Antioxidant and Biological Activities of Leptadenia Hastataethyl Acetate Roots Extracts

Chemicals

All chemicals used in this research were obtained from SIGMA. Standard antibacterial agent (30μg) tetracycline, antimicrobial susceptibility test discs and Nutrient agar (CM0003) were obtained from Oxoid Ltd, Wade Road, Basingstoke, Hants, RG2 8PW, UK.

Preparation of Test Samples

The crude extracts (Ethyl acetate) of Leptadenia hastata was used in antibacterial and antioxidant activity, the ethyl acetate crude extracts were tested by disc diffusion method on nutrient agar medium as described by Pundir & Jain [15]. Exactly 3 mg of the crude sample was dissolved homogeneity in 3mL of methanol giving a stock solution of 1000μg/ mL. Different volumes from the stock solution were taken, amounted to 25, 50, 100, 250, 500, 1000ppm each, and dissolved in 5mL of methanol to make final concentration respectively.

Preparation of Agar Plates

Preparation of agar plates was performed based on method described by Pundir & Jain [15] as reported by Umaru IJ, et al. [14]. Nutrient agar was prepared according to manufacturer's instruction with 14 g of dried agar dissolved in 500mL distilled water. The agar solution was heated until boiling followed by sterilization in autoclave at 121°C. The agar solution was then poured into a sterile petri plate and allowed to cool down and forming a gel. The plate was divided into eight sections by making a line marking on the outside surface of the plate. The eight sections were for each test samples namely the 25, 50, 100, 250, 500, 1000ppm samples, tetracycline 30μg (positive control) and methanol (negative control). The plate was sealed using parafilm and keep chilled at 4°C upon bacteria inoculation.

Preparation of Bacteria Broth

Several selected bacteria were used to evaluate the antibacterial activities of the crude extracts of Leptadenia hastata, Escherichia coli, Salmonella typhi, Staphylococcus aureus and Kliebselia pneumonia were obtained from the stock culture provided by Virology Laboratory, University Malaysia Sarawak, the nutrient broth was prepared according to manufacturer's instruction, with 2.6 g of the dried broth dissolved in 200mL distilled water followed by sterilization in autoclave at 121°C. The bacterial was sub cultured in a 10mL of broth, each in universal glass bottle for 16 hours inside an incubator equipped with shaker at 37°C [16]. After 16 hours’ incubation, turbidity (optical density/OD) of the bacterial broth was measured by using UV mini spectrophotometer (model 1240 of Shimadzu brand), comparable to that of nutrient broth standard tube for further use [17]. Measurement was performed at wavelength 575 nm and the bacterial broth was ready to be used when its turbidity was between OD 0.6 to 0.9. Nutrient broth was used to adjust the turbidity until the desired value was obtained.

Plate Inoculation

Inoculation of the bacteria was carried out in a biohazard cabinet and the procedure was based on method described by Umaru IJ, et al. [14]. Approximately 1mL of the ready bacterial broth was transferred into mini centrifuge tubes. A sterile cotton swap was dipped into the mini centrifuge tube containing bacteria broth and streaked over entire of the agar plate surface, performed in 4 different directions. The agar plate was then left for 5-10 minutes before applying the test samples. The disc used was 6 mm diameter. A volume of 10μL of the test samples of concentration 25, 50, 100, 250, 500, 1000ppm were each pupated onto the discs and placed onto the agar plate by using sterile forceps and gently pressed to ensure contact. Next to be placed on the agar plate was the disc pupated with methanol as negative control, followed by 30μg of tetracycline as standard antibacterial agent (positive control). The plates were left at room temperature for 10 minutes to allow the

Results

diffusion of the test samples and the standards into the agar. Each crude extract was tested in triplicate for each bacterium used. The plate samples were then incubated at 37°C for 24 hours before the inhibition zone around every sample disc being examined. The inhibition zone was measured in diameter to indicate the presence of antibacterial activity for each sample, as compared to the positive control.

Results

The issue of antibacterial resistance emanating has led to the search for new antimicrobial agents. Due to the problem of microbial resistance to antibiotics, attention is given toward biologically active components isolated from plant species commonly used as herbal medicine, as they may offer a new source of antimicrobial activities [14]. Our search for antioxidant and antibacterial activity from this plant Leptadenia hastata ethyl acetate extract revealed an antioxidant and antibacterial activity. The results of antioxidant and antibacterial tests of the plant extracts are as listed in Tables 1 & 2 below.

Discussion

The study revealed in DPPH radical scavenging activity model, ethyl acetate extracts showed high activity than standard compound. IC50 values of the samples was 1.889μg/mL while the IC50 values for ascorbic acid, was 1.763μg/mL. The polyphenolic content in the extracts may correlate to their good DPPH radical scavenging activity. It has been assumed that phenol and flavonoid can quench DPPH radical via hydrogen- or electron- donation mechanism and changes its colour from violet to yellow. Antioxidant is defined as any substances which are able to perform this reaction.

The antibacterial activity of the extract against the tested species of microorganisms (Escherichia coli, Salmonella typhi, Staphylococcus aureus and Kliebselia pneumonia) between concentration ranges for 25 to 1000ppm. The studies revealed that the extracts possess antibacterial activity in a concentration dependent manner against the test organisms and were comparable with the standard drug.

According to the results of disc diffusion assay, this root extract of the plant Leptadenia hastata has active compounds that are effective for the prevention of infections caused by these bacteria Escherichia coli, Salmonella typhi, Staphylococcus aureus and Kliebselia pneumonia. The inhibition yielded at concentration 25- 250ppm was found to be active against all tested strains under study. However, the extract showed higher significant antibacterial activity against Escherichia coli, and Staphylococcus aureus over the other pathogens with an inhibition value of 1.20 ± 0.10mm and 1.23 ± 0.12mm respectively. Though, the extract showed moderate activity at all concentration on the bacterial species, the potency exhibited less significant activity against Escherichia coli and Kleibsilia pneumonia. However, the antioxidant potential of this which showed to be high than the control may accounts for the potent antibacterial activity of the extract. This implies that it may also possess potent antibacterial activity.