Assessment and identification of bioactive metabolites from terrestrial Lyngbya spp. responsible for antioxidant, antifungal, and anticancer activities.

Lyngbya from fresh and marine water produces an array of pharmaceutically bioactive therapeutic compounds. However, Lyngbya from agricultural soil is still poorly investigated. Hence, in this study, the bioactive potential of different Lyngbya spp. extract was explored. Intracellular petroleum ether extract of L. hieronymusii K81 showed the highest phenolic content (626.22 ± 0.65 µg GAEs g-1 FW), while intracellular ethyl acetate extract of L. aestuarii K97 (74.02 ± 0.002 mg QEs g-1 FW) showed highest flavonoid content. Highest free radical scavenging activity in terms of ABTS•+ was recorded in intracellular methanolic extract of Lyngbya sp. K5 (97.85 ± 0.068%), followed by L. wollei K80 (97.22 ± 0.059%) while highest DPPH• radical scavenging activity observed by intracellular acetone extract of Lyngbya sp. K5 (54.59 ± 0.165%). All the extracts also showed variable degrees of antifungal activities against Fusarium udum, F. oxysporum ciceris, Colletotrichum capsici, and Rhizoctonia solani. Further, extract of L. wollei K80 and L. aestuarii K97 showed potential anticancer activities against MCF7 (breast cancer) cell lines. GC-MS analyses of intracellular methanolic extract of L. wollei K80 showed the dominance of PUFAs with 9,12,15-octadecatrienoic acid, methyl ester, (Z,Z,Z) as the most abundant bioactive compound. On the other hand, the extracellular ethyl acetate extract of L. aestuarii K97 was rich in alkanes and alkenes with 1-hexyl-2-nitrocyclohexane as the most predominant compound. Extracts of Lyngbya spp. rich in novel secondary metabolites such as PUFAs, alkanes, and alkenes can be further explored as an alternative and low-cost antioxidant and potential apoptogens for cancer therapy.

Improvement of rooting and growth in kiwifruit (Actinidia deliciosa) cuttings with organic biostimulants.

Seaweed extracts have shown profoundly positive effects on crop growth, quality and reproduction in diverse agricultural and horticultural crops. Seaweed extracts can be used to promote the rooting and growth of cuttings in perennial fruit species like kiwifruit (Actinidia deliciosa). In this study, the cuttings were treated with 1, 5, 10 and 50% solutions of G Sap (Gracilaria edulis), K Sap (Kappaphycus alvarezii), AN (Ascophyllum nodosum), EM (Ecklonia maxima), HA (Humic acid) and control (water) for 6 h as base dipping. Subsequently, the treatments of G Sap, K Sap, AN, EM, HA and control were repeated every 15 days for a period of six months as application of 50 ml solutions in the potted cuttings. All the treatments exhibited significant effects on the rooting percent in all the kiwifruit cultivars, namely 'Monty', 'Abott', 'Hayward', 'Allison' and 'Bruno' (P ≤ 0.01) as compared to the control. Shoot and root growth parameters including leaf number per cutting, number of roots per cutting, number of branches, plant height, shoot diameter, root length, root diameter and root weight were all positively increased with the application of seaweed extracts (P ≤ 0.05). Cuttings treated with seaweed extract exhibited significantly higher levels of pigments (chlorophyll a, chlorophyll b and total carotenoids), metabolites (total carbohydrates and soluble phenols) and less electrolyte leakage as compared to the control cuttings. Significant positive and negative correlations were observed between biochemical parameters combined with plant nutrient concentration. Principal component analysis (PCA) revealed that PC1 and PC2 (first two principal components) accounted for 75% of the entire variation. While, PC1 accounted for 63% of the total variation, PC2 accounted for 11% of the total variation. The leaves and the roots of kiwifruit cultivar 'Hayward' treated with G Sap at 10%, K Sap at 10%, AN at 10%, EM at 10%, HA at 10% exhibited higher expression of all four root promoting candidate genes (GH3-3, LBD16, LBD29 and LRP1) compared to the control. Therefore, it can be concluded that, seaweed extract and humic acid can be used as a suitable alternative to synthetic hormones for promoting the rooting and growth of kiwifruit cuttings.

In-depth evaluation of nutritive, chemical constituents and anti-glycemic properties of jackfruit (Artocarpus heterophyllus Lam) clonal accessions with flake colour diversity from Eastern Sub-Himalayan plains of India.

The study was designed to elucidate the potential of jackfruit clonal accessions having diverse flake colours from nutritional and medicinal perspectives. Jack fruit accessions with deep yellow flakes were found to contain the highest flavonoids, antioxidant activity, ascorbic acid, and α-glucosidase inhibition whereas, orange-red flakes exhibited the highest ß-carotene, phenol, minerals (iron and zinc) and better inhibition of α-amylase and ß-glucosidase enzymes. Phenolic compounds profiling revealed the presence of higher sinapic acid, ferulic acid and quercetin contents in the orange-red-coloured flakes. Metabolite analysis revealed presence of anti-diabetic compounds (n-Hexadecanoic acid, tridecane, 2-Heptadecenal etc.) in deep yellow and orange-red coloured jack flakes with lower glycemic load. Considering the abundant health benefits as evident from the present study, orange-red and deep yellow-coloured flakes may be recommended for consumption to manage the hyperglycemic condition.

A liquid chromatographic method for determination of acetamiprid and buprofezin residues and their dissipation kinetics in paddy matrices and soil.

The present study was conducted to investigate the residue status of two insecticides (acetamiprid and buprofezin) and their dissipation kinetics in three matrices viz. paddy grain, straw, and soil. The extraction procedure for residues of these two insecticides was executed using acetonitrile solvent. The analytical method was validated, which showed good linearity with the limit of quantification (LOQ) value of 0.01 and 0.02 mg kg-1 for acetamiprid and buprofezin, respectively. The recovery range was 79.67-98.33 % concerning all the matrices in both the insecticides. Acetamiprid (20% SP) and Buprofezin (25% SC) were applied separately in the paddy field in two doses: single dose (recommended dose) and double dose along with untreated control throughout the experiment. Residue analysis of these two insecticides in paddy (grain and straw) and soil was accomplished employing high-performance liquid chromatography (HPLC) with ultraviolet (UV) detector and confirmed by ultra-performance liquid chromatography (UPLC) coupled with mass spectrometry (UPLC-MS/MS). The dissipation data showed that acetamiprid exhibited higher dissipation in comparison with buprofezin. However, their persistence was found slightly higher in soil. The dissipation dynamics in the rice and soil were discussed with biological half-lives of both the insecticides. Consumer risk assessment study was also made considering its fate to the consumers.

Evaluation of Quality Parameters of Seven Processing Type Potato (Solanum tuberosum L.) Cultivars in the Eastern Sub-Himalayan Plains.

The eastern sub-Himalayan plain of India is a popular potato growing belt in which vast scope exists to introduce processing grade cultivars. The selection and introduction of a better quality processing grade cultivar in this region may pave the way for the processing industries. Keeping these in the backdrop, this study was conducted at Instructional Farm of Uttar Banga Krishi Viswavidyalaya (UBKV), Pundibari, Cooch Behar, West Bengal, India under eastern sub-Himalayan plains during winter seasons of 2016-17 and 2017-18 in which seven processing type potato cultivars (Kufri Chipsona-1, Kufri Chipsona-3, Kufri Chipsona-4, Kufri Frysona, Kufri Himsona, Kufri Surya and Kufri Chandramukhi) were evaluated in terms of different quality parameters pre-requisite for chips processing viz., dry matter content, specific gravity, starch content, chips colour score, crispiness and hardness of chips through randomised complete block design (RCBD). The study revealed wide variation in all quality parameters amongst the cultivars. Cultivar 'Kufri Frysona' showed the highest specific gravity (1.121) as well as dry matter content (23.35%) followed by 'Kufri Chipsona-3'. The cultivar 'Kufri Frysona' showed the highest starch content (28.52%) too. Chips prepared from 'Kufri Chipsona-1' were recorded to be crispier with a relatively lower value of deformation before the first break and less hardness value. All processing type potato cultivar reflected the chips colour score <3 (evaluated, based on 1-10 scale, 10 being the darkest and least desirable) though 'Kufri Frysona' had the lowest chips colour score (1.50) signifying its superiority for the region. 'Kufri Frysona' cultivation could be recommended in this agro-climatic region particularly for chips manufacturing potato industries.

Evaluation of indigenous aromatic rice cultivars from sub-Himalayan Terai region of India for nutritional attributes and blast resistance.

Indigenous folk rice cultivars often possess remarkable but unrevealed potential in terms of nutritional attributes and biotic stress tolerance. The unique cooking qualities and blissful aroma of many of these landraces make it an attractive low-cost alternative to high priced Basmati rice. Sub-Himalayan Terai region is bestowed with great agrobiodiversity in traditional heirloom rice cultivars. In the present study, ninety-nine folk rice cultivars from these regions were collected, purified and characterized for morphological and yield traits. Based on traditional importance and presence of aroma, thirty-five genotypes were selected and analyzed for genetic diversity using micro-satellite marker system. The genotypes were found to be genetically distinct and of high nutritive value. The resistant starch content, amylose content, glycemic index and antioxidant potential of these genotypes represented wide variability and 'Kataribhog', 'Sadanunia', 'Chakhao' etc. were identified as promising genotypes in terms of different nutritional attributes. These cultivars were screened further for resistance against blast disease in field trials and cultivars like 'Sadanunia', 'T4M-3-5', 'Chakhao Sampark' were found to be highly resistant to the blast disease whereas 'Kalonunia', 'Gobindabhog', 'Konkanijoha' were found to be highly susceptible. Principal Component analysis divided the genotypes in distinct groups for nutritional potential and blast tolerance. The resistant and susceptible genotypes were screened for the presence of the blast resistant pi genes and association analysis was performed with disease tolerance. Finally, a logistic model based on phenotypic traits for prediction of the blast susceptibility of the genotypes is proposed with more than 80% accuracy.

Genome wide molecular evolution analysis of begomoviruses reveals unique diversification pattern in coat protein gene of Old World and New World viruses.

Begomoviruses (Family-Geminiviridae) are plant infecting single stranded DNA viruses known to evolve very fast. Here, we have analysed the DNA-A sequences of 302 begomoviruses reported as 'type isolates' from different countries following the list of International Committee on Taxonomy of Viruses till 2017. Phylogenetic analysis was performed which revealed two major evolutionarily distinct groups namely Old World (OW) and New World (NW) viruses. Our work present evidence that cp gene has varied degree of diversification among the viruses reported from NW and OW. The NW viruses are more conserved in their cp gene sequences than that of OW viruses irrespective of host plant families. Further analysis reveals that cp gene differs in its recombination pattern among OW and NW viruses whereas rep gene is highly recombination prone in both OW and NW viruses. The sequence conservation in cp gene in NW viruses is a result of meagre recombination and subsequent low substitution rate in comparison to OW viruses. Our results demonstrated that the cp gene in NW viruses is less likely to possess nuclear localisation sequences than OW cp gene. Further we present evidence that the NW-cp is under the influence of strong purifying selection. We propose that the precoat protein (pcp) gene present exclusively in the 5' of cp gene in OW viruses is highly diversified and strong positive selection working on pcp gene might be attributing largely to the diversity of OW-cp gene.

Garcinol sensitizes human head and neck carcinoma to cisplatin in a xenograft mouse model despite downregulation of proliferative biomarkers.

Platinum compounds such as cisplatin and carboplatin are frequently used as the first-line chemotherapy for the treatment of the head and neck squamous cell carcinoma (HNSCC). In the present study, we investigated whether garcinol, a polyisoprenylated benzophenone can chemosensitize HNSCC to cisplatin. We found that garcinol inhibited the viability of a panel of diverse HNSCC cell lines, enhanced the apoptotic effect of cisplatin, suppressed constitutive as well as cisplatin-induced NF-κB activation, and downregulated the expression of various oncogenic gene products (cyclin D1, Bcl-2, survivin and VEGF). In vivo study showed that administration of garcinol alone (0.5 mg/kg body weight, i.p. five times/week) significantly suppressed the growth of the tumor, and this effect was further increased by cisplatin. Both the markers of proliferation index (Ki-67) and microvessel density (CD31) were downregulated in tumor tissues by the combination of cisplatin and garcinol. The pharmacokinetic results of garcinol indicated that good systemic exposure was achievable after i.p. administration of garcinol at 0.5 mg/kg and 2 mg/kg with mean peak concentration (Cmax) of 1825.4 and 6635.7 nM in the mouse serum, respectively. Overall, our results suggest that garcinol can indeed potentiate the effects of cisplatin by negative regulation of various inflammatory and proliferative biomarkers.

Naphthoquinone-mediated inhibition of lysine acetyltransferase KAT3B/p300, basis for non-toxic inhibitor synthesis.

Hydroxynaphthoquinone-based inhibitors of the lysine acetyltransferase KAT3B (p300), such as plumbagin, are relatively toxic. Here, we report that free thiol reactivity and redox cycling properties greatly contribute to the toxicity of plumbagin. A reactive 3rd position in the naphthoquinone derivatives is essential for thiol reactivity and enhances redox cycling. Using this clue, we synthesized PTK1, harboring a methyl substitution at the 3rd position of plumbagin. This molecule loses its thiol reactivity completely and its redox cycling ability to a lesser extent. Mechanistically, non-competitive, reversible binding of the inhibitor to the lysine acetyltransferase (KAT) domain of p300 is largely responsible for the acetyltransferase inhibition. Remarkably, the modified inhibitor PTK1 was a nearly non-toxic inhibitor of p300. The present report elucidates the mechanism of acetyltransferase activity inhibition by 1,4-naphthoquinones, which involves redox cycling and nucleophilic adduct formation, and it suggests possible routes of synthesis of the non-toxic inhibitor.

In vitro kinetics of soybean lipoxygenase with combinatorial fatty substrates and its functional significance in off flavour development.

Lipoxygenase (Lox) mediated oxidation of polyunsaturated fatty acids (PUFA) in mature soya seeds results in objectionable flavour. In the present study, Lox isozymes were purified to near homogeneity (107-fold). Lox-2 and 3 displayed remarkable kinetic preference (1.7 and 1.5-fold, respectively) for low PUFA ratios (LA/LeA) (PRs) among the selected PUFA combinations. Lox-1 displayed no specific preference. Pure Lox-1 displayed unbiased response towards substrates with marginal preference (1.2-fold) for linoleic acid at its optimum pH. Volatile compounds profiling showed a direct relationship between PRs and hexanal to trans-2-hexenal (1.47, 2.24 and 18.90 for 2, 7 and 15 PRs, respectively) ratio. The off-flavour determining parameters like TBA value, carbonyl value and lipid hydroperoxides (LHPODs) exhibited significant negative correlation (0.76, 0.74, 0.72; p<0.0001) in selected soya genotypes displaying varied PRs and significant positive correlation (0.89, 0.81. 0.89; p<0.0001) with ratio of PI (polyene index) to PRs - suggesting the plausible significance of PUFA ratios in biological lipid peroxidation.

Molecular cloning, characterization and expression of lipoxygenase 2 (lox-2) isozyme from Indian soybean [Glycine max (L.) Merrill] cv. Pusa 16.

The consumption of soybean is limited worldwide, despite being highly nutritious and having versatile uses due to the presence of grassy, beany and rancid off-flavour. The lipoxygenase-2 (LOX-2) is the key enzyme responsible for the production of volatiles released from the beans, which cause off-flavour in soy products. In this study, a 2.6-kb full-length lox2 gene (NCBI accession No. JQ929619.1) was isolated and cloned from soybean (Glycine max L. Merril) cv. Pusa 16. The cloned cDNA sequence of lox2 gene showed the complete open reading frame (ORF) of a putative protein, having 866 amino acids with start codon present at the foremost position and stop codon at the end. The theoretical pI of predicted protein was 6.22. A hydropathy profile calculated from the amino acid sequence resembled those of dicot LOXs, suggesting conservation of the secondary structure of these enzymes. The LOX-2 showed conserved six Histidine residues within a span of 520 to 590 amino acid position, a signature element for the enzyme activity. The lox2 gene was expressed using pET vector in prokaryotic expression system. The recombinant LOX-2 protein was purified after induction with IPTG (isopentyl thiogalactoside). A prominent band of 97 kDa was observed, when affinity purified fractions were analyzed by SDS-PAGE. The purified protein was characterized for the enzyme activity, substrate preference and K(m). Inhibitor studies with natural antioxidant molecules present in soybean revealed alpha-tocopherol to be the most effective inhibitor of LOX-2.

Ubiquitin-like protein from human placental extract exhibits collagenase activity.

An aqueous extract of human placenta exhibits strong gelatinase/collagenase activity in zymography. 2-D gel electrophoresis of the extract with gelatin zymography in the second dimension displayed a single spot, identified as ubiquitin-like component upon MALDI/TOF MS/MS analysis. Immunoblot indicated presence of ubiquitin and absence of collagenase in the extract. Collagenase activity of the ubiquitin-like component was confirmed from the change in solubility of collagen in aqueous buffer, degradation of collagen by size-exclusion HPLC and atomic force microscopy. Quantification with DQ-gelatin showed that the extract contains 0.04 U/ml of collagenase activity that was inhibited up to 95% by ubiquitin antibody. Ubiquitin from bovine erythrocytes demonstrated mild collagenase activity. Bioinformatics studies suggest that placental ubiquitin and collagenase follow structurally divergent evolution. This thermostable intrinsic collagenase activity of placental extract might have wide physiological relevance in degrading and remodeling collagen as it is used as a drug for wound healing and pelvic inflammatory diseases.

Thermal detoxification of the venom from Daboia russelli russelli of Eastern India with restoration of fibrinolytic activity.

The fibrinolytic components of venom have been evaluated for long in the enzymatic treatment of thrombosis. Russell's viper venom has fibrinolytic activity that is associated with hemorrhagic activity. Here it has been investigated whether the crude venom could be detoxified by thermal denaturation retaining fibrinolytic activity. The venom at 0.05 mg/ml in 20 mM K-phosphate, pH 7.5 when exposed to 100 °C for 5 min followed by cooling at 25 °C for 1 h led to its detoxification, while 80-85% of the fibrinolytic activity was recovered. Assessment of toxicity of the renatured venom in mice after injection at 5 fold excess of the lethal dose showed no lethality including hemorrhage, myotoxicity, cytotoxicity and liver toxicity. This simple method for preparation of fibrinolytic component for therapeutic use may be developed.

Two L-amino acid oxidase isoenzymes from Russell's viper (Daboia russelli russelli) venom with different mechanisms of inhibition by substrate analogs.

Two isoforms, L(1) and L(2), of L-amino acid oxidase have been isolated from Russell's viper venom by Sephadex G-100 gel filtration followed by CM-Sephadex C-50 ion exchange chromatography. The enzymes, with different isoelectric points, are monomers of 60-63 kDa as observed from size exclusion HPLC and SDS/PAGE. Partial N-terminal amino acid sequencing of L(1) and L(2) showed significant homology with other snake venom L-amino acid oxidases. Both the enzymes exhibit marked substrate preference for hydrophobic amino acids, maximum catalytic efficiency being observed with L-Phe. Inhibition of L(1) and L(2) by the substrate analogs N-acetyltryptophan and N-acetyl-L-tryptophan amide has been followed. The initial uncompetitive inhibition of L(1) followed by mixed inhibition at higher concentrations suggested the existence of two different inhibitor-binding sites distinct from the substrate-binding site. In the case of L(2), initial linear competitive inhibition followed by mixed inhibition suggested the existence of two nonoverlapping inhibitor-binding sites, one of which is the substrate-binding site. An inhibition kinetic study with O-aminobenzoic acid, a mimicking substrate with amino, carboxylate and hydrophobic parts, indicated the presence of three and two binding sites in L(1) and L(2), respectively, including one at the substrate-binding site. An inhibitor cross-competition kinetic study indicated mutually excluding binding between N-acetyltryptophan, N-acetyl-L-tryptophan amide and O-aminobenzoic acid in both the isoforms, except at the substrate-binding site of L(1). Binding of substrate analogs with different electrostatic and hydrophobic properties provides useful insights into the environment of the catalytic sites. Furthermore, it predicts the minimum structural requirement for a ligand to enter and anchor at the respective functional sites of LAAO that may facilitate the design of suicidal inhibitors.

Ability of a small, basic protein isolated from Russell's viper venom (Daboia russelli russelli) to induce renal tubular necrosis in mice.

Russell's viper venom (RVV) induced acute renal failure involves both direct and indirect nephrotoxic actions where the specific component/s are yet to be identified. A basic cytotoxin of 7.2kDa (RVV-7) has been identified as potential nephrotoxin. Autoradiographic experiments demonstrated that only RVV-7 among RVV toxins binds specifically to mice kidney membrane. Homogeneous preparation of RVV-7 confirmed its necrotic cell killing property having EC(50) of 4.79+/-3.28microM. Tissue distribution kinetics of RVV-7 in mice showed its higher localization in kidney compared to blood and liver. Role of inherent factor responsible for its localization in kidney was assessed after chemical inactivation of its cytotoxic activity. Cytotoxicity was neutralized by histidine modification but consequent alteration of in vivo distribution was insignificant. Classical concept of glomerular capillary wall (GCW) permselectivity barrier denotes that apart from size selectivity, GCW also restricts anionic proteins from filtration. Reducing the pI of RVV-7 by chemical manipulation of its surface positive charges resulted to decreased accumulation in kidney. Histological observations of kidney from mice treated in vivo with RVV-7 showed degenerated tubular epithelium. These findings indicate that basic character and small size of RVV-7 are favorable for its rapid accumulation in kidney leading to necrotic destruction of tubular epithelium.

Purification and characterization of a low molecular weight multifunctional cytotoxic phospholipase A2 from Russell's viper venom.

A basic toxin from Russell's viper venom of 7.2 kDa (RVV-7) has been purified to homogeneity after partial unfolding by 4 M urea followed by filtration through Centricon-30 membrane. Its N-terminal sequence showed strong homology with snake venom cytotoxins. Cytotoxic activity of RVV-7 has been demonstrated with B16F10 melanoma cells. PLA2 activity was observed in cytotoxin (CX3) from Naja kauthia bearing sequence homology with RVV-7. Phospholipase A2 and trypsin inhibitory activities were also observed with RVV-7. Chemical modification and inhibition studies suggested independent functional sites for these activities. A qualitative assessment of tumor growth inhibition by RVV-7 has been made.