Extraction And Purification Of Superoxide Dismutase From Phyllanthus Emblica L.and Effects Of Flavonoids And Light On Activity

China has an abundant resource of Phyllanthus emblica Linn.,so it has broad potential of development.However,it cannot be directly used as food,due to cool nature,sweet,sour and bitter flavor.For this reason,the development and utilization of Phyllanthus emblica Linn.as natural resource has been neglected.Most of the existing researches focus on process,while few studies on physiologically active ingredients,health functions and its development and utilization remain in the infancy stage,but subsequent work is needed.Findings show that Phyllanthus emblica Linn.is rich in superoxide dismutase?SOD?,which can catalyze the disproportionation of superoxide free radicals?O₂^-?in bodies and function as a natural eliminator,also credited as“body garbage scavenge”.In this study,Phyllanthus emblica Linn.was used as raw material,the extraction and purification techniques of SOD were examined,and the in vivo and in vitro activity studies were performed with the extracted and purified SOD.Main results are described below:?1?The yield rate of SOD was used as observation value,main factors were screened out of microwave power,microwave time,solid-liquid ratio,ultrasonic power,ultrasonic time,and heat deflection temperature?HDT?using Plackett-Burman's experimental design,the main factors were found as HDT,ultrasonic time,and ultrasonic power.The process parameters for extraction of SOD from Phyllanthus emblica Linn.were optimized using Box-Benhnken's CCD experimental design.When the ultrasonic time was 12min,ultrasonic power was 350W;when the HDT was 52.5?,the maximum yield rate was 107.15mg/100g.?2?In this experiment,SOD was separated and purified out of SOD by the fractional precipitation,dialysis and ultrafiltration with ammonium sulfate,in conjunction with SephadexG-100 column chromatography.The activity of SOD precipitated under the saturation of 20%-90%ammonium sulfate was examined here,when the saturation was 50%and 80%,the activity of SOD in Phyllanthus emblica Linn.was enhanced.Therefore,the saturation of 50%and 80%was the parameter of fractional precipitation,as the theoretical evidence for the next step of experiment.Then,the ammonium sulfate with the saturation of 50%and 80%was subjected to fractional precipitation to obtain the purification factor as 1.77X and the recovery rate as 69%.When the saturation was 90%,no protein completely precipitated out of SOD.Obviously,the precipitation with ammonium sulfate cannot completely purify SOD,so further purification process should be needed.SOD can be further extracted and purified through dialysis and ultrafiltration,the process of 8h dialysis and 15min ultrafiltration was operated to obtain the purification factor as 7.93X and the recovery rate as 59%.Advantages of macroporous resin include strong adsorption and affordable price,so SOD was purified using SephadexG-100 column chromatography to obtain the purification factor as 64.64 and the recovery rate as up to 37%.?3?Phyllanthus emblica Linn.was inferred to contain Fe-SOD and Mn-SOD by UV absorption spectroscopy.The effect of separating and purifying SOD out of Phyllanthus emblica Linn.was better if the fractional precipitation,dialysis and ultrafiltration with ammonium sulfate was used in conjunction with SephadexG-100 column chromatography.After the purification process,bands mainly concentrated at 40KD,Phyllanthus emblica Linn.was thus inferred to contain Fe-SOD.According to atomic absorption spectrum?AAS?,the content of Fe,Mn in SOD of Phyllanthus emblica Linn.was 8.95mg/kg and 0.31mg/kg respectively.Obviously,Phyllanthus emblica Linn.mainly contains Fe-SOD and only has a few Mn-SOD.?4?The effects of different amounts of flavonoids on the enzymatic activity of SOD were analyzed,and results show that the higher mass ratio of flavonoids means the more enhanced residual activity of SOD in Phyllanthus emblica Linn.Obviously,the content of flavonoids has a certain influence on the residual activity of SOD.The effects of temperature on the activity of SOD and the mixture of SOD and flavonoids were analyzed,and results show that when the temperature was 0?-60?,the addition of SOD into flavonoids had no obvious influence on the enzymatic activity of SOD;over 60?,the addition of SOD into flavonoids had a certain influence on the enzymatic activity of SOD.The effects of flavonoids on the enzymatic activity of SOD under high temperature were analyzed,and results show that flavonoids may damage the enzymatic activity of SOD under high temperature.The effects of PH on the enzymatic activity of SOD and the mixture of SOD and flavonoids,and the effects of flavonoids on the enzymatic activity of SOD under acidic condition were analyzed,and results show that no obvious change was found in pH5.0-12.0,while under strong acid condition,flavonoids can protect the enzymatic activity of SOD.The effects of flavonoids on the enzymatic activity of SOD in the simulated digestion process were analyzed,and results show that under simulated gastric fluid,simulated intestinal fluid and artificial gastrointestinal fluid,flavonoids would give a certain protection on the enzymatic activity of SOD in Phyllanthus emblica Linn.A set of experiments as described above not only paved a theoretical foundation for the subsequent work about the effects of flavonoids on enzymatic activity of SOD in Phyllanthus emblica Linn,but also provided some data support for industrial products.?5?In this experiment,the effects of different lighting qualities on the enzymatic activity of SOD were examined,results show that green,blue and red light can promote the enzymatic activity of SOD in Phyllanthus emblica Linn.,while yellow and purple light would suppress the enzymatic activity of SOD in Phyllanthus emblica Linn.Box-Benhnken model was constructed by screening out certain lighting conditions,i.e.:lighting temperature:30?,35?and 40?,lighting duration:30min,60min and 90min,and lighting intensity:1500lux,2000lux and2500lux.The regression equation was analyzed using Design Expert8.0 to indicate that the best lighting condition is:lighting duration:63min,lighting intensity:2150lux,lighting temperature:31?.The residual activity of SOD in Phyllanthus emblica Linn.was 90.37%.