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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

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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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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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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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