CN104090049B - A kind of extracting method for oligosaccharides in the rice embryo of HPLC detection - Google Patents
A kind of extracting method for oligosaccharides in the rice embryo of HPLC detection Download PDFInfo
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- CN104090049B CN104090049B CN201410320770.4A CN201410320770A CN104090049B CN 104090049 B CN104090049 B CN 104090049B CN 201410320770 A CN201410320770 A CN 201410320770A CN 104090049 B CN104090049 B CN 104090049B
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Abstract
The invention discloses the extracting method of oligosaccharides in a kind of rice embryo detected for HPLC, comprising: get embryo, add after grinding concentration be 75 ~ 85% ethanolic solution carry out mechanical shaking extraction, mechanical shaking extraction terminates rear centrifugal, obtains the first supernatant and residue; Add in described residue concentration be 75 ~ 85% ethanolic solution carry out ultrasonic extraction, after ultrasonic extraction terminates, centrifugal, obtain the second supernatant; The first supernatant described in merging and the second supernatant, concentrate drying.The present invention needs sample few, and loss solvent is few, and extraction rate is fast, improves detection efficiency.
Description
Technical field
The invention belongs to technical field of analytical chemistry, particularly relate to a kind of extracting method for oligosaccharides in the rice embryo of HPLC detection.
Background technology
Meet unbroken wet weather or hot and humid weather, it is too high that hybrid rice seeds there will be water cut, even Spike sprouting phenomenon, and Spike sprouting greatly reduces seed quality, and water cut is too high will increase the cost of seeds processing.Therefore be necessary in the dehydration of seed maturity later stage appropriateness.
The seed that Desiccation-tolerance is strong and kind that dehydration tolerance seed is not sugary and quantitative difference very large, and there is significant correlativity in the change of Desiccation-tolerance and stage of development and maturity.The number of sugar content is correlated with the vitrifacation process of dehydration cell, therefore studies seed dehydration patience, needs to carry out careful research to oligosaccharides.
Have the detection method of oligosaccharides for a long time: thin-layered chromatography, vapor-phase chromatography, high performance liquid chromatography.Thin-layered chromatography has the advantage that cost is low, speed is fast, duration of run is short, but with gas chromatography and liquid chromatography ratio, have measuring error more greatly, not easily carry out the shortcoming of quantitative test.Gas chromatography has efficiently, at a high speed, high sensitivity and the few advantage of amount of samples, but the high temperature of injection port makes the derivant of sugar decompose, even by the catalysis of injection port residue sometimes.High performance liquid chromatography is because the advantages such as favorable reproducibility, detectability are low, good separating effect are widely used.And good sample pretreating method is the key link successfully utilizing HPLC to analyze determinand.
The current method for the pre-treatment of seed oligosaccharides is comparatively complicated, as (VINEETKUMAR such as VINEETKUMAR, ANITARANI, etal.SucroseandRaffinoseFamilyOligosaccharides (RFOs) inSoybeanSeedsAsInfluencedbyGenotypeandGrowingLocation.J .Agric.FoodChem.2010, 58, 5081 – 5085) when extracting the oligosaccharides in soya seeds, need first soya seeds to be processed 3 hours in hexane, the water-bath lixiviate 4 hours in the ethanol of 80% of seed after process, extract lead acetate is purified twice, the supernatant finally obtained carries out HPLC detection after thickening filtration.This kind of extracting method required time is long, and uses a large amount of toxic reagent such as hexane, lead acetate in leaching process, and in addition, the component difference of soya seeds and rice paddy seed is very large.Therefore, a kind of rice paddy seed oligosaccharides extracting method of quick, economic environmental protection is provided to seem very necessary.
Summary of the invention
The invention provides a kind of extracting method for oligosaccharides in the rice embryo of HPLC detection, be applicable to the situation that sample size is less, quick, accurate, environmental protection.
For an extracting method for oligosaccharides in the rice embryo that HPLC detects, comprising:
(1) get embryo, add after grinding concentration be 75 ~ 85% ethanolic solution carry out mechanical shaking extraction, mechanical shaking extraction terminates rear centrifugal, obtains the first supernatant and residue;
(2) add in described residue concentration be 75 ~ 85% ethanolic solution carry out ultrasonic extraction, after ultrasonic extraction terminates, centrifugal, obtain the second supernatant;
(3) the first supernatant described in merging and the second supernatant, concentrate drying.
When the present invention extracts the oligosaccharides in rice embryo, first carry out mechanical shaking extraction with ethanolic solution, and then adopt ultrasonic further extraction, as much as possiblely can extract the oligosaccharides in rice embryo, ensure the accuracy detected, and mechanical shaking extraction and ultrasonic extraction match, and can shorten extraction time greatly, and required sample size is few, also can meet the sample requirement that HPLC detects simultaneously.Oligosaccharides of the present invention is one or more in sucrose, gossypose, fructose and glucose.
Suitable solid-to-liquid ratio is conducive to the extraction of oligosaccharides in embryo, and preferably, during described mechanical shaking extraction, solid-to-liquid ratio (or solid-liquid ratio) is 1:100 ~ 200, is more preferably 1:150.Preferably, during described ultrasonic extraction, solid-to-liquid ratio is 1:50 ~ 100, is more preferably 1:100.
It will be understood by those in the art that solid-to-liquid ratio is the ratio of the quality (g) of embryo and the volume (ml) of ethanolic solution.
Extracting temperature and time also can affect the release of oligosaccharides in embryo, thus affect extraction effect, temperature is too low, molecular motion is slow, and oligosaccharides release is slow, and temperature is too high, easily causes oligosaccharides to be degraded, in addition, time is too short, can not be extracted by oligosaccharides completely, and overlong time then can make impurity leaching content increase, and increases the interference detected follow-up HPLC.
Preferably, the temperature of described mechanical shaking extraction is 75 ~ 85 DEG C, and the time is 25 ~ 35min.Preferred, the temperature of described mechanical shaking extraction is 80 DEG C, and the time is 30min.
Preferably, the temperature of described ultrasonic extraction is 15 ~ 25 DEG C, and the time is 25 ~ 35min.Preferred, the temperature of described ultrasonic extraction is 20 DEG C, and the time is 30min.During ultrasonic extraction, ultrasonic power is generally 50 ~ 100W.
The first supernatant merged and the second supernatant are the solution containing oligosaccharides, and usually need concentrate drying, be beneficial to constant volume and carry out HPLC analysis, the method for described concentrate drying is that nitrogen dries up.Nitrogen dries up and is applicable to the less solution drying of inventive samples amount, avoids oligosaccharides that larger loss occurs in dry run.The temperature that described nitrogen dries up is 35 ~ 45 DEG C.
Compared with prior art, beneficial effect of the present invention is:
The mode that the present invention adopts mechanical shaking extraction and ultrasonic extraction to combine, required sample is few.Do not use the organic solvent of contaminative, yet environmental protection more while reducing costs.The more important thing is, the present invention can save extraction time greatly.
Extracting method of the present invention is reliable, can extract different sucrose, gossypose, fructose and the glucose four kinds of oligosaccharides of molecular size range accurately simultaneously, can meet the requirement of high performance liquid chromatography to sample preparation.
Embodiment
The present invention is explained further below in conjunction with embodiment.
Instrument of the present invention comprises: Nitrogen evaporator, sonic oscillation instrument, thermostatic water bath vibrator, supercentrifuge.
Comparative example 1
(1) first time extracts supernatant: get the fresh embryo of the rear paddy rice of gathering for 22 days of pollination 10, with 80% ethanolic solution mixed grinding, solid-to-liquid ratio (rice embryo quality g: volumes of aqueous ethanol ml) is 1:150; After grinding, mixed liquor is placed in 80 DEG C of thermostatic water bath vibrator mechanical shaking extraction 30min, then the centrifugal 15min of 16000r/min, obtains extracting supernatant and solid residue for the first time;
(2) second time extracts supernatant: the fixed residue after being extracted first time mixes with 80% ethanolic solution, solid-to-liquid ratio is 1:100, the centrifugal 15min of 16000r/min after 80 DEG C of mechanical shaking extraction 30min, obtains second time and extracts supernatant and solid residue;
(3) third time extracts supernatant: the fixed residue after second time being extracted mixes with 80% ethanolic solution, and solid-to-liquid ratio is 1:50, the centrifugal 15min of 16000r/min after 80 DEG C of mechanical shaking extraction 30min, obtains extracting supernatant and solid residue for the third time;
(4) merge the supernatant extracted for three times, after ambient temperature under nitrogen dries up, obtain oligosaccharides powder;
(5) in oligosaccharides powder, add ultrapure water dissolve, after 0.45 μm of membrane filtration, measure the content of oligosaccharides (sucrose, gossypose, fructose, glucose) by HPLC method.
Chromatographic condition: WATERS2410 differential refraction detector detects, and XtimateSugar-Ca chromatographic column, mobile phase is deionized water, column temperature 80 DEG C, flow velocity 0.5ml/min, and sampling volume is 50 μ l.
Adopt repeatedly the method that ethanolic solution extracts, the leaching process of whole oligosaccharides needs 5.0 hours, HPLC measurement result shows, it is 14.920 μ g/ grains that this extracting method records cane sugar content in rice embryo, gossypose is 2.739 μ g/ grains, fructose is 0.780 μ g/ grain, and glucose is 0.312 μ g/ grain.If after direct for first time extract nitrogen is dried up, dissolution filter detects, recording cane sugar content in rice embryo is 12.681 μ g/ grains, gossypose is 2.314 μ g/ grains, fructose is 0.487 μ g/ grain, and glucose is 0.253 μ g/ grain, although illustrate merely through once extract can shorten extraction time, but the oligosaccharides amount extracted is few, accurately can not reflect the oligosaccharide content in rice paddy seed.
Embodiment 1
(1) 10 fresh embryos of the rear paddy rice of gathering for 22 days of pollination are got, be ground into powder under normal temperature after weighing, add 80% ethanolic solution 1ml (solid-to-liquid ratio is 1:150) grinding evenly, be then placed in 80 DEG C of constant temperature oscillation water-bath mechanical shaking extraction 30min.After mechanical shaking extraction terminates, potpourri is put to normal temperature, the centrifugal 15min of 16000r/min, obtain and extract supernatant and solid residue for the first time.
(2) 80% ethanolic solution 0.5ml (solid-to-liquid ratio is 1:100) is added in the solid residue after extracting to first time, mix to be placed in sonic oscillation instrument and extract 30min, ultrasonic power is 80W, after ultrasonic extraction terminates, the centrifugal 15min of 16000r/min, obtains second time and extracts supernatant.
(3) merge extracted twice supernatant, ambient temperature under nitrogen dries up (about two hours), obtains oligosaccharides powder.
(4) in oligosaccharides powder, add deionized water and be settled to 100 μ l, after 0.45 μm of membrane filtration, measure the content of oligosaccharides (sucrose, gossypose, fructose, glucose) by HPLC method.
In step (2), ultrasonic temperature established 10 DEG C, 20 DEG C and 45 DEG C totally 3 levels extract respectively.
Chromatographic condition: WATERS2410 differential refraction detector detects, and XtimateSugar-Ca chromatographic column, mobile phase is deionized water, column temperature 80 DEG C, flow velocity 0.5ml/min, and sampling volume is 50 μ l.
Result is as shown in table 1, and ultrasonic temperature is that 20 DEG C of extraction effects to oligosaccharides are better, the oligosaccharide content extracted and embodiment 1 result without significant difference, but extraction time greatly shorten, used time shortening about 3 hours.
The impact that table 1 ultrasonic temperature extracts rice embryo oligosaccharides
* CK refers to the result without ultrasonic process in embodiment 1, the significance of difference (α=0.05, LSD) between the process that lowercases different in table represents same oligosaccharides.
Embodiment 2
During ultrasonic extraction, ultrasonic temperature is 20 DEG C, and ultrasonic time arranges 20min, 30min, 50min tri-levels, and all the other oligosaccharides leaching process are with embodiment 1.
The impact that table 2 ultrasonic time extracts rice embryo oligosaccharides
* CK refers to the result without ultrasonic process in embodiment 1, the significance of difference (α=0.05, LSD) between the process that lowercases different in table represents same oligosaccharides.
Result is as shown in table 2, and ultrasonic time is that 30min is better to the extraction effect of oligosaccharides, and the oligosaccharide content extracted and embodiment 1 result without significant difference, and continue to increase ultrasonic time, and the oligosaccharide content extracted is not significantly increased.
Embodiment 3
During ultrasonic extraction, ultrasonic temperature is 20 DEG C, and ultrasonic time is 30min, and nitrogen dries up temperature and arranges 25 DEG C, 35 DEG C and 45 DEG C of three levels, and all the other processes are with embodiment 1.
Table 3 nitrogen blowing temp concentrates the impact of speed on leaching liquor
* CK refers to that ambient temperature under nitrogen dries up required time, and lowercases different in table represents that leaching liquor concentrates the significance of difference (α=0.05, LSD) between process consuming time.
The impact that table 4 nitrogen blowing temp extracts rice embryo oligosaccharides
* CK refers to that ambient temperature under nitrogen dries up process, the significance of difference (α=0.05, LSD) between the process that lowercases different in table represents same oligosaccharides.
As shown in table 3, table 4, in certain temperature range, the stability of the temperature that nitrogen dries up to oligosaccharides has no significant effect, and dries up the time used shorter when temperature is 35 DEG C, 45 DEG C, and comparatively room temperature under nitrogen dries up the used time and shortens 1.5 hours (table 3).
Embodiment 4
With reference to the method for embodiment 1, the temperature setting ultrasonic extraction is 20 DEG C of oligosaccharides extracting in the fresh embryo of the rear paddy rice of gathering for 28 days of pollination, and HPLC measures oligosaccharides (sucrose, gossypose, fructose and glucose) content.
The content adopting sucrose and gossypose in the inventive method practical measurement maturation pollination latter 28 days rice embryos is 15.000 μ g/ grains (fresh seed) and 4.290 μ g/ grains (fresh seed) respectively, and the content of fructose and glucose is 0.170 μ g/ grain (fresh seed) and 0.130 μ g/ grain (fresh seed) respectively.
Carry out recovery of standard addition experiment, the average recovery rate of sucrose, gossypose is respectively 94.1%, 95%, and the average recovery rate of fructose and glucose is respectively 95% and 90%, meets oligosaccharides and measures requirement.
Claims (4)
1., for an extracting method for oligosaccharides in the rice embryo of HPLC detection, it is characterized in that, comprising:
(1) get embryo, be the ethanolic solution that 1:150 adds that concentration is 75 ~ 85% according to solid-to-liquid ratio after grinding, be carry out mechanical shaking extraction 30min under the condition of 80 DEG C in temperature, mechanical shaking extraction terminates rear centrifugal, obtains the first supernatant and residue;
(2) being the ethanolic solution that 1:100 adds that concentration is 75 ~ 85% according to solid-to-liquid ratio in described residue, is carry out ultrasonic extraction 30min under the condition of 20 DEG C in temperature, after ultrasonic extraction terminates, centrifugal, obtains the second supernatant;
(3) the first supernatant described in merging and the second supernatant, concentrate drying.
2. extracting method as claimed in claim 1, it is characterized in that, the method for described concentrate drying is that nitrogen dries up.
3. extracting method as claimed in claim 2, it is characterized in that, the temperature that described nitrogen dries up is 35 ~ 45 DEG C.
4. extracting method as claimed in claim 1, it is characterized in that, described oligosaccharides is one or more in sucrose, gossypose, fructose and glucose.
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| CN103554284A (en) * | 2013-10-11 | 2014-02-05 | 青岛农业大学 | Extraction and separation process for peony stamen polysaccharide |
| CN103788221A (en) * | 2013-10-11 | 2014-05-14 | 青岛农业大学 | Araban in peony stamens and extraction separation method thereof |
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| WO2010135822A1 (en) * | 2009-05-28 | 2010-12-02 | Mcgill University | Determination of fractional compositions using nonlinear spectrophonometry |
| CN103554284A (en) * | 2013-10-11 | 2014-02-05 | 青岛农业大学 | Extraction and separation process for peony stamen polysaccharide |
| CN103788221A (en) * | 2013-10-11 | 2014-05-14 | 青岛农业大学 | Araban in peony stamens and extraction separation method thereof |
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