Identification of clovanediol: A rare sesquiterpene from the stem bark of canella winterana L. (Canellaceae), using spectrophotometric methods

D.W. KIOY,* A. I. GRAY,** and P. G. WATERMAN**

* Kenya Medical Research Institute (TMDRC),
P.O. BOX 54840, Nairobi, Kenya.

**Phytochemistry Research Laboratories,
Department of Pharmacy, University of Strathclyde
Glasgow G1 IXW, U.K.

ABSTRACT

The Canellaceae is a small plant family found in continental Africa, Madagascar and America. In Kenya the two species (Warburgia ugandensis and W. stuhlmannii) that belong to this family are used traditionally as medicines against many aliments. Canella winterana are trees with an aromatic and pungent bark, found in Florida and the West Indies. Its stem bark has been used as a flavouring agent, as spices and as medicine. Previous investigations of the plant have reported the occurrence of monoterpenes, sesquiterpenes, phenylpropanoids and mannitol in the plant. In a re-investigation of the plant, ground stem bark was macerated with petrol, ethyl acetate and methanol. The separation of the extracts chromatographically, that is, column chromatography, vacuum liquid chromatography and high performance liquid chromatography (HPLC) etc., yielded a number of compounds. Of these compounds, one was identified as clovanediol, with the help of nuclear magnetic resonance (NMR), infrared (IR), ultraviolet (UV).

Introduction

The Canellaceae is a small plant family of glabrous, aromatic trees and has been described (Good, 1971 and 1974) as a discontinuous family occurring in America, Africa and Madagascar. The Warburgia species are found in East and Central Africa, and are used traditionally as medicines and spices (Kokwaro, 1976; Watt and Breyer-Brandwijk, 1962; Dale and Greenway, 1961). Canella is a genus consisting of one species, C. winterana and is found in Southern Florida, through the Caribbean, and in Colombia (Hutchinson, 1964). It has been used traditionally as a spice and as medicine (BPC, 1934).

Earlier investigations of the stem bark of Canella reported the occurrence of monoterpenes, eugenol and mannitol (Claus, 1956 and Gibbs, 1974), drimane sesquiterpenes [canellal = muzigadial], 3-methoxy-4, 5-methylenedioxycinnamolide (El-Feraly, 1978 and 1979), and 4, 13-a-epoxymuzigadial (Al-Said et al., 1989). During our re-investigation of the stem bark, we reported on the isolation and identification of myristicin, eugenol, warburganal, mukaadial and 9a-hydroxycinnamolide (Kioy et al., 1989). We now report on the further identification of a tricyclic sesquiterpene, clovanediol (Aebi et al., 1953), using spectroscopic methods.

Materials and method

Plant material

The stem bark of Canella winterana was collected from the coastal bluffs at East End Grand Cayman (Kenya) in August 1981.

Extraction and isolation

Ground stem bark (85 g) was macerated in the cold using petroleum ether (boiling range 40-60°), ethyl acetate, and methanol, in succession. Comparative thin layer chromatography (TLC) of ethyl acetate and methanol extracts showed similar chromatogams, and they were mixed together and separated by means of Vacuum Liquid Chromatography (VLC). Silica gel (Merck, 60 G) chromatography (chloroform, and then a gradient of chloroform and methanol) gave a fraction which contained one major compound. This was purified by HPLC eluting with methanol/chloroform (2:100 v/v) and then by preparative HPLC to yield 18 mg of pure clovanediol.

Physio-chemical measurements

Melting points were determined using a Reichert sub-stage microscope melting point apparatus, and are uncorrected. Specific rotations, [a]_D were measured using a Perkin-Elmer model 241 polarimeter. The infra-red (IR) spectrum was recorded as a KBr disc on a Perkin-Elmer model 781 infra-red spectrophotometer. The Proton Nuclear Magnetic Resonance (¹H-NMR) spectrum was recorded on a Bruker WH-360 operating at 360 MHz instrument, and the carbon-13 nuclear magnetic resonance (¹³C-NMR) spectrum was recorded on a Bruker WH-360 instrument operating at 90.56 MHz. High resolution electron impact mass spectral data were obtained on an AEI-MS 902 double focussing instrument by direct probe insertion.

Discussion

The structure of clovanediol was established on the basis of the spectral data, and eventual comparison with literature information. Accurate mass measurements gave the molecular ion at m/z 238, which is consistent with formula C₁₅H₂₆O₂. The (¹³C-NMR) spectrum contained 15 carbon resonances, while Distortionless Enhancement by Polarisation Transfer (DEPT) experiments revealed that these consisted of three methyl, six methylene, three methine and three quaternary carbons.

Combined spectroscopic analysis and extensive single frequency irradiations and nuclear overhauser enhancement (NOE) experiments ultimately established that the isolated compound was clovanediol.

The relative stereochemistry was established by considering the magnitudes of the coupling constants, and by NOE experiments. The melting point was in agreement with the previously reported value of 152-153° (Aebi et al., 1953). This, together with the specific rotation of +6° [reported: +5° (Aedi et al., 1953)], confirmed the structure of clovanediol.

Scheme 2.4b: Proposed fragmentation pattern for clovanediol (Gupta and Dev. 1971)

Conclusion

The most logical approach towards the discovery of new drugs is through investigation of medicinal plants. This paper discusses an example on how compounds isolated from medicinal plants are identified. Although different physical-chemical methods may be used, the steps outlined in this paper are essential. In some plants, the active compounds are present in very small amounts which would otherwise be difficult to be investigated using other methods. But the use of modern spectroscopic methods has made it possible to carry out complete identification of compounds, even when they are in minute amounts.

The biological activity of clovanediol has not been investigated. However, it would be interesting to see if this compound has any activity.

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