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viz., All rights reserved Allrights www.ijddr.in g and Ferric reducing ities ities Streptomyces Int. J. Drug Dev. & Res & Dev. Drug J. Int. que, que, was selected for nt nt biological activities. death death of worms were alli, Karnataka (state icate icate that the soils of . Anthelmintic effect of olate olate SRDP- Open Access Contents Access Open The radical s s. Antioxidant activity of instar larvae of Streptomyces Copyright © 2013 By IYPF By 2013 © Copyright actinomycete actinomycete isolates for tes. activities. activities. The isolate was nd filtrate wasfiltrate extracted with te te extract of lating the serially diluted soil of bioactive actinomycetes gative bacteria. The extract ecticidal activity ecticidal of the extract A. A. aegypti 07 was identified as a species of . . They are known to play key role in [5-15] s iss Access Open an article permits which unrestricted tes were recovered on Starch casein agar by serial endophytes) and others. They are involved in the decomposition of organic matter in soil, including lignin and other recalcitrant polymers, and can degrade agricultural and urban wastes actinomycetes and their bioactive metabolites have shown to possess antimicrobial, cytotoxic, plant growth promotory, antiviral, insecticidal, antiprotozoal, antioxidant, anthelmintic, enzyme inhibitory, plant growth promoting and herbicidal agents noncommercialprovided use, properly is work original the | July-September 2013 | Vol. 5 | Issue 3 | ISSN 0975-9344 | 0975-9344 ISSN | 3 Issue | 5 Vol. | 2013 July-September | eeIYPF. Thi Int.Drug J. Dev. Res.,& July-September 2013, 26 5(3): species SRDP-07 isolated from mintic mintic activity of the extract was performed by determining 07 07 is under progress. Further screening programmes in soi by cultural and microscopic characteristics. Gram posi

Western Ghats, Thirthahalli, Actinomycetes, Western Ghats are one of the biodiversity hotspots in the : : tate. Antibacterial activity of ethyl acetate extract was tested

. Anthel inent inent inhibition of test bacteria in cross streak techni bstract: bstract: eywords and reducing potential reference of standard. The extract results was of Thirthahalli are known the to also contain present actinomycetes with pote study weaker ind Isolation of when active principles present in the ethyl co aceta species SRDP- Western Ghats of Karnataka can be in fruitful isolation metaboli novel of production for exploited be can which K activities grown grown in for broth casein starch days10 and the culture ethyl ace panel of 6 bacteria by Agar well diffusion assay. Ins was evaluated in terms of aegypti larvicidal effect against 2 taken for paralysis and death of adult Indian earthworm the extract was determined by DPPH free radical scavengin assay. A total of 9 actinomyce dilution-plating technique. The isolate Streptomyces SRDP- were more susceptible to showed dose extract dependent mortality of larvae than of Gram ne extract was lesser and the time considerably higher than takenthat of reference anthelmintic. for paralysis and A present study was undertaken to investigate biological activ insecticidal, anthelmintic and antioxidant activities SRDP-07 isolated of from a a soil sample India. Isolation of actinomycetes was collected carried out by p in Thirthah sample on Starch casein agar. Primary screening of the antibacterial activity was done by Cross streak method. Is prom further characterization and for determining biological Streptomyces

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1 . . Actinomycetes are high G+C containing, [1] © 2013 © Prashith RT. Kekuda. licens and publisher al, et NTRODUCTION filamentous, Gram positive widespread bacteria in that nature. They are are distributed in variety a of ecological habitats such as soil, water, fresh marine environment, plants (as Natural products produced from plants, animals and microbes have been used for several years. However, the scientific knowledge products has been known over of the last 100 years natural or so I

Department of Post of Department Department of Microbiology, of Department Junaid Syed R Onkarappa N K. Rakesh. GraduateStudies and Microbiology,in Research Science Sahyadri College (Autonomous),Shivamogga- 577203, Karnataka,India Authors: Corresponding PrashithKekuda. T. R E-mail:[email protected] S.R.N.M.N College of Applied Sciences,NES Campus, Balraj Urs Road, Shivamogga- Karnataka, India Karnataka,India 2 1 Prashith Kekuda T. R T. Kekuda Prashith N Dileep Mesta C Sunita CoveredEmbase,&Scopus in Elsevier

soil ofThirthahalli,soil Karnataka, India

Biologicalactivities of

Page 268 bioremediation of dyes, petroleum products and Collection of soil sample pesticides and biosorption of heavy metals [16-20] . The soil sample was collected at Thirthahalli, The bioactive metabolites from actinomycetes Shivamogga (district), Karnataka (state), India are also active against antibiotic resistant during the month of July 2012. The soil was bacteria and plant pathogenic fungi [21-25] . collected in a sterile plastic cover from a depth of 15cm, brought to the laboratory and dried at Among actinomycetes, the genus Streptomyces is 40 oC under aseptic conditions [35] . represented in nature by the largest number of species and varieties. These organisms differ in Isolation of actinomycetes their morphology, physiology and biochemical For isolation of actinomycetes, the soil sample was characteristics. They have been distributed in subjected to serial dilution followed by plating on every possible ecological niche. Species of the Starch casein nitrate agar amended with genus Streptomyces are dominating in soil in terms antibiotic Fluconazole (to prevent fungal of number and the bioactive compounds what contamination). The plates were incubated they produce. These organisms have been aerobically at 30 oC for 10 days. Colonies of considered as potential sources of actinomycetes were selected on the basis of pharmaceutically and agriculturally important typical colony morphology. The isolates were

rd bioactive agents. They have provided 2/3 of subcultured on Starch casein agar slants and Page 269 Page species SRDP-07 isolated from soil of Thirthahalli, Thirthahalli, of soil from SRDP-07 isolated species naturally occurring antibiotics, such as maintained in refrigerator [35] . tetracyclines, aminoglysides, macrolides etc., discovered so far [22,26-30] . Western Ghats are one Primary screening for antibacterial potential of the of the biodiversity hotspots in the world. These are actinomycetes isolates grandly known for their diverse flora and fauna. Cross streak method was employed to screen Streptomyces

Karnataka, India Karnataka, However, a very few microbiological studies have antibacterial efficacy of the actinomycete been done in this region. Few studies have been isolates. The actinomycete isolates were streaked carried out on the actinomycetes of Western at the centre of the sterile Starch casein agar Ghats of Karnataka and report antimicrobial, plates and the plates was incubated at 30 oC for 5 antioxidant, enzyme inhibitory, cytotoxic, days. After 5 days test bacteria were streaked anthelmintic, analgesic, anti-inflammatory and perpendicular to the growth of the actinomycete antipyretic activity [31-39] . The present study isolates. The extent of growth inhibition of the test describes isolation and the biological activities bacteria was observed after 24 hours of viz., antibacterial, insecticidal, anthelmintic and incubation. The absence of growth or a less dense antioxidant activity of a Streptomyces species growth of test bacteria near the actinomycete SRDP-07 from a soil sample collected at isolate was considered positive for production and Thirthahalli, Shivamogga (District), Karnataka secretion of antibacterial metabolite by the (State), India. isolates [40] . One isolate (designated as isolate SRDP-07) displaying marked inhibition of test bacteria was selected for further identification MATERIALS AND METHODS and for assessing biological activities.

Covered in Scopus & Embase, Elsevier Int. J. Drug Dev. & Res., July-September 2013, 5 (3): 268-285 Prashith Kekuda T. R et al; Biological activities of Biologicalactivities of al; R et Kekuda T. Prashith © 2013 Prashith Kekuda. T. R et al, publisher and licensee IYPF. This is an Open Access article which permits unrestricted noncommercial use, provided the original work is properly cited.

Characteristics of the isolate SRDP-07 Fermentation and extraction of metabolite from The isolate SRDP-07 was further subjected for isolate SRDP-07 cultural, microscopic, staining and biochemical Sterile Starch casein broth containing Erlenmeyer characteristics. flasks were inoculated with the spore supsenion of well sporulated actinomycete culture and the Cultural characteristics flasks were incubated aerobically at 28 oC for 10 The isolate SRDP-07 was grown on various media days. After incubation, the broth was aseptically viz., Starch casein nitrate agar (SCA), Inorganic filtered through sterilized Whattman No. 1 filter salt-starch agar (ISSA), Actinomycetes isolation paper [35] . The culture filtrate was centrifuged and agar (AIA) and Tryptone yeast extract agar the supernatant was subjected solvent extraction Length Full (TYEA). The color of substrate and aerial mycelium in separation funnel and extracted using ethyl was noted. The plates were observed for the acetate. Equal volume (1:1) of supernatant and production of diffusible pigments. ethyl acetate were taken in a separation funnel and agitated for about 30 minutes. Solvent layer Microscopic characteristic was separated and the supernatant was again The cover slip method was followed to observe extracted with ethyl acetate. The solvent layers

characteristic spore arrangement in isolate SRDP- were pooled and evaporated to dryness at 40 C Original 07. Thin blocks of SCA were cut and placed on [29] . The solvent extract was screened for biological Page 270 Page sterile glass slides. The culture of SRDP-07 was activities. inoculated all over the agar block surface using sterile inoculation loop, a cover slip was placed Antibacterial activity of ethyl acetate extract of over the block, and the slide was placed in a SRDP-07 Research PaperResearch sterile moist chamber and incubated until good The efficacy of ethyl acetate extract of SRDP-07 to growth of the isolate was observed. The cover slip inhibit bacteria was tested against two Gram was removed, placed on a drop of dilute crystal positive bacteria viz., Staphylococcus aureus and violet stain on a clean glass slide and observed Bacillus cereus and four Gram negative bacteria under oil immersion objective in order to study the viz., Escherichia coli , Shigella flexneri , Klebsiella arrangement of spores [35] . pneumoniae and Vibrio cholerae by Agar well diffusion method [43] . The test bacteria were Staining and biochemical characteristics inoculated into sterile nutrient broth (HiMedia, Staining techniques viz., Gram’s and Acid-fast Mumbai) tubes and incubated for 24 hours at

staining and biochemical tests viz., starch 37 C. The broth cultures of test bacteria were hydrolysis, gelatin liquefaction, casein hydrolysis, swabbed aseptically on sterile nutrient agar catalse test, oxidase test, citrate test, cellulose (HiMedia, Mumbai) plates with the help of sterile

hydrolysis, hydrogen sulfide (H 2S) production test cotton swabs. Using a sterile cork borer, wells of and sugar fermentation tests were performed for 6mm diameter were punched in the inoculated the isolate SRDP-07 [41,42] . plates and 100µl of ethyl acetate extract (5mg/ml of 10% DMSO), standard (Streptomycin, 1mg/ml) and DMSO (10%) were transferred into labelled Covered in Scopus & Embase, Elsevier Int. J. Drug Dev. & Res., July-September 2013, 5 (3): 268-285 © 2013 Prashith Kekuda. T. R et al, publisher and licensee IYPF. This is an Open Access article which permits unrestricted noncommercial use, provided the origina l work is properly cited. wells. The plates were then incubated at 37 oC for DPPH free radical scavenging activity 24 hours and the zone of inhibition formed was In order to determine radical scavenging efficacy measured. The experiment was repeated twice of ethyl acetate extract, we have employed and the average value was recorded. DPPH free radical scavenging assay [31] . In brief, 2ml of DPPH solution (0.002% in methanol) was Insecticidal activity of ethyl acetate extract of mixed with 2ml of different concentrations (1- SRDP-07 100µg/ml) of extract and standard separately. The The insecticidal activity in terms of larvicidal effect tubes were incubated in dark at room of different concentrations (1 and 2mg/ml) of temperature for 30 minutes and the optical ethyl acetate extract was screened against II density was measured at 517 nm using UV-Vis instar larvae of Aedes aegypti mosquito. Briefly, 20 spectrophotometer. The absorbance of the DPPH larvae were transferred into beakers containing control was also noted. Ascorbic acid was used as different concentrations of extract. A control was reference standard. The scavenging activity of kept without adding extract. The larvicidal effect the extract was calculated using the formula: was determined by counting the number of dead Scavenging activity (%) = [(A – B) / A] x 100, where larvae after 24 hours. Larvae that failed to move A is absorbance of DPPH and B is absorbance of after probing with a needle in siphon or cervical DPPH and extract/standard combination.

region were identified as dead larvae. The Page 271 Page species SRDP-07 isolated from soil of Thirthahalli, Thirthahalli, of soil from SRDP-07 isolated species experiment was repeated two times and average Ferric reducing activity mortality (%) was noted [32] . The reducing potential of ethyl acetate extract was determined by Ferric reducing assay. Here, Anthelmintic activity of ethyl acetate extract of different concentrations (1-100µg/ml) of extract SRDP-07 and ascorbic acid (reference standard) in 1ml of Streptomyces

Karnataka, India Karnataka, The anthelmintic effect of extract was screened methanol were mixed separately with 2.5ml of against adult Indian earthworms ( Pheretima phosphate buffer (200 mM, pH 6.6) and 2.5 ml of posthuma ). The worms were washed with normal 1% potassium ferricyanide. The tubes were saline (0.85%) to remove extraneous matter. In incubated in water bath for 20 minutes at 50 oC, brief, 6 worms of equal size (6cm long) were cooled rapidly and mixed with 2.5ml of 10% transferred into normal saline containing standard trichloroacetic acid and 0.5 ml of 0.1% ferric (1%) and different concentrations of extract (1 chloride. The amount of iron (II)-ferricyanide and 2mg/ml of saline). The time taken for paralysis complex formed was determined by measuring and death of worms was noted. Piperazine citrate the formation of Perl’s Prussian blue at 700 nm was used as standard anthelmintic. Normal saline after 10 minutes. An increase in absorbance on served as control. The experiment was repeated increase in concentration indicates increased twice and average paralysis and death time was reducing power [31] . noted [31] . UV absorption of ethyl acetate extract of SRDP-07 Antioxidant activity of ethyl acetate extract of The crude bioactive extract was dissolved in ethyl SRDP-07 acetate solvent and subjected to UV absorption

studies. The absorption spectrum of ethyl acetate against a panel of 6 bacteria by cross streak extract was determined in the UV region (200- method. Absence of growth of test bacteria or 400nm) by using a UV-visible spectrophotometer reduced growth of test bacteria in the vicinity of (Shimadzu UV 2554) [44] . the growth of actinomycetes was considered as positive for antibacterial activity. All the isolates showed inhibition of at least one of the test RESULTS bacteria. Isolates SRDP-05 and SRDP-08 inhibited A total of 9 actinomycete isolates (SRDP-01 to only one among six test bacteria. Prominent SRDP-09) were recovered from the soil sample by inhibitory activity was demonstrated by the isolate

serial dilution-plating method. All the isolates were SRDP-07. Table 1 shows the extent of inhibition of Length Full subjected for assessment of antagonistic property test bacteria.

Table 1: Primary screening of actinomycete isolates for antibacterial activity

Extent of inhibition of test bacteria Isolate E.coli S.flexneri K.pneumoniae B.cereus V.cholerae S.aureus SRDP-01 +++ ++ ++ + ++ +++ SRDP-02 + + ++ ++ + +++

SRDP-03 + + +++ + ++ + Original SRDP-04 + + + + - + Page 272 Page SRDP-05 - - + - - - SRDP-06 +++ + + + + + SRDP-07 +++ ++++ ++++ ++ ++++ ++++ SRDP-08 + - - - - - SRDP-09 + + - ++ - - PaperResearch The growth characteristics of the isolate SRDP-07 The isolate SRDP-07 was Gram positive and non- was studied on four media viz., SCA, ISSA, AIA acid fast. The spore arrangement was found to and TYEA. The color of both substrate and aerial be closed spiral type. Biochemically, the isolate mycelium was yellow on SCA and ISSA. On TYEA, was found positive for amylase, cellulose, the substrate and aerial mycelium was yellow catalase and citrase. The isolate was not found and grey respectively. Both substrate and aerial to produce gelatinase, caseinase, oxidase and

mycelia were grey on AIA. Diffusible pigments H2S. The isolate fermented glucose, fructose and were not observed in any of these media (Table galactose with acid production and maltose and 2) . lactose with alkali production. No gas production

was observed (Table 3; Figure 1) . Table 2: Cultural characteristics of isolate SRDP-07 on various media

Substrate Aerial Diffusible Media mycelium mycelium pigment SCA Yellow Yellow - ISSA Yellow Yellow - AIA Grey Grey -

TYEA Yellow Grey -

Test Characteristic absorption maximum of ethyl acetate extract Gram’s staining Gram positive showed a single peak at 214nm. Acid fast staining Non-acid fast Spore arrangement Closed spiral Table 4: Antibacterial activity of ethyl acetate Starch hydrolysis + extract of isolate SRDP-07 Cellulose hydrolysis + Gelatin liquefaction - Zone of inhibition in cm Test bacteria Casein hydrolysis - Extract Standard DMSO Catalase + E. coli 1.2 2.8 0.0 Oxidase - S. flexneri 1.6 3.6 0.0 Citrate test + K. pneumoniae 1.0 3.4 0.0

H2S production - B. cereus 2.3 3.8 0.0 Glucose- Acid V. cholerae 2.0 3.5 0.0 Fructose- Acid S .aureus 2.4 4.1 0.0 Fermentation Galactose- Acid Maltose- Alkali Lactose- Alkali Ethyl acetate extract of isolate SRDP-07 was found to exhibit marked insecticidal activity against II instar larvae of A. aegypti . A dose

dependent larvicidal effect was observed in this Page 273 Page species SRDP-07 isolated from soil of Thirthahalli, Thirthahalli, of soil from SRDP-07 isolated species study. At 2mg/ml concentration, the mortality of larvae was 100% (Figure 2) .

Figure 2: Larvicidal activity of ethyl acetate Figure 1 : Isolate SRDP-07 (Culture and spore extract of isolate SRDP-07

Streptomyces arrangement) Karnataka, India Karnataka, Ethyl acetate extract obtained after solvent extraction of culture filtrate was checked for antibacterial activity by Agar well diffusion method in which positive result was observed as the presence of zone of inhibition around the well. It was found that the extract was effective against all the test bacteria with zone of inhibition ranging from 1 cm to 2.4 cm. Susceptibility to Anthelmintic effect of ethyl acetate extract and extract was higher in case of Gram positive piperazine citrate was determined on the basis of bacteria than Gram negative bacteria. Among time taken for causing paralysis and death of the test bacteria, S. aureus and V. cholerae were adult Indian earthworms. The anthelmintic effect highly susceptible Gram positive and Gram was observed as loss of motility and no response negative bacteria respectively. The extract was to external stimuli which eventually progressed to least effective against K. pneumoniae . Inhibition death of worms. The extract caused dose of test bacteria by standard antibiotic was higher dependent paralysis and death of worms.

Standard anthelmintic piperazine citrate different concentrations of extract and ascorbic exhibited stronger anthelmintic activity than that acid. The absorbance at 700nm was found to of ethyl acetate extract (Table 5) . increase with the increase in concentration of extract indicating reducing potential of extract. Table 5: Anthelminthic activity of ethyl acetate The reducing potential of extract was lesser when extract of isolate SRDP-07 compared with the reference standard (Figure Time in minutes 4) . Treatment Concentration Paralysis Death time time Saline 0.85% - - Figure 4: Ferric reducing activity of ethyl acetate Piperazine extract of isolate SRDP-07

1% 16 26 Length Full citrate 2mg/ml 145 185 Solvent extract 1mg/ml 250 325

Radical scavenging ability of different concentrations of ethyl acetate extract and ascorbic acid was evaluated using DPPH free radical assay. The extract exhibited antioxidant activity by scavenging DPPH* (free radical) and Original Original converting into DPPHH and the activity was

Page 274 Page found to be dose dependent. However, the scavenging potential of extract was found to be DISCUSSION much lesser when compared with reference Natural products are the most important source standard i.e., ascorbic acid. Lower of new compounds having chemical diversity still

concentrations of extract viz., 1 and 2.5µg/ml did PaperResearch unmatched by combinatorial chemistry not shown scavenging of free radicals (Figure 3) . approaches. These natural compounds have

been used in the synthesis of several drugs. It is Figure 3: DPPH radical scavenging activity of well known that microorganisms, in particular ethyl acetate extract of isolate SRDP-07 bacteria and fungi are virtually an unexhaustible source of natural compounds having several therapeutic applications [14,45,46] . Among microorganisms, the filamentous bacteria i.e., actinomycetes account for a significant fraction of microbial metabolites and Streptomyces is the

most prolific genus producing wide range of bioactive metabolites among actinomycetes [47]. The genus Streptomyces comprises Gram The reducing potential of ethyl acetate extract positive, spore forming, aerobic actinomycetes and ascorbic acid was determined by employing having high DNA G-C% content (69~78 mol%) ferric reducing assay in which the reduction of and classified in the family Streptomycetaceae Fe3+ to Fe2+ was investigated in the presence of on the basis of morphological and cell-wall

Covered in Scopus & Embase, Elsevier Int. J. Drug Dev. & Res., July-September 2013, 5 (3): 268-285 © 2013 Prashith Kekuda. T. R et al, publisher and licensee IYPF. This is an Open Access article which permits unrestricted noncommercial use, provided the original work is properly cited. chemotaxonomic characters. The members Streptomycete species. The life cycle of a produce extensively branched substrate Streptomycete provides 3 features for mycelium and aerial hyphae. With more than 500 microscopic characterization viz., vegetative validly described species and subspecies, the mycelium, aerial mycelium baring chains of taxon currently contains the largest number of spores and the characteristics of spores species in the domain Bacteria [27] . themselves. The latter two features produce most Soil is one of the rich reservoirs of microorganisms. diagnostic information [53,54] . Information on Rhizosphere is the fraction of soil which is in cultural features and characteristic spore vicinity to plant roots and is thought to be of arrangement together with biochemical great importance to plant health and soil fertility. properties assists classification of actinomycetes The root exudates of plants stimulate the growth as members of the genus Streptomyces . Saadoun of microbial populations in soil and hence, et al. [55] recovered nine different isolates of microbial activity is greatest in rhizosphere region aquatic actinomycetes and identified them as [48] . Actinomycetes forms a key part of soil Streptomyces spp. on the basis of morphological microbiota. In general, Streptomyces species are and cultural characteristics. Singh et al. [56] saprophytic and are commonly associated with isolated two strains of Streptomyces species and soils, where they significantly contribute to the identified them as Streptomyces albovinaceous

turnover of complex biopolymers such as based on cellular morphology and physiology. In Page 275 Page species SRDP-07 isolated from soil of Thirthahalli, Thirthahalli, of soil from SRDP-07 isolated species cellulose, lignin etc. and antibiotics [49] . It has a study by Savic et al. [57] , morphological and been found that about 90% of soil actinomycetes phenotypic properties of actinomycete isolate are reported to be Streptomyces species [22,50] . In MS405 T were consistent with its classification as a this study, we have isolated nine actinomycetes Streptomyces strain. Similarly, other studies by from the rhizosphere soil sample of Thirthahalli, Rifaat et al. [58] , Sahin et al. [59] and Moncheva et Streptomyces

Karnataka, India Karnataka, Shivamogga, Karnataka. Preliminary screening for al. [60] have assigned the certain actinomycetes antibacterial activity, carried out by cross streak strains under the genus Streptomyces on the basis method, showed varied antagonistic potential of of characteristics such as cultural, microscopic the isolates. Cross streak method is used to and biochemical features. In the present study, determine antagonstic potential of the cultural and microscopic characteristics of actinomycetes. It has become one of the the isolate SRDP-07 were consistent with its routinely used methods and has been employed classification as a member of the genus by several authors to study antimicrobial Streptomyces . potential of actinomycete isolates [35,37,40,44,51,52] . Infectious diseases are caused by a number of One isolate, designated SRDP-07 displayed bacteria, viruses, parasites and fungi which marked inhibition of test bacteria and hence, devastated mankind before the development of selected for further characterization to genus chemotherapeutic agents mainly antibiotics. The level and to determine biological activities. discovery of antibiotics and their subsequent use Morphology plays an important role in has eradicated a lot of infections. However, distinguishing Streptomyces from other sporing traditional antibacterial therapy using antibiotics actinomycetes and in the characterization of from microbial sources or their synthetic

analogues is facing problems due to absorption band was observed in an early study development of resistance in existing agents to by Ezra et al . [74] for coronamycins, peptide antimicrobials. Staphylococcus aureus is one of antibiotics produced by a verticillate the first pathogens that became resistant to Streptomyces sp. (MSU-2110). almost all known antibiotics posing a global Mosquitoes have a predominant role in the threat. Vancomycin resistant enterococci, transmission of several diseases such as malaria, multidrug resistant tuberculosis, antibiotic resistant yellow fever, dengue fever, filariasis, and others. E. coli , P. aeruginosa are among other antibiotic These mosquitoes spread more diseases than any resistant bacteria against which most of the other group of arthropods and also cause allergic antibiotics are not effective. Moreover, these responses that include local skin and systemic Length Full bacteria have the tendency to transmit the reactions [75,76,77] . Aedes aegypti is responsible for resistance gene and this has become a serious transmission of one of the serious arboviral issue in the field of medicine [61-64] . The need for diseases dengue. The control measures taken new antibiotics is increasing day by day due to against arthropod-borne diseases target larval the development and spread of antibiotic- stages and adult mosquitoes. Control of the resistant pathogens which cause life-threatening larvae of the mosquito is one of the widely

[49,65]

infections and patient sensitivity . employed strategies and is frequently done by Original Actinomycetes have provided and continue to the application of synthetic insecticides and Page 276 Page provide a number of antibiotics that are clinically insect growth regulators. However, the raising relevant in the treatment of various diseases. They cost, possible toxicity hazards and insects have the capability to produce a variety of showing resistance highlighted search for the compounds having diverse pharmacological development of alternatives for mosquito control Research PaperResearch activities [66] . Actinomycetes have shown to [75,78] . Actinomycetes are shown to be promising produce metabolites that are active against sources of bioactive metabolites displaying antibiotic resistant bacteria [24,67] . In the present potent insecticidal activity [32,35,79-82] . In the study, the ethyl acetate extract of SRDP-07 present study, we determined insecticidal activity showed marked inhibition of test bacteria. It was of ethyl acetate extract of SRDP-07 against 2 nd found that Gram positive bacteria have shown to instar larvae of A. aegypti . The extract displayed be highly susceptible than Gram negative a dose dependent mortality of larvae. Similar bacteria. Similar results were observed in the results have been noticed in earlier studies. The earlier studies [68-71] . The low antibacterial activity butanol extracts of two Streptomyces species

of ethyl acetate extract against the gram from Agumbe region produced dose dependent negative bacteria could be ascribed to the mortality of 2 nd instar larvae of A. aegypti [32] . In presence of an outer membrane that possess another study, the crude extract of a hydrophilic polysaccharides chains and forms an Streptomyces species isolated from Agumbe additional barrier for extract as well as antibiotics region of Western ghats of Karnataka displayed a [72,73] . UV spectral studies of ethyl acetate extract dose dependent mortality of 3 rd instar larvae of A. of isolate SRDP-07 showed a single peak with aegypti [35] . absorbance maximum at 214nm. Similar Covered in Scopus & Embase, Elsevier Int. J. Drug Dev. & Res., July-September 2013, 5 (3): 268-285 © 2013 Prashith Kekuda. T. R et al, publisher and licensee IYPF. This is an Open Access article which permits unrestricted noncommercial use, provided the origi nal work is properly cited. Helminthic infections are one of the major A well maintained balance exists between diseases worldwide in particular tropical antioxidant defence mechanisms and countries. It is the major cause of morbidity and is generation of free radicals in a normal healthy often fatal in extreme conditions. Parasitic worms individual. During oxidative stress, this balance, also infect livestock and crops, affecting food however, shift towards the excessive production production resulting in low economy. Many of free radicals or deficit in antioxidant defence factors influence susceptibility of an individual mechanism. This oxidative stress is implicated in such as lack of sanitation and supply of pure over hundreds of pathophysiological conditions water together with poverty and illiteracy. such as diabetes, cardiovascular diseases, Helminthic infections contribute to the neurological disorders, cancer, aging etc. prevalence of malnutrition, anemia, eosinophilia, Antioxidant is a substance that significantly and pneumonia. Anthelmintic drugs that expel inhibits or delay oxidative processes. Endogenous parasitic worms from the body have some major antioxidants such as ascorbic acid, tocopherols, drawbacks such as resistance development in glutathione, uric acid, thiols etc., and antioxidant gastro-intestinal helminthes, high cost, adverse enzymes such as superoxide dismutase and effects etc. This situation led to the discovery and catalase protect the body against oxidative development of anthelmintic agents from natural stress. However, in pathophysiological conditions,

[83-86] [91- sources . Actinomycetes have shown to there is additional requirement for antioxidants Page 277 Page species SRDP-07 isolated from soil of Thirthahalli, Thirthahalli, of soil from SRDP-07 isolated species possess marked anthelmintic activity in terms of 95] . Strong restrictions have been placed on the inhibition of plant and animal parasites [12,31,87,88,89] . use of synthetic antioxidants such as BHT, BHA, In this study, the anthelmintic activity of ethyl gallates due to their suspected carcinogenic acetate extract of SRDP-07 was determined using potential [96] . This led to an increasing interest in adult Indian earthworms due to their ready natural products having antioxidant properties. Streptomyces

Karnataka, India Karnataka, availability and anatomical and physiological Actinomycetes have been shown to be resemblance to the human intestinal roundworm promising sources of antioxidants. A number of parasite [84,90] . The standard anthelmintic studies have been carried out on the antioxidant piperazine citrate showed higher activity than the efficacy of extracts and purified compounds extract. The predominant effect of piperazine from actinomycetes [14,31,97-102] . citrate on the worm is to cause a flaccid paralysis DPPH free radical scavenging assay is one of the that result in expulsion of the worm [83,84] . The widely used protocol to arrive the radical extract of SRDP-07 not only demonstrated this scavenging ability of compounds. DPPH is a property but also killed the worms, however, the stable organic free radical having the absorption time taken for this was much higher when maximum around 515-528nm. On accepting an compared with standard. In an earlier study, electron or hydrogen atom it becomes a stable Kekuda et al . [31] showed dose dependent diamagnetic molecule. The effect of antioxidants mortality and death of earthworms by butanol on scavenging DPPH radical is due to their extracts of two Streptomyces species isolated hydrogen donating ability. In this assay, the from western ghat soil of Agumbe, Karnataka. compounds (antioxidants) reduce the purple colored DPPH radical to a yellow colored

compound diphenylpicrylhydrazine and the potential and could serve as electron donors, extent of reaction depends on the hydrogen terminating the radical chain reactions [104] . donating ability of the antioxidants [103,104,105] . In Similar results have been observed in previous the present study, the absorption of DPPH in the studies of Kekuda et al . [31] and Manasa et al . [36] presence of various concentrations of ethyl where the actinomycete extracts have displayed acetate extract of SRDP-07 was measured at weaker reducing potential. 517nm. It was observed that the radical scavenging activities of the extract increased CONCLUSION with increasing concentrations. Although the scavenging abilities of extract were much lesser In the present study, we screened the Length Full than that of ascorbic acid, it was evident that the bioefficacies of an actinomycete isolate extract showed hydrogen donating ability and recovered from a soil sample collected from the extract could serve as free radical Western Ghat region Karnataka, India. One scavengers, acting possibly as primary isolate Streptomyces species SRDP-07 showed antioxidants [104] . The results obtained are in good antibacterial and insecticidal activity. The justification with earlier studies which also reveal results of the present study highlighted that the

[31,36,38] soils of Thirthahalli are reservoirs of potent

weaker scavenging activity of extracts . Original Ferric reducing assay is one of the commonly actinomycetes and hence further screening can Page 278 Page used antioxidant assays which measures total be fruitful for isolation of bioactive actinomycetes antioxidant activity. The assay is performed in which might be exploited in industries for order to measure the reducing power of the production of novel metabolites. compounds. In the present study, we Research PaperResearch +3 +2 investigated the Fe /Fe transformation in the ACKNOWLEDGEMENTS presence of ethyl acetate extract of SRDP-07. In The authors express thanks to Head, Department this assay, the reductants (antioxidants) would of Microbiology, Principal, SRNMN College of cause the reduction of Fe +3 to Fe +2 by donating Applied Sciences, Shivamogga and NES, an electron. The amount of Fe +2 complex formed Shivamogga for giving support to conduct work. can be monitored by measuring the formation of

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Article History:------

Date of Submission: 12-07-2013

Date of Acceptance: 29-07-2013

Conflict of Interest: NIL

Source of Support: NONE

Page 285 Page species SRDP-07 isolated from soil of Thirthahalli, Thirthahalli, of soil from SRDP-07 isolated species Streptomyces Karnataka, India Karnataka,