Differential Impact In Vivo of Pf4-ΔCre-Mediated and Gp1ba-ΔCre-Mediated Depletion of Cyclooxygenase-1 in Platelets in Mice.

BACKGROUND: Low-dose aspirin is widely used for the secondary prevention of cardiovascular disease. The beneficial effects of low-dose aspirin are attributable to its inhibition of platelet Cox (cyclooxygenase)-1-derived thromboxane A2. Until recently, the use of the Pf4 (platelet factor 4) Cre has been the only genetic approach to generating megakaryocyte/platelet ablation of Cox-1 in mice. However, Pf4-ΔCre displays ectopic expression outside the megakaryocyte/platelet lineage, especially during inflammation. The use of the Gp1ba (glycoprotein 1bα) Cre promises a more specific, targeted approach. METHODS: To evaluate the role of Cox-1 in platelets, we crossed Pf4-ΔCre or Gp1ba-ΔCre mice with Cox-1flox/flox mice to generate platelet Cox-1-/- mice on normolipidemic and hyperlipidemic (Ldlr-/-; low-density lipoprotein receptor) backgrounds. RESULTS: Ex vivo platelet aggregation induced by arachidonic acid or adenosine diphosphate in platelet-rich plasma was inhibited to a similar extent in Pf4-ΔCre Cox-1-/-/Ldlr-/- and Gp1ba-ΔCre Cox-1-/-/Ldlr-/- mice. In a mouse model of tail injury, Pf4-ΔCre-mediated and Gp1ba-ΔCre-mediated deletions of Cox-1 were similarly efficient in suppressing platelet prostanoid biosynthesis. Experimental thrombogenesis and attendant blood loss were similar in both models. However, the impact on atherogenesis was divergent, being accelerated in the Pf4-ΔCre mice while restrained in the Gp1ba-ΔCres. In the former, accelerated atherogenesis was associated with greater suppression of PGI2 biosynthesis, a reduction in the lipopolysaccharide-evoked capacity to produce PGE2 (prostaglandin E) and PGD2 (prostanglandin D), activation of the inflammasome, elevated plasma levels of IL-1ß (interleukin), reduced plasma levels of HDL-C (high-density lipoprotein receptor-cholesterol), and a reduction in the capacity for reverse cholesterol transport. By contrast, in the latter, plasma HDL-C and α-tocopherol were elevated, and MIP-1α (macrophage inflammatory protein-1α) and MCP-1 (monocyte chemoattractant protein 1) were reduced. CONCLUSIONS: Both approaches to Cox-1 deletion similarly restrain thrombogenesis, but a differential impact on Cox-1-dependent prostanoid formation by the vasculature may contribute to an inflammatory phenotype and accelerated atherogenesis in Pf4-ΔCre mice.

Identification and quantification of eight alkaloids in Aconitum heterophyllum using UHPLC-DAD-QTOF-IMS: A valuable tool for quality control.

INTRODUCTION: Aconitum spp. are prime medicinal plants rich in alkaloids and have been used as the main constituents of traditional medicine in India and China. The whole plant can be toxic and creates pathophysiological conditions inside the human body. Therefore, simultaneous quantification of alkaloids within plant parts and herbal medicines associated with this genus is essential for quality control. OBJECTIVE: We aimed to develop and validate methods using ultra-high-performance liquid chromatography-diode array detector-quadrupole time-of-flight ion mobility mass spectrometry (UHPLC-DAD-QTOF-IMS) and to develop an analytical strategy for the identification and quantification of alkaloid compounds (aconitine, hypaconitine, mesaconitine, aconine, benzoylmesaconitine, benzoylaconine, bulleyaconitine A, and deoxyaconitine) from Aconitum heterophyllum. METHODOLOGY: We developed a simultaneous identification and quantification method for eight alkaloids using UHPLC-DAD-QTOF-IMS. The method was validated as per International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines and also in IMS mode. RESULTS: The developed method has good linearity (r2 = 0.997-0.999), LOD (0.63-8.31 µg/mL), LOQ (0.63-2.80 µg/mL), recovery (86.01-104.33%), reproducibility, intra- and inter-day variability (<3.25%), and stability. Significant qualitative and quantitative variations were found among different plant parts (flower, leaf, stem, root, and tuber) and five market products of A. heterophyllum. Furthermore, a total of 21 metabolites were also profiled based on the fragmentation pattern of MS2 using the validated method. CONCLUSION: An appropriate mobile phase using acetonitrile and water in a gradient elution gave a satisfactory chromatographic separation of eight Aconitum alkaloids with their adjacent peaks. Therefore, this method could provide a scientific and technical platform for quality control assurance.

Multi-residue determination of pesticides in vegetables and assessment of human health risks in Western Himalayan region of India.

This study was aimed to determine pesticides concentrations in fresh vegetables and assess human health risks in North-Western Himalayan region of India. Vegetable samples (n = 300) collected randomly from different agro-climatic zones were analyzed for 19 pesticides using gas chromatography. Pesticide residues were detected in 116 samples, of which 49 samples exceeded maximum permissible limits established by European Commission. Hexaconazole was most frequently detected in 9.3% samples followed by aldrin (8.3%), alachlor (5.3%), bifenthrin (4.3%), chlorpyrifos (3.7%), metribuzin (2.7%), ß-endosulfan, ethion, ß-HCH (2%, each), γ-HCH (1.3%), α-HCH, δ-HCH, malathion, heptachlor (1%, each), and α-endosulfan, pendimethalin in 0.7% samples. Human health risk assessment revealed that the percent contribution to acceptable daily intakes of pesticides via dietary intake of vegetables ranged from 0.014 to 39.4% in children and 0.003 to 9.85% in adults. Although hazard index values were < 1 but considering the concentrations of detected pesticide in samples, children were found to be at more risk. Since pragmatic investigations on occurrence of pesticides in vegetables and human health risk assessment from study area have not yet been worked out, so, this study highlights the importance of adopting good agricultural practices, awareness on food safety, monitoring of harmful chemicals in food commodities, and execution of food safety regulations to safeguard environmental and human health.

Evaluating Peptides of Picrorhiza kurroa and Their Inhibitory Potential against ACE, DPP-IV, and Oxidative Stress.

Picrorhiza kurroa Royle ex Benth. is a high-altitude plant having great medicinal value. However, its medicinal value at the peptide level is still unknown, which limits its utility in the development of peptide-based therapeutics. Here, we identify 65 peptides fromP. kurroa hydrolysate. Sequence analysis suggests that one novel bioactive peptide, ASGLCPEEAVPRR (BP1), has antioxidant potential and shows angiotensin-converting enzyme (ACE) and dipeptidyl peptidase-IV (DPP-IV) inhibitory activities. The molecular docking study showed that BP1 has a lower binding energy and strong affinity toward active pockets of ACE and DPP-IV, which explains its higher ACE [IC50 = 59.90 ± 9.52 µg/mL (43.40 µM)] and DPP-IV [IC50 = 3.04 ± 0.26 µg/mL (2.2 µM)] inhibitory activities. BP1 protects HEK293 cells from H2O2-induced oxidative damage by inhibiting intracellular reactive oxygen species (ROS) and malondialdehyde accumulation and activating the intrinsic antioxidant defense system. Additionally, phase-contrast microscopy studies revealed that pre-treatment of BP1 to HEK293 cells before exposure to H2O2 retains the normal morphology and blocks apoptosis. Furthermore, it also suppresses ROS-induced mitochondrial apoptosis via restoring the mitochondrial membrane potential (ΔΨm) and inhibiting caspase 3/7 activity. Therefore, BP1 has antioxidant potential and ACE and DPP-IV inhibitory activities that could be used for peptide-based formulation(s) in pharmaceuticals to treat diabetes, cardiovascular diseases, and other diseases associated with ROS.

In-depth assembly of organ and development dissected Picrorhiza kurroa proteome map using mass spectrometry.

BACKGROUND: Picrorhiza kurroa Royle ex Benth. being a rich source of phytochemicals, is a promising high altitude medicinal herb of Himalaya. The medicinal potential is attributed to picrosides i.e. iridoid glycosides, which synthesized in organ-specific manner through highly complex pathways. Here, we present a large-scale proteome reference map of P. kurroa, consisting of four morphologically differentiated organs and two developmental stages. RESULTS: We were able to identify 5186 protein accessions (FDR < 1%) providing a deep coverage of protein abundance array, spanning around six orders of magnitude. Most of the identified proteins are associated with metabolic processes, response to abiotic stimuli and cellular processes. Organ specific sub-proteomes highlights organ specialized functions that would offer insights to explore tissue profile for specific protein classes. With reference to P. kurroa development, vegetative phase is enriched with growth related processes, however generative phase harvests more energy in secondary metabolic pathways. Furthermore, stress-responsive proteins, RNA binding proteins (RBPs) and post-translational modifications (PTMs), particularly phosphorylation and ADP-ribosylation play an important role in P. kurroa adaptation to alpine environment. The proteins involved in the synthesis of secondary metabolites are well represented in P. kurroa proteome. The phytochemical analysis revealed that marker compounds were highly accumulated in rhizome and overall, during the late stage of development. CONCLUSIONS: This report represents first extensive proteomic description of organ and developmental dissected P. kurroa, providing a platform for future studies related to stress tolerance and medical applications.

Deciphering key regulators involved in epilepsy-induced cardiac damage through whole transcriptome and proteome analysis in a rat model.

OBJECTIVE: Sudden unexpected death in epilepsy (SUDEP) is a major outcome of cardiac dysfunction in patients with epilepsy. In continuation of our previous work, the present study was envisaged to explore the key regulators responsible for cardiac damage associated with chronic seizures using whole transcriptome and proteome analysis in a rat model of temporal lobe epilepsy. METHODS: A standard lithium-pilocarpine protocol was used to induce recurrent seizures in rats. The isolated rat heart tissue was subjected to transcriptomic and proteomic analysis. An integrated approach of RNA-Seq, proteomics, and system biology analysis was used to identify key regulators involved in seizure-linked cardiac changes. The analyzed differential expression patterns and network interactions were supported by gene and protein expression studies. RESULTS: Altogether, 1157 differentially expressed genes and 1264 proteins were identified in the cardiac tissue of epileptic animals through RNA-Seq and liquid chromatography with tandem mass spectrometry-based proteomic analysis, respectively. The network analysis revealed seven critical genes-STAT3, Myc, Fos, Erbb2, Erbb3, Notch1, and Mapk8-that could play a role in seizure-mediated cardiac changes. The LC-MS/MS analysis supported the activation of the transforming growth factor ß (TGF-ß) pathway in the heart of epileptic animals. Furthermore, our gene and protein expression studies established a key role of STAT3, Erbb, and Mapk8 to develop cardiac changes linked with recurrent seizures. SIGNIFICANCE: The present multi-omics study identified STAT3, Mapk8, and Erbb as key regulators involved in seizure-associated cardiac changes. It provided a deeper understanding of molecular, cellular, and network-level operations of the identified regulators that lead to cardiac changes in epilepsy.

A Comprehensive Search of the Primary and Secondary Metabolites and Radical Scavenging Potential of Trillium govanianum Wall. ex D. Don.

Trillium govanianum rhizomes are traditionally consumed as a raw powder and decoction for the treatment of health complications. Hence, the present study aimed to investigate whether aqueous and alcoholic extracts of T. govanianum rhizomes under hot and cold extraction conditions have similar or dissimilar chemical, nutrient, and antioxidant profiles. The total phenolics, flavonoids, carbohydrates, proteins, fats, and energy values were estimated in all the conditionally prepared samples. The total phenolics (21.23±1.4 mg GAE/g extract), flavonoids (70.57±3.24 mg RE/g extract) were found higher in hot ethanolic extract (TGHEt), while cold water extract (TGGC) showed higher nutrients including amino acids (10.545±0.219 mg/g) and nucleosides (1.803±0.018 mg/g). The nutrient energy value (2.60 and 2.49 Kcal/g extract) was higher in cold and hot ethanolic extracts. Further, TGHEt scavenged the DPPH. (IC50 ; 870±22 µg/mL) and ABTS.+ (IC50 ; 80±1.49 µg/mL) effectively and proved its highest antioxidant activity compared to other samples. In LC/MS/MS-based metabolite profiling, twenty-six metabolites (fatty acids, steroidal saponins, triterpene saponins, ecdysteroid hormones) were confirmed with mass fragmentation and literature, while one hundred nine metabolites were identified using the METLIN database. The principal component analysis showed clustering of hot condition extracts while cold extracts were differentially located in quadrants. The heatmaps exhibited the associations and differences between metabolite composition, solvents, and extraction conditions. The identified metabolites speculatively predicted the biosynthesis pathway of T. govanianum. Findings also illustrated that T. govanianum is a source of bioactive nutritional components and saponins. The current metabolite profiling of T. govanianum will help in its agricultural and biotechnological interventions for higher quality produce.

Hydrogen peroxide regulates antioxidant responses and redox related proteins in drought stressed wheat seedlings.

Hydrogen peroxide plays pivotal role as a potent regulator in signalling pathways when the plant is under stress. The current study appraised the potential of hydrogen peroxide through seed pre-treatment on the seedling growth and defense responses of three wheat cultivars i.e. PBW 644 (tolerant), PBW 621 and HD 2967 (sensitive) grown under drought stress. Imposition of drought stress reduced seedling growth of all the three wheat cultivars. Pre-treatment of seeds with 60 mM H2O2 alleviated water stress induced growth inhibition in all the three wheat cultivars. Further, it enhanced the drought tolerance of PBW 644 by upregulating SOD, POX, APX and GR enzymes accompanied by an increase in total phenols and ascorbate content. H2O2 treatment also protected the sensitive cultivars from drought stress by increasing CAT, POX, APX, MDHAR and GR enzymes. The contents of osmolytes were comparable or slightly higher as compared to stressed seedlings. The levels of MDA content were reduced in the treated seedlings of all the cultivars which further revealed the role of H2O2 pre-treatment in alleviating membrane damage. The comprehensive scrutiny of proteins differentially expressed in control, stressed and H2O2 primed stressed seedlings revealed that drought stress enhanced the expression of proteins involved in photosynthesis, protein biosynthesis and degradation, carbohydrate metabolism, fatty acid metabolism, nucleic acid metabolism, phytohormone response, defense and regulation, whereas H2O2 pre-treatment led to over expression of proteins which had functions in processes such as defense, redox homeostasis and photosynthesis. SUPPLEMENTARY INFORMATION: The online version of this article (10.1007/s12298-021-00937-z).

Metabolic signatures provide novel insights to Picrorhiza kurroa adaptation along the altitude in Himalayan region.

INTRODUCTION: Along the altitude, environmental conditions vary significantly that might influence plant performance and distribution. Adaptation to these changing conditions is a complex biological process that involves reprogramming of genes, proteins and metabolites. The metabolic response of medicinal plants along the altitude has been less explored yet. OBJECTIVES: In the present study, we investigated the adaptation strategies of Picrorhiza kurroa Royle ex Benth. along the altitude in organ specific manner using metabolomic approach. METHODS: Picrorhiza kurroa plants at flowering stage were randomly sampled from three altitudes viz. 3400, 3800 and 4100 masl in the Himalayan region. Leaf, root and rhizome were used for LC-MS based non-targeted metabolite profiling and targeted analysis of sugars, amino acids, picrosides and their corresponding phenolic acids. RESULTS: A total of 220, primary and secondary metabolites (SMs) were identified (p < 0.05) representing an extensive inventory of metabolites and their spatial distribution in P. kurroa. Differential accumulation of metabolites suggests source-sink carbon partitioning, occurrence of partial TCA cycle, ascorbate metabolism, purine catabolism and salvage route, pyrimidine synthesis, lipid alteration besides gibberellins and cytokinin inhibition might be an adaptive strategy to alpine environmental stress along the altitude. Further, marked differences of organ and altitude specific SMs reflect alteration in secondary metabolic pathways. Significant accumulation of picrosides suggests their probable role in P. kurroa adaptation. CONCLUSION: This study provides a platform that would be useful in deciphering the role of metabolites considered to be involved in plant adaptation.

Identification of host factors limiting the overexpression of recombinant Cu, Zn superoxide dismutase in Escherichia coli.

OBJECTIVE: Superoxide dismutase (SOD) enzyme has implications in modulating the cell's redox state. The study aims to explore the host genetic factors that limit the heterologous expression of a thermostable SOD from Potentilla atrosanguinea (Pa-SOD) in E. coli. RESULTS: It was observed that the heterologous expression of Pa-SOD in E. coli did not exhibit any enhancement after 1 h of induction. This led to the alteration in cell morphology and an increase in the doubling time of E. coli cells expressing Pa-SOD. Label-free quantification and MALDI-TOF/TOF-MS/MS analysis suggested differential expression of 81 proteins, of which 77 proteins were found to be downregulated and 4 were found to be upregulated in Pa-SOD expressing cells as compared to uninduced E. coli cells. Functional analysis of downregulated proteins shows involvement in molecular function, biological process, and were the part of a cellular component. The STRING database revealed interaction of an essential autoregulatory protein, RNase E with other proteins involved in biosynthetic processes, protein biosynthesis and folding, and cell division. Further, validation of RNase E protein revealed upregulation of rne at transcript level and downregulation of RNase E at protein level as compared to uninduced cells. CONCLUSIONS: The observations suggested the operation of multifaceted mechanisms with a key role of RNase E that regulated the expression of Pa-SOD at the physiological and molecular level. Since Pa-SOD has commercial applications, identification and manipulation of these networked genetic factors could lead to improvement of host strain for large-scale production of biologically active Pa-SOD and other heterologous proteins.

Quantitative analysis of flavonols, flavonol glycoside and homoisoflavonoids in Polygonatum verticillatum using UHPLC-DAD-QTOF-IMS and evaluation of their antioxidant potential.

INTRODUCTION: Polygonatum is widely used as a part of food in different regions of the world which covers five main categories such as drinks, vegetables, snacks, staple and seasoning foods. Presently, no analytical method is available for the quality control of Polygonatum. OBJECTIVE: Development and validation of a method using ultrahigh-performance liquid chromatography diode array detector quadrupole time-of-flight (UHPLC-DAD/QTOF) technique for the estimation of six compounds including a flavonol glycoside [rutin (1)], two flavonols [quercetin (2) and kaempherol (3)] and three homoisoflavonoids [5,7-dihydroxy-3-(2-hydroxy-4-methoxybenzyl)-chroman-4-one (4), 5,7-dihydroxy-3-(2-hydroxy-4-methoxybenzyl)-8-methylchroman-4-one (5) and 5,7-dihydroxy-3-(4-methoxybenzyl)-8-methylchroman-4-one (6)]. In addition, screening of extract, fractions and compounds of P. verticillatum for antioxidant activity was also determined. METHODOLOGY: The separation was achieved on C-18 column using acetonitrile and water containing 0.1% formic acid. The method was validated as per ICH (International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use) guidelines. The validated method was applied for the simultaneous identification and quantification of compounds 1-6 in extract (E) and fractions (F1-F4) of P. verticillatum. Furthermore, antioxidant potential of E, F1 and F2 and compounds was evaluated using DPPH (2,2-diphenyl-1-picrylhydrazyl) assay. RESULTS: The method was within the linear range (r2 ) of 0.982 to 0.999, precise (intra- and inter-day percentage relative standard deviations < 2.72 and 2.26) and accurate with recoveries (89.1-98.3%). The limit of detection and limit of quantification were in the ranges 0.02-0.16 and 0.06-0.48 ng/mL, respectively. Compounds 1-6 were quantified in all the samples. Compounds 1, 2 and 5 showed higher activity with half maximal inhibitory concentration (IC50 ) values 0.41, 0.39, 0.72 at 10, 20 and 30 µg/mL, respectively. CONCLUSION: Developed method will be helpful to assess the quality of P. verticillatum raw material and their derived products.

Regulation of color transition in purple tea (Camellia sinensis).

MAIN CONCLUSION: Comparative proteomics and metabolomics study of juvenile green, light purple and dark purple leaf to identify key proteins and metabolites that putatively govern color transition in Camellia sinensis. Color transition from juvenile green to dark purple leaf in Camellia sinensis is a complex process and thought to be regulated by an intricate balance of genes, proteins and metabolites expression. A molecular-level understanding of proteins and metabolites expression is needed to define metabolic process underpinning color transition in C. sinensis. Here, purple leaf growth of C. sinensis cultivar was divided into three developmental stages viz. juvenile green (JG), light purple (LP) and dark purple (DP) leaf. Scanning electron microscope (SEM) analysis revealed a clear morphological variation such as cell size, shape and texture as tea leaf undergoing color transition. Proteomic and metabolomic analyses displayed the temporal changes in proteins and metabolites that occur in color transition process. In total, 211 differentially expressed proteins (DEPs) were identified presumably involved in secondary metabolic processes particularly, flavonoids/anthocyanin biosynthesis, phytohormone regulation, carbon and nitrogen assimilation and photosynthesis, among others. Subcellular localization of three candidate proteins was further evaluated by their transient expression in planta. Interactome study revealed that proteins involved in primary metabolism, precursor metabolite, photosynthesis, phytohormones, transcription factor and anthocyanin biosynthesis were found to be interact directly or indirectly and thus, regulate color transition from JG to DP leaf. The present study not only corroborated earlier findings but also identified novel proteins and metabolites that putatively govern color transition in C. sinensis. These findings provide a platform for future studies that may be utilized for metabolic engineering/molecular breeding in an effort to develop more desirable traits.

Prunus cerasoides fruit extract ameliorates inflammatory stress by modulation of iNOS pathway and Th1/Th2 immune homeostasis in activated murine macrophages and lymphocytes.

The present investigation assessed the potential of Prunus cerasoides fruit extract (PCFE) in alleviation of inflammatory stress in response to lipopolysaccharide (LPS) and interferon-γ (IFN-γ)-stimulated murine peritoneal macrophages as well as in concanavalin A (Con A)-activated splenic lymphocytes. We observed a strong inhibition in production of nitric oxide (NO), reactive oxygen species (ROS), inflammatory cytokines (TNF-α/IL-6/IL-1ß), inducible nitric oxide synthase (iNOS), and NF-kB in macrophages treated with PCFE. Splenic lymphocytes treated with PCFE also showed a reduction in Con-A-induced cell proliferation and numbers of CD3+CD4+ T cells. Furthermore, PCFE treatment to Con A-stimulated lymphocytes decreased the production of inflammatory cytokines (TNF-α/IL-6/IL-1ß) with a concomitant increase in IL-10 suggesting its possible role in alleviation of inflammation-driven Th1/Th2 immune imbalance. PCFE appeared to influence innate immune response even at lower concentrations (25 and 50 µg/ml), while such effects were more pronounced in lymphocytes only at higher concentrations (100 and 200 µg/ml). UPLC-ESI-MS of PCFE revealed the presence of major bioactive phenolics including catechin, naringin as well as ascorbic acid which could have contributed in above findings. Overall, it is indicative that P. cerasoides fruit could be a valuable source for the development of anti-inflammatory functional foods and nutraceuticals.

Characterization of phenolics, amino acids, fatty acids and antioxidant activity in pulp and seeds of high altitude Himalayan crab apple fruits (Malus baccata).

Phytochemicals in fruits and vegetables have achieved immense significance owing to the increasing evidence which signifying their activity for antioxidant and prevention of chronic diseases. The amount of phloretin (88.39 µg mg-1) and phloridzin (83.03 µg mg-1) were found to be higher among other phenolics determined using UPLC. DPPH, ABTS+, metal chelating and ·OH radical assays were used to evaluate antioxidant activity. Malus baccata pulp portion showed higher antioxidant activity than seed portion. HPLC analysis for free amino acids showed that serine (9.06 µg mg-1), alanine (8.03 µg mg-1), tyrosine (10.33 µg mg-1), and cysteine (76.86 µg mg-1) were only detected in pulp portion while seed comprised of histidine (3.96 µg mg-1) only. Seed portion was also determined for their fatty acid composition including palmitic acid (0.89%), ethyl palmitate (0.56%), methyl petroselinate (0.90%) and linolein (3.93%) using GC-MS analysis. HPAEC technique detected fructose and sucrose in a fair amount of 21 and 17.3 mg g-1 in pulp, while 9.4 and 4.24 mg g-1 in seed portion, respectively. The present study suggested that M. baccata fruit is a rich source of phenolic and other chemical components which can be used in food products and nutraceutical formulations.

Anthocyanins enriched purple tea exhibits antioxidant, immunostimulatory and anticancer activities.

Purple coloured tea shoot clones have gained interest due to high content of anthocyanins in addition to catechins. Transcript expression of genes encoding anthocyanidin reductase (ANR), dihydroflavonol-4-reductase (DFR), anthocyanidin synthase (ANS), flavonol synthase (FLS) and leucoantho cyanidin reductase (LAR) enzymes in three new purple shoot tea clones compared with normal tea clone showed higher expression of CsDFR, CsANR, CsANS and lower expression of CsFLS and CsLAR in purple shoot clones compared to normal clone. Expression pattern supported high content of anthocyanins in purple tea. Four anthocyanins (AN1-4) were isolated and characterized by UPLC-ESI-QToF-MS/MS from IHBT 269 clone which recorded highest total anthocyanins content. Cyanidin-3-O-ß-d-(6-(E)-coumaroyl) glucopyranoside (AN2) showed highest in vitro antioxidant activity (IC50 DPPH = 25.27 ± 0.02 µg/mL and IC50 ABTS = 10.71 ± 0.01 µg/mL). Anticancer and immunostimulatory activities of cyanidin-3-glucoside (AN1), cyanidin-3-O-ß-d-(6-(E)-coumaroyl) glucopyranoside (AN2), delphinidin-3-O-ß-d-(6-(E)-coumaroyl) glucopyranoside (AN3), cyanidin-3-O-(2-O-ß-xylopyranosyl-6-O-acetyl)-ß-glucopyranoside (AN4) and crude anthocyanin extract (AN5) showed high therapeutic perspective. Anthocyanins AN1-4 and crude extract AN5 showed cytotoxicity on C-6 cancer cells and high relative fluorescence units (RFU) at 200 µg/mL suggesting promising apoptosis induction activity as well as influential immunostimulatory potential. Observations demonstrate potential of purple anthocyanins enriched tea clone for exploitation as a nutraceutical product.

Osmotin-expressing transgenic tea plants have improved stress tolerance and are of higher quality.

Drought is a major stress that affects the yield and quality of tea, a widely consumed beverage crop grown in more than 20 countries of the world. Therefore, osmotin gene-expressing transgenic tea plants produced using earlier optimized conditions were evaluated for their tolerance of drought stress and their quality. Improved tolerance of polyethylene glycol-induced water stress and faster recovery from stress were evident in transgenic lines compared with the normal phenotype. Significant improvements in growth under in-vitro conditions were also observed. Besides enhanced reactive oxygen species-scavenging enzyme activity, the transgenic lines contained significantly higher levels of flavan-3-ols and caffeine, key compounds that govern quality and commercial yield of the beverage. The selected transgenic lines have the potential to meet the demands of the tea industry for stress-tolerant plants with higher yield and quality. These traits of the transgenic lines can be effectively maintained for generations because tea is commercially cultivated through vegetative propagation only.

Excess electrons bound to molecular systems with a vanishing dipole but large molecular quadrupole.

Electron attachment properties of covalent molecules and ion clusters with vanishing dipole moments but large quadrupoles are studied with coupled cluster ab initio methods. Selection of the molecules studied is driven by two goals, finding a paradigm quadrupole-bound anion and investigating whether there is a correlation between the magnitude of the molecular quadrupole and the vertical attachment energy. Out of all examined species, only the ion clusters and four of the covalent molecules are found to support bound anions. The shapes and spatial extents of the associated excess electron distributions are qualitatively and quantitatively characterized, respectively. Two of the four covalent systems are especially promising as paradigm systems because of advantageous trade-offs regarding the number of isomers and conformers as well as synthetic closeness to commercial sources. No correlation was found between the vertical attachment energy and molecular quadrupole in an analysis that included the newly identified bound anions, those molecules, which were found not to support bound anions, and succinonitrile, which had been studied before. Moreover, there is clearly no such thing as a "critical quadrupole moment". There are, however, very strong electron correlation effects involved in the binding of the excess electrons, and similar to succinonitrile, for five out of six anions identified here, the molecular quadrupole of the neutral itself is too weak to bind an excess electron, and electron correlation in the form of dynamic polarization is required to do so.

Deciphering the metabolic signatures of Trigonella microgreens as a function of photoperiod and temperature using targeted compound analysis and non-targeted UHPLC-QTOF-IMS based approach.

Trigonella foenum-graecum L. (Fenugreek) is an annual herb that belongs to Fabaceae family. The compositional make-up of microgreens depends on prevailing environmental conditions. So, Trigonella microgreens were cultivated under different photoperiod and temperature conditions and evaluated for plant height, total chlorophyll content (TCC), targeted compound analysis and non-targeted UHPLC-QTOF-IMS based metabolomic profile. The plant height and TCC of Trigonella microgreens increased by approximately 22 % and 20 %, respectively under T1 conditions (longer photoperiod of 22 h with 22 °C in light and 17 °C in dark). The targeted phenolic profile analysis revealed the dominant presence of gallic acid, p-coumaric acid and apigenin in Trigonella microgreens. Also, the concentration of p-coumaric acid concentration raised from 3.51 mg/g to 5.83 mg/g as a response of T1 conditions. The sugar profile revealed augmented concentration of myo-inositol, glucose, fructose, xylose, maltose, and sucrose in longer photoperiod with T1 conditions. The microgreens were also rich in amino acids like aspartic acid, glutamic acid, leucine, isoleucine, and phenylalanine. Notably, the concentration of proline increased from 10.40 mg/g to 16.92 mg/g as a response to T1 growth conditions. The concentration of these metabolites varied significantly under different photoperiod and temperature conditions. The comprehensive non-targeted UHPLC-QTOF-IMS analysis of microgreens revealed different class of metabolites like organic compounds, alkaloids, coumarin-derivatives, phenolic and flavonoid derivatives, terpenoids, sugars, amino acids and few nucleic acid derivatives. The multivariate PLS-DA explained different expression level of metabolites under different growing conditions. The T1 growing condition resulted in the increased biosynthesis of phenolic compounds and various metabolites. The expression level of terpenoid derivatives specifically of Trigonelloside C and Trigoneoside XIIa/b increased under T1 conditions. The substantial alteration in the metabolites due to growing conditions may alter the microgreen's dietary benefits. So, additional research may be warranted.

Biochemical characterization of glutaminase-free L-asparaginases from Himalayan Pseudomonas and Rahnella spp. for acrylamide mitigation.

L-asparaginase having low glutaminase activity is important in clinical and food applications. Herein, glutaminase-free L-asparaginase (type I) coding genes from Pseudomonas sp. PCH182 (Ps-ASNase I) and Rahnella sp. PCH162 (Rs-ASNase I) was amplified using gene-specific primers, cloned into a pET-47b(+) vector, and plasmids were transformed into Escherichia coli (E. coli). Further, affinity chromatography purified recombinant proteins to homogeneity with monomer sizes of ~37.0 kDa. Purified Ps-ASNase I and Rs-ASNase I were active at wide pHs and temperatures with optimum activity at 50 °C (492 ± 5 U/mg) and 37 °C (308 ± 4 U/mg), respectively. Kinetic constant Km and Vmax for L-asparagine (Asn) were 2.7 ± 0.06 mM and 526.31 ± 4.0 U/mg for Ps-ASNase I, and 4.43 ± 1.06 mM and 434.78 ± 4.0 U/mg for Rs-ASNase I. Circular dichroism study revealed 29.3 % and 24.12 % α-helix structures in Ps-ASNase I and Rs-ASNase I, respectively. Upon their evaluation to mitigate acrylamide formation, 43 % and 34 % acrylamide (AA) reduction were achieved after pre-treatment of raw potato slices, consistent with 65 % and 59 % Asn reduction for Ps-ASNase I and Rs-ASNase I, respectively. Current findings suggested the potential of less explored intracellular L-asparaginase in AA mitigation for food safety.

Hydroethanolic extract of Gentiana kurroo Royle rhizome ameliorates ethanol-induced liver injury by reducing oxidative stress, inflammation and fibrogenesis in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Gentiana kurroo Royle is a medicinal plant mentioned as Traymana in Ayurveda. In the folklore, it is used to cure fever, stomach ache, skin diseases and liver disorders. However, limited reports are available on the therapeutic potential of Gentiana kurroo Royle against alcohol-induced liver damage. AIM OF THE STUDY: To assess the effectiveness of the hydroethanolic extract of Gentiana kurroo Royle rhizome (GKRE) against alcohol-induced liver injury and explore the mechanism of action. MATERIALS AND METHODS: GKRE was characterized using UHPLC-QTOF-MS/MS. The binding affinity of the identified compound was studied in silico. In vitro studies were performed in the Huh-7 cell line. An acute oral toxicity study (2 g/kg BW) of GKRE was done in rats following OECD 420 guidelines. In the efficacy study, rats were treated with 50% ethanol (5 mL/kg BW, orally) for 4 weeks, followed by a single intraperitoneal dose of CCl4 (30%; 1 mL/kg BW) to induce liver injury. After 4th week, the rats were treated with GKRE at 100, 200 and 400 mg/kg BW doses for the next fifteen days. The biochemical and antioxidant parameters were analyzed using commercial kits and a biochemistry analyzer. Histopathology, gene and protein expressions were studied using qRT PCR and western blotting. RESULTS: Thirteen compounds were detected in GKRE. Few compounds showed a strong interaction with the fibrotic and inflammatory proteins in silico. GKRE reduced (p < 0.05) the ethanol-induced ROS production and inflammation in Huh-7 cells. The acute oral toxicity study revealed no adverse effect of GKRE in rats at 2 g/kg BW. GKRE improved (p < 0.05) the body and liver weights in ethanol-treated rats. GKRE improved (p < 0.05) the mRNA levels of ADH, SREBP1c and mitochondrial biogenesis genes in the liver tissues. GKRE also improved (p < 0.05) the liver damage markers, lipid peroxidation and levels of antioxidant enzymes in the liver. A reduced severity (p < 0.05) of pathological changes, fibrotic tissue deposition and caspase 3/7 activity were observed in the liver tissues of GKRE-treated rats. Further, GKRE downregulated (p < 0.05) the expression of fibrotic (TGFß, αSMA and SMADs) and inflammatory markers (TNFα, IL6, IL1ß and NFκB) in the liver. CONCLUSION: GKRE showed efficacy against alcohol-induced liver damage by inhibiting oxidative stress, apoptosis, inflammation and fibrogenesis in the liver.