A method for detecting a kidney-tonifying and body-strengthening tablet

Abstract

This invention provides a method for detecting kidney-tonifying and body-strengthening tablets, including thin-layer chromatography (TLC) to identify the effective components of Epimedium, Ligustrum lucidum, and Cuscuta chinensis in the tablets. The chromatographic identification method of this invention produces clear spots with good separation, eliminates interference from impurities, improves the controllability of the quality standards for kidney-tonifying and body-strengthening tablets, and further ensures the product's intrinsic quality and efficacy.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine testing, specifically to a method for detecting a kidney-tonifying and body-strengthening tablet. Background Technology

[0002] The current standard for Bu Shen Qiang Shen Pian (a traditional Chinese medicine preparation) is listed in Volume 15 of the Ministry of Health's Drug Standards "Traditional Chinese Medicine Compound Preparations" (standard number: WS3-B-2907-98) published in 1997. This product is composed of five traditional Chinese medicines: Epimedium, Cuscuta, Rosa laevigata, Ligustrum lucidum, and Cibotium barometz (processed). It has the effect of tonifying the kidneys and strengthening the body, and currently has 98 approved drug registration numbers. However, the current quality standard only includes one microscopic identification item, one thin-layer chromatography identification item for Epimedium, and two physicochemical colorimetric reactions with weak specificity. It also suffers from problems such as poor specificity of quality control methods, few detection indicators, and a low overall standard level, failing to meet the product's quality control requirements. Summary of the Invention

[0003] The purpose of this invention is to provide a testing method for kidney-tonifying and body-strengthening tablets, thereby improving the controllability of the quality standards for these tablets and further ensuring the product's intrinsic quality and efficacy. This objective is achieved through the following technical solution:

[0004] A method for detecting kidney-tonifying and body-strengthening tablets, using thin-layer chromatography to identify the effective components of Epimedium, Ligustrum lucidum, and Cuscuta chinensis in the tablets.

[0005] The detection method of the present invention, wherein when the object of identification is the effective component of Epimedium, the thin-layer chromatography identification includes:

[0006] (1) Preparation of the test solution

[0007] Take the kidney-tonifying and body-strengthening tablets, remove the coating, grind them into a fine powder, add anhydrous ethanol, sonicate, filter, evaporate the filtrate to dryness, dissolve the residue in methanol, and use it as the test solution.

[0008] (2) Preparation of reference solution

[0009] Take the reference standard of Astragalus membranaceus C, dissolve it in methanol to prepare the reference solution;

[0010] (3) Thin-layer chromatography identification

[0011] Perform the thin-layer chromatography test (General Rule 0502). Apply the two solutions mentioned above separately to the same silica gel G thin-layer plate. Use ethyl acetate-butanone-methanol-water (9-11:0.8-1.2:2.5-3.5:0.8-1.2) as the developing solvent. Develop the plate, remove it, air dry it, spray it with aluminum trichloride test solution, and heat it at 105°C until the spots are clearly visible. Examine it under ultraviolet light (365nm). In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference sample.

[0012] Furthermore, in the aforementioned detection method, when the object of identification is the effective component of Epimedium, the thin-layer chromatography identification includes:

[0013] (1.1) Preparation of the test solution

[0014] Take the kidney-tonifying and body-strengthening tablets, remove the coating, grind them into a fine powder, add anhydrous ethanol, sonicate, filter, evaporate the filtrate to dryness, dissolve the residue in methanol, and use it as the test solution.

[0015] (1.2) Preparation of reference solution

[0016] Take the reference standard of Astragalus membranaceus C, dissolve it in methanol to prepare the reference solution;

[0017] (1.3) Thin-layer chromatography identification

[0018] Perform the thin-layer chromatography test (General Rule 0502). Take 5 μl of each of the two solutions mentioned above and spot them separately on the same silica gel G thin-layer plate. Use ethyl acetate-butanone-methanol-water (10:1:3:1) as the developing solvent. Develop the plate, remove it, air dry it, spray it with aluminum trichloride test solution, and heat it at 105℃ until the spots are clearly visible. Examine it under ultraviolet light (365nm). In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference sample.

[0019] Furthermore, in the aforementioned detection method, when the object of identification is the effective component of Epimedium, the thin-layer chromatography identification includes:

[0020] (2.1) Preparation of the test solution

[0021] Take the kidney-tonifying and body-strengthening tablets, remove the coating, grind them into a fine powder, add 30 ml of anhydrous ethanol, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, add 1 ml of methanol to dissolve the residue, and use it as the test solution.

[0022] (2.2) Preparation of reference solution

[0023] Take the reference standard of Astragalus membranaceus C and prepare a solution containing 1 mg per 1 ml with methanol as the reference solution;

[0024] (2.3) Thin-layer chromatography identification

[0025] Perform the thin-layer chromatography test (General Rule 0502). Take 5 μl of each of the two solutions mentioned above and spot them separately on the same silica gel G thin-layer plate. Use ethyl acetate-butanone-methanol-water (10:1:3:1) as the developing solvent. Develop the plate, remove it, air dry it, spray it with aluminum trichloride test solution, and heat it at 105℃ until the spots are clearly visible. Examine it under ultraviolet light (365nm). In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference sample.

[0026] The detection method of the present invention, wherein when the object of identification is the effective component of privet fruit, the thin-layer chromatography identification includes:

[0027] (1) Preparation of the test solution

[0028] Take the kidney-tonifying and body-strengthening tablets, remove the coating, grind them into a fine powder, add water and sonicate, centrifuge, take the supernatant, extract with ether, discard the ether solution, extract with water-saturated n-butanol, combine the n-butanol solutions, evaporate to dryness, dissolve the residue in methanol, and use it as the test solution.

[0029] (2) Preparation of reference solution

[0030] Take the reference standard of ligustrum lucidum and dissolve it in methanol to prepare the reference solution;

[0031] (3) Thin-layer chromatography identification

[0032] Perform the thin-layer chromatography test (General Rule 0502). Apply the two solutions mentioned above separately to the same silica gel G thin-layer plate. Use ethyl acetate-acetone-water (3.5-4.5:4.5-5.5:0.8-1.2) as the developing solvent. Develop the plate, remove it, air dry it, spray it with 1% vanillin-sulfuric acid ethanol solution, and heat it at 105°C until the spots are clearly visible. Examine it under sunlight. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference sample.

[0033] Furthermore, in the aforementioned detection method, when the object of identification is the effective component of privet fruit, the thin-layer chromatography identification includes:

[0034] (3.1) Preparation of the test solution

[0035] Take the kidney-tonifying and body-strengthening tablets, remove the coating, grind them into a fine powder, add water and sonicate, centrifuge, take the supernatant, extract with ether, discard the ether solution, extract with water-saturated n-butanol, combine the n-butanol solutions, evaporate to dryness, dissolve the residue in methanol, and use it as the test solution.

[0036] (3.2) Preparation of reference solution

[0037] Take the reference standard of ligustrum lucidum and dissolve it in methanol to prepare the reference solution;

[0038] (3.3) Thin-layer chromatography identification

[0039] Perform the thin-layer chromatography test (General Rule 0502). Take 5 μl of each of the two solutions mentioned above and spot them separately on the same silica gel G thin-layer plate. Use ethyl acetate-acetone-water (4:5:1) as the developing solvent. Develop the plate, remove it, air dry it, spray it with 1% vanillin-sulfuric acid ethanol solution, and heat it at 105°C until the spots are clearly visible. Examine it under sunlight. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference sample.

[0040] Furthermore, in the aforementioned detection method, when the object of identification is the effective component of privet fruit, the thin-layer chromatography identification includes:

[0041] (4.1) Preparation of the test solution

[0042] Take the kidney-tonifying and body-strengthening tablets, remove the coating, grind them into a fine powder, add 30ml of water, sonicate for 60 minutes, centrifuge for 10 minutes, take the supernatant, extract with ether 3 times, 20ml each time, discard the ether solution, then extract with water-saturated n-butanol 3 times, 20ml each time, combine the n-butanol solutions, evaporate to dryness, add 1ml of methanol to dissolve the residue, and use it as the test solution;

[0043] (4.2) Preparation of reference solution

[0044] Prepare a reference solution by dissolving ligustrin in methanol to a concentration of 1 mg per ml.

[0045] (4.3) Thin-layer chromatography identification

[0046] Perform the thin-layer chromatography test (General Rule 0502). Take 5 μl of each of the two solutions mentioned above and spot them separately on the same silica gel G thin-layer plate. Use ethyl acetate-acetone-water (4:5:1) as the developing solvent. Develop the plate, remove it, air dry it, spray it with 1% vanillin-sulfuric acid ethanol solution, and heat it at 105°C until the spots are clearly visible. Examine it under sunlight. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference sample.

[0047] The detection method of the present invention, wherein when the object of identification is the effective component of dodder seed, the thin-layer chromatography identification includes:

[0048] (1) Preparation of the test solution

[0049] Take the kidney-tonifying and body-strengthening tablets, remove the coating, grind them into a fine powder, add petroleum ether (60-90℃) and sonicate, discard the petroleum ether, add ethanol to the residue and sonicate, filter, evaporate the filtrate to dryness, add ethanol to the residue to dissolve it, and use it as the test solution.

[0050] (2) Preparation of control herbal solution

[0051] Take 1g of Cuscuta chinensis reference material and prepare it in the same way as the test solution preparation method in step (1) to serve as the reference material solution;

[0052] (5.3) Thin-layer chromatography identification

[0053] According to the thin-layer chromatography method (General Rule 0502), take the above two solutions and spot them separately on the same silica gel G thin-layer plate. Use ethyl acetate-butanone-formic acid-water (27~33:9~11:3.5~4.5:3.5~4.5) as the developing solvent, develop, remove, air dry at room temperature, and examine immediately under ultraviolet light (365nm). In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference medicinal material.

[0054] Furthermore, in the aforementioned detection method, when the object of identification is the effective component of Cuscuta chinensis, the thin-layer chromatography identification includes:

[0055] (5.1) Preparation of the test solution

[0056] Take the kidney-tonifying and body-strengthening tablets, remove the coating, grind them into a fine powder, add petroleum ether (60-90℃) and sonicate, discard the petroleum ether, add ethanol to the residue and sonicate, filter, evaporate the filtrate to dryness, add ethanol to the residue to dissolve it, and use it as the test solution.

[0057] (5.2) Preparation of control herbal solution

[0058] Take 1g of Cuscuta chinensis reference material and prepare it in the same way as the test solution preparation method in (5.1) to serve as the reference material solution;

[0059] (5.3) Thin-layer chromatography identification

[0060] According to the thin-layer chromatography method (General Rule 0502), take 5 μl of each of the above two solutions and spot them separately on the same silica gel G thin-layer plate. Use ethyl acetate-butanone-formic acid-water (30:10:4:4) as the developing solvent, develop, remove, air dry at room temperature, and examine immediately under ultraviolet light (365 nm). In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference medicinal material.

[0061] Furthermore, in the aforementioned detection method, when the object of identification is the effective component of dodder seed, the thin-layer chromatography identification includes:

[0062] (6.1) Preparation of the test solution

[0063] Take the kidney-tonifying and body-strengthening tablets, remove the coating, grind them into a fine powder, add 50 ml of petroleum ether (60-90℃), sonicate for 30 min, discard the petroleum ether, add 40 ml of ethanol to the residue, sonicate for 30 min, filter, evaporate the filtrate to dryness, add 1 ml of ethanol to the residue to dissolve it, and use it as the test solution.

[0064] (6.2) Preparation of control herbal solution

[0065] Take 1g of Cuscuta chinensis reference material and prepare it in the same way as the test solution preparation method in (6.1) to serve as the reference material solution;

[0066] (6.3) Thin-layer chromatography identification

[0067] According to the thin-layer chromatography method (General Rule 0502), take 5 μl of each of the above two solutions and spot them separately on the same silica gel G thin-layer plate. Use ethyl acetate-butanone-formic acid-water (30:10:4:4) as the developing solvent, develop, remove, air dry at room temperature, and examine immediately under ultraviolet light (365 nm). In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference medicinal material.

[0068] The beneficial effects of this invention: The test solution preparation method used in this invention, combined with a specific ratio of developing solvent, is a technical solution developed by the inventors through in-depth research for the thin-layer identification of Bushen Qiangshen tablets, a compound traditional Chinese medicine oral liquid with complex components and severe negative interference. Compared with existing technologies, this method has better separation effect and stronger specificity, providing a basis for further quality control of Bushen Qiangshen tablets. Attached Figure Description

[0069] Figure 1 Chromatogram of Example 1, wherein 1-2 are test solutions, 3 is ascorbic acid C reference solution, 4-6 are test solutions, and 7 is an epimedium-deficient negative control solution.

[0070] Figure 2 Chromatogram of Example 2, containing 1-Ligustrum lucidum negative control solution, 2-5 test solution, and 6-Ligustrum lucidum reference solution.

[0071] Figure 3 Chromatogram of Example 3, wherein 1-dodder seed negative control solution, 2-5 test solution, and 6-dodder seed reference medicinal material solution.

[0072] Figure 4 Comparative Example 1 chromatogram, wherein 1-Epimedium-deficient negative control solution (Method 1), 2-Epimedium reference herb solution (Method 1), 3-Test solution (Method 1), 4-Epimedium-deficient negative control solution (Method 2), 5-Epimedium reference herb solution (Method 2), 6-Test solution (Method 2), 7-Epimedium glycoside and Ascorbic acid C reference solution

[0073] Figure 5 Comparative Example 2 chromatogram, where 1-icariin, ascorbic acid C reference solution, 2-test solution (Method 3), 3-epimedium-deficient negative control solution (Method 3), 4-test solution (Method 4), 5-epimedium-deficient negative control solution (Method 4), 6-test solution (Method 5), and 7-epimedium-deficient negative control solution (Method 5).

[0074] Figure 6 Comparative Example 3 is a chromatogram with ethyl acetate-methanol-water (10:2:1) as the developing solvent, containing 1-2 test solutions, 3-icariin C reference solution, 4-icariin reference solution, and 5-icariin-deficient negative control solution.

[0075] Figure 7 Chromatogram of Comparative Example 3 with ethyl acetate-methanol-formic acid-water (10:2.5:0.5:1) as the developing solvent; 1-3 test solutions; 4-Epimedium-deficient negative control solution; 5-Icariin C reference solution; 6-Epimedium reference solution.

[0076] Figure 8 Comparative Example 3 uses ethyl acetate-butanone-methanol-water (10:1:3:1) as the developing solvent. The chromatogram lacks the following solutions: Epimedium negative control solution, 2-4 test solution, 5-icariin C reference solution, and 6-icariin reference solution.

[0077] Figure 9 Comparative Example 4 chromatograms, including 1-negative control solution lacking Ligustrum lucidum (Method 1), 2-test solution (Method 1), 3-petrolein reference solution, 4-negative control solution lacking Ligustrum lucidum (Method 2), 5-test solution (Method 2), and 6-Ligustrum lucidum reference medicinal material solution.

[0078] Figure 10 Comparative Example 5 chromatograms, including 1-hyperoside reference solution, 2-cucumber-deficient negative control solution (Method 1), 3-test solution (Method 1), 4-cucumber reference medicinal material solution (Method 1), 5-cucumber-deficient negative control solution (Method 4), 6-test solution (Method 4), 7-cucumber reference medicinal material solution (Method 4); 8-cucumber-deficient negative control solution (Method 3), 9-test solution (Method 3), 10-cucumber reference medicinal material solution (Method 3), 11-cucumber-deficient negative control solution (Method 2), 12-test solution (Method 2). Detailed Implementation

[0079] The kidney-tonifying and body-strengthening tablets used below are those listed in Volume 15 of the 1997 Ministry of Health Drug Standards "Traditional Chinese Medicine Compound Preparations" (Standard No.: WS3-B-2907-98). In each example or comparative example, a negative control sample is provided (other medicinal materials lacking identification items are weighed according to the prescription under the kidney-tonifying and body-strengthening tablets section to prepare a negative control sample lacking identification items; a negative control sample solution lacking identification items is prepared using the same method as the test solution preparation method in each example or comparative example, and the same volume as the test solution is spotted on a thin-layer chromatography plate for thin-layer identification). The thin-layer identification results of the negative control sample demonstrate that the method of this invention is negative and interference-free, and has strong specificity.

[0080] Example 1

[0081] A method for identifying Epimedium in a kidney-tonifying and body-strengthening tablet includes the following steps:

[0082] (1) Preparation of the test solution

[0083] Take 5 tablets of Kidney-Strengthening and Body-Nourishing Tablets, remove the coating, grind them into a fine powder, add 30 ml of anhydrous ethanol, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, add 1 ml of methanol to dissolve the residue, and use it as the test solution.

[0084] (2) Preparation of reference solution

[0085] Take the reference standard of Astragalus membranaceus C and prepare a solution containing 1 mg per 1 ml with methanol as the reference solution;

[0086] (3) Preparation of negative control solution

[0087] A negative control sample lacking Epimedium was prepared according to the method for preparing kidney-tonifying and body-strengthening tablets, and then a negative control solution lacking Epimedium was prepared using the same method as the test solution.

[0088] (4) Thin-layer chromatography identification

[0089] Perform the thin-layer chromatography test (General Rule 0502). Apply 5 μl of each of the three solutions described above to the same silica gel G thin-layer plate. Develop the plate using ethyl acetate-butanone-methanol-water (10:1:3:1) as the developing solvent. Remove the plate, air dry, spray with aluminum trichloride reagent, and heat at 105°C until the spots are clearly visible. Examine under ultraviolet light (365 nm). The chromatogram of the test sample should show fluorescent spots of the same color at the corresponding positions as the reference sample, with no negative interference, indicating good specificity. See the chromatogram below. Figure 1 .

[0090] Example 2

[0091] A method for identifying privet fruit in a kidney-tonifying and body-strengthening tablet includes the following steps:

[0092] (1) Preparation of the test solution

[0093] Take 10 tablets of Bushen Qiangshen Pian (a traditional Chinese medicine), remove the coating, grind into a fine powder, add 30ml of water, sonicate for 60 minutes, centrifuge for 10 minutes, take the supernatant, extract with 20ml of ether three times, discard the ether solution, and then extract with 20ml of water-saturated n-butanol three times, combine the n-butanol solutions, evaporate to dryness, dissolve the residue in 1ml of methanol, and use it as the test solution.

[0094] (2) Preparation of reference solution

[0095] Prepare a reference solution by dissolving ligustrin in methanol to a concentration of 1 mg per ml.

[0096] (3) Preparation of negative control solution

[0097] A negative control sample of Ligustrum lucidum lacking alcohol was prepared according to the method for preparing kidney-tonifying and body-strengthening tablets, and then a negative control solution of Ligustrum lucidum lacking alcohol was prepared in the same way as the test solution.

[0098] (4) Thin-layer chromatography identification

[0099] Perform the thin-layer chromatography test (General Rule 0502). Apply 5 μl of each of the three solutions described above to the same silica gel G thin-layer plate. Develop the plate using ethyl acetate-acetone-water (4:5:1) as the developing solvent. Remove the plate, air dry, spray with 1% vanillin-sulfuric acid ethanol solution, and heat at 105°C until the spots are clearly visible. Examine the plate under sunlight. The chromatogram of the test sample should show spots of the same color at the corresponding positions as the chromatogram of the reference sample, with no negative interference, indicating good specificity. See the chromatogram below. Figure 2 .

[0100] Example 3

[0101] A method for identifying dodder seed in a kidney-tonifying and body-strengthening tablet includes the following steps:

[0102] (1) Preparation of the test solution

[0103] Take 10 tablets of Bu Shen Qiang Shen Pian (a traditional Chinese medicine), remove the coating, grind into a fine powder, add 50 ml of petroleum ether (60-90℃), sonicate for 30 min, discard the petroleum ether, add 40 ml of ethanol to the residue, sonicate for 30 min, filter, evaporate the filtrate to dryness, add 1 ml of ethanol to the residue to dissolve, and use as the test solution.

[0104] (2) Preparation of control herbal solution

[0105] Take 1g of Cuscuta chinensis reference material and prepare it using the same method as the test sample solution to serve as the reference material solution;

[0106] (3) Preparation of negative control solution

[0107] A negative control sample lacking Epimedium was prepared according to the method for preparing kidney-tonifying and body-strengthening tablets, and then a negative control solution lacking Epimedium was prepared using the same method as the test solution.

[0108] (4) Thin-layer chromatography identification

[0109] Perform thin-layer chromatography (General Rule 0502). Apply 5 μl of each of the three solutions mentioned above to the same silica gel G thin-layer plate. Use ethyl acetate-butanone-formic acid-water (30:10:4:4) as the developing solvent. Develop, remove, and air-dry at room temperature. Examine immediately under ultraviolet light (365 nm). In the chromatogram of the test sample, spots of the same color as those in the chromatogram of the reference medicinal material should appear at the corresponding positions, with no negative interference, indicating good specificity. See the chromatogram below. Figure 3 .

[0110] Comparative Example 1

[0111] A method for identifying Epimedium in a kidney-tonifying and body-strengthening tablet includes the following steps:

[0112] (1) Preparation of reference solution

[0113] Prepare a reference solution by dissolving icariin and ascorbic acid C reference standards in methanol to a concentration of 1 mg / ml.

[0114] (2) Preparation of control herbal solution

[0115] Take 0.5g of Epimedium, add 30ml of anhydrous ethanol, sonicate for 30min, filter, recover the solvent from the filtrate until dry, add 0.5ml of methanol to dissolve the residue, and use it as the reference solution.

[0116] (3) Preparation of the test solution

[0117] Method 1: Take 10 tablets of Bu Shen Qiang Shen Pian (a traditional Chinese medicine), add 30 ml of methanol, sonicate for 30 minutes, filter, recover the solvent from the filtrate until dry, add 2 ml of methanol to dissolve the residue, and use it as the test solution.

[0118] Method 2: Take the kidney-tonifying and body-strengthening tablets, remove the coating, grind them into a fine powder, take about 5g, add 30ml of hot water, sonicate for 30min, cool, centrifuge, collect the supernatant, extract with ethyl acetate 3 times, 20ml each time, combine the ethyl acetate solutions, evaporate to dryness, add 1ml of ethanol to dissolve the residue, and use it as the test solution.

[0119] (4) Preparation of negative control solution

[0120] A negative control sample lacking Epimedium was prepared according to the method for preparing kidney-tonifying and body-strengthening tablets, and then a negative control solution lacking Epimedium was prepared using the same method as the test solution.

[0121] (5) Thin-layer chromatography identification

[0122] Perform the thin-layer chromatography test (General Rule 0502). Apply 5 μl of each of the above solutions separately to the same silica gel G thin-layer plate. Develop the plate using ethyl acetate-butanone-formic acid-water (10:1:1:1) as the developing solvent. Remove the plate, air dry, spray with 1% aluminum trichloride solution, and heat at 105°C until the spots are clearly visible. Examine the plate under ultraviolet light (365 nm). The chromatogram is shown below. Figure 4 As can be seen from the graph, this method is subject to interference.

[0123] Comparative Example 2

[0124] A method for identifying Epimedium in a kidney-tonifying and body-strengthening tablet includes the following steps:

[0125] (1) Preparation of reference solution

[0126] Prepare a reference solution by dissolving icariin and ascorbic acid C reference standards in methanol to a concentration of 1 mg / ml.

[0127] (2) Preparation of the test solution

[0128] Method 3: Take 5 tablets of Bu Shen Qiang Shen Pian (a traditional Chinese medicine), remove the coating, grind into a fine powder, add 30 ml of anhydrous ethanol, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1 ml of methanol, and use it as the test solution.

[0129] Method 4: Take 5 tablets of Bu Shen Qiang Shen Pian (a traditional Chinese medicine), remove the coating, grind into a fine powder, add 30 ml of ethanol, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1 ml of methanol, and use it as the test solution.

[0130] Method 5: Take the kidney-tonifying and body-strengthening tablets, remove the coating, grind them into a fine powder, take about 5g, add 30ml of ethanol, extract with ultrasound for 30 minutes, evaporate the ethanol solution to dryness, add 20ml of water to redissolve, extract with ethyl acetate 3 times, 25ml each time, combine the ethyl acetate solutions, evaporate to dryness, add 1ml of methanol to dissolve the residue, and use it as the test solution.

[0131] (3) Preparation of negative control solution

[0132] A negative control sample lacking Epimedium was prepared according to the method for preparing kidney-tonifying and body-strengthening tablets, and then a negative control solution lacking Epimedium was prepared using the same method as the test solution.

[0133] (4) Thin-layer chromatography identification

[0134] Perform the thin-layer chromatography test (General Rule 0502). Apply 5 μl of each of the above solutions separately to the same silica gel G thin-layer plate. Develop using chloroform-methanol-water (14:6:2) as the developing solvent. Remove the plate, air dry, spray with 10% sulfuric acid ethanol solution, and heat at 105°C until the spots are clearly visible. Examine under ultraviolet light (365 nm). The chromatogram is shown below. Figure 5 As can be seen from the chromatogram, at the corresponding positions of the chromatogram of icariin reference standard, except for the test solution prepared by method 3 which has good specificity, the other preparation methods all have certain negative interference phenomena. At the same time, the characteristic spots are lighter in color and the RF value is also smaller under the conditions of the developing solvent and color reagent. Therefore, the above methods are not suitable for the identification of icariin in this product.

[0135] Comparative Example 3

[0136] A method for identifying Epimedium in a kidney-tonifying and body-strengthening tablet includes the following steps:

[0137] (1) Preparation of reference solution

[0138] Prepare a solution containing 1 mg / ml of icariin reference standard by dissolving it in methanol. Prepare a solution containing 1 mg / ml of oxytocin C reference standard by dissolving it in methanol.

[0139] (2) Preparation of test solution: Take 5 tablets of Bu Shen Qiang Shen Pian, remove the coating, grind into a fine powder, add 30 ml of anhydrous ethanol, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, add 1 ml of methanol to dissolve the residue, and use it as test solution.

[0140] (3) Preparation of negative control solution

[0141] A negative control sample lacking Epimedium was prepared according to the method for preparing kidney-tonifying and body-strengthening tablets, and then a negative control solution lacking Epimedium was prepared using the same method as the test solution.

[0142] (4) Thin-layer chromatography identification

[0143] Perform the thin-layer chromatography test (General Rule 0502). Apply 5 μl of each of the above solutions to the same silica gel G thin-layer plate. Develop the plate using ethyl acetate-methanol-water (10:2:1), ethyl acetate-methanol-formic acid-water (10:2.5:0.5:1), or ethyl acetate-butanone-methanol-water (10:1:3:1) as the developing solvent. Remove the plate, air dry, spray with 1% aluminum trichloride solution, and heat at 105°C until the spots are clearly visible. Examine the plate under ultraviolet light (365 nm). The chromatogram is shown below. Figure 6-8 As can be seen from the chromatograms, under different developing solvent conditions, the chromatogram of the test sample always has similar colored spots interfering with the corresponding positions of the chromatogram of icariin reference standard, which can easily cause false positives and seriously affect the judgment. In contrast, the specificity of the icariin C reference standard is better and it is more suitable as a reference for the thin-layer identification of icariin.

[0144] Comparative Example 4

[0145] A method for identifying privet fruit in a kidney-tonifying and body-strengthening tablet includes the following steps:

[0146] (1) Preparation of reference solution

[0147] Prepare a reference solution by dissolving ligustrin in methanol to a concentration of 1 mg per ml.

[0148] (2) Preparation of control herbal solution

[0149] Take 1g of Ligustrum lucidum, add 20ml of anhydrous ethanol, sonicate for 30min, filter, recover the solvent from the filtrate until dry, add 1ml of methanol to dissolve the residue, and use it as the reference solution.

[0150] (3) Preparation of the test solution

[0151] Method 1: Take 10 tablets of Bu Shen Qiang Shen Pian (a traditional Chinese medicine), remove the coating, grind into a fine powder, add 40 ml of ethanol, sonicate for 30 minutes, filter, evaporate to dryness, dissolve the residue in 1 ml of methanol, and use as the test solution.

[0152] Method 2: Take 10 tablets of Bushen Qiangshen Pian (a traditional Chinese medicine), remove the coating, grind into a fine powder, add 40 ml of 50% ethanol, sonicate for 30 min, cool, filter, evaporate the filtrate to dryness, add 20 ml of water to reconstitute, add ether to the solution and shake to extract twice, 20 ml each time, discard the ether solution, then extract twice with water-saturated n-butanol, 20 ml each time, combine the n-butanol solutions, evaporate to dryness, add 1 ml of methanol to dissolve the residue, and use as the test solution.

[0153] (4) Preparation of negative control solution

[0154] A negative control sample lacking Ligustrum lucidum was prepared according to the method for preparing kidney-tonifying and body-strengthening tablets, and then a negative control solution lacking Ligustrum lucidum was prepared in the same way as the test solution.

[0155] (5) Thin-layer chromatography identification

[0156] Perform thin-layer chromatography (General Rule 0502). Apply 5 μl each of the above-mentioned test solution and its corresponding negative sample solution, reference solution, and reference medicinal material solution to the same silica gel G thin-layer plate. Develop the plate using ethyl acetate-acetone-water (4:5:1) as the developing solvent. Remove the plate, air dry, spray with 1% vanillin-sulfuric acid ethanol solution, and heat at 105℃ until the spots are clearly visible. Examine the plate under sunlight. The chromatogram is shown below. Figure 9In the chromatograms of the test samples, the characteristic spots at the same positions as those in the chromatogram of the ligustrum lucidum reference sample prepared by Method 1 were not very obvious, while the chromatogram of the test sample prepared by Method 2 had more background interference, making the target spots difficult to observe. Therefore, Methods 1 and 2 are not the best methods for preparing the test sample solutions. Furthermore, a comparison of the chromatograms of the test samples and the chromatograms of the privet fruit reference material showed that, apart from the characteristic spots of ligustrum lucidum, no other obvious characteristic spots of the same color were observed. Therefore, the reference material cannot be used as a reference for the identification of privet fruit in this product.

[0157] Comparative Example 5

[0158] A method for identifying dodder seed in a kidney-tonifying and body-strengthening tablet includes the following steps:

[0159] (1) Preparation of reference solution

[0160] Take hyperoside reference standard and add methanol to prepare a solution containing 1 mg per 1 ml, which is used as the reference solution.

[0161] (2) Preparation of control herbal solution

[0162] Take 1g of Cuscuta chinensis reference material and prepare it using the same method as the test sample solution to serve as the reference material solution.

[0163] (3) Preparation of the test solution

[0164] Method 1: Take 5 tablets of Bu Shen Qiang Shen Pian (a traditional Chinese medicine), remove the coating, grind into a fine powder, add 25 ml of methanol, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, add 1 ml of methanol to dissolve the residue, and use it as the test solution.

[0165] Method 2: Take 5 tablets of Bu Shen Qiang Shen Pian (a traditional Chinese medicine), remove the coating, grind into a fine powder, add 25 ml of acetone, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1 ml of methanol, and use it as the test solution.

[0166] Method 3: Take 5 tablets of Bu Shen Qiang Shen Pian (a traditional Chinese medicine), remove the coating, grind into a fine powder, add 20 ml of ethanol, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1 ml of methanol, and use it as the test solution.

[0167] Method 4: Take 10 tablets of Bushen Qiangshen Pian (a traditional Chinese medicine), remove the coating, grind into a fine powder, add 50ml of petroleum ether (60-90℃), sonicate for 30min, discard the petroleum ether, add 40ml of ethanol to the residue, sonicate for 30min, filter, evaporate the filtrate to dryness, add 1ml of ethanol to dissolve the residue, and use it as the test solution.

[0168] (4) Preparation of negative control solution

[0169] A negative control sample of Cuscuta chinensis was prepared according to the method for preparing kidney-tonifying and body-strengthening tablets, and then a negative control solution of Cuscuta chinensis was prepared using the same method as the test solution.

[0170] (5) Thin-layer identification

[0171] Perform thin-layer chromatography (General Rule 0502). Take 5 μl each of the test solution prepared by the different methods described above, as well as the corresponding negative sample solution, reference solution, and reference medicinal material solution, and spot them onto the same polyamide thin-layer plate or silica gel G thin-layer plate. Develop with ethyl acetate-formic acid-water (33:4:2) as the developing solvent. Remove the plate, air dry, and examine under ultraviolet light (365 nm). Then spray with 10% sulfuric acid ethanol solution and heat at 105℃ until the spots are clearly visible. Examine under ultraviolet light (365 nm). The thin-layer results are shown in the figure. Figure 10 As can be seen from the chromatograms, the characteristic spots are easier to observe without the addition of a colorimetric reagent. The test solution samples prepared by different methods all exhibited significant negative interference at the same positions as the characteristic spots of hyperoside in their chromatograms. Therefore, hyperoside reference standard is not suitable as an indicator component for the thin-layer identification of Cuscuta chinensis in the Bushen Qiangshen tablet standard. In the chromatogram of the test sample, two characteristic spots of the same color can be observed at the corresponding positions as in the chromatogram of the reference medicinal material, with no negative interference. Therefore, the reference medicinal material is suitable for the thin-layer identification of Cuscuta chinensis in the Bushen Qiangshen tablet. The test solution prepared by method 4, with the same processing steps as in Example 3, shows clearer and easier-to-observe characteristic spots in its chromatogram compared to test solutions prepared by other methods, and exhibits better specificity.

Claims

1. A method for detecting a kidney-tonifying and body-strengthening tablet, characterized in that, Thin-layer chromatography was used to identify the active ingredients of Epimedium, Ligustrum lucidum, and Cuscuta chinensis in Bushen Qiangshen tablets.

2. The detection method according to claim 1, wherein, When the object of identification is the effective component of Epimedium, the thin-layer chromatography method described above includes: (1) Preparation of the test solution Take the kidney-tonifying and body-strengthening tablets, remove the coating, grind them into a fine powder, add anhydrous ethanol, sonicate, filter, evaporate the filtrate to dryness, dissolve the residue in methanol, and use it as the test solution. (2) Preparation of reference solution Take the reference standard of Astragalus membranaceus C, dissolve it in methanol to prepare the reference solution; (3) Thin-layer chromatography identification Perform the thin-layer chromatography test (General Rule 0502). Apply the two solutions mentioned above separately to the same silica gel G thin-layer plate. Use ethyl acetate-butanone-methanol-water (9-11:0.8-1.2:2.5-3.5:0.8-1.2) as the developing solvent. Develop the plate, remove it, air dry it, spray it with aluminum trichloride test solution, and heat it at 105°C until the spots are clearly visible. Examine it under ultraviolet light (365nm). In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference sample.

3. The detection method according to claim 1 or 2, wherein, When the object of identification is the effective component of Epimedium, the thin-layer chromatography method described above includes: (1.1) Preparation of the test solution Take the kidney-tonifying and body-strengthening tablets, remove the coating, grind them into a fine powder, add anhydrous ethanol, sonicate, filter, evaporate the filtrate to dryness, dissolve the residue in methanol, and use it as the test solution. (1.2) Preparation of reference solution Take the reference standard of Astragalus membranaceus C, dissolve it in methanol to prepare the reference solution; (1.3) Thin-layer chromatography identification Perform the thin-layer chromatography test (General Rule 0502). Take 5 μl of each of the two solutions mentioned above and spot them separately on the same silica gel G thin-layer plate. Use ethyl acetate-butanone-methanol-water (10:1:3:1) as the developing solvent. Develop the plate, remove it, air dry it, spray it with aluminum trichloride test solution, and heat it at 105℃ until the spots are clearly visible. Examine it under ultraviolet light (365nm). In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference sample.

4. The detection method according to claim 3, wherein, When the object of identification is the effective component of Epimedium, the thin-layer chromatography method described above includes: (2.1) Preparation of the test solution Take the kidney-tonifying and body-strengthening tablets, remove the coating, grind them into a fine powder, add 30 ml of anhydrous ethanol, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, add 1 ml of methanol to dissolve the residue, and use it as the test solution. (2.2) Preparation of reference solution Take the reference standard of Astragalus membranaceus C and prepare a solution containing 1 mg per 1 ml with methanol as the reference solution; (2.3) Thin-layer chromatography identification Perform the thin-layer chromatography test (General Rule 0502). Take 5 μl of each of the two solutions mentioned above and spot them separately on the same silica gel G thin-layer plate. Use ethyl acetate-butanone-methanol-water (10:1:3:1) as the developing solvent. Develop the plate, remove it, air dry it, spray it with aluminum trichloride test solution, and heat it at 105℃ until the spots are clearly visible. Examine it under ultraviolet light (365nm). In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference sample.

5. The detection method according to claim 1, wherein, When the object of identification is the effective component of privet fruit, the thin-layer chromatography method described above includes: (1) Preparation of the test solution Take the kidney-tonifying and body-strengthening tablets, remove the coating, grind them into a fine powder, add water and sonicate, centrifuge, take the supernatant, extract with ether, discard the ether solution, extract with water-saturated n-butanol, combine the n-butanol solutions, evaporate to dryness, dissolve the residue in methanol, and use it as the test solution. (2) Preparation of reference solution Take the reference standard of ligustrum lucidum and dissolve it in methanol to prepare the reference solution; (3) Thin-layer chromatography identification Perform the thin-layer chromatography test (General Rule 0502). Apply the two solutions mentioned above separately to the same silica gel G thin-layer plate. Use ethyl acetate-acetone-water (3.5-4.5:4.5-5.5:0.8-1.2) as the developing solvent. Develop the plate, remove it, air dry it, spray it with 1% vanillin-sulfuric acid ethanol solution, and heat it at 105°C until the spots are clearly visible. Examine it under sunlight. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference sample.

6. The detection method according to claim 1 or 5, wherein, When the object of identification is the effective component of privet fruit, the thin-layer chromatography method described above includes: (3.1) Preparation of the test solution Take the kidney-tonifying and body-strengthening tablets, remove the coating, grind them into a fine powder, add water and sonicate, centrifuge, take the supernatant, extract with ether, discard the ether solution, extract with water-saturated n-butanol, combine the n-butanol solutions, evaporate to dryness, dissolve the residue in methanol, and use it as the test solution. (3.2) Preparation of reference solution Take the reference standard of ligustrum lucidum and dissolve it in methanol to prepare the reference solution; (3.3) Thin-layer chromatography identification Perform the thin-layer chromatography test (General Rule 0502). Take 5 μl of each of the two solutions mentioned above and spot them separately on the same silica gel G thin-layer plate. Use ethyl acetate-acetone-water (4:5:1) as the developing solvent. Develop the plate, remove it, air dry it, spray it with 1% vanillin-sulfuric acid ethanol solution, and heat it at 105°C until the spots are clearly visible. Examine it under sunlight. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference sample.

7. The detection method according to claim 6, wherein, When the object of identification is the effective component of privet fruit, the thin-layer chromatography method described above includes: (4.1) Preparation of the test solution Take the kidney-tonifying and body-strengthening tablets, remove the coating, grind them into a fine powder, add 30ml of water, sonicate for 60 minutes, centrifuge for 10 minutes, take the supernatant, extract with ether 3 times, 20ml each time, discard the ether solution, then extract with water-saturated n-butanol 3 times, 20ml each time, combine the n-butanol solutions, evaporate to dryness, add 1ml of methanol to dissolve the residue, and use it as the test solution; (4.2) Preparation of reference solution Prepare a reference solution by dissolving ligustrin in methanol to a concentration of 1 mg per ml. (4.3) Thin-layer chromatography identification Perform the thin-layer chromatography test (General Rule 0502). Take 5 μl of each of the two solutions mentioned above and spot them separately on the same silica gel G thin-layer plate. Use ethyl acetate-acetone-water (4:5:1) as the developing solvent. Develop the plate, remove it, air dry it, spray it with 1% vanillin-sulfuric acid ethanol solution, and heat it at 105°C until the spots are clearly visible. Examine it under sunlight. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference sample.

8. The detection method according to claim 1, wherein, When the object of identification is the effective component of dodder seed, the thin-layer chromatography method described above includes: (1) Preparation of the test solution Take the kidney-tonifying and body-strengthening tablets, remove the coating, grind them into a fine powder, add petroleum ether (60-90℃) and sonicate, discard the petroleum ether, add ethanol to the residue and sonicate, filter, evaporate the filtrate to dryness, add ethanol to the residue to dissolve it, and use it as the test solution. (2) Preparation of control herbal solution Take 1g of Cuscuta chinensis reference material and prepare it in the same way as the test solution preparation method in step (1) to serve as the reference material solution; (5.3) Thin-layer chromatography identification According to the thin-layer chromatography method (General Rule 0502), take the above two solutions and spot them separately on the same silica gel G thin-layer plate. Use ethyl acetate-butanone-formic acid-water (27~33:9~11:3.5~4.5:3.5~4.5) as the developing solvent, develop, remove, air dry at room temperature, and examine immediately under ultraviolet light (365nm). In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference medicinal material.

9. The detection method according to claim 1 or 8, wherein, When the object of identification is the effective component of dodder seed, the thin-layer chromatography method described above includes: (5.1) Preparation of the test solution Take the kidney-tonifying and body-strengthening tablets, remove the coating, grind them into a fine powder, add petroleum ether (60-90℃) and sonicate, discard the petroleum ether, add ethanol to the residue and sonicate, filter, evaporate the filtrate to dryness, add ethanol to the residue to dissolve it, and use it as the test solution. (5.2) Preparation of control herbal solution Take 1g of Cuscuta chinensis reference material and prepare it in the same way as the test solution preparation method in (5.1) to serve as the reference material solution; (5.3) Thin-layer chromatography identification According to the thin-layer chromatography method (General Rule 0502), take 5 μl of each of the above two solutions and spot them separately on the same silica gel G thin-layer plate. Use ethyl acetate-butanone-formic acid-water (30:10:4:4) as the developing solvent, develop, remove, air dry at room temperature, and examine immediately under ultraviolet light (365 nm). In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference medicinal material.

10. The detection method according to claim 9, wherein, When the object of identification is the effective component of dodder seed, the thin-layer chromatography method described above includes: (6.1) Preparation of the test solution Take the kidney-tonifying and body-strengthening tablets, remove the coating, grind them into a fine powder, add 50 ml of petroleum ether (60-90℃), sonicate for 30 min, discard the petroleum ether, add 40 ml of ethanol to the residue, sonicate for 30 min, filter, evaporate the filtrate to dryness, add 1 ml of ethanol to the residue to dissolve it, and use it as the test solution. (6.2) Preparation of control herbal solution Take 1g of Cuscuta chinensis reference material and prepare it in the same way as the test solution preparation method in (6.1) to serve as the reference material solution; (6.3) Thin-layer chromatography identification According to the thin-layer chromatography method (General Rule 0502), take 5 μl of each of the above two solutions and spot them separately on the same silica gel G thin-layer plate. Use ethyl acetate-butanone-formic acid-water (30:10:4:4) as the developing solvent, develop, remove, air dry at room temperature, and examine immediately under ultraviolet light (365 nm). In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference medicinal material.

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