CN1304024C - Method for preparing extracts of rabdosia effective ingredient - Google Patents
Method for preparing extracts of rabdosia effective ingredient Download PDFInfo
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- CN1304024C CN1304024C CN 200410053607 CN200410053607A CN1304024C CN 1304024 C CN1304024 C CN 1304024C CN 200410053607 CN200410053607 CN 200410053607 CN 200410053607 A CN200410053607 A CN 200410053607A CN 1304024 C CN1304024 C CN 1304024C
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- rabdosia rubescens
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- rubescensine
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Abstract
The present invention provides a method for quantitatively preparing the extracts of the effective components of rabdosia, which belongs to the technical field of Chinese medicines. The method uses a proceduring integration technology and is a latest result in the technical field of modern extraction and separation, i.e. the proportional extracts of the active components of rabdosia are collectively prepared in a continuous technical process of separation and purification. The method has the advantages of continuous processes, simple technology, high integration level, good quality controllability and small residual solvent quantity, and is suitable for scale production.
Description
Technical field:
The invention belongs to technical field of Chinese medicines.Be specifically related to a kind of preparation method of Rabdosia rubescens active ingredient extract quantification.
Background technology:
Rabdosia rubescens has another name called Rabdosia rubescens, and the formal name used at school inferior Rabdosia rubescens (Hem sl.) that cracks rice is a Labiatae Rabdosia perennial herb, and ground is economized in Henan, Hebei and the Shanxi etc. that are distributed widely in China, records in " first one of Chinese pharmacopoeia version in 1977.The traditional Chinese medical science is thought the Rabdosia rubescens sweet-bitter flavor, and cold nature has clearing away heat and expelling pathogen in the exterior, the effect of promoting blood circulation and stopping pain.Doctor trained in Western medicine thinks that Rabdosia rubescens has anti-inflammation and antineoplastic action, is a kind of natural antibiotic.Medicines such as compound ' Donglingcao ' sheet, Rabdosia rubescens syrup preparation have been arranged in the market, be mainly used in diseases such as treatment acute and chronic tonsillitis, pharyngolaryngitis and stomatitis clinically.
Research data in recent years confirms, the Chinese crude drug Rabdosia rubescens also has tangible anti-tumor activity, through chemical constituent and pharmacological evaluation analysis, wherein contain rubescensine A (Oridonin) and derivant, rubescensine B diterpene-kind compounds such as (Ponicidin) and triterpenes components such as ursolic acid (Ursolic acid) and oleanolic acid.Its molecular structural formula such as figure below:
Rubescensine A
Rubescensine B
Ursolic acid
Ursolic acid (Ursolic acid) has another name called maloic acid, ursolic acid, is a kind of triterpenoid compound that is present in the natural plants, has killing and suppresses the Streptococcus mutans group and the periodontal pathogen, suppress oral cavity carcinoma of tongue cytosis.A lot of in recent years R﹠D institutions find that ursolic acid has certain anti-tumor activity effect.The Cancer Hospital of Chinese Academy of Medical Sciences research worker finds that ursolic acid has carcinogenesis, anti-promoting, induces differentiation of F9 teratocarcinoma cell and blood vessel formation against function, probably becomes low toxicity new type anticancer medicine efficiently.
The research of carrying out for Rabdosia rubescens at present mainly concentrates on rubescensine A and derivant, rubescensine B, the chemical constitution of kaurene class diterpene and their antitumaous effect.Find from the retrieval of patent information, the patent that relates to the Rabdosia rubescens effective ingredient mainly contain following shown in:
1. Zhengzhou University has applied for " Oridonin derivative and preparation method thereof ", and application number is 99101179; Reach the novel antitumor drug of searching by the molecular structural formula that changes rubescensine A;
2. Chinese Academy of Sciences Wuhan plant has been applied for " a kind of preparation method of rubescensine A ", and application number is 02139049.5, relates to extract preparation rubescensine A composition wherein;
3. Baihai Guofa Marine Organism Industry Co., Ltd. has applied for " Chinese medicine Rabdosia rubescens effective site antitumor drug extract and preparation method thereof ", application number is 03125250.8, relate to Chinese medicine Rabdosia rubescens active component composition and preparation method, the total terpene content of its Rabdosia rubescens is more than 50%;
4. " Rabdosia rubescens extract and its production and application " of Shanxi Prov. Medicine Inst. application, application number is 02151503.4, content relates to Rabdosia rubescens extract and includes crude extract, effective site and single component, and single component is rubescensine A and second element;
Above-mentioned patent relates generally to a kind of effective ingredient or the effective part extract in the Rabdosia rubescens, is used for the therapeutical effect of anti-tumor aspect etc.
A large amount of pharmacological researches show that Rabdosia rubescens has anti-inflammation, the effect of relieving pain and activating blood circulation.The recent findings Rabdosia rubescens has obvious antineoplastic, mainly acts on tumors such as the esophageal carcinoma, carcinoma of gastric cardia and hepatocarcinoma.Up to the present, be raw material with the Rabdosia rubescens medical material, developed medicines such as Dong ling cao tablet, Rabdosia rubescens syrup and Rabdosia rubescens terpene buccal tablet, these all are slightly to be extracted as main active with Rabdosia rubescens.
But only be to prepare the Rabdosia rubescens effective part extract in the above-mentioned disclosed content, wherein active ingredient is lacked the integrated extraction process of continuous process that is fit to industrially scalable with traditional handicraft, poor to the quality controllability of Chinese patent medicine.
Summary of the invention:
Technical problem to be solved by this invention is to use Chinese medicine extraction and analytical technology, the quantification extract of a plurality of active ingredients of preparation Rabdosia rubescens.
The invention provides a kind of integrated preparation method of continuous process of Rabdosia rubescens active ingredient extract.
The inventive method is to have used " process integrated technology " principle.This process integrated technology is to utilize in the successive technological process, the multiple active ingredient of disposable in proportion continuous preparation.
Method of the present invention comprises the following steps:
(1) draw materials:
Choosing the Rabdosia rubescens raw material of 1000g pulverizes, add 5-8 and doubly measure ethanol extraction 3-4 time of 80-95%, each extraction time 2-4 hour, merge the Rabdosia rubescens extracting solution, obtain Rabdosia rubescens extractum after reclaiming ethanol, adding 4-6 doubly measures dehydrated alcohol and dissolves, and the back adds ethyl acetate and extracts, and obtains extract.
(2) the continuous separation and purification of processization:
Get acetic acid ethyl ester extract, adding 3-5 petroleum ether doubly precipitates, obtain precipitate, dissolve to wherein adding 100ml ethyl acetate or ethanol, silicagel column on the lysate, on equipment, adopted the multicolumn system that homemade centre can continuous sampling, adopt petroleum ether: ethyl acetate=(10: 1)-(1: 5) mixed solvent carries out gradient elution with the flow velocity of 10-15ml/min, collect according to every part of 200ml, do TLC and HPLC on-line tracing simultaneously, at different parts separate collection eluent, 4: 1-1: in 2 the eluate, 10-22 part has the part white crystals to separate out, and merges for the composition of needs, collects and obtains eluent.
(3) the concentrated finished product that obtains
With the eluent that collect at each position, reclaim eluent to 1/3～1/4 volume respectively, to wherein adding powdered activated carbon (raw material: active carbon=40: 1-100: 1, mass ratio) decolouring, sucking filtration, dry then, obtain the ratio extract.
(4) check
Content detection through TLC and HPLC, compare with reference substance, analyze the relation of ratio extract, wherein contain rubescensine A (Oridonin) content between 5～75%, rubescensine B (Ponicidin) content is controlled in 5～10% scopes, and ursolic acid (Ursolic acid) content is between 5～10%.
Method technology of the present invention is simple; can obtain the ratio extract of Rabdosia rubescens active component; be rubescensine A: rubescensine B: ursolic acid=(5～75): (5～10): (5～10); this extraction process; organic solvent residual is few; be applicable to large-scale production, can effectively improve the active component content in the Rabdosia rubescens.
Testing conditions of the present invention:
(1) testing conditions of rubescensine A:
Mobile phase: methanol: water=55: 45
Detect wavelength: 242nm
Flow velocity: 0.8ml/min
Chromatographic column: day island proper Tianjin C
18ODS, post specification 250*4.6mm; 5um
Sensitivity: 0.005 AUFS
Column temperature: 25 ℃
(2) testing conditions of rubescensine B:
Mobile phase: water: second eyeball=75: 25
Detect wavelength: 252nm
Flow velocity: 0.8ml/min
Chromatographic column: day island proper Tianjin C
18ODS 250*4.6mm; 5um
Sensitivity: 0.005 AUFS
Column temperature: 25 ℃
(3) testing conditions of ursolic acid:
Mobile phase: methanol: acetonitrile: water: phosphoric acid=30: 70: 10: 0.5
Detect wavelength: 220nm
Flow velocity: 0.85ml/min
Chromatographic column: Waters C
18ODS 250*4.6mm; 5um
Sensitivity: 0.005 AUFS
Column temperature: 25 ℃
The present invention compared with prior art, it is as follows to have outstanding substantive distinguishing features and obvious improvement:
1. using a kind of new extracting method one " process integrated technology ", is the newest fruits in the modern extraction and separation technology field, i.e. the ratio extract of centralized preparation Rabdosia rubescens active ingredient in a successive separation purifying technique flow process.Method flow of the present invention is continuous, and technology is simple, the integrated degree height of equipment, and quality controllability is good, and solvent residual amount is little, is suitable for large-scale production.
2. production technology is easy, and the filler of use is a silica gel, adopts column chromatography separation method, is suitable for suitability for industrialized production.
At present first the proportion of utilization preparation method produce and to obtain effective ingredient in the Rabdosia rubescens, and can be controlled in the suitable proportion, its production response rate is higher than present 0.2%.
4. the solvent that adopts in this preparation method has solved dissolvent residual and pollution problem, and is workable.
Description of drawings:
Accompanying drawing contains the flow chart of Rabdosia rubescens active component ratio extract for the present invention's preparation.
The specific embodiment:
Embodiment 1:
Choose the Rabdosia rubescens raw material of 200g, pulverize, adds the ethanol extraction 3 times of 5 times of amounts 85%, at every turn 2 hours extraction times, obtain the Rabdosia rubescens extracting solution.Reclaim the Rabdosia rubescens extractum 12.3g that ethanol obtains green and brown color, add the 50ml dehydrated alcohol and dissolve, the back adds ethyl acetate and extracts, and obtains extract.
Get acetic acid ethyl ester extract, the petroleum ether that adds 4 times precipitates, obtain precipitate, dissolve to wherein adding 50ml ethanol, get silicagel column on the lysate, adopt petroleum ether: ethyl acetate=(10: 1)-(1: 1) mixed solvent carries out gradient elution with 12ml/min, do TLC and HPLC on-line tracing simultaneously, different parts separate collection eluent merges for the composition of needs, obtains eluent.Eluent with collect at each position reclaims eluent to 1/3 volume respectively, to wherein adding the powdered activated carbon decoloring of 4g, and sucking filtration, dry then, obtain ratio extract finished product 0.49g.
Content detection through TLC and HPLC, compare with reference substance, analyze the relation of ratio extract, wherein containing rubescensine A (Oridonin) content is 40.8%, rubescensine B (Ponicidin) content is 8.2%, and ursolic acid (Ursolic acid) content is 7.5%.
Embodiment 2:
Choose the Rabdosia rubescens raw material of 1000g, pulverize, adds the ethanol extraction 3 times of 6 times of amounts 90%, at every turn 3 hours extraction times, obtain the Rabdosia rubescens extracting solution.Reclaim the Rabdosia rubescens extractum 62.8g that ethanol obtains green and brown color, add the 300ml dehydrated alcohol and dissolve, the back adds ethyl acetate and extracts, and obtains extract.
Get acetic acid ethyl ester extract, the petroleum ether that adds 4 times precipitates, obtain precipitate, dissolve to wherein adding 300ml ethanol, get silicagel column on the lysate, adopt petroleum ether: ethyl acetate=(8: 1)-(1: 4) mixed solvent carries out gradient elution with 10ml/min, do TLC and HPLC on-line tracing simultaneously, at different parts separate collection eluent, merge for the composition of needs, obtain eluent.Eluent with collect at each position reclaims eluent to 1/3 volume respectively, to wherein adding the powdered activated carbon decoloring of 10g, and sucking filtration, dry then, obtain ratio extract finished product 2.86g.
Through the content detection of TLC and HPLC, wherein containing rubescensine A (Oridonin) content is 70.6%, and rubescensine B (Ponicidin) content is 8.2%, and ursolic acid (Ursolic acid) content is 8.5%.
Embodiment 3:
Choose the Rabdosia rubescens raw material of 3000g, pulverize, adds the ethanol extraction 4 times of 5 times of amounts 95%, at every turn 3 hours extraction times, obtain the Rabdosia rubescens extracting solution.Reclaim the Rabdosia rubescens extractum 192.6g that ethanol obtains green and brown color, add the 1000ml dehydrated alcohol and dissolve, the back adds ethyl acetate and extracts, and obtains extract.
Get acetic acid ethyl ester extract, the petroleum ether that adds 5 times precipitates, obtain precipitate, dissolve to wherein adding the 800ml ethyl acetate, get silicagel column on the lysate, adopt petroleum ether: ethyl acetate=(4: 1)-(1: 3) mixed solvent carries out gradient elution with 15ml/min, does TLC and HPLC on-line tracing simultaneously, at different parts separate collection eluent, obtain eluent.Eluent with collect at each position reclaims eluent to 1/4 volume respectively, to wherein adding the powdered activated carbon decoloring of 50g, and sucking filtration, dry then, obtain ratio extract finished product 8.21g.
Through the content detection of TLC and HPLC, wherein containing rubescensine A (Oridonin) content is 60%, and rubescensine B (Ponicidin) content is 7.5%, and ursolic acid (Ursolic acid) content is 6.8%.
Claims (2)
1, a kind of Rabdosia rubescens active ingredient preparation method of extract is characterized in that this method comprises the following steps:
(1) draw materials:
Choosing the Rabdosia rubescens raw material of 1000g pulverizes, add 5-8 and doubly measure ethanol extraction 3-4 time of 80-95%, each extraction time 2-4 hour, merge the Rabdosia rubescens extracting solution, obtain Rabdosia rubescens extractum after reclaiming ethanol, adding 4-6 doubly measures dehydrated alcohol and dissolves, and the back adds ethyl acetate and extracts, and obtains extract;
(2) the continuous separation and purification of processization:
Get acetic acid ethyl ester extract, adding 3-5 petroleum ether doubly precipitates, obtain precipitate, dissolve to wherein adding 100ml ethyl acetate or ethanol, silicagel column on the lysate, on equipment, adopted the multicolumn system that homemade centre can continuous sampling, adopt petroleum ether: ethyl acetate=10: 1-1: 5 mixed solvents carry out gradient elution with the flow velocity of 10-15ml/min, collect according to every part of 200ml, do the on-line tracing of TLC and HPLC simultaneously, at different parts separate collection eluent, 4: 1-1: in 2 the eluate, 10-22 part has the part white crystals to separate out, and merges for the composition of needs, collects and obtains eluent;
(3) the concentrated finished product that obtains
With the eluent that collect at each position, reclaim eluent to 1/3～1/4 volume respectively, to wherein adding powdered activated carbon, raw material: active carbon=40: 1-100: 1, mass ratio, decolouring, sucking filtration, dry then, obtain the ratio extract;
(4) check
Content detection and reference substance through TLC and HPLC compare, and analyze the relation of ratio extract, wherein contain rubescensine A content between 5-75%, and rubescensine B content is controlled in the 5-10% scope, and ursolic acid content is between 5-10%.
2, a kind of Rabdosia rubescens active ingredient preparation method of extract according to claim 1 is characterized in that the content detection condition of HPLC in the check of wherein said step (4) is:
(1) testing conditions of rubescensine A:
Mobile phase: methanol: water=55: 45
Detect wavelength: 242nm
Flow velocity: 0.8ml/min
Chromatographic column: day island proper Tianjin C
18ODS, post specification 250*4.6mm; 5um
Sensitivity: 0.005AUFS
Column temperature: 25 ℃
(2) testing conditions of rubescensine B:
Mobile phase: water: second eyeball=75: 25
Detect wavelength: 252nm
Flow velocity: 0.8ml/min
Chromatographic column: day island proper Tianjin C
18ODS 250*4.6mm; 5um
Sensitivity: 0.005 AUFS
Column temperature: 25 ℃
(3) testing conditions of ursolic acid:
Mobile phase: methanol: acetonitrile: water: phosphoric acid=30: 70: 10: 0.5
Detect wavelength: 220nm
Flow velocity: 0.85ml/min
Chromatographic column: Waters C
18ODS 250*4.6mm; 5um
Sensitivity: 0.005 AUFS
Column temperature: 25 ℃.
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| CN 200410053607 CN1304024C (en) | 2004-08-10 | 2004-08-10 | Method for preparing extracts of rabdosia effective ingredient |
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|---|---|---|---|
| CN 200410053607 CN1304024C (en) | 2004-08-10 | 2004-08-10 | Method for preparing extracts of rabdosia effective ingredient |
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| CN1304024C true CN1304024C (en) | 2007-03-14 |
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Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101347501B (en) * | 2007-07-20 | 2011-10-05 | 天津天士力制药股份有限公司 | Effective component of Rabdosia amethystoides and preparation and use thereof |
| CN102242163A (en) * | 2011-04-29 | 2011-11-16 | 郑州大学 | Biological transformation and synthesis method of rubescensin |
| CN103626653B (en) * | 2013-08-14 | 2015-06-24 | 河南大学 | Rabdosia rubescens procoagulant effective components, and preparation method and application thereof |
| CN111166752B (en) * | 2020-02-13 | 2021-08-17 | 黑龙江中医药大学 | Pharmaceutical composition for treating cervical cancer and preparation method thereof |
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