Chemicals
Methanol, hydrochloric acid, Folin-Ciocalteu reagent, sodium carbonate, aluminium chloride, sodium hydroxide, ascorbic acid, alpha-tocopherol, butylatedhydroxytoluene (BHT), dimetylsulfoxide (DMSO), KOH and trifluoride (BF3) were purchased from Fisher Scientifics, USA. The other chemicals used in this study were bought from Merck.
Plant material
Seedlings of Labisia pumila varieties alata, pumila, and lancelota were, respectively, collected from places of origin at Sungkai, Perak; Hulu Langat, Selangor, and Kota Tinggi, Johore, and raised under similar glasshouse condition for 18 months before use in the study. The GPS location details were 3° 0'35.27"N latitude and 101°4219.38"E longitude. Healthy and uniform seedlings in term of leaf numbers were selected from the three varieties. The leaves of three varieties of Labisa pumila Benth. were cleaned, separated, and freeze dried for further analysis.
Microwave Assisted Extraction (MAE)
MAE was performed on microwave apparatus using a closed vessel system with pressure (ETHOS® T Microwave digestion/extraction system, Milestone Co., Italy) based on the method described by Xiao et al. [18] with some modification. One gram of leaf part of three varieties of Labisia pumila was weighed using a clean aluminum container, then transferred into the vessel of the Ethos E Microwave Extraction System and extracted with 30 ml of water as solvent for 2 min (p = 750 w). The extraction temperature was applied to 80°C. After extraction, the vessels were allowed to cool at room temperature before opening. Then the extracts were filtered and stored in refrigerator.
Total phenolics determination
For total phenol determination, briefly 0.5 ml of each methanolic extract, 2 ml of 7.5% sodium carbonate and 2.5 ml Folin-Ciocalteu reagent were mixed together. The mixture was then vortex and incubated for 90 min at room temperature [19]. The absorbance was read using a spectrophotometer (Novaspec II Visiblespectro, Japan) at 765 NM. The total phenol results were expressed as mg gallic acid equivalents (GAE)/g dry weight (DW).
Total flavonoids determination
For total flavonoid compounds 0.1 ml of methanolic extracts was added to 0.3 ml sodium nitrite (5%) and incubated for 5 min at room temperature, then 0.3 ml 10% (w/v) AlCl3 and 2 ml 1 N NaOH was added and the total volume was made up to 5 ml with distilled water [19]. The absorbance was measured at 510 nm by using visible spectrophotometer (Novaspec II Visiblespectro, Japan) at 510 nm. The results were expressed as mg rutin equivalents/g DW.
Fatty acid profile determination
The total fatty acids of the leaves were extracted according to the method of Folch et al. [20] with some modifications as described by Ebrahimi et al. [21], using chloroforms: methanol 2:1 (v/v) which contained butylated hydroxy toluene to prevent the oxidation during fatty acid extraction. Extracted fatty acids Trans methylated to the fatty acid methyl esters (FAME) using KOH in methanol and 14% boron trifluoride (BF3) in methanol. The FAME were separated using gas liquid chromatography (Agilent 7890A), using a Supelco SP 2560 capillary column of 100 m × 0.25 mm ID × 0.2 μm film thickness (Supelco, Inc., Bellefonte, PA, USA). One microliter was injected into the gas chromatography, equipped with an injector and a flame ionization detector. The nitrogen was the carrier gas at a flow rate of 1.2 ml/min. The split ratio was 1: 10. The temperature of the injector was 250°C and the detector temperature was 270°C. The column temperature program started runs at 150°C, for 2 min, warmed to 158°C at 1°C/min, held for 28 min, warmed to 220°C at 1°C/min, and then held for 20 min. A reference standard (C4-C24 methyl esters; Sigma-Aldrich, Inc., St. Louis, Missouri, USA), was used to determine correction factors for the determination of individual fatty acid composition. The data are expressed as g/100 g of detecting total identified fatty acids.
Anti-oxidant activity assay
Nitric oxide (NO) scavenging activity
The nitric oxide (NO) scavenging activity of each plant extract was determined by the method of Tsai et al. [22]. Vitamin C, BHT and α-tocopherol were used as controls. The NO scavenging activity was calculated according to the formula: [(A0 - A1)/A0] × 100%; where A0 was the absorbance of the control reaction and A1 was the absorbance in the presence of the sample.
ABTS radical cation-scavenging
The ABTS was evaluated by Giao et al.[23] method. ABTS was dissolved in water, to a 7 mm concentration. ABTS radical cation (ABTS.+) was produced by reacting ABTS stock solution with 2.45 mM K2S2O8 and allowing the mixture to stand at room temperature (dark place) overnight before utilization.
Anti-bacterial activity assay
The antibacterial assay of the leaf extracts of three varieties of L. pumila was carried out by the disc diffusion method as described by Boussaada et al.[24] against Staphylococcus aureus S1431, Escherichia coli E256, Pseudomonas aeruginosa PI96, Micrococcus luteus, Klibsiella pneumonia K36, Bacillus subtilis B145, Bacillus cereus B43 and Enterococcus aeruginosa. All the bacteria were purchased from the Institute of Malaysian Research (IMR) and maintained in the department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia. In this assay, the positive control without extracts (solvent) and reference control used kanamycin as the standard antibiotic agent. The extracts inhibitions were corrected based on positive control values. The experiments were run in triplicate.
Statistical analysis
All data are presented as means (± SEM) of at least three replicates (n = 3). The total phenolic and flavonoid contents, fatty acid, anti-oxidant and anti-bacterial properties were analyzed using analysis of variance (ANOVA) with the Statistical Analysis System (SAS) Version 9.1 (SAS Institute, Cary, NC). Significant differences among means from triplicate analyses (p < 0.05) were determined by Duncan’s Multiple Range Test. The level of significance was set at p < 0.05 for all statistical tests.