Chromatography Research Articles

Online ligand screening for cytochrome C from herbal medicines through “four-in-one” measurement

Affinity-oriented online ligand screening with LC coupled to different detectors is widely popular to capture active compounds from herbal medicines (HMs). However, false-positive extensively occurs because insufficient information is recorded for the existence and stability of ligand-protein complex. Here, efforts were made to advance the hit confidences via configuring post-column infusion-LC–energy-resolved-affinity MS (PCI-LC–ER-AMS) to achieve “four-in-one” monitoring of: 1) response decrement of potential ligands; 2) response decrement of protein; 3) ions relating to ligand-protein complexes; and 4) ligand-protein binding strength. Ligand fishing for Cyt C from HMs was conducted as a proof-of-concept. For utility justification, a mimic sample containing twelve well-defined ligands and two negative controls underwent LC separation and met Cyt C prior to Qtof-MS measurements. Compared to Cyt C- or ligand-free assay, twelve ligands instead of negative controls showed response decrements that were consistent with twelve negative peaks observed at retention times corresponding to the ligands in Cyt C ion current chromatogram. Serial ions correlating to each ligand-Cyt C complex were observed. After recording breakdown graphs, optimal collision energy (OCE) corresponding to the non-covalent bond dissociation was positively correlated with binding strength. Two HMs including Scutellariae Radix (SR) and Aconiti Lateralis Radix Preparata were investigated. Consequently, 24 compounds were merely fished from SR, and particularly, flavonoid glycosides exhibited greater OCEs and also binding strengths over aglycones. Affinity assays and cellular evaluations consolidated the significant interactions between each captured compound and Cyt C. Overall, PCI-LC–ER-AMS is eligible for confidence-enhanced online ligand screening for Cyt C from HMs through “four-in-one” measurement.

Mechanochemical "Cage-on-MOF" Strategy for Enhancing Gas Adsorption and Separation through Aperture Matching.

Post-modification of porous materials with molecular modulators has emerged as a well-established strategy for improving gas adsorption and separation. However, a notable challenge lies in maintaining porosity and the limited applicability of the current method. In this study, we employed the mechanochemical "Cage-on-MOF" strategy, utilizing porous coordination cages (PCCs) with intrinsic pores and apertures as surface modulators to improve the gas adsorption and separation properties of the parent MOFs. We demonstrated the fast and facile preparation of 28 distinct MOF@PCC composites by combining 7MOFs with 4 PCCs with varying aperture sizes and exposed functional groupsthrough a mechanochemical reactionin 5 mins. Only the combinations of PCCs and MOFs with closely matched aperture sizes exhibited enhanced gas adsorption and separation performance. Specifically, MOF-808@PCC-4 exhibited a significantly increased C2H2 uptake (+64%) and a longer CO2/C2H2 separation retention time (+40%). MIL-101@PCC-4 achieved a substantial C2H2 adsorption capacity of 6.11 mmol/g. This work not only highlights the broad applicability of the mechanochemical "Cage-on-MOF" strategy for thefunctionalization of a wide range of MOFs but also establishes potential design principles for the development of hybrid porous materials with enhanced gas adsorption and separation capabilities, along with promising applications in catalysis and intracellular delivery.

PSVIII-21 Meta-analysis of nutritive factors influencing mean retention time of digesta in the gastrointestinal tract of broiler chickens

Abstract Broiler chicken meat is an important source of food protein for human consumption globally. Over the last several decades, dramatic changes have been made to broiler genetics, management, and nutrition to increase production efficiency. Different feeds with varying nutrient composition have been researched to target goals such as improving growth and feed conversion ratio. Part of understanding and further improving these feeding programs involves understanding the effects of the feed on nutrient digestibility and animal performance. As such, models are a key tool in the wholistic evaluation of diet, genetics, and management interactions. Towards developing a mechanistic digestion model for broilers, knowledge of mean retention time (MRT, min) of digesta in the gastrointestinal tract (GIT) across a variety of diets is required. To understand the relationship between MRT in broilers and their diet, a systematic literature search and meta-analysis was conducted where 34 articles were found to meet the inclusion criteria. MRT in these studies was measured in various parts of the GIT where 7 studies reported MRT in just one segment (e.g. jejunum), 12 studies reported MRT in multiple combined segments (e.g. jejunum + ileum) and 21 studies reported MRT of the entire GIT. Birds used in these studies included those in the starter, grower, and finisher phases as well as breeders. Nutritional composition of the diets was represented by data on metabolizable energy (ME, kcal/kg), crude fiber (% DM), crude fat (% DM), crude protein (% DM) and calcium (% DM) that was consistently reported across studies. A mixed model (PROC MIXED, in SAS), treating study as a random effect, and using a subset of the data collected where MRT was reported for the entire GIT, and limited to grower and finisher phases, was used to examine the relationship between the diet composition and MRT. Only ME (F1,28.1 = 4.25; P = 0.0486) was found to be significantly related to MRT, where an increase in one kcal/kg of ME resulted in a decrease of MRT by 0.091 minutes (within the range of 2,749 to 3,507 kcal/kg ME). Other nutritional components of the diet were found to have no significant relation to MRT (all P > 0.05), though next steps will examine how the source of ME (fat vs starch vs fiber) impacts this relationship. Although other factors (such as age and feed intake) are still to be considered, the initial results from this study provide valuable insights into how nutritional composition of poultry feeds relates to MRT. Further investigation will also quantify this relationship within segment of the GIT. This work not only provides valuable information to nutritionists, but also lays the groundwork for empirical model development to predict MRT of poultry feed, essential to poultry nutritional models.

Mechanism of charge self-reversal nano-nutrient delivery system to overcome the mucosal barrier

When delivering hydrophobic nutrients such as curcumin (Cur) in the gastrointestinal tract (GIT), oral protein nanoparticles (NPs) will encounter several physiological barriers, including gastric acidity, digestive enzymes, mucus, and the epithelial layer. It is worth noting that overcoming both mucus and epithelial barriers requires NPs with completely opposite surface properties. To address this challenge, this study developed enzyme-responsive NPs using chitosan (CS) and tripolyphosphate (TPP) co-modification of zein (Cur/ZCT NPs). Three types of ZCT NPs were studied: positively charged NPs (Cur/ZCT0.2), neutrally charged NPs (Cur/ZCT0.8), and negatively charged NPs (Cur/ZCT3.0), which exhibit good stability in GIT while preserving the bioactivity of Cur. ZCT0.8 (0.86 ± 3.86 mV) and ZCT3.0 (–32.75 ± 1.07 mV) demonstrated superior mucus penetration capabilities. ZCT NPs were shown to effectively undergo charge reversal in the presence of intestinal alkaline phosphatase (IAP). The enhanced cellular uptake of ZCT NPs compared to zein NPs can be attributed to two factors: (1) enzyme-responsive dephosphorylation leading to a hydrophobic and positively charged surface, and (2) the ability of NPs to enter cells via transcellular and paracellular pathways. In vivo experiments revealed that ZCT0.8 and ZCT3.0 NPs exhibited extended retention times in the small intestine and higher uptake by epithelial cells compared to other samples. In conclusion, the enzyme-responsive charge reversal mechanism in ZCT3.0 NPs results in enhancing stability and enabling effective traversal of mucus and epithelial barriers. Consequently, they exhibit significant potential as a nutrient delivery system.

Validation of a rapid GC–MS method for forensic seized drug screening applications

With the lack of standardized validation protocols across the forensic chemistry community, validation of instrumentation can be a challenging and time-consuming task. However, this process is crucial to understanding the associated capabilities and limitations, especially for nascent technologies. Rapid GC–MS is one such emerging analytical technique being increasingly implemented in forensic laboratories due to its fast and informative screening capabilities. However, a full validation for forensic samples has yet to be published since its debut. This work presents the results of a comprehensive validation of a rapid GC–MS system for seized drug screening through the assessment of nine components: selectivity, matrix effects, precision, accuracy, range, carryover/contamination, robustness, ruggedness, and stability. Single- and/or multi-compound test solutions of commonly encountered seized drug compounds were used to assess method and system performance. Results met the designated acceptance criteria for a majority of components. For example, retention time and mass spectral search score % RSDs were ≤10 % for precision and robustness studies. Limitations were identified for components that did not meet the acceptance criteria (e.g., inability to differentiate some isomers). The study design is part of a larger validation package developed for rapid GC–MS that includes a validation plan and automated workbook. The template, available for adoption by laboratories, ultimately aims to reduce the barrier of implementation for rapid GC–MS technology.

Molecular profiling of triacylglycerols in bovine and goat milk powders via ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry

Triacylglycerols (TAG) play several nutritional, technological, and functional roles in the dairy industry, representing approximately 98 % of milk fat. In the present study, the total fatty acid composition and the sn-2 fatty acid positional distribution of bovine and goat milk powders fat were analyzed by using the gas chromatography-flame ionization detector. Furthermore, the identification of TAG molecular species from milk powders was conducted using ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS). The separation of milk TAG was accomplished by using isopropanol/acetonitrile (90:10, v/v) as mobile phase A, and water/acetonitrile (60:40, v/v) as mobile phase B. Myristic, palmitic, stearic, and oleic acids were identified as the most abundant fatty acids making up 11.55 and 10.71 %, 33.23 and 30.64 %, 10.86 and 12.52 %, and 26.74 and 21.80 % in the bovine and goat milk powders, respectively. In bovine milk, 221 molecular species of TAG were identified, with the predominant molecular species (LaCyO, CaLaPa, CyPaM, OCaCa, CaGCy, GLaCo) detected at a retention time of 6.362 min, with a relative abundance of 8.28 %. In goat milk, 202 molecular species were identified, with the major molecular species (CaLaM, CaPCa, CaCyS, CaCoAra, CaBuBe, LaPCy, LaCoS, LaBuAra, MPCo, MCyM, MBuS, PBuP, CyCoBe) detected at a retention time of 6.106 min, making up 8.14 % of the total TAG detected. These results provide valuable insights into milk fat composition for the dairy sector, aiding in the development of specialized dairy products.

Determination of ursolic acid in ethanolic extract of Rotheca serrata (L.) Steane & Mabb. by HPLC method

Rotheca serrata (L.) Steane & Mabb. a plant having significant medicinal value is used in treating a fever, rheumatism, inflammation, pain, and malarial fever. Ursolic acid has a five-ringed pentacyclic structure belongs to the family of triterpenoids. It has antioxidant, antitumor, and hepato-protective properties. The method in HPLC was developed on a SHIMADZU, Shimpack ODS (25 cm × 4.6 mm, 5 µm) column. The mobile phase consisted of a 70:25:5 (v/v/v) mixture of methanol, acetonitrile, and 0.02 M ammonium acetate buffer (pH 3.5). A flow rate of 1.2 mL/min and λmax of 215 nm. Ursolic acid from the extract and standard was eluted at the same retention time of 4.1 min. 20–120 µg/mL was the linearity range chosen and was found to be linear (R 2=0.996). The value of LOD was 0.85 µg/mL and LOQ was 1.96 µg/mL. The method for analysis of ursolic was found to be accurate with good repeatability.

A Systematic Methodology for the Identification of the Chemical Composition of the Mongolian Drug Erdun-Uril Compound Utilizing UHPLC-Q-Exactive Orbitrap Mass Spectrometry.

The traditional Mongolian medicine Erdun-Uril is a conventional combination of 29 herbs commonly used for the treatment of cerebrovascular ailments. It has the effects of reducing inflammation, counteracting oxidative stress, and averting strokes caused by persistent cerebral hypoperfusion. Prior research on Erdun-Uril has predominantly concentrated on its pharmacodynamics and mechanism of action; however, there has been a lack of systematic and comprehensive investigation into its chemical constituents. Therefore, it is crucial to establish an efficient and rapid method for evaluating the chemical constituents of Erdun-Uril. In this study, Erdun-Uril was investigated using UHPLC-Q-Exactive Orbitrap MS combined with parallel reaction monitoring for the first time. Eventually, a total of 237 compounds, including 76 flavonoids, 68 phenolic compounds, 19 alkaloids, 7 amino acids, etc., were identified based on the chromatographic retention time, bibliography data, MS/MS2 information, neutral loss fragments (NLFs), and diagnostic fragment ions (DFIs). And of these, 225 were reported for the first time in this study. This new discovery of these complex components would provide a reliable theoretical basis for the development of pharmacodynamics and quality standards of the Mongolian medicine Erdun-Uril.

Leveraging Strengths of green chromatography in Developing eco-friendly HPLC and HPTLC methods for the simultaneous analysis of Lamivudine, Dolutegravir and tenofovir alafenamide

The development of green analytical methodologies is a critical challenge in the field of pharmaceutical analysis. The ternary mixture investigated in this study consists of the antiretroviral drugs Lamivudine (LMV), Tenofovir alafenamide fumarate (TAF), and Dolutegravir (DTG), which are all approved antiretroviral drugs for the treatment of HIV-1 infection. While LMV was approved in 1995, DTG and TAF were more recently approved in 2013 and 2016, respectively, as newer generation HIV medications with improved efficacy and safety profiles compared to earlier antiretroviral drugs. This study presents three eco-friendly approaches for the simultaneous determination of the ternary mixture. Two HPLC methods were applied: an organic-solvent free mixed-micellar HPLC technique was developed as (method A), using a Cyano column (150 × 4.6 mm,5.0 µm) with a mobile phase consisting of 0.03 M Brij-35, 0.09 M sodium dodecyl sulfate, and 0.02 M ammonium acetate in water, adjusted to pH 3.0. Green solvents were applied in (method B), namely, phosphate buffer 25 mM adjusted to pH 3.0 and ethanol (45:55, v/v) using an Inertsil-C18 column (250 × 4.6 mm,5.0 µm,). Peaks were detected at 258 nm, and the retention times for (method A) were 3.10, 3.95 and 5.03 min, and for (method B) were 2.53, 3.63 and 4.86 min for LMV, TAF and DTG, respectively. Additionally, a green thin-layer chromatographic (TLC) method was developed using a water-isopropanol-formic acid mobile phase (65/35/10,v/v/v) and RP-18 silica gel 60 HPTLC-plates. The drugs were successfully resolved, with retardation factor (Rf) values of 0.26, 0.54 and 0.82 for DTG, TAF, and LMV, respectively. The novel methods were validated and applied successfully in marketed dosage forms. Their greenness profiles were assessed using two recent metrics.

48 Dietary functional fiber properties for improved health and disease outcomes in pigs

Abstract In swine production, using feed antibiotics as antimicrobial growth promotants has been reduced; thus, feed alternatives to manage gut health are required. Dietary fiber and similar carbohydrate structures such as resistant starch, oligosaccharides, and exopolysaccharides are nutritional tools that may enhance gut health in pigs. Dietary fiber may have a role to alleviate diarrheal diseases including in pigs post-weaning but can also reduce constipation in sows, but specific functionality depends on fiber properties. Antibiotics are hypothesized to influence gut health via modulation of intestinal microbial profiles; fermentation and intestinal inflammation are considered important mechanisms. Dietary fiber sources differ in a key functional property: fermentability. Rapid fermentation of fiber and oligosaccharides is associated with changes in microbial profiles and increased metabolite production. Recently, microbial composition was hypothesized to be less important and combined output of metabolites should be the focus. Fiber properties may also manipulate retention time and physico-chemical properties of the undigested residue; hence, non-fermentable fiber may prevent constipation in sows. The fiber-like structure resistant starch is not digested and acts as fermentable fiber but has unique properties, because it specifically increases intestinal abundance of bifidobacteria, which are associated with improved gut health. Using crop breeding, feed processing, and feed additives, the structure of fiber and starch can be altered thereby changing its degradation kinetics and affecting its prebiotic activity. Finally, fiber-like structures such as exopolysaccharides from Limosilactobacillus reuteri and unique oligosaccharides may serve as receptor analogues for pathogenic bacteria, e.g., enterotoxigenic Escherichia coli (ETEC). These receptor analogues block lectin domains of bacterial adhesins and thus prevent adherence of pathogens to the gut wall, thereby avoiding initiation of post-weaning diarrhea. In conclusion, dietary fiber and similar structures possess important functional properties that may also provide important solutions to maintain gut health in pigs.

491 Processing and stomach pH influenced in vitro gastric protein solubilization kinetics in sunflower meal

Abstract The study aimed to evaluate the effect of processing and pH on gastric protein solubilization kinetics in sunflower meal (SFM) products using porcine digestion simulation model. The SFM samples were derived from diverse processing approaches: 1) dehulled-cold pressed (SFM_CP), 2) dehulled-solvent-extracted (SFM_SE), and 3) application of electrostatic separation technology on SFM_SE to generate high crude protein (CP) version (SFM_SEP). Soybean meal (SBM) was included for reference comparison (Table 1). Considering the complex factors influencing retention time, gastric pH, and pepsin secretion, the study employed multiple sampling times, and pH levels. A total of 500 mg CP equivalent of each sample (in triplicates) was mixed with 25 mL of 0.1 M phosphate buffer (pH 6.0) and 2 mL of pepsin (27 mg/mL). The pH was adjusted to either 1.5, 2.5, 3.5 or 4.5 using 1 M HCl and placed in incubator shaker (200 rpm) at 39°C. Samples were taken at 0, 30, 60, and 90 min, snap-frozen in liquid nitrogen, stored frozen, later thawed at 4°C, centrifuged (15,000 × g for 10 min) and supernatants analyzed for CP. The data were analyzed using Proc GLIMIXX in SAS while Gompertz equation was used to describe the gastric protein solubilization kinetics. The model included the fixed effects of protein source or processing, stomach pH and their interaction on the gastric CP solubility at 90 min and gastric protein solubility kinetics (A-maximum solubilized CP, Wo- initial CP solubility, Ku- solubilization rate, Ti- time to inflection). The analyzed CP SBM, SFM_CP, SFM_SE, and SFM_SEP, were 514.4, 524.4, 393.5, and 484.2 g/kg DM, respectively. Gastric CP solubility increased with lower pH and longer incubation time (P < 0.05), which corresponded well with the optimum pH of pepsin activity around 2. Compared with SFM, SBM had the least (P < 0.05) gastric CP solubility at pH 4.5 and 3.5, and the least predicted maximum gastric CP solubility (A) at pH 4.5, 3.5 and 2.5 (P < 0.05; Table 2). SFM_CP showed the greatest predicted initial gastric CP solubility at 0 h (Wo) and A (P < 0.05). Electrostatic fractionation of SFM_SE (SFM_SEP) enhanced gastric CP solubility at pH 3.5 and 2.5 and reduced the solubility time to inflection point (Ti; P < 0.05). In conclusion, data indicated that cold pressing SFM increased gastric CP solubility relative to solvent extraction. However, electrostatic fractionation of SFM_SE improved protein gastric solubility. While SBM remains a standard, incorporating the studied sunflower meals with their fast CP digestion kinetics in feed formulation could improve protein utilization in young animals with high stomach pH. In vivo studies, particularly on ileal amino acid digestibility, are crucial to validate these findings. Sunflower meal tables_FMN.pdf

First demonstration of 2T0C-FeDRAM: a-ITZO FET and double gate a-ITZO/a-IGZO FeFET with a record-long multibit retention time of >4-bit and >2000 s.

Conventional DRAM, consisting of one transistor and one capacitor (1T1C), requires periodic data refresh processes due to its limited retention time and data-destructive read operation. Here, we propose and demonstrate a novel 3D-DRAM memory scheme available with a single transistor and a single ferroelectric field-effect transistor (FeFET) DRAM (2T0C-FeDRAM), which offers extended retention time and non-destructive read operation. This architecture uses a back-end-of-line (BEOL)-compatible amorphous oxide semiconductor (AOS) that is suitable for increasing DRAM cell density. Notably, the device structures of a double gate a-ITZO/a-IGZO FeFET, used for data storage and reading, are engineered to achieve an enlarged memory window (MW) of 1.5 V and a prolonged retention time of 104 s. This is accomplished by a double gate and an a-ITZO/a-IGZO heterostructure channel to enable efficient polarization control in hafnium-zirconium oxide (HZO) layers. We present successful program/erase operations of the double gate a-ITZO/a-IGZO FeFET through incremental step pulse programming (ISPP), demonstrating multi-level states with remarkable retention characteristics. Most importantly, we perform 2T0C-FeDRAM operations by electrically connecting the double gate a-ITZO/a-IGZO FeFET and the a-ITZO FET. Leveraging the impressive performance of the double gate a-ITZO/a-IGZO FeFET technology, we have effectively showcased an exceptionally record-long retention time exceeding 2000 s and 4-bit multi-level states, positioning it as a robust contender among emerging memory solutions in the era of artificial intelligence.

Experimental characterization and prediction of Escherichia coli host cell proteome retention during preparative chromatography.

Purification of recombinantly produced biopharmaceuticals involves removal of host cell material, such as host cell proteins (HCPs). For lysates of the common expression host Escherichia coli (E. coli) over 1500 unique proteins can be identified. Currently, understanding the behavior of individual HCPs for purification operations, such as preparative chromatography, is limited. Therefore, we aim to elucidate the elution behavior of individual HCPs from E. coli strain BLR(DE3) during chromatography. Understanding this complex mixture and knowing the chromatographic behavior of each individual HCP improves the ability for rational purification process design. Specifically, linear gradient experiments were performed using ion exchange (IEX) and hydrophobic interaction chromatography, coupled with mass spectrometry-based proteomics to map the retention of individual HCPs. We combined knowledge of protein location, function, and interaction available in literature to identify trends in elution behavior. Additionally, quantitative structure-property relationship models were trained relating the protein 3D structure to elution behavior during IEX. For the complete data set a model with a cross-validated R2 of 0.55 was constructed, that could be improved to a R2 of 0.70 by considering only monomeric proteins. Ultimately this study is a significant step toward greater process understanding.

Isolation and quantification of human urinary exosomes using a Tween-20 elution solvent from polyester, capillary-channeled polymer fiber columns

BackgroundExosomes, a subset of extracellular vesicles (EVs), are a type of membrane-secreted vesicle essential for intercellular communication. There is a great deal of interest in developing methods to isolate and quantify exosomes to study their role in intercellular processes and as potential therapeutic delivery systems. Polyester, capillary-channeled polymer fiber columns and spin-down tips are highly efficient, low-cost means of exosome isolation. As the methodology evolves, there remain questions as to the optimum elution solvent for specific end-uses of the recovered vesicles; fundamental biochemistry, clinical diagnostics, or therapeutic vectors. ResultsWhile both acetonitrile and glycerol have been proven highly successful in terms of EV recoveries in the hydrophobic interaction chromatography workflow, many biological studies entail the use of the non-ionic detergent, Tween-20, as a working solvent. Here we evaluate the use of Tween-20 as the elution solvent for the recovery of exosomes. A novel 10-min, two-step gradient elution method, employing 0.1 % v/v Tween-20, efficiently isolated EVs at a concentration of ∼1011 EV mL−1 from a 100 μL urine injection. Integration of absorbance and multi-angle light scattering detectors in standard HPLC instrumentation enables a comprehensive single-injection determination of eluted exosome concentration and sizes. Transmission electron microscopy verifies the retention of the vesicular structure of the exosomes. The micro-bicinchoninic acid protein quantification assay confirmed high-purity isolations of exosomes (∼99 % removal of background proteins) SignificanceThe effective use of Tween-20 as an elution solvent for exosome isolation/purification using capillary-channeled polymer fiber columns adds greater versatility to the portfolio of the approach. The proposed method holds promise for a wide range of fundamental biochemistry, clinical diagnostics, and therapeutic applications, marking a significant advancement in EV-based methodologies.

Chromatographic fingerprinting of epiphytic fungal strains isolated from Withania somnifera and biological evaluation of isolated okaramine H.

Medicinal plants are "goldmines" of natural products, and continue to provide key scaffolds for drug development. They have immense therapeutic potential, encapsulating a plethora of metabolites within them, which have yet to be explored. Withania somnifera (L.) Dunal is one such medicinal plant known since time immemorial for its therapeutic activity in the Ayurveda system of medicine. Studies have revealed Nature's marvel of these medicinal plants harbouring endophytic and epiphytic microorganisms from phyllosphere to rhizosphere. Chromatographic fingerprinting was carried out using HPTLC and HPLC on five epiphytic strains isolated from the leaves, stem and fruits of Withania somnifera. Out of five filamentous fungi, one fungus identified as Aspergillus aculeatus S20 was well explored. An indole alkaloid, okaramine H, was isolated using systematic chromatographic investigation at a retention time of 26.278 min showing λmax at 206, 236, 284 and 370 nm. Confirmation was achieved using NMR and mass spectrometry (MS) as analytical techniques. Structure elucidation was done by studying the fragmentation pattern using MS/MS and an accurate mass was determined using HR-ESI-QTOF-MS showing m/z of 521.2546 [M + H]+. The percentage purity of isolated okaramine H was found to be >90. Well known for its insecticidal activity, okaramine H was explored for its antileishmanial activity against the Leishmania donovani parasite for the first time. Under in vitro conditions, the compound showed an inhibitory effect on Leishmania donovani promastigotes with an IC50 of 147 μg mL-1.

Degradation of Azo-dye (Disperse Red) Using Rhizosphere Bacterial Consortium

This study investigates the degradation of the azo dye (Disperse Red) using a rhizosphere bacterial consortium. Standard microbiological and molecular techniques were employed to isolate and identify organisms from rhizosphere soil. Degradation of azodye was carried out in a fabricated anoxic and oxic chambers with hydraulic retention time of 40hrs. Initial identification of the bacterial isolates through Gram’s reaction and biochemical tests revealed the presence of organisms belonging to the genera Pseudomonas, Lysinibacillus, and Citrobacter. Molecular and phylogenetic analyses confirmed the isolates as Pseudomonas aeruginosa, Lysinibacillus sphaericus, Pseudomonas chengduensis, and Citrobacter freundii. During the preliminary testing, the degradation efficiency was assessed under varying glucose concentrations. Higher decolorization rate of 56.17% was observed in the medium with 10% glucose after 72 hours, while the medium with 5% glucose achieved a 44.17% colour reduction. Notably, lower degradation rates recorded were 11.96% and 12.85% for the 5% and 10% dye enhance glucose mineral salt media, respectively. However, During the actual degradation testing in a double-chamber system enhanced with biochar, the first anaerobic cycle achieved a maximum decolorization of 71.95% after 94 hours, with the first aerobic cycle further enhancing degradation to 90.51%. The second anaerobic cycle increased degradation to 94.78%, and the final aerobic cycle achieved a decolorization of 98.47%. These results show that the rate of Disperse Red degradation is highly dependent on glucose levels and alternating anaerobic-aerobic conditions. This study demonstrates the potential of using rhizosphere bacterial consortia to bioremediate wastewater contaminated with azo dyes, offering an efficient and sustainable method of environmental management. The results underline the need of optimizing ambient conditions to increase microbial degradation processes.

Carcinomembrane-Camouflaged Perfluorochemical Dual-Layer Nanopolymersomes Bearing Indocyanine Green and Camptothecin Effectuate Targeting Photochemotherapy of Cancer.

Photochemotherapy has been recognized as a promising combinational modality for cancer treatment. However, difficulties such as off-target drug delivery, systemic toxicity, and the hypoxic nature of the tumor microenvironment remain hindrances to its application. To overcome these challenges, cancer cell membrane camouflaged perfluorooctyl bromide (PFOB) dual-layer nanopolymersomes bearing indocyanine green (ICG) and camptothecin (CPT), named MICFNS, were developed in this study, and melanoma was exploited as the model for MICFNS manufacture and therapeutic application. Our data showed that MICFNS were able to stabilize both ICG and CPT in the nanocarriers and can be quickly internalized by B16F10 cells due to melanoma membrane-mediated homology. Upon NIR irradiation, MICFNS can trigger hyperthermia and offer enhanced singlet oxygen production due to the incorporation of PFOB. With ≥10/2.5 μM ICG/CPT, MICFNS + NIR can provide comparable in vitro cancericidal effects to those caused by using an 8-fold higher dose of encapsulated CPT alone. Through the animal study, we further demonstrated that MICFNS can be quickly brought to tumors and have a longer retention time than those of free agents in vivo. Moreover, the MICFNS with 40/10 μM ICG/CPT in combination with 30 s NIR irradiation can successfully inhibit tumor growth without systemic toxicity in mice within the 14 day treatment. We speculate that such an antitumoral effect was achieved by phototherapy followed by chemotherapy, a two-stage tumoricidal process performed by MICFNS. Taken together, we anticipate that MICFNS, a photochemotherapeutic nanoplatform, has high potential for use in clinical anticancer treatment.

Digitally Enabled Generic Analytical Framework Accelerating the Pace of Liquid Chromatography Method Development for Vaccine Adjuvant Formulations.

The growing use of adjuvants in the fast-paced formulation of new vaccines has created an unprecedented need for meaningful analytical assays that deliver reliable quantitative data from complex adjuvant and adjuvant-antigen mixtures. Due to their complex chemical and physical properties, method development for the separation of vaccine adjuvants is considered a highly challenging and laborious task. Reversed-phase liquid chromatography (RPLC) is among the most important tests in the (bio)pharmaceutical industry for release and stability indicating measurements including adjuvant content, identity, and purity profile. However, the time constraints of developing "on-demand" robust quantitative methods prior to each change in formulation can easily lead to sample analysis becoming a bottleneck in vaccine development. Herein, a simple and efficient generic analytical framework capable of chromatographically resolving the most commonly used non-aluminum-based adjuvants across academic and industrial sectors is introduced. This was designed to seek a more proactive approach for fast-paced assay development endeavors that evolved from extensive stationary phase screening in conjunction with multifactorial in silico simulations of adjuvant retention time (RT) as a function of gradient time, temperature, organic modifier blending, and buffer concentration. The multifactorial retention models yield 3D resolution maps with excellent baseline separation of all adjuvants in a single run, which was found to be very accurate, with differences between experimental and simulated retention times of less than 1%. The analytical framework described here also includes the introduction of a more versatile approach to method development by introducing a dynamic RT database for adjuvants covering the entire library of adjuvants with broad mechanisms of action across numerous vaccine formulations with excellent linearity, accuracy, precision, and specificity. The power of this framework was also demonstrated with numerous analytical assays that can be generated rapidly from simulations guiding vaccine processes in the development of new adjuvant formulations. Analytical assay in this work covers content, purity profile by LC with diode array detector (DAD) and charged aerosol detector (CAD), and component identification by LC with mass spectrometry (MS) across complex vaccine formulations, including the use of surfactants (e.g., polysorbates) as well as their separation from adjuvant targets.

Comparative phytochemical, antioxidant, and hemostatic studies of fractions from raw and roasted sea buckthorn seeds in vitro

Various seeds, including sea buckthorn (Hippophae rhamnoides L.) seeds, are sources of different bioactive compounds. They can show anti-inflammatory, hypoglycemic, anti-hyperlipidemic, antibacterial, antioxidant, or other biological properties in in vitro and in vivo models. Our preliminary in vitro results have demonstrated that the extracts from raw (no thermal processing) and roasted (thermally processed) sea buckthorn seeds have antioxidant potential and anticoagulant activity. However, it was unclear which compounds were responsible for these properties. Therefore, in continuation of our previous study, the extracts were fractionated by C18 chromatography. Phytochemical analysis of three fractions (a, b, and c) from raw sea buckthorn seeds and four fractions (d, e, f, and g) from roasted sea buckthorn seeds were performed. Several in vitro assays were also conducted to determine the antioxidant and procoagulant/anticoagulant potential of the fractions and two of their major constituents—isorhamnetin 3-O-β-glucoside7-O-α-rhamnoside and serotonin. LC–MS analyses showed that serotonin is the dominant constituent of fractions c and f, which was tentatively identified on the basis of its HRMS and UV spectra. Moreover, fractions c and f, as well as b and e, contained different B-type proanthocyanidins. Fractions b and e consisted mainly of numerous glycosides of kaempferol, quercetin, and isorhamnetin. The results of oxidative stress assays (measurements of protein carbonylation, lipid peroxidation, and thiol groups oxidation) showed that out of all the tested fractions, fraction g (isolated from roasted seeds and containing mainly dihexoses, and serotonin) demonstrated the strongest antioxidant properties.

Platinum(II) complexes based on derivatives of natural chlorins with pyridine-containing chelate groups as prototypes of drugs for combination therapy in oncology

Objectives. To synthesize Pt-containing derivatives of natural chlorins as potential agents for the combination therapy in oncology. Platinum compounds are known to occupy an important place as chemotherapeutic agents in the treatment of oncological diseases. However, Pt(II) complexes are highly toxic to the body and are not selectively accumulated in tumor cells. If photodynamic and chemotherapy methods are combined in a single drug, the pigments are responsible for the selectivity of conjugate accumulation in the tumor, while a chemotherapeutic agent based on Pt(II) complexes is responsible for the cytotoxic effect on tumor cells. This will not affect healthy cells and thereby minimize the systemic toxicity of the drug to the body.Methods. Methods for the synthesis of pyridine-containing derivatives of natural chlorins and their metal complexes for use as potential binary agents in oncology were applied. As part of the study, the structures of the compounds obtained were confirmed by mass spectrometry, nuclear magnetic resonance spectroscopy, ultraviolet spectroscopy, and high-resolution chromatography-mass spectrometry. Preparative methods, including thin-layer and column chromatography, centrifugation and recrystallization, were used to isolate and purify the compounds obtained.Results. Platinum(II) complexes of pyridine-containing derivatives of natural chlorins were obtained for application in combination therapy in oncology. The schemes for synthesizing the target photosensitizers were optimized, in order to increase the yields and for subsequent transfer to industrial sites.Conclusions. It was found that pyridine-containing derivatives of natural chlorins could be obtained in high yields, that they possess chelating properties for platinum, and can be considered as binary agents in cancer therapy after successful preclinical trials.