26 lsa saranya.pdf

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Life Science Archives (LSA)

ISSN: 2454-1354

Volume – 1; Issue - 3; Year – 2015; Page: 186 - 191

©2015 Published by JPS Scientific Publications Ltd. All rights reserved

Research Article

SCREENING AND PRODUCTION OF ANTIOXIDANT AND

ANTIPROLIFERATIVE SECONDARY METABOLITES FROM

ACTINOMYCETES

K. Saranya* and K. Vanmathi selvi

PG and Research Department of Microbiology, Sri Akilandeswari Women’s College, Wandiwash,

Thiruvannamalai, Tamil Nadu, India

Abstract

The rapid emergence of antioxidant among pathogens has led to a renewed interest to search for

novel antioxidant potential of actinomycetes. Actinomycetes are enthralling resource due to their ability to

produce novel bioactive secondary metabolites. They have proven to be an inexhaustive mine of

Antioxidant, especially those potent against pathogenic organisms. Microbial secondary metabolites,

especially those from actinomycetes have been a phenomenal success for the discovery of novel drugs. They

produce a wide range of secondary metabolites and more than 70% of the naturally derived antibiotics are

currently in clinical use. They remain a fundamental source of new chemical diversity and an important part

of drug discovery. In the present study, actinomycetes were successfully isolated from soil sample using

SCA medium. These organisms could be isolated with high frequency, without any contamination from the

SCA. Pure culture was maintained in SCA. About eight strains of actinomycetes were isolated. Secondary

metabolite extraction was performed by inoculating eight strains of actinomycetes in minimal media and

incubating the flask for 10 days. The media was separated from the mycelia and extraction was done with

ethyl acetate. The extract was collected and kept for air drying at room temperature for 2 days. The extract

was screened for antioxidant and anticancer assay against MCF cancer lines. The extract showed high

significant activity against the cancer cells.

Article History Received : 08.04.2015

Revised : 15.05.2015

Accepted : 20.05.2015

Key words: Antioxidant, Actinomycetes, Secondary metabolites, Anticancer and MCF

cancer lines.

1. Introduction

The actinomycetes are Gram positive

bacteria having high G + C (>55%) content in

their DNA. The name ‘Actinomycetes’ was

derived from Greek ‘aktis’ (a ray) and ‘mykes’

(fungus) and given to these organisms form initial

observation of their morphology. The

actinomycetes are noteworthy as antibiotic

producers, making three quarters of all known

* Corresponding author: K. Saranya, PG and

Research Department of Microbiology, Sri

Akilandeswari Women’s College, Wandiwash

products; the Streptomyces are especially prolific

and can produce a great many antibiotics and other

class of biologically active secondary metabolites.

They are free living, saprophytic bacteria,

and a major source for production of antibiotics.

They play a major role in recycling of organic

matter, production of novel pharmaceuticals,

nutritional materials, cosmetics, enzymes,

antitumour agents, enzyme inhibitors, immune-

modifiers and vitamins. Streptomyces are

especially prolific and can produce a great many

K. Saranya/ Life Science Archives (LSA), Volume – 1, Issue – 3, Page – 186 - 191, 2015 187


antibiotics (around 80% of the total antibiotic

production) and active secondary metabolites.

Actinomycetes, the filamentous bacteria, are

primarily saprophytic microorganisms of the soil,

where they contribute significantly to the turnover

of complex biopolymers, such as lignocellulose,

hemicellulose, pectin, etc. (Vijayakumar et al.,

2007).

The microbial fibrinolytic enzymes,

especially those from actinomycetes, have the

potential to be developed as drugs to prevent

cardiovascular diseases (Sasirekha et al., 2012).

Early evidence supporting the existence of marine

actinomycetes came from the description of

Rhodococcus marinonascene the first marine

actinomycetes species to be characterized

(Helmke and Weyland, 1984). They produce

numerous substances essential for health such as

antibiotics, enzymes, immunomodulators, etc. The

productivity of Streptomyces strains as antibiotic

producers remains unique amongst Actinomycetes

strains. Early evidence supporting the existence of

marine actinomycetes came from the description

of Rhodococcus marinonascene, the first marine

actinomycetes species to be characterized. The

aim of this work was to isolate and characterize

specific antibiotic producing actinomycetes strain

from marine soil sample.

2. Materials and Methods

Sample collection

The samples were collected from the coastal

region of Bay of Bengal near the shore regions of

Kanathur of Chennai, India. . The samples were

kept for drying in room temperature

Isolation, Characterization and identification

Actinomycetes

Observation of isolation plates

Observing the soil isolation plate under a

high power magnifying lens, the colony

morphology was noted with respect to colour,

aerial mycelium, size and nature of colony,

reverse side colour and feeling the consistency

with a sterile loop.

Microscopic observation wet mount

With clean grease free glass slide placing 1

- 2 drops of water suspended Actinomycetes

colony and cover slip was placed then it was

observed under microscope. It is used to study the

shape, size spores, motility, etc.

Coverslip culture Technique

Starch casein agar plate was prepared and

3 to 4 sterile cover slips were inserted at an angle

of 45o

the Actinomycetes culture was slowly

released at the intersection of medium and cover

slip. The plates were incubated at 28 oC for 4 - 8

days. After 2 days the cover slip was removed air

dried and observed under high power

magnification. It is used to study the

morphological features of spores presence of

sporangia as well as to distinguish between aerial

mycelium and substrate mycelium.

Gram Staining (Cappuccino and Sherman,

1966), Acid fast staining (Ziehl- Neelsen method),

Diffusible pigments test - glycerol/asparagines

agar (Shirling and Gotlllieb, 1996), Melanin

pigment production was carried out with Tyrosine

agar medium, Biochemical characterizations were

carried out according to the method described by

Cappuccino and Sherman (1996).

Maintenance and preservation of

Actinomycetes

The marine actinomycetes were isolated as

pure culture by routine microbiological methods

and maintained on SCA slants. The spores were

stored in 15% glycerol under low temperature of -

20 oC (Williams and Cross, 1971).

Compound production from the Actinomycetes

strains

The medium used for secondary metabolite

production was ISP- 2 (International

Streptomycetes project) also called Yeast Malt

Agar. Yeast Malt Broth was prepared according to

the composition using sea water and sterilized by

autoclaving at 121°C at 15 lbs for 20 mins. The



medium was cooled and the loop full of individual

strains was taken and inoculated separately into

the Yeast Malt Broth. That broth was kept in an

orbital shaker for incubation for 10 days to obtain

for maximum growth of actinomycetes. The

culture was filtered and centrifuged at 10,000 rpm

for 10 min. The supernatant was collected and

addition of equal volume of ethyl acetate was

added. The mixture was kept in separating funnel

and shaken for 1 hour. After 24 hours the upper

layer was collected and kept in condensation at 40

ºC in round bottom flask. The extract was obtained

and it was carefully poured on to sterile petriplates

allowed for drying for 2 days. The plates were

scrapped using 1 ml of ethyl acetate and the

extract was transferred to sterile tubes.

Partial purification of secondary metabolites

The concentrated ethyl acetate extract was

subjected to TLC analysis using mobile phase

ethyl acetate: hexane in the ratio. The crude

extract was spotted on pre-coated silica sheet (5

cm × 1.5 cm) using capillary tube. After

separation of compounds, the TLC sheet was air

dried and visualized under UV and iodine.

Screening of antioxidant activity of

Actinomycetes

Estimation of Radical Scavenging Activity

(RSA) using DPPH assay

The RSA activity of the compound was

determined using DPPH assay according to

Nenadis and Tsimidou (2002), with small

modification. The decrease of the absorption at

517 nm of the DPPH solution after addition of the

antioxidant was measured in a cuvette containing

1 ml of 0.1 mM ethanolic DPPH solution was

mixed with 20 – 100 µg/ml of the extract (from

stock: 10 mg/ml of 5% DMSO). Two ml of DPPH

solution served as control. The ability to scavenge

DPPH radical was calculated by the following

equation.

% RSA =𝐴𝑏𝑠 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 − 𝐴𝑏𝑠 𝑠𝑎𝑚𝑝𝑙𝑒

𝐴𝑏𝑠 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 × 100

Abs control is the absorbance of DPPH

radical + ethanol: Abs sample is the absorbance of

DPPH radical + extract.

Phosphomolybdenum assay

The antioxidant activity of the compound

was evaluated by the green phosphomolybdenum

complex formation according to the method of

Prieto et al. (1999). An aliquot of 100 μl of sample

solution (20 – 100 µg/ml) was combined with 1 ml

of reagent solution (0.6 M sulphuric acid, 28 mM

sodium phosphate and 4 mM ammonium

molybdate) in a 4 ml vial. The vials were capped

and incubated in a water bath at 95 °C for 90 min.

After the samples had cooled to room temperature,

the absorbance of the mixture was measured at

695 nm against a blank.

Metal chelating activity

The chelating of ferrous ions by the

compound was estimated by the method of Dinis

et al. (1994). The sample solution (20 – 100

µg/ml) was added to a solution of 2 m mol/L

FeCl2 (0.05 ml). The reaction was initiated by the

addition of 5 m mol/L ferrozine (0.2 ml) and the

mixture was shaken vigorously and left standing at

room temperature for 10 min. Absorbance of the

solution was then measured

spectrophotometrically at 562 nm.

Hydroxyl radical scavenging activity

The scavenging activity of compound on

hydroxyl radical was measured according to the

method of Klein et al. (1992). The sample solution

(20 – 100 µg/ml were added with 1.0 ml of iron ‐ EDTA solution) (0.13% ferrous ammonium

sulfate and 0.26% EDTA), 0.5 ml of EDTA

solution (0.018 %), and 1.0 mL of dimethyl

sulphoxide (DMSO) (0.85% v/v in 0.1 M

phosphate buffer, pH 7.4). The reaction was

initiated by adding 0.5 ml of ascorbic acid (0.22%)

and incubated at 80 – 90 °C for 15 min in a water

bath. After incubation, the reaction was terminated

by the addition of 1.0 ml of ice‐cold TCA (17.5% w/v). Three milliliters of Nash reagent was added

and left at room temperature for 15 min. The

reaction mixture without sample was used as



control. The intensity of the color formed was

measured spectroscopically at 412 nm against

reagent blank. The % hydroxyl radical scavenging

activity was calculated by the following formula:

% HRSA = from [(A0 ‐ A1)/A0] × 100, where A0 is the absorbance of the control and A1 is the

absorbance of the extract/standard.

Cytotoxicity Assay on cancer cell lines

Chemicals and reagents

MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-

diphenyl tetrazolium bromide) in vitrogen, USA.

Acridine orange were obtained from Sigma, USA.

All other fine chemicals were obtained from

Sigma–Aldrich, St. Louis.

Cell culture

MCF 7 cells obtained from NCCS

(National Centre For Cell Science, Pune) were

cultured in Rose well Park Memorial Institute

medium (RPMI), supplemented with 10% fetal

bovine serum, penicillin/streptomycin (250 U/ml),

gentamycin (100 µg/ml) and amphotericin B (1

mg/ml) were obtained from Sigma Chemicals,

MO, USA. All cell cultures were maintained at

37◦C in a humidified atmosphere of 5% CO2.

Cells were allowed to grow to confluence over 24

hrs before use.

Cell growth inhibition studies by MTT assay

Cell viability was measured with the

conventional MTT reduction assay, as described

previously with slight modification. Briefly, MCF

7 cells were seeded at a density of 5 × 103

cells/well in 96 - well plates for 24 hrs, in 200 µl

of RPMI with 10% FBS. Then, culture supernatant

was removed and RPMI containing various

concentrations (1 – 100 µg/mL) of test compound

was added and incubated for 48 hrs. After

treatment cells were incubated with MTT (10 µl, 5

mg/ml) at 37 ◦C for 4 hrs and then with DMSO at

room temperature for 1 hr. The plates were read at

595 nm on a scanning multi-well

spectrophotometer. Data represented the mean

values for six independent experiments (Evelyn et

al., 2012).

Cell viability (%) = (Average test OD/Control

OD) × 100

3. Results and Discussion

In the present study, five strains of

actinomycetes were chosen from soil habitats. It

was possible to classify actinomycetes colonies to

the genus level while they are still in the primary

culture after sub culturing to obtain pure isolates,

the bulk of the isolates were classified in groups

having the following characters.

a) Formation of aerial mycelium, long chains

spores no sporangia

b) Formation of aerial mycelium, hyphae

having single spores, no sporangia.

c) Formation of substrate and aerial hyphae

forming spore chains, no sporangia.

All the isolates of actinomycetes are gram

positive, branched filamentous in nature producing

spores are in chains. All the isolates of

actinomycetes are non acid fast bacteria. The cells

are blue colour.

Diffusible pigment of Actinomycetes

isolates produce brown, yellow, pink and not

clear. The melanin pigment was demonstrated

following the procedure given in materials and

methods.

The biochemical characteristics were

studied and the actinomycetes strains were

maintained on starch casein agar slants by sub

culturing .

Screening of antioxidant activity

Estimation of Radical Scavenging Activity

(RSA) using DPPH assay

The results of DPPH assay are depicted.

The results showed that among the 8 strains

screened strain-8 showed maximum antioxidant

activity with percentage RSA of 81 %. Hence, the

strain - 8 was selected for other in vitro assays.



Phospho molybdenum assay

The ethyl acetate extract of strain - 8 was

used to determine its antioxidant capacities by the

formation of light green phosphomolybdenum

complex. We can observe from the table that

sample has moderate phosphomolybdenum

reducing potential with an increase in the OD to

value from 0.117 to 0.171.

Table – 1: Phosphomolybdenum reducing potential

S.

No

Concentration

(μg/ml)

OD

1 20 0.117

2 40 0.134

3 60 0.149

4 80 0.156

5 100 0.171

6 Control 0.093

Metal ion chelating assay

The decrease in concentration and

dependent colour formation in the presence of the

extract indicates that it has chelating activity. The

metal ion chelating activity was in the range of 24

– 77%.

Table – 2: Metal chelating potential of ABTRI 1008

S.

No

Concentration

(μg/ml)

% MCA

1 20 24.83

2 40 29.11

3 60 51.69

4 80 77.42

HRSA assay

The potential of the compounds to

scavenge hydroxyl radical was observed to be in

the range of 38 – 57 %.

Table – 3: Hydroxyl radical Scavenging potential

S.

No

Concentration

(μg/ml)

%HRSA

1 20 38.31

2 40 39.25

3 60 48.13

4 80 53.27

5 100 57.94

Production of antioxidant secondary

metabolites from strain-8

Secondary metabolite production media

was filtered and centrifuged at 10,000 rpm for 10

min. The supernatant was collected and equal

volume of ethyl acetate was added and kept in a

separating funnel & shaken for 1 hour. After 24

hours the upper layer was collected kept in

condensation for round bottom flask to obtain the

crude compound.

Partial purification of secondary metabolites –

Thin Layer Chromatography

The chromatogram developed with 10%

ethyl acetate in hexane revealed the presence of 2

major compounds at Rf value of 0.625 and 0.325

as visualized under UV illumination.

MTT Assay method

The results of MTT assay suggest that the

extract was capable of reducing cell viability of

selected cancer cell line. Also, the IC50 of the

selected extract was found to be 100 µg where the

cell viability was recorded as 52.24%.

S.

No

Concentration

(μg/ml)

Cell viability

(%)

1 1 96.66

2 10 95.37

3 20 84.34

4 50 64.26

5 100 52.24

Priya et al. (2012) explained about the

evaluation of antioxidant activity of secondary

metabolites of Streptomyces species isolated from

marine soil sample collected from the Bay of

Bengal coast of Puducherry, India. A similar study

was carried out with different isolates and the

isolates were evaluated for its antioxidant

activities. The sample was serially diluted and

plated on Starch casein agar and actinomycetes

were selectively isolated. The broth culture of the

International Streptomyces Project-1 (ISP-1)

medium was used for fermentation process and

extracellular metabolites were extracted using the

solvent ethyl acetate. The brown colored extract

obtained was dissolved in DMSO and screened for

DPPH radical scavenging activity. The secondary



metabolite showed 83 % inhibition at 0.2 mg/ml,

and the inhibition was compared with the standard

antioxidant ascorbic acid which showed 84%

inhibition at 0.2mg/ ml concentration. The

potential strain was identified as Streptomyces

species and designated as Streptomyces sp.

However, the secondary metabolite showed 81%

inhibition at 10 mg/ml.

4. Conclusion

Because of the immense biological

diversity in the sea as a whole, it is increasingly

recognized that a large number of novel chemical

entities exists in the oceans. As marine

microorganisms, particularly actinomycetes, have

evolved the greatest genomic and metabolic

diversity, efforts should be directed towards

exploring marine actinomycetes as a source for the

discovery of novel secondary metabolites. In this

respect, future success relies on our ability to

isolate novel actinomycetes from the marine

environments. Recent investigations using

enrichment techniques, new selection methods and

media have led to the isolation of novel

actinomycetes from sediment samples. Improved

recovery yields of marine actinomycetes from

sponges using nutrient supplements and enzymes

have been reported. These recent successes are the

first step in the right direction. Further

development work in improving isolation

strategies in the recovery of marine actinomycetes

is of utmost importance for ensuring success in

this area.

5. References

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6) Hayakawa, M. and Nonomura, H. 1987.

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