CN101244096B - Method for preparing total alkaloid - Google Patents
Method for preparing total alkaloid Download PDFInfo
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- CN101244096B CN101244096B CN2007100802394A CN200710080239A CN101244096B CN 101244096 B CN101244096 B CN 101244096B CN 2007100802394 A CN2007100802394 A CN 2007100802394A CN 200710080239 A CN200710080239 A CN 200710080239A CN 101244096 B CN101244096 B CN 101244096B
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Abstract
The invention discloses a preparation method of a total alkaloid, which is characterized in that plant extracts are purified using weak acidic cation exchange resin, strong acidic cation exchange resin and neutral alumina to obtain the total alkaloids. According to the basic experiment, the general effective preparation method of total alkaloids has an advantage of effectively increasing the content of the total alkaloids.
Description
Technical field
The invention belongs to alkaloid separating and purifying technology field, be specifically related to a kind of method for preparing of total alkaloids.
Background technology
Alkaloid generally is meant the nitrogenous organic compound (protein, peptide class, amino acid except) in the plant.Alkaloid is human research's one type of natural product the earliest.Alkaloid mostly has stronger activity.
Alkaloid is a most complicated and the hugest compounds in the natural product; The alkaloid monomer chemical compound that has been found that now has more than 10000, and this compounds is distributed widely in Taxus genus, Pinus, Picea, oily shirt genus, Ephedra, tricuspid shirt genus and Liliaceae, Amaryllidaceae and Stemonaceae, Ranunculaceae, Magnoliaceae, Berberidaceae, Menispermaceae, Aristolochiaceae, Papaveraceae, Fructus Lycopersici esculenti branch section, Rutaceae, Gentianaceae, Apocynaceae, Loganiaceae, Rubiaceae, Solanaceae, Boraginaceae and the feverfew.
Alkaloidal research is remained one of focus of natural product research.The application of alkaloid in medicine is divided into 2 kinds of forms: the one, separate obtaining the alkaloid monomer chemical compound as medicine, and according to incompletely statistics, in the medicine in natural product source, have 42.8% to derive from alkaloid; Though but the activity of monosomic alkali is stronger, its toxic and side effects is also bigger, and this administrated method also can't agree with theory of Chinese medical science mutually, can't bring into play the collaborative effect of multiple alkaloid performance in the plant extract, thereby have certain limitation.A kind of in addition be from the plant extract separating total alkaloids as medicine, but in present separation and extraction technology, still can not obtain the total alkaloids of higher degree.Though current alkaloid extraction and isolation technics have had a lot of progress, still can only reach 50% by content for the total alkaloids in the plant, comparatively ripe at present and general total alkaloids preparation method in Chinese medicine and the technical field of Chinese patent medicine now.Can not satisfy safe, effective, the controlled requirement in the modernization of Chinese medicine process.Current alkaloid method for preparing mainly contains:
1. organic solvent extraction: organic solvent extraction is to utilize that each composition partition coefficient difference in two kinds of immiscible solvents reaches isolating method in the extract; The big more separation efficiency of the partition coefficient of component in two-phase solvent is high more during extraction, and separating effect is good more.For the lipotropy alkaloid, utilize nonpolar and low polar organic solvent such as benzene, ether, chloroform etc. to carry out liquid-liquid extraction with water; For water-soluble alkaloid, utilize the bigger organic solvent of polarity such as ethyl acetate, butanols etc. and aqueous solution extraction.Sometimes available multiple solvent is configured to biphase immiscible solvent and extracts.
2. chromatography is also claimed chromatography, is a kind of physical separation method, can be used for separation and purification and identify pharmaceutically active ingredient.Chromatography comprises paper chromatography, thin layer chromatography and column chromatography, and adsorpting column chromatogram purifying biological alkali composition wherein commonly used generally uses adsorbent to be silica gel and aluminium oxide.
2.1 silica gel column chromatography: utilize silica gel as adsorbent, about separation and purification work more than 90% all can be used this method.Silica gel is neutral no coloured particles, stable performance, and separation efficiency is relevant with factors such as its granularity, aperture and surface areas.The silica gel column chromatography scope of application is wide, can be used as polarity and nonpolar alkaloidal purification, and cost is low, easy to operate.
2.2 aluminum oxide column chromatography: with the chromatography method of aluminium oxide as adsorbent; According to aluminium oxide preparation and processing method difference; Be divided into alkalescence, neutrality and acid 3 kinds, its neutral and alkali and neutral aluminium oxide be applicable to separates acid greatly, the higher alkaloids composition of activation temperature.
2.3 ion exchange resin: ion exchange resin is mainly selected absorption through electrostatic attraction and Van der Waals force, is divided into polytype according to self character.Select strongly acidic cation-exchange for use to alkaloidal character, through resin, the cation exchange that makes alkaloid salt is to resin and separate with impurity with other compositions with acidifying alkaloids extract.Obtain the free state alkaloid through the resin after the ion exchange with the ammonia alkalization, wait resin to dry the back and extract with corresponding solvent according to alkaloidal lipophilic or hydrophilic nmature and obtain total alkaloids.
The conventional method of existing these total alkaloidss preparation, having a problem is exactly that efficiency of pcr product is low, the impurity content height, often can not through this method directly obtain can be medicinal total alkaloids.
Organic solvent extraction is the classical technology of alkaloid purification, is widely used, and have advantage simple to operate, easy amplification, but separation efficiency and purity is lower, use a large amount of organic solvents, and processing safety is not good.
Ion-exchange-resin process is the method for preparing of the recent total alkaloids of using always, but this method can not be used for the preparation poor effect of total alkaloids separately, and must combine with other method and could use.
The patent documentation of the method for preparing of relevant total alkaloids has a lot: CN02148608.5, CN03159674.6, CN02123967.3, CN200310103945.8, CN03118261.5, CN200310109641.2, CN
200410018658.1、CN200310122686.3、CN200410090205.X、CN200410013988.1、CN200410065893.4、CN200510122258.X、CN200510077440.8、CN200510040909.0、CN200510037754.5、CN200510044345.8、CN200510032062.1、CN200510015970.X、CN200510095455.7、CN200410061471.X。These patents all are the method for preparinies to the total alkaloids of a certain or several plant, do not have a kind of method for preparing of general total alkaloids.
Sum up in the point that a bit also there is not at present a kind of method for preparing of the general total alkaloids that extensively is suitable for.And though present method can make total alkaloid contents be higher than 50%, yield is low, and purity is also difficult to be improved.This all proposes higher requirement to the method for preparing of total alkaloids in the plant.The medicine resource maximum for this type of total alkaloids patent medicine probability, that domestic resource is the abundantest needs a kind of generally and extensive, and the method for preparing of developing a kind of total alkaloids with mark post meaning is extremely important.
Summary of the invention
The objective of the invention is to provides a kind of raising total alkaloid content to the method for preparing that does not also have at present a kind of general total alkaloids that extensively is suitable for, and can have the method for preparing of general meaning to alkaloid extraction.
The method for preparing of particularly, inventing described total alkaloids is: plant extract is obtained total alkaloids with weak-type cation exchange resin, strongly acidic cation-exchange and neutral alumina purification successively.
Above-mentioned preferred manufacturing procedure is: plant extract is added on the weak-type cation exchange resin column; Get component A with water elution; Reuse ammonia eluting gets B component; Component A added to eluting gets component C on the strongly acidic cation-exchange post, B component and C are merged obtain total alkaloids with the neutral alumina column purification.
Above-mentioned preferred method for preparing is: plant extract is added on the weak-type cation exchange resin column; Get component A with water elution; Reuse ammonia eluting gets B component; With component A adjust pH is to add to that eluting gets component C on the strongly acidic cation-exchange post behind the 1-3, B component and C is merged obtain total alkaloids with the neutral alumina column purification.
The concentration of above-mentioned ammonia is 3-9%.
Above-mentioned weak-type cation exchange resin column is selected from the D151 of Nankai, Nankai's 101 type weak-type cation exchange resin columns.
Above-mentioned strongly acidic cation-exchange post is selected from Nankai 001 * 1, Nankai 001 * 2, Nankai 001 * 3, Nankai 001 * 14.5, D151, D018, D019, D020, YWD12V and nuclear Yadong 732 type strongly acidic cation-exchanges.
The method for preparing of above-mentioned total alkaloids is characterized in that described strongly acidic cation-exchange post is selected from Nankai 001 * 1, Nankai 001 * 2, Nankai 001 * 3, Nankai 001 * 14.5, D151 and nuclear Yadong 732 type strongly acidic cation-exchanges.
Above-mentioned plant extract is obtained with solvent extraction by one of Herba Leonuri, Rhizoma Corydalis, the Radix Stemonae, Semen daturae, Rhizoma Menispermi, Herba Dendrobii, Radix Tripterygii Wilfordii, Radix Tinosporae, Semen Hyoscyami, Cacumen Securinegae Suffruticosae, Rhizoma Coptidis, Herba Ephedrae, Radix Stephaniae Tetrandrae, Cortex Phellodendri, Ramulus Uncariae Cum Uncis, Bulbus Fritillariae Uninbracteatae, Bulbus Fritillariae Thunbergii, Fructus Camptothecae Acuminatae, Fructus Evodiae, Cortex Cinchonae, Semen Arecae, Herba Catharanthi Rosei, Radix Sophorae Flavescentis, Radix Sophorae Tonkinensis, Radix Gentianae Macrophyllae, Radix Rauvolfiae, the Radix Linderae, Radix Aconiti, Radix Aconiti Lateralis Preparata, Herba Lobeliae Chinensis and Cortex Dictamni.
The inventive method obtains to the process of the groping expansion of total alkaloids method for preparing in the Herba Leonuri.Find that through testing us plant extract is had the universal meaning that alkaloid is prepared with the method for preparing that weak-type cation exchange resin, strongly acidic cation-exchange and neutral alumina column purification obtain this total alkaloids of total alkaloids successively, the preparation of total alkaloids contained in other plant also is superior to prior art.
Below be the process of groping to the Herba Leonuri total alkaloids method for preparing:
1. the separating effect of storng-acid cation exchange resin: with storng-acid cation exchange resin on the Herba Leonuri extract; Find through the thin layer check: storng-acid cation exchange resin can be adsorbed on the resin column by the alkaloid that Herba Leonuri is all; Stachydrine just can desorption with weak ammonia after absorption, and leonurine all can not desorption with ammonia, sodium hydroxide.Conclusion: storng-acid cation exchange resin can not be used for this Alkaloid of enriching and purifying leonurine, and this dead absorption is the irreversible loss of effective ingredient, and this also is the greatest drawback of current existing public technology.
2. the separating effect of weak-acid cation-exchange resin: with weak-acid cation-exchange resin on the Herba Leonuri extract; Find through the thin layer check: weak-acid cation-exchange resin can be adsorbed on leonurine on the resin column; Stachydrine directly passes through resin column; Leonurine can be adsorbed on the resin column preferably, just can desorption with the ammonia eluting.Thereby use the dead absorption problem of the leonurine Alkaloid that weak-acid cation-exchange resin can solve; But can not this Alkaloid of enrichment stachydrine.
3. the separating effect of neutral alumina post: Herba Leonuri is extracted with neutral alumina post column purification.Experimental result: observe with outward appearance through thin layer check and to find: the neutral alumina post all less than adsorbing, still can be removed the impurity of pigment and non-alkaloid to leonurine and stachydrine.
4. the Herba Leonuri extracting solution is obtained total alkaloids with weak-type cation exchange resin, strongly acidic cation-exchange and neutral alumina column purification successively.Find through the thin layer check: contain leonurine and stachydrine two Alkaloids in the gained Herba Leonuri total alkaloids, avoided the loss of two Alkaloids in the preparation process, total alkaloid contents is also brought up to more than 80%.
In the research through adopting methods such as water, sour water, organic solvent extraction method and ion-exchange-resin process and the present invention that method contrast discovery is provided; When technical scheme of the present invention is generalized to the preparation of total alkaloids of other plant; Have the effect that improves alkaloid greatly, be superior to the preparation method of present total alkaloids.Thereby the present invention is a kind of method for preparing of the total alkaloids that is of universal significance.This is the most active to present research, and the preparation that method for preparing remains at insufficient total alkaloids has important practical significance.
The specific embodiment
Further describe the present invention with embodiment below, help understanding, but said experimental example and embodiment only are used to explain the present invention rather than restriction the present invention the present invention and advantage thereof, better effects if.
The preparation of embodiment 1-Herba Leonuri total alkaloids
Get Herba Leonuri 300g, decocte with water three times, 2 hours for the first time, 1.5 hours for the second time, 1 hour for the third time; Filter, merging filtrate is concentrated into about 250ml, cools off, and adds the ethanol of equivalent; Stir, left standstill 24 hours, filter, decompression filtrate recycling ethanol also is concentrated into the thick paste shape, and thin up is to 500ml; Cold preservation 24 hours filters, and filtrating is added on the D151 of Nankai weak-type cation exchange resin column, gets component A with water elution, and reuse 4-5% ammonia eluting gets B component.With component A adjust pH is to add to that eluting gets component C on the D151 strongly acidic cation-exchange post after 3, B component and C is merged promptly obtain Herba Leonuri total alkaloids with flinging to solvent behind the neutral alumina column purification.
The preparation of embodiment 2--Herba Leonuri total alkaloids
Get Herba Leonuri 300g, decocte with water three times, 2 hours for the first time, 1.5 hours for the second time, 1 hour for the third time; Filter, merging filtrate is concentrated into about 250ml, cools off, and adds the ethanol of equivalent; Stir, left standstill 24 hours, filter, decompression filtrate recycling ethanol also is concentrated into the thick paste shape, and thin up is to 500ml; Cold preservation 24 hours filters, and filtrating is added on the GDX104 type macroporous resin column, gets component A with water elution, and reuse 50% ethanol elution gets B component.With component A adjust pH is to add to that eluting gets component C on the D018 strongly acidic cation-exchange post after 1, B component and C is merged promptly obtain Herba Leonuri total alkaloids with flinging to solvent behind the neutral alumina column purification.
The preparation of embodiment 3-Herba Leonuri total alkaloids
Get Herba Leonuri 300g, decocte with water three times, 2 hours for the first time, 1.5 hours for the second time, 1 hour for the third time; Filter, merging filtrate is concentrated into about 250ml, cools off, and adds the ethanol of equivalent; Stir, left standstill 24 hours, filter, decompression filtrate recycling ethanol also is concentrated into the thick paste shape, and thin up is to 500ml; Cold preservation 24 hours filters, and filtrating is added on the D151 of Nankai weak-type cation exchange resin column, gets component A with water elution, and reuse 4-5% ammonia eluting gets B component.With component A adjust pH is to add to that eluting gets component C on Nankai's 001 * 1 type strongly acidic cation-exchange post after 2.5, B component and C is merged promptly obtain Herba Leonuri total alkaloids with flinging to solvent behind the neutral alumina column purification.
The preparation of embodiment 4--Herba Leonuri total alkaloids
Get Herba Leonuri 300g, decocte with water three times, 2 hours for the first time, 1.5 hours for the second time, 1 hour for the third time; Filter, merging filtrate is concentrated into about 250ml, cools off, and adds the ethanol of equivalent; Stir, left standstill 24 hours, filter, decompression filtrate recycling ethanol also is concentrated into the thick paste shape, and thin up is to 500ml; Cold preservation 24 hours filters, and filtrating is added on the GDX104 type macroporous resin column, gets component A with water elution, and reuse 50% ethanol elution gets B component.With component A adjust pH is to add to that eluting gets component C on Nankai's 001 * 2 type strongly acidic cation-exchange post after 3, B component and C is merged promptly obtain Herba Leonuri total alkaloids with flinging to solvent behind the neutral alumina column purification.
The preparation of embodiment 5-Herba Leonuri total alkaloids
Get Herba Leonuri 300g, decocte with water three times, 2 hours for the first time, 1.5 hours for the second time, 1 hour for the third time; Filter, merging filtrate is concentrated into about 250ml, cools off, and adds the ethanol of equivalent; Stir, left standstill 24 hours, filter, decompression filtrate recycling ethanol also is concentrated into the thick paste shape, and thin up is to 500ml; Cold preservation 24 hours filters, and filtrating is added on the D151 of Nankai weak-type cation exchange resin column, gets component A with water elution, and reuse 4-5% ammonia eluting gets B component.With component A adjust pH is to add to that eluting gets component C on the D151 strongly acidic cation-exchange post after 3, B component and C is merged promptly obtain Herba Leonuri total alkaloids with flinging to solvent behind the neutral alumina column purification.
The preparation of embodiment 6-Cortex Dictamni total alkaloids
Get Cortex Dictamni 300g, add 1% sulphuric acid and decoct three times, 2 hours for the first time, 1.5 hours for the second time, 1 hour for the third time; Filter, merging filtrate is concentrated into about 250ml, cools off, and adds the water of equivalent; Stir, left standstill 24 hours, filter, filtrate decompression reclaims solvent and is concentrated into the thick paste shape, and thin up is to 500ml; Cold preservation 24 hours filters, and filtrating is added on Nankai's 101 weak-type cation exchange resin columns, gets component A with water elution, and reuse 50% ethanol elution gets B component.With component A adjust pH is to add to that eluting gets component C on the D018 strongly acidic cation-exchange post after 1, B component and C is merged promptly obtain the Cortex Dictamni total alkaloids with flinging to solvent behind the neutral alumina column purification.
The preparation of embodiment 7-Semen daturae total alkaloids
Get Semen daturae 300g, add 95% ethanol and decoct three times, 2 hours for the first time, 1.5 hours for the second time, 1 hour for the third time; Filter, merging filtrate is concentrated into about 250ml, cools off, and adds 2% sulphuric acid of equivalent; Stir, left standstill 24 hours, filter, decompression filtrate recycling ethanol also is concentrated into the thick paste shape, and thin up is to 500ml; Cold preservation 24 hours filters, and filtrating is added on Nankai's 101 type weak-type cation exchange resin columns, gets component A with water elution, and reuse 4-5% ammonia eluting gets B component.With component A adjust pH is to add to that eluting gets component C on the D019 strongly acidic cation-exchange post after 2.6, B component and C is merged to obtain total alkaloids subsequent use with flinging to solvent behind the neutral alumina column purification.
The preparation of embodiment 8-Rhizoma Coptidis total alkaloids
After the 300g Rhizoma Coptidis soaked 30 minutes, decoct 2 times, each 2 hours, collecting decoction filter clear liquor I, clear liquor I is added on the D151 of Nankai weak-type cation exchange resin column, get component A with water elution, reuse 4-5% ammonia eluting gets B component.With component A adjust pH is to add to that eluting gets component C on the YWD12V strongly acidic cation-exchange post after 3.0, B component and C is merged obtain total alkaloids with flinging to solvent behind the neutral alumina column purification.
The preparation of embodiment 9-Radix Sophorae Flavescentis total alkaloids
After the 300g Radix Sophorae Flavescentis soaked 30 minutes, add 95% methanol and decoct three times, 2 hours for the first time, 1.5 hours for the second time, 1 hour for the third time; Filter, merging filtrate is concentrated into about 250ml, cools off, and adds the water of equivalent; Stir, left standstill 24 hours, filter, decompression filtrate recycling ethanol also is concentrated into the thick paste shape, and thin up is to 500ml; Cold preservation 24 hours filters, and filtrating adds on the D151 of Nankai weak-type cation exchange resin column, gets component A with water elution, and reuse 50% ethanol elution gets B component.With component A adjust pH is to add to that eluting gets component C on the YWD12V strongly acidic cation-exchange post after 1.8, B component and C is merged obtain total alkaloids with flinging to solvent behind the neutral alumina column purification.
The preparation of embodiment 10-Cortex Phellodendri total alkaloids
Get Cortex Phellodendri 300g, decocte with water three times, 2 hours for the first time, 1.5 hours for the second time, 1 hour for the third time; Filter, merging filtrate is concentrated into about 250ml, cools off, and adds the ethanol of equivalent; Stir, left standstill 24 hours, filter, decompression filtrate recycling ethanol also is concentrated into the thick paste shape, and thin up is to 500ml; Cold preservation 24 hours filters, and filtrating is added on Nankai's 101 weak-type cation exchange resin columns, gets component A with water elution, and reuse 4-5% ammonia eluting gets B component.With component A adjust pH is to add to that eluting gets component C on Nankai's 001 * 1 type strongly acidic cation-exchange post after 2.5, B component and C is merged promptly obtain Cortex Phellodendri total alkaloids with flinging to solvent behind the neutral alumina column purification.
The alkaloidal preparation of embodiment 11-tripterygium total
Get Radix Tripterygii Wilfordii 300g, decocte with water three times, 2 hours for the first time, 1.5 hours for the second time, 1 hour for the third time; Filter, merging filtrate is concentrated into about 250ml, cools off, and adds the ethanol of equivalent; Stir, left standstill 24 hours, filter, decompression filtrate recycling ethanol also is concentrated into the thick paste shape, and thin up is to 500ml; Cold preservation 24 hours filters, and filtrating is added on the D151 of Nankai weak-type cation exchange resin column, gets component A with water elution, and reuse 50% ethanol elution gets B component.With component A adjust pH is to add to that eluting gets component C on Nankai's 001 * 2 type strongly acidic cation-exchange post after 3, B component and C is merged promptly obtain the tripterygium total alkaloid with flinging to solvent behind the neutral alumina column purification.
The preparation of embodiment 12-Rhizoma Corydalis total alkaloids
Get Rhizoma Corydalis 300g, decocte with water three times, 2 hours for the first time, 1.5 hours for the second time, 1 hour for the third time; Filter, merging filtrate is concentrated into about 250ml, cools off, and adds the ethanol of equivalent; Stir, left standstill 24 hours, filter, decompression filtrate recycling ethanol also is concentrated into the thick paste shape, and thin up is to 500ml; Cold preservation 24 hours filters, and filtrating is added on Nankai's 101 weak-type cation exchange resin columns, gets component A with water elution, and reuse 4-5% ammonia eluting gets B component.With component A adjust pH is to add to that eluting gets component C on Nankai's 001 * 14.5 type strongly acidic cation-exchange post after 1, B component and C is merged promptly obtain Rhizoma Corydalis total alkaloids with flinging to solvent behind the neutral alumina column purification.
The preparation of embodiment 13-Radix Stephaniae Tetrandrae total alkaloids
Get Radix Stephaniae Tetrandrae 300g, decocte with water three times, 2 hours for the first time, 1.5 hours for the second time, 1 hour for the third time; Filter, merging filtrate is concentrated into about 250ml, cools off, and adds the ethanol of equivalent; Stir, left standstill 24 hours, filter, decompression filtrate recycling ethanol also is concentrated into the thick paste shape, and thin up is to 500ml; Cold preservation 24 hours filters, and filtrating is added on the D151 of Nankai weak-type cation exchange resin column, gets component A with water elution, and reuse 50% ethanol elution gets B component.With component A adjust pH is to add to that eluting gets component C on the 732 type strongly acidic cation-exchange posts of nuclear Yadong after 2, and merging promptly obtains the Radix Stephaniae Tetrandrae total alkaloids with flinging to solvent behind the neutral alumina column purification with C with B component.
The preparation of embodiment 14-Fructus Evodiae total alkaloids
Get Fructus Evodiae 300g, decocte with water three times, 2 hours for the first time, 1.5 hours for the second time, 1 hour for the third time; Filter, merging filtrate is concentrated into about 250ml, cools off, and adds the ethanol of equivalent; Stir, left standstill 24 hours, filter, decompression filtrate recycling ethanol also is concentrated into the thick paste shape, and thin up is to 500ml; Cold preservation 24 hours filters, and filtrating is added on Nankai's 101 weak-type cation exchange resin columns, gets component A with water elution, and reuse 50% ethanol elution gets B component.With component A adjust pH is to add to that eluting gets component C on the 732 type strongly acidic cation-exchange posts of nuclear Yadong after 2, and merging promptly obtains Fructus Evodiae total alkaloids with flinging to solvent behind the neutral alumina column purification with C with B component.
The preparation of Comparative Examples 1-Herba Leonuri total alkaloids
Get Herba Leonuri 300g, decocte with water three times, 2 hours for the first time, 1.5 hours for the second time, 1 hour for the third time; Filter, merging filtrate is concentrated into about 250ml, cools off, and adds the ethanol of equivalent; Stir, left standstill 24 hours, filter, decompression filtrate recycling ethanol also is concentrated into the thick paste shape; Thin up is to 500ml, and cold preservation 24 hours filters, and the adjust pH of will filtrating is to add on the D151 strongly acidic cation-exchange post eluting behind the 1-3 to get component and obtain Herba Leonuri total alkaloids.
The preparation of Comparative Examples 2-Herba Leonuri total alkaloids
Get Herba Leonuri 300g, decocte with water three times, 2 hours for the first time, 3 hours for the second time, 1 hour for the third time; Filter, merging filtrate is concentrated into about 250ml, cools off, and adds the ethanol of equivalent; Stir, left standstill 24 hours, filter, decompression filtrate recycling ethanol also is concentrated into the thick paste shape; Thin up is to 500ml, and cold preservation 24 hours filters, and the adjust pH of will filtrating is to add on the D018 strongly acidic cation-exchange post eluting behind the 1-3 to get component and obtain Herba Leonuri total alkaloids.
The preparation of Comparative Examples 3-Herba Leonuri total alkaloids
Get Herba Leonuri 300g, decocte with water three times, 2 hours for the first time, 1.5 hours for the second time, 1 hour for the third time; Filter, merging filtrate is concentrated into about 250ml, cools off, and adds the ethanol of equivalent; Stir, left standstill 24 hours, filter, decompression filtrate recycling ethanol also is concentrated into the thick paste shape; Thin up is to 500ml, and cold preservation 24 hours filters, and the adjust pH of will filtrating is to add on Nankai's 001 * 1 strongly acidic cation-exchange post eluting behind the 1-3 to get component and obtain Herba Leonuri total alkaloids.
The preparation of Comparative Examples 4-Herba Leonuri total alkaloids
Get Herba Leonuri 300g, decocte with water three times, 2 hours for the first time, 3 hours for the second time, 1 hour for the third time; Filter, merging filtrate is concentrated into about 250ml, cools off, and adds the ethanol of equivalent; Stir, left standstill 24 hours, filter, decompression filtrate recycling ethanol also is concentrated into the thick paste shape; Thin up is to 500ml, and cold preservation 24 hours filters, and the adjust pH of will filtrating is to add on Nankai's 001 * 2 strongly acidic cation-exchange post eluting behind the 1-3 to get component and obtain Herba Leonuri total alkaloids.
The preparation of Comparative Examples 5-Herba Leonuri total alkaloids
Get Herba Leonuri 300g, decocte with water three times, 2 hours for the first time, 1.5 hours for the second time; 1 hour for the third time, filter, merging filtrate is concentrated into about 250ml; Cool off, add the ethanol of equivalent, stir, left standstill 24 hours; Filter, decompression filtrate recycling ethanol also is concentrated into the thick paste shape, and thin up is to 500ml, cold preservation 24 hours; Filter, filtrating is added to eluting gets component C on the D151 strongly acidic cation-exchange post, merging promptly obtains Herba Leonuri total alkaloids with flinging to solvent behind the neutral alumina column purification with C with B component.
The preparation of Comparative Examples 6-Cortex Dictamni total alkaloids
Get Cortex Dictamni 300g, add 1% sulphuric acid and decoct three times, 2 hours for the first time, 1.5 hours for the second time, 1 hour for the third time; Filter, merging filtrate is concentrated into about 250ml, cools off, and adds the water of equivalent; Stir, left standstill 24 hours, filter, filtrate decompression reclaims solvent and is concentrated into the thick paste shape; Thin up is to 500ml, and cold preservation 24 hours filters, and filtrating added to eluting gets the Cortex Dictamni total alkaloids on the D018 strongly acidic cation-exchange post.
The preparation of Comparative Examples 7-Semen daturae total alkaloids
Get Semen daturae 300g, add 95% ethanol and decoct three times, 2 hours for the first time, 1.5 hours for the second time; 1 hour for the third time, filter, merging filtrate is concentrated into about 250ml; Cool off, add 2% sulphuric acid of equivalent, stir, left standstill 24 hours; Filter, decompression filtrate recycling ethanol also is concentrated into the thick paste shape, and thin up is to 500ml, cold preservation 24 hours; Filter, filtrating is added to eluting gets component C on the D019 strongly acidic cation-exchange post, to obtain total alkaloids subsequent use with flinging to solvent behind the neutral alumina column purification with B component and C merging.
The preparation of Comparative Examples 8-Rhizoma Coptidis total alkaloids
After the 300g Rhizoma Coptidis soaked 30 minutes, decoct 2 times, each 2 hours, collecting decoction filter filtrating, filtrating added to eluting obtains total alkaloids on the YWD12V strongly acidic cation-exchange post.
The preparation of Comparative Examples 9-Radix Sophorae Flavescentis total alkaloids
After the 300g Radix Sophorae Flavescentis soaked 30 minutes, add 95% methanol and decoct three times, 2 hours for the first time, 1.5 hours for the second time; 1 hour for the third time, filter, merging filtrate is concentrated into about 250ml; Cool off, add the water of equivalent, stir, left standstill 24 hours; Filter, decompression filtrate recycling ethanol also is concentrated into the thick paste shape, and thin up is to 500ml, cold preservation 24 hours; Filter, filtrating adds to that eluting gets component C on the YWD12V strongly acidic cation-exchange post, and merging obtains total alkaloids with flinging to solvent behind the neutral alumina column purification with C with B component.
The preparation of Comparative Examples 10-Cortex Phellodendri total alkaloids
Get Cortex Phellodendri 300g, decocte with water three times, 2 hours for the first time, 1.5 hours for the second time; 1 hour for the third time, filter, merging filtrate is concentrated into about 250ml; Cool off, add the ethanol of equivalent, stir, left standstill 24 hours; Filter, decompression filtrate recycling ethanol also is concentrated into the thick paste shape, and thin up is to 500ml, cold preservation 24 hours; Filter, the pH value of will filtrating is to add to that eluting gets component C on Nankai's 001 * 1 type strongly acidic cation-exchange post after 2.5, component C is flung to solvent after with the neutral alumina column purification promptly obtain Cortex Phellodendri total alkaloids.
The alkaloidal preparation of Comparative Examples 11-tripterygium total
Get Radix Tripterygii Wilfordii 300g, decocte with water three times, 2 hours for the first time, 1.5 hours for the second time; 1 hour for the third time, filter, merging filtrate is concentrated into about 250ml; Cool off, add the ethanol of equivalent, stir, left standstill 24 hours; Filter, decompression filtrate recycling ethanol also is concentrated into the thick paste shape, and thin up is to 500ml, cold preservation 24 hours; Filter, the pH value of will filtrating is to add to that eluting gets component C on Nankai's 001 * 2 type strongly acidic cation-exchange post after 3, component C is flung to solvent after with the neutral alumina column purification promptly obtain the tripterygium total alkaloid.
The preparation of Comparative Examples 12-Rhizoma Corydalis total alkaloids
Get Rhizoma Corydalis 300g, decocte with water three times, 2 hours for the first time, 1.5 hours for the second time; 1 hour for the third time, filter, merging filtrate is concentrated into about 250ml; Cool off, add the ethanol of equivalent, stir, left standstill 24 hours; Filter, decompression filtrate recycling ethanol also is concentrated into the thick paste shape, and thin up is to 500ml, cold preservation 24 hours; Filter, the adjust pH of will filtrating is to add to that eluting gets component C on Nankai's 001 * 14.5 type strongly acidic cation-exchange post after 1, component C is flung to solvent after with the neutral alumina column purification promptly obtain Rhizoma Corydalis total alkaloids.
The preparation of Comparative Examples 13-Radix Stephaniae Tetrandrae total alkaloids
Get Radix Stephaniae Tetrandrae 300g, decocte with water three times, 2 hours for the first time, 1.5 hours for the second time; 1 hour for the third time, filter, merging filtrate is concentrated into about 250ml; Cool off, add the ethanol of equivalent, stir, left standstill 24 hours; Filter, decompression filtrate recycling ethanol also is concentrated into the thick paste shape, and thin up is to 500ml, cold preservation 24 hours; Filter, the adjust pH of will filtrate is to add to after 2 to examine that eluting gets component C on the 732 type strongly acidic cation-exchange posts of Yadong, component C is flung to solvent after with the neutral alumina column purification promptly obtain the Radix Stephaniae Tetrandrae total alkaloids.
The preparation of Comparative Examples 14-Fructus Evodiae total alkaloids
Get Fructus Evodiae 300g, decocte with water three times, 2 hours for the first time, 1.5 hours for the second time; 1 hour for the third time, filter, merging filtrate is concentrated into about 250ml; Cool off, add the ethanol of equivalent, stir, left standstill 24 hours; Filter, decompression filtrate recycling ethanol also is concentrated into the thick paste shape, and thin up is to 500ml, cold preservation 24 hours; Filter, the adjust pH of will filtrate is to add to after 2 to examine that eluting gets component C on the 732 type strongly acidic cation-exchange posts of Yadong, component C is flung to solvent after with the neutral alumina column purification promptly obtain Fructus Evodiae total alkaloids.
The thin layer of the leonurine of embodiment and Comparative Examples contrast among experimental example 1-the present invention
1. sample:
Embodiment 1-4, Comparative Examples 1-4.
2. experimental technique:
With embodiment 1-4, Comparative Examples 1-4 with respectively get 10g, after the pulverizing, the apparatus,Soxhlet's of packing into, filters after 1 hour with methanol eddy, and is for use.Leonurine, stachydrine sample and embodiment 1-6 methanol solution are put separately on lamellae, launched, develop the color with bismuth potassium iodide with ethyl acetate-n-butyl alcohol-hydrochloric acid (3:2:1) system.
3. experimental result:
Find that commercially available Comparative Examples 1-4 does not have Herba Leonuri alkali and detects.And contain leonurine among the embodiment 1-4.
4. conclusion
This description of test, the alkaloid method for preparing that realizes through the present invention can be avoided the loss of leonurine, is superior to prior art and commercially available product.
The liquid chromatograph of the leonurine content of embodiment and Comparative Examples contrast among experimental example 2-the present invention
1. sample:
Embodiment 1-4, Comparative Examples 1-4.
2. experimental technique:
With embodiment 1-4 and Comparative Examples 1-4 gained total alkaloids, respectively get 10g, after the pulverizing, the apparatus,Soxhlet's of packing into, filters after 1 hour with methanol eddy, changes in the 100ml volumetric flask, and is after methanol constant volume, to be measured.Leonurine detects chromatographic condition: chromatograph of liquid: Tianjin, island LC-20AB, detector: SPD-M20A, chromatographic column: C
184.6 * 250mm5 μ m, column temperature: 30 ℃, mobile phase: acetonitrile-0.05mol/L potassium dihydrogen phosphate 14:86, detect wavelength: 276nm.
3. experimental result:
| Sample | Leonurine content (content in total alkaloids, %) |
| Embodiment 1 | 7.24％ |
| Embodiment 2 | 5.63％ |
| Embodiment 3 | 3.69％ |
| Embodiment 4 | 5.89％ |
| Comparative Examples 1 | 0.17％ |
| Comparative Examples 2 | 0.05％ |
| Comparative Examples 3 | 0.13％ |
| Comparative Examples 4 | 0.25％ |
4. conclusion
Comparative Examples 1-4 is current representative method for the Herba Leonuri total alkaloids extraction separation; Through with contrast experiment's explanation of the present invention; The alkaloid method for preparing that the present invention realizes can be avoided the loss of leonurine; Preserving the important effective ingredient leonurine in the Herba Leonuri greatly, thereby more effectively must guarantee that medicine gets quality, is a kind of novel more and total alkaloids method for preparing that is superior to prior art.
Experimental example 3-total alkaloids with contrast prior art
Sample: embodiment 5-14, Comparative Examples 5-14.
Measure two kinds of each total alkaloid contents of 2 batches with following method.
The preparation precision of reference substance solution takes by weighing monosomic alkali (the representative alkaloid of each plant) 5mg that is dried to constant weight, and in the 25ml volumetric flask, the hydrochloric acid solution that adds 0.1mol/L makes dissolving and is diluted to scale, shakes up, and promptly gets.
The preparation precision of standard curve is measured reference substance solution 2.0,4.0,6.0,8.0,10.0ml, and in the volumetric flask as for 25ml, all the hydrochloric acid solution with 0.1mol/L is diluted to 10ml respectively; Placed 1 hour; Decompression filters, and measures trap in the certain wave strong point, is vertical coordinate with the trap; Concentration is abscissa, the drawing standard curve.
It is an amount of that the algoscopy precision takes by weighing sample, in the 100ml volumetric flask, adds the 0.1mol/L hydrochloric acid solution and be diluted to scale, shakes up; Precision is measured diluent 7.0ml again, puts in the 25ml beaker, adds the 0.1mol/L hydrochloric acid solution and is diluted to 10ml; The sighting target directrix curve prepare the method under the item, from " putting in the ice bath ", measure trap; From standard curve, read the content in the need testing solution, calculate, promptly get.The assay result sees table 1.
Table 1 Determination of Total Alkaloid relatively
| Sample | Total alkaloid content (%) | Sample | Total alkaloid content (%) | Monosomic alkali (the representative alkaloid of each plant) |
| Embodiment 5 | 82.7 | Comparative Examples 5 | 56.4 | Stachydrine |
| Embodiment 6 | 83.0 | Comparative Examples 6 | 51.5 | Dictamine |
| Embodiment 7 | 65.2 | Comparative Examples 7 | 48.1 | Atropina |
| Embodiment 8 | 81.0 | Comparative Examples 8 | 52.0 | Berberine |
| Embodiment 9 | 72.1 | Comparative Examples 9 | 55.5 | Matrine |
| Embodiment 10 | 65.0 | Comparative Examples 10 | 47.5 | Berberine |
| Embodiment 11 | 76.7 | Comparative Examples 11 | 50.1 | Wilfordine |
| Embodiment 12 | 59.3 | Comparative Examples 12 | 33.0 | The Rhizoma Corydalis first is plain |
| Embodiment 13 | 78.1 | Comparative Examples 13 | 58.6 | Tetrandrine |
| Embodiment 14 | 68.7 | Comparative Examples 14 | 52.0 | Rutaecarpin |
Can reach a conclusion thus: the present invention has the purity that is superior to routine techniques for the preparation of the total alkaloids in the medicine; Significantly improving alkaloid and be prepared in the demand of safe, controlled and effective aspect, is a kind of method for preparing with innovative significance that is of universal significance.
Claims (4)
1. the method for preparing of a total alkaloids; It is characterized in that described method for preparing is: plant extract is added on the weak-type cation exchange resin column; Get component A with water elution, reuse ammonia eluting gets B component, is to add to that eluting gets component C on the strongly acidic cation-exchange post behind the 1-3 with component A adjust pH; B component and C merging are obtained total alkaloids with the neutral alumina column purification, and the concentration of described ammonia is 3-9%.
2. the method for preparing of total alkaloids according to claim 1 is characterized in that described weak-type cation exchange resin column is selected from the D151 of Nankai, Nankai's 101 type weak-type cation exchange resin columns.
3. the method for preparing of total alkaloids according to claim 1 is characterized in that described strongly acidic cation-exchange post is selected from the 001X1 of Nankai, the 001X2 of Nankai, the 001X3 of Nankai, the 001X14.5 of Nankai, D018, D019, D020, YWD12V or nuclear Yadong 732 type strongly acidic cation-exchanges.
4. the method for preparing of total alkaloids according to claim 1 is characterized in that described plant extract is obtained by the extraction of one of Herba Leonuri, Rhizoma Corydalis, the Radix Stemonae, Semen daturae, Rhizoma Menispermi, Herba Dendrobii, Radix Tripterygii Wilfordii, Radix Tinosporae, Semen Hyoscyami, Cacumen Securinegae Suffruticosae, Rhizoma Coptidis, Herba Ephedrae, Radix Stephaniae Tetrandrae, Cortex Phellodendri, Ramulus Uncariae Cum Uncis, Bulbus Fritillariae Uninbracteatae, Fructus Camptothecae Acuminatae, Fructus Evodiae, Cortex Cinchonae, Semen Arecae, Herba Catharanthi Rosei, Radix Sophorae Flavescentis, Radix Sophorae Tonkinensis, Radix Gentianae Macrophyllae, Radix Rauvolfiae, the Radix Linderae, Radix Aconiti, Radix Aconiti Lateralis Preparata, Herba Lobeliae Chinensis and Cortex Dictamni.
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| CN102772638A (en) * | 2012-08-10 | 2012-11-14 | 宁波杰顺生物科技有限公司 | Crude dry extract of thunberg fritillary bulb total alkaloids |
| CN102772639A (en) * | 2012-08-10 | 2012-11-14 | 宁波杰顺生物科技有限公司 | Preparation method of thunberg fritillary bulb total alkaloid extract |
| CN104255815A (en) * | 2014-08-29 | 2015-01-07 | 北京市思达尔化工新技术有限责任公司 | Preparation method of botanical insecticide |
| CN107970641A (en) * | 2016-10-24 | 2018-05-01 | 张家港市金港镇宏业海绵复合厂 | The line separation of alkaloid |
| CN109293668B (en) * | 2018-11-01 | 2020-03-31 | 仲恺农业工程学院 | Method for extracting alkaloid |
| CN114315718A (en) * | 2022-01-13 | 2022-04-12 | 山西中医药大学 | Preparation method of cyclanoline |
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