Enhancing plant uptake of polychlorinated biphenyls and cadmium using tea saponin

The potential of natural surfactant tea saponin to enhance uptake of polychlorinated biphenyls (PCBs) and cadmium (Cd) by Zea Mays L. and Saccharum officinarum L. was investigated. With addition of tea saponin at 0.01% in solution culture, the concentrations of PCB 14, PCB 18, PCB 77 and PCB 156 in root of corn seedling were 2.72, 2.68, 1.94 and 2.40 times as those of treatments without adding any surfactant, respectively. Application of tea saponin to the soil significantly elevated PCB 5 accumulation in shoots and roots (p < 0.05) by sugarcanes. With addition of 0.3% tea saponin, Cd concentration was increased by 96.9% in roots, 156.8% in stems and 30.1% in leaves compared with the treatment without addition of surfactant in sugarcane grown in soil. Tea saponin had potential of assisting the uptake of PCBs and Cd by plants from water solution and soil.     

Preparative separation of alkaloids from Nelumbo nucifera leaves by conventional and pH-zone-refining counter-current chromatography

Two modes of high-speed counter-current chromatography (HSCCC) were successfully applied to the separation of alkaloids from crude extract of Nelumbo nucifera leaves. The conventional HSCCC separations were performed with a two-phase solvent system composed of tetrachloromethane–CHCl₃–methanol–0.1 M HCl at a volume ratio of 1:3:3:2 (v/v/v/v), and 120 mg crude extract could be successfully separated. pH-Zone-refining CCC was performed with a two-phase solvent system composed of petroleum ether (60–90 °C)–ethyl acetate–methanol–water (5:5:2:8, v/v/v/v) where triethylamine (10 mM) was added to the upper organic stationary phase as a retainer and hydrochloric acid (5 mM) to the aqueous mobile phase as an eluent. From 4.0 g of the crude extract, 120 mg N-nornuciferine, 1020 mg nuciferine and 96 mg roemerine were obtained in a single run each with a purity of over 98% as determined by HPLC. The structures of the isolated compounds were identified by ESI-MS, ¹H NMR and ¹³C NMR.     

Statistical modelling and optimization of substrate composition for bacterial growth and cadmium removal using response surface methodology

In this study, substrate composition was optimized for the growth of Achromobacter xyloxidans and biosorption of Cd(II) from aqueous solution. Response surface methodology (RSM) was used to investigate the function of three independent operating variables, namely, peptone (2.5-10 g/L), beef extract (2.5-5.0 g/L) and incubation time (24-96 h), on dependent variables, i.e. sorption of Cd(II) ions, protein content and biomass growth of A. xyloxidans. The maximum Cd(II) removal efficiency of 69.2%, protein content 1.9 mg/L and growth 0.354 optical density was found at optimal conditions of peptone 10 g/L, incubation time 60 h and beef extract 2.5 g/L. The significance of independent variables and interactions between variables were tested by means of the analysis of variance (ANOVA) with 95% confidence limits and values of “Prob > F” less than 0.0500 indicate that model terms are significant. Fourier transfer infrared (FTIR) analysis was used to investigate sorption mechanism and involved functional groups in Cd(II) binding.     

Fed-batch fermentation of Tuber melanosporum for the hyperproduction of mycelia and bioactive Tuber polysaccharides

For the first time, a fed-batch fermentation process of Tuber melanosporum was developed for the efficient production of bioactive mycelia and Tuber polysaccharides. Each 1.67 g/L of peptone and 8.33 g/L of yeast extract were added on day 3, 6, and 9, respectively, and sucrose was fed to maintain its concentration around 35–5 g/L when its residual level decreased to 10–5 g/L. Then, the maximal biomass, the production of extracellular polysaccharides (EPS) and intracellular polysaccharides (IPS) reached 53.72 ± 2.57 g DW/L, 7.09 ± 0.62 and 4.43 ± 0.21 g/L, respectively. Compared with the batch culture conducted in the enriched medium, the biomass, the production of EPS and IPS were enhanced by 55.8%, 222.3% and 103.2%, respectively. Not only the cell density but also the production of EPS and IPS were the highest ever reported in truffle fermentation, and the biomass was also the highest as ever reported in mushroom fermentation.     

Seasonal variation in antimicrobial and phytochemical properties of frequently used medicinal bulbous plants from South Africa

The growing popularity of traditional medicine and the unrestricted collection of medicinal plants from the wild have put many of the slow growing bulbous plant species at the risk of over-exploitation and extinction in South Africa. This study was aimed at comparing the phytochemical composition and biological (antibacterial and anticandidal) activities of bulb and leaf extracts of Tulbaghia violacea, Hypoxis hemerocallidea, Drimia robusta and Merwilla plumbea between spring, summer, autumn and winter seasons, with the view of promoting the use of leaves, as a conservation strategy. Antibacterial and anticandidal activities of petroleum ether (PE), dichloromethane (DCM), 80% ethanol and water extracts of bulbs and leaves were tested against Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae bacteria and the fungus Candida albicans using the microdilution bioassay. Spectrophotometric methods were used to evaluate saponin and phenolic compositions for the four seasons. Leaf and bulb extracts exhibited comparable anticandidal activity (MIC < 1 mg/ml) in all the plant species in all seasons. Only ethanol and water extracts of H. hemerocallidea corms (autumn and winter) showed correspondingly good fungicidal activity amongst the bulbs tested. Antibacterial activity was fairly comparable between bulbs and leaves with at least one extract of each plant species showing some good MIC values in most of the seasons. The best antimicrobial activities were recorded in winter and autumn seasons, with MIC values as low as 0.2 mg/ml in the DCM bulb extracts of T. violacea (winter) against K. pneumoniae and S. aureus. The amounts of total phenolic compounds in all plant samples were generally higher in spring compared to the other seasons. Condensed tannin, gallotannin and flavonoid levels, depending on the sample, were either higher in spring or winter except for H. hemerocallidea (corm) which had higher gallotannin levels in autumn. Total saponin levels were higher in winter in all plant samples. Although variation was observed in the phytochemical concentrations between the bulbs and leaves of each plant species, their antimicrobial activities were fairly comparable. Leaves may be used as substitutes for bulbs in the treatment of bacterial and fungal ailments.     

Teratogenic effects of tetrabromobisphenol A on Xenopus tropicalis embryos

Tetrabromobisphenol A (TBBPA) is the most widely used brominated flame retardant and a known thyroid disruptor. We reported exposing Xenopus tropicalis embryos (NF10) to 0.01, 0.1 or 1 mg/L of TBBPA with or without 70 µg/L triiodothyronine (T₃). Compared with the controls, 1 mg/L of TBBPA significantly reduced the body length of embryos after 24, 36 and 48 h of exposure. Embryos treated with TBBPA showed multiple malformations, including: abnormal eyes, skin hypopigmentation, enlarged proctodaeum, narrow fins and pericardial edemas. The effect of abnormal eyes manifested itself in the loss of pigmentation, reduction in size, or absence of external eyes. The degree of eye malformations was quantified with the index of eye malformations (IEM) with 0 being normal and 3 being severe. In the 1 mg/L TBBPA treatment groups, the incidence of total malformations (ITM) was 68–93%, and IEM was 0.8–0.9. T₃ showed no teratogenic effects on embryos, but it significantly enhanced TBBPA-induced teratogenic effects. In the T₃ + 1 mg/L TBBPA treatment groups, ITM was 91–99%, and IEM was 1.8–1.9. Histological observations showed that the retinas were generally smaller, and the lenses were underdeveloped or even absent. These results indicate that TBBPA at relatively high concentration has teratogenic effects on X. tropicalis embryos. The results also suggest that thyroid hormone signaling might be involved in TBBPA induced-teratogenicity.     

Bioaccumulation du Cd et du Zn chez les plants de tomates (Lycopersicon esculentum L.)

This work aims at evaluating the accumulation of cadmium (Cd) and zinc (Zn) (trace elements) in the organs of young tomato plants (Lycopersicon esculentum L. var. Rio Grande) and their effects on the rate of chlorophyll and enzyme activities involved in the antioxidant system: catalase (CAT), glutathion-S-transferase (GST) and peroxysase ascorbate (APX). Plants previously grown on a basic nutrient solution were undergoing treatment for 7 days, either by increasing concentrations of CdCl₂ or ZnSO₄ (0, 50, 100, 250, 500 μM) or by the combined concentrations of Cd and Zn (100/50, 100/100, 100/250, 100/500 μM). The results concerning the determination of metals in the various compartments of tomato plants as a function of increasing concentrations of Cd or Zn, suggest a greater accumulation of Cd and Zn in the roots compared to leaves. The combined treatment (Cd/Zn) interferes with the absorption of the two elements according to their concentrations in the culture medium. The presence of Zn at low concentrations (50 μM of Zn/100 μM Cd) has little influence on the accumulation of Cd in the roots and leaves, while the absorption of these two elements in the leaves increases and decreases in roots when their concentrations are equivalent (100/100 μM) compared to treatment alone. When the concentration of Zn is higher than that of Cd (500 μM of Zn/100 μM Cd) absorption of the latter is inhibited in the roots while increasing their translocation to the leaves. Meanwhile, the dosage of chlorophylls shows that they tend to decrease in a dose-dependent for both treatments (Cd or Cd/Zn), however, treatment with low concentrations of Zn (50 and 100 μM) stimulates chlorophyll synthesis. However, treatment with different concentrations of Cd seems to induce the activity of the enzymes studied (CAT, APX, GST). It is the same for treatment with different concentrations of Zn and this particularly for the highest concentrations. Finally, the combined treatment (Zn/Cd) also appears to cause enzyme inductions: CAT, APX and GST.     

Characterization and transcriptional regulation of thioredoxin reductase 1 on exposure to oxidative stress inducing environmental pollutants in Chironomus riparius

We characterized thioredoxin reductase 1 (TrxR1) from Chironomus riparius (CrTrxR1) and studied its expression under oxidative stress. The full-length cDNA is 1820 bp long and contains an open reading frame (ORF) of 1488 bp. The deduced CrTrxR1 protein has 495 amino acids and a calculated molecular mass of 54.41 kDa and an isoelectric point of 6.15. There was a 71 bp 5’ and a 261 bp 3' untranslated region with a polyadenylation signal site (AATAAA). Homologous alignments showed the presence of conserved catalytic domain Cys-Val-Asn-Val-Gly-Cys (CVNVGC), the C-terminal amino acids ‘CCS’ and conserved amino acids required in catalysis. The expression of CrTrxR1 is measured using quantitative real-time PCR after exposure to 50 and 100 mg/L of paraquat (PQ) and 2, 10 and 20 mg/L of cadmium chloride (Cd). CrTrxR1 mRNA was upregulated after PQ exposure at all conditions tested. The highest level of CrTrxR1 expression was observed after exposure to 10 mg/L of Cd for 24 h followed by 20 mg/L for 48 h. Significant downregulation of CrTrxR1 was observed after exposure to 10 and 20 mg/L of Cd for 72 h. This study shows that the CrTrxR1 could be potentially used as a biomarker of oxidative stress inducing environmental contaminants.     

Kinetics of an extracellular exo-inulinase production from a 5-flourocytosine resistant mutant of Geotrichum candidum using two-factorial design

In the present study, eight different strains of Geotrichum candidum were isolated and screened for an extracellular exo-inulinase production using chemically enriched sucrose–mineral media. The isolate (Zool-3i) with a better enzyme activity (1.38 IU/ml) was subjected to induced mutagenesis using methyl methane sulphonate (MMS) and a mutant with an enzyme activity of 32.06 IU/ml was obtained. Further exposure to ethyl methane sulphonate (EMS) and ultraviolet (UV) radiations yielded a mutant exhibiting an improved activity of 39.34 IU/ml. The potential mutant was cultured overnight and plated on 5fc–YPR agar medium and thus made resistant against 5-flourocytocine. Over 50-fold enhancement in enzyme production (71.85 IU/ml) was achieved when the process parameters including incubation period (48 h), sucrose concentration (5.0 g/L), pH (6.0), inoculum size (2.0%, 16 h old) and urea (0.2%) were identified using Plackett–Burman design. On the basis of kinetic variables, notably Q_p (0.723 U/g/h), Y_p/s (2.036 U/g) and q_p (0.091 U/g cells/h), the mutant MEU-5fc-6 was found to be a hyper producer of exo-inulinase (HS, LSD 0.045, p ? 0.05).     

Separation and purification of echinacoside from Penstemon barbatus (Can.) Roth by recycling high-speed counter-current chromatography

Echinacoside is an important bioactive compound extracted from Cistanche tubulosa which was endangered by overexploitation. It is imperative to find an alternative source. Echinacoside was isolated from Penstemon barbatus (Can.) Roth for the first time. The peak contents of echinacoside are 9.09 ± 0.32 mg/g and 7.25 ± 0.36 mg/g respectively in the leaves and roots annually. The methanolic extracts from 20 g of dried powder of the roots of P. barbatus were pre-purified by AB-8 resin and the fraction containing echinacoside was further purified by conventional high-speed counter-current chromatography (HSCCC) and recycling HSCCC with the solvent system n-butanol–water (1:1, v/v). Totally 42.0 mg echinacoside with a purity of 96.3% was recovered. The recovery rate of echinacoside by recycling HSCCC reached 91.0%. The structure of our echinacoside confirmed by IR, ¹H NMR and ¹³C NMR is identical to the standard sample. This indicates that P. barbatus might be ideal source for preparation of large scale of echinacoside.     

Agrobacterium-mediated transformation of leaf base derived callus tissues of popular indica rice (Oryza sativa L. sub sp. indica cv. ADT 43)

A simple and efficient protocol for the Agrobacterium-mediated transformation of an agronomically useful abiotic sensitive popular indica rice cv. ADT 43 has been developed. Initiation of calli were best achieved from the leaf bases of 4 days old rice seedlings on LS medium supplemented with 2.5 mg/L 2,4-D and 1.0 mg/L thiamine-HCl. Rice calli immersed in Agrobacterium suspension (strain EHA 105, OD₆₀₀ = 0.8) were co-cultured on LS30-AsPC medium for 2 days at 25 ± 2 °C in the dark. Based on GUS expression analysis, 10 min co-cultivation time with 100 μM acetosyringone was found optimum for the delivery of gus gene. Calli were proved to be very sensitive to Agrobacterium infection and we found that the level of necrotic response can be minimized after co-cultivation with 30% LS, 10 g/L PVP, 10% coconut water and 250 mg/L timentin which improved the final transformation efficiency to 9.33%. Molecular and genetic analysis of transgenic plants reveals the integration, expression and inheritance of transgene in the progeny (T₁) of these plants. The copy number of transgenes has been found to vary from 1 to 2 in transgenic plants (T₀ and T₁).     

Phytoaccumulation of cadmium through Azolla from aqueous solution

Azolla, which is an aquatic fern, has proved to be effective in the uptake and accumulation of metals from polluted waters. Azolla spp., namely A. microphylla cv. MH3 and A. caroliniana Willd, were chosen as model plants so that Cd(II) could be accumulated from aqueous solution. An increase in uptake time and the concentration of Cd(II) in aqueous solution resulted in more Cd(II) accumulation in both species. Modified Michaelis-Menten equation was employed to describe the concentration-dependent kinetics of Cd(II) uptake through the roots of A. microphylla cv. MH3, and the values of K_m and V_max were found to be 0.23 mg/L and 16.49 μg/(g.f.wt.h), respectively. Cd(II) uptake by A. microphylla cv. MH3 occurs partly through Ca(II) channels and has the potential to be mediated by Zn(II) transporters.     

Accumulation, speciation, and coordination of arsenic in an inbred line and a wild type cultivar of the desert plant species Chilopsis linearis (Desert willow)

This study investigated the absorption of arsenic (As), sulfur (S), and phosphorus (P) in the desert plant Chilopsis linearis (Desert willow). A comparison between an inbred line (red flowered) and wild type (white flowered) plants was performed to look for differential responses to As treatment. One month old seedlings were treated for 7 days with arsenate (As₂O₅, As^V) at 0, 20, and 40 mg As^V L⁻¹. Results from the ICP-OES analysis showed that at 20 mg As^V L⁻¹, red flowered plants had 280 ± 11 and 98 ± 7 mg As kg⁻¹ dry wt in roots and stems, respectively, while white flowered plants had 196 ± 30 and 103 ± 13 mg As kg⁻¹ dry wt for roots and stems. At this treatment level, the concentration of As in leaves was below detection limits for both plants. In red flowered plants treated with 40 mg As^V L⁻¹, As was at 290 ± 77 and 151 ± 60 mg As kg⁻¹ in roots and stems, respectively, and not detected in leaves, whereas white flowered plants had 406 ± 36, 213 ± 12, and 177 ± 40 mg As kg⁻¹ in roots, stems, and leaves. The concentration of S increased in all As treated plants, while the concentration of P decreased in roots and stems of both types of plants and in leaves of red flowered plants. X-ray absorption spectroscopy analyses demonstrated partial reduction of arsenate to arsenite in the form of As-(SX)₃ species in both types of plants.     

Preparative separation of isoquinoline alkaloids from Stephania yunnanensis by pH-zone-refining counter-current chromatography

In this paper, five isoquinoline alkaloids were successfully separated from a crude extract of Stephania yunnanensis using pH-zone-refining counter-current chromatography in single-step. With a two-phase solvent system composed of methyl-tert-butyl ether (MtBE)–acetonitrile–water (2:2:3, v/v) where triethylamine (10 mM) was added to the upper organic phase as a retainer and hydrochloric acid (5 mM) to the aqueous mobile phase as an eluter. From 1.4 g crude extract, 68.7 mg isocorydine, 78.2 mg corydine, 583.4 mg tetrahydropalmatine, 36.3 mg N-methylasimilobine, and 47.3 mg anonaine were separated with purities over 90%. Their structures were identified by ¹H NMR, ¹³C NMR, ESI-MS data.     

Micropropagation of Albuca bracteata and A. nelsonii — Indigenous ornamentals with medicinal value

In this study, two species from the genus Albuca (Hyacinthaceae) with ornamental and medicinal properties were micropropagated. Adventitious bulblets of Albuca bracteata were cut into quarters and used as explants to examine the effect of temperature (10, 15, 20, 25, 30 or 35 °C), carbohydrates (glucose, fructose or sucrose at 0, 87.5, 175, 262.5 or 350 mM) and hormones (BA, mTR, NAA, IAA, GA₃, ABA or methyl jasmonate each at 0, 0.1, 1.0 or 5.0 mg/L) on the induction and growth of bulblets. Temperatures above 35 °C completely inhibited bulb formation, while induction at all other temperatures was high. Heaviest and largest bulbs formed at 20 °C. Low concentrations (87.5 mM) of all tested carbohydrates increased bulb induction compared to media without a carbohydrate source, while higher levels decreased bulblet induction. The cytokinins mTR and BA inhibited bulb induction, diameter and mass at moderate (1.0 mg/L) and high (5.0 mg/L) concentrations. GA₃, NAA and particularly IAA promoted bulblet induction, while ABA and methyl jasmonate had no significant effect on the induction or bulblet growth. Leaf material and young inflorescences of A. nelsonii were removed, decontaminated, and dissected into seven explant types: leaves, peduncles, pedicels, whole flowers, tepals, ovaries and anthers. These were placed on MS media without hormones, or containing 0.5 mg/L mTR, 0.5 mg/L NAA or 0.5 mg/L mTR + 0.5 mg/L NAA to establish which explant type and hormone combination promoted shoot formation. Some tepal and pedicel explants were capable of shoot production on media with both mTR and NAA, but peduncle explants produced the most shoots when mTR and NAA were both present in the culture medium. Flowers, leaves, ovaries and anthers were completely unresponsive, irrespective of medium composition. These techniques will aid the further horticultural development of these plants, and can be easily adjusted for other species within the genus to promote conservation.     

Early responses to cadmium of two poplar clones that differ in stress tolerance

Soil cadmium (Cd) contamination is becoming a matter of great global concern. The identification of plants differentially sensitive to Cd excess is of interest for the selection of genotype adaptive to grow and develop in polluted areas and capable of ameliorating or reducing the negative environmental effects of this toxic metal. The two poplar clones I-214 (Populus × canadensis) and Eridano (Populus deltoides × maximowiczii) are, respectively, tolerant and sensitive to ozone (O₃) exposure. Because stress tolerance is mediated by an array of overlapping defence mechanisms, we tested the hypothesis that these two clones differently sensitive to O₃ stress factor also exhibit different tolerance to Cd. With this purpose, an outdoor pot experiment was designed to study the responses of I-214 and Eridano to the distribution of different Cd solutions enriched with CdCl₂ (0, 50 and 150 μM) for 35 days. Changes in leaf area, biomass allocation and Cd uptake, photosynthesis, chlorophyll fluorescence, leaf concentration of nutrients and pigments, hydrogen peroxide (H₂O₂) and nitric oxide (NO) production and thiol compounds were investigated. The two poplar clones showed similar sensitivity to excess Cd in terms of biomass production, photosynthesis activity and Cd accumulation, though physiological and biochemical traits revealed different defence strategies. In particular, Eridano maintained in any Cd treatment the number of its constitutively wider blade leaves, while the number of I-214 leaves (with lower size) was reduced. H₂O₂ increased 4.5- and 13-fold in I-214 leaves after the lowest (L) and highest (H) Cd treatments, respectively, revealing the induction of oxidative burst. NO, constitutively higher in I-214 than Eridano, progressively increased in both clones with the enhancement of Cd concentration in the substrate. I-214 showed a more elevated antioxidative capacity (GSH/GSSG) and higher photochemical efficiency of PSII (F_v/F_m) and de-epoxidation degree of xantophylls-cycle (DEPS). The glutathione pool was not affected by Cd treatment in both clones, while non-protein thiols and phytochelatins were reduced at L Cd treatment in I-214. Overall, these two clones presented high adaptability to Cd stress and are both suitable to develop and growth in environments contaminated with this metal, thus being promising for their potential use in phytoremediation programmes.     

Heavy metal impact on bacterial biomass based on DNA analyses and uptake by wild plants in the abandoned copper mine soils

The metals contamination in surface soils and their accumulation in wild plants from the abandoned Burra and Kapunda copper mines located in South Australia were assessed, and the predominant bacterial diversity in the contaminated surface soils from these two abandoned copper mine sites were evaluated through polymerase chain reaction–denaturing gradient gel electrophoresis (PCR-DGGE) analysis. The results showed the average concentration of Cu in soils was 3821.59 mg/kg while wild plants accumulated up to 173.44 mg/kg. The concentration of Cu in shoots of spear grass (Stipa uitida) and berry saltbush (Afriplex semibaccata) was higher than that of roots. The concentration of total and extractable As, Cd, Cu and Pb in soils slightly correlated with of these elements in the corresponding wild plants. The toxicity of Cu in heavily contaminated soils impacted on the quantities of specific microbial populations and no significant change in the microbial diversity of highly contaminated soils.     

Determination of cefazedone in human plasma by high performance liquid chromatography–tandem mass spectrometry: Application to a pharmacokinetic study on Chinese volunteers

A rapid, sensitive and simple high performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) method was developed for determination of cefazedone in human plasma using metronidazole as internal standard (IS). The chromatographic separation was achieved on an Ultimate XB-CN column (2.1 mm × 150 mm, 5 μm) with an isocratic mobile phase of acetonitrile and 20 mM ammonium acetate in 0.1% formic acid in water (15:85, v/v). Detection was performed using electrospray ionization in positive ion multiple reaction-monitoring mode (SRM), monitoring the transitions m/z 548.2 → 344.1 for cefazedone and m/z 172.2 → 128.1 for IS. Calibration curves were linear over a wide range of 0.20–401.12 μg/mL for cefazedone in plasma. The lower limit of quantification (LLOQ) was 0.20 μg/mL. The intra- and inter-day precisions were less than 7.2%. The average recovery of cefazedone was 90.8–91.0%. The validated method was successfully applied to the pharmacokinetic study of cefazedone in Chinese healthy volunteers following intravenous (IV) administration of 500, 1000 and 2000 mg cefazedone injection.