A thin-layer identification method of belamcanda in gynecological antipruritic tablets

Abstract

The application discloses a thin-layer identification method of Belamcanda chinensis in gynecological itching-relieving tablets and relates to the technical field of traditional Chinese medicine quality control. The thin-layer identification method is characterized in that ursolic acid and oleanolic acid are used as the control samples, the developing agent is composed of ethyl ether and dichloromethane in a volume ratio of 0-2:6-9, and the color developing agent is 10% sulfuric acid ethanol solution.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine quality control technology, and in particular to a thin-layer chromatography method for identifying Hedyotis diffusa in gynecological antipruritic tablets. Background Technology

[0002] Gynecological antipruritic tablets are a traditional Chinese medicine for treating vaginal inflammation. The main ingredients include Patrinia scabiosaefolia, Hedyotis diffusa, Taraxacum mongolicum, Rhizoma Cyathulae, Radix Rubiae, Caulis Spatholobi, Radix Angelicae Sinensis, and Rhizoma Corydalis, which have the effects of clearing heat and drying dampness, killing parasites and relieving itching. Clinically, it is used for patients with vaginitis of the damp-heat type.

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

[0004] The 2020 edition of the Chinese Pharmacopoeia, Part I, uses thin-layer chromatography (TLC) identification of Hedyotis diffusa in prepared medicines for qualitative analysis, using petroleum ether-ethyl acetate-glacial acetic acid as the developing solvent and 10% sulfuric acid in ethanol for color development. For content determination, high-performance liquid chromatography (HPLC) is used to determine the content of ursolic acid and oleanolic acid in Hedyotis diffusa. The State Food and Drug Administration standard, number YBZ0232009, discloses a TLC identification method for Hedyotis diffusa in gynecological antipruritic tablets: Take 20 tablets of this product, remove the coating, grind finely, weigh 5g, add 50ml of methanol, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1ml of ethanol to prepare the test solution. Separately, take 2g of Hedyotis diffusa reference material, add 20ml of methanol, and prepare a reference material solution using the same method. According to the thin-layer chromatography method (Chinese Pharmacopoeia 2010 Edition, Part I, Appendix VI B), 10 μl of each of the above two solutions were spotted separately onto the same silica gel G thin-layer plate with sodium carboxymethyl cellulose as a binder. Ethyl acetate-methanol-water (5:1.5:1) was used as the developing solvent. After development, the plate was removed, dried, sprayed with 10% sulfuric acid ethanol solution, and heated at 105℃ until the spots were clearly visible. In the chromatogram of the test sample, spots of the same color appeared at the corresponding positions as in the chromatogram of the reference medicinal material. In actual operation, due to the presence of many impurities, the thin-layer identification test had a long development time and unsatisfactory development effect, resulting in poor spot separation or indistinct spots. Furthermore, the repeatability was not ideal. Therefore, a stable and rapid qualitative method for the identification of Hedyotis diffusa in gynecological antipruritic tablets is lacking. Summary of the Invention

[0005] To address the above shortcomings, this invention provides a thin-layer chromatography method for identifying Hedyotis diffusa in gynecological antipruritic tablets, which can quickly and accurately perform qualitative analysis of Hedyotis diffusa in gynecological antipruritic tablets. The specific technical solution is as follows:

[0006] A thin-layer chromatography method for identifying Hedyotis diffusa in a gynecological antipruritic tablet includes the following steps:

[0007] (1) Preparation of test solution: Take gynecological antipruritic tablets, add ether, heat to reflux, filter, evaporate the filtrate to dryness, add toluene to dissolve the residue at 60°C, load onto silica gel column, elute with petroleum ether, discard the eluent, elute with ethanol-petroleum ether, collect the eluent, evaporate to dryness, dissolve the residue in ethanol to obtain the test solution.

[0008] (2) Preparation of reference solutions: Take ursolic acid and oleanolic acid reference standards and add methanol to prepare reference solutions respectively;

[0009] (3) Preparation of reference medicinal material solution: Take the reference medicinal material of Oldenlandia diffusa and prepare it according to the preparation method of the test solution described above;

[0010] (4) Spot the above test solution, reference solution and reference medicinal material solution onto the same silica gel thin layer plate, develop with a mixture of ether and dichloromethane as the developing solvent, remove, air dry, spray with 10% sulfuric acid ethanol test solution, heat until color develops, and examine.

[0011] Preferably, the specific operation method of step (1) is as follows: Take 10g of gynecological antipruritic tablets, add ether and heat under reflux twice, filter, combine the filtrates, evaporate the filtrate to dryness, add 20ml of hot toluene to dissolve the residue, pour into a silica gel column (100-200 mesh, 2-5g), elute with petroleum ether at 30-60℃ until no waxy substance is found, discard the eluent, add 15-60ml of ethanol-petroleum ether mixture, elute at 60-90℃, collect the eluent, evaporate to dryness, dissolve the residue in 1ml of ethanol, and use as the test solution.

[0012] Preferably, the amount of ether added during the heating and reflux is 20-50 ml each time, and the reflux time is 20-60 minutes each time.

[0013] Preferably, the volume ratio of ethanol to petroleum ether is (1-5):(95-99).

[0014] Preferably, the volume ratio of ethanol to petroleum ether is 2:98.

[0015] Preferably, in step (2), the reference solution is prepared by dissolving 0.1-0.3 mg of ursolic acid and oleanolic acid in 1 ml of methanol.

[0016] Preferably, in step (4), 10-20 μL of each of the test solution, reference solution, and reference medicinal material solution are spotted onto the same silica gel thin-layer plate.

[0017] Preferably, in step (4), the volume ratio of diethyl ether to dichloromethane is (0-2):(6-9).

[0018] Preferably, the volume ratio of the diethyl ether to dichloromethane is 1:8.

[0019] Compared with the prior art, the beneficial effects of the present invention are:

[0020] This invention achieves short development time, good spot separation, and high reproducibility by focusing on optimizing the developing solvent, using a mixed solution of diethyl ether and dichloromethane. Furthermore, this invention offers advantages such as simple operation and accurate qualitative analysis, providing a reliable guarantee for effectively controlling the quality of gynecological antipruritic tablets. Attached Figure Description

[0021] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below.

[0022] Figure 1 This is a thin-layer chromatogram of Example 1 of the present invention;

[0023] Figure 2 This is a thin-layer chromatogram of Example 2 of the present invention;

[0024] Figure 3 This is a thin-layer chromatogram of Example 3 of the present invention;

[0025] Figure 4 This is a thin-layer chromatogram of Comparative Example 1 of the present invention. Detailed Implementation

[0026] The specific embodiments of the present invention will be described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments.

[0027] Example 1

[0028] Preparation of the test solution: Take 10g of gynecological antipruritic tablets, add ether and heat under reflux twice, 20ml each time for 60 minutes. Filter, combine the filtrates, evaporate the filtrate to dryness, add 10ml of toluene to dissolve the residue at 60℃, pour into a silica gel column (100-200 mesh, 2g), elute with petroleum ether (30-60℃) until no waxy substance remains, discard the eluent, then add 60ml of ethanol-petroleum ether (60-90℃) (1:99) to elute, collect the eluent, evaporate to dryness, add 1ml of ethanol to dissolve the residue, and use as the test solution.

[0029] Preparation of reference solutions: ① Dissolve 0.1 mg of ursolic acid in 1 ml of methanol; ② Dissolve 0.1 mg of oleanolic acid in 1 ml of methanol.

[0030] Preparation of reference herb solution: Take 2g of Hedyotis diffusa reference herb and prepare it according to the preparation method of the test solution.

[0031] Preparation of negative control sample solution: Gynecological antipruritic tablets lacking Hedyotis diffusa were prepared according to the preparation method of the test sample solution.

[0032] According to the thin-layer chromatography method (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502), 10 μL each of the above-mentioned test solution, reference solution, reference medicinal material solution, and negative control sample solution were spotted separately onto the same silica gel G thin-layer plate with sodium carboxymethyl cellulose as a binder. The plate was developed in a pre-saturated developing tank using dichloromethane solution as the developing solvent. After development, the plate was removed, dried, sprayed with 10% sulfuric acid ethanol solution, and heated until the spots were clearly visible. The chromatogram of the test solution showed spots of the same color at the corresponding positions as the chromatogram of the reference solution.

[0033] The method in this embodiment has a fast development speed, requiring only 12 minutes, and its thin-layer chromatogram is as follows. Figure 1 As shown in the figure, 1 is the test solution of the gynecological antipruritic tablet in this embodiment; 2 is the ursolic acid reference solution; 3 is the oleanolic acid reference solution; 4 is the white flower tongue grass reference medicinal material solution in this embodiment; and 5 is the negative control sample solution in this embodiment. The figure shows that the separation effect of this method is good, the spots are clearly displayed, and the method has high reproducibility.

[0034] Example 2

[0035] Preparation of the test solution: Take 10g of gynecological antipruritic tablets, add ether and heat under reflux twice, 35ml each time for 40 minutes. Filter, combine the filtrates, evaporate the filtrate to dryness, add 15ml of toluene to dissolve the residue at 60℃, pour into a silica gel column (100-200 mesh, 3.5g), elute with petroleum ether (30-60℃) until no waxy substance remains, discard the eluent, then add 40ml of ethanol-petroleum ether (60-90℃) (2:98) to elute, collect the eluent, evaporate to dryness, add 1ml of ethanol to dissolve the residue, and use as the test solution.

[0036] Preparation of reference solutions: ① Dissolve 0.1 mg of ursolic acid in 1 ml of methanol; ② Dissolve 0.1 mg of oleanolic acid in 1 ml of methanol.

[0037] Preparation of reference herb solution: Take 2g of Hedyotis diffusa reference herb and prepare it according to the preparation method of the test solution.

[0038] Preparation of negative control sample solution: Gynecological antipruritic tablets lacking Hedyotis diffusa were prepared according to the preparation method of the test sample solution.

[0039] According to the thin-layer chromatography method (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502), 10 μL each of the above-mentioned test solution, reference solution, reference medicinal material solution, and negative control sample solution were spotted separately onto the same silica gel G thin-layer plate with sodium carboxymethyl cellulose as a binder. A 1:8 (v / v) mixture of ether and dichloromethane was used as the developing solvent. The plate was placed in a pre-saturated developing tank, developed, removed, and air-dried. The plate was then sprayed with 10% sulfuric acid ethanol solution and heated until the spots were clearly visible. The chromatogram of the test solution showed spots of the same color at the corresponding positions as the chromatogram of the reference solution.

[0040] The method in this embodiment has a fast development speed, requiring only 10 minutes, and its thin-layer chromatogram is as follows. Figure 2 As shown in the figure, 1 is the test solution of the gynecological antipruritic tablet in this embodiment; 2 is the ursolic acid reference solution; 3 is the oleanolic acid reference solution; 4 is the white flower tongue grass reference medicinal material solution in this embodiment; and 5 is the negative control sample solution in this embodiment. The figure shows that the separation effect of this method is good, the spots are clearly displayed, and the method has high reproducibility.

[0041] Example 3

[0042] Preparation of test solutions: Take 10g of each of the four batches of gynecological antipruritic tablets, add ether and heat under reflux twice, 50ml each time for 20 minutes, filter, combine the filtrates, evaporate the filtrates to dryness, add 30ml of toluene to dissolve the residue at 60℃, pour into a silica gel column (100-200 mesh, 5g), elute with petroleum ether (30-60℃) until no waxy substance remains, discard the eluent, add 15ml of ethanol-petroleum ether (60-90℃) (5:95) to elute, collect the eluent, evaporate to dryness, add 1ml of ethanol to dissolve the residue, to obtain test solution 1, test solution 2, test solution 3, and test solution 4.

[0043] Preparation of reference solutions: ① Dissolve 0.3 mg of ursolic acid in 1 ml of methanol; ② Dissolve 0.3 mg of oleanolic acid in 1 ml of methanol.

[0044] Preparation of reference herb solution: Take 2g of Hedyotis diffusa reference herb and prepare it according to the preparation method of the test solution.

[0045] Preparation of negative control sample solution: Gynecological antipruritic tablets lacking Hedyotis diffusa were prepared according to the preparation method of the test sample solution.

[0046] Perform thin-layer chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502). Apply 20 μL each of the above-mentioned test solution and reference solution to the same silica gel G thin-layer plate using sodium carboxymethyl cellulose as a binder. Use a 2:6 (v / v) mixture of ether and dichloromethane as the developing solvent. Place the plate in a pre-saturated developing tank, develop, remove, air dry, spray with 10% sulfuric acid ethanol solution, and heat until the spots are clearly visible. The chromatogram of the test solution should show spots of the same color at the corresponding positions as the chromatogram of the reference solution.

[0047] The method in this embodiment has a fast development speed, requiring only 15 minutes, and its thin-layer chromatogram is as follows. Figure 3 As shown, 1-4 are the test solutions of four batches of gynecological antipruritic tablets in this embodiment; 5 is the ursolic acid reference solution; 6 is the oleanolic acid reference solution; 7 is the white flower tongue grass reference medicinal material solution in this embodiment; and 8 is the negative control sample solution in this embodiment. The figure shows that although there are many impurities in this method, the separation effect is good, the spots are clearly displayed, and the method has high reproducibility.

[0048] Comparative Example 1

[0049] Identification method of Hedyotis diffusa in patent number CYZ0232009:

[0050] Take 10g of gynecological antipruritic tablets, add 50ml of methanol, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1ml of ethanol, and use it as the test solution.

[0051] Take 2g of Hedyotis diffusa reference material, add 20ml of methanol, and prepare a reference material solution using the same method.

[0052] Take the gynecological antipruritic tablets lacking Hedyotis diffusa and prepare a negative control sample solution according to the preparation method of the test sample solution.

[0053] According to the thin-layer chromatography method in General Chapter 0502 of Part IV of the 2020 edition of the Chinese Pharmacopoeia, 10 μL of the test solution and the reference solution were respectively applied to the same silica gel G thin-layer plate. The plate was developed with a mixed solution of ethyl acetate, methanol and water in a volume ratio of (5:1.5:1)3:1 as the developing solvent. The plate was removed, dried, sprayed with 10% sulfuric acid ethanol color reagent, and heated until the spots were clearly visible. The test sample chromatogram showed spots of the same color at the corresponding positions as the reference sample chromatogram.

[0054] This comparative method has a slow development speed, requiring 30 minutes. Its thin-layer chromatogram is shown below. Figure 2 As shown, 1 is the test solution of the gynecological antipruritic tablet in this embodiment, 2 is the control herb solution of Hedyotis diffusa, and 3 is the negative control sample solution in this embodiment. The method has low reproducibility.

[0055] Depend on Figure 1and Figure 2 A comparison of the chromatograms shows that the chromatogram obtained by the identification method for Hedyotis diffusa in standard number CYZ0232009 has small, unclear spots, poor reproducibility, and unsatisfactory development. The chromatogram obtained by the method of this invention has good reproducibility, clear spots, and good separation effect.

[0056] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A thin-layer chromatography method for identifying Hedyotis diffusa in gynecological antipruritic tablets, characterized in that, Includes the following steps: (1) Preparation of test solution: Take gynecological antipruritic tablets, add ether, heat to reflux, filter, evaporate the filtrate to dryness, add toluene to dissolve the residue, load onto a silica gel column, elute with petroleum ether, discard the eluent, and then elute with ethanol-petroleum ether, wherein the volume ratio of ethanol-petroleum ether is (1~5):(95~99); collect the eluent, evaporate to dryness, dissolve the residue in ethanol to obtain the test solution; (2) Preparation of reference solutions: Take ursolic acid and oleanolic acid reference standards and add methanol to prepare reference solutions respectively; (3) Preparation of reference medicinal material solution: Take the reference medicinal material of Oldenlandia diffusa and prepare it according to the preparation method of the test sample solution; (4) Spot the above test solution, reference solution and reference medicinal material solution onto the same silica gel thin layer plate, develop with a mixture of ether and dichloromethane as the developing solvent, remove, air dry, spray with 10% sulfuric acid ethanol test solution, heat until color develops, and examine.

2. The thin-layer chromatography method for identifying Hedyotis diffusa in a gynecological antipruritic tablet according to claim 1, characterized in that, The specific operation method of step (1) is as follows: Take 10g of gynecological antipruritic tablets, add ether and heat under reflux twice, filter, combine the filtrates, evaporate the filtrate to dryness, add 20ml of hot toluene to dissolve the residue, pour it into a silica gel column, elute with petroleum ether at 30~60℃ until no waxy substance is found, discard the eluent, add 15~60ml of ethanol-petroleum ether mixture, elute at 60~90℃, collect the eluent, evaporate to dryness, dissolve the residue in 1ml of ethanol, and use it as the test solution.

3. The thin-layer chromatography method for identifying Hedyotis diffusa in a gynecological antipruritic tablet according to claim 2, characterized in that, During the heating and reflux process, the amount of ether added each time is 20-50 ml, and the reflux time each time is 20-60 minutes.

4. The thin-layer chromatography method for identifying Hedyotis diffusa in a gynecological antipruritic tablet according to claim 1, characterized in that, The volume ratio of ethanol to petroleum ether is 2:

98.

5. The thin-layer chromatography method for identifying Hedyotis diffusa in a gynecological antipruritic tablet according to claim 1, characterized in that, In step (2), the reference solution is prepared by dissolving 0.1~0.3 mg of ursolic acid and oleanolic acid in 1 ml of methanol.

6. The thin-layer chromatography method for identifying Hedyotis diffusa in a gynecological antipruritic tablet according to claim 1, characterized in that, In step (4), 10-20 μL of each of the test solution, reference solution and reference medicinal material solution are spotted onto the same silica gel thin-layer plate.

7. The thin-layer chromatography method for identifying Hedyotis diffusa in a gynecological antipruritic tablet according to claim 1, characterized in that, In step (4), the volume ratio of diethyl ether to dichloromethane is (0~2):(6~9).

8. The thin-layer chromatography method for identifying Hedyotis diffusa in a gynecological antipruritic tablet according to claim 7, characterized in that, The volume ratio of diethyl ether to dichloromethane is 1:8.

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