Investigations into the antioxidant and anti-inflammatory potentials of the ethanolic extract of Citrus sinensis, Linn. (sweet orange) stem-bark

Abstract

This study was designed to investigate the antioxidant and anti-inflammatory potentials of the ethanolic extract and fractions of Citrus sinensis stem-bark, investigate and to evaluate the hepatoprotective  potential of the most active fraction (EAF) of the ethanolic extract against acetaminophen-induced acute  hepatic injury. The ethanolic extract and the fractions were screened for their secondary metabolites  using established methods. The total phenolics and total flavonoid contents of the ethanolic extract and the fractions the antioxidant capacities namely DPPH radical scavenging assay, Ferric reducing power (FRAP) assay, Total antioxidant assay, Nitric oxide inhibitory assay and ABTS (2, 2-azino-bis-(3- ethylbenzothiazoline-6-sulphonic acid) radical scavenging assay and the in vitro anti-inflammatory  potential of the extract and fractions were all evaluated by established methods, Hepatoprotective  potential of the ethyl acetate fraction was carried out by administering the EAF and standard drug Legalex 70 (Silymarin) (p.o) for 7 consecutive days. The phytochemical screening showed the presence of  tannins, flavonoids, terpenoids, cardiac glycosides, saponins and alkaloids in the extract and fractions.  The result also showed that the ethyl acetate fraction  showed the highest scavenging radical activities in Nitric oxide with an IC₅₀ of 68.85±1.43μg/ml, ABTS with an IC₅₀ of 0.42±0.00mg/ml and FRAP value  1.48±0.02mgAA/g also indicated the ethyl acetate fraction to be the most active. Ethyl acetate fraction gave the highest phenolic content (2.03±0.04 mg GAE/g of  fraction) while dichloromethane fraction showed the highest flavonoid content (0.38±0.01mgQUE/g) and DPPH scavenging radical with an IC₅₀ of  0.15±0.03mg/ml. In membrane stability, ethylacetate fraction also showed the highest stability of 88.69±4.17%. Additionally in the in vivo study, administration of acetaminophen showed significant changes in the level of the plasma and liver parameters measured when compared to the control group and it was indicative of liver damage. Pre-treatment with ethylacetate fraction (250 and 500mg/kg) and 50mg/kg legalex 70 for 7 days significantly (p<0.05) reduced the level of plasma marker enzyme (ALT, AST and GGT), total bilirubin and total protein. Increased antioxidant level (SOD, CAT, GPx and GSH) in liver tissue was also observed. In conclusion, it is probable that the antioxidant, anti-inflammatory and hepatoprotective ability of this plant extract could be attributed to the presence of flavonoids and tannins in the plant extract/fractions.