Carapanolides J–L from the Seeds of Carapa guianensis (Andiroba) and Their Effects on LPS-Activated NO Production

A novel gedunin and two novel phragmalin-type limonoids, named carapanolides J–L (compounds 1–3) as well as a known gedunin-type limonoid 4 were isolated from the seeds of Carapa guianensis (andiroba). Their structures were determined on the basis of 1D and 2D NMR spectroscopy and HRFABMS. Compounds 1–4 were evaluated for their effects on the production of NO in LPS-activated mouse peritoneal macrophages.

the alluvial flats, marshes, and uplands of the Amazon Basin. This tree can also be found wild or under cultivation in Brazil in the Islands region, Tocantins, Rio Solimoes, and near the seaside. It is one of the large-leafed trees of the rainforest and can be identified by its large and distinctively textured leaves. The andiroba tree produces a brown, woody, four-cornered nut with a diameter of 3-4 inches that resembles a chestnut. Andiroba oil is a rich source of essential fatty acids including oleic, palmitic, stearic, and linoleic acids. It yields up to 65% unsaturated fatty acids and can contain approximatoly 9% linoleic acid. Andiroba oil extracts yield up to 65% unsaturated fatty acids and can contain approximately 9% linoleic acid. Extracts from its bark, flowers, and seeds have been used for centuries by the Amazonian people and exhibit various repellent [2], analgesic [3], anti-malarial [4], anti-inflammatory [5], anti-allergic [6], and antiplasmoidal [7] activities, as well as acute and subacute toxicities [8]. Our recent study on the components of the seed oil of Carapa guianasis revealed the structures of two new unusual 9,10-seco-mexicanolide-type limonoids, named carapanolides A and B [9], two novel carbon skeletal limonoids, named guianolides A and B [10], and carapanolides C-I [11]. We herein describe the isolation and structural determination of three novel limonoids 1-3, named carapanolides J-L, and the effects of 1-3 and epoxyazadiradione (4) on the production of NO in LPS-activated mouse peritoneal macrophages. The structures of 1-3 were determined on the basis of NMR spectroscopy, including 1D and 2D ( 1 H, 1 H-COSY, NOESY, HSQC, HMBC) NMR, and FABMS.

Results and Discussion
The seed oil of Carapa guianensis (2.03 kg) was separated by silica gel column chromatography, medium-pressure liquid chromatography (MPLC), and reverse-phase HPLC to obtain three new limonoids 1-3 and a known limonoid 4, which was identified as epoxyazadiradione ( Figure 1) [12].
Carapanolide L (3) was obtained as a colorless amorphous solid, and its molecular formula was established as C33H38O13 ([M + H] + ; m/z 643.2391, calcd. for 643.2391) by HRFABMS, implying 15 degrees of unsaturation. The IR spectrum showed the presence of a hydroxyl at υmax 3352 cm −1 , and ester groups at υmax 1742 cm −1 . The 1 H-and 13 C-NMR data indicated that eight of the 15 units of unsaturation came from two carbon-carbon double bonds and four ester carbonyls, including two lactone carbonyls. Therefore, the remaining degrees of unsaturation required 3 to be nonacyclic. The 1 H-and 13 C-NMR spectra of 3 (Table 2)     Physiological nitric oxide (NO) is involved in blood pressure regulation and blood flow distribution, whereas its overexpression may induce tissue injury, multiple organ dysfunction, and death, as well as systemic inflammatory responses in sepsis, such as hypotension, cardiodepression, and vascular hyporeactivity [15]. In the present study, four limonoids and L-NMMA, an inducible nitric oxide synthase (iNOS) inhibitor, were evaluated for their inhibitory effects on NO production in LPS-stimulated RAW264.7 cells (Table 3). To determine safe concentrations, the cytotoxicities of these limonoids against RAW 264.7 were assessed by the MTT assay. Compounds 1 and 3 showed non-toxicities at 3-100 μM, whereas 4 and 2 exhibited moderate cytotoxicities (IC50 4: 21.3 μM; 2: 15.2 μM). In the inhibitory assay of NO production, compound 1 showed similar inhibitory activities (produced NO 83.4% at 10 μM; 61.8% at 30 μM; 16.8% at 100 μM) to the positive control, L-NMMA (produced NO 79.3% at 10 μM; 58.2% at 30 μM; 39.9% at 100 μM), with no cytotoxicities. Compound 4 exhibited superior inhibitory activities on NO production at non-toxic concentrations (produced NO 74.0% at 3 μM; 30.0% at 10 μM) to those of L-NMMA. These results suggested that compound 1 may be valuable as potential inhibitors of NO production.

Determination of RAW264.7 Cell Proliferation
RAW264.7 cell proliferation was examined according to a method reported previously [16] with some modifications. Briefly, RAW264.7 cells (5 × 10 4 cells in 100 μL) were seeded onto 96-well microplates, and incubated for 24 h. D-MEM (100 μL) containing test samples (final concentration of 100, 30, 10, or 3 μM) dissolved in DMSO (final concentration 0.2%) was added. After the cells had been treated for 24 h, the MTT solution was added. After 3 h of incubation, 20% sodium dodecyl sulfate (SDS) in 0.1 M HCl was added to dissolve the formazan produced by 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%.

Inhibitory Assay of NO Production
An inhibitory assay of nitric oxide production was performed according to a method reported previously [17] with slight modifications. Briefly, RAW264.7 cells (5 × 10 4 cells in 100 μL) were seeded onto 96-well microplates, and incubated for 24h. D-MEM (100 μL) containing test samples (final concentration of 100, 30, 10, or 3 μM) dissolved in DMSO (final concentration 0.2%) and LPS (final concentration of 5 μg/mL) were added. After cells had been treated for 24 h, 50 μL of 0.1% N-(1-naphtyl)ethylenediamine in H2O and 50 μL of 1% sulfanylamide in 5% phosphoric acid were added. After being incubated for 30 min, 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%.