CN114924014A - Method for establishing characteristic spectrum of belladonna herbal medicinal material and application thereof - Google Patents
Method for establishing characteristic spectrum of belladonna herbal medicinal material and application thereof Download PDFInfo
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- 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
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- G—PHYSICS
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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
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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
- G01N30/86—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
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Abstract
A method for establishing belladonna herb material characteristic map comprises the following steps: (1) preparing a reference substance solution; (2) preparing a test solution; (3) performing high performance liquid chromatography analysis; (4) and (3) measuring: respectively sucking 10 μ l of reference solution and sample solution, injecting into high performance liquid chromatograph, measuring with kaempferide-3-OGalactose- (6 → 1) rhamnose-7-O-glucoside as a characteristic map reference substance, kaempferide-3-OGalactose- (6 → 1) rhamnose-7-OA glucoside chromatographic peak is used as a reference peak, fingerprint spectrums of 16 batches of belladonna medicinal materials are determined and generated, and the common characteristic peak of the belladonna medicinal materials is calibrated by adopting relative retention time to obtain the characteristic spectrum of the belladonna medicinal materials; has good specificity, good reproducibility, high precision and good stability, and can effectively identifyThe authenticity of the belladonna grass medicinal material can reflect the quality of the belladonna grass medicinal material, the quality evaluation system of the belladonna grass medicinal material is perfected, and the method has remarkable social and economic benefits.
Description
Technical Field
The invention relates to the technical field of characteristic maps of traditional Chinese medicinal materials, in particular to a method for establishing a characteristic map of belladonna herbal materials and application thereof.
Background
Belladonna is dry whole plant of belladonna Atropa belladonna L. Digging in the period from blooming to fruiting, removing thick stem and silt, cutting and drying. Belladonna mainly contains hyoscyamine alkaloids including hyoscyamine, scopolamine, anisodamine, etc. The alkaloid has spasmolytic, analgesic, and tranquilizing effects, and has high medicinal value, but the components are also present in plants such as Datura and Hyoscyamus, and can be mixed during production of belladonna related extract and preparation product; under the item of belladonna grass in 2020 edition of Chinese pharmacopoeia, in the quality control indexes of belladonna grass, alkaloid is used as a detection object in chemical color development identification, thin-layer chromatography identification and content measurement, so that the problem of repeated evaluation exists; the belladonna grass contains coumarins, glycosides and flavonoids besides tropane alkaloids, the coumarins such as scopoletin, the pharmacological action shows that the compounds have the effects of resisting tumor, resisting inflammation, relieving pain, protecting liver, reducing blood pressure and the like, the glycosides such as scopoletin, quercetin-3-O-galactose- (6 → 1) rhamnose-7-O-glucoside, kaempferide-3-O-galactose- (6 → 1) rhamnose-7-O-glucoside and the like, the flavonoids such as rutin have pharmacological activity of relieving pain, resisting inflammation and the like, in addition, the common characteristics of the non-alkaloid components are that the contents in belladonna leaves and flowers are high, and the contents in belladonna roots, stems and fruits are low or none, while the belladonna grass is a full herbal medicine, and during the harvest period of belladonna, in order to simply improve the alkaloid content and avoid disqualification of the hyoscyami alkaloid content, more seeds are mature after the harvest period is prolonged, basically no flowers exist, and leaves only have a small amount of small leaves at the top, which do not meet the harvest requirement of belladonna grass; meanwhile, only the top leaflet is left, which causes the loss of other active ingredients except the alkaloid and causes resource waste.
At present, with the improvement of living standard and the increase of health demand, the medicine market is constantly flourishing and developing, the flourishing and the expansion of the belladonna extract market are driven, more and more areas for planting belladonna grass in China are developed gradually from main production areas of Shandong, Hunan and the like to places such as Heilongjiang, Shanxi, Xinjiang, Jiangxi, Hubei, Henan and the like, the belladonna grass is a foreign introduced plant, so that no 'road medicinal material' exists, the difference of geographical environment, soil, planting level and the like in China causes the greater difference of the belladonna grass in the aspects of plant morphology, growth cycle, sowing, mature season and the like, and the difference of chemical components representing plant characteristics in the belladonna grass possibly causes the difference of the chemical components, thereby affecting the uniform stability of the quality of the medicinal materials, and the research of characteristic components related to the belladonna grass is not developed in China.
In order to comprehensively and reasonably control the quality of the belladonna grass and reasonably utilize the belladonna grass, a characteristic component map of the belladonna grass is necessary to be established, the quality control deficiency of the belladonna grass is made up, the belladonna grass is more perfect and scientific, the healthy development of the belladonna grass industry is guided, and the technical support is provided for the safety, effectiveness and reliable quality of medicines.
Disclosure of Invention
In view of the above situation, in order to overcome the defects of the prior art, the invention aims to provide a method for establishing a belladonna herbal medicine characteristic map and application thereof, which can effectively solve the problem of insufficient quality control of belladonna herbs, can obviously distinguish the belladonna herbs from other medicinal materials by using the map, can ensure the plant integrity of the belladonna herbs, provides a reference basis for further control of the internal quality of the medicinal materials, improves and perfects the quality control level of the belladonna herbs, standardizes and ensures clinical medication.
In order to achieve the aim, the technical scheme of the invention is that the method for establishing the belladonna herbal medicine characteristic spectrum comprises the following steps:
(1) and preparing a reference substance solution: get eastScopoletin, chlorogenic acid, quercetin-3-O-galactose- (6 → 1) rhamnose-7-O-glucoside, quercetin-3-O-glucose- (6 → 1) rhamnose-7-O-glucoside, kaempferide-3-O-galactose- (6 → 1) rhamnose-7-O-glucoside, kaempferide-3-O-glucose- (6 → 1) rhamnose-7-O-glucoside, scopoletin, rutin and kaempferol-3-O-rhamnose galactoside contrast products are dissolved by methanol with the volume concentration of 50 percent and respectively prepared into the contrast products with the concentration of 10.48 mu g.mL -1 、10.12μg·mL -1 、10.03μg·mL -1 、10.56μg·mL -1 、20.31μg·mL -1 、10.12μg·mL -1 、10.03μg·mL -1 、10.62μg·mL -1 And 10.59. mu.g.mL -1 The solution is filtered by a microporous filter membrane to obtain a reference substance solution;
(2) and preparing a test solution: taking 2g of belladonna grass powder, adding 25mL of 50% methanol by volume concentration, carrying out ultrasonic treatment for 30min under the conditions of 400W of power and 60KHz of frequency, cooling to room temperature, complementing the loss weight with 50% methanol by volume concentration, shaking up, filtering to obtain filtrate, and then filtering with a microporous filter membrane to obtain a test solution;
(3) and high performance liquid chromatography analysis: the chromatographic conditions are as follows: ultimate XB-C 18 Chromatography column (4.6 mm. times.250 mm,5 μm); mobile phase: a is methanol, B is phosphoric acid aqueous solution with the mass concentration of 0.05 percent, and the gradient elution order is as follows: 0-5 min, 3-15% of A and 97-85% of B; 5-60 min, 15-60% of A and 85-40% of B; volume flow rate of 0.8mL/min -1 (ii) a The detection wavelength is 344 nm; the column temperature is 35 ℃; the sample volume is 10 mu L;
(4) and (3) measuring: respectively sucking 10 μ l of reference solution and test solution, injecting into high performance liquid chromatograph, measuring, using kaempferide-3-O-galactose- (6 → 1) rhamnose-7-O-glucoside as characteristic spectrum reference substance, using kaempferide-3-O-galactose- (6 → 1) rhamnose-7-O-glucoside chromatographic peak as reference substance peak, measuring to generate fingerprint of 16 batches of belladonna grass medicinal materials, and calibrating common characteristic peak of belladonna grass by relative retention time to obtain belladonna herb characteristic spectrum.
The characteristic map of the belladonna herbal medicine material has 17 common characteristic peaks, wherein a peak No. 8 is kaempferide-3-O-galactose- (6 → 1) rhamnose-7-O-glucoside, a peak corresponding to a reference peak is an S peak, and the relative retention time of the 17 common characteristic peaks is respectively as follows: peak 1: 0.44 plus or minus 0.05; peak 2: 0.62 plus or minus 0.05; peak 3: 0.70 plus or minus 0.05; peak 4: 0.77 plus or minus 0.05; peak 5: 0.80 plus or minus 0.05; peak 6: 0.91 plus or minus 0.05; peak 7: 0.92 plus or minus 0.05; peak 8 (S): 1.00 plus or minus 0.05; peak 9: 1.03 plus or minus 0.05; peak 10: 1.09 +/-0.05; peak 11: 1.46 plus or minus 0.05; peak 12: 1.58 plus or minus 0.05; peak 13: 1.61 plus or minus 0.05; peak 14: 1.64 plus or minus 0.05; peak 15: 1.71 plus or minus 0.05; peak 16: 1.81 plus or minus 0.05; peak 17: 2.02 +/-0.05.
The belladonna herb characteristic spectrum established by the method is applied to the quality control of belladonna herb.
The method for establishing the belladonna herbal medicine material characteristic map and the characteristic map have good specificity, good reproducibility, high precision and good stability, can effectively identify the true and false of the belladonna herbal medicine material, can reflect the quality of the belladonna herbal medicine material, improve the quality evaluation system of the belladonna herbal medicine material, provide a reference basis for further improving the belladonna herbal medicine material standard, standardize and ensure clinical medication, and have remarkable social and economic benefits.
Drawings
FIG. 1 is the HPLC characteristic spectrum of belladonna herb of the present invention.
FIG. 2 is a diagram showing the common mode patterns of HPLC characteristic spectra of the Belladonnae herb of the present invention (wherein, 3-scopoletin, 4-chlorogenic acid, 6-quercetin-3-O-galactose- (6 → 1) rhamnose-7-O-glucoside, 7-quercetin-3-O-glucose- (6 → 1) rhamnose-7-O-glucoside, 8-kaempferide-3-O-galactose- (6 → 1) rhamnose-7-O-glucoside, 9-kaempferide-3-O-glucose- (6 → 1) rhamnose-7-O-glucoside, 10-scopoletin, 11-rutin, 12-kaempferol-3-O-rhamnose galactoside).
FIG. 3 is a chromatogram of an HPLC mixed control of the present invention (wherein 3-scopoletin; 4-chlorogenic acid; 6-quercetin-3-O-galactose- (6 → 1) rhamnose-7-O-glucoside; 7-quercetin-3-O-glucose- (6 → 1) rhamnose-7-O-glucoside; 8-kaempferide-3-O-galactose- (6 → 1) rhamnose-7-O-glucoside; 9-kaempferide-3-O-glucose- (6 → 1) rhamnose-7-O-glucoside; 10-scopoletin; 11-rutin; 12-kaempferol-3-O-rhamnose galactoside).
FIG. 4 is a dendrogram of belladonna grass cluster analysis in different producing areas of the present invention.
FIG. 5 is a comparison graph of HPLC profiles of different parts of belladonna grass according to the present invention.
FIG. 6 is a comparison graph of HPLC profiles of belladonna grass in normal harvest period and belladonna grass in delayed harvest period in the present invention (wherein, A: belladonna grass in normal harvest period; B: belladonna grass (lack of leaves, flowers) in delayed harvest period).
Detailed Description
The following examples are provided to explain the present invention in detail.
The invention relates to a method for establishing a belladonna herbal medicine material characteristic map, which comprises the following steps of:
(1) and preparing a reference substance solution: dissolving scopoletin, chlorogenic acid, quercetin-3-O-galactose- (6 → 1) rhamnose-7-O-glucoside, quercetin-3-O-glucose- (6 → 1) rhamnose-7-O-glucoside, kaempferide-3-O-galactose- (6 → 1) rhamnose-7-O-glucoside, kaempferide-3-O-glucose- (6 → 1) rhamnose-7-O-glucoside, scopoletin, rutin and kaempferol-3-O-rhamnose galactoside with 50% methanol by volume concentration to respectively prepare 10.48 mu g/mL -1 、10.12μg·mL -1 、10.03μg·mL -1 、10.56μg·mL -1 、20.31μg·mL -1 、10.12μg·mL -1 、10.03μg·mL -1 、10.62μg·mL -1 And 10.59. mu.g.mL -1 Filtering the solution with a 0.22 μm microporous membrane to obtain a reference solution;
(2) and preparing a test solution: taking 2g of belladonna grass powder, adding 25mL of 50% methanol by volume concentration, carrying out ultrasonic treatment for 30min under the conditions of 400W of power and 60KHz of frequency, cooling to room temperature, complementing the loss weight with 50% methanol by volume concentration, shaking up, filtering to obtain filtrate, and filtering with a 0.22 mu m microporous filter membrane to obtain a test solution;
(3) and high performance liquid chromatography analysis: the chromatographic conditions are as follows: ultimate XB-C 18 Chromatography column (4.6 mm. times.250 mm,5 μm); mobile phase: a is methanol, B is phosphoric acid aqueous solution with mass concentration of 0.05 percent，
The gradient elution order was:
time (min) mobile phase A (%) mobile phase B (%)
0～5 3～15 97～85
5～60 15～60 85～40
Volume flow rate of 0.8mL/min -1 (ii) a The detection wavelength is 344 nm; the column temperature is 35 ℃; the sample volume is 10 mu L;
(4) and (3) measuring: respectively absorbing 10 mu l of reference substance solution and test solution, injecting into a high performance liquid chromatograph, measuring, selecting chromatographic peaks with good stability, strong absorption and obvious characteristics as characteristic spectrum common peaks in the test solution, taking chromatographic peaks with good separation degree, high content and no interference around as reference peaks, taking kaempferide-3-O-galactose- (6 → 1) rhamnose-7-O-glucoside as characteristic spectrum reference substances, taking kaempferide-3-O-galactose- (6 → 1) rhamnose-7-O-glucoside chromatographic peaks as reference substance peaks, measuring to generate fingerprint of 16 batches of belladonna medicinal materials, calibrating the common characteristic peaks of the belladonna by adopting relative retention time, wherein the peak 8 is kaempferide-3-O-galactose- (6 → 1) rhamnose-7-O-glucose peak, and the total peaks are 17, and the peak 8 is kaempferide-3-O-galactose- (6 → 1) A glycoside.
The characteristic map of the belladonna herbal medicine material obtained by the method for establishing the characteristic map of the belladonna herbal medicine material has 17 common characteristic peaks, the peak corresponding to the reference peak is the S peak, and the relative retention time of the 17 common characteristic peaks is respectively as follows: peak 1: 0.44 plus or minus 0.05; peak 2: 0.62 plus or minus 0.05; peak 3: 0.70 plus or minus 0.05; peak 4: 0.77 plus or minus 0.05; peak 5: 0.80 plus or minus 0.05; peak 6: 0.91 plus or minus 0.05; peak 7: 0.92 plus or minus 0.05; peak 8 (S): 1.00 plus or minus 0.05; peak 9: 1.03 plus or minus 0.05; peak 10: 1.09 +/-0.05; peak 11: 1.46 plus or minus 0.05; peak 12: 1.58 plus or minus 0.05; peak 13: 1.61 plus or minus 0.05; peak 14: 1.64 plus or minus 0.05; peak 15: 1.71 plus or minus 0.05; peak 16: 1.81 plus or minus 0.05; peak 17: 2.02 +/-0.05. The specific test data are as follows.
Instrument and material
1.1, medicinal materials
16 batches of belladonna herbal medicine samples are collected in different planting areas in China, and are detailed in table 1, 5 batches of medicinal materials with different parts and 2 batches of medicinal materials with different plant integrality are collected in a new county Chinese medicinal material planting demonstration base in Henan, and are detailed in table 2, and are identified as dry whole herbs of belladonna Atropa belladonna L.
TABLE 1 belladonna herbal material sample information
TABLE 2 belladonna grass sample information on different sites
1.2 instruments and reagents
LC-20AD type HPLC apparatus and DGC-20A type on-line degassing system, SIL-20A type automatic sample introduction system, CTO-20AC type column oven, SPD-M20A type diode array detector (Shimadzu corporation, Japan); ultimate XB-C 18 5 μm (4.6 × 250mm,5 μm) (Yuxu scientific Shanghai GmbH); DHG-9075A electric hot blast drying cabinet (Shanghai-Hengscientific instruments, Inc.); 114B swing type high-speed Chinese medicine grinder (Yongcalendar pharmaceutical machinery, Inc. of Ruian city); an XSE204 ten thousandth electronic balance (d 0.1mg) (mettler toledo, switzerland); XPE105 ten thousandth electronic balance (d 0.01mg) (mettler toledo switzerland); KQ-600VDE double-frequency numerical control ultrasonic cleaner (ultrasonic instruments, Inc. of Kunshan). Chromatographic methanol (Tianjin Si you Fine chemicals Co., Ltd., batch No.: 673550), water as water for injection, and other reagents were analytically pure. Chlorogenic acid (batch No. 110753-202006, content 99.1%), rutin (batch No. 100080-202002, content 98.8%), scopoletin (batch No. 110768-200504, content 100.0%), all purchased from China institute for testing food and drug; scopoletin (content 99.2%), quercetin-3-O-galactose- (6 → 1) rhamnose-7-O-glucoside (content of 99.1%), quercetin-3-O-glucose- (6 → 1) rhamnose-7-O-glucoside (content of 98.8%), kaempferide-3-O-galactose- (6 → 1) rhamnose-7-O-glucoside (content of 99.6%), kaempferide-3-O-glucose- (6 → 1) rhamnose-7-O-glucoside (content of 99.5%), kaempferol-3-O-rhamnose galactoside (content of 99.2%), all purchased from beijing middle-school quality inspection biotechnology limited.
Second, Experimental methods
2.1 examination of the kind of extraction solvent
The sampling amount of the medicinal materials is 2g, 30% methanol aqueous solution, 50% methanol aqueous solution and 70% methanol aqueous solution are respectively used as extraction solvents, and the type of the extraction solvents is investigated on the same batch of samples by adopting an ultrasonic extraction method.
The result shows (not shown in the figure), the 30% methanol aqueous solution has fewer chromatographic peaks, the extraction effect of the 50% methanol aqueous solution is equivalent to that of the 70% methanol aqueous solution, the peaks are more, the peak area has no obvious difference, and the 50% methanol aqueous solution is selected as the extraction solvent of the test sample in consideration of environmental protection factors.
2.2 examination of the amount of extraction solvent
The sampling amount of the medicinal materials is 2g, 15ml, 25ml and 35ml of 50% methanol aqueous solution are respectively used as extraction solvents, and the ultrasonic extraction method is adopted to carry out the investigation of the dosage of the extraction solvents on the same batch of samples.
The results show (not shown in the figure), 15ml of 50% methanol water solution can not completely submerge the medicinal materials, after 35ml of 50% methanol water solution extraction samples are injected, the component separation peak with small content is not obvious, and the sample characteristic map comparison is influenced, therefore, 50% methanol water solution is selected as the sample extraction solvent.
2.3 ultrasound Power investigation
The sampling amount of the medicinal materials is 2g, 50% methanol aqueous solution is used as an extraction solvent, an ultrasonic extraction method is adopted, the frequency is 60KHz, the power is respectively selected from 200W, 400W and 600W, and ultrasonic power investigation is carried out on the same batch of test samples.
The result shows (not shown in the figure), after the sample is extracted by 200W power ultrasonic wave and 50% methanol aqueous solution, the component peak with small content is not obvious, the ultrasonic effect of 400W and 600W is not obvious, the sample extraction rate is higher, the peak is obvious, the energy is saved, and 400W is selected as the ultrasonic power.
2.4 ultrasound time survey
The sampling amount of the medicinal materials is 2g, 50% methanol water solution is used as an extraction solvent, the ultrasonic power is 400W, the frequency is 60Khz, and the ultrasonic time is respectively selected from 15min, 30min and 45min, and the ultrasonic power of the same batch of samples is investigated.
The result shows (not shown in the figure), after the sample is extracted by ultrasonic for 15 minutes and is injected by 50 percent methanol aqueous solution, the peak separation of the components with small content is not obvious, the ultrasonic is carried out for 30 and 45 minutes, the peak areas of the samples have no obvious difference, the extraction rate of the samples is higher, the peak separation is obvious, the energy is saved, and 30min is selected as the ultrasonic time.
2.5 selection of chromatography columns
Chromatographic columns of different sources and brands have certain differences, chromatographic columns of the same source have different degrees of differences, column efficiency (theoretical plate number), retention time, separation degree and symmetry factor are different, and under the condition that chromatographic conditions are the same in the experiment, C18 two high-performance liquid chromatographic columns of different manufacturers and the same specification are investigated.
The column specifications were as follows:
(1)、Ultimate XB-C 18 (250 mm. times.4.6 mm,5 μm) batch No. 2101.105.
(2) Agilent ZORBAX SB-C18(250 mm. times.4.6 mm,5 μm) lot B11248.
The results show (not shown) that, in Ultimate XB-C 18 In a chromatographic column (250mm multiplied by 4.6mm,5 μm), the separation degree of each peak is better and the base line is smoother.
2.6 selection of detection wavelength
Literature research shows that main ultraviolet absorption bands exist in the flavone and coumarin components in a 200-400 nm region, a diode array detector is adopted to detect the full wavelength of a sample, and the resolution, the peak area, the retention time and the symmetry factor of chromatographic peaks obtained when the wavelength is 344nm are better, the number of the obtained peaks is more, and the base line is more stable through comparison, so that the research selects to be used for measurement at the wavelength of 344nm (not shown in the figure).
2.7 selection of mobile phase System
And (3) adopting gradient elution modes respectively to investigate the peak appearance condition of the sample:
(1) gradient elution (0-5 min, 3-25% A; 5-100 min, 25-60% A);
(2) gradient elution (0-5 min, 3-15% A; 5-100 min, 15-60% A);
(3) and gradient elution (3-10% A in 0-5 min; 10-60% A in 5-100 min).
The result shows (not shown in the figure), the 1 st gradient mode has partial peak area with low separation degree and no peak separation condition in the sample, the 3 rd condition has the sample with incomplete peak after the detection time of 100 minutes, the analysis time is longer, the 2 nd gradient mode has moderate peak output time, all peaks can be output after 60min, the separation degree of each peak is moderate, the base line is stable, so the 2 nd gradient elution mode is selected, and the elution time is positioned for 60 min.
2.8 selection of column temperature
The column temperature was determined to be 35 ℃ because the peak separation effect at 35 ℃ was the best when the column temperatures were 30 ℃, 35 ℃ and 40 ℃ respectively, and the column temperature was determined to be 35 ℃ under otherwise identical chromatographic conditions (not shown).
2.9 selection of flow Rate
The influence of different flow rates (1mL/min, 0.8mL/min and 0.6mL/min) on the separation effect, retention time, peak area and symmetry factor of the belladonna grass chromatographic peak is respectively considered, and the result shows that the obtained spectrum separation degree is better when the flow rate is 0.8mL/min, so that the flow rate adopted in the research is 0.8mL/min (not shown in the figure).
2.10 determining the chromatographic condition of the fingerprint
According to the results of the above investigation, the chromatographic conditions were: an Ultimate XB-C18 chromatography column (4.6 mm. times.250 mm,5 μm); mobile phase: a is methanol, B is 0.05 percent phosphoric acid water solution, and gradient elution is carried out (0-5 min, 3-15 percent A; 5-60 min, 15-60 percent A); volume flow rate of 0.8mL/min -1 (ii) a The detection wavelength is 344 nm; the column temperature is 35 ℃; the sample size was 10. mu.L.
2.11 selection of reference peaks
In the characteristic map established in the experiment, the chromatographic peaks of kaempferide-3-O-galactose- (6 → 1) rhamnose-7-O-glucoside (peak No. 8) are well separated, have larger peak areas and moderate retention time and are shared by all samples, so the kaempferide-3-O-galactose- (6 → 1) rhamnose-7-O-glucoside is selected as a reference peak.
Third, preparation method
3.1, preparation of a reference solution: dissolving scopoletin, chlorogenic acid, quercetin-3-O-galactose- (6 → 1) rhamnose-7-O-glucoside, quercetin-3-O-glucose- (6 → 1) rhamnose-7-O-glucoside, kaempferide-3-O-galactose- (6 → 1) rhamnose-7-O-glucoside, kaempferide-3-O-glucose- (6 → 1) rhamnose-7-O-glucoside, scopoletin, rutin and kaempferol-3-O-rhamnose galactoside reference substances with the volume concentration of 50% methanol, and respectively preparing the reference substances with the concentration of 10.48 mu g/mL -1 、10.12μg·mL -1 、10.03μg·mL -1 、10.56μg·mL -1 、20.31μg·mL -1 、10.12μg·mL -1 、10.03μg·mL -1 、10.62μg·mL -1 And 10.59. mu.g.mL -1 The solution of (2) is filtered by a 0.22 mu m microporous membrane to obtain a reference solution.
3.2, preparation of a test solution: weighing 2g of belladonna grass powder, precisely weighing, precisely adding 25mL of 50% methanol by volume concentration, weighing, ultrasonically treating for 30min (power 400W, frequency 60KHz), cooling, supplementing the lost weight with 50% methanol, shaking, filtering, collecting the filtrate, and filtering with 0.22 μm microporous membrane.
Fourth, characteristic atlas methodology investigation
4.1 precision test
Precisely weighing 1 part of 2.0g of dry powder (S14) of a Shandong tobacco Tai muyupin Gaoling belladonna medicinal material, preparing the same test sample according to a method for preparing a test sample solution of item 3.2, continuously injecting samples for 6 times under a chromatographic condition of item 2.10, recording a spectrum, taking a peak No. 8 as a reference peak, and respectively ensuring that the relative retention time of 17 common peaks and the RSD of the relative peak area are less than 1.3 percent and 3.5 percent, which indicates that the precision of an instrument used in the research is good, and the results are shown in tables 3-1, 3-2, 4-1 and 4-2.
TABLE 3-1 consensus peak relative retention time data
TABLE 3-2 consensus peak relative retention time data
TABLE 4-1 data of relative peak area of common peaks
TABLE 4-2 data of relative peak area of common peaks
4.2 repeatability test
Precisely weighing 6 parts of dry powder (S14) of a Shandong smoke stand, muping GaoLing and belladonna herb medicinal material in parallel, each 2.0g of the dry powder, preparing a test sample according to a method for preparing a test sample solution of item 3.2, measuring according to chromatographic conditions of item 2.10, recording a map, taking a peak 8 as a reference peak, and respectively ensuring that the relative retention time of 17 common peaks and the RSD of the relative peak area are less than 2.0 percent and 4.5 percent, which shows that the experimental repeatability is good, and the results are shown in tables 5-1, 5-2, 6-1 and 6-2.
TABLE 5-1 consensus peak relative retention time data
TABLE 5-2 consensus peak relative retention time data
TABLE 6-1 consensus peak relative peak area data
TABLE 6-2 data of relative peak area of common peaks
4.3 stability test
Precisely weighing 1 part of 2.0g of dry powder (S14) of a Shandong tobacco Tai muyupin Gaoling town belladonna medicinal material, preparing a test sample according to a method for preparing a test sample solution of '3.2', measuring the test sample solution under chromatographic conditions of '2.10' at 0h, 2h, 4h, 8h, 12h and 24h respectively, recording a spectrum, taking a No. 8 peak as a reference peak, and respectively ensuring that the relative retention time of 17 common peaks and the RSD of the relative peak area are less than 1.3 percent and 3.8 percent, thereby indicating that the stability of the experiment is good, and the results are shown in tables 7-1, 7-2, 8-1 and 8-2.
TABLE 7-1 consensus peak relative retention time data
TABLE 7-2 common Peak relative Retention time data
TABLE 8-1 data on relative peak area of common peaks
TABLE 8-2 data on relative peak area of common peaks
Fifthly, establishing and evaluating belladonna grass characteristic spectrum
5.1 blank examination of the Mobile phase
Under the chromatographic condition of '2.10', a single-run program inspects a chromatogram map for 60min without sample injection, and does not detect any chromatographic peak (not shown in the figure), and the result shows that the flow has no influence on the detection of the characteristic map.
5.2 examination of influence of blank solvent
The chromatogram of the blank solvent (50% methanol aqueous solution) was recorded under the chromatographic condition of "2.10" for 60min, and no chromatographic peak (not shown) was detected, thus indicating that the blank solvent has no influence on the detection of the characteristic spectrum.
5.3, collecting characteristic spectrums of belladonna grass medicinal materials in all producing areas
Selecting 16 batches of belladonna herbal material samples from different production places, preparing test solution of each batch of samples according to the method under the item 3.2, performing determination according to the chromatographic condition under the item 2.10, and recording the chromatogram of each batch of samples. And (3) superposing all chromatograms by adopting software carried by an instrument, selecting common chromatographic peaks of all batches as characteristic peaks according to chromatographic peak distribution in the superposed chromatogram, and establishing a unified processing method to process the chromatograms of all batches of samples so as to ensure that the characteristic peaks of all batches can be integrated.
And then introducing the chromatogram into a traditional Chinese medicine chromatogram fingerprint similarity evaluation system (2012 edition) for characteristic spectrum analysis, taking the chromatogram of any sample as a reference spectrum, setting the time window width to be 0.1min by adopting a median method, performing multi-point correction and chromatographic peak matching, and obtaining a superposition characteristic spectrum and a comparison characteristic spectrum (R) of belladonna grass medicinal materials after full-peak matching, wherein the results show that 16 batches of belladonna grass medicinal materials have 17 common peaks and are sequentially marked as No. 1-17 peaks according to peak output time. See fig. 1 and 2 in detail.
5.4 identification of characteristic peaks
In order to examine whether the characteristic map is representative or not and whether the specificity of the contained components can be represented or not, the mixed reference substance solution under the item of 3.1 is taken, sample injection and determination are carried out according to the chromatographic condition under the item of 2.10, and the chromatogram is recorded. Comparing the obtained chromatogram with the chromatogram of belladonna grass medicinal material under item "5.3", comparing with reference substances, as shown in FIG. 3, determining that the chromatogram peaks with relative retention times of 0.70 + -0.05, 0.77 + -0.05, 0.91 + -0.05, 0.92 + -0.05, 1.00 + -0.05, 1.03 + -0.05, 1.09 + -0.05, 1.46 + -0.05, 1.58 + -0.05 correspond to scoposide (peak 3), chlorogenic acid (peak 4), quercetin-3-O-galactose- (6 → 1) rhamnose-7-O-glucoside (peak 6), quercetin-3-O-glucose- (6 → 1) rhamnose-7-O-glucoside (peak 7), kaempferide-3-O-galactose- (6 → 1) rhamnose-7-O-glucoside (peak 8), and kaempferide-3-O-glucose- (6-O-1) rhamnose-7-O-glucoside (peak 7-O-1) respectively -glucoside (peak 9), scopoletin (peak 10), rutin (peak 11), kaempferol-3-O-rhamnoside galactoside (peak 12).
The identified characteristic peaks are all effective components in the belladonna herbal medicine, so that the established characteristic spectrum is identified to be representative, and the quality control can be carried out on the belladonna herbal medicine. Identifying characteristic peaks by using a reference substance, and selecting a peak No. 8 (kaempferide-3-O-galactose- (6 → 1) rhamnose-7-O-glucoside) as a main effective component as a reference peak (S); the relative retention times (Table 9-1, Table 9-2) and relative peak areas (Table 10-1, Table 10-2) of the 17 common peaks and the S peak were calculated.
TABLE 9-1 relative retention time of common peaks of belladonna herbs from different origins
TABLE 9-2 relative retention time of common peaks of belladonna herbs from different origins
TABLE 10-1 relative peak area of common peaks of belladonna herbs from different origins
TABLE 10-2 relative peak areas of common peaks of belladonna herbs from different origins
The results show that the relative retention time RSD of the characteristic peak of each batch of samples is less than 0.25 percent, which indicates that the peak emergence time of the characteristic peak is relatively stable; the relative peak area RSD result shows that the peak area difference of the characteristic peaks of each batch is large, and the difference of belladonna herb medicinal materials of different provinces and producing areas in China is reflected.
5.5 Cluster analysis
In order to further compare the difference of the belladonna grass medicinal materials in different production areas across the country, 17 common peak areas in the belladonna grass characteristic map are taken as variables, SPSS 22.0 software is introduced, a group-to-group connection method is adopted, the squared Euclidean distance is taken as a measure, Q-type clustering analysis is carried out on the belladonna grass medicinal materials, and the result is shown in figure 4. According to the clustering result, 16 batches of belladonna medicinal materials are divided into 4 categories when the square European distance of 16 batches of samples is 5-10, the Li Jixiang in the fixed county of Henan, Yan mountain river village in Henan, New Tianjicun in Hunan, Maurixiang in the New county of Hunan, New Yongchang town in Hunan, Zhi City district town in Hunan, Chenghui county in Henan New county, Shaanxi Xunyang county, West Hua county in Zhou, and belladonna produced in Zhengyang county in Ma shop; the Ganxian in Jiangxi province, the top-yield belladonna grass in Jiangxi province gather as one kind; belladonna grass produced by Kuerle in Xinjiang, Heilongjiang peony river, etc.; belladonna herb from Tokyo mountain Baobai, Shandong tobacco station Muping GaoLing Zhen is a group. Therefore, the chemical component contents of the medicinal materials in different batches are obviously different due to the influence of environments such as geographical positions and the like.
5.6 comparison of the specific spectra of different parts of belladonna grass
In order to ensure reasonable harvesting time, the invention analyzes and compares the characteristic spectrums of different parts of the belladonna grass, and the result is shown in figure 5, the result shows that the characteristic peak areas in roots, fruits and stems are very small, some characteristic peaks are almost not, the characteristic peak areas in leaves and flowers are relatively large, and the characteristic peaks are complete, which indicates that after the harvesting time is delayed, the characteristic peaks in the characteristic spectrums can cause the disappearance of the related characteristic peaks of the whole characteristic spectrums of the belladonna grass due to the great reduction and deletion of the leaves and flowers, so that other effective components except alkaloid in the belladonna grass are lost, as shown in figure 6. By analyzing the characteristic spectrum of the belladonna grass, the integrity of the whole belladonna grass plant can be effectively judged, and the quality control of the belladonna grass can be enhanced.
5.7, conclusion
The chemical components of the traditional Chinese medicine are complex and various, the effective components are difficult to be determined one by one, and how to control the quality of the medicinal materials on the premise of incomplete and definite chemical components is very necessary. 17 common peaks are extracted from the belladonna HPLC characteristic map established in the research, the relative peak areas of other common peaks are calculated by taking a peak No. 8 (kaempferide-3-O-galactose- (6 → 1) rhamnose-7-O-glucoside) as a reference peak, and the results are subjected to cluster analysis. By optimizing the pretreatment method and the instrument conditions, under the conditions, the chemical components of the belladonna grass are well separated within 60min, and a more stable and effective method is added for the quality control of the belladonna grass. In data analysis, the characteristic map mainly emphasizes characteristic information of characteristic peaks, clustering analysis is performed by using the characteristic peak information, and the clustering analysis method is applied to quality standards and is easier to operate and easy to popularize.
Therefore, the method for establishing the belladonna herbal medicinal material characteristic spectrum and the characteristic spectrum are favorable for comprehensively monitoring the quality of the belladonna herbal medicinal material, and compared with the prior art, the method has the following beneficial effects:
(1) the invention has the characteristics of good specificity, good reproducibility, high precision and good stability,
(2) the belladonna grass HPLC characteristic spectrum of the invention has 17 common characteristic peaks, and the truth of the belladonna grass medicinal material can be effectively identified by comparing the existence of the common peaks in the characteristic spectrum, the quality of the belladonna grass medicinal material can be reflected, and the quality evaluation system of the belladonna grass medicinal material is improved;
(3) the belladonna grass feature map provided by the invention can be used in the quality control of belladonna grass as a standard feature map after being verified by multiple batches of samples, is used for integrally evaluating and controlling the quality of the belladonna grass medicinal material, avoids the one-sidedness caused by judging the integral quality of the belladonna grass by means of a single chemical component, guides the reasonable harvesting time section of the belladonna grass, avoids the phenomena that the belladonna grass pursues the alkaloid content and ignores the content of other effective components, causes too late harvesting period, excessive loss of leaves and flowers and the like of medicinal materials, provides a reference basis for further improving the standard of the belladonna grass, standardizes and ensures clinical medication.
Claims (5)
1. A method for establishing a belladonna herbal material characteristic map is characterized by comprising the following steps:
(1) and preparing a reference substance solution: dissolving scopoletin, chlorogenic acid, quercetin-3-O-galactose- (6 → 1) rhamnose-7-O-glucoside, quercetin-3-O-glucose- (6 → 1) rhamnose-7-O-glucoside, kaempferide-3-O-galactose- (6 → 1) rhamnose-7-O-glucoside, kaempferide-3-O-glucose- (6 → 1) rhamnose-7-O-glucoside, scopoletin, rutin and kaempferol-3-O-rhamnose galactoside reference substances with the volume concentration of 50% methanol, and respectively preparing the reference substances with the concentration of 10.48 mu g/mL -1 、10.12μg·mL -1 、10.03μg·mL -1 、10.56μg·mL -1 、20.31μg·mL -1 、10.12μg·mL -1 、10.03μg·mL -1 、10.62μg·mL -1 And 10.59. mu.g.mL -1 The solution is filtered by a microporous filter membrane to obtain a reference substance solution;
(2) and preparing a test solution: taking 2g of belladonna grass powder, adding 25mL of 50% methanol by volume concentration, carrying out ultrasonic treatment for 30min under the conditions of 400W power and 60KHz frequency, cooling to room temperature, complementing the volume concentration of 50% methanol for loss, shaking up, filtering to obtain filtrate, and then passing through a microporous filter membrane to obtain a test solution;
(3) and high performance liquid chromatography analysis: the chromatographic conditions are as follows: ultimate XB-C 18 A chromatographic column; mobile phase: a is methanol, B is phosphoric acid aqueous solution with the mass concentration of 0.05 percent, and the gradient elution sequence is as follows: 0-5 min, 3-15% of A and 97-85% of B; 5-60 min, 15-60% of A and 85-40% of B; volume flow rate 0.8mL/min -1 (ii) a The detection wavelength is 344 nm; the column temperature is 35 ℃; the sample volume is 10 mu L;
(4) and (3) measuring: respectively sucking 10 μ l of reference solution and sample solution, injecting into high performance liquid chromatograph, measuring with kaempferide-3-OGalactose- (6 → 1) rhamnose-7-OThe glucoside is a characteristic map reference substance and is kaempferide-3-OGalactose- (6 → 1) rhamnose-7-OAnd (4) taking a glucoside chromatographic peak as a reference peak, measuring and generating fingerprint spectrums of 16 batches of belladonna herb medicinal materials, and calibrating common characteristic peaks of the belladonna herb by adopting relative retention time to obtain the characteristic spectrums of the belladonna herb medicinal materials.
2. The method for establishing the belladonna herb feature map of claim 1, wherein the microfiltration membrane used in the steps (1) and (2) is a microfiltration membrane with a pore size of 0.22 μm.
3. The method for establishing the belladonna herbal material feature map as claimed in claim 1, wherein said Ultimate XB-C in step (3) 18 The size of the chromatographic column is 4.6mm multiplied by 250mm,5 μm.
4. The method for establishing the belladonna herbal material feature map as claimed in claim 1, wherein the belladonna herbal material feature map has 17 common feature peaks, wherein peak 8 is kaempferide-3-OGalactose- (6 → 1) rhamnose-7-O-glucoside, the relative retention times of the 17 common characteristic peaks are respectively: peak 1: 0.44 plus or minus 0.05; peak 2: 0.62 plus or minus 0.05; peak(s)3: 0.70 plus or minus 0.05; peak 4: 0.77 plus or minus 0.05; peak 5: 0.80 plus or minus 0.05; peak 6: 0.91 plus or minus 0.05; peak 7: 0.92 plus or minus 0.05; peak 8 (S): 1.00 plus or minus 0.05; peak 9: 1.03 +/-0.05; peak 10: 1.09 +/-0.05; peak 11: 1.46 plus or minus 0.05; peak 12: 1.58 plus or minus 0.05; peak 13: 1.61 plus or minus 0.05; peak 14: 1.64 plus or minus 0.05; peak 15: 1.71 plus or minus 0.05; peak 16: 1.81 plus or minus 0.05; peak 17: 2.02 +/-0.05.
5. Use of a belladonna herb profile as established in any one of claims 1 to 3 in the quality control of belladonna herbs.
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Application publication date: 20220819 |