ETHYL LINALOOL AND DIETHYL PHTHALATE FROM PYCNANTHUS ANGOLENSIS ( WELW . ) WARB .

Pycnanthus angolensis (Welw.) Warb. (Family; Myristiceae) leaves were extracted cold with 50 % ethanol and the obtained aqueous crude extract partitioned with ethyl acetate. Furthermore, the ethyl acetate fraction was subjected to silica gel column chromatography and the isolated chemical compounds tested for antimicrobial activity against Staphylococcus aureus, Escherichia coli and Candida albicans. The identities of two isolates have been revealed to be 3-ethoxy-3,7-dimethyl-1, 6-octadiene (ethyl linalool) and diethyl phthalate (1,2benzenedicarboxylic acid diethyl ester) using the MS and IR spectral techniques. Both compounds showed strong bacteriostatic action against E. coli but were inactive against S. aureus and C. albicans.


INTRODUCTION
Pycnanthus angolensis (Welw.)Warb.syn.3][4] The larvicidal and antitumor potentials of the plant have been studied [5][6] while reports of isolation of flavonoids and terpenes from the bark and roots and quinones from the leaves abound. 7This present investigation aimed at isolating the compound(s) from the ethyl acetate fraction which demonstrated the highest antimicrobial activity in a previous bioactivity-guided fractionation study of the plant. 8In addition, the compounds so obtained will be screened for antimicrobial activity with the aim of confirming or disproving the claims highlighted in traditional medicine especially for the treatment/management of bacterial infections.

MATERIALS AND METHODS
The fresh leaves of P. angolensis were collected around April, 2016 within the precinct of University of Uyo, Akwa Ibom State, Nigeria.The plant had previously been identified in a study. 8Immediately after collection, the plant was dried in a laboratory oven (Gallenkamp, England) at 40 0 C for 48 h and the resultant material powdered on an electric mill (Uniscope, England).

Antimicrobial tests
The microorganisms used in this study, namely; Staphylococcus aureus (ATCC 21824), Escherichia coli (ATCC 2353) and Candida albicans (NCYC 106) were clinically isolated from specimens of diarrheal stool, abscesses, necrotizing fasciitis, urine and wounds obtained from the Medical Laboratory, University of Uyo Health Centre, Uyo.0] These clinical microbes were then refrigerated at -5 0 C. The agar plates used were prepared by adhering to the manufacturer's instructions.The media and plates were sterilized in an autoclave at 121°C for 15 min.
The hole-in-plate agar diffusion method was used observing standard procedures for Nutrient Agar-CM003, Mueller-Hinton-CM037 (Biotech Limited, Ipswich, England) and Sabouraud Dextrose Agar (Biomark, India) in respect of bacteria and fungus respectively.The inoculum of each micro-organism was introduced into each petri-dish (Pyrex, England).Cylindrical plugs were removed from the agar plates using a sterile cork borer (Simax, India) to produce wells with a diameter of approximately 5 millimetres.2] Concentrations of 20 mg mL -1 of crude extract, 10 mg mL -1 of ethyl acetate fraction, 2 mg mL -1 of NG-2 and NG-4c were introduced into the wells.4][15] The experiments were carried out in triplicates.The plates were labelled on the underside and left at room temperature for 2 h to allow for diffusion.The plates were then incubated at 37 ± 2 0 C for 24 to 48 h.Zones of inhibition were measured in millimetres (mm) with the aid of a ruler.

NG
The FTIR spectrum of NG-2 shows absorptions at 1652 and 1087 cm -1 indicating acyclic -C=C and -C-O-C (ether linkage) respectively.The compound, ethyl linalool showed an optical rotation of +3° indicating dextrorotation.

Antimicrobial screening
The spectrum of microbes employed in the sensitivity tests was narrow, encompassing one each of gram positive (S.aureus) and gram negative (E. coli) bacterial strains and a fungus (C.albicans).The results displayed in Table 1 show that the crude extract, ethyl acetate fraction, NG-2, and NG-4c were inactive against S. aureus and C. albicans.However, the two compounds were remarkably bacteriostatic against E. coli.This result was unexpected because gram-negative bacteria are well known for their unique resistance to antimicrobial agents.This resistance is believed to be due to the nature of the cell envelope of these organisms which unlike gram-positive organisms possess a sophisticated three-layered envelope which does not allow permeation of external agents.Also, both compounds demonstrated no antifungal activity against C. albicans.This particular observation was to be expected because of fungal strains especially Candida spp.limit the permeation of substances because of their integral structures which are pleomorphic and facultative in nature hence, resembling those of higher plants. 16It is instructive to mention that derivatization studies are currently on-going in our laboratories with the aim of improving on the observed activity.

CONCLUSION
The isolation of the two compounds is being reported for the first time from the ethyl acetate fraction of the plant.Hence, ethyl linalool and diethyl phthalate are expected to serve as chemotaxonomic markers for this species and the genus, Pycnanthus in general.Furthermore, the results of the antimicrobial sensitivity tests lend some credence to the use of this plant especially in the treatment or management of the bacterial disease.

Table 1 .
Results of antimicrobial screening of crude extract, ethyl acetate fraction, NG-2 (ethyl linalool) and NG-4c (diethyl phthalate) at different concentrations on test microbes in deionized water