Abstract: The antimicrobial activity of essential oil of Pentanema indicum var. sivarajanianum (L.) Ling. was investigated. GC-MS analysis of the hydrodistilled oil resulted in the identification of 21 compounds constituting 99.4% of the total oil. The major constituents are hexadecane, α-humulene, myrecene and β-thujone comprising 51.6% of the oil. The oil strongly exhibited antimicrobial activity against Pseudomonas aeruginosa, Staphylococcus aureus, Micrococcus luteus, Micrococcus roseus, Candida albicans and Candida tropicalis. The present study confirms that the essential oil possess significant antimicrobial properties in vitro.
Introduction
Many hundreds of plants worldwide are used in traditional medicines as treatment for bacterial infections. Particularly essential oils are known to possess antimicrobial activity (Hammer et al., 2002; Unlu et al., 2002; Alvaro et al., 2003; Tatjana et al., 2004) but the efficacy of such herbal medicines has seldom been rigorously tested in controlled clinical trails (Martin and Ernst, 2003). With the adverse reactions to the synthetic drugs and the emergence of resistant microorganisms are causing many problems for both the treatment of patients and infection controls. The resistant microorganisms are increasing and eradication with the current agents is not always successful (May et al., 2000). Efforts are being made to look for the products of natural origin, which have a great variety of bio-dynamic actions.
Pentanema indicum var. sivarajanianum (Asteraceae) is habituated in hill slopes and interior forest plains. It is known as ‘Chiruchamanti’ and the leaf extract has taken orally for several skin ailments by the aboriginal tribes of Eastern Ghats, which are common in tribes due to the lack of sanitation and hygienic food (Jeevan Ram et al., 2004). This is the first report on the biological activity and composition of the essential oil of Pentanema indicum. In the current study the essential oil derived from the aerial parts was screened against various pathological microorganisms in vitro. Our previous studies encouraged us to undertake the present investigations to assess the plant for any further beneficial effect of medicinal importance.
Materials and Methods
Extraction of the Essential Oil
The leaves of the plants were collected from the forests of Nallamalais,
India during February 2002. The plant was identified with the help of regional
flora (Pullaiah et al., 1997) and a voucher specimen (AJR-23816) has
been deposited at the herbarium of Sri Krishnadevaraya University (SKU), Anantapur.
The shade dried leaves were subjected to hydro-distillation for 3 h, using a
Clevenger type apparatus. The oil was dried over anhydrous sodium sulphate and
stored at 4°C until tested and analyzed.
GC-MS Analysis
The analysis was carried out on Shimadzu 17 A GC coupled with Shimadzu QP
5050A (Quadruple) Mass-Spectrometer equipped with EI and a fused silica column
DB-5 (30 m x 0.25 mm i.d.) of 0.25 μm film thickness coated with polysilphenylene-siloxane.
One microliter of the concentrated solvent fraction was injected and the GC
oven temperature kept at 50°C for 5 min and programmed from 50°C-280°C
for 40 min. Helium was used as carrier gas at a flow rate of 2 mL min-1
with a split ratio of 1:30 and ionization voltage of Mass spectral analysis
was run by EI technique at 70 eV. The components were identified by comparing
their relative retention indices with those of standard reference compounds
and available literature data (Masada, 1989; Adams, 2001).
Antimicrobial Screening
The antimicrobial activity of the oil was assayed by using disc diffusion
method (Bauer et al., 1966) and Minimum Inhibitory Concentrations (MICs)
were determined by broth microdilution method (NCCLS, 1999). The assay was performed
individually against Pseudomonas aeruginosa (MTCC 1688), Staphylococcus
aureus (MTCC 737), Micrococcus luteus (1541), Micrococcus roseus
(MTCC 2522), Candida albicans (MTCC 183) and Candida tropicalis
(MTCC 184). These were obtained from Institute of Microbial Technology (IMTECH),
Chandigarh, India. All bacteria were cultured overnight at 37°C in nutrient
agar medium and yeasts were cultured overnight at 30°C in Sabouraud dextrose
agar.
A suspension of the each microorganism (0.1 mL of 108 cells per mL) was seeded on to the respective media plates. Sterile Whatman No.1 filter paper discs (6 mm in diameter) were impregnated with 50 μL of the oil, are placed on the inoculated plates. These plates after staying at 40°C for 2 h, were incubated at 37°C for 24 h for the bacteria and 30°C for 48 h for the yeasts. The diameter of the inhibition zones around the discs were measured in millimeters. Standard antibiotics viz., ampilicin for bacteria and ketoconazole for yeasts were used as positive controls. Triplicates were carried out for each concentration.
The determination of Minimum Inhibitory Concentration was performed in nutrient broth for bacteria and Sabouraud dextrose broth for yeasts, supplemented with Tween-80 detergent. The hydrodistilled oil at starting concentration of 64 mg mL-1 was transferred into the first well and serial dilutions were performed ranging to 0.032 mg mL-1 were prepared in 96-well micro-titer plate, including one growth control (Nutrient broth + Tween-80) and one sterile control (Nutrient broth+ Tween-80+test oil). The plates were incubated at 37°C for 24 h for bacteria and 30°C for 48 h for the yeasts.
Results and Discussion
Dermatophytes (candidiosis, dermatomycosis, ringworm) are the most common forms of fungal infections found in most countries (Ribbon, 1988), cause diseases of the skin, nails and hair. The important reason being that a majority of the people of developing world still relies on indigenous systems of medicine for the treatment of their common ailments (Ali and Azhar, 2000). Anecdotally, the leaf juice known as external remedy for bacterial and fungal infection in particular dermatophytes (Jeevan Ram et al., 2004).
Leaves of Pentanema indicum var. sivarajanianum on hydrodistillation yielded dark pungent yellowish green oil (1.2%v/w). GC analysis of freshly extracted distilled oil revealed the presence of 21 different components representing 99.4% of the total composition (Table 1). The oil consists non-terpenoidal alkanes (23.78%), sesquiterpene hydrocarbons (22.58%), alcohols (19.53%), monoterpene hydrocarbons (11.91%), oxygenated monoterpenes (7.86%) accomplished by relatively smaller amounts of esters (3.14%), monoterpene ketones (2.8%) and oxygenated sesquiterpenes (2.34%). The non-terpenoidal alkanes with hexadecane as the major constituent (21.85%) and accomplished by noticeable amount of tetradecane (1.93%). The sesquiterpene hydrocarbons represented by α-humulene (17.71%) as major component and small amounts of cubebene (4.87%). The alcohols with Pentanol (5.28%) and Hexanol (5.16%), monoterpene hydrocarbons with myrecene (6.58%), oxygenated monoterpenes with β-thujone (5.52%) comprises as major components.
The essential oil exhibited stronger antimicrobial activity against all the tested organisms. The growth of tested microorganisms ranged from 0.25 to 0.5 mg mL-1 (w/v) with the lowest MIC value against Pseudomonas aeruginosa, Candida albicans and C. tropicalis at 0.25 mg mL-1 (w/v). The Table 2 lists the zones (mm) and minimum inhibitory values (MIC) for the bacterial and fungal isolates studies. For Staphylococcus aureus, Micrococcus roseus and M. luteus the essential oil generally follows a concentration dependent antimicrobial activity.
The results show that the plant extract investigated demonstrate antimicrobial activity in their potential for helping to cure different skin diseases. The oil contains terpens, ketones, sesquiterpenes that have antimicrobial properties. Surprisingly the non-terpenoidal alkanes are present as major component which are very common in most of the Asteraceous members (Bohlmann, 1973).
| Table 1: | Chemical constituents of essential oil of Pentanema indicum var. sivarajanianum |
| RRI= Relative Retention Indices, calculated against n-alkanes | |
| Table 2: | Antimicrobial activity of essential oil of Pentanema indicum var. sivarajanianum |
It is evident that some minor compounds viz., camphene should also be taken into consideration for antimicrobial activity in combination. Camphene is known as decongestant, antiseptic (Bruneton, 1995) and is further known for its tropical use as a counter-irritant in fibrositis (Martindale, 1996). In light of the above activity that contribute to cure of different skin ailments it is important to use a battery of in vitro tests to evaluate the efficacy of this crude drug. The further studies aimed at the isolation and identification of novel antimicrobial substances with better therapeutic value.