CN115684455A - Thin-layer chromatography identification method for loofah sponge and processed product thereof - Google Patents

Abstract

The invention provides a thin-layer chromatographic identification method for loofah and processed products thereof. The developer used in the thin-layer chromatographic identification method includes petroleum ether, dichloromethane, ethyl acetate and formic acid. The thin-layer chromatography identification method of loofah and its processed products provided by the invention has the characteristics of high accuracy, good separation effect, and clear stains, and can identify different processed products of loofah block and loofah charcoal, followed by loofah It provides a research basis for the change of chemical composition in the process of processing loofah charcoal from lumps to loofah charcoal. It can also qualitatively identify loofah charcoal medicinal materials, decoction pieces, intermediates, and formula granules, and control the quality of the entire process to ensure uniform quality. ,Stable and reliable.

Description

TLC identification method of loofah and its processed products

technical field

The invention belongs to the technical field of traditional Chinese medicine analysis, and in particular relates to a TLC identification method of loofah and processed products thereof.

Background technique

Luffa is the vascular bundle of the dry ripe fruit of Cucurbitaceae Luffa cylindrical (L.) Roem. It has the effects of dredging the meridian and activating collaterals, clearing heat and reducing phlegm, detoxifying and reducing swelling, strengthening the heart and diuresis, etc. It is mainly used to treat chest and hypochondrium pain, cough due to lung heat, amenorrhea, and milk blockage. Loofah contains a variety of loofah saponins, flavonoids and phenols, proteins, amino acids, oils, inorganic elements and organic acids, etc. Pharmacological tests have proved that it has the effects of lowering blood fat, analgesia and sedation, and has significant clinical effects.

Its main processed products of loofah have three kinds of loofah (block), fried loofah and loofah charcoal. Among them, loofah (block) and loofah charcoal are used clinically more, but there is no quality standard at present, and there is no quality standard for loofah. There are few reports on the difference between the two processed products (block) and loofah charcoal. Since the samples of different dosage forms of loofah charcoal are made of loofah charcoal medicinal materials through a series of processes, some of which will destroy the properties and characteristics of the original medicinal materials, it is necessary to have a good ability to distinguish and identify the existing technology and operate Easier and faster identification methods for better quality control.

Kang Along, Tang Yingshuang, Sun Chengrong, Zhang Xian, Zhang Suheng; Study on the quality standards of three different processed loofah slices; Journal of Shaanxi University of Traditional Chinese Medicine, 2011, July, 34 (4) published the quality of three different processed loofah slices Control method comprises the following steps, the preparation of need testing solution: get luffa charcoal 2g, cut into fritters (length is not more than 0.5cm), add methanol 50mL, heat and reflux 1h, let cool, filter, filtrate is evaporated to dryness, residue Add 10 mL of water to dissolve, extract twice with water-saturated n-butanol, 20 mL each time, combine the n-butanol solution, evaporate to dryness, add 1 mL of methanol to the residue to dissolve, and use it as the test solution. Developing solvent: trichloromethane-methanol-water (28:4:1) lower layer solution placed overnight below 10°C. Test according to thin-layer chromatography (Appendix VIB of Chinese Pharmacopoeia in 2005), draw each 10uL of the above-mentioned solution for the test and the solution of the reference medicinal material, and place them on the same spot with 0.5% sodium carboxymethylcellulose solution as the binder. Spread out on a thin-layer silica gel G plate, take it out, dry it in the air, spray it with 10% sulfuric acid ethanol solution, and heat it at 105°C until the spots are clearly colored. Check under the ultraviolet lamp (365nm), in the chromatogram of the test product, on the position corresponding to the chromatogram of the contrast medicinal material, the spots of the same color are all shown. The test results are shown in Figure 1, where 1 is loofah charcoal decoction pieces (YP202105-21-10) 3uL; 2 is loofah charcoal decoction pieces (YP202105-21-10) 5uL. The results of TLC showed poor resolution, unclear spots, and little chromatographic information.

Therefore how to provide a kind of TLC identification method of luffa and processed product thereof, become the problem demanding prompt solution at present.

Contents of the invention

Aiming at the deficiencies in the prior art, the object of the present invention is to provide a TLC identification method for loofah and its processed products. The invention establishes a highly specific thin-layer identification method for loofah and its processed products, and provides experimental basis for the establishment of its quality standard and follow-up research. The separation degree of the chromatographic result provided by the invention is moderate, and the spots are clear, and the chromatographic information is rich, stable and reliable.

To achieve this purpose of the invention, the present invention adopts the following technical solutions:

In the first aspect, the present invention provides a thin-layer chromatographic identification method for loofah and processed products thereof, wherein the developer used in the thin-layer chromatographic identification method includes petroleum ether, dichloromethane, ethyl acetate and formic acid.

In the present invention, the processed product of loofah includes any one or a combination of at least two of loofah block, fried loofah or charcoal of loofah.

In the present invention, the volume ratio of the 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);

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

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

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

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

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

In the present invention, the thin layer chromatography identification method specifically includes the following steps:

Spot the solution of the test product on the silica gel thin-layer plate, and develop it with a developing agent. After the silica gel thin-layer plate is dried, spray the sulfuric acid ethanol solution, dry it, and then spray the vanillin solution and heat to obtain the test product. The position of each component in the product in the thin-layer plate.

In the present invention, the mass content of sulfuric acid in the sulfuric acid ethanol solution is 8-12% (such as 8%, 9%, 10%, 11%, 12% etc.);

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

Preferably, the heating temperature is 100-110°C (such as 100°C, 101°C, 102°C, 103°C, 104°C, 105°C, 106°C, 107°C, 108°C, 109°C, 110°C, etc. ).

In the present invention, the preparation of need testing solution in the thin layer chromatography identification method comprises the following steps:

(1) Grinding loofah roasted and/or loofah roasted processed products, adding water, ultrasonication, filtering, and collecting solids;

(2) The solid obtained in step (1) is extracted with ethyl acetate, the obtained extract is subjected to precipitation, and then methanol is used to constant volume to obtain the test solution.

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

Wherein, "15-25" can be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 and so on.

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

In the present invention, the solid-to-liquid ratio of the loofah roast and/or loofah roasted processed product to ethyl acetate is 0.5: (15-25) g/mL;

Wherein, "15-25" can be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 and so on.

Preferably, in step (2), the number of extractions is 1-3 times (for example, it can be 1 time, 2 times, 3 times, etc.).

In the present invention, the sample volume in the TLC identification method is 5-8 μL (for example, 5 μL, 6 μL, 7 μL, 8 μL, etc.).

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

The thin-layer chromatography identification method of loofah and its processed products provided by the invention has the characteristics of high accuracy, good separation effect, and clear stains, and can identify different processed products of loofah block and loofah charcoal, and secondly, loofah It can also provide research basis for the change of chemical composition in the process of processing loofah charcoal from block to loofah charcoal. It can also qualitatively identify loofah charcoal medicinal materials, decoction pieces, intermediates, and formula granules, and control the quality of the entire process to ensure uniform quality. ,Stable and reliable.

Description of drawings

Fig. 1 is the thin-layer chromatogram of the loofah charcoal decoction pieces in the prior art; wherein 1 is 3uL loofah charcoal decoction pieces (YP202105-21-10); 2 is 5uL loofah charcoal decoction pieces (YP202105-21-10).

Fig. 2 is the thin-layer chromatogram of loofah charcoal, wherein, 1 is negative control, 2 is loofah charcoal particle, 3 is loofah charcoal intermediate, 4 is loofah charcoal decoction piece, 5 loofah charcoal contrast medical material.

Fig. 3 is 3 batches of loofah charcoal decoction pieces and 3 batches of loofah block thin-layer chromatograms, wherein, 1, 2, 3 are 3 batches of loofah charcoal decoction pieces; 4, 5, 6 are 3 batches of loofah blocks.

Fig. 4 is the thin-layer chromatogram of luffa charcoal freeze-dried powder obtained by different extraction methods, wherein, 1, 2, 3, 4, 5, 6 correspond to need testing solution a, b, c, d, e, f respectively .

Fig. 5 is the luffa charcoal freeze-dried powder thin-layer chromatogram that adopts developer 1 to obtain, and wherein, 1, 2, 3, 4 correspond to need testing solution a, d, e, f respectively.

Fig. 6 is the luffa charcoal freeze-dried powder thin-layer chromatogram that adopts developing agent 2 to obtain, and wherein, 1,2 correspond to need testing solution d, e respectively.

Fig. 7 is the thin-layer chromatogram of the luffa charcoal freeze-dried powder that adopts developing agent 3 to obtain, and wherein, 1,2,3 correspond to need testing solution d, e, f respectively.

Fig. 8 is the thin-layer chromatogram of the luffa charcoal standard decocted freeze-dried powder adopting different sample volumes, wherein, 1 is blank control, 2, 3, 4 is standard decocted freeze-dried powder 202105-21-03 sample volumes are respectively 5μL, 7μL, 10μL; 5, 6, and 7 are the standard fried freeze-dried powder 202105-21-09 The sample volume is 5μL, 7μL, 10μL respectively; 8, 9, 10 are the standard fried freeze-dried powder 202105-21-12 points The sample volumes were 5 μL, 7 μL, and 10 μL; 11, 12, and 13 were loofah charcoal control slices, and the sample volumes were 1 μL, 3 μL, and 5 μL, respectively.

Figure 9 is the thin-layer chromatogram of loofah charcoal fried freeze-dried powder obtained by using the silica gel thin-layer plate provided by Yinlong; among them, the points from left to right are: stigmasterol, loofah charcoal freeze-dried powder 03 batches, loofah 09 batches of Luo charcoal freeze-dried powder, 11 batches of loofah charcoal freeze-dried powder, and control slices of loofah.

Figure 10 is the thin-layer chromatogram of loofah charcoal decocted freeze-dried powder obtained by using the silica gel thin-layer plate provided by Zhifu; among them, the points from left to right are: stigmasterol, loofah charcoal freeze-dried powder 03 batches, loofah 09 batches of charcoal freeze-dried powder, 11 batches of loofah charcoal freeze-dried powder, and control slices of loofah.

Figure 11 is the thin-layer chromatogram of loofah charcoal decocted freeze-dried powder obtained by using the silica gel thin-layer plate provided by Merck; among them, the points from left to right are: stigmasterol, loofah charcoal freeze-dried powder 03 batches, loofah 09 batches of Luo charcoal freeze-dried powder, 11 batches of loofah charcoal freeze-dried powder, and control slices of loofah.

Figure 12 is the thin layer chromatogram of luffa charcoal standard fried freeze-dried powder obtained at normal temperature and humidity (23°C, 46%); wherein, the points are from left to right: stigmasterol, luffa charcoal freeze-dried powder 03 batches , 09 batches of loofah charcoal freeze-dried powder, 11 batches of loofah charcoal freeze-dried powder, loofah control decoction pieces.

Figure 13 is the thin-layer chromatogram of the luffa charcoal standard fried freeze-dried powder obtained at normal temperature and high humidity (23°C, 82%); wherein, the points are from left to right: stigmasterol, luffa charcoal freeze-dried powder 03 batches , 09 batches of loofah charcoal freeze-dried powder, 11 batches of loofah charcoal freeze-dried powder, loofah control decoction pieces.

Fig. 14 is the thin-layer chromatogram of the luffa charcoal standard fried freeze-dried powder obtained at normal temperature and low humidity (23°C, 13%); wherein, the points from left to right are: stigmasterol, luffa charcoal freeze-dried powder 03 batches, Loofah charcoal freeze-dried powder 09 batches, loofah charcoal freeze-dried powder 11 batches, loofah control decoction pieces.

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

Figure 16 is the methodological verification chromatogram of loofah charcoal standard fried freeze-dried powder, wherein, 1 is blank; 2-16 is 15 batches of standard fried freeze-dried powder. Loofah charcoal control drink pieces.

Figure 17 is the thin-layer chromatogram of luffa charcoal particles with different sample volumes, wherein, 1 is a blank control; 2, 3, and 4 are loofah charcoal particles KL19022051 sample volumes are 5 μL, 7 μL, and 10 μL respectively; 5, 6 , 7 are loofah charcoal particles KL19003391, the sample volumes are 5 μL, 7 μL, and 10 μL; 8, 9, and 10 are loofah charcoal particles KL19022051 sample volumes are 5 μL, 7 μL, and 10 μL; 11, 12, and 13 are loofah charcoal The sampling volumes of the control decoction pieces were 1 μL, 3 μL, and 5 μL respectively; the optimal sampling volume was determined to be 7 μl for the test sample and 5 μl for the control decoction pieces.

Figure 18 is the methodological verification chromatogram of loofah charcoal particles; among them, 1 is 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 Pieces.

Detailed ways

The technical solutions of the present invention will be further described below through specific embodiments. It should be clear to those skilled in the art that the embodiments are only for helping to understand the present invention, and should not be regarded as specific limitations on the present invention.

The source of each component in the following examples is as follows:

Example 1

The present embodiment provides a kind of TLC identification method of loofah charcoal, the TLC identification method of described loofah charcoal comprises the following steps:

Take loofah charcoal granules, loofah charcoal intermediate, loofah charcoal decoction pieces 0.5g, grind finely, add 20mL of water, treat with ultrasonic (200W) for 30min, let cool, shake and extract twice with ethyl acetate, 20mL each time, combine Ethyl acetate solution, evaporate the ethyl acetate to dryness, then dilute to 1mL with methanol, as the test solution. Loofah charcoal reference medicinal material: Take 5 g of loofah charcoal medicinal material and add 100 mL of water, heat to boil for 30 minutes, spin dry, and prepare the reference medicinal material solution in the same way. (Note: There is no loofah charcoal reference medicinal material in the National Inspection Institute and the market, so the loofah charcoal medicinal material is used as the reference medicinal material first). According to thin-layer chromatography (Chinese Pharmacopoeia 2015 edition four general rules 0502) test, absorb 7 μ L of the solution of the test product (loofah charcoal particle, loofah charcoal intermediate, loofah charcoal reference medicinal material), and 5 μ L of the test product solution of loofah charcoal decoction pieces , respectively point on the same silica gel G thin-layer plate, using petroleum ether (60-90): dichloromethane: ethyl acetate: formic acid (1.5:1:2:0.1) as the developer, develop, take out, and dry in the air. First spray with 10% sulfuric acid ethanol solution, dry it, then spray with 5% vanillin sulfuric acid solution, heat at 105°C until the spots are clear, and inspect under sunlight. The thin-layer chromatogram of loofah charcoal is shown in 2, wherein, 1 is negative control, 2 is loofah charcoal particles, 3 is loofah charcoal intermediate, 4 is loofah charcoal decoction pieces, and 5 is loofah charcoal control medicinal material. At the corresponding position in the chromatogram of the test product, fluorescent spots of the same color are displayed. It can be seen from Figure 2 that the thin-layer chromatography results have moderate separation, clear spots, and rich chromatographic information. On the corresponding position in the chromatogram of the test product, there are fluorescent spots of the same color.

Example 2

The present embodiment provides a kind of TLC identification method of loofah block, the TLC identification method of described loofah block comprises the following steps:

Take 0.5g loofah block and loofah charcoal decoction slices respectively, grind them finely, add 20mL of water, treat with ultrasonic (400W) for 30min, let it cool, shake and extract twice with ethyl acetate, 20mL each time, combine the ethyl acetate solution, and Ethyl acetate was evaporated to dryness, and then dilute to 1mL with methanol as the test solution. Test according to the thin-layer chromatography (Chinese Pharmacopoeia 2015 edition four general rules 0502), draw loofah block test solution 7 μ L, loofah charcoal slices test solution 5 μ L, point respectively on the same silica gel G thin-layer plate, with petroleum Ether (60～90): dichloromethane: ethyl acetate: formic acid (1.5:1:2:0.1) as developing agent, develop, take out, dry in the air, first spray with 10% sulfuric acid ethanol solution, dry in the air, then Spray with 5% vanillin sulfuric acid solution, heat at 105°C until the spots are clear, and inspect under sunlight. At the corresponding position in the chromatogram of the test product, fluorescent spots of the same color are displayed. As shown in Figure 3 below, 1, 2, and 3 are 3 batches of loofah charcoal decoction pieces; 4, 5, and 6 are 3 batches of loofah cubes. It can be seen from Figure 3 that the TLC results show that the chromatographic information of the loofah charcoal slices and the loofah block are different, and the chromatographic information of the former is richer, which may be due to changes in the chemical composition of the two during the processing.

Because Chinese medicinal materials are made into other dosage forms through a series of processes, their original properties have changed, so the identification method provided by the invention can identify different processed products of loofah block and luffa charcoal, and can be used for loofah block to The change of chemical composition in the process of loofah charcoal processing provides a research basis. The separation degree of the chromatographic result provided by the invention is moderate, and the spots are clear, and the chromatographic information is rich, stable and reliable.

Example 3

Preparation and investigation of the test solution

The present embodiment provides a kind of TLC identification method of loofah charcoal, the TLC identification method of described loofah charcoal comprises the following steps:

Preparation of the test solution:

(a) Take 0.25 g of loofah charcoal freeze-dried powder, add 10 mL of methanol, ultrasonicate for 30 min, filter, and concentrate to 1 mL.

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

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

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

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

(f) Take 0.5g of loofah charcoal freeze-dried powder, add 20mL of methanol, ultrasonicate for 30min, filter, evaporate to dryness, add 20mL of water to dissolve, extract twice with ethyl acetate, each 20mL combined ethyl acetate layer, evaporate to dryness, Dilute to 1mL with methanol.

Take μL of the test solution, respectively spot on the same silica gel G thin-layer plate, use cyclohexane: ethyl acetate (1:2) as developing agent, develop, take out, dry in the air, spray with 10% ethanol sulfate first The solution was dried in the air, sprayed with 5% vanillin sulfuric acid solution, heated at 105°C until the spots were clear, and inspected under sunlight.

The thin-layer chromatograms of the luffa charcoal freeze-dried powder obtained by different extraction methods are shown in Figure 4, wherein, 1, 2, 3, 4, 5, 6 correspond to need testing solution a, b, c, d, respectively. e, f. According to the TLC identification results, it is learned that the TLC effect of the test solution prepared by b and c is poor.

Example 4

Developing agent inspection

The present embodiment provides a kind of TLC identification method of loofah charcoal, the TLC identification method of described loofah charcoal comprises the following steps:

The preparation of need testing solution is with embodiment 3.

Developing agent 1: n-hexane: dichloromethane: ethyl acetate (1:1:2);

Developing agent 2: n-hexane: dichloromethane: ethyl acetate (1:1:2.5);

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

Take 7 μL of the test solution, spot it on the same silica gel G thin-layer plate, develop it with developer 1, 2, and 3 respectively, take it out, dry it in the air, spray it with 10% sulfuric acid ethanol solution, dry it in the spray Use 5% vanillin sulfuric acid solution, heat at 105°C until the spots are clear, and inspect under sunlight.

The thin-layer chromatogram of the luffa charcoal freeze-dried powder obtained by developing agent 1 is shown in Figure 5, wherein, 1, 2, 3, 4 correspond to need testing solution a, d, e, f respectively.

The thin-layer chromatogram of the luffa charcoal freeze-dried powder obtained by developing agent 2 is shown in Figure 6, wherein 1 and 2 correspond to the testing solution d and e respectively.

The thin-layer chromatogram of the luffa charcoal freeze-dried powder that adopts developing agent 3 to obtain is shown in Figure 7, and wherein, 1, 2, 3 correspond to need testing solution d, e, f respectively.

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

Example 5

Inspection of different sample volumes

The present embodiment provides a kind of TLC identification method of loofah charcoal, the TLC identification method of described loofah charcoal comprises the following steps:

Take loofah charcoal decoction freeze-dried powder or 0.5g of loofah charcoal control decoction slices, grind finely, add 20mL of water, ultrasonically treat for 30min, let cool, shake and extract twice with ethyl acetate, 20mL each time, combine ethyl acetate solution , Evaporate ethyl acetate to dryness, and then dilute to 1 mL with methanol as the test solution.

Take 5 μL, 7 μL, and 10 μL of the test solution of loofah charcoal decocted and freeze-dried powder, and 1 μL, 3 μL, and 5 μL of the test solution of loofah charcoal control decoction pieces. -90): dichloromethane: ethyl acetate: formic acid (1.5:1:2:0.1) as developing agent, develop, take out, dry, first spray with 10% sulfuric acid ethanol solution, dry, then spray with 5% vanillin sulfuric acid solution, heated at 105°C until the spots are clear, and inspected under sunlight.

The thin-layer chromatogram of loofah charcoal standard decocted freeze-dried powder with different sample volumes is shown in Figure 8, wherein, 1 is a blank control, and 2, 3, and 4 are standard decocted freeze-dried powders. 5μL, 7μL, 10μL; 5, 6, 7 are the standard decocted freeze-dried powder 202105-21-09 The sampling volume is 5μL, 7μL, 10μL respectively; 8, 9, 10 are the standard fried freeze-dried powder 202105-21-12 The sample volumes were 5 μL, 7 μL, and 10 μL; 11, 12, and 13 were loofah charcoal control slices, and the sample volumes were 1 μL, 3 μL, and 5 μL; the optimal sample volume was determined to be 7 μl for the test sample and 5 μL for the control slices. microliter.

Example 6

Inspection of different manufacturers of silica gel G thin-layer board

The present embodiment provides a kind of TLC identification method of loofah charcoal, the TLC identification method of described loofah charcoal comprises the following steps:

Take loofah charcoal standard fried freeze-dried powder (03, 09, 11 batches) or loofah charcoal control decoction pieces 0.5g, grind finely, add 20mL of water, ultrasonicate for 30min, let cool, shake and extract with ethyl acetate twice, each 20 mL at a time, combined ethyl acetate solution, evaporated ethyl acetate to dryness, and then dilute to 1 mL with methanol, as the test solution.

Absorb loofah charcoal decocted and freeze-dried powder test solution 7 μL, loofah charcoal control decoction pieces test solution 3 μL, respectively, on the same silica gel G thin-layer plate, with petroleum ether (60-90): dichloromethane: Ethyl acetate: formic acid (1.5:1:2:0.1) as developing agent, develop, take out, and dry, first spray with 10% sulfuric acid ethanol solution, dry, then spray with 5% vanillin sulfuric acid solution, Heat at 105°C until the spots are clear, and inspect under sunlight.

Figure 9 is the thin layer chromatogram of loofah charcoal decocted freeze-dried powder obtained by using the silica gel thin layer plate provided by Yinlong, and Figure 10 is the thin layer of loofah charcoal decocted freeze-dried powder obtained by using the silica gel thin layer plate provided by Zhifu Chromatogram, Figure 11 is the thin layer chromatogram of loofah charcoal decocted freeze-dried powder obtained by using the silica gel thin-layer plate provided by Merck; among them, the points from left to right are: stigmasterol, loofah charcoal freeze-dried powder 03 09 batches of loofah charcoal freeze-dried powder, 11 batches of loofah charcoal freeze-dried powder, loofah control decoction pieces. It can be seen from Fig. 9-Fig. 11 that the method provided by the present invention is applicable to the silica gel thin-layer boards of various manufacturers.

Example 7

Investigation of different temperature and humidity

The present embodiment provides a kind of TLC identification method of loofah charcoal, the TLC identification method of described loofah charcoal comprises the following steps:

Take loofah charcoal standard fried freeze-dried powder (03, 09, 11 batches) or loofah charcoal control decoction pieces 0.5g, grind finely, add 20mL of water, ultrasonicate for 30min, let cool, shake and extract with ethyl acetate twice, each 20 mL at a time, combined ethyl acetate solution, evaporated ethyl acetate to dryness, and then dilute to 1 mL with methanol, as the test solution.

Tested at different temperature and humidity respectively, absorb 7 μL of the test solution of loofah charcoal decoction and freeze-dried powder, and 5 μL of the test solution of loofah charcoal control decoction pieces, respectively spot on the same silica gel G thin-layer plate, and use petroleum ether ( 60-90): dichloromethane: ethyl acetate: formic acid (1.5:1:2:0.1) as the developer, unfold, take out, and dry, first spray with 10% sulfuric acid ethanol solution, dry, and then spray Use 5% vanillin sulfuric acid solution, heat at 105°C until the spots are clear, and inspect under sunlight.

Figure 12 is the TLC of the luffa charcoal decocted freeze-dried powder obtained at normal temperature and humidity (23°C, 46%), and Figure 13 is the luffa charcoal decoction obtained at normal temperature and humidity (23°C, 82%) The thin-layer chromatogram of freeze-dried powder, Fig. 14 is the thin-layer chromatogram of fried freeze-dried powder obtained at normal temperature and low humidity (23°C, 13%), and Fig. 15 is at low temperature and low humidity (4°C, 46%) The obtained loofah charcoal standard fried and freeze-dried thin-layer chromatogram; among them, the points from left to right are: stigmasterol, loofah charcoal freeze-dried powder 03 batches, loofah charcoal freeze-dried powder 09 batches, loofah charcoal freeze-dried Powder 11 batches, loofah control decoction pieces. It can be seen from Fig. 12-Fig. 15 that the method provided by the present invention is applicable to various temperatures and humidity.

Example 8

Methodology Validation

The present embodiment provides a kind of TLC identification method of loofah charcoal, the TLC identification method of described loofah charcoal comprises the following steps:

Take loofah charcoal decoction freeze-dried powder (202105-21-01～202105-21-15) or loofah charcoal reference slice 0.5g, grind finely, add 20mL of water, sonicate for 30min, let cool, shake with ethyl acetate Extract 2 times, 20mL each time, combine the ethyl acetate solution, evaporate the ethyl acetate to dryness, and then dilute to 1mL with methanol as the test solution.

Under normal temperature and humidity (23°C, 46%), absorb 7 μL of the test solution of loofah charcoal decoction and freeze-dried powder, and 5 μL of the test solution of loofah charcoal control decoction pieces, and place them on the same silica gel G thin-layer plate respectively. Petroleum ether (60-90): dichloromethane: ethyl acetate: formic acid (1.5:1:2:0.1) as the developing agent, develop, take out, and dry in the air, first spray with 10% sulfuric acid ethanol solution, dry in the air, Then spray with 5% vanillin sulfuric acid solution, heat at 105°C until the spots are clear, and inspect under sunlight.

The methodological verification chromatogram of loofah charcoal standard fried freeze-dried powder is shown in Figure 16, where 1 is blank; 2-16 is 15 batches of standard fried freeze-dried powder Batch number: 202105-21-01～202105-21-15; 17 is loofah charcoal contrast decoction pieces.

Example 9

Inspection of different sample volumes

The present embodiment provides a kind of TLC identification method of loofah charcoal, the TLC identification method of described loofah charcoal comprises the following steps:

Take loofah charcoal granules or loofah charcoal control slices 0.5g, grind finely, add 20mL of water, ultrasonically treat for 30min, let cool, shake and extract twice with ethyl acetate, 20mL each time, combine the ethyl acetate solution, and dilute ethyl acetate The ester was evaporated to dryness, and then dilute to 1mL with methanol as the test solution.

Take 5 μL, 7 μL, and 10 μL of the test solution of loofah charcoal granules, and 1 μL, 3 μL, and 5 μL of loofah charcoal control decoction pieces, respectively, and place them on the same silica gel G thin-layer plate, with petroleum ether (60-90): Dichloromethane: ethyl acetate: formic acid (1.5:1:2:0.1) as the developing agent, develop, take out, and dry, first spray with 10% sulfuric acid ethanol solution, dry, and then spray with 5% vanilla Aldehyde sulfuric acid solution, heated at 105°C until the spots are clear, and inspected under sunlight.

The thin-layer chromatograms of loofah charcoal particles with different sample volumes are shown in Figure 17, wherein, 1 is a blank control, 2, 3, and 4 are loofah charcoal particles KL19022051 sample volumes are 5 μL, 7 μL, and 10 μL respectively; 5 , 6, and 7 are loofah charcoal particles KL19003391 with sample volumes of 5 μL, 7 μL, and 10 μL; 8, 9, and 10 are loofah charcoal particles KL19022051 with sample volumes of 5 μL, 7 μL, and 10 μL; 11, 12, and 13 are loofah The sampling volumes of Luotan control decoction pieces were 1 μL, 3 μL, and 5 μL respectively; the optimal sampling volume was determined to be 7 μl for the test product and 5 μl for the control decoction pieces.

Example 10

Methodology Validation

The present embodiment provides a kind of TLC identification method of loofah charcoal, the TLC identification method of described loofah charcoal comprises the following steps:

Take loofah charcoal granules (KL19022051, KL19003391, KL19022051) or loofah charcoal control slices 0.5g, grind finely, add 20mL of water, ultrasonically treat for 30min, let cool, shake and extract twice with ethyl acetate, 20mL each time, combine acetic acid Ethyl solution, evaporate the ethyl acetate to dryness, then dilute to 1mL with methanol, as the test solution.

Under normal temperature and humidity (23°C, 46%), absorb 7 μL of the test solution of loofah charcoal particles and 5 μL of the test solution of loofah charcoal control decoction pieces, respectively spot on the same silica gel G thin-layer plate, and add petroleum ether (60 -90): dichloromethane: ethyl acetate: formic acid (1.5:1:2:0.1) as developing agent, develop, take out, dry, first spray with 10% sulfuric acid ethanol solution, dry, then spray with 5% vanillin sulfuric acid solution, heated at 105°C until the spots are clear, and inspected under sunlight.

The method verification chromatogram of loofah charcoal granules is shown in Figure 18, in which, 1 is the blank control; 2 is loofah charcoal granules KL19022051; 3 is loofah charcoal granules KL19003391; 4 is loofah charcoal granules KL19022051; 5 is loofah Charcoal control drink pieces.

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

Claims (10)

1. A thin-layer chromatography identification method for loofah sponge and its processed products is characterized in that developing agents adopted in the thin-layer chromatography identification method comprise petroleum ether, dichloromethane, ethyl acetate and formic acid.

2. The thin-layer chromatography identification method of loofah sponge and the processed products thereof according to claim 1, wherein the processed products of loofah sponge comprise any one or a combination of at least two of loofah sponge blocks, fried loofah sponge and loofah sponge charcoal.

3. The thin layer chromatography identification method of vegetable sponge and its processed product according to claim 1 or 2, characterized in that 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).

4. The thin-layer chromatography identification method of loofah sponge and processed products thereof according to any one of claims 1-3, characterized in that the boiling point of the petroleum ether is 60-90 ℃.

5. The thin-layer chromatography identification method of loofah sponge and processed products thereof according to any one of claims 1 to 4, characterized in that the thin-layer chromatography identification method specifically comprises the following steps:

and (2) spotting the test sample solution on a silica gel thin-layer plate, developing by adopting a developing agent, spraying a sulfuric acid ethanol solution after the silica gel thin-layer plate is dried, drying, spraying a vanillin solution, and heating to obtain the positions of the components in the test sample in the thin-layer plate.

6. The thin-layer chromatography identification method for the loofah sponge and the processed products thereof according to claim 5, characterized in that the mass content of sulfuric acid in the sulfuric acid ethanol solution is 8-12%;

preferably, the mass content of vanillin in the vanillin sulfuric acid solution is 3-7%;

preferably, the heating temperature is 100-110 ℃.

7. The thin-layer chromatography identification method of loofah sponge and processed products thereof according to claim 5 or 6, characterized in that the preparation of the test solution in the thin-layer chromatography identification method comprises the following steps:

(1) Grinding the baked towel gourd and/or the baked towel gourd product, adding water, performing ultrasonic treatment, filtering, and collecting solid;

(2) And (2) extracting the solid obtained in the step (1) by using ethyl acetate, desolventizing the obtained extract, and then performing constant volume by using methanol to obtain a test sample solution.

8. The thin-layer chromatography identification method for the loofah sponge and the processed products thereof according to claim 7, characterized in that in the step (1), the solid-to-liquid ratio of the loofah baked and/or the processed products of loofah baked to water is 0.5 (15-25) g/mL;

preferably, in the step (1), the power of the ultrasound is 100-500W, and the time of the ultrasound is 20-40min.

9. The thin-layer chromatography identification method for the loofah sponge and the processed products thereof according to claim 7 or 8, characterized in that the solid-to-liquid ratio of the loofah baked and/or the processed products of loofah baked to ethyl acetate is 0.5 (15-25) g/mL;

preferably, in the step (2), the number of times of extraction is 1 to 3.

10. The thin-layer chromatography identification method of loofah sponge and its processed products according to any one of claims 1 to 9, characterized in that the spot size is 5 to 8 μ L in the thin-layer chromatography identification method.

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