FLAVONOID DIVERSIFICATION IN THE ACHILLEA AGERATIFOLIA AND A. CLAVENNAE GROUPS (ASTERACEAE)

The Achillea ageratifolia and A. clavennae groups (Asteraceae, tribe Anthemideae) were investigated for intra- and interspecific leaf flavonoid variation. Numerous flavonoid glycosides, mostly unidentified, and variously methylated free aglycones based on scutellarein, 6-hydroxyluteolin, quercetagetin, and 6-hydroxy-kaempferol were detected. The distribution of methylated aglycones among individuals of A. ageratifolia was used to model the putative biosynthetic pathways. Several steps in the biosynthesis probably involve enzymes (methyltransferases) with strict positional specificities and broader substrate specificities. Geographical patterns in the flavonoid content of A. ageratifolia were only partly consistent with the current concept of A. ageratifolia. Different flavonoid profiles in A. ageratifolia do not appear to be of crucial importance in its adaptation to different habitats. The species of the A. clavennae group were clearly separable on flavonoid profiles and the presence of a hybrid population was supported by the flavonoid data. Interspecific differences in number of glycosides in relation to the number of free aglycones are discussed.     

Localization and antioxidant capacity of flavonoids from intertidal and subtidal Halophila johnsonii and Halophila decipiens

In this study, flavonoid localization, content and total antioxidant capacity in leaves of subtidal Halophila decipiens were compared to intertidal and subtidal Halophila johnsonii. H. johnsonii leaves had significantly higher flavonoid content (3.5 and 3.8 nmol quercetin equivalent mm⁻² leaf for intertidal and subtidal H. johnsonii, respectively) and antioxidant capacity (101.7 and 224.2 nmol Trolox equivalent mm⁻² leaf for intertidal and subtidal H. johnsonii, respectively) than H. decipiens leaves (1.4 nmol quercetin equivalent mm⁻² leaf and 21.0 nmol Trolox equivalent mm⁻² leaf). Flavonoid content did not significantly differ between intertidal and subtidal H. johnsonii, however, antioxidant capacity was significantly higher in subtidal plants. Confocal laser scanning microscopy of fresh leaf cross sections indicated that both species contained flavonoids in the cuticle, but only H. johnsonii contained intracellular flavonoids. Intracellular flavonoids are better situated to perform antioxidant functions in planta. These results suggest that flavonoid compounds in H. johnsonii are capable of sunscreen and antioxidant functions while an antioxidant role for flavonoids within H. decipiens is not supported.     

Metabolic modifications of birch leaf phenolics by an herbivorous insect: detoxification of flavonoid aglycones via glycosylation

The metabolic modifications of birch (Betula pubescens Ehrh.) leaf phenolics in the digestive tract of its major defoliator, larvae of the autumnal moth Epirrita autumnata, were studied. The main phenolic acids of birch, i.e. chlorogenic and p-coumaroylquinic acids, were isomerised in the alkaline digestive tract. Moreover, only 16 to 92% of the ingested amounts of chlorogenic acid were found in the faeces of individual larvae; the missing portion is possibly being used in the formation of reactive o-quinones. Water-soluble flavonoid glycosides were mostly excreted unaltered. In contrast, lipophilic flavonoid aglycones were not excreted as such, but as glycosides after being detoxified by E. autumnata via glycosylation. When the larvae were fed with leaf-painted acacetin and kaempferide, i.e. two naturally occurring birch leaf flavonoid aglycones, acacetin-7-O-glucoside and kaempferide-3-O-glucoside appeared in larval faeces as major metabolites. However, the efficiency of aglycone glycosylation varied-, ranging from 17 to 33%, depending on the aglycone and its dietary level. There was also large variation in the efficiency of glycosylation--from 2 to 57%--among individual larvae. These results demonstrate a compound-specific metabolism of phenolic compounds, leading to different phenolic profiles in the insect gut compared to its leaf diet.     

FLAVONOIDS OF THE MENYANTHACEAE: INTRA- AND INTERFAMILIAL RELATIONSHIPS

This paper describes our study of the flavonoid chemistry of the Menyanthaceae, which included Menyanthes, Fauria (= Nephrophyllidium), Liparophyllum (all monotypic), nine species of Nymphoides, and 11 species of Villarsia. The flavonoid profiles are based upon kaempferol, 7-O-methylkaempferol, quercetin, 7-O-methylquercetin, isorhamnetin, 3,7-di-O-methylquercetin, and 7,3′-di-O-methylquercetin, although not all taxa exhibit all of these aglycones. These compounds occur as a complex mixture of 3-O-mono- and 3-O-diglycosides; 4′-O-glucosides were restricted to four species of Nymphoides. Some acylated glycosides were also observed. The presence of flavonols and absence of both C-glycosylflavones and xanthones set the Menyanthaceae apart from the Gentianaceae, the family with which it is most often allied. Flavonoid data do not allow assignment of the Menyanthaceae with surety to either the Gentianales or Solanales. The flavonoid profiles of Liparophyllum and Fauria are unique within the family and do not support a close affinity of the latter genus to Menyanthes. A close relationship between Nymphoides and Villarsia, suggested on morphological grounds, is supported by the flavonoid data. Flavonoids support the view, based upon morphology, that the South African Villarsia capensis is more closely related to eastern Australian Villarsia species than to Western Australian ones that are geographically closer. Segregation of Nymphoides fallax from other Meso-American and Caribbean taxa is supported by the flavonoid data. Flavonoid data also support the view that N. indica is a circumtropical taxon not deserving separate species status in the New World.     

A morphological and chemical analysis of geographical variation in Tilia L. of eastern North America

A statistical analysis of morphological variation and a Chromatographic analysis of flavonoid variation were performed to determine taxonomic relationships among the species ofTilia of eastern North America. No apparent morphological discontinuities were seen between populations within the sample area although two characters (involving leaf pubescence and gland length) showed definite patterns of geographical variation. Flavonoid patterns showed definite differences between northern and southern populations with an intermediate zone in the Smoky Mountain region. The continuous nature of the morphological and flavonoid variation suggested that the genus as represented in eastern North America should be regarded as one species,Tilia americana L.     

GC-MS of perdeuteriomethylated flavonoid aglycones

GC-MS of perdeuteriomethylated flavonoid aglycones, singly and in mixtures, yields information about both the aglycone types and their substitution patterns. Fragmentation patterns of flavonoid aglycones are discussed. Acid hydrolysis of perdeuteriomethylated flavonoid glycosides, singly and in mixtures, followed by ethylation with diazoethane provides derivatives suitable for GC-MS; the introduced ethyl groups permit identification of the position of attachment of sugars in flavonoid O-glycosides.     

Flavonoid as chemotaxonomic markers in endemic/endangered species of Rauvolfia from Southern Western Ghats of India: A preliminary study

Preliminary analysis of flavonoid chromatographic migration profiles of endemic/endangered species of Rauvolfia L from Southern Western Ghats of India were carried out. Paper chromatogram showed maximum separation in the solvent system of forestral. In the paper chromatogram, number of flavonoid spots varied from 9 to 12 in the five taxa studied. The main aglycones detected in high performance liquid chromatography (HPLC) analysis were flavones apigenin and luteolin, flavonol kaempferol, myricetin, quercetin and anthocyanidins such as delphinidin and cyanidin. Flavonol Quercetin was detected in all the five species of Rauvolfia giving a chemotaxonomic significance to its presence at the generic level. The two species Rauvolfia serpentina and Rauvolfia tetraphylla could be regarded as the most primitive in the evolutionary line with respect to the flavonoid pattern. Rauvolfia densiflora has the most advanced pattern of flavonoids. The dendrogram generated by unweighted pair group method with arithmetic average (UPGMA) cluster analysis of chemo metric data showed a clear grouping of five species in three clusters. Flavonoid profiles were efficiently used for the identification of Rauvolfia beddomei, which due to morphological similarity, was erroneously suspected to be the medicinally significant species Rauvolfia micrantha. Flavonoid profiling using paper chromatography, in the solvent system of forestral could suggest an easy and quick procedure for identifying adulteration by substitution in Rauvolfia species.     

Flavonoids and the taxonomy of Camarea (Malpighiaceae)

Camarea is a South-American endemic genus comprising eight species. In the present work leaf flavonoids of seven species of Camarea were identified, aiming to evaluate the usefulness of their distribution as a taxonomic aid. A total of 12 flavonoids were isolated and identified. Free aglycones, such as apigenin, chrysoeriol, kaempferol and quercetin, as well as 7-O-glycosides of apigenin and luteolin, 3-O-glycosides of kaempferol and quercetin were identified. Flavonoid distribution in Camarea species, taking into account aglycones and aglycone moieties of glycosides, was used to obtain a phenogram of chemical affinities. Apigenin, chrysoeriol and kaempferol were the main discriminating characters for links establishment. The resultant tree suggests the links: 1) Camarea hirsuta, Camarea affinis and C. affinis × C. hirsuta; 2) Camarea elongata and Camarea axillaris; 3) Camarea sericea and Camarea humifusa. The results are in agreement with morphological similarities and disagree with several points of n-alkane evidence. The results support the recognition of Camarea triphylla as synonymy of C. axillaris.     

Foliar flavonoids of North American Solanum section Solanum

A total of ten flavonoids, all flavonols, were isolated from leaves of eleven species of Solanum sect. Solanum. Most species had relatively few compounds, primarily quercetin 3-O-glycosides. Little intraspecific variation in flavonoids was observed, except in S. americanum were it correlated with previously recognized races. Flavonoid data are of little help in determining ancestries of polyploid species, but do rule out S. sarrachoides as a progenitor of S. villosum and S. nigrum. Solanum sarrachoides is unique among species examined in having free flavonoid aglycones as well as extensive 3-O-methoxylation.     

Exudate flavonoid aglycones of Veronica: Ecological and systematic implications

A survey on the distribution of surface flavonoid aglycones in 52 samples of 29 species of Veronica resulted in detection of eight flavone aglycones. Apigenin and luteolin were the most common constituents of the studied exudates, followed by the methylated derivatives apigenin 4′-methyl ether, apigenin 7,4′-dimethyl ether and luteolin 3′-methyl ether. Some intraspecific variations and the main ecological trends of flavonoid aglycone accumulation in Veronica were discussed. The observed exudate profiles appeared to be characteristic for some related groups within the genus, in agreement with the morphological, karyological, molecular and other chemical data.     

Leaf surface traits: overlooked determinants of birch resistance to herbivores and foliar micro-fungi?

Previous studies on the mechanisms of birch resistance to herbivores and foliar micro-fungi (both pathogenic and endophytic) have focused mainly on the role of internal leaf chemistry. In the present study, we examined genetic correlations between leaf surface traits (glandular trichome density and total concentrations of surface flavonoid aglycones) and occurrence of three species of foliar micro-fungi, one pathogenic rust (Melampsoridium betulinum) and two endophytic fungi (Fusicladium sp. and Melanconium sp.), and performance of autumnal moth larvae (Epirrita autumnata) in two birch species, Betula pubescens ssp. czerepanovii and B. pendula. The performance of autumnal moth larvae on B. pubescens ssp. czerepanovii was negatively correlated with density of glandular trichomes (RGR: r=–0.855; pupal mass: r=–0.709). In addition, rust infection was negatively correlated with trichome density in B. pendula (r=–0.675) and with epicuticular flavonoid aglycones in B. pubescens ssp. czerepanovii (r=–0.855). The frequency of the endophytic fungus Fusicladium sp., was related to epicuticular flavonoid aglycones (r=–0.782), while another endophytic fungus, Melanconium sp., showed no associations with any of the studied variables in B. pubescens ssp. czerepanovii. Our results indicate that leaf surface traits may be at least as important determinants of herbivore performance and micro-fungi abundance in birch as leaf internal chemistry.     

Exkret-Flavonoide bei Höheren Pflanzen arider Gebiete

Flavonoids generally occur in higher plants as water-soluble glycosides. However, lipophilic flavonoid aglycones can be excreted by glandular trichomes or extruded through the cuticle. They are accumulated on the plant surface as constituents of leaf resins or thin epicuticular layers. External flavonoid aglycones are found in various families throughout the higher plants, but appear to be most abundant in theAsteraceae. They occur relatively frequently in plants of (semi-)arid habitats. It is advantageous for chemotaxonomic studies that the structural diversity of exudate flavonoids is normally greater than that of the tissue glycosides in these plants.

Vorgetragen auf der Tagung der Deutschen Botanischen Gesellschaft, Wien, September 1984.     

Flavonoid aglycones from leaf resins of two species of Heterotheca (compositae)

Twenty-nine flavonoid aglycones have been identified from two populations each of Heterotheca grandiflora and H. psammophila. Considerable qualitative variation was found between populations of the same species. Overall, H. grandiflora is more complex in its flavonoid profile, accumulating a total of 24 compounds based on eight skeletal types, compared with 13 compounds based on four skeletal types in H. psammophila.     

Leaf expansion as related to plant water availability in a wild and a cultivated sunflower

This study aimed to determine if two species of sunflower, Helianthus annus L. cv. Hysun 31 (cultivated, single-stemmed genotype) and Helianthus petiolaris Nuttall ssp. fallax (wild, many-hranched genotype) differed in the response of leaf growth to water deficits. Earlier published studies, concerned only with H. annuus, failed to reveal differences in the response of sunflowers to water stress. Plants of the two species were paired in large containers of soil and grown under high radiation in a glasshouse. One batch of plants was irrigated and the other allowed to dry so that predawn leaf water potentials declined at an average of 0.072 MPa day^?1. The dry batch was rewatered when predawn leaf water potentials reached ?0.85 MPa. The stress imposed was sufficient to curtail leaf growth so that plants in the dry treatment had only 60% of the leaf area of irrigated plants at the onset of rewatering. Both species were affected by stress to the same relative extent, though their leaf areas at this stage differed 7-fold. Both genotypes also recovered to the same degree in the long term, finally having leaf areas and gross dry matter distribution patterns which were indistinguishable from plants which were irrigated throughout. However, water stress resulted in different distribution patterns of leaf area: H. annuus produced larger leaves at the top of its single stem which compensated for the reduced area in lower leaves, whereas H. petiolaris compensated in the leaves on its branches. Leaves which emerged after the time of stress were most able to compensate in area subsequently. For example, those leaves of H. annuus which emerged one week after stress-relief were more than three times larger than comparable leaves on plants irrigated continuously. Leaf expansion rates were affected earlier in the stress cycle than leaf conductance in H. annuus, but not in H. petiolaris. But as with other plant responses to water stress, the differences between the two species were small.     

FLAVONOID RACES OF CLAYTONIA VIRGINICA (PORTULACACEAE)

Four flavonoid races have been found in the Claytonia virginica aneuploid complex, differing from one another in the accumulation of 12 kaempferol and quercetin 3-glycosides. Each of these races has a distinctive geographical distribution within the overall range of the species. The three major diploid cytotypes of C. virginica each belong to different races, two of which also include polyploids; a fourth race consists entirely of polyploids. An examination of biosynthetic pathways indicates that a small number of genetic changes are responsible for the observed variation; polyploidy per se does not appear to have contributed significantly to the production of novel compounds. Flavonoid data also suggest strongly that polyploidy within rather than between modern chemical races has been responsible for the bulk of chromosome number variation in the species.     

Foliar phenolic composition of European white birch during bud unfolding and leaf development

We studied the between-tree and within-tree variation in the composition and content of foliar low-molecular-weight phenolics (LMWP) of European white birch ( Betula pendula Roth) during the unfolding of vegetative buds and during early leaf development. In buds, the major groups of phenolic compounds were hydrolysable tannins and flavonoid aglycones, whereas, later during leaf development, the flavonoid glycosides accounted for most of the total LMWP. The content of total LMWP, as well as individual compounds, varied largely among individual trees, while variation within an individual tree was low. The biosynthetic origin of individual compounds or compound groups is discussed in order to explain the main patterns in leaf chemistry during bud unfolding and early leaf development.     

A NUMERICAL ANALYSIS OF FLAVONOID VARIATION IN ARNICA SUBGENUS AUSTROMONTANA (ASTERACEAE)

Species of Arnica subgenus Austromontana produce a total of 23 leaf flavonoids, including simple and methylated flavone and flavonol glycosides as well as highly methylated flavone aglycones and a 6-hydroxylated flavone. Most of the taxa exhibit considerable interpopulational variability, with the number of compounds per population ranging from 2 to 14. Analysis of flavonoid variation in 113 populations representing all 9 species of the subgenus was carried out using cluster analysis, principal components analysis, and binary discriminant analysis. Results indicate the flavonoid profile of the very rare A. viscosa is the most distinctive in the subgenus. Although exhibiting considerable interpopulational variability, all populations of A. gracilis, a hybrid taxon, form a very distinct and cohesive group, supporting its recognition at the specific level. Additionally, chemical diversification from A. cordifolia has taken place largely in the Klamath region of Oregon and California. The range of variability exhibited by A. cordifolia is reflected in these Klamath region derivatives.     

Crystal structure of β‐glucosidase 1A from Thermotoga neapolitana and comparison of active site mutants for hydrolysis of flavonoid glucosides

The β‐glucosidase TnBgl1A catalyses hydrolysis of O‐linked terminal β‐glycosidic bonds at the nonreducing end of glycosides/oligosaccharides. Enzymes with this specificity have potential in lignocellulose conversion (degrading cellobiose to glucose) and conversion of bioactive flavonoids (modification of glycosylation results in modulation of bioavailability). Previous work has shown TnBgl1A to hydrolyse 3, 4′ and 7 glucosylation in flavonoids, and although conversion of 3‐glucosylated substrate to aglycone was low, it was improved by mutagenesis of residue N220. To further explore structure‐function relationships, the crystal structure of the nucleophile mutant TnBgl1A‐E349G was determined at 1.9 Å resolution, and docking studies of flavonoid substrates were made to reveal substrate interacting residues. A series of single amino acid changes were introduced in the aglycone binding region [N220(S/F), N221(S/F), F224(I), F310(L/E), and W322(A)] of the wild type. Activity screening was made on eight glucosylated flavonoids, and kinetic parameters were monitored for the flavonoid quercetin‐3‐glucoside (Q3), as well as for the model substrate para‐nitrophenyl‐β‐d ‐glucopyranoside (pNPGlc). Substitution by Ser at N220 or N221 increased the catalytic efficiency on both pNPGlc and Q3. Residue W322 was proven important for substrate accomodation, as mutagenesis to W322A resulted in a large reduction of hydrolytic activity on 3‐glucosylated flavonoids. Flavonoid glucoside hydrolysis was unaffected by mutations at positions 224 and 310. The mutations did not significantly affect thermal stability, and the variants kept an apparent unfolding temperature of 101°C. This work pinpoints positions in the aglycone region of TnBgl1A of importance for specificity on flavonoid‐3‐glucosides, improving the molecular understanding of activity in GH1 enzymes. Proteins 2017; 85:872–884. © 2016 Wiley Periodicals, Inc.     

The utility of leaf flavonoids as taxonomic markers for some Malaysian species of the tribe Shoreae (Dipterocarpaceae)

A flavonoid survey was carried out on 45 taxa from the genera Shorea, Hopea, Parashorea, Neobalanocarpus, and Dryobalanops of the tribe Shoreae in the Dipterocarpaceae. The study showed significant chemotaxonomic differences in leaf flavonoid aglycone patterns and the presence of tannins in these taxa. The flavonoid patterns are useful in the delimitation of some taxa. For example, the genus Parashorea is distinguished by the universal presence of kaempferol 3‐methyl ether, and the monotypic genus Neobalanocarpus is unique in not producing ellagic and gallo tannins. The presence of chalcones and flavone C‐glycosides supports the separation of the genus Hopea into two sections, section Dryobalanoides and section Hopea in Ashton's classification, which is based on the type of venation. The flavonoid distributions in this study show that they can be very useful for differentiating between the Balau group in the genus Shorea and some scaly barked Hopea species, particularly H. helferi (lintah bukit), H. nutans (giam), and H. ferrea (malut). © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society, 2008, 157 , 755–762.