Novel Isoxazole Derivative Attenuates Ethanol-Induced Gastric Mucosal Injury through Inhibition of H+/K+-ATPase Pump, Oxidative Stress and Inflammatory Pathways.

Isoxazole derivatives are significant enough due to their wide range of pharmacological and therapeutic activities. The purpose of the current study is to use computational, in vitro, in vivo, and extensive molecular approaches to examine the possible anti-ulcer activity of 4-benzylidene-3 methyl-1,2-isoxazol-5(4H)-one (MBO). Biovia Discovery Studio visualizer (DSV) was utilized for virtual screening. A tissue antioxidant investigation, H+/K+-ATPase test, and anti-H. pylori activities were carried out. ELISA, immunohistochemistry, and PCR methods were employed for the proteome analysis. An ethanol-induced stomach ulcer model was used to examine the anti-ulcer potential in rats. The binding affinities for MBO ranged from -5.4 to -8.2 Kcal/mol. In vitro findings revealed inhibitory activity against H. pylori and the H+/K+-ATPase pump. It also enhanced levels of glutathione, catalase, and glutathione-S-transferase and reduced lipid peroxidation levels in gastric tissues of rats. In vivo results showed the gastro-protective effect of MBO (30 mg/kg) in ulcerative rat stomachs. The proteomic study revealed decreased expression of inflammatory markers (cyclooxygenase-2, p-NFkB, and TNF-α). In RT-PCR analysis, the expression levels of H+/K+-ATPase were reduced. Furthermore, ADMET (absorption, distribution, metabolism, excretion and toxicity) studies revealed that MBO has high GIT solubility and has a safer profile for cardiac toxicity. This study suggests that MBO displayed anti-ulcer potential, which may have been mediated through the inhibition of the H+/K+-ATPase pump, as well as antioxidant and anti-inflammatory pathways. It has the potential to be a lead molecule in the treatment of peptic ulcers with fewer adverse effects.

Pharmacological Basis of Rumex hastatus D. Don in Gastrointestinal Diseases with Focusing Effects on H+/K+-ATPase, Calcium Channels Inhibition and PDE Mediated Signaling: Toxicological Evaluation on Vital Organs.

This present study aimed to delineate Rumex hastatus D. Don crude extract (Rh.Cr), n-Hexane, ethyl acetate, aqueous fractions (Rh.n-Hex, Rh.ETAC, Rh.Aq) and rutin for antidiarrheal, antisecretory effects, anti-spasmodic, gastrointestinal transient time, anti H. pylori, antiulcer effects, and toxicology. The preliminary phytochemical analysis of Rumex hastatus showed different phytoconstituents and shows different peaks in GC-MC chromatogram. Rumex hastatus crude extract (Rh.Cr), fractions, and rutin attributed dose-dependent (50-300 mg/kg) protection (0-100%) against castor oil-induced diarrhea and dose-dependently inhibited intestinal fluid secretions in mice. They decreased the distance traversed by charcoal in the gastrointestinal transit model in rats. In rabbit jejunum preparations, Rh.Cr and Rh.ETAC caused a concentration-dependent relaxation of both spontaneous and K+ (80 mM)-induced contractions at a similar concentration range, whereas Rh.n-Hex, rutin, and verapamil were relatively potent against K+-induced contractions and shifted the Ca2+ concentration-response curves (CRCs) to the right, Rh.Cr (0.3-1 mg/mL) and Rh.ETAC (0.1-0.3 mg/mL) shifted the isoprenaline-induced inhibitory CRCs to the left. Rh.n-Hex, Rh.ETAC and rutin showed anti-H. pylori effect, also shows an inhibitory effect against H+/K+-ATPase. Rumex hastatus showed gastroprotective and antioxidant effects. Histopathological evaluation showed improvement in cellular architecture and a decrease in the expression of inflammatory markers such as, cyclooxygenase (COX-2), tumor necrosis factor (TN,F-α) and phosphorylated nuclear factor kappa B (p-NFƙB), validated through immunohistochemistry and ELISA techniques. In RT-PCR it decreases H+/K+-ATPase mRNA levels. Rumex hastatus was found to be safe to consume up to a dose of 2000 mg/kg in a comprehensive toxicity profile. Docking studies revealed that rutin against H+/K+-ATPase pump and voltage-gated L-type calcium channel showed E-values of -8.7 and -9.4 Kcal/mol, respectively. MD simulations Molecular Mechanics Poisson Boltzmann surface area and molecular mechanics Generalized Born surface area (MMPBSA/GBSA) findings are consistent with the in-vitro, in-vivo and docking results.

Carveol mitigates the development of the morphine anti-nociceptive tolerance, physical dependence, and conditioned place preference in mice.

Emergence of analgesic tolerance and dependence to morphine is frequently the limiting factor in the use of this agent in the management of pain. Hence, this study aimed to investigate the beneficial effects of the natural compound carveol (CV) against morphine antinociceptive tolerance, dependence and conditioned place preference (CPP) in mice. Behavioural paradigms included hot plate and tail-flick (for tolerance), observation of withdrawal signs (for dependence) while biochemical tests involved the assays for mRNA expression, nitrite levels, antioxidants, and immunohistochemistry studies. Behavioural tests indicated that treatment with CV significantly attenuated the morphine analgesic tolerance, physical dependence and CPP in mice. It was observed during biochemical analysis that CV-treated animals exhibited reduced mRNA expression of inducible nitric oxide synthase (iNOS) and NR2B (an NMDA subtype). In addition, decreased levels of nitrite were observed in mouse hippocampus following CV treatment than morphine administration only. Further, CV enhanced the neuronal innate antioxidants including Glutathione-S-Transferase (GST), glutathione (GSH) and catalase (CAT), while curtailed lipid peroxidase (LPO) levels in mice brain tissues. Moreover, CV exerted significant anti-inflammatory effects as evidenced by reduced expression of TNF-α and p-NF-κB in these animals than with morphine treatment only. Together, anti-inflammatory and antioxidant effects might confer needed neuro-protection following morphine administration. These observations warrant further investigations of the beneficial role of CV as a novel agent in overcoming the development of tolerance and physical dependence following morphine use.

Anti-pruritic effect of L-carnitine against chloroquine-induced pruritus mediated via nitric oxide pathway.

BACKGROUND: Pruritus, or itching, is a distressing symptom associated with various dermatological and systemic diseases. L-carnitine (ßeta hydroxy-γ-tri methyl amino-butyric acid), is a naturally occurring substance, it controls numerous physiological processes. The present research aims to identify L-carnitine for its anti-pruritic effect via nitric oxide-dependent mechanism. METHODS: Chloroquine-induced pruritus serves as an experimental model to investigate possible therapeutic interventions. In this study, we evaluated the efficacy of L-carnitine in combating oxidative stress, nitric oxide, and inflammatory cytokines in a chloroquine-induced pruritus model. RESULTS: L-carnitine treatment significantly reduced scratching behavior compared to the disease group (***P < 0.001 vs. chloroquine group), indicating its antipruritic potential. The markers of oxidative stress, GST, GSH, Catalase, and LPO were dysregulated in the disease model, but administration of L-carnitine restored GST, GSH, and Catalase levels and decreased LPO levels (***P < 0.001 vs. chloroquine group), thereby alleviating oxidative stress. L-carnitine also reduced nitric oxide synthase (NOS) activity, suggesting that it modulates nitric oxide signaling pathways involved in pruritus. In addition, L-carnitine lowered levels of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), inflammatory marker nuclear factor kappa B (p-NFκB) and also reduces an inflammatory enzyme, cyclooxygenase-2 (COX-2), determined by ELISA (Enzyme-Linked Immunosorbent Assay) (***P < 0.001 vs. chloroquine group). It downregulates nNOS mRNA expression confirmed by real-time polymerase chain reaction (RT-PCR). CONCLUSION: These findings highlight the therapeutic effects of L-carnitine in alleviating chloroquine-induced pruritus.

Regulating the NMDA/NR2B signaling pathway mediates anticonvulsant, antineuroinflammation, and anti-oxidative stress effects of 1,3,benzothiazole derivative 1M in pentylenetetrazole-induced kindling in mice.

Periodic epileptic episodes are the hallmark of epilepsy, a prevalent neurological disorder. Research suggests a significant correlation between neuroinflammation and oxidative stress in a variety of neurological diseases, such as epilepsy. A substantial amount of evidence supports the role of N-methyl-D-aspartate receptors (NMDARs) in the progression of epilepsy. Although several lines of research have disclosed numerous biochemical effects of early seizures, its connection with disturbed NMDAR/NR2B subunit expression remains unclear. 2-Mercaptobenzothiazole (MBT) is a vital scaffold with several biological activities, and its various substitutes show promising anti-inflammatory potential. The current study aimed to investigate the newly synthesized 1,3-(benzothiazole-2-sulfanyl)-1-(morpholine-4-yl)ethan-1-one (1 M), a substituted MBT, for its neuroprotective potential in a mice model of pentylenetetrazole-induced epilepsy (PTZ), by modulating NMDA/NR2B pathway. The compound was tested for docking and simulation analysis, demonstrating a solid and stable bond with the NR2B subunit of NMDA. To ascertain the effects of 1 M, as well as to further illustrate its mechanism of neuroprotection via NMDA/NR2B in PTZ-induced kindling model, mice of either sex were given two doses of test compound, 1 M (10 mg/kg and 20 mg/kg). The behavioral assessments were evaluated using open-field, Y-maze, and elevated-plus maze tests, which indicated improved behavioral alterations caused by PTZ after 1 M treatment. The antioxidant profiling was done by estimating glutathione-S-transferase (GST), catalase (CAT), reduced glutathione (GSH), and LPO (lipid peroxidation) in hippocampal tissues, where the test compound 1 M significantly restored the depleted antioxidants, showcasing its antioxidant potential. Moreover, the cellular morphological damages induced by PTZ were detected by H&E staining, which was rescued after 1 M administration. Furthermore, the activation of the inflammatory pathway was confirmed by quantitative analysis of inflammatory mediators tumor necrotic factor (TNF-α), nuclear factor kappa B (NF-κB), and cylooxegenase2 (COX-2) by enzyme-linked immunosorbent assay (ELISA), where 1 M administration significantly ameliorated their expression. Furthermore, to demonstrate the involvement of the NR2B pathway, NR2B-antagonist ifenprodil was employed, and results were further confirmed through RT-PCR analysis. Our results, when considered collectively, indicate that 1 M may act by inhibiting the NR2B subunit of the NMDA receptor, subsequently mitigating downstream oxidative stress and inflammatory mediators through various pathways.

Emodin alleviates chronic constriction injury-induced neuropathic pain and inflammation via modulating PPAR-gamma pathway.

Neuropathic pain has been characterized as chronic pain resulting from pathological damage to the sensorimotor system. Because of its complex nature, it remains refractory to most of the therapeutic interventions, and surgical intervention and physiotherapy alongside steroidal treatments remain the only treatment protocols with limited success, hence solidifying the need to find efficacious therapeutic alternatives. Emodin was used as a post-treatment for its potential to be neuroprotective in the treatment of chronic constriction injury-induced NP. The first day following surgery, Emodin treatment began, and it lasted until the 21st day. On days 3, 7, 14 and 21, all behavioral investigations were conducted. The sciatic nerve and spinal cord were extracted for further molecular examination. Emodin elevated response latency, was able to delay the onset of mechanical hyperalgesia in rats on days 7, 14, and 21 and reduced the CCI-induced paw deformation. Emodin treatment significantly reduced lipid peroxidation and NO levels while restoring the GST, GSH and catalase. It significantly improved the disorientation of the sciatic nerve and spinal cord confirmed by H & E staining and reduced inflammatory markers as observed by the quantification of COX-2, TNF-α, p-NFκb and up-regulated PPAR-γ levels by ELISA and PCR. According to the findings, Emodin has antinociceptive and anti-hyperalgesic properties, which reduced pain perception and inflammation. We also suggested the involvement of PPAR-γ pathway in the therapeutic potential of emodin in chronic nerve injury.

Pharmacological investigation of brucine anti-ulcer potential.

Gastric ulcer is one of the most common chronic gastrointestinal diseases characterized by a significant defect in the mucosal barrier. The current study has been conducted to evaluate the brucine anti-ulcer effect. Brucine has binding energy values ranging from -2.99 to -8.11 kcal/mol against chosen targets, according to in silico research. Brucine exhibits an inhibitory effect against Helicobacter pylori. In vivo findings revealed that brucine (3 mg/kg) showed effective results in healing ethanol-induced ulcer lesions of the gastric region in rats. Brucine showed an inhibitory effect against H+/K+-ATPase. Levels of glutathione, glutathione-s-transferase, and catalase were enhanced in the gastric rat tissue with the use of brucine, while a significant decrease in lipid peroxide levels was seen. Histopathological evaluation showed improvement in cellular architecture and a decrease in inflammatory indicators like cyclooxygenase, tumor necrosis factor, and nuclear factor kappa B expression, validated through immunohistochemistry, enzyme-linked immunosorbent assay, and Western blot techniques. In the reverse transcription-polymerase chain reaction, brucine decreased H+/K+-ATPase mRNA levels. This study reveals that brucine possesses stable binding affinities against selected targets. Brucine exhibits an anti-ulcer effect, mediated via anti-H. pylori, H+/K+-ATPase inhibition, and antioxidant and anti-inflammatory pathways.

Pharmacological basis of bergapten in gastrointestinal diseases focusing on H+/K+ ATPase and voltage-gated calcium channel inhibition: A toxicological evaluation on vital organs.

Aim and objectives: This study aimed to establish a pharmacological basis for evaluating the effects of bergapten (5-methoxypsoralen) in gastrointestinal diseases and assessment of its toxicological profile. Methods: The pharmacokinetic profile was evaluated using the SwissADME tool. AUTODOCK and PyRx were used for evaluating the binding affinities. The obtained results were further investigated for a post-dock analysis using Discovery Studio Visualizer 2016. The Desmond software package was used to conduct molecular dynamic simulations of best bound poses. Bergapten was further investigated for antidiarrheal, anti-secretory, charcoal meal transit time, anti-ulcer, anti-H. pylori activity. Results: Bergapten at a dose of 50, 100, and 200 mg/kg was proved effective in reducing diarrheal secretions, intestinal secretions, and distance moved by charcoal meal. Bergapten at the aforementioned doses acts as a gastroprotective agent in the ethanol-induced ulcer model that can be attributed to its effectiveness against H. pylori. Bergapten shows concentration-dependent relaxation of both spontaneous and K+ (80 mM)-induced contractions in the isolated rabbit jejunum model; the Ca2+ concentration-response curves (CRCs) were shifted to the right showing potentiating effect similar to papaverine. For molecular investigation, the H+/K+ ATPase inhibitory assay indicated inhibition of the pump comparable to omeprazole. Oxidative stress markers GST, GSH, and catalase showed increased expression, whereas the expression of LPO (lipid peroxidation) was reduced. Histopathological examination indicated marked improvement in cellular morphology. ELISA and western blot confirmed the reduction in inflammatory mediator expression. RT-PCR reduced the mRNA expression level of H+/K+ ATPase, confirming inhibition of the pump. The toxicological profile of bergapten was evaluated by an acute toxicity assay and evaluated for behavioral analysis, and the vital organs were used to analyze biochemical, hematological, and histopathological examination. Conclusion: Bergapten at the tested doses proved to be an antioxidant, anti-inflammatory, anti-ulcer, and antidiarrheal agent and relatively safe in acute toxicity assay.

Anti-diabetic Activity of Brucine in Streptozotocin-Induced Rats: In Silico, In Vitro, and In Vivo Studies.

Diabetes mellitus (DM) is a complex and multiple group of disorders, and understanding the molecular mechanisms is a key role in identifying various markers involved in the diagnosis of the disease. Brucine is derived from the seeds of Strychnos nux-vomica L. (Loganiaceae), which has been used in traditional medicine to cure a variety of ailments, such as chronic rheumatism, nervous system diseases, dyspepsia, gonorrhea, anemia, and bronchitis, and has analgesic, anti-inflammatory, anti-oxidant, anti-snake venom, and anti-diabetic properties. The anti-diabetic potential of brucine was studied utilizing in vitro, in silico, in vivo, and molecular methods, including streptozotocin-induced diabetic rat models, α-glucosidase and α-amylase inhibitory assays, and via Auto-DocVina software. Brucine exhibits binding affinities of -5.0 to -10.1 Kcal/mol against chosen protein targets, according to an in silico investigation. In vitro studies revealed that brucine inhibited the enzymes α-amylase and α-glucosidase, and brucine (20 mg/kg) reduced blood glucose levels, oral glucose tolerance overload, body weight, glycosylated hemoglobin levels, total cholesterol, triglycerides, low-density lipoprotein, alanine transaminase, aspartate aminotransferase, total bilirubin, and alkaline phosphatase and elevated high-density lipoprotein levels in in vivo studies. The brucine binding energy against certain protein targets ranges from -5.0 to -10.1 Kcal/mol. It has anti-diabetic, anti-hyperlipidemic, hepatoprotective, anti-oxidant, and anti-inflammatory properties, which are mediated via inhibition of α-glucosidase and α-amylase.

Proteomic Analysis and In Vivo Studies Reveal the Potential Gastroprotective Effects of CHCl3 and Aqueous Extracts of Ficus palmata.

Ficus palmata is rich in several phytochemicals such as chromone, isoflavones, terpenes, lignans, coumarins, glycosides, and furanocoumarins and have been traditionally used for the management of different gastrointestinal disorders. This research reveals the effects of Ficus palmata fruit extracts-Ficus palmata chloroform (Fp.CHCl3) and Ficus palmata aqueous (Fp.Aq)-on gut activity through in vivo and in vitro analyses. Antidiarrheal and enteropooling assays were analyzed with castor oil-induced diarrhea and intestinal fluid accumulation. Jejunum tissues of rabbits were isolated (antispasmodic) for in vitro experiments. Antimotility was carried out by charcoal meal for determining transient time, and ethanol-induced ulcer assay was used to measure the ulceration of stomach; molecular pathways were assessed through proteomic approach. Fp.CHCl3 and Fp.Aq extracts attributed dose-dependently protection against diarrhea, and intestinal fluid secretions were inhibited dose dependently. Extracts of Fp.CHCl3 and Fp.Aq produced reduction in spontaneous and K+ (at 80 Mm)-induced contractions in isolated jejunum tissues, along with the decreased length covered by charcoal in charcoal meal transient time activity. The extract exhibited gastroprotective outcome in rats and reduced tumor necrotic factor (TNF-α) levels and IL-18, measured by proteomic approach. Morphological studies' results showed that ethanol induced significant gastritis, apoptosis, swelling of mucosa, and hydropic degeneration leading to cellular degeneration and necrosis, observed through staining techniques. Furthermore, ethanol activated the inflammation pathway in all gastric zones by elevating the levels of cyclooxygenase-2, TNF-α, and nuclear factor kappa light-chain enhancer of activated B-cells. Overall results expressed the antidiarrheal, antispasmodic, enteropooling, antimotility, and antiulcer activities of Ficus palmata fruit extract.

Prospects of Wedelolactone as a Chemotherapeutic Agent in Gynecological Cancers; Clue From its In Vitro and In Silico Investigation.

BACKGROUND: Identification and development of new drug candidates to be used singly or in combination therapy is critical in anticancer research. In recent years, accumulating evidence encouraged us to investigate the anti-proliferative effects of a small and emerging phytochemical Wedelolactone (WDL) in estrogen-dependent and independent multiple gynecological tumor models. OBJECTIVE: The aim of this study was to investigate the growth inhibitory effect of WDL on estrogen- dependent and independent gynecological cell lines and to explore its inhibitory potential towards key targets through in silico study. METHODS: Cytotoxicity of WDL was investigated in human breast and ovarian cancer cell lines (MCF-7 and SKOV3) through 3-(4,5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) reduction assay. Epigallocatechingallate (EGCG) was used as reference natural compound while cisplatin was taken as a standard clinical agent. Both WDL and EGCG in combination with cisplatin were also evaluated for their combined growth inhibitory potential in MCF-7 cells. WDL was also evaluated in silico against key factors including braf kinases, CDPK, ERα, aromatase, topoisomerase II and dihydrofolate reductase (DHFR) playing pivotal roles in driving multiple tumors. RESULTS AND DISCUSSION: The IC50 value of WDL was 25.77 ± 4.82 µM and 33.64 ± 1.45 µM in MCF-7 and SKOV-3 respectively. The binding energy order was as follows; WDL: DHFR >Braf kinases > CDPK; aromatase > topoisomerase II> ERα > NFkB > alkaline phosphatase; EGCG dihydrofolatereductase (DHFR) > aromatase >CDPK > topoisomerase II > braf kinases > alkaline phosphatase > CDPK > ERα > NFkB. CONCLUSION: We identified WDL as a cytotoxic agent in breast and ovarian tumor models with the potential to inhibit multiple targets in the oncogenic pathway including estrogen receptor ERα, as depicted through its in silico study. Based on our own research findings and from literature evidence, we conclude that further research should be encouraged to investigate different aspects of wedelolactone as an additional agent to be combined with antiestrogen/endocrine therapy.

In Vivo, Proteomic, and In Silico Investigation of Sapodilla for Therapeutic Potential in Gastrointestinal Disorders.

This study aims to delineate the effects of Manilkara zapota Linn. (Sapodilla) fruit chloroform (Mz.CHCl3) and aqueous (Mz.Aq) extracts tested through different techniques. Antidiarrheal activity and intestinal fluid accumulation were examined by using castor oil-induced diarrhea and castor oil fluid accumulation models. Isolated rabbit jejunum tissues were employed for in vitro experiments. Antimotility and antiulcer were performed through charcoal meal transient time and ethanol-induced ulcer assay, molecular studies were conducted through proteomic analysis, and virtual screening was performed by using a discovery studio visualizer (DSV). Mz.CHCl3 and Mz.Aq extracts attributed dose-dependent (50-300 mg/kg) protection (20-100%) against castor oil-induced diarrhea and dose-dependently (50-300 mg/kg) inhibited intestinal fluid secretions in mice. Mz.CHCl3 and Mz.Aq extracts produce relaxation of spontaneous and K+ (80 Mm) induced contractions in isolated tissue preparations and decreased the distance moved by charcoal in the gastrointestinal transit model in rats. It showed gastroprotective effect in ulcerative stomach of rats and decreased levels of IL-18 quantified by proteomic analysis. Histopathological results showed ethanol-induced significant gastric injury, leading to cloudy swelling, hydropic degeneration, apoptosis, and focal necrosis in all gastric zones using hematoxylin and eosin (H&E) staining. Moreover, ethanol increased the activation and the expression of tumor necrotic factor (TNF-α), cyclooxygenase (COX-2), and nuclear factor kappa-light-chain-enhancer of activated B cells (p-NFκB). In silico results were comparative to in vitro results evaluated through virtual screening. Moreover, ethanol increased the activation and expression of tumor necrotic factor, cyclooxygenase, and nuclear factor kappa-light-chain-enhancer of activated B cells. This study exhibits the gastroprotective effect of Manilkara zapota extracts in the peritoneal cavity using a proteomic and in silico approach which reveals different energy values against target proteins, which mediate the gastrointestinal functions.

Synthesis, characterization, anti-ulcer action and molecular docking evaluation of novel benzimidazole-pyrazole hybrids.

A series of six novel benzimidazole-pyrazole hybrid molecules was synthesized and characterized using elemental analysis (CHN) and spectroscopic methods (1HNMR, FT-IR). All the synthesized compounds were evaluated for their in vivo anti ulcerogenic activity using Albino rats (weighing 180-220 g). The interactions between the compounds and active site residues of H+/K+ ATPase were investigated by molecular docking studies using autodock vina 4.0. SCH28080 was used to validate the docking results. Also the drug likeliness of these compounds was predicted using Molinspiration server in light of Lipinski's rule of five. All the six synthesized compounds exhibited higher anti-ulcer activity as compared to omeprazole. These novel hybrid compounds showed comparable anti-ulcer potential of 72-83% at dose level of 500 µg/kg, whereas omeprazole showed 83% anti-ulcer activity at dose level of 30 mg/kg. The results clearly indicate that these novel benzimidazole-pyrazole hybrids can present a new class of potential anti ulcer agents and can serve as new anti-ulcer drugs after further investigation. Graphical abstract An overveiw of synthesis, in silico and in vivo antiulcer screening of benzimidazole pyrazole hybrids.