Method for measuring content of flavonoid components of scutellaria baicalensis in lung-heat-clearing decoction

Abstract

The invention relates to the technical field of detection methods, and provides a method for determining the content of flavonoid components of scutellaria baicalensis in lung-heat clearing decoction, which comprises the following steps: determining the content of flavonoid components of scutellaria baicalensis in a test solution by high performance liquid chromatography, the flavonoid components of scutellaria baicalensis comprise baicalin, wogonoside, baicalein and wogonin; measurement conditions of the high performance liquid chromatography include that a chromatographic column is a C18 chromatographic column, acetonitrile is used as a mobile phase A, and a 0.08-0.13 v / v% phosphoric acid solution is used as a mobile phase B.

Description

Technical Field

[0001] The present invention relates to the technical field of detection methods, and in particular, to a method for determining the content of flavonoid components in Qingfei Decoction Background Art

[0002] Qingfei Decoction is included in the "Catalogue of Ancient Classical Famous Prescriptions (the First Batch)" announced by the State Administration of Traditional Chinese Medicine in the first batch in 2018, and it comes from "Myriad Diseases Returning to Spring" written by Gong Tingxian, a famous medical scientist in the Ming Dynasty. It is recorded that Qingfei Decoction is mainly used to treat all coughs and excessive phlegm in the upper jiao. This prescription is composed of 15 herbs including Scutellaria baicalensis, Platycodon grandiflorum, Poria cocos, Citrus reticulata Blanco, Fritillaria thunbergii Miq., Morus alba L., Angelica sinensis (Oliv.) Diels, Asparagus cochinchinensis (Lour.) Merr., Gardenia jasminoides Ellis, Prunus armeniaca L. var. ansu Maxim., Ophiopogon japonicus (Thunb.) Ker-Gawl., Schisandra chinensis (Turcz.) Baill., Glycyrrhiza uralensis Fisch., Zingiber officinale Rosc., and Ziziphus jujuba Mill. It has the effects of clearing the lung, resolving phlegm, and relieving cough. Modern clinical practice often uses it to treat respiratory diseases such as pneumonia, bronchitis, cough, and acute respiratory distress syndrome, with significant curative effects and fewer adverse reactions. It is one of the key objects for the research and development of traditional Chinese medicine compound preparations of classical famous prescriptions

[0003] Scutellaria baicalensis, also known as Radix Scutellariae and Radix Scutellariae Amurensis, is a perennial herb of the genus Scutellaria in the Lamiaceae family. The wild and cultivated resources of Scutellaria baicalensis are widely distributed, and the most large-scale main production areas include Chengde in Hebei, Shangluo in Shaanxi, and Chifeng in Inner Mongolia. The flavonoid components in Scutellaria baicalensis are the important pharmacological activity basis and also the most commonly used quality evaluation index of Scutellaria baicalensis. Baicalin and wogonoside in Scutellaria baicalensis have pharmacological activities such as anti-allergy, antipyretic, anti-inflammatory, and immunomodulatory effects. Baicalein and wogonin are two key flavonoid aglycones in Scutellaria baicalensis, which have anti-inflammatory, immunomodulatory, and neuroprotective effects. Research shows that the content of baicalin in Scutellaria baicalensis varies with factors such as planting years, origin, processing methods, and storage conditions. The "Technical Guidelines for the Pharmaceutical Research of Traditional Chinese Medicine Compound Preparations" points out that it is necessary to study and determine the material basis of traditional Chinese medicine compound preparations of ancient classical famous prescriptions, and reasonably determine the fluctuation range of the quantitative values of key quality attributes, so as to determine the quality standards of reference samples. Therefore, only by comprehensively mastering the actual situation of the most critical components of Scutellaria baicalensis, namely baicalin, wogonoside, baicalein, and wogonin, in the preparation process can the quality of the preparation be comprehensively evaluated. However, baicalin and wogonoside, baicalein and wogonin in the flavonoid components of Scutellaria baicalensis have similar structures, and it is difficult to achieve efficient separation and synchronous quantification by traditional methods, which easily leads to peak overlap or baseline interference. Moreover, when detecting the flavonoid components in Qingfei Decoction, there is a problem of insufficient sensitivity

[0004] Therefore, a method for determining the content of 4 flavonoid components, namely baicalin, wogonoside, baicalein, and wogonin, applicable to Scutellaria baicalensis and Qingfei Decoction was studied and established. By investigating the transfer law of the quantitative values of key indicators in the whole process of Scutellaria baicalensis from medicinal materials to decoction pieces and then to Qingfei Decoction in 4 main production areas, a method was provided for the quality evaluation and process optimization of Qingfei Decoction Summary of the Invention

[0005] The present invention provides a method for determining the content of flavonoid components of Scutellaria baicalensis Georgi in Qingfei Decoction, which solves the problems of low detection sensitivity and difficulty in efficient separation of flavonoid substances of Scutellaria baicalensis Georgi in Qingfei Decoction in the related art.

[0006] The technical solution of the present invention is as follows: The present invention provides a method for determining the content of flavonoid components of Scutellaria baicalensis Georgi in Qingfei Decoction, comprising the following steps: using high performance liquid chromatography to determine the content of flavonoid components of Scutellaria baicalensis Georgi in the test solution, and the flavonoid components of Scutellaria baicalensis Georgi include baicalin, wogonoside, baicalein, and wogonin; The determination conditions of the high performance liquid chromatography include: the chromatographic column is a C 18 chromatographic column, acetonitrile is used as mobile phase A, and 0.08 - 0.13 v / v% phosphoric acid solution is used as mobile phase B; the elution method is gradient elution, and the program of the gradient elution is as follows: 。

[0007] As a further technical solution, the determination conditions of the high performance liquid chromatography further include: the column temperature is 30 - 40 °C, for example, it can be 30 °C, 32 °C, 35 °C, 38 °C, 40 °C, preferably 30 °C, 35 °C, 40 °C, more preferably 35 °C, the flow rate is 0.9 - 1.1 mL / min, for example, it can be 0.9 mL / min, 1.0 mL / min, 1.1 mL / min, preferably 1.0 mL / min, the detection wavelength is 276 nm, and the theoretical plate number calculated by baicalin is not less than 10000.

[0008] In the present invention, when detecting baicalin, wogonoside, baicalein, and wogonin, the detection wavelength was explored. The reference substance solutions of baicalin, wogonoside, baicalein, and wogonin were scanned spectroscopically at 200 nm - 400 nm, and all had the maximum absorption near the wavelength of 276 nm.

[0009] As a further technical solution, the filler in the C 18 chromatographic column is octadecylsilyl silica gel, and the C 18 chromatographic column is specifically a SHISEIDO Capcell pak C18 MGⅡ chromatographic column, with a specification of 5 µm, 4.6 mm × 250 mm.

[0010] As a further technical solution, the preparation method of the test solution includes the following steps: taking the reference sample of Qingfei Decoction and methanol for ultrasonic mixing, filtering, and taking the filtrate as the test solution.

[0011] As a further technical solution, the mass - volume ratio of the reference sample of Qingfei Decoction to the methanol is 0.1 g:15 - 25 mL, preferably 0.1 g:20 mL.

[0012] As a further technical solution, the preparation method of the reference sample of the Qingfei Decoction comprises the following steps: Weigh the slices of Scutellaria baicalensis, Platycodon grandiflorum, Poria cocos, Citrus reticulata Blanco cv. Erythrosa, Fritillaria thunbergii Miq., Morus alba L. var. multicaulis (Perrott.) Loud., Angelica sinensis (Oliv.) Diels, Asparagus cochinchinensis (Lour.) Merr., Gardenia jasminoides Ellis, Armeniaca vulgaris Lam., Ophiopogon japonicus (Thunb.) Ker-Gawl., Schisandra chinensis (Turcz.) Baill., Glycyrrhiza uralensis Fisch., Zingiber officinale Rosc., and Ziziphus jujuba Mill., add them to water, soak them, after boiling, continue to decoct, filter to obtain a filtrate, concentrate the filtrate, cool it, pre-freeze it, and vacuum freeze-dry it to obtain the reference freeze-dried powder of the Qingfei Decoction, which is the reference sample of the Qingfei Decoction.

[0013] As a further technical solution, when boiling, close the lid and use strong fire with a power of 1190~1210W, for example, it can be 1190W, 1195W, 1200W, 1205W, 1210W, and preferably 1200W; When decocting, open the lid and use slow fire with a power of 590~610W for 45 min, for example, it can be 590W, 595W, 600W, 605W, 610W, and preferably 600W.

[0014] As a further technical solution, when concentrating the filtrate, concentrate the filtrate to a volume of 150~170 mL, for example, it can be 150 mL, 152 mL, 155 mL, 158 mL, 160 mL, 165 mL, 168 mL, 170 mL, and preferably 160 mL.

[0015] As a further technical solution, the preparation method of the slices of Scutellaria baicalensis comprises the following steps: Collect Scutellaria baicalensis herbs, process them, remove impurities, steam them, slice them, and dry them to obtain the slices of Scutellaria baicalensis.

[0016] In the present invention, the Scutellaria baicalensis herbs are 20 batches of herbs collected from different producing areas, and all these batches of herbs have been identified as genuine products by the Hebei Institute for Drug and Medical Device Control. The processing method is the processing method of the slices of Scutellaria baicalensis under the item of Scutellaria baicalensis in Part I of the Chinese Pharmacopoeia (2020 Edition). Except for the slices of Scutellaria baicalensis, the preparation methods of other slices, including the slices of Platycodon grandiflorum, Poria cocos, Citrus reticulata Blanco cv. Erythrosa, Fritillaria thunbergii Miq., Morus alba L. var. multicaulis (Perrott.) Loud., Angelica sinensis (Oliv.) Diels, Asparagus cochinchinensis (Lour.) Merr., Gardenia jasminoides Ellis, Armeniaca vulgaris Lam., Ophiopogon japonicus (Thunb.) Ker-Gawl., Schisandra chinensis (Turcz.) Baill., Glycyrrhiza uralensis Fisch., Zingiber officinale Rosc., and Ziziphus jujuba Mill., are the same as that of the slices of Scutellaria baicalensis.

[0017] As a further technical solution, the mass-to-volume ratio of the slices of Scutellaria baicalensis to the water is 5.6 g:440~460 mL, and preferably 5.6 g:450 mL; The added amounts of the slices of Platycodon grandiflorum, Poria cocos, Citrus reticulata Blanco cv. Erythrosa, Fritillaria thunbergii Miq., and Morus alba L. var. multicaulis (Perrott.) Loud. are equal, and the added amounts of the slices of Angelica sinensis (Oliv.) Diels, Asparagus cochinchinensis (Lour.) Merr., Gardenia jasminoides Ellis, Armeniaca vulgaris Lam., and Ophiopogon japonicus (Thunb.) Ker-Gawl. are equal; The addition amounts of the Scutellaria baicalensis Georgi decoction pieces, Platycodon grandiflorum decoction pieces, Angelica sinensis decoction pieces, Schisandra chinensis decoction pieces, Glycyrrhiza uralensis Fisch. decoction pieces, Zingiber officinale Rosc. decoction pieces, and Ziziphus jujuba Mill. decoction pieces are 5.6:3.5~3.8:2.5~2.7:1:1.1:2:6. For example, they can be 5.6:3.5:2.5:1:1.1:2:6, 5.6:3.7:2.5:1:1.1:2:6, 5.6:3.7:2.6:1:1.1:2:6, 5.6:3.8:2.7:1:1.1:2:6, and the preferred one is 5.6:3.7:2.6:1:1.1:2:6.

[0018] As a further technical solution, the volume fraction of the methanol is 50%~100%.

[0019] As a further technical solution, the volume fraction of the methanol is 70%.

[0020] As a further technical solution, the following steps are further included: determining the contents of Scutellaria flavonoids in the mixed reference substance solution and the negative sample solution by high performance liquid chromatography.

[0021] As a further technical solution, the preparation method of the mixed reference substance solution includes the following steps: taking reference substances of baicalin, wogonoside, baicalein, and wogonin, accurately weighing them, adding methanol, and respectively preparing mixed solutions containing 20 μg / mL of baicalin, wogonoside, baicalein, and wogonin, thus obtaining the mixed reference substance solution; The preparation method of the negative sample solution includes the following steps: taking the remaining medicinal material components in the Qingfei Decoction except the Scutellaria baicalensis Georgi decoction pieces, preparing the negative sample of the Qingfei Decoction without Scutellaria baicalensis Georgi, then ultrasonically mixing the negative sample of the Qingfei Decoction without Scutellaria baicalensis Georgi and methanol, filtering, and taking the subsequent filtrate as the negative sample solution.

[0022] The working principle and beneficial effects of the present invention are as follows: In the present invention, by determining the Scutellaria flavonoids in the Qingfei Decoction sample by high performance liquid chromatography and adopting a specific elution program, high-efficiency separation of baicalin, wogonoside, baicalein, and wogonin can be achieved, and at the same time, baicalin, wogonoside, baicalein, and wogonin in Scutellaria are determined. The method is simple, rapid, has good separation effect, high detection sensitivity, good result reproducibility, strong specificity, no interference, which provides reliable technical support for the quality control of traditional Chinese medicine compound preparations. Description of the Drawings

[0023] The present invention will be further described in detail below in conjunction with the drawings and specific embodiments.

[0024] Figure 1 It is the ultraviolet absorption spectrogram of baicalin, wogonoside, baicalein, and wogonin, In the figure, 1 is the ultraviolet absorption spectrum of baicalin, 2 is the ultraviolet absorption spectrum of wogonoside, 3 is the ultraviolet absorption spectrum of baicalein, and 4 is the ultraviolet absorption spectrum of wogonin; Figure 2 is the chromatogram of the test solution for Comparative Example 1; Figure 3 is the chromatogram of the test solution for Comparative Example 2; Figure 4 is the chromatogram of the test solution for Comparative Example 3; Figure 5 is the chromatogram of the test solution for Comparative Example 4; Figure 6 is the chromatogram of the negative sample in the specificity test; Figure 7 is the chromatogram of the mixed reference substance solution in the specificity test; In the figure, 1 is baicalin, 2 is wogonoside, 3 is baicalein, and 4 is wogonin; Figure 8 is the chromatogram of Scutellaria baicalensis Georgi pieces in the specificity test; In the figure, 1 is baicalin, 2 is wogonoside, 3 is baicalein, and 4 is wogonin; Figure 9 is the chromatogram of the test solution in the specificity test; In the figure, 1 is baicalin, 2 is wogonoside, 3 is baicalein, and 4 is wogonin; Figure 10 is the linear regression curve of baicalin; Figure 11 is the linear regression curve of wogonoside; Figure 12 is the linear regression curve of baicalein; Figure 13 is the linear regression curve of wogonin. Detailed implementation

[0025] Next, in combination with the embodiments of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts fall within the scope of protection of the present invention.

[0026] In the following examples and comparative examples, baicalin was provided by the National Institutes for Food and Drug Control, batch number 110715 - 201821, and the purity was calculated as 95.4%; wogonoside was provided by the National Institutes for Food and Drug Control, batch number 112002 - 201702, and the purity was calculated as 98.5%; Baicalein was provided by the National Institutes for Food and Drug Control, batch number 111595 - 201808, with a purity of 97.9%; Wogonin was provided by the National Institutes for Food and Drug Control, batch number 111514 - 201706.

[0027] Example 1 1. Determination of the extraction solvent The effects of different extraction solvents on the extraction rates of baicalin, wogonoside, baicalein, and wogonin in Scutellaria baicalensis Georgi tablets were investigated. The extraction solvents included methanol with a volume fraction of 50%, 70% methanol, and pure methanol. The investigation results are shown in Table 1: Table 1 Investigation results of different extraction solvents

[0028] As can be seen from Table 1, when the extraction solvent is methanol with a volume fraction of 70%, the extraction effect is the best. Therefore, methanol with a volume fraction of 70% is selected as the solvent during the preparation of the test solution. Different extraction methods (ultrasonic extraction, heating under reflux) and different extraction times (20 min, 30 min, 40 min) have little effect on the extraction effect.

[0029] 2. Selection of the detection wavelength Weigh 5 mg of each reference substance such as baicalin, wogonoside, baicalein, and wogonin respectively, and prepare a reference substance solution with a concentration of 50 μg / mL using the mobile phase (acetonitrile and 0.1 v / v% phosphoric acid solution, where the volume fraction of acetonitrile is 25% by volume percentage), and perform spectral scanning in the range of 200 nm to 400 nm; The results show that baicalin, wogonoside, baicalein, and wogonin all have the maximum absorption near the wavelength of 276 nm, as Figure 1 shown. On the basis of ensuring the accuracy of the measured values of baicalin, wogonoside, baicalein, and wogonin, for the convenience of the experiment, 276 nm is selected as the detection wavelength. At this wavelength, the analyte and the impurity peaks are well separated, and there is no interference from the negative sample.

[0030] 3. Determination of the content of Scutellaria flavonoids A method for determining the content of Scutellaria flavonoids in a Qingfei Decoction includes the following steps: using high - performance liquid chromatography to determine the content of Scutellaria flavonoids in the test solution, the mixed reference substance solution, and the negative sample solution. The Scutellaria flavonoids include baicalin, wogonoside, baicalein, and wogonin; the determination conditions of high - performance liquid chromatography are as follows: The chromatographic column is SHISEIDO Capcell pak C18 MGⅡ, with a specification of 5 µm, 4.6 mm × 250 mm; Packing material: octadecylsilane chemically bonded silica gel; Mobile phase: By volume percentage, acetonitrile is used as mobile phase A, and 0.1 v / v% phosphoric acid solution is used as mobile phase B. Gradient elution is carried out according to the following procedure:

[0031] Column temperature: 35 °C; Detection wavelength: 276 nm; Flow rate: 1.0 mL / min; The number of theoretical plates calculated by baicalin should be not less than 10,000; The preparation method of the mixed reference substance solution includes the following steps: Take appropriate amounts of reference substances of baicalin, wogonoside, baicalein, and wogonin, weigh accurately, add methanol with a volume fraction of 70%, and prepare mixed solutions containing 20 μg / mL of baicalin, wogonoside, baicalein, and wogonin respectively, that is, the mixed reference substance solution is obtained; The preparation method of the Scutellaria baicalensis Georgi decoction pieces includes the following steps: Collect 20 batches of Scutellaria baicalensis Georgi medicinal materials from different producing areas. According to the processing method, after removing impurities, steam them, cut into thin slices, and dry them to obtain 20 batches of Scutellaria baicalensis Georgi decoction pieces; The information on the producing areas of the 20 batches of Scutellaria baicalensis Georgi medicinal materials is shown in Table 2: Table 2 Information on Scutellaria baicalensis Georgi medicinal materials collected from different producing areas

[0032] The preparation method of the Qingfei Decoction reference sample includes the following steps: Weigh 5.6 g of Scutellaria baicalensis Georgi decoction pieces, 3.7 g of Platycodon grandiflorum decoction pieces, Poria cocos decoction pieces, Citri reticulatae Blanco pericarpium decoction pieces, Fritillaria thunbergii Miq. decoction pieces, and Mori cortex decoction pieces each, 2.6 g of Angelicae sinensis radix decoction pieces, Asparagi radix decoction pieces, Gardeniae jasminoides Ellis decoction pieces, Armeniacae amarum semen decoction pieces, and Ophiopogonis radix decoction pieces each, 1 g of Schisandrae chinensis fructus decoction pieces, 1.1 g of Glycyrrhizae radix et rhizoma decoction pieces, 2 g of Zingiberis officinale Rosc. decoction pieces, and 6 g of Ziziphi jujubae fructus decoction pieces. Add 450 mL of water, soak for 30 min, first cover and boil with high heat of 1200 w, then continue to decoct with low heat of 600 w for 45 min. After decoction, filter with a 200-mesh gauze to obtain the filtrate. Concentrate the filtrate to a volume of 160 mL, cool it, pre-freeze it, and vacuum freeze-dry it at -40 °C and 20 Pa for 72 h to obtain the freeze-dried powder of the Qingfei Decoction reference sample, which is the Qingfei Decoction reference sample; The preparation method of the test solution includes the following steps: Weigh 0.3 g of the Qingfei Decoction reference sample accurately, add 20 mL of methanol with a volume fraction of 70%, tightly stopper it, weigh it, perform ultrasonic treatment, take it out, cool it, make up the lost weight with methanol with a volume fraction of 70%, shake it well, filter it, and take the continuous filtrate to obtain the test solution; The preparation method of the negative sample solution includes the following steps: Take the remaining medicinal material components without Scutellaria baicalensis Georgi decoction pieces, prepare the negative sample of the Qingfei Decoction without Scutellaria baicalensis Georgi according to the formula content in the above Qingfei Decoction reference sample, and then prepare the negative sample solution according to the above preparation method of the test solution.

[0033] Comparative Example 1 The difference between this comparative example and Example 1 is only that in this comparative example, the gradient elution program is different. Specifically: Mobile phase: By volume percentage, acetonitrile is used as mobile phase A, and 0.1 v / v% phosphoric acid solution is used as mobile phase B. Gradient elution is carried out according to the following procedure:

[0034] Comparative Example 2 The difference between this comparative example and Example 1 is only that in this comparative example, the gradient elution program is different. Specifically: Mobile phase: By volume percentage, acetonitrile is used as mobile phase A, and 0.1 v / v% phosphoric acid solution is used as mobile phase B. Gradient elution is carried out according to the following procedure:

[0035] Comparative Example 3 The difference between this comparative example and Example 1 is only that in this comparative example, the gradient elution program is different. Specifically: Mobile phase: By volume percentage, acetonitrile is used as mobile phase A, and 0.1 v / v% phosphoric acid solution is used as mobile phase B. Gradient elution is carried out according to the following procedure:

[0036] Comparative Example 4 The difference between this comparative example and Example 1 is only that in this comparative example, the gradient elution program is different. Specifically: Mobile phase: By volume percentage, acetonitrile is used as mobile phase A, and 0.1 v / v% phosphoric acid solution is used as mobile phase B. Gradient elution is carried out according to the following procedure:

[0037] When the gradient elution programs of Comparative Examples 1 - 4 are adopted, the chromatograms of the test solution are as Figures 2 - 5 shown. It can be seen that when the gradient elution programs of Comparative Examples 1 - 4 are used for detection, the chromatographic peak shapes are asymmetric and the separation is poor, indicating that when acetonitrile and 0.1 v / v% phosphoric acid solution are used as the mobile phase, the separation effect between the detected substance and impurities is the best when the gradient elution in Example 1 is adopted.

[0038] Experimental Example 1 Method Validation The detection method of Example 1 is subjected to specificity test, linearity test, repeatability test, recovery test, stability test and durability test.

[0039] 1 Specificity Test The preparation method of the mixed reference substance solution comprises the following steps: Weigh an appropriate amount of baicalin, wogonoside, baicalein, and wogonin reference substances accurately, add methanol with a volume fraction of 70%, and prepare a mixed solution containing 20 μg / mL of each of baicalin, wogonoside, baicalein, and wogonin respectively, thus obtaining the mixed reference substance solution; The preparation method of the baical skullcap root decoction pieces comprises the following steps: Collect the baical skullcap root medicinal materials, remove impurities according to the processing method, steam, cut into thin slices, and dry, thus obtaining the baical skullcap root decoction pieces; The preparation method of the Qingfei Decoction reference sample comprises the following steps: Weigh 5.6 g of the baical skullcap root decoction pieces, 3.7 g of each of the platycodon root decoction pieces, poria cocos decoction pieces, tangerine peel decoction pieces, fritillaria thunbergii decoction pieces, and mulberry bark decoction pieces, 2.6 g of each of the angelica sinensis decoction pieces, asparagus decoction pieces, gardenia jasminoides decoction pieces, bitter almond decoction pieces, and ophiopogon japonicus decoction pieces, 1 g of the schisandra chinensis decoction pieces, 1.1 g of the licorice root decoction pieces, 2 g of the fresh ginger decoction pieces, and 6 g of the jujube decoction pieces, add 450 mL of water, soak for 30 min, first boil with strong fire at 1200 w with the lid closed, then continue to decoct with gentle fire at 600 w for 45 min. After the decoction is completed, filter with a 200-mesh gauze to obtain the filtrate. Concentrate the filtrate to a volume of 160 mL, let it cool, pre-freeze, and vacuum freeze-dry at -40 °C and 20 Pa for 72 h to obtain the freeze-dried powder of the Qingfei Decoction reference sample, which is the Qingfei Decoction reference sample; The preparation method of the test solution comprises the following steps: Weigh 0.3 g of the Qingfei Decoction reference sample accurately, add 20 mL of methanol with a volume fraction of 70%, tightly stopper, weigh, perform ultrasonic treatment, take out, let it cool, make up the lost weight with methanol with a volume fraction of 70%, shake well, filter, and take the subsequent filtrate to obtain the test solution; The preparation method of the negative sample solution comprises the following steps: Take the remaining medicinal material components lacking baical skullcap root, prepare the Qingfei Decoction negative sample lacking baical skullcap root according to the formula content in the above Qingfei Decoction reference sample, and then prepare the negative sample solution according to the preparation method of the above test solution; The preparation method of the baical skullcap root decoction piece solution comprises the following steps: Weigh 0.3 g of the baical skullcap root decoction pieces accurately, add 20 mL of methanol with a volume fraction of 70%, tightly stopper, weigh, perform ultrasonic treatment, take out, let it cool, make up the lost weight with methanol with a volume fraction of 70%, shake well, filter, and take the subsequent filtrate to obtain the test solution The chromatograms of the negative sample solution, the mixed reference substance solution, the baical skullcap root decoction piece solution, and the test solution are respectively determined by high performance liquid chromatography. The chromatogram baselines are stable, the component peak shapes are symmetrical, the separation is good, there is no interference in the negative sample, and the specificity is good. See Figures 6 - 9 .

[0040] 2 Linear test Precisely measure an appropriate concentration of the mixed reference solution, and prepare a series of concentration mixed reference solutions so that the injection volumes are respectively baicalin (34.24 μg, 68.48 μg, 171.20 μg, 342.41 μg, 684.82 μg, 1712.05 μg), wogonoside (25.86 μg, 51.72 μg, 129.29 μg, 258.58 μg, 517.16 μg, 1292.91 μg), baicalein (5.38 μg, 10.76 μg, 26.90 μg, 53.81 μg, 107.61 μg, 269.03 μg), wogonin (5.65 μg, 11.29 μg, 28.23 μg, 56.46 μg, 112.93 μg, 282.32 μg), and measure according to the detection conditions in Example 1. Using the logarithm of the injection volume (μg) of the reference as the abscissa and the logarithm of the peak area integral value as the ordinate, draw a standard curve, as Figures 10 - 13 shown. The results show that baicalin has a good linear relationship between 34.24 μg and 1712.05 μg, and the regression equation is y = 3628.4x - 7995.0 (R 2 = 1), as Figure 10 shown; wogonoside has a good linear relationship between 25.86 μg and 1292.91 μg, and the regression equation is y = 3931.2x - 545.2 (R 2 = 1), as Figure 11 shown; baicalein has a good linear relationship between 5.38 μg and 269.03 μg, and the regression equation is y = 4780.7x - 2257.9 (R 2 = 0.9998), as Figure 12 shown; wogonin has a good linear relationship between 5.65 μg and 282.32 μg, and the regression equation is y = 6257.9x - 11214.3 (R 2 = 0.9997), as Figure 13 shown, and the test results are shown in Table 3.

[0041] Table 3 Results of linear relationship investigation

[0042] 3 Repeatability test Take the same batch of samples, crush them, take three portions of 0.05 g, 0.10 g, and 0.15 g respectively, accurately weigh them, prepare the test solution according to the preparation method of the test solution, and conduct the determination according to the determination method in Example 1. The results are shown in Table 4 and Table 5, and the RSD% is less than 2.0%, indicating that the method has good repeatability.

[0043] Table 4 Results of repeatability investigation 1

[0044] Table 5 Results of Repeatability Investigation 2

[0045] 4 Recovery Test Take 9 portions of the sample powder with known content, 0.05 g for each portion, weigh accurately, and take every three portions as a group. Add 20 mL of the mixed reference substance solution prepared with 70% methanol to each group, and prepare the test solution according to the preparation method of the test solution in Example 1. Determine according to the determination method in Example 1, calculate the recovery rate, and the results are shown in Tables 6 - 9. The RSD% is less than 2.0%, indicating that the recovery rate of this method is good.

[0046] Table 6 Results of Baicalin Recovery Investigation

[0047] Table 7 Results of Wogonoside Recovery Investigation

[0048] Table 8 Results of Baicalein Recovery Investigation

[0049] Table 9 Results of Wogonin Recovery Investigation

[0050] 5 Stability Test Take the same test solution and start the determination at 0 hour, then determine once at intervals for a period of time, and record the peak area. The RSD values of each component are all less than 1.0%, as shown in Table 10. The results indicate that the test solution is stable for at least 24 hours.

[0051] Table 10 Results of Stability Investigation of Test Solution

[0052] 6 Robustness Test Refer to Part I of the Chinese Pharmacopoeia (2020 Edition). Take the same test solution, and conduct the robustness investigation on the chromatographic columns of different brands and specifications, as well as the changes in column temperature and flow rate for the method, and calculate the content. The RSD% is less than 2.0%, and the results are shown in Table 11. The results indicate that the chromatographic peaks are well separated under slight changes in various conditions, and the determination results are basically the same, indicating that the method has good robustness.

[0053] Table 11 Results of Robustness Investigation

[0054] Among them, Chromatographic Column Ⅰ is SHISEIDO Capcell pak C 18MGⅡ 5 μm (4.6 × 250 mm); Column Ⅱ is Agilent 5 TC-C 18 5 μm (4.6 × 250 mm); Column Ⅲ is SHIMADZU VP-ODS 5 μm (4.6 × 250 mm).

[0055] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for determining the content of flavonoid components in Scutellaria baicalensis Georgi in Qingfei Decoction, characterized in that, It includes the following steps: Determine the content of Scutellaria flavonoids in the test solution by high performance liquid chromatography, and the Scutellaria flavonoids include baicalin, wogonoside, baicalein, and wogonin; The determination conditions of the high performance liquid chromatography include: the chromatographic column is a C 18 chromatographic column, acetonitrile is used as mobile phase A, and 0.08 - 0.13 v / v% phosphoric acid solution is used as mobile phase B; the elution mode is gradient elution, and the program of the gradient elution is as follows: 。 2. The method for determining the content of flavonoid components of Scutellaria baicalensis Georgi in the Qingfei Decoction according to claim 1, characterized in that, The determination conditions of the high performance liquid chromatography also include: the column temperature is 30-40°C, the flow rate is 0.9-1.1 mL / min, the detection wavelength is 276 nm, and the theoretical plate number calculated by baicalin is not less than 10000.

3. The method for determining the content of flavonoids in Scutellaria baicalensis Georgi in the Qingfei Decoction according to claim 1, characterized in that, The said C 18 The filler in the chromatographic column is octadecylsilyl silica gel, and the said C 18 The chromatographic column is specifically a SHISEIDO Capcell pak C18 MGⅡ chromatographic column.

4. The method for determining the content of flavonoids in Scutellaria baicalensis Georgi in the Qingfei Decoction according to claim 1, characterized in that, The preparation method of the test solution includes the following steps: Take the Qingfei Decoction reference sample and methanol for ultrasonic mixing, filter, and take the filtrate as the test solution.

5. The method for determining the content of flavonoid components of Scutellaria baicalensis Georgi in the Qingfei Decoction according to claim 4, wherein, The mass-volume ratio of the Qingfei Decoction reference sample to the methanol is 0.1 g: 15-25 mL.

6. The method for determining the content of flavonoids in Scutellaria baicalensis Georgi in the Qingfei Decoction according to claim 4, characterized in that, The preparation method of the Qingfei Decoction reference sample includes the following steps: Weigh Scutellaria baicalensis Georgi slices, Platycodon grandiflorum slices, Poria cocos slices, Citrus reticulata Blanco cv. Erythrosa slices, Fritillaria thunbergii Miq. var. chekiangensis Hsiao et K. C. Hsia slices, Morus alba L. var. multicaulis (Perrott.) Loud. slices, Angelica sinensis (Oliv.) Diels slices, Asparagus cochinchinensis (Lour.) Merr. slices, Gardenia jasminoides Ellis slices, Armeniaca vulgaris Lam. var. ansu (Maxim.) Yü et Lu slices, Ophiopogon japonicus (Thunb.) Ker-Gawl. slices, Schisandra chinensis (Turcz.) Baill. slices, Glycyrrhiza uralensis Fisch. slices, Zingiber officinale Rosc. slices, and Ziziphus jujuba Mill. slices, add them to water, soak, boil, continue to decoct, filter to obtain a filtrate, concentrate the filtrate, cool, pre-freeze, and vacuum freeze-dry to obtain the Qingfei Decoction reference freeze-dried powder, which is the Qingfei Decoction reference sample.

7. The method for determining the content of baicalin flavonoids in a Qingfei Decoction according to claim 6, wherein, The preparation method of the Scutellaria baicalensis Georgi slices includes the following steps: Collect Scutellaria baicalensis Georgi herbs, process them, remove impurities, steam, slice, and dry to obtain Scutellaria baicalensis Georgi slices.

8. The method for determining the content of flavonoids in Scutellaria baicalensis Georgi in the Qingfei Decoction according to claim 6, characterized in that, The mass-volume ratio of the Scutellaria baicalensis Georgi slices to the water is 5.6 g: 440-460 mL; The addition amounts of the Platycodon grandiflorum slices, Poria cocos slices, Citrus reticulata Blanco cv. Erythrosa slices, Fritillaria thunbergii Miq. var. chekiangensis Hsiao et K. C. Hsia slices, and Morus alba L. var. multicaulis (Perrott.) Loud. slices are equal, and the addition amounts of the Angelica sinensis (Oliv.) Diels slices, Asparagus cochinchinensis (Lour.) Merr. slices, Gardenia jasminoides Ellis slices, Armeniaca vulgaris Lam. var. ansu (Maxim.) Yü et Lu slices, and Ophiopogon japonicus (Thunb.) Ker-Gawl. slices are equal; The addition amounts of the Scutellaria baicalensis Georgi slices, Platycodon grandiflorum slices, Angelica sinensis (Oliv.) Diels slices, Schisandra chinensis (Turcz.) Baill. slices, Glycyrrhiza uralensis Fisch. slices, Zingiber officinale Rosc. slices, and Ziziphus jujuba Mill. slices are 5.6: 3.5-3.8: 2.5-2.7: 1: 1.1: 2: 6; The volume fraction of the methanol is 50%-100%; Preferably, the volume fraction of the methanol is 70%.

9. The method for determining the content of flavonoids in Scutellaria baicalensis Georgi in the Qingfei Decoction according to claim 1, characterized in that, It also includes the following steps: Determine the content of Scutellaria flavonoids in the mixed reference solution and the negative sample solution by high performance liquid chromatography.

10. The method for determining the content of flavonoids in Scutellaria baicalensis Georgi in Qingfei Decoction according to claim 9, characterized in that, The preparation method of the mixed reference solution includes the following steps: Take the reference substance and methanol and mix them to obtain the reference solution; The reference substances include baicalin, wogonoside, baicalein, and wogonin; The preparation method of the negative sample solution includes the following steps: Take the remaining medicinal material components in the Qingfei Decoction except for the Scutellaria baicalensis Georgi slices to prepare the negative sample of the Qingfei Decoction without Scutellaria baicalensis Georgi, and then ultrasonically mix the negative sample of the Qingfei Decoction without Scutellaria baicalensis Georgi and methanol, filter, and take the subsequent filtrate as the negative sample solution.

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