A method for identifying luffa cylindrica by thin layer chromatography

Abstract

The present application belongs to the technical field of traditional Chinese medicine analysis, and relates to a thin layer chromatography identification method of luffa retinervae. The method comprises the following steps: 1) preparation of a test sample solution; 2) preparation of a control medicinal material solution; and 3) identification of the medicinal material by using the thin layer chromatography. The method overcomes the problems existing in the prior art, such as complicated operation, fewer chromatographic spots, inability to comprehensively reflect the components in luffa retinervae, no obvious distinguishing point for genuine and fake products, and the like. The method is simple and fast in operation, the spot information is more abundant and clear, and the method has good reproducibility and wide application range. The method can present the main chemical components of luffa retinervae to a greater extent, and can be used for identification of genuine and fake products.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of traditional Chinese medicine analysis, and relates to a thin layer chromatography identification method of luffa fruit vascular bundle. BACKGROUND

[0002] Luffa fruit vascular bundle is the dried mature fruit vascular bundle of Luffa cylindrica (L.) Roem. of Cucurbitaceae. It tastes sweet and is flat, and is related to the lung, stomach and liver channels. It has the effects of dispelling wind, unblocking collaterals, activating blood and promoting lactation. It is mainly used for treating arthralgia, chest and hypochondrium distending pain, lactation obstruction and mastitis [1] . Modern studies have proved that luffa fruit vascular bundle contains polysaccharides (xylan, mannan, galactan) [2][3] , proteins, amino acids, polypeptides, glycosides (saponins, cardiac glycosides), anthraquinones, phenols, tannins, flavonoids, coumarins, terpene lactones and alkaloids [4] , and has the pharmacological effects of diuresis, swelling, pain, inflammation, analgesia, blood sugar reduction, oxidation resistance and prevention of myocardial ischemia, and is widely used in clinical application [5] .

[0003] According to the records in Chinese Plant Flora, Luffa cylindrica (L.) Roem. and Luffa acutangula (L.) Roem. (or Luffa acutangula (L.) Roem.) are two species under the genus Luffa of Cucurbitaceae, and they are different species in the same genus. Luffa cylindrica (L.) Roem. is mainly planted in the north and south of China and is the main market variety of luffa fruit vascular bundle. There is a situation that the dried mature fruit vascular bundle of Luffa acutangula (L.) Roem. is mixed with luffa fruit vascular bundle in the market. The first volume of Chinese Pharmacopoeia (2020 edition) only records Luffa cylindrica (L.) Roem. as the source of luffa fruit vascular bundle, so Luffa acutangula (L.) Roem. is a pseudo-product of luffa fruit vascular bundle, but Luffa acutangula (L.) Roem. also contains xylan, mannan and galactan [6] , and the main chemical components are similar. In terms of characteristics: the main difference between Luffa acutangula (L.) Roem. and Luffa cylindrica (L.) Roem. is that the fruit of Luffa acutangula (L.) Roem. has 8-10 longitudinal sharp edges and grooves, the seeds have reticular ornamentation, and the base is 2 shallowly split; the fruit of Luffa cylindrica (L.) Roem. is smooth, usually has dark longitudinal stripes, the seeds are smooth, and the edges are narrow wings [7] . The above two kinds can be distinguished by the appearance of the fruit, but luffa fruit vascular bundle is the dried mature fruit vascular bundle, and the appearance of luffa fruit vascular bundle and Luffa acutangula (L.) Roem. after removing the pericarp and seeds is similar, and it is difficult to distinguish. Considering that the conventional inspection items cannot distinguish the pseudo-product, and the sensory identification has the disadvantages of relying on experience and sometimes low resolution, especially the characteristics of luffa fruit vascular bundle and Luffa acutangula (L.) Roem. are similar, which brings difficulties to identification. At present, the luffa fruit vascular bundle medicinal material has only source, characteristics, microscopic and other items, and the existing quality evaluation method is difficult to control the quality of luffa fruit vascular bundle medicinal material.

[0004] Identification of luffa fruit vascular bundle and Luffa acutangula (L.) Roem. and research on the quality standard of luffa fruit vascular bundle; Kang Alon[8] Liu et al. established a TLC method for the identification of Luffa cylindrica (L.) Roem. and its processed products, and the TLC method for the identification of L. cylindrica (L.) Roem. was collected in the Third Edition of Guangdong Provincial Standard of Chinese Herbal Medicines. CN 105203670 B discloses a method for establishing a high performance liquid chromatography (HPLC) fingerprint of L. cylindrica (L.) Roem., and CN 115436520 A discloses a method for constructing and detecting a UPLC characteristic spectrum of L. cylindrica (L.) Roem., both of which establish a characteristic spectrum method for L. cylindrica (L.) Roem. and both have 7 peaks.

[0005] However, the above methods have some defects: (1) The method established by Kang A et al. mainly contains glycosides and alkaloids in the prepared sample solution, which cannot comprehensively reflect the chemical components in L. cylindrica (L.) Roem., and the operation is complicated, the distinguishing point position is lower and closer, which is easily affected by the environment or operation, and the result is not obvious. (2) The TLC method in the Third Edition of Guangdong Provincial Standard of Chinese Herbal Medicines is simple to operate, but the developing agent has small polarity and the chromatographic spot information is less, and there is no obvious distinguishing point between the genuine and counterfeit products. (3) The methods in CN 105203670 B and CN 115436520 A have high requirements for instruments and are complicated to operate, and the experiment personnel are also required to be high.

[0006] TLC method has the characteristics of simple operation, rapidity, good reproducibility, obvious image characteristics, wide development range, etc., and is the main method in the field of traditional Chinese medicine analysis. CN 115684455 A discloses a TLC identification method for L. cylindrica (L.) Roem. and its processed products, but the inventors of the present application found through repeated experiments that the number of spots in this method is less, and the color development effect under ultraviolet light (365 nm) is less clear than under daylight, which cannot effectively identify L. cylindrica (L.) Roem. and the counterfeit product Guangdong L. cylindrica (L.) Roem. (as shown in Figure 1 ), and the sample treatment is complicated and the operation efficiency is low.

[0007] Therefore, how to establish a TLC identification method for L. cylindrica (L.) Roem. which can also be used as a specific identification method for distinguishing L. cylindrica (L.) Roem. and its easily confused product Guangdong L. cylindrica (L.) Roem. has important practical significance for improving the standard of L. cylindrica (L.) Roem. and accurately using medicine in clinic.

[0008] REFERENCES

[0009] [1] State Pharmacopoeia Commission of P. R. China. Chinese Pharmacopoeia (2020 Edition, Volume I) [S]. Beijing: China Medical Science and Technology Press, 2020: 126.

[0010] [2] Jiangsu Institute of Botany. Xinhua Herbal Outline (Volume II) [M]. Shanghai: Shanghai Scientific and Technical Publishers, 1991.

[0011] [3] Yang Youhua, Zhang Shaojie, Guo Peixin, et al. Determination of polysaccharide and amino acid content of luffa fiber from different producing areas [J]. Chinese Herbal Drugs, 2000, 31(1): 27.

[0012] [4] Li Yan, Li Wenjia, Wang Yikui, et al. Chemical composition analysis of luffa fiber [J]. 2011, 24(2): 529-534.

[0013] [5] Yang Hua, Gao Qiang, Zhao Bing, et al. Pharmaceutical research overview of luffa fiber [J]. Anhui Agricultural Sciences, 2011, 39(34): 20990-20991.

[0014] [6] Guangdong Province Chinese Herbal Medicine Standards Third Edition [S]. 2019: 428-431.

[0015] [7] Chinese Academy of Sciences, China Plant Editing Committee. Flora Reipublicae Popularis Sinicae [M]. Beijing: Science Press, 1986, 73(1): 194.

[0016] [8] Kang Along, Tang Yingshuang, Sun Chengrong, et al. Quality standard research of three different processed products of luffa fiber [J]. Journal of Shaanxi University of Chinese Medicine, 2011, 34(4): 87+95. SUMMARY

[0017] Problem to be solved by the invention

[0018] To solve the problems existing in the prior art. The purpose of the present application is to provide a thin layer chromatography identification method for luffa fiber medicinal materials, which is simple and fast in operation, rich and clear in spot, good in reproducibility and wide in application range.

[0019] Solution to the problem

[0020] The present application provides a thin layer chromatography identification method for luffa fiber medicinal materials, which comprises the following steps:

[0021] 1) Preparation of test sample solution:

[0022] The luffa fiber to be tested is extracted in the extraction solvent, and after filtration, the filtrate is evaporated to dryness, and the residue is dissolved with the redissolving solvent to obtain the test sample solution;

[0023] 2) Preparation of control medicinal material solution:

[0024] The luffa fiber control medicinal material is extracted in the extraction solvent, and after filtration, the filtrate is evaporated to dryness, and the residue is dissolved with the redissolving solvent to obtain the control medicinal material solution;

[0025] 3) Medicinal material identification by thin layer chromatography:

[0026] The test solution prepared in step 1) and the control solution prepared in step 2) are spotted on the same silica gel thin layer plate, developed with an aromatic-carboxylic acid ester-carboxylic acid ternary system, taken out, dried, sprayed with sulfuric acid ethanol solution, heated until the spots are clearly colored, and observed under ultraviolet light (365 nm). If the thin layer chromatogram of the test solution and the thin layer chromatogram of the control solution both have fluorescent spots at the same Rf value, the Luffa cylindra medicinal material is determined to be genuine; otherwise, it is determined to be fake.

[0027] Preferably, in the above method, the extraction solvent in step 1) or 2) is selected from any one of water, alcohol, and a mixture thereof, preferably alcohol, and more preferably methanol.

[0028] Preferably, in the above method, the ratio of the Luffa cylindra medicinal material to the extraction solvent in step 1) or 2) is 1 g: 15-35 mL, preferably 1 g: 20-30 mL, and more preferably 1 g: 25 mL.

[0029] Preferably, in the above method, the extraction method in step 1) or 2) is selected from any one of ultrasonic extraction and reflux extraction, preferably ultrasonic extraction.

[0030] Preferably, in the above method, the extraction time in step 1) or 2) is 10-60 min, preferably 20-40 min, and more preferably 30 min.

[0031] Preferably, in the above method, the redissolving solvent in step 1) or 2) is selected from any one of water, alcohol, and a mixture thereof, preferably alcohol, and more preferably methanol.

[0032] Preferably, in the above method, the ratio of the Luffa cylindra medicinal material to the redissolving solvent in step 1) or 2) is 1 g: 0.1-1 mL, preferably 1 g: 0.2-0.8 mL, and more preferably 1 g: 0.5 mL.

[0033] Preferably, in the above method, the stationary phase in step 3) is a silica gel G thin layer plate.

[0034] Preferably, in the above method, the developing agent in step 3) is a toluene-ethyl formate-formic acid ternary system.

[0035] More preferably, in the above method, the developing agent in step 3) is a toluene-ethyl formate-formic acid ternary system with a volume ratio of 10:3:1.

[0036] Preferably, in the above method, the color developing agent in step 3) is a 10% sulfuric acid ethanol solution.

[0037] Preferably, in the above method, the spotting amount in step 3) is 5-10 μL.

[0038] Effects of the invention

[0039] Compared with the prior art, the present application has the following beneficial effects:

[0040] (1) The present application adopts a simple and fast pretreatment method to obtain a test sample solution and a control medicinal material solution, which are spotted on the same thin layer plate, developed with a suitable developing agent, colored with a suitable color developing agent, and observed under ultraviolet light, to obtain a luffa sponge thin layer chromatogram with multiple information.

[0041] (2) The method more greatly presents the main chemical components of luffa sponge, and the chromatographic spot information is more abundant, which can be used for the identification of genuine and fake products. BRIEF DESCRIPTION OF DRAWINGS

[0042] Figure 1 The thin layer chromatogram comparison results of the luffa sponge medicinal material and its fake products obtained by the method in CN 115684455 A (wherein 1-3 correspond to luffa sponge; 4-6 correspond to Guangdong luffa sponge; S corresponds to luffa sponge control medicinal material).

[0043] Figure 2 The thin layer chromatograms of the test sample solutions and the control medicinal material solutions prepared by the four methods (wherein 1-4 correspond to the test sample solutions prepared by method one to method four in turn; S1-S4 correspond to the control medicinal material solutions prepared by method one to method four in turn).

[0044] Figure 3 The luffa sponge medicinal material thin layer chromatogram under the condition of different particle sizes of the test medicinal material (wherein 1 corresponds to the test sample solution of the luffa sponge test medicinal material treated by cutting (the side length is not greater than 0.5 cm); 2 corresponds to the test sample solution of the luffa sponge test medicinal material treated by powdering (passing through a No. 1 sieve); S corresponds to the control medicinal material solution).

[0045] Figure 4 The luffa sponge medicinal material thin layer chromatogram obtained by using different developing agents (wherein 1 corresponds to the test sample solution; S corresponds to the control medicinal material solution).

[0046] Figure 5 The luffa sponge medicinal material thin layer chromatogram obtained by using different color developing agents (wherein 1 corresponds to the test sample solution; S corresponds to the control medicinal material solution).

[0047] Figure 6 The luffa sponge medicinal material thin layer chromatogram under different sample application amounts (wherein S1-S5 correspond to the control medicinal material solution, and the sample application amounts are 1 μL, 3 μL, 5 μL, 10 μL, and 20 μL in turn; 1-5 correspond to the test sample solution, and the sample application amounts are 1 μL, 3 μL, 5 μL, 10 μL, and 20 μL in turn).

[0048] Figure 7 The thin layer chromatogram of the gourd retinervus medicinals is shown in the following table (1 corresponds to 3 test sample solutions arranged in parallel; S corresponds to the control medicinal solution; and F corresponds to the negative control solution).

[0049] Figure 8 The thin layer chromatogram of the gourd retinervus medicinals under different temperature conditions is shown in the following table (1 corresponds to 3 test sample solutions arranged in parallel; S corresponds to the control medicinal solution).

[0050] Figure 9 The thin layer chromatogram of the gourd retinervus medicinals under different humidity conditions is shown in the following table (1 corresponds to 3 test sample solutions arranged in parallel; S corresponds to the control medicinal solution).

[0051] Figure 10 The thin layer chromatogram of the gourd retinervus medicinals under different thin layer plate conditions is shown in the following table (1 corresponds to 3 test sample solutions arranged in parallel; S corresponds to the control medicinal solution).

[0052] Figure 11 The thin layer chromatogram of the gourd retinervus medicinals of different batches is shown in the following table (1-12 correspond to test sample solutions of 12 batches of gourd retinervus medicinals to be tested; and S corresponds to the control medicinal solution).

[0053] Figure 12 The comparison results of the thin layer chromatogram of the gourd retinervus obtained by the method of the present application and the Guangdong gourd retinervus are shown in the following table (1-3 correspond to test sample solutions of 3 batches of gourd retinervus to be tested; 4-7 correspond to test sample solutions of 4 batches of Guangdong gourd retinervus to be tested; and S corresponds to the control medicinal solution). DETAILED DESCRIPTION

[0054] In order to better illustrate the present application, numerous specific details are given in the following detailed description. Those skilled in the art will understand that the present application can be practiced without certain specific details. In some instances, well-known methods, means, instruments and steps have not been described in detail in order to highlight the principles of the present application.

[0055] Example 1: Thin layer chromatography identification method of gourd retinervus medicinals

[0056] 1. Main instruments and reagents

[0057] Instrument: thin layer automatic imager (CAMAG TLC VISUALIZER), ME204E electronic analytical balance (Mettler-Toledo Instruments (Shanghai) Co., Ltd.), KQ-250E ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.), HWS-28 type electric heating constant temperature water bath (Shanghai Yiheng Scientific Instruments Co., Ltd.), silica gel G thin layer plate (Qingdao Haoyang Chemical Co., Ltd.), silica gel G thin layer plate (Chip Silicon Valley), silica gel G thin layer plate (Qingdao Kangyexin Medicinal Silica Gel Co., Ltd.).

[0058] Reagents: methanol, ethanol, cyclohexane, petroleum ether, ethyl formate, ethyl acetate, formic acid, vanillin, aluminum trichloride, all purchased from National Pharmaceutical Group Chemical Reagents Co., Ltd., toluene, acetone, sulfuric acid, all purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd., the above reagents are all analytical pure, and water is pure water.

[0059] 2. Experimental sample

[0060] Luffa cylindrica L. control medicinal materials (380035-202109) were purchased from Chengdu Pusai Biological Technology Co., Ltd.

[0061] The 12 batches of Luffa cylindrica L. and 4 batches of Guangdong Luffa cylindrica L. samples were all from the main producing areas or the native producing areas in China, and the Luffa cylindrica L. all met the requirements of Chinese Pharmacopoeia 2020 edition. The medicinal material information table is shown in Table 1.

[0062] Table 1 Information table of Luffa cylindrica L. and Guangdong Luffa cylindrica L. medicinal material samples

[0063]

[0064] 3. Preparation method of test sample and control medicinal material solution

[0065] 3.1 Preparation of test sample solution

[0066] Method one: cut the product into pieces (the side length is not greater than 0.5 cm), take 2 g, add 50 mL of methanol, ultrasonic treatment for 30 minutes, filter, evaporate the filtrate to dryness, add 1 mL of methanol to the residue to dissolve, and use it as the test sample solution.

[0067] Method two: cut the product into pieces (the side length is not greater than 0.5 cm), take 2 g, add 50 mL of methanol, ultrasonic treatment for 30 minutes, filter, evaporate the filtrate to dryness, add 10 mL of water to the residue to dissolve, shake with ethyl acetate twice, each time 20 mL, combine the ethyl acetate liquid, evaporate to dryness, add 1 mL of methanol to the residue to dissolve, and use it as the test sample solution.

[0068] Method three: cut the product into pieces (the side length is not greater than 0.5 cm), take 2 g, add 50 mL of water, heat reflux for 30 minutes, filter, evaporate the filtrate to dryness, add 1 mL of methanol to the residue to dissolve, and use it as the test sample solution.

[0069] Method four: take the product cut (length not more than 0.5 cm), take 2 g, add water 50 mL, heat reflux 30 minutes, filter, concentrate the filtrate to 20 mL, extract with ethyl acetate 2 times, 20 mL each time, combine the ethyl acetate liquid, evaporate to dryness, add methanol 1 mL to dissolve, as the test sample solution.

[0070] 3.2 Preparation of control medicinal material solution

[0071] Method one: take the control medicinal material of luffa cordia 2 g, add methanol 50 mL, ultrasonic treatment for 30 minutes, filter, evaporate the filtrate to dryness, add methanol 1 mL to dissolve the residue, as the control medicinal material solution.

[0072] Method two: take the control medicinal material of luffa cordia 2 g, add methanol 50 mL, ultrasonic treatment for 30 minutes, filter, evaporate the filtrate to dryness, add water 10 mL to dissolve the residue, extract with ethyl acetate 2 times, 20 mL each time, combine the ethyl acetate liquid, evaporate to dryness, add methanol 1 mL to dissolve, as the control medicinal material solution.

[0073] Method three: take the control medicinal material of luffa cordia 2 g, add water 50 mL, heat reflux for 30 minutes, filter, evaporate the filtrate to dryness, add methanol 1 mL to dissolve the residue, as the control medicinal material solution.

[0074] Method four: take the control medicinal material of luffa cordia 2 g, add water 50 mL, heat reflux for 30 minutes, filter, concentrate the filtrate to 20 mL, extract with ethyl acetate 2 times, 20 mL each time, combine the ethyl acetate liquid, evaporate to dryness, add methanol 1 mL to dissolve, as the control medicinal material solution.

[0075] Take the sample (batch number: S1) and prepare the test sample solution according to the above four preparation methods, four control medicinal material solutions, take 5 μL of the test sample solution and the control medicinal material solution respectively, and point them on the same silica gel G thin layer plate, use toluene-ethyl acetate-formic acid (10:3:1) as the developing agent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105°C until the spots develop clearly, and observe under ultraviolet light (365 nm) respectively.

[0076] The experimental results show (see Figure 2 ), the test sample and the control medicinal material preparation method one and two have more spots, the test sample and the control medicinal material preparation method three and four have fewer spots, therefore, the test sample and the control medicinal material preparation method one is selected as the preparation method of the test sample and the control medicinal material solution.

[0077] 4. Investigation of different particle sizes

[0078] Luffa cylindrica is a kind of medicinal material of fiber bundle, which is light and loose. After being beaten, it becomes fluffy. Therefore, the effects of cutting (the length of each side is not more than 0.5 cm) and pulverizing (passing through a sieve) on Luffa cylindrica were investigated.

[0079] Take Luffa cylindrica medicinal material (batch number: S1) and pulverize it (passing through a sieve) or cut it (the length of each side is not more than 0.5 cm), and then take 2 g of each, add 50 mL of methanol, and ultrasonically treat for 30 minutes. Filter, evaporate the filtrate, add 1 mL of methanol to the residue to dissolve it, and use it as a test solution. Take 2 g of Luffa cylindrica control medicinal material, and prepare a control medicinal material solution in the same way. Test by thin layer chromatography (general rule 0502), take 5 μL of each of the above two solutions, and point them on the same silica gel G thin layer plate. Use toluene-ethyl acetate-formic acid (10:3:1) as a developing agent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105°C until the spots develop clearly, and observe under ultraviolet light (365 nm).

[0080] The experimental results show (see Table 1) Figure 3 ), pulverizing (passing through a sieve) and cutting (the length of each side is not more than 0.5 cm) have no effect on the number of chromatographic spots, clarity, and distribution. Luffa cylindrica medicinal material is a kind of medicinal material of fiber bundle, which is light and loose. After being beaten, it becomes fluffy. It is difficult to sieve. Thin layer identification is mainly a qualitative index. Considering the nature of Luffa cylindrica itself and the simplicity of operation, Luffa cylindrica medicinal material is treated by cutting (the length of each side is not more than 0.5 cm).

[0081] 5. Determination of thin layer chromatography conditions

[0082] 5.1 Investigation of different developing agents

[0083] Take cut Luffa cylindrica (the length of each side is not more than 0.5 cm) (batch number: S1), take 2 g, add 50 mL of methanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate, add 1 mL of methanol to the residue to dissolve it, and use it as a test solution. Take 2 g of Luffa cylindrica control medicinal material, and prepare a control medicinal material solution in the same way. Take 5 μL of each of the above test solution and control medicinal material solution, and point them on the same silica gel G thin layer plate. Use (1) cyclohexane-ethyl acetate (1:2), (2) petroleum ether-acetone (8:1), and (3) toluene-ethyl acetate-formic acid (10:3:1) as developing agents, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105°C until the spots develop clearly, and observe under ultraviolet light (365 nm) respectively.

[0084] The experimental results show (see Table 2) Figure 4), although there were spots, but the number of spots was less, the separation effect was poor, the overall effect was poor; the developing condition was toluene-ethyl formate-formic acid (10:3:1), the test sample solution had more spots under the ultraviolet light (365nm), the distribution was uniform, and the effect was better. Therefore, toluene-ethyl formate-formic acid (10:3:1) was selected as the developing agent of thin layer chromatography.

[0085] 5.2 Investigation of different color reagents

[0086] Take the product cut into pieces (edge length not more than 0.5 cm) (batch number: S1), take 2g, add methanol 50mL, ultrasonic treatment for 30 minutes, filter, evaporate the filtrate to dryness, add methanol 1mL to dissolve as test sample solution. Take another 2g of luffa sponge control medicinal material, prepare the control medicinal material solution in the same way. Take 5μL of the above test sample solution and control medicinal material solution respectively, and point them on the same silica gel G thin layer plate, use toluene-ethyl formate-formic acid (10:3:1) as the developing agent, develop, take out, air dry, and spray with 10% sulfuric acid ethanol solution, 5% vanillin sulfuric acid solution, and aluminum chloride test solution, heat to 105℃ until the spots develop clearly, and observe under the ultraviolet light (365nm).

[0087] The experimental results show (see Figure 5 ), the color reagent of spraying 10% sulfuric acid ethanol solution has more spots under the ultraviolet light (365nm), the distribution is uniform, and the effect is better. Therefore, the color developing condition selected is: spray 10% sulfuric acid ethanol solution, heat to 105℃ until the spots develop clearly, and observe under the ultraviolet light (365nm).

[0088] 5.3 Investigation of different sample application amounts

[0089] Take the sample (batch number: S1) to prepare the test sample solution and control medicinal material solution according to the test sample solution preparation method and control medicinal material solution preparation method determined in the foregoing, point the different test sample solution and control medicinal material solution sample application amounts (1μL, 3μL, 5μL, 10μL, 20μL) on the same silica gel G thin layer plate, use toluene-ethyl formate-formic acid (10:3:1) as the developing agent, develop, take out, air dry, spray with 10% sulfuric acid ethanol solution, heat to 105℃ until the spots develop clearly, and observe under the ultraviolet light (365nm).

[0090] The experimental results show (see Figure 6), the sample solution, the control drug solution, the sample solution, the control drug solution, and the negative control solution were each 5-10 μL, the luffa sponge drug sample chromatogram was relatively clear in the corresponding position of the control drug, had good separation degree, and had no other interference. Considering the differences between different batches of samples, the sample solution and the control drug solution were selected to be 5-10 μL.

[0091] 5.4 Preliminary determination of the thin layer chromatography method

[0092] Take the product, cut it into pieces (with a side length of not more than 0.5 cm), weigh 2 g, add 50 mL of methanol, ultrasonic treatment for 30 minutes, filter, evaporate the filtrate to dryness, add 1 mL of methanol to the residue to dissolve it as a test solution. Take another 2 g of luffa sponge control drug, and prepare a control drug solution in the same way. According to the test of thin layer chromatography (Chinese Pharmacopoeia 2020 edition general 0502), 5-10 μL of the above two solutions were taken and spotted on the same silica gel G thin layer plate, with toluene-ethyl acetate-formic acid (10:3:1) as the developing agent, developed, removed, air dried, sprayed with 10% sulfuric acid ethanol solution, heated until the spots were clearly colored, and observed under ultraviolet light (365 nm). In the test sample chromatogram, the same colored fluorescent spots appeared in the corresponding position of the control drug chromatogram.

[0093] 6, Methodology verification

[0094] 6.1 Specificity test

[0095] Take 5 μL of the test solution (batch number: S1), the control drug solution (S), and the negative control solution (F) and spot them on the same silica gel G thin layer plate, with toluene-ethyl acetate-formic acid (10:3:1) as the developing agent, develop, remove, air dry, spray with 10% sulfuric acid ethanol solution, heat until the spots are clearly colored, and observe under ultraviolet light (365 nm).

[0096] The experimental results show (see Figure 7 ), the luffa sponge test sample chromatogram and the control drug chromatogram show the same colored fluorescent spots in the corresponding position, and the negative control has no interference, indicating that the thin layer method has good specificity.

[0097] 6.2 Durability test

[0098] 6.2.1 Investigation of different temperatures

[0099] Take 5 μL of the test solution (batch number: S1) and the control drug solution and spot them on the same silica gel G thin layer plate, with toluene-ethyl acetate-formic acid (10:3:1) as the developing agent, develop at different temperatures (4℃, 20℃, 30℃), remove, air dry, spray with 10% sulfuric acid ethanol solution, heat until the spots are clearly colored, and observe under ultraviolet light (365 nm).

[0100] The experimental results show (see Figure 8 ), under different temperature conditions, the test chromatogram of Luffa cylindrica (L.) Roem. and the control chromatogram show the same color fluorescent spots at the corresponding positions, and both achieve basic separation. Temperature has no significant effect on the thin-layer chromatography of Luffa cylindrica (L.) Roem., indicating that the thin-layer chromatography method has good durability to different temperatures.

[0101] 6.2.2 Investigation of different humidity

[0102] Take 5 μL of the test solution (batch number: S1) and the control solution of medicinal materials, respectively, and spot them on the same silica gel G thin-layer plate. Develop with toluene-ethyl acetate-formic acid (10:3:1) as the developing agent under different humidity (18%, 37%, 88%), take out, air dry, spray with 10% sulfuric acid ethanol solution, heat until the spots develop clearly, and observe under ultraviolet light (365 nm).

[0103] The experimental results show (see Figure 9 ), under different humidity conditions, the test chromatogram of Luffa cylindrica (L.) Roem. and the control chromatogram show the same color fluorescent spots at the corresponding positions, and both achieve basic separation. Humidity has no effect on the thin-layer chromatography of Luffa cylindrica (L.) Roem., indicating that the thin-layer chromatography method has good durability to different humidity.

[0104] 6.2.3 Investigation of different thin-layer plates

[0105] Take 5 μL of the test solution (batch number: S1) and the control solution of medicinal materials, respectively, and spot them on different brands (Qingdao Haoyang, Kangye Xin, and Xinsilu Valley) of silica gel G thin-layer plates. Develop with toluene-ethyl acetate-formic acid (10:3:1) as the developing agent, take out, air dry, spray with 10% sulfuric acid ethanol solution, heat until the spots develop clearly, and observe under ultraviolet light (365 nm).

[0106] The experimental results show (see Figure 10 ), under different humidity conditions, the test chromatogram of Luffa cylindrica (L.) Roem. and the control chromatogram show the same color fluorescent spots at the corresponding positions, and both achieve basic separation. Humidity has no effect on the thin-layer chromatography of Luffa cylindrica (L.) Roem., indicating that the thin-layer chromatography method has good durability to different humidity.

[0107] 6.3 Final determination of the thin-layer chromatography method

[0108] According to the above research results, the thin-layer chromatography method for Luffa cylindrica (L.) Roem. is finally determined as follows:

[0109] Take the product cut into pieces (edge length not more than 0.5 cm), take 2 g, add methanol 50 mL, ultrasonic treatment for 30 minutes, filter, evaporate the filtrate to dryness, add methanol 1 mL to the residue to dissolve as the test solution. Another 2 g of luffa sponge control medicinal materials were prepared into a control medicinal material solution by the same method. According to the thin layer chromatography (Chinese Pharmacopoeia 2020 edition general 0502) test, 5-10 μL of the above two solutions were taken and spotted on the same silica gel G thin layer plate, with toluene-ethyl acetate-formic acid (10:3:1) as the developing agent, developed, taken out, air dried, sprayed with 10% sulfuric acid ethanol solution, heated until the spots were clearly colored, and observed under ultraviolet light (365 nm).

[0110] Example 2: Thin layer identification method of different batches of luffa sponge medicinal materials

[0111] Take different batches (batch numbers S1-S12) of luffa sponge to be tested medicinal materials, prepare the test solution according to the preparation method of the test solution in Example 1, and develop on the same silica gel G thin layer plate with toluene-ethyl acetate-formic acid (10:3:1) as the developing agent, take out, air dry, spray with 10% sulfuric acid ethanol solution, heat until the spots are clearly colored, and observe under ultraviolet light (365 nm).

[0112] The experimental results show (see Figure 11 ), the test solution chromatogram of different batches of luffa sponge to be tested medicinal materials all showed fluorescent spots of the same color at the corresponding positions of the control medicinal material chromatogram. There was no significant difference between different batches.

[0113] Example 3: Thin layer identification method of luffa sponge and Guangdong luffa sponge

[0114] Take 3 batches (batch numbers S1-S3) of luffa sponge to be tested medicinal materials and 4 batches (S13-S16) of Guangdong luffa sponge to be tested medicinal materials, prepare the test solution according to the preparation method of the test solution in Example 1, take 5 μL of the above two solutions, and spot on the same silica gel G thin layer plate, with toluene-ethyl acetate-formic acid (10:3:1) as the developing agent, develop, take out, air dry, spray with 10% sulfuric acid ethanol solution, heat until the spots are clearly colored, and observe under ultraviolet light (365 nm).

[0115] The experimental results show (see Figure 12 ), the test solution chromatogram of Guangdong luffa sponge medicinal materials did not show spots of the same color at the corresponding positions of the luffa sponge control medicinal material chromatogram and the luffa sponge medicinal material test solution chromatogram, and there were 3 more spots at Rf values of about 0.38-0.62; the test solution chromatogram of the genuine luffa sponge medicinal material showed spots of the same color at the corresponding positions of the luffa sponge control medicinal material chromatogram. The thin layer identification method established in the present application can distinguish between genuine and fake luffa sponge, thereby standardizing the use of luffa sponge and achieving the purpose of clearing up the source.

[0116] The above examples are only used to illustrate several embodiments of the present application, which are described in a more specific and detailed manner, but should not be understood as any limitation to the scope of the present application. It should be clear that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present application, which are all within the protection scope of the present application.

Claims

1. A thin-layer chromatography method for detecting loofah sponge medicinal material, comprising the following steps: 1) preparation of test solution; 2) Preparation of the control herbal solution; and 3) Detection of medicinal materials using thin-layer chromatography; The chromatographic conditions for the thin-layer detection method described in step 3) include: the stationary phase is silica gel G thin-layer plate; the developing solvent is a ternary system of toluene-ethyl formate-formic acid with a volume ratio of 10:3:1; the colorimetric reagent is sulfuric acid ethanol; and the detection wavelength is 365 nm. The test solution in step 1) is prepared by the following method: the loofah sponge to be tested is placed in the extraction solvent for extraction, filtered, the filtrate is evaporated to dryness, and the residue is dissolved in a redissolving solvent to obtain the solution; the extraction solvent is methanol.

2. The method according to claim 1, characterized in that, The chromatographic conditions for the thin-layer detection method also include: a sample spotting volume of 5-10 μL.

3. The method according to claim 1, characterized in that, The reference medicinal material solution mentioned in step 2) is prepared by the following method: the loofah reference medicinal material is placed in the extraction solvent for extraction, filtered, the filtrate is evaporated to dryness, and the residue is dissolved in a redissolving solvent to obtain the solution; the extraction solvent is methanol.

4. The method according to claim 1 or 3, characterized in that, The ratio of the medicinal material to the extraction solvent is 1g:15-35mL.

5. The method according to claim 4, characterized in that, The ratio of the medicinal material to the extraction solvent is 1g:20-30mL.

6. The method according to claim 5, characterized in that, The ratio of the medicinal material to the extraction solvent is 1g:25mL.

7. The method according to claim 1 or 3, characterized in that, The extraction method is selected from either ultrasonic extraction or reflux extraction.

8. The method according to claim 7, characterized in that, The extraction method is ultrasonic extraction.

9. The method according to claim 1 or 3, characterized in that, The extraction time is 10-60 minutes.

10. The method according to claim 9, characterized in that, The extraction time is 20-40 minutes.

11. The method according to claim 10, characterized in that, The extraction time was 30 minutes.

12. The method according to claim 1 or 3, characterized in that, The resolution solvent is selected from water, alcohol, or a mixture thereof.

13. The method according to claim 12, characterized in that, The resolution solvent is an alcohol.

14. The method according to claim 13, characterized in that, The resolution solvent is methanol.

15. The method according to claim 1 or 3, characterized in that, The ratio of the medicinal material to the reconstitution solvent is 1g:0.1-1mL.

16. The method according to claim 15, characterized in that, The ratio of the medicinal material to the reconstitution solvent is 1g:0.2-0.8mL.

17. The method according to claim 16, characterized in that, The ratio of the medicinal material to the reconstitution solvent is 1g:0.5mL.

18. The method according to claim 1, characterized in that, The colorimetric reagent is a 10% (v / v) sulfuric acid ethanol solution.

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