BioNanoScience

https://doi.org/10.1007/s12668-019-00631-1

Biosynthesis of Silver Nanoparticles from Endophytic Fungi, and its

Cytotoxic Activity
Vardhana Janakiraman 1 & Kathiravan Govindarajan 2 & Magesh C R 3

# Springer Science+Business Media, LLC, part of Springer Nature 2019

Abstract
Nanoparticle research is currently an area of intense scientific interest due to a wide variety of potential applications in biomed-
ical, optical, and electronic fields. The biological synthesis of nanoparticles includes the synthesis of nanoparticles using
microbes which have the capability to absorb the nanoparticles that exist in various forms. In the biosynthesis of metal nano-
particles by a fungus Botryodiplodia theobromae, the fungus mycelium is exposed to the metal salt solution. The change in color
of the medium indicates the synthesis of silver nanoparticles. The synthesized AgNPs were structurally characterized by UV-
visible spectroscopy, scanning electron microscope (SEM), and Fourier-transform infrared spectroscopy (FTIR) analysis. The
size of the silver nanoparticles was ranging from 62.77 –103 nm in the fungal mat and 66.75–111.23 nm in the cell filtrate
respectively with the existence of free OH and NH groups, aromatic CH stretch, C–C stretch, unsaturation in the molecule, and
C–Cl stretch. The cytotoxic efficiency of the AgNPs on human MCF7 breast cancer cell lines and A549 human lung cancer cell
lines was appraised by cell viability assay. The synthesized AgNPs inhibited the propagation of cells at an IC50 concentration of
100 μg/mL. Thus, this study reveals that the green synthesis is an eco-friendly method for production of AgNPs from endophytic
fungi which provided a powerful anti-proliferative action on MCF-7 and A549 cell lines, suggesting them as a novel chemo-
therapeutic agent against human breast and lung cancers.

Keywords Endophytic fungi . Botryodiplodia theobromae . Silver nanoparticle . SEM . MCF-7 . A549 . MTT assay .
Cytotoxicity

1 Introduction scientific interest due to a wide variety of potential applica-

Nanoparticles (NPs) showed a great biomedical application,
not only in the pharmaceutical sector but also in novel diag-
nostic and therapeutic approach [1] particularly in the treat-
ment of human cancers. Commonly, owing to their relative
mobility and smaller size, NPs have fairly advanced uptake
intracellularly when compared to larger particles as biological
targets. Nanoparticle research is currently an area of intense
* Vardhana Janakiraman
vardhana88@ymail.com
1
Department of Biotechnology, SRM Arts and Science College,
University of Madras, Kattankulathur, Chennai 603203, India
2
Department of Botany, RKM Vivekananda College, University of
Madras, Chennai 600004, India
3
Ministry of Environment, Forest and Climate change, National
Museum of Natural History, New Delhi, India
tions in biomedical, optical, and electronic fields [2–4].
Thus, nanomaterials are, nowadays, successfully manipu-
lated to develop an innovative drug-delivery method which
could successfully solve the issue of poor water solubility of
existing most promising anticancer drugs. Moreover, the
AgNPs are the emergent field of nano-products that got high
attention in the nanomedicine owing to its tremendous thera-
peutic prospective in handling a wide array of maladies such
as AIDS, diabetes, hepatitis B, cancer, and HIV, in the very
near future. Various methods were used for the AgNPs syn-
thesis like chemical, radiational, electrochemical, and photo-
chemical methods. Biological methods are presently gaining
importance due to their cheaper, environmentally friendly ap-
proach. The usage of non-toxic chemicals for the synthesis
adds to this point [5].
The biological technique includes the synthesis of nanopar-
ticles using microbes which have the capability to absorb the
nanoparticles that exist in various forms. It can be classified
into intracellular and extracellular synthesis according to the
location where nanoparticles are formed [6, 7].

In the biosynthesis of metal nanoparticles by a fungus, the
fungus mycelium is exposed to the metal salt solution. That
prompts the fungus to produce enzymes and metabolites for
its own survival. In this process, the toxic metal ions are re-
duced to the non-toxic metallic solid nanoparticles through the
catalytic effect of the extracellular enzyme and metabolites of
the fungus. AgNPs were synthesized in the form of a film or
produced in solution or accumulated on the surface of its cell
when fungi were employed to the salt solution [8–11].
Breast cancer continues to be a threatening ailment
worldwide. Globally, it is the most communal cancer type,
a leading cause of mortality among women.
Approximately one-third of the women population with
breast cancer develop metastasis, which enhances the
mortality rate [12–14]. Worldwide in 2012, lung cancer
occurred in 1.8 million people and resulted in 1.6 million
deaths [15]. This makes it the most common cause of
cancer-related death in men and the second most common
in women after breast cancer [16].
In this study, we have reported the biological synthesis of
silver nanoparticles using an endophytic fungi Botryodiplodia
theobromae and its cytotoxic effects against breast and lung
cancer cell lines. Earlier, we have reported the synthesis of
silver nanoparticles by an endophytic fungi Pestalotiopsis
pauciseta isolated from the leaves of Psidium guajava Linn
[11]. We also reported the synthesis of silver nanoparticles
from B. theobromae isolated from a weed Euphorbia hirta
and its anticancer activity against human colon cancer cell line
HT-29 [17]. Based on this report, we prove that the synthesis
of silver nanoparticles through biological method is a novel
approach for producing bioactive AgNPs with effective anti-
proliferative action against MCF-7 and A549 human breast
and lung cancer cell line respectively.
2 Materials and Methods
2.1 Collection of Leaf Sample
The healthy leaves from the plant Psidium guajava, the com-
mon guava, yellow guava, or lemon guava (known as Goiaba
in Portuguese and Guayaba in Spanish), which belong to the
family Myrtaceae, have been collected for the present study
from Pallavaram, Chennai.
2.2 Isolation of Endophytes
Fresh leaf samples from the plants are collected and were
placed in sterile plastic bags and returned to the laboratory
and were processed within 24 h of collection for the isolation
of endophytic fungi.
BioNanoSci.
2.3 Surface Sterilization of Plant Material
Samples were cleaned under running tap water and then air-
dried. Surface sterilization was carried out according to the
procedure of Suryanarayan and Thennarasan [3] with slight
modifications. Leaves were surface sterilized by immersion in
70% ethanol for 1 min, 0.1% mercuric chloride solution for
3 mins, and sterile distilled water for 1 min two times. The
surface-sterilized leaves were cut into small pieces using a
sterile blade and placed on sterile potato dextrose agar plates
amended with 120 mg/L of chloramphenicol. The inoculation
was carried under laminar wood chamber and after inocula-
tion, the plates were labeled accordingly and incubated for
25 °C.
2.4 Biosynthesis of Silver Nanoparticles
The fungus was inoculated (app. 3 mm in diameter) in
250-mL Erlenmeyer flasks containing 100 mL potato dextrose
broth at 25 °C in an orbital shaker at 120 rpm for 48 h. After
incubation, mycelial biomass was separated by filtration,
washed with sterile distilled water to remove the traces of
media components, resuspended in 100 mL distilled water,
and incubated at 25 °C. After 24 h, the suspension was filtered
through Whatman filter paper. The cell filtrate was treated
with AgNO3 solution (1 mM) and incubated at room temper-
ature in dark condition. The wet fungal biomass was mixed
with 100 mL aqueous solution of 1 mM AgNO3 and was
placed in a 100-rpm rotating shaker for 72 h.
2.5 Characterizations of AgNPs
AgNPs synthesis was confirmed by UV-160 V-visible spec-
troscopy. The characterization studies on morphology, size,
and the composition of nanoparticles were performed by
FTIR and SEM.
2.5.1 Fourier-Transform Infrared Spectroscopy
Characterization of AgNPs was carried out by FTIR (Perkin-
Elmer FTIR-1600, USA) in the range 500–4000 cm−1 at a
resolution of 4 cm−1 [18].
Change in colour of the fungal mat Change in colour of the cell filtrate from
colourless to brown Indicates the nanoparticle
synthesis
Fig. 1 Synthesis of silver nanoparticles from B. theobromae
BioNanoSci.

Fig. 2 UV spectra of cell

filtrate—B. theobromae control— control
without the presence of 0.8 AgNO3
nanoparticles, AgNO3—with the
presence of nanoparticles

0.6

0.4

Abs
0.2

0.0
300 400 500 600 700 800
nm

2.5.2 Scanning Electron Microscope
The size and surface morphology of the biosynthesized silver
nanoparticles using the filtrate of fungal biomass was charac-
terized using scanning electron microscopy analysis.
2.6 Cell Viability Assay
MCF-7 (breast cancer) and A549 (lung cancer) were procured
from the National Centre for Cell Science, Pune, India. The
cell lines were grown adherently and maintained in DMEM
containing 10% fetal calf serum and 1% antibiotic solution
containing penicillin and streptomycin at 37 °C in 5% CO2
at 37 °C until the monolayer was sub-confluent [19].
The cells were plated separately in 96 well plates at a con-
centration of 1 × 105 cells/well. After 24 h, cells were washed
twice with 100 μL of serum-free medium and starved for an
hour at 37 °C. After starvation, cells were treated with a dif-
ferent test compound for 24 h. At the end of the treatment
period, the medium was aspirated and serum-free medium
containing MTT (0.5 mg/mL) was added and incubated for
4 h at 37 °C in a CO2 incubator [20].

1.60

1.5 BNP
1.4

1.3
3377

1.2

1.1

1.0 1644

0.9

0.8 726
Absorbance
0.7

0.6

0.5

0.4

0.3
1965
2139
0.2

0.1

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

Fig. 3 FTIR band peaks of oxidized biomolecules in the AgNPs synthesis. FTIR spectra of the cell filtrate of B. theobromae
 BioNanoSci.

a b c
Fig. 4 Scanning electron microscopy image of AgNPs by the fungal mat. Silver nanoparticles produced by the fungal mat of B. theobromae. SEM
images at a magnification of a × 5.00 k, b × 250, and c × 25.0 k. c Showing the silver nanoparticle size ranging 62.77–103 nm

The MTT containing medium was then discarded and the characterization of the spores was noted using the standard my-
cells were washed with PBS (200 μL). The crystals were then cological manuals. The identified fungal endophyte was found to
dissolved by adding 100 μL of DMSO and this was mixed be Botryodiplodia theobromae (Lasiodiplodia theobromae).
properly by pipetting up and down. Spectrophotometrical ab-
sorbance of the purple-blue formazan dye was measured in a
3.1.1 Botryodiplodia theobromae (Pat. Ponnappa, 1970;
microplate reader at 570 nm (Bio-Rad 680). Cytotoxicity was
Tandon & Verma, 1964; Petrak 1923)
determined using GraphPad Prism 5 Software.

%cell viability ¼ A570 of treated cells=A570 of control cells  100%:
3 Results and Discussion
3.1 Identification of Endophytic Fungus
Macroscopic appearance of the fungal colonies, their morpho-
logical appearance, the mechanism of spore production, and
The fungal mycelium will appear immersed or superficial,
branched, and septate. The mycelia color is initially white; when
mature, it becomes dark chocolate brown. Conidiomata is pyc-
nidial, usually papillate with prominent ostioles, of size 160–
185 μm and globose in structure. The conidiogenous cells are
holoblastic and annellidic. Conidia hyaline when young looks
ellipsoid to oblong, thick walled with granular contents. Later,
the conidia become two celled and the color changes from cin-
namon to dark brown with size ranging from 23 to 27 × 10–
13 μm with longitudinally running striations.

a b c
Fig. 5 Scanning electron microscopy image of AgNPs by cell filtrate. Silver nanoparticles produced by the cell filtrate of B. theobromae. SEM images at
a magnification of a × 10.0 k, b × 15.0 k, and c × 30.0 k. c Showing the silver nanoparticle size ranging 66.75–111.23 nm
BioNanoSci.
a b
Fig. 6 Cytotoxic effect of AgNPs on MCF 7 and A549 cells. Cytotoxicity of silver nanoparticle synthesized extract from B. theobromae. 100 μg/mL of
the sample on a Vero cell line, b MCF-7 cell line, and c A-549 cell line
3.2 Biosynthesis of Silver Nanoparticles
by B. theobromae
Both the fungal mat and the cell filtrate were treated with
1 mM AgNO3, and it was incubated in room temperature
in dark. The change in color was observed in both the
fungal mat and the cell filtrate of the test fungi. The color
was changed from pale yellow to dark brown (Fig. 1). The
fungal cell filtrate after addition of aqueous AgNO 3
(1 mM) was subjected to optical measurements by UV-
Vis spectrophotometer; this analysis showed an absor-
bance of the peak at 410–420 nm (Fig. 2), which was
specific for the silver nanoparticles.
3.3 Characterization of Synthesized AgNPs
The FTIR measurements of the cell filtrates (Fig. 3)
showed the presence of peaks at 3377 and indicate the
Fig. 7 Cytotoxicity of samples in
MCF-7 and A549 cell line at 24 h
and 48 h at the concentration of
100 μg/mL
c
existence of free OH and NH groups. The peak at 2139
indicates the presence of aromatic CH stretching. The
peak at 1644 indicates the presence of C–C stretching.
The peak at 1965 confirms the presence of unsaturation
in the molecule. The peak at 726 indicates the presence of
C–Cl stretching. Similar results were reported by Nirjanta
Devi et al in the synthesis of silver nanoparticles by an
endophytic fungus [21].
Fungal species were inoculated and incubated in the
silver nitrate amended media and it was characterized by
scanning electron microscope for the presence of silver
nanoparticles. It was found that the cell filtrate and the
fungal mat of the fungi were able to synthesize silver
nanoparticles. The size of the nanoparticle produced from
the fungal mat (Fig. 4) of B. theobromae was ranging
from 62.77–103 nm. The cell filtrate of B. theobromae
(Fig. 5) produced silver nanoparticle of size ranging from
66.75–111.23 nm.

3.4 Cytotoxicity of the Samples on MCF-7 and A549
Cancer Cell Lines
The silver nanoparticle synthesized by the fungal cell fil-
trates were treated on MCF-7, A549 cell lines, and Vero
cell lines (Fig. 6). Dose-Dependent cytotoxicity was ob-
served in AgNPs-treated cancer cells, as the concentration
of AgNPs increases the cell cytotoxicity also significantly
increases. The percentage of cell viability based on IC50
value was observed using Graph Pad Prism 5 Software for
24 h and 48 h at various concentrations from 6 μg/mL,
12 μg/mL, 25 μg/mL, 50 μg/mL, and 100 μg/mL in
MCF-7 and A549 cell lines. The assay clearly evidenced
that on using different concentrations of the samples rang-
ing from 6–100 μg/mL, cytotoxicity was observed when
100 μg/mL (Fig. 7) of the sample was used.
4 Conclusion
The biosynthesis of nanoparticles by microorganisms is
quick, consumes less time; it provides satisfactory biosyn-
thesis of nanoparticles and the whole process is very
cheap and cost-effective without the involvement of haz-
ardous chemicals. Thus, the study exhibits that the silver
nanoparticles produced by B. theobromae endophytic fun-
gi from Psidium guajava were potent for effective drug
delivery. It is known that the nanoparticles are so small in
size that they can pass through the cell membrane easily.
This property is exploited to treat diseases like cancer
which is a fatal disease; chemotherapies which are used
to treat cancer have many side effects, so there is a need
to find some other alternative which could treat it without
causing side effects. However, further research involving
animal models is required to validate the biomedical
applications.
Acknowledgments The authors would like to thank the CNST, Anna
University, Chennai, for providing the instrumentation facility.
Funding Information The authors express their sincere thanks to the
Department of Science and Technology (INSPIRE), India, for providing
necessary funds to carry out the research work.
Compliance with Ethical Standards
Conflict of Interest The authors declare that they have no conflict of
interest.
References
1. Caruthers, S. D., Wickline, S. A., & Lanza, G. M. (2007). Nano
technological applications in medicine. Current Opinion in
BioNanoSci.
Biotechnology, 18, 26–30. https://doi.org/10.1016/j.copbio.2007.
01.006.
2. Hewakuruppu, Y. L., Dombrovsky, L. A., Chen, C., Timchenko, V.,
Jiang, X., Baek, S., & Taylor, R. A. (2013). Plasmonic Bpump–
probe^ method to study semi-transparent nanofluids. Applied
Optics, 52(24), 6041–6050.
3. Suryanarayanan, T. S., & Thennarasan, S. (2004). Temporal varia-
tion in endophyte assemblages of Plumeria rubra leaves. Fungal
Diversity., 15, 197–204.
4. Taylor, R. A., Otanicar, T. P., Herukerrupu, Y., Bremond, F. R. G.,
Hawkes, E. R., Jiang, X., & Coulombe, S. (2013). Feasibility of
nanofluid-based optical filters. Applied Optics, 52(7), 1413–1422.
5. Mulder, W. J., & Fayad, Z. A. (2008). Nanomedicine captures car-
diovascular disease. Arteriosclerosis, Thrombosis and Vascular
Biology, 28(5), 801–802. https://doi.org/10.1161/ATVBAHA.108.
165332.
6. Mann, S. (2001). Biomineralization: principles and concepts in
bioinorganic materials chemistry. Oxford: Oxford University
Press.
7. Senapati, S., Mandal, D., & Ahmad, A. (2004). Fungus mediated
synthesis of silver nanoparticles: a novel biological approach.
Indian Journal of Physics A., 78(1), 101–105.
8. Bhainsa, K. C., & D'Souza, S. F. (2006). Extracellular biosynthesis
of silver nanoparticles using the fungus Aspergillus fumigatus.
Colloids and Surfaces B., 47(2), 160–164.
9. Jain, N., Bhargava, A., Majumdar, S., Tarafdar, J. C., & Panwar, J.
(2011). Extracellular biosynthesis and characterization of silver
nanoparticles using Aspergillus flavus NJP08: a mechanism per-
spective. Nanoscale., 3(2), 635–641.
10. Schulz, B., Wanke, U., Draeger, S., & Aust, H. J. (1993).
Endophytes from herbaceous plants and shrubs: effectivenses of
surfuse sterilization methods. Mycological Research, 97, 1447–
1450.
11. Vardhana, J., & Kathiravan, G. (2015). Biosynthesis of silver nano-
particles by endophytic fungi Pestaloptiopsis pauciseta isolated
from the leaves of Psidium guajava Linn. International Journal of
Pharmaceutical Science Review and Research., 31(1), 29–31.
12. Parkin, D. M., Pisani, P., & Ferlay, J. (1993). Estimates of the
worldwide incidence of eighteen major cancers in 1985.
International Journal of Cancer, 54, 594–606. https://doi.org/10.
1002/ijc.2910540413.
13. Asha Rani, P. V., Mun, G. L. K., Hande, M. P., & Valiyaveettil, S.
(2009). Cytotoxicity and genotoxicity of silver nanoparticles in hu-
man cells. ACS Nano, 3, 279–290. https://doi.org/10.1021/
nn800596w.
14. Russo, J., Yang, X., Hu, Y., Bove, B., Huang, Y., Silva, I. D. C. G.,
Tahin, Q., Wu, Y., Higgy, N., Zekri, A., & Russo, I. (1998).
Biological and molecular basis of human breast cancer. Frontiers
in Bioscience, 3, 944–960.
15. World Cancer Report 2014. World Health Organization. 2014. pp.
Chapter 5.1. ISBN 92-832-0429-8.
16. World Cancer Report 2014. World Health Organization. 2014. pp.
Chapter 1.1. ISBN 92-832-0429-8.
17. Janakiraman, V., Jenifer, M., & Ramarajan, S. (2018). Anti diabetic
and anti cancer effects of silver nanoparticles synthesised from
Botryodiplodia theobromae- an endophytic fungi isolated from
Euphorbia hirta – a weed. International Journal of
Pharmaceutical Science Review and Resesrch., 53(1), 72–76.
18. Raheman, F., et al. (2011). Silver nanoparticles: novel antimicrobial
agent synthesized from an endophytic fungus Pestaloptia sp. iso-
lated from leaves of Syzygium cumini (L). Nano Biomedical
Engineering, 3(3), 174–178. https://doi.org/10.5101/nbe.v3i3.
p174-178.
19. Rose, D. P., & Connolly, J. M. (1990). Effects of fatty acids and
inhibitors of eicosanoid synthesis on the growth of a human breast
cancer cell line in culture. Cancer Research., 50, 7139–7144.
BioNanoSci.
20. Mosmann, T. (1983). Rapid colorimetric assay for cellular growth
and survival: application to proliferation and cytotoxicity assays.
Journal of Immunological Methods., 65, 55–63.
21. Nameirakpam, N. D., Dheeban, S. P., & Sutha, S. (2012).
Biomimetic synthesis of silver nanoparticles from an endophytic
fungus and their antimicrobial efficacy. International Journal of
Biomedical and Advance Research, 3(5), 409–415.
Publisher’s Note Springer Nature remains neutral with regard to jurisdic-
tional claims in published maps and institutional affiliations.