Cytotoxic and Anti-Inflammatory Triterpenoids in the Vines and Leaves of Momordica charantia

The vines and leaves of Momordica charantia L. are used as herbal medicines to treat inflammation-related disorders. However, their safety profile remains uncharacterized, and the constituents in their extracts that exert anti-inflammatory and adverse effects remain unclear. This study isolated the characteristic cucurbitane-type triterpenoid species in the vines and leaves of M. charantia L. and analyzed their cytotoxicity, anti-inflammatory effects, and underlying mechanisms. Four structurally related triterpenoids—momordicines I, II, IV, and (23E) 3β,7β,25-trihydroxycucurbita-5,23-dien-19-al (TCD)—were isolated from the triterpenoid-rich fractions of extracts from the vines and leaves of M. charantia. Momordicine I was cytotoxic on normal cells, momordicine II exerted milder cytotoxicity, and momordicine IV and TCD had no obvious adverse effects on cell growth. TCD had anti-inflammatory activity both in vivo and in vitro. In lipopolysaccharide-stimulated RAW 264.7 cells, TCD inhibited the inhibitor kappa B kinase/nuclear factor-κB pathway and enhanced the expression of nuclear factor erythroid 2-related factor 2, heme oxygenase-1, and glutamate-cysteine ligase modifier subunit through the extracellular signal-regulated kinase1/2 and p38. Thus, the vines and leaves of M. charantia should be used with caution. An extraction protocol that can enrich TCD but remove momordicine I would likely enhance the safety of the extract.     

Identification of triterpenoid feeding deterrent of red pumpkin beetles (Aulacophora foveicollis) fromMomordica charantia

The triterpenoids isolated from the leaves ofMomordica charantia Linn (bitter gourd) were found to elicit feeding-deterrent activity against red pumpkin beetles (Aulacophora foveicollis Lucas). The most abundant triterpenoid which deterred feeding was identified as momordicine II, 23-O--glucopyranoside of 3,7,23-trihydroxycucurbita-5,24-dien-19-al. A concentration of 3200g/ml and above of the triterpenoids caused significant reduction of feeding by red pumpkin beetles in in vitro bioassay experiments, which compared favorably with the levels of triterpenoids inM. charantia leaves found in nature.     

A new method for fast isolation of insect antifeedant compounds from complex mixtures

Bioassay-guided isolation of bioactive natural substances requires monitoring of all fractionation and purification steps using a bioassay system. This is often a long and tedious process, especially with insect feeding bioassays. We report here a new method, based on the principle of bioautography, for a quick isolation of insect antifeedant compounds. TLC plates, after development, are coated with a thin layer of artificial diet and fed toSpodoptera litura larvae. The location of uneaten areas is then compared with theR _fvalues of the TLC spots, in order to determine rapidly the active fractions. This methods allows for a very fast determination of the most active antifeedant compounds in a complex mixture and considerably speeds up the isolation process. This new method was successfully applied in the study of antifeedant activity of several plant samples, and results are presented here for a model plant,Skimmia japonica (Rutaceae). Using this new method, the compounds responsible for the feeding-deterrent activity, three furanocoumarins (bergapten, xanthotoxin, and oxypeucedanin), were quickly and efficiently identified.     

Patterns of Bioactivity and Herbivory on Nothofagus Species from Chile and New Zealand

Nothofagus species from Chile and New Zealand were surveyed in the field for invertebrate abundance and leaf feeding damage and in the laboratory for antifeedant activity against leafrollers (Ctenopsteustis obliquana, Epiphyas postvittana), deterrent activity against pea aphid (Acyrthosiphon pisum), insect growth regulatory activity (Oncopeltus fasciatus), nematicidal activity (Caenorhabditis elegans), antibiotic activity (Pseudomonas solanaciarium), and general toxicity. A data matrix indicated that N. alessandri and N. pumilio most likely have a chemical barrier to insect attack as leaves showed low faunal abundance, low herbivory, and activity in the leafroller antifeedant, aphid deterrent, and nematicidal assays. A chemical examination of N. alessandri that used the leafroller antifeedant test to guide the separation yielded an active fraction containing the flavonoid, galangin, and the stilbene, pinosylvin, which appear to act in concert to deter leafroller feeding. The discovery of the phytoalexin, pinosylvin, in the leaves, raises the possibility that Nothofagus in general, and N. alessandri in particular, may have induced chemical defense mechanisms.     

Responses of maxillary sensilla styloconica inBombyx mori to glucosides fromOsmunda japonica,a nonhost plant

We isolated glucosides from the royal fern,Osmunda japonica, which elicit a deterrent response in larvae ofBombyx mori. These compounds were absent in taro (Colocasia antiquorum) and castor-oil plant (Ricinus communis) leaves and did not evoke responses of sensory cells in the lateral and medial sensilla styloconica ofSpodoptera litura. This glucoside extract of the royal fern leaves stimulates receptors generally associated with deterrent. It is also possible that this compound may act as a behavioral deterrent, and from actual feeding tests, it is suggested that this compound may prevent feeding in some monophagous insects, such asBombyx mori. The deterrent glucoside possesses a noncyclic aglycon.     

Developmental stage of herbivorePseudaletia separata affects production of herbivore-induced synomone by corn plants

The female parasitic waspCotesia kariyai discriminated between the volatiles of corn leaves infested by younger host larvaePseudaletia separata (first to fourth instar) and uninfested leaves in a Y-tube olfactometer; the wasps were attracted to the infested leaves. In contrast, when corn plants were infested by the later stages (fifth and sixth instar) of the armyworm, the wasps did not distinguish between infested corn leaves and uninfested corn leaves in the olfactometer. Mechanically damaged leaves were no more attractive than undamaged leaves, and host larvae or their feces were not attractive to the parasitoid. Through chemical analysis, the herbivore-induced plant volatiles were identified in the headspace of infested corn leaves. The herbivore-induced volatiles (HIVs) constituted a larger proportion of the headspace of corn leaves infested by early instar armyworms than of corn leaves infested by late instar armyworms. Application of third-instar larval regurgitant onto artificially damaged sites of leaves resulted in emission of parasitoid attractants from the leaf, whereas leaves treated with sixth-instar regurgitant did not. The function of this herbivore-stage related specificity of herbivore-induced synomones is discussed in a tritrophic context.     

Insect feeding and oviposition deterrents from western red cedar foliage

The feeding deterrent activity of fractions from the foliage of western red cedar,Thujaplicata Donn, was studied in laboratory bioassays using the white pine weevil,Pissodes strobi Peck, as a test insect. The most active fraction was the volatile mixture that comprises the leaf oil of this tree species. Further fractionation of the leaf oil indicated feeding deterrent activity in the monoterpene hydrocarbon, thujone, and terpene alcohol fractions. When tested alone, both (–)-3-isothujone and (+)-3-thujone, which made up 75–88% and 5–10% of the leaf oil, respectively, deterred feeding by the weevils. Western red cedar leaf oil also showed antifeedant activity with the alder flea beetle,Altica ambiens (Le Conte), and served as an oviposition deterrent for the onion root maggot,Hylemya antiqua Meigen. The leaf oil, however, had no inhibitory effect on the feeding of the leaf roller,Epinotia solandriana L., and the red-backed sawfly,Eriocampa ovata L.Research supported by the Natural Science and Engineering Research Council, Canada, Co-op Grant No. A0243 and Operating Grant Nos. A3881 and A3706, and by the British Columbia Ministry of Labour Youth Employment Program.     

Effect of Trichilia americana Extract on Feeding Behavior of Asian Armyworm, Spodoptera litura

We investigated the desensitization of Asian armyworms, Spodoptera litura, to a crude methanolic extract of wood Trichilia americana. Following repeated exposures to the extract over four days, desensitization was observed under no-choice conditions at low extract concentration (0.5 g/cm²). No desensitization was observed under choice conditions or at a higher concentration (5.0 g/cm²). Repeated exposures of larvae to the extract over 7.5 hr showed that larvae increasingly tolerated the extract due to hunger, rather than showing true desensitization. When larvae were exposed to the extract in test arenas of different sizes, larvae showed reduced feeding and desensitization as arena size increased. Finally, treatment of whole cabbage plants with extract resulted in larvae abandoning the plant. The crude extract was effective at protecting the plants, although desensitization did not occur, because the larvae moved away from the plant.     

Some chemical bases for gypsy moth,Lymantria dispar,larval rejection of green ash,Fraxinus pennsylvanica,foliage as food

Green ash is one of the few tree species rejected as food by larvae of the generalist gypsy moth,Lymantria dispar L. (Lepidoptera: Lymantriidae). Such rejection is based especially on chemicals present in green ash foliage. The gypsy moth larval feeding-inhibitory activity is contained in the ethyl acetate extractables of green ash foliage. Three representative columnchromatographed fractions of the extractables contained antifeedant activity. Individual fractions showed weaker antifeedant activity compared to the total ethyl acetate extractables. Acid hydrolysis of the extractables destroyed antifeedant activity and yielded feeding stimulant activity at higher concentrations. The aqueous extractables were not feeding-inhibitory. Compounds in the green ash extractables were separated by TLC, HPLC, CC, and GC.     

Antifeedant and toxic effects of sesquiterpenes fromSenecio palmensis to colorado potato beetle

A bioassay-guided fractionation of the aerial parts ofSenecio palmensis resulted in the isolation of two sesquiterpenes, 2,10-bisaboladien-1-one and 11-acetoxy-5-angeloyloxy-silphinen-3-one. The bisabolene and the silphinene represented 0.012% and 0.024% of the plant dry weight, respectively. Both compounds showed antifeedant activity againstLeptinotarsa decemlineata larvae and adults in short-term choice and no-choice bioassays. Both compounds were also tested against different species of phytopathogenic fungi. The beetles were more sensitive to these compounds in choice than in no-choice assays, with a gradient of increasing sensitivity from second instars to adults. Bisabolene was 45 times less active as an antifeedant than juglone, which was tested as a positive control. The silphinen was more active than the bisabolene, with a range of activity similar to juglone. Furthermore, exposure of fourth instars to these compounds over a 24-hr period resulted in reduced feeding and growth rates. To distinguish between antifeedant and toxic effects, growth efficiencies were calculated as the slope of the regression of relative growth rate on relative consumption rate. The comparison of these results with those of antifeedant simulation and contact toxicity bioassays indicates that feeding inhibition is the primary mode of action of the bisabolene, while the silphinene shows both antifeedant and toxic effects.     

Laboratory Evaluation of <Emphasis Type="Italic">Artemisia annua</Emphasis> L. Extract and Artemisinin Activity against <Emphasis Type="Italic">Epilachna paenulata</Emphasis> and <Emphasis Type="Italic">Spodoptera eridania</Emphasis>

Ethanolic extract of aerial parts of Artemisia annua L. and artemisinin were evaluated as anti-insect products. In a feeding deterrence assay on Epilachna paenulata Germ (Coleoptera: Coccinellidae) larvae, complete feeding rejection was observed at an extract concentration of 1.5 mg/cm² on pumpkin leaf tissue. The same concentration produced a feeding inhibition of 87% in Spodoptera eridania (Cramer) (Lepidoptera: Noctuidae). In a no-choice assay, both species ate less and gained less weight when fed on leaves treated with the extract. Complete mortality in E. paenulata and 50% mortality in S. eridania were observed with extract at 1.5 mg/cm². Artemisinin exhibited a moderate antifeedant effect on E. paenulata and S. eridania at 0.03–0.375 mg/cm². However, a strong effect on survival and body weight was observed when E. paenulata larvae were forced to feed on leaves treated at 0.03 and 0.075 mg/cm². Artemisia annua ethanolic extract of aerial parts at 1.5 mg/cm² showed no phytotoxic effect on pumpkin seedlings.     

Chemical inhibition of fire-prone grasses by fire-sensitive shrub,Conradina canescens

In an investigation of potential chemical activity of fire-sensitive shrubs in Florida's sand pine scrub community, bioassays of foliar washes ofConradina canescens showed significant inhibitory activity on three native grasses that are known to fuel frequent surface fires; inhibition was concentrated seasonally in spring and summer. Application of runoff fromConradina leaves to one of the grasses caused a 50% reduction in growth over a 20-week period. Isolation of the biologically active fractions from the fresh leaves ofC. canescens yielded numerous monoterpenes, a number of which were identified from a GC-MS reference library and/or MS comparison to authentic compounds: 11 from the diethyl ether extract, 11 from steam distillation, and four from the foliar leaf wash. Numerous other monoterpenes present in the extractions were unknown. The terpenoid fraction completely inhibited seed germination of one of the native grasses and of lettuce. Saturated aqueous solutions of nine of the monoterpenes inhibited germination and radicle growth of two native grasses. SEM views of the leaf surfaces ofConradina reveal secretory trichomes that appear to be the source of the monoterpenes as well as the triterpene, ursolic acid. The biological activity ofC. canescens as a fire-sensitive component of the scrub community is reviewed in light of the chemical evidence.     

The Iridoid Glucoside,Antirrhinoside, from <Emphasis Type="Italic">Antirrhinum majus</Emphasis> L. has Differential Effects on Two Generalist Insect Herbivores

The iridoid glucoside, antirrhinoside, is constitutively distributed throughout Antirrhinum majus L. in a manner consistent with its possible role as an allelochemical, but there is no evidence that it has a defensive function with respect to insect herbivory. To address this question, two generalist herbivores, Lymantria dispar L. (gypsy moth) and Trichoplusia ni Hübner (cabbage looper) were chosen for feeding trials on excised whole leaves of A. majus and in artificial diet assays. In leaf excision feeding trials, fourth instar gypsy moth rejected, without sampling, the leaves of A. majus regardless of what node the leaf was excised from. In contrast, fourth instar cabbage looper readily fed on the excised leaves, and antirrhinoside was not found in their bodies or feces (frass) as determined by thin layer and high-pressure liquid chromatography. In the leaf and diet assays, a second major leaf iridoid in A. majus, antirrhide, was found in both cabbage looper and gypsy moth frass. In diet feeding assays, the growth of gypsy moth and cabbage looper were not inhibited by methanol extracts, iridoid fractions, or pure antirrhinoside at concentrations of 0.6% in diet, but cabbage looper growth was enhanced. At an antirrhinoside concentration of 3.3% in diet, gypsy moth growth was reduced, whereas cabbage looper growth again increased significantly relative to the control. It is likely that antirrhinoside functions as defense against herbivory for one generalist insect herbivore but also, at low concentrations, enhances the growth of another.     

Ecdysteroids fromPycnogonum litorale (Arthropoda,Pantopoda) act as chemical defense againstCarcinus maenas (Crustacea,Decapoda)

Pycnogonum litorale (Ström) is unpalatable to the common shore crabCarcinus maenas, a generalist predator in the pycnogonid's habitat. A feeding bioassay reveals that the crabs are deterred by ecdysteroids that occur in high levels in all developmental stages ofP. litorale. The total ecdysteroids in the pycnogonids reach 5.9×10^–4 M. The 20-hydroxyecdysone 22-acetate (20E22A), which is the predominant ecdysteroid in the pycnogonids, and 20-hydroxyecdysone (20E), the arthropod molting hormone, were tested for their antifeedant effect onC. maenas. When contained in food pellets in homogeneous concentrations, 20E and 20E22A significantly reduced food consumption at 1.25×10^–4 and 5.0×10^–4 molar levels, respectively. The present results demonstrate for the first time chemical defense in arthropods in a marine predator-prey relationship. Furthermore, they provide evidence that ES contained in one animal can act as feeding deterrents on another animal.     

Antifeedants against <Emphasis Type="Italic">Hylobius abietis</Emphasis> Pine Weevils: An Active Compound In Extract of Bark of <Emphasis Type="Italic">Tilia cordata</Emphasis> Linden

Linden (Tilia cordata) bark was shown to contain an antifeedant effective against the large pine weevil, Hylobius abietis. Soxhlet extraction of inner and outer bark resulted in an extract that showed antifeedant activity in a microfeeding assay. The extract was fractionated by chromatography on silica gel using gradient elution with solvents of increasing polarity. The content of the fractions obtained was monitored by thin layer- and gas chromatography. Fractions of similar chemical composition were merged. Two of the 17 fractions showed antifeedant activity in the microfeeding assay. Nonanoic acid was identified in both of these fractions. Subsequent testing in the microfeeding assay showed that nonanoic acid possessed strong antifeedant activity against H. abietis adults.     

Chemical constituents of an unacceptable crucifer,Erysimum cheiranthoides,deter feeding byPieris rapae

The wild cruciferErysimum cheiranthoides was found to contain extractable constituents that deterred feeding by larvae of the crucifer specialistPieris rapae when applied to cabbage leaf disks in both choice and nochoice bioassays. High-performance liquid chromatography was used to separate the extract into several fractions, two of which retained the feeding deterrent activity of the extract. UV-absorption spectra of the fractions suggested that one contained cardenolides similar or identical to those reported to deter oviposition byP. rapae onE. cheiranthoides. The other active fraction evidently contains a compound that deters larval feeding but not adult oviposition. The results suggest that the chemical defense ofE. cheiranthoides depends on two types of compounds acting on separate developmental stages of the insect.     

4-Demethylsterols and triterpene alcohols from two Vanilla bean species: Vanilla fragrans and V. tahitensis

4-Demethylsterol and triterpene alcohol compositions of two Vanilla bean species (V. fragrans and V. tahitensis) were investigated. From retention times and gas chromatography-mass spectrometry, nine 4-demethylsterols were identified in V. fragrans and seven in V. tahitensis. The 4-demethylsterol fraction of V. fragrans was characterized by a high content of 24-methylene cholesterol (27–40%) and of β-sitosterol (35–46%). The 4-demethylsterol fraction of V. tahitensis was characterized by a high content of stigmasterol (27%) and of β-sitosterol (57.5%), and a lower amount of 24-methylene cholesterol (5%). Vanilla tahitensis was also characterized by the presence of ergosta-5,25-dien-3β-ol (2%) and the absence of campesterol, stigmasta-5,22,25-trien-3β-ol, and ergosta-7,24(28)-dien-3β-ol. The beans’ age modified the ratio 24-methylene cholesterol/β-sitosterol in V. fragrans. Combining liquid chromatography and gas chromatography allowed the identification of four other demethylsterols in V. fragrans (brassicasterol, 0.02%; stigmasta-5,23-dien-3β-ol, 1.43%; stigmasten-22-ol, 0.1%; and fucosterol, 0.5%) from the 4-demethylsterol fraction. 24-Methylene cholesterol and β-sitosterol were isolated, and their structures were confirmed by ¹H and ¹³C nuclear magnetic resonance. Four triterpene alcohols were identified in V. fragrans, including cycloartenol (0.9–1.6%) from the triterpene alcohol fraction, 24-dihydrotirucallol (17–23%) from the triterpene alcohol fraction, tirucall-7-en-3β-ol (6–7.5%) from the triterpene alcohol fraction, and in a higher content cyclosadol (66–69%) from the triterpene alcohol fraction. The content ranges were studied as a function of the beans’ age. Demethylsterol and triterpene alcohols profile could be used for origin differentiation.     

Developmental profile of sinalbin (p-hydroxybenzyl glucosinolate) in mustard seedlings,Sinapis alba L., and its relationship to insect resistance

Sinalbin was identified as a chemical component of insect anti-xenosis and antibiosis resistance mechanisms in seedlings ofSinapis alba by DEAE-Sephadex chromatography, HPLC, treatment with sulfatase and myrosinase, various feeding tests using artificial and natural substrates, and by measuring sinalbin concentrations in cotyledons and leaves during seedling development. The effects of sinaibin on feeding were dependent upon the insect species and upon the rapidly changing profile of sinaibin concentrations in the developing seedling. The high concentrations of sinalbin found in young cotyledons (up to 20 mM) and leaves (up to 10 mM) deterred the feeding of the flea beetle,Phyllotreta cruciferae Goeze and larvae of the bertha armyworm,Mamestra configurata Walker. The protection that sinalbin confers upon the vulnerable, newly emerged seedling (and upon tiny, young leaves) appears critical for the first few days of survival ofS. alba under feeding pressure from flea beetles in the field. The lower concentrations of sinaibin found in older cotyledons and leaves (2–3 mM) offer little or no protection againstP. cruciferae and may actually stimulate the feeding of this crucifer specialist. These concentrations of sinaibin, however, are still effective in reducing the level of feeding by larvae of the more generalist feederM. configurata.     

Antifeedant activity of extracts from neem,Azadirachta indica,to strawberry aphid,Chaetosiphon fragaefolii

Leaf disk choice test bioassays demonstrated that formulated neem seed oil (NSO) was equally deterrent to first- and third-instar nymphs and adult strawberry aphids,Chaetosiphon fragaefolii (Cockerell). Concentrations of NSO resulting in 50% feeding deterrence were approximately 1.1% for this species. The rapid disruption of aphid feeding (<1 hr) was not related to the presence of the limonoid azadirachtin, and deterrence likely results from the combined activity of several compounds. Activity toC. fragaefolii disappeared within 12–24 hr following application to strawberry in the greenhouse. NSO was deterrent to only half of the six aphid species tested. The antifeedant properties of neem do not appear to contribute significantly to the control of aphids and the viruses they transmit.     

Allelochemical resistance of bald cypress,Taxodium distichum,heartwood to the subterranean termite,Coptotermes formosanus

The heartwood of bald cypress,Taxodium distichum (L.) Rich., resisted feeding attack by the Formosan subterranean termite,Coptotermes formosanus Shiraki. Hexane-extracted heartwood, however, was consumed at > 12 times the amount of sound heartwood eaten. A bioassay usingT. distichum sapwood as a feeding substrate was employed to assess the antitermitic activity of successive hexane, acetone, and methanol extracts of heartwood shavings and isolates derived from the active hexane extract. Two fractions, eluted from the crude hexane extract by liquid chromatography, significantly reduced termite feeding compared to the parent extract, while a third fraction was less active than the original hexane extract. Each fraction contained one major component. All three components were structurally related diterpenes. The two most active heartwood constituents were identified by GC-MS and NMR as ferruginol and manool, while the third and least active, but most prevalent, compound in heartwood was identified as nezukol. Results of bioassays suggest that these allelochemicals act principally as feeding deterrents with accompanying termite mortality due to starvation.