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.

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.

Gravimetric quantitative validation of botanic impurities in feed.

BACKGROUND: Harmful botanical impurities may contaminate feed and feed materials and be a potential danger to animal or human health, or to the environment. The aim of this study was to establish rapid and sensitive methods that can be used in routine official controls to determine botanical impurities such as Datura stramonium, Ricinus communis, Crotaliaria spp., and Ambrosia spp. in animal feed and raw materials. Claviceps sclerotia were also detected in cereals, due to the similarities of the targets and the analytical procedure. Regulation (EU) 625/2017, which replaces Reg. 2004/882/EC, states that EU member states should conduct official controls in assessed and accredited laboratories and that the analytical methods must be validated before use by considering parameters such as specificity, precision, recovery, and measurement uncertainly. RESULTS AND CONCLUSION: The results demonstrate that all of the methods tested are suitable for the official quantitative analyses required by EU official legislation. © 2020 Society of Chemical Industry.

Crotalaria verrucosa Leaf Extract Mediated Synthesis of Zinc Oxide Nanoparticles: Assessment of Antimicrobial and Anticancer Activity.

In this work, we present an ecofriendly, non-hazardous, green synthesis of zinc oxide nanoparticles (ZnO NPs) by leaf extract of Crotalaria verrucosa (C. verrucosa). Total phenolic content, total flavonoid and total protein contents of C. verrucosa were determined. Further, synthesized ZnO NPs was characterized by UV-visible spectroscopy (UV-vis), X-ray diffractometer (XRD), Fourier transform infra-red (FTIR) Spectra, transmission electron microscope (TEM), and Dynamic light scattering (DLS) analysis. UV-vis shows peak at 375 nm which is unique to ZnO NPs. XRD analysis demonstrates the hexagonal phase structures of ZnO NPs. FTIR spectra demonstrates the molecules and bondings associated with the synthesized ZnO NPs and assures the role of phytochemical compounds of C. verrucosa in reduction and capping of ZnO NPs. TEM image exhibits that the prepared ZnO NPs is hexagonal shaped and in size ranged between 16 to 38 nm which is confirmed by DLS. Thermo-gravimetric analysis (TGA) was performed to determine the thermal stability of biosynthesized nanoparticles during calcination. The prepared ZnO NPs showed significant antibacterial potentiality against Gram-positive (S. aureus) and Gram-negative (Proteus vulgaris, Klebsiella pneumoniae, and Escherichia coli) pathogenic bacteria and SEM image shows the generalized mechanism of action in bacterial cell after NPs internalization. In addition, NPs are also found to be effective against the studied cancer cell lines for which cytotoxicity was assessed using MTT assay and results demonstrate highest growth of inhibition at the concentration of 100 µg/mL with IC50 value at 7.07 µg/mL for HeLa and 6.30 µg/mL for DU145 cell lines, in contrast to positive control (C. verrucosa leaf extract) with IC50 of 22.30 µg/mL on HeLa cells and 15.72 µg/mL on DU145 cells. Also, DAPI staining was performed in order to determine the effect on nuclear material due to ZnO NPs treatment in the studied cell lines taking leaf extract as positive control and untreated negative control for comparison. Cell migration assay was evaluated to determine the direct influence of NPs on metastasis that is potential suppression capacity of NPs to tumor cell migration. Outcome of the synthesized ZnO NPs using C. verrucosa shows antimicrobial activity against studied microbes, also cytotoxicity, apoptotic mediated DNA damage and antiproliferative potentiality in the studied carcinoma cells and hence, can be further used in biomedical, pharmaceutical and food processing industries as an effective antimicrobial and anti-cancerous agent.

LC/MS study of the diversity and distribution of pyrrolizidine alkaloids in Crotalaria species growing in Colombia.

Hepatotoxic and genotoxic pyrrolizidine alkaloids have been involved in the acute poisoning of animals and humans. Crotalaria (Fabaceae) species contain these alkaloids. In this work, the diversity and distribution of pyrrolizidine alkaloids in roots, leaves, flowers, and seeds of Crotalaria pallida, Crotalaria maypurensis, Crotalaria retusa, Crotalaria spectabilis, Crotalaria incana, and Crotalaria nitens were studied. Matrix solid-phase dispersion and ultra-high-performance liquid chromatography coupled with Orbitrap mass spectrometry were successfully employed in pyrrolizidine alkaloids extraction and analysis, respectively. Forty-five pyrrolizidine alkaloids were detected and their identification was based on the mass spectrometry accurate mass measurement and fragmentation pattern analysis. The cyclic retronecine-type diesters monocrotaline, crotaleschenine, integerrimine, usaramine, and their N-oxides were predominantly present. Five novel alkaloids were identified for the first time in Crotalaria species, namely 14-hydroxymonocrotaline, 12-acetylcrotaleschenine, 12-acetylmonocrotaline, 12-acetylintegerrimine, and dihydrointegerrimine. Due to a lack of commercially available standards, the response factor of monocrotaline was used for quantification of pyrrolizidine alkaloids and their N-oxides. Seeds and flowers possessed higher pyrrolizidine alkaloids amounts than roots and leaves. Due to their 1,2-unsaturated pyrrolizidine alkaloids content, the ingestion of Crotalaria plant seeds or other parts through herbal products, infusions, or natural remedies is a serious health threat to humans and livestock.

Studying the link between physiological performance of Crotalaria ochroleuca and the distribution of Ca, P, K and S in seeds with X-ray fluorescence.

This study describes the use of X-ray fluorescence spectroscopy in Crotalaria ochroleuca seed technology. This work evaluated X-ray fluorescence techniques to estimate the physiological performance of different C. ochroleuca seed coat colours based on the concentration and distribution of Ca, P, K, and S in seed structures. The treatments consisted of seeds separated by coat colours (yellow, green, and red) and a control treatment (colour mix according to their natural occurrence in commercial lots), and was carried out in a completely randomized design, with four replications. The physiological performance was evaluated by analyzing the water content, germination, first germination count, germination speed index, electrical conductivity, seedling emergence, and seedling length and dry mass. X-ray fluorescence spectroscopy techniques were carried out with quantitative analyses (Ca, P, K, and S concentration in the seed coat and the whole seed) and qualitative analyses (macronutrient mapping). The EDXRF and µ-XRF techniques are efficient and promising to differentiate the physiological performance of C. ochroleuca seeds, based on the concentration and distribution of Ca, P, K, and S in different structures. Ca is predominant in the seed coat, and K, S, and P are found throughout the embryonic axis. Seeds of yellow and green coats have higher nutrients concentration and distribution in the embryonic axis, revealing high germinative capacity and physiological performance. Seeds of red coat have higher nutrients concentration in the seed coat and lower assimilation, showing less vigour, which interferes directly in the quality of commercial lots.

Preincubation format for a sensitive immunochromatographic assay for monocrotaline, a toxic pyrrolizidine alkaloid.

INTRODUCTION: Monocrotaline (MCT), which is classified as a 1,2-dehydropyrrolizidine alkaloid (DHPA), is a toxic compound that is mainly produced by Crotalaria spp. MCT contamination in cereals and herbs leads to hepatitis, gastroenteritis, pulmonary vasculitis and hypertension, and different types of cancer. The current analytical methods for MCT are complicated and expensive using liquid chromatography equipped with mass spectrometry detection. OBJECTIVE: The aim of this study was to develop a simple and sensitive preincubation format for an immunochromatographic assay (PI-ICA) for MCT detection. METHODOLOGY: We conducted the PI-ICA via incubation of an MCT-containing sample with an anti-MCT monoclonal antibody conjugated with colloidal gold before strip dipping. We compared the PI-ICA detection sensitivity with that of the conventional ICA (Conv-ICA) format. RESULTS: The PI-ICA was sensitive with a limit of detection (LOD) of 0.61 ng/mL, which is a 16-fold improvement over the Conv-ICA format. These results indicated that the PI-ICA method exhibits high binding specificity for MCT and low cross-reactivity towards retronecine, retrorsine, senecionine and heliotrine. Sample solutions from plants containing MCT and related DHPAs produced positive results via PI-ICA analysis. CONCLUSIONS: The proposed PI-ICA system provides a highly sensitive method compared to Conv-ICA. In addition, the developed PI-ICA method is simple and highly effective for detecting MCT contamination.

Anti-Helicobacter pylori Potential of Three Edible Plants Known as Quelites in Mexico.

"Quelites" are edible plants that are part of the traditional agro-ecosystems in Mexico. These plants, despite their already known nutritional properties, are now considered neglected and underutilized species. With the objective of promoting their reinsertion in the markets and mainly, in daily diets, efforts have been made to study them from multidisciplinary approaches to demonstrate their beneficial properties. To generate evidence of an added health-promoting value that would encourage quelites consumption, in the present work, the anti-Helicobacter pylori activity of three representative quelite species, Anoda cristata (Alache), Cnidoscolus aconitifolius (Chaya), and Crotalaria pumila (Chepil), was tested. H. pylori is considered the etiological agent of gastritis, ulcer, and gastric cancer, and represents a public health problem in Mexico and worldwide. Aqueous (AQ) and dichloromethane-methanol (DM) extracts were obtained from the three species of quelites to investigate their effect on H. pylori growth and on two of its colonization factors (adherence and urease activity). DM extracts from Chaya, Chepil, and Alache exert the best inhibitory effect on bacterial growth, with minimum inhibitory concentrations of 62.5, 125, and 250 µg/mL, respectively. AQ and DM extracts inhibit bacterial adhesion by 30% to 50%. None of them has an effect on urease activity. The two flavonoids present in A. cristata, acacetin and diosmetin, inhibit H. pylori growth by ∼90% with 3.9 µg/mL. These results provide new information about the anti-H. pylori potential of three edible quelites, and give an added value, since their routine consumption may impact on the prevention and/or control of H. pylori-associated diseases.

Simultaneous degumming and production of a natural gum from Crotalaria juncea seeds: Physicochemical and rheological characterization.

The oil extracted from Crotalaria juncea (Sunn-hemp) contains 70% of gum. Several methods of degumming are attempted in order to maximize the yield of gum. During appropriate water induced degumming, about 95-98% of phosphatides are separated. The maximum oil yield for two types of degummimg processes are 0.59% and 0.69% corresponding to hot water and pure O-phosphoric acid (19.88 N) treatment respectively. The % oil yield obtained for TOP degumming is about 0.78%. Physico-chemical characteristics of the isolated gum such as moisture, ash, protein, fat and aqueous solubility along with FTIR and TGA analysis are studied in order to evaluate the effect of extraction process. The behaviour of gum on the molecular scale is evaluated through alcohol treatment. Chromatographic analysis determines the monosaccharide content of the gum with glucose: xylose: arabinose::54: 34:1. Rheological characterization shows that the juncea gum solutions are shear rate dependent and the behaviour is shear-thinning (or pseudoplastic). Results show that the temperature dependent viscosity decreases with increasing shear rate.

Structural elucidation of galactomannan from seeds of Crotalaria mucronata Desv. by atomic force microscopy.

A non-ionic water-soluble galactomannan, isolated from the seed endosperm of Crotalaria mucronata Desv., may be an ideal thickener to increase food viscosity. The present study hypothesized that the viscous property of Crotalaria galactomannan may be associated with its spatial structure. Therefore, the structure of Crotalaria galactomannan was elucidated using an atomic force microscope. The results of the present study demonstrated that the polysaccharide consisted of a D­mannose backbone with D­galactose branches, and the D­mannose/D­galactose ratio was 2.375:1. In the three­dimensional structure of Crotalaria galactomannan, the helix was a common secondary structure, containing numerous ring structures of different sizes. In addition, multiple helixes may link together via hydrogen bonding and van der Waals forces, forming aggregations with small rings or spiral windings. The results of the present study indicated that the multiple­branching construction of Crotalaria galactomannan may underlie its viscosity­enhancing properties in the water phase.

Flavonoids, alkaloids from the seeds of Crotalaria pallida and their cytotoxicity and anti-inflammatory activities.

Three flavonoids, cropalliflavones A-C, including two homoisoflavonoids with rare skeletons; three previously undescribed alkaloids, usaramine-N-oxide and cropallins A-B; and sixteen known compounds, were isolated from the seeds of Crotalaria pallida Ait. The absolute configurations of cropalliflavone A and usaramine-N-oxide were established by an ECD calculation and X-ray crystallography, respectively. Additionally, cropalliflavone B showed anti-proliferative activity against the MCF-7 cell line with an IC50 value of 6.77 µM, and cropalliflavone C showed anti-inflammatory activity, with an IC50 value of 16.07 µM.

The anti-inflammatory and antinociceptive activity of albumins from Crotalaria retusa seeds.

Seeds of Crotalaria retusa L. are used in popular medicine because of their pharmacological properties. The albumin fraction obtained from its seeds contains lectin, a protein known to have analgesic and anti-inflammatory properties. Thus, albumins extracted from C. retusa were investigated for their anti-inflammatory and antinociceptive effects. The intraperitoneal administration of different doses of albumins (5, 10 or 20mg/kg) significantly inhibited the mice paw edema induced by carrageenan (maximum inhibition rate of 80.9% at four hours, 20mg/kg), and this event was followed by diminishing paw myeloperoxidase measurements. Albumins (20mg/kg) also inhibited neutrophil migration into the peritoneal cavity induced by carrageenan. However, no effect was observed in the dextran-induced paw edema and abdominal contortions induced by acetic acid. Moreover, albumins (20mg/kg) significantly reduced the second (inflammatory) phase of the licking time induced by formalin. The detection of heammaglutinating activity against human erythrocytes in albumins evidences the presence of lectin in seeds of C. retusa. Our data showed that seeds of C. retusa had anti-inflammatory and antinociceptive properties and such activities are probably due to the inhibitory effect on neutrophil migration of lectin present in albumins.

Synthesis and anti-obesity effects in vivo of Crotadihydrofuran C as a novel PPARγ antagonist from Crotalaria albida.

Crotadihydrofuran C (CC) from the herbs of Crotalaria albida is able to inhibit adipocyte differentiation and lipid accumulation. However, the effects of CC on obesity and metabolic disorders have not yet been elucidated. In our study, the first enantioselective synthesis of the 2-isopropenyl dihydrofuran isoflavone skeleton (CC) is described. The convenient and efficient synthetic protocols developed skilfully solve the problems of the ortho-para directing group and Suzuki coupling reaction using a boronic acid pinacol ester that was more stable and easy to obtain. Furthermore, CC treatment of high-fat diet (HFD)-fed obese mice remarkably reduced their body weight, fat mass, and lipid level as well as improved insulin resistance and non-alcoholic fatty liver disease (NAFLD). A TR-FRET assay showed that CC was specifically bound to PPARγ LBD, which was further confirmed by the molecular docking study. These results suggest that CC could be a useful and potential natural product for treating metabolic diseases, including obesity, hyperlipidemia insulin resistance and NAFLD, without toxic side-effects.

Involvement of astrocytic CYP1A1 isoform in the metabolism and toxicity of the alkaloid pyrrolizidine monocrotaline.

Monocrotaline (MCT) and its pyrrole derivative, dehydromonocrotaline (DHMC), interact with molecular targets in cells of the central nervous system. DHMC presents higher toxicity than MCT indicating that its metabolism of MCT is a critical step of this alkaloid toxicity. This study sought to elucidate the metabolism and the toxicity of MCT in C6 astrocyte cell line and primary cultures of rat astrocytes by investigating metabolic enzymatic mechanisms of the cytochrome P450 (CYP) system and conjugation with glutathione. Treatment with omeprazole (OMP) (20 µM), a non-specific inducer of CYP450 induced approximately 10-fold increase in CYP1A1 activity after 2 h of treatment. Similarly, the 7-Ethoxyresorufin-O-deethylase (EROD) activity was induced by treatment with MCT (100-500 µM), indicating that the P450 CYP1A1 isoform was active and involved in the metabolism of MCT. Analysis of conjugation with glutathione showed a significant depletion of GSH after MCT (500 µM) treatment, and this was partially reversed by pretreatment with a P450 inhibitor (cimetidine 100 µM). These results suggest that not only the alkaloid MCT but, also its metabolite may deplete GSH. Rosenfeld staining showed intense vacuolization after MCT treatment, which was partially inhibited in the presence of a P450 activator. MTT test showed that association of MCT with OMP induced a reduction in cell viability in C6 and primary astrocytic cells. These results demonstrate that MCT is metabolized by astrocytic CYP1A1 to generate metabolites that can deplete GSH. Moreover, changes in the activity of the P450 enzymes interfere with the cytotoxic effects induced by the alkaloid.

Cytotoxicity of flavonoids and isoflavonoids from Crotalaria bracteata.

A new flavonoid, crotakanda (1), and seven known compounds including a flavonoid, four isoflavonoids, a pterocarpan and a cinnamaldehyde were isolated from the stems and roots of Crotalaria bracteata. Their structures were elucidated by a combination of spectroscopic methods. Compound 2 showed cytotoxicity against MCF-7 with an IC50 value of 79.90 µM whereas 2 and 4 exhibited cytotoxicity against the NCI-H187 cell line with IC50 values of 71.57 and 95.47 µM, respectively.

Ultra-Performance Liquid Chromatography Hyphenated with Quadrupole-Orbitrap Mass Spectrometry for Simultaneous Determination of Necine-Core-Structure Pyrrolizidine Alkaloids in Crotalaria sessiliflora L. without all Corresponding Standards.

INTRODUCTION: Crotalaria sessiliflora L. is a Chinese traditional herb for treatment of cutaneum carcinoma and cervical carcinoma. In addition to monocrotaline, coexisting pyrrolizidine alkaloids (PAs) also require further quantification for quality control and pharmaceutical uses of the herb. OBJECTIVE: To establish a UPLC-Q-Orbitrap/MS method of simultaneous determination of coexisting PAs with same parent structure for quality control and comprehensive researches of Crotalaria sessiliflora L. METHODOLOGY: PAs in Crotalaria sessiliflora L. were analysed by UPLC-Q-Orbitrap/MS method. Coexisting PAs were identified by mass data of full MS-dd-MS2 based on the characteristic fragmentation pattern and necine-core structure. Moreover, quantification of PAs was conducted by parallel reaction monitoring (PRM) mode using m/z 138, m/z 120 and m/z 94 from identical necine-core structure as quantitative ions with single monocrotaline standard for accurate calibration. RESULTS: Five PAs, named monocrotaline, retrorsine, senecionine, integerrimine, O-9-angeloylretronecine, were indentified and confirmed. Quantitative ions of m/z 138, m/z 120 and m/z 94 were used for quantification of PAs containing the necine-core structure in Crotalaria sessiliflora L. The results demonstrated that contents, precision and recoveries of the five PAs mentioned earlier were respectively 3.307-30.35 µg/g, 1.1-4.5% and 88.91-92.33% while using m/z 120 as the best quantitative ion. CONCLUSION: The UPLC-Q-Orbitrap/MS method was established for simultaneous determination of five PAs in Crotalaria sessiliflora L. without all corresponding standards, and was proved that it was simple, convenient and effective for comprehensive quality control and pharmaceutical uses. Copyright © 2017 John Wiley & Sons, Ltd.

A Novel Heterodimer from Crotalaria ferruginea.

A new compound was isolated from the traditional Chinese folk herb Crotalaria ferruginea. The structure of the new compound was identified as (±)-crotaferruphenol (1) based on analyses of its spectroscopic data including NMR, MS, and specific rotation values. (±)-Crotaferruphenol was a novel heterodimer characteristic of a unique spiroketal moiety, which was produced by the condensation reaction of a chromanone and a 2-isopropenybenzofuran. (±)-Crotaferruphenol exhibited inhibitory activity (IC50 = 6.57 µM.) against phosphodiesterase-4 (PDE4), a drug target for the treatment of chronic obstructive pulmonary disease and asthma.

Pyrrolizidine Alkaloids Negatively Affect a Generalist Herbivore Feeding on the Chemically Protected Legume Crotalaria pallida.

Plant secondary metabolites can have opposing effects on adapted specialist and non-adapted, generalist herbivores. In this study, we used Heliothis virescens (Fabricius) (Lepidoptera: Noctuidae) as a generalist, non-adapted model herbivore to test the possible effects of Crotalaria pallida (Fabaceae: Papilionoideae) defenses on herbivore performance. Neonate H. virescens larvae were able to consume C. pallida leaves and fruits and grow for a few instars, but none of them survived to pupation. We added isolated pyrrolizidine alkaloids (PAs) to an artificial diet at different concentrations, and PA concentration significantly affected the number of larvae that achieved pupation. Larval survival was not reduced at a PA concentration similar to the concentration on green seeds of C. pallida, but it was significantly reduced at PA concentration 5 and 100 times higher. These results suggest that PAs in isolation are not the defense responsible for the mortality in fresh C. pallida plants, indicating the importance of other possible defenses. The negative effect of PAs on fitness of the non-adapted, generalist herbivore is in agreement with few previous studies, but it is in clear contrast to a previous study on the effects of PAs on the adapted specialist herbivore Utetheisa ornatrix (L.) that were able to sequester PAs with no fitness costs.