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Screening of Actinomycetes for Enzyme and


Antimicrobial Activities from the Soil Sediments
of Northern Tamil Nadu, South India
ARTICLE APRIL 2015
DOI: 10.1080/22311866.2015.1009385

8 AUTHORS, INCLUDING:
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John Poonga Preetam Raj

Loyola College

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I.V.s. Nimal Christhudas

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Available from: Nandagopal Murugan


Retrieved on: 04 September 2015

ISSN Print: 2231-1866


ISSN Online: 2231-1874

Screening of Actinomycetes for Enzyme and Antimicrobial Activities


from the Soil Sediments of Northern Tamil Nadu, South India
P. Praveen Kumar 1, J.P. Preetam Raj 1, I.V.S. Nimal Christhudas 1, R. Sagaya Jansi 1,
N. Murugan 2, P. Agastian*1, C. Arunachalam 3, Sulaiman Ali Alharbi 3
1

Research Department of Plant Biology and Biotechnology,


Loyola College, Chennai - 600 034, India
2
Department of Microbiology, L&T Microbiology Research Centre,
Sankara Nethralaya, Chennai, India
3
Department of Botany and Microbiology, College of Science,
King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia

Abstract: The present study involves on screening of actinomycetes for enzyme and antimicrobial
activities from the soil sediments of Northern Tamil Nadu, South India.Actinomycetes from the soil samples
were isolated by pour plate technique on Actinomycetes Isolation Agar. Morphological characters of the
isolates were studied based on the microscopic appearance. Actinomycetes isolates were screened for the
production of different extracellular enzymes like amylase, protease and lipase. In the screening for enzyme
activityof the actinomycetes strains showed amylase activity, protease activity and lipase activity.Preliminary
screening for antimicrobial activity of actinomycetes isolates were performed by cross streak plate method.
Active from of 82 isolated strains showed activity against one or more bacteria and exhibited potential antifungal
activity. The active strain was subjected to cultural, molecular identification and optimization of fermentation
medium, solvent extraction and showing antimicrobial activity for crude extracts. Based on the primary
antimicrobial proficiency, Loyola AR1 strain was selected for further studies. Ethyl acetate extract was showed
good antimicrobial activity when compared to butanol extract. The partial 16s rRNA sequence analysis of
Loyola AR1 isolate confirms it belongs to Streptomyces sp. and the sequence was submitted in the Gene Bank
(HM163569).
Key words: Soil samples, Enzyme activity, Antimicrobial, Streptomyces sp.
Introduction
Actinomycetes are a group of branching
unicellular organisms, which produce either by
fission or by means of special spores or conidia.
They are closely related to bacteria; frequently,
they are considered as higher, filamentous bacteria
1
. Actinomycetes constitute a considerable proportion of the population from soil, lakes and river
muds. Traditionally actinomycetes have been

isolated from terrestrial sources, although the first


report of mycelium forming actinomycetes being
recovered from marine sediments appeared
several decades ago 2. Actinomycetes have the
capacity to synthesize many different biologically
active secondary metabolites such as cosmetics,
vitamins, nutritional materials, herbicides, antibiotics, pesticides, anti-parasitic and enzymes like
cellulase and xylanase used in waste treatment 3.

*Corresponding author (P. Agastian)


E-mail: < agastianloyolacollege@gmail.com; agastian@loyolacollege.edu > 2015, Har Krishan Bhalla & Sons

Moreover, these are important source for novel


antibiotics and hence having a high pharmacological and commercial interest including control
of infectious diseases 4-6. Most of the known
natural antibiotics are produced from actinomycetes 7. Among actinomycetes, around 7600
compounds are produced by Streptomyces
species. Many of these secondary metabolites are
potent antibiotics, which has made streptomycetes
the primary antibiotic-producing organisms
exploited by the pharmaceutical industry 8, 9.
Streptomyces are saprophytic and are
commonly associated with soils, where they
contribute significantly to the turnover of complex
biopolymers and antibiotics 10. Since the discovery
of actinomycin, the first antibiotic from an
actinomycetes, many commercially important
bioactive compounds and anti-tumor agents has
been produced using actinomycetes 11,12. Most of
the antibiotics in use today are derivatives of
natural products of actinomycetes and fungi 13, 14.
Thus screening and isolation of promising strains
of actinomycetes for potential antibiotics production is a thrust area of search since many
years. As there is a geographic variation in Indian
soil type and their contents, hence it is quite likely
that the distribution of antibiotic producing
actinomycetes is also variable. Therefore,
exploration of unexplored ecosystems for
actinomycetes is necessary for the identification
of novel antimicrobial metabolites. The objective
of the present study was isolation, characterization
of actinomycetes from different soil sediments
(Northern Tamil Nadu, South India) and
preliminary screening for enzyme and antimicrobial activities.
Materials and methods
Sample collection
Soil samples were collected from different
locations in Northern Tamil Nadu, South India. It
includes two main districts Thiruvallur (Ambattur,
Porur and Ennore) and Kancheepuram (St.
Thomas Mount, Vandalur and Vedanthangal). The
soil samples were collected from lake soils, bird
sanctuary, termite and soils under rock. The
collected samples were brought to the laboratory
for isolation of actinomycetes and the locations,
nature of sample were recorded.

Isolation of actinomycetes
Actinomycetes from the soil was isolated by
pour plate technique on Actinomycetes Isolation
Agar (AIA) containing: Sodium caseinate 2g, LAsparagine 0.1g, Sodium propionate 4g,
Dipotassium phosphate 0.5g, Magnesium
sulphate 0.1g Ferrus sulphate 0.001g, Agar 15g
also supplemented with actidione 20 mg/l and
nalidixic acid 100 mg/l to minimize the other
fungal and bacterial growth respectively. Soil
samples were serially diluted up to 10-5 and 0.1
ml of aliquots were spread over actinomycetes
isolation agar plates. The plates were incubated
at 282C for 7 days. Strains of actinomycetes
were picked out and purified by repeated
streaking on yeast extract-malt extract agar
(ISP2) and were preserved in slants at 42C 15
for further identi-fication and antimicrobial
screening study.
Morphological characterization
The microscopic morphology of actinomycetes
strains such as formation of aerial and substrate
mycelium and spore management, which are
highly characteristic and useful in the identification of actinomycetes, were observed by cover
slip technique with light microscopy 16.
Preliminary screening of enzyme activity
The actinomycetes isolates were screened for
the production of different extracellular enzymes
like amylase, protease and lipase.
Amylase activity
Actinomycetes isolates were screened for
amylolytic properties by starch hydrolysis test on
starch agar plate. The streaked actinomycetes
isolates were incubated at 282C for 7 days. After
the incubation 1% iodine solution (freshly prepared) was flooded on the starch agar plates and
a clear zone of hydrolysis were considered as
amylase producers 17.
Protease activity
Actinomycetes isolates were streaked on Skim
milk agar plates (SMA) and plates were incubated
at 282C for 7 days. A clear zone of skim milk
hydrolysis gave an indication of protease producing
organisms 18.

Lipase activity
Peptone tween agar (PTA) plates were prepared according to the method described by
Gopinath et al.19. Actinomycetes isolates were
streaked on Peptone tween agar plates and plates
were incubated at 282C for 7 days. The
appearance of visible opalescent structures was
used as an indication of lipolytic activity.
Preliminary screening for antimicrobial activity
Preliminary screening for antimicrobial activity
of actinomycetes isolates were performed by
cross streak plate method on modified nutrient
glucose agar (MNGA) 20. The actinomycetes
isolates were inoculated in a straight line on MNGA
plates and incubated at 282C for 7 days. The
selected Gram positive Bacillus subtilis (MTCC
441), Staphyloccous aureus (ATCC 25923),
MRSA Clinical pathogen (15DR), Staphyloccous
aureus (Methicilin sensitive), Gram negative
Escherichia coli (MTCC 40), Enterobacter
aerogens (MTCC 111), Vibrio parahaemolyticus
(MTCC 451), Yersinia enterocolitica (MTCC
840), Xanthomonas pv. oryzae (MTCC 2760),
Vibrio fischeri (MTCC 1738), Enterobacter
feacalies (MTCC 29212), Pseudomonas
aeruginosa (ATCC 15380) bacterial and fungal
like Botrytis cinerea (MTCC 359), Aspergillus
niger (MTCC 478), Curvularia lunata (MTCC
2098) and Aspergillus flavus (MTCC 9390). The
plates were then inoculated with the test
organisms by a single streak at 90o angles to the
actinomycetes strains and incubated at 37oC
overnight for bacteria and at 282C for 96 h for
fungus. Antagonism was observed by the inhibition
of test organism.
Cultural characterization
Cultural properties and growth characteristics
of the active Loyola AR1 strain was studied on
various defined culture media such as
Actinomycetes isolation agar (AIA), Modified
nutrient glucose agar (MNGA), Nutrient agar
(NA), Skim milk agar (SMA), Starch casein agar
(SCA), Starch agar (SA), Tryptone yeast extract
agar (ISP1), Yeast extract malt extract agar
(ISP2) and Inorganic salts starch agar (ISP4). The

plates were incubated at 282C and observations


were recorded after 7 days. Colony morphology
including colour of aerial mycelium, reverse side
colour and diffusible pigments of the isolates were
observed as per recommendations by ISP 21.
Scanning electron microscopy (SEM) analysis
The selected Loyola AR1 strain was grown for
7 days in Actinomycetes isolation agar. Cells were
then fixed by adding 3.7 % formalin and washed
three times with two volumes of deionized water.
Resuspended cells were spotted on a glass slide,
flash frozen in a bath of 2-methylbutane in liquid
nitrogen, freeze-dried and then sputter coated with
gold-palladium alloy under vacuum. Sample was
visualized under Scanning Electron Microscope.
Media selection for fermentation
Fermentations were carried out using different
media like Actinomycetes broth, Modified nutrient
glucose medium, B6 Medium, Bennet Medium,
Streptomyces medium, Antibiotic production
medium. A loopful of selected strain was
inoculated in 100 ml fermentation broth and
incubated on an orbital shaker for 120 rpm at
282C for 24h. Later, the fermentation broth was
centrifuged and the supernatant was tested for
antibacterial activity against test organisms.
Secondary screening of antibacterial activity
Test organism
The selected test organisms were MRSA
Clinical pathogen (15DR), Enterobacter
aerogens (MTCC 111), Bacillus subtilis (MTCC
441) and Salmonella typhi (MTCC 733). An
inoculum of each bacterial strain was inoculated
in 3 ml of Muller Hinton Broth (MHB) and
incubated at 37C for 24 h.
Kirby-Bauer method
Antibacterial activities were assayed by KirbyBauer method 22. Petri plates were prepared with
20 ml of sterile Muller Hinton Agar (MHA). The
test cultures (100 l of suspension containing 108
CFU/ml bacteria) were swabbed on the top of
the solidified media and allowed to dry for 10 min
followed by well was made using sterile cork borer
and filled with 0.05 ml of supernatant. The plates

were left for 30 min at room temperature for supernatant diffusion. The plates were incubated for
24 h at 37C. Zone of inhibition was recorded in
millimetres and the experiment was repeated
twice.
Fermentation medium
After the medium optimization, selected strain
was inoculated into 50 ml of Modified nutrient
glucose broth (MNG) flasks and was kept in the
orbital shaker at a speed of 120 rpm at 282C
for 24 h. After that 10 % of grown culture was
used as inoculum for production medium in 250
ml Erlenmeyer flask containing 100 ml of
fermentation medium. The seven days grown
culture broth was collected and filtered through
Whatman No.1 filter paper.
Extraction
The bioactive metabolites were recovered from
the harvested medium by solvent extraction, using
ethyl acetate and butanol. The low polar to high
polar solvent was selected for the organic solvent
extraction. Three folds volume of the solvent was
mixed thoroughly with the broth by shaking them
in 1000 ml capacity and was taken in a separating
funnel and shaken vigorously. Extraction was
continued up to three times with the respective
solvents. The solvents were removed using
vacuum rotary evaporator and extracts were
stored at 4C until further use.
Antibacterial activity
Test organisms
The following test organisms were used for disc
diffusion method against the extracts: Bacillus
subtilis (MTCC 441), Enterobacter aerogens
(MTCC 111), MRSA clinical pathogens (15DR),
Pseudomonas aeruginosa (ATCC 27853),
Staphylococcus aureus (ATCC 25923),
Salmonella typhi (733), Yersinia enterocolitica
(MTCC 840), Vibrio fischery (MTCC 1738),
Enterococcus faecalis (MTCC 29212), Vibrio
parahaemolytics (MTCC 451) were used for the
experiment.
Disc diffusion method
Antibacterial activity of the crude extract was

carried out using disc diffusion method. Petri


plates were prepared with 20 ml of sterile Muller
Hinton agar (MHA, Himedia). The test organisms
were swabbed on the media. The crude extracts
1.25, 2.5, 5 mg/disc concentrations were used.
The loaded discs were placed on the surface of
the medium and streptomycin (10 mg/disc) was
used as positive control. The plates were incubated over night at 37C and the antagonism was
observed by zone of inhibition of test organism 22.
Antifungal activity
Test organisms
The following fungi were used for the experiments: Aspergillus flavus (MTCC 9390),
Aspergillus niger (MTCC 478), Botrytis cinerea
(MTCC 359) Curvularia lunata (MTCC 2098),
Candida albicans (MTCC 227), Trichophyton
rubrum (MTCC 296), Trichophyton mentagrophytes (MTCC 8476) were used to determine
minimum inhibitory concentration (MIC)
Minimum inhibitory concentration
The antifungal activity and minimum inhibitory
concentration (MIC) were performed according
to reference method 23. Extracts were dissolved
in 2 % dimethyl sulphoxide (DMSO). The initial
test concentration (2000 g/ml) was serially diluted
two-fold. Each well was inoculated with 5 l of
suspension containing ~104 spore/ml of fungi. The
antifungal agent fluconazole was included in the
assays as positive control. Plates were incubated
for 48h at 282C. MIC was determined as the
lowest concentration of the extracts inhibiting the
visual growth of the test cultures. Three replications were maintained to confirm the antifungal
activity.
Identification of Actinomycetes strain
Genomic DNA was extracted by using Hi-pura
Streptomyces DNA spin kit-MB 527-20pr from
Hi-media. The 16S ribosomal RNA was amplified
by using the thermo cycler (ependorfep. Gradient)
with Taq DNA polymerase and primers 27F (5
AGTTTGATCCTGGCTCAG 3) and 1492R
(5ACGGCTACC TTGTTACGACTT 3). The
conditions for thermal cycling were as follows:
denaturation of the target DNA at 94C for 4 min

followed by 30 cycles at 94C for 1 min, primer


annealing at 52C for 1 min and primer extension
at 72C for 1 min. At the end of cycling, the
reaction mixture was held at 72C for 10 min and
then cooled to 4C. The PCR product obtained
was sequenced by an automated sequencer
(Genetic Analyzer3130, Applied Biosystems, and
USA). The obtained sequences were subjected
to BLAST at http://www.ncbinlm-nih.gov/search
in NCBI database for phylogenetic relationship.
16S rRNA sequence was then submitted to the
GenBank, NCBI, USA 24.
Results
In the present study, the pharmaceutically
significant actinomycetes were isolated from six
diverse soil samples from Thiruvallur and

Kancheepuram districts (Table 1) and selected


for their antimicrobial activities.
Isolation and morphological identification of
Actinomycetes
A total number of 82 different actinomycetes
were isolated from different places and soil
samples (Fig.1). All the strains were isolated on
AIA media supplemented with Actidione and
Nalidixic acid to inhibit common contaminants like
fungi and bacteria respectively with the suitable
culture conditions. The number of actinomycetes
isolates from the soil samples of Kancheepuram
District (57) was more when compared to
Thiruvallur District (25) (Table 2). The morphological characters of the isolates were studied
based on the microscopic appearance after gram

Fig. 1. Sampling sites from Northern Tamil Nadu


Table 1. Different Soil sample collection from Northern Tamil Nadu
Locations
Ambattur Lake soil
Porur Lake soil
Ennore Lake soil
Vandalur Termite soil
St. Thomas Mount soil
Vedanthangal Bird Sanctuaries soil

Latitude

Longitude

130636" N
130342" N
132175" N
128792" N
130036" N
123244" N

801012" E
801506" E
803216" E
800817" E
801933" E
795121" E

District
Thiruvallur District

Kancheepuram District

Table 2. Soil sampling sites and occurrence of actinomycetes in soil samples


Place

Sample
Code

Ambattur Lake soil


Porur Lake soil
Ennore Lake soil
Vandalur Termite soil
St. Thomas mount soil
Vedanthangal Bird
Sanctuaries soil

Loyola AR
Loyola PR
Loyola EES
Loyola VT
Loyola ST
Loyola VBS

staining which were found to be all gram positive.


Preliminary screening of enzyme activity
The isolated actinomycetes were subjected to
screening for enzyme activities. Amylase,
Protease and Lipase enzymes were screened on
Starch, Skim milk and Peptone tween agar
medium respectively. Amylase producing strains
were identified based on the development of clear
area against dark blue plates. Protease producing
strains were identified by formation of clear area
in skim milk agar plates. Lipase producing strains

Number of
Actinomycetes
08
15
02
15
20
22

District

Thiruvallur District

Kancheepuram District

were identified by formation of opalescent


structures around the colonies. The results of
primary screening of enzymes were showed in
(Fig. 2).
Preliminary screening for antimicrobial
activity
A total of 82 actinomycetes strains were
selected for their antimicrobial activity against
various bacteria and fungi. All the actinomycetes
showed antimicrobial activity against either
bacteria or fungi in preliminary screening. Among

Fig. 2. Preliminary screening of Enzyme activity

the 82 strains, 41 strains (50 %) of the isolates


showed more activity against one or more
bacteria. The maximum antibacterial activity was
exhibited in 22 isolates against Staphylococcus
aureus (26.82 %), 17 isolates exhibited activity
against Bacillus subtilis (20.73 %), 10 isolates
exhibited activity against Enterococcus faecalis,
methicillin resistant Staphylococcus aureus,
Yersinia enterocolitica (12.19 %), 9 isolates
exhibited activity against Enterobacter aerogens
(10.97 %), 8 isolates exhibited activity against
methicillin sensitive Staphylococcus aureus and
Vibrio fischery (9.75 %), 7 isolates exhibited activity against E. coli and Pseudomonas aeruginosa (8.53 %), 5 isolates exhibited activity
against Vibrio parahaemolyticus (6.09 %) and

4 isolates exhibited minimum activity for Erwenia


sp. (4.87 %) (Fig. 3). Out of 82 strains, merely
30.48 % isolates displayed potential antifungal
activity in preliminary screening. The maximum
antifungal activity was exhibited by 19 actinomycetes against Curvularia lunata (23.17 %),
3 isolates showed activity against Aspergillus
flavus (3.65 %), 2 isolates showed activity against
Aspergillus niger (2.43 %) and minimum activity
was recorded in Botrytis cinerea (1.21 %) as
shown in Fig.4. Based on the significant results,
Loyola AR1 strain showed potential activity
against all the tested bacteria and fungi. Hence,
the Loyola AR1 strain was considered for further
exploration to identify the strain and for the
production of bioactive metabolites.

Fig. 3. Preliminary screening of antibacterial activity

Fig. 4. Preliminary screening of antifungal activity

Cultural characteristics and scanning electron microscopic analysis


The antagonistic activity of Loyola AR1 isolate
was used to evaluate cultural characteristics, AR1
strain grew well on AIA, MNGA, NA, SMA,
SCA, SA, ISP1, ISP2 and ISP4 medium. The
occurrence of mycelium and soluble pigments and
growth characteristics of the isolate is shown in
Table 3. Scanning electron microscopic studies
for Loyola AR1 that showed spore ornamentation
(Fig. 5).
Media optimization for fermentation and
solvent extraction
Various media such as Actinomycetes broth
(Shaking and Still culture), Modified nutrient
glucose medium, B6 Medium, Bennet Medium,
Streptomyces medium, Antibiotic production
medium were used for the production for bioactive
metabolites by Loyola AR1 strain. Among these,
MNG broth showed notable activity against tested
bacteria. MRSA15DR showed 24 mm, Enterobacter aerogens showed 28 mm, Bacillus
subtilis showed 38 mm and Salmonella typhi
with 25 mm zone of inhibition respectively (Table
4). Among the media screened, the isolate Loyola
AR1 showed good antimicrobial activity in MNG
broth. Further the isolate Loyola AR1 was mass
produced using the optimized media (MNG broth)
and the culture filtrate was subjected to solvent

extraction using ethyl acetate and butanol.


Antibacterial activity by disc diffusion method
The antibacterial efficacy was tested with ethyl
acetate and butanol extracts. Among this, ethyl
acetate extract produced maximum inhibition zone
against pathogenic bacteria. Ethyl acetate extract
of the Loyola AR1 strain showed maximum
activity against Yersinia enterocolitica (25 mm),
Staphylococcus aureus (18 mm), Bacillus
subtilis (20 mm), Enterobacter aerogens (17
mm), Pseudomonas aeruginosa, Enterococcus
faecalis (15 mm), Proteus vulgaris (14 mm),
MRSA 15DR (12 mm), Salmonella typhi (10 mm)
and Klebsiella pneumoniae (8 mm) that
displayed in Table 5.
Antifungal activity-minimum inhibitory
concentration
Antifungal activity of ethyl acetate and butanol
extracts of Loyola AR1 strain was witnessed to
inhibit the growth of tested fungal pathogens. The
MIC of the ethyl acetate extract exhibited activity
against Botrytis cinerea, Aspergillus flavus,
Trichophyton mentagrophytes, Curvularia
lunata, Aspergillus niger (125 g/ml) and
Trichophyton rubrum (62.5 g/ml). Butanol
extract showed activity against Botrytis cinerea,
Aspergillus flavus, Trichophyton rubrum (125
g/ml), Trichophyton mentagrophytes, Curvu-

Fig. 5. Scanning Electron Microscope analysis of Loyola AR1 strain

Good

Good
Good
Good

Good

Good

Good

Good

Good

AIA

MNGA
NA
SMA

SCA

SA

ISP1

ISP2

ISP4
No pigment

No pigment

No pigment

No pigment

No pigment

No pigment
No pigment
Light yellow

No pigment

Soluble pigment
colour
White substrate
with pink spores
Substrate mycelium
Substrate mycelium
Substrate mycelium
with white spores
Substrate mycelium
with white spores
Substrate mycelium
with white spores
Substrate mycelium
with white spores
Substrate mycelium (Creamy
White) with white spores
Substrate mycelium
with pink spores

Mycelium and
Spore colour

Filamentous

Circular

Irregular

Filamentous

Circular

Circular
Circular
Circular

Circular

Colony
form

Convex

Flat

Pulvinate

Convex

Raised

Light raised
Flat
Raised

filamentous

Colony
elevation

Lobate

Erose
(Serrated)
Entire

Lobate

Erose

Umbonate
Filamentous
Lobate
Curled
Undulate

Colony
margin

NA-No Activity

NA
15
22
12

38
25

Actinomycetes
Broth
(Still culture)

24
28

MNG
Broth

MRSA 15DR
Enterobacter
aerogens
Bacillus subtilis
Salmonella typhi

Test
organisms

NA
NA

NA
22

M6
Broth

25
NA

NA
19

Bennet
Broth

36
10

NA
NA

Streptomyces
Broth

25
NA

NA
23

Antibiotic
production
Broth

35
14

19
26

Actinomycetes
Broth

NA
NA

NA
NA

Control

Table 4. Medium optimization for bioactive compounds in Loyola AR1 strain by Kirby-Bauer method (zone of inhibition in mm)

Growth on
Medium

Culture
Media

Table 3. Cultural characteristics of Loyola AR1 strain on Different Agar medium

Table 5. Antibacterial activity of Ethyl acetate and Butanol


extract of Loyola AR1 by Disc diffusion method
Zone of Inhibition (in mm)
Ehtyl acetate
Butanol extract
extract (mg/disc)
(mg/disc)
1.25
2.5
5
1.25
2.5
5

Test

Bacillus subtilis
Enterobacter aerogens
MRSA 15DR
Pseudomonas aeruginosa
Staphylococcus aureus
Salmonella typhi
Yersinia enterocolitica
Klebsiella pneumoniae
Enterococcus faecalis
Proteus vulgaris

NA
NA
NA
NA
NA
NA
NA
NA
NA
NA

18
15
12
14
18
12
25
8
13
11

laria lunata and Aspergillus niger (62.5 g/ml)


respectively. Fluconazole was used as positive
control (Table 6).
Molecular identification of AR1 strain
Genomic DNA of the Loyola AR1 strain was
isolated and 16S rRNA gene was PCR amplified
with specific forward and reverse primers. The
BLAST search analysis revealed 98 % homology
with the genus Streptomyces sp. The 16S rRNA
sequence of Streptomyces sp. Loyola AR1 was
submitted to the Gen bank and the accession
number was given (HM163569).
Discussion
Actinomycetes are incomparable sources of

20
17
15
18
10
25
8
15
14

NA
NA
NA
NA
NA
NA
12
12
09
NA

11
11
NA
NA
NA
NA
14
NA
12
8

S10

12
12
10
NA
10
NA
14
NA
12
14

many important bioactive compounds with high


commercial value and continue to be regularly
screened for new bioactive compounds 25. This
exploration has been remarkably successful and
many naturally arising antibiotics and enzymes of
therapeutic significance have been isolated from
actinomycetes 26.
Gram positive actinomycetes are of special
interest, since they are capable to produce
chemically diverse compounds with wide range
of biological activities. In the present study 82
actinomycetes strains were isolated from different
parts of Thiruvallur and Kancheepuram districts
of Northern Tamil Nadu, India. All the isolates
were assessed for their antimicrobial activity.
Actinomycetes isolation agar (AIA) media

Table 6. Antifungal activity of Ethyl acetate and Butanol extract of


Loyola AR1 by Minimum inhibitory concentration (MIC)

Test
organisms

MIC 2000 g/ml


Ethyl acetate
Butanol
extract
extract

Botrytis cinerea
125
Aspergillus flavus
125
Trichophyton rubrum
62.5
Trychophyton mentagrophytes 125
Curvularia lunata
125
Aspergillus niger
125

37
29
30
36
27
38
40
8
24
28

125
125
125
62.5
62.5
62.5

Fluconazole
(STD)
25
12.5
25
25
25
12.5

amended with Actidione and Nalidixic acid were


used for the isolation of actinomycetes 15.
Morphological characteristics of actinomycetes
strains were identified according to Bergeys
Manual of Determinative Bacteriology 27. In the
course of preliminary enzyme activity of 82
actinomycetes strains, 29 % of the actinomycetes
strains revealed amylase enzyme activity and 14
% of the strains showed protease enzyme activity
and 57 % of the strains exhibited lipase activity.
Studies on enzymatic activity of the isolates
showed that approximately 90 % of the isolates
produced one or more enzyme activity 28-30. This
states that actinomycetes have latent to create
extensive range of enzymes, which might be the
outcome from natural selection of microorganism
in order to survive in competing environment 31.
In preliminary screening of antimicrobial activity,
out of 82 isolates, one strain exhibited good antimicrobial activity, five strains revealed moderate
activity and other strains showed less activity.
Based on the antimicrobial proficiency Loyola AR1
strain was selected for further studies. Similar
result was report by Sarvana Kumar et al.15 that,
five actinomycetes strains were isolated from
Kancheepuram district, Tamil Nadu and was
screened for their antibacterial activity. This states
that actinomycetes have latent to create extensive
range of enzymes, which might be the outcome
from natural selection of micro-organism in order
to survive in competing environment 31.
Microbes are largely characterized on the basis
of their cultural characters. Cultural characteristics
of Loyola AR1 strain such as colour, consistency,
the absence or presence of mycelium and extent
spore formation, diffusible pigment, colony form,
colony elevation and colony margin on different
media were studied. Scanning electron microscopic photographs of the Loyola AR1 strain
showed a branched mycelium and small globose
sporangia on aerial mycelium. Sporangio spores
are formed by septation of hypha within sporangium.
Cultivation of dissimilar complex media
signalized the capability for secondary metabolite
production. Six different fermentation media were
used for the selection of superlative antimicrobial
metabolite production. Where, MNG broth served
as the best base for the antimicrobial metabolite

production by the isolate Loyola AR1. The


biosynthesis of antibiotic substances was found
to be largely influenced by the composition of the
medium 32 both quantitatively and qualitatively.
The antagonistic isolate Loyola AR1 was grown
on MNG broth for investigating its potency to
produce antimicrobial agents. The bioactive
compounds were extracted from natural sources
through several techniques. Solvent extraction is
usually employed for the extraction of secondary
metabolites from culture filtrates. Different
polarities of organic solvents have been utilized
for the extraction of bioactive compounds from
the isolate of actinomycetes. The extract from
ethyl acetate showed maximum antimicrobial
activity against Gram-positive and Gram-negative
bacteria and also fungi, where as other solvent
extracts showed moderate activity against the
tested organisms 33. Streptomyces CDRIL-312
ethyl acetate extract that showed increased
antimicrobial activity against tested organisms.
Further, genomic DNA preparation and 16S
rRNA PCR amplification followed by sequencing
of the strain designated as Streptomyces sp.
Loyola AR1 which was submitted to the GenBank
(HM163569).
Conclusion
Actinomycetes isolated from Thiruvallur and
Kancheepuram districts of Northern Tamil Nadu,
South India soil samples revealed more antimicrobial properties. All the 82 isolated actinomycetes were screened for antimicrobial and
enzyme activities. Loyola AR1 strain showed more
antimicrobial properties, when grown in MNG
broth. The ethyl acetate extract of Loyola AR1
strain showed maximum inhibition against diverse
bacteria and fungi tested.Actinomycetes are one
of the most attractive sources of antibiotics and
otherbiologically active substances such as antibiotics, anti-tumor agents, herbicides, pesticides
and enzymes with high commercial values. The
present study involves on bioprospecting of
actinomycetes isolated from soil sediments of
northern Tamil Nadu, South India
Conflict of interest
The authors declare that there are no conflicts
of interest.

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RESEARCH ARTICLE

Open Access

In vitro antimicrobial, antioxidant and cytotoxic


properties of Streptomyces lavendulae strain SCA5
Pachaiyappan Saravana Kumar1, Naif Abdullah Al-Dhabi2, Veeramuthu Duraipandiyan2,
Chandrasekar Balachandran1, Panthagani Praveen Kumar3 and Savarimuthu Ignacimuthu1,2,4*

Abstract
Background: Actinomycetes are Gram-positive, often filamentous, bacteria known for their unsurpassed capacity
for the production of secondary metabolites with diverse biological activities. The aim of the present study was to
evaluate the antimicrobial, cytotoxic and antioxidant properties of Streptomyces lavendulae strain SCA5.
Results: The ethyl acetate extract of SCA5 broth (EA-SCA5) showed antimicrobial activity with MIC value of 31.25 g/ml.
EA-SCA5 showed good antioxidant potential by scavenging 2, 2-diphenyl-picrylhydrazyl (DPPH) (IC50 507.61 0.66 g/ml),
hydroxyl radical (IC50 617.84 0.57 g/ml), nitric oxide (IC50 730.92 0.81 g/ml) and superoxide anion radical
(IC50 864.71 1.15 g/ml). The EA-SCA5 also showed strong suppressive effect on rat liver lipid peroxidation
(IC50 838.83 1.18 g/ml). The total phenolic content of SCA5 was 577.12 mg of GAE equivalents/gram extract.
EA-SCA5 exhibited cytotoxic activity on A549 adenocarcinoma lung cancer cell line. It showed 84.9% activity at
500 g/ml with IC50 value of 200 g/ml. The gas chromatography mass spectrometry (GC-MS) analysis revealed the
presence of one major bioactive compound actinomycin C2.
Conclusions: The results of this study indicate that the EA-SCA5 could be probed further for isolating some medically
useful compounds.
Keywords: SCA5, Antimicrobial, Antioxidant, Cytotoxicity

Background
Development of multiple drug resistance in microbes and
tumour cells has become a major problem; there is a need
to search for new and novel anticancer antibiotics [1]. Free
radicals are known to be the major cause of various chronic
and degenerative diseases, including aging, coronary heart
disease, inflammation, stroke, diabetes mellitus and cancer.
Oxidative stress occurs when there is excessive free radical
production and/or low antioxidant defence, which leads to
chemical alterations of biomolecules causing structural and
functional modifications [2]. Currently available synthetic
antioxidants show low solubility, promote negative health
and have moderate antioxidant activity [3]. Over the past
75 years, natural compounds have led to the discovery of
* Correspondence: eriloyola@hotmail.com
1
Division of Microbiology, Entomology Research Institute, Loyola College,
Chennai 600 034, India
2
Department of Botany and Microbiology, Addiriyah Chair for Environmental
Studies, College of Science, King Saud University, P.O. Box. 2455, Riyadh
11451, Saudi Arabia
Full list of author information is available at the end of the article

many drugs in the treatment of numerous human diseases


[4]. By employing sophisticated techniques under various
screening programs, the rate of discovery of natural compounds has exceeded 1 million so far [5]. Out of 22,500 biologically active compounds that have been extracted from
microbes, 45% are from actinobacteria, 38% are from fungi
and 17% are from unicellular bacteria [6]. Microbial secondary metabolites are one of the immense reservoirs of
natural chemical diversity with potent biological activity [7].
Actinomycetes represent one of the most studied and
exploited classes of bacteria for their ability to make a wide
range of biologically active metabolites [8]. However, studies on microorganisms with respect to antioxidant and free
radical scavenging activities are very limited. The studies on
streptomycetes with respect to free radical scavenging activity are very few and most of the streptomycetes isolated are
yet to be screened for bioactive secondary metabolites. The
compounds isolated from marine Streptomyces, 2allyoxyphenol and streptopyyrolidine, have been reported
to possess antioxidant and no cytotoxic activity [9,10]. With
todays interest in new renewable sources of chemicals,

2014 Saravana Kumar et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the
Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public
Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this
article, unless otherwise stated.

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polymers, anticancer, antioxidant and antimicrobial agents,


the extracellular extract from soil microorganisms represent
potential source to be explored. In this study, the antimicrobial, antioxidant and cytotoxic properties of extracellular crude extracts isolated from the fermented broth of
soil-associated Streptomyces lavendulae strain SCA5 were
investigated.

Methods

Page 2 of 12

media for the production of bioactive compounds in an


orbital shaker (150 rpm at 30C): Antibiotic production
media (APM), Fermentation media (FEM), Glucose yeast
extract malt media (GLM), M3 media, Modified nutrient
glucose media (MNGA), M6 media and Yeast peptone
glucose media (YPG). The culture was grown with continuous shaking on a rotary shaker (150 rpm) at 30C for
10 days. The antimicrobial activity was tested for fermented broth against microbes using [13].

Isolation

The actinomycetes used in this work were isolated from


soil samples collected from Vengodu (agricultural field),
Thiruvannamalai district, Tamil Nadu, India (Latitude: 12
580033, North; Longitude: 79 705216, East; Elevation
ft/m 228.6/70.0). The actinomycetes isolation was carried
out using the plating technique with serial dilution.
Aliquots (0.1 ml) of 102, 103, 104, and 105 were spread
on the starch casein agar (Himedia, Mumbai). To minimize
the fungal and bacterial growth, actidione 20 mg/l and nalidixic acid 100 mg/l were added [11].
Microbial organisms

The following Gram positive and Gram negative bacteria


and fungi were used for the experiment. Gram positive:
Staphylococcus aureus MTCC 96, Micrococcus lutues
MTCC 106, Bacillus subtilis MTCC 441, Staphylococcus
epidermis MTTC 3615, and Methicillin resistance Staphylococcus aureus (MRSA). Gram negative: Klebsiella pneumoniae MTCC 109, Enterobacter aerogens MTCC 111, Vibrio
parahaemolyticus MTCC 450, Yersinia enterocolitica MTCC
840, Salmonella typhimurium MTCC 1251, Shigella flexneri
MTCC 1457, Proteus vulgaris MTCC 1771, Salmonella
typhi-B (SPB). Fungi: Aspergillus flavus (AF), Botrytis cinerea
(BC), Candida.krusei (CK), Candida parapsilosis (CP),
Malassesia pachydermatis (MP), Scopulariopsis sp. (57), Trichophyton mentagrophytes (66), Trichophyton rubrum (101),
Candida albicans (227), Aspergillus niger (1344). The reference bacterial cultures were obtained from the Institute of
Microbial Technology (IMTECH), Chandigarh, India-160
036 and all the fungal cultures were obtained from the
Department of Microbiology, Christian Medical College,
Vellore, Tamil Nadu, India. Bacterial inoculums were prepared by growing cells in Mueller Hinton broth (MHB)
(Hi-media) for 24 h at 37C. The filamentous fungi were
grown on Sabouraud dextrose agar (SDA) slants at 28C for
10 days and the spores were collected using sterile double
distilled water and homogenized. Yeast was grown on
Sabouraud dextrose broth (SDA) at 28C for 48 h.
Cross streak method and media Optimization

The antimicrobial activity of actinomycetes isolates was


performed by using cross streak method [12]. Antagonism
was observed by the inhibition of test organism. Streptomyces lavendulae strain SCA5 was grown on the following

Culture characterization

Cultural and morphological features of SCA5 were


characterized by following [14]. Visual observation by
light microscopy and Gram-staining were performed for
further identification [15]. Biochemical reactions, different temperatures, NaCl concentration, pH level, pigment production and acid or gas production were done
following the methods [16]. The total genomic DNA
was extracted by using Hipura Streptomyces DNA spin
kit-MB 527-20pr from Hi-media, according to the manufacturers protocol. The actinomycetes DNA fragments
were amplified using Universal primers 16S rRNA and
PCR reactions were standardized as follows: initial denaturation at 94C for 3 min, followed by 35 cycles of
1 min at 94C, 54C for 1 min, 72C for 2 min and a final
extension at 72C for 810 min, stop at 4C for 1 h. The
PCR products were stored at 4C and visualized by electrophoresis. The gel was photographed in gel documentation system. The amplified product was purified and
sequenced with two fragments of the 27F (5AGT TTG
ATC CTG GCT CAG 3) and 1492R (5ACG GCT
ACC TTG TTA CGA CTT 3) region in both the directions and the sequences obtained were submitted to
Genbank. Phylogenetic tree was constructed using the
neighbour-joining DNA distance algorithm using software MEGA (version 4.0) [17].
Cultivation and extraction of antimicrobial metabolites
from Streptomyces lavendulae strain SCA5

Well grown slant culture of the Streptomyces lavendulae


strain SCA5 was used for the preparation of seed culture. The seed culture was inoculated in 50 ml medium
containing the optimized production media and incubated for 10 days in a rotary shaker (150 rpm) at 30C.
The inoculums (10%) were transferred into 150 ml production medium in 250 ml Erlenmeyer flasks and kept
for fermentation for ten days. After fermentation, the
broth was filtered through blotting paper and the supernatant was separated. The supernatant was extracted
twice with ethyl acetate. After separation, the organic
phase was dried over Na2SO4 (anhydrous). The extract
was then concentrated in a rotary vacuum. The crude
extracts were stored at 4C.

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Antibiogram of Streptomyces lavendulae strain SCA5

The antimicrobial activity of the ethyl acetate extract of


SCA5 (EA-SCA5) was assayed using the standard KirbyBauer disc diffusion method [18]. Petri plates were prepared
with 20 ml of sterile Mueller Hinton agar (MHA) (Himedia, Mumbai). The test cultures were swabbed on the top
of the solidified media and allowed to dry for 10 min. The
tests were conducted at 2.5 mg/disc concentrations of EASCA5. The loaded discs were placed on the surface of the
medium and left for 30 min at room temperature for compound diffusion. Negative control was prepared using respective solvent (DMSO). Streptomycin (25 g/disc) for
bacteria and Ketocanozole (30 g/disc) for fungi was used as
positive controls. The plates were incubated over night at
37C for bacteria and at 28C for fungi and the zones of inhibition were recorded. Diameters of the zones of inhibition
were measured using a zone scale from Hi-media and
expressed in millimetres. When the zone of inhibition was 0
to 4 mm it was considered weak activity; when the zone of
inhibition was 5 to 10 mm it was considered moderate activity; when the zone of inhibition was 11 to 15 mm it was
considered good activity.
Minimal inhibitory concentrations of EA-SCA5

The minimal inhibitory concentrations (MICs) of EASCA5, defined as the lowest concentration in the micro
titter plate with no growth (i.e., no turbidity) of the inoculated microorganism, was carried out as described by
the Clinical and Laboratory Standards Institute (CLSI,
2005) with slight modification [19]. The EA-SCA5 was
dissolved in DMSO in a concentration of 1000 g/ml.
The serial two fold dilutions of the EA-SCA5 (1000, 500,
250, 125, 62.5, 31.2, and 15.6 g/ml) were prepared for
MIC tests. MIC tests were carried out in MullerHinton
Broth for bacteria and Sabouraud dextrose broth for
fungi; the organisms were added to 96 well micro titter
plate containing 0.1 ml broth. The 3 l of log phase culture was introduced into respective wells and the final
inoculum size was 1105 cfu/ml. The plates were incubated at 37C for bacteria and 28C for fungi. Streptomycin for bacteria and Ketocanozole for fungi were used
as positive controls. Negative (water) and solvent controls (DMSO) were also included. 5 l of the test broth
was introduced on plain Mueller Hinton agar for bacteria and Sabouraud dextrose agar plates for fungi to observe the viability of the organism. MIC was determined
as the lowest concentration which inhibited complete
growth.
Antioxidant properties
DPPH radical scavenging assay

DPPH quenching ability of EA-SCA5 was measured according to [20]. A methanol DPPH solution (0.15%) was
mixed with serial dilutions (2001,000 g/ml) of EA-

Page 3 of 12

SCA5 and after 10 min, the absorbance was read at


515 nm. The radical scavenging activity was expressed as
IC50 (g/ml), (the dose required to cause a 50% inhibition). Vitamin C was used as standard. The ability to
scavenge the DPPH radical was calculated by the following formula:
DPPH radical scavenging activity %
A0 A1 A0  100

Where A0 is the absorbance of the control at 30 min


and A1 is the absorbance of the sample at 30 min. All
samples were analyzed in triplicate.
Determination of hydroxyl radical scavenging activity

The hydroxyl radical scavenging assay was performed as


described by the method of [21] with minor changes. All
solutions were prepared freshly. Stock solutions of EDTA
(1 mM), FeCl3 (10 mM), ascorbic acid (1 mM), H2O2
(10 mM) and deoxyribose (10 mM) were prepared in distilled deionized water. The assay was performed by adding
0.1 ml EDTA, 0.01 ml of FeCl3, 0.1 ml of H2O2, 0.36 ml of
deoxyribose, 1.0 ml of extract (2001,000 g/ml) each dissolved in distilled water, 0.33 ml of phosphate buffer
(50 mM, pH 7.4) and 0.1 ml of ascorbic acid in sequence.
The mixture was then incubated at 37C for 1 h. About
1.0 ml portion of the incubated mixture was mixed with
1.0 ml of (10%) TCA and 1.0 ml of (0.5%) TBA (in 0.025 M
NaOH containing 0.025 M NaOH BHA) to develop the
pink chromogen and read at 532 nm. The hydroxyl radical
scavenging activity of the extract was reported as the percentage of inhibition of deoxyribose degradation and was
calculated according to the formula (1).
Nitric oxide radical inhibition assay

Sodium nitroprusside in aqueous solution at physiological


pH spontaneously generates nitric oxide; it interacts with
oxygen to produce nitrite ions, which can be estimated by
the use of Griess Illosvoy reaction [22]. In the present investigation, Griess Illosvoy reagent was modified using
naphthylethylenediamine dihydrochloride (0.1% w/v) instead of 1-naphthylamine (5%). The reaction mixture (3 ml)
containing sodium nitroprusside (10 mM, 2 ml), phosphate
buffer saline (0.5 ml) and EA-SCA5 (2001000 g/ml) or
standard solution (0.5 ml) was incubated at 25C for
150 min. After incubation, 0.5 ml of the reaction mixture
containing nitrite was pipetted and mixed with 1 ml of
sulphanilic acid reagent (0.33% in 20% glacial acetic acid)
and allowed to stand for 5 min for completing diazotization. Then, 1 ml of naphthylethylenediamine dihydrochloride (1%) was added, mixed and allowed to stand for 30 min.
A pink colored chromophore was formed in diffused light.
The absorbance of these solutions was measured at 540 nm
against the corresponding blank. Vitamin C was used as

Saravana Kumar et al. BMC Microbiology 2014, 14:291


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standard. The scavenging activity was calculated using the


formula (1).

Page 4 of 12

calibration curve and the total phenolic content was


expressed as mg of gallic acid equivalents per gram of
extract.

Superoxide scavenging activity

Superoxide scavenging activity of EA-SCA5 was determined by monitoring the competition of those with
NBT for the superoxide anion generated by the PMS
NADH system [23]. Superoxide radicals were generated
in 1 ml of 20 mM TrisHCl buffer pH 8.0 containing
0.05 mM nitroblue tetrazolium (NBT), 0.01 mM phenazine methosulphate (PMS), and different concentrations
(2001,000 g/ml) of EA-SCA5 were pre-incubated for
2 min. The reaction was initiated by the addition of
0.078 mM NADH. Blue chromogen, formed due to NBT
reduction, was read at 560 nm. Results were expressed
as percentage of inhibition of superoxide radicals.
Animals

Male albino Wistar rats bred in the animal house of Entomology Research Institute, weighing 170 5 g were used
in the studies. The animals were kept in polypropylene
cages, under controlled temperature, humidity and 12/12
light/dark cycles. The animals were fed pellet diet (Pranav
Agro Industries Ltd., Maharashtra) and water ad libitum.
This study was carried out with prior approval from Institutional Animal Ethical Committee (IAEC-ERI-LC-04/13).
Inhibition of lipid peroxidation in rat liver homogenate

The inhibition effect of EA-SCA5 on lipid peroxidation


was determined according to the thiobarbituric acid
method [24]. FeCl2H2O2 was used to induce liver homogenate peroxidation. In this method, 0.2 ml of EASCA5 extract (2001000 g/ml) was mixed with 1 ml of
1% liver homogenate (each 100 ml homogenate solution
contains 1 g rat liver); then 50 l of FeCl2 (0.5 mM) and
H2O2 (0.5 mM) was added. The mixture was incubated
at 37C for 60 min; then 1 ml of trichloroacetic acid
(15%) with thiobarbituric acid (0.67%) was added and
the mixture was heated in boiling water for 15 min. The
absorbance was recorded at 532 nm. Vitamin C was
used as positive control. The percentage of inhibition
was calculated using the formula (1).
Total phenolic content (TPC)

Total phenolic content of EA-SCA5 was determined according to the Folin-Ciocalteu spectrophotometric method with
some modifications [25]. Briefly, 0.1 (2001,000 g/ml), EASCA5 was mixed with 1.9 ml of distilled water and 1 ml of
diluted Folin-Ciocalteus phenol reagent and allowed to react
for 5 min. Then, 1 ml of 100 g/l Na2CO3 solution was
added. After 2 h of reaction at 25C, the absorbance at
765 nm was determined. The sample was tested in triplicate
and a calibration curve with six data points for gallic acid
was obtained. The results were compared to gallic acid

Reducing power

The determination was carried out as described by


Oktay, Gu lc_in, and Ku frevioglu [26]. Briefly, different
concentrations of EA-SCA5 (2001000 g/ml) were
mixed with phosphate buffer (2.5 ml, 0.2 mol/l, pH 6.6)
and K3Fe (CN)6 (2.5 ml, 1%). The mixtures were incubated for 20 min at 50C. A portion (2.5 ml) of
trichloroacetic acid solution (10%) was added to the
mixture, which was then centrifuged at 10 000 g for
10 min. The upper layer of solution (2.5 ml) was mixed
with deionized water (2.5 ml) and FeCl3 (0.5 ml, 0.1%),
and the absorbance was measured at 700 nm and was
compared with standard BHT absorbance.
Cell line maintenance and growth conditions

A549 adenocarcinoma lung cancer cell line was obtained


from National Institute of Cell Sciences, Pune and was
maintained in complete tissue culture medium DMEM
(Dulbeccos modified eagles medium) with 10% Fetal Bovine Serum and 2 mM L-Glutamine, along with antibiotics
(about 100 IU/ml of penicillin, 100 g/ml of streptomycin)
with the pH adjusted to 7.2. The cell lines were maintained at 37C at 5% CO2 in CO2 incubator [27]. Cultures
were viewed using an inverted microscope to assess the
degree of confluency and the absence of bacterial and fungal contaminants were confirmed.
Cytotoxic properties

The cytotoxicity was determined according to the


method with some changes [28]. Cells (5000 cells/well)
were seeded in 96 well plates containing medium with
different concentrations such as 100 l, 75 l, 50 l,
25 l, 12.5 l and 6.25 l. The cells were cultivated at
37C with 5% CO2 and 95% air in 100% relative humidity. After various durations of cultivation, the solution in
the medium was removed. An aliquot of 100 l of
medium containing 1 mg/ml of 3-(4, 5-dimethylthiazol2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) was
loaded to the plate. The cells were cultured for 4 h and
then the solution in the medium was removed. An aliquot of 100 l of DMSO was added to the plate, which
was shaken until the crystals were dissolved. The cytotoxicity against cancer cells was determined by measuring the absorbance of the converted dye at 570 nm in an
ELISA reader. Cytotoxicity of each sample was expressed
as IC50 value. The IC50 value is the concentration of test
sample that causes 50% inhibition of cell growth, averaged from three replicate experiments.

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TLC analysis

The TLC profile of the active extract was carried out on


Merck silica gel 60F254 pre coated aluminium plates of
layer thickness 0.2 mm developing system chloroform :
methanol 4:1. The plates were viewed under normal
white light and UV light at 366 nm. Derivatization was
carried out by 10% alcoholic sulphuric acid reagent,
heated at 110C for 5 minutes and also by exposure to
iodine vapours. Phenol was located by 0.1% alcoholic
ferric chloride and quinone was located by 0.1% alcoholic NaOH.
GC-MS analysis

The active ethyl acetate crude was subjected to GC-MS


analysis on GC-MS- 5975 (AGILENT), column DB 5 ms
Agilent, dimension length- 30.0 m, ID- 0.2 mm, flim
thickness- 0.25 m with temperature program- 70300C,
10C/min, injection temperature- 240C, carrier gas- helium, flowrate- 1.51 ml/min, equipped with GC-MS NISTII library.

Page 5 of 12

tentatively attached to the genus Streptomyces sp. Table 2


shows the physiological properties of SCA5. Optimal
growth of strain was observed at 30C and at pH 7 and in
the presence of NaCl in the range of 19% (very good
growth). Strains were able to grow at 13% NaCl (moderate
to good growth) and at 30C (maximum temperature
growth), but were unable to grow at pH 5 and pH3. SCA5
was able to produce enzymes such as amylase, chitinase,
protease, gelatinase, and lipase. Further, DNA preparation
and PCR amplification of SCA5 strain genomic DNA resulted in 1500 bp amplicon approximately. 16S rDNA sequence was determined and Blast analysis was performed,
which confirmed that the isolate belonged to Streptomyces
lavendulae (bases 1 to 1459 linear DNA). The strain SCA
5 had 100% similarity with Streptomyces lavendulae subsp.
lavendulae (AB184731); SCA5 was submitted to the
GenBank, NCBI under the accession number, KC315780;
phylogenetic tree was constructed (data given as
Additional file 1). Based on the antimicrobial activity the
Streptomyces lavendulae strain SCA5 was taken to assess
the cytotoxicity and antioxidant properties.

Statistical analysis

The data for biochemical and physiological parameters


were analyzed and expressed as means SEM. The IC50
values were calculated from linear regression analysis. Results were processed by computer program, Microsoft
Excel (2007).

Results
Isolation, characterization and identification of
antagonistic actinomycetes

Based on the colony morphology and stability in subculturing, 37 suspected Streptomyces cultures were purified
on ISP-2 slants. The isolates were initially screened to determine their ability to produce antimicrobial compounds.
In the initial screening 8% showed good activity, 27%
showed moderate activity, 24% showed weak activity and
40% showed no antagonistic activity against fungi; similarly 10% showed good activity, 27% showed moderate activity, 32% showed weak activity and 30% showed no
activity against bacteria. Among the strains tested, SCA5
showed good antimicrobial activity against the tested
pathogens. So SCA5 strain was identified by morphological, biochemical, physiological characteristics and 16S
rRNA amplification. Table 1 shows the morphological features of the strain. SCA5 grew on all media used and had
a grey to white aerial mycelium and light to dark greyish
white vegetative mycelium. Diffusible pigments were not
observed on the media tested. The isolate produced moderately branching, non-fragmenting substrate hyphae. Aerial hyphae were formed on all media except Muller
Hinton agar, Zobell marine agar and Sabouraud dextrose
agar; moderate growth was observed on skim milk agar.
Based on the morphological characteristics, SCA5 was

Optimization of media, extraction and antibiogram

Of the various antibiotic production medium, M3


medium was highly efficient for Streptomyces lavendulae strain SCA5; the broth showed good activity against
the tested pathogens. SCA5 was mass produced using
optimized medium; the broth was extracted using ethyl
acetate. The antimicrobial activities of EA-SCA5 are
shown in Figure 1. EA-SCA5 showed good activity
against 13 bacterial and 10 fungal pathogens with zones
of inhibition ranging from 10 to 15 mm for bacteria and
1013 mm for fungi (Table 3) [29].
In vitro antimicrobial assay (MIC)

The EA-SCA5 showed broad spectrum of activity against


Gram positive and Gram negative bacteria with the MIC
value of 125 g/ml; the MIC value against fungi was
31.25 g/ml (Table 4). EA-SCA5 showed significant activity against M. lutues, S. flexneri (MIC: 125 g/ml), Methicillin resistant S. aureus (MRSA), S. typhi-B (SPB) (MIC:
250 g/ml), S. aureus, B. subtilis, S. epidermis, K. pneumonia (MIC: 500 g/ml), E. aerogens, V. parahaemolyticus,
Y. enterocolitica, S. typhimurium, P. vulgaris (MIC:
1000 g/ml). EA-SCA5 showed significant antifungal activity against A. flavus (MIC: 31.25 g/ml), M. pachydermatis, T. mentagrophyte (MIC: 250 g/ml), C. parapsilosis,
C. krusei, Scopulariopsis sp. (MIC: 500 g/ml), B. cinerea,
T. rubrum, C. albicans, A. niger. (MIC: 1000 g/ml).
Antioxidant properties
Inhibition of DPPH radical

The EA-SCA5 exhibited significant dose dependent inhibition of DPPH activity, with a 50% inhibition (IC50) at

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Page 6 of 12

Table 1 Morphological features of Streptomyces lavendulae strain SCA5 on different media


Media

Aerial mycelium

Substrate mycelium

Diffusible pigment

Reverse side

Growth

AIA

Grey

Grey

White

+++

Agar

White

White

White

ISP 2

White

White

Brownish yellow

+++

MHA

Slimy yellow

Slimy yellow

SCA

Greyish White

Greyish white

Yellow

+++

SDA

Yellow

Yellow

++

SKM

Yellowish white

Yellowish white

Yellow

++

STP

Dark grey

Dark grey

Yellowish grey

+++

YPG

Whitish yellow

Yellow

Yellow

+++

ZMA

Slimy Yellow

Whitish yellow

+++ Good growth; ++ Moderate growth; + Weak growth: absent.

a concentration of 507.61 0.66 g/ml. The results are


presented in Figure 2A. The IC50 value for vitamin C
was 382.09 0.67 g /ml.

Hydroxyl radical scavenging assay

The results for hydroxyl radical scavenging assay are


shown in Figure 2B. The concentrations for 50% inhibition were found to be IC50 617.84 0.57 g/ml), and
501.49 0.67 g /ml for the EA-SCA5 and vitamin C,
respectively.

Nitric oxide radical inhibition assay

The scavenging of nitric oxide by the extract was increased in a dose-dependent manner as illustrated in
Figure 2C. At the concentration of 730.92 0.81 g/ml
of EA-SCA5, 50% of nitric oxide was scavenged. The
IC50 value for vitamin C was 419.33 0.67 g /ml.
Table 2 Physiological and biochemical characteristics of
Streptomyces lavendulae strain SCA5

Superoxide

Figure 2D shows the superoxide radical scavenging capacity


of EA-SCA5. EA-SCA5 demonstrated a doseresponse inhibition of the superoxide anion radicals. EA-SCA5 exhibited superoxide anion radical scavenging activity at all the
concentrations. It showed 50% inhibition at 864.71
1.15 g/ml. The IC50 value for vitamin C was 641.76
1.09 g /ml.
Lipid peroxidation assay

Activity of EA-SCA5 on lipid peroxidation is shown in


Figure 2E. The extract showed inhibition of peroxidation
at all the concentrations; it showed 50% inhibition at
838.83 1.18 g/ml. The IC50 value for vitamin C was
458.90 1.26 g/ml.
Reducing power

Figure 2F shows the reductive capabilities of the extract


compared to the standard butylated hydroxyl toluene.
The reducing power of EA-SCA5 increased with increasing quantity of the sample.

Characteristics

Results

Gram staining

Positive

Shape and growth

filamentous aerial growth

Cytotoxicity property

Range of temperature for growth

25C to 37C

Optimum temperature

30C

EA-SCA5 showed cytotoxic activity in vitro against


A549 lung adenocarcinoma cancer cell line. It showed
84.9% activity at the dose of 500 g/ml with IC50 value
of 51.9% at 200 g/ml (Figure 3). All the concentrations
used in the experiment decreased cell viability significantly (P < 0.05) in a concentration-dependent manner.

Range of pH for growth

3 to 11

Optimum pH

Growth in the presence of NaCl

1 to 9%

Amylase

+++

Chitinase

Protease

++

Gelatinase

+++

Lipase

++

+++ Good activity; ++ Moderate activity; + Weak activity.

TLC analysis

TLC profile of the active ethyl acetate extract is given in


Figure 4 with various methods of visualization. As
shown in Figures 4C and D the extract contained phenol
and quinone.

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Page 7 of 12

Figure 1 Antimicrobial activity of EA-SCA5 using disc diffusion method.

Table 3 Antimicrobial activity of Streptomyces lavendulae


strain SCA5 ethyl acetate extract using disc diffusion
method
Microbes

Table 4 Minimum inhibitory concentration of the


Streptomyces lavendulae strain SCA5 ethyl acetate extract

Zone of inhibition (mm)


Ketoconazole (30 g/disc)

Microbes

Aspergillus flavus

12

20

Aspergillus flavus

Aspergillus niger

10

10

Aspergillus niger

>1000 g/ml

>12.5 g/ml

Botrytis cinerea

10

12

Botrytis cinerea

>1000 g/ml

>6.25 g/ml

Candida albicans

13

15

Candida albicans

>1000 g/ml

>6.25 g/ml

Candida.krusei

11

16

Candida.krusei

>500 g/ml

>12.5 g/ml

Candida parapsilosis

11

35

Candida parapsilosis

>500 g/ml

>12.5 g/ml

Malassesia pachydermatis

10

24

Malassesia pachydermatis

>250 g/ml

>25 g/ml

Scopulariopsis sp.

12

19

Scopulariopsis sp.

>1000 g/ml

>12.5 g/ml

Trichophyton mentagrophytes

13

13

Trichophyton mentagrophytes

>250 g/ml

>12.5 g/ml

Trichophyton rubrum

10

12

Trichophyton rubrum

>1000 g/ml

Streptomycin (25 g/disc)

Gram positive bacteria

EASCA5 (2.5 mg/disc)

Gram positive bacteria

Ketoconazole
>31.25 g/ml

>12.5 g/ml

>12.5 g/ml
Streptomycin

Staphylococcus aureus

13

27

Staphylococcus aureus

>500 g/ml

>6.25 g/ml

Micrococcus luteus

15

30

Micrococcus luteus

>125 g/ml

>6.25 g/ml

Bacillus subtilis

11

23

Bacillus subtilis

>500 g/ml

>6.25 g/ml

Staphylococcus epidermidis

13

25

Staphylococcus epidermidis

>500 g/ml

>6.25 g/ml

MRSA

12

24

MRSA

>250 g/ml

>25 g/ml

Gram negative bacteria

Gram negative bacteria

Klebsiella pneumonia

11

28

Klebsiella pneumonia

>500 g/ml

Enterobacter aerogens

10

24

Enterobacter aerogens

>1000 g/ml

>25 g/ml

Vibrio parahaemolyticus

10

18

Vibrio parahaemolyticus

>1000 g/ml

>25 g/ml

Yersinia enterocolitica

10

20

Yersinia enterocolitica

>1000 g/ml

>6.25 g/ml

Salmonella typhimurium

14

21

Salmonella typhimurium

>250 g/ml

>30 g/ml

Shigella flexneri

15

29

Shigella flexneri

>125 g/ml

>30 g/ml

Proteus vulgaris

10

18

Proteus vulgaris

>1000 g/ml

>6.25 g/ml

Salmonella typhi-B

13

11

Salmonella typhi-B

>250 g/ml

>6.25 g/ml

EA-SCA5: Ethyl acetate extract, Ketoconazole: Antifungal Agent.


Streptomycin: Antibacterial agent.

Ketoconazole: Antifungal Agent.


Streptomycin: Antibacterial agent.

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Page 8 of 12

Figure 2 Antioxidant activity. a. DPPH radical scavenging activity of different concentrations (2001000 g/ml) of EA-SCA5 and Vitamin C. Each value
presents the mean SEM of triplicate experiments. b. Hydroxyl radical scavenging activity of different concentrations (2001000 g/ml) of EA-SCA5
and Vitamin C. Each value presents the mean SEM of triplicate experiments. c. Nitric oxide radical scavenging activity of different concentrations
(2001000 g/ml) of EA-SCA5 and Vitamin C. Each value presents the mean SEM of triplicate experiments. d. Superoxide anion radical-scavenging
activity of different concentrations (2001000 g/ml) of EA-SCA5 and Vitamin C. Each value presents the mean SEM of triplicate experiments. e. Lipid
peroxidation inhibition of different concentrations (2001000 g/ml) of EA-SCA5 and Vitamin C. Each value presents the mean SEM of triplicate
experiments. f. Reducing power determination of different concentrations (2001000 g/ml) of EA-SCA5 and BHT. Each value presents the mean
SEM of triplicate experiments.

GC-MS analysis

The chemical composition of bioactive extract of EASCA5 was investigated using GC-MS analysis. On comparison of the mass spectra of the constituents with the
NIST library, eleven compounds were identified by retention time, molecular weight and molecular formula
as mentioned in Table 5 and Figure 5. Of the eleven
compounds identified, the most important compound
was actinomycin C2 which constituted 7.12%.

Discussion
The resistance of numerous pathogenic bacteria and
fungi to commonly used bioactive secondary metabolites
necessitates the search for new antifungal and antibacterial molecules to combat these pathogens. Secondary
metabolites produced by microbes continue to attract
the attention, due to their sophisticated chemical structures and highly specific biological activities. Filamentous soil bacteria belonging to the genus Streptomyces are

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Page 9 of 12

Figure 3 Cytotoxic effects of EA-SCA5 on cancer cell line (A549). Data are mean SD of three independent experiments with each
experiment conducted in triplicate.

rich sources of a high number of bioactive natural products with biological activity; they are extensively used in
pharmaceutical and agrochemical industries. These bacteria produce about 75% of commercially and medically
useful antibiotics [30]. Newer or rare soil actinomycetes
are good sources of potent molecules. 37 actinomycetes
were isolated from soil samples collected from Vengodu
(agricultural field); among these isolates SCA5 exhibited
good antimicrobial activity. SCA5 was identified as
Streptomyces lavendulae. It had been found that the

majority of the soil isolates produced aerial coiled mycelia and the spores were arranged in chains [31]. Actinomyces are useful biological tools in the production of
antimicrobials against bacteria and fungi [32]. Streptomyces lavendulae strain SCA5 showed good antimicrobial
activity in solid medium and also in fermented broth.
Our results indicated that the antimicrobial metabolites
were extracellular. Most of the secondary metabolites
and antibiotics are extracellular in nature and extracellular products of actinomycetes show potent antimicrobial

Figure 4 TLC profile of the active crude from soil Streptomyces lavendulae strain SCA5. a. Viewed under normal white light. b. Viewed under
UV 366 nm. c. Derivatization with 10% alcoholic sulphuric acid. d. Exposure to iodine vapour. e. 0.01% ferric chloride. f. 0.01% alcoholic NaOH.

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Page 10 of 12

Table 5 GC-MS profile of the Streptomyces lavendulae strain SCA5 ethyl acetate extract
S.no

Retention time

Compound name

Molecular formula

Molecular weight

Area %

1.

4.68

Octaethylene glycol monododecyl ether

C28H58O9

538

0.48

2.

4.75

2(3H)Furanone,5acetyldihydro

C6H8O3

128

2.62

3.

5.37

1,4Dioxane2,5dione,3,6dimethyl

C6H8O4

144

2.24

4.

10.31

Hexanoic acid, 2phenylethyl ester

C14H20O2

220

0.59

5.

11.77

2,4Dimethyl3pentanol acetate

C9H18O2

158

1.06

6.

11.96

Hexadecanoic acid, 1(hydroxymethyl)1,2ethanediyl ester

C35H68O5

568

0.86

7.

14.31,16.27,17.28,17.68, 27.47

Actinomycin C2

C63H88N12O16

1268

7.12

8.

17.55, 18.00

Hexadecanoic acid

C16H32O2

256

14.75

9.

20.39

Oleic Acid

C18H34O2

282

26.46

10.

20.77

2,5Piperazinedione,3,6bis(2methylpropyl)

C12H22N2O2

226

8.42

11.

22.75,23.27

Ergotaman

C33H35N5O5

581

3.56

activities [33]. From the results obtained, it appeared


that the antimicrobial action of EA-SCA5 was more
pronounced on both Gram positive and Gram negative
bacteria and fungi. These results are consistent with previous reports of Saravana kumar et al. [34] and Vijayakumar et al. [35]. In this study, EA-SCA5 was investigated
for the scavenging abilities on DPPH, hydroxyl radicals,
nitric oxide radical, superoxide and lipid peroxidation.
The presence of good amount of phenolics as shown by
FolinCiocalteau method might have contributed to its
antioxidant potential. DPPH is a useful reagent to evaluate the free radical scavenging ability of the hydrogen
donating antioxidant, which can transfer hydrogen
atoms or electrons to DPPH radicals [36]. EA-SCA5 was
able to reduce the stable radical DPPH to the yellowcolored diphenylpicrylhydrazine. Hydroxyl radical is one
of the reactive oxygen species generated in the body,
and removing hydroxyl radicals is important for

antioxidant defence in living cell systems [37]. The nitric


oxides radical inhibition study showed that EA-SCA5
was a potent scavenger of nitric oxide. EA-SCA5 inhibited nitrite formation by competing with oxygen to react
with nitric oxide directly and also to inhibit its synthesis.
Superoxide anions are precursors to active free radicals
that have potential for reacting with biological macromolecules and thereby inducing tissue damage [38]. Antioxidants are able to inhibit the blue NBT formation
[39]. The inhibition of superoxide radical by EA-SCA5
was lower than vitamin C. In our study, the inhibition of
b-carotene bleaching by EA-SCA5 was lower than the
standard for measurement of reductive ability. The reducing power increased with increasing concentration of
the EA-SCA5. The reducing capacity of a compound may
serve as a significant indicator of its potential antioxidant
activity [40]. The studies on soil actinomycetes with respect
to cytotoxic activity are very limited in the Indian sub-

Figure 5 GC-MS analysis of active crude from soil Streptomyces lavendulae strain SCA5.

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continent. EA-SCA5 showed good activity against A549


lung adenocarcinoma cancer cell. Chandrananimycins, isolated from marine Actinomadura spp. MO48 has been
shown to exhibit antibacterial, antifungal and anticancer activity [41]. Eleven compounds were identified from the GCMS analysis of EA-SCA5. These might be responsible for
the antimicrobial, antioxidant and cytotoxity activities. Similar results were reported by Joseph Selvin et al. [42]. The
compounds such as 2(3H)Furanone, 5acetyldihydro,
1,4 Dioxane, 2, 5 dione, 3, 6 dimethyl, Hexanoicacid,
2phenylethylester, 2, 4 Dimethyl3pentanolacetate, Acti
nomycinC2, 2, 5 Piperazinedione, 3, 6 bis(2methylpropyl), Ergotaman were reported to possess activity. The
constituents such as Octaethylene glycol monododecyl ether;
Hexadecanoic acid and Hexadecanoic acid are straight chain
hydrocarbons or alcohols, which are known to be inactive.
Oleic acid is used as an excipient in pharmaceuticals and as
an emulsifying or solubilising agent in aerosol products [43].
The major compound actinomycin C2 has been reported to
have antimicrobial [44], antioxidant [45] and anti-cancerous
property including Gestational trophoblastic neoplasia [46],
Wilms tumor [47], Rhabdomyosarcoma [48], Ewings sarcoma [49], Malignant hydatidiform mole [50].

Conclusion
The ethyl acetate extract of Streptomyces lavendulae strain
SCA5 displayed significant biological activities against
tested Gram positive and Gram negative bacterial pathogens and filamentous fungal pathogens. It also exhibited
antioxidant and cytotoxic activity against carcinoma cell
lines in vitro. GC-MS showed the presences of actinomycin C2 (7.12%) which may be the active principle.
Additional file
Additional file 1: Radical scavenging activity of di fferent
concentrations (200-1000 g/ml) of EA-SCA5e and standards
(Vitamin C and Butylated hydroxytoluene BHT).
Competing interests
The authors declare that they have no competing interests.
Authors contributions
Conceived and designed the experiments: PSK, NAAD, SI and VD. Performed
the experiments: PSK and AS. Analyzed the data: PSK, CB, PP NAAD, SI and
VD. Prepared the manuscript, PSK, CB and VD. All authors read and approved
the final manuscript.
Acknowledgement
The authors are grateful to Entomology Research Institute, Loyola College
and Chennai for providing lab facility. This project was supported by King
Saud University, Deanship of Scientific Research, Addiriyah Chair for
Environmental Studies.
Author details
1
Division of Microbiology, Entomology Research Institute, Loyola College,
Chennai 600 034, India. 2Department of Botany and Microbiology, Addiriyah
Chair for Environmental Studies, College of Science, King Saud University,
P.O. Box. 2455, Riyadh 11451, Saudi Arabia. 3Department of Plant

Page 11 of 12

Biology and Biotechnology, Loyola College, Chennai 600 034, India.


4
Visiting Professor Programme, College of Science, Deanship of
Scientific Research, King Saud University, Riyadh, Saudi Arabia.
Received: 19 September 2014 Accepted: 10 November 2014

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Cite this article as: Saravana Kumar et al.: In vitro antimicrobial,
antioxidant and cytotoxic properties of Streptomyces lavendulae strain
SCA5. BMC Microbiology 2014 14:291.

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-Glucosidase Inhibition and Antioxidant


Properties of Streptomyces sp.: In Vitro.
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-Glucosidase Inhibition and Antioxidant


Properties of Streptomyces sp.: In Vitro

P.Praveen Kumar, J.P.Preetam Raj,


I.V.S.Nimal Christhudas, R.Sagaya
Jansi, M.Narbert Raj & P.Agastian
Applied Biochemistry and
Biotechnology
Part A: Enzyme Engineering and
Biotechnology
ISSN 0273-2289
Volume 172
Number 3
Appl Biochem Biotechnol (2014)
172:1687-1698
DOI 10.1007/s12010-013-0650-z

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Author's personal copy


Appl Biochem Biotechnol (2014) 172:16871698
DOI 10.1007/s12010-013-0650-z

-Glucosidase Inhibition and Antioxidant Properties


of Streptomyces sp.: In Vitro
P. Praveen Kumar & J. P. Preetam Raj &
I. V. S. Nimal Christhudas & R. Sagaya Jansi &
M. Narbert Raj & P. Agastian

Received: 6 February 2013 / Accepted: 8 November 2013 /


Published online: 19 November 2013
# Springer Science+Business Media New York 2013

Abstract Streptomyces strain isolated from the soil sediment was studied for its in vitro glucosidase and antioxidant properties. Morphological characterization and 16S rRNA partial
gene sequencing were carried out to confirm that the strain Loyola AR1 belongs to genus
Streptomyces sp. Modified nutrient glucose broth was used as the basal medium for growth
and metabolites production. Ethyl acetate extract of Loyola AR1 (EA-Loyola AR1) showed
50 % -glucosidase inhibition at the concentration of 860.502.68 g/ml. Antioxidant
properties such as total phenolic content of EA-Loyola AR1 was 176.831.17 mg of catechol
equivalents/g extracts. EA-Loyola AR1 showed significant scavenging activity on 2,2diphenyl-picrylhydrazyl (50 % inhibition (IC50), 750.501.61 g/ml), hydroxyl (IC50,
690.202.38 g/ml), nitric oxide (IC50, 850.501.77 g/ml), and superoxide (IC50,
880.081.80 g/ml) radicals, as well as reducing power. EA-Loyola AR1 showed
strong suppressive effect on lipid peroxidation (IC50, 670.502.52 g/ml). Antioxidants of carotene linoleate model system reveals significantly lower than butylated hydroxyanisole.
Keywords Soil sediment . -Glucosidase inhibition . Antioxidant properties . Streptomyces sp.

Introduction
Actinomycetes are the most economically and biotechnologically worthful prokaryotes. They
are responsible for the production of about half of the discovered bioactive secondary
metabolites, notably antibiotics [4], antitumor agents [7], immunosuppressive agents [23],

P. Praveen Kumar : J. P. Preetam Raj : I. V. S. Nimal Christhudas : R. Sagaya Jansi : M. Narbert Raj :
P. Agastian
Department of Plant Biology and Biotechnology, Loyola College, Chennai 600034, India
P. Agastian (*)
Research Department of Plant Biology and Biotechnology, School of Life Science, Loyola College,
Chennai 600034, India
e-mail: agastian@loyolacollege.edu

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and enzymes [29]. Among the 140 described actinomycetes genera, only a few are responsible
for the majority of 20,000 microbial natural products identified so far. In particular, the genus
Streptomyces accounts for about 80 % of the actinomycetes natural products reported [6]. In
the course of screening for new metabolites, several studies were carried out in order to isolate
new Streptomyces species from different habitats.
-Glucosidase is a very important enzyme responsible for the hydrolysis of dietary
disaccharides into absorbable monosaccharide in microbial system and in small intestine of
animal digestive system. Glucosidase is not only essential for carbohydrate digestion but it is
also very important for processing of glycoproteins and glycolipids and are also involved in
variety of metabolic disorders and other diseases such as diabetes [19]. There are many articles
related to antidiabetic compounds reported from Streptomyces hygroscopicus-limoneus [9] and
Streptomyces calvus [22].
Recent studies focusing on the response of antioxidant system of bacteria, fungi, and
actinomycetes are important in terms of biotechnology, such as Streptomyces growth in various
oxidative stress conditions [35]. Reactive oxygen species (ROS) are known to be implicated in
many cell disorders and in the development of many diseases including cardiovascular
diseases, atherosclerosis, chronic inflammation, and so on [12]. Synthetic antioxidants are
widely used but their use is being restricted nowadays because of their toxic and carcinogenic
effects. Thus, interest in finding natural antioxidants, without any undesirable effect, has
increased greatly [31]. The present study evaluates in vitro -glucosidase inhibition and
antioxidant properties of Streptomyces sp. Loyola AR1 isolated from the lake soil of Ambattur
Industrial estate, Tamil Nadu, India.

Materials and Methods


Chemicals and Reagents
All the chemicals used for preparation of different media and reagent were used from Himedia,
Merck, Qualigens, etc., 1,1-diphenyl,2-picryl hydrazyl (DPPH), nitro blue tetrazolium (NBT),
nicotinamide adenine dinucleotide phosphate reduced (NADH), phenazinemethosulphate
(PMS), trichloro acetic acid (TCA), ferric chloride, and butylatedhydroxyltoluene (BHT) were
obtained from Sigma Chemical Co., USA. Ascorbic acid was obtained from SD Fine Chem.
Ltd., Biosar, India.
Isolation of Actinomycetes
The Streptomyces strain used in this study was isolated from the Ambattur Lake soil sediments,
Tamil Nadu, India with 130636 N latitude 801012 E longitude. Soils from different
places of lake were brought to the laboratory in aseptic condition. Actinomycetes from the soil
was isolated by pour plate technique on actinomycetes isolation agar (in gram per liter)
containing 2 g sodium casinate, 0.1 g L-asparagines, 4 g sodium propionate, 0.5 g dipotassium
sulphate, 0.1 g magnesium sulphate, 0.001 g ferrous sulphate, 15 g agar, pH 8.1, and 1 L
sterile-distilled water, also supplemented with 20 mg/l actidione and 100 mg/l nalidixic acid
were added to minimize fungal and bacterial growth, respectively. Soil samples were serially
diluted up to 105 and 0.1 ml of aliquots spread over actinomycetes isolation agar plates. The
plates were incubated at 282 C for 7 days. Strains of actinomycetes were picked out and
purified by repeated streaking on yeast extractmalt extract agar (International Streptomyces
Project, ISP2) and were preserved in slants at 42 C.

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Identification of Isolate
The Streptomyces strain was characterized morphologically following methods given in the
ISP [32]. The characters including colony morphology of the strains such as the color of aerial
mycelium, reverse side color, size of the colony, and production of diffusible pigments were
observed after incubation at 282 C for 7 days on actinomycetes isolation agar medium. The
microscopic morphology of strains such as formation of aerial and substrate mycelium and
spore management, which are highly characteristic and useful in the identification of actinomycetes, were observed by cover slip technique [27] with light microscopy.
DNA Preparation
The freshly cultured Loyola AR1 cells were pelleted by centrifuging for 2 min at 12,000 rpm
to obtain 1015 mg (wet weight). The cells were suspended thoroughly in 300 l of lysis
solution; 20 l of RNase A solution was added, mixed, and incubated for 2 min at room
temperature. About 20 l of the proteinase K solution (20 mg/ml) was added to the sample;
mixed and resuspended cells were transferred to Hibead Tube and incubated for 30 min at
55 C. The mixture was vortexed for 57 min and incubated for 10 min at 95 C followed by
pulse vortexing. Supernatant was collected by centrifuging the tube at 10,000 rpm for 1 min at
room temperature. About 200 l of lysis solution was added, mixed thoroughly by vortexing,
and incubated at 55 C for 10 min. To the lysate, 200 l of ethanol (96100 %) was added and
mixed thoroughly by vortexing for 15 s. The lysate was transferred to new spin column and
500 l of prewash solution was added to the spin column and centrifuged at 10,000 rpm for
1 min and supernatant was discarded. The lysate was then washed in 500 l of wash solution
and centrifuged at 10,000 rpm for 3 min. Of the elution buffer, 200 l was pipetted out and
added directly into the column without spilling and incubated for 1 min at room temperature.
Finally, the DNA was eluted by centrifuging the column at 10,000 rpm for 1 min (Hipura
Streptomyces DNA spin kit-MB 527-20 pr from HiMedia, Mumbai, India).
PCR Amplification and Sequencing
The 16S ribosomal RNA was amplified by using the thermo cycler (Eppendorf ep. gradient)
with Taq DNA polymerase and primers 27F (5AGTTTGATCCTGGCTCAG 3) and 1492R
(5ACGGCTACC TTGTTACGACTT 3). The conditions for thermal cycling were as follows:
denaturation of the target DNA at 94 C for 4 min followed by 30 cycles at 94 C for 1 min,
primer annealing at 52 C for 1 min, and primer extension at 72 C for 1 min. At the end of the
cycling, the reaction mixture was held at 72 C for 10 min and then cooled to 4 C. The PCR
product obtained was sequenced by an automated sequencer (Genetic Analyzer 3130, Applied
Biosystems, USA). The same primers as above were used for sequencing. The sequence was
compared for similarity with the reference species of bacteria contained in genomic database
banks using the National Center for Biotechnology Information (NCBI) BLAST available at
http://www.ncbi-nlm-nih.gov/. The DNA sequences were aligned and phylogenetic tree was
constructed by using the Molecular Evolution Genetic Analysis (MEGA) software version 4.0.
16S rRNA sequence was then submitted to the GenBank, NCBI, USA.
Extraction
The selected antagonistic actinomycetes isolate was inoculated into MNG broth separately and
incubated at 282 C at 140 rpm for 7 days. After fermentation, broth was harvested and

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centrifuged to remove cell debris. Filtrate was collected and stored at 4 C for further use [36].
The bioactive metabolites were recovered from the harvested medium by solvent-extraction
method. The filtrate was mixed with ethyl acetate (1:1 v/v) and shaken vigorously for 1 h in a
solvent extraction funnel. Extraction was continued up to three times with the same solvent.
The organic phase was concentrated and used for further studies [2].
Determination of In Vitro -Glucosidase Inhibition and Antioxidant Properties
In Vitro -Glucosidase Inhibition
In order to investigate the inhibition activity of EA-Loyola AR1, an in vitro -glucosidase
inhibition test was performed. -Glucosidase from yeast is used extensively as a screening
material for -glucosidase inhibitors, but the results do not always agree with those obtained in
mammals. Therefore, we used the mouse small-intestine homogenate as -glucosidase solution because we speculated that it would better reflect the in vivo state. The inhibitory effect
was measured using the method slightly modified from [8]. After fasting for 20 h, the small
intestine was incised, rinsed with ice-cold saline, and homogenized with 12 ml of maleate
buffer (100 mM, pH 6.0). The homogenate was used as the -glucosidase solution. The assay
mixture consisted of 100 mM maleate buffer (pH 6.0), 2 % (w/v) sugar substrate solution
(100 l), and the EA-Loyola AR1 (2001,000 g/mL). It was preincubated for 5 min at 37 C,
and the reaction was initiated by adding the crude -glucosidase solution (50 l) to it followed
by incubation for 10 min at 37 C. The rate of carbohydrate decomposition was calculated as
the percentage ratio to the amount of glucose obtained when the carbohydrate was completely
digested. The rate of inhibition was calculated by the following formula:
h
Inhibition% amount of glucose produced by the positive control
amount of glucose produced by the addition of sample
i
=amount of glucose produced by the positive control  100

Antioxidant Properties
Determination of Total Phenolic Content
Total phenolic content of EA-Loyola AR1 was assessed according to the FolinCiocalteau
method [33] with some modifications. Briefly, 0.1 ml of extract (2001,000 g/ml), 1.9 ml of
distilled water, and 1 ml of FolinCiocalteau reagent were seeded in a tube, and then 1 ml of
100 g/l Na2CO3 was added. The reaction mixture was incubated at 25 C for 2 h and the
absorbance of the mixture was read at 765 nm. The sample was tested in triplicate and a
calibration curve with six data points for catechol was obtained. The results were compared to
a catechol calibration curve and the total phenolic content of EA-Loyola AR1 extract was
expressed as milligram of catechol equivalents per gram of extract.
Determination of Reducing Power
Determination of reducing power in EA-Loyola AR1 was evaluated according to the method
of [30]. Different amounts of the extract (2001,000 g/ml) was suspended in distilled water

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and mixed with 2.5 ml of 0.2 M phosphate buffer (pH 6.6) and 2.5 ml of 1 % K3Fe(CN)6. The
mixture was incubated at 50 C for 20 min; 2.5 ml of 10 % TCA was added to the mixture and
centrifuged at 3,000 rpm for 10 min. The upper layer of the solution (2.5 ml) was mixed with
distilled water (2.5 ml) and FeCl3 (0.5 ml, 0.1 %), and the absorbance was measured at
700 nm. BHT was used as standard.
DPPH Radical-Scavenging Activity
DPPH radical-scavenging activity of EA-Loyola AR1 was measured according to [13]. The
methanol DPPH solution (0.15 %) was mixed with serial dilutions (2001,000 g/ml) of
the extract and after 10 min, the absorbance was read at 515 nm. Vitamin C was used as
standard. The antiradical activity was expressed as 50 % inhibition (IC50; in microgram per
milligram). The ability to scavenge the DPPH radical was calculated using the following
equation:
DPPH scavenging effect % A0 A1 =A0  100

Where A0 is the absorbance of the control at 30 min and A1 is the absorbance of the sample
at 30 min. All samples were analyzed in triplicate.
Hydroxyl Radical-Scavenging Activity
The assay was performed as described by the method of [10] with minor changes. All
solutions were prepared freshly. One milliliter of the reaction mixture comprised with
100 l of 28 mM 2-deoxy-2-ribose (dissolved in phosphate buffer, pH 7.4), 500 l
solution of various concentrations of EA-Loyola AR1 (2001,000 g/ml), 200 l of
200 M FeCl3, 1.04 mM EDTA (1:1 v/v), 100 l H2O2 (1 mM), and 100 l ascorbic
acid (1 mM). After an incubation period of 1 h at 37 C, the extent of deoxyribose
degradation was measured by thiobarbituric acid reaction. The absorbance was read at
532 nm against the blank solution. Vitamin C was used as a positive control. The
scavenging activity was calculated using formula (1).
Nitric Oxide Radical Inhibition Activity
Sodium nitroprusside in an aqueous solution at physiological pH spontaneously generates
nitric oxide; it interacts with oxygen to produce nitrite ions, which can be estimated by
the use of GriessIllosvoy reaction [11]. In the present investigation, GriessIllosvoy reagent
was modified using naphthylethylenediamine dihydrochloride (0.1 % w/v) instead of 1naphthylamine (5 %). The reaction mixture (3 ml) containing sodium nitroprusside
(10 mM, 2 ml), phosphate buffer saline (0.5 ml), and different concentrations of EALoyola AR1 (2001,000 g/ml) or standard solution (0.5 ml) was incubated at 25 C for
150 min. After incubation, 0.5 ml of the reaction mixture containing nitrite was pipetted
and mixed with 1 ml of sulphanilic acid reagent (0.33 in 20 % glacial acetic acid) and
allowed to stand for 5 min for completing diazotization. Then, 1 ml of
naphthylethylenediamine dihydrochloride (1 %) was added, mixed, and allowed to stand
for 30 min. A pink-colored chromophore was formed in diffused light. The absorbance of
these solutions was measured at 540 nm. Vitamin C was used as positive control. The
scavenging activity was calculated using the formula (1).

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Superoxide Anion Radical-Scavenging Activity


Superoxide anion radical-scavenging activity of EA-Loyola AR1 was determined by monitoring the competition of those with NBT for the superoxide anion generated by the PMS
NADH system [21]. Superoxide radicals were generated in 1 ml of 20 mM TrisHCl buffer
pH 8.0 containing 0.05 mM NBT, 0.01 mM PMS, and different concentration of extracts
(2001,000 g/ml) were preincubated for 2 min. The reaction was initiated by the addition of
0.078 mM NADH. Blue chromogen, formed due to NBT reduction was read at 560 nm.
Results were expressed as percentage of inhibition of superoxide radicals. Vitamin C was used
as a positive control. The scavenging activity was calculated using the formula (1).
Lipid Peroxidation Inhibition
The inhibition effect of EA-Loyola AR1 on lipid peroxidation was determined according to the
thiobarbituric acid method. FeCl2H2O2 was used to induce liver homogenate peroxidation
[39]. In this method, 0.2 ml of different concentration of extract (2001,000 g/ml) was mixed
with 1 ml of 1 % liver homogenate (each 100 ml homogenate solution contains 1 g rat liver);
then 50 l of FeCl2 (0.5 mM) and H2O2 (0.5 mM). The mixture was incubated at 37 C for
60 min; then 1 ml of trichloroacetic acid (15 %) with thiobarbituric acid (0.67 %) was added
and the mixture was heated in boiling water for 15 min. The absorbance was recorded at
532 nm. Vitamin C was used as positive control. The percentage of inhibition was calculated
using the formula (1).
Antioxidant Activity of EA-Loyola AR1 Using -Carotene Linoleate Model System
The antioxidant activity of EA-Loyola AR1 was evaluated by -carotene linoleate model [26].
A solution of -carotene was prepared by dissolving 2 mg of -carotene in 10 ml of
chloroform. Two milliliters of this solution were pipetted into a 100 ml round-bottom flask.
After chloroform was removed under vacuum, 40 mg of purified linoleic acid, 400 mg of
Tween 40 emulsifier, and 100 ml of aerated distilled water were added to the flask with
vigorous shaking. Aliquots (4.8 ml) of this emulsion were transferred into different test tubes
containing different concentrations of the extracts (2001,000 g/ml). As soon as the emulsion
was added to each tube, the zero time absorbance was measured at 470 nm. The tubes were
then placed at 50 C in a water bath. Butylated hydroxyanisole (BHA) was used as positive
control. Antioxidant activity (AA) was calculated using the following equation:
AA carotene content after 2 h of assay=initial carotene content  100
Statistical Analysis
The data for biochemical and physiological parameters were analyzed and expressed
as meanSD. The IC50 values were calculated from linear regression analysis. Results
were processed by computer program, Microsoft Excel (2007).

Results
The morphological properties of Streptomyces strain showed subsequent changes in
substrate or vegetative mycelium, the nature of aerial mycelium, size, and shape

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Fig. 1 a Pure culture of Loyola AR1 strain. b Gram staining photograph of Loyola AR1 (40)

shown in (Fig. 1a, b; Table 1) and 16S rRNA partial sequences strongly proved that
Loyola AR1 belongs to the genus Streptomyces sp. A phylogenetic tree was constructed based on neighbor-joining method (Fig. 2). In this, bioactive metabolites
recovered from MNG broth and ethyl acetate was used for solvent extraction. The
result for in vitro -glucosidase inhibition assay for EA-Loyola AR1 and acarbose
were shown in (Table 2). Total phenolic content of EA-Loyola AR1 was found to be
176.831.17 mg catechol equivalent/gram extract, respectively. Figure 3a shows the
reductive capability of EA-Loyola AR1 that increased with the quantity of the sample.
The extract could reduce the most Fe3+ ions, which had a lesser reductive activity
than the standard of BHT. EA-Loyola AR1 exhibited a significant dose-dependent
inhibition of DPPH activity with a IC50 at a concentration of 750.501.61 g/ml. The
IC50 value of vitamin C was 440.122.25 g/ml (Fig. 3b). The concentrations for
50 % hydroxyl radical scavenging inhibition were found to be 690.202.38 and
290.102.55 g/ml for the EA-Loyola AR1 and vitamin C shown in (Fig. 3c). The
scavenging of nitric oxide by EA-Loyola AR1 was increased in a dose-dependent
manner, 50 % of nitric oxide was scavenged at the concentration of 850.501.77 g/
ml. The IC50 value of vitamin C was 510.122.34 g/ml (Fig. 3d). The superoxide
anion derived from dissolved oxygen by phenazinemethosulphate/NADH coupling
reaction reduces nitro blue tetrazolium. Superoxide anion radical scavenging activity
of EA-Loyola AR1, vitamin C. The IC50 values, 880.081.80 and 470.302.73 g/

Table 1 Morphological characterization of Loyola AR1 strain

Culture character

Growth response

Growth under anaerobic condition

Gram staining

Growth and shape

Substrate, circular

Spores

Pink color

Motility
Diffusible pigments

Nonmotile

Optimum days for growth

7 days

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Fig. 2 Neighbor-joining phylogenetic tree analysis of Streptomyces sp. Loyola AR1

ml, respectively, shown in (Fig. 3e). EA-Loyola AR1 showed inhibition of lipid peroxidation
effect with 50 % of inhibition at 670.502.52 g/ml. The IC50 value of vitamin C was 560.30
2.52 g/ml, respectively, shown in (Fig. 3f). In the -carotene linoleate system, -carotene
undergoes rapid discoloration in the absence of antioxidants. The addition of extracts
to this system prevents the bleaching of -carotene at different degrees. EA-Loyola
AR1 of -carotene bleaching on a dose-dependent manner. Based on 120 min reaction
time (Fig. 3g), the extract showed 50 % inhibition at 580.201.43 g/ml and the
value for BHA was 230.121.48 g/ml.

Discussion
Actinomycetes are abundant in soils with high organic matter and low moisture
contents [20]. In the present study, Loyola AR1 strain was isolated from Ambattur
Table 2 -Glucosidase inhibition
of EA-Loyola AR1

Sample

EA-Loyola AR1

Acarbose

Concentrations
(g/ml)

% of Inhibition

IC50 (g/ml)

860.502.68

200

15.480.77

400

28.781.51

600

34.171.45

800

47.812.78

1,000

57.742.59

200

32.152.87

400

43.262.38

600
800

56.390.29
70.202.31

1,000

80.472.95

500.501.33

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Lake soil sediments, Tamil Nadu, India. Streptomyces Loyola AR1 was identified
according to Bergeys Manual of Determinative Bacteriology [37]. Further, genomic
DNA preparation and PCR amplification of Loyola AR1 strain resulted in 1,500 bp amplicon.
16S rRNA sequence of the strain Streptomyces sp. Loyola AR1 was submitted to the GenBank
(HM163569).
The drugs for lowering glucose are inhibitors of -glucosidase enzyme. This enzyme
breaks down the carbohydrates into absorbable monosaccharides [34]. Therefore, investigation on such agents from new, unexplored sources has become important. Also, there is
a need to find new, safe, and effective therapeutic agents for the treatment of many
diseases and disorders associated with carbohydrate metabolism. For this purpose, we
investigated the inhibitory effect of EA-Loyola AR1 extract from lake soil-derived
Streptomyces strain Loyola AR1 on -glucosidase. There are reports on enzyme inhibitor
producing actinomycetes [15, 16].
Antioxidant properties of EA-Loyola AR1 were evaluated with varying parameters.
Phenolic compounds were considered as a major group of compounds that contributed
to the antioxidant activity [40]. The reducing power increased with increasing concentration of the EA-Loyola AR1. The reducing capacity of a compound may serve as
a significant indicator of its potential antioxidant activity [25]. DPPH test is usually
used as the reduction of alcoholic DPPH solution in the presence of a hydrogendonating antioxidant, due to the formation of the nonradical form DPPH-H by the
reaction [5]. EA-Loyola AR1 has the ability to reduce the stable radical DPPH to
yellow-colored diphenylpicrylhydrazine. The hydroxyl radical is an extremely reactive
free radical formed in biological systems and has been implicated as a highly
damaging species in free radical pathology, capable of damaging almost every molecule found in living cells [14]. Hydroxyl radical-scavenging capacity of an extract is
directly related to its antioxidant activity [3]. EA-Loyola AR1 inhibited free radicalmediated deoxyribose damage remarkably. It has been reported that reducing power is
associated with antioxidant activity and may serve as a significant reflection of the
antioxidant activity [28]. Compounds with reducing power indicate that they are
electron donors, and can reduce the oxidized intermediates of lipid peroxidation
processes so that they can act as primary and secondary antioxidants [38]. For the
measurements of the reductive ability, we studied the Fe3+ to Fe2+ transformation in
the presence of EA-Loyola AR1. Nitric oxide radical inhibition study showed that the
extract was a potent scavenger of nitric oxide. Scavengers of nitric oxide competed
with oxygen leading to reduced production of nitric oxide [24]. Superoxide, the oneelectron reduced form of molecular oxygen, is a precursor of other ROS such as
hydrogen peroxide, hydroxyl radical, and singlet oxygen that have the potential of
reacting with biological macromolecules and thereby inducing tissue damages [1].
These results clearly indicated that EA-Loyola AR1 was a potent scavenger of
superoxide radicals in a dose-dependent manner. Lipid peroxidation is an oxidative
alteration of polyunsaturated fatty acids in the cell membranes that generates a
number of degradation products. Malondialdehyde, one of the products of lipid
peroxidation, has been studied widely as an index of lipid peroxidation and as a
marker of oxidative stress [17]. -Carotene in this model system undergoes rapid
discoloration in the absence of an antioxidant. This is because of the coupled
oxidation of -carotene and linoleic acid, which generates free radicals [18]. In our
present study, the EA-Loyola AR1 extend -carotene bleaching by neutralizing the linoleatefree radical and other free radicals formed in the system.

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Fig. 3

a Reducing power determination of different concentrations (2001,000 g/ml) of EA-Loyola AR1 and BHT.
Each value presents the mean SEM of triplicate experiments. b DPPH radical scavenging activity of different
concentrations (2001,000 g/ml) of EA-Loyola AR1 and vitamin C. Each value presents the mean SEM of triplicate
experiments. c Hydroxyl radical scavenging activity of different concentrations (2001,000 g/ml) of EA-Loyola AR1
and vitamin C. Each value presents the mean SEM of triplicate experiments. d Nitric oxide radical inhibition activity
of different concentrations (2001,000 g/ml) of EA-Loyola AR1 and vitamin C. Each value presents the mean SEM
of triplicate experiments. e Superoxide anion radical-scavenging activity of different concentrations (2001,000 g/ml)
of EA-Loyola AR1 and vitamin C. Each value presents the mean SEM of triplicate experiments. f Lipid peroxidation
inhibition of different concentrations (2001,000 g/ml) of EA-Loyola AR1 and vitamin C. Each value presents the
mean SEM of triplicate experiments. g -Carotene bleaching effect in different concentrations (2001,000 g/ml) of
EA-Loyola AR1 and BHA. Each value presents the mean SEM of triplicate experiments

Conclusion
Our study suggests that Loyola AR1 strain belongs to Streptomyces sp. possessing glucosidase inhibition and oxidative stress mechanism in its system capability to reflect an
imbalance between the systemic manifestation of reactive oxygen species and a biological
systems ability to readily detoxify the reactive intermediates or to repair the resulting damage.
It might be useful to prevent the progress of various oxidative stress-related diseases. Further
investigation on the isolated component may lead the pharmaceutical industries for clinical use.
Acknowledgments Authors wish to thank the management of the Department of Plant Biology & Plant
Biotechnology and M.Sc. Biotechnology Department, Loyola College, Chennai, Tamil Nadu, India, for providing necessary facility to carry out this research work. Also, we thank to Sunil Christhudas (SRF, ICMR, New
Delhi), N. Murugan (SRF, Sankara Nethralaya, Chennai), and G. Ramesh Kumar (SRF, Aravind Eye Hospital,
Tirunalveli) for their constant support throughout the research work.

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OPTIMIZATION OF GROWTH AND BIOACTIVE


METABOLITE PRODUCTION: FUSARIUM
SOLANI
ARTICLE in ASIAN JOURNAL OF PHARMACEUTICAL AND CLINICAL RESEARCH MAY 2013
Impact Factor: 0.51

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Loyola College

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Retrieved on: 04 September 2015

ISSN - 0974-2441

Vol 6, Suppl 3, 2013

Research Article

OPTIMIZATION OF GROWTH AND BIOACTIVE METABOLITE PRODUCTION: FUSARIUM


SOLANI
J. NOMILA MERLIN1, I.V.S. NIMAL CHRISTHUDAS2, P. PRAVEEN KUMAR2, P. AGASTIAN2*
1Research

and Development Centre, Bharathiar University, Coimbatore, TN, India, 2Department of Plant Biology and Biotechnology,
Loyola College, Chennai, TN, India Email: agastian@loyolacollege.edu
Received: 2 May 2013, Revised and Accepted: 3 May 2013

ABSTRACT
The present study aimed to evaluate optimize the growth and antibacterial activity of endophytic fungi isolated from Tylophora indica. Based on the
morphological and molecular characteristic, the isolate was identified as Fusarium solani. Modified liquid medium (M2D) was used as a basal
medium for growth and antibacterial activity. The growth and metabolite production were optimized with 4% dextrose, 0.05% yeast extract and
aspartic acid (0.01%) as carbon, nitrogen and amino acid respectively. The optimum pH and temperature of the strain were 6.0, 252 C observed
for growth and secondary metabolite production. The 9 th day was found to be optimum incubation period of growth and secondary metabolite
production. The metabolite showed maximum inhibition against Enterococcus faecalis, lowest inhibition zone was against Yersinia enterocolitica.
The ESI-MS analysis of bio active compound shows the peak at 301m/z.
Keywords: Endophytic fungus, Tylophora indica, antibacterial activity, Medium Optimization, ESI -MS
INTRODUCTION

Materials and Methods

Endophytes are microorganisms that reside in living plant tissues,


apparently without inflicting negative effects [2]. Endophytes are
presumably ubiquitous in the plant kingdom, some of which can
improve the ecological adaptability of hosts [23, 27, 30]. Moreover,
certain endophytic fungi capable to synthesizing the medicinal
products produced similarly in plants [38]. At present, much
research has focused on isolation of endophytic fungi from
pharmaceutical plants, such as Camptotheca acuminate [21], pine
[7], and Taxus plants [3, 13, 40], discovering a vast number of
undescribed endophytic fungi species, some of which have potential
to be used in the production of medicine. Recently investigations
have been intensified due to the potentialities of endophytic
microorganisms in production of bioactive metabolites, immunesuppressants, anticancer compounds and bio-control agents [36].
Although the active constituents may occur in lower concentrations,
endophytic fungal pigments may be a better source of antimicrobial
compounds than synthetic drugs. Several microorganisms such as
Monascus, Peacilomyces, Serratia, Cordyceps, Streptomyces and
Penicillium have the ability to produce pigments with high yield [33]
which have been developed as a drug and used to treat the wound
infections and skin diseases caused by the pathogens. Many of the
endophytic fungal strains have attracted special attention because
they have the capability of producing different colour pigments with
high chemical stability [38]. The physical and chemical parameters
like pH, temperature, incubation period, carbon and nitrogen
sources and amino acid plays a major role on production of bioactive
compounds and antimicrobial agents [11]. Since, T. indica is an
endemic plant an attempt is made to conserve the medicinal plant
through exploration of endophytic fungi for growth and bioactive
compound production.

Chemicals

Tylophora indica (Burm. f) Merrill (Asclepiadaceae), an indigenous


medicinal plant of Asian origin, is known to host several metabolites
having insecticidal property [17] and medicinal property [29].Leaf
and stem extracts have shown anti-asthmatic [35] anti-leukemic
[8]anti-inflammatory [10] and anti-tumor [5], activities. Therefore,
the present investigation was aimed to studythe medium
optimization and antibacterial activity of Fusarium strain isolated
from Tylophora indica.

All the analytical grade chemicals and solvents were purchased from
Sigma chemical co., USA. SD fine chem.Ltd, Biosar, India and HiMedia Pvt. Ltd. Mumbai, India.
Isolation and identification
The Fusarium strain was isolated from T. indica as we described
previously [26], and strain was deposited in the GenBank JN786598.
Culture medium and extraction
The medium adjusted to pH 5.5 was composed of (g/L): Potato 200;
glucose 40.0; Yeast extract 0.8; Peptone 0.5; Soytone 1.0; KH 2PO42.0;
MgSO4 0.5; (NH4)2SO43.0;phenyl alanine 0.01. Erlenmeyer flasks
(500 ml) containing 200 ml of medium were incubated at 28C in a
static way for 21 days in dark. After incubation the culture was
passed through four layers of cheese cloth to remove solids and
extracted with dichloromethane (DCM).
Antibacterial activity
Test Organisms
The human pathogenic bacteria used for the test included grampositive and gram negative organisms were: Staphylococcus aureus
ATCC 25923, Klebsiella pneumoniae ATCC 15380, Enterococcus
faecalis ATCC 29212, Yersinia enterocolitica MTCC 840, Erwinia. sp
MTCC 2760, Vibrio parahaemolyticus MTCC 451, Enterobacter
aerogenes MTCC 111, Escherichia coli ATCC 25922 and Proteus
vulgaris MTCC 1771. All cultures were procured from IMTECH,
chandigarh, India.
Disc-diffusion method
The crude extracts were dissolved in the DMSO (Dimethyl
sulphoxide) to a final concentration of 20 mg/ml. Antibacterial tests
were carried out by disc-diffusion method [24] using 100 l of
suspension containing 108 CFU/ml of test bacteria, spread on MHA
(Muller Hinton Agar) medium, respectively. The discs (6 mm) were

Nomila Merlin et al.

impregnated with 20 l of the extracts (1.25, 2.5, 5.0 mg/disc) with


the concentration of 20 mg/ml and placed on the inoculated agar.
Negative controls were prepared using the same solvent.
Streptomycin (10 g/disc) was used as positive control. The
inoculated plates were incubated at 37 C for 24 h for clinical
bacterial strains. Antibacterial activity was evaluated by measuring
the zone of inhibition against the test organisms.
Isolation of active fraction
The crude extract was purified by thin layer chromatography (TLC)
(Merk Ltd.) using dichloromethane : acetone (80:20) as running
solvent system. All the fractions were screened for antibacterial
activity against Gram positive and gram negative bacteria by disc
diffusion method [24]. The each disc were impregnated with 20 l of
each TLC fraction (1mg/ml) and placed on the medium and
incubated at 37 C for 24 h. Streptomycin, 10 g/disc was used as
positive control. The MIC of active fraction was determined using
micro dilution bioassay by Eloff [6]. Further identification of active
fraction was using the Electron Spray Ionization (ESI-MS) technique
with an Agilent 1100 LC/ MSD trap. The nebulizer gas flow rate of
the sample was 2 L min-1 and the capillary voltage was 2.2 kV.
Medium Optimization
Standardization of Basal Medium
Standardization of basal medium for optimum growth and bioactive
metabolite production consists of (g/l) Czapek Dox Broth (CDB)
containing: NaNO3 3.0g, K2HPO4 1.0g, MgSO4 0.5g, KCl 0.5g, FeSO4
0.01g, Sucrose 30.0g, Modified liquid medium-1 (M1D); KNO3
80.0mg, CaNO3 0.5g, KCl 60.0mg, MgSO4 360.0mg, NaH2PO4 20.0mg,
Sucrose 30g, Ammonium tartrate 5.0g, FeCl3 2.0mg, MnSO4 5.0mg,
ZnSO4 3.0mg, H3BO3 1.4mg, KI 0.7mg, Yeast extract 0.25g, Peptone
1.0g, Soytone 1.0g, Modified liquid medium-2 (M2D) Potato 200g,
Glucose 40g, Peptone 0.5g, Yeast extract 0.8g, (NH4)2SO4 3.0g,
KH2PO42.0g, MgSO4 0.5g, Phenylalanine 0.01g,Potato dextrose broth
(PDB) Potato 200.0g, Dextrose 20.0g, Potato dextrose yeast extract
broth (PDYEB) Potato 200.0g, Dextrose 20.0g, Yeast extract 2.0g and
Malt extract (ME) Malt extract 20.0 g, Peptone 1.0 g, glucose 20.0 g
media were used. After 21 days of incubation at 28 2 C with pH
6.4, the biomass and secondary metabolite production were
recorded. Biomass was determined by drying the mycelial mat at 70
C until a constant weight was obtained.
Effect of carbon and nitrogen sources
Various carbon sources such as dextrose, sucrose, starch, galactose,
mannose, fructose, lactose and maltose. The various nitrogen
sources like beef extract, yeast extract, peptone, malt extract,
ammonium tartarate, ammonium nitrite, and ammonium sulphate,
respectively were amended separately into the basal medium (M2D)
at a concentration of carbon source (4%) and nitrogen source
(0.05%). The strain LCPANCF01 was inoculated to the respective
medium and incubated at 28 2 C in dark for 21 days in static
condition and their respective biomass vis-a`-vis metabolite
production were recorded.
Effect of amino acid
The effect of various amino acids on growth and bioactive
metabolite production was compared in basal medium in
combination with dextrose and yeast extract. The medium amended
with amino acids (0.001%), carbon source (4%) and nitrogen source
0.05% respectively. The strain LCPANCF01 was inoculated to the

Asian J Pharm Clin Res, Vol 6, Suppl 3, 2013, 98-103

medium and incubated for 21 days in dark at 28 2 C in stationary


condition. The biomass accumulation and bioactive metabolite
production were estimated.
Effect of pH
Initial pH ranges were adjusted from 3 to 11 at a difference of one to
the basal medium amended with dextrose and yeast extract, were
incubated for 21 days in dark at 28 2 C under still condition. The
biomass and bioactive metabolite production were estimated by
comparing the dry weight of mycelial mat and the UV -max of the
clear supernatant.
Effect of temperature
The strain LCPANCF01 was inoculated into basal medium amended
with dextrose and yeast extract, were grown in various range of
temperature from 15 to 50 C at a difference of 5 C for 21 days in
dark under stationary condition and the growth and secondary
metabolite production were recorded.
Determination of incubation period
The strain LCPANCF01 was inoculated into the basal medium (M2D)
amended with dextrose and yeast extract, incubated up to 21 days in
stationary condition in dark at 28 2 C. Their growth and the
secondary metabolite production was determined everyday by
comparing mycelial weight and UV -max of the clear supernatant.
Effect of NaCl concentration
The effect of salinity on growth and bioactive metabolite production
was carried out by incubating the fungus in various NaCl
concentrations (1% to 10%) into the basal medium amended with
4% dextrose, 0.05% yeast extract as carbon and nitrogen source
respectively, while keeping other parameters at optimum level. The
biomass and bioactive metabolite production for each NaCl
concentration were estimated.
Specific rate of product formation (qp)
The specific rate of active metabolite production (qp) was calculated
according to the following equation:
qp = 1/X(dP/dt),
Where X is the biomass concentration (g/ml), P is antibacterial
agent concentration and t is time respectively. The derivative dP/dt
was calculated according to the method proposed by [20] with
software for graphical analysis Origin PRO-7.5.
Results and Discussion
Antibacterial activity and MIC
Fusarium sp. has been reported as endophytes from several plants
with diverse biological activity [34, 19, 3]. This suggests their
ubiquity as endophytes within the plant kingdom and provides an
opportunity to discover novel bioactive metabolites. Table 1and 2
shows antibacterial activity of isolate, MIC of bioactive compound
was defined as the lowest concentration at which 100% inhibition of
growth compared to antibiotic free control. Lowest MIC value of
62.5 g/ml was found against Enterococcus faecalis (Table 2). Active
compound revealed highest inhibition zone of 25 mm against
Enterococcus faecalis and lowest inhibition zone was against Yersinia
enterocolitica (15 mm). Similar MIC (25 g/ml to 100 g/ml), as well
as inhibition zone, produced by some endophytes was reported by
Lu, et al., [22].

Table 1: In vitro antibacterial activity of TLC fractionation against the test organisms
Rf Value (100 g/ml) (Inhibition of Zone diameter in mm)
0.126
0.247
0.345
0.572
0.761
0.872
Staphylococcus aureus
20 (2)
Klebsiella pneumoniae
17 (2)
Enterococcus faecalis
15
22( 2)
Yersinia enterocolitica
15 (2)
Erwinia sp.
25 (2)
Vibrio parahaemolyticus
12
19 (2)
Enterobacter aerogenes
24 (2)
Escherichia coli
21 (2)
Proteus vulgaris
16 (2)
Test Organism

0.912
-

99

Nomila Merlin et al.

Table 2: Minimum inhibitory concentration of TLC active


fractionate (Rf - 0.872) isolated from the strain Fusarium solani
LCPANCF01
MIC of
Test Organisms
Std
0.872
Staphylococcus aureus
500
100
Klebsiella pneumoniae
1000
100
Enterococcus faecalis
62.5
25
Yersinia enterocolitica
500
50
Erwinia sp
250
<12.5
Vibrio parahaemolyticus
1000
100
Enterobacter aerogenes
500
100
Escherichia coli
250
100
Proteus vulgaris
1000
100

Asian J Pharm Clin Res, Vol 6, Suppl 3, 2013, 98-103

Medium Optimization
Standardization of basal medium
Even though water agar medium was used for the isolation of
Fusarium solani LCPANCF01, further standardization of medium
showed that M2D was suitable to use as basal medium (Fig 3a).
Comparative study of growth vis-a`-vis secondary metabolite
production indicated a statistically significant higher biomass and
bioactive metabolite production in M2D by Fusarium solani
LCPANCF01. Hence, M2D medium was used to optimize different
cultural and environmental parameters for growth and bioactive
secondary metabolite production.

MIC; Minimum Inhibitory Concentration, Std; Standard.


Characterization of the bioactive metabolite
Approximately ~ 500 mg/l crude extract was obtained from the
culture broth in dichloromethane solvent extraction. In the
preparative TLC seven major fractions from the crude compound
were recovered with different Rf values (Table2). Each of the major
fractions from the TLC plates with different Rf values was bio
assayed to determine the active fraction. The pure compound was
soluble in methanol. The active fraction with R f value 0.87 showed
UV -max in methanol at 223 nm (Fig 1). ESI-MS spectrum of the red
solid active fraction showed the peak at 301, m/z. Jittra, [15] also
reported the peak at 303 m/z, hence the compound identified was
javanicin derivatives (Fig 2).

Fig 3a: Standardization of basal media on growth and bioactive


metabolite production, Czapek Dox Broth (CDB), Modified
liquid medium 1 (M1D), Modified liquid medium 2 (M2D),
Potato dextrose broth (PDB), Potato dextrose yeast extract
broth (PDYEB), Malt extract (ME).
Effect of carbon and nitrogen source

223n
m

Fig 1: UV -max of bioactive active fractionation isolated from


dichloromethane extracts of Fusarium solani LCPANCF01.

Fig 2: ESI- MS analysis of TLC active fraction shows the


corresponding peak at 301m/z

Fig. 3b shows the effect of carbon and nitrogen source on the


production of biomass and bioactive metabolites. A statistically
significant higher biomass (3.37 mg/ml) and bioactive metabolite
(17.65 g/ml) were produced by the LCPANCF01 in dextrose
amended medium and it was followed by lactose (15.32 g/ml),
respectively in comparison to control (13.97 g/ml) basal medium.
Sucrose (11.45 g/ml), starch (11.38 g/ml), fructose (10.24 g/ml),
galactose (10.19 g/ml), mannose (10.16 g/ml) and glycerol (10.17
g/ml) showed moderate amounts of biomass and bioactive
metabolite production. This finding suggested that dextrose was a
stable carbon source used by F. solani LCPANCF01 which was also
accordant with former research of Strobel, [37]. Amendment of yeast
extract (18 g/ml) enhanced the secondary metabolite production
while peptone (2.36 mg/ml), Soytone (2.01 mg/ml) and beef extract
(2.26 mg/ml) increase biomass production but not bioactive
metabolite (Fig. 3b).

Fig 3b: Effect of Different carbon and nitrogen sources on


growth and bioactive metabolite production, Beef extract (BE),
Yeast extract (YE), Malt extract (ME), Ammonium tartarate
(NH4)2C4H4O6, Ammonium nitrite NH4NO3, Ammonium sulphate
(NH4)2SO4.
Radu, [28] also reported the maximum production of antifungal and
antitumor compound from endophytic fungi grown in glycerol

100

Nomila Merlin et al.

(4.0%) and yeast extract (0.5%) amended medium. The simple


carbohydrates like glucose, dextrose through metabolic pathway
affects the production of intermediates leading to primary as well as
secondary metabolites in addition to CO 2, water and energy [39].
Addition of glucose resulted highest growth of the fungus, but
significantly less bioactive metabolite production. In many
fermentation processes higher concentration of glucose has a
suppressive effect on production of bioactive metabolites [14].
Effect of amino acid
Amino acid along with dextrose enhanced the growth and secondary
metabolite production of strain LCPANF01 (2.8 mg/ml, 17.69 g/ml)
(Fig 3c). It reflects the amino acid supplement may have some role
by sharing their carbon ring or both carbon and nitrogen skeleton in
to the primary or secondary metabolism processes of
microorganisms [25]. Kim [18] reported the importance of rice oil
for improving the production of cephalosporin-C production by
Cephalosporium acremonium M25.

Fig 3c: Effect of different amino acids on growth and bioactive


metabolite production.
Effect of pH
Medium with initial pH 6 was found to be optimal for growth (3.5
mg/ml) and bioactive metabolites production (14.72 g/ml) by the
isolate (Fig 3d), pH-5 and pH-7 also supports the growth and
bioactive metabolite production of the strain. No growth was
observed at pH < 3 and pH >11. Digrak, [4] reported that highest
production of biomass by F. equiseti was at pH 8, whereas maximum
toxic metabolite was produced at the pH 5. The pH of culture
medium is one of the determining factor for the metabolism and
hence for the biosynthesis of secondary metabolites. The pH is
related to permeability characteristics of the cell wall and membrane
and thus has got effect on either ion uptake or loss to the nutrient
medium [12]. Although literature exists on the growth of fungus in
acidic conditions, in our study it was found LCPANCF01 grows in
between pH 5-7. Rubini, [31] also reported the growth and
antibacterial agent production at neutral pH.

Fig 3d: Effect of pH on growth and bioactive metabolite


production

Asian J Pharm Clin Res, Vol 6, Suppl 3, 2013, 98-103

Effect of temperature
Maximum growth (2.45 mg/ml) and bioactive metabolite production
(13.06 g/ml) by Fusarium solani LCPANCF01 was recorded at 25
C and it was followed by 2.10 mg/ml growth and (11.46 g/ml)
bioactive metabolite production recorded at 30 C (Fig 3e). Less
growth and bioactive metabolite production was found at low (15
C) as well as in high temperature (35 C). An exponential growth
pattern was recorded at temperature between 15 C to 35 C, while
growth ceased at <40 C and >50 C. Study proved that low
temperature may cease the metabolic activity of the fungus and high
temperature kills the cell of the fungus. Similarly, Huang, [13] also
reported the isolation of antifungal and anti-tumor agent from
endophytic fungi at 25 C and 7-9 days of incubation period.

Fig 3e. Effect of temperature on growth and bioactive


metabolite production
Effect of incubation period
The incubation period for 9 days was observed to be optimum for
maximum biomass (3.42 mg/ml) and bioactive metabolite (14.08
g/ml) production (Fig 3f). Moreover the growth (3.39 mg/ml) and
secondary metabolite production (13.89g/ml) was slightly lowered
at 10 day of incubation period. Maximum growth and production of
antibacterial agent was recorded after the fungus reached its
stationary phase and remaining almost constant upto 15 days of
incubation. After 15 days the growth and secondary metabolite
production was significantly very low. Stinson, [36] also reported
similar results in the case of endophyte Gliocladium sp. Many studies
on isolation and characterization of endophytic diversity from
different medicinally important plant species have been done or are
in progress around the world [16]. Some workers have given a brief
record on antifungal and antibacterial activity of endophytes
isolated from different gymnosperms of North East India [1, 32].

Fig 3f: Effect of incubation period on growth and bioactive


metabolite production

101

Nomila Merlin et al.

11.
Effect of NaCl
Regarding the effect of NaCl concentration, 3.0% was found to be
optimum for growth (4.9 mg/ml) and production of the antibacterial
agent (11.90 g/ml) while the concentration above 6% reduces the
growth as well as the metabolite production (Fig 3g).

12.
13.

14.

15.

16.
17.
18.
Fig 3g: Effect of NaCl on growth and bioactive metabolite
production in Fusarium solani LCPANF01
CONCLUSION

19.

Our study reports the optimization of still culture conditions for the
high growth and production of bioactive secondary metabolite
compound from F. solani. Using the statistical procedures, the
optimized culture conditions were successfully established. These
findings will assist in formulating still culture medium which
suitable for producing the antibacterial compound from Fusarium
strain.

20.

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In Vitro -Glucosidase Inhibition and


Antioxidative Potential of an Endophyte
Species (Streptomyces sp. Loyola UGC) Isolated
from Datura stramonium L.
ARTICLE in CURRENT MICROBIOLOGY FEBRUARY 2013
Impact Factor: 1.36 DOI: 10.1007/s00284-013-0329-2 Source: PubMed

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In Vitro -Glucosidase Inhibition and


Antioxidative Potential of an Endophyte
Species (Streptomyces sp. Loyola UGC)
Isolated from Datura stramonium L
I.V.S.Nimal Christhudas, P.Praveen
Kumar & P.Agastian

Current Microbiology
ISSN 0343-8651
Volume 67
Number 1
Curr Microbiol (2013) 67:69-76
DOI 10.1007/s00284-013-0329-2

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Curr Microbiol (2013) 67:6976
DOI 10.1007/s00284-013-0329-2

In Vitro a-Glucosidase Inhibition and Antioxidative Potential


of an Endophyte Species (Streptomyces sp. Loyola UGC) Isolated
from Datura stramonium L
I. V. S. Nimal Christhudas P. Praveen Kumar
P. Agastian

Received: 3 August 2012 / Accepted: 18 January 2013 / Published online: 17 February 2013
Springer Science+Business Media New York 2013

Abstract Endophytic actinomycetes isolated from Datura


stramonium L. was evaluated for its effects against in vitro
a-glucosidase inhibition, antioxidant, and free radical scavenging activities. Based on microbial cultural characteristic
and 16S rRNA sequencing, it was identified as Streptomyces
sp. loyola UGC. The methanolic extract of endophytic actinomycetes (MeEA) shows remarkable inhibition of a-glucosidase (IC50 730.21 1.33 lg/ml), scavenging activity
on 2,2-diphenyl-picrylhydrazyl (DPPH) (IC50 435.31
1.79 lg/ml), hydroxyl radical (IC50 350.21 1.02 lg/ml),
nitric oxide scavenging (IC50 800.12 1.05 lg/ml),
superoxide anion radical (IC50 220.31 1.47 lg/ml), as
well as a high and dose-dependent reducing power. The
MeEA also showed a strong suppressive effect on rat liver
lipid peroxidation. Antioxidants of b-carotene linoleate
model system revels significantly lower than BHA. The total
phenolic content of the extract was 176 mg of catechol
equivalents/gram extract. Perusal of this study indicates
MeEA can be used as natural resource of a-glucosidase
inhibitor and antioxidants.

Introduction
Diabetes mellitus (DM) is a metabolic disorder characterized by hyperglycemia resulting from defects in insulin
I. V. S. Nimal Christhudas  P. Praveen Kumar
Department of Plant Biology and Biotechnology,
Loyola College, Chennai 600 034, India
P. Agastian (&)
Research Department of Plant Biology and Biotechnology,
School of Life Science, Loyola College, Chennai 600 034, India
e-mail: agastian@loyolacollege.edu;
agastianloyolacollege@gmail.com

secretion, insulin action, or both. Along with hyperglycemia and abnormalities in serum lipids, diabetes is associated with micro- and macrovascular complications, which
are the major causes of morbidity and death in diabetic
subjects [15]. The currently available antidiabetic agents
including sulfonylureas, biguanide, and thiazolidinedione
inhibitors are widely used to control the hyperglycemia and
hyperlipidemia, but these drugs fail to significantly alter
the course of diabetic complications and have limited use
because of undesirable side effects and high rates of secondary failure [24]. Thus, it is essential to look for more
effective antidiabetic agents with lesser side effects. There
are many articles related to antidiabetic compounds from
plants and also microbial resources have been proven to be
a rewarding source of antidiabetic compounds reported
from Streptomyces hygroscopicus-limoneus [6, 19], Streptomyces calvus [17]. However, normalizing blood glucose
level is a formidable challenge in clinical practice. The
pharmacological agents with greatest effect on postprandial
hyperglycemia include insulin, lispro, amylin analogs, and
a-glucosidase (acarbose and voglibose) inhibitors [10]. It
has been well acknowledged that plant-derived extracts,
phytochemicals, and microbial sources are potential alternatives to synthetic inhibitors against a-glucosidase.
Datura stramonium L. is a wild-growing herb known as
Jimson weed belongs to the family Solanaceae. The plant
distributed throughout most parts of temperate regions of the
world [1]. Whole plant is used as anti-inflammatory, central
nervous system stimulant [28], for the treatment of respiratory decongestion [35] dental and skin infections, toothache,
and alopecia [5]. It is a hallucinogenic plant that causes
serious poisoning. Consumption of any part of the plant may
result in a severe anticholinergic reaction that may lead to
toxicity and occasionally cause diagnostic difficulties [7]. It
is used recreationally for its anticholinergic effects, resulting

123

Author's personal copy


70

I. V. S. Nimal Christhudas et al.: In Vitro a-Glucosidase Inhibition and Antioxidative Potential

in hallucinations. The entire plant has anticholinergic compounds, but the seeds contain the highest concentration. An
extract made by boiling crushed seeds retains the anticholinergic activity, has a rapid onset of action [3], and thus may
be potentially useful as an alternative to atropine for the
treatment of the muscarinic symptoms of organophosphate
toxicity and some of the central anticholinergic effects [29].
There were overexploitations of this plant by the pharmaceutical industries without making any alternative methods
to conserve this medicinally important plant. Attempt
is made to isolate the endophytic actinomycetes from
D. stramonium L. to evaluate the in vitro a-glucosidase and
antioxidant potential of MeEA (Methanolic extract of
endophytic actinomycetes).

pigments and growth. Visual observation by light microscopy and Gram-staining were performed for further identification [31]. The total genomic DNA was extracted by
CTAB-Method. The actinomycetes DNA fragments were
amplified using Universal primers 16S rRNA and PCR
reaction were standardized as initial denaturation at 94 C
for 3 min, followed by 35 cycle of 1 min at 94 C,
54 C for 1 min, 72 C for 2 min and a final extension at
72 C for 810 min, stop at 4 C for 1 h. The PCR products were stored at 4 C and visualized by electrophoresis.
The gel was photographed in gel documentation. The
amplified product was purified and sequenced with two
fragments of the 27F (5 AGT TTG ATC CTG GCT CAG 3)
and 1492R (5 ACG GCT ACC TTG TTA CGA CTT 3)
region in both the directions and the sequences obtained were
submitted to Genbank for homology search with Blast.

Materials and Methods


Plant Materials
Datura stramonium L. was collected from Irula Tribal
Womens Welfare Society (ITWWS), Chengalpattu, Kanchipuram district, Tamil Nadu, South India, in the month
of January. The species was identified and authenticated by
Dr. D. Narashiman, Department of Botany, Madras Christian
College, Chennai, South India.

Culture Media and Extraction


The spore suspensions of the culture were inoculated on the
Modified Nutrient Glucose Broth (MNGB) medium with
pH 7.0 and incubated at 28 C for 7 days in a shaker. Then,
the mycelium filtered and the culture fluid was extracted
with methanol (1:1). The organic phase was evaporated to
dryness under reduced pressure at 35 C using rotor vacuum evaporator.

Isolation of Actinomycetes
The roots and transition zones of D. stramonium L. were
surface sterilized by the methodology of Johannes et al.,
[14] with some modifications. Samples were thoroughly
washed with running tap water and all the visibly damaged
materials were excluded. Plant parts were rinsed in 0.1 %
Tween 20 for 30 s and followed by bevastin for 23 min to
inhibit the fungal growth, sequentially immersed in 0.1 %
sodium hypochlorite (NaClO) for 30 s and in 75 % ethanol
for 35 min. After each treatment, samples were rinsed
three times in sterile distilled water. Finally the surfacesterilized samples were thoroughly dried in a laminar flow
chamber. The samples was aseptically dissected to expose
cortex region and plated onto actinomycetes isolation
medium, incubated for 1215 days at 28 C. The isolation
medium was supplemented with nalidixic acid and actidion
both to a final concentration of 50 lg/ml to inhibit the
growth of non actinomycetes organisms.
Identification of Actinomycetes
The isolated actinomycetes were dereplicated for cultural
and morphological characteristics, including morphology
and color of aerial mycelium: characteristics of colonies on
the plate, mycelium and spore color, color of diffusible

123

Determination of In Vitro a-Glucosidase Inhibition


and Antioxidant Assay
a-Glucosidase Inhibition Activity
In order to investigate the inhibition activity of MeEA, an
in vitro a-glucosidase inhibition test was performed. a-glucosidase from yeast is used extensively as a screening
material for a-glucosidase inhibitors, but the results do not
always agree with those obtained in mammals. Therefore, we
used the mouse small intestine homogenate as a-glucosidase
solution because we speculated that it would better reflect the
in vivo state. The inhibitory effect was measured by the
method slightly modified method of Dahlqvist [4]. After
fasting for 20 h, the small intestine between the part immediately below duodenum and the part immediately above the
cecum was cut, rinsed with ice-cold saline, and homogenized
with 12 ml of maleate buffer (100 mM, pH 6.0). The
homogenate was used as the a-glucosidase solution. The
assay mixture consisted of 100 mM maleate buffer (pH 6.0),
2 % (w/v) each sugar substrate solution (100 ll), and the
sample extract (2001,000 lg/mL). It was preincubated for
5 min at 37 C and the reaction was initiated by adding the
crude a-glucosidase solution (50 ll) to it, followed by
incubation for 10 min at 37 C. Acarbose was used as a

Author's personal copy


I. V. S. Nimal Christhudas et al.: In Vitro a-Glucosidase Inhibition and Antioxidative Potential

standard. The percentage of inhibition was calculated by the


formula.
Inhibition (%) = [(amount of glucose produced by the
positive control) (amount of glucose produced by the addition
of sample)/(amount of glucose produced by the positive
control)] 9 100.
Total Phenolic Content (TPC)
Total phenolic content was assessed according to the Folin
Ciocalteau method [27] with some modifications. Briefly,
0.1 ml of sample (2001,000 lg/ml), 1.9 ml distilled water,
and 1 ml of FolinCiocalteaus reagent were added in a tube
and then 1 ml of 100 g/l Na2CO3 was added. The reaction
mixture was incubated at 25 C for 2 h and the absorbance of
the mixture was read at 765 nm. The sample was tested in
triplicate and a calibration curve with six data points for
catechol was obtained. The results were compared to a catechol calibration curve and the total phenolic content of
MeEA was expressed as mg of catechol equivalents per gram
of extract.
Reducing Power Activity
The reducing power of MeEA was determined according to
Yen and Duh [32]. Different concentrations of MeEA
(2001,000 lg/ml) were mixed with 2.5 ml of phosphate
buffer (200 mM, pH 6.6) and 2.5 ml of 1 % potassium ferricyanide. The mixtures were incubated for 20 min at 50 C.
After incubation, 2.5 ml of 10 % trichloroacetic acid was
added to each mixture followed by centrifugation at
3,000 rpm for 10 min. The upper layer (5 ml) was mixed
with 5 ml of distilled water and 1 ml of 0.1 % ferric chloride.
The absorbance of the resultant solution was measured at
700 nm and was compared with standard BHT absorbance.
DPPH Radical Scavenging Assay
DPPH quenching ability of MeEA was measured according
to Hanato et al. [11]. A methanol DPPH solution (0.15 %)
was mixed with serial dilutions (2001,000 lg/ml) of the
MeEA and after 10 min, the absorbance was read at
515 nm. The antiradical activity was expressed as IC50 (lg/
ml), (the antiradical dose required to cause a 50 % inhibition). Vitamin C was used as standard. The ability to
scavenge the DPPH radical was calculated by the following
formula:
DPPH radical scavenging activity %
A0  A1 =A0  100

where A0 is the absorbance of the control at 30 min and


A1 is the absorbance of the sample at 30 min. All samples
were analyzed in triplicate.

71

Hydroxyl Radical Scavenging Activity


The hydroxyl scavenging assay was performed as described
by the method of Elizabeth and Rao [8] with minor changes.
All solutions were prepared freshly. One milliliter of the
reaction mixture contained 100 ll of 28 mM 2-deoxy-2ribose (dissolved in phosphate buffer, pH 7.4), 500 ll solution of various concentrations of MeEA (2001,000 lg/ml),
200 ll of 200 lM FeCl3 and 1.04 mM EDTA (1:1 v/v),
100 ll H2O2 (1 mM), and 100 ll ascorbic acid (1 mM).
After an incubation period of 1 h at 37 C, the extent of
deoxyribose degradation was measured by the TBA reaction.
The absorbance was read at 532 nm against the blank solution. Vitamin C was used as a positive control. The scavenging activity was calculated by the formula (1).
Nitric Oxide Scavenging Activity
Sodium nitroprusside in aqueous solution at physiologic pH
spontaneously generates nitric oxide; it interacts with oxygen to produce nitrite ions, which can be estimated by the use
of Griess Illosvoy reaction [9]. In the present investigation,
Griess Illosvoy reagent was modified using naphthyl ethylenediamine dihydrochloride (0.1 % w/v) instead of
1-naphthylamine (5 %). The reaction mixture (3 ml) containing sodium nitroprusside (10 mM, 2 ml), phosphate
buffer saline (0.5 ml), and different concentrations of MeEA
(2001,000 lg/ml) or standard solution (0.5 ml) was incubated at 25 C for 150 min. After incubation, 0.5 ml of the
reaction mixture containing nitrite was pipetted and mixed
with 1 ml of sulphanilic acid reagent (0.33 % in 20 % glacial
acetic acid) and allowed to stand for 5 min for completing
diazotization. Then, 1 ml of naphthyl ethylenediamine
dihydrochloride (1 %) was added, mixed, and allowed to
stand for 30 min. A pink colored chromophore was formed in
diffused light. The absorbance of these solutions was measured at 540 nm against the corresponding blank. Ascorbic
acid was used as standard. The scavenging activity was
calculated by Eq. (1).
Superoxide Scavenging Activity
Superoxide scavenging activity of MeEA was determined by
monitoring the competition of those with NBT for the
superoxide anion generated by the PMSNADH system
[16]. Superoxide radicals were generated in 1 ml of 20 mM
TrisHCl buffer pH 8.0 containing 0.05 mM nitroblue tetrazolium (NBT), 0.01 mM phenazine methosulphate (PMS),
and different concentrations (2001,000 lg/ml) of MeEA
were preincubated for 2 min. The reaction was initiated by
the addition of 0.078 mM NADH. Blue chromogen, formed
due to NBT reduction, was read at 560 nm. Results were
expressed as percentage of inhibition of superoxide radicals.

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72

Table 1 Culture characteristics and morphological identification of the isolated strain Streptomyces sp. loyola UGC on different medium
Medium

Growth

Mycelium & spore color

Pigmentation

Colony formed

Colony elevation

MNGA

???

Substrate Orange

Yellow

Circular

Raised

AIA

Substrate White

Punctiform

Flat

ISP2

Substrate white

Yellow

Punctiform

Flat

ISP3

Substrate Orange

Dark yellow

Punctiform

Flat

ISP4

Substrate white

Yellow

Punctiform

Flat

ISP5

Substrate Orange

Yellow

Punctiform

Flat

ISP7

??

Punctiform

Flat

? Less Growth, ?? Moderate Growth, ??? Very High Growth, - No Response of Growth

Vitamin C was used as positive control. The scavenging


activity was calculated by the formula (1).

Antioxidant activity (AA) was calculated by the following


equation:

Lipid Peroxidation Assay

AA b  carotene content after 2h of assay


= initial b  carotene content  100

The inhibition effect of MeEA on lipid peroxidation was


determined according to the thiobarbituric acid method.
FeCl2H2O2 was used to induce the liver homogenate
peroxidation [33]. In this method, 0.2 ml of MeEA
(2001,000 lg/ml) was mixed with 1.0 ml of 1 % liver
homogenate (each 100 ml homogenate solution contains
1.0 g rat liver), then 50 ll of FeCl2 (0.5 mM) and H2O2
(0.5 mM) was added. The mixture was incubated at 37 C
for 60 min, 1.0 ml of trichloroacetic acid (15 %), and
thiobarbituric acid (0.67 %) was added and the mixture
was heated up in boiled water for 15 min. The absorbance
was recorded at 532 nm. Ascorbic acid was used as the
positive control. The percentage of inhibition effect was
calculated according to the formula (1).
Antioxidant Assay Using b-Carotene Linoleate Model
System
The antioxidant activity of MeEA was evaluated by the bcarotene linoleate model system [21]. A solution of bcarotene was prepared by dissolving 2 mg of b-carotene in
10 ml of chloroform. Two milliliters of this solution was
pipetted into a 100-ml round-bottom flask. After chloroform was removed under vacuum, 40 mg of purified linoleic acid, 400 mg of Tween 40 emulsifier, and 100 ml of
aerated distilled water were added to the flask with vigorous shaking. Aliquot (4.8 ml) of this emulsion was transferred into different test tubes containing different
concentrations of MeEA (2001,000 lg/ml). BHA was
used for comparative purposes. As soon as the emulsion
was added to each tube, the zero time absorbance was
measured at 470 nm. The tubes were then placed at 50 C
in a water bath. Measurement of absorbance was continued
until the color of b-carotene disappeared; a blank, devoid
of b-carotene, was prepared for background subtraction.

123

Statistical Analysis
The data for biochemical and physiologic parameters were
analyzed and expressed as mean SD. The IC50 values
were calculated from linear regression analysis. Results
were processed by computer program, Microsoft Excel
(2007).

Results
Morphological Identification of the Isolated Strain
Seven different medium, AIA, ISP2, ISP3, ISP4, ISP5, ISP7,
and MNGA were selected for the growth and morphological identification. The isolated strain was grown in each
medium and it grew best in the Modified nutrient glucose
agar (MNGA). A yellow pigment with raised colonies is
observed (Table 1). The microscopic observation indicated
that the isolated strain was aerobic with branched aerial
hyphae and showed Gram-positive (Fig. 1). The 16S rRNA
sequence data, whereby the closest match (99 % similarity)
in the NCBI GenBank database was found to be with
Streptomyces sp. loyola UGC (Fig. 2). The sequence of the
strain has been deposited in the NCBI GenBank database
(http://WWW.ncbi.nlm.nih.gov/nuccore/JN863117).
In Vitro a-Glucosidase Inhibition and Antioxidant
Assays
a-Glucosidase Inhibition
The results for a-glucosidase inhibition assay are shown in
(Table 2). The concentration for 50 % inhibition was found

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73

to be 730.21 1.33 lg/ml of extract and all the concentration of standard exhibited above 50 % of inhibition.
Total Phenolic Content (TPC)
The total phenolic content of MeEA was 176 mg catechol
equivalent/gram extract.
Reducing Power Activity
Figure 3a shows the reductive capability of MeEA compared to the standard butylated hydroxyl toluene. The
reducing power of MeEA increased with increasing quantity of the sample.
Fig. 1 Gram stain

DPPH Radical Scavenging Assay


The MeEA exhibited a significant dose dependent inhibition of DPPH activity with a 50 % inhibition (IC50) at a
concentration of 435.31 1.79 lg/ml. The results are
presented in (Fig. 3b). The IC50 value of vitamin C was
490.21 1.82 lg/ml.
Hydroxyl Radical Scavenging Activity
The results for hydroxyl scavenging assay is shown in
(Fig. 3c). The concentrations for 50 % inhibition were
found to be 350.21 1.02 and 210.12 0.62 lg/ml for
the MeEA and vitamin C, respectively.
Nitric Oxide Scavenging Activity

Fig. 2 Phylogenetic tree of strain JN863117 and closest related to the


genus Streptomyces

The scavenging of nitric oxide by MeEA was increased in a


dose-dependent manner as illustrated in (Fig. 3d). At
concentration of 800.12 1.05 lg/ml of extract, 50 % of
nitric oxide generated by incubation was scavenged. The
IC50 value of vitamin C was 510.20 1.02 lg/ml.

Table 2 Shows in vitro a-glucosidase inhibition of MeEA

Superoxide Scavenging Activity

Sample

Concentration
(lg/ml)

% of
inhibition

IC50 (lg/ml)

MeEA

200

22.89 2.78

730.21 1.33

400

37.54 1.27

Acarbose
(Std)

600

44.94 1.33

800

53.03 1.33

1,000
200

65.48 2.27
74.91 1.54

400

81.14 0.29

600

91.75 0.29

800

92.76 0.29

1,000

93.93 0.50

Superoxide anion scavenging activity of MeEA is given in


(Fig. 3e). The 50 % of superoxide anion radical generation
was scavenged at the concentration of 220.31 1.47 lg/ml.
The IC50 value of vitamin C was 240.32 0.69 lg/ml.
Lipid Peroxidation Assay
Activity of extract on lipid peroxidation is shown in
(Fig. 3f). The extract showed inhibition of peroxidation at
all concentration; it showed 50 % inhibition at 840.31
2.18 lg/ml. The IC50 value of vitamin C was 610.30
2.23 lg/ml.

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I. V. S. Nimal Christhudas et al.: In Vitro a-Glucosidase Inhibition and Antioxidative Potential

1.6

Absorbance

1.4

b 100

MeEA
BHT

1.2
1
0.8
0.6
0.4
0.2
0
200

400

600

800

1000

200

100
90
80
70
60
50
40
30
20
10
0

400

MeEA
Vitamin C

90
80

1000

MeEA
Vitamin C

70
60
50
40
30
20

400

600

800

1000

200

400

Concentration (g/ml)

600

800

1000

Concentration(g/ml)

f
MeEA
Vitamin C

80

MeEA
Vitamin C

70

% of Inhibition

80

800

10
200

e 100

600

Concentration (g/ml)

% of Inhibition

% of Inhibition

MeEA
Vitamin C

90
80
70
60
50
40
30
20
10
0

Concentraton (g/ml)

60
40
20

60
50
40
30
20
10
0

0
200

400

600

800

200

1000

90
80
70
60
50
40
30
20
10
0

400

600

800

1000

Concentration (g/ml)

Concentration (g/ml)

% of Inhibition

% of Inhibition

Fig. 3 a Reductive ability of


different concentrations
(2001,000 lg/ml) of MeEA
and BHT. Each value represents
the mean SEM of triplicate
experiments. b DPPH
scavenging effect of different
concentrations
(2001,000 lg/ml) of MeEA
and vitamin C. Each value
represents the mean SEM of
triplicate experiments.
c Hydroxyl radical scavenging
effect of different
concentrations
(2001,000 lg/ml) of MeEA
and vitamin C. Each value
represents the mean SEM of
triplicate experiments. d Nitric
oxide scavenging effect of
different concentrations
(2001,000 lg/ml) of MeEA
and vitamin C. Each value
represents the mean SEM of
triplicate experiments.
e Superoxide scavenging effect
of different concentrations
(2001,000 lg/ml) of MeEA
and vitamin C. Each value
represents the mean SEM of
triplicate experiments.
f Antilipid peroxidation effect
of different concentrations
(2001,000 lg/ml) of MeEA
and vitamin C. Each value
represents the mean SEM of
triplicate experiments.
g Antioxidant activity of
different concentrations
(2001,000 lg/ml) of MeEA in
the b carotene bleaching assay
and butylated hydroxyl anisole
(BHA). Each value represents
the mean SEM of triplicate
experiments

% of Inhibition

74

MeEA
BHA

200

400

600

800

1000

Concentration (g/ml)

Antioxidant Activity Using a b-Carotene Linoleate Model


System
In the b-carotene linoleate system, b-carotene undergoes rapid discoloration in the absence of antioxidants.
The addition of extracts to this system prevents the

123

bleaching of b-carotene at different degrees. The


MeEA hindered the extent of b-carotene bleaching in a
dose-dependent manner. Based on 120-min reaction
time (Fig. 3g), the extract showed 50 % inhibition at
420.12 1.50 lg/ml and the value for BHA was
220.30 1.48 lg/ml.

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I. V. S. Nimal Christhudas et al.: In Vitro a-Glucosidase Inhibition and Antioxidative Potential

Discussion
In the present study, the strain Streptomyces sp. loyola
UGC was isolated from the root/transition zone of D.
stramonium L. Morphological and biochemical characteristics are the two important aspects for the classification in
streptomycetaceae family [30]. There are many reports that
support the use of antioxidant supplementation in reducing
the level of oxidative stress and in slowing or preventing
the development of complications associated with diseases
[25]. In this study, the methanol extract of endophytic
actinomycetes strain isolated from the D. stramonium. L
was tested for different in vitro a-glucosidase inhibition
and antioxidant properties. Agents with a-glucosidase
inhibitory activity have been useful as oral anti hypoglycemic agents for the control of hyperglycemia in patients
with diabetes. There are many natural sources with
a-glucosidase inhibitory activity. These studies suggest that
preventing an excessive postprandial rise of blood glucose
level by a-glucosidase inhibition from natural resources is
effective in real life as well. MeEA effectively reduced
glucose level. In addition, some flavonoids and polyphenols
as well as sugar derivatives were found to be effective on the
inhibitory activities of a-glucosidase [34]. It appears that
this effect is associated with Polyphenols present in MeEA.
In the DPPH test, MeEA was able to reduce the stable
radical DPPH to the yellow-colored diphenylpicrylhydrazine. The method is based on the reduction of alcoholic
DPPH solution in the presence of a hydrogen-donating
antioxidant due to the formation of the non-radical form
DPPH-H by the reaction [2]. Superoxide anions derived
from dissolved oxygen by the riboflavin/methionine/illuminate system will reduce NBT in this system. In this
method, superoxide anion reduces the yellow dye (NBT2?)
to produce the blue formazan. Antioxidants are able to
inhibit the blue NBT formation [23]. The decrease in
absorbance indicates the consumption of superoxide anion
in the reaction mixture. In our study, the inhibition of
superoxide radical by MeEA was lower than the vitamin C.
Hydroxyl radical scavenging capacity of a compound is
directly related to its antioxidant activity [26]. MeEA
inhibited the free radical-mediated deoxyribose damage.
Nitric oxides radical inhibition study showed that the extract
was a potent scavenger of nitric oxide. The extract inhibited
nitrite formation by competing with oxygen to react with
nitric oxide directly and also to inhibit its synthesis. Scavengers of nitric oxide competed with oxygen leading to
reduced production of nitric oxide [18]. MeEA inhibited
free radical-mediated deoxyribose damage. Lipid peroxidation is an oxidative alteration of polyunsaturated fatty
acids in the cell membranes that generates a number of
degradation products. Malondialdehyde (MDA), one of the
products of lipid peroxidation, has been studied widely as an

75

index of lipid peroxidation and as a marker of oxidative


stress [12]. MeEA showed strong inhibition of lipid peroxidation. b-Carotene in this model system undergoes rapid
discoloration in the absence of an antioxidant. This is
because of the coupled oxidation of b-carotene and linoleic
acid, which generates free radicals. As a result, b-carotene
will be oxidized and broken down in part; subsequently, the
system looses its chromophore and characteristic orange
color, which can be monitored spectrophotometrically [13].
In our study, the inhibition of b-carotene bleaching by
MeEA was lower than the standard BHT. For measurement
of reductive ability, we investigated the Fe3? to Fe2?
transformation in the presence of methanol extract. The
reducing power increased with increasing concentration of
the extract. The reducing capacity of a compound may serve
as a significant indicator of its potential antioxidant activity
[20]. Polyphenols are the major plant compounds with
antioxidant activity. This activity is believed to be mainly
due to their redox properties [22] which play an important
role in adsorbing and neutralizing free radicals, quenching
singlet and triplet oxygen, or decomposing peroxides. Our
study revealed the antioxidant property of MeEA by
showing significant various scavenging activities. We
believe that this was due to the presence of good amount of
phenolics as estimated by FolinCiocalteau method. Even
though this method has some limitations (possibility of the
reagent reacting with other non-phenolic reducing compounds leading to the overevaluation of the phenolic content, possible interference of organic acids, sugars, and
amino acids, and possible underestimation of some phenolic
compounds due to low absorption) it has been used by many
workers [22].

Conclusion
This study suggested that MeEA possessed antioxidant
activity which might be helpful in preventing or slowing the
progress of various oxidative stress-related diseases. Further
investigation on the isolation and identification of antioxidant component(s) in the MeEA may lead to chemical
entities for clinical use.
Acknowledgments Authors are thankful to the University Grant
Commission, Government of IndiaUGC Major Research Project
under F-39-266/2010 (SR) for financial assistance.

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In vitro studies on -glucosidase inhibition,


antioxidant and free radical scavenging
activities of Hedyotis biflora L.
ARTICLE in FOOD CHEMISTRY JUNE 2013
Impact Factor: 3.39 DOI: 10.1016/j.foodchem.2012.11.051 Source: PubMed

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Food Chemistry 138 (2013) 16891695

Contents lists available at SciVerse ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

In vitro studies on a-glucosidase inhibition, antioxidant and free radical


scavenging activities of Hedyotis biora L.
I.V.S. Nimal Christhudas a, P. Praveen Kumar a, Christudas Sunil b, S. Vajravijayan a, R. Lakshmi Sundaram c,
S. Jenifer Siril a, P. Agastian a,
a
b
c

Department of Plant Biology and Biotechnology, Loyola College, Chennai 600 034, India
Division of Ethnopharmacology, Entomology Research Institute, Loyola College, Chennai 600 034, India
Central Research Facility, Sri Ramachandra University, Porur, Chennai 600 116, India

a r t i c l e

i n f o

Article history:
Received 25 May 2012
Received in revised form 3 November 2012
Accepted 9 November 2012
Available online 20 November 2012
Keywords:
Hedyotis biora
a-Glucosidase
DPPH
b-Carotene
Metal chelating

a b s t r a c t
Aim of this study was to evaluate the in vitro a-glucosidase inhibition and antioxidant activity of hexane,
ethyl acetate and methanol extracts of Hedyotis biora L. (Rubiaceae). In in vitro a-glucosidase inhibition
and antioxidant activity, the methanol extract showed potent effect compared to hexane and ethyl acetate extracts. The methanol extract of H. biora (HBMe) showed 50% a-glucosidase inhibition at the concentration of 480.20 2.37 lg/ml. The total phenolic content of HBMe was 206.81 1.11 mg of catechol
equivalents/g extract. HBMe showed great scavenging activity on 2,2-diphenyl-picrylhydrazyl (DPPH)
(IC50 520.21 1.02 lg/ml), hydroxyl (IC50 510.21 1.51 lg/ml), nitric oxide (IC50 690.20 2.13 lg/ml)
and superoxide (IC50 510.31 1.45 lg/ml) radicals, as well as high reducing power. HBMe also showed
a strong suppressive effect on lipid peroxidation. Using the b-carotene method, the scavenging values
of HBMe was signicantly lower than BHT, and metal chelating ability of HBMe also showed a strong
inhibition effect when compared to the reference standard. The active compound ursolic acid from HBMe
was identied using various spectroscopical studies. The results obtained in this study clearly indicate
that HBMe has a signicant potential to use as a natural a-glucosidase inhibition, antioxidant agent.
2012 Elsevier Ltd. All rights reserved.

1. Introduction
Diabetes mellitus (DM) is a metabolic disorder characterised by
hyperglycemia resulting from defects in insulin secretion, insulin
action or both. Along with hyperglycemia and abnormalities in serum lipids, diabetes is associated with micro- and macro-vascular
complications, which are the major causes of morbidity and death
in diabetic subjects (Kumar & Murugesan, 2008). There are many
articles related to antidiabetic compounds from plants (Matsui,
Ueda, Oki, Sugita, & Terahara, 2001). However, normalising blood
glucose level is a formidable challenge in clinical practice. The
pharmacological agents with greatest effect on postprandial hyperglycemia include insulin, lispro, amylin analogues, and a-glucosidase (acarbose and voglibose) inhibitors (Goda, Yamada, Hosoya,
& Moriuchi, 1981). It has been well acknowledged that plantderived extracts and phytochemicals are potential alternatives to
synthetic inhibitors against a-glucosidase.
Corresponding author. Address: Research Department of Plant Biology and
Biotechnology, School of Life Science, Loyola College, Chennai 600 034, India. Tel.:
+91 9444433117; fax: +91 44 28175566.
E-mail addresses: agastian@loyolacollege.edu, past_hod@rediffmail.com (P.
Agastian).
0308-8146/$ - see front matter 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodchem.2012.11.051

Oxidative stress is an important contributor to the pathophysiology of a variety of pathological conditions including diabetes,
cardiovascular dysfunctions, atherosclerosis, inammation, carcinogenesis, drug toxicity, reperfusion injury and neurodegenerative
diseases (Aruoma, 1998). Antioxidants help organisms to deal with
oxidative stress, caused by free radical damage. Free radicals are
chemical species, which contains one or more unpaired electrons
due to which they are highly unstable and cause damage to other
molecules by extracting electrons from them in order to attain stability. Reactive oxygen species (ROS) include free radicals such as
superoxide (O2), hydroxyl radical (OH), peroxyl radical (RO2) as
well as nonradical species such as hydrogen peroxide (H2O2)
(Cerutti, 1991). These free radicals are formed as part of the normal
metabolic processes (Halliwell & Gutteridge, 1989). Our body has
multiple mechanisms especially enzymatic and nonenzymatic
antioxidant systems to protect the cellular molecules against reactive oxygen species (ROS) induced damage by antioxidant enzymes, such as superoxide dismutase, catalase, glutathione
dependent enzymes such as glutathione peroxidase, and glutathione reductase, as well as compounds such as ascorbic acid, atocopherol and glutathione (Datta, Sinha, & Chattopadhyay,
2000). However these induced damage disrupted by various pathological phenomena; antioxidant supplements are essential to

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I.V.S. Nimal Christhudas et al. / Food Chemistry 138 (2013) 16891695

counter the oxidative damage. There are many reports that support
the use of antioxidant supplementationin reducing the level of oxidative stress and in slowing or preventing the development of
complications associated with diseases. Many synthetic antioxidant components have shown toxic and/or mutagenic effects.
Hence attention has been given to naturally occurring antioxidants. Numerous plant constituents have shown free radical scavenging or antioxidant activity (Sunil & Ignacimuthu, 2011).
Hedyotis biora (Rubiaceae) is used in traditional Chinese medicine for various purposes, such as tonics, and agents for the treatment of appendicitis, boils, dysentery, hepatitis, and tonsillitis
(Yuan, Zhao, Yang, & Li, 2001). A more direct application is for
the treatment of prostate cancer and other tumours (Liang,
2004). Hence, in the present investigation hexane, ethyl acetate
and methanol extracts of H. biora were evaluated for in vitro aglucosidase inhibition and antioxidant activity.

cut, rinsed with ice-cold saline, and homogenized with 12 ml of


maleate buffer (100 mM, pH 6.0). The homogenate was used as
the a-glucosidase solution. The assay mixture consisted of
100 mM maleate buffer (pH 6.0), 2% (w/v) each sugar substrate
solution (100 ll), and the sample extract (2001000 lg/ml). It
was preincubated for 5 min at 37 C, and the reaction was initiated
by adding the crude a-glucosidase solution (50 ll) to it, followed
by incubation for 10 min at 37 C. The glucose released in the reaction mixture was determined with the kit described above. The
rate of carbohydrate decomposition was calculated as the percentage ratio to the amount of glucose obtained when the carbohydrate
was completely digested. The rate of prevention was calculated by
the following formula: Inhibition (%) = [(amount of glucose produced by the positive control)  (amount of glucose produced by
the addition of sample)/(amount of glucose produced by the positive control)]  100.

2. Materials and methods

2.4.2. Determination of total phenolic content


Total phenolic content of H. biora hexane, ethyl acetate and
methanol extracts were assessed according to the FolinCiocalteau
method (Slinkard & Singleton, 1977) with some modications.
Briey, 0.1 ml of extracts (2001000 lg/ml), 1.9 ml distiled water
and 1 ml of FolinCiocalteaus reagent were seeded in a tube, and
then 1 ml of 100 g/l Na2CO3 was added. The reaction mixture
was incubated at 25 C for 2 h and the absorbance of the mixture
was read at 765 nm. The sample was tested in triplicate and a calibration curve with six data points for catechol was obtained. The
results were compared to a catechol calibration curve and the total
phenolic content of H. biora was expressed as mg of catechol
equivalents per gram of extract.

2.1. Chemicals and reagents


DPPH (1,1-diphenyl,2-picrylhydrazyl), NBT (nitro blue tetrazolium), NADH (nicotinamide adenine dinucleotide phosphate reduced), PMS (phenazine methosulphate), TCA (trichloro acetic
acid), ferric chloride and BHT (butylated hydroxyl toluene) were obtained from Sigma chemical co., USA. Ascorbic acid was obtained
from SD ne chem. Ltd., Biosar, India. b-Carotene, ferrozine, folin
phenol reagent and Tween 40 were purchased from Hi-Media Pvt.
Ltd. Mumbai, India. All the other chemicals were of analytical grade.
2.2. Collection of H. biora
H. biora whole plant was collected from the Loyola College
campus, Chennai, South India in January, 2012. The taxonomical
identity of the plant was conrmed by Dr. D. Narashiman, Department of Botany, Madras Christian College, (Tambaram, South India).
2.3. Extraction of H. biora
The whole plant was shade dried and powdered. The powder
(1 kg) was extracted three times by cold percolation method with
3 l of hexane, ethyl acetate and methanol at room temperature for
72 h. The ltrates were concentrated under reduced pressure at
40 C and stored in a refrigerator at 28 C for use in subsequent
experiments. The percent yield of the hexane, ethyl acetate and
methanol extracts were 0.87%, 1.56% and 6.637% (w/w). The concentrations of extracts for in vitro a-glucosidase inhibition and
antioxidant assays were xed based on the previous studies (Sunil
& Ignacimuthu, 2011).
2.4. Determination of in vitro a-glucosidase inhibition and antioxidant
assays
2.4.1. a-Glucosidase inhibition of H. biora
In order to investigate the inhibition activity of H. biora hexane, ethyl acetate and methanol extracts, an in vitro a-glucosidase
inhibition test was performed. a-glucosidase from yeast is used
extensively as a screening material for a-glucosidase inhibitors,
but the results do not always agree with those obtained in mammals. Therefore, we used the mouse small-intestine homogenate
as an a-glucosidase solution because we speculated that it would
better reect the in vivo state. The inhibitory effect was measured
using the method slightly modied from Dahlqvist (1964). After
fasting for 20 h, the small intestine between the part immediately
below duodenum and the part immediately above the cecum was

2.4.3. Reducing ability assay of H. biora


The reducing power of H. biora hexane, ethyl acetate and
methanol extracts were evaluated according to the method of
Oyaizu (1986). Different amounts of the extracts (2001000 lg/
ml) were suspended in distiled water and mixed with 2.5 ml of
0.2 M phosphate buffer (pH 6.6), and 2.5 ml of 1% K3Fe(CN)6. The
mixture was incubated at 50 C for 20 min; 2.5 ml of 10% TCA
was added to the mixture and centrifuged at 3000 rpm for
10 min. The upper layer of the solution (2.5 ml) was mixed with
distiled water (2.5 ml) and FeCl3 (0.5 ml, 0.1%), and the absorbance
was measured at 700 nm. Increase in absorbance of the reaction
mixture indicated the ability of reducing power. Butylated hydroxy
toluene (BHT) was used as standard.
2.4.4. DPPH radical scavenging assay of H. biora
DPPH quenching ability of H. biora hexane, ethyl acetate and
methanol extracts were measured according to Hanato, Kagawa,
Yasuhara, and Okuda (1988). The methanol DPPH solution
(0.15%) was mixed with serial dilutions (2001000 lg/ml) of the
extracts and after 10 min, the absorbance was read at 515 nm.
The antiradical activity was expressed as IC50 (lg/ml), (the antiradical dose required to cause a 50% inhibition). Vitamin C was used as
standard. The ability to scavenge the DPPH radical was calculated
using the following equation:

DPPH scavenging effect % A0  A1 =A0  100

where A0 is the absorbance of the control at 30 min, and A1 is the


absorbance of the sample at 30 min. All samples were analysed in
triplicate.
2.4.5. Hydroxyl radical scavenging assay of H. biora
The assay was performed as described by the method of
Elizabeth and Rao (1990) with minor changes. All solutions were
prepared freshly. One millilitre of the reaction mixture contained
100 ll of 28 mM 2-deoxy-2-ribose (dissolved in phosphate buffer,

I.V.S. Nimal Christhudas et al. / Food Chemistry 138 (2013) 16891695

pH 7.4), 500 ll solution of various concentrations of H. biora hexane, ethyl acetate and methanol extracts (2001000 lg/ml), 200 ll
of 200 lM FeCl3 and 1.04 mM EDTA (1:1 v/v), 100 ll H2O2 (1 mM)
and 100 ll ascorbic acid (1 mM). After an incubation period of 1 h
at 37 C, the extent of deoxyribose degradation was measured by
the TBA reaction. The absorbance was read at 532 nm against the
blank solution. Vitamin C was used as a positive control. The scavenging activity was calculated using formula (1).
2.4.6. Nitric oxide radical inhibition assay of H. biora
Sodium nitroprusside in an aqueous solution at physiological pH
spontaneously generates nitric oxide; it interacts with oxygen to
produce nitrite ions, which can be estimated by the use of Griess
Illosvoy reaction (Garratt, 1964). In the present investigation, Griess
Illosvoy reagent was modied using naphthylethylenediaminedihydrochloride (0.1% w/v) instead of 1-naphthylamine (5%). The reaction mixture (3 ml) containing sodium nitroprusside (10 mM,
2 ml), phosphate buffer saline (0.5 ml) and different concentration
of H. biora hexane, ethyl acetate and methanol extracts (200
1000 lg/ml) or standard solution (0.5 ml) were incubated at 25 C
for 150 min. After incubation, 0.5 ml of the reaction mixture containing nitrite was pipetted and mixed with 1 ml of sulphanilic acid
reagent (0.33% in 20% glacial acetic acid) and allowed to stand for
5 min for completing diazotization. Then, 1 ml of naphthylethylenediaminedihydrochloride (1%) was added, mixed and allowed to stand for 30 min. A pink coloured chromophore was
formed in diffused light. The absorbance of these solutions was measured at 540 nm against the corresponding blank. Vitamin C was
used as positive control. The scavenging activity was calculated
using the formula (1).
2.4.7. Superoxide scavenging activity of H. biora
Superoxide scavenging activities of H. biora hexane, ethyl acetate and methanol extracts were determined by monitoring the
competition of those with NBT for the superoxide anion generated
by the PMSNADH system (Liu, Ooi, & Chang, 1997). Superoxide
radicals were generated in 1 ml of 20 mM TrisHCl buffer pH 8.0
containing 0.05 mM nitrobluetetrazolium (NBT), 0.01 mM phenazinemethosulphate (PMS) and different concentration of extracts
(2001000 lg/ml) were preincubated for 2 min. The reaction was
initiated by the addition of 0.078 mM NADH. Blue chromogen,
formed due to NBT reduction was read at 560 nm. Results were expressed as percentage of inhibition of superoxide radicals. Vitamin
C was used as a positive control. The scavenging activity was calculated using the formula (1).
2.4.8. Inhibition of lipid peroxidation in rat liver homogenate by H.
biora
The inhibition effect of H. biora hexane, ethyl acetate and
methanol extracts on lipid peroxidation was determined according
to the thiobarbituric acid method. FeCl2H2O2 was used to induce
liver homogenate peroxidation (Yen & Hsieh, 1998). In this method, 0.2 ml of different concentration of extracts (2001000 lg/
ml) was mixed with 1 ml of 1% liver homogenate (each 100 ml
homogenate solution contains 1 g rat liver); then 50 ll of FeCl2
(0.5 mM) and H2O2 (0.5 mM) was added. The mixture was incubated at 37 C for 60 min; then 1 ml of trichloroacetic acid (15%)
with thiobarbituric acid (0.67%) was added and the mixture was
heated in boiling water for 15 min. The absorbance was recorded
at 532 nm. Vitamin C was used as positive control. The percentage
of inhibition was calculated using the formula (1).
2.4.9. Antioxidant activity of H. biora using b-carotene linoleate
model system
The antioxidant activity of H. biora hexane, ethyl acetate and
methanol extracts were evaluated by b-carotene linoleate model

1691

(Miller, 1971). A solution of b-carotene was prepared by dissolving


2 mg of b-carotene in 10 ml of chloroform. Two millilitres of this
solution were pipetted into a 100 ml round-bottom ask. After
chloroform was removed under vacuum, 40 mg of puried linoleic
acid, 400 mg of Tween 40 emulsier, and 100 ml of aerated distiled
water were added to the ask with vigorous shaking. Aliquots
(4.8 ml) of this emulsion were transferred into different test tubes
containing different concentrations of the extracts (2001000 lg/
ml). As soon as the emulsion was added to each tube, the zero time
absorbance was measured at 470 nm. The tubes were then placed
at 50 C in a water bath. Measurement of absorbance was continued until the colour of b-carotene disappeared; a blank devoid of
b-carotene, was prepared for background subtraction. BHA was
used as positive control. Antioxidant activity (AA) was calculated
using the following equation;

AA b-carotene content after 2 h of assay=


initial b-carotene content  100:

2.4.10. Metal chelating activity of H. biora


The chelating of ferrous ions by H. biora hexane, ethyl acetate
and methanol extracts were estimated by the method of Dinis,
Madeira, and Almeida (1994). The different concentrations of extract (2001000 lg/ml) were added to a solution of 2 mM FeCl2
(0.05 ml). The reaction was initiated by the addition of 5 mM ferrozine (0.2 ml), 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. EDTA was used
as a positive control. The percentage inhibition of ferrozineFe2+
complex formation was calculated as using the formula (1).
2.4.11. Preliminary phytochemical analysis of active extract
Preliminary phytochemical screening of methanol extract of H.
biora (MeHB) was carried out to detect the phytoconstituents
using standard conventional protocols (Harborne, 1998).
2.5. Isolation and identication of the active compound
The methanol extract (30 g) was chromatographed over silica
gel column (100200 mesh, Sissco, Mumbai) in hexane. The column was eluted with hexane, hexane: chloroform, chloroform,
chloroform: methanol and methanol. Each fraction wass potted
on a TLC plate over silica gel 60F254 (precoated aluminium plate,
layer thickness 0.2 mm) (Merck). All the fractions with similar
retention factor (Rf) in TLC pattern were pooled together. Finally
four fractions were obtained; from fraction 10 (Chloroform:Methanol 3:1) a colourless compound was isolated (yield 0.8 g). It gave
a single spot on chloroform:methanol (30:1) as mobile phase and
the Rf value was observed as 0.50. It answered Nollers test for
triterpenoids. It showed the melting point 288 C (Lit mp: 287
288 C). The melting point, ultra violet (UV) spectrum, mass spectra (MS), 1H and 13C nuclear magnetic resonance (NMR) and infra
red (IR) spectrum were measured and compared with the literature
data as described by Gayathridevi, Chitra, Anitha, and Gopakumar
(2012). The identity was nally conrmed by comparison with an
authentic sample.
2.6. Statistical analysis
The data for biochemical and physiological parameters were
analysed and expressed as means SD. The IC50 values were calculated from linear regression analysis. Results were processed by
computer program, Microsoft Excel (2007).

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I.V.S. Nimal Christhudas et al. / Food Chemistry 138 (2013) 16891695

3. Results

3.1.2. Total phenolic content


The total phenolic content of hexane ethyl acetate and methanol extracts of H. biora was found to be 86.89 0.46,
106.16 0.57 and 206.81 1.11 mg catechol equivalent/gram extract, respectively.
3.1.3. Reducing power
Fig. 1(a) shows the reductive capabilities of hexane, ethyl acetate and methanol extracts of H. biora compared to butylatedhydroxy toluene. The reducing power of HBMe was very
potent than hexane and ethyl acetate extracts and the power of
the extract was increased with quantity of sample. The plant extract could reduce the most Fe3+ ions, which had a lesser reductive
activity than the standard of butylated hydroxy toluene.

Absorbance

3.1.1. a-Glucosidase inhibition


The results for a-glucosidase inhibition assay of HBMe and
acarbose were shown in Table 1. The concentration for 50% inhibition of HBMe and acarbose were found to be 480.20 2.37 and 260,
32 1.23 lg/ml, respectively. The hexane and ethyl acetate extracts showed less inhibition compared to methanol extract (data
not shown).

2
1.5
1
0.5
0

200

400
600
800
Concentration (g/ml)

1000

Fig. 1a. Reductive ability of different concentrations (2001000 lg/ml) of Hedyotis


biora hexane, ethyl acetate, methanol extracts and BHT. Each value represents the
mean SEM of triplicate experiments.

% of Inhibition

3.1. In vitro a-glucosidase inhibition and antioxidant assays of H.


biora

Hexane
Ehtyl acetate
Methanol
BHT

2.5

100
90
80
70
60
50
40
30
20
10
0

Hexane
Ethyl acetate
Methanol
Vitamin C

3.1.4. DPPH radical scavenging activity


HBMe exhibited a signicant dose dependent inhibition of
DPPH activity compared to hexane and ethyl acetate extracts, with
a 50% inhibition (IC50) at a concentration of 520.21 1.02 lg/ml.
The results are presented in Fig. 1(b). The IC50 value of vitamin C
was 450.12 2.25 lg/ml.

Fig. 1b. DPPH scavenging effect of different concentrations (2001000 lg/ml) of


Hedyotis biora hexane, ethyl acetate, methanol extracts and vitamin C. Each value
represents the mean SEM of triplicate experiments.

3.1.5. Hydroxyl radical scavenging assay


To attack the substrate deoxyribose hydroxyl radicals were generated by reaction of Ferric-EDTA together with H2O2 and ascorbic
acid. When the plant extracts were incubated with the above reaction mixture, it could prevent the damage against sugar. The

results for hydroxyl scavenging assay are shown in Fig. 1(c). The
concentrations for 50% inhibition were found to be 510.21 1.51
and 260.10 1.71 lg/ml for the methanol extract and vitamin C
respectively. Hexane and ethyl acetate extracts showed less effect.

Sample

Concentration (lg/
ml)

% of
Inhibition

IC50 (lg/ml)

Hexane extract

200
400
600
800
1000

7.57 2.52
13.29 2.59
20.70 2.81
33.33 2.67
41.07 1.91

6.32 3.24

Ethyl acetate
extract

200

13.80 2.54

960.10 2.13

400
600
800
1000

21.04 2.87
32.66 2.54
41.58 2.78
53.20 2.54

Methanol extract

200
400
600
800
1000

35.58 0.77
46.88 1.51
57.64 1.45
64.04 1.01
73.80 1.77

480.20 2.37

Acarbose

200
400
600
800
1000

45.91 1.54
61.14 0.29
91.75 0.29
92.76 0.29
93.93 0.50

260.32 1.23

Each value represents the mean SEM of triplicate experiments.

400
600
800
Concentration (g/ml)

1000

3.1.6. Nitric oxide radical inhibition assay


The scavenging of nitric oxide by HBMe was increased in a
dose-dependent manner as illustrated in Fig. 1(d). At concentration
of 690.20 2.13 lg/ml of extract 50% of nitric oxide generated by
incubation was scavenged. The IC50 value of vitamin C was
510.12 1.74 lg/ml.

% of Inhibition

Table 1
a-Glucosidase inhibiton of extracts of Hedyotis biora.

200

100
90
80
70
60
50
40
30
20
10
0

Hexane
Ethyl acetae
Methanol
Vitamin C

200

400
600
800
Concentration (g/ml)

1000

Fig. 1c. Hydroxyl radical scavenging effect of different concentrations (200


1000 lg/ml) of Hedyotis biora hexane, ethyl acetate, methanol extracts and
vitamin C. Each value represents the mean SEM of triplicate experiments.

1693

200

400
600
800
Concentration (g/ml)

% of Inhibition

Hexane
Ethyl acetate
Methanol
Vitamin C

1000

Fig. 1d. Nitric oxide scavenging effect of different concentrations (2001000 lg/
ml) of Hedyotis biora hexane, ethyl acetate, methanol extracts and vitamin C. Each
value represents the mean SEM of triplicate experiments.

3.1.7. Superoxide scavenging activity


The superoxide anion derived from dissolved oxygen by phenazinemethosulphate/NADH coupling reaction reduces nitro blue
tetrazolium. The decrease the absorbance at 560 nm with the plant
extract thus indicates the consumption of superoxide anion in the
reaction mixture. As mentioned in Fig. 1(e), the methanol extract
as well as vitamin C showed the scavenging activity; IC50 values,
510.31 1.45 lg/ml and 290.08 2.51 lg/ml, respectively.
3.1.8. Lipid peroxidation assay
Activity of extracts on lipid peroxidation is shown in Fig. 1(f).
Addition of Fe2+/ascorbate to the liver microsomes cause increase
in lipid peroxidation. HBMe showed inhibition of peroxidation effect in all concentrations compared to hexane and ethyl acetate,
which showed 50% inhibition effect at 530.32 2.00 lg/ml. The
IC50 value of vitamin C was 450.35 1.88 lg/ml.
3.1.9. Antioxidant using a b-carotene linoleate model system
In the b-carotene linoleate system, b-carotene undergoes rapid
discolouration in the absence of antioxidants. The addition of extracts to this system prevents the bleaching of b-carotene at different degrees. HBMe hindered the extent of b-carotene bleaching on
a dose dependent manner compared to hexane and ethyl acetate
extract. Based on 120 min reaction time Fig. 1(g), the extract
showed 50% inhibition at 600.10 1.04 lg/ml and the value for
BHA was 330.12 2.28 lg/ml.

% 0f Inhibition

3.1.10. Metal chelating


Activity of extracts on chelating the ferrous ions is shown in
Fig. 1(h). HBMe showed 50% of chelate ions generation at the concentration of 550.50 2.68 lg/ml. The IC50 value of EDTA was
340.20 2.63 lg/ml.

100
90
80
70
60
50
40
30
20
10
0

100
90
80
70
60
50
40
30
20
10
0

Hexane
Ethyl acetate
Methanol
BHT

200

400
600
800
Concentration (g/ml)

1000

Fig. 1f. Antilipid peroxidation effect of different concentrations (2001000 lg/ml)


of Hedyotis biora hexane, ethyl acetate, methanol extracts and vitamin C. Each
value represents the mean SEM of triplicate experiments.

% of Inhibition

90
80
70
60
50
40
30
20
10
0

100
90
80
70
60
50
40
30
20
10
0

Hexane
Ethyl acetate
Methanol
BHA

200

400
600
800
Concentration (g/ml)

1000

Fig. 1g. Antioxidant activity of different concentrations (2001000 lg/ml) of


Hedyotis biora hexane, ethyl acetate, methanol extracts and vitamin C in the bcarotene bleaching assay and butylated hydroxyl anisole (BHA). Each value
represents the mean SEM of triplicate experiments.

90
80
% of Chelating effect

% of Inhibition

I.V.S. Nimal Christhudas et al. / Food Chemistry 138 (2013) 16891695

70

Hexane
Ethyl acetate
Methanol
EDTA

60
50
40
30
20
10
0
200

400
600
800
Concentration (g/ml)

1000

Fig. 1h. Metal chelating effects of different concentrations (2001000 lg/ml) of


Hedyotis biora hexane, ethyl acetate, methanol extracts and vitamin C. Each value
represents the mean SEM of triplicate experiments.

Hexane
Ethyl acetate
Methanol
Vitamin C

3.1.11. Preliminary phytochemical analysis


The preliminary phytochemical evaluation of HBMe showed the
presence of steroids, triterpenoids and phenolic compounds.
3.2. Identication of compound from HBMe

200

400
600
800
Concentration (g/ml)

1000

Fig. 1e. Superoxide scavenging effect of different concentrations (2001000 lg/ml)


of Hedyotis biora hexane, ethyl acetate, methanol extracts and vitamin C. Each
value represents the mean SEM of triplicate experiments.

The active compound from HBMe whole plant was identied as


ursolic acid (38-hydroxy-urs-12-en-28-oic acid). The compound
was identied based on the following evidences: it showed the
molecular ion at m/z 456, which was corresponding to the molecular formula C30H48O3. 1H NMR (400 MHz, DMSO): dH 0.680.66

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I.V.S. Nimal Christhudas et al. / Food Chemistry 138 (2013) 16891695

(4H, m) 0.75 (3H, s), 0.796 (3H, d, J = 6.4 Hz), 0.920.84 (11H, m)
1.04 (3H, s), 1.311.24 (5H, m) 1.601.46 (9H, m) 1.961.77 (4H,
m), 2.09 (2H, s), 2.12 (1H, s), 3.00 (1H, t, J = 5 Hz), 4.29 (1H, d,
J = 5.2 Hz), 5.13 (1H, s). 13C NMR (100 MHz, DMSO): dc 15.12,
15.97, 16.81, 16.91, 17.89, 20.97, 22.74, 23.17, 23.70, 26.88,
27.43, 30.08, 30.58, 32.60, 36.21, 36.42, 38.13, 38.27, 38.33,
38.40, 41.54, 45.34, 46.72, 46.91, 52.27, 54.67, 76.72, 124.47,
138.08, 178.17. The physical and spectroscopical data were similar
to those reported by Alves, Castra, Freire, Cunha, and Silva (2000).

4. Discussion
Agents with a-glucosidase inhibitory activity have been useful as
oral hypoglycemic agents for the control of hyperglycemia in
patients with diabetes. There are many natural sources with aglucosidase inhibitory activity. These studies suggest that preventing an excessive postprandial rise of blood glucose level by aglucosidase inhibition from natural resources is effective in real life
as well. HBMe effectively reduced the glucose level in a-glucosidase
inhibition assay. The isolated compound ursolic acid was reported as
a-glucosidase inhibitor (Wen-Yi, Yan-Li, & Li, 2011). The activity of
HBMe was mainly due to the presence of ursolic acid. In our study,
total phenolic content estimation showed high amount of polyphenols in methanol extract. Polyphenols are the major plant compounds with antioxidant activity, is believed to be mainly due to
their redox properties (Nitin, Yogendra Kumar, & Asheesh, 2010)
which play an important role in adsorbing and neutralizing free radicals, quenching singlet and triplet oxygen, or decomposing peroxides. In our study the antioxidant property of H. biora extracts
were evaluated with varying parameters. We believe that this was
due to the presence of more of phenolics as estimated by Folin
Ciocalteau method (Nitin et al., 2010).
For the measurements of the reductive ability, we studied the
Fe3+ to Fe2+ transformation in the presence of H. biora extracts,
using the method of Oyaizu (1986). The reducing power increased
with increasing concentration of the extract. The reducing capacity
of a compound may serve as a signicant indicator of its potential
antioxidant activity (Meir, Kanner, Akiri, & Hadas, 1995). DPPH test
is usually used as the substrate to evaluate antioxidative activity of
antioxidants (Oyaizu, 1986). This method is based on the reduction
of alcoholic DPPH solution in the presence of a hydrogen donating
antioxidant, due to the formation of the non-radical form DPPH-H
by the reaction (Brand-Williams, Cuvelier, & Berset, 1995). HBMe
has the ability to reduce the stable radical DPPH to the yellow-coloured diphenyl picrylhydrazine. The hydroxyl radical is an extremely reactive free radical formed in biological systems and has
been implicated as a highly damaging species in free radical
pathology, capable of damaging almost every molecule found in
living cells (Hochestein & Atallah, 1988). Hydroxyl radical scavenging capacity of an extract is directly related to its antioxidant activity (Babu, Shylesh, & Padikkala, 2001). HBMe inhibited free radicalmediated deoxyribose damage remarkably.
Nitric oxide plays an important role in various types of inammatory processes in the animal body. Nitric oxide radical inhibition
study showed that the extract was a potent scavenger of nitric
oxide. The extract inhibited nitrite formation by competing with
oxygen to react with nitric oxide directly and also to inhibit its synthesis. Scavengers of nitric oxide competed with oxygen leading to
reduced production of nitric oxide (Marcocci, Packer, Droy-Lefai,
Sekaki, & Gardes-Albert, 1994). In the PMSNADHNBT system,
superoxide anion derived from the dissolved oxygen by PMS/
NADH coupling reaction reduces NBT. The decrease in the absorbance at 560 nm with antioxidants thus indicates the consumption
of the generated superoxide anion in the reaction. Superoxide, the
one-electron reduced form of molecular oxygen, is a precursor of

other ROS such as hydrogen peroxide, hydroxyl radical, and singlet


oxygen that have the potential of reacting with biological macromolecules and thereby inducing tissue damages (Aruoma, 1998).
These results clearly indicated that HBMe is a potent scavenger
of superoxide radicals in a dose-dependent manner. Lipid peroxidation is an oxidative alteration of polyunsaturated fatty acids in
the cell membranes that generates a number of degradation products. Malondialdehyde (MDA), one of the products of lipid peroxidation, has been studied widely as an index of lipid peroxidation
and as a marker of oxidative stress (Janero, 1990). HBMe showed
a strong inhibition of lipid peroxidation.
b-Carotene in this model system undergoes rapid discoloration
in the absence of an antioxidant. This is because of the coupled oxidation of b-carotene and linoleic acid, which generates free radicals.
The linoleic acid free radical, formed upon the abstraction of a
hydrogen atom from one of its diallylic ethylene groups, attacks
the highly unsaturated b-carotene molecules. As a result, bcarotene will be oxidised and broken down in part; subsequently,
the system loses its chromophore and characteristic orange colour,
which can be monitored spectrophotometrically. The presence of
different antioxidants can hinder the extent of b-carotene bleaching
by neutralising the linoleate-free radical and other free radicals
formed in the system (Jayaprakasha, Singh, & Sakariah, 2001). In
our present study, the HBMe found to hinder the extent of b-carotene bleaching by neutralizing the linoleate-free radical and other
free radicals formed in the system. The chelating of ferrous ions
by the extract was estimated by the method of Dinis et al. (1994).
Ferrozine can quantitatively form complexes with Fe2+. In the presence of other chelating agents, the complex formation is disrupted
with the result that the red colour of the complexes decreases.
Measurement of the rate of colour reduction therefore allows estimation of the chelating activity of the coexisting chelator (Yamaguchi, Ariga, Yoshimira, & Nakazawa, 2000). In our study HBMe
reveals effective capacity for iron binding, suggesting that action
as an antioxidant may be related to its iron binding capacity.
Fractionation of HBMe extract yielded the compound Ursolic
acid, a triterpenoid. Ursolic acid and other triterpenoids have also
been reported as potent a-glucosidase inhibitors and antioxidants.
More over these compounds have benecial effects on cardiovascular system (Somova, Nadar, Rammanan, & Shode, 2003), interaction
with cytochrome P450s (Kim et al., 2004), immunomodulation
(Jeong, Kim, & Hwang, 2005), intracellular redox balance (Ikeda,
Sporn, Honda, Gribble, & Kufe, 2003). It has been shown that they
act at various stages of tumour development to inhibit tumour initiation and promotion (Oguro, Liu, Klaassen, & Yoshisa, 1998), as
well as to induce tumour cell differentiation (Lee, Chung, Kim,
Lee, & Kim, 1994) and apoptosis (Lauthier, Taillet, Trouillas, Delage,
& Simon, 2000). These benecial effects are mainly due to the antioxidant activities of these triterpenoids. HBMe methanol extract
showed potent antioxidant activity, this was due to the presence
of good amount of ursolic acid.
5. Conclusion
This study suggested that the methanol extract of H. biora possessed a-glucosidase inhibition and antioxidant activity which
might be helpful in preventing or slowing the progress of various
oxidative stress-related diseases. Further investigation on the isolated component on a-glucosidase inhibition and antioxidant
activity may lead to chemical entities for clinical use.
Acknowledgement
The author thank University Grant Commission of India for providing junior research fellowship (Sanction no. F-39-266/
2010(SR)).

I.V.S. Nimal Christhudas et al. / Food Chemistry 138 (2013) 16891695

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ANTIOXIDANT ACTIVITY OF ILLICIUM


GRIFFITHI HOOK. F. & THOMS SEEDS -IN
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Academic Sciences Asian Journal of Pharmaceutical and Clinical Research


Vol 6, Suppl 2, 2013

ISSN - 0974-2441

Research Article

ANTIOXIDANT ACTIVITY OF ILLICIUM GRIFFITHI HOOK. F. & THOMS SEEDS - IN VITRO


Vol. 4, Issue 3, 2011
ISSN - 0974-2441 2

A. VIJAYAKUMAR*1, P.PRAVEEN KUMAR2 AND B. JEYARAJ1


1Department

of Chemistry, Loyola College, Chennai-600034, India, Department of Plant biology & Biotechnology, Loyola College, Chennai600034, India,Email: loyolavijayakumar@gmail.com.
Received: 19 March 2013, Revised and Accepted: 4 April 2013

ABSTRACT
The antioxidant activity of hexane, ethyl acetate and methanol extracts of Illicium griffithii (I. graffithii, Family: Schisandraceae) seeds were
determined using 1,1-diphenyl-2- picrylhydrazyl (DPPH), phosphomolybdenum, cupric ions (Cu2+) reducing antioxidant capacity (CUPRAC), ferric
reducing antioxidant power (FRAP), reducing power, lipid peroxidation, hydroxyl and N,N-Dimethyl-p-phenylenediamine (DMPD) methods.
Extracts were analyzed for total phenolic content (TPC) and total flavonoids content (TFC) using a spectrophotometric analysis. Total phenolic
content 164.91 12.67 GAE mg/g (as gallic acid equivalents) and total flavonoids content 63.94 0.16 CE mg/g (as catechin equivalents) were
estimated in the methanol extract of seeds. Among the extracts tested for antioxidant activity, methanol extract showed maximum activity on DPPH
(70.96 1.88), CUPRAC (0.988 0.07), reducing power (0.236 0.02), lipid peroxidation (36.95 2.36), hydroxyl (47.52 1.94) and DMPD (64.30
0.31). It also exhibited high activity at 300 g/ml on total antioxidant activity (0.159 0.04 GAE mg/g) and FRAP (0.297 0.03 mM Fe2+/g). The
results indicated that the methanol extract of I. griffithii seeds is having more of natural antioxidants and it can be considered for further clinical use.
Keywords: Illicium griffithii; antioxidant activity; DPPH; phosphomolybdenum; CUPRAC; FRAP; reducing power; lipid peroxidation;
hydroxyl; DMPD.

INTRODUCTION
Living cells may generate free radicals and other reactive oxygen
species as a result of physiological and biochemical processes. Free
radicals can cause oxidative damage to lipids, proteins and DNA,
eventually leading to many chronic diseases, such as cancer,
diabetes, aging, and other degenerative diseases in humans [1]. In
many parts of the world, medicinal plants are used as a source of
phytochemicals to cure various illnesses such as urinary infections,
cervicitis vaginitis, skin infections, blood infections, and
gastrointestinal disorders [2]. The phytochemicals have been found
to act as antioxidants by scavenging free radicals, and many have
therapeutic potential for free radical associated disorders [3].
Therefore, it is important to assess antioxidant activity of the plants
used in the herbal medicine either to elucidate the mechanism of
their pharmacological action or to provide information on
antioxidant activity of these herbal plants.
Illicium griffithii Hook. f. & Thoms is an important medicinal tree
species of the temperate broad-leaved forests of Northeast India [4].
Fruits of I. griffithii are used in the pharmaceutical and spice
industries. In recent years, scientists also found cancer fighting
properties especially against lung cancer cells [5]. These reports are
sufficient to highlight the use of this species [6]. The main objective
of this study was to estimate the total content of polyphenols,
flavonoids, and the antioxidant activity of hexane, ethyl acetate and
methanol extracts of seeds of I. griffithii.
MATERIALS AND METHODS
Chemicals
All the chemicals and reagents used in this study were obtained from
Himedia, Qualigens and SRL and were of analytical grade. FolinCiocalteu reagent, gallic acid, catechin, thiobarbituric acid (TBA),
1,1-Diphenyl-2-picrylhydrazyl (DPPH), sodium hydroxide (NaOH),
aluminums chloride (AlCl3), TPTZ (2, 4, 6-tripyridyl-s-triazine),
nitroblue tetrazolium (NBT), butylated hydroxytoluene (BHT),
copper chloride (CuCl2), neocuproine, deoxyribose, ammonium
molybdate, trichloroacetic acid (TCA), deoxyribose, potassium
dihydrogen phosphate, phenazine methosulphate (PMS), sodium
nitroprusside (SNP), sodium acetate (CH3COONa), acetic acid
(CH3COOH), sodium nitrate (NaNO2), sulfanilamide, ammonium
acetate, naphthylethylenediamine dihydrochloride (NED), sodium
carbonate (Na2CO3), phosphoric acid (H3PO4) , mono and di basic

sodium phosphate, Ferric chloride (FeCl3), Nicotinamide adenine


dinucleotide (NADH), thiobarbituric acid (TBA), dimethyl sulphoxide
(DMSO), ethylene diamine tetra acetic acid (EDTA), hydrogen
peroxide (H2O2), ascorbic acid (AA), nitroblue tetrazolium (NBT) and
ferrous sulphate (FeSO4), N,N-dimethyl-p-phenylenediamine
(DMPD) were used.
Plant materials
Healthy, disease free seeds of I. griffithii were collected from
Arunachal Pradesh, India and were identified and authenticated by
the taxonomist, Department of Plant Biology and Biotechnology,
Loyola College, Chennai, India. All the seeds were separated from
fruits and were shade dried at room temperature. The dried seeds
were then powdered and stored in airtight containers.
Preparation of crude extract
Seed Powder (1 kg) was soaked serially in hexane (4 L), ethyl acetate
(4 L), and methanol (4 L) for 72 h respectively with intermittent
shaking. The solutions were filtered and the filtrates were
concentrated under reduced pressure using rotary vacuum
evaporator (25 Chexane extract; 35 CEthyl acetate extract; 40
CMethanol extract). The yield of seed extracts were: hexane
extract (IS1, 8.1%, w/w), ethyl acetate extract (IS2, 9.8%, w/w) and
methanol extract (IS3, 11.6% w/w). Finally, the crude extracts were
obtained and stored at 4C.
Phytochemical Analysis
Determination of Total Phenolic Contents (TPC).
The total phenolic content of the samples was determined using the
Folin-Ciocalteus reagent as described by the method of Slinkard and
Singleton [7]. Briefly, a 100 l aliquot of extract was assayed with
250 l of Folin reagent and 500 l of sodium carbonate (20%, w/v).
The mixture was vortexed and diluted with water to a final volume
of 5 ml. After incubation for 30 min at room temperature, the
absorbance was read at 765 nm and total phenols in these extracts
were expressed as gallic acid equivalents (GAE), using a calibration
curve of a freshly prepared gallic acid solution.
Estimation of Total Flavonoids Content (TFC).
The total flavonoids content was determined according to the
method Kareti et al [8]. One hundred micro litter aliquot of extract
was added to a 10 ml volumetric flask containing 4 ml of distilled

A.Vijayakumar et al

water. At zero time, 0.3 ml of 5% NaNO2 was added to the flask. After
5 min, 0.3 ml of 10% AlCl3 was added. At 6 min, 2 ml of 1 M NaOH
was added to the mixture. Immediately, the reaction solution was
adjusted to 10 ml by adding 2.4 ml of distilled water and thoroughly
mixed. Absorbance of the mixture was determined at 510 nm versus
blank. The total flavonoids content was expressed in mg catechin
equivalents (CE)/g of extract.
Antioxidant activity
DPPH Radical Scavenging Capacity
DPPH quenching ability of I. griffithii hexane, ethyl acetate and
methanol extracts were measured by method of Hanato et al [9]. The
reaction mixture contained 50 l of different concentrations (1001000 g/ml) of the sample and 2.95 ml of 0.1 mM DPPH in ethanol.
After 30min incubation at room temperature, the absorbance was
recorded at 517 nm using a spectrophotometer. The experiment was
performed in triplicate. The percentage DPPH scavenging activity at
different concentrations was calculated. Ascorbic acid was used as
standard. The ability to scavenge the DPPH radical was calculated
using the following equation:
DPPH scavenging effect (%) = (A0- A1) /A0 100.(1)
Where A0 is the absorbance of the control at 30 min, and A 1 is the
absorbance of the sample at 30 min. All samples were analyzed in
triplicate.
Cupric-Ion-Reducing Antioxidant Capacity (CUPRAC)
CUPRAC assay was performed according to the method of Apak et al
[10] with some modifications. The test mixture contained 1ml of
10mM of CuCl2, 7.5 mMneocuproine, and 1M ammonium acetate
buffer (pH 7.0). Briefly, 1ml of sample in the concentration range of
100 1000 g/ml was added to the test mixture to achieve final
volume of 4ml. The test mixtures were incubated for 30min at room
temperature and then absorbance at 450 nm was recorded against a
blank. BHT was used as standard.
Ferric Reducing Antioxidant Power (FRAP)
A slightly modified method of Benzie and Strain [11] was adopted
for the FRAP assay. A standard or sample extract (300 g/ml) was
mixed with 300 l of ferric-TPTZ reagent (prepared by mixing 300
mM acetate buffer, pH 3.6, 10 mM TPTZ in 40 mM HCl and 20 mM
FeCl3.6H2O at a ratio of 10:1:1 (v/v/v)). The mixture was incubated
at 37C and the absorbance readings were taken at 593 nm after 4
min. Results were expressed in mM Fe (II)/g dry mass.
Determination of Total Antioxidant Capacity (TAC)
Total antioxidant activity of I. griffithii was determined according to
the method of Kareti et al [12]. Briefly, an aliquot 300 g/ml of
sample was combined with 3ml molybdenum reagent (50 ml of 0.6
M sulfuric acid, 50 ml of 28 mM sodium phosphate and 50 ml of 4
mM ammonium molybdate). The reaction mixture was incubated in
a water bath at 95 C for 90 min. After cooling to room temperature,
the absorbance was measured at 695 nm. The total antioxidant
activity of the sample was expressed as mg gallic acid equivalents
(GAE)/g of extracts.
Reducing Power Ability
The reducing power of hexane, ethyl acetate and methanol extracts
of I. griffithii were evaluated according to the method of Oyaizu [13].
Different concentrations of the extracts (1001000 g/ ml) were
suspended in distilled water and mixed with 2.5 ml of 0.2 M
phosphate buffer (pH 6.6), and 2.5 ml of 1% K3Fe(CN)6. The mixture
was incubated at 50 C for 20 min; 2.5 ml of 10% TCA was added to
the mixture and centrifuged at 3000 rpm for10 min. The upper layer
of the solution (2.5 ml) was mixed with distilled water (2.5 ml) and
FeCl3 (0.5 ml, 0.1%), and the absorbance was measured at 700 nm.
Increased in absorbance of the reaction mixture indicates the ability
of reducing power. Vitamin C was used as standard.
Anti-lipid Peroxidation Assay in rat liver homogenate

Asian J Pharm Clin Res, Vol 6, Suppl 2, 2013, 269-273

The inhibition effect of hexane, ethyl acetate and methanol extracts


of I. griffithii on lipid peroxidation was determined according to the
thiobarbituric acid method [14]. FeCl2H2O2 was used to induce liver
homogenate peroxidation. In this method, 0.2 ml of different
concentrations of extracts (1001000 g/ml) were mixed with 1 ml
of 1% liver homogenate (each 100 ml homogenate solution contains
1 g rat liver); then 50 l of FeCl2 (0.5 mM) and H2O2 (0.5 mM) was
added. The mixture was incubated at 37 C for 60 min; then 1 ml of
trichloroacetic acid (15%) with thiobarbituric acid (0.67%) was
added and the mixture was heated in boiling water for 15 min. The
absorbance was recorded at 532 nm and the percentage of inhibition
was calculated using the formula (1). Vitamin C was used as positive
control.
Hydroxyl radical scavenging assay
The assay was performed as described by Elizabeth and Rao method
[15] with minor changes. All solutions were prepared freshly. 1 ml of
the reaction mixture contained 100 l of 28 mM 2-deoxy-2-ribose
(dissolved in phosphate buffer, pH 7.4), 500 l solution of various
concentrations of I. griffithii hexane, ethyl acetate and methanol
extracts (1001000 g/ml), 200 l of 200 M FeCl3 and 1.04 mM
EDTA (1:1 v/v), 100 l H2O2 (1 mM) and 100 l ascorbic acid (1
mM). After an incubation period of 1 h at 37 C, the extent of
deoxyribose degradation was measured by the TBA reaction. The
absorbance was read at 532 nm against the blank solution. Vitamin C
was used as a positive control. The scavenging activity was
calculated using formula (1).
DMPD Radical scavenging assay
DMPD radical (DMPD) scavenging ability was performed by the
method [16] with slight modification. The coloured radical was
formed by adding ferric chloride to the DMPD solution (Fe 3+: DPMD
ratio 1:10) and the absorbance of this solution was measured at 505
nm. 50 l of the test solution (100-1000 g/ml) was added to 2.95ml
DMPD solution. The absorbance at 505 nm was measured after 10
min at 25C under continuous stirring. The percentage scavenging
activity was calculated.
Statistical analysis
The data were analyzed using Microsoft Excel (2007) and expressed
as mean Standard Deviation.
RESULTS AND DISCUSSION
Phytochemical Analysis
Total Phenolic and flavonoids Contents
Considering the physiological importance of phenolic compounds
and their contribution towards total antioxidant capacity, FolinCiocalteu method was used to estimate the total phenolic content of
extracts. It must be noted that this reagent does not react exclusively
with phenolics, but other reducing agents, for example, ascorbic acid
as well [17, 18]. Hence, results of this test therefore reflect the total
reducing capacity of the extracts and positive controls tested. Total
phenolic compounds in extracts varied widely, ranging from
72.756.41 and 164.9112.67 mg/g expressed as gallic acid
equivalents (GAE) (Table 1). Methanol extract of seeds (IS3)
exhibited the highest total phenolic content. The content of
flavonoids expressed as catechin equivalents, varied from
58.830.32 to 63.940.16 mg catechin equivalent/g extract (Table
1). The IS3 showed the highest amount of flavonoids contents
followed by IS2 and IS1.
Table 1: Total Phenolic and flavonoids Contents of hexane (IS1),
ethyl acetate (IS2) and methanol (IS3) extracts of I. griffithii
seeds. GAE: gallic acid equivalents, CE: catechin equivalents.
Data are presented as the mean Standard deviation of
triplicate measurements.
Extracts TPC mg GAE/g TFC mg CE/g
IS1
72.75 6.41
58.83 0.32
IS2
95.02 5.44
61.65 0.29
IS3
164.91 12.67 63.94 0.16

270

A.Vijayakumar et al

Asian J Pharm Clin Res, Vol 6, Suppl 2, 2013, 269-273

In vitro antioxidant activity


Several techniques have been used to determine the antioxidant
activity in vitro in order to allow rapid screening of substances since
substances that have low antioxidant activity in vitro, will probably
show little activity in vivo [19]. Free radicals are known to play a
definite role in a wide variety of pathological manifestations.
Antioxidants fight against free radicals and protect from various
diseases. They exert their action either by scavenging the reactive
oxygen species or protecting the antioxidant defense mechanisms
[20]. Antioxidant capacity of seeds extracts of I. griffithii was
examined using eight different assays.
DPPH radical scavenging capacity.
DPPH is a stable free radical, due to the delocalization of the spare
electron on the whole molecule. Thus, DPPH does not dimerize, as
happens with most free radicals. The delocalization on the DPPH
molecule determines the occurrence of a purple colour, with an
absorption band with a maximum around 517 nm. When DPPH
reacts with a hydrogen donor, the reduced (molecular DPPH) form is
generated, accompanied by the disappearance of the colour.
Therefore, the absorbance diminution depends linearly on the
antioxidant concentration [21, 22]. A large decrease in the
absorbance of the reaction mixture indicates significant free radical
scavenging activity of the compound under test [23]. Figure 1 shows
the scavenging effects of various extracts of I. griffithii seeds on
DPPH in the following order: IS3> IS2>IS1. Among all the extracts
tested methanol extract of seeds (IS3), showed highest inhibition
percentage and directly correlated with total phenolic content.
Results of this study suggest that the extracts contain phytochemical
constituents that are capable of donating hydrogen to a free radical
to scavenge the potential damage.

Figure 2: Antioxidant capacity of hexane (IS1), ethyl acetate


(IS2) and methanol (IS3) extracts of I. griffithii seeds by CUPRAC
method. Each value represents the mean standard deviation
of triplicate experiments.
Table 2: Antioxidant capacity of hexane (IS1), ethyl acetate (IS2)
and methanol (IS3) extracts of I. griffithii seeds by FRAP and
TAC method. GAE: gallic acid equivalents. BHT-butylated
hydroxytoluene. AA-ascorbic acid. Data are presented as the
mean Standard deviation of triplicate measurements. --- : not
done.
Sample
300 g/ml
IS1
IS2
IS3
BHT
AA

FRAP
mM Fe(II) / g
0.108 0.03
0.142 0.02
0.297 0.03
2.312 0.12
2.270 0.05

TAC
mg GAE/g
0.009 0.01
0.028 0.02
0.159 0.04
--0.043 0.01

Determination of total antioxidant capacity.


The total antioxidant capacity of the extracts was measured
spectrophotometrically through phosphomolybdenum method,
based on the reduction of Mo (VI) to Mo (V) by the test sample and
the subsequent formation of green phosphate-Mo(V) compounds.
The antioxidant capacity of various solvent extracts of seeds of I.
griffithii was found to decrease in this order: IS3 > IS2 > IS1 (Table
2). Recent studies have shown that many flavonoids and related
polyphenols contribute significantly to the phosphomolybdate
scavenging activity of medicinal plants [26].
Figure 1:Antioxidant capacity of hexane (IS1), ethyl acetate
(IS2) and methanol (IS3) extracts of I. griffithii seeds by DPPH
method. BHT-butylated hydroxytoluene. Each value represents
the mean standard deviation of triplicate experiments.
Cupric-ion-reducing antioxidant capacity.
The CUPRAC assay utilizes copper(II)-neocuproine (Cu(II)-Nc)
reagent as the chromogenic oxidizing agent. It is based on the
measurement of absorbance at 450 nm by the formation of stable
complex between neocuproine and copper (I) [24]. The cupric ion
(Cu2+) reducing ability of various extracts of I. griffithii seeds is
shown in Figure 2. Cu2+ reducing capability measured by this method
was found to be concentration-dependent. Cu2+ ions reducing power
of extracts and standard compounds were in the following order:
BHT> IS3>IS2>IS1.

Reducing Power Ability


In reducing power assay, the yellow colour of the test solution
changes to various shades of green and blue, depending on the
reducing power of the sample. The presence of reducing agents
causes the conversion of Fe3+-ferricyanide complex to the ferrous
form that may be followed at 700 nm due to the formation of Perls
Prussian blue Fe4[Fe(CN)6]3. Increasing absorbance at 700 nm
indicates an increase in reducing ability [27]. The antioxidants
present in the extracts of seeds of I. griffithii caused their reduction
of Fe3+-ferricyanide complex to the ferrous form, and thus proved
the reducing power. Figure 3 shows the reducing powers of various
extracts of I. griffithii. It was found that the reducing power
increased with concentration of the sample. The ranking order for
reducing power was IS3 > IS2 > IS1. Significantly higher reducing
power (0.236 0.02 at 1000 g/ml) was evident in IS3.

Ferric reducing antioxidant power.


When a Fe3+-TPTZ complex is reduced to the Fe2+ form by an
antioxidant under acidic conditions, an intense blue color develops
with an absorption maximum at 593 nm. The antioxidant effect
(reducing ability) can be evaluated by monitoring the formation of a
Fe2+-TPTZ complex with a spectrophotometer [25]. The antioxidant
potential of I. griffithii extracts was estimated from their ability to
reduce TPRZ-Fe (III) complex to TPTZ-Fe (II). Increasing absorbance
indicates an increase in reductive ability. The FRAP values of the
studied extracts were calculated and the results are presented in
Table 2. Among all the extracts, the methanol extract of seeds (IS3)
showed the highest FRAP value (2.146 0.23) at 300 g/ml.

Figure 3: Antioxidant capacity of hexane (IS1), ethyl acetate


(IS2) and methanol (IS3) extracts of I. griffithii seeds by
reducing power method. BHT - butylated hydroxytoluene. Each

271

A.Vijayakumar et al

Asian J Pharm Clin Res, Vol 6, Suppl 2, 2013, 269-273

value represents the mean standard deviation of triplicate


experiments.
Anti-lipid peroxidation assay in rat liver homogenate.
Lipid peroxidation induced by free radical species results in
breakdown of membrane integrity, affecting its fluidity and
permeability [28]. The initial step, that is, peroxidation of
polyunsaturated fatty acid components in low-density lipoproteins
of membrane produces several byproducts which can damage
biomolecules. Transition ions may either generate hydroxyl radicals
to initiate the lipid peroxidation process or propagate the chain
process via decomposition of lipid hyperoxides [29].The lipid
peroxidation induced by Fe2+ was estimated by the presence of
thiobarbituric acid reactive substances (TBARS). The ability of the
extracts to inhibit peroxidation of phospholipids in rat liver is shown
in Figure 4. The percentage inhibition of lipid peroxidation by
various solvent extracts of seeds of I. griffithii was found to decrease
in the following order: IS2>IS3>IS1.

Figure 4: Antioxidant capacity of hexane (IS1), ethyl acetate


(IS2) and methanol (IS3) extracts of I.griffithii seeds by Lipid
peroxidation method. AA - Ascorbic acid. Each value represents
the mean standard deviation of triplicate experiments.
Hydroxyl radical scavenging assay.
Hydroxyl radicals (OH) are short-lived species possessing high
affinity toward other molecules. OH is a powerful oxidizing agent
that can react at a high rate with most organic and inorganic
molecules in the cell, including DNA, proteins, lipids, amino acids,
sugars, and metals. OH is considered the most reactive radical in
biological systems; due to its high reactivity, it interacts at the site of
its production with the molecules closely surrounding it. It reacts
with polyunsaturated fatty acid moieties of cell membrane
phospholipids and causes damage to cell [30]. There are several
ways to ascertain the ability to form hydroxyl radicals. One of them
is the deoxyribose method. In this method the OH generated
through Fenton reaction degrades deoxyribose into several
fragments using Fe2+ as an important catalytic component. Some of
these fragments are capable of reacting with TBA after heating and
in an acidic pH, originating a pink pigment that can be quantified by
spectrophotometry [31]. The OH scavenging activity of various
solvent extracts of seeds of I. griffithii can be ranked as IS3 > IS2 >
IS1 (Figure 5). The markedly strong antioxidant activity of IS3 and
IS2 in comparison with ascorbic acid might be helpful in
characterizing the significant sources of natural antioxidants.

Figure 5: Antioxidant capacity of hexane (IS1), ethyl acetate


(IS2) and methanol (IS3) extracts of I. griffithii seeds by
Hydroxyl radical method. AA - Ascorbic acid. Each value
represents the mean standard deviation of triplicate
experiments.

Figure 6: Antioxidant capacity of hexane (IS1), ethyl acetate


(IS2) and methanol (IS3) extracts of I. griffithii seeds by DMPD
method. AA - Ascorbic acid. Each value represents the mean
standard deviation of triplicate experiments.
Conclusions
The results of the study have shown that the methanol extract of
seeds of I. griffithii is potentially a good source of free radical
scavenging compounds. It contains high phenolic and flavonoids and
hence exhibits high antioxidant activities. However, the components
responsible for the antioxidant activity are currently unclear.
Therefore, further investigation is needed to isolate and identify the
antioxidant compounds present in the methanol extract.
Furthermore, the in vivo antioxidant activity of this extract needs to
be assessed prior to clinical use.
Acknowledgments
This study was supported by Loyola Institute of Frontier Energy
(LIFE) and Entomology Research Institute, Loyola College, Chennai.
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ANTIMICROBIAL ACTIVITY AND HPLC


ANALYSIS OF TROPANE ALKALOIDS IN
STREPTOMYCES SPP. ISOLATED FROM DATURA
STRAMONIUM L
ARTICLE in ASIAN JOURNAL OF PHARMACEUTICAL AND CLINICAL RESEARCH NOVEMBER 2012
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Academic Sciences Asian Journal of Pharmaceutical and Clinical Research


Vol 5, Suppl 4, 2012

ISSN - 0974-2441

Vol. 4, Issue 3, 2011


ISSN - 0974-2441

Research Article

ANTIMICROBIAL ACTIVITY AND HPLC ANALYSIS OF TROPANE ALKALOIDS IN


STREPTOMYCES SPP. ISOLATED FROM DATURA STRAMONIUM L.
I.V.S. NIMAL CHRISTHUDAS1, P. PRAVEEN KUMAR1, P. AGASTIAN1*
1Department

of Plant Biology and Biotechnology, Loyola College, Chennai - 600 034; *Assistant professor, Research Department of Plant
Biology and Biotechnology, School of Life Science, Loyola College, Chennai 600 034, India;
Email: agastianloyolacollege@gmail.com, agastian@loyolacollege.edu
Received: 30 October 2012, Revised and Accepted:08 November 2012

ABSTRACT
Aim of the present study is to isolate and identify promising antimicrobial metabolite producing Streptomyces strain from Datura stramonium.
Physiological, biochemical and 16S rRNA studies strongly suggested that this isolate belonged to Streptomyces spp and ability to produce enzymes
such as amylase, lipase and catalase. Maximum biological activity was obtained on Modified Nutrient Glucose Agar (MNGA) medium. Preliminary
screening revealed that the isolate was found to be active against bacteria and fungi. Isolate showed activity against bacteria such as Bacillus subtilis,
Staphylococcus aureus, Enterococcus faecalis, and fungi such as Aspergillus niger, Trycophyton rubrum and Aspergillus flavus. The antibacterial
substances were extracted using methanol from MNGA medium in which isolate had grown for five days at 28C. The antimicrobial activity was
assessed using broth microdilution technique. The lowest Minimum Inhibitory Concentration of methanol fractions of Streptomyces spp. Loyola UGC
against B. subtilis, S. aureus was 250 mg/ml and against Aspergillus flavus was 61.5 mg/ml. The growth parameters such as carbon source, Nitrogen
source, Inoculation period, pH and temperature were optimized. Nutritional and cultural conditions for the production of antimicrobial metabolite
by this organism under shake-flask conditions have been studied. HPLC analysis of methanol extract of actinomycets showed the presence of
Hyoscyamine and scopolamine.
Keywords: Endophyte, Streptomyces, Antimicrobial activity, Medium optimization, HPLC, Tropane alkaloids, Datura stramonium.
INTRODUCTION
The term Endophyte was introduced by De Bary (1866) and it was
assigned to all microorganisms that are inside the living tissues of
the host plant asymptomatically (Glienke-Blanco et al. 2002).
Actinomycets are noteworthy as antibiotic producers, making three
quarters of all known products; the Streptomyces are especially
prolific (Nolan and Cross, 1998). Streptomyces species are widely
recognized as industrially important microorganisms because of
their ability to produce many kinds of novel secondary metabolites
including antibiotics (Bibb, 2005). The discovery of new molecules
from actinomycets has marked an epoch in antibiotic research and
subsequent developments in antibiotic chemotherapy (Ameriga et
al. 2000). Searching for original sources of micro-organisms is also
advisable since the environment can affect microbial metabolism
and therefore, antibiotics producing micro-organisms have been
isolated from the most diverse habitats such as endophytes of
terrestrial plants (Strobel et al. 2004) and from sea organisms
(Gandhimathi et al.2008) among other sources. The problems of
drug resistance, patients sensitivity and inability to control certain
infectious diseases have given an impetus for continuous search of
new antibiotics all over the world. To combat the multidrug resistant
organisms, introduction of new antimicrobial compounds or
antibiotics from new source is essential. Datura stramonium L. is a
wild-growing herb known as Jimson weed belongs to the family
Solanaceae. The plant distributed throughout most parts of
temperate regions of the world (Berkov et al.2006). Whole plant is
used as anti-inflammatory, central nervous system stimulant
(Spring, 1989), dental and skin infections, toothache and alopecia
(De Foe and Senatore, 1993). The entire plant has anti-cholinergic
compounds, but the seeds contain the highest concentration (Chang
et al. 1999). Attempt is made to isolate and identification of the
endophytic actinomycets from Datura stramonium L. to evaluate
their antimicrobial activity. The present study evaluates the
isolation, identification; cultural characteristics and antimicrobial
activity of Streptomyces spp. isolate Loyola UGC recovered from
Datura stramonium, Tamil Nadu, India.
MATERIALS AND METHODS
Plant materials
Datura stramonium L. was collected from the Irula Tribal Womens
Welfare Society (ITWWS), Chengalpattu, Kanchipuram district,
Tamil Nadu, South India. The species was identified and

authenticated by Dr. D. Narashiman, Department of Botany, Madras


Christian College, Chennai, and South India.
Isolation of actinomycets
Roots and transition zones of Datura stramonium L. were surface
sterilized by the methodology of Johannes et al.(2006) with some
modifications. Samples were thoroughly washed with running tap
water and all the visibly damaged material was excluded. Plant parts
were rinsed in 0.1% Tween 20 for 30 sec, followed by bevastin for 2
to 3 min to inhibit the fungal growth, sequentially immersed in 0.1%
sodium hypochlorite for 30 sec and in 75% ethanol for 3 to 5 min.
After each treatment, samples were rinsed three times in sterile
distilled water. Finally surface sterilized samples were aseptically
dissected to expose cortex region and placed onto actinomycets
isolation medium, incubated for 12 to 15 days at 28C in dark. The
isolation medium was supplemented with nalidixic acid and actidion
both to a final concentration of 50g/ml to inhibit the growth of non
actinomycets microorganisms.
Physiological and biochemical characteristics
Media used were those recommended by Shirling and Gottlieb
(1966) in the International Streptomyces Project (ISP) and by
Waksman (1961). Mycelium was observed after incubation at 28C
for two weeks. Colors were determined according to Prauser (1964).
Carbohydrate utilization was determined by growth on carbon
utilization medium (ISP 9) (Shirling and Gottlieb (1966)
supplemented with 1% carbon sources at 28C. Temperature range
for growth was determined on inorganic salts starch agar medium
(ISP 4) using a temperature gradient incubator.
Screening of antimicrobial activity
Streptomyces spp. isolate loyola UGC was inoculated on modified
nutrient glucose agar (MNGA) plates by single streak in the center.
The plates were incubated at 28C for four days. The test pathogenic
bacteria (Bacillus subtilis MTCC 441, Staphylococcus aureus ATCC
25923, Staphylococcus aureus ATCC 29213, Staphylococcus
epidermidis MTCC3615, Escherichia coli ATCC 25922, Enterococcus
faecalis ATCC 29212, Pseudomonas aeruginosa ATCC 27853,
Pseudomonas aeruginosa MTCC 1688, Klebsiela pneumonia ATCC
15380 and Xanthomonas spp.) and pathogenic filamentous fungi
(Aspergillus niger MTCC 1344, Aspergillus flavus, Botyritis cinerea,
Curvularia luenata 46/01, Trycophyton rubrum 57/01and

I.V.S. Nimal Christhudas et al.

Trycophyton mentagrophytes 66/01 ) were cross streaked and


incubated at 28C for 96 h. The microbial inhibitions were observed
by determining the inhibition zone from the antagonist.
Antimicrobial activity of the train was determined by standard cup
plate method using Gram (+) and (-) bacteria and fungi Assay plates
were prepared by inoculating 20 ml of Mueller Hinton agar medium
with test organism. Agar-cups (6 mm diameter) were filled with 100
l of mycelia-free culture filtrate in triplicate and the plates were
incubated at 37C for 24 h. Zone of inhibition was measured by the
diameter (mm).
Media optimization for compound production
Modified Nutrient Glucose Broth (MNGB) was used as the base to
determine the optimal nutritional and cultural conditions for the
growth and antimicrobial compound production. Different carbon
and nitrogen sources were provided to assess the growth effects and
antimicrobial metabolite production. MNGB medium was
supplemented with different carbon and nitrogen sources to study
their effect on growth and antibiotic production. The medium (100
ml in 500 ml Erlenmeyer flask) was inoculated with 5 ml of
homogenous spore suspension (0.6 O.D.), and incubated at 28C on a
rotary shaker (180 rpm) for five days. The effect of cultural
conditions like different incubation temperatures (15, 20, 25, 30, 35
and 50C), initial pH (5.5, 6, 6.5, 7, 7.5, 8, 8.5 and 9) and incubation
period (0, 1, 2, 3, 4 and 8 days) on growth and antibiotic production
was studied.

Asian J Pharm Clin Res, Vol 5,Suppl 4, 2012 , 278-282

The same primers as above were used for this purpose. The
sequence was compared for similarity with the reference species of
Streptomyces contained in genomic database, using the NCBI BLAST
available at http://www.ncbinlm-nih.gov/.
RESULTS AND DISCUSSION
Streptomyces spp. isolate Loyola UGC, was isolated from the
transition zone of Datura stramonium belongs to the family
Solanaceae exhibited antimicrobial activity against some Gram
positive bacteria and fungi. Morphological identification of the
isolate Loyola UGC was Gram-positive, substrate and circular in
shape. A yellow pigment diffused in to the surrounding medium
(Table1). Culture characteristics of Loyola UGC were derived on the
basis of observation made after seven days of incubation on different
media. These characteristic morphological properties strongly
suggested that Loyola UGC belonged to the genus Streptomyces spp.
The isolate Loyola UGC was non motile and aerobic, also showed
exponential growth on medium amended with sodium chloride up to
2.5%; poor growth was absorbed at below 5% of NaCl. The
preliminary screening (cross-streak method) revealed that the
Streptomyces spp. isolate Loyola UGC was a good antibacterial and
antifungal compound producer (Fig. 1).

Extraction of antimicrobial metabolites


Streptomyces spp. isolate Loyola UGC culture broth was collected and
centrifuged at 8000 rpm for 10 min. Antimicrobial compound
containing supernatant was extracted using equal volume of
different solvents such as hexane, ethyl acetate and Methanol.
Solvents were concentrated by vacuum evaporator at 40C.
In vitro antimicrobial test
Crude extract (10 mg) was dissolved in 1 ml of dimethyl sulfoxide
(DMSO): water (1:9) and used for antimicrobial study according to
standard broth microdilution method (NCCLS) and the MIC was
calculated. Mueller Hinton broth (Himedia, Mumbai) was prepared
and sterilized by autoclaving at 121C, 15 lbs for 15 minutes. The
required concentration of the extract (1 mg/ml, 0.5 mg/ml, 0.25
mg/ml, 0.125 mg/ ml, 0.0625 mg/ml, and 0.03125 mg/ml) was
added to the 96 well micro titer plate containing 0.1 ml broth. The
10 l of log phase culture was introduced into the respective wells
and the final inoculum size was 1105cfu/ml. The plates were
incubated at 37C for 18 h. Positive control and solvent control
(DMSO) was also included. 5 ml of the test broth was introduced on
plain nutrient agar plates to observe the viability of the organism.
MIC was determined as the complete growth inhibition at the lowest
concentration of the extract.

Fig 1: a & b shows prelimiminary screening of antibacterial and


antifugal activity, c) Different media extract inhibited the
growth of Bacillus subtilits.
Optimization of antimicrobial compound production was carried out
in batch culture. The strain was able to grow in all the tested carbon
sources. However maximum growth and pigment production were
observed in glucose followed by maltose; and nitrogen source were
observed in Soytone (Fig 2).

High performance liquid chromatography (HPLC) analysis of


extract
HPLC analysis of methanolic extract of endophytic actinomycets
(MeEA) was eluting from a RP- C18 (4.6 x 150 mm, 5m) reverse
phase column with ultraviolet (UV) detector. 20l of the sample was
injected each time detected at 270 nm. The mobile phase was
methanol/acetonitrile/water (25:35:40, by v/v/v) at 1.0 ml min-1.
The sample and mobile phase were filtered through 0.2m PVDF
filter before entering the column.
Amplification of 16s r RNA and sequencing
Genomic DNA Loyola UGC was extracted by Enticknap et al. (2006).
The 16 S ribosomal RNA gene was amplified by using the PCR
method with Taq DNA polymerase and primers 27f (5`AGT TTG ATC
CTG GCT CAG 3`) and 1492 (5`ACG GCT ACC TTG TTA CGA CTT 3`).
The conditions for thermal cycling were as follows: denaturation of
the target DNA at 94C for four minutes followed by 30 cycles at
94C for one minute, primer annealing at 52C for one minute and
primer extension at 72C for one minute. At the end of the cycling,
the reaction mixture was held at 72C for 10 min and then cooled to
4C. The PCR product obtained was sequenced by an automated
sequencer (Genetic Analyzer 3130, Applied Biosystem, and USA).

Fig. 2: Effect of different carbon and nitrogen sources for


antimicrobial metabolite production.
The increased glucose level in the broth leads to the higher
production of antimicrobial compound. The optimum temperature

279

I.V.S. Nimal Christhudas et al.

found to be effective for growth and pigment production. Maximum


antimicrobial activity was obtained at PH 7.5 and an incubation
period of 5 days (Fig .3).

Fig. 3: Effect of pH and incubation time for production of


antimicrobial compound.
The other physical parameters such as NaCl (2.5%), temperature
(28C), were optimized for the production (Table 1).
Table1 : Physiological and Biochemical characteristics of
Culture Character
Growth Response
Growth under anaerobic condition
Gram staining
+
Growth and Shape
Substrate, Circular
Motility
Non Motile
Diffusible pigments
+
Range of temperature for growth
25-30C
Optimum temperature for growth
28C
Range of PH for growth
6-8
NaCl tolerance
2.5%
Catalase production
+
Protease
Amylase
+
Lipase
+
Streptomyces spp. isolate Loyola UGC.
Preliminary screening revealed that MNGA medium was very good
base for the production of antibacterial compounds. The diameter of
inhibition zones produced by the extracellular products of Loyola
UGC was 12 mm for B. subtilis, 20 mm for E. faecalis, 19 mm for S.
aureus, and 12 mm for E. coli (Table .2). Methanol extract of
endophytic actinomycets showed activity against gram positive
bacteria and antifungal activity (Table.3, 4). The MIC of 250 g /ml
was determined for Gram positive bacteria; B. subtilis, E. faecalis and
S. aureus, whereas MIC of 500 g /ml was observed for E. coli, and Y.
enterocolitica Most of the gram negative bacteria showed MIC as
1000 g /ml. The Streptomycin antibiotic used positive control
(Table.3). The MIC of antifungal activity 125 g /ml was showed

Asian J Pharm Clin Res, Vol 5,Suppl 4, 2012 , 278-282

against A.niger and T. rubrum (Table4). The HPLC analysis of MeEA


showed the presence of hyoscyamine at 10.59 and scopolamine at
8.04 compared with authentic standard (sigma) (Fig. 4).

Fig4: HPLC-analysis of tropane alkaloids. a & b- Standard


Hyoscyamine and Scopolamine; c- Methanol extract of
Streptomyces spp. isolate Loyola UGC shows presence of tropane
alkaloids.
The sequencing results revealed that isolate was Streptomyces spp.
Antibiotic producing endophytic actinomycets Loyola UGC has been
isolated, from Datura stramonium and characterized by the
Streptomyces spp. By using a wide range of isolation media, culture
characteristics
combining
physiological
and
biochemical
characteristics, we identified the isolate which was found to produce
pigment. Actinomycets are useful biological tools producing
antimicrobials against bacteria and fungi (Okami and Hotta., 1988).
Two novel antimycin antibiotics urauchimycines A and B were
isolated from a fermentation of Streptomyces spp. Ni-80 (Imamura et
al. 1993). The Streptomyces spp Loyola UGC culture showed good
antimicrobial activity in solid as well as in culture broth unlike
fumaradimycine, which is inactivated in fermentation broth
(Maruyama et al. 1975). The isolate Loyola UGC was growing well in
MNGA medium amended with NaCl. It can be placed in intermediate
tolerance group (Tresner et al. 1968). The normal temperature for
growth of Streptomyces is 28 C. Our isolate Loyola UGC was also
growing well at 28 C. The isolated spp. exhibited antibacterial
activity against Gram positive bacteria similar to another report
where aerobic, filamentous bacterium TN97 isolated from soil with
antimicrobial activity against Gram positive bacteria (Ben Ameur
Mehdi et al. 2006). Our results indicated that the antimicrobial
compounds were extracellular. Most of the secondary metabolites
and antibiotics of microbes are extracellular in nature similarly extra
cellular products of actinomycets showed potent antimicrobial
activities (Hacene et al. 2000). The results indicated the dependence
of the production of antimicrobial compound(s) on medium
constituents. Similar findings have been reported by Holmalahti et
al. (1998). It has been reported that the environmental factors like
temperature, pH and incubation period have profound influence on

280

I.V.S. Nimal Christhudas et al.

antibiotic production (Yoshida et al. 1962). Our results indicate that


the synthesis of antimicrobial metabolites depends on the medium
constituents. In fact, it has been shown that the nature of carbon and
nitrogen sources strongly affects antibiotic production in different
organisms and the antibiotic production was increased by glucose
rich medium (Holmalahti et al. 1998). The HPLC analysis of the
methanol extract showed the presence of tropane alkaloids;
hyoscyamine and scopolamine. The growth of many fungi, yeasts,
bacteria, and viruses were inhibited by the phytochemical

Asian J Pharm Clin Res, Vol 5,Suppl 4, 2012 , 278-282

compounds found in Datura (Youdim et al. 1999). Classes of


alkaloids are among the major powerful poisons known (Fluck
1973). Apart from being poisonous, some alkaloids have also been
proved to be useful in correcting renal disorders (Konkwara 1976);
it therefore, means that the alkaloids of D. stramonium may be a
poison that can be tried on lower or higher organisms. The
secondary metabolites identified in the extract used in this study
could be responsible for antimicrobial activity exhibited by these
Streptomyces spp.

Table2: Shows medium optimization of Streptomyces spp. isolate Loyola UGC.


Name of the medium
Modified Nutrient Glucose Broth
Actinomycets isolation broth
Benet Medium
Antibiotic production medium
Starch casein broth

B.subtilis
12
10
10
8

Zone of Inhibition (mm)


E. faecalis S. aureus E. coli
20
19
12
14
10
8
12
8
10
13
-

E. aerogenes
8
10
10
-

Table3: Antibacterial activities of Streptomyces spp. isolate Loyola UGC


Minimum Inhibitory Concentration (MIC) (g/ml)
Test Organisms
Streptomycin (g/ml)
Hexane
Ethyl acetate
Methanol
Bacillus subtilis MTCC 441
6.25
250
Staphylococcus aureus ATCC25923
6.25
250
Klebsiella pneumonia ATCC 15380
2.5
>1000
1000
Enterococcus faecalis ATCC 29212
<50
250
Yersinia enterocolitica MTCC 840
12.5
>1000
1000
Xanthomonas pvoryzae MTCC 2760
25
1000
Vibrio parahaemolyticus MTCC 451
25
1000
Enterobacter aerogenes MTCC 111
25
>1000
1000
Escherichia coli ATCC 25922
2.5
500
Proteus vulgaris MTCC 1771
6.25
1000
Table4: Antifungal activity of Streptomyces spp. isolates Loyola UGC.
Test Organisms
Aspergillus niger MTCC 1344
Aspergillus flavus
Botyritis cinerea
Curvularia luenata 46/01
Trycophyton rubrum 57/01
Trycophyton mentagrophytes 66/01

Fluconazole
(g/ml)
100
50
100
<12.5
25
25

CONCLUSION
From the present study, it is clear that a novel isolate of
Streptomyces spp. Loyola UGC which produced methanol soluble
extracellular product effective against pathogenic test bacteria and
fungi. In view of the decline in the discovery of new lead compounds
in recent years, further investigations on isolate Loyola UGC would
lead to some useful anti biological products.
ACKNOWLEGEMENT
The authors thankful to University Grant Commission of India-UGC
Major Research Project-under F-39-266/2010(SR) Plan, New Delhi
providing financial support.
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Taxol production by endophytic Fusarium


solani LCPANCF01 from Tylophora indica
ARTICLE OCTOBER 2012

CITATION

5 AUTHORS, INCLUDING:
I.V.s. Nimal Christhudas

P. Praveenkumar

Biozone, Research and Development, Herb

Loyola College

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Available from: P. Praveenkumar


Retrieved on: 04 September 2015

281

J. Acad. Indus. Res. Vol. 1(5) October 2012

ISSN: 2278-5213

RESEARCH MANUSCRIPT

Taxol production by endophytic Fusarium solani LCPANCF01 from Tylophora indica


1

J. Nomila Merlin , I.V.S. Nimal Christhudas , P. Praveen Kumar , M. Kumar and P. Agastian
1
Dept.of Biotechnology, Bharathiar University, Coimbatore-641046, TN, India
2
Dept. of Plant Biology and Biotechnology, Loyola College, Chennai-600034, TN, India
3
Dept. of Plant Biology and Plant Biotechnology, Madras Christian College, Tambaram, Chennai-600059, TN, India
agastian@loyolacollege.edu; +91 9444433117
_____________________________________________________________________________________________

Abstract
Twenty five endophytic fungal isolates were obtained from the roots of Tylophora indica (Burm. f) and
screened for the presence of an anticancer drug, taxol. Among the 25 isolates, Fusarium solani LCPANCF01
was identified based on the micro morphology, cultural characteristics and sequence analysis using internal
transcribed spacer (ITS1 and ITS4). Fusarium solani LCPANCF01 strain was grown in M1D liquid medium
for 21 d and extracted with dichloromethane. The presence of taxol was confirmed by TLC, HPLC, UV, IR,
and ESI-MS spectroscopy analysis by comparing with the standard drug.
Keywords: Tylophora indica, Fusarium solani, dichloromethane, taxol, anticancer drug.

Introduction
Taxol is isolated from the barks of pacific yew tree
(Taxus brevifolia) (George et al., 1994; Dewick, 2009)
and the most important antimitotic agent which is active
against lung, ovarian, breast, head-neck cancer and
advanced forms of Kaposi's sarcoma. Taxol inhibits cell
proliferation by binding to the -subunit of the tubulin
heterodimers, thus promoting its polymerization (Kovacs
et al., 2007). However, a complete course of treatment
per patient may requires 2 g of taxol administered
several times over many months. To obtain 1 kg of taxol,
it requires 3000 yew trees (10,000 kg of bark) and
current demand of taxol is 250 kg per annum (Dewick,
2009). This insufficiency of taxol and environmental issue
of harvesting from trees demands researcher to discover
the alternate technique for the production of taxol.

Major alkaloid tylophorine has immunosuppressive,


anti-inflammatory (Gopalakrishnan et al., 1979),
anti-tumor (Donaldson et al., 1968) and anti-amoebic
(Bhutani et al., 1987) properties. Since, T. indica is an
endemic plant an attempt is made to conserve the
medicinal plant through exploration of endophytic fungi
and screened for its taxol production.

Taxol is now obtained by several methods, e.g. total


chemical
synthesis
(Nicolaou
et
al.,
1994),
semi-synthesis from its precursor (Commercn et al.,
1995), plant tissue or cell culture (Hu et al., 2003).
Endophytic fungus from various taxus species produces
taxol although the amounts are very small. First taxol
producing endophytic fungi T. andreanae is from Taxus
brevifolia (Stierle et al., 1993). Since then, many other
endophytic fungi from other plant species have been
successfully reported to produce taxol (Strobel et al.,
1996; Li et al., 1996). Tylophora indica (Burm. f)
(Asclepiadaceae) is a climbing perennial plant that grows
in India, commonly called as antmool in Ayurveda. The
leaves of Tylophora have been traditionally used as a
folk medicine. It has been used for respiratory problems
such as asthma, allergies, bronchitis and common cold.
The roots and leaves contain 0.2 to 0.46% of
therapeutically important alkaloids namely tylophorine,
tylophorinine and tylophrinidine.

Fig 1. Tylophora indica.

Youth Education and Research Trust (YERT)

Materials and methods


Plant collection: Tylophora indica fresh plants were
collected from Kerala Forest Research Institute (KFRI),
Trissur, Kerala, South India during the month of January
2012 (Fig. 1). The taxonomical identity of the plant was
confirmed by Dr. D. Narashiman, Department of Botany,
Madras Christian College, Tambaram, South India.

Nomila Merlin et al., 2012

J. Acad. Indus. Res. Vol. 1(5) October 2012

Isolation and Identification of endophytic fungi: The


fungus used in this study is one of the 25 endophytic
fungi isolated from the root/transition zone of medicinal
plants in T. indica. The root/transition zone samples were
surface sterilized by the modified method of Dobranic
et al. (1995). The root/transition were thoroughly washed
in running tap water and small pieces of approximately
0.5 to 1 cm diameter were cut and then the pieces were
surface sterilized by immersion in 70% ethanol for 5 sec,
followed by 4% sodium hypochlorite for 90 sec and then
rinsed in sterile distilled water for 10 sec. The excess
moisture was blotted in a sterile filter paper. The surface
sterilized samples was aseptically dissected to expose
cortex region and plated onto petri dishes (9 cm dia)
containing potato dextrose agar (PDA) medium
(amended with chloramphenicol 150 mg L1). The petri
dishes were sealed using Parafilm TM and incubated at
26 1C in a light chamber with 12 h light/ dark cycles.
The petri dishes were monitored every day to check the
growth of endophytic fungal colonies from the root
segments. The hyphal tips, which grew out from sample
segments were isolated and sub-cultured onto PDA and
brought into pure culture. The isolated endophytic fungi
were identified based on colony character and
morphology of spore.
Molecular identification: The total genomic DNA was
extracted using CTAB-Method. The endophytic fungal
DNA fragments were amplified using Universal primers
ITS1F, ITS4R and the PCR reactions were standardised
as initial denaturation at 94C for 4 min, followed by 32
cycle of 4 min at 94C, 50C for 1 min, 72C for 2 min
and a final extension at 72C for 8-10 min. The recation
was stopped at 4C for 1 h. The PCR products were
stored at 4C and visualized by DNA gel electrophoresis.
The amplified product was purified and sequenced with
primers, ITS1F (5` AGT TTG ATC CTG GCT CAG 3`)
and ITS4R (5` ACG GCT ACC TTG TTA CGA CTT 3`)
and the sequences obtained were submitted to GenBank
for homology search with Blast. The immediate concern
is to find one or more fungi that produce more taxol. An
endophytic fungus Fusarium solani LCPANCF01 strain
was identified and tested for taxol production.
Preparation of fungal extracts: The test isolate was
grown in 2 L Erlenmeyer flasks containing 500 mL of
M1D medium supplemented with soytone (Pinkerton and
Strobel, 1976) and incubated for 21 d. After 3 weeks of
still culture at 26C, the culture fluid was passed through
4 layers of cheese cloth to remove solids and extracted
with organic solvent. The extraction and isolation
procedure was followed according to Strobel et al.
(1996). After methylene chloride extraction, the organic
phase was collected and the solvent was then removed
by evaporation under reduced pressure at 35C using
rotary vacuum evaporator. The dry solid residue was
redissolved in methanol for the subsequent separation
and extracts were analyzed by chromatographic
separation and spectroscopic analyses, confirmed with
standard taxol (Paclitaxel) purchased from Sigma, USA.

Youth Education and Research Trust (YERT)

282
Thin layer chromatography analysis: TLC analysis was
carried out on Merck 1 mm (20 20 cm) silica gel plate
developed in solvent A (chloroform : methanol, 7:1 v/v)
followed by solvent B (chloroform : acetonitrile, 7:3 v/v),
solvent C (Ethyl acetate: 2-propanol, 95:5, v/v), solvent D
(methylene chloride: tetrahydrofuran, 6:2 v/v) and solvent
E (methylene chloride:methanol : dimethylformamide,
90:9:1, v/v/v) respectively. The area of the plate
containing putative taxol was carefully removed by
scraping off the silica at the appropriate Rf and eluted
with acetonitrile. Taxol was detected with 1% w/v
vanillin/sulphuric acid reagent after gentle heating. It
appeared as a bluish spot that faded to dark grey after
24 h (Cardellina et al., 1991).
High performance liquid chromatography: Further
confirmation for the presence of taxol was performed by
HPLC. The separation was eluting from a RP-C18 (4.6 x
150 mm, 5 m) reverse phase column with ultraviolet
detector. The sample (20 L) was injected each time
detected at 270 nm. The mobile phase was
methanol/acetonitrile/water (25:35:40, by v/v/v) at 1.0 mL
min-1. The sample and mobile phase were filtered
through 0.2 m PVDF filter before entering the column.
Taxol
was
quantified
using
the
formula:
M=MxV1x106/V2. M(gL-1)-taxol content in fermentation
liquid, M (mg/mL)-taxol content of methanol solution; V1
(mL)-volume of methanol used in redissolving of
residues; V2 (mL)-volume of fermentation broth for
extraction.
UV spectroscopic analysis: After chromatography, the
area of plate containing putative taxol was carefully
removed by scrapping off the silica at the appropriate Rf
value and exhaustively eluting it with methanol. The
purified sample of taxol was analysed by UV absorption,
dissolved in 100% methanol at 273 max in a UV
spectrophotometer (Hitachi) and compared with the
authentic taxol.
IR spectroscopic analysis: The purified taxol was ground
with IR quality potassium bromide (1:10), pressed into
discs under vacuum using spectra lab pelletiser and the
spectrum was recorded (4000-5000 cm1 nm) in a Burker
17S 85 FTIR spectrophotometer.
ESI-MS analysis: Taxol was identified by MS analysis
using the Electro Spray Ionization (ESI) technique with
an Agilent 1100 LC/ MSD trap. The nebulizer gas flow
rate of the sample was 2 L min-1 and the capillary
voltage was 2.2 kV.
Results
Isolation and identification of the endophytic fungi: The
strain LCPANCF01 was isolated from the root/transition
zone of Tylophora indica (Fig. 2). The isolated endophyte
typically possessed small hyphae, as a white colony
mycelium, when young. The mycelia are thread-like,
branched, septate and slow-growing; spores were
cylindrical to oval resembled Fusarium solani.

Nomila Merlin et al., 2012

J. Acad. Indus. Res. Vol. 1(5) October 2012

Fig. 2. Strain LCPANCF01 isolated from T. indica.

283

Fig. 4. HPLC analysis of authentic taxol; (a) Fungal taxol


from F. solani LCPANCF01; (b) The mobile phase was
methanol/acetonitrile/water (25:35:40, v/v/v), flow rate at 1.0
mL/min; retention time of authentic taxol: 10.71 min;
retention time of fungal taxol: 10.73.
a

Fig. 3. Phylogenetic tree of Fusarium solani LCPANCF01.

The ITS rDNA sequence data, whereby the closest


match (99% similarity) in the NCBI Gen Bank database
was found to be Fusarium solani LCPANCF01. The
phylogenetic tree was obtained by applying the
neighbor-joining method (Fig. 3). The ITS rDNA
sequence of the strain has been deposited in the NCBI
Gen Bank database (accession number-JN786598).
Screening of taxol: The extract of the test fungal culture
was examined for the presence of taxol by
chromatographic and spectroscopic analyses. Taxol,
produced by the fungus was detected using a spray
reagent consisting of 1% vanillin (w/v) in sulfuric acid
after gentle heating; it appeared as a bluish spot fading
to dark gray after 24 h. The compound has
chromatographic properties identical to standard taxol in
solvent systems A-E and gave colour reaction with the
spray reagent and they had Rf value of 0.58 identical to
that of standard taxol.
High pressure liquid chromatography: In HPLC analysis,
the fungal extract gave a peak when eluting from a
reverse phase C18 column, with a similar retention time
as standard taxol (Fig. 4a and b). The amount of taxol
produced by this fungus in liquid culture was
157.38 g/L.

Youth Education and Research Trust (YERT)

UV spectroscopic analysis: The UV spectral analysis of


the fungal taxol is given in Fig. 5a and b, the spectrum
was superimposed on that of authentic taxol.
IR spectroscopic and ESI MS analysis: The IR spectral
data of fungal taxol from Fusarium solani LCPANCF01
-1
showed broad peak in the region 3447.19 cm was
ascribed to hydroxyl (-OH) and amide (-NH) groups
stretch. The aromatic ring (C=C) group stretch was
observed in the region of 1632.15 cm-1. A peak observed
in the region 1065.73 cm-1 is due to the presence of
aromatic C, H bends. The IR spectrum of DCM extract of
the test isolate showed similar stretching frequency as
that of authentic taxol (Fig. 6a and b). The ESI mass
spectrum of fungal taxol isolated from Fusarium solani
LCPANCF01 is given in Fig. 7a and b.

Nomila Merlin et al., 2012

284

J. Acad. Indus. Res. Vol. 1(5) October 2012

Fig. 5(a) UV spectra absorption of authentic taxol;


(b) Fungal taxol from F. solani LCPANCF01.

Fig. 7(a) ESI mass spectrum of authentic taxol;


(b) Fungal taxol from F. solani LCPANCF01.
a

b
b

Discussion

Fig. 6(a) IR spectral analysis of authentic taxol;


(b) Fungal taxol from F. solani LCPANCF01.

The anticancer drugs which are available at present


possess enormous side effects and are not effective
against many forms of cancer. Taxol has a unique mode
of action when compared with other anti-cancer
compounds. The aim of this present study is to isolate
and identify the taxol-producing endophytic fungi from
Tylophora indica. Therefore, it was evident that this
fungus showed positive results for taxol production.
Taxol is positively identified via its co-chromatographic
mobilities with authentic taxol in a multitude of thin layer
chromatographic systems (Stierle et al., 1993; Strobel
et al., 1996). The biggest problem of using fungi in
fermentation was less production of taxol, its very low
yield and unstable production. The taxol yield of such
reported fungi varies from 24 ng to 70 ng per litre of
culture medium (Stierle et al., 1993; Strobel et al., 1996).
Although, the amount of taxol produced by most of the
endophytic fungi associated with taxus trees is relatively
small when compared with that of the trees, the short
generation time and high growth rate of fungi make it
worthwhile to continue our investigation of these species.
In general, taxol represents 0.01%-0.02% of the weight
of dry bark. The trace amount of compounds other than
taxol may perturb the absorbance reading, giving weight
estimates for taxol greater than the content which was
actually present.

65.7

64
62
60
2367.95

869.93

2862.74

58

760.83
1429.37 1261.53 1215.86

2929.97

56

1065.73

577.45

1149.65

54
52
%T 50

1632.15

48
46
44
42
40
38
3447.19

36
35.1
4000.0

3600

3200

2800

2400

2000

1800

1600

1400

1200

1000

800

600

400.0

cm-1

Youth Education and Research Trust (YERT)

Nomila Merlin et al., 2012

J. Acad. Indus. Res. Vol. 1(5) October 2012

It was evident that the diterpenoid taxol was much more


complex since its molecular weight from high-resolution
mass spectrometry is C47H51NO14, corresponding to a
molecular weight of 853. Characteristically, standard
taxol yielded +H+ at m/z 854 and +Na+ m/z at 876. By
+
comparison, fungal taxol also yielded a peak +H at m/z
+
854.3and +Na m/z at 876.2 with characteristic fragment
peaks at 569, 551, 509, 286 and 268. Major fragment
ions observed in the mass spectrum of taxol can be
placed into 3 categories which represents major portions
of the molecule (McClure and Schram, 1992). The peaks
corresponding to taxol, exhibited mass-to-charge (m/z)
+
ratios corresponding to the molecular ions (M+H) of
standard taxol (854) confirming the presence of taxol in
the fungal extracts.

Conclusion
As the taxol producing plant has become endangered,
this endophyte can be used as an alternative source for
taxol production. Fusarium solani LCPANCF01 produces
taxol which is confirmed by TLC, HPLC, IR and Mass
spectrum. The isolated fungi can be a good source of
taxol for large scale production by pharmaceutical
industries in near future.

Acknowledgements
The authors are grateful to Dr. Kamalraj for his valuable
suggestions and thank the facilities provided by the
Department of Plant Biology and Biotechnology, Loyola
College, Chennai for carrying out this work.

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