CN106770781B - Method for simultaneously determining five flavones in Chinese herbal medicine or health product - Google Patents
Method for simultaneously determining five flavones in Chinese herbal medicine or health product Download PDFInfo
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- CN106770781B CN106770781B CN201611262282.8A CN201611262282A CN106770781B CN 106770781 B CN106770781 B CN 106770781B CN 201611262282 A CN201611262282 A CN 201611262282A CN 106770781 B CN106770781 B CN 106770781B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
Abstract
The invention discloses a method for simultaneously measuring five flavones in Chinese herbal medicines or health products, which comprises the steps of extracting a dry solid medicinal material or health product sample by using methanol, using acetonitrile and 0.5% (v/v) phosphoric acid as mobile phases by using a high performance liquid chromatography, carrying out gradient elution after sample loading to obtain a chromatogram of puerarin, dihydromyricetin, rutin, quercetin and kaempferol with good separation degree, and calculating the content of the five flavones in the raw material according to the integral area of the characteristic peak of each flavone. The method is particularly suitable for rapidly detecting the identification and quantification of the flavone with large polarity difference in the health care products or the Chinese herbal medicines and evaluating the content of the drug effect substances.
Description
Technical Field
The invention relates to the field of chemical analysis and detection, in particular to a method for simultaneously detecting five flavonoids with large polarity differences by utilizing HPLC (high performance liquid chromatography).
Background
The flavonoid substances are effective components of various health products and medicinal materials, and the flavonoid substances such as puerarin, dihydromyricetin, rutin, quercetin, kaempferol and the like are common flavonoid components and are widely distributed in Chinese herbal medicines. Puerarin has effects of increasing cerebral blood flow, enhancing immunity, improving learning and memory, resisting tumor, reducing blood sugar, and relieving hangover; dihydromyricetin has effects of resisting oxidation, resisting bacteria, relieving inflammation, protecting liver, regulating blood sugar and blood lipid, and smoothing muscle; rutin has effects in reducing capillary permeability and fragility, promoting cell proliferation and preventing hemagglutination, resisting inflammation, promoting urination, relieving spasm, relieving cough, and reducing blood lipid; quercetin has antioxidant, antiinflammatory, antiallergic, antibacterial, antiviral, free radical scavenging, and malignant tumor growth and metastasis inhibiting effects; kaempferol has antioxidant, antiinflammatory, tumor growth inhibiting, and liver cell protecting effects. With the intensive research on the physiological efficacy of the flavone, Chinese herbal medicines containing the flavone with health care functions are added into a plurality of health care products, and the content of the flavone also becomes a detection index for evaluating the effective components of the health care products.
However, different flavone compounds have large polarity difference, so that the detection efficiency is influenced by too long time for simultaneously detecting a plurality of flavones with large polarity difference. In addition, flavones with similar structures often exist in natural medicinal materials, and when one kind of flavone is separated by using one elution system, the other kind of flavone is difficult to separate. Therefore, the method for rapidly and accurately analyzing and measuring the flavonoid components with large polarity difference is established, and has important significance for quality control and scientific evaluation of health care products and Chinese medicinal materials containing various flavones.
Disclosure of Invention
The invention aims to provide a method for simultaneously detecting various polar flavones by using high performance liquid chromatography, aiming at the defects of the prior art, the method is simple, high in accuracy and good in reproducibility, and can provide reliable basis for scientific evaluation and effective control of the internal quality of health care products and Chinese herbal medicines containing the five flavonoids.
The invention aims to realize the following technical scheme that the method for simultaneously measuring five flavonoids in Chinese herbal medicines or health products comprises the following steps of:
(1) sample preparation: freeze drying the health product or Chinese medicinal materials, pulverizing into powder, and sieving with 80 mesh sieve; adding the powder into methanol according to a mass-to-volume ratio (g/mL) of 1:200, shaking for 20min, performing ultrasonic treatment for 60min, centrifuging for 10min at 3000g, filtering the supernatant with a 0.45 μm microporous membrane to obtain a sample, and storing the sample in a sample bottle for sample injection;
(2) analyzing the sample prepared in the step 1 by using a reversed phase high performance liquid chromatography, and determining the peak area y of puerarin, dihydromyricetin, rutin, quercetin and kaempferol in the sample1、y2、y3、y4、y5(ii) a The adopted chromatographic column is a C18 column; the sample introduction volume is 10 mu L, the flow rate is 1.0mL/min, and the column temperature is 30 ℃; detection wavelength: 254nm and 290 nm; mobile phase: the organic phase A is acetonitrile; inorganic B phase contains a volume fraction of 0.5% (v-v) an aqueous solution of phosphoric acid; gradient elution is adopted, and the elution program is as follows according to the volume content of the organic phase A in the mobile phase: 0-10min, 16% A-13% A; 10-15min, 13% A-15% A; 15-20min, 15% A-35% A; 20-35min, 35% A-45% A; 35-43min, 45% A-15% A;
(3) obtaining the mass volume concentration (mg/L) of puerarin, dihydromyricetin, rutin, quercetin and kaempferol in the sample: by the formula y1＝39.977x1-1.251 the Mass volume concentration x of puerarin in the sample1(ii) a By the formula y2＝21.918x2+19.74 Mass volume concentration x of dihydromyricetin in the sample can be obtained2(ii) a By the formula y3＝19.398x3+12.15 the mass volume concentration x of rutin in the sample can be obtained3(ii) a By the formula y4＝30.844x47.8948 Mass volume concentration x of Quercetin in the sample is obtained4(ii) a By the formula y5＝35.457x512.72 Mass volume concentration x of Kaempferol in the sample5；
(4) Obtaining the mass contents (mg/g) of puerarin, dihydromyricetin, rutin, quercetin and kaempferol in the Chinese herbal medicine or the health care product: the puerarin mass content is 0.2x1(ii) a The mass content of dihydromyricetin is 0.2x2(ii) a The rutin content is 0.2x3(ii) a The mass content of quercetin is 0.2x4(ii) a The kaempferol content is 0.2x5。
The method has the beneficial effects that dry solid medicinal material or health-care product samples are extracted by methanol, high performance liquid chromatography is utilized, acetonitrile and 0.5% (v/v) phosphoric acid are taken as mobile phases, the chromatogram of puerarin, dihydromyricetin, rutin, quercetin and kaempferol with good separation degree is obtained by gradient elution after the samples are loaded, and the content of five flavones in the raw material is calculated according to the integral area of the characteristic peak of each flavone. The method is particularly suitable for rapidly detecting the identification and quantification of the flavone with large polarity difference in the health care products or the Chinese herbal medicines and evaluating the content of the drug effect substances. The detection method provided by the invention can separate flavonoid substances such as puerarin, dihydromyricetin, rutin, quercetin and kaempferol from the health care product and the Chinese medicinal materials at one time, and has the advantages of simple operation, accuracy and reliability and time saving.
Drawings
FIG. 1 is a chromatogram of a standard sample of flavones; wherein, the detection wavelength is 254nm, peak 1, puerarin; peak 2, dihydromyricetin; peak 3, rutin; peak 4, quercetin; peak 5, kaempferol;
FIG. 2 is a chromatogram of a standard sample of flavones; wherein, the detection wavelength is 290nm, peak 1, puerarin; peak 2, dihydromyricetin; peak 3, rutin; peak 4, quercetin; peak 5, kaempferol;
FIG. 3 is a chromatogram of a sample of a nutraceutical extract; wherein, the detection wavelength is 254nm, peak 1, puerarin; peak 3, rutin; peak 4, Quercetin.
FIG. 4 is a chromatogram of a sample of a nutraceutical extract; wherein the detection wavelength is 290 nm; peak 2, dihydromyricetin; peak 5, kaempferol.
Detailed Description
The present invention will be described in detail below based on examples, and the object and effect of the present invention will become more apparent.
Examples 1 and 5 qualitative separation and detection of flavonoid sample
1. Sample preparation: preparing mixed solution of 75.8 mu g/mL of dihydromyricetin, 66 mu g/mL of kaempferol, 34 mu g/mL of puerarin, 88.4 mu g/mL of quercetin and 38.6 mu g/mL of rutin;
2. analyzing the sample prepared in the step 1 by using a reversed phase high performance liquid chromatography, wherein the analysis conditions are as follows:
a chromatographic column: zorbax eclipse XDB-C18;
the sample introduction volume is 10 mu L, the flow rate is 1.0mL/min, and the column temperature is 30 ℃;
detection wavelength: 254nm and 290 nm;
mobile phase: the organic phase A is acetonitrile; the inorganic B phase is an aqueous solution containing 0.5 percent (v/v) of phosphoric acid by volume fraction; gradient elution is adopted, and the elution procedure is as follows:
0-10min，16％A-13％A；10-15min，13％A-15％A；15-20min，15％A-35％A；20-35min，35％A-45％A；35-43min，45％A-15％A。
the standard chromatogram obtained is shown in FIGS. 1 and 2: peak 1, puerarin; peak 2, dihydromyricetin; peak 3, rutin; peak 4, quercetin; peak 5, kaempferol. Different flavones are distinguished according to different peak emergence times, wherein dihydromyricetin has no absorption peak at 254nm wavelength, has an absorption peak at 290nm wavelength, the puerarin polarity maximum peak emergence time is the earliest, and other three flavones correspond to specific peak emergence times. The polar component in the system mobile phase elution system rises and then falls so that the non-target detection substance is eluted quickly, and compared with the existing elution system, the detection time is reduced. The flavone with larger polarity difference can be well separated by controlling the gradient elution of the mobile phase.
Example 2 precision, stability, reproducibility of detection method and sample recovery rate evaluation precision test
And respectively taking 6.8 mu g/mL, 13.6 mu g/mL, 20.4 mu g/mL, 27.2 mu g/mL and 34 mu g/mL puerarin reference substances, repeatedly injecting samples for 2 times according to the sample amount of 10 mu L of the reference substances, recording the peak area of the puerarin standard solution, and calculating the Relative Standard Deviation (RSD), wherein the relative standard deviations of different sample amounts are respectively 0.05%, 0.13%, 0.26%, 0.00% and 0.42%.
Sampling dihydromyricetin reference substances of 6.96 mu g/mL, 13.92 mu g/mL, 20.88 mu g/mL, 27.84 mu g/mL and 34.8 mu g/mL respectively for 3 times according to the sampling amount of 10 mu L, recording the peak area of the dihydromyricetin standard solution, and calculating the Relative Standard Deviation (RSD), wherein the relative standard deviations of different sampling amounts are respectively 0.35%, 0.38%, 0.06%, 0.04% and 0.38%.
Taking 10.56 mu g/mL, 21.12 mu g/mL, 31.68 mu g/mL and 42.24 mu g/mL rutin reference substances respectively, repeatedly injecting samples for 3 times according to the sample amount of 10 mu L of the reference substances, recording the peak area of the rutin standard solution, and calculating the Relative Standard Deviation (RSD), wherein the relative standard deviations of different sample amounts are 1.73%, 0.17%, 0.18% and 0.18% respectively.
Taking 8.96 mu g/mL, 17.92 mu g/mL, 26.88 mu g/mL, 35.84 mu g/mL, 44.8 mu g/mL and 89.6 mu g/mL of quercetin reference substances according to the sample amount of 10 mu L of the reference substances, repeatedly injecting samples for 3 times respectively, recording the peak area of the quercetin standard solution, and calculating the Relative Standard Deviation (RSD), wherein the relative standard deviations of different sample amounts are respectively 1.67%, 0.07%, 0.12%, 0.08%, 0.11% and 0.13%.
Sampling 7.92 mu g/mL, 15.84 mu g/mL, 23.76 mu g/mL, 31.68 mu g/mL, 39.6 mu g/mL and 79.2 mu g/mL kaempferol reference substances for 3 times according to the sampling amount of 10 mu L of the reference substances respectively, recording the peak area of the kaempferol standard solution, and calculating the Relative Standard Deviation (RSD), wherein the relative standard deviations of different sampling amounts are 1.75%, 0.23%, 0.15%, 0.01%, 0.09% and 0.09% respectively.
The standard deviation of the detected peak area of each standard substance is less than 0.5 percent, which shows that the method has good precision and can obtain accurate results by adopting the least repeated measurement times.
Repeatability test
Precisely measuring 6 parts of 1mL solution in the same test solution, injecting sample by 10 μ L, recording peak area of dihydromyricetin in the sample, and calculating RSD to be 0.39%.
Precisely measuring 6 parts of 1mL solution in the same test solution, injecting samples of 10 mu L, recording the peak area of puerarin in the samples, and calculating the RSD to be 0.13%.
Precisely measuring 6 parts of 1mL of solution in the same test solution, injecting samples of 10 mu L respectively, recording the peak area of rutin in the samples, and calculating the RSD to be 0.32%.
Precisely measuring 6 parts of 1mL solution in the same test solution, injecting samples of 10 mu L, recording peak areas of quercetin in the samples, and calculating RSD to be 0.22%.
Precisely measuring 6 parts of 1mL of solution in the same test solution, injecting samples of 10 mu L respectively, recording peak areas of kaempferol in the samples, and calculating RSD to be 0.28%.
The peak area standard deviation of six sample injection detections of the same volume and equal concentration sample of each standard substance is less than 0.5 percent, which shows that the method has good repeatability and can obtain accurate results by adopting the least repeated detection times.
Stability of
Precisely measuring 1mL of solution in the sample, measuring at 0, 2.5, 5, 7.5, 10, 12.5 and 15h respectively, recording the peak area of dihydromyricetin in the sample, and calculating RSD to be 0.49%.
Precisely measuring 1mL of solution in the sample, measuring under 0, 2.5, 5, 7.5, 10, 12.5 and 15h respectively, recording the peak area of puerarin in the sample, and calculating RSD to be 0.22%.
Precisely measuring 1mL of solution in a test sample, measuring under 0, 2.5, 5, 7.5, 10, 12.5 and 15h respectively, recording the peak area of rutin in the sample, and calculating the RSD to be 0.46%.
Precisely measuring 1mL of solution in a test sample, measuring under 0, 2.5, 5, 7.5, 10, 12.5 and 15h respectively, recording peak area of quercetin in the sample, and calculating RSD to be 0.38%.
Precisely measuring 1mL of solution in the sample, measuring at 0, 2.5, 5, 7.5, 10, 12.5 and 15h respectively, recording peak area of kaempferol in the sample, and calculating RSD to be 0.25%.
The standard deviation of peak areas of the same volume and concentration samples of each standard substance at different sample injection times is less than 0.5 percent, which shows that the method has good stability and stable and accurate data measured at different times.
Sample recovery rate
Taking 1.25mL of tested sample solution containing 85.112 mug/mL, precisely measuring 5 parts of the tested sample solution, placing the measured sample solution into a 10mL volumetric flask, precisely measuring 0.07mL of 1.74mg/mL dihydromyricetin standard substance, placing five parts of the tested sample solution into the volumetric flask respectively, metering the volume to 10mL by methanol, ultrasonically mixing the solutions, filtering the solutions by a 0.45-micron syringe filter membrane, performing chromatographic determination, recording peak areas of the dihydromyricetin in the samples, and calculating the average recovery rate to be 95.78 +/-0.01%.
Taking 0.5mL of solution containing 130.4 mu g/mL of test sample, precisely measuring 5 parts of the solution, placing the solution in a 10mL volumetric flask, precisely measuring 2mL of 34 mu g/mL puerarin standard substance, placing five parts of the solution in the volumetric flask respectively, fixing the volume to 10mL by methanol, ultrasonically mixing the solution, filtering the solution by a 0.45 mu m syringe filter membrane, performing chromatographic determination, recording the peak area of dihydromyricetin in the sample, and calculating the average recovery rate to be 100.05 +/-0.35%.
Taking 0.5mL of tested sample solution containing 116.33 mu g/mL, precisely measuring 5 parts of each solution, placing the solution into a 10mL volumetric flask, precisely measuring 1mL of rutin standard substance containing 52.8 mu g/mL, placing the five parts of each solution into the volumetric flask respectively, fixing the volume to 10mL by methanol, ultrasonically mixing the solution, filtering the solution by a 0.45 mu m syringe filter membrane, performing chromatographic determination, recording the peak area of rutin in a sample, and calculating the average recovery rate to be 99.99 +/-0.62%.
Taking 0.5mL of a solution containing 82.64 mu g/mL of a test sample, precisely measuring 5 parts of each solution, placing the solution into a 10mL volumetric flask, precisely measuring 1mL of 39.6 mu g/mL kaempferol standard substance, placing the five parts of each solution into the volumetric flask respectively, metering the volume to 10mL by using methanol, ultrasonically mixing the solutions, filtering the solutions by using a 0.45 mu m syringe filter membrane, performing chromatographic determination, recording the peak area of rutin in a sample, and calculating the average recovery rate to be 99.98 +/-0.43%.
Taking 0.50mL of solution containing 99.27 mu g/mL of test sample, precisely measuring 5 parts of each solution, placing the solution in a 10mL volumetric flask, precisely measuring 1.10mL of 44.80 mu g/mL of quercetin standard sample, placing the five parts of each solution in the volumetric flask respectively, metering the volume to 10mL by methanol, ultrasonically mixing the solutions, filtering the solution by a 0.45 mu m syringe filter membrane, performing chromatographic determination, recording the peak area of quercetin in the sample, and calculating the average recovery rate of 100.41 +/-0.40%.
The recovery rate experiment shows that the standard recovery rate error of each target detection substance is within 0.5 percent, the detection substance does not have overload in a linear range, and the experimental method has small system error.
Example 3 creation of Standard Curve
After the obtained standard substance solutions with various concentrations are filtered by a 0.45 mu m organic microporous filter membrane according to the chromatographic conditions of the example 1, sample injection is carried out to precisely absorb 0, 2, 4, 6, 8 and 10 mu L of the mixed standard substance solution for determination, each sample is repeated for 3 times to obtain peak areas corresponding to the standard substances with gradient content, and the specific data are shown in a table 2. Taking the peak area as a vertical coordinate (y) and the mass concentration (x) as a horizontal coordinate, drawing a standard curve, and calculating a regression equation and a correlation coefficient of the standard curve, wherein the result is shown in table 1.
Table 1: linear regression equation of puerarin, dihydromyricetin, rutin, kaempferol and quercetin
| Name of Compound | Regression equation | Coefficient of correlation (R)2) | Linear Range (μ g/mL) |
| Puerarin and its preparation method | y＝39.977x－1.251 | 0.9995 | 6.80-37.00 |
| Dihydromyricetin | y＝21.918x+19.74 | 0.9947 | 6.96-27.84 |
| Rutin | y＝19.398x+12.15 | 0.9986 | 10.56-42.24 |
| Kaempferol | y＝35.457x－12.72 | 1 | 7.92-79.20 |
| Quercetin | y＝30.844x－7.89 | 1 | 8.96-89.60 |
Table 2: peak areas of different sample volumes of puerarin, dihydromyricetin, rutin, kaempferol and quercetin
Based on the detection system established by the method, quantitative standard curves of 5 flavones are established, the linear relation of the standard curves in a linear range is good, the correlation coefficients are all more than 99.9%, and the corresponding flavone content in the sample can be accurately quantified. The invention adopts high-polarity eluent to carry out proportional elution, thereby accelerating the elution time; the gradient elution method with the concentration of the polar eluent rising first and then falling can simultaneously separate five flavonoid substances of puerarin, dihydromyricetin, rutin, quercetin and kaempferol and analogues thereof with larger polarity difference but similar structures in the health care product.
Example 4 detection of flavonoids in certain health products
1. Sample preparation: sample preparation: the health product granule or Chinese herbal medicine is lyophilized, pulverized into powder, and sieved with 80 mesh sieve. Weighing 0.5g of powder, adding 100mL of methanol solution, shaking for 20min, performing ultrasonic treatment for 60min, centrifuging for 10min at 3000 Xg, taking 1mL of supernatant, diluting by 10 times, filtering by a 0.45-micrometer microporous membrane, and storing in a sample bottle for sample injection;
2. analyzing the sample prepared in the step 1 by using a reversed phase high performance liquid chromatography, wherein the analysis conditions are as follows:
a chromatographic column: zorbax eclipse XDB-C18;
the sample introduction volume is 10 mu L, the flow rate is 1.0mL/min, and the column temperature is 30 ℃;
detection wavelength: 254nm and 290 nm;
mobile phase: the organic phase A is acetonitrile; the inorganic B phase is an aqueous solution containing 0.5 percent (v/v) of phosphoric acid by volume fraction; gradient elution is adopted, and the elution procedure is as follows:
0-10min，16％A-13％A；10-15min，13％A-15％A；15-20min，15％A-35％A；20-35min，35％A-45％A；35-43min，45％A-15％A。
the chromatograms obtained are shown in FIGS. 3 and 4.
The puerarin peak area is 1461.94397, the calculated puerarin concentration is 36.60 μ g/mL, and the puerarin concentration is converted according to the sample preparation proportion, wherein each gram of the health care product contains 73.20mg puerarin.
The peak area of the dihydromyricetin is 668.07550, the calculated injection concentration is 29.58 mug/mL, and the dihydromyricetin is obtained by converting according to the preparation proportion of the sample, wherein each gram of the health care product contains 59.16mg of dihydromyricetin.
The peak area of rutin is 268.20365, the standard curve in the embodiment 3 is substituted, the concentration of rutin is calculated to be 13.20 mu g/mL, and 26.40mg of rutin is contained in each gram of health care product after conversion according to the preparation proportion of the sample.
The peak area of the quercetin was 337.14377, which was substituted into the standard curve in example 3, and calculated as 11.19 μ g/mL, and converted according to the sample preparation ratio to obtain a product containing 22.37mg quercetin per gram of the health product.
The peak area of kaempferol is 282.10318, which is substituted into the standard curve in example 3, and the calculated value is 8.56 μ g/mL, and the kaempferol content in each gram of health care product is 17.11mg after conversion according to the sample preparation proportion.
The method can rapidly separate 5 kinds of flavones with large polarity difference at one time, accurately quantify the flavone, and rapidly and accurately quantify the content of the components in the health product.
Claims (1)
1. A method for simultaneously measuring five flavones in Chinese herbal medicines or health products is characterized in that the five flavones are puerarin, dihydromyricetin, rutin, quercetin and kaempferol, and the method comprises the following steps:
(1) sample preparation: freeze drying the health product or Chinese medicinal materials, pulverizing into powder, and sieving with 80 mesh sieve; adding the powder into methanol according to the mass-to-volume ratio of 1:200g/mL, shaking for 20min, performing ultrasonic treatment for 60min, centrifuging for 10min at 3000g, filtering the supernatant with a 0.45-micrometer microporous membrane to obtain a sample, and storing the sample in a sample bottle for sample injection;
(2) analyzing the sample prepared in the step 1 by using a reversed phase high performance liquid chromatography, and determining the peak area y of puerarin, dihydromyricetin, rutin, quercetin and kaempferol in the sample1、y2、y3、y4、y5(ii) a The adopted chromatographic column is a C18 column; the sample introduction volume is 10 mu L, the flow rate is 1.0mL/min, and the column temperature is 30 ℃; detection wavelength: 254nm and 290 nm; mobile phase: the organic phase A is acetonitrile; the inorganic phase B is an aqueous solution containing 0.5% v/v by volume of phosphoric acid; gradient elution is adopted, and the elution program is as follows according to the volume content of the organic phase A in the mobile phase: 0-10min, 16% A-13% A; 10-15min, 13% A-15% A; 15-20min, 15% A-35% A; 20-35min, 35% A-45% A; 35-43min, 45% A-15% A;
(3) obtaining a linear regression equation, and obtaining the mass volume concentration mg/L of puerarin, dihydromyricetin, rutin, quercetin and kaempferol in the sample: by the formula y1＝39.977x1-1.251 obtaining the mass volume concentration x of puerarin in the sample1(ii) a By the formula y2＝21.918x2+19.74 obtaining the mass volume concentration x of dihydromyricetin in the sample2(ii) a By the formula y3＝19.398x3+12.15 obtaining the mass volume concentration x of rutin in the sample3(ii) a By the formula y4＝30.844x4-7.89 obtaining the mass volume concentration x of quercetin in the sample4(ii) a By the formula y5＝35.457x5-12.72 obtaining the mass volume concentration x of kaempferol in the sample5；
(4) Obtaining the Chinese herbal medicine or the health product, wherein the mass contents of puerarin, dihydromyricetin, rutin, quercetin and kaempferol in the Chinese herbal medicine or the health product are as follows: the puerarin mass content is 0.2x1(ii) a The mass content of dihydromyricetin is 0.2x2(ii) a The rutin content is 0.2x3(ii) a The mass content of quercetin is 0.2x4(ii) a The kaempferol content is 0.2x5。
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