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Loyola College
Loyola College
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Nandagopal Murugan
Sankara Nethralaya
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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
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
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
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
Sample
Code
Loyola AR
Loyola PR
Loyola EES
Loyola VT
Loyola ST
Loyola VBS
Number of
Actinomycetes
08
15
02
15
20
22
District
Thiruvallur District
Kancheepuram District
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
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
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
Test
organisms
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
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
References
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Weyland, H. (1969). Actinomycetes in North Sea and Atlantic Ocean Sediments. Nature, 223:
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Ogunmwonyi, I.H., Mazomba, N., Mabinya, L., Ngwenya, E., Green, E., Akinpelu, D.A.,
Olaniran, A.O., Bernard, K., Okoh, A.I. (2010). Studies on the culturable marine actinomycetes
isolated from the Nahoon beach in the Eastern Cape Province of South Africa. African Journal of
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Aghamirian, M.R., Ghiasian, S.A. (2009). Isolation and Characterization of medically important
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Butler, M.S., Buss, A.D. (2006). Natural products: The future scaffolds for novel antibiotics?
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Newman, D.J., Cragg, G.M. (2007). Natural products as sources of new drugs over the last 25
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Saravana Kumar, P., Preetam Raj, J.P., Duraipandiyan, V., Ignasimuthu, S. (2012). Antibacterial activity of some actinomycetes from Tamil Nadu, India. Asian pacific Journal of Tropical
Biomedicine, 2(12): 936-943.
Nolan, R., Cross, T. (1988). Isolation and Screening of actinomycetes, In: Good fellow, M.,
Williams, S.T., Mordarski, M., Editors. Actinomycetes in Biotechnology. Academic Press. 1-32.
Hamilton, L.M., Kelly, C.T., Fogarty, W.M. (1999). Production and properties of the raw
starch-digesting -amylase of Bacillus sp. IMD 435. Process Biochemistry, 35(1-2): 27-31.
Adinarayana, K., Ellaiah, P., Prasad, D.S. (2003). Purification and partial characterization of
thermostable serine alkaline protease from a newly isolated Bacillus subtilis PE-11. AAPS Pharm
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Gopinath, S.C.B., Anbu, P., Hilda, A. (2005). Extracellular enzymatic activity in fungi isolated
from oil rich environments. Mycoscience, 46(2): 119-126.
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(2009). Antagonistic interaction between psychrotrophic cultivable bacteria isolated from Antartic
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33.
RESEARCH ARTICLE
Open Access
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
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.
Methods
Page 2 of 12
Isolation
Culture characterization
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
Page 4 of 12
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
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
TLC analysis
Page 5 of 12
Statistical analysis
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
The EA-SCA5 exhibited significant dose dependent inhibition of DPPH activity, with a 50% inhibition (IC50) at
Page 6 of 12
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
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
Characteristics
Results
Gram staining
Positive
Cytotoxicity property
25C to 37C
Optimum temperature
30C
3 to 11
Optimum pH
1 to 9%
Amylase
+++
Chitinase
Protease
++
Gelatinase
+++
Lipase
++
TLC analysis
Page 7 of 12
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
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
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
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
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.
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
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
C14H20O2
220
0.59
5.
11.77
2,4Dimethyl3pentanol acetate
C9H18O2
158
1.06
6.
11.96
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
Figure 5 GC-MS analysis of active crude from soil Streptomyces lavendulae strain SCA5.
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
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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
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.
1689
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
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
1691
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).
Results
The morphological properties of Streptomyces strain showed subsequent changes in
substrate or vegetative mycelium, the nature of aerial mycelium, size, and shape
1693
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/
Culture character
Growth response
Gram staining
Substrate, circular
Spores
Pink color
Motility
Diffusible pigments
Nonmotile
7 days
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
1695
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.
1697
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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Research Article
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
Chemicals
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
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
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.
223n
m
100
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.
101
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.
REFERENCE
21.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
102
32.
33.
34.
35.
36.
37.
38.
39.
40.
103
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Current Microbiology
ISSN 0343-8651
Volume 67
Number 1
Curr Microbiol (2013) 67:69-76
DOI 10.1007/s00284-013-0329-2
1 23
1 23
Received: 3 August 2012 / Accepted: 18 January 2013 / Published online: 17 February 2013
Springer Science+Business Media New York 2013
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
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.
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
71
123
72
Table 1 Culture characteristics and morphological identification of the isolated strain Streptomyces sp. loyola UGC on different medium
Medium
Growth
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
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
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
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
123
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
% of Inhibition
74
MeEA
BHA
200
400
600
800
1000
Concentration (g/ml)
123
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
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.
References
1. Berkov S, Zayed R, Doncheva T (2006) Alkaloids patterns in
some varieties of Datura stramonium. Fitoterapia 77:179182
2. Brand-Williams W, Cuvelier M, Berset C (1995) Use of a free
radical method to evaluate antioxidant activity. Lebensm-Wiss
Technology 28:2530
123
3. Chang SS, Wu ML, Deng JF, Lee CC, Chin TF, Liao SJ (1999)
Poisoning by Datura leaves used as edible wild vegetables. Vet
Hum Toxicol 41:242245
4. Dahlqvist A (1964) Method for assay of intestinal disaccharidases. Anal Biochem 7:1825
5. De Foe V, Senatore F (1993) Medicinal plants and phytotherapy
in the Amal Fitan Cost, Salerno province Campania, Southern
Italy. J Ethnopharmacol 39:3951
6. De Melo EB, Gomes ADS, Carvalho I (2006) a- and b-Glucosidase inhibitors: chemical structure and biological activity.
Tetrahedron 62(44):1027710302. doi:10.1016/j.tet.08.055
7. Diker D, Markovitz D, Rothman M, Sendovski U (2007) Coma as
a presenting sign of Datura stramonium seed tea poisoning. Eur J
Intern Med 18(4):336338
8. Elizabeth K, Rao MNA (1990) Oxygen scavenging activity of
curcumin. Int J Pharm 58:237240
9. Garratt DC (1964) The Quantitative Analysis of Drugs, vol 3.
Chapman and Hall, Tokyo, pp 456458
10. Goda T, Yamada K, Hosoya N, Moriuchi Y (1981) Effect of
alpha glucosidase inhibitor BAY g 5421 on rat intestinal disaccharidases. EiyoToShokuryo (in Japanese) 34:139143
11. Hanato T, Kagawa H, Yasuhara T, Okuda T (1988) Two new
flavonoids and other constituents in licorice root: their relative
astringency and radical scavenging effects. Chem Pharm Bull
36:20902097
12. Janero DR (1990) Malondialdehyde and thiobarbituric acid
reactivity as diagnostic indices of lipid peroxidation and peroxidative tissue injury. Free Radical Biol Med 9:515540
13. Jayaprakasha GK, Singh RP, Sakariah KK (2001) Antioxidant
activity of grape seed (Vitis vinifera) extracts on peroxidation
models invitro. Food Chem 73:285290
14. Johannes H, Gabriele B, Barbara S (2006) Isolation procedures
for endophytic microorganisms. Springer, Berlin, p 299
15. Kumar G, Murugesan AG (2008) Hypolipidaemic activity of
Helicteres isora L. bark extracts in streptozotocin induced diabetic rats. J Ethnopharmacol 116:161166
16. Liu F, Ooi VEC, Chang ST (1997) Free radical scavenging
activities of mushroom polysaccharide extracts. Life Sci 60(10):
763771
17. Mahmud T (2003) The C7N aminocyclitol family of natural
products. Natural Product Reports 20(1):137166. doi:10.1039/
b205561a
18. Marcocci L, Packer L, Droy-Lefai MT, Sekaki A, Gardes-Albert
M (1994) Antioxidant action of Ginkgo biloba extracts EGb 761.
Methods Enzymol 234:462475
19. Matsui T, Ueda T, Oki T, Sugita K, Terahara N (2001) a-Glucosidase inhibitory action of natural acylated anthocyanins.
Journal Agricultural Food Chemistry 49:19481951
20. Meir S, Kanner J, Akiri B, Hadas SP (1995) Determination and
involvement of aqueous reducing compounds in oxidative defense
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21.
22.
23.
24.
25.
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27.
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34.
35.
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CITATIONS
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P. Praveenkumar
Loyola College
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Loyola College
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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
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
1690
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.
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
1692
3. Results
Absorbance
2
1.5
1
0.5
0
200
400
600
800
Concentration (g/ml)
1000
% of Inhibition
Hexane
Ehtyl acetate
Methanol
BHT
2.5
100
90
80
70
60
50
40
30
20
10
0
Hexane
Ethyl acetate
Methanol
Vitamin C
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
400
600
800
Concentration (g/ml)
1000
% 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
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.
% 0f Inhibition
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
% 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
90
80
% of Chelating effect
% of Inhibition
70
Hexane
Ethyl acetate
Methanol
EDTA
60
50
40
30
20
10
0
200
400
600
800
Concentration (g/ml)
1000
Hexane
Ethyl acetate
Methanol
Vitamin C
200
400
600
800
Concentration (g/ml)
1000
1694
(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
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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
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
270
A.Vijayakumar et al
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
271
A.Vijayakumar et al
2.
3.
4.
5.
6.
272
A.Vijayakumar et al
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8.
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29. Moon JK, Shibamoto T. Antioxidant assays for plant and food
components. J. Agr. Food Chem. 2009; 57(5): 16551666.
30. Khan RA, Khan MR, Sahreen S, Ahmed M. Evaluation of phenolic
contents and antioxidant activity of various solvent extracts of
Sonchus asper (L.) Hill. Chem Central J. 2012; 6: 12.
31. Miguel MG. Antioxidant activity of medicinal and aromatic
plants. Flavour Fragr. J. 2010; 25: 291-312.
32. Fogliano V, Verde V, Randazzo G, Rittieni A. Method for
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273
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P. Praveenkumar
Loyola College
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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
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).
279
280
B.subtilis
12
10
10
8
E. aerogenes
8
10
10
-
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.
REFERENCES
1.
2.
281
282
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ISSN: 2278-5213
RESEARCH MANUSCRIPT
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.
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.
283
284
b
b
Discussion
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
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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