A high performance liquid chromatography method for simultaneously determining contents of oleanolic acid and ursolic acid in gynecological antipruritic tablets

Abstract

The present application relates to the technical field of pharmaceutical analysis, in particular to a high performance liquid chromatography method for simultaneously determining contents of oleanolic acid and ursolic acid in gynecological itching-relieving tablets, wherein the high performance liquid chromatography method is used for simultaneously determining contents of oleanolic acid and ursolic acid in gynecological itching-relieving tablets by liquid chromatography under the same chromatographic conditions. The method has good precision, stability, repeatability and high separation degree, and provides a basis for effectively controlling the quality of gynecological itching-relieving tablets and improving the standard of gynecological itching-relieving tablets.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pharmaceutical analysis, in particular to a high performance liquid chromatography method for simultaneously determining the contents of oleanolic acid and ursolic acid in gynecological itching-relieving tablets. BACKGROUND

[0002] The gynecological itching-relieving tablets have the effects of clearing heat and drying dampness, killing insects and relieving itching, and are used for patients with vaginitis syndrome of the damp-heat type. The main components thereof include Herba Patriniae, Herba Belamcandae, Herba Taraxaci, Herba Ecliptae, Rubia Cordifolia, Spatholobi Caulis, Angelica sinensis, Corydalis, etc. The main drugs thereof are Herba Patriniae, Herba Belamcandae and Herba Taraxaci, but the standard thereof does not include the determination of the content of the components of the three main drugs. Both Herba Patriniae and Herba Belamcandae contain oleanolic acid and ursolic acid. Determining the contents of oleanolic acid and ursolic acid in the gynecological itching-relieving tablets is helpful to control the quality of the gynecological itching-relieving tablets and improve the standard thereof.

[0003] There are many reports on simultaneously determining the contents of oleanolic acid and ursolic acid in medicinal materials in the prior art, such as the documents “HPLC method for rapidly determining oleanolic acid and ursolic acid in eight kinds of traditional Chinese medicines” (Chinese Herbal Drugs, 2017, Vol. 48 (No. 10), 1998-2001), “HPLC method for determining the contents of oleanolic acid and ursolic acid in Herba Belamcandae from different regions of Yunnan” (Guizhou Agricultural Sciences, 2019, Vol. 47 (No. 10), 115-118) and “HPLC for simultaneously determining common honeysuckle saponin, oleanolic acid and ursolic acid in Herba Patriniae” (Chinese Journal of Experimental Traditional Medical Formulae, 2012, Vol. 18 (No. 15), 89-92), etc. The above documents all disclose the determination of the contents of oleanolic acid and ursolic acid from medicinal materials. However, there is no relevant report on simultaneously determining the contents of oleanolic acid and ursolic acid in Chinese patent medicines at present. The reason is that compared with single medicinal materials or decoction pieces, Chinese patent medicines have more complex components, and it is necessary to establish specific test conditions to separate the isomers oleanolic acid and ursolic acid.

[0004] Therefore, if the contents of oleanolic acid and ursolic acid in the gynecological itching-relieving tablets can be simultaneously determined, the quality of the gynecological itching-relieving tablets can be effectively controlled, the standard thereof can be improved, and the clinical curative effect thereof can be ensured. SUMMARY

[0005] The present application aims at the above-mentioned problems, and provides a method for simultaneously determining the contents of oleanolic acid and ursolic acid in gynecological itching-relieving tablets by using high performance liquid chromatography. The method has good precision, good stability, good repeatability and high resolution, and provides a basis for effectively controlling the quality of the gynecological itching-relieving tablets and improving the standard thereof.

[0006] In order to achieve the above-mentioned purposes, the present application adopts the following technical solutions:

[0007] The application discloses a high performance liquid chromatography method for simultaneously determining contents of oleanolic acid and ursolic acid in gynecological itching-relieving tablets.

[0008] Further, the high performance liquid chromatography method is performed according to the following steps:

[0009] (1) Chromatographic conditions:

[0010] A C18 reversed-phase chromatographic column is used.

[0011] A mobile phase A is methanol, and a mobile phase B is 0.1% formic acid solution, gradient elution is performed, and the mobile phase ratio is as follows, wherein the mobile phase ratio is all a volume percentage: the mobile phase A is 90%-95%, and the mobile phase B is 10%-5%.

[0012] A flow rate is 0.2-1.5 mL / min, a detection wavelength is 210 nm-350 nm, and a column temperature is 25-40 DEG C.

[0013] (2) Preparation of a test sample solution: gynecological itching-relieving tablets are taken, methanol is added, ultrasonic extraction is performed, and a filtrate is taken to obtain the test sample solution of the gynecological itching-relieving tablets.

[0014] (3) Preparation of a control sample solution: oleanolic acid and ursolic acid control samples are respectively taken, methanol is added to dissolve the oleanolic acid and ursolic acid control samples to obtain oleanolic acid and ursolic acid control sample solutions.

[0015] (4) Determination method: the determination is performed according to the chromatographic conditions in step (1), and the determination is completed.

[0016] Further, the chromatographic conditions in step (1) are as follows:

[0017] An instrument is a Waters 2695 high performance liquid chromatograph.

[0018] A chromatographic column is a ZORBAX Eclipse XDB-C18 Analytical chromatographic column, and the chromatographic column has a specification of 250*4.6 mm and 5 um.

[0019] A mobile phase A is methanol, a mobile phase B is 0.1% formic acid solution, gradient elution is performed, and the mobile phase ratio is as follows, 0-60 min, the mobile phase A is 92%-95%, and the mobile phase B is 8%-5%, wherein the mobile phase ratio is all a volume percentage.

[0020] A column temperature is 25 DEG C, a sample injection amount is 10 uL, and a detection wavelength is 210 nm.

[0021] Further, the preparation of the test sample solution in step (2) is as follows: 6 g of the Gynecological Itchy Relief Tablets is precisely weighed, placed in a 100 mL conical flask with a stopper, 25 mL of methanol is added, tightly stoppered, the weight is determined, ultrasonically treated for 0.5 h, allowed to cool, re-precisely weighed, the lost weight is made up with methanol, shaken well, filtered, concentrated, the obtained filtrate is filtered with a 0.45 μm microporous filter membrane, and the filtrate is obtained, thereby obtaining the Gynecological Itchy Relief Tablet test sample solution.

[0022] Further, the preparation method of the control sample solution in step (3) is as follows: 1.1 mg of oleanolic acid is dissolved in 10 mL of methanol to prepare a 0.11 mg / mL oleanolic acid standard solution, and then the oleanolic acid standard solution is prepared into an oleanolic acid control sample solution with the content of oleanolic acid being 0.0055-0.0275 mg / mL; 3.5 mg of ursolic acid is dissolved in 7 mL of methanol to prepare a 0.5 mg / mL ursolic acid standard solution, and then the ursolic acid standard solution is prepared into a ursolic acid control sample solution with the content of ursolic acid being 0.005-0.1 mg / mL.

[0023] As described above, the present application has the following beneficial effects due to the adoption of the above technical solutions:

[0024] The present application establishes specific test conditions, which can effectively separate the isomers oleanolic acid and ursolic acid. Meanwhile, the determination method provided by the present application realizes the simultaneous determination of the contents of oleanolic acid and ursolic acid in the Gynecological Itchy Relief Tablets through one-time injection, and the chromatographic peak shape is good, the separation degree is high, and the contents of oleanolic acid and ursolic acid in the Gynecological Itchy Relief Tablets can be accurately determined. The method is simple in operation, good in precision, good in stability, good in repeatability and high in separation degree, which provides a basis for effectively controlling the quality of the Gynecological Itchy Relief Tablets and improving the standard of the Gynecological Itchy Relief Tablets. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 HPLC chromatograms of oleanolic acid and ursolic acid mixed standard with different injection amounts under the gradient condition of item 2.4. The chromatographic peaks correspond to: (1) oleanolic acid, (2) ursolic acid. The injection amounts correspond to: a 20 μL; b 10 μL.

[0026] Figure 2 HPLC chromatograms of oleanolic acid and ursolic acid mixed standard under different mobile phase conditions. The chromatographic peaks correspond to: (1) oleanolic acid, (2) ursolic acid. The mobile phases correspond to: a methanol-0.1% phosphoric acid water; b methanol-pure water-0.1% glacial acetic acid-0.05% triethylamine; c methanol-pure water; d methanol-0.1% formic acid water.

[0027] Figure 3The chromatogram of oleanolic acid and ursolic acid mixed standard at different flow rates. The chromatographic peaks correspond to: (1) oleanolic acid, (2) ursolic acid. The flow rates correspond to: a is 0.8 mL / min; b is 0.6 mL / min; c is 0.5 mL / min; d is 0.3 mL / min.

[0028] Figure 4 The linear relationship diagram of oleanolic acid.

[0029] Figure 5 The linear relationship diagram of ursolic acid.

[0030] Figure 6 The chromatogram of specificity test results. DETAILED DESCRIPTION

[0031] In order to express the present application more clearly, the present application is further illustrated by specific examples below.

[0032] Example 1: High performance liquid chromatography method for determining the content of oleanolic acid and ursolic acid in Gynecological Anti-itch Tablets.

[0033] 1. Instruments and reagents

[0034] High performance liquid chromatograph (Waters 2695) purchased from Waters Technology Co., Ltd.; ultrasonic cleaner (KQ-500E) purchased from Kunshan Ultrasonic Instrument Co., Ltd.; ultrapure water machine (Direct-Q5UV) purchased from Guangxi Nanning Bomei Biological Technology Co., Ltd.; electronic balance (BSA224S) purchased from Sartorius Scientific Instruments (Beijing) Co., Ltd.; chromatographic column (ZORBAX Eclipse XDB-C18 Analytical) purchased from Yue Xun Technology (Shanghai) Co., Ltd.;

[0035] 10 batches of Gynecological Anti-itch Tablet powders, batch numbers are 23081801, 23081802, 23081803, 23081804, 23081805, 23121101, 23121102, 23121104, 23121105; oleanolic acid (G2214309), ursolic acid (F2217053), phosphoric acid (H2212477), acetic acid (2017102401), triethylamine (F2217321) purchased from Aladdin Chemical Reagent Co., Ltd.; acetonitrile (F23N4204), methanol (221019) purchased from Thermo Fisher Scientific.

[0036] 2. Investigation of chromatographic conditions

[0037] 2.1 Investigation of mobile phase

[0038] Respectively, to investigate methanol-pure water, methanol-0.1% formic acid water, methanol-0.1% phosphoric acid water, methanol-pure water-0.1% glacial acetic acid-0.05% triethylamine, by Figure 1 It can be known that, compared with other mobile phases, the baseline of methanol-0.1% formic acid aqueous solution is stable, and oleanolic acid and ursolic acid are well separated, so the mobile phase system selected by the application for oleanolic acid and ursolic acid is methanol-0.1% formic acid aqueous solution.

[0039] 2.2 Investigation of flow rate

[0040] Investigate 0.3 mL / min, 0.5 mL / min, 0.6 mL / min, 0.8 mL / min flow rate, by Figure 2 It can be known that, when the flow rate is 0.3 mL / min, the two peaks are best separated, so the liquid chromatography flow rate for determining oleanolic acid and ursolic acid content is determined to be 0.3 mL / min.

[0041] 2.3 Investigation of mobile phase proportion

[0042] Under the conditions of methanol-0.1% formic acid water as the mobile phase system, flow rate of 0.3 mL / min, wavelength of 210 nm, and injection of 10 uL, four different proportions of 98:2, 96:4, 95:5 and 93:7 of the mobile phase are investigated. When the proportion of methanol increases, the retention time of the chromatographic peaks of oleanolic acid and ursolic acid is delayed, and the separation degree is increased, but when the proportion of methanol is 95%, the separation degree begins to decrease, so the proportion of methanol in methanol-0.1% formic acid water is selected to be 95%, and gradient change is carried out on this basis. The separation effect is better under the condition of 60 min (0-30 min, methanol content is 90%-93%; 30-60 min, methanol content is 93%-95%) gradient elution.

[0043] 2.4 Optimal chromatography conditions

[0044] After the above investigation and test, the optimal chromatography conditions are finally determined as follows:

[0045] A ZORBAX Eclipse XDB-C18 Analytical (250x4.6mm 5um) chromatographic column is used for separation;

[0046] The mobile phase A is methanol, the mobile phase B is 0.1% formic acid aqueous solution, gradient elution is carried out, the mobile phase ratio is as follows, 0-60 min, the mobile phase A is 92%-95%, the mobile phase B is 8%-5%, wherein the mobile phase ratio is volume percent; the column temperature is 25°C; the injection amount is 10 uL; and the detection wavelength is 210 nm. The separation condition test is carried out under the above chromatography conditions, and the results are shown in Table 1 and Figure 1 .

[0047] Table 1 Separation degree of different sample injection amounts under gradient elution conditions of item 2.4

[0048]

[0049] 3. Preparation of test solution

[0050] 3.1 Preparation of test solution of Gynecological Itching-Relieving Tablets

[0051] Take 6 g of Gynecological Itching-Relieving Tablets, accurately weigh, and place in a 100 mL conical flask with a stopper. Accurately add 25 mL of methanol, tightly stopper, weigh, ultrasonically treat for 0.5 h, cool, re-accurately weigh, make up the weight loss with methanol, shake well, filter, concentrate, and filter the obtained filtrate with a 0.45 μm microporous filter membrane to obtain the test solution of Gynecological Itching-Relieving Tablets.

[0052] 3.2 Preparation of control solution

[0053] Take 1.1 mg of oleanolic acid and dissolve in 10 mL of methanol to prepare a 0.11 mg / mL oleanolic acid standard solution. Then, the oleanolic acid standard solution is prepared into an oleanolic acid control solution with the content of oleanolic acid being 0.0055-0.0275 mg / mL. Take 3.5 mg of ursolic acid and dissolve in 7 mL of methanol to prepare a 0.5 mg / mL ursolic acid standard solution. Then, the ursolic acid standard solution is prepared into a ursolic acid control solution with the content of ursolic acid being 0.005-0.1 mg / mL.

[0054] 3.3 Preparation of negative control solution

[0055] Take 5 g of Herba Verbenae, 5 g of Herba Taraxaci, 2 g of Radix Pseudo-Ginseng, 5 g of Radix Rubi, 5 g of Caulis Spatholobi, and 3 g of Radix Angelicae Sinensis, except for Herba Oldenlandiae, in a beaker, and add pure water to decoct twice. Add 300 mL of pure water in the first time of decocting, and add 250 mL of pure water in the second time of decocting. Each time of decocting is for 2 hours. Combine the obtained decocted liquids, filter, and concentrate into a thick state. Then, add 1 g of powdered Rhizoma Corydalis, stir, and dry to obtain a negative control powder. Take 6 g of the negative control powder, accurately weigh, place in a 100 mL conical flask with a stopper, accurately add 25 mL of methanol, tightly stopper, weigh, ultrasonically treat for 0.5 h, cool, re-accurately weigh, make up the weight loss with methanol, shake well, filter, concentrate, and filter the obtained filtrate with a 0.45 μm microporous filter membrane to obtain the negative control solution.

[0056] 4. Linear relationship experiment

[0057] 4.1 Linear relationship of oleanolic acid

[0058] Accurately weigh 1.1 mg of oleanolic acid and dissolve it in 10 mL of methanol to prepare a 0.11 mg / mL oleanolic acid standard solution. Pipette 0.5 mL, 1 mL, 1.5 mL, 2 mL, and 2.5 mL of each solution into 10 mL volumetric flasks and dilute to volume to prepare 0.0055 mg / mL, 0.0110 mg / mL, 0.0165 mg / mL, 0.0220 mg / mL, and 0.0275 mg / mL oleanolic acid reference solutions, respectively. Inject each of these oleanolic acid reference solutions into a liquid chromatograph and determine the peak area under the chromatographic conditions described above. Plot a standard curve based on the peak area and concentration, with concentration on the x-axis and peak area on the y-axis. The results are shown below. Figure 3 As shown, the standard curve equation is: y = 152.7962x - 7929.7, and the correlation coefficient R0 is... 2 =0.9992, linear range 0.001~0.100mg / mL;

[0059] 4.2 Linear relationship of ursolic acid

[0060] Accurately weigh 3.5 mg of ursolic acid and dissolve it in 7 mL of methanol to prepare a 0.5 mg / mL ursolic acid standard solution. Pipette 0.1 mL, 0.5 mL, 1 mL, 1.5 mL, and 2 mL of the solution into 10 mL volumetric flasks and dilute to volume to prepare 0.005 mg / mL, 0.025 mg / mL, 0.050 mg / mL, 0.075 mg / mL, and 0.100 mg / mL ursolic acid reference solutions, respectively. Inject each of these ursolic acid reference solutions into a liquid chromatograph and determine the peak area under the chromatographic conditions described above. Plot a standard curve based on the peak area and concentration, with concentration on the x-axis and peak area on the y-axis. The results are shown below. Figure 4 As shown, the standard curve equation is: y = 504.34494x - 24563.03813, and the correlation coefficient R0 is 1. 2 =0.9992, linear range 0.0055~0.0275mg / mL.

[0061] 4. Specificity test

[0062] Take equal volumes of 0.32 mg / mL oleanolic acid methanol solution and 3.0 mg / mL ursolic acid methanol solution, mix them thoroughly, and filter them through a 0.45 μm filter membrane for later use to obtain a mixed standard solution;

[0063] Take the mixed standard solution, the gynecological antipruritic tablet test solution, and the negative control solution respectively, and determine the peak area according to the above chromatographic conditions. The results are shown in the figure. Figure 5 The results showed that the test solution of the gynecological antipruritic tablets had a chromatographic peak at the retention time of the mixed standard solution, and the control solution had a corresponding peak at the corresponding retention time, indicating that the determination method provided by the present invention has good specificity.

[0064] 5. Precision test

[0065] Take 6 g of Gynecological Itching-Relieving Tablets, accurately weigh, prepare the sample according to the preparation method of the test solution of Gynecological Itching-Relieving Tablets in item 3.1, accurately pipette 10 μl, inject into the liquid chromatograph, and determine the peak area according to the above chromatographic conditions. Replace the peak area into the linear equation (oleanolic acid: y = 152.7962x - 7929.7; ursolic acid: y = 504.34494x - 24563.03813) to determine the content. The test is repeated for 6 times. The experimental results are shown in Table 1. The RSD values of the relative peak areas of oleanolic acid and ursolic acid are 0.77% and 0.87% respectively, which meet the precision requirement, indicating that the method has good precision.

[0066] Table 2. Results of precision test

[0067]

[0068] 6. Stability test

[0069] Take 6 g of Gynecological Itching-Relieving Tablets, accurately weigh, prepare the sample according to the preparation method of the test solution of Gynecological Itching-Relieving Tablets in item 3.1, and inject the sample at 0, 2, 4, 8, 12 and 24 h respectively. Determine the peak area according to the above chromatographic conditions. The experimental results are shown in Table 2. The RSD values of the relative peak areas of oleanolic acid and ursolic acid are 2.59% and 0.68% respectively, which meet the stability requirement, indicating that the method has good stability within 24 hours.

[0070] Table 3. Results of stability test

[0071]

[0072] 7. Reproducibility test

[0073] Take 6 g of Gynecological Itching-Relieving Tablets, accurately weigh, prepare 6 test solutions of Gynecological Itching-Relieving Tablets according to the preparation method of the test solution of Gynecological Itching-Relieving Tablets in item 3.1, accurately pipette 10 μl, inject into the liquid chromatograph, and continuously inject the sample. The experimental results are shown in Table 3. Determine the peak area according to the above chromatographic conditions, replace the peak area into the linear equation (oleanolic acid: y = 152.7962x - 7929.7; ursolic acid: y = 504.34494x - 24563.03813) to determine the content. The RSD values of the relative peak areas of oleanolic acid and ursolic acid are 2.01% and 2.05% respectively, which meet the reproducibility requirement, indicating that the method has good reproducibility.

[0074] Table 4. Results of reproducibility test

[0075]

[0076] 8. Sample addition recovery test

[0077] Take 6 parts of known content of gynecological antipruritic tablets, about 3g each, accurately weigh, respectively, add oleanolic acid and ursolic acid standard each about 0.03mg, 0.11mg, continuously sample respectively. The experimental results are shown in Table 4, Table 5, the average recovery of the relative peak area of oleanolic acid and ursolic acid is 100.1% and 100.2%, and the RSD value is 2.64% and 1.48%, which meets the recovery requirement, indicating that the recovery of this method is good.

[0078] Table 5 Oleanolic acid spiking recovery test results

[0079]

[0080] Table 6 Ursolic acid spiking recovery test results

[0081]

[0082]

[0083] 9. Determination of the content of gynecological antipruritic tablets of different batches

[0084] Take gynecological antipruritic tablets of different batches in Table 6 respectively, prepare the sample according to the preparation method of the gynecological antipruritic tablet test solution in item 3.1, and determine the content of oleanolic acid and ursolic acid according to the above chromatographic conditions, and the experimental results are shown in Table 6.

[0085] Table 7 Oleanolic acid and ursolic acid content in 10 batches of gynecological antipruritic tablets of different batches

[0086]

[0087] It can be seen that the contents of oleanolic acid and ursolic acid in gynecological antipruritic tablets of different batches are different, and the content difference is large. The method provided by the present application can simultaneously determine the contents of oleanolic acid and ursolic acid in gynecological antipruritic tablets, which is helpful to control the quality of gynecological antipruritic tablets and improve its standard.

[0088] The above description is a detailed description of the preferred and feasible embodiments of the present application, but the embodiments are not intended to limit the scope of the patent application of the present application. Any equivalent changes or modifications made under the technical spirit of the present application should be included in the scope of the patent.

Claims

1. A high performance liquid chromatography method for simultaneously determining the contents of oleanolic acid and ursolic acid in gynecological antipruritic tablets, characterized in that, The high performance liquid chromatography method is to simultaneously determine the contents of oleanolic acid and ursolic acid in the gynecological itching-relieving tablets under the same chromatographic conditions; The high performance liquid chromatography method is performed according to the following steps: (1) Chromatographic conditions: Chromatographic column: C18 reversed-phase chromatographic column; Mobile phase: mobile phase A is methanol, and mobile phase B is 0.1% formic acid aqueous solution; gradient elution is performed; the mobile phase ratio is as follows: 0-60 min, wherein the mobile phase ratio is all a volume percentage: mobile phase A is 90%-95%, and mobile phase B is 10%-5%; Flow rate: 0.2-1.5 mL / min; detection wavelength: 210 nm-350 nm; column temperature: 25-40 DEG C; (2) Preparation of the test sample solution: the gynecological itching-relieving tablets are taken, methanol is added, ultrasonic extraction is performed, and the filtrate is taken to obtain the gynecological itching-relieving tablet test sample solution; (3) Preparation of the control sample solution: oleanolic acid and ursolic acid control samples are respectively taken, methanol is added to dissolve, and oleanolic acid control sample solution and ursolic acid control sample solution are obtained; (4) Determination method: determination is performed according to the chromatographic conditions in step (1), and the determination is completed.

2. The method for simultaneously determining the contents of oleanolic acid and ursolic acid in gynecological antipruritic tablets according to claim 1, characterized in that, The chromatographic conditions of step (1) are as follows: Instrument: Waters 2695 high performance liquid chromatograph; Chromatographic column: ZORBAX Eclipse XDB-C18 Analytical chromatographic column, with a column specification of 250*4.6 mm and 5 μm; Mobile phase: mobile phase A is methanol, and mobile phase B is 0.1% formic acid aqueous solution; gradient elution is performed; the mobile phase ratio is as follows: 0-60 min, mobile phase A is 92%-95%, and mobile phase B is 8%-5%, wherein the mobile phase ratio is all a volume percentage; Column temperature: 25 DEG C; injection amount: 10 μL; detection wavelength: 210 nm.

3. The method for simultaneously determining the contents of oleanolic acid and ursolic acid in gynecological antipruritic tablets according to claim 1, characterized in that, In step (2), the test sample solution is prepared as follows: 6 g of the gynecological itching-relieving tablets is accurately weighed, placed in a 100 mL conical flask with a plug, 25 mL of methanol is added, the weight is determined, ultrasonic extraction is performed for 0.5 h, the temperature is allowed to decrease, the weight is accurately determined again, the loss weight is made up with methanol, and then shaken uniformly; filtration is performed, and the obtained filtrate is filtered through a 0.45 μm microporous filter membrane to obtain the gynecological itching-relieving tablet test sample solution.

4. The method for simultaneously determining the contents of oleanolic acid and ursolic acid in gynecological antipruritic tablets according to claim 1, characterized in that, In step (3), the control sample solution is prepared as follows: 1.1 mg of oleanolic acid is dissolved in 10 mL of methanol to prepare a 0.11 mg / mL oleanolic acid standard solution; then the oleanolic acid standard solution is prepared into an oleanolic acid control sample solution with the content of oleanolic acid being 0.0055-0.0275 mg / mL; 3.5 mg of ursolic acid is dissolved in 7 mL of methanol to prepare a 0.5 mg / mL ursolic acid standard solution; then the ursolic acid standard solution is prepared into a ursolic acid control sample solution with the content of ursolic acid being 0.005-0.1 mg / mL.

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