Thin-layer identification method for clove medicinal material and preparation thereof

Abstract

The invention discloses a thin-layer identification method for a clove medicinal material and a preparation thereof. The thin-layer identification method comprises the following steps: (1) preparing a clove reference medicinal material solution, a eugenol reference solution and a test solution; the test solution comprises a clove medicinal material test solution or a clove-containing medicinal preparation test solution; (2) respectively dripping the clove reference medicinal material solution, the eugenol reference substance solution and the test solution on the same thin-layer plate, developing by using a developing solvent, spraying a color developing agent for developing, heating until spots are clearly developed, and obtaining a sunlight thin-layer chromatogram to finish identification; and the developing solvent is cyclohexane-ethyl acetate-methanol-formic acid. According to the method, a test solution, a reference medicinal material solution and a reference substance solution are obtained by adopting a simple, convenient and safe pretreatment method and are respectively dripped on the same thin-layer plate, and a multi-information clove thin-layer chromatogram is obtained under the sunlight condition by using a suitable developing solvent for developing. The method is simple and convenient to operate, clear in chromatographic spot and good in separation degree.

Description

Technical Field

[0001] The present invention relates to the field of quality control of traditional Chinese medicines, and particularly to a thin-layer identification method for cloves and their preparations. Background Art

[0002] Clove is the dried flower bud of the plant Eugenia Cargophyllata Thunb. of the Myrtaceae family, which is picked when the flower bud turns from green to red. Clove has a pungent taste and a warm nature, and has the effects of warming the middle-jiao to arrest reverse flow, and warming the kidney to assist yang. Clinically, it is used to treat hiccups, vomiting, nausea, diarrhea, abdominal cold pain, abdominal mass, hernia, and tinea. The modern pharmacological research on cloves mainly focuses on its effects on the digestive system, blood system, and nervous system. Its main component, eugenol, has various pharmacological activities such as antibacterial, anesthetic, antipyretic, antioxidant, and anti-tumor.

[0003] In the first part of the Chinese Pharmacopoeia 2020 edition under the item of "Clove", a thin-layer identification method using clove reference medicinal material and eugenol reference substance as controls is established for the identification of clove medicinal material powder. However, in actual operation, after coloring with 10% sulfuric acid ethanol solution, the thin-layer chromatographic spots of the clove reference medicinal material are light and fuzzy, and the resolution is poor. At the same time, when the developing agent petroleum ether (60 - 90°C) - ethyl acetate (9:1) in this method is used for the thin-layer identification of clove preparations, the above problems are more prominent, and it is difficult to make the thin-layer chromatographic spots of the test sample consistent with those of the reference medicinal material, and accurate quality evaluation cannot be carried out. In addition, for other thin-layer identification methods reported in the existing literature for some clove preparations, they all use the petroleum ether (60 - 90°C) - ethyl acetate developing system, which is similar to the pharmacopoeia method (He Xiaobei, Ran Yang, He Taotao, etc. Quality control test of clove microemulsion [J]. Chinese Journal of Veterinary Medicine, 2021(001):057.), (Fan Xiaoliang, Ruan Weifeng, Fang Ruihua, etc. Determination and quality control of volatile oil in clove formula granules [J]. China Pharmacist, 2019(2):4.). Summary of the Invention

[0004] Object of the Invention: The object of the present invention is to provide a thin-layer identification method for clove preparations with simple and rapid operation and good resolution of chromatographic spots.

[0005] Technical Solution: The present invention provides a thin-layer identification method for cloves and their preparations, including the following steps:

[0006] (1) Prepare a clove reference medicinal material solution, an eugenol reference substance solution, and a test sample solution; the test sample solution includes a clove medicinal material test sample solution or a test sample solution of a drug preparation containing cloves;

[0007] (2) Apply the clove control drug solution, eugenol reference substance solution, and test solution respectively on the same thin-layer plate, develop with the developing agent, spray with the developer, heat until the spots are clearly developed, and obtain the TLC chromatogram under daylight to complete the identification; the developing agent is cyclohexane - ethyl acetate - methanol - formic acid.

[0008] Furthermore, the preparation method of the clove control drug solution described in step (1) includes the following steps: Take the clove control drug, decoct it with water or reflux it by heating, filter it, extract it by shaking with ethyl acetate solution, take the supernatant after standing and separating the layers, evaporate it to dryness, and dissolve the residue in an organic solvent to obtain the control drug solution.

[0009] Specifically, when decocting the clove control drug with water, the mass - volume ratio of the control drug to water is 1:15 g / mL - 50 g / mL, the processing time of decocting or heating reflux is 15 min - 45 min, and the number of times of decocting or heating reflux is 2 - 3 times.

[0010] Specifically, the volume ratio of the ethyl acetate solution to the aqueous solution of the clove control drug is 1:1 - 2, and the number of times of extracting by shaking with the ethyl acetate solution is 1 - 4 times.

[0011] Furthermore, the preparation method of the eugenol reference substance solution described in step (1) includes the following steps: Take the eugenol reference substance, dissolve it in an alcohol solvent to prepare the reference substance solution.

[0012] Specifically, the concentration of the eugenol reference substance solution is 0.5 - 2 mg / ml.

[0013] Furthermore, when the test solution in step (1) is the clove standard decoction, the preparation method includes the following steps: Take the clove standard decoction sample, extract it by shaking with ethyl acetate solution, take the supernatant after standing and separating the layers, evaporate it to dryness, and dissolve the residue in an organic solvent to obtain the standard decoction test solution.

[0014] Specifically, the volume ratio of the ethyl acetate solution to the clove standard decoction is 1:1 - 2, and the number of times of extracting by shaking with the ethyl acetate solution is 1 - 4 times.

[0015] Furthermore, when the test solution in step (1) is the clove formula granule, the preparation method includes the following steps: Take the clove formula granule sample, dissolve it in water, extract it by shaking with ethyl acetate solution, take the supernatant after standing and separating the layers, evaporate it to dryness, and dissolve the residue in an organic solvent to obtain the formula granule test solution.

[0016] Specifically, when dissolving the clove formula granule in water, the mass - volume ratio of the clove formula granule to water is 1:30 g / mL - 200 g / mL, the volume ratio of the ethyl acetate solution to the clove standard decoction is 1:1 - 2, and the number of times of extracting by shaking with the ethyl acetate solution is 1 - 4 times.

[0017] Further, the volume ratio of the developing agent cyclohexane - ethyl acetate - methanol - formic acid described in step (2) is 10 - 20:1 - 4:1 - 3:0.1.

[0018] Preferably, the volume ratio of the developing agent cyclohexane - ethyl acetate - methanol - formic acid is 16:3:2:0.1.

[0019] Further, the color developing agent described in step (2) is a 10% sulfuric acid - ethanol solution, and the heating temperature is 105°C.

[0020] Further, the thin - layer plate described in step (2) is a silica gel G thin - layer plate.

[0021] Further, the spotting volume of the control crude drug solution in step (2) is 1 μl - 3 μl, and the spotting volumes of the control substance solution and the test sample solution are 2 μl - 6 μl.

[0022] The present invention also provides the application of the thin - layer identification method of the above - mentioned clove crude drug and its preparations in the authenticity identification of cloves.

[0023] Beneficial effects: Compared with the prior art, the present invention has the following remarkable advantages: The present invention uses a simple and safe pretreatment method to obtain the test sample solution, the control crude drug solution and the control substance solution, which are respectively spotted on the same thin - layer plate, developed with a suitable developing agent, and under sunlight conditions, a multi - information thin - layer chromatogram of cloves is obtained. This method is easy to operate, the chromatographic spots are clear and the resolution is good, the detection cost is low, safe and environmentally friendly; the evaluation is carried out by the consistency of the control crude drug, which is more accurate, scientific and more conducive to quality supervision. Description of the Drawings

[0024] Figure 1 is the thin - layer chromatogram of the standard decoction of cloves in Example 1 of the present invention;

[0025] Figure 2 is the thin - layer chromatogram of the clove formula granules in Example 1 of the present invention;

[0026] Figure 3 is the thin - layer chromatogram with different spotting volumes in Example 2 of the present invention;

[0027] Figure 4 is the thin - layer chromatogram at different temperatures in Example 2 of the present invention;

[0028] Figure 5 is the thin - layer chromatogram at different humidities in Example 2 of the present invention;

[0029] Figure 6 is the thin - layer chromatogram of thin - layer plates from different manufacturers in Example 2 of the present invention;

[0030] Figure 7 It is the thin layer chromatogram of developing agents with different ratios in Example 2 of the present invention;

[0031] Figure 8 It is the thin layer chromatogram of Flos Caryophylli in Comparative Example 1 of the present invention (Pharmacopoeia method);

[0032] Figure 9 It is the thin layer chromatogram of Flos Caryophylli formula granules in Comparative Example 2 of the present invention (developing agent: petroleum ether (60 - 90°C) - ethyl acetate (9:1), developer: 5% vanillin sulfuric acid solution);

[0033] Figure 10 It is the thin layer chromatogram of Flos Caryophylli formula granules in Comparative Example 2 of the present invention (developing agent: cyclohexane - ethyl acetate - methanol - formic acid (16:3:2:0.1), developer: 10% sulfuric acid ethanol solution). Detailed implementation manners

[0034] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0035] The instruments and reagents used in the present invention are as follows:

[0036] Instruments: Automatic thin layer imager (CAMAG TLC VISUALIZER), ten-thousandth balance (Mettler Toledo, Switzerland), KQ-250E ultrasonic cleaner (Kunshan Ultrasonic Electronics Co., Ltd.), electronic temperature-controlled electric furnace (Jiangyin Poly Scientific Instruments Co., Ltd.), GKC thermostatic water bath (Nantong Huatai Experimental Instrument Co., Ltd.), silica gel G thin layer plate (Qingdao Marine Chemical Factory, Yantai Xincheng Silica Gel Materials Co., Ltd., Qingdao Kangyexin Medicinal Silica Gel Co., Ltd.).

[0037] Reagents: Diethyl ether (Shanghai Lingfeng Chemical Reagent Co., Ltd.), ethyl acetate (Sinopharm Chemical Reagent Co., Ltd.), methanol (Sinopharm Chemical Reagent Co., Ltd.), petroleum ether (60 - 90°C) (Sinopharm Chemical Reagent Co., Ltd.), cyclohexane (Sinopharm Chemical Reagent Co., Ltd.), formic acid (Shanghai Titan Scientific Co., Ltd.) are all of analytical grade; water is purified water.

[0038] The reference crude drug of Flos Caryophylli (batch number: 121039 - 201906) and the reference substance of eugenol (batch number: 110725 - 202318) are both purchased from the National Institutes for Food and Drug Control.

[0039] The crude drug of Flos Caryophylli (batch numbers: YC14, YC15), the standard decoction of Flos Caryophylli (batch numbers: BT1 - BT16), and the formula granules of Flos Caryophylli (batch numbers: KL14, KL15, KL16) are all prepared by Jiangyin Tianjiang Pharmaceutical Co., Ltd.

[0040] For those without specific experimental procedures or conditions noted in the present invention, the operations or conditions of the conventional experimental procedures described in the literature in this field can be followed. For reagents or instruments without the manufacturer noted, they are all conventional reagent products that can be obtained through commercial purchase.

[0041] Example 1

[0042] Preparation of the test solution of the standard decoction: Take 20 ml of the standard decoction of Syzygium aromaticum, extract twice with ethyl acetate by shaking, 20 ml each time. Combine the ethyl acetate solutions, evaporate to dryness, dissolve the residue in 1 ml of ethyl acetate to obtain the test solution.

[0043] Preparation of the test solution of the formula granules: Take 0.5 g of the formula granules of Syzygium aromaticum, dissolve them in 20 ml of hot water, cool, extract twice with ethyl acetate by shaking, 20 ml each time. Combine the ethyl acetate solutions, evaporate to dryness, dissolve the residue in 1 ml of ethyl acetate to obtain the test solution.

[0044] Preparation of the solution of the control crude drug: Take 1 g of the control crude drug of Syzygium aromaticum, add 20 ml of water, reflux by heating for 30 minutes, filter. Add 20 ml of water to the residue again, reflux by heating for 20 minutes, filter. Combine the two extraction solutions, cool, extract twice with ethyl acetate by shaking, 40 ml each time. Combine the ethyl acetate solutions, evaporate to dryness at low temperature, dissolve the residue in 1 ml of ethyl acetate to obtain the solution of the control crude drug.

[0045] Preparation of the reference substance solution: Take eugenol reference substance, make a solution containing 1 mg per 1 ml with methanol to obtain the reference substance solution.

[0046] Thin-layer identification procedure: Pipette 4 μl of each of the test solution and the reference substance solution, and 2 μl of the solution of the control crude drug, and spot them respectively on the same silica gel G thin-layer plate (Qingdao Marine Chemical Factory). Use cyclohexane - ethyl acetate - methanol - formic acid (16:3:2:0.1) as the developing agent, develop, take out, air dry, spray with 10% sulfuric acid ethanol solution, and heat at 105 °C until the spots are clearly visible.

[0047] Evaluation requirements: In the chromatogram of the test sample, spots of the same color should appear at the corresponding positions of the chromatogram of the control crude drug and the chromatogram of the reference substance.

[0048] Result analysis: Figure 1 1 - 16 in the figure respectively correspond to the chromatograms of the test solutions of the standard decoctions of Syzygium aromaticum with batch numbers BT1 - BT16, S is the chromatogram of the control crude drug of Syzygium aromaticum, and T is the chromatogram of eugenol reference substance. Figure 2 1 - 3 in the figure respectively correspond to the chromatograms of the test solutions of the formula granules of Syzygium aromaticum with batch numbers KL14, KL15, and KL16, S is the chromatogram of the control crude drug of Syzygium aromaticum, and T is the chromatogram of eugenol reference substance. In the chromatogram of the test sample, spots of the same color should appear at the corresponding positions of the chromatogram of the control crude drug and the chromatogram of the reference substance.

[0049] Example 2

[0050] The durability verification was carried out on the thin-layer identification in Example 1, specifically including:

[0051] (1) Investigation of different sample application amounts:

[0052] Take clove formula granules (batch number: KL14), clove control crude drug and eugenol reference substance, and prepare the test solution and control crude drug solution respectively according to the method described in Example 1. Then take 2 μl, 4 μl, 6 μl of the test solution of clove formula granules, 1 μl, 2 μl, 3 μl of the control crude drug solution of clove, and 2 μl, 4 μl, 6 μl of the reference substance solution of eugenol, and spot them on the same silica gel G thin-layer plate (except for the investigation experiment of silica gel G thin-layer plates from different manufacturers, other experiments all use the silica gel G thin-layer plate from Qingdao Marine Chemical Factory). Use cyclohexane - ethyl acetate - methanol - formic acid (16:3:2:0.1) as the developing agent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, and heat at 105 °C until the spots are clearly visible. The results are shown in Figure 3 , Figure 3 In which 1 - 3 respectively correspond to the chromatograms of the test solution of clove formula granules with 2 μl, 4 μl, 6 μl, S1 - S3 respectively correspond to the chromatograms of the control crude drug of clove with 1 μl, 2 μl, 3 μl, and T1 - TT3 respectively correspond to the chromatograms of the reference substance solution of eugenol with 2 μl, 4 μl, 6 μl. It can be seen from Figure 3 that when the sample application amounts of the test substance and the reference substance are 2 - 6 μl and the sample application amount of the control crude drug is 1 - 3 μl, clear spots appear at the corresponding positions in the chromatogram of the clove formula granules, the chromatogram of the control crude drug, and the chromatogram of the reference substance, without other interferences.

[0053] (2) Investigation of different temperatures:

[0054] Take clove formula granules (batch number: KL14), clove control crude drug and eugenol reference substance, and prepare the test solution and control crude drug solution respectively according to the method described in Example 1. Then take 4 μl each of the test solution of clove formula granules and the reference substance solution of eugenol, and 2 μl of the control crude drug solution of clove, and spot them on the same silica gel G thin-layer plate. Use cyclohexane - ethyl acetate - methanol - formic acid (16:3:2:0.1) as the developing agent, and develop respectively under the conditions of low temperature (T: 4 °C, RH: 35%), normal temperature (T: 18.6 °C, RH: 35%), and high temperature (T: 35 °C, RH: 35%). Take out, dry, spray with 10% sulfuric acid ethanol solution, and heat at 105 °C until the spots are clearly visible. The results are shown in Figure 4 , Figure 4 In which 1 - 3 respectively correspond to the chromatograms of the test solution of clove formula granules, S corresponds to the chromatogram of the clove control crude drug, and T corresponds to the chromatogram of the reference substance solution of eugenol. It can be seen from Figure 4It can be seen that under different temperature conditions, the separation effect of the clove formula granules is good, and the chromatogram of the test sample of the clove formula granules shows spots of the same color at the corresponding positions of the chromatogram of the control crude drug and the chromatogram of the reference substance. The experimental results show that temperature has little effect on the thin-layer identification of the clove formula granules, indicating that this thin-layer identification method has good durability for different temperatures.

[0055] (3) Investigation of different humidities:

[0056] Take the clove formula granules (batch number: KL14), the clove control crude drug and the eugenol reference substance, and prepare the test sample solution and the control crude drug solution respectively according to the method described in Example 1. Then take 4 μl each of the test sample solution of the clove formula granules and the eugenol reference substance solution, and 2 μl of the clove control crude drug solution, and spot them on the same silica gel G thin-layer plate. Use cyclohexane-ethyl acetate-methanol-formic acid (16:3:2:0.1) as the developing agent, and develop under different humidity conditions (RH: 18%, 35%, 88%) respectively. Take out, dry, spray with 10% sulfuric acid ethanol solution, and heat at 105 °C until the spots are clearly colored. The results are shown in Figure 5 , Figure 5 In which 1-3 respectively correspond to the chromatogram of the test sample of the clove formula granules, S corresponds to the chromatogram of the clove control crude drug, and T corresponds to the chromatogram of the test sample of the eugenol reference substance. It can be seen from Figure 5 that under different humidity conditions, the separation effect of the clove formula granules is good, and the chromatogram of the test sample of the clove formula granules shows spots of the same color at the corresponding positions of the chromatogram of the control crude drug and the chromatogram of the reference substance. The experimental results show that humidity has little effect on the thin-layer identification of the clove formula granules, indicating that this thin-layer identification method has good durability for different humidities.

[0057] (4) Investigation of thin-layer plates from different manufacturers:

[0058] Take the clove formula granules (batch number: KL14), the clove control crude drug and the eugenol reference substance, and prepare the test sample solution and the control crude drug solution respectively according to the method described in Example 1. Then take 4 μl each of the test sample solution of the clove formula granules and the eugenol reference substance solution, and 2 μl of the clove control crude drug solution, and spot them on thin-layer plates of silica gel G from different manufacturers (Qingdao Ocean, Kangyexin, Yantai Xincheng) respectively. Use cyclohexane-ethyl acetate-methanol-formic acid (16:3:2:0.1) as the developing agent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, and heat at 105 °C until the spots are clearly colored. The results are shown in Figure 6 , Figure 6 In which 1-3 respectively correspond to the chromatogram of the test sample of the clove formula granules, S corresponds to the chromatogram of the clove control crude drug, and T corresponds to the chromatogram of the test sample of the eugenol reference substance. It can be seen from Figure 6It can be seen that when using thin-layer plates from different manufacturers, the separation effect of the Syzygium aromaticum formula granules is good, and the chromatograms of the test samples of the Syzygium aromaticum formula granules show spots of the same color at the corresponding positions as the chromatograms of the control crude drug and the reference substance. The experimental results show that thin-layer plates from different manufacturers have little influence on the thin-layer identification of the Syzygium aromaticum formula granules, indicating that this thin-layer identification method has good durability for thin-layer plates from different manufacturers.

[0059] (5) Investigation of different developing agent ratios:

[0060] Take the Syzygium aromaticum formula granules (batch number: KL14), Syzygium aromaticum control crude drug, and eugenol reference substance, and prepare the test solution and the control crude drug solution according to the method described in Example 1 respectively. Then, take 4 μl of the test solution of the Syzygium aromaticum formula granules and 4 μl of the eugenol reference substance solution, and 2 μl of the control crude drug solution of Syzygium aromaticum, and spot them on the same silica gel G thin-layer plate. Use cyclohexane-ethyl acetate-methanol-formic acid (10:1:1:0.1, 10:3:2:0.1, 12:3:2:0.1, and 20:3:2:0.1) as the developing agent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, and heat at 105 °C until the spots are clearly visible. The results are shown in Figure 7 , Figure 7 Figures 1 - 2 in the figure respectively correspond to the chromatogram of the crude drug, Figures 3 - 4 correspond to the chromatogram of the Syzygium aromaticum formula granules, and T corresponds to the chromatogram of the eugenol reference substance test sample. It can be seen that under the developing agent ratios of this application, the separation effect of the Syzygium aromaticum formula granules is good, and the chromatograms of the test samples of the Syzygium aromaticum formula granules show spots of the same color at the corresponding positions as the chromatograms of the control crude drug and the reference substance.

[0061] Comparative Example 1

[0062] In this comparative example, the thin-layer chromatography detection of Syzygium aromaticum herbs was carried out according to the method in Part I of the Chinese Pharmacopoeia (2020 Edition). The specific process is as follows:

[0063] Take 0.5 g of the powder of Syzygium aromaticum herbs, add 5 ml of ether, shake for several minutes, filter, and use the filtrate as the test solution. Separately, take eugenol reference substance and make a solution containing 16 μl per 1 ml with ether as the reference substance solution. According to the thin-layer chromatography method (General Rule 0502 in the Chinese Pharmacopoeia 2020 Edition), take 5 μl of each of the above two solutions and spot them on the same silica gel G thin-layer plate. Use petroleum ether (60 - 90 °C)-ethyl acetate (9:1) as the developing agent, develop, take out, dry, spray with 5% vanillin sulfuric acid solution, and heat at 105 °C until the spots are clearly visible. In the test sample chromatogram, at the corresponding position of the reference substance chromatogram, spots of the same color appear.

[0064] The results are shown in Figure 8 , Figure 8For the test sample chromatograms of Flos Caryophylli (batch numbers: YC14, YC15) corresponding to 1-2 in the Chinese Pharmacopoeia, and the chromatogram of the eugenol reference substance corresponding to T. It can be seen that in the thin-layer chromatograms of Flos Caryophylli and the eugenol reference substance obtained by the method in Part I of the Chinese Pharmacopoeia (2020 Edition), except for the eugenol spots, the other spots are light and blurred, with poor resolution, which is not conducive to discrimination. In addition, only using the eugenol reference substance as the control without using the Flos Caryophylli reference medicinal material as the control has poor specificity.

[0065] Comparative Example 2

[0066] In this comparative example, the thin-layer developing agent under Flos Caryophylli in Part I of the Chinese Pharmacopoeia (2020 Edition): petroleum ether (60-90°C) - ethyl acetate (9:1) and the developing agent provided by the present invention: cyclohexane - ethyl acetate - methanol - formic acid (16:3:2:0.1) were used for development, and the preparation methods of the test sample and reference medicinal material solutions and the developing agent in the method of the present invention and the Chinese Pharmacopoeia were compared and investigated for the detection of Flos Caryophylli formula granules. The specific process is as follows (the serial numbers correspond to those in Figure 9 and 10 ):

[0067] 1: Take 0.5 g of formula granules (batch number: KL14), grind them finely, add 10 ml of ethyl acetate, shake for 5 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1 ml of ethyl acetate to obtain the test sample solution.

[0068] 2: Take 0.5 g of formula granules (batch number: KL14), grind them finely, add 10 ml of ether, shake for 5 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1 ml of ether to obtain the test sample solution.

[0069] 3: Take 0.5 g of formula granules (batch number: KL14), grind them finely, heat 20 ml of hot water to dissolve them, cool, extract twice with ethyl acetate by shaking, 20 ml each time, combine the ethyl acetate solutions, evaporate to dryness, dissolve the residue in 1 ml of ethyl acetate to obtain the test sample solution.

[0070] S1: Take 1.0 g of Flos Caryophylli reference medicinal material, add 10 ml of ethyl acetate, shake for 5 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1 ml of ethyl acetate to obtain the reference medicinal material solution.

[0071] S2: Take 1.0 g of Flos Caryophylli reference medicinal material, add 10 ml of ether, shake for 5 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1 ml of ether to obtain the reference medicinal material solution.

[0072] S3: Take 1.0 g of clove reference medicinal material, add 20 ml of water, heat and reflux for 30 minutes, filter, add 20 ml of water to the residue, heat and reflux for 20 minutes, filter, combine the aqueous solution, shake and extract twice with ethyl acetate, 40 ml each time, combine the ethyl acetate solution, evaporate to dryness, add 1 ml of ethyl acetate to the residue to dissolve it, and use it as the reference medicinal material solution.

[0073] T: Take eugenol reference substance and add methanol to make a solution containing 1 mg per 1 ml as the reference substance solution.

[0074] According to the thin layer chromatography method (General Rules 0502 of the 2020 edition of the Chinese Pharmacopoeia), 4 μl of the test solution and the reference solution, and 2 μl of the reference medicinal material solution were taken and spotted on the same silica gel G thin layer plate, respectively, with petroleum ether (60-90°C)-ethyl acetate (9:1) as the developing solvent, developed, taken out, dried, sprayed with 5% vanillin sulfuric acid solution, and heated at 105°C until the spots were clearly colored; or, with cyclohexane-ethyl acetate-methanol-formic acid (16:3:2:0.1) as the developing solvent, developed, taken out, dried, sprayed with 10% sulfuric acid ethanol solution, and heated at 105°C until the spots were clearly colored. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions of the chromatogram of the reference medicinal material and the chromatogram of the reference substance.

[0075] Results Figure 9 - 10 , 1 to 3 in the figure are the chromatograms of the test samples of clove formula granules made by the three preparation methods, S1 to S3 are the chromatograms of the clove reference medicinal materials made by the three preparation methods, and T is the chromatogram of the eugenol reference substance. Among them, Figure 9 The developing solvent was petroleum ether (60-90°C)-ethyl acetate (9:1), and the color developer was 5% vanillin sulfuric acid solution; Figure 10 The developing solvent was cyclohexane-ethyl acetate-methanol-formic acid (16:3:2:0.1), and the color developer was 10% sulfuric acid ethanol solution.

[0076] When using petroleum ether (60-90°C)-ethyl acetate (9:1) as a developing agent and 5% vanillin sulfuric acid solution as a color developer, the sample spots have less information, the spots are light and blurred, and the separation is poor. When using cyclohexane-ethyl acetate-methanol-formic acid (16:3:2:0.1) as a developing agent and 10% sulfuric acid ethanol solution as a color developer, when the clove formula granule sample is prepared using method 1 and method 2 (the preparation method of clove medicinal materials in the pharmacopoeia), there are almost no spots in the sample chromatogram, indicating that the sample preparation methods of method 1 and method 2 are not suitable for clove preparations, but only for clove medicinal materials.

[0077] Comprehensive analysis: The present invention provides a thin-layer identification method for clove preparations. The preparation operation of the test sample is relatively simple and rapid, and the standard decoction and the aqueous solution of formula granules only need to be extracted with ethyl acetate. In the test sample process of the present invention, petroleum ether and highly flammable dangerous controlled reagent ether are avoided, which is safer and more environmentally friendly.

[0078] The developing agent in the present invention is applicable to the thin-layer identification method of clove preparations. The chromatographic spots are richer and the resolution is good, overcoming the defects of fuzzy chromatographic spots, poor resolution and no use of control medicinal materials as control in the existing thin-layer identification methods. For the thin-layer identification method of the present invention, the quality evaluation is carried out by "the spots showing the same color at the corresponding positions of the test sample chromatogram, the control medicinal material chromatogram and the reference substance chromatogram". Compared with the prior art, there are more corresponding spots between the sample and the control medicinal material, and the problem of inconsistency between the test sample chromatogram and the control medicinal material chromatogram of the clove preparation is eliminated, which is more comprehensive, accurate and credible.

Claims

1. A thin-layer identification method for cloves medicinal materials and their preparations, characterized in that, It includes the following steps: (1) Prepare the control crude drug solution of Flos Caryophylli, the reference substance solution of eugenol, and the test solution; the test solution includes the test solution of Flos Caryophylli crude drug or the test solution of pharmaceutical preparation containing Flos Caryophylli; (2) Respectively spot the control crude drug solution of Flos Caryophylli, the reference substance solution of eugenol, and the test solution on the same thin-layer plate, develop with the developing agent, spray with the color-developing agent for color development, heat until the spots are clearly developed, and obtain the daylight thin-layer chromatogram to complete the identification; the developing agent is cyclohexane-ethyl acetate-methanol-formic acid.

2. The TLC identification method of the clove medicinal material and its preparation according to claim 1, characterized in that, The preparation method of the control crude drug solution of Flos Caryophylli described in step (1) includes the following steps: Take the control crude drug of Flos Caryophylli, decoct it with water or heat it under reflux, filter it, shake and extract it with ethyl acetate solution, take the supernatant after standing and layering, evaporate it to dryness, and dissolve the residue in an organic solvent to obtain the control crude drug solution.

3. The TLC identification method of the clove medicinal material and its preparation according to claim 1, characterized in that, The preparation method of the reference substance solution of eugenol described in step (1) includes the following steps: Take the reference substance of eugenol, dissolve it in an alcohol solvent to prepare the reference substance solution.

4. The TLC identification method of the clove medicinal material and its preparation according to claim 1, characterized in that, When the test solution described in step (1) is the standard decoction of Flos Caryophylli, the preparation method includes the following steps: Take the sample of the standard decoction of Flos Caryophylli, shake and extract it with ethyl acetate solution, take the supernatant after standing and layering, evaporate it to dryness, and dissolve the residue in an organic solvent to obtain the test solution of the standard decoction.

5. The TLC identification method of the clove medicinal material and its preparation according to claim 1, characterized in that, When the test solution described in step (1) is the formula granule of Flos Caryophylli, the preparation method includes the following steps: Take the sample of the formula granule of Flos Caryophylli, dissolve it in water, shake and extract it with ethyl acetate solution, take the supernatant after standing and layering, evaporate it to dryness, and dissolve the residue in an organic solvent to obtain the test solution of the formula granule.

6. The TLC identification method of the clove medicinal material and its preparation according to claim 1, characterized in that, The volume ratio of cyclohexane-ethyl acetate-methanol-formic acid of the developing agent described in step (2) is 10-20:1-4:1-3:0.

1.

7. The TLC identification method of the clove medicinal material and its preparation according to claim 1, characterized in that, The color-developing agent described in step (2) is 10% sulfuric acid ethanol solution, and the heating temperature is 105 °C.

8. The TLC identification method of the clove medicinal materials and their preparations according to claim 1, characterized in that, The thin-layer plate described in step (2) is a silica gel G thin-layer plate.

9. The TLC identification method for the clove medicinal material and its preparation according to claim 1, characterized in that, The spotting amount of the control crude drug solution in step (2) is 1 μl-3 μl, and the spotting amounts of the reference substance solution and the test solution are 2 μl-6 μl.

10. Application of a thin-layer identification method of Flos Caryophylli crude drug and its preparation according to any one of claims 1-9 in the identification of the authenticity of Flos Caryophylli.

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