CN115524423B - Method for constructing, detecting and identifying characteristic spectrum of negundo chastetree fruit and vitex negundo chastetree fruit - Google Patents
Method for constructing, detecting and identifying characteristic spectrum of negundo chastetree fruit and vitex negundo chastetree fruit Download PDFInfo
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- 244000248021 Vitex negundo Species 0.000 title claims abstract description 96
- 235000013399 edible fruits Nutrition 0.000 title claims abstract description 92
- 235000013427 Vitex negundo var incisa Nutrition 0.000 title claims abstract description 86
- 235000014961 Vitex negundo var negundo Nutrition 0.000 title claims abstract description 86
- 235000014956 negundo chastetree Nutrition 0.000 title claims abstract description 86
- 238000000034 method Methods 0.000 title claims abstract description 44
- 238000001228 spectrum Methods 0.000 title claims abstract description 43
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical group OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims abstract description 66
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 36
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims abstract description 33
- 239000000463 material Substances 0.000 claims abstract description 33
- PLAPMLGJVGLZOV-UHFFFAOYSA-N Epi-orientin Natural products OC1C(O)C(O)C(CO)OC1C1=C(O)C=C(O)C2=C1OC(C=1C=C(O)C(O)=CC=1)=CC2=O PLAPMLGJVGLZOV-UHFFFAOYSA-N 0.000 claims abstract description 29
- 239000000243 solution Substances 0.000 claims abstract description 29
- 239000000523 sample Substances 0.000 claims abstract description 26
- YQUVCSBJEUQKSH-UHFFFAOYSA-N 3,4-dihydroxybenzoic acid Chemical compound OC(=O)C1=CC=C(O)C(O)=C1 YQUVCSBJEUQKSH-UHFFFAOYSA-N 0.000 claims abstract description 24
- FJKROLUGYXJWQN-UHFFFAOYSA-N 4-hydroxybenzoic acid Chemical compound OC(=O)C1=CC=C(O)C=C1 FJKROLUGYXJWQN-UHFFFAOYSA-N 0.000 claims abstract description 24
- 239000007864 aqueous solution Substances 0.000 claims abstract description 24
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- ODBRNZZJSYPIDI-UHFFFAOYSA-N 3',4',5,7-tetrahydroxy-6-C-glucopyranosylflavone Natural products OC1C(O)C(O)C(CO)OC1C1=C(O)C=C(OC(=CC2=O)C=3C=C(O)C(O)=CC=3)C2=C1O ODBRNZZJSYPIDI-UHFFFAOYSA-N 0.000 claims abstract description 17
- WJJFWGUVMIUWGG-UHFFFAOYSA-N Stereolensin Natural products OC1C(O)C(O)C(CO)OC1OC1=C(O)C=C(OC(=CC2=O)C=3C=C(O)C(O)=CC=3)C2=C1O WJJFWGUVMIUWGG-UHFFFAOYSA-N 0.000 claims abstract description 17
- ODBRNZZJSYPIDI-VJXVFPJBSA-N isoorientin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1C1=C(O)C=C(OC(=CC2=O)C=3C=C(O)C(O)=CC=3)C2=C1O ODBRNZZJSYPIDI-VJXVFPJBSA-N 0.000 claims abstract description 17
- UYJGIAWJIRZBNU-UHFFFAOYSA-N isoorientin Natural products OCC1OC(C(O)C(O)C1O)c2cc(O)c(O)c3C(=O)C=C(Oc23)c4ccc(O)c(O)c4 UYJGIAWJIRZBNU-UHFFFAOYSA-N 0.000 claims abstract description 17
- 229940090248 4-hydroxybenzoic acid Drugs 0.000 claims abstract description 12
- PTNJRKBWIYNFSY-UHFFFAOYSA-N Lirinin-O-methyl-ether Natural products COc1ccc-2c(CC3N(C)CCc4cc(OC)c(OC)c-2c34)c1 PTNJRKBWIYNFSY-UHFFFAOYSA-N 0.000 claims abstract description 12
- RBVAFYCFAFADAG-UHFFFAOYSA-N Orientin Natural products OCC1OC(C(O)c2c(O)cc(O)c3C(=O)C=C(Oc23)c4ccc(O)c(O)c4)C(O)C1O RBVAFYCFAFADAG-UHFFFAOYSA-N 0.000 claims abstract description 12
- LQSNPVIQIPKOGP-UHFFFAOYSA-N UNPD159785 Natural products OC1C(O)C(O)C(CO)OC1OC1=C(O)C=C(O)C2=C1OC(C=1C=C(O)C(O)=CC=1)=CC2=O LQSNPVIQIPKOGP-UHFFFAOYSA-N 0.000 claims abstract description 12
- 238000004458 analytical method Methods 0.000 claims abstract description 12
- PEFNSGRTCBGNAN-UHFFFAOYSA-N nephrocizin Natural products OC1C(O)C(O)C(CO)OC1OC1=CC(O)=C2C(=O)C=C(C=3C=C(O)C(O)=CC=3)OC2=C1 PEFNSGRTCBGNAN-UHFFFAOYSA-N 0.000 claims abstract description 12
- PLAPMLGJVGLZOV-VPRICQMDSA-N orientin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1C1=C(O)C=C(O)C2=C1OC(C=1C=C(O)C(O)=CC=1)=CC2=O PLAPMLGJVGLZOV-VPRICQMDSA-N 0.000 claims abstract description 12
- 238000002360 preparation method Methods 0.000 claims abstract description 12
- JMFSHKGXVSAJFY-UHFFFAOYSA-N Saponaretin Natural products OCC(O)C1OC(Oc2c(O)cc(O)c3C(=O)C=C(Oc23)c4ccc(O)cc4)C(O)C1O JMFSHKGXVSAJFY-UHFFFAOYSA-N 0.000 claims abstract description 11
- MOZJVOCOKZLBQB-UHFFFAOYSA-N Vitexin Natural products OCC1OC(Oc2c(O)c(O)cc3C(=O)C=C(Oc23)c4ccc(O)cc4)C(O)C(O)C1O MOZJVOCOKZLBQB-UHFFFAOYSA-N 0.000 claims abstract description 11
- 238000001195 ultra high performance liquid chromatography Methods 0.000 claims abstract description 11
- SGEWCQFRYRRZDC-VPRICQMDSA-N vitexin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1C1=C(O)C=C(O)C2=C1OC(C=1C=CC(O)=CC=1)=CC2=O SGEWCQFRYRRZDC-VPRICQMDSA-N 0.000 claims abstract description 11
- PZKISQRTNNHUGF-UHFFFAOYSA-N vitexine Natural products OC1C(O)C(O)C(CO)OC1OC1=C(O)C=C(O)C2=C1OC(C=1C=CC(O)=CC=1)=CC2=O PZKISQRTNNHUGF-UHFFFAOYSA-N 0.000 claims abstract description 11
- 239000012088 reference solution Substances 0.000 claims abstract description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 41
- 241001643642 Viticis Species 0.000 claims description 41
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 21
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- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 9
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims description 8
- 239000000741 silica gel Substances 0.000 claims description 8
- 229910002027 silica gel Inorganic materials 0.000 claims description 8
- 241000532412 Vitex Species 0.000 claims description 6
- 235000001667 Vitex agnus castus Nutrition 0.000 claims description 6
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- 241000989077 Vitex rotundifolia Species 0.000 description 1
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Classifications
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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
-
- 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
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
- G01N30/8679—Target compound analysis, i.e. whereby a limited number of peaks is analysed
-
- 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
- G01N2030/065—Preparation using different phases to separate parts of sample
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- 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)
- Engineering & Computer Science (AREA)
- Library & Information Science (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention provides a method for constructing a characteristic spectrum of negundo chastetree fruit, which comprises the following steps: preparing a mixed reference solution from protocatechuic acid reference, 4-hydroxybenzoic acid reference, isoorientin reference, orientin reference and vitexin reference; taking a negundo chastetree fruit sample, and preparing a sample solution; respectively carrying out ultra-high performance liquid chromatography analysis on the mixed reference substance solution and the sample solution to construct a characteristic spectrum of negundo chastetree fruit; wherein, the conditions of the ultra performance liquid chromatography include: mobile phase A is acetonitrile, mobile phase B is phosphoric acid aqueous solution; gradient elution is used. The preparation method has the advantages of short time consumption, simple operation, rich map information, strong characteristics and good reproducibility, and is simultaneously applicable to negundo chastetree fruit medicinal materials, standard decoction and formula particles.
Description
Technical Field
The invention relates to the field of traditional Chinese medicine identification, in particular to a method for constructing and detecting a characteristic spectrum of negundo chastetree fruit and identifying the negundo chastetree fruit.
Background
Fructus Viticis negundo is fruit of Vitex negundo L. Of Verbenaceae, has effects of dispelling pathogenic wind, relieving exterior syndrome, relieving cough and asthma, regulating qi-flowing, resolving food stagnation, relieving pain, and can be used for common cold, cough, asthma, stomach ache, acid regurgitation, dyspepsia diarrhea, cholecystitis, cholelithiasis or hernia.
At present, few methods and means applied to the quality analysis of negundo chastetree fruits are described in literature, and research reports are focused on the component research of negundo chastetree fruits, and only a small part of the research reports are about the quality standard evaluation research of negundo chastetree fruits. The traditional method generally adopts high performance liquid chromatography to detect or identify the negundo chastetree fruit medicinal material, the evaluation index is generally quantitative research of negundo chastetree fruit extract or lignans, the research on the dissimilarity of negundo chastetree species is less, and particularly after the negundo chastetree fruit medicinal material is prepared into standard decoction or formula particles, the identification difficulty is further increased on the basis of losing the shape, namely the high performance liquid chromatography detection method which is applicable to the negundo chastetree fruit medicinal material at present is not applicable to the detection of the standard decoction and formula particles. The traditional method adopts a high performance liquid chromatography fingerprint method to evaluate the quality of the Chinese patent medicine containing the negundo chastetree fruits, but the method also has certain applicability problems, and the high performance liquid chromatography method has longer time consumption and lower separation degree.
Disclosure of Invention
Based on the method, the invention provides a method for constructing the characteristic spectrum of the negundo chastetree fruit, which has the advantages of short time consumption, simple operation, rich spectrum information, strong characteristics and good reproducibility, and is simultaneously applicable to negundo chastetree fruit medicinal materials, standard decoction and formula particles.
The invention is realized by the following technical scheme.
A method for constructing a characteristic spectrum of negundo chastetree fruit comprises the following steps:
preparing a mixed reference solution from protocatechuic acid reference, 4-hydroxybenzoic acid reference, isoorientin reference, orientin reference and vitexin reference;
taking a negundo chastetree fruit sample, and preparing a sample solution;
respectively carrying out ultra-high performance liquid chromatography analysis on the mixed reference substance solution and the sample solution to construct a characteristic spectrum of negundo chastetree fruit;
wherein, the conditions of the ultra performance liquid chromatography include: mobile phase A is acetonitrile, mobile phase B is phosphoric acid aqueous solution; gradient elution is adopted; the gradient elution procedure was: 0 min-15 min, the volume percentage of the mobile phase A is changed from 7% to 17%; 15-20 min, wherein the volume percentage of the mobile phase A is changed from 17% to 19%; 20-22 min, wherein the volume percentage of the mobile phase A is changed from 19% to 90%; the volume percentage of the mobile phase A is kept to be 90% within 22-30 min.
In one embodiment, the volume fraction of phosphoric acid in the phosphoric acid aqueous solution is 0.05% -0.3%.
In one embodiment, the conditions of the ultra performance liquid chromatography further comprise: the chromatographic column is octadecylsilane chemically bonded silica gel column; the column temperature is 28-32 ℃; the flow rate is 0.28 mL/min-0.32 mL/min; the wavelength is 240 nm-280 nm.
In one embodiment, preparing the mixed control solution includes the steps of:
mixing the protocatechuic acid reference substance, the 4-hydroxybenzoic acid reference substance, the isoorientin reference substance, the orientin reference substance and the vitexin reference substance with 60% -80% ethanol.
In one embodiment, preparing a test solution includes the steps of:
mixing the negundo chastetree fruit sample with an alcohol-containing solution, carrying out ultrasonic extraction, and then filtering to obtain a filtrate.
In one embodiment, the negundo chastetree seed sample is selected from negundo chastetree fruit medicinal material, negundo chastetree fruit standard decoction or negundo chastetree fruit formula particles.
The invention also provides a detection method of the negundo chastetree fruit, which comprises the following steps:
taking a negundo chastetree fruit sample, and preparing a solution to be detected;
carrying out ultra-high performance liquid chromatography on the solution to be detected;
wherein, the conditions of the ultra performance liquid chromatography include: mobile phase A is acetonitrile, mobile phase B is phosphoric acid aqueous solution; gradient elution is adopted; the gradient elution procedure was: 0 min-15 min, the volume percentage of the mobile phase A is changed from 7% to 17%; 15-20 min, wherein the volume percentage of the mobile phase A is changed from 17% to 19%; 20-22 min, wherein the volume percentage of the mobile phase A is changed from 19% to 90%; the volume percentage of the mobile phase A is kept to be 90% within 22-30 min.
In one embodiment, the volume fraction of phosphoric acid in the phosphoric acid aqueous solution is 0.05% -0.3%.
In one embodiment, the conditions of the ultra performance liquid chromatography further comprise: the chromatographic column is octadecylsilane chemically bonded silica gel column; the column temperature is 28-32 ℃; the flow rate is 0.28 mL/min-0.32 mL/min; the wavelength is 240 nm-280 nm.
The invention also provides a method for identifying negundo chastetree fruit and fructus viticis, which comprises the following steps:
taking an object to be detected, carrying out ultra-high performance liquid chromatography analysis, and comparing the obtained spectrum of the object to be detected with a characteristic spectrum constructed by the characteristic spectrum construction method of negundo chastetree fruits;
wherein, the conditions of the ultra performance liquid chromatography include: mobile phase A is acetonitrile, mobile phase B is phosphoric acid aqueous solution; gradient elution is adopted; the gradient elution procedure was: 0 min-15 min, the volume percentage of the mobile phase A is changed from 7% to 17%; 15-20 min, wherein the volume percentage of the mobile phase A is changed from 17% to 19%; 20-22 min, wherein the volume percentage of the mobile phase A is changed from 19% to 90%; the volume percentage of the mobile phase A is kept to be 90% within 22-30 min.
In one embodiment, the volume fraction of phosphoric acid in the phosphoric acid aqueous solution is 0.05% -0.3%.
In one embodiment, the conditions of the ultra performance liquid chromatography further comprise: the chromatographic column is octadecylsilane chemically bonded silica gel column; the column temperature is 28-32 ℃; the flow rate is 0.28 mL/min-0.32 mL/min; the wavelength is 240 nm-280 nm.
Compared with the prior art, the method for constructing the characteristic spectrum of the negundo chastetree fruit has the following beneficial effects:
the invention adopts ultra-high performance liquid chromatography to establish the characteristic spectrum of the negundo chastetree fruit, and the characteristic spectrum of the negundo chastetree fruit finally constructed by screening acetonitrile and phosphoric acid water solutions respectively as mobile phases and limiting the elution mode can be simultaneously applied to negundo chastetree fruit medicinal materials, negundo chastetree fruit standard decoction and negundo chastetree fruit formula particles. In addition, the characteristic spectrum of the negundo chastetree fruit constructed by the invention has 8 common peaks, is rich in spectrum information, strong in characteristics and good in reproducibility, and has the advantages of simplicity in operation and short time consumption.
Furthermore, the method for constructing the characteristic spectrum of the negundo chastetree fruit can distinguish and identify the negundo chastetree fruit and the fructus viticis.
Drawings
FIG. 1 is a superposition of characteristic maps of 15 batches of fructus viticis medicinal materials;
FIG. 2 is a superposition of characteristic maps of 15 batches of fructus viticis standard decoction provided by the invention;
FIG. 3 is a superimposed graph of characteristic maps of 3 batches of fructus viticis formula particles provided by the invention;
fig. 4 is a superposition diagram of characteristic maps of 12 batches of fructus viticis medicinal materials;
fig. 5 is a superposition of characteristic maps of 3 batches of fructus viticis formula granules provided by the invention;
FIG. 6 is a characteristic comparison map of the negundo chastetree fruit medicinal material provided by the invention;
FIG. 7 is a diagram showing the characteristic comparison of the fructus Viticis negundo standard decoction according to the present invention;
FIG. 8 is a graph of the characteristics of the fructus Viticis negundo formula granule according to the present invention;
FIG. 9 is a characteristic map of the fructus Viticis negundo reference medicinal material provided by the invention;
fig. 10 is a diagram for investigating specificity of a characteristic map of negundo chastetree fruit;
FIG. 11 is a graph showing the peak assignment of protocatechuic acid according to the present invention;
FIG. 12 is a graph showing peak assignments of 4-hydroxybenzoic acid according to the present invention;
fig. 13 is a diagram showing the peak assignment of isoorientin provided by the invention;
fig. 14 is a orientin peak identification chart provided by the invention;
fig. 15 is a diagram of the assignment of castus Jing Ganfeng provided by the present invention;
fig. 16 is a graph showing the comparison of fructus Viticis negundo, fructus Viticis negundo medicinal materials and formulation particles.
Detailed Description
In order that the invention may be readily understood, a more complete description of the invention will be rendered by reference to the appended drawings. The drawings illustrate preferred embodiments of the invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.
The invention provides a method for constructing a characteristic spectrum of negundo chastetree fruit, which comprises the following steps:
preparing a mixed reference solution from protocatechuic acid reference, 4-hydroxybenzoic acid reference, isoorientin reference, orientin reference and vitexin reference;
taking a negundo chastetree fruit sample, and preparing a sample solution;
respectively carrying out ultra-high performance liquid chromatography analysis on the mixed reference substance solution and the sample solution to construct a characteristic map of negundo chastetree fruit;
wherein, the conditions of the ultra performance liquid chromatography include: mobile phase A is acetonitrile, mobile phase B is phosphoric acid aqueous solution; gradient elution is adopted; the gradient elution procedure was: 0 min-15 min, the volume percentage of the mobile phase A is changed from 7% to 17%; 15-20 min, wherein the volume percentage of the mobile phase A is changed from 17% to 19%; 20-22 min, wherein the volume percentage of the mobile phase A is changed from 19% to 90%; the volume percentage of the mobile phase A is kept to be 90% within 22-30 min.
In a specific example, the volume fraction of phosphoric acid in the phosphoric acid aqueous solution is 0.05% to 0.3%.
It is understood that in the present invention, the volume fraction of phosphoric acid in the phosphoric acid aqueous solution includes, but is not limited to, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.12%, 0.14%, 0.16%, 0.18%, 0.2%, 0.25%, 0.3%.
In a specific example, the conditions of the ultra performance liquid chromatography further include: the chromatographic column is octadecylsilane chemically bonded silica gel column; the column temperature is 28-32 ℃; the flow rate is 0.28 mL/min-0.32 mL/min; the wavelength is 240 nm-280 nm.
Preferably, the chromatographic column is Agilent SB C 18 (100mm×2.1mm，1.8μm)。
Preferably, the column temperature is 30 ℃; the flow rate is 0.3mL/min; the wavelength was 258nm.
In a specific example, the conditions of the ultra performance liquid chromatography further include: the sample loading was 1. Mu.L.
In a specific example, preparing the mixed control solution includes the steps of:
mixing protocatechuic acid reference substance, 4-hydroxybenzoic acid reference substance, isoorientin reference substance, orientin reference substance and vitexin reference substance with 60% -80% ethanol.
More specifically, the preparation of the mixed control solution comprises the following steps:
mixing protocatechuic acid reference, 4-hydroxybenzoic acid reference, isoorientin reference, orientin reference, and vitexin reference with 70% ethanol.
In a specific example, the negundo chastetree seed sample is selected from negundo chastetree seed medicinal material, negundo chastetree seed standard decoction or negundo chastetree seed formula particles.
In a specific example, preparing a test solution includes the steps of:
mixing fructus Viticis negundo sample with alcoholic solution, ultrasonic extracting, filtering, and collecting filtrate.
Preferably, the alcoholic solution is selected from one or more of aqueous methanol and aqueous ethanol.
More specifically, the preparation of the test solution includes the steps of:
mixing fructus Viticis negundo with 70% methanol, ultrasonic extracting, filtering, and collecting filtrate; or (b)
Mixing fructus Viticis negundo standard decoction with 70% ethanol, ultrasonic extracting, filtering, and collecting filtrate; or (b)
Mixing fructus Vitics negundo formula granule with 70% ethanol, ultrasonic extracting, filtering, and collecting filtrate.
In one specific example, the conditions of ultrasonic extraction include: the power is 250W-350W, the frequency is 30 kHz-50 kHz, the time is 30 min-60 min, and the temperature is 25-35 ℃.
In a specific example, the method for constructing the feature map of negundo chastetree fruit further comprises the following steps:
mixing fructus Viticis negundo reference material with 70% ethanol, ultrasonic extracting, filtering, and collecting filtrate to obtain reference solution of reference material;
and (5) performing ultra-high performance liquid chromatography analysis on the reference substance solution of the reference medicinal material.
The invention also provides a detection method of the negundo chastetree fruit, which comprises the following steps:
taking a negundo chastetree fruit sample, and preparing a solution to be detected;
carrying out ultra-high performance liquid chromatography on the solution to be detected;
wherein, the conditions of the ultra performance liquid chromatography include: mobile phase A is acetonitrile, mobile phase B is phosphoric acid aqueous solution; gradient elution is adopted; the gradient elution procedure was: 0 min-15 min, the volume percentage of the mobile phase A is changed from 7% to 17%; 15-20 min, wherein the volume percentage of the mobile phase A is changed from 17% to 19%; 20-22 min, wherein the volume percentage of the mobile phase A is changed from 19% to 90%; the volume percentage of the mobile phase A is kept to be 90% within 22-30 min.
In one specific example, preparing the solution to be tested includes the steps of:
mixing fructus Viticis negundo sample with alcoholic solution, ultrasonic extracting, filtering, and collecting filtrate.
More specifically, the preparation of the solution to be tested includes the steps of:
mixing fructus Viticis negundo with 70% methanol, ultrasonic extracting, filtering, and collecting filtrate; or (b)
Mixing fructus Viticis negundo standard decoction with 70% ethanol, ultrasonic extracting, filtering, and collecting filtrate; or (b)
Mixing fructus Vitics negundo formula granule with 70% ethanol, ultrasonic extracting, filtering, and collecting filtrate.
In a specific example, the volume fraction of phosphoric acid in the phosphoric acid aqueous solution is 0.05% to 0.3%.
In a specific example, the conditions of the ultra performance liquid chromatography further include: the chromatographic column is octadecylsilane chemically bonded silica gel column; the column temperature is 28-32 ℃; the flow rate is 0.28 mL/min-0.32 mL/min; the wavelength is 240 nm-280 nm.
Preferably, the chromatographic column is Agilent SB C 18 (100mm×2.1mm，1.8μm)。
Preferably, the column temperature is 30 ℃; the flow rate is 0.3mL/min; the wavelength was 258nm.
In a specific example, the conditions of the ultra performance liquid chromatography further include: the sample loading was 1. Mu.L.
The invention also provides a method for identifying negundo chastetree fruit and fructus viticis, which comprises the following steps:
taking an object to be detected, carrying out ultra-high performance liquid chromatography analysis, and comparing the obtained spectrum of the object to be detected with a characteristic spectrum constructed by the chastetree fruit characteristic spectrum construction method;
wherein, the conditions of the ultra performance liquid chromatography include: mobile phase A is acetonitrile, mobile phase B is phosphoric acid aqueous solution; gradient elution is adopted; the gradient elution procedure was: 0 min-15 min, the volume percentage of the mobile phase A is changed from 7% to 17%; 15-20 min, wherein the volume percentage of the mobile phase A is changed from 17% to 19%; 20-22 min, wherein the volume percentage of the mobile phase A is changed from 19% to 90%; the volume percentage of the mobile phase A is kept to be 90% within 22-30 min.
In a specific example, the volume fraction of phosphoric acid in the phosphoric acid aqueous solution is 0.05% to 0.3%.
In a specific example, the conditions of the ultra performance liquid chromatography further include: the chromatographic column is octadecylsilane chemically bonded silica gel column; the column temperature is 28-32 ℃; the flow rate is 0.28 mL/min-0.32 mL/min; the wavelength is 240 nm-280 nm.
Preferably, the chromatographic column is Agilent SB C 18 (100mm×2.1mm，1.8μm)。
Preferably, the column temperature is 30 ℃; the flow rate is 0.3mL/min; the wavelength was 258nm.
In a specific example, the conditions of the ultra performance liquid chromatography further include: the sample loading was 1. Mu.L.
The method for constructing the characteristic spectrum of the negundo chastetree fruit is further described in detail by combining specific examples. The raw materials used in the following examples are all commercially available products unless otherwise specified.
Example 1
The embodiment provides a method for constructing a characteristic spectrum of negundo chastetree fruit, which comprises the following steps:
1.1 instruments and reagents
Agilent ultra high performance liquid chromatograph (Agilent 1290 Infinicity II), thermo Vanquish ultra high performance liquid chromatograph, agilent ZORBAX SB C 18 Chromatographic column (2.1 mm. Times.100 mm,1.8 μm); AL104, ML204, XPR26DR/a electronic analytical balance (mertrer-tolidol instruments (Shanghai); HH-S6A electric heating constant temperature water bath (Beijing Uyoxing Instrument Co., ltd.), KQ-500DA/KQ-500DB type numerical control ultrasonic cleaner (Kunshan ultrasonic instrument Co., ltd.).
Acetonitrile is chromatographic purity; the water is ultrapure water (self-made in laboratory); the other reagents were all analytically pure. Protocatechuic acid reference (lot number: 110809-201906, content: 97.7%, china food and drug inspection institute), 4-hydroxybenzoic acid reference (lot number: 110792-200503, content: 100.0%, china food and drug inspection institute), isoorientin reference (lot number: 111974-201401, content: 94.0%, china food and drug inspection institute), orientin reference (lot number: 111777-202003, content: 98.0%, china food and drug inspection institute), vitexin reference (lot number: CFN99203, content: 98.0%, chemfaces); fructus Viticis negundo control material (China food and drug inspection institute, lot number: 121277-201802); fructus Viticis negundo prescription granule (source: guangdong party pharmaceutical Co., ltd.); fructus Vitics Simplicifoliae granule (source: guangdong party pharmaceutical Co., ltd.); maltodextrin (FH 2106002-A, jilin Zhongliang Biochemical energy sales Co., ltd./Zhongliang Biochemical energy (princess Lin) Co., ltd.).
The medicine materials used in the invention are identified as the Vitex negundo fruit which is the dry mature fruit of Vitex negundo L.var. Fructus Vitics Simplicifoliae is dry mature fruit of Vitex rotundifolia L.var.simplicifolia Cham of Verbenaceae. The fructus viticis standard decoction is prepared by laboratory. Sample information is shown in tables 1 and 2.
TABLE 1 negundo chastetree fruit related sample information
TABLE 2 Vitex negundo sample information
1.2 methods and results
1.2.1A standard decoction is prepared by decocting fructus Viticis negundo decoction pieces 100g, adding 8 times of water for the first time, soaking for 30min, boiling with strong fire (500W), keeping slight boiling with slow fire (200W) for 30min, filtering the decoction with 350 mesh sieve, and cooling the filtrate with cold water; adding 6 times of water into the second decoction, boiling with strong fire (500W), keeping micro-boiling with slow fire (200W) for 25 min, filtering the decoction with 350 mesh sieve, cooling the filtrate with cold water rapidly, mixing the filtrates, concentrating under reduced pressure to obtain 100ml clear paste, packaging into petri dish (diameter 9 cm), vacuum lyophilizing, taking out, and sealing.
1.2.2 characteristic Spectrum chromatography Condition chromatography column Agilent SB C 18 (100 mm. Times.2.1 mm,1.8 μm) at a flow rate of 0.30mL/min; gradient elution with acetonitrile as mobile phase a and 0.1% phosphoric acid as mobile phase B is shown in table 3; the detection wavelength is 258nm; column temperature is 30 ℃; the sample injection amount was 1. Mu.L.
TABLE 3 gradient elution table
1.2.3 preparation of reference solution protocatechuic acid reference, 4-hydroxybenzoic acid reference, isoorientin reference, orientin reference, and appropriate amount of vitexin reference are precisely weighed, and 70% ethanol is added to prepare mixed solution containing 20 μg per 1ml as reference solution.
1.2.4 preparation of control medicinal material solution 1.0g of fructus Viticis negundo control medicinal material is placed in a conical flask with a plug, 25ml of 70% ethanol is added, ultrasonic treatment (power 300W, frequency 40 kHz) is carried out for 30 minutes, cooling is carried out, shaking is carried out, filtration is carried out, and subsequent filtrate is taken as control medicinal material reference material solution.
1.2.5 preparation of sample powder (sieving with a third sieve) of the sample solution, precisely weighing, placing into a conical flask with a plug, precisely adding 25ml of 70% methanol, weighing, ultrasonic treating (power 300W, frequency 40 kHz) for 60min, cooling, weighing again, supplementing the lost weight with 70% methanol, shaking, filtering, and collecting the filtrate.
1.2.6 preparation of standard decoction sample solution A proper amount of sample is ground, about 0.2g is taken, precisely weighed, placed in a conical flask with a plug, precisely added with 25ml of 70% ethanol, weighed, subjected to ultrasonic treatment (power 300W, frequency 40 kHz) for 30 minutes, cooled, weighed again, and subjected to 70% ethanol to make up the lost weight, shaken uniformly, filtered, and the subsequent filtrate is taken, thus obtaining the decoction.
1.2.7 preparation of sample solution of granule, grinding, taking about 0.2g, precisely weighing, placing into conical flask with plug, precisely adding 70% ethanol 25ml, weighing, ultrasonic treating (power 300W, frequency 40 kHz) for 30min, cooling, weighing again, supplementing the lost weight with 70% ethanol, shaking, filtering, and collecting the filtrate.
1.2.8 methodological validation
1.2.8.1 the sample solution is continuously sampled for 6 times according to the chromatographic condition under the item "1.2.2", the isoorientin peak is used as a reference peak S, and the relative retention time of 8 common peaks and the RSD of the relative peak area are calculated to be less than 2%, so that the sample injector has good precision.
1.2.8.2 repeatability test the same sample solution powder was taken at about 0.2g, 6 parts in parallel, the sample solution was prepared according to the method defined under items "1.2.5", "1.2.6", "1.2.7", the relative retention time of 8 common peaks and RSD of the relative peak area were calculated as measured under chromatographic conditions under item "1.2.2" with isoorientin peak as reference peak S, indicating that the method was good in reproducibility.
1.2.8.3 stability test the same sample solution is taken, and sample injection measurement is carried out at 0, 2, 4, 6, 8 and 12 hours according to the chromatographic conditions under the item "1.2.2", isoorientin peaks are taken as reference peaks S, and the relative retention time of 8 common peaks and the RSD of the relative peak areas are calculated to be less than 2%, so that the sample solution has good stability in 12 hours.
1.2.9 sample measurement is carried out by respectively preparing medicinal materials, standard decoction and prescription granule test sample solutions under the conditions of "1.2.5", "1.2.6" and "1.2.7", respectively precisely sucking the test sample solution and reference substance solution of the reference medicinal material, and carrying out measurement and analysis according to the chromatographic conditions under the condition of "1.2.2". The method comprises the steps of adopting a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012.0 version) recommended by the national formulary Committee to respectively perform data processing on UPLC characteristic maps of 15 batches of negundo chastetree fruit medicinal material samples, 15 batches of negundo chastetree fruit standard decoction samples and 3 batches of negundo chastetree fruit standard decoction samples, and performing common peak identification, wherein the results are shown in figures 1-5; the comparison characteristic spectrum of the negundo chastetree fruit medicinal material, the standard decoction and the formula granule UPLC is established, and the results are shown in figures 6-8. 7 common peaks with known components, larger peak areas and better peak pattern and separation degree are selected as characteristic peaks of negundo chastetree fruit medicinal materials, standard decoction and formula particles, the 7 common peaks can correspond to negundo chastetree fruit reference medicinal materials, the result is shown in figure 9, which shows that the 7 common characteristic peaks can be stably transmitted from negundo chastetree fruit medicinal materials to negundo chastetree fruit standard decoction and negundo chastetree fruit formula particles, so that the 7 common peaks are selected as characteristic peaks of a negundo chastetree fruit standard decoction UPLC characteristic spectrum, the retention time is relatively central, the peak 4 isoorientin chromatographic peak of the reference substance is taken as a reference peak S, the relative retention time of the peak 3, the peak 4 and the reference peak is calculated, and the characteristic peaks are positioned by utilizing the relative retention time.
1.2.10 identifying characteristic peaks, carrying out ultraviolet spectrum analysis on the sample solution, and comparing the ultraviolet spectrum with a reference substance, wherein the results are shown in figures 10-15, and finally determining that the components contained in the negundo chastetree fruits are respectively: peak 1 is protocatechuic acid, peak 2 is 4-hydroxybenzoic acid, peak 4 is isoorientin, peak 5 is orientin, peak 6 is vitexin.
Example 2
The embodiment provides a method for determining and identifying negundo chastetree fruit and fructus viticis, which comprises the following steps:
the method established in example 1 was used to perform characteristic spectrum measurement on 15 batches of fructus viticis medicinal materials and standard decoction thereof, 3 batches of fructus viticis formula particles, 12 batches of fructus viticis medicinal materials and 3 batches of fructus viticis formula particles, respectively, and peak 4 isoorientin peak was used as reference peak S, and calculate the relative retention time and relative peak area of each characteristic peak, and calculate the RSD value. The results are all within the standard limit range, and peak 3 is added in the chastetree fruit compared with the chastetree fruit characteristic map, and meanwhile, the results show partial chastetree fruit missing peak 7 in batches (the comparison chart is shown in figure 16); the method comprises the following steps:
(1) The characteristic spectrum measurement results of 15 batches of negundo chastetree fruit medicinal materials are shown in tables 4 and 5.
TABLE 4 relative retention time of characteristic maps of Vitex negundo and Vitex negundo medicinal materials
TABLE 5 characteristic spectrum relative peak area of the Vitex negundo medicinal materials
(2) The results of the feature map measurement of 12 batches of fructus viticis medicinal materials are shown in table 6 and table 7.
TABLE 6 relative Retention time of the characteristic spectra of the fructus Vitics Simplicifoliae batches 12
TABLE 7 characterization of the fructus Viticis in batch and relative peak area
(3) The results of the characteristic spectrum measurement of the 15 batches of fructus viticis standard decoction are shown in table 8 and table 9.
Table 8 15 characteristics of the decoction of the fructus Viticis negundo (relative retention time)
Table 9 15 characteristics map of the Standard decoction of negundo chastetree fruit (relative peak area)
(4) The characteristic spectrum measurement results of the 3 batches of negundo chastetree fruit formula particles are shown in table 10 and table 11.
TABLE 10 3 characteristics spectrum of the particles of the negundo chastetree seed formula (relative retention time)
Table 11-3 characteristics spectrum of the granule formulation of negundo chastetree fruit (relative peak area)
(5) The characteristic spectrum measurement results of the 3 batches of fructus viticis formula particles are shown in table 12 and table 13.
TABLE 12 3 characterization of the particles of the fructus Viticis formula (relative retention time)
TABLE 13 3 characteristics map of the fructus Viticis formula particles (relative peak area)
The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The above examples merely represent a few embodiments of the present invention, which facilitate a specific and detailed understanding of the technical solutions of the present invention, but are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. It should be understood that, based on the technical solutions provided by the present invention, those skilled in the art may obtain technical solutions through logical analysis, reasoning or limited experiments, which are all within the scope of protection of the appended claims. The scope of the patent is therefore intended to be covered by the appended claims, and the description and drawings may be interpreted as illustrative of the contents of the claims.
Claims (8)
1. The method for constructing the characteristic spectrum of the negundo chastetree fruit is characterized by comprising the following steps of:
preparing a mixed reference solution from protocatechuic acid reference, 4-hydroxybenzoic acid reference, isoorientin reference, orientin reference and vitexin reference;
taking a negundo chastetree fruit sample, and preparing a sample solution;
respectively carrying out ultra-high performance liquid chromatography analysis on the mixed reference substance solution and the sample solution to construct a characteristic spectrum of negundo chastetree fruit;
wherein, the conditions of the ultra performance liquid chromatography include: the chromatographic column is octadecylsilane chemically bonded silica gel column; the wavelength is 240 nm-280 nm; the mobile phase A is acetonitrile, the mobile phase B is phosphoric acid aqueous solution, and the volume fraction of phosphoric acid in the phosphoric acid aqueous solution is 0.05% -0.3%; gradient elution is adopted; the gradient elution procedure was: 0 min-15 min, wherein the volume percentage of the mobile phase A is changed from 7% to 17%; 15-20 min, wherein the volume percentage of the mobile phase A is changed from 17% to 19%; 20-22 min, wherein the volume percentage of the mobile phase A is changed from 19% to 90%; 22-30 min, wherein the volume percentage of the mobile phase A is kept to be 90%;
the preparation of the test solution comprises the following steps:
mixing the negundo chastetree fruit sample with an alcohol-containing solution, performing ultrasonic extraction, and then filtering to obtain a filtrate;
the alcohol-containing solution is selected from one or more of methanol aqueous solution and ethanol aqueous solution.
2. The method for constructing a feature map of negundo chastetree fruit according to claim 1, wherein the volume fraction of phosphoric acid in the phosphoric acid aqueous solution is 0.1%.
3. The method for constructing a characteristic spectrum of chaste tree fruit according to claim 1, characterized in that the conditions of the ultra performance liquid chromatography further comprise: the column temperature is 28-32 ℃; the flow rate is 0.28mL/min to 0.32mL/min.
4. The method for constructing a characteristic spectrum of chaste tree fruit according to claim 1, wherein the preparation of the mixed reference solution comprises the steps of:
and mixing the protocatechuic acid reference substance, the 4-hydroxybenzoic acid reference substance, the isoorientin reference substance, the orientin reference substance and the vitexin reference substance with 60% -80% ethanol.
5. The method for constructing a feature map of negundo chastetree fruit according to any one of claims 1 to 4, wherein the negundo chastetree fruit sample is selected from negundo chastetree fruit medicinal materials, negundo chastetree fruit standard decoction or negundo chastetree fruit formula particles.
6. The identification method of the negundo chastetree fruit and the fructus viticis is characterized by comprising the following steps:
taking an object to be detected, carrying out ultra-high performance liquid chromatography analysis, and comparing the obtained spectrum of the object to be detected with a characteristic spectrum constructed by the characteristic spectrum construction method of the negundo chastetree fruit according to any one of claims 1-5;
wherein, the conditions of the ultra performance liquid chromatography include: the chromatographic column is octadecylsilane chemically bonded silica gel column; the wavelength is 240 nm-280 nm; the mobile phase A is acetonitrile, the mobile phase B is phosphoric acid aqueous solution, and the volume fraction of phosphoric acid in the phosphoric acid aqueous solution is 0.05% -0.3%; gradient elution is adopted; the gradient elution procedure was: 0 min-15 min, wherein the volume percentage of the mobile phase A is changed from 7% to 17%; 15-20 min, wherein the volume percentage of the mobile phase A is changed from 17% to 19%; 20-22 min, wherein the volume percentage of the mobile phase A is changed from 19% to 90%; 22-30 min, wherein the volume percentage of the mobile phase A is kept to be 90%;
the preparation of the object to be detected comprises the following steps: mixing a sample of an object to be detected with an alcohol-containing solution, performing ultrasonic extraction, and then filtering to obtain a filtrate;
the alcohol-containing solution is selected from one or more of 70% methanol aqueous solution and 70% ethanol aqueous solution.
7. The method for identifying negundo chastetree fruit and fructus viticis according to claim 6, wherein the volume fraction of phosphoric acid in the phosphoric acid aqueous solution is 0.1%.
8. The method for identifying negundo chastetree fruit and fructus viticis according to claim 6, wherein the conditions of the ultra performance liquid chromatography further comprise: the column temperature is 28-32 ℃; the flow rate is 0.28mL/min to 0.32mL/min.
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