A method for constructing a feature spectrum of a chaulmoogra and a method for distinguishing a chaulmoogra genuine product from a chaulmoogra fake product

Abstract

The application discloses a construction method of a characteristic spectrum of Toona sinensis peel and a method for distinguishing authentic and fake Toona sinensis peels. The application distinguishes authentic and fake Toona sinensis peels by constructing a UPLC characteristic spectrum of Toona sinensis peel medicinal materials, and the obtained characteristic spectrum has the advantages of strong speciality, high stability, good repeatability and the like, fully exhibits characteristic differences of Toona sinensis peel medicinal materials and fake Toona sinensis peels, can effectively identify authentic and fake Toona sinensis peels, can be used for quality evaluation of the Toona sinensis peels, realizes the purpose of effectively identifying fake Toona sinensis peels, and is helpful to improving the quality standard of the Toona sinensis peel medicinal materials.

Description

TECHNICAL FIELD

[0001] The application relates to a feature map construction method of chunpi and a method for distinguishing genuine and fake chunpi, and belongs to the technical field of traditional Chinese medicine identification. BACKGROUND

[0002] Chunpi is the dried root bark or dry bark of Ailanthus altissima (Mill.) Swingle in the Simaroubaceae family, has the functions of clearing heat and drying dampness, astringing and stopping leucorrhea, stopping diarrhea, and stopping bleeding, and is used for treating red and white leucorrhea, damp-heat diarrhea, chronic diarrhea, chronic dysentery, hematochezia, and metrorrhagia. Chunpi and Xiangchunpi (dried root bark or dry bark of Toona sinensis (Juss.) M. Roem.) were not distinguished in ancient times, and were gradually divided into two kinds of traditional Chinese medicines. Figure 1 However, due to usage habits, some places still mix or substitute the two (Tong Huan, Lv Feng, Zhang Gezhi, et al. Application and identification of Xiangchunpi and chunpi in history [J]. Times Journal of Chinese Medicine, 1998, 9(3): 235-235.).

[0003] At present, the identification researches on chunpi mainly adopt conventional pharmacognosy methods (characteristics, microscopy, thin layer, ultraviolet spectrophotometry) to distinguish and differentiate chunpi and Xiangchunpi by Zhao Lianxing et al. (Zhao Lianxing. Identification and reasonable application of chunpi and Xiangchunpi [J]. Hebei Journal of Traditional Chinese Medicine, 2010, 32(5): 744, 747, F0003.), Zhou Xiaojie et al. (Zhou Xiaojie, Gao Ye, Wu Zeyu. Pharmacognosy research of chunpi [J]. Asia-Pacific Traditional Medicine, 2007, 3(10): 39-41.), and Tong Huan et al. (Tong Huan, Lv Feng, Zhang Gezhi, et al. Application and identification of Xiangchunpi and chunpi in history [J]. Times Journal of Chinese Medicine, 1998, 9(3): 235-235.). 18 Yang Mingxia et al. (Yang Mingxia, Wang Yongli, Nuo Guixin. Establishment of HPLC characteristic chromatogram and content determination method of fried chunpi formula granules [J]. Chinese Modern Chinese Medicine, 2023, 25(5): 1079-1085.) used a Shiseido MG II C -1 (250mm x 4.6mm, 5μm) HPLC column, gradient elution with acetonitrile-0.1% phosphoric acid aqueous solution as the mobile phase, a flow rate of 1.0mL·min , a column temperature of 30℃, a detection wavelength of 254nm, and an injection amount of 10μL, established the characteristic chromatogram of fried chunpi formula granules, identified the common peaks by using reference substances, and evaluated the correlation of fried chunpi decoction pieces, standard decoction and formula granules; the established HPLC characteristic chromatogram of fried chunpi formula granules determined 9 characteristic peaks, identified 3 of them, and the similarity of the characteristic chromatograms of each sample and the control chromatogram was 0.984-1.000.

[0004] The above-mentioned patents and literatures mainly have the following problems in identifying the genuine and fake products of the cortex fraxini:

[0005] (1) The existing technology mainly focuses on the identification of traditional pharmacognosy methods (characteristics, microscopy, thin layer, ultraviolet spectrophotometry), the characteristics are mainly empirical identification, and similar varieties are difficult to identify; the resolution of microscopic identification is low, time-consuming and labor-intensive, and is highly subjective; the resolution of thin layer chromatography is not high, especially when the chemical components of similar species are similar.

[0006] (2) The characteristic spectrum established by the above-mentioned HPLC method provides a reference for the quality evaluation and quality control of the cortex fraxini, but the separation degree of part of the common peaks is poor, and the peak area is relatively small, and the S peak iron shimi ketone is unstable, and the retention time changes greatly.

[0007] (3) The traditional pharmacognosy identification method is mainly empirical identification, and similar varieties are difficult to identify; a single content index cannot comprehensively evaluate the quality of the cortex fraxini. SUMMARY

[0008] The purpose of the present application is to provide a characteristic spectrum construction method of the cortex fraxini and a method for distinguishing the genuine and fake products of the cortex fraxini.

[0009] Technical scheme: In order to solve the above technical problems, the present application provides a characteristic spectrum construction method of the cortex fraxini, comprising the following steps:

[0010] (1) 70% methanol solution is added to the cortex fraxini medicinal material powder, heated and refluxed for 30-120 minutes, cooled, and the weight loss is made up with 70% methanol, filtered, and the filtrate is evaporated to dryness, dissolved with ethyl acetate, shaken and extracted, and the ethyl acetate layer is evaporated to dryness, dissolved with 70% methanol solution, filtered, and the filtrate is obtained, that is, the test sample solution;

[0011] (2) 4-hydroxybenzoic acid, vanillic acid, toona ketone, ferulic acid and iron shimi ketone are added to the solvent to prepare a control sample solution;

[0012] (3) injecting the test sample solution and the control sample solution into the ultra-high performance liquid chromatograph respectively to obtain characteristic chromatograms; the characteristic chromatograms have 15 common peaks, taking 4-hydroxybenzoic acid as a control sample, peak 1 is the chromatographic peak of 4-hydroxybenzoic acid; taking vanillic acid as a control sample, peak 2 is the chromatographic peak of vanillic acid; taking atranol as a control sample, peak 3 is the chromatographic peak of atranol; taking ferulic acid as a control sample, peak 6 is the chromatographic peak of ferulic acid; taking atranolone as a control sample, peak 13 is the chromatographic peak of atranolone; the peak corresponding to the atranol reference peak is the S peak, the relative retention times of peaks 1-2 and peaks 4-15 to the S peak are calculated, and the relative retention times should be within ±10% of the specified value, and the specified values of peaks 1-15 are 0.47, 0.70, 1.08, 1.23, 1.28, 1.35, 1.37, 1.47, 1.70, 1.92, 1.93, 2.15, 2.29 and 2.30 respectively.

[0013] In the step (3), the chromatographic conditions of the ultra-high performance liquid chromatograph are as follows: a chromatographic column: CORTECS T3; an injection amount: 1 μl; a flow rate: 0.25-0.35 ml / min; a column temperature: 25-35 DEG C; a detection wavelength: 254 nm; gradient elution is performed with acetonitrile as a mobile phase A and 0.1% formic acid solution as a mobile phase B.

[0014] In the step (3), the elution gradient of the ultra-high performance liquid chromatograph is as follows: 0-5 min, the volume fraction of the mobile phase A is 5%, and the volume fraction of the mobile phase B is 95%; 5-15 min, the volume fraction of the mobile phase A is 5→11%, and the volume fraction of the mobile phase B is 95→89%; 15-22 min, the volume fraction of the mobile phase A is 11→17%, and the volume fraction of the mobile phase B is 89→83%; 22-25 min, the volume fraction of the mobile phase A is 17%, and the volume fraction of the mobile phase B is 83%; 25-40 min, the volume fraction of the mobile phase A is 17→33%, and the volume fraction of the mobile phase B is 83→67%; 40-42 min, the volume fraction of the mobile phase A is 33→80%, and the volume fraction of the mobile phase B is 67→20%; 42-44 min, the volume fraction of the mobile phase A is 80%, and the volume fraction of the mobile phase B is 20%; 44-45 min, the volume fraction of the mobile phase A is 80→5%, and the volume fraction of the mobile phase B is 20→95%.

[0015] The application further provides a method for establishing a control chromatogram of the Chinese toona tree bark, comprising the following steps:

[0016] (1) respectively preparing multiple batches of test sample solutions of Chinese toona; the preparation method of the test sample solutions comprises the following steps: adding 70% methanol solution into Chinese toona medicinal material powder, heating and refluxing for 30-120 minutes, cooling, supplementing the lost weight with 70% methanol, filtering, evaporating the filtrate, dissolving, extracting with ethyl acetate by shaking, evaporating the ethyl acetate layer, dissolving with 70% methanol solution, filtering, and taking the filtrate, thereby obtaining the test sample solution;

[0017] (2) detecting the test sample solutions according to the construction method of the characteristic chromatogram of Chinese toona, thereby obtaining the characteristic chromatogram of Chinese toona;

[0018] (3) importing the obtained characteristic chromatogram into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system to establish a Chinese toona control chromatogram.

[0019] The application further provides a method for distinguishing genuine and fake Chinese toona, which comprises the following steps:

[0020] (1) preparing a test sample solution: adding 70% methanol solution into the medicinal material powder to be distinguished, heating and refluxing for 30-120 minutes, cooling, supplementing the lost weight with 70% methanol, filtering, evaporating the filtrate, dissolving, extracting with ethyl acetate by shaking, evaporating the ethyl acetate layer, dissolving with 70% methanol solution, filtering, and taking the filtrate, thereby obtaining the test sample solution;

[0021] (2) preparing a control sample solution: taking 4-hydroxybenzoic acid, vanillic acid, toona acuminata ketone, ferulic acid and iron shi ketone, and adding a solvent to prepare the control sample solution;

[0022] (3) ultra-high performance liquid chromatography detection: respectively taking the test sample solution or the control sample solution, injecting into an ultra-high performance liquid chromatograph, performing ultra-high performance liquid chromatography detection, and recording the chromatogram;

[0023] (4) analysis: selecting peaks with stable relative retention time as characteristic peaks in the chromatogram, comparing with the peaks of the control sample solution chromatogram, and determining 15 peaks; taking 4-hydroxybenzoic acid as the control sample, peak 1 is the chromatographic peak of 4-hydroxybenzoic acid; taking vanillic acid as the control sample, peak 2 is the chromatographic peak of vanillic acid; taking toona acuminata ketone as the control sample, peak 3 is the chromatographic peak of toona acuminata ketone; taking ferulic acid as the control sample, peak 6 is the chromatographic peak of ferulic acid; and taking iron shi ketone as the control sample, peak 13 is the chromatographic peak of iron shi ketone;

[0024] (5) distinguishing: respectively comparing the chromatogram of the test sample solution with the chromatogram of the control sample solution; when peak 3 exists in the chromatogram of the test sample solution, the test sample is Chinese toona; and when peak 3 does not exist in the chromatogram of the test sample solution, the test sample is Chinese toona bark.

[0025] Wherein, the peak corresponding to the euphorbia ketone reference peak in step (4) is the S peak, the relative retention time of peaks 1-2, 4-15 and the S peak should be within ±10% of the specified value, and the specified values of peaks 1-15 are 0.47, 0.70, 1.08, 1.23, 1.28, 1.35, 1.37, 1.47, 1.70, 1.92, 1.93, 2.15, 2.29, and 2.30, respectively.

[0026] Wherein, the chromatographic conditions of the ultra performance liquid chromatography are as follows: a chromatographic column: CORTECS T3; a sample injection amount: 1ul; a flow rate: 0.25-0.35ml / min; a column temperature: 25-35 DEG C; a detection wavelength: 254nm; gradient elution is performed with acetonitrile as mobile phase A and 0.1% formic acid solution as mobile phase B.

[0027] Wherein, the elution gradient of the ultra performance liquid chromatography is as follows: 0-5min, the volume fraction of mobile phase A is 5%, and the volume fraction of mobile phase B is 95%; 5-15min, the volume fraction of mobile phase A is 5→11%, and the volume fraction of mobile phase B is 95→89%; 15-22min, the volume fraction of mobile phase A is 11→17%, and the volume fraction of mobile phase B is 89→83%; 22-25min, the volume fraction of mobile phase A is 17%, and the volume fraction of mobile phase B is 83%; 25-40min, the volume fraction of mobile phase A is 17→33%, and the volume fraction of mobile phase B is 83→67%; 40-42min, the volume fraction of mobile phase A is 33→80%, and the volume fraction of mobile phase B is 67→20%; 42-44min, the volume fraction of mobile phase A is 80%, and the volume fraction of mobile phase B is 20%; 44-45min, the volume fraction of mobile phase A is 80→5%, and the volume fraction of mobile phase B is 20→95%.

[0028] Wherein, the stationary phase is octadecylsilane bonded silica gel as a filler.

[0029] Preferably, the chromatographic column in step (3) is CORTECS T3; the flow rate is 0.3ml per minute; and the column temperature is 30 DEG C.

[0030] Advantages: compared with the prior art, the present application has the following remarkable advantages: 1, the present application adopts UPLC chromatographic method, the chromatographic information is rich, and the precision and reproducibility are good; 2, the UPLC characteristic spectrum of the chelidonium majus L. constructed by the present application has the advantages of strong specificity, high stability and good reproducibility, and fully exhibits the characteristic differences of chelidonium majus L. and pseudo-product canarium album; 3, the present application can effectively identify chelidonium majus L. and pseudo-product, and can be used for quality evaluation, realizes the purpose of effectively identifying chelidonium majus L. and pseudo-product, and is helpful to perfect the quality standard of chelidonium majus L. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 Figure 1 is a trait map of Toona sinensis bark and Toona sinensis bark;

[0032] Figure 2 Figure 2 is a map of the influence of different chromatographic columns on separation effect;

[0033] Figure 3 Figure 3 is a map of the influence of different column temperatures on separation effect;

[0034] Figure 4 Figure 4 is a map of the influence of different flow rates on separation effect;

[0035] Figure 5 Figure 5 is a specificity investigation map of Toona sinensis bark;

[0036] Figure 6 Figure 6 is a UPLC map of the investigation of different extraction solvents;

[0037] Figure 7 Figure 7 is a UPLC map of the investigation of different extraction methods;

[0038] Figure 8 Figure 8 is a UPLC map of the investigation of different extraction times;

[0039] Figure 9 Figure 9 is a superimposed map of characteristic chromatograms of Toona sinensis bark;

[0040] Figure 10 Figure 10 is a superimposed map of characteristic chromatograms of Toona sinensis bark;

[0041] Figure 11 Figure 11 is a comparison map of characteristic chromatograms of Toona sinensis bark and Toona sinensis bark;

[0042] Figure 12 Figure 12 is a reference chromatogram of Toona sinensis bark. DETAILED DESCRIPTION

[0043] The technical solutions of the present application will be further described below in combination with the drawings.

[0044] Instruments, reagents and samples

[0045] Instruments: Thermo Vanquish Flex ultra-high performance liquid chromatograph (Thermo Corporation); chameleon 7.2 workstations (Thermo Corporation); ME204E / 02 electronic analytical balance (Mettler-Toledo Instruments (Shanghai) Co., Ltd.); GKC114 temperature-controlled water bath (Nantong Huatai Experimental Instrument Co., Ltd.).

[0046] Reagents: Acetonitrile (chromatographically pure, Thermo Fisher); Milli-Q pure water system (Millipore); formic acid (chromatographically pure, aladdin); water is ultrapure water; ethyl acetate (analytically pure, National Pharmaceutical Group Chemical Reagent Co., Ltd.); methanol (analytically pure, National Pharmaceutical Group Chemical Reagent Co., Ltd.).

[0047] Chloranthus bungeanus control drug material (batch number: 121481-201304) was purchased from China Institute for Drug Control; Chloranthus bungeanus samples were purchased from various local production areas and mainstream markets across the country; both are the dried root bark or dry bark of Ailanthus altissima (Mill.) Swingle of the Chloranthaceae family, see Table 1.

[0048] Table 1: Chloranthus bungeanus sample, Toona sinensis bark sample and source:

[0049]

[0050]

[0051] Reference substance:

[0052] 4-Hydroxybenzoic acid (batch number: 101149-202204) was purchased from China Institute for Drug Control, with a purity of 100%; vanillic acid (batch number: 110776-202204) was purchased from China Institute for Drug Control, with a purity of 99.8%; ailanthone (batch number: 16566) was purchased from Shidande Biotechnology Co., Ltd., with a purity of 98.0%; ferulic acid (batch number: 110773-201915) was purchased from China Institute for Drug Control, with a purity of 99.4%; and ferulic acid (batch number: PS012755) was purchased from Chengdu Pus Biological Technology Co., Ltd., with a purity of 98.0%.

[0053] Preparation of reference solution: 4-Hydroxybenzoic acid, vanillic acid, ailanthone, ferulic acid, and ferulic acid were added to 70% methanol to prepare a solution containing 30 μg of each of 4-hydroxybenzoic acid, vanillic acid, ailanthone, ferulic acid, and ferulic acid per 1 ml.

[0054] Preparation of control drug material solution: 2 g of chloranthus bungeanus control drug material powder was placed in a conical flask with a stopper, 25 ml of 70% methanol was added, the flask was tightly sealed, the weight was determined, and the flask was heated to reflux for 1 hour. After cooling, the weight was determined again, the lost weight was made up with 70% methanol, and the mixture was shaken and filtered. The filtrate was evaporated to dryness, the residue was dissolved in 25 ml of water, and the solution was extracted with 50 ml of ethyl acetate. The ethyl acetate layer was evaporated to dryness, the residue was dissolved in 70% methanol, and the solution was transferred to a 5 ml volumetric flask. The volume was adjusted to the mark with 70% methanol, the mixture was shaken and filtered, and the filtrate was obtained.

[0055] Example 1 System suitability investigation

[0056] 1. Different chromatographic columns

[0057] Take 2 g of Tongpi medicinal material powder with batch number S1, place it in a conical flask with a plug, add 25 ml of 70% methanol, seal it, weigh it, heat it to reflux for 1 hour, cool it, weigh it again, make up the weight loss with 70% methanol solution, shake it well, filter it, take the filtrate, evaporate it to dryness, dissolve the residue in 25 ml of water, extract it with 50 ml of ethyl acetate, take the ethyl acetate layer, evaporate it to dryness, dissolve the residue in 70% methanol, transfer it to a 5 ml volumetric flask, add 70% methanol to the mark, shake it well, filter it, take the filtrate, and you get the Tongpi medicinal material test solution.

[0058] Take the Tongpi medicinal material test solution, and select the commonly used chromatographic columns CORTECS T3 (2.1 mm x 150 mm, 1.6 μm), HSS T3 (2.1 mm x 100 mm, 1.8 μm), and BEH shield RP 18 (2.1 mm x 100 mm, 1.7 μm) respectively, and the UPLC chromatographic conditions are as follows: stationary phase: octadecylsilane bonded silica gel as the filler; mobile phase: acetonitrile as the mobile phase A, 0.1% formic acid solution as the mobile phase B, gradient elution: 0-5 min, 5% A, 5-15 min, 5→11% A, 15-22 min, 11→17% A, 22-25 min, 17% A, 25-40 min, 17→33% A, 40-42 min, 33→80% A, 42-44 min, 80% A, 44-45 min, 80→5% A; detection wavelength: 254 nm; flow rate: 0.30 ml per minute; column temperature: 30°C, injection volume: 1 μl, and determine the separation effect of different chromatographic columns, see Figure 2 .

[0059] The results show that the CORTECS T3 (2.1 mm x 150 mm, 1.6 μm) chromatographic column has the best separation effect on the sample, the peak information is rich, the baseline is smooth, and the spectrum is beautiful, so this chromatographic column is finally selected.

[0060] 2. Different column temperatures

[0061] Take the batch number S1 chun skin medicinal materials, prepared according to the above method of test solution, using the same chromatographic column (CORTECS T3 (2.1 mm x 150 mm, 1.6 μm)) and ultra high performance liquid chromatograph, UPLC chromatographic conditions as follows: stationary phase: octadecylsilane bonded silica gel as the filler; mobile phase: acetonitrile as mobile phase A, 0.1% formic acid solution as mobile phase B, gradient elution: 0-5 min, 5% A, 5-15 min, 5→11% A, 15-22 min, 11→17% A, 22-25 min, 17% A, 25-40 min, 17→33% A, 40-42 min, 33→80% A, 42-44 min, 80% A, 44-45 min, 80→5% A; detection wavelength: 254 nm; flow rate: 0.30 ml per minute, injection volume: 1 μl, respectively, at different column temperature (25 ℃, 30 ℃, 35 ℃) under injection, record chromatogram, see Figure 3 .

[0062] The results show that the column temperature in the range of 25 ℃-35 ℃, each chromatographic peak separation is good, good durability. Combined with the actual situation, choose 30 ℃ column temperature for analysis.

[0063] 3, different flow rate investigation

[0064] Take the batch number S1 chun skin medicinal materials, prepared according to the above method of test solution, using the same chromatographic column (CORTECS T3 (2.1 mm x 150 mm, 1.6 μm)) and ultra high performance liquid chromatograph, UPLC chromatographic conditions as follows: stationary phase: octadecylsilane bonded silica gel as the filler; mobile phase: acetonitrile as mobile phase A, 0.1% formic acid solution as mobile phase B, gradient elution: 0-5 min, 5% A, 5-15 min, 5→11% A, 15-22 min, 11→17% A, 22-25 min, 17% A, 25-40 min, 17→33% A, 40-42 min, 33→80% A, 42-44 min, 80% A, 44-45 min, 80→5% A; detection wavelength: 254 nm; column temperature: 30 ℃, injection volume: 1 μl, respectively, with different flow rate 0.25 ml / min, 0.30 ml / min, 0.35 ml / min, determination, record chromatogram, see Figure 4 .

[0065] The results show that the flow rate in the range of 0.25 ml / min-0.35 ml / min, each chromatographic peak separation is good, good durability. Small flow rate change meets the system applicability requirements, fixed flow rate is 0.30 ml / min.

[0066] In summary, the most preferred conditions for chromatography are determined as follows: CORTECS T3 is used (column length is 150 mm, inner diameter is 2.1 mm, and particle size is 1.6 μm); acetonitrile is used as mobile phase A, and 0.1% formic acid solution is used as mobile phase B, gradient elution is performed according to the provisions in Table 1; the flow rate is 0.3 ml per minute; the column temperature is 30 °C; the detection wavelength is 254 nm; and the injection amount is 1 μl. The theoretical plate number should not be less than 5000 according to the calculation of the jujubone peak.

[0067] Table 2 Gradient elution ratio

[0068]

[0069] 4. Specificity investigation

[0070] Respectively, 1 μL of blank solvent (70% methanol) and test sample solution (2 g of Ailanthus altissima (Mill.) Swingle powder was placed in a conical flask with a plug, 25 ml of 70% methanol was added, the weight was determined, heated to reflux for 1 hour, cooled, the weight was determined again, the lost weight was made up with 70% methanol, shaken, filtered, the filtrate was evaporated to dryness, the residue was dissolved in 25 ml of water, 50 ml of ethyl acetate was used for extraction by shaking, the ethyl acetate layer was evaporated to dryness, the residue was dissolved in 70% methanol, transferred to a 5 ml volumetric flask, 70% methanol was added to the calibration mark, shaken, filtered, and the filtrate was obtained) was injected into a liquid chromatograph (the chromatographic conditions were the most preferred conditions determined above), and the results are shown in Table 5. Figure 5 The solvent does not interfere with the determination of the characteristic chromatogram of Ailanthus altissima (Mill.) Swingle medicinal materials.

[0071] 5. Stability investigation

[0072] 2 g of Ailanthus altissima (Mill.) Swingle powder was placed in a conical flask with a plug, 25 ml of 70% methanol was added, the weight was determined, heated to reflux for 1 hour, cooled, the weight was determined again, the lost weight was made up with 70% methanol, shaken, filtered, the filtrate was evaporated to dryness, the residue was dissolved in 25 ml of water, 50 ml of ethyl acetate was used for extraction by shaking, the ethyl acetate layer was evaporated to dryness, the residue was dissolved in 70% methanol, transferred to a 5 ml volumetric flask, 70% methanol was added to the calibration mark, shaken, filtered, and the filtrate was obtained. The test sample solution was injected at 0, 2, 4, 8, 12, and 24 h, respectively, and the peak corresponding to jujubone was taken as the S peak, and the relative retention time and relative peak area of peaks 1-2 and peaks 4-15 were calculated, and the results are shown in Tables 3 and 4. The chromatographic conditions were the most preferred conditions determined above.

[0073] Table 3 Stability experiment results (relative retention time)

[0074]

[0075]

[0076] Table 4 Stability experiment results (relative peak area)

[0077]

[0078] The results show that the relative retention time RSD of each characteristic peak is less than 1%, and the relative peak area is less than 3%, indicating that the stability of the sample within 24 hours is good.

[0079] 6. Repeatability investigation

[0080] Take about 2 g of Chinese tallow tree bark powder (No. S1) and place it in a conical flask with a stopper. Add 25 ml of 70% methanol, seal it tightly, and weigh it. Heat it to reflux for 1 hour, cool it, and weigh it again. Make up the weight loss with 70% methanol, shake it well, filter it, and evaporate the filtrate to dryness. Dissolve the residue in 25 ml of water, extract it with 50 ml of ethyl acetate, evaporate the ethyl acetate layer to dryness, dissolve the residue in 70% methanol, transfer it to a 5 ml volumetric flask, add 70% methanol to the mark, shake it well, filter it, and take the filtrate as the test sample solution. Prepare 6 test sample solutions in parallel according to the above method, inject 1 μl each time, take the peak corresponding to the eurysolone as the S peak, calculate the relative retention time and relative peak area of peaks 1-2 and peaks 4-15, and the results are shown in Tables 5 and 6. The chromatographic conditions are the above-mentioned optimal conditions.

[0081] Table 5 Repeatability experiment results (relative retention time)

[0082]

[0083]

[0084] Table 6 Repeatability experiment results (relative peak area)

[0085]

[0086] The results show that the relative retention time RSD of each characteristic peak is less than 1%, and the relative peak area is less than 4%, indicating that the repeatability of the sample is good.

[0087] Example 2 Influence of test sample solution preparation

[0088] 1. Investigation of extraction solvent

[0089] Take the powder of the wood of the Chinese Tallow tree (No. S1) about 2g, put it in a conical flask with a stopper, add water, 30% methanol solution, 50% methanol solution, 70% methanol solution and methanol respectively, 25ml each, heat to reflux for 1 hour, cool down, shake well, filter, evaporate the filtrate to dryness, add 25ml water to the residue to dissolve, extract with 50ml ethyl acetate by shaking, evaporate the ethyl acetate layer to dryness, dissolve the residue with 70% methanol, transfer to a 5ml volumetric flask, add 70% methanol to the calibration mark, shake well, filter, take the filtrate as the test sample solution.

[0090] Determine the characteristic chromatogram of the wood of the Chinese Tallow tree under different extraction solvents according to the optimal chromatographic conditions above, see Figure 6 . The results show that the chromatographic peak information is comprehensive and the peak shape is good when the extraction solvent is 70% methanol, so 70% methanol is selected as the extraction solvent.

[0091] 2. Investigation of extraction method

[0092] Take the powder of the wood of the Chinese Tallow tree (No. S1) about 2g, put it in a conical flask with a stopper, add 70% methanol solution 25ml, tightly stopper, extract by ultrasonic treatment (power 250W, frequency 40kHz), shaking and heating to reflux respectively for 1 hour, cool down, shake well, filter, evaporate the filtrate to dryness, add 25ml water to the residue to dissolve, extract with 50ml ethyl acetate by shaking, evaporate the ethyl acetate layer to dryness, dissolve the residue with 70% methanol, transfer to a 5ml volumetric flask, add 70% methanol to the calibration mark, shake well, filter, take the filtrate as the test sample solution.

[0093] Determine the characteristic chromatogram of the wood of the Chinese Tallow tree under different extraction methods according to the optimal chromatographic conditions determined in Example 1, see Figure 7 . The results show that the chromatographic peak information is comprehensive and the peak shape is good when the extraction method is reflux extraction, so reflux extraction is selected as the extraction method.

[0094] 3. Investigation of extraction time

[0095] Take the powder of the wood of the Chinese Tallow tree (No. S1) about 2g, put it in a conical flask with a stopper, add 70% methanol solution 25ml, tightly stopper, extract by reflux for 30 minutes, 60 minutes, 90 minutes or 120 minutes respectively, cool down, shake well, filter, evaporate the filtrate to dryness, add 25ml water to the residue to dissolve, extract with 50ml ethyl acetate by shaking, evaporate the ethyl acetate layer to dryness, dissolve the residue with 70% methanol, transfer to a 5ml volumetric flask, add 70% methanol to the calibration mark, shake well, filter, take the filtrate as the test sample solution.

[0096] Determine the characteristic chromatogram of the wood of the Chinese Tallow tree under different extraction times according to the optimal chromatographic conditions determined in Example 1, see Figure 8The results showed that the extraction efficiency of the four different extraction times was similar. Considering the extraction sufficiency and reasonable time, the extraction time was finally determined to be 1 h.

[0097] In summary, the optimal test sample preparation method was determined as follows: 2 g of the powder was placed in a conical flask with a plug, 25 ml of 70% methanol was added, the weight was determined, and heated to reflux for 1 h. After cooling, the weight was determined again, the lost weight was made up with 70% methanol, and the mixture was shaken and filtered. The filtrate was evaporated to dryness, the residue was dissolved in 25 ml of water, and extracted with 50 ml of ethyl acetate. The ethyl acetate layer was evaporated to dryness, the residue was dissolved in 70% methanol, transferred to a 5 ml volumetric flask, and made up to the mark with 70% methanol. The mixture was shaken and filtered, and the filtrate was obtained.

[0098] Example 3: Assignment of characteristic peaks of Toona sinensis peel and establishment of UPLC characteristic chromatogram

[0099] According to the optimal preparation method determined in Example 2, 2.0 g of each sample was prepared into a test sample, and the sample characteristic chromatogram was determined under the optimal chromatographic conditions determined in Example 1. The characteristic chromatograms of 18 batches of Toona sinensis peel samples were superimposed using the "Chinese Medicine Chromatographic Characteristic Chromatogram Similarity Evaluation System (2012.0 version)" issued by the National Pharmacopoeia Committee, as shown in Table 7 and Table 8 (peak 1, peak 2, peak 3, peak 6, peak 13 are respectively 4-hydroxybenzoic acid, vanillic acid, Toona sinensis peel, ferulic acid, and iron shedmiketone). Figure 9 Figure 10 The characteristic chromatograms of 3 batches of Toona sinensis peel samples were superimposed, as shown in Table 7 and Table 8 (peak 1, peak 2, peak 3, peak 6, peak 13 are respectively 4-hydroxybenzoic acid, vanillic acid, Toona sinensis peel, ferulic acid, and iron shedmiketone).

[0100] Table 7: Determination results of 18 batches of Toona sinensis peel samples (relative retention time)

[0101]

[0102]

[0103] Table 8: Determination results of 18 batches of Toona sinensis peel samples (relative peak area)

[0104]

[0105] ​The test sample chromatography presented 15 characteristic peaks, which should correspond to the retention time of 15 characteristic peaks in the reference chromatography of the control material of chinaberry bark. Peak 3 corresponded to the retention time of the reference chromatography peak of the control substance of chinaberry ketone. The peak corresponding to the reference chromatography peak of the control substance of chinaberry ketone was S peak. The relative retention times of peaks 1-2, 4-15 and S peak were calculated. The relative retention times should be within ±10% of the specified values. The specified values were: 0.47 (peak 1), 0.70 (peak 2), 1.08 (peak 4), 1.23 (peak 5), 1.28 (peak 6), 1.35 (peak 7), 1.37 (peak 8), 1.47 (peak 19), 1.70 (peak 10), 1.92 (peak 11), 1.93 (peak 12), 2.15 (peak 13), 2.29 (peak 14), 2.30 (peak 15).

[0106] Example 4 Identification of chinaberry bark and Chinese toona bark medicinal materials

[0107] According to the method described in Example 3, the medicinal materials of chinaberry bark (batch number: S1) and Chinese toona bark (batch number: S18) were compared using the "Traditional Chinese Medicine Chromatographic Characteristic Similarity Evaluation System (2012.0 version)" issued by the National Pharmacopoeia Committee. The results showed that, by taking chinaberry bark as the reference, the control chromatogram of chinaberry bark medicinal material and the control chromatogram of Chinese toona bark medicinal material were compared. Chinese toona bark had no peak 3 (chinaberry ketone). By the presence or absence of peak 3, chinaberry bark and Chinese toona bark medicinal materials could be distinguished. The comparison of the characteristic chromatograms of chinaberry bark and Chinese toona bark medicinal materials is shown in Figure 11 ; and the identification of the control substance of chinaberry bark is shown in Figure 12 . Among them, chinaberry bark and Chinese toona bark had common peaks of peak 4 and peak 6 (ferulic acid), and the rest were non-common peaks. Chinaberry bark had peaks 1 (4-hydroxybenzoic acid), 2 (vanillic acid), 3 (chinaberry ketone), 5, 7, 8, 10, 10, 11, 12, 13 (ferulic acid), 14, 15, while Chinese toona bark did not have the above peaks. Therefore, the characteristic chromatogram established by this study could distinguish chinaberry bark and Chinese toona bark medicinal materials. Since peak 3, chinaberry ketone, is a characteristic component of chinaberry bark and has strong specificity, chinaberry bark and Chinese toona bark can be distinguished by the presence or absence of peak 3.

Claims

1. A method for constructing a feature map of a tree bark, characterized in that, It comprises the following steps: (1) adding 70% methanol solution to the powder of the Chinese medicinal material of Toona sinensis Roem, heating and refluxing for 30-120 minutes, cooling, supplementing the lost weight with 70% methanol, filtering, evaporating the filtrate, dissolving, extracting with ethyl acetate, evaporating the ethyl acetate layer, dissolving with 70% methanol, filtering, and taking the filtrate as the test sample solution; (2) taking 4-hydroxybenzoic acid, vanillic acid, Toona sinensis ketone, ferulic acid and iron shi ketone, adding solvent to prepare the reference sample solution; (3) injecting the test sample solution and the reference sample solution into the ultra-high performance liquid chromatograph to obtain the characteristic chromatogram; the chromatographic column of the ultra-high performance liquid chromatograph is CORTECS T3, the inner diameter is 2.1 mm, the column length is 150 mm, and the particle size is 1.6 μm; the detection wavelength is 254 nm; acetonitrile is used as the mobile phase A, and 0.1% formic acid solution is used as the mobile phase B; the elution gradient is as follows: 0-5 min, the volume fraction of the mobile phase A is 5%, and the volume fraction of the mobile phase B is 95%; 5-15 min, the volume fraction of the mobile phase A is 5→11%, and the volume fraction of the mobile phase B is 95→89%; 15-22 min, the volume fraction of the mobile phase A is 11→17%, and the volume fraction of the mobile phase B is 89→83%; 22-25 min, the volume fraction of the mobile phase A is 17%, and the volume fraction of the mobile phase B is 83%; 25-40 min, the volume fraction of the mobile phase A is 17→33%, and the volume fraction of the mobile phase B is 83→67%; 40-42 min, the volume fraction of the mobile phase A is 33→80%, and the volume fraction of the mobile phase B is 67→20%; 42-44 min, the volume fraction of the mobile phase A is 80%, and the volume fraction of the mobile phase B is 20%; 44-45 min, the volume fraction of the mobile phase A is 80→5%, and the volume fraction of the mobile phase B is 20→95%; the characteristic chromatogram has 15 common peaks, 4-hydroxybenzoic acid is taken as the reference sample, peak 1 is the chromatographic peak of 4-hydroxybenzoic acid, vanillic acid is taken as the reference sample, peak 2 is the chromatographic peak of vanillic acid, Toona sinensis ketone is taken as the reference sample, peak 3 is the chromatographic peak of Toona sinensis ketone, ferulic acid is taken as the reference sample, peak 6 is the chromatographic peak of ferulic acid, iron shi ketone is taken as the reference sample, and peak 13 is the chromatographic peak of iron shi ketone; the peak corresponding to the Toona sinensis ketone reference peak is the S peak, the relative retention times of peaks 1-2 and peaks 4-15 and the S peak are calculated, and the relative retention times should all be within the range of ±10% of the specified values; the specified values of peaks 1-2 and peaks 4-15 are 0.47, 0.70, 1.08, 1.23, 1.28, 1.35, 1.37, 1.47, 1.70, 1.92, 1.93, 2.15, 2.29, and 2.30, respectively. The injection amount of the ultra-high performance liquid chromatograph in step (3) is 1 μl, the flow rate is 0.25-0.35 ml / min, and the column temperature is 25-35 ℃.

2. The method of claim 1, wherein, It comprises the following steps:

3. A method of establishing a reference map of the bark of a tree, characterized in that, (1) preparing multiple batches of test sample solutions of Toona sinensis Roem respectively; ​ The preparation method of the test sample solution comprises the following steps: adding 70% methanol solution into the powder of the material of Toona sinensis peel, heating and refluxing for 30-120 minutes, cooling, supplementing the lost weight with 70% methanol, filtering, evaporating the filtrate, dissolving, extracting with ethyl acetate, evaporating the ethyl acetate layer, dissolving with 70% methanol, filtering, and taking the filtrate as the test sample solution; (2) The test sample solution is detected according to the construction method of the characteristic spectrum of Toona sinensis peel in claim 1, and the characteristic spectrum of Toona sinensis peel is obtained; (3) The obtained characteristic spectrum is introduced into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system to establish a control spectrum of Toona sinensis peel.

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