CN104297370A - Method for identifying and analyzing pure snake protein powder - Google Patents
Method for identifying and analyzing pure snake protein powder Download PDFInfo
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- CN104297370A CN104297370A CN201410510067.XA CN201410510067A CN104297370A CN 104297370 A CN104297370 A CN 104297370A CN 201410510067 A CN201410510067 A CN 201410510067A CN 104297370 A CN104297370 A CN 104297370A
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Abstract
The invention discloses a method for identifying and analyzing pure snake protein powder. The method is characterized in that by comprising the following steps of extracting protein in a pure snake protein powder sample, preparing liquid to be subjected to liquid chromatography, carrying out liquid chromatography determination, comparing with standard protein fingerprint spectrums of various pure snake protein powder samples under the same chromatographic condition, and judging the variety of the pure protein powder according to the characteristic of the chromatographic peak retention time. The method adopts the liquid chromatography determination to rapidly and conveniently distinguish the variety of the pure protein powder and has the advantages of high stability and good reproducibility.
Description
Technical field
The present invention relates to Chemistry for Chinese Traditional Medicine analytical approach, particularly a kind of identification and analysis method of pure snake powder.
Background technology
Propagating artificially of snake class was developed rapidly in recent years, and breed variety and quantity all reach a certain scale, and general as duck is supported in poultry on Guangxi and other places, this makes the management and use of this wild animal of snake class more legal.Can the snake veriety of breeding scale various, wherein zaocys dhumnade, pallas pit viper etc. have medicine-food two-purpose function, in containing the abundant protein be made up of several amino acids, the snake powder adopting these snake kinds to make or particle be have relax through dredging collateral, supplement human nutrition, improve blood circulation, immunity moderation function, activation histocyte, a functional health-care food that enhances metabolism.Based on this, the demand of snake powder or snake particle and output are also increasing.
The expansion of the snake powder market demand, causes increasing personation snake powder or shoddy snake powder to circulate extensively on the market.And traditional snake powder authentication method whether just debate outward appearance, smell etc. to distinguish by simple naked eyes be snake meal component.The powder of dry grinding as the snake of 80% aridity is just with dried meat floss; The snake of the 100% drying powder of dry grinding out 90% be powder and 10% is wherein muscle, shredded meat.Snake powder color is canescence, and smell and have kind of snake special fishy smell with it, its taste is different from other digested tankages and fish meal.It is water-fast for steeping in water 60%, the particulate material that water-bed portion adularescent powder (snake bone) is hard.Although said method can identify snake powder to a certain extent, absolutely can not determine that qualification snake powder result is correct.If businessman is for reaping staggering profits, utilize common snake kind snake powder to replace the high expensive snake kind snake powder of medical value to adulterate, said method cannot pick out the snake powder that specifically that snake is made at all, also just cannot distinguish the quality of snake powder.Based on the standard of perfection and the method that also do not have pure snake powder kind in the market, so urgently formulate a kind of scientific and effective pure snake powder kind discrimination method.
Summary of the invention
For solving the problems of the technologies described above, the invention provides a kind of identification and analysis method of pure snake powder, the method utilizes the differentiation pure snake powder kind that liquid chromatogram measuring analysis can be fast and convenient, and stability is high, favorable reproducibility.
In order to realize above object, the technical solution used in the present invention is: a kind of identification and analysis method of pure snake powder, it is characterized in that, extract the protein preparation liquid-phase chromatographic analysis liquid to be measured in pure snake powder sample, and carry out liquid chromatogram measuring and analysis, contrast with the standard protein finger-print of pure snake powder sample various under existing identical chromatographic conditions, according to the kind of the pure snake powder of the feature decision of chromatographic peak retention time, described snake powder is snake powder or snake particle.
Wherein in an embodiment, pure snake powder is diluted to concentration for 10-100mg/mL for utilizing pure water by the concrete grammar that described preparative liquid chromatography analyzes liquid to be measured, 50-60 DEG C of heating water bath 25-35min, 5000 turns/min low-temperature centrifugation 10min, get supernatant, cross 0.22 μm of filter membrane, obtain liquid-phase chromatographic analysis liquid to be measured.
Wherein in an embodiment, the concrete grammar of described liquid chromatogram measuring for getting liquid-phase chromatographic analysis liquid 20 to be measured μ L injection liquid chromatography, according to high effective liquid chromatography for measuring, record chromatogram, at least replicate determination 3 times of each sample.
Under better performance, the chromatographic condition of described liquid chromatogram measuring is: permaphase ODS C18 4.6mm × 250mm; With 70% acetonitrile solution of 0.1% trifluoroacetic acid aqueous solution+0.1% trifluoroacetic acid for mobile phase, flow velocity 0.6mL/min, through 60min complex gradient from 0 to 100%B; Detecting device is diode array detector or UV-detector; Determined wavelength 280nm.
Beneficial effect of the present invention is: in the identification and analysis method of a kind of pure snake powder provided by the invention, by extracting the protein preparation liquid-phase chromatographic analysis liquid to be measured in pure snake powder sample, and carry out liquid chromatogram measuring and analysis, contrast, according to the kind determining pure snake powder that the feature of chromatographic peak retention time can be fast and convenient with the standard protein finger-print of pure snake powder sample various under existing identical chromatographic conditions.The method reappearance, good stability, method of operating is easy, and precision is high.
Accompanying drawing explanation
Fig. 1 is the standard HPLC finger-print of zaocys dhumnade powder.
Fig. 2 is the standard HPLC finger-print of zaocys dhumnade particle.
Fig. 3 is the standard HPLC finger-print of agkistrodon acutus powder.
Fig. 4 is the standard HPLC finger-print of agkistrodon acutus particle.
Fig. 5 is that embodiment 1 measures the testing sample chromatogram obtained.
Fig. 6 is that embodiment 2 measures the testing sample chromatogram obtained.
Embodiment
For enabling above-mentioned purpose of the present invention, feature and advantage become apparent more, elaborate to the specific embodiment of the present invention below in conjunction with accompanying drawing.Set forth a lot of detail in the following description so that fully understand the present invention.But the present invention can implement to be much different from other modes described here, those skilled in the art can when without prejudice to doing similar improvement when intension of the present invention, therefore the present invention is by the restriction of following public concrete enforcement.
A kind of identification and analysis method of pure snake powder, by extracting the protein preparation liquid-phase chromatographic analysis liquid to be measured in pure snake powder sample, and carry out liquid chromatogram measuring and analysis, contrast with the standard protein finger-print of pure snake powder sample various under existing identical chromatographic conditions, according to the kind of the pure snake powder of the feature decision of chromatographic peak retention time, described snake powder is snake powder or snake particle.
Wherein, pure snake powder is diluted to concentration for 10-100mg/mL, 50-60 DEG C of heating water bath 25-35min for utilizing pure water by the concrete grammar that preparative liquid chromatography analyzes liquid to be measured, 5000 turns/min low-temperature centrifugation 10min, get supernatant, cross 0.22 μm of filter membrane, obtain liquid-phase chromatographic analysis liquid to be measured.
Preferably, pure snake powder is diluted to concentration for 60mg/mL, 55 DEG C of heating water bath 30min for utilizing pure water by the concrete grammar that preparative liquid chromatography analyzes liquid to be measured, 5000 turns/min low-temperature centrifugation 10min, get supernatant, cross 0.22 μm of filter membrane, obtain liquid-phase chromatographic analysis liquid to be measured.
The concrete grammar of above-mentioned liquid chromatogram measuring for getting liquid-phase chromatographic analysis liquid 20 to be measured μ L injection liquid chromatography, according to high effective liquid chromatography for measuring, record chromatogram, at least replicate determination 3 times of each sample.
Preferably, during liquid chromatogram measuring, each liquid sample parallel to be measured measures 3-6 time.
During above-mentioned liquid chromatogram measuring, preferred chromatographic condition is: permaphase ODS C18 4.6mm × 250mm; With 70% acetonitrile solution of 0.1% trifluoroacetic acid aqueous solution+0.1% trifluoroacetic acid for mobile phase, flow velocity 0.6mL/min, through 60min complex gradient from 0 to 100%B; Detecting device is diode array detector or UV-detector; Determined wavelength 280nm.
In the identification and analysis method of above-mentioned pure snake powder, by extracting the protein preparation liquid-phase chromatographic analysis liquid to be measured in pure snake powder sample, and carry out liquid chromatogram measuring and analysis, contrast, according to the kind determining pure snake powder that the feature of chromatographic peak retention time can be fast and convenient with the standard protein finger-print of pure snake powder sample various under existing identical chromatographic conditions.The method reappearance, good stability, method of operating is easy, and precision is high.
It is below specific embodiment.
Embodiment 1:
A kind of identification and analysis method of pure snake powder: 1) extract the protein preparation liquid-phase chromatographic analysis liquid to be measured in pure snake powder sample: utilize pure water that pure snake powder is diluted to concentration for 10mg/mL, 50 DEG C of heating water bath 25min, 5000 turns/min low-temperature centrifugation 10min, get supernatant, cross 0.22 μm of filter membrane, obtain liquid-phase chromatographic analysis liquid to be measured.
2) liquid chromatogram measuring and analysis is carried out: chromatographic condition is permaphase ODS C18 4.6mm × 250mm; With 70% acetonitrile solution of 0.1% trifluoroacetic acid aqueous solution+0.1% trifluoroacetic acid for mobile phase, flow velocity 0.6mL/min, through 60min complex gradient from 0 to 100%B; Detecting device is diode array detector or UV-detector; Determined wavelength 280nm.Get liquid-phase chromatographic analysis liquid 20 to be measured μ L injection liquid chromatography, according to high effective liquid chromatography for measuring, record chromatogram, each sample parallel measures 6 times, contrast, according to the kind of the pure snake powder of the feature decision of chromatographic peak retention time with the standard protein finger-print of pure snake powder sample various under existing identical chromatographic conditions.
This embodiment measures the testing sample chromatogram that obtains as shown in Figure 5, be analyzed from the zaocys dhumnade powder (particle) shown in size three aspect of appearance time, chromatographic peak, peak width and known Fig. 1-Fig. 4, agkistrodon acutus powder (particle) standard HPLC finger-print, determine that testing sample is agkistrodon acutus particle.
Embodiment 2:
A kind of identification and analysis method of pure snake powder: 1) extract the protein preparation liquid-phase chromatographic analysis liquid to be measured in pure snake powder sample: utilize pure water that pure snake powder is diluted to concentration for 100mg/mL, 60 DEG C of heating water bath 35min, 5000 turns/min low-temperature centrifugation 10min, get supernatant, cross 0.22 μm of filter membrane, obtain liquid-phase chromatographic analysis liquid to be measured.
2) liquid chromatogram measuring and analysis is carried out: chromatographic condition is permaphase ODS C18 4.6mm × 250mm; With 70% acetonitrile solution of 0.1% trifluoroacetic acid aqueous solution+0.1% trifluoroacetic acid for mobile phase, flow velocity 0.6mL/min, through 60min complex gradient from 0 to 100%B; Detecting device is diode array detector or UV-detector; Determined wavelength 280nm.Get liquid-phase chromatographic analysis liquid 20 to be measured μ L injection liquid chromatography, according to high effective liquid chromatography for measuring, record chromatogram, each sample parallel measures 3 times, contrast, according to the kind of the pure snake powder of the feature decision of chromatographic peak retention time with the standard protein finger-print of pure snake powder sample various under existing identical chromatographic conditions.
This embodiment measures the testing sample chromatogram that obtains as shown in Figure 6, be analyzed from the zaocys dhumnade powder (particle) shown in size three aspect of appearance time, chromatographic peak, peak width and known Fig. 1-Fig. 4, agkistrodon acutus powder (particle) standard HPLC finger-print, determine that testing sample is zaocys dhumnade powder.
Embodiment 3:
A kind of identification and analysis method of pure snake powder: 1) extract the protein preparation liquid-phase chromatographic analysis liquid to be measured in pure snake powder sample: utilize pure water that pure snake powder is diluted to concentration for 60mg/mL, 55 DEG C of heating water bath 30min, 5000 turns/min low-temperature centrifugation 10min, get supernatant, cross 0.22 μm of filter membrane, obtain liquid-phase chromatographic analysis liquid to be measured.
2) liquid chromatogram measuring and analysis is carried out: chromatographic condition is permaphase ODS C18 4.6mm × 250mm; With 70% acetonitrile solution of 0.1% trifluoroacetic acid aqueous solution+0.1% trifluoroacetic acid for mobile phase, flow velocity 0.6mL/min, through 60min complex gradient from 0 to 100%B; Detecting device is diode array detector or UV-detector; Determined wavelength 280nm.Get liquid-phase chromatographic analysis liquid 20 to be measured μ L injection liquid chromatography, according to high effective liquid chromatography for measuring, record chromatogram, each sample parallel measures 5 times, contrast, according to the kind of the pure snake powder of the feature decision of chromatographic peak retention time with the standard protein finger-print of pure snake powder sample various under existing identical chromatographic conditions.
Contrast being measured the snake amyloid proteins matter chromatogram obtained by above embodiment, according to the kind of the pure snake powder of the feature decision of chromatographic peak retention time with the standard protein finger-print of pure snake powder sample various under existing identical chromatographic conditions.Accompanying drawing 1-4 citing sets forth the standard protein HPLC finger-print of zaocys dhumnade powder, zaocys dhumnade particle, agkistrodon acutus powder, agkistrodon acutus powder particles.
The above embodiment only have expressed several embodiment of the present invention, and it describes comparatively in detail concrete, but therefore can not be interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that to those skilled in the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (4)
1. the identification and analysis method of a pure snake powder, it is characterized in that, extract the protein preparation liquid-phase chromatographic analysis liquid to be measured in pure snake powder sample, and carry out liquid chromatogram measuring and analysis, contrast with the standard protein finger-print of pure snake powder sample various under existing identical chromatographic conditions, according to the kind of the pure snake powder of the feature decision of chromatographic peak retention time, described snake powder is snake powder or snake particle.
2. the identification and analysis method of a kind of pure snake powder according to claim 1, it is characterized in that, pure snake powder is diluted to concentration for 10-100mg/mL for utilizing pure water by the concrete grammar that described preparative liquid chromatography analyzes liquid to be measured, 50-60 DEG C of heating water bath 25-35min, 5000 turns/min low-temperature centrifugation 10min, get supernatant, cross 0.22 μm of filter membrane, obtain liquid-phase chromatographic analysis liquid to be measured.
3. the identification and analysis method of a kind of pure snake powder according to claim 2, it is characterized in that, the concrete grammar of described liquid chromatogram measuring is for getting liquid-phase chromatographic analysis liquid 20 to be measured μ L injection liquid chromatography, according to high effective liquid chromatography for measuring, record chromatogram, at least replicate determination 3 times of each sample.
4. the identification and analysis method of any one the pure snake powder according to claim 1-3, is characterized in that, the chromatographic condition of described liquid chromatogram measuring is: permaphase ODS C18 4.6mm × 250mm; With 70% acetonitrile solution of 0.1% trifluoroacetic acid aqueous solution+0.1% trifluoroacetic acid for mobile phase, flow velocity 0.6mL/min, through 60min complex gradient from 0 to 100%B; Detecting device is diode array detector or UV-detector; Determined wavelength 280nm.
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Citations (3)
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| CN1509758A (en) * | 2002-12-23 | 2004-07-07 | 江苏康缘药业股份有限公司 | Chinese medicinal composition for treating lung cancer, preparing method and quality controlling method thereof |
| CN101539555A (en) * | 2008-03-18 | 2009-09-23 | 上海医药工业研究院 | Method for establishing snake poison fingerprint maps and fingerprint maps thereof |
| US20130171263A1 (en) * | 2006-01-20 | 2013-07-04 | Pedro J. Lecca | Snake Powder Extract For Treatment Of Cancer |
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2014
- 2014-09-29 CN CN201410510067.XA patent/CN104297370B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1509758A (en) * | 2002-12-23 | 2004-07-07 | 江苏康缘药业股份有限公司 | Chinese medicinal composition for treating lung cancer, preparing method and quality controlling method thereof |
| US20130171263A1 (en) * | 2006-01-20 | 2013-07-04 | Pedro J. Lecca | Snake Powder Extract For Treatment Of Cancer |
| CN101539555A (en) * | 2008-03-18 | 2009-09-23 | 上海医药工业研究院 | Method for establishing snake poison fingerprint maps and fingerprint maps thereof |
Non-Patent Citations (5)
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| BRYAN G. FRY 等: "Analysis of Colubroidea snake venoms by liquid chromatography with mass spectrometry: evolutionary and toxinological implications", 《RAPID COMMUNICATIONS IN MASS SPECTROMETRY》 * |
| 任学聪 等: "PICO•TAG法测定王锦蛇中不同部位游离氨基酸含量", 《中国现代应用药学》 * |
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| C41 | Transfer of patent application or patent right or utility model | ||
| CB03 | Change of inventor or designer information |
Inventor after: Guo Dezhi Inventor after: Guo Degang Inventor after: Hu Mingxing Inventor after: Bai Le Inventor before: Guo Dezhi Inventor before: Guo Degang |
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Effective date of registration: 20160311 Address after: 425006, Yongzhou, Hunan, Lingling snake villa (snake world) Applicant after: Yongzhou City Strange Snake Science & Technology Industrial Co., Ltd. Address before: 425006, Yongzhou, Hunan, Lingling snake villa (snake world) Applicant before: Guo Dezhi |
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