A thin-layer identification method of Prinsepia uniflora seed water extract

Abstract

The application discloses a thin-layer identification method of Prinsepia uniflora water extract. The method prepares a control medicinal material solution and a test sample solution, the test sample solution comprises a formula granule test sample solution and a standard decoction test sample solution, the control medicinal material solution and the test sample solution are spotted on the same thin-layer plate, ethyl acetate-methanol-formic acid is used as a developing agent for development, a color developing agent is sprayed for color development, and a multi-information Prinsepia uniflora thin-layer chromatogram is obtained. The method is simple in operation, good in chromatographic spot separation degree, low in detection cost, green and environment-friendly, and suitable for identification of the Prinsepia uniflora water extract.

Description

Technical Field

[0001] This invention relates to the field of quality control of traditional Chinese medicine, specifically to a thin-layer chromatography method for identifying water extracts of rhubarb kernel. Background Technology

[0002] Prinsepia uniflora Batal. or Prinsepia uniflora Batal. var. serrata Rehd., belonging to the Rosaceae family, are dried, mature fruit kernels, mainly produced in Shanxi, Shaanxi, Gansu, Inner Mongolia, and other regions. They are sweet and slightly cold in nature, and enter the liver meridian. They have the effects of dispelling wind and heat, nourishing the liver and improving eyesight. They are used for red, swollen, and painful eyes, red and inflamed eyelids, and blurred vision with photophobia. Modern research shows that the main chemical components of Prinsepia uniflora are organic acids, flavonoids, lignans, and triterpenoids.

[0003] The Chinese Pharmacopoeia 2020 edition, under the identification of *Rhizoma Arundinaceae* (pitcher), uses ursolic acid as a reference for thin-layer chromatography (TLC). However, ursolic acid is a triterpenoid with poor water solubility, making it difficult to detect in formulations and standard decoctions prepared after water extraction. Therefore, it is not suitable for TLC identification of such preparations. Current literature reports on TLC identification of *Rhizoma Arundinaceae* are all based on detection methods established for the medicinal material (Hu Yingjie, Zhu Xiaoyang, Shang Jiarui, et al. Optimized extraction and TLC identification of gallic acid in *Rhizoma Arundinaceae* [J]. Chemical Engineering & Equipment, 2024, (01): 118-119+168.; Wei Feifei, Lü Rong, He Weiwei, et al. Pharmacognosy of *Rhizoma Arundinaceae* [J]. Chinese Journal of Traditional and Folk Medicine, 2020, 29(02): 22-24.). All of these methods use toxic and harmful solvents such as toluene and chloroform, which pose adverse effects on the environment and the safety of laboratory personnel. Summary of the Invention

[0004] Purpose of the invention: In order to solve the above-mentioned technical problems in the background art, the present invention provides a thin-layer identification method for water extract of Prunus armeniaca seed, which is simple to operate, low in cost and environmentally friendly.

[0005] Technical solution: The thin-layer chromatography method for identifying water extracts of Prunus armeniaca seed according to the present invention includes the following steps:

[0006] (1) Prepare a solution of Rhizoma Prunus thunbergii for testing, wherein the Rhizoma Prunus thunbergii test sample includes Rhizoma Prunus thunbergii granules and / or Rhizoma Prunus thunbergii standard decoction;

[0007] (2) Preparation of a reference solution of *Rhizoma Amaryllis* (pita kernel);

[0008] (3) Spot the test solution of jujube kernel and the reference solution of jujube kernel on the same thin layer plate, develop with ethyl acetate-methanol-formic acid as the developing solvent, remove the plate, spray with ferric chloride ethanol solution as the color developing agent, and examine under sunlight.

[0009] Further, in step (1), the preparation process of the Jinyuteng test sample solution comprises: dissolving the Jinyuteng test sample in water, ultrasonic treatment, adjusting pH, extracting with ethyl acetate by shaking, combining the ethyl acetate layers after static layering, evaporating to dryness, dissolving in ethyl acetate, and obtaining the solution.

[0010] Further, the mass-volume ratio of the Jinyuteng test sample to water is 1:15-1:50 g / mL.

[0011] Further, the ultrasonic treatment is performed at a power of 250-600 W, a frequency of 30-50 kHz, and for a time of 15-60 min.

[0012] Further, the ethyl acetate extraction is performed 2-4 times, and the volume ratio of ethyl acetate to sample aqueous solution is 1:0.5-1:2 during the shaking extraction.

[0013] Further, in step (2), the preparation process of the Jinyuteng reference material solution comprises: heating and refluxing Jinyuteng reference material with petroleum ether (boiling point 30-60℃), discarding the petroleum ether liquid, evaporating the solvent, adding water, decocting or heating and refluxing, filtering, adjusting pH, extracting with ethyl acetate by shaking, combining the ethyl acetate layers after static layering, evaporating to dryness, dissolving in ethyl acetate, and obtaining the solution.

[0014] Further, the mass-volume ratio of the Jinyuteng reference material to petroleum ether is 1:200-1:300 g / mL.

[0015] Further, the heating and refluxing treatment with petroleum ether is performed at a temperature of 30-60℃ for a time of 1-3 h.

[0016] Further, the mass-volume ratio of the Jinyuteng reference material to water is 1:10-1:20 g / mL.

[0017] Further, the decocting or heating and refluxing treatment is performed at a temperature of 100℃ or higher for a time of 1-2 h.

[0018] Further, the ethyl acetate extraction is performed 2-4 times.

[0019] Further, the volume ratio of ethyl acetate to sample aqueous solution is 1:0.5-1:2 during the shaking extraction.

[0020] Further, the pH adjuster is dilute hydrochloric acid, and the pH is adjusted to 1-2.

[0021] Further, in step (3), the thin layer plate used in the thin layer identification method is a polyamide film.

[0022] Further, in step (3), the volume ratio of ethyl acetate, methanol and formic acid in the developing agent is (7-9):(0.5-1.5):0.5.

[0023] Further, in step (3), the sample solution is 2-5 μl, and the sample solution of the control medicinal material is 1-4 μl.

[0024] Further, in step (3), the concentration of the ferric chloride ethanol solution is 2%, and after developing by spraying the color developing agent, the sample solution and the sample solution of the control medicinal material show the same color spots at the same Rf value on the thin layer chromatography.

[0025] Principle analysis: According to the chemical structure and properties of each effective component of traditional Chinese medicine, the extraction principle of similar compatibility is followed, and the sample solution and the sample solution of the control medicinal material are prepared by using a suitable extraction solvent. Then, the sample solution and the sample solution of the control medicinal material are developed by using a suitable developing agent, and various chemical components are well separated on the thin layer plate according to the different adsorption, desorption, re-adsorption and re-desorption capacities of the developing agent. Then, under different viewing conditions, various effective components with similar polarity show different color spots on the same thin layer plate, and a multi-information thin layer chromatogram is obtained.

[0026] Advantages: Compared with the prior art, the present application has the following advantages:

[0027] The sample solution and the sample solution of the control medicinal material are obtained by using a simple and fast pretreatment method, and are spotted on the same thin layer plate. Then, the sample solution and the sample solution of the control medicinal material are developed by using ethyl acetate-methanol-formic acid as a developing agent, and a multi-information thin layer chromatogram of the water extract of Prinsepia uniflora is obtained. At present, there is no report on the thin layer identification of the water extract of Prinsepia uniflora. The present application provides a method for identifying the water extract of Prinsepia uniflora, improves the controllability of the quality, and is more conducive to the quality supervision and management. The method is simple in operation, good in separation degree of chromatographic spots, and suitable for identifying the water extract of Prinsepia uniflora. The method has the characteristics of low detection cost, safe operation, and environmental protection of reagents. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 is a thin layer chromatogram of Prinsepia uniflora formula granules in Example 1 of the present application (wherein 1-3 are sample chromatograms of different batches of Prinsepia uniflora formula granules, and S is a chromatogram of Prinsepia uniflora control medicinal material);

[0029] Figure 2 is a thin layer chromatogram of Prinsepia uniflora standard decoction in Example 1 of the present application (wherein 1-19 are sample chromatograms of different batches of Prinsepia uniflora standard decoction, and S is a chromatogram of Prinsepia uniflora control medicinal material);

[0030] Figure 3is the thin layer chromatogram of different stationary phases in Example 2 of the present application (wherein 1-3 are the test product chromatograms of the Jujubae Fructus Formula Granules, and S is the Jujubae Fructus reference material chromatogram);

[0031] Figure 4 is the thin layer chromatogram of different developing agent proportions in Example 3 of the present application (wherein 1-3 are the test product chromatograms of the Jujubae Fructus Formula Granules, and S is the Jujubae Fructus reference material chromatogram);

[0032] Figure 5 is the thin layer chromatogram of different sample application amounts in Example 3 of the present application (wherein 1-4 are the test product chromatograms of the Jujubae Fructus Formula Granules of different sample application amounts, and S1-S4 are the Jujubae Fructus reference material chromatograms of different sample application amounts);

[0033] Figure 6 is the thin layer chromatogram of different temperatures in Example 3 of the present application (wherein 1-3 are the test product chromatograms of the Jujubae Fructus Formula Granules, and S is the Jujubae Fructus reference material chromatogram);

[0034] Figure 7 is the thin layer chromatogram of different humidities in Example 3 of the present application (wherein 1-3 are the test product chromatograms of the Jujubae Fructus Formula Granules, and S is the Jujubae Fructus reference material chromatogram);

[0035] Figure 8 is the thin layer chromatogram of the Jujubae Fructus Formula Granules test product solution and the reference material solution prepared by different methods in Comparative Example 1 of the present application (wherein 1-3 are the test product chromatograms of the Jujubae Fructus Formula Granules prepared by 3 preparation methods, and S1-S2 are the Jujubae Fructus reference material chromatograms prepared by 2 preparation methods);

[0036] Figure 9 is the thin layer chromatogram of the Jujubae Fructus Formula Granules test product solution and the reference material solution in Comparative Example 2 of the present application (wherein 1-3 are the test product chromatograms of the Jujubae Fructus Formula Granules prepared by 3 preparation methods, and S1-S2 are the Jujubae Fructus reference material chromatograms prepared by 2 preparation methods);

[0037] Figure 10 is the thin layer chromatogram of the Jujubae Fructus Formula Granules using the developing agent reported in Literature 1: toluene-ethyl acetate-formic acid (3:5.4:0.6), and the color developer is 10 g / L ferric trichloride in Comparative Example 3 of the present application (wherein 1-3 are the test product chromatograms of the Jujubae Fructus Formula Granules, S is the Jujubae Fructus reference material chromatogram, and T is the gallic acid reference chromatogram);

[0038] Figure 11 is the thin layer chromatogram of the Jujubae Fructus Formula Granules after iodine vapor fumigation using the developing agent reported in Literature 2: chloroform-ethyl acetate-ethanol (2:1:2) in Comparative Example 3 of the present application (wherein 1-3 are the test product chromatograms of the Jujubae Fructus Formula Granules, S is the Jujubae Fructus reference material chromatogram, and T is the ursolic acid reference chromatogram). DETAILED DESCRIPTION

[0039] The technical solutions of the present application are further described below in combination with the drawings.

[0040] The instruments and reagents used in the present application are as follows:

[0041] Instruments: thin layer automatic imaging instrument (CAMAG TLC VISUALIZER); METTLER TOLEDO XPR2 / A millionth balance (Swiss Mettler-Toledo Instruments (Shanghai) Co., Ltd.); JJ500 electronic balance (Changshu Shuangjie Testing Instrument Factory); PL-J100 mechanical ultrasonic cleaning machine (Dongguan Kangsijie Ultrasonic Technology Co., Ltd.); HH-4 digital constant temperature water bath (Changzhou Guohua Electrical Appliance Co., Ltd.); CenLee 20K table type high speed centrifuge (Hunan Xiangli Scientific Instrument Co., Ltd.); polyamide film (Taizhou Luqiao Sijia Biochemical Plastic Factory, Zhejiang Province); silica gel G thin layer plate (Qingdao Haoyang Chemical Plant); silica gel GF thin layer plate (Qingdao Haoyang Chemical Plant). 254 Thin layer plate (Qingdao Haoyang Chemical Plant).

[0042] Reagents: methanol (Shanghai Titan Science and Technology Co., Ltd.), ethyl acetate (Shanghai Titan Science and Technology Co., Ltd.), formic acid (Shanghai Titan Science and Technology Co., Ltd.), petroleum ether (boiling point 30-60°C) (Shanghai Titan Science and Technology Co., Ltd.), acetone (Shanghai Lingfeng Chemical Reagent Co., Ltd.), toluene (Shanghai Lingfeng Chemical Reagent Co., Ltd.), ethanol (Shanghai Titan Science and Technology Co., Ltd.), hydrochloric acid (Shanghai Lingfeng Chemical Reagent Co., Ltd.), sulfuric acid (Shanghai Lingfeng Chemical Reagent Co., Ltd.), ferric chloride (National Pharmaceutical Group Chemical Reagent Co., Ltd.), all of which are analytical pure;

[0043] Ursolic acid reference substance (batch number: 110742-201823) was purchased from China Institute for Drug Control;

[0044] Gallic acid reference substance (batch number: 110831-201906) was purchased from China Institute for Drug Control;

[0045] Prinsepia utilis reference medicinal material (batch number: PS030261) was purchased from Chengdu Pusai Biological Technology Co., Ltd.;

[0046] Tested product: Prinsepia utilis formula granules (batch number: 24110019, 24110029, 24110039; preparation method: take Prinsepia utilis medicinal materials, remove impurities, wash, dry, and crush into Prinsepia utilis decoction pieces by a traditional Chinese medicine crushing machine. Take 12000 g of Prinsepia utilis decoction pieces, add water to decoct twice, add 8 times the amount of water for the first time, decoct for 1.5 hours; add 6 times the amount of water for the second time, decoct for 0.5 hours, filter the decoction, reduce the pressure to concentrate the filtrate to a clear paste with a relative density of 1.04-1.06, filter, add an appropriate amount of malt dextrin, mix well, spray dry, add an appropriate amount of silicon dioxide, magnesium stearate and malt dextrin, mix well, dry granulation, make 1000 g, and pack, and you get it.), Prinsepia utilis standard decoction (batch number: DG1905054, DG1905055, DG1905056, DG2006013, DG2006014, DG2006015, DG2102029, DG2102030, DG2102031, DG2103239, DG2103240, DG2103241, DG2105032, DG2407066, DG2407067, DG2407068, DG1012409014, DG1012408013, DG1012408014; preparation method: take 200 g of Prinsepia utilis decoction pieces, crush, weigh, soak in a sand pot with 1600 ml of water for 30 min, use YMW mechanical split decoction kettle, first boil with strong fire, then simmer for 30 min with gentle fire, filter hot with a 200 mesh standard sieve; add 1200 ml of water for the second decoction, boil with strong fire, then simmer for 25 min with gentle fire, filter hot with a 200 mesh standard sieve; combine the two filtrates and concentrate under reduced pressure at 65°C; concentrate to a relative density of 1.00-1.02, pack into a Westlin bottle, and freeze-dry in a freeze-drying machine, and you get it.) were provided by Jiangyin Tianjiang Pharmaceutical Co., Ltd.

[0047] The specific experimental steps or conditions not mentioned in the present application can be performed according to the conventional experimental steps described in the literature in the art or the operation or conditions. The reagents or instruments not mentioned by the manufacturer are all conventional reagent products that can be obtained by market purchase.

[0048] Example 1: Thin-layer identification method of Prinsepia utilis water extract

[0049] Preparation of test solution: take 1 g of Yeren formula granules (finely ground) or Yeren standard decoction, add 25 ml of water, ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 minutes, centrifuge, take the supernatant, adjust the pH value to 1-2 with dilute hydrochloric acid, extract with ethyl acetate for 3 times, 25 ml each time, combine the ethyl acetate liquid, evaporate to dryness, add 1 ml of ethyl acetate to dissolve the residue as the test solution. Yeren formula granules were respectively batched as: 24110019, 24110029, 24110039, Yeren standard decoction was respectively batched as: DG1905054, DG1905055, DG1905056, DG2006013, DG2006014, DG2006015, DG2102029, DG2102030, DG2102031, DG2103239, DG2103240, DG2103241, DG2105032, DG2407066, DG2407067, DG2407068, DG1012409014, DG1012408013, DG1012408014, a total of 22 groups of test solution.

[0050] Preparation of reference drug solution: take 4 g of Yeren reference drug, place it in a soxhlet extractor, add petroleum ether (boiling point 30-60℃) as needed, heat and reflux at 45℃ for 2 hours, discard the petroleum ether liquid, dry the residue and filter paper cylinder, place it in a conical flask with a plug, add 50 ml of water, heat and reflux at 100℃ for 1 hour, filter, concentrate the filtrate to 25 ml, cool, adjust the pH value to 1-2 with dilute hydrochloric acid, extract with ethyl acetate for 3 times, 25 ml each time, combine the ethyl acetate liquid, recover the solvent to dryness, add 1 ml of ethyl acetate to dissolve the residue as the reference drug solution.

[0051] Thin layer identification step: refer to the test of thin layer chromatography (Chinese Pharmacopoeia 2020 edition general 0502), take 4 μl of test solution and 3 μl of reference drug solution, respectively, and point them on the same polyamide film, use ethyl acetate-methanol-formic acid (ethyl acetate:methanol:formic acid=8:1:0.5) as the developing agent, develop, take out, dry, spray with 2% ferric trichloride ethanol solution for color development, and observe under daylight. The results are shown in Figures 1-2

[0052] Evaluation requirements: in the test chromatogram, the same color spots appear at the corresponding positions of the reference drug chromatogram.

[0053] Results analysis: Figure 1 is the thin layer chromatogram of Yeren formula granules in Example 1, wherein 1-3 are the test chromatograms of Yeren formula granules of three batches 24110019, 24110029, 24110039 respectively, and S is the reference drug chromatogram of Yeren.​Figure 2 is the thin layer chromatogram of the standard decoction of Prinsepia uniflora Batal. in Example 1 of the present application, wherein 1-19 are the test sample chromatograms of different batches of the standard decoction of Prinsepia uniflora Batal. DG1905054, DG1905055, DG1905056, DG2006013, DG2006014, DG2006015, DG2102029, DG2102030, DG2102031, DG2103239, DG2103240, DG2103241, DG2105032, DG2407066, DG2407067, DG2407068, DG1012409014, DG1012408013, DG1012408014, respectively, and S is the chromatogram of the Prinsepia uniflora Batal. reference material. It can be seen from Figure 1 and Figure 2 that the chromatograms of the standard decoction of Prinsepia uniflora Batal. and the formula granules and the chromatogram of the Prinsepia uniflora Batal. reference material are compared, and the same color spots are shown at the same positions, which are all qualified products.

[0054] Example 2: Investigation of the stationary phase of the thin layer identification method of Prinsepia uniflora Batal. water extract

[0055] Prinsepia uniflora Batal. formula granules (batch number: 24110039) and Prinsepia uniflora Batal. reference material were taken, and test sample solutions and reference material solutions were prepared according to the method described in Example 1. 4 μl of the test sample solution of Prinsepia uniflora Batal. formula granules and 3 μl of the reference material solution of Prinsepia uniflora Batal. were taken, and were spotted on polyamide film and silica gel G plates, respectively, with ethyl acetate-methanol-formic acid (8:1:0.5) as the developing agent, developed, taken out, air-dried, sprayed with 2% ferric trichloride ethanol solution, and observed under daylight.

[0056] The results are shown in Figure 3 , Figure 3 The left graph in the figure uses polyamide film as the stationary phase, and the right graph uses silica gel G thin layer plate as the stationary phase. 1-3 correspond to the test sample chromatograms of Prinsepia uniflora Batal. formula granules, and S corresponds to the chromatogram of the Prinsepia uniflora Batal. reference material. It can be seen from Figure 3 that when the stationary phase is a silica gel G plate, the main spot has a large Rf value, and the spot separation degree is poor; when the stationary phase is a polyamide film, the main spot has a moderate Rf value, and the spot separation effect is good, so polyamide film is selected as the stationary phase in the subsequent experiments.

[0057] Example 3: Durability investigation of the thin layer identification method of Prinsepia uniflora Batal. water extract

[0058] (1) Investigation of different developing agent proportions:

[0059] Take the formula granules (batch number: 24110039) and the control drug material of the fruit of Prinsepia uniflora, respectively, according to the method described in Example 1 to prepare the test solution and the control drug material solution, and respectively take 4 μl of the test solution of the formula granules and 3 μl of the control drug material solution of the fruit of Prinsepia uniflora, and point on the same polyamide film, take ethyl acetate, methanol and formic acid as the developing agent with the volume ratio of 7:0.5:0.5, 8:1:0.5, 9:1.5:0.5, develop, take out, air dry, spray with 2% ferric trichloride ethanol solution, and observe under sunlight.

[0060] The results are shown in Figure 4 , Figure 4 Figures 1-3 correspond to the test chromatogram of the formula granules, and S corresponds to the control chromatogram of the fruit of Prinsepia uniflora, which can be seen from Figure 4 that under different developing agent ratios, the sample chromatogram of the formula granules and the control chromatogram of the fruit of Prinsepia uniflora show the same color spots at the corresponding positions, and when the volume ratio of ethyl acetate, methanol and formic acid is (7-9):(0.5-1.5):0.5, the spot separation degree is better, and the chromatogram is more beautiful. Therefore, ethyl acetate-methanol-formic acid with the volume ratio of 8:1:0.5 is used for further investigation in the subsequent experiment.

[0061] (2) Investigation of different sample amounts

[0062] Take the formula granules (batch number: 24110039) and the control drug material of the fruit of Prinsepia uniflora, respectively, according to the method described in Example 1 to prepare the test solution and the control drug material solution, and respectively take 4 μl of the test solution of the formula granules and 3 μl of the control drug material solution of the fruit of Prinsepia uniflora, and point on the same polyamide film, take ethyl acetate, methanol and formic acid as the developing agent with the volume ratio of 7:0.5:0.5, 8:1:0.5, 9:1.5:0.5, develop, take out, air dry, spray with 2% ferric trichloride ethanol solution, and observe under sunlight.

[0063] The results are shown in Figure 5 , Figure 5 Figures 1-4 correspond to the test chromatogram of the formula granules of 2 μl, 3 μl, 4 μl and 5 μl, and S1-S4 correspond to the control chromatogram of the fruit of Prinsepia uniflora of 1 μl, 2 μl, 3 μl and 4 μl, which can be seen from Figure 5 that when the sample amount of the test solution is 2 μl-5 μl and the sample amount of the control drug material solution is 1 μl-4 μl, the test chromatogram and the control chromatogram show the same color spots at the corresponding positions, without other interference, and can be one-to-one corresponding.

[0064] (3) Investigation of different temperatures

[0065] Take the formula granules of Prinsepia uniflora (Batch No. 24110039) and Prinsepia uniflora control medicinal materials, respectively, prepare the test solution and control medicinal material solution according to the method described in Example 1, and respectively take 4 μl of the test solution of the formula granules of Prinsepia uniflora and 3 μl of the control medicinal material solution of Prinsepia uniflora, spot on the same polyamide film, use ethyl acetate-methanol-formic acid (8:1:0.5) as the developing agent, develop under different temperature (T: 4℃, 19.3℃, 40℃) conditions, take out, air dry, spray with 2% ferric trichloride ethanol solution, and observe under sunlight.

[0066] The results are shown in Table 1. Figure 6 , Figure 6 1-3 in Table 1 correspond to the test chromatogram of the formula granules of Prinsepia uniflora, and S corresponds to the control chromatogram of Prinsepia uniflora, which shows that under different temperature conditions, the sample chromatogram of the formula granules of Prinsepia uniflora and the control chromatogram of Prinsepia uniflora show the same color spots at the corresponding positions. Figure 6 The experimental results show that temperature has no significant effect on the thin layer identification of the formula granules of Prinsepia uniflora, indicating that the thin layer identification method has good durability to different temperatures.

[0067] (4) Investigation of different humidity

[0068] Take the formula granules of Prinsepia uniflora (Batch No. 24110039) and Prinsepia uniflora control medicinal materials, respectively, prepare the test solution and control medicinal material solution according to the method described in Example 1, and respectively take 4 μl of the test solution of the formula granules of Prinsepia uniflora and 3 μl of the control medicinal material solution of Prinsepia uniflora, spot on the same polyamide film, use ethyl acetate-methanol-formic acid (8:1:0.5) as the developing agent, develop under different temperature (T: 4℃, 19.3℃, 40℃) conditions, take out, air dry, spray with 2% ferric trichloride ethanol solution, and observe under sunlight.

[0069] The results are shown in Table 1. Figure 7 , Figure 7 1-3 in Table 1 correspond to the test chromatogram of the formula granules of Prinsepia uniflora, and S corresponds to the control chromatogram of Prinsepia uniflora, which shows that under different temperature conditions, the sample chromatogram of the formula granules of Prinsepia uniflora and the control chromatogram of Prinsepia uniflora show the same color spots at the corresponding positions. Figure 7 The experimental results show that temperature has no significant effect on the thin layer identification of the formula granules of Prinsepia uniflora, indicating that the thin layer identification method has good durability to different temperatures.

[0070] Comparative Example 1:

[0071] This comparative example investigates different test solution and control medicinal material solution preparation methods, as follows:

[0072] (1) Investigation of different preparation methods of the test solution of the formula granules of Prinsepia uniflora (Batch No. 24110039):

[0073] Method 1: Take 1 g of Yiren Dispensing Granules, grind finely, add 25 ml of water, ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 minutes, shake well, centrifuge, take all supernatant, adjust pH value to 1-2 with dilute hydrochloric acid, extract with ethyl acetate for 3 times, 25 ml each time, combine ethyl acetate liquid, recover solvent to dryness, add 1 ml of ethyl acetate to the residue to dissolve, as test solution.

[0074] Method 2: Take 1 g of Yiren Dispensing Granules, grind finely, add 25 ml of water, ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 minutes, shake well, centrifuge, take all supernatant, shake with ethyl acetate for 3 times, 25 ml each time, combine ethyl acetate liquid, recover solvent to dryness, add 1 ml of ethyl acetate to the residue to dissolve, as test solution.

[0075] Method 3: Take 1 g of Yiren Dispensing Granules, grind finely, add 25 ml of methanol, ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 minutes, shake well, centrifuge, take all supernatant, recover solvent to dryness, add 1 ml of methanol to the residue to dissolve, as test solution.

[0076] (2) Investigation of different preparation methods of Yiren reference material solution:

[0077] Method 1: Take 4 g of Yiren reference material, place in a Soxhlet extractor, add petroleum ether (30-60℃) appropriately, heat reflux for 2 hours, discard petroleum ether liquid, after the residue and filter paper cylinder are dried, place in a conical flask with plug, add 50 ml of water, heat reflux for 1 hour, filter, concentrate the filtrate to 25 ml, cool, adjust pH value to 1-2 with dilute hydrochloric acid, shake with ethyl acetate for 3 times, 25 ml each time, combine ethyl acetate liquid, recover solvent to dryness, add 1 ml of ethyl acetate to the residue to dissolve, as reference material solution.

[0078] Method 2: Take 4 g of Yiren reference material, place in a Soxhlet extractor, add petroleum ether (30-60℃) appropriately, heat reflux for 2 hours, discard petroleum ether liquid, after the residue and filter paper cylinder are dried, place in a conical flask with plug, add 50 ml of methanol, ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 minutes, shake well, centrifuge, take all supernatant, recover solvent to dryness, add 1 ml of methanol to the residue to dissolve, as reference material solution.

[0079] According to the thin layer chromatography test of Example 1, take 4 μl of test solution and 3 μl of reference material solution, respectively, and point on the same polyamide film, use ethyl acetate-methanol-formic acid (8:1:0.5) as developing agent, develop, take out, dry, spray with 2% ferric trichloride ethanol solution, and observe under daylight.

[0080] Result analysis: Figure 8M1~M3 are the chromatograms of the test sample of the Yeren formula granules prepared by the three preparation methods, respectively, S1~S2 are the chromatograms of the Yeren control medicinal materials prepared by the two preparation methods, respectively. By Figure 8 It can be seen that the test sample solution prepared by method one has clear spots and good separation effect, the test sample solution prepared by method two and method three is affected by too many pigments, which affects the observation of the spots. The chromatogram spots of the control medicinal material solution prepared by method one are clear and have good separation effect; no obvious spots are observed in the chromatogram prepared by method two. In order to reduce the influence of pigments and considering the convenience of spotting and ensuring the consistency of the preparation methods of the test sample solution and the control medicinal material solution, the test sample solution preparation method one is selected as the best treatment method for the Yeren formula granules, and the control medicinal material solution preparation method one is selected as the best treatment method for the Yeren control medicinal materials.

[0081] Comparative Example 2: In this comparative example, the thin layer method under the Yeren item in the first part of the Chinese Pharmacopoeia 2020 edition is used, and the specific process is as follows:

[0082] Preparation of the test sample solution: the same as the preparation of the test sample solution of the formula granules in Comparative Example 1.

[0083] Preparation of the control medicinal material solution: the same as the preparation of the control medicinal material solution in Comparative Example 1.

[0084] Preparation of the control sample solution: take the ursolic acid control sample, add ethanol to prepare a solution containing 0.2 mg per 1 ml as the control sample solution.

[0085] According to the test of thin layer chromatography (Chinese Pharmacopoeia 2020 edition general 0502), 4 μl of the test sample solution, 3 μl of the control medicinal material solution and 6 μl of the ursolic acid control sample solution are spotted on the same silica gel G thin layer plate, petroleum ether (30~60℃)-acetone (petroleum ether and acetone in a volume ratio of 5:2) is used as the developing agent, and the sample is developed, taken out, dried, sprayed with 10% sulfuric acid ethanol solution, heated at 100℃ until the spots are clearly colored, and observed under daylight and ultraviolet light (365 nm) respectively.

[0086] Evaluation requirements: in the test sample chromatogram, the same colored spots or fluorescent spots appear at the positions corresponding to the control sample chromatogram.

[0087] Result analysis: Figure 9 The left graph is observed under ultraviolet light (365 nm), the right graph is observed under daylight, 1~3 are the chromatograms of the test sample of the Yeren formula granules prepared by the three preparation methods in Comparative Example 1, respectively, S1~S2 are the chromatograms of the Yeren control medicinal materials prepared by the two preparation methods in Comparative Example 1, respectively, T corresponds to the ursolic acid control sample chromatogram. By Figure 9It can be seen that the test product chromatograms 1-3 of Rui- ren formula granules do not have spots at the corresponding positions in the chromatogram of the ursolic acid reference substance, indicating that this thin layer method is not suitable for thin layer identification of Rui- ren water extract.

[0088] Comparative Example 3: This comparative example compares different thin layer detection methods reported in the literature as follows:

[0089] Preparation of test product solution: same as preparation of test product solution of formula granules in Example 1.

[0090] Preparation of control medicinal material solution: same as preparation of control medicinal material solution in Example 1.

[0091] Preparation of reference substance solution: take the gallic acid reference substance, add methanol to prepare a solution containing 2 mg per 1 ml, as the reference substance solution; take the ursolic acid reference substance, add ethanol to prepare a solution containing 0.2 mg per 1 ml, as the reference substance solution.

[0092] (1) Thin layer method reported in Literature 1 (Hu Yingjie, Zhu Xiaoyang, Shang Jiarui, et al. Optimization of Extraction and Thin Layer Chromatography Identification of Gallic Acid in Traditional Chinese Medicine Rui- ren [J]. Chemical Engineering and Equipment, 2024, (01): 118-119+168.):

[0093] According to the above Literature 1 thin layer chromatography method, 4 μl of test product solution, 3 μl of control medicinal material solution, and 1 μl of gallic acid reference substance solution were taken and spotted on the same silica gel GF254 thin layer plate. 254 Toluene-ethyl acetate-formic acid (3:5.4:0.6) was used as the developing agent, and the sample was developed, removed, dried, sprayed with 10 g / L ferric chloride for color development, and observed under ultraviolet light (270 nm).

[0094] Evaluation requirements: the test product chromatogram has the same color (blue) spots at the corresponding positions in the reference substance chromatogram.

[0095] Results analysis: Figure 10 1-3 correspond to the test product chromatograms of Rui- ren formula granules, S corresponds to the Rui- ren control medicinal material chromatogram, and T corresponds to the gallic acid reference substance chromatogram, as shown in the following table: Figure 10 It can be seen that the test product chromatograms of Rui- ren formula granules do not have spots at the corresponding positions in the chromatogram of the gallic acid reference substance, indicating that this thin layer method is not suitable for thin layer identification of Rui- ren water extract.

[0096] (2) Thin layer method reported in Literature 2 (Wei Feifei, Lv Rong, He Weimei, et al. Pharmacognosy of Rui- ren [J]. Chinese Ethnic and Folk Medicine, 2020, 29(02): 22-24.):

[0097] According to the thin layer chromatography method in the above-mentioned document 2, 4 μl of the test sample solution, 3 μl of the control medicinal material solution and 6 μl of the ursolic acid control sample solution were spotted on the same silica gel G thin layer plate, developed with chloroform-ethyl acetate-ethanol (2:1:2) as the developing agent, taken out, dried, fumigated with iodine vapor, and observed under daylight.

[0098] Evaluation requirement: the test sample chromatogram shows the same yellow spot at the position corresponding to the control sample chromatogram.

[0099] Result analysis: Figure 11 From left to right, they are: after fumigation with iodine vapor, observation under daylight; after drying the iodine vapor, spraying with 10% sulfuric acid ethanol solution, heating at 100°C until the spot color is clear, and observation under ultraviolet light (365 nm); after drying the iodine vapor, spraying with 10% sulfuric acid ethanol solution, heating at 100°C until the spot color is clear, and observation under daylight; 1-3 correspond to the test sample chromatogram of the Renshen formula granules, S corresponds to the control medicinal material chromatogram of Renshen, and T corresponds to the control sample chromatogram of ursolic acid. Figure 11 It can be seen that the control sample chromatogram of ursolic acid does not show a spot using the color development method of fumigation with iodine vapor reported in document 2; the test sample chromatogram of the Renshen formula granules does not show a spot at the position corresponding to the control sample chromatogram of ursolic acid using the thin layer color development method of document 2 after drying the iodine vapor, indicating that the thin layer method is not suitable for the thin layer identification of Renshen water extract.

Claims

1. A thin-layer chromatography method for identifying aqueous extracts of *Prunus armeniaca* seeds, characterized in that: Includes the following steps: (1) Preparation of Ruiren test solution, Ruiren test solution includes Ruiren formula granules and / or Ruiren standard decoction: including: taking Ruiren test sample, dissolving in water, sonicating, adjusting pH to 1-2 with dilute hydrochloric acid, extracting with ethyl acetate by shaking, allowing to stand for layering, combining the ethyl acetate layer, evaporating to dryness, and dissolving in ethyl acetate to obtain the solution; (2) Preparation of Ruiren reference medicinal material solution: including: take Ruiren reference medicinal material, add petroleum ether and heat under reflux, discard the petroleum ether solution, evaporate the solvent and add water, decoct or heat under reflux, filter, adjust the pH to 1-2 with dilute hydrochloric acid, add ethyl acetate to the filtrate and shake to extract, let stand to separate the layers and combine the ethyl acetate layers, evaporate to dryness, add ethyl acetate to dissolve to obtain the solution; (3) Take the test solution of jujube kernel and the reference solution of jujube kernel and spot them on the same polyamide film. Develop with ethyl acetate-methanol-formic acid as the developing solvent. Take it out and spray it with ferric chloride ethanol solution as the color developing agent. Examine it under sunlight. The volume ratio of ethyl acetate, methanol and formic acid in the developing solvent is (7-9):(0.5-1.5):0.

5.

2. The thin-layer chromatography method for identifying the aqueous extract of Prunus armeniaca according to claim 1, characterized in that: In step (1), the mass-to-volume ratio of the sample of *Rhizoma Prunus* to water is 1:15~1:50 g / mL, the ultrasonic treatment power is 250~600W, the frequency is 30~50kHz, the time is 15~60min, the number of times the ethyl acetate is shaken for extraction is 2~4 times, and the volume ratio of ethyl acetate to the sample aqueous solution is 1:0.5~1:

2.

3. The thin-layer chromatography method for identifying the aqueous extract of Prunus armeniaca according to claim 1, characterized in that: In step (2), the mass-to-volume ratio of the reference herb *Rhizoma Prunellae* to petroleum ether is 1:200 ~ 1:300 g / mL, the temperature for reflux treatment with petroleum ether is 30 ~ 60℃, the treatment time for reflux treatment with petroleum ether is 1 ~ 3 h, the mass-to-volume ratio of the reference herb *Rhizoma Prunellae* to water is 1:10 ~ 1:20 g / mL, the temperature for decoction or reflux treatment is above 100℃, the treatment time for decoction or reflux treatment is 1 ~ 2 h, the number of times for ethyl acetate shaking extraction is 2 ~ 4, and the volume ratio of ethyl acetate to sample aqueous solution during shaking extraction is 1:0.5 ~ 1:

2.

4. The thin-layer chromatography method for identifying the aqueous extract of Prunus armeniaca according to claim 1, characterized in that: In step (3), the amount of the test solution is 2 to 5 μl, and the amount of the control herbal solution is 1 to 4 μl.

5. The thin-layer chromatography method for identifying the aqueous extract of Prunus armeniaca according to claim 1, characterized in that: In step (3), the concentration of ferric chloride ethanol solution is 2%. After spraying on the color reagent and developing the color, it is dried with hot air and examined under sunlight. If the test solution and the reference medicinal material solution show spots of the same color at the same position of the thin-layer chromatography shift value, it is qualified.

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