Maximum Activity Research Articles

Valorization of coconut (Cocos nucifera L.) testa as a biocolourant

IntroductionCoconut testa, a by-product of the coconut processing industry, is currently underutilised. This study aimed to extract a coconut testa-based food colourant using various organic solvents and physical methods, and to utilise this colourant in food product preparation.MethodsDifferent organic solvents, along with various time and temperature combinations, were employed for colourant extraction using both a laboratory-scale water bath and ultrasonication. The colour coordinate values (CIELab) of the testa-derived colourants were measured, and the colourants were screened for various phytochemicals. The in vitro antioxidant potential of the testa colourant was assessed by quantifying total phenolics, and the phytochemical composition, including monomeric anthocyanins, was evaluated.ResultsThe study determined the optimal combinations of organic solvents, temperature and time to obtain extracts with maximum antioxidant activity and total phenolic content (TPC). Acidified ethanol-based extracts of testa colourants yielded highest polyphenol content (154.39 ± 2.63 mg GAE/g) and flavonoids content (53.65 ± 0.62 mg QE/g). Similarly, ethanol-based extractants of coconut testa produced high anthocyanin content [823.02 ± 1.81 mg Cy-3-glc equivalents (C3GE)/100 g]. Acidified (0.3 M HCl) solvents at relatively high temperature and time combinations exhibited high antioxidant potential of testa colourant, as measured by CUPRAC, FRAP, and DPPH assays. Following the foam mat drying process of the colourant, a mature coconut water-based jelly was prepared by incorporating the testa colourant extracted with acidified ethanol.DiscussionThis study highlights the biochemical and antioxidant potential of the food colorant derived from coconut testa and explores its suitability for functional food applications. Therefore, coconut testa extract serves a dual purpose: it enhances the aesthetic appeal of food as a colourant and provides significant health-promoting properties due to its high anthocyanin content. Insights from this study could help in promoting the valorization of one of the beneficial by products of coconut industry.

Biological analysis of shoulder joint muscle strength during volleyball spike specific physical training

Volleyball spike requires extremely high shoulder joint muscle strength. Traditional research methods are single, with insufficient sample size, lack multidimensional analysis and long-term training effect evaluation, and have not fully combined biomechanical and physiological theories. This article combined biomechanical and physiological theories to collect field data and used a three-dimensional motion capture system and surface electromyography technology to analyze the performance of shoulder joint muscle strength in spiking. The experimental results show that Integrated Biomechanics and Physiology (IBP) is significantly superior to other training methods in improving the maximum strength, endurance, and muscle activation of the shoulder joint. Specific data show that after IBP training, the maximum strength of the shoulder joint increased by 50 N, and the endurance increased by 19.6%, and the muscle activation increased by 13.5%. Studies have shown that the IBP method has great potential for application in optimizing special physical training, providing a scientific basis for athletes’ physical training.

Structural determinants of protein kinase A essential for CFTR channel activation

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), the anion channel mutated in cystic fibrosis (CF) patients, is activated by the catalytic subunit of protein kinase A (PKA-C). PKA-C activates CFTR both noncatalytically, through binding, and catalytically, through phosphorylation of multiple serines in CFTR's regulatory (R) domain. Here, we identify key molecular determinants of the CFTR/PKA-C interaction essential for these processes. By comparing CFTR current activation in the presence of ATP or an ATP analog unsuitable for phosphotransfer, as well as pseudosubstrate peptides of various lengths, we identify two distinct specific regions of the PKA-C surface which interact with CFTR to cause noncatalytic and catalytic CFTR stimulation, respectively. Whereas the "substrate site" mediates CFTR phosphorylation, a distinct hydrophobic patch (the "docking site") is responsible for noncatalytic CFTR activation, achieved by stabilizing the R domain in a "released" conformation permissive to channel gating. Furthermore, by comparing PKA-C variants with different posttranslational modification patterns, we find that direct membrane tethering of the kinase through its N-terminal myristoyl group is an unappreciated fundamental requirement for CFTR activation: PKA-C demyristoylation abolishes noncatalytic, and profoundly slows catalytic, CFTR stimulation. For the F508del CFTR mutant, present in ~90% of CF patients, maximal activation by demyristoylated PKA-C is reduced by ~10-fold compared to that by myristoylated PKA-C. Finally, in bacterial genera that contain common CF pathogens, we identify virulence factors that demyristoylate PKA-C in vitro, raising the possibility that during recurrent bacterial infections in CF patients, PKA-C demyristoylation may contribute to the exacerbation of lung disease.

Molecular characterization and computational analysis of a highly specific L-glutaminase from a marine bacterium Bacillus australimaris NIOT30

An alkaline active L-glutaminase (BALG) producing bacterium was screened and identified from seamount sediment samples of the Arabian Sea. The isolate was confirmed to be Bacillus australimaris NIOT30 based on morphological characteristics and 16 S rRNA gene sequencing. The glutaminase gene, balg was PCR amplified, cloned and expressed in E. coli BL21 (DE3) host. The molecular weight of purified BALG was estimated to be 36 kDa and the enzyme showed a specific activity of 507 ± 27 Umg−1 against L-glutamine under optimal assay conditions of pH 7.0 and temperature at 37 °C for 15 min. The enzyme showed maximum activity at pH 7 and retained 95% activity at pH 10. BALG retained a relative activity of about 82% and 45% at 45 °C and 60 °C respectively. The kinetic parameters of BALG, Km and Kcat/Km were determined to be of 210 ± 11 mM and 4.4 × 102 M s−1 respectively. Homology modeling and substrate ligand interaction studies revealed the stability of the enzyme-substrate complex. The present study highlights the characterization of a highly active L-glutaminase from B. australimaris NIOT30. Further, mutational analyses of ligand binding residues would show insights into the affinity of L-Glutaminase.

Odd-Numbered Agaro-Oligosaccharides Produced by α-Neoagaro-Oligosaccharide Hydrolase Exert Antioxidant Activity in Human Dermal Fibroblasts

Agarases produce agar oligosaccharides with various structures exhibiting diverse physiological activities. α-Neoagaro-oligosaccharide hydrolase (α-NAOSH) specifically cleaves even-numbered neoagaro-oligosaccharides, producing 3,6-anhydro-l-galactose (l-AHG) and odd-numbered agaro-oligosaccharides (OAOSs). In this study, α-NAOSH from the agar-degrading marine bacterium Gilvimarinus agarilyticus JEA5 (Gaa117) was purified and characterized using an E. coli expression system to produce OAOSs and determine their bioactivity. Recombinant Gaa117 (rGaa117) showed maximum activity at pH 6.0 and 35 °C. rGaa117 retained >80% of its initial activity after 120 min at 30 °C. The activity was enhanced in the presence of Mn2+. Km, Vmax, and Kcat/Km values of the enzyme were 22.64 mM, 246.3 U/mg, and 15 s−1/mM, respectively. rGaa117 hydrolyzed neoagarobiose, neoagarotetraose, and neoagarohexaose, producing OAOSs that commonly contained l-AHG. Neoagarobiose and neoagarotetraose mixtures, designated NAO24, and mixtures of l-AHG and agarotriose, designated AO13, were obtained using recombinant rGaa16B (β-agarase) and rGaa117, respectively, and their antioxidant activities were compared. AO13 showed higher hydrogen peroxide-scavenging activity than NAO24 in human dermal fibroblasts in vitro because of structural differences: AOSs have d-galactose at the non-reducing end, whereas NAOSs have l-AHG. In conclusion, OAOSs exhibited high ROS-scavenging activity in H2O2-induced human dermal fibroblasts. They may be applicable in cosmetics and pharmaceuticals for prevention of skin aging.

Enhanced activity of split trehalase biosensors by interspecies domain combineering.

The split trehalase biosensor has potential as a versatile diagnostic technology. Split enzymes are engineered proteins, divided into inactive fragments, which can reassemble and regain activity when brought together by an analyte. The split TreA biosensor requires no sample processing and produces stable signals (in the form of glucose). Split trehalase reagents can function in blood, but periplasmic trehalase of E.coli requires blood acidification for maximal activity. The objective of this study was to obtain split trehalase with near physiological pH optimum. For this purpose, periplasmic trehalases of Cellvibrio spp. with higher activity at neutral pH, were split in analogy with the E. coli TreA into hood and catalytic domains. However, these split trehalases displayed self-complementation due to spontaneous reassembly. In contrast, when catalytic domains of Cellvibrio trehalases were combined with E.coli hood domains, these hybrids displayed conditional complementation capacity when split trehalase fragments fused to immunoglobulin-binding protein G (STIGA) were used to quantify immunoglobulin concentrations. Other hybrid combinations of Cellvibrio spp. had increased activity compared to the cognate pairs, albeit with strong self-complementation. A mutagenesis analysis of residues responsible for self-complementation led to uncoupling of self-complementation from allostery. The Michaelis-Menten kinetics of Cellvibrio enzymes and fragment pairs confirmed improved activity of a mutated hybrid pair of Cellvibrio hood and catalytic domains at physiological pH. In conclusion, the improvements in pH optimum and activity, demonstrated with STIGA, can now be leveraged to enhance other variations of the split trehalase biosensor platform, broadening its utility for testing clinical samples.

Determining conditions for inactivation of multispecies biofilm cells by peracetic acid applying response surface methodology

Biofilms can form on various surfaces and are typically associated with multiple microbial species. Particularly in the food industry, biofilms are often a source of contamination, necessitating mitigation strategies. In this study, Response Surface Methodology (RSM) was utilized to optimize conditions for the inactivation of multispecies biofilm cells, with peracetic acid (PAA) (0.05% to 0.5%), treatment time (5 to 30 minutes), and temperature (25 to 60 ºC) as independent variables. The multispecies biofilm consisted of Gram-positive and Gram-negative bacteria previously isolated from biofilms formed in the presence of raw milk. Stainless steel (SS) coupons were immersed in reconstituted whole milk inoculated with Pseudomonas fluorescens, Rahnella inusitata, Staphylococcus aureus, and Micrococcus aloeverae. Sessile cell counts of approximately 108 CFU/cm2 were obtained after 10 days of incubation at 4 ºC. The response surface model exhibited a good fit, with R2 of 0.949 and adjR2 of 0.883. All independent variables had a positive effect on the inactivation of biofilm cells. The maximum inactivation achieved was approximately 7 log (CFU/cm2), with the highest values observed at 0.5% PAA, for 30 min at 60°C. Epifluorescence microscopy revealed that many cells in the biofilm were dead or injured after treatment at the central point (PAA = 0.275%; Time = 17.5 min; Temperature = 42.5 ºC). RSM helps to predict better conditions for maximum biofilm eradication and proves to be a promising approach for monitoring the inactivation of multispecies biofilm cells, which warrants further exploration.

The Anti-Candidal Activity of Salvia verticillata subsp. verticillata Against Several Candida Species

The examination of Salvia verticillata extract encompassed an exploration of its anti-Candida properties, phytochemical composition, antioxidant activity, and the quantification of total tannins. The use of ethanol as a solvent yielded the highest extraction efficiency at 26.1%. The plant exhibited a substantial tannin content of 6.5 mg/kg, signifying a noteworthy concentration. The inhibitory zones against Candida tropicalis reached a minimum diameter of 17.6 mm, while Candida guilliermondii displayed the most significant inhibition with a zone diameter of 21.8 mm. The minor inhibitory concentration (MIC) findings for all Candida species ranged from 6.25 to 12.5 g/ml. In the case of Candida guilliermondii, the synthetic antifungal activity FLU/25 demonstrated a maximal inhibition zone measuring 39.30 mm. Additionally, the maximum antioxidant activity, recorded at 0.3 ml, reached a value of 98.65. Phytochemical screening unveiled elevated concentrations of phenols and flavonoids, with malic acid (1901.1 g/g), hesperidin (302.4 g/g), and rosmarinic acid (30619.93 g/g) all experiencing an increase in concentration. These findings provide a comprehensive understanding of the diverse bioactive components and properties associated with Salvia verticillata extract.

Paradendryphiella arenariae an endophytic fungus of Centella asiatica inhibits the bacterial pathogens of fish and shellfish

IntroductionAquaculture has been considered a major food-producing sector in the world during the last few decades. The foremost constraint in the development of aquaculture is bacterial disease control and management. Since various fish pathogens are resistant to conservative treatments, it is essential to screen new and effective alternative antibacterial agents. Endophytic fungi are microorganisms that live in the plant’s internal tissues without harming its host. Endophytic fungi have proven themselves as reliable sources of novel bioactive compounds that can be used as antibacterial agents.MethodsIn the present study, fifteen morphologically different endophytic fungi were isolated from the fresh and healthy stem section of Centella asiatica. The active endophytic fungal crude extracts were tested for agar well diffusion assay, Minimum Inhibitory Concentration, Minimum Bactericidal Concentration assays, Time-kill kinetic analysis, Brine shrimp lethality assay.Results and DiscussionAgar plug diffusion and agar well diffusion assays revealed that endophytic fungus CAS1 exhibits maximum antagonistic activity against bacterial fish pathogens. The ethyl acetate crude extract of CAS1 exhibited the maximum zone of inhibitions against Aeromonas hydrophila (21 ± 0.11 mm), Aeromonas caviae (18 ± 0.1 mm), Edwardsiella tarda (23 ± 0.11 mm), Vibrio anguillarum (19 ± 0.05 mm) and Vibrio harveyi (20 ± 0.27 mm). The MIC and MBC values extract varied reliant on the trial pathogens ranging between 12.5-100 μg/mL and 25-100μg/mL correspondingly. The morphological and molecular characterization of potential isolate CAS1 was confirmed as Paradendryphiella arenariae by 18S rRNA ITS gene sequencing with 99.18% identity. This is the foremost findings to study the antagonistic effect of Paradendryphiella arenariae isolated from the stem of Centella asiatica against bacterial fish pathogens which can be used as natural effective antibacterial agents in aquaculture.

Anti-inflammatory Activity of Crude Extract and Zinc- Oxide Nanoparticles from the Ink of Squid (Uroteuthis duvaucelii )

Objectives: Marine natural materials can be utilized to construct a variety of medications and have produced significant leads for therapeutic development. The aim of the present study is to evaluate the anti-inflammatory activity of crude extract and zinc-oxide nanoparticles from the ink of squid. Methods : The anti-inflammatory activity of the synthesized zinc-oxide nanoparticles and crude extract of Uroteuthis duvaucelii was determined by using albumin denaturation assay and anti-proteinase activity. Findings : The albumin denaturation assay of the crude ink extract shows a maximum anti-inflammatory activity of 29% at a concentration of 100µl/ml and a minimum activity of 7% at a concentration of 20µl/ml. The zinc-oxide nanoparticles of U. duvaucelii exhibit anti-inflammatory activity of 8% at 20µl/ml, and 37% at a concentration of 100µl/ml. Anti-proteinase activity of the crude extract exhibits 25% of inhibition at a concentration of 100µl/ml and lowest activity of 2% at a concentration of 20µl/ml. Zinc-oxide nanoparticles of Uroteuthis duvaucelii exhibit maximum inhibition of 28% at 100µl/ml and minimum activity of 5% at a concentration of 20µl/ml. Comparing the two extracts the zinc-oxide of U. duvaucelii shows better albumin denaturation and anti-proteinase action than the crude ink extract. So, this can be used as a source of anti-inflammatory agents for drug development. Novelty : Research suggests that natural products may be a source of anti-inflammatory medications with higher activity and fewer side effects. Therefore, the synthesized nanoparticles from the ink of Uroteuthis duvaucelii can be used as an anti-inflammatory drug. Keywords: Anti-inflammatory activity; Zinc-oxide nanoparticles; Uroteuthis duvaucelii; Anti proteinase; Crude ink

Field and biochemical evaluation of glyphosate tolerant chickpea (Cicer arietinum L.) mutants developed through induced mutagenesis

Weed control in chickpea (Cicer arietinum L.) is challenging due to narrow genetic base of available germplasm and limited herbicide options. In this view, present research was focused on induced mutagenesis in chickpea for development of herbicide (glyphosate) tolerant mutants and subsequent screening under field conditions. Further, objective was to analyze the defence response and biochemical adjustments in selected glyphosate tolerant chickpea mutants. Initially, 376 chickpea mutants (M6 populations developed through EMS and gamma rays) were screened for glyphosate tolerance under filed conditions and scored on a 1 to 5 scale based on plant injury related traits. Among tested mutants, 40 were found highly tolerant (score = 5), 32 as tolerant (score = 4) and 20 as highly sensitive (score = 1) to glyphosate. Chickpea mutants with variable glyphosate tolerance also differed significantly (Tukey test, p < 0.05) in leaf biochemical profiles. For instant, lowest total oxidant status (4175.µM/g f. wt.) was detected in glyphosate tolerant mutant developed from desi chickpea genotype “D3009” using 0.3% EMS and in highly tolerant mutant (1775. µM/g f. wt.) developed from kabuli genotype “K709” using 0.2% EMS. In general, highly tolerant chickpea mutants exhibited highest antioxidant potential (SOD, POD, CAT, TAC) that contributed in glyphosate tolerance. Desi i.e. D1M1HT-2 and Kabuli i.e. KM3HT-2 type mutants with highest seed yield had maximum catalase activity (4200 Units/g f. wt and 540 Units/g f. wt.). Mutants developed from desi type genotypes were comparably superior to mutants derive from Kabuli in terms of herbicide tolerance.

Redox Gradient Shapes the Chemical Composition of Peatland Microbial Communities.

The response of soil carbon to climate change and anthropogenic forcing depends on the relationship between the physicochemical variables of the environment and microbial communities. In anoxic soils that store large amounts of organic carbon, it can be hypothesized that the low amount of catabolic energy available leads microbial organisms to minimize the energy costs of biosynthesis, which may shape the composition of microbial communities. To test this hypothesis, thermodynamic modeling was used to assess the link between redox gradients in the ombrotrophic peatland of the Marcell Experimental Forest (Minnesota, USA) and the chemical and taxonomic composition of microbial communities. The average amino acid composition of community-level proteins, called hereafter model proteins, was calculated from shotgun metagenomic sequencing. The carbon oxidation state of model proteins decreases linearly from -0.14 at 10 cm depth to -0.17 at 150 cm depth. Calculating equilibrium activities of model proteins for a wide range of chemical conditions allows identification of the redox potential of maximum chemical activity. Consistent with redox measurements across peat soils, this model Eh decreases logarithmically from an average value of 300 mV at 10 cm depth, close to the stability domain of goethite relative to Fe2+, to an average value of -200 mV at 150 cm, within the stability domain of CH4 relative to CO2. The correlation identified between the taxonomic abundance and the carbon oxidation state of model proteins enables predicting the evolution of taxonomic abundance as a function of model Eh. The model taxonomic abundance is consistent with the measured gene and taxonomic abundance, which evolves from aerobic bacteria at the surface including Acidobacteria, Proteobacteria, and Verrumicrobia, to anaerobes at depth dominated by Crenarchaeota. These results indicate that the thermodynamic forcing imposed by redox gradient across peat soils shapes both the chemical and taxonomic composition of microbial communities. By providing a mechanistic understanding of the relationship between microbial community and environmental conditions, this work sheds new light on the mechanisms that govern soil microbial life and opens up prospects for predicting geochemical and microbial evolution in changing environments.

Synthesis of nanohybrid consisting of taurine derived carbon dots and nanoceria for anticancer applications

Here, we first synthesized carbon dots (CDs) from taurine and then a nanohybrid with ceria (CeO2) nanoparticles using thermal decomposition method for checking their antineoplastic efficacy against human triple negative mammary carcinoma cells, MDA-MB-231. The in vitro study demonstrated significant dose-dependent antineoplastic activity of the nanohybrids within a range of concentration from 10 to 50 μg/mL after 48 h of treatment. The cellular morphology analysis clearly depicted substantial amount of cell death which seems to stem from increased intracellular reactive oxygen species (ROS) production. However, the maximum anticancer activity of the nanohybrid as compared to bare CDs and CeO2 is supposed to be due to the combined anticancer effect of both CDs and CeO2 (a well-established antitumor agent). Further we have performed molecular docking study to reveal the anticancer mechanism of CDs which exhibited high binding capacity towards several proapoptotic and antiapoptotic protein molecules. The binding affinity values were found to be – 8.7 kcal/mol, – 7.9 kcal/mol, – 9.6 kcal/mol, – 9.5 kcal/mol, – 12 kcal/mol and – 11.1 kcal/mol for BAD, BCl-2, p53, Caspase-8, Caspase-9 and Caspase-3 respectively. Taken together, our synthesized CDs-CeO2 nanohybrid could be thought as a potential anticarcinogenic option in the field of breast cancer therapeutics.

Biocatalyst for carbon veil anode in microbial fuel cells: The effect of precursor and catalyst loading on overall performance

Biocatalysts in anodes have been crucial in improving microbial fuel cell (MFC) performance. Biocatalysts derived from biomass such as coconut wood sawdust (SD-B) and pepper processing residue (PS-B), were used to improve the electrocatalytic properties of Carbon Veil (CV) electrodes. The amorphous graphitic carbon biocatalyst with a mesoporous structure was found to have a surface area of 889.31 m2/g for SD-B and 763.56 m2/g for PS-B. Surface functional groups such as -OH which imparts hydrophilicity and -C=O and COOH which promotes bacterial compatibility have been revealed to be present in both catalysts. Catalyst loading played a major role and at an optimal loading of 2 mg/cm2, the electrodes exhibited maximum electrocatalytic activity in the case of both biocatalysts. The voltammetric capacitances of SD-B and PS-B modified electrodes were 191.6 mF/cm2 and 106.6 mF/cm2 demonstrating their pseudocapacitive behaviour too. The performance of MFC with SD-B revealed a maximum power density of 10.1 W/m3 (Open Circuit Voltage (OCV) of 850 mV), followed by PS-B at 6.89 W/m3 (OCV of 825 mV) whereas the plain CV was at 0.168 W/m3 (OCV of 760 mV). Anode polarization studies indicated reduced slopes for biocatalyst-modified anodes, signifying improved electrode kinetics, particularly notable in the SD-B modified MFC where the cathode became the limiting electrode. Coulombic efficiencies of the biocatalyst-modified MFCs increased to 54.50%, (SD-B 2 mg/cm2) and 37.78% (PS-B 2 mg/cm2) respectively from 12.74% as noticed in the case of unmodified MFC. This study emphasizes the potential of biocatalysts as a simple and low-cost means of enhancing the anode properties – porosity, conductivity, hydrophilicity, and biocompatibility.

Effective control of citrus green mold using plasma-activated water with hydrogen peroxide vapor: A non-thermal approach

In this study, a novel approach for controlling Citrus sinensis Salustiana green mold disease in oranges was developed using plasma-activated water (PAW) generated by a Surface Dielectric Barrier Discharge (SDBD) system reinforced with H2O2 cold vapor. Penicillium digitatum, the primary cause of orange mold, was the target of the treatment. The efficiency of the setup was investigated in three stages. First, a suspension of P. digitatum spores was directly treated with Ar/H2O2 plasma to estimate the optimal plasma exposure time required for maximum fungal inactivation. Second, the impact of Argon/H2O2 plasma-treated water (PTW), generated from various plasma exposure times, on mold growth on orange peels was examined. For this, orange peels were placed in Ar/H2O2-PTW for different durations, and mold occurrence was monitored. Finally, the effect of PTW, generated from various Ar/H2O2 plasma exposure times, on mold growth on whole oranges was investigated. Oranges were treated with Ar/H2O2-PTW for different durations, and mold occurrence was analyzed. The results demonstrated that a 20-min treatment of oranges with PTW produced from a 420-s Ar/H2O2 plasma exposure time led to complete inactivation of P. digitatum. This study showed that this hybrid scenario can be effectively used for fungal decontamination in the food industry.

Heliospheric Effect on Solar Activity Parameters during MaximumPhase of Solar Cycle 24 (2012-2015)

Abstract The time series of daily data of solar activity proxies, namely the sunspot number (SSN), sunspot area (SSA), solar radio flux (F10.7), modified coronal index (MCI), solar flare index (FI), and cosmic ray intensity (CRI), were analyzed to understand the solar activity modulations and short-term periodicities therein. Rieger-type, and other short-term periods, including the solar rotational period that covers the maximum activity phase period (maximum phase of solar cycle 24). The wavelet power spectra and Periodogram of SSN, SSA, F10.7, MCI, FI, and CRI exhibited a significant short-term period. The heliospheric effects exist for a particular period (~27 days) and it is related to the solar activity phenomena. The cross-correlation coefficients and time lags between the cosmic ray intensity and solar activity parameters were estimated to be 200, 46, 281, 39, and 47 days for SSN, SSA, F10.7, MCI, and FI respectively during the time series 2012-2015 (maximum phase of solar cycle 24).

Biological activities of Hypericum spectabile extract optimized using artificial neural network combined with genetic algorithm application

Optimizing extraction conditions can help maximize the efficiency and yield of the extraction process while minimizing negative impacts on the environment and human health. For the purpose of the current study, an artificial neural network (ANN) combined with a genetic algorithm (GA) was utilized for that the extraction conditions of Hypericum spectabile were optimized. In this particular investigation, the main objective was to get the highest possible levels of total antioxidant status (TAS) for the extracts that were obtained. In addition to this, conditions of the extract that exhibited the maximum activity have been determined and the biological activity of the extract that was obtained under these conditions was analyzed. TAS values were obtained from extracts obtained using extraction temperatures of 30–60 °C, extraction times of 4–10 h, and extract concentrations of 0.25-2 mg/mL. The best model selected from the established ANN models had a mean absolute percentage error (MAPE) value of 0.643%, a mean squared error (MSE) value of 0.004, and a correlation coefficient (R) value of 0.996, respectively. The genetic algorithm proposed optimal extraction conditions of an extraction temperature of 59.391 °C, an extraction time of 8.841 h, and an extraction concentration of 1.951 mg/mL. It was concluded that the integration of ANN-GA can successfully be used to optimize extraction parameters of Hypericum spectabile. The total antioxidant value of the extract obtained under optimum conditions was determined as 9.306 ± 0.080 mmol/L, total oxidant value as 13.065 ± 0.112 µmol/L, oxidative stress index as 0.140 ± 0.001. Total phenolic content (TPC) was 109.34 ± 1.29 mg/g, total flavonoid content (TFC) was measured as 148.34 ± 1.48 mg/g. Anti-AChE value was determined as 30.68 ± 0.77 µg/mL, anti-BChE value was determined as 41.30 ± 0.48 µg/mL. It was also observed that the extract exhibited strong antiproliferative activities depending on the increase in concentration. As a result of LC-MS/MS analysis of the extract produced under optimum conditions in terms of phenolic content. The presence of fumaric, gallic, protocatechuic, 4-hydroxybenzoic, caffeic, 2-hydoxycinamic acids, quercetin and kaempferol was detected. As a result, it was determined that the H. spectabile extract produced under optimum conditions had significant effects in terms of biological activity.

Nanochitosan-encapsulated melatonin: an eco-friendly strategy to delay petal senescence in cut gerbera flowers

BackgroundThe preservation of cut flowers, particularly Gerbera jamesonii, is crucial for maintaining their aesthetic value and extending vase life in the floriculture industry. To address this challenge, this study investigated the effects of melatonin (Mel) and encapsulated melatonin with nanochitosan (nCS-Mel) as preservative solutions on cut Gerbera jamesonii cv. ‘Terra kalina’ flowers. In research, we examined various physiological and biochemical parameters, including relative water content, membrane stability index, carbohydrate content, and antioxidant enzyme activities, to evaluate the efficacy of these treatments in prolonging the vase life and quality of cut gerbera flowers under controlled environmental conditions.ResultsOur results demonstrated that cut Gerbera jamesonii flowers maintained in vase solutions containing 0.1 and 0.5 mM nCS-Mel exhibited enhanced preservation of cell membrane integrity and anthocyanin content, while also maintaining higher levels of carbohydrates and total flavonoids in petals at the conclusion of their vase life. A decline in petal relative water content and protein levels was observed concomitantly with petal senescence, whereas total phenolic compounds showed an increase. The hydrogen peroxide (H2O2) content in petals exhibited an upward trend during vase life in control specimens, but this effect was mitigated in treatments containing melatonin. Although malondialdehyde (MDA) content generally increased throughout the vase life period, flowers subjected to either Mel or nCS-Mel treatments displayed reduced MDA accumulation. The activity of catalase (CAT) demonstrated an increasing trend during vase life, with the maximum activity observed in Gerbera flowers treated with 0.1 mM nCS-Mel. A similar upward trend was noted for superoxide dismutase (SOD) activity, with flowers in 0.5 mM nCS-Mel treatment exhibiting peak SOD values on day 12 relative to control and other treatments. Peroxidase (POD) activity also increased across all treatments, with particularly pronounced effects in vase solutions containing 0.1 mM Mel and nCS-Mel. Notably, flowers placed in vase solutions containing 0.1 mM nCS-Mel, followed by 0.5 mM nCS-Mel and 0.1 mM Mel, exhibited the most prolonged vase life, extending up to 12, 10.66, and 10.33 days, respectively, under room temperature conditions.ConclusionsThe application of nanoencapsulated melatonin as a vase solution for cut Gerbera jamesonii flowers demonstrates significant potential in extending vase life and maintaining flower quality through enhanced preservation of cellular integrity, antioxidant activity, and biochemical parameters. This innovative approach not only outperforms conventional treatments but also presents a more environmentally friendly alternative to traditional antimicrobial preservatives and sugars, offering a promising solution for the floriculture industry to improve cut flower longevity and reduce ecological impact.

Heterotropic Allosteric Modulation of CYP3A4 In Vitro by Progesterone: Evidence for Improvement in Prediction of Time Dependent Inhibition for Macrolides.

Predictions of drug-drug interactions resulting from time-dependent inhibition (TDI) of CYP3A4 have consistently overestimated or mis-predicted (i.e. false positives) the interaction that is observed in vivo. Recent findings demonstrated that the presence of the allosteric modulator progesterone (PGS) in the in vitro assay could alter the in vitro kinetics of CYP3A4 TDI with inhibitors that interact with the heme moiety, such as metabolic-intermediate complex (MIC) forming inhibitors. The impact of the presence of 100 µM PGS on the TDI of molecules in the class of macrolides typically associated with MIC formation was investigated. Presence of PGS resulted in varied responses across the inhibitors tested. The TDI signal was eliminated for five inhibitors, and unaltered in the case of one, fidaxomicin. The remaining molecules erythromycin, clarithromycin, and troleandomycin, were observed to have a decrease in both potency and maximum inactivation rate ranging from 1.7-fold to 6.7-fold. These changes in TDI kinetics led to a >90% decrease in inactivation efficiency. In order to determine in vitro conditions that could reproduce in vivo inhibition, varied concentrations of PGS were incubated with clarithromycin and erythromycin. Resulting in vitro TDI kinetics were incorporated into dynamic physiologically-based pharmacokinetic (PBPK) models to predict clinically observed interactions. The results suggested that a concentration of ~45 µM PGS would result in TDI kinetic values that could reproduce in vivo observations and could potentially improve predictions for CYP3A4 TDI. Significance Statement The impact of the allosteric heterotropic modulator progesterone on the CYP3A4 time-dependent inhibition kinetics was quantified for a set of metabolic-intermediate complex forming mechanism-based inhibitors. We identify the in vitro conditions that optimally predict time-dependent inhibition for in vivo drug-drug interactions through dynamic physiologically-based pharmacokinetic modeling. The optimized assay conditions improve in vitro to in vivo translation and prediction of time-dependent inhibition.

Cathepsin B- and L-like Protease Activities Are Induced During Developmental Barley Leaf Senescence

Leaf senescence is a developmental process allowing nutrient remobilization to sink organs. Previously cysteine proteases have been found to be highly expressed during leaf senescence in different plant species. Using biochemical and immunoblotting approaches, we characterized developmental senescence of barley (Hordeum vulgare L. var. ‘GemCraft’) leaves collected from 0 to 6 weeks after the onset of flowering. A decrease in total protein and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunits occurred in parallel with an increase in proteolytic activity measured using the fluorogenic substrates Z-RR-AMC, Z-FR-AMC, and casein labeled with fluorescein isothiocyanate (casein-FITC). Aminopeptidase activity detected with R-AMC peaked at week 3 and then decreased, reaching a low level by week 6. Maximal proteolytic activity with Z-FR-AMC and Z-RR-AMC was detected from pH 4.0 to pH 5.5 and pH 6.5 to pH 7.4, respectively, while two pH optima (pH 3.6 to pH 4.5 and pH 6.5 to pH 7.4) were found for casein-FITC. Compound E-64, an irreversible cysteine protease inhibitor, and CAA0225, a selective cathepsin L inhibitor, effectively inhibited proteolytic activity with IC50 values in the nanomolar range. CA-074, a selective cathepsin B inhibitor, was less potent under the same experimental conditions, with IC50 in the micromolar range. Inhibition by leupeptin and phenylmethylsulfonyl fluoride (PMSF) was weak, and pepstatin A, an inhibitor of aspartic acid proteases, had no effect at the concentrations studied (up to 0.2 mM). Maximal proteolytic activity with the aminopeptidase substrate R-AMC was detected from pH 7.0 to pH 8.0. The pH profile of DCG-04 (a biotinylated activity probe derived from E-64) binding corresponded to that found with Z-FR-AMC, suggesting that the major active proteases are related to cathepsins B and L. Moreover, immunoblotting detected increased levels of barley SAG12 orthologs and aleurain, confirming a possible role of these enzymes in senescing leaves.