Kevin A. Mitchell

HPLC and GC-MS identification of the major organic constituents in New Zealand propolis

Abstract The major organic constituents of New Zealand sourced propolis have been identified and quantified in tincture solutions by a combination of HPLC and GC-mass spectrometry (MS). HPLC was the preferred method for the analysis of flavonoids because of their very low response factors in GC-MS. Flavonoid levels ranged from 30–40 mg ml −1 . A distinctive characteristic of the flavonoids in New Zealand propolis is the unusually high proportion ( ca 70%) of dihydroflavonoids, e.g. pinocembrin, pinobanksin and pinobanksin 3-acetate. Non-flavonoid components analysed by GC-MS comprised a range of aromatic compounds (3–7.5 mg ml −1 ), together with low levels (0.25–0.78 mg ml −1 ) of fatty acids. The former comprised mainly cinnamic acids and their esters, but also included the rare 5-phenyl- trans - trans -2,4-pentadienoic acid and the new natural product, 5-phenyl- trans -3-pentenoic acid. Both were synthesized to confirm their identity.

An increase in the luteolin : Apigenin ratio in Marchantia polymorpha on UV-B enhancement

Abstract The effects of varying the UV-B component in ambient light on the liverwort, Marchantia polymorpha are reported. Plants grown under conditions of ambient light, ambient light lacking UV-B, and ambient light with a 25% enhancement of incident UV-B showed, with increasing levels of UV-B, a decrease in growth rate, a decrease in the production of gemmae cups and an increase in the proportion of dead thallus. Thallus surviving after three months of summer growth under these conditions showed no statistically significant increase in flavonoid levels with increasing UV-B levels. However, HPLC monitoring of individual flavonoids (all of which are apigenin and luteolin glucuronides) revealed a strong correlation between increasing UV-B levels and an increase in the ratio of luteolin to apigenin glycosides. It is considered unlikely that this change has significantly altered the UV-B screening effectiveness of the flavonoids. Rather, an improved level of antioxidant defence, or a more effective dissipation of absorbed UV energy, are proposed as possible UV-B protectant benefits to the plant.

UVB RADIATION INDUCED INCREASE IN QUERCETIN : KAEMPFEROL RATIO IN WILD-TYPE AND TRANSGENIC LINES OF PETUNIA

The use of genetically modified plants offers unique opportunities to study the role of specific flavonoids in plant UVB protection. Along with a parental wild‐type Mitchell Petunia, two transgenic lines with altered flavonoids were also examined; Lc with enhanced levels of antho‐cyanins due to the action of a maize flavonoid regulatory gene Leaf color, and AFLS that carries an antisense fla‐vonol synthase construct and is known to have reduced flavonol levels in flowers. All three lines were grown in near ambient sunlight, sunlight lacking UVB (280–320 nm) radiation and sunlight with 25% added UVB. Ultra‐violet‐B radiation induced significant reductions in the rates of leaf expansion and seedling growth in all three lines. The presence of anthocyanins did not appear to afford Lc plants any special protection from UVB. Ul‐traviolet‐B treatment induced increases in total flavonol content in young plants of all three lines, and this effect decreased with increasing leaf age. Notably, increasing UVB levels led to an increase in the ratio of quercetin: kaempferol with all three cultivars. The AFLS transgenic, contrary to expectations based on its genetic construction, had normal levels of flavonols in the leaves and the highest Q:K ratio of the three cultivars. This transgenic was the least susceptible to UVB, which may indicate an enhanced protective role for quercetin. Because both quercetin and kaempferol have similar UVB screening properties, quercetin may exert this role by other means.

An approach to the characterization of bee pollens via their flavonoid/phenolic profiles

Bee pollen is a mix of bee-collected floral pollens which varies widely in composition. A systematic method for characterising bee pollens in terms of their constituent pollens is needed in view of the growing phytotherapeutic interest in bee pollen products. Studies involving three bee pollen samples collected from Portugal and New Zealand are reported. An approach based on flavonoid/phenolics profiles derived from high pressure liquid chromatography is demonstrated to be more precise and informative than traditional microscopy. This method provides a convenient means for identifying the contributing pollens, and for characterising bee pollens in terms of their predominant constituent pollens. The flavonoid/phenolics profiles obtained in the course of this work also highlight other observations of interest. For example: bees are shown to be highly selective pollen gatherers from the finding that bee pollens comprise pollen from only a few of the available species; pollen from only one floral source is found in each bee pollen pellet; and flavonoids are normally found as glycosides in pollens but are shown to occur naturally as aglycones in Eucalyptus globulus pollen. Two of these aglycones, tricetin and 3-O-methylquercetin, are reported as pollen constituents for the first time.

Supercritical antisolvent fractionation of propolis tincture

Abstract Propolis is used by bees for strengthening and waterproofing a hive, and for sterilizing the hive against microbial infections. Propolis contains a high concentration of flavonoids, which are used in a wide range of cosmetic and health food preparations for their antimicrobial properties. Propolis is usually dissolved in ethanol or ethanol/water mixtures to remove insoluble material such as waxes and detritus from the hive. The resultant solution is a propolis tincture. A new supercritical antisolvent/extraction process has been developed for the fractionation of propolis tincture to obtain flavonoids and essential oil fractions by extraction, and remove high molecular mass components by antisolvent precipitation. Flavonoids are practically insoluble in pure CO2, but sufficiently soluble in CO2+ethanol to enable their separation from high molecular mass and/or more polar components. In the first step of the process, supercritical CO2 is used both as an anti-solvent to precipitate high molecular mass components, and as a solvent to extract the ethanol and soluble components of the propolis. This extract is then fractionated in two separation steps to create a concentrated flavonoid fraction as the primary product, and an essential oil/ethanol fraction as a secondary product. The effects of pressure, temperature, flow rate ratio, tincture composition and tincture concentration on product quality and yield were determined at a laboratory and pilot scale. The tincture concentration of propolis has the greatest effect on the yield and concentration of flavonoids in the product fraction when pure ethanol is used as the solvent. The flow rate ratio becomes important when the tincture also contains water. The process has been successfully scaled up to a demonstration scale using optimized pressure, temperature, flow ratio and tincture concentrations obtained from laboratory and pilot scale trials.

Antioxidant capacities of green and cyanic leaves in the sun species, Quintinia serrata

Anthocyanins scavenge harmful reactive oxygen species (ROS), and may provide antioxidative protection within plant cells. For the shade species Elatostema rugosum A. Cunn., it was recently established that red-leaved morphs held a significant antioxidant advantage over green morphs. It is not known whether anthocyanins confer the same protection in sun species that are exposed to elevated levels of irradiation and thus greater photooxidative stress. Quintinia serrata A. Cunn., a canopy tree species, is polymorphic for anthocyanin expression. Levels of antioxidants in leaves of red and green morphs of Q. serrata were identified, and activities quantified using enzymatic and α,α-diphenyl-β-picrylhydrazyl (DPPH) assays and cyclic voltammetry. Both morphs contained similar levels of superoxide dismutase, catalase, ascorbic peroxidase and hydroxycinnamic acids, and showed similar ranges in DPPH scavenging activity and voltammetric profiles. Anthocyanin concentrations increased with leaf age in red morphs and were positively correlated with the extent of DPPH scavenging. However, hydroxycinnamic acids were the most active methanol-extractable fraction in both morphs, contributing 27% more to the overall low-molecular-weight antioxidant pool in the green morph than in the red. We conclude that anthocyanin biosynthesis can enhance, but is not a prerequisite for, oxidative protection in Q. serrata leaves. The possibility that anthocyanins provide a versatile mechanism for photoprotection is discussed.

Pigment chemistry and colour of Pelargonium flowers

The major factors responsible for colour variation in a range of Pelargonium species and cultivars were shown to be the types and relative levels of pigments present. Variations in pH and copigment levels were not found to contribute significantly. Flowers with colours ranging from cream and pink through to deep purple, including salmon, orange and red, were studied. While either flavonols or carotenoids were responsible for cream/yellow colouration, all other colours resulted from anthocyanin mixes. The pigments were isolated, quantified and identified by combinations of column and HPL chromatography, absorption spectroscopy and NMR. The major anthocyanins were identified as the 3,5-diglucosides and 3-glucoside-5-(6-acetyl)glucosides of pelargonidin, cyanidin, peonidin, delphinidin, petunidin and malvidin. The acetates, four of which have not been reported previously, were found predominantly in the regal Pelargonium cultivars.

The mis-identification of the major antioxidant flavonoids in young barley (Hordeum vulgare) leaves.

Several papers have appeared in the literature since 1992 which refer to a major “isoflavonoid” antioxidant in young green barley leaves (Hordeum vulgare) as 2″-O-glucosylisovitexin. In the present paper the original NMR data supporting this structural assignment are examined and found to have been misinterpreted. HPLC and NMR data are used to prove that the major flavonoid antioxidants in young green barley leaves are in fact the flavone-C-glycosides, saponarin and lutonarin

Luteolin and 6-hydroxyluteolin glycosides from Hebe stricta

Abstract Two flavone glycosides, 6-hydroxyluteolin 7-sambubioside and luteolin 4′-glucoside have been isolated from Hebe stricta var. atkinsonii . This is the first report of the former glycoside and the presence of xylose in the saccharide moiety may be significant in the chemotaxonomic differentiation between Hebe and the closely related genus of Veronica .

A taxonomic revision of the Hebe parviflora complex (Scrophulariaceae), based on morphology and flavonoid chemistry

Abstract Two entities have long been recognised at the rank of either species or variety in the New Zealand endemic Hebe parviflora complex but, because one of the critical type specimens had not previously been examined by New Zealand botsnists, there has been uncertainty regarding the correct application Of names. The two entities Can be distinguished on characters of habit, leaves, flowers, leaf flavonoids, and chromosome number, and are accepted here at species rank. H. parvlflora is the correct name for the tetraploid entity previously known as H. parvrflora var. arborea, and H. stenophylla is the diploid entity previously known as H. parviflora var. angustifolia (the autonym var. parviflora has not been used in any recent treatments). H. stenophylla has several distinct geographic races and new names are provided at varietal rank for two of these: var. hesperia occurs in limestone areas near the north‐west coast of the South Island, between Cape Farewell and the Heaphy River; var. oliveri occurs on exposed bluffs on Stephens Island in Cook Strait. Circumscription of these varieties leaves var. stenophylla comprising a range of morphological forms whose relationships require further elucidation. A comparative study of leaf flavonoids identifies flavonoid characters that clearly distinguish H. parviflora and H. stenophylla, and the profiles of these species are compared with those of the related species H. strictissima and H. traversii. Historical factors affecting the distribution of H. parviflora and H. stenophylla are discussed, and descriptions, distribution maps, and illustrations of diagnostic morphological characters are provided for all taxa.