J.Thi-Qar Sci. Vol.3 (1) Jun./2011

الترقيم الدولي ISSN 1991- 8690 1661 - 0968

Antimicrobial activity test of the crude extracts of some marine Mollusca

against selected pathogenic fungi, in vitro

Zainab Dakhil Degiam

Microbiology Department - Collage of Medicine - University of Thi-Qar.

Abstract Crude extracts of marine Mollusca Sepia sp. ,Tibia insulaechorab-curta and Thais sp.

marked as A, B and C respectively, using 50 % acetic acid were studied for their antifungal

activity using agar well diffusion technique against seven fungi (Aspergillus niger, A. flavus,

Candida albicans , C. krusei, C. tropicalis , C. famata & C. guilliermondii ). No antifungal activity was recorded against A. niger &A. flavus in all concentrations of Thais sp. extract.

Sepia sp. & Tibia insulaechorab-curta extracts exhibited significant differences (P≥0.05) to

good antifungal activity more than Thais sp. extract . An extract has been showed high activity

against C. albicans (inhibition zone=27.6 mm) diameter, and minimum zone of inhibition

(16mm) was recorded against growth A. niger of same extract. In B extract, maximum inhibition zone (26.6mm ) in growth A. flavus & C. albicans and minimum inhibition zone

diameter (15mm) in growth A. niger .Also the minimal inhibitory concentration of all extracts

were detected .The results showed the inhibition zones diameter of Fluconazole as positive

control approximately with the inhibition zones of crude extracts .

J.Thi-Qar Sci. Vol.3 (1) Jun./2011

1- Introduction : from Arabian Gulf water. The collected Molluscs are widely distributed samples were extracted by 50% acetic acid throughout the world and have many (Li et al.,1962) , and the crude extracts representatives in the marine and estuarine (A&B) subjugated to various chemical ecosystem, namely slugs, whelks, clams, tests(Al-hussan,2007;Degiam ,2009) , and C mussels, oysters, scallops, squids and extract subjugated to various chemical tests octopus (Kamboj,1999).The number of too, to get acquaint chemical compound. natural products isolated from marine All fungal strains (Aspergillus niger, organisms increases rapidly, and now A. flavus, Candida albicans , C. krusei , C. exceeds, with hundreds of new compounds tropicalis , C. famata & C. guilliermondii ) being discovered every year (Faulkner, were obtained from Microbiology laboratory 2002; Proksch and Muller, 2006). Large of Medicine college-University of Thi-Qar proportions of these natural compounds that were diagnosed by Traditional have been extracted from marine Laboratory methods (Morphology and invertebrates, especially Molluscs, Sponges, Biochemical tests)(McGinnis,1980;Finegold Ascidians and Bryozoans, and some of them & Baron, 1990). are used in clinical trials (Proksch, et al., Antimicrobial activity assay: 2002). The need for discovery of new and Mollusca crude extracts were tested, in novel antibiotics is imperative because vitro against fungal growth. The antifungal evidence suggests that development and activity of the sample was assayed by the spreads of resistance to any new standard agar well diffusion technique antimicrobial agents is inevitable. From (Nathan et al .,1978 ;NCCLS,1998). All the 1960, approximately 300 bioactive marine fungal strains were inoculated in SDA at natural products were field for patent 28°C for (1-3 days) . 300 mg of lyophilized .Approximately 6,500 bioactive compounds powder was dissolved in 1ml of water ,then have been isolated from the marine 100 µL of the extract was pipetted on a 6mm organisms (Kamboj,1999) .Many classes of well . The plates were incubated at 28°C. bioactive compound exhibiting antitumour, Zones of inhibited fungal growth were antileukemia ,antibacterial & antiviral observed as clear halos (zones) around the activities have been reported world wide well . Antifungal activity was measured as (Pettit et al.,1991) . diameter of zone of inhibition, excluding the The aim of the present study was, first to well diameter. Water was used as negative analyze the antifungal activity from the control (-), and Fluconazole was used as tissues extracts of three species of Mollusca positive control (+) with concentration 50 and to determine the Minimal Inhibitory mg/ml . Concentration (MIC) against seven species Determination of Minimal Inhibitory of fungi. Concentration (MIC) To determine minimal inhibitory Material and Methods concentrations of all extracts, using 5 concentrations: 200, 100, 50, 25 and 12.5 Collection and preparation of samples mg/ml from stock solution (300mg/ml) by The Molluscs Sepia sp., Tibia using agar well diffusion technique (Nathan insulaechorab-curta and Thias sp. marked et al.,1978;NCCLS,1998). Preparing fungal as A, B and C respectively were collected dilutions through comparison with

J.Thi-Qar Sci. Vol.3 (1) Jun./2011

McFarland tube 1 (3×106) (Hammer et al., Antimicrobial assay: 2002). All the results of antifungal activity are The data were analyzed using X2 and P- shown in Table (1). Tibia insulaechorab- value to show significant differences at (p curta extract, the activity were 26.6mm ≥0.05) in comparison with positive control. diameter of zone of inhibition growth against A. flavus and C. albicans ,and Results: minimum inhibition zone (15mm) in growth A. niger . Sepia sp. extract, the maximum Sepia sp. , Tibia insulaechorab-curta inhibition zone 27.6mm in C. albicans and & Thais sp. extraction showed that the Thais (16mm) in A. niger growth, while Thais sp. sp. extract contains Amino acids, Proteins , extract, there was antifungal activity Saponins &Carbohydrates, but the extract observed in A. niger and A. flavus growth, had no Aldehyde ,Ketons ,Flavones whereas showed antifungal activity on all Alkaloids and Glycosides while the Tibia testes yeasts growth and inhibition zones insulaechorab-curta extract contained ranging from (8 -15.3mm) . From the X2 and Proteins , Amino acids and Carbohydrates P-value showed significant differences on only ,whereas Sepia sp. extract contained fungi A. Niger, A. flavus and C. Amino acid, Proteins, Saponins guilliermondi at (p ≥ in comparison ,Carbohydrates ,Aldehydes,Ketons,Flavones 0.05) and Alkaloids . with positive control .

Table (1) Diameters of inhibition zones (mm) of the antifungal activity of

Sepia sp. , Thais sp. and Tibia insulaechorab -curta extracts

*Absence of the antifungal activity

The minimal inhibitory concentration of C. famata &C. krusei , and was 50mg/ml on crude extracts: C. albicans & C. guilliermondii. But the The results of minimal inhibitory MIC of A extract ranged from 100 mg/ml on concentration (Table 2) showed that the A. niger & A. flavus to 50 mg/ml on C. MIC of C extract was 200 mg/ml on all albicans, C. tropicalis, C. krusei & C. yeasts growth and 300 mg/ml on moulds guilliermondii to 25 mg /ml on C. famata . growth. The MIC of B extract was 100 mg/ml on A. niger , A. flavus ,C. tropicalis ,

J.Thi-Qar Sci. Vol.3 (1) Jun./2011

Table (2) The minimal inhibitory concentration of A, B &C extracts

The shadow fields refer to significant differences at (p ≥ 0.05)

Bacillus subtillus (Jaysali et al.,2001) . The Discussion: presence of antimicrobial activity in Mollusca tissues were extracted by 50 Mollusca had been reported from the whole % acetic acid, because this acid has the body meats of Mollusk, Gastropoda Trochus ability to lyses animal tissues (Al-hussan, tentorium was extracted with various 2007). Thais sp. extract contains Amino solvents (Anbuselvi et al., 2009). acids, Proteins, The marine animals contains Prem Anand and Patterson Edward (2002) Proteins, Peptides (Baslow, 1977), reported moderate antibacterial and Dolastatin, a cytotoxic peptide from antifungal activity from extracts of various Dolabella auricularia is an antineoplastic bivalve Mollusks. substance (Pettit et al., 1989). The extract In this study, wide antifungal activity has contains Saponin & Carbohydrate was been recorded in all extracts which is the isolated from Lobophytum spp. collected significant finding of the study ,and showed from Hainan Island (He et al., 2002), the different antifungal activity, possibly due to tissues of Crassostera virginica & presence of different chemical substance in Mercenaria mercenaria contain extracts , the biological activity of crude Carbohydrates substance (Baslow, 1977). extracts, possibly due to the Sepia sp. extract In the present investigation a is proline rich extract (Degiam,2009). Cyclic pronounced antimicrobial activity had been heptapeptide, stylisin 1 proline-rich observed against some fungal strains, cyclopolypeptide exhibited moderate to Similar result was reported in four bivalves good antimicrobial activity against gram against few pathogens and found that negative bacteria Klebsiella pneumoniae , extracts showed significant activity against Pseudomonas aeruginosa, dermatophytes

J.Thi-Qar Sci. Vol.3 (1) Jun./2011 and Candida albicans with minimum activity against human pathogenic fungi. inhibitory concentration (MIC) of 6µg/ml This finding is very significant for the (Dahiya et al., 2009). But the Tibia discovery of new potent drugs against these insulaechorab-curta extract contain cystein pathogenic fungi . which have antimicrobial activity (Al- Acknowledgments : hussan, 2007) , Two antimicrobial peptide The author thankful to Mr. Ali Tahar defensins A & B isolated from the blood of Abbas in Microbiology department of immune challenged Mytilus edulis , a family Medicine college of Thi-Qar university for of cysteine-rich cationic peptides (Charlet et his help in this research. al., 1996) .Whereas Thais sp. extract was contained protein and amino acid ,to get its References : biological activity against microorganisms . •Al - hussan, S.K.J. (2007). Extrac tion of Extract of Mollusk, Drupa protein compound from snail Tibia sp, margariticola, exhibited higher antibacterial Tibia insulaechorab-curta, Pomacea activity against human pathogens bridgesii diffusa, Mollusca: Gastropoda (Chellaram and Edward, 2009).Which is in and study their activity for some agreement with the present work. bacterial species. M. S. C. Thesis. Polysaccharide isolated from Cuttlebone Collage Science. University of Basra. Sepia aculeate and Sepia brevimana have •Amede, P. & Chevolot, L. (1982). broad spectrum against 9 bacterial species Antimicrobial activity of marine sponges and 4 fungi (Shanmugam et al.,2008) . from French Polynesia and Brittany.Mar. Latrunculin-A from Chromodoris Biol., 70: 223-228. elisbenthina that showed inhibition activity •Anbuselvi, S.; Chellaram,C.; Jonesh,S.; against C. albicans (Okuda & Sheuer, 1985). Jayanthi, L. & Edward, J.K.P. (2009). But there are some reports on the isolation Bioactive potential of coral associated of the antifungal compound from marine gastropod, trochus tentorium of gulf of animals such as general: puphenone from mannar, southeastern india. J. Med. Sci., marine sponges (Amede & Chevolot, 1982) 9: 240-244. and Dolastain-10 from tunicate and •Baslow,M.H.(1977).Marine Pharmacology, spongistatin from sponges (Pettit et al., 1998 Rober E. Krieger Publishing a,b) . Company, New York. Thais sp. extract showed no effect on •Charlet, M.; Chernysh, S.; Philippe, H.; molds A. niger & A. flavus and showed the Hetru, C.; Hoffmann, J.A. & Bulet, P. MIC of same extract was high, Ronald (1996).Innate immunity: Isolation of (1997) has reported that the fungi are more several cysteine-rich antimicrobial resistant than the bacterial strains to the peptides from the blood of a mollusk, tested compound, this could be lead to the Mytilus edulis. J. Biol. Chem., 271: nature of fungal cell wall made up of chitin. 21808-21813. The hard cover of the exoskeletons of the •Chellaram, C. & Edward ,J.K.P.( 2009) . arthropods are also made up of chitin, which Anti-nocicetive assets of coral associated is relatively resistant, including microbial gastropod, Drupa margariticola. Int. J. decomposition. Pharmacol., 5: 236 -239. In conclusion in the present study, the •Dahiya, R.; Kumar, A. & Gupta, R.(2009). crude extracts have been extracted from Synthesis,Cytotoxic and Antimicrobial Mollusca showed promising antifungal Screening of a Proline-Rich

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Cyclopolypeptide.J.Chem. Pharm. Bull., topical antimicrobial agent , Burns ,4 57: (2) 214-217 . :177 - 178 . •Degaim, Z.D. (2009). Extraction of protein •(NCCLS) National Committee for Clinical compound from some marine Mollusca Laboratory Standards. species and study their antifungal (1998) .Refrence method for broth activity. M. S. C. Thesis. Collage dilution anti fungal susceptibility Science. University of Basra. pesting of conidium forming filamentous •Faulkner, D.J. (2002). Marine natural fungi : proposed standard M38- products. Nat. Prod. Rep., 19:1-48 . P.Wayne.PA,USA . •Finegold,S.M. & Baron,E.J. (1990). •Okuda,R.K. & Sheuer,P.J. (1985). Diagnostic microbiology. 8th ed . The Lautrunchlin-A, ichthyotoxic of the C.V. Mosby Company. St Louis. nudibranch Chromodorsis elisabethina. Baltimore. Philadelphia. Toronto 861 P. Experientia., 41: 279-281 . •Hammer , K . A .; Carson , C .F .& Riley •Pettit, G.R.; Herald, C.L.; Boyd, M.R.; ,T.V.(2002).In vitro activity alternifolia Leet, J.E.; Burfrese, C.; Doubek, D.L.& of Melaleuca (tea tree ) oil against Schimidt, (1991).Antineoplastic agents. dermatophytes and other filamentous 219. Isolation and structure of the cell fungi . Antimicrobial Chemotherapy, 50: growth inhibitory constituents from the 195-199. Western Pacific marine sponge Axinella •He, X. -X.; Su, J. -Y.; Zeng, L.-M.; Yang, sp. J. med. Chem.. 34 : 3339-3340 . X. -P. & Liang, Y. -J.(2002). Studies on •Pettit, R.K.; Mc Allister, S.C.; Pettit, G.R.; the secondary metabolite of the soft Herald, C.L.; Johnson, J.M. & Cichaz, coral Lobophytum sp. Huaxue Xuebao Z.A.(1998 a).A broad-spectrum (ACTA Chim Sinica).,60, 334-337. antifungal from the marine sponge •Jayaseli, A.A.; Prem Anand, T. & Murugan Hyrtios erecta. Int. J. Antimicrob. (2001). Antimicrobial activity of four Agents Chemother., 42: 2961-2965 . bivalves from Gulf of Mannar. Phuket. •Pettit, R.K.; Pettit, G.R. & Hazen, K.C. Mar. Biol. Cent. Spec. Pup., 25(1): 215- (1998b).Specific activites of dolastatin 217. 10 and peptide derivatives against •Kamboj, V.P. (1999). Bioactive Agents Cryptococcus neoformans. Antimicrob. from the Ocean Biota. In: Ocean Agents Chemother., 42: 2961-2965 . Science Trends and Future Direction, •Pettit, G.R.; Singh, S.B.; Hogan, F.; Lloyd- Somayajalu, B.L.K. (Ed). Indian williams, P. ;Herald, C.L.; Burbett, National Science Academy., New Delhi, D.D.& Clewlow, P.J.(1989). The India, pp: 197-227 . absolute configuration and synthesis of •Li , C . P .; Prescott , B . ; Jahues , W .G . & natural (-)-dolostatin10. J Am Chem Martion,E.C.(1962).Antimicrobial Soc., 70, 5463-5465. agents from mollusks. Trans. N. Y. •Prem Anand, T. & Patterson Edward, J.K. Acard. Sci. Se. II., 24: 504-509 . (2002). Antimicrobial activity in the •McGinnis,M.R. (1980). Laboratory hand tissue extracts of five species of cowries book of medical mycology. Academic Cypraea sp. (Mollusca:Gastropoda) press, New York, USA, 66P. and an ascidian Didemmum •Nathan, PE.; Law,J.& Murphy, DF.(1978). psammathodes (Tunicata: Didemmidae). A laboratory method for the selection of Indian J. Mar. Sci., 31(3): 239-142.

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•Proksch, P.; Edrada, R.A. & Ebel, R. Isolated from the (2002). Drug from the seas-current Cuttlebone of Sepia aculeate (Orbingy, status and microbiological implications 1848) and Sepia brevimana .Appl.Microbiol.Biotechnol.,59:125-134. (Steenstrup, 1875): An Approach to •Proksch, P., & Muller, W.E.G. (2006). Selected Antimicrobial Activity for Frontiers in Marine Biotechnology. Human Pathogenic Microorganism. J. Horizon Bioscience: Norfolk, U.K. Fish Aquat. Sci., 3 (5): 268-274. •Ronald, M.A.(1997).Principles of Microbiology, C.Brown Publishers, pp:1298. •Shanmugam, A.; Mahalakshmi, T.S. & Barwin vino, A. (2008). Antimicrobial Activity of Polysaccharide

اختبار الفعالية الضد مايكروبية للمستخلصات الخام لبعض النواعم البحرية تجاه بعض الفطريات الممرضة ،مختبريا

زينب داخل دغيم

قسم األحياء المجهرية- كلية الطب- جامعة ذي قار

المستخلص

تم راسةما سعاليع ما سعدةميرف عياتا ميت عدةت يلميت سعامبسر سعلخا ما سع مي .Sepia sp ب-Tibia insulaechorab curta ب.Thais sp ادممل ع مميA بB ب C ريمما سعتممبسعس ليةممت رس 05% خمميدا سع ي مما بليةممت رس تلا مما سعخامما ت ممي ةمللا ناممبسط يتا مما Aspergillus niger بA. flavus بCandida albicans ب C. krusei ب C. tropicalis بC. famata بC. guilliermondii. عم ةم دةمت ي .Thais spني يليع ما دةميرف عياتما A. niger ب .A flavus يمس د ما سعتاسز مل سعدةمت ردا دم سعدةمت ي . نم ما سعدةت يلمي -Sepia sp. & Tibia insulaechorab curta تليالمي يمس يليع ت دمي سعدةميرف عياتا ميت رامر دةمتبل سختديع ما P≥ 0.05 ( نزثما دم سعدةمت ي .Thais sp. نم مما سعدةممت ي A نريمما يليع مما خ ب مما ت ممي C. albicans للتمما تثلمم ت 6.72 ديمم ( بن مم داتلمما تثلمم ت 62 ديمم ( ريا ادب A. niger . نم ا سعدةت ي B نريا داتلا تثل ت 6272 دي ( ريما سعاتما C. albicans ب A. flavus بس داتلا تثل ت 60 دي ( ريا ادب سعاتا A. niger . زدي ت تخر ر سعتاز ل سعدثلت سألراا ع د ا سعدةت يلميت . نبةخت سعاتيئج ن ن تيا تثل ت Fluconazole زة تاف دب لا تتلياب د ن تيا تثل ت سعدةت يليت سع ي .