Chemical characterization of Cinachyrella tarentina : Sponge of Atlantic Moroccan Coast

Currently, marine organisms are a very important source of new molecules in pharmacology and thus in the development of new bioactive products. Sponges, in particular, given their very primitive origin and persistence during evolution, have developed a chemical defense system.The chemical study of Cinachyrella tarentina, marine sponge recognized by its antibacterial and antifungal activity was investigated for the first time in Morocco. The screening of Cinachyrella tarentina revels that it contains different levels of primary and secondary metabolites. The dosage of polyphenols was carried out using the reagent Foulin-Ciocalteu. The antioxidant activity was evaluated by the DPPH test. The fatty acid composition determined by Gas chromatography-mass spectrometry (GC/MS) showed a predominance of palmitic and stearic acids. Furthermore, we found the presence of several sterols which cholesterol and sitosterol are the most abundants.


INTRODUCTION
The marine environment represents unquestionably a treasure of organisms generating plethora of primary and secondary metabolites that would benefit from further exploration [1].This environment is very diverse and marine organisms, particularly the most primitive of them, produce a variety of bioactive molecules [2].Marine sponges are the most primitive multicellular animals and contain many primary and secondary metabolites, including lipids, alkaloids, and polyphenols [3].Sponges are sessile marine filter feeders that have developed efficient defense mechanisms against foreign attackers such as viruses, bacteria, or eukaryotic organisms.Marine sponges are among the richest sources of pharmacologically-active chemicals from marine organisms.It is suggested that (at least) some of the bioactive secondary metabolites isolated from sponges are produced by functional enzyme clusters, which originated from the sponges and their associated microorganisms.More than 5,300 different products are known from sponges and their associated microorganisms, and more than 200 new metabolites from sponges are reported each year [4].Most of the bioactive compounds from sponges consist of anti-inflammatory, antitumor, immunosuppressive (or) neurosuppressive, antiviral, antibiotics, antifouling and antimalarial properties [5].The chemical composition of Moroccan sponges has not been extensively investigated.The aim of the present work was to analyse the chemical composition of the marine sponge Cinachyrella tarentina and to investigate the antioxidant activity by evaluating the reduction of 2,2-diphenyl-1-picrylhydrazyl (DPPH) reagent.Also we studied the chemical composition of fatty acid and sterols of iso-octanic extract of the marine sponge Cinachyrella tarentina.

Biological material and sampling site
The studied sponge (Cinachyrella Tarentina) was collected during summer season, in June 2011 to the beach Deauville of El Jadida (N 33 15 140 ; W8° 29 930) at low tide to a depth of 3 to 5 meters (Figure 1, harvesting site).After harvest, marine sponge was washed, frozen and lyophilized.

Chemical composition of the marine sponge
The determination of the chemical composition has consisted in dosage of the primary metabolites and to search some secondary metabolites known for their biological activity.

Compounds belonging to the primary metabolism Dosage of lipids
The dosage of lipids was realized by using a soxhlet.The sample was continuously extracted with hexane which gradually dissolves the fat.The solvent containing the fat returns into the balloon by successive spills.Once the extraction was complete, the solvent was evaporated, and the fat was weighed and the percentage was calculated using following formula:

Dosage of total sugars
The dosage of total sugars was performed using phenol /sulfuric acid method as described previously [6].Briefly, in the presence of concentrated sulfuric acid, the oses were dehydrated and the products condense with phenol to give yellow-orange complex.Then, we measured the optical density at 490nm.A standard was prepared from a solution of glucose with different dilutions ranging from 0 to 0.4 mg /ml.

Study of the chemical composition of the marine sponge (preliminary tests)
The tests were conducted according to the standard techniques described by Paris and Moyse [7], Bouquet [8], and Debray et al [9].These tests consist to highlight the presence of a certain number of chemical groups known to their biological activities [10].

Extraction of phenolic compounds
The polyphenols were extracted by maceration of 1g of powder in 20 ml of absolute ethanol, for 48 hours.After centrifugation, the supernatant containing the polyphenols was recovered.Therefore, we proceed to a second extraction overnight on the base to extract supplementary polyphenols to obtain a dosage more comprehensive.The two supernatants were added to the dosage.

Invention Journal of Research Technology in Engineering & Management
To 0.5 mL of extract (diluted 100 times), 2.5 mL of Folin-Ciocalteu reagent (diluted 10 times) and 2 mL of sodium carbonate (75g / l) were added to the tubes.After agitation, the tubes were incubated 5 min at 50 ° C at the dark, and the absorbance was read at 760 nm by a spectrophotometer.A standard range was prepared by gallic acid in concentrations ranging from 0 to 500 ppm [11].

Evaluation of the antioxidant activity DPPH test
The antioxidant activity was measured by the DPPH (1,1-diphenyl-2-picrylhydrazyl) method.In this test the antioxidants reduce the DPPH from a violet color to a yellow compound, the diphenyl picryl hydrazine, the intensity of the color is inversely proportional to the ability of the antioxidants present in the medium to give protons.The solution of DPPH was prepared by dissolving 4 mg of DPPH in 100 ml of ethanol.0.5 ml of the ethanolic extract at different concentrations [0-10 mg/ml] was added to 1.5 ml of the ethanolic DPPH solution.After agitation by a vortex, the mixture was left in the dark for 30 min at room temperature and discoloration relative to the negative control containing only DPPH solution was measured at 517 nm by a spectrophotometer.The positive control was represented by a standard solution of an antioxidant, ascorbic acid (vitamin C) and the absorbance was measured in the same conditions as the test sample.The antioxidant activity, that expresses the capacity of trapping the free radical, was estimated by the percentage of discoloration of the DPPH dissolved in ethanol [12].Inhibition of DPPH free radical in percent (I%) was calculated as follows: [13] 100 A c is the absorbance of the negative control, and A t is the absorbance of the test compound.Extract concentration providing 50% inhibition (IC 50 ) was calculated from the graph plotting inhibition percentage against extract concentration.

Extraction and isolation of sterols and fatty acids
In the laboratory, the lyophilized biological material, ground and weighed is emerged in a mixture of chloroform-ethanol solvent (CHCl3 / EtOH; v / v) for 48 h.Then the solution obtained is evaporated under reduced pressure at 40°C.The extraction is repeated several times until complete exhaustion of the biological material.The crude extract obtained after concentration is weighed to calculate the yield.It is then taken up in a mixture equivolume of isooctane and methanol to give the methanolic and isooctanic phases.The two phases are then subjected to evaporation of the solvent to achieve the methanolic and isooctanic extracts.The isotonic extract of the studied sponge was subsequently subjected to two specific protocols in order to get fatty acids and sterols.5g of the isotonic extract is saponified in alcoholic potassium hydroxide, the saponifiable fraction contains fatty acids in salt form.
An acidification followed by extraction with diétylic ether leads to obtaining a mixture of fatty acids in free form (0.97g).The obtained fatty acids are methyled by esterification with methanol in the presence of boron trifluoride (20%) in excess and reflux at 100 ° C for 15 min.The obtained methyl esters of fatty acids are analyzed by gas chromatography coupled to mass spectrometry.The analysis of the unsaponifiable fraction of the isooctanic extract of the studied sponge, by thin layer chromatography (TLC) in an eluent system 2,2,4-trimethyl -pentane (TMP) / ethyl acetate (EA) 7/3 shows that it has the same frontal report as cholesterol.Spraying with sulfuric acid (50%) followed by oven drying at110 °C for 10 minutes reveals purple spots, characteristic of sterols.The fractionation of the unsaponifiable fraction (4g) is followed on an open column of silica gel using the same couple of eluent (TMP / EA) in order to get the sterol fraction.The sterol fraction (yield (1.47g); frontal report (0.3); white color) obtain ed by eluent system TMP / EA (8/2) was recrystallized in methanol, silylated with 50μL of pyridine and 40μL of MSTFA at 50 °C for 2h, and injected in gas chromatography coupled to mass spectrometry (CG/MS) After adding 910μL of isooctane.

Chromatographic conditions of analysis by GC / MS
DB-5 column ((30 m × 0.32 mm) Injector: Initial temperature 80 °C and then increase to 100 °C / min up to 300 °C.Column: initial temperature 80 °C (maintained 2min) and then increase to 6 °C / min up to 320 °C (maintained 25min).

Identification of compounds by GC/MS analyses
The identification of a part of compounds was accomplished using computer searches on a NIST MS Data library and another from the mass spectra of reference compounds analyzed.

Compounds belonging to the primary metabolism
The averages levels in lipids and carbohydrates of Cinachyrella tarentina were shown in Table 1: From the Table 1, Cinachyrella tarentina represents a source of primary metabolites with a significant proportion of carbohydrates compared to lipids.The carbohydrates are recognized by their role in the mechanism of recognition and cellular interactions of the sponge.Some sugars may be related to lipids, thus forming glycolipids: molecules implicated in many physiological processes and showed a proven pharmacological activity [14][15].

Compounds belonging to the secondary metabolism:
These substances are known to be responsible for the pharmacological activity.The results of tests of the chemical composition are presented in the table 2. Acording to table 2, we observed the presence of different chemical compounds recognized by their biological activity with high concentration of alkaloids, sterol and saponins substances.Therefore according to our preliminary tests, we found that Cinachyrella tarentina produced molecules with potential biological interest.In the case of particular species of sponges, it seems that the production of these active metabolites does either not directly related to the organisms themselves but rather to heterotrophic endosymbionts (bacteria) or autotrophic (microalgae and cyanobacteria).The ability to produce such metabolites is of obvious interest in the context of the search for new molecules presenting interesting therapeutic potential [16].

Determination of polyphenols
The polyphenols content was determinate from ethanolic extract, expressed in milligram of gallic acid equivalent per gram of dry weight.The polyphenol content was 3.61 % (+/-0.02)mg GAE/g DW.

Evaluation of the antioxidant activity by DPPH Test
The results of antioxidant power show that the percentage of inhibition of ethanolic extract of Cinachyrella tarentina is equal to 77.2% (+/-0.8)at a concentration of 10 mg/ml.The IC50 value determined in mg / ml expressing the inhibitory concentration of extract required to inhibit 50% of the initial DPPH free radical (Table 3).Table 3. Result of the antioxidant test expressing the inhibitory concentration (IC50) in mg / ml Extracts / Standard IC50 Ethanolic extract 1.8 mg/ml (+/-0.5)Ascorbic acid 0,13 mg/ml (+/-0.03)The IC50 of the ethanolic extract of the sponge is 1.8 mg/ml, this value remains low compared to ascorbic acid; and used as a reference is 0.13 mg/ml and thus indicates that this sponge contains the modest antioxidants, which is consistent with the result obtained for the low content of polyphenols in the studied sponge.

Sterols composition
The silylated sterols of Cinachyrella tarentina were investigated by GC/MS (qualitative analysis).The data are reported in Table 4.
The studied sponge contains the usual complex mixtures of sterols for marine invertebrates, all of them identified earlier in different sponges.Cinachyrella tarentina contains therefore principally the unsaturated sterols, with a predominance of cholesterol and sitosterol.

Fatty acids composition
The methyl esters of fatty acids of Cinachyrella tarentina were investigated by GC/MS (qualitative analysis).The data are reported in Table 5.No study has been performed in Morocco on the fatty acid composition of Cinachyrella tarentina.This study allowed us to identify the variety of fatty acids contained in Cinachyrella tarentina with predominance of palmitic and stearic acids.The mixture of sodium salts of these two acids led to the preparation of ordinary hard soap.

CONCLUSION
The dosage of primary metabolites revels that Cinachyrella tarentina represents a source of carbohydrates and lipids with a significant proportion of carbohydrates compared to lipids.The Screening of Cinachyrella tarentina revels that it contains different levels of secondary metabolites.The dosage of polyphenols was carried out using the reagent Foulin-Ciocalteu.The antioxidant activity was evaluated by the DPPH test, the results of this test indicates that Cinachyrella tarentina contains the modest antioxidants, which is consistent with the result obtained for the low content of polyphenols in the studied sponge.The fatty acid composition determined by Gas chromatography-mass spectrometry (GC/MS) showed a predominance of palmitic and stearic acids.Furthermore, we found the presence of several sterols which cholesterol and sitosterol are the most abundants.In the current investigation, the extracts from Cinachyrella tarentina gave good results indicating that it possesses significant amount of chemicals compounds and in vitro antioxidant activity.Phenolic compounds and other chemicals compounds appear to be responsible for the in

Fig. 1 .
Fig. 1.Map showing the harvesting site of marine sponge Cinachyrella tarentina The systematic identification of the marine sponge was carried out by Dr. Maria-Jesús Uriz, Professor at the Centro de Estudios Avanzados de Blanes (CEAB) and Consejo Superior de Investigaciones Científicas (CSIC), Spain.The in situ photo of Cinachyrella tarentina is shown in figure 2.

Table 1 .
Averages levels of lipids and carbohydrates expressed in percentage relative to the dry weight.

Table 2 .
Determination of chemical composition of the studied sponge Invention Journal of Research Technology in Engineering & Management

Table 4 :
Chemical composition of silylated sterols of the studied sponge

Table 5 :
Chemical composition of methyl esters of fatty acids of the studied sponge