Reconsideration of the Structures of Stemara-13(14)-en-18-ol and Related Diterpene Natural Products: Vinylic Hydrogen Chemical Shifts Are Key.

The reported synthesis of stemara-13(14)-en-18-ol, which revealed that the structure of this natural product was misassigned, prompted an investigation using density functional theory methods into the structural reassignment of this natural product and related diterpenoids extracted from Calceolaria plants. 1H and 13C chemical shift predictions led to the reassignment of relative configuration, and in one case the carbon skeleton, of several diterpenoids from Calceolaria. In many of these cases, the chemical shift of the vinylic hydrogen was found to be diagnostic.

Calceolarioside A, a Phenylpropanoid Glycoside from Calceolaria spp., Displays Antinociceptive and Anti-Inflammatory Properties.

Phenylpropanoid glycosides are a class of natural substances of plant origin with interesting biological activities and pharmacological properties. This study reports the antinociceptive and anti-inflammatory effects of calceolarioside A, a phenylpropanoid glycoside previously isolated from various Calceolaria species. In models of acute nociception induced by thermal stimuli, such as the hot plate and tail flick test, calceolarioside administered at doses of 1, 5, and 10 µg in the left cerebral ventricles did not modify the behavioral response of mice. In an inflammatory based persistent pain model as the formalin test, calceolarioside A at the high dose tested (100 µg/paw) reduced the licking activity induced by formalin by 35% in the first phase and by 75% in the second phase of the test. In carrageenan-induced thermal hyperalgesia, calceolarioside A (50 and 100 µg/paw) was able to significantly reverse thermal hyperalgesia induced by carrageenan. The anti-inflammatory activity of calceolarioside A was then assessed using the zymosan-induced paw edema model. Calceolarioside A (50 and 100 µg/paw) induced a significant reduction in the edema from 1 to 4 h after zymosan administration. Measuring IL-6, TNFα, and IL-1ß pro-inflammatory cytokines released from LPS-stimulated THP-1 cells, calceolarioside A in a concentration-dependent manner reduced the release of these cytokines from THP-1 cells. Taken together, our results highlight, for the first time, the potential and selective anti-inflammatory properties of this natural-derived compound, prompting its rationale use for further investigations.

The role of ontogenetic allometry and nonallometric flower shape variation in species-level adaptive diversification - Calceolaria polyrhiza (Calceolariaceae) as a case study.

Organism shape changes predictably during ontogeny, resulting in specific patterns of ontogenetic allometry. In several plant and animal lineages, among-species variation in the shape of mature organisms mirrors variation along their growth trajectories. Hence, ontogenetic allometry is an important bias in evolution. This bias should be stronger at reduced evolutionary time scales, in which among-trait correlations had less time to evolve. Nevertheless, it was shown that adaptation of organism shape frequently involved departures from the ancestral ontogenetic allometry. Moreover, only a moderate fraction of shape variation is correlated with size during ontogeny. Hence, nonallometric variation in shape (NAVSh) is likely to contribute to adaptation, even at reduced evolutionary time scales. We explored the contributions of allometric variation in shape (AVSh), NAVSh, and size variation to adaptive evolution in the angiosperm species Calceolaria polyrhiza. This strongly relies on oil-collecting bees for pollination and experienced transitions in the size of pollinators during the last 2 Ma. Using geometric morphometrics, we described corolla morphology in several populations across its distribution range. Variation in corolla shape was decomposed into an allometric and a nonallometric component, and corolla size was estimated. We then looked for the correlation between these aspects of morphology and the pollinator. Our results suggest that adaptation to pollinators with different sizes relied on NAVSh, which resulted from shifts in the allometric slope and from shape changes that occurred early in flower development. We conclude that NAVSh can contribute to adaptation in flowering plants, even at the species-level.

Stemarane Diterpenes and Diterpenoids.

: In this article the scientific activity carried out on stemarane diterpenes and diterpenoids, isolated over the world from various natural sources, was reviewed. The structure elucidation of stemarane diterpenes and diterpenoids was reported, in addition to their biogenesis and biosynthesis. Stemarane diterpenes and diterpenoids biotransformations and biological activity was also taken into account. Finally the work leading to the synthesis and enantiosynthesis of stemarane diterpenes and diterpenoids was described.

In Silico Studies on Compounds Derived from Calceolaria: Phenylethanoid Glycosides as Potential Multitarget Inhibitors for the Development of Pesticides.

An increasing occurrence of resistance in insect pests and high mammal toxicity exhibited by common pesticides increase the need for new alternative molecules. Among these alternatives, bioinsecticides are considered to be environmentally friendly and safer than synthetic insecticides. Particularly, plant extracts have shown great potential in laboratory conditions. However, the lack of studies that confirm their mechanisms of action diminishes their potential applications on a large scale. Previously, we have reported the insect growth regulator and insecticidal activities of secondary metabolites isolated from plants of the Calceolaria genus. Herein, we report an in silico study of compounds isolated from Calceolaria against acetylcholinesterase, prophenoloxidase, and ecdysone receptor. The molecular docking results are consistent with the previously reported experimental results, which were obtained during the bioevaluation of Calceolaria extracts. Among the compounds, phenylethanoid glycosides, such as verbascoside, exhibited good theoretical affinity to all the analyzed targets. In light of these results, we developed an index to evaluate potential multitarget insecticides based on docking scores.

Inhibition of Breast Cancer Cell Migration by Cyclotides Isolated from Pombalia calceolaria.

Two new bracelet cyclotides from roots of Pombalia calceolaria with potential anticancer activity have been characterized in this work. The cyclotides Poca A and B (1 and 2) and the previously known CyO4 (3) were de novo sequenced by MALDI-TOF/TOF mass spectrometry (MS). The MS2 spectra were examined and the amino acid sequences were determined. The purified peptides were tested for their cytotoxicity and effects on cell migration of MDA-MB-231, a triple-negative breast cancer cell line. The isolated cyclotides reduced the number of cancer cells by more than 80% at 20 µM, and the concentration-related cytotoxic responses were observed with IC50 values of 1.8, 2.7, and 9.8 µM for Poca A (1), Poca B (2), and CyO4 (3), respectively. Additionally, the inhibition of cell migration (wound-healing assay) exhibited that CyO4 (3) presents an interesting activity profile, in being able to inhibit cell migration (50%) at a subtoxic concentration (2 µM). The distribution of these cyclotides in the roots was analyzed by MALDI imaging, demonstrating that all three compounds are present in the phloem and cortical parenchyma regions.

Comparative Study of Different Polar Groups of EPA-Enriched Phospholipids on Ameliorating Memory Loss and Cognitive Deficiency in Aged SAMP8 Mice.

SCOPE: Recent studies have shown that omega-3 PUFAs enriched phospholipids (n-3 PUFA-PLs) have beneficial effects on memory and cognition. However, most reports only attribute the benefit to docosahexaenoic acid (DHA) and pay little attention to eicosapentaenoic acid (EPA). METHODS AND RESULTS: We investigate the effect of EPA-enriched phospholipids on cognitive deficiency in senescence-accelerated prone 8 (SAMP8) mouse. Ten-month-old SAMP8 mice are fed with 2% (w/w) EPA-enriched phosphatidylcholine/phosphatidyl ethanolamine (EPA-PC/PE; EPA:DHA = 46.8:3.01) or 2% EPA-enriched phosphatidylserine (EPA-PS; biosynthesized from EPA-PC/PE) for 8 weeks; we then test the behavioral performances in the Barnes maze test and Morris maze test; the changes of oxidative stress, apoptosis, neurotrophic factors, tau phosphorylation, and Aß pathology are also measured. The results of behavior tests indicate that both EPA-PC/PE and EPA-PS significantly improve memory and cognitive deficiency. It is found that remarkable amelioration of oxidative stress and apoptosis occurs in both EPA-PC/PE and EPA-PS groups. EPA-PS shows more ameliorative effects than EPA-PC/PE on neurotrophic activity by decreasing hyper-phosphorylation of tau and depressing the generation and accumulation of ß-amyloid peptide (Aß). CONCLUSION: These data suggest that EPA-PS exhibits better effects than EPA-PC/PE on ameliorating memory and cognitive function, which might be attributed to the phospholipid polar groups.

Effects of Botanical Insecticides on Hymenopteran Parasitoids: a Meta-analysis Approach.

Botanical insecticides (BIs) are considered a valuable alternative for plant protection in sustainable agriculture. The use of both BIs and parasitoids are presumed to be mutually compatible pest management practices. However, there is controversy on this subject, as various studies have reported lethal and sublethal effects of BIs on hymenopteran parasitoids. To shed new light on this controversy, a meta-analytic approach of the effects of BIs on adult mortality, parasitism, and parasitoid emergence under laboratory conditions was performed. We show that BIs increased mortality, decreased parasitism, and decreased parasitoid emergence. Botanical insecticides derived from Nicotiana tabacum and Caceolaria andina were particulary lethal. Most of the parasitoid groups showed susceptibility to BIs, but the families Scelionidae and Ichneumonidae were not significantly affected. The negative effects of BIs were seen regardless of the type of exposure (topical, ingestion, or residual). In conclusion, this meta-analysis showed that under laboratory conditions, exposure of hymenopteran parasitoids to BIs had significant negative effects on adult mortality, parasitism, and parasitoid emergence.

Efecto antioxidante del extracto hidroalcohólico de Calceolaria engleriana Kraenzl "wawillay" in vivo en ratones con lesión hepática / In vivo antioxidant effect of Calceolaria engleriana Kraenzl "wawillay" hydro-alcoholic extract in mice with hepatic injury

Objetivos. Evaluar el efecto antioxidante del extracto hidroalcohólico de Calceolaria engleriana Kraenzl "wawillay" in vivo mediante el uso de ratones con lesión hepática inducida por tetracloruro de carbono (CCL4). Materiales y métodos. Estudio experimental. Se utilizó cuatro grupos de seis ratones albinos Mus musculus - cepa Balb/c/CNPB: un grupo blanco, un grupo control que recibió vitamina E (Vitesol® E 400 U.I) y dos grupos a los que se les administró extracto hidroalcohólico de Calceolaria engleriana Kraenzl "wawillay" en dosis de 50 y 300 mg/kg. Para la evaluación del efecto antioxidante in vivo se sometió a los ratones a un modelo experimental de hepatotoxicidad por tetracloruro de carbono (CCL4) antes y durante las intervenciones. Mediante la reacción del ácido tiobarbitúrico se cuantificó el malonaldialdehido (MDA) formado y se calculó el porcentaje de inhibición de peroxidación lipídica (POL) con el que se establecieron diferencias por grupos. Resultados. El tamizaje fitoquímico mostro la presencia muy abundante de flavonoides. Los porcentajes de inhibición de la POL obtenidos fueron de un 22 y 45%, para las dosis de 50 y 300 mg/kg del extracto hidroalcohólico. Si bien se encontraron diferencias significativas en la comparación entre grupos (p>0,05), las intervenciones experimentales no superaron los resultados del grupo control con vitamina E (60%). Conclusiones. En las condiciones experimentales, el extracto hidroalcohólico de Calceolaria engleriana Kraenz "wawillay" a dosis de 50 y 300 mg/kg presenta actividad antioxidante, siendo esta aun menor a la mostrada por el estándar (vitamina E).

Objetive. To evaluate the in vivo antioxidant effect of Calceolaria engleriana Kraenzl "wawillay" hydro-alcoholic extract through the use of mice with hepatic injury induced by carbon tetrachloride (CCL4). Materials and methods. An experimental study. Four groups of 6 albino Mus musculus ­ strain Balb/c/CNPB mice were used: a no-intervention group; a control group, which received Vitamin E (VITESOL® E 400 U.I); which Calceolaria engleriana Kraenzl "wawillay" hydro-alcoholic extract in doses of 50 mg/kg and 300 mg/kg of the extract. To evaluate in vivo antioxidant effect, mice were subjected to an experimental model of hepatotoxicity by carbon tetrachloride (CCL4) before and during experimental interventions. Through the thiobarbituric acid reaction, the malonalmaldehyde (MDA) formed was quantified and the percentage inhibition of lipid peroxidation (POL) was calculated. With this data, statistical differences were established by groups. Results. Phytochemicalscreening showed "very abundant" presence of flavonoids. The percentages of POL inhibition obtained were 22% and 45%, for the doses of 50 mg/kg and 300 mg/kg of hydro-alcoholic extract. Although significant differences were found in the comparison between groups (p> 0.05), the experimental interventions did not surpass the results of control group intervened with Vitamin E (60%). Conclusions. Under the experimental conditions, the Calceolaria engleriana Kraenz "wawillay" hydro-alcoholic extract (at doses of 50 mg/kg and 300 mg/kg) has antioxidant activity, but the effect found were lower than the standard (Vitamin E).

(+)-Podocarpic Acid as Chiral Template in the Synthesis of Aphidicolane, Stemodane and Stemarane Diterpenoids †.

In this review the synthetic work in the field of aphidicolane, stemodane and stemarane diterpenoids, in which readily available (+)-podocarpic acid (4) was used as chiral template for the construction of their polycyclic structures, is described as it developed along the years. In the frame of this work (+)-podocarpic acid (4) was a very useful tool in a model study leading to the syntheses of tetracyclic ketones 7 and 8, models of key intermediates 5a and 6 in the syntheses of (+)-aphidicolin (1) and (+)-stemodin (2a), respectively. (+)-Podocarpic acid (4) was also converted into (+)-2-deoxystemodinone (2d), allowing confirmation of the stemodane diterpenoids absolute configuration, into (+)-aphidicol-15-ene (36) and into Stemodia chilensis tetracyclic diterpenoid (+)-19-acetoxystemodan-12-ol (2f), allowing confirmation of its structure. (+)-Podocarpic acid (4) was then extensively used in the work which led to the synthesis of (+)-stemar-13-ene (57) and (+)-18-deoxystemarin (3b). Finally, (+)-4 was converted into (+)-2-deoxyoryzalexin S (66), which made it possible to demonstrate that the structure of (+)-66 could not be attributed to a Chilean Calceolaria isolated diterpenoid to which this structure had been assigned.

Drift effects on the multivariate floral phenotype of Calceolaria polyrhiza during a post-glacial expansion in Patagonia.

Quaternary environmental changes substantially impacted the landscape and promoted rapid evolutionary changes in many species; however, analyses of adaptive phenotypic variation in plants have usually neglected the underlying historical context. Here, we associate phylogeography and phenotypic evolution by analysing the divergence of Calceolaria polyrhiza multivariate floral phenotype after a Pleistocene post-glacial expansion in Patagonia. Phenotypic matrix (P) properties (size, shape, orientation and phenotypic integration) of six refugium and six recent populations from two different phylogroups were compared following different approaches. We found that P-matrix shape and orientation remained stable despite the strong phylogeographic footprint of post-glacial expansion. However, average proportional reductions in matrix size supported the expectation that drift had a significant effect on the floral phenotype in the northern phylogroup. When phylogeographic history was not included in the analyses, the results overestimated phenotypic differences, whereas under explicit phylogeographic control, drift appeared as the best explanation for matrix differences. In general, recent populations showed a larger phenotypic divergence among them, but a lower overall phenotypic variation than refugium populations. Random Skewers analyses indicated a lower potential response to selection in recently colonized populations than in refugium populations. We discuss that the combination of phylogeographic analyses with geographical distribution of functional phenotypic (genotypic) variation is critical not only to understand how historical effects influence adaptive evolution, but also to improve field comparisons in evolutionary ecology studies.

Pollination syndromes in a specialised plant-pollinator interaction: does floral morphology predict pollinators in Calceolaria?

Pollination syndromes are defined as suites of floral traits evolved in response to selection imposed by a particular group of pollinators (e.g., butterflies, hummingbirds, bats). Although numerous studies demonstrated their occurrence in plants pollinated by radically different pollinators, it is less known whether it is possible to identify them within species pollinated by one functional pollinator group. In such a framework, we expect floral traits to evolve also in response to pollinator subgroups (e.g., species, genera) within that unique functional group. On this, specialised pollination systems represent appropriate case studies to test such expectations. Calceolaria is a highly diversified plant genus pollinated by oil-collecting bees in genera Centris and Chalepogenus. Variation in floral traits in Calceolaria has recently been suggested to reflect adaptations to pollinator types. However, to date no study has explicitly tested that observation. In this paper, we quantitatively test that hypothesis by evaluating the presence of pollination syndromes within the specialised pollination system formed by several Calceolaria and their insect pollinators. To do so, we use multivariate approaches and explore the structural matching between the morphology of 10 Calceolaria taxa and that of their principal pollinators. Our results identify morphological matching between floral traits related to access to the reward and insect traits involved in oil collection, confirming the presence of pollinator syndromes in Calceolaria. From a general perspective, our findings indicate that the pollination syndrome concept can be also extended to the intra-pollinator group level.

Efecto hipotensor del extracto acuoso de Calceolaria myriophylla kraenz en ratas hipertensas inducidas por L-NAME / Efecto hipotensor del extracto acuoso de Calceolaria myriophylla kraenz en ratas hipertensas inducidas por L-NAME

El objetivo de este estudio experimental ha sido evaluar el posible efecto hipotensor, diurético y tóxico del extracto acuoso de Calceolaria myriophylla Kraenz (zapatilla). Material y Métodos: se utilizó ratas albinas machos de raza Holtzmann para la determinación de los efectos, a quienes después de una semana de adaptación se les midió la presión arterial basal sistólica, diastólica y media. Para la determinación del efecto hipotensor se realizó la inducción con la administración de N-Nitro-L-Arginina Metíl Ester (L-NAME) a dosis de 40 mg/kg/día por vía oral durante 05 días y nuevamente se les midió la presión arterial postinducción, se dividió en 06 grupos de 08 animales; grupo control positivo (L-NAME) y control negativo (agua destilada), grupos experimentales a dosis de 50 mg/kg, 100 mg/kg, 200 mg/kg y enalapril a dosis de 25 mg/kg. El tratamiento fue durante 30 días midiéndose la presión arterial cada 5 días. El efecto diurético se evaluó utilizando ratas normotensas distribuidas en 05 grupos de 09 animales; grupo control (agua destilada), grupos a dosis de 50 mg/kg, 100 mg/kg, 200 mg/kg y furosemida a dosis de 10 mg/kg. La evaluación de la actividad antioxidante se realizó a los grupos determinados en el efecto hipotensor. El estudio del efecto tóxico se determino en el ensayo a dosis repetidas por 60 días a nivel hematológico, bioquímico y anatomopatológico en ratas normotensas distribuidas en 03 grupos de 06 animales; utilizando dosis de 100 mg/kg, 200 mg/kg y un grupo control (agua destilada). Resultados: El extracto acuoso de Calceolaria myriophylla Kraenz presentó efecto hipotensor y diurético a las dosis indicadas por vía oral. El extracto acuoso elevó los niveles séricos de óxido nítrico y disminuyo los niveles séricos de malondialdehido. No presentó efecto tóxico en el ensayo a dosis repetidas por 60 días. Conclusiones: Se demostró que el extracto acuoso de Calceolaria myriophylla Kraenz presenta efecto hipotensor, diurético y antioxidante sin efectos tóxicos.