SCREENING FOR ENZYME INHIBITORS IN MARINE ...shodhganga.inflibnet.ac.in/bitstream/10603/61606/12/12...SCREENING FOR ENZYME INHIBITORS IN MARINE BACTERIA Introduction Marine sources

SCREENING FOR ENZYME INHIBITORS IN MARINE BACTERIA

Introduction

Marine sources have the highest probability of yielding natural products withunprecedented carbon skeletons and interesting biological activity (Konig & Wright,1996). Microorganisms, in particular the bacteria have had a profound effect on thedevelopment of chemistry and upon medical science (Fenical, 1993).

In the past several decades, microorganisms have provided us with manyantibiotics useful as chemotherapeutic agents. It has been considered that manyenzymes have important roles in the maintenance of homeostasis in living organismsand that disease results form a breakdown of homeostasis. The correlation betweenthe biological functions of several enzymes and disease process have beenclassified (Tanaka et al., 1992).

Some inhibitors of enzymes in which the correlation with disease processeshas been established are very important as pharmacologically active drugs. Manyanalogs of the normal substrates of various enzymes have been synthesizedchemically and subjected to the investigation of structure activity relationships(Jung 1978; Krenistsky and Elm, 1982).

In 1960's Umezawa's and Maraos' groups independently started thescreening of microorganisms for new enzyme inhibitors (Tanaka et al., 1992).. Fromthe results of studies on antibiotics and enzyme inhibitors of animal and plant origin,they speculated that microorganisms also produce inhibitors of various enzymes.From then various reports of enzyme inhibitors of microbial origin was published(Hoyem & Skalberg, 1962; Shimidu and Matsuashima, 1969; Shimidu et al., 1969;Aoyagi et at, 1969; Murao and Satoi, 1970; Umezawa, 1972) and a number of recentreviews concerning enzyme inhibitors of microbial origin has also been published(Schindler, 1980; Fleck, 1981; Umezawa, 1982).

Drug discovery in industry today has evolved to the use of specific assayswith target receptors and enzymes involved in pathogenesis of disease rather thancellular or tissue assays. For example the targeted approach in the antitumor area

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includes enzyme inhibitors assays such as topoisomerase inhibitors (McConnelletal., 1994).

Marine natural products were also been screened for various enzymeinhibitors (lzumida et al., 1997). In this present study, marine Streptomyces sp. werescreened for Angiotensin-converting enzyme inhibitors (ACE inhibitors) andAdenosine deaminase inhibitors employing a simple plate assay technique.

The Angiotensin-converting enzyme inhibitors act as anti hype rte nsive drugs.Many commercially available synthetically developed ACE inhibitors are in marketwhich includes drugs like teprotide and captopril etc. (Cushman et aL, 1981). Twoimportant enzyme systems, the rennin - angiotensin (hypertensive) system, areinvolved in the regulation of blood pressure in mammals. Renin cleaves one peptidebond in angiotensinogen to release the biologically inactive decapeptide angiotensinI. Angiotensin - I converting enzyme causes removal of the C-terminal histidylleucine moiety from angiotensin I to give the octapeptide angiotensin II, which is apotent vasoconstrictor and stimulant of adrenocortical aldosterone secretion. Thesame enzyme inactivates the vasodilator bradykinin by the hydrolytic release of oneor more carboxyl - terminal dipeptide residues. These ACE inhibitors can reducehypertension either by suppression of angiotensin II biosynthesis or by thestimulation of bradykinin breakdown (Tanaka etal., 1992).

Adenosine deaminase inhibitors are responsible for alteration in adenosineand deoxyadenosine levels and lympocytic growth and functions and enhance thechemotherapeutic effects of adenoside analogs which are important in. cancerchemotherapy (Agarwal, 1982).

Materials and Methods

The ACE inhibitors were screened following the plate assay method ofO'Conner and Sommers (1985). This agar plate assay involves two aspects, such asapplying the sample to be tested to an enzyme-containing layer and overlaying with asubstrate-containing layer. The lower agar layer contains 1% agarose, 20 mMHEPES buffer, 100mM Nacl, 200 pm CoCl 2 and ACE of 400 pg protein m1 1 with thepH set at 6.5. The broth cultures of the Streptomyces strains were centrifuged and

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the supernatant was impregnated on to 7mm filter paper discs and allowed to dry.The paper discs were placed onto the first layer of agar and given at 2 h diffusiontime. Then this agar layer with disc was overlaid with the second layer of agarcontaining 1% agrose, 20 mM HEPES buffer, 100mM NacI, 200 pm CoCl2 andNBGCG (p-nitrobenzyl oxycarbonylglycyl - (S-4-nitrobenzo —2-ox-1 ,3-diazole)-L-cystinglycine) at 200 pg ml 1 . The plates were incubated for 2 hrs at 37°C andflooded with 0.1N NaOH for 10 mm. Then the solution on the agar was drained andinhibition zones were noted. The zones initially seen as colourless on an amberbackground turn pink after 30 min and fade completely in 2h. The zones weremeasured and tabulated.

ADA inhibitors were screened using an agar plate method containing a pHindicator developed by Katsuragi et al., (1985). Agar plates containing a medium of 6pg of adenosine deaminase, 0.5 m mol of adenosine, 0.25 m mol of potassiumphosphate buffer, 20 mg of phenol red and 1 g of agar in 100 ml of distilled water atpH 5.9 was used for the assay. The culture broth supernatants were impregnated onto the paper discs and were placed on the agar plates. After few hours of incubationat room temperature, yellow zones developed around the paper discs and wereconsidered positive for ADE inhibitors and while the background colour turned to apurplish red. Colour change by the pH indicator depends on ammonia liberation indeamination reaction and increased pH.

Results

A total of 94 Streptomyces strains were screened for ACE inhibitors and ADEinhibitors. In the initial screening, 10 and 15 strains exhibited ACE and ADE inhibitoractivity but in the second confirmational screening only 8 and 4 strains exhibited ACEand ADE activity respectively. A total of 8 strains out of 94 were positive for ADEinhibitors and were SF1, AH7, CC7, BFC21, GMB 10, SCI-18, STCL 20 and SPE5.The strains SF1 (sponge derived), CC7 (Crab derived), BFC21 (Biofilm derived) gavean inhibition zone of 3mm (+++). The strains GMB10 (Gut microflora) exhibited a 2mm zone (++) around the disc. The other strains A1312, AH7 (algae derived), AH7,SCL 18, STCL20 (Coral derived) and SPE 5 (Cephalopod egg derived) exhibitedtrace activity(+).

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r IN Tota I Strains

ACE+

ADE+

MIW7

Only 4 strains (SA4, AE7, BFB 18, 100

J.and GE 2) were found to be 80

positive for ADE inhibitor. The 60

strains were derived from sponge,40- li

algae, bioflim and gastropod egg

respectively (Table 1& Fig. 1). 2:

Table 1: Enzyme inhibitor activity of marine Streptomyces strains.

(+ + + - 3mm, + + - 2mm, + - trace)

Discussion

The screening of enzyme inhibitors from microorganisms requires the

knowledge and techniques obtained in many fields of natural sciences microbiology,

biochemistry, chemistry and pharmacology. These disciplines concern the isolation

and cultivation of microorganisms, measurement of enzyme reactions, isolation and

characterization of the inhibitors and their evaluation in animals etc. During the past

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three decades, about 300 new bloactive compounds have been discovered duringscreening for enzyme inhibitors. Among them, bestatin has been used as a drug forthe treatment of cancer in Japan. Since 1987, HMG - Co A reductase inhibitors suchas lavastatin, pravastatin and simvastatin have been used for the treatment ofhype rcholestrolemia in the world (Tanaka etal., 1992).

In the present study few Streptomyces strains were found to produce ACEand AIDE inhibitors. In the marine side enzyme inhibitor screening has been in focusfor the past one decade in organizations such as National Cancer Institute, USA,where enzyme inhibition drugs were used to screen for antitumor activity(Mc Connell, 1994). Regarding screening for marine bacteria for ACE and AIDEinhibitor, there is a lack of any prior reports. But from the terrestrial side few workshave been reported by different authors. For ACE inhibitors, compounds codedA58365A and A58365B were discovered using the agar plate assay method fromterrestrial microorganisms. (O'Connor & Sommers, 1985). Other ACE inhibitorsreported from terrestrial microorganisms was Phenacein, K-4, K-13, and K-26(Bush etal., 1984; Koguchi etal., 1986; Kase etal., 1987; Yamato et at, 1986).

Regarding AIDE inhibitors the reports from the terrestrial side was also few.Cot romycin, an AIDE inhibitor was isolated from Streptromyces kaniharaensis (Sawaet al., 1967). Deoxycoformycin another AIDE inhibitor was isolated from strain ofStreptomyces antibioticus (Woo et al., 1974). Omura et al., (1985, 1986a, 1986b)isolated two new AIDE inhibitors adechiorin and adecypenol from culture broths ofActinomadura sp. and Streptomyces sp.

Both ACE as well as AIDE inhibitors reported from the terrestrial side wasfrom actinomycetes. The ACE and AIDE inhibitors positive strains of the marineStreptomyces screening may yield potential novel metabolites of these nature.

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CHEMICAL SCREENING FOR ALKALOIDS IN MARINE BACTERIA4.

Introduction

Marine natural products chemistry is essentially a child of 1970s thatdeveloped rapidly during 1980s and matured in the last decade. Early directionstaken by marine natural product chemist drew as much from the examples providedby insect chemical ecology as from the longer history of phytochemistry. By 1975,there were already three parallel tracks in marine natural products chemistry such asmarine toxins, marine biomedicinals and marine chemical ecology. It is theintegration of the three fields of study that has given marine natural productschemistry its unique character and vigour (Faulkner, 2000).

Because of the diversity of marine organisms and habitats, marine naturalproducts encompass a wide variety of chemical classes including terpenes,shikimates, polyketides, acetogenins, peptides, alkaloids of varying structures and amultitude of compounds of mixed biosynthesis (Wright, 1998). Alkaloids, of whichsome 5,500 are known, comprise the largest single class of secondary plantsubstances. There is no one definition of the term 'alkaloid' which is completelysatisfactory, but alkaloids generally include those basic substances that contain oneor more nitrogen atoms usually in combination as part of a cyclic system. Alkaloidsare often toxic to man and many have dramatic physiological activities, hence theirwide use in medicine. They are usually colourless; often optically active substances;most are crystalline but a few are liquids at room temperatur'e. Chemically, alkaloidsare a very heterogeneous group ranging from simple compounds like coniine to thepentacyclic structure of strychnine. The .functions of alkaloids in plants are stilllargely obscure although individual substances have been reported to be involved asgrowth regulators or as insect repellents or attractants. The theory that they act as aform of nitrogen storage in plant is not now generally accepted (Harbone, 1984).

A large number of biologically active compounds from microorganisms havebeen discovered so far mainly by screening programs based on such biologicalactivities as antibacterial, antitumour, antiviral etc. Screening systems based onbioactivity measurements encounter the following difficult problems such as a great

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deal of research work and time is required for the establishment of a reliablescreening program, variations originating from various factors such as growth media,inorganic ions and high molecular weight metabolites are a hindrance to reliablemeasurements by biological assay techniques and there is usually a great gapbetween the biological activities measured in vitro and in vivo of new compoundsdiscovered by in vitro screening. (Nakagawa, 1992).

Considering the limitation faced by biological screening programs, Zahner et

al., (1982) Umezawa etal., (1970) and Omura etal., (1974; 1986) and other researchgroups have searched systematically for new bioactive compounds by chemicalscreening from microorganisms.

The principle of chemical screening is the isolation and detection of newmetabolites, by specific test such as color reactions, using several reagents to detectfunctional groups present in molecules of known secondary metabolites (Nakagawa,1992). The search for nitrogen containing compounds produced by microorganismshas naturally been actively pursued because alkaloids from plants have played asignificant role in medicine. Many alkaloids that have been obtained from fungi suchas nigragillin, echinulin, fumitremorgen, austamide, and rugulovasine have beenreported independently. Rosenberg et al., (1976) described screening methods foralkaloids from lower fungi using several colour reagents. They also pointed out thatmicrobial alkaloids were detected with the high frequency of 12 to 40% from varioustype cultures of fungal species such as Aspergillus, Fusarium, Pencillium andHelminthosporium.

In this present study the marine bacteria found to be active in various assayswere screened with Dragendroff's reagent to identify if the active constituent is analkaloid.

Material and Methods

Screening for alkaloids in the culture broth of marine bacteria were carriedout following the method of Omura et al., 1974, using Dragendroff's reagent. Thereagent was prepared following the method of Touchstone and Dobbins (1983). Asolution was prepared by dissolving 0.85g bismuth subnitrate in lOmi acetic acid and40rnl H20. Another solution was prepared separately by dissolving 8g of potassium

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iodide in 20m1 H 20. The stock solution was prepared by mixing equal amount of boththe solutions. The screening reagent was prepared by mixing imi of stock solutionwith 2m1 acetic acid and lOmI H20.

Primary screening

A 5-day culture filtrate (290rpm) was divided into parts. After adjusting the pH to 4.0with 0.1N HCI, one part of the culture filtrate was tested with Dragendroff reagentaccording to Munier and Macheboeuf (Breed et a/., 1957). The formation ofprecipitate indicated a positive result.

Secondary screening test

The filtrates exhibited positive results were used for secondary screeningtest and it was conducted using the reserved culture filtrate. 5ml of the filtrate wasmade alkaline with NH 4 OH and extracted with 1 volume of ethyl acetate. Then theethyl acetate later was extracted with 1 ml of 0.1 N HCI and tested with Dragendroff'sreagent.

In the present study, the 170 antibiotic producer strains that exhibitedantibacterial activity and the 94 Streptomyces strains, which were screened forartemia toxicity, insecticidal activity and enzyme inhibitors were subjected tochemical screening for alkaloids.

Results

In the screening for alkaloids among the strains, which exhibited antibacterialactivity, 27 strains were positive for alkaloids (Fig.1). Two sponge-derived strainsSE1 1 and SG5 that exhibited potential activity against human pathogens and fishpathogens respectively were found to be r - - I

Fig. 1positive for alkaloids. The strains AA7, AC3, I

'' L He species

dfrom

2e0

alkaloid positive All these strains exhibiteda range of activity from mild to high againsthuman pathogens. Except AF2 and AK17 allother strains exhibited mild activity against fish pathogens. The alkaloid positivestrain CC6 that exhibited mild activity against both human and fish pathogens was

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isolated from a crab. The strains BDA9, BF1313, BFC3, isolated from biofilm, werefound to be alkaloid positive. The strains BFA9 and BFC3 exhibited high activityagainst human pathogens. The alkaloid positive stains GM132 and GM138 wereisolated from gut microflora, The strain GMB2 exhibited higher activity against bothhuman and fish pathogens, the strain GMB8 exhibited mild activity against humanpathogens and no activity against fish pathogens. The strains ASA2 and ASA 7,isolated from ascidian, and exhibited activity against both human and fish pathogens,were positive for alkaloids also. The strains SQE 1 and SQE2, isolated from squideggs, exhibited higher activity against one human pathogen, and fish pathogens,respectively, were positive for alkaloids. The alkaloid positive strains SCL10, STCL5,STCL13 and STCL15 were isolated from corals. Except STCL13 that exhibitedhigher activity against Bacillus substi/is, all the other strains exhibited mild activityagainst both human and fish pathogens. The strains OBSA2 and OBSB5, isolatedfrom Opisthobranch surface were alkaloid positive. OBSB2 exhibited potentialactivity only against 2 human pathogens. OBS135 exhibited higher activity againstone human pathogen and two fish pathogens.

Out of the 94 Streptomyces strains screened for alkaloids 15 strains werefound to be alkaloid positive (Fig.2). The strains SC8 and SP were isolated fromsponges. Only the strain SJ7, exhibitedmild insecticidal activity and as well asartemia toxicity. The alkaloid positiveStreptomyces AF3 and A112 were isolatedfrom seaweeds, both exhibited insecticidaland artemia toxicity. The alkaloid positivestrain 0013, isolated from a crab, exhibitedno artemia toxicity but very mild insecticidalactivity was noted. The positive strain BFC17, isolated from biofilm, exhibited higherartemia toxicity and moderate insecticidal activity. The positive strain SCU13,isolated from sea cucumber, exhibited significant Artemia toxicity and insecticidalactivity. The strains GMA 7 and GMB8, isolated from gut microflora, exhibitedmoderate to mild artemia toxicity and insecticidal activity. The alkaloid positive strainASA7, was isolated from an ascidian, exhibited moderate artemia toxicity and mildinsecticidal activity. The alkaloid positive strain GE13, isolated from gastropod egg,exhibited potent artemia toxicity as well as insecticidal activity. The alkaloid positivestrain SOLiD was isolated from a coral and it exhibited only mild Artemia toxicity.

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The strain OBSA17, isolated from Opisthobranch surface was alkaloid positive andexhibited moderate artemia toxicity and mild insecticidal activity. The alkaloidpositive strain SPE5 was isolated from sepia egg, and exhibited moderate artemiatoxicity and no insecticidal activity was noted (Tables 1 &2)

Table 1. Alkaloid (+) strains Table 2. Alkaloid (+) Streptomycesexhibiting antibacterial activity strians

Strains Alkaloid positive(+)

SEll +SG5 +AA7 +AC3 +AF2 +

AG1 +H4 +Ill +AK17 +CC6 +BFA9 +BFB13 +BFC3 +SUR3 +GMB2 +GMB8 +GMC9 +ASA2 +ASA7 +SQE1 +SQE2 +SCL12 +STCL5 +STCL13 +STCL15 +08S82 +OBSB5 +

Strains Alkaloid positive(+)

SC8 +

SR +

AF3 +

Al12 +

CC13 +

BFB13 +

BFC22 +

SCU13 +

GMA7 +

GMB8 +

ASA7 +

GE13 +

SCL1O +

OBSA17 +

SPE5 +

126

Discussion

Little of the world's biodiversity has been tested for biological activity yetnatural products have been the single most productive source of drug leads (Harvey,2000). Alkaloids have diverse structures and many show a range of pharmacologicalactivities including antimicrobial activity.

Lewis (2000) in a review on alkaloids lists out many marine derived alkaloids.Many alkaloid compounds have been isolated from marine sponges and ascidians.From sponges, compounds such as Makalavic acid A and B, llanishin, dimethyl (oxy)aaptamine, aaptosine, aaptamine, isoaaptamine, pyridoacridine alkaloids, guanidinealkaloids, agelastatin A and B, Konbu'acidin and Manzamine have been reported.From ascidian alkaloid compounds such as Lissoclin disulphide, ritterazine,plakinidine alkaloids etc. were reported.

In the present study the bacterial strains exhibiting activity were subjected tochemical screening to find out whether the active princip!e is an alkaloid compound.The importance of marine alkaloids can be highlighted by the fact that the marinealkaloid ecteinascidin 743 (ET-743) is by far the most advanced compound underclinical investigation. It is in the late stages of phase II clinical trials as an anticanceragent and is expected to enter the drug market in Europe by the end of 2002 or inearly 2003 (Proksch, 2002). The results of the present investigation shows that outof the 264 strains screened for alkaloids, 42 were found to be positive. Few potentialantibiotic producing strains against human pathogens as well as fish pathogen werefound to be alkaloid positive. The Streptomyces strains which were already screenedfor artemia toxicity and insecticidal activity also yielded few alkaloid positive strains.Among these strains potent artemia toxicity and insecticidal activity was noted in 3strains. The active compound of these potent (antibacterial, artemia toxic andinsecticidal) strains must be alkaloids, which can be confirmed by further separationand purification of active compound. This chemical screening has provided an ideaabout the nature of the active compound present in culture extracts of the marinebacterial strains.

Lewis (2000) reported the isolation of phenazine alkaloids from a marineStreptomyces sp. and a halophilic marine bacterium called culture LL-141352. The

127

phenazine alkaloids were identified as pelagiomicins A and B, and both of thesecompounds were found to be toxic. He also reported the isolation ofHydroxyakalone, an alkaloid and a novel xanthine oxidase inhibitor produced by themarine bacterium Agrobacterium aurantiacum.

The characteristics of the chemical screening methods are both simple andrapid for detection and isolation of new metabolites (Nakagawa, 1992). Zahner et al.,

(1982) suggested that the likelihood of finding new bloactive compounds by chemicalscreening seems to be greater than with ordinary screening, employing biologicalactivity. Even though in this present study, biologically active strains were screenedfor the presence of alkaloids, the straight forward approach of chemical screening forvarious chemical classes of marine microbial metabolites can be carried out toovercome the difficulties and disadvantages involved in biological screeningmethods.

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El

STUDIES IN PARTIAL ISOLATION OF ACTIVE COMPOUNDS

Introduction

Biodiversity is a term commonly used to denote the variety of species andmultiplicity of forms of life. But this variety is deeper than is generally imagined. Inaddition to the processes of primary metabolism that involve essentially the samechemistry across great swathes of life, there are a myriad of secondary metabolitesnatural products usually confined to a particular group of organisms or to a singlespecies or even to a single strain growing under certain conditions. Tens ofthousands of natural products have been described, but in a world where we are noteven close to documenting all the extant species, there are almost certainly manymore thousands of compounds waiting to be discovered (Cannell, 1998).

Marine organisms live in complex communities and in close association withother macro and microorganisms. Some organisms derive their chemistry fromdietary sources, while others synthesize the compounds de novo. Some compoundsmay be produced by associated microorganisms, while others may require anassociation between the host and microorganisms to produce the compounds. Thechemistry of any particular specimen can be affected by the habitat as well as bygeographic and seasonal factors (Paul, 1992).

Because of the diversity of marine organisms and habitats, marine naturalproducts encompass a wide variety of chemical classes including terpenes,shikimates, polyketides, acetogenins, peptides, alkaloids of varying structures and amultitude of compounds of mixed biosynthesis (Wright, 1998). Series of marinenatural products often contain a variety of chemical functionalities (like, OH, OCH3,OAC, 0S03 , Na etc.) Each change in functionality has a potential to radicallychange the polarity of compounds and therefore the method required for purification(Wright, 1998).

There is no single approach or method for the purification and isolation ofmarine natural products. Researchers around the globe use different protocols forisolation employing a variety of liquid-liquid extraction methods to chromatographytechniques. This aspect has been reviewed from 115 reports of marine natural

129

products published in the Journal of American Chemical Society, The Journal ofOrganic Chemistry, Tetrahedron and The Journal of Natural Products, and amultitude of techniques employed by various researchers have been noted. Thesilica gel as stationary phase was used for isolation by 56% of researchers and inwhich 44% used only normal phase or reverse phase silica stationary phases(Wright, 1998).

In the present study few strains exhibiting potential bioactivity were masscultured and partial purification of the active compound was carried out using normalphase silica gel chromatography.

Materials and Methods

Potential strains exhibiting antibacterial activity against human pathogens(ASC1, SCI-3, SQE6, ASA2 and JF4), fish pathogens (ASA7, SCL2, OBSB5, SPE9and AK-14), Artemia toxicity (SH6 and SCU13), insecticidal activity (SH6 andSYCL17), herbicidal activity(SB3 and AB17) and growth promotor activity (CC13 andSTCL1 8) were taken for partial purification of the active compound.

The partial purification was done by adopting the method of Wright (1998).The strains were mass cultured in 1 lit, culture flasks for 7 days at 290 rpm at roomtemperature. The broth was extracted by liquid - liquid extraction method given byGailliot (1998) with slight modifications. Equal volume of ethylaceate was added tothe broth culture and stirred on a magnetic stirrer for 30 minutes. The solvent andthe aqueous phases were separated in a separating funnel. The aqueous phase wasdiscarded and the solvent phase was evaporated and concentrated to obtain thecrude extract.

The crude extract was applied to a normal phase silica gel (Himedia,Bombay) column and subsequently eluted using a step gradient solvent system ofincreasing polarity. The solvent system was 100% heptane -b 20% ethyl acetate +80% heptane , 40% ethyl acetate 60% heptane - 60% ethyl acetate + 40 %heptane -i 80% ethylaceate + 20% heptane -+ 100% ethyl acetate -* 25% methanol+ 75% ethyl acetate -+ 50% methanol + 50% ethyl acetate - p 100% methanol.

130

Results

In the column fractionation of the crude extract of the strain ASC1, the activitywas confined to the non-polar end of the step-gradient with a highest activity of 13mm and 14 mm inhibition zones against Bacillus substilis and Candida albicans

respectively. The column fraction of the crude extract of the strain SCL3 exhibitedactivity at the non-polar range exhibiting a maximum of 25 and 22 mm inhibitionzones against E. coil and Pseudomonas aeruginosa. The column fractions of thestrain SQE6 exhibited highest activity at the intermediate polar fractions exhibiting amaximum of 7.5 mm and 10mm against E. coil and Bacillus substilis respectively. Inthe column fractionation of the strain ASA2, the activity was exhibited at the non-polar range with a maximum zone of 8mm against Pseudomonas aeruginosa andCandida albicans. The column fractions of the strain JF4 exhibited activity at thepolar end of the step gradient, exhibiting a maximum zone of 10 againstStaphylococcus aureus, E.coli (8mm) and Candida albicans (8mm) (Table 1).

In the column fractions of the strain ASA7, the activity was exhibited at thenon-polar end of the step gradient with maximum zones of 13 mm and 10mm againstVibrio haiveyi and V. parahaemolyticus respectively. The strain SCL2 exhibitedhighest activity at the polar end of the step gradient, showing maximum inhibitionzones of 10mm and 8.5 mm against Vibrio harveyi and V. parahaemolyticus

respectively. The strain OBS135 exhibited activity at the non polar end of the stepgradient with the highest inhibition zone of 10 & 8.5 mm against Vibrio harveyi andV.parahaemolyticus. The activity of the strains SPE9 and AK14 was noted at theintermediate range of the step gradient with a maximum zone of 10 and 11 mmagainst Vibrio har.'eyi, Vibrio parahaemolyticus and Aeromonas hydrophilarespectively (Table 2).

In the column fractions of the crude extract of the strains SH6 and SCU13exhibiting Artemia toxicity, the activity of SH6 was noted at the intermediately polarrange of the step gradient and for SCU13 at non-polar range of the step gradient.When the crude extracts of the strains SH6 and STCL17 showing insecticidal activity,were column fractioned, the activity was noted at the intermediately polar fraction(Tables 3, 4 and 5).

131

In the column fractionation of the crude extracts of strains SB3 and AB17exhibiting herbicidal activity, the highest activity was exhibited at the intermediatelypolar fractions for S133 and at the polar end of the step-gradient for AB17 (Table 6).When the crude extracts of strains CC13 and STCL18 exhibiting growth promotoractivity were column fractioned, the highest activity for CC13 was exhibited at thepolar end and for STCL1 8 it was at the non-polar end of the step-gradient (Table 7).

Discussion

Novel secondary metabolites including antibiotics from marine bacteria areattracting attention because of the growing demand for new antibiotics (Levy, 1998).Many unusual antibiotic types have been discovered (James etal., 1996; Yoshikawaet al., 1999), although more have not been structurally characterized (Lemos et al.,

1985; Spragg et al., 1998). Marine organisms provided a seemingly endless paradeof novel structures and new carbon skeletons. Several functional groups areuniquely or predominantly marine (Faulkner, 2000).

In the present study, the crude extracts of the potential bacterial strainsexhibiting antibacterial, arternia toxicity, insecticidal, herbicidal and growth promotoractivities were partially purified using column chromatography. The activity variedfrom non-polar, intermediately polar and to polar fractions. The decrease in activitynoted in some fractions in comparison with crude may be due to the fact that the highactivity in crude extracts was due to the synergistic effect of the different constituentsof the crude extracts. Similar type of fractionation was carried out by McCrain andHemscheidt, (2000) in the isolation of antibiotic found in the marine bacteriumA!teromonas rubra using normal phase column chromatography, reverse phasecolumn chromatography and size exclusion column and reported that the activefractions were observed in the reverse phase column.

The isolation and structure determination of natural products is a time-consuming and expensive process even with modern methods (Dobler et al., 2002).So further purification and characterization of the active compounds can be taken upas a separate study to identify the novel metabolites.

132

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