CN103191133A - Preparation method for extracting cinobufagin and bufalin mixture from venenum bufonis - Google Patents
Preparation method for extracting cinobufagin and bufalin mixture from venenum bufonis Download PDFInfo
- Publication number
- CN103191133A CN103191133A CN2013101423530A CN201310142353A CN103191133A CN 103191133 A CN103191133 A CN 103191133A CN 2013101423530 A CN2013101423530 A CN 2013101423530A CN 201310142353 A CN201310142353 A CN 201310142353A CN 103191133 A CN103191133 A CN 103191133A
- Authority
- CN
- China
- Prior art keywords
- cinobufagin
- venenum bufonis
- ethanol
- preparation
- toadpoison medicine
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention relates to a preparation method for extracting a cinobufagin and bufalin mixture from venenum bufonis. The preparation method is characterized by comprising the following steps of: smashing the venenum bufonis into medium coarse powder, and then adding 70%-95% of ethanol for extraction; and carrying out silica gel column chromatography separation on an extracting solution to obtain the high-purity cinobufagin and bufalin mixture, wherein the content of the cinobufagin and bufalin mixture achieves more than 80%. The total content of the cinobufagin and bufalin mixture extracted by adopting the preparation method disclosed by the invention achieves more than 80%.
Description
Technical field
The present invention relates to a kind of method of from the Chinese medicine Venenum Bufonis, extracting high-purity cinobufagin and Toadpoison Medicine mixture, belong to technical field of Chinese medicines.
Background technology
The Chinese medicine Venenum Bufonis derives from the dry secretions of Bufonidae animal Bufo siccus Bufo bufo gargarizans Cantor or Bufo melanostictus Bufo melanostictus Schneider.Suffering, temperature; Poisonous.The GUIXIN warp has detoxifcation, pain relieving, and the refreshment function of having one's ideas straightened out, poisonous.Be used for the carbuncle furuncle, laryngopharynx swelling and pain, the heatstroke coma, erputive abdominal disease is vomited and diarrhoea.Venenum Bufonis has effects such as antitumor, resisting pathogenic microbes and heart tonifying and has been subjected to attention both domestic and external because modern pharmacological research shows it, the cinobufagin that it is contained and Toadpoison Medicine mixture namely are one of its active component, and are the higher active component of content in the Venenum Bufonis.
Venenum Bufonis is because of place of production difference, processing method difference and collecting season and temporal differences, the content difference of cinobufagin and Toadpoison Medicine mixture is bigger in the Venenum Bufonis, be unfavorable for controlling preferably its drug effect, the active component in the Venenum Bufonis of therefore purifying is the means that guarantee its safety and effectiveness preferably.But the extract of these two kinds of compositions in the prior art, purity are generally all very low.
Summary of the invention
In order to solve the problems of the technologies described above, the invention provides a kind of method of from the Chinese medicine Venenum Bufonis, extracting high-purity cinobufagin and Toadpoison Medicine mixture, the content of cinobufagin and Toadpoison Medicine mixture is greater than 80%.Its extracting method is as follows:
(1) Venenum Bufonis is ground into the coarse powder by 40 mesh sieves, adds 70～95% ethanol of 20～40 times of Venenum Bufonis amounts, and heating and refluxing extraction 2～3 times was extracted 2～4 hours at every turn, and extracting solution filters, and filtrate recycling ethanol gets ethanol extract A to there not being the ethanol flavor;
(2) among the extracting solution A, add 5 times of Venenum Bufonis amounts, granularity is 100～200 purpose silica gel, evaporate to dryness, (blade diameter length ratio of silicagel column is 1:3～10 to add the top of the chromatographic column that silica gel has been housed, loading amount is 20～30 times of Venenum Bufonis amount among the extracting solution A), is eluant with petroleum ether-acetone (9:1) mixed solvent earlier, eluting, discard eluent, continuing with petroleum ether-acetone (8.5:1.5) mixed solvent is eluant, is eluted to cinobufagin and Toadpoison Medicine mix ingredients eluting is complete, gets eluent B;
(3) eluent B heating recovery solvent is to solvent-free flavor, and vacuum drying apparatus inner drying 48h gets highly purified cinobufagin and Toadpoison Medicine mixture extract;
(4) content of mensuration cinobufagin and Toadpoison Medicine.
Be experiment content of the present invention and result below
1. extracting method:
According to the character of Venenum Bufonis medical material, the granularity of medical material is chosen as 40 orders.
Factor according to the influence extraction, investigate with orthogonal experiment and to carry concentration of alcohol (A), extraction time (B), solid-liquid ratio (C) to the influence of extraction effect, factor and the level investigated see Table 1, investigate index and are amount and the extraction extractum amount of cinobufagin and the Toadpoison Medicine mixture of extraction.Investigation factor and level see Table 1.
Table 1 Venenum Bufonis extraction conditions is investigated L
9(3
3) quadrature level design table
Get Venenum Bufonis powder (40 order) 2.0g, totally 9 parts, the accurate title, decide, and puts in the round-bottomed flask, extracts by orthogonal table cloth table pack, and the extracting solution sucking filtration merges filtrate twice, and mixing adds respective concentration ethanol and is settled to 250ml, and mixing gets the Venenum Bufonis extracting solution.Measure the amount of cinobufagin and Toadpoison Medicine mixture in the extracting solution and extract the extractum amount, the results are shown in Table 2, table 3, table 4, table 5.
Table 2 Venenum Bufonis extracts Orthogonal experiment results
Annotate: the percentage composition of three kinds of compositions is the grams of contained each composition in every 100g Venenum Bufonis crude drug, i.e. g/100g.
Table 3 cinobufacin aglucon analysis of variance table
Table 4 Toadpoison Medicine analysis of variance table
Table 5 extractum rate analysis of variance table
From experimental result as can be seen, in the scope of design, each factor and varying level are less to the influence of the extraction effect of cinobufagin and Toadpoison Medicine mixture, but concentration of ethanol is to the extracted amount influence of extractum big (P＜0.01).Concentration of alcohol is more high, and the extractum amount of extraction is more little, extracts in the extractum, and the content of cinobufagin and Toadpoison Medicine mixture is more high, and impurity is more few, and is therefore convenient during for purification, extracts solvent and selects 95% ethanol for use.
Purification process
2.1 the preparation of Venenum Bufonis extracting solution: precision takes by weighing Venenum Bufonis medical material 40g, and accurate the title decides, and puts in the round-bottomed flask, add alcohol heating reflux and extract 2 times, add ethanol 960ml, reflux 1h for the first time, for the second time, add ethanol 640ml, reflux 1h, filter, merging filtrate reclaims ethanol to about 1000ml, put in the 1000ml volumetric flask, add ethanol to scale, shake up, namely.
2.2 the investigation of blade diameter length ratio
The pretreatment of silicagel column: taking by weighing 30g silica gel, is eluant with petroleum ether-acetone (9:1) mixed solvent, wet method dress post, 1.5 column volumes of eluting.The blade diameter length ratio of three groups of silicagel columns is: 2:20,2.5:13.5,3:9.
The preparation of last sample silica gel: precision pipettes 3 parts of 50ml Venenum Bufonis extracting solution, puts respectively in the evaporating dish, and water-bath is flung to solvent to about 2ml, adds 1g silica gel (100 orders～200 orders) mix homogeneously, volatilizes solvent, namely.
Get 3 parts and go up the sample silica gel, respectively on the dry method sample to the good silicagel column of pretreatment, saturated 30min, eluting, elution speed are 2.5ml/min.Earlier with petroleum ether-acetone (9:1) mixed solvent eluting, discard eluent, be eluent with petroleum ether-acetone (8.5:1.5) mixed solvent again, be washed till cinobufagin and the Toadpoison Medicine mixture is complete, the eluent evaporate to dryness, add anhydrous alcohol solution, filter, be transferred to weighing to the clean weighing botle of constant weight, 85 ℃ of oven dry, to vacuum drying apparatus inner drying 48h, get purified product.The amount of cinobufagin and Toadpoison Medicine mixture in the mensuration extract, the purity of calculating cinobufagin and Toadpoison Medicine mixture.The results are shown in Table 6.
The investigation result of the different blade diameter length ratios of table 6
2.3 the investigation of silica gel granularity
The pretreatment of silicagel column: taking by weighing silica gel 100 orders～200 orders, 200 orders～each 30g of 300 orders, is eluant with petroleum ether-acetone (9:1) mixed solvent, wet method dress post, 1.5 column volumes of eluting.The blade diameter length ratio of silicagel column is 2.5:13.5.
The preparation of last sample silica gel: each precision pipettes 50ml Venenum Bufonis extracting solution, and water-bath is flung to solvent to about 2ml, adds 1g silica gel (100 orders～200 orders) mix homogeneously, volatilizes solvent, namely.
Get the above-mentioned sample silica gel of going up, sample is to the good silicagel column of pretreatment on the dry method respectively, and with 2.2 following method eluting, preparation purification thing is measured cinobufagin and Toadpoison Medicine mixture purity.The results are shown in Table 7.
The investigation result of table 7 Different Silicon micelle degree
| Granularity | Cinobufagin and Toadpoison Medicine mixture purity (%) |
| 100～200 | 85.97 |
| 200～300 | 81.89 |
The specific embodiment
Below the present invention is further illustrated.
Embodiment 1
(1) Venenum Bufonis is ground into the coarse powder by 40 mesh sieves, adds 95% ethanol of 30 times of Venenum Bufonis amounts, and heating and refluxing extraction 3 times was extracted 3 hours at every turn, and extracting solution filters, and filtrate recycling ethanol gets ethanol extract A to there not being the ethanol flavor;
(2) among the extracting solution A, add 5 times of Venenum Bufonis amounts, granularity is 100～200 purpose silica gel, evaporate to dryness, (blade diameter length ratio of silicagel column is 2.5:13.5 to add the top of the chromatographic column that silica gel has been housed, loading amount is 20～30 times of Venenum Bufonis amount among the extracting solution A), is eluant with petroleum ether-acetone (9:1) mixed solvent earlier, eluting, discard eluent, continuing with petroleum ether-acetone (8.5:1.5) mixed solvent is eluant, is eluted to cinobufagin and Toadpoison Medicine mix ingredients eluting is complete, gets eluent B;
(3) eluent B heating recovery solvent is to solvent-free flavor, and vacuum drying apparatus inner drying 48h gets highly purified cinobufagin and Toadpoison Medicine mixture extract;
(4) content 85.21% of usefulness high-performance liquid chromatogram determination cinobufagin and Toadpoison Medicine.
Embodiment 2
Venenum Bufonis is ground into the fine powder by 40 mesh sieves, adds 80% ethanol of 40 times of Venenum Bufonis amounts, and heating and refluxing extraction 3 times was extracted 2 hours at every turn, and extracting solution filters, and filtrate recycling ethanol gets ethanol extract A to there not being the ethanol flavor;
(2) among the extracting solution A, add 5 times of Venenum Bufonis amounts, granularity is 100～200 purpose silica gel, evaporate to dryness, (blade diameter length ratio of silicagel column is 1:10 to add the top of the chromatographic column that silica gel has been housed, loading amount is 20～30 times of Venenum Bufonis amount among the extracting solution A), is eluant with petroleum ether-acetone (9:1) mixed solvent earlier, eluting, discard eluent, continuing with petroleum ether-acetone (8.5:1.5) mixed solvent is eluant, is eluted to cinobufagin and Toadpoison Medicine mix ingredients eluting is complete, gets eluent B;
(3) eluent B heating recovery solvent is to solvent-free flavor, and vacuum drying apparatus inner drying 48h gets highly purified cinobufagin and Toadpoison Medicine mixture extract;
(4) content of mensuration cinobufagin and Toadpoison Medicine is 82.47%.
Embodiment 3
Venenum Bufonis is ground into the fine powder by 20 mesh sieves, adds 70% ethanol of 20 times of Venenum Bufonis amounts, and heating and refluxing extraction 2 times was extracted 4 hours at every turn, and extracting solution filters, and filtrate recycling ethanol gets ethanol extract A to there not being the ethanol flavor;
(2) among the extracting solution A, add 5 times of Venenum Bufonis amounts, granularity is 100～200 purpose silica gel, evaporate to dryness, (blade diameter length ratio of silicagel column is 1:3 to add the top of the chromatographic column that silica gel has been housed, loading amount is 20～30 times of Venenum Bufonis amount among the extracting solution A), is eluant with petroleum ether-acetone (9:1) mixed solvent earlier, eluting, discard eluent, continuing with petroleum ether-acetone (8.5:1.5) mixed solvent is eluant, is eluted to cinobufagin and Toadpoison Medicine mix ingredients eluting is complete, gets eluent B;
(3) eluent B heating recovery solvent is to solvent-free flavor, and vacuum drying apparatus inner drying 48h gets highly purified cinobufagin and Toadpoison Medicine mixture extract;
(4) content of mensuration cinobufagin and Toadpoison Medicine is 80.29%.
Claims (4)
1. one kind is extracted cinobufagin and Toadpoison Medicine preparation process of mixture from Venenum Bufonis, it is characterized in that, after Venenum Bufonis is ground into middle coarse powder, add 70～95% ethanol extractions, extracting solution separates through silica gel column chromatography, obtain highly purified cinobufagin and Toadpoison Medicine mixture, the content of cinobufagin and Toadpoison Medicine mixture reaches more than 80%.
2. preparation method according to claim 1 is characterized in that, may further comprise the steps:
(1) Venenum Bufonis is ground into the coarse powder by 40 mesh sieves, adds 70～95% ethanol of 20～40 times of Venenum Bufonis amounts at every turn, and heating and refluxing extraction 2～3 times was extracted 2～4 hours at every turn, and extracting solution filters, and filtrate recycling ethanol gets ethanol extract A to there not being the ethanol flavor;
(2) among the extracting solution A, add 5 times of Venenum Bufonis amounts, granularity is 100～200 purpose silica gel, evaporate to dryness, must go up sample silica gel, the top that adds the chromatographic column that silica gel has been housed is earlier that petroleum ether-acetone mixed solvent of 9:1 is eluant with the volume ratio, eluting, discard eluent, continuing with the volume ratio is that petroleum ether-acetone mixed solvent of 8.5:1.5 is eluant, is eluted to cinobufagin and Toadpoison Medicine mix ingredients eluting is complete, gets eluent B;
(3) eluent B heating recovery solvent is to solvent-free flavor, and vacuum drying apparatus inner drying 48h gets highly purified cinobufagin and Toadpoison Medicine mixture extract;
(4) assay of high-purity cinobufagin and Toadpoison Medicine mixture extract;
The blade diameter length ratio of described silicagel column is 1:3～10, and loading amount is 10～30 times of Venenum Bufonis amount among the extracting solution A.
3. preparation method according to claim 1 and 2 is characterized in that, extracting with concentration of ethanol is 95%.
4. preparation method according to claim 1 and 2 is characterized in that, the blade diameter length ratio of described silicagel column is 2.5:13.5.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310142353.0A CN103191133B (en) | 2013-04-23 | 2013-04-23 | Preparation method for extracting cinobufagin and bufalin mixture from venenum bufonis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310142353.0A CN103191133B (en) | 2013-04-23 | 2013-04-23 | Preparation method for extracting cinobufagin and bufalin mixture from venenum bufonis |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103191133A true CN103191133A (en) | 2013-07-10 |
| CN103191133B CN103191133B (en) | 2014-12-24 |
Family
ID=48714070
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310142353.0A Expired - Fee Related CN103191133B (en) | 2013-04-23 | 2013-04-23 | Preparation method for extracting cinobufagin and bufalin mixture from venenum bufonis |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103191133B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107308169A (en) * | 2017-07-07 | 2017-11-03 | 黄志敏 | A kind of pharmaceutical composition with antivirus action |
| CN113116935A (en) * | 2020-04-13 | 2021-07-16 | 合肥华方医药科技有限公司 | Toad skin sterene total lactone extract and its preparation method and use |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101683359A (en) * | 2008-09-28 | 2010-03-31 | 上海秀新臣邦医药科技有限公司 | Traditional Chinese medicine composition for treating tumours and preparation method thereof |
-
2013
- 2013-04-23 CN CN201310142353.0A patent/CN103191133B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101683359A (en) * | 2008-09-28 | 2010-03-31 | 上海秀新臣邦医药科技有限公司 | Traditional Chinese medicine composition for treating tumours and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| 刘丹等: "蟾酥中蟾毒配基类成分的分离纯化及其体外抗肿瘤活性的研究", 《中成药》, vol. 32, no. 6, 30 June 2010 (2010-06-30), pages 937 - 940 * |
| 沈嘉茵等: "正交法对蟾酥提取工艺影响研究", 《中国实验方剂学杂志》, vol. 14, no. 2, 29 February 2008 (2008-02-29), pages 27 - 28 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107308169A (en) * | 2017-07-07 | 2017-11-03 | 黄志敏 | A kind of pharmaceutical composition with antivirus action |
| CN113116935A (en) * | 2020-04-13 | 2021-07-16 | 合肥华方医药科技有限公司 | Toad skin sterene total lactone extract and its preparation method and use |
| CN113116935B (en) * | 2020-04-13 | 2023-11-14 | 合肥华方医药科技有限公司 | Bufonis venenum total lactone extract and preparation method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103191133B (en) | 2014-12-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101190258A (en) | Total sesquiterpene lactone extract containing rich parthenolide and preparation method and application thereof | |
| CN106727806B (en) | A kind of preparation method of sanchi leaf total saposins | |
| CN102977114B (en) | Inoscavin A as a monomeric component in phellinus as well as prepearation method and application thereof | |
| CN102526315A (en) | Preparation method of extracts of effective fractions of lychee seeds | |
| CN111454154A (en) | Euphorbia lathyris diterpene alkane type compound and extraction method and application thereof | |
| CN103191132B (en) | Preparation method for extracting high-purity resibufogenin from venenum bufonis | |
| JP2022543794A (en) | Method for producing tetragalloyl glucose | |
| CN103191133B (en) | Preparation method for extracting cinobufagin and bufalin mixture from venenum bufonis | |
| CN105669797B (en) | A kind of method that burdock seed oil, arctiin, arctigenin, arctigenin-4'-gentiobioside E and arctigenin-4'-gentiobioside H are separated from great burdock achene | |
| CN103006721B (en) | Method for extracting phenylethanoid glycoside and flavonoid C-glycoside from corallodiscus flabellata | |
| CN103006754B (en) | Manaplant alhagi extract and preparation method thereof | |
| CN113264974B (en) | Preparation of type B cardiac glycoside and anti-angiogenesis application thereof | |
| CN110025677B (en) | Preparation method of rhubarb extract | |
| CN109053756B (en) | Phenylpropanoid ester type catechin and preparation method and application thereof | |
| CN106957310A (en) | The high efficiency preparation method of flavonoids monomer in a kind of leaves of Hawthorn | |
| CN101190259A (en) | Southern magnolia total lactone extract and preparation method and application thereof | |
| CN104027403B (en) | A kind of preparation method of Radix Polygalae sugar ester effective site | |
| CN110974857A (en) | Refined panax notoginseng saponins and purification preparation method for extracting panax notoginseng saponins | |
| CN103083388B (en) | Preparation method of fructus gleditsiae total saponins | |
| CN110836939A (en) | Detection method of rheum officinale traditional Chinese medicine decoction pieces | |
| CN103130817B (en) | Bilobalide B compound and preparation method thereof | |
| CN107721857A (en) | A kind of method that high-purity chlorogenic acid is prepared from Gynura procumbens (Lour.) Merr | |
| CN101618069B (en) | Capsule preparation for treating bruise as well as bleeding and easing pain and preparation method and use thereof | |
| CN102389456A (en) | Method for extracting isodon japonica var.galaucocalyx total diterpenoids or Glaucocalyxin A | |
| CN103142474A (en) | Composition taking high-purity ginkgolide B as active ingredient and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20141224 Termination date: 20150423 |
|
| EXPY | Termination of patent right or utility model |