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376 Asian Pacific Journal of Tropical Biomedicine (2011)376-380 Contents lists available at ScienceDirect Asian Pacific Journal of Tropical Biomedicine journal homepage:www.elsevier.com/locate/apjtb Document heading doi:10.1016/S2221-1691(11)60083-X Analytical characterization and structure elucidation of metabolites from Aspergillus ochraceus MP2 fungi Meenupriya J1, Thangaraj M2 1 2 Department of Biotechnology, Sathyabama University, Chennai-119, India CAS in Marine biology, Annamalai u
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  376 Document heading doi:10.1016/S2221-1691(11)60083-X Analytical characterization and structure elucidation of metabolites from  Aspergillus ochraceus MP2 fungi Meenupriya J 1 , Thangaraj M 2 1  Department of Biotechnology, Sathyabama University, Chennai-119, India 2 CAS in Marine biology, Annamalai university, Parangipettai- 608 502, India Asian Pacific Journal of Tropical Biomedicine (2011)376-380 Asian Pacific Journal of Tropical Biomedicine  journal homepage:www.elsevier.com/locate/apjtb   * C orresponding author: J   M eenupriya, D epartment of  B iotechnology, S athyabama U niversity, C hennai- 119 , I ndia.  T el: + 91   44   24501644   E -mail: meenupriya.j@gmail.com 1. Introduction   M any marine invertebrates produce natural compoundsthat affect the growth, metabolism, reproduction, andsurvival of other types of organisms. H ence, they areconsidered to be bioactive. T hose include potentiallyeffective therapeutic agents with antiviral, antibacterial,and antitumor properties produced by invertebrates fromthe classes P orifera, C nidaria, M ollusca, E chinodermata, B ryozoa, and U rochordata. C lose relations between marineinvertebrate species and microorganisms, includingsymbiotic associations and interactions during larvalsettlement, have been characterized and this providesinsights to the regulation of host-symbiont-microbialcommunity interactions. M any of the compounds isolatedfrom marine organisms, such as sponges, may be producedby associated microbes. M arine sponges are benthic animalsfound in the widerange of marine environments. T hediversity of sponges species is superior in the tropical coralreef environments. T he sponges are also very importantresources for searching the biologically active substances,which are useful to develop pharmaceuticals, agrochemicalsand biochemical reagents and their lead compounds [1] . T he srcins of these biologically active substances arerecently thought to be the metabolites produced by themicroorganisms associated with the sponges. A nd studieshave also suggested that some bioactive compounds isolatedfrom marine organisms have been shown to exhibit anti-cancer, anti-microbial, anti-fungal or anti-inflammatoryand other pharmacological activities [2-9] . T hese marineinvertebrates have evolved chemical defense mechanicsmsagainst other invading organisms, which involve theproduction of secondary metabolites [10] . S ponges are goodhomes not only for macro organisms, such as worms,brittlestars, shrimp, crabs, etc. , but also for a variety of microorganisms such as bacteria, fungi, and microalgae, ARTICLE INFO ABSTRACT  Article history: R eceived 5   M arch 2011 R eceived in revised form 4   A pril 2011 A ccepted 20   A pril 2011 A vailable online 10   M ay 2011  Keywords: M arine sponge derived fungi  Aspergillus ochraceus B ioactive metabolites A ntibacterial activity A nalytical characterization F ungi  Apergillus B ioactive secondary metabolite A ntagonistic human pathogen A ntimicrobial activity B ioactive compounds Objective: T o isolate and characterize the bioactive secondary metabolites from  Aspergillusochraceus (  A. ochraceus )   MP 2 fungi. Methods: T he anti bacterial activity of marine sponge derivedfungi  A. ochraceus   MP 2 was thoroughly investigated against antagonistic human pathogens. T heoptimum inhibitory concentration of the fungi in the elite solvent was also determined. T hepromising extracts that showed good antimicrobial activity were subjected to further analyticalseparation to get individual distinct metabolites and the eluants were further identified by GC   MS instrumental analysis. T he molecular characterization of the elite fungal strains were done byisolating their genomic DNA and amplify the internal transcribed spacer  ( ITS ) region of  5 . 8 s r  RNA  using specific ITS primer. T he novelty of the strain was proved by homology search tools and elitesequences was submitted to GENBANK . Results:   T hree bioactive compounds were characterizedto reveal their identity, chemical formula and structure. T he first elutant was identified as毩- C ampholene aldehyde with chemical formula C 10   H 16   O and molecular weight 152   D a. T he secondelutant was identified as L ucenin- 2 and chemical formula C 27   H 30   O 16 and molecular weight 610   D a. T he third elutant was identified as 6 - E thyloct- 3 -yl- 2 - ethylhexyl ester with C hemicalformula C 26   H 42   O 4   with molecular weight 418   D a. Conclusions:   T he isolated compounds showedsignificant antimicrobial activity against potential human pathogens. M icrobial secondarymetabolites represent a large source of compounds endowed with ingenious structures and potentbiological activities. Contents lists available atScienceDirect  J Meenupriya et al./Asian Pacific Journal of Tropical Biomedicine (2011)376-380 377 which live in the canals, between cells, and even insidethe cell [11] . A variety of antimicrobial substances have beenisolated from various species of marine sponges [12] . U p to 800 antibiotic compounds have been isolated from marinesponges, a number of which corroborates assumptions thatsponges appear to defend themselves against infectionsby producing and/or accumulating secondary metabolites.  Aspergillus ochraceus   (  A. ochraceus ) can produce other secondary metabolites, whose biological activity has notbeen characterized until now. T hese molecules may bebeneficial ( antibiotics ) or harmful ( mycotoxins ) to humanhealth [13-15] . F ungi isolated from marine sponge have a high creativityindex, i.e. , ability to synthesize new and interestingsecondary metabolites. A lthough the natual function is notknown, it is assumed that they play an important role inchemical defense and communication of the organism [16] . M any of them have been suggested to act as pheromones,antifeedants or repellents and regulators in the developmentof organisms. T he secondary metabolite does not occur randomly but is correlated with ecological factors [17] . N evertheless, a growing number of metabolites from sponge-derived fungal strains has been reported in the last years [18] . I t provides an overview of sponge species investigated,taxonomy of isolated fungi, and reported metabolites. T hesestructures suggest most of the metabolites to be derived frommetabolic pathways is also common to terrestrial fungi. S ucha similarity is, for example, obvious for sesquiterpenes of thehirsutane-type [16] . S tudies show that secondary metabolitesin sponges play a crucial role in their survival in the marineecosystem [26] . T hese natural products have interestingbiomedical potential, pharmaceutical relevance anddiverse biotechnological applications [18] . T he biomedicaland pharmaceutical importances of these compoundsare attributed to their antiviral, antitumor, antimicrobialand general cytotoxic properties [12] . I nterestingly, out of the 13 marine natural products that are currently under clinical trials as new drug candidates, 12 are derivedfrom invertebrates. A mong them, P orifera remains themost important phylum, as it provides a greater number of natural products, especially novel pharmacologicallyactive compounds [19] . B iochemical characteristics seem tobe useful taxonomic markers and good indicators of spongephylogeny [20] . T he diversity of biochemical properties of sponges has been demonstrated by the continued discoveryof novel compounds that have pharmacological properties [21] . 2. Materials and methods 2.1. Collection of sponge S pecimens were collected by SCUBA diving using hammer and chisel from G ulf of  M annar, located at 215 kms from K anyakumari D istrict, in the narrow strip of peninsular landalong the south east coast of  T amilnadu state. 2.2. Isolation of fungi   T he sponge sample was washed with sterile water  ( distilledwater: sea water; 1 : 1 ) and ground in a mortar and pestleunder aseptic conditions. S erial dilution was performed andfrom each dilution, plating was done in S abourauds agar byspread plate technique. T he plates were then incubated at 27   曟 for  5 days. A fter  5 days, the plates were examined and the pureculture was isolated on pure agar plate. 2.3. Molecular characterization and identification of elite fungi by ITS sequencing T he fungi were grown in culture in potato dextrose brothat room temperature in the dark for  48 to 72 hours. T hegenomic DNA was isolated and the internal transcribedspacer    ( ITS ) region of  5 . 8 s RNA was amplified using primer  ITS 1   TO   5 ’   TCCGTAGGTGAACCTGCGG   3 ’ and primer  ITS 5   5 ’   TCCTCCGCTTATTGATATGC   3 ’   7 and sequenced usingautomated sequencer. 2.4. Mass cultivation of A. ochraceousA. ochraceus   W ilhelm NRRL   3174 was grown on syntheticagar medium ( SAM ) of the following composition: 3 g/ L   NH 4 NO 3 , 26 g/ L   K 2 HPO 4 , 1 g/ L   KC l, 1 g/ L   M g SO 4 . 7 H 2 O , 10 m L  of mineral solution ( containing distilled water per litre, 70 mg N a 2 B 4 O 7 . 10 H 2 O , 50 mg ( NH 4 ) 6 . M o 7 O 24 . 4 H 2 O , 1   000 mg F e SO 4 . 7 H 2 O , 30 mg C u SO 4 . 5 H 2 O , 11 mg M n SO 4 . H 2 O , and 1   760  mg Z n SO 4 . 7 H 2 O ; the p H was adjusted to 2 with 2 mol/ L   HC l ) , 15 g agar, and 50 g/ L glucose. T he p H of the medium wasadjusted to 6 . 5 by 2 mol/ L   HC l and autoclaved at 120   曟 for  20  minutes. 2.5. Extraction process   T he fungal mycelia were homogenized using sea water. T hen the biomass was subjected to an extraction of biologically active components which were carried outwith different solvents in the order of increase polarity: C holoroform, butanol and ethyl acetate by soaking atambient temperature. T he crude extracts obtained weredried under rotary vacuum evaporator and screened for anti-bacterial activity. 2.6. Antimicrobial assay   A gar diffusion assay is used widely to determine theantibacterial activity of crude extract. T he technique workswell with defined inhibitors. N utrient agar was prepared andwas poured in the petri dish and allowed for solidification, 24 hours growing bacterial culture were swabbed on it. T hewells ( 8 mm diameter  ) were made by using cork borer. T hedifference concentration of the crude extract were loaded inthe well. T he plate was then inculated at 37   曟 for  24 hours.  D ilution assay is a standard method used to compare theinhibition efficiency of the antimicrobial agents. N utrientbroth was inoculated with 24 hours growing bacterial cultureand different concentrations of the extract were inoculated.  J Meenupriya et al./Asian Pacific Journal of Tropical Biomedicine (2011)376-380 378 B acterial culture inoculated in nutrient broth were used ascontrol. T he tubes were incubated at 37 曟 for  24 hours. T heoptimal densities were measured spectrometrically at 600  nm. T he percentage of viable cell was calculated using thefollowing formula:  %   V iable cells= C ontrol OD - T est OD 暳 100  / C ontrol 2.7. Thin layer chromatography TLC is used to separate the compound present in the crudeextract. T he separation of the compound also depends onthe usage of the solvent. T he drug with the concentration of  1 mg/m L was plotted on the TLC plate and dried. I t was thenrun with different solvent ratio the spots were identified bothin the uv light and in the iodine chamber. T he R f  value wascalculated using the formula:   R f  value= D istance travelled by the solute / D istance travelled by the solvent 2.8. Gas chromatography-mass spectrometry (GCMS)analysis   T he crude extract was quantified using gas chromatograph ( GCMS - S himadzu ) equipped with a DB - 5 ms column ( mminner diameter  0 . 25 mm, length 30 . 0 m, film thickness 0 . 25   毺 m ) mass spectrometer  ( ion source 200   曟 , RI   70 e V )  programmed at ( 40 - 650 )   曟 with a rate of  4   曟  /min. I njector temperature was 280   曟 ; carrier gas was H e ( 20 psi ) , columnflow rate was 1 . 4 m L  /min, injection mode -split. 3. Results 3.1. Isolation of fungi   I n the present study, the 10 - 5 dilution of the sponge sampleyielded three different isolates. T he characterization andanalysis was performed for  I solate   1 . P ure culture of  I solate 1   ( F igure 1 a ) was obtained and SEM micrograph ( F igure 1 b ) wastaken to visualize the morphological features of the fungi. 3.2. Molecular characterization and identification of elite fungi Table 1 Z one of inhibition of fungal filtrate in different solvents ( mm ) . P athogen  B utanol (毺 L )   C hloroform (毺 L ) E thyl acetate (毺 L ) 255075100255075100255075100  Pseudomonas ---- 1212 . 514151416 . 51819  Klebsiella ---- 1113151517192023 S. aureus ---- 1114182115172325 Table 2 W ell diffusion assay-standardization ( ethyl acetate-low concentration and high concentration ) . C oncentration (毺 L )  Klebsiella   ( mm )  Pseudomonas ( mm ) Staphylococcus   ( mm )  Micrococcus   ( mm ) L ow concentration 25 ---- 50 - 1111 - 75111212 - 1001213   1311 H igh concentration 2501211211750014162319750161823201   00019202424 Table 3 P ercentage of viable cells of  Staphylococcus and  Micrococcus in varying concentrations of ethyl acetate. C oncentration (毺 L )% of viable cells of  Staphylococcus % of viable cells of   Micrococcus 1007 . 5327 . 39920012 . 72713 . 45230013 . 76616 . 59140026 . 75318 . 16150027 . 53228 . 47560030 . 38934 . 97770046 . 23340 . 132   800 * 52 . 20746 . 188   900 ** 71 . 42854 . 4841   00075 . 06483 . 408 * MIC for  Staphyloccoccus ; ** MIC for   Microccoccus.  J Meenupriya et al./Asian Pacific Journal of Tropical Biomedicine (2011)376-380 379   T he ITS region is now perhaps the most widely sequenced DNA region in fungi, I t is most useful for molecular systematics at the species level, and even within species. I nthe present study, the DNA was isolated from the I solate 1  and the ITS region of  5 . 8 s r  RNA was amplified using specificprimers ITS 1 and ITS 4 and the sequence was determinedusing automated sequencers. B last search sequencesimilarity was found against the existing non redundantnucleotide sequence database thus, identifying the fungi as A spergillus ochraceus. T he percentage of similarity betweenthe fungi and database suggests it as novel strain. T hus,the novel strain was named as  A. ochraceus strain MP 2 andmade publically available in G en B ank with an assignedaccession number . a b Figure 1. a: P ure culture of  I solate 1 ; b: SEM micrograph of  I solate 1 . 3.3. Anti-microbial assay   T he fungi  A. ochraceus   MP 2 was extracted in three solventsof varying polarity ( B utanol, chloroform, ethyl aceteate ) . T hree human pathogens namely  Klebsiella pneumonia   ATCC   15380 , Staphylococcus aureus   ATCC   25923 ,  Pseudomonasaeruginosa   ATCC   27853 were used to test the anti-microbialactivity. T heir zone of inhibition in varying concentrations of the sample is given in T able 1 .  E thyl acetate provided promising results compared to theother solvents. T herefore, the optimum concentration of ethyl acetate producing maximum inhibition of the pathogenwas analyzed using the same well diffusion assay for bothlow concentration [ ( 25 - 100 )   毺 L ] and high concentration[ ( 250 - 1   000 )   毺 L ] of the solvent. T heir zone of inhibition is shownin T able 2 .  H igher concentration of ethyl acetate provided a better inhibition activity compared to their low concentrationcounterpart. T he minimum inhibition concentration ( MIC )  of the elite solvent was standardised for two pathogens Staphylococcus and  Micrococcus using broth dilution assayand the result is in T able 3 .  T he fungal extract subjected to TLC separation revealedthe presence of three bioactive metabolites which wasvisualised in UV short range spectrum of  254 nm ( F igure 2 a ) , UV long range of  365 nm ( F igure 2 b ) and iodine chamber  (   F igure 2 c ) .  T he TLC band was eluted and the bioactive metabolitesin the eluant responsible for the anti bacterial activity werecharacterized using GC - MS . T he chromatogram ( F igure 3 )  revealed the presence of different functional groups in theeluant and the solvent. T hree dominant compounds wereindividually characterized to reveal their identity, chemicalformula and structure. T he first elutant was identified as α - C ampholene aldehyde with C hemical formula C 10   H 16   O and molecular weight 152   D a. T he second elutant wasidentified as L ucenin- 2 with C hemical formula C 27   H 30   O 16  and molecular weight 610   D a. T he third elutant is identifiedas 6 - E thyloct - 3 -yl- 2 - ethylhexyl ester with C hemicalformula C 26   H 42   O 4 and molecular weight 418   D a. a b c Figure 2. TLC strip visulaised in a. UV short range b. UV long rangec. I odine chamber. 4. Discussion   W orld ’ s oceans cover more than 70 % of the earth ’ s surfaceand marine biota are an enormous yet underutilized resourcefor the discovery of neutraceuticals, pharmaceuticalsand other high value, low volume bioactives [22] . M arine-derived fungi have been recognized as a potential source of structurally novel and biologically potent metabolites, anda growing number of marine fungi have been reported toproduce novel bioactive.secondary metabolites [23-35] . O f the 18   000 marine naturalproducts described, over  30 % are from sponges and of theantitumor natural product patent registrations in recentyears over  75 % are from sponges [26,27] . E specially, the genus  Aspergillus has been known to be a major contributor to thesecondary metabolites of marine fungal srcin, for example,four sesquiterpenoids with a unique nitrobenzyl ester from  Aspergillus versicolor (  A. versicolor ) , two modified cytotoxictripeptides from  Aspergillus sp., novel pentacyclic oxindolealkaloid from  Aspergillus tamari [28] , four prenylated indolealkaloids from  Aspergillus sp. [29] and two cyclopentapeptidesfrom  A. versicolor [30] . T his paper reports the isolation of threebioactive compounds from  A. ochraceus . T he antimicrobialactivities of the isolated pure compounds in ethyl acetatesolvent were reported since ethyl acetate extract showedpromising results in  Aspergillus flavus [31] . T he isolatedcompounds showed significant antimicrobial activityagainst potential human pathogens. M icrobial secondarymetabolites has a large source of compounds endowed withingenious structures and potent biological activities. M anyof the products currently used for human or animal therapy,in animal husbandry and in agriculture are producedby microbial fermentation, or derived from chemicalmodification of a microbial product [31] . T he present studyof screening bioactive secondary metabolites revealed  A.ochraceus as a source for the production of three effectivemetabolites. T hese metabolites can be further exploited
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