Complete 2 D NMR assignment and antifungal activity of ishwarane isolated from Hortonia , a genus endemic to Sri Lanka

The tetracyclic sesquiterpene, ishwarane, was isolated from the leaves of the representative species of the genus Hortonia, H. angustifolia, H.floribunda and H. ovalifolia collected in Sr i Lanka. The complete 2 D NMR assignments are reported. Ishwarane exhibited antifungal activity against Cladosporium cladosporioides.


INTRODUCTION
The genus Hortonia is endemic to Sri Lanka and belongs to the Order Laurales and Family Monimiaceae.Members of this family are weeds or shrubs and rarely climbers.This family comprises of about 39 genera and 440 species which are widely spread in the Southern Hemisphere, mainly tropical and subtropical regions of the Americas 1 .Members of Family Monimiaceae are present in Sri Lanka, Oceania, Polynesia, Australia, Malaysia, Madagascar and South America 1,2 .They are very rare in Africa and absent in India.Siparuna and Mollinedia comprising of 150 and 94 species respectively are the two major genera of Monimiaceae 2 .Dassanayake (1996) lists three distinct Monimiaceae species {H.floribunda Wight ex Arn., H. angustifolia (Thw.)Trimen and H. ovalifolia Wight} in Sri Lanka 3 .Some phytogeographers consider the genus Hortonia to have originated in Gondwanaland about 100-200 million years ago 4 .The present study was initiated with a view to isolating the bioactive compounds from the plants of genus Hortonia in Sri Lanka.
Although three species of Hortonia are found in a range of environments from sea level (H.angustifolia) to the montane (H.floribunda and H. ovalifolia) regions, there have been no reports of the medicinal use of these plants in Sri Lanka.It has been previously reported that the bioassay-guided fractionation of the dichloromethane extracts of all three species yielded two mosquitolarvicidal butenolides 5 .Herein we report the isolation, complete 1D and 2D NMR assignment and the antifungal activity of the sesquiterpene hydrocarbon ishwarane from all three species of Hortonia.

METHODS AND MATERIALS
Analytical Thin Layer Chromatography (TLC) was performed on Merck Kieselgel 60 F 254 aluminium foil plates.TLC plates were visualized by spraying with anisaldehyde followed by heating.Medium Pressure Liquid Chromatography (MPLC) and flash chromatography were performed on Merck Kieselgel 60 (230-400 mesh ASTM). 1 H and 13 C NMR, COSY, DEPT, HETCOR, HMQC and HMBC spectra were recorded on a VARIAN ( 1 H 500 and 13 C 125 MHz) in CDCl 3 with TMS as the internal standard.EIMS were recorded on a Fisons VG Autospec mass spectrometer operating at 70 eV (direct insertion).Perfluorokerosine was used as the internal reference for HRMS measurements.Antifungal assay was carried out on pure ishwarane (1 mg).An aqueous solution of benomyl, 50% w.p. {methyl 1-[(butylamino) carbonyl]-H-benzimidazol-2-ylcarbamate} (1 mg) were used in the assays for comparison purposes.TLC plates (silica gel PF 254=366 , 0.5 mm x 20 cm x 20 cm) were spotted with pure ishwarane and the aqueous solution of Benor eluted with CH 2 Cl 2 , and then air dried (overnight) to evaporate all traces of remaining solvents.A spore suspension of Cladosporium cladosporioides in Czapek-dox nutrient solution (CNS) was sprayed on to the plates and incubated in a moist chamber at 28±2 °C for two days.Diameter of the inhibition zones were measured 6 .

Specimens of H. angustifolia
Conidia (5-7 d old) of C. cladosporioides were suspended in sterile distilled water and was passed through glass wool to remove mycelia.The conidia were washed three times by centrifugation (3 min at 3000 rpm) and resuspension of the resulting pellet in sterile distilled water.Conidia in the suspension were adjusted to about 5x10 2 mL -1 .A series of solutions of the compound was prepared in 10% EtOH in water.Drops (10 µL) from each were placed on clean glass slides.Care was taken to apply the drops over a uniform area on the glass slides.The control was prepared without the compound.The glass slides were then incubated in a moist chamber for 6 h at room temperature.Germination was stopped at the end of the incubation period by adding a drop of lactophenol.Four randomly selected areas on each slide were observed under a microscope and germinated and non-germinated conidia were counted.Percentage germination was determined by using average values of the four replicates 7 .

RESULTS AND DISCUSSION
Ishwarane was first isolated from the plants Aristolochia indica 8 and Cembopentalum penduliflorum 9 .It has been isolated from the essential oils of Bixa orellana 10 , Corallocarpus epigaeus 11 and Piper fulvescens 12 .Its structure was elucidated 35 years ago by chemical transformations and spectroscopic techniques 8,9,13 (Figure 1).The oxygenated derivatives ishwarol 14 and ishwarone 15 have been reported from the essential oils of Aristolochia indica and more recently, ishwarol B was isolated from the essential oils of Cedrelopsis grevei 16 .
Ishwarane (C 15 H 24 , M + 204.1) was isolated from the CH 2 Cl 2 extracts of the leaves of all three species of Hortonia in identical yields by MPLC followed by column chromatography and was subjected to a full set of 1D and 2D NMR experiments (Table 1).
Interestingly, it showed antifungal activity against C. cladosporioides (Table 2).A recent report showed that the essential oil from Piper guineense from Nigeria containing ishwarane inhibited the growth of Pseudomonas aeruginosa UCH 655 strain 17 .The occurrence of ishwarane in all the representative species in the genus Hortonia is of chemotaxonomic significance.

Table 2 :
Antifungal activity of ishwarane against C.