| Lycium ruthenicum Murr.is a perennial shrub of the genus Solanaceae(Lycium),which widely distributes in salinized areas of the Qinghai-Tibet Plateau.The major constituents of L.ruthenicum are mainly polyphenols,flavonoids and anthocyanins.As an effective anti-tumor drug,doxorubicin(Dox)is still a potent drug for most chemotherapy regimens in clinical use.However,the organ toxicity associated with clinical use cannot be ignored.Therefore,it is valuable to use new therapies to reduce Dox-induced organ toxicity without affecting anti-tumor activity.The pharmacological activities of anthocyanins from L.ruthenicum mainly include anti-oxidation,scavenging free radicals,and preventing cardiovascular diseases.Therefore,the extraction method of anthocyanins,separation,analysis and biological activity are of great significance for the development of L.ruthenicum.The main researches are as follows:1.Pectinase was used to extract the anthocyanins from L ruthenicum,the effects of extraction temperature,extraction time,pH,pectinase dosage and ratio of material to liquid on the anthocyanins yield were studied.On the basis of single factor,the optimization scheme of the response surface method for the auxiliary extraction of anthocyanins was designed.The optimal extraction conditions were as follows:extraction at 38.21℃ for 37.27 min using pectinase 52.04 mg/100 g rendered an extract with 19.51±0.21 mg/g.The optimal extraction conditions of total polyphenols were as follows:extraction at 50℃ for 45.65 min using pectinase 60 mg/100 g.Under these conditions,the extraction rate of total polyphenols was 268.67 ± 3.52 mg/g.2.A method for separation and purification of anthocyanins from L.ruthenicum was established.The XDA-6 macroporous adsorption resin was determined by static adsorption/desorption experiment.Dynamic adsorption/desorption experiment to obtain the process for separation and purification of anthocyanin from L ruthenicum.The aspect ratio was 1:20,and the concentration of the sample was 0.647 ± 0.024 mg/ml,loading volume was 22.3 BV,flow rate was 1 ml/min;elution process:gradient elution with water(0.1%HCl,v/v)and 70%ethanol water(0.1%HCl,v/v),the amount was for 4 BV,the elution flow rate was 2 BV/h.The obtained product has an anthocyanin content of 16.57%.3.HPLC-DAD and HPLC-ESI-MS/MS were using to analyze anthocyanins from L.ruthenicum.13 anthocyanins were separated.In our results,Petunidin-3-O-rutinose(trans-p-coumaric acid)-5-O-glucoside was determined as the most abundant anthocyanins in L.ruthenicum.4.The antioxidant activities of anthocyanins were determined by DPPH scavenging free radical experiment,hydroxyl radical scavenging experiment and ABTS free radical scavenging experiment.It was proved that the anthocyanin has strong antioxidant activity.5.The study on the protective effect of anthocyanins of L.ruthenicum on Dox-induced cardiotoxicity in mice showed that the anthocyanins from L.ruthenicum could significantly reduce the increase of serum CK-MB and LDH levels induced by Dox.At the same time,the anthocyanins from L.ruthenicum significantly reduced the levels of MDA,TNF-a and IL-1β in cardiac tissues and significantly increased the enzyme activities of SOD and GSH.Cardiac pathological sections showed that the anthocyanins from L.ruthenicum could improve inflammatory cell infiltration and myocardial fiber breakage.The expression of apoptotic proteins showed that anthocyanins could down-regulate the expression of Bax and Caspase-9 in Dox-induced cardiac tissue and up-regulate the expression of Bcl-2.Therefore,the anthocyanins from L.ruthenicum protected against Dox-induced cardiotoxicity by mitigating oxidative stress and inflammatory responses,and modulating Bax/Bcl-2-mediated mitochondrial apoptosis signals to inhibit apoptosis.6.The results of the protective effect of L.ruthenicum anthocyanins against Dox-induced hepatorenal toxicity in mice showed that L.ruthenicum anthocyanins could significantly reduce the serum ALT,AST,Cr and BUN levels.Meanwhile,L.ruthenicum anthocyanins markedly decreased the levels of MDA,TNF-a,IL-1β,and notably increased the levels of SOD and GSH.Histopathological sections of liver and kidney showed that the anthocyanins of L.ruthenicum can improve Dox-induced interstitial vasodilation and inflammatory cell infiltration of liver and kidney.In order to investigate the effect of the anthocyanins on the apoptotic pathway,the expression of proapoptotic proteins Bax,Caspase-9 and anti-apoptotic protein Bcl-2 in liver and kidney tissues were determined by Western blotting.The results showed that the anthocyanins from L.ruthenicum could down-regulate the expression of Bax and Caspase-9 proteins in liver and kidney tissues induced by Dox and up-regulate the expression of Bcl-2.Therefore,the anthocyanins inhibited Dox-induced hepato-renal toxicity by mitigating oxidative stress and inflammatory responses,and modulating Bax/Bcl-2-mediated mitochondrial apoptosis signals to inhibit apoptosis. |
PDF Full Text Request