CN102372723A - Method for extracting arglabin from artemisia myriantha - Google Patents
Method for extracting arglabin from artemisia myriantha Download PDFInfo
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Abstract
The invention relates to a method for extracting arglabin from artemisia myriantha. The method comprises the following steps of: crushing the artemisia myriantha serving as a medicinal material, extracting by using ethanol, performing pre-separation, filtering, concentrating, separating concentrates and purifying to obtain the arglabin, wherein the concentrates are separated by a silica gel column; an elution system comprises a petroleum ether-ethyl acetate system, an n-hexane-ethyl acetate system, a petroleum ether-acetone system and an n-hexane-acetone system; and the pre-separation operation can be performed by alcohol precipitation, water precipitation, alcohol precipitation and water precipitation mixed treatment, alcohol precipitation and organic solvent extraction mixed treatment and the like. In the method, raw materials are abundant in plant sources, and the method is simple, environment-friendly and low in production cost, and is more suitable for large-scale industrial production.
Description
Technical field
The present invention relates to the process for extracting of natural product, be specifically related to a kind of method of from spend more wormwood artemisia, extracting Arglabine.
Background technology
Arglabine (Arglabin, structural formula is suc as formula shown in the I), chemistry 1, the 10 beta epoxide by name-wound that heals-3 (4)-alkene-6,12-lactone.
Arglabine at first is to be obtained by the petal and the leaf extraction of Kazakhstan scientist S.M. Adriano Knopf from the bright green wormwood artemisia of local Art plant, and discovers that this compound has anti-tumor activity.1996; Arglabine dimethylamine hydrochloride freeze-dried prepn is gone on the market in the republic of Kazakhstan by the exploitation of Kazakhstan Tabi Fall company; This medicine successively in some country's listings of the FSU, is mainly used in treatment liver cancer, lung cancer, mammary cancer, ovarian cancer again subsequently.
Having put down in writing with bright green wormwood artemisia among the WO9848789 by the application of Kazakhstan Tabi Fall company is the method that raw material extracts Arglabine.This patent at first to adopt multiple solvent as: water, sherwood oil-ether 1: 1, benzene, ether, chloroform, ethanol etc. are investigated the extraction efficiency of bright green wormwood artemisia, and the result shows: adopt chloroform as solvent, extraction efficiency is the highest; Secondly be ethanol, ether, benzene, sherwood oil-ether, ether, water.This patent working example has been carried out the preparation of Arglabine with chloroform as extracting solvent; Leaching process is following: adopting chloroform is that solvent carries out the adverse current heating and extracting to bright green wormwood artemisia medicinal material; Extract is after alcohol precipitation is handled; The extract that obtains is gone up silicagel column again through the benzene wash-out, the Arglabine bullion that obtains is carried out the normal hexane recrystallization, the Arglabine monomer, yield is 0.27%.The bright green wormwood artemisia of vegetable material of this patent record is distributed in Xinjiang of China, and scarcity of resources can not satisfy the drug development needs; And this patent method need be used special counterflow extraction apparatus, the equipment cost height; The preferred extraction agent of institute, eluent adopt highly toxic chloroform and benzene, are unfavorable for workers ' health and environmental protection.
The Ho-Fao Wong of Hong Kong University and Geoffrey D.Brown are at Dimeric guaianolides and a fulvenoguaianolide from Artemisia myriantha (Journal of Natural Products.2002; 65 (4): reported 481-486) that it is raw material that wormwood artemisia is spent more in employing; Extract with room temperature under the methylene dichloride inflated with nitrogen condition; Silicagel column is through normal hexane, ETHYLE ACETATE, methanol-eluted fractions on the extract that obtains; Gained ETHYLE ACETATE partly flows part through the positive liquid phase separation, gets the Arglabine monomer, and yield is 0.037%.Though it is raw material that this method has adopted the abundant relatively wormwood artemisia of spending more of china natural resources, its yield is too low, and solvent for use methylene dichloride toxicity is big.
Reported that with bright green wormwood artemisia be raw material among the PCT application WO2008035958, (pressure is 150 ± 2-10 with the supercritical carbon dioxide extraction method
5Handkerchief, temperature are 60 ℃) obtain the method for Arglabine.This method can bring up to 0.8% with the content of Arglabine.But this method need be used special extraction equipment, has increased production cost undoubtedly.
Summary of the invention
Report from prior art; Arglabine mainly is present in bright green wormwood artemisia and spends more in the wormwood artemisia; And the content in bright green wormwood artemisia is apparently higher than spending more wormwood artemisia, but bright green wormwood artemisia distributes seldom in China, can not satisfy the needs of drug development; Be the technology that raw material extracts the preparation Arglabine to spend more wormwood artemisia, can not satisfy needs of scale production far away and according to existing.
In order to solve the problem that exists in the prior art, the inventor studies the extraction process of Arglabine so that the abundant relatively wormwood artemisia of spending more is a raw material in the Chinese Plants source.Through great deal of experimental; The contriver finds: under the situation of using common extraction equipment, adopting and spending more wormwood artemisia is plant origin, can be that plant origin obtains the suitable even higher Arglabine of yield with adopting bright green wormwood artemisia; And process for extracting is simple, environmental protection, and production cost is low.
Therefore, the invention provides a kind of method of from spend more wormwood artemisia, extracting Arglabine, said method is with after spending more the wormwood artemisia pulverizing medicinal materials; Use extraction using alcohol, carry out pre-separation then, filter; Concentrate, at last with enriched material separate, purifying, obtain Arglabine; Said separation adopts silicagel column to separate, and used elution system is petroleum ether-ethyl acetate system, normal hexane-ETHYLE ACETATE system, sherwood oil-acetone system, normal hexane-acetone system.
Wherein:
Said ethanol is selected from 30～100% aqueous ethanolic solution, is preferably 95% aqueous ethanolic solution.
The solid-liquid ratio of used ethanol and medicinal material (V/W) is any suitable proportion, is preferably 6～12 times.
Extracting temperature is any optimal temperature, is preferably 20-100 ℃, more preferably 70-90 ℃.
Said elution system is preferably the petroleum ether-ethyl acetate system, the petroleum ether-ethyl acetate system of more preferably 9: 1 (V/V).
Said pre-separation can adopt following any one method to carry out: alcohol precipitation processing, depositing in water processing, depositing in water and alcohol precipitation combination treatment, alcohol precipitation and organic solvent extraction combination treatment; Preferred depositing in water and alcohol precipitation combination treatment and alcohol precipitation and the organic solvent extraction combination treatment of adopting more preferably adopts alcohol precipitation and depositing in water combination treatment.Wherein, said depositing in water and alcohol precipitation combination treatment can be chosen sequencing wantonly and carry out, that is: can ethanol extraction carried out handling through alcohol precipitation after depositing in water is handled again, and also can ethanol extraction be handled through alcohol precipitation earlier and carry out depositing in water again and handle; Said alcohol precipitation and organic solvent extraction combination treatment also can be chosen sequencing wantonly and carry out, that is: can ethanol extraction carried out carrying out organic solvent extraction again after alcohol precipitation is handled, and also can ethanol extraction be carried out organic solvent extraction earlier and carry out alcohol precipitation again and handle.Said alcohol precipitation is handled the alcohol-water mixed solution that used alcohol is any suitable proportion, is preferably 60～80% ethanol, more preferably 70% ethanol; The organic solvent of said extraction is selected from ETHYLE ACETATE, chloroform, the methylene dichloride any one, is preferably ETHYLE ACETATE.
Said purifying can adopt any suitable mode to carry out, and preferably adopts recrystallization method to carry out purifying, and used recrystallization solvent is selected from one or more the mixed solvent in normal hexane, sherwood oil, ethanol, methyl alcohol, Virahol, acetone, the water; When the pre-separation method was alcohol precipitation processing, depositing in water and alcohol precipitation combination treatment, alcohol precipitation and organic solvent extraction combination treatment, recrystallization solvent was normal hexane or sherwood oil; When the pre-separation method is a depositing in water when handling, used recrystallization solvent is that normal hexane or sherwood oil and volume ratio are 1: 1 alcohol-water.
In order to obtain Arglabine process for extracting of the present invention, the contriver has mainly carried out following research:
Gather the wormwood artemisia of spending more of growth in May, dry the back primary crusher and pulverize, subsequent use.
1, extracts choice of Solvent
Get the 10g medicinal material for every part, adopt sherwood oil, normal hexane, methylene dichloride, chloroform, ETHYLE ACETATE, acetone, ethanol, several kinds of solvents of methyl alcohol respectively, each handles two repetitions; Add 15 times to the solvent of medicinal material (V/W); Cold soaking extracts 48h, with extracting liquid filtering, concentrate the back with methanol constant volume to 250ml; HPLC detects its content (g/g crude drug), and experimental result is as shown in Figure 1.Experimental result shows: ethanol and methanol extraction are most effective, consider factors such as solvent toxicity and recycling, select ethanol as extracting solvent.
2, the selection of alcohol concn
Get the 10g medicinal material for every part, the ethanol that adds 100ml 30%, 50%, 70%, 80%, 90% and 95% concentration respectively carries out refluxing extraction, two repetitions of each concentration; Heating and refluxing extraction three times; Each 1h, quantity of solvent is respectively 10,8,8 times to crude drug (V/W), is settled to 250ml after the extracting solution merging is concentrated; HPLC detects, and the result is as shown in Figure 2.Experimental result shows: in 30～95% alcohol concn, 95% extraction using alcohol is most effective.
3, the selection of liquid material ratio
Every part takes by weighing the 10g medicinal material, places round-bottomed flask, and each tests two repetitions; 6 times, 8 times, 10 times and 12 times 95% ethanol adding crude drug (V/W) amount respectively, refluxing extraction 1h, extracting solution is settled to 250ml after concentrating; HPLC detects Arglabine content, and the result is as shown in Figure 3.Experimental result shows: the liquid material is higher than more, and the extraction efficiency of Arglabine is high more.
4, spend more the investigation of wormwood artemisia ethanol extraction pre-separation method
1. alcohol precipitation is handled: get ethanol extraction 77g, adopts 4 times behind the dissolve with ethanol extract of crude drug (V/W) thin up to determining alcohol be 70%, precipitate the 24h after-filtration; Obtain filtrating, evaporated under reduced pressure obtains the about 60.8g of dry-matter; 95% ethanol constant volume, HPLC detects.
2. depositing in water is handled: get ethanol extraction 77g, leave standstill 24h after adopting 2 times of water that extract is dissolved, obtain precipitating about 36g to crude drug, and with 95% dissolve with ethanol constant volume, the HPLC detection.
3. first alcohol precipitation is handled depositing in water again and is handled: get ethanol extraction 77g; Adopt 4 times behind the dissolve with ethanol extract of crude drug (V/W) thin up to determining alcohol be 70%, deposition 24h after-filtration obtains filtrating; Concentrating the back adopts 2 times of water to crude drug to carry out the depositing in water processing; Obtain precipitating about 20g, with 95% dissolve with ethanol constant volume, HPLC detects.
4. first depositing in water is handled alcohol precipitation again and is handled: get ethanol extraction 77g, leave standstill 24h after adopting 2 times of water to crude drug that extract is dissolved, obtain precipitating; Deposition is added after 4 times of ethanol to crude drug dissolve, and thin up to determining alcohol is 70%, leaves standstill the 24h after-filtration; Obtain filtrating, concentrating under reduced pressure is weighed; About 20.5g, 95% ethanol constant volume, HPLC detects.
5. ethyl acetate extraction behind the alcohol precipitation: get ethanol extraction 77g, adopt 4 times behind the dissolve with ethanol extract of crude drug (V/W) thin up to determining alcohol be 70%, deposition 24h after-filtration; Obtain filtrating; Concentrate the back and adopt 2 times of water to crude drug to dissolve, solution adopts the ETHYLE ACETATE of equivalent to extract three times, obtains extraction liquid; Concentrate back (about 20.3g) 95% ethanol constant volume, HPLC detects.
6. alcohol precipitation behind the ethyl acetate extraction: get ethanol extraction 77g, add the suitable quantity of water dissolving after, extract three times with the ETHYLE ACETATE of equivalent; After the extraction liquid that obtains concentrated, adds 4 times behind the dissolve with ethanol extract of crude drug (V/W) thin up to determining alcohol be 70%, precipitate the 24h after-filtration; Obtain filtrating, drying under reduced pressure obtains the 19.8g extract; Behind the 95% ethanol constant volume, HPLC detects.
7. chloroform extraction behind the alcohol precipitation: get ethanol extraction 77g, adopt 4 times behind the dissolve with ethanol extract of crude drug (V/W) thin up to determining alcohol be 70%, deposition 24h after-filtration; Obtain filtrating; Concentrate the back and adopt 2 times of water to crude drug to dissolve, solution adopts the chloroform of equivalent to extract three times, obtains extraction liquid; Concentrate back (about 19.5g) 95% ethanol constant volume, HPLC detects.
8. alcohol precipitation behind the chloroform extraction: get ethanol extraction 77g, add the suitable quantity of water dissolving after, extract three times with the chloroform of equivalent; After the extraction liquid that obtains concentrated, adds 4 times behind the dissolve with ethanol extract of crude drug (V/W) thin up to determining alcohol be 70%, precipitate the 24h after-filtration; Obtain filtrating, drying under reduced pressure obtains the 19.3g extract; Behind the 95% ethanol constant volume, HPLC detects.
Experimental result is as shown in Figure 4; Experimental result shows: in several kinds of pre-separation methods; Only adopt depositing in water to handle and to remove most impurity; Influence the isolating oil substances of Arglabine but this method can not be removed, in subsequent separation process, need carry out again just obtaining Arglabine after the oil removal treatment, can increase the rate of loss of Arglabine undoubtedly; The extractive content that other treatment process obtains is suitable; Though but only adopt the method for alcohol precipitation to remove oil substances; But contain a large amount of water-soluble substanceses in the extract that obtains, increased later separation workload (as the usage quantity that increases silica gel with separate number of times).Therefore, the present invention preferably adopts depositing in water and alcohol precipitation mixed processing method and alcohol precipitation and organic solvent extraction mixed processing method; Consider that extracting process relates to an organic solvent, increased potential safety hazard, therefore, more preferably adopt depositing in water and alcohol precipitation mixed processing method.
5, alcohol concn was investigated during the ethanol extraction alcohol precipitation was handled
Adopt the final determining alcohol of 60%, 70%, 80% conduct that alcohol concn in the alcohol precipitation experiment is investigated respectively.After experiment adopted 4 times of ethanol to crude drug (L/kg) to dissolve, thin up was to corresponding concentration.Room temperature left standstill 1 day, filtered, and HPLC detects Arglabine content in the filtrating, compared with the Arglabine content that carries out in the preceding extract of alcohol precipitation processing, and experimental result is as shown in table 1:
Alcohol concn was to the influence of Arglabine content during table 1 alcohol precipitation was handled
| Determining alcohol (%) | 60 | 70 | 80 | Before alcohol precipitation is handled |
| Arglabine content (%) | 7.28 | 7.42 | 7.25 | 8.45 |
Experimental result shows: 70% alcohol concn is minimum to the rate of loss influence of Arglabine.
6, the investigation of eluting solvent in the separating technology
(1) the eluting solvent polar is selected: according to the isolating result of standard substance, select the primary dcreening operation solvent of petroleum ether-ethyl acetate system as development system for use, carry out polar and select.Adopt the petroleum ether-ethyl acetate of 95: 5,9: 1,8: 2,7: 3,6: 4,5: 5 (V/V) to launch respectively, the thin-layer chromatography result sees Fig. 5.According to the thin-layer chromatography result, petroleum ether-ethyl acetate 9: 1 is suitable as the eluting solvent polarity of column chromatography for separation.
(2) selection of eluting solvent: according to the solvent polarity of confirming in (1), the method that adopts thin-layer chromatography to detect is selected the separation system of Arglabine.Adopt petroleum ether-ethyl acetate (9: 1), sherwood oil-acetone (9: 1), normal hexane-ETHYLE ACETATE (9: 1), normal hexane-acetone (9: 1), sherwood oil-chloroform (9: 1), sherwood oil-methylene dichloride (9: 1), chloroform etc. to carry out thin-layer developing respectively, thin layer plate adopts 10%H
2SO
4-EtOH develops the color, and the thin-layer chromatography result sees Fig. 6.The result shows: chloroform is too big as developping agent polarity, can not select for use; The polar phase of petroleum ether-ethyl acetate, sherwood oil-acetone, normal hexane-acetone, normal hexane-ETHYLE ACETATE system is worked as; But petroleum ether-ethyl acetate (9: 1) system can better separate by the assorted point that polarity is close; And take all factors into consideration the volatility and the toxicity of solvent, select the petroleum ether-ethyl acetate system as eluting solvent.
7, the investigation of recrystallization solvent in the purifying process
The Arglabine bullion that will obtain with column chromatography is with normal hexane, sherwood oil, acetone, 1: 1 alcohol-water (V/V; 1: 1 methanol-water (V/V down together); Down with), 1: 1 acetone-water (V/V, down with), etc. carry out recrystallization, find: when adopting non-depositing in water pre-separation method; The Arglabine bullion of gained is through above-mentioned solvent recrystallization, and the pure article purity of gained Arglabine is more than 99%.Wherein normal hexane and sherwood oil are the highest, reach about 99.5%, and the recrystallization loss are little, and the recovery is more than 85%.The Arglabine bullion that only adopts the depositing in water pre-separation to obtain; Owing to contain more lipid-soluble substance, need through twice recrystallization, the result shows: 1: 1 alcohol-water of recrystallization employing for the first time, 1: 1 methanol-water, 1: 1 acetone-water are recrystallization solvent; Recrystallization adopts normal hexane, sherwood oil etc. for the second time; Products obtained therefrom purity is all more than 99%, but 1: 1 alcohol-water of recrystallization employing for the first time is a recrystallization solvent, and the normal hexane of recrystallization employing for the second time, sherwood oil are recrystallization solvent; Product purity is the highest, can reach about 99.5%.The order of above-mentioned two kinds of recrystallizations also can be changed, and promptly earlier carries out recrystallization with normal hexane, sherwood oil and uses 1: 1 alcohol-water to carry out recrystallization again.
Arglabine process for extracting of the present invention has been realized following beneficial effect:
(1) being employed in the abundant relatively wormwood artemisia of spending more of Chinese resource is plant origin, for this medicine provides competent raw material guarantee in the exploitation listing of China;
(2) adopt nontoxic or low-toxic solvents such as ethanol, water, ETHYLE ACETATE, be more conducive to workers ' health and environmental protection.
(3) solvent load is few, and extraction, alcohol precipitation handle used alcohol solvent and can reclaim, and carries out recycle, has practiced thrift production cost.
(4) use common extraction equipment can realize the high-level efficiency extraction, reduced production cost.
To sum up, Arglabine process for extracting of the present invention, raw material sources are abundant, and method is simple, environmental protection, production cost are low, is more suitable in large-scale industrial production.
Description of drawings
Fig. 1 is different influences of extracting solvent to the Arglabine extraction efficiency.
Fig. 2 is the influence of alcohol concn to the Arglabine extraction efficiency.
Fig. 3 is the influence of solid-liquid ratio to the Arglabine extraction efficiency.
Fig. 4 is the influences of different pretreatment processs to Arglabine content.
Fig. 5 is the thin-layer chromatography result of opposed polarity eluting solvent.
Fig. 6 is the thin-layer chromatography result of different solvents elution system.
Fig. 7 is the 1H-NMR spectrogram of Arglabine standard substance.
Fig. 8 is the 13C-NMR spectrogram of Arglabine standard substance.
Fig. 9 is the mass spectrogram of Arglabine standard substance.
Embodiment
To help to understand the present invention through following embodiment, but not be construed as limiting the invention.
Embodiment 1: the acquisition of Arglabine standard substance
Get drying and spend more wormwood artemisia (collection in March) over-ground part 14kg, the mode that adopts cold soaking to extract is extracted.Add for the first time 14 times to the methylene dichloride (W/W) of spending more the wormwood artemisia amount, extraction time is 24h; For the second time add 12 times, extract 48h to the methylene dichloride of spending more wormwood artemisia.Extracted twice liquid is merged, concentrate, obtain the about 654.9g of extract.
To spend more the wormwood artemisia dichloromethane extract and carry out silica gel (100-200 order) at 1: 1 according to material ratio and mix appearance, oven dry.Carry out silicagel column and separate, elution system is normal hexane-ETHYLE ACETATE system, and stream part of collection merges according to the thin-layer chromatography detected result.The employing normal hexane method of recrystallization repeatedly carries out purifying to sample, obtains the compound Arglabine.The gained sample is carried out hydrogen spectrum, carbon spectrum, mass spectrum, fusing point test, and the result is following:
Hydrogen spectrum data: 1.35 (3H, s, 14-H), 1.97 (3H, s, 15-H), 2.18 (2H, 2-H), 2.73 (1H, d; J=22Hz, 7-H), 2.95 (1H, d, J=10Hz, 5-H), 4.00 (1H, T, J=10Hz, 6-H); 5.58 (1H, s, 3-H), 6.1 (1H, s, 13-H), 5.42 (1H, s, 13-H)
Carbon spectrum data: 18.2 (15-C), 21.4 (8-C), 22.7 (14-C), 33.4 (9-C), 39.7 (2-C); 51.0 (7-C), 52.8 (5-C), 62.6 (10-C), 72.3 (1-C), 82.96 (6-C); 118.2 (13-C), 124.8 (3-C), 139.1 (11-C), 140.4 (4-C), 170.4 (12-C)
Mass-spectrometric data: positive ion mode 515.9 [2M+Na]
+, 269.7 [M+Na]
+, 247.1 [M+H]
+
Fusing point: 100-102 ℃
Reference:
①WO?9848789：Pharmaceutical?compositions?of?arglabin?and?arglabin?derivatives.
②Ho-Fao?Wong?and?Geoffrey?D.Brown.Dimeric?guaianolides?and?a?fulvenoguaianolide?from?Artemisia?myriantha.Journal?of?Natural?Products.2002，65(4)：481-486.
Embodiment 2: the preparation of Arglabine
Get and spend more wormwood artemisia medicinal material (flowering period) 1kg, after the pulverizing, 95% alcohol heating reflux that adds 10L extracts 1.5h, filters, and 95% ethanol of the dregs of a decoction with 8L * 2 is extracted, and extracting solution is merged concentrate, and obtains the about 210g of extract.After extract added 2L water heating for dissolving, room temperature left standstill 24h, filtered, and obtained deposition; After the 95% ethanol heating for dissolving of this deposition with 2L, adding water to determining alcohol is 70%, after room temperature is placed 24h; Filter, the filtrating that obtains is concentrated, obtain about 36.5g extract.This enriched material is carried out the silicagel column separation, and material ratio is 1: 5～1: 10 (extract/silica gel: W/W, down together), and 9: 1 wash-outs of petroleum ether-ethyl acetate are collected stream part, and thin layer detects, the merging same composition.After the Arglabine position concentrated, adds the normal hexane of 2～5 times of amounts (W/W extract, down with), heating for dissolving, room temperature is placed crystallization, filters, drying, the gained solid weight is 3.05g, and yield is about 0.305% (W/W medicinal material, under together), and HPLC purity is 99.56%.Gained is fixedly carried out hydrogen spectrum, carbon spectrum, mass spectrum, fusing point test, and the result is with the Arglabine standard substance.
Embodiment 3: the preparation of Arglabine
With reference to the preparation method of embodiment 2, obtain the about 210g of extract.After extract added 95% ethanol 4L heating for dissolving, adding water to determining alcohol was 70%, and room temperature leaves standstill 24h, filtered, and obtained filtrating, and being concentrated into does not have alcohol, adds water to 2L, heated and stirred, and room temperature leaves standstill 24h, and the sucking-off supernatant obtains precipitating 36.2g.This deposition is carried out silicagel column separate, material ratio is 1: 5～1: 10, and 9: 1 wash-outs of petroleum ether-ethyl acetate are collected stream part, and thin layer detects, and merges same composition.After the Arglabine position concentrated, add the sherwood oil of 2～5 times of amounts, heating for dissolving, room temperature is placed crystallization, filters, drying, the gained solid weight is 3.02g, and yield is about 0.302%, and it is 99.51% that HPLC detects its purity.Gained is fixedly carried out hydrogen spectrum, carbon spectrum, mass spectrum, fusing point test, and the result is with the Arglabine standard substance.
Embodiment 4: the preparation of Arglabine
With reference to the preparation method of embodiment 2, obtain the about 210g of extract.After extract added 4L water heating for dissolving, room temperature left standstill 24h, filtered, and obtained filtrating, concentrated, and obtained extract 80g.This extract is carried out silicagel column separate, material ratio is 1: 5～1: 10, and 9: 1 wash-outs of petroleum ether-ethyl acetate are collected stream part, and thin layer detects, and merges same composition.After the Arglabine position concentrated, add the normal hexane of 2～5 times of amounts, heating for dissolving, room temperature is placed crystallization, filters, and obtains the about 5g of Arglabine bullion (containing oily matter).After the Arglabine bullion being added 95% ethanol heating for dissolving of 15 times of amounts, adding water to determining alcohol is 50%, continues heating, filters; Room temperature is placed 24h, filters drying; The gained solid weight is 2.53g, and yield is about 0.253%, and it is 99.3% that HPLC detects its purity.Gained is fixedly carried out hydrogen spectrum, carbon spectrum, mass spectrum, fusing point test, and the result is with the Arglabine standard substance.
Embodiment 5: the preparation of Arglabine
With reference to the preparation method of embodiment 2, obtain the about 210g of extract.After extract added 95% ethanol 4L heating for dissolving, adding water to determining alcohol was 70%, and room temperature leaves standstill 24h, filtered, and obtained filtrating, was concentrated into driedly, obtained extract 145g.This extract is carried out silicagel column separate, material ratio is 1: 5～1: 10, and 9: 1 wash-outs of petroleum ether-ethyl acetate are collected stream part, and thin layer detects, and merges same composition.After the Arglabine position concentrated, add the sherwood oil of 2～5 times of amounts, heating for dissolving, room temperature is placed crystallization, filters, drying, the gained solid weight is 3.0g, and yield is about 0.3%, and it is 99.52% that HPLC detects its purity.Gained is fixedly carried out hydrogen spectrum, carbon spectrum, mass spectrum, fusing point test, and the result is with the Arglabine standard substance.
Embodiment 6: the preparation of Arglabine
With reference to the preparation method of embodiment 2, obtain the about 210g of extract.After extract added 95% ethanol 4L heating for dissolving, adding water to determining alcohol was 70%, and deposition 24h after-filtration obtains filtrating; Being concentrated into does not have alcohol, adds 2L water and dissolves, and gained solution extracts with the ETHYLE ACETATE of 2L * 3; Obtain extraction liquid, concentrate, obtain the about 35.2g of enriched material.This deposition is carried out silicagel column separate, material ratio is 1: 5～1: 10, and 9: 1 wash-outs of petroleum ether-ethyl acetate are collected stream part, and thin layer detects, and merges same composition.After the Arglabine position concentrated, add the normal hexane of 2～5 times of amounts, heating for dissolving, room temperature is placed crystallization, filters, and obtains Arglabine 2.98g, and it is 99.50% that HPLC detects its purity, and yield is about 0.298%.
Embodiment 7: the preparation of Arglabine
With reference to the preparation method of embodiment 2, obtain the about 210g of extract.After adding the 3L water dissolution, extract with the ETHYLE ACETATE of 3L * 3, after the extraction liquid that obtains was concentrated, after enriched material added 95% ethanol 4L heating for dissolving, adding water to determining alcohol was 70%, and deposition 24h after-filtration obtains filtrating, and is concentrated, obtains the 36.0g extract.This extract is carried out silicagel column separate, material ratio is 1: 5～1: 10, and 9: 1 wash-outs of petroleum ether-ethyl acetate are collected stream part, and thin layer detects, and merges same composition.After the Arglabine position concentrated, add the sherwood oil of 2～5 times of amounts, heating for dissolving, room temperature is placed crystallization, filters, and obtains Arglabine 3.03g, and it is 99.54% that HPLC detects its purity, and yield is about 0.303%.
Can find out from embodiment 2～7 experimental results: several kinds of pre-separation methods, gained Arglabine purity is suitable, is about 99.5%, is starkly lower than other pre-separation method but only adopt depositing in water to handle gained Arglabine yield; Only adopt the pretreatment process of alcohol precipitation, the extract amount that obtains is bigger, the usage quantity that has increased silica gel with separate number of times.Therefore, the present invention preferably adopts depositing in water and alcohol precipitation mixed processing method and alcohol precipitation and organic solvent extraction mixed processing method; Consider that extracting process relates to an organic solvent, increased potential safety hazard, therefore, more preferably adopt depositing in water and alcohol precipitation mixed processing method.
With reference to the preparation method of embodiment 2, will extract solvent replacing is methyl alcohol and acetone, carries out the preparation of Arglabine, and experimental result is as shown in table 2:
Table 2: embodiment 8～9 experimental results
| Embodiment | Extract solvent | Product weight (g) | Yield (%) | Purity (%) |
| 8 | Methyl alcohol | 2.89 | 0.289 | 99.55 |
| 9 | Acetone | 1.53 | 0.153 | 99.48 |
Can find out from table 2 experimental result: adopt methyl alcohol as extracting solvent, the yield of Arglabine and purity and ethanol are suitable as extracting the solvent effect; Adopt acetone as extracting solvent and adopting ethanol as extracting the solvent phase ratio; Product purity and yield descend, but adopt ethanol as extracting solvent, can avoid the use of the organic solvent bigger to human toxicity; And compare other organic solvent; Loss is lacked in spissated process, reclaims solvent and can recycle, and has reduced cost.The present invention has selected ethanol as extracting solvent on the basis of taking all factors into consideration multiple factors such as solvent toxicity, production cost, but carries out the Arglabine extraction and also will fall within the protection domain of the present invention with other solvent such as methyl alcohol, acetone equal solvent.
Claims (10)
1. a method of from spend more wormwood artemisia, extracting Arglabine is characterized in that: after will spending more the wormwood artemisia pulverizing medicinal materials, use extraction using alcohol; Carry out pre-separation then, filter, concentrate; At last with enriched material separate, purifying; Obtain Arglabine, said separation adopts silicagel column to separate, and used elution system is petroleum ether-ethyl acetate system, normal hexane-ETHYLE ACETATE system, sherwood oil-acetone system, normal hexane-acetone system.
2. method according to claim 1 is characterized in that: used elution system is 9: 1 a petroleum ether-ethyl acetate system of volume ratio.
3. method according to claim 1 is characterized in that: said pre-separation method is selected from: any one in alcohol precipitation processing, depositing in water processing, depositing in water and alcohol precipitation combination treatment, alcohol precipitation and the organic solvent extraction combination treatment.
4. method according to claim 3 is characterized in that: said depositing in water and alcohol precipitation combination treatment are: first depositing in water is handled alcohol precipitation again and is handled.
5. method according to claim 3 is characterized in that: said depositing in water and alcohol precipitation combination treatment are: first alcohol precipitation is handled depositing in water again and is handled.
6. method according to claim 3 is characterized in that: said alcohol precipitation and organic solvent extraction combination treatment are: first alcohol precipitation is handled organic solvent extraction again.
7. method according to claim 3 is characterized in that: said alcohol precipitation and organic solvent extraction combination treatment are: first organic solvent extraction alcohol precipitation is again handled.
8. according to claim 3 or 6 or 7 described any one methods, it is characterized in that: the organic solvent of said extraction is an ETHYLE ACETATE.
9. method according to claim 3; It is characterized in that: said purifying carries out purifying with recrystallization method; When the pre-separation method was alcohol precipitation processing, depositing in water and alcohol precipitation combination treatment, alcohol precipitation and organic solvent extraction combination treatment, recrystallization solvent was normal hexane or sherwood oil.
10. method according to claim 3 is characterized in that: said purifying carries out purifying with recrystallization method, and when the pre-separation method is a depositing in water when handling, used recrystallization solvent is that normal hexane or sherwood oil and volume ratio are 1: 1 alcohol-water.
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| CN113185562B (en) * | 2021-04-27 | 2023-09-19 | 中国科学院昆明植物研究所 | Artemisinin A-P and pharmaceutical composition thereof, and preparation method and application thereof |
| CN115300498A (en) * | 2022-09-13 | 2022-11-08 | 大连民族大学 | Application of arglabin in preparation of medicine for treating or preventing diabetic nephropathy and extraction method thereof |
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| CN102372723B (en) | 2014-03-26 |
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