Exploring of the ameliorative effects of Nerium (Nerium oleander L.) ethanolic flower extract in streptozotocin induced diabetic rats via biochemical, histological and molecular aspects.

BACKGROUND: Nerium oleander L. is ethnopharmacologically used for diabetes. Our aim was to investigate the ameliorative effects of ethanolic Nerium flower extract (NFE) in STZ-induced diabetic rats. METHODS: Seven random groups including control group, NFE group (50 mg/kg), diabetic group, glibenclamide group and NFE treated groups (25 mg/kg, 75 mg/kg, and 225 mg/kg) were composed of forty-nine rats. Blood glucose level, glycated hemoglobin (HbA1c), insulin level, liver damage parameters and lipid profile parameters were investigated. Antioxidant defense system enzyme activities and reduced glutathione (GSH) and malondialdehyde (MDA) contents and immunotoxic and neurotoxic parameters were determined in liver tissue. Additionally, the ameliorative effects of NFE were histopathologically examined in liver. mRNA levels of SLC2A2 gene encoding glucose transporter 2 protein were measured by quantitative real time PCR. RESULTS: NFE caused decrease in glucose level and HbA1c and increase in insulin and C-peptide levels. Additionally, NFE improved liver damage biomarkers and lipid profile parameters in serum. Moreover, lipid peroxidation was prevented and antioxidant enzyme activities in liver were regulated by NFE treatment. Furthermore, anti-immunotoxic and anti-neurotoxic effects of NFE were determined in liver tissue of diabetic rats. Histopathogically, significant liver damages were observed in the diabetic rats. Histopathological changes were decreased partially in the 225 mg/kg NFE treated group. SLC2A2 gene expression in liver of diabetic rats significantly reduced compared to healthy rats and NFE treatment (25 mg/kg) caused increase in gene expression. CONCLUSION: Flower extract of Nerium plant may have an antidiabetic potential due to its high phytochemical content.

Comparison of Phytoremediation Potential of Nerium indicum with Inorganic Modifier Calcium Carbonate and Organic Modifier Mushroom Residue to Lead-Zinc Tailings.

At present, the application of phytoremediation technology in the ecological remediation of heavy metal tailings is receiving more and more attention. In this study, the physiological and biochemical response and tolerance mechanism of woody plant Nerium indicum to Pb and Zn under different proportions of inorganic modifier calcium carbonate (C1: 5%, C2: 10%, C3: 15%) and organic modifier mushroom residue (M1: 10%, M2: 20%, M3: 30%) was compared. The results showed that the pH value has a trend of C group > M group > CK group and organic matter has a trend of M group > CK group > C group. Phosphatase activity and catalase activity has a trend of M group > C group > CK group, but catalase was more vulnerable to the calcium carbonate concentration. Both modifiers can promote the transformation of Pb, Zn, Cu, and Cd in tailings to more stable organic bound and residual states. However, the stabilization effect of mushroom residue is better, and its stability is Pb, Zn > Cd, Cu. Both modifiers can increase the biomass of Nerium indicum and the modification effect of mushroom residue is better than calcium carbonate. Pb/Zn content and accumulation in Nerium indicum organs showed root > stem > leaf in all groups. Compared with the CK group, the enrichment coefficient of Pb/Zn in C1 and M1 groups decreased, while the translocation factor of Pb/Zn in C1 and M1 groups increased. With the increase in modifier concentration, the enrichment coefficient increases about 1.75~52.94%, but the translocation factor decreases rapidly (20.01~64.46%). Clearly, both the calcium carbonate and mushroom residue amendment could promote the growth ability of Nerium indicum in lead−zinc tailings and strengthen the phytoremediation potential.

Characterisation of recombinant thermostable manganese-superoxide dismutase (NeMnSOD) from Nerium oleander.

Superoxide dismutase is one of the key antioxidant enzymes accountable for the eradication of free radicals generated during various metabolic processes. This is first study reporting a thermostable MnSOD obtained from a xerophytic plant, Nerium oleander. The full-length gene identified using Rapid amplification of cDNA ends revealed an open reading frame of 699 bp flanked by 5'UTR and 3'UTR of 134 bp and 198 bp respectively. The corresponding NeMnSOD protein was cloned and expressed in Escherichia coli. The purified protein yields a band of 25.4 kDa, which established a specific activity of 2617 units mg-1 of protein and under native condition yield bands of 52 kDa and 110 kDa, confirming the dimeric and tetrameric state of the protein. The Km and Vmax of 0.078 ± 0.008 mM and 1052.3 ± 33.59 units mg-1 of protein, respectively. The purified enzyme demonstrated thermostability by retaining more than 20% activity at a temperature 70 ℃. The enzyme functioned at pH range of 4-9.0 with maximum activity at pH 7.4. Sodium azide, effectively inhibited the activity of enzyme confirming it to be MnSOD. The enzyme activity was least affected on treatment with strong denaturants (Urea, guanidine HCl and SDS) and harsh chemicals (DTT, CHAPS and ß-mercapto-ethanol) These experimental data validated with Insilco analysis revealed that NeMnSOD possessed thermo as well as kinetically stable moiety which can be further exploited with its applications in the field of pharmaceutical, food and cosmetic industry, which urge for such thermostable enzyme.

Phytoremediation of fluoride with garden ornamentals Nerium oleander, Portulaca oleracea, and Pogonatherum crinitum.

Nursery grown plants of Nerium oleander, Pogonatherum crinitum, and Portulaca oleracea were observed to remove fluoride up to 92, 80, and 73%, respectively, from NaF solution at the concentration of 10 mg L-1 within 15 days. Concentration range of 10-50 mg L-1 of fluoride revealed a constant decrease of removal from 92 to 51% within 15 days by N. oleander, while the biomass (one to five plants) showed enhancement in removal from 74 to 98% in 10 days. Translocation and bioaccumulation factors calculated after fluoride contents in roots and leaves of N. oleander, P. crinitum, and P. oleracea were 1.85, 1.19, and 1.43, and 9.8, 3.6, and 2.2, respectively. P . oleracea, P. crinitum, and N. oleander showed reductions in chlorophyll contents by 40, 57 and 25 and 8%, carbohydrates by 50, 44, and 16%, and proteins by 38, 53, and 15%, respectively. Activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX) in the roots of P. oleracea, P. crinitum, and N. oleander were observed to be induced by 400, 383, and 500%; 80, 105, and 424%; and 153, 77, and 71%, respectively, while the leaves showed induction in SOD, CAT, and GPX activities by 550, 315, and 165%; 196, 227, and 243%; and 280, 242, and 184%, respectively. Results endorsed the superiority of N. oleander for fluoride removal over other plant species.

Examination of Correlation between Histidine and Cadmium Absorption by Eleagnus angustifolia L., Vitis vinifera L. and Nerium oleander L. Using HPLC-MS and ICP-MS.

In this study, HPLC-MS and ICP-MS methods wereused for the determination of histidine and cadmium in Eleagnus angustifolia L., Vitis vinifera L. and Nerium oleander L. leaves taken from industrial area including Gaziantep and Bursa cities. To histidine determination by HPLC-MS, flow rate of mobile phase, fragmentor potential, injection volume and column temperature were optimized as 0.2 mL · min⁻¹, 70 V, 15 µL and 20 °C, respectively. For extraction of histidine from plants, distilled water was used by applying on 90 °C and 30 min. The concentrations (as mg · kg⁻¹) of histidine were found to be in range of 8~22 for Eleagnus angustifolia L., 10~33 for Vitis vinifera L. and 6~11 for Nerium oleander L. The concentrations of cadmium were found to be in ranges of 6~21 µg · kg⁻¹ for Vitis vinifera L. 15~110 µg · kg⁻¹ for Eleagnus angustifolia L. and 63~218 µg · kg⁻¹ for Nerium oleander L.

Effect of various growth hormone concentration and combination on callus induction, nature of callus and callogenic response of Nerium odorum.

Nerium odorum, Linn. (Apocynaceae) is an important evergreen shrub. It is heat, salinity and drought tolerant. Plants with milky sap have medicinal value, mainly cardenolides, flavonoids and terpenes. It is used for wastewater purification and for restoration of riparian woodlands. In view of these facts, the study was conducted for micropropagation of N. odorum. Murashige and Skoog (MS) media supplemented with different concentrations (0.5-10.0 mg/l) of 1-naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzylaminopurine (BAP) and kinetin (Kin) were used singly and in combinations. Among all the growth hormones, 2,4-D was the best for callus induction (75% in stem and 79% in leaf) and in combination 2,4-D and BAP (78% in stem and 81% in leaf). The day of callus induction started from the 19th to the 37th day. This variation is due to the differences in culture conditions and the age of explants. The fresh and dry weight and moisture content showed good growth of callus, which is used in further studies of alkaloid production. Micropropagation of this plant allows the production of clones at a fast rate and in continuous manner. This work can lead to the development of an efficient protocol for callus induction and other issues.

Metal uptake and distribution in cultured seedlings of Nerium oleander L. (Apocynaceae) from the Río Tinto (Huelva, Spain).

Nerium oleander L. (Apocynaceae) is a micro-nano phanerophyte that grows in the riverbanks of the Río Tinto basin (Southwest Iberian Peninsula). The waters and soils of the Río Tinto area are highly acidic and have high concentrations of heavy metals. In this environment, N. oleander naturally grows in both extreme acidic (EA) and less extreme acidic (LEA) water courses, excluding, and bioindicating certain metals. In this work, we compared and evaluated the accumulation preferences and capacities, the distribution and processes of biomineralization of metals (Fe, Cu, Zn, Mn, Mg, Ca) in the first stages of growth of EA and LEA oleanders by means of inductively coupled plasma-mass spectrometry, scanning electron microscopy, and energy dispersive X-ray analyzer analysis. Seeds from both environments were grown and treated with a self-made solution simulating the most extreme red waters from the Río Tinto. LEA plants drastically reduces the metal uptake at the beginning, but later reactivates the uptake reaching concentration values in the same range as the EA plants. The results showed high Mn, Zn and Mg concentrations, accumulation of Fe and Cu in plants from both environments, differing from the metal concentrations of field-grown oleanders. Iron bioformations with traces of other metals were present inside and over epidermal cells and inside vascular cells of stems and roots. They were absent of leaves. The accumulation properties of N. oleander in its early stages of development make it a species to take in consideration in phytoremediation processes but optimized conditions are needed to ensure enough biomass production.

Testing a vapour-phase model of stomatal responses to humidity.

This study tests two predictions from a recently proposed model for stomatal responses to humidity and temperature. The model is based on water potential equilibrium between the guard cells and the air at the bottom of the stomatal pore and contains three independent variables: gs(0), Z and Θ. gs(0) is the value of stomatal conductance that would occur at saturating humidity and will vary among leaves and with CO2 and light. The value of Z is determined primarily by the resistance to heat transfer from the epidermis to the evaporating site and the value of Θ is determined primarily by the resistance to water vapour diffusion from the evaporating site to the guard cells. This leads to the two predictions that were tested. Firstly, the values of Z and Θ should be constant for leaves of a given species grown under given conditions, although gs(0) should vary among leaves and with light and CO2. And secondly, the ratio of Z to Θ should be higher in leaves having their stomata in crypts because the distance for heat transfer is greater than that for water vapour diffusion. Data from three species, Nerium oleander, Pastinaca sativum and Xanthium strumarium support these two predictions.

Evaluation of leaf aqueous extract and synthesized silver nanoparticles using Nerium oleander against Anopheles stephensi (Diptera: Culicidae).

Green nanoparticle synthesis has been achieved using environmentally acceptable plant extract and ecofriendly reducing and capping agents. The present study was carried out to establish the larvicidal activity of synthesized silver nanoparticles (AgNPs) using leaf extract of Nerium oleander (Apocynaceae) against the first to fourth instar larvae and pupae of malaria vector, Anopheles stephensi (Diptera: Culicidae). Nanoparticles are being used in many commercial applications. It was found that aqueous silver ions can be reduced by the aqueous extract of the plant parts to generate extremely stable silver nanoparticles in water. The results were recorded from UV-Vis spectrum, Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), and energy-dispersive X-ray (EDX) spectroscopy analysis. The production of the AgNPs synthesized using leaf extract of N. oleander was evaluated through a UV-Vis spectrophotometer in a wavelength range of 200 to 700 nm. This revealed a peak at 440 nm in N. oleander leaf extracts, indicating the production of AgNPs. The FTIR spectra of AgNPs exhibited prominent peaks at 509.12 cm(-1) (C-H bend alkenes), 1,077.05 cm(-1) (C-O stretch alcohols), 1,600.63 cm(-1) (N-H bend amines), 2,736.49 and 2,479.04 cm(-1) (O-H stretch carboxylic acids), and 3,415.31 cm(-1) (N-H stretching due to amines group). An SEM micrograph showed 20-35-nm-size aggregates of spherical- and cubic-shaped nanoparticles. EDX showed the complete chemical composition of the synthesized nanoparticles of silver. Larvicidal activity of aqueous leaf extract of N. oleander and synthesized AgNPs was carried out against Anopheles stephensi, and the results showed that the highest larval mortality was found in the synthesized AgNPs against the first to fourth instar larvae and pupae of Anopheles stephensi with the following values: LC(50) of instar larvae 20.60, 24.90, 28.22, and 33.99 ppm; LC(90) of instar larvae 41.62, 50.33, 57.78, and 68.41 ppm; and LC(50) and LC(90) of pupae 39.55 and 79.10 ppm, respectively. The aqueous leaf extract exhibited larval toxicity against the first to fourth instar larvae and pupae of Anopheles stephensi with the following values: LC(50) of instar larvae 232.90, 273.71, 318.94, and 369.96 ppm; LC(90) of instar larvae 455.95, 563.10, 639.86, and 730.30 ppm; and LC(50) and LC(90) of pupae 426.01 and 805.13 ppm, respectively. The chi-square value was significant at p < 0.05 level. The possible larvicidal activity may be due to penetration of nanoparticles through a membrane. The results could suggest that the use of plant N. oleander to synthesize silver nanoparticles is a rapid, environmentally safer, and greener approach for mosquito control. This could lead us to a new possibility in vector-control strategy.

Flavonoid and cardenolide glycosides and a pentacyclic triterpene from the leaves of Nerium oleander and evaluation of cytotoxicity.

A pentacyclic triterpene, oleanderocioic acid, two flavonoidal glycosides, quercetin-5-O-[α-L-rhamnopyranosyl-(1→6)]-ß-D-glucopyranoside and kaempferol-5-O-[α-L-rhamnopyranosyl-(1→6)-ß-D-glucopyranoside, and a cardenolide, oleandigoside, together with 11 known compounds, were isolated from the leaves of Nerium oleander. Their structures were elucidated on the basis of spectroscopic analysis. The growth inhibitory and cytotoxic activities of eight compounds were evaluated against the MCF-7 human breast cancer cell line using a sulforhodamine B assay. Three compounds, oleandrin, odoroside A and B were further assayed using a panel of 57 human cancer cell lines.

Control of transpiration by radiation.

The terrestrial hydrological cycle is strongly influenced by transpiration--water loss through the stomatal pores of leaves. In this report we present studies showing that the energy content of radiation absorbed by the leaf influences stomatal control of transpiration. This observation is at odds with current concepts of how stomata sense and control transpiration, and we suggest an alternative model. Specifically, we argue that the steady-state water potential of the epidermis in the intact leaf is controlled by the difference between the radiation-controlled rate of water vapor production in the leaf interior and the rate of transpiration. Any difference between these two potentially large fluxes is made up by evaporation from (or condensation on) the epidermis, causing its water potential to pivot around this balance point. Previous work established that stomata in isolated epidermal strips respond by opening with increasing (and closing with decreasing) water potential. Thus, stomatal conductance and transpiration rate should increase when there is condensation on (and decrease when there is evaporation from) the epidermis, thus tending to maintain homeostasis of epidermal water potential. We use a model to show that such a mechanism would have control properties similar to those observed with leaves. This hypothesis provides a plausible explanation for the regulation of leaf and canopy transpiration by the radiation load and provides a unique framework for studies of the regulation of stomatal conductance by CO(2) and other factors.

Change in uptake, transport and accumulation of ions in Nerium oleander (rosebay) as affected by different nitrogen sources and salinity.

BACKGROUND AND AIMS: The source of nitrogen plays an important role in salt tolerance of plants. In this study, the effects of NaCl on net uptake, accumulation and transport of ions were investigated in Nerium oleander with ammonium or nitrate as the nitrogen source in order to analyse differences in uptake and cycling of ions within plants. METHODS: Plants were grown in a greenhouse in hydroponics under different salt treatments (control vs. 100 mm NaCl) with ammonium or nitrate as the nitrogen source, and changes in ion concentration in plants, xylem sap exuded from roots and stems, and phloem sap were determined. KEY RESULTS: Plant weight, leaf area and photosynthetic rate showed a higher salt tolerance of nitrate-fed plants compared with that of ammonium-fed plants. The total amount of Na+ transported in the xylem in roots, accumulated in the shoot and retranslocated in the phloem of ammonium-fed plants under salt treatment was 1.8, 1.9 and 2.7 times more, respectively, than that of nitrate-treated plants. However, the amount of Na+ accumulated in roots in nitrate-fed plants was about 1.5 times higher than that in ammonium-fed plants. Similarly, Cl- transport via the xylem to the shoot and its retranslocation via the phloem (Cl- cycling) were far greater with ammonium treatment than with nitrate treatment under conditions of salinity. The uptake and accumulation of K+ in shoots decreased more due to salinity in ammonium-fed plants compared with nitrate-fed plants. In contrast, K+ cycling in shoots increased due to salinity, with higher rates in the ammonium-treated plants. CONCLUSIONS: The faster growth of nitrate-fed plants under conditions of salinity was associated with a lower transport and accumulation of Na+ and Cl- in the shoot, whereas in ammonium-fed plants accumulation and cycling of Na+ and Cl- in shoots probably caused harmful effects and reduced growth of plants.

Rapid detection of oleander poisoning by Digoxin III, a new Digoxin assay: impact on serum Digoxin measurement.

We studied the potential for detecting oleander with a new immunoassay (Digoxin III, Abbott Laboratories, Abbott Park, IL) by comparing results with those from the fluorescence polarization immunoassay (FPIA) and Digoxin II assay (Abbott). In aliquots of drug-free serum pools supplemented with pure oleandrin or oleander extract, we observed apparent digoxin values using all 3 immunoassays, but values obtained by the Digoxin III were higher than obtained by the other assays. We also observed significant apparent digoxin values in vivo in serum samples of mice 1 and 2 hours after feeding oleander extract. The average half-life of digoxin-like factors was 1.1 hours. In a serum pool (prepared from patients taking digoxin) supplemented with oleander extract, the observed digoxin values were falsely lowered when measured by the Digoxin II but falsely elevated when measured by the Digoxin III and FPIA. Monitoring free digoxin using the Digoxin III cannot eliminate this interference. Digibind neutralized digoxin-like factors of oleander extract; the effect can be monitored by observing a significant reduction in apparent free digoxin levels in the presence of Digibind as measured in protein-free ultrafiltrate using the Digoxin III. The Digoxin III is highly sensitive for measuring oleander.

Are saturating pulses indeed saturating? Evidence for considerable PSII yield underestimation in leaves adapted to high levels of natural light.

The "saturating pulse" method of in vivo Chl fluorescence measurement has been widely used by physiologists and especially ecophysiologists, as it allows a simple, rapid and non-invasive assessment of PSII function and the allocation of absorbed energy into photochemical and non-photochemical processes. It is based on the accurate determination of the so-called Fm('), i.e. the fluorescence signal emitted when a "saturating" light pulse closes all PSII centers. In this methodological investigation, we examined whether the saturating pulse intensities required to obtain maximal fluorescence yields differ between leaves of various species receiving varying actinic light irradiances. It was shown that, in leaves adapted to comparatively high (yet realistic) levels of natural irradiances, the saturating pulses usually applied are not able to close all PSII reaction centers. As a result, there is a high risk of considerable Fm(') underestimation. Accordingly, the derived values of effective PSII yields and linear electron transport rates (ETR) are also underestimated, even at the highest saturation pulse levels afforded by commercial instruments. Since the extent of underestimation increases with actinic irradiance, the ETR versus light curves are considerably distorted. The possible reasons for the apparent inability of "saturating" pulses to close all PSII centers at high actinic light and the practical implications, especially in field work, are discussed.

Cytotoxic effects of leaf, stem and root extracts of Nerium oleander on leukemia cell lines and role of the p-glycoprotein in this effect.

Nerium oleander (No), is a toxic plant. In recent studies, it was determined that the extracts of this plant are effective to treat some types of cancer, but these studies are limited and do not include human leukemia. In the present study, firstly we aimed to investigate in vitro the cytotoxic effects of No on the HL60 and K562 leukemia cell lines. The cells were incubated with six different concentrations of each three extracts. MTT assay was employed as a cytotoxicity test. It was observed that concentrations of 1000, 500 and 50 microg/ml from each extract possess marked antileukemic effects. No leaf and root extracts were seen to be more cytotoxic than the stem extract according to LC50. Secondly, in order to understand the role of P-gp in cytotoxicity, P-gp levels of K562 resistant and sensitive cells were measured by flow cytometry before treatment extracts, and then, the cells were incubated with No leaf, stem and root extracts in 500 microg/ml concentrations overnight. After incubation, measurements showed decreased levels of P-gp in the cells. Hence, it is possible to think contributes to their cytotoxic effects that inhibiting of the P-gp pump by No extracts on leukemia cell lines.

Protein, leucine aminopeptidase, esterase, acid phosphatase and photosynthetic responses of oleander (Nerium oleander L.) during cold acclimation and freezing treatments.

Six-month-old oleander (Nerium oleander L.) pot plants, derived from vegetative propagation by cuttings, were tested for their ability to cold hardening. Damage of the non-acclimated (NA) plants was visible when treated by low freezing temperatures (below -2 degrees C). The responses of total proteins, leucine aminopeptidase (LAP), esterase (EST) and acid phosphatase (ACP) isoforms of NA and cold-acclimated (CA; 4 degrees C for 14 days) plants were compared using polyacrylamide gel electrophoresis. These molecular markers were also compared in NA and CA plants which received for 2h temperatures of 0, -2, -4, -6 and -8 degrees C. A new 38-kDa polypeptide appeared from day 7 to 14 during the acclimation treatment in the bark extracts and on day 14 in the leaf extracts. The above-mentioned polypeptide band (38 kDa) strongly appeared in all freezing treatments (0, -2, -4, -6 and -8 degrees C) in both bark and leaf extracts of the CA plants. Alterations in the number and the intensity of LAP and EST isoforms as well as in the intensity of ACP isoforms were observed in both bark and leaf of the CA oleander plants. A newly expressed EST isoform is proposed as biochemical marker for the cold acclimation treatment. CO2 assimilation rates (A) as well as transpiration rates (E) in NA plants were positive in 0 degrees C and negative in all temperatures below zero in the freezing treatments. In contrast, CO2 assimilation rates (A) and transpiration rates (E) were positive in CA plants in all temperatures of freezing treatment. A significant decrease (P<0.05) in chlorophyll (Chl) a, Chl a+b concentration and Chl a/b ratio were noticed in oleander plants during the acclimation treatment (from day 0 to 14), while Chl b concentration was unchanged at the respective time. On the other hand, no significant (P<0.05) differences were observed in the freezing treatments.

Clinical and pathological aspects of experimental oleander (Nerium oleander) toxicosis in sheep.

Dried Nerium oleander leaves at single lethal dose of 110 mg/kg body weight were administered orally to six native male sheep. Clinical signs of toxicosis in sheep began to appear about 30 min after receiving the oleander and included decrease of the heart rate followed by cardiac pauses and tachyarrhythmias; ruminal atony, mild to moderate tympany, abdominal pain, polyuria and polakiuria. Electrocardiography revealed bradycardia, atrio-ventricular blocks, depression of S-T segments, ventricular premature beats and tachycardia, and ventricular fibrillation. Five sheep died within 4-12 h and one survived. At necropsy there were varying degrees of haemorrhages in different organs and gastroenteritis. Histopathological examination of tissue sections revealed myocardial degeneration and necrosis, degeneration and focal necrosis of hepatocytes, necrosis of tubular epithelium in kidneys, oedema in the lungs, and ischemic changes in the cerebrum.

Stress-induced injuries and trace element concentrations in vascular leaf plants from an urban environment (Palermo, Italy).

We examined leaf injuries and measured trace element concentrations in vascular plants from an urban ecosystem with distinct stress valences (the city of Palermo), and compared them with samples of the same species from sites where the stress potential is lower. Urban pollution influences macro-, micro- and toxic element concentrations in leaves. Therefore these leaves can be used as markers of the chemical and biological effects of atmospheric pollution. We studied the trace element content in the leaves of two species, oleander and oak, both fairly tolerant plants and good indicators and bio-monitors of pollution contaminants. Samples were collected at various sites in different periods.

Multiform biosynthetic pathway of syringyl lignin in angiosperms.

To clarify the pathway for biosynthesis of sinapyl alcohol in angiosperms, tracer experiments using stable isotopes were performed on robinia ( Robinia pseudoacacia L.), oleander ( Nerium indicum Mill.), magnolia ( Magnolia kobus DC.) and Arabidopsis thaliana (L.) Heynh. Precursors used in the experiment were (13)C- and (2)H ( D)-labeled [8-(13)C, 3-OCD(3)]ferulic acid and [8-(13)C, 3,5-OCD(3)]sinapic acid. The incorporation of labeled precursor into lignin was confirmed by gas chromatography-mass spectrometry of the products of derivatization followed by reductive cleavage. Crude extracts of differentiating xylem or stems from these plants were also assayed for 4-coumarate-CoA ligase (4CL; EC 6.2.1.12) activity using sinapic acid and ferulic acid as substrates. In robinia and oleander, 4CL activity toward sinapic acid was detected, and labeled sinapic acids were incorporated into syringyl lignin. These results indicate that robinia and oleander have a pathway that produces sinapyl alcohol from sinapic acid via sinapoyl-CoA. By contrast, in magnolia and Arabidopsis, 4CL activity toward sinapic acid could not be detected, and labeled sinapic acid was not incorporated into lignin. These results suggest that syringyl lignin biosynthesis in angiosperms operates via multiple pathways that depend on the species.

Simultaneous determination of cardenolides by sonic spray ionization liquid chromatography-ion trap mass spectrometry--a fatal case of oleander poisoning.

Simultaneous determination of oleandrin and its three related compounds, desacetyloleandrin, oleandrigenin, and gitoxigenin in blood by using liquid chromatography-three-dimensional quadrupole mass spectrometry (LC-3DQMS) system equipped with sonic spray ionization (SSI) interface was conducted. This analyzing method was suitable for all of these compounds except gitoxigenin. The limits of detection of oleandrigenin and desacetyloleandrin from blood were 2 ng/mL and that of oleandrin was 3 ng/mL. The calibration curves for oleandrin, desacetyloleandrin, and oleandrigenin were linear in the range of 5-100 ng/mL. The coefficients of variation of oleandrin, desacetyloleandrin, and oleandrigenin in the blood were satisfactory ranging from 1.6% to 4.1%. This analysis method was applied to a fatal case of oleander poisoning. As a result of liquid chromatography-mass spectrometry (LC-MS) analysis, oleandrin was detected in heart blood and cerebrospinal fluid. Desacetyloleandrin, oleandrigenin, and gitoxigenin were not detected. In order to make identification of oleandrin reliable, LC-MS-MS analysis was performed. The concentrations of oleandrin found in the heart blood and cerebrospinal fluid were 9.8 and 10.1 ng/mL, respectively.