The Synthesis Process Optimization Of Coptisine And Its Antioxidant Activity Analysis

Oxidative stress is a kind of pathological state in which excess reactive oxygen species(ROS)is produced and exceeds the removal ability of antioxidant defense system to lead to oxidation and antioxidant imbalance.A lot of ROS will attack the biological macromolecules and cause lipid peroxidation damage,protein damage,DNA damage and the subsequent cell apoptosis.To a certain extent,the functional damage caused by oxidative stress in cells and tissues is associated with the occurrence and development of systemic diseases.Oxidative stress is likely to lead to the occurrence of the cardiovascular system disease,nervous system disease,endocrine system disease,respiratory system disease and cancer,and so on.Thus,like the risk factors of human metabolic disease,such as high blood sugar,high cholesterol and high triglycerides,oxidative stress has serious impact on human health,and it is particularly important to look for antioxidant activity substances for improving and controling the human chronic diseases.Rhizoma Coptidis is one of the top 50 in the most commonly used traditional Chinese medicines.Its use has a long history in the treatment of traditional Chinese medicine.And the main active constituents of Rhizoma Coptidis are several kinds of alkaloids,such as berberine,coptisine,palmatine,epiberberine and jatrorrhizine.The five kinds of alkaloids are all quinoline derivatives,are only with the different substituent,and possess similar chemical structure and similar physiological activity.Among them,coptisine presents various pharmacological activities,including: antibacterial,gastric mucosa protection,anti-hypercholesterolemia,anti-hypertriglyceridemia,anti-obesity and antioxidant.The antioxidant activity of coptisine is mainly reported about coptisine's radical scavenging activity in vitro and changing the oxidative stress markers in vivo.But coptisine's resistance effects on the pathological states in oxidative stress animal model and antioxidant potential mechanism are still not clear.Therefore,this project aimed to further explore the antioxidant activity of coptisine and its potential mechanism of action,which will provide the theoretical foundation for comprehensive development of Rhizoma Coptidis.Meanwhile,the actvity study of coptisine needs to prepare this compound.This project also aimed to use the method of chemical synthesis to product coptisine largely,improve synthesis yield of coptisine and get coptisine of high yield by optimizing the synthesis process.We took end product coptisine as the material source of research on pharmacological activity.The main contents and results of this project are as follow:(1)With 2,3-dihydroxybenzaldehyde as the starting material,the coptisine was synthesized after cyclization,condensation and closed-loop.The important improvements included using methanol for recrystallization instead of silica gel column chromatography to separate and purify intermediates in the second step,designing the reaction medium of different acidic properties and acid strength in the third step to screen the best reaction condition,to significantly increase the yield of the total synthesis of coptisine,to shorten the reaction time and avoiding the operation of regulating pH in the purification process of third step.We finally chose formic acid supplemented with 5% phosphoric acid(formic acid volume: phosphoric acid volume = 1:5 %)as the reaction solvent of last step to react for 3 hours at 90 ?,and got coptisine of high yield.The total yield of coptisine was increased from 13.7% of the original document to 43.0%,and the coptisine's purity is as high as 98.5%.(2)Preliminary exploration was conducted about the coptisine's antioxidant effect on oxidative stress zebrafish embryos induced by AAPH.The results showed that the oxidative stress inducer AAPH significantly increased the ROS generation,heart beating rate,lipid peroxidation and cell death in zebrafish embryos.However,after coptisine was given for treatment,we found that it had good resistance effects on these pathological states.Especially,high dose coptisine could effectively decrease ROS production,reduce the heart beating rate,inhibit lipid peroxidation and cell death.Its resistance effects made each indicator was significantly reduced by 41.3%(p < 0.01),24.9%(p < 0.01),26.5%(p < 0.01),30.0%(p < 0.01)respectively,compared with model group.(3)With using real-time fluorescent quantitative PCR to explore the antioxidant mechanism of coptisine on AAPH-induced oxidative stress zebrafish embryos,we found that AAPH-induced oxidative stress made the mRNA expression of antioxidant related genes(NQO1,Nrf2,Akt,JNK,p38 and ERK)significantly reduced in zebrafish embryos,compared with the normal group.However,after high dose coptisine of 10?g/mL was given for treatment,it could significantly up-regulate the expressions of these antioxidant related genes.Compared with model group,the mRNA expression of NQO1,Nrf2,Akt,JNK,p38 and ERK was respectively increased by 44.9%(p < 0.05),75.8%(p < 0.01),50.0%(p < 0.01),31.1%(p < 0.01),16.3%(p < 0.05)and 34.7%(p < 0.01).This suggested that coptisine could up-regulate the gene expression of antioxidant related targets at the transcription level,so as to exert antioxidant activity.(4)We explored the antioxidant effects of coptisine on AAPH-induced oxidative stress in HepG2 cells.Results showed that the stimulation of AAPH led to significantly increase ROS,significantly decrease the content of GSH and significantly reduce SOD and GPx activities in HepG2 cells.But coptisine could significantly inhibit the ROS production,improve the content of endogenous antioxidant GSH and enhance activities of antioxidant enzymes SOD and GPx to exert the antioxidant activity.High dose coptisine respectively reduced ROS,increased the GSH content and enhanced SOD and GPx activities by 40.1%(p < 0.01),19.8%(p < 0.01),18.3%(p < 0.05)and 49.3%(p < 0.01),compared with model group.(5)Using western blot method,we further studied the antioxidant mechanism of coptisine on AAPH-induced oxidative stress in HepG2 cells.Data showed that when HepG2 cells suffered from oxidative stress,its NQO1,Nrf2,p-Akt and p-JNK protein expression level were significantly decreased,compared with the normal group.And after the intervention of high dose coptisine,the protein expression of above-mentioned target were extremely significantly increased by 117.2%,34.6%,358.8% and 221.1% respectively,compared with the model group.At the same time,high dose coptisine could promote the nuclear translocation of Nrf2.We concluded that coptisine activated Akt and JNK signaligs through promoting their phosphorylation,also activated Nrf2 and subsequent signal transduction by promoting Nrf2 protein expression and its nuclear translocation,and ultimately induced NQO1 expression.Finally,as corresponding inhibitors of upstream kinase(Akt,JNK,p38 and ERK)and coptisine were co-treated,we found that the activation of Nrf2/NQO1 pathway was dependent on the activation of the upstream signalings Akt and JNK,namely “Akt and JNK” and Nrf2/NQO1 pathways existed the cascade relationship.In conclusion,this study optimized the synthesis process of coptisine,improved the total yield of coptisine synthesis and got highly pure end product coptisine.Coptisine exerted antioxidant activity through activating Akt and JNK/Nrf2/NQO1 pathway,and its antioxidant activity was mainly characterized by scavenging ROS,reducing the heart beating rate,inhibiting lipid peroxidation and cell death,increasing the GSH content and enhancing the activities of SOD and GPx.