Chemical investigation of the Red Sea tunicate Polyclinum constellatum afforded nine compounds, identified as thymidine (1), uridine (2), adenosine (3), inosine (4), 24-methylene cholesterol (5), dihydrocholesterol (6), cholesterol (7), oleic acid (8) and 1,3-palmityl-2-palmitoleoylglycerol (9). All compounds isolated for the first time from the genus polyclinum. Acetylation of compounds 1, 2, 3, 4 and 5 yielded thymidine 3`,5`-diacetate (1-Ac), uridine 2`,3`,5`-triacetate (2-Ac), adenosine 2`,3`,5`-triacetate (3-Ac), inosine 2`,3`,5`-triacetate (2-Ac) and 24-methylene cholesterol-3-acetate (5-Ac), respectively. Compound 5 showed potent antitrypanocidal activity with IC50 and IC90 values of 3.39 and 6.69µg/mL, comparable to α-difluoromethylornithine (3.58 and 8.53μg/mL). The overall findings of the present study have shown that the Red Sea tunicate Polyclinum constellatum revealed different varieties of secondary metabolites with antiprotozoal and cytotoxic activities.

Keywords: red sea, tunicate, polyclinum constellatum, antimicrobial, antimalarial, antiprotozoal,cytotoxicity, acetylation of compounds, metabolites, cellulose, appendicularia, natural products

1H NMR: Proton nuclear magnetic resonance; 13C NMR: Carbon nuclear magnetic resonance; 2D: Two dimension; δ: Chemical shifts; s: Singlet; d: Doublet; dd: Doublet of doublet; t: Triplet; m: Multiplet; J: Coupling constant; Hz: Hertz; HRESIMS: High resolution electrospray ionization mass spectrometry; GC/MS: Gas chromatographic mass spectrometry; m/z: Mass to charge ratio; MeOH: Methanol; EtOAc: Ethyl acetate; CH₂Cl₂: Methylene chloride; CHCl₃: Chloroform; HCl: Hydrochloric acid; DFMO: α-difluoromethylornithine; Ac: Acetyl; min: Minute(s); h: Hour(s) THP-1: human monocytic cell line derived from an acute monocytic leukemia patient; IC₅₀: Concentration that afford 50% inhibition; IC₉₀: Concentration that afford 90% inhibition

Tunicates are marine invertebrate animals (animals lacking a backbone) that are commonly found attached to rocks, they are three main groups, sessile ascidians, pelagic appendicularians and thaliaceans.¹ They are characterized by the possession of a tunic composed of cellulose.¹ Adult tunicates are filter feeders: the seawater enters a pharynx through an inhalating or oral siphon, in most cases set in motion by ciliary beating, food particles are trapped on a mucous net secreted by the endostyle, and the water and waste exit the body through an exhalating or atrial siphon.¹ Appendicularia, ascidians and some thaliaceans possess a tadpole-like larva with a notochord and metamorphose into sessile adults in the case of ascidians. Tunicates have reversible blood flow.¹ Natural products found in tunicate has began to attract many chemist’s interests; alkaloids, carotenoids, macrolides and tunichromes.² The cytotoxic and antibacterial activities of Polyclinum indicum and Polyclinum madrasensis extracts have been tested at various concentrations and showed the results of highest cytotoxicity assay conducted, indicating the presence of cytotoxic compounds in these ascidians.^3,4 The crude extract of ascidian Polyclinum madrasensis showed hemolytic properties against human, cow, goat and chicken bloods that be considered a valuable source for secondary metabolites which could be of pharmaceutical interest.⁵ Polyclinal a sulfated polyhydroxy benzaldehyde has been isolated from extracts of the temperate colonial ascidian Polyclinum planum.⁶ The highest concentration of this metabolite was found in the zooid-rich outer layers of this ascidian suggesting that it may represent a potential chemical defense against predators.⁶ As part of our research dealing with the isolation and biological evaluation of active compounds from marine origin, the chemistry and the biological properties of the tunicate Polyclinum constellatum collected from the Red Sea was investigated, which led to the isolation of nine compounds (1-9), for the first time from the genus polyclinum.

General experimental procedures

¹H-NMR, ¹³C-NMR and 2D-NMR spectra were recorded using the residual solvent signal as an internal standard on Bruker BioSpin Gm bH 400 and 500 spectrometers (Bruker, Rheinstetten, Germany). High resolution mass spectrometry were measured using a Bruker BioApex FT mass spectrometer (Bruker, Rheinstetten, Germany). GC/MS analysis was carried out using an HP 6890 series GC (Agilent Technologies, Santa Clara, CA, USA), equipped with a split/splitless capillary injector, an HP 6890 series injector autosampler and an Agliant DB-5ms column (30 m x 0.25 mm x 0.25 μm), interfaced to a HP 5973 mass selective detector (MSD). The injector temperature was 250 °C, and 1 μL of sample was injected in the splitless mode, with the splitless time set at 60 s, the split flow set at 50 mL/min and the septum purge valve set to close 60 s after the injection occurred. The oven temperature was raised from 70 to 270 °C (held for 20 min) at a rate of 5 °C/min, for a total run time of 60 min; the transfer line temperature was 280 °C. Chemicals for the pharmacological studies were purchased from Sigma-Aldrich (St. Louis, MO, USA) except the fetal bovine serum (Midwest Scientific, Valley Park, MO, USA) and the Bio-rad Bradford dye (Fisher Scientific, Pittsburg, PA, USA).

Tunicate material, collection and identification

The tunicate Polyclinum constellatum (coll. no. SAA-117) was collected in June, 2015 at depths between 10 and 20 m from the Egyptian Red Sea coast at Safaga. The sample was identified by Dr Tarek Temraz, Marine Science Department, Faculty of Science, Suez Canal University, Ismailia, The sample was directly frozen after collection and stored at -20 °C. A voucher specimen was kept at the Department of Pharmacognosy, Faculty of Pharmacy, Suez Canal University, Ismailia, Egypt, under a registration no. SAA-117.

Extraction and isolation

The fresh material (0.5 kg) was frozen after collection. It was chopped while frozen into small pieces and extracted with MeOH (500 mL x 3) at room temperature. The crude extract was concentrated till dryness under reduced pressure to give a dark green viscous crude extract (3.86 g). A 3.25 g of the extract was subjected to fractionation by vacuum liquid chromatography over a silica gel column (100 g, 9 cm x 30 cm) eluted with hexanes/EtOAc (75:25, 0:100) followed by each EtOAc/MeOH (50:50, 0:100) yielding 4 fractions. Fraction 2 (455.5 mg) was purified on silica gel column (20 g, 34 cm x 2 cm) and eluted with hexanes/EtOAc in gradient manner from 5% to 20% to yield three subfractions (2A-2C). Subraction 2A (90.6 mg) was further purified on silica gel column (5 g, 25 cm x 0.8 cm) using hexanes/EtOAc (95:5) to give compound 9 (15.6 mg). Subraction 2B (202.4 mg) was further purified on an amino column followed by silica gel column (8 g, 25 cm x 0.8 cm) eluted with hexanes/EtOAc in gradient manner and a RP column (10 g, 25 cm x 0.8 cm) using MeOH/H₂O (97.5:2.5) to yield compounds 5 (12.1 mg), 6 (18.8 mg) and 7 (4.2 mg). Fraction 3 (148.3 g) was subjected to silica gel column (7 g, 25 cm x 0.8 cm) eluted with CH₂Cl₂/MeOH (98:2) yielding compound 8 (20.5 mg). Fraction 4 (2.2 g) was subjected to silica gel column (80 g, 46 cm x 2.5 cm) eluted with (CH₂Cl₂/MeOH, 98:2) to give four subfractions (4A-4D). Subfraction 4B (35.5 mg) was subjected to silica gel column (2 g, 25 cm x 0.8 cm) using CHCl₃/MeOH/H₂O (15:6:1) as a mobile phase followed by a RP column (10 g, 25 cm x 0.8 cm) using MeOH/H₂O (50:50) to give compound 1 (21.3 mg). Subfraction 4C (687.1 mg) was subjected to silica gel column (28 g, 34 cm x 2 cm) eluted with CHCl₃/MeOH/H₂O (15:6:1) to yield two fractions (4C-1 and 4C-2). Fraction 4C-1 (554.6 mg) was subjected to sephadex LH-20 (100 g, 46 cm x 2.5 cm) using MeOH as an eluent yielding two subfractions (4C-1-1 and 4C-1-2). Subfraction 4C-1-1 (113.4 mg) was purified on a silica gel column (5 g, 25 cm x 0.8 cm) using CHCl₃/MeOH/H₂O (15:6:1) as a mobile phase to yield compound 2 [24.4 mg). Subfraction 4C-1-2 (20.3 mg) was subjected to sephadex LH-20 (100 g, 46 cm x 2.5 cm) using MeOH as an eluent to give compound 3 (2.2 mg). Fraction 4C-2 (132.5 mg) was subjected to silica gel column (6 g, 25 cm x 0.8 cm) eluted with CHCl₃/MeOH/H₂O (15:6:1) followed by sephadex LH-20 (100 g, 46 cm x 2.5 cm) to give compound 4 [17.2 mg) (Scheme 1).

Scheme 1 Isolation of the compounds 1-9 from the Red Sea tunicate Polyclinum constellatum.

Acetylation of compounds 1, 2, 3, 4 and 5:

A 2 mg of each compound was dissolved in 1 mL of pyridine and then 2 mL of acetic anhydride was added. The reaction mixture was kept at room temperature for 12 h.⁷ The solvent was evaporated under vacuum to give compounds 1-Ac, 2-Ac, 3-Ac, 4-Ac and 5-Ac.

Esterfication of compound 8:

Compound 8 (4 mg) was dissolved in 1 mL of chloroform followed by addition of 1 mL of 20 mM cupric acetate monohydrate in methanol and 5 mL of 0.5 N HCl in methanol. The mixture was stored for 60 min at room temperature, and then extracted with 10 mL of chloroform after addition of 10 mL of water.⁸ The pooled chloroformic extract was washed with water and then evaporated to dryness under a flow of nitrogen to give the corresponding fatty acid methyl ester of compounds 8.

Acid hydrolysis of compound 9:

Compound 9 (3 mg) was added to 2 mL of 1.2% HCl/MeOH and then stored for 20 min at 45 °C. The solution was extracted with hexanes (5 mL). The fatty acid methyl esters in the hexanes layer were identified by GC/MS.⁹

In vitro antiprotozoal assay:

All compounds were tested for their antiprotozoal activities against Leishmania donovani promastigote, L. donovani axenic amastigotes, intracellular L. donovani amastigotes/THP1 cellsand Trypanosoma brucei.¹⁰ The assays have been adapted to 384 well microplate format. In a 384 well micro-plate, the samples with appropriate dilution were added to the cultures of protozoan cells (2 × 106 cell/mL). The plates were incubated at 26 °C for 72 h (37 oC for axenic amastigotes and T. brucei trypomastigotes) and growth of the parasites in cultures were determined by Alamar Blue assay.^10,11 The compounds were tested at concentrations ranging from 0.4-10 μg/mL. The compounds were also tested against L. donovani intracellular amastigotes in THP1 cells employing a parasite-rescue and transformation assay.¹² Pentamidine and Amphoterecin B were tested as the standard antileishmanial agents. DFMO (α-difluoromethylornithine) was tested as a positive control for antitrypanosomal activity. IC₅₀ and IC₉₀ values were computed from dose-response curves using XLfit software.

In vitro antimicrobial assay:

All organisms used for the biological evaluation were obtained from the American Type Culture Collection (Manassas, VA, USA) and including the fungi Candida albicans ATCC 90028, Candida glabrata ATCC 90030, Candida krusei ATCC 6258, Cryptococcus neoformans ATCC 90113, and Aspergillus fumigatus ATCC 90906 and the bacteria methicillin-resistant S. aureus ATCC 43300 (MRSa), Escherichia coli ATCC 35218, Pseudomonas aeruginosa ATCC 27853, and Mycobacterium intracellulare ATCC 23068. Susceptibility testing was performed using a modified version of the CLSI (formerly NCCLS) methods as previously described.^13,14 M. intracellulare was tested using a modified method according to the method described in the literature.¹⁵

In vitro Antimalarial Assay:

Antimalarial activity was determined against chloroquine sensitive (D6, Sierra Leone) and resistant (W2, Indo China) strains of Plasmodium falciparum by measuring plasmodial LDH activity as described earlier.¹³

Cytotoxicity evaluation:

The cytotoxicity of the compounds was determined towards four cancer cell lines (SK-MEL, KB, BT-549, SK-OV-3) and one noncancer kidney epithelial cell line (LLC-PK1) according to a method described earlier.¹⁶

Chemistry:

Compound 1 was isolated as needle crystals from methanol. A molecular formula of C₁₀H₁₄N₂O₅ was determined by HRESIMS at m/z 265.0875 ([M+Na]+, calcd for C₁₀H₁₄N₂O₅Na, 265.0800). From NMR spectroscopic data (Table 1) and comparison with the literature,¹⁷ compound 1 has a deoxyribose moiety attached to a pyrimidine nucleuswith values for deoxyribose moiety at δH 7.04 (1H, t)/δC 85.8, δH 5.05 (1H, m)/δC 71.9, δH 4.48 (1H, dd)/δC 89.3, δH 4.25, 4.15 (2H, dd)/δC 62.8 and δH 2.67 (2H, m)/δC 41.9 while pyrimidine moiety values were at δH 8.17 (2H, s)/δC 137.1 and δH 1.88 (3H, s)/δC 41.9 beside δC 165.5, δC 152.4 and δC 110.9. From the above spectroscopic data and comparing with literature,¹⁷ compound 1 was deduced as thymidine (Figure 1).

Figure 1 Chemical structures of the isolated compounds (1-9) and the acetylated compounds (1Ac-5Ac).

Compound 2 was isolated as yellowish amorphous powders. Its molecular formula (C9H12N2O6) was inferred from HRESIMS (negative ion mode) data [m/z 243.0675 ([M-H]-, calcd 243.0617)]. The NMR spectroscopic data (Table 1) is closely related to compound 1 except the presence of an olefinic proton at δH 5.65 (1H, d, J = 8 Hz) instead of the methyl group at C-5 and additional hydroxyl group at C-2` (δH 4.92 (1H, m)/δC 71.6). Comparing the 13C NMR chemical shifts with those reported in literature,¹⁸ compound 2 was identified as uridine (Figure 1).

Compound 3 was isolated as white amorphous powders. It has the molecular formula of C10H13N5O4 that was determined from the HRESIMS data [m/z 268.1053 [M+H]+, (calcd 268.1046)]. The analysis of NMR spectroscopic data (Table 1) showed the presence of the following; an oxygenated methylene group, six methines (four of them were found to be oxygenated, while two olefinic) and three quaternary carbons. From its spectroscopic data and comparing with literature,¹⁹ compound 3 was identified as adenosine (Figure 1).

Compound 4 was isolated as white crystals (MeOH). Its molecular formula C10H13N5O4 was deduced from HRESIMS (positive ion mode) data [m/z 269.0879 ([M+H]+, calcd for C10H13N4O5, 268.0886)] and (negative ion mode) [m/z 267.0822 ([M-H]-, calcd for C10H11N4O5, 267.0729)]. The NMR spectroscopic data (Table 1) of compound 4 were similar to compound 3 except that C-6 is attached to oxygen instead of nitrogen. By comparing its spectroscopic data with those reported in literature,¹⁷ compound 4 was identified as Inosine (Figure 1).

Compounds 5-9 were identified by comparison of their spectroscopic characteristics with those previously reported in the literature as 24-methylene cholesterol (5),^20,21 dihydrocholesterol (6),²² cholesterol (7),²² oleic acid (8)²³ and 1,3-palmityl-2-palmitoleoylglycerol (9).^24,25 Acetylation of compounds 1, 2, 3, 4 and 5 yielded thymidine 3`,5`-diacetate (1-Ac), uridine 2`,3`,5`-triacetate (2-Ac), adenosine 2`,3`,5`-triacetate (3-Ac), inosine 2`,3`,5`-triacetate (2-Ac), cholesterol-3-acetate (5-Ac) and 24-methylene cholesterol-3-acetate (5-Ac), respectively (Figure 1).

Antiprotozoal activity:

Antiprotozoal activity of all compounds was tested against Leishmania donovani Promastigote, Leishmania donovani Amastigote, Leishmania donovani Amastigote/THP1 and Trypanosoma brucei. 24-methylene cholesterol (5) showed promising antitrypanosomal activity with IC₅₀ and IC₉₀ values of 3.39, 6.69 µg/mL, which was comparable to DFMO (3.58, 8.53 µg/mL), the positive control (Table 2) (Figure 2). Other compounds tested did not show noticeable activity against the protozoan parasites. A recent study has reported 24-Methylenecyclopropane as new class of steroidal inhibitors-mechanism-based suicide substrates with promising activity against T. brucei.²⁶ It would be interesting to investigate 24-methylene cholesterol as inhibitor of ergosterol biosynthesis as potential drug target.²⁷

Figure 2 IC₅₀ and IC₉₀ values for in vitro activity of compound 5 against Trypanosoma brucei..

Antimicrobial activity:

Antimicrobial activity of the compounds was determined against bacterial strains, Methicillin-resistant Staphylococcus aureus (MRSa), Escherichia coli, Pseudomonas aeruginosa and Mycobacterium intracellulare, as well as against pathogenic fungi including Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans with no significant activity.

Antimalarial activity:

All the isolated compounds were tested against chloroquine-sensitive (D6) and chloroquine-resistant (W2) strains of Plasmodium falciparum by measuring plasmodial LDH activity but, none of the tested compounds showed any activity.

Cytotoxic activity:

All the isolated compounds were tested against SK-MEL, KB, BT-549, SK-OV-3 and LLC-PK1. None of the compounds was cytotoxic to all the cancer cell lines up to the highest tested concentration of 25 µg/mL. However, compound 5 showed cytotoxicity to kidney cells with IC₅₀ value of 23 µg/mL.

Nine known compounds were isolated from the Red Sea tunicate Polyclinum constellatum. The structures were elucidated by spectroscopic analysis. The antimicrobial, antimalarial, antiprotzoal, and cytotoxic activities of the isolated compounds were evaluated. Compound 5 displayed good antitrypanocidal activity with IC₅₀ and IC₉₀ of 3.39 and 6.69 µg/mL respectively, and cytotoxicity against LLC-PK­1 with IC₅₀ value of 23 µg/mL.

The authors thank Dr. Bharathi Avula, Dr. Mellisa Jacob and Surendra Jain, National Center for Natural Products Research for assistance with the HRESIMS, antimicrobial and antiprotozoal assays respectively and Dr. Tarek Temraz, Marine Science Department, Faculty of Science, Suez Canal University, Ismailia for the taxonomical identification of the marine organism. Thanks are also due to the Egyptian Environmental Affairs Agency (EEAA) for facilitating sample collection along the coasts of the Red Sea. Financial support was partially provided by the Egyptian government. Antimicrobial and antiprotozoal biological screenings at the NCNPR are partly supported by the cooperative scientific agreement with USDA-ARS (58-6408-2-0009).

Author declares that there is no conflict of interest.

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