Construction method of characteristic spectrum of rhizoma arisaematis and application of construction method in primordial identification of rhizoma arisaematis

Abstract

The invention relates to the technical field of traditional Chinese medicine identification and medicine analysis, in particular to a construction method of a specific chromatogram of rhizoma arisaematis and application of the specific chromatogram in primordial identification of the rhizoma arisaematis. The construction method of the specific chromatogram comprises the following steps: carrying out high performance liquid chromatography detection on a test solution, wherein a T3 chromatographic column is adopted; the mobile phase A is a mixed solution of methanol and acetonitrile, and the volume ratio of methanol to acetonitrile is 1: (0.8-1.2); the mobile phase B is an aqueous solution of acid, and the mass concentration of the contained acid is 0.08-0.12%; gradient elution (the volume concentration of the mobile phase A is maintained at 16% in 0-38 min, the volume concentration of the mobile phase A is increased from 16% to 30% in 38-50 min, and the volume concentration of the mobile phase A is maintained at 30% in 50-65 min) is adopted. The technical scheme provided by the invention has the advantages of intuitiveness, rapidness and accuracy, and the basis of the rhizoma arisaematis can be identified according to whether the characteristic peak exists or not.

Description

Technical Field

[0001] This application relates to the field of identification and analysis technology of traditional Chinese medicine, and in particular to the method of constructing characteristic spectra of Arisaema heterophyllum and its application in the identification of the origin of Arisaema heterophyllum. Background Technology

[0002] Arisaema heterophyllum is pungent, bitter, and warm in nature, and is toxic. It enters the liver, spleen, and lung meridians, and has the effects of dispersing nodules and reducing swelling. Externally, it is used to treat carbuncles, sores, and snake and insect bites. Modern pharmacology has found that it has therapeutic effects on stroke, epilepsy, and tetanus. Chemical composition studies have revealed that Arisaema heterophyllum mainly contains alkaloids, glycosides, amino acids, fatty acids, sterols, flavonoids, and lectins. Establishing a characteristic spectrum of Arisaema heterophyllum is of great significance for comprehensively controlling its internal quality and ensuring its efficacy and medication safety.

[0003] The 2020 edition of the Chinese Pharmacopoeia lists the dried tubers of *Arisaema erubescens* (Wall.) Schott, *Arisaema heterophylum* Bl., and *Arisaema amurense* Maxim. as origins of Arisaema. The quality and chemical composition of Arisaema from different origins vary significantly, and there is currently a situation in the market where Arisaema from different origins is mixed. To better ensure the efficacy and safety of Arisaema medicinal materials, it is necessary to develop methods for identifying the origin of Arisaema medicinal materials. Summary of the Invention

[0004] Based on this, one or more embodiments of this application provide a method for constructing a characteristic spectrum of Arisaema heterophyllum and its application in the identification of the origin of Arisaema heterophyllum. The characteristic spectrum presents the common peaks and characteristic peaks of Arisaema heterophyllum from different origins, and can identify different origins by the presence or absence of characteristic peaks.

[0005] One or more embodiments of this application provide a method for constructing a feature map of Araceae, including the following steps:

[0006] Arisaema heterophyllum was extracted with an extraction solvent to obtain the test solution.

[0007] The test solution was subjected to high performance liquid chromatography to construct a characteristic spectrum of the arisaema.

[0008] The conditions for high-performance liquid chromatography (HPLC) detection include: a T3 column; mobile phase A being a mixture of methanol and acetonitrile, wherein the volume ratio of methanol to acetonitrile is 1:(0.8-1.2); mobile phase B being an aqueous solution of acid, wherein the mass concentration of acid is 0.08%-0.12%; and gradient elution is used.

[0009] The gradient elution procedure includes: 0-38 min, the volume concentration of mobile phase A is maintained at 16%; 38-50 min, the volume concentration of mobile phase A is increased from 16% to 30%; 50-65 min, the volume concentration of mobile phase A is maintained at 30%.

[0010] Optionally, the acid includes at least one of formic acid, glacial acetic acid, and phosphoric acid.

[0011] Furthermore, the chromatographic column has a length of 140mm-160mm, an inner diameter of 2.8mm-3.2mm, and a packing particle size of 2.4μm-2.6μm.

[0012] In some embodiments, the method for constructing the feature map of Araceae satisfies at least one of the following conditions:

[0013] (1) The flow rate is 0.3 mL / min - 0.5 mL / min;

[0014] (2) The detection wavelength is 230nm-265nm;

[0015] (3) The column temperature is 30℃-40℃; and

[0016] (4) The injection volume is 1μL-10μL.

[0017] In some embodiments, the extraction solvent is an aqueous solution of an alcohol; optionally, the alcohol includes at least one of methanol and ethanol.

[0018] In some embodiments, the extraction method is heating reflux or ultrasound.

[0019] In some embodiments, the mass-to-volume ratio of the tested Arisaema heterophyllum to the extraction solvent is 0.8-1.2 g: 25 mL.

[0020] In some embodiments, the characteristic spectrum of the arisaema includes the following characteristic peaks: peak 2, peak 7 and peak 10; wherein peak 10 is isoxafen.

[0021] One or more embodiments of this application also provide the application of the above-described method for constructing the characteristic map of Arisaema in the identification of the origin of Arisaema.

[0022] Furthermore, the origins of the araceae include Arisaema erubescens (Wall.) Schott, Arisaema heterophylum Bl., and Arisaema amurense Maxim.

[0023] The characteristic spectra of different Araceae species include the following characteristic peaks: peak 2, peak 7 and peak 10; among which, peak 10 is isosulfanol.

[0024] When the origin of the arisaema is arisaema, the characteristic spectrum of the arisaema also includes peak 1, which is vesenacin II; further, it also includes peaks 3, 4, 5, 6, 8, 9 and 11; wherein, peak 6 is saftoside and peak 9 is vitexin;

[0025] When the original form of the arisaema is *Arisaema heterophyllum*, the characteristic spectrum of the arisaema also includes peak A; further, it also includes peaks B, C, and D; wherein peak D and peak 11 are of the same component;

[0026] When the origin of the Arisaema is Northeast Arisaema, the characteristic spectrum of the Arisaema also includes peaks a, b, and c.

[0027] The method for constructing the characteristic spectrum of Arisaema in this application is simple, stable, and highly specific, and can be used to evaluate the overall quality and stability of Arisaema.

[0028] The method for constructing the characteristic spectrum of Arisaema in this application has the advantages of being intuitive, fast, and accurate in the identification of the origin of Arisaema. Peak 1 is a unique peak of Arisaema, and peak A is a unique peak of Arisaema heterophyllum. The origin of Arisaema can be directly identified by the presence or absence of characteristic peaks. Attached Figure Description

[0029] 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.

[0030] Figure 1 This is a superimposed image of the characteristic spectra of 19 batches of Arisaema heterophyllum medicinal materials in Example 1;

[0031] Figure 2 The characteristic spectrum of Arisaema heterophyllum obtained in Example 1;

[0032] Figure 3 The total ion chromatogram and ultraviolet absorption chromatogram of the Arisaema heterophyllum test solution in Example 1 are shown below.

[0033] Figure 4 This is a superimposed image of the characteristic atlases of 15 batches of Arisaema heterophyllum in Example 1;

[0034] Figure 5 The characteristic spectrum of Arisaema heterophyllum obtained in Example 1 is shown below.

[0035] Figure 6 This is a superimposed image of the characteristic atlases of 15 batches of Araceae davidii from Northeast China in Example 1;

[0036] Figure 7 The reference feature map of Araceae Radix Australis obtained in Example 1;

[0037] Figure 8 Comparison of characteristic spectra of different primordial Arisaema obtained in Example 1; wherein Arisaema (AE-R), Arisaema heterophyllum (AH-R), and Arisaema davidii (AA-R);

[0038] Figure 9 The results of the specificity examination of the feature map construction method in Example 1;

[0039] Figure 10 This is a comparison chart of chromatographic detection results under different mobile phase B conditions in Example 2;

[0040] Figure 11 This is a comparison chart of chromatographic detection results under different column conditions in Example 3;

[0041] Figure 12 This is a comparison chart of chromatographic detection results under different detection wavelength conditions in Example 4;

[0042] Figure 13 This is a comparison chart of chromatographic detection results under different extraction solvent conditions in Example 5;

[0043] Figure 14 This is a comparison graph of the chromatographic detection results under different mobile phase A conditions in Comparative Example 1;

[0044] Figure 15 This is a comparison chart of the chromatographic detection results under different chromatographic column conditions in Comparative Example 2;

[0045] Figure 16 This is a comparison chart of the chromatographic detection results of different gradient elution programs in Comparative Example 3. Detailed Implementation

[0046] The present application is further described below with reference to embodiments and examples. 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 this application, and these equivalent forms also fall within the protection scope of the appended claims.

[0047] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this 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.

[0048] One or more embodiments of this application provide a method for constructing a feature map of Araceae, including the following steps:

[0049] Arisaema heterophyllum was extracted with an extraction solvent to obtain the test solution.

[0050] The test solution was analyzed by high performance liquid chromatography to construct a characteristic spectrum of Arisaema.

[0051] The conditions for high performance liquid chromatography (HPLC) detection include: a T3 column; mobile phase A is a mixture of methanol and acetonitrile, with a volume ratio of methanol to acetonitrile of 1:(0.8-1.2); mobile phase B is an aqueous solution of acid, containing an acid concentration of 0.08%-0.12% by mass; gradient elution is used.

[0052] The gradient elution program includes: 0-38 min, maintaining the volume concentration of mobile phase A at 16%; 38-50 min, increasing the volume concentration of mobile phase A from 16% to 30%; 50-65 min, maintaining the volume concentration of mobile phase A at 30%.

[0053] Optionally, the acid includes at least one of formic acid, glacial acetic acid, and phosphoric acid.

[0054] Furthermore, the chromatographic column has a length of 140mm-160mm, an inner diameter of 2.8mm-3.2mm, and a packing particle size of 2.4μm-2.6μm.

[0055] In some embodiments, the method for constructing the feature map of Araceae satisfies at least one of the following conditions:

[0056] (1) The flow rate is 0.3 mL / min - 0.5 mL / min;

[0057] (2) The detection wavelength is 230nm-265nm;

[0058] (3) The column temperature is 30℃-40℃; and

[0059] (4) The injection volume is 1μL-10μL.

[0060] In some embodiments, the extraction solvent is an aqueous solution of an alcohol; optionally, the alcohol includes at least one of methanol and ethanol.

[0061] In some embodiments, the extraction method is heating reflux or ultrasound.

[0062] In some embodiments, the mass-to-volume ratio of the tested Arisaema heterophyllum to the extraction solvent is 0.8-1.2 g: 25 mL.

[0063] In some embodiments, the characteristic spectrum of Arisaema includes the following characteristic peaks: peak 2, peak 7 and peak 10; wherein peak 10 is isoxafen.

[0064] In some embodiments, in the characteristic chromatogram of Arisaema heterophyllum, the chromatographic peak of shampooside is used as the reference peak S, and the relative retention time of each characteristic peak and peak S is calculated. The relative retention time should be within ±10% of the specified value, which is 0.72 (peak 2), 1.04 (peak 7) and 1.30 (peak 10).

[0065] One or more embodiments of this application also provide the application of the above-described method for constructing the characteristic map of Arisaema in the identification of the origin of Arisaema.

[0066] Furthermore, the origins of the araceae include Arisaema erubescens (Wall.) Schott, Arisaema heterophylum Bl., and Arisaema amurense Maxim.

[0067] The characteristic spectra of different Araceae species include the following characteristic peaks: peak 2, peak 7 and peak 10; among which, peak 10 is isosulfanol.

[0068] When the original source of Arisaema is Arisaema, the characteristic spectrum of Arisaema also includes peak 1, which is vesenacin II; further, it also includes peaks 3, 4, 5, 6, 8, 9 and 11; among them, peak 6 is saftaglycoside and peak 9 is vitexin;

[0069] When the original form of Arisaema heterophyllum is Arisaema heterophyllum, the characteristic spectrum of Arisaema heterophyllum also includes peak A; further, it also includes peaks B, C and D; among them, peak D and peak 11 are the same component;

[0070] When the original form of Arisaema heterophyllum is Arisaema heterophyllum from the northeast, the characteristic spectrum of Arisaema heterophyllum also includes peaks a, b, and c.

[0071] In some embodiments, when the original form of Arisaema is Arisaema heterophyllum, it exhibits at least 11 characteristic peaks. Using the shampooside chromatographic peak as reference peak S, the relative retention times of each characteristic peak and peak S are calculated. These relative retention times should be within ±10% of a specified value, which is: 0.50 (peak 1), 0.72 (peak 2), 0.78 (peak 3), 0.87 (peak 4), 0.93 (peak 5), 1.04 (peak 7), 1.20 (peak 8), 1.25 (peak 9), 1.30... (Peak 10), 1.57 (Peak 11); Using the isosulfanilidine chromatographic peak as the reference peak S, calculate the relative retention time of each characteristic peak and peak S. The relative retention time should be within ±10% of the specified value. The specified values ​​are: 0.39 (Peak 1), 0.56 (Peak 2), 0.60 (Peak 3), 0.67 (Peak 4), 0.71 (Peak 5), 0.77 (Peak 6), 0.80 (Peak 7), 0.93 (Peak 8), 0.96 (Peak 9), 1.20 (Peak 11).

[0072] In some embodiments, when the origin of Arisaema heterophyllum is Arisaema heterophyllum, it exhibits at least 7 characteristic peaks. Using isosulfanol as a reference peak S, the relative retention time of each characteristic peak and peak S is calculated. The relative retention time should be within ±10% of the specified value, which is: 0.33 (peak A), 0.56 (peak 2), 0.69 (peak B), 0.80 (peak 7), 0.87 (peak C), and 1.20 (peak D).

[0073] In some embodiments, when the origin of Arisaema is Arisaema heterophyllum, it exhibits at least 6 characteristic peaks. Using isosulfanol as a reference peak S, the relative retention time of each characteristic peak and peak S is calculated. The relative retention time should be within ±10% of the specified value, which is: 0.56 (peak 2), 0.80 (peak 7), 0.91 (peak a), 1.03 (peak b), and 1.12 (peak c).

[0074] In some embodiments, the method for constructing the feature map of Arisaema further includes:

[0075] Prepare a reference solution and perform high-performance liquid chromatography (HPLC) on the reference solution; the reference solution consists of a reference standard and a solvent.

[0076] Optionally, the reference standard includes at least one of shamfotaside and isoshamfotaside.

[0077] Optionally, the solvent includes at least one of methanol and ethanol.

[0078] Optionally, the concentration of shamfotaside or isoshamfotaside in the reference solution is 30 μg / mL to 50 μg / mL.

[0079] The following are some specific examples.

[0080] The instruments, reagents, and reagents used in the following specific embodiments are as follows:

[0081] Instruments: Thermo Ultra High Performance Liquid Chromatograph (Vanquish, Thermo Fisher Scientific), Waters Ultra High Performance Liquid Chromatograph (H-Class, Waters Technologies), Thermo Vanquish Flex Ultra High Performance Liquid Chromatograph-Thermo Fisher QE High Resolution Mass Spectrometer (Thermo Fisher Scientific), Waters HSS T3 column (3.0mm×150mm, 2.5μm), 1 / 1000 electronic analytical balance (JJ600, Changshu Shuangjie Test Instrument Factory), 0.001% electronic analytical balance (ME204E, Mettler Toledo), 0.1% electronic analytical balance (XP26, Mettler Toledo), electric thermostatic water bath (HWS-28, Shanghai Yiheng Technology Co., Ltd.), CNC ultrasonic cleaner (KQ-500DE, Kunshan Ultrasonic Instrument Co., Ltd.), ultrapure water system (Milli-Q Direct, Merck AG).

[0082] Reagents: Ethanol (Xilong Technology Co., Ltd.) and methanol (Xilong Technology Co., Ltd.) were analytical grade; phosphoric acid (Tianjin Kemeio Chemical Reagent Co., Ltd.), acetonitrile, and methanol (Merck AG) were all chromatographic grade; formic acid (Merck AG) was liquid chromatography-mass spectrometry grade; and water was ultrapure water (prepared in the laboratory).

[0083] Test reagents: Charfutaside reference standard (batch number: 111912-202204), isocharfutaside reference standard (batch number: DSTDY001901); 19 batches of Arisaema rhizome were identified as dried tubers of Arisaema erubescens (Wall.) Schott, 15 batches were identified as dried tubers of Arisaema heterophylum Bl., and 15 batches were identified as dried tubers of Arisaema amurense Maxim. See Table 1 for details of origin and batch number.

[0084] Table 1 Sample Information Table

[0085]

[0086] Example 1

[0087] 1.1 Preparation of reference solution

[0088] Take an appropriate amount of shamfotaside or isoshamfotaside reference standard and add 50% methanol to prepare a reference solution containing 40 μg of shamfotaside or isoshamfotaside per 1 ml.

[0089] 1.2 Preparation of the test solution

[0090] Take approximately 2.0g of Arisaema heterophyllum powder (passed through a No. 3 sieve), accurately weigh it, place it in a stoppered conical flask, accurately add 50ml of 70% methanol, weigh it, heat under reflux for 30min, cool it, weigh it again, replenish the lost weight with 70% methanol, centrifuge it, take 25ml of the supernatant, evaporate it to dryness, dissolve the residue in 70% methanol, transfer it to a 5ml volumetric flask, and dilute to the mark, shake well, filter it, and take the filtrate to obtain the final product.

[0091] 1.3 Ultra-high performance liquid chromatography detection

[0092] Nineteen batches of *Arisaema heterophyllum*, 15 batches of *Arisaema heterophyllum*, and 15 batches of *Arisaema heterophyllum* from Northeast China were collected from Table 1 to prepare test solutions. Both the reference solution and the test solutions were subjected to ultra-high performance liquid chromatography (UHPLC). A Waters HSS T3 column (3.0 mm × 150 mm, 2.5 μm) was used. Methanol:acetonitrile (1:1) was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B. Gradient elution was employed. The flow rate was 0.4 mL / min, the column temperature was 35℃, the detection wavelength was 265 nm, and the injection volume was 5 μL. The gradient elution program was as follows: 0-38 min, the volume concentration of mobile phase A was maintained at 16%; 38-50 min, the volume concentration of mobile phase A increased from 16% to 30%; 50-65 min, the volume concentration of mobile phase A was maintained at 30%.

[0093] 1.4 Construction of Feature Maps and Their Application in Origin Identification

[0094] 1.4.1 Arisaema erubescens (Wall.) Schott (a type of medicinal herb)

[0095] The overlay image of the characteristic atlases of Arisaema heterophyllum from 19 batches is shown below. Figure 1 Using the chromatographic peak of shampooside as the reference peak S, the relative retention times of each characteristic peak and peak S were calculated. The experimental results are shown in Table 2.

[0096] Table 2. Results of the determination of the characteristic spectra (relative retention times) of 19 batches of Arisaema heterophyllum.

[0097]

[0098] Nineteen batches of Arisaema heterophyllum characteristic chromatograms were matched using the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System," and a control chromatogram was generated based on the average. This established an AE-R (Adaptive Observational-Reflective) chromatogram for Arisaema heterophyllum. Figure 2Based on the test results of 19 batches of Arisaema heterophyllum medicinal materials, it was determined that the characteristic chromatogram of Arisaema heterophyllum medicinal materials should show 11 characteristic peaks. Taking the chromatogram peak of shampooside as the reference peak S, the relative retention time of each characteristic peak and peak S was calculated. The relative retention time should be within ±10% of the specified value. The specified values ​​are: 0.50 (peak 1), 0.72 (peak 2), 0.78 (peak 3), 0.87 (peak 4), 0.93 (peak 5), 1.04 (peak 7), 1.20 (peak 8), 1.25 (peak 9), 1.30 (peak 10), and 1.57 (peak 11). Using the isosulfanol chromatographic peak as the reference peak S, calculate the relative retention time of each characteristic peak and peak S. The relative retention time should be within ±10% of the specified value. The specified values ​​are: 0.39 (peak 1), 0.56 (peak 2), 0.60 (peak 3), 0.67 (peak 4), 0.71 (peak 5), 0.77 (peak 6), 0.80 (peak 7), 0.93 (peak 8), 0.96 (peak 9), and 1.20 (peak 11).

[0099] Compounds in the Araceae Radix test solution were analyzed by liquid chromatography-mass spectrometry (LC-MS), and secondary fragment ion matching was performed with spectral data from the local mass spectrometry database. Unknown compounds were identified by referring to relevant literature. Chromatographic conditions were the same as in "1.3 Ultra-high performance liquid chromatography detection," and mass spectrometry conditions are shown in Table 3.

[0100] Table 3 Mass Spectrometry Conditions

[0101]

[0102] The test solution was analyzed using the above-described liquid chromatography and mass spectrometry conditions. By comparing the retention times of the reference standard in liquid chromatography and by comparing the precise molecular weight and fragment ion content in mass spectrometry, four chemical components—vestaining II, succinyl glycoside, vitexin, and isosuccinyl glycoside—were identified in the characteristic chromatogram of Arisaema heterophyllum. The total ion chromatogram and ultraviolet absorption chromatogram of the test solution are shown below. Figure 3 Compound information is shown in Table 4.

[0103] Table 4 Mass Spectrometry Identification Results of Compounds in Araceae

[0104]

[0105] 1.4.2 Arisaema heterophylum Bl. (Medicinal herb)

[0106] Fifteen batches of Arisaema heterophyllum powder were used to prepare test solutions and injected for analysis. The overlay graph is shown below. Figure 4As shown. Seven common peaks with good peak shape and resolution were selected as characteristic peaks of the Arisaema heterophyllum characteristic chromatogram. The characteristic chromatograms of 15 batches of Arisaema heterophyllum were matched using the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System". The average was used to generate a control chromatogram. The Arisaema heterophyllum control characteristic chromatogram (AH-R) is shown below. Figure 5 As shown.

[0107] Based on the test results of 15 batches of Arisaema heterophyllum medicinal materials, it was determined that the characteristic spectrum of Arisaema heterophyllum medicinal materials should show 7 characteristic peaks. With isoxafen as the reference peak S, the relative retention time of each characteristic peak and peak S was calculated. The relative retention time should be within ±10% of the specified value. The specified values ​​are: 0.33 (peak A), 0.56 (peak 2), 0.69 (peak B), 0.80 (peak 7), 0.87 (peak C), and 1.20 (peak D).

[0108] 1.4.3 Northeastern Arisaema amurense Maxim.

[0109] Fifteen batches of Northeast China's Arisaema heterophyllum medicinal materials were used to prepare test solutions and injected for analysis. The overlay graph is shown below. Figure 6 As shown. Six common peaks with good peak shape and resolution were selected as characteristic peaks of the Northeast Arisaema heterophyllum characteristic chromatogram. The characteristic chromatograms of 15 batches of Northeast Arisaema heterophyllum were matched using the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System," and a control chromatogram was generated based on the average. The Northeast Arisaema heterophyllum control characteristic chromatogram (AA-R) is shown below. Figure 7 As shown.

[0110] Based on the test results of 15 batches of Northeast Arisaema medicinal materials, it was determined that the characteristic spectrum of Northeast Arisaema medicinal materials should show 6 characteristic peaks. With isoxafen as the reference peak S, the relative retention time of each characteristic peak and peak S was calculated. The relative retention time should be within ±10% of the specified value. The specified values ​​are: 0.56 (peak 2), 0.80 (peak 7), 0.91 (peak a), 1.03 (peak b), and 1.12 (peak c).

[0111] 1.4.4 Characteristic Atlas of Arisaema

[0112] Based on the determination results of characteristic maps of 19 batches of *Arisaema heterophyllum*, 15 batches of *Arisaema davidii*, and 15 batches of *Arisaema heterophyllum*, corresponding control characteristic maps were established. Comparisons of control characteristic maps of *Arisaema heterophyllum* from different origins were established, for example... Figure 8 As shown, the results indicate that the characteristic spectra of different primordia of Arisaema show at least three common peaks (peak 2, peak 7, and peak 10); Arisaema heterophyllum and Arisaema primordia also show at least one common peak (peak 11 / peak D).

[0113] 1.4.5 Identification of Arisaema heterophyllum from different origins

[0114] according to Figure 8 Peak 1 is a unique peak of *Arisaema heterophyllum*, and peak A is a unique peak of *Arisaema heterophyllum*. The presence or absence of these characteristic peaks can be used to visually distinguish different primordial *Arisaema* species.

[0115] 1.5 Methodological Examination

[0116] 1.5.1 Specificity Examination

[0117] Accurately pipette 5 μl each of the Arisaema test solution (AE-1), reference solution, and blank solvent, and inject them for analysis according to the chromatographic conditions described in section "1.3". The results show ( Figure 9 The test sample and the reference sample showed the same chromatographic peaks at the corresponding retention times, and the blank solvent did not interfere, indicating that the established method has good specificity.

[0118] 1.5.2 Precision Examination

[0119] Accurately pipette the same sample of Arisaema heterophyllum (AE-1) and repeat the injection and determination six times under the chromatographic conditions described in section "1.3". Using the shampooside peak as the reference peak S, calculate the relative retention time of each characteristic peak relative to peak S, and calculate the RSD value. The results show (Table 5) that the RSD values ​​of the relative retention times of each characteristic peak are all less than 1.0%, indicating good instrument precision.

[0120] Table 5. Results of Precision Study (Relative Retention Time)

[0121]

[0122] 1.5.3 Stability Test

[0123] Accurately pipette the same sample of Arisaema heterophyllum test solution (AE-1) and inject it at 0, 3, 6, 9, 15, and 24 hours according to the chromatographic conditions described in section "1.3". Using the shampoosin peak as the reference peak S, calculate the relative retention time of each characteristic peak relative to peak S, and calculate the RSD value. The results show (Table 6) that the RSD value of the relative retention time of each characteristic peak is less than 1.0%, indicating that the test solution has good stability within 24 hours.

[0124] Table 6. Stability Results of Arisaema Characteristic Spectra (Relative Retention Time)

[0125]

[0126] 1.5.4 Reproducibility Test

[0127] Six test solutions were prepared from the same batch of Arisaema (AE-1) powder according to the method described in section "1.2", and injected for determination under the chromatographic conditions described in section "1.3". Using the shampooside peak as the reference peak S, the relative retention times of each characteristic peak and peak S were calculated, and the RSD values ​​were also calculated. The results (Table 7) show that the RSD values ​​of the relative retention times of each characteristic peak were all less than 1.0%, indicating that the established method has good reproducibility.

[0128] Table 7. Repeatability Results of Arisaema Characteristic Maps (Relative Retention Time)

[0129]

[0130] 1.5.5 Durability Assessment

[0131] The effects of different flow rates (0.40±0.02 ml / min), different column temperatures (35±2℃), the same type of chromatographic column (Waters HSS T3, 3.0 mm × 150 mm, 2.5 μm), and different ultra-high performance liquid chromatographs (Thermo Vanquish, Waters H-Class Plus, Waters H-Class) on the characteristic chromatogram of Arisaema heterophyllum (AE-1) were investigated. Using the shampooside peak as the reference peak S, the relative retention times (RSDs) of each characteristic peak relative to peak S were calculated, and the RSD values ​​were also calculated. The results (Table 8) show that, using the different flow rates, different column temperatures, Waters HSS T3 (3.0 mm × 150 mm, 2.5 μm) chromatographic column, and different types of ultra-high performance liquid chromatographs, the RSD values ​​of the relative retention times of each characteristic peak were all less than 5.0%. The above results indicate that the method is well-suited to minor variations in flow rate and column temperature, as well as to different ultra-high performance liquid chromatographs. The characteristic chromatograms require a fixed Waters HSS T3 (3.0 mm × 150 mm, 2.5 μm) column, and the same type of column exhibits good durability.

[0132] Table 8. Results of the Durability Study of the Characteristic Maps of Arisaema (Relative Retention Time)

[0133]

[0134] Example 2

[0135] The test solution was prepared using the same method as in section 1.2, "Preparation of Test Solution," of Example 1. The *Arisaema heterophyllum* was detected using essentially the same method as in section 1.3, "Detection by Ultra-High Performance Liquid Chromatography," except that 0.1% glacial acetic acid and 0.1% formic acid were used as mobile phase B. The resulting chromatogram was compared with the chromatogram in Example 1 (using 0.1% phosphoric acid as mobile phase B). The comparison results are as follows: Figure 10 .according to Figure 10The comparison results show that the separation between chromatographic peaks is good under various acid conditions. It is preferable to use 0.1% phosphoric acid as mobile phase B, which results in a more stable baseline and better chromatographic peak separation and peak shape.

[0136] Example 3

[0137] The test solution was prepared using the same method as in section 1.2, "Preparation of Test Solution," of Example 1. The *Arisaema heterophyllum* was detected using essentially the same method as in section 1.3, "Detection by Ultra-High Performance Liquid Chromatography," except that a Waters HSS T3 column (3.0 mm × 150 mm, 1.8 μm) was used. The resulting chromatogram was compared with the chromatogram in Example 1 (Waters HSS T3 column (3.0 mm × 150 mm, 2.5 μm)). The comparison results are as follows: Figure 11 .according to Figure 11 The comparison results show that the T3 column can achieve good separation effect, and the Waters HSS T3 (3.0mm×150mm, 2.5μm) is preferred, with better resolution and peak shape of each characteristic peak.

[0138] Example 4

[0139] The test solution was prepared using the same method as in section 1.2, "Preparation of Test Solution," of Example 1. The *Arisaema heterophyllum* was detected using essentially the same method as in section 1.3, "Detection by Ultra-High Performance Liquid Chromatography," except that the detection wavelengths were 210 nm, 230 nm, 250 nm, 265 nm, 280 nm, 295 nm, and 310 nm. The resulting chromatograms were compared with those in Example 1 (detection wavelength 265 nm). The comparison results are as follows: Figure 12 .according to Figure 12 The comparison results show that the chromatographic baseline is relatively stable between 250nm and 310nm. Among them, the overall response of the chromatographic peak is higher at 265nm. Therefore, 265nm was selected as the detection wavelength for establishing the characteristic chromatogram of Arisaema heterophyllum.

[0140] Example 5

[0141] The test solution was prepared using essentially the same method as in section 1.2, "Preparation of Test Solution," of Example 1, except that the extraction solvent was changed to methanol, 50% methanol, or 30% methanol. The *Arisaema heterophyllum* medicinal material was detected using the same method as in section 1.3, "Ultra-High Performance Liquid Chromatography Detection." The resulting chromatograms were compared with those in Example 1 (where the extraction solvent for the test sample was 70% methanol). The comparison results are as follows: Figure 13 The comparison results of peak areas are shown in Table 9. According to... Figure 13It is evident that the characteristic peak shape is better when using an aqueous solution of alcohol as the extraction solvent. According to the comparison results in Table 9, using an aqueous solution of alcohol can achieve efficient extraction, and 70% methanol is preferred as the extraction solvent, resulting in a higher "total peak area / sample weight" value.

[0142] Table 9 Results of the investigation on the extraction solvent of Arisaema heterophyllum.

[0143]

[0144] Example 6

[0145] The test solution was prepared using essentially the same method as in section 1.2, "Preparation of Test Solution," of Example 1, except that the extraction method was ultrasonic extraction, with ultrasonic parameters of 300 W and 40 kHz for 30 minutes. The *Arisaema heterophyllum* medicinal material was detected using the same method as in section 1.3, "Ultra-High Performance Liquid Chromatography Detection." The resulting chromatogram was compared with the chromatogram in Example 1 (where the test sample was extracted by reflux). The peak area comparison results are shown in Table 10. According to the comparison results in Table 10, both reflux and ultrasonic extraction can achieve high-efficiency extraction, with reflux extraction being preferred, resulting in a higher "total peak area / sample weight" value.

[0146] Table 10 Results of the Investigation on the Extraction Methods of Arisaema Feature Maps

[0147]

[0148] Example 7

[0149] The test solution was prepared using essentially the same method as in section 1.2, "Preparation of Test Solution," of Example 1, except that the extraction time was 60 minutes, 90 minutes, and 120 minutes. The *Arisaema heterophyllum* was detected using the same method as in section 1.3, "Ultra-High Performance Liquid Chromatography Detection." The resulting chromatograms were compared with those in Example 1 (where the extraction time was 30 minutes), and the peak area comparison results are shown in Table 11. According to the comparison results in Table 11, the difference in "total peak area / sample weight" with increasing the extraction time is not significant, indicating that extraction was complete at 30 minutes. Therefore, an extraction time of 30 minutes is preferred.

[0150] Table 11 Results of the Investigation on the Extraction Time of Arisaema Feature Maps

[0151]

[0152] Comparative Example 1

[0153] The test solution was prepared using the same method as in section 1.2, "Preparation of Test Solution," of Example 1. The *Arisaema heterophyllum* was detected using essentially the same method as in section 1.3, "Detection by Ultra-High Performance Liquid Chromatography," except that mobile phase A was used with pure methanol, acetonitrile:methanol = 2:1, or acetonitrile:methanol = 4:1. The resulting chromatograms were compared with those in Example 1 (using acetonitrile:methanol = 1:1 as mobile phase A). The comparison results are as follows: Figure 14 .according to Figure 14 The comparison results show that using other mobile phases A leads to poorer resolution of characteristic peaks, fewer peaks, and uneven baselines. When using acetonitrile:methanol = 1:1 as mobile phase A, the peak shape and resolution of each characteristic peak are better, the baseline is stable, and the number of peaks is large.

[0154] Comparative Example 2

[0155] The test solution was prepared using the same method as in section 1.2, "Preparation of Test Solution," of Example 1. The *Arisaema heterophyllum* was detected using essentially the same method as in section 1.3, "Detection by Ultra-High Performance Liquid Chromatography," except that a Waters BEH C18 column (3.0 mm × 150 mm, 2.5 μm) was used. The resulting chromatograms were compared with those in Example 1 (Waters HSS T3 column (3.0 mm × 150 mm, 2.5 μm)) and Example 3 (Waters HSS T3 column (3.0 mm × 150 mm, 1.8 μm)). The comparison results are as follows: Figure 15 .according to Figure 15 The comparison results show that without using the T3 column, the characteristic peak resolution is worse, the number of peaks is reduced, and the baseline is uneven.

[0156] Comparative Example 3

[0157] The test solution was prepared using the same method as in section 1.2, "Preparation of Test Solution," of Example 1. The *Arisaema heterophyllum* was detected using essentially the same method as in section 1.3, "Detection by Ultra-High Performance Liquid Chromatography," except that the gradient elution program was adjusted as follows: 0-10 min, mobile phase A concentration maintained at 5%; 10-21 min, mobile phase A concentration increased from 5% to 19%; 21-42 min, mobile phase A concentration maintained at 19%; 42-56 min, mobile phase A concentration increased from 19% to 40%; 56-60 min, mobile phase A concentration maintained at 40%. The resulting chromatogram was compared with the chromatogram in Example 1 (gradient elution program: 0-38 min, mobile phase A concentration maintained at 16%; 38-50 min, mobile phase A concentration increased from 16% to 30%; 50-65 min, mobile phase A concentration maintained at 30%). The comparison results are as follows: Figure 16 ,according to Figure 16 The comparison results show that when using the gradient elution program of this comparative example, the peak shape and resolution of the chromatographic peaks in the 10-25 minute range are poor. Improving the resolution of these peaks would result in a longer analysis time, and since the peaks in this range are all unknown components, they are not retained. Using the gradient elution program of Example 1, the resolution and peak shape of each characteristic peak are better, and the baseline is more stable.

[0158] 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.

[0159] 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 can be used to interpret the content of the claims.

Claims

1. A method for constructing a characteristic map of Arisaema, characterized in that, The steps include the following: Arisaema heterophyllum was extracted with an extraction solvent to obtain the test solution. The test solution was subjected to high performance liquid chromatography to construct a characteristic spectrum of the arisaema. The conditions for high-performance liquid chromatography (HPLC) detection include: a T3 column; mobile phase A being a mixture of methanol and acetonitrile, wherein the volume ratio of methanol to acetonitrile is 1:(0.8-1.2); mobile phase B being an aqueous solution of acid, wherein the mass concentration of acid is 0.08%-0.12%; and gradient elution is used. The gradient elution procedure includes: 0-38 min, the volume concentration of mobile phase A is maintained at 16%; 38-50 min, the volume concentration of mobile phase A is increased from 16% to 30%; 50-65 min, the volume concentration of mobile phase A is maintained at 30%.

2. The method for constructing the characteristic map of Araceae according to claim 1, characterized in that, The acid includes at least one of formic acid, glacial acetic acid, and phosphoric acid.

3. The method for constructing the characteristic map of Araceae according to claim 1, characterized in that, The chromatographic column has a length of 140mm-160mm, an inner diameter of 2.8mm-3.2mm, and a packing particle size of 2.4μm-2.6μm.

4. The method for constructing the characteristic map of Arisaema according to any one of claims 1-3, characterized in that, At least one of the following conditions must be met: (1) The flow rate is 0.3 mL / min - 0.5 mL / min; (2) The detection wavelength is 230nm-265nm; (3) The column temperature is 30℃-40℃; and (4) The injection volume is 1μL-10μL.

5. The construction method according to any one of claims 1-3, characterized in that, The extraction solvent is an aqueous solution of an alcohol; optionally, the alcohol includes at least one of methanol and ethanol; and / or, The mass-to-volume ratio of the tested Arisaema heterophyllum to the extraction solvent is 0.8-1.2 g: 25 mL.

6. The construction method according to any one of claims 1-3, characterized in that, The extraction methods are heating and reflux or ultrasound.

7. The construction method according to any one of claims 1-3, characterized in that, The extraction time is 30-120 minutes.

8. The construction method according to any one of claims 1-3, characterized in that, The characteristic spectrum of the arisaema includes the following characteristic peaks: peak 2, peak 7 and peak 10; wherein, peak 10 is isoxafen.

9. The application of the method for constructing the characteristic spectrum of Arisaema as described in any one of claims 1-8 in the identification of the origin of Arisaema.

10. The application according to claim 9, characterized in that, The origins of the araceae include Arisaema erubescens (Wall.) Schott, Arisaema heterophylum Bl., and Arisaema amurense Maxim.; The characteristic spectra of different Araceae species include the following characteristic peaks: peak 2, peak 7 and peak 10; among which, peak 10 is isosulfanol. When the origin of the arisaema is arisaema, the characteristic spectrum of the arisaema also includes peak 1, which is vesenacin II; further, it also includes peaks 3, 4, 5, 6, 8, 9 and 11; wherein, peak 6 is saftoside and peak 9 is vitexin; When the original form of the arisaema is *Arisaema heterophyllum*, the characteristic spectrum of the arisaema also includes peak A; further, it also includes peaks B, C, and D; wherein peak D and peak 11 are of the same component; When the origin of the Arisaema is Northeast Arisaema, the characteristic spectrum of the Arisaema also includes peaks a, b, and c.

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