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GREEN SYNTHESIS OF GOLD NANOPARTICLES USING LEAF

EXTRACT OF GARCINIA COMBOGIA, ITS GENOTOXIC EFFECTS

ON E.COLI DNA AND CYTOTOXIC EFFECTS ON HE LA CELL

LINES.

B. Nithya1* and A. Jayachitra

2

*1Research Scholar, Research and Development Centre, Bharathiyar University, Tamil Nadu,

India.

2Assistant Professor, Department of Plant Biotechnology, School of Biotechnology Madurai

Kamaraj University, Tamil Nadu, India.

ABSTRACT

Cancer is a mortiferous disease or a destructive phenomenon emerges

in human beings as a result of genetic factors or disclosure to harmful

chemicals or viral infections etc. Compared to other treatments Radio

therapy is potential in the treatment of cancer. In this therapy, despite

the normal cells are also disintegrated in the fore with tumor cells.

Nanomedicine is a relavent treatment of cancer is an innovative

landing. Progressing consignment of anticancer agents to tumors using

nanoparticles is one of the most protrusive combat zone in the research

field of nanotechnology. It can be known from research that MTT

assay is broadly applied and extensively approved method in finding

invitro anticancer activity of metal nanoparticles. Gold nanoparticles biosynthesis is

upsurging from day to day generating an upshot in all biological applications. Green

synthesized gold nanoparticle inhibit the proliferation of cancer cells by activating the

apoptosis process or changing the cellular chemistry. Present investigation reveal the gold

nanoparticles biosynthesizing capability of the leaf of Garcinia combogia. Newly genre gold

nanoparticles were characterized by involving UV–vis spectroscopy, FTIR, XRD, SEM,

analysis. Strikingly as a result of extensive screening on the application of newly synthesized

gold nanoparticles their anticancer cytotoxicity has been discovered using MTT assay and its

genotoxicity by DNA fragmentation.

World Journal of Pharmaceutical Research SJIF Impact Factor 7.523

Volume 7, Issue 1, 1428-1439. Research Article ISSN 2277– 7105

Article Received on

20 Nov. 2017,

Revised on 11 Dec. 2017,

Accepted on 31 Dec. 2017

DOI: 10.20959/wjpr20181-10588

*Corresponding Author

B. Nithya

Research Scholar, Research

and Development Centre,

Bharathiyar University,

Tamil Nadu, India.

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KEYWORDS: Garcinia combogia, Gold nanoparticles, MTTassay, DNA fragmentation.

INTRODUCTION

Cancer is one of the most serious fatal diseases in today’s world that kills millions of people

every year. It is one of the major health concerns of the 21st century which does not have any

boundary and can affect any organ of people from any place.[1]

Cancer, the uncontrolled

proliferation of cells where apoptosis is greatly disappeared, requires very complex process

of treatment. Because of complexity in genetic and phenotypic levels, it shows clinical

diversity and therapeutic resistance. A variety of approaches are being practiced for the

treatment of cancer each of which has some significant limitations and side effects.[2]

Cancer

treatment includes surgical removal, chemotherapy, radiation and hormone therapy.

Chemotherapy, a very common treatment, delivers anticancer drugs systemically to patients

forquenching the uncontrolled proliferation of cancerous cells.[3]

Unfortunately, due to

nonspecific targeting by anticancer agents, many side effects occur and poor drug delivery of

those agents cannot bring out the desired outcome in most of the cases. Cancer drug

development involves a very complex procedure which is associated with advanced polymer

chemistry and electronic engineering. The main challenge of cancer therapeutics is to

differentiate the cancerous cells and the normal body cells. That is why the main objective

becomes engineering the drug in such a way as it can identify the cancer cells to diminish

their growth and proliferation. Conventional chemotherapy fails to target the cancerous cells

selectively without interacting with the normal body cells. Thus they cause serious side

effects including organ damage resulting in impaired treatment with lower dose and

ultimately low survival rates.[4]

Nanotechnology is the science that usually deals with the size range from a few nanometers

(nm) to several hundred nm, depending on their intended use.[5]

It has been the area of

interest over the last decade for developing precise drug delivery systems as it offers

numerous benefits to overcome the limitations of conventional formulations.[6,7]

It is very

promising both in cancer diagnosis and treatment since it can enter the tissues at molecular

level. Cancer nanotechnology is being enthusiastically evaluated and implemented in cancer

treatment indicating a major advance in detection, diagnosis and treatment of the disease.

Various researches are being carried out in order to discover more accurate nanotechnology

based cancer treatment minimizing the side effects of the conventional ones.[5]

Nanoparticles

are now being designed to assist therapeutic agents to pass through biologic barriers, to

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mediate molecular interactions and to identify molecular changes. They have larger surface

area with modifiable optical, electronic, magnetic and biologic properties compared to

macroparticles. Current nanotechnology based drug delivery systems for cancer treatment,

which are already marketed and under research and evaluation, include liposomes, polymeric

micelles, dendrimers, nanospheres, nanocapsules and nanotubes.[8,9]

Nanotechnology based

formulations that have already been marketed are DOXIL (liposomal doxorubicin) and

Abraxane (albumin bound paclitaxel).[10]

Conventional chemotherapeutic agents work by destroying rapidly dividing cells, which is

the main property of neoplastic cells. This is why chemotherapy also damages normal healthy

cells that divide rapidly such as cells in the bone marrow, macrophages, digestive tract and

hair follicles. The main drawback of conventional chemotherapy is that it cannot give

selective action only to the cancerous cells. This results in common side effects of most

chemotherapeutic agents which include myelosuppression (decreased production of white

blood cells causing immunosuppression), mucositis (inflammation of the lining of the

digestive tract), alopecia (hair loss), organ dysfunction and even anemia or

thrombocytopenia. These side effects sometimes impose dose reduction, treatment delay, or

discontinuance of the given therapy.[11,12]

In case of solid tumors cell division may be

effectively ceased near the center, making chemotherapeutic agents insensitive to

chemotherapy. Furthermore, chemotherapeutic agents often cannot penetrate and reach the

core of solid tumors, failing to kill the cancerous cells.[13]

Nowadays, silver and gold nanoparticles are emerging as promising agents for cancer

therapy. The anticancer activities of nano-sized silver and gold particles have been evaluated

against a variety of human cancer cells. However, very few reports were available against the

breast cancer cells and most of these studies have mainly used chemically made

nanoparticles.[14,15,16]

In the current study, we have reported on the synthesis of AuNP from Garcinia cambogia

belongs to the family Guttiferae (Clusiaceae). It is a wild sub tropical and tropical medicinal

plant. Phytochemical analysis shows that G. cambogia contains phenolic compounds,

steroids, xanthins, benzophenone[17]

tannins, gutiferrins and Saponins. Animal and human

studies revealed that the extracts of G.cambogia exhibit aphrodisiac effects on male

subjects.[18]

G. cambogia extracts have been shown to possess antipyretic, anti inflammatory,

analgesic, antiviral, hepotoprotectiv[19]

, antidepressant, antioxidant[20]

, antidiabetic and

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antithrombotic activities. The present study was aimed to synthesis of gold nanoparticles

using aqueous leaves extract of Garcinia cambogia. The green synthesized GNPs of Garcinia

Combogia were Characterized by UV- VIS Spectroscopy, Fourier transform infrared

spectroscopy (FTIR), X-ray diffraction (XRD) analysis for size and shape and scanning

electron microscopy (SEM)

The antibacterial activity of gold nanoparticles was studied in Different gram negative and

gram positive organisms such as Bacillus subtilis, E.coli, L. monocytogenes, Proteus

vulgaris, Vibryo parahaemolytics and published in the previous paper.

In continuation of this study the cytotoxic effects of biosynthesized gold nano particles of

Garcinia combogia were tested against He La cells by MTT assay and the genotoxic effect

against plasmid DNA were carried out as further research.

MATERIALS AND METHOD

MATERIALS

Healthy and fresh leaves of G. cambogia were collected from the Western Ghats of Idukki

district. Chlo- rauric acid (HAuCl4) was purchased from Hi Media (Mumbai). 3,4,5-

Dimethylthiazol-2-yl-2-5-diphenyl- tetrazoliumbromide (MTT) was purchased from Hi Me-

dia (Mumbai, India) Cell lines were obtained from the National Centre for Cell Sciences

(Pune, India). Other chemicals used were of analytical grade and obtained from Merck

(Mumbai, India).

PREPARATION OF THE LEAVES EXTRACT FOR NANOPARTICLE

BIOSYNTHESIS

The fresh leaves of G. cambogia were collected from the Western Ghats of Idukki district.

Ten grams of freshly collected Garcinia combogia leaves were surface cleaned with running

tap water followed by distilled water and boiled in 100 ml of distilled water at 60o

C for 5

min. Then, the extract was filtered and used for the biogenic synthesis of gold nanoparticles.

BIOGENIC SYNTHESIS AND CHARACTERIZATION OF GOLD

NANOPARTICLES

The biogenic synthesis of gold nanoparticles was performed according to the standard

published procedure with slight modifications.[21]

The methods for the biosynthesis and

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characterization of silver nanoparticles from the leaves extract of Garcinia combogia were

given in our previously published paper.[21]

GENOTOXIC EFFECT OF GOLD NANOPARTICLES OF GARCINIA COMBOGIA

AGAINST PLASMID DNA

Plasmid DNA was extracted from Escherichia coli using alkaline lysis (following the

protocol by Sambrook and Russell.[20]

). We identified the purity of plasmid by

OD260/OD280.

The synthesized gold nanoparticles were used for the DNA strand-breaking activity of the

nano-particle by agarose gel electrophoresis. Plasmid DNA was mixed with plant extract,

synthesized nps and the gold particle respectively & incubated for 3hrs in water bath (370C).

Then they were applied to 1% of agarose gel electrophoresis at 75 V for 30 min. After that,

ethidium bromide were added to tris-borate-EDTA gel buffer. UV irradiation at 300nm were

used in order to visualize the DNA bands. The DNA strand-breaking activity of the

nanomaterial were measured by measuring the reduction of super-coiled DNA to circular

form of DNA.[22,23]

CELL LINE

He La cell lines were procured from National centre for cell sciences, Pune, India. (NCCS).

The cells lined were maintained as a monolayer in Rosewell Park Memorial Institute medium

(RPMI) 1640 supplemented with fetal bovine serum (FBS0 (10% v/v), L-Glutamin (2mM),

penicillin (100 U/mL) and streptomycin (100 μg/mL). Cells were incubated at 370C in a 5%

CO2 humidified atmosphere.

MTT ASSAY

REAGENTS

Methylthiazolyl diphenyl- tetrazolium bromide (MTT) and Dimethyl sulfoxide (DMSO) were

purchased from (Sisco research laboratory chemicals Mumbai). All of other chemicals and

reagents were obtained from Sigma Aldrich Mumbai.

IN VITRO ASSAY FOR CYTOTOXICITY ACTIVITY (MTT ASSAY)

The Cytotoxicity of samples He La cells were determined by the MTT assay. Cells (1 ×

105/well) were plated in 1ml of medium/well in 24-well plates (control plant extract and the

synthesized gold nanoaprticles).[24,25]

After 48 hours incubation the cell reaches the

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confluence. Then, cells were incubated in the presence of various concentrations of the

samples in 0.1% DMSO for 48h at 37°C. After removal of the sample solution and washing

with phosphate-buffered saline (pH 7.2), 250μl/well (5mg/ml) of 0.5% 3-(4,5-dimethyl-2-

thiazolyl)-2,5-diphenyl--tetrazolium bromide cells (MTT) phosphate- buffered saline solution

was added. After 4h incubation, 0.04M HCl/ isopropanol were added. Viable cells were

determined by the absorbance at 570nm. The absorbance at 570 nm was measured with a

UV- VIS Spectrophotometer using wells without sample containing cells as blanks. The

effect of the samples on the proliferation of He La was expressed as the % cell viability,

using the following formula:

% cell viability = A570 of treated cells / A570 of control cells × 100%

RESULTS

In this present study, gold nanoparticles were rapidly synthesized using Garcinia combogia

leaves extract as bio-reductants.

SYNTHESIS OF AuNPs

The immediate change in color of the solution from pale yellow to violet color due to the

surface plasmon resource indicated the preliminary confirmation for the formation of plant

extract mediated synthesis of gold nanoparticles. The result obtained in this investigation is

very interesting in terms of identification of Garcinia combogia for synthesizing the Au

Nps.(fig A).

CHARACTERISATION OF BIOSYNTHESIZED GOLD NANOPARTICLES

The UV – Vis spectra of Au Nps synthesized showed the absorbance maxima of Gold

Surface Plasmon Resonance (SPR) at 550 nm and the intensity increased steadily as a

function of time without any shift in the peak wavelength.[26]

XRD Analysis showed three

distinct diffraction peaks at 38.1°, 44.1° and 64.1°which indexed the planes 1 1 1, 2 0 0 and 2

2 0 of the cubic face centered g. The FTIRspectrum of the bio-reduced gold nanoparticles by

the phytochemicals had the adsorption peaks located at about 3,965, 3,466 cm-1, 3404, 2678,

2074, 1638, 1368, 1233, 664 cm-1.SEM image shows the size of the AuNPs ranging from

40–50 nm. Similar result of the gold nanoparticles size was reported by using Aloe vera

extract[27]

and by using Euphorbia hirta leaves.[28]

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DNA FRAGMENTATION

Agarose gel electrophoresis of plasmid DNA of E.coli treated with of gold nanoparticles of

Garcinia combogia showed a dose-dependent induction of DNA strand break, characterized

by increased degradation of supercoiled form to relaxed circle to linear forms (Figure 2).

DNA strand scission induced by gold nanoparticle leads to gradual degradation in the amount

of both linear and supercoiled DNA and appearance of extra bands lower in the gel which are

the resultant fragmented DNA (Figure 1). Besides their antimicrobial activity, gold

nanoparticles of Garcinia combogia have been shown to be potentially genotoxic by in vivo

and in vitro assays.[29]

Recently the genotoxicity exhibited by silver Nanoparticles of

Macrophomina phaseolina was demonstrated by degradation of plasmid.[29]

Such genotoxic

activities of nanoparticles were also reported earlier in case of carbon nanotubes[30]

where

degree of DNA degradation was directly proportional to the concentration of nanoparticles. A

proposed mechanism of DNA damage is through generation of singlet oxygen as reported in

the case of copper nanoparticles.[31]

CYTOTOXIC ACTIVITY OF BIOLOGICALLY SYNTHESIZED

GOLDNANOPARTICLES OF GARCINIA COMBOGIA AGAINST HE LA CELL

The biogenic gold nanoparticles were tested for their potent cytotoxic activity against, He La

cells. The results of the mechanistic studies indicated that gold nanoparticles induced

apoptosis through caspase-3 activation and DNA fragmentation.

DETERMINATION OF CELL VIABILITY BY MTT ASSAY

Different concentrations(250 μg, 500 μg) of plant extract, gold nanoparticles of Garcinia

combogia and control (untreated cells) were used to study the viability of He La cells and the

toxicity was measured. Interestingly, biosynthesized gold nano particles of Garcinia

combogia treated cells showed much toxic effects in both the concentrations than the plant

extract treated tumour cells.(table: 1) The results of this study also suggest that the

cytotoxicity of biologically synthesized gold nanoparticles showed was increased with the

increasing concentration of nanoparticles.(fig3).

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FIGURE LEGENDS

A B

Fig. 1: Digital photograph of Garinia cambogia leaves (A) Synthesized AuNPs and its

color change (B).

DNA FRAGMENTATION

Fig 2: Agarose gel electrophoresis of plasmid DNA of E.coli treated with of gold

nanoparticles of Garcinia combogia. Lane1, DNA molecular weight marker, Lane 2,

plasmid incubated with control plant extract without gold nano particles, Lane 3,

plasmid incubated with gold nano particles showing disappearance of super coiled

plasmid band and appearance of relaxed circular and linear plasmid bands along with

smaller fragmented DNA. Lane 4 plasmid incubated with gold particles without plant

extract.

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MTT ASSAY

Fig. 3: Cytotoxic effects of biosynthesized gold nano particles of the control and treated

He La cells.

Table 1: Effect of the sample on the proliferation of He La cells expressed as % cell

viability.

Sample Test concentration (μg/ml) Cytotoxicity (% of cell

viability) 250 500

Plant extract 0.289 0.312 38.24; 33.33

Gold Nps 0.325 0.356 30.55; 23. 93

Control 0.468 0.468 100

Fig 5: Graphical representation of Cell Viability of Different concentrations(250 μg, 500

μg) of plant extract, gold nanoparticles of Garcinia combogia and control (untreated

cells). Biosynthesized gold nano particles of Garcinia combogia treated cells showed

much toxic effects in both the concentrations than the plant extract treated tumour

cells.

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CONCLUSIONS

In this present study, silver and gold nanoparticles were rapidly synthesized using aqueous

leaves extract of G. combogia as novel source of bio-reductants. This single step procedure

appears to be suitable for large scale production as it is simple, faster, costeffective,

environmentally benign and safe for clinical research. Further, the plant extract derived

nanoparticles exhibited strong genotoxic against plasmid DNA and cytotoxic effects against

He La cells, which suggest that biologically synthesized gold nanoparticles might be used as

novel anticancer agents for the treatment of cancer. However, the fate, transport and

accumulation of nanoparticles inside the human body must be thoroughly studied prior to the

approval to use as anticancer drug.

ACKNOWLEDGEMENTS

The authors thank the Director, Centre for Bioscience and Nanoscience research for

laboratory facilities. We are grateful to the Tamil Nadu Agricultural university, Ciombatore

for taxonomical identification of the plant sample.

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