Antibacterial and antioxidant activity of naphthofuranquinones from the twigs of tropical mangrove Avicennia officinalis

: Mangrove plants are endowed with various biologically active compounds which have potent antibacterial and antioxidant properties. In present study, a bioactivity-guided fractionation for antibacterial and antioxidant active metabolites from the twigs of Avicennia officinalis collected from Kuala Selangor Nature Park, Selangor, Malaysia gave 13 major fractions. The antibacterial activity of A. officinalis fractions using well-diffusion showed strong selectivity on the Gram-positive bacteria ( Staphylococcus epidermidis , S. aureus and Bacillus subtilis ) with minimum inhibition concentration (MIC) values of 0.156-5.00 mg/mL. However, no antibacterial activities were observed on the Gram-negative bacteria ( Vibrio cholera , Enterobacter cloacae and Escherichia coli ). The active antibacterial fractions were further isolated using several chromatographic techniques to give two naphthofuranquinones, namely, avicenol C ( 1 ) and stenocarpoquinone B ( 2 ). Meanwhile, the antioxidant activity of A. officinalis fractions were evaluated using DPPH radical scavenging assay exhibited low antioxidant activities. Molecular structure of the naphthofuranquinones was elucidated using 1D and 2D NMR spectroscopy.

collected yielding 24 fractions. Each sub-fraction was concentrated using a vacuum rotary evaporator and TLC was carried out to monitor the separation. Sub-fractions with the similar profile were pooled together to give 13 major fractions. The fractions were tested for antibacterial and antioxidant activity and further isolation work was carried out on the active fractions using radial chromatography. The glass plate of radial chromatography was coated with silica gel 60 PF 254 gypsum (Merck,art. 7749) to isolate and purify the active metabolites. Compound 1 (8.8 mg, 0.08%) and 2 (10.8 mg, 0.10%) were obtained after purification on fractions F7 and F8, respectively.

Determination of Antibacterial Activity:
Preparation of nutrient agar and Mueller-Hinton agar: 28 g of nutrient agar powder was weighed and added into 1 L of distilled water; 35 g of Mueller-Hinton agar powder was added into 1 L of distilled water. Both media was then heated and autoclaved at 121 ºC for 20 minutes. The screw cap was left loosely for the autoclave. About 20 mL of agar medium was poured into each plate and let to be cool. The agar plates were sealed with parafilm and stored upside-down in the refrigerator for further use. Mueller-Hinton agar plates must be used within a week. Malaysia. Each bacterium was streaked using inoculation loop on a labeled agar plate and incubated for more than 20 hours. A single colony of bacterium was selected and streaked using inoculation loop to another agar plate for the sub-culture of bacteria.

Agar well diffusion and minimum inhibitory concentration (MIC):
An antibacterial activity of ethyl acetate crude extract from the twigs of A. officinalis was conducted using agar well-diffusion method as described (Balouiri et al. 2016). Prior to assay, wells on Mueller Hinton agar plate were created using 8 mm sterile cork borer. Then, six bacteria-S. epidermidis, S. aureus, B. subtilis, V. cholera, E. cloacae and E. coli which were first adjusted of their concentration at optical density (O.D) 0.5 were swabbed on the Mueller-Hinton agar plates. A series of eight concentrations of each selected fractions diluted in DMSO was prepared with 2-fold dilutions ranging from 5 mg/mL to 0 mg/mL. 50 µL of the serial dilutions was pipetted into the agar well accordingly. Oxytetracycline (OT) antibiotic disc (30 µg) (CT0041B, Thermo Scientific Oxoid, United Kingdom) was placed in the middle of the agar plate as a positive control. All plates were incubated for 24 hours at 37 ºC to allow the test extract to diffuse into the agar medium. Three replicates were done for each fraction to obtain average values. Then, the inhibition zone diameter (IZD) was measured to the nearest millimeters. Minimum inhibitory concentration (MIC) was taken as the lowest concentration of the fraction that shows the inhibition zone.

Determination of Antioxidant Activity:
The antioxidant assay was conducted to evaluate the potential of selected fractions of A. officinalis according to method described by Suvik and Effendy (2016). Approximately 2.3659 mg of DPPH powder (D9132, Sigma Aldrich, United States) was weighed and dissolved in 100 mL of 100% methanol to obtain a stock solution. A series of eight concentrations of fractions F5-F10 was diluted in DMSO with 2-fold dilutions ranging from 0-1.0 mg/mL. 20 µL of each serial dilution was pipetted into respective wells of the 96-well plate and the 0 mg/mL dilution was used as a blank. 200 µL of DPPH solution was added to each well containing the fractions, except the blank. The plate was incubated in dark at room temperature for 30 minutes. Then, the absorbance was read at 520 nm using a multiwell scanning spectrophotometer (Variouskan, USA). Quercetin was used as positive control. The concentrations of fractions require to achieve 50% inhibition concentration of DPPH radical (IC 50 ) was determined from the linear regression curve. All determinations were performed in triplicates to obtain a mean value. The radical scavenging activity will be calculated by the following formula: nhi ition ercenta e Where A 0 = Absorption of blank sample A 1 = Absorption of tested fractions mixed with DPPH

Statistical Analysis:
All data were presented as mean + SD and statistically analyzed with One-Way ANOVA in the comparison between selected fractions using statistical analyses software PRISM Ver. 5.
Data are significantly different at p < 0.05. Table S1. Average inhibition zone diameter (IZD, mm) ± S.D. of fractions F5-F10 from the twigs of A. officinalis a different concentration values of 0-5 mg/mL against Gram-positive bacteria.