Extraction And Separation Of Acylated Anthocyanins From Nitraria Tangutorum Bobr.Fruit And It's Mechanism Of Improving Doxorubicin-induced Acute Heart,Liver,and Kidney Injury In Mice

Doxorubicin is a first-line chemotherapy drug commonly used in clinical treatment for a variety of malignant tumors.However,the clinical application of adriamycin is largely limited by severe side effects on non-target organs,resulting in toxicity of heart,liver,kidney and other organs.Therefore,it is urgent to develop natural products to protect the toxicity of doxorubicin.Nitraria tangutorum Bobr.fruit is an indigenous berry of the shrub belonging to the Zygophyllaceae family which mainly grows at an altitude of over 3000 m in the Tibetan Plateau.Modern pharmacological studies show that anthocyanins from N.tangutorun fruit have antioxidant activities in vitro and in vivo.The current study aimed to explore a systematic approach for extraction,purification and analysis of acylated-anthocyanins from N.tangutorun fruit,and investigate possible protective effects of anthocyanins from N.tangutorum Bobr.fruit against doxorubicin-induced acute cardiotoxicity and hepatornel toxicity in mice.Pharmacological experiments were carried out to explore the molecular mechanism,which laid a scientific foundation for the study of drugs to improve doxorubicin-induced cardiotoxicity,hepatic toxicity and renal toxicity.The main research contents and results are as follows:1.Extraction was a very important step to recover valuable compounds,and ultrasound treatment was an alternative affordable,effective and reproducible technology for improving bioactive compound yield.Six acylated-anthocyanins in N.tangutorun fruit were identified by HPLC-MS/MS in this study.Ultrasonic-assisted extraction conditions of acylated-anthocyanins were optimized using response surface methodology,extraction at 70? for 32 min using 70%methanol solution(0.1%HC1,v/v)rendered an extract with 80.37± 2.66 mg/100 g of cyanidin-3-O-(trans-p-coumaroyl)-? diglucoside and 97.88±4.06 mg/100 g of total acylated-anthocyanins.2.Nine macroporous resins were investigated for preliminary purification of acylated-anthocyanins from N.tangutorum.According to the static/dynamic adsorption and desorption tests,XDA-6 macroporous resin exhibited the maximum potential for preparing acylated-anthocyanins from N.tangutorun.The purity of MA(43.30 mg/g)in purified acylated-anthocyanins was 201.89 times of that of the extract(0.21 mg/g),and the purity of total acylated-anthocyanins increased from 0.36 mg/g to 56.44 mg/g.In addition,compared with the stability of extracts and total anthocyanins,the stability of MA and TA in purified acylated anthocyanins was significantly improved.3.A mouse model of acute heart injury induced by doxorubicin was established.Cardiac injury was determined by detecting the expressions of CK-MB and LDH in serum cardiac pathological indicators and the histopathological sections of cardiac tissue in order to study the cardioprotection of acylated-anthocyanins from N.tangutorum.in Dox-treated mice.The cardioprotective effects of acylated-anthocyanins from N.tangutorun were evaluated by estimation of cardiac MDA,SOD,GSH,TNF-?,and IL-1? contents activity.Moreover,expression of Bcl-2,Bax and caspase-3 in heart was determined by western blot.The results demonstrated that acylated anthocyanins from N.tangutorun significantly reduced serum CK-MB and LDH levels with alleviations of histopathological abrasions in Dox-induced mice.It limited oxidative stress via lowering both cardiac MDA TNF-a,and IL-1? contents and restoring cardiac content of reduced GSH and SOD activity.Studies on proteins in the internal apoptosis pathway showed that acylated anthocyanins in the fruit of N.tangutorum.could up-regulate the expression of anti-apoptotic protein bcl-2,down-regulate the Bax/bcl-2 ratio and the expression of pro-apoptotic proteins Bax,caspase-9 and caspase-3.Therefore,acylated anthocyanins in the fruits of N.tangutorum can protect against adriamycin-induced acute heart injury by regulating oxidative stress and internal apoptosis pathways.4.A mouse model of acute hepatornel injury induced by doxorubicin was established.Hepatornel injury was determined by detecting the expressions of aspartate transaminase(AST),alanine transaminase(ALT),blood urea nitrogen(BUN)and creatinine(Cr)and the histopathological sections of liver and kidney tissue.The hepatornel protective effects of acylated-anthocyanins from N.tangutorun were evaluated by estimation of hepatic and renal MDA,SOD,GSH,TNF-?,and IL-1?contents activity.Moreover,expression of Bcl-2,Bax and caspase-3 in liver and kidney were determined by western blot.The results demonstrated that anthocyanins from N.tangutorun significantly reduced serum AST,ALT,BUN and Cr levels with alleviations of histopathological abrasions in Dox-induced mice.It limited oxidative stress via lowering both hepatic and renal MDA TNF-?,and IL-1? contents and restoring hepatic and renal content of reduced GSH and SOD activity.Studies on proteins in the internal apoptosis pathway showed that acylated anthocyanins in the fruit of N.tangutorum Bobr.could up-regulate the expression of anti-apoptotic protein bcl-2,down-regulate the Bax/bcl-2 ratio and the expression of pro-apoptotic proteins Bax,caspase-9 and caspase-3.Therefore,acylated anthocyanins in the fruits of N.tangutorum Bobr.can protect against adriamycin-induced acute hepatornel toxicity by regulating oxidative stress and internal apoptosis pathways.