Thin-layer chromatography identification method of loofah sponge medicinal material

Abstract

The invention belongs to the technical field of traditional Chinese medicine analysis, and relates to a thin-layer chromatography identification method of a loofah sponge medicinal material. The method comprises the following steps: 1) preparing a test solution; 2) preparing a reference medicinal material solution; and (3) identifying the medicinal materials by utilizing the thin layer chromatography, so that the problems that the operation is tedious, the number of chromatographic spots is small, the components in the loofah sponge cannot be comprehensively reflected, and no obvious distinguishing point exists in the genuine and fake products in the existing method are solved at one time, and the method is simple, convenient and rapid to operate, richer and clearer in spot information, good in reproducibility and high in accuracy. The application range is wide, the main chemical components of the loofah sponge are presented to a greater extent, and the method can be used for identifying genuine and fake products.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine analysis and relates to a thin-layer chromatography identification method for loofah medicinal materials. Background Art

[0002] Luffa is the vascular bundle of the dried mature fruit of Luffa cylindrica (L.) Roem., a plant of the Cucurbitaceae family. It is sweet and mild in nature and enters the lung, stomach and liver meridians. It has the effects of dispelling wind, unblocking meridians, activating blood circulation and promoting lactation. It is mainly used to treat arthralgia, cramps, chest and flank pain, milk blockage, mastitis and swelling and pain, etc. [1] Modern research has shown that loofah contains polysaccharides (xylan, mannan, galactan) [2][3] , proteins, amino acids, peptides, glycosides (saponins, cardiac glycosides), anthraquinones, phenols, tannins, flavonoids, coumarins, terpene lactones and alkaloids [4] It has pharmacological effects such as diuresis, swelling, gout, anti-inflammatory, analgesia, hypoglycemia, anti-oxidation, and prevention of myocardial ischemia, and is widely used in clinical practice. [5] .

[0003] According to the Flora of China, the genus Luffa of the Cucurbitaceae family is divided into two types: luffa and Guangdong luffa (or Yue luffa), which are of the same genus but different species. Luffa is grown in all parts of the north and south, and is the main circulating variety in the luffa market. There are cases in the market where the vascular bundles of the dried mature fruits of Guangdong luffa Luffa acutangula (L.) Roem. are adulterated as luffa. The Chinese Pharmacopoeia (2020 edition) only lists Luffa cylindrica (L.) Roem. as its source under the entry of luffa, so Guangdong luffa is a fake luffa, but Guangdong luffa also contains xylan, mannan and galactan, etc. [6] The main chemical components are similar. In terms of properties: The main difference between Guangdong loofah and loofah is that the fruit of Guangdong loofah has 8 to 10 longitudinal sharp edges and grooves, the seeds have a reticular pattern, no narrow wing-like edges, and two shallow cracks at the base; the fruit of loofah is smooth, usually with dark longitudinal stripes, the seeds are smooth, and the edges are narrow wing-like [7] The above two types can be distinguished by the appearance of the fruit, but loofah is the vascular bundle of dried mature fruit. After removing the peel and seeds, the appearance of loofah and Guangdong loofah is similar and difficult to distinguish. Considering that conventional inspection items cannot distinguish genuine products from fakes, and sensory identification has the disadvantages of relying on experience and sometimes low resolution, especially the similarity of loofah and Guangdong loofah, it brings difficulties to identification. At present, the statutory standards for loofah medicinal materials only include sources, properties, microscopy, etc., and the existing quality evaluation methods are difficult to control the quality of loofah medicinal materials.

[0004] Study on the identification of loofah and Guangdong loofah and the quality standard of loofah; Kang Along[8] et al. established a thin layer chromatography identification method for loofah, loofah blocks, fried loofah and loofah charcoal using loofah as a control medicinal material; the "Guangdong Provincial Traditional Chinese Medicine Standard Volume 3" contains a thin layer chromatography identification method for Guangdong loofah; CN 105203670 B discloses a method for establishing a high performance liquid chromatography fingerprint of loofah, and CN 115436520 A discloses a method for constructing and detecting a UPLC characteristic spectrum of loofah. Both established loofah characteristic spectrum methods, and both have 7 peaks.

[0005] However, the above methods have some defects: (1) The main components of the test solution prepared in the method established by Kang Along et al. are glycosides and alkaloids, which cannot fully reflect the chemical composition of loofah. The operation is cumbersome, the difference points are located lower and closer, and are easily affected by the environment or operation, resulting in unclear results. (2) The thin layer chromatography method in the "Guangdong Provincial Traditional Chinese Medicine Standard Volume 3" is simple to operate, but its developing agent has low polarity, the chromatographic spot information is less, and there is no obvious difference between the genuine and fake products. (3) The methods in CN 105203670 B and CN 115436520 A have high requirements for the required instruments when establishing characteristic spectra, are relatively complex to operate, and have high requirements for experimental personnel.

[0006] The thin layer chromatography method is characterized by simple operation, rapidity, good reproducibility, obvious image characteristics, and wide development range, and is the main method in the field of traditional Chinese medicine analysis. CN 115684455 A discloses a thin layer chromatography identification method for loofah sponge and its processed products. However, the inventor of the present invention has found through repeated experiments that the number of spots in this method is small, and the color development effect under ultraviolet light (365nm) is less clear than that under sunlight, and it is impossible to effectively distinguish loofah sponge from counterfeit Guangdong loofah sponge (such as Figure 1 The sample processing is complicated and the operation efficiency is low.

[0007] Therefore, how to establish a thin layer chromatography identification method for loofah sponge, which can also be used as a specific identification method to distinguish loofah sponge from its easily confused product Guangdong loofah, has important practical significance for improving the standard of loofah sponge and accurate clinical medication.

[0008] References

[0009] [1] Chinese Pharmacopoeia Committee. Pharmacopoeia of the People's Republic of China 2020 Edition Volume 1[S]. Beijing: China Medical Science and Technology Press, 2020: 126.

[0010] [2] Jiangsu Institute of Botany. Xinhua Materia Medica Compendium (Volume 2)[M]. Shanghai: Shanghai Science and Technology Press, 1991.

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

[0012] [4] Li Yan, Li Wenjia, Wang Yikui, et al. Analysis of chemical components of loofah[J]. 2011, 24(2): 529-534.

[0013] [5] Yang Hua, Gao Gao, Zhao Bing, et al. Overview of research on the pharmacy of loofah[J]. Anhui Agricultural Sciences, 2011, 39(34): 20990~20991.

[0014] [6] Guangdong Provincial Standard for Traditional Chinese Medicines Volume 3[S]. 2019:428-431.

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

[0016] [8] Kang A-long, Tang Ying-shuang, Sun Cheng-rong, et al. Study on the quality standards of three different processed pieces of loofah[J]. Journal of Shaanxi College of Traditional Chinese Medicine, 2011, 34(4):87+95. Summary of the invention

[0017] Problem that the invention aims to solve

[0018] To solve the problems existing in the above prior art, the present invention aims to provide a thin layer chromatography identification method for loofah medicinal material, which is simple and quick to operate, has abundant and clear spots, good reproducibility and wide application range.

[0019] Solutions for solving problems

[0020] The present invention provides a thin layer chromatography identification method for loofah medicinal material, which comprises the following steps:

[0021] 1) Preparation of test solution:

[0022] The tested medicinal material of loofah is placed in an extraction solvent for extraction, the filtrate is filtered and evaporated to dryness, and the residue is dissolved in a re-dissolving solvent to obtain a test solution;

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

[0024] The loofah control medicinal material is placed in an extraction solvent for extraction, the filtrate is filtered and evaporated to dryness, and the residue is dissolved in a re-dissolving solvent to obtain a control medicinal material solution;

[0025] 3) Identification of medicinal materials using thin layer chromatography:

[0026] According to the thin layer chromatography method (General Rule 0502), the test solution prepared in the above step 1) and the control medicinal material solution prepared in the step 2) are respectively spotted on the same silica gel thin layer plate, and the aromatic hydrocarbon-carboxylic acid ester-carboxylic acid ternary system is used as the developing agent. The plate is developed, taken out, dried, and then sprayed with sulfuric acid ethanol solution. The plate is heated until the spots are clearly colored and examined under ultraviolet light (365nm). If the thin layer chromatogram of the test solution and the thin layer chromatogram of the control medicinal material solution have fluorescent spots at the same Rf value, the loofah to be tested medicinal material is judged to be authentic, otherwise it is judged to be a counterfeit.

[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 medicinal material to the extraction solvent in step 1) or 2) is 1 g: 15-35 mL, preferably 1 g: 20-30 mL, 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, more preferably 30 min.

[0031] Preferably, in the above method, the re-dissolving 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 dosage ratio of the loofah medicinal material to the re-dissolving solvent in step 1) or 2) is 1 g: 0.1-1 mL, preferably 1 g: 0.2-0.8 mL, 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 ternary system of toluene-ethyl formate-formic acid.

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

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

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

[0038] Effects of the Invention

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

[0040] (1) The present invention adopts a simple and quick pretreatment method to obtain a test solution and a control medicinal material solution, which are respectively spotted on the same thin layer plate, developed with a suitable developer, developed with a suitable developer, and examined under ultraviolet light to obtain a multi-information loofah thin layer chromatogram.

[0041] (2) This method presents the main chemical components of loofah to a greater extent, and the chromatographic spot information is richer, which can be used to identify authentic and counterfeit products. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0043] Figure 2 These are thin layer chromatograms of the test solution and the control medicinal material solution prepared by the four methods (wherein 1 to 4 correspond to the test solution prepared by methods one to four, respectively; S1 to S4 correspond to the control medicinal material solutions prepared by methods one to four, respectively).

[0044] Figure 3 The thin layer chromatograms of loofah medicinal materials under the conditions of different particle sizes of the tested medicinal materials (wherein 1 corresponds to the test solution of the loofah medicinal material to be tested that has been shredded (side length is not greater than 0.5 cm); 2 corresponds to the test solution of the loofah medicinal material to be tested that has been powdered (passed through a No. 1 sieve); S corresponds to the control medicinal material solution).

[0045] Figure 4 The thin layer chromatograms of loofah medicinal material obtained using different developing agents (where 1 corresponds to the test solution; S corresponds to the control medicinal material solution).

[0046] Figure 5 The thin layer chromatograms of Luffa sponge medicinal material obtained using different color developers (where 1 corresponds to the test solution; S corresponds to the control medicinal material solution).

[0047] Figure 6 The thin layer chromatograms of loofah medicinal material under different spotting amounts (where S1 to S5 correspond to control medicinal material solutions, and the spotting amounts are 1 μL, 3 μL, 5 μL, 10 μL, and 20 μL, respectively; 1 to 5 correspond to test sample solutions, and the spotting amounts are 1 μL, 3 μL, 5 μL, 10 μL, and 20 μL, respectively).

[0048] Figure 7 This is the specific chromatogram of the thin layer identification of loofah medicinal material (where 1 corresponds to 3 parallel test sample solutions; S corresponds to the control medicinal material solution; F corresponds to the negative control solution).

[0049] Figure 8 The thin layer chromatograms of loofah medicinal material under different temperature conditions (where 1 corresponds to three test sample solutions set in parallel; S corresponds to the control medicinal material solution).

[0050] Fig. 9 The thin layer chromatograms of loofah medicinal material under different humidity conditions (where 1 corresponds to three test sample solutions set in parallel; S corresponds to the control medicinal material solution).

[0051] Fig.10 The thin layer chromatograms of loofah medicinal material under different thin layer plate conditions (where 1 corresponds to three test sample solutions set in parallel; S corresponds to the control medicinal material solution).

[0052] Fig.11 The thin layer chromatograms of different batches of luffa medicinal materials (wherein, 1 to 12 correspond to the test solution of 12 batches of luffa medicinal materials to be tested; S corresponds to the control medicinal material solution).

[0053] Fig.12 The results of thin layer chromatogram comparison between loofah sponge and Guangdong loofah sponge obtained by the method of the present invention are shown in Figure 1 (wherein 1 to 3 correspond to test solutions of 3 batches of loofah sponge medicinal materials to be tested; 4 to 7 correspond to test solutions of 4 batches of Guangdong loofah sponge medicinal materials to be tested; S corresponds to the control medicinal material solution). DETAILED DESCRIPTION

[0054] In order to better illustrate the present invention, numerous specific details are provided in the following specific embodiments. It should be understood by those skilled in the art that the present invention can be implemented without certain specific details. In other examples, methods, means, equipment and steps well known to those skilled in the art are not described in detail in order to highlight the subject matter of the present invention.

[0055] Embodiment 1: thin layer chromatography identification method of loofah medicinal material

[0056] 1. Main instruments and reagents

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

[0058] Reagents: Methanol, ethanol, cyclohexane, petroleum ether, ethyl formate, ethyl acetate, formic acid, vanillin, and aluminum chloride were purchased from Sinopharm Chemical Reagent Co., Ltd.; toluene, acetone, and sulfuric acid were purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd. All of the above reagents were analytical grade, and water was pure water.

[0059] 2. Experimental samples

[0060] The control medicinal material of loofah (380035-202109) was purchased from Chengdu Pusi Biotechnology Co., Ltd.

[0061] The 12 batches of loofah sponge and 4 batches of Guangdong loofah sponge samples all came from the main production areas or authentic production areas across the country. The loofah sponges all met the requirements of the 2020 edition of the "Chinese Pharmacopoeia". The medicinal material information table is shown in Table 1.

[0062] Table 1 Information of loofah and Guangdong loofah medicinal material samples

[0063]

[0064] 3. Investigation of the preparation methods of test sample and control medicinal material solutions

[0065] 3.1 Preparation of test solution

[0066] Method 1: Cut the product into pieces (side length not more than 0.5 cm), weigh 2 g, add 50 mL of methanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, add 1 mL of methanol to the residue to dissolve it, and use it as the test solution.

[0067] Method 2: Cut the product into pieces (side length not more than 0.5 cm), weigh 2 g, add 50 mL of methanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, add 10 mL of water to the residue to dissolve it, shake and extract with ethyl acetate twice, 20 mL each time, combine the ethyl acetate liquid, evaporate to dryness, add 1 mL of methanol to the residue to dissolve it, and use it as the test solution.

[0068] Method 3: Cut the product into pieces (side length not more than 0.5 cm), weigh 2 g, add 50 mL of water, heat and reflux for 30 minutes, filter, evaporate the filtrate to dryness, add 1 mL of methanol to the residue to dissolve it, and use it as the test solution.

[0069] Method 4: Cut the product into pieces (side length not more than 0.5 cm), weigh 2 g, add 50 mL of water, heat and reflux for 30 minutes, filter, concentrate the filtrate to 20 mL, shake and extract with ethyl acetate twice, 20 mL each time, combine the ethyl acetate solution, evaporate to dryness, add 1 mL of methanol to the residue to dissolve it, and use it as the test solution.

[0070] 3.2 Preparation of control medicinal material solution

[0071] Method 1: Take 2 g of loofah control medicinal material, add 50 mL of methanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, add 1 mL of methanol to the residue to dissolve it, and use it as the control medicinal material solution.

[0072] Method 2: Take 2 g of loofah control medicinal material, add 50 mL of methanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, add 10 mL of water to the residue to dissolve it, shake and extract with ethyl acetate twice, 20 mL each time, combine the ethyl acetate liquid, evaporate to dryness, add 1 mL of methanol to the residue to dissolve it, and use it as the control medicinal material solution.

[0073] Method 3: Take 2 g of loofah control medicinal material, add 50 mL of water, heat and reflux for 30 minutes, filter, evaporate the filtrate to dryness, add 1 mL of methanol to the residue to dissolve it, and use it as the control medicinal material solution.

[0074] Method 4: Take 2 g of loofah control medicinal material, add 50 mL of water, heat and reflux for 30 minutes, filter, concentrate the filtrate to 20 mL, shake and extract with ethyl acetate twice, 20 mL each time, combine the ethyl acetate solution, evaporate to dryness, add 1 mL of methanol to the residue to dissolve it, and use it as the control medicinal material solution.

[0075] Take samples (batch number: S1) and prepare test solution and four control medicinal material solutions according to the above four preparation methods. Pipette 5 μL of the test solution and the control medicinal material solution, respectively, and spot 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, dry, and then spray with 10% sulfuric acid ethanol solution, heat at 105°C until the spots are clearly colored, and examine them under ultraviolet light (365nm).

[0076] The experimental results show that (see Figure 2 ), the test sample and control medicinal material preparation methods one and two had more spots, while the test sample and control medicinal material preparation methods three and four had fewer spots. Because the test sample and control medicinal material preparation method two was more complicated, the test sample and control medicinal material preparation method one was selected as the preparation method for the test sample and control medicinal material solutions.

[0077] 4. Investigation of different particle sizes

[0078] Luffa is a fiber bundle-type medicinal material with a light and loose texture. After being ground into powder, it becomes loose flocs. Therefore, the effects of cutting (side length not exceeding 0.5 cm) and crushing (passing through a No. 1 sieve) the luffa on the results were investigated.

[0079] Take the loofah medicinal material (batch number: S1) and crush (pass through a No. 1 sieve) and cut into pieces (side length not greater than 0.5 cm), weigh 2g respectively, add 50mL of methanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, add 1mL of methanol to the residue to dissolve, and use it as the test solution. Take another 2g of loofah control medicinal material and prepare the control medicinal material solution in the same way. According to the thin layer chromatography method (General Rule 0502), take 5μL of each of the above two solutions and spot 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, dry, and then spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly colored, and inspect under ultraviolet light (365nm).

[0080] The experimental results show that (see Figure 3 ), crushing (passing through a No. 1 sieve) and cutting (side length not greater than 0.5 cm) have no effect on the number, clarity and distribution of chromatographic spots. Because loofah is a fiber bundle, it is light and loose. After being ground into powder, it is a loose flocculent material that is difficult to sieve. Thin layer identification is mainly a qualitative indicator. Considering the properties of loofah itself and the ease of operation, loofah is cut into pieces (side length not greater than 0.5 cm).

[0081] 5. Determination of TLC conditions

[0082] 5.1 Investigation of different developing agents

[0083] Cut the product into pieces (side length not more than 0.5 cm) (batch number: S1), weigh 2 g, add 50 mL of methanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate, and dissolve the residue in 1 mL of methanol to prepare the test solution. Take 2 g of loofah sponge as a control medicinal material and prepare the control medicinal material solution in the same way. Take 5 μL of the above test solution and control medicinal material solution, respectively, and spot them on the same silica gel G thin layer plate, respectively using (1) cyclohexane-ethyl acetate (1:2), (2) petroleum ether-acetone (8:1), (3) toluene-ethyl formate-formic acid (10:3:1) as the developing agent, develop, take out, dry, and then spray with 10% sulfuric acid ethanol solution, heat at 105°C until the spots are clearly colored, and examine them under ultraviolet light (365 nm).

[0084] The experimental results show that (see Figure 4), under the same preparation method of the sample, when the developing conditions were cyclohexane-ethyl acetate (1:2) and petroleum ether-acetone (8:1), although there were spots, the number of spots was small, the separation effect was poor, and the overall effect was not good; when the developing conditions were toluene-ethyl formate-formic acid (10:3:1), the sample solution had more spots under ultraviolet light (365nm), the distribution was uniform, and the effect was good. Therefore, toluene-ethyl formate-formic acid (10:3:1) was selected as the thin layer chromatography developing solvent.

[0085] 5.2 Investigation of different color developers

[0086] Cut the product into pieces (side length not more than 0.5cm) (batch number: S1), weigh 2g, add 50mL of methanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate, add 1mL of methanol to the residue to dissolve, and use it as the test solution. Take another 2g of loofah control medicinal material, and prepare the control medicinal material solution in the same way. Take 5μL of the above test solution and control medicinal material solution, respectively, and spot 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, dry, spray with 10% sulfuric acid ethanol solution, 5% vanillin sulfuric acid solution, aluminum chloride test solution, heat at 105℃ until the spots are clearly colored, and inspect under ultraviolet light (365nm).

[0087] The experimental results show that (see Figure 5 ), spray 10% sulfuric acid ethanol solution as a color developer, and under ultraviolet light (365nm), there are more spots, which are evenly distributed and have a better effect. Therefore, the color development conditions selected are: spray 10% sulfuric acid ethanol solution and heat at 105℃ until the spots are clearly colored, and then inspect under ultraviolet light (365nm).

[0088] 5.3 Investigation of different spotting amounts

[0089] Take the sample (batch number: S1) and prepare the test solution and control medicinal material solution according to the test solution preparation method and control medicinal material solution preparation method determined above, spot them on the same silica gel G thin layer plate respectively, and examine the different test solution and control medicinal material solution spotting amounts (1 μL, 3 μL, 5 μL, 10 μL, 20 μL), use toluene-ethyl formate-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 are clearly colored, and inspect under ultraviolet light (365nm).

[0090] The experimental results show that (see Figure 6), when the sample volume of the test solution and the control medicinal material solution was 5-10 μL, the fluorescence spots of the loofah medicinal material chromatogram at the corresponding positions of the control medicinal material were clearer, with good separation and no other interference. Considering the differences between different batches of samples, the sample volume of the test solution and the control medicinal material solution was selected to be 5-10 μL.

[0091] 5.4 Preliminary determination of thin layer chromatography method

[0092] Cut the product into pieces (side length not more than 0.5cm), weigh 2g, add 50mL of methanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate, and add 1mL of methanol to the residue to dissolve it as the test solution. Take another 2g of loofah control medicinal material and prepare the control medicinal material solution in the same way. According to the thin layer chromatography method (General Rules 0502 of the 2020 Edition of the Chinese Pharmacopoeia), take 5-10μL of each of the above two solutions and spot 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, dry, spray with 10% sulfuric acid ethanol solution, heat until the spots are clearly colored, and examine under ultraviolet light (365nm). In the chromatogram of the test product, fluorescent spots of the same color appear at the corresponding position of the chromatogram of the control medicinal material.

[0093] 6. Methodological Validation

[0094] 6.1 Specificity test

[0095] Take 5 μL of the test solution (batch number: S1), control medicinal material solution (S) and negative control solution (F), respectively, and spot 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, dry, spray with 10% sulfuric acid ethanol solution, heat until the spots are clearly colored, and examine under ultraviolet light (365nm).

[0096] The experimental results show that (see Figure 7 ), the chromatogram of the test loofah and the chromatogram of the control medicinal material showed the same color fluorescent spots at the corresponding positions, and there was no interference from the negative control, indicating that the thin-layer method had good specificity.

[0097] 6.2 Durability test

[0098] 6.2.1 Investigation at different temperatures

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

[0100] The experimental results show that (see Figure 8 ), under different temperature conditions, the chromatograms of the test and control medicinal materials of loofah showed fluorescent spots of the same color at the corresponding positions, and both were basically separated. Temperature had no significant effect on the thin layer identification of loofah medicinal materials, indicating that the thin layer identification method had good durability at different temperatures.

[0101] 6.2.2 Investigation of different humidity

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

[0103] The experimental results show that (see Fig. 9 ), under different humidity conditions, the chromatograms of the test and control medicinal materials of loofah showed fluorescent spots of the same color at the corresponding positions, and both were basically separated. Humidity had no effect on the thin layer identification of loofah medicinal materials, indicating that the thin layer identification method had good durability under different humidity conditions.

[0104] 6.2.3 Investigation of different thin layer plates

[0105] Take 5 μL of the test solution (batch number: S1) and the control medicinal material solution, respectively, and spot them on silica gel G thin layer plates of different brands (Qingdao Ocean, Kangyexin, Core Silicon Valley), use toluene-ethyl formate-formic acid (10:3:1) as the developing agent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, heat until the spots are clearly colored, and examine under ultraviolet light (365nm).

[0106] The experimental results show that (see Fig.10 ), the chromatograms of the test and control medicinal materials of the loofah medicinal material in the thin layer plates of different manufacturers showed fluorescent spots of the same color at the corresponding positions, and the separation effect was good. The thin layer plates of different manufacturers had no significant effect on the thin layer identification of loofah medicinal materials, indicating that the thin layer identification method has good durability for thin layer plates of different manufacturers.

[0107] 6.3 Finalization of the TLC Method

[0108] According to the above research results, the TLC identification method of loofah medicinal materials was finally determined as follows:

[0109] Cut the product into pieces (side length not more than 0.5cm), weigh 2g, add 50mL of methanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate, add 1mL of methanol to the residue to dissolve it as the test solution. Take another 2g of loofah control medicinal material and prepare the control medicinal material solution in the same way. According to the thin layer chromatography method (General Rules 0502 of the 2020 Edition of the Chinese Pharmacopoeia), take 5-10μL of each of the above two solutions and spot 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, dry, spray with 10% sulfuric acid ethanol solution, heat until the spots are clearly colored, and inspect under ultraviolet light (365nm).

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

[0111] Take different batches (batch numbers S1 to S12) of loofah to be tested medicinal materials, and prepare test solutions according to the preparation method of the test solution in Example 1. 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, dry, spray with 10% sulfuric acid ethanol solution, heat until the spots are clearly colored, and inspect under ultraviolet light (365nm).

[0112] The experimental results show that (see Fig.11 ), the chromatograms of the test solution of different batches of loofah sponge showed fluorescent spots of the same color at the corresponding positions of the chromatograms of the control medicinal material. There was no significant difference between different batches.

[0113] Embodiment 3: Thin layer identification method of loofah and Guangdong loofah

[0114] Take 3 batches of loofah medicinal materials to be tested (batch numbers S1-S3) and 4 batches of Guangdong loofah medicinal materials to be tested (S13-S16), and prepare the test solution according to the preparation method of the test solution in Example 1. Take 5 μL of each of the above two solutions, spot 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, dry, spray with 10% sulfuric acid ethanol solution, heat until the spots are clearly colored, and inspect under ultraviolet light (365nm).

[0115] The experimental results show that (see Fig.12 ), the Guangdong loofah medicinal material sample chromatogram does not show spots of the same color at the corresponding positions of the loofah control medicinal material chromatogram and the loofah medicinal material sample chromatogram, and there are 3 more spots at the Rf value of about 0.38-0.62; the loofah authentic medicinal material sample chromatogram shows spots of the same color at the corresponding positions of the loofah control medicinal material chromatogram. The thin layer identification method established by the present invention can distinguish the genuine and counterfeit loofahs, and then standardize the use of loofahs to achieve the purpose of correcting the original source.

[0116] The above embodiments are only used to illustrate several embodiments of the present invention, and the description thereof is relatively specific and detailed, but it should not be understood that any limitation is imposed on the scope of the present invention. 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 invention, and these all fall within the scope of protection of the present invention.

Claims

1. A thin layer chromatography identification method for loofah medicinal material, comprising the following steps: 1) preparing a test solution; 2) Preparation of control medicinal material solution; and 3) identification of medicinal materials using thin layer chromatography; Wherein, the chromatographic conditions of the thin layer identification method in step 3) include: the stationary phase is a silica gel thin layer plate; the developing agent is an aromatic hydrocarbon-carboxylic acid ester-carboxylic acid ternary system, preferably a toluene-ethyl formate-formic acid ternary system; the color developing agent is ethanolic sulfuric acid; the detection wavelength is 365nm; Preferably, the chromatographic conditions of the thin layer identification method also include: the sample spotting volume is 5-10 μL.

2. The method according to claim 1, characterized in that The test solution in step 1) is prepared by the following method: extracting the to-be-tested medicinal material of loofah in an extraction solvent, filtering and evaporating the filtrate to dryness, and dissolving the residue in a re-dissolving solvent to obtain the test solution.

3. The method according to claim 1, characterized in that The control medicinal material solution in step 2) is prepared by the following method: extracting the loofah control medicinal material in an extraction solvent, filtering and evaporating the filtrate to dryness, and dissolving the residue in a re-dissolving solvent to obtain the solution.

4. The method according to claim 2 or 3, characterized in that: The extraction solvent is selected from any one of water, alcohol and a mixture thereof, preferably alcohol, and more preferably methanol.

5. The method according to claim 2 or 3, characterized in that: The dosage ratio of the medicinal material to the extraction solvent is 1g:15-35mL, preferably 1g:20-30mL, and more preferably 1g:25mL.

6. The method according to claim 2 or 3, characterized in that: The extraction method is selected from any one of ultrasonic extraction and reflux extraction, preferably ultrasonic extraction; and / or The extraction time is 10-60 min, preferably 20-40 min, more preferably 30 min.

7. The method according to claim 2 or 3, characterized in that: The re-dissolving solvent is selected from any one of water, alcohol or a mixture thereof, preferably alcohol, and more preferably methanol.

8. The method according to claim 2 or 3, characterized in that: The dosage ratio of the medicinal material to the reconstitution solvent is 1 g:0.1-1 mL, preferably 1 g:0.2-0.8 mL, and more preferably 1 g:0.5 mL.

9. The method according to claim 1, characterized in that: The stationary phase is a silica gel G thin layer plate; and / or The developing agent is a ternary system of toluene-ethyl formate-formic acid in a volume ratio of 10:3:1; and / or The color developer is a sulfuric acid ethanol solution with a volume percentage of 10%.

10. The method according to claim 1, characterized in that The medicinal material identification in step 3) is carried out by the following method: if the thin layer chromatogram of the test solution and the thin layer chromatogram of the control medicinal material solution have fluorescent spots at the same Rf value, the loofah medicinal material to be tested is judged to be authentic, otherwise it is judged to be a counterfeit.

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