Carapanosins A–C from Seeds of Andiroba (Carapa guianensis, Meliaceae) and Their Effects on LPS-Activated NO Production

Two new phragmalin-type limonoids, Carapanosins A and B (1 and 2), and a new gedunin-type limonoid, Carapansin C (3), together with five known limonoids (4–8) were isolated from the oil of Carapa guianensis AUBLET (Meliaceae) seeds, a traditional medicine in Brazil and Latin American countries. Their structures were elucidated on the basis of spectroscopic analyses using 1D and 2D NMR techniques and HRFABMS. Compounds 1–8 were evaluated for their effects on the production of NO in LPS-activated mouse peritoneal macrophages. The NO inhibitory assay suggested that Compounds 3, 6, and 8 may be valuable as potential inhibitors of macrophage activation.


Introduction
Limonoids have mainly been found in Meliaceae and Rutaceae plants, and are modified triterpenoids that originate from a precursor with 4,4,8-trimethyl-17-furylsteroids that typically contains four highly oxidized (A, B, C, and D) rings. Meliaceae plants are distributed in tropical regions throughout the world [1]. Carapa guianensis AUBLET (Meliaceae) is a popular medicinal plant known as "Andiroba" in Brazil, and is in the same family as mahogany. Andiroba is a tall rainforest tree that grows up to 40 m in height. It is in the same family as mahogany and has been called Brazilian mahogany or bastard mahogany due to their similarities. The andiroba tree produces a brown, ligneous, quadrilateral nut that is approximately 3 to 4 in. in diameter and has the appearance of a chestnut. The nut from andiroba contains several oil-rich kernels and seeds that are composed of an~60% pale yellow oil. The seed oil of andiroba was previously reported to exhibit highly efficient analgesic [2], anti-bacterial [3], anti-inflammatory [4], anti-cancerous [5], anti-tumor, anti-fungal [6], and anti-allergic properties [7] and was also found to be effective against wounds, bruises, herpes ulcers, rheumatism, ear infections, and insect bites as a repellent [8,9]. We previously reported Carapanolides A and B [10], guianolide A and B [11], Carapanolides C-I [12], Carapanolides J-L [13], Carapanolides M-S [14], and Carapanolides T-X [15] in the seed oil of andiroba. Our continuing research on the seed oil of andiroba revealed the structures of two new phragmalin-type limonoids, Carapanosins A (1) and B (2), a new gedunin-type limonoid, Carapanosin C (3), and five known limonoids (4)(5)(6)(7)(8). We herein describe the isolation and structural elucidation of the new limonoids as well as their inhibitory effects of NO production.
Carapanosin A (1), a colorless crystal, had the molecular formula of C36H42O16 (m/z 731.2551 [M + H] + , calcd. 731.2551) as determined by HRFABMS. The IR absorption bands indicated the existence of hydroxy group (νmax 3647 cm −1 ) and several carbonyl groups (1751, 1700 and 1652 cm −1 ). The UV spectrum showed a furan ring and an αβ-unsaturated δ-lactone at λmax 208 nm (log ε 3.52) and 235.5 nm (log ε 3.54). 1 H-and 13 C-NMR spectra (   Figure 1). Intense NOESY correlation between H-3 and Me-28, and H-29pro-S; between H-5β and H-6, H-12, H-30β, and Me-28; between H-6 and H-30β; between H-12 and H-5β, H-17β, and H-30β; and between Me-19 and H-6, H-29pro-R, and Me-32 revealed an acetyl group at C-3 in the β orientation, C-12, a hydroxyl group at C-2, and a 2-methylpropanoyl group at C-30 in the α orientation. In addition, significant NOEs were observed between H-6 [δH 6.07 (brs)] and H-11β, H-12β and H-17β; therefore, C-6 was presumed to be in an R-configuration, which was consistent with Carapanolide N 14 .  The IR spectrum showed the presence of hydroxyl, ester groups, and an αβ-unsaturated δ-lactone at ν max 3566, 1734, and 1663 cm −1 ; and the UV spectrum indicated the presence of a furan ring and an αβ-unsaturated δ-lactone at λ max 213 nm (log ε 3.84) and 237.5 nm (log ε 3.62). The 1 H-and 13 C-NMR spectra (Table 1) (Table 1) of 2 were very similar to those of 1, so 2 is estimated to be phragmalin-1,8,9-orthoacetate, except for the absence of a hydroxy group and presence of an acetyl group at C-6 [δ H 6.31 (brd), δ C 71.2 (d)]. In the NOESY spectrum, significant NOEs were observed between H-6 and H-11α, and Me-19, so the configuration of H-6 was determined to have the same R as Compound 1 and Carapanolide N [14], and its relative structure was established, as shown in Figure 2. (log ε 3.84) and 237.5 nm (log ε 3.62). The 1 H-and 13 C-NMR spectra (  [14], and its relative structure was established, as shown in Figure 2.   [18,19]. These findings suggest that the planer structure of 3 was as the same as that of 9. However, major differences were detected in the 1 H-and 13 C-NMR spectra between 3 and 9. These differences between 3 and 9 were particularly prominent in C-12 (δ C 23.2 in 3; δ C 37.2 in 9), C-9 (δ C 37.2 in 3; δ C 45.5 in 9), and C-22 (δ C 125.0 in 3: δ C 110.1 in 9), and slight differences were observed in C-5 (δ C 43. Juss (Neem) [18,19]. These findings suggest that the planer structure of 3 was as the same as that of 9. However, major differences were detected in the 1 H-and 13 C-NMR spectra between 3 and 9. These differences between 3 and 9 were particularly prominent in C-12 (δC 23.2 in 3; δC 37.2 in 9), C-9 (δC 37.     Macrophages may be a potential therapeutic target for inflammatory diseases [20]. Activated macrophages release pro-inflammatory mediators, such as NO, reactive oxygen species, interleukin-1 beta, tumor necrosis factor-alpha, and other inflammatory mediators, which play important roles in biological defense. However, the overexpression of these mediators has been implicated in diseases such as osteoarthritis, rheumatoid arthritis, and diabetes because the increased production of pro-inflammatory mediators has been shown to induce severe or chronic inflammation [21]. Eight limonoids, and L-NMMA, an inducible nitric oxide synthase (iNOS) inhibitor, were evaluated for their inhibitory effects on NO production ( Figure 4). All tested compounds did not exhibit cytotoxicity (Cell viability 92.7%-100.4% at 30 µM). Of these, Compounds 3, 6, and 8 exhibited stronger inhibitory activity on NO production (IC 50 3: 13.7 µM; 6: 4.9 µM; 8: 10.8) than L-NMMA (IC 50 23.9 µM). On the other hand, Compounds 4 and 7 showed moderate activity on NO production (IC 50 4: 25.5 µM; 7: 28.9 µM). Macrophages may be a potential therapeutic target for inflammatory diseases [20]. Activated macrophages release pro-inflammatory mediators, such as NO, reactive oxygen species, interleukin-1 beta, tumor necrosis factor-alpha, and other inflammatory mediators, which play important roles in biological defense. However, the overexpression of these mediators has been implicated in diseases such as osteoarthritis, rheumatoid arthritis, and diabetes because the increased production of pro-inflammatory mediators has been shown to induce severe or chronic inflammation [21]. Eight limonoids, and L-NMMA, an inducible nitric oxide synthase (iNOS) inhibitor, were evaluated for their inhibitory effects on NO production ( Figure 4). All tested compounds did not exhibit cytotoxicity (Cell viability 92.7%-100.4% at 30 μM). Of these, Compounds 3, 6, and 8 exhibited stronger inhibitory activity on NO production (IC50 3: 13.7 μM; 6: 4.9 μM; 8: 10.8) than L-NMMA (IC50 23.9 μM). On the other hand, Compounds 4 and 7 showed moderate activity on NO production (IC50 4: 25.5 μM; 7: 28.9 μM). Each value represents the mean ± the standard error (S.E.) of four determinations. Significant differences from the vehicle control (0 μM) group shown as *: p < 0.05 and **: p < 0.01 in the NO inhibitory assay.

General Experimental Procedures
Melting points were determined on a Yanagimoto micro-melting point apparatus and were uncorrected. Optical rotations were measured with a JASCO DIP-1000 digital polarimeter. IR spectra were recorded on a PerkineElmer 1720X FTIR spectrophotometer (Perkin-Elmer Inc., Wellesley, MA, USA). UV spectra were measured on a HITACHI U-2000 spectrometer using EtOH as a solvent. 1 Hand 13 C-NMR spectra were obtained on an Agilent vnmrs 600 spectrometer (Agilent Technologies, Santa Clara, CA, USA) with standard pulse sequences, operating at 600 and 150 MHz, respectively. CDCl3 was used as the solvent and TMS as the internal standard.

General Experimental Procedures
Melting points were determined on a Yanagimoto micro-melting point apparatus and were uncorrected. Optical rotations were measured with a JASCO DIP-1000 digital polarimeter. IR spectra were recorded on a PerkineElmer 1720X FTIR spectrophotometer (Perkin-Elmer Inc., Wellesley, MA, USA). UV spectra were measured on a HITACHI U-2000 spectrometer using EtOH as a solvent. 1 H-and 13 C-NMR spectra were obtained on an Agilent vnmrs 600 spectrometer (Agilent Technologies, Santa Clara, CA, USA) with standard pulse sequences, operating at 600 and 150 MHz, respectively. CDCl 3 was used as the solvent and TMS as the internal standard.
FABMS were recorded on a JEOL JMS-7000 mass spectrometer (JEOL, Tokyo, Japan). Column chromatography was performed over silica gel (70-230 mesh; Merck, Darmstadt, Germany), while medium pressure liquid chromatography (MPLC) was conducted with silica gel (230-400 mesh, Merck). HPLC was carried out using an ODS column [Cosmosil 5C 18 -MS column (Nacalai Tesque, Inc., Kyoto, was added. After a 3 h incubation, 20% sodium dodecyl sulfate in 0.1 M HCl was added to dissolve the formazan produced in the cells. The absorbance of each well was read at 570 nm using a microplate reader. The optical density of vehicle control cells was assumed to be 100%. Values are expressed as the mean ± standard error of the mean (S.E.M.). One-way ANOVA, followed by Dunnett's test, was used for statistical analysis. Significant differences from the vehicle control (0 µM) group shown as *: p < 0.05 and **: p < 0.01.