CN113252836A

CN113252836A - Thin-layer chromatography identification method for golden cypress and golden cypress wine roasted products

Info

Publication number: CN113252836A
Application number: CN202110544537.4A
Authority: CN
Inventors: 阮文辉; 叶松华; 王晓燕; 冯夏珍; 吴哲; 薛润苗
Original assignee: Shanxi Drug Review Center
Current assignee: Shanxi Drug Review Center
Priority date: 2021-05-19
Filing date: 2021-05-19
Publication date: 2021-08-13

Abstract

The invention discloses a thin-layer chromatography identification method for golden cypress and golden cypress wine-roasted products, which comprises the following steps: (1) grinding a sample to obtain medicinal material powder, mixing the medicinal material powder with a first solvent, and extracting by ultrasonic or heating reflux to obtain an extracting solution; (2) carrying out solid-liquid separation on the extracting solution obtained in the step (1), and adding a second solvent into the obtained liquid to prepare a test solution; (3) and (3) spotting the sample solution and the reference solution obtained in the step (2) on a silica gel plate, developing, taking out, drying and inspecting under an ultraviolet lamp. According to the method provided by the invention, after the development in the developing agent is finished, the observation is directly carried out at 365nm, so that the method is convenient and intuitive; solves the problems that the thin-layer plate needs to be placed in a saturated developing agent in ammonia vapor for development, and the thin potassium bismuth iodide test solution needs to be sprayed for color development after the development is finished, and the operation is complex in the prior art.

Description

Thin-layer chromatography identification method for golden cypress and golden cypress wine roasted products

Technical Field

The invention relates to the field of pharmaceutical analysis, and particularly relates to a thin-layer chromatography identification method for golden cypress and golden cypress wine-roasted products.

Background

Cortex Phellodendri is dried bark of Phellodendron chinense (Phellodendron chinense Schneid.) belonging to Rutaceae. It is commonly called Chuan Huang Bai. Bitter and cold in nature. It enters kidney and bladder meridians. Clearing heat and drying dampness, purging fire and removing steam, detoxicating and treating sore^[1-3]. The main producing areas mainly include Sichuan, Guizhou, Hubei and Yunnan, and the yield of Sichuan and Guizhou is the largest^[4]. Phellodendron bark: the plate shape or the shallow groove shape is different in length and width and 3-6mm in thickness. The outer surface is yellow brown or yellow brown, flat or with longitudinal grooves, and some skin hole marks and residual grey brown rough skins are visible. The inner surface is dark yellow or light brown with fine longitudinal ribs. Light weight, hard weight, fibrous cross section, split sheet type layering, and deep yellow color. Light smell, bitter taste, sticky property when chewed. Turning off cortex phellodendri: the plate shape or the shallow groove shape is different in length and width and 2-4mm in thickness. The outer surface is yellowish green or light brown yellow, relatively flat, irregular longitudinal cracks, small and few skin hole marks and occasional grey-white crude skin residues; the inner surface is yellow or yellowish brown. Light weight, hard weight, fibrous cross section, split-sheet layering, and bright yellow or yellow green color. Light smell, extremely bitter taste, sticky when chewed.

The existing Chinese pharmacopoeia (2020 edition) contains phellodendron bark, salted phellodendron bark and phellodendron charcoal, does not contain wine phellodendron bark, and has no thin-layer chromatography identification standard. However, the wine phellodendron amurense has wide application in China and long history^[5-7]. The first wine-making of Huang Bai was recorded in Yinhai Jing Wei of Tang Dynasty^[8]It is called: "wine cypress". Qing Dynasty' Ben Cao Shi Xin^[9]Yue' unprocessed, reducing excess fire, stir-baked to black to stop metrorrhagia and leukorrhagia, processed with wine for treatment, honeyDuring the treatment, salt treatment controls the lower. In the conventional prescriptions, the raw material is usually used as heat-clearing agent, such as Huang Bai Wan (intestine-clearing and dysentery-stopping)^[10]"Huanglian Jiedu Tang" (clearing fire and removing toxicity)^[11](ii) a The wine is mostly used for clearing the heat of upper energizer, such as "Huanjing Wan" (clearing heat, dispelling wind and improving eyesight)^[12]"Qingshangxiehuo decoction" (clearing deficiency heat and dissipating head wind)^[13]。

Literature reports that the processing methods of phellodendron amurense are various^[14-17]The invention adopts the following processing method of wine phellodendron bark according to the processing standard of Chinese traditional medicine materials in China and provinces, the processing standard of Chinese traditional medicine decoction piece factory expert for wine phellodendron bark and the processing experience of the old pharmacy for decades: taking 100kg of the phellodendron shreds, adding 10kg of yellow wine, uniformly stirring, placing in a jar or a suitable container, moistening for 60 minutes until the wine is completely drunk, taking out, parching to dry (the product temperature is 150-.

Thin layer chromatography is a particularly effective chromatographic method for the rapid separation of substances such as fatty acids, steroids, amino acids, nucleotides, alkaloids and many others. The method has the advantages of low equipment requirement, high separation speed, high efficiency, strong applicability, simple and convenient operation and the like, and is widely applied to the rapid identification of the traditional Chinese medicinal materials^[18-21]. The subject group tries to perform thin-layer chromatography identification on the golden cypress and the wine golden cypress by adopting a thin-layer chromatography method of the golden cypress carried in Chinese pharmacopoeia (2020 edition) for many times, and finds that the experimental steps are many, the repeatability is poor, the requirements on the quality of an experimental reagent and the operation of personnel are high, and the difficulty in rapidly identifying the golden cypress and the wine golden cypress is increased. In addition, in the prior art, as shown in table 1, the identification methods of phellodendron bark and phellodendron bark processed with wine have the defects, so that the invention provides a thin-layer chromatography identification method of phellodendron bark and phellodendron bark processed with wine, which is used for effectively solving the technical problems.

TABLE 1 identification method of phellodendron bark and phellodendron bark in wine

Disclosure of Invention

The purpose of the invention is as follows: the invention aims to solve the technical problem of providing a thin-layer chromatography identification method for golden cypress and golden cypress wine-roasted products aiming at the defects of the prior art.

In order to solve the technical problem, the invention discloses a thin-layer chromatography identification method for golden cypress and golden cypress wine roasted products, which comprises the following steps:

(1) grinding a sample to obtain medicinal material powder, mixing the medicinal material powder with a first solvent, and extracting by ultrasonic or heating reflux to obtain an extracting solution;

(2) carrying out solid-liquid separation on the extracting solution obtained in the step (1), and adding a second solvent into the obtained liquid to prepare a test solution;

(3) and (3) spotting the sample solution and the reference solution obtained in the step (2) on a silica gel plate, developing, taking out, drying and inspecting under an ultraviolet lamp.

In the step (1), the sample is phellodendron bark or a fake product of phellodendron bark.

The fake products of the golden cypress are common fake products in the market, and include, but are not limited to, aspen bark and chinaberry bark.

Wherein the bark of Populus tremula Populus davidiana Dode is obtained by removing the bark of a cork layer, and dyeing with yellow dye. The slices are light and uneven in color, and the fibrous splinters in the cut surface are layered, light in taste and not bitter.

Wherein the cortex Meliae is bark of Melia toosendan Sieb.et Zucc of Meliaceae, with cork layer removed. The sheet is plate-shaped, has different sizes and thicknesses of 1-4 mm, has light yellow-brown outer surfaces and yellow-white inner surfaces, has irregular yellow-white and non-uniform longitudinal textures, is tough, is difficult to break, has fibrous sections and is easy to strip in pieces. Is bitter in taste.

In the step (1), the medicinal material powder is ground into powder and sieved by a 24-40 mesh sieve; preferably, the medicinal material powder is ground and sieved by a 30-mesh sieve.

Wherein, the first solvent and the second solvent are respectively and independently selected from any one or a combination of more of methanol, ethanol and ethyl acetate; preferably, the first solvent and the second solvent are the same; further preferably, the first solvent and the second solvent are both methanol.

In the step (1), when the extraction method is ultrasonic, the dosage ratio of the medicinal material powder to the first solvent is 0.1 g: 2-8 mL; preferably, the dosage ratio of the medicinal material powder to the first solvent is 0.1 g: 3-7 mL; further preferably, the dosage ratio of the medicinal material powder to the first solvent is 0.1 g: 4-6 mL; still more preferably, the dosage ratio of the medicinal material powder to the first solvent is 0.1 g: 5 mL.

In the step (1), when the extraction method is heating reflux, the dosage ratio of the medicinal material powder to the first solvent is 0.5-0.8 g/mL.

In the step (1), the extraction method is preferably ultrasonic extraction.

Wherein the temperature of the ultrasonic wave is 10-30 ℃; preferably, the temperature of the ultrasound is 25-30 ℃; wherein the temperature of the ultrasound is the initial temperature of the ultrasound, and the temperature does not exceed 40 ℃ along with the progress of the ultrasound.

Wherein the power of the ultrasound is 200-300W; preferably, the power of the ultrasound is 225-; further preferably, the power of the ultrasound is 250W.

Wherein the frequency of the ultrasound is 10-70 kHz; preferably, the frequency of the ultrasound is 20-60 kHz; further preferably, the frequency of the ultrasound is 30-50 kHz; still further preferably, the frequency of the ultrasound is 40 kHz.

Wherein the ultrasonic time is 10-50 min; preferably, the time of the ultrasonic treatment is 20-40 min; further preferably, the time of the ultrasound is 30 min.

In the step (1), the temperature of heating reflux is 30-90 ℃; preferably, the temperature of the heating reflux is 20-80 ℃; further preferably, the temperature of the heating reflux is the heating reflux temperature.

In the step (1), the heating reflux time is 20-100 min; preferably, the heating reflux time is 40-80 min; further preferably, the heating reflux time is 60 min.

In step (2), the solid-liquid separation includes, but is not limited to, filtration.

In the step (2), the adding amount of the second solvent is controlled, so that the total volume of the obtained liquid and the second solvent is 3-7 mL; preferably, the amount of the second solvent to be added is controlled so that the total volume of the resulting liquid and the second solvent becomes 5 mL.

In the step (3), the reference solution is one or two of cortex Phellodendri reference medicinal material solution and berberine hydrochloride solution.

The preparation method of the golden cypress reference medicinal material solution comprises the step of preparing the golden cypress reference medicinal material as a raw material according to the preparation method of the test sample solution.

Wherein the berberine hydrochloride solution is a mixed solution of berberine hydrochloride and methanol; the concentration of berberine hydrochloride in the mixed solution is 0.2-0.8 mg/mL; preferably, the concentration of berberine hydrochloride in the mixed solution is 0.5 mg/mL.

In the step (3), the environment temperature of the thin layer chromatography such as spotting and developing is 0-40 ℃.

In the step (3), the silica gel plate is a high-efficiency silica gel plate; preferably, the silica gel plate is a silica gel G plate (jiangyou silica gel development ltd, cigarette tai).

In the step (3), the sample application amount of the sample application is 1-9 muL; preferably, the spot size is 1-5 μ L; further preferably, the amount of spotting is 1-2 μ L.

In the step (3), the developing solvent is ethyl acetate, methanol, chloroform, ammonia water and triethylamine in a volume ratio of 4-8: 1-3: 1-3: 0.5-1.5: 1; preferably, the developing solvent is ethyl acetate, methanol, chloroform, ammonia water and triethylamine in a volume ratio of 6: 2: 2: 1: 1, and (b) a mixed solution.

In the step (3), the unfolding distance is 6-10 cm; preferably, the spread of the spread is 7-8 cm.

In the step (3), the drying includes, but is not limited to, airing.

In the step (3), the ultraviolet lamp inspection is preferably performed under an ultraviolet lamp (365 nm).

In the step (3), when the sample solution and the reference solution have fluorescent spots at the same horizontal position, the sample is cortex Phellodendri or cortex Phellodendri wine-processed product.

In the step (3), when the sample solution has a fluorescent spot at the same horizontal position as the reference solution and only has one fluorescent spot, the sample is phellodendron amurense or phellodendron amurense wine-roasted product; when the sample solution and the reference solution have fluorescence spots at the same horizontal position and two fluorescence spots in the up-down direction, the sample is cortex Phellodendri cortex or cortex Phellodendri wine-processed product.

Wherein the fluorescent spots are bright yellow fluorescent spots.

Has the advantages that: compared with the prior art, the invention has the following advantages:

1. the sample powder sampling amount is 0.1g, and the precision of the method is increased.

2. The extraction solvent is methanol, the addition amount is 5mL, the solvent consumption is greatly reduced, and the solvent preparation link is omitted.

3. The developing agent adopted by the invention is ethyl acetate-methanol-trichloromethane-ammonia water-triethylamine, a layering developing agent is not needed, the developing agent is prepared at present when used, spots are concentrated, the boundary is clear, and the developing agent is stable and has no drift; meanwhile, the thin-layer chromatography identification method provided by the invention is simple and easy to operate, good in repeatability and strong in stability.

4. According to the method provided by the invention, after the development in the developing agent is finished, the observation is directly carried out at 365nm, so that the method is convenient and intuitive; solves the problems that the thin-layer plate needs to be placed in a saturated developing agent in ammonia vapor for development, and the thin potassium bismuth iodide test solution needs to be sprayed for color development after the development is finished, and the operation is complex in the prior art.

5. The method can also be used for rapid thin-layer chromatography identification of cortex Phellodendri and cortex Phellodendri, and the spot separation is clear and stable.

Drawings

The foregoing and/or other advantages of the invention will become further apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings.

FIG. 1 shows the diagram of cortex Phellodendri and cortex Phellodendri with wine (left: cortex Phellodendri and right: cortex Phellodendri with wine).

FIG. 2 shows the effect of different extraction solvents on the identification effect of thin layer chromatography.

FIG. 3 is a graph showing the effect of different developing agents on the identification effect of thin layer chromatography.

FIG. 4 shows the effect of different extraction methods on the identification effect of thin layer chromatography.

FIG. 5 is a graph showing the effect of different spans on the identification effect of thin layer chromatography.

FIG. 6 shows the effect of different sample amounts on the identification effect of thin layer chromatography.

FIG. 7 is a thin layer chromatography specificity study.

FIG. 8 shows the effect of different silica gel plates on the identification effect of thin layer chromatography.

FIG. 9 is a graph showing the effect of temperature on the identification effect of thin layer chromatography.

FIG. 10 is a diagram of a method for detecting fifteen batches of cortex Phellodendri decoction pieces by thin layer detection in the 2020 edition of Chinese pharmacopoeia.

FIG. 11 shows the thin-layer identification of fifteen batches of cortex Phellodendri decoction pieces by the method

FIG. 12 shows the identification of fifteen batches of phellodendron amurense decoction pieces processed by wine by thin layer method.

FIG. 13 thin-layer identification of phellodendron amurense and phellodendron amurense decoction pieces by the method of the present invention.

Detailed Description

The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials are commercially available, unless otherwise specified.

The developing agent described in the following examples refers to a volume ratio.

Example 1

1 Material

1.1 Main instruments and materials

AB135-S type electronic balance (METTLER); SB25-12DTD ultrasonic cleaner (Ningbo Xinzhi); CBIO21 multifunctional dark box ultraviolet analyzer (beijing sebaio); CY type drum type medicine frying machine (Nanjing Teng Yang); double-channel chromatography cylinder (solid glass). Silica gel G plate (batch number: 020190411, Yangttaijiang friend); silica gel GF254 board (batch number: 20061021, Qingdao ocean); microcapillary (1. mu.L, lot # 889892; 3. mu.L, lot # 895254, Germany, HIRSCHMANN).

1.2 herbs

The cortex Phellodendri decoction pieces (cortex Phellodendri shreds) (fifteen batches of cortex Phellodendri crude drugs and three batches of cortex Phellodendri of Guanguan) are purchased from Hebei Anguo, and are respectively identified by Rojinnu principal and senior pharmacist of Shanxi province as dried bark of cortex Phellodendri (Phellodenron chinense Schneid.) of Magnoliaceae and dried bark of cortex Phellodendri (Phellodenron amurense Rupr.) of Rutaceae. Processing the obtained cortex Phellodendri decoction pieces to obtain wine cortex Phellodendri decoction pieces. The information of decoction pieces (phellodendron bark and phellodendron bark processed with wine) is shown in Table 2.

TABLE 2 sources and preparation of Chinese herbal pieces

1.3 drugs and reagents

Berberine hydrochloride, phellodendrine hydrochloride and phellodendron amurense reference medicinal materials (batch numbers: 110713-202015, 111895-201805, 121510-201807, China institute for testing food and drug); jizhou yellow wine (batch: 20200806), Shijiazhuang Koelreuteria, and other reagents were analytically pure.

2 methods and results

2.1 processing cortex Phellodendri with wine

According to the national and multi-provincial Chinese medicinal material processing standard, the processing standard of Chinese medicinal decoction pieces factory's Tonghua phellodendron amurense with wine and the processing experience of the old pharmacy's phellodendron amurense with wine for decades, the following processing method of phellodendron amurense with wine is adopted: taking 100kg of cortex phellodendri shreds, adding 10kg of yellow wine, stirring uniformly, placing in a jar or a suitable container, moistening for 60 minutes until the wine is completely drunk, taking out, parching to dry (the product temperature is 150-. The cortex Phellodendri is processed with wine before and after being processed with wine as shown in figure 1.

2.2 preparation and inspection methods of test solution and reference solution

(1) Test solution: grinding a sample (the phellodendron amurense obtained in the step 2.1, or the phellodendron amurense obtained in the step wine) into powder, sieving the powder with a 30-mesh sieve, taking 0.1g of the medicinal material powder, adding 5mL of an extraction solvent (methanol), carrying out ultrasonic extraction (the power is 250W, the frequency is 40kHz, and the ultrasonic temperature is room temperature) for 30min, filtering the mixture, and making up 5mL of the filtrate with methanol.

(2) Control solution:

a. cortex phellodendri contrast medicinal solution: taking cortex Phellodendri reference medicinal material 0.1g, and preparing with "sample solution".

b. Berberine hydrochloride solution: taking berberine hydrochloride, adding methanol to make into solution containing 0.5mg per 1 mL.

c. Hydrochloric acid phellodendron aqueous alkali: taking phellodendrine hydrochloride, and preparing with berberine hydrochloride solution.

(3) The inspection method comprises the following steps: spotting 1 μ L with capillary, developing, taking out, air drying, and viewing under ultraviolet lamp (365 nm).

Silica gel G plate: jiangyou silica gel development Co., Ltd, Taiwan City.

Developing agent: ethyl acetate: methanol: trichloromethane: ammonia water: triethylamine (6: 2: 2: 1: 1).

And (3) span extension: 7 cm.

Ambient temperature: room temperature, 25 ℃.

2.3 examination of identification conditions for thin layer chromatography

The thin-layer chromatography is used for identifying influence factors and comprises the following steps: extraction method, extraction solvent, unfolding system, unfolding distance, sample application amount and the like. The experiment investigated the above-mentioned influencing factors, respectively. The following methods are based on step 2.2 unless otherwise specified.

2.3.1 examination of extraction solvent

The extraction effect of alcohol phellodendron amurense 1 with methanol, ethanol and ethyl acetate as extraction solvents is examined and compared.

Test solution: taking wine phellodendron amurense 1 as a raw material, and taking methanol, ethanol and ethyl acetate as extraction solvents respectively to prepare three test sample solutions.

Control solution: taking berberine hydrochloride solution and cortex Phellodendri hydrochloride solution as reference substances.

Sample amount of spotting: 3 μ L.

The result is shown in figure 2 (test solution: 1. methanol; 2. ethanol; 3. ethyl acetate; 4. phellodendron hydrochloride aqueous alkali; 5. berberine hydrochloride aqueous alkali), the size and intensity of the fluorescence spot of the methanol extract are the highest, the fluorescence spot is uniform and aggregated, and is obviously higher than that of ethanol and ethyl acetate, and the methanol is used as an extraction solvent, which is beneficial to improving the sensitivity of the detection system.

2.3.2 thin layer chromatography development systems investigation

Five developing agents were investigated: developing agent I: cyclohexane: ethyl acetate: isopropyl alcohol: methanol: water: triethylamine (3: 3.5: 1: 1.5: 0.5: 1); developing agent II: toluene: ethyl acetate: methanol: isopropyl alcohol: water (6: 3: 2: 1.5: 0.3); developing agent III: trichloromethane: methanol: water (30: 15: 4); developing agent IV: cyclohexane: ethyl acetate: isopropyl alcohol: methanol: water: triethylamine (3: 3.5: 1: 1.5: 0.5: 1); developing agent V: ethyl acetate: methanol: trichloromethane: ammonia water: triethylamine (6: 2: 2: 1: 1).

Test solution: taking cortex Phellodendri 1, cortex Phellodendri wine 1, wine cortex Phellodendri 7, and wine cortex Phellodendri 13 as raw materials, respectively, and making into four sample solutions.

Comparison products: taking cortex Phellodendri reference medicinal solution, berberine hydrochloride solution and cortex Phellodendri hydrochloride solution as reference substances.

The results are shown in FIG. 3 (test solution: 1. Phellodendri cortex 1; 2. wine Phellodendri cortex 1; 3. wine Phellodendri cortex 7; 4. wine Phellodendri cortex 13; 5. Phellodendri cortex control solution; 6. Phellodendri cortex alkali hydrochloride solution; 7. Berberine hydrochloride solution), developing agent V: ethyl acetate: methanol: trichloromethane: ammonia water: the fluorescent points of triethylamine (6: 2: 2: 1: 1) are relatively concentrated, the boundary is clear, the triethylamine is not easy to drift, the detection precision is high, and the repeatability is good. Meanwhile, fluorescent points with clear three-color boundary and obvious color development difference appear, and the information content of thin-layer chromatography identification is increased (except for appearing the bright yellow fluorescence of the berberine, blue fluorescence and green fluorescence). The bright green fluorescent point in the thin-layer chromatogram is preliminarily estimated to be phellodendron ketone substances, and the bright blue fluorescent point is caused by the fluorescence of magnoflorine substances and needs to be verified subsequently.

2.3.3 thin layer chromatography extraction method investigation

Two extraction methods were investigated: (1) ultrasonic extraction, namely step 2.2; (2) heating reflux extraction: grinding the sample, sieving with 30 mesh sieve, collecting medicinal powder 0.5g, adding 40mL extraction solvent (methanol), adding a condenser tube on the mouth, introducing into air through the opening of the condenser tube, adding appropriate amount of defatted cotton at the mouth part to prevent methanol leakage, heating to 60 deg.C, evaporating methanol, cooling via the condenser tube to obtain liquid, flowing back into the single-necked flask, heating under reflux for extraction for 60min, filtering, and adding 5mL methanol into the filtrate. .

Preparation of a test solution: taking wine phellodendron amurense 1 as a raw material, and preparing two sample solutions by respectively adopting ultrasonic extraction and heating reflux methods.

Comparison products: taking cortex Phellodendri reference medicinal solution and berberine hydrochloride solution as reference substances.

The results are shown in figure 4 (test solution: 1. wine-processed phellodendron bark 1 (ultrasonic extraction), 2. wine-processed phellodendron bark 1 (reflux extraction), 3. processed phellodendron bark contrast solution (ultrasonic extraction), 4. berberine hydrochloride solution), and it can be seen that the berberine is clear in boundary and bright yellow fluorescent dots after ultrasonic extraction. Thin layer scanning can be performed subsequently for more accurate quantification. Ultrasonic extraction method for berberine (C) as marker substance in cortex Phellodendri₂₀H₁₈NO₄) The extraction rate is higher, impurities influencing thin-layer chromatography identification are not seen, the ultrasonic extraction method is high in extraction speed and strong in repeatability, and the resolution of the thin-layer chromatography can be improved, so that the ultrasonic extraction method is preferably used for preparing the extracting solution of the golden cypress and the golden cypress in wine in the subsequent test.

2.3.4 thin layer chromatography spread Range inspection

Five spread distances were investigated: span length I: 6 cm; and (3) span length II: 7 cm; span length III: 8 cm; span IV: 9 cm; span V: 10 cm.

Test solution: taking cortex Phellodendri 1, cortex Phellodendri 7 and cortex Phellodendri 13 as raw materials, respectively, and making into four test solutions.

The effect of different spreading distances on the spreading effect is examined through experiments, and the test solution is shown in figure 5 (test solution: 1. phellodendron 1; 2. wine phellodendron 1; 3. wine phellodendron 7; 4. wine phellodendron 13; 5. phellodendron control solution; and 6. berberine hydrochloride solution). The result shows that the effect is better when the span is 7-8cm, the tailing phenomenon is less, the span is increased along with the prolonging of the chromatography time, the time and the cost are consumed, the front edge of a solvent and a fluorescent point are easy to drift, and the requirements on the processing of a thin-layer plate and the uniformity of silica gel particles are higher.

2.3.5 thin layer chromatography dot sample volume inspection

Nine sample application amounts were examined: 1-9 μ L.

Test solution: preparing a sample solution by using a phellodendron bark 1 as a raw material.

Comparison products: taking berberine hydrochloride solution and cortex Phellodendri hydrochloride solution as reference substances.

Sample amount of spotting: the sample application amount of the sample solution is 1-9 μ L, and the sample application amount of the reference sample is 1 μ L.

As shown in FIG. 6 (sample solution: 1-9: Phellodendri cortex 1; 10, berberine hydrochloride solution; 11, Phellodendri cortex hydrochloride solution), the sample application amount is 1-2 μ L, the fluorescence spots are concentrated, the boundary is clear, the tailing is less, and the identification requirement of thin layer chromatography can be met.

Through the investigation of the thin-layer chromatography identification influence factors, the preparation and detection processes of the sample are determined as follows:

test solution: grinding the sample, sieving with 30 mesh sieve, collecting medicinal powder 0.1g, adding 5mL of extraction solvent (methanol), ultrasonic extracting (power 250W, frequency 40kHz, temperature at room temperature) for 30min, filtering, and adding methanol to the filtrate to 5 mL;

the inspection method comprises the following steps: at room temperature, 1 μ L was spotted using a capillary tube, and the resultant was coated with a silica gel G plate (jiangyou silica gel development ltd, tai), developing agent: ethyl acetate: methanol: trichloromethane: ammonia water: triethylamine (6: 2: 2: 1: 1), spreading under the condition of spreading distance of 7cm, taking out, drying, and viewing under an ultraviolet lamp (365 nm);

and (4) judging the standard: (1) when the sample solution is similar to the cortex Phellodendri reference medicinal material solution and berberine hydrochloride solution in same position, the sample is cortex Phellodendri or cortex Phellodendri wine-processed product; (2) when the sample solution has fluorescent spots at the same horizontal positions as the cortex Phellodendri reference medicinal material solution and berberine hydrochloride solution, and only one fluorescent spot is detected, the sample is cortex Phellodendri or cortex Phellodendri wine-processed product; when the sample solution has fluorescent spots at the same horizontal position as the cortex Phellodendri reference medicinal material solution and berberine hydrochloride solution, and there are two fluorescent spots in the up-down direction, the sample is cortex Phellodendri cortex or cortex Phellodendri wine-processed product.

Example 2: thin layer chromatography identification specificity investigation

The specificity of the method is examined, and the result is shown in FIG. 7 (sample solution: 1. phellodendron 1; 2. wine phellodendron 1; 3. phellodendron reference drug; 4. phellodendron hydrochloride aqueous solution; 5. berberine hydrochloride aqueous solution; sample amount: 3. mu.L). The sample cortex Phellodendri 1 and wine cortex Phellodendri 1 have the same fluorescent spot at the same position as berberine hydrochloride and cortex Phellodendri reference medicinal material, and the berberine hydrochloride has no fluorescent spot and no other interference spots. The method is proved to have good specificity.

The method is used for identifying and inspecting thin-layer color of cortex Phellodendri (cortex Phellodendri, etc.), and is shown in FIG. 13 (sample solution: 1 cortex Phellodendri, 1: 2. cortex Phellodendri reference solution; 3-5. cortex Phellodendri, 1-3: 6. berberine hydrochloride solution). The phellodendron (phellodendron amurense) only has one bright yellow fluorescent spot on the thin-layer chromatogram, and the closed phellodendron amurense is an upper bright yellow fluorescent spot and a lower bright yellow fluorescent spot, so the thin-layer chromatography identification method can also quickly identify the phellodendron amurense and the closed phellodendron amurense.

Example 3: investigation of durability

(1) Investigation of influence of silica gel plate on thin-layer chromatography identification

The durability of the high-efficiency thin-layer plates and the common thin-layer plates produced by three different manufacturers is examined, and the high-efficiency thin-layer plates and the common thin-layer plates are shown in a figure 8 (a sample solution: 1. phellodendron bark 1; 2. wine phellodendron bark 1; 3. wine phellodendron bark 7; 4. wine phellodendron bark 13; 5. phellodendron bark reference medicinal solution; 6. berberine hydrochloride solution; silica gel G plate: I, Jiangyou river friend high-efficiency plate in cigarette Taiwan, II, Qingdao ocean common plate; III, Qingdao ocean high-efficiency plate), and the durability of the high-efficiency thin-layer plates and the common thin-layer plates produced by the three different manufacturers is obviously found that the high-efficiency plates are uniform in silica gel particles, small in particle size and uniform in spraying, so that the number of separation plates is high, so that the fluorescence points of the berberine hydrochloride are relatively concentrated, the boundaries are clear, and the thin-layer chromatography identification of the phellodendron bark and the wine phellodendron is more beneficial to the subsequent thin-layer scanning quantification.

(2) Investigation of temperature effects on thin layer chromatography

The applicability to temperature environment is shown in figure 9 (environment temperature: I, 4 deg.C; II, 40 deg.C; III, room temperature (21 deg.C), sample solution of 1.

cortex Phellodendri

1, 2.

cortex Phellodendri

1, 3.

cortex Phellodendri

7, 4.

cortex Phellodendri

13, 5. cortex Phellodendri reference solution, and 6. berberine hydrochloride solution). Under the conditions of 4 ℃ (refrigerator) and 21 ℃ (room temperature), the size of berberine fluorescence points is stable, relatively concentrated, the boundary is clear, the span is equivalent, under the condition of 40 ℃ (water bath), the span is increased to some extent, but the influence is small, the fluorescence points are changed obviously, the area of the fluorescence points is about twice of that of the fluorescence points when the temperature is 4 ℃ (refrigerator), but the boundary is clear, and the thin-layer chromatography identification of the golden cypress and the wine golden cypress is not influenced.

Example 4: thin layer chromatography detection of samples

(1) Fifteen batches of phellodendron amurense decoction pieces are identified by applying a thin-layer chromatography method of China pharmacopoeia of 2020 edition

1-15 parts of test solution: 1-15 parts of phellodendron; 16. cortex Phellodendri reference medicinal solution; 17. hydrochloric acid phellodendron aqueous alkali; 18. berberine hydrochloride solution.

Based on 2020 edition' Chinese pharmacopoeia^[1]Fifteen batches of phellodendron amurense decoction pieces are subjected to thin-layer identification by a thin-layer chromatography method: extracting solvent: 1% acetic acid-methanol solution; the extraction method comprises the following steps: carrying out ultrasonic treatment; sucking 3-5 μ L of sample solution, respectively dropping (dropping amount: 3 μ L) on the same silica gel G thin layer plate (Jiangyou silica gel development Co., Ltd., Yangtai city), placing the lower layer solution of chloroform-methanol-water (30: 15: 4) as developing agent (span length: 10cm) in an ammonia vapor saturated developing cylinder, developing, taking out, air drying, and spraying diluted potassium bismuth iodide solution. The following profile was obtained over a number of trials and is shown in figure 10. Spots of the same color appear on the chromatogram of the test solution at the positions corresponding to the chromatograms of the control solution and the reference solution. But the spots have obvious drift, and the color development points are not gathered and are relatively dispersed.

(2) Fifteen batches of phellodendron amurense decoction pieces are identified by applying the thin-layer chromatography method

1-15 parts of test solution: 1-15 parts of phellodendron; 16. cortex Phellodendri reference medicinal solution; 17. berberine hydrochloride solution; 18. hydrochloric acid phellodendron amurense aqueous alkali.

Preparing a sample solution according to the method, and taking berberine hydrochloride solution and phellodendron hydrochloride solution as reference substance solutions. Inspecting by thin layer chromatography (general rule 0502) under 365nm ultraviolet lamp, as shown in FIG. 11, the positions corresponding to the reference medicinal material chromatogram and the reference substance chromatogram show bright yellow fluorescent spots with the same color, the spots are concentrated and uniform, the boundary is clear, and the solvent front edge and the fluorescent spots do not drift visible to naked eyes. The method can well carry out thin-layer chromatography identification and detection on the phellodendron amurense.

(3) Fifteen batches of wine phellodendron amurense decoction pieces are identified by applying thin-layer chromatography of the method

1-15 parts of test solution: 1-15 parts of wine phellodendron amurense; 16. cortex Phellodendri reference medicinal solution; 17. berberine hydrochloride solution; 18. hydrochloric acid phellodendron amurense aqueous alkali.

Preparing solution, chromatography and inspection in the same method step (2). As shown in figure 12, in the chromatogram of the test sample, at the positions corresponding to the chromatogram of the reference drug and the chromatogram of the reference sample, bright yellow fluorescent spots with the same color are displayed, the spots are concentrated and uniform, the boundary is clear, the front edge of the solvent and the fluorescent spots do not drift, because the amount of berberine in the wine-roasting process of phellodendron is reduced in different degrees, under the same extraction method and thin-layer chromatography identification method, the fluorescent spots of wine-roasted phellodendron are smaller than that of phellodendron, and the method can well carry out thin-layer chromatography identification detection on wine-roasted phellodendron.

(4) The thin layer chromatography method for detecting the phellodendron bark and the phellodendron bark

Test solution: 1.1, phellodendron bark; 2. cortex Phellodendri reference medicinal solution; 3-5, 1-3 parts of cortex phellodendri; 6. berberine hydrochloride solution.

Preparing solution, chromatography and inspection in the same method step (2). As shown in fig. 13, the thin-layer chromatogram of cortex phellodendri amurense is composed of upper and lower bright yellow fluorescent spots, the upper bright yellow fluorescent spot is berberine, the lower bright yellow fluorescent spot is bamantin, and the thin-layer chromatogram identification method can be used for rapidly identifying cortex phellodendri amurense and cortex phellodendri amurense.

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The invention provides a thought and a method for identifying phellodendron bark and phellodendron bark wine roasted products by thin layer chromatography, and a method and a way for realizing the technical scheme are many. All the components not specified in the present embodiment can be realized by the prior art.

Claims

1. A thin-layer chromatography identification method for golden cypress and golden cypress wine roasted products is characterized by comprising the following steps:

2. The thin layer chromatography identification method of claim 1, wherein the first solvent and the second solvent are respectively and independently selected from any one or a combination of methanol, ethanol and ethyl acetate.

3. The thin layer chromatography identification method as claimed in claim 1, wherein in step (1), the extraction method is ultrasonic extraction; wherein the power of the ultrasound is 200-300W; the frequency of the ultrasonic wave is 10-70 kHz.

4. The thin layer chromatography identification method of claim 1, wherein in step (3), the reference solution is berberine hydrochloride solution; wherein the berberine hydrochloride solution is a mixed solution of berberine hydrochloride and methanol; the concentration of berberine hydrochloride in the mixed solution is 0.2-0.8 mg/mL.

5. The thin layer chromatography identification method of claim 1, wherein in step (3), the reference solution is a cortex Phellodendri reference solution; the preparation method of the golden cypress reference medicinal material solution comprises the step of preparing the golden cypress reference medicinal material as a raw material according to the preparation method of the test sample solution.

6. The thin layer chromatography identification method according to claim 1, wherein in the step (3), the amount of spotting is 1 to 9 μ L.

7. The thin layer chromatography identification method according to claim 1, wherein in the step (3), the developing solvent is ethyl acetate, methanol, chloroform, ammonia water and triethylamine in a volume ratio of 4-8: 1-3: 1-3: 0.5-1.5: 1, and (b) a mixed solution.

8. The thin layer chromatography identification method as claimed in claim 1, wherein in the step (3), the spread distance of the development is 6 to 10 cm.

9. The thin layer chromatography identification method according to claim 1, wherein in the step (3), when the sample solution has a fluorescent spot at the same level as the control solution, the sample is phellodendron amurense or phellodendron amurense processed with wine.

10. The thin layer chromatography identification method of claim 1, wherein in step (3), when the sample solution has a fluorescent spot at the same horizontal position as the reference solution and only has one fluorescent spot, the sample is phellodendron amurense or phellodendron amurense processed with wine; when the sample solution and the reference solution have fluorescence spots at the same horizontal position and two fluorescence spots in the up-down direction, the sample is cortex Phellodendri cortex or cortex Phellodendri wine-processed product.