CN103630634A - Substance and method for detecting tortoise-shell glue, deer-horn glue and products thereof - Google Patents
Substance and method for detecting tortoise-shell glue, deer-horn glue and products thereof Download PDFInfo
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- CN103630634A CN103630634A CN201310526411.XA CN201310526411A CN103630634A CN 103630634 A CN103630634 A CN 103630634A CN 201310526411 A CN201310526411 A CN 201310526411A CN 103630634 A CN103630634 A CN 103630634A
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Abstract
The invention provides a method for identifying tortoise-shell glue, deer-horn glue and products thereof. The method provided by the invention comprises the step of detecting by using the difference between the specific nucleotide sequence or amino acid sequence of genomes of the tortoise-shell glue, deer-horn glue and products thereof and the specific nucleotide sequence or amino acid sequence of genomes of other imitations, wherein the specific amino acid sequence is shown in SEQ. ID No1. According to the method provided by the invention, fake ingredients, such as donkeys, horses, cattle, pigs or sheep, in the tortoise-shell glue, deer-horn glue and products thereof can be detected rapidly, so as to distinguish the authenticity. The invention further relates a polypeptide and a composition formed by the polypeptide and a corresponding carrier. Furthermore, the invention relates to application of the polypeptide and composition thereof in detection of donkey, horse, cattle, pig or sheep-derived ingredients in the tortoise-shell glue and products thereof.
Description
Technical field
The present invention relates to the detection method of a breeding ass, horse, ox, pig or sheep derived material, specifically can be applicable to the authenticity of colla carapacis et plastri testudinis, deer horn glue and goods thereof, belong to Chinese medicine detection field.
Background technology
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.Deer horn glue is to take deer horn as raw material, through decocting, boils, concentrates the solid gum of making, and property is sweet, salty, warm, returns kidney, Liver Channel.Can warm filling liver kidney, beneficial intensive culture blood, the waist knee acid being mainly used in due to kidney deficiency and liver is cold, impotence and seminal emission, consumptive disease is thin thin, metrostaxis, the hematuria of having blood in stool, cloudy subcutaneous ulcer swells and ache.Along with improving constantly of living standard, colla carapacis et plastri testudinis, deer horn glue become one of first-selection that people nourish.
Due to tortoise plastron and deer horn self famousr and precious, medical value is remarkable, to seriously the catching and killing of various Chelonians and animal in deer family, cause the supply of tortoise plastron, deer horn raw material very limited, and price was high in the last few years, has directly had influence on the production of colla carapacis et plastri testudinis, deer horn glue.On colla carapacis et plastri testudinis, deer horn glue market, have some counterfeit and shoddy goods, it makes raw material is not tortoise plastron, deer horn, 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, deer horn glue 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.These adulterants have seriously hindered the sound development of whole colla carapacis et plastri testudinis, deer horn glue Market of Chinese Materia Medica.
The concentrated glue class Chinese medicine forming of skin, bone, the long-time thermophilic digestion of first-class warp by animal, 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 and differentiates the normal situations such as judgement is inaccurate that exist.Have and report and utilize Xing,Niu source, Xing,Lu source, tortoise source property, donkey derived component Mass Spectrometer Method glue class Chinese medicine from the angle of polypeptide, result accurately and reliably.But also there is no at present the angle from collagen polypeptide, the detection method of adulterant composition in disposable discriminating colla carapacis et plastri testudinis, deer horn glue and goods thereof.
In glue class Chinese medicine, principal ingredient is collagen polypeptide, the collagen, amino acid sequence of different animals there are differences, in different plant species except tortoise, deer, the same polypeptide of the stable existence containing can be used as the characteristic polypeptide of adulterant composition in colla carapacis et plastri testudinis, deer horn glue, by digestion with restriction enzyme, utilize the mass spectrum completely can be to the evaluation of tracing to the source of glue class Chinese medicine.
Summary of the invention
The present invention is directed to the problems referred to above, a kind of detection thing and the authenticity method thereof of colla carapacis et plastri testudinis, deer horn glue and goods thereof is provided, the method is simple to operate, and characteristic is strong, highly sensitive, can be used for the evaluation of tracing to the source of adulterant composition in colla carapacis et plastri testudinis, deer horn glue and goods thereof.
Technical scheme of the present invention is as follows:
First, the invention provides a kind of method of identifying colla carapacis et plastri testudinis, deer horn glue and goods thereof, it utilizes colla carapacis et plastri testudinis, deer horn glue and goods thereof and in other Counterfeit Item genomes, has the different of specificity nucleotide sequence or amino acid sequence and detect, and described specific amino acid is as shown in SEQ.ID No1.
Preferably, described colla carapacis et plastri testudinis, deer horn glue goods are selected from a kind of in food, health products or the medicine that colla carapacis et plastri testudinis or deer horn glue make, and do not contain donkey, horse, ox, pig, sheep derived material in formula.
Preferably, described other Counterfeit Items refer to that one or more in donkey, horse, ox, pig or sheep boil the gelatin substance forming.
More specifically, described method comprises the steps:
(1) choose specific amino acid or amino acid sequence in colla carapacis et plastri testudinis, deer horn glue goods and other Counterfeit Item genomes, described specific amino acid is as shown in SEQ.ID No1;
(2) colla carapacis et plastri testudinis, deer horn glue or its goods are dissolved, or extract the polypeptide constituents of described colla carapacis et plastri testudinis or its goods and dissolve, add subsequently restriction enzyme to carry out enzyme and cut;
(3) solution after enzyme step (2) being obtained is cut is put into LC-MS instrument, take colla carapacis et plastri testudinis sterling as matrix, adds the described amino acid sequence of step (1) in contrast, and selects parent ion m/z441.8 and the daughter ion thereof of this amino acid sequence 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, in described colla carapacis et plastri testudinis or its goods, contains this adulterant composition; If this ion consistent with reference substance retention time, does not contain this adulterant composition.
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.
Further, the invention provides a kind of molecular marked compound that detects colla carapacis et plastri testudinis and goods thereof, the amino acid sequence of this molecular marked compound is as shown in SEQ.ID No1.
Further, the invention provides the gene of the described albumen of coding.
Further, the invention provides described molecular marked compound and whether contain the application in donkey, horse, ox, pig or sheep derived material in detecting colla carapacis et plastri testudinis.
Further, the invention provides a kind of pharmaceutical composition, its described albumen that comprises pharmacy effective dose and one or more pharmaceutically acceptable carriers.
Further, the invention provides described pharmaceutical composition and whether contain the application in donkey, horse, ox, pig or sheep derived material in detecting colla carapacis et plastri testudinis.
Beneficial effect of the present invention is as follows:
Adopt method of the present invention, can fast detecting colla carapacis et plastri testudinis, the adulterant composition such as the donkey in deer horn glue and goods thereof, horse, ox, pig, sheep, 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 whether containing the adulterant compositions such as donkey, horse, ox, pig or sheep in colla carapacis et plastri testudinis, deer horn glue and goods thereof.
The invention still further relates to polypeptide, its amino acid sequence is Gly-Val-Val-Gly-Leu-Pro-Gly-Gln-Arg, and the composition being comprised of described polypeptide and respective carrier.In addition, also comprise the application in donkey, horse, ox, pig or sheep derived material in detecting colla carapacis et plastri testudinis, deer horn glue and goods thereof of described polypeptide and composition thereof.
Accompanying drawing explanation
Fig. 1 selects the mass spectrogram of ion pair m/z441.8 → 456.8,627.8 monitorings under synthetic polypeptide and sterling glue multiple-reaction monitoring scan pattern;
Fig. 2 selects the mass spectrogram of ion pair m/z441.8 → 456.8,627.8 monitorings under synthetic polypeptide and the epoxy glue multiple-reaction monitoring scan pattern that adds 5% deer horn glue;
Fig. 3 is the mass spectrograms that synthetic polypeptide and colla carapacis et plastri testudinis, deer horn glue goods (the celestial oral liquid of tortoise deer two of take is example) are selected ion pair m/z441.8 → 456.8,627.8 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
1 material and reagent
Material: sterling glue comprises donkey-hide gelatin, horse skin glue, oxhide gelatin, pig skin gelatin, sheepskin glue, colla carapacis et plastri testudinis, deer horn glue, decocts and obtains with donkey hide, horse skin, ox-hide, pigskin, sheepskin, 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-Val-Gly-Leu-Pro-Gly-Gln-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 and figure, extract ion m/z441.8, by contrast, find in 10.84min left and right only has colla carapacis et plastri testudinis, deer horn glue not this quasi-molecular ions, and all contain in donkey-hide gelatin, horse skin glue, oxhide gelatin, pig skin gelatin and sheepskin glue.Detection and checking by multiple batches of sample show that above-mentioned conclusion is correct.Illustrate: can, by detecting the ion m/z441.8 in colla carapacis et plastri testudinis, deer horn glue, determine whether to be mixed with donkey, horse, ox, pig or sheep derived material.
(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 (does not have in tortoise, deer, and all contain in donkey, horse, ox, pig or sheep, and be about 882.6 by molecular weight after tryptic digestion), find out the possible sequence Gly-Val-Val-Gly-Leu-Pro-Gly-Gln-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 | 849.5 | 825.5 | 806.5 |
| 228.2 | 256.2 | 275.2 | Val | 750.4 | 726.4 | 707.4 |
| 285.2 | 313.2 | 332.2 | Gly | 651.3 | 627.4 | 608.3 |
| 398.3 | 426.3 | 445.3 | Leu | 594.3 | 570.3 | 551.3 |
| 495.3 | 523.3 | 542.4 | Pro | 481.2 | 457.3 | 438.2 |
| 552.4 | 580.3 | 599.4 | Gly | 384.2 | 360.2 | 341.2 |
| 680.4 | 708.4 | 727.4 | Gln | 327.1 | 303.2 | 284.1 |
| - | - | - | Arg | 199.1 | 175.1 | 156.1 |
Can entirely sweep coincideing in figure with the daughter ion of peculiar ion m/z441.8 in donkey-hide gelatin, horse skin glue, oxhide gelatin, pig skin gelatin or sheepskin glue.
(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 donkey-hide gelatin, horse skin glue, oxhide gelatin, pig skin gelatin and sheepskin glue, entirely sweep figure, find that both fit like a glove, thereby determine that this ion m/z441.8 is polypeptide Gly-Val-Val-Gly-Leu-Pro-Gly-Gln-Arg.
Embodiment 1
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, sheepskin glue, decocts and obtains with donkey hide, horse skin, ox-hide, pigskin, tortoise plastron, deer horn, sheepskin respectively.
Epoxy glue sample (containing the colla carapacis et plastri testudinis of 5% oxhide gelatin, the colla carapacis et plastri testudinis of the colla carapacis et plastri testudinis of 10% oxhide gelatin, 20% oxhide gelatin, the colla carapacis et plastri testudinis of 40% oxhide gelatin) 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-Val-Gly-Leu-Pro-Gly-Gln-Arg.
2 detection methods
(1) preparation of synthetic polypeptide control sample enzymolysis solution
Get 1.0g sterling colla carapacis et plastri testudinis 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/z441.8 → 456.8,627.8 as detecting ion pair.
The results are shown in Figure 1,10.84min place and only have in colla carapacis et plastri testudinis, deer horn glue sterling and detect corresponding quasi-molecular ions, other all do not detect.The detection of epoxy glue sample, the percentage composition of oxhide gelatin of take is horizontal ordinate, take m/z441.8 → 456.8, 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 colla carapacis et plastri testudinis, the adulterant composition in deer horn glue, thereby colla carapacis et plastri testudinis, deer horn glue are carried out to authenticity.
Embodiment 2
1 material and reagent
Material: the deer horn glue sterling of epoxy glue sample in embodiment 1 adds respectively 5% adulterant glue to make, comprise containing the deer horn glue of 5% donkey-hide gelatin, the deer horn glue of 5% horse skin glue, containing the deer horn glue of 5% oxhide gelatin, the deer horn glue of the deer horn glue of 5% pig skin gelatin, 5% sheepskin glue (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-Val-Gly-Leu-Pro-Gly-Gln-Arg.
2 detection methods
(1) preparation of synthetic polypeptide control sample enzymolysis solution
Get 1.0g sterling deer horn glue 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/z441.8 → 456.8,627.8 as detecting ion pair.
The results are shown in Figure the synthetic polypeptide control sample in 2,10.84min place, add in the epoxy glue sample of 5% adulterant glue and all detect corresponding quasi-molecular ions, and all do not detect corresponding quasi-molecular ions in sterling colla carapacis et plastri testudinis in case study on implementation 1, deer horn glue.Visible this method can specific detection colla carapacis et plastri testudinis, the adulterant composition in deer horn glue.
Embodiment 3
1 material and reagent
Material: colla carapacis et plastri testudinis, deer horn glue, the celestial oral liquid of tortoise deer two (Dong-E donkey-hide Gelatin Co., Ltd., Shandong Prov.), add oxhide gelatin the celestial oral liquid of tortoise deer two (self-control), add the celestial oral liquid of tortoise deer two (self-control) of donkey-hide gelatin
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-Val-Gly-Leu-Pro-Gly-Gln-Arg.
2 detection methods
(1) preparation of synthetic polypeptide control sample enzymolysis solution
Get 1.0g tortoise deer two celestial oral liquids and 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/z441.8 → 456.8,627.8 as detecting ion pair.
The results are shown in Figure 3,10.84min place synthesizes polypeptide control sample, adds the celestial oral liquid of tortoise deer two of oxhide gelatin, adds the celestial oral liquid of tortoise deer two of donkey-hide gelatin all to detect corresponding quasi-molecular ions, other do not detect, as seen this method can specific detection colla carapacis et plastri testudinis, the adulterant composition in deer horn glue goods.
Sequence table
<110> Dong-E donkey-hide Gelatin Co., Ltd., Shandong Prov.
Detection thing and the authentication method thereof of <120> a colla carapacis et plastri testudinis, deer horn glue and goods thereof
<130>
<160>1
<170>PatentIn version3.3
<210>1
<211>9
<212>PRT
<213> artificial sequence
<400>1
Gly Val Val Gly Leu Pro Gly Gln Arg
1 5 。
Claims (9)
1. a method of identifying colla carapacis et plastri testudinis, deer horn glue and goods thereof, it is characterized in that, it utilizes colla carapacis et plastri testudinis, deer horn glue and goods thereof and in other Counterfeit Item genomes, has the different of specificity nucleotide sequence or amino acid sequence and detect, and described specific amino acid is as shown in SEQ.ID No1.
2. method according to claim 1, is characterized in that, described colla carapacis et plastri testudinis, deer horn glue goods are selected from a kind of in food, health products or the medicine that colla carapacis et plastri testudinis or deer horn glue make, and do not contain donkey, horse, ox, pig, sheep derived material in formula.
3. method according to claim 1, is characterized in that, described other Counterfeit Items refer to that one or more in donkey, horse, ox, pig or sheep boil the gelatin substance forming.
4. according to the method described in any one in claim 1～3, it specifically comprises the steps:
(1) choose specific amino acid or amino acid sequence in colla carapacis et plastri testudinis, deer horn glue goods and other Counterfeit Item genomes, described specific amino acid is as shown in SEQ.ID No1;
(2) colla carapacis et plastri testudinis, deer horn glue or its goods are dissolved, or extract the polypeptide constituents of described colla carapacis et plastri testudinis or its goods and dissolve, add subsequently restriction enzyme to carry out enzyme and cut;
(3) solution after enzyme step (2) being obtained is cut is put into LC-MS instrument, take colla carapacis et plastri testudinis sterling as matrix, adds the described amino acid sequence of step (1) in contrast, and selects parent ion m/z441.8 and the daughter ion thereof of this amino acid sequence 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 colla carapacis et plastri testudinis, deer horn glue 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. whether molecular marked compound claimed in claim 6 contains the application in donkey, horse, ox, pig or sheep derived material in detecting colla carapacis et plastri testudinis, deer horn glue.
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,
Whether pharmaceutical composition claimed in claim 9 contains the application in donkey, horse, ox, pig or sheep derived material in detecting colla carapacis et plastri testudinis.
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| CN106770825A (en) * | 2016-12-22 | 2017-05-31 | 东阿阿胶股份有限公司 | A kind of composition for detecting donkey hide derived components content in animal glue, kit and its detection method |
| CN106841497A (en) * | 2017-01-20 | 2017-06-13 | 东阿阿胶股份有限公司 | For detecting colla carapacis et plastri testudinis, deer horn glue, donkey-hide gelatin and its composition of donkey hide derived components content, kit and detection method in adulterant glue |
| 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 |
| CN109971868A (en) * | 2019-05-07 | 2019-07-05 | 镇江市食品药品监督检验中心 | One group for identifying the specific primer and deer horn authenticity identification method of deer horn |
| CN111825746A (en) * | 2016-03-18 | 2020-10-27 | 青岛海关技术中心 | Polypeptide for identifying donkey-hide gelatin and bovine-derived components in donkey-hide gelatin product |
| CN113880916A (en) * | 2021-08-19 | 2022-01-04 | 青海瑞肽生物科技有限公司 | Yak skin antioxidant polypeptide and preparation method and application thereof |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111825746A (en) * | 2016-03-18 | 2020-10-27 | 青岛海关技术中心 | Polypeptide for identifying donkey-hide gelatin and bovine-derived components in donkey-hide gelatin product |
| CN106770825A (en) * | 2016-12-22 | 2017-05-31 | 东阿阿胶股份有限公司 | A kind of composition for detecting donkey hide derived components content in animal glue, kit and its detection method |
| CN106770825B (en) * | 2016-12-22 | 2019-12-03 | 东阿阿胶股份有限公司 | It is a kind of for detecting composition, kit and its detection method of donkey hide derived components content in animal glue |
| CN106841497A (en) * | 2017-01-20 | 2017-06-13 | 东阿阿胶股份有限公司 | For detecting colla carapacis et plastri testudinis, deer horn glue, donkey-hide gelatin and its composition of donkey hide derived components content, kit and detection method in adulterant glue |
| CN106841497B (en) * | 2017-01-20 | 2018-08-07 | 东阿阿胶股份有限公司 | Composition, kit and detection method for detecting donkey hide derived components content in colla carapacis et plastri testudinis, deer horn glue, donkey-hide gelatin and its adulterant glue |
| 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 |
| CN113004374A (en) * | 2018-09-03 | 2021-06-22 | 贵州广济堂药业有限公司 | Deer glue characteristic peptide and method for identifying deer glue in sample to be detected |
| CN109187783B (en) * | 2018-09-03 | 2021-06-29 | 贵州广济堂健康药业有限公司 | Deer glue characteristic peptide and method for identifying deer glue in sample to be detected |
| CN109971868A (en) * | 2019-05-07 | 2019-07-05 | 镇江市食品药品监督检验中心 | One group for identifying the specific primer and deer horn authenticity identification method of deer horn |
| CN113880916A (en) * | 2021-08-19 | 2022-01-04 | 青海瑞肽生物科技有限公司 | Yak skin antioxidant polypeptide and preparation method and application thereof |
| CN113880916B (en) * | 2021-08-19 | 2023-06-30 | 青海瑞肽生物科技有限公司 | Yak skin antioxidant polypeptide and preparation method and application thereof |
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