Methanol extract of Muntingia calabura leaves attenuates CCl4-induced liver injury: possible synergistic action of flavonoids and volatile bioactive compounds on endogenous defence system

Abstract

Context: Muntingia calabura L. (Muntingiaceae) exerts antioxidant and anti-inflammatory activities, thus, it might be a good hepatoprotective agent.

Objective: This study investigates the effect of methanol extract of M. calabura leaves (MMCL) on hepatic antioxidant and anti-inflammatory activities in CCl₄-induced hepatotoxic rat.

Materials and methods: Sprague Dawley rats (n = 6) were treated (p.o.) with 10% DMSO (Groups 1 and 2), 50 mg/kg N-acetylcysteine (Group 3) or, 50, 250, or 500 mg/kg MMCL (Groups 4-6) for 7 consecutive days followed by pretreatment (i.p.) with vehicle (Group 1) or 50% CCl₄ in olive oil (v/v) (Groups 2-6) on day 7th. Plasma liver enzymes and hepatic antioxidant enzymes and pro-inflammatory cytokines concentrations were measured while liver histopathology was examined.

Results: MMCL, at 500 mg/kg, significantly (p < 0.05) ameliorated CCl₄-induced hepatotoxicity by decreasing the plasma level of alanine transaminase (429.1 versus 168.7 U/L) and aspartate transaminase (513.8 versus 438.1 U/L) as well as the tissue level of nitric oxide (62.7 versus 24.1 nmol/g tissue). At 50, 250, or 500 mg/kg, MMCL significantly (p < 0.05) reduced the tumour necrosis factor α (87.8 versus 32.7 pg/mg tissue), interleukin-1β (1474.4 versus 618.3 pg/mg tissue), and interleukin-6 (136.7 versus 30.8 pg/mg tissue) while increased the liver catalase (92.1 versus 114.4 U/g tissue) and superoxide dismutase (3.4 versus 5.5 U/g tissue). Additionally, qualitative phytochemicals analysis showed that MMCL contained gallic acid, ferulic acid, quercetin, and genistein.

Discussion and conclusions: MMCL ability to attenuate CCl₄-induced hepatotoxicity could be helpful in the development of hepatoprotective agents with fewer side effects.

Keywords: GCMS; Muntingiaceae; UHPLC-ESI; antioxidant activity; hepatoprotective activity; oxidative stress markers; phytoconstituents; pro-inflammatory mediators.

Figure 1.

Microscopic analysis of liver section of normal and CCl₄-induced hepatotoxic rats following pretreatment with 10% DMSO, NAC or MMCL. (A) Liver section of normal control animal exhibits normal hepatic cells each with well-defined cytoplasm, prominent nucleus and nucleolus and well brought central vein; (B) liver section of CCl₄-induced hepatotoxic rats pretreated with 10% DMSO shows total loss of hepatic architecture with massive centrilobular hepatic necrosis, massive fatty changes vacuolization (steatosis), Kupffer cell hyperplasia and apoptosis; (C) liver section of CCl₄-induced hepatotoxic rats pretreated with 50 mg/kg body weight of NAC demonstrates mild centrilobular hepatic necrosis, mark steatosis and mild haemorrhage; (D) liver section of CCl₄-induced hepatotoxic rats pretreated with 50 mg/kg MMCL exhibits total loss of hepatic architecture with massive centrilobular hepatic necrosis, massive steatosis, kupffer cell hyperplasia, massive inflammation, massive haemorrhage and apoptosis; (E) liver section of CCl₄-induced hepatotoxic rats pretreated with 250 mg/kg MMCL exhibits mark centrilobular hepatic necrosis, mild steatosis, marked inflammation, and mild haemorrhage; and (F) liver section of CCl₄-induced hepatotoxic rats pretreated with 500 mg/kg MMCL shows normal liver architecture with mild centrilobular hepatic necrosis, mild inflammation (100× magnification). CV: central vein; N: necrosis; I: inflammation; S: steatosis.

Figure 2.

Total ion chromatography (TIC) of MMCL obtained using the UHPLC instrument in negative ion mode.

Figure 3.

GCMS profile of MMCL revealed the presence of several peaks. Eight of these peaks belong to volatile compounds namely (1) methanone, 6-phenanthridinylphenyl-, oxime, (E)- (retention time (R_T) = 18.78 min), (2) erucylamide/13-docosenamide, (Z) (R_T = 19.74 min), (3) 1,1′:3′,1″-terphenyl-4′,6′-dimethoxy-2,2″,3,3″,5,5″,6,6″-octamethyl- (R_T = 21.55 min), (4) cholest-4-en-3-one, 2-hydroxy-, (2α)- (R_T = 22.10 min), (5) α-tocopherol (R_T = 24.26 min), (6) 2-(acetoxymethyl)-3-(methoxycarbonyl) biphenylene (R_T = 28.64 min), (7) 6-methyl-2-phenylindole (R_T = 29.11 min), and (8) 5-methyl-2-phenylindolizine (R_T = 30.30 min).