Identification method of oleanolic acid and ursolic acid in gynecological antipruritic tablets

Abstract

The invention discloses a kind of oleanolic acid and ursolic acid identification method in gynecological antipruritic tablets, and relates to the technical field of Chinese medicine quality control. Oleanolic acid and ursolic acid identification method of the present invention include the preparation of test solution, the preparation of reference solution, thin layer plate spotting, developing agent development, spraying display agent color development and the step of inspection, also including in situ pretreatment;The in situ pretreatment is:After the thin layer plate spotting ends, one end of the thin layer plate spotting is immersed in prepared iodine ‑ dichloromethane solution, after soaking spotting point 1.0 ‑ 1.5cm, it is quickly taken out, and covered with a glass plate, the glass plate is removed after being placed for 30 ‑ 50min, the solution remaining on the thin layer plate is waved away, and it is observed that brown on the thin layer plate fades, that is, to obtain final product. The present invention is applicable to the simultaneous identification of oleanolic acid and ursolic acid in Hedyotis diffusa.

Description

Technical Field

[0001] The invention relates to the technical field of quality control of traditional Chinese medicines, and in particular to a method for identifying oleanolic acid and ursolic acid in gynecological itching tablets. Background Art

[0002] Fuke Zhiyan Tablets are a traditional Chinese medicine for treating vaginitis. Its main ingredients include Patrinia patriniae, Hedyotis diffusa, Taraxacum mongolicum, Radix hyssop, Rubia cordifolia, Millettia reticulata, Angelica sinensis, and Corydalis yanhusuo. They have the effects of clearing heat and dampness, killing insects, and relieving itching. Clinically, they are used for vaginitis characterized by damp-heat syndrome.

[0003] The herb Hedyotis diffusa in the gynecological antipruritic tablets is the fresh or dried whole herb of Hedyotis diffusa Willd, a plant of the Rubiaceae family. It has a pungent, slightly bitter flavor and a slightly cold nature. It enters the pericardium and liver meridians, promoting blood circulation, regulating menstruation, and promoting diuresis and reducing swelling. The main chemical components of Hedyotis diffusa Willd are flavonoids, terpenes, anthraquinones, phenylpropanoids, sterols, and phenolic acids. It also contains trace elements such as zinc, calcium, copper, manganese, iron, and magnesium. Hedyotis diffusa Willd also contains acidic compounds, primarily oleanolic acid, ursolic acid, caffeic acid, and ferulic acid.

[0004] The 2020 edition of the Chinese Pharmacopoeia, Part I, uses thin-layer chromatography (TLC) for qualitative analysis of Hedyotis diffusa in patent medicines, using petroleum ether, ethyl acetate, and glacial acetic acid as the developing solvent and 10% ethanolic sulfuric acid solution for color development. Regarding content determination, HPLC was used to determine the content of ursolic acid and oleanolic acid in Hedyotis diffusa. However, simultaneous identification of ursolic acid and oleanolic acid in Hedyotis diffusa is difficult. Therefore, a method for simultaneously identifying oleanolic acid and ursolic acid in Hedyotis diffusa is lacking. Summary of the Invention

[0005] In view of the above shortcomings, the present invention provides a method for identifying oleanolic acid and ursolic acid in gynecological itching tablets, which can simultaneously identify oleanolic acid and ursolic acid in Hedyotis diffusa, and the operation method is simple. The specific technical solution is as follows:

[0006] A method for identifying oleanolic acid and ursolic acid in gynecological itching tablets, comprising the steps of preparing a test solution, preparing a reference solution, spotting on a thin layer plate, developing with a developer, spraying a developer for color development, and inspecting the sample, and also comprising an in-situ pretreatment step;

[0007] The in-situ pretreatment is as follows: after the thin layer plate is spotted, one end of the thin layer plate is immersed in a prepared iodine-dichloromethane solution. After the solution is immersed for 1.0-1.5 cm beyond the spotting point, the plate is quickly taken out and covered with a glass plate. After the solution is left for 30-50 minutes, the glass plate is removed and the solution remaining on the thin layer plate is evaporated. When the brown color on the thin layer plate is observed to fade, the thin layer plate after in-situ pretreatment is obtained.

[0008] Preferably, the test solution is prepared by adding the gynecological antipruritic tablets and anhydrous ethanol in a mass ratio of 1:7-9 into a stoppered conical flask, mixing evenly, ultrasonically extracting for 30-50 minutes, filtering, and concentrating the filtrate to obtain the test solution.

[0009] Preferably, the reference solution is prepared as follows: oleanolic acid reference substance and ursolic acid reference substance are placed in volumetric flasks respectively, and anhydrous ethanol is added to make a solution containing 1 mg per 1 mL, thereby obtaining oleanolic acid reference solution and ursolic acid reference solution.

[0010] Preferably, the thin layer plate spotting is as follows: 4 to 8 μL of the test solution and the reference solution are taken respectively and spotted on the same silica gel G thin layer plate.

[0011] Preferably, the developing solvent is petroleum ether-toluene-ethyl acetate-glacial acetic acid (5-15):(15-30):(4-15):(1-5).

[0012] Preferably, the display agent is 10% sulfuric acid ethanol solution.

[0013] Preferably, the inspection condition is ultraviolet light 365nm.

[0014] Preferably, the mass concentration of the iodine-dichloromethane solution is 1-5%.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] After in-situ pretreatment, the method can better separate oleanolic acid and ursolic acid, and the corresponding positions of Hedyotis diffusa show spots with the same color as those of oleanolic acid and ursolic acid reference substances. The method is suitable for simultaneous identification of oleanolic acid and ursolic acid in Hedyotis diffusa. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments.

[0018] Figure 1 is the thin layer chromatogram of Example 1 of the present invention;

[0019] Figure 2 This is the thin layer chromatogram of Example 2 of the present invention;

[0020] Figure 3 This is the thin layer chromatogram of Example 3 of the present invention;

[0021] Figure 4 This is the thin layer chromatogram of Comparative Example 1 of the present invention;

[0022] Figure 5This is the thin layer chromatogram of Comparative Example 2 of the present invention;

[0023] Figure 6 This is the thin layer chromatogram of Comparative Example 3 of the present invention;

[0024] Figure 7 This is a thin layer chromatogram of an application example of the present invention. DETAILED DESCRIPTION

[0025] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments.

[0026] Example 1

[0027] (1) Preparation of test solution:

[0028] Remove the coating from the gynecological antipruritic tablets, grind them into powder using a mortar, accurately weigh 1.1074 g and place it in a stoppered conical flask, add 9 mL of anhydrous ethanol using a pipette, ultrasonicate for 30 minutes, draw up the extract with a syringe, filter using a 0.45 μm organic phase microporous filter membrane, place the filtrate in a 10 mL beaker, concentrate to the 2 mL mark on a light wave oven, and transfer the resulting solution to a sample tube as the test solution for the gynecological antipruritic tablets.

[0029] (2) Preparation of control solution:

[0030] A. Place 0.0051 g of oleanolic acid reference substance in a 5 mL volumetric flask and add anhydrous ethanol to the mark to make a solution containing 1 mg per 1 mL. This is the oleanolic acid reference solution.

[0031] B. Take 0.0052 g of ursolic acid reference substance and place it in a 5 mL volumetric flask. Add anhydrous ethanol to the mark and mix the two evenly to make a solution containing 1 mg per 1 mL, which is used as the ursolic acid reference substance solution.

[0032] (3) Preparation of 1% iodine-dichloromethane solution:

[0033] Accurately weigh 1 g of iodine and place it in a beaker. Measure 74.7 mL of dichloromethane in a measuring cylinder and mix the two thoroughly to prepare a 1% iodine-dichloromethane solution. Transfer the solution to a brown bottle and store for later use.

[0034] (4) Preparation of 10% sulfuric acid ethanol solution:

[0035] Use a 100mL graduated cylinder to measure 90mL of anhydrous ethanol into a 150mL beaker. Use a 10mL graduated cylinder to measure 10mL of sulfuric acid. Slowly add sulfuric acid while stirring the anhydrous ethanol to mix the two evenly to prepare a 10% sulfuric acid-ethanol solution. Transfer it to a spray bottle for later use.

[0036] Refer to the thin layer chromatography method (General Chapter 0502) in the 2020 edition of the Pharmacopoeia of the People's Republic of China. Use a spotting capillary to draw 4 μL of the test solution, oleanolic acid reference solution, and ursolic acid reference solution, respectively, and spot them on the same silica gel G thin layer plate. Immerse one end of the spot in the prepared 1% iodine-dichloromethane solution. After immersing it 1.0 cm from the spot point, quickly remove it and quickly cover it with a glass plate. After leaving it for 30 minutes, remove the glass plate and use a hair dryer to evaporate the remaining solution on the thin layer plate. After observing that the brown color on the thin layer plate has faded, remove it. Use petroleum ether-toluene-ethyl acetate-glacial acetic acid (10:25:11:2) as the developing agent, use a pipette to draw 1 mL of petroleum ether, 2.5 mL of toluene, 1.1 mL of ethyl acetate, and 0.2 mL of glacial acetic acid into the developing cylinder respectively. After saturation for 10 minutes, put it into the thin layer plate for development. After developing to the front line, take it out and dry it, spray it with 10% sulfuric acid ethanol solution, place it in a 105°C oven for about 5 minutes until the spots are clear, then take it out and inspect it under ultraviolet light at 365nm.

[0037] The thin layer chromatogram of this example is as follows Figure 1 As shown (1-Fuke Zhiyun Tablet test solution, 2-oleanolic acid reference solution, 3-ursolic acid reference solution), after in situ pretreatment, oleanolic acid and ursolic acid in Fuke Zhiyun Tablet can be well separated, and the corresponding position of Hedyotis diffusa shows spots of the same color as the oleanolic acid and ursolic acid reference substances, with high spot separation and no interference.

[0038] Example 2

[0039] (1) Preparation of test solution:

[0040] Remove the coating from the gynecological antipruritic tablets, grind them into powder using a mortar, accurately weigh 1.1074 g and place it in a stoppered conical flask, add 8 mL of anhydrous ethanol using a pipette, ultrasonicate for 50 minutes, draw up the extract with a syringe, filter using a 0.45 μm organic phase microporous filter membrane, place the filtrate in a 10 mL beaker, concentrate to the 2 mL mark on a light wave oven, and transfer the resulting solution to a sample tube as the test solution for the gynecological antipruritic tablets.

[0041] (2) Preparation of control solution:

[0042] A. Place 0.0051 g of oleanolic acid reference substance in a 5 mL volumetric flask and add anhydrous ethanol to the mark to make a solution containing 1 mg per 1 mL. This is the oleanolic acid reference solution.

[0043] B. Take 0.0052 g of ursolic acid reference substance and place it in a 5 mL volumetric flask. Add anhydrous ethanol to the mark and mix the two evenly to make a solution containing 1 mg per 1 mL, which is used as the ursolic acid reference substance solution.

[0044] (3) Preparation of 5% iodine-dichloromethane solution:

[0045] Accurately weigh 1 g of iodine and place it in a beaker. Measure 71.7 mL of dichloromethane with a measuring cylinder and mix the two thoroughly to prepare a 5% iodine-dichloromethane solution. Transfer the solution to a brown bottle and store for later use.

[0046] (4) Preparation of 10% sulfuric acid ethanol solution:

[0047] Use a 100mL graduated cylinder to measure 90mL of anhydrous ethanol into a 150mL beaker. Use a 10mL graduated cylinder to measure 10mL of sulfuric acid. Slowly add sulfuric acid while stirring the anhydrous ethanol to mix the two evenly to prepare a 10% sulfuric acid-ethanol solution. Transfer it to a spray bottle for later use.

[0048] Refer to the thin-layer chromatography method (General Chapter 0502) in the 2020 edition of the Pharmacopoeia of the People's Republic of China. Use a spotting capillary to draw 8 μL of the test solution, oleanolic acid solution, and ursolic acid solution, respectively, and spot them on the same silica gel G thin-layer plate. Immerse one end of the spot in the prepared 5% iodine-dichloromethane solution. After it is immersed 1.5 cm from the spot point, quickly remove it and quickly cover it with a glass plate. After leaving it for 50 minutes, remove the glass plate and use a hair dryer to evaporate the remaining solution on the thin-layer plate. Remove it after observing that the brown color on the thin-layer plate has faded. Use petroleum ether-toluene-ethyl acetate-glacial acetic acid (5:10:6:1) as the developing agent, use a pipette to draw 1 mL of petroleum ether, 2 mL of toluene, 1.2 mL of ethyl acetate, and 0.2 mL of glacial acetic acid into the developing cylinder respectively. After saturation for 10 minutes, put it into the thin layer plate for development. After developing to the front line, take it out and dry it, spray it with 10% sulfuric acid ethanol solution, place it in a 105℃ oven for about 5 minutes until the spots are clear, then take it out and inspect it under ultraviolet light at 365nm.

[0049] The thin layer chromatogram of this example is as follows Figure 2 As shown (1-Fuke Zhiyun Tablet test solution, 2-oleanolic acid reference solution, 3-ursolic acid reference solution), after in situ pretreatment, oleanolic acid and ursolic acid in Fuke Zhiyun Tablet can be well separated, and the corresponding position of Hedyotis diffusa shows spots of the same color as the oleanolic acid and ursolic acid reference substances, with high spot separation and no interference.

[0050] Example 3

[0051] (1) Preparation of test solution:

[0052] Remove the coating from the gynecological antipruritic tablets, grind them into powder using a mortar, accurately weigh 1.1074 g and place it in a stoppered conical flask, add 10 mL of anhydrous ethanol using a pipette, ultrasonicate for 40 minutes, draw up the extract with a syringe, filter using a 0.45 μm organic phase microporous filter membrane, place the filtrate in a 10 mL beaker, concentrate to the 2 mL mark on a light wave oven, and transfer the resulting solution to a sample tube as the test solution for the gynecological antipruritic tablets.

[0053] (2) Preparation of control solution:

[0054] A. Place 0.0051 g of oleanolic acid reference substance in a 5 mL volumetric flask and add anhydrous ethanol to the mark to make a solution containing 1 mg per 1 mL. This is the oleanolic acid reference solution.

[0055] B. Take 0.0052 g of ursolic acid reference substance and place it in a 5 mL volumetric flask. Add anhydrous ethanol to the mark and mix the two evenly to make a solution containing 1 mg per 1 mL, which is used as the ursolic acid reference substance solution.

[0056] (3) Preparation of 2% iodine-dichloromethane solution:

[0057] Accurately weigh 1g of iodine and place it in a beaker. Measure 93.7ml of dichloromethane with a measuring cylinder and mix the two thoroughly to prepare a 2% iodine-dichloromethane solution. Transfer the solution to a brown bottle and store for later use.

[0058] (4) Preparation of 10% sulfuric acid ethanol solution:

[0059] Use a 100mL graduated cylinder to measure 90mL of anhydrous ethanol into a 150mL beaker. Use a 10mL graduated cylinder to measure 10mL of sulfuric acid. Slowly add sulfuric acid while stirring the anhydrous ethanol to mix the two evenly to prepare a 10% sulfuric acid-ethanol solution. Transfer it to a spray bottle for later use.

[0060] Refer to the thin-layer chromatography method (General Chapter 0502) in the 2020 edition of the Pharmacopoeia of the People's Republic of China. Use a spotting capillary to draw 5 μL of the test solution, oleanolic acid solution, and ursolic acid solution respectively, and spot them on the same silica gel G thin-layer plate. Immerse one end of the spot in the prepared 2% iodine-dichloromethane solution. After it is immersed 1.2 cm from the spot point, quickly remove it and quickly cover it with a glass plate. After leaving it for 30 minutes, remove the glass plate and use a hair dryer to evaporate the remaining solution on the thin-layer plate. After observing that the brown color on the thin-layer plate has faded, remove it. Use petroleum ether-toluene-ethyl acetate-glacial acetic acid (13:28:14:4) as the developing agent, use a pipette to draw 1.3 mL of petroleum ether, 2.8 mL of toluene, 1.4 mL of ethyl acetate, and 0.4 mL of glacial acetic acid into the developing cylinder respectively. After saturation for 10 minutes, put it into the thin layer plate for development. After developing to the front line, take it out and dry it, spray it with 10% sulfuric acid ethanol solution, place it in a 105°C oven for about 5 minutes until the spots are clear, then take it out and inspect it under ultraviolet light at 365 nm.

[0061] The thin layer chromatogram of this example is as follows Figure 3 As shown (1-gynecological antipruritic tablets test solution, 2-oleanolic acid reference solution, 3-ursolic acid reference solution), after in situ pretreatment and using 10% sulfuric acid ethanol solution as a color developer, oleanolic acid and ursolic acid in the gynecological antipruritic tablets can be well separated, and the corresponding positions of Hedyotis diffusa show spots of the same color as the oleanolic acid and ursolic acid reference substances, with high spot separation and no interference.

[0062] Comparative Example 1

[0063] This comparative example did not perform in-situ pretreatment on the thin layer plate after spotting. Specifically, the step "After spotting, immerse one end of the thin layer plate into the prepared iodine-dichloromethane solution until it extends 1.0 cm beyond the spotting point, then quickly remove the plate and cover it with a glass plate. After 30 minutes, remove the glass plate, evaporate the remaining solution on the plate, and observe that the brown color on the plate has faded" was omitted. All other steps were the same as in Example 1.

[0064] The thin layer chromatogram of this comparative example is as follows Figure 4 As shown (1-gynecological antipruritic tablet test solution, 2-oleanolic acid reference solution, 3-ursolic acid reference solution), the figure shows that without in situ pretreatment, only 10% sulfuric acid ethanol solution is used as the color developer. Although it can be used to develop the color of oleanolic acid and ursolic acid, it cannot separate oleanolic acid and ursolic acid.

[0065] Comparative Example 2

[0066] The inspection condition of this comparative example is daylight, and the other steps are the same as those of Example 1.

[0067] The thin layer chromatogram of this comparative example is as follows Figure 5As shown in the figure (1-Gynecological Antipruritic Tablets test solution, 2-oleanolic acid reference solution, 3-ursolic acid reference solution), the inspection effect under ultraviolet conditions is better than that under sunlight.

[0068] Comparative Example 3

[0069] In this comparative example, iodine jar was used instead of 10% sulfuric acid ethanol solution for color development, and other steps were the same as those in Example 1.

[0070] The thin layer chromatogram of this comparative example is as follows Figure 6 As shown in the figure (1-Gynecological Antipruritic Tablets test solution, 2-oleanolic acid reference solution, 3-ursolic acid reference solution), the iodine cylinder can only be used to develop color for oleanolic acid, but not for ursolic acid.

[0071] Application examples:

[0072] Application of the identification method of the present invention:

[0073] A. Preparation of test solution: Remove the coating from 10 Fuke Zhisuan tablets, grind them finely in a mortar, accurately weigh 1.1074 g and place it in a stoppered conical flask. Use a pipette to add 10 mL of anhydrous ethanol. Ultrasonicate for 30 minutes. Aspirate the extract with a syringe and filter through a 0.45 μm organic phase microporous filter. Place the filtrate in a 10 mL beaker and concentrate to the 2 mL mark in a microwave oven. Transfer the resulting solution to a sample tube to prepare the test solution for Fuke Zhisuan tablets.

[0074] Preparation of the Hedyotis diffusa test solution: take 1g of Hedyotis diffusa and 10mL of anhydrous ethanol, mix the two in a 50mL stoppered conical flask, and extract by ultrasonication for 30 minutes. Use a syringe to draw up the extract, filter it with a 0.45um organic phase microporous filter membrane, place the filtrate in a 10mL beaker, heat it on a light wave oven and concentrate it to the 2mL mark on the beaker, then stop concentrating. The concentrate is used as the Hedyotis diffusa test solution.

[0075] B. Preparation of negative control solution: Place 5g of Patrinia herba, 5g of Taraxacum mongolicum, 2g of Radix Codonopsis pilosulae, 5g of Rubia cordifolia, 5g of Millettia spatholobi, and 3g of Radix Angelicae sinensis in a beaker and decoct twice with purified water, adding 12 times the amount the first time and 10 times the amount the second time. Decoction for 2 hours each time. Combine the decoctions, filter, and concentrate the filtrate until thick. Add 1g of Corydalis yanhusuo powder, mix well, and dry to obtain the negative control powder. Accurately weigh 1.1055g of the negative control powder into a stoppered conical flask. Use a pipette to add 10mL of anhydrous ethanol. Ultrasonicate for 30 minutes. Aspirate the extract with a syringe and filter through a 0.45μm organic phase microporous filter. The filtrate is placed in a 10mL beaker and concentrated to the 2mL mark on a microwave oven. The resulting solution is transferred to a sample tube as the negative control solution.

[0076] C. Preparation of Reference Solution: Place 0.0051 g of oleanolic acid reference substance in a 5 mL volumetric flask and add anhydrous ethanol to the mark to make a solution containing 1 mg per mL. This is the oleanolic acid reference solution. Place 0.0052 g of ursolic acid reference substance in a 5 mL volumetric flask and add anhydrous ethanol to the mark. Mix the two to make a solution containing 1 mg per mL. This is the ursolic acid reference solution.

[0077] D. Preparation of 1% iodine-dichloromethane solution: Accurately weigh 1g of iodine and place it in a beaker. Measure 74.7mL of dichloromethane in a measuring cylinder. Mix the two thoroughly to prepare a 1% iodine-dichloromethane solution. Transfer the solution to a brown bottle and store for later use.

[0078] E. Refer to the thin layer chromatography test (General Chapter 0502) in the 2020 edition of the "Pharmacopoeia of the People's Republic of China". Use a spotting capillary to draw 8uL each of the test solution of the gynecological antipruritic tablets, the test solution of the white flower snake tongue grass, the negative control solution, the oleanolic acid reference solution, and the ursolic acid reference solution, and spot them on the same silica gel G thin layer plate. Immerse one end of the spot in the prepared 1% iodine-dichloromethane solution (1.0cm above the spot point), then quickly take it out and quickly cover it with a glass plate. After leaving it for 30 minutes, remove the glass plate and use a hair dryer to evaporate the remaining solution on the thin layer plate. After observing that the brown color on the thin layer plate fades, take it out and develop it in a saturated developing agent. After developing to the front line, take it out and dry it. Spray it with 10% sulfuric acid ethanol solution, place it in a 105°C oven for 5 minutes until the spots are clear, then take it out, inspect it under ultraviolet light at 365nm, take pictures and record it, and see the development results. Figure 7 (1- Fuke Zhiyan tablet test solution, 2- Houttuynia cordata test solution, 3- negative control solution, 4- oleanolic acid reference solution, 5- ursolic acid reference solution), the figure shows that oleanolic acid and ursolic acid can be identified simultaneously.

[0079] In summary, the present invention improves the existing technology by combining in-situ pretreatment, 10% sulfuric acid ethanol solution and ultraviolet inspection, and can simultaneously identify oleanolic acid and ursolic acid in gynecological itching tablets. The operation method is simple and very suitable for practice.

[0080] The foregoing descriptions of specific exemplary embodiments of the present invention are for purposes of illustration and description. These descriptions are not intended to limit the invention to the precise forms disclosed, and it is apparent that many variations and modifications are possible in light of the foregoing teachings. The exemplary embodiments have been selected and described for the purpose of explaining the specific principles of the invention and their practical application, thereby enabling those skilled in the art to realize and utilize a variety of exemplary embodiments of the invention and various options and modifications. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A method for identifying oleanolic acid and ursolic acid in gynecological itching tablets, comprising the steps of preparing a test solution, preparing a reference solution, spotting on a thin layer plate, developing with a developer, spraying a display agent for color development, and inspecting the sample, wherein: It also includes the steps of in-situ pretreatment; The in-situ pretreatment comprises: after the sample is spotted on the thin layer plate, immersing one end of the thin layer plate in a prepared iodine-dichloromethane solution with a mass concentration of 1-5%, and quickly taking it out after it is immersed 1.0-1.5 cm from the sample point, and covering it with a glass plate. After leaving it for 30-50 minutes, the glass plate is removed, and the solution remaining on the thin layer plate is evaporated. When the brown color on the thin layer plate is observed to fade, the thin layer plate after in-situ pretreatment is obtained; The test solution is prepared by adding the gynecological antipruritic tablets and anhydrous ethanol in a mass ratio of 1:7-9 into a stoppered conical flask, mixing evenly, ultrasonically extracting for 30-50 minutes, filtering, and concentrating the filtrate to obtain the test solution; The developing solvent is petroleum ether-toluene-ethyl acetate-glacial acetic acid (5-15):(15-30):(4-15):(1-5); The display agent is 10% sulfuric acid ethanol solution.

2. The method for identifying oleanolic acid and ursolic acid in a gynecological itching tablet according to claim 1, characterized in that: The reference solution is prepared as follows: oleanolic acid reference substance and ursolic acid reference substance are placed in a volumetric flask, and anhydrous ethanol is added to make a solution containing 1 mg per 1 mL, thereby obtaining oleanolic acid reference substance solution and ursolic acid reference substance solution.

3. The method for identifying oleanolic acid and ursolic acid in a gynecological itching tablet according to claim 1, characterized in that: The thin layer plate spotting is as follows: 4 to 8 μL of the test solution and the reference solution are taken respectively and spotted on the same silica gel G thin layer plate.

4. The method for identifying oleanolic acid and ursolic acid in a gynecological itching tablet according to claim 1, characterized in that: The inspection condition is ultraviolet light 365nm.

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