CN102477063A - Method for preparing high-purity madecassoside and asiaticoside B - Google Patents
Method for preparing high-purity madecassoside and asiaticoside B Download PDFInfo
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- CN102477063A CN102477063A CN201010558562XA CN201010558562A CN102477063A CN 102477063 A CN102477063 A CN 102477063A CN 201010558562X A CN201010558562X A CN 201010558562XA CN 201010558562 A CN201010558562 A CN 201010558562A CN 102477063 A CN102477063 A CN 102477063A
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- centella asiatica
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- high purity
- asiaticoside
- asiatica glucoside
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Abstract
The present invention relates to a method for preparing high-purity madecassoside and asiaticoside B. The method comprises performing preparative high-performance liquid chromatography by using a mobile phase added with cyclodextrin, collecting two eluates according to chromatographic peaks, respectively, recovering the solvent in the eluates, and extracting to remove cyclodextrin. By adopting preparative high-performance liquid chromatography and cyclodextrin-containing mobile phase, the invention has no need for a special analytical column, is cheap and feasible, and has substantial improvements and advantages compared with other methods.
Description
Technical field
The present invention relates to the method for purification technical field of asiaticoside and its isomers centella asiatica glucoside B.
Background technology
Herba Centellae Centella asiatica (L.) Urban. is the dry herb of umbelliferae Centella plant Herba Centellae, and the beginning is stated from Shennong's Herbal, bitter, cold.Main big heat is disliked sore, ulcer, and body heat is classified as middle article.Herba Centellae is the version " conventional Chinese medicine that Chinese pharmacopoeia is recorded in 2010; Effect with clearing away heat-damp and promoting diuresis, removing toxic substances and promoting subsidence of swelling; Be used to treat wound, tetter more clinically, be used for infectious hepatitis, meningococcal meningitis, acute glomerulonephritis etc. simultaneously.China uses Herba Centellae external application and the existing bimillennial history of orally taken for curing disease, and also use it many countries and regions such as South Africa, India, South East Asia as conventional medicament.The main Chemical Composition of Herba Centellae comprises triterpenes, polyyne alkene class, volatile oil and other compositions.
Herba Centellae total glycosides contains multiple triterpene compound for the efficient part that extraction separation obtains from the herb of Herba Centellae, mainly comprises centella asiatica glucoside, asiaticoside and isomers centella asiatica glucoside B thereof.Have the wound healing of promotion effect, be used to treat operation wound, burn, wound and keloid etc. more clinically.
The existing relevant report of HPLC assay for relevant composition in the Herba Centellae total glycosides; All be under general HPLC condition, to measure; The moving phase of general methanol-water or acetonitrile-water system all can't effectively be separated asiaticoside and its isomers centella asiatica glucoside B, and asiaticoside and its isomers centella asiatica glucoside B are the peak of a mixture.
The brief introduction of Schardinger dextrins moving phase additive method: Schardinger dextrins moving phase additive method is that Schardinger dextrins is made an addition in the moving phase.Beta-cyclodextrin has the specific molecule structure, promptly is closed tubular structure.Beta-cyclodextrin is through α-1 by seven D-(+)-glucopyranose units; 4 glycosidic links combine; The outside is hydrophilic surface; Inner then be a hydrophobic tube chamber of chirality with certain size; Hydrophobic in this uniqueness of beta-cyclodextrin, outer hydrophilic-structure not only makes it show important theory value in fields such as analogue enztme, chiral separation, main-guest chemistry, molecular recognition, spectral probes, more demonstrates important practical value in fields such as medicine, food, makeup, environment protection, especially for the chiral separation field.But do not see the Schardinger dextrins in the cyclodextrin inclusion compound is removed and the pertinent literature that standard substance are purified is reported.
Summary of the invention
The object of the invention is exactly to solve the above-mentioned defective that can't isolate asiaticoside and its isomers centella asiatica glucoside B under the existing HPLC condition, and a kind of simple and easy to do separating high-purity asiaticoside and the method for isomers centella asiatica glucoside B thereof are provided.
The present invention is through the preparative high performance liquid chromatography appearance; Adopt moving phase cyclodextrin additive method to collect isomers centella asiatica glucoside B and the solid sample of asiaticoside cyclodextrin inclusion compound, the i.e. inclusion compound of Schardinger dextrins and standard substance that obtains asiaticoside respectively; Through the Schardinger dextrins in the removal cyclodextrin inclusion compound standard substance are purified then and promptly obtain liquid-phase chromatographic analysis with high purity asiaticoside and isomers centella asiatica glucoside B thereof.
The technical scheme that the present invention takes is following:
The preparation method of high purity asiaticoside and centella asiatica glucoside B; This method is through the preparative high performance liquid chromatography appearance; Add Schardinger dextrins in the moving phase; Collect two kinds respectively according to chromatographic peak and collect liquid, two kinds are collected remove Schardinger dextrins through extraction after liquid reclaims solvent respectively, promptly get the standard substance of centella asiatica glucoside B and asiaticoside.
The preferred acetonitrile of above-mentioned moving phase-cyclodextrin aqueous solution system, the concentration range of cyclodextrin aqueous solution be preferably at 2-8mmol/L, most preferably 2mmol/L.
In the above-mentioned extraction step, preferred butanols, amylalcohol, ETHYLE ACETATE etc. are as organic phase, propyl carbinol most preferably, and propyl carbinol is best relatively to the solubility property of sample as organic phase; The ratio of organic phase and water was selected for use between the scope at 1: 100～100: 1, and wherein the ratio preferable range of organic phase and water is 4: 1-1: 4, and most preferred range is 2: 1; Collect for two kinds and get white powder after liquid reclaims solvent respectively, the ratio of the total amount of white powder sample size and organic phase, water (mg/ml) was selected for use between the scope at 1: 100～100: 1, and wherein preferable range is 10: 1; It is 1～10 time that sample is carried out extraction times, and preferred extraction times is 1 time, and layering obtains organic phase, organic phase is reclaimed the standard substance that promptly get centella asiatica glucoside B and asiaticoside behind the solvent.
Beneficial effect of the present invention:
The present invention utilizes Schardinger dextrins moving phase additive method to isolate the cyclodextrin inclusion compound of centella asiatica glucoside B and asiaticoside on preparative high performance liquid chromatography analytical procedure basis, removes Schardinger dextrins through extraction then and obtains liquid-phase chromatographic analysis with high purity asiaticoside and isomers centella asiatica glucoside B thereof.The inexpensive easy row of this method does not need the special analysis post, just separates purified out centella asiatica glucoside B and asiaticoside, has very big progress and advantage with respect to prior art.
Embodiment
Following examples are in order better the present invention to be described but not are used for limiting the present invention.
Embodiment 1-12
1. preparation: (the sample presentation sample is the Herba Centellae total glycosides raw material)
Utilize the method for preparative high performance liquid chromatography that asiaticoside is separated with its isomers centella asiatica glucoside B through the cyclodextrin additive method. Tianjin, island preparative high performance liquid chromatography appearance comprises Shimadzu Class-VP workstation, the LC-8A pump; The unit of SCL-10Avp pump, SPD-M10Avp detector, FRC-10A automatic collector; Moving phase is selected acetonitrile for use: 2mmol/L cyclodextrin aqueous solution (v/v)=26: 74; Chromatographic column is: and SHIMADZU shim-pack PRC-ODS post (20mm * 250mm), flow velocity: 6ml/min, sample size: 5ml; Detect wavelength: 204nm; Collect the collection liquid of preparative liquid chromatography respectively according to chromatographic peak, will collect liquid and reclaim solvent respectively, obtain white powder.
2. extraction:
Get the above-mentioned white powder 50mg that obtains; Select for use propyl carbinol (water saturated), amylalcohol, ETHYLE ACETATE etc. as organic phase; Extraction according to the volume ratio of organic phase and water 1: 100-100: select for use between 1 scope; The ratio of the total amount (V) of total amount of white powder sample (mg) and organic phase, water is 1: 100-100: select for use between 1 scope, sample is extracted, extract 1-10 time; Layering obtains organic phase, organic phase is reclaimed the standard substance that promptly get centella asiatica glucoside B and asiaticoside behind the solvent.
3. sample purity detects:
Adopt HPLC-ELSD and UV to detect HW Workstation workstation, Alltech ELSD 2000 detectors; Waters 515HPLC Pump, moving phase adopts acetonitrile: water (v/v)=30: 70, chromatographic column are the C18 (post of 4.6mm * 250mm); Flow velocity: 1ml/min, sample size: 20 μ l, detect wavelength: 204nm; According to sample size, the concentration of sample and sample peak area can draw the purity of standard substance.Standard substance according to gained calculate yield.
4. the standard substance content that obtains: >=99%.
5. product appearance: white powder.
Each embodiment extraction step actual conditions and result see the following form 1:
Table 1
| 1 | 25∶1 | 1∶4 | 3 | 19.97 |
| 2 | 25∶1 | 3∶2 | 3 | 49.35 |
| 3 | 25∶1 | 2∶1 | 3 | 36.99 |
| 4 | 25∶1 | 3∶1 | 3 | 23.35 |
| 5 | 10∶1 | 3∶2 | 3 | 31.90 |
| 6 | 10∶1 | 2∶1 | 3 | 63.11 |
| 7 | 10∶1 | 3∶1 | 3 | 45.06 |
| 8 | 10∶1 | 4∶1 | 3 | 19.97 |
| 9 | 25∶4 | 3∶2 | 3 | 30.40 |
| 10 | 25∶4 | 2∶1 | 3 | 60.69 |
| 11 | 25∶4 | 3∶1 | 3 | 39.72 |
| 12 | 25∶4 | 4∶1 | 3 | 22.17 |
Test uses sample size to be 50mg, and yield is that the purity with the gained sample reaches the purity calculating of standard substance and gets.All taking extraction times when single factor is investigated experiment is 3 times, merges the sample that extracts according to purity, calculated yield, and last table 1 is the experiment of single factor data.
The present invention has carried out orthogonal experiment in addition, and having taked extraction times is 1,2,3 time experimental design, obtains extracting 1 time according to the result of orthogonal experiment and is optimal selection.
Experiment has adopted butanols, amylalcohol, ETHYLE ACETATE etc. to extract as organic phase respectively; Wherein water saturated propyl carbinol is as organic phase; Solubility property to sample is best relatively; The standard substance yield is also the highest, and the experiment yield of amylalcohol, ETHYLE ACETATE is lower, so most preferably adopt propyl carbinol as organic phase.
Claims (14)
1. the preparation method of high purity asiaticoside and centella asiatica glucoside B; This method is through the preparative high performance liquid chromatography appearance; The interior Schardinger dextrins that adds of moving phase collects two kinds respectively according to chromatographic peak and collects liquid, two kinds is collected extract the removal Schardinger dextrins after liquid reclaim solvent respectively then.
2. the preparation method of high purity asiaticoside as claimed in claim 1 and centella asiatica glucoside B is characterized in that: said moving phase is acetonitrile-cyclodextrin aqueous solution system.
3. the preparation method of high purity asiaticoside as claimed in claim 2 and centella asiatica glucoside B is characterized in that: said cyclodextrin aqueous solution concentration is 2-8mmol/L.
4. the preparation method of high purity asiaticoside as claimed in claim 3 and centella asiatica glucoside B is characterized in that: said cyclodextrin aqueous solution concentration is 2mmol/L.
5. the preparation method of high purity asiaticoside as claimed in claim 1 and centella asiatica glucoside B is characterized in that: extraction step selects for use butanols, amylalcohol or ETHYLE ACETATE as organic phase.
6. the preparation method of high purity asiaticoside as claimed in claim 3 and centella asiatica glucoside B is characterized in that: select for use propyl carbinol as organic phase during extraction.
7. the preparation method of high purity asiaticoside as claimed in claim 1 and centella asiatica glucoside B is characterized in that: the volume ratio of organic phase and water is 1: 100～100: 1 during extraction.
8. the preparation method of high purity asiaticoside as claimed in claim 7 and centella asiatica glucoside B is characterized in that: the volume ratio of organic phase and water is 1 during extraction: 4-4: 1.
9. the preparation method of high purity asiaticoside as claimed in claim 8 and centella asiatica glucoside B is characterized in that: the volume ratio of organic phase and water is 2: 1 during extraction.
10. the preparation method of high purity asiaticoside as claimed in claim 1 and centella asiatica glucoside B is characterized in that: extraction times is 1～10 time.
11. the preparation method of high purity asiaticoside as claimed in claim 10 and centella asiatica glucoside B is characterized in that: extraction times is 1 time.
12. the preparation method of high purity asiaticoside as claimed in claim 1 and centella asiatica glucoside B; It is characterized in that: during extraction, the total amount (ml) of collecting amount (mg) that liquid reclaims the white powder that obtains behind the solvent and organic phase, water is than being 1: 100～100: 1.
13. the preparation method of high purity asiaticoside as claimed in claim 12 and centella asiatica glucoside B; It is characterized in that: during extraction, the total amount (ml) of collecting amount (mg) that liquid reclaims the white powder that obtains behind the solvent and organic phase, water is than being 25: 1～25: 4.
14. the preparation method of high purity asiaticoside as claimed in claim 13 and centella asiatica glucoside B is characterized in that: during extraction, the total amount (ml) of collecting amount (mg) that liquid reclaims the white powder that obtains behind the solvent and organic phase, water is than being 10: 1.
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| CN201010558562XA CN102477063A (en) | 2010-11-24 | 2010-11-24 | Method for preparing high-purity madecassoside and asiaticoside B |
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| CN201010558562XA CN102477063A (en) | 2010-11-24 | 2010-11-24 | Method for preparing high-purity madecassoside and asiaticoside B |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106336448A (en) * | 2016-08-25 | 2017-01-18 | 桂林益天成生物科技有限公司 | Method for extracting madecassic acid from herba centellae |
| CN106380504A (en) * | 2016-08-25 | 2017-02-08 | 桂林益天成生物科技有限公司 | Method for extraction of madecassoside from Centella asiatica |
| CN108395464A (en) * | 2017-02-08 | 2018-08-14 | 桂林三金药业股份有限公司 | A method of preparing asiaticosid, madecassoside and Asaiticoside B from centella |
| CN108398493A (en) * | 2017-02-08 | 2018-08-14 | 桂林三金药业股份有限公司 | A kind of quality determining method of centella medicinal material and its extract and preparation |
| CN112957379A (en) * | 2021-02-20 | 2021-06-15 | 上海珈凯生物科技有限公司 | Method for extracting centella asiatica extract with high biological safety |
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| CN101182346A (en) * | 2007-12-05 | 2008-05-21 | 浙江大学 | Method for preparing asiaticoside A and B simultaneously from herba centellae total saponins |
| CN101724006A (en) * | 2009-12-22 | 2010-06-09 | 浙江大学 | Method for separating asiaticoside-B, hydroxyl asiaticoside and asiaticoside |
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2010
- 2010-11-24 CN CN201010558562XA patent/CN102477063A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101182346A (en) * | 2007-12-05 | 2008-05-21 | 浙江大学 | Method for preparing asiaticoside A and B simultaneously from herba centellae total saponins |
| CN101724006A (en) * | 2009-12-22 | 2010-06-09 | 浙江大学 | Method for separating asiaticoside-B, hydroxyl asiaticoside and asiaticoside |
Non-Patent Citations (4)
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| JIAN PAN,ET AL.: "Separation and Determination of the Structural Isomers of Madecassoside by HPLC Using β-Cyclodextrin as Mobile Phase Additive", 《CHROMATOGRAPHIA》 * |
| 邵燕等: "积雪草总苷及其制剂的含量研究.Ⅰ.积雪苷霜软膏中积雪草苷和羟基积雪草苷的 HPLC 法测定", 《中国医药工业杂志》 * |
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| 陈云艳等: "积雪草总苷及其制剂的含量研究.Ⅱ.积雪草总苷中积雪草苷、羟基积雪草苷和积雪草苷 B 含量的 HPLC 法测定", 《中国医药工业杂志》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106336448A (en) * | 2016-08-25 | 2017-01-18 | 桂林益天成生物科技有限公司 | Method for extracting madecassic acid from herba centellae |
| CN106380504A (en) * | 2016-08-25 | 2017-02-08 | 桂林益天成生物科技有限公司 | Method for extraction of madecassoside from Centella asiatica |
| CN106380504B (en) * | 2016-08-25 | 2018-01-26 | 桂林益天成生物科技有限公司 | The method that madecassoside is extracted from centella |
| CN108395464A (en) * | 2017-02-08 | 2018-08-14 | 桂林三金药业股份有限公司 | A method of preparing asiaticosid, madecassoside and Asaiticoside B from centella |
| CN108398493A (en) * | 2017-02-08 | 2018-08-14 | 桂林三金药业股份有限公司 | A kind of quality determining method of centella medicinal material and its extract and preparation |
| CN112957379A (en) * | 2021-02-20 | 2021-06-15 | 上海珈凯生物科技有限公司 | Method for extracting centella asiatica extract with high biological safety |
| CN112957379B (en) * | 2021-02-20 | 2022-04-08 | 上海珈凯生物科技有限公司 | Method for extracting centella asiatica extract |
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Application publication date: 20120530 |