CN112345655A - Establishing method of wasp venom fingerprint, wasp venom fingerprint and application of wasp venom fingerprint - Google Patents
Establishing method of wasp venom fingerprint, wasp venom fingerprint and application of wasp venom fingerprint Download PDFInfo
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- 239000002578 wasp venom Substances 0.000 title claims abstract description 197
- 238000000034 method Methods 0.000 title claims abstract description 103
- QZAYGJVTTNCVMB-UHFFFAOYSA-N serotonin Chemical compound C1=C(O)C=C2C(CCN)=CNC2=C1 QZAYGJVTTNCVMB-UHFFFAOYSA-N 0.000 claims abstract description 130
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 67
- 229940076279 serotonin Drugs 0.000 claims abstract description 64
- 238000001228 spectrum Methods 0.000 claims abstract description 52
- 239000012085 test solution Substances 0.000 claims abstract description 49
- 238000004458 analytical method Methods 0.000 claims abstract description 25
- 239000007864 aqueous solution Substances 0.000 claims abstract description 16
- 239000012088 reference solution Substances 0.000 claims abstract description 16
- 238000003908 quality control method Methods 0.000 claims abstract description 8
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 98
- 230000014759 maintenance of location Effects 0.000 claims description 73
- 239000002435 venom Substances 0.000 claims description 46
- 231100000611 venom Toxicity 0.000 claims description 46
- 210000001048 venom Anatomy 0.000 claims description 46
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 27
- 239000000243 solution Substances 0.000 claims description 23
- 238000010828 elution Methods 0.000 claims description 17
- 241000797328 Vespa velutina auraria Species 0.000 claims description 15
- 238000002360 preparation method Methods 0.000 claims description 9
- 238000001514 detection method Methods 0.000 claims description 8
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 7
- 239000000463 material Substances 0.000 claims description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
- 239000006228 supernatant Substances 0.000 claims description 5
- 238000009210 therapy by ultrasound Methods 0.000 claims description 4
- 239000000945 filler Substances 0.000 claims description 2
- 239000003814 drug Substances 0.000 abstract description 10
- 238000011161 development Methods 0.000 abstract description 4
- 230000000694 effects Effects 0.000 abstract description 3
- 229940079593 drug Drugs 0.000 abstract description 2
- 239000002547 new drug Substances 0.000 abstract description 2
- 230000035945 sensitivity Effects 0.000 abstract description 2
- 238000000926 separation method Methods 0.000 abstract description 2
- 238000012360 testing method Methods 0.000 description 28
- 239000012488 sample solution Substances 0.000 description 22
- 241000256861 Vespa mandarinia Species 0.000 description 17
- 238000011156 evaluation Methods 0.000 description 16
- 239000007788 liquid Substances 0.000 description 15
- 239000000523 sample Substances 0.000 description 15
- 238000013112 stability test Methods 0.000 description 12
- 241001164827 Vespa velutina Species 0.000 description 9
- 241000256860 Vespa Species 0.000 description 6
- 238000002347 injection Methods 0.000 description 6
- 239000007924 injection Substances 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- 229910021642 ultra pure water Inorganic materials 0.000 description 6
- 239000012498 ultrapure water Substances 0.000 description 6
- 241000256856 Vespidae Species 0.000 description 5
- 238000010521 absorption reaction Methods 0.000 description 4
- 238000000862 absorption spectrum Methods 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 238000012937 correction Methods 0.000 description 4
- 238000007405 data analysis Methods 0.000 description 4
- 238000011835 investigation Methods 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 239000000203 mixture Substances 0.000 description 3
- 108090000765 processed proteins & peptides Proteins 0.000 description 3
- 238000005303 weighing Methods 0.000 description 3
- NTYJJOPFIAHURM-UHFFFAOYSA-N Histamine Chemical compound NCCC1=CN=CN1 NTYJJOPFIAHURM-UHFFFAOYSA-N 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 239000012982 microporous membrane Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 230000000144 pharmacologic effect Effects 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 101710091342 Chemotactic peptide Proteins 0.000 description 1
- 108091035707 Consensus sequence Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 240000003085 Quassia amara Species 0.000 description 1
- 235000009694 Quassia amara Nutrition 0.000 description 1
- 241000219492 Quercus Species 0.000 description 1
- 241000698291 Rugosa Species 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 239000013566 allergen Substances 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 230000000202 analgesic effect Effects 0.000 description 1
- 230000002429 anti-coagulating effect Effects 0.000 description 1
- 230000002785 anti-thrombosis Effects 0.000 description 1
- 239000003659 bee venom Substances 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 229960001340 histamine Drugs 0.000 description 1
- 210000003630 histaminocyte Anatomy 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 229940013788 quassia Drugs 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000013558 reference substance Substances 0.000 description 1
- 238000003260 vortexing Methods 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
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Abstract
The invention relates to the technical field of medicines, in particular to a method for establishing a wasp venom fingerprint, a wasp venom fingerprint and application of the wasp venom fingerprint. The method provided by the invention comprises the following steps: taking the aqueous solution of the wasp venom as a test solution and the aqueous solution of serotonin as a reference solution, respectively carrying out high performance liquid chromatography analysis on the reference solution and the test solution, and taking the chromatographic peak of the reference solution as a reference to obtain the wasp venom fingerprint. The method provided by the invention is simple and convenient to operate, good in repeatability and high in precision, the obtained wasp venom fingerprint spectrum is good in separation effect and high in sensitivity, and the method can be simply and conveniently applied to quality control of wasp venom, provides guarantee for quality control of the wasp venom, and provides early-stage basis for development and development of related new drugs in the future.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a method for establishing a wasp venom fingerprint, a wasp venom fingerprint and application of the wasp venom fingerprint.
Background
In recent decades, wasp venom has attracted more and more attention, and research shows that active substances in wasp venom mainly include macromolecular proteins (such as various enzymes, allergens and the like), bioactive amines (including histamine, 5-hydroxytryptamine and the like) and small peptide molecules (including mast cell degranulation peptides, chemotactic peptides and the like). Currently, researches on wasp venom mainly focus on chemical components, pharmacological effects and the like of the wasp venom, researchers have purified and identified the small peptide molecules from the venom of various wasps, and research reports show that the wasp venom has various pharmacological effects such as tumor resistance, virus resistance, inflammation resistance, bacteria resistance, radiation resistance and the like. Modern pharmacology and clinical research show that bee venom also has a plurality of biological functions, such as analgesic effect, anticoagulant effect, antithrombotic activity and the like.
The wasp venom has high price and complex chemical components, and factors such as producing area, source, harvesting season and the like have great influence on the chemical components and curative effect of the wasp venom. Therefore, there is a need to develop a quality control method for wasp venom, so as to ensure the system, comprehensively reflect the quality of wasp venom, ensure the effectiveness and controllability of clinical application, distinguish from other fake products, and reduce unnecessary loss.
Disclosure of Invention
The invention aims to provide a method for establishing a wasp venom fingerprint, a wasp venom fingerprint and application of the wasp venom fingerprint.
The invention provides a method for establishing a wasp venom fingerprint, which comprises the following steps:
taking the aqueous solution of the wasp venom as a test solution and the aqueous solution of serotonin as a reference solution, respectively carrying out high performance liquid chromatography analysis on the reference solution and the test solution, and taking the chromatographic peak of the reference solution as a reference to obtain the wasp venom fingerprint;
the chromatographic conditions of the high performance liquid chromatography are as follows:
mobile phase: the mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is an acetonitrile solution of trifluoroacetic acid, and the volume percentage of the trifluoroacetic acid in the mobile phase A is 0.05-0.2%; the mobile phase B is an aqueous solution of trifluoroacetic acid, and the volume percentage of the trifluoroacetic acid in the mobile phase B is 0.05-0.2%;
gradient elution procedure: increasing the content of the mobile phase A from 5-6% to 77-78% in 0-40 min; and (3) increasing the mobile phase A from 77-78% to 95% in 40-58 min.
Preferably, the wasp venom comprises wasp venom, Quercus apiacea venom, wasp venom or Vespa velutina aurantiaca venom.
Preferably, the high performance liquid chromatography is performed by using a DAD detector, and the detection wavelength is 215 nm;
the chromatographic column is a ZORBAX 300SB-C18 chromatographic column with the specification as follows: the length is 250mm, the inner diameter is 4.6mm, and the grain diameter of the filler is 5 mu m; the column temperature is 20-35 ℃;
the flow rate of the mobile phase is 0.8-1.2 mL/min.
Preferably, when the wasp venom is wasp venom, the chromatographic conditions of the high performance liquid chromatography are as follows:
mobile phase: the volume percentage content of the trifluoroacetic acid in the mobile phase A is 0.1 percent, and the volume percentage content of the trifluoroacetic acid in the mobile phase B is 0.1 percent;
gradient elution procedure: increasing the content of the mobile phase A from 5% to 77% in 0-40 min; increasing the content of the mobile phase A from 77% to 95% in 40-58 min;
the column temperature is 25 ℃, and the flow rate of the mobile phase is 1 mL/min;
the obtained wasp venom fingerprint is a wasp venom fingerprint, and contains 12 common fingerprint peaks, the reference peak is a serotonin chromatographic peak, and the relative retention time of the 12 common fingerprint peaks is respectively as follows: 0.481 + -0.005 of No. 1 peak, 0.509 + -0.004 of No. 2 peak, 0.638 + -0.005 of No. 3 peak, 0.790 + -0.004 of No. 4 peak, 1.000 + -0.000 of No. 5 peak, 1.791 + -0.010 of No. 6 peak, 2.594 + -0.016 of No. 7 peak, 3.824 + -0.029 of No. 8 peak, 3.976 + -0.029 of No. 9 peak, 4.224 + -0.029 of No. 10 peak, 4.295 + -0.026 of No. 11 peak, and 4.610 + -0.031 of No. 12 peak.
Preferably, when the wasp venom is the vespid venom, the chromatographic conditions of the high performance liquid chromatography are as follows:
mobile phase: the volume percentage content of the trifluoroacetic acid in the mobile phase A is 0.1 percent, and the volume percentage content of the trifluoroacetic acid in the mobile phase B is 0.1 percent;
gradient elution procedure: increasing the content of the mobile phase A from 6% to 78% in 0-40 min; increasing the content of the mobile phase A from 78% to 95% in 40-58 min;
the column temperature is 27 ℃, and the flow rate of the mobile phase is 1 mL/min;
the obtained wasp venom fingerprint is a female wasp venom fingerprint and contains 13 common fingerprint peaks, the reference peak is a serotonin chromatographic peak, and the relative retention time of the 13 common fingerprint peaks is respectively as follows: 0.476 + -0.012 for peak 1, 0.643 + -0.005 for peak 2, 0.788 + -0.009 for peak 3, 1.000 + -0.000 for peak 4, 2.370 + -0.064 for peak 5, 2.653 + -0.034 for peak 6, 3.105 + -0.073 for peak 7, 3.178 + -0.075 for peak 8, 3.357 + -0.038 for peak 9, 3.703 + -0.055 for peak 10, 3.757 + -0.063 for peak 11, 4.106 + -0.058 for peak 12, 4.349 + -0.06 for peak 13.
Preferably, when the wasp venom is wasp venom with yellow waist, the chromatographic conditions of the high performance liquid chromatography are as follows:
mobile phase: the volume percentage content of the trifluoroacetic acid in the mobile phase A is 0.1 percent, and the volume percentage content of the trifluoroacetic acid in the mobile phase B is 0.1 percent;
gradient elution procedure: increasing the content of the mobile phase A from 5% to 77% in 0-40 min; increasing the content of the mobile phase A from 77% to 95% in 40-58 min;
the column temperature is 25 ℃, and the flow rate of the mobile phase is 1 mL/min;
the obtained wasp venom fingerprint is a wasp venom fingerprint with yellow waist, and contains 10 common fingerprint peaks, the reference peak is a serotonin chromatographic peak, and the relative retention time of the 10 common fingerprint peaks is respectively as follows: 0.493 +/-0.008 for peak 1, 0.642 +/-0.007 for peak 2, 1.000 +/-0.000 for peak 3, 1.723 +/-0.025 for peak 4, 2.523 +/-0.058 for peak 5, 2.925 +/-0.084 for peak 6, 3.927 +/-0.147 for peak 7, 4.124 +/-0.094 for peak 8, 4.458 +/-0.104 for peak 9 and 4.753 +/-0.104 for peak 10.
Preferably, when the wasp venom is a basic wasp venom, the chromatographic conditions of the high performance liquid chromatography are as follows:
mobile phase: the volume percentage content of the trifluoroacetic acid in the mobile phase A is 0.1 percent, and the volume percentage content of the trifluoroacetic acid in the mobile phase B is 0.1 percent;
gradient elution procedure: increasing the content of the mobile phase A from 5% to 77% in 0-40 min; increasing the content of the mobile phase A from 77% to 95% in 40-58 min;
the column temperature is 25 ℃, and the flow rate of the mobile phase is 1 mL/min;
the obtained wasp venom fingerprint is a basic wasp venom fingerprint and contains 12 common fingerprint peaks, the reference peak is a serotonin chromatographic peak, and the relative retention time of the 12 common fingerprint peaks is respectively as follows: 0.449 +/-0.001 of the No. 1 peak, 0.810 +/-0.026 of the No. 2 peak, 1.000 +/-0.000 of the No. 3 peak, 3.111 +/-0.021 of the No. 4 peak, 3.478 +/-0.011 of the No. 5 peak, 3.756 +/-0.012 of the No. 6 peak, 3.934 +/-0.012 of the No. 7 peak, 4.188 +/-0.012 of the No. 8 peak, 4.576 +/-0.014 of the No. 9 peak, 4.715 +/-0.014 of the No. 10 peak, 4.800 +/-0.014 of the No. 11 peak and 5.938 +/-0.017 of the No. 12 peak.
Preferably, the method for preparing the test solution comprises the following steps:
mixing wasp venom with water, performing ultrasonic treatment, centrifuging the obtained ultrasonic material, and taking the obtained supernatant as a test solution; the dosage ratio of the wasp venom to the water is (1-15) mg: (2-80) mL.
The invention provides the wasp venom fingerprint spectrum obtained by the establishing method of the wasp venom fingerprint spectrum in the technical scheme.
The invention provides application of the wasp venom fingerprint spectrum in the technical scheme in wasp venom quality control.
The invention provides a method for establishing a wasp venom fingerprint. The method provided by the invention is simple and convenient to operate, good in repeatability and high in precision, the obtained wasp venom fingerprint spectrum is good in separation effect and high in sensitivity, and the method can be simply and conveniently applied to quality control of wasp venom, provides guarantee for quality control of the wasp venom, and provides early-stage basis for development and development of related new drugs in the future.
Drawings
FIG. 1 is a comparison of Vespa mandarinia venom with serotonin by HPLC;
FIG. 2 is a graph showing the ultraviolet absorption curve of peak S and serotonin in HPLC of Vespa mandarinia venom;
FIG. 3 is an HPLC fingerprint of 20 batches of Vespa mandarinia venom;
FIG. 4 is a common mode comparison spectrum R for similarity evaluation of rhizoma Vespa longissima venom fingerprint;
FIG. 5 is a HPLC comparison of Vespa velutina auraria venom and serotonin;
FIG. 6 is a graph showing the UV absorption of serotonin at the S peak in an HPLC chart of Vespa mandarinia rugosa venom;
FIG. 7 is an HPLC fingerprint of 20 batches of Vespa velutina auraria venom;
FIG. 8 is a common mode control spectrum R for the evaluation of the finger print similarity of Vespa velutina auraria venom;
FIG. 9 is a HPLC comparison of wasp venom and serotonin;
FIG. 10 is a graph showing the UV absorption curve of the peak S and serotonin in the HPLC chart of wasp venom from Huangyao;
FIG. 11 is an HPLC fingerprint of 20 batches of wasp venom;
FIG. 12 is a common mode comparison spectrum R for similarity evaluation of finger prints of wasp venom from Huangyao;
FIG. 13 is a HPLC comparison of Vespa mandarinia venom with serotonin;
FIG. 14 is a graph of the UV absorption of serotonin and the S peak in an HPLC chart of Vespa mandarinia venom;
FIG. 15 is an HPLC fingerprint of 20 batches of Vespa velutina auraria smith venom;
FIG. 16 is a consensus pattern control plot R of the similarity evaluation of the finger prints of Vespa velutina venom.
Detailed Description
The invention provides a method for establishing a wasp venom fingerprint, which comprises the following steps:
taking the aqueous solution of the wasp venom as a test solution and the aqueous solution of serotonin as a reference solution, respectively carrying out high performance liquid chromatography analysis on the reference solution and the test solution, and taking the chromatographic peak of the reference solution as a reference to obtain the wasp venom fingerprint;
the chromatographic conditions of the high performance liquid chromatography are as follows:
mobile phase: the mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is an acetonitrile solution of trifluoroacetic acid, and the volume percentage of the trifluoroacetic acid in the mobile phase A is 0.05-0.2%; the mobile phase B is an aqueous solution of trifluoroacetic acid, and the volume percentage of the trifluoroacetic acid in the mobile phase B is 0.05-0.2%;
gradient elution procedure: increasing the content of the mobile phase A from 5-6% to 77-78% in 0-40 min; and (3) increasing the mobile phase A from 77-78% to 95% in 40-58 min.
In the present invention, the wasp venom preferably comprises wasp venom, vespa velutina aurantiaca venom, wasp venom or vespa velutina aurantiaca venom, specifically venom produced by wasps, vespa velutina aurantiaca, wasps giraldii or vespa velutina aurantiaca of the vespidae genus. The wasp venom provided by the invention specifically refers to crude wasp venom raw materials. In the present invention, the method for preparing the test solution preferably comprises the steps of: mixing wasp venom with water, performing ultrasonic treatment, centrifuging the obtained ultrasonic material, and taking the obtained supernatant as a test solution; the preferable dosage ratio of the wasp venom to the water is (1-15) mg: (2-80) mL, more preferably 15 mg: 2 mL; the water is preferably ultrapure water. In the embodiment of the invention, the solution of the Vespa mandarinia venom is precisely weighed and placed in a conical flask with a plug, ultrapure water is added, the mixture is swirled for 0.5min, the mixture is ultrasonically treated for 10min after being uniformly mixed, and the mixture is placed at room temperature and then centrifuged for 10min at 10000 rpm; filtering the supernatant with 0.45 μm microporous membrane, collecting the filtrate, and shaking to obtain sample solution.
In the present invention, the concentration of the control solution is preferably 1 mg/mL; in the embodiment of the invention, serotonin is precisely weighed and added with ultrapure water to prepare a reference substance solution with the concentration of 1mg/mL for later use.
After obtaining the reference solution and the test solution, respectively carrying out high performance liquid chromatography analysis on the reference solution and the test solution, and obtaining the finger print of the wasp venom by taking the chromatographic peak of the reference solution as a reference. The following description is made with respect to the high performance liquid chromatography conditions and the obtained finger print of each wasp venom:
(1) when the wasp venom is the wasp venom, the chromatographic conditions of the high performance liquid chromatography are as follows:
mobile phase: the mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is an acetonitrile solution of trifluoroacetic acid, and the volume percentage content of the trifluoroacetic acid in the mobile phase A is 0.1%; the mobile phase B is an aqueous solution of trifluoroacetic acid, and the volume percentage content of the trifluoroacetic acid in the mobile phase B is 0.1%;
gradient elution procedure: increasing the content of the mobile phase A from 5% to 77% in 0-40 min; increasing the content of the mobile phase A from 77% to 95% in 40-58 min; the flow rate of the mobile phase is 1 mL/min;
a DAD detector is adopted, and the detection wavelength is 215 nm; the sample injection amount is 5 mu L; a chromatographic column: ZORBAX 300SB-C18(4.6 mm. times.250 mm, 5 μm) with a column temperature of 25 ℃;
the obtained wasp venom fingerprint is a wasp venom fingerprint, and contains 12 common fingerprint peaks, the reference peak is a serotonin chromatographic peak, and the relative retention time of the 12 common fingerprint peaks is respectively as follows: 0.481 + -0.005 of No. 1 peak, 0.509 + -0.004 of No. 2 peak, 0.638 + -0.005 of No. 3 peak, 0.790 + -0.004 of No. 4 peak, 1.000 + -0.000 of No. 5 peak, 1.791 + -0.010 of No. 6 peak, 2.594 + -0.016 of No. 7 peak, 3.824 + -0.029 of No. 8 peak, 3.976 + -0.029 of No. 9 peak, 4.224 + -0.029 of No. 10 peak, 4.295 + -0.026 of No. 11 peak, and 4.610 + -0.031 of No. 12 peak.
(2) When the wasp venom is the vespid venom, the chromatographic conditions of the high performance liquid chromatography are as follows:
mobile phase: the mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is an acetonitrile solution of trifluoroacetic acid, and the volume percentage content of the trifluoroacetic acid in the mobile phase A is 0.1%; the mobile phase B is an aqueous solution of trifluoroacetic acid, and the volume percentage content of the trifluoroacetic acid in the mobile phase B is 0.1%;
gradient elution procedure: increasing the content of the mobile phase A from 6% to 78% in 0-40 min; increasing the content of the mobile phase A from 78% to 95% in 40-58 min; the flow rate of the mobile phase is 1 mL/min;
a DAD detector is adopted, and the detection wavelength is 215 nm; the sample injection amount is 5 mu L; a chromatographic column: ZORBAX 300SB-C18(4.6 mm. times.250 mm, 5 μm) at a column temperature of 27 ℃;
the obtained wasp venom fingerprint is a female wasp venom fingerprint and contains 13 common fingerprint peaks, the reference peak is a serotonin chromatographic peak, and the relative retention time of the 13 common fingerprint peaks is respectively as follows: 0.476 + -0.012 for peak 1, 0.643 + -0.005 for peak 2, 0.788 + -0.009 for peak 3, 1.000 + -0.000 for peak 4, 2.370 + -0.064 for peak 5, 2.653 + -0.034 for peak 6, 3.105 + -0.073 for peak 7, 3.178 + -0.075 for peak 8, 3.357 + -0.038 for peak 9, 3.703 + -0.055 for peak 10, 3.757 + -0.063 for peak 11, 4.106 + -0.058 for peak 12, 4.349 + -0.06 for peak 13.
(3) When the wasp venom is the wasp venom with yellow waist, the chromatographic conditions of the high performance liquid chromatography are as follows:
mobile phase: the mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is an acetonitrile solution of trifluoroacetic acid, and the volume percentage content of the trifluoroacetic acid in the mobile phase A is 0.1%; the mobile phase B is an aqueous solution of trifluoroacetic acid, and the volume percentage content of the trifluoroacetic acid in the mobile phase B is 0.1%;
gradient elution procedure: increasing the content of the mobile phase A from 5% to 77% in 0-40 min; increasing the content of the mobile phase A from 77% to 95% in 40-58 min; the flow rate of the mobile phase is 1 mL/min;
a DAD detector is adopted, and the detection wavelength is 215 nm; the sample injection amount is 5 mu L; a chromatographic column: ZORBAX 300SB-C18(4.6 mm. times.250 mm, 5 μm) with a column temperature of 25 ℃;
the obtained wasp venom fingerprint is a wasp venom fingerprint with yellow waist, and contains 10 common fingerprint peaks, the reference peak is a serotonin chromatographic peak, and the relative retention time of the 10 common fingerprint peaks is respectively as follows: 0.493 +/-0.008 for peak 1, 0.642 +/-0.007 for peak 2, 1.000 +/-0.000 for peak 3, 1.723 +/-0.025 for peak 4, 2.523 +/-0.058 for peak 5, 2.925 +/-0.084 for peak 6, 3.927 +/-0.147 for peak 7, 4.124 +/-0.094 for peak 8, 4.458 +/-0.104 for peak 9 and 4.753 +/-0.104 for peak 10.
(4) When the wasp venom is the basic wasp venom, the chromatographic conditions of the high performance liquid chromatography are as follows:
mobile phase: the mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is an acetonitrile solution of trifluoroacetic acid, and the volume percentage content of the trifluoroacetic acid in the mobile phase A is 0.1%; the mobile phase B is an aqueous solution of trifluoroacetic acid, and the volume percentage content of the trifluoroacetic acid in the mobile phase B is 0.1%;
gradient elution procedure: increasing the content of the mobile phase A from 5% to 77% in 0-40 min; increasing the content of the mobile phase A from 77% to 95% in 40-58 min; the flow rate of the mobile phase is 1 mL/min;
a DAD detector is adopted, and the detection wavelength is 215 nm; the sample injection amount is 5 mu L; a chromatographic column: ZORBAX 300SB-C18(4.6 mm. times.250 mm, 5 μm) with a column temperature of 25 ℃;
the obtained wasp venom fingerprint is a basic wasp venom fingerprint and contains 12 common fingerprint peaks, the reference peak is a serotonin chromatographic peak, and the relative retention time of the 12 common fingerprint peaks is respectively as follows: 0.449 + -0.001 for peak No. 1, 0.810 + -0.026 for peak No. 2, 1.000 + -0.000 for peak No. 3(S), 3.111 + -0.021 for peak No. 4, 3.478 + -0.011 for peak No. 5, 3.756 + -0.012 for peak No. 6, 3.934 + -0.012 for peak No. 7, 4.188 + -0.012 for peak No. 8, 4.576 + -0.014 for peak No. 9, 4.715 + -0.014 for peak No. 10, 4.800 + -0.014 for peak No. 11, 5.938 + -0.017 for peak No. 12.
The invention provides a wasp venom fingerprint obtained by the establishing method of the wasp venom fingerprint in the technical scheme.
The invention provides application of the wasp venom fingerprint spectrum in the technical scheme in wasp venom quality control.
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
Example 1
1. Method for establishing rhizoma Vespa longissima venom fingerprint
(1) Instrument and reagent
High performance liquid chromatograph: an Agilent1260 liquid chromatograph system in America, which is provided with an online degasser, a quaternary gradient pump, a DAD detector, an automatic sample injector and a column incubator; a chromatographic column: ZORBAX 300SB-C18(4.6 mm. times.250 mm, 5 μm);
serotonin (standard substance), trifluoroacetic acid (chromatographic purity), acetonitrile (chromatographic purity), and self-made ultrapure water.
(2) Chromatographic conditions of high performance liquid chromatography
Mobile phase: the mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is an acetonitrile solution of trifluoroacetic acid, and the volume percentage content of the trifluoroacetic acid in the mobile phase A is 0.1%; the mobile phase B is an aqueous solution of trifluoroacetic acid, and the volume percentage content of the trifluoroacetic acid in the mobile phase B is 0.1%;
gradient elution procedure: increasing the content of the mobile phase A from 5% to 77% in 0-40 min; increasing the content of the mobile phase A from 77% to 95% in 40-58 min; the flow rate of the mobile phase is 1 mL/min;
the sample introduction amount is 5 mu L, the column temperature is 25 ℃, and the detection wavelength is 215 nm; the number of theoretical plates is not less than 2000 calculated as serotonin.
(3) Preparation of test solution
Precisely weighing 15mg of Vespa mandarinia venom, placing in a conical flask with a plug, adding 2mL of ultrapure water, vortexing for 0.5min, mixing, performing ultrasonic treatment for 10min, standing at room temperature, and centrifuging at 10000rpm for 10 min; filtering the supernatant with 0.45 μm microporous membrane, collecting the filtrate, and shaking.
(4) Preparation of control solutions
Accurately weighing serotonin, adding ultrapure water, and preparing into a reference solution with the concentration of 1mg/mL for later use.
(5) Determination of a reference
Comparing the HPLC chromatograms of the test solution and the control solution to determine that the retention time of the S peak in the test solution is the same as that of serotonin (as shown in FIG. 1); the DAD detector is used for scanning the spectrums of the sample solution respectively, the result shows that the S peak in the sample solution is the same as the ultraviolet absorption spectrum of the serotonin, and the S peak in the HPLC spectrum of the sample solution is further determined to be the serotonin (as shown in figure 2). The serotonin in the test solution has larger peak area, reasonable peak position and good reproducibility of retention time, so the serotonin is selected as the reference peak (S) of the wasp venom.
2. Methodology investigation
(1) Precision test
Taking the Vespa mandarinia venom to prepare a test solution according to the method in '1, establishing Vespa mandarinia venom fingerprint chromatogram' and carrying out high performance liquid chromatography analysis, continuously feeding samples for 6 times (S1, S2, S3, S4, S5 and S6), taking a serotonin peak (peak S) as a reference peak, and respectively inspecting the relative retention time and the relative peak area of the common fingerprint peak, wherein the results are shown in a table 1 and a table 2. As can be seen from tables 1 and 2, RSD of the relative retention time of each common fingerprint peak was less than 1.0%, and RSD of the relative peak area was less than 13%, indicating that the precision of the instrument was good.
Table 1 precision test relative retention time (n ═ 6)
TABLE 2 relative peak area retention for precision test (n ═ 6)
(2) Repeatability test
Taking 6 parts of the Vespa mandarinia venom, preparing a test solution according to the methods in the '1 and the establishment of the Bespa mandarinia venom fingerprint chromatogram' method, respectively, performing high performance liquid chromatography analysis (R1, R2, R3, R4, R5 and R6), taking a serotonin peak (peak S) as a reference peak, and respectively inspecting the relative retention time and the relative peak area of the common fingerprint peak, wherein the results are shown in a table 3 and a table 4. As can be seen from tables 3 and 4, the RSD of the relative retention time of the common fingerprint peaks is less than 2.00%, and the RSD of the relative peak area is less than 17.00%, which indicates that the method has good repeatability.
Table 3 relative retention time for repeatability tests (n ═ 6)
Table 4 relative peak area for repeatability test (n ═ 6)
(3) Stability test
Randomly taking 1 part of the sample solution in the precision test, respectively carrying out high performance liquid chromatography analysis for 0 h, 2h, 4 h, 10 h, 12h and 16h according to the middle method established by the method of 1 and Vespa mandarinia venom fingerprint chromatogram, respectively taking a serotonin peak (peak S) as a reference peak, and respectively investigating the relative retention time and the relative peak area of a common fingerprint peak, wherein the results are shown in tables 5 and 6. As can be seen from tables 5 and 6, RSD of the relative retention time of each common fingerprint peak is less than 1.00%, and RSD of the relative peak area is less than 27.00%, indicating that the test solution is substantially stable within 16 h.
Table 5 stability test relative retention time (n ═ 6)
Table 6 stability test relative peak area (n ═ 6)
3. Establishment of 20 batches of wasp venom fingerprint and data analysis
20 batches of wasp venom (S1-S20) distributed in 7 different producing areas and different years of Yunnan province, Guangxi province, Guizhou province and the like are taken, and the test solution is prepared according to the method in' 1, establishing the method of wasp venom fingerprint chromatogram, and is subjected to high performance liquid chromatography analysis, so that the high performance liquid chromatogram of 20 batches of samples is obtained.
High performance liquid chromatogram data of 20 batches of Vespa mandarinia venom is introduced by software of traditional Chinese medicine chromatogram fingerprint similarity evaluation system (2012.130723 edition) issued by the State pharmacopoeia Commission, and 12 common peaks are determined by taking peak No. 5 (serotonin) as reference peaks. Performing multi-point correction on sample chromatographic peaks, generating a comparison fingerprint spectrum by a median method, wherein the time window width is 0.5, automatically matching the chromatographic peaks after setting parameters, and establishing the comparison fingerprint spectrum, wherein the fingerprint spectrum of 20 batches of the Vespa mandarinia venom is shown in figure 3, and the fingerprint spectrum common mode comparison spectrum is shown in figure 4.
The relative retention time and relative peak area of each common fingerprint peak were calculated and the results are shown in tables 7 and 8. As can be seen from Table 7, the RSD of each common fingerprint peak relative retention time is less than 1.00%, which shows that 12 common fingerprint peaks of the established Vespa mandarinia venom fingerprint are very consistent, and the fingerprint characteristic peak heights of Vespa mandarinia venom collected at different producing areas and different time are consistent; as can be seen from Table 8, the RSD of the relative peak area of each common fingerprint is large, which indicates that the contents of the constituents of the venom of Vespa mandarinia in different producing areas are different.
TABLE 720 relative retention times for samples of Vespa control venom (n ═ 20)
TABLE 820 relative peak areas for batches of Vespa control (n 20)
4. Similarity evaluation of 20 batches of wasp venom fingerprint
Taking 20 batches of wasp venom (S1-S20) distributed in 7 different producing areas and different years of Yunnan province, Guangxi province, Guizhou province and the like, preparing a sample solution according to the method in '1, establishing a wasp venom fingerprint spectrum method', and carrying out high performance liquid chromatography analysis to obtain high performance liquid chromatograms of 20 batches of samples; the similarity is calculated by computer-aided similarity evaluation software (compiled by the Chinese medicine modernization research center of the university of the south of China) recommended by the Chinese pharmacopoeia committee, and the similarity is shown in a table 9. As can be seen from Table 9, the similarity between 20 batches of the wasp venom samples and the reference map is between 0.986 and 0.999, and is greater than 0.950, which indicates that the quality of the medicinal materials is relatively stable.
TABLE 920 results of similarity evaluation of finger prints of wasp venom samples
Example 2
1. Method for establishing finger print of vespa velutina auraria smith venom
(1) Instruments and reagents: the same as in example 1.
(2) Chromatographic conditions of high performance liquid chromatography: gradient elution: 0-40 min, 6% A-78% A; 40-58 min, 78-95% A; the column temperature is 27 ℃; the other conditions were the same as in example 1.
(3) Preparation of a test solution: the venom of Vespa velutina was weighed precisely 15mg, and a test solution was prepared according to the method of example 1.
(4) Preparation of control solutions: a control solution was prepared according to the method of example 1.
(5) Determination of a reference
By comparing the HPLC chromatograms of the test solution and the control solution, the retention time of the S peak in the test solution is the same as that of serotonin (as shown in FIG. 5); the DAD detector is used for scanning the spectrums of the sample solution respectively, the result shows that the S peak in the sample solution is the same as the ultraviolet absorption spectrum of the serotonin, and the S peak in the HPLC spectrum of the sample solution is further determined to be the serotonin (as shown in figure 6). The serotonin in the test solution has larger peak area, reasonable peak position and good reproducibility of retention time, so the serotonin is selected as the reference peak (S) of the vespa velutina venom.
2. Methodology investigation
(1) Precision test
Taking the wasp venom to prepare a test solution according to the method in the '1 and establishment of the wasp venom fingerprint chromatogram' and carrying out high performance liquid chromatography analysis, carrying out continuous sample injection for 6 times (S1, S2, S3, S4, S5 and S6), taking a serotonin peak (peak S) as a reference peak, and respectively inspecting the relative retention time and the relative peak area of the common fingerprint peak, wherein the results are shown in a table 10 and a table 11. As can be seen from tables 10 and 11, RSD of the relative retention time of each common fingerprint peak was less than 1.0%, and RSD of the relative peak area was less than 5%, indicating that the precision of the instrument was good.
Table 10 precision test relative retention time (n ═ 6)
TABLE 11 precision test relative peak area retention (n. 6)
(2) Repeatability test
Taking 6 parts of vespa velutina auraria smith venom, respectively establishing a middle method for preparing a test solution according to the method of 1 and the method of the vespa velutina auraria smith venom fingerprint, carrying out high performance liquid chromatography analysis (R1, R2, R3, R4, R5 and R6), respectively inspecting the relative retention time and the relative peak area of the common finger print peak by taking a serotonin peak (peak S) as a reference peak, and finding the results in tables 12 and 13. As can be seen from tables 12 and 13, the RSD of the relative retention time of the common fingerprint peaks is less than 1.00%, and the RSD of the relative peak area is less than 5.00%, which indicates that the method of the invention has good repeatability.
Table 12 relative retention time for repeatability tests (n ═ 6)
Table 13 relative peak area for repeatability test (n ═ 6)
(3) Stability test
Randomly taking 1 part of the sample solution in the precision test, respectively carrying out high performance liquid chromatography analysis for 0, 2, 4, 6, 8 and 12h according to the middle method established by the method of 1 and the Queensland wasp venom fingerprint spectrum, respectively taking a serotonin peak (peak S) as a reference peak, and respectively investigating the relative retention time and the relative peak area of a common fingerprint peak, wherein the results are shown in tables 14 and 15. As can be seen from tables 14 and 15, RSD of the relative retention time of each common fingerprint peak was < 2.00%, and RSD of the relative peak area was < 5.00%, indicating that the test solution was substantially stable within 12 hours.
Table 14 stability test relative retention time (n ═ 6)
Table 15 stability test relative peak area (n ═ 6)
3. Establishment and data analysis of 20 batches of wasp venom fingerprints
Taking 20 batches of Vespa velutina auraria smith venom (S1-S20) distributed in 7 different producing areas and different years of Yunnan province, Guangxi province, Guizhou province and the like, preparing a sample solution according to the method in' 1 and establishment of the Vespa velutina auraria smith venom fingerprint chromatogram, and performing high performance liquid chromatography analysis to obtain the high performance liquid chromatogram of 20 batches of samples.
High performance liquid chromatogram data of 20 batches of wasp venom were imported by software of traditional Chinese medicine chromatogram fingerprint similarity evaluation system (2012.130723 edition) issued by the State pharmacopoeia Commission, and 13 common peaks were determined by taking peak No. 4 (serotonin) as a reference peak. Performing multi-point correction on the sample chromatographic peak, generating a comparison fingerprint spectrum by a median method, wherein the time window width is 0.5, automatically matching the chromatographic peak after setting parameters, and establishing the comparison fingerprint spectrum, wherein the fingerprint spectrum of 20 batches of the vespa velutina auraria smith venom is shown in figure 7, and the fingerprint spectrum common mode comparison spectrum is shown in figure 8.
The relative retention time and relative peak area of each common fingerprint peak were calculated and the results are shown in tables 16 and 17. As can be seen from Table 16, the RSD of the relative retention time of each common fingerprint peak is less than 3.00%, which indicates that 13 common fingerprint peaks of the Vespa velutina auraria smith venom fingerprint established by the invention are very consistent, and the fingerprint characteristic peak heights of Vespa velutina auraria smith venom collected at different producing areas and different time are consistent; as can be seen from Table 17, the RSD of the relative peak area of each common fingerprint is large, which indicates that the contents of the ingredients of the wasp venom in different locations are different.
Table 1620 batch Vespa samples relative retention time (n ═ 20)
TABLE 1720 batch Vespa sample relative peak area (n 20)
4. Similarity evaluation of 20 batches of wasp venom fingerprints
Taking 20 batches of Vespa velutina auraria smith venom (S1-S20) distributed in 7 different producing areas and different years of Yunnan province, Guangxi province, Guizhou province and the like, preparing a sample solution according to the method in' 1 and establishment of the Vespa velutina auraria smith venom fingerprint chromatogram, and performing high performance liquid chromatography analysis to obtain the high performance liquid chromatogram of 20 batches of samples; the similarity is calculated by computer-aided similarity evaluation software (compiled by the Chinese medicine modernization research center of the university of the south-of-the-middle school) recommended by the Chinese pharmacopoeia committee, and the similarity is shown in a table 18. As shown in Table 18, the similarity between 20 batches of Vespa velutina poison liquid samples and the contrast map is between 0.957 and 1.000, and is more than 0.950, which indicates that the quality of the medicinal materials is relatively stable.
TABLE 1820 evaluation results of fingerprint similarity of samples
Example 3
1. Method for establishing finger print of yellow waist wasp venom
(1) Instruments and reagents: the same as in example 1.
(2) Chromatographic conditions of high performance liquid chromatography: the same as in example 1.
(3) Preparation of a test solution: the venom of the wasp was weighed 15mg precisely, and the test solution was prepared according to the method of example 1.
(4) Preparation of control solutions: a control solution was prepared according to the method of example 1.
(5) Determination of a reference
By comparing the HPLC chromatograms of the test solution and the control solution, the retention time of the S peak in the test solution is the same as that of serotonin (as shown in FIG. 9); the DAD detector is used for scanning the spectrums of the sample solution respectively, the result shows that the S peak in the sample solution is the same as the ultraviolet absorption spectrum of the serotonin, and the S peak in the HPLC spectrum of the sample solution is further determined to be the serotonin (as shown in figure 10). The serotonin in the test solution has larger peak area, reasonable peak position and good reproducibility of retention time, so the serotonin is selected as the reference peak (S) of the wasp venom.
2. Methodology investigation
(1) Precision test
Taking the wasp venom from the Huangyao wasp to prepare a test solution according to the method in the '1 and the establishment of the fingerprint of the wasp venom', carrying out high performance liquid chromatography analysis, carrying out continuous sample injection for 6 times (S1, S2, S3, S4, S5 and S6), taking a serotonin peak (peak S) as a reference peak, and respectively inspecting the relative retention time and the relative peak area of the common fingerprint peak, wherein the results are shown in a table 19 and a table 20. As can be seen from tables 19 and 20, RSD of the relative retention time of each common fingerprint peak was less than 3.0%, and RSD of the relative peak area was less than 5%, indicating that the precision of the instrument was good.
Table 19 precision test relative retention time (n ═ 6)
TABLE 20 relative peak area retention for precision test (n ═ 6)
(2) Repeatability test
Taking 6 parts of wasp venom, preparing a test solution by a medium method according to the method of' 1 and the method of establishing the finger print of the wasp venom, respectively, and performing high performance liquid chromatography analysis (R1, R2, R3, R4, R5 and R6), taking a serotonin peak (peak S) as a reference peak, and respectively inspecting the relative retention time and the relative peak area of the common fingerprint peak, wherein the results are shown in a table 21 and a table 22. As can be seen from tables 21 and 22, the RSD of the relative retention time of each common fingerprint peak is less than 5.00%, and although the RSD of the relative peak area is large, the repeatability of the relative retention time is good, indicating that the repeatability of the method of the present invention is within the acceptable range.
Table 21 repeatability test relative retention time (n ═ 6)
TABLE 22 relative peak area for repeatability test (n ═ 6)
(3) Stability test
Randomly taking 1 part of the sample solution in the precision test, respectively carrying out high performance liquid chromatography analysis for 0, 2, 4, 6, 8 and 12h according to the middle method established by the method of 1 and yellow waist wasp venom fingerprint spectra, respectively taking a serotonin peak (peak S) as a reference peak, and respectively investigating the relative retention time and the relative peak area of a common fingerprint peak, wherein the results are shown in a table 23 and a table 24. As can be seen from tables 23 and 24, RSD of the relative retention time of each common fingerprint peak was < 3.00%, and RSD of the relative peak area was < 6.00%, indicating that the test solution was substantially stable within 12 hours.
Table 23 stability test relative retention time (n ═ 6)
TABLE 24 stability test relative peak area (n ═ 6)
3. Establishment and data analysis of 20 batches of yellow waist-line wasp venom fingerprint
20 batches of wasp venom (S1-S20) distributed in 7 different producing areas and different years of Yunnan province, Guangxi province, Guizhou province and the like are taken, and the test solution is prepared according to the method in the '1 and wasp venom fingerprint spectrum method establishment' and is subjected to high performance liquid chromatography analysis, so that the high performance liquid chromatogram of 20 batches of samples is obtained.
High performance liquid chromatogram data of 20 batches of wasp venom were imported by software of traditional Chinese medicine chromatogram fingerprint similarity evaluation system (2012.130723 edition) issued by the State pharmacopoeia Commission, and 10 common peaks were determined by taking peak No. 3 (serotonin) as a reference peak. Performing multi-point correction on sample chromatographic peaks, generating a comparison fingerprint spectrum by a median method, wherein the time window width is 0.5, automatically matching the chromatographic peaks after setting parameters, and establishing the comparison fingerprint spectrum, wherein the fingerprint spectrum of 20 batches of wasp venom is shown in figure 11, and the fingerprint spectrum common mode comparison spectrum is shown in figure 12.
The relative retention time and relative peak area of each common fingerprint peak were calculated and the results are shown in tables 25 and 26. As can be seen from Table 25, the RSD of the relative retention time of each common fingerprint peak is less than 3.00%, which indicates that 10 common fingerprint peaks of the wasp venom fingerprint established by the invention are very consistent, and the fingerprint characteristic peak heights of the wasp venom collected at different places of production and different times are consistent; as can be seen from Table 26, the RSD of the relative peak area of each common fingerprint is large, which indicates that the contents of the components of the wasp venom from different producing areas are different.
Table 2520 batch yellow waist wasp venom sample relative retention time (n ═ 20)
TABLE 2620 relative peak areas for samples of wasp venom from Huangyao wasps (n 20)
4. Similarity evaluation of 20 batches of wasp venom fingerprints
Taking 20 batches of wasp venom (S1-S20) distributed in 7 different producing areas and different years of Yunnan province, Guangxi province, Guizhou province and the like, preparing a sample solution according to the method in' 1, establishing the method of wasp venom fingerprint chromatogram, and carrying out high performance liquid chromatography analysis to obtain the high performance liquid chromatogram of 20 batches of samples; the similarity is calculated by computer-aided similarity evaluation software (compiled by the Chinese medicine modernization research center of the university of the south-of-the-middle school) recommended by the Chinese pharmacopoeia committee, and the similarity is shown in a table 27. As can be seen from Table 27, the similarity between the 20 batches of wasp venom samples and the reference map is between 0.989 and 0.999, and is greater than 0.950, which indicates that the quality of the medicinal materials is relatively stable.
Table 2720 batch sample fingerprint spectrum similarity evaluation result
Example 4
1. Method for establishing finger print of wasp venom
(1) Instruments and reagents: the same as in example 1.
(2) Chromatographic conditions of high performance liquid chromatography: the same as in example 1.
(3) Preparation of a test solution: the test solution was prepared by precisely weighing 15mg of the quassia wasp venom and following the procedure of example 1.
(4) Preparation of control solutions: a control solution was prepared according to the method of example 1.
(5) Determination of a reference
By comparing the HPLC chromatograms of the test solution and the control solution, it can be seen that the retention time of the S peak in the test solution is the same as that of serotonin (as shown in fig. 13); the DAD detector is used to scan the spectra, and the results show that the S peak in the sample solution is the same as the ultraviolet absorption spectrum of serotonin, and the S peak in the HPLC spectrum of the sample solution is further determined to be serotonin (as shown in fig. 14). The area of the serotonin peak in the test solution is large, the peak position is reasonable, and the reproducibility of the retention time is good, so that the serotonin is selected as the reference peak (S) of the wasp venom.
2. Methodology investigation
(1) Precision test
Taking the wasp venom, preparing a test solution according to the method in the '1 and establishing the fingerprint of the wasp venom' method, carrying out high performance liquid chromatography analysis, continuously feeding samples for 6 times (S1, S2, S3, S4, S5 and S6), taking a serotonin peak (peak S) as a reference peak, and respectively inspecting the relative retention time and the relative peak area of the common fingerprint peak, wherein the results are shown in a table 28 and a table 29. As can be seen from tables 28 and 29, RSD of the relative retention time of each common fingerprint peak was less than 1.0%, and RSD of the relative peak area was less than 3%, indicating that the precision of the instrument was good.
Table 28 precision test relative retention time (n ═ 6)
TABLE 29 precision test relative peak area retention (n ═ 6)
(2) Repeatability test
Taking 6 parts of the wasp venom, preparing a test solution according to the method in the '1 and the method for establishing the fingerprint of the wasp venom', respectively, performing high performance liquid chromatography analysis (R1, R2, R3, R4, R5 and R6), taking a serotonin peak (peak S) as a reference peak, and respectively inspecting the relative retention time and the relative peak area of the common fingerprint peak, wherein the results are shown in a table 30 and a table 31. As can be seen from tables 30 and 31, the RSD of the relative retention time of the common fingerprint peaks is less than 1.00%, and the RSD of the relative peak area is less than 5.00%, indicating that the method of the present invention has good repeatability.
Table 30 relative retention time for repeatability tests (n ═ 6)
TABLE 31 relative peak area for repeatability test (n ═ 6)
(3) Stability test
Randomly taking 1 part of the sample solution in the precision test, establishing a middle method for high performance liquid chromatography analysis according to the method for 1 and base wasp venom fingerprint spectra at 0, 2, 4, 6, 8 and 12 hours respectively, taking a serotonin peak (peak S) as a reference peak, and respectively inspecting the relative retention time and the relative peak area of the common fingerprint peak, wherein the results are shown in tables 32 and 33. As can be seen from tables 32 and 33, RSD of the relative retention time of each common fingerprint peak was < 1.00%, and RSD of the relative peak area was < 6.00%, indicating that the test solution was substantially stable within 12 hours.
Table 32 stability test relative retention time (n ═ 6)
TABLE 33 stability test relative peak area (n ═ 6)
3. Establishment and data analysis of 20 batches of basic wasp venom fingerprint
Taking 20 batches of wasp venom (S1-S20) distributed in 7 different producing areas and different years, such as Yunnan province, Guangxi province, Guizhou province and the like, preparing a sample solution according to the method of '1, establishing a wasp venom fingerprint chromatogram method' and carrying out high performance liquid chromatography analysis to obtain the high performance liquid chromatogram of 20 batches of samples.
High performance liquid chromatogram data of 20 batches of wasp venom are introduced by software of 'traditional Chinese medicine chromatogram fingerprint similarity evaluation system' (2012.130723 edition) issued by the State pharmacopoeia Committee, and 12 common peaks are determined by taking the peak No. 3 (serotonin) as a reference peak. Performing multi-point correction on the sample chromatographic peak, generating a comparison fingerprint spectrum by a median method, wherein the time window width is 0.5, automatically matching the chromatographic peak after setting parameters, and establishing the comparison fingerprint spectrum, wherein the fingerprint spectrum of 20 batches of wasp venom is shown in figure 15, and the fingerprint spectrum common mode comparison spectrum is shown in figure 16.
The relative retention time and relative peak area of each common fingerprint peak were calculated and the results are shown in tables 34 and 35. As can be seen from Table 34, the RSD of the relative retention time of each common fingerprint peak is less than 5.00%, which indicates that 10 common fingerprint peaks of the wasp venom fingerprint established by the invention are very consistent, and the fingerprint characteristic peak heights of the wasp venom collected at different producing areas and different time are consistent; as can be seen from Table 35, the RSD of the relative peak area of each common fingerprint is large, which indicates that the contents of the components of the wasp venom in different places are different.
TABLE 3420 relative retention time for samples of Vespa venom (n ═ 20)
TABLE 3520 relative peak areas for samples of Vespa venom (n 20)
4. Similarity evaluation of 20 batches of wasp venom fingerprint spectra
Taking 20 batches of wasp venom (S1-S20) distributed in 7 different producing areas and different years, such as Yunnan province, Guangxi province, Guizhou province and the like, preparing a sample solution according to the method in '1, establishing a wasp venom fingerprint spectrum method', and carrying out high performance liquid chromatography analysis to obtain high performance liquid chromatograms of 20 batches of samples; the similarity is calculated by computer-aided similarity evaluation software (compiled by the Chinese medicine modernization research center of the university of south China) recommended by the Chinese pharmacopoeia committee, and the similarity is shown in a table 36. As can be seen from Table 36, the similarity between 20 batches of the wasp venom samples and the reference spectrum is between 0.985 and 1.000, and is greater than 0.950, which indicates that the quality of the medicinal materials is relatively stable.
TABLE 3620 evaluation results of fingerprint similarity of samples
The embodiment shows that the method provided by the invention is simple and convenient to operate, accurate and reliable, has reasonable detection time, can truly reflect the characteristic substances contained in the wasp venom, can be used as a method for determining the chemical components in the wasp venom, can identify the truth of the wasp venom and control the quality of the wasp venom, and can comprehensively and accurately evaluate the quality of the wasp venom.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be construed as the protection scope of the present invention.
Claims (10)
1. A method for establishing a wasp venom fingerprint is characterized by comprising the following steps:
taking the aqueous solution of the wasp venom as a test solution and the aqueous solution of serotonin as a reference solution, respectively carrying out high performance liquid chromatography analysis on the reference solution and the test solution, and taking the chromatographic peak of the reference solution as a reference to obtain the wasp venom fingerprint;
the chromatographic conditions of the high performance liquid chromatography are as follows:
mobile phase: the mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is an acetonitrile solution of trifluoroacetic acid, and the volume percentage content of the trifluoroacetic acid in the mobile phase A is 0.05-0.2%; the mobile phase B is an aqueous solution of trifluoroacetic acid, and the volume percentage of the trifluoroacetic acid in the mobile phase B is 0.05-0.2%;
gradient elution procedure: increasing the content of the mobile phase A from 5-6% to 77-78% in 0-40 min; and (3) increasing the mobile phase A from 77-78% to 95% in 40-58 min.
2. The method for establishing the wasp venom fingerprint spectrum according to claim 1, wherein the wasp venom comprises wasp venom, vespa velutina auraria venom, wasp venom from wasp girls or wasp venom from wasp queen wasp.
3. The method for establishing a wasp venom fingerprint according to claim 1 or 2, wherein the HPLC analysis is performed by using a DAD detector with a detection wavelength of 215 nm;
the chromatographic column is a ZORBAX 300SB-C18 chromatographic column with the specification as follows: the length is 250mm, the inner diameter is 4.6mm, and the grain diameter of the filler is 5 mu m; the column temperature is 20-35 ℃;
the flow rate of the mobile phase is 0.8-1.2 mL/min.
4. The method for establishing the wasp venom fingerprint spectrum according to claim 3, wherein when the wasp venom is wasp venom, the chromatographic conditions of the high performance liquid chromatography are as follows:
mobile phase: the trifluoroacetic acid in the mobile phase A accounts for 0.1 percent by volume, and the trifluoroacetic acid in the mobile phase B accounts for 0.1 percent by volume;
gradient elution procedure: increasing the content of the mobile phase A from 5% to 77% in 0-40 min; increasing the content of the mobile phase A from 77% to 95% in 40-58 min;
the column temperature is 25 ℃, and the flow rate of the mobile phase is 1 mL/min;
the obtained wasp venom fingerprint is a wasp venom fingerprint, and contains 12 common fingerprint peaks, the reference peak is a serotonin chromatographic peak, and the relative retention time of the 12 common fingerprint peaks is respectively as follows: 0.481 + -0.005 of No. 1 peak, 0.509 + -0.004 of No. 2 peak, 0.638 + -0.005 of No. 3 peak, 0.790 + -0.004 of No. 4 peak, 1.000 + -0.000 of No. 5 peak, 1.791 + -0.010 of No. 6 peak, 2.594 + -0.016 of No. 7 peak, 3.824 + -0.029 of No. 8 peak, 3.976 + -0.029 of No. 9 peak, 4.224 + -0.029 of No. 10 peak, 4.295 + -0.026 of No. 11 peak, and 4.610 + -0.031 of No. 12 peak.
5. The method for establishing the wasp venom fingerprint spectrum according to claim 3, wherein when the wasp venom is the vespid venom, the chromatographic conditions of the high performance liquid chromatography are as follows:
mobile phase: the trifluoroacetic acid in the mobile phase A accounts for 0.1 percent by volume, and the trifluoroacetic acid in the mobile phase B accounts for 0.1 percent by volume;
gradient elution procedure: increasing the content of the mobile phase A from 6% to 78% in 0-40 min; increasing the content of the mobile phase A from 78% to 95% in 40-58 min;
the column temperature is 27 ℃, and the flow rate of the mobile phase is 1 mL/min;
the obtained wasp venom fingerprint is a female wasp venom fingerprint and contains 13 common fingerprint peaks, the reference peak is a serotonin chromatographic peak, and the relative retention time of the 13 common fingerprint peaks is respectively as follows: 0.476 + -0.012 for peak 1, 0.643 + -0.005 for peak 2, 0.788 + -0.009 for peak 3, 1.000 + -0.000 for peak 4, 2.370 + -0.064 for peak 5, 2.653 + -0.034 for peak 6, 3.105 + -0.073 for peak 7, 3.178 + -0.075 for peak 8, 3.357 + -0.038 for peak 9, 3.703 + -0.055 for peak 10, 3.757 + -0.063 for peak 11, 4.106 + -0.058 for peak 12, 4.349 + -0.068 for peak 13.
6. The method for establishing the wasp venom fingerprint spectrum according to claim 3, wherein when the wasp venom is wasp venom with yellow waist, the chromatographic conditions of the high performance liquid chromatography are as follows:
mobile phase: the trifluoroacetic acid in the mobile phase A accounts for 0.1 percent by volume, and the trifluoroacetic acid in the mobile phase B accounts for 0.1 percent by volume;
gradient elution procedure: increasing the content of the mobile phase A from 5% to 77% in 0-40 min; increasing the content of the mobile phase A from 77% to 95% in 40-58 min;
the column temperature is 25 ℃, and the flow rate of the mobile phase is 1 mL/min;
the obtained wasp venom fingerprint is a wasp venom fingerprint with yellow waist, and contains 10 common fingerprint peaks, the reference peak is a serotonin chromatographic peak, and the relative retention time of the 10 common fingerprint peaks is respectively as follows: 0.493 +/-0.008 for peak 1, 0.642 +/-0.007 for peak 2, 1.000 +/-0.000 for peak 3, 1.723 +/-0.025 for peak 4, 2.523 +/-0.058 for peak 5, 2.925 +/-0.084 for peak 6, 3.927 +/-0.147 for peak 7, 4.124 +/-0.094 for peak 8, 4.458 +/-0.104 for peak 9 and 4.753 +/-0.104 for peak 10.
7. The method for establishing the wasp venom fingerprint spectrum according to claim 3, wherein when the wasp venom is a basic wasp venom, the chromatographic conditions of the high performance liquid chromatography are as follows:
mobile phase: the trifluoroacetic acid in the mobile phase A accounts for 0.1 percent by volume, and the trifluoroacetic acid in the mobile phase B accounts for 0.1 percent by volume;
gradient elution procedure: increasing the content of the mobile phase A from 5% to 77% in 0-40 min; increasing the content of the mobile phase A from 77% to 95% in 40-58 min;
the column temperature is 25 ℃, and the flow rate of the mobile phase is 1 mL/min;
the obtained wasp venom fingerprint is a basic wasp venom fingerprint and contains 12 common fingerprint peaks, the reference peak is a serotonin chromatographic peak, and the relative retention time of the 12 common fingerprint peaks is respectively as follows: 0.449 +/-0.001 of the No. 1 peak, 0.810 +/-0.026 of the No. 2 peak, 1.000 +/-0.000 of the No. 3 peak, 3.111 +/-0.021 of the No. 4 peak, 3.478 +/-0.011 of the No. 5 peak, 3.756 +/-0.012 of the No. 6 peak, 3.934 +/-0.012 of the No. 7 peak, 4.188 +/-0.012 of the No. 8 peak, 4.576 +/-0.014 of the No. 9 peak, 4.715 +/-0.014 of the No. 10 peak, 4.800 +/-0.014 of the No. 11 peak and 5.938 +/-0.017 of the No. 12 peak.
8. The method for establishing the wasp venom fingerprint spectrum according to claim 1, wherein the preparation method of the test solution comprises the following steps:
mixing wasp venom with water, performing ultrasonic treatment, centrifuging the obtained ultrasonic material, and taking the obtained supernatant as a test solution; the dosage ratio of the wasp venom to the water is (1-15) mg: (2-80) mL.
9. The wasp venom fingerprint obtained by the establishing method of the wasp venom fingerprint according to any one of claims 1-8.
10. Use of the wasp venom fingerprint of claim 9 in quality control of wasp venom.
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