羧甲基化修饰多糖的功能及其生物学活性

羧甲基化修饰多糖的功能及其生物学活性

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The function and activity of Modified polysaccharide

Reporter: Wang Siyu Nov.16,2012

羧甲基化修饰多糖的功能及其生物学活性

Contents1The research progress of polysaccharide biological activity

2

The effects of modification on polysaccharide

3

The experiment progress and future design of experiments

羧甲基化修饰多糖的功能及其生物学活性

The research progress of polysaccharide biological activity

Why do we study polysaccharide?

Polysaccharides are molecular structurecomplex and huge saccharide substances from higher plants,animal cell membrane and microbial cell wall.

Polysaccharides are one of the four basicmaterial to constitute a life

羧甲基化修饰多糖的功能及其生物学活性

The research progress of polysaccharide biological activity

Previous research results antiviral effect antineoplastic activity Polysaccharide biological activity

immunoregulation

anti-inflammatoryAnti-hyperlipidemia hpyerglycemic

Text antioxidation

羧甲基化修饰多糖的功能及其生物学活性

The effects of modification on polysaccharide

Immuno-enhancing activity of sulfated Auricularia auricula polysaccharides

羧甲基化修饰多糖的功能及其生物学活性

The effects of modification on polysaccharide

Extraction of sAAPOne-step ethanol precipitation column chromatography. Separation component

Remove proteins by trichloroacetic acid method

Purification of AAP

AAPt AAP1 AAP2Chlorosulfonic acid-pyridine method Sulfation modification of AAP

sAAPt sAAP1 sAAP2

羧甲基化修饰多糖的功能及其生物学活性

The effects of modification on polysaccharide

Product confirmation(Using FT-IR spectroscopy analysis) It was used to investigate the vibrations of molecules and polar bonds between the different atoms. It is possible to analyze structures of polysaccharides such as monosaccharide types , glucosidic bonds ,and functional groups using an FT-IR spectroscopy.

羧甲基化修饰多糖的功能及其生物学活性

The effects of modification on polysaccharide

Results The Infrared spectroscopy characteristic of sAAP

羧甲基化修饰多糖的功能及其生物学活性

The effects of modification on polysaccharide

Results

羧甲基化修饰多糖的功能及其生物学活性

The effects of modification on polysaccharide

biological activities assay

In vitro test

In vivo test

One part was added with PHA final concentration reaching to 10ug/ml The other part without PHA

180 One-day-old White Roman chickens (male), were randomly pided into six groups, sAAPt, sAAP1, AAPt, AAP1,no adjuvant(NA) and blank control(BC) group.

羧甲基化修饰多糖的功能及其生物学活性

The effects of modification on polysaccharide

In vitro test Safe concentrationsof three sAAPs and three nonsulfated AAPs for chicken embryo fibroblast (CEF) were measured by the MTT-assay The result showed that the A570 values of AAPt at 250ug/mL, AAP1 at 15.63ug/mL,AAP2 and sAAPt at 3.91ug/mL,sAAP1 and sAAP2 at 7.81ug/mL group were not significantly lower than those of corresponding cells control group. Therefore these concentrations could be considered as their maximal safe concentrations. In order to make the comparison at the same level, their maximal safe concentrations were supposed to be as 3.91ug/mL

羧甲基化修饰多糖的功能及其生物学活性

The effects of modification on polysaccharide

In vitro test Three sAAPs and three AAPs were dissolved with RPMI-1640 into five concentrati

ons from 3.91 to 0.244 ug/mL based on above-mentioned determination result of safety concentration. The chicken blood samples (5mL)were collected from the heart and transferred immediately into aseptic capped tubes with sodium heparin, diluted with an equal volume of Hanks’ solution and carefully added on the surface of lymphocytes separation medium.

羧甲基化修饰多糖的功能及其生物学活性

The effects of modification on polysaccharide

In vitro test After 20 min of centrifugation at 2000rpm, a white cloud-like lymphocytes band was collected and washed twice with RPMI-1640 media without fetal bovine serum . The resulting pellet was resuspended with RPMI-1640 media and pided into two parts, one part was added with PHA(final concentration reaching to 10 ug/mL), and respectively inoculated into 96-well culture plates, 100 uL per well. Then, in polysaccharide groups the six polysaccharides at series of concentrations were added.

羧甲基化修饰多糖的功能及其生物学活性

The effects of modification on polysaccharide

In vitro test The plates were incubated at 39.5 C in a humid atmosphere of 5% CO2 , then according to MTTassay the cellular A570 values were determined as the index of peripheral T lymphocytes proliferation.

lymphocytes proliferation equation:

羧甲基化修饰多糖的功能及其生物学活性

The effects of modification on polysaccharide

The lymphocyte proliferation in single stimulation of sAAP

羧甲基化修饰多糖的功能及其生物学活性

The effects of modification on polysaccharide

During the single stimulation, the lymphocyte proliferation rate of sAAP1 group was the highest(41.25%) as compared to those of AAP1(14.75%), sAAP2(12.92%),AAPt(10.49%) and AAP2(0.15%) groups(P<0.05).