CN115586279B - Detection method of bupleurum chinense or preparation thereof and construction method of fingerprint of bupleurum chinense or preparation thereof - Google Patents
Detection method of bupleurum chinense or preparation thereof and construction method of fingerprint of bupleurum chinense or preparation thereof Download PDFInfo
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- 241000510654 Bupleurum chinense Species 0.000 title claims abstract description 115
- 238000002360 preparation method Methods 0.000 title claims abstract description 61
- 238000001514 detection method Methods 0.000 title claims abstract description 33
- 238000010276 construction Methods 0.000 title claims abstract description 11
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 69
- 230000014759 maintenance of location Effects 0.000 claims abstract description 42
- 238000000034 method Methods 0.000 claims abstract description 35
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 31
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims abstract description 10
- 238000010828 elution Methods 0.000 claims abstract description 7
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- 238000000605 extraction Methods 0.000 claims description 33
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- IVTMALDHFAHOGL-UHFFFAOYSA-N eriodictyol 7-O-rutinoside Natural products OC1C(O)C(O)C(C)OC1OCC1C(O)C(O)C(O)C(OC=2C=C3C(C(C(O)=C(O3)C=3C=C(O)C(O)=CC=3)=O)=C(O)C=2)O1 IVTMALDHFAHOGL-UHFFFAOYSA-N 0.000 claims description 32
- FDRQPMVGJOQVTL-UHFFFAOYSA-N quercetin rutinoside Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC=2C(C3=C(O)C=C(O)C=C3OC=2C=2C=C(O)C(O)=CC=2)=O)O1 FDRQPMVGJOQVTL-UHFFFAOYSA-N 0.000 claims description 32
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- ALABRVAAKCSLSC-UHFFFAOYSA-N rutin Natural products CC1OC(OCC2OC(O)C(O)C(O)C2O)C(O)C(O)C1OC3=C(Oc4cc(O)cc(O)c4C3=O)c5ccc(O)c(O)c5 ALABRVAAKCSLSC-UHFFFAOYSA-N 0.000 claims description 32
- 235000005493 rutin Nutrition 0.000 claims description 32
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- REFJWTPEDVJJIY-UHFFFAOYSA-N Quercetin Chemical compound C=1C(O)=CC(O)=C(C(C=2O)=O)C=1OC=2C1=CC=C(O)C(O)=C1 REFJWTPEDVJJIY-UHFFFAOYSA-N 0.000 claims description 26
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- CWVRJTMFETXNAD-KLZCAUPSSA-N Neochlorogenin-saeure Natural products O[C@H]1C[C@@](O)(C[C@@H](OC(=O)C=Cc2ccc(O)c(O)c2)[C@@H]1O)C(=O)O CWVRJTMFETXNAD-KLZCAUPSSA-N 0.000 claims description 18
- CWVRJTMFETXNAD-JUHZACGLSA-N chlorogenic acid Chemical compound O[C@@H]1[C@H](O)C[C@@](O)(C(O)=O)C[C@H]1OC(=O)\C=C\C1=CC=C(O)C(O)=C1 CWVRJTMFETXNAD-JUHZACGLSA-N 0.000 claims description 18
- 229940074393 chlorogenic acid Drugs 0.000 claims description 18
- FFQSDFBBSXGVKF-KHSQJDLVSA-N chlorogenic acid Natural products O[C@@H]1C[C@](O)(C[C@@H](CC(=O)C=Cc2ccc(O)c(O)c2)[C@@H]1O)C(=O)O FFQSDFBBSXGVKF-KHSQJDLVSA-N 0.000 claims description 18
- 235000001368 chlorogenic acid Nutrition 0.000 claims description 18
- BMRSEYFENKXDIS-KLZCAUPSSA-N cis-3-O-p-coumaroylquinic acid Natural products O[C@H]1C[C@@](O)(C[C@@H](OC(=O)C=Cc2ccc(O)cc2)[C@@H]1O)C(=O)O BMRSEYFENKXDIS-KLZCAUPSSA-N 0.000 claims description 18
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- ZVOLCUVKHLEPEV-UHFFFAOYSA-N Quercetagetin Natural products C1=C(O)C(O)=CC=C1C1=C(O)C(=O)C2=C(O)C(O)=C(O)C=C2O1 ZVOLCUVKHLEPEV-UHFFFAOYSA-N 0.000 claims description 13
- HWTZYBCRDDUBJY-UHFFFAOYSA-N Rhynchosin Natural products C1=C(O)C(O)=CC=C1C1=C(O)C(=O)C2=CC(O)=C(O)C=C2O1 HWTZYBCRDDUBJY-UHFFFAOYSA-N 0.000 claims description 13
- MWDZOUNAPSSOEL-UHFFFAOYSA-N kaempferol Natural products OC1=C(C(=O)c2cc(O)cc(O)c2O1)c3ccc(O)cc3 MWDZOUNAPSSOEL-UHFFFAOYSA-N 0.000 claims description 13
- 235000005875 quercetin Nutrition 0.000 claims description 13
- 229960001285 quercetin Drugs 0.000 claims description 13
- GYFFKZTYYAFCTR-JUHZACGLSA-N 4-O-trans-caffeoylquinic acid Chemical compound O[C@@H]1C[C@](O)(C(O)=O)C[C@@H](O)[C@H]1OC(=O)\C=C\C1=CC=C(O)C(O)=C1 GYFFKZTYYAFCTR-JUHZACGLSA-N 0.000 claims description 11
- GYFFKZTYYAFCTR-UHFFFAOYSA-N 5-O-(6'-O-galloyl)-beta-D-glucopyranosylgentisic acid Natural products OC1CC(O)(C(O)=O)CC(O)C1OC(=O)C=CC1=CC=C(O)C(O)=C1 GYFFKZTYYAFCTR-UHFFFAOYSA-N 0.000 claims description 11
- GYFFKZTYYAFCTR-LMRQPLJMSA-N cryptochlorogenic acid Natural products O[C@H]1C[C@@](O)(C[C@H](O)[C@H]1OC(=O)C=Cc2ccc(O)c(O)c2)C(=O)O GYFFKZTYYAFCTR-LMRQPLJMSA-N 0.000 claims description 11
- 239000012085 test solution Substances 0.000 claims description 11
- GWTUHAXUUFROTF-UHFFFAOYSA-N pseudochlorogenic acid Natural products C1C(O)C(O)C(O)CC1(C(O)=O)OC(=O)C=CC1=CC=C(O)C(O)=C1 GWTUHAXUUFROTF-UHFFFAOYSA-N 0.000 claims description 10
- CWVRJTMFETXNAD-NXLLHMKUSA-N trans-5-O-caffeoyl-D-quinic acid Chemical compound O[C@H]1[C@H](O)C[C@](O)(C(O)=O)C[C@H]1OC(=O)\C=C\C1=CC=C(O)C(O)=C1 CWVRJTMFETXNAD-NXLLHMKUSA-N 0.000 claims description 10
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 6
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims description 3
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- 239000000945 filler Substances 0.000 claims description 2
- 238000002474 experimental method Methods 0.000 abstract description 5
- 241000202726 Bupleurum Species 0.000 description 39
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- 241001132281 Bupleurum marginatum Species 0.000 description 9
- 235000017166 Bambusa arundinacea Nutrition 0.000 description 8
- 235000017491 Bambusa tulda Nutrition 0.000 description 8
- 241001330002 Bambuseae Species 0.000 description 8
- 235000015334 Phyllostachys viridis Nutrition 0.000 description 8
- 239000011425 bamboo Substances 0.000 description 8
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- 238000011156 evaluation Methods 0.000 description 6
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical group CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
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- 229910021642 ultra pure water Inorganic materials 0.000 description 2
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- 241001513903 Bupleurum microcephalum Species 0.000 description 1
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- 206010037660 Pyrexia Diseases 0.000 description 1
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- 238000000691 measurement method Methods 0.000 description 1
- 201000009240 nasopharyngitis Diseases 0.000 description 1
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- 239000002994 raw material Substances 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
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- 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)
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
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- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
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- Investigating Or Analysing Biological Materials (AREA)
- Saccharide Compounds (AREA)
Abstract
The invention relates to the detection field, in particular to a detection method of bupleurum chinense or a preparation thereof and a construction method of a fingerprint thereof. The detection method provided by the invention comprises the following steps: detecting bupleuri radix or its preparation by HPLC; the chromatographic conditions of the HPLC are as follows: methanol is used as a mobile phase A, a 0.4% phosphoric acid solution is used as a mobile phase B, and gradient elution is adopted. The method provided by the invention can accurately analyze the bupleurum chinense or the preparation thereof and construct the fingerprint, accurately identify the bupleurum chinense of different types and ensure the uniform and stable quality of the bupleurum chinense preparation. Experiments show that the invention explores the optimal chromatographic conditions of the detection method of the bupleurum chinense or the preparation thereof, and detects the bupleurum chinense or the preparation thereof based on the chromatographic conditions, successfully constructs the fingerprint of the bupleurum chinense or the preparation thereof, wherein the RSD of the characteristic peaks of the fingerprint relative to the retention time is less than 1%, and successfully identifies the bupleurum chinense and the bupleurum chinense.
Description
Technical Field
The invention relates to the detection field, in particular to a detection method of bupleurum chinense or a preparation thereof and a construction method of a fingerprint thereof.
Background
The Chinese medicine bupleurum chinense is one of main varieties of bupleurum chinense, has a cultivation history of more than 30 years in Sichuan province, and has the effects of dispelling heat, soothing liver, relieving depression and raising yang. Can be used for treating common cold, fever, cold and heat, malaria, chest pain, menoxenia, uterine prolapse, and rectocele. The bupleurum chinense is used in clinical recipe and is the main material for Chinese medicine, yinshenfei oral liquid, legiophia medicine, etc. At present, researches such as microscopic identification, content measurement, characteristic spectrum and the like are carried out on medicinal materials of the bupleurum (Bupleurum malconense Shan et Y.Li) of the bamboo leaves, but the bupleurum of the bamboo leaves is various in variety and difficult to identify; moreover, the bupleurum chinense is often applied to clinic in the form of a preparation, and the quality of the active ingredients of the preparation is difficult to accurately control because of the large difference of the quality of the internal ingredients of the bupleurum chinense in different batches, so that no literature is available at present for researching the quality standard of the bupleurum chinense preparation such as formula granules and the like.
Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide a method for detecting bupleurum chinense or its preparation and a method for constructing fingerprint thereof.
The invention provides a detection method of bupleurum chinense or a preparation thereof, which comprises the following steps:
detecting bupleuri radix or its preparation by HPLC;
the chromatographic conditions of the HPLC are as follows: methanol is used as a mobile phase A, 0.4% phosphoric acid solution is used as a mobile phase B, and gradient elution is carried out, wherein the gradient elution comprises the following steps:
| time (minutes) | Mobile phase a (%) | Mobile phase B (%) |
| 0～10 | 0～15 | 100～85 |
| 10～40 | 15～45 | 85～55 |
| 40～53 | 45～60 | 55～40 |
| 53～70 | 60～77.5 | 40～22.5 |
。
In certain embodiments of the invention, the bupleurum phyllum is selected from Bupleurum marginatum wall.ex dc, bupleurum chinense DC, bupleurum microcephalum Diels or Bupleurum malconense Shan et y.li, preferably from Bupleurum marginatum wall.ex dc or Bupleurum chinense DC, most preferably from Bupleurum marginatum wall.ex dc. In certain embodiments of the invention, the formulation comprises decoction pieces, standard decoction pieces, or formulated granules.
In certain embodiments of the invention, the HPLC apparatus is a Waters2695-e2998 type high performance liquid chromatograph, agilent 1260 type high performance liquid chromatograph, or Shimadzu LC-20AD type high performance liquid chromatograph.
In certain embodiments of the invention, the HPLC column is packed with octadecylsilane chemically bonded silica. In one embodiment, the chromatographic column is diamondsil C18 (2), kromasiL C18, or Phenomenex C18. In one embodiment, the column length of the column is 250mm, the inner diameter of the column is 4.6mm, and the particle size of the column is 5 μm. In one embodiment, the column temperature of the chromatographic column is 20 ℃ to 40 ℃, preferably 30 ℃. In one embodiment, the theoretical plate number of the chromatographic column is not less than 3000 calculated according to the characteristic peak of rutin.
In certain embodiments of the invention, the mobile phase has a flow rate of 0.5mL/min to 1.5mL/min, preferably 1mL/min. In certain embodiments of the invention, the HPLC detection wavelength is 240nm to 320nm, preferably 266nm. In certain embodiments of the invention, the HPLC is performed with a sample size of 5 to 10. Mu.L. In certain embodiments of the invention, the HPLC detection time is not less than 70min.
Specifically, the HPLC is adopted to detect the test sample solution of the bupleurum chinense or the preparation thereof according to the chromatographic conditions of the HPLC, and the HPLC spectrum of the bupleurum chinense or the preparation thereof is obtained.
The preparation method of the sample solution comprises the following steps: extracting the bupleurum chinense or the preparation thereof with an extraction solvent to prepare a sample solution of the bupleurum chinense or the preparation thereof. In some embodiments of the present invention, the bupleurum chinense or its preparation is extracted with extracting solvent, and the extracting process includes ultrasonic treatment or reflux, cooling, shaking, filtering and obtaining the filtrate. In one embodiment, 0.8g to 1.3g of bupleurum medicinal material or bupleurum decoction pieces, preferably 1.0g of bupleurum medicinal material or bupleurum decoction pieces are taken, ground into powder, screened by a third sieve, precisely weighed, placed in a conical flask with a plug, 50mL of water is added, decocted for 20min to 40min, filtered, evaporated to obtain residues, 50mL of extraction solvent is added into the residues, and the residues are sealed for extraction, the extraction method is ultrasonic or reflux, cooling, shaking and filtering, and the subsequent filtrate is taken to obtain a sample solution of the bupleurum medicinal material or bupleurum decoction pieces. In one embodiment, 0.1 g-0.5 g of bupleurum chinense formula particles are ground, preferably 0.2g of bupleurum chinense formula particles are precisely weighed, placed in a conical flask with a plug, 50mL of extraction solvent is added and sealed for extraction, the extraction method is ultrasonic or reflux, cooling, shaking up and filtering, and the subsequent filtrate is taken to obtain a sample solution of the bupleurum chinense formula particles. In one embodiment, 0.1 g-0.5 g of the standard decoction of the bupleurum chinense, preferably 0.2g of the standard decoction of the bupleurum chinense, is precisely weighed, placed in a conical bottle with a plug, 50mL of extraction solvent is added and sealed for extraction, the extraction method is ultrasonic or reflux, cooling, shaking, filtering, and obtaining a sample solution of the standard decoction of the bupleurum chinense from the subsequent filtrate. In one embodiment, the extraction solvent is selected from ethanol or 30% to 100% methanol, preferably from 50% to 70% methanol. In one embodiment, the extraction time is 20min to 60min, preferably 30min. In one embodiment, the power of the ultrasound is 400W-800W; the frequency of the ultrasonic wave is 30 kHz-60 kHz.
The detection method is used for detecting the bupleurum chinense or the preparation thereof, so that the HPLC (high performance liquid chromatography) spectrum of the bupleurum chinense or the preparation thereof can be obtained, and the fingerprint spectrum of the bupleurum chinense or the preparation thereof can be established; the HPLC (high performance liquid chromatography) spectrum of the obtained bupleurum chinense or the preparation thereof is compared with the fingerprint spectrum of the bupleurum chinense or the HPLC spectrum of the bupleurum chinense of different types is compared, so that the bupleurum chinense or the preparation thereof can be accurately analyzed, the bupleurum chinense of different types is accurately identified, and the quality uniformity and stability of the bupleurum chinense preparation of the bupleurum chinense are ensured.
The invention provides a construction method of a fingerprint of the bupleurum chinense or the preparation thereof, which comprises the following steps:
detecting different batches of bupleurum chinense or preparation thereof according to the detection method;
calibrating a common peak;
the fingerprint of the bupleurum chinense or the preparation thereof is established by adopting a median method.
Specifically, the method detects the test sample solutions of the bupleurum chinense or the preparation thereof in different batches according to the detection method, marks out the common peak with better repeatability in each HPLC (high performance liquid chromatography) according to three principles of stable relative retention time, detection of samples in each batch and relatively higher peak, selects out the reference peak, and establishes the fingerprint of the bupleurum chinense or the preparation thereof by a median method by adopting a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012 edition).
In certain embodiments of the present invention, different batches of bupleurum chinense (Bupleurum marginatum wall. Ex dc.) or test solutions of formulations thereof are detected according to the above detection methods to obtain HPLC profiles of the different batches of bupleurum chinense or formulations thereof; according to the HPLC spectrum, 7 common peaks are marked according to chromatographic peaks of common chemical substances, rutin is used as a reference peak, a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012 edition) is adopted to establish a fingerprint of the bupleurum chinense or the preparation thereof by a median method, the relative retention time of the rutin in the fingerprint is 1.00, the relative retention time of other common peaks is 0.388+/-0.08, (0.424-0.425) +/-0.08,0.572 +/-0.08, (0.606-0.607) +/-0.08, (0.619-0.621) +/-0.08, and 1.231-1.233) +/-0.08 in sequence. The sample solution is the same as above and will not be described again.
In one embodiment, the fingerprint of the bupleurum (Bupleurum marginatum wall. Ex DC.) medicinal material is obtained according to the above construction method, rutin is used as a reference peak, the relative retention time of rutin in the fingerprint is 1.00, and the relative retention time of other common peaks is 0.388+ -0.08, 0.425+ -0.08, 0.572+ -0.08, 0.607+ -0.08, 0.621+ -0.08, 1.233+ -0.08 in sequence.
In one embodiment, the fingerprint of the bupleurum (Bupleurum marginatum wall. Ex DC.) formula particle is obtained according to the above construction method, rutin is taken as a reference peak, the relative retention time of rutin in the fingerprint is 1.00, and the relative retention time of other common peaks is 0.388+ -0.08, 0.425+ -0.08, 0.572+ -0.08, 0.607+ -0.08, 0.621+ -0.08, 1.233+ -0.08 in sequence.
In one embodiment, the fingerprint of the decoction pieces of bupleurum (Bupleurum marginatum wall. Ex DC) is obtained according to the above construction method, rutin is taken as a reference peak, the relative retention time of rutin in the fingerprint is 1.00, and the relative retention time of other common peaks is 0.388+ -0.08, 0.425+ -0.08, 0.572+ -0.08, 0.607+ -0.08, 0.620+ -0.08, 1.233+ -0.08 in sequence.
In one embodiment, a fingerprint of a standard decoction of bupleurum (Bupleurum marginatum wall. Ex DC) is obtained according to the above construction method, rutin is taken as a reference peak, the relative retention time of rutin in the fingerprint is 1.00, and the relative retention time of other common peaks is 0.388+ -0.08, 0.424+ -0.08, 0.572+ -0.08, 0.606+ -0.08, 0.619+ -0.08, 1.231+ -0.08 in sequence.
The common peak is identified, and the reference substance is combined for comparison, wherein the common peak with retention time of (0.424-0.425) +/-0.08 is neochlorogenic acid, the common peak with retention time of 0.572+/-0.08 is chlorogenic acid, the common peak with retention time of (0.606-0.607) +/-0.08 is cryptochlorogenic acid, and the common peak with retention time of (1.231-1.233) +/-0.08 is quercetin.
The method for identifying the shared peak is as follows: detecting the test solution of the bupleurum chinense or the preparation thereof, the control medicinal material reference solution of the bupleurum chinense and the reference solution of each control material respectively according to the detection method to obtain respective HPLC (high performance liquid chromatography); and comparing the retention time of chromatographic peaks in the HPLC spectrum, and identifying the chromatographic peak corresponding to the retention time as a common peak. The sample solution is the same as above and will not be described again. The reference substances refer to common chemical components in different batches of bupleurum chinense or preparations thereof, and can be selected by a person skilled in the art according to experience. In one embodiment, the controls include rutin, chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, and quercetin independently of one another.
In some embodiments of the present invention, the preparation method of the bupleurum control drug reference solution is as follows: taking a bupleurum control medicinal material, precisely weighing, placing in a conical flask with a plug, precisely adding water, weighing, heating to 92-98 ℃ and keeping for 20-60 min, cooling, supplementing the lost weight with water, filtering, evaporating filtrate, precisely adding an extraction solvent into residues, performing ultrasonic treatment, cooling, shaking, filtering, and obtaining a continuous filtrate to obtain a bupleurum control medicinal material reference solution. The extraction solvent is the same as above, and will not be described again.
In some embodiments of the present invention, the reference solution is prepared by the following method: and (3) taking a proper amount of the reference substance, precisely weighing, adding an extraction solvent to prepare a solution containing 50 mug of the reference substance per 1mL, and obtaining a reference substance solution of the reference substance. The extraction solvent is the same as above, and will not be described again.
The invention provides a detection method of bupleurum chinense or a preparation thereof, which comprises the following steps: detecting bupleuri radix or its preparation by HPLC; the chromatographic conditions of the HPLC are as follows: methanol is used as a mobile phase A, a 0.4% phosphoric acid solution is used as a mobile phase B, and gradient elution is adopted. The method provided by the invention can accurately analyze the bupleurum chinense or the preparation thereof and construct the fingerprint, accurately identify the bupleurum chinense of different types and ensure the uniform and stable quality of the bupleurum chinense preparation. Experiments show that the invention explores the optimal chromatographic conditions of the detection method of the bupleurum chinense or the preparation thereof, and detects the bupleurum chinense or the preparation thereof based on the chromatographic conditions, successfully constructs the fingerprint of the bupleurum chinense or the preparation thereof, wherein the RSD of the characteristic peaks of the fingerprint relative to the retention time is less than 1%, and successfully identifies the bupleurum chinense and the bupleurum chinense.
Drawings
FIG. 1 is a liquid chromatogram comparison diagram of a test solution of herba Lophatheri radix bupleuri granule, a reference solution of herba Lophatheri radix bupleuri reference material, and a reference solution of rutin reference material;
FIG. 2 is a rutin ultraviolet absorption spectrum;
FIG. 3 is an ultraviolet absorption spectrum of chlorogenic acid;
FIG. 4 is a graph of ultraviolet absorption spectrum of chlorogenic acid;
FIG. 5 is an ultraviolet absorption spectrum of the chlorogenic acid;
FIG. 6 is a graph of the ultraviolet absorption spectrum of quercetin;
FIG. 7 is a graph showing the comparison of different wavelengths of the granule formulation of Bupleurum scorzonerifolium;
FIG. 8 is a liquid chromatogram of the granule of the formula of Bupleurum scorzonerifolium at different column temperatures;
FIG. 9 is a liquid chromatogram of the granule of the formula of Bupleurum scorzonerifolium at different flow rates;
FIG. 10 is a liquid chromatogram of a delayed investigation of the formulation particles of Bupleurum scorzonerifolium;
FIG. 11 is a liquid chromatogram of the extraction solvent investigation of the bupleurum chinense formula particles;
FIG. 12 is a liquid chromatogram of a method for extracting the bupleurum chinense formula particles;
FIG. 13 is a liquid chromatogram of the time of extraction investigation of the bupleurum formulation particles;
FIG. 14 is a liquid chromatogram of a sample volume investigation of the bupleurum chinense formula particles;
FIG. 15 is a graph showing the chromatographic peak identification of the granule of the bupleurum chinense formula;
FIG. 16 is a comparative chart of different instruments of the granule formulation of Bupleurum scorzonerifolium;
FIG. 17 is a chart showing comparison of different chromatographic column examinations of the granule of the formula of the bupleurum chinense;
FIG. 18 is a liquid chromatogram of a test method of the present invention using the bupleurum formulation particles;
FIG. 19 is a fingerprint of the bupleurum chinense formula particle;
FIG. 20 is a liquid chromatogram of a verification test of 19 batches of bupleurum medicinal material;
FIG. 21 is a fingerprint of a bupleurum medicinal material;
FIG. 22 is a liquid chromatogram of a verification test of 19 batches of bupleurum decoction pieces;
FIG. 23 is a fingerprint of the decoction pieces of Bupleurum scorzonerifolium;
FIG. 24 is a color chromatogram of a standard decoction validation test of 19 batches of bupleuri radix;
FIG. 25 is a fingerprint of a standard decoction of Bupleurum scorzonerifolium;
FIG. 26 is a liquid chromatogram of the comparison of Bupleurum scorzonerifolium and Bupleurum chinense.
Detailed Description
The invention discloses a detection method of bupleurum chinense or a preparation thereof and a construction method of a fingerprint thereof. Those skilled in the art can, with the benefit of this disclosure, suitably modify the process parameters to achieve this. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced and practiced with modification and alteration and combination of the methods and applications herein without departing from the spirit and scope of the invention.
The invention is further illustrated by the following examples:
example 1
Preparing experimental instruments and materials, setting chromatographic conditions, preparing reference solution and test solution, and performing a system applicability experiment, wherein the specific experimental conditions are as follows (1) - (5):
(1) Experimental instrument and materials:
high performance liquid chromatograph: waters2695-e2998 type high performance liquid chromatograph, agilent 1260 type high performance liquid chromatograph, shimadzu LC-20AD type high performance liquid chromatograph;
an electronic balance: ME204E/02, MS205DU, XP26 (Metrele Tolyduo instruments Co., ltd.);
ultrapure water machine: cell type 1810A (Shanghai mueller scientific instruments limited);
ultrasonic cleaner: KQ5200DB model (600W, 40KHz; kunshan ultrasonic instruments Co., ltd.);
chromatographic column: 5 μm, 4.6X1250 mm Diamond C18 (2), 5 μm, 4.6X1250 mm KromasiL C18, 5 μm, 4.6X1250 mm Phenominex C18;
rutin (Chinese food and drug verification institute, lot number: 100080-201811, content is 91.7%), chlorogenic acid (Chinese food and drug verification institute, lot number: 110753-201415, content is 96.2%), neochlorogenic acid (Vickers biotechnology Co., ltd., lot number: wkq18030107, content is 98%), cryptochlorogenic acid (Vickers biotechnology Co., ltd., lot number: wkq19011612, content is 98%), quercetin reference (Chinese food and drug verification institute, lot number: 100080-201610, content is 99.1%), bupleurum control medicinal material (Chinese food and drug verification institute, lot number: 121343-201802), methanol (SIGMA Co., chromatography); the water is ultrapure water, and other reagents are all analytically pure;
the formula granule of the bupleurum chinense is prepared from the following raw materials: ZYCH-01, ZYCH-02, ZYCH-03, ZYCH-04;
bupleurum medicinal material of bamboo leaf: ZYCH-YC-01, ZYCH-YC-02, ZYCH-YC-03, ZYCH-YC-04, ZYCH-YC-05ZYCH-YC-06, ZYCH-YC-07, ZYCH-YC-08, ZYCH-YC-09, ZYCH-YC-10, ZYCH-YC-11, ZYCH-YC-12, ZYCH-YC-13, ZYCH-YC-14, ZYCH-YC-15, ZYCH-YC-16, ZYCH-YC-17, ZYCH-YC-18, ZYCH-YC-19;
bamboo leaf bupleurum decoction pieces: ZYCH-YP-01, ZYCH-YP-02, ZYCH-YP-03, ZYCH-YP-04, ZYCH-YP-05ZYCH-YP-06, ZYCH-YP-07, ZYCH-YP-08, ZYCH-YP-09, ZYCH-YP-10, ZYCH-YP-11, ZYCH-YP-12, ZYCH-YP-13, ZYCH-YP-14, ZYCH-YP-15, ZYCH-YP-16, ZYCH-YP-17, ZYCH-YP-18, ZYCH-YP-19.
Standard decoction of bupleurum chinense (L.) kurz: ZYCH-BT-01, ZYCH-BT-02, ZYCH-BT-03, ZYCH-BT-04, ZYCH-BT-05ZYCH-BT-06, ZYCH-BT-07, ZYCH-BT-08, ZYCH-BT-09, ZYCH-BT-10, ZYCH-BT-11, ZYCH-BT-12, ZYCH-BT-13, ZYCH-BT-14, ZYCH-BT-15, ZYCH-BT-16, ZYCH-BT-17, ZYCH-BT-18, ZYCH-BT-19.
(2) Chromatographic conditions
The specification of the chromatographic column is as follows: the column length is 250mm, the inner diameter is 4.6mm, the granularity is 5 μm, the chromatographic column uses octadecylsilane chemically bonded silica as filler, methanol as mobile phase A, 0.4% phosphoric acid solution as mobile phase B, and gradient elution is carried out according to the specification in Table 1; the column temperature is 30 ℃; the flow rate is 1.0mL per minute; detection wavelength: 266nm. The theoretical plate number is not less than 3000 calculated according to rutin peak.
TABLE 1
| Time (minutes) | Mobile phase a (%) | Mobile phase B (%) |
| 0～10 | 0～15 | 100～85 |
| 10～40 | 15～45 | 85～55 |
| 40～53 | 45～60 | 55～40 |
| 53～70 | 60～77.5 | 40～22.5 |
(3) Preparation of reference solutions
Taking about 1.0g of the bupleurum control medicinal material, precisely weighing, placing in a conical bottle with a plug, adding 50mL of water, boiling for 30min, filtering, evaporating to dryness, adding 50mL of 70% methanol into residues, sealing, performing ultrasonic treatment, wherein the power of the ultrasonic treatment is 600W, the frequency of the ultrasonic treatment is 40kHz, performing ultrasonic treatment for 30min, cooling, shaking uniformly, filtering, and taking the subsequent filtrate to obtain the bupleurum control medicinal material reference solution.
And (3) taking a proper amount of rutin reference substance, precisely weighing, adding 70% methanol to prepare a solution containing 50 mug per 1mL, and obtaining the rutin reference substance solution.
(4) Preparation of test solutions
Taking a proper amount of the product with the batch number of ZYCH-01, grinding, taking about 0.2g, precisely weighing, placing into a conical bottle with a plug, adding 50mL of 70% methanol, sealing, and carrying out ultrasonic treatment, wherein the power of the ultrasonic treatment is 600W, the frequency of the ultrasonic treatment is 40kHz, the ultrasonic treatment lasts for 30 minutes, cooling, shaking uniformly, filtering, and taking a subsequent filtrate to obtain the test solution of the bupleurum chinense formula particles with the batch number of ZYCH-01.
(5) Measurement method
Respectively precisely sucking 5 μl of each of the bamboo leaf radix bupleuri reference substance solution, the rutin reference substance solution and the bamboo leaf radix bupleuri formula particle sample solution, injecting into a liquid chromatograph, and measuring according to the chromatographic conditions to obtain liquid chromatograms of the sample solution and the reference substance solution, as shown in fig. 1, wherein fig. 1 is a liquid chromatogram comparison diagram of the bamboo leaf radix bupleuri formula particle sample solution, the bamboo leaf radix bupleuri reference substance solution and the rutin reference substance solution. In fig. 1, 7 characteristic peaks are shown and correspond to the retention time of 7 characteristic peaks in the solution chromatogram of the reference medicine of the bupleurum chinense, wherein the peak corresponding to the characteristic peak shown in the solution chromatogram of the reference medicine of the rutin is a reference peak (S peak), the relative retention time of each characteristic peak and the S peak is calculated, the relative retention time is within +/-8% of a specified value, and the specified value is: 0.388 (peak 1), 0.425 (peak 2), 0.572 (peak 3), 0.607 (peak 4), 0.621 (peak 5), 1.233 (peak 7), wherein peak 2 is a characteristic peak of new chlorogenic acid, peak 3 is a characteristic peak of chlorogenic acid, peak 4 is a characteristic peak of cryptochlorogenic acid, peak 6 (S-peak) is a characteristic peak of rutin, and peak 7 is a characteristic peak of quercetin.
On the basis of the experimental conditions set forth in the above (1) - (5), respectively carrying out full-band scanning on rutin, chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid and quercetin test sample solutions by using a diode array detector, and respectively extracting chromatograms of the test sample solutions at wavelengths of 240nm, 257nm, 266nm, 280nm, 300nm and 320nm to determine the optimal detection wavelength points. The results are shown in figures 2-7, wherein figure 2 is a rutin ultraviolet absorption spectrum; FIG. 3 is an ultraviolet absorption spectrum of chlorogenic acid; FIG. 4 is a graph of ultraviolet absorption spectrum of chlorogenic acid; FIG. 5 is an ultraviolet absorption spectrum of the chlorogenic acid; FIG. 6 is a graph of the ultraviolet absorption spectrum of quercetin; FIG. 7 is a graph showing the comparison of different wavelengths of the granule formulation of Bupleurum scorzonerifolium. As is clear from FIGS. 2 to 7, the detection wavelength was determined to be 266nm because the amount of information on the chromatographic peak was large and the base line of the chromatogram was smoother at the detection wavelength of 266nm.
Based on the experimental conditions set forth in the above (1) to (5), the column temperatures were examined at 25℃and 30℃and 35℃respectively to determine the optimum column temperature. The results are shown in fig. 8 and table 2, fig. 8 shows liquid chromatograms of the bupleurum formulation granules at different column temperatures, and table 2 shows the ratio of column temperature investigation to characteristic peak to retention time. As can be seen from FIG. 8 and Table 2, the relative retention time RSD value of each characteristic peak is 1.03% -2.64%, and the chromatogram peak shape is symmetrical and the separation degree is good when the column temperature is 30 ℃, so that 30 ℃ is finally selected as the subsequent investigation column temperature of the characteristic spectrum methodology of the bupleurum chinense formula particle.
TABLE 2
Based on the experimental conditions set forth in the above (1) to (5), the flow rates were examined at 0.8mL/min, 1mL/min, and 1.2mL/min, respectively. The results are shown in fig. 9 and table 3, fig. 9 shows the liquid chromatograms of the bupleurum formulation granules at different flow rates, and table 3 shows the flow rate investigation-characteristic peak relative retention time. As shown in FIG. 9 and Table 3, the relative retention time RSD of each characteristic peak was 0.74 to 7.83% at flow rates of 0.8mL/min, 1.0mL/min and 1.2mL/min, respectively, and the chromatogram peak shape was good at a flow rate of 1.0mL/min, and the degree of separation was moderate, so that the flow rate was determined to be 1.0mL/min.
TABLE 3 Table 3
And (3) prolonging the chromatogram acquisition time to 140min on the basis of the experimental conditions set forth in the above (1) - (5). As shown in fig. 10, fig. 10 is a liquid chromatogram of the delayering investigation of the bupleurum chinense formula granule. As can be seen from fig. 10, the chromatogram was collected for 70 minutes, and thus the chromatogram collection time was determined to be 70 minutes.
Examining the influence of different extraction solvents on the detection result: taking about 0.1g of the bupleurum chinense formula particles ZYCH-01, placing the particles into a conical bottle with a plug, respectively adding 50mL of methanol, 30% of methanol, 50% of methanol, 70% of methanol, water and ethanol, sealing, and carrying out ultrasonic treatment, wherein the power of the ultrasonic treatment is 600W, the frequency of the ultrasonic treatment is 40kHz, and the ultrasonic treatment is carried out for 30 minutes, cooling, shaking uniformly, filtering, and taking subsequent filtrate. The results are shown in fig. 11, and fig. 11 shows a liquid chromatogram of the extraction solvent investigation of the bupleurum chinense formula granule. As can be seen from FIG. 11, the chromatographic peak information obtained by using 70% methanol as the extraction solvent is large and the separation degree is good, so 70% methanol is used as the extraction solvent.
Examine the influence of different extraction modes on the detection result: about 0.1g of the sample is taken and placed in a conical bottle with a plug, 50mL of 70% methanol is added, and the plug is used for respectively examining the extraction method of the sample to be tested as reflux and ultrasonic treatment, wherein the power of the ultrasonic treatment is 600W, and the frequency of the ultrasonic treatment is 40kHz; extracting for 30min, cooling, shaking, filtering, and collecting filtrate. The results are shown in fig. 12, and fig. 12 is a liquid chromatogram of the extraction mode of the bupleurum chinense formula particles. As can be seen from fig. 12, the effects of ultrasonic extraction and reflux extraction on the samples were identical. The ultrasonic extraction operation is simpler, so the method for extracting the sample is determined to be ultrasonic extraction.
Examine the influence of different extraction times on the detection result: about 0.1g of the product is taken, the product is placed in a conical flask with a plug, 50mL of 70% methanol is added, the sealing is carried out, the ultrasonic treatment power is 600W, the ultrasonic treatment frequency is 40kHz, the inspection is carried out when the extraction time of the sample to be tested is 20 minutes, 30 minutes and 40 minutes respectively, the cooling is carried out, the shaking is carried out, the filtering is carried out, and the subsequent filtrate is obtained. The results are shown in fig. 13, and fig. 13 is a liquid chromatogram of the time investigation of the extraction of the bupleurum formulation particles. As can be seen from fig. 13, the extraction time was 30 minutes, and the extraction was sufficient. The test sample extraction time was thus determined to be 30 minutes.
Examine the influence of different sampling amounts on the detection result: respectively taking 0.1g, 0.2g and 0.3g of the product, placing the product into a conical bottle with a plug, adding 50mL of 70% methanol, sealing, performing ultrasonic treatment, wherein the power of the ultrasonic treatment is 600W, the frequency of the ultrasonic treatment is 40kHz, and the ultrasonic treatment lasts for 30 minutes, cooling, shaking uniformly, filtering, and taking a subsequent filtrate. The results are shown in fig. 14, and fig. 14 shows a liquid chromatogram of the sample size investigation of the bupleurum chinense formula particles. As is clear from FIG. 14, the sample amount was 0.2g because the sample was sufficiently extracted when the sample amount was 0.2g.
Example 2
The detection method of the invention is examined in methodology, and is concretely as follows:
(1) Chromatographic peak assignment
Preparation of test solution: test solutions of the formulation granules of bupleurum chinense are prepared according to the experimental conditions of example 1.
Preparation of a reference solution for a control: and (3) taking appropriate amounts of rutin, chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid and quercetin reference substances, precisely weighing, and adding 70% methanol to prepare a solution containing 50 mug per 1 mL.
Preparation of control drug reference solution: 2.0g of bupleurum chinense is taken as a control medicinal material, precisely weighed, placed in a conical bottle with a plug, precisely added with 50mL of water, weighed by weight, heated to 92-98 ℃ for 30min, cooled, complemented by water for the weight loss, filtered, evaporated to dryness, precisely added with 50mL of 70% methanol in residues, sealed, subjected to ultrasonic treatment with the power of 600W, subjected to ultrasonic treatment with the frequency of 40kHz for 30min, cooled, shaken uniformly, filtered, and obtained to obtain subsequent filtrate.
Preparation of negative control solution: a negative control solution of the bupleurum chinense formulation particles was prepared according to the experimental conditions of example 1. The characteristic spectrum peaks of the bupleurum chinense formula particles are positioned, the result is shown in figure 15, and figure 15 is a graph for identifying the chromatographic peaks of the bupleurum chinense formula particles.
(2) Precision test
The formulation of Zhuye bupleurum particles with lot number ZYCH-01 was prepared into a sample solution, and the sample solution was continuously sampled 6 times, 5. Mu.L each time, according to the experimental method set forth in example 1, and the retention time and peak area of each characteristic peak were calculated, and the results are shown in tables 4 and 5.
TABLE 4 Table 4
TABLE 5
The results in tables 4 and 5 show that the method has good precision.
(3) Repeatability investigation
6 parts of bupleurum chinense formula particles with batch number ZYCH-01 are precisely weighed, prepared and measured according to the experimental method set forth in example 1, and the results are shown in tables 6 and 7.
TABLE 6
TABLE 7
The results in tables 6 and 7 show that the method is highly reproducible.
(4) Different instruments for examining intermediate precision
Based on the experimental conditions set forth in example 1, two parts of bupleurum chinense formula particles with batch number ZYCH-01 are respectively and precisely weighed, test solution is prepared, and the test solution is respectively measured on Agilent 1260, shimadzu LC-20AT and Waters2695-e2998 type high performance liquid chromatographs. As shown in fig. 16, table 8 and table 9, wherein fig. 16 is a comparison chart of different instrument surveys of the bupleurum formulation granules.
TABLE 8
TABLE 9
The results in fig. 16, table 8 and table 9 show that RSD of each characteristic peak relative retention time was less than 10% when the test pieces were tested by the above 3 instruments.
(5) Different personnel and time investigation of intermediate precision investigation
Based on experimental conditions set forth in example 1, two parts of bupleurum chinense formula particles with batch number ZYCH-01 are precisely weighed by different personnel A and B AT different times T1 and T2 respectively, test products are prepared and measured, and the results are shown in tables 10 and 11, wherein AT1-1 and AT1-2 are respectively detected by the personnel A AT the time T1; AT2-1 and AT2-2 are two tests performed by person A AT time T2, respectively; BT1-1 and BT1-2 were performed at time T1 for human B.
Table 10
TABLE 11
The results in tables 10 and 11 show that the same sample was measured by different persons at different times, and the detection method of the present invention has good stability.
(6) Durability inspection of chromatographic column
Based on the experimental conditions set forth in example 1, a test of 5 μm 4.6X1250 mm of Diamonsil C18 (2), 5 μm 4.6X1250 mm of Kromasil C18, 5 μm 4.6X1250 mm of Phenomex C18 was performed on each column, and the results are shown in FIG. 17, table 12 and Table 13, and FIG. 17 is a graph showing a test and comparison of different columns of the formula granules of Bupleurum scorzonerifolium.
Table 12
TABLE 13
The results in FIGS. 17, 12 and 13 show that the samples were examined using the above 3 columns, and that the RSD of the characteristic peak relative retention time was 0.42% to 2.39%, and that of the characteristic peak relative peak area was 2.07% to 18.86%.
(7) Stability investigation for durability investigation
Based on the experimental conditions set forth in example 1, the same sample solutions were taken and measured at 0h,2h,4h,8h,12h,24h, respectively, and the results are shown in tables 14 and 15.
TABLE 14
TABLE 15
The results in tables 14 and 15 show that the RSD of the corresponding characteristic peak retention time is 0.05% to 0.62%, and the sample solution is stable for 24 hours.
In conclusion, the RSD of each characteristic peak relative retention time meets the requirements in the above examinations, and the method is good. The above 6 characteristic peaks were incorporated into the subsequent investigation.
Example 3
The results of the detection method of the invention are verified by three batches of Zhych-02, zhch-03 and Zhch-04 of bupleurum chinense formula particles, and are specifically as follows: the characteristic spectrum of 3 batches of samples of the product is measured by adopting the conditions formulated in example 1, the relative retention time and the relative peak area are calculated, the results are shown in fig. 18, table 16 and table 17, and fig. 18 is a liquid chromatogram of the detection method of the invention verified by adopting the bupleurum chinense formula particles. Synthesizing liquid chromatogram of 3 batches of bupleurum chinense formula particles by using a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012 edition), establishing fingerprint of bupleurum chinense formula particles, as shown in figure 19, wherein figure 19 is the fingerprint of bupleurum chinense formula particles; in fig. 19, peak 2 is neochlorogenic acid, peak 3 is chlorogenic acid, peak 4 is cryptochlorogenic acid, peak 6 (S peak) is rutin, and peak 7 is quercetin.
Table 16
TABLE 17
According to the principle that the relative retention time is stable, the samples in each batch can be detected and the peak is relatively high, 7 peaks with better repeatability are selected as characteristic peaks. The result shows that when the peak 6 is taken as an S peak, the characteristic peak relative retention time RSD of the 3 batches of bupleurum formula particles is less than 1%.
Summarizing the methodology survey items and verification results, a relative retention time specification limit was established, see table 18 for details:
TABLE 18
From Table 18, it is clear that the flow rate, the relative retention time of each characteristic peak was greatly influenced by the flow rate, the apparatus and the column, and therefore the flow rate was set to 1.0mL/min, and the relative retention time of each peak was set to 8% for the purpose of increasing the reproducibility and applicability of the method.
Example 4
The characteristic spectrum of 19 batches of bupleurum medicinal material samples of the product is measured by adopting the method formulated in the example 1, the relative retention time and the relative peak area are calculated, the results are shown in fig. 20, table 19 and table 20, and fig. 20 is a liquid chromatogram of a verification test of 19 batches of bupleurum medicinal material. Synthesizing liquid chromatograms of 19 batches of bupleurum medicinal materials by adopting a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012 edition), establishing the fingerprint of the bupleurum medicinal materials, as shown in fig. 21, wherein fig. 21 is the fingerprint of the bupleurum medicinal materials; in fig. 21, peak 2 is neochlorogenic acid, peak 3 is chlorogenic acid, peak 4 is cryptochlorogenic acid, peak 6 (S peak) is rutin, and peak 7 is quercetin.
TABLE 19
Table 20
Example 5
The characteristic spectrum of 19 batches of bupleurum chinense decoction pieces samples of the product is measured by adopting the method formulated in the example 1, the relative retention time and the relative peak area are calculated, the results are shown in fig. 22, table 21 and table 22, and fig. 22 is a liquid chromatogram of a verification test of 19 batches of bupleurum chinense decoction pieces. Synthesizing 19 batches of bupleurum chinense slices by adopting a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012 edition), establishing a fingerprint of the bupleurum chinense slices, as shown in fig. 23, and fig. 23 is the fingerprint of the bupleurum chinense slices; in FIG. 23, peak 2 is neochlorogenic acid, peak 3 is chlorogenic acid, peak 4 is cryptochlorogenic acid, peak 6 (S peak) is rutin, and peak 7 is quercetin.
Table 21
Table 22
Example 6
The characteristic spectrum of 19 batches of bupleurum standard decoction samples of the product is measured by adopting the method formulated in the example 1, the relative retention time and the relative peak area are calculated, the results are shown in fig. 24, table 23 and table 24, and fig. 24 is a verification test hue chromatogram of 19 batches of bupleurum standard decoction. Synthesizing 19 batches of the standard decoction of the bupleurum chinense hand-Mazz by adopting a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012 edition), establishing the fingerprint of the standard decoction of the bupleurum chinense hand-Mazz, as shown in fig. 25, and fig. 25 is the fingerprint of the standard decoction of the bupleurum chinense hand-Mazz; in fig. 25, peak 2 is neochlorogenic acid, peak 3 is chlorogenic acid, peak 4 is cryptochlorogenic acid, peak 6 (S peak) is rutin, and peak 7 is quercetin.
Table 23
Table 24
Example 7
The results of the tests performed on bupleurum chinense (Bupleurum marginatum wall. Ex DC.) and bupleurum chinense using the method outlined in example 1 are shown in FIG. 26, and FIG. 26 is a liquid chromatogram comparison of bupleurum chinense and bupleurum chinense. As can be seen from FIG. 26, the characteristic peaks of Bupleurum scorzonerifolium and Bupleurum scorzonerifolium (Bupleurum scorzonerifolium) differ greatly, so that the method can be used for identifying Bupleurum scorzonerifolium and Bupleurum scorzonerifolium (Bupleurum scorzonerifolium).
The foregoing is only a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art, who is within the scope of the present invention, should make equivalent substitutions or modifications according to the technical scheme of the present invention and the inventive concept thereof, and should be covered by the scope of the present invention.
Claims (6)
1. The method for constructing the fingerprint of the bupleurum chinense or the preparation thereof is characterized by comprising the following steps:
detecting different batches of bupleurum chinense or preparation thereof by adopting HPLC; the chromatographic column of the HPLC takes octadecylsilane chemically bonded silica as a filler; the detection wavelength of the HPLC is 266 nm;
detecting the test solution of the bupleurum chinense or the preparation of the bupleurum chinense in different batches by adopting HPLC; the preparation method of the sample solution comprises the following steps: taking the bupleurum chinense or the preparation thereof, and preparing test sample solutions of the bupleurum chinense or the preparation thereof in different batches by taking 70% methanol as an extraction solvent;
calibrating a common peak; the common peak comprises rutin, chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid and quercetin;
establishing fingerprint of bupleuri radix or its preparation by median method;
the chromatographic conditions of the HPLC are as follows: methanol is used as a mobile phase A, 0.4% phosphoric acid solution is used as a mobile phase B, and gradient elution is carried out, wherein the gradient elution comprises the following steps:
。
2. the method of claim 1, wherein the formulation comprises a decoction piece, a decoction, or a formulated granule.
3. The method of construction according to claim 1 or 2, wherein the chromatographic column is diamondsil C18 (2), kromasiL C18 or Phenomenex C18.
4. The method according to claim 3, wherein the column temperature of the chromatographic column is 20 ℃ to 40 ℃.
5. The method of claim 1 or 2, wherein the mobile phase has a flow rate of 0.5mL/min to 1.5 mL/min.
6. The method according to claim 1 or 2, wherein a fingerprint of bupleurum chinense or a preparation thereof is established with rutin as a reference peak, the relative retention time of rutin is 1.00, the relative retention time of other common peaks in the fingerprint is 0.388+ -0.08, (0.424-0.425) + -0.08,0.572 + -0.08, (0.606-0.607) + -0.08, (0.619-0.621) + -0.08, and (1.231-1.233) + -0.08 in sequence.
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| CN108802259A (en) * | 2018-06-11 | 2018-11-13 | 江西普正制药有限公司 | A kind of method of quality control of HERBA BUPLEURI |
| CN110954616A (en) * | 2019-12-12 | 2020-04-03 | 安徽济人药业有限公司 | Method for establishing bupleurum chinense fingerprint spectrum quality control system |
| CN111289668A (en) * | 2020-03-30 | 2020-06-16 | 山东省中医药研究院 | Method for identifying bupleurum chinense and other bupleurum chinense varieties by using HPLC fingerprint |
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| CN110954616A (en) * | 2019-12-12 | 2020-04-03 | 安徽济人药业有限公司 | Method for establishing bupleurum chinense fingerprint spectrum quality control system |
| CN111289668A (en) * | 2020-03-30 | 2020-06-16 | 山东省中医药研究院 | Method for identifying bupleurum chinense and other bupleurum chinense varieties by using HPLC fingerprint |
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