Stress Degradation Research Articles

Development and validation of capillary zone electrophoresis and high-performance liquid chromatography methods for the determination of oral anticoagulant edoxaban in pharmaceutical tablets.

The incidence of thrombotic complications in SARS-CoV-2 infections has become a global concern; thus, anticoagulants are an integral part of the treatment. Edoxaban (EDX) is an oral anticoagulant suitable for pharmacologic thromboprophylaxis. Herein, two novel analytical methods for EDX determination in tablets are developed and validated using capillary zone electrophoresis (CZE) and high-performance liquid chromatography (HPLC). Operating conditions such as the electrolyte's concentration and pH value, injection time, volume, and the capillary temperature, were optimized. The methods were successfully validated by establishing the linearity, intra- and inter-day precisions (relative standard deviation [%]), accuracy, and robustness. Adequate separation of excipients and degradation products of EDX generated by stress degradation conditions demonstrated the stability-indicating capability of the methods. The analytical procedures were linear in the range of 25-125µg/ml (r>0.999), with the limits of detection and quantification of 3.26 and 10.87µg/ml for CZE and 0.740 and 2.78µg/ml for HPLC. Although both methodologies are suitable for determining EDX in tablets, CZE provides a greener alternative due to low-cost analysis using less organic solvents and minimizing waste generation.

Elucidating the binding and inhibitory potential of p-coumaric acid against amyloid fibrillation and their cytotoxicity: Biophysical and docking analysis

P-Coumaric acid (p-CA) is a plant metabolite with anti-inflammatory and antioxidant effects. Due to its therapeutic potential, p-CA has attracted much attention from the scientific community lately. Oxidative stress, amyloid formation, and impaired proteasomal degradation are hallmarks of neurodegenerative diseases like Alzheimer's (AD) and are targets for developing therapeutics against such conditions. Here, we have investigated the anti-amyloidogenic properties of p-coumaric acid on hen egg white lysozyme (HEWL). Heat, pH, and agitation (55 °C, pH 2.0, 600 rpm) stress were used to induce amyloid formation in lysozyme. The aggregates characterization was done by turbidity, Rayleigh light scattering (RLS), and thioflavin-T (ThT) assays. Moreover, ANS (1-anilino naphthalene sulphate) binding assay and circular dichroism (CD) were employed to unveil protein hydrophobicity and secondary structure perturbation, respectively. Lysozyme demonstrated increased hydrophobicity and transition of α-helix to β-sheet under aggregating conditions. Moreover, co-incubation of lysozyme with p-coumaric acid attenuates the process of amyloid in a concentration dependent manner. At 50 and 200 μM concentrations of p-coumaric acid, lysozyme retained its native-like folded structure. Cytotoxicity protection on human SK-N-SH neuroblastoma cell line was also observed using MTT assay and phase contrast microscopy. In addition, transmission electron microscopy (TEM) reaffirms the fibrillar nature of lysozyme aggregates and their attenuation by p-coumaric acid. The steady state fluorescence revealed that the mode of fluorescence quenching for the HEWL-p-coumaric acid interaction is static rather than dynamic. Moderate strength of binding in order of 104 M−1 exists between HEWL and p-coumaric acid. Thermodynamic parameters (∆H and ∆S) obtained from van't Hoff plot suggested spontaneous reaction with hydrophobic interaction. A slight micro-environmental change in HEWL around Tyr residue was observed during the binding process with the help of synchronous fluorescence. Molecular docking analysis reported the involvement of amino acid residues (TRP63, LEU75, ASP101, LYS97) to form a complex between HEWL-p-coumaric acid. The observed anti-amyloidogenic and inherent antioxidative properties of p-coumaric acid could be helpful to design a neuroprotective agent.

Phase Separation: "The Master Key" to Deciphering the Physiological and Pathological Functions of Cells.

Phase separation is a hot research field at present. It involves almost all aspects of cells and plays a significant role in cells, promising to be "a master key" in unlocking the mysteries of nature. In this review, the factors that affect phase separation are introduced, such as own component, electrostatic interaction, and chemical modification. Furthermore, the physiological roles of phase separation in cells, including molecules transport channel, gene expression and regulation, cellular division and differentiation, stress response, proteins refolding and degradation, cell connections, construction of skin barrier, and cell signals transmission, are highlighted. However, the disorder of phase separation leads to pathological condensates, which are associated with neurodegenerative diseases, tumors, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This relationship is considered the potential target for developing corresponded drugs and therapy. Some drugs targeting phase separation have improved meaningful, such as tankyrase, lipoamide, oligonucleotides, elvitagravir, nilotinib, CVL218, PJ34. All in all, mystery phase separation provides a new viewpoint for researchers to explore cells, and is expected to solve many unknown phenomena in nature.

Effects of prometryn on cardiac and liver cell lines and mouse tissues

BackgroundPesticides are important biological agents that deter or kill pests. Globally, about 2 million tons of pesticides are being utilized each year which highlights their importance. Previous studies have suggested that high levels of pesticide exposure are associated with increased risk of chronic diseases, including cancers, cardiotoxicity, Parkinson’s disease, diabetes mellitus, neurological deficits, birth defects, and reproductive disorders. Prometryn is a commonly used selective triazine herbicide, which is currently used as an alternative to atrazine‐based products. It is widely applied to agriculture as it has lower acute toxicity than atrazine herbicides, stable chemical properties, and a long period of effectiveness. Although past studies have shown that triazine herbicides have carcinogenic potential in humans, the cytotoxic effects of prometryn on cardiac and hepatic systems are not well investigated or understood. The aim of this study was to determine the effects of prometryn on the heart and liver.MethodsH9c2 cardiac and HepG2 liver carcinoma cells were used to study the in‐vitro effects of prometryn. Cells were treated with varying concentrations of prometryn for 24 h. Cell viability, mitochondrial membrane potential, and reactive oxygen species (ROS) levels were assessed. For animal studies, male mice (10 weeks old) (n=24) were randomized to two treatment groups (control and prometryn). The control group received corn oil while the prometryn treated group were exposed to 185mg/kg b.w. prometryn dissolved in corn oil every 48 hours for 7 days. After a total of 4 doses, mice were euthanized, and the heart and liver tissues were collected. Proteasome and immunoproteasome activity were measured in the heart and liver. Protein expression levels of markers for oxidative stress, apoptosis, and proteasomal degradation such as GSTT2, GSTA1/2, Hsp70, Hsp27, NADPH oxidase, PARP‐1, p53, PSMD12, LMP2, DNP, VU‐1 were detected using immunoblotting. Protein oxidation and polyubiquitination were detected as an indicator of oxidative stress. A paired t‐test was used to assess group differences for protein levels and p values <0.05 were deemed statistically significant.ResultWe found that prometryn had a concentration‐dependent effects on H9c2 and HepG2 cell lines when we assessed cell viability, mitochondria function, and total intracellular reactive oxygen species formation. Increasing the concentration of prometryn increased the ROS formation, decreased cell viability and decreased mitochondrial membrane potential in these cell lines. Furthermore, in animal studies we observed changes in the proteasome and immunoproteasome activities in the liver but not in the heart. Changes in protein expression levels of oxidative stress and proteasomal degradation markers were only detected in the liver.ConclusionThese results indicate that H9c2 and HepG2 cells are sensitive to prometryn in‐vitro. However, prometryn effects on the heart and liver tissue in male mice is significantly different. Overall, our data supports the deduction that prometryn affected mitochondrial function, induced oxidative stress in cells, but also alters the ubiquitin‐proteasome system and increases oxidative stress in mice liver. These results help towards elucidation of the mechanism by which prometryn could cause diseases.

Mitochondrial Uncoupling Decreases Sarcopenic Obesity By Activation of Skeletal Muscle Mitochondrial Quality Control and Attenuated ER Stress

BackgroundSarcopenic obesity is a highly prevalent disease with poor survival and ineffective medical interventions. Mitochondrial dysfunction is purported to be central in the pathogenesis of sarcopenic obesity by impairing both organelle biogenesis and quality control. We have previously identified an orally available mitochondrial‐targeted furazano[3,4‐b]pyrazine named BAM15 that selectively lowers respiratory coupling efficiency and protects against diet‐induced obesity in mice. Here, we tested the hypothesis that mitochondrial uncoupling simultaneously attenuates loss of muscle function and weight gain in a mouse model of sarcopenic obesity.Methods80‐week‐old male C57BL/6J mice with obesity were randomized to 10 weeks of high fat diet (CTRL) or BAM15 (BAM15; 0.1% w/w in high fat diet) treatment. Body composition, muscle function, energy expenditure, and locomotor activity were determined after treatment. Skeletal muscle was harvested and evaluated for histology, gene expression, protein signaling, and mitochondrial structure and function.ResultsBAM15 decreased body weight (~25% reduction, P<0.001) which was attributable to increased energy expenditure (~20% increment, P<0.001). BAM15 increased muscle mass (~13% increment, P<0.001), strength (~37% increment, P<0.0001), and locomotor activity (~25% increment, P<0.001). Improvements in physical function were mediated in part by reductions in skeletal muscle inflammation (IL‐6 and gp130, both P<0.05), enhanced mitochondrial function, and improved endoplasmic reticulum homeostasis and reduced inflammation. Specifically, BAM15 activated mitochondrial quality control through AMPK (PINK1‐ubiquitin binding and LC3II, P<0.01), increased electron transport chain activity (citrate synthase and complex II activity, all P<0.05), restricted endoplasmic reticulum (ER) misfolding (decreased oligomer A11 insoluble/soluble ratio, P<0.0001) while limiting ER stress (decreased PERK signaling, P<0.0001), apoptotic signaling (decreased cytochrome C release and Caspase‐3/9 activation, all P<0.001), and muscle protein degradation (decreased 14‐kDa actin fragment insoluble/soluble ratio, P<0.001).ConclusionsMitochondrial uncoupling agents such as BAM15 may mitigate age‐related decline in muscle mass and function by molecular and cellular bioenergetic adaptations that confer protection against sarcopenic obesity through activation of mitochondrial quality control and attenuation of ER stress.

Fluid Resuscitation Aggravates the Cellular Injury in a Hemorrhagic Shock Model

Background: Resuscitation is the initial step for hemorrhagic shock. However, there is still controversy as to which fluid achieves the best results clinically and experimentally. Aim: It was aimed to investigate the effects of 0.9% NaCl (sodium chloride) and 6% HES (hydroxyethyl starch) on the kidney and blood environment. Methods: Twenty-four male Wistar rats were assigned as control, shock, and resuscitated (colloid: 6% HES and crystalloid: 0.9% NaCl) groups. Besides hemodynamics (mean arterial pressure and shock index) monitoring and kidney function evaluation, hemolysis, oxidative stress, inflammation, and glycocalyx degradation were evaluated in the plasma and kidney. Results: (1) Macrohemodynamics were successfully restored by both fluids. (2) Although 3 times more crystalloid volume was applied compared to the colloid resuscitation, similar hematocrit levels were found in both resuscitation strategies (32.8 ± 2.3 vs. 33.3 ± 1.0). (3) NaCl resuscitation led to increases in the hemolytic index, catalytic iron, and sialic acid compared to control, while HES administration increased the levels of malondialdehyde, ischemia-modified albumin, and sialic acid. (4) However, both fluid resuscitation strategies could inhibit inflammation and oxidative stress in the kidney and restore kidney function parameters. Conclusion: Although both NaCl and HES resuscitation showed protection of the kidney function against oxidative stress and inflammation, these fluids initiated the injury process.

Identification and characterization of major degradation products of Eprinomectin drug substance including degradation pathways using LC-HRMS and NMR

Eprinomectin (EPM) is a semi-synthetic potent antiparasitic drug widely used in veterinary medicine. In this study, a comprehensive forced degradation study was carried out on EPM drug substance as per ICH guidelines. Generation of adequate quantities of major degradation products of EPM via forced degradation studies was necessary for identification, structure elucidation, and understanding its degradation mechanism and degradation pathways. EPM drug substance was subjected to acid, base, oxidation (H2O2 and K2Cr2O7), thermal (solid and solution state), and photolytic (solid and solution state) stress degradation. The degradation products (DPs) formed in the stressed degraded samples were successfully separated using a gradient elution on a HALO C18 column (100 × 4.6 mm, 2.7 µm). Mobile phase A consisted of water and mobile phase B consisted of ethanol/isopropanol (98/2, v/v). A total of six major DPs of EPM drug substance formed under various stress conditions. The chemical structures of DPs were determined using liquid chromatography-high resolution mass spectrometry (LC-HRMS) and characterized through comparison of their fragmentation profile with EPM B1a using tandem mass spectrometry (MS/MS). Additionally, two solvates (methanol adduct B1a #1 and methanol adduct B1a #2) were observed during the acid-stressed degradation study of EPM in presence of methanol. To confirm the chemical structure, these products were isolated with semi-preparative HPLC and characterized by using a combination of LC-MS/MS and nuclear magnetic resonance spectroscopy. The elucidated chemical structure of the degradation products of EPM was also justified through mechanistic explanations. Identification and characterization of the DPs including degradation mechanism(s) of EPM should facilitate the understanding of the stability behavior of EPM drug substances as well as aid in the design of new formulations made with EPM.

Proposal of steel stress-strain relationships and simple analytical models of beams considering strain-rate effects at elevated temperatures

PurposeIn this study, engineering stress-strain relationships considering an effect of strain rate on steel materials at elevated temperatures were formulated and a simplified analytical model using a two-dimensional beam element to analytically examine the effect of strain rate on the load-bearing capacity and collapse temperature was proposed.Design/methodology/approachThe stress-strain relationships taking into account temperature, strain, and strain rate were established based on the past coupon test results with strain rate as the test parameter. Furthermore, an elasto-plastic analysis using a two-dimensional beam element, which considered the effect on strain rate, was conducted for both transient- and steady-state conditions.FindingsThe analytical results agreed relatively well with the test results, which used small steel beam specimens with a rectangular cross-section under various heating rates (transient-state condition) and deformation rates (steady-state condition). It was found that the bending strength and collapse temperature obtained from the parametric analyses agreed relatively well with those evaluated using the effective strength obtained from the coupon tests with strain equal to 0.01 or 0.02 under the fast strain rates.Originality/valueThe effect of stress degradation, including the stress-strain relationships at elevated temperature, was mitigated by considering the effect of strain rate on the analytical model. This is an important point to consider when considering the effect of strain rate on steel structural analysis at elevated temperatures to maintain analytical stability unaccompanied by the stress degradation.

Protective Effect of Djulis (Chenopodium formosanum) Extract against UV- and AGEs-Induced Skin Aging via Alleviating Oxidative Stress and Collagen Degradation.

Skin aging is a complex process involving photoaging and glycation stress, which share some fundamental pathways and have common mediators. They can cause skin damage and collagen degradation by inducing oxidative stress and the accumulation of reactive oxygen species (ROS). Chenopodium formosanum (CF), also known as Djulis, is a traditional cereal in Taiwan. This study investigated the protection mechanisms of CF extract against ultraviolet (UV) radiation and advanced glycation end products (AGEs)-induced stress. The results indicated that CF extract had strong antioxidant and free radical scavenging effects. It could reduce UV-induced intracellular ROS generation and initiate the antioxidant defense system by activating the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathway in human skin fibroblasts. CF extract modulated mitogen-activated protein kinase (MAPK) and transformed growth factor-beta (TGF-β) signaling pathways to alleviate oxidative stress-induced skin aging. Moreover, the results revealed that CF extract not only promoted collagen synthesis but also improved aging-induced collagen degradation. CF extract attenuated AGEs-induced ROS production and the upregulation of receptor for AGEs (RAGE). The overall results suggest that CF extract provides an effective anti-aging strategy by preventing skin damage from oxidative stress and collagen loss with potent antioxidant, anti-photoaging, and antiglycation activities.

Study of erdafitinib stress degradation behavior using HPLC-UV analysis and multistage fragmentation ion trap mass spectrometry

Erdafitinib is a fibroblast growth factor receptor inhibitor, which has been recently approved for the treatment of urothelial carcinoma. The stress degradation behavior of erdafitinib was examined using a validated stability-indicating high-performance liquid chromatography method. The study was extended to investigate the fragmentation pathways of erdafitinib using ion trap mass spectrometry. Six degradation products and an impurity were characterized depending on their multistage fragmentation pattern. The degradation products formed under different stress conditions showed molecular masses of 443, 477, 433, 479, 463, and 459. The molecular mass of the detected impurity was 525. The drug and its related substances were efficiently separated on an Eclipse Plus C18 column (4.6 × 100 mm, 3.5 µm). An isocratic mobile phase consisting of acetonitrile and 0.01 M ammonium formate in water containing 0.1% formic acid (26:74, v/v) was used at a flow rate of 0.5 mL/min. Erdafitinib was exposed to oxidative, photolytic, acidic, basic, and thermal hydrolysis as recommended by ICH guidelines. The drug was found to be more sensitive to oxidative hydrolysis and slightly sensitive to acidic and photolytic hydrolysis. The method was fully validated according to ICH guidelines. The limit of detection and quantitation were 0.2 and 0.5 µg/mL, respectively. The relative standard deviations of intraday and interday precisions were not more than 1.99%. The developed method was successfully applied for the determination of erdafitinib in bulk and laboratory-prepared tablets.

A new stability indicating RP-UFLC method for the estimation of Etoricoxib in pharmaceutical dosage forms

Etoricoxib is a selective COX-2 inhibitor and used as a non-steroidal anti-inflammatory drug. A new validated stability indicating RP-UFLC method has been developed for the estimation of Etoricoxib in pharmaceutical dosage forms (Tablets). Shimadzu UFLC system (PDA detector) with Agilent C18 column was chosen for the chromatographic study with mobile phase Acetonitrile: water (50: 50) and flow rate 0.8 mL/min (UV detection at 230 nm). Beer-Lambert’s law was obeyed over a concentration range 0.1-100 µg/mL. The linear regression equation was found to be y = 82642x -13032 (R2 = 0.9998). The LOD and LOQ was found to be and 0.0291 and 0.0894 µg/mL respectively. Stress degradation studies were performed and the method was validated as per ICH guidelines. The proposed method is simple, precise, accurate and robust and can be applied for the quantification of Etoricoxib formulations.

Mitochondrial uncoupling attenuates sarcopenic obesity by enhancing skeletal muscle mitophagy and quality control.

BackgroundSarcopenic obesity is a highly prevalent disease with poor survival and ineffective medical interventions. Mitochondrial dysfunction is purported to be central in the pathogenesis of sarcopenic obesity by impairing both organelle biogenesis and quality control. We have previously identified that a mitochondrial‐targeted furazano[3,4‐b]pyrazine named BAM15 is orally available and selectively lowers respiratory coupling efficiency and protects against diet‐induced obesity in mice. Here, we tested the hypothesis that mitochondrial uncoupling simultaneously attenuates loss of muscle function and weight gain in a mouse model of sarcopenic obesity.MethodsEighty‐week‐old male C57BL/6J mice with obesity were randomized to 10 weeks of high fat diet (CTRL) or BAM15 (BAM15; 0.1% w/w in high fat diet) treatment. Body weight and food intake were measured weekly. Body composition, muscle function, energy expenditure, locomotor activity, and glucose tolerance were determined after treatment. Skeletal muscle was harvested and evaluated for histology, gene expression, protein signalling, and mitochondrial structure and function.ResultsBAM15 decreased body weight (54.0 ± 2.0 vs. 42.3 ± 1.3 g, P < 0.001) which was attributable to increased energy expenditure (10.1 ± 0.1 vs. 11.3 ± 0.4 kcal/day, P < 0.001). BAM15 increased muscle mass (52.7 ± 0.4 vs. 59.4 ± 1.0%, P < 0.001), strength (91.1 ± 1.3 vs. 124.9 ± 1.2 g, P < 0.0001), and locomotor activity (347.0 ± 14.4 vs. 432.7 ± 32.0 m, P < 0.001). Improvements in physical function were mediated in part by reductions in skeletal muscle inflammation (interleukin 6 and gp130, both P < 0.05), enhanced mitochondrial function, and improved endoplasmic reticulum homeostasis. Specifically, BAM15 activated mitochondrial quality control (PINK1‐ubiquitin binding and LC3II, P < 0.01), increased mitochondrial activity (citrate synthase and complex II activity, all P < 0.05), restricted endoplasmic reticulum (ER) misfolding (decreased oligomer A11 insoluble/soluble ratio, P < 0.0001) while limiting ER stress (decreased PERK signalling, P < 0.0001), apoptotic signalling (decreased cytochrome C release and Caspase‐3/9 activation, all P < 0.001), and muscle protein degradation (decreased 14‐kDa actin fragment insoluble/soluble ratio, P < 0.001).ConclusionsMitochondrial uncoupling by agents such as BAM15 may mitigate age‐related decline in muscle mass and function by molecular and cellular bioenergetic adaptations that confer protection against sarcopenic obesity.

Deferoxamine Alleviates Osteoarthritis by Inhibiting Chondrocyte Ferroptosis and Activating the Nrf2 Pathway.

Objective: Osteoarthritis (OA) is a common disease with a complex pathology including mechanical load, inflammation, and metabolic factors. Chondrocyte ferroptosis contributes to OA progression. Because iron deposition is a major pathological event in ferroptosis, deferoxamine (DFO), an effective iron chelator, has been used to inhibit ferroptosis in various degenerative disease models. Nevertheless, its OA treatment efficacy remains unknown. We aimed to determine whether DFO alleviates chondrocyte ferroptosis and its effect on OA and to explore its possible mechanism. Methods: Interleukin-1β (IL-1β) was used to simulate inflammation, and chondrocyte ferroptosis was induced by erastin, a classic ferroptosis inducer. A surgical destabilized medial meniscus mouse model was also applied to simulate OA in vivo, and erastin was injected into the articular cavity to induce mouse knee chondrocyte ferroptosis. We determined the effects of DFO on ferroptosis and injury-related events: chondrocyte inflammation, extracellular matrix degradation, oxidative stress, and articular cartilage degradation. Results: IL-1β increased the levels of ROS, lipid ROS, and the lipid peroxidation end product malondialdehyde (MDA) and altered ferroptosis-related protein expression in chondrocytes. Moreover, ferrostatin-1 (Fer-1), a classic ferroptosis inhibitor, rescued the IL-1β–induced decrease in collagen type II (collagen II) expression and increase in matrix metalloproteinase 13 (MMP13) expression. Erastin promoted MMP13 expression in chondrocytes but inhibited collagen II expression. DFO alleviated IL-1β– and erastin-induced cytotoxicity in chondrocytes, abrogated ROS and lipid ROS accumulation and the increase in MDA, improved OA-like changes in chondrocytes, and promoted nuclear factor E2–related factor 2 (Nrf2) antioxidant system activation. Finally, intra-articular injection of DFO enhanced collagen II expression in OA model mice, inhibited erastin-induced articular chondrocyte death, and delayed articular cartilage degradation and OA progression. Conclusion: Our research confirms that ferroptosis occurs in chondrocytes under inflammatory conditions, and inhibition of chondrocyte ferroptosis can alleviate chondrocyte destruction. Erastin-induced chondrocyte ferroptosis can stimulate increased MMP13 expression and decreased collagen II expression in chondrocytes. DFO can suppress chondrocyte ferroptosis and promote activation of the Nrf2 antioxidant system, which is essential for protecting chondrocytes. In addition, ferroptosis inhibition by DFO injection into the articular cavity may be a new OA treatment.

Development of the Validated Stability-Indicating Method for the Determination of Vortioxetine in Bulk and Pharmaceutical Formulation by HPLC-DAD, Stress Degradation Kinetics Studies and Detection of Degradation Products by LC-ESI-QTOF-MS.

Vortioxetine (VOR) is a new antidepressant drug used to treat major depressive disorder. In this work, a novel, simple, rapid, accurate, precise, selective, stability-indicating, and fully validated high-performance liquid chromatography method with diode array detection (HPLC-DAD) was developed to determine VOR in bulk and pharmaceutical formulations. A Polar-RP column was used, with a mobile phase consisting of acetonitrile (ACN), methanol (MeOH), acetate buffer pH 3.5, and addition of diethylamine (DEA) in the isocratic elution mode. Assessing the stability of the VOR is fundamental to guarantee the efficacy, safety, and quality of drug products. In this study, the VOR active pharmaceutical ingredient (API) and tablets were subjected to a detailed study of forced degradation, using several degrading agents (acid, alkaline, water, heat, light, and oxidation agents). The developed HPLC-DAD method allows the collection of all the essential data to determine degradation kinetics. It was found that the decomposition of vortioxetine is fragile towards oxidative conditions and photolysis, yielding the first-order and second-order kinetic reaction in the above stress conditions, respectively. The degradation products (DPs) were identified by the high-resolution liquid chromatography coupled with electrospray ionization-quadrupole-time of flight-mass spectrometry (LC-ESI-QTOF-MS) method. The HPLC-DAD method was successfully applied for the quantification of VOR in tablets. Additionally, in silico toxicity prediction of the DPs was performed.

Ameliorative effect of gel combination of Centella asiatica extract transfersomes and rosemary essential oil nanoemulsion against UVB-induced skin aging in Balb/c mice

Background: Ultraviolet B (UVB) radiation induces physiological and morphological photoaging of the skin resulting in wrinkles, and loss of elasticity. This study analyzed nanoencapsulation of a gel combination of Centella asiatica (CA) transfersomes and rosemary essential oil (REO) nanoemulsion with lipid-based nanocarriers for the ability of both biological compounds to synergistically prevent UVB radiation, along with ameliorative and anti-aging effects. Methods: To ensure the quality, lipid-based nanocarriers of transfersomes and nanoemulsion were characterized based on physicochemical properties such as particle size distribution, polydispersity index, zeta potential. In vivo studies were used to determine the biological effects of a gel combination of CA transfersomes, and REO nanoemulsion applied topically two weeks before UVB radiation (840 mJ/cm2) in BALB/c hairless mice. Results: Results showed that the optimum lipid-based nanocarriers had a particle size of 43.97 ± 5.6 nm, a polydispersity index of 0.64 ± 0.01, and a zeta potential of -10.91 ± 1.99 mV. In vivo experiments revealed that topical application of a gel combination of CA transfersomes and REO nanoemulsion significantly ameliorated wrinkle formation, epidermal hyperplasia, and collagen fiber arrangement caused by UVB exposure. Further, the gel combining CA transfersomes and REO nanoemulsion suppressed lipid peroxidation by decreasing the expression of malondialdehyde (MDA) and collagen destruction by inhibiting matrix metalloproteinase-9 (MMP-9) expression. Moreover, the gel combination of CA transfersomes and REO nanoemulsion upregulated type I collagen through activation of the transforming growth factor-β (TGF-β)/Smad pathway, thereby recovering the density of collagen fiber reduced by UVB radiation. Conclusions: Overall, these data indicate that topical application of a gel combination of CA transfersomes and REO nanoemulsion could act synergistically and potentially prevents oxidative stress and collagen degradation in the skin from UVB-induced photoaging.

Mesenchymal Stem Cell-Derived Extracellular Vesicles: Immunomodulatory Effects and Potential Applications in Intervertebral Disc Degeneration.

Intervertebral disc (IVD) degenerative disease is a common health problem worldwide. Administration of mesenchymal stem cells (MSCs) in intervertebral disc degeneration (IVDD) has been widely explored in recent years. However, transplantation of MSCs is restricted by several factors. Currently, paracrine signaling is one of the main mechanisms by which MSCs play a therapeutic role in disc regeneration. Extracellular vehicles (EVs) are the main paracrine products of MSCs. They show great potential as an effective alternative to MSCs and play immunomodulation roles such as anti-inflammatory effects, antioxidative stress, antiapoptosis, and antiextracellular matrix (ECM) degradation during treatment of IVDD. This review focuses on the immunomodulatory effect of MSC EVs and their potential applications.

Force degradation studies of relugolix: identification, isolation and structure characterization of stress degradation products by using liquid chromatography-mass spectrometry, auto purification mass mediated preparative-high performance liquid chromatography, high resolution mass spectrometry, nuclear magnetic resonance spectroscopy

To assess the stability of Relugolix under stress conditions, identify and characterize the degradation products, it was subjected to hydrolytic and oxidative stress conditions, according to the International Council for Harmonization guideline Q1A (R2). The drug showed degradation in basic and oxidative conditions, while it was stable in acid conditions. Four degradation products were obtained, two from each basic and oxidative degradation, which was separated on a C18 column by using Auto Purification Mass Spectrometer. The four degradant products have not been reported earlier in any of the literature and are being reported here for the first time. HIGHLIGHTS Present work captures force degradation of Relugolix under acidic, basic, oxidative, photolytic, and thermal conditions as per International Council for Harmonization guidelines. Two degradation products were observed in basic condition marked as Degradation Product-1, Degradation Product-2 and two degradation products were formed in oxidative condition marked as Degradation Product-3, Degradation Product-4. All the four degradants are novel and are not published in any of the literature. Four degradation products were identified and characterized using various high-end analytical techniques such as Liquid Chromatography and Mass Spectrometer, Reversed Phase–High Performance Liquid Chromatography, High Resolution Mass Spectrometer and Nuclear Magnetic Resonance spectroscopy.

Tetrandrine reduces oxidative stress, apoptosis, and extracellular matrix degradation and improves intervertebral disc degeneration by inducing autophagy

ABSTRACT Tetrandrine (TET) was reported to be an autophagy agonist, and the activating autophagy could delay intervertebral disc degeneration (IDD). Our study focused on exploring whether TET attenuated tert butyl hydrogen peroxide (TBHP)-induced nucleus pulposus (NP) cell injury and delayed rat IDD by inducing autophagy. In vitro, cytotoxicity was detected by MTT assay, ROS was measured with DCFH-DA probe, MDA, and SOD content was evaluated through ELISA, NP cell apoptosis was tested by flow cytometry, protein expression was detected by Western blot, in particular, LC3 expression was assessed by immunofluorescence. In vivo, pathological changes were estimated by HE and safranin-O staining, related protein expression was measured by immunohistochemistry, and the apoptosis was detected by TUNEL. Compared with the control group, oxidative stress, apoptosis, and extracellular matrix (ECM) degradation were increased, the expression of cleaved caspase-3,9, aggrecan and collagen II were reduced, and the expression of MMP13 and ADAMTS5 were up-regulated in TBHP-treated NP cells. Moreover, TET could reverse the effect of TBHP on NP cells. Further, TET enhanced autophagy in NP cells by amplifying the LC3 II/LC3 I/ratio and reducing p62 expression, which attenuated oxidative stress, apoptosis, and ECM degradation in TBHP-treated NP cells. In addition, in vivo, TET delayed rat IDD, increased the expression of LC3 and collagen II, and weakened apoptosis. TET inhibited oxidative stress, apoptosis, and ECM degradation in TBHP-treated NP cells by inducing autophagy, and alleviated IDD. These indicated that TET might be a potential candidate drug for the treatment of IDD.

A stability indicating method development and validation of esomeprazole in pharmaceutical dosage form by using RP-HPLC and In Vitro evaluation of nasogastric tube delivery of esomeprazole magnesium delayed-release capsules

A simple, selective and well-defined stability indicating method was developed for the quantitative estimation of esomeprazole in tablet dosage form using XBridge BEH Shield RP18 (4.6 x 250 mm), 5μm with phosphate buffer pH 7.3 and acetonitrile (740:260 %v/v) as a mobile phase and successfully validated as per the ICH guideline. The method was found to be specific, linear, accurate, rugged, and robust. Stress degradation studies were performed by exposing the esomeprazole magnesium delayed release capsules into acidic, alkaline, oxidative, thermal, humidity and photolytic stress conditions as per ICH guidelines. In separate in-vitro experiments, esomeprazole pallets dispersion passed through feeding tubes using gentle syringe pressure to develop a clog-free dispersion-delivery method. Nasogastric tube (8-French [Fr]) and diluents (different pH of water used i.e. pH 5.5, 7.0 and 8.5) were tested. The results showed excellent delivery of esomeprazole pellets using water as a medium for tube delivery. Recovery of esomeprazole pallets dispersion in different pH of water i.e. pH 5.5, 7.0 and 8.5 at “0 and 15” minutes incubation time were nearly 100% in 8-Fr nasogastric tubes. Keywords: Esomeprazole, Stress degradation, Stability indicating assay method, Nasogastric Tube.

A stability indicating method development and validation of apixaban in pharmaceutical dosage form by using RP-HPLC and In-Vitro evaluation of apixaban suspension delivery through enteral feeding tubes

A simple, consistent and sensitive stability indicating reversed phase high performance liquid chromatography (RP-HPLC) method was developed for the determination of Apixaban in pharmaceutical dosage form by using Zorbax C18 (150 mm x 4.6 mm, 5μm particle size) with a mobile phase consisting of Ammonium Formate Buffer: Acetonitrile in a ratio of 65:35% v/v and successfully validated as per the international council for harmonization (ICH) guideline. The method was found to be simple, robust, precise, sensitive, accurate; specific and stress degradation studies were performed with acidic, alkaline, oxidative, thermal, humidity and photolytic stress conditions as per ICH guidelines. In separate in-vitro experiments, Apixaban suspension passed through feeding tubes to develop a clog-free suspension delivery method. Nasogastric tube (8-French [Fr]) and diluents (water, 5% dextrose in water) were tested. Recovery of Apixaban Suspension in water and 5% dextrose in water at “0 and 15” minutes incubation time were nearly 100% in 8-Fr nasogastric (NG) tubes. Keywords: Apixaban, Stress Degradation, Stability Indicating Assay Method, Nasogastric Tube.