Method for identifying tetrastigma planicaule and caulis spatholobi based on thin-layer chromatography

Abstract

The invention relates to the technical field of identification of tetrastigma planicaule, in particular to a method for identifying tetrastigma planicaule and caulis spatholobi based on thin-layer chromatography. The invention provides a method for identifying tetrastigma planicaule and caulis spatholobi based on thin-layer chromatography. The method comprises the following steps: (1) preparing a reference medicinal material solution; preparing a test solution; preparation of a reference substance solution: taking a resveratrol reference substance, and preparing the resveratrol reference substance solution by using methanol; taking a piceatannol reference substance, and adding methanol to prepare a piceatannol reference substance solution; and (2) sucking the test solution, the reference medicinal material solution and the reference substance solution, respectively dripping on the same silica gel G thin-layer plate, developing by using a developing solvent, taking out, airing, and inspecting under ultraviolet light. The method is low in cost and easy to popularize, has both accuracy and efficiency, is suitable for large-scale medicinal material screening and daily inspection work, and has important practical application value.

Description

Technical Field

[0001] This invention relates to the field of identification technology of *Symplocos buergeriana*, and in particular to a method for identifying *Symplocos buergeriana* and *Symplocos macrantha* based on thin-layer chromatography. Background Technology

[0002] *Tetrastigma planicaule* (Hook.f.) Gagnep., belonging to the genus *Tetrastigma* in the Vitaceae family, is mainly distributed in southwestern China and is a commonly used medicinal herb among the Zhuang and Yao ethnic groups. However, the genus *Tetrastigma* contains as many as 45 species, which, while morphologically similar, have varying effects, easily leading to market confusion. Among them, *Spatholobus suberectus* is a common adulterant. Although both have blood-activating and meridian-clearing effects, their origins and chemical compositions differ significantly, and misuse may affect efficacy or even cause adverse reactions. Therefore, establishing accurate identification methods is crucial to ensuring medication safety.

[0003] Currently, the quality standards for *Smilax china* are only included in the *Guangxi Zhuang Medicine Standard* and the *Guangxi Yao Medicine Standard*, mainly covering basic items such as appearance, microscopic identification, and thin-layer chromatography (TLC), lacking qualitative and quantitative control of chemical components. TLC identification only uses reference materials for comparison, without introducing specific active ingredients as reference standards, resulting in insufficient specificity and difficulty in effectively distinguishing closely related species. Furthermore, routine tests such as moisture and ash content cannot reflect changes in the active ingredients. The quality standards urgently need improvement to enhance controllability and scientific rigor.

[0004] To improve the quality control of *Hemiberlesia lingua*, future research can combine modern analytical techniques, such as HPLC fingerprinting, LC-MS component identification, and DNA barcoding molecular identification, to establish a multi-dimensional evaluation system. Simultaneously, its pharmacodynamic material basis (such as flavonoids and triterpenoids) should be clarified, characteristic components should be selected as quality control indicators, and content determination methods should be developed. By integrating traditional experience with modern technology, the quality standards of *Hemiberlesia lingua* can be promoted towards standardization and internationalization, ensuring the safety and efficacy of clinical medication. Summary of the Invention

[0005] The purpose of this invention is to provide a method for identifying *Symplocos buergeriana* and *Spatholobus suberectus* based on thin-layer chromatography.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a method for identifying *Spatholobus suberectus* and *Spatholobus suberectus* based on thin-layer chromatography, comprising the following steps:

[0008] (1) Preparation of reference herb solution: Take the reference herb *Symplocos buergeriana* and mix it with ethanol, sonicate, filter, evaporate to dryness, extract with chloroform 2-3 times, discard the chloroform solution, extract with ether 2-3 times, combine the ether solutions, evaporate to dryness, dissolve the residue in methanol to obtain the solution;

[0009] Preparation of the test solution: Take the sample to be tested, dissolve it in water, extract it with chloroform 2 to 3 times, discard the chloroform solution, extract it with ether 2 to 3 times, combine the ether solutions, evaporate to dryness, and dissolve the residue in methanol to obtain the test solution.

[0010] Preparation of reference solutions: Take resveratrol reference standard and prepare a resveratrol reference solution using methanol; then take paclitaxel reference standard and prepare a paclitaxel reference solution using methanol.

[0011] (2) Take 5-10 μl of the above test solution and reference medicinal material solution, and 1-2 μl of the reference solution, and spot them on the same silica gel G thin layer plate. Develop with developing solvent, remove, air dry, and examine under ultraviolet light.

[0012] Preferably, the ratio of the reference herb *Platycarpus stenoptera* to ethanol in step (1) is 2g: 35-40ml.

[0013] Preferably, the power of the ultrasound in step (1) is 450-550W and the time is 25-35min.

[0014] Preferably, the ratio of the sample to water in step (1) is 0.5g: 8-12ml.

[0015] Preferably, the concentration of the resveratrol reference solution in step (1) is 0.4–0.6 mg / ml.

[0016] Preferably, the concentration of the paclitaxel reference solution in step (1) is 0.05–0.15 mg / ml.

[0017] Preferably, the silica gel G thin film in step (2) is prepared using a 45-55% ethanol solution of 0.8-1.2% sodium dihydrogen phosphate.

[0018] Preferably, the wavelength of the ultraviolet light in step (2) is 365nm.

[0019] Preferably, the developing agent in step (2) is toluene-acetone-ethyl acetate-formic acid, with a volume ratio of 8-12:2-4:1-3:1.

[0020] Preferably, the unfolding distance in step (2) is 10 to 15 cm.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] This invention significantly improves the specificity of identification by introducing resveratrol and paclitaxel as reference standards, combined with thin-layer chromatography comparison of reference medicinal materials, effectively distinguishing between the morphologically similar *Smilax china* and *Spatholobus suberectus*. Furthermore, the method is simple to operate, employing conventional thin-layer chromatography techniques with clear steps, readily available reagents, and no need for complex instruments, making it suitable for widespread application in grassroots laboratories. Through systematic optimization of key parameters such as the developing solvent ratio and development distance, the stability and reproducibility of the method are ensured, yielding consistent chromatographic results under different brands of thin-layer plates, temperature, and humidity conditions.

[0023] Furthermore, the method of this invention exhibits high sensitivity, clearly displaying fluorescent spots of trace components, thus meeting the identification requirements for trace components. Methodological validation has demonstrated the robustness and reliability of this method, providing a scientific basis for improving the quality standards of *Smilax china*. This method is low-cost, easy to implement, and combines accuracy and efficiency, making it suitable for large-scale medicinal material screening and routine inspection work, and possesses significant practical application value. Attached Figure Description

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

[0025] Figure 1 To allow for distance observation; the distance is 8cm.

[0026] Figure 2 To allow for distance observation; the distance is 10cm.

[0027] Figure 3 To allow for distance observation; the distance is 12cm.

[0028] Figure 4 To allow for distance observation; the distance is 15cm.

[0029] Figure 5 For the purpose of spot sample size investigation

[0030] 1-3: Chicken Blood Vine YC-1; 5, 8, 10 μl;

[0031] 4-6: *Smilax china* reference material; 5, 8, 10 μl;

[0032] 7-9: *Symplocos buergeriana* YC-1; 5, 8, 10 μl;

[0033] 10-12: *Smilax china* YC-2; 5, 8, 10 μl;

[0034] 13-14: Resveratrol reference standard; 1, 2 μl;

[0035] 15-16: Paclitaxel reference standard; 1, 2 μl.

[0036] Figure 6 The saturation time was investigated; saturation time was 0 min.

[0037] Figure 7 The saturation time was investigated; saturation time was 10 minutes.

[0038] Figure 8 The saturation time was investigated; saturation time was 20 minutes.

[0039] Figure 9 Comparison of different thin-layer plates; Qingdao Ocean.

[0040] Figure 10 Comparison of different thin-layer plates; Yantai Chemical Industry.

[0041] Figure 11 Comparison of different thin-layer plates; Merck.

[0042] Figure 12 Comparison of different temperatures; 4℃.

[0043] Figure 13 Comparison of different temperatures; 20℃.

[0044] Figure 14 Comparison of different temperatures; 35℃.

[0045] Figure 15 Comparison of different humidity levels; RH = 32%.

[0046] Figure 16 Comparison of different humidity levels; RH = 65%.

[0047] Figure 17 Comparison of different humidity levels; RH = 88%.

[0048] Figure 18 Thin-layer chromatography for identification of *Symplocos buergeriana* medicinal materials Figure 1 .

[0049] Figure 19 Thin-layer chromatography for identification of *Symplocos buergeriana* medicinal materials Figure 2 . Detailed Implementation

[0050] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0051] Example 1

[0052] This study used thin-layer chromatography to control sample quality, with *Symplocos buergeriana* (a type of medicinal herb), resveratrol, and paclitaxel as identification indicators.

[0053] 1. Instruments and reagents

[0054] Electronic balance (BCE1202I-1CNN, Sartorius GmbH, Germany); Electronic balance (XSR205DU, Mettler Toledo Group); CNC ultrasonic cleaner (KQ-600DE, Kunshan Ultrasonic Instrument Co., Ltd.); CNC ultrasonic cleaner (KQ2200E, Kunshan Ultrasonic Instrument Co., Ltd.); Digital display precision constant temperature water bath (DK-98-ⅡA, Tianjin Test Instrument Co., Ltd.); Electric thermostatic drying oven (WGLL-125BE, Tianjin Test Instrument Co., Ltd.); Automatic thin-layer chromatography spotting instrument (ATS 4, CAMAG, Switzerland); Digital imaging system for thin-layer chromatography (TLC Visualizer 2, CAMAG, Switzerland); Pre-fabricated silica gel G-plate for thin-layer chromatography (Merck); Pre-fabricated silica gel G-plate for thin-layer chromatography (Qingdao Ocean Chemical Co., Ltd.); Pre-fabricated silica gel G-plate for thin-layer chromatography (Yantai Chemical Industry Research Institute).

[0055] Resveratrol reference standard (batch number 111535-201703, stored at -20℃, China National Institutes for Food and Drug Control); Paclitaxel reference standard (batch number 2415698, stored at 2-8℃, Shanghai Anpu Cuishi Standard Technical Service Co., Ltd.); *Ipomoea batatas* reference material (Institute of Traditional Chinese Medicine and Ethnic Medicine, Guangxi Zhuang Autonomous Region Institute for Drug Control). All other reagents were of analytical grade.

[0056] 2 Thin-layer chromatography method

[0057] Preparation of the control herbal solution: Take 2g of *Symplocos buergeriana* (a type of vine) as the control herbal material, add 40ml of ethanol, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, add 10ml of water to dissolve, extract twice with 10ml of chloroform each time, discard the chloroform solution, and then extract twice with 10ml of ether each time. Combine the ether solutions, evaporate to dryness, and dissolve the residue in 1ml of methanol to obtain the control herbal solution.

[0058] Preparation of the test solution: Take 0.5g of the powder, add 10ml of water to dissolve, extract twice with 10ml of chloroform each time, discard the chloroform solution, and then extract twice with 10ml of ether each time. Combine the ether solutions, evaporate to dryness, and dissolve the residue in 1ml of methanol to obtain the test solution.

[0059] Preparation of reference solutions: Take resveratrol reference standard and dissolve it in methanol to prepare a solution containing 0.5 mg per ml. Then take paclitaxel reference standard and dissolve it in methanol to prepare a solution containing 0.1 mg per ml.

[0060] The assay was performed according to thin-layer chromatography (General Chapter 0502, Chinese Pharmacopoeia 2020). 8 μl each of the test solution and the reference medicinal material solution, and 2 μl of the reference solution, were spotted separately onto the same silica gel G thin-layer plate prepared with a 1% sodium dihydrogen phosphate solution in 50% ethanol. The plate was developed to 12 cm using toluene-acetone-ethyl acetate-formic acid (volume ratio 10:3:2:1). The plate was then removed, air-dried, and examined under ultraviolet light (365 nm). Fluorescent spots of the same color appeared at the corresponding positions in the chromatogram of the test sample and the reference medicinal material; similarly, fluorescent spots of the same color appeared at the corresponding positions in the chromatogram of the reference solution.

[0061] Example 2

[0062] The following methods are all based on the method in Example 1, and aim to examine the impact of different parameter settings on the detection results:

[0063] 1 Investigation of chromatographic conditions

[0064] 1.1 Expansion Distance Examination

[0065] After spotting, the thin-layer chromatographic plates were developed under the above-mentioned thin-layer chromatographic conditions at development distances of 8 cm, 10 cm, 12 cm, and 15 cm for identification. The results showed that at a development distance of 12 cm, the thin-layer spots of the test sample (*Smilax china*) and the adulterant (*Spatholobus suberectus*) were separated better, effectively distinguishing *Smilax china* and its adulterant. (Chromatographia spp.) Figures 1-4 (1-3: Chicken Blood Vine YC-1~YC-3; 8μl; 4: Common Potentilla Reference Material; 8μl; 5-12: Common Potentilla YC-1~YC-8; 8μl; 13: Resveratrol Reference Standard; 2μl; 14: Paclitaxel Reference Standard; 1μl).

[0066] 1.2 Investigation of Spot Sample Quantity

[0067] Different volumes of the test sample solution, reference medicinal material solution, and reference solution were spotted separately onto the same silica gel G thin-layer plate prepared with a 1% sodium dihydrogen phosphate solution in 50% ethanol. Under the aforementioned thin-layer chromatographic conditions, the results showed that when the sample volume for the test sample solution and reference medicinal material solution was 5–10 μl, and when the sample volume for the reference solution was 1–2 μl, the spots were clear after development, with good resolution and no tailing. For ease of evaluation, an 8 μl sample volume was used for both the reference medicinal material and the test sample. The chromatogram is shown below. Figure 5 .

[0068] 1.3 Saturation Time Examination

[0069] Thin-layer plates were developed and identified under the above-mentioned thin-layer chromatographic conditions at unsaturated, saturated for 10 min, and saturated for 20 min, respectively. The results showed that the saturation time had no effect on the chromatographic results. (Chromatograms are shown below.) Figures 6-8(1-3: Chicken Blood Vine YC-1~YC-3; 8μl; 4: Common Potentilla Reference Material; 8μl; 5-12: Common Potentilla YC-1~YC-8; 8μl; 13: Resveratrol Reference Standard; 2μl; 14: Paclitaxel Reference Standard; 1μl).

[0070] 2. Methodological Validation

[0071] 2.1 Comparison of different thin-layer plates

[0072] Different brands of silica gel G pre-prepared thin-layer plates (Qingdao Ocean, Yantai Chemical, Merck) were selected and prepared using a 1% sodium dihydrogen phosphate solution in 50% ethanol. The plates were then developed and identified according to the aforementioned thin-layer chromatographic conditions. The results showed that all brands of thin-layer plates met the identification requirements. The chromatograms are shown below. Figures 9-11 (1-3: Chicken Blood Vine YC-1~YC-3; 8μl; 4: Common Potentilla Reference Material; 8μl; 5-12: Common Potentilla YC-1~YC-8; 8μl; 13: Resveratrol Reference Standard; 2μl; 14: Paclitaxel Reference Standard; 1μl).

[0073] 2.2 Comparison of different temperatures

[0074] Identification was performed under the above-described thin-layer chromatographic conditions at temperatures of 4℃, 20℃, and 35℃. The results showed little difference in the chromatograms, indicating good robustness of this method to ambient temperatures. (Chromatograms are shown below.) Figures 12-14 (1-3: Chicken Blood Vine YC-1~YC-3; 8μl; 4: Common Potentilla Reference Material; 8μl; 5-12: Common Potentilla YC-1~YC-8; 8μl; 13: Resveratrol Reference Standard; 2μl; 14: Paclitaxel Reference Standard; 1μl).

[0075] 2.3 Comparison of different humidity levels

[0076] Thin-layer plates after sampling were developed in developing tanks with humidity levels of 32%, 65%, and 88%, respectively. The results showed no significant differences in the chromatograms under different humidity conditions, indicating that this method has good adaptability to varying humidity environments. (Chromatograms are shown below.) Figures 15-17 (1-3: Chicken Blood Vine YC-1~YC-3; 8μl; 4: Common Potentilla Reference Material; 8μl; 5-12: Common Potentilla YC-1~YC-8; 8μl; 13: Resveratrol Reference Standard; 2μl; 14: Paclitaxel Reference Standard; 1μl).

[0077] 3. Thin-layer chromatography identification of medicinal materials

[0078] Sixteen batches of *Aristolochia debilis* medicinal material test solutions were prepared according to the "Preparation of Test Solution" in Example 1, and then identified. The results showed that in the chromatograms of the test solutions, fluorescent spots of the same color appeared at the corresponding positions as in the chromatograms of the reference medicinal material; and fluorescent spots of the same color appeared at the corresponding positions as in the chromatograms of the reference substance. (See chromatograms below.) Figures 18-19 Sources of medicinal materials: Chicken Blood Vine YC-1~YC-3: Vietnam; Comparative medicinal material of *Smilax china*: Lipu, Guangxi; *Smilax china* YC-1: Lipu, Guangxi; *Smilax china* YC-2: Rongshui, Guangxi; *Smilax china* YC-3: Gongcheng, Guangxi; *Smilax china* YC-4: Lipu, Guangxi; *Smilax china* YC-5: Lipu, Guangxi; *Smilax china* YC-6: Fuzhou, Fujian; *Smilax china* YC-7: Sanya, Hainan; *Smilax china* YC-8: Jinxiu, Guangxi. Chicken Blood Vine YC-1~YC-3: Vietnam; Comparative Medicinal Herb of *Smilax china*: Lipu, Guangxi; Comparative Medicinal Herb of *Smilax china* YC-9: Pingnan, Guangxi; Comparative Medicinal Herb of *Smilax china* YC-10: Anlong, Guizhou; Comparative Medicinal Herb of *Smilax china* YC-11: Sanya, Hainan; Comparative Medicinal Herb of *Smilax china* YC-12: Baoshan, Yunnan; Comparative Medicinal Herb of *Smilax china* YC-13: Yulin, Guangxi; Comparative Medicinal Herb of *Smilax china* YC-14: Gongcheng, Guangxi; Comparative Medicinal Herb of *Smilax china* YC-15: Gongcheng, Guangxi; Comparative Medicinal Herb of *Smilax china* YC-16: Gongcheng, Guangxi.

[0079] Table 1. Selection and sampling quantity of different medicinal materials

[0080]

[0081]

[0082] Table 2. Selection and sampling quantity of different medicinal materials

[0083]

[0084] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for identifying *Smilax china* and *Spatholobus suberectus* based on thin-layer chromatography, characterized in that, Includes the following steps: (1) Preparation of reference herb solution: Take the reference herb *Symplocos buergeriana* and mix it with ethanol, sonicate, filter, evaporate to dryness, extract with chloroform 2-3 times, discard the chloroform solution, extract with ether 2-3 times, combine the ether solutions, evaporate to dryness, dissolve the residue in methanol to obtain the solution; Preparation of the test solution: Take the sample to be tested, dissolve it in water, extract it with chloroform 2 to 3 times, discard the chloroform solution, extract it with ether 2 to 3 times, combine the ether solutions, evaporate to dryness, and dissolve the residue in methanol to obtain the test solution. Preparation of reference solutions: Take resveratrol reference standard and prepare a resveratrol reference solution using methanol; then take paclitaxel reference standard and prepare a paclitaxel reference solution using methanol. (2) Take 5-10 μl of the above test solution and reference medicinal material solution, and 1-2 μl of the reference solution, and spot them on the same silica gel G thin layer plate. Develop with developing solvent, remove, air dry, and examine under ultraviolet light.

2. The method according to claim 1, characterized in that, In step (1), the ratio of the reference herb *Smilax china* to ethanol is 2g: 35-40ml.

3. The method according to claim 1, characterized in that, The ultrasound power in step (1) is 450-550W and the time is 25-35min.

4. The method according to claim 1, characterized in that, The ratio of the sample to water in step (1) is 0.5g: 8-12ml.

5. The method according to claim 1, characterized in that, The concentration of the resveratrol reference solution in step (1) is 0.4–0.6 mg / ml.

6. The method according to claim 1, characterized in that, The concentration of the paclitaxel reference solution in step (1) is 0.05–0.15 mg / ml.

7. The method according to claim 1, characterized in that, The silica gel G thin film in step (2) is prepared using a 45-55% ethanol solution of 0.8-1.2% sodium dihydrogen phosphate.

8. The method according to claim 1, characterized in that, The wavelength of the ultraviolet light in step (2) is 365nm.

9. The method according to claim 1, characterized in that, The developing solvent in step (2) is toluene-acetone-ethyl acetate-formic acid, with a volume ratio of 8-12:2-4:1-3:

1.

10. The method according to claim 1, characterized in that, The unfolding distance in step (2) is 10-15cm.

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