A method for detecting volatile oil of Bupleurum chinense DC

Abstract

The present invention relates to a detection method for volatile oil of Bupleurum chinense DC. The detection method of the present invention includes the extraction of volatile oil of Bupleurum chinense DC. and the detection by gas chromatography. Among them, the extraction of volatile oil of Bupleurum chinense DC. uses the steam distillation method; gas chromatography is used to qualitatively analyze the chemical components of the volatile oil of Bupleurum chinense DC., and 18 compounds are identified.

Description

Technical Field

[0001] The invention belongs to the technical field of analysis of traditional Chinese medicine components, relates to a method for simultaneously detecting multiple chemical components, and particularly relates to a method for detecting chemical components in bupleurum volatile oil. Background Art

[0002] Bupleurum (Bupleuri Radix) is the dried root of Bupleurum or Bupleurum angustifolium, a plant of the Umbelliferae family. It is produced in Northeast my country, North China, Hubei and Sichuan. Bupleurum is slightly cold, pungent and bitter in nature, and enters the liver, gallbladder and lung meridians. It has the effects of dispersing fever, relieving liver depression, and raising Yang Qi. It is mainly used for colds and fever, alternating cold and heat, chest and rib pain, irregular menstruation, uterine prolapse, rectal prolapse and other diseases.

[0003] The volatile oil of Radix Bupleuri is one of the main active ingredients of Radix Bupleuri, mainly containing fats, aromatics and terpenoids. At present, there is no industry or national standard for the detection of volatile oil of Radix Bupleuri. In the existing reports, the detection methods based on fats are mostly used, and the reports based on terpenes are relatively few. Terpenoids are compounds and their derivatives with isoprene units as basic structural units. There are a large number of isomers. Due to the similarity of isomers in physical and chemical properties, the relative molecular weight is the same, the polarity is not much different, the boiling point is similar, etc., it is difficult to separate in daily detection. Therefore, developing a method that can simultaneously detect multiple chemical components based on terpenes in volatile oil of Radix Bupleuri is of great significance for the basic research and quality control of the active substances of Radix Bupleuri. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a method for detecting the quality of Bupleurum Root, which can realize the simultaneous detection of multiple chemical components of Bupleurum Root.

[0005] The detection method comprises extraction of bupleurum volatile oil and gas chromatography detection.

[0006] Further, the following steps are included:

[0007] Step 1: Extracting the volatile oil of Radix Bupleuri by steam distillation;

[0008] Step 2: Gas chromatography is used to detect the chemical components of the volatile oil of Radix Bupleuri.

[0009] The steam distillation method described in step 1 is a method specified in the Chinese Pharmacopoeia. The specific method is: weigh an appropriate amount of Bupleurum, put it in a round-bottom flask, add an appropriate amount of water, heat and reflux to extract until the amount of oil in the volatile oil extractor no longer increases, and dry it with anhydrous sodium sulfate to obtain a test solution.

[0010] Further, weigh 40 - 80 g of Bupleuri Radix, place it in a round-bottom flask, add 6 - 12 times the amount of water, soak it, heat under reflux for extraction until the amount of oil in the volatile oil extractor no longer increases, and dry it with anhydrous sodium sulfate to obtain the test solution.

[0011] In step 2, the gas chromatography conditions are as follows:

[0012] Chromatographic column: a capillary column with a medium polarity or similar polarity stationary phase, and the stationary phase is selected from 100% polydimethylsiloxane, 5% diphenyl - 95% dimethylpolysiloxane, 50% diphenyl - 50% dimethylpolysiloxane, 6% cyanopropylphenyl - 94% dimethylpolysiloxane;

[0013] Temperature control mode: programmed temperature rise, initial temperature 60 - 90 °C, hold for 3 - 7 min, rise at a rate of 3 - 10 °C / min to 200 - 240 °C, and hold for 10 - 25 min;

[0014] Carrier gas: inert gas;

[0015] Column flow rate: 0.6 - 1.4 mL / min;

[0016] Injection mode: split injection, split ratio 5:1 - 40:1.

[0017] Further, the stationary phase of the chromatographic column is 6% cyanopropylphenyl - 94% dimethylpolysiloxane, and the chromatographic column is selected from DB - 624, TG - 624, TG - 624SilMS, OV - 624, HP - 624, RTX - 624, SPB - 624, AT - 624, ZM - 624, BP - 624, TR - V1, TG - 1301MS, DB - 1301, Rtx - 1301, PE - 1301, HP - 1301, OV - 1301, SPB - 1301.

[0018] Furthermore, the chromatographic column is TR - V1.

[0019] Further, for the programmed temperature rise, the initial temperature is 70 - 90 °C, hold for 3 - 7 min, rise at a rate of 3 - 7 °C / min to 210 - 230 °C, and hold for 15 - 25 min.

[0020] Further, the inert gas is selected from helium and nitrogen.

[0021] Furthermore, the inert gas is helium.

[0022] Further, the column flow rate is 0.8 - 1.2 mL / min.

[0023] Furthermore, the column flow rate is 1.0 mL / min.

[0024] Further, the column flow rate is 0.8 mL / min.

[0025] Further, for the split injection, the split ratio is 5:1 to 30:1.

[0026] Further still, the split ratio is 10:1.

[0027] Further still, the split ratio is 30:1.

[0028] Further, the gas chromatograph detector is selected from a flame ionization detector, a thermal conductivity detector, a photoionization detector, and a mass spectrometry detector.

[0029] Further still, the detector is a mass spectrometry detector, and its mass spectrometry conditions are as follows: ionization mode EI, electron energy 60 - 80 eV, transfer line temperature 220 - 240 °C, ion source temperature 220 - 240 °C.

[0030] The present invention provides a method for detecting the volatile oil of Bupleurum chinense DC., specifically by gas chromatography detection. The gas phase conditions are as described above.

[0031] The volatile oil of Bupleurum chinense DC. includes one or more of the following compounds: n-hexanal, 2-thujene, camphene, β-phellandrene, β-pinene, norpinene, n-octanal, D-limonene, β-ocimene, linalool, (-)-menthone, 4-terpineol, α-terpineol, methyl thymol ether, pulegone, bornyl acetate, β-caryophyllene, trans-ligustilide.

[0032] Compared with the prior art, the beneficial effects of the present invention are as follows: The technical solution of the present invention can comprehensively identify the quality of the Chinese medicinal material Bupleurum chinense DC. by one detection method, providing a simple and reliable method for the quality control of the medicinal material.

[0033] The present invention provides a method that can simultaneously detect various chemical components mainly composed of terpenoids in the volatile oil of Bupleurum chinense DC., and a total of 18 compounds are identified; by controlling the gas chromatography parameters, each component can be well separated, especially the difficult-to-separate terpenoid isomers, such as camphene and β-pinene with the molecular formula of C 10 H 16 and β-phellandrene and D-limonene; and (-)-menthone, 4-terpineol, and α-terpineol with the molecular formula of C 10 H 18 O are completely separated, with good peak shapes and stable baselines; the operation is simple and easy to implement, providing a reference basis for the research on the pharmacodynamic substance basis and quality control of Bupleurum chinense DC. Description of the Drawings

[0034] Figure 1 It is the total ion chromatogram of GC-MS for Example 1.

[0035] Figure 2 It is the total ion current chromatogram of Example 2 for GC-MS.

[0036] Figure 3 It is the total ion current chromatogram of Example 3 for GC-MS. Specific Embodiments

[0037] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, so that the public can have a deeper understanding of the content of the invention. This is not a limitation of the present invention. Any equivalent substitution in the art in accordance with the disclosed content of the present invention falls within the protection scope of the present invention.

[0038] Instruments and Materials:

[0039] Thermo scientific trace 1310ISQ / LT gas chromatography-mass spectrometry (Thermo Fisher Scientific, USA); Thermo TR-V1 chromatographic column (0.25mm x 30m, 1.4μm, Thermo Fisher Scientific, USA); ten-thousandth balance (Mettler-Toledo Instruments (Shanghai) Co., Ltd.); volatile oil extraction device; electronically temperature-controlled heating mantle (Henan Yuhua Instrument Co., Ltd.).

[0040] Bupleurum comes from the authentic producing area; water is pure water; anhydrous sodium sulfate is of analytical grade.

[0041] Example 1:

[0042] 1. Volatile Oil Extraction

[0043] Weigh 60g of Bupleurum, place it in a round-bottom flask, add 10 times the amount of water, soak it, heat under reflux for extraction until the amount of oil in the volatile oil extractor no longer increases, and dry it with anhydrous sodium sulfate to obtain the test solution for gas chromatography-mass spectrometry analysis.

[0044] 2. Detection Conditions

[0045] Gas chromatography conditions: Thermo TR-V1 chromatographic column (0.25mm x 30m, 1.4μm), high-purity helium as the carrier gas, column flow rate 1.0 mL / min, initial temperature 80°C, hold for 5 min, increase the temperature to 220°C at a rate of 5°C / min, hold for 20 min, inlet temperature 200°C, split ratio 10:1, injection volume 0.1 μl.

[0046] Mass spectrometry conditions: Ionization mode EI, electron energy 70 eV, transmission line and ion source temperature are both 230°C, scanning mass range 30 - 500 amu.

[0047] 3. Results

[0048] Determination was carried out according to the test method, and its total ion chromatogram is as Figure 1 shown. The obtained chromatographic peaks were retrieved by the NIST14 mass spectrometry database system and the standard library of the gas chromatography-mass spectrometry workstation, and further confirmed with reference to relevant literature. 18 compounds were identified and classified according to their chemical structures. The specific results are shown in Table 1.

[0049] Table 1 Components Identified in the Volatile Oil of Bupleurum chinense DC. and Their Classification

[0050]

[0051] Figure 1 It shows that under these conditions, each component can be well separated, especially the difficult-to-separate terpene isomers, such as camphene and β-pinene with the molecular formula C 10 H 16 , β-phellandrene and D-limonene; levomenthol, 4-terpineol, and α-terpineol with the molecular formula C 10 H 18 O are completely separated with good peak shapes and a stable baseline.

[0052] Example 2:

[0053] 1. Extraction of Volatile Oil

[0054] Weigh 40 g of Bupleurum chinense DC., place it in a round-bottom flask, add 6 times the amount of water, soak it, and heat under reflux for extraction until the amount of oil in the volatile oil extractor no longer increases. Dry it with anhydrous sodium sulfate to obtain the test solution for gas chromatography-mass spectrometry analysis.

[0055] 2. Detection Conditions

[0056] Gas chromatography conditions: Thermo TR-V1 chromatographic column (0.25 mm x 30 m, 1.4 μm), high-purity helium as the carrier gas, column flow rate 0.8 mL / min, initial temperature 80 °C, hold for 5 min, increase the temperature to 220 °C at a rate of 5 °C / min, hold for 20 min, inlet temperature 200 °C, split ratio 30:1, injection volume 0.1 μL.

[0057] Mass spectrometry conditions: Ionization mode EI, electron energy 60 eV, transfer line temperature 220 °C, ion source temperature 240 °C, scanning mass range 30 - 500 amu.

[0058] 3. Results

[0059] Determination was carried out according to the test method, and its total ion chromatogram is as Figure 2 shown, and the results are shown in Table 2.

[0060] Table 2 Components Identified in the Volatile Oil of Bupleurum chinense DC. and Their Classification

[0061]

[0062] Figure 2 It shows that under this condition, each component can be well separated, especially the terpene isomers that are difficult to separate, such as camphene and β-pinene with the molecular formula C 10 H 16 , β-phellandrene and D-limonene; levomenthol, 4-terpenol, and α-terpineol with the molecular formula C 10 H 18 O can also be completely separated, with good peak shapes and a stable baseline.

[0063] Example 3:

[0064] 1. Volatile oil extraction

[0065] Weigh 80 g of Bupleurum chinense DC., place it in a round-bottom flask, add 12 times the amount of water, soak it, heat under reflux for extraction until the amount of oil in the volatile oil extractor no longer increases, and dry it with anhydrous sodium sulfate to obtain the test solution for gas chromatography-mass spectrometry analysis.

[0066] 2. Detection conditions

[0067] Gas chromatography conditions: Thermo TR-V1 chromatographic column (0.25 mm x 30 m, 1.4 μm), high-purity helium as the carrier gas, column flow rate 1.0 mL / min, initial temperature 40 °C, hold for 5 min, increase the temperature to 220 °C at a rate of 10 °C / min, hold for 10 min, inlet temperature 200 °C, split ratio 10:1, injection volume 0.2 μL.

[0068] Mass spectrometry conditions: Ionization mode EI, electron energy 70 eV, transmission line and ion source temperatures are both 230 °C, scanning mass range 30 - 400 amu.

[0069] 3. Results

[0070] Determination was carried out according to the test method, and its total ion current chromatogram is as Figure 3 shown. Figure 3 It shows that under this condition, the initial temperature is low, and the components flow out slowly; the temperature increase rate is fast in the later stage, and the components flow out quickly, resulting in relatively poor resolution, and the peak information was not tabulated and statistically analyzed. However, camphene and β-pinene with the molecular formula C 10 H 16 , β-phellandrene and D-limonene; levomenthol, 4-terpenol, and α-terpineol with the molecular formula C 10 H 18 O can still be completely separated.

Claims

1. A detection method for volatile oil of Bupleurum chinense DC., characterized in that, The chemical components of the volatile oil of Bupleurum chinense DC. were detected by gas chromatography. The gas chromatography conditions are as follows: Chromatographic column: a capillary column with 6% cyanopropylphenyl-94% dimethylpolysiloxane as the stationary phase; Temperature control mode: programmed temperature rise. The initial temperature is 60-90 °C, held for 3-7 min, then heated at a rate of 3-10 °C / min to 200-240 °C and held for 10-25 min; Carrier gas: nitrogen or helium; Column flow rate: 0.6-1.4 mL / min; Injection mode: split injection, split ratio 5:1-40:1; The chemical components of the volatile oil of Bupleurum chinense DC. include hexanal, 2-thujene, camphene, β-phellandrene, β-pinene, norpinene, octanal, D-limonene, β-ocimene, linalool, (-)-menthone, 4-terpineol, α-terpineol, methyl thymol ether, pulegone, bornyl acetate, β-caryophyllene, trans-ligustilide.

2. The detection method according to claim 1, characterized in that, The chromatographic column is selected from DB-624, TG-624, TG-624SilMS, OV-624, HP-624, RTX-624, SPB-624, AT-624, ZM-624, BP-624, TR-V1, TG-1301MS, DB-1301, Rtx-1301, PE-1301, HP-1301, OV-1301, SPB-1301.

3. The detection method according to claim 1, characterized in that, For the programmed temperature rise, the initial temperature is 70-90 °C, held for 3-7 min, then heated at a rate of 3-7 °C / min to 210-230 °C and held for 15-25 min.

4. The detection method according to any one of claims 1-3, characterized in that, The column flow rate is 0.8-1.2 mL / min.

5. The detection method according to claim 4, characterized in that, The column flow rate is 0.8 mL / min or 1.0 mL / min.

6. The detection method according to any one of claims 1-3, characterized in that, For the split injection, the split ratio is 5:1-30:

1.

7. The detection method according to claim 6, characterized in that, The split ratio is 10:1 or 30:

1.

8. The detection method according to any one of claims 1-3, characterized in that, The gas chromatography detector is selected from flame ionization detector, thermal conductivity detector, photoionization detector, mass spectrometry detector.

9. The detection method according to claim 8, characterized in that, The gas chromatography detector is a mass spectrometry detector.

10. The detection method according to any one of claims 1-3, characterized in that, The carrier gas is helium.

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