Chemical composition and biological evaluation of the volatile constituents from the aerial parts of Nephrolepis exaltata (L.) and Nephrolepis cordifolia (L.) C. Presl grown in Egypt

The essential oil from the aerial parts of Nephrolepis exaltata and Nephrolepis cordifolia obtained by hydro-distillation were analyzed by gas chromatography/ mass spectrometry. The essential oils exhibited potential antibacterial and antifungal activities against a majority of the selected microorganisms. NEA oil showed promising cytotoxicity in breast, colon and lung carcinoma cells. The results presented indicate that NEA oil could be useful alternative for the treatment of dermatophytosis. Comparative investigation of hydro-distilled volatile constituents from aerial parts (A) of Nephrolepis exaltata (NE) and Nephrolepis cordifolia (NC) (Family Nephrolepidaceae) was carried out. Gas chromatography/mass spectrometry revealed that oils differ in composition and percentages of components. Oxygenated compounds were dominant in NEA and NCA. 2,4-Hexadien-1-ol (16.1%), nonanal (14.4%), β-Ionone (6.7%) and thymol (2.7%) were predominant in NEA. β-Ionone (8.0%), eugenol (7.2%) and anethol (4.6%) were the main constituents in NCA. Volatile samples were screened for their antibacterial and antifungal activities using agar diffusion method and minimum inhibitory concentrations. The cytotoxic activity was evaluated using viability assay in breast (MCF-7), colon (HCT-116) and lung carcinoma (A-549) cells by the MTT assay. The results revealed that NEA oil exhibited potential antimicrobial activity against most of the tested organisms and showed promising cytotoxicity.


Introduction
Nephrolepis exaltata (NE) and Nephrolepis cordifolia (NC) (Family Nephrolepidaceae) are terrestrial ferns and commonly cultivated as ornamental plants. NC is used to cure renal, liver and skin disorder (Dhiman 1998). NE acts as a natural air humidifier and purifier (Kobayashi et al. 2007). Rani et al. (2010) reported the phytochemical screening as well as the antibacterial and antifungal effects of ethanol extract of aerial parts of NC grown in India. To evaluate the possible use of the essential oils of NCA and NEA grown in Egypt for topical applications for skin infections, gas chromatography/mass spectrometry (GC/MS) was performed on both extracts to identify their antibacterial, antifungal and cytotoxic activities.

Antimicrobial activity
Volatile oils were screened for their antimicrobial activities using agar diffusion method and minimum inhibitory concentration (MIC) ( Table S1). The MIC values of NEA oil against Gramnegative bacterial strains Salmonella typhimurium, Klebsiella pneumoniae, Shigella flexneri and Escherichia coli were 3.9, 0.24, 0.49 and 1.95 mg/mL, respectively, whereas MIC against Proteus vulgaris was 7.81 mg/mL. MIC values indicated sensitivity of these organisms to NEA oil compared to gentamycin. In spite of the hydrophobic nature of the oil, the highest activity was observed against K. pneumoniae and S. flexneri. NEA showed a potent activity against a majority of the selected organisms except Pseudomonas aeruginosa with an MIC value of 125 mg/mL indicating resistance of the organism to the oil. MIC values against Staphylococcus aureus, Streptococcus pneumoniae and Enterococcus faecalis were 0.98, 1.95 and 1.95 mg/mL, respectively. Our study revealed less activity of NEA oil on Gram-positive bacteria as compared to Ampicillin. Although E. faecalis is known to be an intrinsically resistant Gram-positive bacterium, MIC values of NEA oil showed promising activity. Most of the antimicrobial activities could be associated with the presence of thymol, nonenal, nonanal, p-cymene, a-Ionone, b-ionone as well as anethol which are well-known substances with pronounced antimicrobial properties (Chatterjee and Kundu 1975;Mikhlin et al. 1983;Chao et al. 2000;Edris 2007). The antibacterial activity of NCA showed remarkable activity against K. pneumoniae with MIC 3.9 mg/mL. The oils were also screened for their antifungal activity (Table S2) in comparison to standard Amphoterecin B. NEA oil showed potent activity against Microsporum gypseum, Tricophyton rubrum and Tricophyton metagrophytes with MIC values 0.24, 0.98 and 1.95 mg/mL, respectively. However, NCA showed good activity against M. gypseum and T. rubrum with MIC values of 0.98 and 3.9 mg/mL, respectively. It is evident that there is a relation between the strong antimicrobial activity and the high percentage of oxygenated compounds. This is in agreement with the finding of Kalemba and Kunicka (2003) as well as that of Dorman and Deans (2000). The skin has an extremely diverse ecology of organisms that may produce infections. Empirical therapy for skin infections must include coverage of the commonly encountered pathogen. In addition, antimicrobial therapy could be complicated due to emergence of antibiotic resistance among different pathogens. Accordingly, our study emphasised on the antimicrobial activity of the different oils against different Grampositive and Gram-negative bacteria as well as different dermatophytes.

Cytotoxic activity
The cytotoxic activity of the NCA and NEA oils was evaluated using viability assay on human cancer cell lines colon (HCT-116), breast (MCF7) and lung (A-549) and the IC 50 values were calculated (Table S3). NEA and NCA oils were active against lung carcinoma with IC 50 of 43.5 and 46.2 mg/mL, and breast carcinoma with IC 50 of 44 and 47.9 mg/mL, respectively. This may be attributed to the presence of phytol, a-ionone and b-ionone which have been reported with cytotoxic activities (Dasgupta & Humphrey 1998;Sua et al. 2013). NCA and NEA oils were inactive against human colon cell line (HCT-116).

Conclusion
This study is the first report on the composition of volatile constituents, the antimicrobial and antidermatophyte as well as cytotoxic activities of the aerial parts of NC and NE cultivated in Egypt. NEA oil has displayed potent antibacterial and antifungal activities and could be a useful alternative for the treatment of dermatophytosis. NEA oil also showed potent cytotoxicity against three cell lines. We hope that our results will provide a starting point for investigations designed for a new natural antimicrobial essential oil of this plant species. Additional in vivo studies and clinical trials will also be needed to justify and further evaluate the potential of this oil as an antimicrobial agent in topical or oral applications.

Supplementary material
Experimental details relating to this paper are available online at http://dx.doi. org/10.1080/14786419.2015.1046070.

Disclosure statement
No potential conflict of interest was reported by the authors.