HPLC-DAD elucidation of phenolic compounds from plant organ extracts with inhibitory effect on Escherichia coli replikative DNA polymerase IIIα (DnaE)

Inhibition of replicative bacterial DNA polymerase, antimicrobial activity and chemical compounds of eleven plants were investigated in this Master Thesis. The whole or some parts (such as leaf, flower and fruit) of plants were used in assay. E. coli replicative DNA polymerase IIIα (DnaE2) was selected to research inhibition affects of plant compounds and it was produced as recombinant. The gene coding DnaE2 was amplified from E. coli K12 genom by PCR and inserted into the pET100-TOPO expression vector. It was expressed in E. coli BL21 (DE3) and purified by Ni-affinity chromorography. Methanol and DMSO extracts of eleven plant samples were prepared. Methanol extracts were used to investigate their inhibitory affect on polymerase by primer extension assay and DMSO extracts were used to investigate the antimicrobial activity on selective microorganisms by agar diffusion methods. Flourogenic labeled 45/20-mer synthetic DNA used in primer extension assay. According to results, a strong inhibition against to DnaE2 was determined in flower exract of Rosa canina. At the same way, it was determined that Sorbus caucasica and Sorbus aucuparia had the same affect on DnaE2. Antimicrobial activity of Sorbus aucuparia (fruit and leaf), Sorbus caucasica (fruit and leaf) and Rosa canina (flower and fruit) was investigated for Gram (-) and Gram (+) microorganisms. There was no antimicrobial affects in Sorbus extract against to Gram (+), but fruit extract of Rosa canina had antimicrobial affect against to both Gram (+) and Gram (-). The plant extracts having inhibitory affect on DnaE2 was fractioned into four sub-fractions (in order to dietyl eter, etyl acetate, butanol and liquid part) by liquid-liquid extraction method prepared in water and then phenolic compounds in each fraction were identified by HPLC-DAD system. Gallic acid and its derivate (ellagic acid) were found in flower and fruit extracts of Rosa canina. Clorogenic and rutin compounds were found in leaf and fruit extract of Sorbus aucuparia and Sorbus caucasica.