Flavonoids in Citrus Hybrids

The occurrence and distribution of flavanone glycosides in the leaves and fruits of many kinds of artificial citrus hybrid plants were investigated by polyamide thin-layer chromatography. The citrus hybrids can be divided into two broad categories, a) those containing rutinosyl glycosides, b) those containing neohesperidosyl glycosides in accordance with the case of natural citrus species. The fiavonoid patterns of rutinosyl glycosides are classified into the following groups, a) hesperidin, b) narirutin, c) hesperidin and narirutin, d) didymin and narirutin, e) hesperidin, narirutin and eriocitrin and f) hesperidin and eriocitrin, while the pattern of neohesperidosyl glycosides fall into six groups, a) naringin, b) neohesperidin and naringin, c) neohesperidin, naringin and neoeriocitrin, d) neohesperidin and neoeriocitrin, e) naringin and neoeriocitrin, and f) poncirin, neohesperidin, naringin and neoeriocitrin. It is worthy of note that a hybrid (accession number 1088) between C. unshiu and C. hassaku contains only narirutin. Among the ninty-four hybrids examined, fifty-three varieties were obviously different from female parents in their flavonoid pattern and could be judged as true hybrids by fiavonoids but the others could not.

Additionally, a survey of fiavonoids in newly found natural pummelo- and Daidai hybrids were carried out in connection with their origin.     

Flavanone Glycoside Biosynthesis in Citrus: Chalcone Synthase, UDP-Glucose:Flavanone-7-O-Glucosyl-Transferase and -Rhamnosyl-Transferase Activities in Cell-Free Extracts

Previous indirect evidence suggested that the biosynthesis of flavonoids in Citrus may not proceed via the usual chalcone synthase reaction and that glycosylation occurs during chalcone formation and not afterward, as has been reported in other species. We detected chalcone-synthase and UDP-glucose:flavanone-7-O-glucosyl-transferase activities in cell-free extracts of Citrus. The glucosylated flavanone was further rhamnosylated when exogenous UDP-glucose and NADPH were added to the extract. Chalcone-synthase activity was detected in cell-free extracts derived from young leaves and fruits. Young fruits (2 millimeter diameter) had the highest chalcone synthase activity. UDP-glucose:flavanone-7-O-glucosyl-transferase activity was measured in cell-free extracts derived from young leaves and fruits of Citrus mitis and Citrus maxima. The highest UDP-glucose:flavanone-7-O-glucosyl-transferase activity was found in young C. maxima leaves. These data indicate that Citrus contains a flavonoid pathway similar to that studied in other species.     

The molecular and enzymatic basis of bitter/non‐bitter flavor of citrus fruit: evolution of branch‐forming rhamnosyltransferases under domestication

Domestication and breeding of citrus species/varieties for flavor and other characteristics, based on the ancestral species pummelo, mandarin and citron, has been an ongoing process for thousands of years. Bitterness, a desirable flavor characteristic in the fruit of some citrus species (pummelo and grapefruit) and undesirable in others (oranges and mandarins), has been under positive or negative selection during the breeding process of new species/varieties. Bitterness in citrus fruit is determined by the composition of branched‐chain flavanone glycosides, the predominant flavonoids in citrus. The flavor‐determining biosynthetic step is catalyzed by two branch‐forming rhamnosyltransferases that utilize flavanone‐7‐O‐glucose as substrate. The 1,2‐rhamnosytransferase (encoded by Cm1,2RhaT) leads to the bitter flavanone‐7‐O‐neohesperidosides whereas the 1,6‐rhamnosytransferase leads to the tastelessflavanone‐7‐O‐rutinosides. Here, we describe the functional characterization of Cs1,6RhaT, a 1,6‐rhamnosyltransferase‐encoding gene directing biosynthesis of the tasteless flavanone rutinosides common to the non‐bitter citrus species. Cs1,6RhaT was found to be a substrate‐promiscuous enzyme catalyzing branched‐chain rhamnosylation of flavonoids glucosylated at positions 3 or 7. In vivo substrates include flavanones, flavones, flavonols and anthocyanins. Cs1,6RhaT enzyme levels were shown to peak in young fruit and leaves, and gradually subside during development. Phylogenetic analysis of Cm1,2RhaT and Cs1,6RhaT demonstrated that they both belong to the branch‐forming glycosyltransferase cluster, but are distantly related and probably originated separately before speciation of the citrus genome. Genomic data from citrus, supported by a study of Cs1,6RhaT protein levels in various citrus species, suggest that inheritance, expression levels and mutations of branch‐forming rhamnosyltransferases underlie the development of bitter or non‐bitter species/varieties under domestication.     

Changes in citrus leaf flavonoid concentrations resulting from blight-induced zinc-deficiency

Increased flavonoid concentrations were found to correlate with the elevated levels of leaf phenolic compounds occurring in blight-induced zinc-deficient citrus. In orange (Citrus sinensis L.) leaves, the increases occurred primarily in hesperidin and diosmin, whereas in grapefruit (C. paradisi Macf.) the largest increases occurred in naringin and rhoifolin. Zinc-deficiency occurring in the blighted citrus leaves appeared to be the important contributing factor to the increased flavonoid content. Although the leaves from trees with blight were typically smaller than leaves from unaffected trees, the increased flavonoid content was not significantly due to a concentration effect. Large differences occurred in the percent increases in concentrations of certain citrus leaf flavonoids. While large increases occurred for a number of flavanone and flavone glycosides, much smaller percent increases occurred for other minor flavone glycosides, and the polymethoxyflavone aglycones. The parallel increases occurring in the concentrations of certain flavone glycosides and their flavanone analogs provide a further indication that flavanone glycosides are precursors in the biosynthesis of flavone glycosides in citrus.     

Diet of the brown howler monkeyAlouatta fusca in a semi-deciduous forest fragment of southeastern Brazil

The diet of a group of six brown howlers was studied weekly during 12 months in a reserve of 250 ha of secondary, mesophytic, semi-deciduous forest. The phenology of 186 trees of 72 species and 29 families was monitored simultaneously. Scan sampling was used to record the diet from dawn until dusk on a total of 60 days of observation, yielding 718 hr of animal-observer contact and 2,943 feeding scans. The diet was composed of leaves (73%), flowers (12%), and fruits (5%), from 68 identified plant species.Celtis iguanae, Cassia ferruginea, andInga spp. were the main food sources, accounting for approximately 50% of the diet. Young leaves (59%) were preferred to mature leaves (31%), trees contributing 56% and lianas 41% of the leaf diet. The ingestion of young leaves was correlated to the availability of these items, however, the correlations were not significant for flowers and fruits. The diet was poorer in fruits and richer in young leaves of lianas in comparison to other howler monkey species studies, probably as a consequence of the liana abundance in this forest fragment.     

Phenology of figs in Budongo Forest Uganda and its importance for the chimpanzee diet

This paper reports on the phenological patterns of figs in Budongo Forest, Uganda, and how it relates to chimpanzee food availability in different seasons. In addition, we analysed the dung of chimpanzees to understand the composition of fruits in their diet. The aim of our study was to assess Ficus phenology and how it affects chimpanzee diet. Fifteen species of figs were monitored for fruit (syconium) and leaf phenology between June 2000 and 2001. Ficus fruit production varied significantly between and within species, and also with tree trunk and crown diameters. Fig fruit production was asynchronous and individual fig trees produced crops from one to five times in a year. In addition to fruits, chimpanzees fed on young leaves of some Ficus species. Shedding of old Ficus leaves coincided with the dry season, followed by appearance of young leaves. The dry season in Budongo is a period of general fruit scarcity. The combination of fig fruits and young leaves make up the most important food in the diet of chimpanzees. From the chimpanzee dung, more than 78% of seeds comprised fig ‘seeds’ (nutlets) and the rest of the diaspores were from other tree species. Our findings suggest that chimpanzees disperse large number of diaspores in their dung, thereby serving as important agents of natural forest regeneration.     

Age-dependent shift in response to food element composition in Collembola: contrasting effects of dietary nitrogen

Developing leaves that are soft, with high concentrations of resources, can be particularly vulnerable to herbivore damage. Since a developing leaf cannot be very tough, given the constraints of cell expansion, the major form of protection is likely to be chemical defence. We investigated changes in concentration of herbivore resources (protein, carbohydrates and water) and putative defences (total phenolics, tannin activity, cyanogenic glycosides, alkaloids, cell wall, and leaf mechanics) across five leaf development stages of the soft-leaved Toona ciliata M. Roem. and the tough-leaved Nothofagus moorei (F. Muell.) Krasser. Chemical defences were predicted to be more highly developed in young than expanded leaves of both species, and to decline more in expanded leaves of N. moorei, which become tough and strong at maturity, than in the softer expanded leaves of T. ciliata. Resources and defences were dynamic within the developing leaves. Highest concentrations of protein were recorded in young leaves in both species, and highest levels of non-structural carbohydrate were recorded in young leaves of T. ciliata. Allocation to defence varied in both amount and type across leaf stages. In T. ciliata, there was an increase in chemical defence in expanded leaves (tannin activity, alkaloids). However, in N. moorei, increasing strength and toughness of developing leaves coincided with decreasing chemical defence, consistent with our hypothesis. For phenolics, this decrease was partly due to dilution by cell wall, but cyanogenic glycosides were present in young leaves and absent in fully mature leaves. These results are consistent with leaf toughness acting as an effective anti-herbivore defence, thereby reducing the need for investment in chemical defence.     

Flavanone 3-hydroxylase expression in Citrus paradisi and Petunia hybrida seedlings

Petunia hybrida and Citrus paradisi have significantly different flavonoid accumulation patterns. Petunia sp. tend to accumulate flavonol glycosides and anthocyanins while Citrus paradisi is known for its accumulation of flavanone diglycosides. One possible point of regulation of flavanone metabolism is flavanone 3-hydroxylase (F3H) expression. To test whether this is a key factor in the different flavanone usage by Petunia hybrida and Citrus paradisi, F3H mRNA expression in seedlings of different developmental stages was measured using semi-quantitative RT-PCR. Primers were designed to conserved regions of F3H and used to amplify an approximately 350 bp segment for quantitation by PhosphorImaging. Primary leaves of 32 day old grapefruit seedlings and a grapefruit flower bud had the highest levels of F3H mRNA expression. Petunia seedlings had much lower levels of F3H mRNA expression relative to grapefruit. The highest expression in petunia was in primary leaves and roots of 65 day old seedlings. These results indicate that preferential use of naringenin for production of high levels of flavanone glycosides in young grapefruit leaves cannot be attributed to decreased F3H mRNA expression.     

Naringin and Neohesperidin Levels during Development of Leaves, Flower Buds, and Fruits of Citrus aurantium

The distribution of the flavanones naringin and neohesperidin has been analyzed during the development of the leaves, flower buds, and fruits of Citrus aurantium. These flavonoids are at maximum concentration in the organs studied during the logarithmic phase of growth, gradually decreasing until the organs reach maximum development. However, this decrease in the naringin and neohesperidin concentration in leaves, flower buds, and fruits is due to a dilution of the flavonoids caused by cell growth, because total content per organ continues to increase. The levels of neohesperidin are always greater than those of naringin, although the ratio between the relative concentrations is different in the three organs studied. Leaves have the highest ratios, varying between 8.83 and 5.18, followed by flowers (3.15-1.85), and fruits (2.23-1.02). These observations suggest different relationships between the respective enzymic activities in their biosynthetic pathway.     

Western Purple-faced Langurs (Semnopithecus vetulus nestor) Feed on Ripe and Ripening Fruits in Human-modified Environments in Sri Lanka

Although most colobines feed mainly on leaves and a few feed heavily on seeds, colobine digestive adaptations for folivory are thought to preclude the high use of ripe fleshy fruits. In this long-term study of Semnopithecus vetulus nestor, the endemic western purple-faced langur of Sri Lanka, I investigated the feeding ecology and dietary flexibility for fruit feeding in 2 free-ranging groups (PT1 and R1) living in human-modified environments with abundant cultivated fruit, at Panadura and Piliyandala, for 19 mo and 13 mo respectively, using scan-sampling, vegetation enumeration, and phenological studies. In contrast to folivorous forest-living colobines, including other subspecies of Semnopithecus vetulus, my focal groups used more fruit (>50%) than foliage (PT1: 36%; R1: 34%). Both groups used many plant species (PT1 115; R1 59), but selected their food species, fruits over leaves, and young leaves over mature leaves. Fruit use was independent of young leaf availability. Notably, 78.4% and 83.4% of fruits consumed by PT1 and R1 were fleshy and human-edible, most of which were ripening or ripe (PT1: 72.4%; R1: 94.8%). The main fruit for both groups was Artocarpus heterophyllus (Moraceae; jakfruit), a cultivar with fleshy fruit. These findings differ from previous understanding of colobine diets. I suggest that environmental factors, such as the abundance and nature of available fruits, and the absence of arboreal-primate fruit competitors, could influence the use of ripe fleshy fruits by colobines strongly, highlighting the need to review the dietary and digestive flexibility of this group in changed and changing natural environments to formulate effective conservation action.     

喀斯特石山生境中熊猴的雨季食物组成

开展食物组成研究为人们了解灵长类对栖息地的反应提供了很好的途径,对深刻理解动物的行为可塑性及适应性具有重要意义。猕猴属(Macaca)为果食性灵长类,但是不同种类的食性差异很大;即使便同一物种,其不同地理种群也因其栖息环境不同,食物组成存在差异。一般说来,热带地区的种类比生活在较高纬度的种类采食更多的果实。于2012年7—9月,采用瞬时扫描法对广西弄岗自然保护区石山中的两群熊猴(Macaca assamensis)进行了跟踪和观察,对猴群的雨季食物组成及其日时段变化规律进行了研究。结果表明:研究期间熊猴共采食45种植物,其中乔木30种,灌木3种,藤本11种,草本1种。平均每月采食植物22.3种。嫩叶和果实是熊猴的主要食物,分别占食物组成的52.4%和46.1%(其中未成熟果实占21.3%,成熟果实占24.8%)。另外,花占食物组成的0.9%,成熟叶和其它部位分别占0.3%。石山特有植物芸香竹(Bonia saxatilis)的嫩叶提供了43.8%的食物。9种主要植物分别占食物组成的2%,共为猴群提供了85.5%的食物。分析还发现熊猴并不是严格按照环境中的植物生物量来选择食物。嫩叶在弄岗熊猴的食物中的比例高于其他地理种群,而果实低于其他地理种群,这可能与喀斯特石山中果实的丰富度和可利用性较低有关。熊猴一天中不同时间段的食物组成并不相同,主要表现在:熊猴上午时间段对成熟果实和总果实的采食比例高于下午时间段,而嫩叶的采食比例低于下午时间段。另外,不同时间段的食物组成受外界温度的影响,表现为温度与嫩叶的觅食比例成正比,与成熟果实和总果实的觅食比例成反比。这可能与猴群采取的能量平衡策略有关。对熊猴的食物组成的日时段变化规律进行首次报道,研究结果将有助于深入理解熊猴对喀斯特石山生境的适应策略。     

A New Proteolytic Enzyme from Achromobacter lyticus M497-1

Potent antioxidative hydroxyflavanones were produced with Aspergillus saitoi from hesperidin or naringin, which are flavanone glycosides in citrus fruit with weak antioxidative activity. The hydroxyflavanone produced from hesperidin was identified as 8-hydroxyhesperetin (8-HHE), a novel substance, and those from naringin were identified as carthamidin (6-hydroxynaringenin) and isocarthamidin (8-hydroxynaringenin) by FAB-MS, ¹H-NMR and ¹³C-NMR analyses. The antioxidative activity of these hydroxyflavanones was examined by using the free radical-scavenging system of 1,1-diphenyl-2-picrylhydrazyl (DPPH) and the methyl linoleate oxidation system. The hydroxyflavanones (8-HHE, carthamidin, and isocarthamidin) exhibited stronger activity than the flavanone glycosides (hesperidin or naringin) and their aglycones (hesperetin or naringenin). The activity of 8-HHE and isocarthamidin was comparable to that of α-tocopherol, and that of carthamidin was weaker than that of isocarthamidin. The hydroxyflavanones, which were hydroxylated on A ring of flavanone by Aspergillus saitoi, were obtained as potent antioxidants.     

A malonic acid ester derivative of naringin in grapefruit

A malonic acid ester derivative of the flavanone naringin was abundant in the young leaves and fruits of grapefruit plants, but not in the mature leaves and fruits. After isolation, the structure of this compound was established as naringin 6″-malonate (naringenin 72″-O-- -rhamnosyl)-β- -glucoside 6″-malonate).     

Bioproduction of neohesperidin and naringin in callus cultures of Citrus aurantium

The accumulation of both neohesperidin and naringin as major flavonoids in callus cultures of bitter orange (Citrus aurantium) was demonstrated using high performance liquid chromatography with a diode-array detector. The identity of both compounds was confirmed by their corresponding nuclear magnetic resonance spectra. The levels of neohesperidin are higher than those of naringin in callus culture, as they are in immature fruit, and high concentrations of both are found in young tissues such as immature fruits and the outer zone of calli.Abbreviations DMSO dimethylsulphoxide - DW dry weight - HPLC high performance liquid chromatography - NMR nuclear magnetic resonance - Rt retention time - V/UV visible/ultraviolet     

Flavonoids enantiomer distribution in different parts of goldenrod (Solidago virgaurea L.), lucerne (Medicago sativa L.) and phacelia (Phacelia tanacetifolia Benth.)

Plant material is a rich source of valuable compounds such as flavanones. Their different forms influence bioavailability and biological activity, causing problems with the selection of plant material for specific purposes. The purpose of this research was to determine selected flavanone (eriodictyol, naringenin, liquiritigenin, and hesperetin) enantiomer contents in free form and bonded to glycosides by an RP‐UHPLC‐ESI‐MS/MS method. Different parts (stems, leaves, and flowers) of goldenrod (Solidago virgaurea L.), lucerne (Medicago sativa L.), and phacelia (Phacelia tanacetifolia Benth.) were used. The highest content of eriodictyol was found in goldenrod flowers (13.1 μg/g), where it occurred mainly as the (S)‐enantiomer, and the greatest proportion of the total amount was bonded to glycosides. The richest source of naringenin was found to be lucerne leaves (4.7 μg/g), where it was mainly bonded to glycosides and with the (S)‐enantiomer as the dominant form. Liquiritigenin was determined only in lucerne, where the flowers contained the highest amount (1.2 μg/g), with the (R)‐enantiomer as dominant aglycone form and the (S)‐enantiomer as the dominant glycosylated form. The highest hesperetin content was determined in phacelia leaves (0.38 μg/g), where it was present in the form of a glycoside and only as the (S)‐enantiomer. A comparison of the different analyte forms occurring in different plant parts was performed for the first time.     

Comparative antioxidant-capacity and -content of leaves, bulbs, roots, flowers and fruit of Gethyllis multifolia L. Bolus and G. villosa Thunb. species

The total polyphenol, oxygen radical absorbance capacity (ORAC), ferric reducing antioxidant power (FRAP), radical cation scavenging ability, flavonol and flavanone contents were measured in the leaves, bulbs, roots, flowers and fruit (dry weight) of two natural populations of Gethyllis multifolia (Kukumakranka) and Gethyllis villosa. The flowers and fruit of G. multifolia and G. villosa showed higher, and in some cases significantly (P < 0.05) higher antioxidant activities when compared to the leaves, bulbs and roots. This, however, was not true for the flavanone content in both species. The total polyphenol content in the fruits of G. multifolia (21.54 mg GAE/g) and G. villosa (27.64 mg GAE/g) were found to be in agreement with those of raisins (28.30 mg GAE/g), blueberries (24 mg GAE/g) and strawberries (15.40 mg GAE/g). The FRAP values of G. multifolia flowers (76.66 μmole AAE/g) and fruit (91.51 μmole AAE/g) were found to be significantly (P < 0.05) higher than those of the other plant parts (16.76 to 39.08 μmol AAE/g). On the other hand, the flowers (590.23 μmol TE/g) and fruit (741.16 μmol TE/g) of G. villosa revealed a significantly (P < 0.05) higher ORAC when compared to the other plant parts (251.25 to 410.60 μmol TE/g). A strong correlation was evident in the fruit of both species between the total polyphenols and FRAP (r = 0.95), ORAC (r = 0.95) and flavonol content (r = 0.79).     

The diet and feeding behavior of the silver leaf monkey (Trachypithecus auratus sondaicus) in Indonesia

I describe the diet and feeding behavior of silver leaf monkeys (Trachypithecus auratus sondaicus) in the Pangandaran Nature Reserve, West Java, Indonesia, and compare a group living in old secondary rain forest with a group living in mixed plantation/secondary forest to determine intraspecific variation in feeding behavior and the importance of the plantation species in the diet of the monkeys. Young leaves and leaf buds made up slightly less than half of their diets, with both groups showing a preference for a few species when seasonally available. Fruits and flowers of a few species were also preferentially selected when available. These included sweet, fleshy fruits, which most other colobines tend to avoid. Young leaf intake was greatest in months when fruit intake was low. Mature leaves were rarely eaten. Both groups spent approximately 20% of feeding time foraging on Moraceae species. Differences in the diet of the two groups were related largely to differences in vegetational composition and the availability and abundance of food items for the species common to both sites. Teak (Tectona grandis) was the top food species of the group living in mixed plantation/secondary forest, with the midribs of young leaves preferentially selected. Young leaves ofT. grandis, available throughout the study, provided a staple food and were eaten when preferred foods were scarce. More favored food items were available to the group living in old secondary forest, though none was a staple food.     

Seasonal variation in leaf characteristics and food selectionby larval noctuids on an evergreen Mediterranean shrub

Despite year round availability of foliage, abundance of generalist noctuid larvae (Lepidoptera: Noctuidae) in evergreen-dominated Mediterranean forests has a narrow, distinct spring peak. This restricted larval period has been suggested to result in part from avoidance of the nutritionally poor mature foliage, and preference for nutritionally superior spring-produced young leaves. This study examines this hypothesis by (i) documenting differences in nutritional characteristics between expanding (April) and mature (June) young leaves of the evergreen Mediterranean shrub Daphne laureola L. (Thymelaeaceae), and (ii) experimentally studying the feeding preferences of noctuid larvae for young leaves, old leaves (≥ 1 yr old), and developing fruits of this species in one south-eastern Spanish locality. Young leaves of D. laureola declined in nutrient concentration and specific dry mass from April to June. The responses of noctuid larvae, in terms of both relative preference and total consumption, to this seasonal variation in chemical and physical features of young leaves were also investigated. When noctuid larvae were simultaneously offered young leaves, old leaves and developing fruits, they exhibited similar preferences for young leaves and developing fruits, and rejected old leaves developed during the previous year. Noctuid larvae did not modify their consumption of young leaves relative to old leaves and developing fruits in response to seasonal changes. Food selection patterns exhibited by D. laureola noctuid herbivores, notably the rejection of old leaves in favour of young ones, are consistent with the hypothesis relating restricted larval periods of these generalist consumers with the low food value of the previous season leaves of evergreen Mediterranean plants.     

Three new flavonoids,proanthocyanidin, and accompanying phenolic constituents from Feijoa sellowiana

Our investigation of phenolic constituents of fruits, flower buds, and leaves of Feijoa sellowiana led to the isolation of twenty-one phenolics including three new gossypetin glycosides 1–3, and also the purification of a proanthocyanidin fraction. A high-performance liquid chromatography method for simultaneous analysis of phenolic constituents was established and then used to investigate the phenolic profiles of the parts of the plant species, to show the presence of characteristic flavonoids and ellagic acid derivatives or ellagitannins in the extracts from fruits, flower buds, and leaves. The branch extract profile also suggested the presence of alkylated ellagic acids as characteristic constituents. Inhibitory effects of feijoa flavonoids on mushroom tyrosinase were seen, although in some cases this may have resulted from direct interaction with the enzyme. Cytotoxic effect of the proanthocyanidin fraction was also shown.