CN113820405A - Quality control method for pyrrosia lingua formula granules - Google Patents
Quality control method for pyrrosia lingua formula granules Download PDFInfo
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- CN113820405A CN113820405A CN202110912434.9A CN202110912434A CN113820405A CN 113820405 A CN113820405 A CN 113820405A CN 202110912434 A CN202110912434 A CN 202110912434A CN 113820405 A CN113820405 A CN 113820405A
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- acid
- pyrrosia lingua
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- 239000008187 granular material Substances 0.000 title claims abstract description 69
- 238000003908 quality control method Methods 0.000 title claims abstract description 17
- 239000002245 particle Substances 0.000 claims abstract description 50
- PZIRUHCJZBGLDY-UHFFFAOYSA-N Caffeoylquinic acid Natural products CC(CCC(=O)C(C)C1C(=O)CC2C3CC(O)C4CC(O)CCC4(C)C3CCC12C)C(=O)O PZIRUHCJZBGLDY-UHFFFAOYSA-N 0.000 claims abstract description 43
- 238000001228 spectrum Methods 0.000 claims abstract description 38
- 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 36
- UFCLZKMFXSILNL-RVXRWRFUSA-N 4,5-di-O-caffeoylquinic acid Chemical compound O([C@@H]1C[C@](O)(C[C@H]([C@@H]1OC(=O)\C=C\C=1C=C(O)C(O)=CC=1)O)C(O)=O)C(=O)\C=C\C1=CC=C(O)C(O)=C1 UFCLZKMFXSILNL-RVXRWRFUSA-N 0.000 claims abstract description 36
- QAIPRVGONGVQAS-DUXPYHPUSA-N trans-caffeic acid Chemical compound OC(=O)\C=C\C1=CC=C(O)C(O)=C1 QAIPRVGONGVQAS-DUXPYHPUSA-N 0.000 claims abstract description 36
- 239000000843 powder Substances 0.000 claims abstract description 25
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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 abstract description 24
- 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 abstract description 24
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- KRZBCHWVBQOTNZ-RDJMKVHDSA-M (-)-3,5-Dicaffeoyl quinic acid Natural products O([C@@H]1CC(O)(C[C@H](C1O)OC(=O)\C=C\C=1C=C(O)C(O)=CC=1)C([O-])=O)C(=O)\C=C\C1=CC=C(O)C(O)=C1 KRZBCHWVBQOTNZ-RDJMKVHDSA-M 0.000 claims abstract description 18
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- 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 abstract description 18
- 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 abstract description 18
- 235000004883 caffeic acid Nutrition 0.000 claims abstract description 18
- 229940074360 caffeic acid Drugs 0.000 claims abstract description 18
- QAIPRVGONGVQAS-UHFFFAOYSA-N cis-caffeic acid Natural products OC(=O)C=CC1=CC=C(O)C(O)=C1 QAIPRVGONGVQAS-UHFFFAOYSA-N 0.000 claims abstract description 18
- 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 abstract description 18
- 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 abstract description 18
- 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 abstract description 18
- UFCLZKMFXSILNL-AALYGJCLSA-N 3,4-Dicaffeoylquinic acid Natural products O=C(O[C@@H]1[C@H](OC(=O)/C=C/c2cc(O)c(O)cc2)C[C@](O)(C(=O)O)C[C@@H]1O)/C=C/c1cc(O)c(O)cc1 UFCLZKMFXSILNL-AALYGJCLSA-N 0.000 claims abstract description 17
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- 238000010812 external standard method Methods 0.000 claims abstract description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 93
- 239000000523 sample Substances 0.000 claims description 32
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 21
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- 238000001514 detection method Methods 0.000 claims description 17
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 14
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- 239000000945 filler Substances 0.000 claims description 2
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims description 2
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- CWVRJTMFETXNAD-BMNNCGMMSA-N (1s,3r,4s,5r)-3-[(e)-3-(3,4-dihydroxyphenyl)prop-2-enoyl]oxy-1,4,5-trihydroxycyclohexane-1-carboxylic 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-BMNNCGMMSA-N 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/30—Control of physical parameters of the fluid carrier of temperature
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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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
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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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
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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
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
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- 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
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- 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
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- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
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Abstract
The invention discloses a quality control method of pyrrosia lingua formula granules, which comprises the following steps: (1) accurately weighing pyrrosia lingua formula particle powder, and preparing a pyrrosia lingua formula particle test sample solution; (2) analyzing the sample solution of the pyrrosia lingua formula particle by using a high performance liquid chromatograph to obtain an HPLC characteristic spectrum of the pyrrosia lingua formula particle, and measuring a corresponding peak area; (3) analyzing the reference substance solution by using a high performance liquid chromatograph, and determining a corresponding peak area; (4) and calculating by an external standard method to obtain the product. The characteristic spectrum of the pyrrosia lingua formula particle constructed by the invention fully shows the chemical component characteristics of the pyrrosia lingua formula particle, and the method has good stability, high precision and better reproducibility; the quality of the pyrrosia lingua formula granules is monitored from two aspects of HPLC characteristic spectrum and content determination (protocatechuic acid, caffeic acid, chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, isochlorogenic acid A, isochlorogenic acid B and isochlorogenic acid C), and an effective method is provided for quality monitoring of the pyrrosia lingua formula granules.
Description
Technical Field
The invention belongs to the technical field of traditional Chinese medicine detection, and particularly relates to a quality control method of pyrrosia lingua formula granules.
Background
Pyrrosia lingua is dry leaf of Pyrrosia cottage (Bak.) Ching, Pyrrosia lingua (Thunb) Farwell or Pyrrosia petiolata petiolosa (Christ) Ching of the hydrothorax orthopaedics, and has the effects of inducing diuresis for treating stranguria, clearing lung heat to relieve cough, cooling blood to stop bleeding. Modern pharmacological research shows that main active ingredients of pyrrosia lingua comprise polysaccharides, flavonoids, volatile oils, polyphenols and the like, and the pyrrosia lingua has various pharmacological effects of reducing blood sugar, resisting oxidation, resisting inflammation, promoting urination, protecting the kidney, enhancing immunity, promoting wound healing, inhibiting bacteria and the like.
The formula particle is prepared by carrying out water extraction, concentration, drying, granulation and other processes on single medicinal materials, compared with the traditional medicinal materials, the morphological characteristics of the formula particle are obviously changed, and the authenticity and the quality of the product cannot be observed by naked eyes. Therefore, a quality evaluation system of the traditional Chinese medicine formula granules is established, so that the intrinsic quality of the formula granules is comprehensively reflected, and the method has important practical significance.
At present, the research on the fingerprint spectrum of pyrrosia lingua formula particles is relatively few, for example, the research on UPLC characteristic spectrum of pyrrosia lingua medicinal material [ J ] (Pan Experil, Suo Cai Xian, Qiu Yun Jing, etc.. Guangdong university of pharmacy 2020, 36(06): 784-. The prior art reports on the pyrrosia lingua formula granules only monitor the quality of the pyrrosia lingua formula granules from one angle of fingerprint spectrum, and the peak identification of the characteristic spectrum is less.
At present, the existing detection method cannot reflect the integral internal quality of the pyrrosia lingua formula granules, and therefore the invention provides a quality standard detection method for the pyrrosia lingua formula granules.
Disclosure of Invention
The invention aims to provide a quality control method for pyrrosia lingua formula granules, which is used for controlling the quality of pyrrosia lingua formula granules from multiple angles, has strong specificity and short analysis time, and can effectively improve the quality control level of the pyrrosia lingua formula granules.
The invention is realized by the following technical scheme:
a quality control method for pyrrosia lingua formula granules comprises the following steps:
(1) accurately weighing pyrrosia lingua formula particle powder, and preparing a pyrrosia lingua formula particle test sample solution;
(2) analyzing the sample solution of the pyrrosia lingua formula particle by using a high performance liquid chromatograph to obtain an HPLC characteristic spectrum of the pyrrosia lingua formula particle, and measuring a corresponding peak area;
(3) analyzing the reference substance solution by using a high performance liquid chromatograph, and determining a corresponding peak area;
(4) and calculating by an external standard method to obtain the product.
Preferably, the chromatographic conditions for the high performance liquid chromatograph analysis are as follows: the chromatographic column using octadecylsilane chemically bonded silica as a filler has a column temperature of 25-35 ℃, a flow rate of 0.5-1.5 ml/min, a detection wavelength of 200-350 nm and a sample injection amount of 5-15 mu l.
As a most preferred embodiment, the chromatographic conditions for the hplc analysis are: adopting a Waters T3C 18 chromatographic column with specification of 4.6mm x 250mm and 5 μm, wherein the column temperature is 30 ℃, the flow rate is 1.0ml/min, and the detection wavelength is 0-23min 260 nm; 23-65min, 330nm, and the sample amount is 10 μ l.
As a preferred embodiment, the gradient elution conditions are: and performing gradient elution by using acetonitrile as a mobile phase A and using a phosphoric acid aqueous solution as a mobile phase B.
As a most preferable scheme, acetonitrile is taken as a mobile phase A, and 0.1% phosphoric acid water solution is taken as a mobile phase B for gradient elution; the gradient elution conditions were: the volume fraction of the mobile phase A is changed to 3% -5% and the volume fraction of the mobile phase B is changed to 97% -95% in 0-10 min; 10-18 min, the volume fraction of the mobile phase A is changed to 5% -9%, and the volume fraction of the mobile phase B is changed to 95% -91%; for 18-30 min, the volume fraction of the mobile phase A is changed to 9% -14%, and the volume fraction of the mobile phase B is changed to 91% -86%; 30-35 min, the volume fraction of the mobile phase A is changed to 14% -20%, and the volume fraction of the mobile phase B is changed to 86% -80%; and (3) 35-65 min, wherein the volume fraction of the mobile phase A is changed to 20-35%, and the volume fraction of the mobile phase B is changed to 80-65%.
As a preferable scheme, in the step (2), the characteristic spectrum totally comprises 11 common peaks, and the 11 common peaks comprise characteristic peaks of protocatechuic acid, caffeic acid, chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, isochlorogenic acid a, isochlorogenic acid B, and isochlorogenic acid C.
As a preferred scheme, the test solution is prepared by a method comprising the following steps: grinding the pyrrosia lingua formula particles into powder, precisely weighing 0.05-0.2 g of pyrrosia lingua formula particles, placing the powder into a 10mL volumetric flask, precisely adding a proper amount of 30-60% methanol, carrying out ultrasonic treatment for 20-40 minutes, cooling, fixing the volume to a scale mark by using 30-60% methanol, shaking up, filtering, and taking a subsequent filtrate to obtain the pyrrosia lingua powder.
As a most preferred embodiment, the test solution is prepared by a method comprising the steps of: grinding the pyrrosia lingua formula particles into powder, precisely weighing 0.1g of pyrrosia lingua formula particles, placing the powder into a 10mL volumetric flask, precisely adding a proper amount of 50% methanol, carrying out ultrasonic treatment for 30 minutes, cooling, fixing the volume to a scale mark by using the 50% methanol, shaking up, filtering, and taking a subsequent filtrate to obtain the pyrrosia lingua extract.
As a preferred embodiment, the preparation method of the reference solution comprises: precisely weighing protocatechuic acid, caffeic acid, chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, isochlorogenic acid A, isochlorogenic acid B and isochlorogenic acid C as reference substances, and adding 50% methanol to obtain a solution containing 30-100 μ g of chlorogenic acid per 1 ml.
As a most preferred scheme, the detection components are protocatechuic acid, caffeic acid, chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, isochlorogenic acid a, isochlorogenic acid B and isochlorogenic acid C, and the preparation method of the reference solution comprises the following steps: precisely weighing protocatechuic acid, caffeic acid, chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, and isochlorogenic acid C as reference, and adding 50% methanol to obtain solution containing 50 μ g per 1 ml.
Compared with the prior art, the invention has the following beneficial effects:
(1) the invention adopts an HPLC method to establish a characteristic spectrum of the pyrrosia lingua formula granules, and realizes quantitative and qualitative analysis of effective components in the pyrrosia lingua formula granules;
(2) the characteristic spectrum of the pyrrosia lingua formula particle constructed by the invention fully shows the chemical component characteristics of the pyrrosia lingua formula particle, and the method has the advantages of good stability, high precision and good reproducibility;
(3) the quality of the pyrrosia lingua formula granules is monitored from two aspects of HPLC characteristic spectrum and content determination (protocatechuic acid, caffeic acid, chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, isochlorogenic acid A, isochlorogenic acid B and isochlorogenic acid C), and an effective method is provided for quality monitoring of the pyrrosia lingua formula granules.
Drawings
FIG. 1 is a graphical representation of the identification of characteristic peaks shared by 10 batches of Pyrrosia lingua formulation granules;
FIG. 2 is a folium Pyrrosiae reference medicinal material feature spectrum;
FIG. 3 shows a common pattern of characteristic spectra of folium Pyrrosiae granules;
FIG. 4 is an identification chart of characteristic peaks of a characteristic spectrum of a pyrrosia lingua formula granule;
FIG. 5 is a diagram for investigating the specificity of the content determination of the pyrrosia lingua formula granules;
FIG. 6 is a graph of protocatechuic acid standard curves;
FIG. 7 is a standard graph of caffeic acid;
FIG. 8 is a graph of the standard curve of chlorogenic acid;
FIG. 9 is a graph of the standard curve for neochlorogenic acid;
FIG. 10 is a graph of standard cryptochlorogenic acid curves;
FIG. 11 is a graph of isochlorogenic acid A standard;
FIG. 12 is a B standard graph of isochlorogenic acid;
FIG. 13 is a graph of isochlorogenic acid C standard;
in the figure, 6(S) is a chlorogenic acid reference peak.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are a part of the embodiments of the present invention, but not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
Construction of pyrrosia lingua formula granule characteristic spectrum
1. Main instruments, reagents and reagents
1.1 Main instruments: high performance liquid chromatography (Saimei Fei, Ultimate 3000, USA), Waters T3C 18 column (4.6mm 250mm, 5 μm), electronic analytical balance (Sidolisi SQP, Germany), digital controlled ultrasonic cleaner (Shanghai Ke dao ultrasonic instruments, Inc., China), PURELA-ultrapure water instrument (PURELA classic UV, UK)
1.2 main reagents: methanol (national pharmaceutical group ltd, analytical grade); phosphoric acid (national drug group ltd, chromatographic purity); acetonitrile (fischer, usa, chromatographically pure); the water was ultrapure water (self-made in the laboratory).
1.3 main reagents: protocatechuic acid (batch No. 110809-201906, purity 97.7%), caffeic acid (batch No. 110885-201703, purity 99.7%), chlorogenic acid (batch No. 110753-201716, purity 99.3%) (China drug assay research institute), isochlorogenic acid C (batch No. 250036-202005 purity 99.21%) (Shanghai Honghong Biotech Co., Ltd.), cryptochlorogenic acid (batch No. DST200522-035 purity 98.77%), neochlorogenic acid (batch No. DST200521-015 purity 99.68%), isochlorogenic acid A (batch No. DST 190-036 purity 99.55%), isochlorogenic acid B (batch No. DST191008-037 purity 99.04%) (Chengdidedersi Biotech Co., Ltd.), pyrrosia formula particles (batch No. S1-S10, Hongtang medicine Co., Ltd.)
2. Experimental methods
Preparation of a test solution: grinding the pyrrosia lingua formula particles into powder, precisely weighing 0.1g of pyrrosia lingua formula particles, placing the powder into a 10mL volumetric flask, precisely adding a proper amount of 50% methanol, carrying out ultrasonic treatment for 30 minutes, cooling, fixing the volume to a scale mark by using the 50% methanol, shaking up, filtering, and taking a subsequent filtrate to obtain the pyrrosia lingua extract.
The chromatographic conditions of the high performance liquid chromatograph analysis are as follows: performing gradient elution by using a Waters T3C 18 chromatographic column with specification of 4.6mm × 250mm and 5 μm, at column temperature of 30 ℃, acetonitrile as a mobile phase A and phosphoric acid aqueous solution with volume fraction of 0.1% as a mobile phase B, wherein the flow rate is 1.0ml/min, and the detection wavelength is as follows: 0-23min 260 nm; 23-65min, 330nm, and the sample amount is 10 μ l.
The gradient elution conditions were: the volume fraction of the mobile phase A is changed to 3% -5% and the volume fraction of the mobile phase B is changed to 97% -95% in 0-10 min; 10-18 min, the volume fraction of the mobile phase A is changed to 5% -9%, and the volume fraction of the mobile phase B is changed to 95% -91%; for 18-30 min, the volume fraction of the mobile phase A is changed to 9% -14%, and the volume fraction of the mobile phase B is changed to 91% -86%; 30-35 min, the volume fraction of the mobile phase A is changed to 14% -20%, and the volume fraction of the mobile phase B is changed to 86% -80%; and (3) 35-65 min, wherein the volume fraction of the mobile phase A is changed to 20-35%, and the volume fraction of the mobile phase B is changed to 80-65%.
3. Determination of common characteristic peaks of characteristic spectrum of pyrrosia lingua formula granules
Taking 10 batches of pyrrosia lingua formula particle samples, preparing a sample solution according to a sample solution preparation method, carrying out sample injection determination under specified chromatographic conditions, carrying out common peak identification on 10 batches of pyrrosia lingua formula particle characteristic spectrums by using traditional Chinese medicine chromatogram fingerprint similarity evaluation software (2012 edition), and determining characteristic peaks with better separation degree and higher chromatographic peak purity, wherein as shown in figure 1, pyrrosia lingua reference medicinal material characteristic spectrums are shown in figure 2, and a pyrrosia lingua formula particle characteristic spectrum common mode is shown in figure 3.
4. Identification of characteristic peaks
Preparation of reference solutions: taking a pyrrosia lingua reference medicinal material, grinding into powder, taking 0.1g, precisely weighing, placing into a 10mL volumetric flask, precisely adding a proper amount of 50% methanol, carrying out ultrasonic treatment for 30 minutes, cooling, fixing the volume to a scale mark with 50% methanol, shaking up, filtering, and taking a subsequent filtrate to obtain the pyrrosia lingua capsule.
Preparation of a test solution: grinding the pyrrosia lingua formula particles into powder, precisely weighing 0.1g of pyrrosia lingua formula particles, placing the powder into a 10mL volumetric flask, precisely adding a proper amount of 50% methanol, carrying out ultrasonic treatment for 30 minutes, cooling, fixing the volume to a scale mark by using the 50% methanol, shaking up, filtering, and taking a subsequent filtrate to obtain the pyrrosia lingua extract.
Preparation of control solutions: precisely weighing protocatechuic acid, caffeic acid, chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, and isochlorogenic acid C as reference, and adding 50% methanol to obtain solution containing 50 μ g per 1 ml.
The chromatographic conditions of the high performance liquid chromatograph analysis are as follows: performing gradient elution by using a Waters T3C 18 chromatographic column with specification of 4.6mm × 250mm and 5 μm, at column temperature of 30 ℃, acetonitrile as a mobile phase A and phosphoric acid aqueous solution with volume fraction of 0.1% as a mobile phase B, wherein the flow rate is 1.0ml/min, and the detection wavelength is as follows: 0-23min 260 nm; 23-65min, 330nm, and the sample amount is 10 μ l.
The gradient elution conditions were: the volume fraction of the mobile phase A is changed to 3% -5% and the volume fraction of the mobile phase B is changed to 97% -95% in 0-10 min; 10-18 min, the volume fraction of the mobile phase A is changed to 5% -9%, and the volume fraction of the mobile phase B is changed to 95% -91%; for 18-30 min, the volume fraction of the mobile phase A is changed to 9% -14%, and the volume fraction of the mobile phase B is changed to 91% -86%; 30-35 min, the volume fraction of the mobile phase A is changed to 14% -20%, and the volume fraction of the mobile phase B is changed to 86% -80%; and (3) 35-65 min, wherein the volume fraction of the mobile phase A is changed to 20-35%, and the volume fraction of the mobile phase B is changed to 80-65%.
And (3) determination of a sample: respectively taking 10 mul of protocatechuic acid, caffeic acid, chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C reference substance solution and pyrrosia lingua formula granule test solution, injecting the reference substance solution and the isochlorogenic acid C reference substance solution into a high performance liquid chromatograph, measuring, and finding characteristic peaks with the retention time consistent with that of protocatechuic acid, caffeic acid, chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, isochlorogenic acid A, isochlorogenic acid B and isochlorogenic acid C in a pyrrosia lingua formula granule characteristic spectrum.
5. Evaluation of similarity
And (3) introducing the 10 batches of pyrrosia lingua formula particle characteristic spectrums in a cdf format into software of a traditional Chinese medicine chromatogram characteristic spectrum similarity evaluation system (2012 edition), taking the characteristic spectrum of a sample with the batch number of S1 as a reference spectrum, taking a peak 6 as a Mark peak to carry out retention time correction, carrying out full peak matching, and generating a pyrrosia lingua formula particle comparison characteristic spectrum by using an average method. The similarity between the characteristic spectrum of each sample and the reference characteristic spectrum is calculated respectively and shown in table 1.
TABLE 110 evaluation table for similarity of folium Pyrrosiae formula granules
The result shows that the similarity of the characteristic spectrums of 10 batches of samples is more than 0.95, the similarity is higher, the difference of the quality of 10 batches of pyrrosia lingua formula granules is not large, in order to strictly control the quality of pyrrosia lingua formula granules in production, according to the measurement result of 10 batches of samples, the similarity between the characteristic spectrums of the pyrrosia lingua formula granules to be tested and the reference characteristic spectrums is required to be not less than 0.95 according to the calculation of a traditional Chinese medicine chromatographic fingerprint similarity evaluation system.
Example 2
Methodology investigation of constructing method of HPLC characteristic spectrum of pyrrosia lingua formula granules
(1) Precision investigation, the same lot of pyrrosia lingua formula particles are taken and prepared into a test solution, sample introduction is carried out for 6 times continuously under the chromatographic condition in the method for constructing the characteristic spectrum of the pyrrosia lingua formula particles in example 1, 10 mu L of chlorogenic acid at the peak 6 is taken as a reference peak S, the relative retention time and the relative peak area of other common peaks and the reference peak are calculated, and the results are shown in tables 2 and 3.
TABLE 2 relative retention time of each common peak
TABLE 3 relative peak area of each common peak
The experimental result shows that in the precision investigation, the same sample solution is continuously injected for 6 times, the relative retention time RSD of each characteristic peak is within the range of 0.01-0.08%, the relative peak area RSD is within the range of 0.04-0.54%, and the relative retention time and the relative peak area RSD are both less than 1.0%, which indicates that the instrument has good precision.
(2) In the repeatability examination, 6 parts of the same lot of pyrrosia lingua formula granules are respectively taken, 6 parts of test solution is prepared under the chromatographic conditions in the method for constructing the characteristic spectrum of the pyrrosia lingua formula granules in the example 1, the chlorogenic acid of the 6 th peak is taken as the reference peak S, the relative retention time and the relative peak area of other common peaks and the reference peak are calculated, and the results are shown in tables 4 and 5.
TABLE 4 common peaks relative retention time of common peaks
TABLE 5 relative peak area retention of each common peak
The experimental result shows that the relative retention time RSD of each characteristic peak is within the range of 0.01-0.04%, the relative peak area RSD is within the range of 0.13-0.50%, and the relative retention time and the relative peak area RSD are both less than 1.0% by repeating the measurement for 6 times on the same batch of samples, which indicates that the method has good repeatability.
(3) And (3) stability investigation: taking the same batch of pyrrosia lingua formula particles, preparing a test solution, carrying out sample injection detection for 0, 2, 4, 6, 14 and 18 hours respectively under the chromatographic conditions in the method for constructing the characteristic spectrum of the pyrrosia lingua formula particles in the example 1, carrying out sample injection detection for 10 mu L each time, taking the chlorogenic acid of the peak No. 6 as a reference peak S, and calculating the relative retention time and the relative peak area of other common peaks and the reference peak, wherein the results are shown in tables 6 and 7.
TABLE 6 common peaks relative retention time of common peaks
TABLE 7 relative peak area retention of each common peak
The experimental result shows that the relative retention time RSD of each characteristic peak is within the range of 0.01-0.11%, the relative peak area RSD of each characteristic peak is within the range of 0.08-0.70%, and the relative retention time and the relative peak area RSD are both less than 1.0% when the same sample solution is subjected to sample injection measurement for multiple times within 18 hours, which indicates that the sample solution has good relative stability within 18 hours.
Example 3
On the basis of example 1, this example performs the content determination of active ingredients of pyrrosia lingua formula granules, and includes the following steps:
(1) respectively sucking a reference substance solution and a pyrrosia lingua formula particle sample solution, injecting the reference substance solution and the pyrrosia lingua formula particle sample solution into a high performance liquid chromatograph, and measuring corresponding peak areas;
(2) and calculating by an external standard method to obtain the product.
The detection components are protocatechuic acid, caffeic acid, chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, isochlorogenic acid A, isochlorogenic acid B and isochlorogenic acid C.
Main apparatus, reagents and test were in accordance with example 1
The conditions of the high performance liquid chromatography were the same as in example 1
Preparation of control solutions in accordance with example 1
4. Preparation of test solution
4.1 examination of extraction solvent
Taking pyrrosia lingua formula granules (S1), grinding into powder, taking 0.1g of pyrrosia lingua formula granules, precisely weighing and paralleling 5 groups, placing 2 parts of each group into a 10ml volumetric flask, respectively and precisely adding water, 50% methanol, 50% ethanol, proper amounts of methanol and ethanol, carrying out ultrasonic treatment for 30 minutes, cooling, respectively metering the volume to a scale mark by using corresponding solvents, shaking up, filtering, taking subsequent filtrate, measuring under specified chromatographic conditions, and obtaining experimental results shown in Table 8.
TABLE 8 ingredient determination of Pyrrosia lingua formula granule 8 compounds for different extraction solvent investigation
The results show that: the extraction efficiency of 50% methanol and 50% ethanol is highest, and the two are not obviously different, so that the method selects 50% methanol as extraction solvent, and is consistent with the preparation method of the test solution under the characteristic spectrum item.
4.2 examination of extraction methods
Taking pyrrosia lingua formula granules (S1), grinding into powder, taking 0.1g, precisely weighing, paralleling 2 groups, putting 2 parts of each group into a 10ml volumetric flask, precisely adding a proper amount of 50% methanol, respectively carrying out ultrasonic treatment for 30 minutes, heating and refluxing for 30 minutes, cooling, fixing the volume to a scale line by using 50% methanol, shaking up, filtering, taking subsequent filtrate, and measuring under the specified chromatographic conditions, wherein the experimental results are shown in Table 9.
TABLE 9 determination of the content of 8 compounds in Pyrrosia lingua formula granule by different extraction methods
The experimental result shows that the extraction effects of the ultrasonic extraction mode and the reflux extraction mode are equivalent, but the ultrasonic treatment is relatively simple, so the ultrasonic treatment is selected.
4.3 determination of preparation method of test solution
Grinding the pyrrosia lingua formula particles into powder, precisely weighing 0.1g of pyrrosia lingua formula particles, placing the powder into a 10mL volumetric flask, precisely adding a proper amount of 50% methanol, carrying out ultrasonic treatment for 30 minutes, cooling, fixing the volume to a scale mark by using the 50% methanol, shaking up, filtering, and taking a subsequent filtrate to obtain the pyrrosia lingua extract.
5. Methodology validation
5.1 specialization examination
The auxiliary material added in the pyrrosia lingua formula particle is maltodextrin. The experiment inspects the influence of negative samples lacking the pyrrosia lingua on the determination of the pyrrosia lingua formula particle content. Taking negative samples lacking pyrrosia lingua to prepare negative sample solution according to the test article preparation method. The test solution, the negative solution and the reference solution of the pyrrosia lingua formula granules (S1) are injected into a liquid chromatograph, and the measurement is carried out under the specified chromatographic conditions, and the result is shown in figure 5.
The result shows that no chromatographic peak exists at the retention time corresponding to the reference solution in the negative chromatogram, which indicates that the auxiliary materials and the solvent have no interference to the determination of the 8 compound components, and the determination of the content of the 8 compound components in the pyrrosia lingua formula granules by the method has specificity.
5.2 Linear relationship investigation
Protocatechuic acid (9.46mg), caffeic acid (9.34mg), chlorogenic acid (39.91mg), neochlorogenic acid (16.34mg), cryptochlorogenic acid (22.42mg), isochlorogenic acid A (8.62mg), isochlorogenic acid B (11.16mg) and isochlorogenic acid C (9.50mg) are precisely weighed into 20ml volumetric flasks to prepare mother solutions of control products with different concentrations.
Accurately sucking appropriate amount of each reference substance into 10ml volumetric flasks, adding 50% methanol to dilute to scale mark, shaking, and gradually diluting to obtain 5 reference substances with different concentrations. Precisely sucking 10 μ l of the above 5 reference solutions with different concentrations, analyzing by sample injection under specified chromatographic conditions, and recording chromatographic peak area. The peak areas are plotted as ordinate (y) and the control concentrations as abscissa (x), the results are shown in Table 10, and the standard curves are shown in FIGS. 6 to 13.
TABLE 10 ingredient content determination linear investigation results of 8 compounds of folium Pyrrosiae formula granule
5.3 stability Studies
Grinding folium Pyrrosiae formula granules (S1) into powder, weighing 0.1g precisely, preparing into test solution, injecting sample under specified chromatographic conditions for 0, 2, 4, 6, 14 and 18 hours respectively, detecting, calculating RSD values of peak areas of 8 compounds injected at different time, and the calculation results are shown in Table 11.
Table 11 component content testing stability test results for 8 kinds of compound in pyrrosia lingua formulation granule
The results show that the RSD values of the peak areas of 8 compound components are all less than 1.0% when the same test solution is injected at different times, which indicates that the test solution has good stability within 18 h.
5.4 precision investigation
Grinding the same folium Pyrrosiae formula granule (S1) into powder, collecting 0.1g, precisely weighing, preparing into test solution, continuously injecting sample for 6 needles under specified chromatographic conditions, calculating peak area RSD values of 8 compounds, and the calculation results are shown in Table 12.
Table 12 component content measurement precision investigation results of 8 compounds of pyrrosia lingua formula granules
The result shows that the peak areas of the 8 compound components in the sample are all less than 1.0% when the same sample is continuously injected into 6 needles, which indicates that the precision of the instrument is good.
5.5 repeatability test
Grinding the same pyrrosia lingua formula particle (S1) into powder, taking 0.1g of pyrrosia lingua formula particle, precisely weighing and paralleling 6 parts, preparing 6 parts of test solution, carrying out sample injection detection under specified chromatographic conditions, and calculating the content of 8 compound components in a sample and the RSD value thereof, wherein the detection results are shown in Table 13.
Table 13 folium Pyrrosiae formula granule 8 compound component content detection repeatability investigation result
The result shows that the RSD values of 8 compounds in a sample are less than 1.0 percent after 6 times of measurement of the same batch of pyrrosia lingua formula granules, which indicates that the method has better repeatability.
5.6 accuracy survey
Precisely weighing 50mg to 10ml of pyrrosia lingua formula granules (S1) in a volumetric flask, dividing into three groups by 9 parts in parallel, respectively adding corresponding reference substances according to the proportion of 50%, 100% and 150% of the content of 8 components in a sample, adding a proper amount of 50% methanol, carrying out ultrasonic treatment for 30min, cooling, adding 50% methanol to a constant volume to a scale mark, and shaking up to obtain the pyrrosia lingua formula granules. The samples were tested under the specified chromatographic conditions and the sample recovery rates of the 8 components were calculated, and the results are shown in tables 14-21.
TABLE 14 investigation result of protocatechuic acid sample recovery rate in folium Pyrrosiae granule
TABLE 15 investigation result of sample recovery rate of neochlorogenic acid in folium Pyrrosiae formula granule
TABLE 16 investigation results of sample recovery rate of chlorogenic acid in folium Pyrrosiae formula granules
TABLE 17 investigation result of sample recovery rate of cryptochlorogenic acid in folium Pyrrosiae formula granule
TABLE 18 investigation results of sample recovery of caffeic acid in folium Pyrrosiae formula granules
TABLE 19 examination result of sample recovery rate of isochlorogenic acid A in pyrrosia lingua formula granules
TABLE 20 investigation results of sample recovery rate of isochlorogenic acid B in folium Pyrrosiae formula granules
TABLE 21 investigation result of sample recovery rate of isochlorogenic acid C in pyrrosia lingua formula granules
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are merely illustrative of the principles of the invention, but that various changes and modifications may be made without departing from the spirit and scope of the invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (10)
1. A quality control method for pyrrosia lingua formula granules is characterized by comprising the following steps:
(1) accurately weighing pyrrosia lingua formula particle powder, and preparing a pyrrosia lingua formula particle test sample solution;
(2) analyzing the sample solution of the pyrrosia lingua formula particle by using a high performance liquid chromatograph to obtain an HPLC characteristic spectrum of the pyrrosia lingua formula particle, and measuring a corresponding peak area;
(3) analyzing the reference substance solution by using a high performance liquid chromatograph, and determining a corresponding peak area;
(4) and calculating by an external standard method to obtain the product.
2. The quality control method for pyrrosia lingua formula granules according to claim 1, wherein the chromatographic conditions of the high performance liquid chromatograph analysis are as follows: the chromatographic column using octadecylsilane chemically bonded silica as a filler has a column temperature of 25-35 ℃, a flow rate of 0.5-1.5 ml/min, a detection wavelength of 200-350 nm and a sample injection amount of 5-15 mu l.
3. The quality control method for pyrrosia lingua formula granules according to claim 3, wherein the chromatographic conditions of the high performance liquid chromatograph analysis are as follows: adopting a Waters T3C 18 chromatographic column with specification of 4.6mm x 250mm and 5 μm, wherein the column temperature is 30 ℃, the flow rate is 1.0ml/min, and the detection wavelength is 0-23min 260 nm; 23-65min, 330nm, and the sample amount is 10 μ l.
4. The quality control method for pyrrosia lingua formula granules according to claim 1, wherein the gradient elution conditions are as follows: and performing gradient elution by using acetonitrile as a mobile phase A and using a phosphoric acid aqueous solution as a mobile phase B.
5. The quality control method for pyrrosia lingua formula particles according to claim 4, wherein acetonitrile is used as a mobile phase A, and a 0.1% phosphoric acid aqueous solution is used as a mobile phase B to perform gradient elution; the gradient elution conditions were: the volume fraction of the mobile phase A is changed to 3% -5% and the volume fraction of the mobile phase B is changed to 97% -95% in 0-10 min; 10-18 min, the volume fraction of the mobile phase A is changed to 5% -9%, and the volume fraction of the mobile phase B is changed to 95% -91%; for 18-30 min, the volume fraction of the mobile phase A is changed to 9% -14%, and the volume fraction of the mobile phase B is changed to 91% -86%; 30-35 min, the volume fraction of the mobile phase A is changed to 14% -20%, and the volume fraction of the mobile phase B is changed to 86% -80%; and (3) 35-65 min, wherein the volume fraction of the mobile phase A is changed to 20-35%, and the volume fraction of the mobile phase B is changed to 80-65%.
6. The quality control method for pyrrosia lingua formula granules according to claim 1, wherein in the step (2), the characteristic spectrum totally comprises 11 common peaks, and the 11 common peaks comprise characteristic peaks of protocatechuic acid, caffeic acid, chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, isochlorogenic acid A, isochlorogenic acid B and isochlorogenic acid C.
7. The quality control method for pyrrosia lingua formula granules according to claim 1, wherein the test solution is prepared by a method comprising the following steps: grinding the pyrrosia lingua formula particles into powder, precisely weighing 0.05-0.2 g of pyrrosia lingua formula particles, placing the powder into a 10mL volumetric flask, precisely adding a proper amount of 30-60% methanol, carrying out ultrasonic treatment for 20-40 minutes, cooling, fixing the volume to a scale mark by using 30-60% methanol, shaking up, filtering, and taking a subsequent filtrate to obtain the pyrrosia lingua powder.
8. The quality control method for pyrrosia lingua formula granules according to claim 1, wherein the test solution is prepared by a method comprising the following steps: grinding the pyrrosia lingua formula particles into powder, precisely weighing 0.1g of pyrrosia lingua formula particles, placing the powder into a 10mL volumetric flask, precisely adding a proper amount of 50% methanol, carrying out ultrasonic treatment for 30 minutes, cooling, fixing the volume to a scale mark by using the 50% methanol, shaking up, filtering, and taking a subsequent filtrate to obtain the pyrrosia lingua extract.
9. The quality control method for pyrrosia lingua formula granules according to claim 1, wherein the preparation method for the reference solution comprises the following steps: precisely weighing protocatechuic acid, caffeic acid, chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, isochlorogenic acid A, isochlorogenic acid B and isochlorogenic acid C as reference substances, and adding 50% methanol to obtain a solution containing 30-100 μ g of chlorogenic acid per 1 ml.
10. The quality control method of pyrrosia lingua formula granules according to claim 9, wherein the detection components are protocatechuic acid, caffeic acid, chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, isochlorogenic acid A, isochlorogenic acid B and isochlorogenic acid C, and the preparation method of the reference solution comprises the following steps: precisely weighing protocatechuic acid, caffeic acid, chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, and isochlorogenic acid C as reference, and adding 50% methanol to obtain solution containing 50 μ g per 1 ml.
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