CHEMICAL COMPOSITION AND ANTIBACTERIAL ACTIVITY OF LEAF ESSENTIAL OIL OF EUGENIA COTINIFOLIA SSP. CODYENSIS (MUNRO EX WIGHT) ASHTON

CHEMICAL COMPOSITION AND ANTIBACTERIAL ACTIVITY OF LEAF ESSENTIAL OIL OF EUGENIA COTINIFOLIA SSP. CODYENSIS (MUNRO EX WIGHT) ASHTON

R. Sarvesan ¹, P. Eganathan ^{*2, 3}, J. Saranya ² and P. Sujanapal ⁴

Department of Plant Biology and Plant Biotechnology ¹, Presidency College (Autonomous), Kamarajar Salai, Triplicane, Chennai, Tamilnadu 600005, India

Plant Tissue Culture and Bioprospecting Laboratory ², M. S. Swaminathan Research Foundation, 3^rd Cross Road, Taramani, Chennai, Tamilnadu 600113, India

Meta Procambial Biotech Private Limited ³, Thanneer Pandal Palayam, Peria Semur PO, Erode- 638004, India

Kerala Forest Research Institute ⁴, Peechi P.O, Thrissur District, Kerala, India

ABSTRACT: The study was carried out to investigate the essential oil composition and antibacterial effects of Eugenia cotinifolia ssp. codyensis leaves. The fresh leaf essential oil was extracted hydrodistillation process using Clevanger apparatus. The compounds of the essential oil were analyzed using Gas Chromatography Mass Spectrometry (GC-MS) technique identified a total of 84 numbers of chemical constituents and resulted 99.99%. The essential oil was characterized by high content of sesquiterpene compounds (79.23%) and the major constituent being Germacrene D (17.95%). The leaf oil exhibited higher antibacterial effects against gram negative bacteria compare to gram positive bacteria. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of the oil against the organism ranged between 0.25 – 0.75 mg/ml and 0.5 – 2.5 mg/ml respectively. The essential oil exhibited maximum inhibition at 0.25 mg/ml against Proteus vulgaris and the same inhibited Bacillus subtilis and Staphylococcus aureus at 0.75 mg/ml. The result of essential oil chemical constituents and antibacterial activity showed useful for preparation of commercial products

Eugenia cotinifolia ssp. codyensis, Myrtaceae, germacrene D, sesquiterpene, antibacterial activity

INTRODUCTION: Eugenia cotinifolia ssp. codyensis is one of the species belonging to family Myrtaceae and has been categorized as an endangered tree species under the International Union for Conservation of Nature (IUCN) red list of threatened species¹. This subspecies is known only from a single collection in the south of Karnataka. An additional collection, imprecisely located in the Nilgiris, and a record from the Agastyamalai hills has been reported. Moreover, the species is endemic to India ¹.

Eugenia species exhibits antidiabetic and antiulcer ², anti-inflammatory ³, xanthine oxidase inhibitiory ⁴, antibacterial ⁵ activity activities and also reduces blood pressure ⁶.

Literature survey revealed that no study on essential oil of this species has been conducted thus far. In this context, the present effort is to evaluate the chemical composition and antibacterial activity of the leaf essential oil of Eugenia cotinifolia ssp. codyensis.

MATERIALS AND METHODS:

Collection of plant material: Fresh leaf materials were collected from 75 years old tree at Chembra, Wayanad District, Kerala and specimens collected and further confirmed by Dr P. Sujanapal, Kerala Forest Research Institute (KFRI), Peechi, Kerala and the voucher specimens (KFRI 23374) deposited at the KFRI herbarium.

Extraction of essential oil:

Five hundred grams of fresh leaves coarsely chopped and subjected to hydrodistillation for 4 h using a Clevenger apparatus⁷. Distilled essential oil was dried over anhydrous sodium sulfate and stored at 4°C.

Gas Chromatography-Mass Spectrometry analysis:

GC-MS analyses were conducted using Agilent MSD (5975B-inert XL MSD) apparatus equipped with National Institute of Standards and Technology (NIST) reference libraries; column DB-5MS (J&W Scientific) cross-linked fused-silica capillary column (30 m × 0.25 mm × 0.25 µm thickness), coated with 5% phenyl-polymethylsiloxane; column temperature, 80°C for 0 min, rising to 150°C at 10°C/min, then 250°C at 5°C/min, then rising to 270°C at 20°C held for 6 min. injector temperature 270°C, injection mode, split; split ratio 1:20; volume injected, 2 µL of the oil. Helium was used as a carrier; interface temperature 270°C; acquisition mass range, m/z 55-550. The compounds of the oil were identified by comparing their retention indices (RI), with NIST library. Relative Retention Indices (RRI) of lower and higher homologue was obtained from standard hydrocarbon data calculated as:

Microbial isolates:

Four Gram negative (Pseudomonas aeroginosa, Proteus vulgaris, Klebsiella pneumoniae and Escherishia coli), two Gram positive (Staphylococcus aureus and Bacillus subtilis) bacterial strains were used in this study. The bacterial stock culture were maintained on nutrient agar medium stored at 4°C.

Antibacterial assay:

The oil was tested for their antibacterial activity using disc diffusion method. Bacterial species were sub-cultured on nutrient agar medium which were then incubated at 37°C for 24 h. Test solutions of essential oil at concentrations of 1000 μg, 500 μg, 250 μg, 100 μg/ml were impregnated on sterile discs. Ampicillin were used as positive control. The disc impregnated with Dimethyl sulfoxide (DMSO) was used as negative control. The discs were placed on the surface of the nutrient agar for bacteria and incubated at 37°C for 24 h. Inhibition zones were calculated as the difference between disc diameter (6 mm) and the diameters of inhibition ⁸. Antibacterial activities were evaluated by determining MIC using micro broth dilution assay ⁹.

TABLE 1: ESSENTIAL OIL CHEMCIAL COMPOSITION OF LEAF OIL

 Monoterpene hydrocarbon                  0.05

Sesquiterpene hydrocarbon                 79.23

Alkanes hydrocarbon                          0.09

Others                                                 20.62

*RRI value calculated from DB-5MS column

Germacrene D, a sesquiterpenoid compound that has deterrent effects against herbivores and also reported possess insecticidal activity against mosquitoes ¹⁰ and repellant activity against aphids ¹¹ and ticks ¹². The compound is also suggested to serve as biogenetic precursor to a number of different sequiterpenoid skeletons ^{13, 14}. Essential oil containing large concentration of Germacrene D is reported to be accompanied by the formation of cadinene and muurolene sequiterpenoids ^15-19 which has also been proved from our study in Eugenia cotinifolia ssp. codyensis.

Antibacterial activity:

The major compounds dominant in the leaf oil are sequiterpenoid compounds that make up 79.23% of the total oil. Sequiterpenoid compounds are known to possess high medicinal properties ^20-23 that may make this tree species a highly potent medicinal plant against various diseases. The leaf oil possesses significant antibacterial effects against the tested pathogenic organisms (Table 2).

TABLE 2: ANTIBACTERIAL ACTIVITY OF LEAF ESSENTIAL OIL

Both gram positive and gram negative bacteria were found to be sensitive against the leaf oil. Among the screened micro organism, the oil showed higher activity against Proteus vulgaris and moderate activity against Bacillus subtilis and Staphylococcus aureus.

The oil exhibited MIC values against the organism in the range of 0.25 - 0.75 mg/ml and MBC values in the range of 0.5 - 2.5 mg/ml (Table 3). The essential oil showed maximum inhibition at 0.25 mg/ml against Proteus vulgaris whereas the same oil inhibits the growth of Escherichia coli at 0.3 mg/ml, Pseudomonas aeruginosa and Klebsiella pneumoniae at 0.5 mg/ml. It is also observed that gram positive bacteria (Bacillus subtilis and Staphylococcus aureus) were inhibited at higher concentration of 0.75 mg/ml of the sample. The leaf oil is effective towards P. aeruginosa, E. coli, and K. pneumoniae compared to other Syzygium species like, S. alternifolium and S. samarangense ²⁴.

TABLE 3: MIC AND MBC VALUE FOR LEAF ESSENTIAL OIL

CONCLUSIONS: The leaf essential oil of Eugenia cotinifolia ssp. Codyensis has higher activity against gram negative bacteria and the essential oil can be utilized for such antibacterial formulations and applications. 

ACKNOWLEDGMENTS: The authors thank the Department of Science and Technology, and Department of Biotechnology, Govt. of India for financial support.

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    How to cite this article:

    Sarvesan R, Eganathan P, Saranya J and Sujanapal P: Chemical Composition and Antibacterial Activity of Leaf Essential Oil of Eugenia Cotinifolia Ssp. Codyensis (Munro ex Wight) Ashton. Int J Pharm Sci Res 2015; 6(9): 3981-85.doi: 10.13040/IJPSR.0975-8232.6(9).3981-85.

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