Three New Clerodane Diterpenes from Polyalthia longifolia var. pendula

Three new clerodane diterpenes, (4→2)-abeo-cleroda-2,13E-dien-2,14-dioic acid (1), (4→2)-abeo-2,13-diformyl-cleroda-2,13E-dien-14-oic acid (2), and 16(R&S)-methoxycleroda-4(18),13-dien-15,16-olide (3), were isolated from the unripe fruit of Polyalthia longifolia var. pendula (Annonaceae) together with five known compounds (4–8). The structures of all isolates were determined by spectroscopic analysis. The anti-inflammatory activity of the isolates was evaluated by testing their inhibitory effect on NO production in LPS-stimulated RAW 264.7 macrophages. Among the isolated compounds, 16-hydroxycleroda-3,13-dien-15,16-olide (6) and 16-oxocleroda-3,13-dien-15-oic acid (7) showed promising NO inhibitory activity at 10 µg/mL, with 81.1% and 86.3%, inhibition, respectively.


Results and Discussion
Compounds 1 and 2 were isolated as oils.The UV absorption band at ca. 220 nm and the IR absorption bands at 1,680-1,690 cm −1 indicated the presence of a conjugated carbonyl moiety.The 13 C-NMR data of 1 and 2 revealed the presence of 20 carbons.The DEPT and HMBC experiments showed two sets of conjugated systems for C2-C3-C4 and C16-C13-C14-C15 in 1 and 2 (Table 1).Their 1 H-NMR spectra indicated the presence of a single methyl at ca. δ H 2.00-2.08,two methyls at ca. δ H 0.85-0.96,and a secondary methyl group at δ H 0.87-0.92(d, J = 6.5 Hz) (Table 1).No olefinic protons for the C2-C3-C4 conjugated system were detected in the 1 H-NMR spectra.Accordingly, the data suggested that compounds 1 and 2 belong to the clerodane-type diterpene type with a (4→2) rearranged ring A moiety, similar to that of solidagonal acid (4) [8].
Several studies have evaluated the anti-inflammatory activity of methanolic extract of P. longifolia leaves and roots [7,26].Taking these previous reports on the anti-inflammatory activity of P. longifolia into consideration, we thought that the isolated compounds might possess anti-inflammatory activity.Thus, we investigated the inhibitory effect of the isolated compounds on the NO production in LPS-stimulated macrophage (RAW 264.7 cells) by the Griess reaction.The results indicated that 16-hydroxycleroda-3,13-dien-15,16-olide ( 6) and 16-oxocleroda-3,13( 14)E-diene-15-oic acid (7) reduced NO production in LPS-induced cells in a dose-dependent manner (Figure 4).At 10 µM, compounds 6 and 7 exhibited promising NO inhibitory activity, with 81.1% and 86.3%, inhibition respectively, without affecting cell viability (Figures 4 and 5), and their IC 50 values were approaching 1 μΜ.These results supported a previous report, which demonstrated that 6 could inhibit microglia-mediated inflammation and inflammation-related neuronal cell death [27].

Figure 4.
Effect of 6 and 7 isolated from P. longifolia var.pedula on the expression of RAW 264.7 NO.RAW 264.7 macrophages (5 × 10 5 /mL) were pre-treated with compounds 6 and 7, and DMSO (control) for 30 min, followed by stimulation with LPS (1 µg/mL) for 24 h.NO concentration in the culture medium was assayed by the Griess reaction.The data were expressed as the means ± S.E.from three separate experiments.

Plant Material
The unripe fruits of P. longifolia var.pendula (500 g) were collected in Kaohsiung City, Taiwan, in September, 2005.A voucher specimen (PLP) was deposited in the Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung, Taiwan.

Cell Culture
Murine RAW 264.7 macrophages were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA).Cells were propagated in RPMI-1640 medium supplemented with 10% heated-inactivated FCS and 2 mM L-glutamine (Life Technologies, Inc., Gaithersburg, MD, USA), and incubated in a 5% CO 2 incubator at 37 C [28].

Detection of Nitric Oxide Expression by Griess Reaction
RAW 264.7 cells were seeded in 24-well plate at a density of 5 × 10 5 cells/mL, and then incubated with or without LPS (1 µg/mL) in the absence or presence of the tested compounds (1, 5, 10 µM) for 24 h.The effect of the tested compounds on NO production was measured indirectly by determining the nitrite levels using the Griess reaction [2,28].

Statistical Analysis
All tested compounds were re-purified by reversed-phase HPLC before the bioassay test (purity > 99%).All results are expressed as the means ± S.E.from three independent experiments.Data analysis involved one-way ANOVA with subsequent Scheffé test; p values < 0.05 were considered to be significant.

Figure 5 . 6 ( 1
Figure5.Effect of 6 and 7 isolated from P. longifolia var.pedula on cell viability.RAW 264.7 macrophages (5 × 10 3 /well) were treated with compounds 6 and 7, DMSO (control) in the presence or absence of LPS (1 µg/mL) for 24 h, followed by incubating with MTT reagent.After 30 min of incubation, the absorbance (A 550 − A 690 ) was measured by spectrophotometry[26].The data were expressed as the means ± S.E.from three separate experiments.
), which is different from the aldehyde group in 4.