Protein Denaturation Research Articles

Development of a selective COX-2 inhibitor: from synthesis to enhanced efficacy via nano-formulation.

Non-steroidal anti-inflammatory drugs NSAIDs are widely used for managing various conditions including pain, inflammation, arthritis and many musculoskeletal disorders. NSAIDs exert their biological effects by inhibiting the cyclooxygenase (COX) enzyme, which has two main isoforms COX-1 and COX-2. The COX-2 isoform is believed to be directly related to inflammation. Based on structure-activity relationship (SAR) studies of known selective COX-2 inhibitors, our aim is to design and synthesize a novel series of 2-benzamido-N-(4-substituted phenyl)thiophene-3-carboxamide derivatives. These derivatives are intended to be selective COX-2 inhibitors through structural modification of diclofenac and celecoxib. The compound 2-benzamido-5-ethyl-N-(4-fluorophenyl)thiophene-3-carboxamide VIIa demonstrated selective COX-2 inhibition with an IC50 value of 0.29 μM and a selectivity index 67.24. This is compared to celecoxib, which has an IC50 value of 0.42 μM and a selectivity index 33.8. Molecular docking studies for compound VIIa displayed high binding affinity toward COX-2. Additionally, the suppression of protein denaturation with respect to albumin was performed as an indicative measure of the potential anti-inflammatory efficacy of the novel compounds. Compound VIIa showed potent anti-inflammatory activity with 93% inhibition and an IC50 value 0.54 μM. In comparison, celecoxib achieved 94% inhibition with an IC50 value 0.89 μM. Although molecule VIIa demonstrated significant in vitro anti-inflammatory activity, adhered to Lipinski's "five rules" (RO5) and exhibited promising drug-like properties, it showed indications of poor in vivo activity. This limitation is likely due to poor aqueous solubility, which impacts its bioavailability. This issue could be addressed by incorporating the drug in niosomal nanocarrier. Niosomes were prepared using the thin-film hydration technique. These niosomes exhibited a particle size of less than 200 nm, high entrapment efficiency, and an appropriate drug loading percentage. Transmission electron microscopy (TEM) and differential scanning calorimetry (DSC) studies revealed that the niosomes were spherical and demonstrated compatibility of all of its components. The drug release study indicated that the pure drug had limited practicality for in vivo use. However, incorporating the drug into niosomes significantly improved its release profile, making it more suitable for practical use.

Bioactive phytocompounds profiling and the evaluation of analgesic, anti-inflammatory, and antihyperglycemic potential of Argyreia capitiformis (Poir.) Ooststr.: A combined in vitro, in vivo, and computational investigations

Ethnopharmacological relevanceArgyreia capitiformis (Poir.) Ooststr. (Convolvulaceae) is traditionally used by the Chakma community in the hilly region of Bangladesh to treat minor disorders such as pain. Aim of the studyThis study intended to determine the secondary metabolites to identify bioactive compounds and evaluate antioxidant potential, in vitro anti-inflammatory and in vivo analgesic, anti-inflammatory, and antihyperglycemic activities of A. capitiformis along with in silico investigations. Materials and methodsChemical profiling was carried out using HPLC and GC-MS analysis. The analgesic effect was measured employing tail immersion and acetic acid-induced writhing methods. Following protein denaturation and formalin-induced paw edema, anti-inflammatory activity was studied. The antihyperglycemic potential was assessed using an oral glucose tolerance test (OGTT), while further mechanistic investigation was conducted using an alpha-glucosidase enzyme inhibitory assay. Simulations and molecular docking analyses were performed to ascertain the stability and binding affinities of the drug-protein complex. ResultsA. capitiformis ethanolic extract confirmed the presence of phenolics, alkaloids, flavonoids, terpenoids, tannins, gums, steroids, and reducing sugars. HPLC analysis revealed the presence of eight polyphenolic compounds, the most abundant of which was myricetin (64.10 ± 0.14 mg per 100 g dry extract). Moreover, the GC-MS analysis revealed twenty-four molecules, the most important of which was 2,4-bis (dimethylbenzyl)-6-t-butylphenol (9.19%). The concentrations of total flavonoids, total terpenoids, total phenolics, and total tannins were ascertained to be 142.48 mg QE/g, 173.1 mg UAE/g, 19.35 mg GAE/g, and 13.05 mg GAE/g, respectively. Furthermore, the plant extract had a total antioxidant capacity of 388 mg AAE/g. In the writhing assay, the plant extract suppressed writhing by 59.73% and 76.99% at the doses of 250 and 500 mg/kg, respectively, compared to the standard diclofenac Na 87.17%, and in the tail immersion assay, the plant extract displayed a maximum average reaction time of 1.94 and 2.40 s at the doses of 250 and 500 mg/kg, respectively as compared to the control tramadol 2.84 s at 60 min. In an in vitro anti-inflammatory assay, the plant extract possessed an IC50 of 95.51 μg/ml while diclofenac Na (standard drug) was found to be 69.50 μg/ml. Afterward, in vivo anti-inflammatory activity was observed in mice over a period, particularly after 3 h, the plant extract exerted maximum percent inflammation inhibitions of 36.36% and 45.45% at the doses of 250 and 500 mg/kg, respectively whereas ibuprofen the standard drug (100 mg/kg) exhibited 61.82%. The plant extract demonstrated antihyperglycemic activity, lowering blood sugar levels to 5.7 and 4.62 mM at doses of 250 and 500 mg/kg, respectively, as opposed to 8.58 mM in the control group. Meanwhile, the standard drug glibenclamide (5 mg/kg) dropped blood glucose levels to 2.38 mM in 60 min after glucose administration. Molecular docking (MD) and molecular dynamics simulation (MDS) studies support the stability of the protein complex responsible for exerting pharmacological activities. ConclusionA. capitiformis extract exhibited strong medicinal values supporting its traditional uses.

Chemical profiling and bioactivity analysis of shoots and roots essential oil of Indian Blumea mollis D. Don (Merr.)

Aromatic plants contain essential oils, potent extracts renowned for therapeutic benefits. Essential oils are commonly utilized due to their diverse range of phytochemicals that possess therapeutic properties. The present study analyses the chemical composition of the essential oil obtained from shoots and roots of Blumea mollis (Family: Asteraceae), using GC-FID and GC-MS techniques. A total of 49 and 46 compounds were identified from shoots and roots accounting for 95.6 % and 88.4 % respectively. β-caryophyllene is reported as major compound 36.2 ± 0.50 % in essential oil of the shoots and 33.8 ± 0.55 % in essential oil of the roots. Both essential oils of shoots and roots were dominated by sesquiterpene hydrocarbons (59.2–76.4%), followed by oxygenated sesquiterpene (14.9–20.3%). The in vitro antioxidant activity was determined using DPPH radical scavenging activity, H2O2 radical scavenging activity, and Iron(II) complexing activity. The highest antioxidant activity was observed in root essential oil of B. mollis. The oils also inhibited the activity of α-amylase with IC50 of 3.53 ± 0.04 μg/ml (shoot), 3.45 ± 0.04 μg/ml (root). Root oil also showed good activity for protein denaturation with IC50 of 3.02 ± 0.03 g/ml as compared to shoot essential oil. This is first time that essential oil constituent and biological activity of B. mollis roots and shoots have been characterized from India.

Fluorescent Protein PEGylation for Stable Photon Manipulation in Deep‐Red Light‐Emitting Devices

AbstractPEGylation is a classic strategy to reduce denaturation and immunogenicity in therapeutic proteins, bioimaging, and drug delivery. However, this concept has not been applied to incorporate biogenic materials as functional components in optoelectronics. Herein, PEGylation is rationalized as an effective tool to stabilize fluorescent proteins in solid‐state photon manipulation down‐converters integrated into bio‐hybrid light‐emitting diodes. In short, the archetypal red‐emitting protein mCherry is nonspecifically PEGylated with methoxypolyethylene glycols (mPEG) chains of different lengths (mPEG‐350/‐750/‐2000). These derivatives hold the same photoluminescence figures‐of‐merit of mCherry, but an improved resistance against organic solvents, pH, temperature, and polymers (in solution/dry‐coatings) upon increasing the mPEG length. Indeed, the best‐performing mCherry‐mPEG‐2000 adduct leads to deep‐red devices with threefold enhanced color stability (>300 h) under harsh operation conditions (150 mW cm−2) over the prior art. Different device architectures along with spectroscopic, thermocycling, and electrochemical impedance spectroscopy studies of the prepared coatings aid in rationalizing that PEGylation successfully reduces i) nonreversible thermal denaturation, ii) aggregation in solid‐state, and iii) photo‐induced oxidation and H+‐transfer deactivation of the chromophore, since oxygen diffusivity and H+‐reorganization across the protein skeleton are slowed down by strong H+‐bonding/hydrophobic mCherry‐mPEG interactions. Hence, this simple supramolecular modification is of utmost relevance for future advances in protein‐based optoelectronics.

UV photochemistry of the L-cystine disulfide bridge in aqueous solution investigated by femtosecond X-ray absorption spectroscopy

The photolysis of disulfide bonds is implicated in denaturation of proteins exposed to ultraviolet light. Despite this biological relevance in stabilizing the structure of many proteins, the mechanisms of disulfide photolysis are still contested after decades of research. Herein, we report new insight into the photochemistry of L-cystine in aqueous solution by femtosecond X-ray absorption spectroscopy at the sulfur K-edge. We observe homolytic bond cleavage upon ultraviolet irradiation and the formation of thiyl radicals as the single primary photoproduct. Ultrafast thiyl decay due to geminate recombination proceeds at a quantum yield of >80 % within 20 ps. These dynamics coincide with the emergence of a secondary product, attributed to the generation of perthiyl radicals. From these findings, we suggest a mechanism of perthiyl radical generation from a vibrationally excited parent molecule that asymmetrically fragments along a carbon-sulfur bond. Our results point toward a dynamic photostability of the disulfide bridge in condensed-phase.

Cryoprotective effect of magnetic field-assisted freezing in combination with curdlan on myofibrillar protein of Litopenaeus vannamei: Oxidative aggregation, protein structure and thermal stability

To counteract the negative effects of conventional freezing methods on frozen surimi products, the present study investigated the effects of magnetic field (MF) freezing in combination with different amounts of curdlan (CUR) on oxidative denaturation, structural alterations, and thermal stability of myofibrillar protein (MP) in shrimp surimi. The results indicated that the intervention of magnetic fields alone improved the quality of frozen muscle proteins. Under the dual intervention of magnetic field-assisted freezing and CUR, small ice crystals formed with MF6 treatment resulted in significantly lower solubility, turbidity, and mean particle size than the control group (p < 0.05). Moreover, the oxidation of MP was also significantly slowed down by inhibiting the formation of hydrophobic groups and carbonyl groups, maintaining a high content of sulfhydryl groups. MF6 also exhibited a high Ca2+-ATPase activity. The α-helix content, the increase in fluorescence intensity under this condition also tend to make the secondary and tertiary structures of myofibrillar protein more stable. Meanwhile, electrophoretic profiles and CLSM showed that this condition maintains the integrity of the MP. In addition, the MF6 samples also had high protein thermal stability. However, excess CUR reduces the excellence of MP cryoprotection. As a result, MF6 has better protection and improvement effects on protein function, structure and thermal stability, and CUR was also found to have the potential for cryoprotectant development.

Synergistic effect of zinc oxide-cinnamic acid nanoparticles for wound healing management: in vitro and zebrafish model studies

Wound infections resulting from pathogen infiltration pose a significant challenge in healthcare settings and everyday life. When the skin barrier is compromised due to injuries, surgeries, or chronic conditions, pathogens such as bacteria, fungi, and viruses can enter the body, leading to infections. These infections can range from mild to severe, causing discomfort, delayed healing, and, in some cases, life-threatening complications. Zinc oxide (ZnO) nanoparticles (NPs) have been widely recognized for their antimicrobial and wound healing properties, while cinnamic acid is known for its antioxidant and anti-inflammatory activities. Based on these properties, the combination of ZnO NPs with cinnamic acid (CA) was hypothesized to have enhanced efficacy in addressing wound infections and promoting healing. This study aimed to synthesize and evaluate the potential of ZnO-CN NPs as a multifunctional agent for wound treatment. ZnO-CN NPs were synthesized and characterized using key techniques to confirm their structure and composition. The antioxidant and anti-inflammatory potential of ZnO-CN NPs was evaluated through standard in vitro assays, demonstrating strong free radical scavenging and inhibition of protein denaturation. The antimicrobial activity of the nanoparticles was tested against common wound pathogens, revealing effective inhibition at a minimal concentration. A zebrafish wound healing model was employed to assess both the safety and therapeutic efficacy of the nanoparticles, showing no toxicity at tested concentrations and facilitating faster wound closure. Additionally, pro-inflammatory cytokine gene expression was analyzed to understand the role of ZnO-CN NPs in wound healing mechanisms. In conclusion, ZnO-CN NPs demonstrate potent antioxidant, anti-inflammatory, and antimicrobial properties, making them promising candidates for wound treatment. Given their multifunctional properties and non-toxicity at tested concentrations, ZnO-CN NPs hold significant potential as a therapeutic agent for clinical wound management, warranting further investigation in human models.

Cannabidiol finds dihydrocannabidiol as its twin in anti-inflammatory activities and the mechanism

Ethnopharmacological relevanceThe hemp (cataloged at the “Medicinal Plant Names Services” as Cannabis sativa L.) extracts, cannabinoids have been used for centuries in Southeast Asia as folk medicines and now authorized by about 50 countries for application in medicine, health care products and cosmetics. As the most consumed cannabinoid, cannabidiol (CBD) has been recognized due to its various bioactivities, including anti-inflammatory and antibacterial properties. Aims of the studyThe utilization of CBD is limited due to its potential conversion to psychoactive Δ9-tetrahydrocannabinol in strong acidic environment, demanding to excavate safer alternatives with clarified bioactivities. Yet the anti-inflammatory and antibacterial properties of CBD still remain unknown, in both of the performances and the corresponding mechanisms. Previously, a synthetic CBD analogue, H2CBD (Dihydrocannabidiol) was found to be effective as CBD does towards some antioxidantive activities and mouse seizure mitigation. Therefore, it is wondering if H2CBD also acted similarly as CBD does in the aspect of anti-inflammatory performance and mechanism, and the safety. Material and methodsThe anti-inflammatory properties of CBD and H2CBD were revealed with enzymatic assays, proteins denaturation and lipopolysaccharide (LPS) stimulated RAW264.7 cells model, with epigallocatechin gallate (EGCG) as the positive control. Their anti-inflammatory mechanism was revealed with ELISA and Western blot assay. The antibacterial properties of CBD and H2CBD were also investigated towards E. faecalis and B. cereus along with their synergistic effect with commercial antibiotics. ResultsCBD and H2CBD exhibited almost same (P > 0.05) performance in all the assayed anti-inflammatory properties, yet their anti-inflammatory efficiencies positively correlated to their antioxidantive activity. Moreover, both of CBD and H2CBD presented anti-inflammation to LPS stimulated RAW264.7 cells through NF-κB and AKT pathway. Furthermore, CBD and H2CBD also supplied strong and very similar (P > 0.05) antibacterial activities, comparable to tetracycline in same dose and strength. The erythrocyte hemolytic assay indicates CBD and H2CBD possessing the same safety. All the combinations of H2CBD with other cannabinoids or antibiotics present no antagonism against the bacteria, but nice synergistic or additive effect in some cases. ConclusionCBD and H2CBD presented very similarly in all the assayed anti-inflammatory performances, undergoing same inflammatory mechanism with NF-κB and AKT pathway; they also expressed similar antibacterial activity, like twins. These findings will supply CBD a sustainable, safer and economic alternative with same excellent performances.

Green-synthesized carbon dots from ginger: Multifunctional agents against oral pathogens with biocompatibility in human gingival fibroblast cells

Persistent antibiotic use in treating oral infections often leads to drug resistance in pathogenic bacteria, notably impacting conditions like periodontitis. Addressing this challenge, the study pioneers the use of carbon dots (CDs) synthesized from ginger rhizomes (Zingiber officinale) as a novel biocompatible material. CDs were synthesized via the hydrothermal method, emphasizing a green approach, and comprehensively characterized for their optical properties and structural uniformity. The synthesized CDs showed a zeta potential of −24.9 mV, confirming the formation of stable and well-dispersed particles. Dynamic Light Scattering (DLS) confirmed an average particle size of 2.9 nm, thus validating the formation of CDs. Biomedical assessments demonstrated that the synthesized CDs were non-cytotoxic to human gingival fibroblast cell lines, with effective free radical scavenging activity and high total antioxidant capacity, as indicated by their IC50 values. CDs also exhibited moderate inhibition of protein denaturation compared to the standard. Moreover, they showed significant inhibitory effects against bacterial strains (Pseudomonas aeruginosa, Lactobacillus acidophilus, Escherichia coli, Staphylococcus aureus) and fungal strains (Aspergillus niger, Candida albicans) at minimal concentrations. Notably, CDs inhibited the growth of periodontal pathogens including Aggregatibacter actinomycetemcomitans, Tannerella forsythia, Porphyromonas gingivalis, and Prevotella intermedia. These findings underscore the potential of CDs as multifunctional agents possessing anti-inflammatory, antifungal, antioxidant, and antibacterial properties. Remarkably, they offer a promising alternative to conventional antibiotics, potentially revolutionizing oral healthcare. Their proven biocompatibility and potent bioactivity underscore their innovative potential in biomedical research. Future studies should further assess their efficacy in vivo to fully harness their clinical potential.

Polyphenolic Profile and In vitro, In Silico Study of Stem Extracts from J. Maritimus (Juncaceae) Harvested from Eastern Algeria as Potential Anti-Inflammatory and Antioxidant Agents.

Juncus maritimusan extremophilous microorganism speciesknown for its medicinal properties was collected in the Biskra region with the aim of its valorization. The volume size of polyphenols, flavonoids and condensed tannins was performed using the Folin-Ciocalteu method, aluminum trichloride and vanillin respectively. The volume of Polyphenols was 127.73±0.20 μg EAG/mg ES, the volume offlavonoids was 16.42±0.42 μg EQ/mg ES, and the volume ofcondensed tannins was 10.10±0.35 μg EC/mg ES. The results of the antioxidant activity tests using the DPPH and ABTS methods reveal that the ethyl acetate extract had the highest activity in both tests. The results of the in vitro anti-inflammatory activity, using the BSA protein denaturation assay, showed that the percentage of denaturation inhibition wasproportional to the concentration of the extract. At a concentration of 5 mg/mL, the inhibition percentage of the extract were 82.03 % and 80.23 %, respectively,which wereclose tothose of theanti-inflammatory drug Diclofenac.Furthermore, molecular docking simulations indicated that Berberine has high binding affinity to the targets COX-2 and PLA-2 In fact,bioisosteric replacement is being usedto discover new analogs of Berberine. Finally, ADME-Tox predictions demonstrated that this compound and its analogs were not hepatotoxic. This result may lead to the selection ofBerberine and its analogs as active compounds with anti-inflammatory and antioxidant effects.

Evaluation of the in vitro toxicity and anti-inflammatory activity of the methanolic extract of the leaves of Pistacia lentiscus L. harvested from northwestern Algeria.

Medicinal and aromatic plants have been used for thousands of years for their therapeutic properties, to treat various ailments and maintain health. This study was carried out to test the in vitro toxicity and anti-inflammatory activity of the methanolic extract of the leaves of Pistacia lentiscus L., native to the north-western region of Algeria. Toxicity was studied in vitro by the hemolysis test and anti-hemolytic activity on human red blood cells, while anti-inflammatory activity was assessed in vitro by the protein denaturation method. The study of toxicity by hemolysis of red blood cells showed a high rate of hemolysis at a dose of 200 mg/mL, with a hemolytic percentage of around 88.64%, while a high rate of anti-hemolysis was observed at a dose of 25 mg/mL, with an inhibition percentage of around 83.17%. Evaluation of anti-inflammatory activity revealed a high percentage of inhibition of ovalbumin denaturation, reaching 63.53% at the 2000 ug/mL dose, while the lowest percentage was obtained at the 3500 ug\mL concentration with 10.16%. This extract has a toxic substance but does not exceed the toxicity threshold which achieves significant anti-inflammatory activity with depandant doses.

Antifungal and Antibacterial Potential of Essential Oil of Boswellia sacra Resin in the Biological Control

&lt;p&gt;&lt;span lang="EN-US" style="font-size:12.0pt"&gt;&lt;span style="background:white"&gt;&lt;span style="line-height:115%"&gt;&lt;span style="font-family:&amp;quot;Times New Roman&amp;quot;,&amp;quot;serif&amp;quot;"&gt;This study focuses on extraction of Boswillia sacra resin essential oil that was performed using the hydrodistillation method, resulting in a yield of 2.45%. Phytochemical screening of the essential oil provided information about its major compounds, which include terpenes and anthocyanins. To assess the antioxidant and antimicrobial effects of the essential oil, multiple methods were employed. Positive results were obtained from tests such as DPPH and ABTS free radical scavenging, inhibition of bovine serum albumin protein denaturation, and antifungal and antibacterial assays. These tests confirmed the presence of antifungal, antibacterial and antioxidant activities and &lt;/span&gt;&lt;/span&gt;&lt;/span&gt;&lt;/span&gt;&lt;span lang="EN-US" style="font-size:12.0pt"&gt;&lt;span style="line-height:115%"&gt;&lt;span style="font-family:&amp;quot;Times New Roman&amp;quot;,&amp;quot;serif&amp;quot;"&gt;suggest numerous biotechnological applications of the essential oil.&lt;/span&gt;&lt;/span&gt;&lt;/span&gt;&lt;/p&gt;

Ion-Pairing Propensity in Guanidinium Salts Dictates Their Protein (De)stabilization Behavior.

Since the proposition of the Hofmeister series, guanidinium (Gdm) salts hold a special mention in protein science owing to their contrasting effect on protein(s) depending on the counteranion(s). For example, while GdmCl is known to act as a potential protein denaturant, Gdm2SO4 offers minimal effect on protein structure. Despite the fact that theoretical studies reckon the formation of ion-pairing to be responsible for such behavior, experimental validation of this hypothesis is still in sparse. In this study, we combine electrochemical impedance spectroscopy (EIS) and THz spectroscopy to underline the effect of GdmCl and Gdm2SO4 on a model amide molecule N-methylacetamide (NMA). Molecular dynamics (MD) simulation studies predict that Gdm2SO4 forms heteroion pairing in water, which inhibits Gdm+ ions to approach NMA molecules, while in case of GdmCl, Gdm+ ions directly interact with NMA. The experimental findings on ion hydration, specifically the detailed analysis of the ion-water rattling mode, which appears in the THz frequency domain, unambiguously endorse this hypothesis. Our study establishes the fact that the propensity of ion-pairing in Gdm salts dictates their (de)stabilization effect on proteins.

Assessment of the impact of pasteurisation on protein denaturation in sheep sweet whey by asymmetrical flow field-flow fractionation according to sampling period

Whey recovery is limited in artisanal cheese manufacture. This by-product of cheese manufacture was traditionally considered as waste and is highly polluting. However, interest in the nutritional properties of native whey and its proteins has recently increased. Pretreatment is required to preserve highly perishable whey for processing. Only a few studies have focused on sheep whey, despite its considerable nutritional and technological potential. Here, we investigated the effect of heat treatment on the denaturation and formation of protein aggregates in sweet sheep whey collected in January, April and July. Two pasteurisation protocols were studied (72 °C for 1 min and 80 °C for 15 s). Microbiological quality was assessed by checking for the presence of microbes after five days of storage at 4 °C. Asymmetrical flow field-flow fractionation (AF4) coupled to UV detector, multiangle light scattering (MALS) and refractometer (dRi) is a mild, non-naturing technique for determining the extent of denaturation of whey proteins and the size of whey protein aggregates. Greater whey protein denaturation and larger whey protein aggregates were observed for the 80 °C/15 s protocol than for the 72 °C/1 min protocol, for the January and April samples. A decrease in Immunoglobulin G (IgG) content was observed after heat treatments in the samples from July but not significantly for the other proteins. Moreover, the retention times of the monomeric whey protein peaks on the AF4 fractogram were higher for this period, indicating that the proteins were larger. Microbiological testing showed that both pasteurisation treatments were sufficiently effective to ensure good sanitary quality. The pasteurisation schedule best preserving native proteins was heating at 72 °C for 1 min.

Influence of β-lactoglobulin and κ-casein genotype on whey protein denaturation in heated bovine milk and rennet whey

Whey proteins provide nutritional and functional properties in many dairy products, and their denaturation induced by heat has drawn attention in dairy research and industry. Denatured whey proteins have functional properties that are beneficial for certain applications, such as yogurt, while uncontrolled denatured protein aggregation can impair certain processes, such as rennet coagulation. The possible effect of genetic polymorphism of β-lg and κ-CN on heat-induced denaturation of whey proteins in milk and in rennet whey was investigated in this study. No effect of κ-CN genotype on the denaturation of β-lg and α-lac was detected, while the A variant of β-lg was more heat-stable than the B variant. More denatured β-lg B was shown to interact with casein micelles to form insoluble aggregates compared to the A variant. These results may provide guidance for selection of specific milk genotypes for applications where whey protein denaturation needs to be minimised or else preferred, such as foaming and emulsification.

Investigating the Anti-Inflammatory Activity of Various Brown Algae Species.

This literature review investigated the anti-inflammatory properties of brown algae, emphasizing their potential for dermatological applications. Due to the limitations and side effects associated with corticosteroids and immunomodulators, interest has been growing in harnessing therapeutic qualities from natural products as alternatives to traditional treatments for skin inflammation. This review explored the bioactive compounds in brown algae, specifically looking into two bioactive compounds, namely, fucoidans and phlorotannins, which are widely known to exhibit anti-inflammatory properties. This review synthesized the findings from various studies, highlighting how these compounds can mitigate inflammation by mechanisms such as reducing oxidative stress, inhibiting protein denaturation, modulating immune responses, and targeting inflammatory pathways, particularly in conditions like atopic dermatitis. The findings revealed species-specific variations influenced by the molecular weight and sulphate content. Challenges related to skin permeability were addressed, highlighting the potential of nanoformulations and penetration enhancers to improve delivery. While the in vivo results using animal models provided positive results, further clinical trials are necessary to confirm these outcomes in humans. This review concludes that brown algae hold substantial promise for developing new dermatological treatments and encourages further research to optimize extraction methods, understand the molecular mechanisms, and address practical challenges such as sustainability and regulatory compliance. This review contributes to the growing body of evidence supporting the integration of marine-derived compounds into therapeutic applications for inflammatory skin diseases.

Effects of spray drying and freeze drying on the protein profile of whey protein concentrate.

Whey protein concentrate (WPC) is consumed for its high protein content. The structure and biological functionality of whey proteins in WPC powders may be affected by the drying technique applied. However, the specific impact of spray drying and freeze drying on the overall protein profile of whey protein derived from sweet whey streams at scale is unknown. Herein, we examine the effects of commercial-scale freeze drying and spray drying on WPC to determine which method better preserves bioactive whey proteins, with the goal of helping the dairy industry create high-value products that meet the growing consumer demand for functional dairy products. WPCs were produced from pasteurized liquid whey using either a commercial spray dryer or freeze dryer. A variety of analytical techniques, including enzyme-linked immunosorbent assay, polyacrylamide gel electrophoresis, and bottom-up proteomics using liquid chromatography-tandem mass spectroscopy were used to identify, quantify, and compare the retention of bioactive proteins in WPC before and after spray drying and freeze drying. In addition, the extent of denaturation was studied via solubility testing, differential scanning calorimetry, and hydrophobicity assessment. There was little to no difference in the retention or denaturation of key bioactive proteins between spray-dried and freeze-dried WPC powders. There was a higher percentage of select Maillard modifications in freeze-dried and spray-dried powders than in the control. The lack of significant differences between spray drying and freeze drying identified herein indicates that freeze drying does not meaningfully improve retention of bioactive proteins compared with spray drying when performed after multiple pasteurization steps. PRACTICAL APPLICATION: This study aimed to provide insight into the impacts of spray drying versus freeze drying on whey proteins. Overall, our results indicate that for commercial dairy processing that involves multiple rounds of pasteurization, freeze drying does not meaningfully improve the retention of bioactive proteins compared with spray drying. These findings may help the food and dairy industry make informed decisions regarding the processing of its whey protein products to optimize nutritional value.

B-162 Evaluation of two commercially available stable isotope labeled whole-protein infliximab as internal standard for a clinical infliximab LC-MS/MS assay

Abstract Background Liquid chromatography tandem mass spectrometry (LC-MS/MS) based quantitation of infliximab has been successfully introduced in the clinical laboratory for therapeutic drug monitoring in recent years. Although stable isotope labeled (SIL)-peptide or non-labeled analog protein internal standards have been utilized, SIL-whole protein infliximab is generally considered to be the ideal internal standard for the correction of variations during sample preparation and analysis. In this study, we compared two commercially available SIL infliximab proteins, Sigma SILu™MAb and Promise Proteomics, for use in a clinical infliximab assay. Methods Both labeled infliximab proteins incorporate [13C6, 15N4]-arginine and [13C6, 15N2]-lysine in their sequence and are designed for use as an internal standard for LC-MS/MS based quantitative analysis of infliximab. For the comparison of their performance, a laboratory-developed LC-MS/MS clinical method was utilized. Linearity at 6 levels in triplicate with Remicade and 3 biosimilars (-abda, -axxq, and -dyyb), intra-assay precision at 2 levels (n=10/level), and method comparison (n=15) experiments were performed using each labeled infliximab as the internal standard. For sample preparation, internal standard was mixed with the serum sample, followed by protein precipitation with ammonium sulfate, protein denaturation, and tryptic digestion. The peptides YASESMSGIPSR (YASE) from infliximab and YASESMSGIPSR [13C6, 15N4] (SIL-YASE) from labeled infliximab were monitored by LC-MS/MS (Transcend TLX and TSQ Atlis, Thermo Scientific) and utilized for the quantitation of infliximab. Results For the linearity experiment (1-50 µg/mL), while utilizing the Promise Proteomics internal standard to assay had an accuracy range of 103.4% to 114.1% for Remicade, 100.6% to 108.2% for Infliximab-axxq, 103.0% to 110.9% for Infliximab-abda, 102.7% to 108.3% for Infliximab-dyyb. For the Sigma SILu™Mab SIL infliximab, the accuracy range was 92.1% to 99.6% for Remicade, 94.5% to 107.2% for Infliximab-axxq, 99.0% to 104.2% for Infliximab-abda, and 105.1% to 112.0% for Infliximab-dyyb. The mean overall error for Remicade and biosimilars was 0.203 µg/mL (2.0%) for Sigma SILu™Mab SIL infliximab and 0.193 µg/mL (2.0%) for Promise Proteomics internal standardization. Intra-day precision using each SIL infliximab was evaluated with Remicade at low and high levels (5 and 20 µg/mL). Using Promise Proteomics, the coefficient of variation (CV) was 2.8 and 2.2%, while using Sigma SILu™Mab the precision was 6.8 and 6.5% for the low and high levels, respectively. The regression analysis for the method comparison experiment had a correlation coefficient (r) of 0.9996 with a slope of 1.050 (95% confidence interval, 1.030-1.070) and intercept of -0.318 (95% confidence interval, -0.842 to 0.205). Conclusions The mean overall error for the linearity experiment was the same using Sigma SILu™Mab or Promise Proteomics as the internal standard. The Promise Proteomics product demonstrated a lower intra-assay CV compared to the Sigma SILu™Mab. The 2 products displayed a strong correlation during the method comparison experiment. Both Sigma SILu™Mab and Promise Proteomics labeled standards showed acceptable performance as an internal standard for LC-MS/MS based quantitative analysis of infliximab. Having both products available increases the robustness of LC-MS/MS clinical assays in the event of supply issues.

A comparative analysis of antioxidant, anti-inflammatory, and anticancer activities of the crude and fractionated extracts from Distichochlamys benenica

Abstract Distichochlamys benenica is a newly discovered plant belonging to the Distichochlamys family, which exerts many important biological activities. However, the anti-cancer, anti-inflammatory, and anti-oxidant effects of the crude (ME) and fractioned extracts of D. benenica, such as hexane (HE), chloroform (CE), and ethyl acetate extracts (EAE), have not been elucidated yet. The antioxidant activity of the extracts was evaluated via DPPH free radical scavenging and phosphomolybdate assays. The anti-inflammatory potential of the extracts was assessed via their inhibitory effects on protein denaturation and nitric oxide production. The anti-cancer effect of the extracts against A549 and MDA-MB-231 cell lines was determined via MTT assay. The results revealed that among four extracts, the polyphenol concentration was highest in EAE (350.59 ± 13.26 mg GAE/g) while the flavonoid content was highest in HE and ME. Furthermore, the greatest DPPH radical capture efficiency was demonstrated by EAE (89.76 ± 0.68%), and total antioxidant activity was greatest in HE (204.18 ± 3.51 mg AAE/g). The greatest suppression of NO generation was similarly shown by EAE and HE, with IC50 values ranging from 3.72-4.06 µg/mL, respectively. Additionally, HE and EAE exhibited an inhibitory effect on protein denaturation (ranging from 12.76%-28.81%). HE and EAE had the greatest effectiveness against MDA-MB-231 cell lines in the antitumor test. These findings validate the potent antioxidant, anti-inflammatory, and anti-cancer properties of D. benenica extracts, especially EAE and HE, and pave the way for the application of the extracts in the pharmaceutical industry as promising anti-inflammatory and anti-cancer medicines.

Revolutionizing nutrition: Exploring the transformative impact of amaranth integration on composite millet flakes

This study focuses on the development and comparative analysis of multi-millet flakes comprising amaranth, finger millet, and foxtail millet. Two drying methods, baking and dehydration, were employed to produce the flakes. Physicochemical, structural, and thermal properties were evaluated, along with changes observed during soaking in various media. Baked flakes exhibited larger dimensions and a more porous internal structure compared to dried flakes. Baking induced gelatinization and protein denaturation, enhancing water holding capacity and resulting in lower water activity (0.24) conducive to long-term storage. Dehydrated flakes had slightly higher water activity (0.41) and exhibited higher color values with a glossy outer surface. Flakes soaked in hot milk, milk, and water displayed distinct structural properties, crystallinity shifts, and thermal degradation patterns. The nutritional analysis revealed a significant carbohydrate content (55 %), moderate protein (11 %), and notable levels of fat (4.4 %) and total dietary fiber (6.88 %). Fatty acid profiling indicated low levels of saturated, monounsaturated, and polyunsaturated fats, with no trans-fat or cholesterol. Sensory evaluation favored baked flakes, indicating higher acceptability. This research underscores the potential of multi-millet flakes as a nutritious and palatable food option.