Construction and detection method and application of specific chromatogram of sandalwood medicinal material

Abstract

The invention relates to the technical field of traditional Chinese medicine analysis, in particular to a construction and detection method and application of a specific chromatogram of a sandalwood medicinal material. According to the invention, appropriate chromatographic conditions and detection method are provided, a mobile phase A is acetonitrile, and a mobile phase B is a 0.08-0.12% acid solution; gradient elution is adopted and comprises 0-12 min, the volume percent of a mobile phase A is increased to 7% from 5%, 12 min to 25 min, the volume percent of the mobile phase A is increased to 12% from 7%, 25 min to 35 min, the volume percent of the mobile phase A is increased to 30% from 12%, 35 min to 45 min, and the volume percent of the mobile phase A is increased to 75% from 30%; the prepared specific chromatogram has good specificity, and the specific chromatogram and / or the detection method can be used for identifying the sandalwood medicinal material and can be used for effectively distinguishing sandalwood and similar counterfeit products thereof, so that a theoretical basis is provided for quality control and clinical medicine use of the sandalwood medicinal material.

Description

Technical Field

[0001] This application relates to the technical field of traditional Chinese medicine detection, and particularly to the construction, detection method and application of the characteristic chromatogram of Santalum album L. Background Art

[0002] The traditional Chinese medicine Santalum album L. is the dried heartwood of the trunk of Santalum album L. (a plant of the Santalaceae family). It is a cylindrical wooden section of varying lengths, some of which are slightly curved. Generally, it is about 1 m long and 10 cm - 30 cm in diameter. The outer surface is grayish-yellow or yellowish-brown, smooth and delicate. Some have knots or longitudinal cracks. The cross-section is brownish-yellow, showing oil marks, not easily broken, with a delicate fragrance, and the fragrance is stronger when burned; the taste is light, and it has a slightly pungent feeling when chewed. Santalum album L. has a pungent and warm nature, and enters the spleen, stomach, heart and lung meridians. It promotes qi circulation and warms the middle energizer, and appetizes and relieves pain. It is used for cold coagulation and qi stagnation, discomfort in the chest diaphragm, chest pain, abdominal pain, vomiting and reduced food intake.

[0003] Studying the chemical composition of substances is the key to quality control. To promote the clinical application of traditional Chinese medicine in modern medicine, it is necessary to study the specific composition of Santalum album L. Modern analytical techniques show that the chemical composition of Santalum album L. is mainly sesquiterpenoids, monoterpenoids, phenolic acids and lignans. These chemical components have a wide range of pharmacological activities. For example, sesquiterpenoids, monoterpenoids and lignans are found to have anti-tumor and antibacterial effects, sesquiterpenoids also have a central sedative effect, and phenolic acids are found to have anti-inflammatory, anti-diarrheal and other effects.

[0004] However, the detection methods for Santalum album L. are mostly limited to volatile oils or compound traditional Chinese medicines containing Santalum album L.

[0005] There are few reported quality control methods for single-flavor Santalum album L. The content determination under the item of Santalum album L. in the Chinese Pharmacopoeia (2020 Edition) only stipulates the limit for volatile oils (mainly sesquiterpenoids), and does not limit other components, which has great limitations. There are literatures recording the study of volatile components in Santalum album L. by GC-MS and HPLC methods, and identifying some components such as α-curcumene, β-curcumene, α-santalol, etc. 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 Santalum album L. and more reasonably control the quality of Santalum album L. Summary of the Invention

[0006] The objectives of this application include providing a method for constructing, detecting and applying the characteristic chromatogram of Santalum album L. The technical solution of this application can objectively, scientifically and comprehensively reflect the internal chemical characteristic information of Santalum album L. and be used as the basis for its quality detection.

[0007] Specifically, the method for constructing the characteristic chromatogram includes the following steps:

[0008] Take the test sample, extract it with the extraction solvent to obtain the test sample solution, and the test sample is selected from the Santali Albi Lignum medicinal material;

[0009] Take the test sample solution for liquid chromatography analysis to establish the characteristic chromatogram of the Santali Albi Lignum medicinal material;

[0010] Among them, the conditions for the liquid chromatography analysis include:

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

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

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

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

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

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

[0017] The detection method of the Santali Albi Lignum medicinal material includes the following steps:

[0018] Take the object to be tested, extract it with the extraction solvent to obtain the solution of the object to be tested;

[0019] Take the solution of the object to be tested for liquid chromatography analysis to obtain the characteristic chromatogram of the object to be tested;

[0020] Among them, the conditions for the liquid chromatography analysis include:

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

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

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

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

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

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

[0027] This application also relates to the application of the above characteristic fingerprint construction method and detection method in the identification of Santali Albi Lignum medicinal materials.

[0028] This application conducts liquid chromatography analysis on Santali Albi Lignum by establishing appropriate chromatographic conditions, constructs its characteristic fingerprint. The obtained characteristic fingerprint has good specificity, with good separation effect of each characteristic peak, rich chemical information. This method can save costs, simplify operations, improve efficiency, and has accurate results and strong practicability.

[0029] This application conducts a large number of studies on the characteristic components of Santali Albi Lignum. By establishing appropriate chromatographic conditions, 10 common characteristic peaks are obtained: peak 1, peak 2, peak 3, peak 4, peak 5, peak 6, peak 7, peak 8, peak 9 and peak 10, and protocatechuic acid, isovanillin and isovanillin are identified, clarifying the internal chemical information of Santali Albi Lignum, which can provide important references for its quality detection, identification and clinical medication. Description of the Drawings

[0030] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the following will briefly introduce the drawings required 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 present application. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0031] Figure 1 It is a comparison chart of the chromatograms obtained by detecting Santali Albi Lignum medicinal materials under different mobile phase conditions in Example 1; Figure 1 In it, A is the detection result when acetonitrile is used as mobile phase A, Figure 1 and B in it is the detection result when methanol is used as mobile phase A;

[0032] Figure 2 It is a comparison chart of the chromatograms obtained by detecting Santali Albi Lignum medicinal materials under different detection wavelength conditions in Example 1; Figure 2 In it, A is the detection result when the detection wavelength is 310 nm, Figure 2 and B in it is the detection result when the detection wavelength is 280 nm;

[0033] Figure 3 It is a comparison chart of the chromatograms obtained by detecting Santali Albi Lignum medicinal materials under different chromatographic column conditions in Example 1; Figure 3 In it, A is the detection result when the chromatographic column is Waters BEH C18 (100 mm × 2.1 mm, 1.7 μm), Figure 3In B, the detection results are obtained when the chromatographic column is Waters Cortecs T3 (100 mm × 2.1 mm, 1.6 μm). Figure 3 In C, the detection results are obtained when it is Waters Cortecs T3 (150 mm × 2.1 mm, 1.6 μm).

[0034] Figure 4 It is a comparison chart of the chromatograms obtained by detecting the sandalwood medicinal materials in Example 1 under different elution program conditions.

[0035] Figure 5 It is a comparison chart of the chromatograms obtained by detecting the sandalwood medicinal materials in Example 1 under different column temperature conditions.

[0036] Figure 6 It is a comparison chart of the chromatograms obtained by detecting the sandalwood medicinal materials in Example 1 under different flow rate conditions.

[0037] Figure 7 It is a superimposed chart of the chromatograms obtained by detecting 10 batches of sandalwood medicinal materials in Example 2.

[0038] Figure 8 It is the control characteristic chromatogram of the sandalwood medicinal materials obtained by detection in Example 2.

[0039] Figure 9 It is the comparison chromatogram of the sandalwood sample and protocatechuic aldehyde reference substance obtained by detection in Example 2.

[0040] Figure 10 It is the comparison chromatogram of the sandalwood sample and syringaldehyde reference substance obtained by detection in Example 2.

[0041] Figure 11 It is the comparison chromatogram of the sandalwood sample and isovanillin reference substance obtained by detection in Example 2.

[0042] Figure 12 It is a comparison chart of the chromatographic detections of sandalwood and its similar products, Chinese incense cedar, Chinese thuja, and Taihang Chinese thuja, in Example 3. Specific implementation manners

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

[0045] The term

[0046] Unless otherwise specified or there is a contradiction, the terms or phrases used herein have the following meanings:

[0047] As used herein, the selection scope of the terms "and / or", "or / and", "and / or" includes any one of two or more related listed items, and also includes any and all combinations of the related listed items. The said any and all combinations include combinations of 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, this 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: A, B, and A + B. Another example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, D (that is, the technical solution connected by "logical OR"), and also includes any and all combinations of A, B, C, D, that is, it includes combinations of any two or any three of A, B, C, D, and also includes the combination of the four items A, B, C, D (that is, the technical solution connected by "logical AND").

[0048] In this application, "preferred", "better", "more preferably", etc. are only used to describe embodiments or examples with better effects, and it should be understood that they do not constitute a limitation on the protection scope of this application.

[0049] In this application, "further", "furthermore", "especially", etc. are used for descriptive purposes to indicate differences in content, but should not be construed as a limitation on the protection scope of this application.

[0050] In this application, among the technical features described in an open-ended manner, it includes a closed technical solution composed of the listed features, and also includes an open technical solution containing the listed features.

[0051] In this application, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the selectable numerical values are considered continuous within the above numerical intervals, including the two numerical endpoints of the numerical range (i.e., the minimum value and the maximum value), as well as each numerical value between these two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within the numerical interval, it includes the two endpoint integers of the numerical range, as well as each integer between the two endpoints. In addition, when multiple range descriptions of features or characteristics are provided, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all sub-ranges subsumed therein.

[0052] In this application, the weight can be mass units well-known in the chemical industry such as μg, mg, g, kg, etc.

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

[0054] In one aspect of this application, it relates to a method for constructing a characteristic fingerprint of Santalum album L. medicinal materials, which can objectively, scientifically, and comprehensively reflect the internal chemical characteristic information of Santalum album L. medicinal materials and serve as the basis for its quality inspection.

[0055] In some embodiments of this application, the method for constructing the characteristic fingerprint of Santalum album L. medicinal materials includes the following steps:

[0056] Take the test sample, extract it with an extraction solvent to obtain a test sample solution, and the test sample is selected from Santalum album L. medicinal materials;

[0057] Take the test sample solution for liquid chromatography analysis to establish the characteristic fingerprint of Santalum album L. medicinal materials;

[0058] Among them, the conditions for liquid chromatography analysis include:

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

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

[0061] From 0 to 12 min, the volume percentage of mobile phase A increases from 5% to 7%,

[0062] From 12 min to 25 min, the volume percentage of mobile phase A increases from 7% to 12%.

[0063] From 25 min to 35 min, the volume percentage of mobile phase A increases from 12% to 30%.

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

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

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

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

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

[0069] (3) The column temperature of the chromatographic column is 38°C to 42°C;

[0070] (4) The injection volume is 1 μL to 3 μL;

[0071] (5) The detection wavelength is 300 nm to 320 nm.

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

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

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

[0075] 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 packing is 1.4 μm to 1.8 μm.

[0076] Further, the extraction method is ultrasonic and / or heating under reflux.

[0077] Further, the power of ultrasonic is 200 W to 400 W, and the frequency is 30 kHz to 50 kHz.

[0078] Further, the temperature of heating under reflux is 60°C to 80°C.

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

[0080] In some embodiments, the method for constructing the characteristic chromatogram of sandalwood medicinal materials further includes the steps of preparing a reference substance solution and performing liquid chromatography analysis on the reference substance solution;

[0081] Among them, the conditions for liquid chromatography analysis are the same as those for liquid chromatography analysis in any of the above technical solutions, and will not be elaborated here;

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

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

[0084] Take protocatechuic aldehyde, syringaldehyde, and isovanillin, dissolve them in a solvent to obtain a reference substance solution; the solvent is preferably methanol.

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

[0086] Further, the reference substance 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 chromatogram of sandalwood medicinal materials 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, where Peak 2 is protocatechuic aldehyde, Peak 6 is isovanillin, and Peak 8 is syringaldehyde.

[0088] This application also relates to a detection method for sandalwood medicinal materials, which can detect the contents of various components of sandalwood medicinal materials, evaluate the quality of the medicinal materials more objectively, comprehensively, and accurately. Moreover, the detection method of this application uses less reagent, is simple to operate, has high detection sensitivity, good stability, and accurate and reliable determination results.

[0089] In some embodiments of this application, the detection method for sandalwood medicinal materials includes the following steps:

[0090] Take the test substance and extract it with an extraction solvent to obtain a test sample solution;

[0091] Take the test sample solution for liquid chromatography analysis to obtain the characteristic chromatogram of the test sample;

[0092] Among them, the conditions for liquid chromatography analysis include:

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

[0094] (2) Gradient elution is adopted, and the gradient elution program includes:

[0095] From 0 to 12 min, the volume percentage of mobile phase A increases from 5% to 7%,

[0096] From 12 min to 25 min, the volume percentage of mobile phase A increases from 7% to 12%,

[0097] From 25 min to 35 min, the volume percentage of mobile phase A increases from 12% to 30%,

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

[0099] In some embodiments, in the detection method of sandalwood medicinal materials, the conditions for 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 gradient elution is 0.2 mL / min - 0.4 mL / min;

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

[0103] (4) The injection volume is 1 μL - 3 μL;

[0104] (5) The detection wavelength is 300 nm - 320 nm.

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

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

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

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

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

[0110] The temperature of heating under reflux is 60°C to 80°C.

[0111] In another aspect of the present application, it further relates to the application of the method for constructing and / or detecting the characteristic spectrum described in any of the above technical solutions in the identification of sandalwood medicinal materials.

[0112] In one embodiment, identifying sandalwood medicinal materials includes identifying genuine and fake sandalwood medicinal materials.

[0113] Further, examples of fake sandalwood medicinal materials are Chinese incense cedar, Chinese thuja and Taihang Chinese thuja.

[0114] Further, whether peaks 9 and 10 are contained in the characteristic spectrum of the test substance is used as the basis for identifying the authenticity. If peaks 9 and 10 are not detected, it is identified as a fake.

[0115] The following are some specific embodiments.

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

[0117] Instruments and Reagents

[0118] Instruments: Waters high performance liquid chromatograph (Waters H-Class, Waters Corporation); CORTECS T3 chromatographic column (150 mm × 2.1 mm, 1.6 μm); ten-thousandth analytical balance (ME204E, Mettler-Toledo); millionth analytical balance (XP26, Mettler-Toledo); numerically controlled ultrasonic cleaner (KQ500D, Kunshan Ultrasonic Instruments Co., Ltd.); constant temperature water bath (Shanghai Yiheng Technology Co., Ltd., model: HWS28); ultrapure water system (Merck KGaA, 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 Scientific Co., Ltd., analytical pure); phosphoric acid (Tianjin Fuyu Fine Chemical Co., Ltd., chromatographic pure); acetonitrile (Merck KGaA, chromatographic pure); water is ultrapure water (prepared in the laboratory).

[0120] Test drugs: protocatechuic aldehyde (batch number: 110810 - 201909, National Institutes for Food and Drug Control); the main sources of 10 batches of sandalwood medicinal materials are Guangdong, Tianjin and Guangxi, as shown in Table 1 for details.

[0121] Table 1 Origins of sandalwood medicinal materials

[0122]

[0123] Example 1

[0124] 1.1. Investigation of mobile phase conditions

[0125] Use a CORTECS T3 chromatographic column (100 mm × 2.1 mm, 1.6 μm); use acetonitrile and methanol as mobile phase A respectively, and 0.1% phosphoric acid solution as mobile phase B, and perform gradient elution according to the regulations in Table 2; the flow rate is 0.3 mL / min; the column temperature is 30 °C; the injection volume is 1 μL; the wavelength is 338 nm. The obtained UPLC chromatogram is as Figure 1 shown Figure 1 where A is the detection result when acetonitrile is used as mobile phase A, Figure 1 and B in it is the detection result when methanol is used as mobile phase A.

[0126] Table 2

[0127]

[0128] 1.2. Investigation of detection wavelength conditions

[0129] Use a CORTECS T3 chromatographic column (100 mm × 2.1 mm, 1.6 μm); use acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, and perform gradient elution according to the regulations in Table 3; the flow rate is 0.3 mL / min; the column temperature is 30 °C; the injection volume is 1 μL; the wavelengths are 280 nm and 310 nm respectively. The obtained UPLC chromatogram is as Figure 2 shown. According to Figure 2 it can be seen that the characteristic peak information is rich and the response is good at the detection wavelength of 310 nm.

[0130] Table 3 Gradient elution conditions

[0131]

[0132] 1.3. Investigation of chromatographic column conditions

[0133] Use Waters CORTECS T3 chromatographic column (100 mm × 2.1 mm, 1.6 μm), Waters BEH C18 chromatographic column (100 mm × 2.1 mm, 1.7 μm), and Waters CORTECS T3 chromatographic column (150 mm × 2.1 mm, 1.6 μm) respectively; use acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, and perform gradient elution according to the regulations in Table 3; the flow rate is 0.3 mL / min; the column temperature is 30 °C; the injection volume is 1 μL; the wavelength is 310 nm. The obtained UPLC chromatogram is as Figure 3 , Figure 3In A, the detection result is obtained when the chromatographic column is Waters BEH C18 (100 mm × 2.1 mm, 1.7 μm). Figure 3 In B, the detection result is obtained when the chromatographic column is Waters Cortecs T3 (100 mm × 2.1 mm, 1.6 μm). Figure 3 In C, the detection result is obtained when it is Waters Cortecs T3 (150 mm × 2.1 mm, 1.6 μm); It can be seen by comparison that when using the Waters CORTECS T3 chromatographic column (150 mm × 2.1 mm, 1.6 μm), the resolution of each characteristic peak is better and the peak shape is symmetrical.

[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 conditions. Chromatographic conditions: Using the Waters CORTECS T3 chromatographic column (150 mm × 2.1 mm, 1.6 μm), acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, and gradient elution was carried out according to the provisions of Table 4; The flow rate was 0.3 mL / min; The column temperature was 30 °C; The injection volume was 1 μL; The detection wavelength was 310 nm. The obtained UPLC chromatogram is as Figure 4 , according to Figure 4 it can be seen that the resolution of each characteristic peak under the conditions of Method 4 is better and the distribution is more uniform.

[0136] Table 4 Gradient elution conditions of Method 1, Method 2, Method 3, and Method 4

[0137]

[0138] 1.5. Investigation of column temperature conditions

[0139] Using the CORTECS T3 chromatographic column (150 mm × 2.1 mm, 1.6 μm); Acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, and gradient elution was carried out according to the provisions of Table 3; The flow rate was 0.3 mL / min; The column temperatures were 30 °C, 35 °C, 40 °C, 42 °C, and 45 °C respectively; The injection volume was 1 μL; The wavelength was 310 nm. From Figure 5 it can be seen that when the column temperature is 40 °C, the separation effect of chromatographic peaks is the best, so 40 °C was selected as the column temperature for the characteristic chromatographic conditions of sandalwood medicinal materials.

[0140] 1.6. Investigation of flow rate conditions

[0141] Use a CORTECS T3 chromatographic column (150 mm × 2.1 mm, 1.6 μm); use acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, and perform gradient elution according to the regulations in Table 3; the flow rates are 0.25 mL / min, 0.28 mL / min, 0.3 mL / min, and 0.32 mL / min respectively; the column temperature is 40 °C; the injection volume is 1 μL; the wavelength is 310 nm. It can be seen from Figure 6 that when the flow rate is 0.3 mL / min, the separation effect of the chromatographic peaks is the best, so 0.3 mL / min is selected as the flow rate of the characteristic chromatographic conditions for sandalwood medicinal materials.

[0142] 1.7. Determined chromatographic conditions

[0143] According to the experimental results in Sections 1.1. to 1.6, the final determined chromatographic conditions for liquid phase analysis are as follows: use a CORTECS T3 chromatographic column (150 mm × 2.1 mm, 1.6 μm); use acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, and perform gradient elution according to the regulations in Table 5; the flow rate is 0.3 mL / min; the column temperature is 40 °C; the injection volume is 1 μL; the wavelength is 310 nm. The injection volume is 1 μL.

[0144] Table 5 Determined gradient elution conditions

[0145]

[0146] Example 2

[0147] In this example, multiple batches of sandalwood were detected according to the chromatographic conditions determined under "1.7", and the characteristic fingerprint of sandalwood was constructed.

[0148] 2.1. Preparation of reference substance solution

[0149] Take appropriate amounts of protocatechuic aldehyde, syringaldehyde, and isovanillin reference substances, accurately weigh them, and prepare a mixed solution containing 10 μg per 1 mL with methanol solution, that is, obtain.

[0150] 2.2. Preparation of test solution

[0151] The extraction method and extraction time of the sandalwood test sample were investigated to determine the sample pretreatment method for the characteristic fingerprint of sandalwood medicinal materials.

[0152] 2.2.1. Investigation of extraction method

[0153] In this section, the effects of different extraction methods on the characteristic fingerprint of sandalwood medicinal materials were investigated. Ultrasonic extraction and reflux extraction were used as extraction methods. By observing the peak shapes and resolutions of 10 characteristic peaks, and calculating the "total peak area / sample weight" of 10 characteristic peaks, the effects of different extraction methods on the characteristic fingerprint of sandalwood medicinal materials were compared to select the best extraction method.

[0154] Take an appropriate amount of Santalum album L. (YC01), about 1 g, accurately weighed, in duplicate for 2 groups, with 2 portions in each group. Place in a stoppered conical flask, add 25 mL of methanol, weigh, reflux and ultrasonically treat (power 300 W, frequency 40 kHz) for 30 min respectively, cool, weigh again, make up the lost weight with methanol, filter, evaporate the filtrate to dryness, add 20 mL of water to the residue, extract with ethyl acetate twice, 20 mL each time, combine the ethyl acetate layers, evaporate to dryness, dissolve the residue in 2 mL of methanol, filter, take the continuous filtrate, and inject for analysis under the chromatographic conditions described in item "1.7". The results are shown in Table 6.

[0155] Table 6 Results of investigation on extraction methods of characteristic chromatogram of Santalum album L.

[0156]

[0157] The results showed that there were minor differences between ultrasonic extraction and reflux extraction with different extraction methods, and the deviation values of "total peak area / sample weight" of each characteristic peak were small. For the convenience of operation, ultrasonic extraction was selected.

[0158] 2.2.2. Investigation on extraction time

[0159] In this subsection, the effects of different extraction times on the characteristic chromatogram of Santalum album L. were investigated. Ultrasonic extraction for 15 min, 30 min, and 60 min was used as the extraction times. By observing the peak shapes and resolution of 10 characteristic peaks, and calculating the "total peak area / sample weight" of 10 characteristic peaks, the effects of different extraction times on the characteristic chromatogram of Santalum album L. were compared to select the optimal extraction time.

[0160] Take an appropriate amount of Santalum album L. (YC01), about 1.0 g, accurately weighed, in duplicate for 3 groups, with 2 portions in each group. Place in a stoppered conical flask, add 25 mL of methanol, weigh, ultrasonically treat (power 300 W, frequency 40 kHz) for 15 min, 30 min, and 60 min respectively, cool, weigh again, make up the lost weight with methanol, filter, evaporate the filtrate to dryness, add 20 mL of water to the residue, extract with ethyl acetate twice, 20 mL each time, combine the ethyl acetate layers, evaporate to dryness, dissolve the residue in 2 mL of methanol, filter, take the continuous filtrate, and inject for analysis under the chromatographic conditions described in item "1.7". The results are shown in Table 7.

[0161] Table 7 Results of investigation on extraction time of characteristic chromatogram of Santalum album L.

[0162]

[0163] The results showed that when ultrasonically extracting for 15 min, 30 min, and 60 min respectively, there were no obvious differences in the peak shapes, separation effects, and "total peak area / sample weight" of the 10 characteristic peaks. To ensure complete extraction, the ultrasonic extraction time was selected as 30 min.

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

[0165] According to the experimental results in Sections 2.2.1 and 2.2.2, the pretreatment method of the characteristic chromatogram sample of Santali Albi Lignum is determined as follows: Take an appropriate amount of the sample powder (passed through No. 3 sieve), about 1.0 g, place it in a stoppered conical flask, add 25 mL of methanol, ultrasonically treat (power 300 W, frequency 40 kHz) for 30 min, let it cool, filter, evaporate the filtrate to dryness, add 20 mL of water to the residue, extract with ethyl acetate twice, 20 mL each time, combine the ethyl acetate layers, evaporate to dryness, dissolve the residue in 2 mL of methanol, filter, and obtain the solution.

[0166] 2.3. Specificity investigation

[0167] The results are shown in Figures 9 - 11 , and there are the same chromatographic peaks at the retention times corresponding to the reference solution chromatogram in the chromatogram of the test sample, and there is no interference from the negative sample, indicating that the method has good specificity.

[0168] 2.4. Sample detection and construction of the characteristic chromatogram

[0169] Take 10 batches of Santali Albi Lignum as test samples, and perform liquid chromatography analysis respectively according to the chromatographic conditions determined under item "1.7". The preparation of the test sample solution refers to the preparation method under item "2.2.3". Taking the 2nd peak as the reference peak S, calculate the relative retention time and relative peak area of each characteristic peak with respect to the S peak and calculate the RSD value. The detection results are shown in Tables 8 and 9. The superimposed chromatogram of the characteristic chromatograms of 10 batches of Santali Albi Lignum is shown in Figure 7 .

[0170] Table 8 Characteristic chromatograms of 10 batches of Santali Albi Lignum (relative retention time)

[0171]

[0172] Table 9 Characteristic chromatograms of 10 batches of Santali Albi Lignum (relative peak area)

[0173]

[0174] Match the UPLC characteristic chromatograms of 10 batches of Santali Albi Lignum using the "Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints", generate a reference chromatogram by the average method, and establish a reference characteristic chromatogram for Santali Albi Lignum, such as Figure 8 , and this characteristic chromatogram has a total of 10 characteristic peaks.

[0175] 2.5. Similarity analysis

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

[0177] Table 10 Similarity Results of the Characteristic Spectra of 10 Batches of Sandalwood Samples

[0178]

[0179] According to the comparison results in Table 10, the similarity of the 10 batches of samples to the control pattern is as follows: the similarity of the sandalwood origin is greater than 0.95. Generally speaking, the similarity of the sandalwood medicinal material samples among different batches is relatively high.

[0180] 2.6. Identification of Characteristic Peaks

[0181] 2.6.1. Chromatographic Conditions

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

[0183] Column temperature: 40°C

[0184] Flow rate: 0.3 mL / min

[0185] Injection volume: 2 μL

[0186] Detection wavelength: 310 nm

[0187] Mobile phase ratio and flow rate: Phase A is acetonitrile, Phase B is 0.1% formic acid aqueous solution, and the gradient is 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 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 samples were analyzed by ultra-high performance liquid chromatography-high resolution mass spectrometry (UPLC-Q-TOF / MS). According to the multi-stage mass spectrometry information of the samples, combined with the high-resolution mass spectrometry database of natural products and relevant literature, each target peak was identified (Table 13). Among them, the retention time of the second chromatographic peak was consistent with that of protocatechuic aldehyde reference substance, the retention time of the sixth chromatographic peak was consistent with that of isovanillin reference substance, and the retention time of the eighth chromatographic peak was consistent with that of syringaldehyde 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 in sandalwood

[0198]

[0199] Example 3

[0200] In this example, different adulterants of sandalwood were detected according to the chromatographic conditions determined under item "1.7" in Example 1, and sandalwood and its similar adulterants were identified by the specificity of the characteristic chromatogram.

[0201] Cupressus funebris Endl., Thuja sutchuenensis Franch., and Thuja wilsonii Sprague were used as test samples respectively, and liquid chromatography analysis was carried out according to the chromatographic conditions determined under item "1.7". The preparation method of the test sample solution was referred to the preparation method under item "2.2.3". The results showed (see Figure 12 ) that peaks 9 and 10 in the characteristic chromatogram of the original sandalwood were not detected in Cupressus funebris Endl., Thuja sutchuenensis Franch., and Thuja wilsonii Sprague, indicating that the established method could effectively distinguish sandalwood and related adulterants.

[0202] All the documents mentioned in this application are cited in this application as references, just as each document is cited separately as a reference. Unless it conflicts with the application purpose and / or technical solution of this application, the cited documents involved in this application are cited for all contents and all purposes. When citing documents in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited together. When citing documents in this application, the examples and preferred methods of the relevant technical features cited can also be used as references and incorporated into this application, but only to the extent that this application can be implemented. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or be amended adaptively according to the description in this application.

[0203] The technical features of the above-described embodiments and examples can be combined in any suitable manner. For the sake of brevity of description, not all possible combinations of the various technical features in the above-described embodiments and examples are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered to be within the scope described in this specification.

[0204] The above-described embodiments merely represent several implementation manners of the present application, but should not be construed as limiting the scope of the patent application. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present application, several modifications and improvements can still be made, and these all belong to the protection scope of the present application. In addition, it should be understood that after reading the above teachings 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 protection scope 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 based on the technical solutions provided in the present application are all within the protection scope of the appended claims of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the appended claims, and the specification can be used to interpret the content of the claims.

Claims

1. A method for constructing a characteristic fingerprint of Santali Albi Lignum, characterized in that, It includes the following steps: Take the test sample, extract it with an extraction solvent to obtain a test sample solution, and the test sample is selected from the sandalwood medicinal material; Take the test sample solution for liquid chromatography analysis to establish a characteristic chromatogram of the sandalwood medicinal material; Among them, 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 adopted, and the program of the gradient elution includes: 0 - 12 min, the volume percentage of mobile phase A rises from 5% to 7%, 12 min - 25 min, the volume percentage of mobile phase A rises from 7% to 12%, 25 min - 35 min, the volume percentage of mobile phase A rises from 12% to 30%, 35 min - 45 min, the volume percentage of mobile phase A rises from 30% to 75%.

2. The method for constructing a characteristic fingerprint of Santali Albi Lignum according to claim 1, characterized in that, The conditions for the liquid chromatography analysis further 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 0.2 mL / min - 0.4 mL / min; (3) The column temperature of the chromatographic column is 38°C - 42°C; (4) The injection volume is 1 μL - 3 μL; (5) The detection wavelength is 300 nm - 320 nm.

3. The method for constructing a characteristic fingerprint of Santali Albi Lignum according to claim 2, characterized in that, The chromatographic column is a C18 chromatographic column; and / or, the extraction solvent includes organic alcohol.

4. The method for constructing a characteristic fingerprint of Santali Albi Lignum according to claim 3, characterized in that, The model of the C18 chromatographic column is CORTECS T3 chromatographic column; Further, the column length of the CORTECS T3 chromatographic column is 100 mm - 150 mm, the diameter is 2 mm - 2.2 mm, and the particle size of the packing is 1.4 μm - 1.8 μm.

5. The method for constructing a characteristic fingerprint of Santali Albi Lignum according to claim 3, characterized in that, The extraction method is ultrasonic and / or heating under reflux; The power of the ultrasonic is 200 W - 400 W, and the frequency is 30 kHz - 50 kHz; The temperature of the heating under reflux is 60°C - 80°C.

6. The method for constructing a characteristic fingerprint of Santali Albi Lignum according to claim 1, characterized in that, It further includes the steps of preparing a reference substance solution and taking the reference substance solution for liquid chromatography analysis; The conditions for the liquid chromatography analysis are the same as those in any one of claims 1 - 5 for the liquid chromatography analysis; The reference substance solution includes protocatechuic aldehyde, syringaldehyde, and isovanillin.

7. The method for constructing a characteristic fingerprint of Santali Albi Lignum according to any one of claims 1 to 6, characterized in that, The characteristic chromatogram 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, where Peak 2 is protocatechuic aldehyde, Peak 6 is isovanillin, and Peak 8 is syringaldehyde.

8. A method for detecting Santali Albi Lignum, characterized in that, It includes the following steps: Take the test object, extract it with an extraction solvent to obtain a test object solution; Take the test object solution for liquid chromatography analysis to obtain a characteristic chromatogram of the test object; Among them, 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 adopted, and the program of the gradient elution includes: 0 - 12 min, the volume percentage of mobile phase A rises from 5% to 7%, 12 min - 25 min, the volume percentage of mobile phase A rises from 7% to 12%, 25 min - 35 min, the volume percentage of mobile phase A rises from 12% to 30%, 35 min to 45 min, the volume percentage of the mobile phase A increases from 30% to 75%.

9. The detection method of the sandalwood medicinal material according to claim 8, wherein, The conditions of the liquid chromatography analysis further 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 0.2 mL / min to 0.4 mL / min; (3) The column temperature of the chromatographic column is 38 °C to 42 °C; (4) The injection volume is 1 μL to 3 μL; (5) The detection wavelength is 300 nm to 320 nm.

10. The detection method of the sandalwood medicinal material according to claim 9, wherein, The chromatographic column is a C18 chromatographic column; and / or, the extraction solvent includes organic alcohol.

11. The detection method of the sandalwood medicinal material according to claim 10, wherein, The model of the C18 chromatographic column is CORTECS T3 chromatographic column; Furthermore, 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 packing is 1.4 μm to 1.8 μm.

12. The detection method of the sandalwood medicinal material according to claim 10, wherein, The extraction method is ultrasonic and / or heating under reflux; The power of the ultrasonic is 200 W to 400 W, and the frequency is 30 kHz to 50 kHz; The temperature of the heating under reflux is 60 °C to 80 °C.

13. The application of the method for constructing the characteristic spectrum according to any one of claims 1 to 6 and / or the detection method according to any one of claims 7 to 11 in the identification of sandalwood medicinal materials.

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