1.3 Aims and Objectives
I. To determine the antioxidant activity of leaves of C. nardus
II. To determine the antimicrobial activity of leaves of C. nardus
1.4 Scope and Limitation of the study
The leaves of C. nardus will be subjected to hydrodistillation in Clevenger-type apparatus to obtain Citronella oil. The antioxidant activity of C. nardus leaves will be determined by 2, 2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging methods whereas the antimicrobial activity will be determined using disc diffusion method.
Hydrodistillation is used in the extraction of EOs from the C. nardus leaves. Hydrodistillation divided into three types, which are water distillation, water and steam distillation and direct steam distillation. However, …show more content…
It is because DPPH is stable and long-lived nitrogen radical which is difference from the radicals present in living organism. It is not similar with the highly reactive and transient peroxyl radicals that are involved in lipid peroxidation. (Huang. D, Ou. B and Prior. R.L. 2005) Besides that, some test compounds may have absorbance spectra that overlap with DPPH at 515 nm also is the another limitations of DPPH method (Perez-Jimenez et al. 2008). Steric accessibility is a major factor of the reaction mechanisms. So that, small molecules are better access to the DPPH greater part and have higher apparent antioxidant ability. (Prior. R.L, Wu. X and Schaich. K 2005) Analysis of results is tricky when small molecule reducing agents are present in test samples. Antioxidants may be water soluble, fat soluble, insoluble or bound to cell walls and thus not necessarily freely available to react with DPPH, hence there react at different rates, such as differing kinetics. The reaction may often not be able to reach the completion in a reasonable assay time. Therefore, the sample size that can lower the initial absorbance of DPPH solution by 50% has been chosen as endpoint for measuring the antioxidant activity. (Aruna Prakash, PhD, Fred Rigelhof and Eugene Miller, PhD) The parameter “EC50”, equivalent concentration to give 50% antioxidant effect is used as the interpretation …show more content…
Disk diffusion method is an in-vitro determination of antimicrobial susceptibility. These in-vitro results may not always similar with in vivo efficacy. Susceptibility testing microorganisms by disk diffusion should be carried on uncontaminated culture. We should subculture the microorganism strains and re-test it to ensure that we can get an accurate and reliable result. Besides that, the parameter and standardization of disc diffusion method procedure should be followed to get the accurate and reproducible results. For example, the pH of the Mueller-Hinton agar should be maintained in the range of 7.2 -7.4 and keep it in room temperature after gelling. Otherwise, some antimicrobials will appear to lose potency while other agents may appear to have excessive activity. (S. Karlsmose 2010) One of the crucial steps in disc diffusion method is incubate the inoculums, which included selecting appropriate colonies for incubating in the nutrient broth and standardizing the suspension. 3-5 colonies should be tested instead of one colony for increase the chance to detect resistance of microbial. Only choose the well isolated colonies from the plate to prevent testing mixed
I. To determine the antioxidant activity of leaves of C. nardus
II. To determine the antimicrobial activity of leaves of C. nardus
1.4 Scope and Limitation of the study
The leaves of C. nardus will be subjected to hydrodistillation in Clevenger-type apparatus to obtain Citronella oil. The antioxidant activity of C. nardus leaves will be determined by 2, 2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging methods whereas the antimicrobial activity will be determined using disc diffusion method.
Hydrodistillation is used in the extraction of EOs from the C. nardus leaves. Hydrodistillation divided into three types, which are water distillation, water and steam distillation and direct steam distillation. However, …show more content…
It is because DPPH is stable and long-lived nitrogen radical which is difference from the radicals present in living organism. It is not similar with the highly reactive and transient peroxyl radicals that are involved in lipid peroxidation. (Huang. D, Ou. B and Prior. R.L. 2005) Besides that, some test compounds may have absorbance spectra that overlap with DPPH at 515 nm also is the another limitations of DPPH method (Perez-Jimenez et al. 2008). Steric accessibility is a major factor of the reaction mechanisms. So that, small molecules are better access to the DPPH greater part and have higher apparent antioxidant ability. (Prior. R.L, Wu. X and Schaich. K 2005) Analysis of results is tricky when small molecule reducing agents are present in test samples. Antioxidants may be water soluble, fat soluble, insoluble or bound to cell walls and thus not necessarily freely available to react with DPPH, hence there react at different rates, such as differing kinetics. The reaction may often not be able to reach the completion in a reasonable assay time. Therefore, the sample size that can lower the initial absorbance of DPPH solution by 50% has been chosen as endpoint for measuring the antioxidant activity. (Aruna Prakash, PhD, Fred Rigelhof and Eugene Miller, PhD) The parameter “EC50”, equivalent concentration to give 50% antioxidant effect is used as the interpretation …show more content…
Disk diffusion method is an in-vitro determination of antimicrobial susceptibility. These in-vitro results may not always similar with in vivo efficacy. Susceptibility testing microorganisms by disk diffusion should be carried on uncontaminated culture. We should subculture the microorganism strains and re-test it to ensure that we can get an accurate and reliable result. Besides that, the parameter and standardization of disc diffusion method procedure should be followed to get the accurate and reproducible results. For example, the pH of the Mueller-Hinton agar should be maintained in the range of 7.2 -7.4 and keep it in room temperature after gelling. Otherwise, some antimicrobials will appear to lose potency while other agents may appear to have excessive activity. (S. Karlsmose 2010) One of the crucial steps in disc diffusion method is incubate the inoculums, which included selecting appropriate colonies for incubating in the nutrient broth and standardizing the suspension. 3-5 colonies should be tested instead of one colony for increase the chance to detect resistance of microbial. Only choose the well isolated colonies from the plate to prevent testing mixed