Construction method, detection method and application of specific chromatogram of szechuan lovage rhizome perfume extract

Abstract

The invention relates to the technical field of medicine analysis and traditional Chinese medicine detection, in particular to a construction method, a detection method and application of a specific chromatogram of a szechuan lovage rhizome perfume extract. Wherein the conditions of the liquid chromatographic analysis are as follows: gradient elution is carried out by adopting an aqueous solution of a mobile phase A acetonitrile and a mobile phase B phosphoric acid for 0-5 minutes, the volume percent of the mobile phase A is maintained to be 4% and 5-12 minutes, the volume percent of the mobile phase A is increased to 16% from 4% and 12-19 minutes, the volume percent of the mobile phase A is maintained to be 16% and 19-29 minutes, the volume percent of the mobile phase A is increased to 40% from 16% and 29-31 minutes, and the volume percent of the mobile phase A is maintained to be 4% and 12-19 minutes. The volume percentage of the mobile phase A is increased to 55% from 40%, 31-37 min, and the volume percentage of the mobile phase A is maintained to be 55%. The method can be used for quantitatively determining the water-soluble components of the szechuan lovage rhizome perfume extract, and can also be used for identifying the true and false.

Description

Technical Field

[0001] The present application relates to the technical field of drug analysis and traditional Chinese medicine detection, and in particular to a method for constructing a characteristic spectrum of Sichuan wood fragrance extract, a detection method and applications thereof. Background Art

[0002] Costus root (Vladimiria souliei (Franch.) Ling) or Vladimiria souliei (Franch.) Ling var. cinerea Ling, both of the Asteraceae family, is primarily found in western Sichuan and is a local medicinal material in the province. Its properties are pungent, bitter, and warm, and it enters the spleen, stomach, large intestine, and gallbladder meridians. It promotes qi circulation and relieves pain, and is used to treat chest, flank, and abdominal distension and pain, intestinal rumbling, diarrhea, and tenesmus. Modern chemical research has shown that Costus root (Vladimiria souliei) is rich in active ingredients such as sesquiterpenes, triterpenes, lignans, and organic acids, exhibiting multiple effects, including anti-inflammatory, antispasmodic, anti-tumor, and therapeutic effects in the treatment of digestive tract diseases. Establishing a detection method for Costus root (Vladimiria souliei) will help explore the relationship between the pharmacological activities of its chemical components and the symptomatic effects of traditional Chinese medicines. This will facilitate the integration of traditional Chinese medicine with the knowledge and methods of Western medicine, expanding the application scope of traditional Chinese medicine and improving its clinical efficacy.

[0003] Lv Luyang et al. disclosed a UPLC detection method for Sichuan costus root medicinal materials and established a fingerprint spectrum of Sichuan costus root. 12 common peaks were identified in the spectrum, and the separation effect of each characteristic peak needs to be improved. Hu Yijie et al. disclosed a HPLC detection method for Sichuan costus root medicinal materials and established a fingerprint spectrum of Sichuan costus root. 40 common peaks were identified in the spectrum, but no identification was performed. The spectrum baseline flatness, characteristic peak peak shape, separation, etc. need to be improved. In addition, the above detection method also lacks quantitative research on water-soluble components. Sichuan costus root is mainly used in the form of water extract (decoction obtained by decoction or further concentrated preparation), and water-soluble components are an important material basis for its clinical efficacy. The current "Chinese Pharmacopoeia" and literature all use the content of Sichuan costus root fat-soluble components costunolide and dehydrocostunolide as the quality evaluation index of Sichuan costus root, and there are few reports on the content determination of water-soluble components such as phenolic acids.

[0004] Phenolic acids in Radix Aucklandiae Radix have anti-inflammatory, antibacterial, antioxidant, and anti-tumor pharmacological activities. These activities are closely related to the traditional medicinal functions of Radix Aucklandiae Radix and are of great research value and application potential. However, no method has been found to determine the content of phenolic acids in a standardized decoction of Radix Aucklandiae Radix.

[0005] Sichuan costus root contains multiple chemical components, each of which interacts synergistically. Therefore, measuring the content of a few specific components as a quality evaluation indicator is incomplete. A traditional Chinese medicine fingerprint / characteristic spectrum refers to a chromatogram or spectrum that identifies the chemical characteristics of a traditional Chinese medicine or preparation after appropriate treatment and analytical methods. This allows for a holistic consideration of the composition of the traditional Chinese medicine's ingredients, providing a reliable basis for evaluating the quality of the medicine and verifying its authenticity. Summary of the Invention

[0006] Based on this, one or more embodiments of the present application provide a method for constructing a characteristic spectrum of Sichuan wood fragrance extract, a detection method and its application.

[0007] The technical solution of this application includes the following contents:

[0008] A method for constructing a characteristic spectrum of Sichuan wood fragrance extract comprises the following steps:

[0009] A test sample is taken, and an extraction solvent is added for extraction to prepare a test sample solution; the test sample is an extract of Sichuan wood fragrance;

[0010] Taking the test solution for liquid chromatography analysis to construct a characteristic spectrum of Sichuan wood fragrance extract;

[0011] Wherein, the conditions of the liquid chromatography analysis include:

[0012] (1) Mobile phase A is acetonitrile, and mobile phase B is an aqueous solution of 0.08% to 0.12% by volume phosphoric acid;

[0013] (2) Gradient elution is used, and the procedure of the gradient elution includes:

[0014] 0-5min, maintain the volume percentage of the mobile phase A at 4%,

[0015] 5min~12min, the volume percentage of the mobile phase A increases from 4% to 16%,

[0016] 12min~19min, maintain the volume percentage of the mobile phase A at 16%,

[0017] From 19min to 29min, the volume percentage of the mobile phase A increased from 16% to 40%.

[0018] From 29 to 31 minutes, the volume percentage of the mobile phase A increased from 40% to 55%.

[0019] From 31 min to 37 min, the volume percentage of the mobile phase A was maintained at 55%.

[0020] A method for detecting an extract of Sichuan hibiscus odorifera comprises the following steps:

[0021] Take the Kawaki Fragrance extract to be tested, add the extraction solvent to extract, and prepare the test solution;

[0022] Taking the test solution for liquid chromatography analysis to detect the content of the index component in the test solution, wherein the index component includes at least one of costunolide, dehydrocostus lactone, chlorogenic acid and 4,5-di-O-caffeoylquinic acid;

[0023] Wherein, the conditions of the liquid chromatography analysis include:

[0024] (1) Mobile phase A is acetonitrile, and mobile phase B is an aqueous solution of 0.08% to 0.12% by volume phosphoric acid;

[0025] (2) Gradient elution is used, and the procedure of the gradient elution includes:

[0026] 0-5min, maintain the volume percentage of the mobile phase A at 4%,

[0027] 5min~12min, the volume percentage of the mobile phase A increases from 4% to 16%,

[0028] 12min~19min, maintain the volume percentage of the mobile phase A at 16%,

[0029] From 19min to 29min, the volume percentage of the mobile phase A increased from 16% to 40%.

[0030] From 29 to 31 minutes, the volume percentage of the mobile phase A increased from 40% to 55%.

[0031] From 31 min to 37 min, the volume percentage of the mobile phase A was maintained at 55%.

[0032] A method for constructing a characteristic spectrum and an application of the characteristic spectrum constructed by the method and / or the detection method in identifying Sichuan wood fragrance extracts.

[0033] This application uses liquid chromatography to establish a fingerprint of Sichuan wood fragrance extract to characterize the main components in Sichuan wood fragrance extract. At the same time, a detection method for Sichuan wood fragrance extract is established, which can be used to determine the content of characteristic components such as costunolide, dehydrocostus lactone, chlorogenic acid and 4,5-di-O-caffeoylquinic acid, thereby providing a reference for the identification and quality control of Sichuan wood fragrance extract. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the specific implementation methods of the present application or the technical solutions in the prior art, the following is a brief introduction to the drawings required for use in the specific implementation methods or the description of the prior art. Obviously, the drawings described below are some implementation methods of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0035] Figure 1 This is a liquid chromatogram of the standard decoction of Kawana Radix obtained by detection under gradient elution procedure 1 in Example 1 of the present application;

[0036] Figure 2 This is a liquid chromatogram of the standard decoction of Kawana Radix obtained by detection under gradient elution procedure 2 in Example 1 of the present application;

[0037] Figure 3 This is a liquid chromatogram of the standard decoction of Kawana Radix obtained by detection under gradient elution procedure 3 in Example 1 of the present application;

[0038] Figure 4 This is the test result of 3D scanning of the standard decoction of Sichuan costus root at 210nm to 400nm in Example 1 of the present application;

[0039] Figure 5 Comparison results of the liquid chromatograms of the standard decoction of Sichuan costus root obtained under different chromatographic column conditions in Example 1 of the present application;

[0040] Figure 6 The results are compared in Example 1 of the present application using different extraction solvents to extract the standard decoction of Kasumihara and perform liquid chromatography detection;

[0041] Figure 7 The results are compared in Example 1 of the present application using different extraction methods to extract the standard decoction of Kasumihara and perform liquid chromatography detection;

[0042] Figure 8 The results are compared in Example 1 of the present application using different extraction times for the standard decoction of Radix Aucklandiae Root and performing liquid chromatography detection;

[0043] Figure 9 The results are compared in Example 1 of the present application using different volumes of extraction solvent to extract the standard decoction of Kawana Radix and perform liquid chromatography detection;

[0044] Figure 10 This is an overlay of liquid chromatograms obtained by testing 14 batches of standard decoction of Sichuan costus root in Example 1 of the present application;

[0045] Figure 11 This is the characteristic spectrum of the standard decoction of Sichuan costus root prepared in Example 1 of the present application;

[0046] Figure 12 This is the liquid chromatogram of the reference medicinal material of Sichuan costus root in Example 1 of the present application;

[0047] Figure 13 The mass spectra of chlorogenic acid in the standard decoction of Costusroot Root and the reference solution in Example 1 of the present application (m / z 353.09>191.06);

[0048] Figure 14 The mass spectra of 4,5-di-O-caffeoylquinic acid of the standard decoction of Aucklandia lappa and the reference solution in Example 1 of the present application (m / z 515.12>173.04);

[0049] Figure 15 The mass spectra of costunolide in the standard decoction of Radix Aucklandiae Root and the reference solution in Example 1 of the present application (m / z 233.15>187.15);

[0050] Figure 16 The mass spectra of dehydrocostus lactone of the standard decoction of Radix Aucklandiae Root and the reference solution in Example 1 of the present application (m / z 231.14>185.03);

[0051] Figure 17 The characteristic peak identification results of the characteristic spectrum of the standard decoction of Sichuan costus root in Example 1 of the present application; Figure 17 A in the figure is the comparison spectrum of liquid phase detection of chlorogenic acid (peak 2) and 4,5-di-O-caffeoylquinic acid (peak 8) reference substances. Figure 17 B is the reference spectrum of the liquid phase detection of costunolide (peak 13) and dehydrocostuslactone (peak 14). Figure 17 Middle C is the characteristic spectrum of the standard decoction of Sichuan costus root;

[0052] Figure 18 This is the comparison result of the characteristic spectra of the standard decoction of Sichuan costus root and its counterfeit products, namely, wormwood, green costus root and red costus root in Example 2 of the present application. DETAILED DESCRIPTION

[0053] The present application will be further described below in conjunction with the embodiments, examples and accompanying drawings. It should be understood that these examples are intended only to illustrate the present application and are not intended to limit the scope of the present application. In addition, it should be understood that after reading the content taught in this application, those skilled in the art may make various changes or modifications to the present application, and these equivalent forms also fall within the scope of protection of the claims appended hereto.

[0054] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are for the purpose of describing specific embodiments only and are not intended to limit this application.

[0055] the term

[0056] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0057] The terms "and / or", "or / and", and "and / or" used in this article have a selection scope that includes any one of two or more related listed items, and also includes any and all combinations of the related listed items, and said any and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items.

[0058] In this application, “further”, “particularly”, “preferably”, etc. are used for descriptive purposes to indicate differences in content, but should not be understood as limiting the scope of protection of this application.

[0059] In this application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0060] In this application, when referring to a numerical interval (i.e., a numerical range), unless otherwise specified, the optional numerical distribution within the above numerical interval is considered continuous and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every numerical value between the two numerical endpoints. Unless otherwise specified, when a numerical interval refers only to integers within the numerical interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges subsumed therein.

[0061] In this application, weight can be mass units known in the chemical industry, such as μg, mg, g, and kg.

[0062] In this application, "%" indicates percentage and refers to the ratio by volume or mass; for alcohol, the percentage refers to the ratio by volume at 20°C. In addition, the following symbols may be used as needed: % (g / g) indicates the solute content in grams per 100g of solution; % (mL / mL) indicates the solute content in milliliters per 100mL of solution; % (mL / g) indicates the solute content in milliliters per 100g of solution; and % (g / mL) indicates the solute content in grams per 100mL of solution.

[0063] The horizontal axis of the liquid chromatogram of the present application is time, in minutes (min), and the vertical axis reflects the relative strength of the response signal.

[0064] The Sichuan muxiang extract referred to in this application refers to the Sichuan muxiang standard decoction or its preparation made with pharmaceutically acceptable excipients, and further refers to the Sichuan muxiang standard decoction or its Chinese medicine formula granules. The "standard decoction" of a certain medicinal material described herein is a single-flavor Chinese medicine decoction piece water decoction prepared by a standardized process under the guidance of traditional Chinese medicine theory and based on clinical application, with reference to modern extraction methods. The "Chinese medicine formula granules" of a certain medicinal material described herein are a series of pure Chinese medicine products that are extracted, concentrated, separated, dried, granulated, packaged and refined using modern pharmaceutical technology using traditional Chinese medicine pieces that meet the processing specifications as raw materials. It can be understood that the above-mentioned standard decoctions and Chinese medicine formula granules can basically guarantee all the characteristics of the original Chinese medicine pieces.

[0065] In one aspect of the present application, a method for constructing a characteristic spectrum of a Sichuan wood fragrance extract is provided, comprising the following steps:

[0066] Take the test sample, add the extraction solvent to extract, and prepare the test sample solution; the test sample is the extract of Sichuan wood perfume;

[0067] The test solution was subjected to liquid chromatography analysis to construct a characteristic spectrum of the Sichuan wood fragrance extract;

[0068] Among them, the conditions for liquid chromatography analysis include:

[0069] (1) Mobile phase A is acetonitrile, and mobile phase B is an aqueous solution of 0.08% to 0.12% by volume phosphoric acid;

[0070] (2) Gradient elution is used. The procedure of gradient elution includes:

[0071] 0~5min, maintain the volume percentage of mobile phase A at 4%,

[0072] From 5 to 12 minutes, the volume percentage of mobile phase A increased from 4% to 16%.

[0073] 12min~19min, maintain the volume percentage of mobile phase A at 16%,

[0074] From 19min to 29min, the volume percentage of mobile phase A increased from 16% to 40%.

[0075] From 29min to 31min, the volume percentage of mobile phase A increased from 40% to 55%.

[0076] From 31 min to 37 min, the volume percentage of mobile phase A was maintained at 55%.

[0077] Furthermore, the conditions for liquid chromatography analysis also include at least one of the following:

[0078] (1) Liquid chromatography analysis is ultra-high performance liquid chromatography analysis;

[0079] (2) The flow rate of gradient elution is 0.3 mL / min to 0.4 mL / min;

[0080] (3) The chromatographic column is an octadecylsilane bonded silica gel chromatographic column;

[0081] (4) Column temperature is 35°C to 45°C;

[0082] (5) The injection volume is 1 μL to 3 μL;

[0083] (6) In the gradient elution procedure, the detection wavelength is 320 nm to 330 nm from 0 to 21 min, and the detection wavelength is 220 nm to 230 nm from 21 to 37 min.

[0084] The chromatographic column can be selected from but not limited to Waters HSS T3 (2.1mm×100mm, 1.8μm), Waters BEHC18 (2.1mm×100mm, 1.7μm), Waters CORTECS T3 (2.1mm×100mm, 1.6μm), YMC (2.1mm×100mm, 1.9μm) and Agilent SB C18 (2.1mm×100mm, 1.8μm); preferably, the model of the chromatographic column is Waters CORTECS T3 (2.1mm×100mm, 1.6μm), the peak shape of each characteristic peak is better, the separation and peak response of the chromatographic peaks are higher, and the chromatogram baseline is smoother.

[0085] At a wavelength of 325 nm, the separation of the chromatographic peaks of Sichuan wood fragrance extract by liquid chromatography was good, but under this condition, the chromatographic peaks of costus lactone and dehydrocostus lactone had no absorption. Considering the richness of the characteristic spectrum chromatographic information, the wavelength was switched. In the gradient elution program, the detection wavelength was 320 nm to 330 nm from 0 to 21 min, and the detection wavelength was 220 nm to 230 nm from 21 min to 37 min.

[0086] In one embodiment, the extraction method includes ultrasound;

[0087] Furthermore, the power of the ultrasound is 200W to 400W and / or the frequency of the ultrasound is 30kHz to 50kHz;

[0088] Furthermore, the ultrasonication time is 20 min to 40 min.

[0089] In one embodiment, the extraction solvent is an aqueous solution of alcohol with a volume concentration of 60% to 80%;

[0090] Furthermore, the alcohol is methanol.

[0091] In one embodiment, 0.2 g of Kawaki Fragrance extract is extracted using 10 mL to 100 mL of extraction solvent, preferably 10 mL to 30 mL, to ensure sufficient extraction.

[0092] In one embodiment, the method for constructing a characteristic spectrum of the Kawaki perfume extract further includes the steps of preparing a reference solution and performing liquid chromatography analysis on the reference solution;

[0093] The conditions for liquid chromatography analysis are the same as those described in any of the above technical solutions and will not be repeated here;

[0094] The reference solution includes costunolide reference substance, dehydrocostusolide reference substance, chlorogenic acid reference substance and 4,5-di-O-caffeoylquinic acid reference substance.

[0095] In one embodiment, the method for constructing a characteristic spectrum of the extract of Aucklandia odorata further includes the steps of preparing a Aucklandia odorata reference solution and performing liquid chromatography analysis on the Aucklandia odorata reference solution;

[0096] The conditions for liquid chromatography analysis are the same as those described in any of the above technical solutions and will not be repeated here;

[0097] Preparation of a reference solution of Aucklandia lappa, comprising:

[0098] Take a reference medicinal material of Sichuan costus root, add water, heat under reflux, filter, evaporate the filtrate to dryness, and then add an extraction solvent for extraction;

[0099] The extraction solvent and extraction method are the same as those described in any of the above technical solutions, and will not be repeated here;

[0100] Furthermore, 1 g of the control medicinal material of Sichuan costus root was taken, 20 mL to 30 mL of water was added, and the mixture was heated under reflux for 20 min to 40 min.

[0101] In one embodiment, the characteristic spectrum of the Sichuan rosewood extract includes characteristic peaks of costunolide, dehydrocostuslactone reference substance, chlorogenic acid reference substance, and 4,5-di-O-caffeoylquinic acid.

[0102] In one embodiment, the characteristic spectrum of the standard decoction of Sichuan costus root includes at least 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, peak 13 and peak 14; wherein, peak 2 is the characteristic peak of chlorogenic acid, peak 8 is the characteristic peak of 4,5-di-O-caffeoylquinic acid, peak 13 is the characteristic peak of costunolide, and peak 14 is the characteristic peak of dehydrocostus lactone;

[0103] Furthermore, taking peak 2 as the reference peak, the relative retention times of peak 1, peak 3, peak 4, peak 5, peak 6 and peak 7 are as follows: peak 1: 0.47±10%, peak 3: 1.11±10%, peak 4: 1.34±10%, peak 5: 1.43±10%, peak 6: 1.92±10%, peak 7: 1.98±10%; and / or

[0104] Taking peak 13 as the reference peak, the relative retention times of peaks 9, 10, 11 and 12 were as follows: peak 9: 0.78±10%, peak 10: 0.80±10%, peak 11: 0.96±10%, peak 12: 0.97±10%.

[0105] In another aspect of the present application, a method for detecting an extract of Scutellaria baicalensis is provided, comprising the following steps:

[0106] Take the Kawaki Fragrance extract to be tested, add the extraction solvent to extract, and prepare the test solution;

[0107] Taking the test solution for liquid chromatography analysis, detecting the content of the index component in the test solution and establishing a standard curve for the index component, the index component including at least one of costunolide, dehydrocostus lactone, chlorogenic acid and 4,5-di-O-caffeoylquinic acid;

[0108] Among them, the conditions for liquid chromatography analysis include:

[0109] (1) Mobile phase A is acetonitrile, and mobile phase B is an aqueous solution of 0.08% to 0.12% by volume phosphoric acid;

[0110] (2) Gradient elution is used. The procedure of gradient elution includes:

[0111] 0~5min, maintain the volume percentage of mobile phase A at 4%,

[0112] From 5 to 12 minutes, the volume percentage of mobile phase A increased from 4% to 16%.

[0113] 12min~19min, maintain the volume percentage of mobile phase A at 16%,

[0114] From 19min to 29min, the volume percentage of mobile phase A increased from 16% to 40%.

[0115] From 29min to 31min, the volume percentage of mobile phase A increased from 40% to 55%.

[0116] From 31 min to 37 min, the volume percentage of mobile phase A was maintained at 55%.

[0117] In the above detection method, the conditions of liquid chromatography analysis, the method of extraction, the extraction solvent and time, and other parameters related to sample preparation can all refer to the limitations of the construction part of the characteristic spectrum of this application. Preferably, the conditions of liquid chromatography analysis, the method of extraction, the extraction solvent and time, and other parameters related to sample preparation are the same as those defined in the construction part of the characteristic spectrum.

[0118] Another aspect of the present application also relates to the application of the characteristic spectrum constructed by the method for constructing the characteristic spectrum described in any of the above technical solutions and / or the detection method described in any of the above technical solutions in identifying Sichuan wood fragrance extracts.

[0119] The identification described above includes the analysis of the quality of the water extract of Sichuan wood fragrance, and also includes the authenticity identification of the water extract of Sichuan wood fragrance and its counterfeits. Common Sichuan wood fragrance counterfeits include green wood fragrance, red wood fragrance and earthenware, etc. The identification is achieved by performing chromatographic detection on their water extracts and then comparing them with the characteristic spectrum of Sichuan wood fragrance.

[0120] The following are some specific examples.

[0121] The experimental methods in the following examples are conventional methods unless otherwise specified. The medicinal materials, excipients, reagents, etc. used in the following examples are all commercially available products unless otherwise specified.

[0122] Example 1

[0123] This example establishes a characteristic spectrum of a standard decoction of Radix Aucklandiae Radix, comprising the following steps:

[0124] 1. Preparation of Sichuan Wood Rhizome Decoction Slices

[0125] Sichuan costus root slices were prepared according to the processing regulations under Sichuan costus root in the 2020 edition of the Chinese Pharmacopoeia. The specific processing method is as follows: Take Sichuan costus root, remove the black "oil head" and impurities from the root head, wash with water for 30 to 60 seconds, soak for 4 to 6 hours, cut into thick slices, and dry (50 to 60 degrees Celsius) until the moisture content meets the specified requirements.

[0126] 2. Preparation of Standardized Decoction of Sichuan Costus

[0127] Take 100g of Sichuan wood rose slices, add water and decoct twice, add 9 times the water for the first decoction, soak for 30 minutes, heat and boil over high heat (power 500W), then keep it slightly boiling over low heat (power 200W) for 30 minutes, filter it while hot through a 200 mesh sieve, and cool the filtrate rapidly with cold water; add 7 times the water for the second decoction, heat and boil over high heat (power 500W), then keep it slightly boiling over low heat (power 200W) for 25 minutes, filter it while hot through a 200 mesh sieve, and cool the filtrate rapidly with cold water; combine the two filtrates, concentrate under reduced pressure to a clear paste volume of about 400mL, and vacuum freeze-dry to obtain Sichuan wood rose standard decoction freeze-dried powder. The numbers and origin information of 14 batches of Sichuan wood rose standard decoction freeze-dried powder are shown in Table 1.

[0128] Table 1 Origin information of 14 batches of standard decoction of Sichuan costus root

[0129]

[0130] 3. Establishment of the characteristic spectrum of the standard decoction of Sichuan costus root

[0131] 3.1 Preparation of reference solution

[0132] Take 1 g of Sichuan costus root reference medicinal material, add 25 mL of water, heat and reflux for 30 minutes, filter, take the filtrate and evaporate to dryness, add 20 mL of 70% methanol to the residue, ultrasonically treat (power 250 W, frequency 40 kHz) for 30 minutes, take out, cool, shake well, filter, and take the filtrate as the reference solution of the control medicinal material.

[0133] Take appropriate amounts of costunolide reference substance, dehydrocostus lactone reference substance, chlorogenic acid reference substance and 4,5-di-O-caffeoylquinic acid reference substance, accurately weigh them, and add 70% methanol to make a mixed solution of 25.416 μg of costunolide, 45.158 μg of dehydrocostus lactone, 8.480 μg of chlorogenic acid and 7.897 μg of 4,5-di-O-caffeoylquinic acid per 1 mL.

[0134] 3.2 Optimization of chromatographic conditions

[0135] (1) Optimization of elution gradient

[0136] Take an appropriate amount of Sichuan costus root standard decoction (No.: S8), grind it into powder, take about 0.2g, accurately weigh it, place it in a stoppered conical flask, accurately add 20mL of 70% methanol, weigh it, and ultrasonically treat it (power 250W, frequency 40kHz) for 30 minutes. Let it cool, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate.

[0137] Chromatographic conditions: Waters CORTECS T3 (2.1×100 mm, 1.6 μm) column; acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, gradient elution according to Tables 2 to 4; flow rate, 0.35 mL / min; column temperature, 40°C; wavelengths, 225 nm and 325 nm, respectively; injection volume, 1 μL.

[0138] The detection wavelengths of 225 nm and 325 nm were used to investigate the effects of different elution gradients on the baseline and peak shape of the characteristic chromatogram of the standard decoction of Sichuan woody plant, as well as the separation effect of each chromatographic peak. The test results are as follows: Figures 1 to 3 .

[0139] according to Figures 1 to 3 Under the conditions of gradient elution program 3, the separation of each chromatographic peak is better. Program 3 is selected as the best gradient elution program.

[0140] Table 2 Gradient elution procedure 1

[0141]

[0142] Table 3 Gradient elution procedure 2

[0143]

[0144] Table 4 Gradient elution procedure 3

[0145]

[0146] (2) Selection of detection wavelength

[0147] Select 225nm and 325nm as the detection wavelengths of the standard decoction sample of Sichuan costus root, and perform 3D scanning in the wavelength range of 210nm to 400nm. Record the chromatograms of the sample at 225nm and 325nm and the 3D scanning graph in the range of 210nm to 400nm respectively. The results are as follows Figure 4 .

[0148] according to Figure 3 (Optimal gradient elution program) and Figure 4 By comparison, at a wavelength of 325 nm, the separation of the chromatographic peaks is better, but under this condition, the chromatographic peaks of costusinol and dehydrocostusinol have no absorption. Considering the richness of the characteristic spectrum chromatographic information, the wavelength switching is selected and set in the gradient elution program. From 0 to 21 min, the detection wavelength is 320 nm to 330 nm, and from 21 min to 37 min, the detection wavelength is 220 nm to 230 nm.

[0149] (3) Selection of chromatographic column

[0150] Under the optimized chromatographic gradient program, the effects of different types of chromatographic columns, namely Waters HSS T3 (2.1mm×100mm, 1.8μm), Waters BEH C18 (2.1mm×100mm, 1.7μm), Waters CORTECS T3 (2.1mm×100mm, 1.6μm), YMC (2.1mm×100mm, 1.9μm) and Agilent SB C18 (2.1mm×100mm, 1.8μm), on the separation of characteristic peaks in the characteristic spectrum of the standard decoction of Sichuan costus root were investigated. The test results are shown in Figure 2. Figure 5 .

[0151] according to Figure 5 , a Waters CORTECS T3 (2.1mm×100mm, 1.6μm) column was used. The peak shape of each characteristic peak was good, the separation and peak response of the chromatographic peaks were high, and the chromatographic baseline was relatively stable. Therefore, the Waters CORTECST3 (2.1mm×100mm, 1.6μm) column was selected as the chromatographic column for the determination of the characteristic spectrum of the standard decoction of Sichuan costus root.

[0152] (4) Determination of chromatographic conditions

[0153] The chromatographic conditions for the characteristic spectrum of the standard decoction of Sichuan costus root were determined as follows: a Waters CORTECST3 column; acetonitrile as mobile phase A, 0.1% phosphoric acid as mobile phase B, and gradient elution as specified in Table 4; a flow rate of 0.35 mL per minute; a column temperature of 40°C; in the gradient elution program, the detection wavelength was 320 nm to 330 nm from 0 to 21 min, and the detection wavelength was 220 nm to 230 nm from 21 min to 37 min; and the injection volume was 1 μL.

[0154] 3.3 Investigation of the pretreatment method of the test solution

[0155] The extraction solvent, extraction method, extraction time and extraction solvent dosage in the pretreatment process of the test sample solution of Sichuan costus root standard decoction were investigated to determine the optimal sample pretreatment method of Sichuan costus root standard decoction.

[0156] (1) Investigation of extraction solvent

[0157] Take an appropriate amount of Sichuan costus root standard decoction (No. S8), grind it into powder, take about 0.2g, and mix it in 4 parallel groups. Weigh it accurately and place it in a stoppered conical flask. Add 20mL of methanol, 70% methanol, 50% methanol, and 50% ethanol respectively. Weigh the weight and ultrasonicate (power 250W, frequency 40kHz) for 30 minutes. Let it cool and weigh it again. Make up the lost weight with the corresponding solvent. Shake well and filter. Take the filtrate and analyze it according to the chromatographic conditions determined under "3.2". The test results are shown in Figure 2. Figure 6 ,according to Figure 6 The peak shape and separation degree of the chromatographic peaks of the characteristic spectrum of the standard decoction of Sichuan costus root measured by four different extraction solvents were not significantly different. When 70% methanol was used as the extraction solvent, the "total peak area / sample weight" of each characteristic peak was the largest, so 70% methanol was selected as the extraction solvent.

[0158] (2) Investigation of extraction methods

[0159] Take an appropriate amount of Sichuan costus root standard decoction (No. S8), grind it into powder, take about 0.2g, and mix it in two parallel groups. Weigh it accurately and place it in a stoppered conical flask. Add 20mL of 70% methanol accurately and weigh it. Ultrasonicate it (power 250W, frequency 40kHz) for 30 minutes and reflux it for 30 minutes. Let it cool and weigh it again. Make up the lost weight with 70% methanol. Shake it well and filter it. Take the filtrate and analyze it according to the chromatographic conditions determined under "3.2". The test results are shown in Figure 2. Figure 7 ,according to Figure 7 There was no obvious difference in the peak shape, separation effect and “total peak area / sample weight” of each characteristic peak. Considering the ease of operation, ultrasonic extraction was selected.

[0160] (3) Extraction time investigation

[0161] Take an appropriate amount of Sichuan costus root standard decoction (No. S8), grind it into powder, take about 0.2g, and mix it in 3 parallel groups. Weigh it accurately and place it in a stoppered conical flask. Add 20mL of 70% methanol accurately and weigh it. Ultrasonicate it (power 250W, frequency 40kHz) for 15 minutes, 30 minutes and 45 minutes respectively. Let it cool and weigh it again. Make up the lost weight with 70% methanol. Shake it well and filter it. Take the filtrate and analyze it according to the chromatographic conditions specified under "3.2". The test results are shown in Figure 2. Figure 8 ,according to Figure 8 ,For different ultrasonic times, the “total peak area / sample weight” of the 14 characteristic peaks had no significant difference, indicating that ultrasonic extraction for 15 minutes was sufficient for complete extraction. Considering the influence of the experimental environment, in order to ensure the durability of the method, the ultrasonic extraction time was selected as 30 minutes.

[0162] (4) Investigation of extraction solvent volume

[0163] Take an appropriate amount of Sichuan costus root standard decoction (No. S8), grind it into powder, take about 0.2g, and place it in 4 parallel groups, accurately weigh it, and place it in a stoppered conical flask. Accurately add 10mL, 20mL, 50mL, and 100mL of 70% methanol respectively, weigh it, and ultrasonically treat it (power 250W, frequency 40kHz) for 30 minutes. Let it cool, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate. Inject and analyze it according to the chromatographic conditions determined under "3.2". The test results are shown in Figure 9 ,according to Figure 9Different extraction solvent volumes have little effect on the characteristic spectrum of the standard decoction of Sichuan costus root. In order to ensure sufficient extraction, the extraction solvent volume was selected as 20 mL.

[0164] (5) Determination of the preparation method of the test solution

[0165] According to the above experimental results, the sample pretreatment method of the characteristic spectrum of Sichuan costus root standard decoction can be determined as follows:

[0166] Take an appropriate amount of Sichuan costus root standard decoction, grind it into powder, take about 0.2g, accurately weigh it, place it in a stoppered conical flask, accurately add 20mL of 70% methanol, weigh it, and ultrasonically treat it (power 250W, frequency 40kHz) for 30 minutes. Let it cool, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain it.

[0167] 3.4 Determination of characteristic peaks

[0168] Take 14 batches of standard decoction samples of Sichuan costus root, prepare the test solution according to the test solution preparation method specified in "3.3", accurately pipette the above test solution and reference material solution, respectively, and inject and measure according to the chromatographic conditions specified in "3.2". Figure 10 This is an overlay of liquid chromatograms obtained by testing 14 batches of standard decoction of Sichuan costus root in Example 1 of the present application.

[0169] The "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation Software" was used to identify the common peaks of the characteristic spectra of 14 batches of Sichuan costus root standard decoction. 14 common peaks with known ingredients, good peak shape and separation, and high purity were selected as the characteristic peaks of Sichuan costus root standard decoction. The chlorogenic acid chromatographic peak was used as the reference peak S1, and the relative retention times of peaks 1, 3 to 7 and S1 were calculated. The peak corresponding to the costunolide reference was used as S2, and the relative retention times of peaks 9 to 12 and S2 were calculated. The characteristic peaks were located using the relative retention times. Figure 10 In the analysis, peak 2: chlorogenic acid; peak 8: 4,5-di-O-caffeoylquinic acid; peak 13: costunolide; peak 14: dehydrocostuslactone.

[0170] The characteristic spectra of 14 batches of standard decoctions of Sichuan costus root were analyzed. The peak corresponding to the chlorogenic acid reference was the S1 peak. The relative retention times of peak 1, peaks 3 to 7 and S1 were calculated, and the average relative retention times were 0.47 (peak 1), 1.11 (peak 3), 1.34 (peak 4), 1.43 (peak 5), 1.92 (peak 6), and 1.98 (peak 7). The peak corresponding to the costunolide reference was the S2 peak. The average relative retention times of peaks 9 to 12 and S2 were 0.78 (peak 9), 0.80 (peak 10), 0.96 (peak 11), and 0.97 (peak 12).

[0171] 3.5 Development of feature maps

[0172] The UPLC characteristic spectra of 14 batches of standard decoction of Sichuan costus root were matched using the "Chinese Herbal Medicine Chromatographic Fingerprint Similarity Evaluation System" and the reference spectra were generated by the average method to establish a Figure 11 The control characteristic spectrum of the standard decoction of Sichuan costus root is shown in the figure. The characteristic spectrum has 14 characteristic peaks, which correspond to the 14 characteristic peaks in the chromatogram of the Sichuan costus root reference medicinal material. Take 1 g of Sichuan costus root reference medicinal material, add 25 mL of water, heat and reflux for 30 minutes, filter, take the filtrate and evaporate to dryness, prepare the control medicinal material reference solution according to the test solution preparation method specified in "3.3", accurately aspirate the control medicinal material reference solution and measure it according to the chromatographic conditions specified in "3.2", and the obtained chromatogram is as shown below. Figure 12 .

[0173] Figure 11-12 Among them, peak 2 (S1): chlorogenic acid; peak 8: 4,5-di-O-caffeoylquinic acid; peak 13 (S2): costunolide; peak 14: dehydrocostus lactone.

[0174] According to the test results of 14 batches of Sichuan costus root standard decoction samples, the characteristic spectrum standard of Sichuan costus root standard decoction was determined as follows: 14 characteristic peaks should appear in the chromatogram of the test sample, and the retention times of the 14 characteristic peaks in the chromatogram of the reference medicinal material should correspond to those of the reference medicinal material; peaks 2, 8, and 13-14 should correspond to the retention times of the corresponding reference medicinal material peaks, respectively. The peak corresponding to the chlorogenic acid reference is the S1 peak. The relative retention times of peaks 1, 3-7, and S1 should be calculated, and the relative retention times should be within ±1 of the specified value. 0% range, the specified values ​​are: 0.47 (peak 1), 1.11 (peak 3), 1.34 (peak 4), 1.43 (peak 5), 1.92 (peak 6), 1.98 (peak 7), the peak corresponding to the costunolide reference is the S2 peak, calculate the relative retention time of peaks 9 to 12 and the S2 peak, the relative retention time should be within the range of ±10% of the specified value, the specified values ​​are: 0.78 (peak 9), 0.80 (peak 10), 0.96 (peak 11), 0.97 (peak 12).

[0175] 3.6 Mass Spectral Identification of Characteristic Peaks

[0176] (1) Chromatographic conditions

[0177] Except that the flow item B is 0.1% formic acid, the rest are the same as under "3.2".

[0178] (2) Mass spectrometry conditions

[0179] An ESI ion source was used in positive and negative ion mode scanning, with an ion source temperature of 300°C, a desolvation temperature of 250°C, a desolvation gas flow of 5 L / min, and a capillary voltage of 3.5 KV.

[0180] (3) Preparation of reference solution

[0181] Take appropriate amounts of chlorogenic acid, 4,5-di-O-caffeoylquinic acid, costunolide, and dehydrocostus lactone reference substances, weigh them accurately, and add methanol to prepare a mixed solution containing 10 μg of chlorogenic acid, 10 μg of 4,5-di-O-caffeoylquinic acid, 20 μg of costunolide, and 20 μg of dehydrocostus lactone per 1 mL, as the reference substance solution.

[0182] (4) Preparation of test solution

[0183] Same as under “3.3”.

[0184] (5) Determination method

[0185] Accurately pipette 1 μL of the reference solution and the test solution respectively, inject them into the liquid chromatography-mass spectrometer, and use the above liquid chromatography and mass spectrometry analysis conditions to perform mass spectrometry MRM mode detection on the reference solution and the test solution to obtain the corresponding mass spectra. Figures 13 to 16 In each mass spectrum, the horizontal axis represents the retention time (RT, in min), and the vertical axis represents the signal response value (Relative Abundance). (m / z) 353.09>191.06, indicating that the fragment (m / z 191.06) of chlorogenic acid in the parent fragment (m / z 353.09) is selectively detected. The meanings expressed by other similar formulas can refer to this example.

[0186] according to Figures 13 to 16 , four characteristic peaks were identified in the characteristic spectrum of the test solution, namely chlorogenic acid (peak 2), 4,5-di-O-caffeoylquinic acid (peak 8), costunolide (peak 13) and dehydrocostuslactone (peak 14).

[0187] 3.7 Confirmation of characteristic peaks by reference substances

[0188] (1) Chromatographic conditions

[0189] Same as under “3.2”.

[0190] (2) Preparation of reference solution

[0191] Take appropriate amounts of costunolide reference substance, dehydrocostus lactone reference substance, chlorogenic acid reference substance and 4,5-di-O-caffeoylquinic acid reference substance, accurately weigh them, and add 70% methanol to prepare a mixed solution of 25.416 μg of costunolide, 45.158 μg of dehydrocostus lactone, 8.480 μg of chlorogenic acid and 7.897 μg of 4,5-di-O-caffeoylquinic acid per 1 mL.

[0192] (3) Preparation of test solution

[0193] Same as under “3.3”.

[0194] (4) Determination method

[0195] Accurately pipette 1 μL of the reference solution and test solution respectively, inject them into the liquid chromatography and measure.

[0196] The results of the measurement are as follows Figure 17 ,according to Figure 17 The chromatogram of the test sample showed the same chromatographic peaks at the corresponding retention times as the chromatogram of the reference sample. The four peaks were chlorogenic acid (peak 2), 4,5-di-O-caffeoylquinic acid (peak 8), costunolide (peak 13) and dehydrocostuslactone (peak 14).

[0197] Methodological validation

[0198] (1) Precision inspection

[0199] Take the standard decoction of Sichuan costus root (batch number: S8), grind it into powder, take about 0.2g, accurately weigh it, and prepare the test solution according to the test sample preparation method specified in "3.3". Repeat the injection 6 times according to the chromatographic conditions in "3.2" for sample analysis. Using the chlorogenic acid chromatographic peak as the reference peak S1, calculate the relative retention time and relative peak area of ​​peak 1, peaks 3 to 7 and peak S1. Using the costunolide chromatographic peak as the reference peak S2, calculate the relative retention time and relative peak area of ​​peaks 9 to 12 and peak 14 and peak S2. The relative retention time and relative peak area RSD values ​​are also calculated. The results show that the relative retention time RSD values ​​of each characteristic peak are within the range of 0.05% to 0.19%, and the relative peak area RSD values ​​are within the range of 0.09% to 1.74%, all less than 3.0%, indicating good instrument precision.

[0200] (2) Repeatability study

[0201] The same batch of Sichuan costus root standard decoction (No. S8) was taken and ground into powder. Approximately 0.2 g was taken and accurately weighed. Six replicates were prepared according to the test solution preparation method specified in "3.3". Samples were injected and analyzed according to the chromatographic conditions in "3.2". The chlorogenic acid chromatographic peak was used as the reference peak S1. The relative retention times and peak areas of peaks 1, 3 to 7, and S1 were calculated. The costunolide chromatographic peak was used as the reference peak S2. The relative retention times and peak areas of peaks 9 to 12 and 14 were calculated relative to S2. The relative retention times and relative peak areas RSDs were also calculated. The results showed that the relative retention time RSDs of the characteristic peaks were within the range of 0.03% to 0.14%, and the relative peak area RSDs were within the range of 1.01% to 2.64%, all less than 3.0%, indicating good reproducibility of the method.

[0202] (3) Stability investigation

[0203] Take the standard decoction of Sichuan costus root (No.: S8), grind it into powder, take about 0.2g, weigh it accurately, prepare the test solution according to the test sample preparation method determined under "3.3", and analyze it at 0, 2, 4, 6, 8, and 12 hours according to the chromatographic conditions under "3.2". Take the chlorogenic acid chromatographic peak as the reference peak S1, calculate the relative retention time and relative peak area of ​​peak 1, peak 3 to 7 and peak S1, take the costunolide chromatographic peak as the reference peak S2, calculate the relative retention time and relative peak area of ​​peak 9 to 12 and peak 1 The relative retention time and relative peak area of ​​the S4 and S2 peaks were measured, and the relative retention time and relative peak area RSD values ​​were calculated. The results showed that when the same test solution was analyzed at 0, 2, 4, 6, 8 and 12 hours, the relative retention time RSD values ​​of each characteristic peak were in the range of 0.08% to 0.42%, and the relative peak area RSD values ​​were in the range of 0.51% to 2.55%, all of which were less than 3.0%, indicating that the test solution had good relative stability within 12 hours.

[0204] (4) Intermediate precision inspection

[0205] Other analysts of this project team operated on different dates and different chromatographs, took about 0.2g of the same batch of Sichuan costus root standard decoction (No.: S8), accurately weighed, and paralleled 6 times, prepared the test solution according to item "3.3", and injected the sample for analysis according to the chromatographic conditions under item "3.2". The chlorogenic acid chromatographic peak was used as the reference peak S1 to calculate the relative retention time and relative peak area of ​​peak 1, peak 3 to 7 and peak S1. The costunolide chromatographic peak was used as the reference peak S2 to calculate the relative retention time and relative peak area of ​​peak 9 to 12, peak 14 and peak S2. The RSD values ​​of relative retention time and relative peak area were calculated. The results showed that the RSD values ​​of relative retention time of each characteristic peak were in the range of 0.04% to 0.19%, the RSD values ​​of relative peak area were in the range of 0.58% to 2.80%, the RSD values ​​of relative retention time and 6 data of repeatability test were in the range of 0.05% to 2.59%, and the RSD values ​​of relative peak area and 6 data of repeatability test were in the range of 1.41% to 4.92%. The RSD value was less than 5.0%, indicating that the intermediate precision of this method was good.

[0206] 4. Establishment of analytical method for content determination

[0207] 4.1 Chromatographic conditions

[0208] Same as under “3.2”.

[0209] 4.2 Preparation of reference solution

[0210] Take appropriate amounts of costunolide reference substance, dehydrocostus lactone reference substance, chlorogenic acid reference substance and 4,5-di-O-caffeoylquinic acid reference substance, accurately weigh them, and add 70% methanol to prepare a mixed solution of 25.416 μg of costunolide, 45.158 μg of dehydrocostus lactone, 8.480 μg of chlorogenic acid and 7.897 μg of 4,5-di-O-caffeoylquinic acid per 1 mL.

[0211] 4.3 Investigation of test solution pretreatment methods

[0212] (1) Investigation of extraction solvent

[0213] Take an appropriate amount of Sichuan costus root standard decoction (No.: S7), grind it into powder, take about 0.2 g, and mix it in 4 parallel groups. Accurately weigh it and place it in a stoppered conical flask. Accurately add 20 mL of methanol, 70% methanol, 50% methanol, and 50% ethanol respectively, weigh the weight, and ultrasonically treat (power 250 W, frequency 40 kHz) for 30 minutes. Let it cool and weigh it again. Make up the lost weight with the corresponding solvent, shake it well, filter it, and take the filtrate. Sample injection and analysis are carried out according to the chromatographic conditions under "4.1". Record the chromatographic peak area and calculate the content of costunolide and dehydrocostusolide, chlorogenic acid and 4,5-di-O-caffeoylquinic acid. The experimental results are shown in Table 5.

[0214] Table 5 Results of different extraction solvents for content determination of standard decoction of Radix Aucklandiae Radix

[0215]

[0216] According to Table 5, by comparing the total content of costunolide and dehydrocostus lactone, chlorogenic acid and 4,5-di-O-caffeoylquinic acid in the four extraction solvent samples, it can be found that 70% methanol has a higher extraction rate for the total content of costunolide and dehydrocostus lactone, chlorogenic acid and 4,5-di-O-caffeoylquinic acid in the standard decoction of Sichuan costus root. Combined with the characteristic chromatographic peak shape, 70% methanol was selected as the extraction solvent for the content determination of the standard decoction of Sichuan costus root.

[0217] (2) Investigation of extraction methods

[0218] Take an appropriate amount of Sichuan costus root standard decoction (No.: S7), grind it into powder, take about 0.2g, and mix it in two parallel groups. Accurately weigh it and place it in a stoppered conical flask. Accurately add 20mL of 70% methanol and weigh it. Ultrasonicate it (power 250W, frequency 40kHz) for 30 minutes and heat it under reflux for 30 minutes. Let it cool and weigh it again. Make up the lost weight with 70% methanol. Shake it well and filter it. Take the filtrate and analyze it according to the chromatographic conditions under "4.1". Record the chromatographic peak area and calculate the content. The experimental results are shown in Table 6.

[0219] Table 6 Results of different extraction methods for content determination of standard decoction of Sichuan costus root

[0220]

[0221] According to Table 6, different extraction methods had little effect on the extraction rate of costunolide and dehydrocostuslactone, chlorogenic acid and 4,5-di-O-caffeoylquinic acid in the standard decoction of Sichuan costus root. Considering the convenience of operation, ultrasound was selected as the extraction method.

[0222] (3) Extraction time investigation

[0223] Take an appropriate amount of Sichuan costus root standard decoction (No.: S7), grind it into powder, take about 0.2 g, and mix it in 3 parallel groups. Accurately weigh it and place it in a stoppered conical flask. Accurately add 20 mL of 70% methanol and weigh it. Ultrasonicate it (power 250 W, frequency 40 kHz) for 15, 30, and 45 minutes respectively. Let it cool and weigh it again. Make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate. Sample injection and analysis are carried out according to the chromatographic conditions under "4.1". Record the chromatographic peak area and calculate the content of costunolide and dehydrocostusolide, chlorogenic acid and 4,5-di-O-caffeoylquinic acid. The experimental results are shown in Table 7.

[0224] Table 7 Results of different extraction times for determination of costus lactone and dehydrocostus lactone contents in standard decoction of Sichuan costus root

[0225]

[0226] According to Table 7, different extraction times had little effect on the contents of costunolide and dehydrocostusolide, chlorogenic acid and 4,5-di-O-caffeoylquinic acid. To ensure sufficient extraction, the extraction time was selected as 30 minutes.

[0227] (4) Investigation of extraction solvent dosage

[0228] Take an appropriate amount of Sichuan costus root standard decoction (No.: S7), grind it into powder, take about 0.2 g, and divide it into 4 parallel groups. Accurately weigh it and place it in a stoppered conical flask. Accurately add 10 mL, 20 mL, 50 mL, and 100 mL of 70% methanol, weigh it, and ultrasonically treat it (power 250 W, frequency 40 kHz) for 30 minutes. Let it cool, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate. Sample injection and analysis are carried out according to the chromatographic conditions under "4.1". Record the chromatographic peak area, and calculate the content of costunolide and dehydrocostusolide, chlorogenic acid, and 4,5-di-O-caffeoylquinic acid. The results are shown in Table 8.

[0229] Table 8 Results of content determination of standard decoction of Radix Aucklandiae at different material-liquid ratios

[0230]

[0231] Different extraction solvent dosages had little effect on the extraction rates of costunolide and dehydrocostuslactone contents, chlorogenic acid, and 4,5-di-O-caffeoylquinic acid contents in the standard decoction of Rhizoma Aucklandiae (Rhizoma Aucklandiae). To ensure more sufficient extraction, the extraction solvent volume of 20 mL was selected.

[0232] (5) Determination of the preparation method of the test solution

[0233] According to the results of the sample pretreatment investigation experiment, the preparation method of the test sample can be determined as follows: take an appropriate amount of Sichuan costus root (Sichuan costus root) standard decoction, grind it into powder, take about 0.2 g, accurately weigh it, put it in a stoppered conical flask, accurately add 20 mL of 70% methanol, weigh it, ultrasonically treat it (power 250 W, frequency 40 kHz) for 30 minutes, let it cool, weigh it again, make up the lost weight with 70% methanol, shake it evenly, filter it, and take the filtrate to obtain it.

[0234] 4.4 Methodological Validation

[0235] (1) Precision inspection

[0236] Take the mixed reference solution under item "4.2" and inject it continuously for 6 times according to the chromatographic conditions under item "4.1". Record the chromatographic peak areas of costunolide, dehydrocostus lactone, chlorogenic acid and 4,5-di-O-caffeoylquinic acid, and calculate the peak area RSD values ​​to be 0.20%, 0.14%, 1.24% and 1.20%, respectively, indicating that the precision of the injector is good.

[0237] (2) Linear relationship investigation

[0238] Accurately weigh 6.386 mg of costunolide reference substance, 11.312 mg of dehydrocostus lactone reference substance, 2.206 mg of chlorogenic acid reference substance, and 2.073 mg of 4,5-di-O-caffeoylquinic acid, place them in a 25 mL volumetric flask, and add 70% methanol to prepare a mixed reference substance stock solution containing 254.163 μg of costunolide, 451.575 μg of dehydrocostus lactone, 84.799 μg of chlorogenic acid, and 78.857 μg of 4,5-di-O-caffeoylquinic acid per 1 mL. Accurately measure 4.0mL, 2.0mL, 1.0mL, 0.4mL, 0.2mL, and 0.1mL of the mixed reference substance stock solution into 10mL volumetric flasks, add 70% methanol to the mark, and prepare a series of mixed reference substance application solutions. Accurately pipette the mixed reference substance stock solution and application solution, and analyze them in sequence according to the chromatographic conditions under "4.1". Record the chromatographic peak areas. Plot a standard curve with peak area as the ordinate (Y) and reference substance concentration as the abscissa (X). The standard curve equation is shown in Table 9. According to Table 9, the peak area of ​​each chemical component within the corresponding concentration range has a good linear relationship with the reference substance concentration.

[0239] Table 9 Linear relationships of various components

[0240]

[0241] (3) Stability investigation

[0242] Accurately aspirate the test solution of Sichuan costus root standard decoction (No.: S7), and according to the chromatographic conditions under "4.1", sample injection and analysis were performed at 0h, 2h, 4h, 6h, 8h, 10h, 12h and 24h, respectively. The chromatographic peak areas of costunolide, dehydrocostus lactone, chlorogenic acid and 4,5-di-O-caffeoylquinic acid of the same test solution at different time points were recorded, and the peak area RSD values ​​were calculated. The results showed that the chromatographic peak area RSD values ​​of costunolide, dehydrocostus lactone, chlorogenic acid and 4,5-di-O-caffeoylquinic acid of the same test solution at different time points were 0.35%, 0.46%, 0.87% and 1.62%, respectively, all less than 3.0%, indicating that the test solution had good stability within 24 hours.

[0243] (4) Repeatability study

[0244] Take an appropriate amount of the same batch of Sichuan costus root standard decoction (No.: S7), grind it into powder, take about 0.2 g, weigh it accurately, and weigh 6 portions in parallel. According to the method under "2.2.3", 6 test solutions were prepared. The samples were injected and analyzed according to the chromatographic conditions under "4.1". The RSD values ​​of costunolide, dehydrocostus lactone, chlorogenic acid and 4,5-di-O-caffeoylquinic acid were calculated. The results showed that the RSD values ​​of costunolide, dehydrocostus lactone, chlorogenic acid and 4,5-di-O-caffeoylquinic acid were 0.35%, 1.02%, 1.13% and 0.47%, respectively, indicating that the method has good reproducibility.

[0245] (6) Sample recovery rate investigation

[0246] Accurately weigh 4.232 mg of costunolide reference substance, 7.576 mg of dehydrocostus lactone reference substance, 2.150 mg of chlorogenic acid reference substance, and 2.005 mg of 4,5-di-O-caffeoylquinic acid reference substance, place them in a 50 mL volumetric flask, and add methanol to make a mixed reference solution containing 84.217 μg of costunolide, 151.217 μg of dehydrocostus lactone, 41.323 μg of chlorogenic acid, and 38.135 μg of 4,5-di-O-caffeoylquinic acid per 1 mL.

[0247] Accurately draw 1.0mL, 2.0mL and 3.0mL of the mixed reference solution above, in parallel 3 groups, 3 portions in each group, put them into a conical flask and blow dry the solvent with nitrogen, add about 0.1g of the freeze-dried powder of Sichuan costus root standard soup (No.: S7) of known content and accurate weight respectively, prepare 9 portions of test solution according to the method under "4.3", and inject and measure according to the chromatographic conditions under "4.1", calculate the recovery rate and RSD value of the sample. The results show that the average recovery rate of costus lactone is The average recovery rate of dehydrocostus lactone was 100.11% with an RSD of 0.49%, the average recovery rate of chlorogenic acid was 99.57% with an RSD of 2.98%, and the average recovery rate of 4,5-di-O-caffeoylquinic acid was 97.95% with an RSD of 0.79%, all of which were in compliance with the provisions of General Chapter 9101 of Part IV of the 2020 edition of the "Chinese Pharmacopoeia", indicating that the method has good accuracy.

[0248] (7) Sample measurement

[0249] According to the test solution preparation method under "4.3" and the chromatographic conditions under "4.1," the contents of costunolide, dehydrocostuslactone, chlorogenic acid, and 4,5-di-O-caffeoylquinic acid in 14 batches of standard decoctions of Radix Aucklandiae Radix were determined using the external standard method. The total contents of costunolide and dehydrocostuslactone, as well as the total contents of chlorogenic acid and 4,5-di-O-caffeoylquinic acid, were calculated (see Table 10). The results showed that the standard decoction prepared from Radix Aucklandiae Radix raw materials from Aba, Sichuan, had the highest total contents of costunolide and dehydrocostuslactone, and the lowest total contents of chlorogenic acid and 4,5-di-O-caffeoylquinic acid. This may be related to the different growth years and processing methods of the Radix Aucklandiae Radix. The total contents of costunolide and dehydrocostuslactone, as well as chlorogenic acid and 4,5-di-O-caffeoylquinic acid, in standard decoctions prepared from Radix Aucklandiae Radix raw materials from the other three production areas were generally similar, indicating relatively consistent quality.

[0250] Table 10 Content determination results of 14 batches of standard decoction of Sichuan costus root

[0251]

[0252] Example 2

[0253] This Example 2 provides an example of identifying Sichuan costus root and its counterfeit standard decoction. For Sichuan costus root's common counterfeit products, Inula, Aucklandia sinensis and Aucklandia rossi, freeze-dried powder of the standard decoction was prepared according to the method under "2" and the characteristic spectrum was determined according to the method under "3". The results are shown in FIG. Figure 18 ,according to Figure 18The characteristic spectra of the standard decoctions of common counterfeit Sichuan costus root products, namely, wormwood, green costus root and red costus root, are significantly different from the UPLC characteristic spectrum profile of the standard decoction of Sichuan costus root. The characteristic spectrum of the standard decoction of red costus root has fewer chromatographic peaks. Although the fingerprint spectra of wormwood and green costus root have more chromatographic peaks, they have fewer chromatographic peaks shared with Sichuan costus root. This characteristic spectrum method can better distinguish Sichuan costus root and its counterfeit products.

[0254] All documents mentioned in this application are cited as references in this application, just as each document is cited as reference separately. Unless they conflict with the application purpose and / or technical solution of this application, the cited documents involved in this application are cited in their entirety and for all purposes. When cited documents are involved in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When cited documents are involved in this application, the examples and preferred embodiments of the cited relevant technical features may also be incorporated into this application as references, but are limited to the ability to implement this application. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be adaptively amended according to the description in this application.

[0255] The various technical features of the above-mentioned implementation modes and examples can be combined in any appropriate manner. In order to make the description concise, not all possible combinations of the various technical features in the above-mentioned implementation modes 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 the description in this specification.

[0256] The embodiments described above only express several implementation methods of the present application, but they should not be understood as limiting the scope of the patent application. It should be pointed out that, for those of ordinary skill in the art, without departing from the concept of the present application, several variations and improvements can be made, which all fall within the scope of protection of the present application. In addition, it should be understood that after reading the above-mentioned teaching content of the present application, those skilled in the art can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the scope of protection of the present application. It should also be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the scope of protection of the claims attached to the present application. Therefore, the scope of protection of the patent in this application shall be based on the attached claims, and the description and drawings may be used to explain the contents of the claims.

Claims

1. A method for constructing a characteristic spectrum of Kawaki Fragrance Extract, characterized in that: The steps include: A test sample is taken, and an extraction solvent is added for extraction to prepare a test sample solution; the test sample is an extract of Sichuan wood fragrance; Taking the test solution for liquid chromatography analysis to construct a characteristic spectrum of Sichuan wood fragrance extract; Wherein, the conditions of the liquid chromatography analysis include: (1) Mobile phase A is acetonitrile, and mobile phase B is an aqueous solution of 0.08% to 0.12% by volume phosphoric acid; (2) Gradient elution is used, and the procedure of the gradient elution includes: 0-5min, maintain the volume percentage of the mobile phase A at 4%, 5min~12min, the volume percentage of the mobile phase A increases from 4% to 16%, 12min~19min, maintain the volume percentage of the mobile phase A at 16%, From 19min to 29min, the volume percentage of the mobile phase A increased from 16% to 40%. From 29 to 31 minutes, the volume percentage of the mobile phase A increased from 40% to 55%. From 31 min to 37 min, the volume percentage of the mobile phase A was maintained at 55%.

2. The method for constructing a characteristic map according to claim 1, wherein: The conditions for the liquid chromatography analysis also include at least one of the following: (1) The liquid chromatography analysis is ultra-high performance liquid chromatography analysis; (2) The flow rate of the gradient elution is 0.3 mL / min to 0.4 mL / min; (3) The chromatographic column is an octadecylsilane bonded silica gel chromatographic column; (4) Column temperature is 35°C to 45°C; (5) The injection volume is 1 μL to 3 μL; (6) In the gradient elution procedure, the detection wavelength is 320 nm to 330 nm from 0 min to 21 min, and the detection wavelength is 220 nm to 230 nm from 21 min to 37 min.

3. The method for constructing a characteristic spectrum according to claim 1 or 2, wherein: Extraction methods include ultrasound; Furthermore, the power of the ultrasound is 200W to 400W and / or the frequency of the ultrasound is 30kHz to 50kHz; Furthermore, the ultrasonication time is 20 min to 40 min.

4. The method for constructing a characteristic spectrum according to claim 1 or 2, wherein: The extraction solvent is an aqueous solution of alcohol with a volume concentration of 60% to 80%; Furthermore, the alcohol is methanol.

5. The method for constructing a characteristic map according to claim 1 or 2, wherein: The characteristic spectrum of the Sichuan wood fragrance extract includes characteristic peaks of costus lactone, dehydrocostus lactone, chlorogenic acid and 4,5-di-O-caffeoylquinic acid.

6. A method for detecting the extract of Kawaki Fragrance, characterized in that: The steps include: Take the Kawaki perfume extract to be tested, add extraction solvent for extraction, and prepare the test solution; Taking the test solution for liquid chromatography analysis, detecting the content of the index component in the test solution and establishing a standard curve for the index component, wherein the index component includes at least one of costunolide, dehydrocostus lactone, chlorogenic acid, and 4,5-di-O-caffeoylquinic acid; Wherein, the conditions of the liquid chromatography analysis include: (1) Mobile phase A is acetonitrile, and mobile phase B is an aqueous solution of 0.08% to 0.12% by volume phosphoric acid; (2) Gradient elution is used, wherein the gradient elution procedure includes: 0-5min, maintain the volume percentage of the mobile phase A at 4%, 5min~12min, the volume percentage of the mobile phase A increases from 4% to 16%, 12min~19min, maintain the volume percentage of the mobile phase A at 16%, From 19min to 29min, the volume percentage of the mobile phase A increased from 16% to 40%. From 29 to 31 minutes, the volume percentage of the mobile phase A increased from 40% to 55%. From 31 min to 37 min, the volume percentage of the mobile phase A was maintained at 55%.

7. The detection method according to claim 6, wherein The conditions for the liquid chromatography analysis also include at least one of the following: (1) The liquid chromatography analysis is ultra-high performance liquid chromatography analysis; (2) The flow rate of the gradient elution is 0.3 mL / min to 0.4 mL / min; (3) The chromatographic column is an octadecylsilane bonded silica gel chromatographic column; (4) Column temperature is 35°C to 45°C; (5) The injection volume is 1 μL to 3 μL; (6) In the gradient elution procedure, the detection wavelength is 320 nm to 330 nm from 0 min to 21 min, and the detection wavelength is 220 nm to 230 nm from 21 min to 37 min.

8. The detection method according to claim 6 or 7, wherein Extraction methods include ultrasound; Furthermore, the power of the ultrasound is 200W to 400W and / or the frequency of the ultrasound is 30kHz to 50kHz; Furthermore, the ultrasonication time is 20 min to 40 min.

9. The detection method according to claim 6 or 7, wherein: The extraction solvent is an aqueous solution of alcohol with a volume concentration of 60% to 80%; Furthermore, the alcohol is methanol.

10. Use of the characteristic spectrum constructed by the method for constructing the characteristic spectrum according to any one of claims 1 to 5 and / or the detection method according to any one of claims 6 to 9 in identifying an extract of Kawaki perfume.

Images