Isolation and molecular characterization of causal agent of blue mold on Allium cepa L. and its control by Pennisetum flaccidum Griseb

Blue mold pathogen, isolated from infected Allium cepa L., was identified as a Penicillium species through morphological and molecular characterisation. Internal Transcribed spacer (ITS) region of ribosomal DNA (rDNA) was utilised for DNA sequencing. Basic Local Alignment Search Tool (BLAST) analysis has found the maximum similarity index of the fungus to be 82.39% with the Uncultured Penicillium clone (Accession: MF535522). So, the isolated Penicillium specie is the first reported specie of the genus that infects onion. A phylogenetic tree was constructed to establish a relationship of the isolated fungus with the most relevant species reported on GenBank. Extracts of Pennisetum flaccidum Griseb. were evaluated against the isolated fungus as a potential biocontrol agent. Among the five tested methanol concentrations (0.5%, 1%, 1.5%, 2% and 2.5%) of each plant part (root, inflorescence and foliage), 0.5% root extract showed maximum growth retardation, i.e. 89%. For bioassay-guided fractionation, the root extract was partitioned in n-hexane, chloroform, n-butanol and ethyl acetate. Ethyl acetate (1%) was proved to be the most potent one. Phytochemical screening has confirmed the occurrence of terpenoids, tannins, saponins and alkaloids. The applied molecular approach has deduced that the Penicillium specie collected from Pakistan might be novel. This study can be concluded that P. flaccidum contains potent phytochemicals which might be used as antifungal agent against Penicillium species.     

Calcium changes in ovules and embryo sacs of Plumbago zeylanica L.

Calcium was localized in ovules of Plumbago zeylanica from 1 day before anthesis to 3 days after anthesis using potassium antimonate and transmission electron microscopy in pollinated and emasculated flowers. At 1 day before anthesis, embryo sacs (containing an egg cell, a central cell and zero to three accessory cells) appear mature and contain abundant calcium precipitates (ppts), in contrast to nucellar cells. At anthesis, the vacuoles of nucellar cells have enlarged, and micropylar cells, in particular, are heavily labeled with calcium ppts. As pollen tubes elongate through ovular tissues, ppts diminish in ovular cells and become concentrated in the pollen tube cell wall. After fertilization, the calcium ppts sharply diminish in fertilized ovules; in unfertilized ovules, calcium ppts remain abundant up to 3 days after anthesis (when unfertilized ovules are shed). The distribution of calcium in the ovule changes in apparent response to fertilization, suggesting that calcium content may be related to the attraction and receipt of the pollen tube. In contrast with conventionally-organized embryo sacs with synergids, Plumbago accumulates calcium in the egg cell. Received: 30 December 1999 / Revision accepted: 24 March 2000     

Freeze-fracture of sperm of Plumbago zeylanica L. in pollen and in vitro

 Sperm of Plumbago zeylanica are dimorphic with regard to numbers of mitochondria and plastids. In most cases examined, the plastid-rich sperm fused with the egg while the sperm with fewer plastids fused with the central cell. However, plastids cannot be directly responsible for fusion because fusion occurs between the plasma membranes of egg and sperm. The question is whether sperm cell membranes are distinctive and possibly dimorphic. Sperm in whole pollen grains and isolated sperm were freeze-fractured. In pollen, freeze-fractured sperm appeared only in cross fractures. No extended membrane fracture faces of sperm were found. Among isolated sperm, two sizes of sperm with different organelles were observed. Isolated sperm were assigned to two categories based on cell diameter and on size and density of organelles. Membrane particles on most sperm were arranged without distinctive pattern. Some hexagonal arrays were observed. In sperm that had been maintained at 4°C, particle-free areas, a probable consequence of lipid phase separations, appeared on plasma membrane fracture faces. No unique fracture patterns and no patterns of dimorphism were detected on freeze-fractured plasma membranes of Plumbago sperm. Received: 14 January 1997 / Revision accepted: 6 June 1997     

Phytochemical,antioxidant and mineral composition of hydroalcoholic extract of chicory (Cichorium intybus L.) leaves

The phytochemical, antioxidant and mineral composition of hydroalcoholic extract of leaves of Cichorium intybus L., was determined. The leaves were found to possess comparatively higher values of total flavonoids, total phenolic acids. The phytochemical screening confirmed the presence of tannins, saponins, flavonoids, in the leaves of the plant. The leaf extract was found to show comparatively low value of IC₅₀ for 2,2-diphenyl-1-picrylhydrazyl (DPPH) inhibition. The IC₅₀ value of chicory leaves extract was found to be 67.2 ± 2.6 μg/ml. The extracts were found to contain high amount of mineral elements especially Mg and Zn. Due to good phytochemical and antioxidant composition, C. intybus L., leaves would be an important candidate in pharmaceutical formulations and play an important role in improving the human health by participating in the antioxidant defense system against free radical generation.     

Influence of NaCl and mannitol on peroxidase activity and lipid peroxidation in Centaurea ragusina L. roots and shoots

Centaurea ragusina L. is a Croatian endemic plant species growing on cliffs above the Adriatic Sea, but there is no information about its physiological behavior or stress tolerance. To investigate the response of C. ragusina plants to salinity and drought, we have analysed soluble peroxidase (POD; EC 1.11.1.7) activity, anionic isoperoxidase pattern, levels of malondialdehyde (MDA) and hydrogen peroxide in C. ragusina plants exposed to these stresses. Rooted plantlets grown on MS 1/2 nutrient medium supplemented with mannitol (300 mM) and different concentrations of NaCl (150, 300, 450 or 600 mM) were harvested after 5, 10 and 15 days. Both osmotic treatments significantly increased MDA and hydrogen peroxide contents in C. ragusina shoots after 10 days of stress, while in roots these parameters showed no significant difference compared to control in overall. POD activity of salt-stressed plants changed with respect to different saline treatments and plant organs - in shoots enzymatic activity markedly increased in response to lower saline treatments, especially 300 mM NaCl; otherwise it was similar as in control plants while in roots of plants grown under 450 and 600 mM NaCl it significantly decreased. Drought increased POD activity of both shoots and roots especially after 10 days of experiment. Generally, change in the POD isoenzyme pattern of treated plants was in accordance with the activity change in time. Several POD isoforms (P3, P4 and P9) were specifically induced by salinity and drought.     

Therapeutic efficacy of the root tubers of Aconitum heterophyllum and its substitute Cyperus rotundus in the amelioration of pylorus ligation induced ulcerogenic and oxidative damage in rats

The phenomenal increase in the demand of herbal drugs, leads to over exploitation of medicinal plants which ultimately resulted in the scarcity and endangerment of many valuable plant species. On observing the difficulties in procuring genuine herbal drugs arose the concept of substitution which was documented in many classical Ayurvedic texts. The present study made a comparative evaluation of the gastroprotective potential of hydroalcoholic extracts of an original drug Aconitum heterophyllum (HAAH) and its substitute Cyperus rotundus (HACR) in the treatment of gastric ulcer under in vivo experimental conditions. The anti-ulcer property of the plant extracts was investigated against pylorus ligation induced ulcer in Wistar albino rats. The results confirmed that both A. heterophyllum and C. rotundus deliver comparable significant protection against gastric ulcer, indicated by a decrease in the free and total acidity, volume of gastric content, total proteins and increase in pH of gastric content, total carbohydrates and total carbohydrates to total proteins ratio. The observed anti-ulcer potential of both the drugs is attributed mainly to prevention of the generation of damaging free radical cascades and oxidant radical release.     

Investigating the antiangiogenic potential of Rumex vesicarius (humeidh), anticancer activity in cancer cell lines and assessment of developmental toxicity in zebrafish embryos

Recent trends in anticancer therapy is to use therapeutic agents which not only kill the cancer cell, but are less toxic to surrounding normal cells/tissue. One approach is to cut the nutrient supply to growing tumor cells, by blocking the formation of new blood vessels around the tumor. As the phytochemicals and botanical crude extracts have proven their efficacy as natural antiangiogenic agents with minimum toxicities, there is need to explore varieties of medicinal plants for novel antiangiogenic compounds.Rumex vesicarius L. (Humeidh), is an annual herbal plant with proven medicinal values. The antiangiogenic potential, and developmental toxicity of humeidh in experimental animal models has never been studied before. The crude extracts were prepared from the roots, stems, leaves and flowers of Rumex vesicarius L. in methanol, chloroform, ethyl acetate and n-hexane. The developmental toxicity screening in zebrafish embryos, has revealed that Rumex vesicarius was not toxic to zebrafish embryos. The chloroform stem extract showed significant level of antiangiogenic activity in zebrafish angiogenic assay on a dose dependent manner. Thirty five (35) bioactive compounds were identified by gas chromatography mass spectrophotometry (GC–MS) analysis in the stem extract of Rumex vesicarius. Propanoic acid, 2-[(trimethylsilyl)oxy]-, trimethylsilyl ester, Butane, 1,2,3-tris(trimethylsiloxy), and Butanedioic acid, bis(trimethylsilyl) ester were identified as major compound present in the stem of R. vasicarius.The anticancer activity of roots, stem, leaves and flowers crude extract was evaluated in human breast cancer (MCF7), human colon carcinoma (Lovo, and Caco-2), human hepatocellular carcinoma (HepG2) cell lines. Most of the crude extracts did not show significant level of cytotoxicity in tested cancer cells line, except, chloroform extract of stem which exhibited strong anticancer activity in all tested cancer cells with IC₅₀ values in micro molar range.Based on these results, it is recommended that formulation prepared from R. vesicarius can further be tested in clinical trials in order to explore its therapeutic potential as an effective and safe natural anticancer product.     

The potential role of pomegranate and its nano-formulations on cerebral neurons in aluminum chloride induced Alzheimer rat model

The oxidative stress leading to degenerative changes in the brain of Alzheimer’s disease (AD) is evident. Our aim was to evaluate the therapeutic and protective effects of pomegranate extract (PE) and pomegranate extract-loaded nanoparticles (PE nano) in an AlCl 3-induced AD rat model. Nanoparticles were synthesized with a PE load of 0.68% w/w, and 70 male Wistar rats were divided into 7 groups: Group I was the control, Group II received PE., Group III received PE nano for 2 weeks, Group IV received AlCl 3 (50 mg/kg) daily orally for 4 weeks, Group V received PE for 2 weeks, Group VI received PE nano for 2 weeks, and Groups V and VI were started after AlCl 3 administration was stopped. Group VII received PE for 2 weeks and was stopped before AlCl 3 was administered. The Results revealed that the discrimination index in the novel object recognition test was the least in AD rat model but increased in cases protected with PE treated with PE nano. Similar results were shown based on calculating the brain weight/body weight percent. The biomarkers of antioxidant activity (catalase, glutathione and total antioxidant activity) in brain homogenate were significantly increased in groups treated with either PE or PE nano. The thiobarbituric acid reactive substance measured to estimate lipid peroxidation was significantly increased in AD rat model and decreased in groups protected with PE or treated with PE nano. Histopathological studies using hematoxylin and eosin, cresyl violet, and silver stains revealed hyaline degeneration, chromatolysis, and hallmarks of AD; neurofibrillary tangles and the senile plaques in brains of AD rat model. Restoration of the histological architecture, Nissl granules, and minimal appearance of hallmarks of AD characterized brains treated with PE or PE nano. In conclusion, PE was more effective as a protectant than a therapeutic measure in alleviating the antioxidant, lipid peroxidative effects and histopathological hallmarks in AD brains. But, the therapeutic PE-loaded nanoparticles increased the efficacy of active components and produced similar results as the protective PE.     

Pharmacognostic evaluation of Artemisia maritima L. a highly medicinal specie of genus Artemisia

The light and scanning electron microscopic observations were carried out for anatomical features of leaf, pollens and powder.Microscopic studies provide useful information for identification and authentication of adulteration in A. maritima. Nutritional analysis of A. maritima revealed that life fundamental macromolecules such as carbohydrates (49.63 %) crude proteins (13.17 %) and crude fibers (21.06 %) were present in sufficient quantity while crude fats (4.11 %) reported in low quantity. The life essential elements such as Mg (9.472 ± 0.011), Ca (4.152 ± 0.135) and Fe (4.112 ± 0.002) were found in high concentration while heavy metals reported under the safety threshold of WHO. These observations favored A. maritima an alternative of food.Appreciable quantity of phenolics (17.64 ± 0.574) and flavonoids (7.67 ± 0.069) were found while qualitatively active phytochemicals were reported.The FTIR characterization of A. maritima crude powder revealed chromatogram in 3328.61 to 408.68 frequency range and 24 characteristic peaks on the basis of which different compounds of biological importance were classified. HPLC-UV technique quantifiedand identified six phenolic compounds morin,epigallocatechin gallate, catechin hydrate,ellagic acid, pyrogallol andrutin. Identification of compounds through GC–MS chromatogram revealed the presence of 46 compounds in methanolic fraction however 17 compounds of biological importance were selected.In-vitro biological evaluation of A. maritima for antioxidant, antimicrobial, antidiabetic (12.61 ± 0.113 %) and cytotoxic activities (LC₅₀ = 20 μg/ml) suggested that methanolic fractions exhibited the highest activity as compared to chloroform and ethyl acetate fractions. The MIC values of 10 or 15 mg/ml were recorded for most of the fungal pathogens. Antibacterial activity revealed 3.75 mg/ml of MIC values against B. subtilis and 1.87 mg/ml against S. aureus, E. coli and P. aeruginosa. In-vivo biological evaluation revealed thatmaximum inhibition was observed for crude extract at 250 mg/kg body weight. The mechanism underlined in-vivo analgesic responses was carried out which revealed that naloxone (morphine and tramadol antagonist) showed no prominent effect while Glibenclamide pretreatment minutely modified the analgesic action. These observations clearly indicted the absence of opiod receptors and involvement of ATP sensitive potassium channels.     

The Phenolics of the Ornithogalum umbellatum L. (Hyacinthaceae): Phytochemical and Ecological Characterization

Ornithogalum umbellatum L. is a widely distributed species in Europe, that exhibits considerable variability at ecological, morphological, anatomical, and karyological level. Previous reports of the chemical investigations among Ornithogalum species indicate significant diversity of the secondary metabolites, as well. Knowing that environment affects the phenolic composition in plants to a large extent, the main objective of the research was to define relationship between phytochemical and ecological characters. To estimate an environmental influence on these results, plant material was collected at four habitats that differ in ecological factors and belong to two biogeographical regions: the Balkan Peninsula and the Pannonian Plane. Measured phytochemical characters are yield of dry extract, total phenolic and flavonoid contents, the presence of selected phenolic compounds as well as free‐radical scavenging activity (neutralization of DPPH and OH radicals). Results revealed that all analyzed phytochemical parameters differ between investigated O. umbellatum samples. The moisture level of habitat has the highest correlation, either positive or negative, with most of phytochemical characters, and is followed by temperature and soil reaction. Light intensity and nitrogen level have mostly moderate correlation coefficient with phytochemical characters. More complex correlation is revealed between ecological factors and nine phenolic compounds, with three observed patterns of relationship.     

Phytochemical profiling of Azima tetracantha Lam. leaf methanol extract and elucidation of its potential as a chain-breaking antioxidant,anti-inflammatory and anti-proliferative agent

Azima tetracantha, a traditional medicinal plant included in the order Brassicales and family Salvadoraceae, is widely used as a dietary supplement in folklore medicines. The plant is also used for the treatment of rheumatism, diarrhea and other inflammatory disorders. The present investigation focused on the phytochemical composition, radical scavenging, reducing potential and anti-proliferative activities of the A. tetracantha leaves. Quantitative estimation of the polyphenols and flavonoids revealed significantly elevated levels in the methanol extract. Corroborating with this, methanol extract exhibited higher in vitro anti-radical scavenging effect against 2,2-diphenyl-1- picrylhydrazyl (34.14 ± 2.19 μg/mL), and hydrogen peroxide (44.96 ± 1.77 μg/mL), as well as ferric reducing properties (58.24 ± 6.98 μg/mL). The methanolic extract also showed strong lipoxygenase (71.42 ± 6.36 μg/mL) and nitric oxide inhibitory activities (94.23 ± 8.11 μg/mL). Cytotoxic activity against MCF7 cells was found to be higher (IC50= 37.62 ± 2.94 μg/mL), than that of MDAMB231 cells (IC50= 69.11 ± 5.02 μg/mL). The qPCR-based analysis indicated dose-dependent increase in the expression of the pro-apoptotic genes such as executioner caspases and apoptotic protease activating factor-1. Overall, the results indicated the possible use of methanol extract of A. tetracantha leaves as a chain-breaking antioxidant molecule and are capable of inhibiting inflammatory enzymes and the proliferative potential of breast cancer cells.     

In vivo assessment of reversing Cisplatin-Induced nephrotoxicity using Jatropha mollissima crude extract and its potential cytotoxicity

Jatropha mollissima is one of the ancient plants that known in Africa, Asia and Latin America for its high medicinal value. Previously we showed that the ethanolic leaves extract of J. mollissima was able to reverse the aminoglycoside antibiotics induced nephrotoxicity in only two weeks of administration. Here, we evaluated the phytochemicals, antioxidant and in vivo cytotoxicity of the ethanolic leaves extract in addition to the ability of reversing Cisplatin-induced nephrotoxicity in wistar albino rats. The results of phytochemical analysis showed the presence of flavonoids, phenols, tannins and saponins, with significantly high antioxidant activity. The treated rats did not show any cytotoxic signs; no anatomical, physiological and/or histopathological changes compared with the control group. Kidney, spleen and liver tissues appeared normal after two weeks administration of the maximum dose, with a possible alteration in distal tubules, proximal tubules and glomerulus of the kidney tissues. The results of nephrotoxicity and kidney function suggest promising potential for J. mollissima in kidney damage treatment.     

Pre-clinical evaluation of cinobufotalin as a potential anti-lung cancer agent

Lung cancer is a major cause of cancer-related mortality in the United States and around the world. Due to the pre-existing or acquired chemo-resistance, the current standard chemotherapy regimens only show moderate activity against lung cancer. In the current study, we explored the potential anti-lung cancer activity of cinobufotalin in vivo and in vitro, and studied the underlying mechanisms. We demonstrated that cinobufotalin displayed considerable cytotoxicity against lung cancer cells (A549, H460 and HTB-58 lines) without inducing significant cell apoptosis. Our data suggest that mitochondrial protein cyclophilin D (Cyp-D)-dependent mitochondrial permeability transition pore (mPTP) opening mediates cinobufotalin-induced non-apoptotic death of lung cancer cells. The Cyp-D inhibitor cyclosporine A (CsA), the mPTP blocker sanglifehrin A (SfA), and Cyp-D shRNA-silencing significantly inhibited cinobufotalin-induced mitochondrial membrane potential (MMP) reduction and A549 cell death (but not apoptosis). Using a mice xenograft model, we found that cinobufotalin inhibited A549 lung cancer cell growth in vivo. Thus, cinobufotalin mainly induces Cyp-D-dependent non-apoptotic death in cultured lung cancer cells. The results of this study suggest that cinobufotalin might be further investigated as a novel anti-lung cancer agent.     

Phytochemical composition,antioxidant, in vitro and in silico studies of active compounds of Curculigo latifolia extracts as promising elastase inhibitor

Curculigo latifolia is a plant in the Hypoxidaceae family commonly used in herbal medicine. The study objective was to evaluate the antioxidant and anti-elastase properties of C. latifolia extracts in vitro and silico as a candidate for antiaging active ingredients. This study identified secondary metabolites of the hexane (HE), ethyl acetate (EAE), and ethanol extracts (EE) from the root (R), stem (S), and leaf (L) organs by LC-ESI-MS and evaluated in vitro antioxidant and inhibitor elastase activity. An antioxidant evaluation was performed using ABTS, Beta Carotene Bleaching (BCB), and Ferric Reduction Antioxidant Power (FRAP). Evaluation of anti-elastase was carried out using elastase and followed by an in silico study of molecular docking using the target protein elastase (1B0F). Fifteen C. latifolia metabolites were identified in C. latifolia extracts, most of which were phenolic compounds. In antioxidant testing, REE, REAE, SEE, and SEAE extracts showed potent antioxidant activity based on the ABTS, BCB, and FRAP methods. In anti-elastase testing, it was found that SEE, REE, REAE, and RHE extracts gave powerful inhibition of elastase activity (in the ranges of 16.89 to 27.91 µg/mL). The in-silico study demonstrated the potential of the identified metabolites to bind to the target protein 1B0F involved in remodeling the skin aging process. This research concludes that the extracts from C. latifolia have the potential to serve as an active antiaging source.     

The structural mimicry of membrane sterols by tamoxifen: evidence from cholesterol coefficients and molecular-modelling for its action as a membrane anti-oxidant and an anti-cancer agent

The anti-cancer drug tamoxifen is a potent inhibitor of lipid peroxidation induced by Fe(III)-ascorbate in ox-brain phospholipid liposomes. Similar anti-oxidant effects, but with varying potencies, are also shown by 4-hydroxytamoxifen, cholesterol, ergosterol and 17-β-oestradiol. We now describe a computer-graphic fitting technique that demonstrates a structural similarity between the five compounds. In addition, we have quantified the differences (relative to cholesterol) between the anti-oxidant activities of the compounds in terms of a novel expression reffered to here as the cholesterol coefficient (C_c) Finally, we discuss how the inhibitory effect of tamoxifen on lipid peroxidation may result from a membrane stabilization that is associated with a decrease in membrane fluidity. This action may be related to the anti-proliferative effect exerted by tamoxifen on cancer and fungal cells.     

Properties of a mini 9R-lipoxygenase from Nostoc sp. PCC 7120 and its mutant forms

Lipoxygenases (LOXs) consist of a class of enzymes that catalyze the regio- and stereospecific dioxygenation of polyunsaturated fatty acids. Current reports propose that a conserved glycine residue in the active site of R-lipoxygenases and an alanine residue at the corresponding position in S-lipoxygenases play a crucial role in determining the stereochemistry of the product. Recently, a bifunctional lipoxygenase with a linoleate diol synthase activity from Nostoc sp. PCC7120 with R stereospecificity and the so far unique feature of carrying an alanine instead of the conserved glycine in the position of the sequence determinant for chiral specificity was identified. The recombinant carboxy-terminal domain was purified after expression in Escherichia coli. The ability of the enzyme to use linoleic acid esterified to a bulky phosphatidylcholine molecule as a substrate suggested a tail-fist binding orientation of the substrate. Site directed mutagenesis of the alanine to glycine did not cause alterations in the stereospecificity of the products, while mutation of the alanine to valine or isoleucine modified both regio- and enantioselectivity of the enzyme. Kinetic measurements revealed that substitution of Ala by Gly or Val did not significantly influence the reaction characteristics, while the A162I mutant showed a reduced vmax. Based on the mutagenesis data obtained, we suggest that the existing model for stereocontrol of the lipoxygenase reaction may be expanded to include enzymes that seem to have in general a smaller amino acid in R and a bulkier one in S lipoxygenases at the position that controls stereospecificity.     

Ascorbylated 4-hydroxy-2-nonenal as a potential biomarker of oxidative stress response

Oxidative stress, resulting from the generation of reactive oxygen species, contributes to the development of a multitude of age-related diseases. Current methods of assessing oxidative stress levels range from the detection of lipid peroxidation products, such as F(2)-isoprostanes and malondialdehyde, to monitoring the redox status of glutathione. While useful, traditional biomarkers of oxidative stress are not without their drawbacks, including low in vitro concentrations and possible artifact formation. In the present study, we utilize liquid chromatography coupled with tandem mass spectrometry for investigation into the use of a novel compound, ascorbylated 4-hydroxy-2-nonenal, as a potential biomarker of oxidative stress.     

Synthesis and evaluation of stilbene derivatives as a potential imaging agent of amyloid plaques

Fluorescence probes that can detect Aβ (β-amyloid peptide) plaque are important tools for diagnosis of Alzheimer’s disease (AD), and 4-N-methylamino-4′-hydroxystilbene (SB-13) is one of the promising candidate molecules. We report here the synthesis of SB-13 derivatives that consist of various electron donating/withdrawing moieties and distinct size of N-substituents. The synthesized compounds were screened for detection of Aβ40 fibrils in vitro. Four compounds exhibited more than sixfold intensity increase, and they were further analyzed for detail bindings and Aβ plaque imaging. Among these molecules, compound 42 meets two critical requirements for imaging agent; high fluorescence responsiveness and strong binding affinity. This compound showed more than 25-fold increase with the dissociation constant of 1.13 ± 0.37 μM. In AD mouse brain tissue, 42 selectively stained Aβ plaque, more specifically peripheral regions of Aβ plaque. This finding demonstrated its potential use as brain-imaging agents for AD studies.