32 JOURNAL OF COSMETIC SCIENCE of bee honey in comparison to many antioxidants and antibiotics. Bee honey has a strong antibacterial effect against pathogenic and nonpathogenic microorganisms, fungi, and yeasts, even in those who are initially resistance to many antibiotics (5). Nanobiotechnology has contributed significantly to the development of a new generation of antimicrobials used to fight antimicrobials, using silver nanoparticles due to their specific characteristics, such as penetration, contact, and microorganism specificity (6). Biological materials are used instead of harsh reagents in the synthesis of nanoparticles in an attempt to obtain dynamic conditions that allow for their use in biological applications. In such cases, syntheses are termed “green syntheses” (7). The current study therefore aimed to synthesize and characterize antimicrobial activity in silver nanoparticles (AgNPs) that have been reduced and stabilized with bee honey and incorporated into serum formulation, followed by testing for its antimicrobial activity against Staphylococcus aureus. METHODS GENERAL EXPERIMENTAL PROCEDURES All chemicals were in the process of analytical grade and were used as received without further purification. Ultrapure water (resistivity not less than 18.2 MΩ cm at 298 K) from the Milli-Q water purification system (MilliporeSigma, Burlington, MA, USA) was used in all tests. Ethanol, 2.2-diphenyl-1-picrylhydrazyl radical (DPPH), 2,4,6-tripyridyl- s-triazine (TPTZ), and butylated hydroxytoluene (BHT) were purchased from Sigma- Aldrich Corporation (St. Louis, MO, USA) and Folin–Ciocalteu reagent was purchased from Merck (Darmstadt, Germany). Silver nitrate (AgNO 3 ),ferrous sulfate heptahydrate (FeSO 4 .7H 2 O), iron (III), and chloride (FeCl 3 )were the catalysts for analysis. All were provided by Merck. Glycerin. Tween 80 and poly (ethylene glycol) were all purchased from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). The following instruments were used: spectrophotometer (UVmini-1240, Shimadzu Scientific Instruments, Columbia, MD, USA) used to scan the spectra of the sample pH meter (model pH 900, Precisa, Dietikon, Switzerland) water bath and shaker (model SB-1000, Eyela, Japan) Ultrasonicator (model 889, Cole-Parmer, Vernon Hills, IL, USA) rotary evaporator (EYELA N-1200B series, Eyela, Bohemia, NY, USA polytron (PT-MR 3000, Kinematica AG, Malters, Switzerland) and high-pressure homogenizer (EmulsiFlex-C3, Avestin, Ottowa, Canada). The Fourier transform infrared (FTIR) spectra were performed using a Nicolet iS5 FTIR spectrometer (Thermo Scientific, Waltham, MA, USA) it was operated in the range of 4,000 to 400 cm−1. PREPARATION OF BEE HONEY MATERIAL In this study, three samples of bee honey were randomly obtained from local markets around Thailand. The samples (A, B, and C) were bee honey from longan flowers, wildflowers, and benjaphan flowers, respectively. PHYSICOCHEMICAL ANALYSIS OF BEE HONEY Each bee honey sample had undergone some physicochemical analyses by Bogdanov et al. (8). Water content was expressed in percentage, using a refractometer for measuring

33 Silver Nanoparticles of Bee Honey the refractive index at 20°C. The pH value was determined by a pH meter on a solution composed of 10 g of bee honey and 75 ml of distilled water. Electrical conductivity was measured (in μS/cm) using a conductometer device at 20°C of the test solution that consisted of 20% bee honey weighed as dry matter dissolved in distilled water and brought to a volume of 1/5. BIOSYNTHESIS OF AgNPs In the silver nanoparticles biosynthesis, 100 ml of freshly prepared solution of 1 mM AgNO 3 was added to 100 g bee honey in 250-ml Erlenmeyer flasks. The composition solution was stirred and incubated for 8 hours. After incubation, the mixture containing the nanoparticles was centrifuged and the pellets were lyophilized. The lyophilized AgNPs–bee honey nanoparticles were stored for further analysis. The samples of AgNPs– bee honey nanoparticles (A, B, and C) were bee honey from longan flowers, wildflowers, and benjaphan flowers, respectively. CHARACTERIZATION OF AGNPS–BEE HONEY NANOPARTICLES UV-visible spectroscopy. The nanoparticles of AgNPs–bee honey nanoparticles were demonstrated using a UV-visible spectrophotometer by taking absorption samples at 200 to 800 nm after 30 minutes to 8 hours incubation. FTIR analysis. FTIR analysis of AgNPs–bee honey nanoparticles was performed using a durable diamond single-reflection mode. The spectrum was recorded using Thermo Scientific (Nicolet IS5) FT-IR spectroscopy using transmittance mode and operating with 4 cm−1 correction. Particle size distribution. Particle size distribution is measured from a tangible material provided by nanoparticles. The size distribution profile of AgNPs–bee honey nanoparticles was measured using dynamic light scattering (DLS) (Malvern Panalytical Ltd., Malvern, UK). Water samples of the nanoparticles (5 ml) were diluted with double distilled water (50 ml) using sodium chloride as electrolyte suspending solution (2 × 10−2 M NaCl). The pH was then adjusted to the required value. Samples were stirred for 30 minutes. After stirring, the measuring pH was recorded, and the particle size distribution of the metal particles was measured. In each case, an average of three ratings was reported. ANTIOXIDATION ACTIVITY DPPH–free radical scavenging. The antiradical activity of bee honey samples was measured according to the procedure of Ali et al. (9). A bee honey sample was dissolved in distilled water in concentrations from 125 to 500 mg/ml, and 0.2 ml of each solution was combined with 1.8 ml of 130 µM DPPH (final concentration 83.3 µM) dissolved in ethanol complete with 1 ml of acetate buffer solution (100 mM, pH 5.5). The mixtures were stirred vigorously and left for 30 minutes at room temperature in the dark, after which the remaining DPPH absorption was measured at 517 nm compared to blank to eliminate the effect of bee honey color. The blank sample was bee honey, in the same concentration as described above, and acetate buffer without DPPH solution. In each bee honey concentration tested, the