| Aging process is a progressive decline of various physiological functions and stress resistance ability,which accompanines with increasing risk of various age-related diseases and death.Pharmacologic interventions that prolong health span have been shown to alleviate age-related functions decline and enhance organism resistance ability of environmental stress.Natural polysaccharides from plant have become the anti-aging drugs reserve pool,which are due to their structural complexity and low side effect.Panax notoginseng（Burk）F.H.Chen has been historically used as a medicine food homology species in China.In this study,three crude polysaccharides from main root,branch root and fibrous root of P.notoginseng were further isolated and purified to get homogeneous polysaccharide fractions.We used Caenorhabditis elegans to establish the anti-aging compounds screening system to obtain P.notoginseng polysaccharide fraction with the lifespan-extension activity,lifespan-extension molecular mechanism of polysaccharide fraction was further be elucidated.This study will pave the way for the deep processing and developing of P.notoginseng the in functional foods and medicine industry.The main invetigated results were as following:1.Main root polysaccharide（MRP）,branch root polysaccharide（BRP）and fibrous root polysaccharide（FRP）were obtained by hot water extract and ethanol precipitation.The yield rates of MRP,BRP and FRP were separate 15.60,10.40 and 6.1%.Carbohydrate contents of MRP,BRP and FRP were separate 73.31,74.24 and 74.52%.Protein content of MRP,BRP and FRP were separate 1.03,1.22 and1.52%.Uronic acid contents were 7.65,5.43 and 7.12%,respectively.2.MRP,BRP and FRP significantly extended the mean lifespan of wild type N2 worms by 21,10.5 and 5.2%,respectively.Additionally,MRP,BRP and FRP increased heat shock resistant abilities of N2 worms by 17.00,9.00 and10.00%,respectively.There crude polysaccharides also enhanced antioxidant enzymes activities（SOD and CAT）,and reduced MDA contents of C.elegans under heat stress.Above results suggested that MRP possessed the highest bioactive and could be used the further investigation.3.MRP was purified by Cellulose-52 and Sephadex G-100 to yield two fractions named MRP5（90.8%）and MRP5A（91.2%）.The contents of carbohydrate,protein and uronic acid in MRP5 were separate 97.50,0.80 and 0.40%.The average molecular weight of MRP5 was91.6 kDa.The monosaccharide molar ratio of MRP5 was 3.60（rhamnose）:15.50（arabinose）:51.20（glucose）:29.70（galactose）.The backbone chain of MRP5 was composed of→3)-Glcp-（1→,→3,6）-Glcp-（1→,→6）-Glcp-（1→,→3,6）-Galp-（1→and→3）-Galp-(1→.The contents of carbohydrate,protein and uronic acid in MRP5A were separate 87.30,1.40and 9.40%.The average molecular weight of MRP5A was 113.8 kDa.Its monosaccharide molar ratio was 7.70（rhamnose）:6.60（arabinose）:69.70（glucose）:15.90（galactose）.4.The O2-·and·OH free radicals scavenging abilities as well as ferrous ion chelating ability of MRP5 and MRP5A were concentration-dependent manners from 0 to 1.0 mg/mL.The concentrations of MRP5 inhibited 50%O2-·and·OH activity were 122.9 and 315.0μg/mL,respectively.The IC50 of ferrous ion chelating ability was 195.5μg/mL.In addition,the concentrations of MRP5A inhibited 50%of O2-·and·OH activity were separate 575.00and 393.00μg/mL,while the IC50 of ferrous ion chelating ability was 250.70μg/mL.0.5mg/mL MRP5 could prolong the mean lifespan of N2 worms by 21.7%（P<0.001）,increased oxidative stress resistance by 25.30%（P<0.001）,and enhanced gst-4::GFP expression.MRP5A extended the mean lifespan of N2 worms by 14.10%（P<0.05）and increased oxidative stress resistance by 14.20%（P<0.01）.MRP5 had higher bioactive than those of MRP5A in vitro and in vivo.Therefore,MRP5 was selected to elucidate structure and its underlay molecular mechanism of lifespan-extension on C.elegans.5.Under the normal growth condition,0.5 mg/mL MRP5 fast increased ROS content in C.elegans and then reduced ROS level.Under paraquat-induced oxidative stress condition,MRP5 significantly decreased ROS content in C.elegans.Additionally,MRP5 significantly reduced protein carbonyl and MDA content,inhibited polyQ40 aggregation,increased motility of worms and reduced their offspring,but could not affect body size of worms.MRP5 could significantly increase mean lifespan of N2 worms by 18.01%（P<0.0001）under the heat-killed OP50 food sources condition.Above results indicated MRP5 could extend health span of C.elegans,and its lifespan-extension effect was not dependent antibacterial properties.6.MRP5 could promote DAF-16 transcription factor from cell cytoplasm into nucleus,and upregulated expression of daf-16 targeted antioxidant enzyme genes including sod-3 and ctl-2.MRP5 increased mean lifespan of daf-16 loss-of-function mutant by 8.66%（P<0.05）,but could not improve the survival rate of C.elegans under oxidative stress.The results suggested that the lifespan-extension effect of MRP5 on worms acted partially via daf-16gene.7.MRP5 increased the mean lifespan of eat-2（ad1116）II.by 15.3%（P<0.001）,suggested that the effect of MRP5 on lifespan-extension did not act through dietary restriction pathway.MRP5 increased the mean lifespan of hsf-1（sy4411）I.and hif-1（ia4）V.by 6.7%（P<0.05）and 8.3%（P<0.05）,respectively,suggesting that lifespan-extension effect of MRP5was partially dependent on transcription factors HSF-1 and HIF-1.8.RNA-seq results showed 711 genes were differentially expression,of which 284genes were up-regulated and 427 genes were down-regulated.MRP5 significantly affected the monounsaturated fatty acid synthesis pathway.MRP5 not only induced the expression ofΔ9 desaturase genes including fat-6 and fat-7,but also increased the mean lifespan of fat-6（tm331）IV.and fat-7（wa36）V.by 9.30%（P<0.05）and 6.7%（P<0.05）.However,MRP5could not increase the lifespan of fat-6;fat-7 loss-of-function mutant.These findings demonstrated that the lifespan-extension effect of MRP5 was dependent onΔ9 desaturase genes fat-6 and fat-7. |
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