Inhibition of carrageenan-induced acute inflammation in mice by oral administration of anthocyanin mixture from wild mulberry and cyanidin-3-glucoside

Abstract

Anthocyanins are flavonoids which demonstrated biological activities in in vivo and in vitro models. Here in the anti-inflammatory properties of an anthocyanin-enriched fraction (AF) extracted from wild mulberry and the cyanidin-3-glucoside (C3G), the most abundant anthocyanin in diet, were studied in two acute inflammation experimental models, in the peritonitis and in the paw oedema assays, both of which were induced by carrageenan (cg) in mice. In each trial, AF and C3G (4 mg/100 g/animal) were orally administered in two distinct protocols: 30 min before and 1 h after cg stimulus. The administration of both AF and C3G suppresses the paw oedema in both administration times (P < 0.05). In the peritonitis, AF and C3G reduced the polymorphonuclear leukocytes (PMN) influx in the peritoneal exudates when administered 1 h after cg injection. AF was more efficient reducing the PMN when administered 30 min before cg. Both AF and C3G were found to suppress mRNA as well as protein levels of COX-2 upregulated by cg in both protocols, but the inhibitory effect on PGE2 production in the peritoneal exudates was observed when administered 30 min before cg (P < 0.05). Our findings suggest that AF and C3G minimize acute inflammation and they present positive contributions as dietary supplements.

Figure 1

HPLC-DAD of cyanidin-3-glucoside (C3G) at 525 nm (a) and anthocyanin profile of AF at 525 nm (b) and 270 nm (c). Peaks were identified by MS/MS as C3G (structure showed), cyanidin-3-rutinoside (C3R), and rutin. Abbreviations: Hydroxycinnamic acid derivate (HcAc derivate) and pelargonidin (Plg).

Figure 2

Effect of C3G and AF on carrageenan-induced paw oedema. Footpad oedema was induced by injection of cg (0.5% w/v in saline, i.pl.) and was evaluated by plethysmometry. C3G or AF (4 mg/100 g body weight) or indomethacin (1 mg/kg, i.v.) or saline (control oedema) was orally administered in two different times: 30 min before (a) and 1 h after (b) i.pl. injection of cg. The increase paw size was measured 1, 2, 3, 4, and 5 h after cg injection. The time zero corresponds to cg injection. The results were expressed as mean ± EPM of 8 mice. Statistically significant difference regarding saline (control group) and C3G and AF and Indomethacin groups is expressed as *P < 0.05.

Figure 3

Effect of C3G and AF on carrageenan-induced leukocyte influx into peritoneal cavity. Groups of mice received C3G or AF (4 mg/100 g body weight) or indomethacin (4 mg/100 g body weight) or saline (control) by gavage in two different times: 30 min before (a, b, and c) and 1 h after (d, e, and f) cg or saline injection into the peritoneal cavity. Total leukocyte (a, d), PMN (b, e) and MN (c, f) cell counts were determined in peritoneal washes collected 3 h after cg or saline i.p. injection, as described in Section 2. Values are mean ± EPM of 8 animals. ^# P < 0.05 when compared with the corresponding group without cg stimulus (saline + saline). *P < 0.05 when compared with the corresponding control group (saline + cg).

Figure 4

Effect of C3G and AF on carrageenan-induced cyclooxygenase-2 expression in peritoneal leukocytes. Groups of mice received C3G or AF (4 mg/100 g body weight) or saline by gavage in two different times: 30 min before or 1 h after cg (0.3% w/v) or saline injection into the peritoneal cavity. Peritoneal leukocytes were collected 3 h after i.p. administration of either cg or saline and whole cells were analyzed for COX-2 expression by RT-PCR and western blotting performed, as described in Section 2. (a and c) RT-PCR of COX-2, and β-actin (loading control); Bar graph shows densitometric analysis of mRNA COX-2. (b and d) Western blotting of COX-2, and β-actin (loading control) of leukocytes present in the inflammatory exudates; bar graph shows densitometric analysis of protein COX-2. The densities (in densitometry units) were normalized with those of β-actin. Results were expressed as mean ± EPM from 8 mice. ^# P < 0.05 when compared with the corresponding group without cg stimulus (saline + saline). *P < 0.05 when compared with the corresponding control group (saline + cg).

Figure 5

Effect of C3G and AF on carrageenan-released PGE₂ in peritonitis. Groups of mice received C3G or AF (4 mg/100 g body weight) or indomethacin (4 mg/100 g body weight) or saline (control) by gavage in two different times: 30 min before (a) or 1 h after (b) cg or saline (control) injection into the peritoneal cavity. PGE₂ was quantified in peritoneal exudates collected after 3 h of cg or saline administration. Values are mean ± EPM of 8 mice. ^# P < 0.05 when compared with the corresponding group without cg stimulus (saline + saline). *P < 0.05 when compared with the corresponding control group (saline + cg).