Antibacterial Activity of Boswellia Sacra Flueck. Oleoresin Extract Against Porphyromonas Gingivalis Periodontal Pathogen.

The study was carried out on 30 systemically healthy patients of both sexes their ages ranged from 25 to 50 years old, diagnosed with moderate to severe chronic periodontitis (grade III stage b periodontitis), and selected from the clinic of Periodontology, Faculty of Dentistry, Tanta University

Gingival crevicular fluid samples (GCF) were collected from pockets having ≥ 5 mm depth and positive bleeding on probing (active disease) with a paper point. Samples were obtained from the periodontal pockets after removing the supragingival plaque from the teeth to be sampled. The subgingival plaque samples were then inoculated into 2 ml brain heart infusion (BHI) broth supplemented with 5 μg/mL hemin and 1 μg/mL menadione (vitamin K1). They were then diluted and cultured onto blood agar supplemented with 5% sheep blood, hemin (5 μg/mL) and vitamin K1 (0.5 μg/mL). These plates were incubated in duplicate for 7-10 days at 37 °C in an anaerobic atmosphere. The bacteria grown were finally selected based on their color, size, and shape. The black-pigmented colonies and Gram-negative rods (when examined microscopically) were examined by a fluorescence test using longwave UV light. The absence of fluorescence distinguishes between P. gingivalis and other anaerobic, black-pigmented, Gram-negative rods. The identification of P. gingivalis isolates was then confirmed using API 20A (BioMérieux, France).

4.8. Antibacterial screening It was performed using the agar diffusion method [40]. In brief, bacterial suspension was distributed onto blood agar plate surfaces supplemented with 5% sheep blood, 1% (v/v) hemin, and 1% (v/v) vitamin K1, then sterilized 6 mm blank filter paper discs were impregnated with 25 µl of frankincense oleoresin extract with different concentrations ranging from 0.5 to 1000 µg/ml. Discs loaded with 10% DMSO was used as negative controls and tetracycline disc (30 µg) was used as a positive control. All Petri dishes were anaerobically incubated at 37°C for 48 hrs. Inhibition zone diameters were measured and the concentrations of frankincense extract which showed clear zones (inhibition zone) around the discs were regarded to have an inhibitory effect on the tested bacteria.

4.9. Determination of MIC values The MIC values of frankincense extract against P. gingivalis isolates were estimated using the broth microdilution method. Briefly, cultures of P. gingivalis were grown overnight in BHI broth containing hemin (5 µg/mL) and vitamin K1 (1 µg/mL). Then, 100 µL of bacteria plus 100 µL of serial dilutions (two-fold) of the frankincense extract (starting from 2000 µg/mL) in BHI were mixed in the wells of the microtitration plate. Each microtitration plate had a negative control well-containing BHI without bacteria and a positive control well containing only bacteria without the extract. After anaerobic incubation at 37°C for 24 hrs., bacterial growth was inspected visually. The MIC values were recorded as the lowest concentrations that caused an inhibition of the bacterial growth. All the following experiments were performed before and after treatment of P. gingivalis isolates with sub-inhibitory concentrations (0.5 MIC) of the frankincense extract.

4.10. Bacterial growth curve P. gingivalis isolates were cultured in blood agar plates, supplemented with 5% sheep blood, hemin (5 μg/mL), and vitamin K1 (0.5 μg/mL), for 5-7 days under anaerobic conditions at 37°C [43]. Then, a single colony of each isolate was inoculated into BHI broth, containing hemin (5 µg/mL) and vitamin K1 (1 µg/mL), and grown anaerobically for 24 hrs. The optical density (OD) values at 600 nm were detected every 2 hrs. using UV-VIS spectrophotometer (Shimadzu, Japan), and the growth curves were constructed via plotting the log OD against the sampling time (hrs.).