CN113185527A - Method for separating tetrandrine and tetrandrine - Google Patents
Method for separating tetrandrine and tetrandrine Download PDFInfo
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- CN113185527A CN113185527A CN202110506346.9A CN202110506346A CN113185527A CN 113185527 A CN113185527 A CN 113185527A CN 202110506346 A CN202110506346 A CN 202110506346A CN 113185527 A CN113185527 A CN 113185527A
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- C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
- C07D491/12—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains three hetero rings
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Abstract
The invention relates to a method for separating tetrandrine and tetrandrine. The separation method of tetrandrine and tetrandrine comprises the following steps: providing a sample containing tetrandrine and tetrandrine; mixing the sample with 75-85% ethanol at 3-15 deg.C, and stirring thoroughly; filtering to obtain filter residue and filtrate. The separation method provided by the invention has high separation efficiency, does not use toxic and harmful solvents, and is beneficial to industrial production.
Description
Technical Field
The invention relates to a method for separating tetrandrine and tetrandrine.
Background
Stephania tetrandra (scientific name: Sinomenium acutum (Thunb.) rehd. et Wils.) is a plant of genus Euonymus of family Menispermaceae. It is reported that the effective components of tetrandrine are alkaloids, mainly tetrandrine and fangchinoline. The total content of alkaloid is 1.5% -2.3%, mainly comprises tetrandrine, and the content is about 1%; tetrandrine, content about 0.5%; cycloarterine with content of 0.2%; and several other trace alkaloids.
Tetrandrine (Tetrandrine, and Tetrandrine) is colorless needle crystal, is insoluble in water and petroleum ether, is easily soluble in organic solvent such as ethanol, acetone, ethyl acetate, diethyl ether and chloroform, and diluted acid water, is soluble in benzene, has double melting point at mp 216 deg.C, and is crystallized from acetone, melted at about 150 deg.C, heated and solidified, and remelted at 213 deg.C. The dissolution behavior of Fangchinoline (also known as Fangchinoline, nortetrandrine) is similar to that of tetrandrine, and because of a phenolic hydroxyl group, the polarity is slightly higher than that of tetrandrine, and the solubility in benzene is lower than that of tetrandrine and higher than that in ethanol.
In the prior art, the tetrandrine and tetrandrine are separated by using benzene. Benzene has limited use due to its high toxicity and is also a carcinogen.
CN105968120A discloses a method for preparing tetrandrine and tetrandrine, which is prepared by adopting lipophilic tertiary amine total alkali through 2 steps, 1) separating by using a medium-pressure silica gel chromatographic column method, and eluting by using petroleum ether-ethyl acetate-diethylamine; 2) purifying with preparative high performance liquid chromatography column with methanol-water as mobile phase, and concentrating and evaporating eluate respectively. Because the mixture of the tetrandrine and the tetrandrine contains a large amount of impurities, the silica gel chromatographic column is inconvenient to clean and reuse, and a mixed solvent of petroleum ether, ethyl acetate and diethylamine in a certain proportion is used in the desorption process, so that certain difficulty exists in solvent recovery and reuse, the flash point of the petroleum ether is extremely low, and safety production accidents are easy to happen.
Therefore, the separation method of tetrandrine and tetrandrine still needs to be improved.
Disclosure of Invention
The tetrandrine and fangchinoline are difficult to separate because of their close structure and property. Especially in the case of a close content of the two, the separation is particularly difficult.
The inventor finds that the effective separation of the tetrandrine and the tetrandrine can be realized under the condition of lower temperature by taking 75-85% ethanol as a solvent.
A method for separating tetrandrine and fangchinoline comprises:
providing a sample containing tetrandrine and tetrandrine;
mixing the sample with 75-85% ethanol at 3-15 deg.C, and stirring thoroughly;
filtering to obtain filter residue and filtrate.
Wherein, the tetrandrine content in the filter residue is obviously reduced and is enriched in the filtrate; the content of tetrandrine in the filtrate is remarkably reduced, and the tetrandrine is enriched in the filter residue.
And further removing ethanol from the filtrate to obtain the tetrandrine with higher purity.
According to the embodiment of the invention, the separation method further comprises the step of drying the obtained filter residue so as to obtain the tetrandrine with higher content or purity. Drying can also remove ethanol.
According to the embodiment of the invention, the separation method further comprises the step of removing ethanol from the obtained filtrate so as to obtain the tetrandrine with higher content or purity. And further drying. Ethanol may be removed or dried using methods conventional in the art.
According to the embodiment of the invention, the sample containing tetrandrine and tetrandrine is a tetrandrine extract and can be prepared by adopting a conventional method in the prior art.
According to the embodiment of the invention, the weight ratio of the tetrandrine to the tetrandrine in the sample is (45-55): (55-45), for example, 45:53, 47:51, 48:50, 50:48, 51:47, 53:45, 55: 43.
According to the embodiment of the invention, the purity ratio of the tetrandrine to the tetrandrine in the sample is (45-55): (55-45), for example, 45:53, 47:51, 48:50, 50:48, 51:47, 53:45 and 55: 43.
According to an embodiment of the present invention, the purity of tetrandrine in the sample is 45-55%, such as 45%, 46%, 47%, 48%, 50%, 51%, 53%, 55%.
According to an embodiment of the present invention, the purity of tetrandrine in the sample is 45-55%, such as 45%, 46%, 47%, 48%, 50%, 51%, 53%, 55%.
According to the embodiment of the present invention, the sum of the purity of tetrandrine and the purity of fangchinoline in the sample is 90-99%, such as 98-99%, specifically, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%.
According to the embodiment of the invention, the purity of the tetrandrine in the sample is 45% -47%, and the purity of the tetrandrine in the sample is 50% -52%.
According to the embodiment of the invention, the purity of the tetrandrine in the sample is 46.78%, and the purity of the tetrandrine in the sample is 51.67%.
Herein, the above samples are also referred to as intermediates of tetrandrine and fangchinoline halves.
The existing method is particularly difficult to separate the samples of the tetrandrine and the tetrandrine within the proportion range. The method of the invention can realize good separation effect.
According to an embodiment of the invention, the concentration of ethanol is 75-85%, such as 75%, 80% or 85%, preferably 80%. Experiments show that better separation effect can be realized by adopting 80% ethanol.
According to an embodiment of the invention, the sample is mixed with 75-85% ethanol at 3-15 ℃, e.g. 3 ℃, 5 ℃, 6 ℃, 8 ℃, 10 ℃ or 15 ℃, preferably 6 ℃.
The inventors have surprisingly found that temperature has a very important influence on the separation of tetrandrine and tetrandrine, especially in the case of close content of the two. Wherein, when the temperature is 6 ℃, the separation effect is best.
According to the embodiment of the invention, the weight ratio of the sample to the ethanol is 1 (3-9), preferably 1 (5-7), and more preferably 1: 6. Researches show that the ratio of the feed to the liquid also has important influence on the separation of the tetrandrine and the tetrandrine, especially under the condition that the content of the tetrandrine and the tetrandrine is close to that of the tetrandrine and the tetrandrine.
According to the embodiment of the invention, the sample is mixed with 80% ethanol at the temperature of 6 ℃, and the weight ratio of the sample to the ethanol is 1 (5-7), preferably 1: 6.
According to the embodiment of the invention, the sample is mixed with 75-85% of ethanol at the temperature of 6 ℃, and the weight ratio of the sample to the ethanol is 1: 6.
According to the embodiment of the invention, the sample is mixed with 75-85% ethanol at the temperature of 3-15 ℃, and then the mixture is fully stirred, so that more tetrandrine is dispersed into the ethanol, and the separation of the tetrandrine and the ethanol is promoted.
According to the embodiment of the invention, the stirring is performed until the dispersion is uniform, and the stirring time can be 20-60min, such as 20min, 30min, 40min, 50min, 60 min.
According to the embodiment of the present invention, the filtration can be performed by a conventional method.
According to the embodiment of the invention, the obtained filter residue can be continuously separated by adopting the method, namely, the obtained filter residue is mixed with 75-85% of ethanol at the temperature of 3-15 ℃, and the mixture is fully stirred; filtering to obtain filter residue and filtrate again.
Furthermore, the separation method can be repeated for a plurality of times so as to better realize the separation of the tetrandrine and the tetrandrine. However, it was found that the first separation effect was the best for the samples containing tetrandrine and tetrandrine in the above ratio range.
According to the embodiment of the invention, the half-and-half intermediate of tetrandrine and tetrandrine is stirred and mixed with 80% ethanol with 6 times of the weight of the half-and-half intermediate and the ethanol with 6 ℃ so that the separation effect of the tetrandrine and the tetrandrine is the best.
According to the embodiment of the invention, the method for separating the tetrandrine and the tetrandrine comprises the following steps:
providing a sample containing tetrandrine and tetrandrine; wherein, the purity of the tetrandrine in the sample is 45-47%, and the purity of the tetrandrine is 50-52%;
mixing the sample with 80% ethanol 6 times the weight of the sample at 6 ℃, and fully stirring;
filtering to obtain filter residue and filtrate.
Wherein, the tetrandrine content in the filter residue is obviously reduced and is enriched in the filtrate; the content of tetrandrine in the filtrate is remarkably reduced, and the tetrandrine is enriched in the filter residue.
The separation method provided by the invention has high separation efficiency, does not use toxic and harmful solvents, and is beneficial to industrial production. The invention is improved by process technology, the separation effective rate in the purification process reaches 100%, the finished product is rapidly produced, the yield is improved, the energy is saved, and the emission is reduced. The method utilizes ethanol for separation, has low toxicity, needs 80 percent ethanol for subsequent process impurity removal, does not increase the use of new solvents for the process, and is convenient to operate.
Drawings
FIG. 1 is an HPLC chromatogram of the hemiintermediate of tetrandrine and tetrandrine used in the examples of the present invention.
FIG. 2 is an HPLC chromatogram of the filtered residue of example 1 of the present invention.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or instruments used are conventional products available from regular distributors, not indicated by the manufacturer.
The following detection methods were used:
1, appearance: a2.0 g sample was visually observed on a white paper and should be a yellow or pale yellow crystalline powder.
2 loss on drying
2.1 weighing 10g of the product, drying for 3h in a drying oven at 80 ℃, measuring according to the operation procedure of the drying weight loss measurement method, and calculating.
2.2 calculation formula:
W0: empty weighing bottle constant weight, g; w1: sample weight, g;
W2: after drying, empty weighing bottle + sample constant weight, g;
3 purity, related substances: liquid Chromatography (HPLC)
3.1 reagent: acetonitrile (chromatographically pure), methanol (chromatographically pure), triethylamine, perchloric acid
3.2 chromatographic condition chromatography: agilent1100 or the like;
a chromatographic column: c18;
column temperature: 25 ℃;
flow rate: 1.0 ml/min;
wavelength: 280 nm;
sample introduction amount: 20 mu l of the mixture;
3.3 solution preparation
Mobile phase: water-acetonitrile-methanol-triethylamine was mixed at a volume ratio of 650:300:50:3, adjusted to pH 2.0 with perchloric acid, and filtered through a 0.45 μm organic phase filter.
Control solution: taking 10mg of each of tetrandrine and fangchinoline reference substances, dissolving with mobile phase to 25ml, and shaking.
Test solution preparation: about 20mg of the test sample is precisely weighed, dissolved in a 50ml measuring flask by using the mobile phase and diluted to the scale mark. (direct injection of the control solution).
3.4 procedures
Sampling the mobile phase, the reference solution and the test solution for 1 time respectively, and recording the retention time of the chromatogram until the main peak is 3 times.
3.5 purity, related substances: calculated as peak area by area normalization.
The tetrandrine and fangchinoline semi-intermediates are generated by purifying the tetrandrine extract and fangchinoline semi-intermediates, and are used as the following samples containing the tetrandrine and fangchinoline. The results of the above tests are shown in FIG. 1. FIG. 1 is HPLC chromatogram of half-and-half intermediate of tetrandrine and fangchinoline, wherein the purity of fangchinoline is 51.67% at retention time of 9.098min and 46.78% at retention time of 12.257 min.
Example 1
Taking 100g of a sample containing tetrandrine and tetrandrine (wherein the purity of tetrandrine is 46.78%, and the purity of tetrandrine is 51.67%), adding 80% ethanol, controlling the temperature of the system, stirring for 30min, and filtering to obtain filter residue and filtrate. And (6) detecting. The ethanol used (i.e., weight times the sample), system temperature, and separation results are shown in Table 1 below.
Wherein, when the 80% ethanol is 6 times and the temperature of the system is 6 ℃, the HPLC chromatogram of the filtered residue is shown in figure 2, the tetrandrine content with the retention time of 9.314min is 93.12% and the tetrandrine content with the retention time of 12.774min is 4.33% by area normalization method.
TABLE 1
Note: in Table 1, "A46.78% and B51.67%" means "the purity of tetrandrine is 46.78% and the purity of tetrandrine is 51.67%" in the sample; the rest is similar.
Example 2
Taking 100g of a sample containing tetrandrine and tetrandrine (wherein the purity of tetrandrine is 46.78%, and the purity of tetrandrine is 51.67%), adding 75% ethanol, controlling the temperature of the system, stirring for 30min, and filtering to obtain filter residue and filtrate. And (6) detecting. The ethanol used (i.e., weight times the sample), system temperature, and separation results are shown in Table 2 below.
TABLE 2
Note: in Table 2, "A46.78% and B51.67%" means "the purity of tetrandrine is 46.78% and the purity of tetrandrine is 51.67%" in the sample; the rest is similar.
Example 3
Taking 100g of a sample containing tetrandrine and tetrandrine (wherein the purity of tetrandrine is 46.78%, and the purity of tetrandrine is 51.67%), adding 85% ethanol, controlling the temperature of the system, stirring for 30min, and filtering to obtain filter residue and filtrate. And (6) detecting. The ethanol used (i.e., weight times the sample), system temperature, and separation results are shown in Table 3 below.
TABLE 3
Note: in Table 3, "A46.78% and B51.67%" means "the purity of tetrandrine is 46.78% and the purity of tetrandrine is 51.67%" in the sample; the rest is similar.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (10)
1. A method for separating tetrandrine and fangchinoline comprises:
providing a sample containing tetrandrine and tetrandrine;
mixing the sample with 75-85% ethanol at 3-15 deg.C, and stirring thoroughly;
filtering to obtain filter residue and filtrate.
2. The method for separating tetrandrine and tetrandrine according to claim 1, wherein the weight ratio of tetrandrine to tetrandrine in the sample is (40-55): (55-40).
3. The method for separating tetrandrine and fangchinoline according to claim 1, wherein the purity of tetrandrine in the sample is 45-55%; the sum of the purity of tetrandrine and the purity of tetrandrine is 90-99%.
4. The method for separating tetrandrine and fangchinoline according to claim 1, wherein the purity of tetrandrine in the sample is 45% -47%; the purity of the tetrandrine is 50% -52%.
5. The method for separating tetrandrine and fangchinoline according to any one of claims 1-4, wherein the concentration of ethanol is 75%, 80% or 85%.
6. The method for separating tetrandrine and fangchinoline according to any one of claims 1-4, wherein the sample is mixed with 75-85% ethanol at 3 ℃, 5 ℃, 6 ℃, 8 ℃, 10 ℃ or 15 ℃.
7. The method for separating tetrandrine and tetrandrine according to any one of claims 1 to 6, wherein the weight ratio of the sample to the ethanol is 1 (3-9), preferably 1 (5-7), and more preferably 1: 6.
8. The method for separating tetrandrine and fangchinoline according to any one of claims 1-7, wherein the stirring time is 20-60 min.
9. The method for separating tetrandrine and tetrandrine according to any one of claims 1 to 8, wherein the sample is mixed with 80% ethanol at 6 ℃, and the weight ratio of the sample to the ethanol is 1 (5-7), preferably 1: 6; alternatively, the first and second electrodes may be,
mixing the sample with 75-85% ethanol at 6 ℃, wherein the weight ratio of the sample to the ethanol is 1: 6.
10. The method for separating tetrandrine and tetrandrine according to claim 1, comprising:
providing a sample containing tetrandrine and tetrandrine; wherein, the purity of the tetrandrine in the sample is 45-47%, and the purity of the tetrandrine is 50-52%;
mixing the sample with 80% ethanol 6 times the weight of the sample at 6 ℃, and fully stirring;
filtering to obtain filter residue and filtrate.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115144497A (en) * | 2022-06-30 | 2022-10-04 | 浙江金华康恩贝生物制药有限公司 | Detection method of tetrandrine substances |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1903856A (en) * | 2006-08-03 | 2007-01-31 | 西安皓天生物工程技术有限责任公司 | Preparation method of Tetrandrine and Fangchino-kine |
| CN102382119A (en) * | 2011-10-19 | 2012-03-21 | 陕西省西安植物园 | Extraction method of tetrandrine and demethyltetrandrine |
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- 2021-05-10 CN CN202110506346.9A patent/CN113185527B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1903856A (en) * | 2006-08-03 | 2007-01-31 | 西安皓天生物工程技术有限责任公司 | Preparation method of Tetrandrine and Fangchino-kine |
| CN102382119A (en) * | 2011-10-19 | 2012-03-21 | 陕西省西安植物园 | Extraction method of tetrandrine and demethyltetrandrine |
Non-Patent Citations (1)
| Title |
|---|
| 董小萍等: "《天然药物化学实验》", 28 February 2015, 中国医药科技出版社 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115144497A (en) * | 2022-06-30 | 2022-10-04 | 浙江金华康恩贝生物制药有限公司 | Detection method of tetrandrine substances |
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