O-Acylated Flavones in the Alpine Daisy Celmisia viscosa: Intraspecific Variation.

Flavonoids acylated on their core phenolic groups are rare. The Aotearoa New Zealand endemic alpine daisy Celmisia viscosa is widespread, but its flavonoids have not previously been identified. Leaf extracts yielded a series of 8-O-acylated flavones with combinations of 3-methylbutanoate, 2-methylbutanoate, and 2-methylpropanoate groups and one, two, or three O-methyls, all previously unreported. Regiochemistries of 8-(3″-methylbutanoyl)-5-hydroxy-6,7,4'-trimethoxyflavone (5) and 8-(2″-methylbutanoyl)-5,7,4'-trihydroxy-6-methoxyflavone (10) were defined by X-ray crystallography. LC analyses of leaf extracts from the full geographic range of C. viscosa showed intraspecific variation of these flavones: most had high concentrations of trimethoxy 8-O-acylated flavones, but dimethoxy 8-O-acylated flavones were the most abundant flavonoids in two individuals. Three other viscid (sticky leaved) Celmisa species also contained these rare flavones, but four nonviscid Celmisa had none detectable.

Halimane Diterpenes in the Alpine Daisy Celmisia viscosa: Absolute Configuration, 2,6-Dideoxyhexopyran-3-ulosides, Conformational Flexibility, and Intraspecific Variation.

The methyl-migrated bicyclic skeleton of the halimane diterpenes has been found in a wide range of organisms, including flowering plants, liverworts, marine animals, and bacteria. The discovery of halima-1(10),14-dien-13-ol (3) from the Aotearoa New Zealand endemic alpine daisy Celmisia viscosa is now reported. The full configuration was assigned for the first time by X-ray crystallography, enantiomeric to that of a liverwort isolate. The absolute configuration at C-5 of the halimane is opposite to that at C-5 of the labdane epimanool (1) found in some C. viscosa specimens. Two new 2,6-dideoxyhexopyran-3-uloside halimane derivatives (4 and 5) were also found, and the absolute configuration of 5 was determined by 1H NMR analysis of the Mosher esters. Line broadening in the 13C NMR spectra of these halim-1(10)-enes was due to conformational exchange in the decalin ring A, as shown by molecular modeling and DFT calculations. 1H NMR and GC analyses of leaf extracts of individual plants from across the full geographic range of C. viscosa revealed intraspecific variation of diterpenes: 37 samples had halimadienol as the main diterpene in large amounts and 2 specimens had predominantly epimanool, again in large amounts. Three other viscid (sticky leaved) Celmisia species also contained diterpenes, but none was detectable in four nonviscid Celmisia species.

Analytical artefacts: H2 carrier gas hydrogenation of plant volatiles during headspace solid-phase microextraction gas chromatography.

INTRODUCTION: Hydrogen is the most efficient and economical carrier gas for gas chromatography (GC). However, there are rare reports of artefact formation by hydrogenation of unsaturated compounds on GC. Head space solid-phase microextraction (HS-SPME) GC conditions for hydrogenation were studied. METHODOLOGY: HS-SPME-GC-mass spectrometry (MS) analyses of common classes of plant volatiles were carried out using hydrogen (H2 ) and helium (He) carrier gases with different SPME fibre coatings, GC inlet temperatures, and desorption times. RESULTS: Common phenylpropanoids, monoterpenes, and green leaf volatiles were hydrogenated to varying degrees on HS-SPME-GC with H2 carrier gas and SPME fibres coated with polydimethylsiloxane (PDMS)/Carboxen (CAR), PDMS/divinylbenzene (DVB), and PDMS/CAR/DVB. No artefacts were detected using PDMS-only coated fibres or He carrier gas. CONCLUSION: Unsaturated plant volatiles may be hydrogenated on HS-SPME-GC when using H2 carrier gas with SPME fibre coatings containing DVB polymer or CAR porous particles. Parallel analyses with He and H2 carrier gases are recommended when developing HS-SPME-GC methods for plant volatiles, or use of PDMS-only coated fibres.

Fast Sampling, Analyses and Chemometrics for Plant Breeding: Bitter Acids, Xanthohumol and Terpenes in Lupulin Glands of Hops (Humulus lupulus).

INTRODUCTION: The valuable secondary metabolites in hops (bitter acids, xanthohumol, volatile monoterpenes and sesquiterpenes) are sequestered in lupulin glands (extracellular trichomes) which can be collected and analysed with little or no sample preparation. OBJECTIVES: To determine whether high throughput screening of lupulin glands composition, by fast analyses and chemometrics, could be used for breeder selection of hops with key flavour attributes. METHODS: Lupulin glands from 139 plants (39 cultivars/advanced selections) were analysed by Raman and 1 H NMR spectroscopy, and head-space solid-phase microextraction (HS-SPME) GC-FID. The digital X,Y-data were subjected to principal component analysis (PCA) and the results compared with conventional analyses of extracts of whole hops from the same plants. Quantitative 1 H NMR analyses were also done for the bitter acids. RESULTS: Raman spectroscopy rapidly identified hops cultivars with high xanthohumol concentrations and high α:ß bitter acid ratios. 1 H NMR spectroscopy was slower, requiring a solvent extraction, but distinguished cultivars by cohumulone content as well as α:ß acid ratios. HS-SPME-GC rapidly distinguished aroma hops with high myrcene and farnesene contents, and pinpointed a novel selection with unusual sesquiterpenes. The quantitative NMR analyses showed correlations between bitter acid concentrations related to biosynthetic pathways. CONCLUSIONS: Analysis of lupulin glands gave reliable results for the main quality indicators used by hops breeders, potentially avoiding harvesting, drying and solvent extracting whole hops. PCA of digital X,Y-data rapidly discriminated different hops chemotypes, and highlighted plants with potential for new flavourcultivars. Copyright © 2016 John Wiley & Sons, Ltd.

Sweet Poisons: Honeys Contaminated with Glycosides of the Neurotoxin Tutin.

Poisonings due to consumption of honeys containing plant toxins have been reported widely. One cause is the neurotoxin tutin, an oxygenated sesquiterpene picrotoxane, traced back to honeybees (Apis mellifera) collecting honeydew produced by passionvine hoppers (Scolypopa australis) feeding on sap of the poisonous shrub tutu (Coriaria spp.). However, a pharmacokinetic study suggested that unidentified conjugates of tutin were also present in such honeys. We now report the discovery, using ion trap LC-MS, of two tutin glycosides and their purification and structure determination as 2-(ß-d-glucopyranosyl)tutin (4) and 2-[6'-(α-d-glucopyranosyl)-ß-d-glucopyranosyl]tutin (5). These compounds were used to develop a quantitative triple quadrupole LC-MS method for honey analysis, which showed the presence of tutin (3.6 ± 0.1 µg/g honey), hyenanchin (19.3 ± 0.5), tutin glycoside (4) (4.9 ± 0.4), and tutin diglycoside (5) (4.9 ± 0.1) in one toxic honey. The ratios of 4 and 5 to tutin varied widely in other tutin-containing honeys. The glycosidation of tutin may represent detoxification by one or both of the insects involved in the food chain from plant to honey.

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.

Chemical diversity of kanuka: Inter- and intraspecific variation of foliage terpenes and flavanones of Kunzea (Myrtaceae) in Aotearoa/New Zealand.

Kunzea (Myrtaceae) trees and shrubs, generally called kanuka, grow across most of Aotearoa/New Zealand (NZ). With the exception of K. sinclairii, an offshore island endemic, kanuka had been treated as an Australasian species K. ericoides. However, a 2014 taxonomic revision recognized ten species, all endemic to NZ. Kanuka chemistry is less studied than that of its closest relative in NZ, manuka (Leptospermum scoparium), which shows very distinct regional foliage chemotypes. We have used a miniaturized method with GC and 1H NMR to analyze foliage chemistry of voucher specimens from across the geographic ranges of the ten NZ Kunzea species. We found common mono- and sesquiterpenes, with α-pinene dominant in all samples, but only traces of antimicrobial triketones. Two unusual flavanones, with unsubstituted B-rings and known bioactivity against Phytophthora, did distinguish some of the samples. 5,7-Dihydroxy-6,8-dimethyl flavanone was only found at high concentrations in the three K. sinclairii samples in this study's sample set, but this compound has separately been reported in K. robusta samples from a nearby region. Therefore none of the NZ Kunzea species was distinguished by the chemistry analyzed in this study, but there is a possibility of regional flavonoid chemotypes cutting across the species boundaries.

Synthesis and anti-inflammatory structure-activity relationships of thiazine-quinoline-quinones: inhibitors of the neutrophil respiratory burst in a model of acute gouty arthritis.

Sixteen new thiazine-quinoline-quinones have been synthesised, plus one bicyclic analogue. These compounds inhibited neutrophil superoxide production in vitro with IC(50)s as low 60 nM. Compounds with high in vitro anti-inflammatory activity were also tested in a mouse model of acute inflammation. The most active compounds inhibited both neutrophil infiltration and superoxide production at doses 2.5 micromol/kg, highlighting their potential for development as novel NSAIDs.

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.

Quantitative Raman spectroscopy for the analysis of carrot bioactives.

Rapid quantitative near-infrared Fourier transform Raman analyses of the key phytonutrients in carrots, polyacetylenes and carotenoids, are reported here for the first time. Solvent extracts of 31 carrot lines were analyzed for these phytonutrients by conventional methods, polyacetylenes by GC-FID and carotenoids by visible spectrophotometry. Carotenoid concentrations were 0-5586 µg g(-1) dry weight (DW). Polyacetylene concentrations were 74-4846 µg g(-1) DW, highest in wild carrots. The polyacetylenes were falcarinol, 6-1237 µg g(-1) DW; falcarindiol, 42-3475 µg g(-1) DW; and falcarindiol 3-acetate, 27-649 µg g(-1) DW. Strong Raman bands for carotenoids gave good correlation to results by visible spectrophotometry. A chemometric model capable of quantitating carotenoids from Raman data was developed. A classification model for rapidly distinguishing carrots with high and low polyacetylene (limit of detection = 1400 µg g(-1)) concentrations based on Raman spectral intensity in the region of 2250 cm(-1) was produced.

Seasonal variation of biomass and bioactive alkaloid content of goldenseal, Hydrastis canadensis.

Seasonal variations in biomass and alkaloid contents of goldenseal (Hydrastis canadensis) were investigated. Five-year-old plants gave 5x the yield of roots and rhizomes of two-year-old plants, and summer growth gave significant increases in root biomass but not rhizomes. Berberine contents of roots plus rhizomes did not vary significantly and were >3.4% in all samples. Hydrastine contents of 5 y roots plus rhizomes showed significant seasonal variation. These variations were due to significant changes in the hydrastine contents of the roots (1.3-1.9%), but not the rhizomes (2.2-2.8%). Goldenseal leaves plus stems had lower contents of hydrastine (0.4-0.8%) and berberine (1.0-1.5%).

Chemotaxonomy of Pseudowintera: sesquiterpene dialdehyde variants are species markers.

Two sesquiterpene dialdehydes, the 1beta-E-coumaroyl-5alpha-hydroxypolygodial plus the known 1beta-E-cinnamoyl-5alpha-hydroxypolygodial, were isolated from the recently described species Pseudowintera insperata. This discovery is a further example of the rare sesquiterpene dialdehyde coumarate/cinnamate combination being found exclusively in the family Winteraceae. Another sesquiterpene dialdehyde, isopaxidal, with the rare rearranged drimane skeleton, was isolated from Pseudowintera axillaris. The sesquiterpene dialdehyde contents of leaves of 25 individual plants of the four Pseudowintera species, all endemic to New Zealand, were measured by HPLC. P. insperata individuals all had high levels (3.0-6.9% of leaf dry wt.) of the coumarate, P. axillaris had high levels (2.2-6.9%) of paxidal, and Pseudowintera colorata from different areas of New Zealand contained varying levels of polygodial (1.4-2.9%) and 9-deoxymuzigadial (0-2.9%). Therefore the sesquiterpene dialdehydes are good species markers.

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.

An antiproliferative bis-prenylated quinone from the New Zealand brown alga Perithalia capillaris.

Bioactivity-directed isolation work on the endemic New Zealand brown alga Perithalia capillaris, seeking anti-inflammatory compounds, led to a new bis-prenylated quinone ( 4). This compound inhibited superoxide production by human neutrophils in vitro (IC 50 2.1 microM), but was more potent at inhibiting proliferation of HL60 cells (IC 50 0.34 microM). Two related bis-prenylated phenols were also isolated, one known ( 2) and one new ( 5), with weaker biological activities. This report extends the examples of bis-prenylated phenols as chemotaxonomic markers for brown algae of the order Sporochnales.