Herbicidal ß-triketones are compartmentalized in leaves of Leptospermum species: localization by Raman microscopy and rapid screening.

The New Zealand manuka shrub, Leptospermum scoparium, and the Australian L. morrisonii produce herbicidal ß-triketones in their leaves. The localization of these potential self-toxicants has not been proven. We investigated the localization of these compounds in leaves using Raman microscopy. The results are presented as heat maps derived from principal component analysis (PCA) of the Raman spectra from sampling grids of leaf sections. This approach used undirected, data-driven analysis to qualitatively distinguish localized plant chemistry. The presence of ß-triketones and lipophilic flavonoids was confirmed by GC-MS and (1) H NMR spectroscopy. Grandiflorone was compartmentalized within the leaf oil glands of L. morrisonii. Leptospermum scoparium also contained high concentrations of grandiflorone, previously reported as only a trace component in essential oils, localized in the oil glands in the leaves of varieties from diverse geographical locations. Raman microscopy was used to probe the chemistry of oil glands in several ornamental manuka varieties, revealing high concentrations of bioactive flavonoids localized in these glands. The compartmentalization of ß-triketones within oil glands inside leaves of Leptospermum shrubs may defend the plants against herbicidal activity.

Cytotoxic Amides from Fruits of Kawakawa, Macropiper excelsum.

Cytotoxic amides have been isolated from the fruits of the endemic New Zealand medicinal plant kawakawa, Macropiper excelsum (Piperaceae). The main amide was piperchabamide A and this is the first report of this rare compound outside the genus Piper. Eleven other amides were purified including two new compounds with the unusual 3,4-dihydro-1(2H)-pyridinyl group. The new compounds were fully characterized by 2D NMR spectroscopy, which showed a slow exchange between two rotamers about the amide bond, and they were chemically synthesized. In view of the antitumor activity of the related piperlongumine, all of these amides plus four synthetic analogs were tested for cytotoxicity. The most active was the piperine homolog piperdardine, with an IC50 of 14 µM against HT 29 colon cancer cells.

Chemosystematic analyses of Gingidia volatiles.

The leave volatiles of six Gingidia species from New Zealand and Australia and the seed volatiles of G. grisea were characterized by solid-phase microextraction (SPME)-GC/MS analysis. This technique, using a small quantity of samples and automated extraction, gave repeatable results, with maximum sensitivity for medium volatility compounds. The major monoterpenes among the volatiles, i.e., ß-phellandrene (4), limonene (6), and γ-terpinene (5), and phenylpropanoids, i.e., estragole (3), (E)-anethole (7), and myristicin (1), showed to be useful chemotaxonomic markers. For G. grisea leaves and seeds, similar compositions were detected, characterized by high contents of 4. As leaves were more readily available for study than seeds, they were used for further investigations. The G. grisea leaf volatiles showed infraspecific variation in the ratio of 4/5 between and within sites of collection. The G. montana leaf volatiles also showed infraspecific variation, with high contents of 3 at one site and high contents of 7 at another. The SPME-GC/MS analysis of G. montana herbarium voucher specimens resulted in the identification of further chemotypes for this species. The volatiles of the G. amphistoma samples were all dominated by 7 and those of the G. haematitica samples were rich in 5. Moreover, single plants of two Australian Gingidia species were analyzed; the volatiles of G. harveyana showed high concentrations of 5 and 7, whereas those of G. rupicola were dominated by 5 and 1.

Sesquiterpene lactones in Arnica montana: helenalin and dihydrohelenalin chemotypes in Spain.

An analytical RPLC method for sesquiterpene lactones in Arnica montana has been extended to include quantitative analyses of dihydrohelenalin esters. LC-ESI-MS-MS distinguished the isomeric helenalin and dihydrohelenalin esters. The dihydrohelenalin esters have lower response factors for UV detection than do helenalin esters, which must be taken into account for quantitative analyses. Analyses of flowers from 16 different wild populations of A. montana in Spain showed differing proportions of helenalin and dihydrohelenalin esters. For the first time a chemotype with high levels of helenalin esters (total helenalins 5.2-10.3 mg/g dry weight) is reported in Spanish A. montana. These samples were from heath lands at high altitude (1330-1460 m), whereas samples from meadows and peat bogs at lower altitudes were the expected chemotype with high levels of dihydrohelenalin esters (total dihydrohelenalins 10.9-18.2 mg/g). The phenolic compounds, both flavonoid glycosides and caffeoylquinic acids, in Spanish A. montana are reported for the first time. The levels of several of these compounds differed significantly between samples from heath lands and samples from peat bogs or meadows, with the heath land samples being most similar to central European A. montana in their phenolic composition.

Flavor, glucosinolates, and isothiocyanates of nau (Cook's scurvy grass, Lepidium oleraceum) and other rare New Zealand Lepidium species.

The traditionally consumed New Zealand native plant nau, Cook's scurvy grass, Lepidium oleraceum, has a pungent wasabi-like taste, with potential for development as a flavor ingredient. The main glucosinolate in this Brassicaceae was identified by LC-MS and NMR spectroscopy as 3-butenyl glucosinolate (gluconapin, 7-22 mg/g DM in leaves). The leaves were treated to mimic chewing, and the headspace was analyzed by solid-phase microextraction and GC-MS. This showed that 3-butenyl isothiocyanate, with a wasabi-like flavor, was produced by the endogenous myrosinase. Different postharvest treatments were used to create leaf powders as potential flavor products, which were tasted and analyzed for gluconapin and release of 3-butenyl isothiocyanate. A high drying temperature (75 °C) did not give major glucosinolate degradation, but did largely inactivate the myrosinase, resulting in no wasabi-like flavor release. Drying at 45 °C produced more pungent flavor than freeze-drying. Seven other Lepidium species endemic to New Zealand were also analyzed to determine their flavor potential and also whether glucosinolates were taxonomic markers. Six contained mostly gluconapin, but the critically endangered Lepidium banksii had a distinct composition including isopropyl glucosinolate, not detected in the other species.

Alkaloid variation in New Zealand kowhai, Sophora species.

Alkaloid contents of leaf and seed samples of eight species of Sophora native to New Zealand, plus Sophora cassioides from Chile are reported. Fifty-six leaf and forty-two seed samples were analysed for alkaloid content by proton nuclear magnetic resonance spectroscopy, which showed major alkaloids as cytisine, N-methyl cytisine and matrine. GC analyses quantified these and identified further alkaloid components. The alkaloids identified were cytisine, sparteine, and matrine-types common to Sophora from other regions of the world. Cytisine, N-methyl cytisine, and matrine were generally the most abundant alkaloids across all species with seeds containing the highest concentrations of alkaloids. However, there was no clear taxonomic grouping based on alkaloid composition. A quantitative analysis of various parts of two Sophora microphylla trees showed that the seeds were the richest source of alkaloids (total 0.4-0.5% DM), followed by leaf and twig (0.1-0.3%) and then bark (0.04-0.06%), with only low amounts (<0.02%) found in the roots. This study represents the most comprehensive phytochemical investigation of New Zealand Sophora species to date and presents data for three species of Sophora for which no prior chemistry has been reported.

Essential oils from New Zealand manuka: triketone and other chemotypes of Leptospermum scoparium.

The triketone chemotype of manuka, Leptospermum scoparium (Myrtaceae), is commercially important because of its antimicrobial activity. Oils from 36 individual plants on the East Cape of New Zealand all showed similar high triketone contents (>20% total triketones) with little seasonal variation. Analyses of oils from 261 individual manuka plants collected from 87 sites throughout New Zealand showed that the high triketone chemotype was localised on the East Cape, although oils with triketone levels up to 20% were found in the Marlborough Sounds area of the South Island. Cluster analysis revealed other chemotypes localised on other areas. Ten further chemotypes are described: alpha-pinene; sesquiterpene-rich with high myrcene; sesquiterpene-rich with elevated caryophyllene and humulene; sesquiterpene-rich with an unidentified sesquiterpene hydrocarbon; high geranyl acetate; sesquiterpene-rich with high gamma-ylangene + alpha-copaene and elevated triketones; sesquiterpene-rich with no distinctive components; sesquiterpene-rich with high trans-methyl cinnamate; high linalol; and sesquiterpene-rich with elevated elemene and selinene. Some of the chemotypes contained aroma compounds at relatively high levels, with a geranyl acetate-rich oil being most notable. Possible origins for this complex array of chemotypes are proposed.

Betalains in red and yellow varieties of the Andean tuber crop ulluco (Ullucus tuberosus).

The betalain pigments in ulluco (Ullucus tuberosus), a tuberous crop native to the Andes, have been investigated for the first time using LC-DAD-ESI-MS-MS(2) analyses. Five red, yellow, and red-spotted accessions introduced into New Zealand as a new food crop plus two red tetraploid lines were investigated. Thirty-two different betalains were identified. Both the yellow and red tubers were rich in yellow betaxanthins, and the most prominent among the 20 identified were histidine-betaxanthin, arginine-betaxanthin and glutamine-betaxanthin. Arginine-betaxanthin has been reported to occur naturally only once before and was found in yellow ulluco but not in the red tubers. Twelve betacyanins were found in red tubers, with roughly 50% of this content being betanin/isobetanin. Betacyanin levels were up to 70 microg/g fresh weight in red tubers, but were below quantifiable levels in yellow tubers. Betaxanthin levels were up to 50 microg/g fresh weight in yellow tubers. Interference by betacyanins in measuring levels of betaxanthins by visible spectrophotometry is discussed. Low concentrations of betalains were detected in leaves, whereas stems contained total levels similar to the tubers, with dopamine-betaxanthin and betanin being the major pigments. This is the first report describing both the betacyanin and betaxanthin patterns in a plant from the Basellaceae family.

Sesquiterpene lactones in Arnica montana: a rapid analytical method and the effects of flower maturity and simulated mechanical harvesting on quality and yield.

A rapid extraction, clean-up and RPLC procedure suitable for routine quantitative analyses of sesquiterpene lactones (SLs) in Arnica montana is described. Seven SLs were isolated of which tigloyl and methacryloyl esters of helenalin made up over 50 % of the total. This method was applied to analyses of replicated samples of different flower parts, different stages of flower maturity, and herb from different harvest methods. The mean total SL levels were higher in the disk flowers (0.872 % w/w) than the ray flowers (0.712 %), lower in the flower receptacles (0.354 %) and lowest in stems (0.028 %). Relative levels of individual SLs varied significantly between flower parts, especially acetyldihydrohelenalin which had its highest concentration in stems. The total SL contents increased progressively as the flowers matured, from 0.512 % in buds to 0.943 % in withered flowers. Harvesting a range of flower maturities at one time in a simulated mechanical harvest, followed by mechanical separation of low quality stem material gave the same quality as hand harvested A. montana flowers (over 0.8 % total SLs) and the flower yields from the two processes were similar when adjusted for harvesting technique (320 kg dry matter/ha by hand, 295 kg/ha mechanical). Delaying flower harvest until the flower petals had withered greatly improved the SL concentration of the drug.