Selective simultaneous ultra-performance liquid chromatographic quantification of some benzodiazepines drug residues in pharmaceutical industrial wastewater

Purpose: To investigate the sensitivity and selectivity of ultra-performance liquid chromatographic (UPLC) quantification of bromazepam (BRZ) and diazepam (DZP) in pharmaceutical industrial wastewater. Methods: Wastewater samples were collected from the effluents of a pharmaceutical industrial plant producing BRZ and DZP in tablet dosage forms. The quantification of BRZ and DZP was done after their solid-phase extraction. The resolution process was performed on WatersTM column as the stationary phase. The mobile phase was acetonitrile: methanol: 0.05 M phosphate buffer (pH 6.5), at a volume ratio of 5:2:3, with a flow rate of 0.7 mL/min. Detection was carried out at 240 nm in a concentration range of 10 – 250 ng/mL. The method was fully validated in line with ICH-Q2B regulations. Results: The UPLC method was validated for the quantification of BRZ and DZP. The relative percentage recoveries were 99.55 ± 0.48 (n = 5) and 101.34 ± 0.86 (n = 5), for BRZ and DZP, respectively, in spiked distilled water, and 99.16 ± 0.77 (n = 5) and 99.32 ± 0.56 (n = 5), in tap water, respectively. The UPLC revealed effluent content ranging from 20.68 – 44.77 mg/mL for BRZ and 22.77 – 41.83 ng/mL for DZP. These values were not significantly different from their reference standards (p > 0.05). Conclusion: A sensitive and selective UPLC-method has been developed for the reproducible determination of BRZ and DZP in industrial wastewater samples. The effective monitoring of the pharmaceutical industrial pollutant will help to conserve the environment and minimize the hazardous effects of these pollutants

Sequence specific assignment and determination of OSR1 C-terminal domain structure by NMR

The binding of SPAK and OSR1 kinases to their upstream WNK kinases is mediated by the interaction of their highly conserved SPAK and OSR1 C-terminal domain (CTD) to RFx [V/I] peptide sequences from WNK kinases. A SPAK CTD knock-in mouse, where SPAK was unable to bind WNK kinases, exhibited low blood pressure. This highlighted the inhibition of SPAK and OSR1 kinases binding to their upstream WNK kinases as a plausible strategy in the discovery of new antihypertensive agents. To facilitate such endeavour, we herein report the optimisation and expression of isotopically labelled OSR1 CTD in E.coli and a structural model based on the sequence specific NMR assignments giving insights into the structure of apo OSR1 CTD. Additionally, we identified the OSR1 CTD amino acid residues that are important for the binding of an 18-mer RFQV peptide derived from human WNK4. Collectively, the NMR backbone assignments and the generated OSR1 CTD 3D model reported in this work will be a powerful resource for the NMR-based discovery of small molecule OSR1 (and SPAK) kinase inhibitors as potential antihypertensive agents

Bioinformatics and network pharmacology-based study to elucidate the multi-target pharmacological mechanism of the indigenous plants of Medina valley in treating HCV-related hepatocellular carcinoma

The incidence of Hepatocellular Carcinoma (HCC) in Saudi Arabia is not surprising given the relatively high prevalence of hepatitis C virus (HCV) infection. Hepatitis C is also common in Saudi Arabia with a prevalence rate of 1% to 3% of the population, which further increases the risk of HCC. The incidence of HCC has been increasing in recent years, with HCV-related HCC accounting for a significant proportion of cases. Traditional medicine has long been a part of Saudi Arabian culture, and many medicinal plants have been used for centuries to treat various ailments, including cancer. Following that, this study combines network pharmacology with bioinformatics approaches to potentially revolutionize HCV-related HCC treatment by identifying effective phytochemicals of indigenous plants of Medina valley. Eight indigenous plants including Rumex vesicarius, Withania somnifera, Rhazya stricta, Heliotropium arbainense, Asphodelus fistulosus, Pulicaria incise, Commicarpus grandiflorus, and Senna alexandrina, were selected for the initial screening of potential drug-like compounds. At first, the information related to active compounds of eight indigenous plants was retrieved from public databases and through literature review which was later combined with differentially expressed genes (DEGs) obtained through microarray datasets. Later, a compound-target genes-disease network was constructed which uncovered that kaempferol, rhazimol, beta-sitosterol, 12-Hydroxy-3-keto-bisnor-4-cholenic acid, 5-O-caffeoylquinic acid, 24-Methyldesmosterol, stigmasterone, fucosterol, and withanolide_J decisively contributed to the cell growth and proliferation by affecting ALB and PTGS2 proteins. Moreover, the molecular docking and Molecular Dynamic (MD) simulation of 20 ns well complemented the binding affinity of the compound and revealed strong stability of predicted compounds at the docked site. But the findings were not validated in actual patients, so further investigation is needed to confirm the potential use of selected medicinal plants towards HCV-related HC

C-terminal phosphorylation of SPAK and OSR1 kinases promotes their binding and activation by the scaffolding protein MO25

SPAK and OSR1 are two protein kinases that play important roles in regulating the function of numerous ion co-transporters. They are activated by two distinct mechanisms that involve initial phosphorylation at their T-loops by WNK kinases and subsequent binding to a scaffolding protein termed MO25. To understand this latter SPAK and OSR1 regulation mechanism, we herein show that MO25 binding to these two kinases is enhanced by serine phosphorylation in their highly conserved WEWS motif, which is located in their C-terminal domains. Furthermore, we show that this C-terminal phosphorylation is carried out by WNK kinases in vitro and involves WNK kinases in cells. Mutagenesis studies revealed key MO25 residues that are important for MO25 binding and activation of SPAK and OSR1 kinases. Collectively, this study provides new insights into the MO25-mediated activation of SPAK and OSR1 kinases, which are emerging as important players in regulating ion homeostasis

Structure-Based Virtual Screening and Molecular Dynamics of Phytochemicals Derived from Saudi Medicinal Plants to Identify Potential COVID-19 Therapeutics

Coronavirus disease 2019 (COVID-19) has affected almost every country in the world by causing a global pandemic with a high mortality rate. Lack of an effective vaccine and/or antiviral drugs against SARS-CoV-2, the causative agent, has severely hampered the response to this novel coronavirus. Natural products have long been used in traditional medicines to treat various diseases, and purified phytochemicals from medicinal plants provide a valuable scaffold for the discovery of new drug leads. In the present study, we performed a computational screening of an in-house database composed of ~1000 phytochemicals derived from traditional Saudi medicinal plants with recognised antiviral activity. Structure-based virtual screening was carried out against three druggable SARS-CoV-2 targets, viral RNAdependent RNA polymerase (RdRp), 3-chymotrypsin-like cysteine protease (3CLpro) and papain like protease (PLpro) to identify putative inhibitors that could facilitate the development of potential anti-COVID-19 drug candidates. Computational analyses identified three compounds inhibiting each target, with binding affinity scores ranging from-9.9 to -6.5 kcal/mol. Among these, luteolin 7-rutinoside, chrysophanol 8-(6-galloylglucoside) and kaempferol 7-(6’’-galloylglucoside) bound efficiently to RdRp, while chrysophanol 8-(6galloylglucoside), 3,4,5-tri-O-galloylquinic acid and mulberrofuran G interacted strongly with 3CLpro, and withanolide A, isocodonocarpine and calonysterone bound tightly to PLpro. These potential drug candidates will be subjected to further in vitro and in vivo studies and may assist the development of effective anti-COVID-19 drugs

Structural basis of SARS-CoV-2 3CLpro and anti-COVID-19 drug discovery from medicinal plants

The recent outbreak of coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 in December 2019 raised global health concerns. The viral 3-chymotrypsin-like cysteine protease (3CLpro) enzyme controls coronavirus replication and is essential for its life cycle. 3CLpro is a proven drug discovery target in the case of severe acute respiratory syndrome coronavirus (SARS-CoV) and middle east respiratory syndrome coronavirus (MERS-CoV). Recent studies revealed that the genome sequence of SARS-CoV-2 is very similar to that of SARS-CoV. Therefore, herein, we analysed the 3CLpro sequence, constructed its 3D homology model, and screened it against a medicinal plant library containing 32,297 potential anti-viral phytochemicals/traditional Chinese medicinal compounds. Our analyses revealed that the top nine hits might serve as potential anti- SARS-CoV-2 lead molecules for further optimisation and drug development process to combat COVID-19

HPLC Method Validation for the Estimation of Lignocaine HCl, Ketoprofen and Hydrocortisone: Greenness Analysis Using AGREE Score

In the current study, the reversed-phased high-pressure liquid chromatography (RP-HPLC) method was proposed for the estimation of lignocaine hydrochloride (LIG), hydrocortisone (HYD) and Ketoprofen (KET) according to International Conference for Harmonization (ICH) Q2 R1 guidelines, in a gel formulation. The chromatographic evaluation was executed using Shimadzu RP-HPLC, equipped with a C8 column and detected using UV at 254 nm wavelength, using acetonitrile and buffer (50:50) as a mobile phase and diluent, at flow rate 1 mL/min and n injection volume of 20 μL. The retention time for LIG, HYD, and KET were 1.54, 2.57, and 5.78 min, correspondingly. The resultant values of analytical recovery demonstrate accuracy and precision of the method and was found specific in identification of the drugs from dosage form and marketed products. The limit of detection (LOD) for LIG, HYD, and KET were calculated to be 0.563, 0.611, and 0.669 ppm, while the limit of quantification (LOQ) was estimated almost at 1.690, 1.833, and 0.223 ppm, respectively. The AGREE software was utilized to evaluate the greenness score of the proposed method, and it was found greener in score (0.76). This study concluded that the proposed method was simple, accurate, precise, robust, economical, reproducible, and suitable for the estimation of drugs in transdermal gels

Resveratrol-Loaded Chia Seed Oil-Based Nanogel as an Anti-Inflammatory in Adjuvant-Induced Arthritis

Natural anti-inflammatory nutraceuticals may be useful in preventing rheumatoid arthritis from worsening. Resveratrol (RV) and chia seed oil, having antioxidant potential, can assist in avoiding oxidative stress-related disorders. This investigation developed and evaluated resveratrol-loaded chia seed oil-based nanoemulsion (NE) gel formulations through in vitro and in vivo studies. The physical stability and in vitro drug permeability of the chosen formulations (NE1 to NE10) were studied. The optimized RV-loaded nanoemulsion (NE2) had droplets with an average size of 37.48 nm that were homogeneous in shape and had a zeta potential of −18 mV. RV-NE2, with a permeability of 98.21 ± 4.32 µg/cm2/h, was gelled with 1% carbopol-940P. A 28-day anti-arthritic assessment (body weight, paw edema, and levels of pro-inflammatory mediators including TNF-α, IL-6, IL-1β, and COX-2) following topical administration of RV-NE2 gel showed significant reversal of arthritic symptoms in arthritic Wistar rats induced by Freund’s complete adjuvant injection. Therefore, RV-NE2 gel demonstrated the potential to achieve local therapeutic benefits in inflammatory arthritic conditions due to its increased topical bioavailability and balancing of pro-inflammatory mediators

Resveratrol-Loaded Chia Seed Oil-Based Nanogel as an Anti-Inflammatory in Adjuvant-Induced Arthritis

Natural anti-inflammatory nutraceuticals may be useful in preventing rheumatoid arthritis from worsening. Resveratrol (RV) and chia seed oil, having antioxidant potential, can assist in avoiding oxidative stress-related disorders. This investigation developed and evaluated resveratrol-loaded chia seed oil-based nanoemulsion (NE) gel formulations through in vitro and in vivo studies. The physical stability and in vitro drug permeability of the chosen formulations (NE1 to NE10) were studied. The optimized RV-loaded nanoemulsion (NE2) had droplets with an average size of 37.48 nm that were homogeneous in shape and had a zeta potential of −18 mV. RV-NE2, with a permeability of 98.21 ± 4.32 µg/cm2/h, was gelled with 1% carbopol-940P. A 28-day anti-arthritic assessment (body weight, paw edema, and levels of pro-inflammatory mediators including TNF-α, IL-6, IL-1β, and COX-2) following topical administration of RV-NE2 gel showed significant reversal of arthritic symptoms in arthritic Wistar rats induced by Freund’s complete adjuvant injection. Therefore, RV-NE2 gel demonstrated the potential to achieve local therapeutic benefits in inflammatory arthritic conditions due to its increased topical bioavailability and balancing of pro-inflammatory mediators