Method for constructing fingerprint of Artemisia argyi medicinal material and its decoction pieces, standard decoction and formula granules

Abstract

The invention discloses a method for constructing a fingerprint of wormwood medicinal materials and its decoction pieces, standard decoctions and formula granules. The method comprises: extracting the wormwood medicinal materials or its decoction pieces, standard decoctions and formula granules with 20 vol% to 90 vol% methanol to obtain a test solution; dissolving neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, schaftoside, isochlorogenic acid B, 1,5-O-dicaffeoylquinic acid, isochlorogenic acid A, isochlorogenic acid C, brown cyanidin and isoeupatorium flavonoids with 80 vol% to 100 vol% methanol to obtain a reference solution; measuring with a liquid chromatograph to obtain a fingerprint of the wormwood medicinal materials or its decoction pieces, standard decoctions and formula granules; the fingerprint constructed by the invention is rich in information, can quickly and comprehensively reflect the quality of the wormwood medicinal materials or its decoction pieces, standard decoctions and formula granules, and is convenient for distinguishing wormwood from various counterfeit products.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine identification, and in particular to a method for constructing a fingerprint spectrum of wormwood leaf medicinal materials and decoction pieces, standard decoctions and formula granules thereof. Background Art

[0002] Artemisia argyi Levi, et Vant. is a dried leaf of the Asteraceae plant Artemisia argyi Levi, et Vant. It has the functions of warming the meridians and stopping bleeding, dispelling cold and relieving pain. It is used for vomiting blood, abalone blood, metrorrhagia, menorrhagia, fetal bleeding, cold pain in the lower abdomen, irregular menstruation, and infertility caused by cold uterus. Vinegar and wormwood charcoal is a processed product of wormwood, which has the effect of warming the meridians and stopping bleeding and is often used for bleeding caused by deficiency and cold. There are many varieties of wormwood, which are widely distributed and have abundant wild resources. Affected by the environmental and climatic conditions in different regions, the quality of wormwood varies. In history, there are famous and excellent varieties such as northern wormwood, sea wormwood, Qi wormwood, and Qi wormwood. In addition, it was found through investigation that wild small wormwood is often mixed in the wormwood sold on the market, which has similar properties to wormwood medicinal materials and is very easy to confuse, especially after being processed into vinegar and wormwood charcoal, the pieces are irregular fragments, which are difficult to accurately distinguish with the naked eye. Summary of the invention

[0003] The technical problem to be solved by the present invention is to provide a method for constructing a fingerprint of mugwort medicinal materials and their decoction pieces, standard decoctions, and formula granules, which has rich characteristic peak information and can comprehensively reflect the quality characteristics of mugwort in various forms, making it easy to distinguish it from various counterfeits.

[0004] In order to solve the above technical problems, the present invention provides a method for constructing a fingerprint of Artemisia argyi medicinal materials and their decoction pieces, standard decoctions, and formula granules, which comprises:

[0005] Extract the wormwood leaf medicinal material or its decoction pieces, standard decoction, or formula granules with 20 vol% to 90 vol% methanol to obtain a test solution;

[0006] Dissolve neochlorogenic acid reference substance, chlorogenic acid reference substance, cryptochlorogenic acid reference substance, caffeic acid reference substance, schaftoside reference substance, isochlorogenic acid B reference substance, 1,5-O-dicaffeoylquinic acid reference substance, isochlorogenic acid A reference substance, isochlorogenic acid C reference substance, cyanidin reference substance, and isoeupretinoic acid reference substance in 80 vol% to 100 vol% methanol to obtain a reference substance solution;

[0007] The test solution and the reference solution are measured by liquid chromatography to obtain the fingerprint of the Artemisia argyi medicinal material or its decoction pieces, standard decoction, and formula granules;

[0008] The chromatographic column of the liquid chromatograph uses octadecylsilane bonded silica gel as the stationary phase, acetonitrile as the mobile phase A, and 0.05 vol% to 0.2 vol% formic acid aqueous solution as the mobile phase B for gradient elution. The gradient elution curve is:

[0009] 0min~4min, mobile phase A from 8%→10%, mobile phase B from 92%→90%;

[0010] 4min~7min, mobile phase A from 10%→15%, mobile phase B from 90%→85%;

[0011] 7min-12min, mobile phase A from 15% to 18%, mobile phase B from 85% to 82%;

[0012] 12min~18min, mobile phase A changes from 18%→19%, mobile phase B changes from 82%→81%;

[0013] 18min-22min, mobile phase A from 19% to 21%, mobile phase B from 81% to 79%;

[0014] 22min-27min, mobile phase A from 21% to 30%, mobile phase B from 79% to 70%;

[0015] 27min-30min, mobile phase A from 30% to 36%, mobile phase B from 70% to 64%;

[0016] 30min-36min, mobile phase A from 36% to 45%, mobile phase B from 64% to 55%;

[0017] 36min-38min, mobile phase A from 45% to 65%, mobile phase B from 55% to 35%;

[0018] 38min-40min, mobile phase A from 65% to 95%, mobile phase B from 35% to 5%;

[0019] 40min-45min, mobile phase A is 95%, mobile phase B is 5%;

[0020] 45min-46min, mobile phase A from 95% to 8%, mobile phase B from 5% to 92%;

[0021] 46min-50min, mobile phase A is 8%, mobile phase B is 92%;

[0022] As an improvement of the above technical solution, the column length of the chromatographic column is 100 mm to 150 mm, the column diameter is 2 mm to 3 mm, the column temperature is 20° C. to 40° C., and the particle size of the stationary phase is 1.5 μm to 2.1 μm;

[0023] The flow rate of the liquid chromatograph is 0.2 mL / min to 0.35 mL / min, the injection volume of the test solution is 0.5 μL to 3 μL, and the injection volume of the reference solution is 0.5 μL to 3 μL;

[0024] The detection wavelength of the liquid chromatograph is 320nm-340nm.

[0025] As an improvement of the above technical solution, the column length of the chromatographic column is 150 mm, the column diameter is 2.1 mm, the column temperature is 25° C., and the particle size of the stationary phase is 1.7 μm;

[0026] The flow rate of the liquid chromatograph was 0.25 mL / min, the injection volume of the test solution was 1 μL, and the injection volume of the reference solution was 1 μL;

[0027] The detection wavelength of the liquid chromatograph is 325 nm.

[0028] As an improvement of the above technical solution, the mobile phase B is 0.1 vol% formic acid.

[0029] As an improvement of the above technical solution, the preparation method of the test solution is:

[0030] Take 1 g of mugwort leaf medicinal material or its decoction pieces, standard decoction, or formula granule powder, accurately weigh it, place it in a stoppered conical flask, accurately add 50 mL of 80 vol% methanol, weigh it, use an ultrasonic treatment with a power of 300 W and a frequency of 45 kHz for 30 min, take it out, cool it, weigh it again, make up the lost weight with 80 vol% methanol, shake it evenly, filter it through a 0.22 μm microporous filter membrane, and take the filtrate to obtain the product.

[0031] As an improvement of the above technical solution, the fingerprint spectrum of the wormwood medicinal material includes 17 common peaks, among which peak 1 is neochlorogenic acid, peak 2 is chlorogenic acid, peak 3 is cryptochlorogenic acid, peak 4 is caffeic acid, peak 5 is schaftoside, peak 6 is isochlorogenic acid B, peak 7 is 1,5-O-dicaffeoylquinic acid, peak 8 is isochlorogenic acid A, peak 9 is isochlorogenic acid C, peak 13 is cyanidin, and peak 15 is isocyanidin.

[0032] As an improvement of the above technical solution, the fingerprint spectrum of the wormwood slices includes 15 common peaks, among which peak 1 is neochlorogenic acid, peak 2 is chlorogenic acid, peak 3 is cryptochlorogenic acid, peak 4 is caffeic acid, peak 5 is schaftoside, peak 6 is isochlorogenic acid B, peak 7 is 1,5-O-dicaffeoylquinic acid, peak 8 is isochlorogenic acid A, peak 9 is isochlorogenic acid C, peak 13 is cyanidin, and peak 15 is isoeupretinoic acid.

[0033] The mugwort decoction piece is vinegar mugwort charcoal.

[0034] Correspondingly, the present invention also discloses a method for identifying wormwood products, which is used to identify wormwood and vinegar wormwood charcoal, and comprises:

[0035] Provide the substance to be identified;

[0036] Constructing the fingerprint of the substance to be identified by using the fingerprint construction method of the above-mentioned Artemisia argyi medicinal material and its decoction pieces, standard decoction, and formula granules;

[0037] If the fingerprint spectrum presents peaks 1 to 15, but not peaks 16 and 17, the substance to be identified is vinegar wormwood charcoal; if the fingerprint spectrum presents peaks 1 to 17, the substance to be identified includes or is wormwood leaf.

[0038] As an improvement of the above technical solution, the vinegar and moxa charcoal includes vinegar and moxa charcoal pieces, or vinegar and moxa charcoal standard decoction and vinegar and moxa charcoal formula granules obtained by processing vinegar and moxa charcoal pieces;

[0039] The mugwort includes mugwort medicinal materials, mugwort decoction pieces obtained by removing impurities or frying the mugwort medicinal materials, or mugwort standard decoctions and mugwort formula granules obtained by processing the mugwort decoction pieces.

[0040] Correspondingly, the present invention also discloses a method for identifying mugwort and its counterfeit, which is used to identify mugwort medicinal materials and wild mugwort medicinal materials; the method comprises:

[0041] Provide the substance to be identified;

[0042] Constructing the fingerprint of the substance to be identified by using the fingerprint construction method of the above-mentioned Artemisia argyi medicinal material and its decoction pieces, standard decoction, and formula granules;

[0043] If the peak area / sampling volume of peak 12 and peak 13 in the fingerprint spectrum is less than 2500 and 3900 respectively, or the fingerprint spectrum lacks peak 12 and peak 13 and has at least one obvious characteristic peak, then the substance to be identified includes or is wild Artemisia argyi medicinal material; if the fingerprint spectrum presents peaks 1 to 17, and the peak area / sampling volume of peak 12 and peak 13 is greater than 14000 and 59500 respectively, then the substance to be identified is Artemisia argyi medicinal material.

[0044] As an improvement of the above technical solution, if the characteristic spectrum lacks peaks 12 and 13, and there is an additional obvious characteristic peak between peaks 6 and 7, the substance to be identified includes or is Guangxi wild Artemisia argyi medicinal material;

[0045] If the peak areas / sampling amounts of peaks 12, 13, and 14 in the characteristic spectrum are less than 2500, 3900, and 2800, respectively, and there are two more obvious characteristic peaks between peaks 15 and 16, then the substance to be identified includes or is Shandong wild Artemisia argyi medicinal material;

[0046] If the characteristic spectrum shows peaks 1 to 17, and the peak areas / sampling volumes of peak 12, peak 13, and peak 14 are greater than 14,000, 59,500, and 6,800, respectively, the substance to be identified is mugwort leaf medicinal material.

[0047] Correspondingly, the present invention also discloses a method for identifying vinegar moxa charcoal and its counterfeits, which comprises:

[0048] Provide the substance to be identified;

[0049] Constructing the fingerprint of the substance to be identified by using the fingerprint construction method of the above-mentioned Artemisia argyi medicinal material and its decoction pieces, standard decoction, and formula granules;

[0050] If the peak areas / sampling volumes of peak 12 and peak 13 in the fingerprint are less than 600 and 1400 respectively, then the substance to be identified includes or is wild wormwood vinegar and wormwood charcoal slices prepared from wild wormwood medicinal materials; if the fingerprint shows peaks 1 to 15, and the peak areas / sampling volumes of peak 12 and peak 13 are greater than 880 and 3100 respectively, then the substance to be identified is vinegar and wormwood charcoal slices prepared from wormwood medicinal materials.

[0051] As an improvement of the above technical solution, if the characteristic spectrum lacks peak 5, peak 10, peak 12 and peak 13, the substance to be identified includes or is Guangxi wild wormwood vinegar and charcoal slices prepared from Guangxi wild wormwood medicinal materials;

[0052] If the peak areas / sampling amounts of peaks 12 and 13 in the characteristic spectrum are less than 600 and 1400, respectively, and there is an additional obvious characteristic peak after peak 15, then the substance to be identified includes or is the Shandong wild wormwood vinegar and charcoal slices prepared from Shandong wild wormwood medicinal materials;

[0053] If the characteristic spectrum shows peaks 1 to 15, and the peak areas / sampling volumes of peaks 12 and 13 are greater than 880 and 3100 respectively, the substance to be identified is the vinegar and charcoal mugwort slices prepared from mugwort medicinal materials.

[0054] The implementation of the present invention has the following beneficial effects:

[0055] The invention is based on the analysis of the material basis of wormwood, constructs a wormwood fingerprint spectrum, can provide more abundant characteristic peak information, thereby more comprehensively reflecting the sample quality, and the method is stable, with good precision and reproducibility. At the same time, it can effectively identify the processed products of wormwood and vinegar wormwood charcoal, wormwood and wild wormwood, and wormwood and wild wormwood. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 is a comparison chart of chromatographic results under different detection wavelength conditions in Example 1;

[0057] Figure 2is a comparison chart of the chromatographic results under different gradient elution conditions in Example 1;

[0058] Figure 3 is a comparison chart of chromatographic results under different mobile phase conditions in Example 1;

[0059] Figure 4 It is a comparison chart of the chromatographic results under different column temperature conditions in Example 1;

[0060] Figure 5 is a comparison chart of the chromatographic results under different flow rate conditions in Example 1;

[0061] Figure 6 It is the specific property investigation diagram in Example 1;

[0062] Figure 7 In Example 1 Figure 6 Results of chromatographic peak identification Figure 1 ;

[0063] Figure 8 In Example 1 Figure 6 Results of chromatographic peak identification Figure 2 ;

[0064] Fig. 9 In Example 1 Figure 6 Results of chromatographic peak identification Figure 3 ;

[0065] Fig.10 It is the fingerprint superposition diagram of 16 batches of Artemisia argyi medicinal materials in Example 2;

[0066] Fig.11 It is the fingerprint superposition diagram of 16 batches of vinegar and charcoal pieces of mugwort in Example 3;

[0067] Fig.12 It is a comparison diagram of the fingerprints of the wormwood leaf medicinal material and the vinegar wormwood charcoal decoction piece in Example 4;

[0068] Fig.13 It is a comparison diagram of the fingerprints of the Artemisia argyi medicinal material in Example 5 and the wild Artemisia argyi medicinal material in Guangxi;

[0069] Fig.14 It is a comparison diagram of the fingerprints of the Artemisia argyi medicinal material in Example 5 and 3 batches of Shandong wild Artemisia argyi medicinal materials;

[0070] Fig.15 It is a fingerprint comparison diagram of the vinegar and charcoal mugwort slices prepared from mugwort medicinal materials in Example 6 and the vinegar and charcoal mugwort slices prepared from wild mugwort leaves in Guangxi;

[0071] Fig.16 It is a fingerprint comparison diagram of the vinegar and charcoal pieces of mugwort prepared from mugwort medicinal materials in Example 6 and the vinegar and charcoal pieces of mugwort prepared from 3 batches of wild mugwort leaves in Shandong;

[0072] Among them: peak 1 is neochlorogenic acid, peak 2 is chlorogenic acid, peak 3 is cryptochlorogenic acid, peak 4 is caffeic acid, peak 5 is schaftoside, peak 6 is isochlorogenic acid B, peak 7 is 1,5-O-dicaffeoylquinic acid, peak 8 is isochlorogenic acid A, peak 9 is isochlorogenic acid C, peak 13 is cyanidin, and peak 15 is isoeupretinoic acid. DETAILED DESCRIPTION

[0073] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings and specific implementation methods.

[0074] The identification methods of mugwort in the prior art are difficult to effectively distinguish mugwort from various counterfeits, and are also difficult to distinguish various mugwort products. Specifically, there are four common processing methods for mugwort slices, namely: (1) Mugwort: Take the original medicinal material, remove impurities and stems, and sieve out the ash. (2) Vinegar mugwort: Take clean mugwort, add rice vinegar and mix well, steam until thoroughly moistened, put it in a pot, heat it over a low heat, fry it until dry, take it out, spread it out in time, and cool it thoroughly. Use 15 kg of rice vinegar for every 100 kg of mugwort. (3) Mugwort charcoal: Take clean mugwort, put it in a frying container, heat it over medium heat, fry it until the surface is charred black, spray a little water to extinguish the sparks, fry it slightly dry, take it out and spread it out to dry. (4) Vinegar mugwort charcoal: Take clean mugwort, fry it over high heat until the surface is charred black, spray rice vinegar to extinguish the sparks, fry it until dry, take it out, spread it out in time, and cool it thoroughly. Use 15 kg of rice vinegar for every 100 kg of mugwort. These pieces of medicine completely lose their taste, appearance and other characteristics after being processed into standard decoctions (the pieces of medicine are prepared by water decoction, solid-liquid separation, concentration and drying) and formula granules (the pieces of medicine are prepared by water heating extraction, separation, concentration, drying and granulation), making them even more difficult to identify. In addition, some counterfeit wormwood products are also difficult to identify after being processed into vinegar wormwood charcoal. To this end, the present invention proposes a method for constructing a fingerprint of wormwood medicinal materials and their pieces of medicine, standard decoctions and formula granules. The characteristic peak information in the obtained characteristic spectrum is rich, which provides a good basis for rapid and accurate identification. Specifically, the instruments, materials and sources of medicinal materials used in the following embodiments are as follows:

[0075] (1) Instruments: Waters ultra-high performance liquid chromatograph (Waters), PDA detector (Waters), Waters workstation; ME204E 1 / 10,000 balance (Mettler Toledo); XP26 1 / 10,000 balance (Mettler Toledo); 111B 2-liang high-speed Chinese medicine pulverizer (Zhejiang Ruian Yongli Pharmaceutical Machinery Co., Ltd.); CNC KQ-500DE ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); MiliQ Direct 8 ultrapure water machine (Merck Millipore).

[0076] (2) Materials: Neochlorogenic acid (batch number: 14332, content: 98%, Shanghai Shidande Standard Technology Service Co., Ltd.); Chlorogenic acid (batch number: 110753-201817, content: 96.3%, China Food and Drug Inspection Institute); Cryptochlorogenic acid (batch number: DST220104-035, content: 98%, Chengdu Lemeitian Pharmaceutical Technology Co., Ltd.); Caffeic acid (batch number: 110885-201703, content: 99.7%, China Food and Drug Inspection Institute); Schaftoside (batch number: 111912-202204, content: 94.9%, China Food and Drug Inspection Institute); Isochlorogenic acid B (batch number: wkq210222206, content: 99.85%, Sichuan Weikeqi Biotechnology Co., Ltd.); 1,5-O-dichlorobenzene Caffeoylquinic acid (batch number: MUST-19092911, content: 98.52%, Chengdu Mansite Biotechnology Co., Ltd.); isochlorogenic acid A (batch number: 111782-202208, content: 95.9%, China Food and Drug Inspection Institute); isochlorogenic acid C (batch number: DSTDY003802, content: 99.58%, Desite Biotechnology Co., Ltd.); brown cyanidin (batch number: Z05801909018, content: 98%, Chengdu Ruifensi Biotechnology Co., Ltd.); isozeupetin (batch number: 3670, content: 98%, Shanghai Shidande Standard Technology Service Co., Ltd.); acetonitrile is chromatographically pure (Merck), formic acid is chromatographically pure (Tianjin Komiou Chemical Reagent Co., Ltd.), water is ultrapure water, and other reagents are analytically pure. Artemisia argyi (16 batches) and wild Artemisia argyi (4 batches), detailed information is shown in Table 1.

[0077] Table 1 Information on the source of medicinal materials

[0078]

[0079] Example 1 Construction of Artemisia argyi fingerprint

[0080] 1. Preparation of test solution

[0081] Take about 1 g of sample powder (passed through No. 3 sieve), weigh accurately, place in a stoppered conical flask, accurately add 50 mL of 80% methanol, weigh the weight, ultrasonically treat (power 300 W, frequency 45 kHz) for 30 min, take out, cool, weigh again, make up the lost weight with 80% methanol, shake well, pass through a 0.22 μm microporous filter membrane, and take the filtrate to obtain.

[0082] 2. Preparation of reference solution

[0083] Take appropriate amount of neochlorogenic acid reference substance, chlorogenic acid reference substance, cryptochlorogenic acid reference substance, caffeic acid reference substance, schaftoside reference substance, isochlorogenic acid B reference substance, 1,5-O-dicaffeoylquinic acid reference substance, isochlorogenic acid A reference substance, isochlorogenic acid C reference substance, brown cyanidin reference substance, and isoeupretinoic acid reference substance, accurately weigh them, and add 100 vol% methanol to make a solution containing 43.45 μg of neochlorogenic acid, 87.73 μg of chlorogenic acid, 33.32 μg of cryptochlorogenic acid, 43.55 μg of caffeic acid, 20.60 μg of schaftoside, and isochlorogenic acid B per 1 ml. The reference solution contained 20.81μg, 1,5-O-dicaffeoylquinic acid 20.54μg, isochlorogenic acid A 21.33μg, isochlorogenic acid C 11.59μg, cyanidin 21.25μg, and isoeupretinoic acid 22.54μg.

[0084] 3. Chromatographic conditions

[0085] Chromatographic column: ACQUITY UPLC HSS T3 Column (2.1 mm×150 mm, 1.7 μm); mobile phase: acetonitrile (A)-0.1% formic acid aqueous solution (B); elution mode: gradient elution; flow rate: 0.25 mL / min, column temperature: 25°C, detection wavelength: 325 nm. The mobile phase gradient elution program is shown in Table 2.

[0086] Table 2 Elution program

[0087]

[0088] 4. Determination

[0089] Accurately pipette 1 μl of reference solution and 1 μl of test solution respectively, inject into liquid chromatograph, and measure to obtain the result.

[0090] 5. Methodological investigation

[0091] (1) Investigation of extraction solvents

[0092] Take three portions of sample powder (pass through No. 3 sieve) of about 1g each, weigh accurately, place in a stoppered conical bottle, accurately add 80% methanol 25mL, 50mL, 100mL, weigh, ultrasonic treatment (power 300W, frequency 45kHz) for 30min, take out, cool, weigh again, make up the lost weight with 80% methanol, shake well, pass through 0.22μm microporous filter membrane, take the filtrate, and get it. According to the chromatographic conditions determined under "3", inject and measure, the results are shown in Table 3. The results show that when the amount of extraction solvent is 25mL, 50mL, and 100mL, the numerical difference of the total characteristic peak area / sampling amount × solvent amount of the three characteristic spectra is not large. Based on the consideration of saving solvent, the amount of extraction solvent is determined to be 50mL.

[0093] Table 3 Results of investigation on different solvent dosages

[0094]

[0095] (2) Investigation of extraction methods

[0096] Take two portions of sample powder (pass through No. 3 sieve) of about 1g each, weigh accurately, place in a stoppered conical flask, accurately add 80% methanol 50mL, weigh, ultrasonically treat (power 300W, frequency 45kHz) for 30min and heat reflux for 30min, take out, cool, weigh again, make up the lost weight with 80% methanol, shake well, filter, take the filtrate, and get it. According to the chromatographic conditions determined under "3", inject and measure, the results are shown in Table 4. The results show that there is no significant difference in "total peak area / sample weight" of different extraction methods. Considering the simplicity of operation, the extraction method is determined to be ultrasonic.

[0097] Table 4 Results of investigation of different extraction methods

[0098]

[0099] (3) Extraction time

[0100] Take three portions of sample powder (pass through No. 3 sieve) of about 1g each, weigh accurately, place in a stoppered conical flask, accurately add 50mL of 80% methanol, weigh, and ultrasonically treat (power 300W, frequency 45kHz) for 15min, 30min, and 45min, respectively, take out, cool, weigh again, make up the lost weight with 80% methanol, shake well, filter, and take the filtrate. According to the chromatographic conditions determined under "3", sample injection and determination are shown in Table 5. The results show that there is no significant difference in "total peak area / sample weight" at different ultrasonic times. In order to ensure complete extraction, the extraction time is preferably determined to be 30min.

[0101] Table 5 Results of investigation at different extraction times

[0102]

[0103] (4) Wavelength investigation

[0104] Take about 1g of sample powder (pass through No. 3 sieve), weigh accurately, put it in a stoppered conical flask, add 50mL of 80% methanol accurately, weigh the weight, ultrasonically treat (power 300W, frequency 45kHz) for 30min, take it out, let it cool, weigh it again, make up the lost weight with 80% methanol, shake it well, filter it, and take the filtrate. Elute according to the chromatographic gradient, column temperature, and flow rate determined under "3", and record the chromatograms of the test solution at 325nm, 330nm, and 340nm respectively. Compare the collected chromatograms to determine the best wavelength. The results are as follows: Figure 1 The results show that the peak shapes of the chromatographic peaks at 325 nm are better than those at 330 nm and 340 nm, and the absorption intensity of the main chromatographic peaks is high, so the wavelength of 325 nm is selected.

[0105] (5) Mobile phase gradient investigation

[0106] Take the same test solution, use acetonitrile (A)-0.1% formic acid solution (B) as the mobile phase, and elute according to the gradient in Tables 6-9. Column temperature 25℃; detection wavelength 325nm; flow rate 0.25mL / min, injection volume 1μL. Results are shown in Figure 2 The results showed that the separation of the chromatographic peaks under gradient 4 was relatively better than that under gradient 1, gradient 2 and gradient 3, so gradient 4 with better separation was finally determined as the elution gradient of the characteristic spectrum.

[0107] Table 6 Gradient elution table

[0108]

[0109] Table 7 Gradient elution table

[0110]

[0111] Table 8 Gradient elution table

[0112]

[0113] Table 9 Gradient elution table

[0114]

[0115] (6) Investigation of mobile phase types

[0116] Take the same test solution and examine the mobile phases of acetonitrile-0.1% formic acid solution (B) and methanol-0.1% formic acid solution (B) respectively. According to the chromatographic conditions determined under "3", inject the sample and measure. The results are as follows: Figure 3 As shown in the figure, the acetonitrile-0.1% formic acid solution (B) system has relatively more peak information, better separation effect of each peak, and good peak shape of each component, so the acetonitrile-0.1% formic acid solution (B) system is selected.

[0117] (7) Column temperature investigation

[0118] Take the same test solution and test it at column temperatures of 20℃, 25℃ and 30℃ respectively. According to the chromatographic conditions determined under "3", inject the sample and measure. The results are as follows: Figure 4 The results show that when the column temperature is 25℃, the chromatogram peak shape is good and the separation is moderate. Therefore, the column temperature is determined to be 25℃.

[0119] (8) Flow rate investigation

[0120] Based on the experimental conditions proposed above, the flow rates were investigated at 0.23 mL / min, 0.25 mL / min, and 0.27 mL / min. The results are as follows: Figure 5 The results show that when the flow rate is 0.25mL / min, the chromatogram peak shape is good and the separation is moderate. Therefore, the flow rate is determined to be 0.25mL / min.

[0121] 6. Methodological Validation

[0122] (1) Specificity inspection

[0123] Accurately pipette 1 μl of the test solution and blank solvent, inject into the liquid chromatograph, and measure according to the chromatographic conditions under "3". Figure 6 The experimental results show that there are no chromatographic peaks in the blank solvent chromatogram at the retention time corresponding to the characteristic peaks of the test sample chromatogram, indicating that the blank solvent has no interference, indicating that the method has good specificity.

[0124] (2) Precision inspection

[0125] Take the same test solution and inject it continuously for 6 times according to the chromatographic conditions under item "3", record the retention time and peak area, take the chromatographic peak of isochlorogenic acid A as the reference peak (S), and calculate the RSD values ​​of the relative retention time and relative peak area of ​​each characteristic peak, which are all less than 3%, indicating that the precision of the instrument is good.

[0126] (3) Repeatability study

[0127] Take 6 portions of the same batch of wormwood powder, weigh them accurately, prepare the test solution according to the method under "1", and measure them according to the chromatographic conditions under "3". Take the chromatographic peak of isochlorogenic acid A as the reference peak (S), and calculate the RSD values ​​of the relative retention time and relative peak area of ​​each characteristic peak, which are all less than 3%, indicating that the method has good repeatability.

[0128] (4) Stability test

[0129] Accurately aspirate the same test solution, and according to the chromatographic conditions under item "3", inject and measure at 0, 2, 4, 6, 8, 10, 12, 18, and 24 h, respectively, record the retention time and peak area, take the isochlorogenic acid A chromatographic peak as the reference peak (S), and calculate the RSD of the relative retention time and relative peak area of ​​each characteristic peak, which are all less than 3%, indicating that the test solution has good stability within 24 h.

[0130] 7. Identification of chromatographic peaks

[0131] Neochlorogenic acid reference substance, chlorogenic acid reference substance, cryptochlorogenic acid reference substance, caffeic acid reference substance, schaftoside reference substance, isochlorogenic acid B reference substance, 1,5-O-dicaffeoylquinic acid reference substance, isochlorogenic acid A reference substance, isochlorogenic acid C reference substance, brown cyanidin reference substance, and isoeupretinoic acid reference substance were used for positioning. The results showed that peak 1 was neochlorogenic acid, peak 2 was chlorogenic acid, peak 3 was cryptochlorogenic acid, peak 4 was caffeic acid, peak 5 was schaftoside, peak 6 was isochlorogenic acid B, peak 7 was 1,5-O-dicaffeoylquinic acid, peak 8 was isochlorogenic acid A, peak 9 was isochlorogenic acid C, peak 13 was brown cyanidin, and peak 15 was isoeupretinoic acid. Results are shown in Figure 7 to Figure 9 .

[0132] Example 2 Fingerprint of Artemisia argyi medicinal material

[0133] Take 16 batches of Artemisia argyi medicinal materials, and sample and measure according to the chromatographic conditions under item "3" in Example 1 and the test solution preparation method determined under item "1", and obtain the fingerprints of 16 batches of Artemisia argyi. The results are shown in Fig.10 The results showed that the fingerprints of the 16 batches of Artemisia argyi all showed the same 17 common peaks. Therefore, the above 17 common peaks were selected as the characteristic peaks of the fingerprint of Artemisia argyi, and peak 8 (isochlorogenic acid A) was used as the reference peak to calculate the relative retention time and relative peak area of ​​each characteristic peak and the S peak, as shown in Tables 10 and 11.

[0134] Table 10 Relative retention time of fingerprints of 16 batches of Artemisia argyi medicinal materials

[0135]

[0136] Table 11 Relative peak areas of fingerprints of 16 batches of Artemisia argyi medicinal materials

[0137]

[0138] Embodiment 3 vinegar moxa charcoal fingerprint

[0139] Take 16 batches of vinegar moxa charcoal samples respectively, prepare the test solution according to the method under "1" in Example 1, and measure according to the chromatographic conditions under "3" to obtain the fingerprints of 16 batches of vinegar moxa charcoal. The results are shown in Fig.11 The results showed that the fingerprints of the 16 batches of vinegar and wormwood charcoal all showed the same 15 common peaks. Therefore, the above 15 common peaks were selected as the characteristic peaks of the vinegar and wormwood charcoal fingerprint, and peak 8 (isochlorogenic acid A) was used as the reference peak to calculate the relative retention time and relative peak area of ​​each characteristic peak and the S peak, as shown in Tables 12 and 13.

[0140] Table 12 Relative retention time of vinegar and moxa charcoal fingerprints of 16 batches

[0141]

[0142] Table 13 Relative peak areas of vinegar and moxa charcoal fingerprints of 16 batches

[0143]

[0144] Example 4 Identification of Folium Artemisiae Argyi and Vinegar Artemisiae Argyi Charcoal

[0145] Take about 1g of wormwood leaf powder and vinegar wormwood charcoal powder respectively, accurately weigh them, prepare the test solution according to the method under "1" in Example 1, accurately take 1μL of the reference solution and different test solutions respectively, and measure them according to the method under "3" in Example 1 to obtain fingerprints. Compare the differences between the fingerprints of wormwood leaf and vinegar wormwood charcoal. The results are shown in Fig.12 .

[0146] As can be seen from the figure, the fingerprint of mugwort medicinal material presents 17 characteristic peaks, and the fingerprint of vinegar and mugwort charcoal slices presents 15 characteristic peaks, among which peaks 16 and 17 disappear after the mugwort medicinal material is processed into vinegar and mugwort charcoal slices.

[0147] Based on the above-mentioned Example 4, it can be seen that the fingerprint spectrum can effectively identify the mugwort medicinal material and the vinegar mugwort charcoal slices. Accordingly, since the processing methods such as removing impurities and frying will not affect the main components of the mugwort medicinal material, and the preparation process of the standard decoction and formula granules will not affect the main components of the mugwort medicinal material and the vinegar mugwort charcoal slices. Therefore, the following substances (1) and (2) can also be identified through the fingerprint spectrum:

[0148] (1) Artemisia argyi medicinal materials, Artemisia argyi medicinal pieces obtained by removing impurities or frying Artemisia argyi medicinal materials, or Artemisia argyi standard decoctions and Artemisia argyi formula granules obtained by processing the Artemisia argyi medicinal pieces,

[0149] (2) Vinegar and mugwort charcoal slices, or vinegar and mugwort charcoal standard decoction and vinegar and mugwort charcoal formula granules obtained by processing the vinegar and mugwort charcoal slices.

[0150] Example 5 Identification of Artemisia argyi and wild Artemisia argyi medicinal materials

[0151] Take about 1g of wormwood leaf powder and wild wormwood leaf powder respectively, weigh them accurately, prepare the test solution according to the method under "1" in Example 1, accurately take 1μL of the reference solution and different test solutions respectively, and measure them according to the method under "3" in Example 1 to obtain fingerprints. Compare the differences in fingerprints between wormwood and wild wormwood leaves from different origins. The results are shown in Figure 13-14 .

[0152] As can be seen from the figure, there is an additional obvious chromatographic peak between peak 6 and peak 7 in the fingerprint of wild wormwood leaves in Guangxi, named a, and the fingerprint of wild wormwood leaves in Guangxi lacks peaks 12 and 13; compared with the wormwood fingerprint characteristic spectrum, the fingerprint spectra of the three batches of wild wormwood leaves in Shandong have two more obvious chromatographic peaks between peak 15 and peak 16, named b and c, and the peaks 12-14 are particularly small (the peak area of ​​peak 12 / sampling volume <2500, the peak area of ​​peak 13 / sampling volume <3900, and the peak area of ​​peak 14 / sampling volume <2800).

[0153] Example 6 Identification of Vinegar and Charcoal Pieces of Artemisia argyi and Wild Artemisia argyi

[0154] Take about 1g of vinegar and charcoal powder of mugwort prepared from mugwort medicinal materials and about 1g of vinegar and charcoal powder of wild mugwort medicinal materials from different origins, accurately weigh them, prepare the test solution according to the method under "1" in Example 1, and closely pipette 1μL of the reference solution and different test solutions, respectively, and measure them according to the method under "3" in Example 1 to obtain the fingerprint. The results are shown in Figure 15-16 .

[0155] As can be seen from the figure, compared with the vinegar and wormwood charcoal fingerprint characteristic spectrum, the fingerprint spectrum of Guangxi wild wormwood vinegar charcoal lacks peaks 5, peak 10, peak 12, and peak 13; compared with the vinegar and wormwood charcoal fingerprint characteristic spectrum, the fingerprint spectra of the three batches of Shandong wild wormwood vinegar charcoal all have an additional obvious chromatographic peak after peak 15, named d, and the peaks 12-13 are very low (the peak area of ​​peak 12 / sampling volume <600, the peak area of ​​peak 13 / sampling volume <1400).

[0156] The above is a preferred embodiment of the invention. It should be pointed out that a person skilled in the art can make several improvements and modifications without departing from the principle of the invention. These improvements and modifications are also considered to be within the scope of protection of the invention.

Claims

1. A method for constructing fingerprints of Artemisia argyi medicinal materials and their decoction pieces, standard decoctions, and formula granules, characterized in that: include: Extract the wormwood leaf medicinal material or its decoction pieces, standard decoction, and formula granules with 20 vol% to 90 vol% methanol to obtain a test solution; Dissolve the neochlorogenic acid reference substance, chlorogenic acid reference substance, cryptochlorogenic acid reference substance, caffeic acid reference substance, schaftoside reference substance, isochlorogenic acid B reference substance, 1,5-O-dicaffeoylquinic acid reference substance, isochlorogenic acid A reference substance, isochlorogenic acid C reference substance, cyanidin reference substance, and isoeupretinoic acid reference substance in 80 vol% to 100 vol% methanol to obtain a reference substance solution; The test solution and the reference solution are measured by liquid chromatography to obtain the fingerprint of the Artemisia argyi medicinal material or its decoction pieces, standard decoction, and formula granules; The chromatographic column of the liquid chromatograph is an ACQUITY UPLC HSS T3 chromatographic column, with a column length of 150 mm, a column diameter of 2.1 mm, and a particle size of the stationary phase of 1.7 μm. The liquid chromatograph uses acetonitrile as mobile phase A and 0.05 vol% to 0.2 vol% formic acid aqueous solution as mobile phase B for gradient elution. The gradient elution curve is: 0min~4min, mobile phase A from 8%→10%, mobile phase B from 92%→90%; 4min~7min, mobile phase A from 10% to 15%, mobile phase B from 90% to 85%; 7min~12min, mobile phase A from 15% to 18%, mobile phase B from 85% to 82%; 12min~18min, mobile phase A from 18% to 19%, mobile phase B from 82% to 81%; 18min~22min, mobile phase A changed from 19% to 21%, and mobile phase B changed from 81% to 79%; 22min~27min, mobile phase A from 21% to 30%, mobile phase B from 79% to 70%; 27min~30min, mobile phase A from 30% to 36%, mobile phase B from 70% to 64%; 30min~36min, mobile phase A from 36% to 45%, mobile phase B from 64% to 55%; 36min~38min, mobile phase A from 45% to 65%, mobile phase B from 55% to 35%; 38min~40min, mobile phase A from 65% to 95%, mobile phase B from 35% to 5%; 40min~45min, mobile phase A is 95%, mobile phase B is 5%; 45min~46min, mobile phase A from 95% to 8%, mobile phase B from 5% to 92%; 46min~50min, mobile phase A is 8%, mobile phase B is 92%; The detection wavelength of the liquid chromatograph is 320nm~340nm; The fingerprint spectrum of the wormwood medicinal material includes 17 common peaks, among which peak 1 is neochlorogenic acid, peak 2 is chlorogenic acid, peak 3 is cryptochlorogenic acid, peak 4 is caffeic acid, peak 5 is schaftoside, peak 6 is isochlorogenic acid B, peak 7 is 1,5-O-dicaffeoylquinic acid, peak 8 is isochlorogenic acid A, peak 9 is isochlorogenic acid C, peak 13 is cyanidin, and peak 15 is isoeupretinoic acid.

2. The method for constructing the fingerprint of the Artemisia argyi medicinal material and its decoction pieces, standard decoctions, and formula granules as claimed in claim 1, characterized in that: The column temperature of the chromatographic column is 20°C to 40°C; The flow rate of the liquid chromatograph is 0.2 mL / min~0.35 mL / min, the injection volume of the test solution is 0.5 μL~3 μL, and the injection volume of the reference solution is 0.5 μL~3 μL.

3. The method for constructing the fingerprint of the Artemisia argyi medicinal material and its decoction pieces, standard decoction, and formula granules as claimed in claim 1 or 2, characterized in that: The column temperature of the chromatographic column is 25°C; The flow rate of the liquid chromatograph was 0.25 mL / min, the injection volume of the test solution was 1 μL, and the injection volume of the reference solution was 1 μL.

4. The method for constructing the fingerprint of the Artemisia argyi medicinal material and its decoction pieces, standard decoctions, and formula granules as claimed in claim 1, characterized in that: The mobile phase B is 0.1 vol% formic acid.

5. The method for constructing the fingerprint of the Artemisia argyi medicinal material and its decoction pieces, standard decoctions, and formula granules as claimed in claim 1, characterized in that: The preparation method of the test solution is: Take 1 g of mugwort leaf medicinal material or its decoction pieces, standard decoction, or formula granule powder, accurately weigh it, place it in a stoppered conical flask, accurately add 50 mL of 80 vol% methanol, weigh it, use ultrasonic treatment with a power of 300 W and a frequency of 45 kHz for 30 min, take it out, cool it, weigh it again, make up the lost weight with 80 vol% methanol, shake it evenly, filter it through a 0.22 μm microporous filter membrane, and take the filtrate to obtain it.

6. The method for constructing the fingerprint of the Artemisia argyi medicinal material and its decoction pieces, standard decoctions, and formula granules as claimed in claim 1, characterized in that: The fingerprint spectrum of the wormwood slices includes 15 common peaks, among which peak 1 is neochlorogenic acid, peak 2 is chlorogenic acid, peak 3 is cryptochlorogenic acid, peak 4 is caffeic acid, peak 5 is schaftoside, peak 6 is isochlorogenic acid B, peak 7 is 1,5-O-dicaffeoylquinic acid, peak 8 is isochlorogenic acid A, peak 9 is isochlorogenic acid C, peak 13 is cyanidin, and peak 15 is isoeupretinoic acid.

7. A method for identifying wormwood medicinal materials and vinegar wormwood charcoal pieces, characterized in that: include: Provide the substance to be identified; Constructing the fingerprint of the substance to be identified by the method for constructing the fingerprint of the Artemisia argyi medicinal material and its decoction pieces, standard decoction, and formula granules according to any one of claims 1 to 6; If the fingerprint spectrum presents peaks 1 to 15, but not peaks 16 and 17, the substance to be identified is vinegar and charcoal mugwort slices; if the fingerprint spectrum presents peaks 1 to 17, the substance to be identified is mugwort leaf medicinal material.

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