CN103630646A - Method for detecting tortoise-derived ingredients in glue type traditional Chinese medicines and products thereof - Google Patents
Method for detecting tortoise-derived ingredients in glue type traditional Chinese medicines and products thereof Download PDFInfo
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Abstract
The invention provides a method for detecting tortoise-derived ingredients in glue type traditional Chinese medicines and products thereof. The method provided by the invention comprises the step of detecting by using the difference of a specific nucleotide sequence or amino acid sequence in a genome of a tortoise, wherein the specific amino acid sequence is shown in SEQ ID No. 1. By adopting the method provided by the invention, whether the glue type traditional Chinese medicines contain the tortoise-derived ingredients or not can be detected rapidly, so as to distinguish the authenticity; although polypeptides of collagen in the glue type traditional Chinese medicines are subjected to certain hydrolysis and destruction, the tortoise-derived ingredients in the glue type traditional Chinese medicines and products thereof can be identified on the basis that differential polypeptides in a main ingredient, namely collagen, of the glue type traditional Chinese medicines are high in content and little in damaged extent.
Description
Technical field
The present invention relates to a kind of detection method of turtle-derived component, be specifically related to the detection method of turtle-derived component in a kind of glue class Chinese medicine and goods thereof, belong to Chinese medicine detection field.
Background technology
Tortoise plastron, carapace and the plastron of Testudinidae animal tortoise (Chinemys reevesii), belong to traditional rare Chinese medicine.Chinese Pharmacopoeia according to latest edition is recorded, and taking tortoise plastron can be nourishing and suppressing Yang, the strong bone of kidney-nourishing, the bushing of nourishing blood.In the last few years, along with the raising of biology level, the correlative study of tortoise plastron also deepened continuously.There are some researches show, tortoise plastron powder and extract thereof not only can strengthen liver function, release the pressure, and also have effect anticancer and that regulate Immunoresistance.The two series products Guiling Jellies that the tortoise plastron of take is made as raw material and colla carapacis et plastri testudinis on whole Market of Chinese Materia Medica in occupation of important position.
According to version Chinese Pharmacopoeia in 2010, record, colla carapacis et plastri testudinis is to take tortoise plastron as raw material, through decocting, boils, concentrates the solid gum of making, and property is salty, sweet, cool, returns liver, kidney, the heart channel of Hang-Shaoyin.Can enriching yin, nourish blood, stop blooding, be mainly used in deficiency of Yin hectic fever, hectic fever due to yin night sweat, soreness and weakness of waist and knees, the deficiency of blood is sallow, uterine bleeding band is inferior.Long-term taking colla carapacis et plastri testudinis also helps and strengthens body self-regulation, can promote longevity, and is also beneficial to hyperactivity of yang due to yin deficiency patient's physical rehabilitation.Along with improving constantly of living standard, similar with donkey-hide gelatin, colla carapacis et plastri testudinis also becomes one of first-selection that people nourish.
Because tortoise plastron self is famousr and precious, medical value is remarkable, and tortoise meat is also usually edible for people for cook various kinds tonic, in the last few years to various Chelonians to catch and kill phenomenon very serious, some Chelonian kind is endangered even.Therefore, the supply of tortoise plastron raw material is very limited, and price is high, has also directly had influence on the production of colla carapacis et plastri testudinis.On colla carapacis et plastri testudinis market, have some counterfeit and shoddy goods, it makes raw material is not tortoise plastron, but is replaced by the assorted skin of other animals, broken bone, comprises pigskin, ox-hide, the skin of animals died of illness even, dirty skin, rotten skin etc.Once such counterfeit and shoddy goods are shaped, outward appearance, color and luster, smell all to certified products colla carapacis et plastri testudinis and similar, true and false difficulty is distinguished.Take these adulterants not only without any curative effect, also probably harmful to health, consequence is very serious.
Glue class Chinese medicine forms gelatin substance by skin, bone, first-class the boiling of animal, and comprises donkey-hide gelatin, colla carapacis et plastri testudinis, deer horn glue etc., has the traditional Chinese medicine of typical national characters.Glue class Chinese medicine is general to be concentrated and forms through long-time thermophilic digestion, principal ingredient production technology very close and different manufacturers there are differences, and adopts the methods such as infrared, near infrared, X-diffraction to carry out the true and false to glue class Chinese medicine sterling and differentiates the normal situations such as judgement is inaccurate that exist.The discriminating of glue class Chinese medicament turtle-derived component, has scholar and differentiates from DNA angle, but just qualitative, has false negative and false positive phenomenon, and operation is comparatively complicated.Have and report and utilize Mass Spectrometer Method glue class Chinese medicament turtle-derived component from the angle of polypeptide, result accurately and reliably.At present, also not to the method for tracing to the source in the goods of glue class Chinese medicine and identifying.
In glue class Chinese medicine, principal ingredient is collagen polypeptide, the collagen, amino acid sequence of different animals there are differences, the otherness polypeptide of same species stable existence can be used as the characteristic polypeptide of these species, by digestion with restriction enzyme, utilize mass spectrum completely likely accurately glue class Chinese medicine and goods thereof to be traced to the source and identified and content detection.
Summary of the invention
The present invention is directed to the problems referred to above, the detection method of turtle-derived component in a kind of glue class Chinese medicine and goods thereof is provided, the method is simple to operate, and characteristic is strong, highly sensitive, can be used for the evaluation of turtle-derived component in glue class Chinese medicine and goods thereof.
Technical scheme of the present invention is as follows:
First, the invention provides the detection method of turtle-derived component in a kind of glue class Chinese medicine and goods thereof, it utilizes the difference in tortoise genome with specificity nucleotide sequence or amino acid sequence to detect, and described specific amino acid is as shown in SEQ.ID No1.
Preferably, described glue class Chinese medicine is selected from donkey-hide gelatin, oxhide gelatin, colla carapacis et plastri testudinis or deer horn glue.
Preferably, the described choosing of glue class Chinese herbal product food, health products or medicine that freely corresponding glue class Chinese medicine is made.
Particularly, the method comprises the steps:
(1) choose specific amino acid or amino acid sequence in tortoise genome, described specific amino acid is as shown in SEQ.ID No1;
(2) glue class Chinese medicine or its goods are dissolved, or extract polypeptide constituents dissolving wherein, add restriction enzyme to carry out enzyme and cut;
(3) put into subsequently LC-MS instrument, take negative sample to be detected as matrix, add step (1) gained amino acid sequence or colla carapacis et plastri testudinis sterling in contrast, select parent ion m/z569.1 and the daughter ion thereof of this polypeptide to monitor.
If it is consistent with reference substance to detect the retention time of this ion, and its daughter ion is consistent with the daughter ion of reference substance, and described glue class Chinese medicine or its goods contain turtle-derived component; If this ion consistent with reference substance retention time, does not contain turtle-derived component.
Preferably, described in step (2), dissolving reagent used is that mass percent is 1%, the NH of pH value 8.0
4hCO
3solution; Described restriction enzyme is trypsase.
Adopt method of the present invention, can in fast detecting glue class Chinese medicine, whether contain turtle-derived component, thereby minute evident, although collagen polypeptide has certain hydrolysis and destruction in glue class Chinese medicine, but the present invention is based on the otherness polypeptide in its principal ingredient collagen, its content is high, and the degree that is damaged is very little, can identify the turtle-derived component in glue class Chinese medicine and goods thereof.
The invention still further relates to polypeptide, its amino acid sequence is Gly-Val-Gln-Gly-Ala-Hyp-Gly-Pro-Gln-Gly-Pro-Arg, and the composition being comprised of described polypeptide and respective carrier.In addition, also comprise the application in turtle-derived component in detecting glue class Chinese medicine and goods thereof of described polypeptide and composition thereof.
Accompanying drawing explanation
Fig. 1 selects the mass spectrogram of ion pair m/z569.1 → 724.4,795.4,852.4 monitorings under synthetic polypeptide and sterling glue multiple-reaction monitoring scan pattern;
Fig. 2 selects the mass spectrogram of ion pair m/z569.1 → 724.4,795.4,852.4 monitorings under synthetic polypeptide and the epoxy glue multiple-reaction monitoring scan pattern that adds 5% colla carapacis et plastri testudinis;
Fig. 3 is the mass spectrogram that synthetic polypeptide and colla carapacis et plastri testudinis goods (the celestial oral liquid of tortoise deer two of take is example) are selected ion pair m/z569.1 → 724.4,795.4,852.4 monitorings under multiple-reaction monitoring scan pattern.
Embodiment
Below in conjunction with specific embodiment, further describe the present invention, advantage and disadvantage of the present invention will be more clear along with description.But embodiment is only exemplary, scope of the present invention is not formed to any restriction.It will be understood by those skilled in the art that lower without departing from the spirit and scope of the present invention and can the details of technical solution of the present invention and form be modified or be replaced, but these modifications and replacement all fall within the scope of protection of the present invention.
The acquisition of specificity peptide chain in the present invention:
1 material and reagent
Material: sterling glue comprises donkey-hide gelatin, horse skin glue, oxhide gelatin, pig skin gelatin, colla carapacis et plastri testudinis, deer horn glue, decocts and obtains with donkey hide, horse skin, ox-hide, pigskin, tortoise plastron, deer horn respectively.
Reagent: ammonium bicarbonate (analyzing pure), trypsase (sequence is pure, purchased from National Institute for Food and Drugs Control), synthetic polypeptide Gly-Val-Gln-Gly-Ala-Hyp-Gly-Pro-Gln-Gly-Pro-Arg.
The detection pre-treatment of 2 samples
(1) preparation of glue sample enzymolysis solution
Get 1.0g glue sample to be measured in 500mL measuring bottle, add a small amount of 1%NH
4hCO
3solution, the ultrasonic sample that makes dissolves completely, uses 1%NH
4hCO
3solution (pH8.0) is diluted to scale, shakes up, and filtering with microporous membrane, precision measures the trypsin solution 100 μ L that subsequent filtrate 1mL adds 2mg/mL, 37 ℃ of constant temperature enzymolysis 6h.
(2) preparation of synthetic polypeptide control sample enzymolysis solution
Take a certain amount of synthetic polypeptide, add 1%NH
4hCO
3solution (pH8.0), shakes up, and be mixed with concentration and be about 0.3 μ g/mL, filtering with microporous membrane, standby.
The discovery of 3 polypeptide
(1) discovery of ion
Get 5 μ L enzymolysis solution and put into the detection of LC-MS instrument.Liquid-phase condition: C
18reverse-phase chromatographic column (2.1mm * 100mm, 1.8 μ m), mobile phase A is 0.1% formic acid solution (volume fraction), Mobile phase B is acetonitrile, flow velocity 0.3mL/min; Gradient elution: 0～40min, 2%～50% Mobile phase B (the percentage here represents from 0 minute to the 40th minute, flow B by 2% linear gradient be 50%, mobile phase A fades to 50% by 98%).Mass spectrum condition: electron spray positive ion mode (ESI
+) carry out one-level and entirely sweep, sweep limit m/z400-m/z1000.
From the mass spectrum of glue sample separately, entirely sweep figure and extract ion m/z569.1, by contrast, find to contain this quasi-molecular ions in 4.06min left and right only has colla carapacis et plastri testudinis, and all do not have in donkey-hide gelatin, horse skin glue, oxhide gelatin, pig skin gelatin and deer horn glue.Detection and checking by multiple batches of sample show that above-mentioned conclusion is correct.Illustrate: can, by detecting the ion m/z569.1 in colla carapacis et plastri testudinis, determine whether to contain turtle-derived component.
(2) amino acid sequence of polypeptide is tentatively inferred
Utilize triple quadrupole bar mass spectrum, the above-mentioned ion of finding out is carried out to daughter ion scanning, confirm this ion band double charge, by sequence assembly, infer the partial sequence that polypeptide.Partial sequence by inference, at NCBI(http: //www.ncbi.nlm.nih.gov/) database carries out sequence retrieval, feature in conjunction with it (has in tortoise, and all do not have in donkey, horse, ox, pig or deer, and be about 1137.2 by molecular weight after tryptic digestion), derive the possible sequence Gly-Val-Gln-Gly-Ala-Hyp-Gly-Pro-Gln-Gly-Pro-Arg of this polypeptide.According to the CID fracture theory of peptide chain, calculate the possible daughter ion of this polypeptide as follows:
| a | b | c″ | Res: | x | y″ | z |
| 30.0 | 58.0 | 77.1 | Gly | - | - | - |
| 129.1 | 157.1 | 176.1 | Val | 1103.5 | 1079.6 | 1060.5 |
| 257.2 | 285.2 | 304.2 | Gln | 1004.5 | 980.5 | 961.5 |
| 314.2 | 342.2 | 361.2 | Gly | 876.4 | 852.4 | 833.4 |
| 385.2 | 413.2 | 432.3 | Ala | 819.4 | 795.4 | 776.4 |
| 498.3 | 526.3 | 545.3 | Hyp | 748.3 | 724.4 | 705.3 |
| 555.3 | 583.3 | 602.3 | Gly | 635.3 | 611.3 | 592.3 |
| 652.3 | 680.3 | 699.4 | Pro | 578.3 | 554.3 | 535.3 |
| 780.4 | 808.4 | 827.4 | Gln | 481.2 | 457.3 | 438.2 |
| 837.4 | 865.4 | 884.5 | Gly | 353.2 | 329.2 | 310.2 |
| 934.5 | 962.5 | 981.5 | Pro | 296.1 | 272.2 | 253.1 |
| - | - | - | Arg | 199.1 | 175.1 | 156.1 |
Can entirely sweep coincideing in figure with the daughter ion of peculiar ion m/z569.1 in colla carapacis et plastri testudinis.
(3) sequence is determined
Amino acid sequence by inference, entrusts polypeptide Synesis Company (the biochemical company limited of gill) to carry out the synthetic of this polypeptide.Then the daughter ion that utilizes LC-MS instrument to synthesize polypeptide is swept entirely, and in its testing conditions and glue sample, the daughter ion of corresponding polypeptide is swept term harmonization entirely.By contrasting ion appearance time, the daughter ion of synthetic polypeptide and colla carapacis et plastri testudinis, entirely sweep figure, find that both fit like a glove, thereby determine that this ion m/z569.1 is polypeptide Gly-Val-Gln-Gly-Ala-Hyp-Gly-Pro-Gln-Gly-Pro-Arg.
1 material and reagent
Material: sterling glue comprises donkey-hide gelatin, horse skin glue, oxhide gelatin, pig skin gelatin, colla carapacis et plastri testudinis, deer horn glue, decocts and obtains with donkey hide, horse skin, ox-hide, pigskin, tortoise plastron, deer horn respectively.
Epoxy glue sample (containing the oxhide gelatin of 5% colla carapacis et plastri testudinis, the oxhide gelatin of the oxhide gelatin of 10% colla carapacis et plastri testudinis, 20% colla carapacis et plastri testudinis, the oxhide gelatin of 40% colla carapacis et plastri testudinis) by above-mentioned sterling glue, be accurately mixed and made into (percentage is massfraction).
Reagent: ammonium bicarbonate (analyzing pure), trypsase (sequence is pure, purchased from National Institute for Food and Drugs Control), synthetic polypeptide Gly-Val-Gln-Gly-Ala-Hyp-Gly-Pro-Gln-Gly-Pro-Arg.
2 detection methods
(1) preparation of synthetic polypeptide control sample enzymolysis solution
Get 1.0g sterling oxhide gelatin sample in 500ml measuring bottle, add a small amount of 1%NH
4hCO
3solution, the ultrasonic sample that makes dissolves completely, uses 1%NH
4hCO
3solution (pH8.0) is diluted to scale, shakes up, and filtering with microporous membrane, precision measures subsequent filtrate 1ml and adds synthetic polypeptide 0.3 μ g, then adds the trypsin solution 100 μ l of 2mg/ml, 37 ℃ of constant temperature enzymolysis 6h.
(2) preparation of glue sample enzymolysis solution
Get 1.0g glue sample to be measured in 500ml measuring bottle, add a small amount of 1%NH
4hCO
3solution, the ultrasonic sample that makes dissolves completely, uses 1%NH
4hCO
3solution (pH8.0) is diluted to scale, shakes up, and filtering with microporous membrane, precision measures the trypsin solution 100 μ l that subsequent filtrate 1ml adds 2mg/ml, 37 ℃ of constant temperature enzymolysis 6h.
(3) detect
Get 5 μ l enzymolysis solution and put into the detection of LC-MS instrument.Liquid-phase condition: C
18reverse-phase chromatographic column (2.1mm * 100mm, 1.8 μ m), mobile phase A is 0.1% formic acid solution (volume fraction), Mobile phase B is acetonitrile, flow velocity 0.3ml/min; Gradient elution: 0～40min, 2%～50% Mobile phase B (the percentage here represents from 0 minute to the 40th minute, flow B by 2% linear gradient be 50%, mobile phase A fades to 50% by 98%).Mass spectrum condition: electron spray positive ion mode (ESI
+) select to carry out many reaction detection, select m/z569.1 → 724.4,795.4,852.4 as detecting ion pair.
The results are shown in Figure Isosorbide-5-Nitrae .06min place and only have in synthetic polypeptide control sample and colla carapacis et plastri testudinis and detect corresponding quasi-molecular ions, other all do not detect.The detection of epoxy glue sample, the percentage composition of colla carapacis et plastri testudinis of take is horizontal ordinate, take m/z569.1 → 724.4, to extract chromatographic peak area in ion flow graph be ordinate, drawing standard curve, R
2be more than 0.999, linear good.Visible this method can specific detection turtle-derived component, comprises that with other various animal derived materials donkey, horse, ox, pig, deer etc. distinguish.
1 material and reagent
Material: the sterling glue sample of epoxy glue sample in embodiment 1 adds respectively 5% colla carapacis et plastri testudinis to make, comprise containing the donkey-hide gelatin of 5% colla carapacis et plastri testudinis, the horse skin glue of 5% colla carapacis et plastri testudinis, containing the oxhide gelatin of 5% colla carapacis et plastri testudinis, the pig skin gelatin of 5% colla carapacis et plastri testudinis, containing the deer horn glue of 5% colla carapacis et plastri testudinis.
Reagent: ammonium bicarbonate (analyzing pure), trypsase (sequence is pure, purchased from National Institute for Food and Drugs Control), synthetic polypeptide Gly-Val-Gln-Gly-Ala-Hyp-Gly-Pro-Gln-Gly-Pro-Arg.
2 detection methods
(1) preparation of synthetic polypeptide control sample enzymolysis solution
Get 1.0g sterling oxhide gelatin to be measured sample in 500ml measuring bottle, add a small amount of 1%NH
4hCO
3solution, the ultrasonic sample that makes dissolves completely, uses 1%NH
4hCO
3solution (pH8.0) is diluted to scale, shakes up, and filtering with microporous membrane, precision measures subsequent filtrate 1ml and adds synthetic polypeptide 0.3 μ g, then adds the trypsin solution 100 μ l of 2mg/ml, 37 ℃ of constant temperature enzymolysis 6h.
(2) preparation of glue sample enzymolysis solution
Get 1.0g testing sample in 500ml measuring bottle, add a small amount of 1%NH
4hCO
3solution, the ultrasonic sample that makes dissolves completely, uses 1%NH
4hCO
3solution (pH8.0) is diluted to scale, shakes up, and filtering with microporous membrane, precision measures the trypsin solution 100 μ l that subsequent filtrate 1ml adds 2mg/ml, 37 ℃ of constant temperature enzymolysis 6h.
(3) detect
Get 5 μ l enzymolysis solution and put into the detection of LC-MS instrument.Liquid-phase condition: C
18reverse-phase chromatographic column (2.1mm * 100mm, 1.8 μ m), mobile phase A is 0.1% formic acid solution (volume fraction), Mobile phase B is acetonitrile, flow velocity 0.3ml/min; Gradient elution: 0～40min, 2%～50%B(percentage here represents from 0 minute to the 40th minute, flow B by 2% linear gradient be 50%, mobile phase A fades to 50% by 98%).Mass spectrum condition: electron spray positive ion mode (ESI
+) select to carry out many reaction detection, select m/z569.1 → 724.4,795.4,852.4 as detecting ion pair.
The results are shown in Figure 2,4.06min place synthesizes polypeptide control sample, add in the epoxy glue sample of 5% colla carapacis et plastri testudinis and all detect corresponding quasi-molecular ions, and all do not detect corresponding quasi-molecular ions in sterling donkey-hide gelatin in case study on implementation 1, horse skin glue, oxhide gelatin, pig skin gelatin, deer horn glue.The turtle-derived component of visible this method in can specific detection glue class Chinese medicine.
1 material and reagent
Material: colla carapacis et plastri testudinis, the celestial oral liquid of tortoise deer two (Dong-E donkey-hide Gelatin Co., Ltd., Shandong Prov.), not containing the celestial oral liquid of tortoise deer two (self-control) of colla carapacis et plastri testudinis
Reagent: trichloroacetic acid (analyzing pure), ammonium bicarbonate (analyzing pure), trypsase (sequence is pure, purchased from National Institute for Food and Drugs Control), synthetic polypeptide Gly-Val-Gln-Gly-Ala-Hyp-Gly-Pro-Gln-Gly-Pro-Arg.
2 detection methods
(1) preparation of synthetic polypeptide control sample enzymolysis solution
Get 1.0g and containing the celestial oral liquid sample of tortoise deer two of colla carapacis et plastri testudinis, do not add 1%NH
4hCO
3solution 24ml dissolves, and adds 4g trichloroacetic acid, places 10min for 4 ℃, and the centrifugal 5min of 10000rpm, abandons supernatant, and sediment is proceeded in 100ml measuring bottle, uses 1%NH
4hCO
3solution (pH8.0) dissolves and is settled to scale, shakes up, and filtering with microporous membrane, precision measures subsequent filtrate 1ml and adds synthetic polypeptide 0.3 μ g, then adds the trypsin solution 100 μ l of 2mg/ml, 37 ℃ of constant temperature enzymolysis 6h.
(2) preparation of testing sample enzymolysis solution
Get 1.0g testing sample and add 1%NH
4hCO
3solution 25ml dissolves, and adds 4g trichloroacetic acid, places 10min for 4 ℃, and the centrifugal 5min of 10000rpm, abandons supernatant, and sediment is proceeded in 100ml measuring bottle, uses 1%NH
4hCO
3solution (pH8.0) dissolves and is settled to scale, shakes up, and filtering with microporous membrane, precision measures the trypsin solution 100 μ l that subsequent filtrate 1ml adds 2mg/ml, 37 ℃ of constant temperature enzymolysis 6h.
(3) detect
Get 5 μ l enzymolysis solution and put into the detection of LC-MS instrument.Liquid-phase condition: C
18reverse-phase chromatographic column (2.1mm * 100mm, 1.8 μ m), mobile phase A is 0.1% formic acid solution (volume fraction), Mobile phase B is acetonitrile, flow velocity 0.3ml/min; Gradient elution: 0～40min, 2%～50%B(percentage here represents from 0 minute to the 40th minute, flow B by 2% linear gradient be 50%, mobile phase A fades to 50% by 98%).Mass spectrum condition: electron spray positive ion mode (ESI
+) carry out many reaction detection, select m/z569.1 → 724.4,795.4,852.4 as detecting ion pair.
The results are shown in Figure 3, the celestial oral liquid of tortoise deer two that 4.06min place synthesizes polypeptide control sample, colla carapacis et plastri testudinis and contains colla carapacis et plastri testudinis detects corresponding quasi-molecular ions, other do not detect, the turtle-derived component of visible this method in can specific detection colla carapacis et plastri testudinis goods, and comprise that with other various animal derived materials donkey, horse, ox, pig, deer etc. distinguish.
Sequence table
<110> Dong-E donkey-hide Gelatin Co., Ltd., Shandong Prov.
The detection method <130> of turtle-derived component in <120> glue class Chinese medicine and goods thereof
<160>1
<170>PatentIn version3.3
<210>1
<211>12
<212>PRT
<213> artificial sequence
<400>1
Gly Val Gln Gly Ala Hyp Gly Pro Gln Gly Pro Arg
1 5 10。
Claims (10)
1. a detection method for turtle-derived component in glue class Chinese medicine and goods thereof, it utilizes the difference in tortoise genome with specificity nucleotide sequence or amino acid sequence to detect, and described specific amino acid is as shown in SEQ.ID No1.
2. method according to claim 1, is characterized in that, described glue class Chinese medicine is selected from donkey-hide gelatin, oxhide gelatin, colla carapacis et plastri testudinis or deer horn glue.
3. method according to claim 1, is characterized in that, the described choosing of glue class Chinese herbal product food, health products or medicine that freely corresponding glue class Chinese medicine is made.
4. according to the method described in claim 1～3 any one, it specifically comprises the steps:
(1) choose specific amino acid or amino acid sequence in tortoise genome, described specific amino acid is as shown in SEQ.ID No1;
(2) glue class Chinese medicine or its goods are dissolved, or extract polypeptide constituents dissolving wherein, add restriction enzyme to carry out enzyme and cut;
(3) put into subsequently LC-MS instrument, take negative sample to be detected as matrix, add step (1) gained amino acid sequence or colla carapacis et plastri testudinis sterling in contrast, select parent ion m/z569.1 and the daughter ion thereof of this polypeptide to monitor.
5. method according to claim 4, is characterized in that, dissolves reagent used and be mass percent and be 1%, the NH of pH value 8.0 described in step (2)
4hCO
3solution; Described restriction enzyme is trypsase.
6. detect a molecular marked compound for turtle-derived component in glue class Chinese medicine and goods thereof, the amino acid sequence of this molecular marked compound is as shown in SEQ.ID No1.
Coding albumen claimed in claim 6 gene.
8. the application of molecular marked compound claimed in claim 6 turtle-derived component in detecting glue class Chinese medicine and goods thereof.
9. a pharmaceutical composition, is characterized in that, the albumen claimed in claim 6 that comprises pharmacy effective dose and one or more pharmaceutically acceptable carriers.
10. the application of pharmaceutical composition claimed in claim 9 turtle-derived component in detecting glue class Chinese medicine and goods thereof.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108828108A (en) * | 2018-09-05 | 2018-11-16 | 湖南省药品检验研究院(湖南药用辅料检验检测中心) | The method of many animals derived component in product containing gelatin crude drug is detected simultaneously |
| CN109187783A (en) * | 2018-09-03 | 2019-01-11 | 贵州广济堂药业有限公司 | Deer horn glue feature peptide and identification sample to be tested in whether include deer horn glue method |
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