INVESTIGATIONS INTO THE ANTIOXIDANT AND ANTI-INFLAMMATORY POTENTIALS OF THE ETHANOLIC EXTRACT OF CITRUS SINENSIS, LINN (SWEET ORANGE) STEM-BARK.
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Date
2014
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Abstract
This study investigated the antioxidant and anti-inflammatory potentials of the ethanolic extract and fractions of Citrus sinensis stem-bark, its acute toxicity level and the hepatoprotective potential of the most active fraction of the ethanolic extract against acetaminophen induced acute hepatic injury in rats. This was with view to exploring theusefulness in the management of oxidative stress and inflammatory conditions.
The powdered plant material (500 g) was suspended in 2 litres of 70% (v/v) of ethanol for 72 hours with occasional agitation at room temperature. The ethanolic extract was concentrated to dryness invacuoat 40oC in a rotary evaporator, followed by partitioning with dichloromethane (DCM), ethylacetate (EA), and butanol (B) to obtain the solvent fractions (F):dichloromethane fraction (DCMF), ethylacetate fraction (EAF), butanol fraction (BF) and aqueous fraction (AF). Phytochemical screening, total phenolics and total flavonoid contents of the ethanolic extract and the test fractions were determined using established methods while the antioxidant capacities of the extract and fractions were evaluated using2, 2-diphenyl-2-picrylhydrazyl hydrate (DPPH) radical scavenging assay, Ferric reducing power (FRAP) assay, total antioxidant assay, nitric oxide inhibitory assay and2, 2’-azinobis 3-ethyl-benzothiazoline 6-sulfonate (ABTS) radical scavenging assay. The in vitro anti-inflammatory potential of the extract and fractions was evaluated by determining its ability to protect red blood cells exposed to both hypotonic and heat-induced lyses. Hepatoprotective potential was carried `out by administering the EAF at 250 and 500 mg/kg, and the standard drug Legalex 70 (Silymarin) 50 mg/kg, (p.o) for seven(7)consecutive days. Hepatic injury was then induced on seventh day by oral administration of a single acute dose of acetaminophen (2 g/kg body weight). Blood and liver tissues were collected for the assessment of plasma levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyltransferase (GGT), total protein and total bilirubin. The antioxidant enzymes and metabolite (superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione (GSH)) were measured in the liver.
The phytochemical screening showed the presence of tannins, flavonoids, terpenoids, cardiac glycosides, saponins and alkaloids in the extract and fractions. EAF showed the highest scavenging radical activities in nitric oxide assay (IC50 of 68.85 ± 1.43 µg/ml), ABTS (IC50 of 0.42 ± 0.00 mg/ml) and FRAP value (1.48 ± 0.02 mgAA/g) indicating that it was the most active fraction. The highest phenolic content was obtained in EAF(2.03 ± 0.04 mg GAE/g) while DCMF possessed the highest flavonoid content(0.38 ± 0.01 mgQUE/g) and DPPH scavenging radical of IC50 of 0.15 ± 0.03 mg/ml. In membrane stability, EAF also showed the highest membrane stability of 88.69 ± 4.17%. In the in vivo study, administration of acetaminophen showed significant changes in the level of the plasma and liver ALT, AST and GGT measured when compared to the control group, indicating liver damage. Pre-treatment with 250 and 500 mg/kg EAF and 50 mg/kg Legalex 70 for seven days significantly (p<0.05) reduced the level of plasma marker enzyme (ALT, AST and GGT), total bilirubin and total protein. Increase in antioxidant level (SOD, CAT, GPx and GSH) in liver tissue was also observed.
The study concluded that the antioxidant, anti-inflammatory and hepatoprotective ability of this plant could be attributed to the presence of flavonoids and tannins in the plant extract and fractions.
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Keywords
Antioxidant, Anti-inflammatory, Potentials, Extract of Citrus