Establishment and application of GC-MS fingerprint of Juanbi decoction substances

Abstract

The invention discloses a method for establishing a GC-MS fingerprint spectrum of a Juanbi decoction substance benchmark. The method comprises the steps of: preparing a test solution; GC-MS determination and analysis; establishing a GC-MS fingerprint spectrum common peak database. The standard GC-MS fingerprint spectrum of the Juanbi decoction substance benchmark volatile oil established by the present invention can effectively characterize the composition and content of volatile substances in the Juanbi decoction substance benchmark, determine and identify the names of 35 common peak compounds, further improve the quality control method of the Juanbi decoction preparation and its intermediates, improve the quality evaluation system of the Juanbi decoction preparation and its intermediates, promote the standardization of traditional Chinese medicine, provide a theoretical and practical basis for comprehensive and effective control of the quality of the Juanbi decoction preparation and its intermediates, and provide an accurate and reliable basis for the safety and effectiveness of clinical medication.

Description

Technical Field

[0001] The invention relates to a quality control method for a traditional Chinese medicine preparation and an intermediate thereof, and in particular to an establishment method and application of a GC-MS fingerprint spectrum of a Juanbi decoction substance benchmark. Background Art

[0002] Qubi Decoction is a prescription included in the "Catalogue of Ancient Classic Prescriptions (First Batch)" issued by the State Administration of Traditional Chinese Medicine in 2018. It is composed of 11 herbs: Qianghuo, Duhuo, Angelica, Morus alba, Caulis Gentianae, Ligusticum chuanxiong, Frankincense, Aucklandia lappa, Cinnamon and Licorice. Qianghuo and Duhuo are the main herbs, while Angelica, Morus alba, Aucklandia lappa, Cinnamon, Aucklandia lappa, Ligusticum chuanxiong and Caulis Gentianae are the ministers, and frankincense and Licorice are used as envoys to harmonize the herbs. The whole prescription is warm but not dry, and is unblocked but not harmful. It is widely used in the clinical practice of traditional Chinese medicine to treat arthralgia caused by the combination of wind, cold and dampness, and has the effects of dispelling wind and dampness, dispersing cold, activating blood circulation and unblocking collaterals.

[0003] Traditional Chinese medicine fingerprint refers to a chromatogram or spectrum that can indicate the chemical characteristics of certain Chinese medicinal materials or Chinese medicine preparations after appropriate treatment using certain analytical methods. It can more comprehensively reflect the types and quantities of chemical components contained in medicinal materials, effectively reflect the integrity and comprehensive effects of Chinese medicine components, and has been widely used in Chinese medicine analysis and identification and quality control due to its rapid and accurate characteristics. There are also technicians in the prior art who have conducted research on the fingerprint construction method of Qubi Decoction. For example, the patent name is a method for constructing a fingerprint of a Qubi Decoction composition, a detection method and an application, and the application number is 202111461176.3. It provides a method for constructing a UPLC fingerprint of a Qubi Decoction composition, uses a liquid chromatograph for chromatographic analysis, and formulates a standard fingerprint of a Qubi Decoction composition, which can fully reflect the quality information of Qubi Decoction, thereby achieving the purpose of more comprehensive and effective control of the product quality of Qubi Decoction preparations. Another example is the invention named "A method for determining the HPLC fingerprint of a substance benchmark for Juanbi Decoction and its determination", with application number 202211204954.5. It provides a method for determining the HPLC fingerprint of a substance benchmark for Juanbi Decoction, and establishes the fingerprint of Juanbi Decoction. This method has strong specificity, good reproducibility and stability, and can better control the intrinsic quality of the product.

[0004] However, the above methods all use HPLC fingerprints for detection and analysis, and the seven herbs in the Qubi Decoction, namely, Qianghuo, Duhuo, Angelica, Chuanxiong, Frankincense, Aucklandia, and Cinnamon, all contain volatile oil components, and all have certain pharmacological activities. The volatile oils of the Qubi Decoction material base mainly contain terpenes, phthalides, and organic acid compounds. Modern pharmacology shows that the volatile oils of Angelica, Qianghuo, Chuanxiong, Cinnamon, and Frankincense have anti-inflammatory, analgesic, and antibacterial effects. In addition, cinnamaldehyde in cinnamon has anti-tumor effects; Frankincense has anti-ulcer, anti-asthma, and antioxidant pharmacological activities. At present, there is no research on the fingerprint of volatile oils in the Qubi Decoction preparation. Therefore, it is of great significance to conduct qualitative and quantitative analysis of the volatile oils of the Qubi Decoction material base and establish a volatile oil fingerprint for its component analysis and identification and quality control.

[0005] The disclosure of the above background technology content is only used to assist in understanding the inventive concept and technical solution of the present invention. It does not necessarily belong to the prior art of this patent application. In the absence of clear evidence that the above content has been disclosed on the filing date of this patent application, the above background technology should not be used to evaluate the novelty and creativity of the present application. Summary of the invention

[0006] In order to further study and analyze the components and contents in the Juanbi Decoction substance benchmark, improve the quality standards and quality control methods of the Juanbi Decoction preparation and its intermediates, promote the standardization of traditional Chinese medicine, and provide an accurate and reliable basis for the safety and effectiveness of clinical medication, the present invention provides a method for establishing a GC-MS fingerprint spectrum of the Juanbi Decoction substance benchmark, and obtains the Juanbi Decoction substance benchmark GC-MS fingerprint spectrum by this method.

[0007] The technical solution adopted by the present invention is as follows:

[0008] A method for establishing a GC-MS fingerprint spectrum of a substance benchmark of Juanbi decoction, the method comprising the following steps:

[0009] (1) Preparation of test solution: Take the whole prescription of Juanbi Decoction, add water, decoct for 0.5-1h, filter, and set aside the filtrate to prepare the standard water decoction of Juanbi Decoction; take the standard water decoction of Juanbi Decoction base substance, extract the volatile oil, add 1mL of n-hexane from the upper end of the condenser, heat and extract for 5h, collect the cyclohexane solution, put it in a 10mL volumetric flask, make up to volume with ethyl acetate, shake well, and obtain the test solution;

[0010] (2) GC-MS analysis: Accurately pipette 1 μL of the test solution and inject it into the GC-MS chromatograph to obtain the GC-MS fingerprint of the Juanbi Decoction substance standard;

[0011] GC chromatographic conditions: Agilent HP-5MS flexible quartz capillary column (0.25 mm × 30 m, 0.25 μm), injection port temperature 250 °C; split ratio 300:1, carrier gas: helium; flow rate: 1.0 mL min -1 ; Temperature program: initial temperature 120℃, hold for 9min; at 10℃·min -1 The temperature was raised to 131℃ at a rate of 8℃·min and maintained for 22min. -1 The temperature was raised to 140℃ at a rate of 18℃·min -1 The rate was raised to 157°C and maintained for 32 min; then the temperature was increased at 15°C·min -1 Raise to 165°C, hold for 23 min, and finally raise the temperature to 280°C, hold for 5 min;

[0012] MS mass spectrometry conditions: ionization mode: electron impact ion source (EI source); ion source temperature: 230°C; quadrupole temperature: 150°C; electron energy: 70 eV; solvent delay time: 3 min; scanning range: 50-650 m / z; scanning number: 1.3 times per second;

[0013] (4) Establishment of a GC-MS fingerprint common peak database: The GC-MS fingerprint obtained in step (2) is compared with the mass spectrometry retrieval standard library NIST 11, and matching data with a matching degree ≥ 90% are selected to obtain common peaks, and a reference spectrum is generated to establish a standard GC-MS fingerprint of the benchmark volatile oil of Juanbi Decoction.

[0014] In the above-mentioned method for establishing the material benchmark GC-MS fingerprint of Qunbi Decoction, in the "preparation of test solution" step, the amount of water added is preferably 6-14 times the amount of the whole recipe of Qunbi Decoction medicinal materials; the amount of water added is further preferably 8-10 times the amount of the whole recipe of Qunbi Decoction medicinal materials; the most preferred amount of water added is 8 times the amount of the whole recipe of Qunbi Decoction medicinal materials.

[0015] The method for establishing the material benchmark GC-MS fingerprint of the above-mentioned Juanbi Decoction comprises 35 common peaks in the material benchmark GC-MS fingerprint of the Juanbi Decoction: peak 1 with a retention time of 5.78 min is cinnamaldehyde; peak 2 with a retention time of 6.93 min is 4-Acetoxy-3-methoxystyrene; peak 3 with a retention time of 8.29 min is 1,4-cyclohexadiene-1,2-dicarboxylic anhydride; peak 4 with a retention time of 18.38 min is lauric acid; peak 5 with a retention time of 19.14 min is 4-hydroxy-β-dihydrodamascone; peak 6 with a retention time of 25.72 min is fosseene; peak 7 with a retention time of 26.91 min is (2R,4 R)-p-Mentha-[1(7),8]-diene,2-hydroperoxide; Peak 8 retention time 27.85min, 3-n-butenylphthalide; Peak 9 retention time 29.96min, 2,4,5,5,8a-Pentamethyl-4a,5,6,7,8,8a-hexahydro-2H-chromene; Peak 10 retention time 33.03min, 1(3H)-Isobenzofuranone; Peak 11 retention time 33.56min, yangchuanxiong lactone; Peak 12 retention time 34.08min, (3S,3aS)-3-Butyl- 3a,4,5,6-tetrahydroisobenzofuran-1(3H)-one; peak 13, retention time 35.03min, ligustilide; peak 14, retention time 38.35min, 1-benzoyl-2-phenylhydrazine; peak 15, retention time 41.67min, (E)-Ligustilide; peak 16, retention time 54.60min, 4,8,13-Cyclotetradecatriene-1,3-diol,1,5,9-trimethyl-12-(1-methylethyl)-; peak 17, retention time 56.76min, 4H-1,3- Benzodioxin-4-one,5-butyl-2-(1,1-dimethylethyl)hexahydro-4a-methyl-,[2s-(2π4aπ5π8aπ]-; Peak 18 retention time 57.95min, 1,5,9-cyclotetratriene, 1,5,9-trimethyl-12-(1-methylvinyl)-,(1E,5E,9E,12R)-; Peak 19 retention time 60.99min, dehydrocostus lactone; Peak 20 retention time 61.93min, .alpha.-Isonootkatol; Peak 21 retention time 63.15min, bicyclo[9.3.1] pentadeca-4,14-diene, 4,14,15,15-tetramethyl-8-methylene-, (4E, 11S); Peak 22 retention time 64.24min, (1E, 3E, 7E, 11E)-1,7,11-trimethyl-4-isopropyl-1,3,7,11-cyclotetradecatetraene; Peak 23 retention time 68.51min, Phosphonic acid; peak 24, retention time 69.54min, is β-ionone; peak 25, retention time 70.11min, is ISOCEMBROL; peak 26, retention time 71.79min, is Azulene, 1,2,3,5,6,7,8,8a-octahydro-1,4-dimethyl-7-(1-methylethenyl)-, [1S-(1π7π8aπ]-; peak 27, retention time 76.00min, is anthracene; peak 28, retention time 77.46min, is geranyl linalool; peak 29, retention time 79.67min, is alpha-bulnesene; peak 30, retention time 82.45min, is (9β)-9,19-Cyclolanostan-3β-ol Peak 31, retention time 84.39min, is (3E, 7E, 11E)-1-isopropyl-4,8,12-trimethylcyclotetradecane-3,7,11-trienylglycerol; Peak 32, retention time 87.13min, is Formic acid, 3,7,11-trimethyl-1,6,10-dodecatrien-3-yl ester; peak 33, retention time 87.74min, 3,20-allopregnanedione; peak 34, retention time 90.69min, Azulene, 1,2,3,4,5,6,7,8-octahydro-1,4-dimethyl-7-(1-methylethenyl)-, [1S-(1π4π7π]-; peak 35, retention time 92.19min, 2-nonen-1-ol 2-methyl. Peak 1 is cinnamaldehyde, a characteristic component of cinnamon, peak 4 is lauric acid, a characteristic component of frankincense, peak 13 is ligustilide, a characteristic component of angelica and ligusticum chuanxiong, and peak 19 is dehydrocostus lactone, a characteristic component of costus root.

[0016] The above-mentioned method for establishing the standard GC-MS fingerprint of the Juanbi Decoction substance is applied to perform cluster analysis on the common peak database of the standard GC-MS fingerprint of the Juanbi Decoction substance to judge the authenticity of the Juanbi Decoction preparation and its intermediates, or to control the quality of the Juanbi Decoction preparation and its intermediates.

[0017] The beneficial effects of the present invention are:

[0018] 1. The present invention establishes a method for establishing a GC-MS fingerprint of the material benchmark of Juanbi Decoction, by which the GC-MS fingerprint of the material benchmark of Juanbi Decoction can be obtained, which can effectively characterize the composition and content of volatile substances in the material benchmark of Juanbi Decoction, further improve the quality control method of Juanbi Decoction preparations and their intermediates, improve the quality evaluation system of Juanbi Decoction preparations and their intermediates, promote the standardization of traditional Chinese medicine, provide a theoretical and practical basis for comprehensive and effective control of the quality of Juanbi Decoction preparations and their intermediates, and provide an accurate and reliable basis for the safety and effectiveness of clinical medication. The method of the present invention has the advantages of being fast, accurate, high in precision, good in repeatability and stability, rich in chromatographic peak information and good in separation effect.

[0019] 2. Juanbi Decoction is a classic prescription. According to the research specification, the development of the preparation of the classic prescription can be exempted from the evaluation of drug efficacy and clinical practice. In order to smoothly develop it into a modern Chinese medicine preparation that is easy to take, the preparation process of its industrial production needs to be as close as possible to the method recorded in the original prescription, so as to avoid the possibility of causing safety and efficacy inconsistent with the original prescription. Therefore, the present invention adopts the Juanbi Decoction material reference and extracts volatile oil to prepare the test solution. The GC-MS fingerprint of the Juanbi Decoction material reference obtained is suitable for the authenticity and quality identification of the medicinal materials of the Juanbi Decoction prescription, and the quality control of the modern Chinese medicine preparations such as mixtures, oral liquids, pills, granules, capsules, tablets, etc. made by water extraction close to the original prescription of the Juanbi Decoction and their intermediates.

[0020] 3. The GC-MS fingerprint of the material benchmark of Juanbi Decoction established in the present invention determined and identified the names of the compounds with 35 common peaks, and the relative content of each component was determined by the area normalization method. Among them, lauric acid, cinnamaldehyde, ligustilide, and dehydrocostuslactone are the characteristic components of frankincense, cinnamon, angelica, Chuanxiong, and costusroot, respectively. The medicinal ingredients and sources of the volatile substances in the Juanbi Decoction preparation and its intermediates can be further explored. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 GC fingerprints of 15 batches of volatile oils (S1-S15) of Juanbi Decoction and the superposition of the reference fingerprint (R)

[0022] Figure 2 GC-MS fingerprint of the water-extracted intermediate of Juanbi Decoction Granules DETAILED DESCRIPTION

[0023] The present invention is further described below in conjunction with specific embodiments, but the protection scope and application scope of the present invention are not limited thereto.

[0024] 1. Establishment of the GC-MS fingerprint of the Juanbi Decoction substance standard

[0025] The present invention establishes a fingerprint of the volatile oil of Juanbi Decoction by GC-MS, evaluates the similarity of the fingerprints of 15 batches, and uses GC-MS to identify the components of the volatile oil, infers the source of each component, in order to provide a reference for its quality control and evaluation. The test results are as follows:

[0026] 1. Instruments and Reagents

[0027] 1.1 Instrument

[0028] 7890B gas chromatograph, 5977B-MSD gas mass spectrometer, Agilent HP-5 capillary column (30m×0.32mm, 0.25μm); ME204 / E electronic analytical balance, 98-1-B electronic temperature control heating mantle, etc.

[0029] 1.2 Reagents

[0030] The sources of Chinese medicinal materials for Juanbi Decoction are shown in Table 1. They have been identified to be in compliance with the requirements for each medicine under the 2020 edition of the "Chinese Pharmacopoeia"; methanol, n-hexane (chromatographic grade).

[0031] Table 1 Sources of medicinal materials for Juanbi Decoction

[0032]

[0033] 1.3 Preparation of the material standard of Juanbi Decoction

[0034] The medicinal materials in Table 1 were mixed and homogenized according to different years, different origins, and different batches to obtain 15 batches of Qubi Decoction material reference medicinal materials, as shown in Table 2. According to the decoction method in ancient books and the preparation process obtained in previous studies, the whole prescription medicinal materials were weighed according to the prescription ratio: 10.08g of Qianghuo, 10.08g of Duhuo, 30.0g of Angelica, 7.08g of Chuanxiong, 10.08g of Gentiana, 30.0g of Morus, 20.16g of Kadzu, 5.05g of Cinnamon, 8.0g of Aucklandia, 8.09g of Frankincense, and 5.05g of Licorice, that is, 3 times the amount of medicinal materials in the prescription, 8 times the amount of water was added, decocted for 0.5h, filtered, and the filtrate was the Qubi Decoction material reference water decoction.

[0035] The experiment also compared the addition of 5, 6, 8, 10, 12, 14, and 16 times of water, and the decoction time of 0.5, 1, 1.5, and 2 hours. Among them, adding 6-14 times the amount of water of the whole recipe of Qubi Decoction is feasible; adding 8-10 times the amount of water of the whole recipe of Qubi Decoction is better; adding 8 times the amount of water of the whole recipe of Qubi Decoction is the best. The decoction time of 0.5-1.5 hours is feasible, the decoction time of 0.5-1 hour is better, and the decoction time of 0.5 hour is the best. The peak shape and separation effect of each peak of the GC-MS fingerprint spectrum obtained by "adding 8 times the amount of water and decocting for 0.5h" are the best.

[0036] Table 215: Information on the combination of base medicinal materials for batch Qunbi Decoction

[0037]

[0038] 1.4 Preparation of Juanbi Decoction Test Solution

[0039] Take the standard decoction of Juanbi Decoction prepared under "1.3", extract the volatile oil, add 1 mL of n-hexane from the upper end of the condenser, heat and extract for 5 h, collect the cyclohexane solution, put it in a 10 mL volumetric flask, make up to volume with ethyl acetate, shake well, and the test solution is obtained; prepare 15 batches of test solution in the same way, with batch numbers S1 to S15.

[0040] 2. Methods and Results

[0041] 2.1 Gas chromatography conditions

[0042] Agilent HP-5MS flexible quartz capillary column (0.25 mm × 30 m, 0.25 μm), injection port temperature 250 °C; injection volume 1 μL; split ratio 300:1, carrier gas: helium; flow rate: 1.0 mL min -1 ; Temperature program: initial temperature 120℃, hold for 9min; at 10℃·min -1 The temperature was raised to 131℃ at a rate of 8℃·min and maintained for 22min. -1 The temperature was raised to 140℃ at a rate of 18℃·min -1 The rate was raised to 157°C and maintained for 32 min; then the temperature was increased at 15°C·min -1 Raise to 165℃ and maintain for 23min, and finally the temperature rises to 280℃ and maintain for 5min.

[0043] In order to obtain a fingerprint spectrum with better peak shape and more peaks during the experiment, the inventors conducted comparative tests. (1) The following split ratios were compared: 500:1, 400:1, 300:1, 100:1. Among them, the spectrum of the split ratio of 300:1 was the best, so the split ratio of 300:1 was selected for further investigation. (2) The following injection port temperatures were compared: 240℃, 250℃, 260℃, 270℃. Among the injection port temperatures, the injection port temperature of 250℃ had the best separation effect on the sample, so the injection port temperature of 250℃ was selected. (3) The heating conditions of more programs were compared, such as: ① Initial temperature 85℃, maintained for 3min; ① 5℃·min -1 ② The initial temperature was 110℃ and maintained for 3min; the temperature was raised to 200℃ at a rate of 5℃·min -1 The temperature was raised to 145℃ at a rate of 5℃·min and maintained for 10min. -1③ The initial temperature was 95℃, and it was kept for 1min; the temperature was raised to 175℃ at a rate of 8℃·min -1 The temperature was raised to 132℃ at a rate of 6℃·min and maintained for 21.5min. -1 The temperature was raised to 140℃ at a rate of 17℃·min -1 The rate was raised to 155℃ and maintained for 53min. ④ Initial temperature was 120℃ and maintained for 9min; -1 The temperature was raised to 131℃ at a rate of 8℃·min and maintained for 22min. -1 The temperature was raised to 140℃ at a rate of 18℃·min -1 The rate was raised to 157°C and maintained for 32 min; then the temperature was increased at 15°C min -1 The temperature was raised to 165℃ and maintained for 23min, and finally the temperature was raised to 280℃ and maintained for 5min. Method ④ has the best separation effect, so this method is selected.

[0044] 2.2 Mass spectrometry conditions

[0045] Ionization mode: electron bombardment ion source (EI source); ion source temperature: 230°C; quadrupole temperature: 150°C; electron energy: 70 eV; solvent delay time: 3 min; scanning range: 50-650 m / z; scanning number: 1.3 times per second.

[0046] 2.3 Methodological investigation

[0047] 2.3.1. Instrument precision test

[0048] Take the test solution under "1.4" and inject it continuously for 6 times according to the chromatographic conditions under "2.1". Take the chromatographic peak No. 13 (ligustilide) as the reference peak (S). The relative retention time RSD of each common peak is calculated to be <0.3%, and the relative peak area RSD is <3%. According to the evaluation of "Similarity Evaluation System of Chromatographic Fingerprint of Traditional Chinese Medicine (2012 Edition)", the similarity of 6 injections is 1.00, indicating that the precision of the instrument is good. See Table 3 and Table 4 for details.

[0049] Table 3 Relative retention time and RSD value of common peaks of the standard volatile oil samples of Juanbi Decoction in the precision test

[0050]

[0051] Table 4 Relative peak areas and RSD values ​​of the common peaks of the volatile oil samples of the Juanbi Decoction substance reference in the precision test

[0052]

[0053] 2.3.2 Stability test

[0054] Take the test solution under "1.4" and analyze it according to the chromatographic conditions under "2.1" at 0, 2, 4, 8, 10, 12, and 24 hours respectively. Take the chromatographic peak No. 13 (ligustilide) as the reference peak (S). The relative retention time RSD of each common peak is calculated to be <0.4%, and the relative peak area RSD is <3%. According to the evaluation of "Similarity Evaluation System of Chromatographic Fingerprint of Traditional Chinese Medicine (2012 Edition)", the similarity of 6 injections is 1.00, indicating that the components in the test solution have good stability within 24 hours. See Table 5 and Table 6 for details.

[0055] Table 5 Relative retention time and RSD value of each common peak of the standard volatile oil samples of Juanbi Decoction in the stability test

[0056]

[0057] Table 6 Relative peak areas and RSD values ​​of the common peaks of the volatile oil samples of the Juanbi Decoction substance reference in the stability test

[0058]

[0059] 2.3.3 Repeatability test

[0060] Six test sample solutions were prepared according to the method under "1.4" and analyzed according to the chromatographic conditions under "2.1". The chromatographic peak No. 13 (ligustilide) was used as the reference peak (S). The relative retention time RSD of each common peak was calculated to be <0.3%, and the relative peak area RSD was <3%. According to the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine (2012 Edition)", the similarity of the six injections was 1.00, indicating that the method has good repeatability. See Tables 7 and 8 for details.

[0061] Table 7 Relative retention time and RSD value of each common peak of the standard volatile oil samples of Juanbi Decoction in the repeatability test

[0062]

[0063] Table 8 Relative peak areas and RSD values ​​of the common peaks of the volatile oil samples of the Juanbi Decoction substance standard in the repeatability test

[0064]

[0065] 2.4 Establishment and evaluation of the fingerprint of the benchmark volatile oil of Juanbi Decoction

[0066] The GC-MS chromatograms of the 15 batches of Juanbi Decoction substance benchmarks collected were imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)", and the GC-MS overlays of the volatile oils of the 15 batches of Juanbi Decoction substance benchmarks were obtained. The reference spectrum S8, time window 0.1min, median method, and multi-point correction were set to match 35 common peaks and generate a reference spectrum, and the GC-MS fingerprints of the volatile oils of the 15 batches of Juanbi Decoction substance benchmarks and the reference fingerprint overlays were obtained, see Figure 1 ; The similarity was calculated based on the generated reference fingerprint. The similarities between the GC-MS fingerprints of 15 batches of Juanbi Decoction reference volatile oils and the reference fingerprints were 0.936, 0.995, 0.995, 0.982, 0.936, 0.968, 0.991, 0.971, 0.977, 0.997, 0.926, 0.993, 0.978, 0.942 and 0.939, respectively. The similarities were all >0.926, indicating that the quality of each batch of Juanbi Decoction volatile oil was relatively stable, and the overall characteristics of the chemical components were slightly different, which met the analysis requirements of the fingerprint and reflected the integrity and homogeneity of the samples.

[0067] 2.5 GC-MS component analysis of volatile oil

[0068] According to the above mass spectrometry conditions, the sample was injected and compared with the mass spectrometry retrieval standard library NIST 11. The matching data with a matching degree ≥ 90% were selected. When there were multiple options with high matching degrees for the same component, the highest matching value was selected. The 35 common peaks were identified through the corresponding reference materials, and their compound names were determined. The relative content of each component was determined by the area normalization method. The results are shown in Table 9.

[0069] Table 9 Identification results of 35 common peaks of volatile oils from 15 batches of Juanbi decoction

[0070]

[0071]

[0072] In the above-mentioned GC-MS fingerprint of the material benchmark of Juanbi Decoction, peak 1 is cinnamaldehyde, a characteristic component of cinnamon, peak 4 is lauric acid, a characteristic component of frankincense, peak 13 is ligustilide, a characteristic component of angelica and Chuanxiong, and peak 19 is dehydrocostus lactone, a characteristic component of costus root. Among them, ligustilide has the highest percentage. According to relevant literature reports, as a phthalide, ligustilide has good anti-inflammatory and anti-tumor pharmacological effects, which can hinder the transmission of inflammatory signals through (COX-2, ERK2), PKC, JAK1, JAK2, JAK3, NF-кB, β (IKKβ), TNF-α, affect the expression of downstream proteins, inhibit the production of NO and PGE2, and selectively block the Prx / TLR signaling pathway to exert anti-inflammatory effects; and exert anti-tumor effects by selectively blocking the Prx / TLR signaling pathway. The GC-MS fingerprint of the volatile oil of the Juebi Decoction established in the present invention has a simple method and reliable results, which provides a reference for the study of volatile substances in classic prescriptions, and also provides a theoretical basis for the standard quality control of the substances of the Juebi Decoction, and provides a reference for further exploring the effective components and sources of the volatile oil of the Juebi Decoction.

[0073] 2. Application

[0074] In order to facilitate patients to carry and use, the inventor developed the Qunbi Decoction into a modern Chinese medicine granule. The optimal water extraction process of the Qunbi Decoction granules has been optimized through orthogonal experiments in the early stage. Now it is planned to apply the Qunbi Decoction substance benchmark GC-MS fingerprint established by the present invention to the quality control of the water extraction intermediates of the Qunbi Decoction granules.

[0075] 1. Preparation of the water-extracted intermediate of Juanbi Decoction Granules

[0076] Take the medicinal materials of the whole prescription of Qunbi Decoction: 10.08g of Qianghuo, 10.08g of Duhuo, 30.0g of Angelica, 7.08g of Chuanxiong, 10.08g of Qinjiao, 30.0g of Morus alba, 20.16g of Piper mesenteriae, 5.05g of Cinnamon bark, 8.0g of Aucklandia lappa, 8.09g of Frankincense, and 5.05g of Licorice, which is 3 times the amount of the whole prescription, add 8 times the amount of water, decoct 3 times, each time for 1 hour, filter, and combine the filtrate to obtain the water-extracted intermediate of Qunbi Decoction granules.

[0077] 2. Preparation of test solution

[0078] Take the water-extracted intermediate of the Juanbi Decoction granules prepared above, extract the volatile oil, add 1 mL of n-hexane from the upper end of the condenser, heat and extract for 5 h, collect the cyclohexane liquid, put it in a 10 mL volumetric flask, make up to volume with ethyl acetate, shake well, and the test solution is obtained.

[0079] 3. GC-MS analysis

[0080] Accurately pipette 1 μL of the test solution and inject it into the GC-MS chromatograph.

[0081] GC chromatographic conditions: Agilent HP-5MS flexible quartz capillary column (0.25 mm × 30 m, 0.25 μm), injection port temperature 250 °C; split ratio 300:1, carrier gas: helium; flow rate: 1.0 mL min -1 ; Temperature program: initial temperature 120℃, hold for 9min; at 10℃·min -1 The temperature was raised to 131℃ at a rate of 8℃·min and maintained for 22min. -1 The temperature was raised to 140℃ at a rate of 18℃·min -1 The rate was raised to 157°C and maintained for 32 min; then the temperature was increased at 15°C min -1 Raise to 165℃ and maintain for 23min, and finally the temperature rises to 280℃ and maintain for 5min.

[0082] MS mass spectrometry conditions: ionization mode: electron impact ion source (EI source); ion source temperature: 230°C; quadrupole temperature: 150°C; electron energy: 70 eV; solvent delay time: 3 min; scanning range: 50-650 m / z; scanning number: 1.3 times per second.

[0083] 4. Results Analysis

[0084] The GC-MS fingerprint of the water-extracted intermediate of the Juanbi Decoction granules obtained is shown in Figure 2 , the figure is consistent with the GC-MS fingerprint of the Qubi Decoction material benchmark established by the present invention, and there are also 35 common peaks. Therefore, the preferred water extraction process of the Qubi Decoction granules intermediates, the volatile substances are similar to the volatile substances of the Qubi Decoction material benchmark, indicating that the GC-MS fingerprint of the Qubi Decoction material benchmark established by the present invention can be used to judge the authenticity of the Qubi Decoction preparation and its intermediates, or control the quality of the Qubi Decoction preparation and its intermediates; especially the quality control of modern Chinese medicine preparations such as mixtures, oral liquids, pills, granules, capsules, tablets, etc. made by water extraction close to the original Qubi Decoction recipe and their intermediates. The present invention improves the quality evaluation system of the Qubi Decoction preparation and its intermediates, promotes the standardization of traditional Chinese medicine, provides a theoretical and practical basis for comprehensive and effective control of the quality of the Qubi Decoction preparation and its intermediates, and provides an accurate and reliable basis for the safety and effectiveness of clinical medication.

Claims

1. A method for establishing a GC-MS fingerprint spectrum of a substance benchmark of Juanbi Decoction, characterized in that: The following steps are involved: (1) Preparation of test solution: Take the whole prescription of Juanbi Decoction, add water, decoct for 0.5-1h, filter, and set aside the filtrate to prepare the standard water decoction of Juanbi Decoction; take the standard water decoction of Juanbi Decoction base substance, extract the volatile oil, add 1mL of n-hexane from the upper end of the condenser, heat and extract for 5h, collect the cyclohexane solution, put it in a 10mL volumetric flask, make up to volume with ethyl acetate, shake well, and obtain the test solution; (2) GC-MS analysis: Accurately pipette 1 μL of the test solution and inject it into the GC-MS chromatograph to obtain the GC-MS fingerprint of the Juanbi Decoction substance standard; GC chromatographic conditions: Agilent HP-5MS flexible quartz capillary column 0.25 mm × 30 m, 0.25 μm, injection port temperature 250 °C; split ratio 300:1, carrier gas: helium; flow rate: 1.0 mL min -1 ; Temperature program: initial temperature 120℃, hold for 9min; at 10℃·min -1 The temperature was raised to 131℃ at a rate of 8℃·min -1 The temperature was raised to 140℃ at a rate of 18℃·min -1 The rate was raised to 157°C and maintained for 32 min; then the temperature was increased at 15°C·min -1 Raise to 165°C, hold for 23 min, and finally raise the temperature to 280°C, hold for 5 min; MS mass spectrometry conditions: ionization mode: electron impact ion source (EI source); ion source temperature: 230°C; quadrupole temperature: 150°C; electron energy: 70 eV; solvent delay time: 3 min; scanning range: 50-650 m / z; scanning number: 1.3 times per second; (3) Establishment of a GC-MS fingerprint common peak database: The GC-MS fingerprint obtained in step (2) is compared with the mass spectrometry retrieval standard library NIST 11, and matching data with a matching degree ≥ 90% are selected to obtain common peaks, and a reference spectrum is generated to establish a standard GC-MS fingerprint of the benchmark volatile oil of Juanbi Decoction.

2. The method for establishing the GC-MS fingerprint spectrum of the Juanbi Decoction substance benchmark according to claim 1 is characterized in that: In the step of preparing the test solution, the amount of water added is 6-14 times the amount of the medicinal materials in the whole prescription of Qunbi Decoction.

3. The method for establishing the GC-MS fingerprint spectrum of the Juanbi Decoction substance standard according to claim 1 or 2, characterized in that: There are 35 common peaks in the GC-MS fingerprint of the Juanbi Decoction substance benchmark: The retention time of peak 1 is 5.78min, which is cinnamaldehyde; the retention time of peak 2 is 6.93min, which is 4-Acetoxy-3-methoxystyrene; the retention time of peak 3 is 8.29min, which is 1,4-cyclohexadiene-1,2-dicarboxylic anhydride; the retention time of peak 4 is 18.38min, which is lauric acid; the retention time of peak 5 is 19.14min, which is 4-hydroxy-β-dihydrodamascone; the retention time of peak 6 is 25.72min, which is fosseene; the retention time of peak 7 is 26.91min in, which is (2R,4R)-p-Mentha-[1(7),8]-diene,2-hydroperoxide; the retention time of peak 8 is 27.85min, which is 3-n-butenylphthalide; the retention time of peak 9 is 29.96min, which is 2,4,5,5,8a-Pentamethyl-4a,5,6,7,8,8a-hexahydro-2H-chromene; the retention time of peak 10 is 33.03min, which is 1(3H)-Isob enzofuranone; peak 11 retention time 33.56min, is ligusticum lactone; peak 12 retention time 34.08min, is (3S,3aS)-3-Butyl-3a,4,5,6-tetrahydroisobenzofuran-1(3H)-one; peak 13 retention time 35.03min, is ligusticum lactone; peak 14 retention time 38.35min, is 1-benzoyl-2-phenylhydrazine; peak 15 retention time 41.67min n, (E)-Ligustilide; Peak 16, retention time 54.60min, 4,8,13-Cyclotetradecatriene-1,3-diol,1,5,9-trimethyl-12-(1-methylethyl)-; Peak 17, retention time 56.76min, 4H-1,3-Benzodioxin-4-one,5-butyl-2-(1,1-dimethylethyl) hexahydro-4a-methyl-,[2s-(2π4aπ5π8aπ]-; peak 18 retention time 57.95min, 1,5,9-cyclotetratriene, 1,5,9-trimethyl-12-(1-methylvinyl)-, (1E, 5E, 9E, 12R)-; peak 19 retention time 60.99min, dehydrocostus lactone; peak 20 retention time 61.93min, .alpha.-Isonootk atol; peak 21 retention time 63.15min, for bicyclo [9.3.1] pentadeca-4,14-diene, 4,14,15,15-tetramethyl-8-methylene-, (4E, 11S); peak 22 retention time 64.24min, for (1E, 3E, 7E, 11E)-1,7,11-trimethyl-4-isopropyl-1,3,7,11-cyclotetradecatetraene; peak 23 retention time 68.51min, for Phosphonic acid; peak 24, retention time 69.54min, is β-ionone; peak 25, retention time 70.11min, is ISOCEMBROL; peak 26, retention time 71.79min, is Azulene,1,2,3,5,6,7,8,8a-octahydro-1,4-dimethyl-7-(1-methylethenyl)-,[1S-(1π7π8aπ]-; peak 27, retention time 76.00min, is anthracene; peak 28, retention time 7 The peak retention time was 7.46min, which was geranyl linalool; the peak retention time of peak 29 was 79.67min, which was alpha-bulnesene; the peak retention time of peak 30 was 82.45min, which was (9β)-9,19-Cyclolanostan-3β-olacetate; the peak retention time of peak 31 was 84.39min, which was (3E, 7E, 11E)-1-isopropyl-4,8,12-trimethylcyclotetradecane-3,7,11-trienylglycerol; the peak retention time of peak 32 was 87.13min, which was Formic acid,3,7,11-trimethyl-1,6,10-dodecatrien-3-yl ester; peak 33, retention time 87.74min, 3,20-allopregnanedione; peak 34, retention time 90.69min, Azulene,1,2,3,4,5,6,7,8-octahydro-1,4-dimethyl-7-(1-methylethenyl)-,[1S-(1π4π7π]-; peak 35, retention time 92.19min, 2-nonen-1-ol 2-methyl.

4. The method for establishing the GC-MS fingerprint spectrum of the Juanbi Decoction substance standard according to claim 3 is characterized in that: In the material benchmark GC-MS fingerprint of the Juanbi Decoction, peak 1 is cinnamaldehyde, a characteristic component of cinnamon, peak 4 is lauric acid, a characteristic component of frankincense, peak 13 is ligustilide, a characteristic component of angelica and Chuanxiong, and peak 19 is dehydrocostus lactone, a characteristic component of costus root.

5. The application of the method for establishing the GC-MS fingerprint spectrum of the Juanbi Decoction substance standard as described in any one of claims 1 to 4, characterized in that: Cluster analysis was performed on the common peak database of the benchmark GC-MS fingerprint spectra of the Juanbi Decoction substances to determine the authenticity of the Juanbi Decoction preparations or to control the quality of the Juanbi Decoction preparations.

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