Chemical Composition and Antimicrobial Activity of Essential Oil from Eupatorium triplinerve Vahl. Aerial Parts

Eupatorium triplinerve Vahl belongs to the Asteraceae family, popularly known as Ayapana .It is a perennial shrub native to Amazon rainforests of South America. Its leaves are used through infusions, decoctions, baths, and tea. It is largely used in Brazilian folk medicine as sedative, febrifuge, stimulant, tonic and anti-inflammatory . The essential oil from aerial parts of Eupatorium triplinerve was analysis by gas chromatography mass spectroscopy (GC-MS). Thirty compounds representing 98.24 % of the oil were identified. The major compounds were 2-tert- butyl – 1, 4- methoxybenzene (74.3 %) and b – Selinene (8.6 %). The antibacterial activity of the essential oil was evaluated against ten bacteria (including Gram positive and Gram negative) and six phytopathogenic fungi. The oil exhibited moderated antibacterial and strong antifungal activity against all the test pathogens. The highest zone inhibition was recorded against Salamella typhae (21mm) followed by Shigella sonnei (18mm) at a concentration of 20µl/ disc on the other standard Ampicillin 20mg/ disc showed on average of 14-34mm diameter of zone inhibition against the test organism. The lowest antifungal activities were recorded against Macrophomina phaseoline , and Botryodiplodia theobromae . The highest Alternaria alternate (12000ppm).Further pharmacological and toxicology studies will be required to establish the therapeutic uses of this oil.

1998). Eupatorium triplinerve (Compositae) is a slender herb with narrow lanceolate leaves and large number of pedicelled flower-heads at the top of the branch, cultivated in Chittagong and Chittagong Hill Tracts. Extract of the plant is used as antiseptic, and in the treatment of various ulcers and hemorrhages (Ghani 1998). This paper reports the result of antimicrobial activity of E. triplinerve on folk usages by traditional practitioners in Tribal areas of Bangladesh. Ayapana is a rich source of naturally occuring coumarin chemicals and is used in herbal medicine as antitumor remedy. It was reported that a coumarin chemical named hernarin (7-methoxycoumarin) was toxic to cancer cells including multi drug resistant cancer cells (Canales et al., 2005; Yadava and Saini, 1980) and leukemic cells (Garg and Nigam, 1970). Garg and Nigam (1970) reported the presence of thymohydroquinone dimethylether, coumarin, borneol and bornyl acetate along with seven monoterpenoids in the leaf oil. Later,( Garg and Nakhare,1993) analyzed the flower oil of E. triplinerve and found thymohydroquinone dimethylene (50.3%), followed by borneol (5.81%) and isoeugenol (4%) as the major constituent, along with other minor constituents. The essential oil of the flowers has yielded much better antimicrobial results than the plant itself. Ayapana flowers essential oil possess strong antifungal activity against 10 strains of fungi in vitro (Sharma and Singh, 1979). It also possesses antibacterial (against staph, cholera, pneumonia and shigella) as well as antiparasitic (Ascaris) and anthelminitic (Taenia) actions (Garg and Nakhare, 1993). In an early animal study, the flower essential oil injected into mice was reported to have CNS depressant, analgesic and sedative effects (as well as an in vitro antibacterial effect) (Kokate and Verma, 1971). Ghani (2003) reported the presence of coumarin, ayapanin and ayapin having pronounced haemostatic properties. There is no report on the essential oil of E. triplinerve of local origin and its antimicrobial properties. So, the aim of the present study was to investigate the chemical composition and antimicrobial activity of the essential oil of E. triplinerve aerial parts growing in Bangladesh in order to find out new natural antimicrobial agents. Extraction of essential oil: The experiment was conducted when the plants were healthy and well-grown. For extracting of essential oil aerial parts (5 Kg) were subjected to hydro distillation using a modified Clevenger apparatus (Clevenger, 1928) for 4 hours. The oil sample was stored at freeze in air-tight containers after drying them over anhydrous sodium sulfate for GC-MS analysis.

Collection of Plant material
Test organisms: The essential oil obtained from E. triplinerve aerial parts was tested for its antibacterial activity against ten pathogenic bacteria, viz.,

International Letters of Natural Sciences Vol. 31 15
Antibacterial activity: The in vitro antibacterial activity of the oil was determined by disc diffusion method (Bauer and Kirby, 1966). Nutrient agar medium was used for culture of bacteria. Each experiment was repeated thrice. All the results were compared with the standard antibacterial antibiotic Ampicillin (20 µ g/disc).
Antifungal activity: The in vitro antifungal activity of the essential oil of E. triplinerve was determined by poisoned food technique (Grover and Moore, 1962). Potato dextrose agar medium was used for culture of fungi. Each experiment was repeated thrice. Linear mycelia growth of fungus was measured after 3-5 days of incubation. The percentage inhibition of radial

Determination of minimum inhibitory concentration (MIC) against bacteria and minimum bacterial concentration (MBC)
Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the essential oil against ten test bacterial isolates were determined by micro and macro dilution broth technique (Jones et al., 1985) using Muller Hinton medium. The MIC and MBC experiments were carried out using 100-25000 ppm concentrations of the essential oil.

Determination of minimum inhibitory concentration (MIC) against fungus and minimum fungicidal concentration (MFC)
The MIC and MFC of essential oil against six test fungi were determined by micro and macro-dilution broth technique (Jones et al., 1985) using Sabouraud medium. The MIC and MFC experiments were carried out using 100 to 12,000 ppm concentrations of the essential oil.

GC-MS analysis
The essential oil from aerial parts of E. triplinerve was analyzed by GC-MS electron impact ionization (EI) method on GC-17A gas chromatograph (Shimadzu) coupled to a GC-MS QP 5050A Mass Spectrometer (Shimadzu); fused silica capillary column (30m x 0.25mm; 0.25 mm film thickness), coated with DB-5 (J&W); column temperature 100 oC (2 min) to 250oC at the rate of 3oC/min; carrier gas, helium at constant pressure of 90Kpa. Acquisition parameters full scan; scan range 40-350 amu.
Identification of the constituents Constituent's identification was done by comparing the NIST library data of the peaks with those reported in literature. Percentage composition was computed from GC peak areas on DB-5 column without applying correction factors.

ILNS Volume 31
The results of the in vitro antibacterial activity of the essential oil (2, 5, 10, 15 and 20µl/disc) against the ten bacterial strains (including Gram-positive and Gram-negative) are summarized in (Table 2.).It was observed that the essential oil was moderately effective against the test bacterial stains even at the lowest concentration (2µl/ disc) of the essential oil tested 10-25mm diameter of zone of inhibition was recorded in 20µl /disc concentration. The highest zone inhibition was recorded against Salamella typhi (21mm) followed by Shigella sonnei (18mm) at a concentration of 20µl/ disc (Table) on the other standard Ampicillin 20mg/ disc showed an average of 14-34mm diameter of zone inhibition against the test organism.
The MIC value of the essential oil varied between 17000-21000ppm against the test bacteria ( Table 3). The oil showed the lowest MIC (17000ppm) against Shigella typhae followed by 18000ppm against Shidella sonnei, Shigella dysenterae and Pseudomonas and highest (21000ppm) against Staphylococcus aureus, Bacillus cereus, Bacillus subtilis, Bacillus megaterium, Vibro cholera and E.coli. The oil exhibited the lowest MBC value (22000ppm) against Shigella sonnei, Shigella dysenterae, Pseudomonas and Salmonella typhae. The in vitro antifungal activity of essential oil against six phytopathogenic fungi was recorded and presented in ( Table  4). The oil exhibited strong pathogen tested. In most of the cases, the essential oil exhibited better antifungal activity than the standard antibiotic, Nystain ( Table 4). The MIC value of the essential oil was found of very between 6000-9000ppm against the test fungi (Table). The lowest MIC value (6000ppm) was recorded against M .phaseoline and C. corchori. MFC value of the essential oil were found to very between 8000-12000ppm ( Table 5). The oil exhibited the lowest MFC value (8000ppm) against M phaseoline and B. thebromae. Similar antibacterial and antifungal activity of essential oil of other plant have also been reported. Medicinal as well as aromatic plant is an important source of potentially useful new compound for the discovery and development of chemotherapeutic against. E.triplinerve is a medicinal plant and also an important source of essential oil which contains 2-tert butyl-1, 4 methoxybenzene as major constituent (74.3 %) followed by b-selinene (8.6%) is more antifungal compared to antibacterial agent. Further pharmacological and toxicological studies will be required to establish the therapeutic uses of this oil.