Construction method of specific chromatogram of trogopterus dung medicinal material and application of specific chromatogram in identification of trogopterus dung medicinal material

Abstract

The invention relates to the technical field of traditional Chinese medicine detection, in particular to a construction method of a specific chromatogram of a trogopterus dung medicinal material and application of the specific chromatogram in identification of the trogopterus dung medicinal material. The construction method of the specific chromatogram comprises the following steps: carrying out heat reflux extraction on a trogopterus dung medicinal material by using alcohol, filtering, and taking filtrate to prepare a test solution; performing high performance liquid chromatography detection on the test solution, and formulating a characteristic spectrum of the trogopterus dung medicinal material; the conditions of the high performance liquid chromatography detection are as follows: acetonitrile is adopted as a mobile phase A, and an aqueous solution of formic acid is adopted as a mobile phase B; gradient elution is adopted; the detection wavelength is 260 nm to 270 nm; the specific chromatogram shows the key characteristic peaks of the trogopterus dung medicinal material, can be visually used for identifying trogopterus dung medicinal materials of different specifications, and provides objective reference for identification of trogopterus dung and counterfeit products thereof.

Description

Technical Field

[0001] This application relates to the technical field of traditional Chinese medicine detection, and particularly to a method for constructing a characteristic chromatogram of Wulingzhi medicinal materials and its application in the identification of Wulingzhi medicinal materials. Background Art

[0002] The 1990 edition of the Chinese Pharmacopoeia records that Wulingzhi is the dried feces of the flying squirrel Trogopterus xanthipes Milne-Edwards of the family Sciuridae, and has the effects of promoting blood circulation, removing blood stasis, and relieving pain. It can be used to treat chest and hypochondrium pain, abdominal pain, dysmenorrhea, amenorrhea, postpartum blood stasis pain, contusion and swelling pain, snake and insect bites, etc. Modern research shows that Wulingzhi has very important medicinal value, and its components such as flavonoids, organic acids, triterpenes, and diterpenes have a wide range of pharmacological activities, such as anti-atherosclerosis, anti-inflammatory, anti-cerebral ischemia, anti-gastric ulcer, antithrombin, pain relief, etc.

[0003] The sources of Wulingzhi medicinal materials are widely distributed and the quality is uneven. To ensure clinical efficacy, it is necessary to control the quality of Wulingzhi medicinal materials. However, the existing quality control methods mainly stipulate the appearance of Wulingzhi, which has great limitations. At present, the Wulingzhi medicinal materials sold and used in the market are the dried feces of more than 10 animals of the families Sciuridae, Bovidae, and Ochotonidae. In addition, they are also divided into "Wulingzhi blocks" and "Wulingzhi grains" according to specifications. These medicinal materials with different sources and specifications need to be distinguished during actual application to ensure efficacy and medication safety. Summary of the Invention

[0004] Based on this, one or more embodiments of this application provide a method for constructing a characteristic chromatogram of Wulingzhi medicinal materials and its application in the identification of Wulingzhi medicinal materials. The characteristic chromatogram of this application presents the key characteristic peaks of Wulingzhi medicinal materials, which can be directly used to identify different specifications of Wulingzhi medicinal materials and provide an objective reference for the identification of Wulingzhi and its counterfeits.

[0005] One or more embodiments of this application provide a method for constructing a characteristic chromatogram of Wulingzhi medicinal materials, including the following steps:

[0006] Heat reflux extract the Wulingzhi medicinal materials with alcohol, filter, take the filtrate, and prepare a test solution.

[0007] Perform high performance liquid chromatography detection on the test solution to obtain the characteristic chromatogram of the Wulingzhi medicinal materials.

[0008] The detection conditions of the high performance liquid chromatography include:

[0009] Use acetonitrile as mobile phase A and an aqueous solution of formic acid as mobile phase B; the volume concentration of the aqueous solution of formic acid is 0.1% - 0.3%.

[0010] Gradient elution is used, and the gradient elution procedure includes:

[0011] From 0 to 10 minutes, the volume percentage of the mobile phase A increased from 6% to 10%.

[0012] Within 10 to 25 minutes, the volume percentage of the mobile phase A increased from 10% to 23%.

[0013] Between 25 and 35 minutes, the volume percentage of mobile phase A increased from 23% to 26.5%.

[0014] Over 35 to 40 minutes, the volume percentage of mobile phase A increased from 26.5% to 35%.

[0015] Over 40-50 minutes, the volume percentage of mobile phase A increased from 35% to 40%.

[0016] Between 50 and 52 minutes, the volume percentage of mobile phase A increased from 40% to 42%.

[0017] The volume percentage of mobile phase A was maintained at 42% for 52 to 60 minutes.

[0018] The detection wavelength is 260nm-270nm;

[0019] The characteristic spectrum of the Wulingzhi medicinal material shows at least four characteristic peaks: peak 1, peak 2, peak 3 and peak 4; among which, peak 1 is the characteristic peak corresponding to protocatechuic acid, peak 2 is the characteristic peak corresponding to 4-hydroxybenzoic acid, and peak 4 is the characteristic peak corresponding to benzoic acid.

[0020] Further, the mass-to-volume ratio of the *Wulingzhi* herb to the alcohol is 1.5 g: 20-30 mL; and / or,

[0021] The alcohol includes at least one of methanol and ethanol.

[0022] Furthermore, the step of hot reflux extraction of the *Wulingzhi* herb using alcohol includes:

[0023] The medicinal material of *Wulingzhi* and the alcohol were mixed and extracted under heating conditions of 80℃-90℃ in a water bath.

[0024] Optionally, the extraction time is 20-40 minutes.

[0025] Furthermore, the detection conditions of the high-performance liquid chromatography also include at least one of the following conditions:

[0026] (1) The chromatographic column is a T3 column;

[0027] (2) The flow rate is 0.28 mL / min - 0.32 mL / min;

[0028] (3) The column temperature is 28℃-32℃; and

[0029] (4) The injection volume is 1μL-3μL.

[0030] Furthermore, the chromatographic column has a length of 80mm-120mm, an inner diameter of 2mm-2.2mm, and a packing particle size of 1.7-1.9μm.

[0031] Furthermore, the specifications of the Wulingzhi medicinal material are Wulingzhi blocks or Wulingzhi rice;

[0032] The characteristic spectrum of the Lingzhi block shows the following characteristic peaks: peak 1, peak 2, peak 3 and peak 4;

[0033] The characteristic spectrum of the Lingzhi rice shows the following characteristic peaks: peak 1, peak 2, peak 3, peak 4, peak 5, peak 6, peak 7, peak 8, peak 9, peak 10, peak 11, and peak 12; among which, peak 8 is the characteristic peak corresponding to Taxus chinensis biflavonoids, and peak 12 is the characteristic peak corresponding to Platycladus orientalis biflavonoids.

[0034] Furthermore, peak 4 is peak S1. The relative retention times of peaks 1-3, peaks 5-6 and peak S1 are calculated. The relative retention times are within ±10% of the specified values, which are: peak 1: 0.212; peak 2: 0.362; peak 3: 0.950; peak 5: 1.238; peak 6: 1.462.

[0035] Peak 8 is the S2 peak. The relative retention times of peaks 7, 9-12 and the S2 peak are calculated. The relative retention times are within ±10% of the specified values, which are: peak 7: 0.934; peak 9: 1.046; peak 10: 1.216; peak 11: 1.248; peak 12: 1.297.

[0036] Furthermore, the method for constructing the feature map also includes the following steps:

[0037] The reference solution was analyzed by high performance liquid chromatography to obtain a chromatogram of the reference solution; the reference solution included protocatechuic acid, 4-hydroxybenzoic acid and benzoic acid.

[0038] The chromatogram of the reference compound and the characteristic chromatogram of the *Wulingzhi* medicinal material are compared to identify the characteristic peaks in the characteristic chromatogram of the *Wulingzhi* medicinal material.

[0039] Furthermore, when the medicinal material Wulingzhi is Lingzhi rice, the reference solution also includes Taxus chinensis biflavonoids and Platycladus orientalis biflavonoids.

[0040] One or more embodiments of this application also provide the application of the above-described method for constructing feature maps in the identification of the medicinal material *Wulingzhi*.

[0041] The method for constructing the characteristic spectrum of the medicinal material *Wulingzhi* in this application can present the internal components of *Wulingzhi* more comprehensively and objectively, with high separation, stable baseline, good peak shape, and the characteristic spectrum showing at least 4 characteristic peaks: peak 1, peak 2, peak 3, and peak 4; wherein, peak 1 is the characteristic peak corresponding to protocatechuic acid, peak 2 is the characteristic peak corresponding to 4-hydroxybenzoic acid, and peak 4 is the characteristic peak corresponding to benzoic acid.

[0042] The method for constructing the characteristic spectrum of Wulingzhi medicinal material in this application is applicable to Wulingzhi medicinal material of different specifications—Lingzhi rice and Lingzhi blocks. In addition to the above 4 common peaks, the characteristic spectrum of Lingzhi rice also presents 8 other characteristic peaks: peak 5, peak 6, peak 7, peak 8, peak 9, peak 10, peak 11, and peak 12. Among them, peak 8 is the characteristic peak corresponding to Taxodium biflavonoids, and peak 12 is the characteristic peak corresponding to Platycladus orientalis biflavonoids. The specifications of Wulingzhi can be directly identified by the presence or absence of characteristic peaks.

[0043] The method for constructing the characteristic map of this application also provides an important reference for the identification of Wulingzhi and other common counterfeits (including dried feces of more than 10 other animals from the flying squirrel family, bovine family, and pika family). Attached Figure Description

[0044] 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.

[0045] Figure 1 This is a comparison of the chromatograms of the blank solvent (pure methanol), the reference solution, and the test solution in Example 1 of this application;

[0046] Figure 2 This is a superimposed chromatogram of 24 batches of Wulingzhi (Lingzhi rice) in Example 1 of this application;

[0047] Figure 3 The characteristic spectrum of the five-spirit ash (spirit ash rice) formulated in Example 1 of this application;

[0048] Figure 4 This is a comparison of the chromatograms of Wulingzhi (Lingzhi rice) obtained using different chromatographic columns in Example 2 of this application;

[0049] Figure 5This is a comparison of the chromatograms of Wulingzhi (Lingzhi rice) obtained at different column temperatures in Example 3 of this application;

[0050] Figure 6 This is a comparison of the chromatograms of Wulingzhi (Lingzhi rice) obtained by different hot reflux extraction times in Example 4 of this application;

[0051] Figure 7 This is a superimposed image of the chromatograms of four batches of Lingzhi blocks in Example 5 of this application;

[0052] Figure 8 The comparison results of the characteristic spectra of Lingzhi rice and Lingzhi blocks in Example 5 of this application;

[0053] Figure 9 This is a superimposed chromatogram of the three batches of red and white flying squirrel feces in Example 6 of this application;

[0054] Figure 10 The comparison results of the characteristic spectra of red and white flying squirrel feces and Lingzhi rice in Example 6 of this application;

[0055] Figure 11 This is a comparison of the chromatograms of Wulingzhi (Lingzhi rice) obtained using different extraction solvents in Comparative Example 1 of this application;

[0056] Figure 12 This is a comparison of the chromatograms of Wulingzhi (Lingzhi rice) obtained by different extraction methods in Comparative Example 1 of this application;

[0057] Figure 13 This is a comparison of the chromatograms of Wulingzhi (Lingzhi rice) measured using different mobile phases B in Comparative Example 1 of this application;

[0058] Figure 14 This is a comparison of the chromatograms of Wulingzhi (Lingzhi rice) obtained using different elution procedures in Comparative Example 1 of this application;

[0059] Figure 15 This is a comparison of the chromatograms of Wulingzhi (Lingzhi rice) measured using different detection wavelengths in Comparative Example 1 of this application. Detailed Implementation

[0060] 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.

[0061] 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.

[0062] The terms "and / or," "or / and," and "and / or" as used herein include 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 encompass 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 by 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+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0063] In this document, terms such as "preferred," "better," and "more preferred" are merely descriptions of implementation methods or examples that achieve better results, and should be understood as not constituting a limitation on the scope of protection of this application.

[0064] 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.

[0065] 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.

[0066] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, optional numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval points to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges may 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.

[0067] In this application, weight can be a well-known unit of mass in the chemical industry, such as μg, mg, g, or kg.

[0068] In this application, "%" represents percentage, referring to the proportion by weight; however, the percentage of a solution, unless otherwise specified, refers to the number of grams of solute contained in 100 mL of the solution; the percentage of alcohol refers to the proportion by volume at 20°C. Furthermore, the following symbols may be used as needed: %(g / g) indicates the number of grams of solute contained in 100 g of solution; %(mL / mL) indicates the number of milliliters of solute contained in 100 mL of solution; %(mL / g) indicates the number of milliliters of solute contained in 100 g of solution; %(g / mL) indicates the number of grams of solute contained in 100 mL of solution.

[0069] One or more embodiments of this application provide a method for constructing a characteristic spectrum of the medicinal material *Wulingzhi*, including the following steps:

[0070] The medicinal material of Wulingzhi was extracted by hot reflux with alcohol, filtered, and the filtrate was collected to prepare the test solution.

[0071] The test solution was subjected to high performance liquid chromatography to obtain the characteristic chromatogram of the Wulingzhi medicinal material.

[0072] The detection conditions for high performance liquid chromatography include:

[0073] Acetonitrile was used as mobile phase A, and an aqueous solution of formic acid was used as mobile phase B; the volume concentration of the aqueous formic acid solution was 0.1%–0.3%.

[0074] Gradient elution is used, and the gradient elution procedure includes:

[0075] From 0 to 10 minutes, the volume percentage of mobile phase A increased from 6% to 10%.

[0076] From 10 to 25 minutes, the volume percentage of mobile phase A increased from 10% to 23%.

[0077] Between 25 and 35 minutes, the volume percentage of mobile phase A increased from 23% to 26.5%.

[0078] Between 35 and 40 minutes, the volume percentage of mobile phase A increased from 26.5% to 35%.

[0079] Between 40 and 50 minutes, the volume percentage of mobile phase A increased from 35% to 40%.

[0080] Between 50 and 52 minutes, the volume percentage of mobile phase A increased from 40% to 42%.

[0081] From 52 to 60 minutes, the volume percentage of mobile phase A was maintained at 42%.

[0082] The detection wavelength is 260nm-270nm;

[0083] The characteristic spectrum of the medicinal material Wulingzhi shows at least four characteristic peaks: peak 1, peak 2, peak 3 and peak 4; among them, peak 1 is the characteristic peak corresponding to protocatechuic acid, peak 2 is the characteristic peak corresponding to 4-hydroxybenzoic acid, and peak 4 is the characteristic peak corresponding to benzoic acid.

[0084] In some embodiments, the volume concentration of the aqueous formic acid solution is 0.1%-0.3%, for example, 0.1%, 0.2%, 0.3%, etc. When the aqueous formic acid solution at this volume concentration is used as mobile phase B, a stable baseline and good resolution can be obtained. However, when 0.2% formic acid is used as mobile phase B, the peak shapes of each chromatographic peak are better and the resolution is higher.

[0085] In some embodiments, the detection wavelength is 260nm-270nm, such as 260nm, 265nm, 270nm, etc. At this detection wavelength, peak information is relatively rich, the response values ​​of each characteristic peak are large, the baseline is relatively stable, and interference is small.

[0086] Furthermore, the mass-to-volume ratio of the medicinal material *Wulingzhi* to alcohol is 1.5 g: 20-30 mL; and / or,

[0087] The alcohol includes at least one of methanol and ethanol, preferably methanol, and the chromatographic peaks in each interval of the chromatogram can be detected, and the peak shapes are good.

[0088] Optionally, the mass-to-volume ratio of the medicinal material *Wulingzhi* to alcohol is 1.5g:20-30mL, for example, 1.5g:20mL, 1.5g:25mL, 1.5g:30mL, etc.

[0089] In this article, thermal reflux extraction refers to heating the mixture in a container equipped with a reflux device until it reaches the reflux temperature and is then maintained thereafter. The temperature of thermal reflux extraction is related to the boiling point of the solvent in the mixture. For example, when the solvent is methanol, the temperature of thermal reflux extraction is between 80℃ and 90℃.

[0090] Furthermore, the step of hot reflux extraction of the *Wulingzhi* herb using alcohol includes:

[0091] The medicinal material of Wulingzhi was mixed with alcohol and extracted under the heating conditions of a water bath at 80℃-90℃.

[0092] Optionally, the extraction time is 20-40 minutes. In this embodiment, extracting for an appropriate time at a suitable temperature can yield abundant characteristic peaks.

[0093] In some embodiments, the chromatographic column is a T3 column; using a T3 column can achieve better peak resolution.

[0094] Furthermore, the chromatographic column has a length of 80mm-120mm, an inner diameter of 2mm-2.2mm, and a packing particle size of 1.7-1.9μm.

[0095] In some embodiments, the chromatographic column can be selected from Agilent ZORBA SB C18 (2.1mm × 100mm, 1.8μm), Agilent ZORBA RRHD StableBond C18 (2.1mm × 100mm, 1.8μm), Agilent ZORBA RRHD Eclipse Plus C18 (2.1mm × 100mm, 1.8μm), and Waters ACQUITY UPLC HSS T3 (2.1mm × 100mm, 1.8μm). Good peak resolution can be obtained under each chromatographic column condition. Further, Waters ACQUITY UPLC HSS T3 (2.1mm × 100mm, 1.8μm) can be used, as the peak shape and resolution of the chromatographic peaks measured by the column are even better.

[0096] In some embodiments, the flow rate is 0.28 mL / min to 0.32 mL / min. Under these flow rate conditions, good peak separation, good peak shape, and stable baseline can be obtained.

[0097] In some embodiments, the column temperature is 28℃-32℃, under which good peak resolution can be obtained. Preferably, it is 30℃, which yields even better peak resolution.

[0098] In some embodiments, the injection volume is 1 μL-3 μL, for example 1 μL, 2 μL, 3 μL, etc.

[0099] Furthermore, the specifications for the medicinal material Wulingzhi are Wulingzhi blocks or Wulingzhi rice;

[0100] The characteristic spectrum of the Lingzhi block shows the following characteristic peaks: peak 1, peak 2, peak 3 and peak 4;

[0101] The characteristic spectrum of Lingzhi rice shows the following characteristic peaks: peak 1, peak 2, peak 3, peak 4, peak 5, peak 6, peak 7, peak 8, peak 9, peak 10, peak 11, and peak 12; among them, peak 8 is the characteristic peak corresponding to Taxodium biflavonoids, and peak 12 is the characteristic peak corresponding to Platycladus orientalis biflavonoids.

[0102] Furthermore, peak 4 is peak S1. The relative retention times of peaks 1-3, peaks 5-6 and peak S1 are calculated. The relative retention times are within ±10% of the specified values, which are: peak 1: 0.212; peak 2: 0.362; peak 3: 0.950; peak 5: 1.238; peak 6: 1.462.

[0103] Peak 8 is the S2 peak. Calculate the relative retention times of peaks 7, 9-12 and the S2 peak. The relative retention times are within ±10% of the specified values. The specified values ​​are: peak 7: 0.934; peak 9: 1.046; peak 10: 1.216; peak 11: 1.248; peak 12: 1.297.

[0104] Furthermore, the method for constructing the feature map also includes the following steps:

[0105] The reference solution was analyzed by high performance liquid chromatography to obtain a chromatogram of the reference solution; the reference solution included protocatechuic acid, 4-hydroxybenzoic acid and benzoic acid.

[0106] The chromatograms of the reference compound and the characteristic chromatograms of the medicinal material *Wulingzhi* were compared to identify the characteristic peaks in the characteristic chromatograms of the medicinal material *Wulingzhi*.

[0107] Furthermore, when the medicinal material of Wulingzhi is Lingzhi rice, the reference solution also includes Taxus chinensis biflavonoids and Platycladus orientalis biflavonoids.

[0108] One or more embodiments of this application also provide the application of the above-described method for constructing feature maps in the identification of the medicinal material *Wulingzhi*.

[0109] The following are some specific examples.

[0110] For experimental parameters not specified in the following specific embodiments, please refer to the guidelines provided in this application, or consult experimental manuals or other known experimental methods in the field, or refer to the manufacturer's recommended experimental conditions. Raw materials and reagents not mentioned can be obtained commercially, or can be prepared by those skilled in the art using known methods.

[0111] The instruments, reagents, reagents, and samples used in the following examples or comparative examples are as follows:

[0112] Instruments: Agilent ultra-high performance liquid chromatograph (Model 1290, Agilent Technologies), Waters ultra-high performance liquid chromatograph (H-Class Plus, Waters Corporation), ten-thousandth balance (ME204E, Mettler Toledo), millionth balance (XP26, Mettler Toledo), laboratory ultra-pure water system (Milli-Q Direct, Merck KGaA). Reagents: Methanol (Xilong Science Co., Ltd.) is of analytical grade; Phosphoric acid (Tianjin Kemiou Chemical Reagent Co., Ltd.), Acetonitrile (Anke Chemical Co., Ltd., USA) are of chromatographic grade, and water is ultra-pure water (prepared in the laboratory).

[0113] Reagents: Methanol (Xilong Science Co., Ltd.) and Ethanol (Xilong Science Co., Ltd.) are both of analytical grade; Acetonitrile (Merck KGaA), Methanol (Merck KGaA), and Formic acid (Merck KGaA) are of chromatographic grade, and water is ultra-pure water (prepared in the laboratory).

[0114] Test drugs: Protocatechuic acid (Batch No.: 110809 - 201906, purity: 97.7%), 4-Hydroxybenzoic acid (Batch No.: 101149 - 201903, purity: 100.0%), Amentoflavone (Batch No.: 111902201603, purity: 97.7%) are all purchased from National Institutes for Food and Drug Control; Reference substance Biapigenin (Chengdu Refines Biological Technology Co., Ltd., Batch No.: RFS-B0511808009, purity 97.0%); Water is ultra-pure water (prepared in the laboratory); Acetonitrile (Merck KGaA, chromatographically pure), Acetic acid, and Formic acid (Tianjin Kemiou Chemical Reagent Co., Ltd., chromatographically pure) are of chromatographic grade; Rice vinegar is from Guangdong Meiweixian Flavoring Co., Ltd.

[0115] Samples: Control crude drug of Trogopterus Dung (Guangdong Yifang Pharmaceutical Co., Ltd.); Batch numbers of 24 batches of Trogopterus Dung (Trogopterus feces) crude drugs (WLZ01 - WLZ24), 4 batches of Trogopterus feces lumps (LZK01 - LZK04), and 3 batches of feces of Petaurista alborufus (HB01 - HB03) are shown in Table 1.

[0116] Table 1 Information of Samples for Authenticity Identification of Trogopterus Dung

[0117]

[0118] Example 1

[0119] This example provides a method for constructing the characteristic chromatogram of Trogopterus Dung crude drug of the present application, which includes the following steps:

[0120] 1. Preparation of reference substance solution

[0121] Take appropriate amounts of protocatechuic acid reference standard, 4-hydroxybenzoic acid reference standard, benzoic acid reference standard, paclitaxel flavonoid reference standard, and cypress flavonoid reference standard, dissolve them in methanol, and prepare a mixed reference solution containing 10 μg each of protocatechuic acid, 4-hydroxybenzoic acid, benzoic acid, paclitaxel flavonoid, and cypress flavonoid per 1 mL. This solution will serve as the reference standard solution.

[0122] 2. Preparation of reference solution of control medicinal material

[0123] Take about 1.5g of the reference herb *Wulingzhi* and place it in a stoppered conical flask. Add 25mL of methanol, reflux for 30 minutes, cool, weigh again, and replenish the lost weight with methanol. Shake well, filter through a 0.22μm microporous membrane, and collect the filtrate. This is used as the reference solution for the reference herb.

[0124] 3. Preparation of the test solution

[0125] Take about 1.5g of Wulingzhi powder (passed through a No. 3 sieve), accurately weigh it, place it in a stoppered conical flask, add 25mL of methanol, reflux for 30 minutes, cool, weigh it again, replenish the lost weight with methanol, shake well, filter through a 0.22μm microporous membrane, and collect the filtrate.

[0126] 4. Chromatographic detection

[0127] 4.1 Chromatographic detection conditions

[0128] An ACQUITY UPLC HSS T3 (2.1 mm × 100 mm, 1.8 μm) column was used; acetonitrile was used as mobile phase A, and 0.1% formic acid solution was used as mobile phase B, with gradient elution performed according to the specifications in Table 2. The flow rate was 0.30 mL / min; the column temperature was 30 °C; and the detection wavelength was 265 nm. The injection volume was 2 μL.

[0129] Table 2 Gradient Elution Table

[0130]

[0131] 4.2 Determination Method

[0132] Accurately pipette 2 μL of each of the reference solution, the reference medicinal material solution, and the test solution, and inject them into the liquid chromatograph for determination.

[0133] 5. Methodological Examination

[0134] 5.1 Specificity Examination

[0135] Prepare the reference solution according to the method under item "2"; take an appropriate amount of Trogopterus Dung (serial number: WLZ-01), about 1.5 g, and prepare the test solution according to the method under item "3". Precisely pipette 2 μL of the blank solvent (pure methanol), the reference solution and the test solution respectively, and inject them for analysis under the chromatographic conditions under item "4.1". The comparison chart of the chromatographic detection results is as Figure 1 . The results show that the test solution chromatogram has the same chromatographic peaks at the retention times corresponding to the reference solution chromatogram, and the blank solvent has no interference, indicating that this method has good specificity.

[0136] 5.2 Investigation of precision

[0137] Take an appropriate amount of Trogopterus Dung (serial number: WLZ-01), about 1.5 g, and prepare the test solution according to the method under item "3". Inject the sample repeatedly 6 times under the chromatographic conditions under item "4.1". Take benzoic acid as the reference peak S1 and amentoflavone as the reference peak S2, calculate the relative retention time and relative peak area of each characteristic peak with respect to the S peak, and calculate the RSD value. The RSD values of the relative retention times of 12 common peaks are within the range of 0.14% - 1.35%, and the RSD values of the relative peak areas are within the range of 0.90% - 3.87%, indicating that the instrument has good precision.

[0138] 5.3 Investigation of repeatability

[0139] Take an appropriate amount of Trogopterus Dung (serial number: WLZ-01), about 1.5 g, and prepare 6 test solutions according to the method under item "3". Inject the samples for determination under the chromatographic conditions under item "4.1". Take benzoic acid as the reference peak S1 and amentoflavone as the reference peak S2, calculate the relative retention time and relative peak area of each characteristic peak with respect to the S peak, and calculate the RSD value. The RSD values of the relative retention times of 12 common peaks are within the range of 0.09% - 0.26%, and the RSD values of the relative peak areas are within the range of 1.08% - 3.66%, indicating that this method has good repeatability.

[0140] 5.4 Investigation of stability

[0141] Take an appropriate amount of Trogopterus Dung (serial number: WLZ-01), about 1.5 g, and prepare the test solution according to the method under item "3". Inject the samples for determination under the chromatographic conditions under item "4.1" at 0, 2, 4, 6, 8, 12, and 24 hours respectively. Take benzoic acid as the reference peak S1 and amentoflavone as the reference peak S2, calculate the relative retention time and relative peak area of each characteristic peak with respect to the S peak, and calculate the RSD value. The RSD values of the relative retention times of 7 common peaks are within the range of 0.01% - 1.12%, and the RSD values of the relative peak areas are within the range of 0.31% - 2.69%, indicating that the test solution is relatively stable within 24 hours.

[0142] 6. Establish the characteristic chromatogram of Trogopterus Dung

[0143] 6.1 Determination of the characteristic chromatogram of Wulingzhi sample and establishment of the control characteristic chromatogram

[0144] Twenty-four batches of *Wulingzhi* from Table 1 were used. Test solutions were prepared according to the method described in section "3," and the samples were injected and analyzed under the chromatographic conditions described in section "4.1." The chromatograms of the 24 batches of *Wulingzhi* were recorded. The integrated characteristic chromatograms of the 24 batches of *Wulingzhi* were exported in CDF format and imported into the *Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation Software (Version 2012.130723)* for peak matching and multi-point correction. The results showed that 12 common peaks were identified in the characteristic chromatograms of 18 batches of *Wulingzhi*. The overlay diagram of the characteristic chromatograms of the 24 batches of *Wulingzhi* is shown below. Figure 2 As shown. A characteristic chromatogram of *Wulingzhi* was generated using the mean method and compared with the chromatogram of the reference solution of the control herb. The comparison results are as follows. Figure 3 As shown.

[0145] 6.2 Establishment of the Standard for the Characteristic Atlas of Wulingzhi

[0146] Based on the above research results, the standard for determining the characteristic chromatogram of *Wulingzhi* is as follows: the chromatogram of the test sample should show 12 characteristic peaks, and the retention times should correspond to the 12 characteristic peaks in the chromatogram of the reference medicinal material; the peak corresponding to the benzoic acid reference standard is the S1 peak, and the relative retention times of peaks 1-3 and 5-6 with the S1 peak are calculated; the peak corresponding to the *Taxodium flavonoids* reference standard is the S2 peak, and the relative retention times of peaks 7, 9-12 with the S2 peak are calculated. The relative retention times should be within ±10% of the specified values, which are: 0.212 (peak 1), 0.362 (peak 2), 0.950 (peak 3), 1.238 (peak 5), 1.462 (peak 6), 0.934 (peak 7), 1.046 (peak 9), 1.216 (peak 10), and 1.297 (peak 12).

[0147] Example 2

[0148] This embodiment investigates the effect of different chromatographic columns on chromatographic detection results. An appropriate amount of Wulingzhi powder (No.: WLZ-01), approximately 1.5g, was taken and prepared according to the method described in "3. Preparation of the test solution" of Example 1. Chromatographic detection was performed using a method essentially the same as described in "4.1 Chromatographic Detection Conditions" of Example 1, except that Agilent ZORBA SB C18 (2.1mm × 100mm, 1.8μm, denoted as BH-335), Agilent ZORBA RRHDStableBond C18 (2.1mm × 100mm, 1.8μm, denoted as BH-323), and Agilent ZORBA RRHD Eclipse Plus C18 (2.1mm × 100mm, 1.8μm, denoted as BH-246) columns were used, respectively, and compared with the Waters ACQUITY UPLCHSS column of Example 1. The chromatograms of columns T3 (2.1 mm × 100 mm, 1.8 μm, denoted as BH-406) were compared, and the results are as follows: Figure 4 The results showed that good peak resolution could be obtained under various column conditions. Among them, the peak shape and resolution of the chromatographic peaks measured using the Waters ACQUITY UPLCHSS T3 (2.1mm×100mm, 1.8μm) column were better.

[0149] Example 3

[0150] This embodiment investigates the effect of different column temperatures on chromatographic detection results. Approximately 1.5g of Wulingzhi powder (item number: WLZ-01) was taken and prepared according to the method described in section "3. Preparation of the test solution" of Example 1. Chromatographic detection was performed using a method essentially the same as that described in section "4.1 Chromatographic Detection Conditions" of Example 1, except that chromatograms were obtained at 28℃ and 32℃, respectively, and compared with the chromatograms obtained in Example 1 at a column temperature of 30℃. The results are as follows: Figure 5 The results show that good peak resolution can be obtained under various column temperature conditions, with the chromatographic peak resolution being even better when measured at a column temperature of 30℃.

[0151] Example 4

[0152] This embodiment investigates the effect of different hot reflux extraction times on chromatographic detection results. An appropriate amount of *Wulingzhi* powder (item number: WLZ-01), approximately 1.5 g, was taken and prepared according to the same method as under "3. Preparation of the test solution" in Example 1, except that the hot reflux extraction time was 15 minutes and 45 minutes. Chromatographic detection was performed using the method described in "4.1 Chromatographic Detection Conditions" of Example 1, and the results were compared with the chromatograms obtained in Example 1 with a hot reflux extraction time of 30 minutes. The results are as follows: Figure 6 The results show that detection under the specified time conditions of thermal reflux extraction yields relatively rich characteristic peak information.

[0153] Example 5

[0154] This embodiment provides the application of the above-mentioned method for constructing feature maps in the specification identification of Wulingzhi medicinal material.

[0155] Four batches of Lingzhi blocks were taken according to Table 1, and characteristic chromatograms were constructed according to the method in Example 1. The overlay of the chromatograms of the four batches of Lingzhi blocks is shown in Figure 7. The comparison results between the constructed characteristic chromatograms of Lingzhi blocks and the characteristic chromatograms of Lingzhi rice constructed above are shown in Figure 7. Figure 8 The characteristic spectrum of Wulingzhi (Lingzhi block) medicinal material shows that under the judgment criteria, there are only 4 common characteristic peaks (peak 1-peak 4). It can be seen that the specifications of Wulingzhi, namely Lingzhi rice or Lingzhi block, can be intuitively distinguished by the number of characteristic peaks or their presence or absence.

[0156] Example 6

[0157] This embodiment provides the application of the above-mentioned method for constructing feature maps in the identification of the authenticity of Wulingzhi medicinal material.

[0158] Three batches of red and white flying squirrel feces adulterants were taken according to Table 1, and characteristic chromatograms were constructed according to the method in Example 1. The overlay chromatograms of the three batches of red and white flying squirrel feces are shown in the figure. Figure 9 The comparison results between the constructed feature map of red and white flying squirrel feces (adult product) and the feature map of Lingzhi rice constructed above are as follows: Figure 10 The results show that the number of characteristic peaks and retention time of the counterfeit product are significantly different from those of the original Wulingzhi medicinal material. Only peaks 1, 2, 4, and 8 are common characteristic peaks. Therefore, this method can serve as an important reference for distinguishing the counterfeit red and white flying squirrel from the original Wulingzhi medicinal material.

[0159] Comparative Example 1

[0160] This comparative study investigated the effect of different extraction solvents on chromatographic detection results. An appropriate amount of *Wulingzhi* powder (item number: WLZ-01), approximately 1.5 g, was taken and prepared according to the same method as under "3. Preparation of the test solution" in Example 1, except that 70% methanol, 30% methanol, ethanol, 70% ethanol, and 30% ethanol were used for hot reflux extraction. Chromatographic detection was performed using the method described in "4.1 Chromatographic Detection Conditions" of Example 1. The results were compared with those of the test solution extracted with pure methanol using hot reflux in Example 1. The results are as follows: Figure 11The results showed that using 30% ethanol or 30% methanol reduced the abundance of chromatographic peaks in the 30-55 min interval, using 70% methanol or 70% ethanol reduced the abundance of chromatographic peaks in the 15-30 min interval, and using pure ethanol reduced the abundance of chromatographic peaks in the 5-15 min interval. However, when using methanol, chromatographic peaks in all intervals could be detected, and the peak shapes were better.

[0161] Comparative Example 2

[0162] This comparative study investigated the effect of different extraction methods on chromatographic detection results. An appropriate amount of *Wulingzhi* powder (item number: WLZ-01), approximately 1.5 g, was taken and prepared according to the same method as under "3. Preparation of the test solution" in Example 1, except that ultrasonic extraction was used: the *Wulingzhi* herb was mixed with alcohol and ultrasonically extracted at a power of 300 W and a frequency of 40 kHz for 30 min. The results are as follows. Figure 12 The results show that different extraction methods can yield relatively rich chromatographic peaks, but the reflux extraction method extracts more completely, provides richer information about the chromatographic peaks, has a more suitable peak response intensity, and can better construct characteristic spectra.

[0163] Comparative Example 3

[0164] This comparative study investigated the effect of different mobile phases B on chromatographic detection results. Approximately 1.5g of Wulingzhi powder (product number: WLZ-01) was taken and prepared according to the method described in "3. Preparation of the test solution" of Example 1. Chromatographic detection was performed using a method essentially the same as described in "4.1 Chromatographic Detection Conditions" of Example 1, except that 0.1% acetic acid, 0.1% phosphoric acid, 0.1% formic acid, 0.2% acetic acid, and 0.2% phosphoric acid were used as mobile phase B. The chromatograms were compared with those of the mobile phase B obtained in Example 1 using 0.2% formic acid. The results are as follows: Figure 13 The results show that when 0.1% or 0.2% phosphoric acid is used as mobile phase B, the response intensity of some chromatographic peaks is too low, which is not conducive to constructing accurate characteristic chromatograms. When 0.1% or 0.2% acetic acid is used as mobile phase B, the baseline is not stable and there are many impurity peaks. When 0.1% formic acid is used as mobile phase B, a more stable baseline can be achieved, but when 0.2% formic acid is used as mobile phase B, the peak shapes of each chromatographic peak are better and the resolution is higher.

[0165] Comparative Example 4

[0166] This comparative study investigated the effect of different elution programs on chromatographic detection results. Approximately 1.5g of Wulingzhi powder (No.: WLZ-01) was taken and prepared according to the method described in "3. Preparation of Test Solution" of Example 1. Chromatographic detection was performed using a method essentially the same as described in "4.1 Chromatographic Detection Conditions" of Example 1, except that gradient optimization 1-3 from Table 3-5 was used as the elution program. The chromatograms were compared with those from Example 1 using the elution program described in Table 1 (referred to as gradient optimization 4 in this example). The results are as follows: Figure 14 The results show that gradient optimization 1-3 all resulted in a significant increase in the number of extraneous peaks in the chromatogram, a weakening of the target peak's response intensity, poor resolution, and uneven baseline. Gradient optimization 4, on the other hand, can obtain rich characteristic peak information, with good resolution, a stable baseline, and moderate response intensity.

[0167] Table 3 Gradient Optimization 1

[0168]

[0169] Table 4 Gradient Optimization 2

[0170]

[0171] Table 5 Gradient Optimization 3

[0172]

[0173] Comparative Example 5

[0174] This comparative study investigated the effect of different detection wavelengths on chromatographic detection results. An appropriate amount of Wulingzhi powder (item number: WLZ-01), approximately 1.5 g, was taken and prepared as a test solution according to the method described in "3. Preparation of the test solution" of Example 1. Chromatographic detection was performed using a method essentially the same as described in "4.1 Chromatographic Detection Conditions" of Example 1, except that 225 nm, 230 nm, 295 nm, and 335 nm were used as detection wavelengths. The results were compared with the chromatograms obtained in Example 1 at a detection wavelength of 265 nm. The results are as follows: Figure 15 The results show that fewer peaks were detected at wavelengths of 225nm, 230nm, and 335nm, while the response values ​​of each characteristic peak were low at wavelength of 295nm, and the baseline fluctuations were more obvious. When 265nm was the detection wavelength, the peak information was richer, the response values ​​of each characteristic peak were larger, the baseline was more stable, and the interference was less.

[0175] 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.

[0176] 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 the characteristic spectrum of the medicinal material *Wulingzhi*, characterized in that, It includes the following steps: Perform hot reflux extraction on the Wulingzhi medicinal material with alcohol, filter, take the filtrate, and prepare a test solution; Perform high performance liquid chromatography (HPLC) detection on the test solution to obtain the characteristic chromatogram of the Wulingzhi medicinal material; The detection conditions of the HPLC include: Use acetonitrile as mobile phase A and an aqueous solution of formic acid as mobile phase B; the volume concentration of the aqueous solution of formic acid is 0.1% - 0.3%; Perform gradient elution, and the gradient elution program includes: From 0 to 10 min, the volume percentage of mobile phase A increases from 6% to 10%; From 10 min to 25 min, the volume percentage of mobile phase A increases from 10% to 23%; From 25 min to 35 min, the volume percentage of mobile phase A increases from 23% to 26.5%; From 35 min to 40 min, the volume percentage of mobile phase A increases from 26.5% to 35%; From 40 min to 50 min, the volume percentage of mobile phase A increases from 35% to 40%; From 50 min to 52 min, the volume percentage of mobile phase A increases from 40% to 42%; From 52 min to 60 min, the volume percentage of mobile phase A remains at 42%; The detection wavelength is 260 nm - 270 nm; The characteristic chromatogram of the Wulingzhi medicinal material shows at least 4 characteristic peaks: Peak 1, Peak 2, Peak 3, and Peak 4; among them, Peak 1 is the characteristic peak corresponding to protocatechuic acid, Peak 2 is the characteristic peak corresponding to 4-hydroxybenzoic acid, and Peak 4 is the characteristic peak corresponding to benzoic acid.

2. The method for constructing a feature map according to claim 1, characterized in that, The mass-volume ratio of the Wulingzhi medicinal material to the alcohol is 1.5 g: 20 - 30 mL; and / or, The alcohol includes at least one of methanol and ethanol.

3. The method for constructing a feature map according to claim 1, characterized in that, The step of performing hot reflux extraction on the Wulingzhi medicinal material with alcohol includes: Mix the Wulingzhi medicinal material and the alcohol, and perform extraction under the heating condition of a water bath at 80°C - 90°C; Optionally, the extraction time is 20 min - 40 min.

4. The method for constructing a feature map according to claim 1, characterized in that, The detection conditions of the HPLC further include at least one of the following conditions: (1) The chromatographic column is a T3 chromatographic column; (2) The flow rate is 0.28 mL / min - 0.32 mL / min; (3) The column temperature is 28°C - 32°C; and (4) The injection volume is 1 μL - 3 μL.

5. The method for constructing a feature map according to claim 4, characterized in that, The specifications of the chromatographic column are a length of 80 mm - 120 mm, an inner diameter of 2 mm - 2.2 mm, and a particle size of the packing of 1.7 μm - 1.9 μm.

6. The method for constructing a feature map according to any one of claims 1-5, characterized in that, The specifications of the Wulingzhi medicinal material are Wulingzhi lumps or Wulingzhi grains; The characteristic chromatogram of the Wulingzhi lumps shows the following characteristic peaks: Peak 1, Peak 2, Peak 3, and Peak 4; The characteristic chromatogram of the Wulingzhi grains shows the following characteristic peaks: Peak 1, Peak 2, Peak 3, Peak 4, Peak 5, Peak 6, Peak 7, Peak 8, Peak 9, Peak 10, Peak 11, Peak 12; among them, Peak 8 is the characteristic peak corresponding to amentoflavone, and Peak 12 is the characteristic peak corresponding to hinokiflavone.

7. The method for constructing a feature map according to claim 6, characterized in that, Peak 4 is peak S1. The relative retention times of peaks 1-3, peaks 5-6 and peak S1 are calculated. The relative retention times are within ±10% of the specified values, which are: peak 1: 0.212; peak 2: 0.362; peak 3: 0.950; peak 5: 1.238; peak 6: 1.

462. Peak 8 is the S2 peak. The relative retention times of peaks 7, 9-12 and the S2 peak are calculated. The relative retention times are within ±10% of the specified values, which are: peak 7: 0.934; peak 9: 1.046; peak 10: 1.216; peak 11: 1.248; peak 12: 1.

297.

8. The method for constructing a feature map according to any one of claims 1-5 and 7, characterized in that, It also includes the following steps: The reference solution was analyzed by high performance liquid chromatography to obtain a chromatogram of the reference solution; the reference solution included protocatechuic acid, 4-hydroxybenzoic acid and benzoic acid. The chromatogram of the reference compound and the characteristic chromatogram of the *Wulingzhi* medicinal material are compared to identify the characteristic peaks in the characteristic chromatogram of the *Wulingzhi* medicinal material.

9. The method for constructing a feature map according to claim 8, characterized in that, When the medicinal material Wulingzhi is Lingzhi rice, the reference solution also includes Taxus chinensis flavonoids and Platycladus orientalis flavonoids.

10. The application of the method for constructing the characteristic spectrum according to any one of claims 1-9 in the identification of the medicinal material *Wulingzhi*.

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