Rafat, Arash (2012) Effect of selected bacteria on the antibacterial and antioxidant activities of Centella asiatica / Arash Rafat. PhD thesis, University of Malaya.
Abstract
The role of selected naturally distributed bacteria on the antibacterial and antioxidant activities and production of phenolic compounds in one of the most important Asian medicinal plants namely Centella asiatica was investigated based on the effects of beneficial and pathogenic bacteria on the metabolic pathways of plants as reported in literature. Firstly, antibacterial and antioxidant properties and production of phenolic compounds of two subspecies of C. asiatica which are commonly used in Malaysia (University of Malaya Herbarium Voucher Specimens: KLU047364 and KLU047552) were evaluated and the most potent of them was chosen for the rest of the study. Different antioxidant assays were used to investigate the antioxidant activities of different samples while a disc diffusion method was applied to measure the antibacterial activity of these samples. Total phenolic contents of different parts of C. asiatica were also evaluated using Folin-Ciocalteu method. The selected C. asiatica subspecies was then treated with selected pathogenic and beneficial bacteria. Antibacterial and antioxidant activities and production of phenolic compounds in different parts of the plant inoculated with the pathogenic bacteria (Enterobacter sp.) were studied at early and late stages of infection. The antibacterial and antioxidant capacities as well as production of phenolic compounds were also investigated in different parts of the selected subspecies of C. aisiatica after treatment with the beneficial bacteria (Pseudomonas sp.). Production of phenolic compounds in the treated plants was also analyzed using high performance liquid chromatography (HPLC) and then compared to the non-treated plants (controls). Based on the importance of endophytic bacteria and their interactions with the host plant, some endophytic bacteria associated with C. asiatica were also isolated and identified using 16S rDNA sequencing method. Antibacterial and antioxidant potential of the cell-free supernatant of these endophytic bacterial cultures were also evaluated. A multiple callus-subculturing method was used to produce endophytic bacteria-free calli after optimization of an in vitro callus induction protocol for C. asiatica. The results confirmed the promising antibacterial and antioxidant activities of both examined subspecies of C. asiatica. However, the subspecies KLU047364 showed better antibacterial and antioxidant activities compared to KLU047552 and thus selected for the rest of the study. C. asiatica in early response to Enterobacter sp. infection increased the production of phenolic compounds. Both antibacterial and antioxidant activities of the plant were also enhanced in early stage of infection while the production of phenolic compounds and antioxidant activity of plant was reduced in late stage of infection. Antibacterial and antioxidant activities as well as total phenolics production of C. asiatica were increased after treatment with the beneficial Pseudomonas sp. bacteria. HPLC results showed irregular changes in amount of phenolic compounds produced in C. asiatica after treatment with the bacteria. All isolated endophytic bacteria could inhibit the growth of Pseudomonas aeruginosa except Bacillus gibsonni. The isolated endophytic bacteria also showed fair antioxidant potentials. The best result for callus production from leaf explant of C. asiatica was obtained from the combination of 6-benzylaminopurine at concentration of 3mg/ml and 1-naphthaleneacetic acid at a concentration of 3mg/ml. The multiple callus-subculturing method resulted in 75% endophytes-free callus.
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