Method for establishing fingerprint spectrum of volatile components of caulis spatholobi traditional Chinese medicine compound and fingerprint spectrum of caulis spatholobi traditional Chinese medicine compound

Abstract

The invention relates to the technical field of quality standards and detection of traditional Chinese medicine prescriptions, in particular to a method for establishing a fingerprint spectrum of volatile components of a caulis spatholobi traditional Chinese medicine compound and the fingerprint spectrum of the caulis spatholobi traditional Chinese medicine compound. The invention discloses a method for establishing a fingerprint spectrum of volatile components of a caulis spatholobi traditional Chinese medicine compound, germacrone, curcumenol, beta-elemene and curdione are used as reference substances, the fingerprint spectrum is established through gas chromatography-mass spectrometry, and the fingerprint spectrum has 24 common peaks. The fingerprint spectrum of the volatile components of the caulis spatholobi traditional Chinese medicine compound has the advantages of being good in stability, high in precision, good in repeatability and the like, the volatile components of the caulis spatholobi traditional Chinese medicine compound can be accurately analyzed, and a new method is provided for comprehensively and objectively evaluating the quality of the volatile components of the caulis spatholobi traditional Chinese medicine compound.

Description

Technical Field

[0001] The present invention belongs to the field of quality standards and detection technology of traditional Chinese medicine prescriptions, and more specifically, relates to a method for establishing a fingerprint spectrum of volatile components of a Spatholobus suberectus traditional Chinese medicine compound and the fingerprint spectrum thereof. Background Art

[0002] The Spatholobus suberectus TCM compound is a clinically proven anti-tumor Chinese medicine formula optimized by combining established treatment principles and methods based on tumor etiology. Through preliminary evaluation of the entire compound, a compound consisting of Spatholobus suberectus, Paris polyphylla, Curcuma zedoaria, and Bupleurum chinense was selected. The volatile oils of Spatholobus suberectus, Paris polyphylla, Curcuma zedoaria, and Bupleurum chinense are among the primary active ingredients in these herbs. Although the volatile oil compositions of individual herbs in the Spatholobus suberectus TCM compound have been investigated, one study, for example, identified over 130 components in the volatile oil of Curcuma zedoaria, including terpenes (monoterpenes and sesquiterpenes), ketones (aldehydes), alcohols (phenols), alkanes (alkenes), acids (esters), ethers, and aromatic compounds, exhibiting antitumor, anti-inflammatory, antibacterial, antiviral, antithrombotic, hepatoprotective, neuroprotective, and antioxidant activities, is currently lacking. However, there is currently no unified quality standard for controlling this compound.

[0003] Therefore, establishing the fingerprint of the volatile components of the Millettia reticulata TCM compound is of great significance for the comprehensive and objective evaluation of the quality of the Millettia reticulata TCM compound. Summary of the Invention

[0004] The present invention aims to provide a method for establishing a fingerprint spectrum of volatile components of a Spatholobus suberectus Chinese medicine compound, and comprehensively and objectively evaluate the quality of the volatile components of the Spatholobus suberectus Chinese medicine compound by using the fingerprint spectrum.

[0005] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0006] The present invention provides a method for establishing a fingerprint spectrum of volatile components of a Spatholobus suberectus Chinese medicine compound, comprising the following steps:

[0007] S1. Take germacrone, curcumol, β-elemene, and curcuma-dione as reference substances and prepare reference substance solutions respectively using anhydrous ethanol as solvent;

[0008] S2. Take different batches of volatile oil from the Millettia repens Chinese medicine compound and dissolve it in anhydrous ethanol to obtain the test solution;

[0009] S3. Take the reference solution and each test solution in steps S1 and S2, respectively, and perform gas chromatography-mass spectrometry detection to record the chromatogram of each sample;

[0010] S4 using Chinese medicine chromatographic fingerprint similarity evaluation system to analyze the chromatograms of each sample obtained in step S3 to generate a fingerprint;

[0011] The volatile oil of the Spatholobus suberectus Chinese medicine compound is extracted from the following raw materials in parts by weight: 50-70 parts of Spatholobus suberectus, 40-50 parts of Paris polyphylla, 40-50 parts of Curcuma zedoaria, and 40-50 parts of Bupleurum chinense;

[0012] The conditions of gas chromatography-mass spectrometry in step S3 are as follows:

[0013] Gas chromatography conditions are:

[0014] The injection port temperature was 240-260°C; the column temperature was programmed; the carrier gas was helium with a split ratio of (18-22):1 and a carrier gas flow rate of 0.5-1.5 mL / min;

[0015] The mass spectrometry conditions are as follows: the ion source is an electron bombardment source, the ion source temperature is 260-300°C, the energy is 60-80 eV, the mass spectrometry transmission line temperature is 260-300°C, the solvent delay is 3-5 min, the mass spectrometry monitoring mode is the MSI scanning mode, the scanning range is 50-550 m / z, and the gain is set to 4-6.

[0016] Specifically, the above-mentioned reference substances, germacrone, curcumol, β-elemene and curcumadione, are active ingredients of the volatile oil of the Millettia repens traditional Chinese medicine compound.

[0017] As an optional embodiment, the stationary phase of the gas chromatography column is 5% phenyl-95% methyl polysiloxane.

[0018] As an optional embodiment, the gas chromatographic column is a HP-5MS capillary chromatographic column with a specification of 30m×0.25mm×0.25μm.

[0019] As an optional embodiment, in step S1, the concentration of the germacron reference solution is 0.3-0.4 mg / mL, the concentration of the curcumol reference solution is 2.5-3.5 mg / mL, the concentration of the β-elemene reference solution is 1-1.2 mg / mL, and the concentration of the curcuma-dione reference solution is 0.3-0.5 mg / mL.

[0020] As an optional embodiment, in step S1, the concentration of the germacron reference solution is 0.383 mg / mL, the concentration of the curcumol reference solution is 2.988 mg / mL, the concentration of the β-elemene reference solution is 1.125 mg / mL, and the concentration of the curcumadione reference solution is 0.424 mg / mL.

[0021] As an optional embodiment, the preparation method of the volatile oil of the Millettia reticulata Chinese medicine compound described in step S2 is: take 50-70 parts of Millettia reticulata, 40-50 parts of Paris polyphylla, 40-50 parts of Curcuma zedoaria, and 40-50 parts of Bupleurum chinense, add 3600-4400 parts of water, soak for 20-40 minutes, and extract by steam distillation for 3-5 hours to obtain the volatile oil of the Millettia reticulata Chinese medicine compound.

[0022] As an optional implementation scheme, the preparation method of the volatile oil of the Millettia reticulata Chinese medicine compound described in step S2 is: take 62 parts of Millettia reticulata, 46 parts of Paris polyphylla, 46 parts of Curcuma zedoaria, and 46 parts of Bupleurum chinense, add 4000 parts of water, soak for 30 minutes, and extract by steam distillation for 4 hours to obtain the volatile oil of the Millettia reticulata Chinese medicine compound.

[0023] Specifically, in the test solution, the concentration of volatile oil of the Millettia reticulata Chinese herbal compound is 0.4-0.6% (preferably 0.5%).

[0024] As an optional embodiment, the preparation method of the test solution is: take 1 μL of the volatile oil of the Millettia reticulata Chinese medicine compound, add 190-210 times (preferably 200 times) of anhydrous ethanol to dissolve it, and then filter it to obtain the test solution.

[0025] As an optional embodiment, the filtration is performed using a 0.2-0.3 μm (preferably 0.25 μm) microporous membrane.

[0026] As an optional embodiment, in the gas chromatography conditions, the programmed temperature program is as follows: initial temperature 45-55°C, heated to 100°C at a rate of 2-4°C / min, maintained for 4-6 min, heated to 120°C at a rate of 1-3°C / min, maintained for 4-6 min, heated to 122°C at a rate of 0.5-1.5°C / min, maintained for 9-11 min, heated to 125°C at a rate of 0.5-1.5°C / min, maintained for 2-4 min, heated to 127°C at a rate of 0.5-1.5°C / min, maintained for 7-9 min, heated to 130°C at a rate of 0.5-1.5°C / min, maintained for 5-7 min, heated to 133°C at a rate of 0.5-1.5°C / min, maintained for 4-6 min, heated to 160°C at a rate of 1-3°C / min, and then heated to 250°C at a rate of 2-4°C / min, maintained for 1-3 min.

[0027] As an optional embodiment, the gas chromatography conditions are: the injection port temperature is 250°C; the split ratio is 20:1, and the carrier gas flow rate is 1 mL / min;

[0028] As an optional embodiment, the mass spectrometry conditions are: ion source temperature of 280°C, energy of 70eV, mass spectrometry transmission line temperature of 280°C, solvent delay of 4min, scanning range of 50-550m / z, and gain setting of 5.

[0029] As an optional embodiment, the gas chromatography-mass spectrometry detection is performed using an Agilent 7890B-5977A gas chromatography-mass spectrometer.

[0030] Specifically, the fingerprint spectrum includes 24 common peaks, among which peak No. 7 corresponds to β-elemene, peak No. 16 corresponds to curcumol, peak No. 17 corresponds to germacrone, and peak No. 18 corresponds to curdione.

[0031] Specifically, the fingerprint includes 24 common peaks, and the retention time of each common peak is:

[0032] Peak 1: retention time 10.39-10.42 min;

[0033] Peak 2: retention time 13.27-13.29 min;

[0034] Peak 3: retention time 15.09-15.11 min;

[0035] Peak 4: retention time 15.63-15.64 min;

[0036] Peak 5: retention time 22.63-22.78 min;

[0037] Peak 6: retention time 26.26-26.30 min;

[0038] Peak 7: retention time 29.91-29.94 min;

[0039] Peak 8: retention time 31.52-31.53 min;

[0040] Peak 9: retention time 32.63-32.64 min;

[0041] Peak 10: retention time 33.84-33.86 min;

[0042] Peak 11: retention time 37.26-37.28 min;

[0043] Peak 12: retention time 42.97–42.99 min;

[0044] Peak 13: retention time 45.92-45.93 min;

[0045] Peak 14: retention time 46.73-46.75 min;

[0046] Peak 15: retention time 48.03-48.07 min;

[0047] Peak 16: retention time 49.78-50.11 min;

[0048] Peak 17: retention time 61.66-61.83 min;

[0049] Peak 18: retention time 65.61-65.77 min;

[0050] Peak 19: retention time 71.11-71.17 min;

[0051] Peak 20: retention time 72.22-72.42 min;

[0052] Peak 21: retention time 77.71-77.82 min;

[0053] Peak 22: retention time 79.22-79.28 min;

[0054] Peak 23: retention time 81.15-81.22 min;

[0055] Peak 24: retention time is 92.41~92.43min.

[0056] More specifically, the fingerprint spectrum includes 24 common peaks, and the retention time of each common peak is as follows: Peak 1: retention time is 10.404 min;

[0057] Peak 2: retention time 13.279 min;

[0058] Peak 3: retention time 15.101 min;

[0059] Peak 4: retention time 15.636 min;

[0060] Peak 5: retention time 22.702 min;

[0061] Peak 6: retention time 26.279 min;

[0062] Peak 7: retention time 29.925 min;

[0063] Peak 8: retention time 31.524 min;

[0064] Peak 9: retention time 32.633 min;

[0065] Peak 10: retention time 33.851 min;

[0066] Peak 11: retention time 37.271 min;

[0067] Peak 12: retention time 42.98 min;

[0068] Peak 13: retention time 45.925 min;

[0069] Peak 14: retention time 46.744 min;

[0070] Peak 15: retention time 48.052 min;

[0071] Peak 16: retention time 49.947 min;

[0072] Peak 17: retention time 61.744 min;

[0073] Peak 18: retention time 65.692 min;

[0074] Peak 19: retention time 71.14 min;

[0075] Peak 20: retention time 72.317 min;

[0076] Peak 21: retention time 77.768 min;

[0077] Peak 22: retention time 79.248 min;

[0078] Peak 23: retention time 81.183 min;

[0079] Peak 24: retention time is 92.421 min.

[0080] Specifically, the fingerprint spectrum includes 24 common peaks, and the peak area values ​​of the common peaks are as follows: Peak 1: peak area value is 1052527903~1297651629mPA*s;

[0081] Peak 2: peak area value is 313967730.9~366057677.1mPA*s;

[0082] Peak 3: peak area value is 806290225.7~874181274.3mPA*s;

[0083] Peak 4: peak area value is 624017034.1~723169999.9mPA*s;

[0084] Peak 5: peak area value is 780376497.2~833305258.8mPA*s;

[0085] Peak 6: peak area value is 116253908.7~154481789.3mPA*s;

[0086] Peak 7: peak area value is 902130045.4~1141894075mPA*s;

[0087] Peak 8: peak area value is 87003100.73~117277483.3mPA*s;

[0088] Peak 9: peak area value is 66793910.94~90757477.06mPA*s;

[0089] Peak 10: peak area value is 182283063.1~258599904.9mPA*s;

[0090] Peak 11: peak area value is 105211925.4~145651034.6mPA*s;

[0091] Peak 12: peak area value is 216937138.1~286689415.9mPA*s;

[0092] Peak 13: Peak area value is 112350105.2~148232302.8mPA*s.

[0093] Peak 14: Peak area value is 195768756.9~266113411.1mPA*s.

[0094] Peak 15: Peak area value is 412032827.2~474345202.8mPA*s.

[0095] Peak 16: Peak area value is 11153518518~14721712936mPA*s.

[0096] Peak 17: Peak area value is 2329056443~2869730261mPA*s.

[0097] Peak 18: Peak area value is 912778482~1055901518mPA*s.

[0098] Peak 19: Peak area value is 66704724.81~77381515.19mPA*s.

[0099] Peak 20: Peak area value is 157405088.7~218577447.3mPA*s.

[0100] Peak 21: Peak area value is 71966020.09~76616657.91mPA*s.

[0101] Peak 22: Peak area value is 47593487.3~66130496.7mPA*s.

[0102] Peak 23: Peak area value is 81430974.87~86105235.13mPA*s.

[0103] Peak 24: Peak area value is 100830223.2~134589396.8mPA*s.

[0104] More specifically, the fingerprint spectrum includes 24 common peaks, and the peak area values ​​of the common peaks are as follows: Peak 1: peak area value is 1175089766 mPA*s;

[0105] Peak 2: peak area value is 340012704mPA*s;

[0106] Peak 3: peak area value is 840235750 mPA*s;

[0107] Peak 4: peak area value is 673593517 mPA*s;

[0108] Peak 5: peak area value is 806840878 mPA*s;

[0109] Peak 6: peak area value is 135367849 mPA*s;

[0110] Peak 7: peak area value is 1022012060mPA*s;

[0111] Peak 8: peak area value is 102140292 mPA*s;

[0112] Peak 9: peak area value is 78775694 mPA*s;

[0113] Peak 10: peak area value is 220441484 mPA*s;

[0114] Peak 11: peak area value is 125431480 mPA*s;

[0115] Peak 12: peak area value is 251813277 mPA*s;

[0116] Peak 13: Peak area value is 130291204 mPA*s.

[0117] Peak 14: Peak area value is 230941084 mPA*s.

[0118] Peak 15: Peak area value is 443189015 mPA*s.

[0119] Peak 16: Peak area value is 12937615727 mPA*s.

[0120] Peak 17: Peak area value is 2599393352 mPA*s.

[0121] Peak 18: Peak area value is 984340000 mPA*s.

[0122] Peak 19: Peak area value is 72043120 mPA*s.

[0123] Peak 20: Peak area value is 187991268 mPA*s.

[0124] Peak 21: Peak area value is 74291339 mPA*s.

[0125] Peak 22: Peak area value is 56861992 mPA*s.

[0126] Peak 23: Peak area value is 83768105 mPA*s.

[0127] Peak 24: Peak area value is 117709810 mPA*s.

[0128] The present invention has the following beneficial effects:

[0129] 1. The present invention establishes for the first time a fingerprint method for detecting the volatile components of the Millettia reticulata Thunb using gas chromatography-mass spectrometry technology, and optimizes the gas chromatography and mass spectrometry conditions so that it can detect more comprehensive target peaks and better present the overall spectrum characteristics of the volatile components of the Millettia reticulata Thunb.

[0130] 2. The fingerprint of the volatile components of the Spatholobus suberectus Chinese medicine compound of the present invention can quickly and accurately judge the quality of the Chinese medicinal materials in the Spatholobus suberectus Chinese medicine compound, especially the Curcuma zedoaria medicinal materials, and is suitable for product quality control of the Spatholobus suberectus Chinese medicine compound.

[0131] 3. The fingerprint of the volatile components of the Caulis Spatholobi Thunb traditional Chinese medicine compound of the present invention can be used to perform quality detection on the Caulis Spatholobi Thunb traditional Chinese medicine compound. The detection method has the advantages of good stability, high precision, and good repeatability.

[0132] 4. The fingerprint of the Chinese medicinal compound of Millettia spatholobi provided by the present invention has 24 common peaks, and the characteristic components are preserved intact. BRIEF DESCRIPTION OF THE DRAWINGS

[0133] Figure 1 Figure 2 is the chromatogram of the reference substance (Figure a is the chromatogram of the β-elemene reference substance, Figure b is the chromatogram of the curcumol reference substance, Figure c is the chromatogram of the germacrone reference substance, and Figure d is the chromatogram of the curcumadione reference substance).

[0134] Figure 2 The chromatogram of the test product.

[0135] Figure 3 This is the common pattern diagram of the fingerprints of 10 batches of test products (i.e., the control fingerprint). DETAILED DESCRIPTION

[0136] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0137] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0138] The instruments used in the following examples are: Agilent 7890B-5977A gas chromatograph-mass spectrometer (Agilent 7890B gas chromatograph and 5977A mass spectrometer detector), HP-5MS capillary chromatographic column (30 m×0.25 mm×0.25 μm), FA2004B electronic balance, ME 104E / 02 electronic balance.

[0139] The reagents used in the following examples: anhydrous ethanol (analytical grade), Millettia reticulata (batch number 202212523-1~10), Bupleurum chinense (batch number 202404527-1~10), Curcuma zedoaria (batch number 202303591-1~10), Paris polyphylla (batch number 202303506-1~10) (Guangdong Yifang Pharmaceutical Co., Ltd.), curcumol reference substance (batch number 100185-201908) (China Food and Drug Inspection Institute), curcuma dione reference substance (batch number D18GB171806), germacronone reference substance (batch number D02D11S132892), β-elemene reference substance (batch number M20IB215514) were purchased from Shanghai Yuanye Biotechnology Co., Ltd., anhydrous ethanol (analytical grade).

[0140] Example 1 Method for Establishing the Fingerprint of Volatile Components of the Millettia Spatholobi Traditional Chinese Medicine Compound

[0141] 1. Method for establishing fingerprint of volatile components of Millettia repens TCM compound

[0142] 1) Preparation of reference solution:

[0143] Accurately weigh the reference substances of germacrone, curcumol, β-elemene, and curcumadione, and add anhydrous ethanol to prepare reference substance solutions of 0.383 mg / ml, 2.988 mg / ml, 1.125 mg / ml, and 0.424 mg / ml, respectively.

[0144] 2) Preparation of volatile component test solution of Millettia repens TCM compound

[0145] Weigh 10 batches of Spatholobus suberectus, Paris polyphylla, Curcuma zedoaria, and Bupleurum chinense (62g Spatholobus suberectus, 46g Paris polyphylla, 46g Curcuma zedoaria, and 46g Bupleurum chinense) in water (20 times the total weight of the ingredients) and soak for 30 minutes. Extract by steam distillation for 4 hours, and collect the volatile oil from the distiller. Accurately measure 1μL of the volatile oil, dissolve it in anhydrous ethanol, and dilute it 200-fold. Shake well, and filter through a 0.25μm microporous filter to obtain 10 batches of test solution.

[0146] 3) Determination

[0147] Accurately pipette the reference solution prepared according to step 1) and 10 batches of test solution prepared according to step 2) into a gas chromatograph-mass spectrometer, and record the chromatograms.

[0148] Gas chromatography conditions were as follows: chromatographic column: HP-5MS capillary column (30 m × 0.25 mm × 0.25 μm), inlet temperature 250 °C, temperature program as shown in Table 1 , carrier gas: high-purity helium (>99.999%), split ratio: 20:1, carrier gas flow rate: 1 ml / min;

[0149] The mass spectrometry conditions were as follows: electron impact source (EI) ion source, ion source temperature 280°C, energy 70 eV, mass spectrometry transmission line temperature 280°C, solvent delay 4 min, mass spectrometry monitoring mode MSI scanning mode, scanning range 50-550 m / z, gain 5.

[0150] Table 1 Heating program

[0151]

[0152]

[0153] 4) Fingerprint evaluation

[0154] The chromatographic results of the volatile components of the 10 batches of Millettia repens TCM compound obtained in step 3) were imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012 edition) for fingerprint similarity evaluation.

[0155] 2. Experimental Results

[0156] The chromatogram of the reference substance is as follows Figure 1 As shown, the chromatogram of the test sample is as follows Figure 2 As shown in Figure 2, the fingerprint patterns of the 10 batches of test products (control fingerprint) are as follows: Figure 3 As shown in the figure, the results showed that there were 24 common peaks with good separation effect and relatively large response values ​​determined in the fingerprint, which were marked as peaks 1-24. Four of the common peaks were selected as the characteristic peaks of the fingerprint of the volatile components of the Millettia reticulata Chinese medicine compound. Among the four common peaks, the reference substances β-elemene, curcumacol, germacrone and curcumadione were peaks 7, 16, 17 and 18, respectively, and their retention times were 29.925, 49.947, 61.744 and 65.692 minutes, respectively.

[0157] The common peak retention times of the volatile components of 10 batches of Millettia reticulata Chinese medicine compound are shown in Table 2, the common peak areas are shown in Table 3, and the similarity evaluation results are shown in Table 4. According to the fingerprint similarity evaluation results, the volatile components of different batches of Millettia reticulata Chinese medicine compound have certain differences, indicating that the fingerprint of the volatile components of the Millettia reticulata Chinese medicine compound provided by the present invention can be used to distinguish the volatile components of different batches of Millettia reticulata Chinese medicine compound, and can be used for quality control of the volatile components of the Millettia reticulata Chinese medicine compound.

[0158] Table 2 Common peak retention time of volatile components of the Millettia repens TCM compound (min)

[0159]

[0160]

[0161]

[0162] Table 3 Common peak areas (mPA*s) of volatile components of the Spatholobus suberectus TCM compound

[0163]

[0164]

[0165]

[0166] Table 4 Similarity of fingerprints of volatile components of different batches of Millettia repens TCM compound

[0167]

[0168] Example 2 Precision Experiment

[0169] 1. Solution preparation

[0170] Accurately weigh the reference substances of germacrone, curcumol, β-elemene and curcuma-21, and add anhydrous ethanol to prepare reference substance solutions of 0.383 mg / ml, 2.988 mg / ml, 1.125 mg / ml and 0.424 mg / ml, respectively, i.e., 0.383 mg / ml germacrone reference substance solution, 2.988 mg / ml curcuma-21 reference substance solution, 1.125 mg / ml β-elemene reference substance solution and 0.424 mg / ml curcuma-21 reference substance solution.

[0171] 2. Experimental Methods and Results

[0172] Different reference substance solutions prepared in this example were accurately drawn and continuously sampled 6 times according to the gas chromatography and mass spectrometry conditions of Example 1. The measurement results are shown in Tables 5 and 6. The results show that the RSDs of the reference substance peak retention times are all less than 0.1%, and the RSDs of the peak areas are all less than 2.0%, indicating that the fingerprint of the volatile components of the Spatholobus suberectus Chinese medicine compound established by the present invention has good precision.

[0173] Table 5 Retention time of each reference peak in precision experiment (min)

[0174]

[0175]

[0176] Table 6 Peak area of ​​each reference peak in the precision experiment (mPA*s)

[0177] Injection number β-elemene Curcumol Germacron Curcumadione 1 6503000000 1950000000 1310000000 1447500000 2 6460000000 1920000000 1340000000 1421000000 3 6589000000 1982000000 1282000000 1411000000 4 6481000000 1886000000 1326000000 1398360000 5 6612390000 1904000000 1318459000 1410000000 6 6425680000 1917000000 1328000000 1408658300 average value 6511845000 1926500000 1317409833 1416086383 RSD / % 1.13 1.78 1.52 1.20

[0178] Example 3 Stability Test

[0179] 1. Experimental Methods

[0180] The volatile component test solution of the Millettia repens TCM compound was prepared according to the method of Example 1. The same test solution was taken and sampled and measured at 0, 4, 8, 12, 24, and 48 hours according to the gas chromatography and mass spectrometry conditions of Example 1.

[0181] 2. Experimental Results

[0182] The determination results are shown in Tables 7 and 8. The results show that the RSDs of the retention times of the main common peaks are all less than 0.3%, and the RSDs of the peak areas are all less than 2.0%, indicating that the test solution is stable within 48 hours.

[0183] Table 7 Retention time of common peaks in stability experiment (min)

[0184]

[0185]

[0186] Table 8 Peak area of ​​each common peak in the stability experiment (mPA*s)

[0187]

[0188]

[0189] Example 4 Repeatability Experiment

[0190] 1. Experimental Methods

[0191] The same batch of Millettia repens, Paris polyphylla, Bupleurum chinense and Curcuma zedoaria were taken respectively, and 6 test solutions were prepared according to the method of Example 1, and the samples were injected and determined according to the gas chromatography and mass spectrometry conditions of Example 1.

[0192] 2. Experimental Results

[0193] The measurement results are shown in Tables 9 and 10. The results show that the RSDs of the retention times of the main common peaks are all less than 0.3%, and the RSDs of the peak areas are all less than 2.0%, indicating that the fingerprints of the volatile components of the Millettia repens Chinese medicine compound established by the present invention have good repeatability.

[0194] Table 9 Retention time of each common peak in the repetitive experiment (min)

[0195]

[0196]

[0197] Table 10 Peak area of ​​each common peak in the reproducibility experiment (mPA*s)

[0198]

[0199]

[0200] In summary, the experimental results show that the fingerprint spectrum of the volatile components of the Millettia spatholobi Chinese medicine compound provided by the present invention has good stability, high precision and good repeatability, and can comprehensively and objectively evaluate the quality of the volatile components of the Millettia spatholobi Chinese medicine compound, which is of great significance for ensuring clinical efficacy.

[0201] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A method for establishing a fingerprint of volatile components of a Spatholobus suberectus Chinese medicine compound, characterized in that: The following steps are involved: S1. Take germacrone, curcumol, β-elemene, and curcuma-dione as reference substances and prepare reference substance solutions respectively using anhydrous ethanol as solvent; S2. Take different batches of volatile oil from the Millettia repens Chinese medicine compound and dissolve it in anhydrous ethanol to obtain the test solution; S3. Take the reference solution and each test solution in steps S1 and S2, respectively, and perform gas chromatography-mass spectrometry detection to record the chromatogram of each sample; S4 using Chinese medicine chromatographic fingerprint similarity evaluation system to analyze the chromatograms of each sample obtained in step S3 to generate a fingerprint; The volatile oil of the Spatholobus suberectus Chinese medicine compound is extracted from the following raw materials in parts by weight: 50-70 parts of Spatholobus suberectus, 40-50 parts of Paris polyphylla, 40-50 parts of Curcuma zedoaria, and 40-50 parts of Bupleurum chinense; The conditions of gas chromatography-mass spectrometry in step S3 are as follows: Gas chromatography conditions are: The injection port temperature was 240-260°C; the column temperature was programmed; the carrier gas was helium with a split ratio of (18-22):1 and a carrier gas flow rate of 0.5-1.5 mL / min; The mass spectrometry conditions are as follows: the ion source is an electron bombardment source, the ion source temperature is 260-300°C, the energy is 60-80 eV, the mass spectrometry transmission line temperature is 260-300°C, the solvent delay is 3-5 min, the mass spectrometry monitoring mode is the MSI scanning mode, the scanning range is 50-550 m / z, and the gain is set to 4-6.

2. The method according to claim 1, wherein: The stationary phase of the gas chromatography column is 5% phenyl-95% methyl polysiloxane.

3. The method according to claim 1, wherein: In step S1, the concentration of the germacron reference solution is 0.3-0.4 mg / mL, the concentration of the curcumol reference solution is 2.5-3.5 mg / mL, the concentration of the β-elemene reference solution is 1-1.2 mg / mL, and the concentration of the curcumadione reference solution is 0.3-0.5 mg / mL.

4. The method according to claim 1, wherein: The preparation method of the volatile oil of the Millettia spatholobi Chinese medicine compound described in step S2 is as follows: take 50-70 parts of Millettia spatholobi, 40-50 parts of Paris polyphylla, 40-50 parts of Curcuma zedoaria, and 40-50 parts of Bupleurum chinense, add 3600-4400 parts of water, soak for 20-40 minutes, and extract by steam distillation for 3-5 hours to obtain the volatile oil of the Millettia spatholobi Chinese medicine compound.

5. The method according to claim 1, wherein: In the test solution, the concentration of volatile oil of the Millettia reticulata TCM compound is 0.4-0.6%.

6. The method according to claim 1, wherein: The programmed temperature increase procedure is as follows: initial temperature 45-55°C, heating to 100°C at a rate of 2-4°C / min, maintaining for 4-6 min, heating to 120°C at a rate of 1-3°C / min, maintaining for 4-6 min, heating to 122°C at a rate of 0.5-1.5°C / min, maintaining for 9-11 min, heating to 125°C at a rate of 0.5-1.5°C / min, maintaining for 2-4 min, heating to 127°C at a rate of 0.5-1.5°C / min, maintaining for 7-9 min, heating to 130°C at a rate of 0.5-1.5°C / min, maintaining for 5-7 min, heating to 133°C at a rate of 0.5-1.5°C / min, maintaining for 4-6 min, heating to 160°C at a rate of 1-3°C / min, and then heating to 250°C at a rate of 2-4°C / min, maintaining for 1-3 min.

7. The method according to claim 1, wherein: The gas chromatography conditions are as follows: the injection port temperature is 250° C.; the split ratio is 20:1, and the carrier gas flow rate is 1 mL / min.

8. The method according to claim 1, wherein: The mass spectrometry conditions are as follows: ion source temperature of 280° C., energy of 70 eV, mass spectrometry transmission line temperature of 280° C., solvent delay of 4 min, scanning range of 50-550 m / z, and gain setting of 5.

9. The method according to claim 1, wherein: The fingerprint spectrum includes 24 common peaks, among which peak No. 7 corresponds to β-elemene, peak No. 16 corresponds to curcumol, peak No. 17 corresponds to germacrone, and peak No. 18 corresponds to curdione.

10. The establishment method according to claim 1, characterized in that: The fingerprint spectrum includes 24 common peaks, and the retention time of each common peak is: Peak 1: retention time 10.39-10.42 min; Peak 2: retention time 13.27-13.29 min; Peak 3: retention time 15.09-15.11 min; Peak 4: retention time 15.63-15.64 min; Peak 5: retention time 22.63-22.78 min; Peak 6: retention time 26.26-26.30 min; Peak 7: retention time 29.91-29.94 min; Peak 8: retention time 31.52-31.53 min; Peak 9: retention time 32.63-32.64 min; Peak 10: retention time 33.84-33.86 min; Peak 11: retention time 37.26-37.28 min; Peak 12: retention time 42.97–42.99 min; Peak 13: retention time 45.92-45.93 min; Peak 14: retention time 46.73-46.75 min; Peak 15: retention time 48.03-48.07 min; Peak 16: retention time 49.78-50.11 min; Peak 17: retention time 61.66-61.83 min; Peak 18: retention time 65.61-65.77 min; Peak 19: retention time 71.11-71.17 min; Peak 20: retention time 72.22-72.42 min; Peak 21: retention time 77.71-77.82 min; Peak 22: retention time 79.22-79.28 min; Peak 23: retention time 81.15-81.22 min; Peak 24: retention time is 92.41~92.43min.

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