In situ separation and visualization of isomeric auxin derivatives in Arabidopsis by ion mobility mass spectrometry imaging.

In situ separation and visualization of synthetic and naturally occurring isomers from heterogeneous plant tissues, especially when they share similar molecular structures, are a challenging task. In this study, we combined the ion mobility separation with desorption electrospray ionization mass spectrometry imaging (DESI-IM-MSI) to achieve a direct separation and visualization of two synthetic auxin derivatives, auxinole and its structural isomer 4pTb-MeIAA, as well as endogenous auxins from Arabidopsis samples. Distinct distribution of these synthetic isomers and endogenous auxins in Arabidopsis primary roots and hypocotyls was achieved in the same imaging analysis from both individually treated and cotreated samples. We also observed putative metabolites of synthetic auxin derivatives, i.e. auxinole amino acid conjugates and hydrolysed 4pTb-MeIAA product - 4pTb-IAA, based on their unique drifting ion intensity patterns. Furthermore, DESI-IM-MSI-revealed abundance of endogenous auxins and synthetic isomers was validated by liquid chromatography-mass spectrometry (LC-MS). Our results demonstrate that DESI-IM-MSI could be used as a robust technique for detecting endogenous and exogenous isomers and provide a spatiotemporal evaluation of hormonomics profiles in plants.

Synthetic and analytical routes to the L-amino acid conjugates of cis-OPDA and their identification and quantification in plants.

Cis-(+)-12-oxophytodienoic acid (cis-(+)-OPDA) is a bioactive jasmonate, a precursor of jasmonic acid, which also displays signaling activity on its own. Modulation of cis-(+)-OPDA actions may be carried out via biotransformation leading to metabolites of various functions. This work introduces a methodology for the synthesis of racemic cis-OPDA conjugates with amino acids (OPDA-aa) and their deuterium-labeled analogs, which enables the unambiguous identification and accurate quantification of these compounds in plants. We have developed a highly sensitive liquid chromatography-tandem mass spectrometry-based method for the reliable determination of seven OPDA-aa (OPDA-Alanine, OPDA-Aspartate, OPDA-Glutamate, OPDA-Glycine, OPDA-Isoleucine, OPDA-Phenylalanine, and OPDA-Valine) from minute amount of plant material. The extraction from 10 mg of fresh plant tissue by 10% aqueous methanol followed by single-step sample clean-up on hydrophilic-lipophilic balanced columns prior to final analysis was optimized. The method was validated in terms of accuracy and precision, and the method parameters such as process efficiency, recovery and matrix effects were evaluated. In mechanically wounded 30-day-old Arabidopsis thaliana leaves, five endogenous (+)-OPDA-aa were identified and their endogenous levels were estimated. The time-course accumulation revealed a peak 60 min after the wounding, roughly corresponding to the accumulation of cis-(+)-OPDA. Our synthetic and analytical methodologies will support studies on cis-(+)-OPDA conjugation with amino acids and research into the biological significance of these metabolites in plants.

3,5,7-Substituted Pyrazolo[4,3-d]Pyrimidine Inhibitors of Cyclin-Dependent Kinases and Cyclin K Degraders.

3,5,7-Trisubstituted pyrazolo[4,3-d]pyrimidines have been identified as potent inhibitors of cyclin-dependent kinases (CDKs), which are established drug targets. Herein, we describe their further structural modifications leading to novel nanomolar inhibitors with strong antiproliferative activity. We determined the crystal structure of fully active CDK2/A2 with 5-(2-amino-1-ethyl)thio-3-cyclobutyl-7-[4-(pyrazol-1-yl)benzyl]amino-1(2)H-pyrazolo[4,3-d]pyrimidine (24) at 1.7 Å resolution, confirming the competitive mode of inhibition. Biochemical and cellular assays in lymphoma cell lines confirmed the expected mechanism of action through dephosphorylation of retinoblastoma protein and RNA polymerase II, leading to induction of apoptosis. Importantly, we also revealed an interesting ability of compound 24 to induce proteasome-dependent degradation of cyclin K both in vitro and in a patient-derived xenograft in vivo. We propose that 24 has a dual mechanism of action, acting as a kinase inhibitor and as a molecular glue inducing an interaction between CDK12 and DDB1 that leads to polyubiquitination of cyclin K and its subsequent degradation.

Sulfated phenolic acids in plants.

MAIN CONCLUSION: Sulfated phenolic acids are widely occurring metabolites in plants, including fruits, vegetables and crops. The untargeted UHPLC-QTOF-MS metabolomics of more than 50 samples from plant, fungi and algae lead to the discovery of a small group of sulfated metabolites derived from phenolic acids. These compounds were detected in land plants for the first time. In this study, zosteric acid, 4-(sulfooxy)benzoic acid, 4-(sulfoooxy)phenylacetic acid, ferulic acid 4-sulfate and/or vanillic acid 4-sulfate were detected in a number of edible species/products, including oat (Avena sativa L.), wheat (Triticum aestivum L.), barley (Hordeum vulgare L.), tomato (Solanum lycopersicum L.), carrot (Daucus carota subsp. Sativus Hoffm.), broccoli (Brassica oleracea var. Italica Plenck), celery (Apium graveolens L.), cabbage (Brassica oleracea convar. sabauda L.), banana tree (Musa tropicana L.), pineapple fruit (Ananas comosus L.), radish bulb (Raphanus sativus L.) and olive oil (Olea europaea L.). The structural identification of sulfated compounds was performed by comparing retention times and mass spectral data to those of synthesized standards. In addition to above-mentioned compounds, isoferulic acid 3-sulfate and caffeic acid 4-sulfate were putatively identified in celery bulb (Apium graveolens L.) and broccoli floret (Brassica oleracea var. Italica Plenck), respectively. While sulfated phenolic acids were quantified in concentrations ranging from 0.34 to 22.18 µg·g-1 DW, the corresponding non-sulfated acids were mostly undetected or present at lower concentrations. The subsequent analysis of oat symplast and apoplast showed that they are predominantly accumulated in the symplast (> 70%) where they are supposed to be biosynthesized by sulfotransferases.

Procyanidin C1 from Viola odorata L. inhibits Na+,K+-ATPase.

Members of the Viola genus play important roles in traditional Asian herbal medicine. This study investigates the ability of Viola odorata L. extracts to inhibit Na+,K+-ATPase, an essential animal enzyme responsible for membrane potential maintenance. The root extract of V. odorata strongly inhibited Na+,K+-ATPase, while leaf and seeds extracts were basically inactive. A UHPLC-QTOF-MS/MS metabolomic approach was used to identify the chemical principle of the root extract's activity, resulting in the detection of 35,292 features. Candidate active compounds were selected by correlating feature area with inhibitory activity in 14 isolated fractions. This yielded a set of 15 candidate compounds, of which 14 were preliminarily identified as procyanidins. Commercially available procyanidins (B1, B2, B3 and C1) were therefore purchased and their ability to inhibit Na+,K+-ATPase was investigated. Dimeric procyanidins B1, B2 and B3 were found to be inactive, but the trimeric procyanidin C1 strongly inhibited Na+,K+-ATPase with an IC50 of 4.5 µM. This newly discovered inhibitor was docked into crystal structures mimicking the Na3E1∼P·ADP and K2E2·Pi states to identify potential interaction sites within Na+,K+-ATPase. Possible binding mechanisms and the principle responsible for the observed root extract activity are discussed.

The Phenolics and Antioxidant Properties of Black and Purple versus White Eggplant Cultivars.

The total phenolic content, anthocyanins, phenolic acids, antioxidant capacity and α-amylase inhibitory activity of black (Aydin Siyahi), purple (Kadife Kemer) and white (Trabzon Kadife) eggplants grown in Turkey were subjected to a comparative investigation. The black cultivar exhibited the highest total phenolic (17,193 and 6552 mg gallic acid equivalent/kg fw), flavonoid (3019 and 1160 quercetin equivalent/kg fw) and anthocyanin (1686 and 6167 g delphinidin-3-O-glucoside equivalent/kg fw) contents in crude extracts of the peel and pulp. The majority of the caffeic acid was identified in the ester (2830 mg/kg fw) and ester-bound (2594 mg/kg fw) forms in the peel of 'Kadife Kemer' and in the glycoside form (611.9 mg/kg fw) in 'Aydin Siyahi', as well as in the pulp of these two eggplants. 'Kadife Kemer' (purple eggplant) contained the majority of the chlorogenic acid in free form (27.55 mg/kg fw), compared to 'Aydin Siyahi' in the ester (7.82 mg/kg fw), glycoside (294.1 mg/kg dw) and ester-bound (2.41 mg/kg fw) forms. The eggplant cultivars (peel and pulp, mg/kg fw) exhibited a relatively high delphinidin-3-O-rutinoside concentration in the peel of 'Aydin Siyahi' (avg. 1162), followed by 'Kadife Kemer' (avg. 336.6), and 'Trabzon Kadife' (avg. 215.1). The crude phenolic extracts of the eggplants exhibited the highest antioxidant capacity values (peel and pulp, µmoL Trolox equivalent/kg fw) of 2,2-diphenyl-1-picrylhydrazyl (DPPH, 8156 and 2335) and oxygen radical absorbance capacity (ORAC, 37,887 and 17,648). The overall results indicate that black and purple eggplants are the cultivars with greater potential benefits in terms of their phenolics and antioxidant values than the white eggplant.

In situ characterisation of phytohormones from wounded Arabidopsis leaves using desorption electrospray ionisation mass spectrometry imaging.

Phytohormones (plant hormones) are a group of small signalling molecules that act as important endogenous regulators in plant development and stress responses. Previous research has identified the phytohormone species, jasmonates, auxins and abscisic acid, and their related compounds in stressed leaf extracts. However, in situ visualisations of endogenous phytohormones from intact plant tissues remain elusive without the usage of labels or reporters. Mass spectrometry imaging is a label-free analytical technique that has been successfully applied for the direct detection of plant proteins, lipids, carbohydrates and many other biomolecules. In this study, desorption electrospray ionisation mass spectrometry imaging (DESI-MSI) was used for high throughput visualisation and evaluation of wound-induced phytohormones inside Arabidopsis thaliana leaves. The results showed higher levels of jasmonates, salicylic acid, abscisic acid and indole-3-acetic acid in their ion intensity maps established from wounded leaves compared to control leaves, which have been validated in the parallel liquid chromatography-mass spectrometry quantification, and the untainted distributions of the identified phytohormones in leaves were confirmed by mass spectrometry imaging of instant leaf imprinted thin-layer chromatography plate samples. Further statistical analysis has not only demonstrated a significant increase of jasmonic acid and its precursor compounds in wounded leaves/regions but also highlighted a potential correlation in different phytohormone species. Our results suggest that DESI-MSI can be used to in situ characterise multiple phytohormone compounds from intact leaves with 200 µm spatial resolution to provide insight into phytohormone distributions in wounded leaves, along with their correlated precursors and metabolites under mechanical stress.

Interactions between zinc and Phomopsis longicolla infection in roots of Glycine max.

Phomopsis. longicolla is a hemibiotrophic fungus causing significant soybean yield loss worldwide. To reveal the role of zinc in plant-pathogen interactions, soybean seedlings were grown hydroponically with a range of Zn concentrations, 0.06 µM (deficient, Zn0), 0.4 µM (optimal growth), 1.5 µM, 4 µM, 12 µM, and toxic 38 µM, and were subsequently inoculated with P. longicolla via the roots. In vivo analysis of metal distribution in tissues by micro-X-ray fluorescence showed local Zn mobilization in the root maturation zone in all treatments. Decreased root and pod biomass, and photosynthetic performance in infected plants treated with 0.4 µM Zn were accompanied with accumulation of Zn, jasmonoyl-L-isoleucine (JA-Ile), jasmonic acid, and cell wall-bound syringic acid (cwSyA) in roots. Zn concentration in roots of infected plants treated with 1.5 µM Zn was seven-fold higher than in the 0.4 µM Zn treatment, which together with accumulation of JA-Ile, cwSyA, cell wall-bound vanilic acid and leaf jasmonates contributed to maintaining photosynthesis and pod biomass. Host-pathogen nutrient competition and phenolics accumulation limited the infection in Zn-deficient plants. The low infection rate in Zn 4 µM-treated roots correlated with salicylic and 4-hydroxybenzoic acid, and cell wall-bound p-coumaric acid accumulation. Zn toxicity promoted pathogen invasion and depleted cell wall-bound phenolics. The results show that manipulation of Zn availability improves soybean resistance to P. longicolla by stimulating phenolics biosynthesis and stress-inducible phytohormones.

Cytokinin Plant Hormones Have Neuroprotective Activity in In Vitro Models of Parkinson's Disease.

Cytokinins are adenine-based phytohormones that regulate key processes in plants, such as cell division and differentiation, root and shoot growth, apical dominance, branching, and seed germination. In preliminary studies, they have also shown protective activities against human neurodegenerative diseases. To extend knowledge of the protection (protective activity) they offer, we investigated activities of natural cytokinins against salsolinol (SAL)-induced toxicity (a Parkinson's disease model) and glutamate (Glu)-induced death of neuron-like dopaminergic SH-SY5Y cells. We found that kinetin-3-glucoside, cis-zeatin riboside, and N6-isopentenyladenosine were active in the SAL-induced PD model. In addition, trans-, cis-zeatin, and kinetin along with the iron chelator deferoxamine (DFO) and the necroptosis inhibitor necrostatin 1 (NEC-1) significantly reduced cell death rates in the Glu-induced model. Lactate dehydrogenase assays revealed that the cytokinins provided lower neuroprotective activity than DFO and NEC-1. Moreover, they reduced apoptotic caspase-3/7 activities less strongly than DFO. However, the cytokinins had very similar effects to DFO and NEC-1 on superoxide radical production. Overall, they showed protective activity in the SAL-induced model of parkinsonian neuronal cell death and Glu-induced model of oxidative damage mainly by reduction of oxidative stress.

Antibacterial, Mutagenic Properties and Chemical Characterisation of Sugar Bush (Protea caffra Meisn.): A South African Native Shrub Species.

Protea caffra is used as a diarrhoeal remedy in South African herbal medicine, however, its pharmacological properties remain largely unknown. In the present study, extracts from different Protea caffra organs were screened against drug-sensitive and -resistant diarrhoeagenic pathogens using the microdilution assay (minimum inhibitory concentration, MIC). Twig extracts (70% methanol, MeOH) of the plant were purified and the resultant fractions screened for antibacterial properties (MIC). The chemical profiles of the fractions were determined by Gas Chromatography-Mass Spectrometry (GC-MS), while ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was used to quantify the phenolic acids in the plant. The mutagenic properties of bioactive extracts were assessed using the Ames test. The extracts demonstrated weak-moderate antibacterial properties (MIC: 0.3-0.6 mg/mL). A cold ethyl acetate fraction of MeOH twig extract exhibited significant antibacterial properties (MIC = 0.078 mg/mL) against Enterococcus faecalis. The presence of antibacterial compounds (1-adamantane carboxylic acid, heptacosanol, levoglucosan, nonadecanol) in the plant was putatively confirmed based on GC-MS analysis. Furthermore, UHPLC-MS/MS analysis revealed varying concentrations of phenolic acids (0.08-374.55 µg/g DW). Based on the Ames test, the extracts were non-mutagenic thereby suggesting their safety. To a certain degree, the current study supports the traditional use of Protea caffra to manage diarrhoea among local communities in South Africa.

Aromatic Cytokinin Arabinosides Promote PAMP-like Responses and Positively Regulate Leaf Longevity.

Cytokinins are plant hormones with biological functions ranging from coordination of plant growth to the regulation of biotic and abiotic stress-related responses and senescence. The components of the plant immune system can learn from past elicitations by microbial pathogens and herbivores and adapt to new threats. It is known that plants can enter the primed state of enhanced defense induced by either natural or synthetic compounds. While the involvement of cytokinins in defense priming has been documented, no comprehensive model of their action has been provided to date. Here, we report the functional characterization of two aromatic cytokinin derivatives, 6-benzylaminopurine-9-arabinosides (BAPAs), 3-methoxy-BAPA and 3-hydroxy-BAPA, that proved to be effective in delaying senescence in detached leaves while having low interactions with the cytokinin pathway. An RNA-seq profiling study on Arabidopsis leaves treated with 3-methoxy-BAPA revealed that short and extended treatments with this compound shifted the transcriptional response markedly toward defense. Both treatments revealed upregulation of genes involved in processes associated with plant innate immunity such as cell wall remodeling and upregulation of specific MAP kinases, most importantly MPK11, which is a MAPK module involved in stress-related signaling during the pathogen-associated molecular patterns (PAMPs) response. In addition, elevated levels of JA and its metabolites, jasmonate/ethylene-driven upregulation of PLANT DEFENSIN 1.2 (PDF1.2) and other defensins, and also temporarily elevated levels of reactive oxygen species marked the plant response to 3-methoxy-BAPA treatment. Synergistic interactions were observed when plants were cotreated with 3-hydroxy-BAPA and the flagellin-derived bacterial PAMP peptide (flg22), leading to the enhanced expression of the PAMP-triggered immunity (PTI) marker gene FRK1. Our data collectively show that some BAPAs can sensitively prime the PTI responses in a low micromolar range of concentrations while having no observable negative effects on the overall fitness of the plant.

Phytochemical Profiles and Antioxidant Activity of Grasses Used in South African Traditional Medicine.

Grasses are a valuable group of monocotyledonous plants, used as nourishing foods and as remedies against diseases for both humans and livestock. Phytochemical profiles of 13 medicinal grasses were quantified, using spectrophotometric methods and ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS), while the antioxidant activity was done using DPPH and ferric-reducing-power assays. The phytochemical analysis included the total soluble phenolic content, flavonoids, proanthocyanidins, iridoids and phenolic acids. Among the 13 grasses, the root methanolic extracts of Cymbopogon spp., Cymbopogon nardus and Cenchrus ciliaris contained the highest concentrations of total soluble phenolics (27-31 mg GAE/g DW) and flavonoids (4-13 mg CE/g DW). Condensed tannins and total iridoid content were highest (2.3 mg CCE/g DW and 3.2 mg HE/g DW, respectively) in Cymbopogon nardus. The most common phenolic compounds in the grass species included ρ-coumaric, ferulic, salicylic and vanillic acids. In the DPPH radical scavenging assay, the EC50 values ranged from 0.02 to 0.11 mg/mL for the different grasses. The best EC50 activity (lowest) was exhibited by Cymbopogon nardus roots (0.02 mg/mL) and inflorescences (0.04 mg/mL), Cymbopogon spp. roots (0.04 mg/mL) and Vetiveria zizanioides leaves (0.06 mg/mL). The highest ferric-reducing power was detected in the whole plant extract of Cynodon dactylon (0.085 ± 0.45; r2 = 0.898). The observed antioxidant activity in the various parts of the grasses may be due to their rich pool of phytochemicals. Thus, some of these grasses provide a source of natural antioxidants and phytochemicals that can be explored for their therapeutic purposes.

New aromatic 6-substituted 2'-deoxy-9-(ß)-d-ribofuranosylpurine derivatives as potential plant growth regulators.

Cytokinins are naturally occurring substances that act as plant growth regulators promoting plant growth and development, including shoot initiation and branching, and also affecting apical dominance and leaf senescence. Aromatic cytokinin 6-benzylaminopurine (BAP) has been widely used in micropropagation systems and biotechnology. However, its 9-glucoside (BAP9G) accumulates in explants, causing root inhibition and growth heterogenity. To overcome BAP disadvantages, a series of ring-substituted 2'-deoxy-9-(ß)-d-ribofuranosylpurine derivatives was prepared and examined in different classical cytokinin bioassays. Amaranthus, senescence and tobacco callus bioassays were employed to provide details of cytokinin activity of 2'-deoxy-9-(ß)-d-ribosides compared to their respective free bases and ribosides. The prepared derivatives were also tested for their recognition by cytokinin receptors of Arabidopsis thaliana AHK3 and CRE1/AHK4. The ability of aromatic N6-substituted adenine-2'-deoxy-9-(ß)-d-ribosides to promote plant growth and delay senescence was increased considerably and, in contrast to BAP, no loss of cytokinin activity at higher concentrations was observed. The presence of a 2'-deoxyribosyl moiety at the N9-position led to an increase in cytokinin activities in comparison to the free bases and ribosides. The antioxidant capacity, cytotoxicity and effect on the MHV-68 gammaherpesvirus strain were also examined.

Potential of Smoke-Water and One of Its Active Compounds (karrikinolide, KAR1) on the Phytochemical and Antioxidant Activity of Eucomis autumnalis.

Eucomis autumnalis (Mill.) Chitt. subspecies autumnalis is a popular African plant that is susceptible to population decline because the bulbs are widely utilized for diverse medicinal purposes. As a result, approaches to ensure the sustainability of the plants are essential. In the current study, the influence of smoke-water (SW) and karrikinolide (KAR1 isolated from SW extract) on the phytochemicals and antioxidant activity of in vitro and greenhouse-acclimatized Eucomis autumnalis subspecies autumnalis were evaluated. Leaf explants were cultured on Murashige and Skoog (MS) media supplemented with SW (1:500, 1:1000 and 1:1500 v/v dilutions) or KAR1 (10-7, 10-8 and 10-9 M) and grown for ten weeks. In vitro regenerants were subsequently acclimatized in the greenhouse for four months. Bioactive phytochemicals in different treatments were analyzed using ultra-high performance liquid chromatography (UHPLC-MS/MS), while antioxidant potential was evaluated using two chemical tests namely: DPPH and the ß-carotene model. Smoke-water and KAR1 generally influenced the quantity and types of phytochemicals in in vitro regenerants and acclimatized plants. In addition to eucomic acid, 15 phenolic acids and flavonoids were quantified; however, some were specific to either the in vitro regenerants or greenhouse-acclimatized plants. The majority of the phenolic acids and flavonoids were generally higher in in vitro regenerants than in acclimatized plants. Evidence from the chemical tests indicated an increase in antioxidant activity of SW and KAR1-treated regenerants and acclimatized plants. Overall, these findings unravel the value of SW and KAR1 as potential elicitors for bioactive phytochemicals with therapeutic activity in plants facilitated via in vitro culture systems. In addition, it affords an efficient means to ensure the sustainability of the investigated plant. Nevertheless, further studies focusing on the use of other types of antioxidant test systems (including in vivo model) and the carry-over effect of the application of SW and KAR1 for a longer duration will be pertinent. In addition, the safety of the resultant plant extracts and their pharmacological efficacy in clinical relevance systems is required.

Involvement of Phenolic Acids in Short-Term Adaptation to Salinity Stress is Species-Specific among Brassicaceae.

Salinity is a major abiotic stress negatively affecting plant growth and consequently crop production. The effects of short-term salt stress were evaluated on seedlings of three globally important Brassica crops-Chinese cabbage (Brassica rapa ssp. pekinensis), white cabbage (Brassica oleracea var. capitata), and kale (Brassica oleracea var. acephala)-with particular focus on phenolic acids. The physiological and biochemical stress parameters in the seedlings and the levels of three main groups of metabolites (total glucosinolates, carotenoids, and phenolics) and individual phenolic acids were determined. The salt treatments caused a dose-dependent reduction in root growth and biomass and an increase in stress parameters (Na+/K+ ratio, reactive oxygen species (ROS) and glutathione (GSH)) in all seedlings but most prominently in Chinese cabbage. Based on PCA, specific metabolites grouped close to the more tolerant species, white cabbage and kale. The highest levels of phenolic acids, particularly hydroxycinnamic acids, were determined in the more tolerant kale and white cabbage. A reduction in caffeic, salicylic, and 4-coumaric acid was found in Chinese cabbage and kale, and an increase in ferulic acid levels was found in kale upon salinity treatments. Phenolic acids are species-specific among Brassicaceae, and some may participate in stress tolerance. Salt-tolerant varieties have higher levels of some phenolic acids and suffer less from metabolic stress disorders under salinity stress.

Exogenous N-Acetylcysteine alleviates heavy metal stress by promoting phenolic acids to support antioxidant defence systems in wheat roots.

N-acetylcysteine (N-Acetyl L-cysteine, NAC) is a thiol compound derived from the addition of the acetyl group to cysteine amino acid. NAC has been used as an antioxidant, free radical scavenger, and chelating agent for reducing the deleterious effects on plants of biotic and abiotic environmental stresses. It can also relieve heavy metal (HM) toxicity, although its alleviating mechanism remains unknown. In this study, we compared HM-stressed (Cu, Hg, Cd and Pb, 100 µM each) wheat seedlings without NAC treatment and in combination with NAC (1 mM). In comparison to HMs alone, NAC treatment in combination with HMs (Cu, Cd, Hg and Pb, respectively) stimulated root growth (1.1-, 1.5-, 10.5- and 1.9-fold), and significantly increased fresh (1.3-, 1.5-, 4.3- and 1.4-fold) and dry (1.2-, 1.5-, 2.5- and 1.2-fold) mass. Combination treatment also led to significant reductions in HM concentrations (1.3-, 1.4-, 4- and 1.1-fold, respectively). GSH (1.1 - 1.8-fold), TBARS (1.4 - 2.7-fold) and H2O2 (1.6 - 1.8-fold) contents in treatment with HMs alone were significantly mitigated by the NAC combination. Some of the antioxidant enzyme activities increased or reduced by some HM treatments alone were stimulated by a combination of NAC with HMs, or remained unchanged or changed only insignificantly, supported by the phenolic pool of the plant. Ferulic, p-comaric and syringic acids were the major phenolic acids (PAs) in the roots in free, ester, glycoside and ester-bound forms, and their concentrations were increased by HM treatments alone, in comparison to the control seedlings, while PAs concentrations were relatively reduced by NAC in combination with HMs. These results indicate that NAC can alleviate HM toxicity and improve the growth of HM-stressed wheat seedlings by coordinated induction of the phenolic pool and the antioxidant defence system.

Identification of Narciclasine as an in Vitro Anti-Inflammatory Component of Cyrtanthus contractus by Correlation-Based Metabolomics.

In this study, an extract from the bulbs of Cyrtanthus contractus showed strong anti-inflammatory activity in vitro. The extract was partially separated into 14 fractions and analyzed by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry metabolomics, and the correlation coefficients were calculated between biological activities and metabolite levels. As a result, the top-scoring metabolite narciclasine (1) is proposed as the active principle of C. contractus. This was confirmed by comparing the biological effect of crude extract with that of an authentic standard.

Bioactive molecules derived from smoke and seaweed Ecklonia maxima showing phytohormone-like activity in Spinacia oleracea L.

Bioactive compounds such as karrikinolide (KAR1 from smoke) and eckol (from the seaweed Ecklonia maxima) show promising effects on several important crop plants. These plant growth-stimulating organic biomolecules, along with crude extracts (smoke-water and Kelpak® product prepared from Ecklonia maxima), were tested on spinach plants. Eckol sprayed at 10-6 M significantly increased all the growth and biochemical parameters examined compared to control spinach plants. All tested plant growth biostimulants significantly increased total chlorophyll, carotenoids and protein content of spinach leaves. The cytokinin profile of spinach plants was also determined. Cis-zeatin, dihydrozeatin and isopentenyladenine types of cytokinins were promoted by both smoke- and seaweed-based biostimulants. In comparison to the control plants, the level of free sinapic acid was greater in all spinach plants treated with these biostimulants. The application of these biostimulants can help spinach crop by improving growth, yield and nutritional quality; moreover, they are organic and cost-effective.

Identification and characterization of potential bioactive compounds from the leaves of Leucosidea sericea.

ETHNOPHARMACOLOGICAL RELEVANCE: Leucosidea sericea is a southern African tree used for treating different diseases including microbial infections and inflammatory-related conditions. AIM OF THE STUDY: To isolate and identify the chemicals in Leucosidea sericea which possibly account for the diverse therapeutic effects of the species. MATERIALS AND METHODS: Leaf material was extracted using 20% methanol and subsequently partitioned with different solvents (hexane, dichloromethane, ethyl acetate and butanol). Resultant fractions were subjected to bioactive (antimicrobial)-guided isolation and the structural elucidation was conducted using NMR (1D and 2D) spectroscopic methods. Given the broad uses of Leucosidea sericea in traditional medicine, the extract, fractions and isolated compounds were evaluated in five (5) biological assays in vitro (antimicrobial, antioxidant, acetylcholinesterase (AChE) and anti-inflammatory inhibition as well as cytotoxicity effect). RESULTS: As the most active fractions, from ethyl acetate yielded 5,7-dihydroxychromone (1); 1-hydroxy-2-oxopomolic acid (2); 3,5,7,3´,4´-pentahydroxyflavone (3) and Tiliroside (4). For the first time, these four (4) compounds were isolated from leaves of Leucosidea sericea. These aforementioned compounds demonstrated broad-spectrum antibacterial activity (1.95-125 µg/mL) and noteworthy antifungal (3.9-250 µg/mL) potential. In addition to its noteworthy antimicrobial activity, compound 3 also demonstrated significant antioxidant (EC50 = 14 µg/mL in DPPH assay) and anti-inflammatory (inhibited the level of ELAM by approximately 36% and decreased also the viability of endothelial cells) activities in vitro. Overall, AChE inhibition activity and cytotoxic response was generally weak for the extracts, fractions and isolated compounds. CONCLUSION: The pool of chemicals in Leucosidea sericea were enriched with the isolation and identification of four (4) compounds obtained from the leaf extract. Among these compounds, the significant antimicrobial activity of compound 3 provides strong evidence that support the use of Leucosidea sericea for microbial-related infections in folk medicine.

New cytokinin derivatives possess UVA and UVB photoprotective effect on human skin cells and prevent oxidative stress.

Eleven 6-furfurylaminopurine (kinetin, Kin) derivatives were synthesized to obtain biologically active compounds. The prepared compounds were characterized using 1H NMR, mass spectrometry combined with HPLC purity determination and elemental C, H, N analyses. The biological activity of new derivatives was tested on plant cells and tissues in cytokinin bioassays, such as tobacco callus, detached wheat leaf chlorophyll retention bioassay and Amaranthus bioassay. The selected compounds were subsequently tested on normal human dermal fibroblasts (NHDF) and keratinocyte cell lines (HaCaT) to exclude possible phototoxic effects and, on the other hand, to reveal possible UVA and UVB photoprotective activity. The protective antioxidant activity of the prepared cytokinin derivatives was further studied and compared to previously prepared antisenescent compound 6-furfurylamino-9-(tetrahydrofuran-2-yl)purine (Kin-THF) using induced oxidative stress (OS) on nematode Caenorhabditis elegans damaged by 5-hydroxy-1,4-naphthoquinone (juglone), a generator of reactive oxygen species. The observed biological activity was interpreted in relation to the structure of the prepared derivatives. The most potent oxidative stress protection of all the prepared compounds was shown by 6-(thiophen-2-ylmethylamino)-9-(tetrahydrofuran-2-yl)purine (6) and 2-chloro-6-furfurylamino-9-(tetrahydrofuran-2-yl)purine (9) derivatives and the results were comparable to Kin-THF. Compounds 6 and 9 were able to significantly protect human skin cells against UV radiation in vitro. Both the derivatives 6 and 9 showed higher protective activity in comparison to previously known structurally similar compounds Kin and Kin-THF. The obtained results are surprising due to the fact that the prepared compounds showed to be inactive in the ORAC assay which proved that the compounds did not act as direct antioxidants as they were unable to directly scavenge oxygen radicals.