrifuge for 10min by a centrifuge device, and obtain an extract.
6. The method for extracting and separating cyclopamine, an anticancer active ingredient of veratrum, according to claim 1, wherein the rotational speed of the centrifugal device is 7000 revolutions.
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| DE60113733T2 (en) * | 2001-07-02 | 2006-06-29 | Tas, Sinan | USE OF CYCLOPAMINE FOR THE TREATMENT OF BASAL CELL CARCINOMA AND OTHER TUMORS |
| CA2636807A1 (en) * | 2008-07-04 | 2010-01-04 | Steven Splinter | Methods for obtaining cyclopamine |
| CN101798307B (en) * | 2010-03-26 | 2012-02-22 | 巴塞利亚药业（中国）有限公司 | Preparation method of cyclopamine |
| CN108379431A (en) * | 2018-04-09 | 2018-08-10 | 陕西师范大学 | A method of total alkaloid in black false hellebore is extracted using ultrasonic extraction |
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