International Journal of Molecular Sciences Article Hepatoprotective Limonoids from Andiroba (Carapa guianensis) Kiyofumi Ninomiya 1,2, Seiya Miyazawa 1, Kaiten Ozeki 1, Natsuko Matsuo 1, Osamu Muraoka 1,2,3, Takashi Kikuchi 4, Takeshi Yamada 4, Reiko Tanaka 4,* and Toshio Morikawa 1,2,* 1 Pharmaceutical Research and Technology Institute, Kindai University, 3-4-1 Kowakae, Higashi-osaka, Osaka 577-8502, Japan; [email protected] (K.N.); [email protected] (S.M.); [email protected] (K.O.); [email protected] (N.M.) 2 Antiaging Center, Kindai University, 3-4-1 Kowakae, Higashi-osaka, Osaka 577-8502, Japan 3 Laboratory of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Kindai University, 3-4-1 Kowakae, Higashi-osaka, Osaka 577-8502, Japan; [email protected] 4 Laboratory of Medicinal Chemistry, Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan; [email protected] (T.K.); [email protected] (T.Y.) * Correspondences: [email protected] (R.T.); [email protected] (T.M.); Tel.: +81-72-690-1084 (R.T.); +81-6-4307-4306 (T.M.); Fax: +81-72-690-1084 (R.T.); +81-6-6729-3577 (T.M.) Academic Editor: Maurizio Battino Received: 15 January 2016; Accepted: 14 April 2016; Published: 19 April 2016 Abstract: 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. Keywords: hepatoprotective effect; limonoid; andiroba; Carapa guianensis; Meliaceae; structural requirement 1. Introduction The Meliaceae family is recognized as a rich source of limonoids, which have attracted attention from biogenetic and synthetic perspectives [1–3]. Carapa guianensis Aublet (Meliaceae), known locally as andiroba and Brazilian mahogany, is distributed in the tropical rainforests of countries such as Brazil and Colombia, etc. The woody four-cornered andiroba nut has four cells, each of which contains two to three seeds with oil-rich kernels. Extracts of the bark, flowers, and seeds have been used for centuries by the Amazonian people and exhibit various effects: anti-bacterial [4], anti-cancerous [5], anti-tumor [6], anti-fungal [6], insect repellent [7], analgesic [8], anti-malarial [9], anti-inflammatory [10], antiallergic [11], and anti-plasmoidal effects [12], as well as acute and subacute toxicity [13]. In the course of our studies on the chemical constituents from C. guianensis Int. J. Mol. Sci. 2016, 17, 591; doi:10.3390/ijms17040591 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2016, 17, 591 2 of 11 Int. J. Mol. Sci. 2016, 17, 591 2 of 11 from(Carapa the guianensis flower oil)[ [14–17],14–23], carapanolides we have isolated A–X several[18–22], limonoids,and guianolides including A and andirolides B [23] from A–Y the fromseed oil.the flowerWe have oil [14also–17 ],reported carapanolides that A–Xseveral [18 –22limonoids], and guianolides from C. Aguianensis and B [23 ]showed from the cytotoxic seed oil. [14,16,18,19,23],We have also reported antimalarial that several [15], limonoids anti-inflammatory from C. guianensis showed[17,20,22], cytotoxic and [14,16triglyceride,18,19,23], metabolism-promotingantimalarial [15], anti-inflammatory activities [21]. [17 We,20 ,22further], and evaluated triglyceride the metabolism-promoting principal gedunin-type activities limonoids [21]. fromWe further the evaluatedflower oil the of principal C. guianensis gedunin-type, gedunin limonoids (1) from[15], the6α flower-acetoxygedunin oil of C. guianensis (2) [14],, gedunin and 7-deacetoxy-7-oxogedunin(1)[15], 6α-acetoxygedunin ( (32))[ [14],14], andwhich 7-deacetoxy-7-oxogedunin were found to have protective (3)[14], effects which against were found liver to injury have inducedprotective by effects D-galactosamine against liver injury(D-GalN)/lipopolysaccharide induced by D-galactosamine (LPS) (D -GalN)/lipopolysaccharidein mice. To characterize (LPS) the mechanismsin mice. To characterize of action of the limonoids mechanisms and of the action stru ofctural limonoids requirements and the structuralfor their hepatoprotective requirements for effects,their hepatoprotective 17 related effects,limonoids 17 relatedisolated limonoids from isolatedthe flower from theoil flower of oilC. ofguianensisC. guianensis, such, such as as 7-deacetoxy-7α-hydroxygedunin (4(4)[) 21[21],], andirolide andirolide H (5H)[ (155)], [15], 6α-hydroxygedunin 6α-hydroxygedunin (6)[15 (],6) and [15], methyl and methylangolensate angolensate (7)[15], as(7 well) [15], as limonoidsas well as isolated limonoids from theisolated seed oilfrom including the seed epoxyazadiradione oil including epoxyazadiradione(8), 17β-hydroxyazadiradione (8), 17β-hydroxyazadiradione (9)[21], carapanolides (9) C[21], (10 )[carapanolides19], R (11)[21 C], (10 S ()12 [19],)[21 R], M(11 ()13 [21],)[21 S], (Q(12)14 [21],)[21 M], and(13) O[21], (15 Q)[ 21(14],) guianolide[21], and O A (15 (16) [21],)[23 ],guianolide and carapanolide A (16) [23], A ( 17and)[18 carapanolide] (Figure1). ThisA (17 study) [18] (Figurereports the1). This hepatoprotective study reports effects the hepatoprotective and possible mechanisms effects and of possible action of mechanisms1–3, as well of as action the structural of 1–3, asrequirements well as the structural for their hepatoprotective requirements for effects. their hepatoprotective effects. FigureFigure 1. 1. LimonoidsLimonoids ( (11––1717)) from from seed seed and and flower flower oils oils of C. guianensis .. 2. Results and Discussion 2. Results and Discussion 2.1. Isolation Isolation InIn previous previous studies, studies, compounds compounds 11––77 werewere isolated isolated from from the the flower flower oil oil of of C.C. guianensis guianensis [14–17],[14–17], whereas compounds 8–1717 were obtained from the seed oil [18 [18–22].–22]. In In the the present present study, study, principal limonoidslimonoids (1(–13–)3 were) were identified identified from from the seed the oil seed using oil normal using phase normal silica gelphase column silica chromatography gel column chromatographyfollowed by HPLC followed or recrystallization. by HPLC or recrystallization. 2.2. Protective Protective Effects Effects of Principal Limonoids ( 1,, 2, and 3) on Liver Injury Induced by D-GalN/LPS in Mice DD-GalN/LPS-induced-GalN/LPS-induced liver liver injuriesinjuries areare known known to to develop develop through through immunological immunological responses responses [24] [24]that progressthat progress via two via steps. two steps. First, expressionFirst, expression of inhibitors of inhibitors of apoptosis of apoptosis proteins proteins (IAPs) is (IAPs) inhibited 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 Int. J. Mol. Sci. 2016, 17, 591 3 of 11 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 mediators [nitric oxide (NO) and TNF-α from LPS-activated macrophages (Kupffer’s cells) occurs. Apoptosis of hepatocytes induced by TNF-α plays an important role in D-GalN/LPS-induced liver injury [25]. In our previous investigation of compounds from natural medicines possessing hepatoprotective activity, we reported that sesquiterpenes and diarylheptanoids from Curcuma zedoaria [26–29], saponins from Panax notoginseng [30], coumarins from Angelica furcijuga [31], acid amides from Piper chaba [32–34], acylated phenylethanoids from Cistanche tubulosa [35], and triterpenes from Potentilla anserina [36] exhibited significant protective effects against liver injuries induced by D-GalN/LPS in mice. First, the in vivo hepatoprotective effects of the principal limonoid constituents, gedunin (1), 6a-acetoxygedunin (2), and 7-deacetoxy-7-oxogedunin (3), were evaluated. As shown in Table1, 1–3 at a dose of 25 mg/kg, p.o. clearly prevented mortalities and significantly inhibited the increase in serum levels of aspartate aminotransaminase (sAST) and alanine transaminase (sALT), which served as markers of acute liver injury [37–39]. Notably, 1–3 were more potent than positive compounds curcumin [36,40–42] and silybin [43,44], which are well recognized as naturally-occurring hepatoprotective products. Table 1. Inhibitory effects of gedunin (1), 6α-acetoxygedunin