CN103529156B - Method for extracting and measuring ABA (abscisic acid) of strawberry - Google Patents
Method for extracting and measuring ABA (abscisic acid) of strawberry Download PDFInfo
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- CN103529156B CN103529156B CN201310454156.2A CN201310454156A CN103529156B CN 103529156 B CN103529156 B CN 103529156B CN 201310454156 A CN201310454156 A CN 201310454156A CN 103529156 B CN103529156 B CN 103529156B
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- 238000000034 method Methods 0.000 title claims abstract description 32
- 235000011363 Fragaria x ananassa Nutrition 0.000 title claims abstract description 31
- 235000016623 Fragaria vesca Nutrition 0.000 title claims abstract description 30
- JLIDBLDQVAYHNE-YKALOCIXSA-N (+)-Abscisic acid Chemical compound OC(=O)/C=C(/C)\C=C\[C@@]1(O)C(C)=CC(=O)CC1(C)C JLIDBLDQVAYHNE-YKALOCIXSA-N 0.000 title description 49
- FCRACOPGPMPSHN-UHFFFAOYSA-N desoxyabscisic acid Natural products OC(=O)C=C(C)C=CC1C(C)=CC(=O)CC1(C)C FCRACOPGPMPSHN-UHFFFAOYSA-N 0.000 title description 5
- 240000009088 Fragaria x ananassa Species 0.000 title description 2
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 36
- 241000220223 Fragaria Species 0.000 claims abstract description 29
- 238000000605 extraction Methods 0.000 claims abstract description 23
- 239000007788 liquid Substances 0.000 claims abstract description 23
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims abstract description 22
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims abstract description 15
- 239000002904 solvent Substances 0.000 claims abstract description 13
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims abstract description 11
- 235000019253 formic acid Nutrition 0.000 claims abstract description 11
- 239000012528 membrane Substances 0.000 claims abstract description 5
- 238000000967 suction filtration Methods 0.000 claims abstract description 5
- 238000004704 ultra performance liquid chromatography Methods 0.000 claims description 35
- 239000000284 extract Substances 0.000 claims description 22
- 239000012086 standard solution Substances 0.000 claims description 13
- 239000012452 mother liquor Substances 0.000 claims description 11
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 8
- 238000002360 preparation method Methods 0.000 claims description 7
- 239000013558 reference substance Substances 0.000 claims description 7
- 239000000243 solution Substances 0.000 claims description 7
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 6
- 239000000706 filtrate Substances 0.000 claims description 4
- 230000010354 integration Effects 0.000 claims description 4
- 229910052757 nitrogen Inorganic materials 0.000 claims description 4
- 238000010829 isocratic elution Methods 0.000 claims description 3
- 238000005070 sampling Methods 0.000 claims description 3
- 238000001514 detection method Methods 0.000 claims description 2
- 238000004458 analytical method Methods 0.000 abstract description 5
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 5
- 230000035945 sensitivity Effects 0.000 abstract description 4
- 235000013399 edible fruits Nutrition 0.000 abstract description 3
- 238000005259 measurement Methods 0.000 abstract 4
- 238000004090 dissolution Methods 0.000 abstract 1
- 238000001195 ultra high performance liquid chromatography Methods 0.000 abstract 1
- 238000012360 testing method Methods 0.000 description 9
- 239000012071 phase Substances 0.000 description 7
- 241000196324 Embryophyta Species 0.000 description 5
- 238000011084 recovery Methods 0.000 description 5
- 238000004128 high performance liquid chromatography Methods 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 229940088597 hormone Drugs 0.000 description 2
- 239000005556 hormone Substances 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- 244000012254 Canarium album Species 0.000 description 1
- 235000009103 Canarium album Nutrition 0.000 description 1
- 241000594182 Sarcophaga sigma Species 0.000 description 1
- HLVPIMVSSMJFPS-UHFFFAOYSA-N abscisic acid beta-D-glucopyranosyl ester Natural products O1C(CO)C(O)C(O)C(O)C1OC(=O)C=C(C)C=CC1(O)C(C)=CC(=O)CC1(C)C HLVPIMVSSMJFPS-UHFFFAOYSA-N 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000015709 bud dormancy process Effects 0.000 description 1
- 210000005252 bulbus oculi Anatomy 0.000 description 1
- 230000032823 cell division Effects 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000004345 fruit ripening Effects 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 238000005220 pharmaceutical analysis Methods 0.000 description 1
- 230000008635 plant growth Effects 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000004451 qualitative analysis Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000005070 ripening Effects 0.000 description 1
- 230000014284 seed dormancy process Effects 0.000 description 1
- 230000007226 seed germination Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
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- Sampling And Sample Adjustment (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
Abstract
The invention provides a method for extracting and measuring ABA of a strawberry. The method comprises the steps as follows: a mixed liquid of methyl alcohol, ethyl acetate and formic acid in a volume ratio of 50: 50:1 is taken as an extraction solvent, an ABA component in strawberry fruit pulp can be extracted in shorter time, residue is removed through suction filtration, an obtained filter liquor is decompressed and rotationally steamed to be dried at the temperature of 35 DEG C, and then, a flowing phase of an ultra-high performance liquid chromatography is added for dissolution, a solution is collected, and an extracting solution of strawberry ABA is obtained after filtered by a 0.22 mu m filter membrane; and the obtained extracting solution is injected into an ultra-high performance liquid chromatographic instrument under the set chromatographic condition for measurement and analysis. The method has the characteristics of high extraction speed, measurement accuracy and sensitivity and good repeatability, further, a measurement result is accurate and reliable, and the dosage of a reagent is small, so that the measurement speed is greatly increased, and the solvent consumption is reduced.
Description
[technical field]
The present invention relates to the method for composition extraction and determination in a Plants, relate in particular to the method for ABA extraction and determination in a kind of strawberry.
[background technology]
Abscisic acid (Abscisic acid, ABA) is one of important endogenous hormones of coordinate plant growth, extensively exists in higher plant.Abscisic acid can suppress plant cell division and seed germination, promotes bud and seed dormancy, and promotes the aging of leaf and fruit and come off etc.Strawberry (Fragaria ananassa Duch) belongs to typical non-climacteric type fruit, and its Mature Regulation and post-harvest fresh-keeping difficulty are difficult problems in production, transportation and sales process always.Whether ABA plays to Strawberry ripening process the focus that crucial regulating and controlling effect is current research.The content of ABA in Accurate Determining strawberry, studies its Changing Pattern, and for exploring, fruit maturation and preservation and freshness are significant.
From plant tissue, extracting ABA is it to be carried out to the first step of qualitative and quantitative analysis, at present main extracting method is with the lixiviate of spending the night of 80% methanol solution, but it is long that extraction exists extraction time, generally need 18-23 hour, extraction step is complicated, too much operating influence the shortcomings such as the recovery of ABA.So it is not high that methyl alcohol extraction extracts ABA overall efficiency, therefore improving extraction step is one of important research content of ABA being carried out explication de texte.The method of measuring plant endogenous hormones is numerous, and that relatively more conventional was high-efficient liquid phase chromatogram technique analysis (High performance liquid chromatography, HPLC) in recent years.Ultra Performance Liquid Chromatography (Ultra performance liquid chromatography, UPLC) be a kind of new liquid chromatography technology of releasing on HPLC basis, have than the higher velocity of separation of HPLC and sensitivity, be mainly used at present numerous high-end fields such as protein science, Pharmaceutical Analysis, environmental monitoring.But utilize in the research of UPLC determination and analysis strawberry ABA, have not been reported.
[summary of the invention]
Technical matters to be solved by this invention is to provide the method for ABA extraction and determination in a kind of strawberry, not only has higher extraction efficiency, and has set up the system that adopts Ultra Performance Liquid Chromatography method mensuration ABA.
The present invention solves the problems of the technologies described above by the following technical programs: a kind of method of ABA extraction and determination in strawberry, and the method concrete operations are as follows:
(1) preparation of standard solution: take ABA reference substance, adopt afterwards Chromatographic Pure Methanol to dissolve and constant volume to obtain the mother liquor of standard solution, the concentration of this mother liquor is 2.5mg/mL, then mother liquor is diluted to the standard solution of variable concentrations gradient;
(2) preparation of extract: take strawberry pulp, adopt liquid nitrogen to be ground to Powdered, put afterwards in tool plug conical flask, and add extraction solvent, and every gram of strawberry pulp need add 40mL to extract solvent, concuss extracts after 5min, suction filtration discards residue, gained filtrate is placed in to vacuum rotary steam at 35 ℃ and, to dry, then adds the mobile phase of Ultra Performance Liquid Chromatography to dissolve, collect the extract that solution obtain strawberry ABA, gained extract is crossed to 0.22 μ m filter membrane and obtain liquid to be measured;
Wherein, the component of described extraction solvent and volume ratio thereof are: methyl alcohol: ethyl acetate: formic acid=50:50:1;
(3) Ultra Performance Liquid Chromatography detects:
In chromatographic column, methyl alcohol-acetonitrile-0.1% formic acid that the volume ratio of take is 20:10:70 is mobile phase, and isocratic elution is isolated ABA, and chromatographic condition is as follows:
Detecting instrument: Ultra Performance Liquid Chromatography instrument;
Chromatographic column: ACQUITY UPLC BEH C18 specification is 2.1mm * 100mm, the chromatographic column of 1.7u m;
Mobile phase: methyl alcohol-acetonitrile-0.1% formic acid;
Flow velocity: 0.3ml/min;
Detect wavelength: 265nm;
Sampling volume: 10ul;
Column temperature: 30 ℃;
(4) measure: get respectively the standard solution and the liquid to be measured that prepare, and adopt above-mentioned chromatographic condition to carry out sample detection, record chromatogram, through chromatographic apparatus, carry Software Integration and obtain peak area, calculate the content of ABA in liquid to be measured.
Further, described mother liquor is stored at-20 ℃ stand-by.
In a kind of strawberry of the present invention, the beneficial effect of the method for ABA extraction and determination is: provide a kind of strawberry ABA that is applicable to extract and adopt Ultra Performance Liquid Chromatography method to carry out method for measuring to the ABA extract extracting, and the method has shortened the time of extracting ABA from strawberry pulp greatly, improved extraction efficiency, and under the chromatographic condition of setting applicant, carry out Ultra Performance Liquid Chromatography method and measure ABA extract, there is the precision of mensuration high, sensitivity is strong, reproducible feature, and the result of measuring accurately and reliably, reagent dosage is few, thereby greatly improved finding speed and reduced solvent consumption.
[accompanying drawing explanation]
The invention will be further described in conjunction with the embodiments with reference to the accompanying drawings.
Fig. 1 is the Ultra Performance Liquid Chromatography figure of ABA standard solution in the present invention.
Fig. 2 is the Ultra Performance Liquid Chromatography figure of embodiment mono-liquid to be measured in the present invention.
Fig. 3 is the Ultra Performance Liquid Chromatography figure of embodiment bis-liquid to be measured in the present invention.
[embodiment]
In conjunction with following embodiment, the method for ABA extraction and determination in a kind of strawberry of the present invention is further described.And the instrument and the reagent that in each embodiment, adopt are as follows: Ultra Performance Liquid Chromatography Yi Wei U.S. WATERS ACQUITY UPLC H-Class, comprises quaternary solvent manager, automatic sampler, column oven, PDA detecting device and Empower chromatographic work station; ABA reference substance is U.S. Sigma company product; Second eyeball and methyl alcohol are chromatographically pure, from Fisher company; It is pure that other reagent are analysis.
Embodiment mono-
1, the preparation of standard solution: take 25mg ABA reference substance, add 10mL Chromatographic Pure Methanol to dissolve and constant volume, take and make the standard solution mother liquor that concentration is 2.5mg/mL and (it should be noted that, when the mother liquor being made into is not used immediately, need be positioned at-20 ℃ and be preserved), then mother liquor is diluted to respectively to 7 concentration gradients of 0.1,0.25,0.5,0.75,1,2.5 and 5 μ g/mL;
2, the preparation of extract: take 5g strawberry Chinese olive pulp, adopt liquid nitrogen to be ground to Powdered, put afterwards in tool plug conical flask, and add 200mL to extract solvent (methyl alcohol, ethyl acetate, formic acid mix by 50:50:1 volume ratio), concuss extracts after 5min, suction filtration discards residue, gained filtrate is placed in to vacuum rotary steam at 35 ℃ extremely does, then add the mobile phase of Ultra Performance Liquid Chromatography to dissolve, collect the extract that solution obtain strawberry ABA, gained extract is crossed to 0.22 μ m filter membrane and obtain liquid to be measured;
(3) Ultra Performance Liquid Chromatography detects:
In chromatographic column, methyl alcohol-acetonitrile-0.1% formic acid that the volume ratio of take is 20:10:70 is mobile phase, and isocratic elution is isolated ABA, and chromatographic condition is as follows:
Detecting instrument: Ultra Performance Liquid Chromatography instrument;
Chromatographic column: ACQUITY UPLC BEH C18 specification is 2.1mm * 100mm, the chromatographic column of 1.7u m;
Mobile phase: methyl alcohol-acetonitrile-0.1% formic acid;
Flow velocity: 0.3ml/min;
Detect wavelength: 265nm;
Sampling volume: 10ul;
Column temperature: 30 ℃;
4, measure: get respectively the Ultra Performance Liquid Chromatography instrument that the standard solution for preparing and liquid to be measured injects above-mentioned chromatographic condition and analyze mensuration, obtain the regression equation of ABA reference substance and the Ultra Performance Liquid Chromatography figure of sample, particularly, regression equation: y=98200x-2160, R
2=0.9987 (x---sample introduction concentration, y---peak area); The Ultra Performance Liquid Chromatography figure of standard solution as shown in Figure 1, the Ultra Performance Liquid Chromatography figure of liquid to be measured is (a in Fig. 2 is transversal) as shown in Figure 2, and through chromatographic apparatus, carry Software Integration and obtain peak area, the content that calculates ABA in liquid to be measured is 0.23ug/g.
In order not repeat burden, following examples are only set forth to explanation for difference.
Embodiment bis-
The preparation of extract: take 10g strawberry haw pulp, adopt liquid nitrogen to be ground to Powdered, put afterwards in tool plug conical flask, and add 400mL to extract solvent (methyl alcohol, ethyl acetate, formic acid mix by 50:50:1 volume ratio), concuss extracts after 5min, suction filtration discards residue, gained filtrate is placed in to vacuum rotary steam at 35 ℃ extremely does, then add the mobile phase of Ultra Performance Liquid Chromatography to dissolve, collect the extract that solution obtain strawberry ABA, gained extract is crossed to 0.22 μ m filter membrane and obtain liquid to be measured;
Measure: get liquid to be measured that the present embodiment prepares and inject the Ultra Performance Liquid Chromatography instrument consistent with embodiment mono-chromatographic condition and analyze mensuration, obtain the Ultra Performance Liquid Chromatography figure of liquid to be measured, particularly, the Ultra Performance Liquid Chromatography figure of sample liquid is (b in Fig. 3 is transversal) as shown in Figure 3, and through chromatographic apparatus, carry Software Integration and obtain peak area, the content that calculates ABA in liquid to be measured is 1.46ug/g.
In addition, in order to verify that instrument that the present invention adopts has good precision and the reliability of the inventive method, applicant has carried out following test:
Test an instrument precision test
Take ABA reference substance, and adopt Chromatographic Pure Methanol to dissolve and constant volume be take and made concentration as the ABA standard items of 1 μ g/mL, in the Ultra Performance Liquid Chromatography instrument (chromatographic condition is consistent with embodiment's mono-) adopting in the inventive method, repeat sample introduction 5 times, calculate respectively mean value and the corresponding coefficient of variation of retention time and peak area, also calculate and learn after measured: its retention time mean value is 9.503min, and the coefficient of variation is 0.13%; Peak area mean value is 96040.03, and the coefficient of variation is 0.36%; Thereby the repeatability that instrument is described is good, show that the precision of instrument is good.
Test two average recovery tests
Get need testing solution (the 1 μ g/mL) 1mL of concentration known, add the ABA reference substance solution of variable concentrations, by the chromatographic condition consistent with embodiment mono-, measure, calculate average recovery and the coefficient of variation, the related data of test is as shown in table 1.
Table 1ABA recovery experiment table
As shown in Table 1, the average average recovery of this test is 102.2%, and the coefficient of variation is 4.87%, and test figure shows reliable results and meets analysis requirement, thereby has illustrated that the inventive method has good reliability.
To sum up, the invention provides a kind of strawberry ABA that is applicable to extracts and adopts Ultra Performance Liquid Chromatography method to carry out method for measuring to the ABA extract extracting, and the method has shortened the time of extracting ABA from strawberry pulp greatly, improved extraction efficiency, and under the chromatographic condition of setting applicant, carry out Ultra Performance Liquid Chromatography method and measure ABA extract, there is the feature that precision is high, sensitivity is strong, reproducible of measuring, and the result of measuring accurately and reliably, reagent dosage is few, thereby greatly improved finding speed and reduced solvent consumption.
In addition; it should be noted that; the specific embodiment of the present invention has more than been described; but being familiar with those skilled in the art is to be understood that; our described specific embodiment is illustrative; rather than for the restriction to scope of the present invention, those of ordinary skill in the art are in equivalent modification and the variation done according to spirit of the present invention, all should be encompassed in the scope that claim of the present invention protects.
Claims (2)
1. a method for ABA extraction and determination in strawberry, is characterized in that: the method concrete operations are as follows:
(1) preparation of standard solution: take ABA reference substance, adopt afterwards Chromatographic Pure Methanol to dissolve and constant volume to obtain the mother liquor of standard solution, the concentration of this mother liquor is 2.5mg/mL, then mother liquor is diluted to the standard solution of variable concentrations gradient;
(2) preparation of extract: take strawberry pulp, adopt liquid nitrogen to be ground to Powdered, put afterwards in tool plug conical flask, and add extraction solvent, and every gram of strawberry pulp need add 40mL to extract solvent, concuss extracts after 5min, suction filtration discards residue, gained filtrate is placed in to vacuum rotary steam at 35 ℃ and, to dry, then adds the mobile phase of Ultra Performance Liquid Chromatography to dissolve, collect the extract that solution obtain strawberry ABA, gained extract is crossed to 0.22 μ m filter membrane and obtain liquid to be measured;
Wherein, the component of described extraction solvent and volume ratio thereof are: methyl alcohol: ethyl acetate: formic acid=50:50:1;
(3) Ultra Performance Liquid Chromatography detects:
In chromatographic column, methyl alcohol-acetonitrile-0.1% formic acid that the volume ratio of take is 20:10:70 is mobile phase, and isocratic elution is isolated ABA, and chromatographic condition is as follows:
Detecting instrument: Ultra Performance Liquid Chromatography instrument;
Chromatographic column: ACQUITY UPLC BEH C18 specification is 2.1mm * 100mm, the chromatographic column of 1.7 μ m;
Mobile phase: methyl alcohol-acetonitrile-0.1% formic acid;
Flow velocity: 0.3ml/min;
Detect wavelength: 265nm;
Sampling volume: 10 μ l;
Column temperature: 30 ℃;
(4) measure: get respectively the standard solution and the liquid to be measured that prepare, and adopt above-mentioned chromatographic condition to carry out sample detection, record chromatogram, through chromatographic apparatus, carry Software Integration and obtain peak area, calculate the content of ABA in liquid to be measured.
2. the method for ABA extraction and determination in a kind of strawberry according to claim 1, is characterized in that: described mother liquor is stored at-20 ℃ stand-by.
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| CN201310454156.2A CN103529156B (en) | 2013-09-29 | 2013-09-29 | Method for extracting and measuring ABA (abscisic acid) of strawberry |
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| CN101738441A (en) * | 2010-01-22 | 2010-06-16 | 大连民族学院 | Method for testing plant endogenous hormones |
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Effective date of registration: 20190102 Address after: 353100 Shop No. 05, Dongmen Waicheng East Building Complex Building, Jianou City, Nanping City, Fujian Province (1st floor) Patentee after: Jianou Mengyuan Agricultural Development Co., Ltd. Address before: 350000 Xindianpu Party in Fuzhou suburb of Fujian Province Patentee before: Crop Research Institute, Fujian Academy of Agricultural Sciences |