Construction method and application of characteristic chromatogram of water extract of acetoxy five-lingzhi fat

Abstract

The application discloses a construction method and application of a characteristic spectrum of a water extract of vinegar five-ingredient fat. The construction method of the characteristic spectrum of the water extract of the vinegar five-ingredient fat comprises the following steps: mixing the water extract of the vinegar five-ingredient fat with an extraction solvent to perform extraction, and preparing a test sample solution; performing high performance liquid chromatography analysis on the test sample solution to construct the characteristic spectrum of the water extract of the vinegar five-ingredient fat. The construction method of the characteristic spectrum of the water extract of the vinegar five-ingredient fat has high precision, repeatability and stability, can provide more rich characteristic peak information, and thus can more comprehensively reflect the quality of samples.

Description

Technical Field

[0001] This application belongs to the field of traditional Chinese medicine detection and analytical chemistry technology, and relates to a method for constructing a characteristic spectrum of a water extract of vinegar-processed *Wulingzhi* and its application. Background Technology

[0002] Vinegar-processed Wulingzhi is a traditional Chinese medicine prepared by processing Wulingzhi with vinegar. It is used to promote blood circulation, remove blood stasis, and relieve pain. Due to differences in the source, processing methods, and extraction process of Wulingzhi, the components of different batches of vinegar-processed Wulingzhi water extract vary greatly, affecting the stability of its therapeutic effects. Therefore, it is necessary to further improve and standardize the quality standards for vinegar-processed Wulingzhi preparations. Summary of the Invention

[0003] Based on this, one embodiment of this application provides a method for constructing a characteristic spectrum of the aqueous extract of vinegar-processed ginseng and its application, which can be used as a basis for quality detection and identification.

[0004] In some embodiments, a method for constructing a characteristic spectrum of a water extract of vinegar-processed Trogopterus xanthipes is provided, comprising the following steps:

[0005] The aqueous extract of vinegar-processed Trogopterus xanthipes was mixed with an extraction solvent to prepare a test solution.

[0006] The test solution was analyzed by high performance liquid chromatography to construct the characteristic chromatogram of the vinegar-processed Trogopterus dung water extract.

[0007] The conditions for high performance liquid chromatography analysis include: gradient elution; the mobile phase includes mobile phase A and mobile phase B, wherein mobile phase A is methanol or acetonitrile, and mobile phase B is a 0.2% formic acid aqueous solution or water;

[0008] The gradient elution procedure includes: 0 min to 5 min, the volume percentage of mobile phase A changes from 2% to 5%; 5 min to 15 min, the volume percentage of mobile phase A changes from 5% to 15%; 15 min to 25 min, the volume percentage of mobile phase A changes from 15% to 20%; 25 min to 50 min, the volume percentage of mobile phase A changes from 20% to 40%.

[0009] In some embodiments, the method for constructing the characteristic spectrum of the vinegar-processed Trogopterus xanthipes aqueous extract satisfies one or more of the following conditions:

[0010] (1) The extraction method is ultrasonic extraction or reflux extraction. Optionally, the ultrasonic extraction power is 200W~300W and the frequency is 30kHz~50kHz.

[0011] (2) The extraction time is 15 min to 45 min; and

[0012] (3) The extraction solvent includes one or more of methanol, a methanol aqueous solution with a mass fraction of 65% to 75%, a methanol aqueous solution with a mass fraction of 45% to 55%, and a methanol aqueous solution with a mass fraction of 25% to 35%.

[0013] In some embodiments, the method for constructing the characteristic spectrum of the vinegar-processed quinquefolia extract is provided, wherein the mass-volume ratio of the vinegar-processed quinquefolia extract to the extraction solvent is 0.2 g: (5~15) mL.

[0014] In some embodiments, the method for constructing the characteristic chromatogram of the vinegar-processed Trogopterus dung water extract includes high-performance liquid chromatography (HPLC) analysis under the following conditions: injection volume of 1 μL to 10 μL.

[0015] In some embodiments, the method for constructing the characteristic spectrum of the vinegar-processed Trogopterus dung water extract includes high-performance liquid chromatography (HPLC) analysis under the following conditions: a detection wavelength of 254 nm to 300 nm.

[0016] In some embodiments, the method for constructing the characteristic chromatogram of the vinegar-processed quinquefolium extract includes high-performance liquid chromatography (HPLC) analysis under the following conditions: column temperature of 30°C to 40°C.

[0017] In some embodiments, the method for constructing the characteristic spectrum of the vinegar-processed Trogopterus xanthipes water extract includes high-performance liquid chromatography (HPLC) analysis under the following conditions: flow rate of 0.25 mL / min to 0.35 mL / min.

[0018] In some embodiments, the method for constructing the characteristic spectrum of the vinegar-processed Trogopterus xanthipes aqueous extract includes the following steps:

[0019] Mix the vinegar-processed five-spice powder with water, soak, decoct, and filter to obtain the filtrate.

[0020] The filtrate was concentrated and dried to prepare the vinegar-based extract of *Wulingzhi*.

[0021] In some embodiments, the method for constructing the characteristic spectrum of the vinegar-processed quinquefolium extract includes a characteristic spectrum containing nine characteristic peaks.

[0022] In some embodiments, the method for constructing the characteristic spectrum of the vinegar-processed Trogopterus dung water extract is provided for its application in identifying vinegar-processed Trogopterus dung or its vinegar-processed Trogopterus dung water extract.

[0023] The aforementioned method for constructing the characteristic spectrum of the vinegar-processed quinquefolium extract has high precision, repeatability, and stability, and can provide more abundant characteristic peak information, thus more comprehensively reflecting the sample quality. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments and examples of this application, and to more completely understand this application and its beneficial effects, the accompanying drawings used in the description of the embodiments or examples will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of this application. Those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0025] Figure 1 Figure showing the results of the solvent extraction investigation;

[0026] Figure 2 The results of the extraction method examination are shown in the figure;

[0027] Figure 3 A graph showing the results of the time-based investigation;

[0028] Figure 4 The results of the investigation into the amount of extraction solvent are shown in the figure;

[0029] Figure 5 The results of the mobile phase investigation are shown in the figure.

[0030] Figure 6 The results of the wavelength detection investigation are shown in the figure.

[0031] Figure 7 The results of the column temperature test are shown in the figure.

[0032] Figure 8 The graph shows the results of the flow velocity investigation.

[0033] Figure 9 The results of the chromatographic column investigation are shown in the figure.

[0034] Figure 10 This is a superimposed image of the characteristic spectra of the water extract of vinegar-processed Trogopterus xanthipes.

[0035] Figure 11 This is a comparative chromatogram of the characteristic chromatogram of the water extract of vinegar-processed quinoa extract. Detailed Implementation

[0036] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Preferred embodiments of the invention are shown in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the invention.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0038] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0039] The terms "and / or," "or / and," and "and / or" as used in this application encompass any one of two or more 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. It should be noted that when at least three items are connected using at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes solutions connected by "logical AND," and also undoubtedly includes solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and "a combination of A and B."

[0040] In this application, the terms "multiple", "various", "multiple times", "multi-dimensional", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0041] The terms “combinations thereof,” “any combination thereof,” and “any combination thereof” as used in this application include all suitable combinations of any two or more of the listed items.

[0042] In this application, the term "suitable" as used in "suitable combination", "suitable method", "any suitable method", etc., refers to the ability to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0043] In this application, terms such as "preferred," "better," "more suitable," and "ideal" are merely used to describe implementation methods or embodiments that achieve better results, and should be understood not to limit the scope of protection of this application.

[0044] 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.

[0045] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0046] In this invention, the terms "first aspect," "second aspect," "third aspect," and "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," and "fourth," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0047] 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.

[0048] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected 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. In this document, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. 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.

[0049] 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 ±5℃, ±4℃, ±3℃, ±2℃, or ±1℃.

[0050] In this application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass-volume percentage.

[0051] In this application, "room temperature" generally refers to 5℃~30℃, and more preferably 25±5℃.

[0052] This application discloses a method for constructing a characteristic chromatogram of an aqueous extract of *Wu Ling Zhi* (a type of herb) and its application. The method for constructing the characteristic chromatogram of the aqueous extract includes the following steps: mixing the aqueous extract of *Wu Ling Zhi* with an extraction solvent for extraction to prepare a test solution; performing high-performance liquid chromatography (HPLC) analysis on the test solution to construct the characteristic chromatogram of the aqueous extract. The provided method for constructing the characteristic chromatogram of the aqueous extract of *Wu Ling Zhi* has high precision, repeatability, and stability, and can provide more abundant characteristic peak information, thus more comprehensively reflecting the sample quality.

[0053] In some embodiments, a method for constructing a characteristic spectrum of a water extract of vinegar-processed Trogopterus xanthipes is provided, comprising the following steps:

[0054] The aqueous extract of vinegar-processed Trogopterus xanthipes was mixed with an extraction solvent to prepare a test solution.

[0055] The test solution was analyzed by high performance liquid chromatography to construct the characteristic chromatogram of the vinegar-processed Wulingzhi water extract.

[0056] The conditions for high performance liquid chromatography analysis include: gradient elution; the mobile phase includes mobile phase A and mobile phase B, where mobile phase A is methanol or acetonitrile, and mobile phase B is a 0.2% formic acid aqueous solution or water.

[0057] The gradient elution program includes: 0 min to 5 min, the volume percentage of mobile phase A changes from 2% to 5%; 5 min to 15 min, the volume percentage of mobile phase A changes from 5% to 15%; 15 min to 25 min, the volume percentage of mobile phase A changes from 15% to 20%; 25 min to 50 min, the volume percentage of mobile phase A changes from 20% to 40%.

[0058] In some embodiments, the method for constructing the characteristic spectrum of the vinegar-processed Trogopterus xanthipes water extract involves extraction by ultrasonic extraction or reflux extraction.

[0059] In some embodiments, the method for constructing the characteristic spectrum of the vinegar-processed Trogopterus dung water extract uses ultrasonic extraction with a power of 200W~300W and a frequency of 30kHz~50kHz.

[0060] In some embodiments, the method for constructing the characteristic spectrum of the vinegar-processed quinquefolia extract involves an extraction time of 15 min to 45 min.

[0061] In some embodiments, the method for constructing the characteristic spectrum of the vinegar-derived Trogopterus dung water extract includes one or more of the following extraction solvents: methanol, a methanol aqueous solution with a mass fraction of 65% to 75%, a methanol aqueous solution with a mass fraction of 45% to 55%, and a methanol aqueous solution with a mass fraction of 25% to 35%.

[0062] In some embodiments, the method for constructing the characteristic spectrum of the vinegar-processed quinquefolia extract is provided in which the mass-volume ratio of the vinegar-processed quinquefolia extract to the extraction solvent is 0.2 g: (5~15) mL.

[0063] In some embodiments, the method for constructing the characteristic chromatogram of the vinegar-processed Trogopterus dung water extract includes high-performance liquid chromatography (HPLC) analysis under the following conditions: injection volume of 1 μL to 10 μL.

[0064] In some embodiments, the method for constructing the characteristic spectrum of the vinegar-processed Trogopterus dung water extract includes high-performance liquid chromatography (HPLC) analysis under the following conditions: a detection wavelength of 254 nm to 300 nm.

[0065] In some embodiments, the method for constructing the characteristic chromatogram of the vinegar-processed quinquefolium extract includes high-performance liquid chromatography (HPLC) analysis under the following conditions: column temperature of 30°C to 40°C.

[0066] In some embodiments, the method for constructing the characteristic spectrum of the vinegar-processed Trogopterus xanthipes water extract includes high-performance liquid chromatography (HPLC) analysis under the following conditions: flow rate of 0.25 mL / min to 0.35 mL / min.

[0067] In some embodiments, the method for constructing the characteristic spectrum of the vinegar-processed Trogopterus xanthipes aqueous extract includes the following steps:

[0068] Mix the vinegar-processed five-spice powder with water, soak, decoct, and filter to obtain the filtrate.

[0069] The filtrate was concentrated and dried to prepare a water extract of vinegar-processed five-spice powder.

[0070] In some embodiments, the method for constructing the characteristic spectrum of the vinegar-processed quinquefolium extract includes a characteristic spectrum containing nine characteristic peaks.

[0071] In some embodiments, the method for constructing a characteristic spectrum of the vinegar-processed Trogopterus dung water extract is used in the identification of vinegar-processed Trogopterus dung or the vinegar-processed Trogopterus dung water extract.

[0072] The following are specific embodiments. They are intended to provide a more detailed description of this application to help those skilled in the art and researchers better understand it. The technical conditions described do not constitute any limitation on this application. Any modifications made within the scope of the claims of this application are protected by the claims.

[0073] Unless otherwise stated, all raw materials and reagents used in the following examples are commercially available or can be prepared by known methods. Experimental methods not specifying particular conditions in the examples were performed under conventional conditions, such as those described in literature, books, or methods recommended by the manufacturer.

[0074] The instruments used in the examples are as follows: high-resolution mass spectrometer (UPLC-Q-ExactiveOrbitrap-MS, Thermo Fisher Scientific, USA), ultra-high performance liquid chromatograph (Waters H-Class, Waters Corporation, USA), 0.001 g balance (ME204E, Mettler Toledo), 0.1 g balance (XP26, Mettler Toledo), ultrapure water system (Milli-QDirect8 / 16 system, Merck), CNC ultrasonic cleaner (KQ5500DE, Kunshan Ultrasonic Instrument Co., Ltd.), mechanical split decoction pot (YMW, Chaozhou Gangdian Crafts Manufacturing Factory), rotary evaporator (Buchi R-100, Buchi GmbH, Switzerland), circulating water vacuum pump (SHZ-DШ, Gongyi Yuhua Instrument Co., Ltd.). Dissecting microscope (SZX7, OLYMPUS, Japan); CCD camera (VertA1, Shanghai Tusen Vision Technology Co., Ltd., China); Precision electronic balance (CP214, OHAUS, USA); 6-well plate (Zhejiang Beilanbo Biotechnology Co., Ltd., China); High-speed refrigerated centrifuge (Heraeus Fresco17, ThermoFisher, Germany); Cardiac blood flow analyzer (ZebraBlood 3.4, ViewPoint Life Sciences, France); Fully automated rapid sample grinder (JXFSTPRP-24L, Shanghai Jingxin Laboratory Equipment Technology Department, China).

[0075] The reagents used in the examples are as follows: dimethyl sulfoxide (DMSO, batch number I2229063, Shanghai Aladdin Biochemical Technology Co., Ltd., China); arachidonic acid (batch number H2303046, Shanghai Aladdin Biochemical Technology Co., Ltd., China); o-anisidine (batch number MKBX3619V, Sigma, USA); anhydrous sodium acetate (batch number E2317841, Shanghai Aladdin Biochemical Technology Co., Ltd., China); methylcellulose (batch number C2004046, Shanghai Aladdin Biochemical Technology Co., Ltd., China).

[0076] Aspirin Enteric-coated Tablets (batch number BJ72163, Bayer Healthcare Co., Ltd.); Verapamil Hydrochloride (batch number A2007039, Shanghai Aladdin Biochemical Technology Co., Ltd., China); Compound Danshen Dripping Pills (batch number 190203, Tasly Pharmaceutical Group Co., Ltd.). The processed Wulingzhi pieces were provided by Guangdong Yifang Pharmaceutical Co., Ltd. and identified by Professor Huang Haibo of Guangzhou University of Chinese Medicine. After being tested and meeting the pharmacopoeia standards, they were stored in the warehouse. Methanol and acetonitrile were of chromatographic grade, and other reagents were of analytical grade. All the reference standards used were purchased from institutions such as the National Institutes for Food and Drug Control, and their purity was greater than 98%. The grades of the reagents used all met the analytical requirements.

[0077] Example 1 Preparation of the aqueous extract of processed Wulingzhi

[0078] 1 Sample preparation:

[0079] Take 100 g of the processed Wulingzhi pieces, place them in an electric ceramic pot, and decoct with water twice. For the first decoction, add 8 times the amount of water, soak for 30 minutes, bring to a boil over high heat (500 W), and then keep simmering over low heat (200 W) for 30 minutes. Filter the decoction while it is hot through a 200-mesh sieve, and quickly cool the filtrate with cold water. For the second time, add 6 times the amount of water, bring to a boil over high heat (500 W), and then keep simmering over low heat (200 W) for 25 minutes. Filter the decoction while it is hot through a 200-mesh sieve, and quickly cool the filtrate with cold water. Combine the two decoctions. Transfer the decoction to a 5000 mL round-bottom flask, and concentrate it under reduced pressure and low temperature (temperature: 50 °C; vacuum: -0.10 MPa) using a rotary evaporator to obtain a 150 mL fluid extract; transfer it to a vacuum freeze dryer for freeze-drying, seal it, and determine the extraction rate.

[0080] 2 Determination of the extraction rate:

[0081] Precisely weigh 5 g of the concentrated solution, place it in a pre-weighed evaporating dish, evaporate it to dryness in a water bath, dry it at 105 °C for 3 hours, cool it in a desiccator for 30 minutes, quickly weigh it, and calculate the extraction rate according to the following formula.

[0082] Calculation formula: .

[0083] Take 10 batches of the processed Wulingzhi pieces to prepare samples according to the preparation method under "Sample preparation", and determine the extraction rate. The results are expressed as mean ± standard deviation (SD), which is 14.54 ± 3.00 %.

[0084] 3 Determination of the total terpene acid content

[0085] Preparation of the reference solution: Take an appropriate amount of ursolic acid reference substance, and make a solution containing about 0.3 mg of ursolic acid reference substance per 1 mL with 80% ethanol.

[0086] Preparation method of test solution: Take 0.2g of vinegar-processed quinoa extract, accurately weigh it, put it in a 100 mL Erlenmeyer flask, accurately add 50 mL of 80% ethanol, weigh it, sonicate (power 250W, frequency 50kHz) for 30 min, cool, filter, and take the filtrate to obtain the test solution.

[0087] Establishing the standard curve:

[0088] Accurately transfer 0.10, 0.20, 0.25, and 0.30 mL of the reference solution into 10 mL stoppered graduated test tubes, evaporate the solvent in a water bath, accurately add 0.2 mL of 5% vanillin-acetic acid solution and 0.8 mL of perchloric acid solution, shake well, heat in an 80 ℃ water bath for 15 min, cool under running water, accurately add 5 mL of acetic acid, shake well, and use 80% ethanol as a blank. Measure the absorbance at 545 nm wavelength using a UV spectrophotometer. Calculate the regression equation with ursolic acid mass (mg) on ​​the x-axis and absorbance on the y-axis.

[0089] Sample determination:

[0090] Take 2 mL of the test solution, and follow the standard curve establishment method to measure the absorbance. Substitute the absorbance into the regression equation to calculate the total terpene acid content. The results show that the total terpene acid content in the sample is 417.4 mg·g⁻¹. -1 Up to 610.1 mg·g -1 .

[0091] 4. Determination of total flavonoid content

[0092] Preparation of reference solution: Accurately weigh an appropriate amount of rutin reference standard and dissolve it in methanol to prepare a rutin reference standard solution containing approximately 0.2 mg per mL.

[0093] Preparation method of test solution: Accurately weigh 0.2g of vinegar-processed quinoa extract, add 50mL of water, sonicate for 30min, filter, and collect the filtrate to obtain the test solution.

[0094] Establishing the standard curve:

[0095] Accurately pipette 1, 2, 3, 4, 5, and 6 mL of the reference solution into separate 25 mL volumetric flasks. Add water to a final volume of 6 mL, then add 1 mL of 5% sodium nitrite, shake well, and let stand for 6 min. Add 1 mL of 10% aluminum nitrate, shake well, and let stand for 6 min. Add 10 mL of 4% sodium hydroxide, shake well, and let stand for 15 min. Using the corresponding reagent as a blank, measure the absorbance at a wavelength of 595 nm. The concentration (X μg·mL) is used as the reference standard. -1 Plot a standard curve with absorbance (Y) on the x-axis and absorbance (Y) on the y-axis to obtain the regression equation.

[0096] Sample determination:

[0097] Take an appropriate amount of the test solution, operate according to the standard curve establishment method, measure the absorbance, and substitute it into the regression equation to calculate the total terpene acid content. The results show that the total flavonoid content in the sample is 89.2 mg / g. -1 Up to 311.4 mg·g -1 between.

[0098] Example 2: Detection method of characteristic chromatogram of Vinegar-processed Trogopterus dung water extract

[0099] 1. Investigation of pretreatment methods for test sample solutions

[0100] The sample pretreatment methods for the fingerprint spectrum of the aqueous extract of *Typhonium quinquefolium* were investigated, mainly focusing on the extraction solvent, extraction method, extraction time, and amount of extraction solvent.

[0101] (1) Investigation of extraction solvent

[0102] Take 5 mL each of methanol, 70% methanol, 50% methanol, and 30% methanol, add 0.2 g of sample, and extract by sonication for 30 minutes. Filter, collect the filtrate, and inject for analysis to compare the extraction effects of the three solvents. Results are as follows: Figure 1 As shown, Figure 1 The results showed that 50% methanol had the highest extraction efficiency, so 50% methanol was chosen as the extraction solvent.

[0103] (2) Examination of extraction methods

[0104] Both ultrasonic extraction and reflux extraction were performed in parallel to compare their extraction effects. The results are as follows: Figure 2 As shown, Figure 2 The results showed that the characteristic peak areas of the samples obtained by ultrasonic extraction (300W power, 40kHz frequency, 30 minutes) and reflux extraction were not significantly different, and ultrasonic extraction was simple to operate. Therefore, ultrasonic extraction was determined to be the best extraction method.

[0105] (3) Examination of extraction time

[0106] Extraction times were set to 15 minutes, 30 minutes, and 45 minutes respectively. The experimental results are as follows: Figure 3 As shown, Figure 3 The results showed that the characteristic peak area reached its maximum value under extraction conditions of 30 minutes and 45 minutes, indicating optimal extraction efficiency. Under ultrasonic extraction conditions of 30 minutes, the extraction process was complete; therefore, the optimal extraction time was determined to be 30 minutes.

[0107] (4) Investigation on the amount of extraction solvent used

[0108] Take 5 mL, 10 mL, and 15 mL of 50% methanol respectively, add 0.2 g of sample, and extract by ultrasonication for 30 minutes. The results are as follows. Figure 4 As shown, Figure 4 The results showed that the extraction effect was best with 5 mL of solvent, with a large number of chromatographic peaks and clear peak shapes. Therefore, 5 mL of 50% methanol was selected as the optimal extraction solvent.

[0109] (5) Determination of the preparation method of the test solution

[0110] Based on the above findings, the recommended method for preparing the test solution is as follows: Take 0.2 g of sample, add 5 mL of 50% methanol, extract ultrasonically for 30 minutes, filter, collect the filtrate, and inject for analysis. This method is simple to operate, has high extraction efficiency, and is suitable for the characteristic chromatographic analysis of the aqueous extract of *Wu Ling Zhi* (a type of plant extract).

[0111] 2. Establishment of detection methods

[0112] Using the above-mentioned method for preparing the test solution, a characteristic chromatogram of the vinegar-processed Trogopterus dung water extract was established. The selection of the mobile phase, optimization of the elution gradient, and determination of the detection wavelength were systematically investigated, and the optimal chromatographic conditions were finally determined.

[0113] (1) Determination of the selection of mobile phase

[0114] Three mobile phase systems were compared: methanol-water, acetonitrile-0.2% formic acid, and methanol-0.2% formic acid. The results are as follows: Figure 5 As shown, Figure 5 The results showed that the methanol-water mobile phase system had few chromatographic peaks and poor peak resolution, indicating poor elution performance. While the acetonitrile-0.2% formic acid mobile phase system had more chromatographic peaks than the methanol-water system, the peak resolution remained unsatisfactory. The methanol-0.2% formic acid mobile phase system, however, exhibited a larger number of peaks, better resolution, and symmetrical peak shapes. Therefore, the methanol-0.2% formic acid mobile phase system was selected as the mobile phase system for the characteristic chromatogram of the aqueous extract of *Wulingzhi* (a type of spice).

[0115] (2) Selection of detection wavelength

[0116] The effects of different UV absorption wavelengths on sample response and chromatographic peak shape were analyzed, and the optimal detection wavelength was selected based on the changes in acquisition wavelength. The results are as follows: Figure 6 As shown, by comparing the chromatograms at different wavelengths (254nm, 280nm, 300nm), it was found that the chromatographic peak response values ​​at 300nm were higher, the peak shapes were clearer, and the baselines were stable. Therefore, 300nm was determined to be the optimal detection wavelength.

[0117] (3) Selection of column temperature

[0118] The column temperature of a chromatographic column has a certain impact on the resolution of chromatographic peaks. To address variations in column temperature, the optimal column temperature needs to be selected. Three column temperature conditions—30℃, 35℃, and 40℃—were investigated, and the results are as follows: Figure 7 As shown, Figure 7 The results showed that chromatographic peak resolution was optimal at 35℃, with symmetrical peak shapes and stable baselines. Therefore, 35℃ was determined to be the optimal column temperature.

[0119] (4) Selection of flow rate

[0120] Variations in the flow rate of the analytical instrument have a certain impact on the elution time and resolution of chromatographic peaks. To determine the optimal flow rate, three flow rates—0.25 mL / min, 0.3 mL / min, and 0.35 mL / min—were investigated. The results are as follows: Figure 8 As shown, Figure 8 The chromatographic peak resolution, retention time, and baseline stability were found to be optimal at a flow rate of 0.3 mL / min. Therefore, 0.3 mL / min was determined to be the optimal flow rate.

[0121] (5) Selection of chromatographic column

[0122] Different chromatographic columns exhibit varying separation effects on the same sample. Using the number of peaks, peak shape, and resolution as evaluation indicators, the effects of different column types on the characteristic chromatographic results of the aqueous extract of *Wu Ling Zhi* were compared. Waters CORTECS T3, YMC Triart C18, and Waters HSS T3 columns were selected for the investigation. Figure 9 The results showed that the Waters HSS T3 column had the best separation performance, with symmetrical peaks, high resolution, and stable baseline, thus it was determined to be the best column.

[0123] (6) Determination of chromatographic conditions

[0124] Based on the optimization results of the above parameters, the elution gradient was explored and finally determined as follows: a Waters HSS T3 column (2.1 mm × 150 mm, 1.8 μm) was used; the mobile phase was methanol (A) - 0.2% formic acid (B) gradient elution: 0 min ~ 5 min, 2% ~ 5% (A); 5 min ~ 15 min, 5% ~ 15% (A); 15 min ~ 25 min, 15% ~ 20% (A); 25 min ~ 50 min, 20% ~ 40% (A). The flow rate was 0.3 mL / min; the column temperature was 35℃; the detection wavelength was 300 nm; and the injection volume was 1 μL.

[0125] 3. Methodology

[0126] To ensure the stability and reproducibility of the spectra, methodological validation was performed, including precision, repeatability and stability tests.

[0127] (1) Precision experiment

[0128] Take the water extract of Trogopterus dung, prepare the test solution according to the preparation method of the test solution and inject it for analysis. Repeat the determination 6 times. Take benzoic acid as the reference peak (S), calculate the relative retention time and the RSD value of the relative peak area of the common peaks, and investigate the precision of the instrument. As shown in the results of Table 1 and Table 2, after injecting samples continuously for 6 times, the RSDs of the retention time and peak area of each chromatographic peak are less than 3%, indicating that the precision of this method is good and meets the analysis requirements.

[0129] Table 1 Precision Results (Relative Retention Time)

[0130]

[0131] Table 2 Precision Results (Relative Peak Area)

[0132]

[0133] (2) Repeatability Experiment

[0134] Take the water extract of Trogopterus dung (Trogopterus feces granules), prepare the test solution according to the preparation method of the test solution, make 6 parallel samples, and prepare and inject samples for analysis respectively. Take benzoic acid as the reference peak (S), calculate the RSD values of the relative retention time and relative peak area of each common peak and the S peak. As shown in the results of Table 3 and Table 4, after preparing 6 parallel samples, the RSDs of the retention time and peak area of each chromatographic peak are less than 2%, indicating that the repeatability of this method is good and meets the experimental requirements.

[0135] Table 3 Repeatability Experiment Results (Relative Retention Time)

[0136]

[0137] Table 4 Repeatability Experiment Results (Relative Peak Area)

[0138]

[0139] (3) Stability Experiment

[0140] Take the water extract of Trogopterus dung (Trogopterus feces granules), prepare the test solution according to the preparation method of the test solution, and inject samples for analysis at 0, 2, 4, 8, 10, 12, and 24 h respectively. Take benzoic acid as the reference peak (S), calculate the RSD values of the relative retention time and relative peak area of the common peaks, and investigate the stability of sample determination. As shown in the results of Table 5 and Table 6, the RSDs of the retention time and peak area of each chromatographic peak of the sample are less than 2% within 24 hours, proving that the sample has good stability and is suitable for long-term analysis.

[0141] Table 5 Stability Results (Relative Retention Time)

[0142]

[0143] Table 6. Stability results (relative peak area)

[0144]

[0145] 4. Establishment of feature maps and similarity evaluation

[0146] Based on the above verification results, a characteristic chromatogram of the aqueous extract of *Wu Ling Zhi* (a type of traditional Chinese medicine) was constructed. The data was processed using the 2012 version of the *Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System* software, with the following parameter settings: a time window width of 0.1 s was set, and the chromatogram of sample S1 was used as a reference (due to its obvious peak shape and moderate peak signal intensity). The average method was used to generate a common pattern, resulting in a UPLC superimposed characteristic chromatogram of the aqueous extract of *Wu Ling Zhi*. (See attached image). Figure 10 A control fingerprint (R) was generated using the mean method, and nine common characteristic peaks were identified on the control fingerprint. Figure 11 As shown in Table 7, the similarity of all samples was higher than 0.90, indicating that the method is reliable and suitable for quality control. The similarity of the characteristic chromatograms of the aqueous extract of *Wulingzhi* was calculated using the chromatographic fingerprint similarity evaluation system for traditional Chinese medicine (2012 version). The results, as shown in Table 7, were all greater than 0.90, indicating good consistency between batches.

[0147] Table 7. Similarity Evaluation of Characteristic Maps of Vinegar-processed Trogopterus Dextrose Aqueous Extract

[0148]

[0149] Nine common peaks were identified in the characteristic chromatograms of 10 batches of vinegar-processed Trogopterus dung water extract. Three of these peaks were identified using reference solutions, including p-hydroxybenzoic acid (peak 1), m-hydroxybenzoic acid (peak 3), and protocatechuic acid (peak 4). Using protocatechuic acid as the reference peak S, the relative peak area and relative retention time ratio of the 10 batches of vinegar-processed Trogopterus dung water extract were calculated. The specific results are shown in Tables 8 and 9 below.

[0150] Table 8. Relative retention times of 10 batches of Wulingzhi water extract

[0151]

[0152] Table 9. Relative peak areas of 10 batches of vinegar-processed *Wulingzhi* water extract

[0153]

[0154] Based on the determination results of 10 batches of water extracts of vinegar-processed Trogopterus dung (Tortoise Shell Granules), the characteristic chromatographic standard of the water extract of vinegar-processed Trogopterus dung (Tortoise Shell Granules) was determined as follows: the chromatogram of the test sample should show 9 characteristic peaks. Taking protocatechuic acid as the reference peak, the relative retention times of peaks 1 to 3 and peaks 5 to 11 were calculated. The relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.164 (peak 1), 0.554 (peak 2), 0.790 (peak 3), 1.052 (peak 5), 1.474 (peak 6), 1.675 (peak 7), 1.926 (peak 8), and 1.975 (peak 9).

[0155] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments 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.

[0156] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention 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 spectrum of an aqueous extract of *Wu Ling Zhi* (a type of plant extract), characterized in that, Includes the following steps: The aqueous extract of vinegar-processed five-spice powder is mixed with an extraction solvent for extraction to prepare a test solution. The extraction solvent includes one or more of the following: a methanol aqueous solution with a mass fraction of 65% to 75%, a methanol aqueous solution with a mass fraction of 45% to 55%, and a methanol aqueous solution with a mass fraction of 25% to 35%. The test solution was analyzed by high performance liquid chromatography to construct a characteristic spectrum of the vinegar-based extract of *Wulingzhi*. The characteristic peaks of the characteristic spectrum include p-hydroxybenzoic acid peak, m-hydroxybenzoic acid peak and protocatechuic acid peak. The conditions for high-performance liquid chromatography (HPLC) analysis include the following: (1) Gradient elution is adopted; the mobile phase includes mobile phase A and mobile phase B, wherein mobile phase A is methanol and mobile phase B is a 0.2% formic acid aqueous solution; (2) The gradient elution program includes: 0 min to 5 min, the volume percentage of mobile phase A changes from 2% to 5%; 5 min to 15 min, the volume percentage of mobile phase A changes from 5% to 15%; 15 min to 25 min, the volume percentage of mobile phase A changes from 15% to 20%; 25 min to 50 min, the volume percentage of mobile phase A changes from 20% to 40%. (3) The chromatographic column was a Waters HSS T3 column; (4) The detection wavelength is 300nm.

2. The method for constructing the characteristic spectrum of a water extract of vinegar-processed *Wu Ling Zhi* as described in claim 1, characterized in that, One or more of the following conditions must be met: (1) The extraction method is ultrasonic extraction or reflux extraction; and (2) The extraction time is 15 min to 45 min.

3. The method for constructing the characteristic spectrum of a water extract of vinegar-processed *Wu Ling Zhi* as described in claim 2, characterized in that, The ultrasonic extraction power is 200W~300W, and the frequency is 30kHz~50kHz.

4. The method for constructing the characteristic spectrum of a water extract of vinegar-processed *Wu Ling Zhi* as described in claim 1, characterized in that, The mass-volume ratio of the vinegar-processed five-spice oil water extract to the extraction solvent is 0.2 g: (5~15) mL.

5. The method for constructing the characteristic spectrum of a water extract of vinegar-processed *Wu Ling Zhi* according to claim 1, characterized in that, The conditions for high performance liquid chromatography analysis include: injection volume of 1 μL to 10 μL.

6. The method for constructing the characteristic spectrum of a water extract of vinegar-processed *Wu Ling Zhi* according to claim 1, characterized in that, The conditions for high performance liquid chromatography analysis include a column temperature of 30℃~40℃.

7. The method for constructing the characteristic spectrum of a water extract of vinegar-processed *Wu Ling Zhi* according to claim 1, characterized in that, The conditions for high performance liquid chromatography analysis include a flow rate of 0.25 mL / min to 0.35 mL / min.

8. The method for constructing the characteristic spectrum of a water extract of vinegar-processed *Wu Ling Zhi* according to any one of claims 1 to 7, characterized in that, The preparation method of the vinegar-processed Trogopterus xanthipes water extract includes the following steps: Mix the vinegar-processed five-spice powder with water, soak, decoct, and filter to obtain the filtrate. The filtrate was concentrated and dried to prepare the vinegar-based extract of *Wulingzhi*.

9. A method for constructing a characteristic spectrum of an aqueous extract of *Wu Ling Zhi* (a type of plant extract) according to any one of claims 1 to 7, characterized in that, The feature spectrum contains 9 characteristic peaks.

10. The method for constructing a characteristic spectrum of a vinegar-processed ginseng extract according to any one of claims 1 to 9 is used in the identification of vinegar-processed ginseng extract or its aqueous extract.

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