Method for simultaneously determining contents of baicalein and baicalin in scutellaria baicalensis

Abstract

The invention discloses a method for simultaneously measuring the contents of baicalein and baicalin in scutellaria baicalensis, which comprises the following steps: taking a scutellaria baicalensis sample to be measured, crushing, mixing with a solvent, carrying out ultrasonic extraction and centrifugation, taking supernate, filtering, and adding methanol to a constant volume to obtain a test solution; the method comprises the following steps: respectively weighing baicalein and baicalin standard substances, transferring the standard substances into a volumetric flask, and adding methanol to a constant volume to prepare reference substance solutions of corresponding compounds; and respectively detecting the test solution and the reference solution by adopting high performance liquid chromatography (HPLC) to obtain component substance peaks, and analyzing the component substance peaks. The determination method disclosed by the invention is simple to operate and high in detection efficiency, and methodological investigation of linearity, stability, precision, repeatability and the like shows that the content determination method is accurate, reliable and high in practicability.

Description

Technical Field

[0001] The invention relates to the technical field of liquid phase detection, and in particular to a method for simultaneously quantitatively determining baicalein and baicalin in scutellaria baicalensis. Background Art

[0002] Scutellaria baicalensis is the dried root of Scutellaria baicalensis Georgi, a plant of the Lamiaceae family. It is one of the traditional Chinese medicinal materials in my country and is included in the 2010 edition of the Chinese Pharmacopoeia (Volume 1). It is widely used in clinical Chinese medicine and has the effects of clearing away heat and dampness, purging fire and detoxifying, stopping bleeding and stabilizing pregnancy. The main active ingredients of Scutellaria baicalensis include baicalin and baicalein, etc. These two chemical components are considered to be the main active ingredients of Scutellaria baicalensis and have important pharmacological activities, especially in antibacterial, anti-tumor, and anti-inflammatory effects. At present, the quality control of Scutellaria baicalensis mainly depends on the determination of baicalin and baicalin. Accurate and rapid determination of the content of baicalin and baicalin in Scutellaria baicalensis not only helps to ensure the quality stability of Scutellaria baicalensis, but also helps to guide clinical medication. However, due to the similarity in chemical structure between baicalein and baicalin, they often interfere with each other during separation and quantitative analysis, which makes it difficult to simultaneously determine the contents of these two components. Commonly used methods for determining the contents of baicalein and baicalin mainly include ultraviolet spectrophotometry, thin layer chromatography and high performance liquid chromatography (HPLC). Among them, HPLC has become the standard method for quantitative analysis of baicalein and baicalin due to its high efficiency, sensitivity and strong selectivity. However, the existing HPLC method often requires the determination of the contents of baicalein and baicalin separately. Although this method is accurate, it is cumbersome and time-consuming. In order to improve the efficiency and accuracy of analysis, a new method is urgently needed that can effectively separate baicalein and baicalin and complete simultaneous quantitative analysis in a short time.

[0003] Therefore, the present invention proposes a new method based on high performance liquid chromatography (HPLC), which can accurately determine the contents of baicalein and baicalin in Scutellaria baicalensis in the same analysis. This method not only provides an effective means for the quality control of Scutellaria baicalensis, but also has a certain reference significance for the component analysis of other Chinese medicinal materials. Summary of the invention

[0004] The object of the present invention is to provide a method for simultaneously determining the contents of baicalein and baicalin in Scutellaria baicalensis by HPLC, which method has the advantages of fast detection speed, high sensitivity, high accuracy and good repeatability.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides a method for simultaneously determining the contents of baicalein and baicalin in Scutellaria baicalensis using HPLC, comprising the following steps: (1) The scutellaria baicalensis sample to be tested was crushed, mixed with a solvent, subjected to ultrasonic extraction, and centrifuged. The supernatant was filtered and fixed to volume with methanol to serve as the test solution; (2) Weigh baicalein and baicalin standards respectively, transfer them into a volumetric flask, and add methanol to make up to volume to prepare reference solutions of the corresponding compounds; (3) The test solution and the reference solution are respectively tested by high performance liquid chromatography (HPLC) and analyzed after the component peaks are obtained.

[0006] Preferably, in step (1), the mass volume ratio of the crushed Scutellaria baicalensis sample to be tested and the solvent is (1-2) g / (20-30) mL.

[0007] Preferably, the solvent in step (1) is ethanol with a volume fraction of 70-75%.

[0008] Preferably, the dissolving method in step (1) is ultrasonic dissolving until the solution is completely dissolved.

[0009] Preferably, the power of the ultrasonic extraction in step (1) is 150-250 W, the frequency of the ultrasonic extraction is 35-40 kHz, the temperature of the ultrasonic extraction is 30-40° C., the time of the ultrasonic extraction is 30-40 min / time, and the number of ultrasonic extractions is 1-2 times.

[0010] Preferably, the centrifugal speed in step (1) is 3000-4000 r / min, and the centrifugal time is 15-20 min.

[0011] Preferably, the chromatographic conditions in the detection process of step (3) are: Chromatographic column: Agilent Eclipse XDB-C18 column (4.6 mm × 250 mm, 5 μm); Mobile phase A: acetonitrile, mobile phase B: 0.1% phosphoric acid aqueous solution; Column temperature: 35-40°C; Injection volume: 6-10 μL; Flow rate: 0.8-1.0 mL / min.

[0012] Preferably, the gradient elution procedure in the detection process of step (3) is: 0~10min, 15%→25%A, 85%-75%B; 10~15min, 25%→35%A, 75%→65%B; 15~20min, 35%→45%A, 65%→55%B.

[0013] Preferably, the detector used in the detection process of step (3) is an ultraviolet absorption detector with a detection wavelength of 280 nm.

[0014] Technical effects: 1. In the technical scheme provided by the present invention, ethanol aqueous solution is used as the extracting solution, and the extracting solution has good solubility for the active substances in Scutellaria baicalensis. Under the action of ultrasound, baicalein and baicalin in Scutellaria baicalensis can be extracted to the maximum extent, and then HPLC detection is performed using a C18 chromatographic column, a phosphoric acid aqueous solution as the aqueous phase and acetonitrile as the organic phase as the mobile phase, and HPLC-grade baicalein and baicalin are used as standard reference substances to achieve accurate content determination of baicalein and baicalin in Scutellaria baicalensis at the same time. 2. The method is simple to operate, fast in detection speed, high in detection efficiency, and good in repeatability; stability, precision, accuracy, etc. all meet the requirements of methodological verification, and it has strong practicality. In addition, the reference substance, solvent and mobile phase used in HPLC are easily available and low in cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 A high performance liquid chromatogram of a reference solution provided by the present invention; Figure 2 The linear relationship diagram between the concentration x of baicalein and the peak area y of the standard curve solution prepared in the embodiment of the present invention; Figure 3 This is a linear relationship diagram between the concentration x of baicalin and the peak area y of the standard curve solution prepared in the embodiment of the present invention. DETAILED DESCRIPTION

[0016] In order to make the purpose, technical solutions and advantages of the present application clearer, the technical solutions provided by the present invention will be clearly and completely described below in conjunction with embodiments.

[0017] Example 1: Determination of the content of Scutellaria baicalensis samples 1. Chromatographic conditions Chromatographic column: Agilent Eclipse XDB-C18 column (4.6 mm × 250 mm, 5 μm); Mobile phase A: acetonitrile, mobile phase B: 0.1% phosphoric acid aqueous solution; Column temperature: 40°C; Injection volume: 10 μL; Flow rate: 1.0mL / min.

[0018] 2. Preparation of reference solution Accurately weigh appropriate amounts of baicalin and baicalin standard reference substances into a volumetric flask, add methanol to each of them to make a solution containing 5 mg of baicalin and 5 mg of baicalin per 10 mL, shake well, and obtain the solution.

[0019] 3. Preparation of test solution Accurately weigh 0.05 g of Scutellaria baicalensis powder and place it in a 10 mL centrifuge tube. Add 2.5 mL of 75% ethanol aqueous solution, make up to the scale, weigh the weight, ultrasonically treat at 30°C for 40 min, cool to room temperature, make up the lost weight with 75% ethanol aqueous solution, shake well, centrifuge at 4000 r / min for 20 min, take the supernatant, pass it through a 0.22 μm microporous filter membrane, make up to 10 mL with methanol, and take the filtrate as the test solution.

[0020] 4. Determination Method Accurately pipette 10 μL of the reference solution and the test solution respectively, inject into the liquid chromatograph, and determine the peak areas of baicalein, baicalin and the test solution.

[0021] Example 2: Investigation of the linear relationship of baicalein content 1. Chromatographic conditions Chromatographic column: Agilent Eclipse XDB-C18 column (4.6 mm × 250 mm, 5 μm); Mobile phase A: acetonitrile, mobile phase B: 0.1% phosphoric acid aqueous solution; Column temperature: 40°C; Injection volume: 10 μL; Flow rate: 1.0mL / min.

[0022] 2. Preparation of reference solution Accurately weigh an appropriate amount of baicalin standard reference substance into a volumetric flask, add methanol to make solutions containing 5 mg, 4 mg, 3 mg, 2 mg, and 1 mg of baicalin per 10 mL, shake well, and obtain.

[0023] 3. Determination method Accurately pipette 10 μL of standard reference solution of different concentrations respectively, inject into liquid chromatograph, determine the peak area and retention time corresponding to different contents of baicalin, and calculate the content.

[0024] 4. Result record: Table 1 Results of peak areas corresponding to different concentrations of baicalin

[0025] 5. Results Analysis With the peak area as the ordinate (y) and the concentration (x) as the abscissa, the regression equation of baicalein was obtained as: y = 5.797*10 9 x – 1.256*10 6, R² = 0.9914, linear range: 0.1 mg / mL-0.5 mg / mL. The results show that when the HPLC determination scheme provided by the present invention is used, the baicalein content and the peak area show a good linear relationship.

[0026] Example 3: Investigation of the linear relationship of baicalin content 1. Chromatographic conditions Chromatographic column: Agilent Eclipse XDB-C18 column (4.6 mm × 250 mm, 5 μm); Mobile phase A: acetonitrile, mobile phase B: 0.1% phosphoric acid aqueous solution; Column temperature: 40°C; Injection volume: 10 μL; Flow rate: 1.0mL / min.

[0027] 2. Preparation of reference solution Accurately weigh an appropriate amount of baicalin standard reference substance into a volumetric flask, add methanol to make solutions containing 5 mg, 4 mg, 3 mg, 2.5 mg, and 1.25 mg of baicalin per 10 ml, shake well, and obtain.

[0028] 3. Determination method Accurately pipette 10 μL of standard reference solution of different concentrations respectively, inject into liquid chromatograph, determine the peak area and retention time corresponding to different contents of baicalin, and calculate the content.

[0029] 4. Record the results Table 2 Results of peak areas corresponding to different concentrations of baicalin

[0030] 5. Results Analysis With the peak area as the ordinate (y) and the concentration (x) as the abscissa, the regression equation of baicalin was obtained as: y = 3.921*10 9 x + 2.273*10 5 , R² = 0.9983371897, linear range: 0.125 mg / mL-0.5 mg / mL. The results show that when the HPLC determination scheme provided by the present invention is used, the baicalin content and the peak area show a good linear relationship.

[0031] Example 4: Precision Test The reference solution was prepared according to the preparation method of the reference solution in Example 1, and 6 injections were performed continuously according to the chromatographic conditions in Example 1 to obtain 6 chromatograms. The peak area and retention time of each characteristic peak were recorded, and the RSD value was calculated. The results are shown in Table 3. The RSDs of the peak areas of baicalein and baicalin were 2.180% and 1.512%, respectively, and the RSDs of the retention times were 2.426% and 1.863%, respectively, indicating that the precision of the content determination method was good.

[0032] Table 3 Precision test results (n=6)

[0033] Example 5: Stability Test The standard reference solution was prepared according to the preparation method of the reference solution in Example 1, and was injected into a high performance liquid chromatograph for determination at 0, 2, 4, 8, 12, and 24 hours after preparation according to the chromatographic conditions in Example 1 to obtain 6 chromatograms. The chromatographic peak area and retention time were recorded to investigate the change in the peak area of ​​the scutellaria solution after placement. The results are shown in Table 4. The RSDs of the peak areas of baicalein and baicalin were 1.536% and 1.804%, respectively, and the RSDs of the retention times were 1.096% and 2.043%, respectively, indicating that the reference solution was stable within 24 hours.

[0034] Table 4 Stability test results (n=6)

[0035] Example 6: Repeatability test Six reference solutions were prepared according to the preparation method of the reference solution in Example 1, and the samples were injected according to the chromatographic conditions in Example 1 to obtain six chromatograms. The peak area and retention time of each characteristic peak were recorded, and the RSD value was calculated. The results are shown in Table 5. The RSDs of the peak areas of baicalein and baicalin were 1.893% and 0.976%, respectively, and the RSDs of the retention times were 1.291% and 2.077%, respectively, indicating that the repeatability of the content determination method was good.

[0036] Table 5 Repeatability test results (n=6)

[0037] In summary, the HPLC detection method for simultaneously determining the contents of baicalein and baicalin in Scutellaria baicalensis provided by the present invention is simple to operate, has high detection efficiency, small error, good reproducibility, relatively stable test method, and is affected by fewer factors; the stability, precision, repeatability, etc. of the method of the present invention all meet the requirements of methodological validation and are very practical.

[0038] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for simultaneous quantitative determination of baicalein and baicalin in Scutellaria baicalensis, characterized in that: (1) The scutellaria baicalensis sample to be tested was crushed, mixed with a solvent, subjected to ultrasonic extraction, and centrifuged. The supernatant was filtered and fixed to volume with methanol to serve as the test solution; (2) Weigh baicalein and baicalin standards respectively, transfer them into a volumetric flask, and add methanol to make up to volume to prepare reference solutions of the corresponding compounds; (3) The test solution and the reference solution are respectively tested by high performance liquid chromatography, and qualitative and quantitative analyses are performed after the component peaks are obtained.

2. The measuring method according to claim 1, characterized in that In step (1), the mass volume ratio of the crushed Scutellaria baicalensis sample to be tested and the solvent is 0.05 g: (2.5-5) mL; the solvent is ethanol with a volume fraction of 75%.

3. The measuring method according to claim 1, characterized in that The power of the ultrasonic treatment in step (1) is 360W, the ultrasonic frequency is 40kHz, the ultrasonic time is 30-40min, and the number of ultrasonic times is 1-2 times.

4. The measuring method according to claim 1, characterized in that The centrifugal speed in step (1) is 3000-4000 r / min, and the centrifugal time is 15-20 min.

5. The measuring method according to claim 1, characterized in that The chromatographic conditions during the detection process of step (3) are: Chromatographic column: Agilent Eclipse XDB-C18 column (4.6 mm × 250 mm, 5 μm); Mobile phase A: acetonitrile, mobile phase B: 0.1% phosphoric acid aqueous solution; Column temperature: 35-40°C; Injection volume: 6-10 μL; Flow rate: 0.8-1.0 mL / min.

6. The method according to claim 1, characterized in that The gradient elution procedure in the detection process of step (3) is: 0~10min, 15%→25%A, 85%-75%B; 10~15min, 25%→35%A, 75%→65%B; 15~20min, 35%→45%A, 65%→55%B.

7. The method according to claim 1, characterized in that The detector in the detection process of step (3) is an ultraviolet absorption detector with a detection wavelength of 280nm.

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