A method for simultaneously identifying Schisandra chinensis medicinal materials and their preparations

Abstract

The application discloses a method for simultaneously identifying Radix Ampelopsis and its preparation, and relates to the technical field of traditional Chinese medicine quality control. The thin-layer identification method of the Radix Ampelopsis-containing traditional Chinese medicine preparation is as follows: (1) taking the traditional Chinese medicine preparation containing Radix Ampelopsis as a sample to be tested, adding an ethanol solution, performing ultrasonic treatment, filtering, evaporating the filtrate, dissolving the residue in an acidic aqueous solution, extracting with petroleum ether, discarding the petroleum ether layer, adjusting the pH of the acidic water layer, filtering, extracting the filtrate with ethyl acetate, combining the ethyl acetate layers, evaporating, dissolving the residue in methanol, and obtaining a test sample solution; (2) preparing a Radix Ampelopsis medicinal material solution, a control medicinal material solution and a negative sample solution without Radix Ampelopsis; (3) taking the four solutions above and spotting them on a thin-layer plate, developing with a developing agent, drying, and observing. The thin-layer identification method can not only identify single Radix Ampelopsis medicinal material, but also can better identify traditional Chinese medicine compound preparations, has good separation degree of components, has no interference, has short identification time, and reduces the production inspection cost.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of quality control of traditional Chinese medicine, and particularly relates to a method for simultaneously identifying Radix Ampelopsis delavayanae and its preparation. BACKGROUND

[0002] The main components of Radix Ampelopsis delavayanae are flavonoids, tris-iron saponins, organic acids, alkaloids, lactones, volatile oils and sugars, etc., which have the effects of dispelling wind and dampness, relieving swelling and pain, and treating contusions and injuries. The existing technology discloses the following thin-layer identification method of Radix Ampelopsis delavayanae and its traditional Chinese medicine preparation:

[0003] The article “Liao J N et al., Quality Standard of Yao Medicine Ampelopsis delavayanae [J]. Chinese Journal of Traditional Chinese Medicine, 2017, 32(06)” discloses a thin-layer identification method of the Yao medicine Ampelopsis delavayanae, wherein the thin-layer chromatography identification steps are as follows: 1 g of powder of the product is added to 45 mL of ethyl acetate, ultrasonic treatment is performed for 30 min, filtration is performed, the filtrate is concentrated to dryness, 1 mL of ethyl acetate is added to dissolve, and the solution is used as a test solution. 1 g of a control medicinal material is added to prepare a control medicinal material solution in the same manner. 1 mg of schizandrol A is added to methanol to prepare a solution, and the solution is used as a control solution. 6 μL of the test solution, 6 μL of the control medicinal material solution and 4 μL of the control solution are taken, and are spotted on the same silica gel GF254 thin-layer plate. Cyclohexane-ethyl acetate (3:1) is used as a developing agent, and the plate is developed, taken out, dried, and observed under ultraviolet light (254 nm). However, the method cannot be used to identify the preparation, and the spot is not clear.

[0004] The article “Rao W Y et al., Quality Standard Research of Wusongtongtong Tincture [J]. Chinese Medicine Guide, 2013, 19(12)” discloses a thin-layer identification method of Radix Ampelopsis delavayanae in Wusongtongtong Tincture, wherein the thin-layer chromatography identification steps are as follows: 30 mL of the product is evaporated to dryness, 10 mL of chloroform-methanol (8:2) is added to dissolve, filtration is performed, the filtrate is evaporated to dryness, 1 mL of methanol is added to dissolve, and the solution is used as a test solution. 30 mL of a negative control sample of Radix Ampelopsis delavayanae is added to prepare a negative control solution in the same manner. 0.5 g of a Radix Ampelopsis delavayanae control medicinal material is added to 10 mL of chloroform-methanol (8:2) to perform ultrasonic treatment for 20 min, filtration is performed, the filtrate is evaporated to dryness, 1 mL of methanol is added to dissolve, and the solution is used as a control medicinal material solution. 10 μL of each of the three solutions is taken, and is spotted on the same silica gel G thin-layer plate with sodium carboxymethyl cellulose as a binder. Toluene-ethyl acetate-formic acid (8:1.5:0.5) is used as a developing agent, and the plate is developed, taken out, dried, sprayed with 2% ferric trichloride ethanol solution, and heated at 105 DEG C until the spot is colored and clear. However, the method cannot be reproduced in the preparation of the present application, and the spot is not clear and tailing.

[0005] The article "Jiang Liqun, Research on Quality Standards of Zhongfeng Tongluo Tablets [D]. Guangxi University of Traditional Chinese Medicine. 2017" discloses a thin-layer chromatography (TLC) identification method for Schisandra chinensis in Zhongfeng Tongluo Tablets. The TLC identification steps are as follows: Take 20 tablets of this product, grind them into a fine powder, take 0.3g of the powder, place it in an Erlenmeyer flask, add 50ml of methanol, sonicate (power 90W, frequency 40kHz) for 25 minutes, let it stand for 12 hours, take 20ml of the supernatant, add 3 times the amount of ether, let it stand for 1 hour, discard the supernatant, add 50ml of 10% hydrochloric acid solution to the precipitate, reflux in a boiling water bath for 1 hour, filter, dissolve the precipitate in 10ml of methanol, concentrate the methanol solution to 1ml, and use it as the test solution. Take about 2g of Schisandra chinensis reference material and prepare a reference material solution according to the test solution preparation method. Prepare a negative control sample lacking Schisandra chinensis according to the process, take 20 tablets of the negative sample, and prepare a negative control solution according to the test solution preparation method. According to the thin-layer chromatography method, 10 μl each of the test solution and negative control solution, and 5 μl of the reference herbal material solution were spotted separately onto the same silica gel G thin-layer plate. Hexane-chloroform-acetone-acetic acid (0.2:8:4:0.5) was used as the developing solvent. After development, the plate was removed, dried, and sprayed with 8% vanillin anhydrous ethanol-sulfuric acid solution (7-10). The plate was then heated at 105°C until the spots were clearly visible and examined under sunlight. However, this method is time-consuming (preparation of the test solution takes more than 12 hours) and cannot be reproduced in the formulation of this invention; the spots are unclear and show tailing.

[0006] The thin-layer chromatography identification of the above-mentioned Schisandra chinensis medicinal materials and their preparations still has certain limitations, such as unclear spots, trailing, interference from negative samples, inability to reproduce, and long time consumption, which are not conducive to its application in production practice. Summary of the Invention

[0007] To address the above shortcomings, this invention provides a method for simultaneously identifying Schisandra chinensis medicinal material and its preparations. This method solves the problems of unclear spots, tailing, interference from negative samples, lack of reproducibility, and long processing time encountered in thin-layer chromatography (TLC) identification. The TLC identification method of this invention can not only identify Schisandra chinensis as a single medicinal material but also effectively identify compound preparations of traditional Chinese medicine. The components are well separated with no interference, and the identification time is effectively shortened, reducing production and testing costs. The specific technical solution is as follows:

[0008] A method for simultaneously identifying Schisandra chinensis medicinal material and its preparations includes the following steps:

[0009] (1) Preparation of test solution: Take the Chinese medicine preparation containing Schisandra chinensis as the test sample, add ethanol solution, sonicate, filter, evaporate the filtrate to dryness, dissolve the residue in acidic aqueous solution, extract with petroleum ether, discard the petroleum ether layer, adjust the pH of the acidic aqueous layer, filter, extract the filtrate with ethyl acetate, combine the ethyl acetate layers, evaporate to dryness, dissolve the residue in methanol to obtain the test solution.

[0010] (2) Preparation of medicinal material solution: take the Wujiteng medicinal material to prepare the medicinal material solution;

[0011] (3) Preparation of control medicinal material solution: take the Wujiteng control medicinal material to prepare the medicinal material solution according to the preparation method of the medicinal material solution;

[0012] (4) Preparation of Wujiteng negative sample solution: take the Wujiteng negative sample to prepare the medicinal material solution according to the preparation method of the medicinal material solution;

[0013] (5) Take the test sample solution, medicinal material solution, control medicinal material solution and Wujiteng negative sample solution and point them on the thin layer plate respectively, and then develop, dry, color and observe.

[0014] Preferably, in the step (1), the Chinese medicine preparation containing Wujiteng is Didi Zhuanzhengling tincture.

[0015] Preferably, in the step (1), the mass concentration of the ethanol solution is 50%-80%.

[0016] Preferably, in the step (1), the ultrasonic power is 300-320W, and the ultrasonic time is 20-40min.

[0017] Preferably, in the step (1), the pH of the acidic aqueous solution is 1-4.

[0018] Preferably, in the step (1), the boiling range of the petroleum ether is 30-60℃.

[0019] Preferably, in the step (1), the pH of the acid water layer is adjusted to 7-8.

[0020] Preferably, in the step (1), the time for evaporating the filtrate is 1-2h, the time for extracting the petroleum ether is 20-40min, the time for extracting the ethyl acetate is 20-40min, and the time for evaporating the combined ethyl acetate layer is 20-40min.

[0021] Preferably, in the step (2), the preparation method of the medicinal material solution is: take the Wujiteng medicinal material, add methanol, ultrasonic treatment, filter, evaporate the filtrate, and then dissolve it with methanol to obtain the medicinal material solution.

[0022] Preferably, in the step (5), the developing agent is cyclohexane-ethyl acetate (3-5∶2), and the observation condition is 254nm ultraviolet light.

[0023] Compared with the prior art, the present application has the following advantages:

[0024] The application improves the processing method of the sample to be tested, and forms a new thin layer identification method together with a thin layer plate, a developing agent (cyclohexane-ethyl acetate), and 254 nm ultraviolet light, which can not only identify the single medicinal material of Radix Ampelopsis, but also can better identify the compound preparation of Radix Ampelopsis, and the separation degree of each component is good without interference. Compared with the traditional identification method, the whole identification of the application takes 5-6 hours (the traditional method takes about 20 hours), which effectively shortens the identification time and reduces the production inspection cost. BRIEF DESCRIPTION OF DRAWINGS

[0025] In order to more clearly illustrate the technical solutions of the embodiments of the application, the following will briefly introduce the drawings needed to be used in the embodiment description.

[0026] Figure 1 It is the thin layer chromatogram of Radix Ampelopsis in Example 1.

[0027] Figure 2 It is the thin layer chromatogram of Radix Ampelopsis in Example 2.

[0028] Figure 3 It is the thin layer chromatogram of Radix Ampelopsis in Example 3.

[0029] Figure 4 It is the thin layer chromatogram of Radix Ampelopsis in Comparative Example 1.

[0030] Figure 5 It is the thin layer chromatogram of Radix Ampelopsis in Comparative Example 2. DETAILED DESCRIPTION

[0031] The specific embodiments of the application will be described in detail below, but it should be understood that the protection scope of the application is not limited by the specific embodiments.

[0032] Example 1

[0033] (1) Preparation of test solution: 3g of fine powder of test product Dieda Zhuanling Tincture (raw material ratio of Dieda Zhuanling Tincture: Shuaitiancai 17.7%, Chui Fengsan 10.6%, Wumeiteng 15.9%, Guizhi 5.3%, Liangmianzhien 13.3%, Shengcaowu 2.7%, Legewang 17.7%, Jiulongchuan 3.5%, Dafengai 13.3%) was added into 30ml of ethanol with a mass concentration of 70% and ultrasonically treated for 20min under 320W, filtered, and the filtrate was evaporated for 1.5h. The residue was dissolved in 20ml of water solution with a pH value of 2, filtered, and extracted with petroleum ether (30-60℃) twice, 20ml each time, for 20min each time. The petroleum ether layer was discarded, the acid water layer was adjusted to a pH value of 7 with sodium hydroxide solution, filtered, and extracted with ethyl acetate twice, 20ml each time, for 10min each time. The ethyl acetate layers were combined and evaporated for 30min. The residue was dissolved in 1ml of methanol as the test solution.

[0034] (2) Drug solution and control drug solution preparation: Take 0.5g of each of the test drug and the control drug, add 20ml of methanol, and treat under ultrasonic waves at 320W for 30min. Filter, evaporate the filtrate, dissolve in 1ml of methanol, and prepare the drug solution and the control drug solution.

[0035] (3) Preparation of negative sample solution of missing test drug: Take 2.5g of the negative sample of missing test drug (17.7% of Pothos chinensis, 10.6% of Poria cocos, 5.3% of Ramulus cinnamomi, 13.3% of Zanthoxylum bungeanum, 2.7% of Aconitum leucostomum, 17.7% of Wikstroemia indica, 3.5% of Pteroxygonum giraldii, and 13.3% of Artemisia sacrorum), and prepare the negative sample solution of missing test drug according to the preparation method of the test drug.

[0036] Thin layer chromatography conditions: Take 10μL of each of the test drug solution, the drug solution, the control drug solution, and the negative sample solution of missing test drug, and point on the same silica gel GF254 thin layer plate. Use cyclohexane-ethyl acetate (3:2) as the developing agent, develop, take out, dry, and observe under ultraviolet light (254nm).

[0037] The total time of this example is 5.8h, and the thin layer chromatogram is shown in Figure 1 The test drug chromatogram shows the same color spots at the corresponding positions of the control drug chromatogram, the separation degree of each spot is good, there is no interference between them, no tailing phenomenon, and no negative interference.

[0038] Example 2

[0039] (1) Preparation of test drug solution: Take 3g of the fine powder of test drug Didi Zhuanzhengling tincture (raw material ratio: 17.7% of Pothos chinensis, 10.6% of Poria cocos, 15.9% of Wikstroemia indica, 5.3% of Ramulus cinnamomi, 13.3% of Zanthoxylum bungeanum, 2.7% of Aconitum leucostomum, 17.7% of Wikstroemia indica, 3.5% of Pteroxygonum giraldii, and 13.3% of Artemisia sacrorum), add 30ml of 50% ethanol, and treat under ultrasonic waves at 310W for 30min. Filter, evaporate the filtrate for 1h, dissolve the residue in 20ml of water solution with pH value of 4, filter, extract with petroleum ether (30-60℃) twice, 20ml / time, 10min each time, discard the petroleum ether layer, adjust the acid water layer to pH value of 8 with sodium hydroxide solution, filter, extract the filtrate with ethyl acetate twice, 20ml / time, 10min each time, combine the ethyl acetate layers, evaporate for 20min, dissolve the residue in 1ml of methanol, and prepare the test drug solution.

[0040] (2) Preparation of drug solution and control drug solution: Take 0.5g of each of the test drug and the control drug, add 20ml of methanol, and treat under ultrasonic waves at 310W for 30min. Filter, evaporate the filtrate, dissolve in 1ml of methanol, and prepare the drug solution and the control drug solution.

[0041] (3) Preparation of the negative sample solution of the absence of Radix Witherina: 2.5 g of the negative sample of the absence of Radix Witherina (17.7% of P. multiflora, 10.6% of P. multiflora, 5.3% of C. officinalis, 13.3% of Zanthoxylum bungeanum, 2.7% of Aconitum kusnezoffii, 17.7% of Cassia occidentalis, 3.5% of P. multiflora, and 13.3% of Artemisia vulgaris) was prepared into the negative sample solution of the absence of Radix Witherina according to the preparation method of the test sample.

[0042] Thin layer chromatography condition: 10 μL of each of the test sample solution, the medicinal material solution, the control medicinal material solution, and the negative sample solution of the absence of Radix Witherina was spotted on the same silica gel GF254 thin layer plate, and developed with cyclohexane-ethyl acetate (3:2) as the developing agent. After being taken out, dried, and observed under the ultraviolet light (254 nm), the results were recorded.

[0043] The whole process of this example took 5.4 h, and the thin layer chromatogram is shown in FIG. 1. As shown in the figure, the test sample chromatogram showed the same color spots at the positions corresponding to the control medicinal material chromatogram, and each spot was well separated and did not interfere with each other without tailing phenomenon and negative interference. Figure 2

[0044] Example 3

[0045] (1) Preparation of the test sample solution: 3 g of the fine powder of the test sample Didi Zhunzhangleng Tincture (the raw material ratio of Didi Zhunzhangleng Tincture: 17.7% of P. multiflora, 10.6% of P. multiflora, 15.9% of Radix Witherina, 5.3% of C. officinalis, 13.3% of Zanthoxylum bungeanum, 2.7% of Aconitum kusnezoffii, 17.7% of Cassia occidentalis, 3.5% of P. multiflora, and 13.3% of Artemisia vulgaris) was added with 30 ml of 80% ethanol, and ultrasonically treated for 40 min at 300 W. The filtrate was evaporated for 1 h, and the residue was dissolved in 20 mL of water solution with pH value of 1. The filtrate was extracted with petroleum ether (30-60°C) twice, 20 ml each time, for 10 min each time. The petroleum ether layer was discarded, and the acid water layer was adjusted to pH value of 7 with sodium hydroxide solution, filtered, and extracted with ethyl acetate twice, 20 ml each time, for 10 min each time. The ethyl acetate layer was combined, evaporated for 30 min, and the residue was dissolved in 1 mL of methanol as the test sample solution.

[0046] (2) Preparation of the medicinal material solution and the control medicinal material solution: 0.5 g of Radix Witherina and the control medicinal material were added with 20 ml of methanol, and ultrasonically treated for 20 min at 320 W. The filtrate was evaporated, dissolved in 1 ml of methanol, and prepared into the medicinal material solution and the control medicinal material solution.

[0047] (3) Preparation of the negative sample solution of the absence of Radix Witherina: 2.5 g of the negative sample of the absence of Radix Witherina (17.7% of P. multiflora, 10.6% of P. multiflora, 5.3% of C. officinalis, 13.3% of Zanthoxylum bungeanum, 2.7% of Aconitum kusnezoffii, 17.7% of Cassia occidentalis, 3.5% of P. multiflora, and 13.3% of Artemisia vulgaris) was prepared into the negative sample solution of the absence of Radix Witherina according to the preparation method of the test sample.​

[0048] Thin layer chromatography conditions: 10 μL of the above test sample solution, medicinal material solution, control medicinal material solution, and negative sample solution of S. sinica were spotted on the same silica gel GF254 thin layer plate, and developed with cyclohexane-ethyl acetate (3:2) as the developing agent. After being taken out, dried, and placed under UV light (254 nm) for inspection.

[0049] The total time for this example was 5.6 h, and the thin layer chromatogram is shown in Figure Figure 3 As shown in the figure, the test sample chromatogram showed the same color spots at the positions corresponding to the control medicinal material chromatogram, and each spot was well separated and did not interfere with each other, without tailing phenomenon, and without negative interference.

[0050] Comparative Example 1

[0051] 1 g of the same test sample powder as in Example 1 was taken, 45 mL of ethyl acetate was added, and ultrasonic extraction was performed for 30 min at 320 W. The filtrate was concentrated to dryness, 1 mL of ethyl acetate was added to dissolve it, and it was used as the test sample solution. 1 g of the same control medicinal material as in Example 1 was taken, and the test sample solution preparation method was used to prepare the control medicinal material solution. The same negative sample of S. sinica as in Example 1 was taken, and the test sample solution preparation method was used to prepare the negative sample solution of S. sinica. 6 μL of the above test sample solution and medicinal material solution, and 4 μL of the negative sample solution of S. sinica were spotted on the same silica gel GF254 thin layer plate, and developed with cyclohexane-ethyl acetate (3:1) as the developing agent. After being taken out, dried, and placed under UV light (254 nm) for inspection.

[0052] The total time for this comparative example was about 3 h, and the thin layer chromatogram is shown in Figure Figure 4 As shown in the figure, the spots were not clear, i.e., it could only be used for identifying medicinal materials, and could not be used for identifying Chinese medicinal preparations containing S. sinica.

[0053] Comparative Example 2

[0054] The same test sample of 30 mL as in Example 1 was taken, evaporated to dryness, 50 mL of methanol was added, and ultrasonic treatment (power 90 W, frequency 40 kHz) was performed for 25 minutes, and then the sample was left to stand for 12 hours. 20 mL of supernatant was taken, 3 times the amount of ether was added, and the sample was left to stand for 1 hour. The supernatant was decanted, and the precipitate was dissolved in 10 mL of methanol. The methanol solution was concentrated to 1 mL to obtain a test sample solution. The same sample of 2 g of Radix Morindae Officinalis as in Example 1 was taken, and a control sample solution was prepared according to the preparation method of the test sample solution. A negative sample of Radix Morindae Officinalis was prepared according to the process for preparing a sample, and 20 pieces of the same negative sample as in Example 1 were taken, and a negative control solution was prepared according to the preparation method of the test sample solution. The test sample solution, the negative control solution, and the control sample solution were subjected to thin layer chromatography, 10 μL of each of the test sample solution and the negative control solution, and 5 μL of the control sample solution were spotted on the same silica gel G thin layer plate, and developed with n-hexane-chloroform-acetone-acetic acid (0.2:8:4:0.5) as the developing agent. After being taken out, dried, and sprayed with 8% vanillin anhydrous ethanol-sulfuric acid solution (7-10), the sample was heated at 105°C until the spots were clearly colored. The sample was observed under daylight.

[0055] The whole process of the comparative example took about 17 hours, and the thin layer chromatogram is shown in Figure 5 The spots are not clear, and the tailing is not reproducible.

[0056] In summary, the present application improves the treatment method of the sample to be tested, and forms a new thin layer identification method together with the thin layer plate, the developing agent (cyclohexane-ethyl acetate), and 254 nm ultraviolet light. The method can not only identify Radix Morindae Officinalis single medicinal material, but also can better identify Radix Morindae Officinalis compound preparations, has good separation degree of each component, no interference, and can effectively shorten the identification time and reduce the production inspection cost.

[0057] The foregoing description of specific exemplary embodiments of the application is intended to be illustrative only. These descriptions are not intended to be limiting to the precise forms disclosed. Rather, these descriptions are intended to be illustrative of the specific form of the application which is described in sufficient details to enable others skilled in the art to employ the application. Numerous modifications and changes can be made by others skilled in the art which follow from the teachings without departing from the spirit of the application. It is intended that the scope of the application be defined by the claims and their equivalents.

Claims

1. A method for simultaneously identifying Radix Ampelopsis and its preparations, characterized in that, It comprises the following steps: (1) Preparation of test sample solution: take the Chinese medicine preparation containing Radix Sargentodoxae as the test sample, add ethanol solution, ultrasonic treatment, filter, evaporate the filtrate, dissolve the residue in an acidic aqueous solution with pH 1-4, extract with petroleum ether, discard the petroleum ether layer, adjust the pH of the acidic water layer to 7-8, filter, extract the filtrate with ethyl acetate, combine the ethyl acetate layers, evaporate, dissolve the residue in methanol to obtain the test sample solution; the Chinese medicine preparation containing Radix Sargentodoxae is Dizhituduanshengling Tincture; (2) Preparation of medicinal material solution: take Radix Sargentodoxae medicinal material to prepare the medicinal material solution; (3) Preparation of control medicinal material solution: take Radix Sargentodoxae control medicinal material to prepare the medicinal material solution according to the preparation method of the medicinal material solution; (4) Preparation of Radix Sargentodoxae-free negative sample solution: take Radix Sargentodoxae-free preparation as the test sample and prepare it according to the method of the test sample solution; (5) Take the test sample solution, medicinal material solution, control medicinal material solution, and Radix Sargentodoxae-free negative sample solution and point them on the thin layer plate respectively, develop with cyclohexane-ethyl acetate (3-5:2) as the developing agent, dry, color, and observe under 254 nm ultraviolet light. 2.The method for simultaneously identifying Radix Saururi and its preparations according to claim 1, characterized in that, In step (1), the mass concentration of the ethanol solution is 50%-80%. 3.The method for simultaneously identifying Radix Ampelopsis and its preparations according to claim 1, characterized in that, In step (1), the ultrasonic power is 300-320 W, and the ultrasonic time is 20-40 min.

4. The method according to claim 1, characterized in that, In step (1), the boiling range of the petroleum ether is 30-60℃.

5. The method according to claim 1, characterized in that, In step (1), the evaporation time of the filtrate is 1-2 h; the petroleum ether extraction time is 20-40 min; the ethyl acetate extraction time is 20-40 min; and the evaporation time of the combined ethyl acetate layer is 20-40 min.

6. The method according to claim 1, characterized in that, In step (2), the preparation method of the medicinal material solution is as follows: take Radix Sargentodoxae medicinal material, add methanol, ultrasonic treatment, filter, evaporate the filtrate, and then dissolve it in methanol to obtain the medicinal material solution.

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