Cytotoxicity And Mechanisms Of Water Extract Of Rhizoma Coptis And Berberine

Rhizoma Coptis(RC)has been a Chinese herbal medicine used commonly for a long time."Shen Nong’s Herbal Classic" records it serves as the medicinal part with bitter taste,cold nature and tropism to the channels of heart,liver,stomach and large intestine.It belongs to the antipyretic category ascribed to its role played in clearing away the damp-heat,purging fire away from the heart and eliminating toxic materials.Multiple bioactivities of RC and its main component berberine has been demonstrated by modern pharmacological researchers,such as the effects of antimicrobial,anti-inflammatory,antineoplastic,anti-Alzheimer,hepatoprotective,analgesic and antihyperglycemic.However,with the continuous occurrence of adverse reactions in recent years,RC and berberine security issues caused widespread concern,mainly seen in the cases of extrapyramidal reactions,cardiac arrhythmia,and even death,but the toxicity and the relevant mechanisms of RC and berberine,especially researches at the cellular and genetic levels,are still not quite clear.In this experiment,we used L929,BRL,GES-1 and NRK four kinds of cells in vitro as the research objects.Morphological changes were observed by inverted microscope,cell viabilities were determined by CCK-8 assay,cell cycle distribution and reactive oxygen species(ROS)were detected by flow cytometry,comet assay was used for DNA damage.These preliminary studies tried to explore the cytotoxicity and mechanisms of RC and berberine.The specific test results are as follows:(1)Observation results of cell morphology: concentrations of RC higher than 1 mg/mL,0.25 mg/mL,0.25 mg/mL and 3 mg/mL could significantly change the L929,BRL,GES-1 and NRK cells normal morphologies,respectively.And even made these cells off or floating.(2)Results of cell viability test: RC at concentrations above 1 mg/mL,0.1 mg/mL,0.25 mg/mL and 3 mg/mL could significantly inhibit L929,BRL,GES-1 and NRK cell viability,respectively.The survival rate was in a dose-dependent manner;compared with control group,treatment groups of berberine at concentrations above 0.05 mg/mL,0.04 mg/mL,0.05 mg/mL and 0.25 mg/mL has significant difference(P<0.01),respectively;Meanwhile,before showing the effect of inhibition on proliferation,RC and berberine first exhibited the effect of promoting cell growth at low concentrations.(3)Results of cell cycle distribution test: RC at concentrations of 2 mg/mL,1 mg/mL,2 mg/mL and 3 mg/mL,berberine at 0.1 mg/mL,0.05 mg/mL,0.05 mg/mL and 0.25 mg/mL could cause cell cycle of L929,BRL,GES-1 and NRK arrest,respectively.And in the role of the two drugs separately,in addition to BRL showed G0/G1 phase arrest,the other three cells all occurred G2/M phase of the cell cycle arrest.(4)Results of ROS level test: RC at concentrations of 2 mg/mL,0.25 mg/mL,0.25 mg/mL and 1 mg/mL,berberine at 0.05 mg/mL,0.005 mg/mL,0.005 mg/mL and 0.05 mg/mL could raised intracellular ROS levels of L929,BRL,GES-1 and NRK,respectively.(5)Results of DNA damage detection: RC at concentrations of 0.1 mg/mL,0.05 mg/mL,0.05 mg/mL and 1.5 mg/mL,berberine at 0.0125 mg/mL,0.004 mg/mL,0.005 mg/mL and 0.025 mg/mL could significantly induce DNA damage of L929,BRL,GES-1 and NRK cells,showing a certain dose-dependent manner.Conclusion:(1)RC and berberine in lower concentrations can promote the growth of L929,BRL,GES-1,NRK in vitro;(2)RC and berberine at higher concentrations can significantly inhibit the proliferation of four cells and demonstrate the cytotoxicity,both on the size of four cells toxicity are expressed as: BRL> GES-1> L929>NRK,showing a cell-specificity;(3)The cytotoxicity of RC and berberine on L929,BRL,GES-1 and NRK these four cells was a comprehensive implementation related to cell cycle arrest,DNA damage and accumulation of intracellular ROS.