Haliscosamine: a new antifungal sphingosine derivative from the Moroccan marine sponge Haliclona viscosa
© El-Amraoui et al.; licensee Springer. 2013
Received: 14 January 2013
Accepted: 28 March 2013
Published: 4 June 2013
In the aim of searching for new antifungal products from marine origin, we have isolated a sphingosine derivative, (9Z)-2-amino-docos-9-ene-1,3,13,14-tetraol (Haliscosamine) from the Moroccan sea sponge Haliclona viscosa using bio-guided (antifungal) HPLC methods. The molecular structure of this compound was elucidated by spectrometric techniques IR, UV, MS and NMR. The isolated metabolite showed a significant antifungal activity against Cryptococcus and Candida species and a weak general toxicity in the brine shrimp lethality test. Further research is needed to study its in vivo activity, as well as to elucidate the mechanism underlying its activity in the hope of a future use in medical mycology.
Our laboratory aims to find new antifungal metabolites from marine origin for use in human medicine on one hand, and on the other hand in phytopathology. Marine invertebrates of Moroccan Atlantic coast are our preferred source of producers of active substances, specifically sponges, known generally to contain secondary metabolites with interesting biological activities (Faulkner 2002) including antimicrobial (Baker et al. 2009), antifungal (Clark et al. 2001), antileishmanial (Dube et al. 2007), antioxidant (Regoli et al. 2004) and cytotoxic activities (Ayyad et al. 2009 Fusetani et al. 1989 Erickson et al. 1997 Rashid et al. 2000). In the first work (El-Amraoui et al. 2010), we screened antifungal activity in hydroalcoholic and organic extracts of 14 sponges and showed that three species of them are active against pathogenic fungi and bacteria: Haplosclerida adocia, Cinachyrella tarentina and Haliclona viscosa. This latter species being shown the most active, we chose it to isolate the active compound. Kupchan partitioning, then multistep HPLC from the organic extract provided a pure active product. We determined its structure and evaluated its antifungal potential and its toxicity.
Isolation of the active product
Lyophilized sponge (800 g) was extracted with EtOH, the extract partitioned between CH2Cl2 and H2O, and the organic solution submitted to a Kupchan liquid partition procedure. EtOAc and MeOH:H2O fractions were pooled, then the mixture was successively separated by three steps of HPLC to yield 47 mg of an amorphous pale yellow product. All steps of this isolation were bio-guided by antifungal (agar disc-diffusion) test. The total mass of the product (taking into account other fractions containing isolated compound) was estimated to be 80 mg from 800 g of dry sponge (0.01%).
Molecular structure of the product isolated from Haliclona viscosa
NMR spectroscopic data for haliscosamine
δ 1H ppm J(Hz)
(J = 11.7, 4.0)
1b, 2, 3
(J = 11.7, 4.0)
1a, 2, 3
(J = 6.8, 4.0)
1, 3, 4
(J = 6.8, 4.0)
1, 2, 4, 5
1, 2, 4, 6
2, 3, 5
2, 3, 5
7, 9, 10
8, 9, 11
10, 12, 13
10, 12, 13
11, 12, 14, 15
10, 11, 12, 15
13, 15, 16
13, 14, 16
13, 14, 16
(J = 6.5)
The optical rotation was [α]20 D = 22,3 (c = 0,76, MeOH).
Biological activities of haliscosamine
In vitro antifungal activity of haliscosamine and nystatin against pathogenic yeasts
Growth inhibition diameter (mm)
Haliscosamine (100 μg)
Nystatin (100 μg)
(-)-untenospongin B (100 μg)
Minimum Inhibitory Concentration (MIC 90 ) of haliscosamine and nystatin against pathogenic yeasts
Minimum inhibitory concentration (μg/mL)
0.2 – 0.4
3.12 – 6.25
0.4 – 0.8
3.12 – 6.25
0.4 – 0.8
The in vitro antifungal activity by the diffusion method showed that haliscosamine was more active than nystatin in inhibiting the growth of Cryptococcus neoformans (ATCC 11576), but showed the same activity as nystatin in inhibiting the growth of C. albicans (ATCC 10231). On the other hand, haliscosamine was active against C. tropicalis (R2 CIP 1275.81), an amphotericin B and nystatin resistant strain.
The MIC90 of haliscosamine is less than that of nystatin on C. neoformans. and C. albicans, and the same level as the previous in C. tropicalis.
Lethality concentration (24 h-LC50 value) for haliscosamine is 664.86 μg/mL. This general toxicity activity is considered weak when the LC50 was between 500 and 1000 μg/mL, moderate when the LC50 was between 100 and 500 μg/mL, and designated as strong when the LC50 ranged from 0 to 100 μg/mL (Padmaja et al. 2002Canales et al. 2007). Consequentely, the toxicity of the haliscosamine may be considered as weak by this test.
Haliscosamine isolated from the Moroccan marine sponge Haliclona viscosa is a new derivative of sphingosine with an original molecular structure ((Z)-2-amino-docos-9-ene-1,3,13,14-tetraol, C22H45NO4). However, the relative and absolute stereochemistries of the molecule remain to be determined, with confirmation by total synthesis.
The high content (0.01%) of the haliscosamine in the sponge, as its presence in the free state and not involved in a ceramide or other complex lipid is remarkable. The haliscosamine is added to products already isolated from sponges of the genus Haliclona, such the alkaloids viscosamine (Volk and Kock 2003), viscosaline (Volk and Kock 2004) and haliclamines A, B, C and D (Fusetani et al. 1989Volk et al. 2004). The activity of this product also shows the bioactive potential of marine sponges from the Atlantic Coast of Morocco, and encourages them to continue our sorting activity. The only study that has been done before has been the isolation of untenospongin B (antimicrobial) from the marine sponge Hippospongia communis (Rifai et al. 2004) and of fasciculatin (cytotoxic and inhibitor of lymphocyte proliferation) isolated from Ircinia variabilis (Rifai et al. 2005).
Haliscosamine has a remarkable antifungal effect. Sphingolipids are already known for their antiseptic and antifungal activity (Bibel et al. 1995), and sphingosine and its derivatives are natural antimicrobial agents, protecting the human skin from bacterial colonization (Bibel et al. 1993) as well as being anti-inflammatory agents (Radhika et al. 2005).
Compared to nystatin, haliscosamine showed in vitro significant activity against Candida albicans (ATCC 10231), Candida tropicalis (CIP 1275.81) and Cryptococcus neoformans (ATCC 11576). These three yeasts are often involved in human mycology especially C. tropicalis that is resistant to nystatin and amphotericin B.
Comparing the antifungal activity of haliscosamine with (-)-untenospongin B isolated from the marine sponge Hippospongia communis collected from the Atlantic Coast of Morocco (Rifai et al. 2004), it was found that haliscosamine was more active than untenospongin B in inhibiting the growth of C. tropicalis (R2 CIP 1275.81), C. albicans (ATCC 10231) and C. neoformans (ATCC 11576) (Table 2). So, haliscosamine is an interesting product, thanks to its antifungal potential against strains involved in human mycology, including those resistant to nystatin. Further studies are still needed to determine in vivo activity and toxicity of haliscosamine in the animal model as well as to elucidate the mechanism underlying its activity. The lethal concentration (24 h-LC50 = 664.86 μg/mL) of haliscosamine is greater than 500 μg/mL, so the general toxicity activity was regarded weak. Therefore, if the activity persists in vivo, then the product could be a candidate to assess more fully for a development of a new drug for the treatment of fungal infections.
Materials and methods
Biological marine material
The marine sponge (Ref. EM14) H. viscosa(Topsent,1888) was collected from the Atlantic coast of El-Jadida city, Morocco (N 33 15 422, W 008° 29 722)(El-Amraoui et al. 2010). The sponge was identified by Dr. Maria-Jesús Uriz, Research Professor at the Centro de Estudios Avanzados de Blanes(CEAB), Spain. After collection, the sponge was immediately cut into small pieces, washed with sterile distilled water, then frozen at -30°C for two days and immediately freeze-dried to give a lyophilized material ready for extraction.
General experimental procedures
Freeze-dryer was a FreeZone2.5Plus type (Labconco, USA).
TLC was carried out on precoated Macherey-Nagel Alugram silica, and spots were visualized by spraying with iodine vapor or ninhydrine reagents.
The UV spectrum was recorded on a Helios Omega spectrophotometer (Thermo Scientific, France).
The IR spectrum was obtained on a spectrometer IR-FT Paragon 1000 PC (Perkin-Elmer, USA).
The NMR spectra (1H, 13C, HSQC, HMBC, COSY and TOCSY experiments) were recorded on a Bruker 500 with a TXI cryosonde (PRISM, Rennes University, France) and a Bruker Avance 500 (CRMPO, Rennes University, France) for in-deep 2D experiments and irradiations tests.
HRESIMS data were recorded on a Micromass Zab Spec Tof (CRMPO, Rennes University, France). ESIMS data of the acetylated product was recorded on a Finnigan LCQ mass spectrometer (IFREMER, Nantes, France).
Optical rotation was measured on a Schmidt + Haensch Polartronic NH8 polarimeter.
Acetylation: 1 mg of isolated product was treated with 1 mL of acetic anhydride (Ac2O):pyridine (1:1 v/v) for 24 h at ambient temperature, then 40 mL of +4°C water was added and the mixture extracted with diethyl-ether (4 × 10 mL) to afford 1.2 mg of acetylated product.
Extraction and isolation
The sample (800 g of lyophilized sponge) was homogenized with ethanol 80% (1 × 1000 mL), allowed to stand in a dark chamber for 24 h and filtered. The residue was again extracted with absolute ethanol (2 × 1000 mL). Both ethanolic extracts were combined, and then evaporated at reduced pressure until total evaporation of ethanol. The resulting aqueous suspension was completed with distilled water to 1000 mL as final volume and extracted with CH2Cl2 (3 × 500 mL). The CH2Cl2 solutions were combined, dried on anhydrous sodium sulphate (Na2SO4), filtered and concentrated at reduced pressure to give a dichloromethane extract (3 g).
This extract was fractioned by modified Kupchan method: 3 g were dissolved in 300 mL of MeOH:H2O (9:1 v/v) and extracted with 3 × 150 mL of hexane (A) (1.5 g). The remaining solution was adjusted with distilled water to get the proportions 6:4 v/v, then extracted with 3 × 150 mL of dichloromethane (B) (0.3 g) and then with 3 × 150 mL of ethyl acetate (C) (0.7 g). The residue was lyophilized (D) (0.5 g).
Fractions C and D were combined (1.2 g), applied on silica gel 60 (25 g) column, and eluted with successive mixtures (110 mL) of CHCl3: MeOH (10:0; 9:1; 8:2; 7:3; 6:4; 4:6 and 0:10 v/v) to yield nine fractions. Fractions 4, 5 and 6 were pooled (260 mg), then separated on a semi-preparative HPLC diol column (Inertsil Diol L10OH.25R, 10 × 250 mm, 10 μm) by a CH2CL2: MeOH (8.5:1.5 v/v) isocratic mixture. Purification of subfractions 2 (95 mg) by HPLC C18 column (Inertsil ODS-3, 4.6 × 250 mm, 5 μm) with isocratic MeOH:H2O (6.5:3.5 v/v) gave 47 mg of a pure active product.
The fungal species obtained from the Fungi Culture Collection (FCC) of the National Cultures Collection of Microorganisms of the Pasteur Institute, Paris, France, from the Collection of Institut Pasteur (CIP) and from the American Type Culture Collection (ATCC) were used as the antifungal test strains: Candida albicans (ATCC 10231), Candida tropicalis (R2 CIP 1275.81, an amphotericin B and nystatin resistant strain), Cryptococcus neoformans (ATCC 11576). The yeasts were maintained on the Sabouraud’s agar medium at 28°C.
The antifungal activity was assessed in vitro by agar disc-diffusion test. The minimum inhibitory concentration was evaluated by the microdilution method.
Agar disc-diffusion test
This test uses Yeast Morphological Agar (YMA) as medium [yeast nitrogen base (Difco) 60.5 g/L; asparagin (Prolabo) 1.5 g/L; glucose (Merck) 10 g/L and agar (Merck) 20 g/L]. The suspensions of yeast were adjusted in sterile water to match the density of a 0.5 McFarland Standard. Each disk received 100 μg of sponge extract and was applied on the test media which were previously inoculated with each test strain. Plates were first kept at 4°C for at least two hours to allow the diffusion of chemicals, and then incubated at 28°C. Inhibition zones were measured after 24 h of incubation (Galeano and Martínez 2007). Standard disks of nystatin (100 μg) served as positive antifungal control. All the assays were carried out in triplicate.
Minimum inhibitory concentration (MIC)
The MIC90 (the lowest concentration causing at least 90% of growth inhibition when compared to drug-free control) of the isolated compound was measured using the method described by Rifai et al. (2005). Yeast Morphological Broth medium was used as test media. Tests were performed in 96-well round bottom sterile culture plates. The suspensions of yeast were adjusted in sterile water to match the density of a 0.5 McFarland Standard. The wells of a microdilution plate were inoculated with 180 mL of the culture medium containing a final inoculum of 0.5 - 2.5 103 CFU/mL. The test drug and positive control (nystatin) previously solubilized in dimethylsulphoxide (DMSO) were serially diluted two fold in the liquid medium to give a range of concentration from 640 to 0.1 μg/mL. Twenty μL of each concentration were added to wells containing culture suspension except the growth control well. The final concentration ranged from 64 to 0.01 μg/mL. Plates were incubated at 35°C for 48 h. Fungal growth was assessed at 494 nm by measuring the optical density in each well using an enzyme immunoassay multiwell reader (Sigma diagnostic). The test was carried out in triplicate.
Brine shrimp toxicity test
Brine shrimp eggs (Artemia salina) are hatched in artificial seawater prepared by dissolving sea salt in distilled water (38 g/L) during 48 h incubation in a warm room (22 - 29°C). Seawater is placed in a small unequally divided tank and shrimp eggs are added to the larger compartment of the tank which is covered by aluminium foil to darken it. The illuminated compartment attracts shrimp larvae (nauplii) through perforations in the dam (Meyer et al. 1982).
Brine shrimp microwell toxicity assay
The toxicity of haliscosamine was monitored by the brine shrimp lethality test. Samples were dissolved in DMSO and diluted with sea water so that the final concentration of DMSO did not exceed 0.05%. Serial dilutions (2000, 200 and 20 μg/mL) of samples were made in wells of 96-well microplates in triplicate in 100 μL of sea water (Rahman et al. 2001). The last row was left with sea water and DMSO only served as the drug free control. 100μL of suspension of nauplii containing about 10 larvae were added into each well and incubated for 24 h at 22-29°C. The plates were then examined under a binocular microscope (× 12.5) and the number of dead nauplii in each well was counted. One hundred μL of methanol were then added and after 10 min, the total numbers of shrimp in each well was counted and recorded. Lethality concentration fifties (LC50 values) for each assay were calculated by taking the average of the three experiments using a Finney Probit analysis program on an IBM computer.
We thank Dr. Maria-Jesús Uriz, Research Professor at the Centro de Estudios Avanzados de Blanes (CEAB), Spain for sponge identification, Pr. Sourisak Sinbandhit and Pr. Philippe Jehan from CRMPO and Pr. Arnaud Bondon from PRISM platform at Rennes University, France for the NMR and MS, and the CNRST of Morocco for the support of the mobility of the researchers of this project.
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