Chemicals
The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] tetrazolium salt, DPPH [2,2-diphenyl-1-picrylhydrazyl], ascorbic acid (purity = 99%), vincristine sulphate (purity = ≥ 95%), nutrient agar, Folin – ciocalteu, sabouraud dextrose agar, ketoconazole (purity = ≥ 99%), streptomycin (purity = 90%), gallic acid (purity = 90%).
Plant material
The plant parts used for this study (Table 1) were collected and identified at the botanical garden of the University of Ibadan, Ibadan, Nigeria. Authentication of the plant material was done at the herbarium of the Forest Research Institute of Nigeria, Ibadan Nigeria, where voucher specimens were also deposited. The plant materials were dried at room temperature and pulverized into powder. Freshly crushed endosperm of Cocos nucifera was used in this study.
Extraction
Each plant material (200 g) was macerated in 80% methanol for 78 h at room temperature. Crushed endosperm of Cocos nucifera (200 g) was extracted in n-hexane. Extracts were filtered through filter paper (Whatman No. 1) and concentrated to dryness in vacuo.
DPPH radical scavenging assay
The free radical scavenging activities of the extracts against 2,2-diphenyl-1, picrylhydazyl (DPPH) was evaluated following the method of (18) with slight modification. Extracts and ascorbic acid were made into various concentrations (1.56, 3.13, 6.25, 12.5, 25, 50 and 100 µg/mL) in methanol while a 0.004% (w/v) solution of DPPH in methanol was freshly prepared. Each concentration of the extract (1 mL) was mixed with 3 ml of the DPPH solution and incubated in the dark for 30 min at 27± 2 ⁰C. In the control experiment, 1 mL of methanol was used in the place of the extracts. Absorption of the reaction was measured at 517 nm. The experiment was repeated three times. The concentration at which there is 50% inhibition of the DPPH• (IC50) was determined using Graph pad prism (5.0) while the percentage inhibition of the extracts was calculated using the formula;
% Inhibition = Absorbance of control - Absorbance of test sample x 100
Absorbance of control
Total phenolic content (TPC) assay
The total phenolic content of the extracts was determined using Folin – Ciocalteu (FC) reagent following the method of (19) with slight modification. The extracts were made into 100 µg/mL while 10% FC (v/v) in methanol was freshly prepared. The FC reagent (25 µL) was added to 50 µL of the extracts in 96 – well plates and allowed to stand for 3 min. For the blank, methanol was used in place of the extracts. A solution of 7.5% Na2CO3 (125 µL) was added into each well and afterwards incubated in the dark for 2 h at 25 ± 2 ⁰C. The absorbance was recorded with Thermo Fisher Scientific microplate reader at 758 nm. The experiment was carried out in triplicates. The total phenolic content was therefore expressed as Gallic acid equivalents (GAE) (18).
In vitro antimicrobial assay
Test organisms
Reference bacterial and fungi strains were obtained from the Department of Medical Microbiology and Parasitology, University College Hospital, Ibadan Nigeria. The bacterial strains include: Escherichia coli ATCC 25923, Pseudomonas aeruginosa ATCC 10145, and Salmonella typhi ATCC 24683 while the fungi strain used was Candida albicans ATCC 24433. Nutrients broth and sabouraud dextrose broth was used for the maintenance of the bacterial and fungal strains respectively at 4 ⁰C.
Preparation of inoculums
A small piece of colony from a day-old cultures of each test organisms was adjusted to cell density of 1x108 CFU/mL in sterile distilled water using McFarland Standard No. 0.5.
Spectrophotometric growth inhibition method
The method of (20) was adopted with some modifications. Extracts and standard drugs (streptomycin and ketoconazole) were made into concentrations 1000, 500, 250, 125, 62.50, 31.25 and 15.63 µg/mL in freshly prepared nutrient or sabouraud dextrose broth. An aliquot of 75 µL of each test concentration was gently mixed together with 75 µL of the inoculum in wells of 96 – well plate. Sterile distilled water was used in the control experiment. The absorbance at 540 nm was taken before and after 24 h of incubation at 37 ⁰C. Differences in optical densities were taken as microbial growth index. The experiment was carried out in triplicates. The concentration at which there is 50% microbial inhibition (IC50) was determined using Graph pad prism (5.0) while the percentage microbial inhibition was calculated by using the equation:
% Inhibition = ∆Absorbance of control - ∆Absorbance of test sample x 100
∆Absorbance of control
Cytotoxicity assay
Cell culture
Culture of human larynx epithelioma (Hep 2), Human Rhabdomyosarcoma (RD) and cervical adenocarcinoma (HeLa) cell lines were obtained from the Department of Virology, University College Hospital (UCH) affiliated to the University of Ibadan, Ibadan, Nigeria. The cells were maintained in Eagle’s Minimum Essential Medium (MEM) supplemented with 10% fetal bovine serum (FBS) (v/v), 100 units/mL of penicillin, 100 µg/mL of streptomycin, 0.07% NaHCO3 (w/v), 2 mM L-glutamine and 1% non-essential amino acids.
MTT Assay
The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl tetrazolium bromide] viability assay was carried out following a mildly modified method of (21). Each cell line was seeded into different 96 well plate and incubated at 37°C for 24 h. Extracts and positive control (vincristine sulphate) were freshly made into concentrations of 1000, 100, 10, 1, 0.1 and 0.01 ug/mL with 5% (v/v) DMSO in maintenance medium. At the expiration of 24 h, medium in wells with confluent monolayer cells were carefully replaced with 200 uL of various concentrations of the extracts and were further incubated at 37 °C for 72 h. Cytopathic effects of the extracts at various concentrations after 72 h was evaluated and scored under microscope. Medium in wells was carefully replaced with 25 uL of 2% (w/v) MTT dye in PBS and incubated at 37 °C for 2 h. DMSO (125 uL) was added into each well and left on a shaker for 30min to ease solubility and evenness of the colour formed. Absorbance of each well at 492 nm was recorded with a spectrophotometer. The experiment was performed in triplicate while the CC50 was determined using graph pad prism 5.0. Percentage cytotoxicity of the extracts at various concentrations was calculated using the formula;
% Cytotoxicity (CC) = (A - B) x 100
A
Where: A = the optical density of untreated cells
B = the optical density of cells treated with plant extracts/ control drug
Statistical analysis
Graphpad Prism, version 7.01 was used for the statistical analysis of data. The data obtained were expressed as Mean ± SD (Standard deviation) values of three independent assessments. The IC50 and CC50 values of all test samples were determined with nonlinear regression plot of log (cytotoxic concentration) against normalized percentage cytotoxicity. One-way at P < 0.05 followed by Tukey’s test was used test for the significant difference between the extracts and the standard drugs.