Investigating the involvement of the NLRP3/ASC/caspase-1 and NF-κb/MAPK pathways in the pathogenesis of gouty arthritis: Insights from irradiated and non-irradiated Trifolium alexandrium L. extracts and some metabolites.

ETHNOPHARMACOLOGICAL RELEVANCE: Trifolium alexandrinum L. (TA), has traditionally been used in folk medicine for its anti-inflammatory properties against hyperuricemia and gout. However, the specific mechanisms of action of TA have not been thoroughly studied. AIM OF THE WORK: This study aimed to evaluate the protective effects of irradiated (TR25) and non-irradiated (TR0) Trifolium alexandrinum L. aqueous extract (TAAE), along with two isolated compounds, caffeine (CAF) and saponin (SAP), in a rat model of acute gouty arthritis (GA). MATERIALS AND METHODS: The GA model was established by injecting a monosodium urate (MSU) suspension into the knee joint. Synovial tissue pathology was assessed, and levels of TNF-α, IL-6, IL-1ß, NF-κB, mTOR, AKT1, PI3K, NLRP3, and ASC were measured by ELISA. mRNA expression of ERK1, JNK, and p-38 MAPK was detected using qRT-PCR, and Caspase-1 protein expression was assessed by immunohistochemical analysis. Knee swelling, uric acid levels, liver and kidney function, and oxidative stress markers were also evaluated. RESULTS: TAAE analysis identified 170 compounds, with 73 successfully identified using LC-HR-MS/MS, including caffeine citrate and theasapogenol B glycoside as the main constituents. The studied materials demonstrated significant protective effects against GA. TR25 administration significantly mitigated knee joint circumference compared to other treatments. It demonstrated potential in alleviating hyperuricemia, renal and hepatic impairments induced by MSU crystals. TR25 also alleviated oxidative stress and reduced levels of IL1ß, IL-6, TNF-α, and NF-κB. Weak Caspase-1 immune-positive staining was observed in the TR25 group. TR25 decreased NLRP3 and ASC expression, reducing inflammatory cytokine levels in GA. It effectively inhibited the PI3K, AKT, and mTOR signaling pathways, promoting autophagy. Additionally, TR25 suppressed ERK1, JNK, and p-38 MAPK gene expression in synovial tissue. These effects were attributed to various components in TAAE, such as flavonoids, phenolic acids, tannins, alkaloids, and triterpenes. CONCLUSION: Importantly, irradiation (25 KGy) enhanced the antioxidant effects and phtchemical contents of TAAE. Additionally, TR0, TR25, CAF, and SAP exhibited promising protective effects against GA, suggesting their therapeutic potential for managing this condition. These effects were likely mediated through modulation of the NLRP3/ASC/Caspase-1 and ERK/JNK/p-38 MAPK signaling pathways, as well as regulation of the PI3K/AKT/mTOR pathway. Further research is warranted to fully elucidate the underlying mechanisms and optimize their clinical applications.

Ipomoea carnea mitigates ethanol-induced ulcers in irradiated rats via Nrf2/HO-1 pathway: an in vivo and in silico study.

The aim of this study was to investigate the potential of Ipomoea carnea flower methanolic extract (ICME) as a natural gastroprotective therapy against ethanol-induced gastric ulcers, particularly in individuals exposed to ionizing radiation (IR). The study focused on the Nrf2/HO-1 signaling pathway, which plays a crucial role in protecting the gastrointestinal mucosa from oxidative stress and inflammation. Male Wistar rats were divided into nine groups, the control group received distilled water orally for one week, while other groups were treated with ethanol to induce stomach ulcers, IR exposure, omeprazole, and different doses of ICME in combination with ethanol and/or IR. The study conducted comprehensive analyses, including LC-HRESI-MS/MS, to characterize the phenolic contents of ICME. Additionally, the Nrf2/HO-1 pathway, oxidative stress parameters, gastric pH, and histopathological changes were examined. The results showed that rats treated with IR and/or ethanol exhibited histopathological alterations, increased lipid peroxidation, decreased antioxidant enzyme activity, and reduced expression levels of Nrf2 and HO-1. However, pretreatment with ICME significantly improved these parameters. Phytochemical analysis identified 39 compounds in ICME, with flavonoids, hydroxybenzoic acids, and fatty acids as the predominant compounds. Virtual screening and molecular dynamics simulations suggested that ICME may protect against gastric ulceration by inhibiting oxidative stress and inflammatory mediators. In conclusion, this study demonstrates the potential of ICME as a natural gastroprotective therapy for preventing gastric ulcers. These findings contribute to the development of novel interventions for gastrointestinal disorders using natural plant extracts particularly in individuals with a history of radiation exposure.

Anti-Helicobacter pylori, anti-biofilm activity, and molecular docking study of citropten, bergapten, and its positional isomer isolated from Citrus sinensis L. leaves.

Introduction: Citrus sinensis L. is a candidate plant with promising antimicrobial potential. In the current study, the phytochemical investigation of C. sinensis leaf extract led to the isolation of three coumarins, namely bergapten, xanthotoxin, and citropten. Methods: The chemical structures of the isolated coumarins were elucidated using NMR and ESI-MS techniques. The total aqueous ethanol leaf extract and the isolated coumarins were evaluated for their antimicrobial effects against Helicobacter pylori using the MTT-micro-well dilution method and its anti-biofilm activity using MBEC assay, as compared to clarithromycin. Results: The results showed that citropten scored the lowest MIC value at 3.9 µg/mL and completely inhibited the planktonic growth of H. pylori. In addition, it completely suppressed H. pylori biofilm at 31.25 µg/mL. These findings have been supported by molecular docking studies on the active sites of the H. pylori inosine 5'-monophosphate dehydrogenase (HpIMPDH) model and the urease enzyme, showing a strong binding affinity of citropten to HpIMPDH with seven hydrogen bonds and a binding energy of -6.9 kcal/mol. Xanthotoxin and bergapten showed good docking scores, both at -6.5 kcal/mol for HpIMPDH, with each having four hydrogen bondings. Furthermore, xanthotoxin showed many hydrophobic interactions, while bergapten formed one Pi-anion interaction. Concerning docking in the urease enzyme, the compounds showed mild to moderate binding affinities as compared to the ligand. Thus, based on docking results and good binding scores observed with the HpIMPDH active site, an in-vitro HpIMPDH inhibition assay was done for the compounds. Citropten showed the most promising inhibitory activity with an IC50 value of 2.4 µM. Conclusion: The present study demonstrates that C. sinensis L. leaves are a good source for supplying coumarins that can act as naturally effective anti-H. pylori agents.

Antimicrobial and antiviral evaluation of compounds from Holoptelea integrifolia: in silico supported in vitro study.

Holoptelea integrifolia, also known as the Indian Elm Tree, has been used in Ayurvedic medicine for its medicinal properties. In this study, two biologically active metabolites, 5(6) dihydrostigmast 22en 3-O-ß-glucoside (DHS) and 1-O-eicosanoyl glycerol-2'-O-ß-galactouronic (EGG), were isolated for the first time from the n-butanol fraction of H. integrifolia using a chromatographic technique and identified by NMR, and HRESI-MS. The antiviral and multidrug-resistant activities of these metabolites were evaluated as well as the n-butanol fraction. The n-butanol fraction of H. integrifolia exhibited weak antiviral effects, but DHS and EGG demonstrated significant antiviral activity against herpes simplex type-1 (HSV-1) and Coxsackie (CoxB4) viruses. Both metabolites showed lower IC50 values than the standard antiviral drug acyclovir, indicating their potency in inhibiting viral replication. EGG showed potent antiviral activity with minimal cytotoxicity at the highest concentration tested, presenting a selectivity index (SI) of 18.18 and 15.58 against HSV-1 and CoxB4 viruses, respectively. A preliminary assessment of the antibacterial activity of the n-butanol fraction and metabolites revealed that DHS had the highest inhibitory potency against drug-resistant strains, including MRSA and Carbapenem-resistant Klebsiella pneumonia. It also exhibited significant inhibitions against Fluconazole-resistant Candida albicans and ESBL - Escherichia coli. DHS displayed the lowest minimum inhibitory concentration (MIC) values, indicating its superiority as an antibacterial agent compared to EGG and the n-butanol fraction. Molecular docking analysis confirmed the antiviral and antibacterial actions of DHS and EGG by demonstrating their strong binding.

Polyphenolic Profile of Herniaria hemistemon Aerial Parts Extract and Assessment of Its Anti-Cryptosporidiosis in a Murine Model: In Silico Supported In Vivo Study.

Herniaria hemistemon J.Gay is widely used in folk medicine to treat hernia. The present study aimed to annotate the phytoconstituents of H. hemistemon aerial-part extract and investigate its in vivo anticryptosporidial activity. The chemical characterization was achieved via the LC-ESI-MS/MS technique resulting in the annotation of 37 phytocompounds comprising flavonoids and phenolic acids. Regarding the anticryptosporidial activity, fifty dexamethasone-immunosuppressed mice were separated into five groups: GI, un-infected (normal control); GII, infected but not treated (model); GIII, infected and received NTZ, the reference drug; GIV, infected and received H. hemistemon extract (100 mg/kg); and GV, infected and received H. hemistemon extract (200 mg/kg). When GIII, GIV, and GV were compared to GII, parasitological analyses displayed highly significant differences in the mean numbers of Cryptosporidium parvum oocysts in the stool between the different groups. GV demonstrated the highest efficacy of 79%. Histopathological analyses displayed improvement in the small intestine and liver pathology in the treated groups (GIII, IV, and V) related to the model (GII), with GV showing the highest efficacy. Moreover, the docking-based study tentatively highlighted the potential of benzoic acid derivatives as lactate dehydrogenase inhibitors. The docked compounds showed the same binding interactions as oxamic acid, where they established H-bond interactions with ARG-109, ASN-140, ASP-168, ARG-171, and HIS-195. To sum up, H. hemistemon is a promising natural therapeutic agent for cryptosporidiosis.

Ameliorative Effects of Thunbergia erecta L. Leaves Against the Initiation of Hepatocarcinogenesis Induced by Diethylnitrosamine in the Rat Model.

Thunbergia erecta L. contains cytotoxic and liver-protective compounds. Thunbergia erecta L. leaves were macerated in 70% aqueous ethanol, then fractionated with ethyl acetate (9.3 g) and butanol (12.7 g), and attenuated Den-induced liver cancer in a Wistar rat experimental model. Ethyl acetate and butanol fractions were chromatographed using column chromatography and solid-phase extraction (SPE); Vicenin-II (1), kaempferol (2), biochanin A, sissotrin 7-O-ß-glucopyranoside (3), gentianose (4), acacetin 7-O-ß-glucopyranoside (5), apigenin 7-O-ß-glucopyranoside (6), and rosmarinic acid (7) were extracted, and their structures were determined using NMR spectroscopy and ESI-mass spectrometry. Sixty rats were divided into six groups (ten each): control group, Den group, doxorubicin/Den-treated group, butanol fraction/Den-treated group, and isolated acacetin 7-O-ß-glucopyranoside/Den-treated group. The liver enzymes and proinflammatory biomarkers were used to estimate the liver function. In addition, liver tissues were collected for analysis of oxidative stress markers, gene expression, and histopathology. There is a significant increase in the levels of liver enzymes, AFP, and TNF-ἁ. This was conveyed by a significant increase of IL-1 and caspase-3, elevation of MDA and reduction of GSH, and suppression of Bcl2 and elevation of Bax expression. All parameters in butanol, ethyl acetate fractions, and isolated acacetin 7-O-ß-glucopyranoside (major constituents) of T. erecta L. were significantly improved to values close to those of the control group.

Protective effects of Saraca indica L. leaves extract (family Fabaceae) against gamma irradiation induced injury in the kidney of female albino rats.

This work was designed to estimate the protective effect of Saraca indica L. leaves ethanolic exract against γ-irradiation induced renal damage in rats. Phytochemical examinations of S. indica L. leaves extract resulted in the separation of three flavanone glycosides: Astilibin (1), Neoastilbin (2), and Eriodictyol-7-O-α-l-rhamnopyranoside (3); two flavonols: Quercetin (4) and Quercetin-3-O-α-l-arabinopyranosyl-(1'''-6'')-O-ß-D-galactopyranoside (5) in addition of Gallic acid (6) and methyl gallate (7). Their structures elucidated by chemical evidences and spectroscopic analysis (1 and 2D-NMR, -ESI-MS, UV). Female rats were used and classified into: control, Ext (200 mg/kg body wt/day orally for 7 days), IRR (8Gy), Ext + IRR, and Sily+IRR groups (received silymarin 50 mg/kg b.wt orally as reference drug). Results showed that S. indica L. leaves extract ameliorated the kidney function tests, hs-CRP, IL-1ß, ACE, TNF-α, GSH, and MDA as well as, decreased the histopathological changes of kidney. In conclusion, S. indica L. leaves extract had a renoprotective activity against irradiation induced renal injury due to its flavononid contents.

Chemical Profiling of Polyphenolics in Eucalyptus globulus and Evaluation of Its Hepato-Renal Protective Potential Against Cyclophosphamide Induced Toxicity in Mice.

Cyclophosphamide (CP) is a potent anti-neoplastic and immunosuppressive agent; however, it causes multi-organ toxicity. We elucidated the protective activities of Eucalyptus globulus (EG) leaf extract against CP-induced hepato-renal toxicity. Mice were treated with EG for 15 days plus CP on day 12 and 13 of the experiment. Using HPLC-DAD-ESI-MS/MS, 26 secondary metabolites were identified in EG leaf extract. Out of them, 4 polyphenolic compounds were isolated: (1) 4-(O-ß-d-xylopyranosyloxy)-3,5-di-hydroxy-benzoic acid, (2) 4-(O-α-l-rhamnopyranosyloxy)-3,5-di-hydroxy-benzoic acid, (3) gallic acid, and (4) methyl gallate. Effects of EG extract on biochemical parameters, gene expression, and immune-histopathological changes were assessed in comparison to mesna positive control. Results showed that EG improved CP-increased serum ALT, AST, creatinine, and blood urea nitrogen levels. The hepatic and renal tissue levels of MDA, nitric oxide, protein carbonyl, TNF-α, IL-6, and immunohistochemical expression of nuclear factor kappa-B (NF-kB) and caspase-3 were reduced. Also, hepatic and renal GSH contents, and nuclear factor E2-related factor 2 (NRf2)/ hemoxygenase-1 (HO-1) signaling levels were increased. Histopathological findings supported our findings where hepatic and renal architecture were almost restored. Results revealed the protective effects of EG against CP-induced hepato-renal toxicity. These effects may be related to EG antioxidant, anti-inflammatory, and anti-apoptotic properties coupled with activation of Nrf2/HO-1 signaling.