CN101708234A - Finger print quality detecting method by intermediates of schisandra chinensis and cordycepin powder for strengthening body resistance and dissolving stasis - Google Patents
Finger print quality detecting method by intermediates of schisandra chinensis and cordycepin powder for strengthening body resistance and dissolving stasis Download PDFInfo
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- CN101708234A CN101708234A CN200910201482A CN200910201482A CN101708234A CN 101708234 A CN101708234 A CN 101708234A CN 200910201482 A CN200910201482 A CN 200910201482A CN 200910201482 A CN200910201482 A CN 200910201482A CN 101708234 A CN101708234 A CN 101708234A
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Abstract
The invention relates to a finger print quality detecting method by intermediates of schisandra chinensis and cordycepin powder for strengthening body resistance and dissolving stasis. The method comprises the following steps of: carrying out mobile phase gradient elution on prepared testing intermediate products of schisandra chinensis and cordycepin powder respectively under 254nm and 261nm; selecting 10 different batches of intermediates of schisandra chinensis and cordycepin powder for strengthening body resistance and dissolving stasis for carrying out high-efficiency liquid chromatogram finger print analysis according to the two detecting methods; obtaining two finger prints under different wavelengths in each batch of drugs so as to determine 8 common peaks; and respectively comparing a sample HPLC finger print with standard finger prints under 254nm and 261nm, wherein a similarity obtained by calculating the 8 common peaks cannot be less than 0.8. The detecting method can comprehensively control the quality of the intermediates of schisandra chinensis and cordycepin powder for strengthening body resistance and dissolving stasis so as to guarantee the stable quality of the products.
Description
Technical field
The invention belongs to Fructus Schisandrae Chinensis, fermented Cordyceps powder intermediate quality testing field, particularly relate to a kind of supporting vital QI and dispersing blood stasis Fructus Schisandrae Chinensis, fermented Cordyceps powder intermediate finger print quality detecting method.
Background technology
Medicine compositions for strengthening healthy qi to remove blood stasis is made up of Radix Salviae Miltiorrhizae, fermented Cordyceps powder, Semen Persicae, Pollen Pini, Herb Gynostemmae Pentaphylli, Fructus Schisandrae Chinensis Six-element medicine.Go up two kinds of dosage forms of municipalization at present: (1) FUZHENG HUAYU JIAONANG: obtained China national New Drug Certificate (traditional Chinese medicines card word Z20020053) in 2002, and approval national drug standards WS
3-459 (Z-79)-2005 (Z); (2) supporting vital QI and dispersing blood stasis sheet: obtained national New Drug Certificate (traditional Chinese medicines card word Z20050564) in 2005, and approval national drug standards YBZ19332005-2009Z.Supporting vital QI and dispersing blood stasis has effects such as soothing liver-QI is invigorated blood circulation, beneficial intensive culture liver, dissolving blood stasis and detoxication, hard masses softening and resolving, enhancing human body immunity function, cures mainly chronic hepatitis Bhepatic fibrosis, early stage liver cirrhosis.
The effective ingredient nature difference of considering supporting vital QI and dispersing blood stasis preparation Six-element medical material is bigger, therefore selects different extracting method for use at different flavour of a drug in process of production.Wherein Fructus Schisandrae Chinensis and fermented Cordyceps powder adopt alcohol extraction process, specific as follows: with about 1g: the 2g ratio, get Fructus Schisandrae Chinensis and fermented Cordyceps powder is an amount of, extract 2 times with 70% alcohol heating reflux, 1.5h for the first time, 1h for the second time, merge extractive liquid,, be concentrated into relative density 1.1-1.2 (50 ℃-55 ℃), get the alcohol extraction intermediate.According to this extraction process, mainly contain Fructus Schisandrae Chinensis lignin composition in the alcohol extraction intermediate, and the ucleosides composition in the fermented Cordyceps powder.
In recent years, fingerprint pattern technology has become the important means of Chinese medicine compound quality control and evaluation, consults the document of delivering both at home and abroad, does not up to the present have the report of supporting vital QI and dispersing blood stasis preparation alcohol extraction intermediate fingerprint map analyzing.Therefore the present invention will remedy the blank of supporting vital QI and dispersing blood stasis preparation alcohol extraction intermediate finger printing research.
Summary of the invention
Technical problem to be solved by this invention provides a kind of supporting vital QI and dispersing blood stasis Fructus Schisandrae Chinensis, fermented Cordyceps powder intermediate finger print quality detecting method, this method can be controlled the quality of supporting vital QI and dispersing blood stasis Fructus Schisandrae Chinensis, fermented Cordyceps powder alcohol extraction intermediate comprehensively, to guarantee the steady quality of product.
A kind of supporting vital QI and dispersing blood stasis Fructus Schisandrae Chinensis of the present invention, fermented Cordyceps powder intermediate finger print quality detecting method comprise:
(1) detection method A
(1) Fructus Schisandrae Chinensis, the preparation of fermented Cordyceps powder alcohol extraction intermediate test sample
Take by weighing Fructus Schisandrae Chinensis, fermented Cordyceps powder alcohol extraction intermediate 1.00 grams, put in the 10mL volumetric flask, add 50% methanol, 5～8ml, supersound process 20min, add 50% methanol to scale, shake up, cross the 0.45um microporous filter membrane, get the need testing solution of subsequent filtrate as Fructus Schisandrae Chinensis, fermented Cordyceps powder alcohol extraction intermediate;
(2) gradient elution mobile phase
With octadecylsilane chemically bonded silica is filler, and condition of gradient elution is:
| Time (min) | A phase: 0.05% phosphoric acid-water (%) | B phase: acetonitrile (%) |
| ??0 | ??95 | ??5 |
| ??60 | ??60 | ??40 |
| ??120 | ??10 | ??90 |
Chromatographic column C18 post, mobile phase A are 0.05% phosphoric acid-water mutually, and B is acetonitrile mutually; Gradient elution: A phase 0-60-120min, 95-60-10%, B phase 5-40-90%, flow velocity 0.8-1.2ml/min detects wavelength 254nm, and column temperature 20-40 ℃, sample size 10-20ul;
(2) detection method B
(1) Fructus Schisandrae Chinensis, the preparation of fermented Cordyceps powder alcohol extraction intermediate test sample
Take by weighing Fructus Schisandrae Chinensis, fermented Cordyceps powder alcohol extraction intermediate 0.50 gram, put in the 10mL volumetric flask, add water 5～8ml, supersound process 20min adds water to scale, shakes up, cross the 0.45um microporous filter membrane, get the need testing solution of subsequent filtrate as Fructus Schisandrae Chinensis, fermented Cordyceps powder alcohol extraction intermediate;
(2) gradient elution mobile phase
With octadecylsilane chemically bonded silica is filler, and condition of gradient elution is:
| Time (min) | A phase: water (%) | B phase: acetonitrile (%) |
| ??0 | ??100 | ??0 |
| ??30 | ??93 | ??7 |
Chromatographic column C18 post, mobile phase A are water mutually, and B is acetonitrile mutually; Gradient elution: A phase 0-30min, 100-93%, B phase 0-7%, flow velocity 0.8-1.2ml/min detects wavelength 261nm, and column temperature 20-40 ℃, sample size 10-20ul;
(3) foundation of standard finger-print
Choose medicine compositions for strengthening healthy qi to remove blood stasis Fructus Schisandrae Chinensis, the fermented Cordyceps powder alcohol extraction intermediate of 10 batches of different batches, carry out the efficient liquid-phase chromatograph finger print atlas analysis according to above-mentioned two kinds of detection methods, the medicine of each batch obtains the finger printing under 2 different wave lengths, all peaks in the HPLC finger printing are compared, determined 8 total peaks, and be numbered (Fig. 1) by appearance time precedence;
Wherein, among the finger printing a that under the 254nm wavelength, obtains according to detection method A, 4 total peaks (1～No. 4 peak) have been determined; Among the finger printing b that under the 261nm wavelength, obtains according to detection method B, 4 total peaks (5～No. 8 peaks) have been determined;
Through the qualitative evaluation of reference substance, confirm in the total peak, No. 2 peak schisandrins, No. 3 peaks are schisantherin B, and No. 4 peaks are deoxyschizandrin, and No. 6 peaks are uridnine, and No. 7 peaks are guanosine, No. 8 peaks are adenosine;
(4) quality control of finger printing
Under the 254nm wavelength, obtain finger printing a according to detection method A, adopt Chinese medicine fingerprint area of computer aided similarity evaluation software (Chinese Pharmacopoeia Commission), the HPLC finger printing and the standard finger-print (or common pattern finger printing) of sample are compared, should be with the similarity that 4 total peaks calculate less than 0.8;
Under the 261nm wavelength, obtain finger printing b according to detection method B, adopt Chinese medicine fingerprint area of computer aided similarity evaluation software (Chinese Pharmacopoeia Commission), the HPLC finger printing and the standard finger-print (or common pattern finger printing) of sample are compared, should be with the similarity that 4 total peaks calculate less than 0.8.
The present invention adopts two kinds of sample-pretreating methods and chromatographic condition, has set up 2 HPLC finger printing.Wherein finger printing 1 has been expressed the distribution of Fructus Schisandrae Chinensis lignin composition in the supporting vital QI and dispersing blood stasis preparation alcohol extraction intermediate emphatically, and finger printing 2 has been expressed the distribution of Cordyceps ucleosides composition in the supporting vital QI and dispersing blood stasis preparation alcohol extraction intermediate emphatically.By the optimization of chromatographic condition, chromatographic column, mobile phase kind, eluent gradient, detection wavelength, column temperature isochromatic spectrum condition have been determined.
Beneficial effect
1, uses stratographic fingerprint characteristic,, more effectively control the quality of product intermediate by the quantization parameter that finger printing obtains;
2, investigated the stability of need testing solution, the result shows, at room temperature, within 24 hours, medicine compositions for strengthening healthy qi to remove blood stasis Fructus Schisandrae Chinensis, fermented Cordyceps powder are carried the intermediate need testing solution and are kept stable, and precision and replica test show that all similarity is more than 0.8;
3, through the finger printing similarity of 10 batches of supporting vital QI and dispersing blood stasis preparation Fructus Schisandrae Chinensis, fermented Cordyceps powder alcohol extraction intermediate is calculated, the similarity of finding Fructus Schisandrae Chinensis, fermented Cordyceps powder alcohol extraction intermediate is between 0.8-1.0.Supporting vital QI and dispersing blood stasis preparation Fructus Schisandrae Chinensis, the fermented Cordyceps powder alcohol extraction intermediate finger printing quality control method set up are described, good reproducibility, specificity is strong, easy and simple to handle, method is reliable, can carry out science, estimate effectively supporting vital QI and dispersing blood stasis Fructus Schisandrae Chinensis, fermented Cordyceps powder alcohol extraction intermediate quality, and confirm to apply to quality control in the actual production, satisfy the demand of enterprise control of product quality.
4, provide condition for the supporting vital QI and dispersing blood stasis preparation carries out the finger printing quality control, also guaranteed the steady quality of supporting vital QI and dispersing blood stasis preparation simultaneously.
Description of drawings
Fig. 1 is supporting vital QI and dispersing blood stasis preparation Fructus Schisandrae Chinensis, fermented Cordyceps powder alcohol extraction intermediate standard HPLC finger printing;
Wherein, a is 254nm, and b is 261nm;
Wherein, No. 2 peak schisandrins, No. 3 peaks are schisantherin B, and No. 4 peaks are deoxyschizandrin, and No. 6 peaks are uridnine, and No. 7 peaks are guanosine, No. 8 peaks are adenosine;
Fig. 2 is supporting vital QI and dispersing blood stasis Fructus Schisandrae Chinensis, fermented Cordyceps powder alcohol extraction intermediate sample HPLC fingerprint image spectrogram;
Wherein, a is 254nm, and b is 261nm.
The specific embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after the content of having read the present invention's instruction, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
The preparation of finished product, reagent and instrument:
Finished product: supporting vital QI and dispersing blood stasis alcohol extraction intermediate source: Shanghai Huang Hai Pharmaceutical Co. Ltd
Extraction process: with about 1g: the 2g ratio, get Fructus Schisandrae Chinensis and fermented Cordyceps powder is an amount of, extract 2 times with 70% alcohol heating reflux, 1.5h for the first time, 1h for the second time, merge extractive liquid, is concentrated into relative density 1.1-1.2 (50 ℃-55 ℃), the alcohol extraction intermediate.According to this extraction process, mainly contain Fructus Schisandrae Chinensis lignin composition in the alcohol extraction intermediate, and the ucleosides composition in the fermented Cordyceps powder;
Reagent: methanol (analytical pure) source: Shanghai development chemical industry one factory;
Phosphoric acid (analytical pure) source: Shanghai amalgamation factory;
Acetonitrile (chromatographically pure) source: Tedia company Inc;
Water is pure water.
Standard substance: schisantherin A source: Chengdu Cisco China Bioisystech Co., Ltd;
Schisantherin B source: Chengdu Cisco China Bioisystech Co., Ltd;
Schisandrin source: Nat'l Pharmaceutical ﹠ Biological Products Control Institute;
The adenosine source: Chinese pharmaceutical biological product is identified institute;
The uridnine source: Chinese pharmaceutical biological product is identified institute;
Guanosine source: Xinxiang Tuoxin Biochemical Technology Co., Ltd..
Instrument
(1) detection method of finger printing (254nm)
Take by weighing Fructus Schisandrae Chinensis, fermented Cordyceps powder alcohol extraction intermediate 1.00 grams, put in the 10mL volumetric flask, add 50% methanol, 5～8ml, supersound process 20min, add 50% methanol to scale, shake up, cross the 0.45um microporous filter membrane, get the need testing solution of subsequent filtrate as Fructus Schisandrae Chinensis, fermented Cordyceps powder alcohol extraction intermediate;
Chromatographic condition:
Column temperature: 30 ℃, detect wavelength: 254nm, sample size: 10 μ l, chromatographic column: Alltima (250mm * 4.6mm, 5 μ m).Elution requirement sees Table 1,
The elution requirement of table 1 finger printing 1
| Time (min) | A phase: 0.05% phosphoric acid-water (%) | B phase: acetonitrile (%) |
| ??0 | ??95 | ??5 |
| ??60 | ??60 | ??40 |
| ??120 | ??10 | ??90 |
(2) detection method of finger printing (261nm)
Take by weighing Fructus Schisandrae Chinensis, fermented Cordyceps powder alcohol extraction intermediate 0.50 gram, put in the 10mL volumetric flask, add water 5～8ml, supersound process 20min adds water to scale, shakes up, cross the 0.45um microporous filter membrane, get the need testing solution of subsequent filtrate as Fructus Schisandrae Chinensis, fermented Cordyceps powder alcohol extraction intermediate;
Chromatographic condition:
Column temperature: 30 ℃, detect wavelength: 261nm, sample size: 10 μ l, chromatographic column: Alltima (250mm * 4.6mm, 5 μ m).Elution requirement sees Table 2,
The elution requirement of table 2 finger printing 2
| Time (min) | A phase: water (%) | B phase: acetonitrile (%) |
| ??0 | ??100 | ??0 |
| ??30 | ??93 | ??7 |
After finger printing 1 condition determination is determined, by at least three batch samples are measured, calculate relevant parameter, and propose the suitable parameters condition of highly effective liquid phase chromatographic system on this basis.The chromatographic peak area repeatability (RSD) that is schisandrin reference substance solution (100 μ g/ml) is not higher than 3%, and retention time repeatability (RSD) is not higher than 2%; The chromatographic column post is imitated and is no less than 15,000 theoretical cam curves in schisandrin.
After determining fingerprint pattern condition 2 is determined, by at least three batch samples are measured, calculate relevant parameter, and propose the suitable parameters condition of highly effective liquid phase chromatographic system on this basis.The chromatographic peak area repeatability (RSD) that is adenosine reference substance solution (100 μ g/ml) is not higher than 3%, and retention time repeatability (RSD) is not higher than 2%; The chromatographic column post is imitated and is no less than 15,000 theoretical cam curves in adenosine.
Medicine compositions for strengthening healthy qi to remove blood stasis Fructus Schisandrae Chinensis, fermented Cordyceps powder alcohol extraction intermediate sample fingerprint image spectrogram are seen Fig. 2.
Supporting vital QI and dispersing blood stasis alcohol extraction intermediate to 10 batches carries out fingerprint map analyzing, and each intermediate obtains 2 HPLC finger printing.Adopt Chinese medicine fingerprint area of computer aided similarity evaluation software (2004A version, Chinese Pharmacopoeia committee) respectively the similarity of 2 kinds of finger printing to be calculated.Adopt total peak pattern, the setting-up time window width is 0.1min, generate reference fingerprint with the average method, 2 HPLC finger printing and corresponding standard finger printing with sample compares respectively, the result shows, 2 finger printing of 10 batches of supporting vital QI and dispersing blood stasis alcohol extraction intermediate and the similarity of corresponding standard finger printing are all between 0.8-1.0.The supporting vital QI and dispersing blood stasis alcohol extraction intermediate finger printing quality control method of setting up is described, easy and simple to handle, method is reliable, and confirmation can apply to quality control in the actual production, satisfies the demand of enterprise to control of product quality.
Claims (1)
1. a supporting vital QI and dispersing blood stasis Fructus Schisandrae Chinensis, fermented Cordyceps powder intermediate finger print quality detecting method comprise:
(1) detection method A
(1) Fructus Schisandrae Chinensis, the preparation of fermented Cordyceps powder alcohol extraction intermediate test sample
Take by weighing Fructus Schisandrae Chinensis, fermented Cordyceps powder alcohol extraction intermediate 1.00 grams, put in the 10mL volumetric flask, add 50% methanol, 5～8ml, supersound process 20min, add 50% methanol to scale, shake up, cross the 0.45um microporous filter membrane, get the need testing solution of subsequent filtrate as Fructus Schisandrae Chinensis, fermented Cordyceps powder alcohol extraction intermediate;
(2) gradient elution mobile phase
With octadecylsilane chemically bonded silica is filler, and condition of gradient elution is: mobile phase A is 0.05% phosphoric acid-water mutually, and B is acetonitrile mutually; Gradient elution: A phase 0-60-120min, 95-60-10%, B phase 5-40-90%, flow velocity 0.8-1.2ml/min detects wavelength 254nm, and column temperature 20-40 ℃, sample size 10-20ul;
(2) detection method B
(1) Fructus Schisandrae Chinensis, the preparation of fermented Cordyceps powder alcohol extraction intermediate test sample
Take by weighing Fructus Schisandrae Chinensis, fermented Cordyceps powder alcohol extraction intermediate 0.50 gram, put in the 10mL volumetric flask, add water 5～8ml, supersound process 20min adds water to scale, shakes up, cross the 0.45um microporous filter membrane, get the need testing solution of subsequent filtrate as Fructus Schisandrae Chinensis, fermented Cordyceps powder alcohol extraction intermediate;
(2) gradient elution mobile phase
With octadecylsilane chemically bonded silica is filler, and condition of gradient elution is: mobile phase A is water mutually, and B is acetonitrile mutually; Gradient elution: A phase 0-30min, 100-93%, B phase 0-7%, flow velocity 0.8-1.2ml/min detects wavelength 261nm, and column temperature 20-40 ℃, sample size 10-20ul;
(3) foundation of standard finger-print
Choose medicine compositions for strengthening healthy qi to remove blood stasis Fructus Schisandrae Chinensis, the fermented Cordyceps powder alcohol extraction intermediate of 10 batches of different batches, carry out the efficient liquid-phase chromatograph finger print atlas analysis according to above-mentioned two kinds of detection methods, the medicine of each batch obtains the finger printing under 2 different wave lengths, all peaks in the HPLC finger printing are compared, determined 8 total peaks, and be numbered by appearance time precedence;
Wherein, among the finger printing a that under the 254nm wavelength, obtains according to detection method A, 4 total peaks (1～No. 4 peak) have been determined; Among the finger printing b that under the 261nm wavelength, obtains according to detection method B, 4 total peaks (5～No. 8 peaks) have been determined;
Confirm in the total peak, No. 2 peak schisandrins, No. 3 peaks are schisantherin B, and No. 4 peaks are deoxyschizandrin, and No. 6 peaks are uridnine, and No. 7 peaks are guanosine, No. 8 peaks are adenosine;
(4) quality control of finger printing
Under the 254nm wavelength, obtain finger printing a according to detection method A,, the HPLC finger printing and the standard finger-print of sample compared, should be with the similarity that 4 total peaks calculate less than 0.8;
Under the 261nm wavelength, obtain finger printing b according to detection method B,, the HPLC finger printing and the standard finger-print of sample compared, should be with the similarity that 4 total peaks calculate less than 0.8.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200910201482A CN101708234A (en) | 2009-12-18 | 2009-12-18 | Finger print quality detecting method by intermediates of schisandra chinensis and cordycepin powder for strengthening body resistance and dissolving stasis |
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|---|---|---|---|
| CN200910201482A CN101708234A (en) | 2009-12-18 | 2009-12-18 | Finger print quality detecting method by intermediates of schisandra chinensis and cordycepin powder for strengthening body resistance and dissolving stasis |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103484522A (en) * | 2012-06-13 | 2014-01-01 | 中国海洋大学 | Establishing method for cordyceps fungus fingerprints and standard fingerprints |
| CN113820411A (en) * | 2021-09-09 | 2021-12-21 | 上海现代中医药股份有限公司 | Method for measuring contents of schizandrol A and schizandrol B in preparation for strengthening body resistance and removing blood stasis |
-
2009
- 2009-12-18 CN CN200910201482A patent/CN101708234A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103484522A (en) * | 2012-06-13 | 2014-01-01 | 中国海洋大学 | Establishing method for cordyceps fungus fingerprints and standard fingerprints |
| CN103484522B (en) * | 2012-06-13 | 2016-08-17 | 中国海洋大学 | The method for building up of Cordyceps sinensis fungus finger printing and standard finger-print thereof |
| CN113820411A (en) * | 2021-09-09 | 2021-12-21 | 上海现代中医药股份有限公司 | Method for measuring contents of schizandrol A and schizandrol B in preparation for strengthening body resistance and removing blood stasis |
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Application publication date: 20100519 |