CN105548404A - Method for identifying varieties of dendrobium huoshanense and dendrobium officinale based on metabonomics - Google Patents
Method for identifying varieties of dendrobium huoshanense and dendrobium officinale based on metabonomics Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 38
- 241001678082 Dendrobium huoshanense Species 0.000 title abstract 3
- 241001076416 Dendrobium tosaense Species 0.000 title abstract 3
- 238000004458 analytical method Methods 0.000 claims abstract description 12
- 240000004638 Dendrobium nobile Species 0.000 claims description 54
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 51
- 230000004060 metabolic process Effects 0.000 claims description 34
- 241000026010 Dendrobium candidum Species 0.000 claims description 31
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- 235000007164 Oryza sativa Nutrition 0.000 claims description 30
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- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 claims description 21
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 16
- JUJWROOIHBZHMG-UHFFFAOYSA-N pyridine Substances C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims description 14
- 238000000513 principal component analysis Methods 0.000 claims description 12
- 238000012360 testing method Methods 0.000 claims description 11
- 230000002503 metabolic effect Effects 0.000 claims description 10
- WTDHULULXKLSOZ-UHFFFAOYSA-N Hydroxylamine hydrochloride Chemical compound Cl.ON WTDHULULXKLSOZ-UHFFFAOYSA-N 0.000 claims description 9
- 230000009514 concussion Effects 0.000 claims description 9
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- 239000000463 material Substances 0.000 claims description 8
- 229910052757 nitrogen Inorganic materials 0.000 claims description 8
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 claims description 7
- 238000001514 detection method Methods 0.000 claims description 6
- 239000000284 extract Substances 0.000 claims description 6
- 150000002500 ions Chemical class 0.000 claims description 6
- 238000005070 sampling Methods 0.000 claims description 6
- 238000001212 derivatisation Methods 0.000 claims description 5
- 238000000605 extraction Methods 0.000 claims description 5
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- 230000014759 maintenance of location Effects 0.000 claims description 5
- 238000004949 mass spectrometry Methods 0.000 claims description 5
- 238000000491 multivariate analysis Methods 0.000 claims description 5
- JVWLUVNSQYXYBE-UHFFFAOYSA-N Ribitol Natural products OCC(C)C(O)C(O)CO JVWLUVNSQYXYBE-UHFFFAOYSA-N 0.000 claims description 4
- HEBKCHPVOIAQTA-UHFFFAOYSA-N meso ribitol Natural products OCC(O)C(O)C(O)CO HEBKCHPVOIAQTA-UHFFFAOYSA-N 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- HEBKCHPVOIAQTA-ZXFHETKHSA-N ribitol Chemical compound OC[C@H](O)[C@H](O)[C@H](O)CO HEBKCHPVOIAQTA-ZXFHETKHSA-N 0.000 claims description 4
- 239000012159 carrier gas Substances 0.000 claims description 3
- 150000001875 compounds Chemical class 0.000 claims description 3
- 238000001035 drying Methods 0.000 claims description 3
- 238000000227 grinding Methods 0.000 claims description 3
- 239000001307 helium Substances 0.000 claims description 3
- 229910052734 helium Inorganic materials 0.000 claims description 3
- SWQJXJOGLNCZEY-UHFFFAOYSA-N helium atom Chemical compound [He] SWQJXJOGLNCZEY-UHFFFAOYSA-N 0.000 claims description 3
- 230000008676 import Effects 0.000 claims description 3
- 239000012188 paraffin wax Substances 0.000 claims description 3
- 238000012372 quality testing Methods 0.000 claims description 3
- 239000010453 quartz Substances 0.000 claims description 3
- 238000012216 screening Methods 0.000 claims description 3
- 238000007789 sealing Methods 0.000 claims description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 3
- 241001523681 Dendrobium Species 0.000 abstract description 12
- 239000002207 metabolite Substances 0.000 abstract description 6
- 238000001303 quality assessment method Methods 0.000 abstract description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 8
- 238000005516 engineering process Methods 0.000 description 5
- 239000003814 drug Substances 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- IJOOHPMOJXWVHK-UHFFFAOYSA-N chlorotrimethylsilane Chemical compound C[Si](C)(C)Cl IJOOHPMOJXWVHK-UHFFFAOYSA-N 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 238000012797 qualification Methods 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 2
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- 239000012071 phase Substances 0.000 description 2
- QWUWMCYKGHVNAV-UHFFFAOYSA-N 1,2-dihydrostilbene Chemical group C=1C=CC=CC=1CCC1=CC=CC=C1 QWUWMCYKGHVNAV-UHFFFAOYSA-N 0.000 description 1
- NMEZJSDUZQOPFE-UHFFFAOYSA-N Cyclohex-1-enecarboxylic acid Chemical compound OC(=O)C1=CCCCC1 NMEZJSDUZQOPFE-UHFFFAOYSA-N 0.000 description 1
- LKDRXBCSQODPBY-VRPWFDPXSA-N D-fructopyranose Chemical compound OCC1(O)OC[C@@H](O)[C@@H](O)[C@@H]1O LKDRXBCSQODPBY-VRPWFDPXSA-N 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- JPIJQSOTBSSVTP-STHAYSLISA-N L-threonic acid Chemical compound OC[C@H](O)[C@@H](O)C(O)=O JPIJQSOTBSSVTP-STHAYSLISA-N 0.000 description 1
- XCOBLONWWXQEBS-KPKJPENVSA-N N,O-bis(trimethylsilyl)trifluoroacetamide Chemical group C[Si](C)(C)O\C(C(F)(F)F)=N\[Si](C)(C)C XCOBLONWWXQEBS-KPKJPENVSA-N 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- GZCGUPFRVQAUEE-SLPGGIOYSA-N aldehydo-D-glucose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O GZCGUPFRVQAUEE-SLPGGIOYSA-N 0.000 description 1
- 229930013930 alkaloid Natural products 0.000 description 1
- 150000003797 alkaloid derivatives Chemical class 0.000 description 1
- WQZGKKKJIJFFOK-PQMKYFCFSA-N alpha-D-mannose Chemical compound OC[C@H]1O[C@H](O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-PQMKYFCFSA-N 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000000202 analgesic effect Effects 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
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- 238000012850 discrimination method Methods 0.000 description 1
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- 239000012530 fluid Substances 0.000 description 1
- 239000003205 fragrance Substances 0.000 description 1
- 229960002737 fructose Drugs 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 238000009863 impact test Methods 0.000 description 1
- 229940056902 l- threonic acid Drugs 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
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- 238000012913 prioritisation Methods 0.000 description 1
- 238000003672 processing method Methods 0.000 description 1
- 238000004451 qualitative analysis Methods 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 230000003014 reinforcing effect Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 238000010998 test method Methods 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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/067—Preparation by reaction, e.g. derivatising the sample
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
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Abstract
The invention discloses a method for identifying the varieties of dendrobium huoshanense and dendrobium officinale based on metabonomics. The method comprises the following specific steps: (1) collecting dendrobium samples; (2) preparing dendrobium metabonomics samples; (3) detecting metabolites of the dendrobium samples; and (4) performing metabonomics data processing and analysis. The method provided by the invention has the following advantages: a lot of endogenous metabolite information of dendrobium is acquired by means of metabonomics; among the endogenous metabolites, the characteristic metabolites for identification of dendrobium huoshanense and dendrobium officinale are selected out, and then such two dendrobium varieties are distinguished from each other on the whole; the technical mode is advanced and the experimental result is reliable; and a good means is provided for dendrobium variety identification and dendrobium quality assessment.
Description
Technical field
The present invention relates to Chinese crude drug and analyze detection field, relate in particular to a kind of method differentiating Huoshan rice dry measure used in former times and Huoshan dendrobium candidum kind based on metabolism group.
Background technology
The stem of noble dendrobium is a kind of conventional rare traditional Chinese medicine, has the effects such as nourishing Yin and clearing heat, reinforcing stomach reg fluid, voiceless sound improving eyesight, anti-inflammatory analgetic.Studies on chemical constitutents from plants of Dendrobium Sw is complicated, containing polysaccharide, alkaloid, luxuriant and rich with fragrance class, bibenzyl, amino acid and some other medicinal active ingredient.But because Dendrobium abundant species, plesiomorphism degree are high, cause stem of noble dendrobium source, market complicated, adulterate, phenomenon of mixing the spurious with the genuine often has generation, the clinical efficacy of the stem of noble dendrobium and drug safety are caused to having a strong impact on.
The discrimination method of the classical Chinese medicine stem of noble dendrobium has Ji Yuan discriminating, proterties discriminating, microscopical characters and physics and chemistry discriminating etc.These identification marks are essentially the heredity phenotype of biosome, be subject to the impact of the factors such as heredity, environmental baseline, Processing methods, there is very large variability and randomness, there is certain subjectivity and experience dependence unavoidably, bring certain impact to the reliability of qualification result.Therefore, one analytical approach is fast and effectively badly in need of, for discriminating and the quality assessment of stem of noble dendrobium kind.
Metabolism group is the new subject of of growing up the mid-90 in 20th century, is a part for systems biology, can carries out quantitative and qualitative analysis research by all low-molecular-weight metabolic products in a certain specific physiological periods to a certain biology or cell.Metabolism group research can detect main active substance and the change of some mesostates, not only reflects the difference of plant genotype, and reflects the effect of environmental factor.As can be seen here, metabonomic technology can be utilized to carry out discriminating and the quality assessment of stem of noble dendrobium kind.
Identify that the research of different stem of noble dendrobium kind has no report based on metabolism group method method at present, the metabolism group research method especially based on the different stem of noble dendrobium kind of GC-MS technical appraisement is had never heard of especially.
Summary of the invention
In order to solve the technical matters that above-mentioned background technology proposes, the present invention aims to provide a kind of method differentiating Huoshan rice dry measure used in former times and Huoshan dendrobium candidum kind based on metabolism group.
In order to achieve the above object, the present invention adopts following technical scheme:
Differentiate a method for Huoshan rice dry measure used in former times and Huoshan dendrobium candidum kind based on metabolism group, comprise the following steps:
(1) collection of stem of noble dendrobium sample;
(2) preparation of stem of noble dendrobium metabolism group sample;
(3) detection of stem of noble dendrobium sample metabolic product;
(4) treatment and analyses of metabolism group data.
As a kind of preferred version, the collection of step of the present invention (1) stem of noble dendrobium sample comprises the collection of Huoshan rice dry measure used in former times and Huoshan dendrobium candidum sample, preserves under putting-70 DEG C of ultralow temperature after described sample liquid nitrogen flash freezer.
As a kind of prioritization scheme of the embodiment of the present invention, the process of described step (2) comprises extraction and the derivatization of stem of noble dendrobium sample metabolin, and concrete steps are as follows:
A) extraction of stem of noble dendrobium sample metabolin: adopt the method for liquid nitrogen grinding by stem of noble dendrobium sample mill to Powdered, six repetitions got by often kind of stem of noble dendrobium sample.Then take 100 ~ 150mg stem of noble dendrobium sample and be placed in 10mL centrifuge tube, add the methyl alcohol that 1.5mL is chilled to-20 DEG C in advance, after vortex concussion 1min, add the ribitol (0.2mg/mL of 60 μ L, dissolve with methyl alcohol) mark as in methanol/water phase, vortex concussion 30s again, then the centrifugal mouth of pipe is sealed with paraffin wax film, ultrasonic process 30min under 40 ~ 45 DEG C of conditions, centrifugal 15 ~ the 20min of 8000rpm/min, get 1mL upper solution and be placed in 1.5mL centrifuge tube, the methanol/water obtaining stem of noble dendrobium sample metabolin extracts solution mutually.
B) derivatization of stem of noble dendrobium sample metabolin: the methanol/water drying up stem of noble dendrobium sample metabolin with nitrogen extracts solution mutually, add 60 μ L oxammonium hydrochloride pyridine solution (20mg/mL, oxammonium hydrochloride is dissolved in pyridine), 40 DEG C of temperature bath 120min after vortex concussion 30s.Add 60 μ LBSTFA (containing volume fraction 1%TMCS) subsequently, at sealing rearmounted 37 DEG C, react 120min, obtain described stem of noble dendrobium metabolism group sample.
The concrete testing conditions of step of the present invention (3) is:
Chromatographic condition: DB-5 quartz capillary column (30m × 250 μm × 0.25 μm); Sampling volume is 1 μ L; Split sampling, split ratio is 20:1; Injector temperature 280 DEG C; Carrier gas is helium, flow velocity 1mL/min; Temperature programme: initial temperature is 100 DEG C, keeps 2min, is warmed up to 260 DEG C with 4 DEG C/min, keeps 1min, then is warmed up to 280 DEG C with 20 DEG C/min, keeps 3min;
Mass Spectrometry Conditions: interface temperature and ion source temperature are 250 DEG C; Electron impact ion source (EI); Full scan mode, electron energy 70eV, quality testing scope m/z40-600.
The detailed process of step of the present invention (4) is:
The Huoshan rice dry measure used in former times of acquisition and Huoshan dendrobium candidum GC-MS raw data AgilentMSD workstation are opened, and convert CDF formatted file to, then import the pre-service (comprise the calibration of retention time, filter hot-tempered and peak match) that R2.7.2 Bootload XCMS routine package carries out data, obtain retention time and the peak area data file of material.The normalized of data is carried out by internal standard compound matter, after normalized, data importing simca-p11.5 software carries out multivariate statistics Epidemiological Analysis, chooses PCA and OPLS-DA model and carries out principal component analysis (PCA) and otherness metabolin screening (VIP>1 and p<0.05).Utilize NIST11 database to identify to obtained otherness metabolin, namely obtain the characteristic metabolic product for the identification of Huoshan rice dry measure used in former times and Huoshan dendrobium candidum.
Compared with the prior art, beneficial effect of the present invention is embodied in:
(1) the present invention adopts metabolism group method to differentiate stem of noble dendrobium kind, especially based on GC-MS technology platform first.Simultaneously, the preparation method of stem of noble dendrobium metabolism group sample is optimized, a large amount of endogenous metabolites of the stem of noble dendrobium can be detected to greatest extent, avoid the one-sidedness of traditional analysis means, improve the accuracy of result, for the discriminating of stem of noble dendrobium kind provides new method.
(2) Dendrobidium huoshanness of acquisition combines with multivariate statistics method with Huoshan dendrobium candidum GC-MS raw data by the present invention, obtain characteristic metabolic product for differentiating these two kinds of stem of noble dendrobium kinds, for the qualification of stem of noble dendrobium kind and quality control provide scientific basis.
Accompanying drawing explanation
Fig. 1 is annual Huoshan rice dry measure used in former times and Huoshan dendrobium candidum PCA shot chart (A) and load diagram (B) in embodiment 1, and wherein (Fe) represents Huoshan dendrobium candidum, and (M) represents Huoshan rice dry measure used in former times.
Fig. 2 is annual Huoshan rice dry measure used in former times and dendrobium candidum OPLS-DA shot chart (A) and load diagram (B) in embodiment 1, and wherein (Fe) represents Huoshan dendrobium candidum, and (M) represents Huoshan rice dry measure used in former times.
Fig. 3 is the TIC spectrogram that in embodiment 2, battery of tests obtains.
Fig. 4 is the TIC spectrogram that in embodiment 2, second group of test obtains.
Fig. 5 is the TIC spectrogram that in embodiment 2, the 3rd group of test obtains.
Fig. 6 is the TIC spectrogram that in embodiment 2, the 4th group of test obtains.
Embodiment
Below in conjunction with drawings and Examples, the invention will be further described:
General description is carried out to the material used in the present invention's experiment and test method in this part.Although for realizing many materials that the object of the invention uses and method of operating is well known in the art, the present invention describes in detail as far as possible at this.It will be apparent to those skilled in the art that hereinafter, if not specified, material therefor of the present invention, equipment and method of operating are well known in the art.
Embodiment 1
Differentiate a method for Huoshan rice dry measure used in former times and Huoshan dendrobium candidum kind based on metabolism group, comprise the following steps:
1. instrument and reagent
1.1 instruments: Agilent7890A-5975C gas phase-chromatographic mass spectrometry instrument.
1.2 reagents: Huoshan rice dry measure used in former times and Huoshan officinal dendrobium stem plantation seedling are provided by Agricultural University Of Anhui's Life Science College, and methyl alcohol, pyridine are chromatographic grade, other reagent is pure for analyzing.
2. the preparation of stem of noble dendrobium metabolism group sample:
The extraction of 2.1 stem of noble dendrobium sample metabolins
Get annual Huoshan rice dry measure used in former times and Huoshan officinal dendrobium stem plantation seedling leaf as experiment material, be milled to Powdered by the method for liquid nitrogen grinding, six repetitions got by often kind of stem of noble dendrobium sample.Then take 100 ~ 150mg stem of noble dendrobium sample and be placed in 10mL centrifuge tube, add the methyl alcohol that 1.5mL is chilled to-20 DEG C in advance, after vortex concussion 1min, add ribitol (the ribitol concentration 0.2mg/mL of 60 μ L, dissolve with methyl alcohol) mark as in methanol/water phase, vortex concussion 30s again, then the centrifugal mouth of pipe is sealed with paraffin wax film, ultrasonic process 30min under 40 ~ 45 DEG C of conditions, centrifugal 15 ~ the 20min of 8000rpm/min, get 1mL upper solution and be placed in 1.5mL centrifuge tube, the methanol/water obtaining stem of noble dendrobium sample metabolin extracts solution mutually.
The derivatization of 2.2 stem of noble dendrobium sample metabolins
The methanol/water drying up stem of noble dendrobium sample metabolin with nitrogen extracts solution mutually, adds 60 μ L oxammonium hydrochloride pyridine solutions (oxammonium hydrochloride is dissolved in pyridine, oxammonium hydrochloride concentration 20mg/mL), 40 DEG C of temperature bath 120min after vortex concussion 30s.Add 60 μ LBSTFA (containing volume fraction 1%TMCS) subsequently, at sealing rearmounted 37 DEG C, react 120min, obtain described stem of noble dendrobium metabolism group sample.
3. the detection of stem of noble dendrobium sample metabolic product
Chromatographic condition: DB-5 quartz capillary column (30m × 250 μm × 0.25 μm); Sampling volume is 1 μ L; Split sampling, split ratio is 20:1; Injector temperature 280 DEG C; Carrier gas is helium, flow velocity 1mL/min; Temperature programme: initial temperature is 100 DEG C, keeps 2min, is warmed up to 260 DEG C with 4 DEG C/min, keeps 1min, then is warmed up to 280 DEG C with 20 DEG C/min, keeps 3min;
Mass Spectrometry Conditions: interface temperature and ion source temperature are 250 DEG C; Electron impact ion source (EI); Full scan mode, electron energy 70eV, quality testing scope m/z40-600.
4. the treatment and analyses of metabolism group data
GC-MS raw data AgilentMSD workstation step 3 obtained is opened, and convert CDF formatted file to, then import the pre-service (comprise the calibration of retention time, filter hot-tempered and peak match) that R2.7.2 Bootload XCMS routine package carries out data, obtain retention time and the peak area data file of material.The normalized of data is carried out by internal standard compound matter, after normalized, data importing simca-p11.5 software carries out multivariate statistics Epidemiological Analysis, chooses PCA and OPLS-DA model and carries out principal component analysis (PCA) and otherness metabolin screening (VIP>1 and p<0.05).Utilize NIST11 database to identify to obtained otherness metabolin, namely obtain the characteristic metabolic product for the identification of Huoshan rice dry measure used in former times and Huoshan dendrobium candidum.From PCA and OPLS-DA shot chart (Figure 1A and Fig. 2 A), on the score value figure of the first and second major components, Huoshan rice dry measure used in former times and Huoshan dendrobium candidum present obvious separation trend, illustrate that these two kinds of stems of noble dendrobium exist obvious difference on Metabolite, metabonomic technology can be utilized to distinguish different stem of noble dendrobium kind.Further utilization PCA and OPLS-DA load diagram (Figure 1B and Fig. 2 B), filter out VIP>1 component (table 1) and check in conjunction with statistics t, wherein there are 6 kinds of materials of significant difference (p<0.05) to be: sucrose, D-(+)-mannose, D-(-)-fructose, 1-cyclohexene-1-carboxylic acid, D-(+)-glucose and L-threonic acid, namely can be used as the characteristic metabolic product of qualification Huoshan rice dry measure used in former times and Huoshan dendrobium candidum.
Certain step 4 of the present invention only provides a kind for the treatment of and analyses method of metabolism group data, and those skilled in the art also can obtain the characteristic metabolic product of Huoshan rice dry measure used in former times and Huoshan dendrobium candidum by other existing known method.
The methyl alcohol aqueous phase VIP component of table 1 Huoshan rice dry measure used in former times and the annual leaf of Huoshan dendrobium candidum
Embodiment 2
The impact test that different derivative reaction condition detects stem of noble dendrobium metabolism group
1. instrument and reagent
1.1 instruments: Agilent7890A-5975C gas phase-chromatographic mass spectrometry instrument.
1.2 reagents: Huoshan officinal dendrobium stem plantation seedling is provided by Agricultural University Of Anhui's Life Science College, methyl alcohol, pyridine are chromatographic grade, and other reagent is pure for analyzing.
2. different derivative reaction condition impact that stem of noble dendrobium metabolism group is detected
Test is divided into four groups, four groups what adopt is all obtain Huoshan dendrobium candidum annual chrysomelid alcohol/aqueous phase metabolism group sample according to the method close with embodiment 1, the method of these four groups tests and the difference of embodiment 1 are only the difference of derivative reaction condition, and the derivative reaction condition of the present embodiment four groups test is as shown in table 2 below.
The derivative reaction condition of four groups, table 2 test
Oximate reagent in table 2 is oxammonium hydrochloride pyridine solution (oxammonium hydrochloride is dissolved in pyridine, oxammonium hydrochloride concentration 20mg/mL); Silylating reagent is BSTFA (containing volume fraction 1%TMCS);
The present embodiment all adopts the GC-MS detection method identical with embodiment 1 to detect stem of noble dendrobium metabolism group sample to four groups of tests.
Result is see Fig. 3 to Fig. 6, as can be seen from the TIC spectrogram detecting gained, the Detection results impact of different derivative reaction conditions on Huoshan dendrobium candidum metabolin is larger, wherein the number at first group to the 4th group metabolin peak obtained is respectively 153,156,248,481, result shows, select the derivative reaction condition of the 4th group, more metabolin can be made to be detected, and entirety carries out metabonomic analysis comprehensively as far as possible.
Claims (8)
1. differentiate a method for Huoshan rice dry measure used in former times and Huoshan dendrobium candidum kind based on metabolism group, comprise the steps:
(1) collection of stem of noble dendrobium sample;
(2) preparation of stem of noble dendrobium metabolism group sample;
(3) detection of stem of noble dendrobium sample metabolic product;
(4) treatment and analyses of metabolism group data.
2. a kind of method differentiating Huoshan rice dry measure used in former times and Huoshan dendrobium candidum kind based on metabolism group as claimed in claim 1, it is characterized in that, in described step (1), the collection of stem of noble dendrobium sample comprises the collection of Huoshan rice dry measure used in former times and Huoshan dendrobium candidum sample, preserves under putting-70 DEG C of ultralow temperature after described sample liquid nitrogen flash freezer.
3. a kind of method differentiating Huoshan rice dry measure used in former times and Huoshan dendrobium candidum kind based on metabolism group as claimed in claim 1, it is characterized in that, the process of described step (2) comprises extraction and the derivatization of stem of noble dendrobium sample metabolin.
4. a kind of method differentiating Huoshan rice dry measure used in former times and Huoshan dendrobium candidum kind and quality based on metabolism group as claimed in claim 3, it is characterized in that, the extraction step of described stem of noble dendrobium sample metabolin is: adopt the method for liquid nitrogen grinding by stem of noble dendrobium sample mill to Powdered, six repetitions got by often kind of stem of noble dendrobium sample; Then take the Powdered stem of noble dendrobium sample of 100 ~ 150mg and be placed in 10mL centrifuge tube, add the methyl alcohol that 1.5mL is chilled to-20 DEG C in advance, after vortex concussion 1min, the ribitol adding 60 μ L is marked as in methanol/water phase, then vortex concussion 30s, then seals the centrifugal mouth of pipe with paraffin wax film, ultrasonic process 30min under 40 ~ 45 DEG C of conditions, centrifugal 15 ~ the 20min of 8000rpm/min, gets 1mL upper solution and is placed in 1.5mL centrifuge tube, and the methanol/water obtaining stem of noble dendrobium sample metabolin extracts solution mutually.
5. a kind of method differentiating Huoshan rice dry measure used in former times and Huoshan dendrobium candidum kind based on metabolism group as claimed in claim 4, it is characterized in that, the derivatization of described stem of noble dendrobium sample metabolin, concrete steps are: the methanol/water drying up stem of noble dendrobium sample metabolin with nitrogen extracts solution mutually, add 60 μ L oxammonium hydrochloride pyridine solutions, 40 DEG C of temperature bath 120min after vortex concussion 30s; Add 60 μ LBSTFA subsequently, at sealing rearmounted 37 DEG C, react 120min, obtain described stem of noble dendrobium metabolism group sample.
6. a kind of method differentiating Huoshan rice dry measure used in former times and Huoshan dendrobium candidum kind based on metabolism group as claimed in claim 1, it is characterized in that, the testing conditions of described step (3) is: chromatographic condition: DB-5 quartz capillary column (30m × 250 μm × 0.25 μm); Sampling volume is 1 μ L; Split sampling, split ratio is 20:1; Injector temperature 280 DEG C; Carrier gas is helium, flow velocity 1mL/min; Temperature programme: initial temperature is 100 DEG C, keeps 2min, is warmed up to 260 DEG C with 4 DEG C/min, keeps 1min, then is warmed up to 280 DEG C with 20 DEG C/min, keeps 3min;
Mass Spectrometry Conditions: interface temperature and ion source temperature are 250 DEG C; Electron impact ion source (EI); Full scan mode, electron energy 70eV, quality testing scope m/z40-600.
7. a kind of method differentiating Huoshan rice dry measure used in former times and Huoshan dendrobium candidum kind based on metabolism group as claimed in claim 1, it is characterized in that, the treatment and analyses of described step (4) metabolism group data comprises: Huoshan rice dry measure used in former times step (3) obtained and Huoshan dendrobium candidum GC-MS raw data AgilentMSD workstation are opened, and convert CDF formatted file to, then import the pre-service that R2.7.2 Bootload XCMS routine package carries out data, obtain retention time and the peak area data file of material.Carry out the normalized of data by internal standard compound matter, after normalized, data importing simca-p11.5 software carries out multivariate statistics Epidemiological Analysis.
8. a kind of method differentiating Huoshan rice dry measure used in former times and Huoshan dendrobium candidum kind based on metabolism group as claimed in claim 7, it is characterized in that, the particular content of described multivariate statistics Epidemiological Analysis is: choose PCA and OPLS-DA model and carry out principal component analysis (PCA) and the screening of otherness metabolin; Utilize NIST11 database to identify to obtained otherness metabolin, namely obtain the characteristic metabolic product for the identification of Huoshan rice dry measure used in former times and Huoshan dendrobium candidum.
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