Bioactive compounds and antioxidant activity during ripening of Malpighia glabra fruits.

Malpighia glabra (Malpighiaceae) is a cherry fruit popularly known as acerola or West Indian cherry, with nutraceuticals in each ripening stage. The changes in the phytoconstituents, pigments, sugars, organic acids, and antioxidants were investigated during the fruit ripening and expressed in fresh weight (FW). Gentisic acid was present in the highest concentration in IMGL fruits (11.43 mg/100 g), which was reduced to 0.362 mg/100 g over-ripening. The major flavonoid present was epicatechin, and the concentration increased from 2.11 mg/100 g in immature green large (IMGL) fruits to 19.52 mg/100 g in ripe fruits. Ascorbic acid was the most abundant organic acid present, and the highest concentration was found in the IMGL fruits (2030 mg/100 g). Fructose and galactose were found in the highest concentrations in overripe fruits (2290 mg/100 g and 1460 mg/100 g, respectively). The IMGL fruits showed the highest total antioxidant activity of 5.48% and 5.34% ascorbic acid equivalent in methanolic and aqueous extracts, respectively. Mineral quantification showed that the fruits were rich in potassium and calcium (150.43 and 12.90 mg/100 g, respectively). This study could identify the appropriate stage of acerola fruit for developing functional foods with maximum utilization of phytoconstituents in all stages.

Genome-wide identification of MATE, functional analysis and molecular dynamics of DcMATE21 involved in anthocyanin accumulation in Daucus carota.

Anthocyanins are a subclass of flavonoids that are synthesized in the endoplasmic reticulum and then transported to the vacuole in plants. Multidrug and toxic compound extrusion transporters (MATE) is a family of membrane transporters that transport ions and secondary metabolites, such as anthocyanins, in plants. Although various studies on MATE transporters have been carried out on different plant species, this is the first comprehensive report to mine the Daucus carota genome to identify the MATE gene family. Our study identified 45 DcMATEs through genome-wide analysis and detected five segmental and six tandem duplications from the genome. The chromosome distribution, phylogenetic analysis, and cis-regulatory elements revealed the structural diversity and numerous functions associated with the DcMATEs. In addition, we analyzed RNA-seq data obtained from the European Nucleotide Archive to screen for the expression of DcMATEs involved in anthocyanin biosynthesis. Among the identified DcMATEs, DcMATE21 correlated with anthocyanin content in the different D. carota varieties. In addition, the expression of DcMATE21 and anthocyanin biosynthesis genes was correlated under abscisic acid, methyl jasmonate, sodium nitroprusside, salicylic acid, and phenylalanine treatments, which were substantiated by anthocyanin accumulation in the in vitro cultures. Further molecular membrane dynamics of DcMATE21 with anthocyanin (cyanidin-3-glucoside) identified the binding pocket, showing extensive H-bond interactions with 10 crucial amino acids present in the transmembrane helix of 7, 8, and 10 of DcMATE21. The current investigation, using RNA-seq, in vitro cultures, and molecular dynamics studies revealed the involvement of DcMATE21 in anthocyanin accumulation in vitro cultures of D. carota.

Structure-based screening of natural product libraries in search of potential antiviral drug-leads as first-line treatment to COVID-19 infection.

The study focuses on identifying and screening natural products (NPs) based on their structural similarities with chemical drugs followed by their possible use in first-line treatment to COVID-19 infection. In the present study, the in-house natural product libraries, consisting of 26,311 structures, were screened against potential targets of SARS-CoV-2 based on their structural similarities with the prescribed chemical drugs. The comparison was based on molecular properties, 2 and 3-dimensional structural similarities, activity cliffs, and core fragments of NPs with chemical drugs. The screened NPs were evaluated for their therapeutic effects based on their predicted in-silico pharmacokinetic and pharmacodynamics properties, binding interactions with the appropriate targets, and structural stability of the bound complex using molecular dynamics simulations. The study yielded NPs with significant structural similarities to synthetic drugs currently used to treat COVID-19 infections. The study proposes the probable biological action of the selected NPs as Anti-retroviral protease inhibitors, RNA-dependent RNA polymerase inhibitors, and viral entry inhibitors.

Biosynthesis and regulation of anthocyanin pathway genes.

Anthocyanins are the phenolic compounds responsible for coloring pigments in fruits and vegetables. Anthocyanins offer a wide range of health benefits to human health. Their scope has expanded dramatically in the past decade, making anthocyanin control, influx, and outflow regulation fascinating for many researchers. The main culprit is anthocyanin stability and concentration form, which demands novel ways because these are critical in the food industry. This review aims to examine anthocyanin synthesis via triggering transcription genes that code for anthocyanin-producing enzymes. The balance between production and breakdown determines anthocyanin accumulation. Thus, increasing the anthocyanin content in food requires the stability of molecules in the vacuolar lumen, the pigment fading process, and a better understanding of the mechanism. The promising option is biosynthesis by metabolically engineered microorganisms with a lot of success. This study aims to look into and evaluate the existing literature on anthocyanin production, namely the biosynthesis of anthocyanin pathway genes, production by microbial cell factories, and the regulatory factors that can modulate the production of anthocyanins. Understanding these mechanisms will provide new biotechnological approaches.Key points• Factors affecting the regulation of anthocyanins• Focus on degradation, biosynthesis pathway genes, and alternative systems for the production of anthocyanins• Microbial cell factories can be used to produce large amounts of anthocyanins.

Chemometric evaluation of functional components and anti-quorum sensing activity of mulberry leaves from Indian cultivars: a potential contribution to the food industry.

BACKGROUND: Potential use of many native, easily available vegetal materials for human consumption and value addition is not well recognized. Mulberry, being a traditional industrial crop rich in nutrients and nutraceuticals can be of great importance for the food industry. However, mulberry leaves are mainly being utilized in sericulture and are not exploited for their functional components. Thus, the selection of promising mulberry cultivars, rich in bioactive compounds, like resveratrol and 1-deoxynojirimycin, increase their potential use in functional foods. RESULTS: Chlorogenic acid, myricetin and kaempferol were the major polyphenols present in the nine selected cultivars, in the range 0.001-0.086, 0.003-0.079 and 0.003-0.163 g kg-1 fresh weight (FW), respectively. Protocatechuic acid, epicatechin and rutin were predominantly present in cultivars V-1, G-2 and ML (0.103, 0.080 and 0.121 g kg-1 FW, respectively). Similarly, resveratrol and 1-deoxynojirimycin were highest in cultivars ML and K-2 (0.078 and 0.079 g kg-1 FW, respectively). Leaf extracts of cultivars G-2 and ML were able to effectively inhibit the violacein production with 64.08% and 70.04%, respectively at the concentration of 6 mg mL-1 presumably due to a higher content of polyphenols. Chemometric evaluation of chromatographic data showed the intraspecific variability and secondary metabolite co-existence in different cultivars. CONCLUSIONS: Considering phytoconstituents, cultivars G-2, ML, K-2 and V-1 could contribute efficiently to the rational utilization of mulberry in agro-food industries. Furthermore, cultivars G-2 and ML leaves can be a new source of quorum sensing inhibitory agents. © 2021 Society of Chemical Industry.

Salt Stress-Induced Anthocyanin Biosynthesis Genes and MATE Transporter Involved in Anthocyanin Accumulation in Daucus carota Cell Culture.

Anthocyanins biosynthesis is a well-studied biosynthesis pathway in Daucus carota. However, the scale-up production at the bioreactor level and transporter involved in accumulation is poorly understood. To increase anthocyanin content and elucidate the molecular mechanism involved in accumulation, we examined D. carota cell culture in flask and bioreactor for 18 days under salt stress (20.0 mM NH4NO3/37.6 mM KNO3) at 3 day intervals. The expression of anthocyanin biosynthesis and putative MATE (multidrug and toxic compound extrusion) transporter expression was analyzed by qRT-PCR. It was observed that there was a significant enhancement of anthocyanin in the bioreactor compared to the control culture. A correlation was observed between the expression of MATE and the anthocyanin biosynthesis genes (CHS, C4H, LDOX, and UFGT) on the 9th day in a bioreactor, where maximum anthocyanin accumulation and expression was detected. We hypothesize the involvement of MATE in transporting anthocyanin to tonoplast in D. carota culture under salt stress.

Physicochemical Composition and Characterization of Bioactive Compounds of Mulberry (Morus indica L.) Fruit During Ontogeny.

Mulberry fruit is well recognized as one of the richest sources of bioactive compounds. We investigated the physicochemical composition and characterized the bioactive compounds during different ripening stages of mulberry (Morus indica) fruit and evaluated their anti-quorum sensing activity on Chromobacterium violaceum. The proximate components such as carbohydrates, proteins and lipids were found to be high in the ripe fruit compared to unripe and mid-ripe fruit. The ripe fruit contained higher content of total phenolics and flavonoids (336.05 and 282.55 mg/100 g fresh weight (FW), respectively). Epicatechin and resveratrol were the major polyphenols detected in the fruit with the range 5.13-19.46 and 4.07-14.45 mg/100 g FW, respectively. Chlorogenic acid and myricetin were predominant in the unripe and mid-ripe fruit (7.14 and 1.84 mg/100 g FW, respectively). The fruit was found to be an excellent source of anti-diabetic compound 1-deoxynojirimycin. The highest content of 1-deoxynojirimycin was present in the mid-ripe fruit, with a content of 2.91 mg/100 g FW. Furthermore, fruit extracts exhibited anti-quorum sensing activity against Chromobacterium violaceum by effectively inhibiting violacein production. Ripe fruit extracts showed the highest activity of 76.30% at 1 mg/mL and thus, could be used as a potent anti-quorum sensing agent. The results could be promising in the selection of appropriate developmental stages for M. indica fruit commercial exploitation in the food formulations rich in potential health components.

Evolutionary selectivity of amino acid is inspired from the enhanced structural stability and flexibility of the folded protein.

AIM: The present study attempts to decipher the site-specific amino acid alterations at certain positions experiencing preferential selectivity and their effect on proteins' stability and flexibility. The study examines the selection preferences by considering pair-wise non-bonded interaction energies of adjacent and interacting amino acids present at the interacting site, along with their evolutionary history. MATERIALS AND METHODS: For the study, variations in the interacting residues of spike protein (S-Protein) receptor-binding domain (RBD) of different coronaviruses were examined. The MD simulation trajectory analysis revealed that, though all the variants studied were structurally stable at their native and bound confirmations, the RBD of 2019-nCoV/SARS-CoV-2 was found to be more flexible and more dynamic. Furthermore, a noticeable change observed in the non-bonded interaction energies of the amino acids interacting with the receptor corroborated their selection at respective positions. KEY FINDINGS: The conformational changes exerted by the altered amino acids could be the reason for a broader range of interacting receptors among the selected proteins. SIGNIFICANCE: The results envisage a strong indication that the residue selection at certain positions is governed by a well-orchestrated feedback mechanism, which follows increased stability and flexibility in the folded structure compared to its evolutionary predecessor.

Pleiotropic nature of curcumin in targeting multiple apoptotic-mediated factors and related strategies to treat gastric cancer: A review.

Gastric cancer (GC) is one of the major reasons for cancer-associated death and exhibits the second-highest mortality rate worldwide. Several advanced approaches have been designed to treat GC; however, these strategies possess many innate complications. In view of this, the upcoming research relying on natural products could result in designing potential anticancer agents with fewer side effects. Curcumin, isolated from the rhizomes of Curcuma longa L. has several medicinal properties like antiinflammatory, antioxidant, antiapoptotic, antitumor, and antimetastatic. Such pleiotropic nature of curcumin impedes the invasion and proliferation of GC by targeting several oncogenic factors like p23, human epidermal factor receptor2 including Helicobacter pylori. The side effect of chemotherapy, that is, chemotherapeutic resistance and radiotherapy could be reduced combination therapy of curcumin. Moreover, the photodynamic therapy of curcumin destroys the cancer cells without affecting normal cells. However, further more potential studies are required to establish the potent efficacy of curcumin in the treatment of GC. The current review details the anticancer activities of curcumin and related strategies which could be employed to treat GC with additional focus on its inhibitory properties against viability, proliferation, and migration of GC cells through cell cycle arrest and stimulation by apoptosis-mediated factors.

Micro and nanoemulsions of Carissa spinarum fruit polyphenols, enhances anthocyanin stability and anti-quorum sensing activity: Comparison of degradation kinetics.

The low stability of anthocyanins is a constraint in the food industry. The present work has been carried out to overcome this low stability by encapsulating fruit concentrate of underutilized plant Carissa spinarum (CS) with polyphenols in microemulsions (CSME) and nanoemulsions (CSNE). Increasing the amount of CS reduced the particle size from 1154 to 70-300 nm whereas addition of Tween 80 reduced it optimally to 5-25 nm. Degradation of anthocyanins in control and ME/NE proceeded with zero- and first-order reaction rates, respectively, at 28 °C (half-life 6, 25 and 40 days, respectively). The degradation kinetics of phenolics-flavonoids were also studied. CSNE exhibited higher anti-quorum sensing (QS) activity than CSME against Chromobacterium violaceum (73.7%); it inhibited biofilm formation by 70.1 and 64.4% in Pseudomonas aeruginosa, and Yersinia enterocolitica, respectively. This is the first report of using the more stable ME/NE to study anti-QS activity, an alternative to conventional antibiotics.

Exogenous Serotonin and Melatonin Regulate Dietary Isoflavones Profoundly through Ethylene Biosynthesis in Soybean [Glycine max (L.) Merr.].

Serotonin and melatonin are important signaling and stress mitigating molecules. However, their role and molecular mechanism in the accumulation of isoflavones are not clearly defined. To elucidate their functions, serotonin and melatonin were applied to in vitro cultures of soybean at different concentrations and analyzed to assess the accumulation of isoflavone content followed by transcript levels of biosynthesis genes at different time intervals. Increased total phenolics, total flavonoids, and different forms of isoflavone content were observed in the treatments. Expression levels of critical genes in isoflavone, ethylene, jasmonic acid, abscisic acid, and melatonin biosynthesis and related transcription factor were quantified. A correlation was observed between the expression of ethylene biosynthesis genes (S-adenosylmethionine synthase and 1-aminocyclopropane-1-carboxylate oxidase) and isoflavone biosynthesis genes (chalcone synthase, chalcone reductase, and isoflavone synthase). We hypothesize that, under serotonin and melatonin treatments, ethylene biosynthesis may play a role in the increase/decrease in isoflavone content in soybean culture.

Identification and Characterization of Polyphenols from Carissa spinarum Fruit and Evaluation of Their Antioxidant and Anti-quorum Sensing Activity.

Quorum sensing controls bacterial virulence through signaling molecules, which plays a vital role in managing foodborne pathogens that cause food spoilage and human infections. Though many synthetic compounds have been used to control infection, antibiotic resistance has become a global issue. Targeting the bacterial quorum sensing using the bioactive compounds could be an alternative strategy to combat their resistance. In this context, polyphenols from an unexplored unripe and ripe fruits of Carissa spinarum were evaluated for anti-quorum sensing activity. The study aimed at determining nutritional, phytochemical composition, and polyphenol profiling to evaluate their antioxidant potential of the fruit. Furthermore, the violacein inhibition, anti-biofilm, and effect on the motility of foodborne pathogens were also studied. The phytochemical content of C. spinarum fruit showed the phenolic and flavonoid content 273.20 mg GAE/100 g and 453.78 mg QE/100 g fresh weight , respectively, in ripe fruit. HPLC characterization of fruit extracts showed high content of syringic acid, resveratrol, and quercetin in ripe, whereas it showed epicatechin and gentisic acid in unripe fruit. The antioxidant activity of the ripe fruit extract exhibited a higher potential to scavenge DPPH radicals with IC50 4.69 mg/mL. Further, the anti-quorum sensing activity was higher in ripe fruit extract at 1.8 mg/mL inhibited with 78.65% violacein production in Chromobacterium violaceum, swimming motility, and biofilm formation in Pseudomonas aeruginosa and Yersinia enterocolitica (66.25% and 59.36% respectively at 1.2 mg/mL). Hence C. spinarum fruit bioactive could be a natural plant source for anti-quorum sensing activity to manage foodborne pathogens over synthetic compounds.

Evaluation of Various Drying Methods on Bioactives, Ascorbic Acid and Antioxidant Potentials of Talinum triangulare L., foliage.

The aim of this study was to assess the efficiency of different drying methods (room, sun, oven, microwave, cross-flow, infra-red, dehumidifier, and freeze-drying) on maximum retention of the nutritional and bioactive compounds profiling of Talinum triangulare, which is a less-known perishable leafy-vegetable. The evaluation of various drying methods is to learn the best appropriate strategy for a post-harvest drying method for retaining all the potential benefits with minor loss. Microwave and freeze-dried samples held the maximum ascorbic acid quantified by HPLC with 1.36 and 1.11 g/100 g DW, respectively. The main carotenoid compounds identified were violaxanthin, lutein, zeaxanthin, ß-carotene isomers, trans-ß-carotene, and cis-ß-carotenes. Gallic, protocatechuic, catechin, para-coumaric, ferulic, rutin, trans-cinnamic, and quercetin are the significant phenolics and flavonoids identified and quantified by liquid chromatography. The efficiency of different solvents on bioactive extractions uncovered that the methanol and 80% aqueous ethanol were good for retention of total phenolics, total flavonoids, and antioxidant compounds, which was affirmed through phosphomolybdate, DPPH, and FRAP assays. Dried T. triangulare foliage could be productively utilized as a promising raw material for food and pharma businesses because of its rich bioactive composition.

Suppression of N-glycan processing enzymes by deoxynojirimycin in tomato (Solanum lycopersicum) fruit.

The present study investigated the potential of a small molecule inhibitor, 1-deoxynojirimycin (DNJ), to extend the shelf life of tomatoes. The optimum concentration of DNJ and the proper ripening stage for treatment were standardized using response surface methodology, following a central composite design. The concentration of DNJ used for the analysis was 0.15 mM, and 0.30 mM and the ripening stages of the tomato fruit analysed were immature green, mature green, breaker, ripen and over-ripen. Analysis of the influence of the DNJ treatment of the fruit using quadratic multiple regression models considering the factors colour, texture, and free sugars revealed significant responses. A DNJ concentration of 0.30 mM and fruit-ripening stage of mature green was found to be optimal for the treatment. DNJ-treatment maintained fruit firmness throughout ripening with a significant reduction in reducing sugar formation. Enzyme activity of the N-glycan processing enzymes involved in cell wall softening, α-mannosidase and ß-d-N-acetylhexosaminidase revealed a significant reduction in their activity by 2 and 3.5-fold, respectively. Down-regulation of expression of important ripening-related and softening process-associated genes, aminocyclopropane carboxylic synthase-4, aminocyclopropane carboxylic oxidase, polygalacturonase and pectin methylesterases at 4, 5, 6 and 5-fold, respectively, was also observed. The present results showed that the treatment of mature green tomato fruit with DNJ at a concentration of 0.30 mM can delay the ripening of the tomato fruit by inhibiting cell wall and N-glycan processing enzymes.

Expression profile of phenylpropanoid pathway genes in Decalepis hamiltonii tuberous roots during flavour development.

To explore the transcriptional control of phenylpropanoid pathway (PPP) involved in vanillin flavour metabolites production in tuberous roots of Decalepis hamiltonii, four PPP key genes expressed during the tuber development were identified and their mRNA expression profiles were evaluated using quantitative real-time PCR. Flavour metabolite quantification by HPLC analysis confirmed 10, 170 and 500 µg/g 2-hydroxy-4-methoxy benzaldehyde and 4, 20 and 40 µg/g vanillin in first- (3-month-old plant), second- (18-month-old plant) and third-stage tubers (60-month-old matured plant), respectively. The expression of all four genes phenylalanine ammonia lyase (DhPAL), cinnamate-4-hydroxylase (DhC4H), caffeic acid-O-methyltransferase (DhCOMT) and vanillin synthase (DhVAN) increased with flavour development from first stage to second stage. A decrease in expression from 1.9-folds to 0.1-folds and 19.2-folds to 5.2-folds for DhCOMT and DhVAN was recorded for second stage to third stage, respectively. However, a gradual increase in expression of DhPAL (up to 26.4-folds) and a constant expression pattern for DhC4H (up to 7.1-folds) was evident from second stage to third stage of flavour development. The decrease in the expression levels of DhCOMT and DhVAN in third stage shows that the second-stage tubers are more transcriptionally active towards flavour biosynthesis.

Augmentation of pyrethrins content in callus of Chrysanthemum cinerariaefolium and establishing its insecticidal activity by molecular docking of NavMS Sodium Channel Pore receptor.

Pyrethrins are effective food-grade bio-pesticides obtained from the flowers of Chrysanthemum cinerariaefolium and this crop cannot be cultivated widely in India due to its specific agro-climatic requirement. Hence pyrethrins are mostly imported from Kenya. Therefore, the present study aims to develop a process for augmentation of pyrethrin contents in C. cinerariaefolium callus and establish the correlation between early knockdown effects through docking on grain storage insect. In vitro seedlings were used as explants to induce callus on MS medium with different concentrations of auxins and cytokinins. Pyrethrin extracted from the callus was estimated by RP-HPLC. In callus, total pyrethrin was found to be 17.5 µg/g, which is higher than that found in natural flowers of certain Pyrethrum cultivars. The concentrations of cinerin II, pyrethrin II and jasmoline II were quite high in callus grown on solid medium. Bio-efficacy of pyrethrum extracts of flower and callus on insect Tribolium sp., showed higher repellency and early knock-down effect when compared with pure compound pestanal. Further, the rapid knockdown effect of all pyrethrins components was established by molecular docking studies targeting NavMS Sodium Channel Pore receptor docking followed by multiple ligands simultaneous docking, performed to investigate the concurrent binding of different combinations of pyrethrin. Among the six pyrethrin components, the pyrethrin I and II were found to be a more efficient, binding more firmly to the target, exhibiting higher possibilities of insecticidal effect by an early knockdown mechanism.

Medium composition potentially regulates the anthocyanin production from suspension culture of Daucus carota.

In the present study, an effort has been made to optimize various culture conditions for enhanced production of anthocyanin. Nutrient content of MS medium (ammonium to potassium nitrate ratio and phosphate concentration) had a profound influence on the cell biomass and anthocyanin accumulation in cell suspension cultures of Daucus carota. Suspension cultures were carried out in shake flasks for 18 days and examined for cell growth, anthocyanin synthesis, anthocyanin yield and development of pigmented cells in relation to the uptake of total sugar, extracellular phosphate, nitrate and ammonia. The addition of NH4NO3 to KNO3 ratio (20.0 mM: 37.6 mM) in the suspension culture media resulted in a 2.85-fold increase in anthocyanin content at day 3. Similarly, a lower concentration of KH2PO4 (0.45 mM) in the MS medium resulted in 1.63-fold increase in anthocyanin content at day 9. The total sugar uptake was closely associated with a significant increase in anthocyanin accumulation. Total sugar and nitrate were consumed until 9-12 days, while ammonia and phosphate were completely consumed within 3 days after inoculation. After 9 days, cell lysis was observed and resulted in the leakage of intracellular substances. These observations suggest that anthocyanin was synthesized only by viable pigmented cells and degraded rapidly after cell death and lysis. This study signifies the utility of D. carota suspension culture for further up-scaling studies of anthocyanin.

Enhanced production of vanillin flavour metabolites by precursor feeding in cell suspension cultures of Decalepis hamiltonii Wight & Arn., in shake flask culture.

The flavour rich tuberous roots of Decalepis hamiltonii are known for its edible and medicinal use and have become endangered due to commercial over-exploitation. Besides 2-Hydroxy-4-methoxy benzaldehyde (2H4MB), other flavour metabolites in tuberous roots include vanillin, 4-Methoxy Cinnamic acid derivatives, aromatic alcohols etc. So far, there are no reports on the pathway of 2H4MB biosynthesis nor there is an organized work on biotransformation using normal and cell suspension cultures for obtaining these metabolites using precursors. The main aim of the study is to develop a method for enhanced production of flavour attributing metabolites through ferulic acid (FA) feeding to the D. hamiltonii callus culture medium. Biomass of D. hamiltonii cell suspension cultures was maximum (200.38 ± 1.56 g/l) by 4th week. Maximum production of 2H4MB was recorded on 4th week (0.08 ± 0.01 mg/100 g dry weight) as quantified by HPLC. Addition of 0.1-1.5 mM ferulic acid as precursor in the culture medium showed significant (p < 0.001) effect on suspension cultures biomass and respective phenylpropanoid metabolites content and 2H4MB accumulation. The maximum accumulation of vanillin, 2H4MB, vanillic acid, ferulic acid were of 0.1 ± 0.02 mg/100 g, 0.44 ± 0.01 mg/100 g, 0.52 ± 0.04 mg/100 g, 0.18 ± 0.02 mg/100 g DW respectively in 4 weeks of cultured cells supplemented with 1 mM ferulic acid as a precursor. The results indicate that, substantial increase in the levels of flavour metabolites in D. hamiltonii callus suspension culture was achieved. This would be having implications in biosynthesis of respective vanilla flavour attributing metabolites at very high levels for their large scale production.

Effect of different drying methods on chlorophyll, ascorbic acid and antioxidant compounds retention of leaves of Hibiscus sabdariffa L.

BACKGROUND: Use of the indigenous, easily accessible leafy vegetable roselle (Hibiscus sabdariffa L.) for value addition is gaining impetus as its nutritive and nutraceutical compounds are exposed by investigations. Being a perishable, storage is challenging, hence different methods of drying have been an attractive alternative for its postharvest usage in foods without much compromising its quality and antioxidant potential. RESULTS: Room- and freeze-dried samples were found to have best quality in terms of colour, total flavonoid content (18.53 ± 2.39 and 18.66 ± 1.06 g kg(-1) respectively), total phenolic content (17.76 ± 1.93 and 18.91 ± 0.48 g kg(-1)), chlorophyll content (1.59 ± 0.001 and 1.55 ± 0.001 g kg(-1)) and ascorbic acid content (11.11 ± 1.04 and 8.92 ± 0.94 g kg(-1)) compared with those subjected to infrared, crossflow, microwave, oven or sun drying. Samples treated by room and freeze drying retained maximum antioxidant potential as shown by the phosphomolybdate method and the 2,2-diphenyl-1-picrylhydrazyl free radical-scavenging activity and ferric-reducing antioxidant power assays. Cold water and hot water extracts showed significantly higher total phenolic content and total antioxidant activity owing to the greater solubility of phenolics and destruction of cellular components in polar solvents than in organic solvents. CONCLUSION: The data obtained show the potential for retaining quality parameters of roselle leaf under suitable drying methods.

Differences in outcome according to Clostridium difficile testing method: a prospective multicentre diagnostic validation study of C difficile infection.

BACKGROUND: Diagnosis of Clostridium difficile infection is controversial because of many laboratory methods, compounded by two reference methods. Cytotoxigenic culture detects toxigenic C difficile and gives a positive result more frequently (eg, because of colonisation, which means that individuals can have the bacterium but no free toxin) than does the cytotoxin assay, which detects preformed toxin in faeces. We aimed to validate the reference methods according to clinical outcomes and to derive an optimum laboratory diagnostic algorithm for C difficile infection. METHODS: In this prospective, multicentre study, we did cytotoxigenic culture and cytotoxin assays on 12,420 faecal samples in four UK laboratories. We also performed tests that represent the three main targets for C difficile detection: bacterium (glutamate dehydrogenase), toxins, or toxin genes. We used routine blood test results, length of hospital stay, and 30-day mortality to clinically validate the reference methods. Data were categorised by reference method result: group 1, cytotoxin assay positive; group 2, cytotoxigenic culture positive and cytotoxin assay negative; and group 3, both reference methods negative. FINDINGS: Clinical and reference assay data were available for 6522 inpatient episodes. On univariate analysis, mortality was significantly higher in group 1 than in group 2 (72/435 [16·6%] vs 20/207 [9·7%], p=0·044) and in group 3 (503/5880 [8·6%], p<0·001), but not in group 2 compared with group 3 (p=0·4). A multivariate analysis accounting for potential confounders confirmed the mortality differences between groups 1 and 3 (OR 1·61, 95% CI 1·12-2·31). Multistage algorithms performed better than did standalone assays. INTERPRETATION: We noted no increase in mortality when toxigenic C difficile alone was present. Toxin (cytotoxin assay) positivity correlated with clinical outcome, and so this reference method best defines true cases of C difficile infection. A new diagnostic category of potential C difficile excretor (cytotoxigenic culture positive but cytotoxin assay negative) could be used to characterise patients with diarrhoea that is probably not due to C difficile infection, but who can cause cross-infection.