Identification of Antiprotozoal Compounds from Buxus sempervirens L. by PLS-Prediction.

Various nor-triterpene alkaloids of Buxus (B.) sempervirens L. have shown remarkable in vitro activity against the causative agents of tropical malaria and East African sleeping sickness. To identify further antiprotozoal compounds of this plant, 20 different fractions of B. sempervirens L., exhibiting a wide range of in vitro bioactivity, were analyzed by UHPLC/+ESI-QqTOF-MS/MS. The analytical profiles were investigated by partial least squares regression (PLS) for correlations between the intensity of LC/MS signals, bioactivity and cytotoxicity. The resulting models highlighted several compounds as mainly responsible for the antiprotozoal activity and thus, worthwhile for subsequent isolation. These compounds were dereplicated based on their mass spectra in comparison with isolated compounds recently reported by us and with literature data. Moreover, an estimation of the cytotoxicity of the highlighted compounds was derived from an additional PLS model in order to identify plant constituents with strong selectivity. In conclusion, high levels of antitrypanosomal and antiplasmodial activity were predicted for eight and four compounds, respectively. These include three hitherto unknown constituents of B. sempervirens L., presumably new natural products.

Topographical structures in planting design of living walls affect their ability to immobilise traffic-based particulate matter.

Traffic-generated particulate matter (PM) pollution is a serious threat to human health and the environment, especially in urban settings. Recent studies have revealed the effectiveness of living walls in the reduction of this pollution; these systems use variable planting designs and their topographical dynamics might have an impact on PM dry deposition. This present study, employing an experimentally manipulable living wall system using box (Buxus sempervirens L.) plants, examined whether plants arranged in a design with heterogeneous topography have a differential PM removal capacity compared to plants in a design with homogenous topography. Two planting designs using 'short' and 'tall' plants, were simultaneously used on this living wall and equally exposed to traffic-based PM for 5 consecutive days. PM accumulation on leaves was estimated using an Environmental Scanning Electron Microscope and ImageJ image analysis software. The experiment was replicated four times changing the position of each design on the wall, and any variation in PM capture levels on leaves belonging to different designs were identified using a Generalised Linear Mixed-effect Models (GLMM). The planting design with topographical heterogeneity resulted in significantly higher PM densities (PM10, PM2.5 and PM1) on leaf surfaces compared to a design with homogenous topography, indicating that topographical heterogeneity has a strong positive impact on the ability of plants to immobilise PM.

Effect of amendments on contaminated soil of multiple heavy metals and accumulation of heavy metals in plants.

The contamination of soil with heavy metals is a severe problem due to adverse impact of heavy metals on environmental safety and human health. It is essential to remediate soil contaminated with heavy metals. This study has evaluated the effects of pine biochar, kaolin, and triple super phosphate (TSP) on multiple heavy metals (Ni, Zn, Cu, and Cd) in contaminated soil and accumulation of heavy metals in plants. The amendments can reduce availability of heavy metals in soil by increasing pH, adsorption, complexation, or co-precipitation. Different amendments have variable effects on accumulation of heavy metals in plants and in soil due to its diverse mechanism of stability. The results showed that application of triple super phosphate (TSP) has significant reduced soil Cd exchangeable (EXC) fraction from 58.59 to 21.30%. Bound to carbonates (CAR) fraction decreased from 9.84 to 5.11%, and bound to Fe-Mn oxides (OX) fraction increased from 29.61 to 69.86%. The triple super phosphate (TSP) has the ability to stabilize Cu and especially Cd. However, triple super phosphate (TSP) has enhanced ecological risk of Zn and Ni. Application of pine biochar has significantly enhanced soil pH. The kaolin has significantly reduced EXC fraction of Cd and increased OX fraction of Cu. The amendments and heavy metals have not caused significant effect on SPAD value of Buxus microphylla Siebold & Zucc (B. microphylla). The triple super phosphate (TSP) has significant decreased biomass of B. microphylla and bamboo-williow (Salix sp.) by 24.91 and 57.43%, respectively. Pine biochar and kaolin have increased the accumulation of Zn and Cd in plants. It is concluded that triple super phosphate (TSP) was effective in remediation of Cd and kaolin was effective in remediation of Cd and Cu. Pine biochar was effective in remediation of Cd, Cu, and Zn.

Altitudinal and seasonal changes of phenolic compounds in Buxus sempervirens leaves and cuticles.

The variation in the leaf content of phenolic compounds has been related to the UV-B changes of the environment in which plants grow. In this context, we aimed to investigate: a) whether the seasonal and altitudinal changes in the content of phenolic compounds of Buxus sempervirens L. leaves and cuticles could be related to the natural fluctuations in UV-B levels and b) the possible use of specific phenolic compounds as biomarkers of ambient UV-B levels. To achieve these goals we sampled, every three months during one year, leaves of B. sempervirens along an altitudinal gradient. At the lowest and the highest altitudes, we also conducted a UV-exclusion experiment to discern whether the observed changes could be attributed to the natural variation in UV-B. Results show that total phenolic content of leaves was lower in June than in the other sampling dates, which suggests a leaf ontogenic rather than a UV-B effect on the leaf content of these compounds. Regarding the elevational gradient, the overall amount of phenolic acids and neolignan of entire leaves increased with altitude while the total amount of flavonoids in leaf cuticles decreased. However, the lack of a significant effect of our UV-exclusion treatment on the content of these compounds suggests that the observed variations along the altitudinal gradient would respond to other factors rather than to UV-B. Concomitantly, we did not find any phenolic compound in leaves or cuticles of B. sempervirens that could be considered as a biomarker of ambient UV-B levels.

Measurement of carbon flux through the MEP pathway for isoprenoid synthesis by (31)P-NMR spectroscopy after specific inhibition of 2-C-methyl-d-erythritol 2,4-cyclodiphosphate reductase. Effect of light and temperature.

The methylerythritol 4-phosphate (MEP) and the mevalonate pathways are the unique synthesis routes for the precursors of all isoprenoids. An original mean to measure the carbon flux through the MEP pathway in plants is proposed by using cadmium as a total short-term inhibitor of 2-C-methyl-d-erythritol 2,4-cyclodiphosphate (MEcDP) reductase (GcpE) and measuring the accumulation rate of its substrate MEcDP by (31) P-NMR spectroscopy. The MEP pathway metabolic flux was determined in spinach (Spinacia oleracea), pea (Pisum sativum), Oregon grape (Mahonia aquifolium) and boxwood (Buxus sempervirens) leaves. In spinach, flux values were compared with the synthesis rate of major isoprenoids. The flux increases with light intensity (fourfold in the 200-1200 µmol m(-2) s(-1) PPFR range) and temperature (sevenfold in the 25-37 °C range). The relationship with the light and the temperature dependency of isoprenoid production downstream of the MEP pathway is discussed.

Plasticity of photoprotective mechanisms of Buxus sempervirens L. leaves in response to extreme temperatures.

Summer 2003 was unusually hot in Western Europe, with local droughts and an intense heatwave, that led to a massive damage in vegetation. Since high temperatures are supposed to generate photooxidative stress, we analysed photoprotective responses in leaves of the evergreen boxtree (Buxus sempervirens L.) during summer 2003. All the photoprotective compounds analysed (alpha-tocopherol, beta-carotene, and xanthophylls cycle pigments) were simultaneously induced in parallel with a reduction in photochemical efficiency (Fv/Fm). To characterise these responses, we compared these data with other data obtained during cold stress periods (2003, 2005) and with an unstressful summer (2002). Photoprotective responses observed during the heatwave were also induced by low temperature stress, and in both situations, this effect was exacerbated by light. In parallel with such induction the accumulation of red retro-carotenoids and xanthophyll esters was also observed under unfavourable conditions, suggesting a photoprotective role for both groups of carotenoids. This is the first report showing that in any species (Buxus sempervirens L.), the same retro-carotenoids can be induced in response to winter and summer stress. Present results demonstrate that the same mechanisms are induced as response to sub- and supraoptimal temperatures and the plasticity of such responses plays a critical role in plant acclimation to extreme temperatures, an ability that is specially important in the context of any future climate warming.

Role of red carotenoids in photoprotection during winter acclimation in Buxus sempervirens leaves.

The red leaf coloration of several plant species during autumn and winter is due to the synthesis of phenolic compounds such as anthocyanins or red carotenoids. The latter occur very rarely and are non-ubiquitous and taxonomically restricted compounds. The present study shows that the leaves of common box ( Buxus sempervirens L.) accumulate red carotenoids (eschscholtzxanthin, monoanhydroeschscholtzxanthin, anhydroeschscholtzxanthin) as a response to photoinhibitory conditions during winter acclimation. These compounds are produced in a coordinated manner with the operation of other photoprotective systems: accumulation and sustained deepoxidation of VAZ pigments with a concomitant decrease in maximal photochemical efficiency, accumulation of alpha-tocopherol and a gradual decrease on chlorophyll content. All these processes were reversed when the photosynthetic tissues were transferred from photoinhibitory winter conditions to room temperature for 9 days. Buxus leaves showed a large degree of phenotype variation in the degree of reddening, ranging from green to orange. The differences in colour pattern were mainly due to differences in the accumulation of red carotenoids and xanthophyll esters. Red pigments were mainly anhydroeschscholtzxanthin and esters of eschscholtzxanthin. Conversely to fruit or petal chromoplasts, the plastids of red leaves in this species are not the terminal differentiated state but are able to redifferentiate again to chloroplasts. Their photoprotective role during winter as a light screen system or as antioxidants, in a similar way to other red pigments, and their implications on the wide ecological tolerance of this evergreen species are discussed.