Hot alkali-extracted antioxidative crude polysaccharide from a novel mushroom enhances immune response via TLR-mediated NF-κB activation: A strategy for full utilization of a neglected tribal food.

Mushroom polysaccharides have been revered for decades as incredibly versatile compounds suitable for application in biomedical, pharmaceutical, food, and cosmetic industries. In that essence Russula alatoreticula, a traditionally appraised unique macrofungus, has earlier been explored to contain functional macromolecules elicited by hydrothermal and cold alkali systems. Remnants of that multistep extraction could further be utilized not only to isolate high-value components, but also to reduce waste material. With this in mind, we decided to recycle leftover residue of the tribal delicacy, rather than undervalue, by following heated NaOH reflux. Intriguingly, the effort resulted productive isolation of carbohydrate fraction (RualaHap) enriched in ß-glucan that showed excellent antioxidant activity in terms of radical scavenging, chelating ability, and reducing power with EC50 of 207-5000 µg/ml. Besides strong immune boosting prowess was also witnessed as exposure of RAW 264.7 cells to the metabolites influenced macrophage viability and phagocytosis in a time-dependent manner. Induction of intracellular NO and ROS generation was also enumerated along with aggrandized production of pseudopods representing hallmark of monocyte stimulation. Moreover, the treatment drastically triggered level of TLR-2, TLR-4, NF-κB, Iκ-Bα, iNOS, COX-2, TNF-α, IFN-γ, and IL-10 maintaining balance between M1 and M2 cytokines via TLR coordinated NF-κB pathway. Conclusively the research offers a roadmap for better utilization of a species boasted in traditional practice aiding future implementation of RualaHap as ingredients of nutraceuticals or functional foods. PRACTICAL APPLICATIONS: At present, macrofungal polysaccharides are regarded as an ideal resource for a range of safe uses encompassing direct contact or ingestion by human. In this context, RualaHap isolated from the wild edible mushroom, R. alatoreticula, could have a broad application prospect, particularly in development of natural therapeutics to ameliorate radical-induced ailment and immune-deficiency condition. The biopolymers could thus be utilized in functional diet or complementary medicine to improve health that in turn might raise local cost of the abandoned taxon boosting rural empowerment.

Characterization and Inception of a Triterpenoid Astrakurkurol, as a Cytotoxic Molecule on Human Hepatocellular Carcinoma Cells, Hep3B.

Mushrooms are customary influential sources of pharmaceutically active metabolites. Usually lanostane-type triterpenoids from mushrooms had prospective for cancer disease treatments. Recently, a triterpenoid, astrakurkurol obtained from the fresh basidiocarps of the edible mushroom Astraeus hygrometricus, drew attention as a new cytotoxic therapeutic. The structural stability of this triterpenoid had been established with the amalgamation of density functional theory (DFT) calculations and study of single-crystal X-ray diffraction. To successfully manifest astrakurkurol as a potent cytotoxic therapeutics, a wide apprehension on the molecular and cellular mechanisms underlying their action is prerequisite. On this account, our study was directed to scrutinize the influence of this triterpenoid on human hepatocellular cancer cell model Hep3B. Encapsulating all experimental facts revealed that astrakurkurol had significantly decreased cell viability in a concentration-dependent manner. This effect was unveiled to be apoptosis, documented by DNA fragmentation, chromatin condensation, nuclear shrinkage, membrane blebing, and imbalance of cell cycle distribution. Astrakurkurol persuaded the expression of death receptor associated proteins (Fas), which triggered caspase-8 activation following tBid cleavage. Moreover, tBid mediated ROS generation, which triggered mitochondrial dysfunction and activated the mitochondrial apoptotic events. Astrakurkurol cytotoxicity was based on caspase-8-mediated intrinsic apoptotic pathway and was associated with inhibition at Akt and NF-κB pathway. Astrakurkurol had also inhibited the migration of Hep3B cells, indicating its antimigratory potential. These findings led us to introduce astrakurkurol as a feasible and natural source for a safer cytotoxic drug against hepatocellular carcinoma.

Expanding knowledge on Russula alatoreticula, a novel mushroom from tribal cuisine, with chemical and pharmaceutical relevance.

Since antiquity, numerous macrofungi are being worshiped as food and natural medicine especially in Asian tribal communities. Recent investigation has correlated these medicinal properties with bioactive components including phenols and flavonoids. However, research on mushrooms is not satisfactory; as several traditionally prized members remain undiscovered or poorly explored yet. This backdrop tempted us to unveil secondary metabolites empowered with therapeutics from an ethnic delicacy, Russula alatoreticula that was justified as a novel macrofungus in our previous publication. Accordingly, methanol extract was prepared from dried basidiocarps that was found to be enriched with phenolic compounds (pyrogallol > cinnamic acid > p-coumaric acid) and ascorbic acid. As a result, the fraction exhibited strong antioxidant activity evident by the ability of quenching free radicals, chelating Fe2+ ion and reducing components with EC50 of 263-2382 µg/ml. Besides, effective antibacterial potential against six investigated microbes was also noticed where MIC value ranged from 99 to 2673.74 µg/ml. Furthermore, the extract revealed promising anticancer property as it induced apoptosis of Hep3B cell (IC50 358.57 µg/ml) by imparting morphological changes, interfering cell cycle, depleting MMP and alleviating ROS through Bax, Bcl2, caspases 9 and 3 intrinsic mitochondrial pathway. Overall study implied that the immense bioactive potential of R. alatoreticula could possibly be utilized as a good source of natural supplement to combat against free radicals, pathogenic bacteria and hepatocellular carcinoma as well as in food safety industry.

Syntheses, crystal structures, DFT calculations, protein interaction and anticancer activities of water soluble dipicolinic acid-imidazole based oxidovanadium(iv) complexes.

Three novel water soluble neutral mononuclear oxidovanadium(iv) complexes 1-3, [VOLB2] (where H2L = dipicolinic acid (DPA) and B = imidazole (1)/1-methylimidazole (2)/1-allylimidazole (3)), were synthesized by the reaction of [VOL(H2O)2] with imidazole/1-methylimidazole/1-allylimidazole in ethanol. The complexes were thoroughly characterized by elemental analysis, IR, UV-Vis and EPR spectroscopy, magnetic susceptibility, cyclic voltammetry and single crystal X-ray diffraction techniques. In all the complexes the vanadium(iv) centre assumes a distorted octahedral environment. All the three complexes have similar structures and contain a range of intramolecular interactions such as hydrogen bonding, C-Hπ, and ππ stacking dominating their supramolecular architectures. A thermal study of the complexes was carried out to analyze their stability. The energy of non-covalent interactions and frontier orbitals for the complexes were also calculated by DFT. In order to investigate the binding interactions and conformational changes of the secondary structure of bovine serum albumin (BSA) with the complexes, absorption, fluorimetric titration and circular dichroism measurements in aqueous medium were carried out. Molecular docking studies have also been carried out to understand the binding modes and interaction patterns of the oxidovanadium(iv) complexes with BSA. The anticancer activities of the ligand and complexes 1-3 were tested against the human hepatic carcinoma cell line Hep3B. The complexes showed prominent cytotoxicity towards cancer cells.

Biochemical basis of improvement of defense in tomato plant against Fusarium wilt by CaCl2.

The objective of this study was to investigate the effectiveness of calcium chloride (CaCl2), as potential elicitor, on tomato plants against Fusarium oxysporum f. sp. lycopersici. Foliar application of CaCl2 showed significant reduction of wilt incidence after challenge inoculation. Increased production of defense and antioxidant enzymes was observed in elicitor treated sets over control. Simultaneously, altered amount of phenolic acids were analyzed spectrophotometrically and by using high performance liquid chromatography. Significant induction of defense-related genes expressions was measured by semi-quantitative RT-PCR. Greater lignifications by microscopic analysis were also recorded in elicitor treated plants. Simultaneously, generation of nitric oxide (NO) in elicitor treated plants was confirmed by spectrophotometrically and microscopically by using membrane permeable fluorescent dye. Furthermore, plants treated with potential NO donor and NO modulators showed significant alteration of all those aforesaid defense molecules. Transcript analysis of nitrate reductase and calmodulin gene showed positive correlation with elicitor treatment. Furthermore, CaCl2 treatment showed greater seedling vigor index, mean trichome density etc. The result suggests that CaCl2 have tremendous potential to elicit defense responses as well as plant growth in co-relation with NO, which ultimately leads to resistance against the wilt pathogen.

Introducing a novel mushroom from mycophagy community with emphasis on biomedical potency.

Mushrooms have been prized by humankind as medicine and culinary wonder since antiquity. Though several species are ethnically valued; many prospective species are still being discovered. One such wild macrofungus has recently been discovered during subsequent field surveys in West Bengal, India which in turn exposed as a traditionally consumed popular myco-food. The collected taxon was found to be unique with regard to its morphological as well as genetical features. After detailed characterizations, the fungus was identified as a novel taxon belonging to the genus Russula (Russulaceae, Basidiomycota). Besides, the investigation was further extended in search of new functional ingredients and in this context, a water soluble crude polysaccharide rich extract (Rusalan) was isolated from dried basidiocarps. Accumulating evidences from GC-MS, HPTLC, FT-IR along with several spectrophotometric methods postulated that the fraction consisted mainly of carbohydrate in triple helical conformation, where glucose was the major monosaccharide mostly with ß-type glycosidic linkage. Conversely, Rusalan showed pronounced antioxidant activity in six in vitro assay systems with EC50 value ranging from 190-1328 µg/ml concentration. The crude polysaccharide was also evaluated against six bacterial strains using microdilution method and the growth of Staphylococcus aureus and Bacillus subtilis were found to be inhibited effectively. In addition, immune-stimulatory assays demonstrated that Rusalan could evidently promote proliferation, induce phagocytosis, release NO, produce intracellular ROS and upregulate mRNA expression of iNOS, TNF-α, COX-2, as well as IL-6 genes in in mouse macrophage cells. Therefore, aim of the present study was not only to describe a new taxon to the world mycoflora but also to introduce a potent therapeutic agent that could be explored for food and pharmaceutical purposes. However, isolation of active component and in vivo studies need to be designed further.

Chitosan-induced immunity in Camellia sinensis (L.) O. Kuntze against blister blight disease is mediated by nitric-oxide.

Blister blight disease, caused by an obligate biotrophic fungal pathogen, Exobasidium vexans Massee is posing a serious threat for tea cultivation in Asia. As the use of chemical pesticides on tea leaves substantially increases the toxic risks of tea consumption, serious attempts are being made to control such pathogens by boosting the intrinsic natural defense responses against invading pathogens in tea plants. In this study, the nature and durability of resistance offered by chitosan and the possible mechanism of chitosan-induced defense induction in Camellia sinensis (L.) O. Kuntze plants against blister blight disease were investigated. Foliar application of 0.01% chitosan solution at 15 days interval not only reduced the blister blight incidence for two seasons, but also maintained the induced expressions of different defense related enzymes and total phenol content compared to the control. Defense responses induced by chitosan were found to be down regulated under nitric oxide (NO) deficient conditions in vivo, indicating that the observed chitosan-induced resistance is probably activated via NO signaling. Such role of NO in host defense response was further established by application of the NO donor, sodium nitroprusside (SNP), which produced similar defense responses accomplished through chitosan treatment. Taken together, our results suggest that increased production of NO in chitosan-treated tea plants may play a critical role in triggering the innate defense responses effective against plant pathogens, including that causing the blister blight disease.

Abiotic elicitors mediated elicitation of innate immunity in tomato: an ex vivo comparison.

Improvement of the host resistance by using hazard free chemical elicitors is emerging as an alternative approach in the field of plant disease management. In our present work, we have screened the efficacy and possible mechanism of abiogenic elicitors like Dipotassium hydrogen orthophosphate (K2HPO4), Oxalic acid (OA), Isonicotinic acid (INA), Salicylic acid (SA), Acetylsalicylate (AS), Arachidonic acid (AA) and Calcium chloride (CaCl2) to stimulate innate immune responses in Lycopersicum esculentum Mill. Excised tomato leaves, treated with elicitors at three different concentrations, were found to stimulate defense and antioxidative enzymes, total phenol and flavonoid content after 24 h of incubation. CaCl2 (0.5 %) followed by INA (2.5 mM) were found most effective in activation of all such defense molecules in tomato leaves. Furthermore, nitric oxide (NO), a key gaseous mediator in plant defense signaling, was also measured after subsequent elicitor application. Higher doses of elicitors showed an elevated level of reactive oxygen species (ROS) generation, enhanced lipid peroxidation rate and proline content, which indicates the extent of abiotic stress generation on the leaves. However, ROS production, lipid peroxidation rate and proline concentration remain significantly reduced as a result of CaCl2 (0.5 %) and INA (2.5 mM) application. A sharp increase of total chlorophyll content was also recorded due to treatment of CaCl2 (0.5 %). These results demonstrate the effects of different abiogenic elicitors to regulate the production of defense molecules. Results also suggest that among all such chemicals, CaCl2 (0.5 %) and INA (2.5 mM) can be used as a potential elicitor in organic farming of tomato.

Chitosan nanoparticles: A positive modulator of innate immune responses in plants.

The immunomodulatory role of the natural biopolymer, chitosan, has already been demonstrated in plants, whilst its nanoparticles have only been examined for biomedical applications. In our present study, we have investigated the possible ability and mechanism of chitosan nanoparticles (CNP) to induce and augment immune responses in plants. CNP-treatment of leaves produced significant improvement in the plant's innate immune response through induction of defense enzyme activity, upregulation of defense related genes including that of several antioxidant enzymes as well as elevation of the levels of total phenolics. It is also possible that the extracellular localization of CNP may also play a role in the observed upregulation of defense response in plants. Nitric oxide (NO), an important signaling molecule in plant defense, was also observed to increase following CNP treatment. However, such CNP-mediated immuno-stimulation was significantly mitigated when NO production was inhibited, indicating a possible role of NO in such immune induction. Taken together, our results suggest that CNP may be used as a more effective phytosanitary or disease control agent compared to natural chitosan for sustainable organic cultivation.

Sublethal heavy metal stress stimulates innate immunity in tomato.

Effect of sublethal heavy metal stress as plant biotic elicitor for triggering innate immunity in tomato plant was investigated. Copper in in vivo condition induced accumulation of defense enzymes like peroxidase (PO), polyphenol oxidase (PPO), phenylalanine ammonia-lyase (PAL), and ß-1,3 glucanase along with higher accumulation of total phenol, antioxidative enzymes (catalase and ascorbate peroxidase), and total chlorophyll content. Furthermore, the treatment also induced nitric oxide (NO) production which was confirmed by realtime visualization of NO burst using a fluorescent probe 4,5-diaminofluorescein diacetate (DAF-2DA) and spectrophotometric analysis. The result suggested that the sublethal dose of heavy metal can induce an array of plant defense responses that lead to the improvement of innate immunity in plants.

Apoptogenic effects of Tricholoma giganteum on Ehrlich's ascites carcinoma cell.

This study explored the efficacy of Fa fraction of Tricholoma giganteum against Ehrlich's ascites carcinoma (EAC). Mechanisms of apoptogenic effect of the fraction were delineated. The flow cytometric analysis of EAC cells, showed an increase in number of cells in sub-G(0)/G(1) population and reduction in the G(2)/M phase due to the treatment thus suggesting apoptosis. The induction of apoptosis has also been confirmed by nuclear staining that demonstrated distinctive morphological features of apoptosis. Our data also revealed an increase in the expression of pro-apoptotic protein p53 in EAC and induced factors contributing to apoptosis. Pro-apoptotic gene Bax was up-regulated during p53-mediated apoptosis. No significant change in the expression of anti-apoptotic protein Bcl-2 was observed ensuing in decrease of the Bcl-2/Bax ratio. p53-mediated growth arrest involves p21 as a major effecter, which interestingly showed moderate elevation. All these observations indicate that Fa fraction of T. giganteum induces apoptogenic signal in EAC.