Thin-layer identification method of Speranskia tuberculata and its application

Abstract

The present invention provides a thin-layer identification method for Speranskia tuberculata and its application. The thin-layer identification method comprises the following steps: developing, coloring and inspecting the reference substance, reference medicinal material and test sample on a thin-layer plate. In the chromatogram of the test sample, spots of the same color are shown at the positions corresponding to the chromatograms of the reference substance and the reference medicinal material; in the thin-layer identification method, the developing agent comprises ethyl acetate and petroleum ether; the reference substance comprises β-sitosterol. In the present invention, the thin-layer identification method can clearly and efficiently separate and identify the active ingredients in Speranskia tuberculata, with a moderate Rf value; and can accurately enrich and purify the reference substance, and has a simple process, does not require the use of toxic reagents, and has a low cost. It can effectively identify the Speranskia tuberculata medicinal material, its standard decoction and its formula granules, and has good specificity and good durability for temperature and humidity. It can be effectively identified using different thin-layer plates.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a thin-layer identification method of Speranskia tuberculata and its application. Background Art

[0002] Speranskia tuberculata was first recorded in "Ben Cao Yuan Shi", which states: "The seedlings of Speranskia tuberculata grow in the fields in spring, about one foot high, with round stems and serrated leaf tips. In summer, it grows three or four spikes, with yellow flowers, and the fruits are triangular, similar to castor beans. It is collected in May." This describes the plant morphology of Speranskia tuberculata, which is consistent with the current "Speranskia tuberculata" (i.e., Speranskia tuberculata Bunge), and it is one of the mainstream varieties of traditional medicinal Speranskia tuberculata. Speranskia tuberculata is the dried whole herb of the plant Speranskia tuberculata Bunge of the Euphorbiaceae family. It is warm in nature, pungent and bitter in taste, and belongs to the liver meridian. It has the effects of dispelling wind-dampness, relaxing tendons, promoting blood circulation, relieving pain, and detoxifying. It is used for rheumatic pain, muscle and bone spasms, limb numbness, sores and ulcers, skin diseases, scrotal eczema and other symptoms. Its chemical components include pyrimidine compounds, organic acids, volatile oil compounds, sterols, etc.

[0003] As a commonly used medicinal material in Shanxi, Speranskia tuberculata is currently included in the "Shanxi Chinese Medicinal Materials Standard" (2011 edition). The quality control mainly focuses on inspection items such as morphological identification, microscopic identification, and extract content, and there are no relevant index components to identify and evaluate its quality. In addition, in the existing technology, the sample preparation methods for Speranskia tuberculata drugs mostly use vacuum recovery, heating under reflux, Soxhlet extraction, and extraction, etc. The methods are complex and the extraction efficiency is low; when performing thin-layer identification, the developing solvents mostly use chloroform, toluene, etc., but chloroform, toluene, etc. are drugs and explosives precursors with great harm; in addition, the control of Speranskia tuberculata drugs mostly uses reference crude drugs, and there is no reference substance of active ingredients as a control.

[0004] In addition, in recent years, plant sterols have received extensive attention due to their safety and effective anti-inflammatory activity. β-sitosterol belongs to tetracyclic triterpenoid compounds and is a plant steroid with a chemical structure similar to mammalian cholesterol. It has various biological activities such as cholesterol-lowering, blood sugar-lowering, antioxidant, anti-inflammatory, antibacterial, and hormone-like functions, and has become a research hotspot in various fields of life science, especially in the medical field. And β-sitosterol is also an active ingredient in Speranskia tuberculata. Therefore, it is also of great significance to efficiently recover and enrich β-sitosterol in Speranskia tuberculata drugs.

[0005] For example, CN 103105450 A discloses a thin-layer identification method for a formula granule of Speranskia tuberculata, using toluene-ethyl acetate-formic acid as the developing agent to develop and identify the test sample and the reference crude drug; the developing agent of the thin-layer identification method includes toluene, which has great harm, poor separation effect, and is not suitable for the enrichment of β-sitosterol.

[0006] Therefore, it is an urgent problem to be solved in this field to develop a thin-layer identification method with high sample preparation efficiency, good separation effect, good universality, which can efficiently identify the active ingredients in Herba Phrymae Leptostachyae, its standard decoction and formula granules, accurately recover and enrich β-sitosterol, and has a simple process, low cost, non-toxic and environmental protection. Summary of the Invention

[0007] Aiming at the deficiencies of the existing technology, the purpose of the present invention is to provide a thin-layer identification method and its application of Herba Phrymae Leptostachyae. The thin-layer identification method can clearly and efficiently separate and identify the active ingredients in Herba Phrymae Leptostachyae, with a moderate Rf value; and can accurately enrich and purify the reference substance, and has a simple process, does not require the use of toxic reagents, and has a low cost. It can effectively identify Herba Phrymae Leptostachyae herbs, standard decoctions and their formula granules, has good specificity, has good durability for temperature and humidity, and can be effectively identified using different thin-layer plates.

[0008] To achieve this purpose, the present invention adopts the following technical solutions:

[0009] In the first aspect, the present invention provides a thin-layer identification method of Herba Phrymae Leptostachyae, and the thin-layer identification method includes the following steps:

[0010] Develop and color the reference substance, reference herb and test sample on a thin-layer plate, and examine. In the chromatogram of the test sample, at the positions corresponding to the chromatograms of the reference substance and the reference herb, spots of the same color are shown; in the thin-layer identification method, the developing agent includes ethyl acetate and petroleum ether; the reference substance includes β-sitosterol.

[0011] In the present invention, on the basis of using the reference herb as a reference, the thin-layer identification method adds an active ingredient reference substance as a control, and at the same time adopts a specific developing agent, which has a stronger elution ability for β-sitosterol, is more conducive to its development, has a good separation effect, clear thin-layer chromatogram spots, and a moderate Rf value, realizing the efficient identification of the active ingredients in Herba Phrymae Leptostachyae; the identification method has good repeatability and specificity, is not affected by the environment, and can be effectively identified at different temperatures and humidities; and can accurately enrich and purify the reference substance, has good commercial value; the process is simple, does not require the use of toxic reagents, has a low cost, has good universality, and can effectively identify Herba Phrymae Leptostachyae herbs, standard decoctions and their formula granules.

[0012] Preferably, the volume ratio of ethyl acetate to petroleum ether is 1:(0.25 - 6), for example, it can be 1:0.25, 1:0.3, 1:0.5, 1:0.8, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.1, 1:2.2, 1:2.25, 1:2.3, 1:2.4, 1:2.5, 1:2.8, 1:3.2, 1:3.6, 1:4, 1:4.2, 1:4.4, 1:4.6, 1:4.8, 1:5, 1:5.2, 1:5.6, 1:6, etc.; more preferably 1:(1.5 - 2.2).

[0013] In the present invention, within the above - defined range of the volume ratio of ethyl acetate to petroleum ether, the elution ability for β - sitosterol is stronger and the development effect is better, thereby improving the separation efficiency, making the thin - layer chromatography clearer and the identification efficiency higher. If it is not within the above - defined range, it will affect the polarity of the developing agent, resulting in the polarity of the developing agent being too large or too small, and both too large and too small polarities are not conducive to spot separation, thus affecting the identification result.

[0014] Preferably, the boiling range of the petroleum ether is 60 - 90 °C.

[0015] Preferably, before the development of the thin - layer plate, it further includes preparing a reference substance solution, a reference crude drug solution and a test sample solution respectively.

[0016] Preferably, the preparation method of the reference substance solution includes: mixing the reference substance with a first solvent to obtain the reference substance solution.

[0017] Preferably, the first solvent includes an aqueous methanol solution.

[0018] Preferably, the volume concentration of the aqueous methanol solution is 80 - 100%, for example, it can be 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 100%, etc.

[0019] In the present invention, a volume concentration of 100% means that the solvent is methanol.

[0020] Preferably, the concentration of the reference substance solution is 0.1 - 4 mg / mL, for example, it can be 0.1 mg / mL, 0.2 mg / mL, 0.4 mg / mL, 0.6 mg / mL, 0.8 mg / mL, 1 mg / mL, 1.2 mg / mL, 1.4 mg / mL, 1.6 mg / mL, 1.8 mg / mL, 2 mg / mL, 2.2 mg / mL, 2.4 mg / mL, 2.6 mg / mL, 2.8 mg / mL, 3 mg / mL, 3.2 mg / mL, 3.4 mg / mL, 3.6 mg / mL, 3.8 mg / mL, 4 mg / mL, etc.

[0021] Preferably, the preparation method of the reference crude drug solution includes: mixing the reference crude drug with a second solvent to obtain the reference crude drug solution.

[0022] Preferably, the reference crude drug includes Speranskia tuberculata.

[0023] Preferably, the second solvent includes methanol.

[0024] Preferably, the mass - to - volume ratio of the reference crude drug to the second solvent is (0.5 - 6):25 g / mL, where the specific values in (0.5 - 6) can be, for example, 0.5, 0.6, 0.8, 1, 1.2, 1.4, 1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.2, 4.4, 4.6, 4.8, 5, 5.2, 5.4, 5.6, 5.8, 6, etc.; more preferably (1 - 3):25 g / mL.

[0025] Preferably, the mixing of the reference crude drug and the second solvent is carried out under ultrasonic conditions.

[0026] Preferably, the ultrasonic time is 15 - 45 min, for example, it can be 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, etc.; more preferably 25 - 35 min.

[0027] Preferably, after the mixing, it further includes the steps of filtration and / or concentration.

[0028] Preferably, the preparation method of the test solution includes: mixing Speranskia tuberculata with a third solvent and performing ultrasonic extraction to obtain the test solution.

[0029] Preferably, the Speranskia tuberculata includes Speranskia tuberculata crude drug, freeze - dried powder of standard decoction of Speranskia tuberculata, or formula granules of Speranskia tuberculata.

[0030] Preferably, the third solvent includes methanol and / or ethyl acetate, preferably methanol.

[0031] Preferably, the mass-volume ratio of the pearl phryma leptostachya subsp. asiatica to the third solvent is (0.5 - 6):25 g / mL. The specific values in (0.5 - 6) can be, for example, 0.5, 0.6, 0.8, 1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.2, 4.4, 4.6, 4.8, 5, 5.2, 5.4, 5.6, 5.8, 6, etc.; more preferably (1.2 - 2.2):25 g / mL.

[0032] In the present invention, when the mass-volume ratio of the pearl phryma leptostachya subsp. asiatica to the third solvent is within the above-defined range, the third solvent has a better extraction effect on the pearl phryma leptostachya subsp. asiatica, and thus it is easier to separate and identify the active ingredients in the pearl phryma leptostachya subsp. asiatica.

[0033] Preferably, the time for ultrasonic extraction is 15 - 45 min, for example, it can be 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, etc.; more preferably 25 - 35 min.

[0034] Preferably, after the ultrasonic extraction, steps of filtration and / or concentration are further included.

[0035] In the present invention, using methanol as the extraction agent, and with the methanol and pearl phryma leptostachya subsp. asiatica in a specific ratio, combined with ultrasonic extraction, preparing the test solution is more conducive to subsequent thin-layer identification, can improve the thin-layer identification efficiency, and has simple sample preparation, less sample loss, low cost, and is safe and environmentally friendly.

[0036] Preferably, the spotting amounts of the reference substance, reference crude drug, and test sample are 1 - 25 μL, for example, it can be 1 μL, 2 μL, 3 μL, 4 μL, 5 μL, 6 μL, 7 μL, 8 μL, 9 μL, 10 μL, 11 μL, 12 μL, 13 μL, 14 μL, 15 μL, 16 μL, 17 μL, 18 μL, 19 μL, 20 μL, 21 μL, 22 μL, 23 μL, 24 μL, 25 μL, etc.; more preferably 10 - 20 μL.

[0037] Preferably, the spotting concentration of the test sample is 0.2 - 0.8 g / mL, for example, it can be 0.2 g / mL, 0.25 g / mL, 0.3 g / mL, 0.35 g / mL, 0.4 g / mL, 0.45 g / mL, 0.5 g / mL, 0.55 g / mL, 0.6 g / mL, 0.65 g / mL, 0.7 g / mL, 0.75 g / mL, 0.8 g / mL, etc.

[0038] In the present invention, when the spotting concentration and spotting volume of the test sample are within the above-defined ranges, the thin-layer chromatography spots are clearer and the resolution is better.

[0039] Preferably, the developer for color development includes a sulfuric acid ethanol solution.

[0040] Preferably, the concentration of the sulfuric acid ethanol solution is 5-15%, and for example, it can be 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, etc.

[0041] In the present invention, the concentration of the sulfuric acid ethanol solution refers to the mass-volume ratio; when the developer is a sulfuric acid ethanol solution with a specific concentration, it can increase the spot information and is easy to observe after color development.

[0042] Preferably, the color development is carried out under heating conditions.

[0043] Preferably, the heating temperature is 100-110°C, and for example, it can be 100°C, 101°C, 102°C, 103°C, 104°C, 105°C, 106°C, 107°C, 108°C, 109°C, 110°C, etc.; the heating time is 3-5 min, and for example, it can be 3 min, 4 min, 5 min, etc.

[0044] Preferably, the inspection is carried out under the irradiation of an ultraviolet lamp.

[0045] Preferably, the wavelength of the ultraviolet lamp is 360-370 nm, and for example, it can be 360 nm, 362 nm, 365 nm, 368 nm, 370 nm, etc.

[0046] As a preferred technical solution of the present invention, the thin-layer identification method includes:

[0047] (1) Mix β-sitosterol with an aqueous methanol solution to obtain a reference substance solution; mix the reference crude drug with methanol at a mass-volume ratio of (0.5-6):25 g / mL, ultrasonicate for 15-45 min, filter and concentrate to obtain a reference crude drug solution; mix the Herba Phrymae Leptostachyae with a third solvent at a mass-volume ratio of (0.5-6):25 g / mL, ultrasonically extract for 15-45 min, filter and concentrate to obtain a test sample solution; the third solvent includes methanol and / or ethyl acetate;

[0048] (2) Spot the reference substance solution, reference crude drug solution, and test sample solution obtained in step (1) on the same thin-layer plate respectively, develop with ethyl acetate and petroleum ether with a volume ratio of 1:(0.25 - 6) as the developing agent, then spray the surface of the thin-layer plate with a sulfuric acid ethanol solution with a concentration of 5 - 15%, heat at 100 - 110°C for 3 - 5 min until the spots are clearly developed, and examine under an ultraviolet lamp. In the test sample chromatogram, at the positions corresponding to the reference substance chromatogram and the reference crude drug chromatogram, spots of the same color are shown.

[0049] In the present invention, the thin-layer plate includes a silica gel G thin-layer plate.

[0050] In the second aspect, the present invention provides an application of the thin-layer identification method as described in the first aspect in analyzing the active ingredients in Speranskia tuberculata (Bunge) Baill.

[0051] The numerical ranges described in the present invention not only include the above-listed point values, but also any point values between the above numerical ranges not listed. Due to space limitations and for the sake of brevity, the present invention does not exhaustively list the specific point values included in the ranges.

[0052] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0053] (1) The thin-layer identification method provided by the present invention uses the reference substance of the active ingredient β-sitosterol as a control. It can not only accurately identify the β-sitosterol active ingredient in Speranskia tuberculata (Bunge) Baill., but also enrich, separate, and purify it at the corresponding Rf position on the silica gel plate, having good commercial value;

[0054] (2) The thin-layer identification method provided by the present invention can quickly and effectively identify the active ingredients in Speranskia tuberculata (Bunge) Baill., and has good repeatability and specificity. It has good durability for temperature and humidity, can be effectively identified using different thin-layer plates, greatly reduces the identification cost, improves the identification efficiency, and is of great significance for the identification of traditional Chinese medicines and formula granules containing the same substance components, having good application prospects;

[0055] (3) The thin-layer identification method provided by the present invention has high safety, low cost, and simple process. Description of the Drawings

[0056] Figure 1 It is the thin-layer chromatogram obtained by Method 1 in Test Example 1 under the condition of no color developer;

[0057] Figure 2 It is the thin-layer chromatogram obtained by Method 1 in Test Example 1 under the condition that the color developer is 5% aluminum trichloride solution;

[0058] Figure 3It is the thin-layer chromatogram obtained by Method 1 in Test Example 1 under the condition that the developer is 10% sulfuric acid ethanol solution;

[0059] Figure 4 It is the thin-layer chromatogram obtained by Method 2 in Test Example 1 under the condition of no developer;

[0060] Figure 5 It is the thin-layer chromatogram obtained by Method 2 in Test Example 1 under the condition that the developer is 5% aluminum trichloride solution;

[0061] Figure 6 It is the thin-layer chromatogram obtained by Method 2 in Test Example 1 under the condition that the developer is 10% sulfuric acid ethanol solution;

[0062] Figure 7 It is the thin-layer chromatogram obtained by Method 3 in Test Example 1 under the condition of no developer;

[0063] Figure 8 It is the thin-layer chromatogram obtained by Method 3 in Test Example 1 under the condition that the developer is 5% aluminum trichloride solution;

[0064] Figure 9 It is the thin-layer chromatogram obtained by Method 3 in Test Example 1 under the condition that the developer is 10% sulfuric acid ethanol solution;

[0065] Figure 10 It is the thin-layer chromatogram of the reference substance β-sitosterol solution, the reference crude drug solution and the test solution under the condition that the volume ratio of ethyl acetate to petroleum ether is 1:2.5;

[0066] Figure 11 It is the thin-layer chromatogram of the reference substance β-sitosterol solution, the reference crude drug solution and the test solution under the condition that the volume ratio of ethyl acetate to petroleum ether is 1:1.2;

[0067] Figure 12 It is the thin-layer chromatogram under different extraction solvents in Test Example 2;

[0068] Figure 13 It is the thin-layer chromatogram with different sample application amounts for the test solution prepared with methanol and ethyl acetate;

[0069] Figure 14 It is the thin-layer chromatogram of the test solutions of different concentrations of Phryma leptostachya L. var. asiatica Hara in Test Example 3;

[0070] Figure 15 It is the thin-layer chromatogram with different sample application amounts of the test solutions in Test Example 4;

[0071] Figure 16 It is the thin-layer chromatogram for the specificity investigation in Test Example 5;

[0072] Figure 17The thin-layer chromatogram at a temperature of 6.5 °C and a RH of 66% for Test Example 6;

[0073] Figure 18 The thin-layer chromatogram at a temperature of 28.4 °C and a RH of 28% for Test Example 6;

[0074] Figure 19 The thin-layer chromatogram at a temperature of 28.8 °C and a RH of 88% for Test Example 6;

[0075] Figure 20 The thin-layer chromatogram with the thin-layer plate No. 20200707 for Test Example 6;

[0076] Figure 21 The thin-layer chromatogram with the thin-layer plate No. 1.05553.0001 for Test Example 6;

[0077] Figure 22 The thin-layer chromatogram with the thin-layer plate No. 20190307 for Test Example 6;

[0078] Figure 23 The thin-layer chromatogram of the freeze-dried powder of Zhenzhu Tougucao Biaotang;

[0079] Figure 24 The thin-layer chromatogram of the medicinal material of Zhenzhu Tougucao;

[0080] Figure 25 The thin-layer chromatogram of the formula granules of Zhenzhu Tougucao. Detailed implementation manners

[0081] The technical solution of the present invention will be further described below through specific implementation manners. Those skilled in the art should understand that the embodiments are only for helping to understand the present invention and should not be regarded as specific limitations on the present invention.

[0082] The instruments and materials used in the present invention are as follows:

[0083] Instruments: Good Look-1000 thin-layer imaging system (Shanghai Kezhe Biochemical Technology Co., Ltd.), JJ500 electronic balance (Changshu Shuangjie Testing Instrument Factory), MSA6.6S-OCE-DM electronic balance (Sartorius Scientific Instruments (Beijing) Co., Ltd.), DK-S26 constant temperature water bath (Zhongyi Guoke (Beijing) Technology Development Co., Ltd.), KQ-100DE ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.), silica gel G plate (Merck Chemical Technology Co., Ltd., Qingdao Marine Chemical Factory);

[0084] Materials: β-sitosterol, ferulic acid, myricetin, rutin, caffeic acid, luteolin, quercetin, kaempferol, vanillic acid.

[0085] Medicinal material of Zhenzhu Tougucao: 20210506-15;

[0086] Pearl Tougucao Standard Decoction Freeze-dried Powder: 20210506-1, 20210506-2, 20210506-3, 20210506-4, 20210506-5, 20210506-6, 20210506-7, 20210506-8, 20210506-9, 20210506-10, 20210506-11, 20210506-12, 20210506-13, 20210506-14, 20210506-15;

[0087] β-sitosterol reference substance (National Institutes for Food and Drug Control, batch number: 110851-201909, purity: 92.7%);

[0088] Ferulic acid reference substance (National Institutes for Food and Drug Control, batch number: 110773-201614, purity: 99.0%);

[0089] Myricetin reference substance (Chengdu Efa Biotechnology Co., Ltd., batch number: AF20081652, purity: 98.0%);

[0090] Rutin reference substance (National Institutes for Food and Drug Control, batch number: 100080-201811, purity: 92.4%);

[0091] Caffeic acid reference substance (National Institutes for Food and Drug Control, batch number: 110885-200102, purity: 99.7%);

[0092] Luteolin reference substance (National Institutes for Food and Drug Control, batch number: 111520-201605, purity: 99.6%);

[0093] Quercetin reference substance (National Institutes for Food and Drug Control, batch number: 100081-201610, purity: 99.6%);

[0094] Kaempferol reference substance (National Institutes for Food and Drug Control, batch number: 110861-201611, purity: 95.5%);

[0095] Vanillic acid reference substance (National Institutes for Food and Drug Control, batch number: 110776-201503, purity: 99.8%);

[0096] Reagents: Methanol, ethanol, ethyl acetate, chloroform, petroleum ether (60-90 °C), formic acid, toluene, butyl acetate, sulfuric acid, aluminum trichloride are of analytical grade.

[0097] Example 1

[0098] This example provides a thin-layer identification method for Speranskia tuberculata, which specifically includes the following steps:

[0099] (1) Mix 0.5 mg of β-sitosterol reference substance with 1 mL of methanol to obtain a reference substance solution; mix 1.5 g of Speranskia tuberculata reference medicinal material with 25 mL of methanol, ultrasonicate for 30 min, filter, and concentrate the filtrate to 3 mL to obtain a reference medicinal material solution; mix 1.5 g of Speranskia tuberculata test sample with 25 mL of methanol, ultrasonically extract for 30 min, filter, and concentrate the filtrate to 3 mL to obtain a test sample solution.

[0100] (2) Take 15 μL each of the reference substance solution, reference medicinal material solution, and test sample solution obtained in step (1) and spot them on the same silica gel G thin-layer plate respectively. Use ethyl acetate and petroleum ether (60 - 90 °C) with a volume ratio of 1:2 as the developing agent for development. Subsequently, spray a 10% sulfuric acid ethanol solution on the surface of the thin-layer plate, heat at 105 °C for 4 min until the spots are clearly developed, and place it under a 365 nm ultraviolet lamp for inspection. In the test sample chromatogram, at the positions corresponding to the reference substance chromatogram and the reference medicinal material chromatogram, spots of the same color are shown, thereby identifying the active ingredients in Speranskia tuberculata.

[0101] Example 2

[0102] This example provides a thin-layer identification method for Speranskia tuberculata, which specifically includes the following steps:

[0103] (1) Mix 0.75 mg of β-sitosterol reference substance with 1 mL of 90% methanol aqueous solution to obtain a reference substance solution; mix 1.2 g of Speranskia tuberculata reference medicinal material with 25 mL of methanol, ultrasonicate for 30 min, filter, and concentrate the filtrate to 1.6 mL to obtain a reference medicinal material solution; mix 1.2 g of Speranskia tuberculata test sample with 25 mL of methanol, ultrasonically extract for 30 min, filter, and concentrate the filtrate to 1.6 mL to obtain a test sample solution.

[0104] (2) Take 18 μL each of the reference substance solution, reference medicinal material solution, and test sample solution obtained in step (1) and spot them on the same silica gel G thin-layer plate respectively. Use ethyl acetate and petroleum ether (60 - 90 °C) with a volume ratio of 1:1.8 as the developing agent for development. Subsequently, spray a 10% sulfuric acid ethanol solution on the surface of the thin-layer plate, heat at 100 °C for 5 min until the spots are clearly developed, and place it under a 365 nm ultraviolet lamp for inspection. In the test sample chromatogram, at the positions corresponding to the reference substance chromatogram and the reference medicinal material chromatogram, spots of the same color are shown, thereby identifying the active ingredients in Speranskia tuberculata.

[0105] Example 3

[0106] This embodiment provides a thin-layer identification method for Speranskia tuberculata, which specifically includes the following steps:

[0107] (1) Mix 0.6 mg of β-sitosterol reference substance with 1 mL of methanol to obtain a reference substance solution; mix 1.8 g of the reference medicinal material of Speranskia tuberculata with 25 mL of methanol, ultrasonically extract for 30 min, filter, and concentrate the filtrate to 3 mL to obtain a reference medicinal material solution; mix 1.8 g of the test sample of Speranskia tuberculata with 25 mL of methanol, ultrasonically extract for 30 min, filter, and concentrate the filtrate to 3 mL to obtain a test sample solution;

[0108] (2) Take 18 μL each of the reference substance solution, reference medicinal material solution, and test sample solution obtained in step (1) and spot them on the same silica gel G thin-layer plate. Use ethyl acetate and petroleum ether (60 - 90 °C) with a volume ratio of 1:2.1 as the developing agent for development. Subsequently, spray a 10% sulfuric acid ethanol solution on the surface of the thin-layer plate, heat at 105 °C for 3 min until the spots are clearly visible, and view under a 365 nm ultraviolet lamp. In the test sample chromatogram, at the positions corresponding to the reference substance chromatogram and the reference medicinal material chromatogram, spots of the same color are shown, thereby identifying the active ingredients in Speranskia tuberculata.

[0109] Based on the thin-layer identification method provided in Example 1, the following tests are carried out:

[0110] Test Example 1

[0111] Compare the thin-layer identification effects of different reference substances, developing agents, and color-developing agents on the test sample of Speranskia tuberculata. The method is as follows:

[0112] Method 1: The same as Example 1, observe the thin-layer chromatograms under the conditions of no color-developing agent, color-developing agent being 5% aluminum trichloride solution, and color-developing agent being 10% sulfuric acid ethanol solution. The results are as Figure 1 (no color-developing agent), Figure 2 (5% aluminum trichloride solution), Figure 3 (10% sulfuric acid ethanol solution) shown;

[0113] Method 2: The difference from Example 1 is only that the upper layer solution of toluene - ethyl acetate - formic acid (volume ratio of 3.5:1.2:1.5) is used as the developing agent for development. Observe the thin-layer chromatograms under the conditions of no color-developing agent, color-developing agent being 5% aluminum trichloride solution, and color-developing agent being 10% sulfuric acid ethanol solution. The results are as Figure 4 (no color-developing agent), Figure 5 (5% aluminum trichloride solution), Figure 6 (10% sulfuric acid ethanol solution) shown;

[0114] Method 3: The difference from Example 1 is only that chloroform-methanol (volume ratio 30:1) is used as the developing agent for development, and the thin-layer chromatograms are observed under the conditions of no developer, 5% aluminum trichloride solution as the developer, and 10% sulfuric acid-ethanol solution as the developer. The results are as Figure 7 (no developer), Figure 8 (5% aluminum trichloride solution), Figure 9 (10% sulfuric acid-ethanol solution) as shown.

[0115] Using the above three methods, with different reference substances and reference crude drugs as references, the active ingredients in the test sample of Speranskia tuberculata were identified. Among them, in thin-layer chromatography, from left to right, they are: 1-rutin reference substance solution; 2-ferulic acid reference substance solution; 3-myricetin reference substance solution; 4-caffeic acid reference substance solution; 5-β-sitosterol reference substance solution; 6-luteolin reference substance solution; 7-quercetin reference substance solution; 8-kaempferol reference substance solution; 9-vanillic acid reference substance solution; 10-freeze-dried powder test sample solution of Speranskia tuberculata; 11-reference crude drug solution of Speranskia tuberculata.

[0116] It can be Figures 1 - 9 seen that using β-sitosterol as the reference substance solution and ethyl acetate-petroleum ether (60-90 °C) (1:2) as the developing agent for development, it can be clearly seen that the test sample solution shows spots of the same color as the reference substance solution and the reference crude drug solution at the corresponding positions, and the spots are clear, the Rf value meets the requirements, and the separation effect is good; when using the upper layer solution of toluene-ethyl acetate-formic acid (volume ratio 3.5:1.2:1.5) as the developing agent for development, although the reference substance solutions are all developed, there are no spots corresponding to the test sample solution, and the spots of the test sample solution and the reference crude drug solution are blurred; when using chloroform-methanol (volume ratio 30:1) as the developing agent for development, the test sample solution shows spots of the same color as the β-sitosterol reference substance solution and the reference crude drug solution at the corresponding positions, but the Rf value is larger, and the number of points corresponding to the reference crude drug solution is less than that when using ethyl acetate-petroleum ether (60-90 °C) (volume ratio 1:2) as the developing agent. Therefore, using ethyl acetate-petroleum ether (60-90 °C) (volume ratio 1:2) as the developing agent and β-sitosterol as the reference substance solution has a better separation and identification effect.

[0117] When investigating different developers, it can be Figures 1 - 3 , Figures 4 - 6 as well as Figures 7 - 9 all seen that there is no obvious change in the spot information before and after the development with 5% aluminum trichloride solution, and the spot information increases significantly after the development with 10% sulfuric acid-ethanol solution and is easy to observe. Therefore, 10% sulfuric acid-ethanol is selected as the developer, and the observation effect is better.

[0118] In addition, when ethyl acetate and petroleum ether with different volume ratios were used as the developing agents, the thin-layer chromatograms of the reference substance β-sitosterol, the reference medicinal material, and the test sample were observed (except for the different ratios of the developing agents, others were the same as in Example 1); the results were as Figure 10 (the volume ratio of ethyl acetate to petroleum ether was 1:2.5) and Figure 11 (the volume ratio of ethyl acetate to petroleum ether was 1:1.2) as shown; among them, Figure 10 、 11 in, from left to right in the thin-layer chromatogram were the reference substance β-sitosterol solution, the reference medicinal material solution, and the test sample solution; it can be seen that when the proportion of petroleum ether was too large, the spots were not well developed and the Rf value was small; when the proportion of petroleum ether was too small, the red spots in the reference medicinal material (i.e., the spots below the same position as the spots of the reference substance β-sitosterol solution) overlapped with the target spots, which was not conducive to observation.

[0119] Test Example 2

[0120] Compare the influence of different extraction solvents on the thin-layer chromatogram when preparing the test sample solution

[0121] The extraction solvents were water, 50% ethanol, ethanol, 50% methanol, methanol, and ethyl acetate respectively. Observe the separation effect of the thin-layer chromatogram. The results were as Figure 12 shown, among which, from left to right were: 1 - test sample solution (water); 2 - reference medicinal material solution (water); 3 - test sample solution (50% ethanol); 4 - reference medicinal material solution (50% ethanol); 5 - test sample solution (ethanol); 6 - reference medicinal material solution (ethanol); 7 - β-sitosterol reference substance solution; 8 - test sample solution (50% methanol); 9 - reference medicinal material solution (50% methanol); 10 - test sample solution (methanol); 11 - reference medicinal material solution (methanol); 12 - test sample solution (ethyl acetate); 13 - reference medicinal material solution (ethyl acetate).

[0122] It can be Figure 12 seen that when water, 50% ethanol, and 50% methanol were used as solvents, there was less spot information; when ethanol was used as the solvent, the separation effect of the spots was poor; when methanol and ethyl acetate were used as solvents, the chromatographic behaviors were consistent, but due to the small sample application amount, the observation was not comprehensive. Therefore, methanol and ethyl acetate were selected as solvents to increase the sample application amount and observe the spot color development situation. The chromatogram was as Figure 13 shown, among which, from left to right were: 1, 14: β-sitosterol reference substance solution; 2, 4, 6: test sample solution prepared with methanol (sample application amounts were 2 μL, 4 μL, 6 μL respectively); 3, 5, 7. reference medicinal material solution prepared with methanol (1 μL, 2 μL, 3 μL); 8, 10, 12, 15: test sample solution prepared with ethyl acetate (2 μL, 4 μL, 6 μL, 10 μL); 9, 11, 13: reference medicinal material solution prepared with ethyl acetate (1 μL, 2 μL, 3 μL).

[0123] As can be seen from Figure 13 it, when preparing the test solution with methanol and ethyl acetate respectively, and increasing the sample application amount, the permeability of both is good. Considering the safety of the experiment, methanol was selected as the extraction solvent.

[0124] Test Example 3

[0125] To compare the effects of different concentrations of the test solution of Speranskia tuberculata on thin-layer chromatography identification

[0126] When preparing the test solution, the weighed amounts were 0.5 g, 1 g, 1.5 g, and 2 g respectively. According to the method of Example 1, it was concentrated to 3 mL to obtain the test solutions of Speranskia tuberculata with concentrations of 0.17 g / mL, 0.33 g / mL, 0.5 g / mL, and 0.67 g / mL respectively. The thin-layer chromatogram is as shown in Figure 14 the figure. Among them, from left to right are 1. β-sitosterol reference substance solution; 2. reference crude drug solution of Speranskia tuberculata; 3-6. test solutions, and the concentrations of Speranskia tuberculata are 0.17 g / mL, 0.33 g / mL, 0.5 g / mL, and 0.67 g / mL respectively.

[0127] As can be seen from Figure 14 it, on the premise of ensuring the separation and identification effect and using less sampling amount, when the concentration of the test solution of Speranskia tuberculata is 0.5 g / mL, the spots are clear and the resolution is good.

[0128] Test Example 4

[0129] Investigation of sample application amount

[0130] To compare the effects of different sample application amounts of the test solution on the thin-layer identification method, the results are as shown in Figure 15 the figure. Among them, from left to right are 1. β-sitosterol reference substance solution; 2. reference crude drug solution of Speranskia tuberculata; 3-9. test solutions with different sample application amounts (8 μL, 10 μL, 12 μL, 15 μL, 18 μL, 20 μL, 22 μL in sequence).

[0131] As can be seen from Figure 15 it, on the premise of ensuring the separation and identification effect and using less sampling amount, when the sample application amount of the test solution is 15 μL, the thin-layer chromatogram spots are clear and the resolution is excellent.

[0132] Test Example 5

[0133] Investigation of specificity

[0134] Take the Speranskia tuberculata medicinal material (test sample 20210506-15) and prepare the test solution according to the method of Example 1. Spot the reference substance solution, test solution, reference crude drug solution of Speranskia tuberculata and methanol solution on the same silica gel G thin-layer plate respectively. Other thin-layer chromatography conditions are the same as those in Example 1. The results are as shown inFigure 16 As shown in the figure, from left to right are 1. methanol solution; 2. β-sitosterol reference substance solution; 3. reference crude drug solution of Phryma leptostachya L. var. asiatica Hara; 4. test solution.

[0135] It can be seen from Figure 16 that the test solution and the reference crude drug solution show spots of the same color at the corresponding positions in the thin-layer chromatography, and there are no spots in the methanol solution, indicating that this method has good specificity.

[0136] Test Example 6

[0137] Durability investigation

[0138] (1) Investigation under different temperature and humidity

[0139] The reference substance solution, the reference crude drug solution, and the test solution were respectively spotted on the same silica gel G thin-layer plate. After spotting, the thin-layer plate was taken, and this experiment was carried out by selecting 2 temperatures in the temperature environment of 4 - 35 °C; 2 humidities were selected in the humidity environments of 10 - 30% and 75% - 90% respectively, and other thin-layer chromatography conditions were the same as those in Example 1. The results are as Figure 17 (temperature is 6.5 °C, RH is 66%), Figure 18 (temperature is 28.4 °C, RH is 28%), Figure 19 (temperature is 28.8 °C, RH is 88%) as shown. Among them, from left to right are 1. β-sitosterol reference substance solution; 2. reference crude drug solution of Phryma leptostachya L. var. asiatica Hara; 3. test solution.

[0140] It can be seen from Figures 17 - 19 that under different temperature and humidity conditions, the spots of the test solution, the reference crude drug solution, and the reference substance solution are all relatively clear, and the separation effect of each spot in the test solution chromatogram is good, indicating that this thin-layer identification method has good durability to temperature and humidity.

[0141] (2) Investigation with different thin-layer plates

[0142] Silica gel G thin-layer plates produced by different manufacturers were respectively used and carried out according to the method of Example 1.

[0143] Trade name of the thin-layer plate: silica gel plate, manufacturer: Qingdao Ocean Chemical Industry Co., Ltd., specification: 100 * 100 mm, model: silica gel G plate, batch number: 20200707;

[0144] Trade name of the thin-layer plate: silica gel plate, manufacturer: Qingdao Ocean Chemical Industry Co., Ltd., specification: 50 * 50 mm, model: silica gel G plate, batch number: 20190307;

[0145] TLC plate trade name: silica gel plate, manufacturer: Merck Chemical Technology Co., Ltd., specification: 20*20cm, model: silica gel G plate, batch number: 1.05553.0001;

[0146] Sample application method: contact sample application;

[0147] Developing agent dosage: ethyl acetate - petroleum ether (60 - 90 °C) (1:2) as the developing agent;

[0148] Developing tank specification: double - groove;

[0149] Developing method and developing distance: ascending development, developing distance 8cm;

[0150] Saturation method: pre - equilibrate the TLC plate for 15 minutes.

[0151] The results are as Figure 20 (the TLC plate is 20200707), Figure 21 (the TLC plate is 1.05553.0001), Figure 22 (the TLC plate is 20190307) shown. Among them, in the thin - layer chromatography, from left to right are β - sitosterol reference substance solution; reference medicinal material solution of Phryma leptostachya Turcz. var. asiatica Hara; test solution.

[0152] It can be seen from Figures 20 - 22 that for silica gel G TLC plates produced by different manufacturers and different batches, the chromatograms of the test solutions obtained show that all spots can be well separated, indicating that this thin - layer identification method has good durability for TLC plates produced by different manufacturers.

[0153] Test example 7

[0154] Perform thin - layer identification tests on freeze - dried powder of standard decoction of Phryma leptostachya Turcz. var. asiatica Hara, medicinal materials of Phryma leptostachya Turcz. var. asiatica Hara, and formula granules of Phryma leptostachya Turcz. var. asiatica Hara in different batches. The method is the same as that in Example 1. The results are as Figure 23 (15 batches of freeze - dried powder of standard decoction of Phryma leptostachya Turcz. var. asiatica Hara), Figure 24 (15 batches of medicinal materials of Phryma leptostachya Turcz. var. asiatica Hara), Figure 25 (3 batches of formula granules of Phryma leptostachya Turcz. var. asiatica Hara) shown; among them, Figure 23From left to right are: 1. Solution of the reference crude drug of Speranskia tuberculata; 2. Solution of the reference substance of β-sitosterol; 3. Test solution (20210506-1); 4. Test solution (20210506-2); 5. Test solution (20210506-3); 6. Test solution (20210506-4); 7. Test solution (20210506-5); 8. Test solution (20210506-6); 9. Test solution (20210506-7); 10. Test solution (20210506-8); 11. Test solution (20210506-9); 12. Test solution (20210506-10); 13. Test solution (20210506-11); 14. Test solution (20210506-12); 15. Test solution (20210506-13); 16. Test solution (20210506-14); 17. Test solution (20210506-15). Figure 24 From left to right are: DP. Solution of the reference substance of β-sitosterol; 1. Test solution (20210506-1); 2. Test solution (20210506-2); 3. Test solution (20210506-3); 4. Test solution (20210506-4); 5. Test solution (20210506-5); 6. Test solution (20210506-6); 7. Test solution (20210506-7); 8. Test solution (20210506-8); 9. Test solution (20210506-9); 10. Test solution (20210506-10); 11. Test solution (20210506-11); 12. Test solution (20210506-12); 13. Test solution (20210506-13); 14. Test solution (20210506-14); 15. Test solution (20210506-15). Figure 25 From left to right are: 1. Solution of the reference substance of β-sitosterol; 2. Solution of the reference crude drug of Speranskia tuberculata; 3-5. Test solutions (batch numbers of the formula granules of Speranskia tuberculata are: KL20210506-03, KL20210506-08, KL20210506-09).

[0155] It can be seen from Figures 23 - 25 that when the thin-layer identification method provided by the present invention is used to identify the freeze-dried powder of the standard decoction of Speranskia tuberculata, the medicinal material of Speranskia tuberculata, and the formula granules of Speranskia tuberculata, it has clear spots, good resolution, and moderate Rf values. The spots of the samples and the reference substances are clear and have good correspondence, meeting the requirements of the thin-layer identification method and having good universality.

[0156] In summary, the thin-layer identification method provided by the present invention uses β-sitosterol active ingredient reference substance as a control, and at the same time adopts a developing agent of specific types and ratios, which can clearly and efficiently separate and identify the active ingredients in the medicinal material of Phryma leptostachya L. subsp. asiatica Hara, with a moderate Rf value; and can accurately enrich and purify the reference substance, and the process is simple, without using toxic reagents and with low cost. It can effectively identify the medicinal material of Phryma leptostachya L. subsp. asiatica Hara, its standard decoction and its formula granules, and has good specificity, good durability to temperature and humidity, and can be effectively identified using different thin-layer plates.

[0157] The applicant declares that the above is only the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by any person skilled in the art within the technical scope disclosed by the present invention fall within the protection scope and the disclosure scope of the present invention.

Claims

1. A thin-layer identification method for Speranskia tuberculata, characterized in that, The thin-layer identification method includes the following steps: Develop, color, and examine the reference substance solution, reference crude drug solution, and test sample solution on a silica gel G thin-layer plate. In the chromatogram of the test sample solution, spots of the same color should be shown at the corresponding positions of the chromatograms of the reference substance solution and the reference crude drug solution; In the thin-layer identification method, the developing agent includes ethyl acetate and petroleum ether; the volume ratio of ethyl acetate to petroleum ether is 1:(1.5 - 2.2); the boiling range of the petroleum ether is 60 - 90 °C; The reference substance includes β-sitosterol; Before the development of the thin-layer plate, it also includes the preparation of the reference substance solution, reference crude drug solution, and test sample solution respectively; The preparation method of the test sample solution includes: mixing Speranskia tuberculata with a third solvent, methanol, at a mass-to-volume ratio of (1.2 - 2.2):25 g / mL, and performing ultrasonic extraction to obtain the test sample solution; The Speranskia tuberculata includes Speranskia tuberculata medicinal materials, freeze-dried powder of the standard decoction of Speranskia tuberculata, or formula granules of Speranskia tuberculata; The spotting volume of the reference substance solution, reference crude drug solution, and test sample solution is 10 - 20 μL; the spotting concentration of the test sample solution is 0.2 - 0.8 g / mL; The color-developing agent for color development includes sulfuric acid ethanol solution; the examination is carried out under ultraviolet lamp irradiation.

2. The thin-layer identification method according to claim 1, characterized in that, The preparation method of the reference substance solution includes: mixing the reference substance with a first solvent to obtain the reference substance solution.

3. The thin-layer identification method according to claim 2, characterized in that, The first solvent includes methanol aqueous solution.

4. The thin-layer identification method according to claim 3, characterized in that, The volume concentration of the methanol aqueous solution is 80 - 100%.

5. The thin-layer identification method according to claim 2, characterized in that, The concentration of the reference substance solution is 0.1 - 4 mg / mL.

6. The thin-layer identification method according to claim 1, characterized in that, The preparation method of the reference crude drug solution includes: mixing the reference crude drug with a second solvent to obtain the reference crude drug solution.

7. The thin-layer identification method according to claim 6, characterized in that, The reference crude drug includes Speranskia tuberculata.

8. The thin-layer identification method according to claim 6, characterized in that, The second solvent includes methanol.

9. The thin-layer identification method according to claim 6, characterized in that, The mass-to-volume ratio of the reference crude drug to the second solvent is (0.5 - 6):25 g / mL.

10. The thin-layer identification method according to claim 9, characterized in that, The mass-to-volume ratio of the reference crude drug to the second solvent is (1 - 3):25 g / mL.

11. The thin-layer identification method according to claim 6, characterized in that, The mixing of the reference crude drug and the second solvent is carried out under ultrasonic conditions.

12. The thin-layer identification method according to claim 11, characterized in that, The time of ultrasonic treatment is 15 - 45 min.

13. The thin-layer identification method according to claim 12, characterized in that, The time of ultrasonic treatment is 25 - 35 min.

14. The thin-layer identification method according to claim 6, characterized in that, After the mixing, it also includes the steps of filtration and / or concentration.

15. The thin-layer identification method according to claim 1, characterized in that, The time of ultrasonic extraction is 15 - 45 min.

16. The thin-layer identification method according to claim 15, wherein, The time of ultrasonic extraction is 25 - 35 min.

17. The thin-layer identification method according to claim 1, wherein, After the ultrasonic extraction, it also includes the steps of filtration and / or concentration.

18. The thin-layer identification method according to claim 1, wherein, The concentration of the sulfuric acid ethanol solution is 5 - 15%.

19. The thin-layer identification method according to claim 1, wherein, The color development is carried out under heating conditions.

20. The thin-layer identification method according to claim 19, wherein, The heating temperature is 100 - 110 °C, and the heating time is 3 - 5 min.

21. The thin-layer identification method according to claim 1, wherein, The wavelength of the ultraviolet lamp is 360 - 370 nm.

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