Bioactive Clerodane Diterpenoids from the Leaves of Casearia coriacea Vent.

Casearia coriacea Vent., an endemic plant from the Mascarene Islands, was investigated following its antiplasmodial potentialities highlighted during a previous screening. Three clerodane diterpene compounds were isolated and identified as being responsible for the antiplasmodial activity of the leaves of the plant: caseamembrin T (1), corybulosin I (2), and isocaseamembrin E (3), which exhibited half maximal inhibitory concentrations (IC50) of 0.25 to 0.51 µg/mL. These compounds were tested on two other parasites, Leishmania mexicana mexicana and Trypanosoma brucei brucei, to identify possible selectivity in one of them. Although these products possess both antileishmanial and antitrypanosomal properties, they displayed selectivity for the malaria parasite, with a selectivity index between 6 and 12 regarding antitrypanosomal activity and between 25 and 100 regarding antileishmanial activity. These compounds were tested on three cell lines, breast cancer cells MDA-MB-231, pulmonary adenocarcinoma cells A549, and pancreatic carcinoma cells PANC-1, to evaluate their selectivity towards Plasmodium. This has not enabled us to establish selectivity for Plasmodium, but has revealed the promising activity of compounds 1-3 (IC50 < 2 µg/mL), particularly against pancreatic carcinoma cells (IC50 < 1 µg/mL). The toxicity of the main compound, caseamembrin T (1), was then evaluated on zebrafish embryos to extend our cytotoxicity study to normal, non-cancerous cells. This highlighted the non-negligible toxicity of caseamembrin T (1).

Unveiling antiplasmodial alkaloids from a cumulative collection of Strychnos extracts by multi-informative molecular networks.

Malaria, a disease known for thousands of years and caused by parasites of the Plasmodium genus, continues to cause many deaths throughout the world today, particularly due to the emergence of parasite resistance to the current therapeutic arsenal. Plants of the Strychnos genus, remarkable due to their multiple traditional uses as well as their alkaloid content, are promising candidates to develop new antimalarial treatments. Indeed, previous research on this plant group has shown promising (≤ 5 µg/ml) or good (between 5 and 15 µg/ml) antiplasmodial activities. Using the chloroquine-sensitive strain of Plasmodium falciparum (3D7), and artemisinin as positive control, a screening of antiplasmodial activities from 43 crude methanolic extracts from 28 species of the Strychnos genus was carried out in three independent assays. A total of 12 extracts had good (6 extracts) or promising (6 extracts) antiplasmodial activities. These results allowed both to confirm known activities but also to detect new ones. These extracts were then analyzed by HPLC-ESI(+)-Q/TOF, and the processed MS/MS data allowed to generate a molecular network in which the antiplasmodial activities were implemented as metadata. The exploration of the molecular network revealed the presence of alkaloids still unknown, and potentially active against malaria, in particular alkaloids close to usambarensine and its derivatives. This study shows that the emergence of molecular networking offers new leads for identifications of alkaloids from the Strychnos genus. The presence of unknown alkaloids potentially active against malaria confirms all the interest to continue in studying the Strychnos genus. Bioassay- and mass-guided fractionations as well as various dereplication tools would allow to identify and characterize these interesting alkaloids further.

Exploration by molecular networking of Strychnos alkaloids reveals the unexpected occurrence of strychnine in seven Strychnos species.

INTRODUCTION: Plants of the Strychnos genus, which include about 200 species, are used for multiple traditional purposes as hunting poison, for example, and have shown interesting pharmacological properties, especially curarizing and tetanizing, but also against malaria. Many monoterpene indole alkaloids have already been isolated and identified. Among them, there is strychnine, a famous alkaloid that can cause death by asphyxiation. OBJECTIVE: Investigate alkaloidic molecular diversity from Strychnos genus using molecular networking technique and study the Strychnos genus from a chemotaxonomic point of view. MATERIAL AND METHODS: Twenty-eight different species and different plant parts were ground into powder using a grinder. The methanolic extracts were carried out using a pressurized solvent extraction and the alkaloid extract was performed manually with a separating funnel. The extracts were analyzed by HPLC-ESI(+)-Q/TOF. The data were processed using MZmine 2 software and the molecular network was generated on the GNPS platform. The study of the generated molecular network allowed the detection of various alkaloids. Among these is the famous strychnine which has been detected in 7 new Strychnos species not yet described as strychnine producers. This identification was investigated using orthogonal approaches, namely TLC, NMR, HPLC-UV and UHPLC-ESI(+)-Q/TOF analyses. The LOD by HPLC-UV of strychnine was also determined. RESULTS: Further analyses allowed to confirm the presence of strychnine in S. densiflora trunk barks but also to show the presence of strychnine with high probability in the trunk barks of S. camptoneura, S. congolana, S. boonei, and S. tchibangensis, and in the leaves of S. usambarensis. About the trunk barks of S. tricalyisoides, the probability of a strychnine content remains low. CONCLUSION: This work exemplified the efficiency of molecular networking in identifying known metabolites (major and minor alkaloids) involved in the chemotaxonomic study of plants from Strychnos genus.

What, how, and how much do herbivores eat? The Continuous Bite Monitoring method for assessing forage intake of grazing animals.

Determining herbage intake is pivotal for studies on grazing ecology. Direct observation of animals allows describing the interactions of animals with the pastoral environment along the complex grazing process. The objectives of the study were to evaluate the reliability of the continuous bite monitoring (CBM) method in determining herbage intake in grazing sheep compared to the standard double-weighing technique method during 45-min feeding bouts; evaluate the degree of agreement between the two techniques; and to test the effect of different potential sources of variation on the reliability of the CBM. The CBM method has been used to describe the intake behavior of grazing herbivores. In this study, we evaluated a new approach to this method, that is, whether it is a good proxy for determining the intake of grazing animals. Three experiments with grazing sheep were carried out in which we tested for different sources of variations, such as the number of observers, level of detail of bite coding grid, forage species, forage allowance, sward surface height heterogeneity, experiment site, and animal weight, to determine the short-term intake rate (45 min). Observer (Pexp1  = 0.018, Pexp2  = 0.078, and Pexp3  = 0.006), sward surface height (Pexp2  < 0.001), total number of bites observed per grazing session (Pexp2  < 0.001 and Pexp3  < 0.001), and sward depletion (Pexp3  < 0.001) were found to affect the absolute error of intake estimation. The results showed a high correlation and agreement between the two methods in the three experiments, although intake was overestimation by CBM on experiments 2 and 3 (181.38 and 214.24 units, respectively). This outcome indicates the potential of CBM to determining forage intake with the benefit of a greater level of detail on foraging patterns and components of the diet. Furthermore, direct observation is not invasive nor disrupts natural animal behavior.

In Vitro Antiplasmodial and Cytotoxic Activities of Compounds from the Roots of Eriosema montanum Baker f. (Fabaceae).

Malaria remains one of the leading causes of death in sub-Saharan Africa, ranked in the top three infectious diseases in the world. Plants of the Eriosema genus have been reported to be used for the treatment of this disease, but scientific evidence is still missing for some of them. In the present study, the in vitro antiplasmodial activity of the crude extract and compounds from Eriosema montanum Baker f. roots were tested against the 3D7 strain of Plasmodium falciparum and revealed using the SYBR Green, a DNA intercalating compound. The cytotoxicity effect of the compounds on a human cancer cell line (THP-1) was assessed to determine their selectivity index. It was found that the crude extract of the plant displayed a significant antiplasmodial activity with an IC50 (µg/mL) = 17.68 ± 4.030 and a cytotoxic activity with a CC50 (µg/mL) = 101.5 ± 12.6, corresponding to a selective antiplasmodial activity of 5.7. Bioactivity-guided isolation of the major compounds of the roots' crude extract afforded seven compounds, including genistein, genistin and eucomic acid. Under our experimental conditions, using Artemisinin as a positive control, eucomic acid showed the best inhibitory activity against the P. falciparum 3D7, a well-known chloroquine-sensitive strain. The present results provide a referential basis to support the traditional use of Eriosema species in the treatment of malaria.

Pentas longiflora Oliv. (Rubiaceae), a plant used in the treatment of Pityriasis Versicolor in Rwanda: Chemical composition and standardization of leaves and roots.

In Rwanda, the roots of Pentas longiflora Oliv. (Rubiaceae) have been used for a long time to treat Pityriasis versicolor. However, many people reported the use of leaves instead of roots. This research was conducted to compare the phytochemical composition and establish chromatographic methods for the standardization of roots and leaves extracts of P. longiflora. During this process, three new pentalongin glycosides (pentalonginoside A, pentalonginoside B, and pentalonginoside C) and two known glycosides of the same type (harounoside and clarinoside), as well as rutin, luteolin-7-rutinoside were isolated from methanol extract of leaves. In addition, pentalongin and psychorubrin, previously isolated from ethylacetate roots extract, were also identified in Pentas longiflora ethylacetate leaves extract. The presence of the antifungal compound pentalongin in leaves may explain the traditional use of leaves in the treatment of Pytiriasis versicolor. Furthermore, harounoside, psychorubrin, and pentalongin were selected as markers for HPLC fingerprints of MeOH extract. The accuracy and risk profile demonstrated the reliability of the validated method. In general, considerable variations of concentration in plant metabolites, including pentalongin, were observed between samples from different sites. The content in pentalongin (expressed as juglone) in collected samples ranged between 1.7 and 70.0 mg/100 g. The highest concentration (70.0 ± 17 mg/100 g) was registered in the cultivated samples from Mukoni. This important variation of pentalongin concentrations according to sampling sites, shows that in order to guarantee equivalent efficacy, finished products with P. longiflora should be standardized based on their pentalongin content.

Potential of grazing management to improve beef cattle production and mitigate methane emissions in native grasslands of the Pampa biome.

We tested the hypothesis that improving sward structure through adjustments in forage allowance results in greater forage intake and live weight (LW) gains by beef cattle and lower CH4 emissions per unit LW gain and unit area in a native grassland ecosystem of the Pampa biome. The experiment was carried out during 2012 and 2013 in southern Brazil. The experimental design was a randomized complete block with two replicates. Treatments consisted of five contrasting forage allowances of a native grassland managed under continuous stocking: 4, 8, 8-12, 12, and 16 kg of dry matter (DM) 100 kg LW-1 day-1 (or % LW). The 8-12% LW treatment had a variable forage allowance of 8% LW in spring and 12% LW in summer, autumn, and winter. Forage allowance was controlled by changes in stocking rate (kg LW ha-1). Average daily gain (kg LW day-1) was high for forage allowances of 12 and 16% LW but decreased at 8%, reaching the lowest value at 4% LW treatment (p < 0.001). Live weight gain ha-1 year-1 was the greatest at forage allowance of 8-12% LW (p < 0.001). Forage DM intake peaked at a forage allowance of 12% LW (p = 0.005). Individual CH4 emissions remained constant around 150 g day-1 for the two highest forage allowances and decreased to 118 and 107 g day-1 under forage allowances of 8 and 4% LW, respectively (p = 0.002). Emissions per unit LW gain and unit area were driven by animal productivity changes and decreased with increasing forage allowance (p = 0.001 and p = 0.040, respectively). We propose that the combination of 8% LW forage allowance during spring and 12% LW during the rest of the year should be targeted to best balance animal production and environmental impact in the Pampa biome.

Does grazing management provide opportunities to mitigate methane emissions by ruminants in pastoral ecosystems?

Agriculture, and livestock production in particular, is criticized for being a contributor to global environmental change, including emissions of greenhouse gases (GHG). Methane (CH4) from grazing ruminants accounts for most of livestock's carbon footprint because a large share of them are reared under suboptimal grazing conditions, usually resulting in both low herbage intake and animal performance. Consequently, the CH4 quota attributed to animal maintenance is spread across few or no animal outputs, increasing the CH4 intensity [g CH4/kg live weight (LW) gain or g CH4/kg milk yield]. In this review, the generalized idea relating tropical pastures with low quality and intrinsically higher CH4 intensity is challenged by showing evidence that emissions from animals grazing tropical pastures can equal those of temperate grasses. We demonstrate the medium-to-high mitigation potential of some grazing management strategies to mitigate CH4 emissions from grazing ruminants and stress the predominant role that sward canopy structure (e.g., height) has over animal behavioral responses (e.g., intake rate), daily forage intake and resulting CH4 emissions. From this ecological perspective, we identify a grazing management concept aiming to offer the best sward structure that allows animals to optimize their daily herbage intake, creating opportunities to reduce CH4 intensity. We show the trade-off between animal performance and CH4 intensity, stressing that mitigation is substantial when grazing management is conducted under light-to-moderate intensities and optimize herbage intake and animal performance. We conclude that optimizing LW gain of grazing sheep and cattle to a threshold of 0.14 and 0.7 kg/day, respectively, would dramatically reduce CH4 intensity to approximately 0.2 kg CH4/kg LW gain, as observed in some intensive feeding systems. This could represent a mitigation potential of around 55% for livestock commodities in pasture-based systems. Our results offer new insights to the debate concerning mitigation of environmental impacts of pastoral ecosystems.

Mechanisms and implications of a type IV functional response for short-term intake rate of dry matter in large mammalian herbivores.

The functional response (i.e. the relationship between consumers' intake rate and resource density) is central in plant-herbivore interactions. Its shape and the biological processes leading to it have significant implications for both foraging theory and ecology of grazing systems. A type IV functional response (i.e. dome-shaped relationship) of short-term intake rate of dry matter (intake while grazing) has rarely been reported for large herbivores and the conditions that can lead to it are poorly understood. We report a type IV functional response observed in heifers grazing monocultures of Cynodon sp. and Avena strigosa. The mechanisms and consequences of this type of functional response for grazed system dynamics are discussed. Intake rate was higher at intermediate than at short or tall sward heights in both grass species. The type IV functional response resulted from changes in bite mass instead of a longer time needed to encounter and process bites. Thus, the decrease of intake rate of dry matter in tall swards is not explained by a shift from process 3 (potential bites are concentrated and apparent) to process 2 (potential bites are apparent but dispersed, Spalinger & Hobbs 1992). Bite mass was smaller in tall than in intermediate swards due to a reduction of bite volume possibly caused by the greater proportion of stem and sheath acting as a physical barrier to bite formation. It is generally accepted that potential bites are abundant and apparent in most grassland and meadow systems, as they were in the present experiments. Therefore, a type IV response of intake rate not directly related to digestive constraints may determine the dynamics of intake and defoliation under a much larger set of conditions than previously thought. These results have implications for foraging theory and stability of grazing systems. For example, if animals prefer patches of intermediate stature that yield the highest intake rate, grazing should lead to the widely observed bimodal distribution of plant mass per unit area, even when tall patches are not of significantly lower digestive quality than the pasture average.