Protective Effects Of Laminaria Japonica Extract Against Paraquat-induced Oxidative Stress Damage In Caenorhabditis Elegans

Reactive oxygen species （ROS） is the intermediate product of the normal metabolism inorganism. The life survival in the aerobic organism inevitably exposed to reactiveoxygen species. ROS of normal life could maintain the balance of oxygen and redox,which mainly depended on the antioxidant system. Oxidation and reduction was out ofbalance when the body was exposed to a variety of stimulation. When a large amount ofROS was producing beyond the capacity of antioxidant, the biological macromoleculessuch as proteins, fat and nucleic acid was attacked, resulted oxidative stress damage inthe body tissue, a variety of neurodegenerative diseases such as PD and AD will beinduced. Therefore, an important strategy for prevention and treatment of this kind ofdisease was to remove the excess ROS in the body. Many studies found that somenatural plant extracts had potential antioxidant activity. Several natural plant extractswere selected for evaluation of antioxidant activity in this study, one of those extractswas chosen to evaluate the antioxidant activity in complete life. The main results wereas follows:1. Comparison of antioxidant/reduction capacity and free radical scavengingactivity of four natural plant extractsObjects: To compare the differences of the capacity of total antioxidant, reducing andscavenging superoxide anion ability in vitro, we purpose to choose the strongestantioxidant from Laminaria japonica Extract （ELJ）, HericiumerinaceusPolysaccharides （HEP）, Sargassum pallidum Polysaccharides （SPP） and Achyranthesbidentata Glycopeptide （ABG）.Methods: The total antioxidant capacity assay kit （FRAP method） was used to evaluatethe total antioxidant capacity. The principle is that antioxidant could reduce Ferric-tripyridyltriazine (Fe³⁺-TPTZ) to Fe²⁺-TPTZ under the conditions of acidity. Thesample’s total antioxidant was obtained in593nm. The potassium ferricyanide reducingwas used to determine the capacity of reducing. The reduction ability of antioxidantswas determined by the ability of conversion from Fe³⁺to Fe²⁺. The NBT-photoreductionmethod was used to detect the ability of superoxide anion scavenging. VB2and EDTAreacted under illumination, superoxide anion was produced, superoxide anion and NBTwas reacted, and the maximum absorption peak was read at560nm.Results: The order of total antioxidant activity of the four kinds of natural plant extractsare ELJ，HEP，SPP，ABG.The order of reducing capacity are HEP，ELJ，ABG，SPP.The order of scavenging supeioxide anion are ELJ，HEP，ABG，SPP. ELJ had theoverall strongest antioxidant activity.2. Protective effects of Laminaria japonica extract against paraquat inducedoxidative stress damage in Caenorhabditis elegansObjects: To determine the antioxidant activity of ELJ in complete life such as modelorganisms. To explore the changes of antioxidant activity in vivo.Methods: The paraquat-induced oxidative stress model of Caenorhabditis elegans wasprepared. After ELJ intervention, the antioxidant effective was evaluated by the bodysize, adult rate, brood size and et al. The level of ROS, apoptosis and expression ofstress-related genes were examined to reveal the mechanism of protection effect of ELJ.Results: The ELJ could restore the body size of C. elegans after PQ-injured, improvethe adult rate, brood size, reduce the ROS level, decrease the number of apoptotic germcell, and also assist the expression of the stress-related genes daf-16/sod withantioxidant effect. 3. Eeffect of the inactive bacteria OP50wi th different methods on the growth anddevelopment of Caenorhabditis elegansObjective: To find a suitable method for preparing the inactivated Escherichia coliOP50based on the influence of the growth and development of Caenorhabditis elegans.Methods: The E. coli strain OP50was inactivated by heat treatment （65℃） and60Coirradiation （2000Gy） respectively following with the observation of the growth of C.elegans. The activity of succinate dehydrogenase was further analyzed by the MTTmethod to determine the effect of these two operations on protein activity of OP50.Results: There is no difference on the growth of C. elegans between the60Co irradiationgroup and control group, however the development of heat treatment group wassignificantly delayed compared to the control group.3-（4,5）-dimethylthiahiazo（-z-y1）-3,5-di-phenytetrazoliumromide （MTT） analysis showed that after heat treatment, thesuccinate dehydrogenase activity of OP50was significantly lower than that of thecontrol group and60Co irradiation group. The inactivated method of60Co irradiation is asuitable treatment method of OP50in the C. elegans experiment. The different effect of60Co irradiation and heat treatment of OP50on C. elegans growth may be due to thedeterioration of bacterial proteins activity. Our study provides important reference forimproving the reliability of studies using C. elegans by eliminating the potential factorsthat might affect the growth and development of C. elegans through OP50indirectly.