Synthesis and Structural Activity Relationship Study of Ursolic Acid Derivatives as Antitubercular Agent.

OBJECTIVE: The chemical transformation of ursolic acid (UA) into novel C-3 aryl ester derivatives and in vitro and silico assessment of their antitubercular potential. BACKGROUND: UA is a natural pentacyclic triterpenoid with many pharmacological properties. Semisynthetic UA analogs have demonstrated enhanced anticancer, antimalarial, and antifilarial properties in our previous studies. METHOD: The C-30 carboxylic group of previously isolated UA was protected, and various C-3 aryl ester derivatives were semi-synthesized. The agar dilution method was used to evaluate the in vitro antitubercular efficacy of Mycobacterium tuberculosis (Mtb) H37Ra. In silico docking studies of the active derivative were carried out against Mtb targets, catalase peroxidase (PDB: 1SJ2), dihydrofolate reductase (PDB: 4M2X), enoyl-ACP reductase (PDB: 4TRO), and cytochrome bc1 oxidase (PDB: 7E1V). RESULTS: The derivative 3-O-(2-amino,3-methyl benzoic acid)-ethyl ursolate (UA-1H) was the most active among the eight derivatives (MIC1 2.5 µg/mL) against Mtb H37Ra. Also, UA-1H demonstrated significant binding affinity in the range of 10.8-11.4 kcal/mol against the antiTb target proteins, which was far better than the positive control Isoniazid, Ethambutol, and co-crystallized ligand (HEM). Moreover, the predicted hit UA-1H showed no inhibition of Cytochrome P450 2D6 (CYP2D6), suggesting its potential for favorable metabolism in Phase I clinical studies. CONCLUSION: The ursolic acid derivative UA-1H possesses significant in vitro antitubercular potential with favorable in silico pharmacokinetics. Hence, further in vivo assessments are suggested for UA-1H for its possible development into a secure and efficient antitubercular drug.

Acylphloroglucinol-monoterpene meroterpenoids from Eucalyptus tereticornis and their inhibitory activity against ATP citrate lyase.

Five undescribed enantiomeric pairs of acylphloroglucinol-monoterpene meroterpenoids ((+)-/(-)-eucateretins A-E) resolved by chiral-phase HPLC were obtained from the leaves of Eucalyptus tereticornis Smith, along with nine known analogues. Their structures were elucidated by spectroscopic methods and ECD calculations. This is the first report of meroterpenoid enantiomers from this plant. Some of the isolates, (-)-eucateretin A, (+)-/(-)-eucateretins E, 7'α-eucalrobusone X, eucalrobusone X, and robustadial B, exhibited inhibitory effects on ATP citrate lyase, and 7'α-eucalrobusone X significantly suppressed the hepatocyte lipogenesis.

Chemical composition, in vitro and in silico evaluation of essential oil from Eucalyptus tereticornis leaves for lung cancer.

Chemical composition of the essential oil (EO) of Eucalyptus tereticornis leaves was studied by gas chromatography-mass spectrometry. Forty-five constituents were identified in the oil hydrodistilled from the sample collected from Ghudda Village, Bathinda (Pb), India of which eucalyptol (34.39%) and ledol (9.92%) were the major constituents. In vitro antioxidant and anticancer potential of EO was analysed by DPPH 2,2-diphenylpicrylhydrazyl (DPPH) and MTT assay. The percentage free radical scavenging activity was found to be 63.77%. The antiproliferative activity was analysed using MTT assay in adenocarcinomic human alveolar basal epithelial A549 cancer cell line and showed IC50 value of 47.14 µg/ml. In silico study of EO, constituents were performed using Maestro 12.9 against EGFR (PDB ID-2RGP). Five constituents from EO showed high dockscore as compared to standard Mobicertinib which indicated the effectiveness of oil constituents against lung cancer.

The temperature optima for tree seedling photosynthesis and growth depend on water inputs.

Understanding how tree growth is affected by rising temperature is a key to predicting the fate of forests in future warmer climates. Increasing temperature has direct effects on plant physiology, but there are also indirect effects of increased water limitation because evaporative demand increases with temperature in many systems. In this study, we experimentally resolved the direct and indirect effects of temperature on the response of growth and photosynthesis of the widely distributed species Eucalyptus tereticornis. We grew E. tereticornis in an array of six growth temperatures from 18 to 35.5°C, spanning the climatic distribution of the species, with two watering treatments: (a) water inputs increasing with temperature to match plant demand at all temperatures (Wincr ), isolating the direct effect of temperature; and (b) water inputs constant for all temperatures, matching demand for coolest grown plants (Wconst ), such that water limitation increased with growth temperature. We found that constant water inputs resulted in a reduction of temperature optima for both photosynthesis and growth by ~3°C compared to increasing water inputs. Water limitation particularly reduced the total amount of leaf area displayed at Topt and intermediate growth temperatures. The reduction in photosynthesis could be attributed to lower leaf water potential and consequent stomatal closure. The reduction in growth was a result of decreased photosynthesis, reduced total leaf area display and a reduction in specific leaf area. Water availability had no effect on the response of stem and root respiration to warming, but we observed lower leaf respiration rates under constant water inputs compared to increasing water inputs at higher growth temperatures. Overall, this study demonstrates that the indirect effect of increasing water limitation strongly modifies the potential response of tree growth to rising global temperatures.

Elevated CO2 does not affect stem CO2 efflux nor stem respiration in a dry Eucalyptus woodland, but it shifts the vertical gradient in xylem [CO2 ].

To quantify stem respiration (RS ) under elevated CO2 (eCO2 ), stem CO2 efflux (EA ) and CO2 flux through the xylem (FT ) should be accounted for, because part of respired CO2 is transported upwards with the sap solution. However, previous studies have used EA as a proxy of RS , which could lead to equivocal conclusions. Here, to test the effect of eCO2 on RS , both EA and FT were measured in a free-air CO2 enrichment experiment located in a mature Eucalyptus native forest. Drought stress substantially reduced EA and RS , which were unaffected by eCO2 , likely as a consequence of its neutral effect on stem growth in this phosphorus-limited site. However, xylem CO2 concentration measured near the stem base was higher under eCO2 , and decreased along the stem resulting in a negative contribution of FT to RS , whereas the contribution of FT to RS under ambient CO2 was positive. Negative FT indicates net efflux of CO2 respired below the monitored stem segment, likely coming from the roots. Our results highlight the role of nutrient availability on the dependency of RS on eCO2 and suggest stimulated root respiration under eCO2 that may shift vertical gradients in xylem [CO2 ] confounding the interpretation of EA measurements.

Elevated CO2 did not affect the hydrological balance of a mature native Eucalyptus woodland.

Elevated atmospheric CO2 concentration (eCa ) might reduce forest water-use, due to decreased transpiration, following partial stomatal closure, thus enhancing water-use efficiency and productivity at low water availability. If evapotranspiration (Et ) is reduced, it may subsequently increase soil water storage (ΔS) or surface runoff (R) and drainage (Dg ), although these could be offset or even reversed by changes in vegetation structure, mainly increased leaf area index (L). To understand the effect of eCa in a water-limited ecosystem, we tested whether 2 years of eCa (~40% increase) affected the hydrological partitioning in a mature water-limited Eucalyptus woodland exposed to Free-Air CO2 Enrichment (FACE). This timeframe allowed us to evaluate whether physiological effects of eCa reduced stand water-use irrespective of L, which was unaffected by eCa in this timeframe. We hypothesized that eCa would reduce tree-canopy transpiration (Etree ), but excess water from reduced Etree would be lost via increased soil evaporation and understory transpiration (Efloor ) with no increase in ΔS, R or Dg . We computed Et , ΔS, R and Dg from measurements of sapflow velocity, L, soil water content (θ), understory micrometeorology, throughfall and stemflow. We found that eCa did not affect Etree , Efloor , ΔS or θ at any depth (to 4.5 m) over the experimental period. We closed the water balance for dry seasons with no differences in the partitioning to R and Dg between Ca levels. Soil temperature and θ were the main drivers of Efloor while vapour pressure deficit-controlled Etree , though eCa did not significantly affect any of these relationships. Our results suggest that in the short-term, eCa does not significantly affect ecosystem water-use at this site. We conclude that water-savings under eCa mediated by either direct effects on plant transpiration or by indirect effects via changes in L or soil moisture availability are unlikely in water-limited mature eucalypt woodlands.

A common thermal niche among geographically diverse populations of the widely distributed tree species Eucalyptus tereticornis: No evidence for adaptation to climate-of-origin.

Impacts of climate warming depend on the degree to which plants are constrained by adaptation to their climate-of-origin or exhibit broad climatic suitability. We grew cool-origin, central and warm-origin provenances of Eucalyptus tereticornis in an array of common temperature environments from 18 to 35.5°C to determine if this widely distributed tree species consists of geographically contrasting provenances with differentiated and narrow thermal niches, or if provenances share a common thermal niche. The temperature responses of photosynthesis, respiration, and growth were equivalent across the three provenances, reflecting a common thermal niche despite a 2,200 km geographic distance and 13°C difference in mean annual temperature at seed origin. The temperature dependence of growth was primarily mediated by changes in leaf area per unit plant mass, photosynthesis, and whole-plant respiration. Thermal acclimation of leaf, stem, and root respiration moderated the increase in respiration with temperature, but acclimation was constrained at high temperatures. We conclude that this species consists of provenances that are not differentiated in their thermal responses, thus rejecting our hypothesis of adaptation to climate-of-origin and suggesting a shared thermal niche. In addition, growth declines with warming above the temperature optima were driven by reductions in whole-plant leaf area and increased respiratory carbon losses. The impacts of climate warming will nonetheless vary across the geographic range of this and other such species, depending primarily on each provenance's climate position on the temperature response curves for photosynthesis, respiration, and growth.

Coupled response of stomatal and mesophyll conductance to light enhances photosynthesis of shade leaves under sunflecks.

Light gradients within tree canopies play a major role in the distribution of plant resources that define the photosynthetic capacity of sun and shade leaves. However, the biochemical and diffusional constraints on gas exchange in sun and shade leaves in response to light remain poorly quantified, but critical for predicting canopy carbon and water exchange. To investigate the CO2 diffusion pathway of sun and shade leaves, leaf gas exchange was coupled with concurrent measurements of carbon isotope discrimination to measure net leaf photosynthesis (An ), stomatal conductance (gs ) and mesophyll conductance (gm ) in Eucalyptus tereticornis trees grown in climate controlled whole-tree chambers. Compared to sun leaves, shade leaves had lower An , gm , leaf nitrogen and photosynthetic capacity (Amax ) but gs was similar. When light intensity was temporarily increased for shade leaves to match that of sun leaves, both gs and gm increased, and An increased to values greater than sun leaves. We show that dynamic physiological responses of shade leaves to altered light environments have implications for up-scaling leaf level measurements and predicting whole canopy carbon gain. Despite exhibiting reduced photosynthetic capacity, the rapid up-regulation of gm with increased light enables shade leaves to respond quickly to sunflecks.

Does physiological acclimation to climate warming stabilize the ratio of canopy respiration to photosynthesis?

Given the contrasting short-term temperature dependences of gross primary production (GPP) and autotrophic respiration, the fraction of GPP respired by trees is predicted to increase with warming, providing a positive feedback to climate change. However, physiological acclimation may dampen or eliminate this response. We measured the fluxes of aboveground respiration (Ra ), GPP and their ratio (Ra /GPP) in large, field-grown Eucalyptus tereticornis trees exposed to ambient or warmed air temperatures (+3°C). We report continuous measurements of whole-canopy CO2 exchange, direct temperature response curves of leaf and canopy respiration, leaf and branch wood respiration, and diurnal photosynthetic measurements. Warming reduced photosynthesis, whereas physiological acclimation prevented a coincident increase in Ra . Ambient and warmed trees had a common nonlinear relationship between the fraction of GPP that was respired above ground (Ra /GPP) and the mean daily temperature. Thus, warming significantly increased Ra /GPP by moving plants to higher positions on the shared Ra /GPP vs daily temperature relationship, but this effect was modest and only notable during hot conditions. Despite the physiological acclimation of autotrophic respiration to warming, increases in temperature and the frequency of heat waves may modestly increase tree Ra /GPP, contributing to a positive feedback between climate warming and atmospheric CO2 accumulation.

Using models to guide field experiments: a priori predictions for the CO2 response of a nutrient- and water-limited native Eucalypt woodland.

The response of terrestrial ecosystems to rising atmospheric CO2 concentration (Ca ), particularly under nutrient-limited conditions, is a major uncertainty in Earth System models. The Eucalyptus Free-Air CO2 Enrichment (EucFACE) experiment, recently established in a nutrient- and water-limited woodland presents a unique opportunity to address this uncertainty, but can best do so if key model uncertainties have been identified in advance. We applied seven vegetation models, which have previously been comprehensively assessed against earlier forest FACE experiments, to simulate a priori possible outcomes from EucFACE. Our goals were to provide quantitative projections against which to evaluate data as they are collected, and to identify key measurements that should be made in the experiment to allow discrimination among alternative model assumptions in a postexperiment model intercomparison. Simulated responses of annual net primary productivity (NPP) to elevated Ca ranged from 0.5 to 25% across models. The simulated reduction of NPP during a low-rainfall year also varied widely, from 24 to 70%. Key processes where assumptions caused disagreement among models included nutrient limitations to growth; feedbacks to nutrient uptake; autotrophic respiration; and the impact of low soil moisture availability on plant processes. Knowledge of the causes of variation among models is now guiding data collection in the experiment, with the expectation that the experimental data can optimally inform future model improvements.

The capacity to cope with climate warming declines from temperate to tropical latitudes in two widely distributed Eucalyptus species.

As rapid climate warming creates a mismatch between forest trees and their home environment, the ability of trees to cope with warming depends on their capacity to physiologically adjust to higher temperatures. In widespread species, individual trees in cooler home climates are hypothesized to more successfully acclimate to warming than their counterparts in warmer climates that may approach thermal limits. We tested this prediction with a climate-shift experiment in widely distributed Eucalyptus tereticornis and E. grandis using provenances originating along a ~2500 km latitudinal transect (15.5-38.0°S) in eastern Australia. We grew 21 provenances in conditions approximating summer temperatures at seed origin and warmed temperatures (+3.5 °C) using a series of climate-controlled glasshouse bays. The effects of +3.5 °C warming strongly depended on home climate. Cool-origin provenances responded to warming through an increase in photosynthetic capacity and total leaf area, leading to enhanced growth of 20-60%. Warm-origin provenances, however, responded to warming through a reduction in photosynthetic capacity and total leaf area, leading to reduced growth of approximately 10%. These results suggest that there is predictable intraspecific variation in the capacity of trees to respond to warming; cool-origin taxa are likely to benefit from warming, while warm-origin taxa may be negatively affected.

Hybrid purity assessment in Eucalyptus F1 hybrids using microsatellite markers.

The worldwide expansion of hybrid breeding and clonal forestry is to meet the demands of paper pulp and bioenergy. Although India was one of the pioneers in hybrid production of eucalypts only recently the hybrid clonal forestry is gaining momentum. Inter-specific hybrids are being produced to exploit the hybrid vigor of F1 individuals. Quality control genotyping for hybrid purity and parentage confirmation at the early stage is one of the essential criteria for clonal propagation and field trails for the assessment of growth performance. Eucalyptus being a obligatory outcrossed species with potential to self pollination, possibilities of pollen contamination are high. Hence, in the present study, Eucalyptus camaldulensis × E. tereticornis inter-specific hybrids were genotyped using 25 fluorescent labeled microsatellite markers available in public domain. Multiplex loading of PCR products was performed successfully for most of the microsatellite loci. Hybrid purity index was calculated and parentage was confirmed. Hybrid purity values ranged from 85 to 100 % showed the efficiency of controlled pollination techniques. A subset of six fully informative simple sequence repeats was identified for routine quality control genotyping for these hybrids. Detection of non-essential genotypes observed among the hybrid seedlings proved the significance of hybrid purity tests and the false hybrids were removed at the seedling stage. The hybrids with proven hybridity will be used for generation of genetic linkage, discovery of quantitative trait loci and the individuals with high productivity can enter into mass clonal multiplication.

Actividad insecticida de una emulsión aceite/agua del aceite esencial de Eucalyptus tereticornis / Insecticide activity of oil/water emulsion of Eucalyptus tereticornis essential oil

Introducción: el aceite esencial extraído de especies del género Eucalyptus, entre estas Eucalyptus tereticornis, ofrece un importante potencial para el control de hongos patógenos e insectos plaga y de importancia médica. Objetivo: evaluar la actividad anti-insecto (aguda, crónica y repelente) de una emulsión aceite/agua del aceite esencial de Eucalyptus tereticornis, en función de su composición química y fraccionamiento. Métodos: se prepararon emulsiones aceite/agua con aceite esencial previamente extraído, separado y caracterizado, y 2 de sus fracciones (A y B). Se evaluó su actividad anti-insecto mediante bioensayos con Drosophila melanogaster. Se determinó la fitotoxicidad foliar sobre plántulas de Phaseolus vulgaris. Resultados: el aceite completo mostró considerable actividad repelente e insecticida aguda que guarda relación con su composición química. La actividad insecticida aguda de las fracciones disminuyó de manera notable con respecto a la mostrada por el aceite completo, sin embargo, no se observaron efectos significativos sobre la actividad repelente. En ningún caso se presentó fitotoxicidad foliar sobre frijol. Conclusión: el aceite esencial de esta especie de eucalipto posee actividad anti-insecto considerable y podría formularse directamente como insecticida para el control de insectos de interés agrícola y médico.

Introduction: the essential oil extracted from Eucalyptus species, including Eucalyptus tereticornis, offers significant potential for control of fungal pathogens and insect pests and medical importance. Objectives: to evaluate the anti-insect activity (acute, chronic and repellent) of an oil/water emulsion of Eucalyptus tereticornis essential oil in terms of their chemical composition. Methods: oil/water emulsion was prepared with essential oil previously extracted, separated and characterized, and two of its fractions (A and B). Their anti-insect activity was evaluated through bioassays on Drosophila melanogaster. Foliar phytotoxicity was evaluated on seedlings of Phaseolus vulgaris. Results: the complete oil showed considerable acute repellent and insecticide activity related to its chemical composition. The acute insecticidal activity of the fractions decreased significantly if compare with that of the whole oil , but neither significant effects on the repellent activity nor phytotoxicity on the bean leaves were observed. Conclusions: the essential oil of this eucalyptus species has considerable anti-insect properties and could be formulated as an insecticide for the control of insects of agricultural and medical significance.

Fungicidal activity of Eucalyptus tereticornis essential oil on the pathogenic fungus Fusarium oxysporum / Actividad antimicótica del aceite esencial a partir de Eucalyptus tereticornis sobre el hongo patógeno Fusarium oxysporum

The objective of present paper was to determine the antifungal activity of the Eucalyptus tereticornis (Myrtaceae) essential oil and two fractions on the Fusarium oxysporum mushroom, a pathogen with clinical and agricultural significance. The total citronelal (44.8 percent) and geraniol (9.78 percent) essential oil had a fungicidal effect at a 3 g/L concentration and a fungicidal activity at small concentrations. The A and B fractions composed most of p-mentane-3,8-diol (18.95 percent) and geraniol acetate (24.34 percent), respectively were more active than the total extract. The observations at microscopic level showed damages and changes in hyphae and chlamydospores, as well as a decrease in the number of conidia. The observed fungicidal activity and the morphologic damages were dependent on the concentration.

El objetivo de este trabajo fue determinar la actividad antifúngica del aceite esencial de Eucalyptus tereticornis (Myrtaceae) y 2 fracciones sobre el hongo Fusarium oxysporum, patógeno de importancia tanto clínica como agrícola. El aceite esencial total, compuesto principalmente por citronelal (44,8 por ciento), citronelol (9,78 por ciento) presentó un efecto fungicida a una concentración de 3 g/L y actividad fungistática a concentraciones menores. La fracciones A y B compuestas en su mayoría por p-mentano-3,8-diol (18,95 por ciento) y acetato de citronelol (24,34 por ciento) respectivamente fueron más activas que el extracto total. Las observaciones a nivel microscópico mostraron daños y cambios en hifas y clamidosporas, así como disminución en el número de conidias. La actividad fungistática observada y los daños morfológicos fueron dependientes de la concentración.

Component Analysis of Essential Oil Extracted with Supercritical CO_2 from Eucalyptus tereticornis by GC-MS / 中国药房

OBJECTIVE:To analyze the chemical components extracted with supercritical CO2(SFE-CO2)from Eucalyptus tereticornis.METHODS: Volatile oil was extracted from E.tereticornis with SFE-CO2.The chemical component analyzed by GC-MS and its proportion was determined by normalization method.RESULTS: 28 compounds which account for 88.13% of the total peak area were separated and identified.The main components of volatile oil were eucalyptol (33.99%),borneol(8.88%),?-pinene (5.39%),caryophyllene (4.51%),(+)-4-carene (4.19%).CONCLUSION:This study can be served as a scientific basis for the further exploitation and utilization of E.tereticornis.

Micropropagation of 4-year-old elite Eucalyptus tereticornis trees.

A protocol for the micropropagation of mature Eucalyptus tereticornis Smith has been developed using regenerated shoots from axillary bud explants. The trees were selected on the basis of their better growth rate, physical and phenotypic characteristics and freedom from disease. Regeneration was obtained in modified Murashige and Skoog (1962) medium. Evaluation of explant regeneration throughout the year indicated that the incidence of browning of explants was maximum during the month of February, while dominance of the microbes in endogenously infected explants peaked in August-September. Regeneration from primary explants was maximum during the months of March-April. Subcultures were carried out every 4 weeks. Effects of hormones and media composition on regeneration and growth were studied. Phytagel induced vitrification, while calcium chloride dihydrate reduced vitrification and induced the elongation of shoots. Best rooting was obtained with half-strength, modified MS medium supplemented with 1.0 mg/l indolebutyric acid. Plantlets were hardened in a nonsterile potting mix at high humidity and gradually exposed to the ambient environment over a period of 6 weeks, and upon transfer to field conditions the survival rate varied from 84% to 100%.

The effects of elevated CO2 atmospheres on the nutritional quality of Eucalyptus foliage and its interaction with soil nutrient and light availability.

Seedlings of Eucalyptus tereticornis (Smith) were grown under two levels of availability each of CO2 (352 and 793 µmol mol-1), soil nutrients (1/24 and 1/4 Hoagland's solution) and light (full and 30% sunlight). Low soil nutrient availability or high light increased the C:N ratio of leaves, leading to lower leaf nitrogen concentrations, higher leaf specific weights and higher levels of both total phenolics and condensed tannins. These results were consistent with other studies of the effect of environmental resource availability on foliage composition. Similar results were observed when the C:N ratio of leaves was increased under elevated CO2. The changes in leaf chemistry induced by the treatments affected the performance of 4th-instar larvae of Chrysophtharta flaveola (Chapuis) fed on the leaves. Increased C:N ratios of leaves reduced digestive efficiencies and pupal body sizes and increased mortality. Below a threshold nitrogen concentration of approximately 1% dry mass, severe reductions in the performance of larvae were recorded. Such changes may have significant consequences for herbivores of Eucalyptus, particularly in view of projected increases in atmospheric CO2.