CN104714037A - Method for testing content of monosaccharide in polysaccharide in pear peel tissue - Google Patents
Method for testing content of monosaccharide in polysaccharide in pear peel tissue Download PDFInfo
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Abstract
The invention relates to a method for testing the content of monosaccharide in polysaccharide in pear peel tissue. The method comprises the following steps: preparing a sample to be tested, namely, weighing a fixed amount of pear peel, extracting with petroleum ether, performing heating extraction to the residues by using deionized water, filtering by using a membrane of 0.45mu m, putting the filtrate into an ampoule bottle, adding trifluoroacetic acid, introducing N2, sealing the tube to react for 0.5-5 hours, adding a NaOH solution and a PMP methanol solution, and uniformly mixing to react; preparing a standard monosaccharide solution, and quantitatively analyzing the content of different monosaccharide components in polysaccharide in the pear peel by using a high performance liquid chromatography method. The change rules of the content of the different monosaccharide components in polysaccharide in the peel in different periods of fruits can be compared. The method is small in sample amount, capable of effectively increasing the extraction rate of the content of the monosaccharide components in polysaccharide, and high in accuracy and repeatability.
Description
Technical field
The present invention relates to a kind of sugared content assaying method, be specifically related to the assay method of contents of monosaccharides in a kind of pears pericarp tissue polysaccharide.
Background technology
At present, the method and technology measuring monosaccharide component in plant polyose mainly concentrates on Radix Angelicae Sinensis, cactus, matrimony vine, white fungus and longan pulp part, little about the assay method of contents of monosaccharides in pericarp tissue polysaccharide.And sample size is many needed for these methods, complex steps and required sample starting material are many, and obtain the peel sample that pears young fruit period produces time-consuming, take a lot of work, bring difficulty to wanting to explore the effect of different monosaccharide component in pears different times pericarp in Changing Pattern, pericarp structure and function in polysaccharide.
The third-largest fruit industry of Li Shi China is one of favorite fruit of consumer.In the operatic circle skin polysaccharide, monosaccharide component assay not only contributes to the research of pericarp structure, also for analysis pericarp Middle nutrition composition and value provide theoretical foundation.At present, in working sample polysaccharide, monosaccharide component needs examination material more, if sample is less, causes the amount of extraction Thick many candies less and cannot carry out follow-up work.Therefore, develop a kind of method that is efficient, accurate and that need sample amount to measure monosaccharide component in the operatic circle skin polysaccharide less and become particularly important.
Summary of the invention
The present invention utilizes high performance liquid chromatography, measures the content of different monosaccharide component in the operatic circle skin polysaccharide, and the method not only effectively improves monosaccharide component content extraction ratio in polysaccharide, and accuracy is high, reproducible.
One aspect of the present invention relates to the assay method of contents of monosaccharides in a kind of pears pericarp tissue polysaccharide, it is characterized in that, comprises the steps:
1) take quantitative pericarp, extract with sherwood oil, filter, collect filter residue, residue deionized water heat is extracted, and supernatant is evaporated to 1/3 of initial volume, adds absolute ethyl alcohol, mixing, leaves standstill 24h, centrifugally must to precipitate; Precipitation is made into aqueous solution, adds the concussion of Sevage reagent, centrifugal, through vacuum freeze drying, obtains sample I;
2) sample I is dissolved in pure water, crosses 0.45 μm of film, get filtrate and be placed in ampoule bottle, add trifluoroacetic acid, fill N
2rear tube sealing, reacts 0.5-5h under 100-120 DEG C of condition, is cooled to room temperature, adds methyl alcohol evaporate to dryness again, repetitive operation 2-5 time after evaporated under reduced pressure in ampoule bottle, redissolves with pure water, crosses 0.45 μm of film, obtains sample II for subsequent use;
3) sample thief II, be placed in container, add NaOH solution, PMP methanol solution, reaction after mixing, reaction terminates rear test tube and is cooled to room temperature, by HCl neutralization reaction system, adds chloroform and fully shakes, leave standstill, discard lower floor's organic phase, repeat 2-5 time, after crossing 0.45 μm of filter membrane after thin up, carry out HPLC analysis.
Preferably, described assay method also comprises standard solution preparation steps, be specially the standard reserving solution preparing glucose, rhamnose, galactose, arabinose, galacturonic acid, mannose 6 kinds of monose standard items compositions respectively, save backup under 4 DEG C of conditions, standard items working fluid is diluted further by storing solution and obtains 6 concentration, carries out HPLC analysis after crossing 0.45 μm of film.
Preferably, in described assay method, the analysis condition of HPLC is mobile phase is 20mmol/L mixture of acetonitrile-phosphate buffer, and preferably, its volume ratio is 83: 17; Preferably, flow velocity is 1mL/min, determined wavelength 245nm, column temperature 30 DEG C, sample size 20 μ L, detection time: 30min.
Preferably, described pears are ' Dangshan pear ' (shagreen pears) and brown skin bud mutation ' rust is crisp ' (brown skin pears) thereof.
Preferably, described step 1) in, the temperature that sherwood oil extracts is 70 DEG C; Described step 1) in, the temperature that heat is extracted is 80 DEG C;
Preferably, described step 1) be, take quantitative pericarp, 1-4h is extracted with the sherwood oil 70 DEG C of 4-8 times of volume, filter, collect filter residue, the deionized water of residue 8-12 times volume carries out heat and extracts 2-5 time, bath temperature 80 DEG C, each extraction time 1-4h, extract is centrifugal, merge supernatant, put that Rotary Evaporators 40-60 DEG C is evaporated to initial volume 1/3, add the absolute ethyl alcohol of 2-5 times of concentrate volume, mixing, leave standstill 24h, centrifugally must to precipitate, precipitation is made into the aqueous solution of 5%, add the Sevage reagent of 1/3 volume, concussion 25min, centrifugal, through vacuum freeze drying, obtain sample I.
Most preferably, described step 1) be, take quantitative pericarp, 2h is extracted with the sherwood oil 70 DEG C of 6 times of volumes, filter, collect filter residue, residue carries out heat with the deionized water of 10 times of volumes and extracts 3 times, bath temperature 80 DEG C, each extraction time 2h, extract is centrifugal, merge supernatant, put Rotary Evaporators 50 DEG C and be evaporated to 1/3 of initial volume, add the absolute ethyl alcohol of 4 times of concentrate volumes, mixing, leave standstill 24h, centrifugally must to precipitate, precipitation is made into the aqueous solution of 5%, add the Sevage reagent of 1/3 volume, concussion 25min, centrifugal, through vacuum freeze drying, obtain sample I.
Preferably described step 2) be that Thick many candies sample is dissolved in pure water, cross 0.45 μm of film, get filtrate and be placed in ampoule bottle, add 4mol/L trifluoroacetic acid, fill N
2rear tube sealing, reacts 2h under 110 DEG C of conditions, is cooled to room temperature, adds methyl alcohol evaporate to dryness again after evaporated under reduced pressure in ampoule bottle, repetitive operation 3 times, redissolves with pure water, crosses 0.45 μm of film, obtains sample II for subsequent use.
Preferably, described step 3) be, sample thief II, be placed in 5mL tool plug test tube, add 0.3mol/L NaOH solution, 0.5mol/L PMP methanol solution, mix rear 70 DEG C of water-bath 2h, reaction terminates rear test tube and is cooled to room temperature, by 0.3mol/L HCl neutralization reaction system, adds chloroform and fully shakes, leave standstill, discard lower floor's organic phase, repeat 3 times, after crossing 0.45 μm of filter membrane after thin up, carry out HPLC analysis.
Beneficial effect of the present invention
(1) the present invention adopts sherwood oil first to extract when processing sample, adopts liquid nitrogen grinding, adopts Sevage reagent to carry out aftertreatment, uses freeze drying processing sample, effectively keeps raw-material composition and extraction efficiency is high.
(2) the present invention utilizes methyl alcohol to dissolve evaporate to dryness after the process of employing TFA highly acid, with various monosaccharide components after PMP process, there is uv absorption, detected and above reagent dosage detect time peak shape better, adopt column temperature 30 DEG C, mobile phase is phosphate buffer solution (PH=6.8)-acetonitrile (volume ratio is 83: 17), flow velocity is 1ml/min, determined wavelength is 245nm, sample size is 20 μ L, good separating resulting is reached to monose, makes final detection result accuracy rate high.
Accompanying drawing explanation
Fig. 1 is the appearance time of different monose in homopolysaccharide not;
Fig. 2, that is, each monosaccharide component content in 2A-2F different times ' Dangshan pear ' and ' rust crisp ' pericarp polysaccharide;
Embodiment
Embodiment 1
1 instrument and sample
1.1 instruments: Agilent 1260 HPLC system (Agilent Technologies, USA).Chromatographic column: ZORBAX SB-C18column (4.6m*150mm*5 μm, Agilent, USA).
Sample: the pericarp of rear 25d, 50d, 75d, 100d, 125d and 150d is spent in ' Dangshan pear ' (shagreen pears) and brown skin bud mutation ' rust is crisp ' (brown skin pears) thereof.
2 methods and result
2.1 take different times pears skin 2g, extract 2h, filter, collect filter residue with the sherwood oil 70 DEG C of 6 times of volumes, and residue carries out heat with the deionized water of 10 times of volumes and extracts 3 times, bath temperature 80 DEG C, each extraction time 2h.Extract is centrifugal, merge 3 supernatants, puts Rotary Evaporators 50 DEG C and is evaporated to 1/3 of initial volume, add the absolute ethyl alcohol of 4 times of concentrate volumes, and mixing leaves standstill 24h, centrifugally must precipitate.Precipitation is made into the aqueous solution of 5%, and the Sevage reagent (chloroform: normal butyl alcohol=4:1) adding 1/3 shakes 25min, centrifugal, through vacuum freeze drying, obtains crude product.
Crude product 3mg is dissolved in 3mL pure water by 2.2, crosses 0.45 μm of film, gets 1mL filtrate and be placed in ampoule bottle, add 4mol/L trifluoroacetic acid 1mL, fill N
2rear tube sealing, reacts 2h under 110 DEG C of conditions, is cooled to room temperature, adds 500 μ L methyl alcohol evaporate to dryness again, repetitive operation 3 times after evaporated under reduced pressure in ampoule bottle, the trifluoroacetic acid that removing is residual, redissolves with 1mL pure water, crosses 0.45 μm of film, obtains sample liquid for subsequent use.
2.3 standard solution prepare the standard reserving solution that compound concentration is respectively the glucose of 6mmol/L, rhamnose, galactose, arabinose, galacturonic acid, mannose 6 kinds of monose standard items compositions, save backup under 4 DEG C of conditions, standard items working fluid is diluted further by storing solution and obtains 6 concentration, carries out HPLC analysis after crossing 0.45 μm of film.
2.4 get 250 μ L hybrid standard product solution, sample liquid, be placed in 5mL tool plug test tube, add 0.3mol/L NaOH solution 250 μ L, 0.5mol/L PMP methanol solution 250 μ L, mix rear 70 DEG C of water-bath 2h, reaction terminates rear test tube and is cooled to room temperature, by 250 μ L 0.3mol/L HCl neutralization reaction systems, add 1mL chloroform fully to shake, leave standstill, discard lower floor's organic phase, repeat 3 times, unreacted PMP in removing reaction system, carries out liquid-phase chromatographic analysis after crossing 0.45 μm of filter membrane after adding 100 μ L water dilutions.
In 2.5 polysaccharide, monose Identification of Species and quantitative test condition mobile phase are 20mmol/L phosphate buffer (pH 6.7)-acetonitrile (83: 17), flow velocity is 1mL/min, determined wavelength 245nm, column temperature 30 DEG C, sample size 20 μ L, detection time: 30min.
In this test, because the few and separating effect in assorted peak is better, different monose kind (Fig. 1) can be identified according to appearance time and peak sequence.The concentration that the relative response of the standard solution gained under each concentration quantitatively can be utilized to be ordinate (Y) and standard solution of different monose for standards calibration curve that horizontal ordinate (X) is set up respectively detect actual sample time, the relative response of gained per sample, then to read in sample various contents of monosaccharides in polysaccharide respectively from typical curve.Utilize statistics software SPSS to carry out multiple comparison analyse to result, it is all variant that result shows two kinds different times after namely pears skin tissue development process is spent.
In this example assay result (Fig. 2), in the pericarp development forming process of two kinds all variant and in most polysaccharide different contents of monosaccharides 50d or 75d difference after Sheng is spent maximum, illustrate that this period is formation critical period of ' rust crisp ' pericarp brown, this, pericarp membrane wall construction occurred obviously to change in period.
Claims (10)
1. the assay method of contents of monosaccharides in pears pericarp tissue polysaccharide, is characterized in that, comprise the steps:
1) take quantitative pericarp, extract with sherwood oil, filter, collect filter residue, residue deionized water heat is extracted, and supernatant is evaporated to 1/3 of initial volume, adds absolute ethyl alcohol, mixing, leaves standstill 24h, centrifugally must to precipitate; Precipitation is made into aqueous solution, adds the concussion of Sevage reagent, centrifugal, through vacuum freeze drying, obtains sample I;
2) sample I is dissolved in pure water, crosses 0.45 μm of film, get filtrate and be placed in ampoule bottle, add trifluoroacetic acid, fill N
2rear tube sealing, reacts 0.5-5h under 100-120 DEG C of condition, is cooled to room temperature, adds methyl alcohol evaporate to dryness again, repetitive operation 2-5 time after evaporated under reduced pressure in ampoule bottle, redissolves with pure water, crosses 0.45 μm of film, obtains sample II for subsequent use;
3) sample thief II, be placed in container, add NaOH solution, PMP methanol solution, reaction after mixing, reaction terminates rear test tube and is cooled to room temperature, by HCl neutralization reaction system, adds chloroform and fully shakes, leave standstill, discard lower floor's organic phase, repeat 2-5 time, after crossing 0.45 μm of filter membrane after thin up, carry out HPLC analysis.
2. assay method according to claim 1, also comprise standard solution preparation steps, be specially the standard reserving solution preparing glucose, rhamnose, galactose, arabinose, galacturonic acid, mannose 6 kinds of monose standard items compositions respectively, save backup under 4 DEG C of conditions, standard items working fluid is diluted further by storing solution and obtains 6 concentration, carries out HPLC analysis after crossing 0.45 μm of film.
3. assay method according to claim 1, wherein the analysis condition of HPLC is mobile phase is 20mmol/L mixture of acetonitrile-phosphate buffer, and volume ratio is 83: 17, flow velocity is 1mL/min, determined wavelength 245nm, column temperature 30 DEG C, sample size 20 μ L, detection time: 30min.
4. assay method according to claim 1, described pears are that Dangshan pear and rust are crisp.
5. assay method according to claim 1, described step 1) temperature extracted of PetroChina Company Limited.'s ether is 70 DEG C.
6. assay method according to claim 1, described step 1) in the temperature extracted of heat be 80 DEG C.
7. assay method according to claim 1, described step 1) be, take quantitative pericarp, 1-4h is extracted with the sherwood oil 70 DEG C of 4-8 times of volume, filter, collect filter residue, the deionized water of residue 8-12 times volume carries out heat and extracts 2-5 time, bath temperature 80 DEG C, each extraction time 1-4h, extract is centrifugal, merge supernatant, put that Rotary Evaporators 40-60 DEG C is evaporated to initial volume 1/3, add the absolute ethyl alcohol of 2-5 times of concentrate volume, mixing, leave standstill 24h, centrifugally must to precipitate, precipitation is made into the aqueous solution of 5%, add the Sevage reagent of 1/3 volume, concussion 25min, centrifugal, through vacuum freeze drying, obtain sample I.
8. detection method according to claim 1, described step 1) be, take quantitative pericarp, 2h is extracted with the sherwood oil 70 DEG C of 6 times of volumes, filter, collect filter residue, residue carries out heat with the deionized water of 10 times of volumes and extracts 3 times, bath temperature 80 DEG C, each extraction time 2h, extract is centrifugal, merge supernatant, put Rotary Evaporators 50 DEG C and be evaporated to 1/3 of initial volume, add the absolute ethyl alcohol of 4 times of concentrate volumes, mixing, leave standstill 24h, centrifugally must to precipitate, precipitation is made into the aqueous solution of 5%, add the Sevage reagent of 1/3 volume, concussion 25min, centrifugal, through vacuum freeze drying, obtain sample I.
9. assay method according to claim 1, described step 2) be that sample I is dissolved in pure water, cross 0.45 μm of film, get filtrate and be placed in ampoule bottle, add 4mol/L trifluoroacetic acid, fill N
2rear tube sealing, reacts 2h under 110 DEG C of conditions, is cooled to room temperature, adds methyl alcohol evaporate to dryness again after evaporated under reduced pressure in ampoule bottle, repetitive operation 3 times, redissolves with pure water, crosses 0.45 μm of film, obtains sample II for subsequent use.
10. assay method according to claim 1, described step 3) be, sample thief II, be placed in 5mL tool plug test tube, add 0.3mol/L NaOH solution, 0.5mol/L PMP methanol solution, mix rear 70 DEG C of water-bath 2h, reaction terminates rear test tube and is cooled to room temperature, by 0.3mol/LHCl neutralization reaction system, adds chloroform and fully shakes, leave standstill, discard lower floor's organic phase, repeat 3 times, after crossing 0.45 μm of filter membrane after thin up, carry out HPLC analysis.
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| CN105136951A (en) * | 2015-08-07 | 2015-12-09 | 华中农业大学 | Rapid quantitative method for tea polysaccharide monosaccharide composition |
| CN106290205A (en) * | 2016-11-14 | 2017-01-04 | 河南农业大学 | The assay method of glycolipid content and application thereof in a kind of flour |
| CN106770732A (en) * | 2016-11-30 | 2017-05-31 | 无锡艾科瑞思产品设计与研究有限公司 | A kind of method of sugar in detection grain |
| CN107966502A (en) * | 2017-11-01 | 2018-04-27 | 广西壮族自治区食品药品检验所 | The method for measuring Radix Salviae Miltiorrhizae polysaccharide hydrolysate-galacturonic acid |
| CN108956792A (en) * | 2017-05-29 | 2018-12-07 | 青岛大学附属医院 | The high performance liquid chromatography detection of free serum mannose and glucose |
| CN109212103A (en) * | 2018-11-13 | 2019-01-15 | 湖北科技学院 | A kind of high efficiency extraction measures the column front derivation HPLC method of polysaccharide monosaccharide component in fritillaria |
| CN110261494A (en) * | 2018-11-01 | 2019-09-20 | 青岛大学附属医院 | A kind of method and its detection kit for identifying thyroid malignancy biomarker |
| CN112461948A (en) * | 2020-11-03 | 2021-03-09 | 广西壮族自治区亚热带作物研究所（广西亚热带农产品加工研究所） | Method for determining polysaccharide components and monosaccharide compositions in saw palmetto fruits |
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| CN106290205A (en) * | 2016-11-14 | 2017-01-04 | 河南农业大学 | The assay method of glycolipid content and application thereof in a kind of flour |
| CN106290205B (en) * | 2016-11-14 | 2018-11-20 | 河南农业大学 | Application of the measuring method of glycolipid content in wheat flour in a kind of flour |
| CN106770732A (en) * | 2016-11-30 | 2017-05-31 | 无锡艾科瑞思产品设计与研究有限公司 | A kind of method of sugar in detection grain |
| CN108956792A (en) * | 2017-05-29 | 2018-12-07 | 青岛大学附属医院 | The high performance liquid chromatography detection of free serum mannose and glucose |
| CN108956792B (en) * | 2017-05-29 | 2020-06-16 | 青岛大学附属医院 | High performance liquid chromatography detection of serum free mannose and glucose |
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| CN107966502A (en) * | 2017-11-01 | 2018-04-27 | 广西壮族自治区食品药品检验所 | The method for measuring Radix Salviae Miltiorrhizae polysaccharide hydrolysate-galacturonic acid |
| CN107966502B (en) * | 2017-11-01 | 2021-01-01 | 广西壮族自治区食品药品检验所 | Method for determining polysaccharide hydrolysate-galacturonic acid in salvia miltiorrhiza bunge |
| CN110261494A (en) * | 2018-11-01 | 2019-09-20 | 青岛大学附属医院 | A kind of method and its detection kit for identifying thyroid malignancy biomarker |
| CN109212103A (en) * | 2018-11-13 | 2019-01-15 | 湖北科技学院 | A kind of high efficiency extraction measures the column front derivation HPLC method of polysaccharide monosaccharide component in fritillaria |
| CN112461948A (en) * | 2020-11-03 | 2021-03-09 | 广西壮族自治区亚热带作物研究所（广西亚热带农产品加工研究所） | Method for determining polysaccharide components and monosaccharide compositions in saw palmetto fruits |
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Application publication date: 20150617 |