A multi-information rapid TLC overall identification method for Dendrobium officinale Yangwei granules

Abstract

The present invention discloses a multi-information rapid TLC overall identification method for Dendrobium officinale nourishing stomach granules. Utilizing the principle that each component in the medicinal material has different adsorption capacity for the same adsorbent, methanol extraction and water-saturated n-butanol-ether extraction are used in sequence, and characteristic spots of three medicinal materials, namely, Bletilla striata, Panax notoginseng and Licorice, are simultaneously inspected for the first time. This method only requires the preparation of a test solution, and the use of the same thin layer plate and one inspection condition can obtain characteristic spot information of a variety of medicinal materials, and the spot color differentiation is high, the overlap is low, and the repeatability is strong, thus avoiding the inconvenience caused by different inspection conditions. Compared with conventional TLC in which a method can only identify a single medicine, this method has better separation and stronger durability, greatly improves the controllability of the quality standard of Dendrobium officinale nourishing stomach granules, improves inspection efficiency, and is an important guarantee for achieving the effectiveness and safety of the preparation.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine component detection, and particularly relates to a multi-information rapid TLC overall identification method for Dendrobium officinale stomach-nourishing granules. Background Art

[0002] Stable and controllable quality is the fundamental measure to ensure the safety and effectiveness of Chinese herbal compound preparations. The efficacy of Chinese herbal compound preparations is the result of the combined action of multiple ingredients, and the integrity of their quality evaluation has become an inevitable trend.

[0003] TLC identification is a key quality control method for traditional Chinese medicines (TCMs), herbal extracts, Chinese patent medicines, and chemical pharmaceuticals. Its advantages include simple equipment requirements, ease of operation, and rapid separation speed, leading to its widespread use. However, due to the multiplicity of medicinal ingredients in TCM compounds, the complexity of their components, which can interfere with each other and hinder separation, a single TLC method is often only effective for identifying a single ingredient, failing to meet the requirements of modern TCM preparation research, which relies on holistic identification. Overcoming the challenges of overall quality assessment, efficient and rapid identification, reflecting the overall quality characteristics of a preparation with a relatively small number of TLC tests, is crucial for the overall quality control of TCM compounds.

[0004] Dendrobium officinale stomach-nourishing granules, composed of multiple Chinese medicinal herbs including Dendrobium officinale, Bletilla striata, Panax notoginseng, and Licorice root, are known to invigorate the spleen and stomach, stop bleeding, and promote tissue regeneration. Modern pharmacological studies have shown that Dendrobium officinale stomach-nourishing granules have a significant preventive and therapeutic effect on gastric mucosal damage. Bletilla striata, Panax notoginseng, and Licorice root are the three medicinal herbs in Dendrobium officinale stomach-nourishing granules. However, currently, no suitable method exists for the comprehensive identification of these three herbs, and existing methods for identifying individual herbs are complex and inefficient. Therefore, it is of great significance to establish a simple and reproducible method for the comprehensive detection of the multiple ingredients in Dendrobium officinale stomach-nourishing granules. Summary of the Invention

[0005] In response to the above technical problems, the purpose of the present invention is to provide a multi-information rapid TLC overall identification method for Dendrobium officinale Yangwei Granules. This method only requires the preparation of a test solution, and can be used under one development condition and one inspection condition to comprehensively identify the three medicinal materials, Bletilla striata, Panax notoginseng, and Licorice, from the Dendrobium officinale Yangwei Granule system through thin layer chromatography.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A multi-information rapid TLC overall identification method for Dendrobium officinale stomach-nourishing granules comprises the following steps:

[0008] 1) Preparation of test solution: Weigh Dendrobium officinale Yangwei Granules, extract with methanol, filter, evaporate the filtrate to dryness, dissolve the residue in water, extract with water-saturated n-butanol, take the n-butanol layer, wash with water saturated with n-butanol, discard the water, extract with ether, take the upper layer, evaporate to dryness, and dilute the residue to volume with methanol to prepare the test solution;

[0009] 2) Preparation of reference solution: Take Bletilla striata, Panax notoginseng, and Glycyrrhiza uralensis as reference herbs, and extract them according to the method for preparing the test solution in step 1) to prepare the reference solution;

[0010] 3) Thin layer chromatography identification: Pipette the same amount of test solution, Bletilla striata reference solution, Panax notoginseng reference solution and Glycyrrhiza uralensis reference solution, respectively, and spot them on the same silica gel G thin layer plate prepared with 1% sodium hydroxide solution, develop with cyclohexane-ethyl acetate-methanol as the developing solvent, take out, dry, spray with 10% sulfuric acid ethanol solution, heat until the spots are clear, and examine under 365nm ultraviolet light. In the chromatogram of the test sample, the corresponding position of the Bletilla striata reference will show the same green fluorescent spot under ultraviolet light, the corresponding position of the Panax notoginseng reference will show the same reddish-pink fluorescent spot under ultraviolet light, the corresponding position of the Glycyrrhiza uralensis reference will show the same yellow spot under visible light, and the same green and blue spots under ultraviolet light.

[0011] Furthermore, the extraction method in step 1 and step 2) is ultrasonic extraction for 10-120 minutes.

[0012] Furthermore, in step 1), the mass ratio of the Dendrobium officinale nourishing stomach granules in step 1) to the amount of methanol added for extraction is 5:1-50 by volume; the mass ratio of the Dendrobium officinale nourishing stomach granules to the amount of water-saturated n-butanol solution added is 5:1-50 by volume, and the number of extractions is 1-10 times; the amount of the n-butanol-saturated aqueous solution added is 1-8 times that of the water-saturated n-butanol solution; the mass ratio of the Dendrobium officinale nourishing stomach granules to the amount of ether added is 5:1-50 by volume, with mass units being g and volume units being mL.

[0013] Furthermore, in step 3), 0.1 to 15 μL of each of the test solution, Bletilla striata reference solution, Panax notoginseng reference solution, and Licorice reference solution was aspirated.

[0014] Furthermore, in step 3), the volume ratio of cyclohexane-ethyl acetate-methanol is 5-40:1-35:0.5-15.

[0015] Furthermore, the expansion distance described in step 3) is 6 to 12 cm.

[0016] Furthermore, in step 3), the volume ratio of cyclohexane-ethyl acetate-methanol is 14:8:3.

[0017] Compared with traditional TLC identification, the present invention has the following advantages:

[0018] (1) The processing method for preparing the test sample solution of the present invention is simple and easy to operate. By using the same test sample and the same developing agent, the white radix schizonepetae, the white radix notoginseng and the white licorice in the Dendrobium officinale stomach-nourishing granules can be identified separately on a thin layer chromatography plate, which shortens the thin layer chromatography identification time, improves the identification efficiency, reduces the consumption of reference substances and reagents, and can identify the three drugs as a whole without step-by-step color development and inspection. It is easy to control, greatly reduces the detection cost, and can meet the production detection requirements.

[0019] (2) The three control medicinal materials in the present invention are treated with the same method, which can meet the requirements for the identification of the three medicinal materials in the Dendrobium officinale Yangwei Granule system. The identification characteristic spots in the present invention are clear, the separation is good, and the negative is free of interference. Experiments have found that Bletilla striata can be identified under a 365 nm ultraviolet lamp after being sprayed with 10% sulfuric acid ethanol and heated, and Panax notoginseng and Licorice can be identified under visible light and a 365 nm ultraviolet lamp after being sprayed with 10% sulfuric acid ethanol and heated. This method has strong specificity and good repeatability, and can more accurately and comprehensively control the quality of Dendrobium officinale Yangwei Granules. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Thin-layer plate for the common identification of Bletilla striata, Panax notoginseng, and Glycyrrhiza uralensis - characteristic spots for identification of Bletilla striata, Panax notoginseng, and Glycyrrhiza uralensis (sprayed with 10% sulfuric acid ethanol and then heated, 365 nm UV light), where 1 is Bletilla striata reference solution, 2 is Bletilla striata negative control solution, 3-5 are test solution, 6 is Glycyrrhiza uralensis reference solution, 7 is Glycyrrhiza uralensis negative control solution, 8 is Panax notoginseng reference solution, and 9 is Panax notoginseng negative control solution;

[0021] Figure 2 Thin layer plate for the common identification of Bletilla striata, Panax notoginseng and Licorice - characteristic spots for the identification of Bletilla striata, Panax notoginseng and Licorice (sprayed with 10% sulfuric acid ethanol and then heated, visible light), where 1 is the Bletilla striata reference solution, 2 is the Bletilla striata negative control solution, 3-5 are the test solution, 6 is the Licorice reference solution, 7 is the Licorice negative control solution, 8 is the Panax notoginseng reference solution, and 9 is the Panax notoginseng negative control solution. DETAILED DESCRIPTION

[0022] The present invention will be further described below with reference to specific embodiments and the accompanying drawings, but the scope of protection of the present invention is not limited thereto. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0023] The thin layer plates used in the examples of the present invention were purchased from Qingdao Ocean Chemical Co., Ltd.

[0024] Sample and control herbs:

[0025] Three batches of Dendrobium officinale stomach-nourishing granules were produced by the Yangtze River Delta Green Pharmaceutical Collaborative Innovation Center of Zhejiang University of Technology. The control medicinal materials of Bletilla striata (121262-201706), Panax notoginseng (120941-201810), and Glycyrrhiza uralensis (120904-202021) were all purchased from the China Food and Drug Inspection Institutes. Example 1

[0026] (1) Preparation of test solution: Weigh 5 g of Dendrobium officinale stomach-nourishing granules, add 30 mL of methanol, sonicate for 60 min, let cool to room temperature, filter, evaporate the filtrate in a water bath, dissolve it in 20 mL of water, extract it with 30 mL of water-saturated n-butanol, seal it tightly, shake it vigorously, and let it stand to separate; take the n-butanol layer, add an equal amount of water saturated with n-butanol, shake it well, and let it stand to separate; take the n-butanol layer, add 30 mL of ether, seal it tightly, shake it vigorously, let it stand to separate, collect the upper layer solution, evaporate it to dryness, and make up to 1 mL with methanol.

[0027] (2) Preparation of negative control solution: The preparation method of negative control solution lacking Bletilla striata, Panax notoginseng and Glycyrrhiza uralensis is the same as that of test sample solution.

[0028] (3) Preparation of control medicinal material solution: Take 1 g of Bletilla striata control medicinal material, 0.5 g of Panax notoginseng control medicinal material, and 0.3 g of Glycyrrhiza uralensis control medicinal material, and prepare the control medicinal material solution in the same way as the test solution.

[0029] (4) Thin layer chromatography identification: 3 μL each of the test solution, negative control solution lacking Bletilla striata, Bletilla striata control medicinal material solution, negative control solution lacking Panax notoginseng, Panax notoginseng control medicinal material solution, negative control solution lacking Licorice, and Licorice control medicinal material solution were taken and spotted on the same silica gel G thin layer plate prepared with 1% sodium hydroxide solution by mass concentration, and cyclohexane-ethyl acetate-methanol (volume ratio of 14:8:3) was used as the developing solvent. Develop 8 cm, take out, dry, spray with 10% sulfuric acid ethanol solution by mass concentration, heat until the spots are clear, and place under 365nm ultraviolet light for inspection. The results are as follows Figure 1 and Figure 2 As shown, in the chromatogram of the test sample, at the corresponding position with the Bletilla striata reference substance, the same green fluorescent spot appeared under ultraviolet light, and there was no interference in the negative test; at the corresponding position with the Panax notoginseng reference substance, the same red-purple spot appeared under visible light, and the same red-pink fluorescent spot appeared under ultraviolet light, and there was no interference in the negative test; at the corresponding position with the Licorice reference substance, the same yellow spot appeared under visible light, and the same color spot appeared under ultraviolet light, and there was no interference in the negative test. Example 2

[0030] (1) The test solution was prepared in the same manner as in Example 1;

[0031] (2) The negative control solution was prepared in the same manner as in Example 1;

[0032] (3) The preparation method of the control medicinal material solution is the same as that of Example 1;

[0033] (4) Thin layer chromatography identification: 3 μL each of the test solution, negative control solution lacking Bletilla striata, Bletilla striata control medicinal material solution, negative control solution lacking Sanqi, Sanqi control medicinal material solution, negative control solution lacking Licorice, and Licorice control medicinal material solution were taken and spotted on the same silica gel G thin layer plate prepared with 1% sodium hydroxide solution by mass concentration, and 8 cm was developed with cyclohexane-ethyl acetate-methanol (volume ratio of 5:3:2). The plate was taken out, dried, and sprayed with 10% sulfuric acid ethanol solution by mass concentration. The plate was heated until the spots were clear and then placed under 365 nm ultraviolet light for inspection. In the chromatogram of the test sample, at the position corresponding to the Bletilla striata control, the corresponding spot R f The value is too large and the separation is poor; at the corresponding position with the Panax notoginseng reference substance, the same color of red-purple spots appear under visible light, the same red-pink fluorescent spots appear under ultraviolet light, and there is no negative interference; at the corresponding position with the licorice reference substance, the same yellow spots appear under visible light, the same color spots appear under ultraviolet light, but the corresponding spots cannot be completely separated. Example 3

[0034] (1) The test solution was prepared in the same manner as in Example 1;

[0035] (2) The negative control solution was prepared in the same manner as in Example 1;

[0036] (3) The preparation method of the control medicinal material solution is the same as that of Example 1;

[0037] (4) Thin layer chromatography identification: 3 μL each of the test solution, negative control solution lacking Bletilla striata, Bletilla striata control medicinal material solution, negative control solution lacking Panax notoginseng, Panax notoginseng control medicinal material solution, negative control solution lacking Glycyrrhiza uralensis, and Glycyrrhiza uralensis control medicinal material solution were aspirated and spotted on the same silica gel G thin layer plate prepared with 1% sodium hydroxide solution by mass concentration, and developed with cyclohexane-ethyl acetate-methanol (volume ratio of 12:5:3) for 8 cm, taken out, dried, sprayed with 10% sulfuric acid ethanol solution by mass concentration, heated until the spots were clear, and inspected under 365 nm ultraviolet light. In the chromatogram of the test sample, at the corresponding position with the Bletilla striata reference substance, the same green fluorescent spot appeared under ultraviolet light, but the corresponding spots could not be completely separated; at the corresponding position with the Panax notoginseng reference substance, the same red-purple spot appeared under visible light, the same red-pink fluorescent spot appeared under ultraviolet light, and there was no interference under negative test; at the corresponding position with the Licorice reference substance, the same yellow spot appeared under visible light, the same color spot appeared under ultraviolet light, and there was no interference under negative test. Example 4

[0038] (1) The test solution was prepared in the same manner as in Example 1;

[0039] (2) The negative control solution was prepared in the same manner as in Example 1;

[0040] (3) The preparation method of the control medicinal material solution is the same as that of Example 1;

[0041] (4) Thin layer chromatography identification: 3 μL each of the test solution, negative control solution lacking Bletilla striata, Bletilla striata control medicinal material solution, negative control solution lacking Panax notoginseng, Panax notoginseng control medicinal material solution, negative control solution lacking Glycyrrhiza uralensis, and Glycyrrhiza uralensis control medicinal material solution were taken and spotted on the same silica gel G thin layer plate prepared with 1% sodium hydroxide solution by mass concentration. The plate was developed with cyclohexane-ethyl acetate-methanol (volume ratio of 14:8:3) and developed to 8 cm. The plate was taken out, dried, and then placed in the same developing agent for secondary development to 8 cm. The plate was taken out, dried, and sprayed with 10% sulfuric acid ethanol solution. The plate was heated until the spots were clear and examined under a 365 nm ultraviolet lamp. In the chromatogram of the test sample, the corresponding position with the Bletilla striata reference showed the same green fluorescent spot under UV light, and there was no interference in the negative assay. The corresponding position with the Panax notoginseng reference showed the same reddish-purple spot under visible light and the same reddish-pink fluorescent spot under UV light, and there was no interference in the negative assay. The corresponding position with the Licorice reference showed the same yellow spot under visible light and the same color spot under UV light, and there was no interference in the negative assay. Although the secondary development method achieved better separation results, a single development method was sufficient for separation under these development conditions. Example 5

[0042] (1) The test solution was prepared in the same manner as in Example 1;

[0043] (2) The negative control solution was prepared in the same manner as in Example 1;

[0044] (3) The preparation method of the control medicinal material solution is the same as that of Example 1;

[0045] (4) Thin layer chromatography identification: 3 μL each of the test solution, negative control solution lacking Bletilla striata, Bletilla striata control medicinal material solution, negative control solution lacking Sanqi, Sanqi control medicinal material solution, negative control solution lacking Licorice, and Licorice control medicinal material solution were aspirated and spotted on the same silica gel G thin layer plate prepared with 1% sodium hydroxide solution by mass concentration. The plate was developed with toluene-ethyl acetate-methanol (volume ratio of 16:4:1) and developed to 8 cm. The plate was removed, dried, sprayed with 10% sulfuric acid ethanol solution by mass concentration, heated until the spots were clear, and examined under 365 nm ultraviolet light. In the chromatogram of the test sample, at the position corresponding to the Bletilla striata control, the same green fluorescent spot appeared under ultraviolet light, and there was no negative interference; at the position corresponding to the Sanqi control, Sanqi could not be effectively identified; at the position corresponding to the Licorice control, the same yellow spot appeared under visible light and the same color spot appeared under ultraviolet light, but the corresponding spots were not clear and could not be completely separated. Example 6

[0046] (1) The test solution was prepared in the same manner as in Example 1;

[0047] (2) The negative control solution was prepared in the same manner as in Example 1;

[0048] (3) The preparation method of the control medicinal material solution is the same as that of Example 1;

[0049] (4) Thin layer chromatography identification: 3 μL each of the test solution, negative control solution lacking Bletilla striata, control medicinal material solution of Bletilla striata, negative control solution lacking Sanqi, control medicinal material solution of Sanqi, negative control solution lacking Licorice, and control medicinal material solution of Licorice were taken and spotted on the same silica gel G thin layer plate prepared with 1% sodium hydroxide solution by mass concentration. The plate was developed with chloroform-ethyl acetate-methanol-water (volume ratio of 3:8:5:2) and developed to 8 cm. The plate was removed, dried, sprayed with 10% sulfuric acid ethanol solution by mass concentration, heated until the spots were clear, and examined under 365 nm ultraviolet light. In the chromatogram of the test sample, Bletilla striata, Sanqi, and Licorice could not be effectively identified.

[0050] In summary, the thin layer chromatography method for overall identification of three single medicinal materials provided in the embodiment of the present invention can overall identify Bletilla striata, Panax notoginseng, and Licorice in Dendrobium officinale Yangwei Granules, indicating that the method has high accuracy and strong repeatability.

[0051] The embodiments described above are some, but not all, embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but rather merely represents selected embodiments of the present invention. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without creative effort are intended to fall within the scope of protection of the present invention.

Claims

1. A multi-information rapid TLC overall identification method for Dendrobium officinale nourishing stomach granules, characterized in that The steps include: 1) Preparation of test solution: Weigh Dendrobium officinale Yangwei Granules, extract with methanol, filter, evaporate the filtrate to dryness, dissolve the residue in water, extract with water-saturated n-butanol, take the n-butanol layer, wash with water saturated with n-butanol, discard the water, extract with ether, take the upper layer, evaporate to dryness, and dilute the residue to volume with methanol to prepare the test solution; 2) Preparation of reference solution: Take Bletilla striata, Panax notoginseng, and Glycyrrhiza uralensis as reference herbs, and extract them according to the method for preparing the test solution in step 1) to prepare the reference solution; 3) Thin layer chromatography identification: Pipette the same amount of test solution, Bletilla striata reference solution, Panax notoginseng reference solution and Glycyrrhiza uralensis reference solution, respectively, and spot them on the same silica gel G thin layer plate prepared with 1% sodium hydroxide solution, develop with cyclohexane-ethyl acetate-methanol as the developing solvent, take out, dry, spray with 10% sulfuric acid ethanol solution, heat until the spots are clear, and examine under 365nm ultraviolet light. In the chromatogram of the test sample, the corresponding position of the Bletilla striata reference will show the same green fluorescent spot under ultraviolet light, the corresponding position of the Panax notoginseng reference will show the same reddish-pink fluorescent spot under ultraviolet light, the corresponding position of the Glycyrrhiza uralensis reference will show the same yellow spot under visible light, and the same green and blue spots under ultraviolet light; In step 3), the volume ratio of cyclohexane-ethyl acetate-methanol is 5-40:1-35:0.5-15.

2. The multi-information rapid TLC overall identification method of Dendrobium officinale nourishing stomach granules according to claim 1 is characterized in that The extraction method in step 1 and step 2) is ultrasonic extraction for 10-120 minutes.

3. The multi-information rapid TLC overall identification method of Dendrobium officinale Yangwei granules according to claim 1 is characterized in that The mass ratio of the Dendrobium officinale nourishing stomach granules in step 1) to the amount of methanol added for extraction is 5:1-50 by volume; the mass ratio of the Dendrobium officinale nourishing stomach granules to the amount of water-saturated n-butanol solution added is 5:1-50 by volume, and the number of extractions is 1-10 times; the amount of the n-butanol-saturated aqueous solution added is 1-8 times that of the water-saturated n-butanol solution; the mass ratio of the Dendrobium officinale nourishing stomach granules to the amount of ether added is 5:1-50 by volume, with mass units being g and volume units being mL.

4. The multi-information rapid TLC overall identification method of Dendrobium officinale Yangwei granules according to claim 1 is characterized in that In step 3), aspirate 0.1-15 μL each of the test solution, Bletilla striata reference solution, Panax notoginseng reference solution, and Licorice reference solution.

5. The multi-information rapid TLC overall identification method of Dendrobium officinale Yangwei granules according to claim 1 is characterized in that In step 3), the expansion distance is 6~12 cm.

6. The multi-information rapid TLC overall identification method of Dendrobium officinale Yangwei granules according to claim 1 is characterized in that In step 3), the volume ratio of cyclohexane-ethyl acetate-methanol is 14:8:3.

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