Antimicrobial activity of different fractions (500mg/6mm disc) of
1. Introduction
Reactive oxygen species (ROS) are various forms of activated oxygen, which include free- radicals, e.g., superoxide anions (O2-), hydroxyl radicals (.OH), non-free-radical compounds (H2O2) and singlet oxygen (1O2), which can be formed by different mechanisms in living organisms. Oxidative damage of DNA molecules associated with electron-transfer reactions is an important phenomenon in living cells, which can lead to mutations and contribute to carcinogenesis and the aging processes. ROS species are considered as important causative factors in the development of certain diseases such as diabetes, stroke, arteriosclerosis, cancer and cardiovascular diseases, in addition to the aging process. Prior administration of antioxidant provides a close relationship between FRSA and the involvement of endocrinological responses, which help to reverse the effect [1, 2]. Plants are rich sources of phytochemicals such as saponin, tannin, flavanoids, phenolic and alkaloids, which possess a variety of biological activities including antioxidant potential. Antioxidants provide protection to living organisms from damage caused by uncontrolled production of ROS and concomitant lipid peroxidation, protein damage and DNA stand breaking. Natural antioxidants are in high demand for application as bio-pharmaceuticals, nutraceuticals and food additives.
Terrestrial plants are considered potent sources of bioactive compounds and pharmacologically active compounds, however, little is known about the therapeutical potential of mangrove plants. Exploration of the chemical constituents of mangrove plants is necessary to find new therapeutic agents and this information is very important to the local community. Important reasons for studying the chemical constituents of mangrove plants are first, mangroves are a type of tropical forest that grows easily and has not as yet been widely utilized. Secondly, the chemical aspects of mangrove plants are very important because of the potential to develop compounds of agrochemical and medical value.
The plants of the genus
In this study we investigated the antimicrobial and antioxidant potential of methanol extract of
2. Material and methods
2.1. Plant materials and extraction procedure
The plants of
3. Determination of antimicrobial activities
3.1. Microorganisms
Microbial cultures
3.2. Antimicrobial activity by Disc-diffusion assay
The dried plant extracts were dissolved in the same solvent (methanol and distilled water) to a final concentration of 100 mg/ml and sterilized by filtration by 0.45 μm Millipore filters. Antimicrobial tests were then carried out by disc-diffusion method [4] using 100 μl of suspension containing 108 CFU/ml of bacteria spread on nutrient agar (NA). The discs (6 mm in diameter) were impregnated with 5 μl of the extracts (500 μg/disc) at the concentration of 100 mg/ml and placed on the inoculated agar. Negative controls were prepared using the same solvents employed to dissolve the plant extracts. Ampicillin (10 μg/disc) was used as a positive reference standard to determine the sensitivity of one strain/isolate in each microbial species tested. The inoculated plates were incubated at 37 oC for 24 h for clinical bacterial strains. Antimicrobial activity was evaluated by measuring the zone of inhibition against the test organisms. Each assay in this experiment was repeated twice.
3. Evaluation of antioxidant activity
3.1. Reducing power assay
The reducing power of
3.2. Metal chelating effect
The chelation of ferrous ions by the extract was estimated as per the method of Dinis [6]. Different concentrations of the extract (100-2000 μg/μl) were added to a solution of 1 mM FeCl2 (50 μl). The reaction was initiated by the addition of 1 mM ferrozine (0.1 ml) and the mixture was finally quantified to 1 ml with methanol, shaken vigorously and left standing at room temperature for 10 min. After the mixture had reached equilibrium, the absorbance of the solution was measured spectrophotometrically at 562 nm. All analyses were done in triplicate and average values were taken. The percentage of inhibition of ferrozine-Fe2+ complex formation was calculated using the formula given below: % Inhibition [(
3.3. Nitric oxide radical inhibition activity
Nitric oxide, generated from sodium nitroprusside in an aqueous solution at physiological pH, interacts with oxygen to produce nitrite ions which were measured by Griess reaction [7]. The reaction mixture (3 ml) containing sodium nitroprusside (10 mM) in a phosphate buffer saline and the extract (100 – 2000 μg/μl) were incubated at 25°C for 150 min. After incubation, 0.5 ml of the reaction mixture was removed and 0.5 ml of Griess reagent (1 % (w/v) sulfanilamide, 2 % (v/v) H3PO4 and 0.1 % (w/v) naphthylethylene diamine hydrochloride were added. The absorbance of the chromophore formed was measured at 546 nm.
3.4. Lipid peroxidation and thiobarbituric acid reaction
A modified TBARS assay [8] was used to measure the lipid peroxide formed using egg yolk homogenate as lipid rich media [9]. Egg homogenate (0.5 ml of 10 %, v/v) and 0.1 ml of extract were added to a test tube and made up to 1 ml with distilled water, 0.05 ml of FeSO4 (0.07 M) was added to induce lipid peroxidation and the mixture was incubated for 30 min. Then, 1.5 ml of 20 % acetic acid (pH 3.5) and 1.5 ml of 0.8 % (w/v) thiobarbituric acid in 1.1 % sodium dodecyl sulphate were added, the resulting mixture was vortexed and then heated at 95 °C for 1 h. After cooling, 5.0 ml of butanol was added to each tube and centrifuged at 3000 rpm for 10 min. The absorbance of the organic upper layer was measured at 532 nm. Inhibition of lipid peroxidation percent by the extract was calculated as 100- [
3.5. Determination of DPPH radical scavenging capacity
Quantitative estimation of the free-radical scavenging activity was measured by DPPH assay [10]. The reaction mixture contained a different concentration (100-2000μg/μl) of test extract and 2.9 ml of DPPH (60 μM) in methanol. These reaction mixtures were taken in test tubes and incubated at 37 oC for 30 min, the absorbance was measured at 517 nm. The percentage of radical scavenging activity by the sample treatment was determined by comparison with the methanol treated control group. BHT and ascorbic acid was used as a positive control. The DPPH radical concentration was calculated using the following equation: scavenging effect (%) = (DPPH∙)
3.6. Total antioxidant activity
The assay is based on the reduction of Mo (VI) to Mo (V) by the extract and subsequent formation of a green phosphate / Mo (V) complex at the acid pH [11]. The tubes containing 0.1 ml of the extract and the 1 ml of reagent solution (0.6 M sulphuric acid, 28 mM sodium phosphate and 4 mM Ammonium molybedate) were incubated at 95 oC for 90 min. After the mixture was cooled to room temperature, absorbances were taken at 695 nm against the blank. The antioxidant capacity was expressed as AAE.
3.7. DNA nicking induced by hydroxyl radical
The DNA damage protective activity of
3.8. Phytochemical analysis
Chemical tests were carried out on the aqueous extract of the powdered specimens using standard procedures to identify the constituents as described by Harborne [13].
4. Results and discussion
4.1. Antimicrobial activity
This paper illustrates the antimicrobial, antioxidant and DNA protective effect of
Microbes Sample | +ve | 1 | 2 | 3 | 4 |
17 | NI | NI | NI | NI | |
NI | NI | NI | NI | NI | |
13 | 12 | NI | NI | 10 | |
15 | 12 | NI | NI | 11 | |
"/>10 | 12 | NI | NI | 10 | |
36 | 11 | 10 | NI | 10 | |
10 | 11 | 10 | NI | 10 | |
15 | 10 | NI | NI | 10 |
4.2. Reducing power assay
The reducing power of
4.3. Metal chelating effect
It has been proposed that transition metals catalyse the formation of the first few radicals to start the propagation of radical chain reaction in lipid peroxidation. Chelating agents may inhibit lipid oxidation by stabilizing transition metals. Ferrozine can quantitatively form complexes with Fe2+. In the presence of other chelating agents, the complex formation is disrupted with the result that the red colour of the complex is decreased. As shown in Figure 1B, the ferrozine - Fe2+ complex is not complete in presence of the plant extract, indicating its ability to chelate the iron. The absorbance of ferrozine-Fe2+ complex decreased linearly in a dose dependent manner (100– 2000μg/μl) and the IC50 value is estimated as 2.47 μg. The metal chelating activity of
4.4. Nitric oxide radical inhibition activity
The antioxidant system protects the pathogens against the ROS-induced oxidative damage. Nitric oxide radical generated from the sodium nitropruside is measured by the Greiss reduction. Sodium nitropruside at physiological pH spontaneously generates nitric oxide, which thereby interacts with oxygen to produce nitrate ions that can be estimated using Greiss reagents. Thus, the scavengers of nitric oxide compete with the oxygen, leading to reduced production of nitric oxide. The chromophore formed during diazotization of nitrite with sulphanilamide and its subsequent coupling with naphthyl ethylene diamine was read at 546 nm. The methanolic leaf extract of
4.5. Lipid peroxidation and thiobarbituric acid reaction
Egg yolk lipids undergo rapid non-enzymatic peroxidation when incubated in the presence of ferrous sulphate with subsequent formation of malonodialdehyde (MDA) and other aldehydes that form pink chromogen with TBA absorbing at 532 nm [18]. Peroxidation of lipids has been shown to be the cumulative effect of reactive oxygen species, which disturb the assembly of the membrane causing changes in fluidity and permeability, alterations of ion transport and inhibition of metabolic processes [19]. The extract of
4.6. Determination of DPPH radical scavenging capacity
Methanolic leaf extract of
4.7. Total antioxidant activity
The total antioxidant potential of
4.8. DNA nicking induced by hydroxyl radical
Hydroxyl radical is the most reactive among reactive oxygen species, it has the shortest half life compared with others and is considered to be responsible for much of the biological damage in free-radical pathology. The radical has the capacity to cause strand breakage in DNA, which contributes to carcinogenesis, mutagenesis and cytotoxicity [11]. The DNA protective effect of hexane water and methanol extract of
5. Conclusion
In conclusion, the result obtained in the present study shows that the methanolic extract of
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