CN105891196A - Simple determining method for content of sialic acid in complex sample - Google Patents
Simple determining method for content of sialic acid in complex sample Download PDFInfo
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- CN105891196A CN105891196A CN201410631238.4A CN201410631238A CN105891196A CN 105891196 A CN105891196 A CN 105891196A CN 201410631238 A CN201410631238 A CN 201410631238A CN 105891196 A CN105891196 A CN 105891196A
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- sialic acid
- acid content
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- acid
- complex sample
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Abstract
The invention discloses a simple method for determining the content of sialic acid in a complex sample. The method utilizes acid hydrolysis to release the saliva acid in a combination state with proteins in the sample, and the saliva acid in a free state and in an oligosaccharide combination state is separated from saccharides by a polyacrylamide gel column. The sialic acid is subjected to a reaction with resorcinol under a synergistic catalytic effect of Cu<2+> and Cr<3+>, a generated colored material is extracted with butyl acetate-n-butanol, the absorbance is determined at 610 nm, and then the content of the sialic acid is obtained. The method for determining the content of the saliva acid in the complex sample greatly simplifies the process, reduces the cost, and fills the blank of sialic acid determination methods at home and abroad.
Description
Sialic acid (Sialic Acid) is the common name of neuraminic acid derivatives, and formal name used at school is called " N-acetyl-neuraminate ", is
A kind of naturally occurring carbohydrate, is widely present in multiple biological tissue, is to constitute glycoprotein and the weight of glycolipid on cell membrane
Want composition, participate in the different physiological roles of cell surface.Sialic acid has antiviral and bacterial-infection resisting, raising immunity, neutralization
Toxin and antiinflammatory, the function of protection respiratory system, be developed into the new of prophylactic treatment respiratory virus infection and influenza the most
Type medicine, therefore sialic acid and the most potential newtype drug becoming prevention and treatment infective virus of derivant thereof.It is reported,
Sialic domestic and international market annual requirement is more than 100 tons, and recent world market share, more than 500,000,000 yuans, has long-range
Market prospect and huge market potential.
Sialic acid content in complex sample is measured and is studied by the present invention, constructs the simple measuring side of a kind of sialic acid content
Method.This invention utilizes the sialic acid in acid hydrolysis release sample with protein bound state, will be free by Polyacrylamide Gel Column
The sialic acid of state and oligosaccharide bonding state is separated with saccharide.Sialic acid under Cu2+ and Cr3+ concerted catalysis with isophthalic two
Phenol reacts, the coloring matter of generation butyl acetate-n-butanol extraction, and measures absorbance at 610nm, and then draws sample
Sialic acid content in product.
Most preferred embodiment:
1. preparation
The sialic acid standard solutions of a series of variable concentrations is prepared with sialic acid standard (Sigma, USA)；Preparation developer: contain
0.25mmol/L copper sulfate, 0.15mmol/L Chlorizate chromium, 2g/L resorcinol, 30% hydrochloric acid solution；Preparation extractant: butyl acetate
-n-butyl alcohol (volume ratio 85: 15)；Prepare Polyacrylamide Gel Column.
2. make sialic acid standard curve
In the glass centrifuge tube of 10mL band plug, it is separately added into the titer of 2mL variable concentrations, 2mL developer, makees sky with distilled water
In vain, vibration, in boiling water bath, heat micro-boiling 15min, after taking-up, be cooled to room temperature with flowing water immediately.Each 4mL extractant that adds, rotation
Whirlpool mixes, and under 1000r/min, centrifugal 3min, takes upper strata extracting solution respectively, use 0.5cm cuvette, surveys absorbance at 610nm, will
Result carries out linear regression analysis, obtains the regression equation between absorbance and sialic acid mass concentration.
3. the sialic acid content with protein bound measures
Accurately weigh 50.0g sample suitable quantity of water dissolve and be settled to 500mL, pipette 10mL solution in centrifuge tube, at 4 DEG C,
Centrifugal 10min under the conditions of 10000r/min.After taking-up, upper strata oils and fats is removed, adds isopyknic 10%TCA solution protein precipitation,
Vibration, ice bath 10min, 4 DEG C, 10000r/min be centrifuged 10min.After taking-up, clear liquid is removed, add the cold 5%TCA of 10mL
Washing precipitation, vibration, at 4 DEG C, centrifugal 10min under 10000r/min.Being removed by clear liquid after taking-up, merge twice clear liquid, clear liquid is pressed
4 step process.Precipitation moves in conical flask, hydrolyzes with 0.10mol/L sulfuric acid solution 30mL, is heated to reflux 1.5h, takes out, uses immediately
Flowing water cools down, and is filtered by hydrolyzed solution, precipitates with distilled water wash.Gained filtrate evaporation and concentration on Rotary Evaporators, is settled to
25mL.Accurately pipette 2.0mL filtrate with pipet, record absorbance by step 2 method, go out corresponding matter by regression equation calculation
Amount concentration, and then draw the sialic acid content Y1 of protein bound.
4. sialic acid content that is free and that be combined with oligosaccharide measures
Clear liquid step 3 obtained is settled to 50mL, all moves in conical flask, hydrolyzes with 0.10mol/L sulfuric acid solution 50mL, add
Hot reflux 1.5h, takes out, and cools down with flowing water immediately, and on Rotary Evaporators, evaporation and concentration is to about 20mL, fixed after regulation pH to 6.0
Hold to 25mL.All adding in gel column, make eluent with 3～4 DEG C of water, controlling its flow velocity is 10mL/h, collects washing liquid 50mL.In
Evaporation and concentration on Rotary Evaporators, is settled to 25mL.Accurately pipette 2mL to operate by step 2 method, record absorbance, by recurrence side
Journey calculates corresponding mass concentration, and then draws sialic acid content Y2 that is free and that be combined with oligosaccharide.
5. Total silicic acid content in sample
In sample, Total silicic acid content is the sialic acid content Y1 with protein bound, saliva that is free and that be combined with oligosaccharide
Acid content Y2 sum.
Claims (5)
1. the portable measuring method for measuring of the sialic acid content in a complex sample, it is characterised in that through acid hydrolysis, separates, chemical combination
Reaction, extraction, measure sialic acid content in the step measurements samples such as absorbance.
The most according to claim 1, the method measuring sialic acid content is characterized in that, utilize acid hydrolysis release sample in egg
The sialic acid of white matter bonding state.
The most according to claim 1, the method measuring sialic acid content is characterized in that, will trip by Polyacrylamide Gel Column
Sialic acid from state with oligosaccharide bonding state separates with saccharide, makees eluent with 3～4 DEG C of water.
The most according to claim 1, the method measuring sialic acid content is characterized in that, sialic acid is worked in coordination with at Cu2+ and Cr3+
Catalysis is lower to react with resorcinol, the coloring matter of generation butyl acetate-n-butanol extraction.
The most according to claim 1, the method measuring sialic acid content is characterized in that, the coloring matter that catalytic reaction generates exists
Measure absorbance at 610nm, and then draw sialic acid content.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410631238.4A CN105891196A (en) | 2014-11-11 | 2014-11-11 | Simple determining method for content of sialic acid in complex sample |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410631238.4A CN105891196A (en) | 2014-11-11 | 2014-11-11 | Simple determining method for content of sialic acid in complex sample |
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| CN105891196A true CN105891196A (en) | 2016-08-24 |
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| CN201410631238.4A Pending CN105891196A (en) | 2014-11-11 | 2014-11-11 | Simple determining method for content of sialic acid in complex sample |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106770243A (en) * | 2016-12-21 | 2017-05-31 | 张静 | A kind of assay method of poly sialic acid |
| CN107991294A (en) * | 2017-11-28 | 2018-05-04 | 陕西慧康生物科技有限责任公司 | A kind of detection method of poly sialic acid |
| CN109030470A (en) * | 2018-05-29 | 2018-12-18 | 中国水产科学研究院珠江水产研究所 | A kind of measurement reagent of bacterial eapsular polysaccharide terminal sialic acid content and its preparation method and application |
-
2014
- 2014-11-11 CN CN201410631238.4A patent/CN105891196A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106770243A (en) * | 2016-12-21 | 2017-05-31 | 张静 | A kind of assay method of poly sialic acid |
| CN107991294A (en) * | 2017-11-28 | 2018-05-04 | 陕西慧康生物科技有限责任公司 | A kind of detection method of poly sialic acid |
| CN107991294B (en) * | 2017-11-28 | 2020-11-06 | 陕西慧康生物科技有限责任公司 | Method for detecting polysialic acid |
| CN109030470A (en) * | 2018-05-29 | 2018-12-18 | 中国水产科学研究院珠江水产研究所 | A kind of measurement reagent of bacterial eapsular polysaccharide terminal sialic acid content and its preparation method and application |
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Application publication date: 20160824 |
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| WD01 | Invention patent application deemed withdrawn after publication |