Seco-Taondiol, an Unusual Meroterpenoid from the Chilean Seaweed Stypopodium flabelliforme and Its Gastroprotective Effect in Mouse Model

Ten known meroterpenoids and the new meroterpenoid 7 were isolated from the Chilean seaweed Stypopodium flabelliforme as their acetylated derivatives. Furthermore, the known metabolite taondiol has been isolated for the first time from this species. The molecular structure of the new metabolite was determined by spectroscopic methods based on 1D- and 2D-NMR. Isolation of 7 represents a key step toward a better understanding of the biogenesis of this class of meroterpenoids. Among the meroditerpenoids isolated, stypodiol, isoepitaondiol, epitaondiol and sargaol exhibited gastroprotective activity on the HCl/Ethanol-induced gastric lesions model in mice. Regarding the mode of gastroprotective action, the activity of epitaondiol was reversed significantly when animals were pretreated with indomethacin, N-ethylmaleimide and N-nitro-l-arginine methyl ester (L-NAME) suggesting that prostaglandins, sulfhydryl groups and nitric oxide are involved in their mode of gastroprotective action. In the case of sargaol the gastroprotective activity was attenuated with indomethacin and N-ethylmaleimide, which suggests that prostaglandins and sulfhydryl groups are also involved in the mode of action using this model.

action, the activity of epitaondiol was reversed significantly when animals were pretreated with indomethacin, N-ethylmaleimide and N-nitro-L-arginine methyl ester (L-NAME) suggesting that prostaglandins, sulfhydryl groups and nitric oxide are involved in their mode of gastroprotective action. In the case of sargaol the gastroprotective activity was attenuated with indomethacin and N-ethylmaleimide, which suggests that prostaglandins and sulfhydryl groups are also involved in the mode of action using this model. Keywords: Stypopodium flabelliforme; meroditerpenoids; gastroprotective; gastric ulcer; seaweed
A collection of Stypopodium flabelliforme from Papua New Guinea showed a different pattern of metabolites with respect to a Chilean one [1,4,5]. This variation in metabolic profile could be linked to the different stage of their life cycle, collection places or/and environmental conditions. On the other hands, both collections produced these three known marker compounds: 2-geranylgeranyl-6-methyl-1,4-benzohydroquinone, stypodiol and stypotriol [1,4,5].
This work describes the isolation and structural elucidation by NMR of a key metabolite in the biogenesis of the meroterpenoids known as taondiol's family, and the mode of gastroprotective action of the pure compounds epitaondiol and sargaol.

Results and Discussion
Compound 7 was isolated as an acetylated derivative from S. flabelliforme to avoid the over oxidation of some meroditerpenoids such as stypotriol and tetraprenylhydroquinones ( Figure 1). The 13 C-NMR and mass spectral data indicated that 7 had the molecular formula C32H46O5 indicating ten degree of unsaturation. The 1 H-NMR spectrum showed signals for two meta-coupled aromatic protons at δ 6.64 (d, J = 2.8 Hz) and 6.55 (d, J = 2.8 Hz), a doublet at δ 5.52 (brd, J = 5.5 Hz) assigned to the olefinic proton, a methine proton at δ 4.72 brs assigned to a secondary alcohol, a methoxy group at δ 3.76 s, an aromatic methyl group at δ 2.11 s, five methyl groups at δ 0.75 s, δ 0.80, δ 0.96 d (J = 5.7 Hz), δ 1.07 s, δ 1.09 s, and two methyl groups at δ 2.01 s and 2.34 s assigned to the acetyl groups. The 13 C-NMR spectrum including DEPT 135 (Distortionless Enhancement by Polarization Transfer) showed the presence of eight quaternary carbons (five olefinic), seven methine (two aromatic and an olefinic), six methylenes, six methyl groups, a methoxy, and two acetate groups. Comparison of the spectroscopic data of 7 with those of epitaondiol diacetate and isoepitaondiol diacetate [1,3,5,6] indicated that the ring D is open in compound 7. This fact was confirmed by HSQC (Heteronuclear Simple Quantum Correlation) and HMBC (Heteronuclear Multiple Bond Correlation) spectra which showed correlations of protons H-6' and H-2 with C-1, and methyl H3-16 with C-2, C-3 and C-4 ( Figure 2). Further, HMBC cross-peaks between H3-18 and C-6, C-10 and C-11, H3-19 and C-10, C-14 and C-15, H-9 and C-7, C-8, C-10, C-11 and C-15 allowed the assignment of double bond and secondary alcohol at C-9 and C-14 respectively. Heteronuclear couplings between H-1 and C-1', C-2' and C-6', and between H3-7' and C-2, C-3' and C-4' completed the assignment of the aromatic ring. Thus the planar structure of compound 7 was established. Analysis of the NOESY (Nuclear Overhauser Effect Spectroscopy) spectrum clarified the relative configuration ( Figure 2). A NOE effect between H-14 and H3-19 was observed. H3-19 had NOE with H3-18, while H3-18 showed cross peaks with H-6 and H3-17 in the NOESY spectra indicating that these groups are on the same face of the molecule. A similar situation was observed between H3-17 with H3-16. No NOE cross peak was observed between H3-16 and H-2, which implies that both are on the opposite face of the molecule. It is well known that H-14 in epitaondiol, 2β,3α-epitaondiol, isoepitaondiol and taondiol is on the α face of the molecule whose splitting pattern is a doublet doublet (J = 11.7; 5.0 Hz) [1,[3][4][5][6]. In our case, H-14 is on the β face of the molecule due to its coupling pattern which is a broad singlet; therefore, H-14, H3-19, H3-18, H-6, H3-17 and H3-16 are on the β face of the molecule. The above considerations support the proposed unprecedented syn-cis-anti arrangement for the A/B/C ring system for compound 7. Thus, the structure of compound 7 was elucidated as O,C(3)-seco-9-ene-6β-taondiol.
Other authors have previously reported a possible explanation the biosynthesis of Stypopodium meroterpenoids, which may occur thorough the cyclization of 2-geranylgeranyl-6-methylhydroquinone in different folding patterns to give different classes of metabolites related to taondiol's family [3,4,6,7,16,17]. Our compound isolated is key to a better understanding of the biogenetic pathway, which was suggested for the first time by Gonzalez et al. [16].
PGs are involved in the protection of the gastric mucosa against necrotizing agents via induction of endogenous PGs [24,25]. In our study, pre-treatment with indomethacin attenuated the gastroprotective effect of 1a and 3a ( Figure 3). This fact suggested that PGs participate in the protective activity of 1a and 3a. Table 1. Gastroprotective effect of compound 1a, 3a, 4a, 5a, 9, 11 and lansoprazole at 30 mg/kg on HCl/EtOH-induced gastric lesions in mice and cytotoxicity towards (human epithelial gastric cells (AGS) and human fibroblast.  Depletion of endogenous SHs has been related with gastric damage induced by ethanol [26]. Endogenous Sulfhydryls such as glutathione is known to protect the integrity and permeability of the cell membrane and may act as antioxidants, scavengers of free radicals, maintenance of immune function, regulation of protein synthesis and degradation, and the maintenance of important surface protein structures [26,27]. In this study, pre-treatment with N-ethylmaleimide (NEM, SH-blocker) C CARB 1a+IND 1a+NAME 1a+NEM 1a+RR reduced the gastroprotective effect showed by 1a and 3a ( Figure 3). This fact indicates that endogenous SHs participate in the protective effect of 1a and 3a.
NO participates in gastric defense by the regulation of the gastric mucosal blood flow, angiogenesis and gastric mucus secretion [28,29]. In our study, pretreatment with N G -nitro-L-arginine methyl ester (L-NAME, an inhibitor of NO synthase) has not attenuated the gastroprotective effect showed by 1a. This finding suggests that endogenous NO have null participation in the gastroprotective effect. In the case of the Compound 3a, the gastroprotective effect was reduced (Figure 3). This fact indicates that endogenous SHs participate in the protective effect of 3a.
Capsaicin-sensitive sensory neurons via VR on the gastrointestinal tract participate in gastric defense mechanisms (by the regulation of gastric motility, acid secretion, gastric blood flow through the action of calcitonin gene-related peptide (CGRP) and by the stimulation of gastric mucus production and bicarbonate) [30,31]. In this study (Figure 3), pre-treatment with ruthenium red (a vanilloid receptor antagonist), did not reduce the lesion index suggesting that the gastroprotection of 1a and 3a have no relationship with capsaicin-sensitive sensory neurons via VR.

Instrumentation
Measurements of NMR spectra of meroterpenoids used a Bruker Avance AM-400 spectrometer (Bruker, Bremen, Germany) equipped with 5 mm probes. All compounds were individually dissolved in 0.6 mL of CDCl3 containing tetramethylsilane (TMS) as internal standard. Chemical shifts (δ) were reported in ppm and coupling constants (J) in Hertz. IR spectra were recorded on a Vector 22 FT-IR spectrometer (Bruker, Bremen, Germany). Optical rotations were obtained in CHCl3 on a Polax-2L ATAGO, polarimeter (Atago Co., Tokio, Japan). ESIMS (Electrospray ionization mass spectrometry) was recorded on a Waters/Micromass Q-TOF micro high-resolution hybrid quadrupole orthogonal time-of-flight mass spectrometer (Waters Co., Milford, MA, USA) with a constant nebulizer temperature of 100 °C. The experiments were carried out in positive ion mode, and the cone and extractor potentials were set at 10 and 3.0 V, respectively, with a scan range of m/z 100-600. The samples were infused directly into the ESI source, via a syringe pump, at flow rates of 5 µL min −1 , through the instrument's injection valve.

Plant Material
The brown seaweed, Stypopodium flabelliforme, was collected by hand using scuba diving near to the coastline of "playa Anakena" in Easter Island, Chile, in May 2013. A voucher specimen (No. SF 15052013) was deposited in the Museo Nacional De Historia Natural, Santiago, Chile while its identity was confirmed by M. Eliana Ramirez from the Museo de Historia Natural de Santiago, Chile.
Compounds 1a, 3a, 4a and 5a were obtained by hydrolysis of 1, 3-5 (see Supplementary Information). The 1 H-and 13 C-NMR data of compound 7 are presented below, whereas the structure of compounds isolated from S. flabelliforme is given in the Figure 1.

Animals
Swiss albino mice (30 ± 3 g) were purchased from the Instituto de Salud Pública de Chile, Santiago, Chile. Mice were fed on certified Champion diet with free access to water under standard conditions of 12-h dark-light cycle and 22 °C room temperature. The protocols were approved in July 2, 2010 (expiration date: October 31, 2015) by the Ethics Committee of the University of Chile (Chairman Marco Mé ndez) that follows the recommendations of the Canadian Council on Animal Care and with the ethical guidelines for investigations in conscious animals [37].

HCl/EtOH-Induced Lesions in Mice
The gastroprotective activity of the compounds 4a-5a, and 9, 11 was assessed in the HCl/EtOH-induced lesion model as described previously [38,39]. Mice were randomly distributed into groups of seven animals each and fasted for 12 h with free access to water prior to the experiment. Fifty min after oral administration of the meroditerpenoids (40 mg/kg), lansoprazole (30 mg/kg) or 1% Tween 80 (10 mL/kg), all groups were orally treated with 0.2 mL of a solution containing 0.3 M HCl/60% ethanol (HCl/EtOH) for gastric lesion induction. Animals were sacrificed 1 h after the administration of HCl/EtOH, and the stomachs were excised and inflated by injection of saline (1 mL). The ulcerated stomachs were fixed in 5% formalin for 30 min and opened along the greater curvature. Gastric damage visible to the naked eye was observed in the gastric mucosa as elongated black-red lines, parallel to the long axis of the stomach similar to the HCl/EtOH-induced lesions in rats. The length (mm) of each lesion was measured, and the lesion index was expressed as the sum of the length of all lesions.

HCl/EtOH-Induced Gastric Lesions in Indomethacin-Pretreated Mice
To investigate the involvement of endogenous prostaglandins in the gastroprotective effect of 1a and 3a, indomethacin s.c. (30 mg/kg, an inhibitor of the prostaglandin synthesis was dissolved in 5% NaHCO3) was injected 30 min before administration of 1a, 3a or vehicle (IND-treated) [38,39]. Fifty min after oral administration of 1a (35 mg/kg), 3a (40 mg/kg) or vehicle, all groups were orally treated with 0.2 mL of a solution containing 0.3 M HCl/60% ethanol (HCl/EtOH) for gastric lesion induction. Animals were sacrificed 1 h after the administration of HCl/EtOH, and the stomachs were excised and inflated by injection of saline (1 mL). The gastric mucosal lesions were induced and the length of gastric lesions was measured as described above.

HCl/EtOH-Induced Gastric Lesions in N-Ethylmaleimide (NEM)-Pretreated Mice
To investigate the involvement of sulfhydryl compounds (SHs) in the gastroprotective effect of 1a and 3a, NEM s.c. (10 mg/kg, an SH blocker was dissolved in saline) was injected 30 min before administration of 1a, 3a or vehicle (NEM-treated) [38,39]. Fifty min after oral administration of 1a (35 mg/kg), 3a (40 mg/kg) or vehicle, all groups were orally treated with 0.2 mL of a solution containing 0.3 M HCl/60% ethanol (HCl/EtOH) for gastric lesion induction. Animals were sacrificed 1 h after the administration of HCl/EtOH, and the stomachs were excised and inflated by injection of saline (1 mL). The gastric mucosal lesions were induced and the length of gastric lesions was measured as described above.

HCl/EtOH-Induced Gastric Lesions in N-Nitro-L-Arginine Methyl Ester (L-NAME)-Pretreated Mice
To investigate the involvement of endogenous nitric oxide (NO) in the gastroprotective effect of 1a and 3a, L-NAME i.p. (70 mg/kg, an inhibitor of NO synthase was dissolved in saline) was injected 30 min before administration of 1a, 3a or vehicle (L-NAME-treated) [38,39]. Fifty min after oral administration of 1a (35 mg/kg), 3a (40 mg/kg) or vehicle, all groups were orally treated with 0.2 mL of a solution containing 0.3 M HCl/60% ethanol (HCl/EtOH) for gastric lesion induction. Animals were sacrificed 1 h after the administration of HCl/EtOH, and the stomachs were excised and inflated by injection of saline (1 mL). The gastric mucosal lesions were induced and the length of gastric lesions was measured as described above.

HCl/EtOH-Induced Gastric Lesions in Ruthenium Red (RR)-Pretreated Mice
To investigate the involvement of vanilloid receptor in the gastroprotective effect of 1a and 3a, RR s.c. (3.5 mg/kg, a vanilloid receptor antagonist was dissolved in saline) was injected 30 min before administration of 1a, 3a or vehicle (RR-treated) [38,39]. Fifty min after oral administration of 1a (35 mg/kg), 3a (40 mg/kg) or vehicle, all groups were orally treated with 0.2 mL of a solution containing 0.3 M HCl/60% ethanol (HCl/EtOH) for gastric lesion induction. Animals were sacrificed 1 h after the administration of HCl/EtOH, and the stomachs were excised and inflated by injection of saline (1 mL). The gastric mucosal lesions were induced and the length of gastric lesions was measured as described above.

Cytotoxicity Assay
The cytotoxic assay expressed as cell viability was conducted using MTT assay method [40]. Cells at a density of 3 × 10 4 of MRC fibroblasts or AGS cells were plated in 96-well culture dishes. Compounds were assayed at concentrations ranging from 0 up to 500 μM. Incubated at 37 °C in humidified CO2 incubator for 24 h. After incubation, various concentrations in DMSO solvent of the compounds were added. Each compound was tested in quadruplicate and repeated three times. After 48 h incubation, assay was stop by adding MTT reagent (3-(4,5dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) and the incubation continue for next 4 h before the addition of MTT stop solution containing sodium dodecyl sulphate (SDS), the incubation continue for next 24 h. optical density was measured using microplate reader at 550 nm. IC50 value obtained from the plotted graph between percentage live cells compared to control.

Statistical Analysis
Results of statistical analysis were expressed as the mean ± s.e.m. In all experiments, statistical differences between treatments and their respective control were determined by one-way analysis of variance (ANOVA) followed by Dunnett's test. The level of significance was set at p < 0.01. All statistical analyses were performed using the software GraphPad Prism 5 for Windows.

Conclusions
A new unusual compound was isolated from S. flabelliforme along with ten known compounds, which were identified using mainly NMR. O,C(3)-seco-9-ene-6β-taondiol possesses an unprecedented syn-cis-anti arrangement for the A/B/C ring system not previously encountered in nature. We have isolated taondiol for the first time from this species. Regarding the mode of gastroprotective action at an oral dose of 35 mg/kg, the gastroprotective activity of 1a was reversed significantly when animals were pretreated with IND and NEM, which suggests that prostaglandins and sulfhydryl groups are involved in the mode of gastroprotective action of sargaol. Finally, the gastroprotective activity of 3a was reversed significantly when the animals were pretreated with IND, NEM and L-NAME, which suggests that prostaglandins, sulfhydryl groups, and NO are involved in the mode of gastroprotective action of epitaondiol.