A thin layer chromatography identification method for loofah and its processed products

Abstract

The present invention provides a thin layer chromatography identification method for loofah and its processed products, wherein the developing agent used in the thin layer chromatography identification method includes petroleum ether, dichloromethane, ethyl acetate and formic acid. The thin layer chromatography identification method for loofah and its processed products provided by the present invention has the characteristics of high accuracy, good separation effect and clear color spots, and can identify different processed products of loofah blocks and loofah charcoal, and secondly can provide a research basis for the change of chemical components in the process of processing from loofah blocks to loofah charcoal, and can also qualitatively identify loofah charcoal medicinal materials, decoction pieces, intermediates and formula granules, and control the quality of the entire process to ensure the uniformity, stability and reliability of its quality.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine analysis, and particularly relates to a thin layer chromatography identification method for loofah sponge and processed products thereof. Background Art

[0002] Luffa is the vascular bundle of the dried mature fruit of Luffa cylindrical (L.) Roem., a plant of the Cucurbitaceae family. It has the effects of promoting blood circulation, clearing away heat and resolving phlegm, detoxifying and reducing swelling, strengthening the heart and promoting urination, and is mainly used to treat chest and flank pain, coughing up phlegm due to lung heat, amenorrhea, and milk stagnation. Luffa contains a variety of luffa saponins, flavonoids and phenols, proteins, amino acids, oils and inorganic elements, and organic acids. Pharmacological experiments have shown that it has the effects of lowering blood lipids, relieving pain and calming the nerves, and has significant clinical effects.

[0003] The main processed products of loofah include loofah (block), stir-fried loofah and loofah charcoal. Among them, loofah (block) and loofah charcoal are more commonly used in clinical practice, but there are currently no quality standards, and there are few reports on the differences between the two processed products. Since samples of different dosage forms of loofah charcoal are made from loofah charcoal medicinal materials through a series of processes, some of which will destroy the properties of the original medicinal materials, an identification method with good differentiation and identification capabilities in the existing technology and simpler and faster operation is needed to better control the quality.

[0004] Kang A-long, Tang Ying-shuang, Sun Cheng-rong, Zhang Xian, Zhang Su-heng; Study on the quality standard of three different processed slices of luffa; Journal of Shaanxi University of Traditional Chinese Medicine, 2011, 7, 34(4) The quality control method of three different processed slices of luffa includes the following steps: preparation of the test solution: take 2g of luffa charcoal, cut into small pieces (no longer than 0.5cm), add 50mL of methanol, heat and reflux for 1h, cool, filter, evaporate the filtrate to dryness, add 10mL of water to dissolve the residue, extract twice with saturated n-butanol with 20mL each time, combine the n-butanol solution, evaporate to dryness, add 1mL of methanol to dissolve the residue, and use it as the test solution. Developing agent: chloroform-methanol-water (28:4:1) The lower layer solution is placed below 10℃ overnight. According to the thin layer chromatography method (Appendix VI B of Part I of the 2005 edition of the Chinese Pharmacopoeia), 10uL of the test solution and the control medicinal material solution were taken and spotted on the same silica gel G thin layer plate made of 0.5% sodium carboxymethyl cellulose solution as an adhesive, then taken out, dried, sprayed with 10% sulfuric acid ethanol solution, and heated at 105°C until the spots were clearly colored. When examined under ultraviolet light (365nm), spots of the same color appeared in the chromatogram of the test sample at the corresponding positions of the chromatogram of the control medicinal material. The test results are as follows: Figure 1As shown, 1 is 3uL of Luffa charcoal slices (YP202105-21-10); 2 is 5uL of Luffa charcoal slices (YP202105-21-10). The TLC results have poor separation, unclear spots, and little chromatographic information.

[0005] Therefore how to provide a thin layer chromatography identification method for loofah sponge and its processed products has become a problem that needs to be solved urgently. Summary of the invention

[0006] In view of the shortcomings of the prior art, the object of the present invention is to provide a thin layer chromatography identification method for loofah and its processed products. The present invention establishes a thin layer chromatography identification method with strong specificity for loofah and its processed products, providing an experimental basis for the establishment of its quality standard and subsequent research. The chromatographic result provided by the present invention has moderate separation, clear spots, rich, stable and reliable chromatographic information.

[0007] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a thin layer chromatography identification method for loofah sponge and its processed products, wherein the developing solvent used in the thin layer chromatography identification method includes petroleum ether, dichloromethane, ethyl acetate and formic acid.

[0009] In the present invention, the prepared loofah product comprises any one of loofah blocks, fried loofah or loofah charcoal, or a combination of at least two of them.

[0010] In the present invention, the volume ratio of petroleum ether, dichloromethane, ethyl acetate and formic acid is (1.3-1.7):(0.8-1.2):(1.8-2.2):(0.08-0.12);

[0011] Among them, "1.3-1.7" can be 1.3, 1.4, 1.5, 1.6, 1.7, etc.;

[0012] "0.8-1.2" can be 0.8, 0.9, 1, 1.1, 1.2, etc.;

[0013] "1.8-2.2" can be 1.8, 1.9, 2, 2.1, 2.2, etc.;

[0014] "0.08-0.12" can be 0.08, 0.09, 0.1, 0.11, 0.12, etc.

[0015] In the present invention, the boiling point of the petroleum ether is 60-90°C (for example, it may be 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, etc.).

[0016] In the present invention, the thin layer chromatography identification method specifically comprises the following steps:

[0017] The test sample solution is spotted on a silica gel thin layer plate and developed with a developing agent. After the silica gel thin layer plate is dried, it is first sprayed with sulfuric acid ethanol solution, dried, and then sprayed with vanillin solution and heated to obtain the position of each component in the test sample in the thin layer plate.

[0018] In the present invention, the mass content of sulfuric acid in the sulfuric acid ethanol solution is 8-12% (for example, it can be 8%, 9%, 10%, 11%, 12%, etc.);

[0019] Preferably, the mass content of vanillin in the vanillin sulfuric acid solution is 3-7% (for example, 3%, 4%, 5%, 6%, 7%, etc.).

[0020] Preferably, the heating temperature is 100-110°C (for example, it may be 100°C, 101°C, 102°C, 103°C, 104°C, 105°C, 106°C, 107°C, 108°C, 109°C, 110°C, etc.).

[0021] In the present invention, the preparation of the test solution in the thin layer chromatography identification method comprises the following steps:

[0022] (1) grinding luffa lao and / or luffa lao processed product, adding water, sonicating, filtering, and collecting solids;

[0023] (2) The solid obtained in step (1) is extracted with ethyl acetate, the obtained extract is desolventized, and then the volume is fixed with methanol to obtain a test solution.

[0024] In the present invention, in step (1), the solid-liquid ratio of the luffa lao and / or luffa lao processed product to water is 0.5:(15-25) g / mL;

[0025] Among them, "15-25" can be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, etc.

[0026] Preferably, in step (1), the power of the ultrasound is 100-500 W (for example, it can be 100 W, 150 W, 200 W, 250 W, 300 W, 350 W, 400 W, 450 W, 500 W, etc.), and the time of ultrasound is 20-40 min (for example, it can be 20 min, 22 min, 24 min, 26 min, 28 min, 30 min, 32 min, 34 min, 36 min, 38 min, 40 min, etc.).

[0027] In the present invention, the solid-liquid ratio of the luffa lao and / or luffa lao processed product to ethyl acetate is 0.5:(15-25) g / mL;

[0028] Among them, "15-25" can be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, etc.

[0029] Preferably, in step (2), the extraction is performed 1-3 times (for example, 1 time, 2 times, 3 times, etc.).

[0030] In the present invention, the sample spotting volume in the thin layer chromatography identification method is 5-8 μL (for example, it can be 5 μL, 6 μL, 7 μL, 8 μL, etc.).

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] The thin layer chromatography identification method of luffa and its processed products provided by the present invention has the characteristics of high accuracy, good separation effect and clear color spots, and can identify different processed products of luffa blocks and luffa charcoal. Secondly, it can provide a research basis for the change of chemical composition in the process of processing from luffa blocks to luffa charcoal, and can also perform qualitative identification of luffa charcoal medicinal materials, decoction pieces, intermediates and formula granules, and control the quality of the entire process to ensure the uniformity, stability and reliability of its quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 It is a thin layer chromatogram of the luffa charcoal decoction piece in the prior art; wherein 1 is the luffa charcoal decoction piece (YP202105-21-10) 3uL; 2 is the luffa charcoal decoction piece (YP202105-21-10) 5uL.

[0034] Figure 2 It is a thin layer chromatogram of luffa charcoal, wherein 1 is a negative control, 2 is luffa charcoal granules, 3 is a luffa charcoal intermediate, 4 is a luffa charcoal slice, and 5 is a luffa charcoal reference medicinal material.

[0035] Figure 3 These are thin layer chromatograms of 3 batches of luffa charcoal slices and 3 batches of luffa blocks, among which 1, 2, and 3 are 3 batches of luffa charcoal slices; 4, 5, and 6 are 3 batches of luffa blocks.

[0036] Figure 4 The thin layer chromatograms of luffa charcoal freeze-dried powder obtained by different extraction methods, among which 1, 2, 3, 4, 5, and 6 correspond to the test solutions a, b, c, d, e, and f, respectively.

[0037] Figure 5 This is the thin layer chromatogram of luffa charcoal freeze-dried powder obtained using developer 1, where 1, 2, 3, and 4 correspond to the test solutions a, d, e, and f, respectively.

[0038] Figure 6The thin layer chromatogram of luffa charcoal freeze-dried powder obtained by using developing agent 2, wherein 1 and 2 correspond to the test solution d and e, respectively.

[0039] Figure 7 This is the thin layer chromatogram of luffa charcoal freeze-dried powder obtained using developer 3, where 1, 2, and 3 correspond to the test solutions d, e, and f, respectively.

[0040] Figure 8 The thin layer chromatograms of luffa charcoal standard decoction freeze-dried powder with different spotting amounts, among which 1 is the blank control, 2, 3, 4 are standard decoction freeze-dried powder 202105-21-03, with spotting amounts of 5μL, 7μL, 10μL respectively; 5, 6, 7 are standard decoction freeze-dried powder 202105-21-09, with spotting amounts of 5μL, 7μL, 10μL respectively; 8, 9, 10 are standard decoction freeze-dried powder 202105-21-12, with spotting amounts of 5μL, 7μL, 10μL respectively; 11, 12, 13 are luffa charcoal control pieces, with spotting amounts of 1μL, 3μL, 5μL respectively.

[0041] Fig. 9 This is a thin layer chromatogram of luffa charcoal standard decoction freeze-dried powder obtained using the silica gel thin layer plate provided by Yinlong; among them, the points from left to right are: stigmasterol, luffa charcoal freeze-dried powder batch 03, luffa charcoal freeze-dried powder batch 09, luffa charcoal freeze-dried powder batch 11, and luffa control decoction piece.

[0042] Fig.10 This is a thin layer chromatogram of luffa charcoal standard decoction freeze-dried powder obtained using the silica gel thin layer plate provided by Zhifu; among them, the points from left to right are: stigmasterol, luffa charcoal freeze-dried powder batch 03, luffa charcoal freeze-dried powder batch 09, luffa charcoal freeze-dried powder batch 11, and luffa control decoction piece.

[0043] Fig.11 This is a thin layer chromatogram of luffa charcoal standard decoction freeze-dried powder obtained using the silica gel thin layer plate provided by Merck; among them, the points from left to right are: stigmasterol, luffa charcoal freeze-dried powder batch 03, luffa charcoal freeze-dried powder batch 09, luffa charcoal freeze-dried powder batch 11, and luffa control decoction piece.

[0044] Fig.12 It is a thin layer chromatogram of luffa charcoal standard decoction freeze-dried powder obtained at room temperature and humidity (23°C, 46%); wherein, the points from left to right are: stigmasterol, luffa charcoal freeze-dried powder batch 03, luffa charcoal freeze-dried powder batch 09, luffa charcoal freeze-dried powder batch 11, luffa control decoction piece.

[0045] Fig.13 It is a thin layer chromatogram of luffa charcoal standard decoction freeze-dried powder obtained at room temperature and high humidity (23°C, 82%); wherein, the points from left to right are: stigmasterol, luffa charcoal freeze-dried powder batch 03, luffa charcoal freeze-dried powder batch 09, luffa charcoal freeze-dried powder batch 11, luffa control decoction piece.

[0046] Fig.14 It is a thin layer chromatogram of luffa charcoal standard decoction freeze-dried powder obtained at room temperature and low humidity (23°C, 13%); among them, the points from left to right are: stigmasterol, luffa charcoal freeze-dried powder batch 03, luffa charcoal freeze-dried powder batch 09, luffa charcoal freeze-dried powder batch 11, and luffa control decoction piece.

[0047] Fig.15 It is a thin layer chromatogram of luffa charcoal standard decoction freeze-dried powder obtained at low temperature and normal humidity (4°C, 46%); wherein, the points from left to right are: stigmasterol, luffa charcoal freeze-dried powder batch 03, luffa charcoal freeze-dried powder batch 09, luffa charcoal freeze-dried powder batch 11, luffa control decoction piece.

[0048] Fig.16 This is the chromatogram of the methodological validation of Luffa charcoal standard decoction and freeze-dried powder, among which 1 is blank; 2-16 are 15 batches of standard decoction and freeze-dried powder batch numbers: 202105-21-01~202105-21-15; 17 is the Luffa charcoal control decoction piece.

[0049] Fig.17 The thin layer chromatograms of loofah charcoal particles with different spotting amounts, among which 1 is the blank control; 2, 3, 4 are loofah charcoal particles KL19022051 with spotting amounts of 5μL, 7μL, 10μL respectively; 5, 6, 7 are loofah charcoal particles KL19003391 with spotting amounts of 5μL, 7μL, 10μL respectively; 8, 9, 10 are loofah charcoal particles KL19022051 with spotting amounts of 5μL, 7μL, 10μL respectively; 11, 12, 13 are loofah charcoal control pieces with spotting amounts of 1μL, 3μL, 5μL respectively; the optimal spotting amount was determined to be 7 μL for the test sample and 5 μL for the control piece.

[0050] Fig.18 This is the chromatogram of the methodology validation of loofah charcoal particles; among them, 1 is the blank control; 2 is loofah charcoal particles KL19022051; 3 is loofah charcoal particles KL19003391; 4 is loofah charcoal particles KL19022051; 5 is loofah charcoal control decoction piece. DETAILED DESCRIPTION

[0051] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0052] The sources of the components in the following examples are as follows:

[0053]

[0054] Example 1

[0055] The present embodiment provides a thin layer chromatography identification method of luffa charcoal, and the thin layer chromatography identification method of luffa charcoal comprises the following steps:

[0056] Take 0.5g of luffa charcoal particles, luffa charcoal intermediates, and luffa charcoal slices, grind them into powder, add 20mL of water, treat with ultrasound (200W) for 30min, cool, extract with ethyl acetate twice by shaking, 20mL each time, combine the ethyl acetate solution, evaporate the ethyl acetate, and then dilute to 1mL with methanol as the test solution. Luffa charcoal reference medicinal material: Take 5g of luffa charcoal medicinal material and add 100mL of water, heat and boil for 30min, spin dry, and prepare the reference medicinal material solution in the same way. (Note: There is no luffa charcoal reference medicinal material in the China National Institute for Food and Drug Control and the market, so luffa charcoal medicinal material is used as the reference medicinal material first). According to the thin layer chromatography method (Chinese Pharmacopoeia 2015 edition Part IV General Rules 0502), 7 μL of the test sample (luffa charcoal granules, luffa charcoal intermediates, luffa charcoal control medicinal materials) solution and 5 μL of the luffa charcoal slice test sample solution were respectively spotted on the same silica gel G thin layer plate, and petroleum ether (60-90): dichloromethane: ethyl acetate: formic acid (1.5:1:2:0.1) was used as the developing agent. The plate was developed, taken out, dried, first sprayed with 10% sulfuric acid ethanol solution, dried, and then sprayed with 5% vanillin sulfuric acid solution. Heat at 105°C until the spots are clear and inspected under sunlight. The luffa charcoal thin layer chromatogram is shown in Figure 2, where 1 is the negative control, 2 is the luffa charcoal granules, 3 is the luffa charcoal intermediate, 4 is the luffa charcoal slice, and 5 is the luffa charcoal control medicinal material. Fluorescent spots of the same color appear at the corresponding positions in the chromatogram of the test sample. From Figure 2 It can be seen that the TLC results have moderate separation, clear spots, rich chromatographic information, and fluorescent spots of the same color appear at corresponding positions in the chromatogram of the test sample.

[0057] Example 2

[0058] The present embodiment provides a thin layer chromatography identification method for a loofah sponge block, and the thin layer chromatography identification method for the loofah sponge block comprises the following steps:

[0059] Take 0.5g of loofah block and loofah charcoal slices respectively, grind them, add 20mL of water, treat them with ultrasound (400W) for 30min, let them cool, shake and extract them with ethyl acetate twice, 20mL each time, combine the ethyl acetate solution, evaporate the ethyl acetate, and then dilute it to 1mL with methanol as the test solution. According to the thin layer chromatography method (Chinese Pharmacopoeia 2015 Edition Four General Rules 0502), take 7μL of loofah block test solution and 5μL of loofah charcoal slice test solution, and spot them on the same silica gel G thin layer plate, use petroleum ether (60~90): dichloromethane: ethyl acetate: formic acid (1.5:1:2:0.1) as the developing agent, develop, take out, dry, first spray with 10% sulfuric acid ethanol solution, dry, then spray with 5% vanillin sulfuric acid solution, heat at 105℃ until the spots are clear, and inspect under sunlight. Fluorescent spots of the same color appear at the corresponding positions in the chromatogram of the test sample. as follows Figure 3 As shown in the figure, 1, 2, and 3 are 3 batches of luffa sponge charcoal slices; 4, 5, and 6 are 3 batches of luffa sponge blocks. Figure 3 It can be seen that the thin layer chromatography results show that the chromatographic information of luffa charcoal slices and luffa blocks are different. The chromatographic information of the former is richer, which may be due to changes in the chemical composition of the two during the processing process.

[0060] Since Chinese herbal medicines are made into other dosage forms through a series of processes, their original properties have changed, so the identification method provided by the present invention can identify different processed products of loofah blocks and loofah charcoal, and can provide a research basis for the changes in chemical components during the processing of loofah blocks to loofah charcoal. The chromatographic results provided by the present invention have moderate separation, clear spots, rich, stable and reliable chromatographic information.

[0061] Example 3

[0062] Preparation of test solution

[0063] The present embodiment provides a thin layer chromatography identification method of luffa charcoal, and the thin layer chromatography identification method of luffa charcoal comprises the following steps:

[0064] Preparation of test solution:

[0065] (a) Take 0.25 g of luffa charcoal freeze-dried powder, add 10 mL of methanol, sonicate for 30 min, filter, and concentrate to 1 mL.

[0066] (b) Take 0.25 g of luffa charcoal freeze-dried powder, add 10 mL of petroleum ether, sonicate for 30 min, filter, evaporate to dryness, and dilute to 1 mL with methanol.

[0067] (c) Take 0.25 g of luffa charcoal freeze-dried powder, add 10 mL of ethyl acetate, sonicate for 30 min, filter, and concentrate to 1 mL.

[0068] (d) Take 0.5 g of luffa charcoal freeze-dried powder, add 20 mL of water, ultrasonicate for 30 min, filter, extract twice with n-butanol, each time with 20 mL, combine the n-butanol layers, evaporate to dryness, and dilute to 1 mL with methanol.

[0069] (e) Take 0.5 g of luffa charcoal freeze-dried powder, add 20 mL of water, sonicate for 30 min, filter, extract twice with ethyl acetate, each time with 20 mL, combine the ethyl acetate layers, evaporate to dryness, and dilute to 1 mL with methanol.

[0070] (f) Take 0.5 g of luffa charcoal freeze-dried powder, add 20 mL of methanol, sonicate for 30 min, filter, evaporate to dryness, add 20 mL of water to dissolve, extract twice with ethyl acetate, each time with 20 mL, combine the ethyl acetate layers, evaporate to dryness, and dilute to 1 mL with methanol.

[0071] Pipette μL of the test solution and spot them on the same silica gel G thin layer plate. Use cyclohexane:ethyl acetate (1:2) as the developing solvent, develop, take out, dry, first spray with 10% sulfuric acid ethanol solution, dry, then spray with 5% vanillin sulfuric acid solution, heat at 105℃ until the spots are clear, and inspect under sunlight.

[0072] The thin layer chromatograms of luffa charcoal freeze-dried powder obtained by different extraction methods are shown in the figure. Figure 4 As shown, 1, 2, 3, 4, 5, and 6 correspond to the test solutions a, b, c, d, e, and f, respectively. According to the TLC identification results, the TLC results of the test solutions prepared by b and c are poor.

[0073] Example 4

[0074] Developing agent investigation

[0075] The present embodiment provides a thin layer chromatography identification method of luffa charcoal, and the thin layer chromatography identification method of luffa charcoal comprises the following steps:

[0076] The preparation of the test solution is the same as in Example 3.

[0077] Developing solvent 1: hexane: dichloromethane: ethyl acetate (1:1:2);

[0078] Developing solvent 2: n-hexane: dichloromethane: ethyl acetate (1:1:2.5);

[0079] Developing solvent 3: petroleum ether 60-90: dichloromethane: ethyl acetate: formic acid (1.5:1:2:0.1).

[0080] Pipette 7 μL of the test solution and spot them on the same silica gel G thin layer plate, develop them with developing agents 1, 2, and 3 respectively, take out, dry, spray with 10% sulfuric acid ethanol solution first, dry, spray with 5% vanillin sulfuric acid solution, heat at 105°C until the spots are clear, and inspect under sunlight.

[0081] The thin layer chromatogram of the freeze-dried powder of loofah charcoal obtained by using developing agent 1 is as follows Figure 5 As shown, 1, 2, 3, and 4 correspond to the test solutions a, d, e, and f, respectively.

[0082] The thin layer chromatogram of the freeze-dried powder of loofah charcoal obtained by using developing agent 2 is as follows Figure 6 As shown, 1 and 2 correspond to the test solution d and e, respectively.

[0083] The thin layer chromatogram of the freeze-dried powder of loofah charcoal obtained by using developing agent 3 is as follows Figure 7 As shown, 1, 2, and 3 correspond to the test solutions d, e, and f, respectively.

[0084] By comparison Figure 4-Figure 7 It can be seen that the chromatogram result of developer 3 is the best, so developer 3 (petroleum ether 60-90: dichloromethane: ethyl acetate: formic acid (1.5:1:2:0.1)) is used as the developer of luffa charcoal. Inspection: first spray with 10% sulfuric acid ethanol solution, dry, then spray with 5% vanillin sulfuric acid solution, heat until the spots are clear, and inspect under sunlight.

[0085] Example 5

[0086] Different spot sampling amount investigation

[0087] The present embodiment provides a thin layer chromatography identification method of luffa charcoal, and the thin layer chromatography identification method of luffa charcoal comprises the following steps:

[0088] Take 0.5 g of luffa charcoal standard decoction freeze-dried powder or luffa charcoal control slices, grind it into powder, add 20 mL of water, ultrasonically treat it for 30 min, cool it, and extract it twice with ethyl acetate by shaking, 20 mL each time, combine the ethyl acetate solutions, evaporate the ethyl acetate, and then make up to 1 mL with methanol as the test solution.

[0089] 5 μL, 7 μL and 10 μL of the test solution of luffa charcoal standard decoction freeze-dried powder and 1 μL, 3 μL and 5 μL of the test solution of luffa charcoal control slices were respectively spotted on the same silica gel G thin layer plate, and petroleum ether (60-90): dichloromethane: ethyl acetate: formic acid (1.5:1:2:0.1) was used as the developing agent. The plate was developed, taken out, dried, sprayed with 10% sulfuric acid ethanol solution first, dried, and then sprayed with 5% vanillin sulfuric acid solution. The plate was heated at 105°C until the spots were clear and inspected under sunlight.

[0090] Different amounts of luffa charcoal-decoction freeze-dried powder were used for thin layer chromatography. Figure 8 As shown, 1 is the blank control, 2, 3, 4 are the standard decoction freeze-dried powder 202105-21-03, the sampling volumes are 5μL, 7μL, 10μL respectively; 5, 6, 7 are the standard decoction freeze-dried powder 202105-21-09, the sampling volumes are 5μL, 7μL, 10μL respectively; 8, 9, 10 are the standard decoction freeze-dried powder 202105-21-12, the sampling volumes are 5μL, 7μL, 10μL respectively; 11, 12, 13 are the loofah charcoal control pieces, the sampling volumes are 1μL, 3μL, 5μL respectively; the optimal sampling volume is determined to be 7 microliters for the test sample and 5 microliters for the control piece.

[0091] Example 6

[0092] Inspection of different manufacturers of silica gel G thin layer sheets

[0093] The present embodiment provides a thin layer chromatography identification method of luffa charcoal, and the thin layer chromatography identification method of luffa charcoal comprises the following steps:

[0094] Take 0.5 g of luffa charcoal standard decoction freeze-dried powder (batches 03, 09, and 11) or luffa charcoal control slices, grind it into powder, add 20 mL of water, ultrasonically treat it for 30 min, cool it, and extract it twice with ethyl acetate by shaking, 20 mL each time. Combine the ethyl acetate solutions, evaporate the ethyl acetate, and then make up to 1 mL with methanol as the test solution.

[0095] 7 μL of luffa charcoal standard decoction freeze-dried powder test solution and 3 μL of luffa charcoal control decoction sample solution were respectively spotted on the same silica gel G thin layer plate, and petroleum ether (60-90): dichloromethane: ethyl acetate: formic acid (1.5:1:2:0.1) was used as the developing agent. The plate was developed, taken out, and dried. First, it was sprayed with 10% sulfuric acid ethanol solution, dried, and then sprayed with 5% vanillin sulfuric acid solution. The plate was heated at 105°C until the spots were clear and inspected under sunlight.

[0096] Fig. 9 This is the thin layer chromatogram of luffa charcoal decoction freeze-dried powder obtained using the silica gel thin layer plate provided by Yinlong. Fig.10 This is the thin layer chromatogram of luffa charcoal decoction freeze-dried powder obtained using the silica gel thin layer plate provided by Zhifu. Fig.11 This is a thin layer chromatogram of luffa charcoal standard decoction freeze-dried powder obtained using a silica gel thin layer plate provided by Merck; among them, the points from left to right are: stigmasterol, luffa charcoal freeze-dried powder batch 03, luffa charcoal freeze-dried powder batch 09, luffa charcoal freeze-dried powder batch 11, luffa control decoction piece. Figure 9-11 It can be seen that the method provided by the present invention is applicable to silica gel thin layer plates from various manufacturers.

[0097] Example 7

[0098] Investigation at different temperatures and humidity

[0099] The present embodiment provides a thin layer chromatography identification method of luffa charcoal, and the thin layer chromatography identification method of luffa charcoal comprises the following steps:

[0100] Take 0.5 g of luffa charcoal standard decoction freeze-dried powder (batches 03, 09, and 11) or luffa charcoal control slices, grind it into powder, add 20 mL of water, ultrasonically treat it for 30 min, cool it, and extract it twice with ethyl acetate by shaking, 20 mL each time. Combine the ethyl acetate solutions, evaporate the ethyl acetate, and then make up to 1 mL with methanol as the test solution.

[0101] The detection was carried out under different temperatures and humidities respectively. 7 μL of the test solution of luffa charcoal standard decoction freeze-dried powder and 5 μL of the test solution of luffa charcoal control slices were respectively spotted on the same silica gel G thin layer plate, and petroleum ether (60-90): dichloromethane: ethyl acetate: formic acid (1.5:1:2:0.1) was used as the developing agent. The plate was developed, taken out, dried, sprayed with 10% sulfuric acid ethanol solution first, dried, and then sprayed with 5% vanillin sulfuric acid solution, heated at 105°C until the spots were clear, and inspected under sunlight.

[0102] Fig.12 This is the thin layer chromatogram of luffa sponge charcoal decoction freeze-dried powder obtained at room temperature and humidity (23°C, 46%). Fig.13 This is the thin layer chromatogram of luffa sponge charcoal decoction freeze-dried powder obtained at room temperature and high humidity (23°C, 82%). Fig.14 This is the thin layer chromatogram of luffa charcoal decoction freeze-dried powder obtained at room temperature and low humidity (23°C, 13%). Fig.15 This is a thin layer chromatogram of luffa charcoal freeze-dried powder obtained at low temperature and normal humidity (4°C, 46%); the points from left to right are: stigmasterol, luffa charcoal freeze-dried powder batch 03, luffa charcoal freeze-dried powder batch 09, luffa charcoal freeze-dried powder batch 11, luffa control decoction piece. Figure 12-Figure 15 It can be seen that the method provided by the present invention is applicable to various temperatures and humidities.

[0103] Example 8

[0104] Methodological validation

[0105] The present embodiment provides a thin layer chromatography identification method of luffa charcoal, and the thin layer chromatography identification method of luffa charcoal comprises the following steps:

[0106] Take 0.5 g of luffa charcoal standard decoction freeze-dried powder (202105-21-01~202105-21-15) or luffa charcoal control slices, grind it into powder, add 20 mL of water, ultrasonically treat it for 30 min, cool it, and extract it with ethyl acetate by shaking twice, 20 mL each time, combine the ethyl acetate solutions, evaporate the ethyl acetate, and then make up to 1 mL with methanol as the test solution.

[0107] At room temperature and humidity (23°C, 46%), 7 μL of the test solution of luffa charcoal standard decoction freeze-dried powder and 5 μL of the test solution of luffa charcoal control slices were respectively spotted on the same silica gel G thin layer plate, and petroleum ether (60-90): dichloromethane: ethyl acetate: formic acid (1.5:1:2:0.1) was used as the developing agent. The plate was developed, taken out, dried, first sprayed with 10% sulfuric acid ethanol solution, dried, and then sprayed with 5% vanillin sulfuric acid solution. The plate was heated at 105°C until the spots were clear and inspected under sunlight.

[0108] Chromatogram of the method validation of luffa charcoal decoction freeze-dried powder Fig.16 As shown, 1 is blank; 2-16 are 15 batches of standard decoction freeze-dried powder batch numbers: 202105-21-01~202105-21-15; 17 is the loofah charcoal control decoction piece.

[0109] Example 9

[0110] Different sampling quantities

[0111] The present embodiment provides a thin layer chromatography identification method of luffa charcoal, and the thin layer chromatography identification method of luffa charcoal comprises the following steps:

[0112] Take 0.5 g of luffa charcoal granules or luffa charcoal control slices, grind them into powder, add 20 mL of water, ultrasonically treat for 30 min, cool, and extract with ethyl acetate by shaking twice, 20 mL each time, combine the ethyl acetate solutions, evaporate the ethyl acetate, and then make up to 1 mL with methanol as the test solution.

[0113] 5μL, 7μL, and 10μL of the test solution of loofah charcoal particles and 1μL, 3μL, and 5μL of the test solution of loofah charcoal control pieces were respectively spotted on the same silica gel G thin layer plate, and petroleum ether (60-90): dichloromethane: ethyl acetate: formic acid (1.5:1:2:0.1) was used as the developing agent. The plate was developed, taken out, and dried. First, it was sprayed with 10% sulfuric acid ethanol solution, dried, and then sprayed with 5% vanillin sulfuric acid solution. It was heated at 105°C until the spots were clear and inspected under sunlight.

[0114] The thin layer chromatograms of luffa charcoal particles with different sample amounts are shown in Figure 2. Fig.17As shown, 1 is a blank control, 2, 3, 4 are luffa charcoal particles KL19022051 with spotting volumes of 5μL, 7μL, 10μL respectively; 5, 6, 7 are luffa charcoal particles KL19003391 with spotting volumes of 5μL, 7μL, 10μL respectively; 8, 9, 10 are luffa charcoal particles KL19022051 with spotting volumes of 5μL, 7μL, 10μL respectively; 11, 12, 13 are luffa charcoal control pieces with spotting volumes of 1μL, 3μL, 5μL respectively; the optimal spotting volumes were determined to be 7 μL for the test sample and 5 μL for the control piece.

[0115] Example 10

[0116] Methodological validation

[0117] The present embodiment provides a thin layer chromatography identification method of luffa charcoal, and the thin layer chromatography identification method of luffa charcoal comprises the following steps:

[0118] Take 0.5 g of luffa charcoal granules (KL19022051, KL19003391, KL19022051) or luffa charcoal control slices, grind them into powder, add 20 mL of water, ultrasonically treat for 30 min, cool, and extract with ethyl acetate by shaking twice, 20 mL each time, combine the ethyl acetate solutions, evaporate the ethyl acetate, and then make up to 1 mL with methanol as the test solution.

[0119] At room temperature and humidity (23°C, 46%), 7 μL of the test solution of loofah charcoal particles and 5 μL of the test solution of loofah charcoal control slices were respectively spotted on the same silica gel G thin layer plate, and petroleum ether (60-90): dichloromethane: ethyl acetate: formic acid (1.5:1:2:0.1) was used as the developing agent. The plate was developed, taken out, dried, first sprayed with 10% sulfuric acid ethanol solution, dried, and then sprayed with 5% vanillin sulfuric acid solution. The plate was heated at 105°C until the spots were clear and inspected under sunlight.

[0120] The chromatogram of the validation of the method of loofah charcoal particles is as follows Fig.18 As shown, 1 is a blank control; 2 is luffa charcoal granules KL19022051; 3 is luffa charcoal granules KL19003391; 4 is luffa charcoal granules KL19022051; 5 is luffa charcoal control decoction piece.

[0121] The applicant declares that the present invention illustrates the process method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned process steps, that is, it does not mean that the present invention must rely on the above-mentioned process steps to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of the raw materials selected by the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A thin layer chromatography identification method for loofah and its processed products, characterized in that, The developing solvent used in the thin layer chromatography identification method includes petroleum ether, dichloromethane, ethyl acetate and formic acid in a volume ratio of (1.3-1.7):(0.8-1.2):(1.8-2.2):(0.08-0.12); The thin layer chromatography identification method specifically comprises the following steps: The test sample solution is spotted on a silica gel thin layer plate and developed with a developing agent. After the silica gel thin layer plate is dried, it is first sprayed with sulfuric acid ethanol solution, dried, and then sprayed with vanillin solution and heated to obtain the position of each component in the test sample on the thin layer plate; The preparation of the test solution in the thin layer chromatography identification method comprises the following steps: (1) grinding luffa lao and / or luffa lao processed product, adding water, ultrasonicating, filtering, and collecting solids; (2) The solid obtained in step (1) is extracted with ethyl acetate, the obtained extract is desolventized, and then the volume is fixed with methanol to obtain a test solution.

2. the thin layer chromatography identification method of loofah and processed product thereof according to claim 1, is characterized in that, The prepared loofah product comprises any one of loofah blocks, fried loofah or loofah charcoal, or a combination of at least two of them.

3. The thin layer chromatography identification method of loofah and processed product thereof according to claim 1, is characterized in that, The boiling point of the petroleum ether is 60-90°C.

4. The thin layer chromatography identification method of loofah and processed product thereof according to claim 1, wherein The mass content of sulfuric acid in the sulfuric acid ethanol solution is 8-12%.

5. The thin layer chromatography identification method of loofah and processed product thereof according to claim 1, is characterized in that, The mass content of vanillin in the vanillin solution is 3-7%.

6. The thin layer chromatography identification method of loofah and processed product thereof according to claim 1, wherein The heating temperature is 100-110°C.

7. The thin layer chromatography identification method of loofah and processed product thereof according to claim 1, characterized in that, In step (1), the solid-liquid ratio of the luffa scoparia and / or luffa scoparia processed product to water is 0.5:(15-25) g / mL.

8. The thin layer chromatography identification method of loofah and processed product thereof according to claim 1, characterized in that, In step (1), the power of the ultrasound is 100-500 W, and the time of the ultrasound is 20-40 min.

9. The thin layer chromatography identification method of loofah and processed product thereof according to claim 1, wherein The solid-liquid ratio of the luffa lao and / or luffa lao processed product to ethyl acetate is 0.5:(15-25) g / mL.

10. The thin layer chromatography identification method of loofah and its processed product according to claim 1, characterized in that, In step (2), the extraction is performed 1 to 3 times.

11. The thin layer chromatography identification method of the loofah and its processed product according to any one of claims 1 to 10, characterized in that: The sample volume in the thin layer chromatography identification method is 5-8 μL.

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