A thin layer identification method suitable for different harvest periods of artemisia herb / standard decoction / formula granules

Abstract

The present application belongs to the technical field of traditional Chinese medicine detection, and particularly relates to a thin layer identification method suitable for different harvest periods of Chinese thorowax root medicinal materials / standard decoction / formulated granules. The thin layer identification method comprises the following steps: 1) taking Chinese thorowax root samples and preparing test sample solutions; 2) spotting the test sample solutions on a polyamide thin layer plate, using a toluene-butyl acetate-formic acid-water upper solution with a volume ratio of 1:(9-12):(3.5-4.5):(2.5-3.5) as a developing agent, developing, taking out, air-drying, and observing under ultraviolet light to obtain a thin layer chromatogram, and performing quality analysis on the Chinese thorowax root samples according to the thin layer chromatogram; the different harvest periods are spring / summer / autumn; and the Chinese thorowax root samples include Chinese thorowax root medicinal materials, Chinese thorowax root standard decoction, or Chinese thorowax root formulated granules. The present application can clearly distinguish Chinese thorowax root samples harvested in spring / summer / autumn, is suitable for various dosage forms, is simple to operate, has strong durability and specificity, and is efficient and stable in detection.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine detection, and particularly relates to a thin-layer identification method applicable to different harvesting periods of Artemisia capillaris Thunb. medicinal materials / standard decoctions / formula granules. Background Art

[0002] Artemisia capillaris Thunb. is the dried aerial part of the plant Artemisia scoparia Waldst. & Kit. Artemisia scoparia Waldst. et Kit. or Artemisia capillaris Thunb. Artemisia capillaris Thunb. It is harvested when the spring seedlings are 6 - 10 cm tall or when the autumn flower buds grow to the initial blooming stage, the impurities and old stems are removed, and then it is dried in the sun. The one harvested in spring is commonly called "Mianyinchen", and the one harvested in autumn is called "Huayinchen". Artemisia capillaris Thunb. was first recorded in "Shennong Ben Cao Jing" and listed as a superior grade. It has the effects of clearing away damp-heat and promoting bile flow to reduce jaundice, and is used for symptoms such as less urine with jaundice, damp-warm in summer and dampness, itching of damp sores, etc. Due to historical reasons, people commonly use Mianyinchen, and Huayinchen is rarely seen in the market. Traditional Chinese medicine theory believes that the medicinal value of Huayinchen is lower than that of Mianyinchen. Modern pharmacological research believes that both types of Artemisia capillaris Thunb. have obvious medicinal properties. The main component of Artemisia capillaris Thunb. (Mianyinchen) is chlorogenic acid, and the main component of Artemisia capillaris Thunb. (Huayinchen) is coumarin compounds, which have good anti-inflammatory, hepatoprotective and analgesic effects. Therefore, Artemisia capillaris Thunb. medicinal materials of different harvesting periods can be selected according to different disease types to obtain better clinical efficacy.

[0003] Due to different harvesting periods, the characters of Mianyinchen and Huayinchen medicinal materials are quite different and are very easy to distinguish. However, for the freeze-dried powder of the standard decoction obtained by decocting or the formula granules obtained through steps such as modern process extraction, concentration, drying, granulation, etc., the unique character attributes of the medicinal materials are lost in the finished product state, and professional personnel need to use different detection methods for analysis. And it is very necessary to quickly and accurately identify the samples. Through relevant literature research, among the three literatures "Determination of Chlorogenic Acid Content in Artemisia capillaris Thunb. at Different Flowering Stages", "Determination of the Content of Scoparone in Artemisia capillaris Thunb. Medicinal Materials at Different Harvesting Periods and Different Parts and Suggestions on Resource Utilization", and "Comparison of Chlorogenic Acid Content in Wild Artemisia capillaris Thunb. and Huayinchen", high-performance liquid chromatography methods are used to compare the content of chlorogenic acid or scoparone in Mianyinchen and Huayinchen medicinal materials. The chlorogenic acid content in Mianyinchen medicinal materials is high, and the scoparone content in Huayinchen medicinal materials is high. The judgment basis requires professional personnel to accurately analyze the detection results.

[0004] For the 2020 Edition of "Chinese Pharmacopoeia", there are different thin-layer chromatography identification methods for Mianyinchen and Huayinchen under Artemisia capillaris Thunb. medicinal materials, which are means for detecting after the known harvesting periods of Artemisia capillaris Thunb. medicinal materials (spring, autumn). For unknown samples, multiple tests are required, with a large workload and serious waste of resources. It cannot accurately judge the Artemisia capillaris Thunb. harvested in summer, and this method only targets the medicinal materials and cannot identify the Artemisia capillaris Thunb. preparations (freeze-dried powder of Artemisia capillaris Thunb. standard decoction, Artemisia capillaris Thunb. formula granules) obtained by water extraction process. Summary of the Invention

[0005] Technical issues

[0006] This invention aims to establish a thin-layer chromatography (TLC) identification method that can differentiate between Artemisia capillaris (Yin Chen Hao) medicinal materials and its standard decoction freeze-dried powder and formulation granules from different harvesting periods. The TLC results show that Artemisia capillaris medicinal materials and their preparations have obvious fluorescent spots in the ultraviolet light at 365 nm, which can distinguish them from Artemisia capillaris samples from different harvesting periods. The method is simple, easy to implement, and yields obvious results. The methodological verification shows that the results are stable and feasible. It can be used as a TLC detection method for Artemisia capillaris medicinal materials and their preparations, as well as an identification method for Artemisia capillaris samples from different harvesting periods.

[0007] Technical solution

[0008] This invention provides a thin-layer chromatography (TLC) identification method applicable to different harvesting periods of Artemisia capillaris (Yin Chen) medicinal materials / standard decoctions / formulated granules. The TLC identification method includes the following steps: 1) Take Artemisia capillaris samples and prepare test solution; 2) Spot the test solution onto a polyamide TLC plate, using a toluene-butyl acetate-formic acid-water upper layer solution with a volume ratio of 1:(9-12):(3.5-4.5):(2.5-3.5) as the developing solvent for development, remove and dry the plate, and examine it under ultraviolet light to obtain a TLC spectrum. The quality of the Artemisia capillaris samples is analyzed based on the TLC spectrum. The different harvesting periods are spring, summer, and autumn. The Artemisia capillaris samples include Artemisia capillaris medicinal materials, Artemisia capillaris standard decoctions, or Artemisia capillaris formulated granules.

[0009] In some embodiments, the volume ratio of the developing agent is 1:10:4:3.

[0010] In some embodiments, the sample volume of the test solution is 0.5~2 μL, preferably 1 μL.

[0011] In some embodiments, the wavelength of the ultraviolet lamp is 365 nm.

[0012] In some embodiments, the preparation method of the test solution in step 1) is as follows: weigh an appropriate amount of Artemisia capillaris sample, extract and filter it, and take the filtrate to obtain the test solution.

[0013] In some embodiments, the solvent used for extraction is methanol, and the extraction is performed by ultrasound for 20-40 min, preferably 30 min; when the Artemisia capillaris sample is Artemisia capillaris herb, the material-to-liquid ratio for extraction is 1:(20-60), preferably 1:40; when the Artemisia capillaris sample is Artemisia capillaris standard decoction or Artemisia capillaris formula granules, the material-to-liquid ratio for extraction is 1:(80-120), preferably 1:100.

[0014] In some embodiments, the thin-layer identification method further includes the step of preparing a reference solution, and the step of developing a thin-layer spectrum using the same identification method as the test solution; wherein the reference solution is a *Artemisia capillaris* reference medicinal material solution and a chlorogenic acid reference solution, and the *Artemisia capillaris* reference medicinal material solution and the chlorogenic acid reference solution are developed on the same plate.

[0015] In some embodiments, the chlorogenic acid reference solution is prepared by taking chlorogenic acid reference standard and adding methanol to prepare a solution containing 1 mg per 1 mL, which is used as the reference solution.

[0016] In some embodiments, the preparation method of the Artemisia capillaris reference herb solution is as follows: take an appropriate amount of Artemisia capillaris reference herb, add water and boil, filter, evaporate the filtrate to dryness to obtain an extract, extract the extract, filter, and use the filtrate as a reference solution.

[0017] In some embodiments, the boiling time is 50-80 min; the solvent used for extraction is methanol; the material-to-liquid ratio for extraction is 1:(15-25), preferably 1:20; the extraction is performed by ultrasound for 20-40 min, preferably 30 min.

[0018] In some embodiments, the thin-layer chromatography identification method includes the following steps: 1) Weighing the Artemisia capillaris sample, ultrasonically extracting it with methanol for 20-40 min, preferably 30 min, filtering, and using the filtrate as the test solution; wherein, when the Artemisia capillaris sample is Artemisia capillaris medicinal material, the extraction material-to-liquid ratio is 1:(20-60), preferably 1:40; when the Artemisia capillaris sample is Artemisia capillaris standard decoction or Artemisia capillaris formula granules, the extraction material-to-liquid ratio is 1:(80-120), preferably 1:100; 2) Spotting 0.5-2 μL, preferably 1 μL, of the test solution onto a polyamide thin-layer plate at a volume ratio of 1:(9-12):(3.5-4.5):(2.5-3.5), preferably 1:10:4: The upper layer of a 3% toluene-butyl acetate-formic acid-water solution was used as the developing solvent for development. After drying, the solution was examined under ultraviolet light to obtain a thin-layer chromatography (TLC) spectrum. The quality of the Artemisia capillaris samples was analyzed based on the TLC spectrum. The different harvesting periods were spring, summer, and autumn. The Artemisia capillaris samples included Artemisia capillaris raw materials, Artemisia capillaris standard decoction, or Artemisia capillaris formula granules.

[0019] In some embodiments, it was identified that spring Artemisia capillaris has no characteristic fluorescent spots near Rf 0.17 and Rf 0.83; summer Artemisia capillaris has yellow fluorescent spots near Rf 0.17; and autumn Artemisia capillaris has blue fluorescent spots near Rf 0.83 and yellow fluorescent spots near Rf 0.17.

[0020] Technical effect 1) High versatility: This method can be applied to the thin-layer identification of Artemisia capillaris, freeze-dried powder of standard decoction and formula granules. It breaks through the limitation of pharmacopoeia methods that are only applicable to medicinal materials. One method covers samples of the whole chain and can be used as a qualitative evaluation method in the production process of Artemisia capillaris formula granules, thus initially ensuring the consistency of qualitative analysis of the whole industry chain.

[0021] 2) Can distinguish samples from different harvesting periods: The method can clearly distinguish between spring Artemisia capillaris, summer Artemisia capillaris and autumn Artemisia capillaris. It achieves rapid identification through characteristic fluorescent spots, and the results are clear and concise. It makes up for the shortcomings of existing methods that cannot distinguish summer-harvested samples and the disadvantages of different analysis methods for medicinal materials in spring and autumn.

[0022] 3) The method is simple and durable: the preparation steps of the test sample, reference medicinal material and reference solution are simple and do not require complicated pretreatment; the thin-layer operation process is simple and has good durability for temperature and humidity and thin-layer plates from different manufacturers. The spots are clear and the separation is good. Ordinary laboratory personnel can complete the test smoothly.

[0023] 4) High specificity: The identification is accurate. The reference medicinal material and chlorogenic acid reference standard are used for double-comparison positioning. Negative results are free from interference. The results are intuitive and reliable. It can initially ensure the uniformity and stability of the medicinal material to the finished product and avoid confusion in clinical use. Attached Figure Description

[0024] Figure 1 Thin-layer chromatography (TLC) spectral analysis of different spotting amounts of Artemisia capillaris formula granules; Figure 2 Thin-layer chromatography (TLC) spectra for different batches of Artemisia capillaris formula granules with different spotting amounts; Figure 3 For the specificity study of Artemisia capillaris formula granules; Figure 4 Thin-layer chromatography (TLC) images of Artemisia capillaris granules at room temperature and humidity. Figure 5 Thin-layer chromatography (TLC) images of Artemisia capillaris granules under low temperature and normal humidity conditions (T: 6.4℃, RH: 33%). Figure 6 Thin-layer chromatography identification of Artemisia capillaris formula granules under normal temperature and high humidity (T: 27℃, RH: 79%); Figure 7 Thin-layer chromatography identification of Artemisia capillaris formula granules under normal temperature and low humidity (T: 27℃, RH: 12%); Figure 8 Thin-layer diagram of polyamide thin-layer board manufactured by Jingjing Times; Figure 9 Thin-layer diagram of polyamide thin-layer board manufactured by Xinsirui; Figure 10 Thin-layer diagram of polyamide thin-layer board from Luqiao Sijia manufacturer; Figure 11Thin-layer chromatography identification spectrum of freeze-dried powder of Artemisia capillaris standard decoction (polyamide plate from Taizhou, Zhejiang). Figure 12 Thin-layer chromatography for identification of Artemisia capillaris formula granules at different harvesting periods (thin-layer chromatography method for Artemisia capillaris under the Artemisia capillaris section of the Pharmacopoeia). Figure 13 Thin-layer chromatography method for identifying Artemisia capillaris granules from different harvesting periods (Artemisia capillaris thin-layer chromatography method under Artemisia capillaris in the Pharmacopoeia). Figure 14 Thin-layer chromatography (TLC) images of Artemisia capillaris formula granules at different harvesting periods (TLC method in the literature "Identification of Artemisia capillaris Formula Granules by Thin-layer Chromatography and Derivative Spectroscopy"; a: UV 254 nm; b: UV 365 nm). Figure 15 Thin-layer chromatography for the identification of Artemisia capillaris and Artemisia capillaris flowers harvested at different times; Figure 16 Thin-layer chromatography of freeze-dried powder of standard decoction for Artemisia capillaris at different harvesting periods; Figure 17 Thin-layer chromatography of granules formulated with Artemisia capillaris at different harvesting periods. Detailed Implementation

[0025] To facilitate the implementation of the technical solutions applied for, the terms and expressions involved in this invention will first be explained and defined in general terms and expressions below.

[0026] The terms “comprising,” “including,” or any other variations thereof are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitation, an element defined by the phrase “comprising one…” does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0027] The terms "one embodiment," "some embodiments," "exemplary," "specific example," or "some examples," etc., used in this invention refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the invention. In this document, the illustrative expressions of the above terms are not necessarily directed at the same embodiment or example.

[0028] 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.

[0029] The sources of the reagents, materials, or instruments used in the following examples are as follows: 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, polyamide thin-layer plates (produced by Wuhan Xinsirui Technology Co., Ltd., Luqiao Sijia Biochemical Plastics Factory of Taizhou City, Zhejiang Province, and Beijing Jingshidai Biotechnology Co., Ltd.).

[0030] Test reagents: Artemisia capillaris reference material (variety: Artemisia capillaris (Mian Yin Chen), batch number: 121555-201602, specification: 1.5g / bottle, source: China National Institutes for Food and Drug Control); chlorogenic acid reference standard (batch number: 110753-201817, specification: 20 mg / bottle, source: China National Institutes for Food and Drug Control); artemisinin reference standard (batch number: B-025-181216, specification: 20 mg / bottle). mg / bottle, manufacturer: Chengdu Ruifensi Biotechnology Co., Ltd.); Artemisia capillaris (Artemisia capillaris (Mian Yin Chen)) formula granules (batch numbers: KL20200101, KL20200102, KL20200105, manufacturer: Beijing Kangrentang Pharmaceutical Co., Ltd.); Artemisia capillaris (Artemisia capillaris (Mian Yin Chen)) standard decoction freeze-dried powder (batch numbers: BJ20200101, BJ20200102, BJ20200105, manufacturer: Beijing Kangrentang Pharmaceutical Co., Ltd.); Spring-harvested Artemisia capillaris (Artemisia capillaris (Mian Yin Chen)) medicinal materials (batch number: YC20200101, place of origin: Qin'an County, Tianshui City, Gansu Province); Batch No.: YC20200102, Origin: Kongtong District, Pingliang City, Gansu Province; Batch No.: YC20200105, Origin: Zhengning County, Qingyang City, Gansu Province); Summer-harvested Artemisia capillaris (Batch No.: HYC01, Origin: Anguo City, Hebei Province) was used to prepare granules and standard decoctions of summer-harvested Artemisia capillaris (Batch No.: HYC02, Origin: Anguo City, Hebei Province) and autumn-harvested Artemisia capillaris (Batch No.: HYC02, Origin: Anguo City, Hebei Province) was used to prepare granules and standard decoctions of autumn-harvested Artemisia capillaris (Batch No.: HYC02, Origin: Anguo City, Hebei Province) and the preparation methods are described in Example 1.

[0031] Reagents: Toluene, butyl acetate, formic acid, and methanol were all of analytical grade, and water was purified water.

[0032] Accuracy testing of medicinal materials: The collected Artemisia capillaris medicinal materials from different harvest periods were subjected to DNA barcoding technology, RCR amplification of the ITS2 sequence, and sequencing. The sequencing results showed a 100% match rate with Artemisia capillaris Thunb. in the Global Pharmacopoeia Genome Database.

[0033] The following is a further explanation of the thin-layer chromatography method for different harvesting periods of Artemisia capillaris, standard decoctions, and formulation granules provided by this invention.

[0034] Example 1: Establishment of a thin-layer identification method for polyamide thin-layer plates (1) Preparation of test solution 1) Preparation of test solution of Artemisia capillaris Take about 0.5 g of Artemisia capillaris (Artemisia capillaris var. mongolica) (passed through a No. 3 sieve), add 20 mL of methanol, sonicate for 30 min, filter, and take the filtrate as the test solution.

[0035] 2) Preparation of test solution of standard decoction lyophilized powder Take Artemisia capillaris slices, place them in a clay pot, soak for 30 minutes. For the first decoction, add 12 times the amount of water as the slices, bring to a boil over high heat (500 W), then simmer over low heat (200 W) for 30 minutes. Filter while hot, cool quickly, and set aside. For the second decoction, add 8 times the amount of water as the slices, bring to a boil over high heat (500 W), then simmer over low heat (200 W) for 20 minutes. Filter while hot, cool quickly, and set aside. Combine the filtrates, concentrate (65℃) until the material-to-liquid ratio is about 1:1 (relative density of 1.05~1.10 (60℃)), freeze-dry, and the product is obtained.

[0036] Take an appropriate amount of the above standard decoction lyophilized powder test sample, grind it into a fine powder, take about 0.2 g, add 20 mL of methanol, sonicate for 30 min, filter, and take the filtrate as the test sample solution.

[0037] 3) Preparation of the test solution for the formulated granules Take 3700 g of Artemisia capillaris slices, add 12 times the amount of water for the first decoction, boil (100℃) and extract for 1.0 h, add 10 times the amount of water for the second decoction, boil (100℃) and extract for 0.5 h, filter through a 150-mesh filter cloth while hot, concentrate under reduced pressure (70℃) until the relative density is 1.10~1.15 (60℃), spray dry, add appropriate amount of excipients, mix well, granulate to make 1000 g.

[0038] Take an appropriate amount of the above-mentioned granules for testing, grind them into a fine powder, take about 0.2 g, add 20 mL of methanol, sonicate for 30 min, filter, and take the filtrate as the test solution.

[0039] (2) Preparation of reference solution 1) Preparation of control herbal solution Take 0.5 g of Artemisia capillaris (Artemisia capillaris) as a reference herb, add 50 mL of water, boil for 1 h, filter, evaporate the filtrate to dryness to obtain an extract, add 10 mL of methanol and sonicate for 30 min, filter, and use the filtrate as the reference herb solution.

[0040] 2) Preparation of chlorogenic acid reference solution Take an appropriate amount of chlorogenic acid reference standard and add methanol to prepare a solution containing 1 mg per 1 mL, which is used as the reference solution.

[0041] (3) Establishment of specific methods Preparation of test solution: According to the preparation method of test solution of Artemisia capillaris / standard decoction freeze-dried powder / formulation granules recorded in (1), take an appropriate amount of this product (Artemisia capillaris / standard decoction freeze-dried powder / formulation granules), grind it finely, take about 0.2 g, add 20 mL of methanol, sonicate for 30 min, filter, and take the filtrate as test solution.

[0042] Preparation of reference solution: Prepare reference medicinal material solution and chlorogenic acid reference solution simultaneously as described in (2).

[0043] Detection method: Perform thin-layer chromatography (General Chapter 0502, Chinese Pharmacopoeia 2020 Edition). Apply 1 μL each of the above-mentioned test solution, reference medicinal material solution, and chlorogenic acid reference solution to the same polyamide thin-layer plate. Use the upper layer of toluene-butyl acetate-formic acid-water (1:10:4:3) as the developing solvent. Develop, remove, and air-dry the plate, then examine under ultraviolet light (365 nm). In the chromatogram of the test sample, fluorescent spots of the same color should appear at the corresponding positions as in the chromatograms of the reference medicinal material and the chlorogenic acid reference solution.

[0044] Example 2: Sample Size Investigation Methods: In this study, the preparation methods for the test sample, reference medicinal material, and chlorogenic acid reference solution described in Example 1 were followed. Three batches of Artemisia capillaris [Artemisia capillaris (Mian Yin Chen)] formula granules (KL20200101, KL20200102, KL20200105) were taken to prepare test sample solutions. 0.5 μL, 1 μL, and 2 μL of the test sample solution, reference medicinal material solution, and reference solution were taken, and the sample spotting amount was investigated according to the method described in Example 1.

[0045] Result: See Figure 1 and Figure 2 ,in, Figure 1 Test sample No. 1 is the formula granule test sample KL20200101 (0.5 μL, 2 μL); test sample No. 2 is the reference medicinal material 121555-201602 (0.5 μL, 2 μL); test sample No. 3 is the chlorogenic acid reference standard 110753-201817 (1 μL, 2 μL). Figure 2No. 1 is Artemisia capillaris formula granules KL20200101 (1 μL); No. 2 is Artemisia capillaris formula granules KL20200102 (1 μL); No. 3 is Artemisia capillaris formula granules KL20200105 (1 μL); No. 4 is reference medicinal material 121555-201602 (1 μL); No. 5 is chlorogenic acid reference standard 110753-201817 (1 μL).

[0046] Conclusion: Through the examination of the sample size, such as Figure 1 and Figure 2 As shown, when the spotting volume of Artemisia capillaris test solution, Artemisia capillaris reference medicinal material solution, and chlorogenic acid reference solution is 1 μL, the thin-layer chromatographic spots are clear, the spot size is moderate, the resolution is good, and the Rf is moderate. Therefore, the spotting volume of test solution, reference medicinal material solution, and chlorogenic acid reference solution in the Artemisia capillaris thin-layer method is determined to be 1 μL.

[0047] Example 3 Durability Test (1) Specificity of Artemisia capillaris formula granules Methods: The test solution of Artemisia capillaris formula granules, the reference herb solution and the chlorogenic acid reference solution were prepared according to the method of Example 1. Methanol was used as the extraction solvent as a blank control. Thin-layer chromatography was carried out under the conditions of Example 1. The samples were taken out, dried and examined under ultraviolet light (365 nm).

[0048] Results: Thin-layer chromatograms are shown below. Figure 3 ,in, Figure 3 No. 1 is Artemisia capillaris formula granules (KL20200101); No. 2 is Artemisia capillaris formula granules (KL20200102); No. 3 is Artemisia capillaris formula granules (KL20200105); No. 4 is Artemisia capillaris reference material (121555-201602); No. 5 is chlorogenic acid reference standard (110753-201817); No. 6 is solvent methanol (negative control).

[0049] Conclusion: The above chromatographic results show that, on the chromatogram of the test sample solution, fluorescent spots of the same color and position as Artemisia capillaris (Artemisia capillaris var. mongolica) and chlorogenic acid reference standard are present, and there is no interference from the negative control, which meets the requirements of thin-layer chromatography analysis.

[0050] (2) Study on the effect of different temperature and humidity on the thin-layer chromatography of Artemisia capillaris formula granules Methods: Following the method in Example 1, 1 μL each of the Artemisia capillaris formula granules, Artemisia capillaris reference herb solution, and chlorogenic acid reference solution were spotted onto the same polyamide thin-layer plate (Jing Shidai). The development was investigated under the thin-layer conditions in Example 1, with two different temperatures (5-35°C) and two different relative humidities (15%-30% and 75%-90%). The plates were then removed, dried, and examined under ultraviolet light (365 nm).

[0051] Results: Thin-layer chromatograms are shown below. Figures 4-7 .in, Figure 4 This is a thin-layer chromatography (TLC) identification image of Artemisia capillaris formula granules under normal temperature and humidity (T: 27℃, RH: 32%). In the image, 1 is Artemisia capillaris formula granule (KL20200101); 2 is Artemisia capillaris formula granule (KL20200102); 3 is Artemisia capillaris formula granule (KL20200105); 4 is Artemisia capillaris reference material (121555-201602); 5 is chlorogenic acid reference standard (110753-201817); and 6 is methanol (negative control). Figure 5 This is a thin-layer chromatography (TLC) identification image of Artemisia capillaris formula granules under low temperature and normal humidity conditions (T: 6.4℃, RH: 33%). In the image, 1 is Artemisia capillaris formula granule (KL20200101); 2 is Artemisia capillaris formula granule (KL20200102); 3 is Artemisia capillaris formula granule (KL20200105); 4 is Artemisia capillaris reference material (121555-201602); 5 is chlorogenic acid reference standard (110753-201817); and 6 is methanol (negative control). Figure 6 This is a thin-layer chromatography (TLC) identification image of Artemisia capillaris formula granules under normal temperature and high humidity conditions (T: 27℃, RH: 79%). In the image, 1 is Artemisia capillaris formula granule (KL20200101); 2 is Artemisia capillaris formula granule (KL20200102); 3 is Artemisia capillaris formula granule (KL20200105); 4 is Artemisia capillaris reference material (121555-201602); 5 is chlorogenic acid reference standard (110753-201817); and 6 is methanol (negative control). Figure 7 The image shows a thin-layer chromatography (TLC) identification diagram of Artemisia capillaris formula granules under normal temperature and low humidity conditions (T: 27°C, RH: 12%). In the diagram, 1 represents Artemisia capillaris formula granules (KL20200101); 2 represents Artemisia capillaris formula granules (KL20200102); 3 represents Artemisia capillaris formula granules (KL20200105); 4 represents Artemisia capillaris reference material (121555-201602); 5 represents chlorogenic acid reference standard (110753-201817); and 6 represents methanol as a solvent (negative control).

[0052] Conclusion: From Figures 4-7 It can be seen that the separation effect of the Artemisia capillaris formula granules is good under different temperature and humidity conditions, and spots of the same color appear at the same positions as the control herb and chlorogenic acid standard. The experimental results show that this thin-layer identification method has good durability under temperature and humidity.

[0053] (3) Investigation of different thin-layer plates 1) Investigation of different thin-layer plates for Artemisia capillaris formula granules Methods: Polyamide thin-layer plates from three different manufacturers—Wuhan Xinsirui Technology Co., Ltd., Luqiao Sijia Biochemical Plastics Factory of Taizhou City, Zhejiang Province, and Beijing Jingjing Times Biotechnology Co., Ltd.—were developed under the thin-layer conditions of Example 1.

[0054] Result: See Figures 8-10 . Figure 8 The images show thin-layer chromatography (TLC) images of polyamide thin-layer plates manufactured by Jingjing Times. In the images, 1 represents Artemisia capillaris formula granules (KL20200101); 2 represents Artemisia capillaris formula granules (KL20200102); 3 represents Artemisia capillaris formula granules (KL20200105); 4 represents Artemisia capillaris reference material (121555-201602); 5 represents chlorogenic acid reference standard (110753-201817); and 6 represents methanol as a solvent (negative control). Figure 9 The image shows a thin-layer chromatography (TLC) image of polyamide thin-layer plates manufactured by Xinsirui. In the image, 1 represents Artemisia capillaris formula granules (KL20200101); 2 represents Artemisia capillaris formula granules (KL20200102); 3 represents Artemisia capillaris formula granules (KL20200105); 4 represents Artemisia capillaris reference material (121555-201602); 5 represents chlorogenic acid reference standard (110753-201817); and 6 represents methanol as a solvent (negative control). Figure 10 The image shows the thin-layer chromatography (TLC) images of polyamide thin-layer plates from Luqiao Sijia manufacturer. In the image, 1 represents Artemisia capillaris formula granules (KL20200101); 2 represents Artemisia capillaris formula granules (KL20200102); 3 represents Artemisia capillaris formula granules (KL20200105); 4 represents Artemisia capillaris reference material (121555-201602); and 5 represents chlorogenic acid reference standard (110753-201817).

[0055] Conclusion: From Figures 8-10 It can be seen that the Artemisia capillaris formulation particles can be well separated under polyamide thin-layer plates produced by different manufacturers, and this thin-layer identification method has good durability for thin-layer plates produced by different manufacturers.

[0056] 2) Investigation of different thin-layer plates for Artemisia capillaris standard decoction freeze-dried powder Method: The freeze-dried powder of Artemisia capillaris standard decoction was developed under the identification conditions of Example 1.

[0057] Result: See Figure 11 , Figure 11Thin-layer chromatography (TLC) chromatograms of the freeze-dried powder of Artemisia capillaris standard decoction (Zhejiang Taizhou polyamide plate) are shown. No. 1 is the freeze-dried powder of Artemisia capillaris standard decoction (BJ20200101); No. 2 is the freeze-dried powder of Artemisia capillaris standard decoction (BJ20200102); No. 3 is the freeze-dried powder of Artemisia capillaris standard decoction (BJ20200105); No. 4 is the reference material of Artemisia capillaris [Artemisia capillaris (Mian Yin Chen)] (121555-201602); and No. 5 is the chlorogenic acid reference standard (110753-201817).

[0058] Conclusion: It is evident that the freeze-dried powder of Artemisia capillaris standard decoction and the granules of Artemisia capillaris formula have the same material composition and are both suitable for this thin-layer chromatography identification method.

[0059] Example 4: Identification Study of Artemisia capillaris Samples at Different Harvest Periods (1) Comparative study of other thin-layer methods 1) Thin-layer chromatography method for Artemisia capillaris under the Artemisia capillaris section of the 2025 edition of the Chinese Pharmacopoeia Method: Take 0.5 g of the powder, add 20 mL of 50% methanol, sonicate for 30 min, centrifuge, and take the supernatant as the test sample. Separately, take chlorogenic acid reference standard, add methanol to prepare a solution containing 0.1 mg per mL, as the reference solution. Perform thin-layer chromatography (General Rule 0502), take 2 μL of each of the above two solutions, and spot them separately on the same silica gel G thin-layer plate. Use the upper layer solution of butyl acetate-formic acid-water (7:2.5:2.5) as the developing solvent, develop, remove, air dry, and examine under ultraviolet light (365 nm).

[0060] Result: As Figure 12 As shown in the figure, item 1 is the freeze-dried powder of standard decoction of Artemisia capillaris (Artemisia capillaris var. ...

[0061] Conclusion: Based on the thin-layer chromatogram, the thin-layer chromatographic method of Artemisia capillaris under the Artemisia capillaris subsection was used to identify Artemisia capillaris formula granules. The thin-layer spots were few and the material information was incomplete. Artemisia capillaris flower granules had one more bright spot at the solvent front than Artemisia capillaris cotton granules, but it was impossible to distinguish between Artemisia capillaris samples harvested in autumn and those harvested in summer.

[0062] 2) Thin-layer chromatography method for Artemisia capillaris flower under the Artemisia capillaris section of the 2025 edition of the Chinese Pharmacopoeia. Method: Take 0.4 g of the powder, add 10 mL of methanol, sonicate for 30 min, filter, recover the solvent from the filtrate to dryness, dissolve the residue in 2 mL of methanol to obtain the test solution. Separately, take artemisinin reference standard, add methanol to prepare a solution containing 0.4 mg per mL to obtain the reference solution. Perform thin-layer chromatography (General Rule 0502), apply 5 μL of each of the above two solutions separately to the same silica gel G thin-layer plate, develop with petroleum ether (60~90℃)-ethyl acetate-acetone (6:3:0.5) as the developing solvent, remove, air dry, and examine under ultraviolet light (365 nm). In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference standard.

[0063] Result: As Figure 13 As shown in the figure, No. 1 is the Artemisia capillaris (Artemisia capillaris) formula granule KL20200101; No. 2 is the Artemisia capillaris (Artemisia capillaris) formula granule KL20200102; No. 3 is the Artemisia capillaris (Artemisia capillaris) formula granule KL20200105; No. 4 is the Artemisia capillaris (Artemisia capillaris) reference material; No. 5 is the formula granule prepared from Artemisia capillaris (Artemisia capillaris) harvested in summer; No. 6 is the formula granule prepared from Artemisia capillaris (Artemisia capillaris) harvested in autumn; No. 7 is the formula granule prepared from Artemisia capillaris (Artemisia capillaris) harvested in autumn; No. 8 is chlorogenic acid reference standard; and No. 9 is artemisinin reference standard.

[0064] Conclusion: According to the thin-layer chromatography method of Artemisia capillaris, there was basically no spot information in the thin-layer chromatography of Artemisia capillaris [Artemisia capillaris (Mian Yin Chen)] formulation granules. This indicates that this thin-layer chromatography method is only suitable for the detection of Artemisia capillaris (Artemisia capillaris) medicinal material, but cannot be used as a thin-layer identification method for Artemisia capillaris (Mian Yin Chen) medicinal material and its preparations. Its scope of application is relatively narrow.

[0065] 3) The Artemisia capillaris formula granule samples were analyzed according to the method described in the literature "Identification of Artemisia capillaris Formula Granules by Thin Layer Chromatography and Derivative Spectroscopy".

[0066] Methods: Thin-layer chromatography (Appendix VIB, Part I, Chinese Pharmacopoeia (2010 Edition)) was performed. 2 μL of the test solution and 5 μL of the reference herb solution were spotted separately onto the same silica gel G thin-layer plate prepared with 1% sodium hydroxide solution. Ethyl acetate-methanol-water (17:2:1) was used as the developing solvent. After development, the plate was removed, dried, and examined under ultraviolet light (254 nm). 10% sulfuric acid ethanol solution was sprayed on the plate, and the plate was heated until the spots were clearly visible. The plates were then examined under ultraviolet light (365 nm).

[0067] Result: As Figure 14(a: UV 254 nm; b: UV 365 nm) As shown in the figure, No. 1 is the Artemisia capillaris (Artemisia capillaris) formula granule KL20200101; No. 2 is the Artemisia capillaris (Artemisia capillaris) formula granule KL20200102; No. 3 is the Artemisia capillaris (Artemisia capillaris) formula granule KL20200105; No. 4 is the Artemisia capillaris (Artemisia capillaris) reference material; No. 5 is the formula granule prepared from Artemisia capillaris (Artemisia capillaris) harvested in summer; No. 6 is the formula granule prepared from Artemisia capillaris (Artemisia capillaris) harvested in autumn; No. 7 is the formula granule prepared from Artemisia capillaris (Artemisia capillaris) harvested in autumn; No. 8 is the chlorogenic acid reference standard; No. 9 is the artemisinin reference standard.

[0068] Conclusion: In Figure 14 In the thin-layer chromatography (TLC) images of Artemisia capillaris formulation granules, severe spot tailing was observed, and the TLC results showed limited material information, making it impossible to effectively distinguish Artemisia capillaris formulations harvested in spring.

[0069] (2) Application of the thin-layer method of the present invention 1) Thin-layer foliar application of Artemisia capillaris at different harvesting periods Methods: Thin-layer chromatography was performed on Artemisia capillaris from different harvesting periods, as well as Artemisia capillaris var. velutipes and Artemisia capillaris var. florida, according to the method in Example 1.

[0070] Result: As Figure 15 As shown, No. 1 is Artemisia capillaris; No. 2 is Artemisia capillaris; No. 3 is Artemisia capillaris (Artemisia capillaris) control material; No. 4 is Artemisia capillaris (Artemisia capillaris) harvested in summer; No. 5 is Artemisia capillaris (Artemisia capillaris) harvested in autumn; No. 6 is Artemisia capillaris (Artemisia capillaris) harvested in autumn; and No. 7 is chlorogenic acid control.

[0071] Conclusion: According to the thin-layer chromatography results above, at the Rf value of 0.83 within the red box at the top of the thin-layer plate, the autumn-harvested Artemisia capillaris has one more bright blue fluorescent spot than the Artemisia argyi. Artemisia capillaris labeled 4, due to its harvesting period being close to that of Artemisia argyi, has no bright fluorescent spot, but at an Rf value of 0.17, it has one more yellow fluorescent spot than Artemisia argyi. Therefore, the thin-layer chromatography method of this invention can distinguish Artemisia capillaris from different harvesting periods.

[0072] 2) Thin-layer chromatography of Artemisia capillaris standard decoction freeze-dried powder and Artemisia capillaris formula granules at different harvesting periods Methods: Thin-layer chromatography was performed on the freeze-dried powder of Artemisia capillaris standard decoction and the granules of Artemisia capillaris formula from different harvesting periods, respectively, according to the method in Example 1.

[0073] Result: As Figure 16 and Figure 17 As shown. Among them, Figure 16Thin-layer chromatography (TLC) of freeze-dried powders of Artemisia capillaris from different harvesting periods is shown in the figure. Particle 1 is freeze-dried powder of standard decoction of Artemisia capillaris (Artemisia capillaris var. ... Figure 17 The figures show thin-layer chromatography of Artemisia capillaris formulation granules harvested at different times. Granule 1 in the figure is Artemisia capillaris formulation granule [Artemisia capillaris (Mian Yin Chen)] KL20200101; Granule 2 is Artemisia capillaris formulation granule [Artemisia capillaris (Mian Yin Chen)] KL20200102; Granule 3 is Artemisia capillaris formulation granule [Artemisia capillaris (Mian Yin Chen)] KL20200105; Granule 4 is Artemisia capillaris [Artemisia capillaris (Mian Yin Chen)] reference material; Granule 5 is Artemisia capillaris formulation granule harvested in summer; Granule 6 is Artemisia capillaris [Artemisia capillaris (Mian Yin Chen)] formulation granule (harvested in autumn); Granule 7 is Artemisia capillaris [Artemisia capillaris (Hua Yin Chen)] formulation granule harvested in autumn; and Granule 8 is chlorogenic acid reference standard.

[0074] Conclusion: 1. Based on Figure 16 The thin-layer chromatography results show that, within the red frame at the top of the thin-layer plate, the freeze-dried powder of the standard decoction of Artemisia capillaris (Artemisia capillaris flower) harvested in autumn has one more bright blue fluorescent spot than the freeze-dried powder of the standard decoction of Artemisia capillaris var. mongolica, consistent with the thin-layer chromatography results of the medicinal materials. This invention's thin-layer chromatography method can distinguish the freeze-dried powders of standard decoctions from different harvesting periods of Artemisia capillaris. 2. The thin-layer chromatography results under ultraviolet 365 nm examination of three batches of Artemisia capillaris formula granules (…) Figure 17 In the thin-layer chromatography (TLC) results, two distinguishing features were observed in the formulation granules from different harvesting periods. Within the first red box along the solvent edge, autumn-harvested Artemisia capillaris (flowering variety) exhibited one more bright blue fluorescent spot than Artemisia capillaris (soft variety), while summer-harvested Artemisia capillaris lacked this fluorescent spot. Within the second red box along the lower edge of the TLC plate, autumn-harvested Artemisia capillaris (flowering variety) and summer-harvested Artemisia capillaris (soft variety) exhibited one more bright yellow spot than Artemisia capillaris (soft variety). In summary: Spring-harvested Artemisia capillaris: no Rf 0.83 blue spot, no Rf 0.17 yellow spot; Summer Artemisia capillaris: no Rf 0.83 blue spot, present Rf 0.17 yellow spot; Autumn-harvested Artemisia capillaris (flowering variety): present Rf 0.83 blue spot, present Rf 0.17 yellow spot. Based on these TLC results, Artemisia capillaris (flowering variety) from different harvesting periods, its decoction, and granules can be differentiated. This TLC method can be incorporated into the main text of the Artemisia capillaris (flowering variety) granule quality standard to ensure product quality.

[0075] The above specific embodiments further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above are merely specific embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made on the basis of the technical solution of the present invention should be included within the scope of protection of the present invention.

Claims

1. A thin-layer chromatography method for different harvesting periods of Artemisia capillaris, standard decoctions, and formulated granules, characterized in that... The thin-layer identification method includes the following steps: 1) Take a sample of Artemisia capillaris and prepare a test solution; 2) Spot the test solution onto a polyamide thin-layer plate, and develop it using the upper layer of a toluene-butyl acetate-formic acid-water solution with a volume ratio of 1:(9-12):(3.5-4.5):(2.5-3.5). After drying, examine the plate under ultraviolet light to obtain the thin-layer spectrum. Analyze the quality of the Artemisia capillaris sample based on the thin-layer spectrum. The different harvesting periods are spring, summer, and autumn. The Artemisia capillaris samples include Artemisia capillaris raw material, Artemisia capillaris standard decoction, or Artemisia capillaris formula granules.

2. The thin-layer chromatography method for different harvesting periods of Artemisia capillaris / standard decoction / formulated granules according to claim 1, characterized in that, The sample volume of the test solution is 0.5~2 μL.

3. The thin-layer chromatography method for different harvesting periods of Artemisia capillaris / standard decoction / formulated granules according to claim 1, characterized in that, The wavelength of the ultraviolet lamp is 365 nm.

4. The thin-layer chromatography method for different harvesting periods of Artemisia capillaris / standard decoction / formulated granules according to claim 1, characterized in that, 1) The preparation method of the test solution in the step is as follows: weigh an appropriate amount of Artemisia capillaris sample, extract and filter it, and take the filtrate to obtain the test solution.

5. The thin-layer chromatography method for different harvesting periods of Artemisia capillaris / standard decoction / formulated granules according to claim 4, characterized in that, The solvent used for extraction is methanol, and the extraction is carried out by ultrasound for 20-40 min. When the Artemisia capillaris sample is Artemisia capillaris herb, the material-to-liquid ratio for extraction is 1:(20-60). When the Artemisia capillaris sample is Artemisia capillaris standard decoction or Artemisia capillaris formula granules, the material-to-liquid ratio for extraction is 1:(80-120).

6. The thin-layer chromatography method for different harvesting periods of Artemisia capillaris / standard decoction / formulated granules according to claim 1, characterized in that, The thin-layer identification method further includes the step of preparing a reference solution, and the step of developing a thin-layer spectrum using the same identification method as the test solution; wherein the reference solution is a reference medicinal material solution of Artemisia capillaris and a reference solution of chlorogenic acid, and the reference medicinal material solution of Artemisia capillaris and the reference solution of chlorogenic acid are developed on the same plate.

7. A thin-layer chromatography method for different harvesting periods of Artemisia capillaris / standard decoction / formulated granules according to claim 6, characterized in that, The method for preparing the chlorogenic acid reference solution is as follows: take chlorogenic acid reference standard, add methanol to prepare a solution containing 1 mg per 1 mL, and use it as the reference solution.

8. A thin-layer chromatography method for different harvesting periods of Artemisia capillaris / standard decoction / formulated granules according to claim 6, characterized in that, The preparation method of the reference herb solution of Artemisia capillaris is as follows: take an appropriate amount of Artemisia capillaris reference herb, add water and boil, filter, evaporate the filtrate to dryness to obtain extract, extract the extract, filter, and use the filtrate as reference solution.

9. A thin-layer chromatography method for different harvesting periods of Artemisia capillaris / standard decoction / formulated granules according to claim 8, characterized in that, The boiling time is 50-80 min; the solvent used for extraction is methanol; the material-to-liquid ratio for extraction is 1:(15-25); the extraction is performed using ultrasound for 20-40 min.

10. A thin-layer chromatography method for different harvesting periods of Artemisia capillaris / standard decoction / formulated granules according to any one of claims 1-9, characterized in that, The thin-layer identification method includes the following steps: 1) Weigh the Artemisia capillaris sample, extract it with methanol using ultrasound for 20-40 min, filter it, and use the filtrate as the test solution; wherein, when the Artemisia capillaris sample is Artemisia capillaris medicinal material, the material-to-liquid ratio of the extraction is 1: (20-60); when the Artemisia capillaris sample is Artemisia capillaris standard decoction or Artemisia capillaris formula granules, the material-to-liquid ratio of the extraction is 1: (80-120). 2) Spot 0.5~2 μL of the test solution onto a polyamide thin-layer plate. Use the upper layer of toluene-butyl acetate-formic acid-water with a volume ratio of 1:(9-12):(3.5-4.5):(2.5-3.5) as the developing solvent for development. After drying, examine the plate under ultraviolet light to obtain the thin-layer spectrum. Perform quality analysis on the Artemisia capillaris sample based on the thin-layer spectrum. The different harvesting periods are spring, summer, and autumn. The Artemisia capillaris samples include Artemisia capillaris raw material, Artemisia capillaris standard decoction, or Artemisia capillaris formula granules.

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