Green manure (Crotalaria juncea L.) enhances Origanum vulgare L. biomass accumulation, essential oil yield, and phytochemical properties.

Green manure (GM) may reduce the use of chemical fertilizers, been an ecologically appropriate strategy to cultivation of medicinal plants. Crotalaria juncea, is one of the most used because it adapts to different climatic and high nitrogen content. Origanum vulgare. is widely used in cooking, pharmaceutical, cosmetic industries and food products. The objectives of this study were to evaluate the GM on biomass, essential oil (EO), phenolic and antioxidant. The experiment consisted: control; 150, 300, 450, and 600 g (Sh= leaves+steam) more 200 g roots (R); 600 g aerial part; 200 g roots; and soil with 300 g cattle manure per pot. The highest dry weights were observed in the presence of GM and cattle manure (90 days). The control had an EO production 75% lower in relation to the dose of 450 g GM (Sh+R). Principal component analysis showed that GM and cattle manure positively influenced the dry weight, content, yield, and EO constituents, and total flavonoids. The GM contributed to the accumulation of the major EO compounds (trans-sabinene hydrate, thymol, terpinen-4-ol). The GM management may be beneficial for cultivating, because it can increase the production of biomass and the active components, in addition to being an inexpensive resource.

An insight into seed priming response of Crotalaria ochroleuca and Crotalaria spectabilis during storage.

The proper establishment of plants is essential for the efficient use of resources such as water and light. Besides, even after seed storage and sowing the uniform establishment of plants is essential for their success. Crotalaria ochroleuca and Crotalaria spectabilis are important medicinal plants with poor seed germination rate, occasionally. The effects of seed priming in both C. ochroleuca and C. spectabilis were evaluated in seed performance even after seeds storage for up 90-days. Experimental assays were performed in a randomized design with gibberellic acid (GA3, 100 ppm), polyethylene glycol (PEG 6000, -0.2 MPa) and PEG (-0.2 MPa) + GA3 (100 ppm) solutions during seed priming in four replicates. Seeds not submitted to priming treatments constituted control. Seeds physiological performance were evaluated immediately and even after 30, 60 and 90-days seed dry-storage. The data obtained in each experiment were submitted to variance analysis (ANOVA) adopting a confidence level of 95%. The effects of seed priming with PEG and GA3 during seed ageing were significant for germination variables of C. ochroleuca and C. spectabilis. During dry storage, seed viability of both species gradually decreased and the first symptoms were delayed seed germination, especially more evident for C. ochroleuca, even in primed or non-primed seeds. Afterwards, C. ochroleuca seeds previously GA3 primed had higher results of root protrusion (86%), hypocotyls elongation (76%) and complete seedlings (75%) than non-primed seeds (control). These findings shown a good potential of hormopriming to attenuate damage during the seed aging of C. ochroleuca.

Purification and characterization of a new trypsin-like protease from Crotalaria stipularia.

Proteases are the main enzymes traded worldwide-comprising 60% of the total enzyme market-and are fundamental to the degradation and processing of proteins and peptides. Due to their high commercial demand and biological importance, there is a search for alternative sources of these enzymes. Crotalaria stipularia is highlighted for its agroecological applications, including organic fertilizers, nematode combat, and revegetation of areas contaminated with toxic substances. Considering the pronounced biotechnological functionality of the studied species and the necessity to discover alternative sources of proteases, we investigated the extraction, purification, and characterization of a protease from seeds of the C. stipularia plant. Protease isolation was achieved by three-phase partitioning and single-step molecular exclusion chromatography in Sephacryl S-100, with a final recovery of 47% of tryptic activity. The molecular mass of the isolated enzyme was 40 kDa, demonstrating optimal activities at pH 8.0 and 50 °C. Enzymatic characterization demonstrated that the protease can hydrolyze the specific trypsin substrate, BApNA. This trypsin-like protease had a Km, Vmax, Kcat, and catalytic efficiency constant of 0.01775 mg/mL, 0.1082 mM/min, 3.86 s-1, and 217.46, respectively.

Monocrotaline presence in the Crotalaria (Fabaceae) plant genus and its influence on arthropods in agroecosystems / Presença de monocrotalina em plantas do gênero Crotalaria (Fabaceae) e a sua influência sobre artrópodes nos agroecossistemas

Abstract Crotalaria (Fabaceae) occurs abundantly in tropical and subtropical regions and has about 600 known species. These plants are widely used in agriculture, mainly as cover plants and green manures, in addition to their use in the management of phytonematodes. A striking feature of these species is the production of pyrrolizidine alkaloids (PAs), secondary allelochemicals involved in plant defense against herbivores. In Crotalaria species, monocrotaline is the predominant PA, which has many biological activities reported, including cytotoxicity, tumorigenicity, hepatotoxicity and neurotoxicity, with a wide range of ecological interactions. Thus, studies have sought to elucidate the effects of this compound to promote an increase in flora and fauna (mainly insects and nematodes) associated with agroecosystems, favoring the natural biological control. This review summarizes information about the monocrotaline, showing such effects in these environments, both above and below ground, and their potential use in pest management programs.

Resumo Crotalaria (Linnaeus, 1753) (Fabaceae) ocorre abundantemente em regiões tropicais e subtropicais e tem cerca de 600 espécies conhecidas. Estas plantas são amplamente utilizadas na agricultura, principalmente como cobertura e adubos verdes, além da sua utilização no manejo de fitonematoides. Uma característica marcante destas espécies é a produção de alcalóides pirrolizidinicos (APs), aleloquímicos secundários envolvidos na defesa das plantas contra os herbívoros. Nas espécies de Crotalaria, a monocrotalina é a AP predominante, que tem muitas atividades biológicas relatadas, incluindo citotoxicidade, tumorigenicidade, hepatotoxicidade e neurotoxicidade, além de uma vasta gama de interações ecológicas. Assim, estudos têm procurado elucidar os efeitos desse composto para promover um incremento na flora e fauna (principalmente insetos e nematoides) associados aos agroecossistemas, favorecendo o controle biológico natural. Esta revisão compila informações sobre a monocrotalina, mostrando tais efeitos nesses ambientes, tanto acima como abaixo do solo e a sua potencial utilização em programas de manejo de pragas.

Selectivity of herbicides used in corn on Crotalaria ochroleuca G. Don.

In the cropping systems that integrate the corn crop, the insertion of Crotalaria ochroleuca G. Don is predominantly intercropped. In this context, there is a need to observe herbicides that present selectivity for this sunn hemp species. The objective of this study was to evaluate the selectivity of pre and post-emergent herbicides on C. ochroleuca. Two field experiments were conducted in randomized blocks with four replications, involving the pre-emergence and post-emergence application of different herbicide treatments. For the pre-emergent ones, amicarbazone, atrazine and flumioxazin provided phytotoxicity higher than 90% and, consequently, low plant biomass. On the other hand, acetochlor and s-metolachlor did not cause phytotoxicity and did not affect the dry mass of crotalaria. In post-emergence, atrazine + mesotrione showed phytotoxicity >95%, followed by nicosulfuron and 2.4-D with phytotoxicity between 50-60%, whereas tembotrione did not cause injury to the plants. Thus, it was found that among the pre-emergent, acetochlor and s-metolachlor were selective, and for the emerging powders, only tembotrione was the most selective for all parameters analyzed.

Antileishmanial effects of Crotalaria spectabilis Roth aqueous extracts on Leishmania amazonensis.

Fifteen polar extracts from leaf, seed, pod, stem, flower and root of Crotalaria spectabilis were prepared using aqueous systems, based on the principles of green chemistry, and showed different protease inhibitor (PI) activities on trypsin, papain, pepsin and the extracellular L. amazonensis serine protease (LSPIII). The most pronounced inhibitory effect on LSPIII was observed in leaf (CS-P), root, stem, flower (CS-FPVPP) and pod (CS-VA) extracts. Crotalaria extracts exhibited low cytotoxicity on macrophages; however, they decreased the viability of L. amazonensis promastigotes and amastigotes, as observed in leaf (CS-AE, CS-P, CS-T and CS-PVPP), seed (CS-ST), flower and root (CS-RA) extracts. CS-P was chosen to study PI and secondary metabolites and a 10-12 kDa protein, analyzed by mass spectrometry, was identified as a serine PI homologous with papaya latex serine PI. Glycosylated flavonoids, such as quercetins, vitexin and tricin were the major secondary metabolites of CS-P. The presence of PIs in C. spectabilis is a new finding, especially in other organs than seeds since PIs have been reported only in seed legumes. Besides, this is the first report of antileishmanial activity of C. spectabilis extracts and the identification of serine polypeptide PI and glycosylated flavonoids from leaf.

Different nanocellulose morphologies (cellulose nanofibers, nanocrystals and nanospheres) extracted from Sunn hemp (Crotalaria Juncea).

Nanocellulose of different morphologies was extracted from Sunn Hemp (Crotalaria Juncea) using acid hydrolysis. The work focused on two objectives: first, to valorize the Sunn Hemp fibers for nanocellulose (NC) production, and second, to study the effects of acid concentration on different morphologies of NC and their properties. The study extracted nanocellulose at five different concentrations of H2SO4: 16 %, 32 %, 48 %, 64 %, and 72 %. Obtained nanocellulose was characterized by Scanning Electron Microscopy (FE-SEM), Atomic Force Microscopy (AFM), Fourier Transform Infrared Spectroscopy (FTIR), X-ray Diffraction (XRD) and Thermogravimetric Analysis (TGA). AFM and FE-SEM confirmed the production of three different morphologies of nanocellulose. The NC-32 had a web-like structure typically observed for cellulose nanofibrils (CNF), whereas NC-48 and NC-64 were observed as cellulose nanocrystals (CNC) with rod-like and needle-like shapes, respectively, and NC-72 displayed spherical particles termed cellulose nanospheres (CNS). The total crystallinity index of NC was calculated using FTIR, and a similar trend of crystallinity was also observed from XRD analysis. NC-32 was obtained with the highest yield of 94.83 %, followed by 91.40 % and 81.70 % for NC-48 and NC-64, respectively, whereas NC-72 yielded the lowest yield of 12.03 %. NC-72 had the highest thermal stability among other NC morphologies.

BBD optimized antioxidants of Crotalaria candicans and its nanoconjugates, exert potent in vivo anti-biofilm effects against MRSA.

Crotalaria genus is extensively dispersed in tropical and subtropical provinces, and it is found to harbor antioxidant flavonoids. Response surface methodology-based optimization was carried out for the purpose of efficient extraction involving a suitable solvent which can maximize the yield along with higher total phenolic content and total flavonoid content (TFC). Optimization conditions for extraction of C.candicans flavonoids (CCF) based on variables such as solvent, solid-solvent ratio and extraction temperature were evaluated. The optimized conditions were found as Solvent i.e., Aqueous-ethanol (53.42%), Solid-solvent ratio (1:15.83 w/v) and temperature (44.42 °C) and resulted to obtain the TFC as 176.23 mg QRET/g C. candicans extract with the yield 27.42 mg CCF/g (C. candicans dry weight). LC-MS analysis of CCF, revealed the presence of seven major flavonoids. The antioxidant flavonoids were further used to functionalize the zero-valent silver (ZVAgF) and copper (ZVCuF) nanoparticles. The ZVAgF and ZVCuF were investigated using UV-Vis spectrophotometry, FT-IR spectroscopy and X-ray diffractometry to confirm the presence of the zero valent metals and possible functional groups which capped the elemental metal. Further transmission electron microscopy, dynamic light scattering method and zeta-potential studies were done to understand their respective structural and morphological properties. The efficacy of the as-prepared ZVAgF/ZVCuF as antibiofilm agents on Methicillin-resistant Staphylococcus aureus (MRSA) with the mechanism studies have been explored. The MRSA-colony count from the infection zebrafish (in vivo) model, portrayed a reduction of > 1.9 fold for ZVCuF and > twofold for ZVAgF, with no alteration in liver morphology when treated with ZVAgF, implying that the nanoparticles were safe and biocompatible.

Crotalaria juncea L. enhances the bioremediation of sulfentrazone-contaminated soil and promotes changes in the soil bacterial community.

Sulfentrazone (STZ) is an efficient tool for the pre- and post-emergence control of monocotyledonous and dicotyledonous weeds in fields of crops such as pineapple, coffee, sugarcane, citrus, eucalyptus, tobacco, and soybean. However, this herbicide persists in the soil, causing phytotoxicity in the subsequent crop. Therefore, it is important to use efficient strategies for the remediation of STZ-contaminated areas. The aim of this study was to evaluate the effects of Crotalaria juncea L. on the remediation of STZ-contaminated soil and on the microbial activity and bacterial community structure therein. The study was conducted in three stages: (i) cultivation of C. juncea in soil contaminated with 200, 400, and 800 g ha-1 STZ; (ii) determination of the soil microbial activity (basal respiration, microbial biomass carbon, and bacterial community structure); and (iii) cultivation of a bioindicator species and determination of the residual fraction of STZ. The soil microbial activity was impacted by the soil type and STZ dose. Soil previously cultivated with C. juncea (rhizospheric soil) displayed higher CO2 and lower qCO2 values than non-rhizospheric soil (no previous C. juncea cultivation). Increasing doses of STZ reduced the activity and lowered the diversity indices of the soil microorganisms. The bacterial community structure was segregated between the rhizospheric and non-rhizospheric soils. Regardless of soil type, the bioindicator of remediation (Pennisetum glaucum R.Br.) grew only at the STZ dose of 200 g ha-1, and the plant intoxication level was also lower in rhizospheric soil treated with this herbicide dose. All P. glaucum plants died in the soils treated with 400 and 800 g ha-1 STZ. Previous cultivation of C. juncea in soils contaminated with 200, 400, and 800 g ha-1 STZ reduced the residual fraction of the herbicide by 4.8%, 12.5%, and 17.4%, respectively, compared with that in the non-rhizospheric soils. In conclusion, previous cultivation with C. juncea promoted increases in the soil bacterial activity and diversity indices, mitigated the deleterious effects of STZ on the bioindicator crop, and reduced the residual fraction of the herbicide in the soil.

Molecular and Biological Characterization of a Novel Tobamovirus Infecting Sunn Hemp (Crotalaria juncea) in Hawaii.

Sunn hemp (Crotalaria juncea L.) cultivar Tropic Sun plants, stunted and displaying mottle and mosaic symptoms on foliage, were observed at a seed farm in Maui County, Hawaii. Lateral flow assays indicated the presence of either tobacco mosaic virus or a serologically related virus. High-throughput sequencing results coupled with real-time PCR experiments recovered the 6,455-nucleotide genome of a virus with an organization typical of tobamoviruses. Nucleotide and amino acid sequence comparisons and phylogenetic analyses indicated that this virus was most closely related to sunn-hemp mosaic virus but represents a distinct species. Sunn-hemp mottle virus (SHMoV) is being proposed as the common name of this virus. Transmission electron microscopy of virus extracts purified from symptomatic leaves revealed rod-shaped particles approximately 320 by 22 nm in size. In inoculation studies, the experimental host range of SHMoV appeared limited to members of the plant families Fabaceae and Solanaceae. Greenhouse experiments demonstrated plant-to-plant transmission of SHMoV that increased with ambient wind speed. Seeds from SHMoV-infected Tropic Sun were collected and were either surface disinfested or directly planted. A total of 924 seedlings germinated; 2 were positive for the virus, resulting in a seed transmission rate of 0.2%. Both infected plants came from the surface disinfestation treatment, suggesting that the virus might be unaffected by the treatment.

Investigation of Dielectric and Mechanical Properties of Pretreated Natural Sunn Hemp Fiber-Reinforced Composite in Correlation with Macromolecular Structure of the Fiber.

The present study attempts to establish a correlation between the macromolecular parameters obtained from small angle X-ray scattering (SAXS) analysis of pretreated sunn hemp (Crotalaria juncea) fibers with the dielectric and mechanical properties of their composites. Sunn hemp fiber is pretreated chemically by dewaxing and alkalization methods and physically by microwave irradiation. The treatment effect is structurally investigated using a correlation function from SAXS data and correlated with the mechanical as well as electrical properties of composites. The macromolecular parameters are observed to be influenced by pretreatment methods. Macromolecular structural modifications are seen in dewaxed fiber (DSHC), fiber treated with 10% of alkali soaked for 6 h (10K6C), and fiber microwave irradiated with 800 watts for 6 min (800W6M), improving the mechanical as well as electric properties of the reinforced composites.

Cadmium-resistant Streptomyces stimulates phytoextraction potential of Crotalaria juncea L. in cadmium-polluted soil.

This work evaluated the competence of two strains of cadmium (Cd)-resistant Streptomyces, namely Streptomyces rapamycinicus K5PN1, an indole-3-acetic acid (IAA) producer, and Streptomyces cyaneus 11-10SHTh, a siderophore producer on promoting Cd phytoextraction by sunn hemp. The results showed that S. rapamycinicus improved root elongation of sunn hemp seedlings under Cd stress conditions. S. rapamycinicus and S. cyaneus were colonized on the root surface of sunn hemp at concentrations of 2.3 × 104 and 6.4 × 103 CFU g-1 root fresh weight, respectively. The results of pot-culture experiments showed that S. rapamycinicus increased the root and shoot lengths, and dry biomass of sunn hemp planted in high Cd-contaminated soil. The Cd concentration in the leaves of sunn hemp inoculated with S. cyaneus (73.82 ± 2.20 mg kg-1 plant dry wt) was higher than that of plants with S. rapamycinicus inoculation and the uninoculated control. The phytoextraction of Cd by sunn hemp was significantly increased with Cd-resistant Streptomyces inoculation. In conclusion, both strains of Cd-resistant Streptomyces had potential on enhancing Cd phytoextraction efficiency of sunn hemp. Our study suggests the application of Cd-resistant Streptomyces can improve Cd phytoextraction by sunn hemp for restoration of Cd-polluted sites.

Our study demonstrated the potential of two strains of Cd-resistant Streptomyces to stimulate Cd phytoextraction from soil by sunn hemp. Cadmium-resistant Streptomyces strongly stimulated Cd uptake and accumulation in sunn hemp planted in high level of Cd-polluted soil, supporting sunn hemp to be a superior Cd accumulator.

Genetic diversity and population structure of wild and cultivated Crotalaria species based on genotyping-by-sequencing.

Crotalaria is a plant genus that is found all over the world, with over 700 species of herbs and shrubs. The species are potential alternative food and industrial crops due to their adaptability to different environments. Currently, information on the genetic diversity and population structure of these species is scanty. Genotyping-by-sequencing (GBS) is a cost-effective high-throughput technique in diversity evaluation of plant species that have not been fully sequenced. In the current study, de novo GBS was used to characterize 80 Crotalaria accessions from five geographical regions in Kenya. A total of 9820 single nucleotide polymorphism (SNP) markers were obtained after thinning and filtering, which were then used for the analysis of genetic diversity and population structure in Crotalaria. The proportion of SNPs with a minor allele frequency (maf) > = 0.05 was 45.08%, while the Guanine-Cytosine (GC) content was 0.45, from an average sequence depth of 455,909 reads per base. The transition vs transversion ratio was 1.81 and Heterozygosity (He) ranged between 0.01-0.07 in all the sites and 0.04 to 0.52 in the segregating sites. The mean Tajima's D value for the population was -0.094, suggesting an excess of rare alleles. The fixation index (Fst) between the different populations based on the Wright Fst (1943) ranged from 0.0119 to 0.066 for the Eastern-Western and Nairobi-Western populations. Model based techniques of population structure analysis including structure, k-means and cross-entropy depicted eight clusters in the study accessions. Non-model based techniques especially DAPC depicted poor population stratification. Correspondence Analysis (CA), Principal coordinate analyses (PCoA) and phylogenetic analysis identified a moderate level of population stratification. Results from this study will help conservationists and breeders understand the genetic diversity of Crotalaria. The study also provides valuable information for genetic improvement of domesticated species.

Effect of Cosmos, Crotalaria, Foeniculum, and Canavalia species, single-cropped or mixes, on the community of predatory arthropods.

Some plants can attract natural enemy by offering resources such as alternative food and refuge. However, studies need to be conducted before agricultural landscape diversification is implement. Our objective was to determine the best floristic compositions of cosmos (Cosmos sulphureus-Asteraceae), showy rattlepod (Crotalaria spectabilis-Fabaceae), fennel (Foeniculum vulgare-Apiaceae), and jack bean (Canavalia ensiformis-Fabaceae) to attract and maintain predatory arthropods, and know the potential of these treatments for future use in diversifying agricultural systems. The experimental design consisted in seven treatments of four species in single-crop, intercrops in three densities called mix1, mix2, and mix3, and the control (weeds). For the arthropod families classified as very frequent and constant, population dynamics in intercropping treatments was plotted according to the plant phenology. We conclude that all plants cultivated in single-cropping and intercropping treatments showed high predator richness and can potentially be used to diversify cultivated areas. Sulfur cosmos as a single crop and three mixes attracts higher numbers and greater family richness. Spider families-Oxyopidae, Araneidae and Thomisidae-and insects-Chrysopidae and Coccinellidae are more frequents. The dynamics of the predator populations varied according to the mixes treatment.

Metal nanoparticles functionalized with nutraceutical Kaempferitrin from edible Crotalaria juncea, exert potent antimicrobial and antibiofilm effects against Methicillin-resistant Staphylococcus aureus.

Kaempferitrin (KF), a flavonol glycoside, was isolated from the edible plant Crotalaria juncea. Optimization for the synthesis of silver (AgNPs) and copper (CuNPs) nanoparticles using C. juncea extract and kaempferitrin were attempted for the first time. A detailed study on size and stability analysis have been reported. Efficacy of KF@AgNPs and KF@CuNPs against biofilm formation and planktonic mode of growth on methicillin-resistant Staphylococcus aureus (MRSA) along with possible mechanisms has been explored. Release of Cu(II) upon prolonged treatment with KF@CuNPs in the presence of MRSA was quantified through Alizarin red test, indicating the antibacterial effect is initiated by the CuNPs itself. Time kill curve depicted both the NPs have similar kill kinetics to curtail the pathogen and imaging with Crystal violet assay, Fluorescent live dead imaging and SEM analysis revealed a 60% reduction in biofilm formation at the Sub-MIC concentration of KF@AgNPs and KF@CuNPs. Furthermore, the membrane permeability and cell surface hydrophobicity were altered in the presence of both the NPs. The colony count from the in vivo infection zebrafish model in the treatment group showed a decline of > 1.8 fold for KF@AgNPs and > two fold for KF@CuNPs. Toxicity studies did not reveal any abnormality in liver and brain enzyme levels. Liver morphology images show no severe cytological alterations when treated with KF@AgNPs and were almost similar to the normal liver. Thus, KF@AgNPs was nontoxic and caused significant reduction in biofilm formation in MRSA, also reduced bacterial bioburden in the infected zebrafish, which has the potential to be explored in higher animal models.

Monocrotaline presence in the Crotalaria (Fabaceae) plant genus and its influence on arthropods in agroecosystems.

Crotalaria (Fabaceae) occurs abundantly in tropical and subtropical regions and has about 600 known species. These plants are widely used in agriculture, mainly as cover plants and green manures, in addition to their use in the management of phytonematodes. A striking feature of these species is the production of pyrrolizidine alkaloids (PAs), secondary allelochemicals involved in plant defense against herbivores. In Crotalaria species, monocrotaline is the predominant PA, which has many biological activities reported, including cytotoxicity, tumorigenicity, hepatotoxicity and neurotoxicity, with a wide range of ecological interactions. Thus, studies have sought to elucidate the effects of this compound to promote an increase in flora and fauna (mainly insects and nematodes) associated with agroecosystems, favoring the natural biological control. This review summarizes information about the monocrotaline, showing such effects in these environments, both above and below ground, and their potential use in pest management programs.

Experimental validation under controlled conditions of real time PCR to quantify arbuscular mycorrhizal colonization in root.

Mycorrhizal colonization of roots is traditionally evaluated by empirical methods, such as root microscopy. We compared this method with data from using a real time PCR technique, and determined the correlation between methods, indicating particularities of a promising system for a quick and accurate molecular diagnostic of arbuscular mycorrhization.

Volatiles Emission by Crotalaria nitens after Insect Attack.

Plants are known to increase the emission of volatile organic compounds upon the damage of phytophagous insects. However, very little is known about the composition and temporal dynamics of volatiles released by wild plants of the genus Crotalaria (Fabaceae) attacked with the specialist lepidopteran caterpillar Utetheisa ornatrix (Linnaeus) (Erebidae). In this work, the herbivore-induced plant volatiles (HIPV) emitted by Crotalaria nitens Kunth plants were isolated with solid phase micro-extraction and the conventional purge and trap technique, and their identification was carried out by GC/MS. The poly-dimethylsiloxane/divinylbenzene fiber showed higher affinity for the extraction of apolar compounds (e.g., trans-ß-caryophyllene) compared to the Porapak™-Q adsorbent from the purge & trap method that extracted more polar compounds (e.g., trans-nerolidol and indole). The compounds emitted by C. nitens were mainly green leaf volatile substances, terpenoids, aromatics, and aldoximes (isobutyraldoxime and 2-methylbutyraldoxime), whose maximum emission was six hours after the attack. The attack by caterpillars significantly increased the volatile compounds emission in the C. nitens leaves compared to those subjected to mechanical damage. This result indicated that the U. ornatrix caterpillar is responsible for generating a specific response in C. nitens plants. It was demonstrated that HIPVs repelled conspecific moths from attacked plants and favored oviposition in those without damage. The results showed the importance of volatiles in plant-insect interactions, as well as the choice of appropriate extraction and analytical methods for their study.

Pyrrolizidine Alkaloid-Induced Hepatotoxicity Associated with the Formation of Reactive Metabolite-Derived Pyrrole-Protein Adducts.

Pyrrolizidine alkaloids (PAs) with 1,2-unsaturated necine base are hepatotoxic phytotoxins. Acute PA intoxication is initiated by the formation of adducts between PA-derived reactive pyrrolic metabolites with cellular proteins. The present study aimed to investigate the correlation between the formation of hepatic pyrrole-protein adducts and occurrence of PA-induced liver injury (PA-ILI), and to further explore the use of such adducts for rapidly screening the hepatotoxic potency of natural products which contain PAs. Aqueous extracts of Crotalaria sessiliflora (containing one PA: monocrotaline) and Gynura japonica (containing two PAs: senecionine and seneciphylline) were orally administered to rats at different doses for 24 h to investigate PA-ILI. Serum alanine aminotransferase (ALT) activity, hepatic glutathione (GSH) level, and liver histological changes of the treated rats were evaluated to assess the severity of PA-ILI. The levels of pyrrole-protein adducts formed in the rats' livers were determined by a well-established spectrophotometric method. The biological and histological results showed a dose-dependent hepatotoxicity with significantly different toxic severity among groups of rats treated with herbal extracts containing different PAs. Both serum ALT activity and the amount of hepatic pyrrole-protein adducts increased in a dose-dependent manner. Moreover, the elevation of ALT activity correlated well with the formation of hepatic pyrrole-protein adducts, regardless of the structures of different PAs. The findings revealed that the formation of hepatic pyrrole-protein adducts-which directly correlated with the elevation of serum ALT activity-was a common insult leading to PA-ILI, suggesting a potential for using pyrrole-protein adducts to screen hepatotoxicity and rank PA-containing natural products, which generally contain multiple PAs with different structures.

Pyrrolizidine-Derived Alkaloids: Highly Toxic Components in the Seeds of Crotalaria cleomifolia Used in Popular Beverages in Madagascar.

Seeds of Crotalaria cleomifolia (Fabaceae) are consumed in Madagascar in preparation of popular beverages. The investigation of extracts from the seeds of this species revealed the presence of high amounts of alkaloids from which two pyrrolizidine-derived alkaloids were isolated. One of them was fully characterized by spectroscopic and spectrometric methods, which was found to be usaramine. Owing to the high toxicity of these alkaloids, issuing a strong warning among populations consuming the seeds of Crotalaria cleomifolia must be considered.