Anti-oxidative Stress Effect And Its Mechanism Of Bilberry (Vaccinium Myrtillus L.) Extract

Object:This experiment,having oxidative stress as a main line,researched the changes of antioxidants and antioxidative enzymes in mice,the variety of mitochondria function and mitochondrial membrane potential.It would discuss the possible mechanism of Bilberry(Vaccinium myrtillus L.) extract improving the status of oxidative stress.Method:7-week old Kunming mice were used in this experiment.(1) The contents of bilberry extract were analyzed with HPLC-ESI-MS.And antioxidative effect in vitro was evaluated with ORAC and DPPH methods.(2) The effect of bilberry extract on survival time and oxygen consumption were observed in mice suffered from acute normobaric hypoxia,and the malondialdehyde(MDA),Oxygen radical absorbance capacity(ORAC) and NO content in plasma,brain and liver tissue were determined respectively.(3) A single ip administration of KBrO₃ at a dose of 200mg·kg^-1 induced severe kidney damage. And then ORAC in kidney was determined by automated multifunction microplate reader.Serum creatinine level was determined by HPLC.Spectrophotometric method was performed to detect the levels of blood urea nitrogen(BUN),kidney MDA.Griess method was performed to detect NO levels in kidney.(4) After 18 h restraint stress,the ALT activity in plasma,MDA and ORAC value in plasma and liver,Glutathione(GSH) and vitamin C content,Xethione(XOD) and NO level in liver were determined.(5) We observed mitochondrial function through optical microscope,examined the respiratory chain complex,cytochrom content and Na⁺-K⁺-ATPnase through spectrophotometry instrument, measured mitochondria membrane potential through the Flow Cytometer,examined the changes of the expression of liver SDHA-D and COâ…ª-â…¢through the method of RT-PCR.Result:(1) In the HPLC-ESI-MS analysis,bilberry extract contained 420.33mg of anthocyanins expressed as cyanidin-3-glucoside.(2) ORAC value indicated that antioxidative capacity of bilberry extract is stronger than that of Vitamin C in vitro.(3) Bilberry extract prolonged the survival time and decreaed oxygen consumption in mice suffered from acute normobaric hypoxia.It also enhanced ORAC and decreased MDA content in plasma,brain cortex,and liver tissues,and modulated levels of NO in plasma,brain cortex and liver tissues.(4) Our experiment showed that administration of 200mg·kg^-1 KBrO₃ could induce serious kidney damage,with an increase in BUN and creatinine levels.Five-day oral administration of anthocyanin extract resulted in a reversal in serum BUN and creatinine to normal levels and decreased kidney MDA,NO and XOD levels.Also,bilberry extracts improved ORAC levels in kidney tissue,which showed that bilberry extract reduced the degree of oxidative stress and kidney damage induced by KBrO₃.(5) Our experiments showed that 18h restraint stress could induce serious liver damage,with an increase in plasma ALT activity.Meanwhile,we observed increased MDA levels and lowered ORAC values in plasma and liver of restraint stress mice compared with normal mice.Bilberry extract was oral administrated to mice for 5d,which remarkedly decreased ALT activity,and protect the liver damage induced by oxidative stress.In addition,Bilberry extract could effectively increase the ORAC value,GSH content and reduce the MDA and NO levels in liver.(6) Bilberry extract improved mitochondrial function:mitochondrial membrane potential,the activities of respiratory complexs,Na⁺-K⁺-ATPase and the expression of related gene through scavenging ROS。 Conclusion:Bilberry extract play important roles in improving oxidative stress by scavenging free radical,keeping the balance of antioxidant and oxidant,protecting mitochondrial function.This study showed the beneficial effects of bilberry extract through its antioxidative action.Novel Points1 Anti-oxidative stress effect of bilberry extract was studied with these anima models in the first time.2 Restraint stress selectively affected the second chain of respiratory chain.3 Bilberry extract alleviated the status of oxidative stress through improving mitochondrial function.