Investigation on pharmacological activities of secondary metabolite extracted from a mangrove associated actinobacterium Streptomyces olivaceus (MSU3)

Author’s Accepted Manuscript INVESTIGATION ON PHARMACOLOGICAL ACTIVITIES OF SECONDARY METABOLITE EXTRACTED FROM A MANGROVE ASSOCIATED ACTINOBACTERIUM STREPTOMYCES OLIVACEUS (MSU3) M. Sanjivkumar, D. Ramesh Babu, A.M. Suganya, T. Silambarasan, R. Balagurunathan, G. Immanuel

PII: DOI: Reference:

www.elsevier.com/locate/bab

S1878-8181(16)30036-6 http://dx.doi.org/10.1016/j.bcab.2016.03.001 BCAB369

To appear in: Biocatalysis and Agricultural Biotechnology Received date: 28 November 2015 Accepted date: 1 March 2016 Cite this article as: M. Sanjivkumar, D. Ramesh Babu, A.M. Suganya, T. Silambarasan, R. Balagurunathan and G. Immanuel, INVESTIGATION ON PHARMACOLOGICAL ACTIVITIES OF SECONDARY METABOLITE EXTRACTED FROM A MANGROVE ASSOCIATED ACTINOBACTERIUM STREPTOMYCES OLIVACEUS (MSU3), Biocatalysis and Agricultural Biotechnology, http://dx.doi.org/10.1016/j.bcab.2016.03.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

INVESTIGATION ON PHARMACOLOGICAL ACTIVITIES OF SECONDARY METABOLITE EXTRACTED FROM A MANGROVE ASSOCIATED ACTINOBACTERIUM STREPTOMYCES OLIVACEUS (MSU3) Sanjivkumar1, M., Ramesh Babu1, D., Suganya1, A.M., Silambarasan2, T., Balagurunathan2, R., Immanuel, G.* 1

MNP-Laboratory, Centre for Marine science and Technology, M.S. University, Rajakkamangalam- 629502, Tamilnadu, India. 2

Department of Microbiology, Periyar University, Periyar Palkalai Nagar, Salem – 636 011, Tamil Nadu, India. *Corresponding author. email: [email protected]

Abstract The present study was undertaken to evaluate pharmacological activities of secondary metabolite of Streptomyces olivaceus (MSU3) isolated from the sediment sample of a mangrove ecosystem. The isolated strain was screened for its preliminary antagonistic property against various clinical pathogens and its secondary metabolite was extracted by using ethyl acetate. The 30µl concentration of the crude extract of the isolate expressed the maximum zone of inhibition of 27±2.44mm against the bacterial pathogen Streptococcus mutant with the MIC and MBC values of 0.625 and ≤ 5µg/ml concentrations, respectively. Further different concentrations of extract were tested for in-vitro antioxidant, antiinflammatory and in-vivo cytotoxicity studies. It expressed the maximum percentage of in-vitro total antioxidant activity of 87%, DPPH scavenging activity of 62.06%, reducing power effect of 32.51%, hydroxyl radical scavenging activity of 47.99 % and nitric oxide activity of 33.20% at 100µg/ml concentration respectively. Further the extract exhibited 96.63% of inhibition of in-vitro antiinflammatory activity, 49.60 %

of total hemolytic activity and also has 42.11% of total phenolic content at respective concentration of 500µg/ml. The cytotoxic effect of the crude extract was analyzed by MTT assay on MCF-7 and HT-29 cell lines and the cell viabilities were observed as 24.00 and 39.17% at 250µg/ml concentration with the respective IC50 values of 88.26 and 104.81µg/ml. From the results, it is evident that the ethyl acetate crude extract of S. olivaceus (MSU3) has potent antimicrobial, antioxidant, antiinflammatory and cytotoxic activity and suggested that the isolated strain could be a candidate for the nature resource of pharmaceutical. Keywords: Actinobacteria; Streptomyces sp; crude extract; DPPH; anticancer activity.

1. Introduction Free radicals are highly reactive particles produced by the body either as a by-product during normal biochemical process like enzyme activation. Under normal condition, the body is capable of neutralizing these particles and maintains them at a safe minimum level. Excess or abnormal formation of free radicals is potentially dangerous and can lead to oxidation and even irreversible damage of body tissues (Sato et al., 1996; Badmus et al., 2011). An antioxidant acts as a free radical scavenger and neutralizes these reactive particles by binding to their free electrons. By destroying free radicals, antioxidants help to detoxify and protect the vital body tissues and organs. Cells have a comprehensive array of antioxidant defense mechanisms to reduce free radical formation or limit their damaging effect (Sato et al.,1996). These mechanisms are not sufficient when the balance shifts to the side of free radical generation, thus body requires antioxidant supplements to reduce oxidative damage. Antioxidants are molecules capable of preventing oxidative damage. Recent investigation suggested that antioxidant capacity of putative antioxidants can be attributed to various mechanisms such as prevention of chain initiation, binding of transition metal ion catalysts, decomposition of peroxides, prevention of continued hydrogen abstraction and radical scavenging in body cells alleviating lipid, protein oxidation and may reduce potential mutations (Sheikh et al., 2009). Therefore the antioxidants help to prevent degenerative diseases and other pathologies (Gautham and Onkarappa, 2013). Basically there are two types of antioxidants, as natural and synthetic antioxidants. Many studies reported that the synthetic antioxidants are suspected to have carcinogenic probability; therefore, natural antioxidants are the most preferred type (Mousumi and Dayanand, 2013). Antioxidants

from natural sources play a paramount role in helping endogenous antioxidants to neutralize oxidative stress. Antioxidants therefore are used to reverse the harmful effects of the free radicals by scavenging the free radicals and detoxifying the physiological system (Farnet et al., 2005; Gautham and Onkarappa, 2013). Microbial based natural products are notable not only for their potent therapeutic activities, but also for the fact that they frequently possess the desirable pharmacokinetic properties required for clinical development; many microbial natural products reach market without any chemical modifications, a testimony to the remarkable ability of microorganisms to produce drug like small molecules (Farnet et al., 2005). Members of the actinomycetes genus, especially Streptomyces sp. have been recognized as prolific producer of useful bioactive compounds with broad spectrum of activities that produce about 75% of commercially and medically useful antibiotics (Moncheva, 2002). Among various genera of actinomycetes, Streptomyces, Saccharopolyspora, Amycolatopsis, Micromonospora and Actinoplanes are the major producers of secondary metabolites (Karthik et al., 2011). These secondary metabolites produced by actinomycetes have a broad spectrum of biological activities (Karthik et al., 2013). Streptomycetes and related actinomycetes continued to be useful sources of novel secondary metabolites with a range of biological activities that may ultimately find applications as antiinfective, anti-cancer agents or other pharmaceutically useful compounds (Forar et al., 2006). The metabolites like benthocyanins and carquinostatin produced by S. prunicolor and S. exfoliates showed maximum antioxidant activity (Karthik et al., 2013). S. chibaensis AUBN1/7 (Gorajana et al., 2007) and Streptomyces spp. KR-5 exhibited cytotoxic activity against the growth of human breast cancer cell line (Sateesh et al., 2011). Whereas, Streptomyces spp. SRDP-H03 (Rakesh et al., 2013) and BI244 (Kiruthika et al., 2013) exhibited both antiinflammatory and antioxidant activities. Considering the importance of the above, the present study was focused on the antimicrobial, antioxidant, anti-inflammatory and cytotoxic properties of secondary metabolite extracted from a novel actinobacterium Streptomyces olivaceus (MSU3) isolated from the rhizosphere sediment samples of Manakudy estuary, Kanyakumari District, South India. 2. Materials and Methods 2.1. Sample collection

The culture S. olivaceus (MSU3) was isolated from rhizosphere soil of mangrove plant Rhizophora mucronata of Manakudy estuary, Kanyakumari District, South India and it was identified and confirmed up to species level through 16S rRNA partial sequencing method described by Nathan et al. (2004). The isolated strain was sub-cultured in sterile ISP2 media (Yeast extract-Malt extract agar) and incubated at 28°C for 7 days. After incubation, the isolate was kept in MNP laboratory at Centre for Marine Science and Technology, M.S. University, as reference strain and it was used as a working strain for further studies.

2.2. Test organisms The clinical pathogens used in this study were Streptococcus mutant (NCIM2063), Escherichia

coli

(ATCC25922), Klebsiella pneumoniae

(ATCC10273),

Streptococcus

pneumoniae (ATCC49619) and Vibrio cholerae (MTCC3905) obtained from MNP laboratory of Centre for Marine Science and Technology, M.S. University. 2.3. Preliminary screening of antagonistic activity of S. olivaceus (MSU3) through cross streak method The antagonistic activity of the selected actinobacterial strain S. olivaceus (MSU3) was screened by using cross streak method (Devi et al., 2012; Alexander et al., 1977). The isolated strain was streaked as a straight line at the middle of the petriplate containing Modified nutrient glucose agar (NA+1% Glucose) medium (MNGA). After inoculation, the plate was incubated at 28°C for 7 days for their growth. After incubation, 24h old pathogenic bacterial strains were individually inoculated perpendicular to the growth line of selected actinobacterium in the same plate. The cross streaked plate was further incubated at 37°C for 24 - 48h and the extent of inhibition was observed. The absence of growth or a less dense growth of test bacteria near the actinobacterial isolate was considered positive for production and secretion of antibacterial metabolite by the isolate.

2.4. Extraction of secondary metabolite from S. olivaceus (MSU3)

The secondary metabolite of S. olivaceus (MSU3) was extracted by using the method described by Zhong et al. (2011). The spore suspension of the actinobacterium S. olivaceus (MSU3) was inoculated into the ISP2 agar plates and incubated at 28°C for 7 days. Then the aerial mycelia were scraped and soaked in 80% ethyl acetate for 24h. It was centrifuged (4000 rpm, 10 min) and extracted twice more. The supernatant was dried and dissolved with distilled water and then it was extracted with three half volume of ethyl acetate. The crude ethyl acetate extract was dried in vacuum at 40°C and the dried powder was used for further studies.

2.5. Secondary screening of antagonistic activity of the crude extract of S. olivaceus (MSU3) through agar well diffusion method Antibacterial activity of the crude extract of S. olivaceus (MSU3) was determined by agar well diffusion method (Kekuda et al., 2012; Rakesh et al., 2013). Wells of 5mm diameter were made using sterilized cork borer on Mueller Hinton Agar. 0.1ml each of inoculum of test pathogens were spread on the plates and different concentrations (10 to 30μl) of the crude extract was tested for their activity against the test pathogens and chloramphenicol (25μg/ml) was used as positive control. Plates were incubated at 37°C for 24h and the zone of inhibition was measured. 2.6. Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) The Minimum Inhibitory Concentration (MIC) of the extract against the individual test pathogens was determined by using the method of Akinjogunla et al. (2010) with slight modification. 2ml of sterile nutrient broth with different concentrations of the extract (0.313, 0.625, 1.25, 2.5, 5, 10 and 20µg/ml) were taken in test tubes and to this 0.1ml each of test pathogens were added. Then the test tubes were incubated at 37°C for 24h. Similar test tube sets containing chloramphenicol at the concentration of 25μg/ml were used as control. After incubation, the tubes were examined for microbial growth by observing the turbidity. The tubes containing the least concentration of extract showing no visible sign of growth was considered as the minimum inhibitory concentration. To determine the MBC of the extract, 0.1ml each of the culture broth was collected and inoculated onto sterile nutrient agar. The plates were then

incubated at 37°C for 24h. After incubation, the lowest concentration yielding negative subculture was considered as Minimum Bactericidal Concentration (MBC). Both MIC and MBC for the test bacteria were determined in triplicate assays.

2.7. Determination of total phenolic content The total phenolic content of the crude extract of S. olivaceus (MSU3) was determined by employing Folin–Ciocalteu Reagent (FCR) method (Slinkard and Singelton, 1977). 0.5ml of crude extract was taken in a test tube containing 2ml of Folin-Ciocalteu reagent with 4ml of sodium carbonate solution (75% w/v). Then the reaction mixture was incubated at room temperature for 30min with intermittent shaking for colour development. The absorbance of the resulting blue colour was measured at 765nm using a UV spectrophotometer (UV2301II model). The amount of total phenolic content was expressed as mg/g gallicacid equivalent (GAE) as a standard.

2.8. Total hemolytic activity of crude extract of S. olivaceus (MSU3)

Hemolytic activity was performed according to Fischer et al. (2003) with slight modification. The hemolytic activity of the crude extract of S. olivaceus (MSU3) on erythrocytes was tested by using the washed erythrocytes (RBCs) from human blood under in-vitro condition in 96-well plates. Each well received 100µl of 0.85% NaCl solution containing 10mM CaCl2. The first well served as negative control contained only distilled water and in the second well, 100µl of crude extract of various concentrations (100 - 500µg/ml) were added. The last well served as positive control containing 20µl of 0.1% Triton X-100 in 0.85% saline. Then, each well received 100µl of a 2% suspension of human erythrocytes in 0.85% saline containing 10 mM CaCl2 and incubated for 30 min at room temperature. After incubation the test solution was centrifuged and the supernatant was used to measure the absorbance of the liberated hemoglobin at 540 nm. The average value was calculated from triplicate assay. The level on percentage of haemolysis by the extract was calculated according to the following formula: Haemolysis (%) = [(At – An) (Ac – An)] × 100

At is the absorbance of test sample

An is the absorbance of saline control Ac is the absorbance of distilled water control

2.9. Evaluation of in-vitro anti-inflammatory activity of crude extract of S. olivaceus (MSU3) Anti-inflammatory activity of the crude extract of S. olivaceus (MSU3) was evaluated by protein denaturation method as described by Kamleshiya et al. (2010) with slight modification. Diclofenacsodium, a powerful non steroidal anti-inflammatory drug was used as a standard drug. The reaction mixture contain 2ml of different concentrations of actinobacterium extract (100 – 500µg/ml) or standard diclofenac sodium (100 and 200µg/ml) and 2.8ml of phosphate buffered saline (pH 6.4) were mixed with 2 ml of egg albumin (from fresh hen’s egg) and incubated at 27 ± 1°C for 15min. Denaturation was induced by keeping the reaction mixture at 70°C in a water bath for 10 min. After cooling, the absorbance was measured at 660 nm by using double distilled water as blank. The percentage inhibition of protein denaturation was calculated by the following formula: Inhibition (%) = [(At - Ac) Ac] ×100 Where, At = absorbance of test sample; Ac = absorbance of control

2.10. Antioxidant assays of crude extract of S. olivaceus (MSU3)

The crude extract of S. olivaceus (MSU3) was tested for its antioxidant properties by using five different assays (in-vitro antioxidant, DPPH scavenging, total reducing power, hydroxyl radical scavenging and nitric oxide assays). Different concentrations of the extract such as 12.5, 25, 50 and 100µg/ml were used for the assays. Standards were also used in their respective concentrations.

2.10.1. In-vitro total antioxidant activity

0.1ml each of different concentrations (12.5, 25, 50, 75 and 100µg/ml) of crude extract were taken in test tubes and were mixed with1.9ml of reagent solution containing 0.6 M sulfuric acid, 28mM sodium phosphate and 4 mM ammonium molybdate. The tubes were incubated at

95°C for 90min under water bath. Further the absorbance of all the sample mixtures was measured at 695nm. Total antioxidant activity was calculated using standard graph of ascorbic acid and denoted as the equivalents of ascorbic acid in µg/ml of dry weight of extract (Raghava Rao and Raghava Rao, 2013).

2.10.2. DPPH scavenging assay The radical scavenging activity of crude extract was determined by using DPPH assay (Chang et al. 2001). Different concentrations (12.5, 25, 50 and 100 µg/ml) of crude extract were taken in test tubes, made up to 40µl with DMSO and 2.96ml of DPPH (0.1mM) solution was added in test tubes. Then the reaction mixtures were incubated in dark condition at room temperature for 20 min. After incubation, the absorbance of the mixtures was read at 517 nm. 3.0 ml of DPPH was taken simultaneously as control and the IC50 (concentration providing 50% inhibition) was estimated graphically using a calibration curve versus percentage of inhibition. The DPPH activity was calculated by the following formula: Inhibition (%) = [(control – test) control] ×100 2.10.3. Total reducing Power

The reducing power of crude extract was determined by the method of Yen and Duh (1993) with slight modification. Different concentrations (12.5, 25, 50 and100µg/ml) of crude extract were taken in test tubes, mixed with 2.5 ml of phosphate buffer (0.2M, pH 6.6) and 2.5 ml of 1% potassium ferric cyanide. The test tubes containing reagents were boiled for 20 min at 50°C. After that, 2.5ml of TCA was added and centrifuged for 10min at 2000rpm. The supernatant was collected, to this 1ml of distilled water was added, followed by 250µl of 0.1% ferric chloride was added. Finally the reaction mixture was measured at 700 nm and the reducing power of the extract was calculated by the formula Reducing power (%) = [(Atest Ablank) –1]×100

2.10.4. Hydroxyl radical scavenging activity

The hydroxyl radical scavenging activity of the extract was determined by the method of Parejo et al. (2000) with minor change. All solutions were prepared freshly (Stock solutions of EDTA (1mM), FeCl3 (10mM), ascorbic acid (1mM), H2O2 (10 mM) and deoxyribose (10mM) were prepared in distilled deionized water). The assay was performed by adding 0.1ml of EDTA, 0.01ml of FeCl3, 0.1ml of H2O2, 0.36ml of deoxyribose and 1.0ml each of different concentrations of extract (12.5, 25, 50 and 100µg/ml) dissolved in distilled water. To this 0.33 ml of phosphate buffer (50mM, pH7.4) and 0.1ml of ascorbic acid were also added simultaneously. The mixture was then incubated at 37°C for 1h. About 1.0ml portion of the incubated mixture was mixed with 1.0 ml of (10 %) TCA and 1.0 ml of (0.5 %) TBA to develop pink colour and the colour intensity was read at 532nm. The hydroxyl radical scavenging activity of the extract was reported as the percentage inhibition of doxyribose degradation and it was calculated according to the formula Inhibition (%) = [ABS (control)-ABS (standard) / ABS (control)] × 100 ABS (control) – Absorbance of the control ABS (standard) – Absorbance of the extract 2.10.5. Nitric oxide assay The nitric oxide assay was estimated by the method of Green et al. (1982) with minor modification. 0.5 ml each of different concentrations of extract (12.5, 25, 50 and100 µg/ml) were mixed with 0.1 ml of sulphosalicylic acid taken in test tubes and vortexed well for 30 min. The samples were centrifuged at 5,000 rpm for 15 min. After centrifugation, 200 μl of supernatant solution was taken and it was mixed with 30 μl of 10 % NaOH, followed by 300 μl of Tris-HCl buffer and mixed well. To this, 530 μl of Griess reagent was added and incubated in the dark for 10 –15 min. Then the reaction mixture was read at 546nm against a Griess reagent blank. Sodium nitrite solution was used as the standard and the nitric oxide assay was calculated by using the formula Inhibition (%) = [(control – test) control] ×100

2.11. Determination of cytotoxic properties of crude extract of S. olivaceus (MSU3) 2.11.1. Collection of cell-lines

In this study, MCF-7 (breast) and HT – 29 (colon) carcinoma cell lines were obtained from National Centre for Cell Science, Pune, India and these two cell lines were further maintained in Cell Culture Laboratory, Centre for Biological Sciences, Pondicherry for cytotoxic study 2.11.2. Cytotoxic activity The cytotoxicity of different concentrations (10, 25, 50, 100 and 250µg/ml) of ethyl acetate extract of S. olivaceus (MSU) against human breast adenocarcinoma (MCF-7) and colon carcinoma (HT-29) cell lines were tested by using MTT assay method described by Mosmann (1983). Cyclophosphamide (20µg/ml) was used as the positive control. The tests were done in triplicates to ensure accuracy.

2.12. Statistical analysis

The data obtained in the present study were expressed as Mean ± SD and were analyzed using one-way ANOVA test and subsequently conducted post hoc multiple comparison with SNK test at 5 % level of significance using computer software STATISTICA 6.0 (Statosoft, Bedford, UK).

3. Results The working strain of this study S. olivaceus (MSU3) was isolated from the rhizosphere soil of mangrove environment of Manakudy estuary and it was identified and confirmed by using 16S rRNA partial sequencing method. Further it was deposited in the gene bank with the accession number KM212958. The strain was appeared as powdery, grey coloured colony with smooth and spiral spore chain (Figure 1).The results on the antagonistic and antioxidant properties of the secondary metabolite of the working strain are summarized below. 3.1. Preliminary screening of antagonistic activity of S. olivaceus (MSU3) through cross streak method The preliminary screening study on antagonistic activity of the working strain S. olivaceus (MSU3) against the pathogenic bacteria was performed by using Modified nutrient

agar. After incubation, the plate was observed. The isolate showed antagonistic activity against all the five tested pathogens (Figure 2). From this strain, the secondary metabolite, the crude ethyl acetate extract was prepared. This crude extract was used for further studies. 3.2. Secondary screening of antagonistic activity of crude extract of S.olivaceus (MSU3) through agar-well diffusion method The antagonistic activity of the crude extract of S. olivaceus (MSU3) was determined by agar-well diffusion method using Muller Hinton agar. After incubation, the plates were observed and the zone of inhibition was recorded (Table 1). The result indicated that the crude extract of S. olivaceus (MSU3) exhibited the maximum (27 ± 2.05mm) zone of inhibition against S. mutant at 30µl concentration, whereas the same organism displayed the minimum inhibition of 20 ± 1.86mm at 10µl concentration (Figure 3). At the same time, the extract comparatively inhibited minimum level (12 to19mm) against V. cholerae. The remaining test pathogens like K. pneumoniae exhibited 17 to 23mm, S. pneumoniae showed 16 to 21mm and E. coli displayed 21 to 25mm at 10 to 30µl concentrations respectively. However, the positive control chloramphenicol exhibited 2 to 12 mm zone of inhibition against all the tested pathogens. 3.3. Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) The ethyl acetate crude extract of S. olivaceus (MSU3) showed maximum activity against S. mutant with the MIC and MBC values of 0.625 and ≤ 5µg/ml respectively, when compared to chloramphenicol (25µg/ml). It also showed the MIC and MBC values of 2.5 and >5µg/ml against E. coli, 2.5 and 5µg/ml against S. pneumoniae, 1.25 and 2.5µg/ml each against K. pneumoniae and V. cholerae respectively (Table 2). 3.4. Determination of total phenolic content The total phenolic content of ethyl acetate crude extract of S. olivaceus (MSU3) was determined as 0.0421 mg GAE/g of dry weight of extract. 3.5. Total hemolytic activity of crude extract of S. olivaceus (MSU3) The total hemolytic activity of different concentrations of ethyl acetate crude extract of S. olivaceus (MSU3) was determined by using human erythrocyte. The result showed with highest

level of inhibition of 49.60 % at 500µg/ml concentration, but the lowest level of inhibition of 11.66% was noted at 100µg/ml concentration. Here the total hemolytic activity of the crude extract was gradually increased from100µg/ml to 500µg/ml concentration of extract (Fig. 4). 3.6. Evaluation of in-vitro anti-inflammatory activityof crude extract of S. olivaceus (MSU3)

The anti-inflammatory effect of the crude extract of S.olivaceus (MSU3) was determined by protein denaturation method. The different concentrations of the crude extract (100, 200, 300, 400 and 500µg/ml) exhibited the percentage inhibition of 34.08, 53.08, 70.71, 80.46 and 96.63 % respectively. The percentage inhibition was gradually increased from 100 to 500µg/ml concentration of the crude extract (Fig. 4). The One-way ANOVA test revealed that the variation between different concentrations of ethyl acetate crude extract on anti-inflammatory activity was statistically more significant (P< 0.05). 3.7. Antioxidant assays of crude extract of S. olivaceus (MSU3)

The antioxidant properties of the crude extract of S. olivaceus (MSU3) were tested by using five different assays. The different concentrations of the crude extract used for the assay were 12.5, 25, 50 and 100µg/ml. In in-vitro antioxidant assay, the crude extract exhibited maximum ascorbic acid equivalent of 87µg/ml and it was observed at100µg/ml, whereas minimum equivalent of 32µg/ml was noted at 12.50µg/ml concentration of extract (Fig. 5). In DPPH scavenging assay, the metabolite showed increasing percentage of inhibition with increasing concentration of extract. The DPPH scavenging activity of the crude extract exhibited as 38.5, 44.52, 57.36 and 62.06 % against 12.5, 25, 50 and 100µg/ml concentrations respectively (Fig. 6) with the IC50 of 75.21µg/ml. The reducing power of the extract was noted at different concentrations of the extract. It exhibited the level of inhibition of 7.27, 15.07, 26.93 and 32.51% against the respective concentrations of extract such as 12.5, 25, 50 and100µg/ml with IC50 value of 39.75µg/ml. Based on this reducing power, the crude extract was capable to reduce metal ion complexes to their lower oxidation state or to take part in any electron transfer reaction. In hydroxyl radical scavenging assay, the crude extract of the potent strain exhibited the maximum inhibition of

47.19% at 100µg/ml concentration, whereas the minimum inhibition of 24.45% was observed at 12.5µg/ml concentration with the IC50 of 71.46µg/ml. The metabolite inhibited the nitric oxide in a dose depended manner. The percentage inhibition observed was 7.67, 10.60, 20.67 and 33.20 % at the extract concentrations of 12.5, 25, 50 and 100µg/ml respectively with the IC50 of 48.02µg/ml (Fig. 6). The statistical One-way ANOVA result revealed that the differences between in-vitro antioxidant, DPPH scavenging, hydroxyl radical scavenging, total reducing power and nitric oxide assay were highly significant (P< 0.05).

3.8. Cytotoxic activities of crude extract of S. olivaceus (MSU3) against MCF- 7 and HT- 29 cell lines by MTT assay method In MTT assay, the cytotoxic activities of the ethyl acetate crude extract of S. olivaceus (MSU3) against breast (MCF-7) and colon (HT-29) cancer cell lines were analyzed and the result obtained is represented in Figure 7(A & B). It expressed the percentage cell viabilities of both cell lines were gradually decreased (92.11 to 24 and 89.20 to 39.17%) and in turn the percentage cell inhibition of both cancer cell lines was increased respectively with the increasing concentrations (10 to 250µg/ml) of crude extract. The IC50 values of the extract against the breast and colon cancer cell lines were recorded with 88.26 and 104.81µg/ml respectively. The positive control cyclophosphamide (20µg/ml) showed the percentage inhibition of 83.47%. 4. Discussion Actinobacteria constantly hold a special significance in the research area during the last five decades as the members of this group, especially Streptomycetes are known to produce an array of bioactive compounds with diverse biological properties (Williams, 2009). Actinomycetes form 10 % of the total bacteria colonizing marine aggregates. Marine habitat has been proven as an outstanding and fascinating resource for innovating new and potent bioactive substances producing microorganisms. However only very few reports are available on the occurrence and distribution of actinomycetes from marine environment having antagonistic property. Recent investigations indicated the tremendous potential of marine actinomycetes, particularly Streptomyces species as a useful and sustainable source of new bioactive natural products. In the present study, the working actinobacterium strain S. olivaceus (MSU3) was isolated from rhizosphere sediment soil samples of Manakudy estuary, Tamilnadu, South India.

The working strain was screened for their antagonistic properties against various clinical pathogens such as S. mutant (NCIM 2063), S. pneumoniae (ATCC 49619), K. pneumoniae (ATCC 10273), E.coli (ATCC 25922) and V. cholerae (MTCC 3905) using cross streak method. After incubation S. olivaceus (MSU3) showed antagonistic activity against all the five tested pathogens. In accordance with these, Nithya et al. (2012) isolated 238 morphologically different marine and terrestrial actinobacterial strains from 185 sediments/rhizosphere soil samples collected from different sites of Manakudy estuary of south-west coastal regions and Yercaud hills of Eastern ghats of Tamilnadu, India. These isolates were used for screening of antagonistic activity against human pathogens and cancerous cell lines. Patil et al. (2001) isolated a total of 133 actinomycetes from the marine sediment samples in Southeast coast of India, out of these, 129 actinobacterial strains possessed high antagonistic activity. Oskay et al. (2004) isolated 50 different actinomycete strains from farming soil samples of Manisa province. Among these 50 strains, 34% of isolates were shown antimicrobial activity against various tested pathogens such as K. pneumoniae (ATCC 1003), B .subtilis (ATCC 6633), Sarcinalutea (ATCC 9341) and E. coli (ATCC 29998). Wu et al. (2013) examined a new antimicrobial compound namely napyradiomycin, isolated from a marine derived Streptomyces sp. SCSIO 10428 and it displayed antibacterial activity against the gram-positive bacteria Staphylococcus and Bacillus strains with MIC value ranging from 0.25 to 32μg/ml. In the present study also, the ethyl acetate crude extract from S. olivaceus (MSU3) was prepared and the efficiency of the extract was tested against five different pathogens and it exhibited the maximum zone of inhibition against S. mutant (27 ± 2.05mm) at 30µl concentration, and the minimum zone of inhibition (20 ± 1.86mm) was noted at 10µl concentration with the MIC and MBC values noted respectively at 0.625 and ≤ 5µg/ml concentrations when compared to chloramphenicol (25µg/ml). Saravanakumar et al. (2014) portrayed the ethyl acetate extract of soil actinobacterium Streptomyces lavendulae (SCA5) showed antimicrobial activity with MIC value of 31.25µg/ml. Ramasamy et al. (2010) isolated 192 morphologically distinct actinomycete strains from near shore marine environment of Point Colimere, east coast of India; out of which 68 strains displayed the maximum zone of inhibition against B. subtilis (16mm), E. coli (15mm) and also against a fungal pathogen Candida albicans (16mm). Saurav and Kannabiran (2011) isolated 164 actinobacterial strains from 39 sediment samples collected from Bay of Bengal coast of Puducherry and Marakkanam, India. Among them, the isolate VITSUK9 showed

antibacterial activity against Bacillus subtilis (18mm) and antifungal activity against Aspergillus niger (17mm). Kiruthika et al. (2013) demonstrated In-vitro antimicrobial and antioxidant profile of Streptomyces sp isolated from Coromandel Coast region and the crude extract from the isolated strain showed the highest zone of inhibition with 17mm against S. aureus and the lowest value of 7mm against K. pneumoniae. The DPPH scavenging assay of the same extract exhibited IC50 value of 3.5mg/ml. In the present study, the crude extract of S. olivaceus (MSU3) exhibited the anti-inflammatory effect and it was gradually increased at respective increasing concentrations of the extract. The hemolytic property of the crude extract expressed the highest percentage of inhibition at 500µg/ml and the lowest inhibition value recorded at 100µg/ml concentration. Invitro antioxidant property of the crude extract exhibited 87µg/ml of ascorbic acid equivalent at 100µg/ml and also the DPPH radical scavenging activity of the crude extract showed 62.06% inhibition at 100µg/ml concentration. Similarly Gautham and Onkarappa, (2013) studied the invitro antioxidant activity of secondary metabolite from S. fradiae strain (GOS), expressed the percentage inhibition of DPPH radicals and TBHQ with 33.33 and 68.4% respectively at the concentration of 100µg/ml. Similarly Thenmozhi and Kannabiran (2012) reported that the ethyl acetate extract of Streptomyces spp UITSTK7 isolated from sediment samples of Bay of Bengal coast of Puducherry showed the DPPH scavenging activity of 43.20% and Fe+3 reducing activity of 3.65 % at 100µg/ml concentration. In accordance with these, in the present study also the crude extract of S. olivaceus (MSU3) exhibited maximum reducing power (32.51%), hydroxyl radical scavenging effect (47.19%) and inhibition of nitric oxide (33.20%) at 100µg/ml concentration. Likewise Jemimah Naine et al. (2014) evaluated the bioactive properties of ethyl acetate crude extract of S. parvulus (VITJS11), which exhibited the maximum reducing power activity at the concentration of 50µg/ml with 85% inhibition. Saravanakumar et al. (2014) reported that the soil actinobacterium S. lavendulae (SCA5) had antimicrobial activity and the ethyl acetate crude extract of this isolate exhibited good antioxidant properties, which was achieved by DPPH scavenging activity (IC50 507.61µg/ml), hydroxyl radical scavenging activity (IC50 617.84µg/ml), nitric oxide scavenging activity (IC50 730.92µg/ml) and superoxide anion radicals (IC50 838.83µg/ml) assay. The results of the present study expressed that increasing percentage of inhibition with increasing concentration of the crude extract. The statistical result of the present study revealed that the differences between various concentrations of crude extract

on in-vitro antioxidant, DPPH, hydroxyl radical scavenging, reducing power and nitric oxide activities were more significant (P < 0.001). Ignacimuthu et al. (2014) studied the cytotoxic property of ethyl acetate extract of Streptomyce sp (ERINLG-01) against A549 lung adenocarcinoma cancer cell line, it expressed 82.4 % activity at the dose of 1000μg/ml with the IC50value of 600μg/ml and also expressed the various concentrations used in this experiment decreased the cell viability significantly (P<0.05) in a concentration-dependent manner. In the present investigation, the ethyl acetate extract of S. olivaceus (MSU3) against breast (MCF-7) and colon (HT-29) carcinoma cell lines predicted maximum percentage of inhibition observed at 250 μg/ml concentration with the respective IC50 values of 88.26 and 104.81μg/ml, compared with cyclophosphamide (20μg/ml) as positive control. Jemimah Naine et al. (2014) also evaluated a bioactive properties of ethyl acetate crude extract of S. parvulus (VITJS11) and it exhibited the cytotoxic effect with the IC50 value of 500/ml on HepG2 cell lines. Hong et al. (2009) found that 20% of the bacteria isolated from different marine environments showed activity against colon cancer cells (HCT-116), and that the genus Streptomyces showed the highest activity in the in vitro assays. Kharat et al. (2009) reported the cytotoxic activity of Streptomyces spp from Lonar lake, against human carcinoma cell line (A549) and the cells were died at 1/500 dilution and the cell viability was greatly reduced at 48 h of exposure. From this present findings, it could be concluded that the working actinobacterial strain S. olivaceus (MSU3) isolated from rhizosphere sediment soil samples of Manakudy estuary, has antagonistic properties against clinical pathogens and its ethyl acetate crude extract showed high antioxidant, anti-inlammatory as well as cytotoxic activities. Acknowledgement The authors gratefully acknowledge the University Grants Commission (UGC), New Delhi, Govt. of India, for its financial support in the form of Special Assistance Programme (SAP) [UGC No. F.3-24/2012(SAPII) dtd October2012].

References Akinjogunla, O.J., Eghafona, N.O., Enabulele, I.O., Mboto, C.I., Ogbemudia, F.O., 2010. Antibacterial activity of ethanolic extracts of Phyllanthus amarus against extended spectrum β-

lactamase producing E. coli isolated from stool samples of HIV sero-positive patients with or without diarrhea. Afr. J. Pharm. Pharmacol. 4, 402–407. Alexander, M., 1977. Introduction to soil microbiology. 2nd Eds. John Wiley & Sons, New York, pp. 207. Badmus, J.A., Adedosu, T.O., Fatoki, J.O., Adegbite, V.A., Odunola, O.A., 2011. Lipid peroxidation inhibition and anti-radical scavenging activities of some leaf fractions of Mangifera indica. Act. Poloniac Pharma. Drug Res. 68, 23-29. Chang, H.B., Kim, J.H., 2001. Antioxidant properties of dihydroherbimycin-A from a newly isolated Streptomyces sp. Biotechnol. Let. 29, 599- 603. Devi, S., Jemimah Naine, S., Nasimunislam, N., Vaishnavi, B., Mohanasrinivasan, V., 2012. Isolation of soil actinomycetes inhabiting Amrithi forest for the potential source of bioactive compounds. Asian J. Pharma. Clinic. Res. 5, 189–192. Farnet, C.M., Zazopoulos, E., 2005. Improving drug discovery from microorganisms. In: Zhang, L., Demain, A.L., (Eds). Natural products: Drug discovery and therapeutic medicine. Totowa, New Jersey, Humana press Inc, pp. 95–106. Fischer, D., Li, Y., Ahlemeyer, B., Krieglstein, J., Kissel, T., 2003. In-vitro cytooxicity testing of polycations: influence of polymer structure on cell viability and hemolysis. Biomet.24, 1121– 1131. Forar, L.R., Amany, K., Bengraa, C.H., 2006. Screening, isolation and characterization of actinomycetes from marine sediment samples. Arab. J. Biotechnol. 9, 427–436. Gautham, S.A., Onkarappa, R., 2013. In-vitro antioxidant activity of metabolite from Streptomyces fradiae strain GOS1. Inter. J. Drug Develop. Res. 5, 235–244. Gorajana, A., Venkatesan, M., Vinjamuri, S., Peela, S., Jangam, P., 2007. Resistoflavine, cytotoxic compound from a marine actinomycete, Streptomyces chibaensis AUBN1/7. Microbiol. Res.162, 322-327. Green, L.C., Wagner, D.A., Glogowski, J., Tannenbaum, S.R., 1982. Analysis of nitrate and 15N nitrate in biological fluids. Anal. Biochem. 126, 131–136.

Hong, K., Gao, A.H., Xie, Q.Y., Gao, H., Zhuang, H., Goodfellow, M., Ruan, J.S., 2009. Actinomycetes for marine drug discovery isolated from mangrove soil and plants in China. Marine Drug. 7, 24–44. Ignacimuthu, S., Balachandran, C., Duraipandiyan, V., Valan Arasu, M., 2014. Antimicrobial, antioxidant and cytotoxic properties of Streptomyces sp. (ERINLG-01) isolated from Southern Western ghats. Inter. J. Pharm. Pharmaceu. Sci. 6, 189–196. Jemimah Naine, S., Devi, C.S., Mohana Srinivasan, V., Vaishnavi, B., 2014. Antimicrobial, antioxidant and cytotoxic activity of marine Streptomyces parvulus VITJS11crude extract. Braz. Arch. Biol. Technol. 3, 1–10. Kamleshiya, P., Meshram, V.G., Ansari, A.H., 2010. Comparative evaluation of antioxidant and free-radical scavenging activity of aqueous and methanolic spice extracts. Inter. J. Life Sci. Pharma Res. 2, 118–125. Karthik, L., Gaurav, K., Bhaskara Rao, K.V., 2013. Antioxidant activity of newly discovered lineage of marine actinobacteria. Asia. Pacific J. Tropic. Medi. 325–332. Karthik, L., Gaurav, K., Rao, B.K.V., Rajakumar, G., Rahuman, A.A., 2011. Larvicidal, repellent and ovicidal activity of marine actinobacteria against Culex tritaeniorhynchus and Culex gelidus. Parasitol. Res.108, 1447–1455. Kekuda, P.T.R., Shobha, K.S., Onkarappa, R., Gautham, S.A., Raghavendra, H.L., (2012). Screening of biological activities of a Streptomyces species isolated from soil of Agumbe, Karnataka, India. Inter. J. Drug Develop. Res. 4, 104–114. Kharat, K.R., Kharat, A., Hardikar, B.P., 2009. Antimicrobial and cytotoxic activity of Streptomyces sp from Lonar lake. Afr. J. Biotechnol. 8, 6645–6648. Kiruthika, P., Nisshanthini, S.D., Angayarkanni, J., 2013. In-vitro antimicrobial and antioxidant profile of Streptomyces sp. isolated from coromandel coast region, India. Inter. J. Pharm. Biosci. 4, 127-136. Moncheva, P., Tishkov, S., Dimitrova, N., Chipeva, V., Bogatzevska, N., 2002. Characteristics of soil actinomycetes from Antarctica. J. Culture Collec. 3, 3-14.

Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J. Immunol. Method. 5, 53–63. Mousumi, D., Dayanand, A., 2013. Production and antioxidant attribute of L-glutaminase from Streptomyces enissocaesilis DMQ-24. Inter. J. Latest Res. Sci. Technol. 2, 1–9. Nathan, A., Jessica, M., Bernan, V., Sherman, D.H., 2004. Isolation and characterization of novel marine derived actinomycete taxa rich in bioactive metabolites. Appl. Environ. Microbiol. 70, 7520-7529. Nithya, B., Ponmurugan, P., Fredimoses, M., 2012. 16SrRNA phylogenetic analysis of actinomycetes isolated from Eastern Ghats and marine mangrove associated with antibacterial and anticancerous activities. Afr. J. Biotechnol. 11, 12379-12388. Oskay, M., Usame Tamer, A., Azeri, C., 2004. Antibacterial activity of some actinomycetes isolated from farming soils of Turkey. Afr. J. Biotechnol. 3, 441-446. Parejo, I., Codina, C., Petrakis, C., Kefalosp, T., 2000. Evaluation of scavenging activity assessed by Co(II)/EDTA induced luminal chemiluminescence and DPPH free radical assay. J. Pharmacol.Toxicol. method. 44, 507–512. Patil, R., Jeyasekaran, G., Shanmugam, S.A., Shakila, J.R., 2001. Control of bacterial pathogens, associated with fish diseases, by antagonistic marine actinomycetes isolated from marine sediments. Ind. J. Marine Sci. 30, 264–267. Raghava Rao, K.V., Raghava Rao, T., 2013. Molecular characterization and its antioxidant activity of a newly isolated Streptomycetes coelicoflavus BC01 from mangrove soil. J. Young Pharmacis.5, 121-126. Rakesh, K.N., Syed, J., Dileep, N., Prashith, K.T.R., 2013. Antibacterial and antioxidant activities of Streptomyces sp SRDP-H03 isolated from soil of Household, Karnataka, India. J. Drug Deliver. Therapeu.3, 47-53. Ramasamy, V., Subban, M., Annamalai, P., 2010. Isolation, characterization and antimicrobial activity of actinobacteria from Point Calimere coastal region, East coast of India. Inter. Res. J. Pharm.1, 358–365. Saravanakumar, P., 2014. In-vitro antimicrobial, antioxidant and cytotoxic properties of Streptomyces lavendulae strain SCA5. Biomedi. Microbiol. 14, 1–12.

Sateesh, V., Naikpatil, P., Rathod, J.L., 2011. Antimicrobial and cytotoxic activity of actinomycetes from Karwar Coast, West of India. World J. Sci.Technol.1, 7-10. Sato, M., Ramarathnam, N., Suzuki, Y., Ochi, H., 1996. Varietal differences in the phenolic content and superoxide radical scavenging potential of wines from different sources. J. Agri. Food Chem. 44, 37-41. Saurav, K., Kannabiran, K., 2012. Cytotoxicity and antioxidant activity of 5-(2,4dimethylbenzyl) pyrrolidin-2-one extracted from marine Streptomyces VITSVK5 spp. Saudi J. Biol. Sci.19, 81-86. Sheikh, T.Z.B., Yong, C.L., Lian, M.S., 2009. In-vitro antioxidant activity of the hexane and methanolic extract of Streptomyces baccularia and Cladophora patentiramea. J. Appl. Sci. 9, 2490–2493. Shiling, E.B., Gottlieb, D., 1966. Methods for characterization of Streptomyces species. Inter. J. Syst. Bacteriol.16, 312-340. Slinkard, K., Singelton, V.L., 1977. Total phenol analysis automation and comparison with manual methods. Am. J. Enol. Vitic. 28, 49–55. Thenmozhi, M., Kannabiran, K., 2012. Antimicrobial and antioxidant properties of marine actinomycetes Streptomyces sp VITSTK7. Oxi. Antioxi. Medi. Sci.1, 51-57. Williams, P.G., 2009. Panning for chemical gold: marine bacteria as a source of new therapeutics. Trends Biotechnol. 27, 45-52. Wu, Y., Huang, A.E., Yi-Zhong Cai, A.E., Kevin, D., Sun, M., 2007. Endophytic fungi from Nerium oleander L (Apocynaceae): main constituents and antioxidant activity. World J. Microbiol. Biotechnol.23, 1253–1263. Yen, G.C., Duh, P.D., 1993. Antioxidant properties of methonolic extracts from peanut hull. J. American Oil Chem. Soci.70, 383-386. Zhong, K., Gao, X.L., XU, Z.J., Gao, H., Yamaguchi, I.. Li. L.H., Chen, R.J., 2011. Antioxidant activity of a novel Streptomyces strain Eri12 isolated from the rhizosphere of Rhizomacurcum aelongae. Cur. Res. Biotechnol. 4, 63-72.

Table 1. Preliminary screening of antagonistic activity of extract of S. olivaceus (MSU3) against test pathogens through agar-well diffusion method Clinical pathogens

Zone of inhibition (mm) in diameter Concentrations of crude extract (µl) 10

20

30

P.c

S. mutant (NCIM 2063)

20 ± 1.86

24 ± 2.02

27 ± 2.05

06 ± 0.04

E. coli (ATCC 25922)

21 ± 1.44

23 ± 1.49

25 ± 2.87

12 ± 1.26

S. pneumoniae (ATCC 49619)

16 ± 1.90

19 ± 1.05

21 ± 1.45

08 ± 0.05

K. pneumoniae (ATCC 10273)

17 ± 1.80

20 ± 1.49

23 ± 1.26

02 ± 0.02

V. cholerae (MTCC 3905)

12 ± 1.86

15 ± 1.26

19 ± 1.68

05 ± 0.04

P.c = Positive control (Chlorophenicol - 25 µg/ml); Each value is the Mean ± SD of triplicate analysis

Table 2. Determination of minimum inhibitory and minimum bactericidal concentrations of ethyl acetate extract of S. olivaceus (MSU3) against test pathogens Clinical pathogens

MIC (µg/ml)

MBC (µg/ml)

S. mutants(NCIM 2063)

0.625

≤ 5.00

E. coli(ATCC 25922)

2.50

>5.00

K. pneumoniae (ATCC 10273)

1.25

2.50

S. pneumoniae (ATCC 49619)

2.50

5.00

V. cholerae (MTCC 3905)

1.25

≤ 2.50

Fig.1 Actinobacterial strain S. olivaceus (MSU3)

Fig. 2 Cross streak method (MNA plate)

Fig. 3 Antagonistic activity of crude extract against test pathogens

a) S.mutant (NCIM2063), b) E. coli (ATCC25922), c) S. pneumoniae (ATCC49619), d) K.pneumoniae (ATCC10273), e) V. cholerae (MTCC3905).

Fig. 4 Total hemolytic and in-vitro anti-inflammatory activities of ethyl acetate extract of S. olivaceus (MSU3)

Inhibition (%)

Conc. of extract (µg/ml) In-vitro anti-inflammatory activity

Total hemolytic activity

Ascorbic acid equivalents (µg/ml)

Fig. 5 Total antioxidant activity of ethyl acetate extract of S. olivaceus (MSU3)

e d

a

b

c

Conc. of extract (µg/ml)

Fig. 6 In-vitro antioxidant activity of ethyl acetate extract of S. olivaceus (MSU3)

d

Inhibition (%)

c b

a

b2

c1

a2 a1

d2

c2

b1

a3

d1

d3

c3

b3

Conc. of ethyl acetate extract DPPH assay

Total reducing power

Hydroxyl radical scavenging assay

Nitric oxide assay

Growth (% )

Fig.7 (A&B) Cytotoxic effects of ethyl acetate extract of S. olivaceus (MSU3) on MCF-7 and HT-29 cells

Conc. of Extract (µg/ml) MCF-7 Carcinoma cell line

HT-29 Carcinoma cell line

Cyclophosphamide

A. Growth percentage of MCF-7 and HT-29 cells when treated with ethyl acetate extract of S. olivaceus (MSU3)

B. (a & c) Normal MCF-7 and HT-29 cells; (b & d) S. olivaceus (MSU3) ethyl acetate extract treated MCF-7 and HT-29 cells