CN114276342A - Method for separating and purifying anisodamine monomer from plant extract - Google Patents
Method for separating and purifying anisodamine monomer from plant extract Download PDFInfo
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- CN114276342A CN114276342A CN202111647013.4A CN202111647013A CN114276342A CN 114276342 A CN114276342 A CN 114276342A CN 202111647013 A CN202111647013 A CN 202111647013A CN 114276342 A CN114276342 A CN 114276342A
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- WTQYWNWRJNXDEG-LEOABGAYSA-N anisodamine Chemical compound C1([C@@H](CO)C(=O)O[C@@H]2C[C@H]3[C@@H](O)C[C@@H](C2)N3C)=CC=CC=C1 WTQYWNWRJNXDEG-LEOABGAYSA-N 0.000 title claims abstract description 168
- WTQYWNWRJNXDEG-UHFFFAOYSA-N 6-Hydroxy-hyoscyamin Natural products CN1C(C2)CC(O)C1CC2OC(=O)C(CO)C1=CC=CC=C1 WTQYWNWRJNXDEG-UHFFFAOYSA-N 0.000 title claims abstract description 162
- 239000000178 monomer Substances 0.000 title claims abstract description 75
- 239000000419 plant extract Substances 0.000 title claims abstract description 59
- 238000000034 method Methods 0.000 title claims abstract description 37
- 239000003480 eluent Substances 0.000 claims abstract description 44
- 239000007788 liquid Substances 0.000 claims abstract description 23
- 239000003960 organic solvent Substances 0.000 claims abstract description 18
- 239000002253 acid Substances 0.000 claims abstract description 10
- WORJEOGGNQDSOE-UHFFFAOYSA-N chloroform;methanol Chemical compound OC.ClC(Cl)Cl WORJEOGGNQDSOE-UHFFFAOYSA-N 0.000 claims abstract description 10
- 239000003513 alkali Substances 0.000 claims abstract description 7
- 238000004587 chromatography analysis Methods 0.000 claims abstract description 6
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 81
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 42
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 36
- 238000010828 elution Methods 0.000 claims description 32
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 24
- 239000000741 silica gel Substances 0.000 claims description 24
- 229910002027 silica gel Inorganic materials 0.000 claims description 24
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 20
- 239000000706 filtrate Substances 0.000 claims description 16
- 238000004809 thin layer chromatography Methods 0.000 claims description 15
- 239000012043 crude product Substances 0.000 claims description 13
- 238000000605 extraction Methods 0.000 claims description 12
- 239000000047 product Substances 0.000 claims description 10
- NBEMQPLNBYYUAZ-UHFFFAOYSA-N ethyl acetate;propan-2-one Chemical group CC(C)=O.CCOC(C)=O NBEMQPLNBYYUAZ-UHFFFAOYSA-N 0.000 claims description 8
- 238000001914 filtration Methods 0.000 claims description 8
- 238000000227 grinding Methods 0.000 claims description 8
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 6
- 241000602542 Anisodus acutangulus Species 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- 238000010898 silica gel chromatography Methods 0.000 claims description 3
- 239000006228 supernatant Substances 0.000 claims description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims 2
- 229910021529 ammonia Inorganic materials 0.000 claims 1
- 238000012824 chemical production Methods 0.000 abstract description 2
- 239000000203 mixture Substances 0.000 abstract description 2
- 239000010410 layer Substances 0.000 description 38
- 229930013930 alkaloid Natural products 0.000 description 22
- 239000012535 impurity Substances 0.000 description 20
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 13
- 235000011114 ammonium hydroxide Nutrition 0.000 description 13
- 150000003797 alkaloid derivatives Chemical class 0.000 description 11
- 239000000126 substance Substances 0.000 description 11
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 6
- 241000242873 Scopolia Species 0.000 description 6
- 239000012044 organic layer Substances 0.000 description 6
- 238000000746 purification Methods 0.000 description 6
- 238000000926 separation method Methods 0.000 description 6
- 239000003814 drug Substances 0.000 description 5
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 241000999530 Anisodus Species 0.000 description 3
- 241000208292 Solanaceae Species 0.000 description 3
- 239000000284 extract Substances 0.000 description 3
- CPELXLSAUQHCOX-UHFFFAOYSA-N Hydrogen bromide Chemical compound Br CPELXLSAUQHCOX-UHFFFAOYSA-N 0.000 description 2
- RKUNBYITZUJHSG-UHFFFAOYSA-N Hyosciamin-hydrochlorid Natural products CN1C(C2)CCC1CC2OC(=O)C(CO)C1=CC=CC=C1 RKUNBYITZUJHSG-UHFFFAOYSA-N 0.000 description 2
- 241000208125 Nicotiana Species 0.000 description 2
- 241000208128 Nicotiana glauca Species 0.000 description 2
- 235000002637 Nicotiana tabacum Nutrition 0.000 description 2
- 208000002193 Pain Diseases 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000011010 flushing procedure Methods 0.000 description 2
- 210000000936 intestine Anatomy 0.000 description 2
- 230000036407 pain Effects 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 238000001953 recrystallisation Methods 0.000 description 2
- 238000002137 ultrasound extraction Methods 0.000 description 2
- RKUNBYITZUJHSG-FXUDXRNXSA-N (S)-atropine Chemical compound C1([C@@H](CO)C(=O)O[C@H]2C[C@H]3CC[C@@H](C2)N3C)=CC=CC=C1 RKUNBYITZUJHSG-FXUDXRNXSA-N 0.000 description 1
- 208000004998 Abdominal Pain Diseases 0.000 description 1
- 206010000087 Abdominal pain upper Diseases 0.000 description 1
- 229930003347 Atropine Natural products 0.000 description 1
- KMYQCELRVANQNG-YXGOVGSCSA-N Br.CN1[C@@H]2C[C@H](O)[C@H]1C[C@H](C2)OC(=O)[C@H](CO)c1ccccc1 Chemical compound Br.CN1[C@@H]2C[C@H](O)[C@H]1C[C@H](C2)OC(=O)[C@H](CO)c1ccccc1 KMYQCELRVANQNG-YXGOVGSCSA-N 0.000 description 1
- 230000005526 G1 to G0 transition Effects 0.000 description 1
- 241001529553 Scoparia <angiosperm> Species 0.000 description 1
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- 206010042674 Swelling Diseases 0.000 description 1
- WTQYWNWRJNXDEG-YFVFXCHESA-N [(1s,3r,5s)-6-hydroxy-8-methyl-8-azabicyclo[3.2.1]octan-3-yl] (2s)-3-hydroxy-2-phenylpropanoate Chemical compound C1([C@@H](CO)C(=O)O[C@H]2C[C@H]3C(O)C[C@@H](C2)N3C)=CC=CC=C1 WTQYWNWRJNXDEG-YFVFXCHESA-N 0.000 description 1
- OIPILFWXSMYKGL-UHFFFAOYSA-N acetylcholine Chemical compound CC(=O)OCC[N+](C)(C)C OIPILFWXSMYKGL-UHFFFAOYSA-N 0.000 description 1
- 229960004373 acetylcholine Drugs 0.000 description 1
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- 230000001078 anti-cholinergic effect Effects 0.000 description 1
- RKUNBYITZUJHSG-SPUOUPEWSA-N atropine Chemical compound O([C@H]1C[C@H]2CC[C@@H](C1)N2C)C(=O)C(CO)C1=CC=CC=C1 RKUNBYITZUJHSG-SPUOUPEWSA-N 0.000 description 1
- 229960000396 atropine Drugs 0.000 description 1
- GHQPBDDZGPAVJP-UHFFFAOYSA-N azanium;methanol;hydroxide Chemical compound N.O.OC GHQPBDDZGPAVJP-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 235000019658 bitter taste Nutrition 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
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- 238000009835 boiling Methods 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
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- 239000013078 crystal Substances 0.000 description 1
- 238000003795 desorption Methods 0.000 description 1
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- 229940079593 drug Drugs 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000004817 gas chromatography Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 229930005342 hyoscyamine Natural products 0.000 description 1
- 229960003210 hyoscyamine Drugs 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 238000010829 isocratic elution Methods 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 238000002386 leaching Methods 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 230000009965 odorless effect Effects 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 235000019633 pungent taste Nutrition 0.000 description 1
- 238000004451 qualitative analysis Methods 0.000 description 1
- 239000013558 reference substance Substances 0.000 description 1
- 210000002460 smooth muscle Anatomy 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Medicines Containing Plant Substances (AREA)
- Nitrogen Condensed Heterocyclic Rings (AREA)
Abstract
The invention provides a method for separating and purifying anisodamine monomers from plant extract, and relates to the technical field of chemical production. The method comprises the following steps of extracting a plant extract by using an organic solvent, taking upper-layer liquid, eluting the upper-layer liquid by using eluent, performing chromatography to obtain a crude anisodamine product, adding an acid solution into the crude anisodamine product, acidifying the mixture by using an alkali solution to obtain a treatment liquid, extracting the treatment liquid by using a chloroform-methanol solution, taking lower-layer liquid, and recrystallizing the lower-layer liquid to obtain the anisodamine monomer.
Description
Technical Field
The invention relates to the technical field of chemical production, and particularly relates to a method for separating and purifying anisodamine monomers from plant extract.
Background
The anisodus acutangulus, the Lei Shao Zi, the Lijiang anisodus, the Scopolia cirrhosa and the Scopolia denticulata and the like are roots or leaves of solanaceae plants, and are called Dayaoshu, Yexerophyll tobacco, wild tobacco and the like, and the traditional Chinese medicine has bitter and pungent taste, warm property, spleen, stomach and kidney channels, has the effects of relieving spasm and pain, dispelling wind and eliminating dampness and the like, is mainly used for treating symptoms of rheumatalgia, traumatic injury, swelling and pain, stomachache and abdominal pain and the like, and mainly plays a role due to chemical components of anisodamine and the like contained in the traditional Chinese medicine.
Anisodamine, commonly used as its hydrobromide, is a white crystal or crystalline powder, odorless. Is very soluble in water. It is easily soluble in ethanol. The hyoscyamine has obvious peripheral anticholinergic effectIt can resist the contraction of smooth muscle of intestine and bladder and the decrease of blood pressure caused by acetylcholine, and can reduce the tension of intestine in vivo, and the action intensity is similar to that of atropine. Alias: 7-hydroxy hyoscyamine; racanisodamine hydrobromide; anisodamine hydrobromide. Chemical name: an alkaloid extracted from the special solanaceae plant anisodamine in China is often called as 654 for short, and the natural product of the alkaloid is 654-1. The product made by the artificial synthesis method is '654-2'. CAS number: 55869-99-3. The molecular formula is as follows: c17H23NO4. Molecular weight: 305.36900.
the anisodamine monomer has higher medicinal value and is a common nerve blocking medicament, and the anisodamine monomer separated by the prior common purification method of the anisodamine monomer has more impurities, and is unstable and modified into other substances, thereby reducing the yield of the anisodamine.
Therefore, there is an urgent need to provide a method for improving the separation, extraction and purification of anisodamine monomer from acutangular anisodus root.
Disclosure of Invention
The invention aims to provide a method for separating and purifying a anisodamine monomer from a plant extract, which is simple to operate and convenient for operators to implement, and can ensure the purity of the extracted anisodamine so that the anisodamine monomer can be used for preparing medicaments.
The technical problem to be solved by the invention is realized by adopting the following technical scheme.
The embodiment of the application provides a method for separating and purifying a anisodamine monomer from a plant extract, which comprises the following steps of extracting the plant extract by using an organic solvent, taking an upper layer liquid, eluting the upper layer liquid by using an eluent, carrying out chromatography to obtain a crude anisodamine product, adding an acid solution into the crude anisodamine product, acidifying the acidified anisodamine product by using an alkali solution to obtain a treatment solution, extracting the treatment solution by using a chloroform-methanol solution, taking a lower layer liquid, and recrystallizing the lower layer liquid to obtain the anisodamine monomer. The method comprises the steps of extracting alkaloid in the plant extract by using an organic solvent, mutually dissolving anisodamine and other alkaloids in one part of organic layers in the organic solvent, mutually dissolving other organic matters in the plant extract and the other part of organic layers in the organic solvent, separating anisodamine monomers from most of organic matters in the plant extract so as to achieve the purpose of primary purification, adding acid liquor so as to react most of anisodamine insoluble substances and other impurities into anisodamine salt, adding ammonia water for alkalization so as to salinize the anisodamine, generating more anisodamine so as to improve the yield of the final anisodamine, eluting so as to remove the residual other alkaloids which are difficult to remove, finally obtaining the anisodamine monomers, improving the purity of the finally obtained anisodamine monomers, and finally recrystallizing to enable the anisodamine monomers to settle at the bottom, removing the upper liquid to obtain high-purity anisodamine monomer.
Compared with the prior art, the embodiment of the invention has at least the following advantages or beneficial effects:
the embodiment of the invention provides a method for separating and purifying a anisodamine monomer from a plant extract, which comprises the steps of extracting alkaloid in the plant extract by using an organic solvent, mutually dissolving the anisodamine and other alkaloids in one part of organic layers in the organic solvent, mutually dissolving other organic matters in the plant extract and the other part of organic layers in the organic solvent, separating the anisodamine monomer from most of the organic matters in the plant extract so as to achieve the aim of primary purification, reacting most of anisodamine insoluble substances and other impurities into anisodamine salt by adding acid liquor, alkalifying the anisodamine salt by adding ammonia water so as to generate more anisodamine, so as to improve the yield of the final anisodamine, removing the other residual alkaloids which are difficult to remove by elution, finally obtaining the anisodamine monomer and improving the purity of the finally obtained anisodamine monomer, and finally, the anisodamine monomer is settled at the bottom through recrystallization, and the liquid at the upper part is removed, thus obtaining the anisodamine monomer with higher purity.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present invention will be described in detail below with reference to specific examples.
The embodiment provides a method for separating and purifying a anisodamine monomer from a plant extract, which is characterized by comprising the following steps of extracting the plant extract by using an organic solvent, taking an upper layer liquid, eluting the upper layer liquid by using an eluent, carrying out chromatography to obtain a crude anisodamine product, adding an acid solution into the crude anisodamine product, acidifying the mixture by using an alkali solution to obtain a treatment solution, extracting the treatment solution by using a chloroform-methanol solution, taking a lower layer liquid, and recrystallizing the lower layer liquid to obtain the anisodamine monomer. The method comprises the steps of extracting alkaloid in the plant extract by using an organic solvent, mutually dissolving anisodamine and other alkaloids in one part of organic layers in the organic solvent, mutually dissolving other organic matters in the plant extract and the other part of organic layers in the organic solvent, separating anisodamine monomers from most of organic matters in the plant extract so as to achieve the purpose of primary purification, adding acid liquor so as to react most of anisodamine insoluble substances and other impurities into anisodamine salt, adding ammonia water for alkalization so as to salinize the anisodamine, generating more anisodamine so as to improve the yield of the final anisodamine, eluting so as to remove the residual other alkaloids which are difficult to remove, finally obtaining the anisodamine monomers, improving the purity of the finally obtained anisodamine monomers, and finally recrystallizing to enable the anisodamine monomers to settle at the bottom, removing the upper liquid to obtain high-purity anisodamine monomer.
In some embodiments of the present invention, the preparation method of the plant extract comprises the following steps of sequentially grinding trisection, extracting with alcohol, and filtering to obtain a filtrate, and concentrating the filtrate under reduced pressure to obtain the plant extract. The method discloses a preparation method of a plant extract, wherein part of impurities which are difficult to remove can be primarily removed by alcohol extraction. The anisodus acutangulus, the jingle bell, the Lijiang anisodus, the Scopolia scoparia, the Scopolia denticulata and the like are roots or leaves of solanaceae plants, are named as Dazaoshu, Yerihisha tobacco, wild tobacco and the like, and contain rich anisodamine, anisodamine and the like, so that the extract contains rich raw materials of the Scopolia denticulata, and the content of the finally prepared anisodamine can be effectively improved by taking the Scopolia acutangulus as a main raw material.
In some embodiments of the present invention, the alcohol concentration of the alcohol extraction is 90-95%, and the temperature of the concentration under reduced pressure is 55-65 ℃. The concentration interval and the temperature interval can reduce the impurities in the prepared plant extract, when the alcohol extraction concentration is higher than the interval, more volatilization of ethanol can be caused during alcohol extraction, so that the alcohol extraction efficiency is reduced, and when the alcohol extraction concentration is lower than the interval, the amount of ethanol during alcohol extraction is less, so that the content of the prepared anisodamine monomer is lower.
In some embodiments of the invention, the organic solvent is an acetone-ethyl acetate solution. Wherein, the alkaloid such as anisodamine monomer contained in the plant extract can be effectively dissolved in ethyl acetate solution, and other macromolecular organic matters in the plant extract can be dissolved in acetone, and the acetone and the ethyl acetate are layered, wherein the ethyl acetate is positioned at the upper layer, and the alkaloid containing the anisodamine monomer can be effectively obtained by taking the upper layer liquid.
In some embodiments of the present invention, the elution includes a first elution, a second elution, and a third elution, and the first elution includes a step of passing the supernatant through a silica gel chromatography column of 80-120 mesh, and then sequentially eluting the supernatant with chloroform and ethyl acetate to obtain a first eluate. Elution is the process of continuously changing the concentration ratio of a mobile phase to a certain extent in the same analysis period and is called elution. In gas chromatography, temperature-programmed methods are widely used in order to improve the separation of wide boiling range samples and to shorten the analysis period. In liquid chromatography, elution is used for samples with complex components. In the same analysis period, the concentration ratio of the mobile phase is changed continuously according to a certain program, which is called elution. Therefore, the components with larger property difference in a complex sample can achieve good separation according to the respectively proper capacity factor k. Whether the elution is performed isocratic or isocratic is mainly seen in the case of impurities. When the same substance has two conditions of elution and isocratic elution, the recognition degree of elution is higher. The degree of separation of impurities from impurities, main peaks and solvent peaks must be good (if not good, elution is required). (1) The polarity difference between the impurities and the main component is large, and the impurities can be eluted completely (sometimes, the impurities cannot be eluted after being flushed for two or three hours under the condition of equal temperature, and the use of gradient is required to be considered). (2) The polarity difference between the impurities and the main component is large, the impurity peak emergence is too late (the sample detection time can be shortened by gradient after two hours of isocratic flushing) (3) the impurity polarity is larger than that of the main peak, the proper time of the main peak is required to be fully piled in front when isocratic flushing is carried out, the impurities are separated from the main peak and the peak emergence is too late, and the elution is also considered in the condition. In the invention, the peak difference between the anisodamine and other alkaloids in the alkaloid extract is larger, so that the anisodamine monomer can be better eluted by selecting elution, so as to improve the purity of the anisodamine monomer. The ammonia water-methanol is mainly selected as a mobile phase for elution, and firstly, the ammonia water can create alkaline relief for elution, so that the desorption of the anisodamine monomer is facilitated, and the higher purity of the eluted anisodamine monomer is ensured. The methanol can destroy functional hydrogen bonds formed between the anisodamine and other alkaloids, and can effectively eliminate charge action generated between the anisodamine and other alkaloids so as to elute the anisodamine, and the methanol is selected as an eluent to achieve a better elution effect because of the higher polarity of the anisodamine. The weight ratio of ammonia water to methanol is preferably 1 (4-10), and the interval not only can ensure the elution speed, but also can ensure the elution effect so as to improve the purity of the finally prepared anisodamine monomer as much as possible.
In some embodiments of the present invention, the second elution includes a step of passing the primary eluate through a silica gel column of 80-120 mesh, and then sequentially eluting with chloroform and ethyl acetate to obtain a second eluate. The anisodamine, anisodamine and the like contained in the anisodamine can be further and completely removed through secondary elution, so that the purity of the prepared anisodamine is further improved, the content of the anisodamine is further improved, and the chromatographic column in the mesh interval can effectively remove the anisodamine and the anisodamine, so that the purity of the finally prepared anisodamine is further improved.
In some embodiments of the invention, the three elutions include the step of applying the second eluate to a thin layer chromatography silica gel Kiesegel60 silica gel column and eluting with ethyl acetate to obtain a third eluate. The small molecular impurities contained in the anisodamine monomer can be further removed by three times of elution, thereby improving the purity of the prepared anisodamine monomer.
In some embodiments of the present invention, the chromatography comprises separating the three eluates by thin layer chromatography to obtain crude anisodamine, wherein the thin layer used in the thin layer chromatography is silica gel thin layer. Thin Layer Chromatography (TLC) is carried out by applying a suitable stationary phase to a glass plate, plastic or aluminum substrate in a thin, uniform layer. After spotting and developing, comparing the specific shift value (Rf) with that of chromatogram obtained by the same method based on the appropriate reference substance, and performing drug identification, impurity inspection or content determination. Thin layer chromatography is an important experimental technique for rapid separation and qualitative analysis of small amounts of substances, and is also used to follow the progress of the reaction. The method can effectively improve the purity of the anisodamine monomer and improve the content of the finally prepared anisodamine monomer.
In some embodiments of the present invention, the acid solution comprises sulfuric acid and/or hydrochloric acid, and the alkali solution is concentrated ammonia water. The sulfuric acid and/or the hydrochloric acid can react with the anisodamine and the anisodamine, the anisodamine and the anisodamine are acidified, and then the anisodamine can be further reacted into the anisodamine through alkali lye alkalization, so that the content of the finally prepared anisodamine monomer can be improved, and the purity of the finally prepared anisodamine monomer can also be improved.
In some embodiments of the invention, the pH of the treatment fluid is 8 to 10. The pH interval can ensure that the prepared anisodamine monomer can stably exist in the solution and can not react with other substances, when the pH interval is lower than the pH interval, the content of the prepared anisodamine monomer is lower, and when the pH interval is higher than the pH interval, the alkalinity of the prepared anisodamine monomer is stronger, the stability is poorer, and the reaction with the outside is easy to occur.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
The embodiment provides a method for separating and purifying anisodamine monomers from a plant extract, which comprises the following steps of sequentially grinding anisodamine acutangula, extracting with ethanol (90% ethanol) and filtering to obtain a filtrate, concentrating the filtrate at 55 ℃ under reduced pressure to obtain a plant extract, extracting the plant extract with an acetone-ethyl acetate solution, taking an upper layer solution, passing the upper layer solution through an 80-mesh silica gel chromatographic column, sequentially eluting with chloroform and an ethyl acetate solution to obtain a primary eluent, passing the primary eluent through an 80-mesh silica gel chromatographic column, sequentially eluting with chloroform and an ethyl acetate solution to obtain a secondary eluent, passing the secondary eluent through a Kiesegel60 silica gel column, eluting with ethyl acetate to obtain a tertiary eluent, treating the tertiary eluent with a thin layer chromatography to obtain a anisodamine crude product, adding sulfuric acid into the anisodamine crude product, alkalifying with ammonia water until the pH is 8, extracting the treated solution with chloroform-methanol solution, collecting the lower layer solution, and recrystallizing the lower layer solution to obtain anisodamine monomer.
Example 2
The embodiment provides a method for separating and purifying anisodamine monomers from a plant extract, which comprises the following steps of sequentially grinding anisodamine acutangula, extracting with ethanol (95% ethanol) and filtering to obtain a filtrate, concentrating the filtrate at 65 ℃ under reduced pressure to obtain a plant extract, extracting the plant extract with an acetone-ethyl acetate solution, taking an upper layer solution, passing the upper layer solution through a 120-mesh silica gel chromatographic column, sequentially eluting with chloroform and an ethyl acetate solution to obtain a primary eluent, passing the primary eluent through a 120-mesh silica gel chromatographic column, sequentially eluting with chloroform and an ethyl acetate solution to obtain a secondary eluent, passing the secondary eluent through a Kiesegel60 silica gel column, eluting with ethyl acetate to obtain a tertiary eluent, treating the tertiary eluent with a thin layer chromatography to obtain a anisodamine crude product, adding sulfuric acid into the anisodamine crude product, alkalifying with ammonia water until the pH is 10, extracting the treated solution with chloroform-methanol solution, collecting the lower layer solution, and recrystallizing the lower layer solution to obtain anisodamine monomer.
Example 3
The embodiment provides a method for separating and purifying anisodamine monomers from a plant extract, which comprises the following steps of sequentially grinding anisodamine acutangula, extracting with ethanol (92% ethanol) and filtering to obtain a filtrate, concentrating the filtrate at 60 ℃ under reduced pressure to obtain a plant extract, extracting the plant extract with an acetone-ethyl acetate solution, taking an upper layer solution, passing the upper layer solution through a 100-mesh silica gel chromatographic column, sequentially eluting with chloroform and an ethyl acetate solution to obtain a primary eluent, passing the primary eluent through a 100-mesh silica gel chromatographic column, sequentially eluting with chloroform and an ethyl acetate solution to obtain a secondary eluent, passing the secondary eluent through a Kiesegel60 silica gel column, eluting with ethyl acetate to obtain a tertiary eluent, treating the tertiary eluent with a thin layer chromatography to obtain a anisodamine crude product, adding sulfuric acid into the anisodamine crude product, alkalifying with ammonia water until the pH is 9, extracting the treated solution with chloroform-methanol solution, collecting the lower layer solution, and recrystallizing the lower layer solution to obtain anisodamine monomer.
Example 4
The embodiment provides a method for separating and purifying anisodamine monomers from a plant extract, which comprises the following steps of sequentially grinding anisodamine acutangula, extracting with ethanol (91% ethanol) and filtering to obtain a filtrate, concentrating the filtrate at 58 ℃ under reduced pressure to obtain a plant extract, extracting the plant extract with an acetone-ethyl acetate solution, taking an upper layer solution, passing the upper layer solution through a 90-mesh silica gel chromatographic column, sequentially eluting with chloroform and an ethyl acetate solution to obtain a primary eluent, passing the primary eluent through a 90-mesh silica gel chromatographic column, sequentially eluting with chloroform and an ethyl acetate solution to obtain a secondary eluent, passing the secondary eluent through a Kiesegel60 silica gel column, eluting with ethyl acetate to obtain a tertiary eluent, treating the tertiary eluent with a thin layer chromatography to obtain a anisodamine crude product, adding sulfuric acid into the anisodamine crude product, alkalifying with ammonia water until the pH is 8.5, extracting the treated solution with chloroform-methanol solution, collecting the lower layer solution, and recrystallizing the lower layer solution to obtain anisodamine monomer.
Example 5
The embodiment provides a method for separating and purifying anisodamine monomers from a plant extract, which comprises the following steps of sequentially grinding anisodamine acutangula, extracting with ethanol (94% ethanol) and filtering to obtain a filtrate, concentrating the filtrate at 61 ℃ under reduced pressure to obtain a plant extract, extracting the plant extract with an acetone-ethyl acetate solution, taking an upper layer solution, passing the upper layer solution through a 110-mesh silica gel chromatographic column, sequentially eluting with chloroform and an ethyl acetate solution to obtain a primary eluent, passing the primary eluent through a 110-mesh silica gel chromatographic column, sequentially eluting with chloroform and an ethyl acetate solution to obtain a secondary eluent, passing the secondary eluent through a Kiesegel60 silica gel column, eluting with ethyl acetate to obtain a tertiary eluent, treating the tertiary eluent with a thin layer chromatography to obtain a anisodamine crude product, adding sulfuric acid into the anisodamine crude product, alkalifying with ammonia water until the pH is 9.3, extracting the treated solution with chloroform-methanol solution, collecting the lower layer solution, and recrystallizing the lower layer solution to obtain anisodamine monomer.
Example 6
The embodiment provides a method for separating and purifying anisodamine monomers from a plant extract, which comprises the following steps of sequentially grinding anisodamine acutangula, extracting with ethanol (93% ethanol) and filtering to obtain a filtrate, concentrating the filtrate at 62 ℃ under reduced pressure to obtain a plant extract, extracting the plant extract with an acetone-ethyl acetate solution, taking an upper layer solution, passing the upper layer solution through a 95-mesh silica gel chromatographic column, sequentially eluting with chloroform and an ethyl acetate solution to obtain a primary eluent, passing the primary eluent through a 105-mesh silica gel chromatographic column, sequentially eluting with chloroform and an ethyl acetate solution to obtain a secondary eluent, passing the secondary eluent through a Kiesegel60 silica gel column, eluting with ethyl acetate to obtain a tertiary eluent, treating the tertiary eluent with a thin layer chromatography to obtain a anisodamine crude product, adding sulfuric acid into the anisodamine crude product, alkalifying with ammonia water until the pH is 10.5, extracting the treated solution with chloroform-methanol solution, collecting the lower layer solution, and recrystallizing the lower layer solution to obtain anisodamine monomer.
Test examples
Taking the anisodamine samples of examples 1-6 to perform high performance liquid chromatography analysis to obtain chromatographic peaks of the anisodamine samples in each example, comparing the chromatographic peaks with a standard substance, substituting the area of the chromatographic peaks obtained in each example into a formula of a standard curve to calculate the content, and obtaining the purity of the anisodamine in the samples through content calculation, wherein the specific data are as follows: (in the test, the standard was calibrated)
TABLE 1 statistical table of results
| Examples | 1 | 2 | 3 | 4 | 5 | 6 |
| Purity of | 70％ | 65％ | 85％ | 81％ | 88％ | 60％ |
As is clear from Table 1, the anisodamine obtained in example 5 had the best purity. In example 5, the procedure was substantially the same and the operating conditions were different from those of examples 1 to 4, and the purity of the obtained anisodamine was improved by adding sulfuric acid to the first residue, extracting with dichloromethane, and removing the dichloromethane layer, thereby further removing impurities in the alkaloid and thus the purity of the obtained anisodamine was improved, which indicates that the anisodamine obtained under the operating conditions of example 5 had the best purity. Example 5 compared with example 6, in example 5, the number of times of ultrasonic extraction and chloroform extraction was increased, and the number of times of treatment of the first residue was also increased, and the treatment of the first residue enabled the purity of anisodamine to be improved, and the number of times of ultrasonic extraction and chloroform extraction was increased, and multiple extractions allowed further improvement of the separation effect of anisodamine from the solution, and further improved purity of the obtained anisodamine. Therefore, the invention can separate and extract anisodamine with higher purity from the alkaloid leaching paste.
In summary, the embodiments of the present invention provide a method for separating and purifying anisodamine monomer from plant extract, the method comprises extracting alkaloid in the plant extract with an organic solvent, mutually dissolving anisodamine and other alkaloids in one part of the organic solvent, mutually dissolving other organic matters in the plant extract with another part of the organic solvent, so as to separate the anisodamine monomer from most of the organic matters in the plant extract, thereby achieving the purpose of primary purification, adding an acid solution to react the anisodamine insoluble substances and other impurities as much as possible into anisodamine salt, adding ammonia water for alkalization to alkalize the anisodamine salt, thereby generating more anisodamine, so as to improve the final yield of anisodamine, and eluting to remove other remaining difficult-to-remove alkaloids, thereby finally obtaining the anisodamine monomer and improving the purity of the finally obtained anisodamine monomer, and finally, the anisodamine monomer is settled at the bottom through recrystallization, and the liquid at the upper part is removed, thus obtaining the anisodamine monomer with higher purity.
The embodiments described above are some, but not all embodiments of the invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Claims (10)
1. A method for separating and purifying a anisodamine monomer from a plant extract is characterized by comprising the following steps of extracting the plant extract by using an organic solvent, taking an upper layer liquid, eluting the upper layer liquid by using an eluent, carrying out chromatography to obtain a crude anisodamine product, adding an acid solution into the crude anisodamine product, acidifying the crude anisodamine product by using an alkali solution to obtain a treatment solution, extracting the treatment solution by using a chloroform-methanol solution, taking a lower layer liquid, and recrystallizing the lower layer liquid to obtain the anisodamine monomer.
2. The method for separating and purifying anisodamine monomer from plant extract as claimed in claim 1, wherein the preparation method of the plant extract comprises the following steps of grinding anisodus acutangulus, extracting with alcohol and filtering in sequence to obtain filtrate, and concentrating the filtrate under reduced pressure to obtain the plant extract.
3. The method for separating and purifying anisodamine monomer from plant extract as claimed in claim 2, wherein the alcohol concentration of the alcohol extraction is 90-95%, and the temperature of the vacuum concentration is 55-65 ℃.
4. The method for separating and purifying anisodamine monomer from plant extract as claimed in claim 1, wherein the organic solvent is acetone-ethyl acetate solution.
5. The method for separating and purifying anisodamine monomers from plant extracts as claimed in claim 1, wherein the elution comprises a primary elution, a secondary elution and a third elution, wherein the primary elution comprises the following steps of passing the supernatant through a silica gel chromatography column of 80-120 meshes, and then sequentially eluting with chloroform and ethyl acetate solution to obtain a primary eluent.
6. The method for separating and purifying anisodamine monomer from plant extract as claimed in claim 5, wherein the secondary elution comprises the following steps of passing the primary eluent through a silica gel chromatography column of 80-120 meshes, and then sequentially eluting with chloroform and ethyl acetate solution to obtain a secondary eluent.
7. The method as claimed in claim 6, wherein the third elution comprises subjecting the second eluate to thin layer chromatography on a Kiesegel60 silica gel column, and eluting with ethyl acetate to obtain a third eluate.
8. The method of claim 7, wherein the chromatography comprises separating the three eluates by thin layer chromatography to obtain the crude product of anisodamine, wherein the thin layer chromatography is silica gel thin layer.
9. The method for separating and purifying anisodamine monomer from plant extract as claimed in claim 1, wherein the acid solution comprises sulfuric acid and/or hydrochloric acid, and the alkali solution is concentrated ammonia.
10. The method for separating and purifying anisodamine monomer from plant extract as claimed in claim 1, wherein the pH of the treatment solution is 8-10.
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| CN113549061A (en) * | 2021-08-27 | 2021-10-26 | 锦州拾正生物科技有限公司 | Anisodine separated and purified from alkaloid extract and preparation method thereof |
| CN113698397A (en) * | 2021-08-28 | 2021-11-26 | 锦州拾正生物科技有限公司 | Anisodamine and purification process thereof |
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