CN115420832B - GC-MS fingerprint spectrum and multi-index content determination method for litsea coreana oil - Google Patents
GC-MS fingerprint spectrum and multi-index content determination method for litsea coreana oil Download PDFInfo
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- 241000587213 Litsea coreana Species 0.000 title claims abstract description 87
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 title claims abstract description 35
- 238000000034 method Methods 0.000 title claims description 27
- 238000001228 spectrum Methods 0.000 title abstract description 17
- 239000003921 oil Substances 0.000 claims abstract description 123
- 235000013399 edible fruits Nutrition 0.000 claims abstract description 59
- 238000000605 extraction Methods 0.000 claims abstract description 51
- 238000005303 weighing Methods 0.000 claims abstract description 45
- 239000007788 liquid Substances 0.000 claims abstract description 34
- 238000002791 soaking Methods 0.000 claims abstract description 32
- 241001081179 Litsea Species 0.000 claims abstract description 25
- 235000012854 Litsea cubeba Nutrition 0.000 claims abstract description 25
- 239000002245 particle Substances 0.000 claims abstract description 16
- 239000000341 volatile oil Substances 0.000 claims abstract description 14
- 238000007789 sealing Methods 0.000 claims abstract description 10
- 238000002360 preparation method Methods 0.000 claims abstract description 8
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims abstract description 7
- 238000004458 analytical method Methods 0.000 claims abstract description 6
- 239000000463 material Substances 0.000 claims abstract description 5
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 63
- 239000000523 sample Substances 0.000 claims description 27
- UFWIBTONFRDIAS-UHFFFAOYSA-N Naphthalene Chemical compound C1=CC=CC2=CC=CC=C21 UFWIBTONFRDIAS-UHFFFAOYSA-N 0.000 claims description 26
- JHIVVAPYMSGYDF-UHFFFAOYSA-N cyclohexanone Chemical compound O=C1CCCCC1 JHIVVAPYMSGYDF-UHFFFAOYSA-N 0.000 claims description 26
- 239000000243 solution Substances 0.000 claims description 22
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- 150000002500 ions Chemical class 0.000 claims description 15
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- RXBQNMWIQKOSCS-UHFFFAOYSA-N (7,7-dimethyl-4-bicyclo[3.1.1]hept-3-enyl)methanol Chemical compound C1C2C(C)(C)C1CC=C2CO RXBQNMWIQKOSCS-UHFFFAOYSA-N 0.000 claims description 8
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- KQAZVFVOEIRWHN-UHFFFAOYSA-N alpha-thujene Natural products CC1=CCC2(C(C)C)C1C2 KQAZVFVOEIRWHN-UHFFFAOYSA-N 0.000 claims description 8
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- UAHWPYUMFXYFJY-UHFFFAOYSA-N beta-myrcene Chemical compound CC(C)=CCCC(=C)C=C UAHWPYUMFXYFJY-UHFFFAOYSA-N 0.000 claims description 8
- NPNUFJAVOOONJE-UONOGXRCSA-N caryophyllene Natural products C1CC(C)=CCCC(=C)[C@@H]2CC(C)(C)[C@@H]21 NPNUFJAVOOONJE-UONOGXRCSA-N 0.000 claims description 8
- NVEQFIOZRFFVFW-RGCMKSIDSA-N caryophyllene oxide Chemical compound C=C1CC[C@H]2O[C@]2(C)CC[C@H]2C(C)(C)C[C@@H]21 NVEQFIOZRFFVFW-RGCMKSIDSA-N 0.000 claims description 8
- 229960005233 cineole Drugs 0.000 claims description 8
- 229930006696 sabinene Natural products 0.000 claims description 8
- 229940099369 (+)- limonene Drugs 0.000 claims description 7
- KMRMUZKLFIEVAO-IUCAKERBSA-N (1s,5r)-6,6-dimethylbicyclo[3.1.1]hept-3-ene-4-carbaldehyde Chemical compound C1[C@@H]2C(C)(C)[C@H]1CC=C2C=O KMRMUZKLFIEVAO-IUCAKERBSA-N 0.000 claims description 7
- IICQZTQZQSBHBY-HWKANZROSA-N (e)-non-2-ene Chemical compound CCCCCC\C=C\C IICQZTQZQSBHBY-HWKANZROSA-N 0.000 claims description 7
- IICQZTQZQSBHBY-UHFFFAOYSA-N 2t-nonene Natural products CCCCCCC=CC IICQZTQZQSBHBY-UHFFFAOYSA-N 0.000 claims description 7
- KMRMUZKLFIEVAO-RKDXNWHRSA-N Myrtenal Natural products C1[C@H]2C(C)(C)[C@@H]1CC=C2C=O KMRMUZKLFIEVAO-RKDXNWHRSA-N 0.000 claims description 7
- KGEKLUUHTZCSIP-HOSYDEDBSA-N [(1s,4s,6r)-1,7,7-trimethyl-6-bicyclo[2.2.1]heptanyl] acetate Chemical compound C1C[C@]2(C)[C@H](OC(=O)C)C[C@H]1C2(C)C KGEKLUUHTZCSIP-HOSYDEDBSA-N 0.000 claims description 7
- 238000001514 detection method Methods 0.000 claims description 7
- 238000007865 diluting Methods 0.000 claims description 7
- NVEQFIOZRFFVFW-UHFFFAOYSA-N 9-epi-beta-caryophyllene oxide Natural products C=C1CCC2OC2(C)CCC2C(C)(C)CC21 NVEQFIOZRFFVFW-UHFFFAOYSA-N 0.000 claims description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 5
- 241000633855 Litsea pungens Species 0.000 claims description 5
- 238000004090 dissolution Methods 0.000 claims description 5
- 229960001867 guaiacol Drugs 0.000 claims description 5
- 238000004949 mass spectrometry Methods 0.000 claims description 5
- 239000002904 solvent Substances 0.000 claims description 5
- WTARULDDTDQWMU-IUCAKERBSA-N (-)-β-pinene Chemical compound C1[C@@H]2C(C)(C)[C@H]1CCC2=C WTARULDDTDQWMU-IUCAKERBSA-N 0.000 claims description 4
- 125000000590 4-methylphenyl group Chemical group [H]C1=C([H])C(=C([H])C([H])=C1*)C([H])([H])[H] 0.000 claims description 4
- AANMVENRNJYEMK-UHFFFAOYSA-N 4-propan-2-ylcyclohex-2-en-1-one Chemical compound CC(C)C1CCC(=O)C=C1 AANMVENRNJYEMK-UHFFFAOYSA-N 0.000 claims description 4
- RXBQNMWIQKOSCS-RKDXNWHRSA-N Myrtenol Natural products C1[C@H]2C(C)(C)[C@@H]1CC=C2CO RXBQNMWIQKOSCS-RKDXNWHRSA-N 0.000 claims description 4
- 150000001299 aldehydes Chemical class 0.000 claims description 4
- VYBREYKSZAROCT-UHFFFAOYSA-N alpha-myrcene Natural products CC(=C)CCCC(=C)C=C VYBREYKSZAROCT-UHFFFAOYSA-N 0.000 claims description 4
- RSYBQKUNBFFNDO-UHFFFAOYSA-N caryophyllene oxide Natural products CC1(C)CC2C(=C)CCC3OC3(C)CCC12C RSYBQKUNBFFNDO-UHFFFAOYSA-N 0.000 claims description 4
- 239000011802 pulverized particle Substances 0.000 claims description 4
- WUOACPNHFRMFPN-VIFPVBQESA-N (R)-(+)-alpha-terpineol Chemical compound CC1=CC[C@H](C(C)(C)O)CC1 WUOACPNHFRMFPN-VIFPVBQESA-N 0.000 claims description 3
- 235000011869 dried fruits Nutrition 0.000 claims description 3
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 3
- 239000012498 ultrapure water Substances 0.000 claims description 3
- WUOACPNHFRMFPN-SECBINFHSA-N (S)-(-)-alpha-terpineol Chemical compound CC1=CC[C@@H](C(C)(C)O)CC1 WUOACPNHFRMFPN-SECBINFHSA-N 0.000 claims description 2
- OVKDFILSBMEKLT-UHFFFAOYSA-N alpha-Terpineol Natural products CC(=C)C1(O)CCC(C)=CC1 OVKDFILSBMEKLT-UHFFFAOYSA-N 0.000 claims description 2
- 229940088601 alpha-terpineol Drugs 0.000 claims description 2
- 238000010813 internal standard method Methods 0.000 claims 1
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- 238000002474 experimental method Methods 0.000 description 2
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- 238000001256 steam distillation Methods 0.000 description 2
- 229940126680 traditional chinese medicines Drugs 0.000 description 2
- 206010002383 Angina Pectoris Diseases 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 241000723347 Cinnamomum Species 0.000 description 1
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- HSRJKNPTNIJEKV-UHFFFAOYSA-N Guaifenesin Chemical compound COC1=CC=CC=C1OCC(O)CO HSRJKNPTNIJEKV-UHFFFAOYSA-N 0.000 description 1
- 241000218195 Lauraceae Species 0.000 description 1
- FAMPSKZZVDUYOS-UHFFFAOYSA-N alpha-Caryophyllene Natural products CC1=CCC(C)(C)C=CCC(C)=CCC1 FAMPSKZZVDUYOS-UHFFFAOYSA-N 0.000 description 1
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- 239000012088 reference solution Substances 0.000 description 1
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- 230000000630 rising effect Effects 0.000 description 1
- 239000012085 test solution Substances 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
- G01N30/8686—Fingerprinting, e.g. without prior knowledge of the sample components
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N2030/042—Standards
- G01N2030/045—Standards internal
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Abstract
The invention discloses a GC-MS fingerprint spectrum and multi-index content measurement method of large fruit litsea oil, which belongs to the field of large fruit litsea oil quality, and comprises the following steps: s1: accurately weighing 20g of litsea coreana, and placing in a flask, and respectively observing the influence of four factors of crushed particle size, feed-liquid ratio, soaking time and extraction time on oil yield; s2: adding proper amount of anhydrous sodium sulfate into the collected volatile oil, sealing overnight, centrifuging at 8000r/min for 10min in the next day, collecting upper layer oil layer, weighing for 3 times, and taking average value to calculate oil rate; s3: the solution preparation and analysis result can be realized, the chemical components in the litsea coreana oil can be researched by adopting a GC-MS technology, the material basis is primarily defined, and the foundation is laid for subsequent researches such as quality control and the like.
Description
Technical Field
The invention relates to the field of oil quality of litsea coreana, in particular to a GC-MS fingerprint spectrum and multi-index content measuring method of litsea coreana.
Background
The big fruit litsea coreana is a dry mature fruit of Cinnamomum migao of Lauraceae, also called Maodan, qingdao, and Dysosma, and is mainly produced in Guizhou, guangxi and Yunnan places in China, the big fruit litsea coreana oil is volatile oil obtained from the dry fruit of big fruit litsea coreana by steam distillation, is a common medicine for Guizhou minority nationality, and is mainly used for treating cardiovascular diseases such as coronary heart disease and angina pectoris and gastrointestinal diseases clinically.
In the aspect of quality control of the traditional Chinese medicine volatile oil, qualitative and quantitative research on chemical components of the volatile oil is mainly carried out by adopting a GC-MS or GC technology. In the traditional Chinese medicine quality evaluation system, fingerprint spectrum technology is mostly adopted to reflect the integrity of traditional Chinese medicines. The fingerprint spectrum can be combined with chemical pattern recognition to carry out deeper mining and discussion on fingerprint spectrum data, and is widely used for identification and quality evaluation of traditional Chinese medicines.
At present, the literature reports at home and abroad on the litsea coreana oil mainly concentrate on the aspects of extraction and purification, chemical components and pharmacological activity, and the research on pharmacokinetics and tissue distribution of the litsea coreana oil is not seen, so that in order to make the related preparation of the litsea coreana oil better applied to clinic, an analysis method combining fingerprint spectrum with multi-index content measurement is necessary to be established, and scientific basis is provided for quality control and quality evaluation of the litsea coreana oil.
Disclosure of Invention
1. Technical problem to be solved
Aiming at the problems existing in the prior art, the invention aims to provide the GC-MS fingerprint spectrum and the multi-index content measurement method of the large fruit litsea oil, which can be realized by researching chemical components in the large fruit litsea oil by adopting a GC-MS technology, so that the material basis is primarily defined, and the foundation is laid for subsequent quality control and other researches.
2. Technical proposal
In order to solve the problems, the invention adopts the following technical scheme.
The method for determining the GC-MS fingerprint of the litsea coreana oil comprises the following steps of:
s1: accurately weighing 20g of litsea coreana, and placing in a flask, and respectively observing the influence of four factors of crushed particle size, feed-liquid ratio, soaking time and extraction time on oil yield;
s2: adding proper amount of anhydrous sodium sulfate into the collected volatile oil, sealing overnight, centrifuging at 8000r/min for 10min in the next day, collecting upper layer oil layer, weighing for 3 times, and taking average value to calculate oil rate;
s3: establishing a GC-MS fingerprint;
s4: solutions were prepared and analyzed for results.
Further, the influence on the oil yield of the litsea coreana comprises the following factors:
influence of the pulverized particle size on the oil yield: crushing the litsea coreana seeds by using a crusher, respectively inspecting 10, 30, 60, 80 and 100 meshes of crushed litsea coreana seeds powder, accurately weighing 20g, soaking for 0h with a feed-liquid ratio of 1:12, extracting for 5h, comparing oil yield, and determining the optimal crushing particle size;
influence of feed liquid ratio on oil yield: crushing the large fruit litsea fruits by using a crusher, sieving the crushed large fruit litsea fruits by using a 60-mesh sieve, accurately weighing 20g, respectively observing the feed liquid ratio of 1:6, 1:8, 1:10, 1:12 and 1:14, soaking for 0h, extracting for 5h, comparing the oil yield, and determining the optimal feed liquid ratio;
influence of soaking time on oil yield: crushing the large fruit litsea fruits by using a crusher, sieving the crushed large fruit litsea fruits by using a 60-mesh sieve, accurately weighing 20g, respectively observing and soaking for 0h, 0.5h, 1h, 1.5h and 2h according to a feed-liquid ratio of 1:12, extracting for 5h, comparing oil yield, and determining the optimal soaking time;
influence of extraction time on oil yield: crushing the litsea coreana seeds by using a crusher, sieving the crushed litsea coreana seeds powder by a 60-mesh sieve, accurately weighing 20g, respectively inspecting and extracting for 2h, 3h, 4h, 5h and 6h according to a feed-liquid ratio of 1:12, comparing oil yields, and determining the optimal extraction time.
Further, carrying out an extraction test of the oil of the large fruit litsea coreana on the result of the single-factor test, accurately weighing 20g of crushed large fruit litsea coreana, calculating the oil yield, and carrying out 3 times in parallel;
the calculation method comprises the following steps:
further, accurately weighing the extracted large fruit litsea oil into a volumetric flask of 50mg to 50mL, adding ethyl acetate for dilution to a scale, shaking uniformly, accurately weighing the extracted large fruit litsea oil into a volumetric flask of 1mL to 50mL, adding ethyl acetate for dilution to the scale, and shaking uniformly to obtain a sample solution.
Further, the establishing of the GC-MS fingerprint comprises the following steps:
a1: GC-MS conditions:
chromatographic conditions: the split ratio is 50:1, and the temperature of a sample inlet is 220 ℃; sample introduction without diversion; the sample injection amount is 1 mu L;
mass spectrometry conditions: ion source temperature 200 ℃; the interface temperature is 230 ℃; ionization means are electron bombardment ions (EI); detection voltage: 0.2KV; delaying the solvent for 3min; mass number scanning range: 30-400 amu; SCAN full SCAN mode;
a2: the extraction method comprises the following steps:
respectively taking 22 batches of dried fruits of litsea coreana, crushing, sieving with a 60-mesh sieve, accurately weighing 20g of powder, respectively and rapidly filling into 250mL round-bottomed flasks, adding 160mL of ultrapure water, sealing and soaking for 0.5h, connecting an extraction device for extraction for 5h, standing for 1h after extraction, collecting volatile oil, adding a proper amount of anhydrous sodium sulfate, sealing overnight, centrifuging at 8000r/min for 10min the next day, collecting an upper layer of oil layer, and preserving at minus 20 ℃ in a dark place;
a3: preparing a solution:
accurately weighing 0.1g of litsea coreana oil respectively, placing in a 20mL volumetric flask to obtain stock solution, accurately weighing 1.5mL of stock solution in the 20mL volumetric flask, and adding ethyl acetate to fix volume to obtain sample solution;
a4: and (5) conclusion is drawn through experiments, and a fingerprint is established.
The multi-index content determination method of the litsea coreana oil comprises the following steps of:
s1: GC-MS conditions
Chromatographic conditions: chromatographic column: SH-Stabilwax capillary column; the temperature of the sample inlet is 220 ℃; the split ratio is 50:1; the sample injection amount is 1 mu L;
mass spectrometry conditions: ion source temperature 200 ℃; the interface temperature is 230 ℃; the ionization mode is electron bombardment ion; detection voltage: 0.2KV; delaying the solvent for 3min; mass number scanning range: 30-400 amu; a SIM mode;
s2: preparing a reference substance solution;
s3: preparing an internal standard solution;
s4: preparing a sample solution;
s5: and (5) examining, analyzing and testing.
Further, the preparation method of the reference substance solution in the step S2 comprises the following steps: precisely weighing appropriate amount of L-beta-pinene, sabinene, myrcene, (+) -limonene, eucalyptol, gamma-terpinene, terpinolene, 2-nonene, 4-biotinol, borneol acetate, beta-caryophyllene, 4-terpene enol, (-) -myrtenal, 4-isopropylcyclohex-2-ene-1-one, alpha-terpinol, cumyl aldehyde, myrtenol, 2- (4-methylphenyl) propan-2 alcohol, caryophyllene oxide and guaiacol, placing into a 10mL volumetric flask, adding ethyl acetate, dissolving and diluting to scale, and making into L-beta-pinene, sabinene, myrtene, (+) -limonene, eucalyptol, gamma-terpinene, p-cymene, terpinolene, 2-nonene, 4-biotinol, borneol acetate, beta-caryophyllene, 4-terpene enol, (-) -myrtenal, 4-isopropyl-2-methyl-alkenol, alpha-2- (4-methyl-alkenol, and guaiac-2-methyl-alkenol, storing in refrigerator, and storing at 20 ℃ for use.
Further, the preparation method of the internal standard solution in the step S3 comprises the following steps: accurately weighing cyclohexanone and naphthalene reference substances, placing into a 10mL volumetric flask, adding ethyl acetate for dissolution, and diluting to scale to obtain internal standard stock solution of cyclohexanone (3.300 mg.mL-1) and naphthalene (2.710 mg.mL-1). Respectively precisely measuring appropriate amount of internal standard stock solution into 100mL volumetric flask, diluting with ethyl acetate to scale, preparing into internal standard solution of cyclohexanone (150 μg/mL-1) and naphthalene (3 μg/mL-1), and storing in (-20deg.C) refrigerator.
Further, the preparation method of the sample solution in the step S4 comprises the following steps: accurately weighing 0.1g of litsea coreana oil, placing in a 20mL volumetric flask, adding ethyl acetate for dissolution and dilution to scale, accurately weighing 1mL of solution, placing in a 10mL volumetric flask, respectively accurately adding 1mL of cyclohexanone and naphthalene internal standard solution, adding ethyl acetate for dilution to scale, shaking uniformly, and obtaining a sample solution for 2 times.
3. Advantageous effects
Compared with the prior art, the invention has the advantages that:
(1) According to the technical scheme, firstly, chemical components in the large fruit litsea coreana oil are researched by adopting a GC-MS technology, the material basis is primarily clarified, a foundation is laid for subsequent quality control and other researches, secondly, the multi-index component content of large fruit litsea coreana oil samples of different production places is analyzed by adopting a GC-MS SIM technology in combination with chemical pattern recognition, so that the foundation is laid for comprehensively and effectively controlling the overall quality of the large fruit litsea coreana oil, the quality difference mark components of the large fruit litsea coreana oil are screened, and the basis and research thought are provided for improving and perfecting the quality standard of the large fruit litsea coreana oil.
(2) According to the technical scheme, the extraction method of the litsea coreana oil by the steam distillation method is examined, and the optimal extraction condition is determined to be that the crushed particle size is 60 meshes, the feed-liquid ratio is 1:8 (g: mL), the soaking time is 0.5h, and the extraction time is 5h. The verification test result shows that the extraction method is simple to operate, low in cost, stable and feasible.
(3) According to the technical scheme, the GC-MS fingerprint spectrum of 22 batches of the litsea coreana oil is established by taking the comparison fingerprint spectrum of the litsea coreana oil as a comparison. Proved by methodology, the method has good precision, stability and repeatability, and can be used for fingerprint research of the litsea coreana oil.
(4) The GC-MS SIM method is combined with chemical pattern recognition, so that researches on the quality of the oil of the large fruit litsea in different batches are systematically, comprehensively and integrally carried out, and the method is scientific, accurate, reliable and easy to implement, and can provide references for controlling and identifying the quality of the oil of the large fruit litsea.
Drawings
FIG. 1 is a GC-MS total ion flow diagram of the inventive large fruit litsea oil;
FIG. 2 is a graph showing the relative percentages of various types of compounds in the oil of the present invention;
FIG. 3 is a graph of the inventive accuracy test chromatogram of the Litsea coreana oil and a comparison fingerprint;
FIG. 4 is a graph of a repetitive test chromatogram of the inventive Litsea coreana oil and a control fingerprint;
FIG. 5 is a graph of a stability test chromatogram of the inventive Litsea coreana oil and a comparison fingerprint;
FIG. 6 is a graph of the inventive Litsea coreana oil against fingerprints;
FIG. 7 is a superimposed graph of the fingerprint of 22 batches of Litsea coreana oil according to the present invention;
FIG. 8 is a graph showing the effect of the size of the crushed particles on the oil yield of the litsea coreana;
FIG. 9 shows the effect of feed liquid comparison on oil yield of Litsea coreana;
FIG. 10 is a graph showing the effect of soaking time on oil yield of Litsea coreana of the present invention;
FIG. 11 shows the effect of the extraction time on the oil yield of Litsea coreana.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention; it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments, and that all other embodiments obtained by persons of ordinary skill in the art without making creative efforts based on the embodiments in the present invention are within the protection scope of the present invention.
Example 1:
referring to fig. 1-11, the method for determining GC-MS fingerprint of the litsea coreana oil comprises the following steps:
s1: accurately weighing 20g of litsea coreana, and placing in a flask, and respectively observing the influence of four factors of crushed particle size, feed-liquid ratio, soaking time and extraction time on oil yield;
s2: adding proper amount of anhydrous sodium sulfate into the collected volatile oil, sealing overnight, centrifuging at 8000r/min for 10min in the next day, collecting upper layer oil layer, weighing for 3 times, and taking average value to calculate oil rate;
s3: establishing a GC-MS fingerprint;
s4: solutions were prepared and analyzed for results.
The influence on the oil yield of the litsea coreana comprises the following factors:
influence of the pulverized particle size on the oil yield: crushing the litsea coreana seeds by using a crusher, respectively inspecting 10, 30, 60, 80 and 100 meshes of crushed litsea coreana seeds powder, accurately weighing 20g, soaking for 0h with a feed-liquid ratio of 1:12, extracting for 5h, comparing oil yield, and determining the optimal crushing particle size;
as shown in fig. 8, under the extraction conditions of a feed-liquid ratio of 1:12, a soaking time of 0h and an extraction time of 5h, when the crushed particle size is 10 to 100 mesh, the oil yield of the litsea coreana is increased with the increase of the crushed particle size, and when the crushed particle size is more than 60 mesh, the oil yield of the volatile oil is reduced. Therefore, it is preferable to select 60-mesh pulverized particle size.
Influence of feed liquid ratio on oil yield: crushing the large fruit litsea fruits by using a crusher, sieving the crushed large fruit litsea fruits by using a 60-mesh sieve, accurately weighing 20g, respectively observing the feed liquid ratio of 1:6, 1:8, 1:10, 1:12 and 1:14, soaking for 0h, extracting for 5h, comparing the oil yield, and determining the optimal feed liquid ratio;
as can be seen from fig. 9, under the extraction conditions of 60 mesh crushed particle size, 0h soaking time and 5h extraction time, the oil yield of the large fruit litsea coreana increases and decreases with the increase of the feed liquid ratio, and the oil yield of the large fruit litsea coreana is highest when the feed liquid ratio is 1:8, so the feed liquid ratio is 1:8 (g: mL).
Influence of soaking time on oil yield: crushing the large fruit litsea fruits by using a crusher, sieving the crushed large fruit litsea fruits by using a 60-mesh sieve, accurately weighing 20g, respectively observing and soaking for 0h, 0.5h, 1h, 1.5h and 2h according to a feed-liquid ratio of 1:12, extracting for 5h, comparing oil yield, and determining the optimal soaking time;
as shown in fig. 10, under the conditions of 60 mesh crushed particle size, 1:12 feed-liquid ratio and 5h extraction time, the oil yield of the large fruit litsea pungens reached the maximum value when the soaking time was 0.5h with the extension of the soaking time. Thus, a 0.5h soak was selected.
Influence of extraction time on oil yield: crushing the litsea coreana seeds by using a crusher, sieving the crushed litsea coreana seeds powder by a 60-mesh sieve, accurately weighing 20g, respectively inspecting and extracting for 2h, 3h, 4h, 5h and 6h according to a feed-liquid ratio of 1:12, comparing oil yields, and determining the optimal extraction time.
As shown in FIG. 11, under the extraction conditions of 60 mesh crushed particle size, 1:12 feed-liquid ratio (g: mL) and 0h soaking time, the oil yield of the Litsea coreana was gradually decreased after rising upward with the increase of the extraction time, and the oil yield was highest at 5h. The extraction time is too long, and the volatile oil can be lost slightly. Therefore, the extraction time is selected to be 5h based on the principle of saving time.
Carrying out an extraction test of the oil of the litsea coreana on the result of the single-factor test, accurately weighing 20g of crushed litsea coreana, calculating the oil yield, and carrying out 3 times in parallel;
the calculation method comprises the following steps:
accurately weighing the large fruit litsea oil extracted from the solution into a volumetric flask of 50mg to 50mL, adding ethyl acetate for dilution to a scale, shaking uniformly, accurately weighing the large fruit litsea oil into the volumetric flask of 1mL to 50mL, adding ethyl acetate for dilution to the scale, and shaking uniformly to obtain the sample solution.
Verification test:
according to the test result, 3 parts of crushed litsea coreana are taken for carrying out an extraction verification test of volatile oil. The extraction condition is that the particle size is 60 meshes, 20g of the litsea coreana powder is accurately weighed, the feed-liquid ratio is 1:8 (g: mL), the soaking time is 0.5h, the extraction time is 5h, volatile oil is collected, a proper amount of anhydrous sodium sulfate is added for sealing overnight, centrifugation is carried out for 10min at 8000r/min in the next day, the upper layer oil layer is collected, and the oil ratio is weighed and calculated. The extraction rates of 3 parts of the litsea coreana oil are respectively 11.23%, 11.29% and 11.37%, the average value is 11.30%, and the RSD is 0.62%, which indicates that the method is stable and reliable for extracting the litsea coreana oil.
And (3) chemical component identification:
taking a litsea coreana volatile oil sample obtained by the above factor processing method, taking 1 mu L, detecting by using a GC-MS combined instrument, comparing the analysis result with a NIST 2020 standard mass spectrum library, selecting a peak with a matching degree higher than 80, and determining the total ion flow diagram of each chemical component by combining related literature and manual analysis of the spectrum, wherein the relative percentage of each compound is shown in figure 1 and figure 2.
The extraction method of the litsea coreana oil is examined, and the optimal extraction condition is determined to be that the crushed particle size is 60 meshes, the feed-liquid ratio is 1:8 (g: mL), the soaking time is 0.5h, and the extraction time is 5h. The verification test result shows that the extraction method is simple to operate, low in cost, stable and feasible.
The establishment of the GC-MS fingerprint comprises the following steps:
a1: GC-MS conditions:
chromatographic conditions: the split ratio is 50:1, and the temperature of a sample inlet is 220 ℃; sample introduction without diversion; the sample injection amount is 1 mu L; the temperature programming is shown in the following table:
column incubator temperature program
Mass spectrometry conditions: ion source temperature 200 ℃; the interface temperature is 230 ℃; ionization means are electron bombardment ions (EI); detection voltage: 0.2KV; delaying the solvent for 3min; mass number scanning range: 30-400 amu; SCAN full SCAN mode;
a2: the extraction method comprises the following steps:
respectively taking 22 batches of dried fruits of litsea coreana, crushing, sieving with a 60-mesh sieve, accurately weighing 20g of powder, respectively and rapidly filling into 250mL round-bottomed flasks, adding 160mL of ultrapure water, sealing and soaking for 0.5h, connecting an extraction device for extraction for 5h, standing for 1h after extraction, collecting volatile oil, adding a proper amount of anhydrous sodium sulfate, sealing overnight, centrifuging at 8000r/min for 10min the next day, collecting an upper layer of oil layer, and preserving at minus 20 ℃ in a dark place;
a3: preparing a solution:
accurately weighing 0.1g of litsea coreana oil respectively, placing in a 20mL volumetric flask to obtain stock solution, accurately weighing 1.5mL of stock solution in the 20mL volumetric flask, and adding ethyl acetate to fix volume to obtain sample solution;
a4: and (5) conclusion is drawn through experiments, and a fingerprint is established.
Test example:
precision test:
taking the same sample solution, continuously sampling and measuring for 6 times according to the GC-MS conditions of each factor to obtain a fingerprint (see figure 3), and performing similarity calculation by adopting a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012.130723 edition). Comparing the 6 times of fingerprints with the 1 st time of fingerprints by taking the 1 st time of fingerprints as a reference. The results show that the similarity of the 6 times of fingerprints is greater than 0.996, and the precision is good, and the detailed table is shown below:
similarity calculation result of fingerprint precision test
Repeatability test:
taking 6 parts of the same batch of sample solution, detecting according to the GC-MS conditions of each factor to obtain a fingerprint (see figure 4), and performing similarity calculation by adopting a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012.130723 edition). And comparing the fingerprint of the 6 samples with the fingerprint of the 1 st sample by taking the fingerprint of the 1 st sample as a reference. The results show that the fingerprint similarity of 6 samples is greater than 0.996, and the repeatability is good, and the detailed table is shown below:
similarity calculation result of fingerprint repeatability test
Stability test:
taking the same batch of sample solutions, sampling for 0, 2, 4, 8, 12 and 24 hours respectively, measuring according to GC-MS condition under the condition of 1.2.1 to obtain a fingerprint (see figure 5), and calculating the similarity by adopting a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012.130723 edition). And comparing the fingerprint spectrum of the sample introduction for 0h with the fingerprint spectrum of the sample introduction for 0h for 6 times. The results show that the similarity of fingerprints of 6 times is greater than 0.996, and the stability is good, and the detailed table is shown below:
finger print stability similarity calculation result
Fingerprint establishment:
and (3) using a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012.130723 edition), taking a sample spectrum as a reference spectrum, adopting an average method, enabling the time window width to be 0.1, automatically matching full spectrum peaks to generate a reference fingerprint (see figure 6), and overlapping 22 batches of large fruit litsea pungens oil with the reference fingerprint (see figure 7).
The study establishes 22 batches of GC-MS fingerprints of the litsea coreana oil by taking the comparison fingerprints of the litsea coreana oil as a comparison. Proved by methodology, the method has good precision, stability and repeatability, and can be used for fingerprint research of the litsea coreana oil. The quality condition of the large fruit litsea pungens oil in Guizhou, yunnan and Guangxi is primarily known by analyzing 22 batches of samples through a similarity evaluation method, and the fingerprint method can provide a reference for the quality standard of the large fruit litsea pungens oil.
The multi-index content determination method of the litsea coreana oil comprises the following steps of:
s1: GC-MS conditions
Chromatographic conditions: chromatographic column: SH-Stabilwax capillary column; the temperature of the sample inlet is 220 ℃; the split ratio is 50:1; the sample injection amount is 1 mu L (see the upper graph);
mass spectrometry conditions: ion source temperature 200 ℃; the interface temperature is 230 ℃; the ionization mode is electron bombardment ion; detection voltage: 0.2KV; delaying the solvent for 3min; mass number scanning range: 30-400 amu; SIM mode, each component detects ion information as follows:
ion information detection method for multi-index components of litsea coreana oil
S2: preparing a reference substance solution;
precisely weighing appropriate amount of levo-beta-pinene, sabinene, myrcene, (+) -limonene, eucalyptol, gamma-terpinene p-cymene, terpinolene, 2-nonene, 4-biotinol, borneol acetate, beta-caryophyllene, 4-terpene enol, (-) -myrtenal, 4-isopropylcyclohex-2-ene-1-one, alpha-terpinol, cumyl aldehyde, myrtenol, 2- (4-methylphenyl) propan-2 alcohol, caryophyllene oxide and guaiacol, placing into a 10mL volumetric flask, adding ethyl acetate to dissolve and dilute to scale, and making into levo-beta-pinene, sabinene, myrtene, (+) -limonene, eucalyptol, gamma-terpinene, p-cymene, terpinolene, 2-nonene, 4-biotinol, borneol acetate, beta-caryophyllene, 4-terpene enol, (-) -myrtenal, 4-isopropyl-2-methyl-alkenol, alpha-2- (4-methyl-alkenol, and guaifenesin a refrigerator, storing the liquid of the formula;
s3: preparing an internal standard solution;
accurately weighing cyclohexanone and naphthalene reference substances, placing into a 10mL volumetric flask, adding ethyl acetate for dissolution, and diluting to scale to obtain internal standard stock solution of cyclohexanone (3.300 mg.mL-1) and naphthalene (2.710 mg.mL-1). Respectively precisely measuring an appropriate amount of internal standard stock solution into a 100mL volumetric flask, diluting with ethyl acetate to scale, preparing into internal standard solutions of cyclohexanone (150 mug.mL-1) and naphthalene (3 mug.mL-1), and storing in a (-20 ℃) refrigerator for standby;
s4: preparing a sample solution;
accurately weighing 0.1g of litsea coreana oil, placing in a 20mL volumetric flask, adding ethyl acetate for dissolution and dilution to scale, accurately weighing 1mL of solution, placing in a 10mL volumetric flask, respectively accurately adding 1mL of cyclohexanone and naphthalene internal standard solution, adding ethyl acetate for dilution to scale, shaking uniformly, and obtaining a sample solution for 2 times.
S5: and (5) examining, analyzing and testing.
Test example:
accurately sucking a proper amount of reference substance solution extracted under the condition of each factor, preparing a series of mixed reference substance solutions, and carrying out sample injection measurement according to the GC-MS condition. And drawing a standard working curve by taking the concentration of each reference substance as an abscissa (X) and the peak area as an ordinate (Y), and calculating a regression equation, wherein the table is shown below. R2 is greater than 0.9997, which shows that 21 components have good linear relationship in the corresponding concentration range.
Taking mixed reference substance solution, carrying out sample injection analysis according to GC-MS conditions under the conditions of each factor, continuously injecting sample for 6 times in 1 day, recording peak area, and calculating the daily precision of the mixed reference substance solution, wherein the result is shown in the following table 1; the same mixed reference solution was precisely sucked, continuously injected for 3 days, measured 3 times a day, and peak areas were recorded to obtain the daytime precision, and the results are shown in Table 2 below. The daily precision and the daytime precision of 21 components are less than 3 percent, which indicates that the instrument precision is good.
TABLE 1 within-day precision test of 21 ingredients in Litsea coreana oil
TABLE 2 daytime precision test of 21 ingredients in Litsea coreana oil
Stability test:
the sample solutions prepared into the sample solutions were precisely sucked, and the peak areas of 21 components were measured by sampling at 0, 2, 4, 6, 8, 12 and 24 hours under the GC-MS conditions, and the results are shown in the following table. The peak area RSD of 21 components is between 1.67 and 2.63 percent, which shows that the stability of the test sample solution is good within 24 hours.
Stability test of 21 ingredients in Litsea coreana oil
Repeatability test:
taking 0.1g of litsea coreana oil, precisely weighing, preparing 6 parts of test solution in parallel, carrying out sample injection measurement according to GC-MS conditions, recording peak area, calculating content, and obtaining the results shown in the following table. RSD of the 21 component contents is less than 3%, which shows that the method has good repeatability.
Repeatability test of 21 ingredients of litsea coreana oil
The GC-MS SIM method is combined with chemical pattern recognition, so that researches on the quality of the oil of the large fruit litsea in different batches are systematically, comprehensively and integrally carried out, and the method is scientific, accurate, reliable and easy to implement, and can provide references for controlling and identifying the quality of the oil of the large fruit litsea. The method screens out 6 components of eucalyptol, p-cymene, sabinene, L-beta-pinene, alpha-terpineol and oxidized caryophyllene which are main quality sign components possibly generating differences among the large fruits Jiang Ziyang of Guizhou, yunnan and Guangxi.
The above description is only of the preferred embodiments of the present invention; the scope of the invention is not limited in this respect. Any person skilled in the art, within the technical scope of the present disclosure, may apply to the present invention, and the technical solution and the improvement thereof are all covered by the protection scope of the present invention.
Claims (3)
1. The method for measuring the multi-index content of the litsea coreana oil is characterized by comprising the following steps of: the method comprises the following measuring steps:
s1: extracting the sample to be detected, namely, the litsea coreana oil:
s11: respectively examining the influence of four factors of the crushing grain diameter, the feed-liquid ratio, the soaking time and the extraction time on the oil yield, and optimizing to obtain the optimal extraction condition, wherein the optimal extraction condition is that the crushing grain diameter is 60 meshes, the feed-liquid ratio is 1g to 8mL, the soaking time is 0.5h, and the extraction time is 5h;
s12: under the optimal extraction condition, the extraction method of the large fruit litsea pungens oil comprises the following steps: pulverizing dried fruit of litsea coreana, sieving with 60 mesh sieve, weighing 20g powder, loading into 250mL round bottom flask, adding 160mL ultrapure water, sealing and soaking for 0.5h, connecting extraction device for 5h, standing for 1h after extraction, collecting volatile oil, adding appropriate amount of anhydrous sodium sulfate, sealing overnight, centrifuging at 8000r/min for 10min the next day, and collecting upper layer oil layer;
s2: measuring the content of multiple indexes;
wherein the multi-index component is 21 kinds of L-beta-pinene, sabinene, myrcene, (+) -limonene, eucalyptol, gamma-terpinene, p-cymene, terpinolene, 2-nonene, 4-biotyl alcohol, borneol acetate, beta-caryophyllene, 4-terpene enol, (-) -myrtenal, 4-isopropyl cyclohex-2-ene-1-one, alpha-terpineol, cumyl aldehyde, myrtenol, 2- (4-methylphenyl) propan-2 alcohol, caryophyllene oxide and guaiacol;
s21: preparing a mixed reference substance solution;
the preparation method of the mixed reference substance solution comprises the following steps: precisely weighing levo-beta-pinene, sabinene, myrcene, (+) -limonene, eucalyptol, gamma-terpinene, p-cymene, terpinolene, 2-nonene, 4-biotinol, borneol acetate, beta-caryophyllene, 4-terpene alcohol, (-) -myrtenal, 4-isopropylcyclohex-2-ene-1-one, alpha-terpinol, cumyl aldehyde, myrtenol, 2- (4-methylphenyl) propan-2 alcohol, caryophyllene oxide and guaiacol reference, placing in a 10mL volumetric flask, adding ethyl acetate to dissolve and dilute to scale, and making into levo-beta-pinene, sabinene, myrtene, (+) -limonene, eucalyptol, gamma-terpinene, p-cymene, terpinolene, 2-nonene, 4-biotinol, borneol acetate, beta-caryophyllene, 4-terpene alcohol, (-) -myrtenal, 4-isopropyl-2-methyl-alkenol, alpha-2- (4-methyl phenyl) propan-2-ol, and guaiacol, storing at a temperature of 20 ℃ for use; precisely sucking the stock solutions of the reference substances to prepare a series of mixed reference substance solutions with concentration;
s22: preparing an internal standard solution;
the preparation method of the internal standard solution comprises the following steps: accurately weighing cyclohexanone and naphthalene reference substances, placing the cyclohexanone and naphthalene reference substances into a 10mL volumetric flask, adding ethyl acetate for dissolving and diluting to a scale, preparing internal standard stock solutions of 3.300 mg/mL-1 of cyclohexanone and 2.710 mg/mL-1 of naphthalene, accurately weighing a proper amount of the internal standard stock solutions into a 100mL volumetric flask respectively, diluting to the scale by using ethyl acetate, preparing internal standard solutions of 150 mug/mL-1 of cyclohexanone and 3 mug/mL-1 of naphthalene, and placing the internal standard stock solutions into a refrigerator at a temperature of minus 20 ℃ for later use;
s23: preparing a sample solution;
the preparation method of the sample solution comprises the following steps: precisely weighing 0.1g of large fruit litsea oil extracted from a sample to be measured, placing the large fruit litsea oil into a 20mL volumetric flask, adding ethyl acetate for dissolution and dilution to scale, precisely weighing 1mL of solution, placing the solution into a 10mL volumetric flask, precisely adding 1mL of cyclohexanone and naphthalene internal standard solution respectively, adding ethyl acetate for dilution to scale, and shaking uniformly to obtain a sample solution;
s3: sample analysis:
respectively carrying out sample injection detection on the prepared serial concentration mixed reference substance solution and the sample solution in a GC-MS combined instrument, drawing a standard working curve, and leading the correlation coefficient to be more than 0.9997; obtaining the content of 21 components in a sample to be tested according to a drawn standard working curve by adopting an internal standard method;
wherein the GC-MS conditions include:
chromatographic conditions: chromatographic column: SH-Stabilwax capillary column; the temperature of the sample inlet is 220 ℃; the split ratio is 50:1; the sample injection amount is 1 mu L;
mass spectrometry conditions: ion source temperature 200 ℃; the interface temperature is 230 ℃; the ionization mode is electron bombardment ion; detection voltage: 0.2KV; delaying the solvent for 3min; mass number scanning range: 30-400 amu; SIM mode, each component detects ion information as follows:
2. the method for measuring the multi-index content of the litsea coreana oil according to claim 1, wherein the method is characterized by comprising the following steps of: the method for optimizing the extraction conditions comprises the following specific steps of:
influence of the pulverized particle size on the oil yield: crushing the litsea coreana fruits by using a crusher, sieving the crushed litsea coreana fruits by using 10, 30, 60, 80 and 100-mesh sieves respectively, accurately weighing 20g, soaking for 0h according to the feed liquid ratio of 1g to 12mL, extracting for 5h, comparing oil yield, and determining the optimal crushing particle size;
influence of feed liquid ratio on oil yield: crushing the large fruit litsea fruits by using a crusher, sieving the crushed large fruit litsea fruits by a 60-mesh sieve, accurately weighing 20g, soaking for 0h according to the feed liquid ratio of 1g to 6mL, 1g to 8mL, 1g to 10mL, 1g to 12mL and 1g to 14mL respectively, extracting for 5h, comparing the oil yield, and determining the optimal feed liquid ratio;
influence of soaking time on oil yield: crushing the litsea coreana fruits by using a crusher, sieving the crushed litsea coreana fruits by a 60-mesh sieve, accurately weighing 20g, respectively soaking the materials in a feed liquid ratio of 1g to 12mL for 0h, 0.5h, 1h, 1.5h and 2h, extracting for 5h, comparing oil yield, and determining the optimal soaking time;
influence of extraction time on oil yield: crushing the litsea coreana seeds by using a crusher, sieving the crushed litsea coreana seeds powder by a 60-mesh sieve, accurately weighing 20g, extracting the materials for 2h, 3h, 4h, 5h and 6h respectively according to the feed liquid ratio of 1g to 12mL, comparing oil yield, and determining the optimal extraction time.
3. The method for measuring the multi-index content of the litsea coreana oil according to claim 2, which is characterized by comprising the following steps: the calculated oil ratio formula is:
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