Sequestration of host-plant-derived flavonoids by lycaenid butterflyPolyommatus icarus

Larvae of the lycaenid butterflyPolyommatus icarus were reared on inflorescences ofCoronilla varia andMedicago sativa, which are rich in flavonoids. Twelve different flavonoids (five compounds from the former and nine from the latter), including aglycones andO-glycosides of kaempferol, quercetin, and myricetin were isolated and identified by spectroscopic means. NMR and MS data for the new acylated glycoside kaempferol 3-O-6-(3-hydroxy-3-methylglutaroyl)--d-glucopyranoside are reported. Comparative HPLC analysis of the respective host plants and of larvae, pupae, and imagines ofP. icarus indicated selective uptake and accumulation of kaempferol vs. quercetin and myricetin derivatives. The latter were excreted largely unchanged through the feces. Irrespective of the larval host plant kaempferol 3-O-glucoside was found as the major flavonoid in larvae, pupae, and imagines ofP. icarus, accounting for approximately 83–92% of all soluble flavonoids in adult butterflies. Within the imagines, approximately 80% of all flavonoids are stored in the wings (especially in the orange submarginal lunules), whereas the remaining 20% reside in the bodies. Feeding experiments with artificial diet demonstrated that the insects are able to form kaempferol 3-O-glucoside by glucosylation of dietary kaempferol. Possible functions of the sequestered flavonoids, especially for mate recognition ofP. icarus, are discussed.     

Effect of Flavonoids on Feeding Preference and Development of the Crucifer Pest Mamestra configurata Walker

Thirty-seven flavonoid compounds (9 flavones, 18 flavonols, 8 flavanones, and 2 flavanonols) were investigated for their effect on feeding choice with bertha armyworm (Mamestra configurata Walker; BAW). Feeding choice was dependent upon subtle differences in biochemical structure. Unsubstituted flavone and flavanone were the strongest feeding deterrents in the choice bioassay, while 7.4'-dihydroxyflavone and dihydroquercetin stimulated BAW to feed. The constitutive flavonoids of Brassica napus, isorhamnetin-3-sophoroside-7-glucoside and kaempferol-3,7-diglucoside, were effective deterrents when supplemented at concentrations higher than endogenous levels. In a no-choice bioassay, flavone reduced both larval weight as well as larval and pupal development time.     

Evaluation of flavonoids inGossypium arboreum (L.) cottons as potential source of resistance to tobacco budworm

Asiatic cottons [Gossypium arboreum (L.)] have been investigated as a source of resistance to the tobacco budworm [Heliothis virescens (Fab.)] because their diversely colored petals have been presumed to contain various allelochemicals. However, we found that larvae fedG. arboreum squares (buds) grew about equally compared with those fed squares from commercialG. hirsutum lines. The best source of resistance was found in severalG. hirsutum double-haploid (DH) lines. In our investigation of allelochemicals, G.arboreum lines were found to contain much less gossypol in leaves, squares (buds), and petals thanG. hirsutum L. lines. Flavonoids were significantly higher inG. arboreum lines only in petals. Of 22G. arboreum lines from which squares were gathered and fed to tobacco budworm (TBW) larvae in the laboratory, larval growth was not significantly decreased on any, but larval survival was decreased on six. When the square flavonoids were isolated and incorporated in laboratory diets for the TBW, moderate toxicity was observed. However, the estimated toxicities were not greater than those of the same flavonoid isolates fromG. hirsutum lines. The most prevalent flavonoids, all previously found in G.arboreum plant tissues, were gossypetin 8-0-glucoside and gossypetin 8-0-rhamnoside, neither of which were present inG. hirsutum tissue. Quercetin 3-0-glucoside, quercetin-3-0-glucoside, and quercetin 7-0-glucoside were also present in significant amounts in both species. Gossypetin 8-0-rhamnoside and gossypetin 8-0-glucoside were the most toxic flavonoids tested (the ED₅₀% was estimated to be 0.007 and 0.024) and therefore may prove to be contributing factors of resistance to TBW feeding.Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable.     

Flavonoid Metabolites in the Hemolymph of European Pine Sawfly (Neodiprion sertifer) Larvae

Flavonoids in the hemolymph of European pine sawfly (Neodiprion sertifer) larvae that were feeding on Pinus sylvestris needles were identified. HPLC-ESI-MS analysis revealed that the main components in the hemolymph were flavonol di- and triglucosides and a catechin monoglucoside. These compounds were isolated from the larval hemolymph and their structures were established by HPLC-MS, GC-MS, and NMR spectroscopy. The isolated flavonoids were identified as (+)-catechin 7-O-β-glucoside, isorhamnetin 3,7,4'-tri-O-β-glucoside, kaempferol 3,7,4'-tri-O-β-glucoside, and quercetin 3,7,4'-tri-O-β-glucoside. The combined concentration of these four compounds in the hemolymph was 3.7 mg/ml. None of these compounds was present in the needles of P. sylvestris. Therefore, we propose that the flavonoid glucosides were produced by the larvae from flavonoid monoglucosides and (+)-catechin obtained from the pine needles.     

Flavonoid pigments in marbled white butterfly (Melanargia galathea) are dependent on flavonoid content of larval diet

Analyses of two-dimensional chromatographic flavonoid patterns of butterflies reared on different grass species have shown that the flavonoid pattern ofMelanargia galathea is dependent on the flavonoid content of the larval diet. This confirms the dietary origin of flavonoid pigments inM. galathea. The flavonoid patterns of butterflies reared on different grass species differ from each other and from the larval food plants;M. galathea reared on the same grass species have identical flavonoid patterns. Differences in the butterfly and larval food plant flavonoid patterns indicate that the ingested flavonoids are metabolized byM. galathea or its gut flora before sequestration. The distinct flavonoid patterns of butterflies reared on different larval food plants have been defined as the flavonoid fingerprint profiles for each grass species. Similarity between theFestuca rubra flavonoid fingerprint profile and the constant flavonoid pattern characteristic of wild-capturedMelanargia suggests thatMelanargia larvae are not generalist grass feeders, but are specific toF. rubra or toF. rubra and a few closely related grass species in the wild.     

Flavonoids released by carrot (Daucus carota) seedlings stimulate hyphal development of vesicular-arbuscular mycorrhizal fungi in the presence of optimal CO2 enrichment

Carbon dioxide has been previously identified as a critical volatile factor that stimulates hyphal growth ofGigaspora margarita, a vesiculararbuscular mycorrhizal fungus, and we determined the optimal concentration at 2.0%. The beneficial effect of CO₂ on fungal development is also visible in the presence of stimulatory (quercetin, myricetin) or inhibitory (naringenin) flavonoids. Sterile root exudates from carrot seedlings stimulate the hyphal development ofG. margarita in the presence of optimal CO₂ enrichment. Three flavonols (quercetin, kaempferol, rutin or quercetin 3-rutinoside) and two flavones (apigenin, luteolin) were identified in carrot root exudates by means of HPLC retention time. Flavonols like quercetin and kaempferol are known to have stimulatory effects on hyphal growth ofG. margarita.     

The Influence of Tissue Handling on the Flavonoid Content of the Aquatic Plant Posidonia oceanica

In recent times, more and more studies have focused on flavonoids as biomarkers of environmental quality in aquatic plants, in particular, Posidonia oceanica (Linnaeus) Delile. It is therefore of interest to determine how different prehandling methods can affect flavonoid concentrations. The methods tested were (1) immediate extraction of fresh samples, (2) extraction after 48 hr chilling, (3) freeze-drying, and (4) oven drying. Chilling and freeze-drying considerably altered the quantity of flavonoids measured, but not their profile. The effect of oven drying was not significant. Chilling led to a loss of 57% of total (pro)anthocyanidins, 39% of total flavonols, and 48% of all simple flavonols (myricetin, quercetin, isorhamnetin, and kaempferol). Freeze-drying caused a loss of 71% of total (pro)anthocyanidins, 87% of total flavonols, and 95% of all simple flavonols.     

HPLC法测定牙膏中银杏叶提取物总黄酮苷的含量

以槲皮素、山奈素和异鼠李素为指标,建立HPLC法测定牙膏中银杏叶提取物总黄酮苷的含量。采用Agilent ZORBAX Eclipse XDB-C18色谱柱（Analytical 4．6×250mm 5-Micron）;流动相为甲醇-0．4％磷酸（55／45,V/V）;流速为1．0mL／min;检测波长为360nm;柱温为40℃;进样量为20μL。结果表明,槲皮素、山奈素和异鼠李素浓度分别在2．1～21．0、1．8～18．0和0．42～4．2μg／mL范围内呈良好的线性关系,相关系数分别为：0．9999、0．9999和0．9998;槲皮素、山奈素和异鼠李素回收率分别为：97．7％、98．3％和97．6％。该方法简单、灵敏、准确,可用于牙膏中银杏叶提取物总黄酮苷含量的测定。     

Nutrient Composition of Larval Nectar Secretions from Three Species of Myrmecophilous Butterflies

A comparative chemical analysis of the larval nectar secretions and hemolymph from three unspecifically and facultatively ant-attended lycaenid species (Polyommatus coridon, P. icarus, and Zizeeria knysna) was performed by using high-performance liquid chromatography techniques. Sucrose was the main sugar component in all three species. In half of the samples of P. coridon, it was accompanied by glucose, whereas other sugars occurred only rarely. In P. icarus and Z. knysna, melezitose was the second-most important component, followed by fructose and glucose. Total sugar contents were 43.6 ± 14.8 g/l (mean ± SD) for P. coridon, 74.2 g/l for P. icarus, and 68.3 ± 22.6 g/l for Z. knysna. Up to 14 different identified amino acids were found in P. coridon nectar, with a total content of 9.7 ± 3.4 g/l. Leucine was always the major component (contributing 50% of overall amino acid content). Other important amino acids were tyrosine, proline, arginine, and phenylalanine. P. icarus nectar contained up to six amino acids with a total content of 1.2 g/l, dominated by tyrosine and phenylalanine. Z. knysna nectar contained alanine and proline, with only 0.3 ± 0.17 g/l total content. In the hemolymph of all species, up to 16 different amino acids occurred relatively regularly, with histidine dominating, followed by serine and proline. The amino acid pattern in hemolymph was considerably different from that of the nectar secretions. Larval diet weakly influenced P. coridon nectar sugars, and with a semisynthetic diet, a more homogeneous amino acid pattern was detected. Comparison with reports from other lycaenid species shows that secretions rich in amino acids are related to intimate, often obligate ant associations, whereas facultative, unspecific myrmecophiles rely on carbohydrates.     

Variability in Flavonoid Compounds of Four Tribulus Species: Does it Play a Role in Their Identification by Desert Locust Schistocerca gregaria?

We examined how variability in phenolic metabolism of four closely related Tribulus taxa might affect palatability to Schistocerca gregaria. Plants were collected in the Mauritanian breeding areas of the locust. Eighteen flavonoids (caffeoyl derivatives, quercetin glycosides, including rutin, and kaempferol glycosides) were detected with high-performance liquid chromatography in leaf extracts. Principal component analysis (PCA) differentiated relative abundance of flavonoids of Tribulus terrestris from T. longipetalus, T. ochroleucus, and T. bimucronatus. Flavonoid content of T. terrestris leaves varied between northern and southern Mauritanian sites. Dual choice tests with plants whose flavonoid profiles had been determined previously were analyzed by multiple regression. The data suggest that the desert locust prefers T. terrestris plants rich in quercetin glycosides.     

Antioxidant effects of flavonoids ofAnthriscus sylvestris in lard

Ethanol/water (7∶3) extracts of the plant speciesAnthriscus sylvestris possess antioxidant activity. Separation and identification of antioxidant components by thin-layer and column chromatography and spectral analysis demonstrated that quercetin and apigenin appeared to be the main flavonoid species inAnthriscus sylvestris. Rutin was one of the major quercetin glycosides. Structures of the isolated compounds were determined by infrared and¹H nuclear magnetic resonance spectroscopies. Ethanolic extract (70%) ofA. sylvestris showed concentration-dependent, strong antioxidant activity as determined by the Schaal Oven test of lard at 60°C. Rancimat analysis at 100°C showed that the antioxidant activity of 70% ethanolic extract ofA. sylvestris was superior to quercetin, apigenin, or a tocopherol mixture.     

Chemical composition of North American bee propolis and biological activity towards larvae of greater wax moth (Lepidoptera: Pyralidae)

Bee propolis is a sticky amalgamation of plant resins collected by honeybees (Apis mellifera L.) and used in the hive for filling cracks and repairing combs. Propolis contains a diversity of compounds of plant origin, and is reported to have medicinal, antimicrobial, insecticidal, and phytotoxic properties. We examined the physical and chemical composition of North American samples of bee propolis from several sites in North America and tested for bioactivity against larvae of the greater wax moth (Galleria mellonella L.), a common apiary pest. The amount of methanol-extractable resin in samples from Ohio and Georgia ranged from 24% to 79% by weight. Propolis collected from hives in Ohio was more chemically diverse (over 30 compounds detected by paper chromatography) than material from south Georgia (fewer than 10 major compounds) and contained a lower proportion of methanol-insoluble beeswax. The paper chromatographic surveys revealed little variation in the chemical profile of specific hives over a six-month period and no differences between propolis from adjacent hives. Four flavonoids were identified from propolis collected in Ohio: kaempferol, galangin, 3,3-dimethoxyquercetin and 3-methoxykaempferol. When mixed into artificial diet, fractionated propolis reduced larval growth of the greater wax moth, but not dramatically. An array of phenolics reported from propolis (caffeic acid, chrysin, ferulic acid, galangin, kaempferol, and quercetin) were bioassayed individually for effects on larvae, but none reduced larval growth at the concentrations tested, suggesting that wax moths are tolerant of some phenolics in their diet.     

Acquired chemical defense in the lycaenid butterfly,Eumaeus atala

The lycaenid butterfly,Eumaeus atala, was found to contain cycasin, an azoxyglycoside, by thin-layer chromatography (TLC). Quantification of cycasin content in 10 individual freshly killed and frozen males and females, using capillary gas chromatography (GC), showed that cycasin contents of individual butterflies ranged from 0.21 to 0.51 mg (1.24–2.75% dry weight). A museum specimen ofE. atala of unknown age had undetectable amounts of cycasin by GC. GC showed that larval frass contained about 0.10% cycasin, which was not detectable by TLC. Cycasin in the host plant was not detectable by TLC but was detected by GC and found to be 0.02% dry weight. There was no macrozamin, another azoxyglycoside characteristic of many cycads, in the butterfly or plant. Feeding trials with a colony of the ant,Camponotus abdominalis floridanus, showed that both cycasin and the adult ofE. atala were deterrent to the ants.     

LC-electrospray ionization and LC-FABMS study of flavonoid glycosides extracted from peanut meal

HPLC, HPLC-electrospray ionization (LC-ESI), and LC-FABMS were used to characterize flavonoid glycosides in the methanol extract from peanut meal. Five isoflavones, diadzin, glycitin, genistin, daidzein, and genistein, were separated by HPLC and characterized by comparison with known standards using ESI-MS. The flavonoid methylquercetin (rhamnetin) was present in the methanol extract from peanuts and identified by ESI-MS. Four other flavonoids, two quercetin diglycosides, one quercetin monoglucoside, and isorhamnetin glucoside, were found to be present in the methanol extract based on their reversed-phase elution pattern, mass ions, and fragment ions using flow-FABMS.     

Scale-up purification for rutin hyrdrolysates by high-performance counter-current chromatography coupled with semi-preparative high-performance liquid chromatography

The study made the isolation of three constituents from rutin and exemplified how to achieve quercetin separation and quantification in rapid and scale-up processes by high-performance counter-current chromatography (HPCCC). Meanwhile, we also isolated the other two constituents, kaempferol and isorhamnetin, by semi-preparative high-performance liquid chromatography. After systematic optimization of solvent systems, sample concentration and flow rate on analytical Mini-DE centrifuge, the same conditions were scale-up by 50-fold in preparative Midi-DE centrifuge to purify quercetin, and the maximum sample loading achieved 1.65 g. Eventually, we successfully isolated quercetin, kaempferol and isorhamnetin with the purity of 99.80%, 99.84% and 99.95%, respectively. The process, therefore, provided an efficient method of obtaining sufficient quantities of highly purified quercetin, as well as kaempferol and isorhamnetin.     

Newly Hatched Neonate Larvae Can Glycosylate: The Fate of Betula pubescens Bud Flavonoids in First Instar Epirrita autumnata

Betula pubescens bud flavonoid aglycones reportedly have negative effects on the performance of first instar Epirrita autumnata and, thus, may defend birch leaves from larval defoliation. We hypothesized that the detrimental effects of these lipophilic flavonoids on larvae are due to their high levels in birch buds and/or the inability of naïve neonates to glycosylate them, which we have shown to occur in fifth instars. To test the latter hypothesis, we investigated the biochemical transformation of bud flavonoids in first instar E. autumnata. We found that newly hatched larvae have the ability to glycosylate birch bud/leaf flavonoid aglycones into corresponding glycosides. Moreover, we suggest that glycosylation may depend upon the chemical character of the aglycone and is an important factor in the performance of first instars.     

Enhanced extraction efficiency of bioactive compounds and antioxidant activity from Hippophae rhamnoides L. by-products using a fast and efficient extraction method

In this study, a novel method named high-speed homogenization coupled with ultrasound-assisted extraction (HSH-UAE) followed by high-performance liquid chromatography (HPLC) was employed and investigated for the extraction and determination of main flavonoids rutin, quercetin-3-O-glucoside, quercetin, kaempferol and isorhamnetin from Hippophae rhamnoides L. by-products. Optimal conditions for HSH-UAE, proposed by the single factor and the Box–Behnken design (BBD) tests coupled with response surface methodology (RSM), were 68% ethanol concentration, ultrasonic power 250 W, homogenate speed 9000 rpm, liquid/solid ratio 22 mL/g and extraction time 12 min. The extraction yields of five main flavonoids were significantly improved by HSH-UAE than UAE and high-speed homogenization-assisted extraction (HSHAE). Furthermore, the antioxidant activity of HSH-UAE extracts was assessed by 1,1-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing/antioxidant power (FRAP) assays with IC₅₀ value of 0.091 mg/mL and 2.837 mmol FeSO₄/g DW. Good linear ranges were obtained for five main flavonoids in the range of 2.5–500 μg/mL with the coefficient higher than 0.99. The recoveries of the five flavonoids were in the range of 97.87–99.26%. The limit of detection (LOD) and the limit of quantification (LOQ) for the main flavonoids were within the ranges of 0.28–0.46 and 0.89–1.74 μg/mL, respectively. The present results showed that the proposed method HSH-UAE could be developed as a fast and efficient method for extracting the active ingredients from medical edible plant materials for potential application.     

The Effects of Ants on the Entomophagous Butterfly Caterpillar Feniseca tarquinius, and the Putative Role of Chemical Camouflage in the Feniseca–Ant Interaction

Butterfly caterpillars in the lycaenid subfamily Miletinae are predators of ant-tended Homoptera, yet they lack specialized secretory and call-production organs crucial to ant association in other lycaenids. Here, we address the question of how miletine caterpillars have invaded the ant–Homoptera symbiosis through a study of the only New World miletine, Feniseca tarquinius, a predator of the wooly aphid Prociphilus tesselatus. Previous interpretations have suggested that F. tarquinius and other miletine caterpillars avoid ant aggression by concealing themselves under silken webs. In contrast, our field data indicate that F. tarquinius caterpillars are less likely to be concealed in the presence of the ants Camponotus pennsylvanicus and Formica obscuriventris than in the absence of ants, although caterpillar and ant behaviors vary between years. Chemical analysis and behavioral assays suggest that chemical camouflage, not physical concealment, is responsible for the ants’ failure to detect and remove F. tarquinius caterpillars from aphid colonies. Analyses by gas chromatography indicate that the cuticular lipid composition of caterpillars are similar to that of their aphid prey, although it varies with prey species. Behavioral assays confirm that solvent extracts of F. tarquinius caterpillars and P. tesselatus aphids evoke similar behavioral responses in C. pennsylvanicus ants. Chemical camouflage is well known in social parasites of ants, but the present study represents one of a few documented cases where chemical deceit is important to interactions with ants outside the nest.     

Concentrations of Foliar Quercetin in Natural Populations of White Birch (Betula pubescens) Increase with Latitude

We investigated latitudinal and regional variations in the composition and concentrations of foliar flavonoids and condensed tannins in wild populations of white birch (Betula pubescens EHRH) in a large climatic transect in Finland. Concentrations of quercetin derivatives were correlated positively with latitude. By contrast, the concentrations of apigenin and naringenin derivatives were correlated negatively with latitude. These compound-specific latitudinal gradients compensated each other, resulting in no changes in the concentration of total flavonoids. Our results thus demonstrate a qualitative, but not quantitative, latitude-associated gradient in the foliar flavonoids in white birch. Due to higher antioxidant capacity of the quercetin derivatives in relation to other flavonoids, the qualitative change can reflect higher adaptation to light in the north than south. An investigation on a regional scale in the northern boreal zone showed that the temperature sum was correlated positively and soil P concentration was correlated negatively with the concentrations of foliar flavonoid, while the concentration of condensed tannins was correlated with slope. The variation in concentrations of flavonoids at large-scale geographical patterns is in line with the conjecture that foliar flavonoids are synthesized for protection against photooxidative stress.     

Defining the Role of Flavonoid Structure on Cottonseed Oil Stabilization: Study of A- and C-Ring Substitution

Structure—activity relationships of flavonoids against cottonseed oil oxidation are the objective of this work, which focuses on the contribution of C-ring and 5,7-di-OH A-ring. Flavonoids with a catecholic B-ring, namely quercetin, fisetin, luteolin, taxifolin, (+)-catechin and eriodictyol were added in increasing concentrations to cottonseed oil and the induced decrease in the rate constant of peroxide formation (k) and peroxide values (PV) were determined. The flavonoid depletion was monitored during the autoxidation with high-performance liquid chromatography-diode array detector (HPLC-DAD) analysis. The fully substituted C-ring was established as the most significant element for maximal antioxidant activity; quercetin even decreased k by 86% in comparison with the control and presented the highest decrease of PV per mmole of antioxidant. Flavonoids missing the 2,3-double bond or the 3-OH (taxifolin, luteolin) resulted in a smaller decrease of k, and the lack of both of the above structural elements (eriodictyol) resulted in the smallest decrease. Lack of the 4-carbonyl in the C-ring had the least negative effect on antioxidant activity since (+)-catechin presented the strongest protection after quercetin and fisetin. The activity of the latter flavonoid proved that A-ring hydroxyls, despite the unfavorable m-configuration, participate in the stabilization of the lipid substrate, although their significance was secondary.