Construction of characteristic chromatogram of sandalwood medicinal material, detection method and application thereof

Abstract

The application relates to the technical field of traditional Chinese medicine analysis, in particular to a construction method and a detection method of a characteristic spectrum of sandalwood medicinal materials and application thereof. The application provides suitable chromatographic conditions and a detection method, mobile phase A is acetonitrile, and mobile phase B is an acid solution with a concentration of 0.08%-0.12%; gradient elution is adopted, including: 0-12 min, the volume percentage of mobile phase A is increased from 5% to 7%, 12 min-25 min, the volume percentage of mobile phase A is increased from 7% to 12%, 25 min-35 min, the volume percentage of mobile phase A is increased from 12% to 30%, and 35 min-45 min, the volume percentage of mobile phase A is increased from 30% to 75%; the prepared characteristic spectrum has good specificity, the characteristic spectrum and / or the detection method can be used for identifying sandalwood medicinal materials and can be used for effectively distinguishing sandalwood and similar counterfeit products, thereby providing a theoretical basis for quality control and clinical medication.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of traditional Chinese medicine detection, in particular to the construction of a characteristic chromatogram of sandalwood medicinal materials, a detection method and application thereof. BACKGROUND

[0002] Sandalwood medicinal materials are the dried heartwood of Santalum album L. of Santalaceae, in the form of cylindrical wood segments of varying lengths, some of which are slightly curved, generally about 1 m long and 10-30 cm in diameter. The outer surface is grayish yellow or yellowish brown, smooth and delicate, with some knots or longitudinal cracks. The cross-section is brownish yellow, with visible oil marks, and it is not easy to break. It has a clear fragrance, and the fragrance is even stronger when burned. It tastes bland and slightly spicy when chewed. Sandalwood is pungent and warm in nature, and is related to the spleen, stomach, heart and lung meridians. It can warm the middle and open the stomach to relieve pain. It is used for cold stagnation, chest and diaphragm discomfort, chest pain, stomach and abdominal pain, and vomiting with little food.

[0003] Researching the chemical composition of a substance is the key to quality control. In order to promote the clinical application of traditional Chinese medicine in modern medicine, it is necessary to study the specific composition of sandalwood. Modern analytical techniques show that the chemical composition of sandalwood mainly includes sesquiterpenes, monoterpenes, phenolic acids and lignans. These chemical components have a wide range of pharmacological activities, such as sesquiterpenes, monoterpenes and lignans, which have been found to have anti-tumor and antibacterial effects, and sesquiterpenes also have a calming effect on the central nervous system. Phenolic acids have been found to have anti-inflammatory, antidiarrheal and other effects.

[0004] However, most of the detection methods for sandalwood medicinal materials are limited to volatile oils or complex traditional Chinese medicines containing sandalwood.

[0005] Few quality control methods for single-ingredient sandalwood medicinal materials have been reported. The content determination of sandalwood medicinal materials in the Chinese Pharmacopoeia (2020 edition) only limits the volatile oil (mainly sesquiterpenes) and does not limit other components, which has great limitations. Some literature has recorded the use of GC-MS and HPLC methods to study the volatile components in sandalwood medicinal materials and identify some components such as α-curcumene, β-curcumene and α-sandalol. However, this method is also limited to volatile components and has limited applications. Therefore, it is necessary to develop a detection method that can more comprehensively reflect the internal chemical information of sandalwood and more reasonably control the quality of sandalwood medicinal materials. SUMMARY

[0006] The purpose of the present application includes providing a construction method of a characteristic chromatogram of sandalwood medicinal materials, a detection method and application thereof. The technical solution of the present application can objectively, scientifically and comprehensively reflect the internal chemical characteristic information of sandalwood medicinal materials as a basis for quality detection.

[0007] Specifically, the construction method of the characteristic chromatogram comprises the following steps:

[0008] Taking a test sample, extracting with an extraction solvent to prepare a test sample solution, the test sample is selected from sandalwood medicinal materials;

[0009] Taking the test sample solution to perform liquid chromatography analysis to establish a characteristic chromatogram of sandalwood medicinal materials;

[0010] The conditions of the liquid chromatography analysis include:

[0011] (1) the mobile phase A is acetonitrile, and the mobile phase B is an acid solution with a volume concentration of 0.08% to 0.12%;

[0012] (2) gradient elution is used, and the program of the gradient elution includes:

[0013] 0-12 min, the volume percentage of the mobile phase A is increased from 5% to 7%,

[0014] 12 min-25 min, the volume percentage of the mobile phase A is increased from 7% to 12%,

[0015] 25 min-35 min, the volume percentage of the mobile phase A is increased from 12% to 30%,

[0016] 35 min-45 min, the volume percentage of the mobile phase A is increased from 30% to 75%.

[0017] The detection method of the sandalwood medicinal materials includes the following steps:

[0018] Taking a test sample, extracting with an extraction solvent to prepare a test sample solution;

[0019] Taking the test sample solution to perform liquid chromatography analysis to establish a characteristic chromatogram of sandalwood medicinal materials;

[0020] The conditions of the liquid chromatography analysis include:

[0021] (1) the mobile phase A is acetonitrile, and the mobile phase B is an acid solution with a volume concentration of 0.08% to 0.12%;

[0022] (2) gradient elution is used, and the program of the gradient elution includes:

[0023] 0-12 min, the volume percentage of the mobile phase A is increased from 5% to 7%,

[0024] 12 min-25 min, the volume percentage of the mobile phase A is increased from 7% to 12%,

[0025] 25 min-35 min, the volume percentage of the mobile phase A is increased from 12% to 30%,

[0026] 35 min~45 min, the volume percentage of the mobile phase A is increased from 30% to 75%.

[0027] The application also relates to application of the construction method and the detection method of the above characteristic spectrum in identification of sandalwood medicinal materials.

[0028] The application establishes suitable chromatographic conditions to perform liquid chromatography analysis on sandalwood, constructs a characteristic spectrum thereof, and the obtained characteristic spectrum has good specificity, good separation effect of each characteristic peak, and rich chemical information, the method can save cost, simplify operation, improve efficiency, and has accurate results and strong practicability.

[0029] The application performs a large amount of research on characteristic components of sandalwood, establishes suitable chromatographic conditions, obtains 10 common characteristic peaks: peak 1, peak 2, peak 3, peak 4, peak 5, peak 6, peak 7, peak 8, peak 9 and peak 10, and identifies protocatechuic acid, isovanillin and isovanillin, and the internal chemical information of sandalwood is clear, which can provide important reference for quality detection, identification and clinical medication of sandalwood. BRIEF DESCRIPTION OF DRAWINGS

[0030] In order to more clearly illustrate the specific embodiments of the application or the technical solutions in the prior art, the following will briefly introduce the drawings needed to be used in the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the application, and other drawings can also be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0031] Figure 1 A comparison chart of the chromatograms of sandalwood medicinal materials in Example 1 detected under different mobile phase conditions; Figure 1 A is the detection result when acetonitrile is used as the mobile phase A, Figure 1 B is the detection result when methanol is used as the mobile phase A;

[0032] Figure 2 A comparison chart of the chromatograms of sandalwood medicinal materials in Example 1 detected under different detection wavelength conditions; Figure 2 A is the detection result when the detection wavelength is 310 nm, Figure 2 B is the detection result when the detection wavelength is 280 nm;

[0033] Figure 3 A comparison chart of the chromatograms of sandalwood medicinal materials in Example 1 detected under different chromatographic column conditions; Figure 3 A is the detection result when the chromatographic column is Waters BEH C18 (100 mm x 2.1 mm, 1.7 μm), Figure 3The detection results when the chromatographic column is Waters Cortecs T3 (100 mm x 2.1 mm, 1.6 μm), Figure 3 The detection results when the chromatographic column is Waters Cortecs T3 (150 mm x 2.1 mm, 1.6 μm);

[0034] Figure 4 A comparison chart of chromatograms of sandalwood medicinal materials detected under different elution procedure conditions in Example 1;

[0035] Figure 5 A comparison chart of chromatograms of sandalwood medicinal materials detected under different column temperature conditions in Example 1;

[0036] Figure 6 A comparison chart of chromatograms of sandalwood medicinal materials detected under different flow rate conditions in Example 1;

[0037] Figure 7 A superimposed chart of chromatograms of 10 batches of sandalwood medicinal materials detected in Example 2;

[0038] Figure 8 A sandalwood medicinal material control characteristic chromatogram detected in Example 2;

[0039] Figure 9 A comparison chromatogram of a sandalwood sample and a protocatechuic aldehyde control sample detected in Example 2;

[0040] Figure 10 A comparison chromatogram of a sandalwood sample and a syringaldehyde control sample detected in Example 2;

[0041] Figure 11 A comparison chromatogram of a sandalwood sample and an isovanillin control sample detected in Example 2;

[0042] Figure 12 A comparison chart of chromatographic detection of sandalwood and its approximate products, cypress, thuja, and Taihang thuja in Example 3. DETAILED DESCRIPTION

[0043] The application will be further described below in conjunction with the embodiments and examples. It should be understood that these examples are only used to illustrate the application and not used to limit the scope of the application. Furthermore, it should be understood that those skilled in the art can make various modifications or changes to the application after reading the content taught by the application, and these equivalent forms also fall within the protection scope of the appended claims of the application.

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description herein is for describing specific embodiments only and is not intended to be limiting of the application.

[0045] The terms

[0046] Unless otherwise indicated, or unless the context clearly indicates otherwise, the terms or phrases used in this application have the following meanings:

[0047] The term "and / or", "or / and", "and / or" used herein is a selective scope including any one of two or more related listed items, and also including any and all combinations of the related listed items, the any and all combinations of the related listed items including any two related listed items, any more related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", "and / or", it should be understood that in this application, the technical solution undoubtedly includes the technical solution connected by "logical and", and also undoubtedly includes the technical solution connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For another example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C and D (i.e. the technical solution connected by "logical or"), and also includes any and all combinations of A, B, C and D, i.e. includes the combination of any two or any three of A, B, C and D, and also includes the four-item combination of A, B, C and D (i.e. the technical solution connected by "logical and").

[0048] Herein, "preferably", "more preferably", "even more preferably" and the like are merely used to describe the embodiments or examples with better effects, and it should be understood that they do not constitute a limitation on the protection scope of the present application.

[0049] In the present application, "further", "even further", "in particular" and the like are used for the purpose of description, and should not be understood as a limitation on the protection scope of the present application.

[0050] In the present application, the technical features described in an open manner include both the closed technical solution consisting of the listed features, and the open technical solution containing the listed features.

[0051] In the present application, when a numerical interval (i.e. a numerical range) is involved, unless otherwise specified, the optional numerical values are considered to be continuous within the numerical interval, and include both numerical end points (i.e. the minimum value and the maximum value) of the numerical range, and every numerical value between the two numerical end points. Unless otherwise specified, when a numerical interval refers only to integers within the numerical interval, both end point integers of the numerical range, and every integer between the two end points are included. In addition, when multiple ranges are provided to describe a feature or a characteristic, the ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein are to be understood as including any and all sub-ranges subsumed therein.

[0052] In the present application, weight can be μg, mg, g, kg, and other mass units commonly known in the chemical industry.

[0053] In the present application, "%" represents percentage, which refers to the proportion of weight; however, the percentage of a solution refers to the number of grams of solute contained in 100 mL of the solution, unless otherwise specified; the percentage of alcohol refers to the proportion of volume at 20°C. In addition, the following symbols can be used as needed: %(g / g) refers to the number of grams of solute contained in 100 g of the solution; %(mL / mL) refers to the number of milliliters of solute contained in 100 mL of the solution; %(mL / g) refers to the number of milliliters of solute contained in 100 g of the solution; and %(g / mL) refers to the number of grams of solute contained in 100 mL of the solution.

[0054] In one aspect of the present application, a method for constructing a characteristic chromatogram of sandalwood medicinal material is provided, which can objectively, scientifically, and comprehensively reflect the inherent chemical characteristic information of sandalwood medicinal material, and serve as a basis for quality detection.

[0055] In some embodiments of the present application, the method for constructing a characteristic chromatogram of sandalwood medicinal material comprises the following steps:

[0056] A test sample is taken, and an extraction solvent is added for extraction to prepare a test sample solution. The test sample is selected from sandalwood medicinal material;

[0057] The test sample solution is taken for liquid chromatography analysis to establish a characteristic chromatogram of sandalwood medicinal material;

[0058] The conditions for liquid chromatography analysis include:

[0059] (1) The mobile phase A is acetonitrile, and the mobile phase B is an acid solution with a volume concentration of 0.08% to 0.12%;

[0060] (2) Gradient elution is used, and the gradient elution program includes:

[0061] 0~12min, the volume percentage of mobile phase A is increased from 5% to 7%,

[0062] 12 min~25 min, the volume percentage of the mobile phase A is increased from 7% to 12%,

[0063] 25 min~35 min, the volume percentage of the mobile phase A is increased from 12% to 30%,

[0064] 35 min~45 min, the volume percentage of the mobile phase A is increased from 30% to 75%.

[0065] In some embodiments, the acid solution of the mobile phase B refers to a phosphoric acid solution.

[0066] In some embodiments, the conditions of the liquid chromatography analysis further comprise at least one of the following:

[0067] (1) the liquid chromatography analysis is an ultra-high performance liquid chromatography analysis;

[0068] (2) the flow rate of the gradient elution is 0.2 mL / min~0.4 mL / min;

[0069] (3) the column temperature of the chromatographic column is 38℃~42℃;

[0070] (4) the injection volume is 1 μL~3 μL;

[0071] (5) the detection wavelength is 300 nm~320 nm.

[0072] In some specific embodiments, the liquid chromatography analysis is an ultra-high performance liquid chromatography analysis, the flow rate of the gradient elution is 0.3 mL / min, the column temperature of the chromatographic column is 40℃, the injection volume is 1 μL, and the detection wavelength is 310 nm.

[0073] In some of the embodiments, the chromatographic column is a C18 chromatographic column; and / or, the extraction solvent comprises an organic alcohol.

[0074] Further, the model of the C18 chromatographic column is CORTECS T3 chromatographic column.

[0075] Further, the CORTECS T3 chromatographic column has a column length of 100 mm~150 mm, a diameter of 2 mm~2.2 mm, and a particle size of the filler of 1.4 μm~1.8 μm.

[0076] Further, the extraction is performed by ultrasonic and / or heating reflux.

[0077] Further, the ultrasonic has a power of 200 W~400 W and a frequency of 30 kHz~50 kHz.

[0078] Further, the heating reflux has a temperature of 60℃~80℃.

[0079] In some embodiments, the extraction solvent is methanol, further, the extraction method is ultrasonic and / or heating reflux, further, the ultrasonic power is 200W-400W, the ultrasonic frequency is 30kHz-50kHz, and the heating reflux temperature is 60°C-80°C.

[0080] In some embodiments, the method for constructing the characteristic spectrum of the sandalwood medicinal material further comprises the steps of preparing a reference solution and taking the reference solution for liquid chromatography analysis.

[0081] In some embodiments, the liquid chromatography analysis conditions are as described in any one of the technical solutions above, and will not be repeated here.

[0082] The reference solution comprises protocatechuic aldehyde, syringaldehyde and isovanillin.

[0083] In some embodiments, the reference solution is prepared by a preparation method comprising the following steps:

[0084] The protocatechuic aldehyde, syringaldehyde and isovanillin are dissolved in a solvent to prepare the reference solution; the solvent is preferably methanol.

[0085] In some embodiments, the mass concentration of the reference solution is 5μg / mL-15μg / mL.

[0086] Further, the reference solution contains 1μg / mL-5μg / mL of protocatechuic aldehyde, 1μg / mL-5μg / mL of syringaldehyde and 1μg / mL-5μg / mL of isovanillin.

[0087] In some embodiments, the characteristic spectrum of the sandalwood medicinal material comprises the following 10 common peaks: peak 1, peak 2, peak 3, peak 4, peak 5, peak 6, peak 7, peak 8, peak 9 and peak 10, wherein peak 2 is protocatechuic aldehyde, peak 6 is isovanillin, and peak 8 is syringaldehyde.

[0088] The present application also relates to a detection method for sandalwood medicinal material, which can detect the content of each component of the sandalwood medicinal material, more objectively, comprehensively and accurately evaluate the quality of the medicinal material, and the detection method of the present application has the advantages of less reagent usage, simple operation, high detection sensitivity, good stability, accurate and reliable determination results.

[0089] In some embodiments of the present application, the detection method for sandalwood medicinal material comprises the following steps:

[0090] The test sample is taken and extracted with an extraction solvent to prepare a test sample solution;

[0091] The test sample solution is taken for liquid chromatography analysis to obtain a characteristic spectrum of the test sample;

[0092] In some embodiments of the present application, the liquid chromatography analysis conditions comprise:

[0093] (1) the mobile phase A is acetonitrile, and the mobile phase B is an acid solution with a volume concentration of 0.08% to 0.12%;

[0094] (2) gradient elution is used, and a gradient elution procedure includes:

[0095] 0-12 min, the volume percentage of the mobile phase A is increased from 5% to 7%,

[0096] 12 min-25 min, the volume percentage of the mobile phase A is increased from 7% to 12%,

[0097] 25 min-35 min, the volume percentage of the mobile phase A is increased from 12% to 30%,

[0098] 35 min-45 min, the volume percentage of the mobile phase A is increased from 30% to 75%.

[0099] In some embodiments, in the detection method of the sandalwood medicinal material, the conditions of the liquid chromatography analysis further include at least one of the following:

[0100] (1) the liquid chromatography analysis is ultra-high performance liquid chromatography analysis;

[0101] (2) the flow rate of the gradient elution is 0.2 mL / min to 0.4 mL / min;

[0102] (3) the column temperature of the chromatographic column is 38°C to 42°C;

[0103] (4) the injection volume is 1 μL to 3 μL;

[0104] (5) the detection wavelength is 300 nm to 320 nm.

[0105] In some embodiments, in the detection method of the sandalwood medicinal material, the chromatographic column is a C18 chromatographic column; and / or, the extraction solvent includes an organic alcohol.

[0106] In some embodiments, in the detection method of the sandalwood medicinal material, the model of the C18 chromatographic column is a CORTECS T3 chromatographic column;

[0107] Further, the column length of the CORTECS T3 chromatographic column is 100 mm to 150 mm, the diameter is 2 mm to 2.2 mm, and the particle size of the filler is 1.4 μm to 1.8 μm.

[0108] In some embodiments, in the detection method of the sandalwood medicinal material, the extraction method is ultrasonic and / or heating reflux;

[0109] The power of the ultrasonic is 200 W to 400 W, and the frequency is 30 kHz to 50 kHz;

[0110] The temperature of the heating reflux is 60-80℃.

[0111] Still another aspect of the present application relates to the use of the feature map construction method and / or the detection method of any of the technical solutions above in identifying sandalwood medicinal materials.

[0112] In one embodiment, identifying sandalwood medicinal materials includes identifying authentic products and fake products of sandalwood medicinal materials.

[0113] Further, the fake products of sandalwood medicinal materials are exemplified by cypress, Thuja sutchuenensis and Thuja sutchuenensis var. sutchuensis.

[0114] Further, according to whether the feature map of the measured object contains peak 9 and peak 10, if peak 9 and peak 10 are not detected, it is identified as a fake product.

[0115] The following are some specific embodiments.

[0116] In the following examples, the experimental methods are all conventional methods unless otherwise specified. In the following examples, the medicinal material raw materials, excipients, reagents, etc. are all commercially purchased products unless otherwise specified.

[0117] Instruments and reagents

[0118] Instrument: Waters high performance liquid chromatograph (Waters H-Class, Waters Corporation); CORTECS T3 chromatographic column (150 mm x 2.1 mm, 1.6 μm); one-hundredth analytical balance (ME204E, Mettler-Toledo); one-millionth analytical balance (XP26, Mettler-Toledo); digital ultrasonic cleaner (KQ500D, Kunshan Ultrasonic Instrument Co., Ltd.); constant temperature water bath (Shanghai Yiheng Scientific Instrument Technology Co., Ltd., Model: HWS28); ultrapure water system (Merck, Milli-Q Direct).

[0119] Reagents: ethanol (Tianjin Fuyu Fine Chemical Co., Ltd., analytical pure); methanol (Tianjin Fuyu Fine Chemical Co., Ltd., analytical pure); ethyl acetate (Xilong Technology Co., Ltd., analytical pure); phosphoric acid (Tianjin Fuyu Fine Chemical Co., Ltd., chromatographically pure); acetonitrile (Merck, chromatographically pure); water is ultrapure water (laboratory self-made).

[0120] Reagents: protocatechuic aldehyde (batch number: 110810-201909, China Institute for Drug Control); 10 batches of sandalwood medicinal materials mainly come from Guangdong, Tianjin and Guangxi, as shown in Table 1.

[0121] Table 1 Origin of sandalwood medicinal materials

[0122]

[0123] Example 1

[0124] 1.1. Investigation of mobile phase conditions

[0125] A CORTECS T3 column (100 mm x 2.1 mm, 1.6 μm) was used; acetonitrile and methanol were used as mobile phase A and 0.1% phosphoric acid solution was used as mobile phase B, gradient elution was performed according to the provisions in Table 2; the flow rate was 0.3 mL / min; the column temperature was 30°C; the injection volume was 1 μL; the wavelength was 338 nm. The obtained UPLC spectrum is shown in Figure 1 Figure 1 The detection results when acetonitrile was used as mobile phase A are shown in Table A, Figure 1 The detection results when methanol was used as mobile phase A are shown in Table B.

[0126] Table 2

[0127]

[0128] 1.2. Investigation of detection wavelength conditions

[0129] A CORTECS T3 column (100 mm x 2.1 mm, 1.6 μm) was used; acetonitrile was used as mobile phase A and 0.1% phosphoric acid solution was used as mobile phase B, gradient elution was performed according to the provisions in Table 3; the flow rate was 0.3 mL / min; the column temperature was 30°C; the injection volume was 1 μL; the wavelength was 280 nm and 310 nm. The obtained UPLC spectrum is shown in Figure 2 According to the results, it can be known that the characteristic peak information is rich and the response is better at the detection wavelength of 310 nm. Figure 2

[0130] Table 3 Gradient elution conditions

[0131]

[0132] 1.3. Investigation of column conditions

[0133] A CORTECS T3 column (100 mm x 2.1 mm, 1.6 μm), a BEH C18 column (100 mm x 2.1 mm, 1.7 μm), and a CORTECS T3 column (150 mm x 2.1 mm, 1.6 μm) were used respectively; acetonitrile was used as mobile phase A and 0.1% phosphoric acid solution was used as mobile phase B, gradient elution was performed according to the provisions in Table 3; the flow rate was 0.3 mL / min; the column temperature was 30°C; the injection volume was 1 μL; the wavelength was 310 nm. The obtained UPLC spectrum is shown in Figure 3 Figure 3 ​​​In Figure A, the detection result was obtained using a Waters BEH C18 column (100 mm × 2.1 mm, 1.7 μm). Figure 3 Figure B shows the detection results when the chromatographic column was Waters Cortecs T3 (100 mm × 2.1 mm, 1.6 μm). Figure 3 C represents the detection results using a Waters Cortecs T3 column (150 mm × 2.1 mm, 1.6 μm). The comparison shows that using a Waters Cortecs T3 column (150 mm × 2.1 mm, 1.6 μm) results in better resolution of characteristic peaks and symmetrical peak shapes.

[0134] 1.4. Investigation of elution gradient conditions

[0135] Based on the separation effect of chromatographic peaks, the chromatographic conditions were optimized to determine the optimal elution gradient. Chromatographic conditions: A Waters CORTECS T3 column (150 mm × 2.1 mm, 1.6 μm) was used, with acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, and gradient elution was performed according to the specifications in Table 4; the flow rate was 0.3 mL / min; the column temperature was 30℃; the injection volume was 1 μL; and the detection wavelength was 310 nm. The obtained UPLC chromatogram is shown below. Figure 4 ,according to Figure 4 It can be seen that the characteristic peaks under the conditions of Method 4 have good separation and are relatively uniformly distributed.

[0136] Table 4. Gradient elution conditions for Methods 1, 2, 3, and 4

[0137]

[0138] 1.5. Investigation of Column Temperature Conditions

[0139] A CORTECS T3 column (150 mm × 2.1 mm, 1.6 μm) was used; acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B, with gradient elution performed according to the specifications in Table 3; the flow rate was 0.3 mL / min; the column temperatures were 30℃, 35℃, 40℃, 42℃, and 45℃; the injection volume was 1 μL; and the wavelength was 310 nm. Figure 5 It is known that the chromatographic peak separation effect is best when the column temperature is 40℃, so 40℃ is selected as the column temperature for the characteristic chromatographic conditions of sandalwood.

[0140] 1.6. Investigation of Flow Velocity Conditions

[0141] A CORTECS T3 column (150 mm x 2.1 mm, 1.6 μm) was used; acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B, gradient elution was performed according to the provisions in Table 3; the flow rates were 0.25 mL / min, 0.28 mL / min, 0.3 mL / min, and 0.32 mL / min, respectively; the column temperatures were 40°C, respectively; the injection volume was 1 μL; and the wavelength was 310 nm. From Figure 6 It can be seen that when the flow rate is 0.3 mL / min, the chromatographic peak separation effect is the best, so 0.3 mL / min is selected as the flow rate of the characteristic chromatographic condition of sandalwood medicinal materials.

[0142] 1.7. Determined chromatographic conditions

[0143] According to the experimental results in sections 1.1-1.6, the chromatographic conditions for liquid analysis were finally determined as follows: a CORTECS T3 column (150 mm x 2.1 mm, 1.6 μm) was used; acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B, gradient elution was performed according to the provisions in Table 5; the flow rate was 0.3 mL / min; the column temperature was 40°C; the injection volume was 1 μL; and the wavelength was 310 nm. The injection volume was 1 μL.

[0144] Table 5. Determined gradient elution conditions

[0145]

[0146] Example 2

[0147] In this example, a plurality of batches of sandalwood were detected according to the chromatographic conditions determined in item 1.7, and a characteristic chromatogram of sandalwood was constructed.

[0148] 2.1. Preparation of reference solution

[0149] An appropriate amount of protocatechuic aldehyde, syringaldehyde, and isovanillin reference substances were accurately weighed and dissolved in methanol to prepare a mixed solution containing 10 μg per 1 mL, thereby obtaining the reference solution.

[0150] 2.2. Preparation of test solution

[0151] The pre-treatment of sandalwood test samples was investigated in terms of extraction method and extraction time to determine the sample pre-treatment method for the characteristic chromatogram of sandalwood medicinal materials.

[0152] 2.2.1. Investigation of extraction method

[0153] In this subsection, the effects of different extraction methods on the characteristic chromatogram of sandalwood medicinal materials were investigated. Ultrasonic extraction and reflux extraction were used as the extraction methods. The peak shape and separation degree of 10 characteristic peaks were observed, and the "total peak area / sample weight" of the 10 characteristic peaks was calculated to compare the effects of different extraction methods on the characteristic chromatogram of sandalwood medicinal materials, and the best extraction method was selected.

[0154] Take sandalwood medicinal materials (YC01) appropriate amount, take about 1g, precision weighing, parallel 2 groups, 2 groups each, in the conical flask with plug, add methanol 25ml, weigh, reflux, ultrasonic treatment (power 300w, frequency 40kHz) 30min, cool, weigh again, make up the weight loss with methanol, filter, filter the filtrate, add water 20ml to the residue, extract with ethyl acetate twice, 20ml each time, combine the ethyl acetate layer, dry, add 2ml methanol to dissolve the residue, filter, take the filtrate, sample analysis according to the chromatographic conditions under item "1.7", the results are shown in table 6.

[0155] Table 6 sandalwood medicinal materials characteristic chromatogram extraction mode investigation results

[0156]

[0157] The results show that the ultrasonic and reflux extraction have little difference, the "total peak area / weight" deviation value of each characteristic peak is small, and it is convenient to operate, so the ultrasonic extraction is selected.

[0158] 2.2.2. Extraction time investigation

[0159] This section investigates the influence of different extraction times on the characteristic chromatogram of sandalwood medicinal materials, taking ultrasonic extraction for 15min, 30min and 60min as the extraction time, by observing the peak shape and separation degree of 10 characteristic peaks, and calculating the "total peak area / weight" of 10 characteristic peaks, the influence of different extraction times on the characteristic chromatogram of sandalwood medicinal materials is compared, and the best extraction time is selected.

[0160] Take sandalwood medicinal materials (YC01) appropriate amount, take about 1.0g, precision weighing, parallel 3 groups, 2 groups each, in the conical flask with plug, add methanol 25ml, weigh, ultrasonic treatment (power 300w, frequency 40kHz) 15min, 30min, 60min respectively, cool, weigh again, make up the weight loss with methanol, filter, filter the filtrate, add water 20ml to the residue, extract with ethyl acetate twice, 20ml each time, combine the ethyl acetate layer, dry, add 2ml methanol to dissolve the residue, filter, take the filtrate, sample analysis according to the chromatographic conditions under item "1.7", the results are shown in table 7.

[0161] Table 7 sandalwood medicinal materials characteristic chromatogram extraction time investigation results

[0162]

[0163] The results show that the ultrasonic extraction for 15min, 30min and 60min respectively, the peak shape, separation effect and "total peak area / weight" of 10 characteristic peaks have no obvious difference, in order to ensure complete extraction, the ultrasonic extraction time is selected as 30min.

[0164] 2.2.3. Determination of preparation method of test sample

[0165] According to the experimental results of 2.2.1 and 2.2.2, the sample pretreatment method of sandalwood characteristic map was determined as follows: taking the sample powder (passing through No. 3 sieve), about 1.0 g, was placed in a conical flask with a plug, 25 mL of methanol was added, and ultrasonic treatment (power 300 W, frequency 40 kHz) was performed for 30 min. After cooling, filtration was performed, the filtrate was evaporated to dryness, 20 mL of water was added to the residue, and extraction was performed twice with 20 mL of ethyl acetate each time. The ethyl acetate layers were combined and evaporated to dryness. The residue was dissolved in 2 mL of methanol, filtered, and the filtrate was obtained.

[0166] 2.3. Investigation of specificity

[0167] The results are shown in Table 7. Figures 9 to 11 There were identical chromatographic peaks of each characteristic peak in the test sample chromatogram at the retention time corresponding to the reference solution chromatogram, and the negative sample had no interference, indicating that the method had good specificity.

[0168] 2.4. Sample detection and characteristic map construction

[0169] Ten batches of sandalwood were taken as test samples, and liquid chromatography was performed according to the chromatographic conditions determined in “1.7”. The preparation of test sample solution was according to the preparation method in “2.2.3”. Taking peak No. 2 as the reference peak S, the relative retention time and relative peak area of each characteristic peak to S peak were calculated, and the RSD value was calculated. The detection results are shown in Tables 8 and 9. The superimposed chromatogram of the characteristic map of 10 batches of sandalwood is shown in Figure 7 .

[0170] Table 8 Characteristic map of 10 batches of sandalwood (relative retention time)

[0171]

[0172] Table 9 Characteristic map of 10 batches of sandalwood (relative peak area)

[0173]

[0174] The UPLC characteristic maps of 10 batches of sandalwood were matched using the “Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System”, and the control map was generated by the average method. The control characteristic map of sandalwood was established, as shown in Figure 8 , and the characteristic map had 10 characteristic peaks.

[0175] 2.5. Similarity analysis

[0176] The peak area of the fingerprint of 10 batches of samples was matched by the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012B) software, and the common peak was identified by the average method. The similarity calculation results are shown in Table 10.

[0177] Table 10 Similarity results of characteristic chromatograms of 10 batches of sandalwood samples

[0178]

[0179] According to the comparison results in Table 10, the similarity of 10 batches of samples with the control mode is: the similarity of sandalwood base is greater than 0.95. Overall, the similarity of sandalwood medicinal material samples between different batches is high.

[0180] 2.6. Characteristic peak identification

[0181] 2.6.1. Chromatographic conditions

[0182] Chromatographic column: Waters Cortecs T3 (2.1 × 150 mm, 1.6 μm)

[0183] Column temperature: 40℃

[0184] Flow rate: 0.3 mL / min

[0185] Injection volume: 2 μL

[0186] Detection wavelength: 310 nm

[0187] Mobile phase ratio and flow rate: A phase acetonitrile, B phase 0.1% formic acid aqueous solution, gradient as shown in Table 11.

[0188] Table 11 Gradient elution conditions

[0189]

[0190] 2.6.2. Mass spectrometry conditions

[0191] The mass spectrometry detection mode is ESI ion source Negative / Positive ion mode, and the detection parameters are as shown in Table 12.

[0192] Table 12 Mass parameters (Agilent 6545 Q-TOF LC-MS)

[0193]

[0194] 2.6.3. Experimental results

[0195] The sandalwood sample was analyzed by using ultra-high performance liquid chromatography-high resolution mass spectrometry (UPLC-Q-TOF / MS). According to the mass spectrum information of the sample, combined with the high resolution mass spectrometry database of natural products and related literature, each target peak was identified (Table 13). The peak No. 2 was consistent with the peak position of the protocatechuic aldehyde reference substance, the peak No. 6 was consistent with the peak position of the isovanillin reference substance, and the peak No. 8 was consistent with the peak position of the syringic aldehyde reference substance.

[0196] The data acquisition software was MassHunter Workstation Software LC / MS Data Acquisition for 6200 series TOF / 6500 series Q-TOF (version B.06.01), and the data processing software was MassHunter Workstation Software Qualitative Analysis (version B.07.00).

[0197] Table 13 Identification results of target components of sandalwood

[0198]

[0199] Example 3

[0200] In this example, the different pseudo-sandalwoods were detected according to the chromatographic conditions determined in item “1.7” of Example 1, and the characteristic chromatogram was used to identify the sandalwood and its approximate pseudo-sandalwoods.

[0201] Cupressus funebris, Thuja sutchuenensis and Thuja sutchuenensis var. orientalis were taken as test samples, and were subjected to liquid chromatography analysis according to the chromatographic conditions determined in item “1.7”. The preparation of the test sample solution was performed according to the preparation method in item “2.2.3”. The results showed (see Figure 12 ), and the peaks 9 and 10 of the original sandalwood characteristic chromatogram were not detected in Cupressus funebris, Thuja sutchuenensis and Thuja sutchuenensis var. orientalis, indicating that the established method can effectively distinguish sandalwood and related pseudo-sandalwoods.

[0202] All the documents mentioned in the present application are incorporated by reference in the present application, as if each document is incorporated by reference individually. Unless and to the extent that the documents mentioned in the present application conflict with the application purpose and / or technical scheme of the present application, the documents mentioned in the present application are incorporated by reference in the present application in the whole content and purpose. When the present application refers to the documents mentioned in the present application, the definitions of the related technical features, terms, names, phrases, etc. in the documents are also incorporated by reference. When the present application refers to the documents mentioned in the present application, the examples and preferred modes of the related technical features incorporated by reference can also be incorporated by reference in the present application, but are limited by the implementability of the present application. It should be understood that when the incorporated content conflicts with the description in the present application, the present application is used for reference or is modified according to the description in the present application.

[0203] Each technical feature in the above-described embodiments and examples can be combined in any suitable manner, and the present application can be applied to other combinations or modifications of the above-described embodiments and examples. The scope of the application is not limited by the above-described embodiments and examples.

[0204] The above embodiments are merely exemplary, and do not limit the scope of the application. Any variation or modification of the above-described embodiments and examples can be made without departing from the scope of the application. Accordingly, the scope of the application should be determined by the following claims, and all changes which come within the meaning and range of equivalents of the claims are intended to be embraced therein. BRIEF DESCRIPTION OF THE DRAWINGS Furthermore, it should be understood that the application can be used in any number of embodiments, including the embodiments specifically described herein and modifications which are within the spirit of the application. For purposes of conveying the essence of the application, the specification and drawings have been simplified in that supporting art has been omitted, and portions have been presented in a generalized or schematic form. It should be further understood that the application can be used in alternative embodiments which employ structures, components, concepts, and / or other changes from those described above without departing from the spirit and scope of the underlying technological concept.

Claims

1. A method for constructing a characteristic spectrum of sandalwood medicinal material, characterized in that, Includes the following steps: Take 1g of the test sample, add 25mL of methanol, sonicate for 30min, filter, and collect the filtrate; evaporate the filtrate to dryness, add 20mL of water to the residue, extract twice with 20mL of ethyl acetate each time, and combine the ethyl acetate layers; evaporate the combined ethyl acetate layers to dryness, add 2mL of methanol to the residue to dissolve, filter, and prepare the test sample solution, wherein the test sample is selected from sandalwood medicinal material. A reference solution is prepared, wherein the reference solution comprises protocatechuic aldehyde, syringaldehyde, and isovanillin; The test solution and the reference solution were subjected to liquid chromatography analysis to establish a characteristic chromatogram of sandalwood. The conditions for the liquid chromatography analysis include: (1) Mobile phase A is acetonitrile, and mobile phase B is an acid solution with a volume concentration of 0.08%~0.12%; (2) Gradient elution is employed, wherein the gradient elution procedure includes: From 0 to 12 minutes, the volume percentage of the mobile phase A increased from 5% to 7%. Between 12 and 25 minutes, the volume percentage of mobile phase A increased from 7% to 12%. Between 25 and 35 minutes, the volume percentage of mobile phase A increased from 12% to 30%. Over 35 to 45 minutes, the volume percentage of mobile phase A increased from 30% to 75%. (3) The liquid chromatography analysis is ultra-high performance liquid chromatography analysis; (4) The detection wavelength is 300nm~320nm; (5) A CORTECS T3 column was used, with a column length of 150 mm, a diameter of 2.1 mm, and a particle size of 1.6 μm for the packing material.

2. The method for constructing the characteristic spectrum of sandalwood medicinal material as described in claim 1, characterized in that, The conditions for the liquid chromatography analysis also include at least one of the following: (1) The flow rate of the gradient elution is 0.2 mL / min to 0.4 mL / min; (2) The column temperature of the chromatographic column is 38℃~42℃; (3) The injection volume is 1 μL to 3 μL.

3. The method for constructing the characteristic spectrum of sandalwood medicinal material as described in claim 2, characterized in that, The ultrasound power is 300W and the frequency is 40kHz.

4. The method for constructing the characteristic spectrum of sandalwood medicinal material as described in any one of claims 1 to 3, characterized in that, The characteristic spectrum of the sandalwood medicinal material includes the following 10 common peaks: peak 1, peak 2, peak 3, peak 4, peak 5, peak 6, peak 7, peak 8, peak 9 and peak 10, wherein peak 2 is protocatechuic aldehyde, peak 6 is isovanillin, and peak 8 is syringaldehyde.

5. A method for detecting sandalwood medicinal materials, characterized in that, Includes the following steps: Take 1g of the sample to be tested, add 25mL of methanol, sonicate for 30min, filter, and take the filtrate; evaporate the filtrate to dryness, add 20mL of water to the residue, extract twice with 20mL of ethyl acetate each time, and combine the ethyl acetate layers; evaporate the combined ethyl acetate layers to dryness, add 2mL of methanol to the residue to dissolve, filter, and prepare the sample solution. A reference solution is prepared, wherein the reference solution comprises protocatechuic aldehyde, syringaldehyde, and isovanillin; The test sample solution and the reference solution were subjected to liquid chromatography analysis to obtain the characteristic chromatogram of the test sample. The conditions for the liquid chromatography analysis include: (1) Mobile phase A is acetonitrile, and mobile phase B is an acid solution with a volume concentration of 0.08%~0.12%; (2) Gradient elution is employed, wherein the gradient elution procedure includes: From 0 to 12 minutes, the volume percentage of the mobile phase A increased from 5% to 7%. Between 12 and 25 minutes, the volume percentage of mobile phase A increased from 7% to 12%. Between 25 and 35 minutes, the volume percentage of mobile phase A increased from 12% to 30%. Over 35 to 45 minutes, the volume percentage of mobile phase A increased from 30% to 75%. (3) The liquid chromatography analysis is ultra-high performance liquid chromatography analysis; (4) The detection wavelength is 300nm~320nm; (5) A CORTECS T3 column was used, with a column length of 150 mm, a diameter of 2.1 mm, and a particle size of 1.6 μm for the packing material.

6. The method for detecting sandalwood medicinal material as described in claim 5, characterized in that, The conditions for the liquid chromatography analysis also include at least one of the following: (1) The flow rate of the gradient elution is 0.2 mL / min to 0.4 mL / min; (2) The column temperature of the chromatographic column is 38℃~42℃; (3) The injection volume is 1 μL to 3 μL.

7. The method for detecting sandalwood medicinal material as described in claim 6, characterized in that, The ultrasound power is 300W and the frequency is 40kHz.

8. The application of the method for constructing the characteristic spectrum according to any one of claims 1 to 4 and / or the detection method according to any one of claims 5 to 6 in the identification of sandalwood medicinal materials.

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