Disruption of the PGE 2 synthesis / response pathway restrains atherogenesis in programmed cell death-1 (Pd-1) deficient hyperlipidemic mice.

Immune checkpoint inhibitors (ICIs) that target programmed cell death 1 (PD-1) have revolutionized cancer treatment by enabling the restoration of suppressed T-cell cytotoxic responses. However, resistance to single-agent ICIs limits their clinical utility. Combinatorial strategies enhance their antitumor effects, but may also enhance the risk of immune related adverse effects of ICIs. Prostaglandin (PG) E 2 , formed by the sequential action of the cyclooxygenase (COX) and microsomal PGE synthase (mPGES-1) enzymes, acting via its E prostanoid (EP) receptors, EPr2 and EPr4, promotes lymphocyte exhaustion, revealing an additional target for ICIs. Thus, COX inhibitors and EPr4 antagonists are currently being combined with ICIs potentially to enhance antitumor efficacy in clinical trials. However, given the cardiovascular (CV) toxicity of COX inhibitors, such combinations may increase the risk particularly of CV AEs. Here, we compared the impact of distinct approaches to disruption of the PGE 2 synthesis /response pathway - global or myeloid cell specific depletion of mPges-1 or global depletion of Epr4 - on the accelerated atherogenesis in Pd-1 deficient hyperlipidemic (Ldlr -/- ) mice. All strategies restrained the atherogenesis. While depletion of mPGES-1 suppresses PGE 2 biosynthesis, reflected by its major urinary metabolite, PGE 2 biosynthesis was increased in mice lacking EPr4, consistent with enhanced expression of aortic Cox-1 and mPges-1. Deletions of mPges-1 and Epr4 differed in their effects on immune cell populations in atherosclerotic plaques; the former reduced neutrophil infiltration, while the latter restrained macrophages and increased the infiltration of T-cells. Consistent with these findings, chemotaxis by bone-marrow derived macrophages from Epr4 -/- mice was impaired. Epr4 depletion also resulted in extramedullary lymphoid hematopoiesis and inhibition of lipoprotein lipase activity (LPL) with coincident spelenomegaly, leukocytosis and dyslipidemia. Targeting either mPGES-1 or EPr4 may restrain lymphocyte exhaustion while mitigating CV irAEs consequent to PD-1 blockade.

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.

Proteomic and metabolomic insights into seed germination of Ferula assa-foetida.

Cold stratification is known to affect the speed of seed germination; however, its regulation at the molecular level in Ferula assa-foetida remains ambiguous. Here, we used cold stratification (4 °C in the dark) to induce germination in F. assa-foetida and adopted a proteomic and metabolomic approach to understand the molecular mechanism of germination. Compared to the control, we identified 209 non-redundant proteins and 96 metabolites in germinated F. assa-foetida seed. Results highlight the common and unique regulatory mechanisms like signaling cascade, reactivation of energy metabolism, activation of ROS scavenging system, DNA repair, gene expression cascade, cytoskeleton, and cell wall modulation in F. assa-foetida germination. A protein-protein interaction network identifies 18 hub protein species central to the interactome and could be a key player in F. assa-foetida germination. Further, the predominant metabolic pathways like glucosinolate biosynthesis, arginine and proline metabolism, cysteine and methionine metabolism, aminoacyl-tRNA biosynthesis, and carotenoid biosynthesis in germinating seed may indicate the regulation of carbon and nitrogen metabolism is prime essential to maintain the physiology of germinating seedlings. The findings of this study provide a better understanding of cold stratification-induced seed germination, which might be utilized for genetic modification and traditional breeding of Ferula assa-foetida. SIGNIFICANCE: Seed germination is the fundamental checkpoint for plant growth and development, which has ecological significance. Ferula assa-foetida L., commonly known as "asafoetida," is a medicinal and food crop with huge therapeutic potential. To date, our understanding of F. assa-foetida seed germination is rudimentary. Therefore, studying the molecular mechanism that governs dormancy decay and the onset of germination in F. assa-foetida is essential for understanding the basic principle of seed germination, which could offer to improve genetic modification and traditional breeding.

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.

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.

Wild Edible Flowers of Western Himalayas: Nutritional Characterization, UHPLC-QTOF-IMS-Based Phytochemical Profiling, Antioxidant Properties, and In Vitro Bioaccessibility of Polyphenols.

Four edible flowers commonly consumed in the Western Himalayan region, namely, Bauhinia variegata (Kachnar), Tropaeolum majus (Nasturtium), Matricaria chamomilla (Chamomile), and Tagetes erecta (Marigold), were characterized for their nutritional and phytochemical composition. Through the UHPLC-QTOF-IMS-based metabolomics approach, 131 compounds were tentatively identified consisting of phenolic acids, flavonoid glycosides, terpenoids, amino acids, and fatty acid derivatives. Kaempferol and quercetin glycosides for Kachnar, apigenin glycosides and caffeoylquinic acid derivatives for Chamomile, patulin and quercetin derivatives for Marigold, cyanidin and delphinidin glycosides for Nasturtium were the predicted marker metabolites identified through non-targeted metabolomics. Kachnar and Chamomile scored best in terms of macronutrients and essential micronutrients, respectively. Nasturtium contained high concentrations of α-linolenic acid, anthocyanins, and lutein. Kachnar contained the highest total phenolic acids (63.36 ± 0.38 mg GAE g-1), while Marigold contained the highest total flavonoids (118.90 ± 1.30 mg QUE g-1). Marigolds possessed excellent free radical scavenging and metal chelation activities. Chamomile exhibited strong α-glucosidase inhibition activity, followed by Nasturtium. The in vitro gastrointestinal digestibility of flower extracts indicated that the bioaccessibility of phenolic acids was higher than that of flavonoids. Polyphenols from Nasturtium and Chamomile showed the highest bioaccessibility. The study is an attempt to characterize traditionally consumed edible flowers and promote their wider utilization in gastronomy and nutraceuticals.

Systematic analyses with genomic and metabolomic insights reveal a new species, Ophiocordyceps indica sp. nov. from treeline area of Indian Western Himalayan region.

Ophiocordyceps is a species-rich genus in the order Hypocreales (Sordariomycetes, Ascomycota) depicting a fascinating relationship between microbes and insects. In the present study, a new species, Ophiocordyceps indica sp. nov., is discovered infecting lepidopteran larvae from tree line locations (2,202-2,653 m AMSL) of the Kullu District, Himachal Pradesh, Indian Western Himalayan region, using combinations of morphological and molecular phylogenetic analyses. A phylogeny for Ophiocordyceps based on a combined multigene (nrSSU, nrLSU, tef-1α, and RPB1) dataset is provided, and its taxonomic status within Ophiocordycipitaceae is briefly discussed. Its genome size (~59 Mb) revealed 94% genetic similarity with O. sinensis; however, it differs from other extant Ophiocordyceps species based on morphological characteristics, molecular phylogenetic relationships, and genetic distance. O. indica is identified as the second homothallic species in the family Ophiocordycipitaceae, after O. sinensis. The presence of targeted marker components, viz. nucleosides (2,303.25 µg/g), amino acids (6.15%), mannitol (10.13%), and biological activity data, suggests it to be a new potential source of nutraceutical importance. Data generated around this economically important species will expand our understanding regarding the diversity of Ophiocordyceps-like taxa from new locations, thus providing new research avenues.

Metabolomic approach for phytochemical assessment of Murraya koenigii fruits during different maturity stages.

A detailed metabolomic study was performed on various maturation stages of Murraya koenigii fruit pulps, seed, and leaf. Among the fruit pulps, stage 6 had the highest TPC (13.27 mg/g of GAE) and TFC content (6.16 mg/g RE). The extracts also showed promising free radical scavenging activity, especially in the seed (IC50DPPH 427 µg/mL). Metabolomics study revealed the identification of 133 metabolites in fruit pulps, seeds and leaves using the METLIN database. In silico PASS software analysis predicted the antimutagenic property of myricetin and bismurrayaquinone A. Pathway analysis revealed the phenylpropanoid biosynthesis pathway as one of the major pathways present in the fruit pulps. This detailed metabolic report of M. koenigii fruit maturation report brings a new insight into phytochemicals and their distribution in seed, pulps and leaves along with nutritive values and can be considered for nutritive and therapeutic purposes.

Assessment of nutritional properties and phenolic characterization of freshly harvested Dendrocalamus hamiltoni shoots and processed bamboo candy.

The free and bound phenolic constituents in Dendrocalamus hamiltonii shoots were evaluated and compared to processed bamboo candy. Preliminary proximate analysis revealed a percent reduction in moisture and protein with a less significant change in fibre content. The fresh free phenolic extract (FFPE) exhibited a total phenolics of 131.22 mg GAE/g and recovered 48.29 mg GAE/g phenolic content in bound fraction (FBPE). Results demonstrated higher loss of free phenolics after processing compared to bound fraction (CBPE). Although similar results were observed in total flavonoid content. Antioxidant activity was reduced after candy processing, with fresh shoots having the lowest percent inhibition (IC50) against DPPH· and ABTS· radicals. Although both free and bound fractions of candy demonstrated effective antioxidant activity. HPLC analysis revealed that FFPE contained more chlorogenic acid (0.14 mg/10 g) and cinnamic acid (0.75 mg/10 g) than CFPE. Quercetin was undetected in all free fractions but was found in bound form.

Comparative phytochemical analysis of Ferula assa-foetida with Ferula jaeschkeana and commercial oleo-gum resins using GC-MS and UHPLC-PDA-QTOF-IMS.

Ferula assa-foetida is an important species of the genus Ferula, best known for its oleo-gum resin, mainly used as a flavoring agent. Ferula jaeschkeana is another Himalayan medicinal plant of this genus, known for its contraceptive effect but not used in food applications. This study aimed to do a detailed phytochemical analysis of F. assa-foetida growing under controlled conditions in India using GC-MS/headspace and UHPLC-PDA-QTOF-IMS. Further, a comparative analysis of F. assa-foetida was performed with F. jaeschkeana (collected from its natural habitat) and commercial samples of F. assa-foetida oleo-gum resin (collected from the local market). UHPLC-QTOF-IMS profiling of F. assa-foetida led to the identification of foetisulfide C, assafoetidnol A, gumosin, flabellilobin (A/B), and foetisulfide A. In total, 141 metabolites were identified, including vitamins, nucleosides, sulfur compounds, flavonoids, sugars derivatives, and others, using METLIN database. Serine, arginine, asparagine, isoleucine, and phenylalanine were major amino acids quantified among the samples for the nutritional aspect. Characteristic sulfurous compounds (n-propyl-sec-butyl disulfide, trans-propenyl-sec-butyl disulfide, cis-propenyl-sec-butyl disulfide, and bis[1-(methylthio)propyl] disulfide) were identified in all samples except F. jaeschkeana. PCA and cluster analysis showed a significant difference in the volatile constituents of rhizomes of both species. Metabolomics studies also revealed the association of sesquiterpenoid and triterpenoid biosynthesis, phenylpropanoid, flavon, and flavanol biosynthesis. The current study demonstrates, "why only F. assa-foetida is used in culinary applications instead of F. jaeschkeana"?

Catechins prevent obesity-induced kidney damage by modulating PPARγ/CD36 pathway and gut-kidney axis in rats.

Obesity is an epidemic and a growing public health concern worldwide. It is one of the significant risk factors for developing chronic kidney disease. In the present study, we evaluated the preventive effect of green tea catechins (GTC) against obesity-induced kidney damage and revealed the underlying molecular mechanism of action. Various green tea catechins were quantified in the catechins-rich fraction using HPLC. In vitro, the palmitic and oleic acid-treated NRK-52E cells showed reduced fat accumulation and modulated expressions of PPARγ, CD36, and TGFß after GTC treatment. In vivo, rats were fed with a high-fat diet (HFD), and the effect of GTC was assessed at 150 and 300 mg/kg body weight doses. HFD-fed rats showed a significant reduction in weight gain and improved serum creatinine, urea, and urine microalbumin levels after GTC treatment. The improved adipokines and insulin levels in GTC treated groups indicated the insulin-sensitizing effect. Histopathology revealed reduced degenerative changes, fibrous tissue deposition, and mesangial matrix proliferation in GTC treated groups. GTC treatment also downregulated the gene expressions of lipogenic and inflammatory factors and improved the altered expressions of CD36 and PPARγ in the kidney tissue. Further, GTC prevented gut dysbiosis in rats by promoting healthy microbes like Akkermansia muciniphila and Lactobacillus reuteri. Faecal metabolome revealed reduced saturated fatty acids, and improved amino acid levels in the GTC treated groups, which help to maintain gut health and metabolism. Overall, GTC prevented obesity-induced kidney damage by modulating PPARγ/CD36 signaling and maintaining gut health in rats.

Volatile and aroma profiling of flower and rhizome of Hedychium flavescens growing in North Western Himalayan region.

The present investigation was carried out for a comparative volatile study and aroma profiling of Hedychium flavescens. The headspace gas chromatography mass spectrometry (GC-MS) analysis of flowers (HS-F) and rhizome (HS-R); GC-MS analysis of flower essential oil (EO-F), flower absolute (AB-F) and rhizome essential oil (EO-R) revealed 27, 19, 19, 15 and 12 compounds which constitute 96.22%, 96.93% 97.43%, 86.79% and 97.62% composition, respectively. The identification results demonstrated that flowers and rhizome were rich in ß-pinene, 1,8-cineol, linalool and E-ß-caryohyllene components. ß-Pinene was the most abundant component in HS-R (38.99%), EO-R (26.61%); linalool in HF-F (25.34%) and EO-F (25.99%) and ρ-vinyl-guaiacol in AB-F (32.19%), respectively. The aroma profile of H. flavescens was dominated with floral and jasmine (flowers); spicy, earthy and herbal (EO-F); floral and balsamic (AB-F); herbal, pungent, spicy and earthy (rhizome and EO-R) notes. Based on aroma profile, AB-F was evaluated as potential ingredient for perfume industry.

Comparative metabolomics of Himalayan crab apple (Malus baccata) with commercially utilized apple (Malus domestica) using UHPLC-QTOF-IMS coupled with multivariate analysis.

A comprehensive UHPLC-QTOF-IMS based metabolomics investigation in skin and pulp of Malus domestica and Malus baccata was performed. M. domestica fruit parts had higher phenolic contents (25.75-43.05 mg GAE/g) as compared to M. baccata (18.10-28.37 mg GAE/g) and flavonoid content (1.34-9.59 mg RE/g) followed by promising antioxidant activity (MD_Skin DPPH 119.41 µg/mL and MB_Skin DPPH 148.24 µg/mL). Targeted metabolomics quantified higher chlorogenic acid in MD_pulp (929.54 mg/100 g) and phloridzin in MD_skin (722.54 mg/100 g). Amino acids, sugars, flavonoids, vitamins, nucleosides, quinones, fatty acids, and derivatives are among the 248 distinctive metabolites identified using non-targeted metabolomics. Multivariate data analysis, VIP projection and pathway interaction studies demonstrated the metabolic changes and differential distribution in both fruits. Using KEGG pathway enrichment analysis, the biosynthesis of flavone and flavonols is the most prevalent in both fruits. The current study found that M. baccata has a comparable metabolite distribution and should be considered for health-beneficial products.

Acrylamide mitigation in foods using recombinant L-asparaginase: An extremozyme from Himalayan Pseudomonas sp. PCH182.

Acrylamide has received worldwide attention due to its existence in commonly consumed foods. L-asparaginase reduces acrylamide formation in foods by hydrolyzing available L-asparagine. Herein, L-asparaginase (Ps-ASNase II) of Pseudomonas sp. PCH182 was expressed in Escherichia coli (E. coli), purified, and evaluated for acrylamide reduction in food samples. The monomeric 37 kDa Ps-ASNase II protein was purified to homogeneity with a 70 % yield. The enzyme was active at a wide pH range (5.0-11.0) and temperature (10-80 °C) with optimum activity at 45 °C in 50 mM Tris-HCl (pH 8.5) after 10 min. The Km and Vmax for L-asparagine were 0.52 ± 0.06 mM and 42.55 ± 4.0 U/mg, respectively. Also, the half-life and Kd value of the enzyme at 37 °C was 458 min and 1.51 × 10-3/min, suggesting its higher stability. Consistently, the enzyme retained 62 % residual activity after 60 days of storage at 4 °C. The Ps-ASNase II enzyme (5 U/mL) treatment of raw potato chips resulted in 90 % asparagine hydrolysis exhibiting high efficiency. Ps-ASNase II (5 U/mL) treated potato chips significantly reduced acrylamide content by 73 % at 37 °C within 24 min compared to untreated controls. Collectively, these findings verified Ps-ASNase's effectiveness and capability to lower acrylamide formation in fried potato chips without altering the food product's nutritional profile.

De novo transcriptome based insights into secondary metabolite biosynthesis in Malaxis acuminata (Jeevak)-A therapeutically important orchid.

Malaxis acuminata D. Don [=Crepidium acuminatum (D. Don) Szlach.] is an endangered medicinal orchid of the Ashtvarga group of plants in Ayurveda (Indian system of traditional medicine). Using a combination of aromatic cytokinin [meta-Topolin (mT)], plant biostimulant (chitosan), auxin [indole-3-butyric acid (IBA)], and a phenolic elicitor [phloroglucinol (PG)], plants of M. acuminata were regenerated in vitro for mass multiplication. The present research reveals the first-ever transcriptome of M. acuminata. A total of 43,111 transcripts encoding 23,951 unigenes were assembled de novo from a total of 815.02 million reads obtained from leaf and pseudobulb of in vitro raised M. acuminata. Expression analysis of genes associated with ß-sitosterol and eugenol biosynthesis in leaf and pseudobulb provided vital clues for differential accumulation of metabolites in M. acuminata. Ultra-performance liquid chromatography (UPLC) confirmed higher amounts of ß-sitosterol and eugenol content in the leaf as compared to the pseudobulb. Differential expression of transcripts related to starch and sucrose metabolism, plant hormone signal transduction, diterpenoid biosynthesis, phenylalanine metabolism, stilbenoid, diarylheptanoid, and gingerol biosynthesis suggested the operation of differential metabolic pathways in leaf and pseudobulb. The present research provides valuable information on the biosynthesis of secondary metabolites in M. acuminata, which could be used for advanced metabolite bioprospection using cell suspension culture and bioreactor-based approaches. Data also suggested that leaf tissues rather than pseudobulb can be used as an alternate source of bioactive metabolites thereby shifting the need for harvesting the pseudobulb. This will further facilitate the conservation and sustainable utilization of this highly valued medicinal orchid.

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.

A comparative metabolomic investigation in fruit sections of Citrus medica L. and Citrus maxima L. detecting potential bioactive metabolites using UHPLC-QTOF-IMS.

The current study focused on targeted and non-targeted metabolomics of Citrus fruit parts (exocarp, mesocarp, endocarp, and seeds) to gain a comprehensive metabolomic insight. Sections of the Citrus fruit were preliminarily examined for proximate compositions (moisture, ash, fibre, fat, and protein). Whereas ultrasonication-assisted solvent extraction revealed a higher phenolic and flavonoid content at 80% (v/v) ethanolic medium, with the highest amount in the exocarp. Using targeted metabolomics, hesperidin (3307.25 mg/100 g), naringin (4803.73 mg/100 g) were detected in C. medica and C. maxima at greater levels, respectively. Further quantitative analysis revealed the presence of phenolic acids (gallic acid, trans-ferulic acid, p-coumaric acid, trans-cinnamic acid), and polymethoxyflavones (nobiletin, and tangeretin) and detected in the order of exocarp > mesocarp > endocarp > seeds. Using an untargeted metabolomics approach, metabolite discriminations among Citrus fruit sections were illustrated by Venn-diagram, heatmap, PCA, o-PLSDA, correlation matrices, and S-plot. UHPLC-QTOF-IMS revealed 48 metabolites including phenolics, vitamins, and amino acids. Furthermore, the METLIN database leads to the identification of 202 unknown metabolites. The metabolite biosynthesis and corresponding metabolite presence in Citrus fruit sections were confirmed using pathway enrichment and mass fragmentation analysis. Finally, potential biological activities were determined using in silico PASS software approach, and free radical scavenging potential was confirmed using in vitro assays for future preventive and therapeutic applications of the identified metabolites.

Advances of Ion Mobility Platform for Plant Metabolomics.

Metabolomics aims to profile the extensive array of metabolites that exists in different types of matrices using modern analytical techniques. These techniques help to separate, identify, and quantify the plethora of chemical compounds at various analytical platforms. Hence, ion mobility spectrometry (IMS) has emerged as an advanced analytical approach, exclusively owing to the 3D separation of metabolites and their isomers. Furthermore, separated metabolites are identified based on their mass fragmentation pattern and CCS (collision cross-section) values. The IMS provides an advanced alternative dimension to separate the isomeric metabolites with enhanced throughput with lesser chemical noise. Thus, the present review highlights the types, factors affecting the resolution, and applications of IMMS (Ion mobility mass spectrometry) for isomeric separations, and ionic contaminants in the plant samples. Furthermore, an overview of IMS-based applications for the identification of plant metabolites (volatile and non-volatile) over the last few decades has been discussed, followed by future assumptions for creating IM-based databases. Such approaches could be significant to accelerate and improve our knowledge of the vast chemical diversity found in plants.