Construction method of characteristic chromatogram of aqueous extract of melia azedarach l. bark and identification method thereof

Abstract

The application relates to the technical field of traditional Chinese medicine detection and analytical chemistry, in particular to a characteristic spectrum construction method of water extracts of Melia toosendan fruits and a distinguishing method thereof, and specifically relates to a characteristic spectrum construction method of water extracts of Melia toosendan fruits with same or different origins, a distinguishing method between water extracts of Melia toosendan fruits with different origins, and a distinguishing method between water extracts of Melia toosendan fruits and water extracts of other similar medicinal materials. The characteristic spectrum of the technical scheme can objectively reflect the chemical characteristic information of the water extracts of Melia toosendan fruits, meets the basic requirements of integrity, characteristic and stability, and solves the problems of characteristic loss and difficulty in distinguishing after the medicinal materials are prepared into water extracts.

Description

Technical Field

[0001] This application relates to the field of traditional Chinese medicine detection and analytical chemistry technology, and in particular to the construction method of characteristic chromatograms of water extracts of neem bark and their identification methods. Specifically, it relates to the construction method of characteristic chromatograms of water extracts of neem bark from the same or different origins, the identification method between water extracts of neem bark from different origins, and the identification method between water extracts of neem bark and water extracts of other similar medicinal materials. Background Technology

[0002] The traditional Chinese medicine neem bark is the dried bark and root bark of *Melia toosenclan* Sieb. et Zucc. or *Melia azedarach* L., belonging to the Lepidaceae family. It is bitter and cold in nature, and enters the liver, spleen, and stomach meridians. It has insecticidal and antifungal effects, and is commonly used for ascariasis, pinworm infection, abdominal pain due to intestinal parasites, and externally for scabies and itching. The main chemical components of neem bark include azadirachtin, azadirachtinone, azadirachtin lactone, azadirachtin alcohol lactone, azadirachtin ketone, and methyl azadirachtin acid, as well as coumarins, polysaccharides, tannins, resins, and catechins. Azadirachtin plays a major role in its anthelmintic effect.

[0003] As the main active ingredient in neem bark that exerts its anthelmintic effect, azadirachtin needs to be clearly presented in the characteristic spectrum and fingerprint spectrum of neem bark. However, the characteristic ultraviolet absorption wavelength of azadirachtin is about 210 nm, which is terminal absorption. It is difficult to analyze accurately using traditional ultraviolet detectors. The detection method specified in the Pharmacopoeia (2020 edition) uses a single quadrupole mass spectrometer, which has higher sensitivity, but this detector is expensive and difficult to put into application.

[0004] Studies have shown that, in the identification results of thin-layer chromatography, the fruit of *Melia toosenclan* Sieb. et Zucc. contains one more component (developing solvent: chloroform / methanol = 16:2) than the fruit of *Melia azedarach* L. In the results of reversed-phase high-performance liquid chromatography, the azadirachtin content of *Melia toosenclan* is higher than that of *Melia azedarach*. This demonstrates that the medicinal active ingredients and their contents may vary significantly among plants of the same family but different genera, and should be identified and differentiated for medicinal use.

[0005] Literature reports a study on the quality evaluation of neem bark from different origins using HPLC-ELSD fingerprinting combined with statistical methods. This method can identify the origin of neem bark to a certain extent. The constructed characteristic chromatogram identified eight common peaks and identified azadirachtin, catechin, and epicatechin. However, observation of the characteristic chromatogram revealed that the baseline of the chromatogram was uneven in the retention time range of 24 min to 40 min, which may have obscured key chemical information. The characteristicity and stability of the fingerprint chromatogram of this method still need to be improved.

[0006] The aqueous extract of neem bark is an important raw material for traditional Chinese medicine preparations such as patent medicines and herbal granules. However, because it has lost the characteristics of the medicinal material, it cannot be effectively identified. Therefore, there is an urgent need to provide an identification method that can effectively distinguish the aqueous extract of neem bark that has lost its medicinal characteristics. Summary of the Invention

[0007] In view of the above problems, the technical content of this application is hereby proposed.

[0008] One aspect of this application relates to a method for constructing a characteristic spectrum of water extract of neem bark. The characteristic spectrum prepared by this method can objectively reflect the chemical characteristic information of water extract of neem bark and can be used to distinguish neem bark medicinal materials of different origins, thus solving the problem of characteristic loss and difficulty in identification after the medicinal material is prepared into water extract.

[0009] In some embodiments, the method for constructing the characteristic spectrum of the water extract of neem bark includes the following steps:

[0010] The aqueous extract of neem bark was extracted with an extraction solvent to prepare the test solution;

[0011] The test solution was taken and detected by a high-performance liquid chromatograph equipped with an evaporative light scattering detector to obtain the characteristic chromatogram of the water extract of the neem bark;

[0012] The conditions for high-performance liquid chromatography include the following characteristics:

[0013] (1) Mobile phase A is acetonitrile, and mobile phase B is water;

[0014] (2) The elution process includes:

[0015] From 0 to 16 minutes, the volume percentage of the mobile phase A increased from 10% to 12%.

[0016] Between 16 and 20 minutes, the volume percentage of the mobile phase A increased from 12% to 18%.

[0017] Between 20 and 33 minutes, the volume percentage of the mobile phase A increased from 18% to 30%.

[0018] Between 33 and 43 minutes, the volume percentage of the mobile phase A increased from 30% to 60%.

[0019] Between 43 and 45 minutes, the volume percentage of the mobile phase A increased from 60% to 90%.

[0020] For 45 to 50 minutes, maintain the volume percentage of the mobile phase A at 90%.

[0021] This application, through extensive research on the characteristic components of the aqueous extract of neem bark, established suitable chromatographic conditions, and the resulting characteristic chromatograms met the basic requirements of integrity, specificity, and stability.

[0022] Another aspect of this application relates to a method for identifying water extracts of neem bark. The traditional Chinese medicine neem bark can originate from either Sichuan neem or neem, and there is currently no effective method for differentiation. Furthermore, clinically used neem bark is generally an water extract, and the lack of morphological characteristics in the processed form makes identification even more difficult. This application uses a suitable fingerprinting method to obtain a characteristic chromatogram of neem bark water extracts. This chromatogram can accurately reflect the chemical characteristics of water extracts from different neem bark origins, effectively distinguishing neem bark water extracts from different sources, and providing a reference for the clinical use of neem bark.

[0023] In some embodiments, the identification method for the water extract of neem bark includes the following steps:

[0024] The aqueous extract of neem bark was extracted with an extraction solvent to prepare the test solution;

[0025] Take the analyte and extract it with an extraction solvent to prepare an analyte solution;

[0026] Take the analyte solution and the test sample solution, and perform analysis using a high-performance liquid chromatograph equipped with an evaporative light scattering detector. Construct the chromatogram of the analyte and the characteristic chromatogram of the water extract of neem bark, and compare the chromatogram of the analyte with the characteristic chromatogram of the water extract of neem bark.

[0027] The conditions for high-performance liquid chromatography include the following characteristics:

[0028] (1) Mobile phase A is acetonitrile, and mobile phase B is water;

[0029] (2) The elution process includes:

[0030] From 0 to 16 minutes, the volume percentage of the mobile phase A increased from 10% to 12%.

[0031] Between 16 and 20 minutes, the volume percentage of the mobile phase A increased from 12% to 18%.

[0032] Between 20 and 33 minutes, the volume percentage of the mobile phase A increased from 18% to 30%.

[0033] Between 33 and 43 minutes, the volume percentage of the mobile phase A increased from 30% to 60%.

[0034] Between 43 and 45 minutes, the volume percentage of the mobile phase A increased from 60% to 90%.

[0035] For 45 to 50 minutes, maintain the volume percentage of the mobile phase A at 90%.

[0036] The identification method described in this application can effectively distinguish between the water extract of neem bark and water extracts of similar medicinal materials, providing a basis for the quality testing of neem bark water extract. Attached Figure Description

[0037] To more clearly illustrate the technical solutions in the specific embodiments of this application or the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0038] Figure 1 This is a chromatogram comparison of the detection under different extraction solvent conditions as described in Section 2.2.1 of Example 1 of this application;

[0039] Figure 2 The above are chromatographic comparison diagrams of detection under different gradient elution conditions in Section 3.1 of Example 1 of this application, where 2A is a comparison diagram of detection results under elution gradients 2 and 3, and 2B is a comparison diagram of detection results under elution gradients 3 and 4.

[0040] Figure 3 This is a chromatogram comparison of the detection under different mobile phase conditions as described in Section 3.2 of Example 1 of this application;

[0041] Figure 4 This is a chromatogram comparison of the detection performed under different column temperature conditions in Section 3.3 of Example 1 of this application;

[0042] Figure 5 This is a chromatogram comparison of the detection under different flow rate conditions in Section 3.4 of Example 1 of this application;

[0043] Figure 6 This is a chromatographic comparison of the column temperature durability test in Section 3.5.4 of Example 1 of this application;

[0044] Figure 7This is a chromatographic comparison of the flow rate robustness test in Section 3.5.4 of Example 1 of this application;

[0045] Figure 8 This is a chromatographic comparison of the column type durability test in Section 3.5.4 of Example 1 of this application;

[0046] Figure 9 This is a chromatogram comparison of detection under different detector conditions in Section 4 of Example 1 of this application;

[0047] Figure 10 This is a chromatogram overlay of the results from the detection of 17 batches of water extracts of neem bark in Section 5.1.1 of Example 1 of this application;

[0048] Figure 11 The characteristic spectrum of the water extract of neem bark obtained in Section 5.1.1 of Example 1 of this application;

[0049] Figure 12 This is a chromatogram overlay of the results from the detection of six batches of water extracts of neem bark (Chuanlian) in Section 5.1.2 of Example 1 of this application;

[0050] Figure 13 The characteristic spectrum of the water extract of neem bark (Chuanlian) obtained in Section 5.1.2 of Example 1 of this application;

[0051] Figure 14 This is a comparison of the specificity chromatograms of each reference standard in Section 5.2.1 of Example 1 of this application and the characteristic chromatogram of the water extract of neem bark.

[0052] Figure 15 This is a comparison of the specificity chromatograms of each reference standard in Section 5.2.2 of Example 1 of this application and the characteristic chromatogram of the water extract of Melia azedarach bark (Chuanlian).

[0053] Figure 16 This is a comparison diagram of the characteristic spectra of the aqueous extracts of Acacia bark, Sapindus mukorossi bark, and Melia azedarach bark (Melia azedarach) in Example 3 of this application, as well as the specificity spectrum of azadirachtin.

[0054] Figure 17 This is a chromatogram overlay of 17 batches of water extracts of neem bark (neem) from Comparative Example 1 of this application.

[0055] Figure 18 This is a characteristic spectrum of the water extract of neem bark obtained in Comparative Example 1 of this application. Detailed Implementation

[0056] The present application is further described below with reference to the embodiments, examples, and accompanying drawings. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the application. Furthermore, it should be understood that after reading the teachings of this application, those skilled in the art can make various alterations or modifications to the application, and these equivalent forms also fall within the protection scope of the appended claims.

[0057] 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 application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0058] the term

[0059] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0060] As used herein, the term "and / or" encompasses any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. For example, "A and / or B" includes three parallel options: A, B, and A+B.

[0061] In this document, terms such as "preferred," "better," and "more preferred" are merely descriptions of implementation methods or examples that achieve better results, and should be understood as not constituting a limitation on the scope of protection of this application.

[0062] In this application, terms such as "further," "even further," and "particularly" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0063] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0064] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, optional numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0065] Unless otherwise specified, the temperature parameters in this application are permitted to be either constant-temperature treatment or variations within a certain temperature range. It should be understood that the constant-temperature treatment allows temperature fluctuations within the precision range of the instrument control, such as ±0.5℃, ±0.4℃, ±0.3℃, ±0.2℃, and ±0.1℃.

[0066] In this application, weight can be a well-known unit of mass in the chemical industry, such as μg, mg, g, or kg.

[0067] One aspect of this application relates to a method for constructing a characteristic spectrum of a water extract of neem bark.

[0068] The traditional Chinese medicine neem bark can be derived from the dried bark and root bark of plants such as Melia toosenclan Sieb. et Zucc. or Melia azedarach L. Currently, there is very little research on the characteristic chromatograms / fingerprints of neem bark, and few components can be observed on chromatography, making it difficult to comprehensively reflect the overall quality of neem bark. Furthermore, traditional Chinese medicine is typically used clinically as a decoction, and current research focuses on the neem bark material itself rather than its water extract, thus lacking a basis for clinical medicinal quality evaluation.

[0069] The characteristic chromatograms obtained by the method for constructing the characteristic chromatograms of the water extract of neem bark in this application can objectively and accurately reflect the chemical characteristic information of the water extract of neem bark, and can be used as the basis for quality detection and identification of the water extract of neem bark.

[0070] In one embodiment, the aqueous extract of neem bark is prepared by the following steps: taking the neem bark medicinal material, soaking it in water, adding 10 times the amount of water to the neem bark medicinal material for a first decoction, filtering and collecting the first filtrate; then adding 8 times the amount of water to the neem bark medicinal material for a second decoction, filtering and collecting the second filtrate, combining the first filtrate and the second filtrate, concentrating, and freeze-drying. In this application, "times" refers to a mass ratio; for example, adding 10 times the amount of water means that the mass ratio of the added water to the medicinal material is 10:1.

[0071] In one embodiment, the water extract of neem bark is prepared by the following steps: taking neem bark slices, soaking them for 30 minutes, adding 10 times the amount of water to the neem bark slices, decocting for 30 minutes, filtering while hot through a 350-mesh sieve, collecting the first filtrate, then adding 8 times the amount of water to the neem bark slices, decocting for 25 minutes, filtering while hot through a 350-mesh sieve, collecting the second filtrate, combining the first and second filtrates, concentrating, and transferring to a vacuum freeze dryer for freeze drying.

[0072] In one embodiment, the method for constructing the characteristic spectrum of the water extract of neem bark includes the following steps:

[0073] S100: Take the water extract of neem bark, extract it with an extraction solvent, and prepare the test solution;

[0074] S200: Take the test solution and the reference solution separately, and use a high performance liquid chromatograph equipped with an evaporative light scattering detector to detect them and obtain the characteristic chromatogram of the water extract of neem bark.

[0075] S100 Preparation of test solution

[0076] In step S100, the aqueous extract of neem bark is taken and extracted with an extraction solvent to obtain a test solution.

[0077] In one embodiment, the extraction method is extraction, and the extract includes ethyl acetate. Further, the aqueous extract of neem bark is dispersed in water, extracted with ethyl acetate, the organic phase is collected, dried to obtain a solid, and the solid is dissolved in a suitable organic solvent to obtain a test solution.

[0078] In one embodiment, in step S100, the water extract of neem bark is first finely ground, 0.5g is placed in a stoppered conical flask, 10mL of water is added, the mixture is shaken to dissolve, and then transferred to a separatory funnel. The extract is extracted three times with ethyl acetate, 20mL each time. The ethyl acetate layers are combined, evaporated to dryness, and the residue is dissolved in methanol and transferred to a 2mL volumetric flask. The residue is diluted to the mark with methanol, shaken well, filtered, and the filtrate is collected to obtain the test solution.

[0079] S200 High Performance Liquid Chromatography Analysis

[0080] In step S200, the test solution is taken and analyzed by high performance liquid chromatography (HPLC) equipped with an evaporative light scattering detector (ELSD) to construct a characteristic chromatogram of the water extract of neem bark.

[0081] During the construction of the characteristic spectrum, the researchers used an ultraviolet detector for analysis and found that the baseline of the ultraviolet detection spectrum fluctuated significantly. This is speculated to be because the characteristic ultraviolet absorption wavelength of azadirachtin is approximately 210 nm, close to the far ultraviolet region (10-200 nm). Therefore, only the end of this peak can be detected in the near ultraviolet region (200-400 nm), resulting in reduced detection sensitivity and the appearance of a non-peaked signal response value. Using a single-stage quadrupole mass spectrometer to detect azadirachtin can avoid this problem; however, the application of single-stage quadrupole mass spectrometers in characteristic spectrum and fingerprint spectrum analysis is currently limited, and they are relatively expensive.

[0082] Through extensive experimental research, researchers discovered that under suitable chromatographic and detection conditions, analysis using a high-performance liquid chromatograph equipped with an evaporative light scattering detector can yield accurate and reliable characteristic spectra.

[0083] In one embodiment, the conditions for high-performance liquid chromatography include the following characteristics:

[0084] (1) Mobile phase A is acetonitrile, and mobile phase B is water;

[0085] (2) The elution process includes:

[0086] From 0 to 16 minutes, the volume percentage of the mobile phase A increased from 10% to 12%.

[0087] Between 16 and 20 minutes, the volume percentage of the mobile phase A increased from 12% to 18%.

[0088] Between 20 and 33 minutes, the volume percentage of the mobile phase A increased from 18% to 30%.

[0089] Between 33 and 43 minutes, the volume percentage of the mobile phase A increased from 30% to 60%.

[0090] Between 43 and 45 minutes, the volume percentage of the mobile phase A increased from 60% to 90%.

[0091] For 45 to 50 minutes, maintain the volume percentage of the mobile phase A at 90%.

[0092] In some embodiments, the conditions for high-performance liquid chromatography include one or more of the following characteristics:

[0093] (1) The flow rate is 0.4 ml / min to 0.6 ml / min;

[0094] (2) The injection volume is 0.5 μL to 1.5 μL;

[0095] (3) The chromatographic column is an octadecylsilane-bonded silica gel column;

[0096] (4) The column temperature is 28℃~32℃;

[0097] (5) The column length is 150 mm, the inner diameter is 4.6 mm, and the particle size is 2.7 μm.

[0098] Furthermore, the column eluent separated by high performance liquid chromatography enters the ELSD detector and undergoes SO1 nebulization, SO2 evaporation and SO3 detection in sequence.

[0099] S01 Nebulization: The eluent is first mixed with a stable nebulizing gas (such as nitrogen) in the nebulizer to form an aerosol. The aerosol is composed of uniformly distributed droplets. The droplet size depends on the gas flow rate used in the analysis. The lower the gas flow rate, the larger the droplets, and the more light is scattered by the droplets, thus improving the analytical sensitivity. However, larger droplets are more difficult to evaporate in the drift tube. Each method has an optimal gas flow rate that produces the best signal-to-noise ratio.

[0100] In one embodiment, the gas flow rate is 2.5 L / min to 3.5 L / min, for example 2.5 L / min, 3 L / min, 3.5 L / min, etc.

[0101] In one embodiment, the atomizing gas is compressed air.

[0102] SO2 evaporation: The aerosol formed by atomization evaporates the solvent in a heated drift tube, leaving behind non-volatile particles. The evaporation temperature depends on the composition and flow rate of the mobile phase, as well as the volatility of the sample.

[0103] In one embodiment, the evaporation temperature is 90°C to 100°C, for example, 90°C, 95°C, 100°C, etc.

[0104] S03 Detection: The remaining non-volatile particles after evaporation are detected in a light scattering detection cell. In the detection cell, the sample particles scatter the light emitted by the excitation source. The scattered light is detected by a silicon photodiode, generating an electrical signal that is transmitted to the analog signal output port and used for data acquisition at the workstation.

[0105] In one embodiment, in the method for constructing the characteristic spectrum of the water extract of neem bark, catechin reference standard, epicatechin reference standard, and azadirachtin reference standard are used as references, and the references are dissolved in a suitable solvent to prepare a reference solution.

[0106] Furthermore, a reference solution was taken and analyzed using the liquid chromatography conditions described in any of the above technical solutions. The analysis results were then compared with the characteristic chromatograms of the water extract of neem bark.

[0107] Furthermore, the preparation steps of the reference solution include: taking appropriate amounts of catechin reference standard, epicatechin reference standard, and azadirachtin reference standard, accurately weighing them, and adding methanol to prepare a mixed solution containing 0.5 mg of each per 1 ml.

[0108] In one embodiment, the method for constructing the characteristic spectrum of the water extract of neem bark includes the following steps:

[0109] Preparation of water extract S010: Take neem bark slices, soak for 30 minutes, and decoct twice with water. For the first decoction, add 10 times the amount of water and decoct for 30 minutes. Filter the decoction while hot through a 350-mesh sieve. For the second decoction, add 8 times the amount of water and decoct for 25 minutes. Filter the decoction while hot through a 350-mesh sieve. Combine the two decoctions. Transfer to a vacuum freeze dryer for freeze-drying. Remove and obtain the product.

[0110] S020 Preparation of reference solution: Take appropriate amounts of catechin reference standard, epicatechin reference standard and azadirachtin reference standard, accurately weigh them, add methanol to prepare a mixed solution containing 0.5 mg of each per 1 ml, as the reference solution;

[0111] Preparation of the test solution for S030: Take an appropriate amount of this product, grind it into a fine powder, take 0.5g, place it in a stoppered conical flask, add 10ml of water, shake to dissolve, transfer to a separatory funnel, extract with ethyl acetate 3 times, 20ml each time, combine the ethyl acetate layers, evaporate to dryness, dissolve the residue in methanol and transfer to a 2ml volumetric flask, dilute to the mark with methanol, shake well, filter, and take the filtrate to obtain the test solution;

[0112] S040 High Performance Liquid Chromatography Analysis: Accurately pipette 1 μl of the reference solution and the test solution into the liquid chromatograph and detect them with an evaporative light scattering detector.

[0113] The high-performance liquid chromatography (HPLC) conditions included: octadecylsilane-bonded silica gel as the packing material (column length 150 mm, inner diameter 4.6 mm, particle size 2.7 μm); acetonitrile as mobile phase A; and water as mobile phase B. The elution program included: 0–16 min, increasing the volume percentage of mobile phase A from 10% to 12%; 16–20 min, increasing the volume percentage of mobile phase A from 12% to 18%; 20–33 min, increasing the volume percentage of mobile phase A from 18% to 30%; 33–43 min, increasing the volume percentage of mobile phase A from 30% to 60%; 43–45 min, increasing the volume percentage of mobile phase A from 60% to 90%; and 45–50 min, maintaining the volume percentage of mobile phase A at 90%. The flow rate was 0.5 mL / min; and the column temperature was 30 °C.

[0114] The conditions for an evaporative light scattering detector include:

[0115] The theoretical plate number, calculated based on the azadirachtin peak, is no less than 3000;

[0116] Based on the analysis results, a characteristic spectrum of the water extract of neem bark was constructed.

[0117] In one embodiment, the basis of the water extract of neem bark is neem;

[0118] The characteristic spectrum of the aqueous extract of neem bark includes at least the following 9 common peaks: peak 1, peak 2, peak 3, peak 4, peak 5, peak 6, peak 7, peak 8 and peak 9;

[0119] Among them, peak 1 is (+)-galactoatechin, peak 2 is proanthocyanidin B3, peak 3 is catechin, peak 4 is epicatechin, peak 7 is iso-neemin, and peaks 8 and 9 together are neemin.

[0120] Among them, with peak 3 as the reference peak, the relative retention times of peaks 1 and 2 are as follows: peak 1: 0.49±10%; peak 2: 0.86±10%; with peak 8 as the reference peak, the relative retention times of peaks 5, 6 and 7 are as follows: peak 5: 0.81±10%; peak 6: 0.90±10%; peak 7: 0.95±10%.

[0121] It should be noted that the statement in this application that "peak 8 and peak 9 are both azadirachtin" is not due to the failure of the chromatographic method development. The "Precautions" section of the instructions for use of the national drug standard substance "azadirachtin" states that azadirachtin exhibits isomerism in solution, and two chromatographic peaks are displayed in the liquid chromatogram. Therefore, the appearance of two peaks in azadirachtin is reasonable.

[0122] Furthermore, in the characteristic spectrum of the water extract of neem bark, which is derived from neem, peak 8 is used as the reference peak, and the relative peak area of ​​peak 3 is greater than 1.6.

[0123] In one embodiment, the source of the water extract of neem bark is Sichuan neem;

[0124] The characteristic spectrum of the aqueous extract of neem bark includes at least the following 6 common peaks: peak 1, peak 2, peak 3, peak 6, peak 8 and peak 9;

[0125] Among them, peak 1 is (+)-galactoatechin, peak 2 is proanthocyanidin B3, peak 3 is catechin, and peaks 8 and 9 together are neemin;

[0126] Among them, with peak 3 as the reference peak, the relative retention times of peak 1 and peak 2 are: peak 1: 0.49±10%; peak 2: 0.86±10%; with peak 8 as the reference peak, the relative retention time of peak 6 is: 0.90±10%.

[0127] Furthermore, in the characteristic spectrum of the water extract of Melia toosendan bark, with peak 8 as the reference peak, the relative peak area of ​​peak 3 is less than or equal to 1.6.

[0128] Identification of water extracts from neem bark

[0129] Another aspect of the present application relates to a method for identifying the aqueous extract of Cortex Meliae. The method for identifying the aqueous extract of Cortex Meliae in the present application has high accuracy and stability, and the detection process is rapid and simple. The identification result can be obtained without quantitative characterization, calculation or secondary analysis.

[0130] Specifically, the present application relates to a method for identifying the aqueous extracts of Cortex Meliae with different origins, and a method for identifying the aqueous extract of Cortex Meliae and the aqueous extracts of other similar medicinal materials.

[0131] Origin Identification

[0132] In traditional Chinese medicine, the origin generally refers to the origin (source) of Chinese medicine species (varieties). The identification of the origin of traditional Chinese medicine is the basis of the identification of traditional Chinese medicine, and also the main basis for the inheritance, research, production, development and utilization of traditional Chinese medicine.

[0133] The traditional source of Cortex Meliae can be the plant Melia toosendan Sieb. et Zucc. or Melia azedarach L. However, there is currently no relevant research on distinguishing the two origins of Cortex Meliae (Melia azedarach) and Cortex Meliae (Melia toosendan) and their preparations.

[0134] The identification method of the present application can effectively distinguish the aqueous extracts of Cortex Meliae from different sources, providing a reference for the clinical use of Cortex Meliae.

[0135] Identification of medicinal material raw materials

[0136] The identification method of the present application can effectively distinguish the aqueous extract of Cortex Meliae and the aqueous extracts of other medicinal materials similar to Cortex Meliae, providing a basis for the authenticity identification and quality detection of the aqueous extract of Cortex Meliae. Chinese medicinal materials similar to Cortex Meliae can be other medicinal materials that are similar in appearance and easy to be confused, such as Acacia bark, Sapindus mukorossi Gaertn. bark, etc.

[0137] In one embodiment, the method for identifying the aqueous extract of Cortex Meliae comprises the following steps: [[ID=二十]] [[ID=二十一]]

[0138] Take the aqueous extract of Cortex Meliae, extract it with an extraction solvent to obtain a test solution;

[0139] Take the test substance, extract it with an extraction solvent to obtain a test substance solution;

[0140] Take the test substance solution and the test solution, and analyze them respectively with a high performance liquid chromatograph equipped with an evaporative light scattering detector to construct the chromatogram of the test substance and the characteristic chromatogram of the aqueous extract of Cortex Meliae, and compare the chromatogram of the test substance with the characteristic chromatogram of the aqueous extract of Cortex Meliae.

[0141] Understandably, the high-performance liquid chromatography (HPLC) analysis process in the identification method of this application is similar to the HPLC analysis process in the method for constructing the characteristic spectrum of the water extract of neem bark described in any of the above technical solutions. The preparation of the analyte solution is the same as the preparation of the test sample solution described in any of the above technical solutions, and will not be repeated here.

[0142] Understandably, in the identification method of this application, the water extract of neem bark can be water extract of neem bark from different origins, such as water extract of neem bark from the origin of Sichuan neem (Melia toosenclan Sieb. et Zucc.) or water extract of neem bark from the origin of neem (Melia azedarach L.), and corresponding characteristic spectra can be constructed for each, and used to compare with the characteristic spectra of the analyte.

[0143] Understandably, the chromatograms of the analytes and the characteristic chromatograms of the water extracts of *Melia azedarach* bark constructed in the identification method of this application can be used for direct comparison or for data analysis as needed. For example, a single analyte can be detected and compared with the characteristic chromatograms of water extracts of *Melia azedarach* bark from different origins to obtain the attribution, authenticity, or quality test results of the single analyte; or multiple batches of analytes can be detected, and the characteristic chromatograms of multiple batches can be compared, with data analysis or without data analysis, extracting common features among the multiple batches of analytes. The water extracts of *Melia azedarach* bark from different origins mentioned in the identification method of this application can be from *Melia toosenclan* Sieb. et Zucc. or *Melia azedarach* L.

[0144] In some embodiments, the analyte origin is selected only from *Melia azedarach* or *Melia azedarach*, and the identification method can be selected from any of the following:

[0145] (1) If the peak of the different chinaberry element is missing, the original source is identified as chinaberry; otherwise, it is chinaberry.

[0146] (2) If the peak response value of catechin is lower than that of neemin, the identification source is neem; otherwise, it is neem.

[0147] (3) If the peak response value of catechin is higher than that of neem, the identification source is neem; otherwise, it is neem.

[0148] (4) If the relative peak area of ​​catechin peak is between 0.003 and 0.425, the identification source is Sichuan pepper; otherwise, it is neem.

[0149] (5) Taking the peak of Sichuan chinaberry as a reference, the relative peak area of ​​catechin peak is in the range of 2.309 to 10.885. If the identification source is chinaberry, otherwise it is Sichuan chinaberry.

[0150] (6) If the relative peak area of ​​the catechin peak and the neem peak is greater than 1.6, the identification source is neem; otherwise, it is neem.

[0151] (7) If the relative peak area of ​​the catechin peak and the neem peak is less than or equal to 1.6, the identification source is neem; otherwise, it is neem.

[0152] In some embodiments, it is uncertain whether the origin of the analyte is selected solely from *Melia azedarach* or *Melia azedarach*, and the identification method may be selected from any of the following:

[0153] (1) If the characteristic chromatogram of the water extract of neem bark has the following characteristics, the original source is determined to be neem: The chromatogram of the test sample shows 9 characteristic peaks, of which 4 peaks should correspond to the retention times of the corresponding reference peaks. The peak corresponding to the catechin reference peak is peak S1. Calculate the relative retention times of peak 1, peak 2 and peak S1. Their relative retention times should be within ±10% of the specified value. The specified values ​​are: 0.49 (peak 1) and 0.86 (peak 2). The peak corresponding to the main reference peak of neem is peak S2. Calculate the relative retention times of peak 5, peak 6, peak 7 and peak S2. Their relative retention times should be within ±10% of the specified value. The specified values ​​are: 0.81 (peak 5), 0.90 (peak 6), and 0.95 (peak 7). The relative peak area of ​​the catechin peak and peak S2 is greater than 1.6.

[0154] (2) If the characteristic chromatogram of the water extract of neem bark has the following characteristics, then the origin is determined to be neem: The chromatogram of the test sample shows 6 characteristic peaks, of which 3 peaks should correspond to the retention times of the corresponding reference peaks. The peak corresponding to the catechin reference peak is peak S1. Calculate the relative retention times of peak 1, peak 2 and peak S1. Their relative retention times should be within ±10% of the specified value. The specified values ​​are: 0.49 (peak 1) and 0.86 (peak 2). The peak corresponding to the neem reference peak is peak S2. Calculate the relative retention time of peak 6 and peak S2. Their relative retention time should be within ±10% of the specified value. The specified value is: 0.90 (peak 6). The relative peak area of ​​the catechin peak and peak S2 is less than or equal to 1.6.

[0155] The following are some specific examples.

[0156] Example 1: Construction of a characteristic spectrum of neem bark extract

[0157] 1. Instruments and reagents

[0158] 1.1 Instruments

[0159] Agilent HPLC system (1260, Agilent Technologies), Waters HPLC system (ACQUITYArc, Waters Corporation), evaporative light scattering detector (Alltech 6000, Alltech Technology Co., Ltd.), Thermo Vanquish Flex UHPLC-Thermo Fisher QE HMI mass spectrometer (Thermo Fisher Scientific), Poroshell 120EC-C18 column (150mm × 4.6mm, 2.7μm), 0.001% balance (ME204E, Mettler Toledo), 0.1% balance (XP26, Mettler Toledo), CNC ultrasonic cleaner (KQ500DE, Kunshan Ultrasonic Instrument Co., Ltd.), constant temperature water bath (HWS28, Shanghai Yiheng Technology Co., Ltd.), ultrapure water system (Milli-Q Direct, Merck KE Ltd.).

[0160] 1.2 Reagents

[0161] Methanol (Xilong Scientific Co., Ltd.) was analytical grade, ethanol (Xilong Scientific Co., Ltd.) was analytical grade, acetonitrile (Merck, Inc.) for liquid chromatography was HPLC grade, and water was ultrapure water (prepared in the laboratory).

[0162] 1.3 Drug Test

[0163] Aqueous extract of neem bark: The aqueous extract of neem bark in this study was prepared in-house. The neem bark was processed according to standard processing procedures, made into slices, and then the aqueous extract was prepared using the following method: Preparation method: Soak the neem bark slices for 30 minutes, then decoct twice. For the first decoction, add 10 times the amount of water to the slices and decoct for 30 minutes. For the second decoction, add 8 times the amount of water to the slices and decoct for 25 minutes. Filter the decoction while hot through a 350-mesh sieve. Combine the two filtrates, concentrate, transfer to a vacuum freeze dryer, and freeze-dry. The neem bark information used in the above preparation method is shown in Table 1.

[0164] Reference standards: Catechin reference standard (batch number: 110877-202005, content: 95.1%), epicatechin reference standard (batch number: 110878-201703, content: 99.7%), azadirachtin reference standard (batch number: 111842-201804, content: 96.9%), and neem bark (batch number: 121111-201704) were purchased from the China National Institutes for Food and Drug Control; proanthocyanidin B3 (batch number: 21060706, content: 97.6%, Chengdu Pufeide Biotechnology Co., Ltd.); isoazadirachtin (batch number: DST210629580, content: 98%, Chengdu Lemeitian Biotechnology Co., Ltd.).

[0165] Table 1 Information on Neem Bark (a medicinal material)

[0166]

[0167]

[0168] 2. Sample preparation

[0169] 2.1 Preparation of reference solution

[0170] Take appropriate amounts of catechin reference standard, epicatechin reference standard, and azadirachtin reference standard, accurately weigh them, and add methanol to prepare a mixed solution containing 0.5 mg of each per 1 mL, which is used as the reference solution.

[0171] 2.2 Preparation of test solution

[0172] 2.2.1. Investigation of extraction solvent

[0173] This experiment investigated the effects of different extraction solvents on the characteristic chromatograms of the aqueous extract of neem bark. The main solvents used were dichloromethane, n-butanol, and ethyl acetate. The peak shape and resolution of nine characteristic peaks were observed, and the effects of different extraction solvents on the characteristic chromatograms of the aqueous extract of neem bark were compared.

[0174] Take an appropriate amount of water extract from neem bark (Melia azedarach), grind it finely, and accurately weigh about 0.5g. Perform three parallel extractions, two portions per group. Add 10ml of water to each group, shake to dissolve, transfer to a separatory funnel, and extract three times each with 20ml of dichloromethane, n-butanol, and ethyl acetate. Combine the organic layers, evaporate to dryness, redissolve the residue in 70% methanol, transfer to a 2ml volumetric flask, dilute to the mark with 70% methanol, shake well, filter, and collect the filtrate. The experimental results are shown in Table 2. Figure 1 .

[0175] Table 2. Characteristic spectrum of neem bark (Melia azedarach) formulation granules and results of extraction solvent investigation.

[0176]

[0177] The results showed that when ethyl acetate and n-butanol were used as extraction solvents, the "total peak area / sample weight" was not significantly different. However, the chromatogram baseline was more stable and the characteristic peaks were better separated after extraction with ethyl acetate. Therefore, ethyl acetate was chosen as the extraction solvent.

[0178] 2.2.2. Established method for preparing test samples

[0179] Grind the water extract of neem bark into a fine powder. Take 0.5g of the powder and place it in a stoppered conical flask. Add 10mL of water, shake to dissolve, and transfer to a separatory funnel. Extract the powder three times with 20mL of ethyl acetate each time. Combine the ethyl acetate layers, evaporate to dryness, dissolve the residue in methanol, and transfer to a 2mL volumetric flask. Dilute to the mark with methanol, shake well, filter, and collect the filtrate.

[0180] 3. Determination of chromatographic conditions

[0181] 3.1 Elution gradient

[0182] Compare the effects of different gradient conditions on the characteristic spectrum of water extract of neem bark.

[0183] Take an appropriate amount of water extract from neem bark (Melia azedarach), grind it finely, and accurately weigh about 0.5g. Prepare the test solution according to the method of this invention, except for using different elution gradients; other chromatographic conditions are the same as those of this invention. Inject and analyze. The elution gradients are shown in Tables 3-6; the results are shown in... Figure 2 .

[0184] Table 3 Elution gradient 1

[0185]

[0186]

[0187] Table 4 Elution gradient 2

[0188]

[0189] Table 5 Elution gradient 3

[0190]

[0191] Table 6 Elution gradient 4

[0192]

[0193] The results showed that both gradients 1 and 2 detected nine chromatographic peaks of neem bark, but peak 2 contained other impurity peaks, resulting in a resolution of less than 1.5, which did not meet the separation requirements for characteristic chromatographic peaks. In gradient 3, peaks 8 and 9 were connected, and the resolution also failed to meet the 1.5 requirement. By adjusting the elution gradient, the gradient method of this patented invention (gradient 4) achieved good resolution for all nine chromatographic peaks, meeting the requirements for characteristic chromatographic analysis. Therefore, elution gradient 4 was selected as the elution procedure for subsequent studies of the characteristic chromatographic profile of neem bark.

[0194] 3.2. Mobile phase conditions

[0195] The effects of different mobile phases (acetonitrile-water, acetonitrile-0.2% formic acid, acetonitrile-0.2% glacial acetic acid) on the characteristic chromatograms of water extracts from neem bark were compared.

[0196] Take the test solution from the "Comparison of Different Elution Gradients" test. Except for using different mobile phases (acetonitrile-water, acetonitrile-0.2% formic acid, acetonitrile-0.2% glacial acetic acid) for elution, use the same chromatographic conditions as described in this invention, and perform injection analysis. Results are shown in [Figure Number]. Figure 3 .

[0197] The results showed that using different aqueous phases as mobile phases had little impact on the separation effect of the nine chromatographic peaks. To reduce the use of organic solvents, acetonitrile-water was used as the mobile phase for subsequent studies of the characteristic chromatograms of neem bark.

[0198] 3.3. Column temperature conditions

[0199] The effects of different column temperatures (25℃, 30℃, and 40℃) on the characteristic spectra of water extracts from neem bark were compared.

[0200] The test solution used in the "Comparison of Different Elution Gradients" was subjected to the same chromatographic conditions as described in this invention, except that different column temperatures (25℃, 30℃, and 40℃) were used for control. The results are shown below. Figure 4 .

[0201] The results showed that different column temperatures had little effect on the separation of the nine chromatographic peaks. Therefore, the column temperature set in general experimental studies (30℃) was sufficient. Thus, 30℃ was chosen as the column temperature for subsequent studies of the characteristic chromatograms of the water extract of neem bark.

[0202] 3.4. Flow velocity conditions

[0203] The effects of different flow rates (0.35 ml / min, 0.50 ml / min, and 0.70 ml / min) on the characteristic chromatograms of the water extract of neem bark were compared.

[0204] The test solution used for the "comparison of different elution gradients" was analyzed under the same chromatographic conditions as described in this invention, except that different flow rates (0.35 ml / min, 0.50 ml / min, and 0.70 ml / min) were used for comparison. Results are shown in [Figure number missing]. Figure 5 .

[0205] The results showed that different flow rates had little impact on the response and separation of the nine chromatographic peaks. Considering the inner diameter and particle size of the chromatographic column and the pump pressure of the instrument, and to avoid affecting the reproducibility and relative peak area due to excessively low or high flow rates in different laboratory environments, a flow rate of 0.50 ml / min, close to the middle value, was selected as the flow rate for subsequent studies on the characteristic chromatograms of the water extract of neem bark.

[0206] 3.5. Methodological Examination

[0207] 3.5.1 Precision

[0208] Aqueous extracts of neem bark were taken and a test solution was prepared according to the method for preparing the test solution of this invention. The solution was then injected and analyzed under the chromatographic conditions of this invention. The same test solution was injected six times consecutively. Using peak 3 (catechin) as the reference peak S1, the relative retention times and relative peak areas of peaks 1, 2, and 4 with respect to peak S1 were calculated. Using peak 8 (neemin) as the reference peak S2, the relative retention times and relative peak areas of peaks 5, 6, 7, and 9 with respect to peak S2 were calculated, and the RSD values ​​were also calculated. The experimental results are shown in Tables 7 and 8.

[0209] Table 7. Precision Study Results of Characteristic Chromatography of Water Extract from Neem Bark (Relative Retention Time)

[0210]

[0211] Table 8. Precision Study Results of Characteristic Chromatography of Water Extract from Neem Bark (Relative Peak Area)

[0212]

[0213]

[0214] The results showed that when the same sample solution was injected six times consecutively, the relative retention time RSD of each characteristic peak was in the range of 0.00% to 0.22%, and the relative peak area RSD was in the range of 0.66% to 3.79%, indicating that the instrument precision was good.

[0215] 3.5.2 Repeatability

[0216] Aqueous extracts of neem bark were taken, and six test solutions were prepared according to the test solution preparation method of this invention. These solutions were then injected and analyzed under the chromatographic conditions of this invention. Using peak 3 (catechin) as reference peak S1, the relative retention times and relative peak areas of peaks 1, 2, and 4 with respect to peak S1 were calculated. Using peak 8 (neemin) as reference peak S2, the relative retention times and relative peak areas of peaks 5, 6, 7, and 9 with respect to peak S2 were calculated, and the RSD values ​​were also calculated. The experimental results are shown in Tables 9 and 10.

[0217] Table 9. Results of Repeatability Study of Characteristic Chromatography of Water Extract from Neem Bark (Relative Retention Time)

[0218]

[0219] Table 10. Results of Repeatability Study of Characteristic Chromatographic Images of Water Extracts from Neem Bark (Relative Peak Area)

[0220]

[0221] The results showed that when the same batch of samples was measured repeatedly 6 times, the relative retention time RSD of each characteristic peak was in the range of 0.01% to 0.25%, and the relative peak area RSD was in the range of 1.13% to 4.30%, indicating that the characteristic spectral method has good repeatability.

[0222] 3.5.3. Stability Assessment

[0223] Aqueous extracts of neem bark were taken and a test solution was prepared according to the method of the present invention. The solution was then analyzed under the chromatographic conditions of the present invention at 0, 3, 6, 13, 19, and 24 hours. Using peak 3 (catechin) as reference peak S1, the relative retention times and relative peak areas of peaks 1, 2, and 4 with respect to peak S1 were calculated. Using peak 8 (neemin) as reference peak S2, the relative retention times and relative peak areas of peaks 5, 6, 7, and 9 with respect to peak S2 were calculated, and the RSD values ​​were calculated. The experimental results are shown in Tables 11 and 12.

[0224] Table 11. Results of the stability study of the water extract of neem bark (neem) (relative retention time)

[0225]

[0226]

[0227] Table 12. Results of the stability study of the characteristic spectra of water extracts of neem bark (relative peak areas).

[0228]

[0229] The results showed that when the same test solution was analyzed at 0, 3, 6, 13, 19 and 24 hours, the relative retention time RSD of each characteristic peak was in the range of 0.01% to 0.59%, and the relative peak area RSD was in the range of 1.35% to 4.64%, indicating that the test solution was relatively stable within 24 hours.

[0230] 3.5.4. Durability Assessment

[0231] ① Investigation at different column temperatures

[0232] The effects of different column temperatures (28℃, 30℃, and 32℃) on the characteristic spectra of water extracts from neem bark were compared.

[0233] Take an appropriate amount of water extract from neem bark (Melia azedarach), grind it finely, and accurately weigh about 0.5g. Prepare the test solution according to the method of this invention, except that the column temperatures are 28℃, 30℃, and 32℃ respectively. Other chromatographic conditions are the same as those of this invention. Inject and analyze the sample. Using peak 3 (catechin) as the reference peak S1, calculate the relative retention times and relative peak areas of peaks 1, 2, and 4 with peak S1. Using peak 8 (neemin) as the reference peak S2, calculate the relative retention times and relative peak areas of peaks 5, 6, 7, and 9 with peak S2, and calculate the RSD values. The experimental results are shown in Tables 13 and 14. Figure 6 .

[0234] Table 13. Characteristic spectrum of water extract of neem bark (Melia azedarach) under different column temperatures (relative retention time)

[0235]

[0236] Table 14. Characteristic spectrum of water extract of neem bark (neem) under different column temperatures (relative peak area)

[0237]

[0238] The results showed that, at different column temperatures, with peak 3 (catechin) as reference peak S1 and peak 8 (aristolide) as reference peak S2, the relative retention time (RSD) of each characteristic peak was in the range of 0.02% to 0.63%, and the relative peak area (RSD) was in the range of 0.72% to 4.37%, all less than 5%, indicating good durability at a column temperature of ±2℃.

[0239] ② Investigation of different flow velocities

[0240] The effects of different flow rates (0.45 ml / min, 0.50 ml / min, and 0.55 ml / min) on the characteristic chromatograms of the water extract of neem bark were compared.

[0241] Take the test solution from the "Different Column Temperatures Investigation" section. Except for flow rates of 0.45 ml / min, 0.50 ml / min, and 0.55 ml / min, other chromatographic conditions are the same as those of this invention. Inject and analyze the sample. Using peak 3 (catechin) as reference peak S1, calculate the relative retention times and relative peak areas of peaks 1, 2, and 4 with peak S1. Using peak 8 (aridine) as reference peak S2, calculate the relative retention times and relative peak areas of peaks 5, 6, 7, and 9 with peak S2, and calculate the RSD values. Experimental results are shown in Tables 15 and 16. Figure 7 .

[0242] Table 15. Characteristic spectrum of water extract of neem bark (Melia azedarach) under different flow rates (relative retention time)

[0243]

[0244] Table 16. Characteristics of water extracts from neem bark (neem) under different flow rates (relative peak area)

[0245]

[0246] The results showed that, at different flow rates, with peak 3 (catechin) as reference peak S1 and peak 8 (aristolide) as reference peak S2, the relative retention time RSD of each characteristic peak was in the range of 0.03% to 1.47%, and the relative peak area RSD was in the range of 1.74% to 5.14%, exceeding 5%. This indicates that when the flow rate is ±0.05 ml / min, the change in flow rate has little effect on the relative retention time of each characteristic peak, but a greater effect on the relative peak area of ​​each characteristic peak. Therefore, it is recommended to fix the flow rate at 0.50 ml / min.

[0247] ③ Investigation of different brands of chromatographic columns

[0248] The effects of different brands of chromatographic columns on the characteristic chromatograms of water extracts of neem bark were compared. The effects of four chromatographic columns—Shim-pack Scepter C18-120 (4.6 mm × 250 mm, 3 μm), Agilent Poroshel 120EC-C18 (4.6 mm × 250 mm, 2.7 μm), Agilent Poroshel 120SB-C18 (4.6 mm × 250 mm, 2.7 μm), and Agilent ZORBAX SB-C18 (4.6 mm × 250 mm, 5 μm)—on the characteristic chromatograms of water extracts of neem bark were investigated.

[0249] Take the test solution from the "Different Column Temperatures Investigation" section. Except for the chromatographic column, use the same chromatographic conditions as described in this invention. Inject and determine the sample. Using peak 3 (catechin) as reference peak S1, calculate the relative retention times and relative peak areas of peaks 1, 2, and 4 with peak S1. Using peak 8 (aridine) as reference peak S2, calculate the relative retention times and relative peak areas of peaks 5, 6, 7, and 9 with peak S2. Calculate the RSD values. The experimental results are shown in Tables 17 and 18. Figure 8 .

[0250] Table 17. Characteristic chromatograms of water extracts of neem bark (Melia azedarach) obtained using different chromatographic columns (relative retention times).

[0251]

[0252] Table 18. Result of Chromatographic Examination of Water Extracts of Melia azedarach Bark Using Different Columns (Relative Peak Area)

[0253]

[0254] The results showed that, using columns of different brands and particle sizes, the relative retention time RSD values ​​of each characteristic peak ranged from 0.09% to 3.73%, and the relative peak area RSD values ​​ranged from 2.30% to 6.86%, exceeding 5%. This indicates that different brands and particle sizes of columns have little impact on the relative retention time of each characteristic peak, but a significant impact on the relative peak area of ​​some characteristic peaks, such as peaks 6 and 7. The baseline of the ZORBAX SB-C18 column was relatively unstable, showing a bulge in the 35–45 minute range, which may affect the integration of peaks 6 and 7, which have smaller peak areas. Therefore, it is recommended to use columns with a particle size of 2.7–3.0 μm.

[0255] 3.6. Determination of chromatographic conditions

[0256] Based on the above investigation, the determined chromatographic conditions are as follows: a Poroshell 120EC-C18 column (150 mm in length, 4.6 mm in inner diameter, and 2.7 μm in particle size) is used as the chromatographic column; acetonitrile is used as mobile phase A and water is used as mobile phase B, with gradient elution performed according to the specifications in Table 19; the flow rate is 0.5 ml / min; the column temperature is 30 °C; and the injection volume is 1 μl.

[0257] Table 19 Gradient Elution Table

[0258]

[0259] 4. Detector confirmation

[0260] 4.1. Evaporation Light Scattering Detector

[0261] The detection conditions were as follows: a Poroshell 120EC-C18 column (150 mm length, 4.6 mm inner diameter, 2.7 μm particle size) was used; acetonitrile was used as mobile phase A and water as mobile phase B, with gradient elution performed according to the specifications in Table 20; the flow rate was 0.5 mL / min; the column temperature was 30 °C; and the injection volume was 1 μL. Evaporative light detector: drift tube temperature was 95 °C, and carrier gas flow rate was 3.0 L / min.

[0262] Table 20 Gradient Elution Table

[0263]

[0264]

[0265] 4.2. Ultraviolet Detector (Instrument: Waters PDA)

[0266] The detection conditions were as follows: a Poroshell 120EC-C18 column (150 mm length, 4.6 mm inner diameter, 2.7 μm particle size) was used; acetonitrile was used as mobile phase A and water as mobile phase B, with gradient elution performed according to the specifications in Table 20; the flow rate was 0.5 mL / min; the column temperature was 30 °C; and the injection volume was 1 μL. The UV detector wavelengths were: 3D scanning (200 nm–400 nm) and 210 nm.

[0267] The comparison results of the above tests are shown in the figure. Figure 9 ,according to Figure 9 It can be seen that, using the same chromatographic conditions and comparing different detectors, 3D scanning with an ultraviolet detector revealed that the main absorption wavelengths of the components of neem bark are between 200nm and 220nm. At 210nm, the absorption of each chromatographic peak is relatively balanced, maximizing the presentation of each absorption peak, but the ultraviolet response of peaks 5 to 9 is extremely low or absent. Characteristic chromatograms extracted at 210nm showed that (+)-gallic acid, proanthocyanidin B3, catechin, and epicatechin in neem bark could be detected, but the absence of obvious iso-neemin and azadirachtin chromatographic peaks is detrimental to analysis.

[0268] Since azadirachtin is the main characteristic component of neem bark and the component that exerts its main medicinal effect, an evaporative light detector was selected to present its characteristic information more clearly.

[0269] 5. Characteristic Peak Confirmation and Identification

[0270] 5.1. Characteristic Peak Confirmation

[0271] 5.1.1. Confirmation of characteristic peaks of water extract of neem bark (Melia azedarach)

[0272] The data of 17 batches of water extracts of neem bark were processed using the "Similarity Evaluation System for Chromatographic Characteristic Magnetism of Traditional Chinese Medicine (Version 2012.0)". The resulting overlay chromatograms are shown below. Figure 10 Nine common peaks with relatively stable relative retention time and relative peak area RSD were selected as characteristic peaks, and a control characteristic spectrum was generated (see [reference]). Figure 11 .

[0273] 5.1.2. Confirmation of characteristic peaks of the water extract of Melia toosendan bark (Chuanlian)

[0274] The data of six batches of water extracts of Melia toosendan bark (Chuanlian) were processed using the "Similarity Evaluation System for Chromatographic Characteristic Magnetism of Traditional Chinese Medicine (Version 2012.0)". The resulting overlay chromatograms are shown below. Figure 12 Six common peaks with relatively stable relative retention time and relative peak area (RSD) were selected as characteristic peaks, and a control characteristic spectrum was generated (see [reference]). Figure 13 .

[0275] 5.2. Characteristic Peak Identification

[0276] 5.2.1. Identification of characteristic peaks in the water extract of neem bark (Melia azedarach)

[0277] By comparing with references and relevant standards, the chemical components of its nine common peaks were assigned. The specificity chromatograms of the aqueous extract of *Melia azedarach* bark and the standards are shown below. Figure 14 By matching the retention time with the reference standard, a total of 7 components were identified, including peak 1 ((+)-gallocatechin), peak 2 (proanthocyanidin B3), peak 3 (catechin), peak 4 (epicatechin), peak 7 (iso-neemin), and peaks 8 / 9 (neemin).

[0278] 5.2.2. Identification of characteristic peaks in the water extract of Melia toosendan bark (Chuanlian)

[0279] By comparing with references and relevant standards, the chemical components of its six common peaks were assigned. The specificity chromatograms of the water extract of *Melia azedarach* bark (Sichuan chinaberry) and the standards are shown below. Figure 15 By matching the retention time with the reference standard, a total of 5 components were identified, including peak 1 ((+)-gallic catechin), peak 2 (proanthocyanidin B3), peak 3 (catechin), and peaks 8 / 9 (neemin).

[0280] Example 2: Identification of the Root Cause

[0281] This embodiment uses the characteristic spectra of water extracts of neem bark from two different origins constructed in Example 1. The peak areas and retention times of the characteristic peaks of the water extracts of neem bark from different origins are compared. The comparison process and the identification method obtained are as follows:

[0282] 1. Comparison of the characteristic chromatograms of water extracts of Melia azedarach bark (Sichuan neem) and Melia azedarach bark (neem)

[0283] The characteristic chromatogram of the water extract of neem bark includes the following features:

[0284] (1) It includes at least the following 9 common peaks: peak 1, peak 2, peak 3, peak 4, peak 5, peak 6, peak 7, peak 8 and peak 9; wherein, peak 1 is (+)-gallic acid catechin, peak 2 is proanthocyanidin B3, peak 3 is catechin, peak 4 is epicatechin, peak 7 is iso-neemin, and peaks 8 and 9 are both neemin;

[0285] (2) With peak 3 (S1) as the reference peak, the relative retention times of peak 1 and peak 2 are as follows: peak 1: 0.49±10%; peak 2: 0.86±10%; with peak 8 (S2) as the reference peak, the relative retention times of peak 5, peak 6 and peak 7 are as follows: peak 5: 0.81±10%; peak 6: 0.90±10%; peak 7: 0.95±10%;

[0286] (3) The response value of peak 3 is higher than that of peak 8;

[0287] (4) With peak 8 (S2) as a reference, the relative peak area of ​​peak 3 ranges from 2.309 to 10.885.

[0288] The characteristic chromatogram of the water extract of neem bark (Chuanlian) includes the following features:

[0289] (1) It includes at least the following 6 common peaks: peak 1, peak 2, peak 3, peak 6, peak 8 and peak 9; wherein, peak 1 is (+)-gallic catechin, peak 2 is proanthocyanidin B3, peak 3 is catechin, and peak 8 and peak 9 are neemin together;

[0290] (2) With peak 3 (S1) as the reference peak, the relative retention times of peak 1 and peak 2 are: peak 1: 0.49±10%; peak 2: 0.86±10%; with peak 8 (S2) as the reference peak, the relative retention time of peak 6 is: 0.90±10%; (3) The response value of peak 3 is lower than that of peak 8;

[0291] (3) With peak 8 (S2) as a reference, the relative peak area of ​​peak 3 ranges from 0.003 to 0.425.

[0292] The differences between the characteristic chromatogram of the water extract of Melia azedarach bark (Sichuan Melia) and the characteristic chromatogram of the water extract of Melia azedarach bark (Melia) include:

[0293] (1) Different response values ​​for peak 7 (isozane): The isozane peak is missing in the characteristic spectrum of the water extract of neem bark (neem);

[0294] (2) The relative response values ​​of peak 8 (neemin) and peak 3 (catechin) are different: the response value of peak 3 in the characteristic spectrum of water extract of neem bark is higher than that of peak 8, while the response value of peak 3 in the characteristic spectrum of water extract of neem bark is lower than that of peak 8.

[0295] (3) In the characteristic spectrum of the water extract of neem bark (neem), with peak 8 (neemin) as the reference, the relative peak area of ​​peak 3 ranges from 2.309 to 10.885; in the characteristic spectrum of the water extract of neem bark (neem), with peak 8 (neemin) as the reference, the relative peak area of ​​peak 3 ranges from 0.003 to 0.425.

[0296] 2. Identification methods

[0297] Based on the above analysis results, the proposed identification method is as follows:

[0298] A: The analyte's origin is selected only from *Melia azedarach* or *Melia azedarach*, and the identification method can be selected from any of the following:

[0299] (1) If the peak of the different chinaberry element is missing, the original source is identified as chinaberry; otherwise, it is chinaberry.

[0300] (2) If the peak response value of catechin is lower than that of neemin, the identification source is neem; otherwise, it is neem.

[0301] (3) If the peak response value of catechin is higher than that of neem, the identification source is neem; otherwise, it is neem.

[0302] (4) If the relative peak area of ​​catechin peak is between 0.003 and 0.425, the identification source is Sichuan pepper; otherwise, it is neem.

[0303] (5) Taking the peak of Sichuan chinaberry as a reference, the relative peak area of ​​catechin peak is in the range of 2.309 to 10.885. If the identification source is chinaberry, otherwise it is Sichuan chinaberry.

[0304] (6) If the relative peak area of ​​the catechin peak and the neem peak is greater than 1.6, the identification source is neem; otherwise, it is neem.

[0305] (7) If the relative peak area of ​​the catechin peak and the neem peak is less than or equal to 1.6, the identification source is neem; otherwise, it is neem.

[0306] B: If it is uncertain whether the origin of the analyte is selected solely from *Melia azedarach* or *Melia azedarach*, the identification method can be selected from any of the following:

[0307] (1) If the characteristic chromatogram of the aqueous extract of neem bark has the following characteristics, the original substance is determined to be neem: The chromatogram of the test sample shows 9 characteristic peaks, which are counted as peak 1, peak 2, peak 3, peak 4, peak 5, peak 6, peak 7, peak 8 and peak 9 respectively; among them, 4 peaks should correspond to the retention times of the corresponding reference peaks, and the peak corresponding to the catechin reference peak is peak S1. Calculate the relative retention times of peak 1, peak 2 and peak S1. Their relative retention times should be within the specified range. The values ​​should be within ±10% of the specified values: 0.49 (peak 1) and 0.86 (peak 2). The peak corresponding to the main reference peak of azadirachtin is peak S2. Calculate the relative retention times of peaks 5, 6, and 7 with peak S2. The relative retention times should be within ±10% of the specified values: 0.81 (peak 5), 0.90 (peak 6), and 0.95 (peak 7). The relative peak area of ​​the catechin peak with peak S2 should be greater than 1.6.

[0308] (2) If the characteristic chromatogram of the water extract of neem bark has the following characteristics, then the origin is determined to be neem: The chromatogram of the test sample shows 6 characteristic peaks, of which 3 peaks should correspond to the retention times of the corresponding reference peaks. The peak corresponding to the catechin reference peak is peak S1. Calculate the relative retention times of peak 1, peak 2 and peak S1. Their relative retention times should be within ±10% of the specified value. The specified values ​​are: 0.49 (peak 1) and 0.86 (peak 2). The peak corresponding to the neem reference peak is peak S2. Calculate the relative retention time of peak 6 and peak S2. Their relative retention time should be within ±10% of the specified value. The specified value is: 0.90 (peak 6). The relative peak area of ​​the catechin peak and peak S2 is less than or equal to 1.6.

[0309] Example 3 Identification of Similar Substances

[0310] Based on literature review and field surveys, two similar products of neem bark were found: Acacia farnesiana bark and Sapindus mukorossi bark. Acacia farnesiana bark is the bark of the legume Acacia farnesiana (Linn.) Willd., produced in Zhejiang, Taiwan, Fujian, Guangdong, Guangxi, Yunnan, and Sichuan provinces. Its roots and pods contain tannins, which can be used as a black dye; medicinally, it has astringent and heat-clearing properties. Sapindus mukorossi bark is the bark of the Sapindaceae plant Sapindus saponaria Linnaeus, listed in the 2006 edition of the *Dictionary of Traditional Chinese Medicine*, and has detoxifying, throat-soothing, wind-dispelling, and insecticidal effects. This embodiment analyzes these two similar products using the method described in Example 1, and obtains the characteristic spectra of the corresponding aqueous extracts of Acacia farnesiana bark and Sapindus mukorossi bark.

[0311] The characteristic spectra of the water extracts of the two similar substances mentioned above were compared with the characteristic spectra of the water extract of neem bark. The comparison results are as follows: Figure 16 .according to Figure 16 The characteristic spectra of the aqueous extracts of Acacia bark and Sapindus mukorossi bark differed significantly from those of Melia azedarach bark. Neither the characteristic spectra of Acacia bark nor Sapindus mukorossi bark showed the corresponding characteristic peaks of azadirachtin, the main characteristic component of Melia azedarach bark. This indicates that the established characteristic spectra method can distinguish Melia azedarach bark from its two similar products.

[0312] Comparative Example 1

[0313] The HPLC-ELSD fingerprinting method for neem bark was established based on the references. The method was analyzed and evaluated using water extract samples of neem bark.

[0314] Instruments, reagents and reagents: Agilent high performance liquid chromatograph (1260, Agilent Technologies), evaporative light scattering detector (Alltech ELSD 6000, Alltech Technology Co., Ltd.); analytical electronic balance of 0.001 g (ME204E, Mettler Toledo), analytical electronic balance of 0.1 g (XP26, Mettler Toledo), ultrapure water system (Milli-QDirect, Merck KGaA).

[0315] Reagents: Methanol (Xilong Scientific Co., Ltd., analytical grade); formic acid (Tianjin Kemeio Chemical Reagent Co., Ltd., chromatographic grade), acetonitrile (Merck, Inc., chromatographic grade), methanol (Merck, Inc., chromatographic grade); water was ultrapure water (prepared in the laboratory).

[0316] Test reagents: Catechin reference standard (batch number: 110877-201604, content: 99.2%, China National Institutes for Food and Drug Control); epicatechin reference standard (batch number: 110878-200102, China National Institutes for Food and Drug Control); azadirachtin reference standard (batch number: 111842-201804, content: 96.9%, China National Institutes for Food and Drug Control); detailed information on the water extract of neem bark is provided in Example 1.

[0317] Chromatographic conditions and system suitability test: Thermo Acclaim C18 column (4.6 mm × 250 mm, 5 μm); acetonitrile as mobile phase A, 0.1% formic acid as mobile phase B, gradient elution as specified in Table 21, flow rate 1.0 mL / min; column temperature 30 °C; evaporative light scattering detector. The theoretical plate number, calculated based on the azadirachtin peak, should be no less than 3000.

[0318] Table 21 Gradient Elution Table

[0319]

[0320] Preparation of the test solution: Take an appropriate amount of water extract of neem bark (neem), grind it into a fine powder, take about 0.5g, place it in a stoppered conical flask, add 50ml of 70% methanol, heat under reflux for 30 minutes, cool, centrifuge (4000R / min, 5 minutes), transfer 25ml of the supernatant to an evaporating dish and evaporate to dryness, dissolve the residue in 70% methanol in a 2ml volumetric flask, add 70% methanol to the mark, shake well, filter, and take the filtrate to obtain the test solution.

[0321] For the assay, accurately pipette 20 μl each of the reference solution and the test solution and inject them into the liquid chromatograph. Measure the solution to obtain the final result.

[0322] Results Analysis: Seventeen batches of water extracts from *Melia azedarach* bark were analyzed using the methods described in the literature. The overlay plot of data from the seventeen batches is shown below. Figure 17 See the comparison chart. Figure 18 The results were compared with the chromatogram in Example 1. The results showed that the overall profiles obtained by the two methods were similar. However, the method in the literature, using an acetonitrile-0.1% formic acid mobile phase system, exhibited a severe baseline bulge in the middle section, affecting the symmetry of the chromatographic peaks and providing less peak information. It was unable to identify the three components (+)-gallic acid, proanthocyanidin B3, and isohyetalin. Furthermore, the literature did not investigate the relevant origins. The method invented in this patent uses ethyl acetate extraction in the sample preparation method, removing the influence of impurities, including a large number of impurities in the solvent peak position, resulting in a flat chromatogram baseline. Due to the concentration process, the method in Example 1 could also detect isohyetalin, fully demonstrating the characteristic components of the water extract of neem bark. Moreover, the selection of an acetonitrile-water mobile phase reduces the use of organic solvents. Through comparison, the method invented in this patent yields better overall results.

[0323] The technical features of the above-described embodiments and examples can be combined in any suitable manner. For the sake of brevity, not all possible combinations of the technical features in the above-described embodiments and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0324] The embodiments described above merely illustrate several implementation methods of this application and should not be construed as limiting the scope of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Furthermore, it should be understood that after reading the above teachings, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the protection scope of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the protection scope of the appended claims. Therefore, the protection scope of this patent application should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A method for constructing a characteristic chromatogram of Melia azedarach L. water extract, characterized in that, The method comprises the following steps: Take the aqueous extract of Melia azedarach bark to prepare a test solution by extraction with an extraction solvent; the aqueous extract of Melia azedarach bark is an aqueous extract of Melia azedarach bark from different origins, wherein the different origins refer to Melia azedarach or Melia toosendan; the extraction method is extraction; and the extraction solvent comprises ethyl acetate; Take the test solution and analyze it by using a high performance liquid chromatograph equipped with an evaporative light scattering detector to construct a characteristic chromatogram of the aqueous extract of Melia azedarach bark; The conditions of the high performance liquid chromatograph comprise the following characteristics: (1) The mobile phase A is acetonitrile, and the mobile phase B is water; (2) The elution program comprises: 0-16 min, the volume percentage of the mobile phase A is increased from 10% to 12%; 16 min-20 min, the volume percentage of the mobile phase A is increased from 12% to 18%; 20 min-33 min, the volume percentage of the mobile phase A is increased from 18% to 30%; 33 min-43 min, the volume percentage of the mobile phase A is increased from 30% to 60%; 43 min-45 min, the volume percentage of the mobile phase A is increased from 60% to 90%; 45 min-50 min, the volume percentage of the mobile phase A is kept at 90%; (3) The chromatographic column is an octadecylsilane-bonded silica gel chromatographic column; (4) The chromatographic column has a column length of 150 mm, an inner diameter of 4.6 mm, and a particle size of 2.7-3 μm; When the origin of the aqueous extract of Melia azedarach bark is Melia toosendan, the characteristic chromatogram of the aqueous extract of Melia azedarach bark comprises the following nine common peaks: peak 1, peak 2, peak 3, peak 4, peak 5, peak 6, peak 7, peak 8, and peak 9; wherein peak 1 is (+)-gallocatechin, peak 2 is procyanidin B3, peak 3 is catechin, peak 4 is epicatechin, peak 7 is isomeliaazedarachin, and peaks 8 and 9 are both toosendanin; When the origin of the aqueous extract of Melia azedarach bark is Melia azedarach, the characteristic chromatogram of the aqueous extract of Melia azedarach bark comprises the following six common peaks: peak 1, peak 2, peak 3, peak 6, peak 8, and peak 9; wherein peak 1 is (+)-gallocatechin, peak 2 is procyanidin B3, peak 3 is catechin, and peaks 8 and 9 are both toosendanin.

2. The method of constructing a feature map according to claim 1, wherein, The conditions of the high performance liquid chromatograph comprise one or more of the following characteristics: (1) The flow rate is 0.4-0.6 ml / min; (2) The injection volume is 0.5-1.5 μL; (3) The column temperature is 28-32℃.

3. The method of constructing a feature map according to claim 1, wherein, The conditions of the evaporative light scattering detector comprise the following characteristics: (1) The drift tube temperature is 90-100℃; (2) The carrier gas flow rate is 2.5-3.5 L / min.

4. The method of constructing a feature map according to claim 1, wherein, The aqueous extract of Melia azedarach bark is prepared by the following preparation steps: Take Melia azedarach medicinal materials, soak them in water, perform first decoction by adding 8-12 times the amount of water to the Melia azedarach medicinal materials, collect the first filtrate by filtration, then perform second decoction by adding 6-10 times the amount of water to the Melia azedarach medicinal materials, collect the second filtrate by filtration, and combine the first filtrate and the second filtrate, and then freeze-dry.

5. The method of constructing a feature map according to any one of claims 1 to 4, wherein, When the base origin of the Melia toosendan Sieb et Zucc. water extract is Melia azedarach L., the relative retention times of peaks 1 and 2 are 0.49±10% and 0.86±10% respectively, with peak 3 as the reference peak, among the 9 common peaks included in the characteristic pattern of the Melia toosendan Sieb et Zucc. water extract.

6. The method of constructing a feature map according to claim 5, wherein, The relative peak area of peak 3 is greater than 1.6, with peak 8 as the reference peak.

7. The method of constructing a feature map according to any one of claims 1 to 4, wherein, When the base origin of the Melia toosendan Sieb et Zucc. water extract is Melia toosendan Sieb et Zucc., the relative retention times of peaks 1 and 2 are 0.49±10% and 0.86±10% respectively, with peak 3 as the reference peak, among the 6 common peaks included in the characteristic pattern of the Melia toosendan Sieb et Zucc. water extract. The relative retention time of peak 6 is 0.90±10%, with peak 8 as the reference peak.

8. The method of constructing a feature map according to claim 7, wherein, The relative peak area of peak 3 is less than or equal to 1.6, with peak 8 as the reference peak.

9. A method for identifying the origin of the aqueous extract of Melia azedarach bark, characterized by, The method comprises the following steps: The Melia toosendan Sieb et Zucc. water extract is extracted with an extraction solvent to prepare a test sample solution; the Melia toosendan Sieb et Zucc. water extract is of different base origins, i.e., the base origin is Melia toosendan Sieb et Zucc. or Melia azedarach L.; the extraction is performed by extraction; and the extraction solvent comprises ethyl acetate; The test sample is extracted with an extraction solvent to prepare a test sample solution; the test sample is a Melia toosendan Sieb et Zucc. water extract of different base origins, i.e., the base origin is Melia toosendan Sieb et Zucc. or Melia azedarach L.; the extraction is performed by extraction; and the extraction solvent comprises ethyl acetate; The test sample solution and the test sample solution are analyzed by high performance liquid chromatography equipped with an evaporative light scattering detector, respectively, to construct a pattern of the test sample and a characteristic pattern of the Melia toosendan Sieb et Zucc. water extract, and the pattern of the test sample is compared with the characteristic pattern of the Melia toosendan Sieb et Zucc. water extract; The conditions of the high performance liquid chromatography comprise the following characteristics: (1) the mobile phase A is acetonitrile, and the mobile phase B is water; (2) the elution program comprises: 0-16 min, the volume percentage of the mobile phase A is increased from 10% to 12%; 16 min-20 min, the volume percentage of the mobile phase A is increased from 12% to 18%; 20 min-33 min, the volume percentage of the mobile phase A is increased from 18% to 30%; 33 min-43 min, the volume percentage of the mobile phase A is increased from 30% to 60%; 43 min-45 min, the volume percentage of the mobile phase A is increased from 60% to 90%; 45 min-50 min, the volume percentage of the mobile phase A is kept at 90%; (3) the chromatographic column is an octadecylsilane bonded silica gel chromatographic column; (4) the length of the chromatographic column is 150 mm, the inner diameter is 4.6 mm, and the particle size is 2.7-3 μm; When the base of the Melia azedarach L. water extract is Melia azedarach, the characteristic spectrum of the Melia azedarach L. water extract comprises 9 common peaks: peak 1, peak 2, peak 3, peak 4, peak 5, peak 6, peak 7, peak 8 and peak 9; wherein, peak 1 is (+)-gallocatechin, peak 2 is procyanidin B3, peak 3 is catechin, peak 4 is epicatechin, peak 7 is isofagarine, and peak 8 and peak 9 are fagarine together. When the base of the Melia azedarach L. water extract is Melia azedarach, the characteristic spectrum of the Melia azedarach L. water extract comprises 6 common peaks: peak 1, peak 2, peak 3, peak 6, peak 8 and peak 9; wherein, peak 1 is (+)-gallocatechin, peak 2 is procyanidin B3, peak 3 is catechin, and peak 8 and peak 9 are fagarine together.

10. The authentication method of claim 9, wherein, The conditions of the high performance liquid chromatography comprise one or several of the following characteristics: (1) the flow rate is 0.4 ml / min-0.6 ml / min; (2) the injection amount is 0.5 μL-1.5 μL; (3) the column temperature is 28 ℃-32 ℃.

11. The authentication method of claim 9, wherein, The conditions of the internal detection of the evaporative light scattering detector comprise the following characteristics: (1) the drift tube temperature is 90 ℃-100 ℃; (2) the carrier gas flow rate is 2.5 L / min-3.5 L / min.

12. The authentication method of claim 9, wherein, The Melia azedarach L. water extract is prepared by the following preparation steps: Melia azedarach L. medicinal materials are taken, soaked with water, and subjected to first decoction with 8-12 times the amount of water of the Melia azedarach L. medicinal materials, and the first filtrate is collected by filtration; then the Melia azedarach L. medicinal materials are subjected to second decoction with 6-10 times the amount of water of the Melia azedarach L. medicinal materials, and the second filtrate is collected by filtration, and the first filtrate and the second filtrate are combined, concentrated, and freeze-dried.

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