Biogenic synthesis of gold nanoparticles using Commiphora wightii and their cytotoxic effects on breast cancer cell line (MCF-7)

Journal Pre-proof Biogenic synthesis of gold nanoparticles using Commiphora wightii and their cytotoxic effects on breast cancer cell line (MCF-7) M. Uzma, N. Sunayana, Vinay B. Raghavendra, C.S. Madhu, Rajasree Shanmuganathan, Kathirvel Brindhadevi

PII:

S1359-5113(19)31329-7

DOI:

https://doi.org/10.1016/j.procbio.2020.01.019

Reference:

PRBI 11904

To appear in:

Process Biochemistry

Received Date:

1 September 2019

Revised Date:

14 January 2020

Accepted Date:

18 January 2020

Please cite this article as: Uzma M, Sunayana N, Raghavendra VB, Madhu CS, Shanmuganathan R, Brindhadevi K, Biogenic synthesis of gold nanoparticles using Commiphora wightii and their cytotoxic effects on breast cancer cell line (MCF-7), Process Biochemistry (2020), doi: https://doi.org/10.1016/j.procbio.2020.01.019

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Biogenic synthesis of gold nanoparticles using Commiphora wightii and their cytotoxic effects on breast cancer cell line (MCF-7) M. Uzma a, N. Sunayana b, Vinay B. Raghavendra a*, C. S. Madhu c, Rajasree Shanmuganathan d, Kathirvel Brindhadevi e

Teresian Research Foundation, Siddarthanagar, Mysore - 570011, India

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Department of Biochemistry, Indian Institute of Science, Bengaluru - 560012, India

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Department of Biochemistry, Yuvarajas College, Mysore - 570 005, India

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Institute of Research and Development, Duy Tan University, Da Nang 550000, Vietnam

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Innovative Green Product Synthesis and Renewable Environment Development Research

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Group, Faculty of Environment and Labour Safety, Ton Duc Thang University, Ho Chi Minh City, Vietnam. Email: [email protected]

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Corresponding Author Address

Assistant Professor PG Department of Biotechnology

Mysore- 570011. India.

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Email: [email protected]

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Teresian College

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Dr. Vinay B. Raghavendra

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Graphical abstract

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Highlights

Eco-friendly and simple biosynthesis of Cw@AuNPs using Commiphora wightii leaf extract.

Characterization by spectroscopic and microscopic studies.

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Cw@AuNPs exhibit remarkable cytotoxicity against MCF-7 cells by causing

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apoptotic cell death.

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Abstract

The current study aimed at developing gold nanoparticles (AuNPs) using the aqueous

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extract of the medicinal plant Commiphora wightii. The phytosynthesized gold nanoparticles (Cw@AuNPs) were evaluated for their anticancer activity against MCF-7 breast cancer cell model. The formation of AuNPs by Commiphora wightii leaf extract was confirmed by UV– Visible spectra where their surface plasmon resonance was found at 533 nm. Further characterization of Cw@AuNPs was done by transmission electron microscopy (TEM), Xray diffraction (XRD), energy dispersive X-ray (EDX) analysis, and fourier-transform infrared spectroscopy (FTIR) analysis. In vitro anticancer potential of thus obtained AuNPs was evaluated against MCF-7 and where the IC50 was found to be 66.11 µg/mL Further, 2

apoptotic studies were carried out using ethidium bromide dual staining, DNA fragmentation, comet assay, and flow cytometry studies. Results revealed that Cw@AuNPs at higher concentration significantly increased the apoptotic cells when compared to control cells. Cell cycle analysis of MCF-7 cells confirmed the cell cycle arrest at G2/M phase. These results demonstrate that the biosynthesized Cw@AuNPs appear to be promising for therapeutical applications against breast cancer. Keywords: Gold nanoparticles; Cytotoxic; Apoptosis; DNA damage.

1. Introduction

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Nanotechnology is a burgeoning field of research which plays a central role in the development of effective carrier systems for site-specific drug delivery [1-3]. Green nanoparticles comprising metals such as gold, silver, copper, titanium, platinum, zinc, and iron prepared from diverse biological agents are known [3-17]. Among those, gold nanoparticles (AuNPs) have found vast applications due to their remarkable Plasmon

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resonance optical parameters [18-20]. Chemical and physical methods used for the preparation of AuNPs are associated with biological hazards and environmental toxicity [21].

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Unlike these methods, biologically synthesized nanoparticles are considered eco-friendly, safe and non-toxic [22]. Use of extracts from medicinal plants for the synthesis of

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nanoparticles is recognized as a simple eco-friendly method against the chemical methods. Such biosynthesized nanoparticles display great potentials in various applications, including therapeutic and other medical uses [23, 24]. More importantly, plant-based AuNPs are proven

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to be biocompatible and non-toxic to cells [20]. Recently the synthesis of AuNPs has been reported using medicinal plant Halymenia dilatata owing to their non-toxic effects and improved drug delivery [25].

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Breast carcinoma is recognized as the second most common reason for cancer-related deaths globally. Among the Indian females, it occupies the first position in age-standardised

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mortality rates [26]. Though wide varieties of chemotherapeutic agents have been used in the treatment of breast cancer, they are often associated with various risk factors [27]. Hence, there is a need to identify novel strategies to combat cancer. Among many such approaches, nanomolecules are of interest due to their specificity in target-oriented drug delivery and nontoxicity. Earlier pre-clinical reports have shown that the plant-based nanoparticles hold great promise for better management of cancer with negligible side effects [28]. Commiphora wightii, commonly known as the Guggul tree, is a medicinal plant having various therapeutic applications including antimicrobial, anticancer and anti3

inflammatory activities [29]. C.wightii has been used in traditional medicine to treat various ailments owing to its rich content of numerous phytoconstituents viz., terpenoids, flavonoids, steroids, carbohydrates, sterols, and ferulates among others [30]. Previous studies have reported the Commiphora wightii mediated silver nanoparticles having antibacterial activity [31]. Recently, Sunayana et al. reported Vitex negundo mediated synthesis of AuNPs that displayed in vitro and in vivo anti-inflammatory activities [32]. The present study aimed at the synthesis of gold nanoparticles using Commiphora wightii aqueous extract and evaluating their in vitro anticancer efficacy against MCF-7 cell lines.

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2. Materials and methods 2.1 Collection of plant material

Plant leaves of C. wightii were collected from Chamundi hill region (Latitude 12.2732o N; Longitude 76.6707o E) of Mysore district of Karnataka state in India during the

Botany, University of Mysore, Karnataka, India.

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2.2 Preparation of the extract

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summer season and the material was identified by experts at the Department of Studies in

Fresh C. wightii leaves were washed thoroughly with distilled water dried on blotters

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under lab conditions and powdered using a blender. An aliquot (5g) of C. wightii powder was extracted in 100 mL of water using the microwave method, followed by two cycles of 10 mins each at 100o C. The extract obtained was cooled, filtered through Whatman No. 1 filter

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paper twice and was preserved in a refrigerator for future use.

2.3 Synthesis of Cw@AuNPs

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C. wightii leaf aqueous extract (50 mL) was mixed with 1mM Chloroauric acid (HAuCl4) solution (50 mL) at room temperature. The bioreduction reaction was completed

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within 5 min while the color of the solution changed from pale yellow to purple wine, which in turn indicated the formation of nanogold in solution. This was confirmed by visual inspection and also with the aid of UV-Visible spectrophotometer by recording the spectra from 200-800 nm. The resultant suspension was further subjected to centrifugation at 25000 rpm for 15 min and washed thrice using double-distilled water. The obtained pellet was dissolved using sonication in double distilled water for biological experiments (1 mg/mL).

2.4 Characterization of Cw@AuNPs 4

FTIR spectrum of the synthesized nanoparticles sample was analyzed by PerkinElmer Spectrum Version 10.03.09 at a resolution of 4 cm-1. XRD patterns were obtained on a desktop X-ray diffractometer operating at a voltage of 30Kv and a current of 15Ma with Cu radiation. TEM analysis of the synthesized nanoparticles was done by preparing samples on carbon-coated copper TEM grids. TEM measurements (FEI-Titan-Themis 3391) were obtained at the voltage of 300 KV. HRTEM images and SAED pattern were also analyzed to confirm the nature of the biosynthesized gold nanoparticles. EDX was done to find out the elemental composition of the nanoparticles sample using Instrument Bruker Nano GmbH

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Berlin, Germany, Esprit 1.9.

2.5 Cytotoxic effects of Cw@AuNPs

MCF-7 human breast cancer cell lines were procured from ATCC. Cytotoxic property was examined in a concentration dependent manner with the aid of MTT assay [33]. Briefly, a fixed number of cells (1×104 cells) were seeded in a 96 well plate followed by

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incubation for 24 h at 37° C. Different concentrations of gold nanoparticles (0-320 μg/mL) were added and incubated for 24 h. After incubation, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-

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diphenyltetrazolium bromide (MTT) (5 mg/mL of MTT in Phosphate buffer saline) was added to each well following incubation for 4 h. The supernatant was discarded, 100 µL of

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dimethyl sulfoxide (DMSO) was added and the plates were gently shaken to dissolve the formazan produced. The absorbance was measured at 590 nm using a microplate reader. The percentage inhibition was calculated using the formula mentioned below; the concentration of

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nanoparticles needed to inhibit 50% of the cell growth (IC50) values was determined and was considered for further studies.

 OD of sample  100    OD of control

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% Inhibition

  X 100 

------------------------------------ (1)

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Based on the IC50 concentrations (IC50×1 and IC50×2), doses were selected for further studies.

2.6 Acridine orange/Ethidium bromide dual staining assay The effect of Cw@AuNPs on cell morphology was assessed by fluorescent

microscopic studies as described earlier using Acridine orange and ethidium bromide stains. Cells treated with or without nanoparticles were fixed using the fixative solution and stained with AO/EtBr at a concentration of 50 µg/mL about 2 min. The excess stain was removed and the cells were observed in a fluorescence microscope with the aid of a fluorescein filter [34]. 5

2.7 DNA fragmentation assay For assessing apoptosis, DNA fragmentation was carried out using agarose gel electrophoresis. Cells treated with or without Cw@AuNPs were subjected to whole genomic DNA isolation as described earlier by using phenol: chloroform: isoamyl alcohol method. The resulting precipitated DNA was quantified using nano spectrophotometer and analyzed by agarose gel electrophoresis [35].

2.8 Flow cytometry analysis Apoptosis of MCF-7 cells was investigated using the Annexin-V FITC/PI staining kit

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(Sigma) as per the manufacturer’s instructions. Briefly, 1 X 106 cells per dish in P-35 dishes using Dulbecco's modified eagle medium (DMEM) media were plated a day before induction of apoptosis and the media were then replaced with Cw@AuNPs. The treated cells were incubated for 24 h at normal culture conditions, harvested, transferred to sterile FACS

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(Fluorescence activated cell sorting) tubes, spun at 2000 rpm for 5 min and the supernatants were decanted. Cells were washed twice with cold PBS following centrifugation and

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resuspended in binding buffer at a concentration of ~1 x 106 cells/mL About 100 μL of the cell suspension (~1 x 105 cells), 5 μL of Annexin V and 10 μL of PI (0.05 mg/mL) were

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added to the tubes, gently mixed and incubated for 15 min in dark at room temperature. Finally, cell analysis was performed using flow cytometry [36].

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2.9 Alkaline comet assay

Comet assay was performed to analyze the level of DNA damage caused due to the effect of biosynthesized gold nanoparticles as described earlier [37] with minor

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modifications. Briefly, cells were subjected to centrifugation, supernatants were discarded and washed with PBS to obtain the yield of 1x105cells/mL Cells treated with Cw@AuNPs

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were mixed with LMPA (Low melting point agarose) and were spread on fully frozen microscopic slides containing NMA (Normal melting agarose). The slides were then immersed in the lysis solution for nearly 2 to 24 h at 4 °C, arranged side by side on a gel electrophoresis apparatus and were allowed to sit in an alkaline buffer for around 20 min for the unwinding of DNA and the expression of alkaline labile DNA damage. Further, the electric field was applied by adjusting the current of 300Ma and electrophoresis was performed for 30 min. Slides were washed using distilled water followed by chilled 70% ethanol and were air dried. Finally, the slides were stained with 80 µl of ethidium bromide 6

and kept for 5 min. Further, the slides were observed under a fluorescent microscope. Comet images obtained were analyzed for olive moments using open comet plugin in Image J software.

2.10 Cell cycle arrest Cell cycle changes were analyzed using flow cytometry to detect the proportions of cells in different cell cycle phases. In this order, 1x106 cells were seeded and cultured for 24 h in a 6-well plate supplemented with serum free media. Cells were then treated with desired concentrations of Cw@AuNPs prepared in complete media following the incubation for

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another 24 h. Cells were then harvested, spun at 2000 rpm in a centrifuge for 5 min at room temperature and the supernatants were discarded. Cell pellets were washed in PBS twice, fixed by addition of chilled 70% ethanol with continuous gentle shaking and then, stored at 4 °C overnight. Post fixing, the cells were spun at 2000 rpm for 5 min followed by washing with cold PBS twice and resuspended in 450µL of sheath fluid containing 0.05 mg/mL PI and

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0.05 mg/mL RNaseA and incubated for 15 min in dark. The levels of cells in different phases of cell cycle in treated and untreated populaces were resolved utilizing FACS Calibre (BD

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Biosciences, San Jose, CA) using Cell quest pro software.

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2.11 Statistical analysis

Statistical analysis was performed using Graph pad prism 5.1 software. Experiments were conducted in triplicates and data were presented as mean ± standard deviation.

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Statistical significance was denoted as **P<0.005.

3. Results and discussion

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3.1 Synthesis and characterization of nanoparticles Biosynthesis of nanoparticles, particularly, using medicinal plants is gaining more

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attention due to its non-toxic and eco-friendly nature [38]. Therefore, the present report mainly emphasizes on the bioreduction of ionic gold into nanogold using an aqueous leaf extract. The formation of gold nanoparticles (Cw@AuNPs) in the solution of plant extract was confirmed by visual change in color from yellow to purple (Fig. 1 inset). The color appeared was in order of the excitation of Surface Plasmon Resonance (SPR), exhibited by the gold nanoparticles [39]. The characteristic SPR band was centered at the wavelength of 533nm as shown in the UV-Vis spectrum (Fig. 1). The Surface Plasmon band is known to occur in the range of 510-560 nm in the aqueous medium for gold nanoparticles synthesis 7

[40]. The reduction of metal ions and the formation of corresponding nanoparticles occurred due to the secondary metabolites such as alkaloids, amino acids, flavonoids, saponins, steroids, glycosides, carbohydrates, tannins and phenolic groups present in the leaf extract. The reaction time required for synthesis was found to be 5 min without extreme environmental conditions. The results obtained were in the closest proximity to those of the study conducted earlier for gold nanoparticles synthesis using C. guianensis aqueous flower extract [28]. Further, the stability of Cw@AuNPs in such solutions was tested and found to be stable for more than 4 months at room temperature although with minor aggregation of particles in the solution. The stability of the nanoparticles formed was assumed to be due to

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the presence of a wide variety of phytoconstituents present in the sample. Further, XRD was performed to depict the structural elucidation of the biosynthesized gold nanoparticles (Fig. 2a). The spectrum had intense peaks at 2θ= 38.17o, 44.30o, 64.52o indexed to (111), (200), (220) planes confirming their face-centered cubic structure. The XRD pattern clearly revealed the crystalline nature of gold nanoparticles. The results

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obtained were in agreement with the Joint Committee on Powder Diffraction Standards database (JCPDS No 04-0784). The elevated level of purity of the crystalline Cw@AuNPs

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was witnessed by the XRD pattern obtained, which revealed the absence of other crystalline peaks. The reported peak values also matched with the XRD patterns of gold nanoparticles

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obtained by different green synthesis methods [41]. Debye Scherer equation was utilized to calculate the average crystallite size of Cw@AuNPs, which was found to be 27.91nm. The spot-profile EDX of the biosynthesized gold nanoparticles showed strong signals for gold

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atoms (Fig. 2b). The spectrum confirmed the presence of elemental gold, signifying a strong absorption peak. The results obtained were in close agreement with the studies conducted earlier [42].

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The biosynthesized nanoparticles were further analyzed for their shape and size using TEM. The TEM images showed different shapes of nanoparticles. The Cw@AuNPs were

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mostly polydispersed with spherical, triangular and hexagonal morphologies (Fig. 2c) with the average size ranging from 20.2±6.6 nm. A similar observation was also found in the synthesis of gold nanoparticles using the aqueous leaf extract of Acalypha indica [43]. The clear lattice fringes, which appeared in the HRTEM images (Fig. 2e), have also confirmed the crystalline nature of gold nanoparticles. The SAED pattern obtained from TEM images (Fig. 2d) for the biosynthesized nanoparticles revealed brilliant circular rings relating to the (111), (200), (220) and (311) planes, which in turn, affirmed the crystalline property of biosynthesized gold nanoparticles established by the XRD results obtained. 8

The functional groups present in the plant extract had the potential for the reduction of nanoparticles as well as for maintaining their stability, which were analyzed using FTIR spectra at diverse vibrational stretches in the range of 400 - 4000 cm-1 (Fig. 3 a, b). FTIR spectra of both the plant extract and the biosynthesized gold nanoparticles demonstrated almost similar peaks with a minute shift in the spectra. Cw@AuNPs and plant extract exhibited major peaks at 3367.69 cm-1 and 3359.27 cm-1, signifying the carboxylic CO-OH and phenolic O-H groups. This was due to the presence of amines, alcohols, aldehydes, proteins, and metabolites present in the C. wightii leaves. The band obtained at 1636 cm-1 for both the spectra corresponded to amide I linkage due to the carbonyl stretch in the proteins.

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Thus, the amide linkages in the proteins present in the plant extract could have been responsible for the reduction of gold ions into nanoparticles. The minute drift observed in the intensity of the FTIR spectra of the extract and the synthesized nanoparticles might have been the result of phytochemical coordination with the metal surface [44]. The FTIR results indicated the role of proteins and other functional groups in reduction, capping and

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3.2 Anticancer studies

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stabilization of the biosynthesized AuNPs.

While exploring the applications of the newly synthesized AuNPs, anticancer activity

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against breast cancer cell lines MCF-7 was considered where a series of in vitro experiments were conducted including MTT, dual staining, flow cytometry analysis of apoptosis, DNA fragmentation followed by Comet assay and Cell cycle analysis.

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Cytotoxicity of Cw@AuNPs was tested using MTT assay against MCF-7 and 3T3-L1 (embryonic cells) and IC50 was found to be 66.11 µg/mL (Fig. 4) and 306.41 µg/mL (Supplementary Data Fig. 1a and 1b), respectively. Thus, the cytotoxicity results revealed

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that the biosynthesized nanoparticles significantly inhibited MCF-7 cell lines by way of apoptosis. Results indicated that anticancer activity increased with the increase in the

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treatment concentrations. Studies have reported that the enhanced cytotoxicity of the gold nanoparticles might occur due to the secondary metabolites present in the medium used for their synthesis [45]. Based on the IC50 concentrations, doses of Cw@AuNPs (IC50 ×1 and IC50 ×2) were selected for further studies. Apoptosis is a widely used phenomenon used to detect cell death, morphological as well as molecular changes [46]. Apoptosis studies were carried out to evaluate the apoptotic cell deaths in treated groups of cells using fluorescence microscopy, DNA fragmentation and flow cytometry analysis. To comprehend the impacts of Cw@AuNPs on the morphological 9

changes in MCF-7 cells after treatment, AO/EtBr dual staining was carried out. As revealed that in the double staining method, control cells were apparent to have intact DNA as well as nuclear membranes resulting in fluorescent green color, whereas the treated cells having DNA with late apoptotic and necrotic cells emitted red fluorescence [47]. These results revealed that the treated cells showed early apoptosis marked with green granulation within the cells representing early nuclear condensation. The late apoptotic cells had AO/EtBr incorporation in the cells fluorescing green with AO and granulation within the nucleus representing fragmented DNA fluorescing orange with EtBr whereas the control cells showed a fluorescent green color (Fig. 5a). These results demonstrated that the biosynthesized AuNPs

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had the potential to induce apoptosis in MCF-7 cells when used at higher concentrations. AuNPs synthesized using Nerium oleander leaf extract have also been reported for their cytotoxic effects against MCF-7 cell lines and found to be highly effective in inducing apoptosis [48]. Further, to support the evidence that the synthesized nanoparticles have induced apoptosis in MCF-7 cells, DNA fragmentation assay was carried out. The treatment

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with Cw@AuNPs showed significant DNA fragmentation in MCF-7 cells whereas the untreated cells exhibited a single band (Data not shown here). Thus, the cell death occurring

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as a result of cytotoxicity of Cw@AuNPs was detected. The appearance of fragmented DNA was more in higher treatment concentrations.

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The amount of externalization of phosphatidylserine (PS) from the MCF-7 cell surfaces as a result of apoptotic cell damage was further assayed using Annexin V FITC/PI staining. The cell lines treated with Cw@AuNPs showed 0.74% early apoptotic & 1.21% late

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apoptotic cells at IC50×1 µg/mL while at IC50× 2 µg/mL, 2.29 % were early apoptotic and 1.95% was late apoptotic cells (Fig. 5b). Significant increase in apoptosis was observed in the cells treated at higher concentration in comparison to the control. The overall results

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suggested apoptotic cell death in MCF-7 cells demonstrating the capacity of biosynthesized gold nanoparticles in inducing such a process. Similar studies have been conducted for the

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green synthesized AuNPs mediated by Zataria multiflora leaves, which exhibited a dosedependent apoptotic activity [49]. Further, alkaline comet assay was used to detect the DNA strand breaks in the MCF-7

cells following exposure to the Cw@AuNPs. The levels of DNA damage in the cells treated at different concentrations and the untreated control cells were compared. The results revealed that the cells treated at IC50×2 µg/mL had a significant DNA damage with 24.97 ± 19.77 olive moments compared to the control with 3.10 ± 5.12 olive moments whereas cells treated at IC50×1 µg/mL caused minimum DNA damage with 4.06 ± 7.67 olive moments 10

(Fig. 6). Taken together, biosynthesized AuNPs caused significant damages to DNA at increasing concentrations with tail formations (Fig. 5c), instigating apoptosis in MCF-7 cell lines. To examine whether the inhibition of the AuNPs treated cells also involved changes in the cell cycle, the phase distribution of the cell cycle was analyzed using flow cytometry. Cell cycle exhibits three distinct phases’ viz., G0/G1, S, and G2/M phases. In cells treated with substances which can induce apoptosis, a population of cells in the sub-G1 phase of the cell cycle should increase because of the after-effect of endonuclease activation and subsequent spillage of DNA from the cell. Since a necrotic cell does not demonstrate the

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immediate decrease in DNA content, the differentiation between apoptotic and necrotic cells can be made. In the present study, the results of cell cycle analysis showed increased G2M arrest from 10.33% (Control) to 14.57% and 18.29%, respectively. Thus, the treatment of MCF-cells with Cw@AuNPs has shown a modest increase in cell arrest at the G2M phase in comparison to the control (Fig. 7). In this regard, comparable outcomes have been obtained

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wherein gold nanoparticles have arrested the cell cycle at G2M Phase and induced apoptosis

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[50].

4. Conclusion

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The present study was specially designed to investigate the role of AuNPs synthesized using Commiphora wightii and to explore their anticancer efficacy. As this is a green chemistry approach, the method does not pose any environmental hazard. In this regard, the

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present report has clearly emphasized the rapid synthesis, characterization, and evaluation of anticancer efficiency of Cw@AuNPs on MCF-7 cell line. Cw@AuNPs was found to have significant effects on the induction of apoptosis in MCF-7 cells. Cytotoxic effects were

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proven by cell morphology, DNA fragmentation, and flow cytometry analysis followed by comet assay. Significant amount of apoptotic cells were observed upon exposure to

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Cw@AuNPs. Further validation of these findings is in progress through studies evaluating molecular mechanisms and the in vivo effects of Cw@AuNPs. Cw@AuNPs synthesized and characterized in the present study appears useful for several therapeutic applications.

Acknowledgments

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Uzma M is highly grateful to GOKDOM (Government of Karnataka Department of Minorities) for providing fellowship to carry out the research. Authors are thankful to IISc, Bangalore for providing necessary facility for nanoparticle characterization. Authors are grateful to SKANDA Life sciences Pvt Ltd for gifting the ATCC cell line for the present study. Authors are grateful to Dr. Bhagyalakshmi Neelwarne (Retd. Head and Chief Scientist, Plant Cell Biotechnology Department, CFTRI, Mysore), for her critical comments

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and evaluation of the manuscript.

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Figure Legends Figure. 1 UV-Vis spectra of Cw@AuNPs synthesized from Commiphora wightii; inset shows yellow to purple colour of AuNPs Figure. 2 Characterization of Cw@AuNPs: (a) XRD pattern; (b) EDX analysis; (c) TEM image at 100 nm magnification; (d) SAED pattern; (e) HR-TEM image of Cw@AuNPs Figure. 3 Fourier Transform Infrared Radiation (FTIR) analysis: FTIR spectra of (a) Commiphora wightii aqueous extract; (b) Phytosynthesized gold nanoparticles (Cw@AuNPs)

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Figure. 4 In vitro cytotoxic assay: Cytotoxic effect of Cw@AuNPs on MCF-7 (n=3) Figure. 5 Apoptotic studies: (a) AO/EtBr staining assay using fluorescent microscopy; (b) Flow Cytometry analysis using Annexin-V/FITC/PI staining; (c) Alkaline comet assay for dsDNA break analysis.

Figure. 6 Comet assay: Bar graph represents mean olive tail moment of the comet. Data

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expressed as mean ± SD. Statistical significance was determined using one-way ANOVA followed by Dunnet’s multiple comparison test(***p<0.0001)

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Figure. 7 Cell cycle analysis: Effect of Cw@AuNPs on MCF-7 cell cycle (n=3).

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