Isolation and Identification of Acylphloroglucinols in the Medicinal Plant, Melaleuca alternifolia (Australian Tea Tree).

Melaleuca alternifolia (tea tree), family Myrtaceae, is endemic to the northern rivers of NSW, Australia. Since 1925, the volatile components of the hydro- and steam-distilled oils of the leaves have been studied in detail. However, the less-volatile compounds have not been investigated. Using an ethanolic extract of the seedling leaves, the non-volatile components were studied using gas chromatography-mass spectrometry (GC/MS) and liquid chromatography- mass spectrometry (LC/MS). Four of these less-volatile components were isolated by preparative-HPLC from young seedling leaves and identified as the acylphloroglucinols 1-(2,6-dihydroxy-4-methoxy-3-methylphenyl)-2-methylpropan-1-one, callisalignone A, 1-(2,6-dihydroxy-4-methoxyphenyl)-3-methylbutan-1-one and pulverulentone B described here for the first time from M. alternifolia. These compounds change in concentration in the leaf sets as later seedling leaves mature on the seedling.

1H and 13C NMR assignments for the sesquiterpene aldehydes, lepidozenal and isobicyclogermacrenal, from Eucalyptus dawsonii.

Lepidozenal and isobicyclogermacrenal were isolated from the leaves of Eucalyptus dawsonii and a complete assignment of their 1H and 13C NMR spectra was carried out using 2D NMR methods.

25 years of natural product R&D with New South Wales agriculture.

Following recent NSW Government restructuring, the Department of Agriculture now exists in a composite form along with Forestry, Fisheries and Minerals in the new NSW Department of Primary Industries. This paper outlines some of the highlights of secondary metabolite R&D accomplished in the 25 years since the essential oil research unit was transferred from the Museum of Applied Arts & Sciences, Sydney to NSW Agriculture's Wollongbar Agricultural Institute on the NSW north coast. The essential oil survey was continued, typing the Australian flora as a suitable source of isolates such as myrtenal (Astartea), myrtenol (Agonis), methyl chavicol(Ochrosperma), alpha-phellandren-8-ol (Prostanthera), methyl myrtenate (Darwinia), methyl geranate (Darwinia), kessane (Acacia), cis-dihydroagarofuran (Prosthanthera), protoanemonin (Clematis), isoamyl isovalerate (Micromyrtus), methyl cinnamate (Eucalyptus) and bornyl acetate (Boronia). Many of these components are used, or have potential use in the fragrance, flavour, medicinal plant or insect attraction fields. Two weeds toxic to livestock in the Central West of the State are also harvested commercially as medicinal plants. Measurement of hypericin concentrations in the various plant parts of St John's Wort (Hypericum perforatum) over two seasons has shown that the weed can be effectively managed by grazing sheep during the winter months when toxin levels are low. Syntheses of beta-carbolines tribulusterine and perlolyrine have shown that the former alkaloid was misidentified in the literature and hence not the toxic principle responsible for Tribulus staggers in sheep. Poor quality (high 1,8-cineole - low terpinen-4-ol) oil bearing tea tree (Melaleuca alternifolia) plantations have been established to the detriment of many a tea tree farmer. Analytical methods developed to check leaf quality at an early age indicated precursor sabinene constituents that convert to the active terpinen-4-ol both as the leaf matures or as the precursors are distilled for oil production. Tea tree's major insect pest, pyrgo beetle (Paropsisterna tigrina), was seen to selectively metabolize only 1,8-cineole from it's monoterpenoid-rich diet. Characterization of these and other metabolites from myrtaceous herbivores showed a species specific production of predominately ring hydroxylated products, some of which were attractive when bioassayed against adult beetles.

Differential metabolism of 1,8-cineole in insects.

In order to compare the metabolism of 1,8-cineole in the pyrgo beetle, Paropsisterna tigrina, three other herbivorous insect species, Faex nigroconspersa, Chrysophtharta bimaculata, and Oxyops vitiosa, were fed 1,8-cineole leaf diets. F. nigroconspersa adults excreted predominantly 9-hydroxy-1,8-cineole (36.2% of the volatile constituents) with some 2alpha-hydroxy-1,8-cineole (11.4%). In contrast, larvae excreted predominantly 2alpha-hydroxy-1,8-cineole (27.4%) and smaller proportions of 9-hydroxy-1,8-cineole (5.2%) and 3alpha-hydroxy-1,8-cineole (4.3%). C. bimaculata adults excreted predominantly 3alpha-hydroxy-1,8-cineole (16.5%). Oxyops vitiosa adults, on a lower 1,8-cineole diet, excreted predominantly 2alpha,9-dihydroxy-1,8-cineole (4.2%) and 2alpha-hydroxy-1,8-cineole (3.5%), with smaller proportions of 3alpha-hydroxy-1,8-cineole (1.1%) and 9-hydroxy-1,8-cineole (0.5%). This is the first reported occurrence of a dihydroxycineole as an insect metabolite. Gas chromatographic and mass spectral data for hydroxycineoles are recorded and interspecific metabolite variation discussed.

Monoterpenoid accumulation in 1,8-cineole, terpinolene and terpinen-4-ol chemotypes of Melaleuca alternifolia seedlings.

Individual leaves of the three most common chemotypes of Melaleuca alternifolia were examined both quantitatively and qualitatively for volatile constituents from the emergence of the first true leaves, through to 6-week-old tenth leaf set material. The 1,8-cineole and terpinolene chemotypes were investigated and compared with the recently reported commercial terpinen-4-ol chemotype. The 1,8-cineole chemotype was found to accumulate 1,8-cineole and associated p-menthanes limonene, terpinen-4-ol and alpha-terpineol gradually with increasing leaf set number. As with the terpinen-4-ol variety, higher than expected concentrations of the pinenes and terpinolene were found only in the early leaf sets. The terpinolene variety showed two stages of terpinolene accumulation, the first at leaf sets 2-3 similar to the unexpected biosynthesis of terpinolene in the terpinen-4-ol chemotype and the second at leaf sets 8-9 which is characteristic of the terpinolene variety.

Volatile oil comparison of cotyledon leaves of chemotypes of Melaleuca alternifolia.

Oil quantity and quality were measured for the cotyledon leaves of the commercial terpinen-4-ol chemical variety of Melaleuca alternifolia. Oil yield obtained by ethanolic extraction was 3.8 micrograms per leaf or 2.6% (dry weight basis). The major components of the oil were alpha-pinene (7.4%), beta-pinene (12.0%) and terpinolene (27.3%). The non-commercial terpinolene chemical variety was found to be rich in 1,8-cineole (12.5%) and terpinolene (25.4%). The non-commercial 1,8-cineole chemical variety was rich in 1,8-cineole (37%) with significant quantities of alpha-pinene (15.5%), beta-pinene (23.3%) and terpinolene (10.9%). The cotyledon leaf composition, when compared with that of mature leaf from the same chemical variety, was found to be biased toward pinene and terpinolene biogenetic pathway constituents and hence not a good indicator of mature tree quality especially for the commercial terpinen-4-ol chemical variety. The implications of these analyses for the determination of M. alternifolia plantation quality and the understanding of oil formation, are discussed.