Thin layer identification method of shexie formula granules

Abstract

The present application relates to the technical field of analytical detection, and particularly relates to a thin layer identification method of Shechuan formula granules. The present application provides a thin layer identification method of Shechuan formula granules, comprising: step 1, taking a sample to be detected, obtaining a test solution after pretreatment; taking Shechuan control medicinal materials, obtaining a Shechuan control medicinal material solution after pretreatment; step 2, taking the test solution and the Shechuan control medicinal material solution in step 1 to perform thin layer chromatography detection; the thin layer plate for the thin layer chromatography detection is a silica gel G thin layer plate, and the developing agent is n-butanol-glacial acetic acid-water; step 3, inspection, if there are the same color spots in the test chromatogram corresponding to the position of the Shechuan control medicinal material chromatogram, then the sample is Shechuan formula granules. The thin layer identification method of Shechuan formula granules established by the present application is used for quickly and effectively identifying Shechuan formula granules by observing whether the formula granules and the control medicinal materials have the same color spots at the same position in the thin layer chromatogram.

Description

Technical Field

[0001] The invention relates to the technical field of analysis and detection, in particular to a thin layer identification method for snake slough formula granules. Background Art

[0002] This invention uses dried epidermal membranes shed by Zaocys dhumnades (Cantor), a colubrid snake, as research subjects. The sloughs are collected in late spring, early summer, or early winter, desilted, and dried. They have the effects of dispelling wind, calming convulsions, removing cataracts, and detoxifying. Clinically, they are primarily used to treat pediatric convulsions, convulsions, cataracts, laryngeal paralysis, carbuncle, and itchy skin.

[0003] Snake sloughs contain collagen, amino acids, fatty acids, sterols, and trace elements. Current research on snake slough identification methods focuses primarily on microscopic identification, trait identification, and DNA molecular identification. For example, Zhang Ji's study compared the traits of nine snake sloughs. While simple, it requires high identification skills from the operator. Furthermore, the harvesting and processing methods are crude, and the medicinal materials are often broken, making identification difficult. Shi Linchun used DNA barcoding technology to identify snake sloughs. While the experiment yielded high accuracy in determining their origin, the equipment was expensive, the procedures were cumbersome, and the time and labor costs were high, making it difficult to apply to actual production testing. Yang Jiuyan used thin-layer chromatography (TLC) to identify snake sloughs using glycine as a reference. While this method is simple, glycine is widely present in animal medicines and cannot fully identify the species. Snake slough formula granules are prepared by processing, extracting, concentrating, drying, and granulating the medicinal materials. Its properties and DNA information are destroyed during the production process, and it is difficult to qualitatively identify it using the above-mentioned identification methods. No physical and chemical identification methods for snake slough formula granules have been found yet. Summary of the Invention

[0004] In view of this, the present invention provides a thin layer identification method for snake slough formula granules, comprising: step 1, taking a sample to be tested, and obtaining a test solution after pretreatment; taking a snake slough control medicinal material, and obtaining a snake slough control medicinal material solution after pretreatment; step 2, taking the test solution and the snake slough control medicinal material solution described in step 1 for thin layer chromatography detection; the thin layer plate for thin layer chromatography detection is a silica gel G thin layer plate, and the developing agent is n-butanol-glacial acetic acid-water; step 3, inspection, in the test sample chromatogram, if a spot of the same color appears at a position corresponding to the snake slough control medicinal material chromatogram, then it is a snake slough formula granule. The thin layer identification chromatography method for snake slough formula granules established by the present invention is used to quickly and effectively identify snake slough formula granules by observing whether the formula granules have spots of the same color as the control medicinal material in the same position in the thin layer chromatogram, thereby filling the gap in the qualitative identification of snake slough formula granules, improving the quality standard of snake slough formula granules, and ensuring drug safety.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a thin layer identification method for snake slough formula granules, comprising:

[0007] Step 1: Take the sample to be tested and obtain the test solution after pretreatment;

[0008] Take snake slough control medicinal material, and after pretreatment, obtain snake slough control medicinal material solution;

[0009] Step 2, taking the test solution and the snake skin control medicinal material solution described in step 1 for thin layer chromatography detection; the thin layer chromatography plate for the thin layer chromatography detection is a silica gel G thin layer plate, and the developing solvent is n-butanol-glacial acetic acid-water;

[0010] Step 3: Check. If a spot of the same color appears in the chromatogram of the test sample at a position corresponding to the chromatogram of the snake slough control medicinal material, the sample is the snake slough formula granules.

[0011] In some specific embodiments of the present invention, the pretreatment in step 1 comprises: mixing the sample to be tested with 70% ethanol and hydrochloric acid, refluxing for hydrolysis, filtering, and evaporating to dryness to obtain a first residue; dissolving the first residue with water, extracting with petroleum ether by shaking, evaporating the water to dryness to obtain a second residue, dissolving the second residue with 70% ethanol, and filtering to obtain the test solution;

[0012] Take a snake slough control medicinal material, add water to decoct, filter, evaporate the filtrate, mix it with 70% ethanol and hydrochloric acid, reflux hydrolysis, filter, and evaporate to dryness to obtain a third residue; dissolve the third residue in water, extract it with petroleum ether through shaking, evaporate the water to dryness to obtain a fourth residue, dissolve the fourth residue in 70% ethanol, filter, and obtain the snake slough control medicinal material solution.

[0013] In some specific embodiments of the present invention, the mass volume ratio of the test sample, the 70% ethanol, and the hydrochloric acid, measured in g / mL / mL, comprises: 0.5:10:2;

[0014] In terms of g / mL / mL, the mass volume ratio of the snake slough control medicinal material, the 70% ethanol and the hydrochloric acid is 2:10:2.

[0015] In some specific embodiments of the present invention, in terms of g / mL, the mass volume ratio of the snake slough control medicinal material to the water in the decoction of the snake slough control medicinal material is 2:50, and the decoction time is 30 minutes.

[0016] In some specific embodiments of the present invention, the temperature of the reflux hydrolysis is 90-100° C., and the time is 2 hours.

[0017] In some specific embodiments of the present invention, the boiling point of the petroleum ether is 60 to 90°C;

[0018] The volume ratio of the petroleum ether to the water in the petroleum ether shaking extraction is 2:3;

[0019] The petroleum ether shaking extraction was performed twice.

[0020] In some specific embodiments of the present invention, the thin layer chromatography detection specifically includes: aspirating the test sample solution and the snake skin control medicinal material solution, respectively spotting them on the same silica gel G thin layer plate, developing with a developing agent, taking out, drying, spraying with ninhydrin solution, heating to develop color, and inspecting.

[0021] The silica gel G thin layer plate of the present invention is selected from Qingdao Yuminyuan, batch number: 20180527, Tianjin Silida Technology Co., Ltd., batch number: 191016, and Merck, batch number: HX87183353.

[0022] In some specific embodiments of the present invention, the volume ratio of n-butanol, glacial acetic acid and water in the n-butanol-glacial acetic acid-water in step 2 is 4:1:1.

[0023] In some specific embodiments of the present invention, the sample spotting volume for thin layer chromatography detection comprises 10 μL.

[0024] In some specific embodiments of the present invention, the inspected temperature in step 3 is 4°C to 25°C.

[0025] In some specific embodiments of the present invention, the humidity under inspection is 32% to 75%.

[0026] In some specific embodiments of the present invention, step 3 includes: in the chromatogram of the test sample, if the ratio transfer value Rf1 is in the range of 0.571 to 0.63 and the ratio transfer value Rf2 is in the range of 0.412 to 0.51, and the spots show the same color as the chromatogram of the snake slough control medicinal material, then it is a snake slough formula granule.

[0027] The present invention includes but is not limited to providing the following beneficial effects:

[0028] The present invention discloses the construction and application of a thin-layer chromatography method for identifying snake slough formula granules. By observing the chromatogram of the test sample in the thin-layer chromatography spectrum and determining whether there are spots of the same color at the corresponding position of the chromatogram of the control medicinal material, the authenticity of the snake slough formula granules can be determined. The established thin-layer chromatography method can quickly and effectively identify snake slough formula granules. The method is simple to operate, highly sensitive, and stable, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for describing the embodiments or the prior art.

[0030] Figure 1 Indicates different systematic investigations, among which 1 is the snake slough control medicinal material; 2 to 4 are snake slough formula granules;

[0031] Figure 2 The sample volume is shown in Figure 1, where 1 to 4 are the sample volumes of the snake slough control medicinal material, 3 μL, 5 μL, 7 μL, and 10 μL; 4 to 8 are the sample volumes of the snake slough formula granules, 3 μL, 5 μL, 7 μL, and 10 μL;

[0032] Figure 3 The specificity test is shown in Figure 1, where 1 is a negative solution; 2 is a snake slough control medicinal material; 3 is a snake slough formula granule;

[0033] Figure 4 The figure shows the comparison of different thin layer plates (Tianjin Silida), where 1 is the control medicinal material of snake skin; 2 is the formula granule of snake skin;

[0034] Figure 5 Different thin layer plate tests (Merck) are shown, among which 1 is the control medicinal material of snake skin; 2 is the formula granule of snake skin;

[0035] Figure 6 Different thin layer plates were examined (Qingdao Ocean), among which 1 is the control medicinal material of snake skin; 2 is the formula granule of snake skin;

[0036] Figure 7 Shows the comparison of different temperatures (4°C), where 1 is the snake skin control medicinal material; 2 is the snake skin formula granule;

[0037] Figure 8 Shows the comparison of different temperatures (25°C), where 1 is the snake skin control medicinal material; 2 is the snake skin formula granule;

[0038] Figure 9 Shows the comparison of different humidity (32%), among which 1 is the snake skin control medicinal material; 2 is the snake skin formula granule;

[0039] Figure 10 Shows the comparison of different humidity (75%), among which 1 is the snake skin control medicinal material; 2 is the snake skin formula granule;

[0040] Figure 11 Figure 1 shows different batches of validation, where 1 is the snake slough control medicinal material; 2-4 are snake slough formula granules 2306051, 2306052, and 2306053 respectively;

[0041] Figure 12 The result diagram of comparative example 1 of the present invention is shown, wherein 4 is a snake slough control medicinal material; 1 to 3 are snake slough formula granules;

[0042] Figure 13 The result diagram of comparative example 2 of the present invention is shown, wherein 1-2 are snake slough control medicinal materials; 3-4 are snake slough formula granules;

[0043] Figure 14 The result diagram of Comparative Example 3 of the present invention is shown, wherein 1 is glycine; 2 is snake slough formula granules. DETAILED DESCRIPTION

[0044] The present invention discloses a thin layer identification method for snake skin formula particles. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously change or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0045] The present invention establishes a thin-layer method capable of identifying snake slough formula granules, uses snake slough reference medicinal materials with stronger specificity, and is more convincing in terms of identification.

[0046] The instruments, reagents and reagents used in the present invention are as follows:

[0047] 1. Instruments

[0048] Heating plate, mortar, thin layer imaging system: CAMAG TLC Visualizer, silica gel G thin layer plate (Qingdao Yuminyuan, batch number: 20180527, Tianjin Silida Technology Co., Ltd., batch number: 191016, Merck, batch number: HX87183353)

[0049] 2. Reagents

[0050] Ethanol, glacial acetic acid, hydrochloric acid, methanol, and n-butanol were all of analytical grade, and water was ultrapure water (prepared in the laboratory).

[0051] 3. Drug testing

[0052] Snake skin (Dieselgut) control medicinal material: (Shanghai Hongyong Biotechnology Co., Ltd., batch number: 380140-202301). Snake skin formula granules (Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch numbers: 2306051, 2306052, 2306053).

[0053] Unless otherwise specified, the raw materials and reagents used in the thin-layer identification method of the snake slough formula granules provided by the present invention can be purchased from the market.

[0054] The present invention will be further described below in conjunction with the embodiments:

[0055] Example 1 Establishment of the Thin Layer Method for Snake Skin Formula Granules

[0056] Take 0.5 g of this product, grind it into powder, add 10 mL of 70% ethanol and 2 mL of hydrochloric acid (AR grade), reflux and hydrolyze at 90-100 ° C for 2 hours, cool, filter, evaporate to dryness, dissolve the residue in 10 mL of water, and extract twice with 30 mL of petroleum ether (60-90 ° C), 15 mL each time, discard the petroleum ether liquid, evaporate the water liquid, dissolve the residue in 5 mL of 70% ethanol, filter, and obtain the filtrate, which is used as the test solution (snake skin formula granule solution).

[0057] Take 2 g of snake skin control medicinal material, add 50 mL of water, decoct for 30 minutes, filter, evaporate the filtrate to dryness, add 10 mL of 70% ethanol and 2 mL of hydrochloric acid, reflux hydrolysis for 2 hours, cool, filter, evaporate to dryness, dissolve the residue in 10 mL of water, and extract twice with 30 mL of petroleum ether (60-90 ° C), 15 mL each time, discard the petroleum ether liquid, evaporate the water liquid, dissolve the residue in 5 mL of 70% ethanol, filter, and obtain a filtrate, which is used as the snake skin control medicinal material solution.

[0058] According to the thin layer chromatography method (Chinese Pharmacopoeia 2020 Edition, General Chapter 0502), 10 μL of each of the above solutions was applied to the same silica gel G thin layer plate. Develop the plate with n-butanol-glacial acetic acid-water (4:1:1 by volume). Remove the plate, air dry, spray with ninhydrin test solution, and heat at 105°C until the spots are clearly colored. In the chromatogram of the test sample, a spot of the same color appears at the corresponding position in the chromatogram of the control medicinal material.

[0059] Example 2 Investigation of different deployment systems

[0060] The effects of three different development systems on the detection effect were investigated: n-butanol-glacial acetic acid-water (4:1:1), n-butanol-glacial acetic acid-water (6:1:0.5), and n-butanol-glacial acetic acid-water (3:1:1). The results showed that ( Figure 1 ), using n-butanol-glacial acetic acid-water (4:1:1) as the developing solvent, the separation effect is better, and the snake skin formula particles can be clearly identified.

[0061] Example 3 Methodological Investigation

[0062] 3.1 Sampling quantity inspection

[0063] Under the experimental conditions proposed above, 3 μL, 5 μL, 7 μL and 10 μL of snake skin control medicinal material solution and snake skin formula granule solution were spotted on the same silica gel G thin layer plate. The results were shown in the following table. Figure 2As can be seen from the figure, when 10 μL of the control herbal solution and 10 μL of the test solution were applied, the TLC spots were clearly colored. In the test sample chromatogram, spots of the same color appeared at positions corresponding to those in the control herbal solution. This method can be used for TLC identification of snake slough granules.

[0064] 3.2 Specificity Investigation

[0065] Prepare snake slough control medicinal material solution, snake slough formula granule solution, and negative solution (prepare the excipients in the same way as the test sample preparation method) according to the above test sample preparation method, spot them on the same thin layer plate, develop them according to the proposed thin layer chromatography conditions, and inspect them. The results are shown in Figure 3 The results showed that the negative sample did not interfere with the snake slough formula granules test sample, indicating that the method has good specificity. In the chromatogram of the test sample, spots of the same color appeared at the corresponding positions in the chromatogram of the control medicinal material. This method can be used for thin-layer chromatography identification of snake slough formula granules.

[0066] 3.3 Durability Investigation

[0067] 3.3.1 Comparison of different thin layer plates

[0068] Tianjin Silida, Merck, and Qingdao Yuminyuan silica gel G plates were selected and tested according to the proposed test methods. Figures 4-6 The results showed that all three brands of TLC plates met the identification requirements, indicating that the method is robust. The test sample chromatogram showed spots of the same color at positions corresponding to those in the control chromatogram. This method can be used for TLC identification of snake slough granules.

[0069] 3.3.2 Comparison of different temperatures

[0070] Take the spotted thin layer plate and develop it at low temperature 4℃ and normal temperature 25℃ respectively. Figures 7-8 It can be seen that this method has good durability at different temperatures. In the chromatogram of the test sample, spots of the same color appear at positions corresponding to the chromatogram of the control medicinal material. This method can be used for thin layer chromatography identification of snake slough formula granules.

[0071] 3.3.3 Comparison of different humidity levels

[0072] Take the spotted thin layer plate and develop it in 32% and 75% humidity environment respectively. Figures 9-10 As can be seen from the figure, this method has good durability to different humidity levels. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions in the chromatogram of the control medicinal material. This method can be used for thin-layer chromatography identification of snake slough formula granules.

[0073] 3.3.4 Verification

[0074] Three batches of snake slough formula granules were subjected to thin layer identification and verification, and the experimental results are shown in Figure 11 In the chromatogram of the test sample, spots of the same color appeared at the corresponding positions in the chromatogram of the control medicinal material. This method can be used for thin-layer chromatography identification of snake slough formula granules.

[0075] Comparative Example 1

[0076] Take 1 g of snake skin granules, grind them into powder, add 40 mL of 50% ethanol, heat and reflux for 30 minutes, filter, evaporate the filtrate to dryness, add 2 mL of methanol to dissolve the residue, and use it as the test solution.

[0077] Take another 1g of snake skin control medicinal material, add 50mL of water, boil for 30 minutes, cool and filter, evaporate the filtrate to dryness, add 40mL of 50% ethanol to the residue, heat and reflux for 30 minutes, filter, evaporate the filtrate to dryness, add 2mL of methanol to the residue to dissolve it, and prepare a control medicinal material solution.

[0078] According to the thin-layer chromatography method (General Method 0502), 10 μl of each of the two solutions was applied to the same silica gel G thin-layer plate. Develop the plate using n-butanol-glacial acetic acid-water (4:1:1) as the developing solvent. Remove the plate, allow it to air dry, spray it with ninhydrin test solution, and heat it at 105°C until the spots are clearly colored. When examined under sunlight, the test sample chromatogram should show a spot of the same color at the corresponding position in the control herbal chromatogram.

[0079] The results showed that ( Figure 12 ), using the above-mentioned thin-layer identification method, there were obvious differences between the spots of the snake slough formula granules and the snake slough control medicinal materials, indicating that this method cannot effectively perform effective qualitative identification of the snake slough formula granules.

[0080] Comparative Example 2

[0081] Take the snake skin formula granules, grind them into powder, add 25 mL of 50% methanol, soak for 30 minutes, ultrasonically treat for 25 minutes, cool, shake well, transfer to a 50 mL centrifuge tube, centrifuge (at 3000 revolutions per minute) for 10 minutes, and take the supernatant as the test solution.

[0082] Preparation method of control medicinal material solution: Take 1 g of control medicinal material and prepare the control medicinal material solution in the same way as the above-mentioned snake slough granules.

[0083] According to the thin layer chromatography method (General Method 0502), 10 μl of the test solution and the control medicinal material solution were taken and spotted on the same thin layer silica gel plate. The plate was developed with n-butanol-glacial acetic acid-water (4:1:1) as the developing solvent. The plate was taken out, dried, sprayed with ninhydrin test solution, and heated at 105°C until the spots were clearly colored. When examined under sunlight, spots of the same color appeared in the chromatogram of the test sample at the corresponding position in the chromatogram of the control medicinal material. The experimental results are shown in Figure 13 .

[0084] The results showed that there were differences in the thin layer chromatography of snake slough formula granules and the thin layer chromatography of snake slough control medicinal materials using the above-mentioned thin layer chromatography identification method, indicating that the method cannot effectively perform effective qualitative identification of snake slough formula granules.

[0085] Comparative Example 3

[0086] Take 4 g of the powder of this product and add 60 mL of 50% ethanol, heat under reflux in a water bath for 1 hour, filter, evaporate the filtrate to dryness, add 1 mL of methanol to dissolve it, and use it as the test solution.

[0087] Separately, add dilute ethanol to a glycine reference solution to prepare a solution containing 0.5 mg of glycine per 1 mL as the reference solution.

[0088] According to the thin layer chromatography method (Appendix VI B of the first part of the Chinese Pharmacopoeia 2005 edition), 5 μL of each of the two upper layer solutions was taken and spotted on the same silica gel G thin layer plate with sodium carboxymethyl cellulose as the adhesive. The plate was developed with n-butanol-ethanol-glacial acetic acid-water (4:1:1:1.5) as the developing solvent. The plate was taken out, dried, sprayed with ninhydrin test solution, and heated at 105°C until the spots were clearly colored. Spots of the same color appeared at the corresponding positions of the chromatogram of the reference substance. The experimental results are shown in Figure 14 .

[0089] The above-mentioned thin layer identification method can only prove that the medicinal material contains glycine but cannot prove that it is the snake skin medicinal material, let alone that it is the snake skin formula granules. The specificity is not strong, indicating that this method cannot be used for effective qualitative identification of the snake skin formula granules.

[0090] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A thin layer identification method for snake skin formula granules, characterized in that: include: Step 1: Take the sample to be tested and obtain the test solution after pretreatment; Take snake slough control medicinal material, and after pretreatment, obtain snake slough control medicinal material solution; Step 2, taking the test solution and the snake skin control medicinal material solution described in step 1 for thin layer chromatography detection; the thin layer chromatography plate for the thin layer chromatography detection is a silica gel G thin layer plate, and the developing solvent is n-butanol-glacial acetic acid-water; Step 3: Check: If a spot of the same color appears in the chromatogram of the test sample at a position corresponding to the chromatogram of the control medicinal material, the sample is the snake slough formula granules; The pretreatment in step 1 comprises: mixing the sample to be tested with 70% ethanol and hydrochloric acid, refluxing and hydrolyzing, filtering, and evaporating to dryness to obtain a first residue; Dissolve the first residue in water, extract with petroleum ether through shaking, evaporate the water to dryness to obtain a second residue, dissolve the second residue in 70% ethanol, filter, and obtain the test solution; A snake slough control medicinal material was decocted with water, filtered, and the filtrate was evaporated to dryness, mixed with 70% ethanol and hydrochloric acid, refluxed for hydrolysis, filtered, and evaporated to dryness to obtain a third residue; the third residue was dissolved in water, extracted with petroleum ether by shaking, and the water was evaporated to dryness to obtain a fourth residue, which was dissolved in 70% ethanol and filtered to obtain the snake slough control medicinal material solution; The volume ratio of n-butanol, glacial acetic acid and water in the n-butanol-glacial acetic acid-water in step 2 is 4:1:

1.

2. The thin layer identification method according to claim 1, characterized in that: In terms of g / mL / mL, the mass volume ratio of the sample to be tested, the 70% ethanol, and the hydrochloric acid comprises: 0.5:10:2; In terms of g / mL / mL, the mass volume ratio of the snake slough control medicinal material, the 70% ethanol and the hydrochloric acid is 2:10:

2.

3. The thin layer identification method according to claim 1, characterized in that: In terms of g / mL, the mass volume ratio of the snake slough control medicinal material to the water in the decoction of the snake slough control medicinal material is 2:50, and the decoction time is 30 minutes.

4. The thin layer identification method according to claim 1, characterized in that: The temperature of the reflux hydrolysis is 90-100° C., and the time is 2 hours.

5. The thin layer identification method according to claim 1, characterized in that: The boiling point of the petroleum ether is 60-90°C; The volume ratio of the petroleum ether to the water in the petroleum ether shaking extraction is 2:3; The petroleum ether shaking extraction was performed twice.

6. The thin layer identification method according to claim 1, characterized in that: The sample spotting volume for the thin layer chromatography assay comprised 10 µL.

7. The thin layer identification method according to claim 1, characterized in that: The temperature of the inspection in step 3 includes 4°C to 25°C.

8. The thin layer identification method according to claim 1, characterized in that: The humidity ranges from 32% to 75%.

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