39 Silver Nanoparticles of Bee Honey ANTIMICROBIAL ACTIVITY In the present study, AgNPs–bee honey and bee honey were tested against S aureus and E coli. The zone of inhibition in diameter was discerned by disk diffusion method. For antibacterial function testing, S aureus, as well as E coli were used as the bacilli. The clear zone diameter of the bacterial inhibition zone was correlated to antibiotic activity (oxacillin and gentamicin). The clear zone diameter increased as the concentration of AgNPs–bee honey increased because of the bactericidal activity of Ag (15). The AgNPs–bee honey showed higher activity against gram-positive than gram-negative. The clear zones diameter of the bacterial inhibition zone for S aureus and E coli at a concentration of 1,000 µg/ml were 5.0±0.2 mm and 3.0±0.5 mm, respectively. AgNPs–bee honey exhibited effective zone of inhibition against S aureus and E coli more than the bee honey sample. However, AgNPs– bee honey showed better activities compared to bee honey. The highest zone of inhibition was observed for S aureus even at high concentration (≥250 µg/ml). The mechanism of the inhibition of the bacteria is still unknown, but some hypothetical mechanisms show that the inhibition is due to ionic binding of the silver nanoparticles on the surface of the bacteria, which creates a great intensity of the proton motive force, and the one hypothesis from the research states that the silver nanoparticles invade the bacterial cell and bind to the vital enzymes containing thiol groups (16). SERUM FORMULATION The F1-nano serum formulation containing 2% bee honey and AgNPs–bee honey nanoparticles was selected as best due to good physical properties such as homogeneity and fluidity, which certainly would make the serum application easier. The stability test of serum formulation was investigated. The obtained serums were submitted for 3 months and taken to accelerated stability study by heating/cooling for 3 cycles (kept at 45°C for 1 month and 4°C for 1 month/1 cycle) according to previous reports (17). The serums were then characterized by pH value and viscosity (18). The results showed that the formulations of F0-nano and F1-nano were not changed visually. These serums had a brawny and smooth texture with a bee honey odor. Thus, these formulations were used to evaluate characteristics Figure 6. Ferric reducing antioxidant power assay of bee honey samples and the AgNPs–bee honey nanoparticles at different concentrations.

40 JOURNAL OF COSMETIC SCIENCE including pH value, viscosity, and phase separation. The viscosities (50–200 revolutions per minute) of F0-nano and F1-nano were 180 to 272 cP and 413 to 768 cP, respectively (Figure 7). F1-nano showed higher viscosity than F0-nano, but no difference was found when compared to base serums. The viscosity of formulations (F0-nano–F4-nano) compared between before and after accelerated stability (15, 30, and 45°C), showed in Figure 8a, shows AgNPs–bee honey nanoparticles serums and control serums. It was found that all serums appeared yellow to brawny in color, smooth in texture, and had bee honey odor. The formulations of F-0 to F-2 and F0-nano to F2-nano showed smooth texture, whereas F-3 to F-4 and F3-nano to F4-nano showed viscous texture. The formulation of F1-nano was in good accordance with the results of serum formulation. After the accelerated stability test, the result accorded with the centrifugation test after preparation. After the centrifugation to characterize the phase separation, F-2 and F4-nano occurred in the phase separation except for F-1 and F1-nano, which were in good accordance with the results of serum formulation as shown in Figure 8b. The pH of the serum formulation should have acidic Control serums Nanoparticles serums 45°C Nanoparticles serums 30°C Nanoparticles serums 15°C a b Figure 8. Appearance of control serums (F0–F4) and AgNPs–bee honey nanoparticles serums (F0-nano to F4-nano) after stored at various temperature. Figure 7. Viscosity values of serum from AgNPs–bee honey nanoparticles.