Received date: July 10, 2015 Accepted date: July 20, 2015 Published date: July 27, 2015
Citation: Tommonaro G, Iodice C, Abd El-Hady FK, Guerriero G, Pejin B (2015) The Mediterranean Sponge Dysidea avara as a 40 Year Inspiration of Marine Natural Product Chemists. J Biodivers Endanger Species S1:001. doi:10.4172/2332-2543.S1-001
Copyright: © 2015, Tommonaro G, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Marine sponges have been intensively studied by organic chemists, biologists and pharmacologists during last 60 years, due to great biodiversity of their secondary metabolites. A couple of very promising bioactivities of these compounds have been reported. Indeed, some of these metabolites are considered as the leads for the future drugs targeting tumours, viral and bacterial diseases, malaria and inflammations. Herein we briefly report key bioactivities of avarol, the main secondary metabolite of the marine sponge Dysidea avara (Schmidt, 1862).
Marine sponges; Dysidea avara; Avarol; Bioactivity; Antimicrobial; Cytotoxic activities; Alzheimer's disease; Antipsoriatic
The first marine invertebrate group to be studied in any comprehensive way by those searching for new organic compounds was the sponges (sedentary filter-feeding metazoans belonging to the phylum Porifera) [1,2]. Confronted to other organisms, these animals are found to be a rich source of biologically active compounds exhibiting, inter alia , cytotoxic, immune-modulatory [3,4] and antifouling effects . Marine sponges are the most ancient metazoan phylum which taxonomy depends on the composition of their skeletal materials  and morphological characteristics . Actually, the sponges are divided into three main classes: Calcarea (calcareous spicules), Hexactinellidae (siliceous spicules) and Demospongiae (spicules made of proteinaceous sponging fibres). This phylum encompasses ≈ 6000 taxonomically validated species, with the new ones still being discovered [2,8]. Among the sponges, the class Demospongiae represents the most various and ecologically remarkable group, covering 90% of all species . These sponge species are asymmetric and brightly coloured (being orange, green, yellow, purple or red), due to the presence of pigment granules in their amoebocytes.
The sponges of the genus Dysidea belong to the class Demospongiae, order Dictyoceratida and family Dysideidae. Usually lack mineral spicules and have the main skeleton consisting of reticulation of sponging fibers . By the year 2001, this genus was reported to include 17 species .
Dysidea avara (Schmidt, 1862), a violet-colored, encrusting and heavily conulated dictyoceratid sponge, has been mostly observed at sciaphilic microhabitats of the rocky sublittoral Mediterranean to 80- m depths . This species produces the sesquiterpene hydroquinone avarol (as the main secondary metabolite) and its quinone form avarone , as well as some other minor derivatives . Avarol has showed numerous bioactivities such as antitumour, antileukemic and antipsoriasiatic [13-16]. Indeed, high pharmacological potential of avarol has prompted a number of researchers worldwide to address the issue of its obtaining at a large scale, by means of chemical synthesis  or the sponge cultivation [18-20].
Different factors have been described to regulate the production of secondary metabolites in marine sessile organisms, primarily in the sponges, both abiotic (temperature, light, salinity and pollutants) and biotic (fouling, space competition, predation, and presence/absence of bacterial symbionts) [21-24]. The spatial variation of these compounds in the sponges has been reported between different localities [25-27] and within the localities in opposing habitats differing in the amount of irradiance  or predation pressure . Additionally, the sponge cells that biosynthesise and store these compounds have been identified in a number of cases [30-34]. Interestingly, the concentration of target metabolites is usually higher at the sponge periphery. The favoured storage of bioactives out of the sponge centre zone has been ascribed to a defensive role against the foulers or predators [35-41].
Avarol (1) was firstly isolated from the Mediterranean sponge D. avara along with a minor amount of its oxidised derivative avarone (2) (Figure 1) . Its chemical structure was firstly established with circular dichroism and spectroscopic studies , and later on by chemical synthesis . Avarol and its derivatives are among the most important marine metabolites because of their broad spectrum bioactivity and very low toxicity.
Over 100 articles on avarol and/or avarone recorded in Chemical Abstracts show the high level of interest in these compounds. In a mini-review article, it is almost impossible to cover all the bio-effects reported for both compounds so far.
As a consequence, herein the attention will be paid only to more significant ones.
Avarone and to a lesser extent avarol are active against a variety of Gram-positive bacterial species. The highest activity was observed for Streptococcus pneumoniae and Erysipelothrix rhusiopathiae (MIC 0.78 μg/ml) . Recently, it has been reported that avarol exhibits much higher antibacterial activity towards Bacillus cereus , Salmonella typhimurium and Enterobacter cloacae with minimum inhibitory and bactericidal concentrations ranging between 2-8 μg/mL and 4-16 μg/mL, respectively . On the other hand, avarol primarily displayed antifungal activity against Trichophyton mentagrophytes (MIC 7.8 μg/ml), T. rubrum (MIC 15.6 μg/ml) and Microsporum canis (MIC 15.6 μg/ml) . This compound also showed considerable effect on Aspergillus versicolor and Penicillium verrucosum var. cyclopium exhibiting MIC and MFC values ranging between 4-15 μg/mL and 8-30 μg/mL, respectively.
In addition, avarol proved to be more active both than ketoconazole and bifonazole . Avarol and avarone also showed potent cytostatic activities on L-5178Y (ED50 0.93 and 0.62 μM, respectively) , L-1210 (ED50 13.9 and 15.6 μM, respectively) and C-8166 (ED50 9.2 and 12.9 μM, respectively) cells .
Both compounds exhibited good antileukaemic activity in vivo as well, using L-5178Y cells in NMRI mice. At doses of 10 mg/kg applied to the mice bearing ≈ 108 leukaemia cells during 5 days, avarone was more curative than avarol (70 and 20%, respectively) . Moreover, these compounds are effective antimutagenic agents displaying similar activity as the known cytochrome P-450-dependent mono-oxygenase inhibitor, benzoflavone . The cytotoxicity of avarol was in vitro screened by MTT assay against four human tumour cell line [breast (MCF-7), colon (HT-29), prostate (PC-3) and skin (HS-294T)], too. The colon HT-29 tumour cells practically were the only sensitive ones towards this compound (IC50 <7 μM), while no toxicity was found against the fetal lung fibroblast MRC-5 cells at the concentrations tested. In comparison with doxorubicin, used as a positive control, avarol actually exhibited at least 588-fold less toxicity towards normal MRC-5 cells. Finally, comet assay indicated that DNA fragmentation was almost 5-fold higher upon the treatment with doxorubicin, compared to avarol . The obtained results have actually confirmed that avarol scaffold may contribute to development of new cytostatics inspired by Nature.
Both avarol and avarone showed a dose-related inhibitory effect on HIV (HTLV-III) replication in human H-9 cells measured by determination of RT activity and the expression of HTLV-III gag proteins p24 and p17 by monoclonal antibodies . At the administered dose of 3 mg/kg, no side effects of avarol were observed on healthy test subjects .
Afterwards, it was established that these compounds exhibited a topical anti-inflammatory activity, with ID50 values of 97 and 397 μg/ ear, respectively, higher than indomethacin, both in the Tetradecanoyl-Phorbol Acetate (TPA) induced ear oedema and the oedema induced by carrageenan (ID50 9.2 and 4.6 mg/kg p.o., respectively) . Avarol also moderately inhibited the human recombinant synovial PLA2 (IC50 158 μM) and COX (IC50 1.4 μM), displaying more effective activity against LO (IC50 0.6 μM) . Furthermore, avarol showed high antioxidant activity (IC50 18 μM), evaluated by the free-radical scavenging assay using DPPH ; this compound inhibited in vitro induced microsomial lipid peroxidation, too . At the first place, avarone exerted antiplatelet activity both on platelet-rich plasma and washed platelets. It was a stronger inhibitor of platelet aggregation induced by arachidonic acid or A23187 vs. adenosine-5'-diphosphate, platelet-activating factor or U46619 .
On the other hand, avarol inhibited tumour factor-α (TNF-α) generation in stimulated human monocytes (IC50 1 μM) and TNF-α- induced activation of nuclear factor-κB (NF-κB)-DNA binding in keratinocytes.
In the psoriasis-like model of TPA-induced mouse epidermal hyperplasia, topical administration of avarol (0.6-1.2 μmol/site) reduced oedema, myeloperoxidase activity, IL-1β, IL-2 and eicosanoid levels in skin. Avarol was also found to suppress NF-κB nuclear translocation in vivo , determined in mouse skin . Psoriasis is a known skin disease typified by epidermal hyperplasia, inflammation of the dermis and epidermis and leukocyte infiltration . It is firstly a keratinocyte proliferation disorder caused by the pathological development of a hypertrophic physical defence barrier. The classic topical treatments are founded mainly on anti-proliferative treatment or differentiation modifying activity . NF-κB is a crucial factor for the immuno-inflammatory responses which is also implicated in different skin diseases including psoriasis . A crucial link between high levels of TNF-α and NF-κB activation has been found in psoriatic patients. Indeed, the potential mechanism of action of TNF-targeting agents lies in the down-regulation of NF-κB transcriptional activity .
The anti-inflammatory treatments based on the blocking of TNF-α signalling have been recently shown to be valid and highly promising alternative for this skin condition.
The bioactivity of both compounds has been mainly correlated with their redox chemistry and ability to affect radical production. In other words, the terpenoid moiety is believed to play just a marginal role in these processes [59-61].
The interesting pharmacological properties of avarol and avarone together with previous findings that avarone, the quinone of avarol, reacts towards protein sulfhydryl groups, have prompted the researchers to prepare further sulfhydryl derivatives of these compounds and extend the bioactivity evaluation to other effects [52,62,63].
Avarol-3'-thiosalicylate (3) (Figure 2) is a potent inhibitor both of superoxide generation in human neutrophils and PGE2 generation in the human keratinocyte HaCaT cell line (IC50 2.5 μM) . This compound also reduced LTB4 (IC50 1.79 μM), prostaglandin E2 (IC50 17.30 nM) and TNF-α (IC50 4.18 μM) production in activated leukocytes, in a concentration-dependent manner. Its oral administration in the mouse air pouch model gave a dose-dependent reduction of all aforementioned inflammatory mediators. The same compound inhibited human synovial recombinant PLA2 (IC50 5.9 μM) as well as the binding of NF-κB to DNA (at 5 μM) in HaCaT keratinocytes. These findings have indicated that avarol-3'- thiosalicylate represents a promising antipsoriatic agent due to both in vitro and in vivo inhibition of different biomarkers related to the inflammatory response of psoriatic skin .
A number of semisynthetic thio-avarol derivatives have been assayed on the inhibition of the enzyme acetylcholinesterase (AChE) .
The AChE inhibition test pointed out a moderate inhibitory activity (1μg) for all thio-avarol derivatives with a carboxylic acid group in the molecule, namely avarol-3'-thiosalicylate (3), avarol-3'-thiolactate (4), avarol-4'-thiolactate (5), avarol-3'-thioglycolate (6), avarol-4'- thioglycolate (7) and avarol-3'-thiobenzoate (8) (Figure 2). In comparison, the alkaloid galanthamine used clinically for the treatment of Alzheimer's disease (AD) inhibited the enzyme at 0.01 μg .
In AD, the most common cause of senile dementia in later life, a deficiency in cholinergic neurotransmission has been observed. AChE is the enzyme involved in the metabolic hydrolysis of acetylcholine at cholinergic synapses in the central and peripheral nervous systems. Currently, AChE inhibitors are still the best drugs used for the management of this disease . Most inhibitors of AChE are alkaloids that often possess several side effects; therefore it is important to search for new AChE inhibitors not belonging to this chemical class.
Recently, Sladić and co-workers have reported the synthesis and biological activity of further thio-avarone derivatives. After preliminary cytotoxic bioassays, only 4'-isobutylthio-avarone (9), 3',4'- ethylenedithio-avarone (10) and 4'-phenylthio-avarone (11) (Figure 3) were selected by NIH-NCI for in vitro screening in a panel of human tumour cell lines (Table 1) .
|Mean GI50 (µM) of compounds*|
Table 1: Cytotoxicity of Avarol derivatives against a panel of human tumour cells. *(9) 4'-Isobutylthio-avarone, (10) 3',4'-Ethylenedithioavarone and (11) 4'-Phenylthio-avarone.
The finding that 3'-methylamino-avarone (12) and 4'-methylaminoavarone (13) (Figure 3) inhibited the cell cleavage of fertilized eggs from the sea-urchin Sphaerechinus granularis , has prompted a research group to prepare a series of amino derivatives of avarone and estimate their biological activity [45,70]. 4'-Methylamino-avarone (13) and 4'-ethylamino-avarone (15) (Figure 3) were found to be the most toxic among the compounds tested in brine shrimp lethality assay (as an indicator of cytotoxicity), with the activity (LC50 0.23 and 0.34 ppm, respectively) comparable to the avarol one (LC50 0.18 ppm) . The brine shrimp assay is an in-house assay substituting the KB-9, PS-9, L-5178y and L-1210 cytotoxicity .
The amino derivatives of avarol were also tested in vitro as cytostatic and antiviral agents (Table 2) . Generally, the introduction of a methylamino or an ethylamino group in the 3' position of the quinone ring of avarone has resulted in the chemical structures endowed with higher potency against L-1210 and B- and Tlymphoblast cells.
|Cytostatic||Mean ID50 (µM)|
|Antiviral||Mean ED50 (µM)|
Table 2: Cytostatic and antiviral activities of Avarol derivatives. *Avarol (1), Avarone (2), 3'-Methylamino-avarone (12), 4'- Methylamino-avarone (13); 3’-Ethylamino-avarone (14); 4’- Ethylamino-avarone (15); 4'-Leucine-avarone (16); 4'-Serine-avarone (17).
On the other hand, the same introduction in the 4' position has offered the compounds (13 and 15) with cytotoxicity comparable to the avarone one (2). The modifications in the quinone ring of avarone have always resulted in the loss of anti-HIV activity with two exceptions, 4'-leucine-avarone (16) and 4'-serine-avarone (17). Indeed, both avarone deivatives have retained the potency of the parent one .
A couple of amino-avarone derivatives were also tested as potential antipsoriatic agents. 3'-methylamino-avarone (12) displayed the best antiproliferative profile, inhibiting the 3H-thymidine incorporation in HaCaT cells (IC50 4.5 μM) with potency similar to anthralin, followed by lack of any cell toxicity. Due to its antioxidant properties, the same compound (12) was also able to decrease the undesirable effects derived from respiratory bursts of neutrophils infiltrated in the psoriatic skin .
The additional derivatives were isolated from the sponge D. avara (collected from different places in the Gulf of Naples, Italy), namely 5'- acetyl avarol (18) , diacetyl avarol (19) and 6'-hydroxy-5'-acetyl avarol (20) (Figure 4) .
Monoacetyl avarol (18) showed a cytotoxicity (LD50 0.09 ppm) twice of the avarol one (1) , while diacetyl avarol (19) (LD50 0.15 ppm) and the compound 20 (LD50 1.30 ppm) exhibited the activity comparable to avarol, in the brine shrimp assay . Afterwards, Shen and co-workers reported the synthesis and cytotoxicity of several acylated derivatives of avarol. Diacetyl avarol (19) displayed a marked antitumour activity against KB cells (IC50 1.35 μg/ml), while di-pbromobenzoyl avarol (21) (Figure 4) was active against Hepa cells (IC50 1.30 μg/ml) .
Till date the sponges have been in the focus for the reason of two main and often interrelated factors: they are a rich source of biologically active secondary metabolites and form close association with a wide variety of microorganisms. Indeed, the surfaces and internal spaces of marine sponges make a unique microhabitat for these organisms. As more rich in nutrient than seawater and most sediment, the sponge environments do represent a very promising niche for the isolation of diverse bacteria and fungi.
In fact, high microbial diversity within some marine sponges and intriguing 'host-bacterial symbiont' distributions have been recently reported .
Certain demosponges are known to be associated with enormous amounts of microorganisms contributing up to 40-60% of the animal biomass, with density in excess of 109 microbial cells per mL of the sponge tissue, several orders of magnitude higher than those typical for sea water . The vast majority of these microorganisms are located extracellularly within the mesohyl matrix .
It appears that a given sponge species contains a mixture of generalist and specialist microorganisms with the associated microbial communities being fairly stable both in space and time . To date, the bacterial roles in the sponge biology and ecology have not been elucidated in details. More precisely, a few reports describe the contribution of symbiotic microbes to the sponge well-being or survival . The symbiont community may serve as a source of nutrition by transferring products of metabolite processes to the host. For example, symbiotic cyanobacteria have been shown to transfer organic carbon obtained through photosynthesis to the host . The other suspected benefits of the symbionts include contribution to the structural rigidity of the sponges and production of halogenated marine natural products .
Generally, little is known about the mechanisms that regulate association and communication between the sponges and associated microorganisms. Recently, it has been demonstrated that multispecies bacterial communities can communicate with each other via small secreted molecules, such as N-acyl homoserine lactones (AHLs) and diketopiperazines (DKPs), through the process known as quorum sensing (QS) [81,82].
The specimen of D. avara sponge (collected in the Gulf of Naples, Italy) was used for the isolation of bacteria. The identified strains by PCR analysis were found to belong to the genera Vibrio and Pseudoalteromonas . These strains were tested for AHL production by means of a TLC-overlay (with Agrobacterium tumefaciens NTL4) and Lux screen assays. The dichloromethane extract of cell-free medium of Vibrio sp. (isolated from the sponge) activated QS both in TLC overlay and Lux screen assays. Furhermore, Pseudoalteromonas sp. inhibited QS in a 'T' streak assay with Chromobacterium violaceum . More precisely, a negative gradient of violacein production (that is under control of AHL molecules) was observed. This indicated either AHL inhibition or degradation by Pseudolateromonas sp. To identify the AHL bioreporter activating signal, the aforementioned extract of Vibrio sp. was purified by means of chromatographic techniques affording four compounds characterised by NMR (1H, 13C) . The isolated compounds displayed typical spectra of cyclic dipeptides and were identified as cyclo-(L-prolyl-L-phenylalanine) (22), cyclo-(cis-4- hydroxy-D-prolyl-L-leucine) (23), cyclo-(trans-4-hydroxy-L-prolyl-Lphenylalanine) (24) and cyclo-(L-prolyl-L-leucine) (25) (Figure 5) . The finding of two different bacteria associated with D. avara which display opposed responses on AHL synthesis adds an interesting dimension to the study of signalling interactions in the sponge microcosms. This peculiar behaviour may be part of the mutual control of the growth of different microorganisms in the same host. In addition, other cyclic dipeptides, namely cyclo-(L-prolyl-Lleucine) and cyclo-(L-prolyl-L-phenylalanine), isolated from Vibrio sp. associated with the marine sponge D. avara , were also able to activate QS bioreporters. These experimental data should be useful for studying cross-kingdom chemical communication .
Although the marine environment is a plentiful source of novel marine natural products with pharmaceutical potential, only a few of them have reached the stage of commercial production so far. Arabinofuranosyladenine (Ara-A, isolated from the gorgonian Eunicella cavolini) , classifying among the most potent antiviral drugs, is one of the compounds currently in clinical use . Avarol [11,87] has been also commercialised, as a cream against skin disorders including psoriasis.
Nowadays the patent applications on these natural products represent less than 10% of the total number of the relevant papers. Additionally, the number is very little compared with that one covering the patents on terrestrial organisms. The limited availability of larger quantities of a particular organism to be extracted is one of the major causes for low commercial utilisation. On the other hand, the recovery from marine organisms is unacceptable, due to the devastating impact on the natural environment. Actually, four different strategies may be attempted to obtain marine bioactives in enhanced amounts: chemical synthesis, aquaculture, cultivation of marine organisms in bioreactor and cell culture.
Generally, pharmaceutical companies need a strong patent position before starting the long and rather expensive path of a drug development. This approach has successfully been undertaken especially for the compounds with industrial application that can be easily synthesised. High structural and stereochemical complexity of the marine metabolites make their chemical synthesis not realistic, primarily for the numerous steps followed by low yield.
The first effort for in situ aquaculture of marine sponges (bath sponges) was achieved in Adriatic Sea in 1870, but no detailed statement of the applied methods was reported. Indeed, such reports were firstly published during the late 19th and early 20th centuries [88,89]. The technique is based on the capacity of sponges to regenerate themselves and create new colonies starting with small fragments. Later on, the large-scale sponge aquaculture has been developed in more countries [90-92].
Some researchers have considered the possibility of producing sponge biomass under controlled conditions, because the in situ conditions in the aquaculture cannot be affected. The supply of an adequate food source and accumulation of waste products represent the main difficulties. Recently, Osinga and co-workers  have reported about the growth of the sponge Pseudosuberites andrewsi in a closed system, using the microalgae Chlorella sorokiniana and Rhodomonas sp. as food source. These two microalgae were selected due to ingestion and digestion by the sponge cells (microscopic observation). High growth rates of this sponge species indicate a promising future for the sponge cultivation in the closed systems.
The sponge cells are characterised by significant proliferation capacity which may facilitate the establishing of their cell cultures in vitro . In analogy with the production of bioactive metabolites from bacteria and fungi, the sponge secondary metabolites might be produced in bioreactor using their cell cultures. In the last few years, the production of axenic sponge cell cultures has been developed. However, till date the maintenance of sponge cells has been reported only for in vitro conditions [94-96]. Primary cell cultures have been obtained from several sponges, with a low cell density in the cultures. This low proliferation could depend on the culture condition and/or relevant experimental approach. The optimisation of some physical parameters (pH, temperature and light) together with the improved commercial medium (using different supplements, such as cholesterol, fatty acids and glucose) has led to the promotion of the sponge cell proliferation [97,98].
The single cells in suspension do not proliferate readily , since they loose telomerase activity and consequently the potency for cell division . The formation of multicellular aggregates (primmorphs) from dissociated single sponge cells both regains telomerase activity and their growth potential [100,101].
Another promising method for the cultivation of sponges is based on the fragmentation of the whole organisms. Actually, in vitro cultivation of sponge fragments (without additional dissociation and reaggregation) has been reported. However, there is a limitation – only the species with high capability of wound healing may be used .
The morphological changes observed in all aforementioned methods might indicate non optimal culture conditions. Therefore, additional ecological parameters have to be considered in the optimisation of the sponge cell culture conditions and bioreactor design. Other studies have demonstrated the ability of these cultures to produce secondary metabolites [18,103,104]. If an appropriate growth medium and bioreactor system for primmorphs can be developed, this system may represent the most promising biotechnological approach.
The Mediterranean sponge D. avara has been attracting the attention of a marine research community since 1974, with Dr. Salvatore De Rosa (CNR-ICB, Pozzuoli-Naples, Italy) playing one of very special roles in the field. In terms of sourcing novel therapeutics, marine sponges are currently recognised as one of the most promising source of bioactive substances for drug discovery research. As previously stated, avarol and avarone, two sesquiterpenes (hydroquinone and quinone, respectively) possessing a rearranged drimane skeleton, were firstly isolated from this marine species. Their penetration of the blood-brain barrier and lack of neurotoxicity, together with a broad range bioactivity in vitro and in vivo , make this scaffold an excellent platform for AD drug development. The rationale for selecting a drug development platform based on derivatives and analogues of avarol and avarone, and in particular thio- and aminoderivatives, for the prevention and treatment of AD rests on several basic elements including the forementioned anti-AChE and antioxidant activities.
This mini-review article is dedicated to the memory of Dr. S. De Rosa, Marine Natural Product Chemist and Research Professor (Dirigente di Ricerca) of CNR-ICB, Pozzuoli-Napoli (I). This work was supported by Ministero degli Affari Esteri e della Cooperazione Internazionale, Direzione Generale per la Promozione del Sistema Paese, Italia - Progetto di Grande Rilevanza ''Applicazioni biotecnologiche di microorganismi associati alle spugne marine e valutazione biologica di composti bioattivi'' Italia-Egitto 2013-2016.
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