CN103630635B - MS (Mass Spectrometry) detection method for tortoise-derived ingredients in glue type traditional Chinese medicines and products thereof - Google Patents
MS (Mass Spectrometry) detection method for tortoise-derived ingredients in glue type traditional Chinese medicines and products thereof Download PDFInfo
- Publication number
- CN103630635B CN103630635B CN201310526412.4A CN201310526412A CN103630635B CN 103630635 B CN103630635 B CN 103630635B CN 201310526412 A CN201310526412 A CN 201310526412A CN 103630635 B CN103630635 B CN 103630635B
- Authority
- CN
- China
- Prior art keywords
- glue
- amino acid
- chinese medicine
- tortoise
- class chinese
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 239000003292 glue Substances 0.000 title claims abstract description 62
- 241000270708 Testudinidae Species 0.000 title claims abstract description 28
- 238000001514 detection method Methods 0.000 title claims abstract description 24
- 238000004949 mass spectrometry Methods 0.000 title abstract description 9
- 239000004615 ingredient Substances 0.000 title abstract description 7
- 229940126680 traditional chinese medicines Drugs 0.000 title abstract 6
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 27
- 229920001184 polypeptide Polymers 0.000 claims abstract description 21
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 21
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 13
- 238000000034 method Methods 0.000 claims abstract description 13
- 241001550206 Colla Species 0.000 claims description 39
- 239000003814 drug Substances 0.000 claims description 38
- 229920000159 gelatin Polymers 0.000 claims description 29
- 108010010803 Gelatin Proteins 0.000 claims description 28
- 239000008273 gelatin Substances 0.000 claims description 28
- 235000019322 gelatine Nutrition 0.000 claims description 28
- 235000011852 gelatine desserts Nutrition 0.000 claims description 28
- 241000270666 Testudines Species 0.000 claims description 21
- 241000282994 Cervidae Species 0.000 claims description 18
- 239000003153 chemical reaction reagent Substances 0.000 claims description 10
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 claims description 7
- 150000001413 amino acids Chemical class 0.000 claims description 6
- 235000013305 food Nutrition 0.000 claims description 6
- 108091008146 restriction endonucleases Proteins 0.000 claims description 5
- 108090000623 proteins and genes Proteins 0.000 claims description 3
- 108090000790 Enzymes Proteins 0.000 claims description 2
- 102000004190 Enzymes Human genes 0.000 claims description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 2
- 239000011159 matrix material Substances 0.000 claims description 2
- 102000004169 proteins and genes Human genes 0.000 claims description 2
- 150000001875 compounds Chemical class 0.000 claims 5
- 239000008194 pharmaceutical composition Substances 0.000 claims 2
- 239000003937 drug carrier Substances 0.000 claims 1
- 102000008186 Collagen Human genes 0.000 abstract description 6
- 108010035532 Collagen Proteins 0.000 abstract description 6
- 229920001436 collagen Polymers 0.000 abstract description 6
- 230000007062 hydrolysis Effects 0.000 abstract description 2
- 238000006460 hydrolysis reaction Methods 0.000 abstract description 2
- 230000006378 damage Effects 0.000 abstract 1
- 239000002075 main ingredient Substances 0.000 abstract 1
- 239000002773 nucleotide Substances 0.000 abstract 1
- 125000003729 nucleotide group Chemical group 0.000 abstract 1
- 150000002500 ions Chemical class 0.000 description 30
- 239000000523 sample Substances 0.000 description 25
- 230000015572 biosynthetic process Effects 0.000 description 21
- 238000003786 synthesis reaction Methods 0.000 description 21
- 239000012071 phase Substances 0.000 description 18
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical group CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 12
- 239000000463 material Substances 0.000 description 10
- 238000001914 filtration Methods 0.000 description 8
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 8
- 239000012982 microporous membrane Substances 0.000 description 8
- 238000002360 preparation method Methods 0.000 description 8
- 102000004142 Trypsin Human genes 0.000 description 7
- 108090000631 Trypsin Proteins 0.000 description 7
- 239000013068 control sample Substances 0.000 description 7
- 239000000706 filtrate Substances 0.000 description 7
- 239000012588 trypsin Substances 0.000 description 7
- 241001465754 Metazoa Species 0.000 description 6
- 241000283074 Equus asinus Species 0.000 description 5
- 229920006335 epoxy glue Polymers 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 4
- ATRRKUHOCOJYRX-UHFFFAOYSA-N Ammonium bicarbonate Chemical compound [NH4+].OC([O-])=O ATRRKUHOCOJYRX-UHFFFAOYSA-N 0.000 description 4
- 229910000013 Ammonium bicarbonate Inorganic materials 0.000 description 4
- 235000012538 ammonium bicarbonate Nutrition 0.000 description 4
- 239000001099 ammonium carbonate Substances 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 238000010828 elution Methods 0.000 description 4
- 235000019253 formic acid Nutrition 0.000 description 4
- 239000007791 liquid phase Substances 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 239000002994 raw material Substances 0.000 description 4
- 239000007921 spray Substances 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 239000008280 blood Substances 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 230000029087 digestion Effects 0.000 description 3
- 238000001819 mass spectrum Methods 0.000 description 3
- 238000012544 monitoring process Methods 0.000 description 3
- 238000012797 qualification Methods 0.000 description 3
- 239000013558 reference substance Substances 0.000 description 3
- 238000011895 specific detection Methods 0.000 description 3
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 3
- 208000010392 Bone Fractures Diseases 0.000 description 2
- 206010037660 Pyrexia Diseases 0.000 description 2
- 210000000988 bone and bone Anatomy 0.000 description 2
- 230000007812 deficiency Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000013049 sediment Substances 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 108020004414 DNA Proteins 0.000 description 1
- 241000034042 Mauremys reevesii Species 0.000 description 1
- 206010046788 Uterine haemorrhage Diseases 0.000 description 1
- 208000031971 Yin Deficiency Diseases 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 208000013403 hyperactivity Diseases 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 210000003127 knee Anatomy 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 230000003908 liver function Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000002932 luster Substances 0.000 description 1
- 239000002398 materia medica Substances 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 206010029410 night sweats Diseases 0.000 description 1
- 230000036565 night sweats Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000001256 tonic effect Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention provides an MS (Mass Spectrometry) detection method for 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 No1. 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 field of traditional Chinese medicine detection.
Background technology
Tortoise plastron, namely the carapace of Testudinidae animal tortoise (Chinemys reevesii) and plastron, 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, bushing of nourishing blood.In the last few years, along with the raising of biology level, the correlative study of tortoise plastron also deepens continuously.There are some researches show, tortoise plastron powder and extract thereof can not only strengthen liver function, release the pressure, and also have effect that is anticancer and immunity moderation resistibility.With tortoise plastron be the two series products Guiling Jellies made of raw material and colla carapacis et plastri testudinis on whole Market of Chinese Materia Medica in occupation of important position.
Record according to version Chinese Pharmacopoeia in 2010, colla carapacis et plastri testudinis take tortoise plastron as raw material, and through soak by water, the concentrated solid gum made, 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 be sallow, uterine bleeding band is inferior.Long-term taking colla carapacis et plastri testudinis also helps and strengthens body self-regulation, can promote longevity, also be beneficial to the physical rehabilitation of hyperactivity of yang due to yin deficiency patient.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 also usually eats for people for cook various kinds tonic, in the last few years to various Chelonian 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.Colla carapacis et plastri testudinis market exists some counterfeit and shoddy goods, its make raw material be not tortoise plastron, but by other animals mix skin, broken bone replace, comprise pigskin, ox-hide, even the skin of animals died of illness, dirty skin, rotten skin etc.Such counterfeit and shoddy goods once be 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, be also probably harmful to health, consequence is very serious.
Glue class Chinese medicine forms gelatin substance by the skin of animal, bone, first-class boiling, and comprises donkey-hide gelatin, colla carapacis et plastri testudinis, deer horn glue etc., has the traditional Chinese medicine of typical ethnic characteristic.Glue class Chinese medicine generally concentrates through long-time thermophilic digestion and forms, the very close and production technology of different manufacturers of principal ingredient there are differences, and adopts infrared, the method such as near infrared, X-diffraction to carry out True-false distinguish to glue class Chinese medicine sterling and often exists and judge the situations such as inaccurate.The discriminating of glue class Chinese medicament turtle-derived component, existing scholar differentiates from DNA angle, but just qualitative, has false negative and false positive phenomenon, and operation is comparatively complicated.Have and report from the angle of polypeptide and utilize Mass Spectrometer Method glue class Chinese medicament turtle-derived component, result accurately and reliably.At present, trace to the source in the goods also not to glue class Chinese medicine qualification method.
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 accurate to glue class Chinese medicine and goods thereof trace to the source qualification and content detection.
Summary of the invention
The present invention is directed to the problems referred to above, provide the detection method of turtle-derived component in a kind of glue class Chinese medicine and goods thereof, the method is simple to operate, and characteristic is strong, highly sensitive, can be used for the qualification 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 specific nucleotide sequences 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, described glue class Chinese herbal product is selected from food, health products or the medicine be made up of corresponding glue class Chinese medicine.
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 protein and peptide component dissolves wherein, add restriction enzyme and carry out enzyme and cut;
(3) put into LC-MS instrument subsequently, with negative sample to be detected for matrix, add step (1) gained amino acid sequence or colla carapacis et plastri testudinis sterling in contrast, select the parent ion m/z442.0 of this polypeptide and daughter ion thereof to monitor.
If the retention time detecting this ion is consistent with reference substance, and its daughter ion is consistent with the daughter ion of reference substance, then described glue class Chinese medicine or its goods contain turtle-derived component; If this not consistent with reference substance retention time ion, then not containing turtle-derived component.
Preferably, to dissolve reagent used described in step (2) be mass percent is 1%, the NH of pH value 8.0
4hCO
3solution; Described restriction enzyme is trypsase.
Adopt method of the present invention, whether can detect fast in glue class Chinese medicine containing turtle-derived component, thus point evident, although collagen polypeptide has certain hydrolysis and destroys in glue class Chinese medicine, but the otherness polypeptide that the present invention is based 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 Val-Gly-Pro-Thr-Gly-Pro-Ala-Gly-Ala-Arg, and the composition be made up of described polypeptide and respective carrier.In addition, described polypeptide and the application of composition in detection glue class Chinese medicine and goods thereof in turtle-derived component thereof is also comprised.
Accompanying drawing explanation
Fig. 1 be under improvement on synthesis and sterling glue SRM scan pattern Selective ion mode to m/z442.0 → 471.3,528.3, the mass spectrograms of 629.3 monitorings;
Fig. 2 be under improvement on synthesis and the epoxy glue SRM scan pattern adding 5% colla carapacis et plastri testudinis Selective ion mode to m/z442.0 → 471.3,528.3, the mass spectrograms of 629.3 monitorings;
Fig. 3 be improvement on synthesis and colla carapacis et plastri testudinis goods (for the celestial oral liquid of tortoise deer two) under SRM scan pattern Selective ion mode to m/z442.0 → 471.3,528.3, the mass spectrograms of 629.3 monitorings.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But embodiment is only exemplary, does not form any restriction to scope of the present invention.It will be understood by those skilled in the art that and can modify to the details of technical solution of the present invention and form or replace down without departing from the spirit and scope of the present invention, but these amendments and replacement all fall within the scope of protection of the present invention.
The acquisition of specificity peptide chain in 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 obtain 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), improvement on synthesis Val-Gly-Pro-Thr-Gly-Pro-Ala-Gly-Ala-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, filtering with microporous membrane, and precision measures the trypsin solution 100 μ L that subsequent filtrate 1mL adds 2mg/mL, 37 DEG C of constant temperature enzymolysis 6h.
(2) preparation of improvement on synthesis control sample enzymolysis solution
Take a certain amount of improvement on synthesis, add 1%NH
4hCO
3solution (pH8.0), shakes up, and is mixed with concentration and is about 0.3 μ g/mL, filtering with microporous membrane, for subsequent use.
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 μm), mobile phase A is 0.1% formic acid solution (volume fraction), and Mobile phase B is acetonitrile, flow velocity 0.3mL/min; Gradient elution: 0 ~ 40min, 2% ~ 50% Mobile phase B (percentage here represents from 0 minute to the 40th minute, flowing B by 2% linear gradient be 50%, mobile phase A then fades to 50% by 98%).Mass Spectrometry Conditions: electron spray positive ion mode (ESI+) carries out one-level and entirely sweeps, sweep limit m/z400-m/z1000.
Entirely sweep figure from the mass spectrum of respective glue sample and extract ion m/z442.0, find to only have containing this quasi-molecular ions in colla carapacis et plastri testudinis at about 4.45min by contrast, and all do not have in donkey-hide gelatin, horse skin glue, oxhide gelatin, pig skin gelatin and deer horn glue.Show that above-mentioned conclusion is correct by the detection of multiple batches of sample and checking.Illustrate: by detecting the ion m/z442.0 in colla carapacis et plastri testudinis, can determine whether containing turtle-derived component.
(2) the amino acid sequence initial guess of polypeptide
Utilize triple quadrupole bar mass spectrum, daughter ion scanning is carried out to the above-mentioned ion found out, confirm this ion band double charge, by sequence assembly, infer the partial sequence 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 883.0 by molecular weight after tryptic digestion), find out the sequence Val-Gly-Pro-Thr-Gly-Pro-Ala-Gly-Ala-Arg that this polypeptide is possible.According to the CID fracture theory of peptide chain, the daughter ion calculating this polypeptide possible is as follows:
| a | b | c" | Res: | x | y" | z |
| 72.1 | 100.1 | 119.1 | Val | - | - | - |
| 129.1 | 157.1 | 176.1 | Gly | 807.4 | 783.4 | 764.4 |
| 226.2 | 254.2 | 273.2 | Pro | 750.4 | 726.4 | 707.3 |
| 327.2 | 355.2 | 374.2 | Thr | 653.3 | 629.3 | 610.3 |
| 384.2 | 412.2 | 431.3 | Gly | 552.3 | 528.3 | 509.2 |
| 481.3 | 509.3 | 528.3 | Pro | 495.2 | 471.3 | 452.2 |
| 552.3 | 580.3 | 599.4 | Ala | 398.2 | 374.2 | 355.2 |
| 609.3 | 637.3 | 656.4 | Gly | 327.1 | 303.2 | 284.1 |
| 680.4 | 708.4 | 727.4 | Ala | 270.1 | 246.2 | 227.1 |
| - | - | - | Arg | 199.1 | 175.1 | 156.1 |
Coincideing in figure can be entirely swept with the daughter ion of peculiar ion m/z442.0 in colla carapacis et plastri testudinis.
(3) sequence is determined
Amino acid sequence by inference, entrusts Peptide systhesis company (the biochemical company limited of gill) to carry out the synthesis of this polypeptide.Then the daughter ion utilizing LC-MS instrument to carry out improvement on synthesis is swept entirely, and in its testing conditions and glue sample, the daughter ion of corresponding polypeptide is swept consistent entirely.Entirely sweep figure by the ion appearance time, the daughter ion that contrast improvement on synthesis and colla carapacis et plastri testudinis, both discoveries fit like a glove, thus determine that this ion m/z442.0 is polypeptide Val-Gly-Pro-Thr-Gly-Pro-Ala-Gly-Ala-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, decocts obtain with donkey hide, horse skin, ox-hide, pigskin, tortoise plastron, deer horn respectively.
Epoxy glue sample (oxhide gelatin, the oxhide gelatin of 10% colla carapacis et plastri testudinis, the oxhide gelatin of 20% colla carapacis et plastri testudinis, the oxhide gelatin of 40% colla carapacis et plastri testudinis containing 5% colla carapacis et plastri testudinis) is accurately mixed (percentage is massfraction) by above-mentioned sterling glue.
Reagent: ammonium bicarbonate (analyzing pure), trypsase (sequence is pure, purchased from National Institute for Food and Drugs Control), improvement on synthesis Val-Gly-Pro-Thr-Gly-Pro-Ala-Gly-Ala-Arg.
2 detection methods
(1) preparation of improvement on synthesis 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, filtering with microporous membrane, and precision measures subsequent filtrate 1ml and adds improvement on synthesis 0.3 μ g, then adds the trypsin solution 100 μ l of 2mg/ml, 37 DEG C 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, filtering with microporous membrane, and precision measures the trypsin solution 100 μ l that subsequent filtrate 1ml adds 2mg/ml, 37 DEG C 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 μm), mobile phase A is 0.1% formic acid solution (volume fraction), and Mobile phase B is acetonitrile, flow velocity 0.3ml/min; Gradient elution: 0 ~ 40min, 2% ~ 50% Mobile phase B (percentage here represents from 0 minute to the 40th minute, flowing B by 2% linear gradient be 50%, mobile phase A then fades to 50% by 98%).Mass Spectrometry Conditions: electron spray positive ion mode (ESI+) is selected to carry out many reaction detection, selects m/z442.0 → 471.3,528.3,629.3 as detecting ion pair.
The results are shown in Figure Isosorbide-5-Nitrae .45min place to only have in improvement on synthesis 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, with the percentage composition of colla carapacis et plastri testudinis for horizontal ordinate, with chromatographic peak area in m/z442.0 → 528.3 extraction ion flow graph for ordinate, drawing standard curve, R
2be more than 0.999, linearly well.This method visible can specific detection turtle-derived component, comprises donkey, horse, ox, pig, deer etc. distinguish with other various animal derived materials.
Embodiment 2
1 material and reagent
Material: epoxy glue sample adds 5% colla carapacis et plastri testudinis respectively by the sterling glue sample in embodiment 1 and makes, comprises donkey-hide gelatin, the horse skin glue of 5% colla carapacis et plastri testudinis, the oxhide gelatin containing 5% colla carapacis et plastri testudinis, the pig skin gelatin of 5% colla carapacis et plastri testudinis, the deer horn glue containing 5% colla carapacis et plastri testudinis containing 5% colla carapacis et plastri testudinis.
Reagent: ammonium bicarbonate (analyzing pure), trypsase (sequence is pure, purchased from National Institute for Food and Drugs Control), improvement on synthesis Val-Gly-Pro-Thr-Gly-Pro-Ala-Gly-Ala-Arg.
2 detection methods
(1) preparation of improvement on synthesis 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, filtering with microporous membrane, and precision measures subsequent filtrate 1ml and adds improvement on synthesis 0.3 μ g, then adds the trypsin solution 100 μ l of 2mg/ml, 37 DEG C 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, filtering with microporous membrane, and precision measures the trypsin solution 100 μ l that subsequent filtrate 1ml adds 2mg/ml, 37 DEG C 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 μm), mobile phase A is 0.1% formic acid solution (volume fraction), and 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, flowing B by 2% linear gradient be 50%, mobile phase A then fades to 50% by 98%).Mass Spectrometry Conditions: electron spray positive ion mode (ESI
+) select to carry out many reaction detection, select m/z442.0 → 471.3,528.3,629.3 as detecting ion pair.
The results are shown in Figure 2,4.45min place's improvement on synthesis control sample, add 5% colla carapacis et plastri testudinis epoxy glue sample in 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.This method visible can turtle-derived component in specific detection glue class Chinese medicine.
Embodiment 3
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), improvement on synthesis Val-Gly-Pro-Thr-Gly-Pro-Ala-Gly-Ala-Arg.
2 detection methods
(1) preparation of improvement on synthesis control sample enzymolysis solution
Get 1.0g and do not add 1%NH containing the celestial oral liquid sample of tortoise deer two of colla carapacis et plastri testudinis
4hCO
3solution 24ml dissolves, and adds 4g trichloroacetic acid, places the centrifugal 5min of 10min, 10000rpm, abandons supernatant, proceeded to by sediment in 100ml measuring bottle, use 1%NH for 4 DEG C
4hCO
3solution (pH8.0) dissolves and is settled to scale, shakes up, filtering with microporous membrane, and precision measures subsequent filtrate 1ml and adds improvement on synthesis 0.3 μ g, then adds the trypsin solution 100 μ l of 2mg/ml, 37 DEG C 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 the centrifugal 5min of 10min, 10000rpm, abandons supernatant, proceeded to by sediment in 100ml measuring bottle, use 1%NH for 4 DEG C
4hCO
3solution (pH8.0) dissolves and is settled to scale, shakes up, filtering with microporous membrane, and precision measures the trypsin solution 100 μ l that subsequent filtrate 1ml adds 2mg/ml, 37 DEG C 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 μm), mobile phase A is 0.1% formic acid solution (volume fraction), and 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, flowing B by 2% linear gradient be 50%, mobile phase A then fades to 50% by 98%).Mass Spectrometry Conditions: electron spray positive ion mode (ESI+) carries out many reaction detection, selects m/z442.0 → 471.3,528.3,629.3 as detecting ion pair.。
The results are shown in Figure 3,4.45min place improvement on synthesis control sample, colla carapacis et plastri testudinis and the celestial oral liquid of tortoise deer two containing colla carapacis et plastri testudinis detect corresponding quasi-molecular ions, other do not detect, this method visible can turtle-derived component in specific detection colla carapacis et plastri testudinis goods, and comprises donkey, horse, ox, pig, deer etc. with other various animal derived materials and distinguish.
Claims (8)
1. the detection method of turtle-derived component in glue class Chinese medicine and goods thereof, it utilizes the difference in tortoise genome with specific nucleotide sequences or amino acid sequence to detect, and described specific amino acid is as shown in SEQ.ID No1; Described glue class Chinese medicine is selected from donkey-hide gelatin, oxhide gelatin, colla carapacis et plastri testudinis or deer horn glue.
2. method according to claim 1, is characterized in that, described glue class Chinese herbal product is selected from food, health products or the medicine be made up of corresponding glue class Chinese medicine.
3. the method according to claim 1 ~ 2 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 protein and peptide component dissolves wherein, add restriction enzyme and carry out enzyme and cut; To dissolve reagent used described in step (2) be mass percent is 1%, the NH of pH value 8.0
4hCO
3solution; Described restriction enzyme is trypsase;
(3) put into LC-MS instrument subsequently, with negative sample to be detected for matrix, add step (1) gained amino acid sequence or colla carapacis et plastri testudinis sterling in contrast, select the parent ion m/z442.0 of this polypeptide and daughter ion thereof to monitor.
4. 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.
5. the gene of coding molecular marked compound according to claim 4.
6. molecular marked compound according to claim 4 is detecting the application of turtle-derived component in glue class Chinese medicine and goods thereof.
7. a pharmaceutical composition, is characterized in that, comprises molecular marked compound according to claim 4 and one or more pharmaceutically acceptable carriers of pharmacy effective dose.
8. pharmaceutical composition according to claim 7 is detecting the application of turtle-derived component in glue class Chinese medicine and goods thereof.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310526412.4A CN103630635B (en) | 2013-10-30 | 2013-10-30 | MS (Mass Spectrometry) detection method for tortoise-derived ingredients in glue type traditional Chinese medicines and products thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310526412.4A CN103630635B (en) | 2013-10-30 | 2013-10-30 | MS (Mass Spectrometry) detection method for tortoise-derived ingredients in glue type traditional Chinese medicines and products thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103630635A CN103630635A (en) | 2014-03-12 |
| CN103630635B true CN103630635B (en) | 2015-06-24 |
Family
ID=50211906
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310526412.4A Active CN103630635B (en) | 2013-10-30 | 2013-10-30 | MS (Mass Spectrometry) detection method for tortoise-derived ingredients in glue type traditional Chinese medicines and products thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103630635B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105837676B (en) * | 2016-03-18 | 2020-07-31 | 青岛海关技术中心 | Polypeptide for identifying bovine-derived components in donkey-hide gelatin and products thereof |
| 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 |
| CN111060622B (en) * | 2019-12-26 | 2021-06-29 | 北京化工大学 | A labeled peptide of tuna-derived components and its application in the detection of collagen degradation products and their products |
| CN114487207A (en) * | 2022-02-17 | 2022-05-13 | 杭州市食品药品检验研究院(杭州市食品药品审核查验服务中心、杭州市药品与医疗器械不良反应监测中心) | Liquid chromatography-mass spectrometry combined use method for simultaneously identifying 10 Chinese medicines of tortoise-turtle shell |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101838684A (en) * | 2009-12-25 | 2010-09-22 | 华东理工大学 | PCR identification method of Chinese medicament turtle-derived component |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2446886A1 (en) * | 2001-04-06 | 2002-10-17 | Biovision Ag | Method for detecting chronic dementia diseases, and corresponding peptides and detection reagents |
-
2013
- 2013-10-30 CN CN201310526412.4A patent/CN103630635B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101838684A (en) * | 2009-12-25 | 2010-09-22 | 华东理工大学 | PCR identification method of Chinese medicament turtle-derived component |
Non-Patent Citations (1)
| Title |
|---|
| 明胶制备工艺及溯源方法研究;王前;《中国优秀硕士学位论文全文数据库》;20100715(第7期);第56-65页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103630635A (en) | 2014-03-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103630620B (en) | Method for detecting deer-derived ingredients in glue type traditional Chinese medicines and products thereof | |
| CN103630621B (en) | Method for detecting sheep-derived ingredients in glue type traditional Chinese medicines and products thereof | |
| Sherpa et al. | How the ends signal the end: Regulation by E3 ubiquitin ligases recognizing protein termini | |
| CN103630634B (en) | The detection thing of a kind of colla carapacis et plastri testudinis, deer horn glue and goods thereof and detection method thereof | |
| Park et al. | Evaluation of polyphenols from Broussonetia papyrifera as coronavirus protease inhibitors | |
| Feng et al. | Purification, identification, and sensory evaluation of kokumi peptides from agaricus bisporus mushroom | |
| CN104280468B (en) | The detection method of horse kind and mule kind derived component in a kind of glue class Chinese medicine and goods thereof | |
| CN103383383B (en) | The detection method of pig derived component in a kind of glue class Chinese medicine and goods thereof | |
| CN103630619B (en) | Detection substance for tortoise-shell glue and products thereof and MS (Mass Spectrometry) detection method thereof | |
| CN103630635B (en) | MS (Mass Spectrometry) detection method for tortoise-derived ingredients in glue type traditional Chinese medicines and products thereof | |
| CN103630644B (en) | The LC-MS detection method of turtle-derived component in a kind of glue class Chinese medicine and goods thereof | |
| CN112048000B (en) | A kind of buffalo horn characteristic peptide segment and detection method thereof | |
| CN105255978A (en) | Coilia mystus Maillard peptide with memory improvement function as well as preparation method and application of coilia mystus Maillard peptide | |
| Zhang et al. | Characterization of peanut protein hydrolysate and structural identification of umami-enhancing peptides | |
| Lu et al. | Assessment of antibacterial properties and the active ingredient of plant extracts and its effect on the performance of crucian carp (Carassius auratus gibelio var. E'erqisi, Bloch) | |
| CN114113381A (en) | A kind of Shu's sea dragon characteristic polypeptide and its application and identification method of comfortable sea dragon | |
| Ambrosio et al. | Natural agents as novel potential source of proteasome inhibitors with anti-tumor activity: focus on multiple myeloma | |
| CN103630646B (en) | The detection method of turtle-derived component in a kind of glue class Chinese medicine and goods thereof | |
| Cheong et al. | Cloning, overexpression, purification, and modeling of a lectin (Rhinocelectin) with antiproliferative activity from tiger milk mushroom, Lignosus rhinocerus | |
| Bernier et al. | Enzymatic hydrolysis of water lentil (duckweed): An emerging source of proteins for the production of antihypertensive fractions | |
| Ippoushi et al. | Absolute quantification of Pru av 2 in sweet cherry fruit by liquid chromatography/tandem mass spectrometry with the use of a stable isotope-labelled peptide | |
| CN103630623A (en) | Substance and method for detecting tortoise-shell glue and products thereof | |
| CN108828108A (en) | The method of many animals derived component in product containing gelatin crude drug is detected simultaneously | |
| Gębalski et al. | Eleutherococcus divaricatus fruits decrease hyaluronidase activity in blood serum and protect from oxidative damages in in vitro model | |
| Kowalczewski et al. | Bioactive substances of potato juice reveal synergy in cytotoxic activity against cancer cells of digestive system studied in vitro |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |