A detection method of a kidney-tonifying and essence-filling oral liquid

Abstract

The application provides a detection method of a kidney-tonifying and essence-filling oral liquid, which comprises thin layer chromatography for identifying effective components of astragalus, ginseng, epimedium, lycium, angelica and black ginseng in the kidney-tonifying and essence-filling oral liquid, and high performance liquid chromatography for identifying effective components of cistanche. The chromatographic identification method of the application has clear spots, good separation degree, and can exclude impurity interference; the high performance liquid chromatography has good specificity and high chromatographic peak separation degree, and improves the controllability of the quality standard of the kidney-tonifying and essence-filling oral liquid, and further ensures the internal quality and curative effect of the product.

Description

TECHNICAL FIELD

[0001] The application relates to a detection method of traditional Chinese medicine, in particular to a detection method of Bushen Tianjing oral liquid. BACKGROUND

[0002] The Bushen Tianjing oral liquid is composed of fifteen medicinal materials such as ginseng and pilose antler, and is used for treating kidney yang deficiency, cold pain of waist and knees, cold hands and feet, impotence, cold essence, mental atrophy and fatigue, etc. The current standard is the national drug standard (revision) issued by the batch number ZGB2017-40, and only the thin layer chromatography identification of radix astragali, angelica sinensis, medlar and ginseng is provided in the current standard, and the impurities interfere, which cannot meet the increasingly strict demand of the existing traditional Chinese medicine preparation standard. SUMMARY

[0003] The purpose of the application is to provide a detection method of Bushen Tianjing oral liquid, improve the controllability of the quality standard of Bushen Tianjing oral liquid, and further ensure the internal quality and curative effect of the product. The purpose of the application is realized by the following technical scheme:

[0004] A detection method of Bushen Tianjing oral liquid, characterized in that the detection method comprises thin layer chromatography for identifying the effective components of radix astragali, ginseng, epimedium, medlar, angelica sinensis and black smooth tablets, and high performance liquid chromatography for determining the effective components of cistanche. The steps of thin layer chromatography for identifying radix astragali and ginseng are as follows:

[0005] (1) Preparation of test solution: the Bushen Tianjing oral liquid is extracted by shaking with water-saturated n-butanol, the n-butanol solution is washed with ammonia test solution, the ammonia washing solution is discarded, the solution is washed with water, the water solution is discarded, the residue is dissolved in methanol as the test solution;

[0006] (2) Preparation of control medicinal material solution: the radix astragali control medicinal material is boiled with water, cooled and filtered, and the filtrate is prepared into the radix astragali control medicinal material solution by step (1); the ginseng control medicinal material is boiled with water, cooled and filtered, and the filtrate is prepared into the ginseng control medicinal material solution by step (1);

[0007] (3) Preparation of control solution: the astragaloside A control sample is prepared into the astragaloside A control solution by adding methanol; the ginsenoside Re, ginsenoside Rb1 and ginsenoside Rg1 control samples are prepared into the ginseng mixed control solution by adding methanol;

[0008] (4) Preparation of negative control solution: the negative sample without radix astragali is prepared into the negative control solution without radix astragali by step (1); the negative sample without ginseng is prepared into the negative control solution without ginseng by step (1);

[0009] (5) Thin layer identification: according to the thin layer chromatography (Chinese Pharmacopoeia 2020 edition four general test 0502) test, 5-10 μl of the above test solution, 5-10 μl of the astragalus membranaceus control drug solution, 5-10 μl of the astragalus membranaceus glycoside control solution, 5-10 μl of the negative control solution without astragalus membranaceus, and 5-10 μl of the above test solution, 5-10 μl of the ginseng control drug solution, 5-10 μl of the ginseng mixed control solution, and 5-10 μl of the negative control solution without ginseng were spotted on the same silica gel G thin layer plate, and the lower solution was placed at 10 ℃ for 4 hours or more (preferably 4-12 hours) to develop the chromatogram, and then the chromatogram was taken out, dried, sprayed with 10% sulfuric acid ethanol solution, heated at 105 ℃ until the spots were clearly colored, and observed under daylight and under ultraviolet light (365 nm), respectively;

[0010] (6) Analysis of results: in the test chromatogram, whether the same color fluorescent spots appear at the corresponding positions of the astragalus membranaceus control drug and the astragalus membranaceus glycoside control under ultraviolet light (365 nm); in the test chromatogram, whether the same color spots appear at the corresponding positions of the ginseng control drug and the ginseng mixed control under daylight and ultraviolet light (365 nm).

[0011] Preferably, the steps of identifying astragalus membranaceus and ginseng by thin layer chromatography are as follows:

[0012] (1) Preparation of test solution: 30-50 ml of Bushen Tianjing Oral Liquid was added to water-saturated n-butanol and shaken to extract 3 times, 30-50 ml each time, the n-butanol was combined, washed with ammonia solution 2 times, 40-50 ml each time, the ammonia washing solution was discarded, washed with water 2 times, 40-50 ml each time, the water was discarded, the n-butanol was combined and evaporated to dryness, the residue was dissolved in 1-2 ml of methanol as the test solution;

[0013] (2) Preparation of control drug solution: 1 g of astragalus membranaceus control drug was added to 50 ml of water, decocted for 30-60 min, cooled and filtered, and the filtrate was prepared into astragalus membranaceus control drug solution according to step (1); 1 g of ginseng control drug was added to 50 ml of water, decocted for 30-60 min, cooled and filtered, and the filtrate was prepared into ginseng control drug solution according to step (1);

[0014] (3) Preparation of control solution: 1 mg of astragalus membranaceus glycoside control was added to methanol to prepare 1 ml of astragalus membranaceus glycoside control solution; 1 mg of ginsenoside Re, ginsenoside Rb1 and ginsenoside Rg1 control was added to methanol to prepare 1 ml of mixed solution, which was used as ginseng mixed control solution;

[0015] (4) Negative control solution preparation: Take 30-50 ml of negative sample of Huangqi, and prepare the negative control solution of Huangqi according to the method of step (1); take 30 ml of negative sample of Renshen, and prepare the negative control solution of Renshen according to the method of step (1);

[0016] (5) Thin layer identification: According to the test of thin layer chromatography (Chinese Pharmacopoeia 2020 edition four chapters 0502), 5-10 μl of the above test solution, 5-10 μl of Huangqi control drug solution, 5-10 μl of astragaloside control solution, 2-5 μl of the above test solution, 2-5 μl of Renshen control drug solution, 2-5 μl of Renshen mixed control solution and 2-5 μl of negative control solution of Renshen were taken and spotted on the same silica gel G thin layer plate. The lower solution of chloroform-methanol-water (volume ratio 65:35:10) was used as developing agent, which was placed at 10℃ for more than 4 hours (preferably 4-12 hours), then taken out, dried, sprayed with 10% sulfuric acid ethanol solution, heated at 105℃ until the spots were clear, and observed under daylight and ultraviolet light (365 nm) respectively;

[0017] (6) Result analysis: In the test solution chromatogram, whether the same color fluorescent spots appear at the corresponding positions of Huangqi control drug and astragaloside control solution under ultraviolet light (365 nm) was determined; in the test solution chromatogram, whether the same color spots appear at the corresponding positions of Renshen control drug and Renshen mixed control solution under daylight and ultraviolet light (365 nm) was determined.

[0018] The steps of the thin layer chromatography method for identifying Herba Epimedii are as follows:

[0019] (1) Preparation of test solution: After the Shu Shenan filling oral liquid was extracted with water-saturated n-butanol and washed with water, the n-butanol was combined and evaporated, the residue was dissolved in methanol and added to a neutral alumina column, eluted with 40% methanol, the eluate was discarded, and then eluted with 40% methanol, the eluate was collected, evaporated, and the residue was dissolved in methanol as the test solution;

[0020] (2) Preparation of control drug solution: Take Herba Epimedii, add water and decoct, cool and filter, and prepare the control drug solution according to the method of step (1);

[0021] (3) Preparation of control solution: Take Epimedoside and Chomodine C control samples, add methanol to prepare a mixed solution as the control solution;

[0022] (4) Preparation of negative control solution: Take negative sample of Herba Epimedii, and prepare the negative control solution of Herba Epimedii according to the method of step (1);

[0023] (5) Thin layer identification: according to the thin layer chromatography (Chinese Pharmacopoeia 2020 edition four general rules 0502) test, 2-5 μl of each of the above test solution, epimedium control drug solution, epimedium control solution, and epimedium negative control solution was taken and spotted on the same silica gel G thin layer plate, methanol- butanone-chloroform-water (volume ratio of 4:6:6:1) was used as developing agent, developed, taken out, dried, sprayed with 3% aluminum chloride solution, heated at 105°C, and observed under ultraviolet lamp (365 nm);

[0024] (6) Analysis of results: whether the same color fluorescent spots appear in the test chromatogram at the corresponding positions of the control drug and control chromatogram under ultraviolet lamp (365 nm) was determined.

[0025] Preferably, the steps of identifying epimedium by thin layer chromatography are as follows:

[0026] (1) Preparation of test solution: 30-50 ml of Bushen Fillian oral liquid was taken and extracted with water-saturated n-butanol for 3 times, 30-50 ml each time, the n-butanol was combined and washed with water for 2 times, 30-50 ml each time, the n-butanol was combined and evaporated to dryness, the residue was dissolved in 2-5 ml of methanol. It was added to a neutral alumina column (100-200 mesh, 5 g, 1.5 cm inner diameter), eluted with 40% methanol 30 ml, the eluate was discarded, and then eluted with 40% methanol 50-100 ml, the eluate was collected, evaporated to dryness, and the residue was dissolved in 1-2 ml of methanol as the test solution;

[0027] (2) Preparation of control drug solution: 1 g of epimedium medicinal material was taken and added to 50 ml of water, decocted or ultrasonicated for 30-60 min, cooled and filtered, and the filtrate was prepared into a control drug solution according to step (1);

[0028] (3) Preparation of control solution: epimedium glycoside and chomilofendin C control were taken and dissolved in methanol to prepare a mixed solution containing 1 mg per 1 ml as the control solution;

[0029] (4) Preparation of negative control solution: 30-50 ml of negative sample without epimedium was taken and prepared into a negative control solution without epimedium according to the method of step (1);

[0030] (5) Thin layer identification: according to the thin layer chromatography (Chinese Pharmacopoeia 2020 edition four general rules 0502) test, 2-5 μl of each of the above 4 solutions was taken and spotted on the same silica gel G thin layer plate, methanol- butanone-chloroform-water (volume ratio of 4:6:6:1) was used as developing agent, developed, taken out, dried, sprayed with 3% aluminum chloride solution, heated at 105°C, and observed under ultraviolet lamp (365 nm);

[0031] (6) Result analysis: In the test sample chromatogram, whether the same color fluorescent spots appear at the corresponding position of the control drug and control substance chromatogram under ultraviolet light (365 nm) is determined.

[0032] The steps of the thin layer chromatography method for identifying medlar and angelica are as follows:

[0033] (1) Preparation of test sample solution: Take Bushen Tianjing Oral Liquid, extract with ethyl acetate, combine the ethyl acetate extract, evaporate to dryness, and dissolve the residue with ethyl acetate as the test sample solution.

[0034] (2) Preparation of control drug solution: Take the medlar control drug, decoct with water, filter, and prepare the control drug solution according to step (1); take the angelica control drug, decoct with water, filter, and prepare the control drug solution according to step (1);

[0035] (3) Preparation of negative control solution: Take the negative sample of medlar, and prepare the negative control solution of medlar according to step (1); take the negative sample of angelica, and prepare the negative control solution of angelica according to step (1);

[0036] (4) Thin layer identification: According to the thin layer chromatography method (Chinese Pharmacopoeia 2020 edition four general test 0502), the above test sample solution, medlar control drug solution and medlar negative control solution, and the above test sample solution, angelica control drug solution and angelica negative control solution are respectively spotted on the same silica gel G thin layer plate, and developed with toluene-ethyl acetate-formic acid (volume ratio of 9:21:0.5), taken out, dried, and observed under ultraviolet light (365 nm);

[0037] (5) Result analysis: In the test sample chromatogram, whether the same color spots appear at the corresponding position of the medlar control drug chromatogram under ultraviolet light (365 nm) is determined; in the test sample chromatogram, whether the same color spots appear at the corresponding position of the angelica control drug chromatogram under ultraviolet light (365 nm) is determined.

[0038] Preferably, the steps of the thin layer chromatography method for identifying medlar and angelica are as follows:

[0039] (1) Preparation of test sample solution: Take Bushen Tianjing Oral Liquid 15-30 ml, extract with ethyl acetate 2 times, 30 ml each time, combine the ethyl acetate extract, evaporate to dryness, and dissolve the residue with 2 ml of ethyl acetate as the angelica test sample solution; dilute 1 ml of the angelica test sample solution to the mark with ethyl acetate in a 10 ml capacity bottle, shake well, and use as the medlar test sample solution;

[0040] (2) Preparation of control medicinal material solution: take 1 g of control medicinal material of Fructus Lycii, add 50 ml of water, ultrasonic or decoct for 15-30 min, filter, and the filtrate is prepared into the control medicinal material solution of Fructus Lycii according to step (1); take 1 g of control medicinal material of Angelica sinensis, add 50 ml of water, ultrasonic or decoct for 15-30 min, filter, and the filtrate is prepared into the control medicinal material solution of Angelica sinensis according to step (1);

[0041] (3) Preparation of negative control solution: take 15-30 ml of negative sample of Fructus Lycii, and prepare into the negative control solution of Fructus Lycii according to the method of step (1); take 30 ml of negative sample of Angelica sinensis, and prepare into the negative control solution of Angelica sinensis according to the method of step (1);

[0042] (4) Thin layer identification: according to the test of thin layer chromatography (Chinese Pharmacopoeia 2020 edition four chapters 0502), 1-2 μl of the Fructus Lycii test sample solution, the control medicinal material solution of Fructus Lycii and the negative control solution of Fructus Lycii, and 1-2 μl of the Angelica sinensis test sample solution, the control medicinal material solution of Angelica sinensis and the negative control solution of Angelica sinensis are taken, respectively, and are spotted on the same silica gel G thin layer plate, toluene-ethyl acetate-formic acid (volume ratio is 9:21:0.5) is used as developing agent, development, take out, dry, and observe under ultraviolet lamp (365 nm);

[0043] (5) Result analysis: whether the same color fluorescent spot appears in the test sample chromatogram at the corresponding position of the control medicinal material chromatogram under ultraviolet lamp (365 nm) is judged.

[0044] The steps of the thin layer chromatography for identifying He Shun Pian are as follows:

[0045] (1) Preparation of test sample solution: take the Bushen Chongjing Oral Liquid, add ammonia test solution, shake with chloroform, shake with dilute hydrochloric acid, adjust the pH value to 10 with concentrated ammonia solution, shake with chloroform, evaporate to dryness, add an appropriate amount of anhydrous ethanol to dissolve, and use as the test sample solution;

[0046] (2) Preparation of control solution: take benzoyl neo-lappaconitine and benzoyl lappaconitine control, add isopropyl alcohol-dichloromethane (volume ratio is 1:1) mixed solution to prepare a mixed solution, which is used as the control solution;

[0047] (3) Thin layer identification: according to the test of thin layer chromatography (Chinese Pharmacopoeia 2020 edition four chapters 0502), 10 μl of the above two solutions are taken, respectively, and are spotted on the same silica gel G thin layer plate, n-hexane-ethyl acetate-methanol (volume ratio is 6.4:5:1) is used as developing agent, and is placed in an ammonia vapor saturated developing cylinder for 15-30 min, development, take out, dry, and spray with dilute potassium iodide solution;

[0048] (4) Result analysis: In the test sample chromatogram, whether the same color fluorescence spot is shown at the corresponding position of the control sample chromatogram is determined.

[0049] Preferably, the step of identifying He Shun Pian by thin layer chromatography is as follows:

[0050] (1) Preparation of test sample solution: 100 ml of Bushen Chongjing Oral Liquid was precisely measured, 15 ml of ammonia test solution was added, and chloroform was used for shaking extraction for 5 times, 30 ml each time, 15-30 ml of dilute hydrochloric acid was added for shaking extraction for 5 times, 30 ml of chloroform was added for shaking extraction for 3-5 times, and the PH value was adjusted to 10 with concentrated ammonia solution. After being evaporated to dryness, the residue was dissolved with an appropriate amount of anhydrous ethanol 0.5 ml to serve as the test sample solution;

[0051] (2) Preparation of control sample solution: benzoyl neotuberostemonine and benzoyl tuberostemonine control samples were taken, and isopropyl alcohol-dichloromethane (volume ratio 1:1) mixed solution was used to prepare a mixed solution containing 1 mg per 1 ml, which served as the control sample solution;

[0052] (3) Thin layer identification: according to the test of thin layer chromatography (Chinese Pharmacopoeia 2020 edition four parts general test 0502), 10-15 μl of the above two solutions were taken and spotted on the same silica gel G thin layer plate. N-hexane-ethyl acetate-methanol (volume ratio 6.4:5:1) was used as the developing agent, and the developing cylinder was saturated with ammonia vapor for 15-30 min. After development, it was taken out, air-dried, and sprayed with dilute potassium iodide solution;

[0053] (4) Result analysis: In the test sample chromatogram, whether the same color fluorescence spot is shown at the corresponding position of the control sample chromatogram is determined.

[0054] The steps of identifying Cistanche by high performance liquid chromatography are as follows:

[0055] (1) Preparation of test sample solution: 15-30 ml of the product was taken, extracted with n-butanol for 3 times, 30 ml each time, the n-butanol liquid was combined, evaporated to dryness, and dissolved with methanol to a 25 ml volumetric flask, and shaken well to obtain the test sample solution;

[0056] (2) Preparation of control sample solution: an appropriate amount of Echinacoside control sample was accurately weighed and measured, and 50% methanol was used to prepare a mixed solution containing 0.2 mg per 1 ml, which was obtained;

[0057] (3) Preparation of negative control solution: 15-30 ml of negative sample of Cistanche was taken, and the negative control solution of Cistanche was prepared by the method of step (1);

[0058] (4) Determination method: 10 μl of each of the above three solutions was precisely taken and injected into a liquid chromatograph for determination, and the following conditions were used: octadecylsilane-bonded silica gel as the filler; acetonitrile as the mobile phase A and 0.1% formic acid solution as the mobile phase B, with a flow rate of 1.0 ml / min, gradient elution according to the following table; detection wavelength was 330 nm; column temperature was 30-40°C;

[0059]

[0060]

[0061] (5) Result analysis: in the chromatogram of the test sample, there was a corresponding chromatographic peak at the corresponding position of the chromatogram of the control sample.

[0062] Preferably, the steps of the high performance liquid chromatography for identifying Cistanche are as follows:

[0063] (1) Preparation of test sample solution

[0064] 25 ml of the product was extracted with n-butanol for three times, 30 ml each time, the n-butanol was combined and evaporated to dryness, and then dissolved in methanol to a 25 ml volumetric flask, and shaken to obtain the test sample solution;

[0065] (2) Preparation of control sample solution

[0066] An appropriate amount of Echinacoside control sample was precisely weighed and dissolved in 50% methanol to obtain a mixture solution containing 0.2 mg per ml, to obtain the control sample solution;

[0067] (3) Preparation of negative control solution

[0068] 25 ml of the negative sample of Cistanche deficiens was prepared into a negative control solution of Cistanche deficiens according to the method of step (10.1);

[0069] (4) High performance liquid chromatography determination

[0070] 10 μl of each of the above three solutions was precisely taken and injected into a liquid chromatograph for determination, and the following conditions were used: octadecylsilane-bonded silica gel as the filler; acetonitrile as the mobile phase A and 0.1% formic acid solution as the mobile phase B, with a flow rate of 1.0 ml / min, gradient elution according to the following table; detection wavelength was 330 nm; column temperature was 35°C;

[0071] (5) Result analysis: in the chromatogram of the test sample, there was a corresponding chromatographic peak at the corresponding position of the chromatogram of the control sample.

[0072] The present application has the beneficial effects: the technical scheme of the present application further improves the detection of the Bushen Chixian oral liquid, the chromatographic identification method adopted has clear spots, good separation degree, and excludes impurity interference, the high performance liquid chromatography method adopted has good specificity and high chromatographic peak separation degree, the controllability of the quality standard of the Bushen Chixian oral liquid is improved, the internal quality and efficacy of the product are further ensured, and it is of great significance to ensure the safety of patients using the medicine.

[0073] The present application will be described in more detail below in combination with embodiments. BRIEF DESCRIPTION OF DRAWINGS

[0074] Figure 1 It is a thin layer identification diagram of Huangqi in Example 1, wherein 1 is a negative sample solution of Huangqi; 2 is a Huangqi control medicinal material solution; 3 is a Huangqi glycoside control solution; 4 is a test product solution 20200701; 5 is a test product solution 20200904; and 6 is a test product solution 20200905.

[0075] Figure 2 It is a thin layer identification diagram of Renshen under sunlight inspection in Example 1, 1 is a negative sample solution of Renshen; 2 is a Renshen control medicinal material solution; 3 is a Renshen saponin mixed control solution; 4 is a test product solution 20200701; 5 is a test product solution 20200904; and 6 is a test product solution 20200905.

[0076] Figure 3 It is a thin layer identification diagram of Renshen under ultraviolet lamp inspection in Example 1, 1 is a negative sample solution of Renshen; 2 is a Renshen control medicinal material solution; 3 is a Renshen saponin mixed control solution; 4 is a test product solution 20200701; 5 is a test product solution 20200904; and 6 is a test product solution 20200905.

[0077] Figure 4 It is a thin layer identification diagram of Yiyangyao in Example 1, 1 is a negative sample solution of Yiyangyao; 2 is a Icariin control solution; 3 is a Tsushamin C control solution; 4 is a Yiyangyao control medicinal material solution; 5 is a test product solution 20200701; 6 is a test product solution 20200904; and 7 is a test product solution 20200905.

[0078] Figure 5 It is a thin layer identification diagram of Gouqizi in Example 1, 1 is a negative sample solution of Gouqizi; 2 is a Gouqizi control medicinal material solution; 3 is a test product solution 20200701; 4 is a test product solution 20200904; and 5 is a test product solution 20200905.

[0079] Figure 6For Angelica thin-layer identification chart in Example 1, 1- Angelica negative sample solution; 2- Angelica control drug solution; 3- Test sample solution 20200701; 4- Test sample solution 20200904; 5- Test sample solution 20200905.

[0080] Figure 7 For black smooth sheet thin-layer identification chart in Example 1, 1- black smooth sheet negative sample solution; 2- Test sample solution 20200904; 3- Test sample solution 20200905; 4- Test sample solution 20200701; 5- Mixed control solution, wherein the mixed control solution from top to bottom is benzoyl protoaconitine, benzoyl aconine, benzoyl neoaconitine.

[0081] Figure 8 For Echinacoside control chromatogram in Example 1 Cistanche identification

[0082] Figure 9 For Test sample 20200904 high performance liquid chromatogram in Example 1 Cistanche identification

[0083] Figure 10 For Test sample 20200905 high performance liquid chromatogram in Example 1 Cistanche identification

[0084] Figure 11 For Test sample 20200701 high performance liquid chromatogram in Example 1 Cistanche identification

[0085] Figure 12 For negative sample solution without Cistanche in Example 1 Cistanche identification high performance liquid chromatogram

[0086] Figure 13 For Astragalus thin-layer identification chart under ultraviolet lamp in Comparative Example 1, wherein 1- Astragalus control drug solution; 2- Astragaloside control solution; 3- Astragalus negative sample solution; 4- Test sample solution 20200701; 5- Ginseng control drug solution; 6- Ginseng mixed control solution; 7- Ginseng negative sample solution.

[0087] Figure 14 For Astragalus thin-layer identification chart under daylight in Comparative Example 1, wherein 1- Astragalus control drug solution; 2- Astragaloside control solution; 3- Astragalus negative sample solution; 4- Test sample solution 20200701; 5- Ginseng control drug solution; 6- Ginseng mixed control solution; 7- Ginseng negative sample solution.

[0088] Figure 15For the thin layer identification chart-a of Epimedium in Example 2, 1-test sample solution 1; 2-test sample solution 2; 3-test sample solution 3; 4-icariin reference solution; 5-chaohuding C reference solution; 6-epimedium reference material solution 2; 7-epimedium reference material solution 1.

[0089] Figure 16 For the thin layer identification chart-b of Epimedium in Example 2, 1-test sample solution 4; 2-test sample solution 5; 3-icariin reference solution; 4-chaohuding C reference solution; 5-test sample solution 2; 6-test sample solution 3; 7-epimedium reference material solution 2.

[0090] Figure 17 For the thin layer identification chart of Heishunpian in Example 3, 1-aconitine reference solution; 2-test sample solution 20200701; 3-test sample solution 20200904; 4-test sample solution 20200905.

[0091] Figure 18 For the reference chromatogram of Echinacoside in the identification of Cistanche in Example 4

[0092] Figure 19 For the high performance liquid chromatogram of test sample solution in the identification of Cistanche in Example 4

[0093] Figure 20 For the high performance liquid chromatogram of negative sample solution of Cistanche in the identification of Cistanche in Example 4 DETAILED DESCRIPTION

[0094] The technical solutions of the present application will be further illustrated below in combination with some examples, and the following examples do not constitute any limitation to the present application. A negative control sample is set in the examples, and the thin layer identification result of the negative control sample can show that the method of the present application has no interference and strong specificity.

[0095] The materials and reagents used in the following examples are as follows:

[0096] The Bushen Chongjing Oral Liquid in the following examples is the Bushen Chongjing Oral Liquid collected in the National Drug Standard (Revision) issued by the batch number ZGB2017-40.

[0097] Bushen Chongjing Oral Liquid (batch number: 20200701, 20200904, 20200905, Jiuzhitang Co., Ltd.);

[0098] Radix Astragali reference material (batch number: 120974-201813, China Institute for Drug Control);

[0099] Radix Ginseng reference material (batch number: 120917-201712, China Institute for Drug Control);

[0100] Epimedium herb (batch number: 121632-201502, China Institute for Drug Control);

[0101] Medlar fruit (batch number: 121072-201611, China Institute for Drug Control);

[0102] Angelica sinensis (batch number: 120927-201617, China Institute for Drug Control);

[0103] Astragaloside IV reference substance (batch number: 110781-201717, China Institute for Drug Control);

[0104] Ginsenoside Re reference substance (batch number: 110754-202028, China Institute for Drug Control);

[0105] Ginsenoside Rb1 reference substance (batch number: 110704-202028, China Institute for Drug Control);

[0106] Ginsenoside Rg1 reference substance (batch number: 110703-201933, China Institute for Drug Control);

[0107] Icaritin reference substance (batch number: 110737-202017, China Institute for Drug Control);

[0108] Tsushamin C reference substance (batch number: 111780-201905, China Institute for Drug Control);

[0109] Matatabiin reference substance (batch number: 111670-201907, China Institute for Drug Control);

[0110] Benzoyl protopine (batch number: 111796-201906, China Institute for Drug Control);

[0111] Benzoyl aconine (batch number: 111794-201705, China Institute for Drug Control);

[0112] Benzoyl mesaconine (batch number: 111795-201805, China Institute for Drug Control);

[0113] Thin layer plate: High-performance thin layer chromatography silica gel GF254 plate, produced by Merck, Germany.

[0114] Detection method of the kidney-tonifying and essence-filling oral liquid of Example 1

[0115] 1.1 Identification of Astragalus and Panax by thin layer chromatography

[0116] (1) Preparation of test solution: Take 30 ml of Bushen Fillian Oral Liquid, extract 3 times with n-butanol saturated with water, 30 ml each time, combine the n-butanol liquid, wash 2 times with ammonia test solution, 40 ml each time, discard the ammonia wash liquid, wash twice with water, 40 ml each time, discard the water, evaporate the n-butanol liquid to dryness, add 2 ml of methanol to the residue to dissolve it as the test solution;

[0117] (2) Preparation of control medicinal material solution: Take 1 g of Huangqi control medicinal material, add 50 ml of water, decoct for 30 min, cool and filter, take the filtrate to prepare the Huangqi control medicinal material solution according to step (1); take 1 g of Renshen control medicinal material, add 50 ml of water, decoct for 30 min, cool and filter, take the filtrate to prepare the Renshen control medicinal material solution according to step (1);

[0118] (3) Preparation of control solution: Take the Astragaloside A control, add methanol to prepare a solution containing 1 mg of Astragaloside A control per 1 ml; take the ginsenoside Re, ginsenoside Rb1, and ginsenoside Rg1 controls, add methanol to prepare a mixed solution containing 1 mg of each per 1 ml as the Renshen mixed control solution;

[0119] (4) Preparation of negative control solution: Take 30 ml of negative sample without Huangqi, prepare the negative control solution without Huangqi according to step (1); take 30 ml of negative sample without Renshen, prepare the negative control solution without Renshen according to step (1);

[0120] (5) Thin layer identification: according to the thin layer chromatography (Chinese Pharmacopoeia 2020 edition four general test 0502), take 5 μl of the above test solution, Huangqi control medicinal material solution, Astragaloside A control solution, and negative sample solution without Huangqi, and 2 μl of the above test solution, Renshen control medicinal material solution, Renshen mixed control solution, and negative sample solution without Renshen, respectively, and point them on the same silica gel G thin layer plate, use the lower layer solution of chloroform-methanol-water (volume ratio of 65:35:10) placed at 10 ℃ for 5 hours as the developing agent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105 ℃ until the spots develop clear color, identify Huangqi under ultraviolet lamp (365 nm); identify Renshen under daylight and ultraviolet lamp (365 nm);

[0121] (6) Analysis of results: in the test chromatogram, the same colored fluorescent spots appear at the corresponding positions of the Huangqi control medicinal material and Astragaloside A control under ultraviolet lamp (365 nm), see Figure 1 ; in the test chromatogram, the same colored spots appear at the corresponding positions of the Renshen control medicinal material and Renshen mixed control under daylight and ultraviolet lamp (365 nm), see Figure 2 , Figure 3 .

[0122] 1.2 Identification of Epimedium by TLC

[0123] (1) Preparation of Test Solution: Take 50 ml of Bushen Tianjing Oral Liquid, extract with water-saturated n-butanol for 3 times, 50 ml each time, combine the n-butanol liquid, wash with water for 2 times, 30 ml each time, combine the n-butanol liquid, evaporate to dryness, dissolve the residue with 5 ml of methanol. Add to a neutral alumina column (100-200 mesh, 5 g, 1.5 cm in diameter), elute with 30 ml of 40% methanol, discard the eluate, elute with 50 ml of 40% methanol, collect the eluate, evaporate to dryness, dissolve with 2 ml of methanol as the test solution;

[0124] (2) Preparation of Control Drug Solution: Take 1 g of Epimedium medicinal material, add 50 ml of water, decoct for 30 min, cool, filter, take the filtrate to prepare the control drug solution according to step (1);

[0125] (3) Preparation of Control Solution: Take the control samples of icariin and epimedin C, add methanol to prepare a mixed solution containing 1 mg per 1 ml as the control solution;

[0126] (4) Preparation of Negative Control Solution: Take 50 ml of negative sample without Epimedium, prepare the negative control solution without Epimedium according to the method of step (1);

[0127] (5) TLC Identification: According to the test of TLC (Chinese Pharmacopoeia 2020 Edition Part IV General Test 0502), take 2-5 μl of the above 4 solutions respectively, point on the same silica gel G thin layer plate, use methanol-butanone-chloroform-water (volume ratio of 4:6:6:1) as the developing agent, develop, take out, dry, spray with 3% aluminum chloride solution, heat at 105°C, and observe under ultraviolet lamp (365 nm);

[0128] (6) Analysis of Results: In the test chromatogram, the same color fluorescent spots appear at the corresponding positions of the control drug and control solution, see Figure 4 .

[0129] 1.3 Identification of Fructus Lycii and Angelica by TLC

[0130] (1) Preparation of Test Solution: Take 30 ml of Bushen Tianjing Oral Liquid, extract with ethyl acetate for 2 times, 30 ml each time, combine the ethyl acetate liquid, evaporate to dryness, obtain the residue, add 2 ml of ethyl acetate to dissolve the residue as the test solution of Angelica; take 1 ml from the test solution of Angelica to a 10 ml capacity bottle, dilute to the mark with ethyl acetate, shake well, as the test solution of Fructus Lycii;

[0131] (2) Preparation of control medicinal material solution: Take 1 g of control gouqi medicinal material, add 50 ml of water, decoct for 30 min, filter, and the filtrate is prepared into the control gouqi medicinal material solution according to step (1); take 1 g of control danggui medicinal material, add 50 ml of water, decoct for 30 min, filter, and the filtrate is prepared into the control danggui medicinal material solution according to step (1);

[0132] (3) Preparation of negative control solution: take 30 ml of negative sample of gouti, and prepare into the negative control solution of gouti according to the method of step (1); take 30 ml of negative sample of danggui, and prepare into the negative control solution of danggui according to the method of step (1);

[0133] (4) Thin layer identification: according to the test of thin layer chromatography (Chinese Pharmacopoeia 2020 edition volume four general test 0502), take 1 μl of each of the above-mentioned gouti test sample solution, control gouti medicinal material solution and negative control solution of gouti, and point them on the same silica gel G thin layer plate; take 2 μl of each of the above-mentioned danggui test sample solution, control danggui medicinal material solution and negative control solution of danggui, and point them on the same silica gel G thin layer plate. Take toluene-ethyl acetate-formic acid (volume ratio of 9:21:0.5) as developing agent, develop, take out, dry, and observe under ultraviolet lamp (365 nm);

[0134] (5) Analysis of results: in the test sample chromatogram, the same color fluorescent spots appear at the corresponding positions of the control medicinal material chromatogram, respectively Figure 5 、 Figure 6 .

[0135] 1.4 Identification of Heishun tablets by thin layer chromatography

[0136] (1) Preparation of test sample solution: accurately take 100 ml of Shu- jinkanli oral liquid, add 15 ml of ammonia test solution, extract with chloroform for 5 times, 30 ml each time, combine the chloroform liquid, add dilute hydrochloric acid and shake to extract 4 times, 15 ml each time, combine the acid liquid, adjust the PH value to 10 with concentrated ammonia solution, extract with chloroform for 4 times, 25 ml each time, combine the chloroform liquid, evaporate to dryness, add an appropriate amount of anhydrous ethanol 0.5 ml to dissolve, as the test sample solution;

[0137] (2) Preparation of control solution: take benzoyl neopine and benzoyl opine control, add isopropyl alcohol-dichloromethane (volume ratio of 1:1) mixed solution to prepare a mixed solution containing 1 mg per 1 ml as the control solution;

[0138] (3) Thin layer identification: according to the thin layer chromatography (Chinese Pharmacopoeia 2020 edition four general test 0502), 10 μl of the above two solutions were taken and spotted on the same silica gel G thin layer plate, n-hexane-ethyl acetate-methanol (volume ratio of 6.4:5:1) was used as the developing agent, and the developing tank was saturated with ammonia vapor for 20 min, then developed, taken out, air dried, and sprayed with dilute potassium iodide solution;

[0139] (4) Results analysis: in the test sample chromatogram, there were the same color fluorescent spots at the corresponding positions of the control sample chromatogram, and Figure 7 .

[0140] 1.5 Identification of Cistanche tubulosa by high performance liquid chromatography

[0141] (1) Preparation of test sample solution: 25 ml of the product was extracted with n-butanol for 3 times, 30 ml each time, the n-butanol solution was combined, evaporated to dryness, and dissolved with methanol to a 25 ml volumetric flask, and shaken well.

[0142] (2) Preparation of control sample solution: an appropriate amount of Echinacoside control sample was accurately weighed, and 50% methanol was added to prepare a mixed solution containing 0.2 mg per 1 ml, and then the control sample solution was obtained.

[0143] (3) Preparation of negative control solution: 25 ml of negative sample of Cistanche tubulosa was prepared according to the method of step (1) to prepare the negative control solution of Cistanche tubulosa;

[0144] (4) Determination method: 10 μl of each of the above three solutions was accurately taken and injected into the liquid chromatograph for determination, wherein the chromatographic conditions were as follows: octadecylsilane bonded silica gel as the filler; acetonitrile as the mobile phase A, and 0.1% formic acid solution as the mobile phase B, the flow rate was 1.0 ml / min, gradient elution was performed according to the following table; the detection wavelength was 330 nm; the column temperature was 35℃;

[0145]

[0146] (5) Results analysis: in the test sample chromatogram, there were corresponding chromatographic peaks at the corresponding positions of the control sample chromatogram, and the method was specific, and Figures 8-12 .

[0147] Example 1 Identification of Astragalus and Panax ginseng in Bushen Tianjing Oral Liquid by thin layer chromatography

[0148] This comparative example is the comparison of 1.1 in Example 1.

[0149] (1) Preparation of test solution: Take 30 ml of Bushen Tianjing Oral Liquid, extract 3 times with n-butanol saturated with water, 30 ml each time, combine the n-butanol liquid, wash twice with ammonia test solution, 40 ml each time, wash twice with water, 40 ml each time, discard the water, evaporate the n-butanol liquid to dryness, add 2 ml of methanol to dissolve the residue as the test solution;

[0150] (2) Preparation of control medicinal material solution: Take 1 g of each of Astragalus and ginseng control medicinal materials, add 50 ml of water respectively, decoct for 30 min, cool and filter, take the filtrate to step (1) to prepare Astragalus and ginseng control medicinal material solutions respectively;

[0151] (3) Preparation of control solution: Take the astragaloside A control, add methanol to prepare a solution containing 1 mg of astragaloside A per 1 ml; take the ginsenoside Re, ginsenoside Rb1 and ginsenoside Rg1 controls, add methanol to prepare a mixed solution containing 1 mg of each per 1 ml, as the ginseng mixed control solution;

[0152] (4) Preparation of negative control solution: Take 30 ml of negative sample without Astragalus, prepare the negative control solution without Astragalus by the method of step (1); take 30 ml of negative sample without ginseng, prepare the negative control solution without ginseng by the method of step (1);

[0153] (5) Thin layer identification: according to the thin layer chromatography method (Chinese Pharmacopoeia 2020 edition four chapters 0502) test, take 5 μl of each of the above 5 solutions, point on the same silica gel G thin layer plate, use the lower layer solution of chloroform-ethyl acetate-methanol-water (volume ratio 15:40:22:10) placed overnight at 10℃ as the developing agent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spots develop clearly, observe under daylight and ultraviolet light (365 nm) respectively;

[0154] (6) Analysis of results: it can be seen from Figure 13 、 14 that under daylight or ultraviolet light, the test chromatogram can also show the same color spots or fluorescent spots at the corresponding positions of Astragalus control medicinal material, astragaloside A control, ginseng control medicinal material and ginseng mixed control, but under this developing condition, the separation degree is poor for Astragalus identification, and the ginseng control spots are more serious than those of Example 1.1, which are not conducive to the judgment of ginseng and Astragalus identification results.

[0155] Example 2 Identification of Epimedium in Bushen Tianjing Oral Liquid by Thin Layer Chromatography

[0156] This comparative example is the comparison of 1.2 in Example 1.

[0157] A method for identifying Herba Epimedii in Bushen Tianjing Oral Liquid by thin layer chromatography, the steps are as follows:

[0158] (1) Preparation of test solution: take 50 ml of Bushen Tianjing Oral Liquid, extract with water-saturated n-butanol for 3 times, 50 ml each time, combine the n-butanol liquid, wash with water for 2 times, 50 ml each time, combine the n-butanol liquid, evaporate to dryness, dissolve the residue with 5 ml of methanol as test solution 1; take 50 ml of Bushen Tianjing Oral Liquid, extract with water-saturated n-butanol for 3 times, 50 ml each time, combine the n-butanol liquid, wash with water for 2 times, 50 ml each time, combine the n-butanol liquid, evaporate to dryness, dissolve the residue with 5 ml of methanol, add to a neutral alumina column (100-200 mesh, 5 g, 1.5 cm inner diameter), elute with 40% methanol 30 ml, discard the eluate, elute with 40% methanol 50 ml, collect the eluate, evaporate to dryness, dissolve the residue with 2 ml of methanol as test solution 2; take 50 ml of Bushen Tianjing Oral Liquid, extract with water-saturated n-butanol for 3 times, 50 ml each time, combine the n-butanol liquid, wash with water for 2 times, 50 ml each time, combine the n-butanol liquid, evaporate to dryness, dissolve the residue with 5 ml of methanol, add to a neutral alumina column (100-200 mesh, 5 g, 1.5 cm inner diameter), elute with 40% methanol 80 ml, discard the eluate, elute with 100% methanol 100 ml, collect the eluate, evaporate to dryness, dissolve the residue with 2 ml of methanol as test solution 3.

[0159] Take 50 ml of Bushen Tianjing Oral Liquid, extract with ethyl acetate for 3 times, 50 ml each time, combine the ethyl acetate liquid, evaporate to dryness, dissolve the residue with 5 ml of methanol into two equal parts, one part passes through a neutral alumina column (200-300 mesh, 5 g, 1.5 cm inner diameter), elute with 40% methanol 80 ml, collect the eluate, evaporate to dryness, dissolve the residue with 2 ml of methanol as test solution 4; one part passes through a neutral alumina column (200-300 mesh, 2.5 g, 1.5 cm inner diameter), elute with 40% methanol 80 ml, collect the eluate, evaporate to dryness, dissolve the residue with 2 ml of methanol as test solution 5.

[0160] (2) Preparation of control medicinal material solution: take 1 g of Herba Epimedii, add 50 ml of water, decoct for 30 min, cool and filter, take the filtrate to prepare control medicinal material solution 1 according to the preparation method of test solution 1 in step (1); take 1 g of Herba Epimedii, add 50 ml of water, decoct for 30 min, cool and filter, extract with ethyl acetate for 3 times, 50 ml each time, combine the ethyl acetate liquid, evaporate to dryness, dissolve the residue with 5 ml of methanol as control medicinal material solution 2.

[0161] (3) Preparation of control solution: take icariin and epimediin C control samples, add methanol to prepare a mixed solution containing 1 mg per 1 ml as control solution;

[0162] (4) Thin layer identification: according to the thin layer chromatography (Chinese Pharmacopoeia 2020 edition four general test 0502) test, 5 μl of each of the above test solution 1-3 and the control medicinal material solution, 2 μl of each of the control solution, were spotted on the same silica gel G thin layer plate; 5 μl of each of the above test solution 2 and the control medicinal material solution, 2 μl of each of the control solution, were spotted on the same silica gel G thin layer plate, with methanol-butanone-chloroform-water (volume ratio of 4:6:6:1) as the developing agent, developed, taken out, dried, sprayed with 3% aluminum chloride solution, heated at 105°C, and observed under ultraviolet light (365 nm);

[0163] (5) Result analysis: it can be seen from Figure 15 that the test solution 1 without column treatment and the control medicinal material chromatogram have serious interference of mixed peaks, and the corresponding target spots have poor separation effect; it can be seen from Figure 16 that the test solution 4 and 5 prepared by ethyl acetate extraction and using 200-300 mesh neutral alumina column have unclear characteristic spot and poor separation effect, and the test solution 2 prepared by the same method as the test solution for identifying epimedium in example 1 has clear spot and good separation effect, which can meet the identification requirements. Through the above comparison, it can be known that the method for identifying epimedium in example 1.2 can effectively remove the interference of mixed peaks, so that the characteristic spot is clear and obvious and has better separation effect.

[0164] Example 3: thin layer chromatography for identifying black ginseng in shenjian filling oral liquid

[0165] This comparative example is the comparative example of 1.4 in example 1.

[0166] A method for identifying black ginseng in shenjian filling oral liquid by thin layer chromatography, the steps are as follows:

[0167] (1) Preparation of test solution: accurately take 100 ml of shenjian filling oral liquid, add 15 ml of ammonia test solution, extract with chloroform for 5 times, 30 ml each time, combine the chloroform liquid, add dilute hydrochloric acid and shake to extract 4 times, 15 ml each time, combine the acid solution, adjust the PH value to 10 with concentrated ammonia solution, extract with chloroform for 4 times, 25 ml each time, combine the chloroform liquid, evaporate to dryness, add an appropriate amount of anhydrous ethanol 1 ml to dissolve, as the test solution;

[0168] (2) Preparation of control solution: take aconite original control, add anhydrous ethanol to prepare a solution containing 1 mg per 1 ml, as the control solution;

[0169] (3) Thin layer identification: according to the thin layer chromatography (Chinese Pharmacopoeia 2020 edition four general test 0502) test, 10 ul of the above two solutions was taken and spotted on the same-silica gel G thin layer plate, with toluene-ethyl acetate-diethylamine (volume ratio of 14:4:1) as the developing agent, placed in the sulfuric acid saturated developing tank, developed for 10 cm, taken out, dried, sprayed with dilute bismuth potassium iodide solution and sodium nitrite solution, and observed under daylight.

[0170] (4) Results analysis: from Figure 17 It can be found that the test chromatogram has no same color spots at the corresponding position of the reference chromatogram, indicating that aconitine is not suitable as a control for the identification of He Shun tablets; even after developing for 10 cm, the main characteristic spots of the test chromatogram are mostly concentrated at the lower end of the plate, with a low Rf value, which may be due to the weak polarity of the existing standard developing agent, so this method cannot meet the TLC identification of He Shun tablets; the identification method of He Shun tablets in Example 1.4 has better developing effect, and the Rf value is within the required range, which can be applied to the TLC identification of He Shun tablets in Bushen Tianjing Oral Liquid.

[0171] Example 4 High performance liquid chromatography for identifying Cistanche deserticola in Bushen Tianjing Oral Liquid

[0172] This example is the counterexample of 1.5 in Example 1.

[0173] A method for identifying Cistanche deserticola in Bushen Tianjing Oral Liquid by high performance liquid chromatography, the steps are as follows:

[0174] (1) Preparation of test solution: take 25 ml of the product, extract with n-butanol for 3 times, each time for 30 ml, combine the n-butanol liquid, evaporate to dryness, and dissolve with methanol to 25 ml in a volumetric flask, shake well, and get it;

[0175] (2) Preparation of reference solution: take appropriate amount of Echinacoside reference substance, accurately weigh, add 50% methanol to prepare a mixed solution containing 0.2 mg per 1 ml, and get it;

[0176] (3) Preparation of negative control solution: take 25 ml of negative sample without Cistanche deserticola, and prepare the negative control solution of Cistanche deserticola according to the method of step (1);

[0177] (4) Determination method: accurately take 10 ul of the above three solutions respectively, inject into the liquid chromatograph, and determine, wherein the chromatographic conditions are: octadecylsilane bonded silica gel as the filler; acetonitrile-0.1% formic acid solution (15:85) as the mobile phase, the flow rate is 1.0 ml / min; the detection wavelength is 330 nm; the column temperature is 35℃;

[0178] (5) Results analysis: from Figures 18-20It can be seen that the chromatogram of the test sample has corresponding chromatographic peaks at the same retention time as the chromatogram of the reference substance, but the negative sample also has a larger interference peak. The identification method of Cynomorium songaricum in Example 1.5 is good in specificity and high in chromatographic peak separation degree.

Claims

1. A detection method of Bushen Chengan oral liquid, the preparation raw materials of the Bushen Chengan oral liquid include ginseng, astragalus, medlar, angelica, herba epimedii, cistanche and black slice, characterized in that, The effective components of Astragalus, ginseng, medlar, angelica, herba epimedii and black antler are identified by thin layer chromatography, and the effective components of cistanche are detected by high performance liquid chromatography; wherein, when the effective components of Astragalus and ginseng are identified, the thin layer chromatography identification comprises the following steps: 1.1 Preparation of test sample solution The kidney filling oral liquid is extracted by water-saturated n-butanol, and the n-butanol solution is washed by ammonia solution, and then the ammonia washing solution is discarded. The water is washed and discarded, and the residue is dissolved in methanol as the test sample solution; 1.2 Preparation of control medicinal material solution The Astragalus control medicinal material is boiled with water, cooled and filtered, and the filtrate is prepared into the Astragalus control medicinal material solution according to step 1.

1. The ginseng control medicinal material is boiled with water, cooled and filtered, and the filtrate is prepared into the ginseng control medicinal material solution according to step 1.1; 1.3 Preparation of control sample solution The astragaloside control sample is prepared into the astragaloside control sample solution by adding methanol. The ginsenoside Re, ginsenoside Rb1 and ginsenoside Rg1 control samples are prepared into the mixed control sample solution by adding methanol as the ginseng mixed control sample solution; 1.4 Preparation of negative control solution The negative sample without Astragalus is prepared into the negative control solution without Astragalus according to step 1.

1. The negative sample without ginseng is prepared into the negative control solution without ginseng according to step 1.1; 1.5 Identification by thin layer chromatography According to the thin layer chromatography test, 5-10 μl of the test sample solution, the Astragalus control medicinal material solution, the astragaloside control sample solution and the negative control solution without Astragalus, and 2-5 μl of the test sample solution, the ginseng control medicinal material solution, the ginseng mixed control sample solution and the negative control solution without ginseng are spotted on the same silica gel G thin layer plate. The lower layer solution of chloroform-methanol-water (65:35:10, by volume) is used as the developing agent, and the plate is developed, taken out, dried, sprayed with 10% sulfuric acid ethanol solution, heated at 105°C until the spots are clear, and observed under daylight and ultraviolet light 365 nm respectively; 1.6 Result analysis: whether the same color fluorescent spots appear in the test sample chromatogram at the corresponding positions of the Astragalus control medicinal material and the astragaloside control sample under ultraviolet light 365 nm; whether the same color spots appear in the test sample chromatogram at the corresponding positions of the ginseng control medicinal material and the ginseng mixed control sample under daylight and ultraviolet light 365 nm; When the effective components of herba epimedii are identified, the thin layer chromatography identification comprises: 3.1 Preparation of test sample solution The kidney filling oral liquid is extracted by water-saturated n-butanol, and the n-butanol solution is washed by ammonia solution, and then the ammonia washing solution is discarded. The water is washed and discarded, and the residue is dissolved in methanol as the test sample solution; 3.2 Preparation of herba epimedii control medicinal material solution The herba epimedii medicinal material is boiled with water, cooled and filtered, and the filtrate is prepared into the control medicinal material solution according to step 3.1; 3.3 Preparation of epimedoside and desmosdumortioidin C control sample solution The epimedoside and desmosdumortioidin C control samples are prepared into the mixed solution by adding methanol as the control sample solution; ​ 3.4 Preparation of Epimedium sagittatum negative control solution Take the negative sample of Epimedium sagittatum, and prepare the Epimedium sagittatum negative control solution according to the method of step 3.1; 3.5 Thin layer chromatography identification According to the thin layer chromatography test, 2-5 μl of each of the above four solutions is spotted on the same silica gel G thin layer plate, and a developing agent of methanol-ketone-chloroform-water with a volume ratio of 4:6:6:1 is used for development, removal, air drying, spraying with 3% aluminum chloride solution, heating at 105°C, and observation under a UV lamp at 365 nm; 3.6 Result analysis: whether the same color spots appear in the test product chromatogram at the corresponding positions of the Epimedium sagittatum control medicinal material and control product chromatogram under the UV lamp at 365 nm; When the effective components of Medlar and Angelica are identified, the thin layer chromatography identification includes: 5.1 Preparation of test product solution Take the Shenshen Chengan oral liquid, extract with ethyl acetate, combine the ethyl acetate extract, evaporate to dryness, and dissolve the residue in ethyl acetate to obtain the test product solution; 5.2 Preparation of control medicinal material solution Take the Medlar control medicinal material, decoct with water, filter, and prepare the Medlar control medicinal material solution according to the method of step 5.1; take the Angelica control medicinal material, decoct with water, filter, and prepare the Angelica control medicinal material solution according to the method of step 5.1; 5.3 Preparation of negative control solution Take the negative sample of Medlar, and prepare the Medlar negative control solution according to the method of step 5.1; take the negative sample of Angelica, and prepare the Angelica negative control solution according to the method of step 5.1; 5.4 Thin layer chromatography identification According to the thin layer chromatography test, 2-5 μl of each of the above four solutions is spotted on the same silica gel G thin layer plate, and a developing agent of methanol-ketone-chloroform-water with a volume ratio of 4:6:6:1 is used for development, removal, air drying, spraying with 3% aluminum chloride solution, heating at 105°C, and observation under a UV lamp at 365 nm; 5.5 Result analysis: whether the same color spots appear in the test product chromatogram at the corresponding positions of the Epimedium sagittatum control medicinal material and control product chromatogram under the UV lamp at 365 nm; When the effective components of Medlar and Angelica are identified, the thin layer chromatography identification includes: 7.1 Preparation of test product solution Take the Shenshen Chengan oral liquid, extract with ethyl acetate, combine the ethyl acetate extract, evaporate to dryness, and dissolve the residue in ethyl acetate to obtain the test product solution; 7.2 Preparation of control product solution Take the benzoyl new aconitum alkaloid and benzoyl aconitum alkaloid control product, and prepare a mixed solution of isopropyl alcohol-dichloromethane with a volume ratio of 1:1 as the control product solution; 7.3 Thin layer chromatography identification The above two solutions were each taken 10-15 μl and spotted on the same silica gel G thin layer plate, and developed with a developing agent of n-hexane-ethyl acetate-methanol (6.4:5:1) in a developing cylinder saturated with ammonia vapor for 15-30 min, taken out, air-dried, and sprayed with dilute potassium iodide solution; 7.4 Result analysis: in the test sample chromatogram, whether there is a same color spot at the corresponding position of the control sample chromatogram is determined; When the object of identification is the effective component of Cistanche, the high performance liquid chromatography detection includes: 9.1 Preparation of test sample solution Take the Bushen Tianjing Oral Liquid 15-30 ml, extract with n-butanol for 3 times, 30 ml each time, combine the n-butanol liquid, evaporate to dryness, and dissolve in methanol to a 25 ml volumetric flask, shake well, and obtain the test sample solution; 9.2 Preparation of control sample solution Take the Echinacoside control sample, accurately weigh, add 50% methanol to prepare a mixed solution containing 0.2 mg per 1 ml, and obtain the control sample solution; 9.3 Preparation of negative control solution Take the negative sample of Cistanche 15-30 ml, and prepare the negative control solution of Cistanche according to step 9.1; 9.4 High performance liquid chromatography detection Accurately take 10 μl of the test sample solution, control sample solution and negative control solution respectively, inject into the high performance liquid chromatograph, and detect; wherein the chromatographic conditions are: octadecylsilane bonded silica gel as the filler; acetonitrile as the mobile phase A, and 0.1% formic acid solution as the mobile phase B, the flow rate is 1.0 ml / min, gradient elution is performed according to the following table; the detection wavelength is 330 nm; and the column temperature is 30-40℃; 9.5 Result analysis: in the test sample chromatogram, whether there is a corresponding chromatographic peak at the corresponding position of the control sample chromatogram is determined.

2. The detection method of claim 1, wherein, When the object of identification is the effective component of Astragalus and Panax, the thin layer chromatography identification includes: 2.1 Preparation of test sample solution Take the Bushen Tianjing Oral Liquid 30-50 ml, add water-saturated n-butanol to extract 3 times, 30-50 ml each time, combine the n-butanol liquid, wash with ammonia solution 2 times, 40-50 ml each time, discard the ammonia washing liquid, wash with water 2 times, 40-50 ml each time, discard the water liquid, combine the n-butanol liquid, evaporate to dryness, add 1-2 ml of methanol to dissolve the residue, and obtain the test sample solution; 2.2 Preparation of control medicinal material solution Take Astragalus control medicinal material 1 g, add water 50 ml, decoct for 30-60 min, cool, filter, take the filtrate, and prepare the Astragalus control medicinal material solution according to step 2.1; take Panax control medicinal material 1 g, add water 50 ml, decoct for 30-60 min, cool, filter, take the filtrate, and prepare the Panax control medicinal material solution according to step 2.1; 2.3 Preparation of Astragaloside control sample solution Take the Astragaloside control sample, dissolve in methanol to prepare an Astragaloside control sample solution containing 1 mg per 1 ml; take the Panax Saponin Re, Panax Saponin Rb1, and Panax Saponin Rg1 control samples, dissolve in methanol to prepare a mixed solution containing 1 mg per 1 ml, as the Panax mixed control sample solution; 2.4 Preparation of negative control solution Take the negative sample of Cistanche 15-30 ml, and prepare the negative control solution of Cistanche according to step 9.1; Take the negative sample of Huangqi 30-50ml, and make the negative control solution of Huangqi according to the method of step 2.1; Take the negative sample of Renshen 30-50ml, and make the negative control solution of Renshen according to the method of step 2.1; 2.5 Identification by thin layer chromatography According to the thin layer chromatography, take 5-10ul of the test solution, the control solution of Huangqi, the control solution of Astragaloside, the negative control solution of Huangqi, and 2-5ul of the test solution, the control solution of Renshen, the control solution of Renshen, the negative control solution of Renshen, respectively, and point them on the same silica gel G thin layer plate. Take the lower solution of chloroform-methanol-water (65:35:10) at 10℃ for 4-12 hours as the developing agent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clear, and observe under daylight and ultraviolet light 365nm respectively; 2.6 Analysis of results: In the test chromatogram, whether the same color fluorescent spots appear at the corresponding positions of the control solution of Huangqi and the control solution of Astragaloside under ultraviolet light 365nm; In the test chromatogram, whether the same color spots appear at the corresponding positions of the control solution of Renshen and the control solution of Renshen under daylight and ultraviolet light 365nm.

3. The detection method of claim 1, wherein, When the effective components of Herba Epimedii are identified, the thin layer chromatography includes: 4.1 Preparation of test solution Take the Bushen Fillian Oral Liquid 30-50ml, and extract with water-saturated n-butanol for 3 times, 30-50ml each time, combine the n-butanol solution, wash with water for 2 times, 30-50ml each time, combine the n-butanol solution, evaporate to dryness, dissolve the residue with 2-5ml of methanol, and add to the neutral alumina column. The neutral alumina column is selected to be 100-200 mesh, 5g, and 1.5cm in diameter. Wash with 30ml of 40% methanol, discard the eluent, wash with 50-100ml of 40% methanol, collect the eluent, evaporate to dryness, and dissolve with 1-2ml of methanol to obtain the test solution; 4.2 Preparation of Herba Epimedii control solution Take 1g of Herba Epimedii control solution, add 50ml of water, and boil or ultrasonic for 30-60min. After cooling, filter, and prepare the control solution according to step 4.1; 4.3 Preparation of Icaritin and Epimedoside C control solution Take the Icaritin and Epimedoside C control solution, dissolve with methanol, and prepare the control solution containing 1mg of Icaritin and Epimedoside C per 1ml; 4.4 Preparation of negative control solution of Herba Epimedii Take the negative sample of Herba Epimedii 30-50ml, and prepare the negative control solution of Herba Epimedii according to step 4.1; 4.5 Identification by thin layer chromatography According to the thin layer chromatography test, 2-5 μl of the test solution, the Epimedium herb control solution, the icariin and epimedin C control solution, and the negative control solution without Epimedium are respectively spotted on the same silica gel G thin layer plate, and developed with methanol-ketone-chloroform-water (4:6:6:1, by volume) as the developing agent. After development, the plate is taken out, dried, sprayed with 3% aluminum chloride solution, heated at 105°C, and observed under UV light at 365 nm. 4.6 Result analysis: whether the same color spots appear in the test chromatogram at the corresponding positions of the Epimedium herb control and the control at UV light 365 nm.

4. The detection method of claim 1, wherein, When the effective components of Fructus Lycii and Angelica are identified, the thin layer chromatography identification includes: 6.1 Preparation of test solution The kidney-tonifying and essence-filling oral liquid 15-30 ml is extracted with ethyl acetate for 2 times, 30 ml each time, and the ethyl acetate extracts are combined and evaporated to dryness to obtain a residue. The residue is dissolved in 2 ml of ethyl acetate to serve as the Angelica test solution. 1 ml of the Angelica test solution is diluted to the mark of a 10 ml volumetric flask with ethyl acetate to serve as the Fructus Lycii test solution. 6.2 Preparation of control solution 1 g of the Fructus Lycii control is added with 50 ml of water, and ultrasonically treated or decocted for 15-30 min. The filtrate is prepared into the Fructus Lycii control solution according to step 6.

1. 1 g of the Angelica control is added with 50 ml of water, and ultrasonically treated or decocted for 15-30 min. The filtrate is prepared into the Angelica control solution according to step 6.

1. 6.3 Preparation of negative control solution 30 ml of the negative sample without Fructus Lycii is prepared into the negative control solution according to step 6.

1. 30 ml of the negative sample without Angelica is prepared into the negative control solution according to step 6.

1. 6.4 Thin layer chromatography identification According to the thin layer chromatography test, 1-2 μl of the Fructus Lycii test solution, the Fructus Lycii control solution, and the negative control solution without Fructus Lycii, and 1-2 μl of the Angelica test solution, the Angelica control solution, and the negative control solution without Angelica are respectively spotted on the same silica gel G thin layer plate, and developed with toluene-ethyl acetate-formic acid (9:21:0.5, by volume) as the developing agent. After development, the plate is taken out, dried, and observed under UV light at 365 nm. 6.5 Result analysis: whether the same color spots appear in the test chromatogram at the corresponding positions of the Fructus Lycii control at UV light 365 nm; whether the same color spots appear in the test chromatogram at the corresponding positions of the Angelica control at UV light 365 nm.

5. The detection method of claim 1, wherein, When the effective components of Heishunpian are identified, the thin layer chromatography identification includes: 8.1 Preparation of test solution Precise measuring the kidney filling essence oral liquid 100 ml, add ammonia test solution 15 ml, extract with chloroform 5 times, 30 ml each time, combine chloroform liquid, add dilute hydrochloric acid and shake, extract 5 times, 15-30 ml each time, combine the acid solution, adjust the PH value to 10 with concentrated ammonia solution, extract with chloroform 3-5 times, 30 ml each time, combine chloroform liquid, evaporate to dryness, add anhydrous ethanol 0.5 ml to dissolve, as the test solution; 8.2 Preparation of reference solution Take benzoyl new aconitine and benzoyl aconine reference substance, add isopropyl alcohol-dichloromethane mixed solution with a volume ratio of 1:1 to prepare a mixed solution containing 1 mg per 1 ml, as the reference solution; 8.3 Thin layer chromatography identification Take 10-15 μl of the test solution and the reference solution respectively, and point on the same silica gel G thin layer plate, use n-hexane-ethyl acetate-methanol mixed solution with a volume ratio of 6.4:5:1 as developing agent, place in the developing cylinder saturated with ammonia vapor for 15-30 min, develop, take out, air dry, and spray with dilute potassium iodide solution; 8.4 Result analysis: in the test chromatogram, judge whether the same color spots appear at the corresponding positions of the reference chromatogram.

6. The detection method of claim 1, wherein, When identifying the effective components of Cistanche, the high performance liquid chromatography detection includes: 10.1 Preparation of test solution Take 25 ml of the product, extract with n-butanol 3 times, 30 ml each time, combine n-butanol liquid, evaporate to dryness, dissolve with methanol to 25 ml in a volumetric flask, shake well, and the test solution is obtained; 10.2 Preparation of reference solution Take appropriate amount of Echinacoside reference substance, accurately weigh and add 50% methanol to prepare a mixed solution containing 0.2 mg per 1 ml, and the reference solution is obtained; 10.3 Preparation of negative control solution Take 25 ml of negative sample of Cistanche, and prepare the negative control solution of Cistanche according to the method of step 10.1; 10.4 High performance liquid chromatography detection Accurately take 10 μl of each of the above three solutions, inject into the liquid chromatograph, and measure, wherein the chromatographic conditions are: octadecylsilane bonded silica gel as the filler; acetonitrile as the mobile phase A, and 0.1% formic acid solution as the mobile phase B, the flow rate is 1.0 ml / min, gradient elution is performed according to the following table; the detection wavelength is 330 nm; the column temperature is 35℃; 10.5 Result analysis: in the test chromatogram, judge whether there are corresponding chromatographic peaks at the corresponding positions of the reference chromatogram.

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