In vitro Cytotoxic, Antibacterial and Antiviral Activities of Triterpenes from the Red Sea Sponge, Siphonochalina siphonella

Purpose: To study the phytochemical composition of Siphonochalina siphonella sponge from the western coast of the Red Sea and to evaluate the isolates for possible in vitro cytotoxic, antibacterial and antiviral activities. Methods: The compounds obtained were isolated and purified by different chromatographic means. Their structures were established by means of spectral analysis including 1D 1 H and 13 C and 2D correlation nuclear magnetic resonance (NMR) and high resolution mass spectroscopy (HR-MS). Crystal violet staining method (CVS) was used for the assessment of the cytotoxic activity against HepG-2 and MCF-7 human cell lines, while agar-well diffusion method was employed to measure antimicrobial activity against two Gram-positive and two Gram-negative bacteria. The antiviral activity was determined by the inhibition of cytopathic effect (CPE) in susceptible mammalian cells. Results: Four triterpenes, possessing two different skeletons, were isolated and identified as sipholenone A, sipholenol A, neviotine A and sipholenol L. All four compounds were significantly cytotoxic to MCF-7 and HepG-2 cancer cell lines (p < 0.05) in a concentration-dependent manner with IC50 (the inhibitory concentration required to reduce cell survival by 50 %) in range 2.8 - 19.2 μg/mL. The highest antibacterial activity was observed for neviotine A (Compound 3) against Bacillis subtilis (17.2 ± 0.58). On the other hand, all the compounds showed moderate to weak anti-viral activity. Conclusion: The findings reveal the strong cytotoxic activity of all the isolated triterpenes from the Red Sea Sponge, S. siphonella. Sipholenone A (Compound 1) displayed significant cytotoxic activity towards MCF-7 and HepG-2 cancer cell lines with IC50 = 3.0 and 2.8 μM, respectively.


INTRODUCTION
Sea organisms are a rich source of unique and diverse natural products with potent pharmacological activities, some of which are currently in preclinical or clinical trials [1]. Sponges (Porifera) are a predominantly marine phylum living in the intertidal and the abyssal (deepest ocean) zones. Worldwide, there are approximately 8,500 described species of sponges, with around eleven described from genus Siphonochalina [2]. Species of the Red Sea sponge belonging to genus Siphonochalina are distributed throughout the Gulfs of Aqaba and Suez [3]. A potent hemiasterlin class of antitumor agents has been isolated from Siphonochalina spp. while an antifungal and antibacterial polyacetylene diol, siphonodiol, was reported from the marine sponge Siphonochalina truncate [4]. On the other hand, acetylated bile acid derivatives were obtained from the organic extracts of the sponge S. fortis, collected from Argentina shores [5]. Siphonochalina siphonella, a grey colonial tube-like sponge, is one of the few sponges known to produce squalene-derived cyclic ether triterpenes [3]. So far, about thirty compounds belonging to four different skeletal, namely, the sipholanes [6], siphonellanes [7], neviotanes [8] and dahabanes [3] have been reported. Several studies have described remarkable anti-proliferative activity of sipholane triterpenoids and some of their semisynthetic derivatives against a number of highly malignant cell lines [9]. Sipholenol A was found to potently reverse P-gp-mediated MDR (permeability glycoprotein-mediated multiple drug resistance) to colchicine, paclitaxel, and vinblastine in the resistant KB-C2 and KB-V1 cells overexpressing P-gp [10]. Biocatalysis of sipholenol A and sipholenone A were useful in getting new similar triterpenes with potent antiproliferative activity [11]. To the best of our knowledge, this is the first report assessing the antiviral activity; as well as the cytotoxic activity against HepG-2 cancer cell line, of S. siphonella triterpenoids from the Red Sea shores. Moreover, we report here the complete assignments of 1 H and 13 C NMR data of the isolated triterpenes.

Animal material
The marine sponge Siphonochalina siphonella was collected from Sharm Obhur, Jeddah, at the Saudi Arabian Red Sea coast by Scuba divers in 2012. The sponge was deep-frozen immediately after collection and then freeze-dried to give the dry material.

General experimental procedures
The 1D-NMR and 2D-NMR spectra were recorded on a Bruker AMX-500 spectrometer with tetramethylsilane (TMS) as an internal standard.
Both n-hexane and dichloromethane extracts were pooled together based on their close similarity on TLC. The combined extracts were further purified by application on top of a silica gel packed column (Merck), eluted with nhexane. Polarity was gradually increased with ethyl acetate to obtain six main fractions (A-F). Direct crystallization of fraction A eluted by 10 % ethyl acetate/n-hexane yielded compound 1 (350 mg).

Cytotoxic activity assay
The cells were propagated in Dulbecco's modified Eagle's Medium (DMEM) supplemented with 10 % heat-inactivated fetal bovine serum, 1 % L-glutamine, HEPES buffer and 50 μg/mL gentamicin. Cells were maintained at 37 °C in a humidified atmosphere with 5 % CO 2 and were sub-cultured two times a week. The cytotoxic activity was evaluated by the crystal violet staining (CVS) method [12]. Briefly, the cells were seeded in a 96-well tissue culture microplate, at a concentration of 1 × 10 4 cells per well in 100 μL of growth medium at 37 °C. After 24 h of seeding, fresh medium containing various concentrations of the tested compounds (50, 25, 12.5, 6.25, 3.125 and 1.56 µg) were added to the microtiter plates (each compound was tested in triplicate for all concentrations). Next, the microtiter plates were incubated at 37 °C in a humidified incubator with 5 % CO 2 . Control cell were incubated without test sample and with or without DMSO. The little percentage of DMSO present in the wells was found not to affect the experiment.
After 48 h incubation period, viable cells yield was determined calorimetrically. In brief, the media were aspirated and the crystal violet solution (1 %) was added to each well for 30 min. The plates were rinsed after removing the stain by distilled water. Glacial acetic acid (30 %) was then added to all wells and mixed thoroughly. The evaluation of the fixed cells was performed calorimetrically by measuring absorbance at 595 mm in an automatic Microplate reader (TECAN, Inc.). The effect on cell growth was calculated as the presence and absence of the tested compounds. A dose-response curve was plotted to the concentration at which the growth of cells was inhibited to 50 % of the control (IC 50 ). The standard anti-tumor drug used was Doxorubicin®.

Antimicrobial assay
The anti-bacterial activity was evaluated using the agar-well diffusion method. Mueller-Hinton agar was used as media for bacteria. Bacterial suspension was prepared using the sterile saline water equivalent to a 0.5 McFarland standard and was cultivated on agar medium. Afterwards, 6 mm diameter wells were pressed in the used agar plates. Zone of inhibitions were compared with the standard drugs Ampicillin® and Gentamicin® (30 μg/ mL) against Gram-positive and Gram-negative bacteria, respectively. The bacterial plates were incubated at 37 °C for 24 h. After incubation, the diameter of the inhibition zone was measured to evaluate the anti-microbial activity. Each test was performed twice and the average of the results was calculated. The extraction solvents were used as negative control [13].

Antiviral activity assay
The assay used is based on measuring the inhibition capacity of the tested samples on the virus-induced cytopathic effect (CPE) in susceptible mammalian cells (kidney epithelial cells of monkey) [14]. Monolayers of 10,000 vero cells, adhered at the bottom of a 96-well microtiter plate wells, were incubated with 5 % CO 2 for 24 h in a humidified incubator at 37 o C. The plates were washed with fresh Dulbecco's Modified Eagle's Medium (DMEM) and challenged with 10 4 Herpes simplex type II virus doses and instantaneously treated with two-fold serial dilutions of tested compounds in fresh medium. The plates were incubated at 37 o C for two days. At the same time, both an infection and an untreated vero cells controls, were conducted in the absence of the tested samples. The virus in the control wells was monitored every 24 h by an inverted microscope, until complete viralinduced cytopathic effect (CPE) was shown. The inhibition of cytopathic effect by the tested sample was used as a tool to measure their antiviral activity [15]. The monolayers were fixed with formalin and stained with a 0.1 % crystal violet solution. Three independent experiments were assessed each containing four replicates per treatment. Acyclovir® was used as a positive control under this assay system [16].

Statistical analysis
Results of the study were based on three independent experiments that were performed in triplicate. Data were expressed as mean ± standard deviation (SD). Four-parameter logistic regression analysis was used to determine the medium inhibitory concentration at half-maximal (IC 50 ) of the compounds. Unpaired Student t-test was conducted on the results using GraphPad InStat (ISI Software) computer program. Differences were considered significant at p < 0.05.

Identification of triterpenes
The identification of the isolated triterpenes was based on extensive analyses of their HR-MS, 1D and 2D NMR (COSY, HSQC, HMBC) spectroscopic data and comparison with literature data. The structures of the isolated compounds are presented in Fig. 1.

Antimicrobial activity
In the current study, the anti-microbial activity of the isolated compounds was evaluated by determining the zone of inhibition against two Gram-positive and two Gram-negative bacteria. Results of antimicrobial activity are presented in Table 3. Compound 3 exhibited the highest antimicrobial activity (12.7 ± 0.58 -17.2 ± 0.58 mm, while compound 4 revealed moderate to low activity against Gram positive bacteria only (12.3 ± 0.72 -14.5 ± 0.72 mm. Compound 2 was totally ineffective against all tested microorganisms at 20 mg/mL. Furthermore, the Gram-negative bacteria Pseudomonas aeruginosa was resistant to all compounds. The anti-microbial activity of each of tested compounds is less than that of the standard. Therefore, no minimum inhibitory concentration was determined.

Antiviral activity
All four S. siphonella triterpenes were tested against two viruses, the hepatitis A virus (HAV-10) and the herpes simplex virus (HSV-1). The results showed moderate anti-viral activity for compound 2 against HAV-10 virus, whereas compound 4 was weakly active against both viruses. Compound 3 showed weak activity against HAV-10 virus only.

DISCUSSION
Compound 1 was obtained as white crystals. It displayed a m/z of 474.7156 suggesting a molecular formula C 30 H 50 O 4 with 6 degrees of unsaturation. Its 13 C-NMR and DEPT spectrum exhibited 30 carbons, including eight singlet methyl signals, nine methylenes, six methines one of which is olefinic and seven quaternary carbons. Moreover, the presence of a double bond at δ H 5.45 and δ C 143.0, 121.5 and a ketonic group (δ C 213.4) in the NMR data suggested a tetracyclic structure. Comparing the above data with those of the previously isolated compounds from Siphonochalina revealed the basic skeleton of squalene-derived cyclic ether triterpene. These data confirm the structure of compound 1 to be sipholenone A [6,9,10].
Compound 2 was also isolated as white crystals and analyzed for C 30 H 52 O 4 . The NMR data were identical with those of sipholenone (compound 1), except for the absence of the carbonyl carbon which is replaced by oxymethine group at C-4 (δ H 3.81, dd 6.5, 3.5) and δ C (76.8). The MS and NMR spectral data confirmed compound 2 to be sipholenol A [3,6,9,10].
Compound 3 was also isolated as white crystals. HRMS displayed a molecular ion peak at m/z of 506.7143 suggesting a molecular formula C 30 H 50 O 6 with 6 degrees of unsaturation. DEPT experiments of 3 revealed the presence of seven methyl groups (rather than eight in the sipholanes), two of which were doublets (δ H 0.89 and 0.87, J = 6.6), nine methylenes, seven methines and seven quaternary carbons which account together for thirty carbon atoms and indicating a neviotane triterpenoidal skeleton rather than sipholane-type. Both H-3 (δ H 4.16) and H-5 (δ H 5.03) appeared as doublets with coupling constants of 4.0 and 8.0 Hz consequently, due to coupling with the hydroxyl groups attached at the same positions. Comparing the MS and NMR spectral data of 3 with those reported for neviotine A, previously isolated from S. siphonella [8,17] suggested that both compounds are identical. This is the third report for isolation of neviotine A from S. siphonella sponge, or elsewhere, which indicates the chemical variability of the triterpenoidal constituents of this sponge from different areas of the Red Sea [3].
Compound 4, isolated as white crystals, displayed a molecular ion peak at m/z of 476.4865 calculated for C 30 H 52 O 4 . The NMR data revealed a squalene-type triterpene with rings A and B exactly similar to those of compounds 1 and 2, while rings C and D were fused to form a cis-decaline system. Compound 4 was identified as sipholenol L, by comparing its physical and spectral data including MS, 1 H NMR and 13 C NMR data with those reported for sipholenone L [18].
From the results shown above, it could be concluded that all compounds possessed a dose dependent cytotoxic effect against the two tested cell lines. However, sipholenone A (compound 1) showed the highest cytotoxic activities against both MCF-7 and HepG-2 tumor cell lines (IC 50 = 3.0 and 2.8 μg/mL, respectively), while sipholenol L (compound 4) was significantly active against MCF-7 cell line (IC 50 = 4.0 μg/mL). Thus, both sipholenone A and sipholenol L could be potential candidates to develop promising drugs to treat cancer. This is the first report for anti-microbial evaluation of neviotane-type triterpenes. A previously published study by Gab-Alla et al, revealed that the total methanolic extract of S. siphonella was inactive against some tested gram positive and gram negative bacterial strains [19]. However, the results of the present study are partly in agreement with those published by Aqil et al which showed that sipholenone A and sipholenol A are inactive against the Gram-positive bacteria S. aureus and B. subtilis; and the Gram-negative bacteria P. aeruginosa and E. coli [20].

CONCLUSION
Four sipholane and neviotane-type triterpenes have been isolated and identified from the Red Sea sponge S. siphonella. Compound 1 has significant cytotoxic activity against MCF-7 and HepG-2.