Bioactive Guided Fractions of Annona reticulata L. bark: Protection against Liver Toxicity and Inflammation through Inhibiting Oxidative Stress and Proinflammatory Cytokines

Abstract

Herbal medicine is popularized worldwide due to its ability to cure the diseases with lesser or no side effects. North Eastern part of India comes under one of the world biodiversity hotspots which is very rich in traditional herbal medicine. Annona reticulata L. (Annonaceae) is one such plant used for the treatment of inflammatory diseases, liver ailments and diabetes by traditional healers. The present study was aimed to scientifically validate this folk knowledge and to develop an herbal remedy through evaluating bioactive guided fractions of A. reticulata (AR) bark against hepatotoxicity and inflammation using in vitro and in vivo models. Results of this study demonstrates that among all fractions of AR bark, methanol extract and its water fraction possess strong anti-oxidant ability and showed protection against CCl4 induced toxicity in HepG2 cell lines and rats. Both the fractions also exhibit dose dependent anti-inflammatory activity against carrageenan induced inflammation in rats. Water fraction showed potent response in the entire tests conducted than methanol extract, which states that polar components of the AR bark methanol extract were responsible for these activities. Further, from the experiments conducted to elucidate the mechanism of action, the results revealed that AR bark showed liver protection and anti-inflammatory response through inhibiting the oxidative stress and inflammatory cytokines.

Keywords: antioxidant; cytokines; inflammation; liver toxicity; oxidative stress.

FIGURE 1

Schematic representation of bioactive guided fractionation of Annona reticulata bark. ARBME, A. reticulata bark methanol extract; ARBHF, A. reticulata bark hexane fraction; ARBCF, A. reticulata bark chloroform fraction; ARBEAF, A. reticulata bark ethyl acetate fraction; ARBWF, A. reticulata bark water fraction; ARBMR, A. reticulata bark methanol residue.

FIGURE 2

Diphenyl picryl hydrazine (DPPH) radical scavenging activity of different fractions of A. reticulata bark. ARBME, A. reticulata bark methanol extract; ARBHF, A. reticulata bark hexane fraction; ARBCF, A. reticulata bark chloroform fraction; ARBEAF, A. reticulata bark ethyl acetate fraction; ARBWF, A. reticulata bark water fraction; ARBMR, A. reticulata bark methanol residue; Vc, ascorbic acid. All the results were expressed in mean ± SD (n = 3).

FIGURE 3

Reducing power ability of different fractions of A. reticulata bark. ARBME, A. reticulata bark methanol extract; ARBHF, A. reticulata bark hexane fraction; ARBCF, A. reticulata bark chloroform fraction; ARBEAF, A. reticulata bark ethyl acetate fraction; ARBWF, A. reticulata bark water fraction; ARBMR, A. reticulata bark methanol residue; BHT, butylated hydroxyl toluene. All the results were expressed in mean ± SD (n = 3).

FIGURE 4

Effect of different drug treatment on serum TNF-α levels. All the results were expressed in mean ± SD (n = 6). ^$$$P < 0.001 in comparison of CCl₄ alone treated animals with normal animals. ^∗∗∗P < 0.001, ^∗∗P < 0.01, and ^∗P < 0.05 in comparison of drug treated animals with CCl₄ alone treated animals. ARBME, A. reticulata bark methanol extract; ARBWF, A. reticulata bark water fraction.

FIGURE 5

Effect of different drug treatment on serum IL-1β levels. All the results were expressed in mean ± SD (n = 6). ^$$$P < 0.001 in comparison of CCl₄ alone treated animals with normal animals. ^∗∗∗P < 0.001 and ^∗∗P < 0.01 in comparison of drug treated animals with CCl₄ alone treated animals. ARBME, A. reticulata bark methanol extract; ARBWF, A. reticulata bark water fraction.

FIGURE 6

Effect of different drug treatment on serum IL-10 levels. All the results were expressed in mean ± SD (n = 6). ^$$$P < 0.001 in comparison of CCl₄ alone treated animals with normal animals. ^∗∗∗P < 0.001, ^∗∗P < 0.01, and ^∗P < 0.05 in comparison of drug treated animals with CCl₄ alone treated animals. ARBME, A. reticulata bark methanol extract; ARBWF, A. reticulata bark water fraction.

FIGURE 7

Effect of drug treatment on liver histopathology of CCl₄ intoxicated rats. (A) Liver of control rat with no CCl₄ treatment showing normal hepatocytes with no inflammatory cell infiltrate; x10. (B) Liver of rat treated with CCl4 showing formation of bridging necrosis with chronic inflammatory cells; x10. (C) Liver of rat treated with CCl4 and 100 mg/kg silymarin showing normal hepatocytes with no inflammation and necrosis; x10. (D) Liver of rat treated with CCl4 and 400 mg/kg of ARBME showing mild periportal inflammation and no necrosis; x10. (E) Liver of rat treated with CCl4 and 100 mg/kg ARBWF showing absence of inflammatory cells or necrosis; x10. ARBME, A. reticulata bark methanol extract; ARBWF, A. reticulata bark water fraction.

FIGURE 8

Effect of A. reticulata bark fractions on carrageenan induced paw edema over 5 h. All the results were expressed in mean ± SD (n = 6). S P < 0.05 in comparison of drug treated animals with saline treated animals. ARBME, A. reticulata bark methanol extract; ARBWF, A. reticulata bark water fraction.

FIGURE 9

Effect of different drug treatment on serum IL-1β levels of carrageenan induced paw edema rats. All the results were expressed in mean ± SD (n = 6). ^∗∗∗P < 0.001 and ^∗P < 0.05 in comparison of drug treated animals with saline treated animals. ARBME, A. reticulata bark methanol extract; ARBWF, A. reticulata bark water fraction.

FIGURE 10

Effect of different drug treatment on serum TNF-α levels of carrageenan induced paw edema rats. All the results were expressed in mean ± SD (n = 6). ^∗∗∗P < 0.001 and ^∗P < 0.05 in comparison of drug treated animals with saline treated animals. ARBME, A. reticulata bark methanol extract; ARBWF, A. reticulata bark water fraction.