Method for simultaneous thin-layer identification of ginseng and glossy privet fruit in cordyceps sinensis capsule

Abstract

The invention discloses a method for simultaneous thin-layer identification of ginseng and glossy privet fruit in cordyceps sinensis capsules, and belongs to the field of health food quality control. Aiming at the defects of separate detection, tedious process and low efficiency in the prior art, the method comprises the following steps: preparing a test solution through water ultrasonic extraction, water saturated n-butyl alcohol extraction and washing with a 5-8% ammonia water solution, matching with a ginseng / glossy privet fruit reference medicinal material and a mixed reference solution, and adopting a trichloromethane-methanol-underwater layer solution as a developing solvent. Synchronous identification of two components is realized through double processes of iodine fumigation sunlight inspection and sulfuric acid ethanol spray ultraviolet 365nm inspection. The method is good in separation degree, clear in spots, excellent in specificity and durability, efficient in operation and low in reagent toxicity, the detection cost is greatly reduced, reliable support is provided for cordyceps sinensis capsule quality control, and a technical normal form is provided for synchronous thin-layer identification of multiple traditional Chinese medicinal materials.

Description

Technical Field

[0001] This invention relates to the field of quality control technology for health food, and more specifically, to a simultaneous thin-layer chromatography method for identifying ginseng and privet fruit in Cordyceps sinensis capsules. Background Technology

[0002] Cordyceps sinensis capsules are a health food product developed based on the formula in the Qing Dynasty book "Siheting Jifang". The formula contains ingredients such as Cordyceps sinensis, ginseng, deer antler, Dendrobium officinale, and Ligustrum lucidum. It has the effects of relieving physical fatigue and enhancing immunity. Ginseng and Ligustrum lucidum are the key components that exert the core pharmacological effects: ginsenosides (Rg1, Re, Rf, Rb1, etc.) in ginseng have the effects of regulating immunity and anti-fatigue, while ligustrazine in Ligustrum lucidum has the effects of immune regulation and anti-oxidation. The quality control of both is directly related to the safety and efficacy of the product.

[0003] Thin-layer chromatography (TLC) is a commonly used qualitative analysis technique in food and drug quality control. With its advantages of simple equipment, convenient operation, small sample volume, and fast analysis speed, it is widely used in the identification of components in traditional Chinese medicine. However, the thin-layer chromatography (TLC) identification technology for ginseng and privet fruit in Cordyceps sinensis capsules still has significant shortcomings: existing technologies (such as Chinese patent CN101036774A and "Quality Standard Improvement of Xianlu Oral Liquid and its Network Pharmacological Study on the Treatment of Oligospermia") all adopt the mode of "preparing test solutions separately, using different developing systems, and detecting twice". This is not only cumbersome and inefficient, but also has the problems of large amounts of organic reagents and high costs. More importantly, due to the differences in physicochemical properties between ginsenosides (moderately polar and requiring fluorescence development) and privetin (moderately polar and requiring iodine fuming development), existing technologies have formed a technical bias of "different components need to be adapted to different processes". A solution that can simultaneously meet the separation and identification needs of the two has not been explored, resulting in problems such as spot tailing, blurred color development, and insufficient specificity in some methods, which makes it difficult to meet the needs of efficient and accurate quality control of products. Summary of the Invention

[0004] In view of this, the present invention proposes a method for simultaneous thin-layer chromatography identification of ginseng and privet fruit in Cordyceps sinensis capsules with good separation, high speed and efficiency, and strong specificity. It aims to overcome the shortcomings of existing technologies, such as the need for separate detection, cumbersome process, and poor separation effect, and to provide reliable technical support for product quality control.

[0005] The technical solution of this invention is achieved as follows: This invention provides a method for simultaneous thin-layer chromatography identification of ginseng and privet fruit in Cordyceps sinensis capsules, including four core steps: preparation of test solution, preparation of control solution, thin-layer chromatography operation, and double inspection. Through a unified extraction-separation-identification system, the simultaneous qualitative analysis of the two components is achieved.

[0006] In some embodiments, the test solution is prepared by an integrated process of sequential ultrasonic water extraction, water-saturated n-butanol extraction, ammonia solution washing, and methanol dissolution, wherein the ultrasonic water extraction time is 40-60 min, and the weight-to-volume ratio of the contents of the Cordyceps sinensis capsule to water is (1-2) g: (30-60) mL.

[0007] Ultrasonic extraction utilizes the cavitation effect of ultrasound to rapidly disrupt the cell wall structure of the raw materials in the capsule contents, allowing target components such as ginsenosides and ligustrazine to fully dissolve. Water, as the extraction solvent, is suitable for the water solubility of both components, avoiding the problem of incomplete extraction of a single component. Specific extraction times and material-to-liquid ratios are defined because excessively short extraction times lead to insufficient dissolution, while excessively long times may introduce more impurities. A reasonable material-to-liquid ratio balances extraction efficiency and subsequent processing costs, ensuring that the concentration of the target component remains within a suitable detection range.

[0008] In some embodiments, the water-saturated n-butanol is used for extraction 2-4 times, and the volume of each extraction is (20-40) mL : (1-2) g of the contents of the Cordyceps sinensis capsule.

[0009] By utilizing the principle of "like dissolves like," water-saturated n-butanol, with a polarity between that of water and organic solvents, can selectively enrich ginsenosides and ligustrazines with isopolar to moderately polar polarity, while repelling water-soluble polysaccharides, inorganic salts, and other impurities. The design of multiple extractions is to ensure that the target components are fully transferred to the organic phase, avoiding component loss caused by a single extraction. The limited volume ratio of extract to sample is to ensure the stability and repeatability of the extraction process, avoiding waste due to excessive solvent usage or affecting the extraction effect due to insufficient solvent usage.

[0010] In some embodiments, the concentration of the ammonia solution is 5-8%, the number of washes is 2-4, and the volume used for each wash is (20-40) mL: (1-2) g of the contents of the Cordyceps sinensis capsule.

[0011] Residual acidic impurities (such as organic acids) in the sample extract will adsorb onto the silica gel G thin-layer plate (weakly acidic), causing the target component to tail in spots. However, a 5-8% ammonia solution can react with the acidic impurities to form water-soluble salts, which are then transferred to the aqueous layer for removal. At the same time, the mild alkalinity of ammonia will not damage the structural stability of ginsenosides and ligustrum lucidum glycosides, achieving the effect of "removing impurities without damaging the target components". Multiple washings ensure that impurities are fully removed, laying the foundation for good separation results in the future.

[0012] In some embodiments, the control solution includes ginseng control solution, privet fruit control solution, ginseng mixed control solution, and privetin reference solution. The concentrations of ginsenosides Rg1, Re, Rf, and Rb1 in the ginseng mixed control solution are all 0.8-1.8 mg / mL, and the concentration of privetin reference solution is 0.8-1.8 mg / mL. The preparation process of the control solution is completely consistent with that of the test solution.

[0013] Using the same preparation process as the test sample avoids inconsistencies in chromatographic behavior due to differences in pretreatment, ensuring the reliability of identification results. The limitation of the concentration of the reference material is to match the chromatographic response values ​​of the target components in the reference material and the test sample, facilitating the comparison of spot positions and colors, and avoiding misjudgment due to concentration differences.

[0014] In some embodiments, thin-layer chromatography is performed using silica gel G thin-layer plates, with the developing solvent being a lower layer solution of chloroform-methanol-water in a volume ratio of (12.5-13.5):(6.5-7.5):(1.5-2.5), and the spotting bands are 0.8 mm wide and spaced 0.5 cm apart.

[0015] Silica gel G thin-layer plates have stable adsorption properties and are commonly used stationary phases in thin-layer chromatography, providing a uniform adsorption-desorption environment for target components. The core innovation lies in the design of the developing solvent ratio: chloroform provides weakly polar elution, methanol adjusts to moderate polarity, and water supplements strong polarity. The precise ratio of these three components ensures that the ratio shift (Rf) of ginsenosides and ligustrazine is within the optimal separation range of 0.3-0.7. This avoids spot concentration due to insufficient elution of ligustrazine and spot diffusion due to over-elution of ginsenosides, achieving complete separation of the two components in a single development. The lower layer solution is chosen because the mixture will separate into layers at this ratio. The lower layer solution has more stable polarity, reducing the impact of temperature and humidity on the development effect and improving the durability of the method.

[0016] In some embodiments, the double inspection step is as follows: the thin-layer plate is fumigated with iodine and then inspected under sunlight, heated until the spots fade, then sprayed with 10% sulfuric acid ethanol solution, heated at 105°C and then inspected under ultraviolet light at 365nm.

[0017] This process is designed based on the complementary colorimetric properties of the two components: privetin has no fluorescent properties and cannot be directly colored under ultraviolet light, but iodine fuming can cause it to show clear spots under sunlight through charge transfer reaction; ginsenosides show weak color under iodine fuming, but after being sprayed and heated with 10% sulfuric acid ethanol solution, they undergo dehydration and oxidation reactions to generate fluorescent derivatives, which can show characteristic fluorescence under ultraviolet light at 365nm; the heating fading step can completely eliminate the interference of iodine fuming on the fluorescence detection of ginsenosides, enabling the sequential identification of the two components on the same thin-layer plate, avoiding the problem of asynchronous detection caused by conflicting colorimetric methods.

[0018] In some embodiments, the sample volume of the test solution is 10 μL, and the sample volume of the ginseng reference material solution, the ginseng mixed reference solution, the privet fruit reference material solution, and the privetin reference solution is 3 μL.

[0019] Excessive spotting volume can cause spots to spread and overlap, affecting resolution; insufficient spotting volume will result in spots that are too light in color and difficult to identify. The concentration of the target component in the test solution is relatively lower than that in the control solution, so a larger spotting volume is set to ensure clear spots; the concentration of the control solution is appropriate, so a smaller spotting volume is sufficient for comparison, while avoiding excessively large spots that could interfere with the chromatography of the test sample.

[0020] In some implementations, the ultrasonic extraction power is 500W and the frequency is 40KHz.

[0021] The combination of 500W power and 40KHz frequency can generate a sufficiently strong ultrasonic cavitation effect, effectively destroying the cell walls of the raw materials and accelerating the dissolution of the target components. At the same time, it avoids the impact of excessive power on local temperature rise or improper frequency on extraction efficiency, ensuring the stability and repeatability of the extraction process.

[0022] The present invention has the following advantages over the prior art:

[0023] Compared to the cumbersome process of existing technologies requiring two sample preparations and two development and detection steps, this invention optimizes the entire process—synergistic extraction, targeted impurity removal, unified development, and dual inspection—to achieve simultaneous identification of ginseng and privet fruit components in a single operation, significantly simplifying the process and improving detection efficiency. Targeted impurity removal with ammonia solution and precisely proportioned developing solvent effectively solves the problems of spot tailing and poor separation in existing technologies, resulting in clear, interference-free spots of the target components and stronger specificity. The method avoids the use of high-risk reagents such as ether, employing only low-toxicity reagents such as n-butanol and methanol, and significantly reducing reagent usage, thus lowering detection costs and improving operational safety. Furthermore, the method is unaffected by temperature, humidity, and different manufacturers' thin-layer chromatography plates, exhibiting excellent durability and adaptability to various detection environments. This approach overcomes the technical bias of requiring separate detection of components with different physicochemical properties, providing a universal technical paradigm for the simultaneous identification of multiple components in compound preparations containing saponins and iridoid glycosides, expanding the application scenarios of thin-layer chromatography in multi-component quality control, and possessing significant industry promotion value. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 The thin-layer chromatography identification results of the test solution preparation method in Example 1 of the present invention are optimized; the left figure shows the identification results under visible light after iodine fuming, and the right figure shows the identification results under ultraviolet light at 365 nm.

[0026] Figure 2 The images show the results of thin-layer chromatography identification for the specificity investigation of Example 3 of the present invention; the left image shows the identification results under visible light after iodine fuming, and the right image shows the identification results under ultraviolet light at 365 nm.

[0027] Figure 3 The results of thin-layer chromatography identification for the sample quantity robustness test in Example 4 of the present invention are shown; the left figure shows the identification results under visible light after iodine fuming, and the right figure shows the identification results under ultraviolet light at 365 nm.

[0028] Figure 4 The thin-layer chromatography identification results are for the temperature durability test of Example 4 of the present invention; the left figure is the identification result under visible light after iodine fuming, and the right figure is the identification result under ultraviolet light at 365nm.

[0029] Figure 5The results of thin-layer chromatography identification for the high humidity durability test in Example 4 of the present invention are shown; the left figure shows the identification results under visible light after iodine fuming, and the right figure shows the identification results under ultraviolet light at 365 nm.

[0030] Figure 6 The following are the thin-layer chromatography identification results for the low-humidity durability test of Example 4 of the present invention; the left figure shows the identification results under visible light after iodine fuming, and the right figure shows the identification results under ultraviolet light at 365nm.

[0031] Figure 7 The following are the thin-layer chromatography identification results for the durability test of thin-layer plates of different brands in Example 4 of the present invention; wherein Figure A shows the identification results of the thin-layer plate produced by Qingdao Marine Chemical Plant Branch (left figure under visible light after iodine fuming, right figure under ultraviolet 365nm), and Figure B shows the identification results of the thin-layer plate produced by Yantai Chemical Industry Research Institute (left figure under visible light after iodine fuming, right figure under ultraviolet 365nm).

[0032] Figure 8 The results of thin-layer chromatography identification of ginseng are shown in Comparative Example 1 of this invention.

[0033] Figure 9 The results of thin-layer chromatography identification of Ligustrum lucidum in Comparative Example 2 of this invention are shown. Detailed Implementation

[0034] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0035] Example 1: Optimization of the preparation method of the test solution 1. Preparation steps Step 1: Preparation of the test solution. Take three portions of the contents of Cordyceps sinensis capsules, each 1g, and process them as follows: Test sample ①: Add 30 mL of water, sonicate (power 500 W, frequency 40 kHz) for 60 minutes, centrifuge at 8000 r / min for 10 min and take the supernatant; extract twice with 20 mL of water-saturated n-butanol each time, combine the n-butanol layers, evaporate to dryness and dissolve in 1 mL of methanol to obtain the test sample ①.

[0036] Test sample ②: Add 30 mL of water, sonicate (power 500 W, frequency 40 kHz) for 60 minutes, centrifuge at 8000 r / min for 10 min and take the supernatant; extract twice with ethyl acetate, 20 mL each time, combine the ethyl acetate layers, evaporate to dryness and dissolve in 1 mL of methanol to obtain the test sample.

[0037] Test sample ③: Add 30 mL of water, sonicate (500 W power, 40 kHz frequency) for 60 minutes, centrifuge at 8000 r / min for 10 minutes and collect the supernatant; extract twice with 20 mL of water-saturated n-butanol each time, and combine the n-butanol layers; wash twice with 20 mL of 5% ammonia solution each time, discard the aqueous layer, evaporate the n-butanol layer to dryness and dissolve it in 1 mL of methanol to obtain the test sample.

[0038] Step 2: Preparation of control solutions. Take ginsenoside reference standard and dissolve it in methanol to prepare a ginsenoside reference standard solution containing 1 mg per 1 mL; take ginsenoside Rb1, Re, Rg1, and Rf reference standards and dissolve them in methanol to prepare a mixed ginseng reference standard solution containing 1 mg of each per 1 mL. Take 0.5 g each of ginseng and privet fruit reference materials, add 30 mL of water to each, sonicate (500 W, 40 kHz) for 60 minutes, centrifuge at 8000 r / min for 10 minutes, and collect the supernatant; extract twice with 20 mL of water-saturated n-butanol each time, combine the n-butanol layers; wash twice with 20 mL of 5% ammonia solution each time, discard the aqueous layer, evaporate the n-butanol layer to dryness, and dissolve it in 1 mL of methanol to obtain ginseng reference material solution and privet fruit reference material solution, respectively.

[0039] 2. Performance Verification Step 1: Thin-layer chromatography. Take 3 μL each of the following: privetin reference solution, privet fruit reference material solution, 10 μL each of test solutions ①-③, 3 μL of ginseng reference material solution, and 3 μL of mixed ginseng reference solution, and spot them onto the same silica gel G thin-layer plate (spot width 0.8 mm, spacing 0.5 cm). Use a chloroform-methanol-water mixture (volume ratio 13:7:2) as the developing solvent (after mixing, allow to stand for 30 min and then collect the lower layer). After development, remove the plate and air dry.

[0040] Step 2: Double inspection. Place the thin-layer plate in iodine vapor for 2 minutes until the spots are clear, and inspect it under sunlight; after heating to fade the spots, spray it with 10% sulfuric acid ethanol solution, heat it at 105℃ until the spots are clear, and inspect it under ultraviolet light at 365nm.

[0041] Step 3: Evaluation of indicators. Record the resolution (the degree of separation between the target component and adjacent impurities), spot clarity (no tailing / slight tailing / severe tailing) and identification results (whether ginseng / privet fruit components were identified) for each test sample.

[0042] 3. Results Statistics:

[0043] Example 2: Establishment of the identification method 1. Preparation steps Step 1: Preparation of the test solution. Take 1g of the contents of Cordyceps sinensis capsules, add 30mL of water, sonicate (500W power, 40KHz frequency) for 60 minutes, centrifuge at 8000r / min for 10 minutes and collect the supernatant; extract twice with 20mL of water-saturated n-butanol each time, combine the n-butanol layers; wash twice with 20mL of 5% ammonia solution each time, discard the aqueous layer, evaporate the n-butanol layer to dryness and dissolve it in 1mL of methanol to obtain the test solution.

[0044] Step 2: Preparation of the control solution. Same as Step 2 in Example 1.

[0045] 2. Performance Verification Step 1: Thin-layer chromatography. Take 3 μL of privetin reference solution, 3 μL of privet fruit reference material solution, 10 μL of the test solution, 3 μL of ginseng reference material solution, and 3 μL of mixed ginseng reference solution, and spot them onto the same silica gel G thin-layer plate (spot width 0.8 mm, spacing 0.5 cm). Use a chloroform-methanol-water solution (v / v) of 13:7:2 as the developing solvent. After development, remove the plate and allow it to air dry.

[0046] Step 2: Double inspection. Same as Step 2 in Example 1.

[0047] Step 3: Evaluation of indicators. Record the separation, spot clarity, identification accuracy, and operation time.

[0048] 3. Results Statistics:

[0049] Example 3: Specificity Investigation 1. Preparation steps Step 1: Preparation of negative control solutions. Take 1g each of the Cordyceps sinensis capsule negative control standard lacking ginseng and the negative control standard lacking privet fruit, and follow the test solution preparation steps in Example 2 to obtain the negative control solutions lacking ginseng and privet fruit respectively.

[0050] Step 2: Preparation of the test solution and control solution. Same as steps 1 and 2 in Example 2.

[0051] 2. Performance Verification Step 1: Thin-layer chromatography. Take 10 μL of the negative control solution lacking privet fruit, 3 μL of the privetin reference solution, 3 μL of the privet fruit reference material solution, 10 μL of the test solution, 3 μL of the ginseng reference material solution, 3 μL of the mixed ginseng reference solution, and 10 μL of the negative control solution lacking ginseng, and spot them onto the same silica gel G thin-layer plate (spotting band width 0.8 mm, spacing 0.5 cm); develop and dry the plate according to the chromatographic conditions of Example 2.

[0052] Step 2: Double inspection. Same as Step 2 in Example 1.

[0053] Step 3: Indicator Evaluation. Observe whether the negative control solution shows spots corresponding to the target component (to determine if there is interference), and whether the test sample accurately identifies the two components.

[0054] 3. Results Statistics:

[0055] Example 4: Comprehensive Durability Assessment 1. Preparation steps Step 1: Preparation of test solution, control solution and negative control solution. Same as the preparation steps in Examples 2 and 3.

[0056] 2. Performance Verification Different spotting volumes were investigated. 5 μL, 10 μL, and 15 μL of the test solution were respectively pipetted, and the remaining control solution and negative control solution were pipetted according to the predetermined volumes. The chromatographic conditions and inspection methods of Example 2 were followed to evaluate the clarity and resolution of the spots.

[0057] Stability was evaluated at different temperatures. The chromatographic conditions and inspection methods described in Example 2 were followed, and the stability was assessed at both room temperature (23.4°C, RH 64.4%) and low temperature (5.0°C, RH 57.2%).

[0058] Stability was evaluated under different humidity conditions, including high humidity (24.8°C, RH 88.3%) and low humidity (25.6°C, RH 35.6%), following the chromatographic conditions and inspection methods described in Example 2.

[0059] Thin-layer plates from different manufacturers were tested. Qingdao G plates and Yantai G plates were used respectively, and the chromatographic conditions and inspection methods of Example 2 were followed to evaluate suitability.

[0060] 3. Results Statistics:

[0061] Example 5: Verification of the Synergistic Effect of Innovation Points 1. Preparation steps Step 1: Preparation of the test solution. Take 3 portions of the contents of Cordyceps sinensis capsules, each 1g, and process them as follows: Group 1 (lacking ammonia washing): same preparation steps as test sample ① in Example 1.

[0062] Group 2 (deviation of developing solvent ratio): Same as the sample preparation steps in Example 2, except that the developing solvent is adjusted to chloroform-methanol-water = 10:5:1 (lower layer).

[0063] Group 3 (Synergistic Innovation): Same test sample preparation steps as in Example 2.

[0064] Step 2: Preparation of the control solution. Same as Step 2 in Example 1.

[0065] 2. Performance Verification Step 1: Thin-layer chromatography operation. Follow the same developing solvent conditions as in Step 1 of Example 2.

[0066] Step 2: Double inspection. Same as Step 2 of Example 1 (Group 2 uses the same inspection method).

[0067] Step 3: Evaluation of indicators. Record the separation degree, spot state, and simultaneous identification effect.

[0068] 3. Results Statistics:

[0069] Example 6: An Attempt at Combining Existing Technologies 1. Preparation steps Step 1: Preparation of the test solution (simulating existing technology combination). Take 1g of the contents of Cordyceps sinensis capsules and process it according to the ginseng extraction process of Chinese patent CN101036774A (extract by shaking with 30mL of ethyl acetate, discard the ethyl acetate; extract the aqueous layer twice with 30mL of water-saturated n-butanol each time, combine the n-butanol solutions; wash with 40mL of 0.1mol / L sodium hydroxide solution, then wash twice with water, evaporate to dryness and dissolve in 1mL of methanol). At the same time, process it according to the privet fruit extraction process studied by Li Jingfei (sonicate with 25mL of dilute ethanol for 30 minutes, filter). Mix the two extracts to prepare the combined test solution.

[0070] Step 2: Preparation of the control solution. Same as Step 2 in Example 1.

[0071] Step 3: Developing solvent and inspection method. Two existing developing solvents were mixed (chloroform-ethyl acetate-methanol-water = 8:4:5:2), and the inspection method was dual-wavelength inspection at 254nm + 365nm.

[0072] 2. Performance Verification Step 1: Thin-layer chromatography operation. Take 10 μL of the combined test solution and the predetermined volume of each control solution, and spot them on the same silica gel G thin-layer plate (spotting band width 0.8 mm, spacing 0.5 cm). Develop with mixed developing solvent and then air dry.

[0073] Step 2: Dual-wavelength inspection. Observe the spot state under ultraviolet light at 254nm and 365nm respectively.

[0074] Step 3: Evaluation of indicators. Record the separation degree, spot overlap, and identification effect.

[0075] 3. Results Statistics:

[0076] Comparative Example 1: Existing technology for single ginseng identification 1. Preparation steps Step 1: Preparation of the test solution. Take 1g of the contents of Cordyceps sinensis capsules, add 30mL of ethyl acetate, shake to extract, and discard the ethyl acetate; extract the aqueous layer twice with 30mL of water-saturated n-butanol each time, and combine the n-butanol solutions; wash with 40mL of 0.1mol / L sodium hydroxide solution, discard the washings, wash with 30mL of water saturated with n-butanol, and then wash with 30mL of water; evaporate the n-butanol solution to dryness in a water bath, dissolve the residue in 1mL of methanol, and obtain the test solution.

[0077] Step 2: Preparation of control solution. Take 1g of ginseng reference material, add 80mL of ether and reflux for 1 hour, then filter; after the ether has evaporated from the residue, add 80mL of water-saturated n-butanol and sonicate for 30 minutes, then filter; wash with 40mL of 0.1mol / L sodium hydroxide solution, discard the washings, and then wash with 40mL of water; evaporate the n-butanol layer to dryness, dissolve the residue in 1mL of methanol to obtain the ginseng reference material solution. Take ginsenosides Rb1, Re, and Rg1 reference standards, add methanol to prepare a mixed solution containing 2.0mg of each per 1mL, to obtain the ginseng reference standard solution.

[0078] Step 3: Chromatographic operation. Take 10 μL of the test solution and 5 μL each of the reference solution / medicinal material solution and spot them on the same silica gel G thin-layer plate; use chloroform-ethyl acetate-methanol-glacial acetic acid-water = 10:4:5:2:2 (lower layer below 10℃) as the developing solvent, develop and air dry; spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clear, and examine under sunlight.

[0079] 2. Performance Verification Step 1: Follow the above preparation steps and chromatographic conditions to observe the state of ginsenoside spots and their identification effect.

[0080] Step 2: Evaluation indicators include resolution, types of identified components, operation time, and reagent toxicity.

[0081] 3. Results Statistics:

[0082] Comparative Example 2: Existing technology for single identification of Ligustrum lucidum 1. Preparation steps Step 1: Preparation of the test solution. Take 1g of the contents of a Cordyceps sinensis capsule, add 25mL of dilute ethanol, sonicate for 30 minutes, and filter to obtain the test solution.

[0083] Step 2: Preparation of control solution. Take 0.5g of privet fruit reference material, add 50mL of dilute ethanol, sonicate for 30 minutes, filter, and obtain privet fruit reference material solution. Take privetin reference standard, add methanol to prepare a solution containing 0.25mg per 1mL, and obtain privetin reference standard solution.

[0084] Step 3: Chromatographic operation. Take 15 μL of the test solution, 5 μL of the reference herb solution, and 10 μL of the reference solution and spot them on the same silica gel GF254 plate; use chloroform-methanol-ethanol-water = 10:3:0.1:1 (lower layer) as the developing solvent, develop and air dry; examine under UV light at 254 nm.

[0085] 2. Performance Verification Step 1: Follow the above preparation steps and chromatographic conditions to observe the state of the ligustrazine spots and its recognition effect.

[0086] Step 2: Evaluation indicators include separation degree, identification of component types, and spot clarity.

[0087] 3. Results Statistics:

[0088] Comparative Example 3: Comparative Example Lacking Ammonia Washing 1. Preparation steps Step 1: Preparation of the test solution. Same as the preparation steps for test sample ① in Example 1 (washing with ammonia-free water).

[0089] Step 2: Preparation of the control solution. Same as Step 2 in Example 1.

[0090] Step 3: Chromatographic operation and inspection. Same conditions as in Example 2.

[0091] 2. Performance Verification The performance verification steps are the same as in Example 1.

[0092] 3. Results Statistics:

[0093] Comparative Example 4: Comparative Example with Replaced Extractant 1. Preparation steps Step 1: Preparation of the test solution. Same as the preparation steps for test sample ② in Example 1 (ethyl acetate replaces water-saturated n-butanol).

[0094] Step 2: Preparation of the control solution. Same as Step 2 in Example 1.

[0095] Step 3: Chromatographic operation and inspection. Same conditions as in Example 2.

[0096] 2. Performance Verification The performance verification steps are the same as in Example 1.

[0097] 3. Results Statistics:

[0098] Comparative Example 5: Comparative Example of Existing Technology Combinations 1. Preparation steps Step 1: Preparation of test solution. Prepare the test solution according to the ginseng extraction process in CN101036774A and the developing system of Li Jingfei (chloroform-methanol-ethanol-water = 10:3:0.1:1).

[0099] Step 2: Preparation of the control solution. Same as Step 2 in Example 1.

[0100] Step 3: Inspection method. Dual inspection using sunlight + 254nm ultraviolet light.

[0101] 2. Performance Verification Steps The performance verification steps are the same as in Example 2 (the developing agent is replaced with Li Jingfei's system).

[0102] 3. Results Statistics:

[0103] The key performance indicators of the examples and comparative examples are summarized in the table below:

[0104] In summary, this invention systematically verifies the scientific validity and practicality of the method for simultaneous thin-layer chromatography (TLC) identification of ginseng and privet fruit in Cordyceps sinensis capsules through a series of examples. Example 1 clarifies that the test sample solution requires a synergistic process of ultrasonic extraction with water, extraction with water-saturated n-butanol, and washing with 5% ammonia solution. The omission of any step leads to problems such as spot tailing and incomplete extraction, confirming that this preparation process is crucial for the effective enrichment and purification of the two components. Example 2 establishes a complete identification method that achieves simultaneous identification of two core components in a single operation, demonstrating simplicity and efficiency. Example 3, through a negative control experiment, verifies the method's good specificity and absence of matrix interference. Example 4, considering factors such as sample volume, temperature and humidity, and the type of TLC plate used, confirms that this method maintains clear spots and excellent separation under different detection conditions, exhibiting outstanding durability. Compared to comparative examples, existing technologies require separate preparation of test samples and two separate detections using different developing systems, resulting in cumbersome and inefficient processes. Some methods also use highly toxic reagents such as ether, posing safety hazards. Furthermore, the simple combinations of existing technologies cannot solve the problem of simultaneous separation and identification of the two components. This invention overcomes the technical bias of "different polarity components need to be detected separately" through synergistic innovation in extraction process, development system and dual inspection mode. It has the advantages of good separation, strong specificity, high efficiency, safety and low toxicity and excellent durability. It provides reliable technical support for the quality control of Cordyceps sinensis capsules and provides an effective paradigm for the simultaneous thin-layer identification of multiple Chinese medicinal materials.

[0105] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for simultaneous thin-layer chromatography identification of ginseng and privet fruit in Cordyceps sinensis capsules, characterized in that, Includes the following steps: (1) Preparation of test solution: Take the contents of Cordyceps sinensis capsules, extract with water, extract with water-saturated n-butanol, wash the obtained n-butanol layer with ammonia solution, evaporate to dryness and dissolve in methanol to obtain test solution; (2) Preparation of control solutions: Take ginseng and privet fruit control materials respectively, and prepare ginseng control material solution and privet fruit control material solution according to the method in step (1); take ginsenoside Rg1, Re, Rf, Rb1 control standards and privet glycoside control standard respectively, and add methanol to prepare ginseng mixed control standard solution and privet glycoside control standard solution. (3) Thin-layer chromatography operation: Take the test solution, ginseng reference material solution, ginseng mixed reference solution, privet fruit reference material solution, and privetin reference solution, and spot them on the same silica gel G thin-layer plate. Develop the plate with the lower layer of chloroform-methanol-water as the developing solvent, and then air dry. (4) Double inspection: After fuming with iodine, the thin-layer plate is inspected under sunlight, heated until the spots fade, then sprayed with sulfuric acid ethanol solution, heated and inspected under ultraviolet light at 365nm to achieve simultaneous identification of ginseng and privet fruit.

2. The method according to claim 1, characterized in that, The water extraction in step (1) is ultrasonic extraction, with an extraction time of 40-60 min. The weight-to-volume ratio of the contents of the Cordyceps sinensis capsule to water is (1-2) g: (30-60) mL.

3. The method according to claim 2, characterized in that, The ultrasonic extraction power is 500W and the frequency is 40KHz.

4. The method according to claim 1, characterized in that, The water-saturated n-butanol extraction in step (1) is performed 2-4 times, and the volume of water-saturated n-butanol used in each extraction is (20-40) mL: (1-2) g of the contents of the Cordyceps sinensis capsule.

5. The method according to claim 1, characterized in that, The concentration of the ammonia solution in step (1) is 5-8%, the number of washings is 2-4, and the volume of the ammonia solution used in each washing is (20-40) mL: (1-2) g of the contents of the Cordyceps sinensis capsule.

6. The method according to claim 1, characterized in that, In step (2), the concentrations of ginsenosides Rg1, Re, Rf, and Rb1 in the ginseng mixed reference solution are all 0.8-1.8 mg / mL; the concentration of the ligustrazine reference solution is 0.8-1.8 mg / mL.

7. The method according to claim 1, characterized in that, The volume ratio of the developing agent in step (3) is chloroform:methanol:water = (12.5-13.5):(6.5-7.5):(1.5-2.5).

8. The method according to claim 1, characterized in that, The concentration of the sulfuric acid ethanol solution in step (4) is 10%, and the heating temperature after spraying is 105°C until the spots are clear.

9. The method according to claim 1, characterized in that, In step (2), the weight-to-volume ratio of ginseng reference material, privet fruit reference material and water is (0.5-1) g: 30 mL, and the ultrasonic extraction time is 40-60 min.

10. The method according to claim 1, characterized in that, The sample volume of the test solution in step (3) is 10 μL, and the sample volumes of the ginseng reference material solution, the ginseng mixed reference solution, the privet fruit reference material solution, and the privetin reference solution are all 3 μL.

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