P. speciosa, A. subintegra, and P. minus were collected around Tanjung Malim, Perak while S. aromaticum, C. verum and P. nigrum were purchased from the local market in Behrang, Perak in 2013. Plant species and their parts used in this study were summarized in Table 1. Plant materials were separated, cut into small pieces and oven-dried at 40˚C. The dried plant materials were ground into powder using grinder machine.

Powdered samples were sequentially extracted with hexane, dichloromethane and methanol by maceration tech-

nique at room temperature for 72 hours and then extract with water by reflux technique for two hours to avoid deterioration of the chemical compounds. The organic extracts (18 extracts) were evaporated in vacuum by using rotary evaporator at 40˚C while the water extracts (6 extracts) were freeze-dried. The dried extracts were stored in glass bottle at 4˚C for further analysis.

The minimal inhibitory concentration was performed on extracts which showed zone of inhibition in the preliminary screening by using dilution method according to Nascimento et al. [12] . MIC of selected extracts was determined by measuring the optical density at 620 nm by comparing the reading of nutrient broth added with extract and inoculated with tested bacteria, with the nutrient broth added with extract without the tested bacteria as control. Each MIC determination was carried out in triplicate.

The disc diffusion method was used to evaluate the antibacterial activity. In this study, 18 organic extracts and 6 water extracts from P. speciosa, A. subintegra, P. minus, S. aromaticum, P. nigrum and C. verum were tested against Gram negative bacteria, E. coli, S. typhi, P. aeruginosa and Gram positive bacteria, B. cereus and S. aureus which commonly found in food such as chilli sauce. Four out of six herbs and spices tested in this study have shown antibacterial activity by using disc diffusion method (Table 2). Nor organic neither water extracts from P. speciosa and P. nigrum showed any antibacterial activity. Only methanol extracts of A. subintegra at higher concentration (75 mg/ml and 100 mg/ml) showed inhibition zone to only P. aeruginosa. Aqueous extracts of P. minus did not show any antibacterial activities against all bacteria tested.

A study carried out on pure piperine isolated from P. nigrum’s drupes showed zone of inhibition against E. coli, P. aeruginosa and S. aureus [13] . P. nigrum’s extracts in this study were found not active against all tested bacteria. The difference result obtained probably caused by antagonistic effect of the compound where the pure single compound inhibition effect is greater than the combination of compound mixture in an extract [14] .

Other than environmental condition, genetic factor, solvent used for extraction also affects the degree of antibacterial activity [15] [16] . Previous study of water extract from P. speciosa showed inhibition against S. aureus and other bacteria including Staphylococcus xylosus and Micrococcus species [17] . However, P. speciosa from this study were found not active against all the tested bacteria. Method of extraction in this study might be affecting the antibacterial activity of P. speciosa because the compound had been extracted serially in different polarity solvents. The composition of bioactive compounds from plant material will depend on the types and

Key: Hex = Hexane, DCM = Dichloromethane, MeOH = Methanol (n = 3), (-) = No zone of inhibition.

polarity of solvent used for extraction. A polar compound will extracted by polar solvent and non-polar compound will extracted by non-polar solvent. Thus, the biological activity shown may be different between extracts since different compounds will be extracted [18] . For example, Qader et al. [19] reported that P. minus have antibacterial properties against H. pylori when extract using petroleum ether, chloroform and methanol. However, aqueous extract of P. minus showed no inhibition against H. pylori.

Interestingly, hexane extract of C. verum and S. aromaticum showed antibacterial properties against all five tested organisms while hexane extract from P. minus gave positive result against three bacteria; S. aureus, B. cereus and P. aeruginosa. Even though essential oil is usually extracted by steam distillation technique, in this study, essential oil, waxes and fats might be dissolved in non polar solvent such as hexane [10] (Peter and Amala, 1998) and showed good antibacterial activity based on the zone of inhibition exhibited, range from 7 mm to 11 mm in diameter (Table 2). In previous study, ethanol extract of P. minus was found to be active as antimicrobial against Gram-positive bacteria; Bacillus cereus and Bacillus megaterium, two Gram-negative bacteria; Escherichia coli and Pseudomonas aeruginosa, and two fungi; Aspergillus ochraceous and Cryptococcus neoformans [20] (Macken et al., 1997). In present study, dichloromethane extracts from P. minus showed inhibition against B. cereus and P. aeruginosa while S. aromaticum showed positive results against E. coli, S. aureus and P. aeruginosa. In addition, dichloromethane extract from C. verum gave positive results against S. aureus, S. typhi, B. cereus and P. aeruginosa. Methanol extracts from C. verum and P. minus showed antibacterial activity against S. aureus, B. cereus and P. aeruginosa while methanol extract of S. aromaticum showed no inhibition against all the tested bacteria. Water extract from C. verum showed inhibition against S. aureus, B. cereus and P. aeruginosa.

Previous studies indicate that eugenol and cinnamaldehyde, were two major chemical components found in the spice oil of Cinnamom species and S. aromaticum that showed inhibition against gram negative and gram positive bacteria [21] [22] . Eugenol is a phenolic compound with-OH group, which gives hydrophobicity of the molecule that enables to penetrate the lipopolysaccharide of the gram negative bacterial cell membrane and disturbed the cell structures [23] . Hence, this lead to the leakage of ions and other cell contents, which consequently inhibit the bacterial growth.

The results from disc diffusion methods show three extracts that exhibited very active antibacterial activity; dichloromethane extract of C. verum, hexane extract of S. aromaticum and hexane extract of P. minus Huds. leaves (Table 2). They were further tested to determine the Minimal Inhibitory Concentration (MIC). The MIC values for the extracts were shown in Table 3. Plant extract of C. verum in dichloromethane showed MIC of 25

mg/ml against tested bacteria S. aureus, B. cereus and P. aeruginosa, whereas 50 mg/ml for S. typhi. Hexane extract of S. aromaticum showed MIC of 25 mg/ml against E. coli, S. aureus and P. aeruginosa, whereas 50 mg/ml against B. cereus and 75 mg/ml against S. typhi. The MIC of 25 mg/ml was obtained against S. aureus, B. cereus and P. aeruginosa when tested with hexane extract of P. minus Huds. From the results, antibacterial activity of the selected herbs and spices against both gram positive and gram negative bacteria show the presence of broad-spectrum antibacterial properties. Therefore, it is hypothesized that this local herbs and spices can be used as biopreservative agent in food products.