CN105548404B - It is a kind of that Huoshan rice dry measure used in former times and the method for huoshan dendrobium candidum kind are differentiated based on metabolism group - Google Patents
It is a kind of that Huoshan rice dry measure used in former times and the method for huoshan dendrobium candidum kind are differentiated based on metabolism group Download PDFInfo
- Publication number
- CN105548404B CN105548404B CN201610029560.9A CN201610029560A CN105548404B CN 105548404 B CN105548404 B CN 105548404B CN 201610029560 A CN201610029560 A CN 201610029560A CN 105548404 B CN105548404 B CN 105548404B
- Authority
- CN
- China
- Prior art keywords
- stem
- noble dendrobium
- huoshan
- sample
- metabolism group
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 230000004060 metabolic process Effects 0.000 title claims abstract description 34
- 241000026010 Dendrobium candidum Species 0.000 title claims abstract description 30
- 238000000034 method Methods 0.000 title claims abstract description 28
- 235000007164 Oryza sativa Nutrition 0.000 title claims abstract description 25
- 235000009566 rice Nutrition 0.000 title claims abstract description 25
- 240000007594 Oryza sativa Species 0.000 title 1
- 240000004638 Dendrobium nobile Species 0.000 claims abstract description 59
- 241000209094 Oryza Species 0.000 claims abstract description 24
- 238000004458 analytical method Methods 0.000 claims abstract description 13
- 239000002207 metabolite Substances 0.000 claims abstract description 13
- 238000001514 detection method Methods 0.000 claims abstract description 8
- 238000012545 processing Methods 0.000 claims abstract description 7
- 238000002360 preparation method Methods 0.000 claims abstract description 5
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 51
- 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
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- 230000009514 concussion Effects 0.000 claims description 9
- 239000000463 material Substances 0.000 claims description 8
- 229910052757 nitrogen Inorganic materials 0.000 claims description 8
- 238000001212 derivatisation Methods 0.000 claims description 6
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 claims description 6
- 150000002500 ions Chemical class 0.000 claims description 6
- 238000005070 sampling Methods 0.000 claims description 6
- 238000010792 warming Methods 0.000 claims description 6
- 239000000284 extract Substances 0.000 claims description 5
- 238000000605 extraction Methods 0.000 claims description 5
- 239000007788 liquid Substances 0.000 claims description 5
- 238000004949 mass spectrometry Methods 0.000 claims description 5
- 238000000491 multivariate analysis Methods 0.000 claims description 5
- IJOOHPMOJXWVHK-UHFFFAOYSA-N chlorotrimethylsilane Chemical compound C[Si](C)(C)Cl IJOOHPMOJXWVHK-UHFFFAOYSA-N 0.000 claims description 4
- 230000014759 maintenance of location Effects 0.000 claims description 4
- JVWLUVNSQYXYBE-UHFFFAOYSA-N Ribitol Natural products OCC(C)C(O)C(O)CO JVWLUVNSQYXYBE-UHFFFAOYSA-N 0.000 claims description 3
- 239000012159 carrier gas Substances 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
- DTOIYLIABDDGQS-UHFFFAOYSA-N hydroxylamine;pyridine;hydrochloride Chemical compound Cl.ON.C1=CC=NC=C1 DTOIYLIABDDGQS-UHFFFAOYSA-N 0.000 claims description 3
- 230000008676 import Effects 0.000 claims description 3
- 238000011068 loading method Methods 0.000 claims description 3
- HEBKCHPVOIAQTA-UHFFFAOYSA-N meso ribitol Natural products OCC(O)C(O)C(O)CO HEBKCHPVOIAQTA-UHFFFAOYSA-N 0.000 claims description 3
- 239000012188 paraffin wax Substances 0.000 claims description 3
- 238000000513 principal component analysis Methods 0.000 claims description 3
- 238000012372 quality testing Methods 0.000 claims description 3
- 239000010453 quartz Substances 0.000 claims description 3
- HEBKCHPVOIAQTA-ZXFHETKHSA-N ribitol Chemical compound OC[C@H](O)[C@H](O)[C@H](O)CO HEBKCHPVOIAQTA-ZXFHETKHSA-N 0.000 claims description 3
- 238000012216 screening Methods 0.000 claims description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 3
- 238000012360 testing method Methods 0.000 claims description 3
- 241000196324 Embryophyta Species 0.000 claims description 2
- XCOBLONWWXQEBS-KPKJPENVSA-N N,O-bis(trimethylsilyl)trifluoroacetamide Chemical compound C[Si](C)(C)O\C(C(F)(F)F)=N\[Si](C)(C)C XCOBLONWWXQEBS-KPKJPENVSA-N 0.000 claims description 2
- 235000000884 Oryzopsis hymenoides Nutrition 0.000 claims description 2
- 244000194445 Oryzopsis hymenoides Species 0.000 claims description 2
- 238000005119 centrifugation Methods 0.000 claims description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 claims 2
- 150000001875 compounds Chemical class 0.000 claims 1
- 229910052742 iron Inorganic materials 0.000 claims 1
- 238000013441 quality evaluation Methods 0.000 abstract description 3
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 12
- WTDHULULXKLSOZ-UHFFFAOYSA-N Hydroxylamine hydrochloride Chemical class Cl.ON WTDHULULXKLSOZ-UHFFFAOYSA-N 0.000 description 11
- 238000002474 experimental method Methods 0.000 description 11
- 239000003153 chemical reaction reagent Substances 0.000 description 8
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 6
- 239000003814 drug Substances 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- 238000010586 diagram Methods 0.000 description 3
- 239000012071 phase Substances 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 241001523681 Dendrobium Species 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 239000007789 gas 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 class 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
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 description 1
- JPIJQSOTBSSVTP-STHAYSLISA-N L-threonic acid Chemical class OC[C@H](O)[C@@H](O)C(O)=O JPIJQSOTBSSVTP-STHAYSLISA-N 0.000 description 1
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-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
- 239000011149 active material 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
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 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
- 239000008346 aqueous phase Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000012850 discrimination method Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000004438 eyesight Effects 0.000 description 1
- 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
- 238000007689 inspection Methods 0.000 description 1
- 238000011017 operating method Methods 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 238000012913 prioritisation Methods 0.000 description 1
- 238000003672 processing method Methods 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 230000003014 reinforcing effect Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 239000004575 stone Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
Classifications
-
- 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
-
- 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
-
- 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
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
- Medicines Containing Plant Substances (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a kind of method for differentiating Huoshan rice dry measure used in former times and huoshan dendrobium candidum kind based on metabolism group, specific steps include：(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 metabolite；(4) processing and analysis of metabolism group data.The advantage of the invention is that：The substantial amounts of endogenous metabolism thing information of the stem of noble dendrobium is obtained using metabolism group method, and therefrom filter out the characteristic metabolite for differentiating Huoshan rice dry measure used in former times and huoshan dendrobium candidum, and then both stem of noble dendrobium kinds are distinguished on the whole, technical method is advanced, experimental result is reliable, and the cultivar identification and quality evaluation for the stem of noble dendrobium provide a kind of excellent process.
Description
Technical field
Detection field is analyzed the present invention relates to Chinese medicine, is related in particular to a kind of based on metabolism group discriminating Huoshan rice dry measure used in former times
With the method for huoshan dendrobium candidum kind.
Background technology
The stem of noble dendrobium is a kind of conventional rare traditional Chinese medicine, with nourishing Yin and clearing heat, reinforcing stomach reg fluid, voiceless sound improving eyesight, anti-inflammatory analgetic etc.
Effect.Studies on chemical constitutents from plants of Dendrobium Sw is complicated, contains polysaccharide, alkaloid, luxuriant and rich with fragrance class, bibenzyl, amino acid and some other medicine
Use active component.But because Dendrobium species is abundant, form similarity is high, cause market stem of noble dendrobium source complicated, adulterate, with
False purious artefact often has generation, and the clinical efficacy and drug safety of the stem of noble dendrobium are caused to have a strong impact on.
The discrimination method of the classical Chinese medicine stem of noble dendrobium has Ji Yuan discriminatings, character discriminating, microscopical characters and physics and chemistry discriminating etc..These mirror
Determine the heredity phenotype that feature is substantially organism, influenceed by factors such as heredity, environmental condition, Processing methods, had
There is very big variability and randomness, unavoidably in the presence of certain subjectivity and experience dependence, the reliability band to qualification result
Carry out certain influence.Therefore, it is badly in need of a kind of fast and effectively analysis method, discriminating and quality evaluation for stem of noble dendrobium kind.
Metabolism group is the new subject that 1990s mid-term grows up, and is one of systems biology
Point, can all low molecule amount metabolites be carried out qualitative and quantitatively ground in a certain specific physiological periods to a certain biological or cell
Study carefully.Metabolism group research is able to detect that the change of main active material and some mesostates, and not only reflection is planted
The difference of thing genotype, and reflect the effect of environmental factor.As can be seen here, it is possible to use metabonomic technology carries out stone
The discriminating and quality evaluation of dry measure used in former times kind.
The research that metabolism group method method is currently based on to identify different stem of noble dendrobium kinds has no report, is based especially on GC-
The metabolism group research method of MS technical appraisement difference stem of noble dendrobium kind is even more to have never heard of.
The content of the invention
In order to solve the technical problem that above-mentioned background technology is proposed, the present invention is intended to provide a kind of differentiated based on metabolism group
Huoshan rice dry measure used in former times and the method for huoshan dendrobium candidum kind.
In order to achieve the above object, the present invention is adopted the following technical scheme that：
It is a kind of that Huoshan rice dry measure used in former times and the method for huoshan dendrobium candidum kind are differentiated 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 metabolite；
(4) processing and analysis of metabolism group data.
As a kind of preferred scheme, the collection of step (1) stem of noble dendrobium sample of the present invention includes Huoshan rice dry measure used in former times and Huoshan
Put and preserved under -70 DEG C of ultralow temperature after the collection of dendrobium candidum sample, the sample liquid nitrogen flash freezer.
As a kind of prioritization scheme of the embodiment of the present invention, the process of described step (2) includes stem of noble dendrobium sample metabolin
Extraction and derivatization, comprise the following steps that：
A) extraction of stem of noble dendrobium sample metabolin：Stem of noble dendrobium sample is milled to by powdered, Mei Zhongshi using the method for liquid nitrogen grinding
Dry measure used in former times sample takes six repetitions.Then 100~150mg stem of noble dendrobium samples are weighed to be placed in 10mL centrifuge tubes, add 1.5mL be cooled in advance-
20 DEG C of methanol, is vortexed after concussion 1min, the ribitol (0.2mg/mL is dissolved with methanol) for adding 60 μ L is used as methanol/water phase
Internal standard, then be vortexed concussion 30s, then seal the centrifugation mouth of pipe with paraffin wax film, the ultrasonically treated 30min under the conditions of 40~45 DEG C,
8000rpm/min centrifuges 15~20min, takes 1mL upper solutions to be placed in 1.5mL centrifuge tubes, obtains stem of noble dendrobium sample metabolin
Methanol/water mutually extracts solution.
B) derivatization of stem of noble dendrobium sample metabolin：The methanol/water for drying up stem of noble dendrobium sample metabolin with nitrogen mutually extracts solution,
60 μ L hydroxylamine hydrochlorides pyridine solutions (20mg/mL, hydroxylamine hydrochloride is dissolved in pyridine) are added, 40 DEG C of warm bath after the concussion 30s that is vortexed
120min.60 μ LBSTFA (containing volume fraction 1%TMCS) are subsequently added, seals and reacts 120min at rearmounted 37 DEG C, are produced described
Stem of noble dendrobium metabolism group sample.
The specific testing conditions of step (3) of the present invention are：
Chromatographic condition：DB-5 quartz capillary columns (30m × 0.25 μm of 250 μ m)；Sampling volume is 1 μ L；Split sampling,
Split ratio is 20：1；280 DEG C of injector temperature；Carrier gas is helium, flow velocity 1mL/min；Temperature programming：Initial temperature is 100 DEG C,
2min is kept, 260 DEG C are warming up to 4 DEG C/min, 1min is kept, then 280 DEG C are warming up to 20 DEG C/min, 3min is kept；
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/z 40-600.
The detailed process of step (4) of the present invention is：
The Huoshan rice dry measure used in former times of acquisition is opened with huoshan dendrobium candidum GC-MS initial data with Agilent MSD work stations, and
CDF formatted files are converted into, R2.7.2 softwares loading XCMS program bags is then introduced into and carries out the pretreatment of data (including when retaining
Between calibration, filter hot-tempered and peak match), obtain material retention time and peak area data file.Data are carried out with internal standard substance
Normalized, data import simca-p11.5 softwares and carry out multivariate statistics analysis after normalized, choose PCA and
OPLS-DA models carry out principal component analysis and otherness metabolin screening (VIP>1 and p<0.05).To the otherness obtained
Metabolin is identified using NIST11 databases, that is, obtains the characteristic generation for identifying Huoshan rice dry measure used in former times and huoshan dendrobium candidum
Thank to product.
Compared with the prior art, the present invention has the beneficial effect that：
(1) present invention differentiates stem of noble dendrobium kind using metabolism group method first, is based especially on GC-MS technology platforms.
Meanwhile, the preparation method to stem of noble dendrobium metabolism group sample is optimized, and a large amount of endogenous of the stem of noble dendrobium can be detected to greatest extent
Metabolite, it is to avoid the one-sidedness of traditional analysis means, improves the accuracy of result, the discriminating for stem of noble dendrobium kind is provided
New method.
(2) Dendrobidium huoshanness of the invention by acquisition and huoshan dendrobium candidum GC-MS initial data and multivariate statistics method
It is combined, obtains the characteristic metabolite for differentiating both stem of noble dendrobium kinds, is the identification and quality control of stem of noble dendrobium kind
There is provided scientific basis.
Brief description of the drawings
Fig. 1 is annual Huoshan rice dry measure used in former times and huoshan dendrobium candidum PCA shot charts (A) and load diagram (B) in embodiment 1, its
In (Fe) represent huoshan dendrobium candidum, (M) represent 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 charts (A) and load diagram (B) in embodiment 1, its
In (Fe) represent huoshan dendrobium candidum, (M) represent Huoshan rice dry measure used in former times.
Fig. 3 is the TIC spectrograms that battery of tests is obtained in embodiment 2.
Fig. 4 is the TIC spectrograms that second group of experiment is obtained in embodiment 2.
Fig. 5 is the TIC spectrograms that the 3rd group of experiment is obtained in embodiment 2.
Fig. 6 is the TIC spectrograms that the 4th group of experiment is obtained in embodiment 2.
Embodiment
The invention will be further described with reference to the accompanying drawings and examples：
This part carries out general description to the material and test method that are arrived used in present invention experiment.Although being
It is it is known in the art that still the present invention makees as far as possible herein to realize many materials used in the object of the invention and operating method
It is described in detail.It will be apparent to those skilled in the art that hereinafter, if not specified, material therefor of the present invention, equipment and operation
Method is well known in the art.
Embodiment 1
It is a kind of that Huoshan rice dry measure used in former times and the method for huoshan dendrobium candidum kind are differentiated based on metabolism group, comprise the following steps：
1. instrument and reagent
1.1 instrument：Agilent 7890A-5975C gas phases-chromatographic mass spectrometry instrument.
1.2 reagent：Huoshan rice dry measure used in former times and huoshan dendrobium candidum cultivation seedling are provided by Agricultural University Of Anhui's Life Science College, first
Alcohol, pyridine are chromatographic grade, and other reagents are pure to analyze.
2. the preparation of stem of noble dendrobium metabolism group sample：
The extraction of 2.1 stem of noble dendrobium sample metabolins
Annual Huoshan rice dry measure used in former times and huoshan dendrobium candidum cultivation seedling leaf are taken as experiment material, will with the method for liquid nitrogen grinding
It is milled to powdered, and every kind of stem of noble dendrobium sample takes six repetitions.Then 100~150mg stem of noble dendrobium samples are weighed and are placed in 10mL centrifuge tubes
In, the methanol that 1.5mL is cooled to -20 DEG C in advance is added, is vortexed after concussion 1min, adds 60 μ L ribitol (ribose determining alcohol
0.2mg/mL, is dissolved with methanol) as the internal standard of methanol/water phase, then the concussion 30s that is vortexed, then seal centrifuge tube with paraffin wax film
Mouthful, the ultrasonically treated 30min under the conditions of 40~45 DEG C, 8000rpm/min centrifuges 15~20min, takes 1mL upper solutions to be placed in
In 1.5mL centrifuge tubes, the methanol/water for obtaining stem of noble dendrobium sample metabolin mutually extracts solution.
The derivatization of 2.2 stem of noble dendrobium sample metabolins
The methanol/water for drying up stem of noble dendrobium sample metabolin with nitrogen mutually extracts solution, adds 60 μ L hydroxylamine hydrochloride pyridine solutions
40 DEG C of warm bath 120min after (hydroxylamine hydrochloride is dissolved in pyridine, hydroxylamine hydrochloride concentration 20mg/mL), the concussion 30s that is vortexed.It is subsequently added
60 μ LBSTFA (contain volume fraction 1%TMCS), seal and react 120min at rearmounted 37 DEG C, produce the stem of noble dendrobium metabolism group sample
Product.
3. the detection of stem of noble dendrobium sample metabolite
Chromatographic condition：DB-5 quartz capillary columns (30m × 0.25 μm of 250 μ m)；Sampling volume is 1 μ L；Split sampling,
Split ratio is 20：1；280 DEG C of injector temperature；Carrier gas is helium, flow velocity 1mL/min；Temperature programming：Initial temperature is 100 DEG C,
2min is kept, 260 DEG C are warming up to 4 DEG C/min, 1min is kept, then 280 DEG C are warming up to 20 DEG C/min, 3min is kept；
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/z 40-600.
4. the processing and analysis of metabolism group data
The GC-MS initial data Agilent MSD work stations that step 3 is obtained are opened, and are converted into CDF forms text
Part, be then introduced into R2.7.2 softwares loading XCMS program bags carry out data pretreatment (including retention time calibration, filter it is hot-tempered and
Peak match), obtain the retention time and peak area data file of material.The normalized of data is carried out with internal standard substance, is returned
Data import simca-p11.5 softwares and carry out multivariate statistics analysis after one change processing, choose PCA and OPLS-DA models and carry out
Principal component analysis and otherness metabolin screening (VIP>1 and p<0.05).The otherness metabolin obtained is utilized
NIST11 databases are identified, that is, obtain the characteristic metabolite for identifying Huoshan rice dry measure used in former times and huoshan dendrobium candidum.By
PCA and OPLS-DA shot charts (Figure 1A and Fig. 2A) are understood, on the score value figure of the first and second principal components, Huoshan rice dry measure used in former times and suddenly
Obvious separation trend is presented in mountain dendrobium candidum, illustrates that both stems of noble dendrobium have obvious difference on Metabolite, can be with
Different stem of noble dendrobium kinds are distinguished using metabonomic technology.Further use PCA and OPLS-DA load diagrams (Figure 1B and Fig. 2 B), sieve
Select VIP>1 component (table 1) simultaneously combines statistics t inspections, wherein there is significant difference (p<0.05) 6 kinds of materials are：Sucrose,
D- (+)-mannose, D- (-)-fructose, 1- cyclohexene -1- carboxylic acids, D- (+)-glucose and L- threonic acids, you can as identification suddenly
The characteristic metabolite of mountain rice dry measure used in former times and huoshan dendrobium candidum.
Certain step 4 of the present invention only provides processing and the analysis method of a kind of metabolism group data, those skilled in the art
Huoshan rice dry measure used in former times and the characteristic metabolite of huoshan dendrobium candidum can also be obtained by other existing known methods.
The methanol aqueous phase VIP components of the Huoshan of table 1 rice dry measure used in former times and the annual leaf of huoshan dendrobium candidum
Embodiment 2
The influence experiment that different derivative reaction conditions are detected to stem of noble dendrobium metabolism group
1. instrument and reagent
1.1 instrument：Agilent 7890A-5975C gas phases-chromatographic mass spectrometry instrument.
1.2 reagent：Huoshan dendrobium candidum cultivation seedling is provided by Agricultural University Of Anhui's Life Science College, and methanol, pyridine are
Chromatographic grade, other reagents are pure to analyze.
2. the influence that different derivative reaction conditions are detected to stem of noble dendrobium metabolism group
Experiment is divided into four groups, and four groups of uses are that huoshan dendrobium candidum one is made according to the method close with embodiment 1
Year leave methanol/water phase metabolism group sample, the method for four groups of experiments only difference is that derivatization is anti-with embodiment 1
The difference of condition is answered, the derivative reaction condition of four groups of experiments of the present embodiment is as shown in table 2 below.
The derivative reaction condition of 2 four groups of experiments of table
For hydroxylamine hydrochloride pyridine solution, (hydroxylamine hydrochloride is dissolved in pyridine oximate reagent in table 2, hydroxylamine hydrochloride concentration
20mg/mL)；Silylating reagent is BSTFA (containing volume fraction 1%TMCS)；
The present embodiment is to four groups of experiments using GC-MS detection methods same as Example 1 to stem of noble dendrobium metabolism group sample
Product are detected.
As a result referring to Fig. 3 to Fig. 6, different derivative reaction conditions is to suddenly it can be seen from the TIC spectrograms obtained by detection
The Detection results influence of mountain dendrobium candidum metabolin is larger, wherein the number difference at first group of metabolin peak to the 4th group of acquisition
For 153,156,248,481, as a result show, from the 4th group of derivative reaction condition, more metabolin quilts can be made
Detect, it is as overall as possible to carry out metabonomic analysis comprehensively.
Claims (2)
1. a kind of differentiate Huoshan rice dry measure used in former times and the method for huoshan dendrobium candidum kind based on metabolism group, comprise the following steps：
（1）The collection of stem of noble dendrobium sample；
（2）The preparation of stem of noble dendrobium metabolism group sample；
（3）The detection of stem of noble dendrobium sample metabolite；
（4）The processing and analysis of metabolism group data；
The step（2）Process including stem of noble dendrobium sample metabolin extraction and derivatization；
The extraction step of the stem of noble dendrobium sample metabolin is：Stem of noble dendrobium sample is milled to using the method for liquid nitrogen grinding it is powdered, often
Plant stem of noble dendrobium sample and take six repetitions；Then the powdered stem of noble dendrobium samples of 100 ~ 150mg are weighed to be placed in 10mL centrifuge tubes, are added
1.5mL is cooled to -20 DEG C of methanol in advance, is vortexed after concussion 1min, adds 60 μ L ribitol as the internal standard of methanol/water phase, then
Be vortexed concussion 30s, then seals the centrifugation mouth of pipe with paraffin wax film, ultrasonically treated 30min, 8000rpm/ under the conditions of 40~45 DEG C
Min centrifuges 15 ~ 20min, takes 1mL upper solutions to be placed in 1.5mL centrifuge tubes, obtains the methanol/water phase of stem of noble dendrobium sample metabolin
Extract solution；
The derivatization of the stem of noble dendrobium sample metabolin, is concretely comprised the following steps：The methanol/water phase of stem of noble dendrobium sample metabolin is dried up with nitrogen
Solution is extracted, 60 μ L concentration 20mg/mL hydroxylamine hydrochloride pyridine solutions are added, 40 DEG C of warm bath 120min after the concussion 30s that is vortexed；With
60 μ L 1%TMCS containing volume fraction BSTFA is added afterwards, seals and reacts 120min at rearmounted 37 DEG C, produces the stem of noble dendrobium metabolism
Group imitates product；
The step（3）Testing conditions be：Chromatographic condition：DB-5 quartz capillary columns, the μ of capillary column specification 30m × 250
m× 0.25μm；Sampling volume is 1 μ L；Split sampling, split ratio is 20：1；280 DEG C of injector temperature；Carrier gas is helium, stream
Fast 1mL/min；Temperature programming：Initial temperature is 100 DEG C, keeps 2min, and 260 DEG C are warming up to 4 DEG C/min, keeps 1min,
280 DEG C are warming up to 20 DEG C/min again, 3min is kept；
Mass Spectrometry Conditions：Interface temperature and ion source temperature are 250 DEG C；Electron impact ion source；Full scan mode, electron energy
70eV, quality testing scope m/z 40-600；
The step（4）The processing of metabolism group data includes with analysis：By step（3）Huoshan rice dry measure used in former times and the Huoshan iron sheet of acquisition
Stem of noble dendrobium GC-MS initial data is opened with Agilent MSD work stations, and is converted into CDF formatted files, is then introduced into R2.7.2 soft
Part loading XCMS program bags carry out the pretreatment of data, obtain the retention time and peak area data file of material；Use internal standard compound
Matter carries out data after the normalized of data, normalized and imports the progress multivariate statistics analysis of simca-p11.5 softwares；
The particular content of multivariate statistics analysis is：Choose PCA and OPLS-DA models and carry out principal component analysis and difference
Property metabolin screening；The otherness metabolin obtained is identified using NIST11 databases, that is, obtained for identifying suddenly
The characteristic metabolite of mountain rice dry measure used in former times and huoshan dendrobium candidum.
2. a kind of method for differentiating Huoshan rice dry measure used in former times and huoshan dendrobium candidum kind based on metabolism group as claimed in claim 1,
Characterized in that, the step（1）In, the collection of stem of noble dendrobium sample includes the collection of Huoshan rice dry measure used in former times and huoshan dendrobium candidum sample,
Put and preserved under -70 DEG C of ultralow temperature after the sample liquid nitrogen flash freezer.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610029560.9A CN105548404B (en) | 2016-01-15 | 2016-01-15 | It is a kind of that Huoshan rice dry measure used in former times and the method for huoshan dendrobium candidum kind are differentiated based on metabolism group |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610029560.9A CN105548404B (en) | 2016-01-15 | 2016-01-15 | It is a kind of that Huoshan rice dry measure used in former times and the method for huoshan dendrobium candidum kind are differentiated based on metabolism group |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105548404A CN105548404A (en) | 2016-05-04 |
| CN105548404B true CN105548404B (en) | 2017-09-26 |
Family
ID=55827737
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610029560.9A Expired - Fee Related CN105548404B (en) | 2016-01-15 | 2016-01-15 | It is a kind of that Huoshan rice dry measure used in former times and the method for huoshan dendrobium candidum kind are differentiated based on metabolism group |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105548404B (en) |
Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106501437B (en) * | 2016-12-27 | 2017-11-24 | 安徽农业大学 | Method based on UPLC/LTQ Orbitrap Velos MS detection stem of noble dendrobium alkaloid components |
| CN106680400B (en) * | 2017-01-25 | 2019-08-06 | 青岛市食品药品检验研究院 | Static headspace-GC-MS is combined the adulterated method of qualitative, quantitative measurement vegetable oil |
| CN106770796B (en) * | 2017-01-25 | 2019-08-06 | 青岛市食品药品检验研究院 | The method of static headspace-GC-MS combination measurement ready-mixed oil harmonic proportion |
| CN106970161B (en) * | 2017-03-04 | 2019-09-27 | 宁夏医科大学 | A kind of method of the non-target method rapid screening plant otherness metabolin of GC-MS |
| CN110487922B (en) * | 2018-12-17 | 2022-09-27 | 广西中烟工业有限责任公司 | GC-MS analysis method for threonic acid and threonate in tobacco leaves |
| CN111721857A (en) * | 2020-05-27 | 2020-09-29 | 广东省农业科学院果树研究所 | Method for identifying litchi varieties by using extensive targeted metabonomics technology |
| CN111505171B (en) * | 2020-06-08 | 2021-08-27 | 安徽农业大学 | Method for identifying three medicinal polygonatum varieties |
| CN113311076A (en) * | 2021-07-02 | 2021-08-27 | 上海应用技术大学 | Method for rapidly distinguishing different varieties of rice based on aldehyde compounds |
| CN115327013A (en) * | 2022-08-10 | 2022-11-11 | 杭州师范大学 | Traditional Chinese medicine dendrobium species identification method based on two-dimensional ion mobility mass spectrogram |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102478563A (en) * | 2010-11-25 | 2012-05-30 | 中国科学院大连化学物理研究所 | Method for studying metabolic difference of transgenic rice and non-transgenic rice |
| EP2592420A1 (en) * | 2011-11-10 | 2013-05-15 | BIOCRATES Life Sciences AG | Method and use of metabolic compounds for diagnosing stroke |
| CN103983726A (en) * | 2014-05-29 | 2014-08-13 | 西南大学 | Application of glycine or metabolism regulator thereof in improving domestic silkworm silk output and method thereof |
| CN104422742A (en) * | 2013-09-05 | 2015-03-18 | 中国科学院大连化学物理研究所 | Method for discriminating quality of fresh tobacco leaf sample based on research of metabonomics |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006112841A1 (en) * | 2005-04-19 | 2006-10-26 | The Scripps Research Institute | Methods for metabolite profiling |
-
2016
- 2016-01-15 CN CN201610029560.9A patent/CN105548404B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102478563A (en) * | 2010-11-25 | 2012-05-30 | 中国科学院大连化学物理研究所 | Method for studying metabolic difference of transgenic rice and non-transgenic rice |
| EP2592420A1 (en) * | 2011-11-10 | 2013-05-15 | BIOCRATES Life Sciences AG | Method and use of metabolic compounds for diagnosing stroke |
| CN104422742A (en) * | 2013-09-05 | 2015-03-18 | 中国科学院大连化学物理研究所 | Method for discriminating quality of fresh tobacco leaf sample based on research of metabonomics |
| CN103983726A (en) * | 2014-05-29 | 2014-08-13 | 西南大学 | Application of glycine or metabolism regulator thereof in improving domestic silkworm silk output and method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105548404A (en) | 2016-05-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105548404B (en) | It is a kind of that Huoshan rice dry measure used in former times and the method for huoshan dendrobium candidum kind are differentiated based on metabolism group | |
| CN102565231B (en) | Method for determining eight kinds of alkaloid in cured tobacco through gas chromatography-nitrogen chemoluminescence detection method | |
| CN103076411A (en) | Analytical method for determining aromatic constituents in tea | |
| CN101634648A (en) | Method for detecting six trace sweetening agents in white spirit by ultra-high pressure liquid chromatography and time-of-flight mass spectrometry | |
| CN107202836B (en) | Method for rapidly analyzing theanine content in fresh tea sample | |
| CN101487826B (en) | Method for recognizing odor type and/or grade of Chinese spirit | |
| CN103257194A (en) | GC-MS/MS (Gas chromatography - tandem mass spectrometry) method for simultaneously analyzing three polycyclic aromatic hydrocarbons and four tobacco-specific nitrosamines in main stream smoke of cigarettes | |
| CN103499666B (en) | Method for detecting rape honey aroma components by solid phase microextraction | |
| CN109781918A (en) | A kind of gaseous ion migration spectrum discrimination method of the yellow rice wine of difference enterprise production | |
| CN102445505A (en) | Method for quickly determining ethyl carbamate (EC) in alcoholic drink | |
| CN105536746A (en) | Solid-phase micro-extraction capsule and application of same in detection of phenol environmental estrogen in beverage | |
| CN103163241A (en) | Detection and analysis method for trans-2-nonenyl aldehyde potentiality in wort | |
| Rinke | Tradition meets high tech for authenticity testing of fruit juices | |
| Mitsuhashi et al. | Gas chromatographic determination of total iodine in foods | |
| CN112180004A (en) | Method for identifying high-volatility traditional Chinese medicinal materials on site by using surface acoustic wave gas chromatograph | |
| CN108459038B (en) | Nuclear magnetic fingerprint spectrum method for quickly identifying authenticity of poplar type propolis | |
| CN112180003B (en) | Method for identifying volatile Chinese medicinal materials in site by using surface acoustic wave gas chromatograph | |
| CN109696511B (en) | GC-MS-based method for obtaining metabolic difference between transgenic corn and non-transgenic corn | |
| CN110927318A (en) | Method for rapidly identifying thrips by GC-MS | |
| Brookman | Stable isotope dendroclimatology of New Zealand kauri (Agathis australis (D. don) lindl.) and cedar (Libocedrus bidwillii hook. F.) | |
| RU2390771C1 (en) | Method of identifying narcotic and psychoactive substances in biological fluids | |
| CN112180002A (en) | Method for identifying low-volatility traditional Chinese medicinal materials on site by using surface acoustic wave gas chromatograph | |
| CN104535638A (en) | Method for rapidly detecting monosaccharide in tulip tree plant nectar | |
| CN111707746A (en) | Method for detecting different arsenic form contents in food | |
| CN101782568B (en) | Method for comprehensively detecting minute amount of wooden meal in crime scene |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20170926 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |