Method for distinguishing plumbago zeylanica and counterfeit arabia sambac based on thin layer chromatography
By using a mixture of toluene, methanol, glacial acetic acid, and water as the developing solvent, combined with thin-layer chromatography, the problem of distinguishing between *Plumbago semperflorens* and the adulterant *Jasminum nudiflorum* was solved, providing a simple and efficient identification method and improving the quality control capabilities of medicinal materials.
Patent Information
- Application Number
- CN202511311262.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-15
- Publication Date
- 2025-11-14
AI Technical Summary
Existing thin-layer chromatography methods cannot effectively distinguish between Plumbago salsa and the adulterant jasmine, which increases the difficulty of quality control of medicinal materials.
A mixture of toluene, methanol, glacial acetic acid, and water was used as the developing solvent in a volume ratio of 6–8:0.8–1.2:0.4–0.6:0.4–0.6. Thin-layer chromatography was used to distinguish between *Plumbago salsa* and the adulterant *Jasminum nudiflorum*, including ultrasonic extraction, spotting, and development steps, followed by detection under ultraviolet light.
It enables a simple and easy distinction between white peony and counterfeit jasmine, with high detection efficiency, intuitive and clear results, and objective and reliable conclusions, supporting the quality control of medicinal materials.
Smart Images
Figure CN120948686A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of white peony identification technology, and in particular to a method for distinguishing white peony from the counterfeit jasmine based on thin-layer chromatography. Background Technology
[0002] The white peony (Plumbago alatus) was first recorded in *Sheng Cao Yao Xing Bei Yao* (Prepared Medicinal Herbs for Herbal Properties) and has been widely used in folk medicine. The dried roots, stems, leaves, or whole plant of the white peony can all be used medicinally. Experimental studies have shown that white peony contains components such as plumbagoside, isovitexin, vanillic acid, xanthophyllone, peonyxin, plumbagoside ketone, transcinnamic acid, cork xanthophyllone, artemisinin, neoechinulin A, matoquinone, 3-indolecarboxaldehyde, rotenone, peony ketone, 1,4-androstadiene-3,17-dione, and 2,5-dimethyl-7-hydroxychromone. Existing thin-layer chromatography (TLC) methods cannot distinguish between white peony and its adulterant, *Pseudojasminoides var. sarcodactylis*. Therefore, developing a simple and easy-to-use TLC method to differentiate white peony from easily confused adulterants is crucial. Summary of the Invention
[0003] The purpose of this invention is to provide a method for distinguishing between *Plumbago salsa* and counterfeit *Jasminum nudiflorum* based on thin-layer chromatography. This method can distinguish between genuine and counterfeit *Plumbago salsa* using only thin-layer chromatography. The method is simple, easy to implement, and has good adaptability.
[0004] To achieve the above-mentioned objectives, the present invention provides the following technical solution:
[0005] The present invention provides a developing solvent for thin-layer chromatography, wherein the developing solvent is a mixture of toluene, methanol, glacial acetic acid and water, and the volume ratio of toluene, methanol, glacial acetic acid and water is 6-8:0.8-1.2:0.4-0.6:0.4-0.6.
[0006] Preferably, the developing agent is the upper layer solution of the mixture.
[0007] The present invention also provides the application of the aforementioned developing agent in distinguishing between white peony and counterfeit jasmine.
[0008] The present invention also provides a method for distinguishing between *Plumbago salsa* and counterfeit *Jasminum nudiflorum* using the aforementioned developing agent, comprising the following steps:
[0009] (1) The white peony sample, the counterfeit jasmine sample and the sample to be tested were extracted with methanol by ultrasonic extraction, and filtered to obtain the white peony sample solution, the counterfeit jasmine sample solution and the sample to be tested solution.
[0010] (2) Spot the white peony sample solution, the counterfeit jasmine sample solution and the sample solution to be tested onto the same silica gel G thin layer plate;
[0011] (3) Develop the sample on the thin-layer plate using the developing agent described above;
[0012] (4) After removing the silicone G thin film and drying it, test the results.
[0013] Preferably, in step (1), the ratio of the amount of the white peony sample, the counterfeit jasmine sample, the sample to be tested, and methanol is 1g:18-22mL.
[0014] Preferably, the ultrasonic parameters in the ultrasonic extraction process described in step (1) are 35-45 kHz, 340-380 W, and the ultrasonic time is 40-50 min.
[0015] Preferably, the amount of a single sample spotted in step (2) is 4 to 6 μL.
[0016] Preferably, the detection in step (4) is performed under ultraviolet light, and the wavelength of the ultraviolet light is 360-370nm.
[0017] This invention provides a method for distinguishing between *Plumbago alatus* and its adulterant *Jasminum nudiflorum* using thin-layer chromatography (TLC). The developing solvent is a mixture of toluene, methanol, glacial acetic acid, and water, with a volume ratio of 6–8:0.8–1.2:0.4–0.6:0.4–0.6. Compared with the current standards for *Plumbago alatus*, this method can distinguish between *Plumbago alatus* and its adulterant *Jasminum nudiflorum* using only TLC. It is simple, fast, durable, highly efficient, provides clear and intuitive judgments, and yields objective and reliable conclusions. This provides technical support for improving and enhancing the quality standards of *Plumbago alatus* and better controlling the quality of the medicinal material. Attached Figure Description
[0018] Figure 1 The image shows a thin-layer chromatogram of Plumbago 'White Flower', where 1-3 are Plumbago 'White Flower' samples, 4 is the reference material of Plumbago 'White Flower', 5 is the adulterant material, and A is a yellow-green fluorescent spot.
[0019] Figure 2 To illustrate the experimental results of System 1, 1 represents the adulterated medicinal material, 2 represents the control medicinal material of Plumbago salsa, 3-4 represent Plumbago salsa samples, and A represents the yellow-green fluorescent spot.
[0020] Figure 3 To illustrate the experimental results of System 2, 1 represents the adulterated medicinal material, 2 represents the control medicinal material of Plumbago salsa, 3-4 represent Plumbago salsa samples, and A represents the yellow-green fluorescent spot.
[0021] Figure 4 To illustrate the experimental results of System 3, 1 represents the adulterated medicinal material, 2 represents the control medicinal material of Plumbago salsa, 3-4 represent Plumbago salsa samples, and A represents the yellow-green fluorescent spot.
[0022] System 3: Toluene-Methanol-Glacial Acetic Acid-Ethyl Acetate (volume ratio 7:1:0.5:0.25)
[0023] Figure 5 For the sample quantity assessment - Qingdao Marine G plate, 1-4 are the control solution of Plumbago glabra (volumes of 2μL, 5μL, 10μL, and 15μL, respectively), 5-8 are the test solution of Plumbago glabra (volumes of 2μL, 5μL, 10μL, and 15μL, respectively), 9-12 are the adulterant solution (volumes of 2μL, 5μL, 10μL, and 15μL, respectively), and A is the yellow-green fluorescent spot;
[0024] Figure 6 For the thin-layer plates from different manufacturers, 1-3 are the test solutions of Plumbago salsa, 4 is the reference solution of Plumbago salsa, 5 is the adulterant solution, and A is the yellow-green fluorescent spot.
[0025] Figure 7 The results are for different temperatures and humidity levels. 1-3 are the test solutions of Plumbago salsa, 4 is the control solution of Plumbago salsa, 5 is the adulterant solution, and A is the yellow-green fluorescent spot.
[0026] Figure 8 The results of thin-layer chromatography identification of Plumbago 'White Flower' are shown on Qingdao Marine G plate. In this plate, 1 is the reference solution of Plumbago 'White Flower', 2-13 are the test solutions of Plumbago 'White Flower' (samples 1-12), 14 is the adulterant solution, and A is the yellow-green fluorescent spot.
[0027] Figure 9 The results of thin-layer chromatography identification of Plumbago 'White Flower' are shown on Qingdao Ocean G plate. 1 is the reference solution of Plumbago 'White Flower', 2-13 are the test solutions of Plumbago 'White Flower' (samples 13-24), 14 is the adulterant solution, and A is a yellow-green fluorescent spot. Detailed Implementation
[0028] The present invention provides a developing solvent for thin-layer chromatography, wherein the developing solvent is a mixture of toluene, methanol, glacial acetic acid and water, and the volume ratio of toluene, methanol, glacial acetic acid and water is 6-8:0.8-1.2:0.4-0.6:0.4-0.6.
[0029] In this invention, the volume ratio of toluene, methanol, glacial acetic acid and water is preferably 7:1:0.5:0.5.
[0030] In this invention, the developing agent is preferably the upper layer solution of the mixture.
[0031] The present invention also provides the application of the aforementioned developing agent in distinguishing between white peony and counterfeit jasmine.
[0032] The present invention also provides a method for distinguishing between *Plumbago salsa* and counterfeit *Jasminum nudiflorum* using the aforementioned developing agent, comprising the following steps:
[0033] (1) The white peony sample, the counterfeit jasmine sample and the sample to be tested were extracted with methanol by ultrasonic extraction, and filtered to obtain the white peony sample solution, the counterfeit jasmine sample solution and the sample to be tested solution.
[0034] (2) Spot the white peony sample solution, the counterfeit jasmine sample solution and the sample solution to be tested onto the same silica gel G thin layer plate;
[0035] (3) Develop the sample on the thin-layer plate using the developing agent described above;
[0036] (4) After removing the silicone G thin film and drying it, test the results.
[0037] In this invention, the preferred ratio of the amount of the white peony sample, the fake jasmine sample, the sample to be tested, and methanol in step (1) is 1g:18-22mL, and more preferably 1g:20mL.
[0038] In this invention, the ultrasonic parameters in the ultrasonic extraction process described in step (1) are 35-45 kHz and 340-380 W, more preferably 40 kHz and 360 W, and the ultrasonic time is preferably 40-50 min, more preferably 45 min.
[0039] In this invention, the amount of a single sample spotted in step (2) is preferably 4 to 6 μL, and more preferably 5 μL.
[0040] In this invention, the detection in step (4) is preferably performed under ultraviolet light, and the wavelength of the ultraviolet light is preferably 360-370nm, and more preferably 365nm.
[0041] The developing solvent of this invention is an optimization based on previous research on a benzene-ethanol (8:2 volume ratio) developing system. Different developing systems, such as toluene-methanol-formic acid (14:2:0.5 volume ratio) and toluene-methanol-glacial acetic acid-ethyl acetate (7:1:0.5:0.25 volume ratio), showed poor separation results. Finally, the upper layer solution of toluene-methanol-glacial acetic acid-water (7:1:0.5:0.5 volume ratio) was used as the developing solvent, resulting in ideal spot separation. Furthermore, this developing system can distinguish between *Plumbago semperflorens* and the adulterant *Jasminum nudiflorum*. See [link to relevant documentation]. Figure 1 .
[0042] 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.
[0043] Example 1: System Investigation
[0044] This invention examines three deployment systems:
[0045] System 1: Toluene-methanol-formic acid (volume ratio 14:2:0.5);
[0046] Development of System 2: The upper layer of the toluene-methanol-glacial acetic acid-water mixture (volume ratio 7:1:0.5:0.5);
[0047] System 3: Toluene-methanol-glacial acetic acid-ethyl acetate (volume ratio 7:1:0.5:0.25).
[0048] Thin-layer chromatography analysis showed that developing system II produced a clearer spot (A) distinguishing it from the adulterant compared to system I, with a more suitable development distance and better separation. Developing system III, however, failed to produce an ideal spot. Therefore, developing system II was ultimately selected, and the results are as follows: Figures 2-4 As shown.
[0049] Example 2: Investigation of different sample sizes
[0050] Take 2 μL, 5 μL, 10 μL, and 15 μL of the test solution, the reference herb solution, and the adulterant solution, respectively, and spot them onto the same silica gel G thin-layer plate. Use the upper layer solution of toluene-methanol-glacial acetic acid-water (volume ratio 7:1:0.5:0.5) as the developing solvent, place the plate in a developing tank for development, and then remove the thin-layer plate and air dry it. Use a thin-layer digital imaging system to photograph and examine it under ultraviolet light (365 nm).
[0051] pass Figure 5 It is known that when the sample volume of the test solution, the reference medicinal material solution, and the adulterant solution is 5 μL, the size, separation, and clarity of the fluorescent spots are the best under ultraviolet light (365 nm). Therefore, the sample volume of the reference medicinal material solution, the test solution, and the adulterant solution is set at 5 μL.
[0052] Example 3: Investigation of Thin-Layer Laminates from Different Manufacturers
[0053] Three types of thin-layer chromatography silica gel plates—Qingdao Marine G plate, Huanghai G plate, and Merck-G60 plate—were tested. The tested sample, adulterant sample, and control drug solution of the specified volumes were spotted at corresponding positions on the Qingdao Marine G plate, Huanghai G plate, and Merck-G60 plate, respectively. Products from all three manufacturers exhibited identical experimental results, demonstrating the good robustness of the thin-layer development method. Results are as follows: Figure 6 As shown.
[0054] Example 4: Investigation at different temperatures and humidity levels
[0055] The experiment investigated the temperature and humidity conditions for thin-layer chromatography (TLC) development. For the selected developing system: toluene-methanol-glacial acetic acid-water (volume ratio 7:1:0.5:0.5), the upper layer solution was tested at room temperature (28℃), low temperature (4℃), humidity (60%), low humidity (35%), and high humidity (85%). TLC chromatographic analysis showed that under different temperature and humidity conditions, the spots of the test sample, adulterant, and reference material were clear, easily distinguishable, and exhibited good separation. The results are as follows: Figure 7 As shown.
[0056] Test case
[0057] Thin-layer chromatography (TLC) identification experiments were conducted on 24 batches of *Plumbago alatus* and 1 batch of *Pseudojasminoides var. *assamica* (see Table 1) from different origins. 1 g of each of 25 batches of *Plumbago alatus* and *Pseudojasminoides var. *assamica*, plus 1 g of *Plumbago alatus* control material, were added to 20 mL of methanol and sonicated at 40 kHz and 360 W for 45 min. After filtration, *Plumbago alatus* sample solution, *Pseudojasminoides var. *assamica* sample solution, and *Plumbago alatus* control material solution were prepared. 5 μL of each of these three solutions were spotted onto the same silica gel G TLC plate. The upper layer of a toluene:methanol:glacial acetic acid:water (volume ratio 7:1:0.5:0.5) was used as the developing solvent. After development, the plate was removed, dried, and examined under ultraviolet light (365 nm) using a TLC digital imaging system. Figures 8-9 It can be seen that the yellow-green fluorescent spots on the thin-layer plate can distinguish between Plumbago glabra and the counterfeit jasmine.
[0058] Table 1 Sample Collection Information
[0059]
[0060]
[0061]
[0062] As can be seen from the above embodiments, the present invention provides a method for distinguishing between *Plumbago alatus* and its adulterant *Jasminum nudiflorum* based on thin-layer chromatography. The developing solvent is a mixture of toluene, methanol, glacial acetic acid, and water, and the volume ratio of toluene, methanol, glacial acetic acid, and water is 6–8:0.8–1.2:0.4–0.6:0.4–0.6. Compared with the current standards for *Plumbago alatus* medicinal materials, the method of the present invention can distinguish between *Plumbago alatus* and its adulterant *Jasminum nudiflorum* solely through thin-layer chromatography. It is simple, fast, durable, highly efficient, provides intuitive and clear judgment, and yields objective and reliable conclusions. This provides technical support for improving and enhancing the quality standards of *Plumbago alatus* medicinal materials and better controlling the quality of medicinal materials.
[0063] 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 developing solvent for thin-layer chromatography, characterized in that, The developing solvent is a mixture of toluene, methanol, glacial acetic acid, and water, wherein the volume ratio of toluene, methanol, glacial acetic acid, and water is 6–8. 0.8～1.2：0.4～0.6：0.4～0.6。 2. The developing agent according to claim 1, characterized in that, The developing agent is the upper layer solution of the mixture.
3. The application of the developing agent according to claim 1 or 2 in distinguishing between Plumbago salsa and the counterfeit jasmine.
4. A method for distinguishing between *Plumbago alatus* and counterfeit *Jasminum nudiflorum* using the developing agent described in claim 1 or 2, characterized in that, Includes the following steps: (1) The white peony sample, the counterfeit jasmine sample and the sample to be tested were extracted with methanol by ultrasonic extraction, and filtered to obtain the white peony sample solution, the counterfeit jasmine sample solution and the sample to be tested solution. (2) Spot the white peony sample solution, the counterfeit jasmine sample solution and the sample solution to be tested onto the same silica gel G thin layer plate; (3) Develop the sample on the thin-layer plate using the developing agent described in claim 1 or 2; (4) After removing the silicone G thin film and drying it, test the results.
5. The method according to claim 4, characterized in that, In step (1), the ratio of the amount of white peony sample, fake jasmine sample, and test sample to methanol is 1g:18-22mL.
6. The method according to claim 5, characterized in that, In step (1), the ultrasonic parameters during ultrasonic extraction are 35-45 kHz and 340-380 W, and the ultrasonic time is 40-50 min.
7. The method according to claim 6, characterized in that, In step (2), the amount of a single sample to be spotted is 4 to 6 μL.
8. The method according to claim 7, characterized in that, The detection in step (4) is performed under ultraviolet light, and the wavelength of the ultraviolet light is 360-370nm.