Purpose: In the current study, the evaluation of anti-inflammatory (in vitro) activity of chemically synthesized Urolithin-C was examined. Methods: The synthesis of Urolithin-C (3,8,9-trihydroxy-6H-benzo[c]chromen-6-one) was carried out by chemical method and it was characterized using various techniques. The anti-inflammatory efficacy of synthesized Urolithin-C was studied by membrane stabilization, protein denaturation and protease inhibition assays. In addition, MTT (3-[4,5-dimethylthiazol-2-yl] 2,5-diphenyl tetrazolium bromide) assay was employed to evaluate the cytotoxic effect of Urolithin-C. The anti-inflammatory property of Urolithin-C was further examined using LPS (Lipopolysaccharide) induced RAW 264.7 (Mouse macrophage) cells. Furthermore, the anti-inflammatory properties of Urolithin-C was studied by quantifying pro/anti-inflammatory cytokines using ELISA (enzyme-linked immunosorbent assay). The mechanism of action of Urolithin-C on NF-κB (Nuclear Factor-kappa B) translocation was studied using CLSM (confocal laser scanning microscopy). While gene expression pattern was analyzed using RT-qPCR (Reverse Transcription quantitative Polymerase Chain Reaction). Results: In comparison to the positive control aspirin, Urolithin-C showed a strong anti-inflammatory effect by preventing lysosomal degradation, protein denaturation and inhibition of protease. Furthermore, at the higher dose (200 μg/mL), Urolithin-C was found to be toxic to the mouse macrophages; however, at lower concentration (25 μg/mL) it did not cause toxicity to the said. Thus, 25 μg/mL of Urolithin-C was used to assess the anti-inflammatory activity. Interestingly, Urolithin-C efficiently reduced the expression of pro-inflammatory inducible enzyme (Cox-2), cytokines (IL-2, IL-6, and TNF-alpha) and increased the anti-inflammatory cytokine (TGF-beta1), compared to positive control diclofenac (DFC). Urolithin-C effectively abrogated the NF-κB p65 phosphorylation and its translocation to the nucleus as well. Most importantly, Urolithin-C efficiently suppressed the expression of pro-inflammatory genes and elevated the expression of anti-inflammatory gene. Conclusion: Urolithin-C exhibited anti-inflammatory properties by regulating the expression of pro-inflammatory inducible enzyme, cytokines and the translocation of NF-κB p65 to the nucleus.

Ellagitannins (ETs) are the group of tannins found in fruits, nuts and seeds; they undergo hydrolysis in the human digestive tract to form ellagic acid (EA). EA further converted into various forms of Urolithin derivatives (A, B, C, D, M-5, M-6 and M-7) by gut micro biota. Perhaps, the pharmacological properties of said Urolithins were extensively studied, while, little is known about the therapeutic role of synthetic Urolithin-C on oxidative stress induced eryptosis and tissue damage. Therefore, in the current study we investigated the ameliorative role of chemically syn-thesized Urolithin-C on sodium nitrite and Diclofenac (DFC) induced eryptosis and vital organ damage respectively. Urolithin-C was synthesized using chemical method and characterized by High-Performance Liquid Chromatography (HPLC), Nuclear Magnetic Resonance (NMR), Fourier Transform Infrared Spectroscopy (FTIR), and Liquid Chromatography Mass Spectroscopy (LC-MS). The synthesized Urolithin-C scavenged free radicals (DPPH, H2O2) and reduced ferric iron to ferrous iron, revealed its antioxidant activity. The observed antioxidant potential of Uro-lithin-C was investigated in sodium nitrite induced stress in RBC and Diclofenac induced tissue damage in rats. Interestingly, Urolithin-C normalized the sodium nitrate-induced oxidative stress in red blood cells by regulating stress markers, such as lipid peroxidation (LPO), total thiol (TT), and protein carbonyl content (PCC), and endogenous antioxidant enzymes superoxide dis-mutase (SOD) and catalase (CAT) in a dose dependent manner. Similar effect was also observed in the tissue homogenates of liver, kidney, heart and pancreas taken from the Diclofenac injected Sprague Dawley rats. The tissue sections (liver, kidney, heart and pancreas) obtained from the Diclofenac (DFC) received rats showed massive destruction of hepatocytes, nephrons, cardiocytes and pancreas. However, Urolithin-C received rats tissue sections showed normal histology compared to the positive (Silymarin) control treated groups. In addition, Urolithin-C regulated the key tissue specific biochemical markers in DFC injected Sprague Dawley rats serum strengthened its tissue protective role. In conclusion, chemically synthesized Urolithin-C regulated NaNO2 and diclofenac induced oxidative stress in RBC and vital organs through its antioxidant property.