2 increasing recognition of herbal medicine as an alternative form of health care, screening of medicinal plants for biologically active compounds has become an important source of antibiotic prototypes and cancer-related drugs [12–14]. Hence, for selecting crude plant extracts with potential useful properties, in vitro screening methods have been used for further in-depth chemical elucidation and pharmacological investigations [15]. To date, few studies of EI have been reported; specially, its phytochemical content of sterol glucosides forms [16] and C-glycosylﬂavone having anti-inﬂammatory activity [17]. To our knowledge, no other scientiﬁc investigation has been reported in evaluating EI’s therapeutic potential. Therefore, this study was conducted to evaluate the antioxidant, antibacterial and anti-cancer activities of the hexane, dichloromethane (DCM), ethyl acetate (EA) and methanol extracts (MeTH) of EI using total phenolic content (TPC), DPPH, disc diﬀusion and MTT cytotoxicity assay methods of analysis. 2. Methods 2.1. Collection of Plant Materials. EI leaves were collected in July 2007 from the Laboratory of Natural Products, Institute of Bioscience, UPM, Selangor, Malaysia. Authentication was done at the same laboratory where the voucher specimen (EI- L100158) was deposited. 2.2. Extraction Procedure. The leaves were air dried and then oven dried at reduced temperature and then ground into powder before cold maceration. The powdered leaves (300 g) were extracted with diﬀerent solvents in the order of increasing polarity. The solvents used were hexane, DCM, EA and methanol. Extractions were done for 7 days with occasional shaking and the process repeated three times. The residue was air dried overnight and used for next solvent extraction (DCM) as per the above procedure and the same procedure was repeated for next two other solvents (EA and methanol). Finally, the combined extracts for each solvent were ﬁltered through Whatman No. 41 ﬁlter paper (pore size 20–25 µm) and dried under vacuum using a rotary evaporator and kept at 4◦C until required. 2.3. Antioxidant Assays 2.3.1. Amount of Total Phenolic Compounds. TPC of the EI extracts was determined using Folin-Ciocalteu reagent method [18]. Brieﬂy, stock solutions of EI extracts were prepared in a concentration of 20 mg/ml. Fifty microliters of this solution was transferred to test tubes (n = 3). To this, 0.4 ml of Folin-Ciocalteu reagent (1 : 10) was added and mixed thoroughly. After 1 min, 0.8 ml of sodium bicarbonate solution (NaHCO3 7.5%) was added and the mixture was allowed to stand for 30 min with intermittent shaking. Absorbance was measured at 765 nm using a Shimadzu UV- Vis spectrophotometer. The TPC was expressed as gallic acid equivalents (GAE) in mg/g extract, obtained from the standard curve of gallic acid solutions. The gallic acid standard curve was established by plotting concentration Evidence-Based Complementary and Alternative Medicine (mg/ml) versus absorbance (nm) (y = 5.145x + 0.014; R2 = 0.9975), where y is absorbance and x is concentration. 2.3.2. DPPH Radical Scavenging Assay. The radical scav- enging activity of extracts was determined following the method of Changwei et al. [19] with slight modiﬁcation. The radical scavenging activity of the extracts against stable 2,2- diphenyl-2-picrylhydrazyl hydrate (DPPH, Sigma-Aldrich Chemie, Steinheim, Germany) was determined spectropho- tometrically. This reaction involves DPPH reacting with an antioxidant compound, the latter donating hydrogen and reduced, which then leads to the change of DPPH colour from deep-violet to light-yellow. This colour change was monitored at 517 nm wavelength. We selected the DPPH free radical scavenging assay due to its straightforwardness, quickness, sensitivity and reproducibility [20]. Apart from this, the assay is useful to screen large number of samples with diﬀerent polarity. Brieﬂy, EI extracts stock solutions were prepared at 100 mg/ml in ethanol. Since the MeTH was not fully soluble in ethanol (even after sonication for 5 min), the extract was dissolved in dimethyl sulfoxide (DMSO). The working solution was prepared in methanol at concentration of 500 µg/ml. The DPPH solution in methanol (2.5 mg/ml) was freshly prepared before all absorption measurements. Five microliter of this solution was mixed with 100 µl extract solutions in microtiter 96-well plates. The samples were kept in the dark for 30 min at an ambient temperature and the decrease in absorption was measured. Absorption of blank sample containing the same amount of methanol and DPPH solution was prepared and measured daily. The experiments were done in triplicates. The radical scavenging activity was calculated by the following formula: Percentage inhibition = (AB − AA) AB × 100, (1) where AB is absorption of blank sample (t = 0 min), AA is absorption of tested extract solution (t = 30 min). A commercial antioxidant, butylated hydroxytoluene (BHT), was used as the reference standard. 2.4. Antimicrobial Activity of EI 2.4.1. Bacterial Strains. The antimicrobial activity of plant extracts was evaluated using two Gram-positive bacteria, methicillin resistant Staphylococcus aureus (MRSA) and Bacillus subtilis B29, and other two Gram-negative bacteria, Pseudomonas aeruginosa 60690 and Salmonella choleraesuis. All the bacterial strains were obtained from the Laboratory of Molecular Biomedicine, Institute of Bioscience, Universiti Putra Malaysia, Serdang, Malaysia. 2.4.2. Antibacterial Assay. Screening of antibacterial activity was carried out by determining the zone of inhibition using the Paper Disc (6 mm in diameter, Whatman No. 1)