Heavy metal pollution leads to human health problems and represents a constant threat to the environment. Pollutant clean-up using conventional methods are often hampered by high cost and ineffective pollutant removal. Phytoremediation technique is a preferable alternative due to its minimal side effects to the environment in addition to reasonable treatment cost. In this study, we investigated the potential of Centella asiatica and Orthosiphon stamineus as phytoremediation agents. Both species were grown in contaminated soil obtained from industrial land. Plant growth response and their ability to accumulate and translocate zinc, copper and lead were assessed. From this study, root growth of C. asiatica was compromised when grown in contaminated soil. Copper was highly accumulated in C. asiatica roots while the leaves were more concentrated with zinc and lead. Conversely, all three tested metals were highly detected in the roots of O. stamineus, although the root elongation was not adversely affected. Low amount of metals in the stems of both species permits longer stem length. Correlation study showed that the accumulation of zinc, copper and lead in plant tissues varies depending on plant species and the type of metals. Based on the bioaccumulation, translocation and enrichment factor, our study showed that C. asiatica was tolerant towards zinc, copper and lead; hence suitable for phytoextraction. By contrast, O. stamineus acted as a moderate accumulator of the tested metal elements.

Plants need an appropriate amount of copper to sustain their normal growth. However, excess of copper may interrupt plant development and cause stress. In this study, the responses of Orthosiphon stamineus callus culture under various copper concentrations were investigated. Callus cells at 24-days old were treated with 100, 150, 200 and 250 µM of copper solution and harvested after one week. Mitochondria from the calluses were isolated and the total mitochondrial proteins were determined. We used sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to compare the mitochondrial protein profiles in normal medium and copper-containing medium. The activities of cytochrome c oxidase, as well as the total phenolic and flavonoid contents were determined. The results of atomic absorption spectroscopy (AAS) analysis have shown significant amount of copper accumulated in O. stamineus callus cells, depending on the amount of copper supplied. With the BCF values of more than 10, this species can be categorized as copper hyperaccumulator. When the amount of copper supplied increased, the cells exhibit higher cytochrome c oxidase activities and produce more phenolic compounds, in spite of decreasing amount of total mitochondrial proteins and a lesser number of visible bands visualized from SDS-PAGE analysis. Overall, our findings highlight the dynamic responses of O. stamineus cells when exposed to various levels of copper, notably by alteration of biochemical and mitochondrial properties as their protection mechanism under stress.

. (2015), Heavy metal accumulation and antioxidant properties of Nephrolepis biserrata growing in heavy metal-contaminated soil, Global NEST Journal, 17(3), 544-554. HEAVY METAL ACCUMULATION AND ANTIOXIDANT PROPERTIES OF ABSTRACTAntioxidant defense mechanisms are crucial for plants to survive under stress conditions. We investigated the capacity of a wild fern species, Nephrolepis biserrata, growing in the vicinity of industrial land to accumulate heavy metals, and assessed its antioxidative response under metal stress. The soils in this particular area were highly contaminated with zinc followed by lead and copper. As control, N. biserrata located 10 km away from the industrial area were collected and assessed. N. biserrata from the contaminated sites accumulated metals in their tissues in similar descending order of zinc>lead>copper. The values of bioaccumulation factor between 0 to 0.1 indicate N. biserrata as a moderate accumulator for the tested metals. For the enzymatic antioxidant assays, the activities of catalase and ascorbate peroxidase were significantly higher in N. biserrata from contaminated soil compared to control, while the activity of superoxide dismutase was not differ significantly in plants from both sites. We also detected higher contents of total phenolics and total flavonoids in N. biserrata collected from contaminated site compared to control. Our HPLC analysis revealed higher levels of myricetin and kaempferol in plant samples from the contaminated area. Our study verified the capacity of N. biserrata to scavenge oxygen radicals when exposed to heavy metal stress. Such ability to tolerate stressful condition suggests that the plant is a potential metal phytoremediator.

Plants normally change their physiological and biochemical properties when exposed to heavy metal stress. We investigated the response of Orthosiphon stamineus towards different concentrations of Pb (0, 2, 5, 8 mg/L) and Cu (1, 2, 4, 5 mg/L). Heavy metals left in soil, plant physical characteristics, and the level of antioxidants in O. stamineus were determined. Our results showed that the tested Pb concentrations did not significantly affect stem elongation, but at 2 mg/L, Pb increased the leaf growth. Pb at 5 and 8 mg/L increased the total plant biomass. In contrast, 5 mg/L Cu treatment affected stem elongation and the root length of O. stamineus. The concentrations of Pb and Cu in soil were significantly reduced after the plants were harvested. Biochemically, 5 mg/L Pb had significantly increased the activity of catalase, while Cu at 5 mg/L significantly reduced the activity of superoxide dismutase and ascorbate peroxidase. Total flavonoid content increased in Pb-treated plants, but the total phenolics content decreased. Cu treatment at 2 mg/L, on the other hand increased the total phenolics content. Our results demonstrated that O. stamineus adapt to metal stress via physical changes, and scavenge oxygen radicals through enzymatic and non-enzymatic antioxidant productions.

Orthosiphon stamineus is one of the most prominent medicinal herbs widely grown in Southeast Asia. Propagation of O. stamineus using tissue culture technique helps to rapidly produce samples for research purposes, increase the production of secondary metabolites and is considered as one of the strategies in plant genetic improvement. Despite vast information on O. stamineus pharmacological properties, the protein profile of this species is currently underexplored. In the present study, proteins expressed in embryogenic callus developed from O. stamineus leaves in Murashige and Skoog medium supplemented with 1-naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D) were identified via Liquid chromatography- tandem mass spectrometry (LC-MS/MS). The presence of embryogenic callus was confirmed with microscope observation and Evan Blue assay. Using this proteomic approach, we identified peptides that represent 22 proteins localized to different cell compartments and involved in various biological and metabolic functions in plants. This finding is useful to improve our understanding of protein functions, thus their future applications could be further explored. Keywords: Embryogenic, Callus, Herb, Orthosiphon stamineus, Proteins