See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/344486798

(Print) www.rjptonline.org 0974-360X (Online) Cytotoxic Potency of Bioactive

Metabolites from an Endophytic Fungi Pestaloptiopsis breviseta against MCF-7
and A 549 Cancer cell line...

Research  in  Research Journal of Pharmacy and Technology · October 2020

DOI: 10.5958/0974-360X.2020.00824.0

CITATIONS READS

0 15

2 authors, including:

Govindarajan Kathiravan
Ramakrishna mission vivekananda college, chennai, 600 004
50 PUBLICATIONS   486 CITATIONS   

SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Project View project

Synthesis of silver nanoparticles View project

All content following this page was uploaded by Vardhana Janakiraman on 06 October 2020.

The user has requested enhancement of the downloaded file.

 Research J. Pharm. and Tech. 13(10): October 2020

ISSN 0974-3618 (Print) www.rjptonline.org

0974-360X (Online)

RESEARCH ARTICLE

Cytotoxic Potency of Bioactive Metabolites from an Endophytic Fungi

Pestaloptiopsis breviseta against MCF-7 and A 549 Cancer cell lines
Vardhana Janakiraman*, Kathiravan Govindarajan2
1
Department of Biotechnology, Alpha Arts and Science College, University of Madras, Chennai- 600116.
2
Department of Botany, RKM Vivekananda College, Chennai.
*Corresponding Author E-mail: vardhana88@ymail.com
ABSTRACT:
Fungi are organisms which lack chlorophyll and play a major role in plants. Some cause the disease to the plant,
some live inside it as parasites and some as endophytes. Endophytic fungi play a very important role such that
some endophytes are host specific and others just reside in it. In this study, one of the frequently recurring
endophytic fungi isolated were identified as Pesatloptiopsis breviseta. The maximum absorbance was recorded
at 302 nm in the UV spectra. The peaks above 3000 - 3400 cm-1 in the FT-IR analysis, confirms the existence of
free OH, NH, and NO2 groups. The presence of peaks at 1900 - 2900 cm-1 confirms the presence of alkyl groups
attached to an aromatic moiety and confirms the presence of a double bond in the compound. The peak at 1965
cm -1 confirms the presence of unsaturation in the molecule. The NMR spectra confirm the presence of an
aromatic compound. GC-MS analysis showed the maximum m/z valve at 15.5 retention time followed by 23.38
and 18.92 correspond to the compounds Neoisolongifolane, hydroxy-, 1, 2-Benzenedicarboxylic acid, mono(2-
ethylhexyl) ester and 8-Octadecenoic acid, methyl ester respectively. From the MTT assay, the percentage of cell
viability based on IC50 value was observed. From the assay, it was proved that on using different concentrations,
the viability of cells was minimum at 100μl. Thus this study shows that the endophytic fungi can be served as a
source for the production of bioactive metabolites, with which the pharmaceutical products can be developed.

KEYWORDS: Endophytic fungi, Secondary metabolites, MTT assay, MCF-7, A549, Pestaloptiopsis
breviseta.

INTRODUCTION: Some Endophytes are widespread and others are highly

Fungi are organisms which lack chlorophyll and play a
major role in plants. Some cause the disease to the plant,
some live inside it as parasites and some as endophytes.
Endophytic fungi play a very important role such that
some endophytes are host specific and others just reside
in it. These endophytes will not damage the host but will
be a part of its life cycle. The plant produces bioactive
compounds which may be useful in various
pharmaceutical and industrial applications. Natural
products have been playing a major role in the search for
novel drugs for numerous illnesses including cancer [1-8].
Endophytes are now considered as an important
component of biodiversity. The distribution of
endophytic microflora differs with the host.
Received on 11.10.2019 Modified on 19.12.2019
Accepted on 21.02.2020 © RJPT All right reserved
Research J. Pharm. and Tech. 2020; 13(10):4683-4686
DOI: 10.5958/0974-360X.2020.00824.0
4683
specific to a single host in a single environment. These
endophytes protect their hosts from infectious agents and
adverse conditions by secreting bioactive secondary
metabolites[9-11]. The metabolic interactions of
endophytes with its host may favor the synthesis of
biologically active secondary metabolites. There has
been a great interest in endophytic fungi as potential
producers of the novel, biologically active products [12,13].
In this study, one of the frequently recurring endophytes
which were isolated from various plant sources was used
for the analysis of secondary metabolites and the solvent
extract was used to study the cytotoxic potency against
MCF-7 and A549 cell lines.
MATERIALS AND METHODS:
Collection of plant sample:
Healthy leaf samples were collected from Chennai and it
was rinsed in distilled water to remove the dust and the
leaves were dried in tissue paper. After the leaves are
 Research J. Pharm. and Tech. 13(10): October 2020

dried, the leaves are cut over the midrib region. The RESULT AND DISCUSSION:
alternative midrib pieces are taken to avoid colonization Identification of endophytic fungi:
of the same organism. The endophytic fungi isolated were identified as
Pesatloptiopsis breviseta (Sacc, 1949) based on the
Isolation of endophytic fungi: morphological and microscopical observations (Fig 1).
The surface sterilized leaves were trimmed using a Spots definite, circular, gray, pustules small, punctiform,
sterile blade and inoculated into plates containing sterile gregarious, subglobose, largely hypophyllous. Conidia 5-
Potato Dextrose Agar medium. The plates were celled, elliptic fusiform, 18-24 x 6-8μm, hardly
incubated at 28°C. constricted at septa, intermediate colored cells
olivaceous, concolorous, sometimes slightly contrasted,
Extraction of Secondary Metabolites: 12-16μm long, setulae 3, sometimes 2, 3-12μm long,
The test fungus used in the present study was grown in exteriorhyaline cells short, apical cells, conic. Cylindric,
2L Erlenmeyer flasks containing 500ml MID medium the basal cells broad conic, Pedicles short.
supplemented with 1g of soytone L- 1. Three mycelial
agar plugs (0.5cm) were used as inoculum. The organism
was grown at 24±2°C statically for 3-4 weeks. After
incubating the culture for 3–4 weeks, the culture filtrate
was passed through four-layered cheesecloth. The fungal
mycelia were collected and it was fixed in
Glutaraldehyde solution and stored for subsequent
studies. The culture fluid was extracted with two equal
volumes of Methylene chloride or DCM and the organic
A B
phase was evaporated to dryness under reduced pressure Fig 1 Culture and Spores of Pestaloptiopsis breviseta
at 35°C.
Extraction of secondary metabolites:
Cytotoxic study: The test fungal colonies were inoculated in M1D
The cytotoxic study was carried out using the MTT medium. The inoculated flask cultures of the fungal
assay[14]. MCF-7 (Breast cancer) and A549 (Lung species were harvested after 3-4 weeks when it has
cancer) were procured from National Centre for Cell attained the maximum growth. The culture filtrate and
Science, Pune, India. The cell lines were grown the mycelia mat separated were extracted using
adherently and maintained in DMEM containing 10% dichloromethane solvent.
fetal calf serum, and 1% antibiotic solution containing
penicillin and streptomycin at 37°C in 5% CO2 at 37ºC UV Spectroscopic Analysis:
until the monolayer was subconfluent[15]. The cells were The UV spectra 200–400nm using UV-VIS
plated separately in 96 well plates at a concentration of 1 spectrophotometer was recorded. The maximum
× 105 cells/well. After 24 h, cells were washed twice absorbance was recorded at 302nm (Fig 2)
with 100μl of serum-free medium and starved for an
hour at 37oC. After starvation, cells were treated with a 0.6

different test compound for 24 hrs. At the end of the

0.5
treatment period, the medium was aspirated and serum-
free medium containing MTT (0.5mg/ml) was added and 0.4
incubated for 4 h at 37ºC in a CO2 incubator. The MTT
containing medium was then discarded and the cells 0.3
Abs

were washed with PBS (200μl). The crystals were then

dissolved by adding 100μl of DMSO and this was mixed 0.2

properly by pipetting up and down.

0.1
Spectrophotometrical absorbance of the purple-blue
formazan dye was measured in a microplate reader at 0.0
570nm (Biorad 680). Cytotoxicity was determined using 300 400 500 600 700 800

Graph pad prism5 software. nm

Fig 2 UV Spectral analysis of the endophytic fungal extract
% Cell viability = A570 of treated cells / A570 of control
FT-IR spectral analysis:
cells × 100%.
The FT-IR spectra were observed both in the fingerprint
region and functional group region (Fig 3). The peaks
above 3000 - 3400 cm-1 confirms the existence of free

4684
 Research J. Pharm. and Tech. 13(10): October 2020

OH, NH and NO2 groups. The presence of peaks at 1900
- 2900 cm-1 confirms the presence of alkyl groups
attached to an aromatic moiety and confirms the
presence of a double bond in the compound. These peaks
reveal the SP3 hybridized stretching vibration of alkyl
groups attached to an aromatic moiety. The peak at 1965
1.60
cm -1 confirms the presence of unsaturation in the
molecule. The peaks at 1000 - 1500 cm-1 in the
fingerprint region is due to the out of plane (OOP)
bending vibration, further confirms the presence of
aromatic nuclei.

1.5
PSM 1718
1.4 1702

1.3

1.2

1.1

1.0

0.9

0.8 1519 1347

A 1226 1065
1312
0.7 2922 1269
3403 1382 1087
0.6
2953 1402 857

0.5
1470 1152
2852 1606
0.4 1965 1494
998 812 666
0.3 3063 1014 790 696 520
730
904
0.2 3835 2318

0.1

0.00
4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 450.0
cm -1

Fig 3: FT-IR Spectra of the solvent extract of the fungi

NMR Spectral Analysis:
NMR Analysis of the solvent extract was carried out
using the Carbon and Hydrogen Spectra. From the
carbon spectra, it was found that the presence of peaks at
150 – 160 δ indicates the aromatic nuclei in the
compound. Peaks at 160- 165 δ indicates the presence of
a carbonyl group as it is evident from C - 13 spectra.
From the proton spectra, it was evident that the peaks at
1 δ – 3 δ- alkyl groups attached to an aromatic ring. 3.5-
4.5 δ- Benzylic CH and Omethoxy groups. 6- 6.5 δ-
unsaturation in the molecule. 7 – 8 δ- aromatic group.
GC- MS SPECTRA ANALYSIS:
The maximum m/z valve at 15.5 retention time followed
by the peak in 23.38 and 18.92 which supports the
molecular weight of the substance which is not found in
the GC- MS spectra of other retention time. The peaks
correspond to the compounds Neoisolongifolane,
hydroxy-, 1,2-Benzenedicarboxylic acid, mono(2-
ethylhexyl) ester and 8-Octadecenoic acid, methyl ester
respectively. (Fig 4).
From the above results, it could be interpreted that the
extracted compound from Pestalotiopsis breviseta
should be an aromatic compound with the presence of
one or more carbonyl group, benzylic group, O- methoxy
group, and the aliphatic side chain should contain two or
more alkyl group with one or more units of unsaturation.

Fig 4: GC-MS Spectra of the solvent extract

4685
 Research J. Pharm. and Tech. 13(10): October 2020

CYTOTOXIC ASSAY: from microbes is a cost-effective and eco-friendly

The solvent extracted metabolite samples were treated
on human breast cancer (MCF-7) and lung cancer
(A549) cell lines. The samples were taken in different
concentrations from 6μl to 100μl. The MTT assay was
carried out for 24 hrs and 48 hrs interval time (Table 1)
and the percentage of cell viability based on IC50 value
was observed using graph pad prism 5 software. From
the assay, it was proved that on using different
concentrations, the viability of cells was minimum at
100μl (Fig 5).
method.
ACKNOWLEDGMENT:
The authors express sincere thanks to the Department of
Science and Technology (INSPIRE), New Delhi, India,
for providing necessary funds to carry out the research
work. The Authors also thank CNST Anna University
for providing the instrumentation facility.
CONFLICT OF INTEREST:
The authors have no conflict of interest to declare.

REFERENCES:
1. Butler MS, Newman DJ (2008) Mother Nature’s gifts to diseases
of man: the impact of natural products on anti-infective,
anticholestemics and anticancer drug discovery. Prog Drug Res 65:
1, 3-44.
2. Molinari G (2009) Natural products in drug discovery: present
status and perspectives. Adv Exp Med Biol 655: 13-27.
3. Li JW, Vederas JC (2009) Drug discovery and natural products:
A B end of an era or an endless frontier? Science 325: 161-165.
4. Daniel AD, Sylvia U, Ute R (2012) A Historical Overview of
Natural Products in Drug Discovery. Metabolites 2: 303-336.
5. da Rocha AB, Lopes RM, Schwartsmann G (2001) Natural
products in anticancer therapy. Curr Opin Pharmacol 1: 364-369.
6. Cragg GM, Newman DJ (2000) Antineoplastic agents from natural
sources: achievements and future directions. Expert Opin Investig
Drugs 9: 2783-2797.
7. Mishra BB, Tiwari VK (2011) Natural products: an evolving role
in future drug discovery. Eur J Med Chem 46: 4769-4807.
8. Mondal S, Bandyopadhyay S, Ghosh MK, Mukhopadhyay S, Roy
S, et al. (2012) Natural products: promising resources for cancer
C drug discovery. Anticancer Agents Med Chem 12: 49-75.
Fig 5: (A) Vero (B) MCF -7 and (C) A549 cell line treated with 100 9. Azevedo, J.L., Pereira, J.O., and Araújo, W.L. 2000. Endophytic
μl of the solvent extract microorganisms: a review on insect control and recent advances on
tropical plants. Electronic J Biotechnol. 3(1): 40-65.
Table 1 Percentage of cell viability at different concentrations of 10. Carroll, G.C., and Carroll, F.E. 1978.Studies on the incidence of
samples in MCF-7 and A549 cell line at 24 hr and 48 hrs. coniferous needle endophytes in the Pacific Northwest. Can J Bot.
The concentration of MCF 7 A549 56: 3032- 43.
the sample 11. Strobel, G.A., 2003. Endophytes as a source of bioactive products.
Time 24 hrs 48 hrs 24 hrs 48 hrs Microbe Infect 5(6) : 535 44.
6 μg 89 90 93 94 12. Schulz B, Boyle C, Draeger S, Rommert AK, Krohn K. 2002 –
12 μg 82 80 90 81 Endophytic fungi: a source of novel biologically active secondary
25 μg 69 68 73 70 metabolites. Mycological Research. 106, 996–1004.
50 μg 63 60 54 51 13. Strobel G, Daisy B. 2003 – Bioprospecting for microbial
100 μg 43 47 50 46 endophytes and their natural products. Microbiology and
Molecular Biology Reviews. 67(4), 491–502.
DISCUSSION: 14. Mosmann T. 1983. Rapid colorimetric assay for cellular growth
and survival: application to proliferation and cytotoxicity assays.
In the present investigation, the fungal secondary Journal of Immunological Methods. 65: 55-63.
metabolites were extracted using dichloromethane 15. Rose DP and Connolly JM (1990). Effects of Fatty Acids and
solvent and the extract was subjected to spectral analysis Inhibitors of Eicosanoid Synthesis on the Growth of a Human
to find out the group of compounds present in it. The Breast Cancer Cell Line in Culture. Cancer Research. 50: 7139-
7144.
crude solvent extract was treated on MCF-7 and A549
cancer cell lines. It was clearly evidenced that at the
concentration of 100μg/ml of the sample, the cytotoxic
potent was more effective. Thus this study shows that the
endophytic fungi can be served as a source for the
production of bioactive metabolites, with which the
pharmaceutical products can be developed. Also, this
study shows that the production of bioactive metabolites

4686

View publication stats