Hepatoprotective Limonoids from Andiroba (Carapa guianensis)

Three gedunin-type limonoids, gedunin (1), 6α-acetoxygedunin (2), and 7-deacetoxy-7-oxogedunin (3), which were isolated from the seed and flower oils of andiroba (Carapa guianensis Aublet, Meliaceae), exhibited hepatoprotective effects at doses of 25 mg/kg, p.o. against d-galactosamine (d-GalN)/lipopolysaccharide (LPS)-induced liver injury in mice. To characterize the mechanisms of action of 1–3 and clarify the structural requirements for their hepatoprotective effects, 17 related limonoids (1–17) isolated from the seed and/or flower oils of C. guianensis were examined in in vitro studies assessing their effects on (i) d-GalN-induced cytotoxicity in primary cultured mouse hepatocytes, (ii) LPS-induced nitric oxide (NO) production in mouse peritoneal macrophages, and (iii) tumor necrosis factor-α (TNF-α)-induced cytotoxicity in L929 cells. The mechanisms of action of 1–3 are likely to involve the inhibition of LPS-induced macrophage activation and reduced sensitivity of hepatocytes to TNF-α; however, these compounds did not decrease the cytotoxicity caused by d-GalN. In addition, the structural requirements of limonoids (1–17) for inhibition of LPS-induced NO production in mouse peritoneal macrophages and TNF-α-induced cytotoxicity in L929 cells were evaluated.


Protective Effects of Principal Limonoids (1, 2, and 3) on Liver Injury Induced by D-GalN/LPS in Mice
D-GalN/LPS-induced liver injuries are known to develop through immunological responses [24] that progress via two steps. First, expression of inhibitors of apoptosis proteins (IAPs) is inhibited by administration of D-GalN through depletion of uridine triphosphate and increased sensitivity of hepatocytes to tumor necrosis factor-α (TNF-α. Second, release of pro-inflammatory

Animals
Male ddY mice (Kiwa Laboratory Animal Co., Ltd., Wakayama, Japan) were housed at a constant temperature of 23˘2˝C and were fed a standard laboratory chow (MF, Oriental Yeast Co., Ltd., Tokyo, Japan). The animals were fasted for 24 h prior to the beginning of the experiment but were allowed free access to tap water. All experiments were performed with conscious mice unless otherwise noted. The experimental protocol was approved by the Experimental Animal Research Committee of Kindai University (KAPR-26-001, 1 April 2014).

Effects on D-GalN/LPS-induced Liver Injury in Mice
The method described by Tiegs et al. [57] was modified and used for this study [33,35,36]. Curcumin [36] and silybin were used as reference compounds.

Effects on Production of NO in LPS-induced Mouse Peritoneal Macrophages
Assay for NO production in TGC-induced mouse peritoneal macrophages was performed as described previously [33,35,36]. N G -Monomethyl-L-arginine (L-NMMA) and caffeic acid phenethyl ester (CAPE) were used as reference compounds [33,36].

Effects on Cytotoxicity Induced by TNF-α in L929 Cells
Assay for the TNF-α-induced cytotoxicity in L929 cells was performed as described previously [33,35,36]. Silybin was used as a reference compound [36].

Statistics
All data are expressed as means˘S.E.M. The data analysis was performed with an one-way analysis of variance (ANOVA), followed by Dunnett's test. Probability (p) values less than 0.05 were considered significant.