Chemical composition, antimicrobial and antibiofilm activity of the essential oil and methanol extract of the Mediterranean cypress (Cupressus sempervirens L.)

Abstract

Background: Cupressus sempervirens is a medicinal plant traditional, its dried leaves are used in treatment of stomach pain, diabetes, inflammation, toothache, laryngitis and as contraceptive.

Methods: The present study was conducted to evaluate the in vitro antimicrobial, antibiofilm and determination chemical contents of the essential oil (Eo) and methanol extract from Mediterranean C. sempervirens L. The chemical composition of a hydrodistilled Eo of C. sempervirens was analyzed by a GC and GC/MS system.

Results: A total of 20 constituents representing 98.1% of the oil were identified: α-pinene (48.6%), δ-3-carene (22.1%), limonene (4.6%) and α-terpinolene (4.5%) were the main components comprising 79.8% of the oil. The antimicrobial test results showed that the methanol extract of C. sempervirens strongly inhibited the growth of the test bacteria studied, except for yeast species while the Eo had moderate antibacterial, but no anti-candida activity. Klebsiella pneumoniae was proven to be the most susceptible against methanol extract. The exposure time of Eo and methanol extract for complete inhibition of cell viability of K. pneumoniae was found to be 250 μg at 30 min and 500 μg at 120 min, respectively. The antibiofilm potential of the samples was evaluated using methods of PVC microtiter and eradication on biomaterial. Visual results showed visible biofilm eradication from the surface of intravenous infusion tube at 500 μg of Eo and methanol extract.

Conclusions: The results presented here may suggest that the Eo and extracts of C. sempervirens possess antimicrobial and antibiofilm properties, and therefore, can be used as natural preservative ingredients in food and/or pharmaceuticals.

Figure 1

Effect of essential oil and methanol extract from of Mediterranean cypress (C. sempervirens L.) as 0 (control, ♦), 250 μg/ml methanol extract (■), 500 μg/ml methanol extract (▲), 250 μg/ml Eo (x) and 500 μg/ml Eo ( )on viability of K. pneumoniae. Control as no treatment with the essential oil and methanol extract. Values are the average of three individual replicates (means ± S.D). Differences between samples were determined by Student’s t-test and were considered to be significant when p ≤ 0.05 at least.

Figure 2

Showed biofilm formation of K. pneumoniae in PVC microtiter plate wells arranged from low to high biofilm production. Wells from left to right: (A) negative control (nutrient broth); (B) bacterial suspension supplemented by Eo 100 μg (C), 50 μg (D), 25 μg (E); methanol extract 100 μg (F), 50 μg (G), 25 μg (H) and ceftriaxone as reference antibiotic 100 μg (I) of stained with 1% crystal violet solution after 24 h of incubation at 37°C.

Figure 3

Image of intravenous infusion tube colonized by Klebsiella pneumoniae biofilm: from left to right, (A) not colonized negative control, (B) treated positive control, (C) treated with 500 μg/ml methanol extract of C. sempervirens , (D) treated with 500 μg/ml Eo of C. sempervirens and (E) treated with 500 μg/ml ceftriaxone as reference antibiotic.