CN114252521B - Detection method of Chinese herbal medicine Chinese lobelia fingerprint - Google Patents
Detection method of Chinese herbal medicine Chinese lobelia fingerprint Download PDFInfo
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- 241000411851 herbal medicine Species 0.000 title claims abstract description 36
- 240000003146 Lobelia chinensis Species 0.000 title claims abstract description 33
- 238000001514 detection method Methods 0.000 title claims abstract description 26
- 241000208672 Lobelia Species 0.000 claims abstract description 35
- 239000013558 reference substance Substances 0.000 claims abstract description 26
- 239000012488 sample solution Substances 0.000 claims abstract description 24
- 239000000523 sample Substances 0.000 claims abstract description 19
- 239000000126 substance Substances 0.000 claims abstract description 10
- 239000007788 liquid Substances 0.000 claims abstract description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 84
- 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 30
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- 230000014759 maintenance of location Effects 0.000 claims description 22
- 239000000243 solution Substances 0.000 claims description 18
- YFVGIJBUXMQFOF-PJOVQGMDSA-N 5-hydroxy-2-(4-methoxyphenyl)-7-[(2s,3r,4s,5s,6r)-3,4,5-trihydroxy-6-[[(2r,3r,4r,5r,6s)-3,4,5-trihydroxy-6-methyloxan-2-yl]oxymethyl]oxan-2-yl]oxychromen-4-one Chemical compound C1=CC(OC)=CC=C1C(OC1=C2)=CC(=O)C1=C(O)C=C2O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO[C@H]2[C@@H]([C@H](O)[C@@H](O)[C@H](C)O2)O)O1 YFVGIJBUXMQFOF-PJOVQGMDSA-N 0.000 claims description 16
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- 238000000034 method Methods 0.000 claims description 16
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- KZNIFHPLKGYRTM-UHFFFAOYSA-N apigenin Chemical compound C1=CC(O)=CC=C1C1=CC(=O)C2=C(O)C=C(O)C=C2O1 KZNIFHPLKGYRTM-UHFFFAOYSA-N 0.000 claims description 15
- 235000008714 apigenin Nutrition 0.000 claims description 15
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- 229940117893 apigenin Drugs 0.000 claims description 15
- IQPNAANSBPBGFQ-UHFFFAOYSA-N luteolin Chemical compound C=1C(O)=CC(O)=C(C(C=2)=O)C=1OC=2C1=CC=C(O)C(O)=C1 IQPNAANSBPBGFQ-UHFFFAOYSA-N 0.000 claims description 15
- 235000009498 luteolin Nutrition 0.000 claims description 15
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- 229960001285 quercetin Drugs 0.000 claims description 15
- 238000005303 weighing Methods 0.000 claims description 13
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- 229920000061 Geraniin Polymers 0.000 claims description 12
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- 238000002347 injection Methods 0.000 claims description 8
- 239000007924 injection Substances 0.000 claims description 8
- 238000002360 preparation method Methods 0.000 claims description 8
- 238000010828 elution Methods 0.000 claims description 7
- 239000000706 filtrate Substances 0.000 claims description 7
- 238000001704 evaporation Methods 0.000 claims description 5
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- 239000012528 membrane Substances 0.000 claims description 5
- 238000009210 therapy by ultrasound Methods 0.000 claims description 5
- 238000011156 evaluation Methods 0.000 claims description 4
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- QAGGICSUEVNSGH-UHFFFAOYSA-N Diosmetin Natural products C1=C(O)C(OC)=CC=C1C1=CC(=O)C2=CC=C(O)C=C2O1 QAGGICSUEVNSGH-UHFFFAOYSA-N 0.000 claims description 3
- 235000015428 diosmetin Nutrition 0.000 claims description 3
- MBNGWHIJMBWFHU-UHFFFAOYSA-N diosmetin Chemical compound C1=C(O)C(OC)=CC=C1C1=CC(=O)C2=C(O)C=C(O)C=C2O1 MBNGWHIJMBWFHU-UHFFFAOYSA-N 0.000 claims description 3
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- 230000000694 effects Effects 0.000 description 8
- 238000000605 extraction Methods 0.000 description 6
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 4
- 238000004128 high performance liquid chromatography Methods 0.000 description 4
- 238000002137 ultrasound extraction Methods 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
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- 239000000203 mixture Substances 0.000 description 2
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- 241001492658 Cyanea koolauensis Species 0.000 description 1
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- PQLVXDKIJBQVDF-UHFFFAOYSA-N acetic acid;hydrate Chemical compound O.CC(O)=O PQLVXDKIJBQVDF-UHFFFAOYSA-N 0.000 description 1
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 229930013930 alkaloid Natural products 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000007882 cirrhosis Effects 0.000 description 1
- 208000019425 cirrhosis of liver Diseases 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
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- 229930003944 flavone Natural products 0.000 description 1
- 150000002212 flavone derivatives Chemical class 0.000 description 1
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- 238000011835 investigation Methods 0.000 description 1
- 238000010829 isocratic elution Methods 0.000 description 1
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- 230000027939 micturition Effects 0.000 description 1
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- 230000004048 modification Effects 0.000 description 1
- SYSQUGFVNFXIIT-UHFFFAOYSA-N n-[4-(1,3-benzoxazol-2-yl)phenyl]-4-nitrobenzenesulfonamide Chemical class C1=CC([N+](=O)[O-])=CC=C1S(=O)(=O)NC1=CC=C(C=2OC3=CC=CC=C3N=2)C=C1 SYSQUGFVNFXIIT-UHFFFAOYSA-N 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
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- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 229940127557 pharmaceutical product Drugs 0.000 description 1
- 150000007965 phenolic acids Chemical class 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 239000001397 quillaja saponaria molina bark Substances 0.000 description 1
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- 238000012216 screening Methods 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 235000007586 terpenes Nutrition 0.000 description 1
- 150000003505 terpenes Chemical class 0.000 description 1
- 231100000611 venom Toxicity 0.000 description 1
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- VHBFFQKBGNRLFZ-UHFFFAOYSA-N vitamin p Natural products O1C2=CC=CC=C2C(=O)C=C1C1=CC=CC=C1 VHBFFQKBGNRLFZ-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- 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
- G01N30/26—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
-
- 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
- G01N30/8686—Fingerprinting, e.g. without prior knowledge of the sample components
-
- 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
- G01N2030/042—Standards
- G01N2030/047—Standards external
Abstract
The invention discloses a detection method of Chinese medicine lobelia fingerprint, which comprises the following steps: step 1, preparing a Chinese medicinal lobelia sample solution; step 2, preparing a mixed reference substance solution: step 3, respectively precisely sucking the mixed reference substance solutions, injecting the mixed reference substance solutions into a liquid chromatograph, and recording a standard substance chromatogram; and 4, respectively precisely sucking the sample solution, injecting the sample solution into a liquid chromatograph, and recording a sample chromatogram. The Chinese herbal medicine Chinese lobelia fingerprint provided by the invention can comprehensively and objectively represent the quality of Chinese herbal medicine Chinese lobelia. The fingerprint detection method provided by the invention has the advantages of simplicity, convenience, stability, high precision, good reproducibility and the like.
Description
Technical Field
The invention relates to a detection method of traditional Chinese medicine, in particular to a detection method of a traditional Chinese medicine Chinese lobelia fingerprint.
Background
The fingerprint refers to a chromatogram or a spectrogram which is obtained by adopting a certain analysis means after a proper treatment of some complex substances such as traditional Chinese medicine, DNA of a certain organism or a certain tissue or cell and protein and can mark the chemical characteristics of the complex substances. The fingerprint spectrum of the traditional Chinese medicine is a comprehensive and quantifiable identification means, is based on the research of the chemical components of the traditional Chinese medicine, and is mainly used for evaluating the authenticity, superiority and stability of the quality of the traditional Chinese medicine and the traditional Chinese medicine preparation. The traditional Chinese medicine and the preparation thereof are all multi-component complex systems, so that the quality of the traditional Chinese medicine is evaluated by adopting a detection method which is suitable for the traditional Chinese medicine and the preparation thereof and can provide rich identification information, and the establishment of the traditional Chinese medicine fingerprint can more comprehensively reflect the types and the amounts of chemical components contained in the traditional Chinese medicine and the preparation thereof, thereby integrally describing and evaluating the quality of the medicine.
Lobelia chinensis (academic name: lobelia chinensis Lour.) is a perennial herb of the genus Lobelia of the family Campanulaceae. Distributed in middle and downstream of Yangtze river and in south provinces of China; there is also a distribution in other countries of asia, eastern india. The whole herb medicine contains various alkaloids, has the effects of clearing heat and detoxicating, promoting urination and reducing swelling, and has the effects of treating venomous snake bite, ascites due to cirrhosis, ascites due to late schistosomiasis, appendicitis and the like.
Disclosure of Invention
The invention aims to: the invention aims to solve the defects of the prior art and provides a fingerprint detection method of Chinese herbal medicine Chinese lobelia, which can objectively, comprehensively and accurately evaluate the quality of the Chinese herbal medicine Chinese lobelia and has important significance in controlling the quality of the Chinese herbal medicine Chinese lobelia and ensuring clinical curative effect.
The technical scheme is as follows: in order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
step 1, preparing a Chinese medicinal lobelia sample solution:
taking Chinese medicinal lobelia samples of different batches, respectively precisely weighing the Chinese medicinal lobelia samples, placing the Chinese medicinal lobelia samples into a conical flask, adding methanol, performing ultrasonic treatment, filtering, evaporating filtrate to dryness, redissolving the filtrate with methanol, and passing through a microporous filter membrane to obtain a sample solution;
step 2, preparing a reference substance solution:
precisely weighing reference substances of quercetin, luteolin, linarin, apigenin and geraniin, placing into a volumetric flask, metering with methanol to scale, shaking, and making into reference substance solution;
step 3, precisely sucking the reference substance solution in the step 2, injecting the reference substance solution into a high performance liquid chromatograph, and recording a chromatogram of the reference substance;
step 4, precisely sucking each batch of sample solution in the step 1, injecting the sample solution into a high performance liquid chromatograph, and recording a sample chromatogram;
step 5, deriving the fingerprint of the Chinese medicinal lobelia sample solution obtained in the step 4, and introducing the fingerprint into a Chinese medicinal chromatographic fingerprint similarity evaluation system 2004A; selecting chromatographic peaks existing in chromatograms of Chinese medicinal lobelia samples in different batches as common peaks; generating a control fingerprint of Chinese medicinal lobelia by an average value calculation method, and calculating the relative retention time and the relative peak area of each common peak; and labeling the chemical components of peaks in the reference fingerprint according to the retention time of the mixed reference solution chromatogram.
As a preferred scheme, the detection method of the Chinese herbal medicine Chinese lobelia fingerprint, the preparation method of the Chinese herbal medicine Chinese lobelia sample solution in the step 1 is as follows: taking 11 batches of Chinese herbal medicine lobelia, precisely weighing 1-5 g of Chinese herbal medicine lobelia sample, placing in a conical flask, adding 50mL of methanol, performing ultrasonic treatment for 0.5-1 h, filtering, evaporating filtrate to dryness, re-dissolving with methanol, fixing the volume to 5mL, and passing through a 0.22 mu m microporous filter membrane to obtain a sample solution.
As a preferred scheme, the detection method of the Chinese herbal medicine Chinese lobelia fingerprint is characterized in that the reference substance solution is prepared in the step 2: precisely weighing quercetin, luteolin, apigenin and geraniin reference substances, placing in a volumetric flask, metering with methanol to scale, shaking, and making into a solution containing quercetin 503.50 μg/mL, luteolin 20.15 μg/mL, apigenin 10.0394 μg/mL, geraniin 25.46 μg/mL, and mixed reference substances, precisely weighing linarin, placing in a volumetric flask, metering with methanol to scale, shaking, and making into a solution containing linarin reference substances 156.31 μg/mL.
As a preferred scheme, the detection method of the Chinese herbal medicine Chinese lobelia fingerprint comprises the following steps of: chromatographic column: YMC-Pack ODS-A, mobile phase: methanol is phase A, water is phase B, and the ultraviolet detector detects the wavelength: 330nm, column temperature 40 ℃, flow rate 1.0mL/min, sample injection volume: 20 μl, gradient elution procedure is as follows:
| Procedure | time/min | Methanol concentration/% | Water concentration/% |
| 1 | 0.01 | 30 | 70 |
| 2 | 5.00 | 30 | 70 |
| 3 | 15.00 | 35 | 65 |
| 4 | 20.00 | 40 | 60 |
| 5 | 30.00 | 42.5 | 57.5 |
| 6 | 35.00 | 46 | 54 |
| 7 | 45.00 | 48 | 52 |
| 8 | 55.00 | 48 | 52 |
| 9 | 60.00 | 100 | 0 |
。
As a preferred scheme, the detection method of the Chinese herbal medicine Chinese lobelia fingerprint spectrum has 11 total peaks in the fingerprint spectrum; wherein the quercetin retention time is 27.905min, which is peak number 4; the retention time of luteolin is 42.060min, which is peak 6; the retention time of linarin was 43.628min, peak No. 7; the retention time of apigenin is 51.790min, which is peak 9; the retention time of diosmetin was 54.699min, which was peak number 11.
As a preferred scheme, the detection method of the Chinese herbal medicine Chinese lobelia fingerprint is introduced into a Chinese herbal medicine chromatographic fingerprint similarity evaluation system 2004A; the similarity of the 11 batches of Chinese herbal medicine Chinese lobelia is shown in the following table:
optimizing fingerprint detection conditions:
1. in the preparation optimization of sample solution
According to the invention, experimental comparison is carried out on different extraction methods (ultrasonic extraction, reflux extraction, percolation and the like) and different extraction solvents (methanol, water, 70% ethanol water solution, 85% ethanol water solution, 95% ethanol and absolute ethanol), and the result shows that the spectrogram difference between ultrasonic extraction and reflux extraction is smaller, and the ultrasonic extraction efficiency is high, so that the ultrasonic extraction method is adopted; the investigation of the extraction solvent finds that the information content of the chromatograms of the methanol extract is the most and the content of the components is the highest; therefore, methanol is selected for extraction.
2. In optimizing chromatographic conditions
The invention adopts a diode array detector to examine the detection wavelength, extracts chromatograms at 260nm, 280nm and 330nm, and when the detection wavelength is 330nm, the response value of the chromatographic peak is higher, and the detected component is the most, so 330nm is selected as the detection wavelength;
the flow rates (1 mL/min, 0.8mL/min, 0.7mL/min, 0.6mL/min and 0.5 mL/min) are screened, and the components in the Chinese herbal medicine Chinese lobelia are most of isomers and other components with very similar polarities, so that the components cannot be separated at a high flow rate, the separation effect is good at a low flow rate, and finally substances with similar polarities are separated under the condition of multiple equal gradients of the flow rate of 1 mL/min.
The invention compares the elution effects of 5 different elution systems of methanol-water, acetonitrile-water, methanol-0.1% acetic acid water, methanol-0.1% phosphoric acid water and acetonitrile-0.1% phosphoric acid water under different gradients. As a result, the components in Chinese herbal medicine Chinese lobelia can achieve a good separation effect when methanol-water is used as a mobile phase, so that the methanol-water is finally selected as the mobile phase.
After the optimal mobile phase composition is optimized, the Chinese medicinal lobelia contains phenolic acid, terpenes, flavone, saponin and other components. The large isocratic elution mode can not well realize the good separation of each component, so the invention continuously screens the gradient elution mode, and the optimal gradient elution mode obtained by the screening is as follows:
| Procedure | time/min | Methanol concentration/% | Water concentration/% |
| 1 | 0.01 | 30 | 70 |
| 2 | 5.00 | 30 | 70 |
| 3 | 15.00 | 35 | 65 |
| 4 | 20.00 | 40 | 60 |
| 5 | 30.00 | 42.5 | 57.5 |
| 6 | 35.00 | 46 | 54 |
| 7 | 45.00 | 48 | 52 |
| 8 | 55.00 | 48 | 52 |
| 9 | 60.00 | 100 | 0 |
。
The beneficial effects are that:
1. the invention screens out the optimal mobile phase composition through a large number of experiments according to the structural property characteristics of the active ingredients contained in the Chinese medicinal Chinese lobelia, and the analysis conditions such as gradient elution program, flow velocity, detection wavelength, chromatographic column, column temperature and the like, multiple experimental verification shows that the Chinese herbal medicine Chinese lobelia fingerprint detection method provided by the invention can comprehensively, objectively and accurately detect and evaluate the quality of Chinese herbal medicine Chinese lobelia, and has important significance for ensuring the curative effect of clinical Chinese herbal medicine Chinese lobelia.
2. The Chinese medicinal lobelia fingerprint established by the method provided by the invention can effectively represent the quality of Chinese medicinal lobelia, objectively embody the front-back sequence and interrelation of each constituent fingerprint characteristic peak, pay attention to the overall appearance characteristics, not only can avoid judging the unilateral nature of the quality of Chinese medicinal lobelia by measuring individual chemical components, but also can reduce the possibility of human treatment for reaching the quality standard.
3. The detection method of the Chinese herbal medicine Chinese lobelia fingerprint has the advantages of simplicity, convenience, good stability, high precision, good reproducibility and the like.
Drawings
FIG. 1 is a chromatogram of a linarin control of the present invention.
Fig. 2 is a fingerprint of the mixed standard of the present invention.
FIG. 3 is a sample control fingerprint of a Chinese medicinal Lobelia sample of the present invention.
Detailed Description
Embodiments of the present invention will be described in detail with reference to examples, which are not to be construed as specific conditions, either as normal conditions or as recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The instruments and reagents used in the examples were as follows:
experimental equipment
1.1 instruments
The double-wavelength scanning high-performance liquid chromatography system comprises a full-automatic online degassing system, a full-automatic sample injection system SIL-20A, an ultraviolet detector SPD-20A and an automatic Wen Kongzhu incubator CTD-20AC, KH-500E type ultrasonic cleaner (Kunshan He Chuan ultrasonic instrument Co., ltd.), and an ML104/02 electronic analytical balance (Mettler Toledo).
1.2 pharmaceutical products and reagents
11 batches (210401; 210402;210403;210404;210405;210406;210407;210408;210409;210410;210411; traditional Chinese medicine Lobelia sample sources Nanjing Zhongshan pharmaceutical Co., ltd.); the control of quercetin, luteolin, linarin, apigenin and geraniin is purchased from the national food and drug testing institute; methanol (chromatographic purity); water (baby haha purified water).
Embodiment 1 a detection method of Chinese herbal medicine Chinese lobelia fingerprint, comprising the following steps:
step 1, preparing a Chinese medicinal lobelia sample solution:
taking the 11 batches of Chinese medicinal lobelia, precisely weighing 1g of Chinese medicinal lobelia sample, placing in a 100mL conical flask, adding 50mL of methanol, performing ultrasonic treatment for 1h, filtering, evaporating filtrate to dryness, re-dissolving with methanol, fixing the volume to 5mL, and passing through a 0.22 mu m microporous filter membrane to obtain 11 batches of sample solution.
Step 2, preparing a reference substance solution:
precisely weighing reference substances of quercetin, luteolin, apigenin and geraniin, placing into a volumetric flask, metering with methanol to a scale, shaking uniformly to obtain mixed reference substance solution of quercetin with concentration of 503.50 μg/mL, luteolin with concentration of 20.15 μg/mL, apigenin with concentration of 10.0394 μg/mL and geraniin with concentration of 25.46 μg/mL, precisely weighing linarin, placing into volumetric flask, metering with methanol to a scale, shaking uniformly to obtain linarin reference substance solution with concentration of 156.31 μg/mL. The chromatogram of the control is shown in fig. 1 and 2.
Step 3, precisely sucking the 11 batches of Chinese medicinal lobelia sample solution in the step 1 and the reference substance solution in the step 2 respectively, injecting into a high performance liquid chromatograph, and recording chromatograms; the liquid chromatography conditions were: chromatographic column: YMC-Pack ODS-A, mobile phase: methanol is phase A, water is phase B, and the ultraviolet detector detects the wavelength: 330nm, column temperature 40 ℃, flow rate 1.0mL/min, sample injection volume: 20 μl, gradient elution procedure is as follows:
| Procedure | time/min | Methanol concentration/% | Water concentration/% |
| 1 | 0.01 | 30 | 70 |
| 2 | 5.00 | 30 | 70 |
| 3 | 15.00 | 35 | 65 |
| 4 | 20.00 | 40 | 60 |
| 5 | 30.00 | 42.5 | 57.5 |
| 6 | 35.00 | 46 | 54 |
| 7 | 45.00 | 48 | 52 |
| 8 | 55.00 | 48 | 52 |
| 9 | 60.00 | 100 | 0 |
，
And 4, deriving the fingerprint of the 11 batches of Chinese medicinal lobelia sample solutions obtained in the step 3, wherein the Chinese medicinal lobelia has 11 common peaks as a result, and the reference fingerprint is shown in figure 3. Determining that quercetin retention time is 27.905min, which is peak number 4 according to the control; the retention time of luteolin is 42.060min, which is peak 6; the retention time of linarin was 43.628min, peak No. 7; the retention time of apigenin is 51.790min, which is peak 9; the retention time of diosmetin was 54.699min, which was peak number 11.
Step 4, deriving the fingerprint of the 11 batches of Chinese medicinal lobelia sample solutions obtained in the step 3, and introducing the fingerprint into a Chinese medicinal chromatographic fingerprint similarity evaluation system 2004A; the similarity of the 11 batches of Chinese herbal medicine Chinese lobelia is shown in the following table:
forensic study of fingerprint detection method:
1. precision study
Sample number S1 test solution prepared according to the method of example 1 is analyzed according to the detection method of example 1, the sample is injected for 6 times in parallel, the injection amount is 20 mu L, quercetin, luteolin, linarin, apigenin and geraniin are taken as reference peaks, the peak area and retention time of the common peaks of the HPLC fingerprints of the samples are analyzed, the RSD value is calculated, the fingerprint comparison and data analysis are carried out by adopting 'Chinese medicine chromatographic similarity evaluation software 2004A', the similarity is 0.906, and the result shows that the parallel injection precision of the equipment is good.
2. Stability study
Sample number S1 test solution prepared according to the method of example 1 is analyzed according to the detection method of example 1, samples are injected for analysis at different time intervals of 0, 2, 6, 12, 18 and 24 hours, the injection amount is 20 mu L, quercetin, luteolin, linarin, apigenin and geraniin are used as reference peaks, the peak area and retention time of the common peaks of the HPLC fingerprints of the samples are analyzed, and the RSD value is calculated, the similarity is 1.25, so that the chromatographic peaks of the Chinese herbal medicine lobelia test solution within 24 hours are hardly changed, and the stability is very good.
3. Repeatability study
Six samples with the serial number of S1 are precisely weighed in parallel, the weight of each Chinese herbal medicine Chinese lobelia is 1g, 6 parts of the same sample solution is prepared according to the method of the example 1, the chromatographic condition of the example 1 is referred, the sample injection amount is 20 mu L, the peak area and the retention time of the common peak of the HPLC fingerprint of the sample are analyzed by taking quercetin, luteolin, linarin, apigenin and geraniin as reference peaks, the RSD value is calculated, the similarity is 1.99, and the result shows that the sample chromatographic peak reproducibility is good, and the repeatability of the method is good.
The experimental result shows that the Chinese herbal medicine Chinese lobelia fingerprint detection method provided by the invention has the advantages of good stability, high precision and good repeatability, can comprehensively and objectively evaluate the quality of Chinese herbal medicine Chinese lobelia, and has important significance for ensuring clinical curative effect.
The above embodiments are only exemplary embodiments of the present invention and are not intended to limit the present invention, the scope of which is defined by the claims. Various modifications and equivalent arrangements of this invention will occur to those skilled in the art, and are intended to be within the spirit and scope of the invention.
Claims (4)
1. The detection method of the Chinese herbal medicine Chinese lobelia fingerprint is characterized by comprising the following steps:
step 1, preparing a Chinese medicinal lobelia sample solution:
taking Chinese medicinal lobelia samples of different batches, respectively precisely weighing the Chinese medicinal lobelia samples, placing the Chinese medicinal lobelia samples into a conical flask, adding methanol, performing ultrasonic treatment, filtering, evaporating filtrate to dryness, redissolving the filtrate with methanol, and passing through a microporous filter membrane to obtain a sample solution;
step 2, preparing a reference substance solution:
precisely weighing reference substances of quercetin, luteolin, linarin, apigenin and geraniin, placing into a volumetric flask, metering with methanol to scale, shaking, and making into reference substance solution;
step 3, precisely sucking the reference substance solution in the step 2, injecting the reference substance solution into a high performance liquid chromatograph, and recording a chromatogram of the reference substance;
step 4, precisely sucking each batch of sample solution in the step 1, injecting the sample solution into a high performance liquid chromatograph, and recording a sample chromatogram;
step 5, deriving the fingerprint of the Chinese medicinal lobelia sample solution obtained in the step 4, and introducing the fingerprint into a Chinese medicinal chromatographic fingerprint similarity evaluation system 2004A; selecting chromatographic peaks existing in chromatograms of Chinese medicinal lobelia samples in different batches as common peaks; generating a control fingerprint of Chinese medicinal lobelia by an average value calculation method, and calculating the relative retention time and the relative peak area of each common peak; labeling chemical components of peaks in the reference fingerprint according to retention time of the mixed reference solution chromatogram;
in the step 3 and the step 4, the liquid chromatography conditions are as follows: chromatographic column: YMC-Pack ODS-A, mobile phase: methanol is phase A, water is phase B, and the ultraviolet detector detects the wavelength: 330nm, column temperature 40 ℃, flow rate 1.0mL/min, sample injection volume: 20. Mu.L, gradient elution procedure was as follows:
。
2. The method for detecting the fingerprint of Chinese herbal medicine Chinese lobelia according to claim 1, wherein the preparation method of the Chinese herbal medicine Chinese lobelia sample solution in step 1 is as follows: taking 11 batches of Chinese herbal medicine lobelia, precisely weighing 1-5 g of Chinese herbal medicine lobelia sample, placing in a conical flask, adding 50mL of methanol, performing ultrasonic treatment for 0.5-1 h, filtering, evaporating filtrate to dryness, re-dissolving with methanol, fixing the volume to 5mL, and passing through a 0.22 mu m microporous filter membrane to obtain a sample solution.
3. The method for detecting the fingerprint of Chinese herbal medicine Chinese lobelia according to claim 1, wherein the preparation of the mixed reference solution in step 2: precisely weighing reference substances of quercetin, luteolin, apigenin and geraniin, placing into a volumetric flask, fixing volume to scale with methanol, shaking uniformly, and preparing into mixed reference substance solution of quercetin, luteolin, apigenin and geraniin with concentration of 503.50 μg/mL, 20.15 μg/mL, 10.0394 μg/mL and 25.46 μg/mL; accurately weighing linarin, placing into volumetric flask, metering with methanol to scale, shaking, and making into linarin reference solution with concentration of 156.31 μg/mL.
4. The method for detecting the Chinese herbal medicine herba Lobeliae chinensis fingerprint according to claim 1, wherein the total number of peaks in the fingerprint is 11; wherein the quercetin retention time is 27.905min, which is peak number 4; the retention time of luteolin is 42.060min, which is peak 6; the retention time of linarin was 43.628min, peak No. 7; the retention time of apigenin is 51.790min, which is peak 9; the retention time of diosmetin was 54.699min, which was peak number 11.
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Non-Patent Citations (4)
| Title |
|---|
| Chemical Profiling of Lobelia chinensis with High-Performance Liquid Chromatography/Quadrupole Time-of-Flight Mass Spectrometry (HPLC/Q-TOF MS) Reveals Absence of Lobeline in the Herb;Haixing Wang 等;《molecules》;第23卷;第1-13页 * |
| 半边莲的化学成分和药理作用研究进展;周斌 等;《中药材》;第36卷(第4期);第1.1小节 * |
| 半边莲药材HPLC指纹图谱的研究;周颖 等;《中成药》;第29卷(第12期);第2.1-2.3、2.5-2.6小节，表1、5-6 * |
| 离子液体辅助超声萃取-高效液相色谱同时测定半边莲中6种黄酮类化合物;邓永利 等;《食品科学》;第37卷(第20期);第37-41页 * |
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