CN1305866C - Method of extracting phenolic components from chinese medicine red sage root and its freeze dried powder injection agent - Google Patents
Method of extracting phenolic components from chinese medicine red sage root and its freeze dried powder injection agent Download PDFInfo
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- CN1305866C CN1305866C CNB031323820A CN03132382A CN1305866C CN 1305866 C CN1305866 C CN 1305866C CN B031323820 A CNB031323820 A CN B031323820A CN 03132382 A CN03132382 A CN 03132382A CN 1305866 C CN1305866 C CN 1305866C
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Abstract
The present invention relates to an extraction method of phenolic components from traditional Chinese medicine salvia for preventing and treating the diseases of hepatic injury, hepatic fibrosis, etc. and a freeze-dried injection. In the method, salvia is leached in hot water in a short time; after being merged, leaching liquid is centrifugated; supernatant is ultrafiltered; filter liquid passes through a reversed-phase column or water extract after acetic ester extraction and acidification; after organic phases are separated and solvent is recovered, different components are acquired by passing through the reversed-phase column; and salvianolic acid A and salvianolic acid B are proportioned according to the proportion of 1:1 to 5 and prepared into the freeze-dried injection by adding propping agent or not additiong propping agent, and the freeze-dried injection is used for preventing and treating the diseases of hepatic injury, hepatic fibrosis, etc. The extraction method has the advantages of stability and high yield, and the salvia phenolic component prepared by the extraction method has the advantages of high purity and stable performance.
Description
Technical field
The present invention relates to method and lyophilized injectable powder thereof that a kind of control salviamiltiorrhizabung that is used for diseases such as liver injury, hepatic fibrosis extracts each component of phenolic acid.
Background technology
" research and development of natural products " 1998,11 75-80 pages or leaves report: the phenolic acid in the red sage root is the main activeconstituents of treatment cardiovascular disorder." pharmacy circular " 1981,16 24-28 pages or leaves report: phenolic acid mainly comprises salvianolic acid A-C in the red sage root, Salvianic acidA, rosmarinic acid, rancinamycin IV.Recently studies show that phenolic acid has significant preventive and therapeutic effect to liver injury and liver fiber, report as " world Chinese digests magazine " 1999,7 570-572 pages or leaves: salvianolic acid A has good anti-peroxidation liver injury effect; " Chinese hepatopathy magazine " 1996,4 the 86th pages of reports: salvianolic acid B has the collegen filament of inhibition at intrahepatic deposition with to the obvious provide protection of hepatocellular injury tool.
Chinese patent, CN1247855A disclosed and adopts water to put forward the method that phenolic acid in the resin method extraction red sage root is crossed in the back in 2000, because of aqueous extract impurity is many, caused the resin regeneration rate low, and the extract phenolic content is low.On this basis, Chinese patent, CN1384090A, disclosed in 2002 improved water carry the back alcohol precipitation after resin extracting method, though increased alcohol precipitation process, alcohol precipitation causes the loss of phenolic acid to surpass 30%, and water extraction time is all longer, surpass 1 hour, because heating causes the considerable damage of phenolic acid.Chinese patent, CN1425659A disclosed the method for extracting salvianolic acid B from salvianolic acid in 2003, also had above-mentioned extraction defective, and other effective constituent such as salvianolic acid A etc. fail to separate in the salvianolic acid.Because phenolic acid belongs to readily oxidizable substance, easy oxidation destroys when heating and seasoning.Present existing red sage formulation such as Radix Salviae Miltiorrhizae Injection etc. have only content of Danshensu higher, and phenolic content are less, does not have the pharmaceutical formulation of each component of salvianolic acid, and is therefore active on the low side.
Summary of the invention
The present invention be directed to defective and deficiency that above-mentioned prior art exists, provide a kind of and can from salviamiltiorrhizabung, extract active phenolic acid, and separation obtains different phenolic acid components, carry out the method for proportioning composing prescription preparation, and prepare lyophilized injectable powder with this method.
A kind of method of extracting each component of phenolic acid from salviamiltiorrhizabung of the present invention may further comprise the steps:
Step 1, salviamiltiorrhizabung lozenge or crushed material, flooding gets extracting solution;
Precipitation is removed in step 2, separation;
Step 3 A, the supernatant liquor after will separating, through the ultra-filtration membrane ultrafiltration, the molecular weight of holding back is not more than 10000Da;
B, with the liquid concentration mentioned among the step 3 A (or not concentrating), transfer pH2-5 with acid, through organic solvent extraction, separate organic solvent then and must contain drug solns;
Step 4, with the above-mentioned drug solns that contains, cross reversed-phase column.Use solvent elution, after testing, collect isolating each component salvianolic acid, through normal pressure or evaporation under reduced pressure removed recovered solvent, dry then each component salvianolic acid that gets.
Wherein the water extraction time is no more than 30 minutes in the step 1, extracts 80-100 ℃ of temperature; Method is the high speed centrifugation impurity elimination in the step 2, and rotating speed is greater than 10000rpm; What adopt among the step 3 B is acidifying acetate esters extraction process, and used acid is various organic acids or mineral acid, and regulating the pH scope is 2-5, and extracting used solvent can be the acetic ester solvent.
The present invention presses 1 with salvianolic acid A and salvianolic acid B: the 1-5 proportioning, add or do not add the freeze-dried powder that propping agent is made.
The present invention adopts 80-100 ℃ of short period of time lixiviate 2 times, is less than 30 minutes at every turn, can reduce the salviamiltiorrhizabung phenolic acid as far as possible and destroy in hot water, and can improve the rate of transform of salvianolic acid as far as possible.
The present invention adopts supercentrifugal process to remove large particulate matter (rotating speed is greater than 10000rpm) in the extracting solution, has avoided the precipitator method because of precipitating the loss that not exclusively causes.
The present invention adopts ultrafiltration process to remove macromolecular components in the extracting solution, the destruction that heating causes salvianolic acid when having avoided alcohol deposition method to reclaim because of precipitating the loss that not exclusively causes and ethanol.
The present invention adopts the acetate esters extraction method, can remove the composition that major part is insoluble to acetate esters, enrichment salvianolic acid composition.
The present invention crosses reversed-phase column with the solution of the poly-salvianolic acid of richness, adopts same solvent or different solvents wash-out, can obtain the various phenolic acid components behind the purifying.
The present invention carries out different prescriptions with various phenolic acid components by the disease of application of treatment, improves the result of treatment of one-component, reduces the side effect that one-component brings.
The present invention has overcome defective and the deficiency in the existing salvianolic acid extraction.Each Danshen component that obtains with the present invention is active high, and stable performance can be used for the control of diseases such as liver injury, hepatic fibrosis.
The salviamiltiorrhizabung phenolic acid component that the present invention utilizes this extracting method to obtain is made lyophilized injectable powder according to a certain ratio, and LD50 is greater than 2g/Kg in its intravenous injection, and toxicity significantly is lower than single phenolic acid component (LD50 is less than 1g/Kg).The effect of its treatment liver injury and hepatic fibrosis is better than single phenolic acid component, shows the shortening of treatment phase, and it is very fast that every index is recovered.
Clinical treatment hepatic fibrosis does not at present have special-purpose medicaments, is primarily aimed at the cause of disease and treats, and as the Yiganling electuary, careless celestial hepatitis B capsule etc. are used for antiviral therapy.Salvianolic acid component lyophilized injectable powder of the present invention utilizes the anti-oxidant activity of phenolic acid and removes the free radical characteristic, can promote the recovery of liver injury and the absorption of collegen filament faster, is the special-purpose medicaments that is used for liver injury and hepatic fibrosis.
Embodiment
Embodiment one extracts each component of phenolic acid for the present invention from salviamiltiorrhizabung: get red sage root 1Kg, add 100 ℃ of boiling water 10L, timing lixiviate 30 minutes, during keep heating.Filter, filter residue adds 100 ℃ of boiling water 5L again, timing lixiviate 30 minutes, during keep heating.Filter, abandon filter residue, merging filtrate, filtrate is centrifugal through the continuous high speed whizzer, rotating speed 15,000rpm.The supernatant liquor ultrafiltration of centrifugal back, membrane retention molecular weight 3000.Keep filtration fraction, last C8ODS preparative column is gone up sample 5mL at every turn, uses the methanol-water wash-out, and 270nm detects, and with isolating each component vacuum-drying, gets component 1 (salvianolic acid B) 32.4mg, component 2 (salvianolic acid A) 5.6mg, component 3 (rancinamycin IV) 8.63mg.
Embodiment two extracts each component of phenolic acid for another kind of the present invention from salviamiltiorrhizabung: get red sage root 1Kg, add 80 ℃ of boiling water 12L, timing lixiviate 30 minutes, during keep heating.Filter, filter residue adds 80 ℃ of boiling water 6L again, timing lixiviate 30 minutes, during keep heating.Filter, abandon filter residue, merging filtrate, filtrate is centrifugal through the continuous high speed whizzer, rotating speed 15,000rpm.Centrifugal back supernatant liquor is evaporated to 5L.Add the 5L ethyl acetate extraction 2 times, standing demix keeps ethyl acetate layer, the reclaim under reduced pressure ethyl acetate, residue adds the less water dissolving, and last C18ODS preparative column is gone up sample 5mL at every turn, use the methanol-water wash-out, 270nm detects, and with isolating each component vacuum-drying, gets component 1 (salvianolic acid B) 26.2mg, component 2 (salvianolic acid A) 4.2mg, component 3 (rancinamycin IV) 9.26mg.
Embodiment three salvianolic acid component freeze-dried powder preparation methods: get salviol acid A 20g, salvianolic acid B 80g, N.F,USP MANNITOL 20g, 1000 of freeze-dried powders are made in the smart filter freeze-drying of water 24L dissolving back.
Embodiment four salvianolic acid component freeze-dried powder preparation methods: get salviol acid A 40g, salvianolic acid B 60g, 1000 of freeze-dried powders are made in the smart filter freeze-drying of water 20L dissolving back.
Embodiment five salvianolic acid components are used for the treatment of the experimentation on animals of hepatic fibrosis:
Method: 50 of rats, adopt CCl
4Cause liver injury and Liver Fibrosis Model. be divided into normal group, model group, 3 groups of salvianolic acid As and B proportioning group (1: 1,1: 3,1: 5) at random, 10 every group.Begin with 5mgKg from modeling
-1D
-1. dosage is irritated the back experiment of stomach to 6 week and is finished, and model group and normal group are irritated with equivalent distilled water. observe the hepatic pathology form; Serum albumin (Alb) content; Serum alanine transaminase (ALT), Aspartic Acid transaminase (AST) activity; Hepatic tissue mda (MDA).
The result: salvianolic acid component A and B proportioning group all have remarkable reduction ALT than model group, AST, and the content of MDA, Alb has the trend of increasing, and shows that they have the effect of treatment liver injury.Find that in pathological study treatment treated animal hepatic fibrosis degree is obviously improved, and shows the therapeutic action of salvianolic acid component to hepatic fibrosis.3 assembly ratio was with 1: 5 the best.
Table 1 salvianolic acid component is to rat blood serum Alb, ALT, the influence of AST and hepatic tissue MDA (x ± s)
| Group | Alb(g/L) | ALT(U/L) | AST(U/L) | MDA(nmol/L/g) |
| Normal group model group 1: 1AB group 1: 3AB group 1: 5AB group | 31.2±0.8 24.1±2.3 27.1±1.6 28.4±1.9 30.1±1.2 | 45±8 157±63 82±33 72±24 59±12 | 82±11 172±78 106±21 93±23 87±17 | 124±37 332±69 206±43 187±41 162±24 |
Table 2 salvianolic acid component is to the influence of liver tissues of rats collegen filament (x ± s)
| Group | Collegen filament (integration) |
| Normal group | 1.23±0.76 |
| Model group | 3.17±0.93 |
| 1: the 1AB group | 1.73±0.59 |
| 1: the 3AB group | 1.66±0.56 |
| 1: the 5AB group | 1.41±0.62 |
Claims (6)
1. method of from salviamiltiorrhizabung, extracting each component of phenolic acid, this method may further comprise the steps:
Step 1, salviamiltiorrhizabung lozenge or crushed material, flooding gets extracting solution;
Precipitation is removed in step 2, separation;
Step 3, A, the supernatant liquor after will separating, through the ultra-filtration membrane ultrafiltration, the molecular weight of holding back is not more than 10000Da;
B, with the liquid concentration mentioned among the step 3 A or do not concentrate, with sour pH is adjusted to 2-5, through organic solvent extraction, separate organic solvent then and must contain drug solns;
Step 4, with the above-mentioned drug solns that contains, cross reversed-phase column, use solvent elution, after testing, collect isolating each component salvianolic acid, through normal pressure or evaporation under reduced pressure removed recovered solvent, dry then must each component salvianolic acid.
2. extracting method according to claim 1 is characterized in that the water extraction time is no more than 30 minutes in this method steps one, extracts 80-100 ℃ of temperature.
3. extracting method according to claim 1 is characterized in that method is the high speed centrifugation impurity elimination in this method steps two, and rotating speed is greater than 10000rpm.
4. extracting method according to claim 1, what it is characterized in that adopting among this method steps three B is acidifying acetate esters extraction process, and used acid is various organic acids or mineral acid, and regulating the pH scope is 2-5, and extracting used solvent is the acetic ester solvent.
5. extracting method according to claim 1, it is characterized in that these method steps four used reversed-phase columns separate each component of phenolic acid in the red sage root, reversed-phase column is the various reversed-phase columns of C8, C18 type, and the solvent that wash-out adopts is aqueous methanol, aqueous ethanol, aqueous acetone or acetonitrile eluting solvent.
6. salvianolic acid component lyophilized injectable powder is characterized in that salvianolic acid A and salvianolic acid B are by 1: the 1-5 proportioning, add or do not add the freeze-dried powder that propping agent is made.
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| Application Number | Priority Date | Filing Date | Title |
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| CNB031323820A CN1305866C (en) | 2003-08-19 | 2003-08-19 | Method of extracting phenolic components from chinese medicine red sage root and its freeze dried powder injection agent |
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| CNB031323820A CN1305866C (en) | 2003-08-19 | 2003-08-19 | Method of extracting phenolic components from chinese medicine red sage root and its freeze dried powder injection agent |
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| CN1513848A CN1513848A (en) | 2004-07-21 |
| CN1305866C true CN1305866C (en) | 2007-03-21 |
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Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1301707C (en) * | 2005-03-18 | 2007-02-28 | 邹巧根 | Danhong freeze dried powder injection agent and its preparation method |
| CN100420665C (en) * | 2006-04-21 | 2008-09-24 | 王国振 | Method for extracting 'Danfen' phenolic acid-A |
| CN1943569B (en) * | 2006-10-30 | 2010-05-12 | 王煜 | Chinese medicine active component composition and its preparing method and use |
| CN100462072C (en) * | 2007-01-23 | 2009-02-18 | 北京本草天源药物研究院 | Medicine composition used for injection and its preparing method |
| CN102086613A (en) * | 2010-11-17 | 2011-06-08 | 广州市余平图文实业有限公司 | Preparation method of paper sheet deacidification agent |
| CN104434899A (en) * | 2013-09-24 | 2015-03-25 | 天士力制药集团股份有限公司 | Application of danshinolic acid L in preparing medicament for treating or preventing hepatic fibrosis and renal fibrosis |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1247855A (en) * | 1998-09-11 | 2000-03-22 | 中国科学院上海药物研究所 | Preparation and application of tanshinpolyphenolic salt |
| CN1384090A (en) * | 2001-09-26 | 2002-12-11 | 中国医学科学院药物研究所 | Extraction process of tanshin general phenolic acid and its prepn and use |
| CN1425659A (en) * | 2002-12-31 | 2003-06-25 | 南京虹桥医药技术研究所 | Process for preparing danshen salviandic acid |
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2003
- 2003-08-19 CN CNB031323820A patent/CN1305866C/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1247855A (en) * | 1998-09-11 | 2000-03-22 | 中国科学院上海药物研究所 | Preparation and application of tanshinpolyphenolic salt |
| CN1384090A (en) * | 2001-09-26 | 2002-12-11 | 中国医学科学院药物研究所 | Extraction process of tanshin general phenolic acid and its prepn and use |
| CN1425659A (en) * | 2002-12-31 | 2003-06-25 | 南京虹桥医药技术研究所 | Process for preparing danshen salviandic acid |
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