Thin layer detection method of Radix Rehmanniae medicinal materials or formula granules and its application in identification of counterfeit products

Abstract

The present invention relates to a thin-layer detection method for Radix Rehmanniae medicinal materials or formula granules and its application in counterfeit identification. The thin-layer detection method comprises the following steps: extracting Radix Rehmanniae medicinal materials or formula granules with methanol, evaporating the extract to dryness, dissolving the evaporated solid in water, extracting with n-butanol, evaporating the obtained n-butanol phase to dryness to obtain a solid, adding methanol to dissolve, and preparing a test solution; extracting Radix Rehmanniae control medicinal materials with methanol, evaporating the extract to dryness, dissolving the evaporated solid in water, extracting with n-butanol, evaporating the obtained n-butanol phase to dryness to obtain a solid, adding methanol to dissolve, and preparing a control medicinal material solution; taking a Radix Rehmanniae ethyl reference material, adding a solvent to dissolve, and preparing a reference material solution; spotting the test solution, the control medicinal material solution, and the reference material solution on the same thin-layer plate, developing with a mixture of n-butanol, formic acid, and water as a developing agent, drying, developing with a color developer, and inspecting. The thin-layer detection method is simple, efficient, and highly specific.

Description

Technical Field

[0001] The present application relates to the technical field of traditional Chinese medicine detection, and in particular to a thin layer detection method for Radix Rehmanniae Radix or its formula granules and its application in the identification of counterfeit products. Background Art

[0002] Rehmannia root bark is the dried root bark of Lycium chinense Mill. or Lycium barbarum L., both of the Solanaceae family. It is sweet and cold in nature. It enters the lung, liver, and kidney meridians, cooling the blood, eliminating steam, clearing the lungs, and reducing internal heat. It is primarily used for treating yin deficiency-induced hot flashes, night sweats caused by bone steaming, coughs due to lung heat, hemoptysis, epistaxis, and internal heat-induced thirst. It is a commonly used Chinese medicinal herb in clinical practice. Rehmannia root bark contains a variety of chemical components, primarily alkaloids, organic acids and their phenols, peptides, anthraquinones, lignans, sterols, flavonoids, and others. Alkaloids are the primary component, including phenolamides, cyclopentapyrrolidine-type alkaloids, tropane-type alkaloids, and other types. Phenolic amides include tropane ethyl, tropane methyl, dihydrocaffeoylamide, and caffeoylamide. Tropanes include atropine and scopolamine. The bark of Rehmannia glutinosa also contains a variety of alkaloids, including betaine, choline, etc., among which the highest content is lycoside, which is the characteristic component of Rehmannia glutinosa.

[0003] The 2020 edition of the Chinese Pharmacopoeia does not specify the content determination indicators of Radix Rehmanniae under the medicinal materials item. It is only identified with reference to the control medicinal materials in the thin layer identification item. There is no clear component for its specific identification. Moreover, the thin layer chromatogram presented by the pharmacopoeia method has a main blue spot, which has been confirmed to be scopoletin. Since scopoletin is present in many medicinal materials and its content in Radix Rehmanniae is not high, the rationality of choosing it as an identification reference is still worth discussing. However, the existing research and literature materials almost all refer to the pharmacopoeia method, using scopoletin or using Radix Rehmanniae control medicinal materials, and observing under 365nm fluorescence to perform specific identification of Radix Rehmanniae and related preparations. Since a large number of studies have shown that scopoletin is the main component of Radix Rehmanniae and is specific, the Radix Rehmanniae formula granule standard also uses it as a quantitative indicator to establish a content determination method. Furthermore, lycopodiol has the pharmacological effects of lowering blood pressure, lowering blood sugar, reducing fever, and combating stress-induced ulcers, consistent with the "cooling blood and removing steaming" effect of Lycium bark. However, there are currently no reports or standards for the use of lycopodiol to identify Lycium bark and its related preparations.

[0004] Compared to content determination and characteristic chromatographic identification, thin-layer chromatography (TLC) is one of the most widely used methods within planar chromatography. It is easy to master, employs inexpensive equipment, and offers simple and flexible operation. It boasts the following advantages: the ability to readily change developing agents and development directions, as well as derivatization, color development, observation, and detection, rapid method development, high sensitivity and resolution, the ability to simultaneously separate multiple samples, and simplified sample pretreatment. Despite the rapid development of other chromatographic techniques in recent years, the use of TLC has not significantly decreased. On the contrary, with the introduction of new stationary phases and rapidly evolving instrumentation, its application has become increasingly widespread, establishing it as a modern, sensitive, and efficient separation and analysis method.

[0005] Since the currently available rapid identification methods for Radix Rehmanniae and its preparations lack certain specificity, it is urgent to provide an exclusive identification method for Radix Rehmanniae and its formula particles that can be quickly and effectively provided. Summary of the Invention

[0006] Based on this, the present invention provides a thin layer detection method of the Chinese herbal medicine Radix Rehmanniae with simple operation, high efficiency and strong specificity, and its application in the identification of Chinese herbal medicine formula granules.

[0007] The first aspect of the present invention provides a thin layer detection method for the medicinal material of Lycium bark, comprising the following steps:

[0008] The Chinese medicinal material or the formulated granules of Radix Lycii were extracted with methanol, the obtained extract was evaporated to dryness, the evaporated solid was dissolved in water, extracted with n-butanol, the obtained n-butanol phase was evaporated to dryness, and the evaporated solid was dissolved in methanol to prepare the test solution;

[0009] The reference medicinal material of Radix Lycii was extracted with methanol, the obtained extract was evaporated to dryness, the evaporated solid was dissolved in water, extracted with n-butanol, the obtained n-butanol phase was evaporated to dryness, and the evaporated solid was dissolved in methanol to prepare a reference medicinal material solution;

[0010] Take the lycopodine standard substance, add solvent to dissolve it, and prepare the standard solution;

[0011] The test solution, control medicinal material solution and reference substance solution are spotted on the same thin layer plate, developed with a mixture of n-butanol, formic acid and water as a developing agent, dried, developed with a color developer, and inspected.

[0012] In one embodiment, the volume ratio of n-butanol, formic acid and water in the developing agent is (2-4):(0.5-2):1; further, the volume ratio of n-butanol, formic acid and water in the developing agent is (2-4):(1-2):1.

[0013] In one embodiment, the unfolding temperature is below 10°C.

[0014] In one embodiment, the pre-saturation time of the deployment cylinder is 12 minutes to 20 minutes, and the deployment distance is 7 cm to 10 cm.

[0015] In one embodiment, the developer is iodine vapor.

[0016] In one embodiment, the inspection method is observation under fluorescent light.

[0017] In one embodiment, the extraction method is ultrasonic extraction.

[0018] In one embodiment, the power of ultrasonic extraction is 250W to 350W, the frequency is 35kHz to 45kHz, and the time is 20min to 40min.

[0019] In one embodiment, the spotting volumes of the test solution, the control medicinal material solution, and the reference solution are each independently 8 μl to 12 μl.

[0020] The second aspect of the present invention provides the application of the thin layer detection method of the Chinese wolfberry bark medicinal material or the formula granules in the identification of the Chinese wolfberry bark medicinal material and its counterfeit products.

[0021] In one embodiment, the identification method includes: comparing the chromatogram of the test solution to determine whether the same spot exists at a corresponding position in the chromatogram of the control medicinal material solution and / or the chromatogram of the reference substance solution.

[0022] The thin layer detection method of the Chinese wolfberry bark medicinal material or formula granules provided by the present invention has the following beneficial effects:

[0023] The present invention, based on thin-layer chromatography, can detect the Chinese herb Radix Lycoris Root and the Chinese herb Radix Lycoris Root formula granules that have lost their properties by adopting a suitable test sample preparation method and a developing agent, and can achieve separation and detection of lycoside ethyl in the Chinese herb Radix Lycoris Root and the Chinese herb Radix Lycoris Root formula granules, filling the gap in thin-layer identification of lycoside ethyl in the Chinese herb Radix Lycoris Root and the Chinese herb Radix Lycoris Root formula granules, and has strong specificity, providing a reference for the establishment of quality standards for the Chinese herb Radix Lycoris Root and the Chinese herb Radix Lycoris Root formula granules. At the same time, compared with methods such as high-performance liquid chromatography, the present invention is simpler to operate, faster to detect, and low in cost.

[0024] In addition, the thin layer detection method of the above-mentioned Radix Rehmanniae medicinal materials and formula granules can be used to identify Radix Rehmanniae medicinal materials and their counterfeits, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The thin layer chromatograms of different sample amounts of Radix Rehmanniae (T: 25°C, RH: 50%; Merck silica gel G plate);

[0026] Among them, 1. Radix Lycoris Root (DGP079) 5μl; 2. Radix Lycoris Root (DGP079) 10μl; 3. Radix Lycoris Root (DGP079) 15μl; 4. Radix Lycoris Root Control 5μl; 5. Radix Lycoris Root Control 10μl; 6. Radix Lycoris Root Control 15μl; 7. Lycopodiol 2μl; 8. Lycopodiol 5μl; 9. Lycopodiol 10μl;

[0027] Figure 2 This is the TLC identification chromatogram of Radix Rehmanniae at room temperature (T: 25°C, RH: 50%; Merck silica gel G plate)

[0028] Among them, 1. Radix Lycoris Root (DGP079) 10 μl; 2. Radix Lycoris Root (DGP080) 10 μl; 3. Radix Lycoris Root (DGP081) 10 μl; 4. Radix Lycoris Root control 10 μl; 5. Radix Lycoside 10 μl;

[0029] Figure 3 This is the thin layer identification chromatogram of Radix Rehmanniae under low temperature conditions (T: 7°C, RH: 68%; Merck silica gel G plate)

[0030] Among them, 1. Radix Lycoris Root (DGP079) 10 μl; 2. Radix Lycoris Root (DGP080) 10 μl; 3. Radix Lycoris Root (DGP081) 10 μl; 4. Radix Lycoris Root control 10 μl; 5. Radix Lycoside 10 μl;

[0031] Figure 4 This is the TLC chromatogram of Radix Rehmanniae under high humidity conditions (T: 25°C, RH: 85%; Merck silica gel G plate)

[0032] Among them, 1. Radix Lycoris Root (DGP079) 10 μl; 2. Radix Lycoris Root (DGP080) 10 μl; 3. Radix Lycoris Root (DGP081) 10 μl; 4. Radix Lycoris Root control 10 μl; 5. Radix Lycoside 10 μl;

[0033] Figure 5 This is the thin layer identification chromatogram of Radix Rehmanniae under low humidity conditions (T: 25°C, RH: 20%; Merck silica gel G plate)

[0034] Among them, 1. Radix Lycoris Root (DGP079) 10 μl; 2. Radix Lycoris Root (DGP080) 10 μl; 3. Radix Lycoris Root (DGP081) 10 μl; 4. Radix Lycoris Root control 10 μl; 5. Radix Lycoside 10 μl;

[0035] Figure 6 Chromatograms of different manufacturers' (Merck) silica gel G plates (T: 25°C, RH: 50%; Merck silica gel G plates)

[0036] Among them, 1. Radix Lycoris Root (DGP079) 10 μl; 2. Radix Lycoris Root (DGP080) 10 μl; 3. Radix Lycoris Root (DGP081) 10 μl; 4. Radix Lycoris Root control 10 μl; 5. Radix Lycoside 10 μl;

[0037] Figure 7 Chromatograms of silica gel G plates from different manufacturers (Qingdao) (T: 25°C, RH: 52%; marine silica gel G plates)

[0038] Among them, 1. Radix Lycoris Root (DGP079) 10 μl; 2. Radix Lycoris Root (DGP080) 10 μl; 3. Radix Lycoris Root (DGP081) 10 μl; 4. Radix Lycoris Root control 10 μl; 5. Radix Lycoside 10 μl;

[0039] Figure 8 Chromatograms of different manufacturers' (Yinlong) silica gel G plates (T: 25°C, RH: 52%; Yinlong silica gel G plates)

[0040] Among them, 1. Radix Lycoris Root (DGP079) 10 μl; 2. Radix Lycoris Root (DGP080) 10 μl; 3. Radix Lycoris Root (DGP081) 10 μl; 4. Radix Lycoris Root control 10 μl; 5. Radix Lycoside 10 μl;

[0041] Figure 9 Thin layer chromatograms of 5 batches of Radix Rehmanniae Radix (T: 25°C, RH: 55%; Merck silica gel G plate);

[0042] Among them, 1. Radix Lycoris Root Bark (DGP079) 10μl; 2. Radix Lycoris Root Bark (DGP080) 10μl; 3. Radix Lycoris Root Bark (DGP081) 10μl; 4. Radix Lycoris Root Bark (DGP082) 10μl; 5. Radix Lycoris Root Bark (DGP083) 10μl; 6. Radix Lycoris Root Bark Control Material 10μl; 7. Lycopodiol 10μl;

[0043] Figure 10 Thin layer chromatograms of three batches of Radix Rehmanniae formula granules (T: 25°C, RH: 52%; Merck silica gel G plate);

[0044] Among them, 1. Radix Lycoris Formula Granules (CG024) 10μl; 2. Radix Lycoris Formula Granules (CG025) 10μl; 3. Radix Lycoris Formula Granules (CG026) 10μl; 4. Radix Lycoris Control Herb 10μl; 5. Radix Lycoside E 10μl;

[0045] Figure 11 The thin layer chromatograms of three batches of Radix Rehmanniae Radix (pharmacopoeia method) (T: 25°C, RH: 50%; Merck silica gel G plate

[0046] Among them, 1. Radix Lycoris Radiatae (DGP079) 5μl; 2. Radix Lycoris Radiatae (DGP080) 5μl; 3.

[0047] 5 μl of Radix Lycii (DGP081); 5 μl of Radix Lycii control; 5 μl of Scopoletin 2 μl;

[0048] Figure 12 Chromatograms (T: 25°C, RH: 48%; Merck silica gel G plates) were investigated for different sample preparation methods (direct sonication with methanol);

[0049] Among them, 1. Radix Lycoris Root (DGP079) 10 μl; 2. Radix Lycoris Root (DGP080) 10 μl; 3. Radix Lycoris Root (DGP081) 10 μl; 4. Radix Lycoris Root control 10 μl; 5. Radix Lycoside 10 μl;

[0050] Figure 13 Chromatograms (T: 25°C, RH: 50%; Merck silica gel G plates) were investigated for different sample preparation methods (methanol sonication followed by ethyl acetate extraction);

[0051] Among them, 1. Radix Lycoris Root (DGP079) 10 μl; 2. Radix Lycoris Root (DGP080) 10 μl; 3. Radix Lycoris Root (DGP081) 10 μl; 4. Radix Lycoris Root control 10 μl; 5. Radix Lycoside 10 μl;

[0052] Figure 14 Chromatograms were investigated for different chromogenic agents (365 nm) (T: 25 °C, RH: 50%; Merck silica gel G plates);

[0053] Among them, 1. Radix Lycoris Root (DGP079) 10 μl; 2. Radix Lycoris Root (DGP080) 10 μl; 3. Radix Lycoris Root (DGP081) 10 μl; 4. Radix Lycoris Root control 10 μl; 5. Radix Lycoside 10 μl;

[0054] Figure 15 Chromatograms for different color developing agents (potassium bismuth iodide test solution) (T: 25°C, RH: 53%; Merck silica gel G plate)

[0055] Among them, 1. Radix Lycoris Root (DGP079) 10 μl; 2. Radix Lycoris Root (DGP080) 10 μl; 3. Radix Lycoris Root (DGP081) 10 μl; 4. Radix Lycoris Root control 10 μl; 5. Radix Lycoside 10 μl;

[0056] Figure 16 To investigate the chromatograms (T: 25°C, RH: 47%; Merck silica gel G plate) for different color developing agents (modified potassium bismuth iodide test solution);

[0057] Among them, 1. Radix Lycoris Root (DGP079) 10 μl; 2. Radix Lycoris Root (DGP080) 10 μl; 3. Radix Lycoris Root (DGP081) 10 μl; 4. Radix Lycoris Root control 10 μl; 5. Radix Lycoside 10 μl;

[0058] Figure 17 Chromatograms (T: 25°C, RH: 55%; Merck silica gel G plates) were investigated for different color developing agents (5% ferric chloride ethanol solution);

[0059] Among them, 1. Radix Lycoris Root (DGP079) 10 μl; 2. Radix Lycoris Root (DGP080) 10 μl; 3. Radix Lycoris Root (DGP081) 10 μl; 4. Radix Lycoris Root control 10 μl; 5. Radix Lycoside 10 μl;

[0060] Figure 18 Chromatograms (T: 25°C, RH: 52%; Merck silica gel G plates) were investigated for different color developers (10% sulfuric acid ethanol solution);

[0061] Among them, 1. Radix Lycoris Root (DGP079) 10 μl; 2. Radix Lycoris Root (DGP080) 10 μl; 3. Radix Lycoris Root (DGP081) 10 μl; 4. Radix Lycoris Root control 10 μl; 5. Radix Lycoside 10 μl;

[0062] Figure 19 Chromatograms were investigated for different color developing agents (ammonia fumigation) (T: 25°C, RH: 55%; Merck silica gel G plates);

[0063] Among them, 1. Radix Lycoris Root (DGP079) 10 μl; 2. Radix Lycoris Root (DGP080) 10 μl; 3. Radix Lycoris Root (DGP081) 10 μl; 4. Radix Lycoris Root control 10 μl; 5. Radix Lycoside 10 μl;

[0064] Figure 20 Chromatograms (T: 25 °C, RH: 48%; Merck silica gel G plates) were investigated for different developing solvents (ethyl acetate-methanol-formic acid-water 5:2:1:0.5);

[0065] Among them, 1. Radix Lycoris Root (DGP079) 10 μl; 2. Radix Lycoris Root (DGP080) 10 μl; 3. Radix Lycoris Root (DGP081) 10 μl; 4. Radix Lycoris Root control 10 μl; 5. Radix Lycoside 10 μl;

[0066] Figure 21Chromatograms (T: 25 °C, RH: 50%; Merck silica gel G plates) were investigated for different developing solvents (dichloromethane-ethyl acetate-formic acid-water 6:4:2:0.1);

[0067] Among them, 1. Radix Lycoris Root (DGP079) 10 μl; 2. Radix Lycoris Root (DGP080) 10 μl; 3. Radix Lycoris Root (DGP081) 10 μl; 4. Radix Lycoris Root control 10 μl; 5. Radix Lycoside 10 μl;

[0068] Figure 22 The chromatograms were investigated for different developing solvents (acetone-methanol-formic acid 8:1:1) (T: 25°C, RH: 48%; Merck silica gel G plates);.

[0069] Among them, 1. Radix Lycoris Root (DGP079) 10 μl; 2. Radix Lycoris Root (DGP080) 10 μl; 3. Radix Lycoris Root (DGP081) 10 μl; 4. Radix Lycoris Root control 10 μl; 5. Radix Lycoside 10 μl;

[0070] Figure 23 Chromatograms (T: 25 °C, RH: 50%; Merck silica gel G plates) were investigated for different developing solvents (acetone-formic acid-water 6:1:1);

[0071] Among them, 1. Radix Lycoris Root (DGP079) 10 μl; 2. Radix Lycoris Root (DGP080) 10 μl; 3. Radix Lycoris Root (DGP081) 10 μl; 4. Radix Lycoris Root control 10 μl; 5. Radix Lycoside 10 μl;

[0072] Figure 24 Chromatograms (T: 25 °C, RH: 58%; Merck silica gel G plates) were investigated for different developing solvents (n-butanol-glacial acetic acid-water 3:1:1);

[0073] Among them, 1. Radix Lycoris Root (DGP079) 10 μl; 2. Radix Lycoris Root (DGP080) 10 μl; 3. Radix Lycoris Root (DGP081) 10 μl; 4. Radix Lycoris Root control 10 μl; 5. Radix Lycoside 10 μl;

[0074] Figure 25 Chromatograms (T: 25 °C, RH: 52%; Merck silica gel G plates) were investigated for different developing solvents (n-butanol-ethyl acetate-formic acid-water 3:2:2:0.5);

[0075] Among them, 1. Radix Lycoris Radiatae (DGP079) 10 μl; 2. Radix Lycoris Radiatae (DGP080) 10 μl; 3. Radix Lycoris Radiatae (DGP081) 10 μl; 4. Radix Lycoris Radiatae control medicinal material 10 μl; 5. Radix Lycoside E 10 μl. DETAILED DESCRIPTION

[0076] The following is a further detailed description of the thin layer detection method for the Chinese medicinal formula granules of the Chinese herbal medicine of Radix Lycii and its application in counterfeit identification, in conjunction with specific examples. The present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to provide a more thorough and comprehensive understanding of the disclosure of this application.

[0077] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are for the purpose of describing specific embodiments only and are not intended to limit this application.

[0078] In this application, terms such as "first aspect" and "second aspect" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor as implicitly indicating the importance or quantity of the technical features indicated. Furthermore, terms such as "first" and "second" serve only as non-exhaustive enumeration and description and should not constitute a closed-ended limitation on quantity.

[0079] In this application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0080] In this application, when referring to numerical ranges, unless otherwise specified, the numerical ranges are considered continuous and include the minimum and maximum values of the range, as well as every value between such minimum and maximum values. Further, when a range refers to an integer, every integer between the minimum and maximum values of the range is included. In addition, when multiple ranges are provided to describe a feature or characteristic, the ranges can be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges subsumed therein.

[0081] Unless otherwise specified, the percentage contents mentioned in this application refer to mass percentage for solid-liquid mixture and solid-solid mixture, and refer to volume percentage for liquid-liquid mixture.

[0082] The percentage concentrations mentioned in this application, unless otherwise specified, refer to the final concentration, which refers to the percentage of the added component in the system after the addition of the component.

[0083] The temperature parameters in this application, unless otherwise specified, allow for both constant temperature treatment and treatment within a certain temperature range. The constant temperature treatment allows for temperature fluctuations within the precision range of instrument control.

[0084] Normal temperature in this application generally refers to 10°C to 30°C, preferably 25±5°C.

[0085] Currently, the first part of the 2020 edition of the Chinese Pharmacopoeia does not record the specific ingredients of the Chinese herbal medicine Rehmannia glutinosa in the identification and content determination items. The research on the thin layer identification of Chinese herbal medicine Rehmannia glutinosa and the standards of Chinese herbal formula granules in most provinces in the literature is basically based on the Chinese herbal medicine reference material Rehmannia glutinosa. A small number of studies attribute the main spots to scopoletin. However, there is no evidence that the spots presented by the reference material or scopoletin are related to the "cooling blood, removing steam, clearing the lungs and reducing fire" effect of Chinese herbal medicine Rehmannia glutinosa, and scopoletin lacks specificity. Since there is no thin layer identification method for scopoletin in current research, it brings certain difficulties to the establishment of the identification method. There is an urgent need to provide a fast and effective specific identification method for Chinese herbal medicine Rehmannia glutinosa and its Chinese herbal formula granules.

[0086] Therefore, the present invention combines the current research situation and is based on the blank of thin layer chromatography identification of the phytochrome component in the cortex rehmanniae. The present invention takes the phenolamide components of the cortex rehmanniae as the research object, takes the phytochrome content with a higher content as a reference, extracts, purifies and enriches, and uses a simpler, faster and lower-cost thin layer chromatography to establish an identification method for the cortex rehmanniae medicinal material and its traditional Chinese medicine formula granules, which provides a reference for the quality standard of the cortex rehmanniae and can be used to distinguish its counterfeits.

[0087] The technical solution is as follows:

[0088] A thin layer detection method for the medicinal material of Lycium bark comprises the following steps:

[0089] The Chinese medicinal material or the formulated granules of Radix Lycii were extracted with methanol, the obtained extract was evaporated to dryness, the evaporated solid was dissolved in water, extracted with n-butanol, the obtained n-butanol phase was evaporated to dryness, and the evaporated solid was dissolved in methanol to prepare the test solution;

[0090] The reference medicinal material of Radix Lycii was extracted with methanol, the obtained extract was evaporated to dryness, the evaporated solid was dissolved in water, extracted with n-butanol, the obtained n-butanol phase was evaporated to dryness, and the evaporated solid was dissolved in methanol to prepare a reference medicinal material solution;

[0091] Take the lycopodine standard substance, add solvent to dissolve it, and prepare the standard solution;

[0092] The test solution, control medicinal material solution and reference substance solution are spotted on the same thin layer plate, developed with a mixture of n-butanol, formic acid and water as a developing agent, dried, developed with a color developer, and inspected.

[0093] In one embodiment, the extraction method is ultrasonic extraction.

[0094] In one embodiment, the power of ultrasonic extraction is 250W to 350W, the frequency is 35kHz to 45kHz, and the time is 20min to 40min. It is understandable that in the present invention, the power of ultrasonic extraction includes but is not limited to 250W, 260W, 270W, 280W, 290W, 300W, 310W, 320W, 330W, 340W and 350W, the frequency includes but is not limited to 35kHz, 40kHz and 45kHz, and the time includes but is not limited to 20min, 25min, 30min, 35min and 40min. Further, the power of ultrasonic extraction is 300W, the frequency is 40kHz, and the time is 30min.

[0095] In one embodiment, each 1 ml of the test solution contains 0.8 g to 1.2 g of the Chinese wolfberry bark medicinal material.

[0096] In one embodiment, each 1 ml of the test solution contains 0.1 g to 0.3 g of the Radix Rehmanniae formula particles.

[0097] In one embodiment, each 1 ml of the control medicinal material solution contains 0.8 g to 1.2 g of the Chinese wolfberry bark control medicinal material.

[0098] In one embodiment, each 1 ml of the reference solution contains 0.8 mg to 1.2 mg of cefotaxime.

[0099] In one embodiment, the sample volume of the test solution is 8 μl to 12 μl, including but not limited to 8 μl, 9 μl, 10 μl, 11 μl and 12 μl. Further, the sample volume of the test solution is 10 μl.

[0100] In one embodiment, the spotting volume of the control medicinal material solution is 8 μl to 12 μl, including but not limited to 8 μl, 9 μl, 10 μl, 11 μl and 12 μl. Furthermore, the spotting volume of the control medicinal material solution is 10 μl.

[0101] In one embodiment, the spotting volume of the reference solution is 8 μl to 12 μl, including but not limited to 8 μl, 9 μl, 10 μl, 11 μl and 12 μl. Further, the spotting volume of the reference solution is 10 μl.

[0102] In one embodiment, the volume ratio of n-butanol, formic acid and water in the developing agent is (2-4): (0.5-2): 1, including but not limited to 2:0.5:1, 2:0.6:1, 2:0.7:1, 2:0.8:1, 2:0.9:1, 2:1:1, 2:1.1:1, 2:1.2:1, 2:1.3:1, 2:1.4:1, 2:1.5:1, 2:1.6:1, 2:1.7:1, 2:1.8:1, 2:1.9:1, 2:2:1, 2.5:0.5 :1, 2.5:0.6:1, 2.5:0.7:1, 2.5:0.8:1, 2.5:0.9:1, 2.5:1:1, 2.5:1.1:1, 2.5:1.2:1, 2.5:1.3:1, 2.5:1.4:1, 2.5:1.5:1, 2.5:1.6:1, 2.5:1.7:1, 2.5:1.8:1, 2.5:1.9:1, 2.5:2:1, 3:0.5:1, 3:0.6:1, 3:0.7:1, 3:0.8:1, 3:0 .9:1, 3:1:1, 3:1.1:1, 3:1.2:1, 3:1.3:1, 3:1.4:1, 3:1.5:1, 3:1.6:1, 3:1.7:1, 3:1.8:1, 3:1.9:1, 3:2:1, 3.5:0.5:1, 3.5:0.6:1, 3.5:0.7:1, 3.5:0.8:1, 3.5:0.9:1, 3.5:1:1, 3.5:1.1:1, 3.5:1.2:1, 3.5:1.3:1, 3.5:1.4 :1, 3.5:1.5:1, 3.5:1.6:1, 3.5:1.7:1, 3.5:1.8:1, 3.5:1.9:1, 3.5:2:1, 4:0.5:1, 4:0.6:1, 4:0.7:1, 4:0.8:1, 4:0.9:1, 4:1:1, 4:1.1:1, 4:1.2:1, 4:1.3:1, 4:1.4:1, 4:1.5:1, 4:1.6:1, 4:1.7:1, 4:1.8:1, 4:1.9:1 and 4:2:1. Further, in the developing solvent, the volume ratio of n-butanol, formic acid and water is (2-4):(1-2):1. Furthermore, in the developing agent, the volume ratio of n-butanol, formic acid and water is (2.5-3.5):(1-1.5):1.

[0103] In one embodiment, the unfolding temperature is below 10°C. It is understood that, in the present invention, the unfolding temperature includes, but is not limited to, -10°C, -5°C, 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, and 10°C. Furthermore, the unfolding temperature is below 8°C. Furthermore, the unfolding temperature is 7°C.

[0104] In one embodiment, the pre-saturation time of the deployment cylinder is 12 minutes to 20 minutes, including but not limited to 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, and 20 minutes. Furthermore, the pre-saturation time of the deployment cylinder is 14 minutes to 20 minutes. Even more preferably, the pre-saturation time of the deployment cylinder is 15 minutes.

[0105] In one embodiment, the unfolded distance is 7 cm to 10 cm, including but not limited to 7 cm, 8 cm, 9 cm and 10 cm. Further, the unfolded distance is 8 cm.

[0106] In one embodiment, the developer is iodine vapor.

[0107] In one embodiment, the inspection method is observation under fluorescent light.

[0108] In one embodiment, the thin layer detection method of the Chinese medicinal material of Lycium bark comprises the following steps:

[0109] (1) Take 1 g of Radix Rehmanniae (passed through a No. 4 sieve) or 0.25 g of Radix Rehmanniae granules, add 20 ml of methanol, and ultrasonically treat (power 300 W, frequency 40 kHz) for 30 minutes. Filter, evaporate the filtrate to dryness, dissolve the residue in 20 ml of water, and shake and extract twice with 20 ml of saturated n-butanol. Combine the n-butanol extracts, evaporate to dryness, and dissolve the residue in 1 ml of methanol to prepare the test solution.

[0110] (2) Take 1 g of Radix Rehmanniae glutinosae as a control medicinal material, add 20 ml of methanol, and prepare a control medicinal material solution in the same way.

[0111] (3) Take an appropriate amount of scutellariae acetate reference substance and add methanol to prepare a solution containing 1 mg per 1 ml as the reference substance solution.

[0112] (4) Take 10 μl of each of the three solutions mentioned above and spot them on the same silica gel G thin layer plate. Use the upper layer solution of n-butanol-formic acid-water (3:1:1) stored below 10°C as the developing agent, develop, take out and dry.

[0113] (5) Place the sample in iodine vapor until the spots are clearly colored. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions in the chromatograms of the Radix Rehmanniae glutinosae control medicinal material and the Radix Rehmanniae ethyl reference material.

[0114] The present invention also provides application of the thin layer detection method of the Chinese wolfberry bark medicinal material or the formula granules in identifying the Chinese wolfberry bark medicinal material and its counterfeit products.

[0115] It can be understood that the Radix Rehmanniae Root Bark medicinal material and its counterfeit products can be in the form of medicinal materials, decoction pieces, Chinese medicine formula granules or other preparation forms.

[0116] In some examples, the identification method includes: In one embodiment, the identification method includes: comparing the chromatogram of the test solution to see whether the same spots exist at positions corresponding to the chromatogram of the control medicinal material solution and / or the chromatogram of the reference substance solution. If the same spots exist, it is the Chinese herbal medicine or its formula granules; if not, it is a counterfeit. In addition, it can be understood that the reference medicinal material is used as a reference for thin layer chromatography identification, and the basis of identification is upgraded from a single indicator component to authenticity judgment based on a complete thin layer chromatography, which is reflected in the fact that the thin layer chromatography is a chromatographic image with fingerprint significance. Different varieties have their own fixed patterns, which can improve the specificity of identification. Identification using it in combination with the reference substance is more accurate.

[0117] The following are specific examples.

[0118] Example 1

[0119] This embodiment provides a thin layer detection method for the medicinal material of Lycium bark, which is as follows:

[0120] 1. Instruments, reagents and test drugs

[0121] Instruments: Automatic TLC Sampler 4 (Kamar, Switzerland), TLC Visualizer 2 (Kamar, Switzerland), 1 / 10,000 microbalance (ME204E, Mettler, Switzerland), ultrasonic cleaner (KQ-500DE, Kunshan Ultrasonic Instrument Co., Ltd.), double-slot developing cylinder, silica gel G thin layer plate, silica gel G thin layer plate (20 cm × 10 cm, thickness 0.20-0.25 mm, Yantai Institute of Chemical Industry), silica gel G thin layer plate (20 cm × 10 cm, thickness 0.20-0.25 mm, Qingdao Ocean Chemical Co., Ltd.), silica gel G thin layer plate (20 cm × 10 cm, thickness 0.20-0.25 mm, Merck KGaA)

[0122] Reagents: methanol (Tianjin Fuyu Fine Chemical Co., Ltd.), n-butanol (Xilong Scientific Co., Ltd.), formic acid (Xilong Scientific Co., Ltd.), and purified water (self-made in the laboratory).

[0123] Test drugs: Radix Lycii control medicinal material (batch number: 121087-201707, China Food and Drug Inspection Institute), Radix Lycii B-type reference substance (batch number: D04102204013, content: 98%, Chengdu Ruifensi Biotechnology Co., Ltd.), Radix Lycii medicinal material (see Table 1 below).

[0124] Table 1 Information of 5 batches of Radix Rehmanniae

[0125]

[0126]

[0127] 2. Solution Preparation

[0128] 2.1 Preparation of test solution

[0129] Take 1 g of Radix Rehmanniae (pass through No. 4 sieve), add 20 ml of methanol, ultrasonically treat (power 300 W, frequency 40 kHz) for 30 minutes, filter, evaporate the filtrate to dryness, add 20 ml of water to dissolve the residue, add saturated n-butanol with water and shake to extract twice, 20 ml each time, combine the n-butanol extracts, evaporate to dryness, and add 1 ml of methanol to dissolve the residue as the test solution.

[0130] 2.2 Preparation of control medicinal material solution

[0131] Take 1 g of the Radix Rehmanniae control medicinal material, add 20 ml of methanol, and ultrasonically treat (power 300 W, frequency 40 kHz) for 30 minutes, filter, evaporate the filtrate to dryness, add 20 ml of water to dissolve the residue, add water-saturated n-butanol and shake to extract twice, 20 ml each time, combine the n-butanol extracts, evaporate to dryness, and add 1 ml of methanol to dissolve the residue to prepare a control medicinal material solution.

[0132] 2.3 Preparation of reference solution

[0133] Take an appropriate amount of dichotomane-2 standard substance and add methanol to prepare a solution containing 1 mg per 1 ml, which is used as the standard solution.

[0134] 3. Thin layer chromatography conditions

[0135] Thin layer plate: Silicone G thin layer plate.

[0136] Developing agent: n-butanol-formic acid-water (volume ratio 3:1:1) upper layer solution placed below 10°C.

[0137] Spotting method: spray strip spotting.

[0138] Deployment method: A double-slot deployment cylinder is used, the pre-saturation time of the deployment cylinder is 15 minutes, and the deployment distance is about 8 cm.

[0139] Inspection: Place in iodine vapor until the spots are clearly colored, and inspect under sunlight.

[0140] 4. Investigation of different sampling quantities of Radix Rehmanniae

[0141] The test solution of Radix Lycopersicum (DGP079), the control solution of Radix Lycopersicum and the control solution of Radix Lycopersicum ethyl were respectively spotted on the same silica gel G thin layer plate, developed, taken out, dried, and fumigated in iodine vapor until the spots were clearly colored. The experimental results were observed under sunlight. Figure 1 .

[0142] Depend on Figure 1 It can be seen that when the sample volume of the test solution, control medicinal material solution and reference substance solution is 10 μl, the test sample chromatogram shows spots of the same color at the corresponding positions of the control medicinal material chromatogram and the reference substance chromatogram, without tailing and other interference. Therefore, the sample volume of the test sample solution, control medicinal material solution and reference substance solution is set to 10 μl.

[0143] 5. Investigation at different temperatures

[0144] The test solutions of Radix Lycii (DGP079, DGP080, DGP081), Radix Lycii control solutions and Radix Lycii B reference solutions were spotted on the same silica gel G thin layer plate. According to the above thin layer chromatography conditions, the plates were developed at room temperature and low temperature, taken out, dried, and fumigated in iodine vapor until the spots were clearly colored. The plates were examined under sunlight. The experimental results are shown in the table. Figure 2 and Figure 3 .

[0145] Depend on Figure 2 and Figure 3 It can be seen that the separation effect of Radix Lycii was good under both room temperature and low temperature conditions, and the test sample chromatogram showed spots of the same color at the corresponding positions in the control sample chromatogram and the control sample chromatogram. The experimental results show that temperature has no significant effect on the thin layer identification of Radix Lycii, indicating that the thin layer identification method has good durability at different temperatures.

[0146] 6. Investigation of different humidity

[0147] The test solutions of Radix Lycii (DGP079, DGP080, DGP081), Radix Lycii control solutions and Radix Lycii B reference solutions were spotted on the same silica gel G thin layer plate. The plates were developed under high humidity and low humidity conditions according to the above thin layer chromatography conditions. The plates were taken out, dried, and fumigated in iodine vapor until the spots were clearly colored. The plates were examined under sunlight. The experimental results are shown in the table. Figure 4 and Figure 5 .

[0148] Depend on Figure 4 and Figure 5 It can be seen that the chromatographic separation effect of Radix Rehmanniae was good under both high and low humidity conditions, and the test sample chromatogram showed spots of the same color at the corresponding positions in the control sample chromatogram and the control sample chromatogram. The experimental results show that humidity has no significant effect on the TLC identification of Radix Rehmanniae, and the TLC identification method has good durability under different humidity conditions.

[0149] 7Inspection of thin layer boards from different manufacturers

[0150] The samples of the Radix Rehmanniae medicinal material test solution (DGP079, DGP080, DGP081), the Radix Rehmanniae control medicinal material solution and the Radix Rehmanniae ethyl reference solution were respectively spotted on silica gel G thin layer plates of different manufacturers (Silver Dragon Silica G plate, Ocean Silica G plate, Merck Silica G plate), and developed under the same temperature and humidity conditions according to the above-mentioned thin layer chromatography conditions. The plates were taken out, dried, and fumigated in iodine vapor until the spots were clearly colored and examined under sunlight. The experimental results are shown in Figure 6 、 Figure 7 and Figure 8 .

[0151] Depend on Figure 6 、 Figure 7 and Figure 8 As can be seen, using silica gel G thin layer plates from different manufacturers (Yinlong silica gel G plate, Ocean silica gel G plate, Merck silica gel G plate), the spots in the chromatograms of the test sample, the control sample, and the control sample were all clearly colored and well separated, and the spots in the chromatograms of the test sample, the control sample, and the control sample corresponded to each other. This shows that the thin layer identification method is durable for silica gel G thin layer plates from different manufacturers.

[0152] 8 Thin layer identification chromatogram of Radix Rehmanniae

[0153] The samples of the Radix Lycii test samples (DGP079, DGP080, DGP081, DGP082, DGP083), the Radix Lycii control samples and the Radix Lycii ethyl reference solution were spotted on the same silica gel G thin layer plate, developed, taken out, dried, and smoked in iodine vapor until the spots were clearly colored. The plates were inspected under sunlight. The experimental results are shown in the table. Figure 9 .

[0154] Depend on Figure 9 It can be seen that in the chromatograms of the five batches of Radix Rehmanniae medicinal materials test samples, spots of the same color are displayed at the corresponding positions in the chromatograms of the Radix Rehmanniae control medicinal material solution and the Radix Rehmanniae ethyl reference product. Using the Radix Rehmanniae control medicinal material solution and the Radix Rehmanniae ethyl reference product as references can effectively perform thin layer chromatography identification of the Radix Rehmanniae medicinal materials, and all five batches of Radix Rehmanniae medicinal material samples meet the requirements.

[0155] 9. Summary

[0156] In summary, the Rf value of each spot in the chromatogram established by the present invention is moderate, the overall separation effect is good, the spots are clearer, and the test sample chromatogram is in the corresponding position with the control medicinal material chromatogram and the reference substance chromatogram, and the spots of the same color can correspond. According to methodological verification, the specificity, reproducibility and durability of the thin layer chromatography method are good, and the method can be used for effective qualitative identification of the Chinese medicinal material Radix Lycii.

[0157] Example 2

[0158] This embodiment provides a thin layer detection method for the Radix Lycii formula particles, which is as follows:

[0159] 1. Solution Preparation

[0160] 1.1 Preparation of test solution

[0161] Take 0.25 g of Radix Rehmanniae formula granules (batch number: CG024, CG025, CG026, provided by Guangdong Yifang Pharmaceutical Co., Ltd.), add 20 ml of methanol, ultrasonically treat (power 300 W, frequency 40 kHz) for 30 minutes, filter, evaporate the filtrate to dryness, add 20 ml of water to dissolve the residue, add saturated n-butanol with water and shake to extract twice, 20 ml each time, combine the n-butanol extracts, evaporate to dryness, and add 1 ml of methanol to dissolve the residue as the test solution.

[0162] 1.2 Preparation of control medicinal material solution

[0163] Take 1 g of the Radix Rehmanniae control medicinal material, add 20 ml of methanol, and ultrasonically treat (power 300 W, frequency 40 kHz) for 30 minutes, filter, evaporate the filtrate to dryness, add 20 ml of water to dissolve the residue, add water-saturated n-butanol and shake to extract twice, 20 ml each time, combine the n-butanol extracts, evaporate to dryness, and add 1 ml of methanol to dissolve the residue to prepare a control medicinal material solution.

[0164] 1.3 Preparation of reference solution

[0165] Take an appropriate amount of dichotomane-2 standard substance and add methanol to prepare a solution containing 1 mg per 1 ml, which is used as the standard solution.

[0166] 2 Thin layer chromatography conditions

[0167] Thin layer plate: Silicone G thin layer plate.

[0168] Developing agent: n-butanol-formic acid-water (volume ratio 3:1:1) upper layer solution placed below 10°C.

[0169] Spotting method: spray strip spotting.

[0170] Deployment method: A double-slot deployment cylinder is used, the pre-saturation time of the deployment cylinder is 15 minutes, and the deployment distance is about 8 cm.

[0171] Inspection: Place in iodine vapor until the spots are clearly colored, and inspect under sunlight. The test results are shown in the table. Figure 10 .

[0172] 3 Conclusions:

[0173] Depend on Figure 10It can be seen that the thin-layer chromatography identification method using lycium bark ethylene established in Example 1 is also applicable to the identification of lycium bark formula granules that have lost their property characteristics. In the chromatograms of the three batches of lycium bark formula granules, spots of the same color are shown at the corresponding positions of the chromatograms of the lycium bark control medicinal material solution and the lycium bark ethylene reference substance. Using the lycium bark control medicinal material solution and the lycium bark ethylene reference substance as a reference, the lycium bark formula granules can be effectively identified by thin-layer chromatography, and the three batches of lycium bark formula granule samples all meet the requirements.

[0174] Comparative Example 1

[0175] This comparative example provides a thin layer detection method for the Chinese medicinal material of Radix Lycoris Radiatae, which is a method under the Chinese medicinal material of Radix Lycoris Radiatae in the 2020 edition of the Pharmacopoeia, as follows:

[0176] 1. Solution Preparation

[0177] 1.1 Preparation of test solution

[0178] Take 1.5 g of Rehmannia root bark powder, add 15 ml of methanol, and ultrasonically treat (power 300 W, frequency 40 kHz) for 30 minutes. Filter, evaporate the filtrate to dryness, and add 1 ml of methanol to dissolve the residue, which is used as the test solution.

[0179] 1.2 Preparation of control medicinal material solution

[0180] Take 1.5 g of Radix Rehmanniae Root Bark control medicinal material, add 15 ml of methanol, and ultrasonically treat (power 300 W, frequency 40 kHz) for 30 minutes, filter, evaporate the filtrate to dryness, and add 1 ml of methanol to dissolve the residue to prepare a control medicinal material solution.

[0181] 1.3 Preparation of reference solution

[0182] Take an appropriate amount of scopoletin reference substance (110768-202105, China Food and Drug Inspection Institute) and add methanol to make a solution containing 1 mg per 1 ml as the reference substance solution.

[0183] 2. Thin layer chromatography conditions

[0184] Thin layer plate: Silicone G thin layer plate.

[0185] Developing solvent: toluene-acetone-formic acid (volume ratio 10:1:0.1).

[0186] Spotting method: spray strip spotting.

[0187] Deployment method: A double-slot deployment cylinder is used, the pre-saturation time of the deployment cylinder is 15 minutes, and the deployment distance is about 8 cm.

[0188] Inspection: Check under ultraviolet light (365nm), the experimental results are shown in Figure 11 .

[0189] 3. Conclusion:

[0190] Depend on Figure 11 It can be seen that the thin layer identification method under the 2020 edition of the "Chinese Pharmacopoeia" was used for the Radix Rehmanniae medicinal material. 5μl of the test solution and the control medicinal material, and 2μl of the control solution were aspirated and spotted on the same silica gel G thin layer plate. The results showed that the two identical blue spots in the chromatogram of the Radix Rehmanniae medicinal material test solution corresponded to the chromatogram of the Radix Rehmanniae control medicinal material. There were fewer spots and it was not possible to better reflect the overall quality of the Radix Rehmanniae medicinal material. After matching with the reference material, it was found that the spot was scopoletin, but this component is not a specific component of Radix Rehmanniae.

[0191] Comparative Example 2

[0192] This comparative example provides a thin layer detection method for the medicinal material of Lycium bark, and examines the influence of the sample preparation method on the identification method, as follows:

[0193] 1. Solution Preparation

[0194] 1.1 Preparation of test solution

[0195] ① Take 1 g of Radix Rehmanniae (pass through No. 4 sieve), add 20 ml of methanol, and ultrasonically treat (power 300 W, frequency 40 kHz) for 30 minutes. Filter, evaporate the filtrate to dryness, and add 1 ml of methanol to dissolve the residue as the test solution.

[0196] ② Take 1 g of Radix Rehmanniae (passed through No. 4 sieve), add 20 ml of methanol, ultrasonically treat (power 300 W, frequency 40 kHz) for 30 minutes, filter, evaporate the filtrate to dryness, add 20 ml of water to dissolve the residue, add ethyl acetate and shake to extract twice, 20 ml each time, combine the ethyl acetate extracts, evaporate to dryness, and add 1 ml of methanol to dissolve the residue as the test solution.

[0197] 1.2 Preparation of control medicinal material solution

[0198] ① Take 1 g of Radix Rehmanniae Root Bark as a control medicinal material, add 20 ml of methanol, and ultrasonically treat (power 300 W, frequency 40 kHz) for 30 minutes. Filter, evaporate the filtrate to dryness, and add 1 ml of methanol to dissolve the residue to prepare a control medicinal material solution.

[0199] ② Take 1 g of Radix Rehmanniae Root Bark as a control medicinal material, add 20 ml of methanol, and ultrasonically treat (power 300 W, frequency 40 kHz) for 30 minutes. Filter, evaporate the filtrate to dryness, add 20 ml of water to dissolve the residue, add ethyl acetate and shake to extract twice, 20 ml each time, combine the ethyl acetate extracts, evaporate to dryness, and add 1 ml of methanol to dissolve the residue to prepare a control medicinal material solution.

[0200] 1.3 Preparation of reference solution

[0201] Take an appropriate amount of dichotomane-2 standard substance and add methanol to prepare a solution containing 1 mg per 1 ml, which is used as the standard solution.

[0202] 2 Thin layer chromatography conditions

[0203] Thin layer plate: Silicone G thin layer plate.

[0204] Developing agent: n-butanol-formic acid-water (3:1:1) upper layer solution placed below 10℃

[0205] Spotting method: spray strip spotting.

[0206] Deployment method: A double-slot deployment cylinder is used, the pre-saturation time of the deployment cylinder is 15 minutes, and the deployment distance is about 8 cm.

[0207] Inspection: Place in iodine vapor until the spots are clearly colored, and inspect under sunlight. The test results are shown in the table. Figure 12 and 13 .

[0208] 3 Conclusions:

[0209] Depend on Figure 12 It can be seen that referring to the thin layer identification sample preparation method under the item of Rehmannia glutinosa medicinal materials in the 2020 edition of the "Chinese Pharmacopoeia", direct methanol ultrasonic extraction and concentration were performed. The results showed that in the chromatograms of the Rehmannia glutinosa medicinal materials test samples and Rehmannia glutinosa control medicinal materials, the spots at the position corresponding to Rehmannia glutinosa ethyl were tailing, and there were interferences from other components, which had a certain impact on the judgment of the results.

[0210] Depend on Figure 13 It can be seen that purification was carried out on the basis of methanol extraction, and an ethyl acetate extraction step was added. The results showed that the number of spots was reduced after extraction with ethyl acetate, and only dichotomane-2 spots could be seen. The overall information was less than that of the extraction with n-butanol in Example 1. See Example 1 for details.

[0211] After comparing three different sample preparation methods: direct extraction with methanol, extraction with methanol followed by extraction with ethyl acetate, and extraction with methanol followed by extraction with n-butanol, it was found that the chromatographic spots presented by extraction with methanol followed by extraction with n-butanol were rich in information, with clear spots and no tailing phenomenon. This method was finally selected as the sample preparation method.

[0212] Comparative Example 3

[0213] This comparative example provides a thin layer detection method for the medicinal material of Radix Lycii, and examines the influence of the color development method on the identification method, as follows:

[0214] 1. Solution Preparation

[0215] 1.1 Preparation of test solution

[0216] Take 1 g of Radix Rehmanniae (pass through No. 4 sieve), add 20 ml of methanol, ultrasonically treat (power 300 W, frequency 40 kHz) for 30 minutes, filter, evaporate the filtrate to dryness, add 20 ml of water to dissolve the residue, add saturated n-butanol with water and shake to extract twice, 20 ml each time, combine the n-butanol extracts, evaporate to dryness, and add 1 ml of methanol to dissolve the residue as the test solution.

[0217] 1.2 Preparation of control medicinal material solution

[0218] Take 1 g of the Radix Rehmanniae control medicinal material, add 20 ml of methanol, and ultrasonically treat (power 300 W, frequency 40 kHz) for 30 minutes, filter, evaporate the filtrate to dryness, add 20 ml of water to dissolve the residue, add water-saturated n-butanol and shake to extract twice, 20 ml each time, combine the n-butanol extracts, evaporate to dryness, and add 1 ml of methanol to dissolve the residue to prepare a control medicinal material solution.

[0219] 1.3 Preparation of reference solution

[0220] Take an appropriate amount of dichotomane-2 standard substance and add methanol to prepare a solution containing 1 mg per 1 ml, which is used as the standard solution.

[0221] 1.4 Preparation of color developer

[0222] (1) Potassium iodide test solution: Take 0.85 g of bismuth subnitrate / bismuth basic nitrate, dissolve it in 10 ml of glacial acetic acid and 40 ml of water, then add 20 ml of potassium iodide solution (4→10), shake well, and the solution is ready.

[0223] (2) Improved potassium bismuth iodide test solution: Take 1 ml of potassium bismuth iodide test solution, add 2 ml of 0.6 mol / L hydrochloric acid solution, and add water to 10 ml.

[0224] (3) Take 5g of ferric chloride and dilute it to 100ml with ethanol.

[0225] (4) Take 10 ml of sulfuric acid solution and dilute it to 100 ml with ethanol.

[0226] 2 Thin layer chromatography conditions

[0227] Thin layer plate: Silicone G thin layer plate.

[0228] Developing agent: n-butanol-formic acid-water (3:1:1) upper layer solution placed below 10℃

[0229] Spotting method: spray strip spotting.

[0230] Deployment method: A double-slot deployment cylinder is used, the pre-saturation time of the deployment cylinder is 15 minutes, and the deployment distance is about 8 cm.

[0231] Inspection: ①Inspect directly under fluorescent light (365nm), the experimental results are shown in Figure 14 .

[0232] ② Spray with potassium bismuth iodide test solution and inspect under sunlight. The test results are shown in Figure 15 .

[0233] ③ Spray with modified potassium bismuth iodide test solution and inspect under sunlight. The test results are shown in Figure 16 .

[0234] ④ Spray with 5% ferric chloride ethanol solution, heat at 105℃ until the spots are clearly colored, and inspect under sunlight. The experimental results are shown in Figure 17 .

[0235] ⑤ Spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly colored, and inspect under sunlight. The experimental results are shown in Figure 18 .

[0236] ⑥ Place in ammonia vapor until the spots are clearly colored, and inspect under sunlight. The experimental results are shown in Figure 19 .

[0237] 3 Conclusions:

[0238] Depend on Figure 14 It can be seen that when the sample is developed according to the sample preparation method and development conditions of Example 1 and directly examined under a fluorescent lamp (365 nm), no lycoside spots can be observed because lycoside has no ultraviolet absorption at 365 nm.

[0239] Depend on Figure 15 and 16 It can be seen that lycopoeia ethyl is an alkaloid component, so potassium bismuth iodide test solution and modified potassium bismuth iodide test solution, which are more commonly used alkaloid components, were used for color development. The results showed that the spots of the lycopoeia medicinal material test sample, lycopoeia medicinal material control, and lycopoeia ethyl were blurred, and the background interference increased, making it impossible to see the spots clearly. The color developer did not meet the requirements.

[0240] Depend on Figure 17 It can be seen that after spraying with 5% ferric chloride ethanol solution and heating and inspecting under sunlight, the number of spots is the same as that in Example 1, but the color of the spots is lighter and the color development effect is slightly worse than that of iodine fumigation.

[0241] Depend on Figure 18 It can be seen that when the samples were sprayed with 10% sulfuric acid ethanol solution and then heated and examined under sunlight, no cerebroside spots were observed.

[0242] Depend on Figure 19 It can be seen that the spots were clearly colored after being smoked in ammonia vapor. It was found that the other spots above the cypermethrin spots were more blurred and scattered after coloration, and the separation between the spots was poor.

[0243] Comprehensively comparing the above color development methods, the color development using iodine fumigation in Example 1 has the largest number of spots, better separation, and clearer chromatographic spots.

[0244] Comparative Example 4

[0245] This comparative example provides a thin layer detection method for the medicinal material of Radix Lycii, and examines the effect of the developing agent on the identification method, as follows:

[0246] 1. Solution Preparation

[0247] 1.1 Preparation of test solution

[0248] Take 1 g of Radix Rehmanniae (pass through No. 4 sieve), add 20 ml of methanol, ultrasonically treat (power 300 W, frequency 40 kHz) for 30 minutes, filter, evaporate the filtrate to dryness, add 20 ml of water to dissolve the residue, add saturated n-butanol with water and shake to extract twice, 20 ml each time, combine the n-butanol extracts, evaporate to dryness, and add 1 ml of methanol to dissolve the residue as the test solution.

[0249] 1.2 Preparation of control medicinal material solution

[0250] Take 1 g of the Radix Rehmanniae control medicinal material, add 20 ml of methanol, and ultrasonically treat (power 300 W, frequency 40 kHz) for 30 minutes, filter, evaporate the filtrate to dryness, add 20 ml of water to dissolve the residue, add water-saturated n-butanol and shake to extract twice, 20 ml each time, combine the n-butanol extracts, evaporate to dryness, and add 1 ml of methanol to dissolve the residue to prepare a control medicinal material solution.

[0251] 1.3 Preparation of reference solution

[0252] Take an appropriate amount of dichotomane-2 standard substance and add methanol to prepare a solution containing 1 mg per 1 ml, which is used as the standard solution.

[0253] 2 Thin layer chromatography conditions

[0254] Thin layer plate: Silicone G thin layer plate

[0255] Developing solvent: ① Ethyl acetate-methanol-formic acid-water (5:2:1:0.5), experimental results are shown in Figure 20 ;

[0256] ② Dichloromethane-ethyl acetate-formic acid-water (6:4:2:0.1), experimental results see Figure 21 ;

[0257] ③ Acetone-methanol-formic acid 8:1:1, experimental results see Figure 22 ;

[0258] ④ Acetone-formic acid-water 6:1:1, experimental results see Figure 23 ;

[0259] ⑤ n-Butanol-glacial acetic acid-water 3:1:1, experimental results see Figure 24 ;

[0260] ⑥ n-Butanol-ethyl acetate-formic acid-water 3:2:2:0.5, experimental results see Figure 25 ;

[0261] Spotting method: spray strip spotting.

[0262] Deployment method: A double-slot deployment cylinder is used, the pre-saturation time of the deployment cylinder is 15 minutes, and the deployment distance is about 8 cm.

[0263] Inspection: Place in iodine vapor until the spots are clearly colored, and inspect under sunlight.

[0264] 3 Conclusions:

[0265] According to the sample preparation method of Example 1, different developing agents were used for development, and the samples were placed in iodine vapor and smoked until the spots were clearly colored and examined under sunlight. Figure 20 It can be seen that the polarity of the developing agent ① is low and the spots cannot develop upwards. Figure 21 It can be seen that only one scutellarin spot was observed in the developing agent ②, which has less information and the spot has a tail. Figure 22 It can be seen that the effect of developing agent ③ is similar to that of developing agent ②, with spots tailing and Rf value being too low. Figure 23 It can be seen that no spots can be observed in the developing agent ④, which does not meet the requirements. Figure 24 It can be seen that the overall development effect of the developing agent ⑤ is similar to that of Example 1, but the separation degree and tailing phenomenon of the spots are poor. Figure 25 It can be seen that with the developing agent ⑥, only two spots were seen, the spots of lycopodine were severely tailed, and the developing effect was poor.

[0266] A comprehensive comparison of the development effects of the above developing agents shows that the upper layer solution of n-butanol-formic acid-water (volume ratio 3:1:1) placed below 10°C in Example 1 is used for development. The number of spots is the largest, the separation is better, the chromatographic spots have no tailing or diffusion, and the Rf value is moderate.

[0267] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0268] The embodiments described above only express several implementation methods of the present application, which are convenient for understanding the technical solutions of the present application in a specific and detailed manner, but they cannot be understood as limiting the scope of protection of the patent application. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present application, several variations and improvements can be made, which all fall within the scope of protection of the present application. It should be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the scope of protection of the claims attached to the present application. Therefore, the scope of protection of the patent application of this application shall be based on the content of the attached claims, and the description and drawings can be used to interpret the content of the claims.

Claims

1. A thin layer detection method for Radix Lycii Radix or its granules, characterized in that: The steps include: The Chinese medicinal material or the formulated granules of Radix Lycii were extracted with methanol, the obtained extract was evaporated to dryness, the evaporated solid was dissolved in water, extracted with n-butanol, the obtained n-butanol phase was evaporated to dryness, and the evaporated solid was dissolved in methanol to prepare the test solution; The reference medicinal material of Radix Lycii was extracted with methanol, the obtained extract was evaporated to dryness, the evaporated solid was dissolved in water, extracted with n-butanol, the obtained n-butanol phase was evaporated to dryness, and the evaporated solid was dissolved in methanol to prepare a reference medicinal material solution; Take the lycopodine standard substance, add solvent to dissolve it, and prepare the standard solution; The test solution, control medicinal material solution and reference substance solution are spotted on the same thin layer plate, developed with a mixture of n-butanol, formic acid and water as a developing agent, dried, developed with a color developer, and examined; The thin layer plate is a silica gel G thin layer plate; In the developing solvent, the volume ratio of the n-butanol, the formic acid and the water is 3:1:1; The color developer is iodine vapor; The inspection method is to observe under fluorescent light.

2. The thin layer detection method of the Chinese wolfberry bark medicinal material or formula granules according to claim 1, characterized in that: The development temperature is 10°C or lower.

3. The thin layer detection method of the Chinese wolfberry bark medicinal material or formula granules according to claim 2, characterized in that: The pre-saturation time of the expansion cylinder is 12 minutes to 20 minutes, and the expansion distance is 7 cm to 10 cm.

4. The thin layer detection method of the Chinese wolfberry bark medicinal material or the formula granules according to any one of claims 1 to 3, characterized in that: The extraction method is ultrasonic extraction.

5. The thin layer detection method of the Chinese wolfberry bark medicinal material or formula granules according to claim 4, characterized in that: The power of ultrasonic extraction is 250W to 350W, the frequency is 35kHz to 45kHz, and the time is 20min to 40min.

6. The thin layer detection method of the Chinese wolfberry bark medicinal material or the formulated granules according to any one of claims 1 to 3, characterized in that: The spotting volumes of the test solution, the control medicinal material solution, and the reference solution are each independently 8 μl to 12 μl.

7. Use of the thin layer detection method of the Chinese wolfberry bark medicinal material or the formula granules according to any one of claims 1 to 6 in the identification of Chinese wolfberry bark medicinal material and its counterfeit products.

8. The use according to claim 7, characterized in that The identification method includes: comparing the chromatogram of the test solution to determine whether the same spot exists at a position corresponding to the chromatogram of the control medicinal material solution and / or the chromatogram of the reference substance solution.

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