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Biological activities of Streptomyces species SRDP-07 isolated from soil of Thirthahalli, Karnataka, India

Prashith Kekuda T. R1,2*, Dileep N1, Syed Junaid1, Rakesh. K. N1, Sunita C Mesta2 and Onkarappa R2

1Department of Microbiology, S.R.N.M.N College of Applied Sciences, NES Campus, Balraj Urs Road, Shivamogga-577201, Karnataka, India

2Department of Post Graduate Studies and Research in Microbiology, Sahyadri Science College(Autonomous), Shivamogga-577203, Karnataka, India

Corresponding Author:
Prashith Kekuda. T. R
Department of Microbiology
S.R.N.M.N College of Applied Sciences
NES Campus, Balraj Urs Road
Shivamogga-577201, Karnataka, India
E-mail: [email protected]

Received Date: 12-07-2013; Accepted Date: 29-07-2013

Citation: Prashith Kekuda T. R, Dileep N, Syed Junaid, Rakesh. K. N, Sunita C Mesta, Onkarappa R. “Biological activities of Streptomyces species SRDP-07 isolated from soil of Thirthahalli, Karnataka, India”. Int. J. Drug Dev. & Res., July-September 2013, 5 (3): 268-285

Copyright: © 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.

 

Abstract

Western Ghats are one of the biodiversity hotspots in the world. The present study was undertaken to investigate biological activities viz., antibacterial, insecticidal, anthelmintic and antioxidant activities of a Streptomyces species SRDP-07 isolated from a soil sample collected in Thirthahalli, Karnataka (state), India. Isolation of actinomycetes was carried out by plating the serially diluted soil sample on Starch casein agar. Primary screening of the actinomycete isolates for antibacterial activity was done by Cross streak method. Isolate SRDP-07, showing prominent inhibition of test bacteria in cross streak technique, was selected for further characterization and for determining biological activities. The isolate was grown in starch casein broth for 10 days and the culture filtrate was extracted with ethyl acetate. Antibacterial activity of ethyl acetate extract was tested against a panel of 6 bacteria by Agar well diffusion assay. Insecticidal activity of the extract was evaluated in terms of larvicidal effect against 2nd instar larvae of Aedes aegypti. Anthelmintic activity of the extract was performed by determining time taken for paralysis and death of adult Indian earthworms. Antioxidant activity of the extract was determined by DPPH free radical scavenging and Ferric reducing assay. A total of 9 actinomycetes were recovered on Starch casein agar by serial dilution-plating technique. The isolate SRDP-07 was identified as a species of Streptomyces by cultural and microscopic characteristics. Gram positive bacteria were more susceptible to extract than Gram negative bacteria. The extract showed dose dependent mortality of larvae of A. aegypti. Anthelmintic effect of extract was lesser and the time taken for paralysis and death of worms were considerably higher than that of reference anthelmintic. The radical scavenging and reducing potential of extract was also weaker when compared with reference standard. The results of the present study indicate that the soils of Thirthahalli are known to contain actinomycetes with potent biological activities. Isolation of active principles present in the ethyl acetate extract of Streptomyces species SRDP-07 is under progress. Further screening programmes in soils of Western Ghats of Karnataka can be fruitful in isolation of bioactive actinomycetes which can be exploited for production of novel metabolites.

Keywords

Western Ghats, Thirthahalli, Actinomycetes, Streptomyces, Biological activities

Introduction

Natural products produced from plants, animals and microbes have been used for several years. However, the scientific knowledge of natural products has been known over the last 100 years or so [1]. Actinomycetes are high G+C containing, filamentous, Gram positive bacteria that are widespread in nature. They are distributed in a variety of ecological habitats such as soil, fresh water, marine environment, plants (as 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 [2,3,4]. The actinomycetes and their bioactive metabolites have shown to possess antimicrobial, cytotoxic, plant growth promotory, antiviral, antioxidant, insecticidal, antiprotozoal, anthelmintic, enzyme inhibitory, plant growth promoting and herbicidal agents [5-15]. They are known to play key role in bioremediation of dyes, petroleum products and pesticides and biosorption of heavy metals [16-20]. The bioactive metabolites from actinomycetes are also active against antibiotic resistant bacteria and plant pathogenic fungi [21-25].

Among actinomycetes, the genus Streptomyces is represented in nature by the largest number of species and varieties. These organisms differ in their morphology, physiology and biochemical characteristics. They have been distributed in every possible ecological niche. Species of the genus Streptomyces are dominating in soil in terms of number and the bioactive compounds what they produce. These organisms have been considered as potential sources of pharmaceutically and agriculturally important bioactive agents. They have provided 2/3rd of naturally occurring antibiotics, such as tetracyclines, aminoglysides, macrolides etc., discovered so far [22,26-30]. Western Ghats are one of the biodiversity hotspots in the world. These are grandly known for their diverse flora and fauna. However, a very few microbiological studies have been done in this region. Few studies have been carried out on the actinomycetes of Western Ghats of Karnataka and report antimicrobial, antioxidant, enzyme inhibitory, cytotoxic, anthelmintic, analgesic, anti-inflammatory and antipyretic activity [31-39]. The present study describes isolation and the biological activities viz., antibacterial, insecticidal, anthelmintic and antioxidant activity of a Streptomyces species SRDP-07 from a soil sample collected at Thirthahalli, Shivamogga (District), Karnataka (State), India.

Materials and Methods

Collection of soil sample

The soil sample was collected at Thirthahalli, Shivamogga (district), Karnataka (state), India during the month of July 2012. The soil was collected in a sterile plastic cover from a depth of 15cm, brought to the laboratory and dried at 40oC under aseptic conditions [35].

Isolation of actinomycetes

For isolation of actinomycetes, the soil sample was subjected to serial dilution followed by plating on Starch casein nitrate agar amended with antibiotic Fluconazole (to prevent fungal contamination). The plates were incubated aerobically at 30oC for 10 days. Colonies of actinomycetes were selected on the basis of typical colony morphology. The isolates were subcultured on Starch casein agar slants and maintained in refrigerator [35].

Primary screening for antibacterial potential of the actinomycetes isolates

Cross streak method was employed to screen antibacterial efficacy of the actinomycete isolates. The actinomycete isolates were streaked at the centre of the sterile Starch casein agar plates and the plates was incubated at 30°C for 5 days. After 5 days test bacteria were streaked perpendicular to the growth of the actinomycete isolates. The extent of growth inhibition of the test bacteria was observed after 24 hours of incubation. The absence of growth or a less dense growth of test bacteria near the actinomycete isolate was considered positive for production and secretion of antibacterial metabolite by the isolates [40]. One isolate (designated as isolate SRDP-07) displaying marked inhibition of test bacteria was selected for further identification and for assessing biological activities.

Characteristics of the isolate SRDP-07

The isolate SRDP-07 was further subjected for cultural, microscopic, staining and biochemical characteristics.

Cultural characteristics

The isolate SRDP-07 was grown on various media viz., Starch casein nitrate agar (SCA), Inorganic salt-starch agar (ISSA), Actinomycetes isolation agar (AIA) and Tryptone yeast extract agar (TYEA). The color of substrate and aerial mycelium was noted. The plates were observed for the production of diffusible pigments.

Microscopic characteristic

The cover slip method was followed to observe characteristic spore arrangement in isolate SRDP- 07. Thin blocks of SCA were cut and placed on sterile glass slides. The culture of SRDP-07 was inoculated all over the agar block surface using sterile inoculation loop, a cover slip was placed over the block, and the slide was placed in a sterile moist chamber and incubated until good growth of the isolate was observed. The cover slip was removed, placed on a drop of dilute crystal violet stain on a clean glass slide and observed under oil immersion objective in order to study the arrangement of spores [35].

Staining and biochemical characteristics

Staining techniques viz., Gram’s and Acid-fast staining and biochemical tests viz., starch hydrolysis, gelatin liquefaction, casein hydrolysis, catalse test, oxidase test, citrate test, cellulose hydrolysis, hydrogen sulfide (H2S) production test and sugar fermentation tests were performed for the isolate SRDP-07 [41,42].

Fermentation and extraction of metabolite from isolate SRDP-07

Sterile Starch casein broth containing Erlenmeyer flasks were inoculated with the spore supsenion of well sporulated actinomycete culture and the flasks were incubated aerobically at 28°C for 10 days. After incubation, the broth was aseptically filtered through sterilized Whattman No. 1 filter paper [35]. The culture filtrate was centrifuged and the supernatant was subjected solvent extraction in separation funnel and extracted using ethyl acetate. Equal volume (1:1) of supernatant and ethyl acetate were taken in a separation funnel and agitated for about 30 minutes. Solvent layer was separated and the supernatant was again extracted with ethyl acetate. The solvent layers were pooled and evaporated to dryness at 40°C [29]. The solvent extract was screened for biological activities.

Antibacterial activity of ethyl acetate extract of SRDP-07

The efficacy of ethyl acetate extract of SRDP-07 to inhibit bacteria was tested against two Gram positive bacteria viz., Staphylococcus aureus and Bacillus cereus and four Gram negative bacteria viz., Escherichia coli, Shigella flexneri, Klebsiella pneumoniae and Vibrio cholerae by Agar well diffusion method [43]. The test bacteria were inoculated into sterile nutrient broth (HiMedia, Mumbai) tubes and incubated for 24 hours at 37°C. The broth cultures of test bacteria were swabbed aseptically on sterile nutrient agar (HiMedia, Mumbai) plates with the help of sterile cotton swabs. Using a sterile cork borer, wells of 6mm diameter were punched in the inoculated plates and 100μl of ethyl acetate extract (5mg/ml of 10% DMSO), standard (Streptomycin, 1mg/ml) and DMSO (10%) were transferred into labelled wells. The plates were then incubated at 37oC for 24 hours and the zone of inhibition formed was measured. The experiment was repeated twice and the average value was recorded.

Insecticidal activity of ethyl acetate extract of SRDP-07

The insecticidal activity in terms of larvicidal effect of different concentrations (1 and 2mg/ml) of ethyl acetate extract was screened against II instar larvae of Aedes aegypti mosquito. Briefly, 20 larvae were transferred into beakers containing different concentrations of extract. A control was kept without adding extract. The larvicidal effect was determined by counting the number of dead larvae after 24 hours. Larvae that failed to move after probing with a needle in siphon or cervical region were identified as dead larvae. The experiment was repeated two times and average mortality (%) was noted [32].

Anthelmintic activity of ethyl acetate extract of SRDP-07

The anthelmintic effect of extract was screened against adult Indian earthworms (Pheretima posthuma). The worms were washed with normal saline (0.85%) to remove extraneous matter. In brief, 6 worms of equal size (6cm long) were transferred into normal saline containing standard (1%) and different concentrations of extract (1 and 2mg/ml of saline). The time taken for paralysis and death of worms was noted. Piperazine citrate was used as standard anthelmintic. Normal saline served as control. The experiment was repeated twice and average paralysis and death time was noted [31].

Antioxidant activity of ethyl acetate extract of SRDP-07

DPPH free radical scavenging activity

In order to determine radical scavenging efficacy of ethyl acetate extract, we have employed DPPH free radical scavenging assay [31]. In brief, 2ml of DPPH solution (0.002% in methanol) was mixed with 2ml of different concentrations (1- 100μg/ml) of extract and standard separately. The tubes were incubated in dark at room temperature for 30 minutes and the optical density was measured at 517 nm using UV-Vis spectrophotometer. The absorbance of the DPPH control was also noted. Ascorbic acid was used as reference standard. The scavenging activity of the extract was calculated using the formula: Scavenging activity (%) = [(A – B) / A] x 100, where A is absorbance of DPPH and B is absorbance of DPPH and extract/standard combination.

Ferric reducing activity

The reducing potential of ethyl acetate extract was determined by Ferric reducing assay. Here, different concentrations (1-100μg/ml) of extract and ascorbic acid (reference standard) in 1ml of methanol were mixed separately with 2.5ml of phosphate buffer (200 mM, pH 6.6) and 2.5 ml of 1% potassium ferricyanide. The tubes were incubated in water bath for 20 minutes at 50oC, cooled rapidly and mixed with 2.5ml of 10% trichloroacetic acid and 0.5 ml of 0.1% ferric chloride. The amount of iron (II)-ferricyanide complex formed was determined by measuring the formation of Perl’s Prussian blue at 700 nm after 10 minutes. An increase in absorbance on increase in concentration indicates increased reducing power [31].

UV absorption of ethyl acetate extract of SRDP-07

The crude bioactive extract was dissolved in ethyl acetate solvent and subjected to UV absorption studies. The absorption spectrum of ethyl acetate extract was determined in the UV region (200- 400nm) by using a UV-visible spectrophotometer (Shimadzu UV 2554) [44].

Results

A total of 9 actinomycete isolates (SRDP-01 to SRDP-09) were recovered from the soil sample by serial dilution-plating method. All the isolates were subjected for assessment of antagonistic property against a panel of 6 bacteria by cross streak method. Absence of growth of test bacteria or reduced growth of test bacteria in the vicinity of the growth of actinomycetes was considered as positive for antibacterial activity. All the isolates showed inhibition of at least one of the test bacteria. Isolates SRDP-05 and SRDP-08 inhibited only one among six test bacteria. Prominent inhibitory activity was demonstrated by the isolate SRDP-07. Table 1 shows the extent of inhibition of test bacteria.

Isolate Extent of inhibition of test bacteria
E.coli S.flexneri K.pneumoniae B.cereus V.cholerae S.aureus
SRDP-01 +++ ++ ++ + ++ +++
SRDP-02 + + ++ ++ + +++
SRDP-03 + + +++ + ++ +
SRDP-04 + + + + - +
SRDP-05 - - + - - -
SRDP-06 +++ + + + + +
SRDP-07 +++ ++++ ++++ ++ ++++ ++++
SRDP-08 + - - - - -
SRDP-09 + + - ++ - -

Table 1: Primary screening of actinomycete isolates for antibacterial activity

The growth characteristics of the isolate SRDP-07 was studied on four media viz., SCA, ISSA, AIA and TYEA. The color of both substrate and aerial mycelium was yellow on SCA and ISSA. On TYEA, the substrate and aerial mycelium was yellow and grey respectively. Both substrate and aerial mycelia were grey on AIA. Diffusible pigments were not observed in any of these media (Table 2).

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

Table 2: Cultural characteristics of isolate SRDP-07 on various media

The isolate SRDP-07 was Gram positive and nonacid fast. The spore arrangement was found to be closed spiral type. Biochemically, the isolate was found positive for amylase, cellulose, catalase and citrase. The isolate was not found to produce gelatinase, caseinase, oxidase and H2S. The isolate fermented glucose, fructose and galactose with acid production and maltose and lactose with alkali production. No gas production was observed (Table 3; Figure 1).

Test Characteristic
Gram’s staining Gram positive
Acid fast staining Non-acid fast
Spore arrangement Closed spiral
Starch hydrolysis +
Cellulose hydrolysis +
Gelatin liquefaction -
Casein hydrolysis -
Catalase +
Oxidase -
Citrate test +
H2S production -
Fermentation Glucose- Acid Fructose- Acid Galactose- Acid Maltose- Alkali Lactose- Alkali

Table 3: Microscopic, staining and biochemical characteristics of the isolate SRDP-07

International-Journal-spore-arrangement

Figure 1: Isolate SRDP-07 (Culture and spore arrangement)

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 extract was higher in case of Gram positive bacteria than Gram negative bacteria. Among the test bacteria, S. aureus and V. cholerae were highly susceptible Gram positive and Gram negative bacteria respectively. The extract was least effective against K. pneumoniae. Inhibition of test bacteria by standard antibiotic was higher than that of ethyl acetate extract. DMSO did not cause inhibition of test bacteria (Table 4). UV absorption maximum of ethyl acetate extract showed a single peak at 214nm.

Test bacteria Zone of inhibition in cm
Extract Standard DMSO
E. coli 1.2 2.8 0.0
S. flexneri 1.6 3.6 0.0
K. pneumoniae 1.0 3.4 0.0
B. cereus 2.3 3.8 0.0
V. cholerae 2.0 3.5 0.0
S .aureus 2.4 4.1 0.0

Table 4: Antibacterial activity of ethyl acetate extract of isolate SRDP-07

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 study. At 2mg/ml concentration, the mortality of larvae was 100% (Figure 2).

International-Journal-ethyl-acetate

Figure 2: Larvicidal activity of ethyl acetate extract of isolate SRDP-07

Anthelmintic effect of ethyl acetate extract and piperazine citrate was determined on the basis of time taken for causing paralysis and death of adult Indian earthworms. The anthelmintic effect was observed as loss of motility and no response to external stimuli which eventually progressed to death of worms. The extract caused dose dependent paralysis and death of worms. Standard anthelmintic piperazine citrate exhibited stronger anthelmintic activity than that of ethyl acetate extract (Table 5).

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 converting into DPPHH and the activity was found to be dose dependent. However, the scavenging potential of extract was found to be much lesser when compared with reference standard i.e., ascorbic acid. Lower concentrations of extract viz., 1 and 2.5μg/ml did not shown scavenging of free radicals (Figure 3).

International-Journal-radical-scavenging

Figure 3: DPPH radical scavenging activity of ethyl acetate extract of isolate SRDP-07

The reducing potential of ethyl acetate extract and ascorbic acid was determined by employing ferric reducing assay in which the reduction of Fe3+ to Fe2+ was investigated in the presence of different concentrations of extract and ascorbic acid. The absorbance at 700nm was found to increase with the increase in concentration of extract indicating reducing potential of extract. The reducing potential of extract was lesser when compared with the reference standard (Figure 4).

International-Journal-Ferric-reducing

Figure 4: Ferric reducing activity of ethyl acetate extract of isolate SRDP-07

Discussion

Natural products are the most important source of new compounds having chemical diversity still unmatched by combinatorial chemistry approaches. These natural compounds have been used in the synthesis of several drugs. It is well known that microorganisms, in particular 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 positive, spore forming, aerobic actinomycetes having high DNA G-C% content (69~78 mol%) and classified in the family Streptomycetaceae on the basis of morphological and cell-wall chemotaxonomic characters. The members produce extensively branched substrate mycelium and aerial hyphae. With more than 500 validly described species and subspecies, the taxon currently contains the largest number of species in the domain Bacteria [27].

Soil is one of the rich reservoirs of microorganisms. Rhizosphere is the fraction of soil which is in vicinity to plant roots and is thought to be of great importance to plant health and soil fertility. The root exudates of plants stimulate the growth of microbial populations in soil and hence, microbial activity is greatest in rhizosphere region [48]. Actinomycetes forms a key part of soil microbiota. In general, Streptomyces species are saprophytic and are commonly associated with soils, where they significantly contribute to the turnover of complex biopolymers such as cellulose, lignin etc. and antibiotics [49]. It has been found that about 90% of soil actinomycetes are reported to be Streptomyces species [22,50]. In this study, we have isolated nine actinomycetes from the rhizosphere soil sample of Thirthahalli, Shivamogga, Karnataka. Preliminary screening for antibacterial activity, carried out by cross streak method, showed varied antagonistic potential of the isolates. Cross streak method is used to determine antagonstic potential of actinomycetes. It has become one of the routinely used methods and has been employed by several authors to study antimicrobial potential of actinomycete isolates [35,37,40,44,51,52]. One isolate, designated SRDP-07 displayed marked inhibition of test bacteria and hence, selected for further characterization to genus level and to determine biological activities.

Morphology plays an important role in distinguishing Streptomyces from other sporing actinomycetes and in the characterization of Streptomycete species. The life cycle of a Streptomycete provides 3 features for microscopic characterization viz., vegetative mycelium, aerial mycelium baring chains of spores and the characteristics of spores themselves. The latter two features produce most diagnostic information [53,54]. Information on cultural features and characteristic spore arrangement together with biochemical properties assists classification of actinomycetes as members of the genus Streptomyces. Saadoun et al. [55] recovered nine different isolates of aquatic actinomycetes and identified them as Streptomyces spp. on the basis of morphological and cultural characteristics. Singh et al. [56] isolated two strains of Streptomyces species and identified them as Streptomyces albovinaceous based on cellular morphology and physiology. In a study by Savic et al. [57], morphological and phenotypic properties of actinomycete isolate MS405T were consistent with its classification as a Streptomyces strain. Similarly, other studies by Rifaat et al. [58], Sahin et al. [59] and Moncheva et al. [60] have assigned the certain actinomycetes strains under the genus Streptomyces on the basis of characteristics such as cultural, microscopic and biochemical features. In the present study, the cultural and microscopic characteristics of the isolate SRDP-07 were consistent with its classification as a member of the genus Streptomyces.

Infectious diseases are caused by a number of bacteria, viruses, parasites and fungi which devastated mankind before the development of chemotherapeutic agents mainly antibiotics. The discovery of antibiotics and their subsequent use has eradicated a lot of infections. However, traditional antibacterial therapy using antibiotics from microbial sources or their synthetic analogues is facing problems due to development of resistance in existing agents to antimicrobials. Staphylococcus aureus is one of the first pathogens that became resistant to almost all known antibiotics posing a global threat. Vancomycin resistant enterococci, multidrug resistant tuberculosis, antibiotic resistant E. coli, P. aeruginosa are among other antibiotic resistant bacteria against which most of the antibiotics are not effective. Moreover, these bacteria have the tendency to transmit the resistance gene and this has become a serious issue in the field of medicine [61-64]. The need for new antibiotics is increasing day by day due to the development and spread of antibioticresistant pathogens which cause life-threatening infections and patient sensitivity [49,65]. Actinomycetes have provided and continue to provide a number of antibiotics that are clinically relevant in the treatment of various diseases. They have the capability to produce a variety of compounds having diverse pharmacological activities [66]. Actinomycetes have shown to produce metabolites that are active against antibiotic resistant bacteria [24,67]. In the present study, the ethyl acetate extract of SRDP-07 showed marked inhibition of test bacteria. It was found that Gram positive bacteria have shown to be highly susceptible than Gram negative bacteria. Similar results were observed in the earlier studies [68-71]. The low antibacterial activity of ethyl acetate extract against the gram negative bacteria could be ascribed to the presence of an outer membrane that possess hydrophilic polysaccharides chains and forms an additional barrier for extract as well as antibiotics [72,73]. UV spectral studies of ethyl acetate extract of isolate SRDP-07 showed a single peak with absorbance maximum at 214nm. Similar absorption band was observed in an early study by Ezra et al. [74] for coronamycins, peptide antibiotics produced by a verticillate Streptomyces sp. (MSU-2110).

Mosquitoes have a predominant role in the transmission of several diseases such as malaria, yellow fever, dengue fever, filariasis, and others. These mosquitoes spread more diseases than any other group of arthropods and also cause allergic responses that include local skin and systemic reactions [75,76,77]. Aedes aegypti is responsible for transmission of one of the serious arboviral diseases dengue. The control measures taken against arthropod-borne diseases target larval stages and adult mosquitoes. Control of the larvae of the mosquito is one of the widely employed strategies and is frequently done by the application of synthetic insecticides and insect growth regulators. However, the raising cost, possible toxicity hazards and insects showing resistance highlighted search for the development of alternatives for mosquito control [75,78]. Actinomycetes are shown to be promising sources of bioactive metabolites displaying potent insecticidal activity [32,35,79-82]. In the present study, we determined insecticidal activity of ethyl acetate extract of SRDP-07 against 2nd instar larvae of A. aegypti. The extract displayed a dose dependent mortality of larvae. Similar results have been noticed in earlier studies. The butanol extracts of two Streptomyces species from Agumbe region produced dose dependent mortality of 2nd instar larvae of A. aegypti [32]. In another study, the crude extract of a Streptomyces species isolated from Agumbe region of Western ghats of Karnataka displayed a dose dependent mortality of 3rd instar larvae of A. aegypti [35].

Helminthic infections are one of the major diseases worldwide in particular tropical countries. It is the major cause of morbidity and is often fatal in extreme conditions. Parasitic worms also infect livestock and crops, affecting food production resulting in low economy. Many factors influence susceptibility of an individual such as lack of sanitation and supply of pure water together with poverty and illiteracy. Helminthic infections contribute to the prevalence of malnutrition, anemia, eosinophilia, and pneumonia. Anthelmintic drugs that expel parasitic worms from the body have some major drawbacks such as resistance development in gastro-intestinal helminthes, high cost, adverse effects etc. This situation led to the discovery and development of anthelmintic agents from natural sources [83-86]. Actinomycetes have shown to possess marked anthelmintic activity in terms of inhibition of plant and animal parasites [12,31,87,88,89]. In this study, the anthelmintic activity of ethyl acetate extract of SRDP-07 was determined using adult Indian earthworms due to their ready availability and anatomical and physiological resemblance to the human intestinal roundworm parasite [84,90]. The standard anthelmintic piperazine citrate showed higher activity than the extract. The predominant effect of piperazine citrate on the worm is to cause a flaccid paralysis that result in expulsion of the worm [83,84]. The extract of SRDP-07 not only demonstrated this property but also killed the worms, however, the time taken for this was much higher when compared with standard. In an earlier study, Kekuda et al. [31] showed dose dependent mortality and death of earthworms by butanol extracts of two Streptomyces species isolated from western ghat soil of Agumbe, Karnataka.

A well maintained balance exists between antioxidant defence mechanisms and generation of free radicals in a normal healthy individual. During oxidative stress, this balance, however, shift towards the excessive production of free radicals or deficit in antioxidant defence mechanism. This oxidative stress is implicated in over hundreds of pathophysiological conditions such as diabetes, cardiovascular diseases, neurological disorders, cancer, aging etc. Antioxidant is a substance that significantly inhibits or delay oxidative processes. Endogenous antioxidants such as ascorbic acid, tocopherols, glutathione, uric acid, thiols etc., and antioxidant enzymes such as superoxide dismutase and catalase protect the body against oxidative stress. However, in pathophysiological conditions, there is additional requirement for antioxidants [91-95]. Strong restrictions have been placed on the use of synthetic antioxidants such as BHT, BHA, gallates due to their suspected carcinogenic potential [96]. This led to an increasing interest in natural products having antioxidant properties. Actinomycetes have been shown to be promising sources of antioxidants. A number of studies have been carried out on the antioxidant efficacy of extracts and purified compounds from actinomycetes [14,31,97-102].

DPPH free radical scavenging assay is one of the widely used protocol to arrive the radical scavenging ability of compounds. DPPH is a stable organic free radical having the absorption maximum around 515-528nm. On accepting an electron or hydrogen atom it becomes a stable diamagnetic molecule. The effect of antioxidants on scavenging DPPH radical is due to their hydrogen donating ability. In this assay, the compounds (antioxidants) reduce the purple colored DPPH radical to a yellow colored compound diphenylpicrylhydrazine and the extent of reaction depends on the hydrogen donating ability of the antioxidants [103,104,105]. In the present study, the absorption of DPPH in the presence of various concentrations of ethyl acetate extract of SRDP-07 was measured at 517nm. It was observed that the radical scavenging activities of the extract increased with increasing concentrations. Although the scavenging abilities of extract were much lesser than that of ascorbic acid, it was evident that the extract showed hydrogen donating ability and the extract could serve as free radical scavengers, acting possibly as primary antioxidants [104]. The results obtained are in justification with earlier studies which also reveal weaker scavenging activity of extracts [31,36,38].

Ferric reducing assay is one of the commonly used antioxidant assays which measures total antioxidant activity. The assay is performed in order to measure the reducing power of the compounds. In the present study, we investigated the Fe+3/Fe+2transformation in the presence of ethyl acetate extract of SRDP-07. In this assay, the reductants (antioxidants) would cause the reduction of Fe+3 to Fe+2 by donating an electron. The amount of Fe+2complex formed can be monitored by measuring the formation of Perl’s Prussian blue at 700nm. Increasing absorbance at 700nm indicates an increase in reductive ability [104]. The reducing capacity of a compound may serve as a significant indicator of its potential antioxidant activity [106]. In our study, the reducing power of the extract also increased with the increase of its concentration. However, the reducing potential of extract was lesser when compared with reference standard. Although the extract was found to possess less reducing power, it is evident that the extract possesses reductive potential and could serve as electron donors, terminating the radical chain reactions [104]. Similar results have been observed in previous studies of Kekuda et al. [31] and Manasa et al. [36] where the actinomycete extracts have displayed weaker reducing potential.

Conclusion

In the present study, we screened the bioefficacies of an actinomycete isolate recovered from a soil sample collected from Western Ghat region Karnataka, India. One isolate Streptomyces species SRDP-07 showed good antibacterial and insecticidal activity. The results of the present study highlighted that the soils of Thirthahalli are reservoirs of potent actinomycetes and hence further screening can be fruitful for isolation of bioactive actinomycetes which might be exploited in industries for production of novel metabolites.

Acknowledgements

The authors express thanks to Head, Department of Microbiology, Principal, SRNMN College of Applied Sciences, Shivamogga and NES, Shivamogga for giving support to conduct work.

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