CN106324145A - Distinguishing method of quality of fresh tobacco leaf sample in tobacco metabonomics based on terpenes - Google Patents
Distinguishing method of quality of fresh tobacco leaf sample in tobacco metabonomics based on terpenes Download PDFInfo
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- CN106324145A CN106324145A CN201610868641.8A CN201610868641A CN106324145A CN 106324145 A CN106324145 A CN 106324145A CN 201610868641 A CN201610868641 A CN 201610868641A CN 106324145 A CN106324145 A CN 106324145A
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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
Abstract
The invention provides a distinguishing method of the quality of a fresh tobacco leaf sample in tobacco metabonomics based on terpenes. The method comprises the following steps: detecting content of phytol and spinacene in fresh tobacco leaves and calculating the ratio of the content of phytol and the content of spinacene; carrying out absolute quantification analysis on the phytol and the spinacene in the fresh tobacco leaf sample in a metabonomics research by adopting gas chromatography-tandem mass spectrometry (GC-MS), so as to provide the basis for selecting qualified metabonomics samples. The distinguishing method provided by the invention has the characteristics of accuracy and reliability, and simplicity and feasibility, and can provide reference for tobacco leaf metabolic characteristic analysis and related tobacco leaf metabonomics researches.
Description
Technical field
The present invention relates to analytical chemistry field, particularly relate to a kind of differentiate the fresh of Nicotiana tabacum L. metabolism group institute
Whether tobacco sample quality meets the method for discrimination of metabolism group research needs.
Background technology
Plant Metabolome be by investigate the change of plant irriate or disturbance later stage metabolite or its in time
Change, studies a kind of technology of plant.In the flow process of metabolism group edge analysis, the collection of typical sample is the most also
And extremely important step, it is thus achieved that qualified samples, with the metabolism state of the reflection sample of objective, is that metabonomic analysis enters
The basis of row and premise.The collection of fresh tobacco leaves sample, needs cancellation at low temperatures to stop its physiological activity to ensure that it stops
The only metabolism state when sampling, after standing procedure is for sample of plucking a plant, tinfoil wraps up, and is placed in-196 DEG C of liquid nitrogen freezings, uses
Dry powder sample analysis after fresh tobacco leaves sample analysis or lyophilizing.Pluck this section of process before analyzing at sample, need to protect
Card fresh tobacco leaves sample preserves in liquid nitrogen and grinds under the conditions of liquid nitrogen, otherwise can cause the enzymatic browning of Nicotiana tabacum L..Due to cigarette
Leaf sample preservation is improper and that cause quality changes, when metabolite level can not reflect the situation of tobacco sample time of day
There is generation.The tobacco sample quality being badly in need of during Plant Metabolome is studied by a kind of analysis method simple, quick differentiates.
Nicotiana tabacum L. terpenoid is closely related with quality of tabacco fragrance, extract before they fragrance that the most still Nicotiana tabacum L. is important
Matter, and one of their catabolite most important fragrance source that is Nicotiana tabacum L..Meanwhile, terpenoid metabolism is the important secondary of Nicotiana tabacum L.
One of metabolic pathway, volatility terpenoid vying each other between the plant adaptation, plant and plant to environment-stress
With coevolution, the harm of plants against insects, Herbivore search for food and pathogenic microorganism the process such as invasion and attack defence in
Play an important role.In recent years about the separation of terpenoid in Nicotiana tabacum L., identify and detect and achieve certain progress, but not
It has been proposed that the terpenoid content in fresh tobacco leaves or content ratio are used for metabolism group fresh tobacco leaves sample quality
Differentiate.
Summary of the invention
It is an object of the invention to provide sentencing of fresh tobacco leaves sample quality in a kind of Nicotiana tabacum L. metabolism group based on terpenoid
Other method.The present invention utilizes in fresh tobacco leaves the ratio of phytol and Squalene content to the fresh tobacco leaves sample in metabolism group research
Quality differentiates, this method is simple to operate quickly, and result is accurately and reliably.
It is an object of the invention to be realized by following technique measures:
In the Nicotiana tabacum L. metabolism group based on terpenoid of the present invention, the method for discrimination of fresh tobacco leaves sample quality is by detecting fresh cigarette
Phytol and Squalene content in leaf, calculate its ratio, uses new in metabolism group research of gas chromatogram tandem mass spectrum GC-MS
In fresh tobacco leaf sample, phytol and Squalene carry out absolute quantification analysis；Concrete preparation method is as follows:
A, weigh 10 ~ 25mg lyophilizing grind tobacco sample, add Extraction solvent normal hexane 0.75 ~ 1.5 mL, ultrasonic under room temperature
Extract 30min, centrifugal, supernatant is transferred in sample bottle, and dries up with nitrogen；Residue after extraction adds 0.6 ~ 1.2 mL
The mixed liquor being made up of by the volume ratio of 4:1 methanol and potassium hydroxide solution, mechanical shaking extraction 30min at 70 DEG C, centrifugal, by supernatant
Liquid is transferred in Eppendorf pipe；Add 0.5 ~ 1.0mL normal hexane, extract；Vortex 6 s, centrifugal 1 min；Shift afterwards
In above-mentioned sample bottle, and dry up with nitrogen；Derivatization: add 50 ~ 100 μ L 20mg/mL methoxamine hydrochlorides-pyridine solution,
40 ~ 60 DEG C are reacted 60 ~ 90 minutes, add 50 ~ 100 μ L N-methyl-N-(trimethyl is silica-based) trifluoroacetyl amine aqueous solutions, 40
React 30 ~ 90 minutes under the conditions of ~ 60 DEG C, enter GC/MS and analyze, use retention time and select ion (SIM) pattern qualitative, detection
The content of terpene substances；
The gas chromatograph parameters of b, GC/MS analysis method: chromatographic column: DB-35MS (30 mm i.d. × 0.25, m × 0.25 μm
d.f. )；Temperature programming: initial temperature 60 DEG C (3 min), the speed of 25 DEG C/min rises to 210 DEG C, the speed liter of 0.8 DEG C/min
To 220 DEG C, the speed of 15 DEG C/min rises to 310 DEG C (13min).Carrier gas: helium；Column flow rate: 1.0 mL/min；Injection port temperature
Degree: 250 DEG C；Sample size 2 L, split sampling, split ratio 10:1；
The mass spectrometry parameters of c, GC/MS analysis method: ionization voltage: 70 ev；Ion source temperature: 250 DEG C；Transmission line temperature: 280
℃；Quota ion is to as follows: farnesol (135 > 107), phytol (123 > 87), deuterated octadecanoid acid (IS) (376 > 136), and α-
Western cypress three enediol (156 > 141), β-Xi Bai three enediol (156 > 141), Squalene (149 > 107), epoxidation zamene (135 >
107), cholesterol (329 > 121), campesterol (255 > 173), stigmasterol (255 > 173), sitosterol (396 > 255), balsam
Alcohol (218 > 203).
It is that the fresh tobacco leaves sample plucked by land for growing field crops is placed in-196 DEG C of liquid nitrogen that tobacco sample is ground in heretofore described lyophilizing
Tank preserves transport, and liquid nitrogen freezing grinds, low-temperature freeze drying, the tobacco slag that-80 DEG C of Refrigerator stores are stand-by.
Heretofore described Extraction solvent is added with 5 ~ 20 μ L deuterated octadecanoid acids of 2mg/mL internal standard.
Phytol in fresh tobacco leaves sample and Squalene are carried out by heretofore described employing gas chromatogram tandem mass spectrum GC-MS
The analytical procedure of absolute quantification analysis is as follows: GC-MS analytical data can phytol in qualitative fresh tobacco leaves and Squalene, by painting
Standard curve processed can carry out absolute quantitation to measured phytol and Squalene, using the ratio of phytol and Squalene content as district
Divide phytol and the ratio of Squalene content in metabolism group qualified samples and the standard of rotten failed test sample, i.e. testing sample
More than 120 for qualified samples, can be used for metabonomic analysis, the ratio of phytol and Squalene content is below 120
Rotten failed test sample, is not useable for metabonomic analysis.
The present invention is through the analysis and research of a large amount of fresh tobacco leaves samples, if it is improper to find that fresh tobacco leaves sample preserves, its
Terpenoid content can change, and the ratio of phytol and Squalene content also can substantially reduce, and this type of sample can not be used
In metabonomic analysis.Therefore can be using the ratio of phytol and Squalene content as a standard in metabolism group research
Fresh tobacco leaves sample quality differentiates.
Beneficial effects of the present invention is as follows:
The present invention utilizes phytol and the ratio of Squalene content and the dependency of sample quality in fresh tobacco leaves sample, to metabolism group
Learn fresh tobacco leaves sample quality differentiate, have the following characteristics that 1 compared to existing technology) simple to operate quickly；2) result is accurate
Reliably；3) amount of samples is few, can be Nicotiana tabacum L. metabolic characteristics analysis and relevant Nicotiana tabacum L. metabolism group research is offered reference.
Detailed description of the invention
The present invention is further described below with reference to embodiment:
Phytol in fresh tobacco leaves sample and Squalene are carried out definitely by heretofore described employing gas chromatogram tandem mass spectrum GC-MS
The analytical procedure of quantitative analysis is as follows: GC-MS analytical data can phytol in qualitative fresh tobacco leaves and Squalene, marked by drafting
Directrix curve can carry out absolute quantitation to measured phytol and Squalene, using the ratio of phytol and Squalene content as distinguishing generation
Thank to the ratio of phytol and Squalene content in group qualified samples and the standard of rotten failed test sample, i.e. testing sample to exist
More than 120 for qualified samples, can be used for metabonomic analysis, the ratio of phytol and Squalene content below 120 for becoming
The failed test sample of matter, is not useable for metabonomic analysis.Hereinafter repeat no more.
Embodiment 1
Use GC-MS method to measure the terpenoid content in fresh tobacco leaves sample, find phytol and the spiny dogfish of brownization sample
Alkene content ratio substantially reduces.
Testing sample includes 6 kinds in province, the fresh cigarettes in 17 places of production such as Fujian, Guizhou, Hunan, Henan, Yunnan
97 parts of leaf sample (each point has repeat 5 ~ 6 biologys).Including 81 parts of qualified samples, (sample is gathering, is transporting and pre-
Processing procedure is all strict controlled in-80 DEG C), 16 parts of brown stain samples are (owing to the reasons such as in storage and transport process liquid nitrogen is not enough are the most complete
Brown stain).
Sample analysis step is as described below:
1) the fresh tobacco leaves sample plucked by land for growing field crops, preserves transport in-196 DEG C of liquid nitrogen containers, and liquid nitrogen freezing grinds, low-temperature freeze drying ,-
80 DEG C of Refrigerator stores are treated follow-up to carry out metabonomic analysis；Weigh 20 ~ 25mg lyophilizing and grind tobacco sample, just add Extraction solvent
The hexane 1.5 mL(deuterated octadecanoid acid of 2mg/mL containing 20 μ L), supersound extraction 30min under room temperature, centrifugal, supernatant is turned
Move on in sample bottle, and dry up with nitrogen；Residue after extraction adds 1.2mL and is pressed the body of 4:1 by methanol and potassium hydroxide solution
The long-pending mixed liquor than composition, mechanical shaking extraction 30min at 70 DEG C, centrifugal, supernatant is transferred in Eppendorf pipe, add 1mL
Normal hexane, extracts；Vortex 6 s, transfers in above-mentioned sample bottle after centrifugal 1 min, and dries up with nitrogen；Derivatization: add
Entering 50 ~ 100 μ L 20mg/mL methoxamine hydrochlorides-pyridine solution, 40 ~ 60 DEG C are reacted more than 60 minutes, add 50 ~ 100 μ L
N-methyl-N-(trimethyl is silica-based) trifluoroacetyl amine aqueous solution, 40 ~ 60 DEG C are reacted more than 30 minutes, enter GC/MS and analyze, use and protect
Stay the time and select ion (SIM) pattern qualitative, the content of detection terpene substances；
2) gas chromatograph parameters of GC/MS analysis method: chromatographic column: DB-35MS (30 mm i.d. × 0.25, m × 0.25 μm
d.f. )；Temperature programming: initial temperature 60 DEG C (3 min), the speed of 25 DEG C/min rises to 210 DEG C, the speed liter of 0.8 DEG C/min
To 220 DEG C, the speed of 15 DEG C/min rises to 310 DEG C (13min).Carrier gas: helium；Column flow rate: 1.0 mL/min；Injection port temperature
Degree: 250 DEG C；Sample size 2 L, split sampling, split ratio 10:1.
3) mass spectrometry parameters of GC/MS analysis method: ionization voltage: 70 ev；Ion source temperature: 250 DEG C；Transmission line temperature
Degree: 280 DEG C；Quota ion is to as follows: farnesol (135 > 107), phytol (123 > 87), deuterated octadecanoid acid (IS) (376 >
136), α-Xi Bai three enediol (156 > 141), β-Xi Bai three enediol (156 > 141), Squalene (149 > 107), epoxidation shark
Alkene (135 > 107), cholesterol (329 > 121), campesterol (255 > 173), stigmasterol (255 > 173), sitosterol (396 >
255), Amyrin (218 > 203).
Depict the standard curve of phytol (0.12-29.6 μ g/mL) and Squalene (0.31-12.32 μ g/mL).With mesh
Mark thing peak area is vertical coordinate Y with the ratio of internal standard substance peak area (deuterated octadecanoid acid), mass concentration X(μ g/mL) it is horizontal seat
Mark, calculates its standard curve and correlation coefficient, and measurement result is as follows: Y(phytol)=231.8X, Y(Squalene)=860.4X-
323.5, correlation coefficient is respectively 0.995 and 0.999.
Obtain the absolute content of 11 kinds of terpenoids such as phytol in 97 parts of fresh tobacco leaves under these conditions, find to plant
Alcohol is changed significantly with the ratio of Squalene content, and detailed data is shown in Table 1.
The ratio of tobacco sample information and phytol and Squalene content in table 1 embodiment 1
| Sample number into spectrum | The place of production | Kind | Repeat number | Phytol/Squalene | The most qualified |
| C23Y | Guizhou Bijie | Cloud 97 | 6 | 52.2-99.1 | Defective |
| C01K | Kunming, Yunnan | K326 | 5 | 397.9-635.8 | Qualified |
| C02K | Yunnan Yuxi | K326 | 6 | 200.3-263.5 | Qualified |
| C03C | Qujing of Yunnan | Cloud 87 | 6 | 250.5-367.4 | Qualified |
| C04Y | Honghe, Yunnan | Cloud 97 | 6 | 149.1-267.7 | Qualified |
| C05Y | Pu'er, Yunnan | Cloud 87 | 6 | 176.2-576.6 | Qualified |
| C06Y | Dali | Cloud 87 | 5 | 282.1-399.6 | Qualified |
| C09Y | Southwest Guizhou | Cloud 97 | 6 | 246.8-444.5 | Qualified |
| C12C | Table mountain, Henan | Zhongyan-100 | 6 | 279.3-481.8 | Qualified |
| C13C | Nanyang, henan | Zhongyan-100 | 5 | 151.9-352.7 | Qualified |
| C14C | Henan Zhumadian | Zhongyan-100 | 6 | 179.3-375.6 | Qualified |
| C15K | Shaoguan, Guangdong | K326 | 6 | 280.3-385.7 | Qualified |
| C17K | Chenzhou, Hunan Province | K326 | 6 | 165.4-338.8 | Qualified |
| C25N | Tongren district Guizhou Province | River 3, south | 6 | 174.1-242.5 | Qualified |
| C26Y | Enshi | Cloud 87 | 6 | 145.2-190.2 | Qualified |
| C29K | Zhangjiajie, Hunan | K326 | 5 | 41.7-116.8 | Defective |
| C24B | The south of Guizhou Province, Guizhou | Your cigarette 2 | 5 | 97.9-112.6 | Defective |
Through above experiment, inventor finds that phytol in brownization sample significantly reduces with the ratio of Squalene content.The present invention
Using the ratio of phytol and Squalene content as the standard distinguishing metabolism group normal specimens and rotten sample.I.e. in testing sample
The ratio of phytol and Squalene content more than 120 for normal specimens, can be used for metabonomic analysis, phytol contains with Squalene
The ratio of amount below 120 for rotten sample, be not useable for metabonomic analysis.
Claims (4)
1. the method for discrimination of fresh tobacco leaves sample quality in a Nicotiana tabacum L. metabolism group based on terpenoid, it is characterised in that: pass through
Phytol and Squalene content in detection fresh tobacco leaves, calculate its ratio, uses gas chromatogram tandem mass spectrum GC-MS to metabolism group
In fresh tobacco leaves sample in research, phytol and Squalene carry out absolute quantification analysis；Concrete preparation method is as follows:
Tobacco sample is ground in a, the lyophilizing weighing 10 ~ 25g, adds as normal hexane 0.75 ~ 1.5 mL of Extraction solvent, under room temperature
Supersound extraction 30min, centrifugal, supernatant is transferred in sample bottle, and dries up with nitrogen；Residue after extraction adds 0.6 ~
The mixed liquor that 1.2mL is made up of by the volume ratio of 4:1 methanol and potassium hydroxide solution, mechanical shaking extraction 30min at 70 DEG C, centrifugal,
Supernatant is transferred in Eppendorf pipe；Add 0.5 ~ 1.0mL normal hexane, extract；Vortex 6 s, centrifugal 1 min；It
After transfer in above-mentioned sample bottle, and dry up with nitrogen；Derivatization: add 50 ~ 100 μ L 20mg/mL methoxamine hydrochlorides-pyrrole
Pyridine solution, 40 ~ 60 DEG C are reacted 60 ~ 90 minutes, add 50 ~ 100 μ L N-methyl-N-(trimethyl is silica-based) trifluoroacetamides molten
Liquid, reacts 30 ~ 90 minutes under the conditions of 40 ~ 60 DEG C, enters GC/MS and analyzes, and uses retention time and selects ion (SIM) pattern fixed
Property, the content of detection terpene substances；
The gas chromatograph parameters of b, GC/MS analysis method: chromatographic column: DB-35MS (30 mm i.d. × 0.25, m × 0.25 μm
d.f. )；Temperature programming: initial temperature 60 DEG C (3 min), the speed of 25 DEG C/min rises to 210 DEG C, the speed liter of 0.8 DEG C/min
To 220 DEG C, the speed of 15 DEG C/min rises to 310 DEG C (13min)；Carrier gas: helium；Column flow rate: 1.0 mL/min；Injection port temperature
Degree: 250 DEG C；Sample size 2 L, split sampling, split ratio 10:1；
The mass spectrometry parameters of c, GC/MS analysis method: ionization voltage: 70 ev；Ion source temperature: 250 DEG C；Transmission line temperature: 280
℃；Quota ion is to as follows: farnesol (135 > 107), phytol (123 > 87), deuterated octadecanoid acid (IS) (376 > 136), and α-
Western cypress three enediol (156 > 141), β-Xi Bai three enediol (156 > 141), Squalene (149 > 107), epoxidation zamene (135 >
107), cholesterol (329 > 121), campesterol (255 > 173), stigmasterol (255 > 173), sitosterol (396 > 255), balsam
Alcohol (218 > 203).
Method of discrimination the most according to claim 1, it is characterised in that: described employing gas chromatogram tandem mass spectrum GC-MS
The analytical procedure that phytol in fresh tobacco leaves sample and Squalene carry out absolute quantification analysis is as follows: GC-MS analytical data can be determined
Property fresh tobacco leaves in phytol and Squalene, by draw standard curve measured phytol and Squalene can be carried out the most fixed
Amount, using the ratio of phytol and Squalene content as distinguishing metabolism group qualified samples and the standard of rotten failed test sample,
I.e. in testing sample the ratio of phytol and Squalene content more than 120 for qualified samples, can be used for metabonomic analysis, plant
The ratio of alcohol and Squalene content below 120 for rotten failed test sample, be not useable for metabonomic analysis.
Method of discrimination the most according to claim 1, it is characterised in that: described Extraction solvent is added with 5 ~ 20 μ L 2mg/
The deuterated octadecanoid acid of mL internal standard.
Method of discrimination the most according to claim 1, it is characterised in that: described lyophilizing grinds tobacco sample for be plucked by land for growing field crops
Fresh tobacco leaves sample be placed in-196 DEG C of liquid nitrogen containers and preserve transport, liquid nitrogen freezing grinds, and low-temperature freeze drying ,-80 DEG C of Refrigerator stores are treated
Tobacco slag.
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| CN108828097A (en) * | 2018-07-24 | 2018-11-16 | 北京红太阳药业有限公司 | Method that is a kind of while measuring three kinds of liposoluble constituent contents in red seven preparations |
| CN109001306A (en) * | 2018-06-01 | 2018-12-14 | 南昌大学 | The prediction technique of squalene and sterol index in a kind of tea oil |
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