3.6.1. Antibacterial Disk Diffusion Assay

The qualitative analysis of the antibacterial activity of *C. pentandra*/Ag-NPs was evaluated based on the diameter of growth inhibition zone against the tested bacteria and the results are shown in Figure 15. The *C. pentandra*/Ag-NPs inhibited the growth of all tested bacteria species in a dose-dependent manner. The results showed that the diameter of the growth inhibition zone increased with an increasing amount of *C. pentandra*/Ag-NPs from 1 to 4 mg as shown in Table 1. The significant differences in the diameter of the growth inhibition zone were also observed between the amounts of *C. pentandra*/Ag-NPs used (*p* < 0.05). The multiple comparison post hoc test value showed no significant difference in the diameter of the growth inhibition zone between the mass from 1 to 2 mg of *C. pentandra*/Ag-NPs (*p* > 0.05). By increasing the mass of *C. pentandra*/Ag-NPs up to 4 mg, the diameter of the growth inhibition zone increased significantly (*p* < 0.05). The finding can be explained due to the more Ag-NPs accumulated on the bacterial surface which can enter the cell and damage the nuclei and eventually causing bacterial death [37]. The suppression of bacterial growth increased with the increase in the amount of Ag-NPs is in agreement with the finding from the research by Sowmyya and Lakshmi [38].

**Figure 15.** The images of inhibition zones growth against (**a**) *E. coli*, (**b**) *P. vulgaris*, (**c**) *E. faecalis* and (**d**) *S. aureus* around the different mass of *C. pentandra* incorporated with Ag-NPs at 1 (1 mg), 2 (2 mg), and 3 (4 mg).

**Table 1.** The diameter of the growth inhibition zone of *C. pentandra*/Ag-NPs against different bacteria species.


NA means no activity; <sup>a</sup> is mean of the triplicate experiment, ±Standard Error (S.E).

For the bacteria species, the order of the strongest antibacterial activity of *C. pentandra*/Ag-NPs was *S. aureus.* The less antibacterial activity of *C. pentandra*/Ag-NPs against *P. vulgaris* might be due to the presence of capsule on the bacterial cell wall and the negatively charged of the outer lipid membrane (lipopolysaccharide) cover [39]. The electrostatic repulsion between the nanoparticles and Gram-negative bacteria hinders particles attachment and penetration into the cells [4]. However, the negatively charged of *C. pentandra*/Ag-NPs can bind electrostatically with the negatively charged of teichoic acid present in Gram-positive bacteria cell leading to the enhancement of cell permeability, cytoplasmic leakage and cell death [4,40].

Further analysis was tested on the significant differences in antibacterial activity between Gram-positive and Gram-negative bacteria. Surprisingly, the difference between these bacteria is not significant (*p* > 0.05). This result approved that the *C. pentandra*/Ag-NPs possessed antibacterial activity against both types of bacteria. According to Pollini et al. [41], a diameter of more than 1.0 mm of the microbial growth inhibition zone can be considered as a good antibacterial product. In this study, the *C. pentandra*/Ag-NPs which exhibited more than 1.0 mm of growth inhibition zone has potential antibacterial application, especially in biomedical, textile, wastewater treatment and food packaging areas. The performance of antibacterial activities of *C. pentandra*/Ag-NPs with other Ag-NPs is shown in Table 2. From the Table 2, it showed that the *C. pentandra*/Ag-NPs have comparable antibacterial activities compare to other Ag-NPs. This result showed *C. pentandra*/Ag-NPs have good antibacterial activities for both Gram-positive and Gram-negative bacteria. Yet, the good dispersion of Ag-NPs on

the *C. pentandra* surface can contact well with bacteria and releasing more Ag<sup>+</sup> ions for the effective antibacterial mechanisms.

**Table 2.** The comparison on the performance of antibacterial activities based on the zone of growth inhibition by *C. pentandra*/Ag-NPs with other Ag-NPs loaded in supporting materials reported in the literature.

