CN113176366A - Evaluation method of secondary refined polysaccharide and flavonoid components based on spectral efficiency relationship - Google Patents
Evaluation method of secondary refined polysaccharide and flavonoid components based on spectral efficiency relationship Download PDFInfo
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Abstract
The invention discloses a method for evaluating polysaccharide and flavonoid components of a second-extract prescription based on a spectral-effect relationship, which belongs to the technical field of traditional Chinese medicine quality standard detection. The evaluation method provided by the invention is accurate and high in sensitivity, provides a basis for the basic and quality evaluation of the glucose-reducing substances of the second-extract prescription, and also provides a reference for the further product development of the second-extract prescription.
Description
Technical Field
The invention relates to the technical field of traditional Chinese medicine quality standard detection, in particular to a method for evaluating polysaccharide and flavonoid components in a second formula based on a spectrum effect relationship.
Background
Diabetes is a disease of endocrine metabolism caused by insulin deficiency or reduced biological effects. In recent years, with the increasing quality of life, the change of eating style and structure, life rhythm and life style, and other factors, the incidence of diabetes is rapidly increasing, and diabetes may become the third disease in the world. Diabetic patients also develop various complications due to disorders in sugar, lipid and protein metabolism, wherein peripheral vascular disease, nephropathy and eye diseases appear more frequently in type II diabetic patients.
The traditional Chinese medicine spectrum effect relationship is a research method aiming at the research of the drug effect substance basis and the action mechanism thereof of the traditional Chinese medicine. It combines the traditional Chinese medicine fingerprint with the effect and the effect system, thereby screening the active ingredients and clarifying the material basis of the traditional Chinese medicine for playing the drug effect. At present, for the quality control of the polysaccharide and flavonoid components with two essences, a standard and a method which are obviously related to the efficacy of the hypoglycemic drug and are specifically quantized are still lacking. Therefore, the quality of the two effective prescriptions with different compatible medicines is difficult to be accurately and effectively evaluated.
Disclosure of Invention
In view of the above, it is necessary to provide a method for evaluating di-essential polysaccharides and flavonoids based on the spectrum efficiency relationship.
A method for evaluating secondary refined polysaccharide and flavonoid components based on a spectrum-effect relationship comprises the following steps:
(1) preparing a second refined concentrated solution; (2) preparing a second-extract polysaccharide test solution; (3) preparing a second-extract formula flavone test solution; (4) preparing a standard solution; (5) establishing HPLC fingerprint of effective components of polysaccharide and flavonoid in the second refined formula by using different compatible medicines, determining characteristic peaks and extracting common peak data; (6) evaluating the hypoglycemic activity of different compatible medicines to the extracts of the two essences through pharmacodynamic experiments; (7) substituting the characteristic peak data of the fingerprint and the data of the hypoglycemic drug effect activity into the constructed spectrum-effect relationship by utilizing a partial least square method and related analysis, and screening out a common peak related to pharmacological activity in chromatographic peaks of polysaccharides and flavonoids serving as two-component hypoglycemic active ingredients;
preferably, the preparation of the second-extract concentrate in the step (1) comprises the following steps: weighing rhizoma Polygonati and fructus Lycii at a predetermined ratio, extracting with hot water under reflux for 3 times (each time for 1 hr) at a ratio of 1:10, filtering, mixing the filtrates, and concentrating to obtain concentrated solution.
Preferably, the preparation of the second-square polysaccharide test solution in the step (2) comprises the following steps:
(2-1) preparation of crude polysaccharide: precipitating the second-refined concentrated solution with alcohol, standing overnight, filtering, rinsing the precipitate with 80% ethanol for 2-3 times, and drying to obtain crude polysaccharide;
(2-2) preparation of Fine polysaccharide: dissolving crude polysaccharide in water, adding chloroform-n-butanol (4:1) mixed solution, centrifuging to obtain supernatant, adding 80% anhydrous ethanol into the supernatant, standing overnight, filtering, washing the obtained precipitate with 95% ethanol, anhydrous ethanol, acetone and diethyl ether, and oven drying to obtain refined polysaccharide;
(2-3) derivatization of polysaccharides: dissolving refined polysaccharide with trifluoroacetic acid, sealing, hydrolyzing in 100 deg.C oven for 8 hr, blow-drying, dissolving residue with methanol, blow-drying, repeating for 3 times, dissolving with water to obtain polysaccharide hydrolysate, adding 1-phenyl-3-methyl-5-pyrazolone methanol solution and sodium hydroxide solution into polysaccharide hydrolysate, mixing, heating in 70 deg.C water bath for 30min, taking out, cooling to room temperature, adding hydrochloric acid solution to adjust pH to neutrality, mixing, extracting with chloroform for 3 times, collecting water layer, filtering with needle cylinder type microporous membrane, and collecting filtrate as test solution of polysaccharide.
Preferably, the preparation method of the second-extract-formula flavone test solution in the step (3) comprises the following steps: degreasing the two concentrated solutions with petroleum ether, adding 80% ethanol into the residue, performing ultrasonic treatment twice at 60 deg.C for 30min each time, filtering, mixing the filtrates, concentrating under reduced pressure, removing water soluble impurities, eluting with 50% ethanol, collecting ethanol eluate, and filtering with a syringe microporous membrane to obtain filtrate as sample solution of flavone.
Preferably, the HPLC column conditions in step (5) are as follows:
(5-1) HPLC column conditions of the second-refined polysaccharide are as follows: an Agilent XDB-C18 (4.6X 250mm, 5 μm) column was used, and the mobile phase was (A) phosphate buffer (pH 6.8): (B) acetonitrile 84: 16, isocratic elution; the flow rate is 1 mL/min-1And the detection wavelength is as follows: 250nm, column temperature 30 deg.C, and sample injection amount of 10 μ L.
(5-2) HPLC chromatographic column conditions of difang flavone are as follows: an Agilent XDB-C18 (4.6X 250mm, 5 μm) chromatographic column was used, the mobile phase was (A) acetonitrile: water: glacial acetic acid 5: 94.5: 0.5 and (B) acetonitrile: water: glacial acetic acid 70: 29.5: 0.5; gradient elution conditions: 0-14 min: 100% -86% (A); 14-22 min: 86% -84% (A); 22-40 min: 84% -70% (A); 40-60 min: 70% -100% (A); the flow rate is 1 mL/min-1And the detection wavelength is as follows: 254nm, the column temperature is 30 ℃, and the sample injection amount is 10 mu L.
Preferably, in the step (5), the polysaccharide components in the second extract side are calibrated to have 10 common peaks accounting for more than 90% of the total area of chromatographic peaks, and mannose and glucose are identified according to retention time by comparing with a standard reference substance; the two-refined flavonoid component has 18 common peaks which account for more than 90% of the total area of chromatographic peaks, and rutin is identified according to the retention time of the two-refined flavonoid component by comparing with a standard reference substance.
Preferably, in the step (6), the medicine pair with the best blood sugar reducing activity of the extracts of the two essences is medlar-rhizoma polygonati with the mass ratio of 1: 1.
Preferably, in the step (7), the correlation analysis result shows that the characteristic peaks 1, 7, 9 and 10 of the polysaccharide components in the second essential formula are closely related to the hypoglycemic effect, the PLS result shows that the characteristic peaks 9 and 10 have large contribution degree to the hypoglycemic effect, and the two kinds of data are combined for screening, which shows that the characteristic peaks 9 and 10 of the polysaccharide components in the second essential formula are effective components playing the hypoglycemic effect; the results of correlation analysis methods show that peaks 1, 9, 10, 11, 12, 14, 16, 17 and 18 of the flavonoids in the second-extract are closely related to the drug effect of reducing blood sugar, the PLS results show that peaks 1, 9, 10, 11, 12, 14, 16, 17 and 18 of the flavonoids in the second-extract have large contribution degree to reducing blood sugar, and the two kinds of data are combined for screening, which shows that peaks 1, 9, 10, 11, 12, 14, 16, 17 and 18 of the flavonoids in the second-extract are effective components playing a role in reducing blood sugar.
By adopting the technical scheme, the invention has the beneficial effects that: the invention is based on the fingerprint of different compatible medicines to the second-extract prescription, takes the drug effect of the second-extract prescription for resisting II type diabetes as the main content, detects the content of polysaccharide and flavonoid hypoglycemic active ingredients of the second-extract prescription through different compatible medicines, constructs a spectrum-effect relationship by adopting a Correlation Analysis (CA) and a partial least squares discriminant analysis (PLS) in cooperation with the hypoglycemic drug effect, and screens out a common peak related to pharmacological activity in chromatographic peaks of polysaccharide and flavonoid active ingredients of the second-extract prescription. The evaluation method provided by the invention is accurate and high in sensitivity, provides a basis for the basic and quality evaluation of the glucose-reducing substances of the second-extract prescription, and also provides a reference for the further product development of the second-extract prescription.
Drawings
FIG. 1 shows fingerprint spectra of polysaccharides of fructus Lycii-rhizoma Polygonati obtained by different extraction methods.
FIG. 2 shows fingerprint chromatogram of polysaccharide from fructus Lycii-rhizoma Polygonati with different extraction methods.
FIG. 3 shows the fingerprint spectra of flavonoids of Lycium chinense Miller and Polygonatum sibiricum Red in different ratios.
FIG. 4 shows fingerprint spectra of different proportions of flavonoids of fructus Lycii-rhizoma Polygonati.
Fig. 5 is a chromatogram of a standard mixture of polysaccharide chemicals.
FIG. 6 is a chromatogram of rutin standard.
FIG. 7 change in fasting plasma glucose values of 1w before administration, 2w, 3w, 4w and 5w after administration in rats of each group.
FIG. 8 is an overview of polysaccharide model with different compatibility ratios of fructus Lycii and rhizoma Polygonati.
Figure 9 is a graph of VIP contribution of each characteristic peak to drug efficacy.
FIG. 10 is an overview of flavonoid component models with different compatibility ratios of fructus Lycii and rhizoma Polygonati.
Figure 11 is a graph of VIP contribution of each characteristic peak to drug efficacy.
Detailed Description
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following detailed description will be given of specific embodiments of the present invention.
The embodiment of the invention provides a method for evaluating secondary refined polysaccharide and flavonoid components based on a spectral efficiency relationship, which comprises the following steps: (1) preparing a second refined concentrated solution; (2) preparing a second-extract polysaccharide test solution; (3) preparing a second-extract formula flavone test solution; (4) preparing a standard solution; (5) establishing HPLC fingerprint of effective components of polysaccharide and flavonoid in the second refined formula by using different compatible medicines, determining characteristic peaks and extracting common peak data; (6) evaluating the hypoglycemic activity of different compatible medicines to the extracts of the two essences through pharmacodynamic experiments; (7) substituting the characteristic peak data of the fingerprint and the data of the hypoglycemic drug effect activity into the constructed spectrum-effect relationship by utilizing a partial least square method and related analysis, and screening out a common peak related to pharmacological activity in chromatographic peaks of polysaccharides and flavonoids serving as two-component hypoglycemic active ingredients;
preparing the two-extract formula extracting solution with different compatibility proportions, and establishing HPLC fingerprint spectra of polysaccharide and flavonoid effective components in the two-extract formula by using different compatible medicines, thereby providing basis for blood sugar reducing substance basis and quality evaluation of the two-extract formula. The hypoglycemic activity of the polysaccharide and the flavonoid extract of the second-extract prescription of different compatibility medicine pairs is evaluated through pharmacodynamic experiments, the second-extract prescription proportion with the optimal compatibility proportion is obtained, the hypoglycemic effect of the polysaccharide and the flavonoid extract of the second-extract prescription with the optimal compatibility proportion is better, the spectrum effect relationship evaluation method established by the partial least square method and the related analysis method is accurate, and the sensitivity is high.
Further, the preparation steps of the second-extract concentrated solution in the step (1) are as follows: weighing rhizoma Polygonati and fructus Lycii at a predetermined ratio, extracting with hot water under reflux for 3 times (each time for 1 hr) at a ratio of 1:10, filtering, mixing the filtrates, and concentrating to obtain concentrated solution.
Further, the preparation of the second-formula polysaccharide test solution in the step (2) comprises the following steps:
(2-1) preparation of crude polysaccharide: precipitating the second-refined concentrated solution with alcohol, standing overnight, filtering, rinsing the precipitate with 80% ethanol for 2-3 times, and drying to obtain crude polysaccharide;
(2-2) preparation of Fine polysaccharide: dissolving crude polysaccharide in water, adding chloroform-n-butanol (4:1) mixed solution, centrifuging to obtain supernatant, adding 80% anhydrous ethanol into the supernatant, standing overnight, filtering, washing the obtained precipitate with 95% ethanol, anhydrous ethanol, acetone and diethyl ether, and oven drying to obtain refined polysaccharide;
(2-3) derivatization of polysaccharides: dissolving refined polysaccharide with trifluoroacetic acid, sealing, hydrolyzing in 100 deg.C oven for 8 hr, blow-drying, dissolving residue with methanol, blow-drying, repeating for 3 times, dissolving with water to obtain polysaccharide hydrolysate, adding 1-phenyl-3-methyl-5-pyrazolone methanol solution and sodium hydroxide solution into polysaccharide hydrolysate, mixing, heating in 70 deg.C water bath for 30min, taking out, cooling to room temperature, adding hydrochloric acid solution to adjust pH to neutrality, mixing, extracting with chloroform for 3 times, collecting water layer, filtering with needle cylinder type microporous membrane, and collecting filtrate as test solution of polysaccharide.
Further, the preparation method of the second-extract formula flavone test solution in the step (3) comprises the following steps: degreasing the two concentrated solutions with petroleum ether, adding 80% ethanol into the residue, performing ultrasonic treatment twice at 60 deg.C for 30min each time, filtering, mixing the filtrates, concentrating under reduced pressure, removing water soluble impurities, eluting with 50% ethanol, collecting ethanol eluate, and filtering with a syringe microporous membrane to obtain filtrate as sample solution of flavone.
Further, the HPLC column conditions in the step (5) are as follows:
(5-1) HPLC column conditions of the second-refined polysaccharide are as follows: an Agilent XDB-C18 (4.6X 250mm, 5 μm) column was used, and the mobile phase was (A) phosphate buffer (pH 6.8): (B) acetonitrile 84: 16, isocratic elution; the flow rate is 1 mL/min-1And the detection wavelength is as follows: 250nm, column temperature 30 deg.C, and sample injection amount of 10 μ L.
(5-2) HPLC color of dijing flavonoidThe spectral column conditions are as follows: an Agilent XDB-C18 (4.6X 250mm, 5 μm) chromatographic column was used, the mobile phase was (A) acetonitrile: water: glacial acetic acid 5: 94.5: 0.5 and (B) acetonitrile: water: glacial acetic acid 70: 29.5: 0.5; gradient elution conditions: 0-14 min: 100% -86% (A); 14-22 min: 86% -84% (A); 22-40 min: 84% -70% (A); 40-60 min: 70% -100% (A); the flow rate is 1 mL/min-1And the detection wavelength is as follows: 254nm, the column temperature is 30 ℃, and the sample injection amount is 10 mu L.
After derivatization of polysaccharide components, phosphate buffer solution is adopted as a mobile phase, so that the peak shape and the separation degree of a target chromatographic peak can be improved; the flavone component adopts acetic acid water solution as mobile phase, and the target chromatographic peak can eliminate tailing phenomenon.
Further, in the step (5), the polysaccharide components in the second extract side are calibrated to have 10 common peaks which account for more than 90% of the total area of chromatographic peaks, and mannose and glucose are identified according to the retention time of the standard reference substance by comparing the standard reference substance with the second extract side; the two-refined flavonoid component has 18 common peaks which account for more than 90% of the total area of chromatographic peaks, and rutin is identified according to the retention time of the two-refined flavonoid component by comparing with a standard reference substance.
Further, in the step (6), the medicine pair with the best blood sugar reducing activity of the extracts of the two essences is medlar-rhizoma polygonati with the mass ratio of 1: 1.
Furthermore, in the step (7), the results of a correlation analysis method show that the characteristic peaks 1, 7, 9 and 10 of the polysaccharide components in the second essential formula are closely related to the hypoglycemic effect, the PLS results show that the characteristic peaks 9 and 10 have large contribution degree to the hypoglycemic effect, and the two kinds of data are combined for screening, which shows that the characteristic peaks 9 and 10 of the polysaccharide components in the second essential formula are effective components playing the hypoglycemic effect; the results of correlation analysis methods show that peaks 1, 9, 10, 11, 12, 14, 16, 17 and 18 of the flavonoids in the second-extract are closely related to the drug effect of reducing blood sugar, the PLS results show that peaks 1, 9, 10, 11, 12, 14, 16, 17 and 18 of the flavonoids in the second-extract have large contribution degree to reducing blood sugar, and the two kinds of data are combined for screening, which shows that peaks 1, 9, 10, 11, 12, 14, 16, 17 and 18 of the flavonoids in the second-extract are effective components playing a role in reducing blood sugar.
The following experimental examples are provided to explain the specific embodiments in detail.
Experimental example:
1. laboratory instruments and materials
1.1 Experimental instruments
KQ-250E ultrasonic device (Ultrastuer, Kunshan, Inc.); a constant temperature water bath (Beijing Long-Source laboratory plant); a circulating water type multipurpose vacuum pump (Zhengzhou great wall science, industry and trade, Ltd.); electronic weighing scales (Xiamen Bailun electronic technology, Inc.); a pulverizer (Stand alone industrial and trade company, ltd); RE-2000A rotary evaporator (Shanghai Yangrong Biochemical Instrument factory); analytical balance (mettler-toledo); high performance liquid chromatography (Agilent 1260).
1.2 materials of the experiment
Medlar (batch number: 070101, producing area: Berlin processing factory in Ningxia, Shijiazhuang); rhizoma polygonati (batch number: 070101, origin: Sichuan, Berlin pharmaceutical processing factory, Shijiazhuang city) standard: d-glucose (China institute for food and drug assay, batch No. 110833-201506); d-galactose (China institute for food and drug assay, batch number: 100226-201506); d-mannose (China institute for testing food and drug, batch No. 140651-201504); l-arabinose (China institute for testing and testing food and drug, batch No. 1506) 200202); l-rhamnose (China institute for food and drug assay, batch No. 111683-201502); d-xylose (China institute for testing food and drug; batch No. 111508-201605); fucose (China institute for testing food and drug, batch No. 112014-201601); liquiritin (China food and drug testing research institute, batch No. 111610-201607); quercetin (China institute for food and drug testing, lot number: 100081) -201610); rutin (China institute for testing food and drug, batch number: 100080-one 200707)
1.3 Experimental reagents
Ethanol (analytical grade, batch No. 20130128, Mimi European Chemicals Co., Tianjin, Ltd.); trichloromethane (analytically pure, Tianjin, Dalochi chemical reagents works, Lot: 20160709); n-butanol (analytical purity, batch number 201340506, Mimi European chemical reagents Co., Tianjin); methanol (analytical grade, batch No. 20151120, Mimi European Chemicals Co., Tianjin); acetonitrile (chromatographically pure, Fisher, usa); methanol (chromatographically pure, Fisher, usa); trifluoroacetic acid (analytically pure, Shanghai, Qin chemical Co., Ltd., Lot: 20170915); 1-phenyl-3-methyl-5-pyrazolone (analytically pure, shinny fine chemical research institute, Tianjin, lot number: 20100729); wahaha water.
2. Preparation of two-essence formula extracting solution by using different compatible medicines
Weighing the medlar and the sealwort according to the mass ratio of 1:0, 1:1, 1:2, 1:3, 3:1, 2:1 and 0:1 respectively to form 7 groups of compatible medicine pairs, respectively carrying out reflux extraction on the 7 groups of compatible medicine pairs with hot water for 3 times, wherein the material-liquid ratio is 1:10 each time, filtering, combining the 3 times of filtrate and concentrating to obtain 7 groups of medicine two-concentrated solution.
3. Preparation of two-component polysaccharide test solution by using different compatible medicines
3.1 preparation of crude polysaccharide
Respectively weighing 7 groups of the five-component medicine pair two-refined prescription concentrated solution with equal mass, concentrating, precipitating with ethanol (the alcohol content reaches about 80%), standing overnight, filtering, rinsing the precipitate with 80% ethanol for 2-3 times, and drying to obtain crude polysaccharide.
3.2 preparation of purified polysaccharide
Dissolving the crude polysaccharide in water, adding chloroform-n-butanol (4:1) mixed solution for deproteinization, shaking vigorously, centrifuging at 3000r/min for 10min to obtain supernatant, adding anhydrous ethanol (to make the alcohol content reach about 80%), standing overnight, filtering, washing the obtained precipitate with 95% ethanol, anhydrous ethanol, acetone and diethyl ether, and oven drying to obtain refined polysaccharide.
3.3 derivatization of polysaccharides
Taking 10mg of the refined polysaccharide, precisely weighing, placing in a screw cap test tube, adding 2mL of trifluoroacetic acid to dissolve, sealing, hydrolyzing in an oven at 100 ℃ for 8h, blow-drying, adding 1mL of methanol to the residue to dissolve, blow-drying, repeating for 3 times, and adding 2mL of water to dissolve to obtain polysaccharide hydrolysates with different drug ratios. Adding 1.2mL of 1-phenyl-3-methyl-5-pyrazolone (PMP) methanol solution and 1mL of 0.3 mol/L-1 sodium hydroxide solution into the hydrolysate, mixing uniformly, heating in a 70 ℃ water bath for 30min, taking out, cooling to room temperature, adding 0.3 mol/L-1 hydrochloric acid solution to adjust the pH to be neutral, mixing uniformly, extracting with trichloromethane for 3 times in equal volume, taking out a water layer, filtering with a syringe type microporous membrane, and taking the filtrate as a sample solution of polysaccharide.
3.4 preparation of polysaccharide test solutions from the alcohol extracts of the two extracts
Weighing the medlar and the sealwort according to the mass ratio of 1:1, adding 75% ethanol, carrying out reflux extraction at 70 ℃ for 1.5h, filtering, carrying out reflux extraction for 2 times, each time for 1.5h, combining the filtrates to obtain an alcohol extract, and preparing the alcohol extract into a polysaccharide test sample solution according to the steps.
4. Preparation of two-component flavone test solution by using different compatible medicines
Respectively weighing 7 groups of compatible medicines with equal mass, degreasing with petroleum ether, adding 80% ethanol into residues, performing ultrasonic treatment twice at 60 ℃ for 30min each time, filtering, combining filtrates, concentrating under reduced pressure, passing the concentrated solution through HPD-600 type macroporous resin, removing water-soluble impurities with distilled water, eluting with 50% ethanol, collecting ethanol eluate, filtering with syringe type microporous membrane, and collecting filtrate as sample solution of flavone.
5. Preparation of Standard solutions
5.1 preparation of polysaccharide reference solutions
Precisely weighing fucose, rhamnose, arabinose, xylose, glucose, mannose and galactose, respectively placing into test tubes, respectively adding 5mL of 0.3 mol.L < -1 > NaOH solution, respectively, oscillating for dissolving, taking 100 μ L of the dissolved solution, adding 50 μ L of 0.5 mol.L < -1 > PMP methanol solution, reacting in 70 deg.C water bath for 30min, cooling to room temperature, adding 100 μ L of 0.3 mol.L < -1 > hydrochloric acid for neutralization, mixing, extracting with chloroform for 3 times, taking water layer, filtering with syringe type microporous membrane, and collecting filtrate as polysaccharide component control solution.
5.2 preparation of flavone reference solutions
Precisely weighing rutin, liquiritin, isoliquiritin and quercetin, respectively placing into 10mL volumetric flasks, adding ethanol for dissolving, and metering to 10 mL. During chromatographic analysis, the reference substance solution is filtered through a syringe type microporous filter membrane, and the subsequent filtrate is the flavonoid component reference substance solution.
6. Establishment of HPLC fingerprint of polysaccharide and flavonoid active ingredients in two fine prescriptions by using different compatible medicines
6.1HPLC chromatographic conditions
Chromatographic conditions for polysaccharide component: agilent XDB-C18 (4.6X 250mm, 5 μm) column, (A) phosphate buffer (pH 6.8): (B) acetonitrile 84: 16, isocratic elution; flow rate of 1 mL. min-1, detection wavelength: 250nm, column temperature 30 deg.C, and sample injection amount of 10 μ L.
Chromatographic conditions of the flavonoid components: agilent XDB-C18 (4.6X 250mm, 5 μm) column with mobile phase (A) acetonitrile: water: glacial acetic acid 5: 94.5: 0.5 and (B) acetonitrile: water: glacial acetic acid 70: 29.5: 0.5; gradient elution conditions: 0-14 min: 100% -86% (A); 14-22 min: 86% -84% (A); 22-40 min: 84% -70% (A); 40-60 min: 70% -100% (A); flow rate of 1 mL. min-1, detection wavelength: 254nm, the column temperature is 30 ℃, and the sample injection amount is 10 mu L.
6.2 results of the experiment
6.2.1 Linear relationship investigation
The standard solution prepared under item 5 is adopted, the standard solution is precisely absorbed, diluted by a dissolving reagent and subjected to constant volume in a volumetric flask. The concentrations of the glucose standard substances are respectively 0.17mg/mL, 0.84mg/mL, 1.67mg/mL, 2.51mg/mL, 3.34mg/mL and 4.18 mg/mL; the concentration of the mannose standard substance is 0.19mg/mL, 0.95mg/mL, 1.90mg/mL, 2.85mg/mL, 3.80mg/mL and 4.75mg/mL respectively; the concentration of rutin standard substance is 0.16mg/mL, 0.32mg/mL, 1.60mg/mL, 3.20mg/mL, 4.80mg/mL, 6.40mg/mL, 8.00 mg/mL. And respectively injecting the standard substance solution with each concentration. After the solutions are subjected to HPLC measurement under selected chromatographic conditions, standard curves are drawn. The linear regression equation, R2 and linear range are shown in Table 1-1.
TABLE 1 wavelength, linear equation, correlation coefficient, and linear range results for each component detection
As can be seen from Table 1, the linear correlation coefficient of the standard curve is 0.9996-1.0000, which indicates that the method has good linear relation.
6.2.2 precision, stability, repeatability and recovery from sample application
Continuously sampling fructus Lycii-rhizoma Polygonati (1:1) polysaccharide component sample and flavonoid component sample solution for 6 times, recording HPLC chromatogram, and determining precision, the results are shown in Table 2.
Sampling fructus Lycii-rhizoma Polygonati (1:1) polysaccharide component sample and flavonoid component test solution at 0, 3, 6, 9, 12, and 24 hr respectively, recording HPLC chromatogram, and determining stability, the results are shown in Table 2.
Taking wolfberry-rhizoma polygonati (1:1) polysaccharide component samples and flavonoid component samples, respectively preparing 6 parts of polysaccharide component test sample and flavonoid component test sample solution in parallel, respectively injecting samples, recording HPLC chromatogram, and judging repeatability, wherein the results are shown in Table 2.
Precisely sucking 1.0mL of the measured sample solution, placing the sample solution in a sample feeding bottle, precisely sucking 1.0mL of the standard solution under the item '5', shaking up, preparing 6 parts in parallel, and calculating the sample feeding recovery rate according to the amount of the added standard solution and the measured amount, wherein the results are shown in Table 2.
TABLE 2 measurement results of precision, stability and repeatability experiment of each component
As can be seen from Table 2, in the precision experiment, RSD of the chromatographic peak areas (A) corresponding to glucose, mannose and rutin is 2.01-2.72%, and RSD of retention time (T) is 0.48-3.36%, which meets the requirement of a characteristic spectrum and shows that the precision of the instrument is good. In the stability experiment, RSD of chromatographic peak areas (A) corresponding to glucose, mannose and rutin is 1.07-2.95%, and RSD of retention time (T) is 0.04-2.96%, so that the stability of the test solution in 24h is shown. In a repeatability experiment, RSD of chromatographic peak areas (A) corresponding to glucose, mannose and rutin is 0.01-3.06%, and RSD of retention time (T) is 0.0004-0.98%, so that the method meets the requirement of a characteristic spectrum, and the repeatability of the method is good. The sample recovery rate is 93.07% -105.11%, and the RSD is 0.009% -0.68%, which shows that the method has good accuracy.
6.3 creation of fingerprint of two prescriptions with different compatibility of medicines and similarity analysis
6.3.1 establishment of HPLC fingerprint of polysaccharide component of two essential formulas with different compatible drugs
Performing HPLC detection according to polysaccharide component test solution prepared under item 3, performing HPLC detection according to chromatographic conditions and gradient conditions under item 6.1, precisely absorbing 10 μ L of test solution, injecting into an HPLC chromatograph, recording HPLC chromatogram, and generating fingerprint spectra of fructus Lycii-rhizoma Polygonati with different compatibility ratios as shown in figure 1, wherein fructus Lycii-rhizoma Polygonati (1:0) is recorded as S1, fructus Lycii-rhizoma Polygonati (0:1) is recorded as S2, fructus Lycii-rhizoma Polygonati (1:1) is recorded as S3, fructus Lycii-rhizoma Polygonati (1:1) alcohol extract is recorded as S4, fructus Lycii-rhizoma Polygonati (1:2) is recorded as S5, fructus Lycii-rhizoma Polygonati (1:3) is recorded as S6, fructus Lycii-rhizoma Polygonati (2:1) is recorded as S7, and fructus Lycii-rhizoma Polygonati (3:1) is recorded as S8.
6.3.2 evaluation of similarity of polysaccharide ingredients in the second formula with different compatibility drugs
Importing the chromatogram into a traditional Chinese medicine fingerprint similarity evaluation system, selecting a chromatogram of No. S1 as a reference chromatogram, automatically matching chromatographic peaks after multi-point correction by software, generating a reference chromatogram by a median method as shown in FIG. 2, wherein a wolfberry-rhizoma polygonati (1:0) is recorded as S1, a wolfberry-rhizoma polygonati (0:1) is recorded as S2, a wolfberry-rhizoma polygonati (1:1) is recorded as S3, a wolfberry-rhizoma polygonati (1:1) alcohol extract is recorded as S4, a wolfberry-rhizoma polygonati (1:2) is recorded as S5, a wolfberry-rhizoma polygonati (1:3) is recorded as S6, a wolfberry-rhizoma polygonati (2:1) is recorded as S7, and a wolfberry-rhizoma polygonati (3:1) is recorded as S8.
Comparing the chromatograms of the test solution, inputting a traditional Chinese medicine fingerprint similarity evaluation system, calibrating 10 common peaks in total, and enabling the sum of the peak areas of the 10 common peaks to be larger than 90%. The peak No. 8 (retention time 10.931min) was designated as a reference peak, and the relative peak areas and RSD values of the relative retention times of the remaining 9 peaks were calculated as shown in tables 3 and 4.
TABLE 3 relative peak area of polysaccharide component common peak of different compatibility medicine
As can be seen from Table 3, the difference of the RSD values of the relative peak areas of the polysaccharide components of the two essential formulas of different compatibility medicines is large, which indicates that the content difference of the polysaccharide components in the wolfberry-rhizoma polygonati with different proportions and extraction methods is large.
TABLE 4 relative retention time of polysaccharide component common peak of two essential formulas
From table 4, it can be seen that the RSD values of the common peaks of the chromatograms of the sample solutions are all less than 3.0%, indicating that the relative retention times are relatively stable and the variation is small.
The similarity evaluation system of the traditional Chinese medicine fingerprint spectrum takes the No. 8 peak as a reference peak, the similarity value of the generated reference spectrum (R) is determined as 1, the similarity of different compatible medicines to the characteristic spectrums of the polysaccharide components of the second formula is respectively calculated, and the similarity evaluation is carried out, and the result is shown in table 5.
TABLE 5 analysis of polysaccharide component similarity of different compatibility herbs
As can be seen from Table 5, the similarity of polysaccharide components of the second formula is close to 1, indicating that the similarity is high.
6.3.3 establishment of HPLC fingerprint of flavonoid component of two effective prescriptions with different compatibility medicines
And (3) carrying out HPLC detection on 7 parts of the flavonoid component test solution of the second-extract formula according to different compatible medicines prepared under the item 4, and carrying out HPLC detection according to chromatographic conditions and gradient conditions under the item 6.1. The chromatographic peaks with the same retention time of the 7 matches were considered as common peaks. Precisely sucking 10 mu L of test solution of 7 parts of rhizoma polygonati-Chinese wolfberry in different proportions, injecting the solution into an HPLC chromatograph, recording an HPLC chromatogram, and generating fingerprint spectrums of different compatibility medicines for flavonoid components of the second formula as shown in figure 3, wherein the wolfberry-rhizoma polygonati (1:0) is recorded as S1, the wolfberry-rhizoma polygonati (0:1) is recorded as S2, the wolfberry-rhizoma polygonati (1:1) is recorded as S3, the wolfberry-rhizoma polygonati (1:2) is recorded as S4, the wolfberry-rhizoma polygonati (1:3) is recorded as S5, the wolfberry-rhizoma polygonati (2:1) is recorded as S6, and the wolfberry-rhizoma polygonati (3:1) is recorded as S7.
6.3.4 evaluation of similarity of flavonoids of two effective prescriptions with different compatibility of medicines
Introducing the chromatogram into a traditional Chinese medicine fingerprint similarity evaluation system, selecting a chromatogram of S1 as a reference chromatogram, performing multi-point correction by software, automatically matching chromatographic peaks, and generating a reference chromatogram by a median method as shown in FIG. 4, wherein the chromatogram is obtained by recording the weight ratio of wolfberry-sealwort (1:0) as S1, the chromatogram is obtained by recording wolfberry-sealwort (0:1) as S2, the chromatogram is obtained by recording wolfberry-sealwort (1:1) as S3, the chromatogram is obtained by recording wolfberry-sealwort (1:2) as S4, the chromatogram is obtained by recording wolfberry-sealwort (1:3) as S5, the chromatogram is obtained by recording wolfberry-sealwort (2:1) as S6, and the chromatogram is obtained by recording wolfberry-sealwort (3:1) as S7.
Comparing the chromatograms of the test solutions, recording into a traditional Chinese medicine fingerprint similarity evaluation system, calibrating 18 common peaks in total, enabling the sum of peak areas of the 18 common peaks to be larger than 90%, designating the peak 1 (with the retention time of 2.471min) as a reference peak, and calculating the RSD values of the relative peak areas and the relative retention times of the rest 17 peaks, as shown in tables 6 and 7.
TABLE 6 relative peak area of common peak of flavonoid components of two effective formulas
As can be seen from Table 6, the difference of the RSD values of the relative peak areas of the flavonoids of the two prescriptions from the different compatibility medicines is also large, and the content of the flavonoids of the Chinese wolfberry and the rhizoma polygonati in different proportions and extraction methods is probably large.
TABLE 7 relative retention time of different compatibility herbs on common peak of flavonoid component of second extract formula
As can be seen from Table 7, the RSD values of the common peaks of the flavonoids of the second formula are relatively large for different compatibility medicines, and the RSD values of the retention times are influenced by the fact that the common peaks of the wolfberry-polygonatum sibiricum (0:1) group and other groups are relatively low in matching.
The similarity evaluation system of the traditional Chinese medicine fingerprint spectrum takes the No. 1 peak as a reference peak, the similarity value of the generated reference spectrum (R) is determined as 1, the similarities of the characteristic spectrums of the medlar-rhizoma polygonati flavonoid components with different proportions and different extraction methods are respectively calculated, and the similarity evaluation is carried out, and the result is shown in a table 8.
TABLE 8 analysis of the similarity of flavonoids in the second formula with different drugs
6.3.5 qualitative analysis of common peaks in HPLC finger prints
Comparing the retention time of the chromatogram of the reference fingerprint generated by the software with the retention time of the chromatogram of the mixed standard reference substance, finding that the No. 9 and No. 10 chromatogram peaks in the reference fingerprint are mannose and the No. 8 glucose peak is PMP solvent peak respectively, and the reference chromatogram of the polysaccharide standard substance is shown in figure 5.
Comparing the retention time of the chromatogram of the comparison fingerprint generated by the software with that of the mixed standard reference substance, finding that the No. 18 chromatographic peak in the comparison fingerprint is rutin, and the rest chromatographic peaks are not identified, and after the identification of the subsequent experimental study, the comparison chromatogram of the flavonoid standard substance is shown in figure 6.
7. Research on drug effect of different compatibility medicines on two-extract polysaccharide and flavone on blood sugar reduction
7.1 instruments and reagents
A stable glucometer (Sannuo biosensing Co., Ltd.) and a special test paper; H1850R centrifuge (Hunan instruments laboratory development Co., Ltd.).
Streptozotocin (STZ) (Sigma, 100 mg/vial, CAS NO: 18883-66-4) was dissolved in 0.1 mol. L-1(pH 4.3) citric acid-sodium citrate buffer for use and ready to use. Metformin hydrochloride tablets (Shanghai Shi Guibao pharmaceutical Co., Ltd., China, lot number: AAU 9951).
The body mass of the SPF SD male rat is 180-200 g.
7.2 methods
7.2.1 establishment of type II diabetic rat model
Continuously feeding high-fat high-sugar feed for 8 weeks, fasting without water for 12h, performing intraperitoneal injection of STZ 35mg/kg, and performing intraperitoneal injection of citric acid buffer solution with the same amount to normal control rats; after 24 hours, the rats were fasted and kept for 12 hours, and were injected with STZ 30mg/kg again in the abdominal cavity, and normal control rats were given an equal amount of citrate buffer solution in the abdominal cavity.
7.2.2 animal grouping and drug intervention
SD rats were divided into Control groups: normal control group (8), corresponding amount of physiological saline was gavaged; model group: the model control group (8), the normal saline of the corresponding volume of the gavage; DMBG group: diabetes plus metformin (250 mg/kg); gouqi group: adding rhizoma Polygonati-fructus Lycii (0:1) water extractive solution for treating diabetes; huanggjing group: adding rhizoma Polygonati-fructus Lycii (1:0) water extractive solution for treating diabetes; HG1-1 group: adding rhizoma Polygonati-fructus Lycii (1:1) water extractive solution for treating diabetes; HG1-1 Et group: adding rhizoma Polygonati-fructus Lycii (1:1) ethanol extractive solution for treating diabetes; HG1-2 group: adding rhizoma Polygonati-fructus Lycii (1:2) water extractive solution for treating diabetes; HG1-3 group: adding rhizoma Polygonati-fructus Lycii (1:3) water extractive solution for treating diabetes; HG2-1 group: adding rhizoma Polygonati-fructus Lycii (2:1) water extractive solution for treating diabetes; HG3-1 group: rhizoma Polygonati-fructus Lycii (3:1) water extractive solution group is added for diabetes, and the administration is performed by intragastric administration at a dose of 3.4 g/kg. After successful molding in week 10, drug intervention was performed once a day for 30 days.
7.3 results of the experiment
Fasting plasma glucose detection, after administration, fasting plasma glucose of rats in each group is detected every week, blood is collected from tail tips, the influence of different compatibility medicines on the fasting plasma glucose of rats is determined, and the result is shown in figure 7, wherein a, medlar-rhizoma polygonati (1:0), b, medlar-rhizoma polygonati (0:1), c, medlar-rhizoma polygonati (1:1), d, medlar-rhizoma polygonati ethanol extract (1:1), e, medlar-rhizoma polygonati (2:1), f, medlar-rhizoma polygonati (3:1), g, medlar-rhizoma polygonati (1:2), h, medlar-rhizoma polygonati (1:3)
As can be seen from FIG. 7, the fasting blood glucose of the rats was decreased to some extent in each treatment group compared with the model group. Wherein, the reduction degree of the medlar-rhizoma polygonati (1:1) water extract group is more obvious (P is less than 0.001).
8. The spectral efficiency relation is established by adopting Correlation Analysis (CA) and partial least squares discriminant analysis (PLS)
8.1 Process software
SPSS 20.0 data processing software; SIMCA-P14.1 data processing software
8.2 hypoglycemic drug effect
8.2.1 the common peak area information and hypoglycemic drug efficacy value of different compatible drugs on HPLC fingerprint characteristic peaks of polysaccharide components of two essences are shown in Table 9, wherein Gouqi is Lycium barbarum-Polygonatum sibiricum (1:0), Huangjing is Lycium barbarum-Polygonatum sibiricum (0:1), GH1-1 is Lycium barbarum-Polygonatum sibiricum (1:1), GH1-1Et is Lycium barbarum-Polygonatum ethanol extraction (1:1), GH1-2 is Lycium barbarum-Polygonatum (1:2) GH1-3 is Lycium barbarum-Polygonatum (1:3), GH2-1 is Lycium barbarum-Polygonatum (2:1), GH3-1 is Lycium barbarum-Polygonatum (3:1), and hypoglycemic drug efficacy value Y is model group FBG-GSFBn.
TABLE 9 common Peak area and FBG (Y) reduction value for polysaccharide of two fine formulas
8.2.2 the information of the common peak area and the hypoglycemic drug efficacy value of the HPLC fingerprint characteristic peak of the flavonoid component of the second formula of the different compatible drugs are shown in the table 10, wherein Gouqi is medlar-rhizoma polygonati (1:0), Huanggjin is medlar-rhizoma polygonati (0:1), GH1-1 is medlar-rhizoma polygonati (1:1), GH1-2 is medlar-rhizoma polygonati (1:2), GH1-3 is medlar-rhizoma polygonati (1:3), GH2-1 is medlar-rhizoma polygonati (2:1), GH3-1 is medlar-rhizoma polygonati (3:1), and the hypoglycemic drug efficacy value Y is shown in the model group FBG-each administration group FBG.
TABLE 10 common Peak area and FBG (Y) value of flavonoid component of two fine formulas obtained from different compatibility herbs
8.3 spectral efficiency analysis results
8.3.1 correlation assay
In order to preliminarily investigate the closeness degree, the correlation size and the change direction between each chromatographic peak and the index of the hypoglycemic drug effect in the fingerprint, the research respectively considers 10 and 18 characteristic peaks in the HPLC fingerprint of polysaccharide components and flavonoid components of different compatible drugs as variables, simultaneously considers the hypoglycemic drug effect of the two essential formulas of medlar-rhizoma polygonati (1:1) as variables, introduces the characteristic peak area and the hypoglycemic effect into SPSS 20.0 data processing software, finds out the characteristic peaks which are obviously related and closely related to the drug effect through the correlation analysis between the two groups of variables, and the polysaccharide component results are shown in Table 11.
TABLE 11 correlation coefficient between each characteristic peak of HPLC fingerprint of polysaccharide component and hypoglycemic drug effect
As can be seen from Table 11, the other characteristic peaks are positively correlated with the hypoglycemic effect, except that the characteristic peaks 3, 4, 5, and 6 are negatively correlated with the hypoglycemic effect. The correlation between each characteristic peak and the hypoglycemic drug effect is different, wherein the correlation coefficient between the characteristic peaks 1, 7, 8, 9 and 10 and the hypoglycemic drug effect is larger and ranges from 0.134 to 0.415, which indicates that the characteristic peaks possibly have larger contribution degree to the drug effect.
The flavonoid content results are shown in table 12.
TABLE 12 correlation coefficient of each characteristic peak of HPLC fingerprint of flavonoid component and hypoglycemic drug effect
As can be seen from Table 12, the other characteristic peaks are positively correlated with the hypoglycemic effect, except that the characteristic peaks 2, 4 and 5 are negatively correlated with the hypoglycemic effect. The correlation between each characteristic peak and the hypoglycemic drug effect is different, wherein the correlation coefficient between the characteristic peaks 1, 9, 10, 11, 12, 14, 16, 17 and 18 and the hypoglycemic drug effect is larger and ranges from 0.607 to 0.803, which indicates that the characteristic peaks possibly have larger contribution degree to the drug effect.
8.3.2PLS analysis
The analysis of PLS in SIMCA-P14.1 statistical software and multiple regression analysis are adopted to establish the spectrum effect analysis of the hypoglycemic drug effect of different compatible drugs on the two-component prescription.
8.3.2.1 auto-fit
Taking the common peak area information of different compatible medicines to the second essential polysaccharide component fingerprint as an X variable and the hypoglycemic drug effective value of different compatible medicines to the second essential polysaccharide component as a Y variable, carrying out PLS model fitting on the two variables, and establishing a prediction model of the influence of the common peak area on the hypoglycemic drug effect, wherein the model overview chart is shown in FIG. 8; the PLS model fitting is carried out on the different compatibility medicines and the effective value of the different compatibility medicines for the flavonoid of the second refined prescription as the variable X and the effective value of the different compatibility medicines for the flavonoid of the second refined prescription as the variable Y, and the outline of the model is shown in figure 9. Wherein R2Y (cum) is a part for explaining the change of the information of the common peak area of all dependent variables after the model is classified each time; q2(cum) is a part for predicting the effect change of the dependent variable sugar-reducing drug by cross validation of a model, for example, the following analysis is carried out by model fitting of the diquan polysaccharide and flavone of wolfberry-rhizoma polygonati (1: 1).
8.3.2.2 variable weight importance index
Variable weight importance index (VIP), variables of VIP >1 are generally considered to be statistically significant.
The results of analysis of the diutan polysaccharides VIP are shown in table 13 and fig. 10.
TABLE 3-5 VIP value of polysaccharide component
As shown in Table 13 and FIG. 10, it can be seen that the VIP values of peaks 8, 9 and 10 are greater than 1, indicating that peaks 8, 9 and 10 of polysaccharides of second formula have prominent effect on lowering blood sugar of diabetic rats.
The results of VIP analysis of the dijing flavonoids are shown in table 14 and fig. 11.
TABLE 14 VIP values of flavonoid components
As can be seen from Table 14 and FIG. 11, the peak numbers 1, 9, 10, 11, 12, 14, 16, 17, and 18 of VIP values are greater than 1, indicating that the peak numbers 1, 9, 10, 11, 12, 14, 16, 17, and 18 of the flavonoids of the second formula have prominent effect of lowering blood sugar in diabetic rats.
While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention.
Claims (8)
1. A method for evaluating a second-extract polysaccharide and a flavonoid component based on a spectrum-effect relationship is characterized by comprising the following steps: the method comprises the following steps: (1) preparing a second refined concentrated solution; (2) preparing a second-extract polysaccharide test solution; (3) preparing a second-extract formula flavone test solution; (4) preparing a standard solution; (5) establishing HPLC fingerprint of effective components of polysaccharide and flavonoid in the second refined formula by using different compatible medicines, determining characteristic peaks and extracting common peak data; (6) evaluating the hypoglycemic activity of the two-component extract by different proportions of the compatible medicines through pharmacodynamic experiments; (7) and substituting the characteristic peak data of the fingerprint and the data of the hypoglycemic drug effect activity into the constructed spectrum-effect relationship by utilizing a partial least square method and related analysis, and screening out the common peak related to the pharmacological activity in the chromatographic peaks of the two-essence hypoglycemic active ingredients, namely polysaccharide and flavonoid.
2. The method for evaluating a di-refined polysaccharide and a flavonoid component based on a spectrum-activity relationship according to claim 1, wherein: the preparation method of the second-extract concentrated solution in the step (1) comprises the following steps: weighing rhizoma Polygonati and fructus Lycii at a predetermined ratio, extracting with hot water under reflux for 3 times (each time for 1 hr) at a ratio of 1:10, filtering, mixing the filtrates, and concentrating to obtain concentrated solution.
3. The method for evaluating di-essential polysaccharide and flavonoid components based on spectrum-effect relationship according to claim 1, wherein the step of preparing the di-essential polysaccharide test solution in the step (2) comprises:
(2-1) preparation of crude polysaccharide: precipitating the second-refined concentrated solution with alcohol, standing overnight, filtering, rinsing the precipitate with 80% ethanol for 2-3 times, and drying to obtain crude polysaccharide;
(2-2) preparation of Fine polysaccharide: dissolving crude polysaccharide in water, adding chloroform-n-butanol (4:1) mixed solution, centrifuging to obtain supernatant, adding 80% anhydrous ethanol into the supernatant, standing overnight, filtering, washing the obtained precipitate with 95% ethanol, anhydrous ethanol, acetone and diethyl ether, and oven drying to obtain refined polysaccharide;
(2-3) derivatization of polysaccharides: dissolving refined polysaccharide with trifluoroacetic acid, sealing, hydrolyzing in 100 deg.C oven for 8 hr, blow-drying, dissolving residue with methanol, blow-drying, repeating for 3 times, dissolving with water to obtain polysaccharide hydrolysate, adding 1-phenyl-3-methyl-5-pyrazolone methanol solution and sodium hydroxide solution into polysaccharide hydrolysate, mixing, heating in 70 deg.C water bath for 30min, taking out, cooling to room temperature, adding hydrochloric acid solution to adjust pH to neutrality, mixing, extracting with chloroform for 3 times, collecting water layer, filtering with needle cylinder type microporous membrane, and collecting filtrate as test solution of polysaccharide.
4. The method for evaluating di-essential polysaccharide and flavonoid components based on spectrum-effect relationship according to claim 1, wherein the method for preparing the di-essential flavone test solution in the step (3) comprises: degreasing the two concentrated solutions with petroleum ether, adding 80% ethanol into the residue, performing ultrasonic treatment twice at 60 deg.C for 30min each time, filtering, mixing the filtrates, concentrating under reduced pressure, removing water soluble impurities, eluting with 50% ethanol, collecting ethanol eluate, and filtering with a syringe microporous membrane to obtain filtrate as sample solution of flavone.
5. The method for evaluating di-refined polysaccharides and flavonoids according to claim 1, wherein the HPLC column conditions in step (5) are as follows:
(5-1) HPLC column conditions of the second-refined polysaccharide are as follows: an Agilent XDB-C18 (4.6X 250mm, 5 μm) column was used, and the mobile phase was (A) phosphate buffer (pH 6.8): (B) acetonitrile = 84: 16, isocratic elution; the flow rate is 1 mL/min-1And the detection wavelength is as follows: 250nm, column temperature 30 deg.C, and sample injection amount of 10 μ L.
(5-2) HPLC chromatographic column conditions of difang flavone are as follows: an Agilent XDB-C18 (4.6X 250mm, 5 μm) chromatographic column was used, the mobile phase was (A) acetonitrile: water: glacial acetic acid = 5: 94.5: 0.5 and (B) acetonitrile: water: glacial acetic acid = 70: 29.5: 0.5; gradient elution conditions: 0-14 min: 100% -86% (A); 14-22 min: 86% -84% (A); 22-40 min: 84% -70% (A); 40-60 min: 70% -100% (A); the flow rate is 1 mL/min-1And the detection wavelength is as follows: 254nm, the column temperature is 30 ℃, and the sample injection amount is 10 mu L.
6. The method for evaluating refined polysaccharides and flavonoids according to claim 1, wherein in step (5), the polysaccharide components of the refined polysaccharides have 10 common peaks in total, which account for 90% or more of the total area of the chromatographic peaks, and mannose and glucose are identified by the retention time thereof by comparing with the standard control; the two-refined flavonoid component has 18 common peaks which account for more than 90% of the total area of chromatographic peaks, and rutin is identified according to the retention time of the two-refined flavonoid component by comparing with a standard reference substance.
7. The method for evaluating polysaccharide and flavonoid ingredients based on spectrum-effect relationship of claim 1, wherein in step (6), the ratio of the compatible drugs with the best hypoglycemic activity of the extract of the second extract to the wolfberry-polygonatum sibiricum is 1:1 by mass.
8. The method for evaluating di-essential polysaccharide and flavonoid components based on spectral efficiency relationship as claimed in claim 1, wherein in the step (7), the correlation analysis result shows that the peaks 1, 7, 9 and 10 characteristic of di-essential polysaccharide components are closely related to the hypoglycemic drug effect, the PLS result shows that the peaks 9 and 10 have large contribution degree to the hypoglycemic drug effect, and the combination of the two data screening shows that the peaks 9 and 10 are effective components playing hypoglycemic effect; the results of correlation analysis methods show that peaks 1, 9, 10, 11, 12, 14, 16, 17 and 18 of the flavonoids in the second-extract are closely related to the drug effect of reducing blood sugar, the PLS results show that peaks 1, 9, 10, 11, 12, 14, 16, 17 and 18 of the flavonoids in the second-extract have large contribution degree to reducing blood sugar, and the two kinds of data are combined for screening, which shows that peaks 1, 9, 10, 11, 12, 14, 16, 17 and 18 of the flavonoids in the second-extract are effective components playing a role in reducing blood sugar.
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| CN115219619A (en) * | 2022-07-05 | 2022-10-21 | 山东中医药大学 | Method for evaluating Shuanghuanglian extract compatibility based on spectrum-effect correlation mode |
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