Advances in the Biosynthesis of Pyranocoumarins: Isolation and 13C-Incorporation Analysis by High-Performance Liquid Chromatography-Ultraviolet-Solid-Phase Extraction-Nuclear Magnetic Resonance Data.

Citrus sinensis and Citrus limonia were obtained by germination from seeds, and isotopic-labeling experiments using d-[1-13C]glucose were performed with the seedlings. After 60 days, the seedlings were analyzed by high-performance liquid chromatography-ultraviolet-solid-phase extraction-nuclear magnetic resonance, data and the 13C enrichment patterns of xanthyletin and seselin indicated that the pyran ring was formed by the methylerythritol phosphate pathway and that the coumarin moiety was derived from the shikimate pathway in both compounds. This information regarding the biosynthetic pathway can be used to increase resistance against phytopathogens, because xanthyletin and seselin are reported to have antimicrobial activity on the growth of Xylella fastidiosa, which causes citrus variegated chlorosis in orange.

New approach for natural products screening by real-time monitoring of hemoglobin hydrolysis using quartz crystal microbalance.

The hemoglobin (Hb) released from erythrocytes is a primary nutritive component for many blood-feeding parasites. The aspartic protease cathepsin D is a hemoglobinase that is involved in the Hb degradation process and is considered an interesting target for chemotherapy intervention. However, traditional enzymatic assays for studying Hb degradation utilize spectrophotometric techniques, which do not allow real-time monitoring and can present serious interference problems. Herein, we describe a biosensor using simple approach for the real-time monitoring of Hb hydrolysis as well as an efficient screening method for natural products as enzymatic inhibitors using a quartz crystal microbalance (QCM) technique. Hemoglobin was anchored on the quartz crystal surface using mixed self-assembled monolayers. The addition of the enzyme caused a mass change (frequency shift) due to Hb hydrolysis, which was monitored in real time. From the frequency change patterns of the Hb-functionalized QCM, we evaluated the enzymatic reaction by determining the kinetic parameters of product formation (k(cat)). The QCM enzymatic assay using immobilized human Hb was shown to be an excellent approach for screening possible inhibitors in complex mixtures, opening up a new avenue for the discovery of novel inhibitors.

Synthesis of a combinatorial library of amides and its evaluation against the fall armyworm, Spodoptera frugiperda.

The fall armyworm Spodoptera frugiperda is a polyphagous pest that causes important damage in different regions of America and mainly affects corn crops in both tropical and subtropical areas. Currently, control relies on both transgenic plants and/or chemical pesticides. In this work we describe the preparation of an indexed combinatorial library of amides and its toxic effect by contact against S. frugiperda . (E)-1-(1-Piperidinyl)-3-[4-(trifluoromethoxy)phenyl]-2-propen-1-one was the most active compound with an LD(50) = 0.793 µg mg(-1) of larva. This amide was also evaluated by ingestion and at the lowest concentration (1 mg kg(-1)) achieved 83.3% mortality.

Anacardic acid derivatives as inhibitors of glyceraldehyde-3-phosphate dehydrogenase from Trypanosoma cruzi.

Chagas' disease, a parasitic infection caused by the flagellate protozoan Trypanosoma cruzi, is a major public health problem affecting millions of individuals in Latin America. On the basis of the essential role in the life cycle of T. cruzi, the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been considered an attractive target for the development of novel antitrypanosomatid agents. In the present work, we describe the inhibitory effects of a small library of natural and synthetic anacardic acid derivatives against the target enzyme. The most potent inhibitors, 6-n-pentadecyl- and 6-n-dodecylsalicilic acids, have IC(50) values of 28 and 55 microM, respectively. The inhibition was not reversed or prevented by the addition of Triton X-100, indicating that aggregate-based inhibition did not occur. In addition, detailed mechanistic characterization of the effects of these compounds on the T. cruzi GAPDH-catalyzed reaction showed clear noncompetitive inhibition with respect to both substrate and cofactor.

Insecticidal activity of synthetic amides on Spodoptera frugiperda.

The phytochemistry of the genus Piper (Piperaceae) has been widely studied due to the biological properties of amides from these plants. In this work, we have synthesized and evaluated the toxic effect of 11 amides against the fall armyworm Spodoptera frugiperda larvae. The naturally occurring piperine was also evaluated. The most active amide was N-[3-(3',4'-methylenedioxyphenyl)-2-(E)-propenoyl]piperidine with a LD50 of 1.07 microg mg(-1) larvae. This amide was also evaluated by ingestion.

Volatile oil from Guarea macrophylla ssp. tuberculata: seasonal variation and electroantennographic detection by Hypsipyla grandella.

GC and GC-MS analyses of the volatile oils from Guarea macrophylla (Meliaceae) collected during three different periods in one year (February, June and October) indicated a seasonal variation in chemical composition. Whilst sesquiterpenes were the predominant class of components present in the leaf oil, a seasonal dependent variation in the degree of oxygenation of these compounds was detected, which seemed to be associated with phenological factors. The leaf oil, and fractions thereof, were subjected to GC coupled with electroantennographic detection employing antennae of females of Hypsipyla grandella, an insect pest that attacks several meliaceous species. Five compounds elicited significant responses and these were identified as ledol, 1-cubenol, guai-6-en-10beta-ol, 1-epi-cubenol, and tau-muurolol. The results suggest that these components could be responsible for the attraction of H. grandella to G. macrophylla.

Electrophysiological responses of female and male Hypsipyla grandella (Zeller) to Swietenia macrophylla essential oils.

The mahogany shoot borer, Hypsipyla grandella, is one of the most economically important Neotropical forest pest insects because it attacks valuable timber of the Meliaceae. Despite detailed information on the life cycle of H. grandella, factors influencing attack by the shoot borer are not well known. Electroantennogram studies of borer antennae indicate that both female and male moths elicited significant antennal responses to essential oil of the terminal shoots, mature and senescent leaves, in three concentrations. Gamma-himachalene, germacrene D, germacrene A, cadina-1,4-diene, hexadecanoic acid, and ethyl hexadecanoate appeared in all essential oils from Swietenia macrophylla. Germacrene-D and gamma-himachalene were also present in the active essential oil of Cedrela odorata and Toona ciliata. These findings suggest that these compounds play a role in attracting H. grandella to oviposit on the leaves of these plants. The GC-EAD of essential oil from S. macrophylla mature leaves to female H. grandella antennae led to the identification of beta-caryophyllene as the main constituent responsible for the antennae response.

Biological activity of astilbin from Dimorphandra mollis against Anticarsia gemmatalis and Spodoptera frugiperda.

Astilbin was isolated in high yield from Dimorphandra mollis, and its insecticidal and growth inhibiting activity by stomach ingestion were evaluated against Anticarsia gemmatalis and Spodoptera frugiperda. The insecticidal activity of astilbin, the weight reduction of the larval phase and the prolongation of the larval and pupal phases were verified for both species. Astilbin was identified on the base of its NMR, MS and physical data.

Triterpenoid constituents of Raulinoa echinata.

A phytochemical investigation of the stems of the South Brazilian endemic species Raulinoa echinata has led to the isolation of two new methoxylated protolimonoid epimers (1 and 2) together with the known melianone and melianodiol. The leaves afforded three glabretal-type triterpene derivatives esterified by N-methylanthranilic acid (3-5). Compounds 1 and 2 displayed weak inhibitory activity when assayed in vitro against trypomastigote forms of Trypanosoma cruzi. Compounds 1-5 were inactive in a brine shrimp (Artemia salina) lethality test.