CN109490446B - Method for simultaneously measuring 5 flavonoid compounds in centipede medicinal materials - Google Patents
Method for simultaneously measuring 5 flavonoid compounds in centipede medicinal materials Download PDFInfo
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Abstract
The invention discloses a method for simultaneously measuring the content of 5 flavonoid compounds in centipede medicinal materials, which comprises the following steps: preparing a test solution, preparing a reference solution, detecting and analyzing by high performance liquid chromatography and the like. The invention simultaneously detects 5 flavonoid active ingredients in the centipede medicinal materials, namely afzerin-3-O-alpha-L-rhamnose, astragalin, multiforin B, afzerin and multiforin A for the first time. The detection method has the advantages of high sensitivity, good repeatability and high recovery rate. The invention establishes the detection method of the centipede medicinal material by taking the main chemical components in the centipede as detection indexes, and has important significance for the quality control research and the guidance of clinical application of the centipede medicinal material.
Description
Technical Field
The invention belongs to the technical field of detection, and particularly relates to a method for simultaneously determining 5 flavonoid compounds in centipede medicinal materials.
Background
Geophilus bracteata is derived from PolypodiodiodiodiodiodiaceaeArthromeris mairei(Brause) Ching. The Yi nationality medicine is named as ' I ' Yitonuo ', which is interpreted as ' medicine for abdominal distension '. It has bitter taste and slightly cold nature, and has effects of activating collaterals, resolving food stagnation, relaxing bowels, and lowering fire, and can be used for treating food stagnation, gastralgia, abdominal distention, constipation, rheumatism bone and muscle pain, ischialgia, headache, and toothache. The plant is mainly distributed in Yunnan, Tibet, Sichuan and the like, as well as Burma and northern India.
The centipede medicinal material is one of prescription medicinal materials of a preparation, namely a bowel-clearing and bowel-relaxing capsule, and is currently collected in the Yunnan province medicinal material standard (a book) (2005 edition). At present, 4 flavonoid glycoside compounds are reported to be separated from centipede, such as: rosaponin, kaempferol-3-O-beta-D-glucopyranose (1 → 4) -alpha-L-rhamnopyranoside, arthromerin A, arthromerin B. In the research process, 5 flavonoid compounds are separated from the centipede medicinal material for the first time: the flavonoid glycoside comprises Multiflorin A, Multiflorin B, astragalin, afzelin and afzelin-3-O-alpha-L-rhamnose, wherein the Multiflorin A, the Multiflorin B, the afzelin-3-O-alpha-L-rhamnose are main components, and the astragalin and the afzelin are flavonoid glycoside components with strong physiological activity. The 5 flavonoid glycoside components are active components of the centipede medicinal material, and the establishment of the detection method of the centipede medicinal material by taking the 5 flavonoid glycoside components as detection indexes has important significance for the quality control of the centipede medicinal material and related preparations.
Disclosure of Invention
The invention aims to provide a method for simultaneously measuring 5 flavonoid compounds in centipede medicinal materials.
The invention realizes the aim by the following method, which comprises the steps of preparing a test solution, preparing a reference solution, detecting and analyzing, and specifically comprises
(1) Preparation of a test solution: taking about 1g of centipede medicinal material powder, precisely weighing, placing in a conical flask, precisely adding 50mL of methanol water, weighing the mass, and heating and refluxing; cooling, weighing, adding solvent to supplement the weight loss, shaking, and filtering; precisely measuring 5mL of filtrate, placing the filtrate in a 25mL volumetric flask, adding methanol water to a constant volume, and shaking up to obtain a test solution;
(2) preparation of a reference solution:
A. precisely weighing afzedin-3-O-alpha-L-rhamnose reference substance, and preparing with 50% methanol to obtain a 20 μ g.mL solution-1Filtering the reference solution with a 0.45-micrometer microporous membrane to obtain a reference solution A for later use;
B. accurately weighing astragalin, Multiflorin B, African glycosides, and Multiflorin A reference substances, respectively, mixing, and making into astragalin with concentration of 0.8 μ g.mL with methanol-1Multiflorin B concentration 54. mu.g.mL-1And the concentration of the afzerin is 1.5 mu g-1 Multiflorin A concentration 20. mu.g.mL-1To obtain a reference substance solution, namely a reference substance solution B for later use;
(3) and (3) detection: injecting the test solution, the reference solution A and the reference solution B into a high performance liquid chromatograph for detection;
(4) quantitative analysis of 5 flavonoid compounds in centipede medicinal materials is carried out by an external standard method: performing regression analysis by comparing the chromatographic peak area of the flavonoid compounds with known concentration with the corresponding concentration to obtain a standard curve, measuring the test solution, measuring the chromatographic peak area of 5 flavonoid compounds in the test solution, substituting into the standard curve, and obtaining the content of the 5 flavonoid compounds in the centipede medicinal material.
The granularity of the centipede medicinal material powder in the step (1) is 65 meshes.
The concentration of the methanol water in the step (1) is 75% methanol.
The heating reflux time in the step (1) is 1 h.
The extraction temperature in step (1) is 70 ℃ +/-2 ℃.
The chromatographic conditions of the high performance liquid chromatograph of the test solution in the step (3) are as follows:
A. a chromatographic column: waters C18（4.6nm×250mm,5 μm), the mobile phase is: v (methanol) = V (water) =35:65 isocratic elution; the flow rate was 1.0mL/min-1The column temperature is 25 ℃, the detection wavelength is 230nm, and the sample injection amount is 10 mu L;
B. a chromatographic column: waters SunAire C18(4.6X 250mm,5 μm), mobile phase 0.2% aqueous phosphoric acid (C) -methanol (D), gradient elution: 0-30 min, C: d =53: 47; 30-40 min, 47% D → 66% D; flow rate 1.0mL/min-1Detecting the wavelength of 265nm, the column temperature of 25 ℃ and the sample injection amount of 10 mu L;
the chromatographic conditions of the high performance liquid chromatograph of the reference substance solution A in the step (3) are as follows:
a chromatographic column: waters C18(4.6 nm X250 mm,5 μm) with mobile phases: v (methanol) = V (water) =35:65 isocratic elution; the flow rate was 1.0mL/min-1The column temperature was 25 ℃, the detection wavelength was 230nm, and the sample volume was 10 μ L.
The chromatographic conditions of the high performance liquid chromatograph of the reference substance solution B in the step (3) are as follows:
a chromatographic column: waters SunAire C18(4.6X 250mm,5 μm), mobile phase 0.2% aqueous phosphoric acid (C) -methanol (D), gradient elution: 0-30 min, C: d =53: 47; 30-40 min, 47% D → 66% D; flow rate 1.0mL/min-1The detection wavelength is 265nm, the column temperature is 25 ℃, and the sample injection amount is 10 mu L.
The invention firstly and definitely determines that the centipede medicinal materials contain 5 flavonoid compounds including Multiflorin A, Multiflorin B, afzerin-3-O-alpha-L-rhamnose, astragalin and afzerin, and the 5 flavonoid compounds are used as detection indexes to respectively establish a method for measuring the afzerin-3-O-alpha-L-rhamnose and a method for simultaneously measuring the Multiflorin A, the Multiflorin B, the astragalin and the afzerin, and have important significance for the quality control research and the guidance clinical application of the centipede medicinal materials and related preparations thereof.
Drawings
FIG. 1 shows the structural formula of 5 flavonoids
Wherein: 1. afumarin-3-O-a-L-rhamnose; 2. (iii) multiforin a; 3. astragalin; 4. (iii) multiforin B; 5. afzelin.
FIG. 2 is a high performance liquid chromatogram of an afurosin-3-O-alpha-L-rhamnose reference substance solution
Wherein: 1. afzeocin-3-O-alpha-L-rhamnose.
FIG. 3 is a high performance liquid chromatogram of a sample solution of Gekko Swinhonis (for determination of afzepin-3-O-alpha-L-rhamnose, chromatographic condition A)
Wherein: 1. afzeocin-3-O-alpha-L-rhamnose.
FIG. 4 is a standard curve of afurosin-3-O-alpha-L-rhamnose.
FIG. 5 shows NMR spectra of afurosin-3-O-alpha-L-rhamnose1H NMR) graph.
FIG. 6 shows NMR spectrum of afurosin-3-O-alpha-L-rhamnose (C:)13C NMR) graph.
FIG. 7 is a high performance liquid chromatogram of a reference solution of a mixture of Multiflorin A, Multiflorin B, astragalin and African glycosides
Wherein: 1. astragalin; 2. (iii) multiforin B; 3. afzelin; 4. multiflorin A.
FIG. 8 is a high performance liquid chromatogram of a sample solution of Geophilus japonicum (simultaneous determination of Multiflorin A, Multiflorin B, astragalin and African glycosides, chromatographic conditions B)
Wherein: 1. astragalin; 2. (iii) multiforin B; 3. afzelin; 4. multiflorin A.
FIG. 9 shows NMR spectra of astragalin (A)1H NMR) graph.
FIG. 10 is the NMR spectrum of astragalin (C)13C NMR) graph.
FIG. 11 is a nuclear magnetic resonance hydrogen spectrum of Multiflorin B (M1H NMR) graph.
FIG. 12 is a nuclear magnetic resonance carbon spectrum of Multiflorin B (C: (M))13C NMR) graph.
FIG. 13 shows NMR spectra of afzelin1H NMR) graph.
FIG. 14 shows NMR spectrum of afuroside (C:)13C NMR) graph.
FIG. 15 is a nuclear magnetic resonance hydrogen spectrum of Multiflorin A (1H NMR) graph.
FIG. 16 is a nuclear magnetic resonance carbon spectrum of Multiflorin A (C: (M))13C NMR) graph.
Detailed Description
The process of the present invention is further illustrated in detail by the following examples, but the scope of the present invention is not limited to the above-described contents, and in the examples, reagents are commercially available or prepared by a conventional method unless otherwise specified.
The invention relates to a method for simultaneously measuring 5 flavonoids compounds in centipede medicinal materials, which realizes the aim by the following method, comprises the steps of preparing a test solution, preparing a reference solution, detecting and analyzing, and specifically comprises
(1) Preparation of a test solution: taking about 1g of centipede medicinal material powder, precisely weighing, placing in a conical flask, precisely adding 50mL of methanol water, weighing the mass, and heating and refluxing; cooling, weighing, adding solvent to supplement the weight loss, shaking, and filtering; precisely measuring 5mL of the filtrate, placing the filtrate in a 25mL volumetric flask, adding methanol water to a constant volume, and shaking up to obtain a test solution.
Grinding the centipede medicinal materials into powder, adding methanol water with the mass volume ratio of 1g to 50mL, and heating and refluxing to ensure that 5 flavonoid compounds can be fully dissolved from the centipede medicinal materials; the mass is weighed after the addition of methanol water and the solvent is added to make up the lost weight after heating, refluxing and cooling, in order to make the sample content determination accurate.
Preferably, the centipede medicinal material powder has the granularity of 65 meshes, the methanol water concentration of 75% methanol, the heating reflux time of 1h and the extraction temperature of 70 +/-2 ℃.
(2) Preparation of a reference solution:
A. precisely weighing afzedin-3-O-alpha-L-rhamnose reference substance, and preparing with 50% methanol to obtain a 20 μ g.mL solution-1Filtering the reference solution with a 0.45-micrometer microporous membrane to obtain a reference solution A for later use;
B. accurately weighing astragalin, Multiflorin B, African glycosides, and Multiflorin A reference substances, mixing, and making into astragalin concentrate with methanolDegree of 0.8. mu.g.mL-1Multiflorin B concentration 54. mu.g.mL-1And the concentration of the afzerin is 1.5 mu g-1 Multiflorin A concentration 20. mu.g.mL-1To obtain a reference substance solution, namely a reference substance solution B for later use;
(3) and (3) detection: injecting the test solution, the reference solution A and the reference solution B into a high performance liquid chromatograph for detection;
the test sample is detected twice, and the chromatographic conditions are as follows:
A. a chromatographic column: waters C18(4.6 nm X250 mm,5 μm) with mobile phases: v (methanol) = V (water) =35:65 isocratic elution; the flow rate was 1.0mL/min-1The column temperature is 25 ℃, the detection wavelength is 230nm, and the sample injection amount is 10 mu L;
B. a chromatographic column: waters SunAire C18(4.6X 250mm,5 μm), mobile phase 0.2% aqueous phosphoric acid (C) -methanol (D), gradient elution: 0-30 min, C: d =53: 47; 30-40 min, 47% D → 66% D; flow rate 1.0mL/min-1Detecting the wavelength of 265nm, the column temperature of 25 ℃ and the sample injection amount of 10 mu L;
the chromatographic condition of the control solution A is A, and the chromatographic condition of the control solution B is A.
(4) Quantitative analysis of 5 flavonoid compounds in centipede medicinal materials is carried out by an external standard method: performing regression analysis by comparing the chromatographic peak area of the flavonoid compounds with known concentration with the corresponding concentration to obtain a standard curve, measuring the test solution, measuring the chromatographic peak area of 5 flavonoid compounds in the test solution, substituting into the standard curve, and obtaining the content of the 5 flavonoid compounds in the centipede medicinal material.
The test solution is simple and convenient to prepare, and the using amount of the organic solvent is small; the detection method has high sensitivity, good repeatability and high recovery rate. The invention firstly defines that the centipede medicinal materials contain 5 flavonoid compounds including Multiflorin A, Multiflorin B, afzerin-3-O-alpha-L-rhamnose, astragalin and afzerin, and respectively establishes a method for measuring the afzerin-3-O-alpha-L-rhamnose and a method for simultaneously measuring the Multiflorin A, the Multiflorin B, the astragalin and the afzerin by taking the 5 flavonoid compounds as detection indexes, thereby having important significance for the quality control research of the centipede medicinal materials and related preparations thereof.
Example 1: determination of afumarin-3-O-alpha-L-rhamnose in centipede medicinal material
1. Apparatus and materials
A high performance liquid chromatograph (Agilent: 1260) is provided with a quaternary pump, an automatic sample injector, a column incubator, an ultraviolet detector, an electric pulverizer, a medicine sieve (No. 4 sieve, 65 meshes), an electronic balance (AUW120D, SHIMADZU), a UpK series ultrapure water purifier (UPC-I10T), volumetric flasks (50 mL, 25 mL) and a conical flask with a plug (50 mL).
The afzeocin-3-O-alpha-L-rhamnose reference substance is separated from the centipede medicinal material in the laboratory, and the purity is 98.23 percent by high performance liquid chromatography (the detection wavelength is 230 nm); the centipede is purchased in the market and identified as the centipede by the assistant professor Yanzhuyan of the Chinese medicine identification and research laboratory of Yunnan traditional Chinese medicine collegeArthromeris mairei(Brause) dried rhizome of Ching.
Water, methanol; methanol (pure chromatography merck ltd) as a reagent for HPLC detection, and ultrapure water as water; water was used for the experiment as purified water, methanol (analytical pure Lianlong Bohuatianjin pharmaceutical chemistry Co., Ltd.).
2. Experimental methods
2.1 chromatographic conditions
A chromatographic column: waters C18 (4.6 nm X250 mm,5 μm), mobile phase: v (methanol):V (water) =35:65 isocratic elution; flow rate: 1.0 mL/min; column temperature: 25 ℃; detection wavelength: 230 nm; the amount of the sample was 10. mu.L. Under the condition of the chromatogram, the chromatogram peak of the afurosin-3-O-alpha-L-rhamnose is better separated from the peaks of other components, and the results are shown in figures 2 and 3.
2.2 preparation of control solutions
2.5mg of a cefuroxime sodium-3-O-alpha-L-rhamnose reference substance is precisely weighed and placed in a 25mL volumetric flask, 50% methanol is added for dissolution, the volume is constant, and the solution is shaken up. Precisely sucking 5mL of constant volume liquid into a 25mL volumetric flask, performing constant volume with 50% methanol, and shaking up. Was prepared at a concentration of 20. mu.g.mL-10.45 mu of the control solutionAnd m, filtering by using a microporous filter membrane for later use.
2.3 preparation of test solutions
Precisely weighing 1g of medicinal powder, precisely weighing, placing in a conical flask, adding 50mL of 75% methanol, and weighing. Heating reflux extraction for 60min at 70 + -2 deg.C. Cooled to room temperature and weighed. The lost weight was made up with 75% methanol. Filtering, precisely measuring 5mL of the subsequent filtrate, placing in a 25mL volumetric flask, adding 75% methanol to constant volume to a scale mark, shaking up, and filtering with a 0.45 μm microporous membrane for later use.
3. Methodology investigation
3.1 Standard Curve and Linear relationship inspection
Accurately sucking 1, 5, 10, 20 and 50 μ L of AffDodin-3-O-alpha-L-rhamnose reference substance to be tested, respectively injecting sample, analyzing according to 2.1 chromatographic conditions, and determining peak area. The measurement results are shown in table 1:
TABLE 1 AffDodin-3-O-alpha-L-rhamnose Linear relationship examination results
Taking the peak area as the Y-axis ordinate and the Affymenin-3-O-alpha-L-rhamnose sample injection amount (mu g) as the X-axis abscissa to obtain a regression equation: y =5022.6X-25.944(r =0.99995), indicating that afzeocin-3-O-alpha-L-rhamnose is well-linearly related in the 0.02104-1.05200 [ mu ] g range (see FIG. 4).
3.2 precision test
Precisely sucking 10 mu L of the reference substance solution of afuropin-3-O-alpha-L-rhamnose, repeatedly injecting samples for 6 times, and analyzing the samples according to the chromatographic condition of '2.1', wherein the corresponding peak areas are shown in Table 2. Calculating the average peak area of the afuropin-3-O-alpha-L-rhamnose as follows: 938.633. the RSD value is: 0.42034 percent. Indicating that the precision of the instrument is good.
TABLE 2 AffDocin-3-O-alpha-L-rhamnoside Density Observation and determination results
3.3 stability test
1 part of test solution is prepared according to the preparation method of the item 2.3, the test solution is subjected to sample injection analysis according to the chromatographic condition of the item 3.1.2 for 0, 2, 4, 8, 12 and 24 hours after being prepared, and the peak area of the afzelechin-3-O-alpha-L-rhamnose in the centipedes is measured, and the result is shown in the table 3. The RSD value of the peak area of the afuropin-3-O-alpha-L-rhamnose is as follows: 0.825%, indicating that the centipede sample solution is stable well within 24 h.
TABLE 3 Affymenin-3-O-alpha-L-rhamnose stability test measurement results
3.4 repeatability experiments
Taking 6 parts of centipede medicinal material powder of the same batch, and numbering in sequence. Respectively prepared according to the preparation method of the '2.3'. And sequentially carrying out sample injection analysis under the chromatographic condition of 2.1, measuring the peak area of the afzelechin-3-O-alpha-L-rhamnose in the centipedes, and calculating the content according to a related formula. The results are shown in Table 4, where the average content of afurosin-3-O-alpha-L-rhamnose is: 0.2999%, the RSD value is: 1.742 percent. The method is proved to have good repeatability for extracting the afzerin-3-O-alpha-L-rhamnose from the centipede.
TABLE 4 AffDodin-3-O-alpha-L-rhamnose repeatability measurements
3.5 sample application recovery test
Weighing 6 parts of test sample, precisely weighing, placing into a conical flask, precisely adding afurosin-3-O-alpha-L-rhamnose solution, volatilizing, introducing sample of the test sample solution prepared under the item '2.2' into 10 mu L, analyzing according to the sample introduction condition of the item '2.1', and calculating the recovery rate according to the related public indication when measuring the peak area. The results are shown in Table 5. The average sample recovery rate was: 100.22%, RSD value 1.99%. The method is proved to be good in accuracy.
TABLE 5 AffDodin-3-O-alpha-L-rhamnose sample recovery assay results
3.6 results of content measurement of sample
Two batches of samples of centipede were taken, 3 parts each. The test solution was prepared according to the preparation method under item "2.3", 10. mu.L of sample was sequentially and precisely aspirated, and the sample injection was measured under the chromatographic condition of item "2.1", and the measurement results are shown in Table 6.
TABLE 6 measurement of the content of Affyllin-3-O-alpha-L-rhamnose from different batches of centipedes (n = 3)
4. Nuclear magnetic data of reference substance afurosin-3-O-alpha-L-rhamnose
1H-NMR( MeOD， 500Hz )，δ5.95( 1H，d，J = 2.3Hz ， H-6 )，δ5.84 ( 1H ， d ，J = 2.3 Hz ， H-8 )，δ7.72 ( 2H ， d ， J = 8.6 Hz ， H-2、，H-3、)，δ6.78（2H ， d ， J = 8.6 Hz ， H-2、，H-6、），δ4.24 ( 1H，d，J = 1.5Hz， H-2)，δ1.23 ( 2H , d , J = 6.25 Hz ， H-4 )；13C-NMR（MeOD，125Hz），δ81.13 ( C-2 )，δ76.20 ( C-3 )，δ28.2 ( C-4 )，δ156.9 ( C-5 )，δ96.4 ( C-6 ), δ157.5 ( C-7 )，δ95.4 ( C-8 )，δ158.5 ( C-9 )，δ100.7 ( C-10 )，δ131.2 ( C-1、)，δ129.4 ( C-2、)，δ116.0 ( C-3、)，δ157.9 ( C-4、)，δ116.0 ( C-5、)，δ129.4 ( C-6、)。
FIG. 5 shows NMR spectra of afurosin-3-O-alpha-L-rhamnose1H NMR chart, FIG. 6 is nuclear magnetic resonance carbon spectrum of afurosin-3-O-alpha-L-rhamnose: (13C NMR) graph. The nuclear magnetic data proves that the reference substance is afurosin-3-O-alpha-L-rhamnose.
Example 2: and simultaneously measuring the Multiflorin A, the Multiflorin B, the astragalin and the afzelin in the centipede medicinal materials.
1. Apparatus and materials
A high performance liquid chromatograph (Agilent: 1260) is provided with a quaternary pump, an automatic sample injector, a column incubator, an ultraviolet detector, an electric pulverizer, a medicine sieve (No. 4 sieve, 65 meshes), an electronic balance (AUW120D, SHIMADZU), a UpK series ultrapure water purifier (UPC-I10T), volumetric flasks (50 mL, 25 mL) and a conical flask with a plug (50 mL).
The control substances of the Multiflorin A, the Multiflorin B, the astragalin and the African odoriferin are separated from the centipede medicinal material in the laboratory, and the purity of the control substances is respectively 98.11%, 99.02%, 98.65% and 98.48% through high performance liquid chromatography (detection wavelength is 265 nm); the centipede is purchased in the market and identified as the centipede by the assistant professor Yanzhuyan of the Chinese medicine identification and research laboratory of Yunnan traditional Chinese medicine collegeArthromeris mairei(Brause) dried rhizome of Ching.
Water, methanol; the reagent methanol for HPLC detection (pure chromatography Merck Co., Ltd.), water ultrapure water, phosphoric acid (Guangdong Guanghua science and technology Co., Ltd.); water was used for the experiment as purified water, methanol (analytical pure Lianlong Bohuatianjin pharmaceutical chemistry Co., Ltd.).
2. Experimental methods
2.1 chromatographic conditions
Waters SunFire C18A chromatography column (4.6X 250mm,5 μm) with a mobile phase of 0.2% aqueous phosphoric acid (C) -methanol (D), gradient elution (0-30 min, C: D =53: 47; 30-40 min, 47% D → 66% D); flow rate 1.0mL/min-1The detection wavelength is 265nm, the column temperature is 25 ℃, and the sample injection amount is 10 mu L. Under the chromatographic conditions, the separation degree of each component is good, and the HPLC graphs of the mixed control and the sample are shown in figures 7 and 8.
2.2 preparation of control solutions
Accurately weighing astragalin reference substance 5.01mg, placing in a 250mL measuring flask, adding methanol to dissolve, fixing volume to scale, shaking up to obtain reference substance stock solution; precisely weighing 13.50mg of a Multiflorin B reference substance, placing the reference substance in a 10mL measuring flask, adding methanol for dissolving, fixing the volume to a scale, and shaking up to obtain a reference substance stock solution; precisely weighing 9.94mg of an afzelin reference substance, placing the afzelin reference substance in a 250mL measuring flask, adding methanol to dissolve the afzelin reference substance, fixing the volume to a scale, and shaking up to obtain a reference substance stock solution; accurately weighing 5.02mg of a Multiflorin A reference substance, placing the reference substance in a 10mL measuring flask, adding methanol for dissolving, fixing the volume to a scale, and shaking up to obtain a reference substance stock solution; precisely measuring 1mL of the reference stock solutions respectively, placing in a 25mL measuring flask, adding methanol to dilute to scale, and shaking up for use.
2.3 preparation of test solutions
Weighing about 1g of the powder, accurately weighing, placing in a conical flask with a plug, accurately adding 50mL of 75% methanol water, weighing, and heating and refluxing for 1 h. Cooling, weighing, adding solvent to supplement the weight loss, shaking, and filtering. Accurately measuring 5mL of the subsequent filtrate, placing the subsequent filtrate in a 25mL measuring flask, adding 75% methanol to a constant volume, and shaking up for later use.
3. Methodology investigation
3.1 System Adaptation
Precisely measuring 10 μ L of each of the mixed reference solution, the sample solution and the blank solution, and analyzing by sample injection under the above chromatographic conditions. The result shows that the chromatogram of the test solution presents a chromatographic peak with the same retention time as the reference substance; the separation degrees of astragalin, Multiflorin B, African glycosides and Multiflorin A and adjacent chromatographic peaks are all greater than 1.5, and the tailing factor is 0.97-1.10.
3.2 Linear Range
Sampling mixed reference substance solutions with series concentrations prepared under the item of 2.2, respectively sampling according to 1 muL, 3 muL, 5 muL, 10 muL, 20 muL, 30 muL and 40 muL, drawing a standard curve by taking a peak area as a vertical coordinate (Y) and a sampling amount as a horizontal coordinate (X), and performing linear regression to obtain regression equations of astragalin, Multiflorin B, African glycosides and Multiflorin A. The results of the experiments show that the components have a good linear relationship in the respective concentration ranges, and the results are shown in Table 7.
Table 7.4 Linear relationship examination results of flavonoid components
3.3 precision test
Precisely sucking 10 mu L of the mixed control solution, continuously injecting samples for 6 times, and calculating RSD of peak areas of astragalin, Multiflorin B, African fordii glycoside and Multiflorin A to be 1.65%, 0.19%, 1.87% and 0.37% respectively (n is 6), which indicates that the precision of the instrument is good.
3.4 stability test
The same test sample solution is sampled by 10 mu L after 0, 2, 4, 8, 12 and 24 hours after preparation, the average values of the peak areas of astragalin, Multiflorin B, afungin and Multiflorin A are respectively 30.79, 1235.6, 53.48 and 445.81, and the RSD values are respectively 1.50%, 0.34%, 1.24% and 0.48%, which shows that the method has good stability.
3.5 repeatability test
Precisely weighing 6 parts of the same centipede herb, each part is about 1g, preparing 6 parts of test solution in parallel according to the method under the item 2.3 and determining according to the method. As a result, the average contents of astragalin, Multiflorin B, afzelin and Multiflorin A were 0.286, 11.075, 0.238 and 7.772mg g, respectively-1RSD values were 2.47%, 1.55%, 2.30%, 1.84%, respectively, indicating good reproducibility of the method.
3.6 sample application recovery test
Precisely weighing 0.5g of sample with known content (each 1g of Gekko Swinhonis medicinal material contains astragalin 0.286mg, Multiflorin B11.075 mg, African glycosides 0.238mg, and Multiflorin A7.772 mg), and precisely adding astragalin (0.143 mg. mL) in 6 parts in parallel-1）、Multiflorin B（5.55mg·mL-1) Afzelin (0.120 mg. mL)-1）、Multiflorin A（3.85mg·mL-1) The mixed reference substance solution is 1mL, volatilized, the sample solution is prepared according to the method under the item 2.3, and the sample injection recovery rate is calculated through sample injection measurement. As a result, the sample recovery rates of astragalin, Multiflorin B, afzelin and Multiflorin A were 95.38%, 97.38%, 100.42% and 105.36%, respectively, and the RSD values were 1.82%, 2.64%, 1.43% and 1.36%, respectively,the recovery rate of the process was good and the results are shown in Table 8.
Table 8.4 average recovery of flavonoids (n =6)
3.7 measurement of sample content
Respectively taking 2 centipede medicinal materials of different batches, precisely weighing 1g, preparing a sample solution according to a method of 2.3, injecting samples under a chromatographic condition of 2.1, and calculating the contents of astragalin, Multiflorin B, afzerin and Multiflorin A, wherein the results are shown in a table 9.
TABLE 9 content determination results of 4 flavonoid components in Scolopendra subspinipes medicinal materials of different batches
According to the results of liquid phase maps, the 4 ingredients including astragalin, Multiflorin B, African fordii glycoside and Multiflorin A can be well separated from other ingredients, and the method has accuracy and feasibility.
4. Nuclear magnetic data of control astragalin, Multiflorin B, African glycosides, and Multiflorin A
Multiflorin A：1H-NMR ( MeOD,500 MHz )，δ7.7(2H, d, J = 8.8Hz, H-2′，H-6′)，δ6.92(2H, d, J = 8.8Hz ，H-3′，H-5′)，δ6.32 (1H, d, J = 1.9Hz, H-8)，δ6.15(1H, d, J = 2.0Hz, H-6)，δ5.33 (1H，s, J = 1.5Hz，H-1′′)，δ4.47（1H，s, J = 7.9Hz H-1′′′），δ2.0（3H，s, CH3COO），δ0.92（3H，d, J = 1.5Hz H-6′′）；13C-NMR (MeOD,100MHz) δ159.3(C-2)，δ136.1 (C-3)，δ179.5(C-4)，δ163.1(C-5)，δ99.9(C-6)，δ165.8 (C-7)，δ94.9(C-8)，δ158.4 (C-9)，δ105.9(C-10)，δ122.6(C-1′)，δ131.9(C-2′，5′)，δ116.5(C-3′，5′)，δ105.6(C-1′′′)，δ83.2(C-5′′′)，δ75.2(C-3′′′)，δ71.8(C-2′′′)，δ71.7(C-4′′′)，δ64.8(C-6′′′)，δ20.81(CH3)，δ172.8(C=O)，δ103.3(C-1′′)，δ78.0(C-5′′)，δ75.9(C-3′′)，δ72.1(C-2′′)，δ70.5(C-4′′)，δ17.8(C-6′′)。
Multiflorin B：1H-NMR (MeOD, 500 MHz)：δ12.6(1H,s,5-OH)，δ7.75(2H, d, J= 8.8Hz, H-2′,6′)，δ6.92(2H, d, J = 8.8Hz ，H-3′,5′)，δ6.43 (1H, d, J = 1.9Hz, H-8)，δ6.22(1H, d, J = 2.0Hz, H-6)，δ5.48（1H，s, H-1′′），δ4.05（1H，s, H-1′′′），δ0.90（3H, d, rha-CH3）；13C-NMR (MeOD,500 MHz)：δ157.3(C-2)，δ134.4(C-3)，δ177.68(C-4)，δ161.3 (C-5)，δ98.8(C-6)，δ164.3 (C-7)，δ93.81(C-8)，δ156.5 (C-9)，δ104.7(C-10)，δ120.4(C-1′)，δ131.6(C-2′，5′)，δ115.5(C-3′，5′)，δ104.1(C-1′′′)，δ82.0(C-5′′′)，δ74.5(C-3′′′)，δ69.8(C-2′′′)，δ69.7(C-4′′′)，δ60.96 (C-6′′′)，δ101.8(C-1′′)，δ76.9(C-5′′)，δ76.6(C-3′′)，δ70.3(C-2′′)，δ68.9(C-4′′)，δ17.3(C-6′′)。
Afzelin:1H-NMR( MeOD， 500Hz )，δ6.15( 1H，d，J = 2.1 Hz ， H-6 )，δ6.31 ( 1H ， d ，J = 2.1 Hz ， H-8 )，δ7.72（2H ， dt ， J = 6.9，2 Hz ， H-2’、，H-6’、），δ6.90（2H ， dt ， J = 6.9，2 Hz ， H-3’、，H-5’、），δ5.36 ( 1H , d , J = 1.65 Hz ， H-1’’ )， 13C-NMR（MeOD，125Hz），δ158.4 ( C-2 )，δ136.2 ( C-3 ), δ179.5 (C-4)，δ163.1 (C-5 )，δ99.8 ( C-6 )，δ165.8 ( C-7 )，δ94.8 ( C-8 )，δ159.2 ( C-9 )，δ105.9 ( C-10 )，δ122.6 ( C-1’、)，δ131.9 ( C-2’，6’、)，δ116.5 ( C-3’，5’、)，δ161.5 ( C-4’、)，δ103.5 ( C1’’)，δ72.1 ( C2’’)，δ72.0 ( C3’’)，δ73.2 ( C4’’)，δ71.9 ( C5’’)，δ17.7 ( C6’’)。
astragalin:1H-NMR( MeOD， 500Hz )，δ6.19( 1H，d，J = 2.1 Hz ， H-6 )，δ6.39 ( 1H ， d ，J = 2.1 Hz ， H-8 )，δ8.04（2H ， dt ， J = 6.9，2 Hz ， H-2’、，H-6’、），δ6.87（2H ， dt ， J = 6.9，2 Hz ， H-3’、，H-5’、），δ5.25 ( 1H , s , H-1’’ )， 13C-NMR（MeOD，125Hz），δ158.5 ( C-2 )，δ135.4 ( C-3 ), δ179.5 ( C-4 )，δ163.1 ( C-5 )，δ99.9 ( C-6 )，δ166.0 ( C-7 )，δ94.7 ( C-8 )，δ159.1 ( C-9 )，δ105.7 ( C-10 )，δ122.8 ( C-1’、)，δ132.3 ( C-2’，6’、)，δ116.1 ( C-3’，5’、)，δ161.6 ( C-4’、)，δ104.0 ( C1’’)，δ75.7 ( C2’’)，δ78.4 ( C3’’)，δ71.3 ( C4’’)，δ78.0 ( C5’’)，δ62.6 ( C6’’)。
FIG. 9 shows NMR spectra of astragalin (A)1H NMR chart, FIG. 10 is nuclear magnetic resonance carbon spectrum of astragalin ((R))13C NMR) pattern; FIG. 11 is a nuclear magnetic resonance hydrogen spectrum of Multiflorin B (M1H NMR chart, FIG. 12 is nuclear magnetic resonance carbon spectrum of Multiflorin B (C:)13C NMR) pattern; FIG. 13 shows NMR spectra of afzelin1H NMR) chart, FIG. 14 is nuclear magnetic resonance carbon spectrum of Affyllin: (13C NMR) pattern; FIG. 15 is a nuclear magnetic resonance hydrogen spectrum of Multiflorin A (1H NMR, FIG. 16 is a nuclear magnetic resonance carbon spectrum of Multiflorin A (13C NMR) graph. The nuclear magnetic data shows that the compounds are astragalin, multiforin B, afzerin and multiforin A respectively.
Claims (5)
1. A method for simultaneously determining the content of 5 flavonoid compounds in centipede medicinal materials is characterized in that the centipede is derived from Polypodia gigas which is a water dragon orthopedic plantArthromeris mairei(Brause) Ching; the method comprises the steps of preparing a test solution, preparing a reference solution, detecting and analyzing, and specifically comprises
(1) Preparation of a test solution: taking 1g of centipede medicinal material powder, precisely weighing, placing in a conical flask, precisely adding 50mL of methanol water, weighing the mass, and heating and refluxing; cooling, weighing, adding solvent to supplement the weight loss, shaking, and filtering; precisely measuring 5mL of filtrate, placing the filtrate in a 25mL volumetric flask, adding methanol water to a constant volume, and shaking up to obtain a test solution;
(2) preparation of a reference solution:
A. precisely weighing afzedin-3-O-alpha-L-rhamnose reference substance, and preparing with 50% methanol to obtain a 20 μ g.mL solution-1Filtering the reference solution with a 0.45-micrometer microporous membrane to obtain a reference solution A for later use;
B. accurately weighing astragalin, Multiflorin B, African glycosides, and Multiflorin A reference substances, respectively, mixing, and making into astragalin with concentration of 0.8 μ g.mL with methanol-1Multiflorin B concentration 54. mu.g.mL-1And the concentration of the afzerin is 1.5 mu g-1Multiflorin A concentration 20. mu.g.mL-1To obtain a reference substance solution, namely a reference substance solution B for later use;
(3) and (3) detection: injecting the test solution, the reference solution A and the reference solution B into a high performance liquid chromatograph for detection; the chromatographic conditions of the high performance liquid chromatograph of the test solution are as follows:
A. a chromatographic column: 4.6X 250mm,5 μm Waters C18The mobile phase is as follows: methanol, water V/V =35:65 isocratic elution; flow rate 1.0mL/min-1The column temperature is 25 ℃, the detection wavelength is 230nm, and the sample injection amount is 10 mu L;
B. a chromatographic column: 4.6X 250mm,5 μm Waters SunAire C18The mobile phase is 0.2 percent phosphoric acid water solution C-methanol D, and the gradient elution is as follows: 0-30 min, C: D V/V =53: 47; 30-40 min, 47% D → 66% D; flow rate 1.0mL/min-1Detecting the wavelength of 265nm, the column temperature of 25 ℃ and the sample injection amount of 10 mu L;
the hplc conditions of the control solution A, B were A, B;
(4) quantitative analysis of 5 flavonoid compounds in centipede medicinal materials is carried out by an external standard method: performing regression analysis by comparing the chromatographic peak area of the flavonoid compounds with known concentration with the corresponding concentration to obtain a standard curve, measuring the test solution, measuring the chromatographic peak area of 5 flavonoid compounds in the test solution, substituting into the standard curve, and obtaining the content of the 5 flavonoid compounds in the centipede medicinal material.
2. The method according to claim 1, wherein the particle size of the centipede powder in step (1) is 65 meshes.
3. The method according to claim 1, wherein the methanol-water concentration in the step (1) is 75% methanol.
4. The method according to claim 1, wherein the heating reflux time in step (1) is 1 hour.
5. The assay method according to claim 1, wherein the extraction temperature in step (1) is 70 ℃ ± 2 ℃.
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