CN101810662B - Elephantopus scaber linn extractive, preparation method and applications thereof - Google Patents
Elephantopus scaber linn extractive, preparation method and applications thereof Download PDFInfo
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- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 3
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- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention provides an elephantopus scaber linn extractive product, preparation method and medical application thereof. The elephantopus scaber linn extractive product is prepared from a perennial herbaceous plant of compositae elephantopus scaber, i.e. elephantopus scaber linn, contains elephantopus scaber linn lactone, sterol and flavonoid compounds. The preparation method comprises the following steps of: thermally extracting or quenching elephantopus scaber linn herbs with 20-95 % ethanol to obtain an extraction solution; and separating and purifying by using macroporous adsorption resin and refining to obtain an elephantopus scaber linn extractive which has main active components of elephantopus scaber linn lactone, sterols, flavone, and other compounds. Proved by animal experiments, the elephantopus scaber linn extractive has obvious anti-tumor effect and liver-protecting effect.
Description
Technical field
The present invention relates to Elephantopus scaber linn extractive and antitumor and the medicine that protects the liver.
Background technology
Scabrous Elephantfoot Herb belongs to the Compositae Elephantopus scaber L., belongs to herbaceos perennial, and another name Herba Elephantopi scaberis, God in charge of the Earth's English etc. originate in ground such as Guangdong, Hainan, Guangxi, Fujian and Taiwan, belong to traditional herbal medicine commonly used.The little suffering of Scabrous Elephantfoot Herb bitter in the mouth, cool in nature, whole plant for medical use has the effect of heat clearing away, removing heat from blood, dampness removing.
Elephantopus scaber L. mainly contains chemical compounds such as lactone, flavone, sterol.In recent years research shows that Scabrous Elephantfoot Herb has obvious antineoplastic and leukocyte is had the obvious suppression effect.In addition, relevant research shows that also Scabrous Elephantfoot Herb also has antibacterial action, refrigeration function, the effect of promotion gastrointestinal motility, hepatoprotective effect etc., for the further development and utilization of Scabrous Elephantfoot Herb provides theoretical foundation.We study with regard to the method for preparing of Elephantopus scaber linn extractive; Its method for distilling is feasible; The extract active constituent content of gained is very high; Mainly contain chemical compounds such as lactone, flavone, sterol in the Elephantopus scaber linn extractive, Elephantopus scaber linn extractive has very significantly antitumor and liver protection effect.
Summary of the invention
First purpose of the present invention is to provide a kind of Elephantopus scaber linn extractive.
Second purpose of the present invention also is to provide a kind of method for preparing of Elephantopus scaber linn extractive.
The 3rd purpose of the present invention is to provide the medical usage of the Elephantopus scaber linn extractive that the present invention relates to.
The present invention provides a kind of Elephantopus scaber linn extractive, is to belong to the herbaceos perennial Scabrous Elephantfoot Herb from the Compositae Elephantopus scaber L. extracting, and contains lactone, flavone and sterol compound.
Elephantopus scaber linn extractive of the present invention is prepared by following method:
(1). after Scabrous Elephantfoot Herb is pulverized, carry or merceration gets extracting solution with ethanol heat;
(2). extracting solution is centrifugal, discards deposition, and it is subsequent use to get supernatant;
(3). supernatant adds activated carbon and adsorbs, and is centrifugal, filters, and must filtrate;
(4). filtrating concentrates, behind the recovery ethanol, the concentrated solution thin up, last resin chromatographic column carries out chromatography, and water and organic solvent eluting are collected the organic solvent eluent successively, reclaim organic solvent, and surplus liquid is centrifugal, gets deposition;
(5). deposition is used the ethanol stirring and dissolving, and is centrifugal, discards deposition, and it is subsequent use to get supernatant;
(6). the supernatant in the step (5) is carried out precipitate with ethanol, centrifugal, discard deposition, get supernatant;
(7) supernatant concentration behind the precipitate with ethanol in the step (6) is extremely done, promptly got Elephantopus scaber linn extractive; Or with after the supernatant concentration behind the precipitate with ethanol, spray drying or lyophilization get Elephantopus scaber linn extractive.
In above-mentioned method for preparing, the alcohol extraction process of step (1) is with ethanol twice, merge extractive liquid,, and each extraction time is more than 1 hour, extracts temperature and be 20 ℃～100 ℃.
The alcoholic acid volumetric concentration of in described step (1), (5), using is 10%～95%, and amount of alcohol added is Scabrous Elephantfoot Herb or corresponding weight of precipitate 3～20 times.
Activated carbon adsorption in described step (3), the consumption of activated carbon (by solution weight) is 2%～10%, the activated carbon adsorption time is more than 2 hours.
Amount of water in the thin up process in described step (4) is 4～20 times of original solution weight, and resin is polyamide, silica gel resin, ion exchange resin or macroporous adsorbent resin.
Employed organic solvent is ethanol, methanol, acetone, chloroform, ethyl acetate or petroleum ether in described step (4).
Precipitate with ethanol in described step (5) is that to adopt volumetric concentration be that 95% ethanol carries out precipitate with ethanol, and through leaving standstill, isolating precipitate after centrifugal, the final volume concentration of alcohol is 30%～95% behind the precipitate with ethanol.
The application of Elephantopus scaber linn extractive of the present invention in preparation antitumor drug or hepatic also is provided.
The present invention adopts modern scientific method, and described Elephantopus scaber linn extractive is extracted purification, and prepared Elephantopus scaber linn extractive product safety is nontoxic; Reliable preparation process, raw material is easy to get, and reaction condition is gentle; Yield is high, and purity is good, environmental protection; Good stability, also very low to preparation equipment and place requirement, be suitable for large-scale industrial production.
Result according to pharmacology, pharmacodynamic study shows that the prepared Elephantopus scaber linn extractive product of the present invention can be developed into antitumor, liver protecting medicine.
The specific embodiment
Through specific embodiment the present invention is described in further detail below, to help understanding content of the present invention.
Embodiment 1: the resin pretreatment
The used resin of the present invention is polyamide, silica gel resin, ion exchange resin or macroporous adsorbent resin.We are that example is come brief account with the macroporous resin:
The pretreatment of resin: spend the night with water or ethanol swelling, wet method dress post, with ethanol or acetone eluting, to reclaiming no white precipitate in back or muddy getting final product, water is washed till does not then have the alcohol flavor, and alternately eluting is 2～3 times.Also usable acid, alkali soak, and then washing is to neutral.
Regeneration of resin: the general water eccysis of non-adsorptivity impurity is gone; Adsorptivity impurity can be removed (NaOH or HCl) with certain density acid or alkali liquor.Add a certain amount of ethanol during preservation.
Embodiment 2: the Elephantopus scaber linn extractive preparation
Get Scabrous Elephantfoot Herb powder 5kg, in 60 ℃ of reflux, extract, 6 hours, filter with 25.0L 95% ethanol (v%, following concentration of alcohol all refers to volumetric concentration); Must filtrate, residue filters with 60 ℃ of reflux, extract, of 15.0L 95% ethanol 4 hours; Must filtrate, merge extracted twice liquid, get the 38.0L extracting solution; Centrifugal, discard deposition, get the 37.0L supernatant.
Supernatant adds the 3.5kg activated carbon and adsorbs, and is centrifugal, filters, and gets 35.0L filtrating; Filtrating concentrates, reclaim ethanol after, in the residual liquid thin up to 15.0L, last macroporous resin chromatography; Elder generation's water is eluted to water white transparency, and reuse 95% ethanol elution is collected 95% ethanol elution 20.0L to water white transparency; Reclaim the ethanol in the eluent, residual liquid is centrifugal, gets deposition.
Deposition uses weight as 20.0L 60% ethanol stirring and dissolving, and is centrifugal, discards deposition, gets the 19.0L supernatant; In supernatant, add 95% ethanol, make the concentration of alcohol in the solution reach 75%, leave standstill; Centrifugal, discard deposition, the supernatant concentration behind the precipitate with ethanol is extremely done; Promptly get Elephantopus scaber linn extractive, about 240g, yield are 4.8%.
Testing result:
1. lactone is differentiated: take by weighing the 0.50g Elephantopus scaber linn extractive, put in the 100ml measuring bottle, add deionized water dissolving and standardize solution.Get Elephantopus scaber linn extractive solution 2ml, add 1% sodium hydroxide solution 2ml, in boiling water bath, heated 3～5 minutes,, add and become muddiness again after the acidify and then contain the Scabrous Elephantfoot Herb lactone like the more preceding clarification of solution;
2. lactone content: 9.61% (with reference to two appendix V of version pharmacopeia in 2005 D).
3. sterol is differentiated: thin layer chromatography (with reference to two appendix V of version pharmacopeia in 2005 B);
4. sterol content: 5.17% (with reference to two appendix V of version pharmacopeia in 2005 D).
5. flavone is differentiated: extracting sample solution point drips 1% aluminum chloride alcoholic solution on filter paper, dry up.Under visible light, be lark, under ultraviolet light, be the yellow fluorescence speckle;
6. flavone: 1.21%, method: ultraviolet spectrophotometry (two appendix IVA of Chinese Pharmacopoeia version pharmacopeia in 2005).
Embodiment 3: the Elephantopus scaber linn extractive preparation
Get Scabrous Elephantfoot Herb powder 5kg, in 60 ℃ of reflux, extract, 4 hours, filter with 30.0L 50% ethanol (v%, following concentration of alcohol all refers to volumetric concentration); Must filtrate, residue filters with 60 ℃ of reflux, extract, of 20.0L50% ethanol 2 hours; Must filtrate, merge extracted twice liquid, get the 48.0L extracting solution; Centrifugal, discard deposition, get the 47.0L supernatant.
Supernatant adds the 0.92kg activated carbon and adsorbs, and is centrifugal, filters, and gets 46.0L filtrating; Filtrating concentrates, reclaim ethanol after, thin up is to 20.0L, last resin chromatography, water is eluted to water white transparency, reuse 95% ethanol elution is collected 95% ethanol elution 19.5.0L to water white transparency, recovery ethanol is centrifugal, gets deposition.
Deposition uses weight as 20.0L 40% ethanol stirring and dissolving, and is centrifugal, discards deposition, gets the 18.8L supernatant; In supernatant, add 95% ethanol, make the concentration of alcohol in the solution reach 80%, leave standstill, centrifugal; Discard deposition,, reclaim ethanol, lyophilization the supernatant concentration behind the precipitate with ethanol; Promptly get Elephantopus scaber linn extractive, about 180g, yield are 3.6%.
Testing result:
1. lactone is differentiated: take by weighing the 0.50g Elephantopus scaber linn extractive, put in the 100ml measuring bottle, add deionized water dissolving and standardize solution.Get Elephantopus scaber linn extractive solution 2ml, add 1% sodium hydroxide solution 2ml, in boiling water bath, heated 3～5 minutes,, add and become muddiness again after the acidify and then contain the Scabrous Elephantfoot Herb lactone like the more preceding clarification of solution;
2. lactone content: 9.78% (with reference to two appendix V of version pharmacopeia in 2005 D).
3. sterol is differentiated: thin layer chromatography (with reference to two appendix V of version pharmacopeia in 2005 B);
4. sterol content: 5.39% (with reference to two appendix V of version pharmacopeia in 2005 D).
5. flavone is differentiated: extracting sample solution point drips 1% aluminum chloride alcoholic solution on filter paper, dry up.Under visible light, be lark, under ultraviolet light, be the yellow fluorescence speckle;
6. flavone: 1.01%, method: ultraviolet spectrophotometry (two appendix IVA of Chinese Pharmacopoeia version pharmacopeia in 2005).
Embodiment 4: the Elephantopus scaber linn extractive preparation
Get Scabrous Elephantfoot Herb powder 5kg,, filter with 60 ℃ of reflux, extract, of 30.0L60% ethanol (v%, following concentration of alcohol all refers to volumetric concentration) 8 hours; Must filtrate, residue filters with 60 ℃ of reflux, extract, of 20.0L60% ethanol 6 hours; Must filtrate, merge extracted twice liquid, get the 47.0L extracting solution; Centrifugal, discard deposition, get the 46.0L supernatant.
Supernatant adds the 2.0kg activated carbon and adsorbs, and is centrifugal, filters, and gets 44.8L filtrating; Filtrating concentrates, reclaim ethanol after, thin up is to 18.0L, last resin chromatography, water is eluted to water white transparency, reuse 95% ethanol elution is collected 95% ethanol elution 19.2L to water white transparency, recovery ethanol is centrifugal, gets deposition.
Deposition uses weight as 30.0L30% ethanol stirring and dissolving, and is centrifugal, discards deposition, gets the 19.0L supernatant; In supernatant, add 95% ethanol, make the concentration of alcohol in the solution reach 70%, leave standstill, centrifugal; Discard deposition, after the supernatant liquid behind the precipitate with ethanol is concentrated, spray drying; Promptly get Elephantopus scaber linn extractive, about 220g, yield are 4.4%.
Testing result:
1. lactone is differentiated: take by weighing the 0.50g Elephantopus scaber linn extractive, put in the 100ml measuring bottle, add deionized water dissolving and standardize solution.Get Elephantopus scaber linn extractive solution 2ml, add 1% sodium hydroxide solution 2ml, in boiling water bath, heated 3～5 minutes,, add and become muddiness again after the acidify and then contain the Scabrous Elephantfoot Herb lactone like the more preceding clarification of solution;
2. lactone content: 10.11% (with reference to two appendix V of version pharmacopeia in 2005 D).
3. sterol is differentiated: thin layer chromatography (with reference to two appendix V of version pharmacopeia in 2005 B);
4. sterol content: 5.28% (with reference to two appendix V of version pharmacopeia in 2005 D).
5. flavone is differentiated: extracting sample solution point drips 1% aluminum chloride alcoholic solution on filter paper, dry up.Under visible light, be lark, under ultraviolet light, be the yellow fluorescence speckle;
6. flavone: 1.32%, method: ultraviolet spectrophotometry (two appendix IVA of Chinese Pharmacopoeia version pharmacopeia in 2005).
Experimental example:
Test of pesticide effectiveness result
1, the antitumor action of Elephantopus scaber linn extractive
(1). Elephantopus scaber linn extractive is to H
22Tumor-bearing mice and S
180The influence of tumor-bearing mice life span
Get 60 Male Kunming strain mice, body weight 18～22g.Aseptic condition extracts the 7th day H of inoculation down
22Tumor-bearing mice ascites is put into sterile chamber, inserts in the ice chest and preserves.Microscopically counting (cancerous cell＞95%) is adjusted cell density to 1x10 with HankJs liquid
6/ ml.Press the inoculation of abdominal cavity, 0.2ml/ sterile working.Subsequently mice is divided into 5 groups at random, 12 every group.Each group beginning gastric infusion (0.9% (w%) sodium chloride solution group 0.2mld behind the 24h
-1, cyclophosphamide group 30mgkg
-1D
-1, Elephantopus scaber linn extractive low dose group 60mgkg
-1D
-1, dose groups 120mgkg in the Elephantopus scaber linn extractive
-1D
-1, Elephantopus scaber linn extractive high dose group 240mgkg
-1D
-1), successive administration 10d observes mouse diing time (life span) after the drug withdrawal, calculate increase in life span.Calculate lotus tumor S with method
180The increase in life span of mice.
Result of the test:
Table 1 Elephantopus scaber linn extractive is to H
22Tumor-bearing mice and S
180The influence of tumor-bearing mice life span
Annotate: compare with the sodium chloride solution group,
*P＜0.05,
*P＜0.01; With the cyclophosphamide group mutually
Relatively,
△P＜0.05,
△ △P＜0.01.
Can find out in the table 1 that Elephantopus scaber linn extractive high dose sample sets is to prolonging H
22Tumor-bearing mice and S
180The effect of tumor-bearing mice life span is very obvious, and is suitable with the effect of matched group cyclophosphamide.
(2). Elephantopus scaber linn extractive is to S
180The influence that the sarcoma mouse tumor is heavy
Get 60 Male Kunming strain mice, body weight 18～22g.Aseptic condition extracts the 7th day S of inoculation down
180Tumor-bearing mice ascites is put into sterile chamber, inserts in the ice chest and preserves.Microscopically counting (cancerous cell＞95%) is adjusted cell density to 1x10 with HankJs liquid
6/ ml.Press 0.2ml/ sterile working's mice right fore armpit subcutaneous vaccination.Mice is divided into 5 groups at random, 12 every group, each group beginning gastric infusion (0.9% (w%) sodium chloride solution group 0.2mld behind the 24h
-1, cyclophosphamide group 30mgkg
-1D
-1, Elephantopus scaber linn extractive low dose group 60mgkg
-1D
-1, dose groups 120mgkg in the Elephantopus scaber linn extractive
-1D
-1, Elephantopus scaber linn extractive high dose group 240mgkg
-1D
-1), successive administration 10d.Mensuration mice body weight, thymus, spleen, tumor weigh, and the result sees table 2, and detect mice TNF-α, SOD, MDA and NO, and the result sees table 3.
The mensuration result of A thymus index, index and spleen index, tumour inhibiting rate.
Table 2 Elephantopus scaber linn extractive is to S
180The influence of sarcoma tumor weight, thymus coefficient and spleen coefficient
Annotate: compare with the sodium chloride solution group,
*P＜0.05,
*P＜0.01; Compare with the cyclophosphamide group,
△P＜0.05，
△△P＜0.01。
Can find out in the table 2 that Elephantopus scaber linn extractive high dose sample sets is to S
180The tumour inhibiting rate effect of sarcoma tumor weight, thymus coefficient and spleen coefficient is very obvious, and is not worse than the effect of matched group cyclophosphamide.
The testing result of B SOD, MDA and NO.
Table 3 Elephantopus scaber linn extractive is to S
180The influence of sarcoma mice TNF-α, SOD, MDA and NO
Annotate: compare with the sodium chloride solution group:
*P＜0.05,
*P＜0.01.
Can find out in the table 3 that Elephantopus scaber linn extractive high dose sample sets is to S
180The influence of sarcoma mice TNF-α, SOD, MDA and NO is very obvious, and is suitable with the effect of matched group cyclophosphamide.
Result of the test can find out that Elephantopus scaber linn extractive high dose group antitumor action is very obvious from table 1-3.
2, the liver protection effect of Elephantopus scaber linn extractive
Get 60 of Kunming male mices, body weight 25～30g is divided into 6 groups at random, blank group, carbon tetrachloride model group, positive controls, Elephantopus scaber linn extractive group (basic, normal, high dose groups).Each sample sets mice gastric infusion every day given the test agent; Blank control group, carbon tetrachloride model group are all irritated normal saline 12ml/kg, and drug group is irritated the isopyknic Elephantopus scaber linn extractive of stomach (low dose group is that 80mg/kg, middle dose groups are that 160mg/kg, high dose group are 320mg/kg), and positive controls is irritated DONGBAO GANTAI 12ml/kg (containing DONGBAO GANTAI 0.01g); One day twice; Continuous irrigation stomach 3 days is irritated behind the stomach first except that the blank group, and all the other group mices subcutaneous injection immediately are the CCl of 0.1% (w%)
40.1mL/10g body weight, experimental period are 60 days.When experiment finishes, fasting 12h, the animal eye socket is got blood behind the 24h.Survey ALT, AST with automatic clinical chemistry analyzer, and win liver, fix with 10% (w%) formalin, do check pathological section, the result sees table 4 to table 6.
The liver protection effect of table 4 Elephantopus scaber linn extractive
The table 5 test mice result of histopathologic examination
The table 6 test mice result of histopathologic examination
Can find out that from table 4-table 6 Elephantopus scaber linn extractive has very significantly hepatoprotective effect, suitable with the hepatoprotective effect of positive controls (DONGBAO GANTAI).
Claims (9)
1. the method for preparing of an Elephantopus scaber linn extractive comprises following process:
(1). Scabrous Elephantfoot Herb is carried or merceration with ethanol heat after pulverizing, and gets extracting solution;
(2). extracting solution is centrifugal, discards deposition, and it is subsequent use to get supernatant;
(3). supernatant adds activated carbon and adsorbs, and is centrifugal, filters, and must filtrate;
(4). filtrating concentrates, reclaim ethanol after, the concentrated solution thin up, last resin chromatographic column carries out chromatography, water and organic solvent eluting are successively collected the organic solvent eluent again, the recovery organic solvent, surplus liquid is centrifugal, gets deposition;
(5). deposition is used the ethanol stirring and dissolving, and is centrifugal, discards deposition, and it is subsequent use to get supernatant;
(6). the supernatant in the step (5) is carried out precipitate with ethanol, centrifugal, discard deposition, get supernatant;
(7) supernatant concentration behind the precipitate with ethanol in the step (6) is extremely done, promptly got Elephantopus scaber linn extractive; Or with after the supernatant concentration behind the precipitate with ethanol, spray drying or lyophilization get Elephantopus scaber linn extractive; This extract extracts from Compositae Elephantopus scaber L. genus herbaceos perennial Scabrous Elephantfoot Herb, contains Scabrous Elephantfoot Herb lactone, sterol and chromocor compound.
2. method for preparing as claimed in claim 1 is characterized in that: the alcohol extraction process of described step (1) is that ethanol extraction is twice, and each time is more than 1 hour, extracts temperature and be 20 ℃～100 ℃.
3. method for preparing as claimed in claim 1 is characterized in that: described step (1) is 10%～95% with the middle ethanol volumetric concentration of using of step (5), and amount of alcohol added is 3～20 times to solid materials weight.
4. method for preparing as claimed in claim 1 is characterized in that: in described step
(3) the activated carbon adsorption process in, the consumption of activated carbon are 2%～10% of supernatant weight, and the activated carbon adsorption time is more than 2 hours.
5. method for preparing as claimed in claim 1; It is characterized in that: the amount of water in the thin up process in described step (4) is 4～20 times of original solution weight, and the used resin of chromatographic column is polyamide, silica gel resin, ion exchange resin or macroporous adsorbent resin.
6. method for preparing as claimed in claim 1 is characterized in that: employed organic solvent is ethanol, methanol, acetone, chloroform, ethyl acetate or petroleum ether in described step (4).
7. method for preparing as claimed in claim 1; It is characterized in that: the precipitate with ethanol in described step (5); Be that to adopt volumetric concentration be that 95% ethanol carries out precipitate with ethanol, and through leaving standstill, isolating precipitate after centrifugal, the final volume concentration of alcohol is 30%～95% behind the precipitate with ethanol.
8. the Elephantopus scaber linn extractive that described method is extracted one of among the claim 1-7 contains Scabrous Elephantfoot Herb lactone, sterol and chromocor compound.
9. the application of the described Elephantopus scaber linn extractive of claim 8 in the preparation hepatic.
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Non-Patent Citations (2)
| Title |
|---|
| 曹晖等.地胆草属药用植物的研究概述.《国外医学（中医中药分册）》.1998,第20卷(第2期),11－15. * |
| 黄婷.地胆草化学成分的研究.《中国优秀硕士论文电子期刊网》.2010, * |
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