Chemical Composition, Antimicrobial Activity and Antioxidant Activity of Litsea cubeba Essential Oils in Different Months

: In this study, the chemical composition, antimicrobial and antioxidant activities of Litsea cubeba essential oils extracted in different months were analysed. Results showed that the essential oil contents of the fruits collected in June, July and August were 3.47%, 5.02% and 5.64%, respectively, and had 13, 17 and 17 components, respectively. Neral and geranial were the main components and accounted for 54.76%. The essential oil extracted from the fruits collected in July had the highest antimicrobial activity against Staphylococcus aureus , Escherichia coli , and Salmonella typhimurium , and was most effective based on the OH· scavenging activity test. The essential oil extracted from the fruits collected in August was the most effective based on the text for DPPH· scavenging activity and ferric-reducing antioxidant power. Considering the contents, chemical compositions, antimicrobial and antioxidant activities, and the appropriate harvest time for L. cubeba essential oils is from July to August.

. Chromatogram profiles of LCFEOs in June, July and August Note: The Numbers represent the identified compositions, and the same Number means same compositions. University.

Antimicrobial activity test
The antimicrobial activity of the LCEOs was evaluated by measuring their minimum inhibitory concentration (MIC) (Benkeblia 2004;Hussain et al. 2008). The LCEOs were diluted to different concentrations (0.5%, 0.8%, 1%, 2% and 4%, v/v) with anhydrous ethanol and were tested for MIC through the disk diffusion method. Then, 0.1 mL portions of a 10 6 CFU/mL suspension of the test strains were uniformly distributed to Petri dishes soaked with essential oils. Then incubated at 37 °C for 24 h.
TBHQ was used as the positive control and anhydrous ethanol as the blank control.
The diameters of the inhibition zones were measured to evaluate the antibacterial activity of the LCEOs. MIC was defined as the lowest concentration of LCEOs that resulted in the complete inhibition of visible growth after incubation for 24 h (Table   S2 and Table S3).
After the addition of 2.5 mL of 10% trichloroacetic acid, the solutions were centrifuged at 3 000 r/min for 10 min. Then, 2.5 mL of the supernatant was added with 2.5 mL of distilled water and 1 mL of 0.1% ferric chloride. Absorbance measurements were recorded at 700 nm to evaluate the reducing power of the essential oils.

DPPH· scavenging activity
The DPPH· scavenging activity of the LCEOs was tested following the method proposed by Gordon et al. (Gordon et al. 2001). The LCEOs were diluted to 0.5%, 1%, 2%, 4% and 6% (v/v) with anhydrous ethanol. A 2 mL sample solution was mixed with 2 mL of methyl alcohol and 2.0 mL of 0.2 mmol/L DPPH. After 30 min of incubation in the dark at 25 °C, the absorbance was obtained at 517 nm by using a spectrophotometer (UV-1600PC). Methyl alcohol without DPPH was used as the blank sample and TBHQ as the positive control. The percentage inhibition rate of the DPPH· scavenging activity of the essential oils was calculated using the following formula: where IC is the inhibition rate of the essential oils, A 0 is the absorbance value of the blank sample (DPPH), A 1 is the absorbance value of the tested sample, and A 2 is the absorbance value of the blank sample (LCEOs).

OH· scavenging activity
The essential oils were diluted to 0.05%, 1%, 0.2%, 0.3%, 0.4% and 0.5% (v/v) with anhydrous ethanol. Solutions (1 mL) were mixed with 1 mL of 9 mmol/L FeSO 4 and 1.0 mL of H 2 O 2 and stored in the dark. After 30 min of incubation in the dark at room temperature, the absorbance was recorded at 510 nm by using a spectrophotometer (UV-1600PC) (Wang et al. 2007). TBHQ was used as the positive control. The hydroxyl radical-scavenging activity of the essential oils was calculated using the following formula: where the IC is the inhibition rate of the essential oils, A 0 is the absorbance value of the blank sample (DPPH), A 1 is the absorbance value of the tested sample, and A 2 is the absorbance value of the blank sample (LCEOs).