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527Govindappa M. et al . / International Journal of Biol ogical & Pharmaceutical Research. 2014; 5(6):527-534.
e- ISSN 0976 - 3651
Print ISSN 2229 - 7480
International Journal of Biological
&Pharmaceutical ResearchJournal homepage: www.ijbpr.com
IDENTIFICATION OF BIOACTIVE METABOLITES BY GC-MS
FROM AN ENDOPHYTIC FUNGUS, ALTERNARIA ALTERNATA
FROM TABEBUIA ARGENTEA AND THEIR IN VITRO CYTOTOXIC
ACTIVITY
Govindappa M*, Channabasava R, Sadananda TS, Chandrappa CP and Umashankar T
Endophytic Natural Product Laboratory, Department of Biotechnology, Shridevi Ins titute of Engineering & Technology,
Tumkur 572 106, Karnataka, India.
ABSTRACT
In this paper, we report a method of extraction, identification of major bioactive metabolites from endophytic fungi
Alternaria alternata ethanol extract using an in vitro antimitotic assay, antiproliferative and DNA fragmentation assays. The
fraction 6 of A. alternata ethanol extract strongly inhibited the onion meristematic cells, inhibited the yeast cells and induced
the DNA fragmentation in yeast cells. To know the bioactive compounds in the 6th
fraction from 7 different peaks found in GC-MS analogues based on retention time, the compounds are 2-benezenedicarboxylic acid, bis (2-methylpropyl) ester,
hexadecanoic acid, methyl ester, 1,2 benzenedicarboxylic acid, butyl 2-methylpropyl ester, 1,4-napththalenedione, 2-hydroxy-
3-(3-methyl-2-butenyl)-, 9-octadecenoic acid (Z), methyl ester, 10,13-octadecadienoic acid, methyl ester and 1,2-
benzenecarboxylic acid. The antimitotic, antiproliferative and DNA fragmentation assay may be due to the presence of potent bioactive compounds may occur in exclusively or combination. Further work is needed to identify the exact compound that may
be us ed for cancer therapy .
Key Words: Tabebuia argentea, Alternaria alternata, Antimitotic, Antiproliferative, DNA fragmentation, GC-MS.
INTRODUCTION
Fungal endophytes are micro-organisms that
colonize living, internal tissues of plants without causing
any immediate and overt negative effects (Bacon and
White, 2000). The fungal endophytes have proven to be
promising sources of many biologically active natural
products (Strobel, 2002).
Tabebuia argentea (Bignoniaceae) is a large andyellow flowering tree and have proven be rich sources of
many organic compounds viz., phenolic, polyphenolic and
lapachol. This plant is able to produce an anticancer agent
Corresponding Author
Govindappa M
Email: [email protected]
(lapachol) is able to interfere with topoisomerase I enzyme
(Wuerzberger et al ., 1998) and also cause an effect on
RNA synthesis (Murray and Pizzorno, 1998). The plant
extract showed many biological activities such as
antimetastic, antimicrobial and antifungal, antiparasitic,
leishmanicidal and mollescidal activities.
Recently, 12 different fungal endophytes wereisolated from different parts of T. argentea. Out of 12, the
Aspergillus niger , Penicillium sp, Trichoderma harzianum
had shown antioxidant activity (Govindappa et al ., 2013).
The endophytic fungus, Aspergillus niger extract exhibited
in vitro antimitotic, antiproliferative and DNA
fragmentation assay (Channabasava and Govindappa,
2013). In the present study we have aimed to identify
major bioactive constituents in endophyte, Alternaria
IJBPR
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528Govindappa M. et al . / International Journal of Biol ogical & Pharmaceutical Research. 2014; 5(6):527-534.
alternata by GC-MS and its in vitro cytotoxicity assay was
performed.
MATERIAL AND METHODFungal materi al
The fungus Alternaria alternata was isolated from
fresh bark of Tabebuia argentea. The plant was collectedin the month of September 2012 near Shridevi Institute of
Engineering & Technology (SIET) campus, Tumkur,
Karnataka, India. A voucher specimen has been deposited
at Department of Biotechnology, SIET, Tumkur. Voucher
specimen was identified by Dr Sharanappa P, Departmentof 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 specimens from bark
were aseptically cut and pressed onto agar plates
containing an antibiotic to suppress bacterial growth (15 g/lmalt extract, 15 g/l agar, 0.2 g/l chloramphenicol in
distilled water pH 7.0-7.2). The plates were incubated at
room temperature (26+20C). The fungal strains growexclusively out of the plant tissue. Pure strains of A.
alternata were isolated by repeated re-inoculation on malt-
agar plates from the growing cultures.
Identification of fungal culture
The fungal strain was identified as A. alternata
based on the colony, hyphal and conidial morphology
(Ellis, 1971; Barnett and Hunter, 1972).
Mass culti vation of the fungus
For isolation and identification of metabolites was
carried out using Potato Dextrose Broth (PDB) containing
Erlenmeyer flasks. The fungus inoculated flasks wereincubated at room temperature under static conditions for
21 days.
Extr action and isolati on
After incubation, fungal mycelia was separated
from liquid culture media and soaked in ethanol overnight.
The cells were disturbed using mortar and pestle for 10
min followed by filtration and exhaustive extraction.
The ethanol extract of A. alternata (0.5g) was
separated on silica gel 60 using stepwise elution with a
mixture of n-hexane. The extract sample to be separated is
placed on the top of the column near the end to collect the
elute. An ethanol extract fraction over Sephadox LH-60(EtOH) fraction 6 gave the clear band.
Antimi totic activity
The method adopted by Shweta et al . (2012) was
used for determination of antimitotic activity using Allium
cepa root with slight modification. Allium cepa were
collected from the Tumkur vegetable market. Allium cepa
bulbs were sprouted in water for 24 h at room temperature.
The uniform root tips of Allium cepa were selected for the
study. These roots were dipped in the extract (200 µl/ml)
for 48h. Water was used for dilution and lapachol was us ed
as a standard for study. After 48h, the root tips were fixed
in the fixing solution of acetic acid and alcohol (1:3).
Squash preparation was made by staining with
acetocarmine stain. Morphology and the number of the
cells were observed under microscope (40X). In all 350-400 cells were counted and cells manifesting different
stages of mitosis i.e., interphase and prophase (P),
metaphase (M), anaphase (A) and telophase (T) were
recorded. The mitotic index was calculated using the
following formula (Shwetha et al ., 2012; Subhadradevi etal ., 2011).
P + M + A + T
Mitotic index=_____________ X 100
Total cells
Anti proli ferative activity
Evaluation of the antiproliferative activity of
endophytic extract was done by yeast Saccharomyces
cerevisiae model according to Shwetha et al . (2012).
Yeast inocul um preparati on
The yeast was inoculated with s terilized PDB and
incubated at 37º C for 24 h and it was referred as seed broth .
Determination of cell viabil ity
The cell viability assay was performed with 2.5
ml of PDB and 0.5 ml of yeast inoculums in four separate
test tubes. In the first test tube distilled water, in second
test tube quercetin (Sigma Aldrich) as standard (1mg/ml),
in third and fourth test tubes endophytic extract (10mg/ml
respectively) were added. All tubes were incubated at 37ºC for 24h. In the above cell suspension, 0.1% methylene
blue dye was added in all tubes and they were obs erved
under low power microscope. The number of viable cells,
those that do not stain and look transparent with oval shape
while dead cells get stained and appeared blue in color,
were counted in 16 chambers of hemocytometer and the
average number of cells was calculated. The percentage of
cell viability was calculated using the formula
(Subhadradevi et al ., 2011).
No. of dead cells
% cytotoxicity = X 100
No. of viable cell +No. of dead cells
DNA f ragmentati on assay
DNA fragmentation assay was performed by the
method of Bicas et al . (2011). Briefly, 0.1 ml of extract
was mixed with 2.5 ml PDB and 0.5 ml of yeast
inoculums. The cell suspension was incubated for 24 h at
37º C. DNA was isolated from the treated cell suspension
with Tris-EDTA buffer and DNA was electrophoresed
(Shwetha et al ., 2012).
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529Govindappa M. et al . / International Journal of Biol ogical & Pharmaceutical Research. 2014; 5(6):527-534.
GC-MS anal yses
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 n-
hexane: EtOH (8:2). The earlier stated 6th
fraction showed
a clear band in TLC and is selected for GC-MS analyses.
GC-MS analyses were performed at CentralInstrumentation 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 Analys is, Library: Nist 2008,
column: HP 5 ms, Dimensions: 30m L X 0.25mm ID x
0.25µm film thickness, initial temperature is 0 to 400C 2
min hold time, ram temperature is 100C to 310
0C 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:1µl, 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 compoundwas compared with the spectrum of the known compounds
in library.
RESULTS AND DISCUSSION
Shown are the natural habitat of T. argentea
(Fig1) and pure culture of isolated A.alternata (Fig 2). The
ethanol extract of A. alternata was found to function in
antimitotic, antiprolerative and DNA fragmentation assays.
The lower concentration of 200 µl was more significant in
reducing cell division and effective in reducing index value
after 48h of treatment. We have found that A. alternata
extract induced antimitotic activity at various levels of cell
cycle viz., (1) arrest of cells at interphase with largenucleoli and binucleoli; (2) metaphase with irregular
chromosomal distribution and inter chromosomal gap; (3)
chromosomal stickiness and bridge at anaphase; (4)
chromosomal stickiness and cell shrinkage at telophas e; (5)
cell shrinkage at prophase surrounded by normal cells; (6)
abnormal anaphase with vagrant chromosomes.
Chromosomal fragmentation at late prophase and
chromosomal separation at anaphase was observed and
compared with normal mitotic phases of interphase,
metaphase, anaphase, prophase and telophase (Figs 3a &
3b). The value decrease dose was consistent with the
standard lapachol. Alternaria alternata mitotic index was
found to be 30.6 whereas the untreated control showed a91.4 mg/ml (Fig 4). Similar results were observed with
plant extracts of Ocimum gratissimum, Morinda lucida
(Bernice et al ., 2009), endophytes Fusarium oxysporum,
Trichothecium sp lectin (Sadananda et al ., 2013) and
Aspergillus niger lapachol (Channabasava and
Govindappa, 2013). The extract showed potential
antimitogenic activity by inducing structural changes to
chromosome.
The A. alternata extract (6th
fraction) was
evaluated against Saccharomyces cerevisiae in
antiproliferative activity and it showed potent inhibition of
yeast cell growth. The number of dead cells was calculated
using above mentioned formula. The A. alternata extract
inhibited the growth of yeast cells above 78.6% whereas
the standard showed more than above 90% (Fig 5). Thisresult confirms the potent bioactive compounds may be
present in the extract. Alternaria alternata extract leads to
death of yeast by inducing toxicity and dead cells with
debris was observed in treated yeast cells. Interest ingly, we
sequentially observed the necrosis from A. alternata treated yeast cells after 24 h of treatment and result the
clearly indicates that our A. al ternata extract acts on yeast
cells (Fig 6). In vitro antiproliferative and cytotoxic assay
was s tudied using yeas t as a model sys tem. Yeast is widely
used as a model organism to study many aspects of
eukaryotic cell biology (Clarke et al ., 1993). Cytotoxicitystudies using yeast has been reported on cytotoxic potential
of Revie hypocrateriformis (Shwetha et al ., 2012),
endophytic lectin (Sadananda et al ., 2013) and endophyticlapachol (Channabasava and Govindappa, 2013).
Apoptotic cells are characterized by the number of
morphological changes such as cell shrinkage, membrane
blebbing, chromatic condensation and formation ofapoptotic bodies (Zimmermann et al ., 2001). Some of the
morphological changes associated apoptosis occur as a
result of activation of endogenous and endonucleolytic,
proteolytic enzymes that in turn mediate the cleavage of
DNA into fragmentation.
DNA fragmentation assay proved the
antiproliferation activity of A. al ternata extract. After 48 h
of treatment with A. alternata extract, breakdown of the
DNA of yeast has occurred. It is one of the methods ofinhibition of DNA replication in cancer therapy. The DNA
fragmentation may be due to inhibition of replication of
topoisomerase enzymes or direct cleavage of cleaved the
DNA (Fig 7). Most of the anticancer drugs of plant origin
and their synthetic counterparts have been known to cause
DNA damage. Suppression of DNA replication instead is
not necessary for killing cells directly but induces
apoptosis.
The GC-MS analyses of A. alternata extract
yielded 7 prominent peaks with retention times of 20.743,
21.361, 21.715, 22,561, 23.027, 23.086 and 26.818 min
(Fig 8). The GC-MS analyzed the compounds compared
with library search (mainlib) and identified the majorcompounds as 1,2-benezenedicarboxylic acid, bis (2-
methylpropyl) ester (1), hexadecanoic acid, methyl ester
(2), 1,2 benzenedicarboxylic acid, butyl 2-methylpropyl
ester (3), 1,4-Napththalenedione, 2-hydroxy-3-(3-methyl-
2-butenyl)- (4), 9-Octadecenoic acid (Z), methyl ester (5),
10,13-octadecadienoic acid, methyl ester (6) and 1,2-
benzenecarboxylic acid (7) from 6th
fraction of ethanol
extract of A. al ternata (Fig 9).
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530Govindappa M. et al . / International Journal of Biol ogical & Pharmaceutical Research. 2014; 5(6):527-534.
The spectrum of unknown compounds was
identified with library based on retention time and mass
spectra. The chemical structure, biological activity and
other sources of the identified compounds are depicted in
Table 1. These are: 1,2-benezenedicarboxylic acid, bis (2-
methylpropyl) ester already proven as cytoxic compound
(Sudha and Masilamani, 2012), hexadecanoic acid, methylester (Amanian and Brindha, 2013), 1,4-napththalenedione,
2-hydroxy-3-(3-methyl-2-butenyl)- (Oliveira et al ., 2002),
10,13-octadecadienoic acid, methyl ester (Hayashi et al .,
1998) and 9-octadecenoic acid (Z), methyl ester (Wei et
al ., 2011). Some of the above experiments were performed
with either single or combination of the bioactive
compounds. Our results confirm with cytotoxicity results.
These potent bioactive compounds not only showed
cytotoxicity or anticancer properties, they also showed
different biological properties. There is currently no reportavailable on cytotoxicity of 1, 2 benzenedicarboxylic acid,
butyl 2-methylpropyl ester.
Table 1. Al ternaria al ternata showing major bioactive compounds in ethanol extract and their biological activity
Sl. No. RT Compound name Biological activity Plant/microbes
1 20.743 1,2- benzene dicarboxylicacid, bis (2-ethylexyl) ester
Ant ifungal Certain Bacteria
Antibacterial agent Burkholderia cepicia
antimicrobial Edible mushrooms
antifouling Kedrostis foetidissima
cytotoxicity actinomycete Streptomyces avidinii
In vitro cytoxicity and
antioxidant
Centratherum punctatum
2 21.361 Hexadecanoic acid, methyl
ester
antibacterial activity Anthemis mixta and A. tomentos
antitumour Marine alga
In vitro cytoxicity,
antioxidant
Centratherum Punctatum
3 21.715 1,2 benzenedicarboxylic acid,
butyl 2-methylpropyl ester
antiviral Fern Acros tichumaureum
Ant imicrobial Edible mushoorms
4 22.561 1,4-Napththalenedione, 2-
hydroxy-3-(3-methyl-2-
butenyl)-
Anticancer cytotoxicity Tabebuia serratifolia
Antileishmanial, cellular
antioxidant, cytotoxic,
Mammea africana
5 23.027 10,13-octadecadienoic acid,
methyl ester
Anticancer activity Rice bran
Antifungal Pseudomonas
Antibacterial Spirulina platensis
Cytotoxicity Euphorbia kansui6 23.086 9-Octadecenoic acid (Z),
methyl es ter
Antifungal Azadirachta indica
Antioxidant properties flowers and roots of Pyrostegia venusta
Anticancer Peperomia pellucida Leaf
7 26.818 1,2-benzenecarboxylic acid Cytotoxicity,
antimicrobial
Ceratonia siliqua
fungitoxic and cytotoxic
activity
Plumbago zeylanica
Cytotoxicity, antioxidant
and antimicrobial
Euphorbia heterophylla
Fig 1. Habitat of Tabebuia argentea Fig 2. Pure culture of endophyte Al ternaria alternate
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Fig 3a. Normal mitotic phases, A) prophase B) Metaphase C)anaphase & D)Telophase
Fig 3b. Chromosomal, nucleolar and cellular abnormalities of polyphenolic fraction from endophytyic Alternaria
alternata of Tabebuia argentea , A) Arrest of cells at interphase with large nucleoli and binucleoli, B) Metaphase with
irregular chromosomal dis tribution and inter chromosomal gap, C) Chromosomal stickiness and bridge at anaphase,
D) Chromosomal stickiness and cell shrinkage at telophase, E) Cell shrinkage at prophase surrounded by normal
cells, F) Abnormal anaphase with vagrant chromosomes, G) Chromosomal fragmentation at late prophase & H)
Chromosomal separation at anaphase
Fig 4. Mitotic index of Al ternaria alternata extract on
Al li um cepa
Fig 5. Antiproliferative activity of Al ternaria alternata
treated Yeast cells
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Fig 6. Sequential process of cel l necrosis in yeast cells induced by A.alternata extract
Fig 7. DNA fragmentation, A) Treated yeast DNA and B)Untreated yeast DNA
Fig 8. GC-MS total ion chromatogram of Alternariaalternata showing 7 different bioactive metabolites
Fig 9. Structures of the 7 different bioactive metabolites in 6 fraction of Al ternaria alternata ethanol extract
1,2- benzene dicarboxylic acid, bis (2-ethylexyl) ester
Hexadecanoic acid, methyl es ter
1,2 benzenedicarboxylic acid, butyl 2-methylpropyl ester 1,4-Napththalenedione, 2-hydroxy-3-(3- methyl-2-
butenyl)-
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10,13-octadecadienoic acid, methyl ester9-Octadecenoic acid (Z), methyl ester
1, 2-benzenecarboxylic acid
CONCLUSION
Based on the above result, the endophytic fungus,
Alternaria alternata ethanol extract have proven to contain
biologically important compounds . In combination, these
bioactive compounds have shown strong antimitotic
activity in onion root meristematic cells and
antiproliferative activity and DNA fragmentation assay in
yeast cells. Further investigation is needed to identify the
exact candidate component compound that causes
cytotoxicity which can be used for treating cancer.
ACKNOWLEDGEMENT
The authors are grateful to Visvesvaraya
Technological University (VTU), Belgaum, Karnataka,
India for providing financial support for this investigation
under research grants activity (Ref No. VTU/Aca./2010-
11/A-9/11339 dated 7 December 2010). We also thank, Dr
Prasad Koka is a Ramalingaswami Fellow of the
Department of Biotechnology, Government of India, New
Delhi.
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