Flash chromatography was performed using guidelines from Weizman and Still et al. About 70g of silica gel was used to pack the column using the wet method; the silica was mixed with hexane to obtain a thick slurry. The slurry was mixed carefully and poured in the column a little at a time while swirling it. Once the column was packed, hexane was flushed carefully through the column by applying suction. Hexane was recycled 3-4 times as necessary until the silica was completely solvated (homogenous appearance and no air bubbles). The surface of the silica was ensured to be uniform and horizontal. Once the column was packed, hexane was drained till it reached about 2 cm above the level of the silica surface. A layer of sand was added to protect the silica surface. Hexane was then brought to the level of the sand.
6mL of essential oil was added on the walls of the column in a circular motion using a Pasteur pipette. The side of the column was then rinsed with a minimum amount of hexane and the sample was allowed to percolate into the silica. 150mL of hexane was then eluted through the column and the extract collected. This was repeated for the remaining solvents (dichloromethane, ethyl acetate and methanol) in order of increasing polarity.
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A new column was set up for each essential oil and the whole process was carried out inside a fume hood. Figure 2 shows the flash chromatography column set up after local ylang oil had been loaded.
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Figure 2: Flash chromatography set up
2.4.2 Concentration of Extracts
The resultant solutions of each of the extracts were evaporated using a Büchi R-114 rotary evaporator. The four extracts (hexane, dichloromethane, ethyl acetate and methanol) were then kept at room temperature, uncovered to ensure that all of the solvent was driven off. Masses were recorded every 2-3 days until a constant mass (to 2 decimal places) was achieved.
2.5 DPPH Free Radical Scavenging Activity
The DPPH free radical scavenging activity was evaluated according to the method of Kondo et al. (2002). 0.1mL of various dilutions of the test sample (essential oils or extracts) in methanol (0.20% to 100%) were prepared. 2mL of DPPH (0.21mM in methanol) was added to each of the solutions. The mixture was shaken, and left for 1 h in the dark at room temperature. A blank was also carried out containing all the reagents except the test material. Any turbid solution was filtered off using a syringe fitted with a micro filter paper as shown in Figure 3.
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Figure 3: Syringe fitted with a micro filter paper
The absorbance of each solution was measured in a quartz cell at 517 nm using a Camspec M105 spectrophotometer. The percentage of DPPH inhibition was calculated using the equation:
where Abscontrol is the absorbance of the control reaction (containing all reagents except the test material) and Abssample is the absorbance of the test material. All determinations were performed in triplicate. The sample concentration (in 1 mL reaction mixture) providing 50% inhibition (EC50) was estimated by plotting the percentage inhibition against log of concentration of samples.
An estimation of IC50 of the antioxidant reagent ascorbic acid was used as a positive control.
The DPPH results were standardized using the antioxidant activity index (AAI):
Samples were classified according to their AAI as follows (Teixeira et al., 2012):
2.6 Assay for Total Phenolics
Phenolic contents were determined using the method of Chandler and Dodds (1983) described by Goze et al. (2009). 1mL of 10%(v/v) of test sample (essential oils or extracts) dissolved in methanol was added to a conical flask and to that 45mL of distilled water followed by 1mL Folin-Ciocalteu reagent were added. The mixture was shaken. After 3 minutes, 3mL of 2% Na2CO3 solution was added and the mixture was allowed to stand for 2 hours with intermittent shaking. The absorbance of the solution was read spectrophotometrically at 760 nm with a Camspec M105 spectrophotometer using a quartz cell. The same procedure was repeated using 1mL gallic acid standard solutions (0-1500μg/mL) and a standard curve was obtained with the following equation:
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Absorbance = 0.0005 gallic acid (μg) (R2=0.9997)
The results were expressed as μg gallic acid equivalence (GAE) per mg sample. All the tests were performed in triplicate and phenolic content as gallic acid equivalents were reported as mean±SD of triplicate determinations.
2.7 Assay for Total Flavonoids
Total flavonoids content was determined according to the method of Dewanto et al. (2002) adapted by Kesraoui et al. (2011). 20μL of 10% (v/v) of sample (essential oils or extracts) was mixed with 30μL of 5% (w/v) NaNO2 solution. After 6 minutes, 60μL of freshly prepared 10% (w/v) AlCl3.6H2O was added. After 5 minutes, 200μL of 1M NaOH and 690μL of distilled water were added. Absorbance was determined at 510 nm by a Camspec M105 spectrophotometer using a quartz cell. The same procedure was repeated for quercetin standard solutions (0-500μg/mL). The concentrations of flavonoid compounds were calculated according to the following equation obtained from the standard quercetin graph:
Absorbance = 0.0001 quercetin (μg) 0.0045 (R2=0.9996)
The results were expressed as μg quercetin equivalence (QE) per mg sample. All the tests were performed in triplicate and flavonoid content as quercetin equivalents were reported as mean±SD of triplicate determinations.
2.8 Antibacterial Activity
2.8.1 General Procedure
All equipment and media used for antibacterial screening tests were autoclaved at 121°C for 1½ hours prior to use. The experiment was carried out aseptically in a Laminar Air Flow.
2.8.2 Bacterial Strains and Growth Conditions
Antibacterial activity tests were carried out on: Bacillus cereus (ATCC 11778), Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922) and Klebsiella pneumonia (ATCC 13883). The test organisms were kindly provided by (?)
The bacteria were cultured in Mueller-Hinton broth (21g/dm3) at 37°C for 24 hours. The cultures were kept in the refrigerator (2 to 8°C) and sub-cultures were made every two weeks in broth.
2.8.3 Disc Diffusion Method
Antibacterial activity of the essential oils and extracts were determined using the disc diffusion method (NCCLS, 2002). Mueller-Hinton agar was prepared according to the manufacturer's recommendations, autoclaved and allowed to cool. The cooled medium was poured in sterile disposable plastic Petri dishes to form a uniform horizontal surface of a depth of about 4mm.
The agar medium was allowed to set and dry aseptically at room temperature. The agar plates were either used on the same day or stored in refrigerator (2 to 8°C) wrapped in plastic.
The selected bacterial suspension was spread on the agar using sterile cotton swabs. Sterile filter paper discs (6mm) were placed on the surface of the agar and impregnated with 5μl of essential oil or extract. Petri dishes were incubated at 37°C for 24h. All determinations were performed in triplicates. Erythromycin (30μg) discs were used as controls.
The plates were examined after incubation. The inhibition zone was the clear area devoid of bacterial growth. Antibacterial activity was evaluated by measuring the inhibition radius to the nearest millimeter. Value of each diameter was measured in triplicate and averaged.