Thin-layer chromatography detection and identification method for a type of Polygonatum bistorta or its adulterants and their formulation granules

Abstract

This invention provides a thin-layer chromatography (TLC) method for detecting and identifying Polygonum bistorta or its adulterants and their formulation granules. The TLC method for detecting Polygonum bistorta or its adulterants includes: spotting the test solution onto a silica gel G TLC plate; developing the plate using a toluene-butyl acetate-formic acid-acetic acid solution at a volume ratio of 10:(8-10):(3-5):(1-2); removing the plate; air-drying the plate; and examining it under ultraviolet light and / or spraying it with a colorimetric reagent and examining it under sunlight. This invention can effectively obtain TLC chromatograms of Polygonum bistorta and its adulterants with a greater number of significant spots. The TLC method is simple to operate, has a rapid detection time, produces a large number of fluorescent spots in the Polygonum bistorta sample, displays rich material information, and provides clear and concise results, allowing for intuitive determination of sample measurement results. The method has strong adaptability; therefore, it can effectively provide the prerequisites for quality control or subsequent identification of Polygonum bistorta and its adulterants.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine testing, specifically to a thin-layer chromatography method for the detection and identification of Polygonum bistorta or its adulterants and their formulation granules. Background Technology

[0002] *Polygonum bistorta* L., a plant in the Polygonaceae family, is a dried rhizome that grows mainly in grassy slopes and damp places. It is produced in Hebei, Shandong, Liaoning, Inner Mongolia, Anhui, Shanxi, and other regions. It is also known as *Shanxiazi*, *Daogencao*, *Zishen*, *Xiashen*, *Huitoushen*, and *Shanliu*. *Polygonum bistorta* has a bitter and astringent taste and is slightly cold in nature. It has the effects of clearing heat and detoxifying, reducing swelling, and stopping bleeding. It is used for dysentery, cough due to lung heat, carbuncles and scrofula, sores in the mouth and tongue, hematemesis due to blood heat, hemorrhoidal bleeding, and snake and insect bites. *Dracaena cochinchinensis* has the effects of dispersing blood stasis, stopping bleeding, and relieving pain. It is mainly used to treat chronic gastritis, duodenal ulcers, food stagnation, abdominal masses, and irregular menstruation. *Polygonum aviculare* has the effects of clearing heat, stopping diarrhea, and promoting blood circulation. It is mainly used to treat diarrhea and anemia.

[0003] Bistorta officinalis, Daemonorops draco, and Polygonum hydropiper belong to the same family and genus, and their rhizomes are quite similar in appearance. Distinguishing between them based on appearance alone requires extensive professional experience. Currently, due to the relatively high price of Bistorta officinalis, adulteration is common, with Polygonum hydropiper being a frequent adulterant. Because these three medicinal materials differ in their functions and indications, careful differentiation is crucial in clinical use to avoid misdiagnosis. Therefore, the ability to identify Bistorta officinalis and its adulterants is essential in clinical practice.

[0004] When studying *Polygonum bistorta* and its adulterants using medicinal properties and microscopic analysis, the small differences between the various medicinal materials necessitate specialized personnel to accurately analyze the test results. Furthermore, when using thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) to identify *Polygonum bistorta* and its adulterants, especially when using the TLC method described in "Identification Study of *Polygonum bistorta* and its Easily Confusing Agents" to differentiate *Polygonum bistorta*, *Polygonum hydropiper*, and *Dracaena cochinchinensis*, the existing publicly available TLC methods show no significant spots in the TLC chromatograms of *Polygonum bistorta*. This requires adulterants as a reference for application, but currently, the China National Institutes for Food and Drug Control does not have reference materials for *Dracaena cochinchinensis* and *Polygonum hydropiper*, making it difficult to incorporate into enterprise internal control standards and hindering its application. Moreover, this method is only applicable to *Polygonum bistorta* raw materials and cannot identify *Polygonum bistorta* preparations obtained through water extraction. Summary of the Invention

[0005] Therefore, the technical problem to be solved by the present invention is to overcome the problem that there are no significant spots in the thin-layer chromatography of Polygonum bistorta obtained by the thin-layer method of the prior art; thereby providing a thin-layer detection method for Polygonum bistorta or its adulterants and their preparations with more significant spots, and the thin-layer chromatography obtained by the thin-layer detection method can effectively realize the identification of Polygonum bistorta and its adulterants.

[0006] To address the above problems, the technical solution provided by this invention is as follows:

[0007] A thin-layer chromatography method for detecting Polygonatum bistorta or its adulterants, comprising:

[0008] Apply the test solution to a silica gel G thin-layer plate. Use a toluene-butyl acetate-formic acid-acetic acid solution with a volume ratio of 10:(8-10):(3-5):(1-2) as the developing solvent. Develop the plate, remove it, air dry it, and examine it under ultraviolet light or / and spray it with a colorimetric reagent and examine it under sunlight.

[0009] The wavelength of the ultraviolet lamp is 365nm;

[0010] The colorimetric reagent is a 5% ferric chloride ethanol solution.

[0011] The test solution includes solutions prepared from medicinal materials or preparations of Bistorta officinalis and its adulterants.

[0012] The preparation is a standard decoction freeze-dried powder or a formulation granule.

[0013] The preparation process of the test solution is as follows:

[0014] Take the test sample, grind it into a fine powder, add methanol, sonicate, filter, and evaporate the filtrate to dryness to obtain residue one; dissolve residue one in water, extract with petroleum ether by shaking to obtain an aqueous solution, extract the aqueous solution with ethyl acetate by shaking to obtain an ethyl acetate solution, evaporate to dryness to obtain residue two, and dissolve residue two in methanol to obtain the test sample solution.

[0015] Add 40 ml of methanol to each gram of the test sample and sonicate.

[0016] For each gram of test sample, 40 ml of water was added to the residue.

[0017] Each gram of test sample yields residue one, and the amount of petroleum ether added each time is 40 ml;

[0018] For each gram of test sample, 40 ml of ethyl acetate is added to the aqueous solution each time.

[0019] The amount of methanol added to the residue 2 obtained per gram of test sample is 2-4 ml.

[0020] The petroleum ether extraction is performed twice by shaking; and / or the ethyl acetate extraction is performed twice by shaking.

[0021] The silica gel G thin-layer plate is also speckled with a reference medicinal material solution and / or a reference standard solution.

[0022] The preparation method of the reference medicinal material solution is as follows: take the reference medicinal material, add water and decoct, centrifuge, take the supernatant and concentrate it to dryness under reduced pressure to obtain dry residue one; take the residue after the supernatant, add methanol, sonicate, filter, evaporate the filtrate to dryness to obtain dry residue two, mix dry residue one and dry residue two and add methanol to prepare the reference medicinal material solution.

[0023] The preparation method of the reference solution is as follows: take gallic acid and chlorogenic acid reference standards respectively, add methanol to prepare the corresponding reference solutions.

[0024] A method for identifying Polygonum bistorta or its adulterants includes: obtaining a thin-layer chromatogram of Polygonum bistorta or its adulterants using the above-mentioned thin-layer detection method, and identifying them based on the spots displayed by the thin-layer chromatogram, wherein the spots include at least the spots corresponding to chlorogenic acid and gallic acid.

[0025] The term "Polygonum bistorta" or its alterations includes medicinal materials or preparations of Polygonum bistorta, Daemonorops draco, and Polygonum bulbiferum.

[0026] The technical solution of this invention has the following advantages:

[0027] This invention provides a thin-layer chromatography (TLC) method for detecting Polygonum bistorta or its adulterants. This method can effectively obtain TLC samples of Polygonum bistorta and its adulterants with more significant spots. The TLC method is simple to operate, has a rapid detection time, and produces a large number of fluorescent spots in the Polygonum bistorta sample, displaying rich material information and providing clear and concise results. It allows for intuitive determination of sample measurement results and has strong adaptability. Therefore, it can effectively provide the prerequisites for quality control or subsequent identification of Polygonum bistorta or its adulterants.

[0028] As a standard testing item for quality control of Polygonum bistorta medicinal materials, standard decoctions of Polygonum bistorta, and formula granules of Polygonum bistorta, it can initially ensure the uniformity and stability of the medicinal materials to the finished products.

[0029] When identifying Polygonum bistorta or its adulterants, it is essential to ensure the accuracy of the original medicinal materials used. It is also crucial to be able to quickly identify finished formula granules that have lost their characteristic appearance, thus avoiding errors in clinical medication and delaying the patient's condition. Attached Figure Description

[0030] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0031] Figure 1 This is a thin-layer chromatogram of the test solution of the medicinal material in Example 1 of the present invention;

[0032] Figure 2 This is a thin-layer chromatogram of the test solution of the standard decoction lyophilized powder in Example 1 of the present invention;

[0033] Figure 3 This is a thin-layer chromatogram of the test solution of the Polygonum bistorta formulation granules in Example 1 of the present invention;

[0034] Figure 4 These are thin-layer chromatograms of the test sample solution under different spotting amounts in Example 2 of the present invention;

[0035] Figure 5 This is a thin-layer chromatogram of the test solution under low temperature and normal humidity in Example 2 of the present invention;

[0036] Figure 6 This is a thin-layer chromatogram of the test solution under normal temperature and humidity in Example 2 of the present invention;

[0037] Figure 7 This is a thin-layer chromatogram of the test solution under normal temperature and high humidity in Example 2 of the present invention;

[0038] Figure 8 This is a thin-layer chromatogram of the test solution under normal temperature and low humidity in Example 2 of the present invention;

[0039] Figure 9 This is a thin-layer chromatogram of Merck's silica gel G plate in Embodiment 2 of the present invention;

[0040] Figure 10 This is a thin-layer chromatogram of the silica gel G plate from Yantai Chemical Industry Research Institute in Example 2 of the present invention;

[0041] Figure 11 This is a thin-layer chromatogram of Jiangyou's silica gel G plate in Embodiment 2 of the present invention;

[0042] Figure 12 This is a thin-layer chromatogram of the test solution in Comparative Example 1 of this invention;

[0043] Figure 13 This is a thin-layer chromatogram of the three medicinal materials in Comparative Example 2 of this invention;

[0044] Figure 14 This is a thin-layer chromatogram of the standard decoction freeze-dried powder of the three medicinal materials in Comparative Example 2 of this invention;

[0045] Figure 15 This is a thin-layer chromatogram of the formulation particles of the three medicinal materials in Comparative Example 2 of this invention;

[0046] Figure 16 These are thin-layer chromatograms under different thin-layer conditions in Example 2 of the present invention. Detailed Implementation

[0047] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0048] Instruments: CAMAG TLC VISUALIZER thin-layer imaging system, ME104E electronic balance (Mettler Toledo), JY20002 electronic balance (Shanghai Sunny Hengping Scientific Instruments Co., Ltd.), BSA124S electronic balance (Sartorius Scientific Instruments (Beijing) Co., Ltd.), BT25S electronic balance (Sartorius Scientific Instruments (Beijing) Co., Ltd.), KQ-300DB ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.), developing cylinder, silicone G thin-layer plates (Merck, Germany; Yantai Chemical Industry Research Institute; Yantai Jiangyou Silicone Development Co., Ltd.).

[0049] Drug trials:

[0050] Bistorta officinalis reference material (batch number: 121569-202203, 0.5g / bottle, China National Institutes for Food and Drug Control).

[0051] Gallic acid reference standard (batch number: 110831-201605, 20mg / vial, China National Institutes for Food and Drug Control).

[0052] Chlorogenic acid reference standard (batch number: 110753-202119, 20mg / vial, China National Institute for Food and Drug Control).

[0053] Quanshen Formula Granules: KL190116-264400-03, KL190125-444200-11, KL190320-273400-18, Source: Beijing Kangrentang Pharmaceutical Co., Ltd.

[0054] Reagents: Toluene, butyl acetate, formic acid, acetic acid, and petroleum ether (60℃~90℃) were all of analytical grade, and water was purified water.

[0055] Example 1

[0056] A thin-layer chromatography method for detecting Polygonatum bistorta or its adulterants and their formulation granules, comprising:

[0057] 1. Preparation of test sample

[0058] We collected medicinal materials from different sources, including Polygonum bistorta, Daemonorops draco, and Polygonum hydropiper. The sources of these medicinal materials are shown in Table 1 below.

[0059] Table 1

[0060]

[0061]

[0062] The collected Polygonum bistorta, Bistorta paleacea, and Bistorta vivipara medicinal materials were used to amplify the ITS2 sequence by PCR using DNA barcoding technology and sequencing. The sequencing results showed a 100% match rate with the records of Polygonum bistorta, Bistorta paleacea, and Bistorta vivipara in the Global Pharmacopoeia Genome Database.

[0063] The above-mentioned medicinal materials were used to prepare standard decoction freeze-dried powder and formulation granules, respectively. The specific preparation methods are as follows:

[0064] Standard decoction freeze-dried powder: Take the Polygonum bistorta medicinal material, place it in a clay pot, soak for 30 minutes, add 8 times the amount of water to the first decoction, bring to a boil over high heat (500W), then simmer over low heat (200W) for 20 minutes, and filter while hot; add 7 times the amount of water to the second decoction, bring to a boil over high heat (500W), then simmer over low heat (200W) for 15 minutes, filter while hot, combine the filtrates, concentrate (65℃) until the remaining liquid is 500ml, and cool quickly for later use; concentrate the remaining liquid to a material-to-liquid ratio of about 1:1 (relative density of 1.05-1.10 (60℃)), freeze-dry, and the product is obtained.

[0065] Formula granules: Take 4000g of Polygonum bistorta medicinal material, add water and decoct, filter, concentrate the filtrate into a clear paste, dry (or dry and pulverize), add appropriate amount of excipients, mix well, granulate to make 1000g, and you will get the product.

[0066] The preparation of standard decoction freeze-dried powder and formulation granules of Polygonum hydropiper and Polygonum aviculare are the same as those of Bistorta officinalis.

[0067] 2. Preparation of test solution and thin-layer chromatography method

[0068] 2.1 Preparation of the test solution

[0069] Test solution for medicinal materials: Take 2.0g of Polygonum bistorta, Daemonorops draco, and Polygonum hydropiper, add 40ml of water, decoct for 40 minutes, centrifuge, and concentrate the supernatant under reduced pressure to dryness to obtain dry residue one. Add 40ml of methanol to dry residue one, and extract twice with petroleum ether (60℃~90℃), 20ml each time. Combine the aqueous solution and extract twice with ethyl acetate, 20ml each time. Combine the ethyl acetate solution and evaporate to dryness to obtain dry residue two. Dissolve dry residue two in 2ml of methanol to obtain the test solution for medicinal materials.

[0070] Test solution for standard decoction lyophilized powder or formula granules: Take an appropriate amount of standard decoction lyophilized powder or formula granules, grind it finely, take about 0.5g, add 20ml of methanol, sonicate for 60 minutes, filter, evaporate the filtrate to dryness to obtain residue one, add 20ml of water to residue one to dissolve it, extract twice with petroleum ether (60℃~90℃) by shaking, 20ml each time, combine the aqueous solution, extract twice with ethyl acetate by shaking, 20ml each time, combine the ethyl acetate solution, evaporate to dryness to obtain residue two, add 2ml of methanol to residue two to dissolve it, and use it as the test solution for lyophilized powder or formula granules.

[0071] Reference herbal solution: Its preparation method is the same as that of the test solution of the herbal material.

[0072] Preparation of reference solutions: Gallic acid and chlorogenic acid reference standards were prepared by adding methanol to prepare solutions containing 1 mg per ml, which were used as reference solutions for gallic acid and chlorogenic acid.

[0073] 2.2 Thin-layer method

[0074] Take 6 μl of the test solution, 8 μl of the reference herb solution, and 1 μl of the reference standard solution, and spot them separately onto the same silica gel G thin-layer plate to form strips. Develop the plate using toluene-butyl acetate-formic acid-acetic acid (volume ratio 10:10:5:1) as the developing solvent. Remove the plate, air dry, and examine it under ultraviolet light (365 nm). Then spray with 5% ferric chloride ethanol solution and heat at 105℃ until the spots are clearly visible. Examine the plate under sunlight.

[0075] In the chromatogram of the test sample examined under ultraviolet light, the test sample shows fluorescent spots of the same color at the corresponding positions as the chromatograms of the reference medicinal material and the reference standard; in the chromatogram of the test sample examined under sunlight, the test sample shows spots of the same color at the corresponding positions as the chromatograms of the reference medicinal material and the reference standard.

[0076] 3. Test Results

[0077] 3.1 Test solution of medicinal materials

[0078] The test solution of the medicinal material, the reference solution of *Polygonum bistorta*, and the reference solutions of gallic acid and chlorogenic acid prepared in section 2.1 were analyzed using the thin-layer chromatography method described in section 2.2. The results are as follows: Figure 1 As shown.

[0079] Figure 1 In the image, (a) shows the thin-layer identification pattern under ultraviolet light at 365 nm, and (b) shows the thin-layer identification pattern under sunlight. Figure 1In the text, number 1 represents *Polygonum bistorta* medicinal material (190116-132100-02), number 2 represents *Polygonum bistorta* medicinal material (190116-264400-03), number 3 represents *Polygonum bistorta* medicinal material (190116-264400-04), number 4 represents *Polygonum bistorta* medicinal material (190117-274600-05), and number 5 represents *Polygonum bistorta* medicinal material (190117-264...). 400-06), No. 6 is Polygonum bistorta medicinal material (190125-444200-11), No. 7 is Polygonum bistorta medicinal material (190125-444200-12), No. 8 is Polygonum bistorta medicinal material (190125-264200-13), No. 9 is Polygonum bistorta medicinal material (190125-276500-14), No. 10 is Polygonum bistorta medicinal material (190 125-264000-15), No. 11 is Polygonum bistorta medicinal material (190125-276800-16), No. 12 is Polygonum bistorta medicinal material (190125-110000-17), No. 13 is Polygonum bistorta medicinal material (190320-273400-18), No. 14 is Polygonum bistorta medicinal material (190320-273400-19), No. 15 is Polygonum bistorta medicinal material (190325-276500-24), 16 is Polygonum bistorta reference medicinal material (121569-202203), 17 is Daemonorops draco medicinal material, 18 is Polygonum bulbiferum medicinal material, 19 is chlorogenic acid reference standard (110753-202119), and 20 is gallic acid reference standard (110831-201906).

[0080] According to the above-mentioned thin-layer chromatography identification method for Polygonum hydropiper, obvious bright blue fluorescent spots can be found at the upper edge (second dotted box) of the thin-layer chromatography pattern (a). Polygonum hydropiper and Daemonorops draco do not have these spots. Comparing the thin-layer chromatography patterns of Polygonum hydropiper and Daemonorops draco, the number of spots in Polygonum hydropiper is slightly more than that in Daemonorops draco. Therefore, this thin-layer chromatography method can distinguish between Polygonum hydropiper and counterfeit medicinal materials.

[0081] 3.2 Test solution of standard decoction lyophilized powder

[0082] The test solution of the standard decoction lyophilized powder prepared in section 2.1, the reference herb solution of *Polygonum bistorta*, and the reference solutions of gallic acid and chlorogenic acid were analyzed using the thin-layer chromatography method described in section 2.2. The results are as follows: Figure 2 and Figure 3 As shown.

[0083] Figure 2 In the image, (a) shows the thin-layer identification pattern under ultraviolet light at 365 nm, and (b) shows the thin-layer identification pattern under sunlight. Figure 2In the text, number 1 represents the standard freeze-dried powder of *Polygonum bistorta* (190116-132100-02), number 2 represents the standard freeze-dried powder of *Polygonum bistorta* (190116-264400-03), number 3 represents the standard freeze-dried powder of *Polygonum bistorta* (190116-264400-04), number 4 represents the standard freeze-dried powder of *Polygonum bistorta* (190117-274600-05), and number 5 represents the standard freeze-dried powder of *Polygonum bistorta* (190117-264400-...). 06) standard decoction freeze-dried powder, No. 6 is standard decoction freeze-dried powder of Polygonum bistorta (190125-444200-11), No. 7 is standard decoction freeze-dried powder of Polygonum bistorta (190125-444200-12), No. 8 is standard decoction freeze-dried powder of Polygonum bistorta (190125-264200-13), No. 9 is standard decoction freeze-dried powder of Polygonum bistorta (190125-276500-14), No. 10 is standard decoction freeze-dried powder of Polygonum bistorta (190) Freeze-dried powder of standard decoction (125-264000-15), No. 11 is freeze-dried powder of standard decoction of Polygonum bistorta (190125-276800-16), No. 12 is freeze-dried powder of standard decoction ... The following are listed: 15 is the freeze-dried standard decoction powder of Polygonum bistorta (190325-276500-24); 16 is the freeze-dried standard decoction powder of Polygonum bistorta reference material (121569-202203); 17 is the freeze-dried standard decoction powder of Dragon's Blood; 18 is the freeze-dried standard decoction powder of Polygonum villosa; 19 is the chlorogenic acid reference standard (110753-202119); and 20 is the gallic acid reference standard (110831-201906).

[0084] 3.3 Test solution of Bistorta officinalis granules

[0085] The test solutions of *Polygonum bistorta* granules (labeled KL190116-264400-03, KL190125-444200-11, and KL190320-273400-18), *Polygonum bistorta* reference herb solutions, and gallic acid and chlorogenic acid reference solutions were analyzed using the thin-layer chromatography method described in section 2.2. The test results are as follows: Figure 3 As shown.

[0086] Figure 3 In the image, (a) shows the thin-layer identification pattern under ultraviolet light at 365 nm, and (b) shows the thin-layer identification pattern under sunlight. Figure 3In the table, number 1 is the Polygonum bistorta formula granule KL190116-264400-03, number 2 is the Polygonum bistorta formula granule KL190125-444200-11, number 3 is the Polygonum bistorta formula granule KL190320-273400-18, number 4 is the Polygonum bistorta reference material (121569-202203), number 5 is the formula granule prepared from the herb Dragon's Blood, number 6 is the formula granule prepared from the herb Polygonum villosa, number 7 is chlorogenic acid (110753-202119), and number 8 is gallic acid (110831-201906).

[0087] According to the above Figure 1-3 Thin-layer chromatography (TLC) results from 15 batches of standard decoction freeze-dried powder and three batches of Polygonum multiflorum granules showed two distinguishing features between the standard decoction and granules of Polygonum multiflorum and its adulterants, Daemonorops draco and Polygonum bulbiferum, under UV 365nm examination. Specifically, within the first dashed box, the adulterants Daemonorops draco and Polygonum bulbiferum had fewer fluorescent spots than the Polygonum multiflorum sample; within the second dashed box, the adulterant Polygonum bulbiferum showed more obvious blue fluorescent spots, while neither Polygonum multiflorum nor the adulterant Daemonorops draco had these spots.

[0088] Based on the above thin-layer chromatography results, it can be seen that the method of the present invention can distinguish between standard decoction and granules of Polygonum bistorta and counterfeit products. This thin-layer chromatography method can be incorporated into the quality standard of Polygonum bistorta granules to distinguish them from adulterated products and ensure product quality.

[0089] Example 2

[0090] A thin-layer chromatography method for detecting Polygonum bistorta or its adulterants and their formulation granules, employing different chromatographic conditions for evaluation, including:

[0091] 1. Sample quantity investigation

[0092] Using the preparation method of the test sample solution in 2.1 of Example 1 above, test sample solutions of Polygonum bistorta granules KL190116-264400-03, KL190125-444200-11, and KL190320-273400-18 were prepared. At the same time, the preparation method of the reference medicinal material solution and reference solution in 2.1 above was used to prepare the Polygonum bistorta reference medicinal material solution and gallic acid and chlorogenic acid reference solutions. The spotting amount of the three was investigated.

[0093] Specifically, 4 μL, 6 μL, and 8 μL of the test sample solution (KL190116-264400-03, KL190125-444200-11, KL190320-273400-18), 1 μL, 2 μL, and 3 μL of the reference standard solution were taken respectively. The sample quantity was investigated using the thin-layer chromatography method described in section 2.2 of Example 1 above. The results are as follows. Figure 4 As shown.

[0094] Figure 4 (a) is the thin-layer chromatography identification spectrum under ultraviolet light at 365 nm, and (b) is the thin-layer chromatography identification spectrum under sunlight; No. 1 is the test sample KL190116-264400-03 (4 / 6 / 8 μL); No. 2 is the test sample KL190125-444200-11 (4 / 6 / 8 μL); No. 3 is the test sample KL190320-273400-18 (4 / 6 / 8 μL); No. 4 is the reference material of Polygonum bistorta (121569-202203) (4 / 6 / 8 μL); No. 5 is chlorogenic acid 140677-201206 (1 / 2 / 3 μL); No. 6 is gallic acid 140687-201905 (1 / 2 / 3 μL).

[0095] according to Figure 4 The results showed that when the spotting volume of the *Polygonum bistorta* test solution was 4 μL, the spots under ultraviolet light were not clear enough. The difference between spotting volumes of 6 μL and 8 μL was not significant. Therefore, the spotting volume of the test solution was determined to be 6 μL. When the spotting volume of the *Polygonum bistorta* reference material was 4 μL and 6 μL, there was no difference in the spots under ultraviolet light. However, gallic acid spots were more likely to appear under sunlight when the spotting volume was 8 μL. Therefore, the spotting volume of the *Polygonum bistorta* reference material was determined to be 8 μL. When the spotting volume of the chlorogenic acid and gallic acid reference solutions was 1 μL, the spots were visible under both ultraviolet light and sunlight. Therefore, the spotting volume of the reference standards was determined to be 1 μL.

[0096] 2. Investigation under different temperatures and humidity levels

[0097] The same test solution, *Polygonum bistorta* reference herb solution, and gallic acid and chlorogenic acid reference solutions were used in the above-mentioned sampling volume investigation. 6 μL of the test solution, 8 μL of the *Polygonum bistorta* reference herb solution, and 1 μL each of the chlorogenic acid and gallic acid reference solutions were spotted onto the same silica gel G plate. Using the thin-layer chromatography conditions described in 2.2 of Example 1 above, the investigation was conducted at two different temperatures (5–35°C) and two different relative humidity conditions (15–30% and 75–90%). The results are as follows: Figures 5-8 As shown.

[0098] Figure 5 This is a thin-layer chromatography identification spectrum under low temperature and normal humidity (5℃, 50%). Figure 6 This is a thin-layer chromatography identification spectrum under normal temperature and humidity (25℃, 50%). Figure 7 This is a thin-layer chromatography identification spectrum under normal temperature and high humidity (25℃, 85%). Figure 8 The thin-layer identification spectrum is obtained under normal temperature and low humidity (25℃, 20%).

[0099] Figure 5-8In the figure, (a) is the thin-layer identification spectrum under ultraviolet light at 365 nm, and (b) is the thin-layer identification spectrum under sunlight; wherein, number 1 is the test sample KL190116-264400-03, number 2 is the test sample KL190125-444200-11, number 3 is the test sample KL190320-273400-18, number 4 is the reference medicinal material of Polygonum bistorta (121569-202203), number 5 is chlorogenic acid 140677-201206, number 6 is gallic acid 140687-201905, and number 7 is the negative control.

[0100] 3. Investigation of different thin-layer plates

[0101] The same test solution, *Polygonum bistorta* reference herb solution, and gallic acid and chlorogenic acid reference solutions were used in the above-mentioned sampling volume investigation. 6 μL of the test solution, 8 μL of the *Polygonum bistorta* reference herb solution, and 1 μL each of the chlorogenic acid and gallic acid reference solutions were respectively spotted onto silica gel G plates from three different manufacturers: Merck, Yantai Jiangyou Silica Gel Development Co., Ltd., and Yantai Chemical Industry Research Institute. The thin-layer chromatography conditions described in 2.2 of Example 1 were used for the investigation, and the results are as follows: Figures 9-11 As shown.

[0102] Figure 9 Thin-layer chromatography for Merck's silicone G plate. Figure 10 Thin-layer chromatography (TLC) images of silica gel G plates from Yantai Chemical Industry Research Institute. Figure 11 Thin-layer chromatography (TLC) of Jiangyou's silicone G-plate.

[0103] Figures 9-11 In the figure, (a) is the thin-layer identification spectrum under ultraviolet light at 365 nm, and (b) is the thin-layer identification spectrum under sunlight; wherein, number 1 is the test sample KL190116-264400-03, number 2 is the test sample KL190125-444200-11, number 3 is the test sample KL190320-273400-18, number 4 is the reference medicinal material of Polygonum bistorta (121569-202203), number 5 is chlorogenic acid 140677-201206, number 6 is gallic acid 140687-201905, and number 7 is the negative control.

[0104] The results above show that the optimized thin-layer chromatography identification method under the original thin-layer chromatography conditions has an increase in fluorescent spots under ultraviolet 365nm and can identify finished products and adulterants. After spraying with a colorimetric reagent, the spots in the thin-layer spectrum under sunlight are clear. Therefore, the optimized thin-layer chromatography can be included in the main text of the quality standard for the identification of Polygonum bistorta formula granules.

[0105] 4. Investigation of thin-layer conditions

[0106] Using the preparation method of the test sample solution in 2.1 of Example 1 above, test sample solutions of the standard decoction freeze-dried powders of Polygonum hydropiper, Polygonum bulbiferum, and Daemonorops draco were prepared. At the same time, the preparation method of the reference medicinal material solution and reference solution in 2.1 above was used to prepare the reference medicinal material solution of Polygonum hydropiper and the reference solution of gallic acid and chlorogenic acid. Thin-layer chromatography was conducted under different conditions than in Example 1.

[0107] Specifically, in this embodiment, a toluene-butyl acetate-formic acid-acetic acid solution with a volume ratio of 10:8:3:2 was used as the developing solvent in thin-layer chromatography, and other conditions were the same as in Example 1. 6 μL of the test solution, 8 μL of the reference herb solution, and 1 μL of the reference standard solution were taken for spotting analysis, and the results are as follows. Figure 16 As shown.

[0108] Figure 16 (a) is the thin-layer chromatography identification spectrum under ultraviolet light at 365 nm, and (b) is the thin-layer chromatography identification spectrum under sunlight; No. 1 is Polygonum hydropiper (190116-132100-02); No. 2 is the freeze-dried standard decoction powder of Polygonum hydropiper (190116-132100-02); No. 3 is Polygonum viviparum; No. 4 is Daemonorops draco; No. 5 is the freeze-dried standard decoction powder of Polygonum viviparum; No. 6 is the reference material of Polygonum hydropiper (121569-202203); No. 7 is chlorogenic acid (110753-202119); No. 8 is gallic acid (110831-201906).

[0109] according to Figure 16 The results show that, based on the above developing agent conditions, it is possible to identify counterfeit products of Polygonum bistorta or its adulterants and preparations, such as formulated granules, thus meeting the quality control requirements of Polygonum bistorta and ensuring the identification results.

[0110] Comparative Example 1

[0111] The test solution of the medicinal material in Example 1 was tested using the pharmacopoeia method (thin-layer chromatography method under the Polygonum bistorta section of the 2020 edition of the Chinese Pharmacopoeia). The test results are as follows: Figure 12 As shown, Figure 12 In the table, number 1 is the test solution of Polygonum villosa, number 2 is the test solution of Dragon's Blood, number 3 is the test solution of Bistorta officinalis, number 4 is the reference solution of Bistorta officinalis, and number 5 is gallic acid.

[0112] According to the thin-layer chromatograms above, when using thin-layer chromatography to identify Polygonum hydropiper, Polygonum bulbiferum, and Daemonorops draco, the thin-layer chromatograms showed the same spots at the corresponding positions as the reference materials Polygonum hydropiper and gallic acid, making it impossible to accurately distinguish the three materials.

[0113] Comparative Example 2

[0114] Thin-layer chromatography (TLC) of the test sample solution was performed using the chromatographic method described in "Identification Study of Gynostemma pentaphyllum and its Easily Confused Substances". The specific method is as follows:

[0115] Take 1g of the herbal powder, add 30ml of ethanol, sonicate for 30min, filter, evaporate the filtrate to dryness, dissolve the residue in 10ml of water, extract twice with 10ml of ethyl acetate each time, combine the ethyl acetate extracts, evaporate to dryness, dissolve the residue in 1ml of methanol, and use this as the test solution. Perform thin-layer chromatography (General Chapter 0502 of the 2015 edition of the Chinese Pharmacopoeia), apply 10-20μl of the test solution separately to the same silica gel G thin-layer plate, develop with chloroform-methanol (19:1) as the developing solvent, remove, air dry, and examine under ultraviolet light (365nm).

[0116] Three types of medicinal materials, freeze-dried powder of standard decoctions of the three types of medicinal materials, and granules of the formula of the three types of medicinal materials were taken and tested respectively. The test results are as follows. Figures 13-15 As shown.

[0117] Figure 13 This is a thin-layer chromatography (TLC) spectrum of the test solution of the medicinal material. Figure 14 Thin-layer chromatography (TLC) chromatograms of the test solution for the standard lyophilized decoction powder. Figure 15 Thin-layer chromatography (TLC) chromatograms of the test solution of the formulation granules. Figure 13 In the table, No. 1 is the test solution of Bistorta officinalis, No. 2 is the test solution of Dracaena cochinchinensis, and No. 3 is the test solution of Polygonum viviparum. Figures 14-15 In the table, numbers 1-3 are test solutions of Bistorta officinalis, number 4 is test solution of Daemonorops draco, number 5 is test solution of Polygonum viviparum, number 6 is reference material of Bistorta officinalis, number 7 is reference standard of gallic acid, and number 8 is reference standard of chlorogenic acid.

[0118] pass Figure 13 It can be seen that the thin-layer chromatography of Polygonum bistorta showed almost no fluorescent spots, while both Daemonorops draco and Polygonum villosa showed bright fluorescent spots at the solvent front of the thin-layer plate. Polygonum villosa clearly had more thin-layer spots than Daemonorops draco. This thin-layer method can be used to preliminarily identify Polygonum bistorta and its adulterants, but it cannot be used as a thin-layer identification method for Polygonum bistorta because it has no spots and there is no reference standard.

[0119] pass Figure 14 As can be seen, when developed with the developing solvent in this comparative example, there are no chlorogenic acid or gallic acid spots in the chromatogram under these thin-layer conditions. The spots of the freeze-dried powder of the Polygonum bistorta standard decoction are not clear. The thin-layer chromatography of Polygonum hydropiper shows blue fluorescent spots, which can be used for identification. The spots of the freeze-dried powder of the Daemonorops draco standard decoction are consistent with those of the Polygonum bistorta reference material and cannot be distinguished.

[0120] pass Figure 15 As can be seen, when developed with the developing solvent in this comparative example, the spectral density of the *Polygonum bistorta* formulation granules under these thin-layer conditions has a total of 3 fluorescent spots, which are consistent with the number and color of the spots of the *Dracaena cochinchinensis* formulation granules and cannot be distinguished. Compared with the *Polygonum hydropiper* formulation granules, there are fewer spots than in the *Polygonum hydropiper* formulation granules, which can be used to identify the *Polygonum hydropiper* formulation granules. However, as a thin-layer method for *Polygonum bistorta* medicinal materials and preparations, the number of fluorescent spots is slightly less, and the material information displayed by the thin-layer results cannot show the material composition of *Polygonum bistorta*.

[0121] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for detecting Gastrodia elata Blume by thin layer chromatography, characterized in that, The method comprises the following steps: The test sample solution is spotted on a silica gel G thin layer plate, and toluene-butyl acetate-formic acid-acetic acid with a volume ratio of 10:(8-10):(3-5):(1-2) is used as a developing agent to develop, take out, air dry, and observe under ultraviolet light or / and spray with a color developing agent to observe under sunlight. The wavelength of the ultraviolet light is 365 nm. The color developing agent is 5% ferric trichloride ethanol test solution. The test sample solution comprises a solution prepared from a preparation of B. wallichii. The preparation is a standard decoction lyophilized powder or formula granules. The preparation process of the test sample solution is as follows: The test sample is finely ground, and then methanol is added for ultrasonic treatment. The filtrate is evaporated to dryness to obtain residue I. Residue I is dissolved in water, and then petroleum ether is added for extraction. The water solution is extracted with ethyl acetate to obtain ethyl acetate solution. Residue II is obtained by evaporation, and then dissolved in methanol to obtain the test sample solution. The silica gel G thin layer plate is also spotted with a control sample solution. The control sample solution is prepared by adding methanol to gallic acid and chlorogenic acid control samples, respectively.

2. The thin layer test method according to claim 1, characterized by Ultrasonic treatment is performed on 40 ml of methanol per gram of test sample; and / or, the amount of water added to residue I obtained from 1 gram of test sample is 40 ml; and / or, the amount of petroleum ether added for each extraction of residue I obtained from 1 gram of test sample is 40 ml; and / or, the amount of ethyl acetate added to the water solution obtained from 1 gram of test sample is 40 ml; and / or, the amount of methanol added to residue II obtained from 1 gram of test sample is 2-4 ml.

3. The thin layer test method according to claim 1 or 2, characterized by, The petroleum ether extraction is performed twice; and / or, the ethyl acetate extraction is performed twice.

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