Method for identifying zanthoxylum bungeanum and zanthoxylum armatum

Abstract

The present application belongs to the technical field of traditional Chinese medicine identification, and provides a method for identifying Zanthoxylum nitidum and Zanthoxylum ailanthoides. The present application extracts Zanthoxylum nitidum samples with 70% methanol to obtain a test solution; extracts Zanthoxylum ailanthoides medicinal materials with 70% methanol, concentrates and dissolves the obtained filtrate after evaporation, filters, injects into a high performance liquid chromatograph, elutes, collects effluent liquid of 20-24 min, and obtains characteristic components after nitrogen blowing dry, mixes with ethanol to obtain a control solution; points the two solutions on the same silica gel G plate, develops twice with petroleum ether-chloroform-methanol and n-butanol-methanol-ammonia solution, inspects under ultraviolet light after iodine vapor fumigation, and if the same color fluorescent spots appear in the test solution chromatogram at the corresponding positions of the control solution chromatogram, it is identified that the sample to be measured is Zanthoxylum ailanthoides instead of Zanthoxylum nitidum. The method is simple, rapid, specific, can accurately identify Zanthoxylum nitidum and Zanthoxylum ailanthoides, and is suitable for the true and false identification and quality control of Zanthoxylum nitidum medicinal materials.

Description

Technical Field

[0001] This invention relates to the field of identification technology of Chinese medicinal materials, and in particular to a method for identifying Zanthoxylum nitidum and Zanthoxylum nitidum with hairy leaves. Background Technology

[0002] Zanthoxylum nitidum (Roxb.) DC., a plant in the Rutaceae family, is the dried root of the Chinese Pharmacopoeia (2015 edition, Part I). This medicinal material possesses the functions of promoting blood circulation and removing blood stasis, regulating qi and relieving pain, dispelling wind and unblocking collaterals, and detoxifying and reducing swelling, making it a commonly used traditional Chinese medicine. Its chemical components include alkaloids, lignans, flavonoid glycosides, and sterols. Modern pharmacological studies have shown that it exhibits various biological activities such as antispasmodic, anti-inflammatory, analgesic, antibacterial, and antitumor effects, demonstrating significant application value. However, with the continuous growth in market demand for Zanthoxylum nitidum, its wild resources are increasingly scarce due to ecological damage and over-harvesting. Furthermore, due to differences in production areas and growing environments, the sources of commercially available Zanthoxylum nitidum are quite mixed, making it difficult to distinguish genuine from counterfeit products.

[0003] Zanthoxylum nitidum var. tomentosum is a variety of Zanthoxylum nitidum and is one of the adulterants used to adulterate Zanthoxylum nitidum. It is now included in the "Quality Standard of Yao Medicinal Materials of Guangxi Zhuang Autonomous Region" (2014 edition). This variety has densely hairy leaves on the underside, making it extremely similar in appearance to the original variety of Zanthoxylum nitidum (i.e., Zanthoxylum nitidum var. tomentosum used as an equivalent to Zanthoxylum nitidum). The two varieties differ by only one character in their names, thus often leading to confusion in circulation and use. Currently, there is a lack of systematic experimental research on this adulterant, Zanthoxylum nitidum var. tomentosum, which has a similar origin and morphology. However, given the scarcity of resources, it has gradually entered the circulation field to replace the genuine product. This variety confusion directly leads to a loss of quality control, not only posing significant difficulties for the quality control of Zanthoxylum nitidum but also potentially threatening the safety and efficacy of clinical use.

[0004] Therefore, establishing an identification method that can accurately distinguish between Zanthoxylum nitidum and Zanthoxylum nitidum has become an urgent need to ensure the quality and safety of Zanthoxylum nitidum medicinal materials and their clinical efficacy. Summary of the Invention

[0005] In view of this, the present invention provides a method for identifying Zanthoxylum nitidum and Zanthoxylum nitidum with hairy leaves, in order to solve the problem that it is difficult to accurately distinguish between Zanthoxylum nitidum and Zanthoxylum nitidum with hairy leaves in the prior art, which leads to the difficulty in effectively controlling the quality of medicinal materials.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a method for identifying Zanthoxylum nitidum and Zanthoxylum nitidum with hairy leaves, comprising the following steps: 1) Preparation of test solution: Extract the powder of Zanthoxylum nitidum from the sample using a 70% methanol solution to obtain the test solution; 2) Preparation of characteristic components of Zanthoxylum nitidum: Zanthoxylum nitidum powder was mixed with a 70% (v / v) methanol solution, and the mixture was subjected to reflux and a first filtration sequentially to obtain a filtrate. The filtrate was then subjected to evaporation to dryness, concentration and dissolution, and a second filtration sequentially. The filtrate was then injected into a high-performance liquid chromatograph for elution. The chromatographic conditions for elution were as follows: Chromatographic column: Shiseido CAPCELL PAK, 4.6 × 250 mm, 5 μm; Flow rate: 1.0 mL / min; Detection wavelength: 220 nm; Injection volume: 20 μL; Mobile phase: Mobile phase A is acetonitrile, and mobile phase B is a mixed aqueous solution of phosphate and triethylamine, wherein the mass-volume ratio of phosphate, triethylamine and water is 0.2 g: 0.25 g: 100 mL; The elution is gradient elution, and the gradient elution procedure is as follows: From 0 to 20 min, mobile phase A increased from 10% to 21%, and mobile phase B increased from 90% to 79%. Over 20-35 minutes, mobile phase A changed from 21% to 25%, and mobile phase B changed from 79% to 75%. 35~50 min, mobile phase A is 25%, mobile phase B is 75%; Simultaneously, the effluent was collected for 20-24 minutes, and then the effluent was dried with nitrogen to obtain the characteristic components of Zanthoxylum bungeanum. 3) Preparation of reference solution: The characteristic components of Zanthoxylum bungeanum obtained in step 2) are mixed with an organic solvent to obtain the reference solution; 4) Development and detection: Take 2 μL each of the test solution and the reference solution and spot them separately on the same silica gel G thin-layer plate. First, use petroleum ether-chloroform-methanol (volume ratio 2:13:1) as the developing solvent for the first development, remove and air dry. Petroleum ether is the fraction with a boiling point range of 60~90℃. Then, use n-butanol-methanol-ammonia solution (volume ratio 5:25:13) as the developing solvent for the second development, remove and air dry. Then, fumigate in iodine vapor. After removal, evaporate the iodine adsorbed on the silica gel G thin-layer plate. Finally, examine under a UV lamp with a wavelength of 365 nm. 5) Result determination: If the test sample chromatogram shows a spot of the same color at the corresponding position as the reference chromatogram, the Zanthoxylum nitidum test sample is Zanthoxylum nitidum; if the test sample chromatogram does not show a spot of the same color at the corresponding position as the reference chromatogram, the Zanthoxylum nitidum test sample is not Zanthoxylum nitidum.

[0007] Preferably, the particle size of the two-sided needle sample powder in step 1) is 342~368 μm.

[0008] Preferably, in step 2), the mass-to-volume ratio of the Zanthoxylum nitidum powder to the methanol solution is 2 g: 45~55 mL; and the particle size of the Zanthoxylum nitidum powder is 342~368 μm.

[0009] Preferably, the temperature of the heating reflux in step 2) is 80~85℃ and the time is 27~33 min.

[0010] Preferably, the concentrated and dissolved solution in step 2) is a methanol solution with a volume fraction of 70%; the mass-to-volume ratio of the evaporated sample to the concentrated and dissolved solution is 0.2 g: 1 mL; and the pore size of the filter membrane for the second filtration is 0.22 μm.

[0011] Preferably, in step 3), the mass-to-volume ratio of the characteristic component of Zanthoxylum bungeanum to the organic solvent is 0.8~1.2 mg:1 mL; the organic solvent includes ethanol and / or methanol.

[0012] Preferably, in step 4), the temperature of the first and second unfolding is 4~30℃ and the humidity is 30~70% independently; the unfolding distance of the first unfolding is 8~10 cm; the unfolding distance of the second unfolding is 8~10 cm; and the fumigation time is 40~60 s.

[0013] As can be seen from the above technical solution, compared with the prior art, the beneficial effects of the present invention are as follows: This invention employs a two-stage development process: first using petroleum ether (60-90℃)-chloroform-methanol as the developing solvent, and then using n-butanol-methanol-ammonia solution as the developing solvent. This allows the chemical components of *Zanthoxylum nitidum* and *Zanthoxylum nitidum* var. *hairylum* to exhibit different spot colors on a thin-layer chromatography plate, effectively distinguishing them. This solves the problem of difficulty in differentiating the two species due to their similar origins and appearance, providing a reliable technical means for the authenticity identification and quality control of *Zanthoxylum nitidum* medicinal materials. This is of great significance for ensuring the safety and efficacy of clinical medication. Furthermore, the method described in this invention is simple to operate, requiring only conventional thin-layer chromatography equipment and reagents, without complex pretreatment or expensive instruments, making it low-cost and easy to promote and apply. Attached Figure Description

[0014] 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.

[0015] Figure 1 The images are inspection views of Zanthoxylum nitidum (L1~L6), Zanthoxylum nitidum (M1~M8), Zanthoxylum nitidum (F1~F3), as well as bamboo leaf pepperin and Zanthoxylum nitidum alkaloid under ultraviolet light (365 nm) in Example 1. Figure 2 The images are inspection views of Zanthoxylum nitidum (L7~L11), Zanthoxylum nitidum (M9~M16), Zanthoxylum nitidum (F4~F7), as well as bamboo leaf pepperin and Zanthoxylum nitidum alkaloids under ultraviolet light (365 nm) in Example 1. Figure 3 The images are inspection views of Zanthoxylum nitidum (L12~L17), Zanthoxylum nitidum (M17~M23), Zanthoxylum nitidum (F8~F11), as well as bamboo leaf pepperin and Zanthoxylum nitidum alkaloid under ultraviolet light (365 nm) in Example 1. Figure 4 The images are inspection views of Zanthoxylum nitidum (L1~L3), Zanthoxylum nitidum (M1~M3), Zanthoxylum nitidum (F1~F3), as well as bamboo leaf pepperin and Zanthoxylum nitidum alkaloid under ultraviolet light (365 nm) in Example 2. Figure 5 The images are inspection views of Zanthoxylum nitidum (L1~L3), Zanthoxylum nitidum (M1~M3), Zanthoxylum nitidum (F1~F3), as well as bamboo leaf pepperin and Zanthoxylum nitidum alkaloids under ultraviolet light (365 nm) in Example 3. Figure 6 The images are inspection views of Zanthoxylum nitidum (L1~L3), Zanthoxylum nitidum (M1~M3), Zanthoxylum nitidum (F1~F3), as well as bamboo leaf pepperin and Zanthoxylum nitidum alkaloids under ultraviolet light (365 nm) in Example 4. Figure 7 The images show inspection views of Zanthoxylum nitidum (L1~L3), Zanthoxylum nitidum (M1~M3), Zanthoxylum nitidum (F1~F3), as well as bamboo leaf pepperin and Zanthoxylum nitidum alkaloids under ultraviolet light (365 nm) in Example 5. Detailed Implementation

[0016] This invention provides a method for identifying Zanthoxylum nitidum and Zanthoxylum nitidum with hairy leaves, comprising the following steps: 1) Preparation of test solution: Extract the powder of Zanthoxylum nitidum from the sample using a 70% methanol solution to obtain the test solution; 2) Preparation of characteristic components of Zanthoxylum nitidum: Zanthoxylum nitidum powder was mixed with a 70% (v / v) methanol solution, and the mixture was subjected to reflux and a first filtration sequentially to obtain a filtrate. The filtrate was then subjected to evaporation to dryness, concentration and dissolution, and a second filtration sequentially. The filtrate was then injected into a high-performance liquid chromatograph for elution. The chromatographic conditions for elution were as follows: Chromatographic column: Shiseido CAPCELL PAK, 4.6 × 250 mm, 5 μm; Flow rate: 1.0 mL / min; Detection wavelength: 220 nm; Injection volume: 20 μL; Mobile phase: Mobile phase A is acetonitrile, and mobile phase B is a mixed aqueous solution of phosphate and triethylamine, wherein the mass-volume ratio of phosphate, triethylamine and water is 0.2 g: 0.25 g: 100 mL; The elution is gradient elution, and the elution procedure for the gradient elution is as follows: From 0 to 20 min, mobile phase A increased from 10% to 21%, and mobile phase B increased from 90% to 79%. Over 20-35 minutes, mobile phase A changed from 21% to 25%, and mobile phase B changed from 79% to 75%. 35~50 min, mobile phase A is 25%, mobile phase B is 75%; Simultaneously, the effluent was collected for 20-24 minutes, and then the effluent was dried with nitrogen to obtain the characteristic components of Zanthoxylum bungeanum. 3) Preparation of reference solution: The characteristic components of Zanthoxylum bungeanum obtained in step 2) are mixed with an organic solvent to obtain the reference solution; 4) Development and detection: Take 2 μL each of the test solution and the reference solution and spot them separately on the same silica gel G thin-layer plate. First, use petroleum ether-chloroform-methanol with a volume ratio of 2:13:1 as the developing solvent for the first development, remove and air dry. Petroleum ether is the fraction with a boiling point range of 60~90℃. Then, use n-butanol-methanol-ammonia test solution with a volume ratio of 5:25:13 as the developing solvent for the second development, remove and air dry. Then, fumigate in iodine vapor. After removal, evaporate the iodine adsorbed on the silica gel G thin-layer plate. Finally, examine under a UV lamp with a wavelength of 365 nm. 5) Result determination: If the test sample chromatogram shows a spot of the same color at the corresponding position as the reference chromatogram, the Zanthoxylum nitidum test sample is Zanthoxylum nitidum; if the test sample chromatogram does not show a spot of the same color at the corresponding position as the reference chromatogram, the Zanthoxylum nitidum test sample is not Zanthoxylum nitidum.

[0017] In this invention, the extraction process described in step 1) is preferably to mix the powder of the two-sided needle sample to be tested with a methanol solution with a volume fraction of 70%, perform a first ultrasonic treatment, filter to obtain a first filtrate and a first filter residue, mix the first filter residue with a methanol solution with a volume fraction of 70%, perform a second ultrasonic treatment, filter to obtain a second filtrate and a second filter residue, wash the second filter residue to obtain a washing solution, mix the washing solution, the second filtrate and the first filtrate, and sequentially perform volume adjustment and concentration to obtain the test solution.

[0018] In this invention, the preferred mass-to-volume ratio of the *Zanthoxylum bungeanum* sample powder to the methanol solution is 0.9-1.1 g:20 mL, more preferably 1 g:20 mL; the preferred mass-to-volume ratio of the first filter residue to the methanol solution is 0.9-1.1 g:20 mL, more preferably 1 g:20 mL; and the preferred particle size of the *Zanthoxylum bungeanum* sample powder is 342-368 μm, more preferably 355 μm.

[0019] In this invention, the power of the first ultrasonic treatment and the second ultrasonic treatment in step 1) are independently preferred to be 180~220 W, more preferably 200 W; the frequency is independently preferred to be 53.1~64.9 kHz, more preferably 59 kHz; and the time is independently preferred to be 27~33 min, more preferably 30 min.

[0020] In this invention, the washing solution in step 1) is preferably a methanol solution with a volume fraction of 70%; the washing is preferably performed twice.

[0021] In this invention, the solution to be brought to volume in step 1) is preferably a methanol solution with a volume fraction of 70%; the volume to be brought to volume is preferably 50 mL; the concentration is preferably to take 4 mL of the solution after bringing to volume and concentrate it to 2 mL to obtain the test solution.

[0022] In this invention, the mass-to-volume ratio of the Zanthoxylum nitidum powder to the methanol solution in step 2) is preferably 2 g: 45~55 mL, more preferably 2 g: 50 mL; the particle size of the Zanthoxylum nitidum powder is preferably 342~368 μm, more preferably 355 μm.

[0023] In this invention, the temperature of the heating reflux in step 2) is preferably 80~85℃, more preferably 83℃; the time is preferably 27~33 min, more preferably 30 min.

[0024] In this invention, the concentrated and dissolved solution in step 2) is preferably a methanol solution with a volume fraction of 70%; the mass-to-volume ratio of the evaporated sample to the concentrated and dissolved solution is preferably 0.2 g: 1 mL; and the pore size of the filter membrane for the second filtration is preferably 0.22 μm.

[0025] In this invention, the mass-to-volume ratio of the characteristic component of Zanthoxylum bungeanum to the organic solvent in step 3) is preferably 0.8~1.2 mg:1 mL, more preferably 1 mg:1 mL; the organic solvent preferably includes ethanol and / or methanol.

[0026] In this invention, the temperatures of the first and second unfoldings in step 4) are preferably 4~30℃, more preferably one of 4℃, 5℃, 8℃, 10℃, 15℃, 20℃, 25℃, and 30℃; the humidity is preferably 30~70%, more preferably one of 30%, 40%, 50%, 60%, and 70%; the unfolding distance of the first unfolding is preferably 8~10cm, more preferably one of 8cm, 9cm, and 10cm; the unfolding distance of the second unfolding is preferably 8~10cm, more preferably one of 8cm, 9cm, and 10cm; the fumigation time is preferably 40~60s, more preferably one of 40s, 42s, 45s, 50s, 55s, and 60s, and more preferably 40s.

[0027] 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.

[0028] Example 1

[0029] The specific sources of Zanthoxylum nitidum powder (L1~L17), Zanthoxylum nitidum powder (M1~M23), and Pterocarya stenoptera blood (F1~F11) are shown in Table 1.

[0030] 1) Preparation of the test solution: 1 g of Zanthoxylum nitidum sample powder (L1~L17) with a particle size of 355 μm was mixed with 20 mL of 70% methanol solution. The mixture was ultrasonically treated at 200 W power and 59 kHz frequency for 30 min. After cooling and filtration, the filtrate was placed in a 50 mL volumetric flask. 1 g of filter residue and filter paper were mixed with 20 mL of 70% methanol solution. The mixture was ultrasonically treated at 200 W power and 59 kHz frequency for 30 min. After cooling and filtration, the filtrate was placed in the same 50 mL volumetric flask. The filter residue was washed twice with 70% methanol solution. The washing solution was placed in the same 50 mL volumetric flask. Then, 70% methanol solution was added to the 50 mL volumetric flask to make up to 50 mL. 4 mL of the solution was then taken and concentrated to 2 mL to obtain the test solution. 2) Preparation of characteristic components of Zanthoxylum bungeanum: 2 g of Zanthoxylum bungeanum powder (M1~M23) with a particle size of 355 μm was mixed with 50 mL of 70% methanol solution. The mixture was heated and refluxed at 83℃ for 30 min. The mixture was then filtered through qualitative filter paper to obtain the filtrate. The filtrate was then evaporated to dryness. 0.2 g of the evaporated sample was dissolved in 1 mL of 70% methanol solution and filtered through a 0.22 μm pore size filter membrane. The filtrate was then injected into a high-performance liquid chromatograph (HPLC) for gradient elution. The eluent was collected for 20–24 min. The chromatographic conditions for gradient elution were as follows: Chromatographic column: Shiseido CAPCELL PAK, 4.6 × 250 mm, 5 μm; Flow rate: 1.0 mL / min; Detection wavelength: 220 nm; Injection volume: 20 μL; Mobile phase: Mobile phase A is acetonitrile, and mobile phase B is a mixed aqueous solution of phosphate and triethylamine, wherein the mass-volume ratio of phosphate, triethylamine and water is 0.2 g: 0.25 g: 100 mL; The gradient elution procedure is as follows: From 0 to 20 min, mobile phase A increased from 10% to 21%, and mobile phase B increased from 90% to 79%. Over 20-35 minutes, mobile phase A changed from 21% to 25%, and mobile phase B changed from 79% to 75%. 35~50 min, mobile phase A is 25%, mobile phase B is 75%; The effluent was then dried with nitrogen to obtain the characteristic components of Zanthoxylum bungeanum. 3) Mix 1 mg of the characteristic component of Zanthoxylum bungeanum obtained in step 2) with 1 mL of ethanol to obtain a reference solution; 4) Development and detection: Take 2 μL of the test solution obtained in step 1) and the reference solution obtained in step 2) respectively, and spot them on the same silica gel G thin-layer plate (manufactured by Qingdao Ocean Chemical Co., Ltd.). First, use petroleum ether (60~90℃)-chloroform-methanol with a volume ratio of 2:13:1 as the developing solvent, and develop at 25℃ and 60% humidity with a development distance of 9 cm. Remove and air dry. Then, use n-butanol-methanol-ammonia test solution with a volume ratio of 5:25:13 as the developing solvent, and develop at 25℃ and 60% humidity with a development distance of 9 cm. Remove and air dry. Then, fumigate in iodine vapor for 40 s. Remove and evaporate the iodine adsorbed on the silica gel G thin-layer plate. Finally, examine under a UV lamp with a wavelength of 365 nm. Meanwhile, the same development and detection methods were used to detect commercially available bamboo leaf pepper alkaloids (i.e., the characteristic component of Zanthoxylum bungeanum, the sample solution for detection was obtained by directly mixing 1 mg of commercially available bamboo leaf pepper alkaloids with 1 mL of ethanol). Meanwhile, the blood of *Pterocarya stenoptera* (F1~F11) was tested using the method described in the 2025 edition of the Chinese Pharmacopoeia under the section on *Zanthoxylum nitidum*. Commercially available Zanthoxylum nitidum extract (i.e., the characteristic component of *Pterocarya stenoptera* blood, the sample solution for testing was obtained by directly mixing 1 mg of commercially available Zanthoxylum nitidum extract with 1 mL of ethanol) was tested using the same method as that for *Pterocarya stenoptera* blood. 5) Result Interpretation: If no spot of the same color appears at the corresponding position in the chromatogram of the test sample as in the chromatogram of the reference sample, the Zanthoxylum nitidum sample is not Zanthoxylum nitidum. Figures 1-3 As shown, the identification of Zanthoxylum nitidum and Zanthoxylum nitidum with hairy leaves is completed; Figure 1 The images show the examination views of Zanthoxylum nitidum (L1~L6), Zanthoxylum nitidum (M1~M8), Zanthoxylum nitidum (F1~F3), as well as bamboo leaf alkaloids and taurenin under ultraviolet light (365 nm). Figure 1 In the sequence, numbers 1-6 correspond to Zanthoxylum nitidum L1-L6, numbers 7-9 correspond to Pteris vittatae (F1-F3), number 10 corresponds to Zanthoxylum nitidum alkaloid, number 11 corresponds to Zanthoxylum nitidum alkaloid, and numbers 12-19 correspond to Zanthoxylum nitidum (M1-M8). Point A in number 10 is a blue fluorescent spot, which is a characteristic spot of Zanthoxylum nitidum alkaloid. Figure 2 The images show the examination views of Zanthoxylum nitidum (L7~L11), Zanthoxylum nitidum (M9~M16), Zanthoxylum nitidum (F4~F7), as well as bamboo leaf alkaloids and trichol under ultraviolet light (365 nm). Figure 2The serial numbers 1-5 correspond to Zanthoxylum nitidum L7-L11, serial numbers 6-9 correspond to Zanthoxylum nitidum (F4-F7), serial number 10 corresponds to Zanthoxylum nitidum alkaloid, serial number 11 corresponds to Zanthoxylum nitidum alkaloid, and serial numbers 12-19 correspond to Zanthoxylum nitidum (M9-M16). Figure 3 The images show the examination of Zanthoxylum nitidum (L12~L17), Zanthoxylum nitidum (M17~M23), Zanthoxylum nitidum (F8~F11), as well as bamboo leaf alkaloids and thorny alkaloids under ultraviolet light (365 nm). Figure 3 The serial numbers 1-6 correspond to Zanthoxylum nitidum L12-L17, serial numbers 7-10 correspond to Zanthoxylum nitidum (F8-F11), serial number 11 corresponds to Zanthoxylum nitidum alkaloid, serial number 12 corresponds to Zanthoxylum nitidum alkaloid, and serial numbers 13-19 correspond to Zanthoxylum nitidum (M17-M23).

[0031] from Figures 1-3 As can be seen from the chromatogram, Zanthoxylum nitidum shows fluorescent spots of different colors at the corresponding positions as Zanthoxylum nitidum, thus completing the identification of Zanthoxylum nitidum and Zanthoxylum nitidum.

[0032] Table 1. Sources of Zanthoxylum nitidum powder (L1~L17), Zanthoxylum nitidum powder (M1~M23), and Pteris multifida blood (F1~F11).

[0033] Example 2

[0034] The only difference between this embodiment and Embodiment 1 is that in step 3), the silica gel G thin film plate is the Qingdao plate produced by Qingdao Ocean Chemical Co., Ltd. The temperature for the first and second developments is 4°C and the humidity is 70%. The samples tested are only Zanthoxylum nitidum powder (L1~L3), Zanthoxylum nitidum powder (M1~M3), Pterocarya scabra blood (F1~F3), as well as bamboo leaf pepper and Zanthoxylum nitidum extract. Other conditions remain unchanged.

[0035] Test results as follows Figure 4 As shown, where, Figure 4 Serial numbers 1-3 correspond to Zanthoxylum nitidum (L1-L3), serial numbers 4-6 correspond to Zanthoxylum nitidum (F1-F3), serial number 7 corresponds to Zanthoxylum nitidum extract, serial numbers 8-10 correspond to Zanthoxylum nitidum (M1-M3), and serial number 11 corresponds to Zanthoxylum nitidum extract; from Figure 4 As can be seen from the chromatograms, the chromatograms of Zanthoxylum bungeanum (M1~M3) show fluorescent spots of different colors at the corresponding positions as those of Zanthoxylum bungeanum (L1~L3), thus completing the identification of Zanthoxylum bungeanum and Zanthoxylum bungeanum.

[0036] Example 3

[0037] The only difference between this embodiment and Embodiment 1 is that in step 3), the silica gel G thin film plate is the Qingdao plate produced by Qingdao Ocean Chemical Co., Ltd. The temperature for the first and second developments is 25°C and the humidity is 30%. The samples tested are only Zanthoxylum nitidum powder (L1~L3), Zanthoxylum nitidum powder (M1~M3), Pterocarya scabra blood (F1~F3), as well as bamboo leaf pepper and Zanthoxylum nitidum alkaloid. Other conditions remain unchanged.

[0038] Test results as follows Figure 5 As shown, where, Figure 5 Serial numbers 1-3 correspond to Zanthoxylum nitidum (L1-L3), serial numbers 4-6 correspond to Zanthoxylum nitidum (F1-F3), serial number 7 corresponds to Zanthoxylum nitidum extract, serial numbers 8-10 correspond to Zanthoxylum nitidum (M1-M3), and serial number 11 corresponds to Zanthoxylum nitidum extract; from Figure 5 As can be seen from the chromatograms, the chromatograms of Zanthoxylum bungeanum (M1~M3) show fluorescent spots of different colors at the corresponding positions as those of Zanthoxylum bungeanum (L1~L3), thus completing the identification of Zanthoxylum bungeanum and Zanthoxylum bungeanum.

[0039] Example 4

[0040] The only difference between this embodiment and Embodiment 1 is that in step 3), the silica gel G thin film plate is a Merck plate manufactured by Merck. The temperature for the first and second unfolding is 25°C and the humidity is 70%. The samples tested are only Zanthoxylum nitidum powder (L1~L3), Zanthoxylum nitidum powder (M1~M3), Pterocarya scabra blood (F1~F3), as well as bamboo leaf pepperin and Zanthoxylum nitidum extract. Other conditions remain unchanged.

[0041] Test results as follows Figure 6 As shown, where, Figure 6 Serial numbers 1-3 correspond to Zanthoxylum nitidum (L1-L3), serial numbers 4-6 correspond to Zanthoxylum nitidum (F1-F3), serial number 7 corresponds to Zanthoxylum nitidum extract, serial numbers 8-10 correspond to Zanthoxylum nitidum (M1-M3), and serial number 11 corresponds to Zanthoxylum nitidum extract; from Figure 6 As can be seen from the chromatograms, the chromatograms of Zanthoxylum bungeanum (M1~M3) show fluorescent spots of different colors at the corresponding positions as those of Zanthoxylum bungeanum (L1~L3), thus completing the identification of Zanthoxylum bungeanum and Zanthoxylum bungeanum.

[0042] Example 5

[0043] The only difference between this embodiment and Embodiment 1 is that in step 3), the silica gel G thin film plate is an MN plate produced by MN Company of Germany. The temperature for the first and second unfolding is 25°C and the humidity is 70%. The samples tested are only Zanthoxylum nitidum powder (L1~L3), Zanthoxylum nitidum powder (M1~M3), Pterocarya scabra blood (F1~F3), as well as bamboo leaf pepperin and Zanthoxylum nitidum alkaloid. Other conditions remain unchanged.

[0044] Test results as follows Figure 7 As shown, where, Figure 7 Serial numbers 1-3 correspond to Zanthoxylum nitidum (L1-L3), serial numbers 4-6 correspond to Zanthoxylum nitidum (F1-F3), serial number 7 corresponds to Zanthoxylum nitidum extract, serial numbers 8-10 correspond to Zanthoxylum nitidum (M1-M3), and serial number 11 corresponds to Zanthoxylum nitidum extract; from Figure 7 As can be seen from the chromatograms, the chromatograms of Zanthoxylum bungeanum (M1~M3) show fluorescent spots of different colors at the corresponding positions as those of Zanthoxylum bungeanum (L1~L3), thus completing the identification of Zanthoxylum bungeanum and Zanthoxylum bungeanum.

[0045] In summary, the identification method described in this invention has excellent durability. Using silica gel G thin-layer plates from different companies and different unfolding temperatures and humidity, it can achieve the ideal effect of distinguishing between Zanthoxylum nitidum and Zanthoxylum nitidum. It provides a reliable technical means for the identification and quality control of Zanthoxylum nitidum medicinal materials, which is of great significance for ensuring the safety and effectiveness of clinical medication.

[0046] 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 distinguishing between Zanthoxylum nitidum and Zanthoxylum nitidum, characterized in that, Includes the following steps: 1) Preparation of test solution: Extract the powder of Zanthoxylum nitidum from the sample using a 70% methanol solution to obtain the test solution; 2) Preparation of characteristic components of Zanthoxylum nitidum: Zanthoxylum nitidum powder was mixed with a 70% (v / v) methanol solution, and the mixture was subjected to reflux and a first filtration sequentially to obtain a filtrate. The filtrate was then subjected to evaporation to dryness, concentration and dissolution, and a second filtration sequentially. The filtrate was then injected into a high-performance liquid chromatograph for elution. The chromatographic conditions for elution were as follows: Chromatographic column: Shiseido CAPCELL PAK, 4.6×250mm, 5μm; Flow rate: 1.0 mL / min; Detection wavelength: 220 nm; Injection volume: 20 μL; Mobile phase: Mobile phase A is acetonitrile, and mobile phase B is a mixed aqueous solution of phosphate and triethylamine, wherein the mass-volume ratio of phosphate, triethylamine and water is 0.2 g: 0.25 g: 100 mL; The elution is gradient elution, and the elution procedure for the gradient elution is as follows: From 0 to 20 min, mobile phase A increased from 10% to 21%, and mobile phase B increased from 90% to 79%. Over 20-35 minutes, mobile phase A changed from 21% to 25%, and mobile phase B changed from 79% to 75%. 35~50 min, mobile phase A is 25%, mobile phase B is 75%; Simultaneously, the effluent was collected for 20-24 minutes, and then the effluent was dried with nitrogen to obtain the characteristic components of Zanthoxylum bungeanum. 3) Preparation of reference solution: The characteristic components of Zanthoxylum bungeanum obtained in step 2) are mixed with an organic solvent to obtain the reference solution; 4) Development and detection: Take 2 μL each of the test solution and the reference solution and spot them separately on the same silica gel G thin-layer plate. First, use petroleum ether-chloroform-methanol with a volume ratio of 2:13:1 as the developing solvent for the first development, remove and air dry. Petroleum ether is the fraction with a boiling point range of 60~90℃. Then, use n-butanol-methanol-ammonia test solution with a volume ratio of 5:25:13 as the developing solvent for the second development, remove and air dry. Then, fumigate in iodine vapor. After removal, evaporate the iodine adsorbed on the silica gel G thin-layer plate. Finally, examine under a UV lamp with a wavelength of 365 nm. 5) Result determination: If the test sample chromatogram shows a spot of the same color at the corresponding position as the reference chromatogram, the Zanthoxylum nitidum test sample is Zanthoxylum nitidum; if the test sample chromatogram does not show a spot of the same color at the corresponding position as the reference chromatogram, the Zanthoxylum nitidum test sample is not Zanthoxylum nitidum.

2. The method for identifying Zanthoxylum nitidum and Zanthoxylum nitidum according to claim 1, characterized in that, The particle size of the two-sided needle sample powder mentioned in step 1) is 342~368 μm.

3. The method for identifying Zanthoxylum nitidum and Zanthoxylum nitidum according to claim 2, characterized in that, In step 2), the mass-to-volume ratio of the powdered Zanthoxylum bungeanum to the methanol solution is 2 g: 45~55 mL. The particle size of the powder of Zanthoxylum bungeanum is 342~368 μm; The heating reflux temperature is 80~85℃, and the time is 27~33 min.

4. A method for identifying Zanthoxylum nitidum and Zanthoxylum nitidum according to any one of claims 1 to 3, characterized in that, The concentrated and dissolved solution mentioned in step 2) is a methanol solution with a volume fraction of 70%; the mass-to-volume ratio of the evaporated sample to the concentrated and dissolved solution is 0.2 g: 1 mL; The filter membrane used in the second filtration has a pore size of 0.22 μm.

5. The method for identifying Zanthoxylum nitidum and Zanthoxylum nitidum according to claim 4, characterized in that, In step 3), the mass-to-volume ratio of the characteristic component of Zanthoxylum nitidum to the organic solvent is 0.8~1.2 mg:1 mL; The organic solvents include ethanol and / or methanol.

6. The method for identifying Zanthoxylum nitidum and Zanthoxylum nitidum according to claim 5, characterized in that, In step 4), the temperature for the first and second unfoldings is independently 4~30℃, and the humidity is independently 30~70%. The first unfolding distance is 8-10 cm; the second unfolding distance is 8-10 cm; the fumigation time is 40-60 s.

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