CN101210002B - Method for separating and preparing salvianolic acid B chemical reference substance - Google Patents
Method for separating and preparing salvianolic acid B chemical reference substance Download PDFInfo
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- CN101210002B CN101210002B CN2006101350972A CN200610135097A CN101210002B CN 101210002 B CN101210002 B CN 101210002B CN 2006101350972 A CN2006101350972 A CN 2006101350972A CN 200610135097 A CN200610135097 A CN 200610135097A CN 101210002 B CN101210002 B CN 101210002B
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Abstract
The invention relates to a separation and preparation method of a salvianolic acid B chemical reference standard, which takes salvianolic acid B extractive, the content of which by weight is 10-97 percent, as raw material, preparative high efficiency liquid chromatography as a separation means and carbinol-acid water solution with certain proportion as an elution system to prepare the salvianolic acid B chemical reference standard, the purity of which is higher than 98 percent. The technical steps are: pretreatment of samples, treatment of chromatographic column, sample feeding, elution, on-line inspection, collection of the eluant containing salvianolic acid B, vacuum concentration of the eluant containing salvianolic acid B till no carbinol exists, and freezing and drying, and the salvianolic acid B chemical reference standard is prepared. The content of the salvianolic acid B can reach over 98 percent based on the inspection through a high efficiency liquid chromatography method.
Description
Technical field
The present invention relates to a kind of preparation method who from Radix Salviae Miltiorrhizae extract, separate to obtain purity greater than 98% salvianolic acid B chemical reference substance.
Background technology
The red sage root is one of common drug in China's ethnopharmacology, and long clinical application history is arranged.Modern pharmacological research shows that salvianolic acid B has very strong antioxygenation, can remove intravital superoxide anion and hydroxyl radical free radical; No matter platelet aggregation and thrombosis that salvianolic acid B causes multiple reason are in the body or in vitro tests, the obvious suppression effect is all arranged, and do not influence the blood coagulation system function, no hemorrhage risk; Salvianolic acid B can be protected the injury of mitochondria due to the perfusion of limitation cerebral ischemia-again and suppress cerebral ischemia---the nerve cell apoptosis that causes of perfusion again.At acute and chronic nerve degenerative diseases such as preventing and treating cerebral ischemia and senile dementia good prospect is arranged.It is the important effective component of red sage compound preparation for treating coronary heart disease, cardiovascular and cerebrovascular diseases.In 2005 editions state-promulgated pharmacopoeia of China salvianolic acid B as measuring composition, and stipulate that its content in red rooted salvia should reach more than 3%.
The extraction and separation method of salvianolic acid B mainly adopts following steps at present: medicinal material extract---removal of impurities---column chromatography process.The difference of different extraction separation processes is used method therefor of removal of impurities and the used chromatographic column difference of column chromatography.Mas Li Lian etc. (Chinese patent CN1384090A, 2002 open) adopt ethanol sedimentation, use macroporous adsorptive resins to separate, and obtain to contain the polyphenol acid extraction thing of 40% salvianolic acid B; Zhang Guoli etc. (Chinese patent CN1528756A, 2004 open) adopt the flocculate with chitosan precipitation, use ethyl acetate extraction again, obtain the salvianolic acid B sample of purity at 50%-96% with silica gel column chromatography at last; Xu Yaming etc. (Chinese patent CN1247855A, 2000 open) adopt macroporous adsorbent resin, and after washing with water, with the methanol-eluted fractions of different concns, obtaining with the salvianolic acid B magnesium is the poly phenolic acid of Radix Salviae Miltiorrhizae extract of principal constituent; Wei Feng etc. (Chinese patent CN1459448A, in December, 2003 is open) adopt polyamide column and macroporous adsorptive resins, collect and obtain total phenolic acid, and total phenolic content is greater than 80%, and wherein content of danshinolic acid B is greater than 50%.Report such as Zhang Fengxia (Chinese patent CN1164582C, 2004 open) adopts the chromatographic column of multiple filler type to obtain purity at the salvianolic acid B more than 90%.
Above method is all being considered the influence of other compositions in the Radix Salviae Miltiorrhizae extract to salvianolic acid B extraction separation process in varying degrees, makes salvianolic acid B and other compositions obtain good separation, obtains the salvianolic acid B of different purity.But, can not stablize acquisition purity at the salvianolic acid B chemical reference substance more than 98% owing to lack post separation method and corresponding method of detection efficiently.
Summary of the invention
For overcoming the defective of prior art, the optimized Separation condition the object of the present invention is to provide the method for separating and preparing of the salvianolic acid B chemical reference substance of a kind of disengaging time weak point, good separating effect, product purity height (purity is more than 98%).
For achieving the above object, the technical solution used in the present invention: the method for separating and preparing of reference substance is a raw material with the salvianolic acid B extract of weight content 10～97%, is separation means with the preparative high performance liquid chromatography, with methyl alcohol-aqueous acid is eluent system, and concrete steps are:
(1) sample pretreatment: with salvianolic acid B extract methyl alcohol, ethanol, water or 50% dissolve with methanol solution of weight content 10～97%, being configured to weight concentration is the salvianolic acid B extract solution of 8～60mg/ml, through 0.22～0.45 μ m filtering with microporous membrane;
(2) chromatographic column is handled: performance liquid chromatographic column is selected the preparative high performance liquid chromatography post, with the eluent system solution equilibria chromatographic column of 5-20 times of column volumes; Eluent system solution is methyl alcohol-aqueous acid, and wherein the volumetric concentration of methyl alcohol in methyl alcohol-aqueous acid is 50-80%; Acid is 0.1～1% in the volumetric concentration of aqueous acid;
(3) sample introduction: the sample solution after will filtering is by six-way valve or pump sample introduction, and sampling volume is 1～100ml;
(4) wash-out: adopt eluent system eluant solution chromatographic column, flow rate control is at 10～600ml/min, and online ultraviolet monitoring is collected the elutriant that contains salvianolic acid B;
(5) obtaining of salvianolic acid B chemical reference substance: the salvianolic acid B elutriant that concentrating under reduced pressure is collected, do not have methyl alcohol, lyophilize to concentrated solution.
The structural formula of salvianolic acid B is as follows:
Described preparation type high performance liquid phase post can be selected the preparation type high performance liquid phase post of commercialization or oneself filling for use; Its filler can be C8 or C18 bonded phase packings; The preparation type high performance liquid phase post type of feed of oneself loading is a wet method dress post, and packing material size is 5～20 μ m, and post is imitated more than 2000; Acid in the eluent system can be formic acid or acetate; Online ultraviolet monitoring, its monitoring wavelength is 254nm～290nm; After the salvianolic acid B elutriant was collected, concentrating under reduced pressure, thickening temperature were controlled at 55～65 ℃.
From Radix Salviae Miltiorrhizae extract, separate the salvianolic acid B chemical reference substance with present method and have following advantage and progress:
1. the present invention is a separation means with preparation type high performance liquid phase, makes salvianolic acid B obtain high efficiency separation, can obtain purity at the salvianolic acid B chemical reference substance more than 98%, shortens disengaging time simultaneously.
2. methyl alcohol of the present invention-sour water eluent system can guarantee effectively that salvianolic acid B is effectively separated with other compositions, thereby guarantees isolating effect.
3. the present invention adopts the online detection of UV-detector, and the separation case of salvianolic acid B can directly be detected, and can selective collection, significantly improves collecting effect.
4. the method for the invention is easy and simple to handle, and mild condition can effectively guarantee the stability of salvianolic acid B.
5. but industry is promoted.When the chromatographic column specification was 50 * 250mm, its scale can reach more than the 10 gram/moons.
Description of drawings
Fig. 1 is the purity check color atlas of the salvianolic acid B chemical reference substance of the embodiment of the invention one preparation.
Embodiment
Below by embodiment and accompanying drawing the present invention is described in further details.
In the present embodiment, the salvianolic acid B extract with 10% is a raw material, is sorbent material with 5 μ mC18 bonded phase packings, is eluting solvent with water (45:55, the volume ratio) solution that contains methyl alcohol-0.1% formic acid, and concrete processing step is as follows:
1. sample pretreatment
Salvianolic acid B extract water dissolution, being configured to concentration is the salvianolic acid B solution of 60mg/ml, through 0.22 μ m filtering with microporous membrane.
2. chromatographic column is handled
Preparation type high performance liquid phase post be chosen as the preparative column that stationary phase that Japanese Sheshido company produces is C18 (5 μ m, 20 * 250mm), with the eluent system solution equilibria chromatographic column of about 5 times of column volumes.
3. go up sample
By the six-way valve sample introduction, sampling volume is 1ml with the sample solution after filtering.
4. wash-out
Flow rate control is at 10ml/min, and online 254nm ultraviolet detection is collected the elutriant that contains salvianolic acid B,
5. the salvianolic acid B chemical reference substance obtains
Concentrating under reduced pressure salvianolic acid B elutriant does not have methyl alcohol to concentrated solution, thickening temperature is controlled at 65 ℃, lyophilize.Obtain purity and be 98.19% salvianolic acid B chemical reference substance; As shown in Figure 1, its analytical results is as shown in table 1:
Table 1
| The peak | Title | Retention time | The A peak | Area percentage | |
| 1 | ? | 3.418 | 5823 | 0.05 | |
| 2 | ? | 5.691 | 4357 | 0.04 | |
| 3 | ? | 7.052 | 2370 | 0.02 | |
| 4 | ? | 7.343 | 107430 | 0.90 | |
| 5 | Salvianolic acid B | 8.425 | 11742656 | 98.19 | |
| 6 | ? | 14.946 | 96478 | 0.81 |
In the present embodiment, being raw material with 50% red sage root acid B extract, is sorbent material with the C18 bonded phase packings of 10 μ m, is eluting solvent with water (50:50, the volume ratio) solution that contains methyl alcohol-1% acetate, and concrete processing step is as follows:
1. sample pretreatment
Salvianolic acid B extract 50% dissolve with methanol, being configured to concentration is the salvianolic acid B solution of 10mg/ml, through 0.45 μ m filtering with microporous membrane.
2. chromatographic column is handled
Preparation type high performance liquid phase post is the preparative column that oneself loads, and filler is that particle diameter is the C18 bonded phase packings of 5 μ m, wet method dress post, and chemical reference substance chromatographic column specification is 50 * 200mm, with the eluent system solution equilibria chromatographic column of about 10 times of column volumes.
3. go up sample
By the six-way valve sample introduction, sampling volume is 8ml with the sample solution after filtering.
4. wash-out
Flow rate control is at 90ml/min, and online 254nm ultraviolet detection is collected the elutriant that contains salvianolic acid B,
5. the salvianolic acid B chemical reference substance obtains
Concentrating under reduced pressure salvianolic acid B elutriant does not have methyl alcohol to concentrated solution, thickening temperature is controlled at 55 ℃, lyophilize.Obtain purity and be 98.51% salvianolic acid B chemical reference substance.
Embodiment 3
In the present embodiment, the salvianolic acid B extract with 60% is a raw material, is sorbent material with the C18 bonded phase packings of 10 μ m, is eluting solvent with water (45:55, the volume ratio) solution that contains methyl alcohol-0.5% formic acid, and concrete processing step is as follows:
1. sample pretreatment
Salvianolic acid B extract dissolve with ethanol, being configured to concentration is the salvianolic acid B solution of 20mg/ml, through 0.45 μ m filtering with microporous membrane.
2. chromatographic column is handled
Preparation type high performance liquid phase post is the preparative column that oneself loads, and filler is that particle diameter is the C18 bonded phase packings of 10 μ m, and the chromatographic column specification is 75 * 250mm, with the eluent system solution equilibria chromatographic column of about 10 times of column volumes.
3. go up sample
By the six-way valve sample introduction, sampling volume is 15ml with the sample solution after filtering.
4. wash-out
Flow rate control is at 150ml/min, and online 254nm ultraviolet detection is collected the elutriant that contains salvianolic acid B,
5. the salvianolic acid B chemical reference substance obtains
Concentrating under reduced pressure salvianolic acid B elutriant does not have methyl alcohol to concentrated solution, thickening temperature is controlled at 65 ℃, lyophilize.Obtain purity and be 98.74% salvianolic acid B chemical reference substance.
Embodiment 4
In the present embodiment, the salvianolic acid B extract with 60% is a raw material, is sorbent material with 20 μ mC18 bonded phase packings, and water (45:55, the volume ratio) solution that contains methyl alcohol-1% acetate is eluting solvent, and concrete processing step is as follows:
1. sample pretreatment
Salvianolic acid B extract water dissolution, being configured to concentration is the salvianolic acid B solution of 8mg/ml, through 0.45 μ m filtering with microporous membrane.
2. chromatographic column is handled
Preparation type high performance liquid phase post is the preparative column that oneself loads, and filler is that particle diameter is the C18 bonded phase packings of 20 μ m, and the chromatographic column specification is 100 * 270mm, with the eluent system solution equilibria chromatographic column of about 10 times of column volumes.
3. go up sample
By the six-way valve sample introduction, sampling volume is 20ml with the sample solution after filtering.
4. wash-out
Flow rate control is at 300ml/min, and online 290nm ultraviolet detection is collected the elutriant that contains salvianolic acid B,
5. the salvianolic acid B chemical reference substance obtains
Concentrating under reduced pressure salvianolic acid B elutriant does not have methyl alcohol to concentrated solution, thickening temperature is controlled at 65 ℃, lyophilize.Obtain purity and be 98.15% salvianolic acid B chemical reference substance.
In the present embodiment, being raw material with 97% red sage root acid B extract, is sorbent material with the C18 bonded phase packings of 20 μ m, is eluting solvent with water (60:40, the volume ratio) solution that contains methyl alcohol-1% acetate, and concrete processing step is as follows:
1. sample pretreatment
Salvianolic acid B extract water dissolution, being configured to concentration is the salvianolic acid B solution of 20mg/ml, through 0.45 μ m filtering with microporous membrane.
2. chromatographic column is handled
Preparation type high performance liquid phase post is the preparative column that oneself loads, and filler is that particle diameter is the C18 bonded phase packings of 10 μ m, wet method dress post, and the chromatographic column specification is 150 * 250mm, with the eluent system solution equilibria chromatographic column of about 10 times of column volumes.
3. go up sample
By the six-way valve sample introduction, sampling volume is 100ml with the sample solution after filtering.
4. wash-out
Flow rate control is at 600ml/min, and online 254nm ultraviolet detection is collected the elutriant that contains salvianolic acid B,
5. the salvianolic acid B chemical reference substance obtains
Concentrating under reduced pressure salvianolic acid B elutriant is not to there being methyl alcohol, and thickening temperature is controlled at 55～65 ℃, lyophilize.Obtain purity and be 98.95% salvianolic acid B chemical reference substance.
In the present embodiment, being raw material with 80% red sage root acid B extract, is sorbent material with the C18 bonded phase packings of 20 μ m, is eluting solvent with water (60:40, the volume ratio) solution that contains methyl alcohol-1% acetate, and concrete processing step is as follows:
1. sample pretreatment
Salvianolic acid B extract water dissolution, being configured to concentration is the salvianolic acid B solution of 20mg/ml, through 0.45 μ m filtering with microporous membrane.
2. chromatographic column is handled
Preparation type high performance liquid phase post is the preparative column that oneself loads, and filler is that particle diameter is the C18 bonded phase packings of 10 μ m, wet method dress post, and the chromatographic column specification is 150 * 250mm, with the eluent system solution equilibria chromatographic column of about 10 times of column volumes.
3. go up sample
By the six-way valve sample introduction, sampling volume is 100ml with the sample solution after filtering.
4. wash-out
Flow rate control is at 600ml/min, and online 254nm ultraviolet detection is collected the elutriant that contains salvianolic acid B,
5. the salvianolic acid B chemical reference substance obtains
Concentrating under reduced pressure salvianolic acid B elutriant is not to there being methyl alcohol, and thickening temperature is controlled at 55～65 ℃, lyophilize.Obtain purity and be 98.95% salvianolic acid B chemical reference substance.
Claims (8)
1. the method for separating and preparing of a salvianolic acid B chemical reference substance, it is characterized in that: the salvianolic acid B extract with weight content 10～97% is a raw material, is separation means with the preparative high performance liquid chromatography, is eluent system with methyl alcohol-aqueous acid, concrete steps are,
(1) sample pretreatment: with salvianolic acid B extract methyl alcohol, ethanol, water or 50% dissolve with methanol solution of weight content 10～97%, being configured to weight concentration is the salvianolic acid B extract solution of 8～60mg/ml, through 0.22～0.45 μ m filtering with microporous membrane;
(2) chromatographic column is handled: performance liquid chromatographic column is selected the preparative high performance liquid chromatography post, with the eluent system solution equilibria chromatographic column of 5-20 times of column volumes; Eluent system solution is methyl alcohol-aqueous acid, and wherein the volumetric concentration of methyl alcohol in methyl alcohol-aqueous acid is 50～80%; Acid is 0.1～1% in the volumetric concentration of aqueous acid;
(3) sample introduction: the sample solution sample introduction after will filtering, sampling volume are 1～100ml;
(4) wash-out: adopt eluent system eluant solution chromatographic column, flow rate control is at 10～600ml/min, and online ultraviolet monitoring is collected the elutriant that contains salvianolic acid B;
(5) obtaining of salvianolic acid B chemical reference substance: the collected salvianolic acid B elutriant of concentrating under reduced pressure is to not having methyl alcohol, lyophilize.
2. according to the method for separating and preparing of the described salvianolic acid B chemical reference substance of claim 1, it is characterized in that: described preparation type high performance liquid phase post can be selected the preparation type high performance liquid phase post of commercialization or oneself filling for use; Its filler can be C8 or C18 bonded phase packings.
3. according to the method for separating and preparing of the described salvianolic acid B chemical reference substance of claim 2, it is characterized in that: described preparation type high performance liquid phase post type of feed of oneself loading is a wet method dress post.
4. according to the method for separating and preparing of the described salvianolic acid B chemical reference substance of claim 3, it is characterized in that: the post of described preparation type high performance liquid phase post is imitated more than 2000.
5. according to the method for separating and preparing of the described salvianolic acid B chemical reference substance of claim 2, it is characterized in that: the packing material size of described preparation type high performance liquid phase post is 5～20 μ m.
6. according to the method for separating and preparing of the described salvianolic acid B chemical reference substance of claim 1, it is characterized in that: the acid in the described eluent system can be formic acid or acetate.
7. according to the method for separating and preparing of the described salvianolic acid B chemical reference substance of claim 1, it is characterized in that: described online ultraviolet monitoring, its monitoring wavelength is 254nm～290nm.
8. according to the method for separating and preparing of the described salvianolic acid B chemical reference substance of claim 1, it is characterized in that: after described salvianolic acid B elutriant was collected, concentrating under reduced pressure, thickening temperature were controlled at 55～65 ℃.
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| CN101638404B (en) * | 2008-07-29 | 2012-06-06 | 王宇 | High-purity salvianolic acid B and preparation method and application thereof |
Citations (5)
| 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 |
| CN1459448A (en) * | 2002-05-23 | 2003-12-03 | 天津天士力制药股份有限公司 | Preparation method of red sageroot total phenolic acid and its use |
| CN1528756A (en) * | 2003-09-29 | 2004-09-15 | 华东理工大学 | Technical method for extracting salvianolic acid B from radix salivae miltiorrhizae |
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Patent Citations (5)
| 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 |
| CN1459448A (en) * | 2002-05-23 | 2003-12-03 | 天津天士力制药股份有限公司 | Preparation method of red sageroot total phenolic acid and its 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 |
| CN1528756A (en) * | 2003-09-29 | 2004-09-15 | 华东理工大学 | Technical method for extracting salvianolic acid B from radix salivae miltiorrhizae |
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