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Govindappa. et al. / International Journal of Biological & Pharmaceutical Research. 2014; 5(11): 861-869.

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International Journal of Biological

&
Pharmaceutical Research
Journal homepage: www.ijbpr.com

IJBPR

CHEMICAL COMPOSITION OF METHANOL EXTRACT OF

ENDOPHYTIC FUNGI, ALTERNARIA SP. OF TEBEBUIA ARGENTEA
AND THEIR ANTIMICROBIAL AND ANTIOXIDANT ACTIVITY
Govindappa M*, Prathap S, Vinay V and Channabasava R
Endophytic Natural Product Laboratory, Department of Biotechnology, Shridevi Institute of Engineering & Technology,
Tumkur 572 106, Karnataka, India.
ABSTRACT
We report the identification of the bioactive compounds in endophytic fungus, Alternaria sp methanol extract in 6th
fraction in GC-MS analyses. Totally 11 peaks were identified, they are acetic acid, phenylmethyl ester (1), phenol, 2,4-bis(1,1dimethylethyl)- (2), diethyl Phthalate (3), dodecanoic acid, 1-methylethyl ester (4), 2-pentadecanone, 6,10,14-trimethyl- (5),
1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester (6), pentadecanoic acid, 14-methyl-, methyl ester (7), 2benzenedicarboxylic acid, butyl 2-methylpropyl ester (8), 10,13-octadecadienoic acid, methyl ester (9), 9-octadecenoic acid
(Z)-, methyl ester (10) and 1,2-benzenedicarboxylic acid, mono(2-ethylhexyl) ester (11). They exhibited strong antioxidant and
antibacterial activity in combination of these compounds. The present approach offers a strategy for accelerating research on
natural products and their pharmacological applications.
Key Words: Tabebuia argentea, endophytes, Alternaria sp, GC-MS, antioxidant, antibacterial.
INTRODUCTION
Natural products are naturally derived
metabolites and/or by-products from microorganisms,
plants or animals (Baker et al., 2000). Endophytes are
microorganisms that reside in the tissues of living plants,
are relatively unstudied and potential sources of novel
natural products for exploitation in medicine, agriculture
and industry. It is noteworthy that, of the nearly 300,000
plant species that exist on the earth, each individual plant
is host to one or more endophytes. Only a few these
plants have ever been completely studied relative to their
endophytic biology (Strobel and Daisy, 2003). Novel
antibiotics, antimycotics,
immunosuppressants
and
anticancer compounds are only a few examples of what
has been found after the isolation, culture, Purification
Corresponding Author
Govindappa M
Email: endophytessiet@gmail.com

and characterization of some choice endophytes in the

recent past. The potential prospects of finding new drugs
that may be effective candidates for treating newly
developing diseases in humans, plants and animals are
great (Strobel and Daisy, 2003). Many endophytes are
producing antibiotics, antiviral, anticancer, antioxidant,
insecticidal, antidiabetic, immunosuppressive compounds
etc.
Tabebuia argentea (Bignoniaceae) is a large
and yellow flowering tree and have proven to be a rich
source of many organic compounds, especially, of
phenolic and poliophenolic nature. The plant able to
produce anticancer agent, lapachol it is ability to interfere
with the bioactivities of enzymes known as,
topoisomerases, a group of enzymes that are critical for
DNA replication in cells (Wuerzberger et al., 1998). The
antitumor activity of lapachol may be due to its interaction
with nucleic acids and the interaction of the
naphthoquinone moiety between base pairs of the DNA

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helix occurs with subsequent inhibition of DNA

replication and RNA synthesis (Murray and Pizzorno,
1998). Other biological activities of lapachol are
antimetastatic (Balassiano et al., 2005), anti-microbial
and antifungal (DaSilva et al., 2003), antiviral (Breger et
al., 2007), anti-inflammatory (Almeida et al., 1990),
antiparasitic (Wuerzberger et al., 1998), leishmanicidal
(Teixeira et al., 2001) and molluscicidal (Silva et al.,
2005). Only one report is available on identification of
lapachol producing endophytes of Tabebuia argentea
from our
lab only (Sadananda et al., 2011).
Channabasava and Govindappa (2014) have studied on
lapachol from Alternaria alternata and its in vitro
antimitotic, antiproliferative and DNA fragmentation
assays. The present investigation was aimed to study the
other endophytic fungal species Alternaria sp. role in
antioxidant and antibacterial activity and identification of
active phytochmical analysis using GC-MS.
MATERIALS AND METHODS
Fungal material
The fungus Alternaria sp. was isolated from fresh
bark of Tabebuia argentea. The plant was collected in the
month of September 2012 near Shridevi Institute of
Engineering & Technology campus, Tumkur, Karnataka,
India. A voucher specimen has been deposited at
Department of Biotechnology, SIET, Tumkur. Voucher
specimen was identified by Dr Sharanappa P, Department
of Studies in Biosciences, University of Mysore, Hema
Gangothri, Hassan, Karnataka, India. The collected bark
was surface sterilized with 70% ethyl alcohol for 1 min and
rinsed in sterile water. Small tissue from bark were
aseptically cut and pressed onto agar plates containing an
antibiotic to suppress bacterial growth (15 g/l malt extract,
15 g/l agar, 0.2 g/l chloramphenicol in distilled water pH
7.0-7.2). Incubated the plates at room temperature
(26+20C) the fungal strain grow exclusively out of the
plant tissue. Pure strains of Alternaria sp were isolated by
repeated re-inoculation on malt-agar plates from the
growing cultures.
Identification of fungal culture
The fungal strain was identified as Alternaria sp
based on the colony, hyphal and conidial morphology
(Ellis, 1971; Barnett and Hunter, 1972)
Mass cultivation of the fungus
For isolation and identification of metabolites was
carried out using Potato Dextrose Broth containing
Erlenmeyer flasks. The fungus inoculated flasks were
incubated at room temperature (26+20C) under static
conditions for 21 days.
Extraction and isolation of the bioactive compounds
After incubation, fungal mycelia was separated
from liquid culture media and soaked in MeOH overnight.

The cells were disturbed using mortar and pestle for 10

min followed by filtration and exhaustive extraction.
The methanol extract of Alternaria sp (0.5g) was separated
on silica gel 60 using stepwise elution with a mixture of nhexane. The extract sample to be separated is placed on the
top of the column near the end to collect the elute. A
methanol extract fraction over Sephadox LH-60 (MeOH)
fraction 6 gave the clear band.
Thin layer chromatography
A thin mark is made at the bottom of the plate
with a pencil to apply the sample spots. Then samples
solutions applied to the spots marked on the line at equal
distances.
The mobile phase (chloroform: methanol, 19:1)
(Houghton et al., 1994) is poured into the TLC chamber
to a level few centimeters above the chamber bottom. Then
the plate prepared with sample spotting is placed in TLC
chamber such that the side of the plate with sample line is
towards the mobile phase. Then the chamber is closed with
a lid.
The plate is immersed such that sample spots are
well above the level of mobile phase, but not immersed in
the solvent for development. Allow sufficient time for
development of spots. Then the plates were removed and
allowed to dry. The sample spots were visualized in the
UV light chamber for the sample.
ANTIBACTERIAL ACTIVITY
Bacteria tested
Total four pathogenic bacterial strains were used
in the investigation, namely Proteus mirabilis (MTCC
3425), Serratia marcescens (MTCC 9527), Klebsiella
pneumonia (MTCC 4032) all are of Gram-ve bacteria and
Gram+ve Staphylococcus aureus (MTCC 3160). All the
bacterial cultures were obtained from Microbial Type
Culture Collection (MTCC), Institute of Microbial
Technology, Chandigarh, India. The young bacterial broth
cultures were prepared before the screening procedure.
Agar well diffusion assay
The antimicrobials present in the endophytic
fungal extracts was allowed to diffuse out into the medium
and interact in a plate freshly seeded with the test
organism. The resulting zone of inhibition will be uniform
circular as there will be confluent lawn of growth. The
diameter of zone of inhibition can be measured in
millimeters. Agar well diffusion assay was performed to
determine the endophytic hydrocarbons derivatives activity
against bacterial strains by the modified method (Ettebong
and Nwafor, 2009). Muller Hinton agar medium plates
were seeded with 24 h bacterial cultures (250 l bacterial
culture with OD600 of about 1 per 100 ml Muller Hinton
broth) and spread uniformly with glass spreader. Five wells
at the distance of 3cm and one well in the center were
made by using 4 mm cork borer. 50 l of extracts were

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Govindappa. et al. / International Journal of Biological & Pharmaceutical Research. 2014; 5(11): 861-869.

added. The plates were then incubated at 370C for 24 h.

The antibacterial activity was assayed by measuring the
diameter of the inhibition zone formed around the well.
ANTIOXIDANT
ACTIVITIES
OF
THE
ENDOPHYTIC EXTRACT
FRAP assay
FRAP reagents were freshly prepared by
mixing 25 ml acetate buffer (300 mM, pH 3.6), 0.5 mL
2,4,6-tris(2- pyridyl)-S-triazine (TPTZ) solution (10 Mm
TPTZ in 40 Mm/I HCl) and 2.5 mL FeCl3 (2 Mm) water
solution. Each sample (150 l) (0.5 mg/ml) dissolved in
methanol was added to 4.5 ml of freshly prepared FRAP
reagent and stirred. After 5 min, absorbance was measured
at 593 nm, using the FRAP working solution as blank
(Tomic et al., 2009). A calibration curve of ferrous
sulphate (100 to 1000 umol/l) was used and results were
2+

expressed in mol Fe /mg dry weight extract. The relative

activity of the sample was compared to L-ascorbic acid.
DPPH radical assay
The hydrogen atom or electron donation ability of
the corresponding extracts and some pure compounds were
measured from the bleaching of purple coloured methanol
solution of DPPH. This spectrophotometric assay uses
stable radical diphenylpicrylhydrazyl (DPPH) as a reagent
(Gulluce et al., 2006). Fifty microlitres of various
concentrations of the extracts in methanol was added to 5
ml of a 0.004% methanol solution of DPPH. After a 30
min incubation period at room temperature the absorbance
was read against s blank at 517 nm. Inhibition free radical
DPPH in percent (I%) was calculated in the following way:
Inhibition Percentage (I%) = A blank (A sample A blank)x100

where A blank is the absorbance of the control

reaction (containing all reagents except the test
compound), and A sample is the absorbance of the test
compound. Extract concentration from the graph plotted
inhibition (IC50) was calculated from the graph plotted
against extract concentration. Synthetic antioxidant reagent
butylatedhy- roxytoulene (BHT) was used in triplicate.
Superoxide Radical Scavenging Activity
Superoxide radical O 2 was generated from the
phototoreduction of riboflavin and was deducted by nitro
blue tetrazolium dye (NBT) reduction method of
m easurement of superoxide anion scavenging activity
(Winterbourne et al., 1975). The assay mixture contained
sample with 0.1 ml of 1.5 mM NBT solution, 0.2 ml of 0.1
M EDTA, 0.05 ml riboflavin (0.12 mM) and 2.55 ml of
0.067 M phosphate buffer. The control tubes were also set
up where in DMSO was added instead of samples. The
reaction mixture was illuminated for 30 min and the
absorbance at 560 nm was measured against the control

samples. Ascorbate was used as the reference compound.

All the tests were performed in triplicate and the results
averaged. The percentage inhibition was calculated by
comparing the results of control and test samples.
Free radical scavenging activity
The ability of m ethanol extract of A l t e r n a r i a
s p to scavenge 1,1-diphenyl-2-picryl-hydrazyl (DPPH)
free radicals was estimated (Jain et al., 2008). Extract (3
ml) with six different concentrations (15.62, 31.25, 62.5,
125, 250 and 500 g/ml) were mixed with 1 ml of a 0.1
mM methanol solution of DPPH. The absorbance was
measured by a spectrophotometer at 517 nm at 30 min
intervals against a blank (pure methanol). The percentage
of radical scavenging activity was calculated using the
following formula:
Radical scavenging % = 1- [( A0 - A1) /A0 x 100]
where A0 is the absorbance of the control and A1
is the absorbance of the sample extracts21. Lower
absorbance values show higher free radical scavenging
activity. Ascorbic acid was used as a reference standard in
different concentrations (1.56, 3.12, 6.25, 12.5, 25 and
50 g/ml). The 50% inhibitory concentration value (IC50) is
indicated as the effective concentration of the sample that
is required to scavenge 50% of the DPPH free radicals.
GC-MS analyses
The bioactive crude extract was separated into
various fractions by column chromatography. The column
was packed with silica gel (mesh 60-120) and run with nhexane: MeOH (8:2). The 6th fraction showed a clear band
in TLC and is selected for GC-MS analyses. GC-MS
analyses were performed at Central Instrumentation
Department, Indian Institute of Sciences (IISc), Bangalore,
India. GC-MS measurements were performed with a
Shimadzu instrument equipped with GC: Aligent 7890 A,
MS: MS detector 5975C, Ionization for MS: Electron
Impact Ionization, Mass Analyzer: Quadrupole, Software:
Data Analysis, Library: Nist 2008, column: HP 5 ms,
Dimensions: 30m L X 0.25mm ID x 0.25m film
thickness, initial temperature is 0 to 400C 2 min hold time,
ram temperature is 100C to 3100C 10 min is the hold time,
total time is 34 min, carrier gas is helium, flow (ml/min) is
1.0, split flow: 1ml/min, injection volume: 1l, Scan mass
range: 30m/z-600m/z and polarity +ve. GC-MS performed
based on the database having more than many patterns.
The spectrum of the unknown compound was compared
with the spectrum of the known compounds in library.
RESULTS
We have selected the 6th fraction of Alternaria sp
methanol extract in TLC (Fig 1) and is very prominent and
collected the same fraction for further studies. In in vitro
antibacterial assay, the 6th fraction of Alternaria sp extract
showed a varying degree of antibacterial activities against
the test bacterial species (Table 1). Proteus mirabilis,

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Govindappa. et al. / International Journal of Biological & Pharmaceutical Research. 2014; 5(11): 861-869.

Klebsiella pneumonia and Staphylococcus aureus were

inhibited strongly (13 mm each), whereas Serratia
marcescens inhibited by 11 mm. Results show that
endophytic fungus Alternaria sp extract strongly inhibited
the growth of all test bacterial species.
Superoxide is a highly reactive molecule that
reacts with various substances produced through metabolic
processes. Superoxidase dismutase enzymes present in
aerobic and anaerobic organisms catalyses the breakdown
of super- oxide radical. Percentage scavenging of
superoxide anion examined at different concentrations of
methanol extract of Alternaria sp (125, 250, 500, and
1000) was depicted in Table 2. The percentage scavenging
of superoxide radical surged with the enhanced
concentration of endophytic extract. The maximum
scavenging activity of endophytes extract and quercetin at
1000 g/ml was found to be 94.71+0.012 and 98.31+0.013
respectively. Superoxide scavenging ability of plant extract
might primarily be due to the presence of bioactive
compounds in 6th fraction of Alternaria sp extract.
The antioxidant potentials of the methanol extract
of Alternaria sp. was estimated from their ability to reduce
TPRZ-Fe (III) complex to TPTZ-Fe (II). The FRAP values
for the 6th fraction methanol endophytic fungus extract
significantly lower that of ascorbic acid but higher that of
plant extract. The Alternaria sp extract marginally lower
than standard in FRAP assay (Table 3).
The antioxidant activity of the methanol extract
was measured by the ability to scavenge DPPH free
radicals, was compared with the standards Butylated
Hydroxy Toluene (BHT). It was observed that methanol
extract of the Alternaria sp. had higher activity. At a
concentration of 0.1 mg/ml, the scavenging activity of
methanol extract reached to 90.08. Though the DPPH
Fig 1. The 5th and 6th fractions of sample 6 (Alternaria
sp.) showing pure band

radical scavenging abilities of the extract were less than

about 0.1 mg/ml, the study showed that the extracts have
the proton donating ability and could serve as free radical
inhibitors or scavenging, acting possibly as primary
antioxidants (Figure 2). The performance of methanol
extract of endophytic fungi, Phyllosticta sp. was higher
than that, the standard -tocopherol22 and from different
endophytes (Phongpaichit et al., 2006).
GC-MS analyses
The GC-MS analyses of Alternaria sp extract
yielded 11 prominent peaks with retention time 11.959,
16.681, 17.710, 18.085, 20.517, 20.744, 21.358, 21.714,
23.027, 23.085 and 26.818 min. The GC-MS analyzed the
compounds compared with library search (mainlib) and
identified the major compounds as acetic acid,
phenylmethyl ester (1), phenol, 2,4-bis(1,1-dimethylethyl)(2), diethyl Phthalate (3), dodecanoic acid, 1-methylethyl
ester (4), 2-pentadecanone, 6,10,14-trimethyl- (5), 1,2benzenedicarboxylic acid, bis(2-methylpropyl) ester (6),
pentadecanoic acid, 14-methyl-, methyl ester (7), 2benzenedicarboxylic acid, butyl 2-methylpropyl ester (8),
10,13-octadecadienoic acid, methyl ester (9), 9octadecenoic acid (Z)-, methyl ester (10) and 1,2benzenedicarboxylic acid, mono(2-ethylhexyl) ester (11)
from 6th fraction of methanol extract of Alternaria sp (Fig
3). The structure of each identified and isolated compounds
were presented in Fig 4.
The spectrum of unknown compounds was
identified with library based on retention time and mass
spectra. From earlier reports, the identified compounds
exhibited many biological activity and isolated from
various sources are depicted in Table 4.
Fig 2. DPPH scavenging activities of methanol Alternaria
sp extract

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Govindappa. et al. / International Journal of Biological & Pharmaceutical Research. 2014; 5(11): 861-869.

Fig 3. GC-MS total ion chromatogram of Alternaria sp. 6th fraction

Fig 4. Structures of the 11 different bioactive metabolites in 6 th fraction of Alternaria sp methanol extract
Phenol, 2,4-bis(1,1-dimethylethyl)Acetic acid, phenylmethyl ester

Diethyl Phthalate

Dodecanoic acid, 1-methylethyl ester

2-Pentadecanone, 6,10,14-trimethyl-ester

1,2-Benzenedicarboxylic acid, bis(2-methylpropyl)

Pentadecanoic acid, 14-methyl-, methyl ester

1,2-Benzenedicarboxylic acid, butyl 2-methylpropyl ester

10,13-Octadecadienoic acid, methyl ester

9-Octadecenoic acid (Z)-, methyl ester

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Govindappa. et al. / International Journal of Biological & Pharmaceutical Research. 2014; 5(11): 861-869.

1,2-Benzenedicarboxylic acid, mono(2-ethylhexyl) ester

Table 1. Antibacterial activity of 6th fraction of methanol extract of endophytic fungus, Alternaria sp
Inhibition in mm
Bacteria tested
Fraction 6
Proteus mirabilis
13
Serratia marcescens
11
Klebsiella pneumonia
13
Staphylococcus aureus
13
Table 2. Effect of Alternaria sp 6 fraction extract on superoxide radical scavenging activity
Concentration (g/ml)
Alternaria sp extract
Querscetin
125
63.31+0.016d
73.88+0.006d
250
79.66+0.033c
91.38+0.012c
b
500
89.12+0.012
93.41+0.026b
a
1000
94.71+0.012
98.31+0.013a
Repeated the each experiment three times. Data represents mean + SD. Different lowercase and upper case letters in the same
column indicate significant difference at < 0.05
Table 3. Total antioxidant activity of methanol extract of Alternaria sp extract
Total antioxidant activity
FRAP
Alternaria sp extract
4322+0.03c
Plant leaf extract
3644+0.03a
Ascorbic acid
4401+0.03b
Repeated the each experiment three times. Data represents mean + SD. Different lowercase and upper case letters in the same
column indicate significant difference at < 0.05
Table 4. Biologically importance of the identified compounds from different sources
Retention
Identified compound
Peak No
Biological activity
time (min)
name

11.959

16.681

17.710

18.085

20.517

Acetic acid, phenylmethyl

ester

Phenol, 2,4-bis(1,1dimethylethyl)-

Diethyl Phthalate
Dodecanoic acid, 1methylethyl ester
2-Pentadecanone, 6,10,14trimethyl-

Flavoring agent in
cosmetics, lotions
Fragrance material
Antifungal activity,
Antimicrobial,
UV stabilizer and an
antioxidant for
hydrocarbon-based
products
antimalarial activity
antibacterial activity
antioxidant
Antioxidant

Source
Jasmine, apple, cherry, guava fruit
and peel, wine grape, white wine,
tea, plum, cooked rice, Bourbon
vanilla, naranjila fruit
Chimonanthus praecox

Avocado roots,
Barleria Montana

Balanites aegyptiaca
Monochaetia kansensis
Scolopendra subspinipes
Scolopendra subspinipes
Abutilon indicum

allelopathic activity

Ecballium elaterium

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Govindappa. et al. / International Journal of Biological & Pharmaceutical Research. 2014; 5(11): 861-869.

antimicrobial activity

20.744

1,2-Benzenedicarboxylic
acid, bis(2-methylpropyl)
ester

21.358

Pentadecanoic acid, 14methyl-, methyl ester

21.714

1,2-Benzenedicarboxylic
acid, butyl 2-methylpropyl
ester

23.027

10,13-Octadecadienoic
acid, methyl ester

10

23.085

9-Octadecenoic acid (Z)-,

methyl ester

11

26.818

1,2-Benzenedicarboxylic
acid, mono(2-ethylhexyl)
ester

antimicrobial
-Glucosidase inhibition
and the in vivo
hypoglycemic effect

Sedum pallidum Bieb. var.

bithynicum (Boiss.) and S.
spurium Bieb
Lobelia pyramidalis
Laminaria japonica
Cassia Sophera Linn
Synurus deltoides

-Glucosidase inhibition
and the in vivo
hypoglycemic effect
antimicrobial
antimicrobial
antimicrobial
anti-oxidant
antimicrobial
Antiviral
antimicrobial
anti-oxidant and antiinflammatory properties

Laminaria japonica
Lobelia pyramidalis
bereberise integrrima
Desmodium gangeticum
Celtis australis
Pyrostegia venusta
Desmodium gangeticum
Psidium guajava Linn.
Desmodium gangeticum
Polygonum chinense L.
Cadaba Trifoliata

DISCUSSION
Channabasava and Govindappa (2014) have
already reported that the enophytic fungi Alternaria sp was
isolated from bark. The methanol extract of Alternaria sp
6th fraction was taken for further study and it has only one
band in the TLC. The extract has shown strong
antibacterial activity in testing bacteria. The results are
supported by the findings of Sadananda et al (2013). The
same extract has shown potent antioxidant activity in three
different methods. Sadananda et al (2014) has reported
endophytic extracts antioxidant activity. Our results are
confirmed by these findings.
In GC-MS, 11 peaks were observed in the fraction
of 6 of methanol extract of Alternaria sp based on retention
time and chromatograms using the library. The identified
compounds are present in various sources are presented in
Table 2. The compounds 2, 3, 10, 11 have already reported
as strong antioxidant molecules and were isolated from
different natural sources (plants) (Yoon et al., 2006; Roy et
al., 2011). But the antibacterial reports are evidenced with
compounds 2, 5, 6, 8, 9, 10 and 1as1 (Yoon et al., 2006;
Yayli et al., 2010; Joshi et al., 2011; Hemlal et al., 2012;
Badoni et al., 2010). Based on the above evidence, the 6th
fraction of Alternaria sp methanol extracts exhibited very
good bioactive natural products and showed strong
antioxidant and antibacterial activity. This confirms may

be due to the effect of single compound or in group

showed the above properties.
CONCLUSIONS
The identification of bioactive compounds in 6 th
fraction of Alternaria sp methanol shows 11 compounds.
These compounds contain extract exhibited strong
antibacterial and antioxidant activity. Therefore, this
strategy may significantly accelerate the discovery process
in natural products research and their role in
pharmacology. The separation of each compound
individually needed and their role should be identified in
the future.
CONFLICTS OF INTEREST
All authors have none to declare.
ACKNOWLEDGEMENT
The authors are grateful to Visvesvaraya
Technological University (VTU), Belgaum, Karnataka,
India
for providing financial support for this
investigation under Research Grants. We thank Dr MR
Hulinaykar, Managing Trustee, Sri Shridevi Charitable
Trust (R.) and Dr Sukumaran K, Principal, SIET,
Tumkur, India for encouragement.

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