A New Atisane-Type Diterpene from the Bark of the Mangrove Plant Excoecaria Agallocha

A new atisane-type diterpene, ent-16α-hydroxy-atisane-3,4-lactone (4) and three known diterpenes, ent-16α-hydroxy-atisane-3-one (1), ent-atisane-3β,16α-diol (2), ent-3,4-seco-16α-hydroxyatis- 4(19)-en -3-oic acid (3) were isolated from the bark of the mangrove plant Excoecaria agallocha. Their structures and relative stereochemistry were elucidated by means of extensive NMR and MS analysis. Compound 3 exhibited significant anti-microfouling activity against the adherence of Pseudomonas pseudoalcaligenes, with an EC50 value of 0.54 ± 0.01 ppm.


Introduction
Excoecaria agallocha L (Euphorbiaceae) is an important mangrove species mainly distributed in China, India, Philippines, and Oceania [1]. This plant is used as a traditional remedy for epilepsy, conjunctivitis, dermatitis, haematuria, leprosy, and toothache [2]. The latex and leaves have been used as a dart poison and fish poison in Sarawak, New Caledonia, and Goa [3][4][5]. The phorbol ester isolated from the leaves and stems has been proved to be cytoprotective in the NCI primary anti-HIV screen OPEN ACCESS [6]. Some diterpenes isolated from the wood of this plant showed anti-tumor-promoting activity [7,8]. However, there is scant information on the isolation of antifouling compounds from mangrove species [9,10].
Biofouling, the undesirable buildup of sessile marine organisms (such as barnacles, mussels, tubeworms, and seaweeds) onto man-made surfaces, causes severe problems in the maritime industry [11]. In many cases, biofouling will consist of microscopic organic impurities or a visible slimy layer containing bacteria and other microorganisms. This category of biofouling is called microfouling, or more commonly biofilm, and occurs everywhere in both natural and industrial environments where surfaces are exposed to water [12]. Once this biofilm has formed, higher ordered organisms such as barnacles, algae, mollusks, tubeworms and sponges can attach. Therefore preventing the attachment of bacteria during the early stages of biofilm formation could greatly reduce the subsequent biofouling problems [13]. Marine organisms use both physical and chemical methods to protect themselves from the harmful process of biofouling [14,15]. The key chemical antifouling mechanism of marine organisms occurs via the production of secondary metabolites (also known as natural products) which deter foulers [16]. Mangroves are unique intertidal woody communities common in tropical and subtropical coastlines [17], and as such, these plants are also affected with marine-fouling organisms [18]. Our investigation on the bark of E. agallocha has resulted in the isolation of a new diterpene, ent-16α-hydroxy-atisane-3,4-lactone (4), along with three known compounds, ent-16α-hydroxy-atisane-3-one (1) [19,20], entatisane-3β,16α-diol (2) [20], and excoecarin V3 [=ent-3,4-seco-16α-hydroxyatis-4(19)-en-3-oic acid (3) [21][22][23] (Figure 1). Compound 3 significantly inhibited the adherence of the marine microorganisms, Pseudomonas pseudoalcaligenes. The isolation, structural elucidation and anti-microfouling activity of these compounds are described in this paper.
Our structure-activity relationship analysis of atisane-type diterpenes suggested that the presence of a free hydroxyl group might be more important in the expression of the activity than a carbonyl group. Compound 2, with a free hydroxyl group at C-3, had stronger activity than compound 1 with a carbonyl group at C-3, and compound 3, a secoatisane-type diterpenoid with a carboxylic acid at C-3 had higher activity than compounds 1, 2 or 4.   The 1 H-, 13 C-and 2D-NMR spectra were recorded with a Bruker Avance-600 FT NMR spectrometer (Germany). Low-resolution ESI mass spectra data were recorded on an AB 3200Q TRAP spectrometer (USA). HR-ESI mass spectra data were recorded on an APEXIII 7.0 TESLA FTMS from Bruker Daltonics, Inc (USA). The optical rotation data were obtained on a Rudolph Autopol IV Polarimeter (USA). Column chromatography was performed with silica gel (200-300 mesh), and GF254 silica gel for TLC was obtained from Qingdao Marine Chemistry Co. Ltd (Qingdao, P.R. China). ODS (Octadecyltrichlorosilane) and Sephadex LH-20 (18-110 μm) were obtained from Pharmacia Co (Sweden).

Extraction and Isolation
The bark of E. agallocha was collected from Hainan Province, China in July 2006. The plant material was indentified by Professor Yi-Ming Lin, Xiamen University. A voucher specimen (HQ-2006-6) is deposited at the herbarium of the Department of Biology, School of Life Sciences, Xiamen University. The air-dried and powdered material (2.1 kg) was extracted with 95% EtOH three times at reflux temperature. Removal of the solvent from the combined EtOH extracts yielded a brown viscous mass (104 g). The extract was suspended in H 2 O, and partitioned with petroleum ether, EtOAc, and n-BuOH. The petroleum ether layer (32 g) was chromatographed on silica gel column with petroleum ether-EtOAc as gradient eluent to obtain eight fractions, 1-8. Fraction 3 (890 mg) was subjected to ODS column chromatography [MeOH-H 2 O (4:1) and MeOH] to yield fractions 3-1 and 3-2. Fraction 3-1 was subjected to silica gel column chromatography (petroleum ether-EtOAc = 18:1) to give compound 1 (27 mg 1) and MeOH] to give two fractions 1-1 and 1-2. Fraction 1-1 was purified by repeated silica gel column chromatography (petroleum ether-ethyl acetate) to yield compounds 2 (32 mg), 3 (7.2 mg) and 4(18 mg).     Table 1.

Anti-microfouling Assay
The experimental method, adapted to screen antifouling agents, was based on bacterial adhesion in natural sterile sea water in a microtiter plate and on total biomass quantification by the fluorescent dye 4,6-diamidino-2-phenylindole (DAPI) [26]. The adhered microorganisms, P. pseudoalcaligenes, were incubated in a 50-mL flask containing 2216E liquid medium at 30 °C for 12 h. The mature biofilms were incubated with different concentrations of compounds in 6-well plates with a cover glass at 30 °C for 3 h. The cover glasses were washed with water. And these glasses were observed by microscope after dying with 4 µg/mL DAPI for 20 min, and assayed. Differences in the settlement percentages of experimental and control treatments were tested for significance by one-way ANOVA. Significance was set at the 1% level. The EC50 (the concentration that reduces the settlement rate by 50% relative to the control) was estimated by using the Spearman-Karber method [27].