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Anticancer activity of gold nanobioconjugates synthesized from Elephantopus scaber (linn.) leaf extract


1 Department of Biotechnology, Institute of Science, Mumbai, Maharashtra, India
2 Department of Botany, Institute of Science, Mumbai, Maharashtra, India

Date of Submission04-Aug-2020
Date of Decision01-Dec-2020
Date of Acceptance25-Dec-2020
Date of Web Publication23-Jul-2021

Correspondence Address:
Vijay D Mendhulkar,
Department of Botany, Institute of Science, 15, Madame Cama Road, Fort, Mumbai-32, Maharashtra
India
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jcrt.JCRT_1043_20

 > Abstract 


Introduction: Medicinal plants are the major natural resources for the treatment of human ailments including cancer therapy. The current cancer treatments such as surgery, radiation, and chemotherapy affect normal cells too. Thus, treatments like synthesized nanoscale particles using plant extracts have proven to be potential anticancer agent.
Aim of the Study: We hypothesize that the gold nanoparticles (AuNPs) synthesized using Elephantopus scaber hydro-methanolic extract may have anti-cancer activity along with their synergistic counterparts with adriamycin (ADR) on human breast cancer MCF-7: human breast cancer (A-549), human oral cancer (squamous cell carcinoma [SCC]-40), and COLO-205: human colon cancer cell lines.
Materials and Methods: The phytosynthesized AuNPs were characterized for ultraviolet-visible (UV-Vis) spectroscopy, nanoparticle tracking analysis (NTA), X-ray diffraction, scanning electron microscopy, transmission electron microscopy (TEM), and Fourier transform infrared (FTIR) analysis. The anticancer ability of the AuNPs against human MCF-7, A-549, SCC-40, and COLO-205 through sulforhodamine B assay has been studied.
Results: The synthesis of AuNPs was confirmed with the UV-Vis spectrophotometer with a peak at 540 nm. The FTIR analysis showed polyphenolic groups were major found to be the reduction and capping agent for AuNPs. According to the results obtained, AuNPs showed good anti-proliferative activity with GI50 <10 μg/ml on MCF-7 cancer cell line. The synergistic effect of AuNPs + ADR was even better for all the four cell lines than that of the AuNPs alone.
Conclusion: The green synthesis of AuNPs is a simple, eco-friendly, and cost-effective method with dominantly spherical morphology ranging from 20 to 40 nm confirmed by NTA and TEM analysis. The study reveals the potent therapeutic value of the AuNPs.

Keywords: A-549, anticancer, COLO-205, Elephantopus scaber, gold nanoparticles, MCF-7, squamous cell carcinoma-40, Sulforhodamine B assay



How to cite this URL:
Shinde AS, Mendhulkar VD. Anticancer activity of gold nanobioconjugates synthesized from Elephantopus scaber (linn.) leaf extract. J Can Res Ther [Epub ahead of print] [cited 2021 Dec 5]. Available from: https://www.cancerjournal.net/preprintarticle.asp?id=322157




 > Introduction Top


For drug formulation and the treatment of human ailments, medicinal plants are used as a major natural source. Synthesized nanoscale particles (SNPs) are one of the recent nanobiotechnology production processes from the medicinal plants. Due to the unique properties such as size, distribution and morphology, synthesis of nanoscale particles has been an economic, eco-friendly, and easily scaled up without the use of any toxic chemicals.[1],[2] SNPs have tremendous applications for drug delivery,[3] gene therapy,[4] catalysts,[5] bio labeling, anti-fungal,[6] and anti-oxidant.[7] The growing need to develop nontoxic and eco-friendly procedures for SNPs have resulted in more researchers to work in biological systems. Using chemical and physical methods, nanoparticles (NPs) are synthesized using various metals such as Au, Ag, Zn, Pd, Cu. Among these NPs, the most attention is seeked by gold NPs (AuNPs) due to their applications in photoimaging, angiogenesis, photothermal therapy, genetic diseases, genetic disorder diagnosis and cancer therapy and imaging.[8] AuNPs has been designed and developed for drug delivery like anticancer drugs (e.g., doxorubicin, methotrexate, and paclitaxel) to reach its specific targets.[9] Chemical methods used for the synthesis of metallic NPs use chemicals such as sodium borohydride, sodium hydroxide, sodium dodecyl sulfate, sodium citrate, trisodium citrates, 2-mercaptobenzimidazole and N, N-dimethyl formamide as reducing and capping agent of the metallic ion. These materials are harmful for both human health as well as environment. Thus, green nanotechnology is most eco-friendly with various methods to design nanomaterials for biomedical applications.[10]

Elephantopus scaber L., a scabrescent, erect, perennial herb belongs to the family Asteraceae, found in Neotropics, Europe, Asia, Africa, and Australia. E. scaber Linn., commonly known as Prickly-leaved elephant's foot, is the lectotype species of stiff wiry herb Elephantopus, L. with around 30 species under the family of Compositae.[11] E. scaber is a common wild weed that forms undergrowth in shady places and grows vigorously in tropical deciduous forest areas.[12] Conventionally, the plant was used as folk medicine by tribal communities across India for the treatment of rheumatism, dysentery, gout, gummosis, toothache, spider, and snakebite.[13] A decoction of the whole plant has been used to treat gonorrhea and colic pain while hot water extract of the root is applied for filariasis.[14] The root extracts of E. scaber L. showed anti-diarrheal activity and PGE2-induced enteropooling in rats.[15] It had also shown the anti-hepatotoxic effect on carbon tetrachloride-induced hepatotoxicity in rats.[16] Even it showed hypoglycemic effects in alloxan-induced diabetic rats.[17] E. scaber L. has mainly been a matter of research interest for many workers due to its importance in particular anti-cancer characteristics.


 > Materials and Methods Top


Experimental system and chemicals

E. scaber L. was used as an experimental system for the phytosynthesis of metal NPs and further to check its effect on cancer cell culture. The plant material of E. scaber (healthy and disease-free plant sample) was collected from Matheran, Raigad district, MS, India. The plant was taxonomically authenticated from Department of Botany, Blatter Herbarium, St. Xavier's College, Mumbai (Voucher specimen no. 2943 of H. Santapau). The studied experimental system was chosen because it has potent polyphenols, flavonoids, sesquiterpenes, sterols, and alkaloids compound which may act as good stabilizing agents during the synthesis of NPs and are well-known for their anticancer activity. The plant material was washed, cleaned, air-dried, and finely powdered for further use. Hydrogen tetrachloroaurate (III) hydrate (HAuCl4) is of Alfa Aesar make with 99.9% purity. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) was purchased from Sigma Aldrich.

Preparation of Elephantopus scaber leaf extract

The soxhlet extraction of E. scaber leaf powder was carried out using solvents as 60% methanol. The extraction was carried out till the solvent appears to be colorless at temperatures 70°C. The obtained extract was further filtered with Whatman filter paper no. 1 and concentrated using a rotary evaporator. It was further dried completely and kept in the refrigerator (4°C) for further analysis.

Experimental synthesis of gold nanoparticles

The synthesis of AuNPs was carried using H-MeOH soxhlet extract. The H-MeOH extract (2 mg/ml) was dissolved in 60% methanol (10 ml) using water bath sonicator for about 15–20 min. This extract was then diluted to 100 ml with distilled water and homogenized. The HAuCl4 salt was added to the solution to obtain 1 mM of concentration. It was kept on a shaker in dark for overnight. The solution turned from yellow color to a red wine color. The change in color indicates the synthesis of AuNPs. The NPs formed were recovered by centrifugation at 10,000 rpm for 30 min at 10°C and washed three times with distilled water by centrifugation. The NPs obtained were dried completely to obtain crystals and were stored at 4°C.[18]

Characterization methods for nanoscale particles

The physical properties such as particle size, distribution, pattern stability, surface morphology, and surface capping of phytoconstituents were focused with priority for the confirmation and characterization of the synthesized nanoscale particles. The bioreduction of Au+ ions in plant extract sample was observed using ultraviolet-visible (UV-Vis) Spectroscopy (Model– Shimadzu UV 1800, Germany). This AuNPs mixture was scanned in a range of 200–1100 nm. Nanosight UK–LM20 instrument was used for NTA analysis by light scattering technique. The diffraction pattern of phytosynthesized AuNPs was recorded from a diffraction angle of 20° to 80° using Rigaku mini flex benchtop X-ray spectrophotometer. The possible phytoconstituents responsible for surface capping and reduction of Au ions into AuNPs using H-MeOH E. scaber leaf extract was determined using Fourier transform infrared (FTIR) analysis (Perkin Elmer FTIR spectrum– 100 model). The size of the AuNPs was determined using scanning electron microscopy (SEM) analysis while transmission electron microscopy (TEM) was used to visualize the morphology of the AuNPs.

Diphenyl-1-picrylhydrazyl scavenging assay

The DPPH assay is well known for natural products antioxidant studies. This method is simple and sensitive. This assay is based on the theory that DPPH accepts hydrogen from an antioxidant compound. DPPH radical scavenging method was used to evaluate the free radicals scavenging activity of different concentration of soxhlet extract of E. scaber leaves, phytosynthesized NPs, and of standard ascorbic acid.[19]

DPPH (0.1 mM) solution in methanol, ascorbic acid (10 mg/ml) in methanol, Tris HCl buffer (100 mM, pH 7.4), and samples (1 mg/ml) were prepared. In 96 well plate, tris HCl buffer was added to the sample (1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 μl) to make up 100 μl. Further, 150 μl of DPPH was added to this mixture. A solution of ascorbic acid (2 μl) with methanol (8 μl), Tris HCl buffer (90 μl), and DPPH (150 μl) solution was used as a positive control. The negative control was used as methanol (10 μl) added to tris HCl buffer (90 μl) and DPPH solution (150 μl). Distilled water was used as a blank. The plate was incubated for 30 min in dark to carry out the reaction. The absorbance was measured spectrophotometrically at 517 nm using the multi-mode reader. The data were triplicated and averaged. The standard deviation (SD) was determined. The scavenging ability of leaf extracts and AuNPs on DPPH was calculated using the following equation:



To determine the concentration of the sample required to scavenge 50% of DPPH-free radical (IC50), the curve of per cent inhibition was plotted against the respective concentration.

Anticancer studies using sulforhodamine B assay

For the present study, four human cancer cell lines were selected namely breast cancer cell line (MCF-7), colon cancer cell line (COLO-205), lung cancer cell line (A-549), and oral cancer cell line (squamous cell carcinoma [SCC]-40) using sulforhodamine B (SRB) assay[20] at Advanced Centre for Treatment, Research and Education in Cancer, Tata Memorial Centre, Mumbai.


 > Results and discussion Top


The present study is regarding the green synthesis of AuNPs, its characterization, assessment of anti-oxidant and anti-cancer activity. The E. scaber plant is an excellent source of secondary metabolites in medicinal viewpoint. Thus providing greater scope for biocompatible production of nanoscale particles. The characteristic and applicative findings of the AuNPs synthesized using H-MeOH E. scaber leaf extract are discussed below.

Characterization of gold nanoparticles

The formation of AuNPs was initially confirmed spectrophotometrically. The color change for the AuNPs was from yellow to wine-red color after the addition of 1 mM concentration of the HAuCl4 salt to the E. scaber H-MeOH leaf extract. Plasmon resonance singularity provides a fitting signature to signify the formation of AuNPs. The color of the reaction mixture is attributable to the excitation of surface plasmon vibrations. The reduction of Au ion to AuNPs was reflected in spectral data obtained using UV-Vis. Spectrophotometer. A sharp peak of AuNPs was observed at 540 nm [Figure 1]. While the Jasminum auriculatum leaf extract-mediated AuNPs revealed UV-Vis absorption spectra with well-defined surface plasmon band centered at 547 nm, thus the presence of homogeneous hydrosol biogenic AuNPs.[21] While the AuNPs synthesized using Marsdenia tenacissima extract showed the highest peak at 542 nm by UV-Vis spectroscopy.[22] The NTA analysis of AuNPs revealed the mean size of 50 nm with a mode of 21 nm and SD of 53 nm. The concentration of the phytosynthesized AuNPs was found to be 2.24 × 108 particles/ml [Figure 2]. The particle size of the Panax notoginseng leaf extract-mediated AuNPs was found to be 100 nm using dynamic light scattering analysis.[23] In the present study, zeta potential analysis of the synthesized AuNPs was found to be-51.93 ± 7.87 mV. Zeta potential shows stability and the high surface charge prevents NPs aggregation due to electrostatic repulsion between NPs. While the zeta potential of the AuNPs synthesized using Hygrophila spinosa extract was to be-27.78 ± 0.66 mV.[10] The X-ray diffraction pattern of the E. scaber H-MeOH extract stabilized AuNPs prepared from gold chloride is shown in [Figure 3]. Four peaks at 2 θ values of 37.98°, 44.06°, 64.5°, and 77.38° corresponding to (111), (200), (220), and (311) planes of gold were observed and were well matched with the standard powder diffraction card of JCPDS, gold file No. 04–0784.[24],[25] No impurity peaks were observed in the XRD pattern indicating the high phase purity. SEM analysis shows high-density and monodisperse distribution AuNPs synthesized by the extract. It shows relatively spherical and uniform AuNPs of a particle in the range of 20–60 nm [Figure 4]. While the Energy dispersive x-ray spectroscopy (EDS) analysis of AuNPs showed the presence of the elemental gold, carbon, nitrogen, and oxygen [Figure 5]. The presence of the gold elements is attributed to the phytosynthesized AuNPs. Whereas, the carbon and oxygen are assigned to the bio-organic compounds present in the outer shell. A strong optical absorption peak is observed at 2.1 keV, which is typical for the absorption of metallic AuNPs. The TEM images of AuNPs synthesized using E. scaber H-MeOH extract are shown in [Figure 6] which predominates with spherical, oval, triangular, and hexagonal morphologies ranging from 20 to 40 nm. According to the Balasubramanian et al., the AuNPs synthesized using Jasminum auriculatum leaf extract are in spherical with 8–37 nm in size from the evidence of TEM analysis.[21] The TEM analysis of Scutellaria barbata extract-mediated AuNPs possess spherical shaped particles with typical size range of 0.4–1 μm.[26] SAED pattern for silver NPs synthesized using plant systems shows concentric rings with intermittent bright dots, an indication that the sample is highly crystalline. The rings are attributed respectively to diffraction from (111), (200), (220), and (311) planes of gold [Figure 7].
Figure 1: Ultraviolet-visible spectrophotometry of Elephantopus scaber H-MeOH leaf extract mediated gold nanoparticles

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Figure 2: Frequency of occurrence of varying sizes of synthesized gold nanoparticles calculated using nanoparticle tracking analyzer

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Figure 3: X-ray diffraction spectra of gold nanoparticles synthesized using Elephantopus scaber H-MeOH leaf extract

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Figure 4: Field emission scanning electron microscopy image of Elephantopus scaber H-MeOH leaf extract - mediated gold nanoparticles at 300 KX

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Figure 5: Energy-dispersive X-ray spectroscopy spectra of gold nanoparticles synthesized using Elephantopus scaber H-MeOH leaf extract

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Figure 6: Transmission electron microscopy image of Elephantopus scaber H-MeOH leaf extract-mediated synthesized gold nanoparticles

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Figure 7: Selected area electron diffraction pattern of gold nanoparticles using Elephantopus scaber H-MeOH leaf extract

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FTIR analysis was carried out to discover the potential biomolecules in E. scaber H-MeOH extract responsible for the reduction and then providing stability to the bioreduced AuNPs. Several absorption peaks were found similar to the peaks of E. scaber H-MeOH extract. The widest absorption peak is observed at 3430.80 cm−1 is due to stretching vibrations of O-H groups in alcohols, water, and phenols and N-H group in primary, secondary amines, and amides in proteins. Comparing the 3394 cm−1 bands of E. scaber H-MeOH extract with 3430.8 cm−1 of AuNPs, this peak–OH group is slightly shifted. This shift in the peak is due to the difference between the force of interaction between the hydrogen bond of two organic shells and water molecules. The bands at 2921.86 and 2854.38 cm−1 correspond to the C-H stretch in alkanes and O-H stretch in a carboxylic acid. The strong band at 1695.66 cm−1 is attributed to the C = O stretch in the amide I in proteins. While 1628.44 cm−1 corresponds to the C = C stretch in hydrocarbons. The 1408.19 cm−1 corresponds to C-C stretch in aromatic and aliphatic compounds. The C-N stretch of amide III in proteins gives the band at 1315.26 and 1253.34 cm−1. While, the C-O stretch of alcohols, carboxylic acids, esters, and ethers gives bands at 1202.20, 1157.45, 1116.90, 1098.98, and 1006.63 cm−1. It is evident from the results that the biomolecules present in the E. scaber H-MeOH extract are responsible for reduction and stabilization/capping of AuNPs [Figure 8] and [Figure 9]. The results of the biosynthesized metal NPs corroborate the stability with the help of free functional groups of various chemical constituents that present in the plant extract.[24],[27] The metal NPs were found stable in solution even after 4 weeks of their synthesis. There were no noticeable variations in the optical properties of the NP solution concerning time.
Figure 8: Fourier transform infrared spectroscopy spectra of Elephantopus scaber H-MeOH leaf extract

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Figure 9: Fourier transform infrared spectroscopy spectra of Elephantopus scaber H-MeOH extract-mediated gold nanoparticles

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Applications of synthesized nanoparticles

The advantages of using NPs for the delivery of the drug were derived from their 2 main properties–First, due to their smaller size, NPs can penetrate through capillaries and are absorbed by the cells. This causes efficient accumulation of the drug at the site of the target. Second, while the preparation of NPs, the use of biodegradable materials allows prolonged dispense of the drug within the target site for days or even weeks.[28] Thus, the synthesized metal NPs using H-MeOH E. scaber leaf extract were further used to analyze their anti-oxidant and anticancer activities.

Anti-oxidant activity

The anti-oxidant activity of AuNPs synthesized using H-MeOH E. scaber leaf extract was evaluated by DPPH radical scavenging assay. This activity was monitored by UV-Vis spectrophotometer. DPPH shows strong absorption at 517 nm which is purple. The scavenging effect of the samples on the DPPH was expressed as per cent radical scavenging activity. Ascorbic acid was used as a positive control. A calibration curve was plotted by percentage DPPH radical scavenging activity against concentration (μg/ml). The regression equation of linearity curve for AuNPs was y = 1.0739x + 46.576 and coefficient of determination (R2 = 0.9754). Dose-dependent scavenging activity of DPPH was observed. AuNPs were found to consist of the lowest IC50 value of 3.19 μg/ml while H-MeOH leaf extracts showed 18.73 μg/ml. According to Aslam et al., 50% ethanolic soxhlet extract of E. scaber had EC50 of about 15.67 μg/ml.[29] While IC50 of methanol and ethanol extracts from successive solvent extraction method of E. scaber are 195 μg/ml and 139.6 μg/ml, respectively.[30] Shabestarian et al. reported the free radical scavenging activity of biosynthesized AuNPs by DPPH scavenging method. The NPs showed dose-dependent activity and DPPH scavenging effect was 13.43% at a concentration of 25 μM and then reached to 85.73% at 800 μM of AuNPs.[31]

Anti-cancer studies

One of the important aims of the present study was to evaluate our compounds and its synergistic effect for anti-proliferative properties in vitro against cancer cell lines. The treatment of AuNPs, AuNPs + ADR, H-MeOH extract and H-MeOH extract + ADR was administered on breast cancer cell line (MCF-7), lung cancer cell line (A-549), oral cancer cell line (SCC-40), and colon cancer cell line (COLO-205) by performing SRB assay. Adriamycin (ADR) drug was used as a reference compound. The susceptibility of cells to the drug exposure was characterized by GI50 (IC50; concentration of drug causing 50% inhibition of cell growth), TGI (concentration of drug causing total inhibition of cell growth), and LC50 (concentration of drug causing 50% cell kill) values [Table 1].
Table 1: LC50, TGI, and GI50 values of MCF 7, A-549, SCC-40 and COLO-205 cell lines after treatment with test drugs

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MCF-7 cancer cells were treated with different concentrations of the samples for 24 h. Lowest anticancer activity was found in E. scaber H-MEOH leaf extract when compared with its synergistic activity. Thus, in this case, the effect was shown by the ADR alone. Percentage control growth is graphically represented in [Figure 10]. Among the AuNPs, the 10 μg/ml concentration exhibits the least per cent control growth of-26.9%. AuNPs alone treatment was superior compared to the AuNPs+ ADR combination treatment especially the 10 μg/ml concentration. The GI50 value for AuNPs, AuNPs + ADR and H-MeOH extract + ADR was <10 μg/ml, which is in similar to that of the positive control, ADR While the TGI value for AuNPs was calculated to have 36.1 μg/ml of concentration [Table 1]. Treated cells showed morphological changes characterized by shrinkage, irregular shape, and cytotoxicity. The microscopic photos of various sample treatments are illustrated in Photoplate [Figure 11]. According to Krishnaraj et al. reported at 100 μg/ml concentration, the Acalypha indica plant extract derived AgNPs and AuNPs exhibited significant cytotoxic effects and apoptotic features were confirmed through caspase-3 activation and DNA fragmentation assays.[32] An in vitro cytotoxicity of Hygrophila spinosa extract-mediated AuNPs was studied by Satpathy et al. against MCF-7 cancer cell line at different concentrations (ranging from 12.5 to 200 μg/ml) using MTT assay. AuNPs exhibited dose-dependent cytotoxicity with percentage cell viability (at 200 μg/ml) was about 43.78%.[10] The in vitro anticancer potential of AuNPs synthesized using Commiphora wightii leaf extract evaluated against MCF-7 with IC50 found to be 66.11 μg/ml.[33]
Figure 10: Growth curve of MCF-7 cancer cell lines for Elephantopus scaber H-MeOH leaf extract, gold nanoparticles and its synergistic activity with adriamycin

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Figure 11: Effect of test drug on MCF-7 as observed under inverted microscope

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The effect of various treatments on A-549 cancer cell line is shown in percentage control growth as graphically represented in [Figure 12] and Photoplate [Figure 13]. AuNPs exhibited-55.8% control growth at the highest concentration of 80 μg/ml. When compared to AuNPs, the synergistic effect of AuNPs + ADR at all four concentrations gives better results. The best results were observed at 10 μg/ml exhibiting-33.0% growth control. The synergistic effect of E. scaber H-MeOH leaf extract with ADR showed a decrease in the percentage growth control. This may be due to the ADR alone. The synergistic effects of AuNPs + ADR and H-MeOH extract + ADR show the GI50 value of less than 10 μg/ml. AuNPs gave GI50 value of 32.0 while the H-MeOH extract shows no effect according to the GI50 value (>80 μg/ml). It is evident from [Figure 14], that the AuNPs + ADR, H-MeOH + ADR and positive control (ADR) leads to the retardation of cell growth followed by the AuNPs and H-MeOH extract. In the context of per cent cell growth inhibition, ADR showed a linear cytotoxic response concerning drug concentration. According to Rajeshkumar, AuNPs were synthesized using marine bacteria Enterococcus sp. showed significant anti-cancer activity against A-549 cells at 100 μg concentration of NPs.[34] In another study, Qian et al. reported the synthesis of AuNPs using Cetuximab (unique agent, targeting epidermal growth factor receptor EGFR-positive cancer). Marsdenia tenacissima plant extract-mediated AuNPs showed dose-dependent cell growth inhibition (IC50 = 15 μg/ml) in in vitro anticancer activity against A-549 cancer cell line.[35] The anticancer efficacy of Magnolia officinalis mediated AuNPs assessed against A-549 cells showed cytotoxic activity and apoptosis caused by apoptotic gene expressions in the A-549 cells.[36] While for Rabdosia rubescens mediated AuNPs, the IC50 dose against A-549 lung carcinoma cell line was detected to be 25 μg/ml using MTT assay with potent anticancer and apoptotic effect.[37]
Figure 12: Growth curve of A-549 cancer cell lines for Elephantopus scaber H-MeOH leaf extract, gold nanoparticles and its synergistic activity with adriamycin

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Figure 13: Effect of test drug on A-549 as observed under inverted microscope

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Figure 14: Growth curve of squamous cell carcinoma-40 cancer cell lines for Elephantopus scaber H-MeOH leaf extract, gold nanoparticles and its synergistic activity with Adriamycin

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The treatments of two samples (AuNPs and E. scaber H-MeOH leaf extract) and their synergistic effects with the standard drug (ADR) through the SRB assay was evaluated on SCC-40 cell line. Results of cytotoxicity on SCC-40 cell line are tabularized in [Table 1] and graphically represented in [Figure 14]. In the case of SCC-40, the AuNPs exhibited low activity for the first three concentrations but the 80 μg/ml concentration range produced good cytotoxicity. While the E. scaber H-MeOH extract showed no such activity. Here, AuNPs + ADR showed maximum activity followed H-MeOH + ADR, AuNPs and H-MeOH extract. The synergistic anti-proliferative activities were evident in SCC-40 cell line. AuNPs and E. scaber H-MeOH extract when combined with ADR, gives better results (<10 μg/ml) compared to the test drugs alone treatments. SCC-40 cells treated with the different treatments for 24 h shows morphological changes [Figure 15]. Kah et al. carried out a study which demonstrated the potential of antibody-conjugated gold NPs to target and eliminates oral cancer cells under a reflectance-based optical imaging system. In particular, they have shown that AuNPs can provide an optical contrast to discriminate between cancerous and normal cells and their conjugation with antibodies also allows them to map the expression of relevant biomarkers for molecular imaging.[38] El-Sayed et al., firstly employed anti-epidermal growth factor receptor antibody conjugated gold nanospheres to image and treat oral tumor cells in vitro, in which a continuous argon laser at 514 nm, the peak absorbance of 40 nm particles, was used. Compared with normal and noncancerous cells, it was observed by these workers that the cancerous cells targeted with NPs were destroyed with 2–3 times lower the laser power.[39]
Figure 15: Effect of test drug on squamous cell carcinoma-40 as observed under inverted microscope

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In case of COLO-205, AuNPs and H-MeOH leaf extract treatments exhibited low activity for first three concentrations (10, 20, and 40 80 μg/ml) but the 80 μg/ml concentration range produces strong cytotoxicity. While the synergistic effects of the samples (AuNPs + ADR and H-MeOH + ADR) showed maximum activity [Table 1]. The test samples when combined with the ADR, gives better results compared to the individual test samples. The GI50 value for the synergistic effects of the test samples showed <10 μg/ml along with the ADR standard. While the GI50 values for AuNPs sample were observed to be 24.7 μg/ml. H-MeOH extract showed GI50 values >80 μg/ml. The results for cytotoxicity on human colon COLO-205 cancer cell line are graphically presented in [Figure 16] and the morphological changes are shown in Photoplate [Figure 17]. Brown et al. tethered the active component of the anticancer drug oxaliplatin to a AuNP for improved drug delivery. Naked AuNPs were functionalized with a thiolated poly (ethylene glycol) (PEG) monolayer capped with a carboxylate group. [Pt (1R,2R-diaminocyclohexane)(H2O)2]2NO3 was added to the PEG surface to yield a supramolecular complex with 280 (20) drug molecules per NP.[40]
Figure 16: Growth curve of COLO-205 cancer cell lines for Elephantopus scaber H-MeOH leaf extract, gold nanoparticles and its synergistic activity with Adriamycin

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Figure 17: Effect of test drug on COLO-205 as observed under inverted microscope

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After SRB staining, the cytotoxic effect of these treatments were visualized under an inverted microscope showing morphological changes such as shrinking of cells, low cell count, and irregular shape which shows the cytotoxicity of the treatments.


 > Conclusion Top


There are several studies carried in nanobiotechnology for medicinal plants. However, there are no reports available on gold nanoscale particles synthesized in E. scaber used in anti-proliferative studies. Thus, we carried out the phytosynthesis of AuNPs using E. scaber H-MeOH extract, its characterization, anti-oxidant, and anti-cancer applications. Visual observations of colour change from yellow to wine red confirmed the synthesis of AuNPs. UV-Vis spectrophotometer confirmed the AuNPs with a peak at 540 nm. NTA analysis revealed the mean size of the AuNPs as 50 nm with the frequency of 2.24 × 108 particles/ml. The spherical morphology and aggregation were confirmed by TEM and SEM analysis, respectively. XRD spectra exhibited four 2 θ peaks which correspond to the planes of gold. FTIR analysis revealed the presence of polyphenols, aromatic, carboxylic acid, esters, ethers, polysaccharide, amino acids, and proteins as the major factors in the reduction and capping of the AuNPs. Dose-dependent scavenging activity of DPPH was shown by AuNPs with the lowest IC50 value of 3.19 μg/ml. The anticancer abilities of AuNPs were studied with human MCF-7 (breast), A-549 (lung), SCC-40 (oral), and COLO-205 (colon) cancer cell lines with reference of standard anticancer drug ADR using the SRB assay. According to the cytotoxic analysis, AuNPs is a good antiproliferative agent on MCF-7 giving GI50 <10 μg/ml. While for A-549, SCC-40, and COLO-205 the GI50 was reported to be 33.0, 28.3, and 24.7 μg/ml, respectively. The synergistic drug combination treatments unearthed better results for all four cell lines with <10 μg/ml. The findings of this study exhibited efficient antiproliferative results of E. scaber H-MeOH-mediated AuNPs on studied human cancer cell lines.

Acknowledgment

We would like to acknowledge Sophisticated Analytical Instrument Facility (SAIF), Indian Institute of Technology, Powai, Mumbai; Officer In-charge of In-vitro SRB assay for anticancer activity evaluation of drugs, Anti-Cancer Drug Screening Facility (ACDSF), ACTREC, Tata Memorial Centre, Kharghar, Navi Mumbai and The Institute of Science, Mumbai, for providing facilities. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10], [Figure 11], [Figure 12], [Figure 13], [Figure 14], [Figure 15], [Figure 16], [Figure 17]
 
 
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