A thin-layer chromatographic identification method for plants of the Musaceae family

Abstract

This invention belongs to the field of identification technology of traditional Chinese medicinal materials, specifically involving a thin-layer chromatography identification method for plants of the Musaceae family. The method includes the following steps: (1) Preparation of test solution and reference medicinal material solution: Take a sample of Musaceae plant, add methanol, sonicate, filter, and use as test solution; take another reference medicinal material of Musaceae plant and prepare a reference medicinal material solution in the same way; (2) Preparation of reference solution: Take β-sitosterol reference standard, add methanol to prepare a solution, and use as reference solution; (3) Take the test solution, reference medicinal material solution, and reference solution, and spot them on the same silica gel G thin-layer plate, use dichloromethane-toluene-methanol as the developing solvent, develop, spray with a colorimetric reagent, and examine. In the chromatogram of the test sample, spots of the same color are displayed at the corresponding positions of the chromatograms of the reference standard and the reference medicinal material. This invention has good separation effect, the spots are round and clear, and the development rate is fast, the results are easy to judge, and it can quickly and accurately identify plants of the Musaceae family.

Description

Technical Field

[0001] This invention belongs to the field of identification technology of traditional Chinese medicinal materials, specifically relating to a thin-layer chromatography identification method for plants of the Musaceae family. Background Technology

[0002] Currently, the standards for medicinal materials related to Musaceae plants include: Fujian Provincial Standards for Traditional Chinese Medicine (2006 edition), Guizhou Provincial Standards for Quality of Traditional Chinese Medicine and Ethnic Medicine (2003 edition), Jiangsu Provincial Standards for Traditional Chinese Medicine (2016 edition), and Guizhou Provincial Standards for Traditional Chinese Medicine and Ethnic Medicine (2019 edition) (Volume 1), which correspond to the following Musaceae plant medicinal materials: banana plant, banana root, banana flower, and banana root, respectively.

[0003] Among them, the Guizhou Provincial Standard for Quality of Traditional Chinese and Ethnic Medicinal Materials (2003 Edition) did not introduce thin-layer chromatography (TLC) for the identification of banana root, resulting in inaccurate identification results. Although the other standards introduced TLC, they all used only reference medicinal materials as controls, without any active ingredient reference standards.

[0004] In recent years, phytosterols have received widespread attention due to their safety and effective anti-inflammatory activities. β-Sitosterol is a tetracyclic triterpenoid compound, similar to cholesterol in mammals, and is a type of plant steroid. β-Sitosterol possesses various biological activities, including lowering cholesterol and blood sugar levels, antioxidant, anti-inflammatory, antibacterial, and hormone-like functions. Therefore, it has become a research hotspot in the life sciences, especially in the medical field. Musaceae plants also contain β-Sitosterol, an effective component. Therefore, adding β-Sitosterol reference standards for thin-layer chromatography identification can ensure the accuracy and quality of components in Musaceae plants and provide a reference for quality control and standardized production of Musaceae plants. Summary of the Invention

[0005] This invention aims to solve the aforementioned technical problems by providing a thin-layer chromatography (TLC) method for identifying Musaceae plants. The method uses β-sitosterol as a reference standard and incorporates reference medicinal materials of Musaceae plants for identification. Using this method to analyze Musaceae plant samples yields excellent TLC separation, with clear and round spots, rapid development, and easily interpretable results, enabling rapid and accurate identification of Musaceae plants.

[0006] The technical solution of this invention is as follows:

[0007] A thin-layer chromatography method for identifying plants of the Musaceae family, characterized by the following steps:

[0008] (1) Preparation of test solution and reference medicinal material solution: Take a sample of Musaceae plant, add methanol, sonicate, filter, and use as test solution; take another reference medicinal material of Musaceae plant and prepare reference medicinal material solution in the same way;

[0009] (2) Preparation of reference solution: Take β-sitosterol reference standard, add methanol to prepare a solution, and use it as the reference solution;

[0010] (3) Take the test solution, the reference medicinal material solution, and the reference solution, and spot them on the same silica gel G thin layer plate. Use dichloromethane-toluene-methanol as the developing solvent, develop the plate, spray with a colorimetric reagent, and examine it. In the chromatogram of the test sample, spots of the same color are displayed at the corresponding positions of the chromatograms of the reference substance and the reference medicinal material.

[0011] Furthermore, in step (1), the volume fraction of methanol is 90%.

[0012] Furthermore, in step (1), the ultrasound time is 40 minutes.

[0013] Further, in step (3), the volume ratio of dichloromethane, toluene and methanol in the dichloromethane-toluene-methanol mixture is 10:5:1.5.

[0014] Further, the method includes the following steps: In step (3), the colorimetric agent is a 10% sulfuric acid ethanol solution.

[0015] Furthermore, in step (3), the inspection is performed under an ultraviolet lamp with a wavelength of 365nm.

[0016] Furthermore, the plant in the Musaceae family is either Saigon banana or plantain.

[0017] Due to the adoption of the above technical solution, the beneficial effects of the present invention are as follows:

[0018] This invention provides a thin-layer chromatography (TLC) identification method for Musaceae plants. This method uses β-sitosterol as a reference standard and combines it with reference medicinal materials of Musaceae plants for identification. The Musaceae plant samples analyzed by this method exhibit good separation in TLC, with round and clear spots, moderate shift values, and good reproducibility. Furthermore, this method is simple to operate, requires low-end equipment, facilitates color development, has a fast development speed, and the results are easy to interpret.

[0019] The identification method of this invention can quickly and accurately identify medicinal plants of the Musaceae family, thus providing a reliable guarantee for the safety of using medicinal plants of the Musaceae family. Attached Figure Description

[0020] Figure 1 This is an illustration of the original plant of the Saigon banana, a plant belonging to the Musaceae family.

[0021] Figure 2 This is a specimen of the herbaceous leaf of the Saigon banana plant (Musaceae family).

[0022] Figure 3 This is a picture of the medicinal material Saigon banana, a plant belonging to the Musaceae family.

[0023] Figure 4 This is a micrograph of a cross-section of the pseudostem of the Musaceae plant, *Musa serrata*. In the image, 1-upper epidermis; 2-fiber bundle; 3-secreting cell; 4-vascular bundle; 5-airway; 6-calcium oxalate needle crystal; 7-calcium oxalate square crystal; 8-lower epidermis.

[0024] Figure 5 This is a powder image of the Saigon banana plant (Musaceae family). In the image, 1-starch granules; 2-fiber bundles; 3-secreting cells; 4-calcium oxalate needle crystal bundles; 5-calcium oxalate square and sandy crystals; 6-vessels.

[0025] Figure 6 TLC images of samples of *Musa serrata* (Musa family): 1, XGJ-1; 2, XGJ-2; 3, XGJ-3; 4, XGJ-4; 5, XGJ-5; 6, *Musa serrata* (Musa family) reference material; 7, β-sitosterol reference standard; 8, XGJ-6; 9, XGJ-7; 10, XGJ-8; 11, XGJ-9; 12, XGJ-10.

[0026] Figure 7 TLC images of *Musa coccinea* samples from the Musaceae family under different extraction methods are shown. In the figures, 1 represents ethanol extraction; 2 represents methanol extraction; 3 represents water extraction; reflux and ultrasonic extraction represent two different extraction methods; 30%, 50%, 70%, and 90% represent different volume fractions of methanol extraction concentration.

[0027] Figure 8 The images show TLC images of *Musa coccinea* samples from the Musaceae family under different polarity developing systems. In the images, a) is the developing system of dichloromethane-acetone-methanol (volume ratio 8.5:1.5:1); b) is the developing system of dichloromethane-toluene-methanol (volume ratio 10:5:1.5); and c) is the developing system of cyclohexane-ethyl acetate-methanol (volume ratio 8:2:1).

[0028] Figure 9 TLC images of *Musa coccinea* samples under different colorimetric reagents. In the images, a) is a 10% sulfuric acid ethanol solution; b) is a 10% aluminum trichloride ethanol solution.

[0029] Figure 10 TLC images of *Musa spp.* samples of *Musa spp.* with different spread distances: a) 13cm spread distance; b) 14cm spread distance; c) 15cm spread distance. Detailed Implementation

[0030] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] Example 1

[0032] [Source] This product is the rhizome and pseudostem of Musa spp. ABB group, Pisang Awak, a plant of the Musaceae family. It can be harvested year-round. Remove fine roots and dirt, wash, slice, and dry.

[0033] 1. Sample Collection Status: A total of 10 batches of samples were received. Details are shown in Table 1 below. Images of the original plant species are shown below. Figure 1 As shown.

[0034] Table 1. Summary of Saigon Banana Sample Information

[0035]

[0036] Note: Sample XGJ-5 of the Musaceae family, Saigon banana, was also prepared as a herbarium (e.g. Figure 2 As shown, the rhizome and pseudostem of *Musas pp. ABB group, Pisang Awak*, a plant in the Musaceae family, were identified by Professor Wei Songji of Guangxi University of Chinese Medicine and Researcher Li Xiaoquan of Guangxi Academy of Agricultural Sciences. This sample was used as a control material for comparison with other samples in the experiment. After sample collection, all 10 samples (approximately 120g) were pulverized and passed through a 40-mesh sieve for later use.

[0037] The results of the morphological identification and microscopic identification of the samples are as follows:

[0038] Identification of characteristics: The rhizome of this product is in irregularly shaped pieces, grayish-brown to grayish-brown on the surface, with residual fine roots or root scars, sunken in the center, gray to grayish-brown, with irregular wrinkles. It is hard in texture. The pseudostem is a long, thin, blocky slice, grayish-white to grayish-yellow on the outside and grayish-yellow on the inside, with coarse longitudinal stripes on both sides. It is tough, light in weight, and has a strong fibrous texture in cross-section. It is easily torn longitudinally but not easily torn transversely. Coarse, vesicular gaps can be seen in the transverse cross-section. It has a slight odor and a bitter taste. See Figure 3 .

[0039] Cross-section of the pseudostem: The outer epidermal cells are arranged in a single row, nearly round, with thickened outer walls. Scattered fiber bundles are present on the inner side. Large, nearly round airways are arranged in the center of the ground tissue, with scattered vascular bundles within the ground tissue between the airways. The inner epidermal cells are small and thin-walled. The parenchyma cells are nearly round or oval, with thin walls; some contain shell-shaped starch grains, calcium oxalate needle crystal bundles, or yellowish-brown secretions. Scattered calcium oxalate crystals are also present. Figure 4 .

[0040] Microscopic identification: The powder is light grayish-yellow. Starch granules are abundant, shell-shaped, broadly ovate, kidney-shaped, or elliptical, 6–60 μm in diameter, some with distinct lamellae and punctate or fissured hilum. Fibers are bundled, with lignified walls, about 10 μm in diameter. Secretory cells contain yellowish-brown to reddish-brown substances. Calcium oxalate needle crystal bundles are present in thin-walled cells or scattered, with needle crystals 30–120 μm long and up to 5 μm in diameter. Calcium oxalate prismatic and sandy crystals are present in sheets in thin-walled cells. Reticulate and spiral vessels are common, 20–80 μm in diameter. Figure 5 .

[0041] 2. Thin-layer chromatographic identification method for *Musa saigonensis*, a plant of the Musaceae family.

[0042] (1) Take 2g of Saigon banana coarse powder, add 10ml of 90% methanol solution, sonicate for 40 minutes, filter, and use as the test solution; take Saigon banana reference material and prepare the reference material solution in the same way.

[0043] (2) Take β-sitosterol reference standard and add methanol to prepare a solution containing 1 mg per 1 ml as the reference solution;

[0044] (3) Perform thin-layer chromatography (General Chapter 0502, Chinese Pharmacopoeia 2020 Edition). Apply 5 μl of the test solution, 5 μl of the reference medicinal material solution, and 3 μl of the reference standard solution to the same silica gel G thin-layer plate. Develop the plate using a dichloromethane-toluene-methanol solution (v / v) at a ratio of 10:5:1.5. Remove the plate, air dry, spray with 10% sulfuric acid ethanol solution, and heat at 105℃ until the spots are clearly visible. Examine the plate under ultraviolet light at a wavelength of 365 nm. In the chromatogram of the test sample, fluorescent spots of the same color should appear at the corresponding positions as in the chromatograms of the reference standard and the reference medicinal material.

[0045] 3. Results and Analysis

[0046] All 10 batches of samples were tested using the above method and met the requirements. The thin-layer chromatography separation effect was good, the spots were round and clear, the specific shift value was moderate, and the reproducibility was good.

[0047] TLC images of 10 batches of Saigon banana samples from different production areas (Musaceae family) are shown below. Figure 6The chromatographic conditions were as follows: silica gel G thin-layer plate, manufacturer: Qingdao Ocean Chemical Plant, batch number: 20220608; size: 20cm×20cm; spotting: round dots, spotting volume: 5μL; temperature: 24.7℃ (±2℃); relative humidity: 46RH% (±4%); developing solvent: dichloromethane: toluene: methanol (10:5:1.5).

[0048] 4. Chromatographic conditions investigation:

[0049] (1) Investigation of different extraction conditions: By comparing three different extraction solvents (methanol, ethanol, and water), different extraction concentrations of methanol (30%, 50%, 70%, and 90% by volume), two different extraction methods (reflux and ultrasonic), and three different extraction times (20 minutes, 40 minutes, and 60 minutes), the extraction conditions were finally determined as follows: Take the crude powder of this product, pass it through a No. 2 sieve, shake well, weigh 2g of crude powder into a 50mL ground glass conical flask, add 10mL of 90% methanol by volume, ultrasonically extract for 40 minutes, filter, and let stand to collect the supernatant as the test solution. Preparation of reference solution: Weigh an appropriate amount of β-sitosterol reference standard, add methanol to prepare a reference solution containing approximately 1mg per 1mL, and the results are as follows. Figure 7 As shown.

[0050] (2) Investigation of developing systems with different polarities: XGJ-1 medicinal material was prepared into a test solution using the method in step (1), and spotted onto a silica gel G plate. The plate was developed using three different polarity developing systems: dichloromethane-acetone-methanol (8.5:1.5:1), dichloromethane-toluene-methanol (10:5:1.5), and cyclohexane-ethyl acetate-methanol (8:2:1). The plates were then removed, dried with hot air, and examined under a 365nm UV lamp. The results showed that when dichloromethane-toluene-methanol (10:5:1.5) was used as the developing solvent, the main spots were clearly visible, and the developing effect was better. The results are as follows: Figure 8 As shown.

[0051] (3) Investigation of different color-developing agents: XGJ-1 medicinal material was prepared into a test solution according to the method in step (1), spotted on a silica gel G plate, and developed with dichloromethane-toluene-methanol (10:5:1.5) as the developing solvent. After removal and drying with hot air, it was sprayed with 10% sulfuric acid ethanol solution and 10% aluminum trichloride ethanol solution for color development, respectively, and examined under a UV lamp with a wavelength of 365nm. The results showed that when 10% sulfuric acid ethanol solution was used as the color-developing agent, the spots were clearly developed. The results are as follows. Figure 9 As shown.

[0052] (4) Investigation of different development distances: 5 μL of each test solution was taken and developed using dichloromethane-toluene-methanol (10:5:1.5) as the developing solvent. The development distances were 13 cm, 14 cm, and 15 cm, respectively. The samples were dried with hot air, sprayed with 10% sulfuric acid ethanol solution for color development, and examined under a 365 nm UV lamp. The results showed that the spots at 15 cm were clearly larger and brighter. Considering all factors, a development distance of 15 cm was selected. The results are as follows: Figure 10 As shown.

[0053] By optimizing and investigating the above chromatographic conditions, the accuracy and repeatability of the identification of this invention have been further improved. The method of this invention fills the gap in thin-layer chromatography identification technology for the medicinal material of Musaceae plant, Saigon banana, and can achieve rapid and accurate identification. This method provides a reliable basis for the identification of Musaceae plants, thereby providing a reliable guarantee for their safe use.

[0054] The above description is a detailed description of the preferred embodiments of the present invention. However, the embodiments are not intended to limit the scope of the patent application of the present invention. All equivalent changes or modifications made under the technical spirit of the present invention should fall within the patent scope covered by the present invention.

Claims

1. A thin-layer chromatographic identification method for plants of the Musaceae family, characterized in that: Includes the following steps: (1) Preparation of test solution and reference medicinal material solution: Take a sample of Musaceae plant, add 90% methanol, sonicate for 40 minutes, filter, and use as test solution; take another Musaceae plant reference medicinal material and prepare reference medicinal material solution in the same way; (2) Preparation of reference solution: Take β-sitosterol reference standard, add methanol to prepare a solution, and use it as the reference solution; (3) Take the test solution, the reference medicinal material solution, and the reference solution, and spot them on the same silica gel G thin layer plate. Use dichloromethane-toluene-methanol with a volume ratio of 10:5:1.5 as the developing solvent, develop the plate, spray with 10% sulfuric acid ethanol solution for color development, and examine it under a UV lamp with a wavelength of 365nm. In the chromatogram of the test sample, spots of the same color are displayed at the corresponding positions of the chromatogram of the reference substance and the chromatogram of the reference medicinal material. The plant in question is either the Saigon banana or the large banana.

Images