CN102372723B - 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 extracting method of natural product, be specifically related to a kind of method of extracting Arglabine from spend more wormwood artemisia.
Background technology
Arglabine (Arglabin, structural formula is suc as formula shown in I), chemistry 1, the 10 beta epoxide by name-wound that heals-3 (4)-alkene-6,12-lactone.
Arglabine first Shi You Kazakhstan scientist S.M. Adriano Knopf extracts and obtains from the petal of local Art plant BG wormwood artemisia and leaf, and research finds that this compound has anti-tumor activity.1996, Arglabine dimethylamine hydrochloride freeze-dried preparation is gone on the market in the republic of Kazakhstan by the exploitation of Kazakhstan Tabi Fall company, this medicine, again successively in some country's listings of USSR (Union of Soviet Socialist Republics), is mainly used in Hepatoma therapy, lung cancer, mammary cancer, ovarian cancer subsequently.
In the WO9848789 of Kazakhstan Tabi Fall company application, recorded and take BG wormwood artemisia and extract the method for Arglabine as raw material.This patent first to adopt multi-solvents as: water, sherwood oil-ether 1: 1, benzene, ether, chloroform, ethanol etc. are investigated the extraction efficiency of BG wormwood artemisia, and 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 is usingd chloroform and has been carried out the preparation of Arglabine as extracting solvent, leaching process is as follows: adopting chloroform is that solvent carries out adverse current heating extraction to BG wormwood artemisia medicinal material, extract is after alcohol precipitation is processed, the extract obtaining is gone up silicagel column again through benzene wash-out, the Arglabine crude product obtaining is carried out to normal hexane recrystallization, obtain Arglabine monomer, yield is 0.27%.The vegetable material BG wormwood artemisia that this patent is recorded is distributed in Xinjiang of China, and scarcity of resources, can not meet drug development needs; And this patent method need use special counterflow extraction apparatus, equipment cost is high; Institute preferably extraction agent, eluent adopts highly toxic chloroform and benzene, is 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): 481-486), reported that it is raw material that wormwood artemisia is spent more in employing, by room temperature under methylene dichloride inflated with nitrogen condition, extract, on the extract obtaining, silicagel column is through normal hexane, ethyl acetate, methanol-eluted fractions, gained ethyl acetate partly flows part through positive liquid phase separation, obtain Arglabine monomer, yield is 0.037%.Although it is raw material that the method has adopted the relatively abundant wormwood artemisia of spending more of china natural resources, its yield is too low, and solvent for use methylene dichloride toxicity is large.
In PCT application WO2008035958, reported and take BG wormwood artemisia as raw material, by supercritical carbon dioxide extraction method, (pressure is 150 ± 2-10
5handkerchief, temperature is 60 ℃) obtain the method for Arglabine.The method can bring up to 0.8% by the content of Arglabine.But the method need to be used special extraction equipment, increased undoubtedly production cost.
Summary of the invention
From prior art, report; Arglabine is mainly present in BG wormwood artemisia and spends more in wormwood artemisia; and the content in BG wormwood artemisia is apparently higher than spending more wormwood artemisia; but BG wormwood artemisia at distribution in China seldom; can not meet the needs of drug development; and according to existing take spend more wormwood artemisia and as raw material extracts, prepare the technique of Arglabine, can not meet the needs of large-scale production far away.
In order to solve problems of the prior art, the inventor be take in Chinese Plants source and relatively abundant is spent more wormwood artemisia as raw material, and the extraction process of Arglabine is studied.Through a large amount of experimental studies, contriver finds: in the situation that using common extraction equipment, adopting and spending more wormwood artemisia is plant origin, can be that plant origin obtains the Arglabine that yield is quite even higher with adopting BG wormwood artemisia, and extracting method is simple, environmental protection, and production cost is low.
Therefore, the invention provides a kind of method of extracting Arglabine from spend more wormwood artemisia, described method is to spend more after wormwood artemisia pulverizing medicinal materials, with extraction using alcohol, then carries out pre-separation, filter, concentrated, finally enriched material is carried out to separation, purifying, obtain Arglabine, the described separated silicagel column that adopts is separated, and elution system used is petroleum ether-ethyl acetate system, normal hexane-ethyl acetate system, sherwood oil-acetone system, normal hexane-acetone system.
Wherein:
Described ethanol is selected from 30～100% aqueous ethanolic solution, is preferably 95% aqueous ethanolic solution.
The solid-liquid ratio of ethanol used 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 ℃.
Described elution system is preferably petroleum ether-ethyl acetate system, the petroleum ether-ethyl acetate system of more preferably 9: 1 (V/V).
Described 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, preferably adopt depositing in water and alcohol precipitation combination treatment and alcohol precipitation and organic solvent extraction combination treatment, more preferably adopt alcohol precipitation and depositing in water combination treatment.Wherein, described depositing in water and alcohol precipitation combination treatment optionally sequencing are carried out, that is: ethanol extraction can be carried out through alcohol precipitation, processing after depositing in water processing again, also ethanol extraction first can be processed and are carried out depositing in water processing again through alcohol precipitation; Described alcohol precipitation and organic solvent extraction combination treatment also optionally sequencing carry out, that is: ethanol extraction can be carried out carrying out again organic solvent extraction after alcohol precipitation processing, also ethanol extraction first can be carried out to organic solvent extraction and carry out again alcohol precipitation processing.Described alcohol precipitation is processed the alcohol-water mixed solution that alcohol used is any suitable proportion, is preferably 60～80% ethanol, more preferably 70% ethanol; The organic solvent of described extraction is selected from ethyl acetate, chloroform, methylene dichloride any one, is preferably ethyl acetate.
Described purifying can adopt any suitable mode to carry out, and preferably adopts recrystallization method to carry out purifying, and recrystallization solvent used is selected from one or more the mixed solvent in normal hexane, sherwood oil, ethanol, methyl alcohol, Virahol, acetone, water; When Isolation method is alcohol precipitation processing, depositing in water and alcohol precipitation combination treatment, alcohol precipitation and organic solvent extraction combination treatment, recrystallization solvent is normal hexane or sherwood oil; When Isolation method is depositing in water while processing, recrystallization solvent used is the alcohol-water that normal hexane or sherwood oil and volume ratio are 1: 1.
In order to obtain Arglabine extracting method of the present invention, contriver mainly carries out the following studies:
Gather the wormwood artemisia of spending more of growth in May, dry rear primary crusher and pulverize, standby.
1, extract the selection of solvent
Get 10g medicinal material for every part, adopt respectively sherwood oil, normal hexane, methylene dichloride, chloroform, ethyl acetate, acetone, ethanol, several solvents of methyl alcohol, each processes two repetitions, add 15 times to the solvent of medicinal material (V/W), cold soaking extracts 48h, by extracting liquid filtering, concentrated after by methanol constant volume to 250ml, HPLC detects its content (g/g crude drug), and experimental result as shown in Figure 1.Experimental result shows: ethanol and methanol extraction are most effective, considers the factors such as solvent toxicity and recycling, selects ethanol as extracting solvent.
2, the selection of alcohol concn
Get 10g medicinal material for every part, add respectively the ethanol of 100ml 30%, 50%, 70%, 80%, 90% and 95% concentration to carry 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), after extracting solution merging is concentrated, is settled to 250ml, HPLC detects, and result 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 ratio
Every part takes 10g medicinal material, is placed in round-bottomed flask, and each tests two repetitions, 6 times, 8 times, 10 times and 95% ethanol of 12 times adding respectively crude drug (V/W) amount, refluxing extraction 1h, is settled to 250ml after extracting solution is concentrated, HPLC detects Arglabine content, and result as shown in Figure 3.Experimental result shows: liquid ratio is higher, and the extraction efficiency of Arglabine is higher.
4, spend more the investigation of wormwood artemisia ethanol extraction Isolation method
1. alcohol precipitation is processed: get ethanol extraction 77g, adopting 4 times, after the dissolve with ethanol extract of crude drug (V/W), to be diluted with water to determining alcohol be 70%, filters after precipitating 24h, obtain filtrate, evaporated under reduced pressure, obtains the about 60.8g of dry-matter, 95% ethanol constant volume, HPLC detects.
2. depositing in water is processed: get ethanol extraction 77g, adopt 2 times of water to crude drug to dissolve rear standing 24h to extract, be precipitated about 36g, with 95% dissolve with ethanol constant volume, HPLC detects.
3. first alcohol precipitation is processed depositing in water processing again: get ethanol extraction 77g, adopting 4 times, after the dissolve with ethanol extract of crude drug (V/W), to be diluted with water to determining alcohol be 70%, after precipitation 24h, filter, obtain filtrate, after concentrated, adopt 2 times of water to crude drug to carry out depositing in water processing, be precipitated about 20g, with 95% dissolve with ethanol constant volume, HPLC detects.
4. first depositing in water is processed alcohol precipitation processing again: get ethanol extraction 77g, standing 24h after adopting 2 times of water to crude drug to dissolve extract, is precipitated, after adding 4 times of ethanol to crude drug to dissolve precipitation, being diluted with water to determining alcohol is 70%, after standing 24h, filter, obtain filtrate, concentrating under reduced pressure, weigh, about 20.5g, 95% ethanol constant volume, HPLC detects.
5. ethyl acetate extraction after alcohol precipitation: get ethanol extraction 77g, adopting 4 times, after the dissolve with ethanol extract of crude drug (V/W), to be diluted with water to determining alcohol be 70%, after precipitation 24h, filter, obtain filtrate, after concentrated, adopt 2 times of water to crude drug to dissolve, solution adopts the ethyl acetate of equivalent to extract three times, is extracted liquid, (about 20.3g) 95% ethanol constant volume after concentrated, HPLC detects.
6. ethyl acetate extracts rear alcohol precipitation: get ethanol extraction 77g, after adding suitable quantity of water to dissolve, by the ethyl acetate of equivalent, extract three times, the extraction liquid obtaining is concentrated after, adding 4 times, after the dissolve with ethanol extract of crude drug (V/W), to be diluted with water to determining alcohol be 70%, after precipitation 24h, filter, obtain filtrate, drying under reduced pressure, obtains 19.8g extract, after 95% ethanol constant volume, HPLC detects.
7. chloroform extraction after alcohol precipitation: get ethanol extraction 77g, adopting 4 times, after the dissolve with ethanol extract of crude drug (V/W), to be diluted with water to determining alcohol be 70%, after precipitation 24h, filter, obtain filtrate, after concentrated, adopt 2 times of water to crude drug to dissolve, solution adopts the chloroform of equivalent to extract three times, is extracted liquid, (about 19.5g) 95% ethanol constant volume after concentrated, HPLC detects.
8. alcohol precipitation after chloroform extraction: get ethanol extraction 77g, after adding suitable quantity of water to dissolve, with the chloroform of equivalent, extract three times, the extraction liquid obtaining is concentrated after, adding 4 times, after the dissolve with ethanol extract of crude drug (V/W), to be diluted with water to determining alcohol be 70%, after precipitation 24h, filter, obtain filtrate, drying under reduced pressure, obtains 19.3g extract, after 95% ethanol constant volume, HPLC detects.
Experimental result as shown in Figure 4, experimental result shows: in several Isolation methods, only adopt depositing in water to process and can remove most impurity, but the method can not be removed the oil substances that affects Arglabine separation, in subsequent separation process, need to carry out again just obtaining Arglabine after oil removal treatment, can increase undoubtedly the rate of loss of Arglabine; The extractive content that other treatment process obtains is suitable, although but only adopt the method for alcohol precipitation to remove oil substances, but in the extract obtaining, contain a large amount of water-soluble substanceses, increased the workload (as increased the usage quantity and separated number of times of silica gel) of later separation.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, in the processing of ethanol extraction alcohol precipitation, alcohol concn is investigated
Adopt respectively 60%, 70%, 80% as final determining alcohol, alcohol concn in alcohol precipitation experiment to be investigated.Experiment is diluted with water to corresponding concentration after adopting 4 times of ethanol to crude drug (L/kg) to dissolve.Standing 1 day of room temperature, filters, and HPLC detects Arglabine content in filtrate, contrasts with the Arglabine content carrying out in the front extract of alcohol precipitation processing, and experimental result is as shown in table 1:
The impact of alcohol concn on Arglabine content in the processing of table 1 alcohol precipitation
| Determining alcohol (%) | 60 | 70 | 80 | Before alcohol precipitation is processed |
| Arglabine content (%) | 7.28 | 7.42 | 7.25 | 8.45 |
Experimental result shows: 70% alcohol concn is minimum on the rate of loss impact of Arglabine.
6, the investigation of eluting solvent in separating technology
(1) selection of eluting solvent polarity: according to the result of standard substance separation, select petroleum ether-ethyl acetate system as the primary dcreening operation solvent of development system, carry out the selection of polarity.Adopt respectively the petroleum ether-ethyl acetate of 95: 5,9: 1,8: 2,7: 3,6: 4,5: 5 (V/V) to launch, thin-layer chromatography the results are shown in Figure 5.According to 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 solvent polarity definite in (1), the method that adopts thin-layer chromatography to detect, selects the separation system of Arglabine.Adopt respectively petroleum ether-ethyl acetate (9: 1), sherwood oil-acetone (9: 1), normal hexane-ethyl 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, thin layer plate adopts 10%H
2sO
4-EtOH develops the color, and thin-layer chromatography the results are shown in Figure 6.Result shows: chloroform is too large as developping agent polarity, can not select; The polar phase of petroleum ether-ethyl acetate, sherwood oil-acetone, normal hexane-acetone, normal hexane-ethyl acetate system is worked as, but petroleum ether-ethyl acetate (9: 1) system can be carried out better separation by the close assorted point of polarity, and consider volatility and the toxicity of solvent, select petroleum ether-ethyl acetate system as eluting solvent.
7, the investigation of recrystallization solvent in purifying process
By the Arglabine obtaining with column chromatography normal hexane, sherwood oil, acetone, 1: 1 alcohol-water (V/V for crude product, 1: 1 methanol-water (V/V down together),, 1: 1 acetone-water (V/V down together),, lower with), etc. carry out recrystallization, find: when adopting non-depositing in water Isolation method, the Arglabine crude product of gained is through above-mentioned solvent recrystallization, and gained Arglabine sterling purity is more than 99%.Wherein normal hexane and sherwood oil are the highest, reach 99.5% left and right, and recrystallization loss is little, and the rate of recovery is more than 85%.The Arglabine crude product that only adopts depositing in water pre-separation to obtain, owing to containing more lipid-soluble substance, need be through twice recrystallization, 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 is for the first time recrystallization solvent, the normal hexane of recrystallization employing for the second time, sherwood oil are recrystallization solvent, product purity is the highest, can reach 99.5% left and right.The order of above-mentioned two kinds of recrystallizations also can be changed, and first with normal hexane, sherwood oil, carries out recrystallization and uses 1: 1 alcohol-water to carry out recrystallization again.
Arglabine extracting method of the present invention has been realized following beneficial effect:
(1) adopting the relatively abundant wormwood artemisia of spending more of Chinese Resources is plant origin, for this medicine provides sufficient raw material guarantee in Chinese exploitation listing;
(2) adopt the nontoxic or low-toxic solvents such as ethanol, water, ethyl acetate, be more conducive to workers ' health and environmental protection.
(3) solvent load is few, and extraction, alcohol precipitation process alcohol solvent used and can reclaim, and carries out recycle, has saved production cost.
(4) use common extraction equipment can realize high-level efficiency and extract, reduced production cost.
To sum up, Arglabine extracting method of the present invention, raw material sources are abundant, and method is simple, environmental protection, production cost are low, is more suitable for producing in large-scale industrial.
Accompanying drawing explanation
Fig. 1 is the impact of different solvents on Arglabine extraction efficiency.
Fig. 2 is the impact of alcohol concn on Arglabine extraction efficiency.
Fig. 3 is the impact of solid-liquid ratio on Arglabine extraction efficiency.
Fig. 4 is the impact of different pretreatments method on 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
By following embodiment, will contribute to understand the present invention, but not be construed as limiting the invention.
Embodiment 1: the acquisition of Arglabine standard substance
Get dry wormwood artemisia (collection in March) the over-ground part 14kg that spends more, 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 wormwood artemisia amount, extraction time is 24h; Add for the second time 12 times to the methylene dichloride of spending more wormwood artemisia, extract 48h.Extracted twice liquid is merged, concentrated, obtain the about 654.9g of extract.
To spend more wormwood artemisia dichloromethane extract and according to material ratio, carry out silica gel (100-200 order) at 1: 1 and mix sample, dry.Carry out silicagel column separation, elution system is normal hexane-ethyl acetate system, and stream part of collection merges according to thin-layer chromatography detected result.The employing normal hexane repeatedly method of recrystallization carries out purifying to sample, obtains compound Arglabine.Gained sample is carried out to hydrogen spectrum, carbon spectrum, mass spectrum, fusing point test, and result is as follows:
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 pulverizing, add 95% alcohol heating reflux of 10L to extract 1.5h, filter, 95% ethanol of 8L * 2 for the dregs of a decoction is extracted, extracting solution is merged and concentrated, obtain the about 210g of extract.Extract is added after 2L water heating for dissolving, and the standing 24h of room temperature, filters, and is precipitated, by this precipitation, with after the 95% ethanol heating for dissolving of 2L, adding water to determining alcohol is 70%, and room temperature is placed after 24h, filter, the filtrate obtaining is concentrated, obtain about 36.5g extract.This enriched material is carried out to silicagel column separation, and material ratio is 1: 5～1: 10 (extract/silica gel: W/W, lower same), and 9: 1 wash-outs of petroleum ether-ethyl acetate, collect stream part, and thin layer detects, merging same composition.After Arglabine position is concentrated, add the normal hexane of 2～5 times of amounts (W/W extract, lower same), heating for dissolving, room temperature is placed crystallization, filters, and dry, gained solid weight is 3.05g, yield is about 0.305% (W/W medicinal material, lower same), and HPLC purity is 99.56%.Gained is fixedly carried out to hydrogen spectrum, carbon spectrum, mass spectrum, fusing point test, and result is with Arglabine standard substance.
Embodiment 3: the preparation of Arglabine
With reference to the preparation method of embodiment 2, obtain the about 210g of extract.Extract is added after 95% ethanol 4L heating for dissolving, and adding water to determining alcohol is 70%, and the standing 24h of room temperature filters, and obtains filtrate, is concentrated into without alcohol, adds water to 2L, heated and stirred, and the standing 24h of room temperature, sucking-off supernatant liquor, is precipitated 36.2g.This precipitation is carried out to silicagel column separation, and 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 Arglabine position is concentrated, add the sherwood oil of 2～5 times of amounts, heating for dissolving, room temperature is placed crystallization, filters, and dry, gained solid weight is 3.02g, and yield is about 0.302%, HPLC, and to detect its purity be 99.51%.Gained is fixedly carried out to hydrogen spectrum, carbon spectrum, mass spectrum, fusing point test, and result is with Arglabine standard substance.
Embodiment 4: the preparation of Arglabine
With reference to the preparation method of embodiment 2, obtain the about 210g of extract.Extract is added after 4L water heating for dissolving, and the standing 24h of room temperature, filters, and obtains filtrate, concentrated, obtains extract 80g.This extract is carried out to silicagel column separation, and 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 Arglabine position is 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 crude product (containing oily matter).Arglabine crude product is added after 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, dry, gained solid weight is 2.53g, and yield is about 0.253%, HPLC, and to detect its purity be 99.3%.Gained is fixedly carried out to hydrogen spectrum, carbon spectrum, mass spectrum, fusing point test, and result is with Arglabine standard substance.
Embodiment 5: the preparation of Arglabine
With reference to the preparation method of embodiment 2, obtain the about 210g of extract.Extract is added after 95% ethanol 4L heating for dissolving, and adding water to determining alcohol is 70%, and the standing 24h of room temperature filters, and obtains filtrate, is concentrated into dryly, obtains extract 145g.This extract is carried out to silicagel column separation, and 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 Arglabine position is concentrated, add the sherwood oil of 2～5 times of amounts, heating for dissolving, room temperature is placed crystallization, filters, and dry, gained solid weight is 3.0g, and yield is about 0.3%, HPLC, and to detect its purity be 99.52%.Gained is fixedly carried out to hydrogen spectrum, carbon spectrum, mass spectrum, fusing point test, and result is with Arglabine standard substance.
Embodiment 6: the preparation of Arglabine
With reference to the preparation method of embodiment 2, obtain the about 210g of extract.Extract is added after 95% ethanol 4L heating for dissolving, and adding water to determining alcohol is 70%, after precipitation 24h, filters, and obtains filtrate, be concentrated into without alcohol, add 2L water to dissolve, gained for solution the ethyl acetate of 2L * 3 extract, be extracted liquid, concentrated, obtain the about 35.2g of enriched material.This precipitation is carried out to silicagel column separation, and 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 Arglabine position is 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.Add after 3L water dissolution, by the ethyl acetate of 3L * 3, extract, after the extraction liquid obtaining is concentrated, enriched material adds after 95% ethanol 4L heating for dissolving, and adding water to determining alcohol is 70%, after precipitation 24h, filters, and obtains filtrate, concentrated, obtains 36.0g extract.This extract is carried out to silicagel column separation, and 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 Arglabine position is 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%.
From embodiment 2～7 experimental results, can find out: several Isolation methods, gained Arglabine purity is suitable, is 99.5% left and right, but only adopt depositing in water to process gained Arglabine yield, be starkly lower than other Isolation method; Only adopt the pretreatment process of alcohol precipitation, the extract amount obtaining is larger, has increased the usage quantity and separated number of times of silica gel.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 |
From table 2 experimental result, can find out: adopt methyl alcohol as extracting solvent, the yield of Arglabine and purity and ethanol are suitable as extracting solvent effect; Adopt acetone as extracting solvent and adopting ethanol as extracting solvent phase ratio, product purity and yield decline, but adopt ethanol as extracting solvent, can avoid using the organic solvent larger to human toxicity, and compare other organic solvent, in concentrated process, loss less, is reclaimed solvent and can be recycled, and has reduced cost.The present invention is considering on the basis of the many factors such as solvent toxicity, production cost, has selected ethanol as extracting solvent, but carries out within Arglabine extraction also will fall into protection domain of the present invention as methyl alcohol, acetone equal solvent with other solvent.
Claims (2)
1. from spend more wormwood artemisia, extract a method for Arglabine, it is characterized in that: will spend more after wormwood artemisia pulverizing medicinal materials, use 95% extraction using alcohol, then carry out pre-separation, filter, concentrated, finally enriched material is carried out to separation, purifying, obtain Arglabine;
Described Isolation 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 ethyl acetate extraction combination treatment;
The described separated silicagel column that adopts is separated, and elution system used is the petroleum ether-ethyl acetate system of volume ratio 9:1.
2. method according to claim 1, is characterized in that: described depositing in water and alcohol precipitation combination treatment are: first depositing in water is processed alcohol precipitation again and processed.
3. method according to claim 1, is characterized in that: described depositing in water and alcohol precipitation combination treatment are: first alcohol precipitation is processed depositing in water again and processed.
4. method according to claim 1, is characterized in that: described alcohol precipitation and ethyl acetate extraction combination treatment are: first alcohol precipitation is processed ethyl acetate extraction again.
5. method according to claim 1, is characterized in that: described alcohol precipitation and ethyl acetate extraction combination treatment are: first ethyl acetate extracts alcohol precipitation again and processes.
6. method according to claim 1, it is characterized in that: described purifying carries out purifying with recrystallization method, when Isolation method is alcohol precipitation processing, depositing in water and alcohol precipitation combination treatment, alcohol precipitation and ethyl acetate extraction combination treatment, recrystallization solvent is normal hexane or sherwood oil.
7. method according to claim 1, is characterized in that: described purifying carries out purifying with recrystallization method, and when Isolation method is depositing in water while processing, recrystallization solvent used is the alcohol-water that normal hexane or sherwood oil and volume ratio are 1:1.
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| CN109045022A (en) * | 2018-07-03 | 2018-12-21 | 厦门大学 | It Ah Calais must be in the purposes in the drug for preparing Stargardt macular degeneration disease |
| CN110496145B (en) * | 2019-10-09 | 2022-12-09 | 中国科学院新疆理化技术研究所 | Preparation method and application of effective part of artemisia sieversiana |
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