A thin-layer chromatographic method for the detection of Cistanche deserticola and Cistanche tubulosa.

Abstract

This invention belongs to the field of quality control technology for traditional Chinese medicinal materials, specifically relating to a method for detecting Cistanche deserticola (C. deserticola Y.C.Ma) and C. tubular Cistanche (C. tubular Cistanche (Schenk.) Wight) based on thin-layer chromatography. The detection method includes the following steps: preparing a test solution, a reference medicinal material solution, and a reference solution respectively; spotting the test solution, reference medicinal material solution, and reference solution onto the same thin-layer chromatography plate; developing the plate with a developing solvent of ethyl acetate:methanol:formic acid:water = 15:2:2:2; spraying a colorimetric reagent for development; and examining the plate under a 365nm ultraviolet lamp. The thin-layer chromatography detection method for Cistanche deserticola described in this invention exhibits clear bands, good separation effect, rich chromatographic information, and can specifically identify Cistanche deserticola and C. tubular Cistanche, which is superior to other existing standards.

Description

Technical Field

[0001] This invention belongs to the field of quality control technology of traditional Chinese medicinal materials, specifically relating to a method for detecting Cistanche deserticola and Cistanche tubulosa based on thin-layer chromatography. Background Technology

[0002] Cistanche deserticola is a perennial parasitic medicinal plant belonging to the genus Cistanche of the family Orobanchaceae. It has extremely high medicinal value and is known as "desert ginseng". There are 4 species and 1 variety of Cistanche deserticola in my country, namely Cistanche deserticola (commonly known as desert Cistanche) C. deserticola YCMa, C. salsa (CAMey.) G.Beck, C. salsa var. albiflora PFTu et ZCLou, C. tubulosa (Schenk) Wight, and C. sinensis G.Beck (see Tu Pengfei et al., Modern Chinese Materia Medica, 2015, 17(4):297-301). Among them, desert Cistanche and tubulosa are officially recognized as the original plants of Cistanche deserticola and are included in the Chinese Pharmacopoeia.

[0003] *Cistanche deserticola* is a flattened cylindrical, slightly curved shape, 3-15 cm long and 2-8 cm in diameter. The surface is brownish-brown or grayish-brown, densely covered with imbricate fleshy scales, usually with broken tips. It is heavy, hard, slightly flexible, and not easily broken. The cross-section is brownish-brown with light brown dotted vascular bundles arranged in wavy rings. It has a faint odor and a sweet, slightly bitter taste. *Cistanche tubulosa* is spindle-shaped, flattened spindle-shaped, or flattened cylindrical, slightly curved, 5-25 cm long and 2.5-9 cm in diameter. The surface is brownish-brown to dark brown. The cross-section is granular, grayish-brown to grayish-brown, with scattered dotted vascular bundles.

[0004] Traditionally, the detection of Cistanche deserticola is mainly based on the methods recorded in the Chinese Pharmacopoeia. Taking the 2020 edition as an example, the specific steps include: taking 1g of the powder, adding 20mL of methanol, sonicating for 15 minutes, filtering, concentrating the filtrate to near dryness, dissolving the residue in 2mL of methanol to obtain the test solution. Separately, prepare reference solutions containing 1mg per 1mL of echinacoside and verbascoside by dissolving them in methanol. Perform thin-layer chromatography (General Rule 0502), applying 2μL of each of the three solutions to the same polyamide thin-layer plate, developing with methanol-acetic acid-water (2:1:7) as the developing solvent, removing the plate, drying it, and examining it under ultraviolet light (365nm). In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference samples.

[0005] However, this detection method cannot distinguish between different species of Cistanche species. Summary of the Invention

[0006] Since traditional thin-layer chromatography (TLC) methods cannot distinguish between different species of Cistanche, this invention provides a TLC-based method for detecting Cistanche and Cistanche tubulosa. The TLC method for detecting Cistanche and Cistanche tubulosa described in this invention produces clear bands, good separation, and rich chromatographic information. Furthermore, it can specifically identify Cistanche and Cistanche tubulosa medicinal materials, which is superior to other existing standards.

[0007] This invention is achieved through the following technical solution:

[0008] In a first aspect, the present invention provides a thin-layer chromatography detection method for Cistanche deserticola and Cistanche tubulosa, the detection method comprising the following steps:

[0009] A test solution, a reference herb solution, and a reference standard solution were prepared separately. These solutions were then spotted onto the same thin-layer chromatography plate, developed in the developing solvent, and subjected to color development using a colorimetric reagent.

[0010] The preparation steps of the test solution include: taking 1g of Cistanche deserticola and Cistanche tubulosa test sample powder, adding 10mL of methanol, sonicating for 20min, filtering, taking 5mL of the filtrate, evaporating to dryness, adding 5mL of water to dissolve the residue, extracting 3 times with 10mL of water-saturated n-butanol each time, combining the n-butanol extracts, evaporating to dryness, adding 5mL of methanol to dissolve the residue, accurately measuring 1mL, placing it in a 10mL volumetric flask, diluting with methanol to the mark, shaking well, filtering, and using it as the test solution;

[0011] Following the steps for preparing the test solution, the reference medicinal material solution was prepared using Cistanche deserticola and Cistanche tubulosa as reference medicinal materials;

[0012] The preparation steps of the reference solution include: taking echinacoside reference standard and verbascoside reference standard, adding methanol to prepare a mixed solution containing 2 mg of each per 10 mL, as the reference solution.

[0013] Alternatively, in the above thin-layer chromatography detection method, the thin-layer chromatography plate is a silica gel thin-layer plate, and the average particle size of the adsorbent is 5-10 μm.

[0014] Preferably, the thin-layer chromatography plate is HPTLC Silica gel 60F. 254 (Merck), HPTLC Silica gel 60 (Merck), TLC Silica gel 60 (Merck) or TLC Silica gel 60G (Merck).

[0015] More preferably, the thin-layer chromatography plate is HPTLC Silica gel 60 (Merck).

[0016] As an optional method, in the above thin-layer chromatography detection method, the developing solvent system is ethyl acetate:methanol:formic acid:water = 15:2:2:2, the spotting volume is 3-5 μL, and the band width is 8 mm.

[0017] Preferably, the sample spotting volume is 4 μL.

[0018] As an optional method, in the above thin-layer chromatography detection method, the colorimetric reagent is a 10% (v / v) sulfuric acid ethanol solution.

[0019] Alternatively, in the above thin-layer chromatography detection method, the detection method includes the following steps:

[0020] The test solution, reference herb solution, and reference standard solution were spotted onto a thin-layer chromatography plate. The plate was developed under saturated conditions using ethyl acetate:methanol:formic acid:water = 15:2:2:2, relative humidity ≤40%, and development distance 7.5 cm. The plate was then removed, air-dried, sprayed with 10% sulfuric acid ethanol solution (v / v), heated at 105 °C for about 3 min, and examined under a 365 nm UV lamp.

[0021] As an optional method, in the above thin-layer chromatography detection method, under ultraviolet light, echinacoside and verbascoside appear as yellow-green fluorescent spots at approximately one-third and one-half of the thin-layer plate, respectively, with RF values ​​of 0.25 and 0.52.

[0022] In a second aspect, the present invention provides the use of the thin-layer chromatography detection method described in the first aspect above in the specific identification of Cistanche deserticola and Cistanche tubulosa medicinal materials.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The thin-layer chromatography detection method for Cistanche deserticola and Cistanche tubulosa described in this invention has clear bands, good separation effect, and rich chromatographic information. It can also specifically identify Cistanche deserticola and Cistanche tubulosa medicinal materials, which is superior to other existing standards. Attached Figure Description

[0025] Figure 1 The experiment investigated whether the test samples were extracted or not. Among them, 1. from top to bottom: verbascoside, echinacoside; 2. *Cistanche tubulosa* reference material; 3. unextracted; 4. extracted; sample volume 4 μL. HPTLC Silica gel 60 (Merck) 10×10cm, 24.6℃, 33.2%, echinacoside RF = 0.29, verbascoside RF = 0.56.

[0026] Figure 2Investigation of different extraction solvents. The solvents used were: 1. verbascoside, echinacoside (from top to bottom); 2. *Cistanche tubulosa* reference material; 3. ethanol; 4. methanol; sample volume 4 μL. HPTLC Silica gel 60 (Merck) 10×10cm, 23℃, 35.2%, echinacoside RF = 0.29, verbascoside RF = 0.58.

[0027] Figure 3 Thin-layer chromatography (TLC) chromatograms for the extraction method were obtained. The TLC chromatograms are as follows: 1. From top to bottom: verbascoside and echinacoside; 2. Control material; 3. Reflux; 4. Sonication; sample volume: 4 μL. HPTLC Silica gel 60 (Merck), 10 × 10 cm, 23.3℃, ​​53.2%, echinacoside RF = 0.26, verbascoside RF = 0.53.

[0028] Figure 4 The effects of different extraction times were investigated. The samples were: 1. verbascoside and echinacoside (from top to bottom); 2. control material; 3. 10 min; 4. 15 min; 5. 20 min; sample volume: 4 μL. HPTLC Silica gel 60 (Merck) 10×10 cm, 23℃, 33.2%, echinacoside RF = 0.28, verbascoside RF = 0.56.

[0029] Figure 5 Investigation of different solvent volumes. Among them, 1. from top to bottom: verbascoside, echinacoside; 2. *Cistanche tubulosa* control material; 3. 10 mL, 4. 20 mL, 5. 30 mL. HPTLC Silica gel 60 (Merck) 10×10 cm, 25℃, 32.3%, echinacoside RF = 0.26, verbascoside RF = 0.54.

[0030] Figure 6 Investigation of different spotting volumes. The sample volumes were 1.1 μL, 2.2 μL, 3.3 μL, 4.4 μL, and 5.5 μL. HPTLC Silica gel 60F254 (Merck) 20×10 cm, 20℃, 38.2%, echinacoside RF = 0.26, verbascoside RF = 0.53.

[0031] Figure 7 Investigation of different spot widths. Among them, 1.2mm, 2.4mm, 3.6mm, 4.8mm, and 5.10mm. HPTLCSilica gel 60 (Merck) 10×10cm, 25℃, 33.8%, echinacoside RF=0.28, verbascoside RF=0.56.

[0032] Figure 8Investigation of different solvent systems. Among them, 1. from top to bottom: verbascoside, echinacoside, 2. Cistanche tubulosa reference material, 3. test sample 6, 4. test sample 9, 5. test sample 10, 6. test sample 11. HPTLC Silica gel60 (Merck) 10×10cm, A: dichloromethane-glacial acetic acid-methanol-water (15:6.5:2:2), 24℃, 35.4%, echinacoside RF=0.16, verbascoside RF=0.33; B: ethyl acetate-methanol-formic acid-water (12:1:1:1), 23.2℃, 32.3%, echinacoside RF=0.16, verbascoside RF=0.42; C: ethyl acetate-methanol-formic acid-water (15:2:2:2), 23.1℃, 31.1%, echinacoside RF=0.30, verbascoside RF=0.60.

[0033] Figure 9 Different unfolding distances were investigated. Among them, 1. from top to bottom: verbascoside and echinacoside; 2. *Cistanche tubulosa* reference material; 3. Test sample 6; 4. Test sample 9; 5. Test sample 10; 6. Test sample 11. HPTLC Silica gel 60 (Merck) 10×10cm, A: 23.8℃, 32%, echinacoside RF=0.30, verbascoside RF=0.60; B: 24.6℃, 19%, echinacoside RF=0.30, verbascoside RF=0.65; C: 24.6℃, 17%, echinacoside RF=0.38, verbascoside RF=0.76.

[0034] Figure 10 Thin-layer chromatography (TLC) spectra examined by the inspection method. Among them, 1. from top to bottom: verbascoside and echinacoside; 2. *Cistanche tubulosa* reference material; 3. Test sample 6; 4. Test sample 9; 5. Test sample 10; 6. Test sample 11. HPTLC Silicagel 60 (Merck), 10×10cm, 23.1℃, 31.1%, echinacoside RF = 0.30, verbascoside RF = 0.60. A: Examination under UV lamp (366nm); B: Examination under UV lamp (365nm) after spraying with 10% sulfuric acid ethanol; C: Examination under sunlight after spraying with 10% sulfuric acid ethanol.

[0035] Figure 11 Thin-layer chromatography (TLC) spectroscopy results after heating following color development. From top to bottom: 1. Verbascoside and echinacoside; 2. *Cistanche tubulosa* reference material; 3. Test sample 9. HPTLC Silica gel 60 (Merck), 10×10cm, 23.3℃, ​​34.2%, echinacoside RF = 0.26, verbascoside RF = 0.56.

[0036] Figure 12 Thin-layer chromatography (TLC) images for specificity studies. From top to bottom: 1. Verbascoside and echinacoside; 2. Control herb; 3. Cistanche tubulosa; 4. Cistanche deserticola; 5. Cistanche salsa; 6. Cistanche aridae. HPTLC Silica gel 60 (Merck), 10×10cm, 23.3℃, ​​34.2%, echinacoside RF = 0.29, verbascoside RF = 0.60.

[0037] Figure 13 Comparison of different precast thin-layer slabs. Among them, 1. from top to bottom: verbascoside, echinacoside; 2. *Cistanche tubulosa* reference material; 3. Test sample 1. A: 18℃, 30%, echinacoside RF = 0.29, verbascoside RF = 0.60; B: 19℃, 25%, echinacoside RF = 0.26, verbascoside RF = 0.56; C: 20℃, 40%, echinacoside RF = 0.29, verbascoside RF = 0.57; D: 20℃, 40%, echinacoside RF = 0.25, verbascoside RF = 0.53.

[0038] Figure 14 Comparison of fluorescent agents. Among them, 1. from top to bottom: verbascoside, echinacoside; 2. *Cistanche tubulosa* reference material; 3. Test sample 9. A: GF254 (365nm), 18℃, 30%, echinacoside RF = 0.29, verbascoside RF = 0.60; B: G (365nm), 19℃, 25%, echinacoside RF = 0.26, verbascoside RF = 0.56.

[0039] Figure 15 Comparison at different temperatures. Among them, 1. from top to bottom: verbascoside and echinacoside; 2. *Cistanche tubulosa* reference material; 3. Test sample 6; 4. Test sample 9; 5. Test sample 10; 6. Test sample 11. HPTLC Silica gel 60 (Merck) 10×10cm, A: 4℃, 40%, echinacoside RF = 0.33, verbascoside RF = 0.63; B: 25℃, 40%, echinacoside RF = 0.26, verbascoside RF = 0.61; C: 40℃, 40%, echinacoside RF = 0.29, verbascoside RF = 0.63.

[0040] Figure 16Comparison of different relative humidity levels. Among them, 1. from top to bottom: verbascoside, echinacoside; 2. *Cistanche tubulosa* reference material; 3. Test sample 6; 4. Test sample 9; 5. Test sample 10; 6. Test sample 11. HPTLC Silica gel 60 (Merck) 10×10cm. A: 25℃, 40%, echinacoside RF = 0.20, verbascoside RF = 0.53; B: 25℃, 75%, echinacoside RF = 0.29, verbascoside RF = 0.64; C: 25℃, 92.5%, echinacoside RF = 0.29, verbascoside RF = 0.68.

[0041] Figure 17 Stability study. Among them, 1. from top to bottom: verbascoside, echinacoside; 2. *Cistanche tubulosa* control material; 3. 0 days, 4. 1 days, 5. 2 days, 6. 3 days. HPTLC Silica gel 60 (Merck) 10×10cm. 24.6℃, 42.5%, echinacoside RF=0.27, verbascoside RF=0.56.

[0042] Figure 18 Identification atlases of multiple batches of Cistanche tubulosa medicinal materials. Among them, 1. from top to bottom: verbascoside and echinacoside; 2. Cistanche tubulosa reference material; 3. Test sample 1; 4. Test sample 2; 5. Test sample 3; 6. Test sample 4; 7. Test sample 5; 8. Test sample 6; 9. Test sample 7; 10. Test sample 8; 11. Test sample 9. HPTLC Silica gel 60 (Merck) 10×20cm. 24.7℃, 48.2%, echinacoside RF=0.25, verbascoside RF=0.52.

[0043] Figure 19 Identification charts of multiple batches of Cistanche tubulosa medicinal materials. Among them, 1. from top to bottom: verbascoside and echinacoside; 2. Cistanche tubulosa reference material; 3. Test sample 10; 4. Test sample 11; 5. Test sample 12; 6. Test sample 13; 7. Test sample 14; 8. Test sample 15; 9. Test sample 16; 10. Test sample 17; 11. Test sample 18. HPTLC Silica gel 60 (Merck) 10×20cm, 26.7℃, 48.1%, echinacoside RF=0.26, verbascoside RF=0.53.

[0044] Figure 20Identification charts of multiple batches of Cistanche deserticola. Among them, 1. from top to bottom: verbascoside and echinacoside; 2. Cistanche deserticola reference material; 3. Test sample 1; 4. Test sample 2; 5. Test sample 3; 6. Test sample 4; 7. Test sample 5; 8. Test sample 6; 9. Test sample 7; 10. Test sample 8; 11. Test sample 9. HPTLC Silica gel 60 (Merck) 10×20cm, 23.5℃, 25.8%, echinacoside RF=0.25, verbascoside RF=0.52.

[0045] Figure 21 Comparison with other pharmacopoeia standards. Among them, 1. from top to bottom are echinacoside, verbascoside (A); verbascoside, echinacoside (C) 2. Cistanche tubulosa reference material, 3. Cistanche tubulosa, 4. Cistanche deserticola, 5. Cistanche salsa, 6. Cistanche deserticola. A: Chinese Pharmacopoeia, polyamide film 10×10cm, 20.7℃, 35.0%, verbascoside RF=0.24, echinacoside RF=0.67, blue fluorescent spots; B: TLC Silica gel 60 (Merck) F254 10×10cm, 20.3℃, 63.0%, not developed; C: The thin-layer chromatography identification method established in this invention, HPTLC Silica gel 60 (Merck) 10×10cm, 23.3℃, ​​34.2%, echinacoside RF=0.29, verbascoside RF=0.63, yellow-green fluorescent spots. Detailed Implementation

[0046] To facilitate understanding of the present invention, a more comprehensive description will be given below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0047] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0048] The technical terms and their meanings involved in the embodiments of this invention are as follows:

[0049] Thin-layer chromatography (TLC) is a rapid, simple, and low-volume separation and analysis method. A suitable stationary phase is coated onto a glass plate, plastic, or aluminum substrate to form a uniform thin layer. After spotting and development, the ratio shift (Rf) value is compared with that of a suitable reference substance obtained using the same method. This method is used for drug identification, impurity detection, or content determination. It is an important experimental technique for the rapid separation and qualitative analysis of small amounts of substances and is also used to track reaction progress. It features short development time, strong separation ability, and high sensitivity. TLC is one of the important testing items for traditional Chinese medicine materials. By comparing the chromatographic behavior of the test sample and the reference material / standard, the authenticity and quality of the medicinal material can be determined.

[0050] Spotting: The process of adding droplets of the sample solution to be separated and identified onto a thin-layer plate is called spotting.

[0051] Development: The process by which the developing solvent carries the sample components across a thin-layer plate by capillary action of the stationary phase.

[0052] Example:

[0053] 1. Instruments and Materials

[0054] Analytical balance: Sartorius BT25S, BS2202S (Germany); Oil bath: EYELA, model SB-1000 (Shanghai Ailang Instrument Co., Ltd.); Ultrasonic cleaner: KQ-500DE CNC, power 500W, frequency 40KHz; Thin-layer chromatography imaging system: CAMAG, AUTOMATIC TLC SAMLER4 and TLC VISUALIZER; YSUK comprehensive drug stability test chamber.

[0055] Thin layer plates: Merck HPLC sillica gel 60F254 (LOT HX03161042), Merck HPTLC sillica gel 60 (LOT HX02721141), Merck TLC Silica gel60G 25Glassplates (LOTHX03290984), Merck TLC Silica gel 60 (LOT HX90656226).

[0056] 2 reagents

[0057] 95% ethanol (AR, LOT20210120). Ethyl acetate (AR, LOT20200912) and (AR, LOT20210105), from Beijing Tongguang Fine Chemical Co., Ltd. Methanol (HPLC, LOT204133), from Fisher Chemical. Sulfuric acid (AR, LOT20190426) and (AR, LOT20201105), from Beijing Chemical Plant. Formic acid (AR, LOT20200819), from Sinopharm Chemical Reagent Co., Ltd., and (AR, LOT20201130), from Beijing Tongguang Fine Chemical Co., Ltd. Reference materials of Cistanche tubulosa (LOT121276-201102) and Cistanche deserticola (desert) (LOT121101-201603), both purchased from the China National Institutes for Food and Drug Control.

[0058] 3. Study on the preparation method of test sample

[0059] 3.1 Chromatographic System

[0060] Test solution: Take 1.0 g of Cistanche tubulosa powder, add 10 mL of methanol, sonicate for 20 min, let stand, take 5 mL of supernatant, evaporate to dryness, add 5 mL of water to dissolve the residue, extract three times with water-saturated n-butanol (10 mL, 10 mL, 10 mL), combine the n-butanol extracts, evaporate to dryness, add 5 mL of methanol to dissolve the residue, measure 1 mL, put it in a 10 mL volumetric flask, add methanol to the mark, shake well, and the test solution is obtained.

[0061] Reference herbal solution: Take 1.0 g of Cistanche tubulosa powder, add 10 mL of methanol, sonicate for 20 min, let stand, take 5 mL of supernatant, evaporate to dryness, add 5 mL of water to dissolve the residue, extract three times with water-saturated n-butanol (10 mL, 10 mL, 10 mL), combine the n-butanol solutions, evaporate to dryness, add 5 mL of methanol to dissolve the residue, measure 1 mL, put it in a 10 mL volumetric flask, add methanol to the mark, shake well, and the solution is obtained.

[0062] Reference solution: Dissolve 1.5 mg each of echinacoside and verbascoside in 10 mL of methanol.

[0063] Thin-layer plates: Silicone thin-layer plates (5-10μm);

[0064] Developing solvent: ethyl acetate-methanol-formic acid-water (15:2:2:2);

[0065] Sample volume: 4 μL, in a band shape 8 mm;

[0066] Relative humidity: less than or equal to 40%;

[0067] Unfolding distance: 7.5cm;

[0068] Heating temperature: 105℃;

[0069] Identification: Identification under a 365nm ultraviolet lamp.

[0070] Analysis: Spot the test solution, control extract solution, control herbal solution, and control solution onto the same thin-layer plate, develop them at a distance of 7.5 cm, remove the plate, spray with 10% sulfuric acid ethanol solution, heat at 105℃ for about 3 min, and examine at 365 nm.

[0071] 3.2 Investigation on whether the test sample is extracted or not

[0072] The effect of extraction versus non-extraction on the test sample during the preparation of the test sample solution was investigated.

[0073] Take 1.0 g of the powder, add 20 mL of methanol, sonicate for 15 min, let stand, and transfer 1 mL to a 10 mL volumetric flask. Add methanol to the mark and shake well to obtain the test solution. Separately, take 5 mL of the supernatant, evaporate to dryness, dissolve the residue in 5 mL of water, and extract three times with water-saturated n-butanol (10 mL, 10 mL, 10 mL). Combine the n-butanol extracts, evaporate to dryness, dissolve the residue in 5 mL of methanol, transfer 1 mL to a 10 mL volumetric flask, add methanol to the mark, shake well, and spot 4 μL onto a silica gel thin-layer plate. The results are shown in the figure. Figure 1 .

[0074] Depend on Figure 1 It is evident that the extracted sample has a clear and uninterrupted background color, and exhibits an enrichment effect on verbascoside components. Therefore, n-butanol extraction was chosen for sample preparation.

[0075] 3.3 Investigation of Extraction Solvent Types

[0076] The extraction efficiency of different solvents used in the preparation of the test sample solution was investigated.

[0077] Take 20 mL each of methanol and ethanol, sonicate for 15 min, let stand, take 5 mL of the supernatant, evaporate to dryness, dissolve the residue in 5 mL of water, and extract three times with water-saturated n-butanol (10 mL, 10 mL, 10 mL each). Combine the n-butanol extracts, evaporate to dryness, dissolve the residue in 5 mL of methanol, measure 1 mL, place in a 10 mL volumetric flask, add methanol to the mark, shake well, and spot 4 μL onto a silica gel thin-layer plate. The results are shown in the figure. Figure 2 .

[0078] Depend on Figure 2 It is evident that when methanol and ethanol are used as extraction solvents, the chromatographic information is identical, and the background color of the sample prepared by methanol extraction is clearer and less interfering. Therefore, methanol is chosen as the extraction solvent.

[0079] 3.4 Examination of Extraction Methods

[0080] The extraction efficiency of different extraction methods used in the preparation of the test sample solution was investigated.

[0081] Take 1.0g of Cistanche deserticola powder, divide into two portions, add 20mL of methanol to each, heat one portion under reflux for 15min, and sonicate the other portion for 15min. Cool and allow to stand, take 5mL of the supernatant, evaporate to dryness, dissolve the residue in 5mL of water, and extract three times with water-saturated n-butanol (10mL, 10mL, 10mL). Combine the n-butanol extracts, evaporate to dryness, dissolve the residue in 5mL of methanol, measure 1mL, place in a 10mL volumetric flask, add methanol to the mark, shake well, and spot 4μL onto a silica gel thin-layer plate. See the results below. Figure 3 .

[0082] Depend on Figure 3 It is evident that different extraction methods yield similar extraction results; therefore, ultrasonic extraction is chosen for its ease of operation.

[0083] 3.5 Examination of extraction time

[0084] The extraction effects of different extraction times (10 min, 15 min, 20 min) were investigated.

[0085] Take 1.0 g of the powder, three portions in total, add 20 mL of methanol, and extract each portion using the ultrasonic extraction time described above. After standing, take 5 mL of the supernatant, evaporate to dryness, dissolve the residue in 5 mL of water, and extract three times with water-saturated n-butanol (10 mL, 10 mL, 10 mL). Combine the n-butanol extracts, evaporate to dryness, dissolve the residue in 5 mL of methanol, measure 1 mL of the solution, place it in a 10 mL volumetric flask, add methanol to the mark, shake well, and spot 4 μL onto a silica gel thin-layer plate. The results are shown in the figure. Figure 4 .

[0086] Depend on Figure 4 It can be seen that chromatographic information is relatively clear when 20 minutes is used as the extraction time. Therefore, 20 minutes is selected as the extraction time.

[0087] 3.6 Investigation of the extraction solvent volume

[0088] The extraction effect of using different volumes (10 mL, 20 mL, 30 mL) of methanol in the preparation of the test solution was investigated.

[0089] Extraction solutions were prepared using 10 mL, 20 mL, and 30 mL of methanol as the extraction solvent, following the same method as for the test solution. 4 μL of each solution was spotted onto a silica gel thin-layer plate. Results are shown below. Figure 5 .

[0090] Depend on Figure 5 It can be seen that different extraction solvent volumes produce similar results. In order to save resources, the extraction solvent volume was chosen to be 10 mL.

[0091] In summary, the optimized method for preparing the test solution is as follows: Take 1.0 g of Cistanche deserticola powder, add 10 mL of methanol, sonicate for 20 min, let stand, take 5 mL of the supernatant, evaporate to dryness, add 5 mL of water to dissolve the residue, extract three times with water-saturated n-butanol (10 mL, 10 mL, 10 mL), combine the n-butanol solutions, evaporate to dryness, add 5 mL of methanol to dissolve the residue, measure 1 mL, place in a 10 mL volumetric flask, add methanol to the mark, and shake well.

[0092] 4. Study of Chromatographic Systems

[0093] 4.1 Investigation of sample volume

[0094] The thin-layer chromatography effect of different spotting volumes (1 μL, 2 μL, 3 μL, 4 μL, 5 μL) was investigated.

[0095] Take the test solution and spot 1 μL, 2 μL, 3 μL, 4 μL, and 5 μL onto the same thin-layer plate, respectively. Develop, visualize, and identify the sample. Results are as follows: Figure 6 As shown.

[0096] Depend on Figure 6 It can be seen that the spots are clear when the sample volume is 3-5 μL, so a sample volume of 3-5 μL is acceptable.

[0097] 4.2 Examination of the width of the sampling strip

[0098] The thin-layer chromatography effect of different spot widths (2, 4, 6, 8, 10 mm) was investigated.

[0099] Take 4 μL of the test solution and spot it onto the same thin-layer plate with widths of 2, 4, 6, 8, and 10 mm. Develop, visualize, and identify the sample. The results are as follows: Figure 7 As shown.

[0100] Depend on Figure 7 It can be seen that a width of 8mm can achieve good separation and clear color bands, so the sampling width of 8mm is selected.

[0101] 4.3 Investigation of the developing solvent system

[0102] The effects of thin-layer chromatography using different solvent systems as developing solvents were investigated.

[0103] Using the same test solution, the thin-layer chromatography performance of three solvent systems as developing solvents was compared. The results are as follows: Figure 8 As shown.

[0104] Depend on Figure 8 To date, the solvent system of dichloromethane-ethyl acetate-methanol-water (15:2:2:2) has the best separation effect.

[0105] 4.4 Conduct a distance survey

[0106] The effect of different developing distances on the thin-layer chromatography performance was investigated.

[0107] Take 4 μL of the same test solution and develop it at distances of 7.5 cm, 10 cm, and 12 cm. The results are as follows: Figure 9 As shown.

[0108] Depend on Figure 9 As is known, the spots gradually become blurred as the unfolding distance increases, so the unfolding distance was chosen to be 7.5cm.

[0109] 4.5 Examination of Inspection Methods

[0110] The effects of thin-layer chromatography using different inspection methods as developing solvents were investigated.

[0111] Using the same test solution, the thin-layer chromatography effects of three different inspection methods were compared. The results are as follows: Figure 10 As shown.

[0112] Depend on Figure 10 As is known, inspection method B provides rich chromatographic information and clear spots. Therefore, inspection method B was selected as the identification method for thin-layer chromatography of Cistanche tubulosa.

[0113] 4.6 Examination of heating time after color development

[0114] The effect of different heating times on the thin-layer chromatography results was investigated.

[0115] Take 4 μL of the test solution and spot it on the same thin-layer plate. Develop the plate, add 10% sulfuric acid ethanol solution for color development, heat at 105℃ for 3 min, and examine. Continue heating for another 2 min and examine again. Continue heating for another 5 min and examine again. The results are as follows: Figure 11 As shown.

[0116] Depend on Figure 11 It is known that the spots are clearer when heated for 3-5 minutes. Therefore, a heating time of 3 minutes was chosen after developing the color with 10% sulfuric acid ethanol solution.

[0117] 5. Methodological Investigation

[0118] 5.1 Exclusivity

[0119] The specificity of the established thin-layer chromatography method was examined.

[0120] 1.0 g each of *Cistanche tubulosa*, *Cistanche deserticola*, *Cistanche salsa*, and *Cistanche aridans* were taken and samples were prepared according to the optimized test solution preparation method. 4 μL of each sample was spotted onto the same thin-layer plate, developed, colored, and examined. The results are as follows: Figure 12 As shown. By Figure 12It can be seen that the thin-layer chromatography information of *Cistanche tubulosa* and *Cistanche salsa* is most similar. In *Cistanche salsa*, there are more detected pinkish-purple spots below the echinacoside spots than in *Cistanche tubulosa*. The thin-layer chromatography of *Cistanche aquaticus* shows the most significant differences from the other three. No yellow-green spots of echinacoside were detected in *Cistanche aquaticus*, while an additional yellow-green spot appeared in the middle of the development distance for echinacoside and verbascoside. Other raw medicinal materials did not detect these components. Due to differences in content, the detection spots of echinacoside and verbascoside in *Cistanche deserticola* were weaker in samples of the same concentration. The biggest difference between *Cistanche deserticola* and *Cistanche tubulosa* is that two distinct blue fluorescent spots appear below the yellow-green spots of echinacoside in *Cistanche deserticola*. Other raw medicinal materials did not detect these components. Therefore, this thin-layer chromatography method can specifically identify *Cistanche tubulosa*.

[0121] 5.2 Study on the applicability of the method

[0122] 5.2.1 Comparison of different precast slabs

[0123] Thin-layer chromatography (TLC) analysis of the same test solution was performed using A. HPTLC Silica gel 60F254 (Merck), B. HPTLC Silica gel 60 (Merck), C. TLC Silica gel 60 (Merck), and D. TLC Silica gel 60G (Merck). The results are shown in the figure. Figure 13 .Depend on Figure 13 It can be seen that all four types of thin-layer plates produced by Merck can achieve good separation results.

[0124] 5.2.2 Examination of Fluorescent Thin-Layer Plates and G Plates

[0125] Thin-layer chromatography analysis of the same test solution was performed using GF254 plates containing fluorescent dye and G plates without fluorescent dye, respectively. The results are shown in the figure. Figure 14 .Depend on Figure 14 It is evident that the presence or absence of fluorescent agents has little effect on chromatographic separation.

[0126] 5.2.3 Comparison of different temperatures

[0127] Thin-layer chromatography analysis of the same test solution was performed at different temperatures (4℃, 25℃, 40℃), and the results are shown in the figure. Figure 15 .Depend on Figure 15 It is evident that temperature has little effect on chromatographic separation.

[0128] 5.2.4 Comparison of different relative humidity levels

[0129] Thin-layer chromatography analysis of the same test solution was performed at different relative humidity levels (40%, 70%, 92.5%), and the results are shown in the figure. Figure 16 .Depend on Figure 16It is evident that the separation efficiency of verbascoside, one of the main components, deteriorates when the relative humidity exceeds 40%. Therefore, a relative humidity of 40% or less is specified.

[0130] 5.2.4 Sample Stability

[0131] To examine the stability of the test solution, the thin-layer chromatography results of the test solution stored at room temperature for 0, 1, 2, and 3 days were compared, such as... Figure 17 As shown. Figure 17 As shown, the thin-layer chromatography results of the test solution for different days were consistent, indicating that the test solution is stable within 3 days of storage at room temperature.

[0132] 6 Confirmed Thin-Layer Chromatography Identification Methods

[0133] Based on the above research results, the final thin-layer chromatographic conditions for Cistanche tubulosa are as follows:

[0134] Adsorbent: Thin-layer plates with an average particle size of 5-10 μm.

[0135] Preparation method of reference solution: Take echinacoside and verbascoside reference standards, add methanol to prepare a mixed solution containing 2 mg of each per 10 mL, as the reference solution.

[0136] Preparation method of the reference herbal solution: Take 1g of the powder, add 10mL of methanol, sonicate for 20min, filter, take 5mL of the filtrate, evaporate to dryness, dissolve the residue in 5mL of water, extract three times with 10mL of water-saturated n-butanol each time, combine the n-butanol extracts, evaporate to dryness, dissolve the residue in 5mL of methanol, accurately measure 1mL, place in a 10mL volumetric flask, dilute to the mark with methanol, shake well, filter, and use as the test solution.

[0137] Preparation of the test solution: Take 1g of the powder, add 10mL of methanol, sonicate for 20min, filter, take 5mL of the filtrate, evaporate to dryness, add 5mL of water to dissolve the residue, extract three times with 10mL of water-saturated n-butanol each time, combine the n-butanol extracts, evaporate to dryness, add 5mL of methanol to dissolve the residue, accurately measure 1mL, place in a 10mL volumetric flask, dilute to the mark with methanol, shake well, filter, and use as the test solution.

[0138] Effervescent solvent system: ethyl acetate-methanol-formic acid-water (15:2:2:2).

[0139] Spotting volume: 3-5 μL, strip width: 8 mm.

[0140] analyze

[0141] Spot the sample onto the corresponding thin-layer chromatography plate and perform the test according to the thin-layer chromatography method (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502). Develop with ethyl acetate-methanol-formic acid-water (15:2:2:2) under saturated conditions (relative humidity ≤40%), with a development distance of 7.5 cm. Remove the plate, air dry, spray with 10% sulfuric acid ethanol solution, and heat at 105℃ for about 3 min. Examine under ultraviolet light (365 nm). Echinacoside and verbascoside appear as yellow-green fluorescent spots at approximately one-third and one-half of the plate, respectively, with RF values ​​of 0.25 and 0.52. In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatograms of the reference medicinal material, echinacoside, and verbascoside reference standards.

[0142] Identification of more than 7 batches of Cistanche tubulosa medicinal materials

[0143] Eighteen batches of *Cistanche tubulosa* samples and nine batches of *Cistanche deserticola* samples were analyzed by thin-layer chromatography using the optimized method described above. The results are shown in the table below. Figure 18-20 .

[0144] 8. Comparison with other pharmacopoeia standards

[0145] The thin-layer chromatography identification methods in the two standards are now compared with the newly established thin-layer chromatography identification method in this paper.

[0146] The results are as follows Figure 21 The results showed that the newly established thin-layer chromatography identification method in this paper had clear bands, good separation effect, and rich chromatographic information. It can also specifically identify Cistanche deserticola and Cistanche tubulosa medicinal materials, which is superior to other existing standards.

[0147] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A thin-layer chromatographic detection method for Cistanche deserticola and Cistanche tubulosa, characterized in that: The detection method includes the following steps: A test solution, a reference herb solution, and a reference standard solution were prepared separately. These solutions were then spotted onto the same thin-layer chromatography plate, developed in the developing solvent, and subjected to color development using a colorimetric reagent. The preparation steps of the test solution include: taking 1 g of Cistanche deserticola and Cistanche tubulosa test sample powder, adding 10 mL of methanol, sonicating for 20 min, filtering, taking 5 mL of the filtrate, evaporating to dryness, adding 5 mL of water to dissolve the residue, extracting 3 times with 10 mL of water-saturated n-butanol each time, combining the n-butanol extracts, evaporating to dryness, adding 5 mL of methanol to dissolve the residue, accurately measuring 1 mL, placing it in a 10 mL volumetric flask, diluting with methanol to the mark, shaking well, filtering, and using it as the test solution; Following the steps for preparing the test solution, the reference medicinal material solution was prepared using Cistanche deserticola and Cistanche tubulosa as reference medicinal materials; The preparation steps of the reference solution include: taking echinacoside reference standard and verbascoside reference standard, adding methanol to prepare a mixed solution containing 2 mg of each per 10 mL, as the reference solution. The developing solvent system was ethyl acetate:methanol:formic acid:water = 15:2:2:2; the spotting volume was 3-5 μL; and the band width was 8 mm. The colorimetric reagent is a 10% sulfuric acid ethanol solution (by volume).

2. The thin-layer chromatography detection method according to claim 1, characterized in that: The thin-layer chromatography plate is a silica gel thin-layer plate, and the average particle size of the adsorbent is 5-10 μm.

3. The thin-layer chromatography detection method according to claim 1 or claim 2, characterized in that: The detection method includes the following steps: The test solution, reference herb solution, and reference standard solution were spotted onto a thin-layer chromatographic plate. The plate was developed under saturated conditions using ethyl acetate:methanol:formic acid:water = 15:2:2:2, relative humidity ≤40%, and development distance 7.5 cm. The plate was then removed, air-dried, sprayed with 10% sulfuric acid ethanol solution (v / v), heated at 105℃ for about 3 min, and examined under a 365 nm UV lamp.

4. The thin-layer chromatography detection method according to claim 3, characterized in that: Under ultraviolet light, echinacoside and verbascoside appeared as yellow-green fluorescent spots at approximately one-third and one-half of the thin-layer plate, with RF values ​​of 0.25 and 0.52, respectively.

5. The use of the thin-layer chromatography detection method according to any one of claims 1 to 4 in the specific identification of Cistanche deserticola and Cistanche tubulosa medicinal materials.

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