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 (sumancas2010@gmail.com)

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.

References

[1] Manivasagan P, Venkatesan J, Kang K H, Sivakumar K,

Park S J and Kim S K 2015 Int. J. Biol. Macromol. 72 71

[2] Ahmad A, Mukherjee P, Senapati S, Mandal D, Khan M I,

Kumar R et al 2003 Coll. Surf. B: Biointerface 28 313

[3] Song J Y and Kim B S 2009 Bioproc. Biosyst. Eng. 32 79

[4] Thrall J 2004 Radiology 230 315

[5] El-Sayed I H, Huang X and El-Sayed M A 2005 Nano Lett. 5829

[6] Prashanth S, Menaka I, Muthezhilan R and Sharma N K

2011 Intern. J. Engine Sci. Tech. 3 6235

[7] Schrofel A, Kratosova G, Safarık I, Safarıkova M, Raska I

and Shor L M 2014 Acta Biomater. 10 4023

[8] Boroumand Moghaddam A, Namvar F, Moniri M, Azizi S

and Mohamad R 2015 Molecule 20 16540

[9] Pereira L, Mehboob F, Stams A J, Mota M M, Rijnaarts H H

and Alves M M 2015 Crit. Rev. Biotech. 35 114

[10] Borhamdin S, Shamsuddin M and Alizadeh A 2016 J. Exp.Nanosci. 11 518

[11] Shedbalkar U, Singh R, Wadhwani S, Gaidhani S and

Chopade B A 2014 Adv. Colloid Interface Sci. 209 40

[12] Adil S F, Assal M E, Khan M, Al-Warthan A, Siddiqui M R

H and Liz-Marzan L M 2015 Dalton Trans. 44 9709

[13] Tahir K, Li B, Khan S, Nazir S, Khan Z U H, Khan A U et al2015 J. Alloys Compd. 651 322

[14] Quester K, Avalos-Borja M and Castro-Longoria E 2013

Micron 54 1

[15] Nazeruddin G M, Prasad N R, Waghmare S R, Garadkar K

M and Mulla I S 2014 J. Alloys Compd. 583 272

[16] Owaid M N and Ibraheem I J 2017 Eur. J. Nanomed. 9 5

[17] Clarance P, Luvankar B, Sales J, Khusro A, Agastian P, Tack

J C et al 2020 Saudi J. Biol. Sci. 27 706

[18] Mandal D, Bolander M E, Mukhopadhyay D, Sarkar G and

Mukherjee P 2006 Appl. Microbiol. Biotechnol. 69 485

[19] Faramarzi M A and Forootanfar H 2011 Coll. Surf. B:Biointerface 87 23

[20] Owaid M N, Rabeea M A, Aziz A A, Jameel M S and

Dheyab M A 2019 Environ. Nanotech. Monit. Manag. 12100270

[21] Rabeea M A, Owaid M N, Aziz A A, Jameel M S and

Dheyab M A 2020 J. Environ. Chem. Eng. 8 103841

[22] Sanghi R, Verma P and Puri S 2011 Adv. Chem. Engine Sci.1 154

[23] Bhat R, Sharanabasava V G, Deshpande R, Shetti U, Sanjeev

G and Venkataraman A 2013 J. Photochem. Photobiol. B:Biol. 125 63

[24] Sen IK,MaityK and IslamS S 2013Carbohydr. Polym. 91 518[25] Sarkar J, Roy S K, Laskar A, Chattopadhyay D and Acharya

K 2013 Mater. Lett. 92 313

[26] Seigneuric R, Markey L, Nuyten D S A, Dubernet C, Evelo

T A C, Finot E et al 2010 Curr. Mol. Med. 10 640

[27] Wasser S P 2005 Encycl. Diet. Suppl. 1 603

[28] Gerhauser C, Zhang W D, Ho-Chong-Line N and Fouraste I

2000 Planta Med. 66 681

[29] Chang S T and Mshigeni K E 2001 Mushrooms and humanhealth: their growing significance as potent dietery supple-ments University of Namibia

[30] Kim H W and Kim B K 2002 Ganoderma 10 19

[31] Suman T Y, Rajasree S R, Ramkumar R, Rajthilak C and

Perumal P 2014 Spectrochem. Acta Part A: Mol. Biomol.Spectrosc. 118 11

[32] Mishell B B, Shiiqi S M and Henry C 1980 Cellularimmunology (San Francisco: Freeman) p 21

[33] Owaid M N, Al-Saeedi S S S and Abed I A 2017 Environ.Nanotech. Monit. Manag. 8 157

[34] Arumugam P and Berchmans S 2011 ACS Appl. Mater.Interface 3 1418

[35] Mahendran G and Ponnuchamy K 2018 Appl. Nanosci. 8 447[36] Ahmad M F 2018 Biomed. Pharmacother. 107 507

[37] Peng Q and Chen R 2019 J. Photochem. Photobiol. B: Biol.192 13

[38] Baharara J, Ramezani T, Divsalar A, Mousavi M and

Seyedarabi A 2016 Avicen. J. Med. Biotech. 8 75

[39] Gong N, Chen S, Jin S, Zhang J, Wang P C and Liang X J

2015 Regen. Biomater. 2 273

[40] Albanese A, Tang P S and Chan W C 2012 Ann. Rev.Biomed. Engine 14 1

[41] Parida U K, Biswal S K and Bindhani B K 2014 Adv. Biol.Chem. 4 360

[42] Tiloke C, Phulukdaree A, Gengan R M and Chuturgoon A A

2016 J. Cell. Biochem. 117 2302

132 Page 6 of 6 Bull. Mater. Sci. (2021) 44:132