A thin layer identification method for lychee seed medicinal material and its application

Abstract

The present invention relates to a thin-layer chromatography (TLC) identification method for lychee seeds and its application, comprising the steps of preparing a test solution, preparing a control medicinal solution and a control solution, and performing TLC identification. The method is simple and rapid to operate, produces abundant and clear spots, has good reproducibility, and has a wide range of applications. It addresses a gap in the field of lychee seed identification technology and improves the quality standards of lychee seeds. Furthermore, the method can effectively distinguish lychee seeds from their mixed counterparts, such as longan seeds.

Description

Technical Field

[0001] The invention belongs to the field of quality control of traditional Chinese medicines, and specifically relates to a thin-layer identification method for a lychee seed medicinal material and an application thereof. Background Art

[0002] Litchi seeds are the dried, mature seeds of the Sapindaceae plant, Litchi chinensis Sonn. Ripe fruits are harvested in summer, the peel and fleshy aril removed, washed, and sun-dried. This product is warm in nature, sweet with a slightly bitter taste, and enters the liver and kidney meridians, boasting the effects of promoting qi circulation, dispersing stagnation, dispelling cold, and relieving pain. Traditional Chinese Medicine believes that raw lychee seeds are particularly effective in treating liver qi stagnation and epigastric pain, while salt-roasted lychee seeds can be used to treat hernia pain by channeling the medicaments into the kidneys. Modern research indicates that lychee seeds, rich in chemical components such as flavonoids, volatile oils, organic acids, and fatty acids, have multiple pharmacological effects, including hypoglycemic and lipid-regulating properties, analgesic, anti-inflammatory, anti-tumor, antioxidant, free radical scavenging, and platelet aggregation inhibition. Guangxi, Guangdong, Fujian, and Hainan are the main lychee-producing regions, blessed with unique geographical and climatic conditions. Currently, the legal standards for lychee seeds as medicinal materials only cover sources, properties, and microscopic features, making it difficult to control the quality of lychee seeds as medicinal materials using existing quality assessment methods.

[0003] After investigation, there is the situation that longan seed (Sapindaceae plant longan Dimocarpus longan Lour.) is served as the litchi seed for sale on the market, but the two components are different, and their effect is also different, and can not be mixed. Considering that routine inspection item can not carry out true and false product distinction, and sensory evaluation (including base original identification, property identification) exists and relies on experience, the shortcoming that resolution is lower sometimes, especially for the identification of property close or variety confusion medicinal material is comparatively difficult, although document 1 (CN105353065A) discloses a kind of establishment method of HPLC fingerprint of litchi seed and its standard fingerprint and purposes, establish litchi seed control fingerprint, a total of 15 common characteristic peaks, but the method is difficult to carry out effective and simple identification to litchi seed and mixed false product longan seed. The thin layer chromatography method disclosed in document 2 (Xiao Bingmei, Pei Gang. Thin layer chromatography and ultraviolet spectroscopic identification of litchi kernel and its counterfeit longan kernel [J]. Journal of Primary Chinese Medicine, 2022, 15(1): 34.) was verified to be unreproducible, with severe tailing, and different batches of litchi kernel or longan kernel samples with different spot positions, which is not suitable for quality evaluation and origin identification.

[0004] Therefore, how to establish a thin-layer chromatography identification method for litchi kernel medicinal materials, which can also serve as a specific identification method for distinguishing litchi kernel from its easily confused product longan kernel, has important practical significance for improving the standard of litchi kernel medicinal materials and ensuring accurate and safe clinical use of drugs. Summary of the Invention

[0005] Problems to be solved by the invention

[0006] In order to solve the problems existing in the above-mentioned prior art, the purpose of the present invention is to provide a thin layer identification method for litchi seed medicinal materials, which is simple and fast to operate, has rich and clear spots, good reproducibility, and a wide range of applications.

[0007] Another object of the present invention is to provide an application of a thin layer identification method for lychee seeds, which can well separate lychee seeds and their mixed and counterfeit products, longan seeds.

[0008] Solutions for solving problems

[0009] The present invention provides the following technical solutions:

[0010] [1] A thin layer chromatography identification method for lychee seed medicinal materials, comprising the following steps:

[0011] 1) Preparation of test solution:

[0012] The steps include extracting the medicinal material to be tested by using an alcohol solvent;

[0013] 2) Preparation of reference medicinal material solution and reference substance solution:

[0014] Take litchi kernel as control medicinal material and prepare control medicinal material solution by the test solution preparation method described in 1); take protocatechuic acid reference substance and prepare reference substance solution;

[0015] 3) Thin layer identification step: comprising taking a control medicinal material solution, a reference substance solution and a test substance solution and spotting them on the same thin layer plate, and developing them with a developing agent, wherein the developing agent is toluene-ethyl formate-glacial acetic acid, and the volume ratio of toluene:ethyl formate:glacial acetic acid is (6-10):(2-5):0.5.

[0016] [2] The thin layer chromatography identification method for lychee seed medicinal materials according to [1], wherein: the step 1) comprises taking the powder of the medicinal material to be tested and treating it with an alcohol solvent, filtering, concentrating the filtrate to dryness, dissolving the residue in water, adding an organic solvent and shaking to extract, evaporating to dryness, and dissolving the residue in an alcohol solvent to prepare a test solution.

[0017] [3] The thin layer chromatography identification method for lychee seed medicinal materials according to [1] or [2], wherein: the alcohol solvent is selected from a lower alcohol solvent with carbon atoms of C1 to C5 or an aqueous solution thereof, and further, the lower alcohol solvent with carbon atoms of C1 to C5 is selected from methanol, ethanol or isopropanol.

[0018] [4] The thin layer chromatography identification method for lychee seed medicinal materials according to any one of the technical solutions [1] to [3], wherein: the organic solvent is selected from ethyl acetate, n-butanol or water-saturated n-butanol.

[0019] [5] The thin layer chromatography identification method for lychee seed medicinal materials according to any one of the technical solutions [1] to [4], wherein the protocatechuic acid concentration of the reference solution is 0.2 to 2 mg / mL.

[0020] [6] The thin layer chromatography identification method for lychee seed medicinal materials according to any one of the technical solutions [1] to [5], wherein the volume ratio of toluene:ethyl formate:glacial acetic acid is (7-9):(2-4):0.5.

[0021] [7] The thin layer chromatography identification method for lychee seed medicinal materials according to any one of the technical solutions [1] to [6], wherein: the sample volume of the control medicinal material solution, the reference substance solution and the test solution in step 3) is 8 μL to 20 μL.

[0022] [8] The thin layer identification method for lychee seed medicinal material according to any one of the technical solutions [1] to [7], wherein: the thin layer identification step also includes the step of unfolding and inspecting under ultraviolet light, and further, the inspection includes taking out the unfolded thin layer plate, drying it, and spraying it with 5% to 15% sulfuric acid ethanol solution or 5% to 25% phosphomolybdic acid ethanol solution for color development.

[0023] [9] The thin-layer chromatography identification method for litchi seed medicinal materials according to any one of the technical solutions [1] to [8], wherein: the thin-layer chromatography identification method further comprises comparing the chromatogram of the test sample with the chromatogram of the reference medicinal material and the chromatogram of the reference substance to determine whether spots of the same color are displayed at corresponding positions, and if spots of the same color are displayed, it is determined that the medicinal material to be tested is litchi seed medicinal material.

[0024]

[10] The use of the thin layer identification method of litchi seed medicinal materials as described in any one of the technical solutions [1] to [9] to distinguish litchi seed medicinal materials from longan seed medicinal materials.

[0025] Effects of the Invention

[0026] The present invention provides a thin layer identification method for litchi kernel medicinal materials, which is characterized by high efficiency, rapidity, and accuracy, is easy to operate, has rich and clear spots, and good reproducibility. It can effectively identify litchi kernels and mixed and counterfeit products, is easy to promote and apply, and provides a basis for accurate clinical medication. The present invention uses alcohol solvents to extract the components with higher polarity in litchi kernel medicinal materials, and uses a developing agent of a specific type and proportion for thin layer development, which fills a gap in the technical field of litchi kernel identification and improves the quality standard of litchi kernel medicinal materials. The present invention uses litchi kernel control medicinal materials and protocatechuic acid reference substances as controls. The operation is simple and easy, the identification characteristics are obvious, the results are easy to determine, and the specificity is good, and it can accurately identify litchi kernel medicinal materials. In some preferred embodiments of the present invention, the thin layer plate of the present invention only requires ordinary silica gel GF 254The thin layer plate can achieve the effect of rich spot information and good separation without using expensive high-efficiency silica gel G thin layer plate. The present invention is more popular and popularizable.

[0027] It should be noted that the above description does not disclose all embodiments of the present invention and all advantages of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the specific embodiments or the description of the prior art. The drawings described below are some embodiments of the present invention. Those skilled in the art can also derive other drawings based on these drawings without inventive effort.

[0029] Figure 1 The characteristics of lychee and longan seeds

[0030] Figure 2 These are thin-layer chromatograms of litchi kernel medicinal materials prepared by different preparation methods for test solution and reference medicinal material solution (1 to 3 are chromatograms of test solution of litchi kernel medicinal materials prepared by three different preparation methods, S1 to S3 are chromatograms of litchi kernel reference medicinal materials prepared by three different preparation methods, and T is the chromatogram of protocatechuic acid reference substance; the left figure is the thin-layer chromatogram at 365 nm, and the right figure is the thin-layer chromatogram at 254 nm)

[0031] Figure 3 These are thin-layer chromatograms of litchi kernel medicinal material obtained with different developing agents (3-1: n-butanol-acetic acid-water (4:1:2), 3-2: chloroform-ethyl formate-glacial acetic acid (6:3:1), 3-3: chloroform-ethyl formate-glacial acetic acid (16:3:1), 3-4: toluene-ethyl formate-glacial acetic acid (8:3:0.5); 1 is the chromatogram of the test sample from batch S1, S is the chromatogram of the litchi kernel reference medicinal material, and T is the chromatogram of the protocatechuic acid reference substance; the left figure is the thin-layer chromatogram at 365 nm, and the right figure is the thin-layer chromatogram at 254 nm).

[0032] Figure 4 This is a thin layer chromatogram specific for identification of litchi kernel medicinal material (1 to 3 are chromatograms of three parallel test samples from batch number S1, S is the chromatogram of litchi kernel control medicinal material, T is the chromatogram of protocatechuic acid reference substance, and F is the negative control solution of litchi kernel; the left picture is the thin layer chromatogram at 365nm, and the right picture is the thin layer chromatogram at 254nm)

[0033] Figure 5These are the thin-layer identification chromatograms of litchi kernel medicinal materials under different temperature conditions (5-1: 4°C, 5-2: 27°C, 5-3: 35°C; 1 to 3 are chromatograms of three parallel samples of the test sample from batch number S1, S is the chromatogram of the litchi kernel reference medicinal material, and T is the chromatogram of the protocatechuic acid reference substance; the left picture is the thin-layer chromatogram at 365nm, and the right picture is the thin-layer chromatogram at 254nm)

[0034] Figure 6 These are thin-layer chromatograms of litchi seed medicinal materials under different humidity conditions (6-1: relative humidity 18%, 6-2: relative humidity 65%, 6-3: relative humidity 88%; 1 to 3 are chromatograms of three parallel samples of the test sample from batch number S1, S is the chromatogram of the litchi seed reference medicinal material, and T is the chromatogram of the protocatechuic acid reference substance; the left picture is the thin-layer chromatogram at 365nm, and the right picture is the thin-layer chromatogram at 254nm).

[0035] Figure 7 This is the thin layer chromatography identification verification chart of different batches of litchi kernel medicinal materials (1 to 15 are the chromatograms of the test samples of batch numbers S1 to S15, S is the chromatogram of the litchi kernel reference medicinal material, and T is the chromatogram of the protocatechuic acid reference substance)

[0036] Figure 8 This is a TLC comparison chart of litchi kernel and longan kernel (1 to 3 are chromatograms of litchi kernel medicinal material test samples from batches S1 to S3, 4 to 6 are chromatograms of longan kernel medicinal material test samples from batches S16 to S18, S is the chromatogram of litchi kernel reference medicinal material, and T is the chromatogram of protocatechuic acid reference substance)

[0037] Figure 9 This is the thin layer identification chromatogram of litchi kernel medicinal material in comparative example 1 (sunlight) (1 to 4 are the chromatograms of litchi kernel medicinal material test samples, and the sample volumes are 0.5 μL, 1 μL, 2 μL, and 3 μL, respectively)

[0038] Figure 10 This is the thin layer identification chromatogram of the longan seed medicinal material of comparative example 1 (sunlight) (1 to 4 are the chromatograms of the longan seed medicinal material test sample, and the sample volume is 0.5μL, 1μL, 2μL, and 3μL respectively)

[0039] Figure 11 Thin layer chromatograms (sunlight) of three batches of litchi kernel medicinal materials and three batches of longan kernel medicinal materials in Comparative Example 1 (1 to 3 are chromatograms of litchi kernel medicinal materials for test; 5 to 7 are chromatograms of longan kernel medicinal materials for test; S: litchi kernel medicinal materials for control) DETAILED DESCRIPTION

[0040] To better illustrate the present invention, numerous specific details are provided in the following detailed description. Those skilled in the art will appreciate that the present invention can be practiced without certain specific details. In other instances, methods, means, equipment, and steps well known to those skilled in the art are not described in detail in order to highlight the main points of the present invention.

[0041] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0042] In this specification, the numerical range expressed using "a numerical value A to a numerical value B" means a range including the endpoints A and B.

[0043] In this specification, the use of "may" includes both the meaning of performing a certain process and the meaning of not performing a certain process.

[0044] It should be understood that the singular article "a," "an," and "the" as used in the specification and claims of this application include plural referents unless the context clearly dictates otherwise.

[0045] In this specification, references to "one or some specific / preferred embodiments / solutions," "another or other specific / preferred embodiments / solutions," "one or another embodiment / solution," "one or another technical solution," etc., mean that the specific elements (e.g., features, structures, properties, and / or characteristics) described in connection with the embodiment are included in at least one embodiment described herein, and may or may not be present in other embodiments. In addition, it should be understood that the elements may be combined in various embodiments in any appropriate manner.

[0046] The term "comprises" and any variations thereof in the specification and claims of the present invention and the accompanying drawings are intended to cover non-exclusive inclusions. For example, a process, method, system, product, or apparatus comprising a series of steps or elements is not limited to the listed steps or elements, but may optionally include steps or elements not listed, or may optionally include other steps or elements inherent to the process, method, product, or apparatus.

[0047] Unless otherwise specified, the term "herbal medicine" used in this article refers to unprocessed or unfinished Chinese medicine raw materials; "control medicinal materials" (or "reference medicinal materials") refer to standard medicinal materials that have completed species identification and are used to identify the samples to be tested.

[0048] Unless otherwise specified, the term "test article" used herein refers to an experimental sample used for detection or identification.

[0049] Unless otherwise specified, the term "thin layer chromatography" used in this article is a trace, rapid and simple separation and analysis method. A suitable stationary phase is coated on a glass plate, plastic or aluminum substrate to form a uniform thin layer. After the sample is spotted and developed, the relative shift value (Rf) is compared with the relative shift value of the chromatogram obtained by the same method with a suitable reference substance, and is used for drug identification, impurity inspection or content determination. This method is a very important experimental technique for rapid separation and qualitative analysis of small amounts of substances, and is also used to track the progress of reactions. It has the characteristics of short development time, strong separation ability and high sensitivity. Thin layer chromatography is one of the important inspection items for traditional Chinese medicines. By comparing the chromatographic behavior of the test sample and the reference medicinal materials and reference substances, the authenticity and quality of the medicinal materials can be judged.

[0050] Unless otherwise specified, the term "spotting" used herein refers to the process of adding drops of sample solution to be separated and identified onto a thin layer plate.

[0051] Unless otherwise specified, the term "development" used herein refers to the process in which the developing agent carries the sample components to migrate a certain distance on the thin layer plate by the capillary action of the stationary phase on the thin layer plate.

[0052] The present invention provides a thin layer identification method for lychee seed medicinal materials. Based on the chemical structure and properties of each effective component of traditional Chinese medicine and following the principle of similar and compatible extraction, a suitable extraction solvent is used to simply and quickly prepare test sample and control medicinal material solutions. Then, a suitable developing agent is used for development. Various chemical components will be well separated on the thin layer plate according to the different abilities of adsorption, desorption, re-adsorption, and re-desorption of the selected developing agent. Then, with the help of various effective components with similar polarity, different color spots are presented on the same thin layer plate under different inspection conditions (sunlight, ultraviolet light or color development with a color developer), thereby obtaining a multi-information thin layer chromatogram.

[0053] The thin layer chromatography identification method of litchi seed medicinal material provided by the present invention comprises the steps of preparing a test solution, preparing a reference medicinal material solution and a reference product solution, and thin layer chromatography identification.

[0054] <Preparation of test solution>

[0055] The preparation of the test solution of the present invention includes the step of extracting the medicinal material to be tested with an alcohol solvent. The use of an alcohol solvent is helpful in extracting the more polar components in the litchi seed medicinal material. In some specific embodiments of the present invention, the alcohol solvent includes a lower alcohol solvent selected from a carbon atom of C1 to C5 or an aqueous solution thereof, and further, the lower alcohol solvent with a carbon atom of C1 to C5 is selected from methanol, ethanol or isopropanol. In some preferred embodiments of the present invention, the alcohol solvent is selected from methanol or a methanol aqueous solution (such as a methanol aqueous solution with a methanol volume percentage of 10 to 80%), and is further preferably selected from methanol. In some specific embodiments of the present invention, the amount of the alcohol solvent used can be 10 to 40 mL relative to 1 g of the medicinal material to be tested, and can further be 15 to 30 mL. As for the extraction method, it can be any one or more of ultrasound, heating reflux, decoction, immersion, and shaking. In some preferred embodiments of the present invention, taking into account the convenience of operation and extraction efficiency, the present invention adopts ultrasonic treatment for extraction. The power and frequency of the ultrasound have little effect on the present invention. In some specific embodiments of the present invention, the ultrasonic power can be 220W to 300W, the frequency can be 35kHz to 45kHz, and the ultrasonic treatment time can be 10 to 45 minutes, and further can be 15 to 30 minutes.

[0056] In some specific embodiments of the present invention, the preparation of the test solution includes taking the powder of the medicinal material to be tested and adding an alcohol solvent for treatment, filtering, concentrating the filtrate to dryness, dissolving the residue in water, adding an organic solvent for shaking extraction, evaporating to dryness, and dissolving the residue in an alcohol solvent to obtain a test solution. This method can better extract the components with larger polarity in the litchi core medicinal material, which helps to obtain more and clearer fluorescent spots. In some specific embodiments of the present invention, the medicinal material to be tested is ground and sieved (such as No. 3 sieve) to prepare the medicinal material powder to be tested, and then an alcohol solvent is added for ultrasonic treatment, followed by filtration, and the filtrate is concentrated to dryness, and the residue is added with water to dissolve. In some specific embodiments of the present invention, the organic solvent for shaking extraction is selected from ethyl acetate, n-butanol or water-saturated n-butanol, which is conducive to extracting the polar components in the litchi core medicinal material more fully. In some preferred embodiments of the present invention, the organic solvent is preferably selected from ethyl acetate, and the fluorescent spot color of the thin layer chromatography obtained using it is relatively clearer and brighter. Regarding the amount of organic solvent used, the amount of organic solvent used for each shaking extraction can be 10 to 40 mL, and further can be 15 to 30 mL, relative to 1 g of the medicinal material to be tested. The present invention is not limited to the number of shaking extractions. In some specific embodiments of the present invention, the shaking extraction can be performed 2 to 3 times. The resulting solutions are then combined and evaporated to dryness. A small amount of the alcohol solvent described above can be added to the residue to dissolve it, thereby obtaining the test solution of the present invention.

[0057] <Preparation of Reference Herbal Solution and Reference Substance Solution>

[0058] The preparation of the control medicinal material solution of the present invention is the same as the preparation method of the test solution described above. The preparation of the control solution of the present invention includes preparing a control solution using a protocatechuic acid control substance. In some embodiments of the present invention, the control solution is prepared by dissolving the protocatechuic acid control substance in an alcoholic solvent. The definition of an alcoholic solvent is the same as described above. Preferably, the protocatechuic acid concentration of the control solution can be 0.2 to 2 mg / mL, and further can be 0.4 to 1.0 mg / mL.

[0059] <Thin layer identification step>

[0060] The thin layer identification step of the present invention includes taking a control medicinal material solution, a reference substance solution and a test substance solution and spotting them on the same thin layer plate respectively, and developing them with a developing agent, the developing agent is toluene-ethyl formate-glacial acetic acid, and the volume ratio is toluene: ethyl formate: glacial acetic acid = (6-10): (2-5): 0.5. The use of a specific type and ratio of developing agents in the present invention helps to obtain a thin layer chromatogram of litchi core medicinal materials with multiple information and clear and rich spots. If the developing agent of the present invention is not used, problems such as no clear spots, less spot information, poor separation or inappropriate spot Rf value may occur. In some preferred embodiments of the present invention, toluene: ethyl formate: glacial acetic acid = (7-9): (2-4): 0.5, more preferably, toluene: ethyl formate: glacial acetic acid = 8:3:0.5. The thin layer plate of the present invention is preferably silica gel GF 254 Thin layer plates can achieve rich spot information and good separation, and are popular and popularizable. In some embodiments of the present invention, the spotting volume of the control medicinal material solution, the reference substance solution, and the test solution is 8 μL to 20 μL. Furthermore, to obtain clearer spots, the spotting volume of the test solution and the control medicinal material solution is preferably 10 μL to 15 μL.

[0061] In some specific embodiments of the present invention, the thin layer identification step further includes a step of inspecting under ultraviolet light after development to obtain an ultraviolet thin layer chromatogram. More specifically, the inspection step includes removing the developed thin layer plate, drying it, spraying it with 5% to 15% sulfuric acid ethanol solution or 5% to 25% phosphomolybdic acid ethanol solution for color development, and heating it until the spots are clearly colored. Compared with ferric chloride and aluminum chloride color developers, the spots using sulfuric acid ethanol solution or phosphomolybdic acid test solution are richer and clearer. In some preferred embodiments of the present invention, the color developer is selected from 10% to 15% sulfuric acid ethanol solution. The color developer can be prepared by conventional methods, such as taking 10mL of sulfuric acid and diluting it to 100mL with 95% ethanol. In the present invention, the wavelength of the ultraviolet lamp is 365nm or 254nm, and the heating temperature is preferably 100 to 120°C, for example, it can be 105°C. The present invention can obtain a multi-information thin-layer chromatogram of the lychee seed medicinal material under the inspection condition of ultraviolet light (365nm), which is conducive to comparison with the chromatogram of the reference medicinal material, while the inspection condition of ultraviolet light (254nm) is conducive to comparison with the chromatogram of the reference substance.

[0062] In some specific embodiments of the present invention, the method further comprises comparing the test sample chromatogram with the control medicinal material chromatogram and the control substance chromatogram to determine whether fluorescent spots of the same color are displayed at corresponding positions. If spots of the same color are displayed, the test medicinal material is determined to be a litchi seed medicinal material. Otherwise, the test medicinal material is determined not to be a litchi seed medicinal material.

[0063] The thin-layer chromatographic identification method for lychee seed medicinal materials provided by the present invention can effectively develop and separate compounds with high polarity in lychee seed medicinal materials. After color development, the spots are rich and clear, the separation is good, the identification results are easy to judge, and the reproducibility is good. In addition, the method of the present invention has good specificity, good durability at different temperatures (for example, the applicable temperature range can be 2°C to 50°C), good durability at different humidity levels (for example, the applicable humidity range can be 10% to 90%), and good durability for thin-layer plates from different manufacturers. The detection cost is low, safe, and environmentally friendly, and it can better control the quality of lychee seed medicinal materials.

[0064] <Purpose>

[0065] The present invention also provides the use of the aforementioned thin-layer identification method of litchi core medicinal materials in distinguishing litchi core medicinal materials from common counterfeit longan core medicinal materials in the market, which is convenient for promotion and application and provides a basis for accurate clinical medication. In some specific embodiments of the present invention, under ultraviolet light (254nm), the chromatogram of the litchi core medicinal material test sample shows spots of the same color at the corresponding position of the protocatechuic acid reference substance chromatogram, while the chromatogram of the longan core medicinal material test sample does not show spots of the same color at the corresponding position of the protocatechuic acid reference substance chromatogram; under ultraviolet light (365nm), the chromatogram of the litchi core medicinal material test sample shows fluorescent spots of the same color at the corresponding position of the litchi core reference medicinal material chromatogram, while the chromatogram of the longan core medicinal material test sample does not show fluorescent spots of the same color at the corresponding position of the litchi core reference medicinal material chromatogram, and lacks blue spots at Rf values ​​of approximately 0.25 to 0.33. According to the method provided by the invention, the blue spots with Rf values ​​of 0.25 to 0.33 under ultraviolet light (254 nm) of the protocatechuic acid reference substance and under ultraviolet light (365 nm) can be used as the distinguishing points for thin-layer identification of litchi seed medicinal materials and longan seed medicinal materials.

[0066] Example

[0067] The technical solutions of the present invention will be further described below with reference to specific embodiments.

[0068] The present invention adopts the following instruments and reagents:

[0069] Instruments: CAMAH TLC VISUALIZER, CAMAG TLCVISUALIZER, ME204E 1 / 10,000 balance (Mettler-Toledo), KQ-250E ultrasonic cleaner (Kunshan Ultrasonic Electronics Co., Ltd.), silica gel GF254 thin layer plate (Qingdao Ocean Chemical Plant, Qingdao Kangyexin Pharmaceutical Silica Gel Desiccant Co., Ltd., Rushan Sun Desiccant Co., Ltd.).

[0070] Reagents: methanol (Sinopharm Chemical Reagent Co., Ltd.), ethyl acetate (Shanghai Titan Technology Co., Ltd.), toluene (Shanghai Lingfeng Chemical Reagent Co., Ltd.), ethyl formate (Sinopharm Chemical Reagent Co., Ltd.), glacial acetic acid (Sinopharm Chemical Reagent Co., Ltd.), sulfuric acid (Shanghai Lingfeng Chemical Reagent Co., Ltd.), ethanol (Sinopharm Chemical Reagent Co., Ltd.), n-butanol (Sinopharm Chemical Reagent Co., Ltd.), chloroform (Shanghai Lingfeng Chemical Reagent Co., Ltd.), cyclohexane (Sinopharm Chemical Reagent Co., Ltd.), acetone (Shanghai Lingfeng Chemical Reagent Co., Ltd.), and vanillin (Sinopharm Chemical Reagent Co., Ltd.) were all of analytical grade.

[0071] Litchi seed control medicinal material (310027-202111) was purchased from Chengdu Pusi Biotechnology Co., Ltd.

[0072] Protocatechuic acid reference substance (110809-201205) was purchased from China Food and Drug Inspection Institute.

[0073] 15 batches of litchi kernels and 3 batches of longan kernels are from major production areas and authentic production areas across the country. The litchi kernels all meet the requirements of the 2020 edition of the Chinese Pharmacopoeia. The medicinal material information is shown in Table 1, and the medicinal material properties are shown in Table 2. Figure 1 .

[0074] Table 1 Litchi and longan kernel sample information

[0075]

[0076]

[0077] If no specific experimental steps or conditions are specified in the present invention, the conventional experimental steps or conditions described in the literature in this field can be followed. If the manufacturer of the reagents or instruments used is not specified, they are all commercially available conventional reagents.

[0078] Investigation of the preparation methods of test and control medicinal material solutions

[0079] Method 1: Take 1 g of the powder of this product, add 20 mL of methanol, ultrasonically treat for 20 minutes, filter, concentrate the filtrate to dryness, dissolve the residue in 20 mL of water, add ethyl acetate and shake to extract twice, 20 mL each time, combine the ethyl acetate solution, evaporate to dryness, and dissolve the residue in 1 mL of methanol as the test solution.

[0080] Method 2: Take 1 g of the powder of this product, add 20 mL of ethyl acetate, ultrasonically treat for 20 minutes, filter, evaporate the filtrate to dryness, and dissolve the residue in 1 mL of ethyl acetate to prepare the test solution.

[0081] Method 3: Take 1g of the powder of this product, add 20mL of methanol, ultrasonically treat for 20 minutes, filter, concentrate the filtrate to dryness, dissolve the residue in 20mL of water, add saturated n-butanol and shake to extract twice, 20mL each time, combine the n-butanol solutions, evaporate to dryness, and dissolve the residue in 1mL of methanol to prepare the test solution. 3.1.2 Preparation of reference medicinal materials and reference solution

[0082] Method 1: Take 1 g of litchi seed control medicinal material, add 20 mL of methanol, sonicate for 20 minutes, filter, and concentrate the filtrate to dryness. Dissolve the residue in 20 mL of water. Extract twice with ethyl acetate by shaking, using 20 mL each time. Combine the ethyl acetate solutions, evaporate to dryness, and dissolve the residue in 1 mL of methanol to prepare the control medicinal material solution. Also, take the protocatechuic acid reference substance and add methanol to a solution containing 0.5 mg per 1 mL. This is the reference substance solution.

[0083] Method 2: Take 1 g of litchi seed (a reference medicinal material) and add 20 mL of ethyl acetate. Ultrasonicate for 20 minutes, filter, evaporate the filtrate to dryness, and dissolve the residue in 1 mL of ethyl acetate to prepare the reference medicinal material solution. Also, take the protocatechuic acid reference substance and add methanol to a solution containing 0.5 mg per 1 mL. This is the reference solution.

[0084] Method 3: Take 1 g of litchi seed control medicinal material, add 20 mL of methanol, sonicate for 20 minutes, filter, and concentrate the filtrate to dryness. Dissolve the residue in 20 mL of water. Shake and extract twice with 20 mL of saturated n-butanol. Combine the n-butanol solutions, evaporate to dryness, and dissolve the residue in 1 mL of methanol to prepare the control medicinal material solution. Also, take the protocatechuic acid reference substance and add methanol to a solution containing 0.5 mg per 1 mL. This is the reference solution.

[0085] Take the sample (batch number: S1) and prepare the test solution, three control medicinal material solutions and reference solution according to the above three preparation methods. Take 10 μl of each of the test solution, control medicinal material solution and reference solution and spot them on the same silica gel GF 254 On the thin layer plate, chloroform-ethyl formate-glacial acetic acid (6:3:1) was used as the developing agent, and the plate was developed, taken out, dried, sprayed with 10% sulfuric acid ethanol solution, heated at 105°C until the spots were clearly colored, and examined under ultraviolet light (365nm, 254nm).

[0086] The experimental results show that (see Figure 2 ): The test sample and control medicinal material preparation method 2 have one less blue fluorescent spot compared with method 1 and method 3. The fluorescent spot color of test sample preparation method 1 is clearer and brighter than that of method 3. Therefore, test sample preparation method 1 and control medicinal material preparation method 1 were selected as the test sample solution and control medicinal material solution preparation methods, respectively. The control substance solution preparation method is to take protocatechuic acid reference substance and add methanol to make a solution containing 0.5 mg per 1 mL.

[0087] Investigation of different developing agents

[0088] Take 1g of this product powder (batch number: S1), add 20mL of methanol, sonicate for 20 minutes, filter, and concentrate the filtrate to dryness. Dissolve the residue in 20mL of water. Extract twice with ethyl acetate by shaking, 20mL each time. Combine the ethyl acetate solutions, evaporate to dryness, and dissolve the residue in 1mL of methanol to prepare the test solution. Separately, prepare a control solution using 1g of litchi kernel as a control medicinal material using the same method. Also prepare a protocatechuic acid reference substance and add methanol to a solution containing 0.5mg per 1mL. This will serve as the reference solution. According to the thin layer chromatography method (General Chapter 0502 of the 2020 edition of the Chinese Pharmacopoeia), 10 μl of each of the three solutions mentioned above were taken and spotted on the same silica gel G thin layer plate, and various developing agents with different composition ratios such as (1) chloroform-ethyl formate-glacial acetic acid (6:3:1) and (16:3:0.5), (2) n-butanol-acetic acid-water (4:1:2), and (3) toluene-ethyl formate-glacial acetic acid (8:3:0.5) were used as developing agents. The plate was developed, taken out, dried, sprayed with 10% sulfuric acid ethanol solution, heated at 105°C until the spots were clearly colored, and inspected under ultraviolet light (365nm, 254nm).

[0089] The experimental results show that (see Figure 3 ): When the developing conditions were n-butanol-acetic acid-water (4:1:2), the chromatogram of the test sample had no clear spots or fluorescent spots at 254nm and 365nm under ultraviolet light; when the developing conditions were chloroform-ethyl formate-glacial acetic acid (6:3:1), the chromatogram of the test sample had clear spots or fluorescent spots at 254nm and 365nm under ultraviolet light, but the Rf values ​​of most spots were high. As the proportion of chloroform in the developing solvent increased, the spots gradually moved downward. When developing with chloroform-ethyl formate-glacial acetic acid (16:3:0.5), most spots were concentrated between Rf values ​​of 0.45 and 0.92, with the Rf values ​​still being on the high side. When developing with toluene-ethyl formate-glacial acetic acid (8:3:0.5), the sample chromatogram showed clear fluorescent spots at both 254 nm and 365 nm under UV light. The spots were abundant and the overall distribution was clear and beautiful, with Rf values ​​for all spots ranging from 0.16 to 0.81. Therefore, toluene-ethyl formate-glacial acetic acid (8:3:0.5) was selected as the developing solvent.

[0090] Investigation of different color developers

[0091] Take 1g of the powder of this product (batch number: S1), add 20mL of methanol, ultrasonically treat for 20 minutes, filter, concentrate the filtrate to dryness, add 20mL of water to dissolve the residue, add ethyl acetate and shake to extract twice, 20mL each time, combine the ethyl acetate solution, evaporate to dryness, add 1mL of methanol to dissolve the residue, and use it as the test solution. Take another 1g of litchi kernel reference medicinal material, and prepare a reference medicinal material solution in the same way. Take the protocatechuic acid reference substance, add methanol to make a solution containing 0.5mg per 1mL, as the reference solution. According to the thin layer chromatography method (General Rules 0502 of the 2020 edition of the Chinese Pharmacopoeia), take 10μl of each of the above three solutions and spot them on the same silica gel GF 254 For the thin layer plate, use chloroform-ethyl formate-glacial acetic acid (6:3:1) as the developing agent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution and aluminum trichloride test solution respectively. The thin layer plate sprayed with 10% sulfuric acid ethanol solution is heated at 105°C until the spots are clearly colored; the thin layer plate sprayed with aluminum trichloride test solution is blown dry with hot air, and they are examined under ultraviolet light (365nm, 254nm) respectively.

[0092] Experimental results show that the spots produced by spraying with a 10% sulfuric acid ethanol solution under ultraviolet light (365nm, 254nm) are clearer and more abundant than those produced by using aluminum chloride. Therefore, the following development conditions were selected: spraying with a 10% sulfuric acid ethanol solution, heating at 105°C until the spots are clearly colored, and then inspecting under ultraviolet light (365nm, 254nm).

[0093] Investigation of different spot sizes

[0094] Take the sample (batch number: S1) and prepare the test solution and control medicinal material solution according to the method determined above. 254 Spot samples on the thin layer plate respectively, and examine the spot amounts of different test solutions, control medicinal material solutions and reference solution (1 μL, 5 μL, 10 μL, 15 μL, 20 μL). Use toluene-ethyl formate-glacial acetic acid (8:3:0.5) as the developing agent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105 ° C until the spots are clearly colored, and inspect under ultraviolet light (365 nm, 254 nm).

[0095] The experimental results show that when the sample solution, control herb solution, and reference substance solution concentrations are 10-20 μL, the spots or fluorescent spots in the litchi seed herb sample chromatogram at the corresponding positions in the control herb chromatogram and reference substance chromatogram are clear, with good resolution and no other interference. Therefore, the sample volume of the test sample solution, control herb solution, and reference substance solution was selected to be 10 μL.

[0096] Methodological validation

[0097] <Specialty Experiment>

[0098] 10 μL each of the test solution (batch number: S1, 3 replicates), control medicinal material solution (S), reference solution, and negative control solution lacking litchi kernel were aspirated and spotted on the same silica gel GF 254 On the thin layer plate, use toluene-ethyl formate-glacial acetic acid (8:3:0.5) as the developing agent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly colored, and examine under ultraviolet light (365nm, 254nm), see Figure Figure 4 (Left image viewed at 365 nm, right image viewed at 254 nm).

[0099] The results showed that the chromatograms of the litchi seed test sample, the control herb, and the control sample showed the same color spots or fluorescent spots at corresponding positions, and there was no interference from the negative control, indicating that the thin-layer chromatography method has good specificity.

[0100] <Durability Test>

[0101] Investigation at different temperatures:

[0102] Pipette 10 μL each of the test solution (batch number: S1), control medicinal material solution, and reference substance solution and spot them on the same silica gel GF 254 On the thin layer plate, toluene-ethyl formate-glacial acetic acid (8:3:0.5) was used as the developing agent and developed at different temperatures (4°C, 27°C, 35°C). The plate was taken out, dried, sprayed with 10% sulfuric acid ethanol solution, heated at 105°C until the spots were clearly colored, and examined under ultraviolet light (365nm, 254nm). Figure 5 .

[0103] The results showed that under different temperature conditions, the chromatograms of the test and control samples of litchi seed medicinal materials showed spots of the same color or fluorescent spots at corresponding positions, and the separation effect was good. The experimental results showed that temperature had no significant effect on the thin-layer chromatography identification of litchi seed medicinal materials, indicating that the thin-layer chromatography identification method has good durability at different temperatures.

[0104] Investigation of different humidity:

[0105] Pipette 10 μL each of the test solution (batch number: S1), control medicinal material solution, and reference substance solution and spot them on the same silica gel GF 254 On the thin layer plate, toluene-ethyl formate-glacial acetic acid (8:3:0.5) was used as the developing agent and developed at different humidity levels (18%, 65%, 88%). The plate was taken out, dried, sprayed with 10% sulfuric acid ethanol solution, heated at 105°C until the spots were clearly colored, and examined under ultraviolet light (365nm, 254nm). Figure 6 .

[0106] The results showed that under different humidity conditions, the chromatograms of the test and control samples of litchi seed medicinal materials showed spots of the same color or fluorescent spots at corresponding positions, and the separation effect was good. The experimental results showed that humidity had no effect on the thin-layer chromatography identification of litchi seed medicinal materials, indicating that the thin-layer chromatography identification method has good durability under different humidity conditions.

[0107] Investigation of different thin layer plates

[0108] 10 μL of each of the test solution (batch number: S1), the control medicinal material solution, and the reference solution were taken and spotted on silica gel GF of different brands (Qingdao Ocean Chemical Plant, Qingdao Kangyexin Pharmaceutical Silica Gel Desiccant Co., Ltd., Rushan Sun Desiccant Co., Ltd.). 254 The thin layer plate was developed with toluene-ethyl formate-glacial acetic acid (8:3:0.5) as the developing agent, taken out, dried, sprayed with 10% sulfuric acid ethanol solution, heated at 105°C until the spots were clearly colored, and examined under ultraviolet light (365nm, 254nm).

[0109] The results showed that the chromatograms of the test sample, the reference sample, and the reference sample on different manufacturers' thin-layer plates showed spots of the same color or fluorescent spots at corresponding positions, and the separation effect was good. The results showed that different manufacturers' thin-layer plates had no significant effect on the thin-layer identification of litchi kernel medicinal materials, indicating that the thin-layer identification method has good durability for thin-layer plates from different manufacturers.

[0110] According to the above research results, the thin layer identification method of litchi seed medicinal material is determined as follows:

[0111] Take 1g of the powder of this product, add 20mL of methanol, ultrasonically treat for 20 minutes, filter, concentrate the filtrate to dryness, add 20mL of water to dissolve the residue, add ethyl acetate and shake to extract twice, 20mL each time, combine the ethyl acetate solution, evaporate to dryness, add 1mL of methanol to dissolve the residue, and use it as the test solution. Take 1g of litchi kernel control medicinal material and prepare a control medicinal material solution in the same way. Take the protocatechuic acid reference substance and add methanol to make a solution containing 0.5mg per 1mL as the reference solution. According to the thin layer chromatography method (General Rules 0502 of the 2020 Edition of the Chinese Pharmacopoeia), take 10μL of each of the above three solutions and spot them on the same silica gel GF 254 Develop the plate with toluene-ethyl formate-glacial acetic acid (8:3:0.5) as the developing solvent. Remove the plate, allow to air dry, spray with 10% sulfuric acid in ethanol, and heat at 105°C until the spots are clearly colored. Examine under ultraviolet light (365 nm, 254 nm). The test sample chromatogram should show spots of the same color or fluorescent spots at corresponding positions in the control herb and reference sample chromatograms.

[0112] <Thin layer chromatography identification and verification of different batches of lychee seeds>

[0113] Take different batches (batch number: S1 ~ S15) of lychee seeds medicinal materials, according to the determined preparation method of the test solution, make the test solution, on the same silica gel GF 254 Spot samples on the thin layer plate, use toluene-ethyl formate-glacial acetic acid (8:3:0.5) as the developing agent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly colored, and examine under ultraviolet light (365nm, 254nm). The results are shown in the figure. Figure 7 .

[0114] The results showed that the chromatograms of the test sample solutions from different batches of litchi seed medicinal materials all displayed spots of the same color or fluorescent spots at corresponding positions in the chromatograms of the control medicinal material and the reference substance. The thin-layer chromatography identification method of the present invention can effectively develop and separate the more polar compounds in litchi seed medicinal materials. The spots after color development are abundant and clear, with good separation, and the identification results are easy to judge.

[0115] <Study on the Identification of Litchi and Longan Seeds>

[0116] Take 3 batches of litchi kernel and longan kernel medicinal materials (litchi kernel batch numbers are: S1, S2, S3; longan kernel batch numbers are: S16, S17, S18), and prepare the test solution according to the determined test solution preparation method. Take 10 μL of each of the above two solutions and spot them on the same silica gel GF 254 On the thin layer plate, use toluene-ethyl formate-glacial acetic acid (8:3:0.5) as the developing agent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly colored, and examine under ultraviolet light (365nm, 254nm). Figure 8 (Among them, 1-3 are litchi seeds, 4-6 are longan seeds, S is litchi seed control medicinal material, and T is protocatechuic acid reference substance).

[0117] The results showed that under ultraviolet light (254nm), the chromatogram of the litchi seed medicinal material test sample showed a spot of the same color at the corresponding position of the chromatogram of the protocatechuic acid reference substance, while the chromatogram of the longan seed medicinal material test sample did not show a spot of the same color at the corresponding position of the chromatogram of the protocatechuic acid reference substance; under ultraviolet light (365nm), the chromatogram of the litchi seed medicinal material test sample showed a fluorescent spot of the same color at the corresponding position of the chromatogram of the litchi seed medicinal material, while the chromatogram of the longan seed medicinal material test sample did not show a fluorescent spot of the same color at the corresponding position of the chromatogram of the litchi seed medicinal material, and lacked a blue spot at an Rf value of approximately 0.25-0.33; therefore, in this method, the protocatechuic acid reference substance under ultraviolet light (254nm) and the blue spot at an Rf value of 0.25-0.33 under ultraviolet light (365nm) can both be used as the distinguishing points for thin-layer identification of litchi seed medicinal material and longan seed medicinal material.

[0118] Comparative Example 1

[0119] This comparative example adopts the following method to carry out thin layer chromatography detection, and the specific process is as follows:

[0120] Materials and reagents: CAMAG TLC VISUALIZER, ME204E electronic analytical balance (Mettler-Toledo Instruments (Shanghai) Co., Ltd.), HH-4 digital constant temperature water bath (Guohua Changzhou Instrument Manufacturing Co., Ltd.), silica gel H thin layer plate (Qingdao Ocean Chemical).

[0121] Ethyl ether (Shanghai Lingfeng Chemical Reagent Co., Ltd.), ethyl acetate (Sinopharm Chemical Reagent Co., Ltd.), vanillin (Sinopharm Chemical Reagent Co., Ltd.), sulfuric acid (Shanghai Lingfeng Chemical Reagent Co., Ltd.), and ethanol (Sinopharm Chemical Reagent Co., Ltd.).

[0122] The reference medicinal material of litchi seed (batch number 310027-202111) was obtained from Chengdu Pusi Biotechnology Co., Ltd.

[0123] Litchi seed medicinal materials and longan seed (litchi seed batch numbers: S1, S2, S3; longan seed batch numbers S16, S17, S18) were all sourced from Jiangyin Tianjiang Pharmaceutical Co., Ltd.

[0124] Method: Take 1g of lychee kernel and longan kernel powder (lychee kernel batch number: S1, longan kernel batch number: S16), add 10mL of ether, reflux for 30 minutes, cool, filter, and concentrate the filtrate to 0.5mL as the test solution. According to the thin layer chromatography method (General Rule 0502), take 0.5μL, 1μL, 2μL, and 3μL of the above solutions, respectively, and spot them on the same silica gel H thin layer plate, use ether-ethyl acetate (8:1) as the developing solvent, develop, take out, dry, spray with 1% vanillin sulfuric acid solution, and heat at 105℃ until the spots are clearly colored. The results are shown in Figure 9 and Figure 10 .

[0125] Depend on Figure 9 and Figure 10 As can be seen, the tailing of the lychee and longan seed medicinal material test solutions remained severe at different sample volumes, and reducing the sample volume did not significantly improve the situation. Furthermore, the spot positions of the same sample varied with different sample volumes, with the larger the sample volume, the higher the tailing spots. The lychee and longan seed medicinal material test solutions exhibited clearer results at sample volumes of 0.5 to 3 μL, with 1 μL being the clearest. Therefore, 1 μL was selected as the sample volume for the comparative study of lychee seeds and counterfeit longan seeds.

[0126] Take 1g of lychee kernel and longan kernel powder, add 10mL of ether, reflux for 30 minutes, cool, filter, and concentrate the filtrate to 0.5mL as the test solution. Take 1g of lychee kernel control medicinal material and prepare the control medicinal solution in the same way. According to the thin layer chromatography method (General Rule 0502), take 1μL of each of the above three solutions and spot them on the same silica gel H thin layer plate, use ether-ethyl acetate (8:1) as the developing agent, develop, take out, dry, spray with 1% vanillin sulfuric acid solution, and heat at 105℃ until the spots are clearly colored. The results are shown in Figure 11 .

[0127] Depend on Figure 11 It can be seen that the three different batches of litchi kernel medicinal materials ( Figure 11 The chromatograms of the test samples were slightly different between batches 1 to 3 (S1, S2, and S3, respectively), and the corresponding spots with the litchi kernel control medicinal materials were not consistent; the three different batches of longan kernel medicinal materials ( Figure 11 The chromatograms of the samples (5-7, corresponding to batches S16, S17, and S18, respectively) also differ slightly. This method has high requirements for sample content, and the locations of the spots vary between batches of lychee or longan seeds, making it less suitable for quality evaluation and pathogen identification.

[0128] The above examples are merely intended to illustrate several embodiments of the present invention. Although the descriptions thereof are relatively specific and detailed, they are not to be construed as limiting the scope of the present invention. It should be understood that those skilled in the art may make various modifications and improvements without departing from the scope of the present invention, and all such modifications and improvements fall within the scope of protection of the present invention.

Claims

1. A thin layer chromatography method for detecting lychee seed medicinal material, characterized by: The steps include: 1) Preparation of test solution: Take the powder of the medicinal material to be tested and extract it with methanol, filter it, concentrate the filtrate to dryness, dissolve the residue in water, add ethyl acetate and shake to extract, evaporate to dryness, dissolve the residue in methanol to prepare the test solution; 2) Preparation of reference medicinal material solution and reference substance solution: Take litchi kernel as control medicinal material and prepare control medicinal material solution by the test solution preparation method described in 1); take protocatechuic acid reference substance and prepare reference substance solution; 3) Thin layer test steps: taking the control medicinal material solution, the reference substance solution and the test solution and spotting them on the same silica gel GF 254 The thin layer plate was developed with a developing agent consisting of toluene-ethyl formate-glacial acetic acid, with a volume ratio of toluene:ethyl formate:glacial acetic acid = 8:3:0.5; The thin layer detection step also includes the step of inspecting under ultraviolet light after unfolding, wherein the inspection includes taking out the unfolded thin layer plate, drying it, and spraying it with 5% to 15% sulfuric acid ethanol solution for color development; the wavelength of the ultraviolet light is 365nm or 254nm.

2. The thin layer chromatography method for detecting the lychee seed medicinal material according to claim 1, wherein: The protocatechuic acid concentration of the reference solution is 0.2-2 mg / mL.

3. The thin layer chromatography detection method for litchi seed medicinal material according to claim 1, characterized in that: In step 3), the spotting volume of the control medicinal material solution, the reference substance solution, and the test solution is 8 μL to 20 μL.

4. The thin layer chromatography method for detecting the medicinal material of litchi seed according to claim 1, characterized in that: The thin layer detection method also includes comparing the chromatogram of the test sample with the chromatogram of the reference medicinal material and the chromatogram of the reference substance to determine whether spots of the same color are displayed at corresponding positions. If spots of the same color are displayed, it is determined that the medicinal material to be tested is litchi seed medicinal material.

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