Immunomodulatory efficacy of Cousinia thomsonii C.B. Clarke in ameliorating inflammatory cascade expressions.

ETHNOPHARMACOLOGICAL RELEVANCE: Cousinia thomsonii is traditionally known for treating various diseases including joint pain, swelling, body ache, asthma, dermatitis, cough and arthritis. AIM OF THE STUDY: This study employs lipopolysaccharide induced inflammatory wistar-rat model to evaluate efficacy of Cousinia thomsonii active-extracts on the expression of crucial inflammatory markers viz. iNOS, PPAR-γ, Rel-A, COX-2 and serum analysis of CRP. MATERIALS AND METHODS: Methanol and aqueous extracts were administered orally at 25, 50, 100 mg/kg doses for 21 days. Serum was collected on 22nd day and rats were sacrificed to extract paw tissues. Dexamethasone (0.5 mg/kg) served as positive control. Immunoblotting and qPCR was used for expression analysis of iNOS, PPAR-γ, Rel-A, COX-2 respectively. ELISA was employed for evaluating CRP levels. Discovery-studio and Auto-Dock-Vina were used to check docking interactions of various identified compounds. RESULTS: Both extracts caused dose-dependent decline in iNOS, Rel-A, COX-2 and CRP levels, while there was a dose-dependent increase in PPAR-γ expression. Methanol extract dominated immunomodulatory potential as compared with the aqueous extract. The results of the GCMS revealed the presence of ten compounds. Some of these compounds include 1-Octacosanol, Ethyl Linoleate, 1-Heptacosanol, 1-Hexadecanol, 1-Dodecanol and Behenic alcohol having strong anti-inflammatory, antimicrobial, anti-acne and anti-viral activities. Molecular Docking scores were calculated between each target protein and selected compounds. The best affinity/interactions were observed between 1-Octacosanol towards iNOS, PPAR-γ, Rel-A, COX-2 and CRP with binding energy of -10.4, -11.1, -8.6, -9.9 and -7.9 (kcal/mol) respectively. These compounds may act as strong inhibitors for iNOS, Rel-A, COX-2 and CRP or as agonists for PPAR-γ; thereby inducing anti-inflammatory/immuno-modulatory activities. CONCLUSIONS: The results indicate that Cousinia thomsonii contains therapeutically active compounds and thus could serve as potential therapeutic regimen against diverse inflammatory diseases.

Toxicological impact of sodium benzoate on inflammatory cytokines, oxidative stress and biochemical markers in male Wistar rats.

Sodium benzoate is a widely used food and pharmaceutical preservative due to its antibacterial and antifungal activity. In the present study effect of different concentrations of sodium benzoate on hepatic antioxidants, inflammatory cytokines (TNF-α, IFN-γ, IL-1ß and IL-6), biochemical markers and histopathology of liver was evaluated. Twenty five adult rats (aged 1-2 months) with 5 rats per group were randomly distributed into 5 groups. Group 1 rats were used as control and all groups (1-5) were provided with water and fed ad libitum. In addition to usual water and food, rats of group 2, 3, 4 and 5 were treated with 70, 200, 400 and 700 mg/kg b.wt of sodium benzoate once a day via oral gavage for 30 days. Our results showed that activity of glutathione peroxidase (GPx), catalase (CAT), glutathione-s-transferase (GST), glutathione reductase (GR) and superoxide dismutase (SOD) in rats decreased significantly when treated with 200, 400 and 700 mg/kg b.wt of sodium benzoate. Increase in the concentration of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, serum total protein, albumin, globulin, urea and creatinine was found to be dose dependent. Severe histopathological damage was observed in the hepatic tissue at higher concentrations of sodium benzoate. It was noticed that high concentrations of sodium benzoate (200, 400 and 700 mg/kg b.wt) produce significant increase in inflammatory cytokine markers (TNF-α, IFN-γ, IL-1ß and IL-6) in comparison to control. Sodium benzoate at concentration of 70 mg/kg b.wt did not produce any significant changes in any of the above studied parameters.

Toxicological analysis of synthetic dye orange red on expression of NFκB-mediated inflammatory markers in Wistar rats.

Orange red is a food and cosmetic coloring agent made by the amalgamation of two azo dyes carmoisine and sunset yellow. The current study demonstrates the effect of different concentrations of orange red on antioxidant status, inflammatory biomarkers (TNFα, IFNγ, IL1ß, IL6, COX-2, iNOS, and NFκB/p65), biochemical enzymes, and liver histology. In totality, 25 male Wistar rats were procured and arbitrarily alienated into 5 different groups each with 5 animals. Group I was taken as the control. Groups II-V were designated as treatment groups. Groups II and III were administered with (5 and 25 mg/kg b.wt.) and groups IV and V with (150 and 300 mg/kg b.wt.) of orange red via oral gavage for 30 days. It was observed that both low and high concentrations of orange red (25, 150, and 300 mg/kg) remarkably augmented the levels of serum inflammatory cytokines (TNFα, IFNγ, IL1ß, and IL6) and the protein and gene expression of COX-2, iNOS, and NFκB/p65. A significant decrease in glutathione reductase, glutathione peroxidase, glutathione-S-transferase, superoxidase dismutase, and catalase activity was observed with increasing concentration of orange red. Furthermore, an increase in the level of several vital biochemical parameters and damage severity to hepatic tissue was also found dose dependent.

Active Cousinia thomsonii Extracts Modulate Expression of Crucial Proinflammatory Mediators/Cytokines and NFκB Cascade in Lipopolysaccharide-Induced Albino Wistar Rat Model.

INTRODUCTION: Chronic inflammation is implicated in a multitude of diseases, including arthritis, neurodegeneration, autoimmune myositis, type 2 diabetes, rheumatic disorders, spondylitis, and cancer. Therefore, strategies to explore potent anti-inflammatory regimens are pivotal from a human-health perspective. Medicinal plants represent a vast unexplored treasure trove of therapeutically active constituents with diverse pharmacological activities, including anti-inflammatory properties. Herein, we evaluated Cousinia thomsonii, an edible medicinal herb, for its anti-inflammatory/immunomodulatory properties. METHODS: Soxhlet extraction was used to obtain different solvent extracts (hexane, ethyl acetate, ethanol, methanol, and aqueous extract) in increasing order of polarity. In vitro anti-inflammatory assays were performed to investigate the effects of extracts on protein denaturation, proteinase activity, nitric oxide surge, and erythrocyte-membrane stabilization. The most effective extracts, ie, ethyl acetate (CTEA) and ethanol (CTE) extracts (150-200 g) were selected for further in vivo analysis using albino Wistar rats. Wistar rats received varying concentrations of CTEA and CTE (25, 50, and 100 mg/kg) for 3 weeks, followed by a single subplantar injection of lipopolysaccharide. Dexamethasone served as positive control. Blood was obtained from the retro-orbital plexus and serum separated for estimation of proinflammatory cytokines (IL6, IL1ß, IFNγ and TNFα). Western blotting was performed to study expression patterns of crucial proteins implicated in the NFκB pathway, ie, NFκB p65, NFκB1 p50, and NFκB2 p52. Histopathological examination was done and gas chromatography-mass spectrometry (GC-MS) carried out to reveal the identity of compounds responsible for ameliorating effects of C. thomsonii. RESULTS: Among five tested extracts, CTEA and CTE showed marked inhibition of protein denaturation, proteinase activity, nitric oxide surge and erythrocyte-membrane hemolysis at 600 µg/mL (P<0.001). Both these extracts showed no toxic effects up to a dose of 2,500 mg/kg. Extracts exhibited concentration-dependent reductions in expression of IL6, IL1ß, IFNγ, TNFα, NFκB-p65, NFκB1, and NFκB2 (P<0.05). Healing effects of extracts were evident from histopathological investigation. GC-MS analysis revealed the presence of important anti-inflammatory compounds, notably stigmast-5-en-3-ol, oleate, dotriacontane, ascorbic acid, n-hexadecanoic acid, and α-tocopherol, in C. thomsonii. CONCLUSION: C. thomsonii possesses significant anti-inflammatory/immunomodulatory potential by virtue of modifying levels of proinflammatory cytokines/markers and NFκB proteins.

Genotoxic effect of two commonly used food dyes metanil yellow and carmoisine using Allium cepa L. as indicator.

Food dyes are important component of food in this fast life. Metanil yellow and carmoisine are two azo dyes which are being used at an alarming rate for increasing visual appearance and consumer validity of food. There is a lot of controversy regarding the genotoxicity of these two dyes. In the present study genotoxicity of two food dyes metanil yellow and carmoisine was evaluated using Allium cepa as indicator. The effect of these two azo dyes was determined at concentration of 0.25 %, 0.50 %, 0.75 % and 1.0 % for 24 h and 48 h of exposure period using root meristematic cells of Allium cepa. Some genotoxicity parameters like mitotic indices and chromosomal aberrations were studied. It was found that both metanil yellow and carmoisine caused a significant reduction in mitotic index and also produce different kinds of chromosomal aberrations mostly at higher concentration and longer exposure period. The different kinds of aberrations that were observed in meristematic cells after treatment with both metanil yellow and carmoisine are disorientation at metaphase, metaphase stickiness, anaphase stickiness, anaphase bridge, c-mitosis and chromosome breaks. The genotoxicity of carmoisine was found very high as compared to metanil yellow at all concentrations and exposure periods. Thus it was concluded from the present study that carmoisine and metanil yellow have genotoxic activities and should be taken in very control and limited doses.