Thin-layer identification method and application of betel nut and preparation thereof

Abstract

The invention discloses a thin-layer identification method of areca nuts and preparations thereof, in the thin-layer chromatography identification analysis step, methanol-ethyl acetate-cyclohexane-ammonia water is used as a developing solvent, and the volume ratio of methanol to ethyl acetate to cyclohexane to ammonia water is (10-20): 10: 10: (0.01-0.1). The thin-layer identification method provided by the invention is strong in specificity, negative and free from interference on the identification method of the areca-nut and the preparation thereof, and can accurately and stably control the quality of the areca-nut and the preparation thereof so as to adapt to the industrial stable production of medicines, and a more convenient and effective method is provided for the quality control, market supervision and basic research of pharmacodynamic substances of the areca-nut and the preparation thereof.

Description

Technical Field

[0001] The invention belongs to the field of drug analysis, and specifically relates to a quality control and identification determination method for betel nut and a preparation thereof. Background Art

[0002] Betel nut (Areca catechu L.) is an evergreen tree of the genus Areca in the palm family. Medicinal betel nut (Arecae Semen, AS) is the dried mature seed of the palm plant Areca nut. It is described in the Chinese Pharmacopoeia as a mature fruit from late summer to early autumn, dried, peeled, and the seeds taken out. Betel nut is one of the "four major southern medicines" in my country. It belongs to the stomach meridian, small intestine meridian and large intestine meridian, and has the effects of killing insects, eliminating accumulation, promoting diuresis and qi, and stopping malaria. The betel nut seeds of immature fruits are called tender betel nut (also known as green fruit betel nut, Arecae Semen Viride, ASV), which has many synonyms, also known as betel nut core, betel nut kernel, etc. Tender betel nut is exactly the same as the source and matrix of medicinal betel nut in the pharmacopoeia, only the maturity is different. The betel nut medicinal material in this patent refers to medicinal betel nut and / or tender betel nut.

[0003] Thin layer chromatography has been widely used in the qualitative identification of Chinese herbal medicines and their preparations. This is mainly because it has the dual functions of separation and analysis, which greatly improves the sensitivity and specificity of identification work and becomes an important method for quality control and identification of Chinese medicines. At present, the thin layer identification methods of the identification methods of "Betel Nut" medicinal materials and their decoction pieces included in the 2020 edition of the "Chinese Pharmacopoeia" and the 2020 edition of the "Chinese Veterinary Pharmacopoeia" only use "arecoline" as the indicator component. After reviewing the existing technology, there are no reports on the thin layer chromatography method for the simultaneous qualitative detection of the four alkaloids of arecoline, norarecoline, arecoline, and norarecoline in betel nut. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0005] The object of the present invention is to provide a thin layer identification method for multiple components in betel nut and its preparation, which has high sensitivity and can effectively and quickly perform quality control and identification detection of betel nut and its preparation. The thin layer identification method comprises the following steps:

[0006] (1) Prepare test solution;

[0007] (2) preparing a reference solution;

[0008] (3) Pipette the test solution and the reference solution onto the same silica gel G thin layer plate, develop it with a developing agent, and perform thin layer chromatography identification analysis.

[0009] Preferably, step (1) comprises at least one of the following methods:

[0010] (1) Take 1-5 g of the sample powder, add 25-50 ml of water, heat and reflux for 0.5-1 h, filter, take 15-50 ml of the filtrate, dry, add 5-20 ml of solvent to the residue, ultrasonicate for 0.5-1 h, filter, and concentrate the filtrate to 2-10 ml.

[0011] (2) Take 1-5 g of the sample powder, add 25-50 ml of solvent, heat to reflux for 0.5-1 h, filter, dry, add 5-15 ml of solvent to dissolve the residue, and take the supernatant as the sample solution.

[0012] (3) Take 1-5 g of the test sample powder, add 25-50 ml of solvent, sonicate for 0.5-1 h, filter, and use the filtrate as the test sample solution.

[0013] Preferably, method (1) is to take 2.5 g of the test sample powder, add 50 ml of water, heat and reflux for 1 hour, filter, take 25 ml of the filtrate, dry, add 10 ml of solvent to the residue, ultrasonicate for 30 minutes, filter, and concentrate the filtrate to 5 ml to obtain; method (2) is to take 2.5 g of the test sample powder, add 50 ml of solvent, heat and reflux for 30 minutes, filter, dry, dissolve the residue in 10 ml of solvent, and take the supernatant as the test sample solution; method (3) is to take 2.5 g of the test sample powder, add 25 ml of solvent, ultrasonicate for 30 minutes, filter, and take the filtrate as the test sample solution.

[0014] Preferably, the solvent in methods (1) to (3) is any one of water, methanol, 80% anhydrous ethanol, and anhydrous ethanol.

[0015] Preferably, step (2) is to dissolve the reference substance in methanol to prepare a solution containing 1.5 mg per 1 ml.

[0016] Preferably, the reference substance is any at least one component of norarecoline, arecoline, norarecoline, and arecoline hydrobromide.

[0017] Preferably, step (3) is to take 3-7 μl of each of the test sample and reference solution, spot them on the same silica gel G thin layer plate, use methanol-ethyl acetate-cyclohexane-ammonia water in a volume ratio of (10-20):10:10:(0.01-0.1) as a developing solvent, place them in a developing cylinder pre-saturated with ammonia vapor, develop, take out, dry, and fumigate in iodine vapor until the spots are clear. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions of the chromatogram of the reference sample.

[0018] Preferably, the solution spotting volume is 5 μL, and the volume ratio of the developing solvent methanol-ethyl acetate-cyclohexane-ammonia water is 17:10:10:0.05;

[0019] Beneficial effects of the present invention:

[0020] The thin-layer identification method provided by the present invention has appropriate selection of developing agent, high identification efficiency and precision, and convenient operation. The identification method for betel nut and its preparations has strong specificity, no negative interference, and can accurately and stably control the quality of betel nut and its preparations to adapt to the industrialized stable production of drugs. It provides a more convenient and effective method for the quality control, market supervision, and basic research on medicinal efficacy substances of betel nut single preparations and traditional Chinese medicine compound preparations containing betel nut. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Chromatogram of the test sample extraction method-1 (samples corresponding to sample numbers 1-11 are shown in Table 1)

[0022] Figure 2 Chromatogram of the test sample extraction method-2 (samples corresponding to sample numbers 1-10 are shown in Table 2)

[0023] Figure 3 Chromatogram of the test sample extraction method-3 (samples corresponding to sample numbers 1-10 are shown in Table 3)

[0024] Figure 4 Developing agent system investigation chromatogram-1 (samples corresponding to spot numbers 1-8 are shown in Table 4)

[0025] Figure 5 Developing agent system investigation chromatogram-2 (samples corresponding to spot numbers 1-10 are shown in Table 5)

[0026] Figure 6 Chromatogram of sample quantity inspection (samples corresponding to sample numbers 1-12 are shown in Table 6)

[0027] Figure 7 Specificity inspection chromatogram (samples corresponding to spot numbers 1-12 are shown in Table 7)

[0028] Figure 8 Repeatability test chromatogram (samples corresponding to spot numbers 1-12 are shown in Table 8)

[0029] Fig. 9 Durability test chromatogram-1 (samples corresponding to spot numbers 1-12 are shown in Table 10)

[0030] Fig.10 Durability test chromatogram-2 (samples corresponding to spot numbers 1-12 are shown in Table 11)

[0031] Fig.11 Durability test chromatogram-3 (samples corresponding to spot numbers 1-12 are shown in Table 12)

[0032] Fig.12 Durability test chromatogram-4 (samples corresponding to spot numbers 1-12 are shown in Table 13)

[0033] Fig.13Durability test chromatogram-5 (samples corresponding to spot numbers 1-12 are shown in Table 14) DETAILED DESCRIPTION

[0034] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0035] Unless otherwise indicated, all technical and scientific terms and abbreviations used herein have the meanings commonly understood by those of ordinary skill in the field of the invention or the field in which the terms are used. Although any methods, conditions, substances or materials similar to or equivalent to those disclosed herein may be used in the practice of the present invention, preferred methods, conditions, substances or materials are described herein.

[0036] The betel nut decoction pieces, betel nut medicinal materials, and reference substances (arecoline, norarecoline, arecoline, and norarecoline) in the embodiments are all commercially available products, and the betel nut extract is provided by Hunan Jiuanhe Biotechnology Co., Ltd.

[0037] Example 1 Investigation of the preparation method of the test sample

[0038] 1.1 Method 1: Weigh the powder of betel nut medicinal materials, powder of betel nut decoction pieces, and powder of betel nut extract respectively, about 2.5g of each test sample, put it in a stoppered conical flask, add 50ml of solvent, reflux, filter, take 25ml of the filtrate, evaporate it in a water bath, add 10ml of methanol to the residue, ultrasonicate for 30min, filter, and concentrate the filtrate to 5ml to obtain the test sample solution. The reference sample is added with methanol to prepare a solution containing 1.5mg per 1ml, which is used as the reference sample solution. Pipette 5μl of the above test sample and reference sample solutions respectively, and spot them on the same silica gel G thin layer plate, use methanol-ethyl acetate-cyclohexane-ammonia water (17:10:10:0.05) as the developing agent, place it in a developing cylinder pre-saturated with ammonia vapor, develop, take out, dry, and fumigate in iodine vapor until the spots are clear. The inspection method and results are shown in Table 1. Figure 1 shown.

[0039] Table 1: Inspection table of extraction methods for test samples-1

[0040]

[0041] 1.2 Method 2: Weigh about 2.5g of betel nut powder, add 50ml of solvent, heat and reflux for 30 minutes, filter, evaporate, add 10ml of methanol to the residue to dissolve it, and take the supernatant as the test solution. Add methanol to the reference substance to make a solution containing 1.5mg per 1ml, which is used as the reference substance solution. Pipette 5μl of the above test sample and reference substance solutions, respectively, and spot them on the same silica gel G thin layer plate, use methanol-ethyl acetate-cyclohexane-ammonia water (17:10:10:0.05) as the developing agent, place it in a developing cylinder pre-saturated with ammonia vapor, develop, take out, dry, and fumigate in iodine vapor until the spots are clear. The inspection method and results are shown in Table 2. Figure 2 shown.

[0042] Table 2 Inspection of extraction methods for test samples-2

[0043] Sample number Sample name Sample number Sample name 1 Mixed reference 7 Areca nut extract (extraction solvent: anhydrous ethanol) 2 Norarecoline 8 Areca nut extract (extraction solvent: methanol) 3 Arecaline 9 Betel nut slices (extraction solvent: water) 4 Norecoline 10 Betel nut medicinal material (extraction solvent: water) 5 Arecoline 11 Mixed reference 6 Mixed reference

[0044] 1.3 Method 3: Weigh about 2.5g of betel nut powder, add 25ml of solvent, ultrasonicate for 30 minutes, filter, and take the filtrate as the test solution. Add methanol to the reference substance to prepare a solution containing 1.5mg per 1ml, which is used as the reference solution. Pipette 5μl of the above test sample and reference solution respectively, and spot them on the same silica gel G thin layer plate, use methanol-ethyl acetate-cyclohexane-ammonia water (17:10:10:0.05) as the developing agent, place it in a developing cylinder pre-saturated with ammonia vapor, develop, take out, dry, and fumigate in iodine vapor until the spots are clear. The inspection method and results are shown in Table 2. Figure 2 shown.

[0045] Table 3: Inspection of extraction methods for test samples-3

[0046]

[0047] 1.4 Test results: Figure 1 , 2 3. From the inspection results, it can be seen that the three methods can all well develop the color of areca alkaloids, and the best method is method 1: take 2.5g of the test sample powder, add 50ml of water, heat reflux for 1h, filter, take 25ml of the filtrate, evaporate to dryness in a water bath, add 10ml of methanol to the residue, ultrasonicate for 30min, filter, and concentrate the filtrate to 5ml.

[0048] Example 2 Determination of the developing agent system

[0049] 1.1 Take 2.5g of the test sample powder, add 50ml of water, heat to reflux for 1h, filter, take 25ml of the filtrate, evaporate to dryness in a water bath, add 10ml of methanol to the residue, ultrasonicate for 30min, filter, and concentrate the filtrate to 5ml to obtain the test sample solution. Add methanol to the reference substance to make a solution containing 1.5mg per 1ml, which is used as the reference substance solution. Pipette 5μl of the above test sample and reference substance solutions, respectively, and spot them on the same silica gel G thin layer plate, use methanol-ethyl acetate-cyclohexane-ammonia water (10:10:10:0.01) as the developing agent, place it in a developing cylinder pre-saturated with ammonia vapor, develop, take out, dry, and fumigate in iodine vapor until the spots are clear. The inspection method and results are shown in Table 4. Figure 4 shown.

[0050] Table 4 Development agent system investigation table-1

[0051]

[0052] The results showed that the four alkaloids in the mixed reference and alcohol-provided samples were all developed, but the development distances of norarecoline and arecoline were small, so the polarity of the developing agent was further increased. The development distance of arecoline was too large, so the proportion of ammonia water was reduced.

[0053] 1.2 Take 2.5g of the test sample powder, add 50ml of water, heat to reflux for 1h, filter, take 25ml of the filtrate, evaporate to dryness in a water bath, add 10ml of methanol to the residue, ultrasonicate for 30min, filter, and concentrate the filtrate to 5ml to obtain the test sample solution. Add methanol to the reference substance to make a solution containing 1.5mg per 1ml, which is used as the reference substance solution. Pipette 5μl of the above test sample and reference substance solutions, respectively, and spot them on the same silica gel G thin layer plate, use methanol-ethyl acetate-cyclohexane-ammonia water (17:10:10:0.05) as the developing agent, place it in a developing cylinder pre-saturated with ammonia vapor, develop, take out, dry, and fumigate in iodine vapor until the spots are clear. The inspection method and results are shown in Table 5. Figure 5 shown.

[0054] Table 5 Development agent system investigation table-2

[0055]

[0056] The results showed that the four alkaloids in the mixed reference and test samples were all developed and the development distance was moderate, so the developing solvent system of this method was confirmed to be methanol: ethyl acetate: cyclohexane: ammonia water = 17:10:10:0.05.

[0057] Example 3 Determination of the amount of sample spotted

[0058] Take the reference solution and the test solution under the item "Study on the preparation method of the test sample" in Example 1, and spot the sample with the developing agent system determined in Example 2, spotting 3μl, 5μl, and 7μl respectively. The results are shown in Table 6. Figure 6shown.

[0059] Table 6 Determination of spotting amount

[0060]

[0061] The results showed that the four alkaloids in the mixed reference and test samples were colored at three spotting volumes, but when the spotting volume was 3μl, some spots were not obvious; when the spotting volume was 7μl, the first spot of the test sample was easily interfered by the origin; therefore, the spotting volume of this method was determined to be: 5μl.

[0062] Example 4 Thin layer chromatography method investigation

[0063] 1.1 Exclusiveness Investigation

[0064] Specificity refers to the ability of an analytical method to correctly determine the analyte in the presence of other components.

[0065] 1.1.1 Specific sample preparation

[0066] Blank solution: methanol.

[0067] Blank auxiliary material: Take 2.5 g of maltodextrin and prepare a blank auxiliary material solution according to the test sample preparation method determined under the item "Investigation of test sample preparation method" in Example 1.

[0068] Reference substance positioning solution: Add methanol to arecoline, norarecoline, arecoline, and norarecoline to make a solution containing 1.5 mg per 1 ml.

[0069] Mixed reference solution: weigh about 3 mg of norarecoline reference substance, about 3 mg of arecoline reference substance, about 3 mg of norarecoline reference substance, and about 3 mg of arecoline hydrobromide reference substance, add methanol to dissolve and dilute to 2 ml, mix well, and obtain.

[0070] Specific test solution: Take betel nut medicinal materials, betel nut decoction pieces, and betel nut extract respectively to prepare the test solution according to the test sample preparation method determined under the item "Investigation of test sample preparation method" in Example 1.

[0071] 1.1.2 Specificity inspection points

[0072] Take 5 μl of the above test and reference solutions, spot them on the same silica gel G thin layer plate, use methanol-ethyl acetate-cyclohexane-ammonia water (17:10:10:0.05) as the developing agent, place it in a developing cylinder pre-saturated with ammonia vapor, develop, take it out, dry it, and fumigate it in iodine vapor until the spots are clear. See Table 7 for the spotting table.

[0073] Figure 7 Specific survey sample table

[0074]

[0075] 1.1.3 Exclusive results

[0076] There is no interference from the blank solution and blank excipients; the 4 spots in the chromatogram of the specific test solution correspond to the corresponding spots in the chromatogram of the 4 reference solution and the mixed reference solution, and there is no other interference. Figure 7 .

[0077] 1.2 Repeatability study

[0078] 1.2.1 Repeatable sample preparation

[0079] Mixed reference solution: same as 4.1.1.

[0080] Repeatable test solution: Prepare the test solution of betel nut medicinal material, betel nut slices and betel nut extract according to the test sample preparation method determined under the item "Study on the test sample preparation method" in Example 1. Prepare 6 portions of the test solution of young betel nut medicinal material and betel nut slices in parallel, and prepare 5 portions of the test solution of betel nut extract in parallel.

[0081] 1.2.2 Repeatability inspection sampling

[0082] Take 5 μl of the above test and reference solutions, spot them on the same silica gel G thin layer plate, use methanol-ethyl acetate-cyclohexane-ammonia water (17:10:10:0.05) as the developing agent, place it in a developing cylinder pre-saturated with ammonia vapor, develop, take it out, dry it, and fumigate it in iodine vapor until the spots are clear. See Table 8 for the spotting table.

[0083] Table 8 Repeatability of spotting

[0084]

[0085] 1.2.3 Repeatability test results

[0086] The four spots in the chromatograms of the 6 repetitive test solutions of betel nut medicinal materials and betel nut decoction pieces and the 5 repetitive test solutions of betel nut extracts all correspond to the spots at the corresponding positions in the chromatogram of the mixed reference solution, and there is no other interference. Figure 8 .

[0087] 1.3 Durability test

[0088] Durability refers to the degree to which the test results are not affected when there are small changes in the test conditions, which provides a basis for the established method to be used for routine testing. The durability change conditions investigated in this verification experiment are shown in Table 9:

[0089] Table 9 Durability Condition Change Table

[0090] Serial number Change conditions Normal conditions Change conditions 1 temperature Room temperature Low temperature (5℃) 2 Thin layer plate Qingdao Ocean 1: Qingdao Dingkang 2: Merk 3 humidity 45％ 1：25％2：75％

[0091] 1.3.1 Durability sample preparation

[0092] Mixed reference solution: same as 4.1.1.

[0093] Repeatable test solution: Betel nut medicinal materials, betel nut decoction pieces, and betel nut extract are respectively prepared according to the test sample preparation method determined under the item "Investigation of test sample preparation method" in Example 1 to prepare test sample solutions, and prepare 3 copies in parallel.

[0094] 1.3.2 Durability test points

[0095] Take 5 μl of the above test and reference solutions, spot them on the same silica gel G thin layer plate, use methanol-ethyl acetate-cyclohexane-ammonia water (17:10:10:0.05) as the developing agent, place it in a developing cylinder pre-saturated with ammonia vapor, develop, take it out, dry it, and fumigate it in iodine vapor until the spots are clear. See Tables 10, 11, 12, 13, and 14 for the spotting table.

[0096] Table 10 Durability test-1 (low temperature)

[0097]

[0098] Table 11 Durability inspection-2 (Qingdao Dingkang thin layer board)

[0099]

[0100] Table 12 Durability Study-3 (Merk Thin Layer Board)

[0101]

[0102] Table 13 Durability Study-4 (RH% = 25%)

[0103]

[0104] Table 14 Durability Study-5 (RH%=75%)

[0105]

[0106] 1.3.3 Durability test results

[0107] like Fig. 9 , 10 As shown in Figures 11, 12, and 13, the spots of norarecoline, arecoline, norarecoline, and arecoline in the chromatograms of the reference solution and the test solution under each condition correspond to each other without interference, which proves that the method has good durability.

[0108] 1.4 Results of thin layer chromatography

[0109] Specificity: There is no interference from blank solvents and excipients; the test sample shows spots of the same color at the corresponding positions of the reference sample and there is no interference from other spots.

[0110] Repeatability: All 6 test samples showed spots of the same color at the corresponding positions of the reference sample.

[0111] Durability: When the TLC conditions are changed within the durability range, the test sample will show spots of the same color at the corresponding positions as the reference sample and there will be no interference from other spots.

[0112] Conclusion: All validation items met the standards and the method was validated.

Claims

1. A thin layer identification method for betel nut and its preparation and its application, characterized in that: The thin layer identification method includes the following steps: (1) Prepare test solution; (2) preparing a reference solution; (3) Pipette the test solution and the reference solution onto the same silica gel G thin layer plate, develop it with a developing agent, and perform thin layer chromatography identification analysis. Wherein, in the step of thin layer chromatography identification analysis, methanol-ethyl acetate-cyclohexane-ammonia water is used as a developing agent, and the volume ratio of methanol, ethyl acetate, cyclohexane and ammonia water is (10-20):10:10:(0.01-0.1).

2. According to claim 1, it is characterized in that: The test samples are betel nut medicinal materials, decoction pieces, processed products, preparation intermediates or finished preparations.

3. According to claim 1, it is characterized in that: The preparation of the test solution in step (1) comprises at least one of the following methods: (1) Take 1-5 g of the sample powder, add 25-50 ml of water, heat and reflux for 0.5-1 h, filter, take 15-50 ml of the filtrate, dry, add 5-20 ml of solvent to the residue, ultrasonicate for 0.5-1 h, filter, and concentrate the filtrate to 2-10 ml. (2) Take 1-5 g of the sample powder, add 25-50 ml of solvent, heat to reflux for 0.5-1 h, filter, dry, add 5-15 ml of solvent to dissolve the residue, and take the supernatant as the sample solution. (3) Take 1-5 g of the test sample powder, add 25-50 ml of solvent, sonicate for 0.5-1 h, filter, and use the filtrate as the test sample solution.

4. According to claim 3, it is characterized in that: Method (1) is to take 2.5 g of the test sample powder, add 50 ml of water, heat and reflux for 1 hour, filter, take 25 ml of the filtrate, dry, add 10 ml of solvent to the residue, ultrasonicate for 30 minutes, filter, and concentrate the filtrate to 5 ml to obtain the product; method (2) is to take 2.5 g of the test sample powder, add 50 ml of solvent, heat and reflux for 30 minutes, filter, dry, dissolve the residue in 10 ml of solvent, and take the supernatant as the test sample solution; method (3) is to take 2.5 g of the test sample powder, add 25 ml of solvent, ultrasonicate for 30 minutes, filter, and take the filtrate as the test sample solution.

5. According to claims 3 to 4, it is characterized in that: The solvent is any one of water, methanol, 80% ethanol and anhydrous ethanol.

6. According to claim 1, it is characterized in that: Step (2) is to dissolve the reference substance in methanol to prepare a solution containing 1.5 mg per 1 ml.

7. According to claim 6, it is characterized in that: The reference substance is at least one component of norarecoline, arecoline, norarecoline, and arecoline hydrobromide.

8. According to claim 1, it is characterized in that: Step (3) is to take 3-7 μl of each of the test sample and reference solution, spot them on the same silica gel G thin layer plate, use methanol-ethyl acetate-cyclohexane-ammonia water in a volume ratio of (10-20):10:10:(0.01-0.1) as a developing solvent, place them in a developing cylinder pre-saturated with ammonia vapor, develop, take out, dry, and fumigate in iodine vapor until the spots are clear. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions of the chromatogram of the reference sample.

9. According to claim 8, it is characterized in that: The solution spotting volume was 5 μl, and the volume ratio of the developing solvent methanol-ethyl acetate-cyclohexane-ammonia water was 17:10:10:0.

05.

10. According to claims 1 to 9, it is characterized in that: The thin layer identification method can effectively detect betel nut alkaloids, which include at least one of the following components: (1) Arecoline; (2) Arecaline; (3) Norecoline; (4) Norarecoline.

Images