Pakistan Journal of Pharmaceutical Sciences

Abstract: Background: Gallic acid is a natural phenolic compound with excellent antioxidant, antimicrobial and anti-inflammatory activity and good skin penetration. Gallic acid demonstrates both stability and solubility problems in topical formulations. A nanoemulgel combines an emulsion for enhanced solubility with a gel base for improved physical stability. Objectives: The main objective of this study is to characterize and assess the stability of a gallic acid-containing nanoemulgel. Methods: Various gallic acid nanoemulsions (GNE) were prepared using optimized ratios of oil, surfactants, gallic acid and water and evaluated for stability. Finally, stable GNE-19 was characterized for particle size and converted to nanoemulgel (GNEG-19). Nanoemulgel was subjected to short-term and accelerated stability studies and characterized for pH, viscosity, spreadability, occlusion effect, FTIR, in-vitro drug release and in-vitro pharmacological activities. Results: The results showed that formulation GNEG-19 showed optimum thermodynamic stability, having no phase separation and color change. The droplet size of freshly prepared GNE and after 28 days was 175.43nm and 183.23nm, respectively. The pH was slightly acidic but still within the human skin range (3.9–4.5). No interaction was found between the gallic acid and other components of nanoemulgel following FTIR analysis. The viscosity of freshly prepared GNEG-19 was 2127.1 cP and after 90 days, it was 2102.3 cP, which showed its stability. The percent drug release of gallic acid from nanoemulgel after 24 hrs was 93.12%. GNEG-19 also showed effective occlusion properties when compared with gel base and petroleum jelly. In-vitro pharmacological activities showed that GNEG-19 had antioxidant activity of 63.77% and a zone of inhibition (ZOI) of 11.4 mm compared to pure gallic acid (9.5 mm). Conclusion: In conclusion, nanoemulgel containing gallic acid showed good physical and chemical stability and was pharmacologically active.

Abstract: Background: HPV-associated cervical cancer remains a leading cause of cancer-related death in women worldwide. The role of bone marrow mesenchymal stem cells (BMSCs) and miR-129 in tumor progression remains unclear. Objectives: To investigate miR-129 expression and its clinical significance in HPV-associated cervical cancer and to explore whether BMSCs regulate malignant phenotypes through miR-129. Methods: miR-129 expression was measured by qRT-PCR in 77 cervical cancer and 30 normal cervical tissues. Associations with clinicopathological features and overall survival (OS) were analyzed. Diagnostic value was assessed by ROC curve. Rat BMSCs were co-cultured with SiHa and HeLa cells. Following miR-129 overexpression or knockdown, cell proliferation (CCK-8, colony formation), invasion (Transwell) and expression of E-cadherin, N-cadherin and MRP1 (Western blot) were examined. Results: miR-129 expression was significantly higher in cervical cancer tissues than in normal controls (3.24±0.56 vs. 1.00±0.32, P<0.05). High miR-129 expression correlated with tumor size ≥3 cm (P=0.009), FIGO stage III-IV (P=0.0034) and lymph node metastasis (P=0.038). High expression was associated with shorter OS (median: 28.4 vs. 46.2 months; HR=2.944, 95%CI:1.484-5.842, P=0.002). The AUC was 0.788 (95%CI:0.693-0.884), with a cut-off of 1.85 (sensitivity:76.9%, specificity:70.4%). BMSC co-culture upregulated miR-129 expression (SiHa: 3.52±0.42 vs. 1.00±0.08, P<0.01). miR-129 overexpression promoted proliferation, colony formation and invasion, while knockdown suppressed these phenotypes. Overexpression of miR-129 reduced E-cadherin (0.42±0.05 vs. 1.00±0.08, P<0.01) and increased N-cadherin (2.85±0.22 vs. 1.00±0.10, P<0.01) and MRP1 (2.46±0.18 vs. 1.00±0.09, P<0.01). Knockdown of miR-129 partially reversed BMSC-induced changes in these proteins. Conclusion: miR-129 is significantly upregulated in HPV-associated cervical cancer and shows potential as a diagnostic and prognostic biomarker. BMSCs upregulate miR-129 in cervical cancer cells and miR-129 modulates proliferation, invasion, EMT marker expression and MRP1 expression. These findings suggest that the BMSCs/miR-129 axis may contribute to cervical cancer progression, but further in-vivo and mechanistic studies are needed.

Abstract: Background: Dysregulated expression of prolyl 4-hydroxylase subunit alpha 3 (P4HA3) has been identified in Colon cancer, breast cancer, head and neck cancers, etc. and is closely associated with tumor progression. However, its specific roles in gastric cancer (GC), particularly regarding alterations in metabolic pathways, are remain unknown. Objectives: This study investigated how P4HA3 influences glycolytic metabolic reprogramming and the proliferation and invasion of gastric cancer cells via the PI3K/AKT/mTOR signaling pathway. Methods: To determine P4HA3 levels in GC tissues and corresponding cell lines, bioinformatics methods, immunohistochemical (IHC) staining and Western blotting (WB) were conducted. Furthermore, gain- and loss-of-function experiments, including cell viability assays (CCK-8), colony-forming tests, scratch wound assays and Transwell invasion experiments, were utilized to explore the influence of P4HA3 on GC cell growth, motility and invasive capacity. Changes in cellular metabolism were assessed using the Seahorse system (under normoxic conditions), including measurements of basal metabolism, glycolytic capacity and mitochondrial respiration (oxidative phosphorylation), as well as Western blot analysis of key glycolytic proteins. Additionally, the involvement of the PI3K/AKT/mTOR signaling axis was examined by gene set enrichment analysis (GSEA), WB analysis and pharmacological inhibition studies. Results: GC tissues and cell lines exhibited markedly increased levels of P4HA3, correlating significantly with decreased overall survival (OS). Experimental data indicated that P4HA3 facilitated GC cell proliferation, migration and invasion. In addition, P4HA3 enhanced glycolytic activity and increased the expression of key glycolysis-related proteins. Unexpectedly, P4HA3 also increased mitochondrial respiration under the assay conditions, suggesting that P4HA3 may induce a broader metabolic activation rather than an exclusively glycolytic shift. Mechanistically, the glycolysis-enhancing effects of P4HA3 were found to require PI3K/AKT/mTOR signaling (as shown by inhibitor studies). Conclusion: P4HA3 promotes glycolysis via the PI3K/AKT/mTOR signaling pathway, thereby promoting the progression of gastric cancer.

Abstract: Background: Bacteria have become more resistant to commonly used antibiotics. Among the many reasons, biofilm-mediated protection plays an indispensable role. To overcome microbial resistance, alternative antibiofilm therapies may be developed to address this challenge and they should be cost-effective and readily available. Objectives: To evaluate the antibiofilm activities of natural compounds Allium Cepa (Onion), Menthe spicata (mint) and Zingiber officinale (Ginger) against Escherichia coli and Pseudomonas Aeruginosa Biofilms. Methods: This cross-sectional study includes 150 patients from various clinics in Karachi, Pakistan. Samples of Escherichia coli and Pseudomonas aeruginosa were collected and the study was carried out to determine the antibiofilm-forming activity of natural product extracts of Allium cepa (Onion) G.H.NO 100929, Mentha spicata (mint) G.H.NO100933 and Zingiber officinale (Ginger) G.H.NO100928, evaluated separately and in different combinations. 96 well plate method was used to evaluate biofilm forming activity of microorganisms and antibiofilm forming activity of Allium Cepa (Onion) Menthe Spicata( mint) Zingiber Officinale (Ginger Peels) Results: Natural compounds Allium Cepa (Onion) Menthe Spicata( mint) Zingiber Officinale (Ginger) has shown antibiofilm activity against Escherichia coli and Pseudomonas aeruginosa biofilms separately but when used in combination only synergistic activity shows ginger and mint combination with FIC 0.45 against Pseudomonas Aeruginosa. Whereas both organisms have shown resistance to conventional antimicrobial agents. Conclusion: This study concludes that Allium Cepa (Onion) Menthe Spicata (mint) Zingiber Officinale (Ginger) having antibiofilm activity when used separately but in combination Zingiber Officinale (Ginger) And Menthe Spicata (mint) against Pseudomonas Aeruginosa only shows synergistic activity where as other combination shows indifferent activity but shows lower concentration of extract as compare to when used separately but not match the criteria of synergism but importantly none of combination shows antagonist activity. These findings emphasize the need to incorporate these natural agents, after drug authority approval, with already available antimicrobial agents.

Abstract: Background: Suaeda fruticosa is a widely distributed plant in Pakistan which has been traditionally used as a remedy for constipation, dyspnoea, headache, nausea, neurosis and vertigo. Objectives: The present study aimed to scientifically validate the traditionally claimed medicinal properties of Suaeda fruticosa. Methods: The experiment involved the preparation of a 70% ethanolic extract of Suaeda fruticosa (Sf.Cr), which was tested ex-vivo in an isolated rabbit jejunum preparation for spasmolytic activity. Moreover, the crude extract was tested on isolated tracheal and aorta preparations to evaluate the effects. Results: Sf.Cr dose-dependently relaxed the jejunum and inhibited K⁺(80 mM)-induced contractions with EC₅₀ value of 0.229 mg/ml and 0.095 mg/ml, respectively. Further, the Sf.Cr caused a rightward shift in Ca2+ concentration response curves (CRCs), similar to verapamil. So, calcium channel-blocking and anticholinergic actions were responsible for the antispasmodic (anti-diarrhoeal) effect. Sf.Cr produced inhibition of carbachol (1 µM)- and K⁺(80 mM)-induced contractions in isolated rabbit tracheal tissues with EC₅₀ values of 0.8339 mg/ml and 1.022 mg/ml, respectively. Sf. Cr also completely abolished the Phenylephrine (1 µM)- and K+(80 mM)-induced constrictions in isolated rabbit aortic rings, with EC50 values of 0.9448 mg/ml and 0.2026 mg/ml, respectively. Molecular docking against calmodulin (PDB ID: 6M7H) showed that 3-hydroxy-2-quinolinone attained the highest glide score (-6.742 kcal.mol⁻¹), establishing hydrogen bonding and Pi–Pi stacking interactions with MET124 and PHE92, surpassing the binding affinity of the standard drug verapamil (-5.464 kcal.mol⁻¹). SwissADME profiling showed that all identified phytoconstituents complied with Lipinski’s rule of five, exhibited high gastrointestinal absorption and were predicted to cross the blood-brain barrier, supporting their oral bioavailability as drug candidates. Conclusion: These findings collectively established Sf.Cr as a source of non-specific calcium channel-blocking phytochemicals with potential for developing antispasmodic, bronchodilator, and vasorelaxant agents.