Comparison of furanocoumarin concentrations of greenhouse-grownRuta chalepensis with outdoor plants later transferred to a greenhouse

Ruta chalepensis contained concentrations of furanocoumarins 25–50% of those found inR. graveolens both in the whole leaf and on its surface. On the leaf surface of plants grown all year indoors in a greenhouse, they increased steadily between November 1 and December 14 on mature upper and lower leaves. New growth upper leaves on December 14 contained less than mature upper leaves. Plants transferred from outdoors to the greenhouse showed decreased concentrations after the first two weeks, followed by recovery both in the whole leaf and on the leaf surface. Proportions of xanthotoxin and bergapten to psoralen changed during the experiment. On the leaf surfaces and in the whole upper leaves of the indoor plants, the proportions were often similar, but in the transferred plants, in most cases, psoralen was less than bergapten or xanthotoxin in the upper leaves and markedly less in the lower leaves. Implications of these findings for possible effects of environmental changes on secondary plant metabolism are discussed.     

Psoralens in senescing leaves ofRuta graveolens

Concentrations of three furanocoumarins, psoralen, xanthotoxin, and bergapten, were measured on the surface and within mature whole leaves of two groups ofRuta graveolens L. late autumn plants, 2 and 6 years old, which contained green, yellow, and dry yellow leaves. Upper green leaves contained higher concentrations of these coumarins than lower green leaves, green leaves contained several times as much as yellow leaves, and dry leaves contained even smaller amounts than yellow ones. The dry yellow leaves contained only a very small percentage of furanocoumarins on the surface, suggesting that extrusion to the surface of yellow leaves was slower or had stopped, while loss from the surface continued. The loss of psoralen was the most dramatic in and on the dry leaves. Bergapten's ratio to the other cournarins increased during senescence. Xanthotoxin was always the predominant furanocoumarin in this species.     

Seasonal change in nagilactone contents in leaves inPodocarpus nagi forest

Nagilactones isolated fromPodocarpus nagi (Thunb.) Zoll. et Moritz. are known by their physiological activities as a plant growth inhibitor or antiherbivory substance. As the first step in clarifying the nagilactone dynamics in a forest canopy, the seasonal variations in nagilactone contents in leaves and mass of nagilactones accumulated in the canopy were examined at Mt. Mikasa, Nara City, central Japan. Nagilactone content in mature leaves dropped sharply in May, when new leaves flushed. The nagilactone content of new leaves was far greater than that of mature leaves, implying a translocation of nagilactones from old leaves to new leaves. The total mass of nagilactones in the canopy was 3.9 kg/ha.     

Characterization of furanocoumarin metabolites in parsnip webworm,Depressaria pastinacella

Although metabolites of furanocoumarins have been characterized in a wide range of organisms, to date they have been identified in only a single insect species, Papilio polyxenes. Depressaria pastinacella, the parsnip webworm, like P. polyxenes a specialist on Apiaceae, routinely consumes plant tissues higher in furanocoumarin content than does P. polyxenes and is capable of faster cytochrome P-450-mediated detoxification of these compounds. In this study, we characterized metabolites of xanthotoxin, a linear furanocoumarin, and sphondin, an angular furanocoumarin, in midguts and frass of parsnip webworms. Two metabolites were isolated and identified from webworms fed artificial diet containing xanthotoxin. LC-ESI-MS analysis resulted in the determination of a MW of 266 for the compound in the frass and one of the compounds in the midgut; ¹H NMR confirmed its structure as 6-(7-hydroxy-8-methoxycoumaryl)-hydroxyacetic acid (HCHA). The second compound from the midgut had a MW of 252 and was identified by ¹H NMR and ¹³C NMR analysis as 6-(7-hydroxy-8-methoxycoumaryl)-hydroxyethanol) (HMCH). Whereas HCHA has been found in frass of Papilio polyxenes fed xanthotoxin, HMCH has not been reported previously in insects. Although the first step of metabolism of xanthotoxin in webworms as well as P. polyxenes is likely the formation of an epoxide on the furan ring, angular furanocoumarin metabolism in webworms appears to differ. The principal metabolite of sphondin was identified as demethylated sphondin (6-hydroxy-2H-furo[2,3-h]-1-benzopyran-2-one) by LC-ESI-MS and confirmed by ¹H NMR and ¹³C NMR analyses. That webworms produce metabolites of xanthotoxin in common not only with other Lepidoptera (e.g., HCHA) but with other vertebrates (e.g., HMCH) suggests a remarkable conservatism in the metabolic capabilities of cytochrome P-450s and raises the possibility that insects may share other detoxification reactions with vertebrates with respect to toxins in foodplants.     

Identification of eight coumarins occurring with psoralen,xanthotoxin, and bergapten on leaf surfaces

Surface extracts of the leaves of five species in the Umbelliferae,Citrus limon (Rutaceae), andPsoralea bituminosa (Leguminosae) were examined for the presence of coumarins, after a previous study had shown the presence of three psoralens. In the current investigation eight more coumarins were identified by mass spectrometric techniques: the simple coumarins scopoletin, scoparone, and osthol, the linear furanocoumarins imperatorin and phellopterin, the angular furanocoumarins angelicin and pimpinellin, and the pyranocoumarin seselin. Five of these occur inApium graveolens, and scopoletin, scoparone, and imperatorin were each found in three of the species examined. The co-occurrence of all these coumarins on the surface may be significant in communication between the plant and its environment.     

Lutein Sequestration and Furanocoumarin Metabolism in Parsnip Webworms Under Different Ultraviolet Light Regimes in the Montane West

Both biotic and abiotic selection pressures can contribute to geographic variation in allelochemical production in plants. We examined furanocoumarin production in western North American populations of Heracleum lanatum and Pastinaca sativa that, at different latitudes and altitudes, experience different ultraviolet (UV) light regimes. Total furanocoumarins and linear furanocoumarins of fruits were negatively correlated with UV irradiance, whereas amounts of angular furanocoumarins, which are generally less phototoxic, were not. Another factor potentially influencing furanocoumarin production is the presence of the parsnip webworm Depressaria pastinacella, (Lepidoptera: Oecophoridae), an herbivore that feeds on reproductive structures of both plant species. These insects sequester lutein from their host plants; this carotenoid acts to ameliorate furanocoumarin toxicity. Although the concentration of lutein in fruits did not vary with UV irradiance, lutein sequestration by sixth instars was positively correlated with UV irradiance. Webworm populations are variably infested with the polyembryonic webworm parasitoid Copidosoma sosares Walker (Hymenoptera: Encyrtidae). H. lanatum fruits from populations with webworms parasitized by C. sosares had lower concentrations of furanocoumarins, with the exception of sphondin, than fruits from plants infested with webworms free from parasitism. Lower levels of these furanocoumarins may reduce negative effects on the fitness of this parasitoid. In contrast with the variation in furanocoumarin content, the ability of webworms to metabolize furanocoumarins by cytochrome P450 did not differ significantly among populations from New Mexico to Alberta.     

Influence of low-intensity ultraviolet radiation on extrusion of furanocoumarins to the leaf surface

Exposure ofRuta graveolens leaves to low intensity 366-nm radiation led to a ca. 20% increase in concentrations of the furanocoumarins psoralen, xanthotoxin and bergapten, as compared to leaves kept in darkness. Both direct and, even more, scattered UV radiation produced increases in total concentrations. Changes in the concentrations of individual coumarins were generally parallel. Extrusion to the surface was increased, especially in lower, older leaves exposed to the scattered radiation, where it exceeded the control by factors of eight or nine. It is suggested that this response could enhance shielding of leaves against penetration of UV into the cells and that irradiation, by exciting the furanocoumarins, could augment protection against potential microbial invaders.     

Extracting Oviposition Stimulants for Carrot Fly from Host-Plant Leaves

Extracts of carrot foliage obtained with various extraction methods were compared for effectiveness in stimulating oviposition in the carrot fly. In choice assays, surrogate leaves treated with a hexane surface extract produced with a new microwave-assisted procedure were almost as acceptable as real host leaves. The high stimulatory activity of this extract was attributable to the raised solvent temperature, since cold hexane extracts were much less stimulatory. The microwave extract elicited about twice as much oviposition as the previously used dichloromethane surface extracts and the diethyl ether fraction of an extract that was obtained by brief immersion of leaves into water near its boiling point. The ovipositional responses to crude methanol and hot water extracts were weak because of the presence of yet unidentified polar deterrent compounds. Total extracts of ground foliage (vacuum distillation and extraction with liquid carbon dioxide) had no net stimulatory effect on oviposition.     

Impact of atmospheric pollution on linear furanocoumarin content in celery

In a series of laboratory studies, a single 4-hr acidic fog at pH levels associated with commercial celery (Apium graveolens L.) production near major population centers in California was found to stimulate development of psoralen, bergapten, xanthotoxin, and isopimpinellin within 24 hr and for at least 120 hr after exposure. At 120 hr posttreatment, the concentrations of phototoxin furanocoumarins (psoralen + bergapten + xanthotoxin) increased 540% in the leaves (to 135 g/g fresh weight) and 440% in the petioles (to 55.56 g/g fresh weight) of celery exposed to a pH 2.0 fog as compared to plants exposed to control fogs (pH 6.3–6.5). Concentrations of these compounds in test plants were 7.5 times higher than the amount known to produce contact dermatitis. The nonphototoxic isopimpinellin increased more than threefold in the leaves (to 39.23 g/g fresh weight, 120 hr) and petioles (to 25.88/gmg/g fresh weight) as compared to control plants. In contrast, a single ozone fumigation at 0.20 ppm for 2 hr generally reduced concentrations of phototoxin furanocoumarin in leaves of celery within 24 hr (ozone-treated plants = 37.9, controls = 69.5 g/g fresh weight), but levels of these chemicals in leaves of ozone-fumigated plants increased rapidly and concentrations were not significantly different at 120 hr. Isopimpinellin concentrations in foliage followed a similar trend (at 24 hr, control = 25.11, ozone treated = 10.96/gmg/g fresh weight, no difference at 120 h). In petioles, none of the linear furanocoumarin levels differed significantly at 120 hr posttreatment.     

Behavior of neonate diamondback moth larvae [Plutella xylostella (L.)] on leaves and on extracted leaf waxes of resistant and susceptible cabbages

NeonatePlutella xylostella moved more rapidly, spent more time walking, and engaged in searching behaviors more often on leaves of NY 8329, a resistant cabbage with glossy leaves, than on Round-Up, a susceptible variety with normal wax bloom. The neonates also spent significantly more time palpating and more time biting and spinning silk on the susceptible cabbage (although the latter two differences were not significant). Very similar differences in neonate behavior occurred on leaf surface wax extracts (hexane and dichloromethane) of the two cabbage genotypes. Leaf surface waxes are thus strongly implicated in eliciting reduced acceptance of the glossy cabbage by neonateP. xylostella. The chemical compositions of the leaf wax extracts were markedly different. Several compounds, including the triterpenols- and-amyrin, were found only in the glossy waxes. The percentages of some major wax constituents differed between wax extracts of the two cabbage types. These differences in wax composition may condition the plant resistance in glossy types.     

Allelochemicals fromPilocarpus goudotianus leaves

The effect on germination, shoot, and root growth by bergapten, xanthotoxin, imperatorin, xanthyletin, xanthoxyletin, luvangetin, donatin and alloxanthoxyletol fromPilocarpus goudotianus leaves, onLactuca sativa var. nigra seedlings has been evaluated. A structure-activity correlation is discussed based on the bioassay results. Furanocoumarins appear to be the most active compounds in comparison with pyrano- and simple coumarins. The presence of an oxygenated function at C-8 decreases the germination effect in furano- and pyranocoumarins, while C-5 substituents do not cause significant changes on the activity.Part 2 in the series:Natural Product Models as Allelochemicals. For Part 1 see: Macíaset al. (1992).     

Artificial defloration and furanocoumarin induction inPastinaca sativa (Umbelliferae)

Damage simulating herbivory was tested as an inducer of furanocoumarins in the floral parts of the wild parsnip,Pastinaca sativa (Umbelliferae). In one experiment, primary umbels ofP. sativa were partially deflorated over the course of nine days, and higher-order umbels as well as the remaining primary umbel floral parts were sampled. Total furanocoumarin concentration was not significantly affected by defloration, but one furanocoumarin, isopimpinellin, increased in one floral stage of the secondary umbel in damaged plants. In a second experiment, primary umbels were completely deflorated and the higher-order umbels allowed to set seed. No significant effect of defloration on furanocoumarin content was found in the seeds of the higher-order umbels.     

The effects of fertilizing on the foliar nutrient concentrations inEucalyptus grandis

The foliar concentrations of macro- and micro-nutrients were studied in four N P K-lime fertilizer experiments withEucalyptus grandis, in the Natal Midlands and Zululand, South Africa. The sampling method and the chemical analysis of the foliar nutrient concentrations are described. Nutrient levels are similar to results found elsewhere and they differ little from those for other eucalypt species. There are many significant changes in foliar nutrient concentrations as a result of fertilizing. The relationship between soil and foliar N and P concentrations are clear as well as the importance of well-balanced N/P and Ca/Mg ratios. The Zn-P and Zn-Mg relationships make it apparent that Zn-enriched fertilizers should be used forE. grandis.     

Phenolics in black oak bark and leaves

Catechin, quercitrin, robinin, quercetin 3-methyl ether, scopoletin, cholorogenic acid, several leucoanthocyanins, and condensed and hydrolyzable tannins were identified in bark and leaves ofQuercus velutina Lamarck. The concentrations of most phenolics in leaves increased as the growing season progressed, whereas those of most phenolics in bark remained essentially unchanged. Qualitative differences in bark and leaf phenolics among different trees were negligible.     

Seasonal and population variation in flavonoid and alliarinoside content of Alliaria petiolata

Pieris napi oleracea, an indigenous butterfly in North America, lays eggs on Alliaria petiolata, an invasive weed that was introduced from Europe. However, larval development on plants from different sources varies considerably. A. petiolata is a compulsive biennial, and its foliage is rich in apigenin flavonoids. We compared the chemistry of different vegetative forms from different populations in the vicinity of Ithaca, NY throughout the year. Significant differences occurred in the number of apigenin derivatives in different populations and vegetative forms, and seasonal variations in the amounts of these compounds were found. We have previously isolated two major compounds, alliarinoside [(2Z)-4-(-D-glucopyranosyloxy)-2-butenenitrile] (1) and isovitexin-6-O-D-glucoside (3), which negatively affect development of P. napi oleracea larvae. Comparative analyses of these compounds in two populations throughout the year showed that their concentrations reached maxima twice annually. Foliage is almost devoid of flavonoids in June-July. Thus, variation in the chemistry of the plant may account for observed variation in development rates and survival of the larvae. Several apigenin compounds were isolated and identified by spectral studies.     

Preservation of salicaceae leaves for phytochemical analyses: Further assessment

The chemistry of the plant family Salicaceae has been of interest to researchers as diverse as chemical ecologists, chemosystematists, and paper chemists. Continuing the debate on proper methods for preservation of plant material prior to analysis, vacuum-drying was recently advocated, because freeze-drying may cause degradation of phenolic glycosides. This study was conducted to clarify the consequences of freeze-drying for foliar secondary chemicals and to evaluate the consequences of vacuum-drying for primary compounds (protein and carbohydrates). Leaves of quaking aspen (Populus tremuloides) were flash-frozen in liquid nitrogen and freeze-dried or vacuum-dried at room temperature. We then analyzed samples for levels of salicortin and tremulacin (phenolic glycosides), condensed tannins, nitrogen, soluble protein, sugars, and starch. Freeze-drying did not alter the concentrations of phenolic glycosides or tannins, relative to vacuum-drying. Freeze-drying did cause a small and inexplicable decline in nitrogen and soluble protein. Vacuum-drying, however, reduced starch concentrations by 38%. We suggest that the vacuum-drying method be used in studies in which carbohydrates are of no interest. For studies measuring carbohydrates, however, freeze-drying is a better alternative, and should effect no changes in levels of secondary compounds if samples are not allowed to thaw during the drying process.     

Seasonal variation in phytotoxicity of bracken (Pteridium aquilinum L. Kuhn)

Laboratory bioassays were used to test for the phytotoxicity of volatile compounds, fresh plant material as a seed bed, and water extracts from bracken [Pteridium aquilinum (L.) Kuhn] pinnules to germination and seedling growth of aspen (Populus tremula L.) and Scots pine (Pinus sylvestris L.). Fronds were sampled from two bracken populations, one in the south and one in the north of Sweden. All three bioassays showed inhibitory effects, and these varied seasonally with the most inhibitory effects occurring in May, June, and September. The peak of inhibition in May and June coincides with the start of the growing season when bracken still is immature and vulnerable to interference from other species. The increase in inhibitory effects in September appears to be due to transformation of natural products or an accumulation of inhibitory compounds that are released during decomposition following frond death. Addition of activated carbon did not remove the inhibitory effects.     

Dried leaves from rocky mountain plants decrease infestation by stored-product beetles

Leaves of two highly aromatic plants,Artemisia tridentata (Nutt.) andMonarda fistulosa L., prepared according to a patented process, inhibited oviposition by the Mexican bean weevil,Zabrotes subfasciatus (Boheman), in beans at concentrations less than 1% w/w. Both plant species were less effective against the rice weevil,Sitophilus oryzae L., in wheat, with onlyM. fistulosa exhibiting any concentration-dependent activity. The maximal control achieved against this species was less than 50% at 3% w/w. Two less aromatic plant species,Balsamorhiza sagittata (Pursh.) Nutt. andGeranium viscosissimum Fisch. and Mey., caused only low levels of inhibition against both insect species. Volatiles probably caused the response toA. tridentata andM. fistulosa, while the asymptotic concentration dependence for the less volatile plant material was likely caused by behavioral factors related to the physical presence of foreign particulate matter in the foodstuff. Chemical analysis indicated that most of the volatile components from the dried leaf material from all species were terpenoids, with camphor (9.7 mg/g) and 1,8-cineole (4.0 mg/g) being most abundant inA. tridentata and carvacrol (26.3 mg/g) being most abundant inM. fistulosa.Portions of this research were conducted by the U.S. Department of Agriculture. All programs and services of the U.S. Department of Agriculture are offered on a nondiscriminatory basis without regard to race, color, national origin, religion, sex, age, marital status, or handicap. This article reports the results of research only. Mention of a proprietary product does not constitute an endorsement or a recommendation for its use by USDA.     

Metabolism and excretion of the furanocoumarin xanthotoxin by parsnip webworm,Depressaria pastinacella

The parsnip webworm,Depressaria pastinacella, feeds on plants containing high concentrations of furanocoumarins. compounds toxic to many organisms. Parsnip webworm larvae were fed radiolabeled xanthotoxin to quantify the detoxification of this furanocoumarin. They metabolized approximately 95% of the ingested xanthotoxin, indicating that metabolic detoxification is important in their tolerance to this allelochemical. Excretion of xanthotoxin and its metabolites was not restricted to the frass but also occurred by means of the silk glands. The silk glands contained half as much of the tritiated compounds as the rest of the body. Because of the feeding habits of this insect, such an excretory pathway may have implications for interactions with predators and pathogens.