Biofabrication of gold nanoparticles using Ganoderma lucidum andtheir cytotoxicity against human colon cancer cell line (HT-29)
D ELUMALAI1, T Y SUMAN2,3,*, M HEMAVATHI4, C SWETHA1, R KAVITHA5, C ARULVASU5
and P K KALEENA6
1PG Department of Zoology, Pachaiyappas College for Men, Kanchipuram 631501, India2School of Civil, Environmental and Chemical Engineering, Changwon National University, Changwon 51140, Republic
of Korea3Ecotoxicology Division, Centre for Ocean Research (DST-FIST Sponsored Centre), Sathyabama Institute of Science and
Technology, Chennai 600119, India4Department of Zoology, Arignar Anna Govt. Arts & Science College for Women, Vellore 632513, India5Department of Zoology, Guindy Campus, University of Madras, Chennai 600 025, India6Department of Zoology, Presidency College, Chennai 600005, India
*Author for correspondence ([email protected])
MS received 15 September 2020; accepted 24 January 2021
Abstract. Ganoderma lucidum (G. lucidum) is a medicinal mushroom, which is rich in flavonoids as well as
polyphenols and contains triterpenes, ganoderic acid. Thus, this research examined the biosynthesis of gold nanoparticles
(GNPs) using G. lucidum fruit body and their cytotoxicity, cell morphology and apoptosis detection against HT-29 colon
cancer cell line. The study results revealed the occurrence of GNPs, as evidenced by ultraviolet–visible spectroscopy
(UV–vis spectroscopy; 520 nm). The X-ray diffraction results show that the synthesized Au-NPs are crystalline.
According to field-emission scanning electron microscope, transmission electron microscopy and atomic force micro-
scope, the synthesized GNPS exhibited shapes such as spherical, oval and irregular in shape and the size ranges in
between 1 and 100 nm. G. lucidum biofabricated GNPs showed inhibited HT-29 colon cancer cell line with an efficacy
IC50 of 84.58 lg ml–1. The results indicated that the apoptotic effects were increased on cells based on the dose-dependent
concentration of GNPs.
Keywords. G. lucidum; biofabrication; gold nanoparticles; cytotoxicity.
1. Introduction
Nanobiotechnology is one of the most exciting fields used for
synthesizing nanoparticles of various types [1,2]. It is one of
the critical areas of experimental medicine with many appli-
cations [3]. The nanoparticles produced by nanotechnology
can be employed to detectmany diseases at themolecular level
[4,5]. Their possible applicability in biotechnology and
biomedical sciences has made its way to various dimensions
[6]. Metal nanoparticles have vast potential for a range of
applications like drug delivery, anticancer, medical imaging
and antimicrobial activity in recent years [7–9].
Researchers in chemical and biological fields have recently
been attracted to gold nanoparticles (GNPs), due to its unique
properties.Numerous findings statedmuch attentionwas paid to
controlling the shape and size of GNPs during synthetic proce-
dures [10]. Traditionally, nanoparticles are made of chemical
and physicalmethods [11]. Furthermore, high energy and scarce
usage of chemical reagents have expressed fears in traditional
methods about the impact of environmental threats [12,13].
Biosynthetic processes are cost-effective and environmentally
friendly, contrary to physical and chemical methods, making
them a better option for metal nanoparticle synthesis [14,15].
Myconanotechnology is a recent green synthesis technique
for nanoparticle synthesis, including mushrooms, yeast and
mould [16]. Endophytic organisms can synthesize antioxidant,
antibacterial, antiviral, insulin-mimetic properties and immuno-
suppressant compounds [17]. For metallic nanoparticles
synthesis mushrooms’ use mycelia, fruiting body [16]. It is
harder to identify nanoparticle synthesis enzymes, proteins,
polysaccharides, amino acids and vitamins [18,19]. Samples
of mushrooms synthesized metal nanoparticles about less
than 75 nm [16]. Few mushrooms were used for GNPs
synthesis, such as Lentinula edodes [20], Flammulina velu-tipes [21], Phaenerochaete chrysosporium (white-rot fungus)
[22], Pleurotus florida [23,24], Pleurotus sapidus [25].
G. lucidum grows at the base and stumps of deciduous trees.G. lucidum possess immunomodulatory, antitumour and car-
diovascular activity [26–28]. The polysaccharide compounds,
triterpenes and ganoderic acid isolated from G. lucidum have
Bull. Mater. Sci. (2021) 44:132 � Indian Academy of Scienceshttps://doi.org/10.1007/s12034-021-02435-0Sadhana(0123456789().,-volV)FT3](0123456789().,-volV)
pharmacological activities [29,30]. This study aimed to syn-
thesize the GNPs usingG. lucidum fruit body and no published
work on the synthesis of GNPs using G. lucidum fruit body is
available. Therefore, the first attempt is made to synthesize
GNPs from the fruit body ofG. lucidum and their cytotoxicity
against the HT-29 colon cancer cell line.
2. Materials and methods
2.1 Mushroom sample
G. lucidum was collected in Kambarajapuram, Kanchipuram,
Tamilnadu, India. Following collection, Dr Vignesh, Depart-
ment ofBotany, Pachaiyappa’sCollege forMen,Kanchipuram,
identified the specimen. The voucher specimen was numbered
and stored inourbotanyherbarium.The fruit bodyofG. lucidumhasbeen thoroughlywashedwithdeionizedwater (DW)andair-
dried. The samples were then chopped into a fine powder and
placed in a dry and dark place until they were used.
2.2 Extract preparation and synthesis of GNPs usingthe fruit body of G. lucidum
The extractwas 5 g ofG. lucidum and 100ml of deionizedwater
in a 250ml Erlenmeyer flask, and themixture boiled for 20min
at 60�C. It was filtered by nylon mesh, followed by the hydro-
philic filter ofmillipore (0.22m) and stored for additional study.
Aqueous chloroauric acid (1 mM) solution was taken and used
for GNPs. In 34ml of 1mM, chloroauric acid aqueous solution,
3 ml of fruit body ofG. lucidum aqueous extract was added for
reduction into GNPs and incubated for 60 min at 28�C [31].
After 30 min, the colour of the solution changed to light purple.
ForUV–visible analysis, amixturewas taken after 30minwhen
light violet, and after 60min final readingwas taken. For further
use, the final colloidal solution was kept at 4�C.
2.3 Characterization of GNPs
GNPs formation was analysed by UV–vis Shimadzu 1800
spectrophotometer (Shimadzu, Kyoto, Japan), and samples
were scanned using a scan of 300 to 700 nm at a speed of
480 mm min–1. The spectrophotometer baseline correction
was conducted with a blank reference. G. lucidum reduced
GNPs were measured in X-ray diffraction (XRD) on films
from the respective solution on a glass substrate on a
Rigaku instrument operated at a current of 30 MA and a
voltage of 9 kW with radiations Cu Ka.
2.4 Morphology analysis
The sample was sputter-coated on the copper stub, and
nanoparticle images were examined using a scanning
electron microscope (SEM, JEOLMudelJfc-1600) and for
transmission electron microscopy analysis were JEOL JEM
model 2100 (HR-TEM). For atomic force microscope
(AFM) analysis, 100 ll of the sample was dropped on the
slide and the thin film was prepared by drying it for 5 min.
The slides were scanned using AFM 5 9 5 lm2 scanner and
a silicon nitride tip attached to a cantilever.
2.5 In-vitro cytotoxicity activity
2.5a Cell viability assay: Dulbecco’s Modified Eagle
medium (DMEM) was used to culture HT-29 colon cancer
cell line (2 9 105 cells ml–1) supplemented with 100 lgml–1 streptomycin, 10% FBS 100 U ml–1 penicillin and
2 mM L-glutamine, for 24 h, under 5% CO2, atmospheric
condition at 37�C. Then the cells were seeded and
incubated for 24 h in 96-well bottom culture plate.
Eventually, different concentrations of AuNPs (25, 50,
75, 100, 125 lg ml–1) were treated kept at 37�C for 24 h,
followed by 10 ll (5 mg ml–1) of MTT solution in each
well. The plates were incubated at 37�C for 4 h and to
dissolve the purple-coloured formazan crystals, dimethyl
sulphoxide (DMSO, 100 ll) was added to each well. In a
multiwell ELISA plate reader, the optical density was read
at 570 nm. The cell viability was calculated using the
following formula:
Viability ¼ OD value of experimental sample
OD value of experimental control� 100
2.5b Apoptosis analysis by PI staining: Propidium
iodide (PI) stain was used to examine the cell death
induced by GNPs. Briefly, cells from cultivation flask
were harvested. After 24 h, 10 ml of PI (1 mg ml–1) was
added [32]. The 20 ml was aliquoted on a slide and placed
under a fluorescence microscope. Then the cell death was
evaluated.
2.5c DNA fragmentation assay: About 0.5 9 106 cells
were lysed with RNase A for 2 h, and then with proteinase
K (20 ll, 0.2 mg ml–1) at 508C overnight. Ammonium
acetate (10 M) solution was added during the incubation
period, and DNA precipitation is done by adding 100%
ice-cold ethanol. Ethidium bromide was added in 2%
agarose gel and electrophoresed for 45 min at 90 V. The
gels were placed in gel documentation system’s UV
transilluminator.
2.6 Statistical analysis
To evaluate the difference between the control and treated
groups, the unpaired t-test was used using GraphPad prism-
8 to measure all statistical values (p\ 0.05).
132 Page 2 of 6 Bull. Mater. Sci. (2021) 44:132
3. Results and discussion
3.1 Optical detection and UV–vis characterizationof GNPs
Figure 1 shows that after 60 min of the reaction time, the
extract’s colour was changed slowly from light yellow to dark
purple (figure 1A). The purple colour reveals a higher GNPs
level in the G. lucidum suspensions. Then the colour intensity
increased with increased time of interaction, which shows that
more AuNPs were formed. When the reaction time increased,
more ions reduced into gold by the fungal compounds present in
interaction solution [33]. As shown in figure 1, UV–vis
absorption with the SPR band indicates the presence of AuNPs
in the final reaction mixture obtained [34,35]. A high SPR band
of approximately 520 nm (figure 1B) represents the formation of
GNPs in UV–spectral findings. The findings show that the for-
mation of nanoparticle was directly proportional to the absor-
bance rate and incubation time. The broad peak indicates a
different particle shape, while the narrow peak reveals the same
nanoparticle shape. The rapid synthesis of GNPs without any
external reducing agent at room temperature indicates that the
G. lucidum extract contains compounds like phenolic and
flavonoid, constituents that may reduce HAuCl4. The phyto-
chemical analysis showed thatG. lucidum fruit body extract has
monoterpenes, triterpenoids and flavonoids [36].
3.2 X-ray diffraction
Figure 2 shows the XRD pattern, indicating the existence of
AuNPs alone. A strong diffraction band at approximately
38� typically applied to facets {111} shows the cubic, face-
centred structure (FCC). A broad diffraction peak at 111 of
the XRD matched GNPs FCC structure. However, the other
two facets of the peaks of diffraction were weaker. Bragg
reflection were mainly associated with AuNPs at 111�,200�, 220� and 311�. Xanthium strumarium fruit extract for
synthesized GNPs is a similar XRD report [37].
3.3 Morphology study and EDX analysis of biosynthesizedGNPs
AFM displayed 2D (figure 3A) and 3D (figure 3B) repre-
sentation of the AuNPs. AFM analysis of biosynthesized
Figure 1. (A) From left to right, G. lucidum fruit body and its extract, 1 mM tetrachloroauric acid
and synthesized AP-AuNPs. (B) UV–visible spectrum of synthesized GNPs.
Bull. Mater. Sci. (2021) 44:132 Page 3 of 6 132
GNPs provided a nominal size range between 1 and 100 nm
(figure 3C). Most of the GNPs have spherical, oval and
irregular shapes, as in FE-SEM and size in the range of
25–29 nm (figure 3D) and TEM images (figure 3E). The
SAED of GNPs shows the spherical shape from synthesized
GNPs, and GNP’s reflective patterns show the crystalline
structure (figure 3F). The EDAX analysis study revealed a
strong signal corresponding to the gold element in G.lucidum assisted synthesized GNPs (figure 3G). The pres-
ence of a strong Au signal supports the SPR peak appear-
ance in the spectrum of UV-absorption.
3.4 In-vitro toxicity study
In this study, the colon cancer cells of HT-29 treated at
25, 50, 75, 100, 125 lg ml–1 concentrations of GNPs, and
dose-dependent manner reduces cells’ viability. In the
presence and absence of GNPs, HT-29 colon cancer cells’
morphology was studied using inverted phase-contrast
microscopy. The results are shown in figure 4A. High cell
layer density has been observed in control (figure 4Ai). A
low density and irregular cell lines due to apoptotic cell
death have been observed for cells treated with GNPs
(figure 4Aii and iii). The IC50 value was found to be at
84.58 lg ml–1 for biofabricated GNPs. Taken from the
results of the study HT-29 colon cancer cells, all the
groups have shown a significant difference in comparison
with control (*p \ 0.05; figure 4B). For high cytotoxic
efficacy, the size and capping of the biomolecules on the
nanoparticles also plays a vital function. The plant
extracts mediated AuNPs against HeLa cells; DNA
damage contributes to caspase activation and induces G2/
M phase cell growth arrest [29]. In cancer treatment,
AuNPs could be a novel agent [2–7]. Also, cancer cells’
death due to apoptosis induction or damage to DNA has
been previously reported [4,5].
3.5 PI staining
The changes in apoptosis was also examined by PI staining.
There was less red fluorescence in control cells (figure 4Ci),
while more red fluorescence revealed the apoptosis with
Figure 2. XRD pattern of synthesized AuNPs.
Figure 3. (A) AFM image of the synthesized GNPs-2D, (B) AFM image of the synthesized GNPs-3D. (C) Size distribution of
biosynthesized GNPs and (D) field-emission scanning electron microscope images of biosynthesized GNPs. (E) TEM images of
biosynthesized GNPs, (F) EDX images showing the strong signal Au and (G) SAED pattern of biosynthesized GNPs.
132 Page 4 of 6 Bull. Mater. Sci. (2021) 44:132
biosynthesized GNPs at the IC50 84.58 lg ml–1 (figure 4Cii)
and a higher concentration (figure 4Ciii). PI can cross
membranes and thus considered as membrane integrity
indicator. Within dead cells, it stains DNA and RNA. Red
fluorescence showed the cell death stage [38].
3.6 DNA ladder assay
DNAfragmentationof the treatment samples revealed apoptosis
on the agarose gel. It demonstrated that the long double-strand
breaks in GNPs treated HT-29 colon cancer cells, resulting in
ladder appearance. The untreated control did not show any band
(figure 4D). The treated cells showed significant apoptotic
activity with higher concentrations (figure 4D). Biosynthesized
GNPs have demonstrated apoptotic changes in HT-29 colon
cancer cells. The cancerous cells have negative charges, while
AuNPs have positive charges for the AuNPs uptake and inter-
nalization due to opposite charges [39,40]. The anticancer
activity has been demonstrated by the AuNPs with ROS over-
production, leading to oxidative stress, DNA damage, and
trigger apoptosis and mitochondrial dysfunction [41,42].
4. Conclusion
Taken together, this studywas able to achieve a simple, fastway
and cheap method to synthesize GNPs with the aqueous extract
of G. lucidum fruit body as a potential capping and reducing
agent. The synthesized nanoparticles are spherical, oval and
Figure 4. (A) Morphology of control and AuNPs-treated HT-29 colon cancer cells (409 magnification); (i) control; (ii) 75 lg ml–1;
(iii) 125 lg ml–1. (B) The significance between control and GNPs treated groups were represented as p\0.05*, p\ 0.01** and p\0.001***. (C) Propidium iodide staining of HT-29 cells in both control and treated with AuNPs (209 magnification); (C) PI staining(i) control; (ii) IC50 concentration (84.58 lg ml–1); (iii) maximum concentration (125 lg ml–1). (D) DNA fragmentation assay.
Bull. Mater. Sci. (2021) 44:132 Page 5 of 6 132
irregularly shaped in the size of 1–100 nm, as observed in
HRSEM, TEM and AFM. The findings from MTT of biosyn-
thesizedGNPs showedpotent cytotoxic activity onHT-29 colon
cancer cells. The biosynthesized nanoparticles can, therefore, be
an effective candidate for cancer therapy.
Acknowledgements
Our thanks are also extended to The Principal, Pachaiyap-
pas College for Men, Kanchipuram, for providing infras-
tructure and research facilities.
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