| Objective:Phellodendri chinensis Cortex(PCC)is a traditional Chinese medicine which widely distributed in China and its resources are very rich.In "Chinese Pharmacopoeia",berberine(BBR)and phellodendrine(PHE)are detected as the main active ingredient,but in industry PCC is often used as material to extract BBR while PHE was throwed away in the industrial waste water,that caused a lot of waste and pollution.In this study,we focused on the extraction of PHE from the waste water by simulation of industrial production process.The antioxidant activity and antioxidant mechanism of PHE were researched in this study.This systemic study of PHE can promote the using of PCC perfectly and provide a theoretical basis for the clinical development and application of PHE.Also it is a protective effect on environment.Methods and results:(1)In this study,PHE was enriched by macroporous resin,and then purified by reversed phase silica gel column chromatography.The whole process was analyzed by TLC and HPLC.The results showed when the volume of effluent was 5.5BV the PHE was found,so the maximum loading was 5BV.The purity of PHE was about 70% after recovery by macroporous resin,and the yield was 71.76%.Silica gel column chromatography by using methanol-water gradient elution,and finally using 60% methanol adding a small amount of ammonia water can quickly wash down most of PHE.After concentrated added a certain amount of methanol to crystallize under 4 ?.The purity of PHE after purification can reach 90% by HPLC analysis.Then used a small amount of concentrated hydrochloric acid to dissolve the extract and added the a methanol to recrystallize,which can obtain PHE with about 98% of purity.The final yield is about 35.59 %.(2)The antioxidant activity of PHE was studied in vitro by using chemical analysis and fluorescence spectrophotometry.First,the results were demonstrated that PHE had better ability to remove ABTS +,DPPH free radicals and O2.-in vitro experiments.And the ability of removing DPPH by PHE was stronger than that of berberine at the same concentration in A549 cell.In this study,the ability of removing ROS by PHE was evaluated in A549.The results showed that PHE at 5,25?g/ml had a good effect in scavenging ROS in A549 cells(P <0.5).(3)The antitoxic activity of PHE was investigated by using zebrafish embryo.The fluorescence was also analyzed by fluorescence spectrophotometry using DCFH-DA,DPPP and AO staining to detect the antioxidant effect of PHE.The results showed that different concentrations of PHE had good protective effect on death of zebrafish embryo and abnormal heartbeat caused by AAPH.Especially 200?g/ml of PHE at 72 h h the survival rate of zebrafish embryos increased to 90.26 ± 1.40%.In addition,PHE can remove ROS caused by AAPH in zebrafish,reduce the lipid peroxidation and cell death and in the removal of ROS there is a dose-dependent relationship.(4)The antioxidant mechanism of PHE was studied by using A549 cells and zebrafish embryos respectively.The main genes and proteins were detected by RT-PCR and western blot.The results showed that the expression of AKT,Nrf2,HO-1 and Bcl-2 in the zebrafish embryo was significantly higher than that in the normal group(P <0.05),while was decreased in NF-?B and P53 by treartment with 100?g/ml of PHE.The addition of pathway inhibitor LY294002 and BAY11-7082 can significantly inhibit the expression of downstream protein of AKT and NF-?B,and PHE had the same effect.Conclusion:The PHE was extracted by the macroporous resin and C18 silica gel that commonly used in the pharmaceutical industry.The ethanol,methanol and water as the main extraction reagents are cheap and easy to recyle and have less environmental pollution.In addition,the study showed that PHE had good antioxidant activity in vitro and in vivo.In vitro,it was found that the effect of PHE removing DPPH stronger than that of berberine.In vivo PHE had a better effect of clearing ROS,anti-lipid peroxidation and improved the survival rate of the embryo.The mechanism was found that AKT / Nrf2 / HO-1 which was the critical anti-oxidation defense system was activated by PHE in zebrafish embryo.On the other hand,NF-?B is not only associated with antioxidant but also an important pathway in inflammatory response,so it has been studied more in A549 cells by western blotting.It has been found that PHE can prevent ROS-induced inflammatory expression and mainly inhibit IKK / NF-?B pathway,and it can also significantly reduce the expression of COX-2. |
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