Cytotoxic and Antiviral Triterpenoids from the Mangrove Plant Sonneratia paracaseolaris

A chemical investigation was conducted on the aerial parts of the mangrove plant Sonneratia paracaseolaris, yielding five new triterpenoid paracaseolins A–E (1–4, and 11) together with twelve known analogues (5–10, 12–17). Their structures were established by extensive spectroscopic methods and comparisons their spectroscopic data with those of the known related compounds. The cytotoxicities against P388, HeLa, A549, and K562 tumor cell lines and anti-H1N1 (Influenza A virus) activities for the isolates were evaluated. Compound 4 showed potent cytotoxicity against the A549 cell line with an IC50 value of 1.89 µM, and compound 1 exhibited significant anti-H1N1 virus activity with an IC50 value of 28.4 µg/mL. A preliminary structure activity relationship was discussed.


Introduction
Mangrove plants of the genus Sonneratia (family Sonneratiaceae) consist of nine species and are generally distributed in the tropical and subtropical regions in the world [1].The fruit, bark, and leaves of several Sonneratia plants have been used as folk medicines for treatment of various diseases, such as asthma, febris, ulcers, hepatitis, piles, sprains, and hemorrhages [2].So far, almost half of the species of the genus have been studied for their chemical constituents, yielding various types of compounds, such as triterpenoids, steroids, alkaloids, flavonoids, aromatics, and tannins, some of which exhibited cytotoxic [3][4][5], anti-HIV [5], and antioxidant [6] activities.
Paracaseolin D (4), a white amorphous powder, had the molecular formula C 39 H 56 O 5 , as determined by HR-ESI-MS data ([M − H] − at m/z 603.4056; calcd.for 603.4044).1D NMR spectra of compound 4 were very similar to those of 2 apart from a slight difference in ring A. The split pattern of H-3 (δ H 4.48, d, J = 10.0 Hz) in 4 showed double peak instead of multiple peak in 2 and the carbon signal at C-3 shifted from δ C 76.7 in 2 to δ C 83.4 in 4 (Table 2), suggesting the ring A of compound 4 to be 2, 3-dioxyged structure which was supported by 1 H-1 H COSY correlation between H-2 (δ H 3.67) and H-3.The O-trans-p-coumaroyl group was also assigned at C-3 on the basis of the HMBC correlation of H-3 with C-9 (δ C 166.7).The half-peak-width value (26.6 Hz) of H-2 further indicated an axial β-orientation.Compound 4 was finally determined as 2α-hydroxy-3β-O-trans-p-coumaroyl betulin.

Biological Evaluations
The cytotoxicities of the isolates were evaluated against the selected K562, P388, HeLa, and A549 tumor cell lines in vitro with adriamycin (ADM) as a positive control, and the results are summarized in Table 3. Almost all of the compounds were active against the P388 cell line and exhibited different levels of cytotoxicities against HeLa and A549 cell lines, but were not sensitive toward the K562 cell line.In particular, new compound 4 showed the strongest cytotoxicity against A549 cells with an IC50 value of 1.89 µM, and compounds 3 and 14 displayed low IC50 values, ranging from 11.04 to 13.10 µM, against A549, HeLa, and P388 cell lines, whereas only 15 showed cytotoxic activity against the K562 cell line with an IC50 value of 16.28 µM.Furthermore, a preliminary structure activity relationships were analyzed.Comparing the cytotoxic data of 1 and 3, 8 and 9, as well as 13 and 14 against HeLa and P388 cell lines, it is suggested that the introduction of an O-p-coumaroyl group at C-3 could increase the cytotoxicities no matter the cis or trans configuration, which is consistent with previous reports [21].Another comparison between 2 and 4, and 7 and 8 implied that 2-OH played significant roles on the cytotoxicities, whereas the OH group at C-1 and the substituent patterns at C-28 showed no effects, which could be predicted from a comparison between compounds 1 and 5-7.

Biological Evaluations
The cytotoxicities of the isolates were evaluated against the selected K562, P388, HeLa, and A549 tumor cell lines in vitro with adriamycin (ADM) as a positive control, and the results are summarized in Table 3. Almost all of the compounds were active against the P388 cell line and exhibited different levels of cytotoxicities against HeLa and A549 cell lines, but were not sensitive toward the K562 cell line.In particular, new compound 4 showed the strongest cytotoxicity against A549 cells with an IC50 value of 1.89 µM, and compounds 3 and 14 displayed low IC50 values, ranging from 11.04 to 13.10 µM, against A549, HeLa, and P388 cell lines, whereas only 15 showed cytotoxic activity against the K562 cell line with an IC50 value of 16.28 µM.Furthermore, a preliminary structure activity relationships were analyzed.Comparing the cytotoxic data of 1 and 3, 8 and 9, as well as 13 and 14 against HeLa and P388 cell lines, it is suggested that the introduction of an O-p-coumaroyl group at C-3 could increase the cytotoxicities no matter the cis or trans configuration, which is consistent with previous reports [21].Another comparison between 2 and 4, and 7 and 8 implied that 2-OH played significant roles on the cytotoxicities, whereas the OH group at C-1 and the substituent patterns at C-28 showed no effects, which could be predicted from a comparison between compounds 1 and 5-7.

Biological Evaluations
The cytotoxicities of the isolates were evaluated against the selected K562, P388, HeLa, and A549 tumor cell lines in vitro with adriamycin (ADM) as a positive control, and the results are summarized in Table 3. Almost all of the compounds were active against the P388 cell line and exhibited different levels of cytotoxicities against HeLa and A549 cell lines, but were not sensitive toward the K562 cell line.In particular, new compound 4 showed the strongest cytotoxicity against A549 cells with an IC50 value of 1.89 µM, and compounds 3 and 14 displayed low IC50 values, ranging from 11.04 to 13.10 µM, against A549, HeLa, and P388 cell lines, whereas only 15 showed cytotoxic activity against the K562 cell line with an IC50 value of 16.28 µM.Furthermore, a preliminary structure activity relationships were analyzed.Comparing the cytotoxic data of 1 and 3, 8 and 9, as well as 13 and 14 against HeLa and P388 cell lines, it is suggested that the introduction of an O-p-coumaroyl group at C-3 could increase the cytotoxicities no matter the cis or trans configuration, which is consistent with previous reports [21].Another comparison between 2 and 4, and 7 and 8 implied that 2-OH played significant roles on the cytotoxicities, whereas the OH group at C-1 and the substituent patterns at C-28 showed no effects, which could be predicted from a comparison between compounds 1 and 5-7. ) correlations of compounds 1 and 11.

Biological Evaluations
The cytotoxicities of the isolates were evaluated against the selected K562, P388, HeLa, and A549 tumor cell lines in vitro with adriamycin (ADM) as a positive control, and the results are summarized in Table 3. Almost all of the compounds were active against the P388 cell line and exhibited different levels of cytotoxicities against HeLa and A549 cell lines, but were not sensitive toward the K562 cell line.In particular, new compound 4 showed the strongest cytotoxicity against A549 cells with an IC 50 value of 1.89 µM, and compounds 3 and 14 displayed low IC 50 values, ranging from 11.04 to 13.10 µM, against A549, HeLa, and P388 cell lines, whereas only 15 showed cytotoxic activity against the K562 cell line with an IC 50 value of 16.28 µM.Furthermore, a preliminary structure activity relationships were analyzed.Comparing the cytotoxic data of 1 and 3, 8 and 9, as well as 13 and 14 against HeLa and P388 cell lines, it is suggested that the introduction of an O-p-coumaroyl group at C-3 could increase the cytotoxicities no matter the cis or trans configuration, which is consistent with previous reports [21].Another comparison between 2 and 4, and 7 and 8 implied that 2-OH played significant roles on the cytotoxicities, whereas the OH group at C-1 and the substituent patterns at C-28 showed no effects, which could be predicted from a comparison between compounds 1 and 5-7.Antiviral activities of all the isolates against influenza A H1N1 virus (IAV) were also evaluated by CPE (the cytopathic effects) assay [27].Only new compound 1 exhibited significant anti-H1N1 virus activity with an IC 50 value of 28.4 µg/mL, very close to the positive control of Ribavirin with an IC 50 value of 24.6 µg/mL.The other compounds showed no activity with inhibition rates less than 50% at 50 µg/mL (Table S2, Supporting Information).

Plant Material
The aerial parts of Sonneratia paracaseolaris were collected in Wenchang, Hainan Province, China, in October 2007, and was identified by Associate Prof. Cairong Zhong (Dongzhai Mangrove Forest National Nature Reserve).A voucher specimen (NO.WC-2007-10) was deposited at the State Key Laboratory of Marine Drugs, Ocean University of China, Qingdao, China.

Cytotoxicity Assay
All the cell lines were purchased from Shanghai Institute of Cell Biology (Shanghai, China).A549, P388, and K562 cell lines were grown in RPMI 1640 while HeLa cell line was maintained in DMEM.Each medium contained 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin.These cell lines were incubated in a humidified atmosphere with 5% CO 2 at 37 • C.

Anti-H1N1 Virus Assay
The antiviral activity against H1N1 was evaluated by the CPE inhibition assay [27].Confluent MDCK cell monolayers were firstly incubated with influenza virus (A/Puerto Rico/8/34 (H1N1), PR/8) at 37 • C for 1 h.After removing the virus dilution, cells were maintained in infecting media (RPMI 1640, 4 µg/mL of trypsin) containing different concentrations of test compounds at 37 • C.After 48 h incubation at 37 • C, cells were fixed with 100 µL of 4% formaldehyde for 20 min at room temperature.After removal of the formaldehyde, the cells were stained with 0.1% crystal violet for 30 min.The plates were washed and dried, and the intensity of crystal violet staining for each well was measured in a microplate reader (Bio-Rad, Hercules, CA, USA) at 570 nm.The IC 50 was calculated as the compound concentration required inhibiting CPE production at 48 h post-infection by 50% Ribavirin (LuKang Cisen, Jining, China, purity > 98%) was used as positive control, and compounds with an inhibition rate of >70%, >50%, and <30% at 50 µg/mL were, respectively, regarded as having strong, moderate, and weak activities.
a By MTT method.b By SRB method.c Positive control.