Studies On The Aging-delay Effect Of Polysaccharide From A Complex Decoction In Caenorhabditis Elegans

Objective:Factors like ROS?reactive oxygen species?will cause irreversible aging and effective strategies and approaches need to be found to cope with the accelerating aging population.Study of the traditional Chinese medicine will contribute to its development and modernization.Many researchers used cells or mice to search for new activities of polysaccharides from the traditional Chinese medicine and proofed their aging-delay activity.However,we studied a complex decoction composed primarily of Astragalus radix and other life-prolonging herbs using the model animal Caenorhabditis elegans?C.elegans?.We focus not only on the overall decoction,but main ingredients to analyze the aging-delay function and physical and chemical properties of the traditional Chinese medicine.We hope our studies provide modernization of the traditional Chinese medicine with more helpful theoretical foundation.Methods:1.The anti-aging activity of the initial complex decoction?0.25,0.5,1,2,4 and 8 mg/mL?was examined by lifespan assay using wild-type C.elegans.2.After precipitation with ethanol for the fraction of polysaccharide,the complex decoction was then extracted into 5 fractions using chemical reagents with different polarities,and the 6 fractions were tested for their effects on C.elegans lifespan.3.Polysaccharides were extracted from main medicine materials included in the complex decoction and used to lifespan assay.4.The C.elegans were firstly administrated with all the drugs respectively,After 3 days,500?M juglone were added to make animal ROS stress and the survival was detected.To test the antioxidative activity of the polysaccharides in vitro,we measured the DPPH radical scavenging activity,ABTS⁺scavenging activity and metal chelating activity.Synchronizing C.elegans were administrated with the whole drugs respectively.After 10 days,50?M DCFH-DA was added to detect the ROS level in C.elegans.5.Lavals in L1 state were administrated with the whole drugs respectively and OD₅₇₀ value of every well was measured at 24-hour intervals in the next 5 days.C.elegans eggs were treated with the whole drugs respectively.After 2 days,animals that hatched out were scored to calculate the hatch rate.After administration,Lavals in L1 state were used for body lenghth measure every 24 hours in the next 96 hours.6.Total carbohydrate was detected by phenol-sulfuric acid method.Residual protein was quantified by BCA kit.Representative compounds in the complex decoction and its fractions were identified by thin layer chromatography.Functional groupes were analyzed by infrared spectroscopy?IR?at a scanning scope of between 4000 and 400 wave numbers and resolution of 4 wave numbers.Gas chromatography?GC?equipped with DB-5 capillary column?30 m×0.25 mm×0.25?m?and hydrogen flame ionization detector was used to analyze the monosaccharide composition of polysaccharides.Results:1.The decoction and its polysaccharide fraction was able to prolong C.elegans lifespan around 16%?P=0.0002?.2.The polysaccharide,petroleum ether extract,ethyl acetate extract,n-butanol extract,ammonia extract and remaining parts of the water layer were extracted from the complex decoction and only the 8 mg/mL polysaccharide show aging-delay effect.3.All polysaccharides extracted from main medicine materials included in the complex decoction showed significant aging-delay effect in C.elegans.Their optimal concentrations were respectively 1 mg/mL?Rehmanniae radix praeparata?,0.25 mg/mL?Astragalus radix?,0.25mg/mL?Mori fructuc?,1 mg/mL?Ecliptae herba?and 0.25 mg/mL?Polygoni multiflori radix?.4.In vivo,8 mg/mL polysaccharide from the complex decoction improved the suvival of C.elegans in juglone stress.Polysaccharide from Rehmanniae radix praeparata showed best effect at 2 mg/mL.Polysaccharide from Astragalus radix showed no effect but speeded up the death of animals.Polysaccharide from Mori fructuc showed effect at 2mg/mL in an hours.Polysaccharide from Ecliptae herba showed obvious effect at the concentration of greater than or equal to 2 mg/mL.Polysaccharide from Polygoni multiflori radix showed effect at 8 mg/mL.,All drugs showed antioxidation in vitro and reduced the relative ROS fluorescence intensity in C.elegans.5.All drugs had no significant effect on food scavenging rate,egg hatchability and body length in C.elegans.6.Extraction rate of polysaccharides were respectively 8.6%?complex decoction?,7.0%?Rehmanniae radix praeparata?,3.7%?Astragalus radix?,3.9%?Mori fructuc?,2.2%?Ecliptae herba?and 1.3%?Polygoni multiflori radix?.IR showed the typical spectroscopy of polysaccharide.GC showed polysaccharide from the complex decoction consisted of Ara?5.1%?,Rha?0.2%?,GalA?8.2%?,GlcA?6.6%?,Man?12.4%?,Gal?30.7%?,Glc?36.9%?.Conclusion:The complex decoction may exert anti-aging activity through its polysaccharide extract,without influence on life quality of C.elegans.It is possible that several polysaccharides from the main medicine materials of complex decoction contribute to the decrease of ROS together.