Detection method for simultaneously determining contents of 12 components in dragon's blood medicinal material

Abstract

The invention provides a detection method for simultaneously determining the contents of 12 components in a dragon's blood medicinal material, which adopts a high performance liquid chromatography method for simultaneously determining the contents of 7, 8-dihydroxy-7, 8-dihydroxy-7, 8-dihydroxy-7, 8-dihydroxy-7 the content of 4, 4 '-dihydroxy flavone, resveratrol, loureirin A, loureirin B, loureirin C, loureirin D, pterostilbene, pinoresinol, licochalcone, 7, 4'-dihydroxy homoisoflavone, liquiritigenin and isoliquiritigenin is measured, and mobile phases comprise a mobile phase A glacial acetic acid aqueous solution and a mobile phase B acetonitrile; the method comprises the following steps: S1, preparing a test solution; s2, preparing a mixed standard reference solution; and S3, high performance liquid chromatography determination. The detection method established by the invention can simultaneously detect 12 medicinal components in the dragon's blood medicinal material, and can provide a basis for quality evaluation and control of the dragon's blood medicinal material. The detection method is stable, feasible, reasonable in design, high in operability, precise and reliable.

Description

Technical Field

[0001] The invention relates to the field of quality detection of traditional Chinese medicine preparations, and in particular to a detection method for simultaneously determining the contents of 12 components in a dragon's blood medicinal material. Background Art

[0002] Dragon's Blood is a typical pure resin-type medicinal material, and its medicinal history can be traced back to the Southern and Northern Dynasties. Its medicinal origins include five species of the genus Dracaena in the Liliaceae family, among which Hainan Dracaena (D. cambodiana Pierre ex Gagn.) or Sword-leaved Dracaena (D. cochinchinensis (Lour.) SCChen) have natural distribution in my country and are included in the Dragon's Blood medicinal material origin standard, and are the most abundant in resource reserves among the five origin species. The other three species D. draco, D. cinnabari and D. ombet have been listed in the Red List of Endangered Species of the World Conservation Union with different degrees of endangered levels. According to existing research reports, Dragon's Blood has multiple biological activities such as hemostasis, blood circulation, analgesia, promotion of epidermal repair, anti-oxidation, anti-inflammatory, and anti-radiation.

[0003] The quality standard of dragon's blood medicinal material has been studied and formulated since 1974 and has undergone many revisions. The latest revised version is the ministerial standard (WS3-082(Z-016)-99(Z)) formulated by the Pharmaceutical Factory of Guangxi University of Traditional Chinese Medicine in 1999, approved by the National Pharmacopoeia Committee and issued by the State Food and Drug Administration. Among them, due to the limited reports on the separation and identification of the chemical components of dragon's blood at that time, the researchers selected the unique dihydrochalcone compound dragon's blood B from more than 30 compounds that have been separated as the quality marker of dragon's blood medicinal material, and limited its content to more than 0.40%. With the deepening of modern research on the separation and purification of dragon's blood medicinal components and the evaluation of pharmacological activity, people have a deeper understanding of the medicinal components of dragon's blood. At present, more than 300 monomer compounds have been separated and identified from dragon's blood, including 23 representative dihydrochalcone compounds, and most of them have good pharmacological activity, such as dragon's blood A, dragon's blood C and dragon's blood D. In addition, it also includes a large number of active flavonoids and stilbene substances. Therefore, the rationality of the limit value of dragon's blood protein B as a quality marker of dragon's blood medicinal materials has been questioned by many scientific researchers.

[0004] In order to further control the quality of medicinal materials and ensure the efficacy of traditional Chinese medicine, multi-component content control technology combined with characteristic spectrum technology has been successfully applied to the formulation of quality standards for multiple medicinal materials. However, there are few research reports on the content determination method of the active ingredients in Dragon's Blood, especially the high performance liquid chromatography method commonly used in medicinal material quality standards. Therefore, in order to promote the improvement of the quality standard of Dragon's Blood medicinal materials, it is of great significance to establish a simultaneous content determination method for multiple active ingredients in Dragon's Blood. Summary of the invention

[0005] In view of this, the present invention proposes a detection method for simultaneously determining the contents of 12 components in the Dragon's Blood Dried Herb.

[0006] The technical solution of the present invention is achieved in this way:

[0007] A method for simultaneously determining the contents of 12 components in dragon's blood medicinal materials, which uses high performance liquid chromatography to simultaneously determine the contents of 7,4'-dihydroxyflavone, resveratrol, dragon's blood A, dragon's blood B, dragon's blood C, dragon's blood D, pterostilbene, pinoresinol, licorice chalcone, 7,4'-dihydroxy homoisoflavone, liquiritigenin and isoliquiritigenin in dragon's blood medicinal materials;

[0008] The mobile phases include: mobile phase A is glacial acetic acid aqueous solution, mobile phase B is pure acetonitrile, and gradient elution is adopted;

[0009] The procedure of the gradient elution includes:

[0010] 0-15min, the volume percentage of the mobile phase A is 75%, the volume percentage of the mobile phase B is 25%,

[0011] 15.1-30min, the volume percentage of the mobile phase A decreased from 75% to 66%, and the volume percentage of the mobile phase B increased from 25% to 34%,

[0012] 30.1-38min, the volume percentage of the mobile phase A is 66%, the volume percentage of the mobile phase B is 34%,

[0013] 38.1-46min, the volume percentage of the mobile phase A decreased from 66% to 62%, and the volume percentage of the mobile phase B increased from 34% to 38%,

[0014] 46.1-70min, the volume percentage of the mobile phase A decreased from 62% to 57%, and the volume percentage of the mobile phase B increased from 38% to 33%,

[0015] 70.1-80min, the volume percentage of the mobile phase A increased from 57% to 75%, and the volume percentage of the mobile phase B decreased from 33% to 25%,

[0016] 80.1-85min, the volume percentage of the mobile phase A is 75%, and the volume percentage of the mobile phase B is 25%;

[0017] The specific steps include:

[0018] S1. Prepare the test solution

[0019] Take the dragon's blood medicinal material to be tested, weigh it accurately, add methanol, ultrasonically treat it, and filter it to obtain the test solution;

[0020] S2. Prepare mixed standard reference solution

[0021] Accurately weigh 7,4'-dihydroxyflavone, resveratrol, loureirin A, loureirin B, loureirin C, loureirin D, pterostilbene, pinoresinol, licorice chalcone, 7,4'-dihydroxy homoisoflavone, liquiritigenin and isoliquiritigenin reference substances respectively, add methanol, shake well to obtain 12 standard reference substance solutions, mix the 12 standard reference substance solutions, add methanol to obtain a mixed standard reference substance solution;

[0022] S3. High performance liquid chromatography

[0023] Accurately pipette the test solution and the mixed standard reference solution, inject them into the high performance liquid chromatograph, record the liquid chromatogram, and obtain the contents of the 12 components.

[0024] Furthermore, the dragon's blood medicinal material comes from Hainan Dragon Blood Tree (D. cambodiana Pierre ex Gagn.) or Sword-leaved Dragon Blood Tree (D. cochinchinensis (Lour.) SCChen).

[0025] Furthermore, the concentration of the mobile phase A, glacial acetic acid aqueous solution, is 0.35-0.65% v / v.

[0026] Furthermore, in step S1, the solid-liquid ratio of the tested dragon's blood medicinal material to methanol is 1:100-200 g / mL.

[0027] Furthermore, in step S1, the ultrasonic treatment is performed at 20-30 KHz for 2-5 min; and the filtration is performed through a 0.22 μm filter membrane.

[0028] Furthermore, in step S3, the high performance liquid chromatograph is Waters e2695, the detector is Waters 2489UV, and the chromatographic column is Waters XBridge-C18 (250 mm×4.6 mm×5 μm).

[0029] Furthermore, in step S3, the detection wavelength of the high performance liquid chromatograph is 250-290 nm, the column temperature is 30-35° C., and the flow rate is 0.8-1.2 mL / min.

[0030] Furthermore, in step S3, the contents of the 12 components are obtained by establishing regression curve standard equations of the 12 active ingredients using mixed standard reference solutions of different concentrations and their peak areas, and by reading the corresponding peak areas in the chromatogram of the sample to be tested and substituting them into the standard curve equation to obtain the contents.

[0031] Furthermore, the peak times of the 12 components in the dragon's blood medicinal material are 7,4'-dihydroxyflavone: 9.425min, resveratrol: 10.497min, liquiritigenin: 13.378min, dragon's blood D: 14.751min, pinoresinol: 17.262min, 7,4'-dihydroxyhomoisoflavone: 20.399min, licorice chalcone: 24.321min, dragon's blood C: 25.068min, isoliquiritigenin: 33.082min, dragon's blood B: 51.782min, dragon's blood A: 53.394min, and pterostilbene: 64.504min.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] The detection method established by the present invention can simultaneously detect 12 kinds of active ingredients in the Dragon's Blood medicinal material, and can provide a basis for the quality evaluation and control of the Dragon's Blood medicinal material. The detection method of the present invention is stable and feasible, and the research results of the methodologies such as linear relationship, precision, repeatability, recovery rate, durability, etc. show that the method is reasonably designed, highly operable, and precise and reliable. The detection method of the present invention has good stability, precision, detection limit, and quantitative limit for the content determination of 12 kinds of active ingredients. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 HPLC spectra of the test solution and the mixed standard reference solution of the present invention;

[0035] Among them, (A) is the sword-leaved dragon's blood, (B) is the Hainan dragon's blood, and (C) is the mixed standard reference solution.

[0036] Figure 2 HPLC spectrum for specificity investigation;

[0037] In the figure, (A) is a mixed standard reference solution, and (B) is an acetonitrile solvent. DETAILED DESCRIPTION

[0038] In order to better understand the technical content of the present invention, specific embodiments are provided below to further illustrate the present invention.

[0039] Unless otherwise specified, the experimental methods used in the embodiments of the present invention are all conventional methods.

[0040] Unless otherwise specified, the materials, reagents, etc. used in the embodiments of the present invention can be obtained from commercial sources.

[0041] The instruments and models used in the present invention are as follows:

[0042]

[0043] The drugs used in the present invention and their purchase sources are as follows:

[0044] Resveratrol China Food and Drug Inspection Institute Dragon's blood A China Food and Drug Inspection Institute Dragon's blood B China Food and Drug Inspection Institute 7,4'-Dihydroxyflavone China Food and Drug Inspection Institute Pterostilbene Chengdu Purifa Technology Development Co., Ltd. Dragon's Blood C Chengdu Purifa Technology Development Co., Ltd. Dragon's Blood D Chengdu Purifa Technology Development Co., Ltd. Pinoresinol Chengdu Pusi Biotechnology Co., Ltd. Licorice Chalcone Chengdu Pusi Biotechnology Co., Ltd. Licorice Chengdu Pusi Biotechnology Co., Ltd. Isoliquiritigenin Chengdu Pusi Biotechnology Co., Ltd. 7,4'-Dihydroxyisoflavonoids Chengdu Pusi Biotechnology Co., Ltd. Methanol Merck KGaA Acetonitrile Merck KGaA glacial acetic acid Xilong Scientific Co., Ltd.

[0045] The raw material of the dragon's blood medicinal material of the present invention comes from Hainan dragon's blood tree (D.cambodiana Pierre ex Gagn.) or sword-leaved dragon's blood tree (D.cochinchinensis (Lour.) SCChen);

[0046] The dragon's blood medicinal material produced by the Hainan Dragon Blood Tree is obtained by ethanol extraction and concentration of the resinous wood of the wild Hainan Dragon Blood Tree in Wanning City, Hainan Province.

[0047] The dragon's blood medicinal material produced by the sword-leaved Dracaena is obtained by ethanol extraction and concentration of the resinous wood of the sword-leaved Dracaena in Xishuangbanna Autonomous Prefecture, Yunnan Province.

[0048] Example 1

[0049] A method for simultaneously determining the contents of 12 components in a dragon's blood medicinal material comprises the following steps:

[0050] S1. Prepare the test solution

[0051] Take the drug material of Dragon's Blood to be tested, accurately weigh 0.20 g, add 35 mL of methanol, ultrasonically treat for 5 min at 20-30 KHz, cool to room temperature, and filter through a 0.22 μm filter membrane to obtain the test solution;

[0052] S2. Prepare mixed standard reference solution

[0053] Accurately weigh 10.0 mg each of 7,4'-dihydroxyflavone, resveratrol, dracotrienol A, dracotrienol B, dracotrienol C, dracotrienol D, pterostilbene, pinoresinol, licorice chalcone, 7,4'-dihydroxyisoflavone, licoricein and isoliquiritigenin reference substances, place them in a 10 mL volumetric flask, add methanol to dissolve and dilute to the scale, shake well to obtain 12 standard reference substance solutions, take 1 mL of each of the 12 standard reference substance solutions, place them in a 20 mL volumetric flask, add methanol to obtain a mixed standard reference substance solution, wherein the mixed standard reference substance solution contains 17.2353 μg / mL of 7,4'-dihydroxyflavone, 17.9715 μg / mL of resveratrol, 98.2450 μg / mL of dracotrienol A, 17.4048 μg / mL of dracotrienol B, and 17.7067 μg / mL of dracotrienol C. 82.3763μg / mL, dragon's blood D79.2901μg / mL, pterostilbene 83.8885μg / mL, pinoresinol 17.3264μg / mL, licorice chalcone 81.3186μg / mL, 7,4'-dihydroxy homoisoflavone 18.3744μg / mL, licorice 17.0692μg / mL and isoliquiritigenin 13.5632μg / mL;

[0054] S3. High performance liquid chromatography

[0055] Accurately pipette 10 μL of the test solution and the mixed standard reference solution, inject into the high performance liquid chromatograph (Waters e2695 high performance liquid chromatograph, Waters 2489UV detector, Empower workstation), and record the liquid chromatogram.

[0056] The regression curve standard equations of 12 kinds of active ingredients were established by using mixed standard reference solutions of different concentrations and their peak areas. The corresponding peak areas in the chromatogram of the sample to be tested were read and substituted into the standard curve equation to obtain the content.

[0057] Parameters of HPLC:

[0058]

[0059]

[0060] The gradient elution procedure includes:

[0061] 0-15min, the volume percentage of the mobile phase A is 75%, the volume percentage of the mobile phase B is 25%,

[0062] 15.1-30min, the volume percentage of the mobile phase A decreased from 75% to 66%, and the volume percentage of the mobile phase B increased from 25% to 34%,

[0063] 30.1-38min, the volume percentage of the mobile phase A is 66%, the volume percentage of the mobile phase B is 34%,

[0064] 38.1-46min, the volume percentage of the mobile phase A decreased from 66% to 62%, and the volume percentage of the mobile phase B increased from 34% to 38%,

[0065] 46.1-70min, the volume percentage of the mobile phase A decreased from 62% to 57%, and the volume percentage of the mobile phase B increased from 38% to 33%,

[0066] 70.1-80min, the volume percentage of the mobile phase A increased from 57% to 75%, and the volume percentage of the mobile phase B decreased from 33% to 25%,

[0067] 80.1-85min, the volume percentage of the mobile phase A is 75%, and the volume percentage of the mobile phase B is 25%.

[0068] Example 2

[0069] According to the detection method of Example 1, the high performance liquid chromatography was performed on the dragon's blood medicinal materials (Dragon's Blood of Hainan and Dragon's Blood of Sword Leaf).

[0070] from Figure 1 It can be seen that the 12 active ingredients to be tested have good separation and peak shape, and have good separation from other ingredients in the two samples, among which peak 1 is 7,4'-dihydroxyflavone (9.425min), peak 2 is resveratrol (10.497min), peak 3 is licorice root (13.378min), peak 4 is dragon blood D (14.751min), peak 5 is pinoresinol (17.262min), Peak 6 is 7,4'-dihydroxyhomoisoflavone (20.399 min), peak 7 is licorice chalcone (24.321 min), peak 8 is dragon's blood C (25.068 min), peak 9 is isoliquiritigenin (33.082 min), peak 10 is dragon's blood B (51.782 min), peak 11 is dragon's blood A (53.394 min), and peak 12 is pterostilbene (64.504 min).

[0071] Test case - methodological investigation

[0072] 1. Linear

[0073] Based on the detection method of Example 1, a gradient dilution method was used to obtain mixed standard reference substance solutions of other concentrations, and mixed standard reference substance solutions of different concentrations were injected repeatedly 3 times, and the Empower workstation was used for automatic peak area integration, and the average value of the 3 repeated peak areas was taken as the ordinate Y, and the concentration of the mixed standard reference substance solution was taken as the abscissa X, a scatter plot was established, and the standard curve equation was simulated. Among them, the concentration of the mixed standard reference substance solution when the ratio of the detection signal height to the baseline noise height (signal-to-noise ratio) was 3:1 was the detection limit (LOD) of the component, and the concentration when the ratio was 10:1 was the quantitative limit (LOQ) of the component.

[0074] The standard curve equations of the 12 active ingredients are shown in Table 1. It can be seen from Table 1 that the 12 linear regression equations have a good linear relationship (R 2 >0.9994).

[0075] Table 1

[0076]

[0077]

[0078] 2. Precision test

[0079] 10 μL of the mixed standard reference solution was accurately pipetted and sampled for 6 consecutive times within 24 hours. The measurement results are shown in Table 2.

[0080] Table 2

[0081] Ingredient name Peak area average Standard Deviation RSD% 7,4'-Dihydroxyflavone 44.92 0.30 0.67 Resveratrol 252.13 1.60 0.64 Licorice 59.97 0.44 0.73 Dragon's Blood D 83.95 0.60 0.71 Pinoresinol 42.31 0.25 0.59 7,4'-Dihydroxyisoflavonoids 50.83 0.30 0.59 Licorice Chalcone 5.73 0.03 0.60 Dragon's Blood C 45.29 0.28 0.61 Isoliquiritigenin 24.23 0.16 0.65 Dragon's blood B 51.75 0.35 0.67 Dragon's blood A 78.20 0.50 0.64 Pterostilbene 127.73 0.79 0.62

[0082] As can be seen from Table 2, the method for simultaneously determining the contents of 12 kinds of medicinal ingredients in the Dragon's Blood medicinal material of the present invention has good precision.

[0083] 3. Test solution stability test

[0084] The test solution prepared in Example 1 was sampled and tested within 0h, 2h, 4h, 6h, 8h, 10h, 12h, 16h, 24h, 36h, and 48h, respectively. The 12 active ingredients were integrated and the relative standard deviation RSD of the peak area was calculated to determine whether the test solution was stable over a period of time. The results are shown in Table 3.

[0085] Table 3

[0086] Ingredient name Peak area average Standard Deviation RSD% 7,4'-Dihydroxyflavone 7.1227 0.1666 2.34 Resveratrol 40.8302 0.7434 1.82 Licorice 10.6976 0.2606 2.44 Dragon's Blood D 24.4929 0.4028 1.64 Pinoresinol 1.3150 0.0308 2.34 7,4'-Dihydroxyisoflavonoids 12.0879 0.3792 3.14 Licorice Chalcone 1.2842 0.0445 3.47 Dragon's Blood C 40.8653 0.5051 1.24 Isoliquiritigenin 12.2547 0.3224 2.63 Dragon's blood B 6.4487 0.1778 2.76 Dragon's blood A 30.5512 0.3040 0.99 Pterostilbene 21.6901 0.6376 2.94

[0087] As can be seen from Table 3, the 12 active ingredients in the test solution have good stability within 48 hours, meeting the time requirement of the determination.

[0088] 4. Specificity test

[0089] Prepare a mixed standard reference solution of 12 components to be tested, and inject an equal volume of methanol solution as a blank solvent into a liquid chromatograph for determination. Figure 2 shown.

[0090] from Figure 2 It can be seen that the absorption of the blank reagent is extremely low and has no interference with the chromatographic analysis of the 12 components to be tested, indicating that the detection method of the present invention has good specificity.

[0091] 5. Repeatability test

[0092] The mixed standard reference solution in Example 1 was accurately weighed and repeatedly injected and analyzed for 12 times. The peaks of 7,4'-dihydroxyflavone, resveratrol, licorice root, dragon's blood D, pinoresinol, 7,4'-dihydroxy homoisoflavone, licorice chalcone, dragon's blood C, isoliquiritigenin, dragon's blood B, dragon's blood A and pterostilbene compound in the chromatograms of the 12 injections were integrated by the Empower workstation, and the relative standard deviation RSD of the peak area of ​​the 12 injections was calculated. The results are shown in Table 4.

[0093] Table 4

[0094] Ingredient name Peak area average Standard Deviation RSD% 7,4'-Dihydroxyflavone 44.81 0.26 0.58 Resveratrol 251.39 1.55 0.62 Licorice 59.79 0.37 0.62 Dragon's Blood D 83.72 0.51 0.61 Pinoresinol 42.20 0.24 0.57 7,4'-Dihydroxyisoflavonoids 50.65 0.32 0.63 Licorice Chalcone 5.73 0.03 0.54 Dragon's Blood C 45.10 0.33 0.73 Isoliquiritigenin 24.16 0.15 0.60 Dragon's blood B 51.59 0.32 0.62 Dragon's blood A 77.95 0.47 0.61 Pterostilbene 127.36 0.75 0.59

[0095] As can be seen from Table 4, it is proved that the determination method of the present invention has good repeatability.

[0096] 6. Recovery rate test

[0097] The samples to be tested were taken for determination of the contents of 12 components to be tested, and the contents were calculated. According to the contents, 0.5, 1.0 and 1.5 times the amount of standard reference solution were added, and the samples were injected 3 times respectively. The contents of the 12 components to be tested in the samples after adding the standard reference solution were calculated, and the sample recovery rate was calculated. The results are shown in Table 5.

[0098] Sample recovery rate (%) = (C1-C2) / C×100%

[0099] Wherein, C1 is the target component content measured after adding the standard reference substance, C2 is the target component content measured in the sample without adding the standard reference substance, and C is the amount of the added standard reference substance.

[0100] Table 5

[0101]

[0102] As can be seen from Table 5, the assay method of the present invention has a good recovery rate.

[0103] Application Examples

[0104] The content of 5 batches of Hainan Dragon Blood and 5 batches of Sword Leaf Dragon Blood were determined by the content detection method of Example 1. The results are shown in Tables 6-7.

[0105] Table 6

[0106]

[0107]

[0108] Table 7

[0109]

[0110]

[0111] It can be seen from Tables 6-7 that the detection method of the present invention can be well applied to the determination of the contents of 12 kinds of active ingredients in two kinds of Dragon's Blood medicinal materials.

[0112] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for simultaneously determining the contents of 12 components in Dragon's Blood, characterized in that: The method uses high performance liquid chromatography to simultaneously determine the contents of 7,4'-dihydroxyflavone, resveratrol, dragon's blood A, dragon's blood B, dragon's blood C, dragon's blood D, pterostilbene, pinoresinol, licorice chalcone, 7,4'-dihydroxy homoisoflavone, licorice and isoliquiritigenin in dragon's blood. The mobile phases include: mobile phase A is glacial acetic acid aqueous solution, mobile phase B is acetonitrile, and gradient elution is adopted; The procedure of the gradient elution includes: 0-15min, the volume percentage of the mobile phase A is 75%, the volume percentage of the mobile phase B is 25%, 15.1-30min, the volume percentage of the mobile phase A decreased from 75% to 66%, and the volume percentage of the mobile phase B increased from 25% to 34%, 30.1-38min, the volume percentage of the mobile phase A is 66%, the volume percentage of the mobile phase B is 34%, 38.1-46min, the volume percentage of the mobile phase A decreased from 66% to 62%, and the volume percentage of the mobile phase B increased from 34% to 38%, 46.1-70min, the volume percentage of the mobile phase A decreased from 62% to 57%, and the volume percentage of the mobile phase B increased from 38% to 33%, 70.1-80min, the volume percentage of the mobile phase A increased from 57% to 75%, and the volume percentage of the mobile phase B decreased from 33% to 25%, 80.1-85min, the volume percentage of the mobile phase A is 75%, and the volume percentage of the mobile phase B is 25%; The specific steps include: S1. Prepare the test solution Take the dragon's blood medicinal material to be tested, weigh it accurately, add methanol, ultrasonically treat it, and filter it to obtain the test solution; S2. Prepare mixed standard reference solution Weigh 7,4'-dihydroxyflavone, resveratrol, dragon's blood A, dragon's blood B, dragon's blood C, dragon's blood D, pterostilbene, pinoresinol, licorice chalcone, 7,4'-dihydroxy homoisoflavone, licorice and isoflavone respectively. Licoricein reference substance, add methanol, shake well to obtain 12 standard reference substance solutions, mix the 12 standard reference substance solutions, add methanol to obtain a mixed standard reference substance solution; S3. High performance liquid chromatography Accurately pipette the test solution and the mixed standard reference solution, inject them into the high performance liquid chromatograph, record the liquid chromatogram, and obtain the contents of the 12 components.

2. A method for simultaneously determining the contents of 12 components in Dragon's Blood medicinal material as claimed in claim 1, characterized in that: The dragon's blood medicinal material comes from Hainan dragon blood tree or sword-leaved dragon blood tree.

3. A method for simultaneously determining the contents of 12 components in Dragon's Blood medicinal material as claimed in claim 1, characterized in that: The concentration of the mobile phase A, glacial acetic acid aqueous solution, is 0.35-0.65% v / v.

4. A method for simultaneously determining the contents of 12 components in Dragon's Blood medicinal material as claimed in claim 1, characterized in that: In step S1, the solid-liquid ratio of the tested dragon's blood medicinal material to methanol is 1:100-200 g / mL.

5. The method for simultaneously determining the contents of 12 components in Dragon's Blood medicinal material as claimed in claim 1, characterized in that: In step S1, the ultrasonic treatment is performed at 20-30 KHz for 2-5 min; and the filtration is performed through a 0.22 μm filter membrane.

6. A method for simultaneously determining the contents of 12 components in Dragon's Blood medicinal material as claimed in claim 1, characterized in that: In step S3, the high performance liquid chromatograph is Waters e2695, the detector is Waters 2489UV, and the chromatographic column is Waters XBridge-C18 (250 mm×4.6 mm×5 μm).

7. The method for simultaneously determining the contents of 12 components in Dragon's Blood medicinal material as claimed in claim 1, characterized in that: In step S3, the detection wavelength of the high performance liquid chromatograph is 250-290 nm, the column temperature is 30-35° C., and the flow rate is 0.8-1.2 mL / min.

8. The method for simultaneously determining the contents of 12 components in Dragon's Blood medicinal material as claimed in claim 1, characterized in that: In step S3, the contents of the 12 components are obtained by establishing regression curve standard equations of the 12 active ingredients using mixed standard reference solutions of different concentrations and their peak areas, and by reading the corresponding peak areas in the chromatogram of the sample to be tested and substituting them into the standard curve equation to obtain the contents.

9. The method for simultaneously determining the contents of 12 components in Dragon's Blood medicinal material as claimed in claim 1, characterized in that: The peak times of the 12 components in the dragon's blood medicinal material are 7,4'-dihydroxyflavone: 9.425min, resveratrol: 10.497min, licorice root: 13.378min, dragon's blood D: 14.751min, Pinoresinol: 17.262min, 7,4'-dihydroxyisoflavonoids: 20.399min, Licorice chalcone: 24.321min, Loureirin C: 25.068min, Isoliquiritigenin: 33.082min, Loureirin B: 51.782min, Loureirin A: 53.394min, Pterostilbene: 64.504min.

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