Infraspecific variation of foliage terpenes of Podocarpus hallii

Journal of Essential Oil Research: JEOR, Jul/Aug 2003 by Clarke, Don B, Weavers, Rex T, Perry, Nigel B

Received: September 2002

Revised: October 2002

Accepted: December 2002

Abstract

A detailed study of the levels of sesquiterpenoids and diterpenoids in essential oils from 36 specimens of Podocarpus hallii from two populations has revealed considerable infraspecific variation both within and between populations. The unusual tetracyclic diterpene laurenene, which has previously been encountered only in Podocarpus totara and Dacrydium cupressinum, was found at low levels in several of the samples.

Key Word Index

Podocarpus hallii, Podocarpaceae, infraspecific variation, essential oil composition, laurenene.

Introduction

Lauren-1-ene (I, Figure 1) is a unique, tetracyclic diterpene containing a triquinane system. It was first reported from Dacrydium cupressinum over 20 years ago (1), but since then, neither it, nor any related compounds, have been reported from natural sources. Its incidence in D. cupressinum is highly variable (2). We have issued a preliminary report of new sources of laurenene from three New Zealand Podocarpus species, which collectively are known as totara (3). These include two large forest trees, Podocarpus totara G. Benn. ex D. Don and P. hallii Kirk, which are distinguished by different bark morphology; and the sub-alpine P. nivalis Hook., which is normally a prostrate shrub (4,5). As communicated previously (3,6), this investigation also revealed a new, laurenene-related diterpene, waihoensene (II, Figure 1) that was found only in P. totara samples. This compound, like laurenene, is tetracyclic and contains a triquinane unit.

The seven endemic New Zealand species previously classified into the genus Podocarpus are now contained in Dacrycarpus, Prumnopitys and Podocarpus, with four species retaining the old genus name Podocarpus (5). Detailed studies of the essential oils from multiple specimens of Prumnopitys taxifolia (matai, previously Podocarpus spicatus) (7) and of Prumnopitys ferruginea (miro, syn. Podocarpus ferrugineus) (8) have revealed a high degree of infraspecific variability, but no study has yet been reported on variability of the oils of any of the totara species. As the diterpenes laurenene (I) and waihoensene (II) contain structural elements more closely allied to known sesquiterpene structures such as silphenene (III, Figure 1) (9), both sesquiterpenes and diterpenes were examined in this study. Cambie's New Zealand phytochemical registers (10) reveal that previous workers have found regular tricyclic and tetracyclic diterpenes in the oils of P. hallii, P. nivalis and P. totara, along with a cadinene from P. totara. In a more recent study of a sample of an oil from bulked P. totara foliage, six sesquiterpenoids and nine diterpenoids were identified by GC/MS (11). These compounds did not include waihoensene, laurenene, or any triquinane sesquiterpene.

The goal of this investigation was to explore the variability of the levels of the terpenes in P. hallii and to determine if any patterns emerged. In addition, we were alert to the possibility of finding either sesquiterpenes or diterpenes containing triquinane sub-units, possible links to laurenene or waihoensene.

Experimental

General: ^sup 1^H-NMR and ^sup 13^C-NMR spectra were determined as CDCl^sub 3^ solutions on a Varian VXR 300 spectrometer operating at 300 MHz and 75 MHz, respectively. ^sup 1^H-NMR spectra were referenced to the residual CHCl^sub 3^ peak ([delta] 7.26) and ^sup 13^C-NMR spectra to the centre line of the CDCl^sub 3^ signal (77.08 ppm). Column chromatography was performed with Kieselgel 60 (Merck Art. 9385) or Kieselgel S (Merck 31607) as the adsorbent. Radial chromatography was performed using a chromatotron 7924T (Harrison Research, Palo Alto, CA) with Kieselgel 60 PF^sub 254^ as the adsorbent. Silica coated plates were prepared according to the manufacturer's instructions. Loadings of 100-200 mg, 300-750 mg and 800-1500 mg of impure samples were separated on glass plates coated with silica of thickness 1, 2 and 4 mm, respectively. For silver nitrate impregnated silica gel, the same procedure of plate preparation was used, this time with the addition of silver nitrate (1 mm-2 g, 2 mm-3 g, 4 mm-5 g). GC was performed on a Perkin-Elmer 8420 capillary gas chromatograph fitted with a flame ionization detector. A 10 m x 0.25 mm DB-1 fused-silica capillary column, film thickness 0.25 [mu]M, supplied by J&W Scientific was used. Injections were 0.05-1.0 [mu]L with a split ratio of ca. 1:50. The injector temperature was 230[degrees]C and the FID temperature was 240[degrees]C. The temperature was programmed (50[degrees]-300[degrees]C with 25[degrees]C/min ramp rate) and the carrier gas used was helium. GC/MS was performed on a Perkin-Elmer Ion Trap Detector mass spectrometer, directly coupled to a Perkin-Elmer 8420 capillary gas chromatograph (column details and operating conditions as above). Statistical analyses used Gen Stat.

Sampling and extraction procedures: Podocarpus hallii foliage was collected from Mt. Cargill (NZMS 1S 164 205797, 17 trees, 8 November 1990) and Oamaru (NZMS 18 23 320636, 19 trees, 20 November 1990) in the South Island of New Zealand. Voucher samples have been lodged in the Plant Extracts Research Unit herbarium in the chemistry department, University of Otago.