RESEARCH ARTICLE

Antioxidant Activity of Nocardiopsis sp., a MarineActinobacterium, Isolated from the Gulf of MannarBiosphere Reserve, India

Subramaniam Poongodi • Valliappan Karuppiah •

Kannan Sivakumar • Lakshmanan Kannan

Received: 17 October 2012 / Revised: 31 July 2013 / Accepted: 22 October 2013 / Published online: 8 February 2014

� The National Academy of Sciences, India 2014

Abstract Six actinobacterial strains (PU1, PU2, PU3,

PU4, PU5 and PU6) were isolated from the sediment

samples of the coral reef environment of the Pullivasal

Island, Gulf of Mannar Biosphere Reserve, India using

Kuster’s agar. Ethyl acetate extracts of these six strains

were screened for antioxidant activity viz. total antioxidant

activity, total reducing power, DPPH (a,a-diphenyl-a-pic-

rylhydrazyl) radical scavenging, Scavenging of hydrogen

peroxide (H2O2) and nitric oxide radical scavenging

activity (NO). Among the six strains, PU3 showed strong

antioxidant activity and PU1 had a week antioxidant

activity. PU3 recorded the following: total antioxidant

activity, 0.662; total reducing power, 0.181; DPPH,

53.6 %; scavenging of hydrogen peroxide (H2O2), 74.2 %

and nitric oxide radical scavenging activity (NO), (56 %)

in 100 lg/ml concentration. Hence, this strain was further

studied and identified as Nocardiopsis sp. based on

chemotaxanomical, micromorphological and molecular

characters.

Keywords Marine actinobacteria �Gulf of Mannar Biosphere Reserve � Antioxidant activity �Nocardiopsis sp.

Many researchers have reported various types of antiox-

idants in different kinds of higher plants and seaweeds.

More recently, actinobacteria have been found to be a

rich source of antioxidant compounds. The compounds

isolated from marine Streptomyces, 2-allyoxyphenol and

streptopyyrolidine have been reported to possess antioxi-

dant property and no cytotoxic activity [1, 2]. To get

insight into the role of the antioxidant response against

H2O2 and paraquat stresses, superoxide dismutase (SOD)

and catalase (CAT) activities and lipid peroxidation LPO

levels in relation to incubation time were studied on

Streptomyces sp. M3004 isolated from Turkish soil [3].

Further, reports on the antioxidant properties of actino-

bacterial extracts from India are very limited. Hence, the

present study.

Sediments from the coral reef environment of Pullivasal

Island of the Gulf of Mannar Biosphere Reserve, south east

coast of India (9�230699N; 79�19010000E) were collected

with a corer. The collected samples were transferred to a

sterile polythene bag and taken immediately to the labo-

ratory. The sediment samples were aseptically air-dried in

laminar air flow and pretreated by incubating them at 55 �C

in a hot air oven for 10 min.

After the pretreatment, tenfold serial dilution of the

sediment sample was prepared, using filtered and sterilized

50 % sea water. Serially diluted samples were plated in the

Kuster’s agar medium in triplicate. To minimize the bac-

terial and fungal contaminations, all the plates were sup-

plemented with Nystatin (20 mg/ml) and Cycloheximide

(100 mg/ml) respectively [4, 5]. The actinobacterial colo-

nies were counted from 14th day onwards upto 28 days and

the colonies were picked up and grown separately by

streaking in yeast extract, malt extract dextrose agar.

Subcultures ensured for their axenicity and were main-

tained in slants.

S. Poongodi � V. Karuppiah � K. Sivakumar (&) � L. Kannan

CAS in Marine Biology, Faculty of Marine Sciences, Annamalai

University, Paragipettai 608 502, Tamilnadu, India

e-mail: oceanactino@gmail.com

S. Poongodi

e-mail: poomicro.acitno@gmail.com

V. Karuppiah

e-mail: karuppiah.vkbiotech@gmail.com

L. Kannan

e-mail: kannanlk69@gmail.com

123

Natl. Acad. Sci. Lett. (January–February 2014) 37(1):65–70

DOI 10.1007/s40009-013-0193-4

Well grown slant culture of the strains was inoculated

into 50 ml of nutrient broth in 250 ml Erlenmeyer flasks

and incubated for 5 days in a rotary shaker (200 rpm) at

35 �C. The inocula (10 %) were transferred into 100 ml of

yeast extract, malt extract, and dextrose broth in 250 ml

Erlenmeyer flasks. The inoculated culture broth was incu-

bated for 5 days on a rotary shaker (200 rpm) at 35 �C.

After fermentation, the broth was centrifuged at

10,000 rpm for 10 min at 4 �C and the supernatant was

separated. The supernatant was extracted twice with equal

volume of ethyl acetate and was concentrated under

reduced pressure. The dried extract was dissolved in

phosphate buffer (pH 6.6) and stored at 4 �C until use.

Total antioxidant activity of the actinobacterial crude

extracts was determined according to the method of Prieto

et al. [6]. 0.3 ml of actinobacterial extract (20–100 lg/ml)

was mixed with 3.0 ml of reagent solution (0.6 M sul-

phuric acid, 28 mM sodium phosphate and 4 mM ammo-

nium molybdate). Reaction mixture was incubated at 95 �C

for 90 min in a water bath. Absorbance of all the sample

mixtures was measured at 695 nm. Ascorbic acid

(20–100 lg/ml) was used as positive control and phosphate

buffer used as a blank.

Total reducing capacity of the actinobacterial extracts

was determined according to the method of Oyaizu [7].

The actinobacterial extracts (20–100 lg/ml) in phosphate

buffer (0.2 M, pH 6.6) were mixed with 1 % potassium

ferricyanide and the mixture was incubated at 50 �C for

20 min; 2.5 ml of 10 % trichloro acetic acid (TCA) was

added to the mixture and centrifuged at 5,000 rpm for

10 min. The upper layer of solution (2.5 ml) was mixed

with 2.5 ml of distilled water and 0.5 ml of 0.1 % FeCl3and the colour developed was measured at 700 nm.

Ascorbic acid (20–100 lg/ml) was used as positive control

and phosphate buffer used as a blank.

Ability of the actinobacterial extracts to scavenge H2O2

was determined according to the method of Ruch et al. [8]

with the slight modification of Green et al. [9]: 40 mM

H2O2 was prepared in phosphate buffer (pH 7.4) and the

H2O2 concentration was determined spectrophotometri-

cally by measuring the absorption with the extinction co-

efficient for H2O2 of 81 M-1 cm-1. Actinobacterial extract

and positive control (20-100 lg/ml) was added to 0.6 ml

of 40 mM H2O2 solution and the absorbance was read at

230 nm after 10 min incubation against a blank solution

containing phosphate buffer without H2O2. The percentage

of scavenging of H2O2 was calculated as follows:

Scavenging effect %ð Þ ¼ Acont�Atestð Þ = Acont � 100

NO generated from sodium nitroprusside in the aqueous

solution at physiological pH interacts with oxygen to

produce nitrite ions, which were measured by the Griess

reaction [8, 10]. In this experiment, 3 ml of the reaction

mixture containing 10 mM sodium nitroprusside and the

actinobacterial extracts (20–100 lg/ml) in phosphate

buffer were incubated at 25 �C for 150 min. After

incubation, 0.5 ml of the reaction mixture was mixed

with 1 ml of sulfanilic acid reagent (0.33 % sulfanilic acid

in 20 % glacial acetic acid) and allowed to stand for 5 min

for complete diazotization. Then, 1 ml of naphthyl

ethylene diamine dihydrochloride (0.1 %) was added and

the solution, mixed. The mixture was allowed to stand for

30 min at 25 �C. A pink coloured chromophore developed

was absorbed at 540 nm against the corresponding blank

solution. Ascorbic acid (20–100 lg/ml) was used as

positive control and phosphate buffer used as a blank.

The NO scavenging activity of the actinobacterial extracts

is reported as % inhibition, and was calculated as in H2O2

scavenging activity.

Free radical scavenging activity of actinobacterial

extracts was measured by DPPH using the method of Blois

[11]; 1 ml of DPPH (0.1 mM) solution in methanol was

added to 3 ml of actinobacterial extracts (20–100 lg/ml) in

phosphate buffer, shaken vigorously, allowed to stand at

room temperature for 30 min and the absorbance was

measured at 517 nm in a UV–visible spectrophotometer

(U-2800 model, Hitachi, Japan). A low absorbance of the

reaction mixture indicated a high free radical scavenging

activity. Ascorbic acid (20–100 lg/ml) was used as posi-

tive control and phosphate buffer used as a blank. The

percentage of DPPH scavenging effect was calculated with

the following equation.

Percentage of inhibtion I %ð Þ ¼ Ablank� Asample

� �= Ablank � 100

The potential antioxidant strain was characterized

chemotaxonamically [12] and morphologically. General

morphology was studied on ISP2 agar and cultural

characteristics were determined [13]. Spore morphology

was noted by direct microscopic examination using the

15 days old cultures by observing under a light. Whole cell

sugar and cell wall amino acids were studied using thin

layer chromatography.

Genomic DNA was extracted from cultures grown on

ISP2 using the method of Ausubel et al. [14]. Each 50 ll of

amplification reaction contained 1 ll template DNA

(50–200 ng), 5 ll 109 PCR buffer with MgCl2, 1 ll each

PCR primer (20 mM) (27F, 1492R), 1 ll dNTP mix

(10 mM), 2.5 U Taq DNA polymerase, 2.5 ll DMSO and

35 ll sterile MilliQ water. The reaction conditions were

initial denaturation at 95 �C for 5 min, followed by 30

cycles of denaturation at 95 �C for 30 s, annealing at 55 �C

for 30 s and extension at 72 �C for 90 s. A final extension

was performed at 72 �C for 10 min. Reaction products

were electrophoresed on a 1 % agarose gel and checked

with ethidium bromide under UV light. The PCR products

66 S. Poongodi et al.

123

were purified and sequenced directly using a Taq Dye

Deoxy Terminator Cycle Sequencing Kit and an ABI Prism

3730 automated DNA sequencer (Applied Biosystems).

Both strands were sequenced as a cross-check by using

forward and reverse primers [15].

The 16S rDNA sequence of the test strain was aligned

manually with available nucleotide sequences retrieved

from the Ribosomal Database Project and EMBL/GenBank

databases. An evolutionary tree was inferred by using tree-

making algorithm, namely the neighbour joining algorithm.

Evolutionary distance matrices were generated using the

MEGA version 4.1 package. A bootstrap analysis of 1,000

replicates was carried out. The root position of the tree

based on the neighbour-joining method was estimated

using Pseudomonas sp. as an out group.

During the present investigation, the actinobacterial

colonies were enumerated from the sediment samples of

the coral reef environment of Pullivasal Island. Population

density of the actinobacteria in Kuster’s medium was

0.3 9 102 CFU/g and a total of six distinct morphological

strains (PU1, PU2, PU3, PU4, PU5 and PU6) were

obtained and used for antioxidant studies. Total antioxidant

activity of the actinobacterial extracts (20–100 lg/ml) and

the standard ascorbic acid (20–100 lg/ml) at 695 nm is

shown in Fig. 1. PU3 showed higher activity (0.622), fol-

lowed by PU4 (0.453) and PU3 (0.622) compared to others.

Similarly, total antioxidant activity of the actinobacterial

extract was investigated by Poongodi et al. [16] who found

total antioxidant activity, varying from 0.1 to 0.5 absor-

bance at 695 nm for 100 lg/ml of different actinobacterial

species.

Total reducing power of the actinobacterial extracts and

the standard ascorbic acid (20–100 lg/ml) at 700 nm is

shown in Fig. 2. The reducing ability of the actinobacterial

extract depends on the presence of reductons in the extract

which exhibit the antioxidative potential by breaking the

free radical chain by donating a hydrogen atom [17].

Among the six extracts, PU3 (20–100 lg/ml) showed a

strong activity of 0.18 absorbance, equivalent to the

100 lg/ml of standard ascorbic acid, whereas others

showed weak activity. Similar trend has been reported by

Zhong et al. [18] in the extract of the actinobacteria, iso-

lated from south west China. The reducing capacity of PU3

is the significant indicator of its potential antioxidant

activity. Since the total reducing power indicates the

potential antioxidant ability, PU3 can be considered as a

potent source of antioxidants.

Ability of actinobacteria to scavenge H2O2 was deter-

mined according to the method of Ruch et al. [8] and

Gulcin et al. [19]. Hydrogen peroxide itself is not very

reactive, but it can sometimes be toxic to cells because it

may give rise to hydroxyl radical in the cells [20]. Figure 3

shows the percentage of scavenging activity of the six

actinobacterial extracts (PU1, PU2, PU3, PU4, PU5 and

PU6) with the standard ascorbic acid (20–100 lg/ml).

Among them, PU3 (20–100 lg/ml) exhibited the maxi-

mum activity of 74.2 % which was significantly higher

than the standard L-ascorbic acid whose scavenging effect

was only 64.2 %. IC50 of the hydrogen peroxide scav-

enging activity of the PU3 was 44.41 lg/ml.

Nitric oxide radicals play an important role in inducing

inflammatory response and their toxicity multiplies only

when they react with O2 radicals to form peroxynitrite and

damage biomolecules like proteins, lipids and nucleic acids

[21]. Actinobacterial extracts inhibit nitrite formation by

competing with O2 to react with nitric oxide directly.

20 40 60 80 1000.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Abs

orba

nce

at 6

95nm

Concentrations (µg/ml )

PU1PU2PU3PU4PU5PU6CONTROL

Fig. 1 Total antioxidant activity of six actinobacterial extracts with

standard ascorbic acid as positive control

20 40 60 80 1000.0

0.1

0.2

0.3

0.4

0.5

Abs

orba

nce

at 7

00nm

Concentrations (µg/ml)

PU1PU2PU3PU4PU5PU6CONTROL

Fig. 2 Total reducing ability of actinobacterial extracts with standard

ascorbic acid as positive control

Antioxidant Activity of Nocardiopsis sp. 67

123

Present results showed that PU3 (20–100 lg/ml) had a

scavenging activity of 56 % of inhibition, which is sig-

nificantly higher than that of the standard L-ascorbic acid

(54 %) at 100 lg/ml (Fig. 4). Other extracts showed

modest activity. IC50 of the nitric oxide scavenging

activity of the PU3 was 56.6 lg/ml. Therefore, PU3 could

be a potent and novel source for obtaining therapeutic

agents for scavenging NO.

DPPH is a stable free radical and accepts an electron or

hydrogen radical to become a stable diamagnetic molecule

[19]. Hence, DPPH is often used as a substrate to evaluate

antioxidative activity of antioxidants [22, 23]. Figure 5

shows the percentage scavenging activity of the six

actinobacterial extracts (PU1, PU2, PU3, PU4, PU5 and

PU6) with the standard ascorbic acid (20–100 lg/ml) as

control. Among them, PU3 exhibited the maximum activity

of 53.6 % inhibition which was significantly higher than

that of the standard L-ascorbic acid whose scavenging

effect was only 59 % and IC50 of the PU3 was 58.2 lg/ml.

Similarly, Kamala [24] has screened the antioxidant

activity of actinobacterial extract (Nocardiopsis sp. NCS1),

which showed radical scavenging activity with the IC50

value of 6.5 lg/ml. Karuppiah [10] has also investigated

the IC50 value of the actinobacterial pigment extract,

which was 13.065 lg/ml, indicating its higher inhibiting

and potential activity against free radicals. The ability of

pigment to scavenge DPPH could also reflect its ability to

inhibit the formation of free radicals.

As PU3 showed potential antioxidant activity, it was

identified based on the chemotaxonomical, micromorpho-

logical and molecular methods. Colony morphology of the

PU3 showed white color aerial mass. Its colonies did not

produce any pigment upon the prolonged incubation for

2 weeks in the ISP2, ISP7, nutrient and actinomycetes

isolation agar. Cell wall of the strain possessed meso-DAP

as cell wall aminoacid and no diagnostic sugars, which

indicates the cell wall type III with sugar pattern C, being

the characters of Nocardiopsis. Comparison of the 16S

rRNA gene sequence (1,440 bp) of the strain PU3 with

previously obtained sequences of Nocardiopsis species

deposited in GenBank (Accession number: JF445308)

indicated that this organism is phylogenetically related to

the members of the genus Nocardiopsis.

A phylogenetic tree based on 16S rRNA gene sequences

of the members of the genus Nocardiopsis was constructed

20 40 60 80 1000

10

20

30

40

50

60

70

80%

of i

nhib

ition

Concentration (µg/ml)

PU1PU2PU3PU4PU5PU6CONTROL

Fig. 3 Percentage scavenging of hydrogen peroxide radicals by

different actinobacterial extracts with ascorbic acid as positive control

20 40 60 80 1000

10

20

30

40

50

60

% o

f inh

ibiti

on

Concentrations (µg/ml)

PU1PU2PU3PU4PU5PU6CONTROL

Fig. 4 Percentage scavenging of nitric oxide by different actinobac-

terial extracts with standard ascorbic acid as positive control

20 40 60 80 1000

10

20

30

40

50

60

% o

f inh

ibiti

on

Concentration (µg/ml)

PU1PU2PU3PU4PU5PU6CONTROL

Fig. 5 Percentage scavenging of DPPH by different actinobacterial

extracts with ascorbic acid as positive control

68 S. Poongodi et al.

123

according to the neighbour-joining method of Saitou and

Nei [25] with CLUSTAL W (version 1.81) and MEGA

version 4 [26]. For the neighbour-joining analysis, a dis-

tance matrix was calculated according to Kimura’s two-

parameter correction model. The rooted phylogenetic tree

indicated that the strain PU3 formed a branch with the

strains of N. alba with 98.2 % similarity (Fig. 6).

It is clear that out of the six strains isolated from the

coral reef environment of the Gulf of Mannar Biosphere

Reserve, only PU3 showed higher antioxidant activity, and

it was identified as Nocardiopsis sp. This strain could be a

good candidate for pursuing work on the industrial pro-

duction of antioxidant products, helpful in preventing or

slowing down the process of various oxidative stress

related disorders and bacterial contaminations. As of now,

there are only few reports on the antioxidant activity of

microbes. Hence, there is a vast scope for further research

on the isolation of marine actinobacteria and studying their

active compounds, having antioxidant activity, for the

benefit of humans.

Acknowledgments Authors thank Prof. K. Kathiresan, Director,

Centre of Advanced Study in Marine Biology and the authorities of

Annamalai University for providing with necessary facilities. They

also thank the Ministry of Environment and Forests, Government of

India for financial support.

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