A thin layer identification method for lotus stamens or its preparations

Abstract

The invention discloses a thin layer identification method for lotus stamens and its preparation, wherein a control medicinal material solution, a medicinal material test solution, a formula granule and a standard decoction test solution are prepared, the control medicinal material solution and the test solution are respectively spotted on the same thin layer plate, after being developed with a developing agent, a color developing agent is sprayed on for color development, and the plate is inspected under an ultraviolet lamp to obtain a thin layer chromatogram under ultraviolet light, thereby completing the identification operation. The method of the invention is simple and fast to operate, has good chromatographic spot separation, low detection cost, safety and environmental protection.

Description

Technical Field

[0001] The invention relates to a quality control method for traditional Chinese medicine, and in particular to a thin layer identification method for lotus stamens and preparations thereof. Background Art

[0002] Lotus stamens are the dried stamens of the lotus plant Nelumbo nucifera Gaertn. of the Nymphaeaceae family. Lotus stamens have the function of strengthening the kidney and astringing semen. They are used to treat spermatorrhea, leucorrhea, and frequent urination. In ancient times, lotus stamens were mainly used to treat spermatorrhea, nocturnal emission, incontinence, chronic hemorrhoids, diarrhea, enuresis, frequent urination, leucorrhea, and hemostasis. In modern times, it has clinical whitening, anti-thrombotic, and anti-hepatitis B virus effects. Modern research shows that lotus stamens contain flavonoids, alkaloids, volatiles and other ingredients, among which flavonoids are its main active ingredients, which have antioxidant, anti-inflammatory, anti-cancer, neuroprotective and cardioprotective effects.

[0003] There are few literature reports on the existing thin-layer identification methods of lotus stamens, which are relatively simple, and mainly include the thin-layer identification methods of lotus stamens included in the 2020 edition of the Chinese Pharmacopoeia. However, the method in the 2020 edition of the Chinese Pharmacopoeia cannot fully display the unique flavonoids and flavonoid glycosides in lotus stamens, lacks specificity, and when applied to lotus stamen formula granules, there are fewer thin-layer spots and poor separation, which cannot accurately evaluate the quality. Summary of the invention

[0004] Purpose of the invention: In view of the shortcomings and defects of the prior art, the present invention provides a thin layer identification method for lotus stamens or its preparations, which presents the specific components of lotus stamens to the greatest extent, is simple and fast to operate, has good chromatographic spot separation, and has low detection cost, safety, and environmental protection.

[0005] Technical solution: The thin layer identification method of lotus stamens or its preparations described in the present invention comprises the following steps:

[0006] (1) taking a lotus stamen reference medicinal material, boiling it with water or heating it under reflux, filtering it, concentrating the filtrate to near dryness, ultrasonically treating it with an alcohol solvent, evaporating it to dryness, dissolving it with water, and then extracting it by shaking with ethyl acetate. After standing and stratifying, the ethyl acetate layers are combined, evaporated to dryness, and the residue is dissolved with an alcohol solvent to obtain a reference medicinal material solution;

[0007] (2) taking lotus stamens and boiling them with water or heating them under reflux, filtering, concentrating the filtrate to near dryness, ultrasonically treating it with an alcohol solvent, evaporating it to dryness, dissolving it with water, and then extracting it by shaking with ethyl acetate. After standing and stratifying, the ethyl acetate layers are combined and evaporated to dryness. The residue is dissolved with an alcohol solvent to obtain a decoction piece test solution;

[0008] and / or, taking a sample of lotus root formula granules and / or standard decoction, adding an alcohol solvent for ultrasonic extraction, filtering, evaporating the filtrate to dryness, dissolving it in water, and then adding ethyl acetate for shaking extraction, standing to separate, combining the ethyl acetate layers, evaporating to dryness, and dissolving the residue in an alcohol solvent to obtain a formula granule test solution and / or a standard decoction test solution;

[0009] (3) Take the control medicinal material solution and the test sample solution and spot them on the same thin layer plate respectively. After developing with a developing agent, spray the plate with a color developer to develop the color. The plate is then inspected under ultraviolet light to obtain an ultraviolet thin layer chromatogram for comparative identification.

[0010] In the thin-layer identification method of lotus stamens or its preparation, when the lotus stamen reference medicinal material is decocted with water in step (1), or when the lotus stamen medicinal material is decocted with water in step (2), the mass volume ratio of the medicinal material to water is 1:50 g / mL to 200 g / mL, and the decocting or heating reflux treatment time is 15 min to 60 min.

[0011] The thin-layer identification method of lotus stamens or its preparations, in the step (2), when the lotus stamen formula particles and the standard decoction are extracted with an alcohol solvent, the mass volume of the lotus stamen formula particles, the standard decoction and the alcohol solvent is 1:40g / mL-200g / mL, the power of the ultrasonic treatment is 250-300W, the frequency is 40kHz, the time is 15min-60min, the number of ethyl acetate shaking extraction is 1-4 times, and the volume ratio of the ethyl acetate solution to the alcohol solution of the sample is 0.5:2-1.

[0012] In the thin-layer identification method of lotus stamens or its preparation, the developing agent in step (3) is a mixed solution of n-butanol, acetone, glacial acetic acid and water.

[0013] In the thin-layer identification method of lotus stamens or its preparation, the developing agent in step (3) is n-butanol-acetone-glacial acetic acid-water in a volume ratio of 2:2:1:6 to 2:2:1:10. Preferably, the volume ratio of n-butanol-acetone-glacial acetic acid-water is 2:2:1:7.

[0014] In the thin layer identification method of lotus stamens or its preparation, the spotting volume of the reference medicinal material solution and the test solution in the step (3) is 1 μl to 4 μl.

[0015] In the thin-layer identification method of lotus stamens or its preparation, the wavelength of the ultraviolet lamp in the step (3) is 360-370nm, the developer is 3% aluminum chloride ethanol solution, and after the developer is sprayed on for color development, it is naturally dried or blown with hot air.

[0016] In the thin layer identification method of lotus stamens or its preparation, the thin layer plate in step (3) is a polyamide film.

[0017] In the thin-layer identification method of lotus stamens or its preparation, the comparative identification of thin-layer chromatograms in step (3) refers to observing whether fluorescent spots of the same color appear at positions corresponding to those of the control medicinal material in the chromatogram of the test solution.

[0018] In the thin layer identification method of lotus stamens or its preparation, the alcohol solvent includes methanol.

[0019] Principle analysis: Based on the chemical structure and properties of each active ingredient of traditional Chinese medicine, follow the principle of similar and compatible extraction, use appropriate extraction solvents to prepare test and control medicinal material solutions simply and quickly. Then use appropriate developing agents for development, and various chemical components will be well separated on the thin layer plate according to the different adsorption, desorption, re-adsorption, and re-desorption abilities of the selected developing agents. Then, with the help of various active ingredients with similar polarity, on the same thin layer plate, under different inspection conditions (sunlight, ultraviolet light or color developer), different color spots are presented, and multi-information thin layer chromatograms are obtained.

[0020] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: the present invention adopts a simple and fast pretreatment method to obtain a test solution and a control medicinal material solution, which are respectively spotted on the same thin layer plate, developed with a suitable developing agent, sprayed with a color developer and inspected under ultraviolet light to obtain a multi-information thin layer chromatogram of lotus stamens. Compared with the pharmacopoeia method and existing literature reports, this method is simple and fast to operate, has good chromatographic spot separation, and can present the unique flavonoid components of lotus stamens, has specificity, low detection cost, and is more conducive to quality supervision. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is a thin layer chromatogram of the lotus root formula granules in Example 1 of the present invention;

[0022] Figure 2 is a thin layer chromatogram of the lotus root standard decoction in Example 1 of the present invention;

[0023] Figure 3 is a thin layer chromatogram of the lotus root medicinal material in Example 1 of the present invention;

[0024] Figure 4 is a thin layer chromatogram of different sample amounts in Example 2 of the present invention;

[0025] Figure 5 is a thin layer chromatogram at different temperatures in Example 2 of the present invention;

[0026] Figure 6 is a thin layer chromatogram of different humidity in Example 2 of the present invention;

[0027] Figure 7It is a thin layer chromatogram of different development systems of the lotus root formula granules in Comparative Example 1 of the present invention;

[0028] Figure 8 It is a thin layer chromatogram of different developing agent ratios of lotus root formula granules in comparative example 2 of the present invention;

[0029] Fig. 9 It is a thin layer chromatogram of different preparation methods of lotus root formula granules and control medicinal materials in comparative example 3 of the present invention. DETAILED DESCRIPTION

[0030] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and specific implementation methods.

[0031] The present invention adopts the following instruments and reagents:

[0032] Instruments: CAMAG TLC VISUALIZER, 1 / 10,000 balance (Mettler-Toledo, Switzerland), GKC temperature-controlled water bath (Nantong Huatai Experimental Instrument Co., Ltd.), silica gel G thin layer plate (Qingdao Ocean Chemical Plant), polyamide film (Taizhou Luqiao Sijia Biochemical Plastic Factory).

[0033] Reagents: methanol (Sinopharm Chemical Reagent Co., Ltd.), ethanol (Sinopharm Chemical Reagent Co., Ltd.), ethyl acetate (Sinopharm Chemical Reagent Co., Ltd.), n-butanol (Sinopharm Chemical Reagent Co., Ltd.), acetone (Shanghai Lingfeng Chemical Reagent Co., Ltd.), glacial acetic acid (Sinopharm Chemical Reagent Co., Ltd.), cyclohexane (Sinopharm Chemical Reagent Co., Ltd.), anhydrous formic acid (Aladdin Chemical Reagent Co., Ltd.) were all of analytical grade; water was pure water.

[0034] The reference medicinal material of lotus stamen (batch number: 121392-202102) was purchased from China Food and Drug Inspection Institutes.

[0035] Lotus stamen medicinal materials, lotus stamen standard decoction, and lotus stamen formula granules are all provided by Jiangyin Tianjiang Pharmaceutical Co., Ltd.

[0036] If no specific experimental steps or conditions are specified in the present invention, the conventional experimental steps or conditions described in the literature in the art can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be purchased commercially.

[0037] Embodiment 1:

[0038] Preparation of medicinal material test solution: Take 1g of lotus stamen medicinal material, add 50ml of water, heat and reflux for 1 hour, filter, concentrate the filtrate to near dryness, add 20ml of methanol to the residue, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, add 20ml of water to the residue to dissolve it, shake and extract with ethyl acetate twice, 20ml each time, combine the ethyl acetate solutions, evaporate to dryness, add 1ml of methanol to the residue to dissolve it, and use it as the test solution.

[0039] Preparation of test solution of formula granules: Take 0.5 g of the product, grind it, add 20 ml of methanol, ultrasonically treat it for 30 minutes, filter, evaporate the filtrate to dryness, add 20 ml of water to dissolve the residue, shake and extract with ethyl acetate twice, 20 ml each time, combine the ethyl acetate liquid, evaporate to dryness, add 1 ml of methanol to dissolve the residue, and use it as the test solution.

[0040] Preparation of standard decoction test solution: Take 0.3 g of the product, grind it into powder, add 20 ml of methanol, ultrasonically treat it for 30 minutes, filter it, evaporate the filtrate to dryness, add 20 ml of water to dissolve the residue, shake it with ethyl acetate for 2 times, 20 ml each time, combine the ethyl acetate solutions, evaporate them to dryness, add 1 ml of methanol to dissolve the residue, and use it as the test solution.

[0041] Preparation of control medicinal solution: Take 1g of lotus stamen control medicinal material, add 50ml of water, heat and reflux for 1 hour, filter, concentrate the filtrate to near dryness, add 20ml of methanol to the residue, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, add 20ml of water to the residue to dissolve it, shake and extract with ethyl acetate twice, 20ml each time, combine the ethyl acetate solutions, evaporate to dryness, add 1ml of methanol to the residue to dissolve it, and use it as the control medicinal solution.

[0042] Thin layer identification steps: According to the thin layer chromatography method (General Rules 0502 of the 2020 edition of the Chinese Pharmacopoeia), 1μ1 of the test sample solution and 2μl of the control drug solution were respectively spotted on the same polyamide film, and developed with n-butanol-acetone-glacial acetic acid-water (2:2:1:7). After development, the film was taken out, dried, sprayed with 3% aluminum chloride ethanol solution, dried with hot air, and inspected under ultraviolet light (365nm).

[0043] Evaluation requirements: In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions in the chromatogram of the control medicinal material.

[0044] Result analysis: Figure 1 1 to 3 are the chromatograms of test samples of different batches of lotus stamen formula granules, and S is the chromatogram of lotus stamen reference medicinal material. Figure 2 1 to 19 are the chromatograms of test samples of different batches of lotus stamen standard decoction, and S is the chromatogram of lotus stamen reference medicinal material. Figure 3 1 to 3 are the chromatograms of the test samples of different batches of lotus stamens, and S is the chromatogram of the reference lotus stamens. The lotus stamens, granules, and standard soups all correspond to the chromatograms of the reference lotus stamens.

[0045] Embodiment 2:

[0046] The durability verification of the thin layer identification in Example 1 specifically includes:

[0047] (1) Investigation of different sample quantities:

[0048] Take lotus stamen formula granules (batch number: 22090059) and lotus stamen control medicinal materials, prepare test solution and control medicinal material solution respectively according to the method described in Example 1, and take 0.5μL, 1μL, 1.5μL, 2μL of lotus stamen formula granules test solution and 1μL, 2μL, 3μL, 4μL of lotus stamen control medicinal material solution respectively, spot on the same polyamide film, use n-butanol-acetone-glacial acetic acid-water (2:2:1:7) as developing agent, develop, take out, dry, spray with 3% aluminum chloride ethanol test solution, dry with hot air, and inspect under ultraviolet light (365nm). Results are shown in Figure 4 , Figure 4 1 to 4 correspond to the chromatograms of 0.5 μL, 1 μL, 1.5 μL, and 2 μL of the lotus stamen formula granule test sample, and 5 to 8 correspond to the chromatograms of 1 μL, 2 μL, 3 μL, and 4 μL of the lotus stamen reference medicinal material, respectively. Figure 4 It can be seen that when the sample volume is 1 μL to 4 μL, the chromatogram of the lotus root formula particles and the chromatogram of the reference medicinal material show clear spots at corresponding positions without other interference.

[0049] Investigation at different temperatures:

[0050] Take lotus stamen formula granules (batch number: 22090059) and lotus stamen control medicinal materials, prepare test solution and control medicinal material solution respectively according to the method described in Example 1, and take 1 μL of lotus stamen formula granules test solution and 2 μL of lotus stamen control medicinal material solution respectively, spot on the same polyamide film, use n-butanol-acetone-glacial acetic acid-water (2:2:1:7) as developing agent, develop at low temperature (T: 4°C, RH: 55.2%), room temperature (T: 19.2°C, RH: 55.2%) and high temperature (T: 32.2°C, RH: 55.2%), take out, dry, spray with 3% aluminum chloride ethanol test solution, blow dry with hot air, and inspect under ultraviolet light (365nm). Results are shown in Figure 5 , Figure 5 1 to 3 correspond to the chromatograms of the test sample of lotus root formula granules, and S corresponds to the chromatogram of the reference medicinal material of lotus root. Figure 5 It can be seen that under different temperature conditions, the same color fluorescent spots appear at the corresponding positions of the chromatograms of the test and control medicinal materials of the lotus root formula granules, and the separation effect is good. The experimental results show that temperature has little effect on the thin layer identification of the lotus root formula granules, indicating that the thin layer identification method has good durability at different temperatures.

[0051] (3) Investigation of different humidity levels:

[0052] Take lotus stamen formula granules (batch number: 22090059) and lotus stamen control medicinal materials, prepare test solution and control medicinal material solution respectively according to the method described in Example 1, and take 1 μL of lotus stamen formula granules test solution and 2 μL of lotus stamen control medicinal material solution respectively, spot on the same polyamide film, use n-butanol-acetone-glacial acetic acid-water (2:2:1:7) as developing agent, develop under different humidity conditions (RH: 18%, 55.2%, 88%), take out, dry, spray with 3% aluminum chloride ethanol test solution, dry with hot air, and inspect under ultraviolet light (365nm). The results are shown in Figure 6 , Figure 6 1 to 3 correspond to the chromatograms of the test sample of lotus root formula granules, and S corresponds to the chromatogram of the reference medicinal material of lotus root. Figure 6 It can be seen that under different humidity conditions, the same color fluorescent spots appear at the corresponding positions of the chromatograms of the test and control medicinal materials of the lotus stamen formula granules, and the separation effect is good. The experimental results show that humidity has little effect on the thin layer identification of the lotus stamen formula granules, indicating that the thin layer identification method has good durability under different humidity conditions.

[0053] Comparative Example 1:

[0054] This comparative example uses lotus root formula particles to compare different developing agent systems. The specific process is as follows:

[0055] Preparation of the test solution: Take 0.5 g of lotus root granules, grind them into powder, add 20 ml of methanol, treat with ultrasound for 30 minutes, filter, evaporate the filtrate to dryness, add 20 ml of water to the residue to dissolve it, shake and extract with ethyl acetate twice, 20 ml each time, combine the ethyl acetate solutions, evaporate to dryness, add 1 ml of methanol to the residue to dissolve it, and use it as the test solution.

[0056] Preparation of control medicinal solution: Take 1g of lotus stamen control medicinal material, add 50ml of water, heat and reflux for 1 hour, filter, concentrate the filtrate to near dryness, add 20ml of methanol to the residue, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, add 20ml of water to the residue to dissolve it, shake and extract with ethyl acetate twice, 20ml each time, combine the ethyl acetate solutions, evaporate to dryness, add 1ml of methanol to the residue to dissolve it, and use it as the control medicinal solution.

[0057] Different development systems: 1) The thin layer developer under the lotus stamen item in the 2020 edition of the "Chinese Pharmacopoeia": cyclohexane-ethyl acetate-anhydrous formic acid (9:3:0.7), the color developer is 10% sulfuric acid ethanol solution; 2) methanol-glacial acetic acid-water (12:1:1), the color developer is 3% aluminum chloride ethanol solution; 3) The developer of the present invention: n-butanol-acetone-glacial acetic acid-water (2:2:1:7), the color developer is 3% aluminum chloride ethanol solution. Absorb the test solution of the lotus stamen formula granules prepared by the above method three and the control medicinal material solution prepared by method one, spot them on the polyamide film, develop them according to the above different thin layer identification conditions, spray them with the specified color developer, and inspect them under ultraviolet light (365nm).

[0058] Result analysis: Figure 7 ① to ③ correspond to three different development systems. ① is the developing agent for thin-layer identification of lotus stamens in the 2020 edition of the Chinese Pharmacopoeia, but the results show that when this developing agent system is applied to formula granules, there are fewer thin-layer spots and poor separation, so it is not suitable for identification of lotus stamens formula granules; at the same time, when the developing system in ② is used, the separation is poor. When ③, i.e., the developing agent system in the method of the present invention, is used, the separation of the test sample of the formula granules is good, the spot information is rich and corresponds one to one with the control medicinal materials.

[0059] Comparative Example 2:

[0060] This comparative example uses lotus root formula particles to compare different proportions of the developing agent. The specific process is as follows:

[0061] Preparation of the test solution: Take 0.5 g of lotus root granules, grind them into powder, add 20 ml of methanol, treat with ultrasound for 30 minutes, filter, evaporate the filtrate to dryness, add 20 ml of water to the residue to dissolve it, shake and extract with ethyl acetate twice, 20 ml each time, combine the ethyl acetate solutions, evaporate to dryness, add 1 ml of methanol to the residue to dissolve it, and use it as the test solution.

[0062] Preparation of control medicinal solution: Take 1g of lotus stamen control medicinal material, add 50ml of water, heat and reflux for 1 hour, filter, concentrate the filtrate to near dryness, add 20ml of methanol to the residue, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, add 20ml of water to the residue to dissolve it, shake and extract with ethyl acetate twice, 20ml each time, combine the ethyl acetate solutions, evaporate to dryness, add 1ml of methanol to the residue to dissolve it, and use it as the control medicinal solution.

[0063] Different developing agent ratios: 1) n-butanol-acetone-glacial acetic acid-water (2:2:1:10); 2) n-butanol-acetone-glacial acetic acid-water (2:2:1:8); 3) n-butanol-acetone-glacial acetic acid-water (2:2:1:7); 4) n-butanol-acetone-glacial acetic acid-water (2:2:1:6), the lotus root formula granule test solution prepared by the above method 3 and the control medicinal material solution prepared by method 1 are taken, spotted on the polyamide film, developed according to the above different thin layer identification conditions, sprayed with the specified developer, and inspected under ultraviolet light (365nm),

[0064] Result analysis: Figure 8 ①~④ correspond to 4 different developing agent ratios. When using developing agent ratio ①, there are fewer thin layer spots and poor separation; when using medium developing agent ratio ②, the separation is poor. When using medium developing agent ratio ③, the separation of the formula granules is good, the spot information is rich and corresponds to the control medicinal materials one by one, and the Rf value is moderate; when using developing agent ratio ④, the chromatographic spots have overlapping tailing phenomenon.

[0065] Comparative Example 3:

[0066] This comparative example compares different developing agent systems, and at the same time compares and examines the preparation methods of the test sample and the control medicinal material solution under the method item of the Chinese Pharmacopoeia under the developing agent of the present invention, and the developing agent used for the detection of lotus root formula granules. The specific process is as follows:

[0067] (1) Preparation method of different lotus stamen formula granules as test samples: 1) Take 0.5 g of lotus stamen formula granules, grind them into powder, add 20 ml of methanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, add 1 ml of methanol to the residue to dissolve it, and use it as the test sample solution.

[0068] 2) Take 0.5 g of lotus root granules, grind them into powder, add 20 ml of water to dissolve them, shake and extract with ethyl acetate twice, 20 ml each time, combine the ethyl acetate solutions, evaporate to dryness, add 1 ml of methanol to dissolve the residue, and use it as the test solution.

[0069] 3) Take 0.5 g of lotus root formula granules, grind them into powder, add 20 ml of methanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, add 20 ml of water to the residue to dissolve it, shake and extract with ethyl acetate twice, 20 ml each time, combine the ethyl acetate solutions, evaporate to dryness, add 1 ml of methanol to the residue to dissolve it, and use it as the test solution.

[0070] (2) Preparation method of different lotus stamen reference medicinal material solutions: S1: Take 1 g of lotus stamen reference medicinal material, add 50 ml of water, heat and reflux for 1 hour, filter, concentrate the filtrate to near dryness, add 20 ml of methanol to the residue, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, add 20 ml of water to the residue to dissolve it, shake and extract with ethyl acetate twice, 20 ml each time, combine the ethyl acetate solutions, evaporate to dryness, add 1 ml of methanol to the residue to dissolve it, and use it as the reference medicinal material solution.

[0071] S2: Take 1 g of lotus stamen reference medicinal material, add 20 ml of methanol, treat with ultrasound for 30 minutes, filter, evaporate the filtrate to dryness, add 1 ml of methanol to the residue to dissolve it, and use it as the reference medicinal material solution.

[0072] According to the thin layer chromatography method (General Rules 0502 of the 2020 edition of the Chinese Pharmacopoeia), 1 μL of each of the three test sample solutions and 2 μL of each of the two control medicinal material solutions were taken and spotted on the same polyamide thin layer plate, and developed with n-butanol-acetone-glacial acetic acid-water (2:2:1:7) as the developing agent. After development, the plate was taken out, dried, sprayed with 3% aluminum chloride ethanol solution, and examined under ultraviolet light (365 nm).

[0073] Result analysis: Fig. 9 1 to 3 are the chromatograms of the test samples of lotus stamen formula granules prepared by the three preparation methods, and S1 to S2 are the chromatograms of the lotus stamen reference medicinal materials prepared by the two preparation methods. When the lotus stamen formula granules were prepared by methods 1 and 2, the sample spots were blurred, and the spots were clear and round when method 3 was used; when the reference medicinal material was prepared by method 1, the spot separation was good, and at this time, the spots of the granules method 3 corresponded one to one.

[0074] Comprehensive analysis: The thin-layer identification method of lotus stamens and its preparation provided by the present invention is simple and fast in sample preparation operation. The developing agent in the present invention can be used for thin-layer identification of lotus stamens medicinal materials and thin-layer identification of lotus stamen preparations. The chromatographic spots are more abundant and the separation is good, which overcomes the problems of few spots of the sample and few spot information of the thin-layer spectrum of lotus stamen preparations in the existing thin-layer identification method. At the same time, aluminum chloride ethanol is used as a color developer, which is a color developer unique to flavonoid components. Therefore, the thin-layer identification method presents the flavonoid components unique to lotus stamens. At the same time, the method of the present invention uses a polyamide film as a thin-layer plate, which greatly reduces the amount of sample spotting, and can achieve the effect of rich spot information and good separation.

Claims

1. A thin layer identification method for lotus stamens or its preparation, characterized in that: The steps include: (1) taking a lotus stamen reference medicinal material, boiling it with water or heating it under reflux, filtering it, concentrating the filtrate to near dryness, ultrasonically treating it with an alcohol solvent, evaporating it to dryness, dissolving it with water, and then extracting it by shaking with ethyl acetate. After standing and stratifying, the ethyl acetate layers are combined, evaporated to dryness, and the residue is dissolved with an alcohol solvent to obtain a reference medicinal material solution; (2) taking lotus stamens and boiling them with water or heating them under reflux, filtering, concentrating the filtrate to near dryness, ultrasonically treating it with an alcohol solvent, evaporating it to dryness, dissolving it with water, and then extracting it by shaking with ethyl acetate. After standing and stratifying, the ethyl acetate layers are combined and evaporated to dryness. The residue is dissolved with an alcohol solvent to obtain a medicinal material test solution; Or, take a sample of lotus root formula granules or standard decoction, add an alcohol solvent to ultrasonically extract, filter, evaporate the filtrate to dryness, dissolve it in water, and then add ethyl acetate to shake and extract, let it stand for stratification, combine the ethyl acetate layer, evaporate to dryness, and dissolve the residue in an alcohol solvent to obtain a formula granule test solution or a standard decoction test solution; (3) Take the control medicinal material solution and the test sample solution and spot them on the same thin layer plate respectively, develop them with a developing agent, spray them with a color developer for color development, and examine them under an ultraviolet lamp to obtain an ultraviolet thin layer chromatogram for comparative identification; the developing agent is a mixed solution of n-butanol, acetone, glacial acetic acid and water, and the volume ratio of n-butanol, acetone, glacial acetic acid and water is 2:2:1:7; the spotting volume of the control medicinal material solution and the test sample solution is 1μl~4μl; the thin layer plate is a polyamide film.

2. The thin layer identification method of lotus stamen medicinal material or its preparation according to claim 1, characterized in that: When the lotus stalk control medicinal material is decocted with water in the step (1), or when the lotus stalk medicinal material is decocted with water in the step (2), the mass volume of the medicinal material and water is 1:50g / mL-200g / mL, and the decocting treatment time is 15min-60min.

3. The thin layer identification method of lotus stamen medicinal material or its preparation according to claim 1, characterized in that: In the step (2), when the lotus stamen formula granules and the standard decoction are extracted with an alcohol solvent, the mass volume of the lotus stamen formula granules, the standard decoction and the alcohol solvent is 1:40g / mL-200g / mL, the power of the ultrasonic treatment is 250-300W, the frequency is 40kHz, the time is 15min-60min, the number of ethyl acetate shaking extraction is 4 times, and the volume ratio of the ethyl acetate solution to the alcohol solution of the sample is 0.5:2-1.

4. The thin-layer identification method of lotus stamen medicinal material or its preparation according to claim 1, characterized in that: In the step (3), the wavelength of the ultraviolet lamp is 360-370nm, the color developer is 3% aluminum chloride ethanol solution, and after the color developer is sprayed on, it is naturally dried or blown dry with hot air.

5. The thin-layer identification method of lotus stamens or its preparation according to claim 1, characterized in that: The comparative identification of the thin layer chromatograms in step (3) refers to observing whether fluorescent spots of the same color appear at positions corresponding to those of the control medicinal material in the chromatogram of the test solution.

6. The thin-layer identification method of lotus stamen medicinal material or its preparation according to claim 1, characterized in that: The alcohol solvent includes methanol.

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