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Eco-friendly synthesis of gold nanoparticles using fruit extracts and in vitro anticancer studies
Journal of Saudi Chemical Society (2019) 23, 753–761
King Saud University
Journal of Saudi Chemical Society www.ksu.edu.sa www.sciencedirect.com
ORIGINAL ARTICLE
Eco-friendly synthesis of gold nanoparticles using fruit extracts and in vitro anticancer studies S. Vijayakumar Department of Science and Humanities, Sri Ramakrishna Institute of Technology, Coimbatore, India Received 12 November 2018; revised 17 December 2018; accepted 20 December 2018 Available online 30 December 2018
KEYWORDS Gold nanoparticles; Aegle marmelos; Eugenia jambolana; Soursop; Anticancer activity
Abstract Gold nanoparticles are biocompatible and are having several applications in biomedical Sciences and Engineering. Integration of nanoscience in medicine leads to the development of biomedical products that helps the Society in a faster and safer manner. In the present research work, bioreduction and biofunctionalization of gold nanoparticles are performed with fruit extracts of Aegle marmelos, Eugenia jambolana and soursop. The nanoparticles are characterized using UV– Vis spectroscopy, Transmission Electron Microscopy, Fourier Transform Infrared Spectroscopy and Zeta potentiometer. The qualitative phytochemical analysis of the fruit extracts shows the presence of alkaloids, amino acid, flavonoids, phenol, proteins, tannin, reducing sugars and total Sugars. The in vitro anticancer activity was confirmed by MTT assay on the human breast cancer cell line MCF-7 at different concentrations. The flavonoids present in the fruit extracts are potential reducing agent which is responsible for the formation of gold nanoparticles. Stabilization of gold nanoparticles are performed by the carboxylate group present in the proteins. Also, the nanoparticles are held apart from each other by the electrostatic repulsions that exist due to the presence of like charges surrounding the gold nanoparticles. This study proves that the fruit extracts can be used for the synthesis and stabilization of gold nanoparticles. Further, the engineered nanoparticles capped with bioactive compounds are potential anticancer agents against breast cancer cell line MCF-7. Ó 2019 King Saud University. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction Nanotechnology is an interdisciplinary domain of technological pool containing Physics, Chemistry, Biology, E-mail address: [email protected] Peer review under responsibility of King Saud University.
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Medicine and Material Science having applications ranging from material science to personal care products [1], Quantum dot-based photoelectric conversion for immunoassays [2] and development of an improved photoelectrochemical sensing platform [3]. The smaller size and unique properties of the nanoparticles have substantially improved their application in different areas such as anti HIV agent [4] and anticancer agent as well as therapy [5–7]. Green synthesis of gold nanoparticles are possible and has better stability over the conventional methods of preparation [8–10]. Different parts of the fruits, plants and biological entities are used for the
https://doi.org/10.1016/j.jscs.2018.12.002 1319-6103 Ó 2019 King Saud University. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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Fig. 1 The schematic illustration of the development of fruit extracts using soxhlet apparatus and the formation of gold nanoparticles using aegle marmelos, eugenia jambolana and soursop fruit extracts.
preparation of nanoparticles [11]. The nanosize and distinctive properties of nanoparticle have considerably improved in the recent years [12]. There is a variety of recent articles highlighting the advantages of nanoscale materials used for the investigation into biomedical applications [13–17]. During the past twenty years, technology shows a convergence of nano-engineering and nanomedicine, giving rise to many new technologies and ideas that can be achieved in medicine [18–21]. Nature has perfected with the art of biology at nanoscale and many of the inner workings cells naturally occur at the nanoscale. Nanoscale materials have a larger surface area to volume ratio than bulk materials. The larger surface area of nanoparticles enhances the chemical reactivity which helps to create better catalytic applications. The preparation of gold nanoparticles using plant extracts providesan efficient and environmentally friendly method to produce well defined size of nanoparticles [22]. Eugenia jambolana has immense health benefits. It is believed to be of a special use in treatment of diabetes and diarrhea. Several studies provide evidence that it has hypoglycemic activity by stimulating insulin secretion with up to 30 per cent reduction in blood sugar [23]. Literature reveals that the Aegle marmelos commonly has a fraction of ethyl acetate suppressed TNF – alpha-mediated, translocation of NF kappa B, ERK phosphorylation and inhibited AKT in tumor xenografts [24]. Soursop fruit extracts has anticancer activities that kill cancer cells and leaving healthy cells untouched. The benefits of soursop fruits are that they alleviate cough, back pain, stress, migraine headache and high blood pressure. Also, it showed cardio depressant activities in animals and anti-
cancer activities and is a more effective agent against multidrug resistant cancer cell lines [25–28]. In the present research work, chloroauric acid is reduced by the flavonoids of fruit extracts. The carboxylate group present in the protein of the fruit extract stabilizes the particles through electrostatic repulsion. Also it proves the possibilities of green synthesis of gold nanoparticles (AuNps) capped with Aegle marmelos, Eugenia jambolana and soursop extracts and in vitro anticancer activity. The schematic methodology for the preparation of gold nanoparticles using fruit extract is shown in Fig. 1. 2. Materials and methods 2.1. Chemicals and reagents The chemicals used for the synthesis of AuNps are hydrogen tetrachloro aurate (HAuCl4H2O) which was purchased from Hi-media, Coimbatore, India. Doubly distilled water was used for the preparation of Aegle marmelos, Eugenia jambolana and soursop extracts. In vitro assay reagents such as Phosphate buffered saline (PBS), Dimethyl sulphoxide (DMSO), trypsin-ethylenediaminetetraacetic acid (EDTA) and Fetal bovine serum (FBS) reagents were purchased from Sigma Aldrich, India. Eugenia jambolana is a very popular and seasonal fruit being sold everywhere in India and as a roadside avenue tree. It was obtained from the local garden near Coimbatore, Aegle marmelos was obtained from the Perur Patteeswarar temple. Soursop was purchased from Kutralam
Synthesis of gold nanoparticles using fruit extracts
Fig. 2
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Schematic visualization of reduction and stabilization of gold nanoparticles using different fruit extracts.
forest base, Thirnelveli District, Tamilnadu. All the chemicals and solvents were used as received. The illustration of reduction and stabilization of nanoparticles by the phytochemical is shown in Fig. 2. 2.2. Preparation of fruit extracts The fruits are washed with deionized water several times and chopped into small pieces. Then, 100 mg of fruit is mixed with 100 ml of deionized water and put inside a glass bowl of soxhlet apparatus. The mixture is boiled continuously for 12 h. The evaporated gases are condensed and recirculated for boiling. The entire system was allowed to cool down to room temperature. The fruit extracts were stored at 4 °C after filtration with Whatman No. 1 filter paper and used within a week. 2.3. Phytochemical qualitative analysis Bioactive compounds like alkaloids, amino acid, flavonoids, phenol, proteins, tannin, reducing sugars and total Sugars present in Aegle marmelos, Eugenia jombolana and soursop are qualitatively analyzed [29–31]. The extracts were treated with various chemical reagents and their color reactions were observed to identify the presence of different phytoconstituents. 2.4. Preparation of gold nanoparticles using Aegle marmelos, Eugenia jambolana and soursop extracts The Aegle marmelos, Eugenia jambolana and soursop fruit extracts were used to obtain phytochemical derived reducing and stabilizing agents for the generation of AuNps. About 1 ml of 5 mM tetra chloroauric acid (HAuCl4) was diluted by adding 50 ml of deionized water and heated until it boils. Then 10 ml of Aegle marmelos, Eugenia jambolana and soursop extracts was added in a separate beaker containing gold chloride solution. The reaction mixture was stirred well continuously using a magnetic stirrer. The color of the solution turned wine red from pale yellow within 5 min of the addition of fruit extracts, which indicates the formation of AuNps. The reaction mixture was allowed to cool after 10 min. AuNps stabilized with Aegle marmelos, Eugenia jambolana and soursop extracts were labeled as S1, S2 and S3.
2.5. Cytotoxicity studies MCF-7 cells are widely studied model for hormone-dependent human breast cancer. They are derived from a pleural effusion of a malignant breast cancer and having estrogen and progesterone receptors. The human breast cancer cell line, Michigan Cancer Foundation-7 (MCF-7) was received from National Centre for Cell Science at Pune. The cell lines are grown in 10% fetal bovine serum (FBS) contained in Eagle minimum essential medium. The cells were maintained in an incubator with 95% air and 100% relative humidity at 37 °C in 5% CO2. The viable cells were counted using a hemocytometer and diluted with medium containing 5% FBS to give a final density of 1 105 cells/mL. The cytotoxicity evaluation of Aegle marmelos, Eugenia jambolana and soursop extract stabilized AuNps was performed using tetrasolium dye MTT assay as described by Mossman [32]. About 1 105 mL 1 cells of MCF-7 in their experimental growth phase was seeded in a flat bottomed 96 well polystyrene coated plate and incubated for cell attachment. The cells were treated with serial concentrations (0, 20, 40, 60, 80, 100, 120) mgm/mL of the test samples after 24 h. About 100 ml of these different sample dilutions was added to the appropriate wells already containing 100 ml of the medium. Further, the plates were incubated for 48 h. Each well was added with phosphate buffered saline (PBS) containing 15 ml of MTT (5 mg/mL) reagent and incubated at 37 °C about 4 h. The medium without samples served as control. The medium with MTT was then flicked off and the formosan crystals were solubilized in 100 ml of DMSO. The absorbance was measured at 570 nm using a micro plate reader. The percentage of cell viability was then calculated with respect to control. 3. Characterization Characterization is a collection of tools to create knowledge on synthesis, properties and applications of nanoparticles. AuNps synthesized using Aegle marmelos, Eugenia jambolana and soursop fruit extracts were characterized by Ultraspec-3000 series UV–Visible spectrophotometer (Department of Nanoscience and Nanotechnology, Sri Ramakrishna Engineering College, Coimbatore, India) to study the peak absorption band. About 1 ml solution of AuNps in water was used.
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The formation of AuNps was confirmed by UV–visible spectroscopy, which is based on localized SPR band exhibited at a particular wavelength. High Resolution Transmission Electron Microscopy, JEOL JEM 2100 operating at an accelerating Voltage of 200 KV with a lattice resolution of 0.14 nm and point to point resolution of 0.19 nm (PSG Institute of Advanced Studies, Coimbatore, India) was used to confirm the formation of nanoparticles, particle distribution and size range. The crystalline nature of nanoparticles can be analyzed with the help of SAED pattern. The AuNps images were formed from the interaction of electron transmitted through the thin film of specimen. The Zeta potentiometer quantifies the effective electric charges on the surface of nanoparticles. The stability of the particles are directly proportional to the magnitude of the Zeta potential. The electrostatic repulsion increases with the increase in zeta potential and stability as well. Zeta potential measurements were performed using a Malvern Instruments Zeta sizer 1000 Hs operating with a variable power of (5–50 mW) He-Ne laser at 633 nm (Sri Ramakrishna Engineering College, Coimbatore). SHIMADZU FTIR spectrometer was used to identify the FTIR spectrum. The FTIR spectra of the sample were recorded in KBr phase in the frequency region of 10,000–400 cm 1. The cytotoxicity assay on AuNps capped with Aegle marmelos, Eugenia jambolana and soursop on breast cancer cell lines were analyzed by MTT assay and was performed in KMCH Pharmacy College, Coimbatore. 4. Results The curative properties of medicinal plants are possibly due to the presence alkaloids, flavonoids, phenols, alkaloids, tannins and proteins. The ethanolic extract of whole fruits was found to have more constituents of phytochemicals. The results of phytochemical analysis of fruit extracts are shown in Table 1. Further it shows that the qualitative phytochemicals present in soursop is more than that of Aegle marmelos and Eugenia jambolana. The reduction of hydrogen tetra chloroaurate into AuNps during exposure to the fruit extracts could be followed by color change in the solution. AuNps exhibit dark pinkish and wine red color in aqueous solution due to the SPR phenomenon. When metal absorbs light of a resonant wavelength, it causes the electron cloud to vibrate, thus dissipating the
Table 1
energy. This process takes place on the surface of nanoparticles and therefore called Surface Plasmon Resonance (SPR). The collective oscillations of electrons of a AuNps upon interaction with light of suitable energy cause the nanoparticles to obtain a specific color. The frequency of oscillation is strongly dependent on the particle size, shape, and aggregation. The SPR were found to be 519 nm, 523 nm and 526 nm for Aegle marmelos, Eugenia jambolana and soursop fruit extracts stabilized AuNps. The SPR peak increases with increase in particle size, which is shown in Fig. 3. The TEM image of gold nanoparticle is shown in Fig. 4 (a), (b) and (c) and SAED patterns are shown in Fig. 4 (d), (e) and (f) respectively. TEM images show the spacing between the AuNps. Several particles with a diameter greater than the specified sizes were also present, but it was suspected that these particles arose from the overlapping of two or more small particles [33]. The phytochemicals present in fruit extracts act as a reducing as well as a stabilizing agent. It helps the nanoparticles to stay apart and thus provides stability to nanoparticles. The diffraction patterns obtained for AuNps are (1 1 1), (2 0 0), (2 2 0) and (3 1 1) reflections which are face-centered-cubic (FCC) structure of nanoparticles. The corresponding d-spacing are calculated as 2.40 A˚, 2.05 A˚, 1.467 A˚ and 1.22 A˚. The SAED shows that the samples have crystalline AuNps as deciphered from diffraction pattern using X-rays. The average size of the AuNps was found to be 18 nm, 28 nm and 16 nm, respectively for Aegle marmelos, Eugenia jambolana and soursop fruit extracts stabilized AuNps. Another characterization is Zeta potential which is a measure of the effective electric charge on the nanoparticle surface and quantifies the charges. This was already in the literature stating that the prepared AuNps are stable without any aggregation [34]. The zeta potential gives information about particle stability. The higher the magnitude of potential exhibits increased electrostatic repulsion and stability. The particle size distribution and zeta potential graph are shown in Fig. 5 (a), (b), (c) and (d), (e), (f) respectively. EDAX is an attachment to electron microscopy instruments and is one of an X-ray technique. The spectra show the peaks relevant to the composition of elements present in the sample. The qualitative analysis can be performed using the instrumental technique. The EDAX of bio functionalized AuNps showed strong signals for gold atoms and the weaker
Result of qualitative analysis of phyto constituents in fruit extracts.
Chemical test
Result
Phytochemical identified
Aegle marmelos
Annona muricata
Eugenia jambolana
Dragendroff’s Ninhydrin Alkaline reagent Ferric chloride Bromine Fehling Ninhydrin Anthrone reagent
Reddish brown precipitate Blue color Intense yellow coloration & disappeared on the addition of dilute HCl Bluish black color
Alkaloids Amino acid Flavonoids
++ + ++
+ + ++
++ + +
Phenol
++
++
+
Decoloration of Bromine Brick red color Violet color Blue-green color
Tannin Reducing sugars Proteins Total Sugars
++ + + +
++ ++ ++ ++
+ ++ ++ ++
Note: + lower, ++ higher.
Synthesis of gold nanoparticles using fruit extracts
Fig. 3 The Surface Plasmon Resonance of gold nanoparticles stabilized with aegle marmelos, eugenia jambolana and soursop fruit extracts.
signals for carbon, oxygen and chloride which is shown in Fig. 6. FTIR analysis was used to identify the chemical components responsible for the reduction of Au+ ions and stabilization. This measures infrared intensity versus wavelength (wavenumber) of light. It is used to determine the nature of associated molecules of plants or their extracts with nanoparticles. The FTIR of the bio-functionalized AuNps using the aqueous extracts of Aegle marmelos, Eugenia jambolana and soursop are shown in Fig. 7. The reduction of chloroauric acid may be due to the presence of flavonoids in the fruit extract [35]. The electrostatic repulsion among the nanoparticles are due to the presence of
757 carbohydrate group. It is found that the aqueous medium of fruit extract has the ability to perform dual functions of reduction and stabilization [26–39]. As noted above, the functional groups include primary amino group, ester moiety, mono substituted benzene ring and carboxylic group. The sharp peaks at 1639.49 cm 1 and at 1546 cm 1 were assigned to the C‚O stretching vibration (amide I) and a mixed vibration of NH deformation and CN stretch (amide II) in amides respectively. In addition, the IR bands at approximately 3371.59 cm 1 could also be observed, which may be assigned to O–H stretching vibration in the carboxyl moiety [40]. By contrast, the absorption band corresponding to amide II on the IR spectrum of AuNPs was broadened and blue shifted to 1560 cm 1, whereas the band corresponding to amide was also broadened but red shifted to 1620 cm 1. Generally, such broadening and shifting of the vibrational spectra have been ascribed to the formation of hydrogenbonded molecular complexes. Another feature of the gold particles was the presence of a strong and broad band centered at 3440 cm 1. On the basis of literature values [41], this band was attributed to be the various O–H stretching vibrations. The association of O–H O hydrogen bonding interaction with the ester moiety stretching was identified. Most importantly the peak at 1380 cm 1 was assigned to the bending of methyl group, 1226 cm 1 was assigned to C–O stretching of ester group and 1260 cm 1 was assigned to C–O stretching of a methoxyl group. Further IR clusters from 2800 cm 1 to 3200 cm 1 are attributed to aromatic CH of monosubstituted benzene. Also the intense band of C–O stretches in the ester moiety were identified at 1735 cm 1 and 1732 cm 1. It was thus concluded that chemical constituents of fruit extracts could serve as a reducing and stabilizing agent.
Fig. 4 (a), (b), (c) are Transmission electron micrograph and (d), (e), (f) are SAED pattern of AuNps stabilized with aegle marmelos, eugenia jambolana and soursop fruit extracts.
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Fig. 5 The Size distribution (a), (b), (c) and Zeta potentials (d), (e), (f) of gold nanoparticles stabilized with aegle marmelos, eugenia jambolana and soursop fruit extracts.
MTT cytotoxicity assay reveals that the sample were capable of metabolizing a dye (3-(4,5-dimethylthiozol-2-yl)-2,5-di phenyl tetrazolium bromide) efficiently. Spectrophotometric analysis was performed for the precipitate dissolved in detergent solution. After 24 h of treatment, it was found that the cell viability depends on the concentration of gold nanoparticles. The results demonstrate that the phytochemicals capping the nanoparticles acted upon the cancer cell lines and destroy them by influencing the chemical reactions. The concentration dependent toxicity of AuNps provides new opportunities for the safe application of nanoparticles in molecular therapy. Soursop capped gold nanoparticles treated MCF-7 cells exhibited cell death higher than the nanoparticles capped with Aegle marmelos and Eugenia jambolana. At higher concentration this shows more cell death which is about 30% more than others. The cell viability analysis is shown in Fig. 8. The result of this study suggests that the cytotoxicity of greener synthesized gold nanoparticles stabilized with soursop was increased with the increasing concentration. Also as per the experimental studies and literature survey the soursop is a fruit which has better anticancer effects. The IC50 values of AuNPs stabilized with Aegle marmelos, Eugenia jambolana and soursop fruit extracts are tabulated in Table 2.
5. Discussions Interestingly gold nanoparticles were formed within 30 min due to the rapid reduction of chloroaurate ions by the fruit extracts. It is clear from these studies that the plant extract mediated biosynthesis is very simple, fast, economic, ecofriendly and safe for human therapeutic use. Thus, the greener method of nanoparticles synthesis has a much reduced impact to the environment and has recently emerged as a viable alternative to conventional physical, chemical and even microbial methods. Research analysis proves that the biomoities present in the fruit extracts are responsible for the reduction and capping of nanoparticles [42–46]. The cytotoxicity of gold nanoparticles was increased with the increasing concentration of nanoparticles. Recent studies shows that the cytotoxicity depends on the stabilizing agent and size of the AuNps [47]. In the current work, MCF-7, human breast cancer cells showed 60% cell death at 120 mg/ ml concentrations of AuNps. In biomedicine, a lower concentration of drug has been used for treatment. Due to the presence of microvasculature in cancerous tumor, the AuNp based targeted drug delivery is possible to destroy the cancer cells by photothermal therapy. Therefore, it might be worth-
Synthesis of gold nanoparticles using fruit extracts
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Fig. 7 FTIR analyses of gold nanoparticles stabilized with aegle marmelos, eugenia jambolana and soursop fruit extracts.
Fig. 6 Elemental analyses of gold nanoparticles stabilized with (a) aegle marmelos, (b) eugenia jambolana and (c) soursop fruit extracts.
while to explore the biosynthesized nanoparticles as a possible source of novel anticancer drugs. 6. Conclusion Simple, economic and non-toxic AuNps were synthesized using Aegle marmelos, Eugenia jambolana and soursop fruit extracts and in vitro anticancer activity was studied. Qualitative tests confirmed the presence of bioactive phytoconstituents in these fruit extracts. FTIR spectra revealed the presence of reducing and stabilizing groups in the extract responsible for AuNp synthesis. The synthesized AuNps were near spherical. The in-vitro anticancer activity was confirmed by MTT assay on MCF-7 cell lines that showed IC50 values of soursop stabilized AuNps as 98 ± 4 lg/mL which is much better than AuNps stabilized with Aegle marmelos and Eugenia jambolana. This study suggests that green synthesized AuNps might be a potential agent in cancer therapy. These particles are easily sol-
Fig. 8 Cell lines in the exponential growth phase were exposed to different concentrations of AuNps stabilized with aegle marmelos, eugenia jambolana and soursop fruit extracts. Cell viability was determined by the MTT assay as described in the experimental section. Each result represents the mean viability ± standard deviation (SD) of three independent experiments and each of these was performed in triplicate. Cell viability was calculated as the percentage of viable cells compared to untreated controls.
Table 2 The summary of physical and chemical properties of gold nanoparticles. Sample
SPR absorption
Particle size
IC50 value (mg mL 1)
S1 S2 S3
519 nm 523 nm 526 nm
18 nm 16 nm 28 nm
172 ± 4 163 ± 4 98 ± 4
760 uble in water and stable for more than 5 months. The toxicity due to the neglected dimensions must be a major concern since AuNps have been widely used in biomedical applications. Further these green synthesized AuNps may be used in developing catalysts, sensors, nanomedicine, target delivery and biomarker applications. Funding This work was supported by the Tamil Nadu State Council for Science and Technology, Government of Tamil Nadu [grant numbers TNSCST/S&T Projects/AR/PHY/2011-2012/2124, 2012]. Acknowledgements This work was funded by the Tamilnadu State Council for Science and Technology, Government of Tamilnadu, India. The author acknowledges the Management, SNR Sons Charitable Trust, Dr. M. Paulraj, Principal, Sri Ramakrishna Institute of Technology; sincere thanks to Dr. S. Harihara Sivakumar, KMCH Pharmacy College, Coimbatore for providing facilities and support to execute the research work successfully. References [1] J. Shu, D. Tang, Current advances in quantum-dots-based photoelectrochemical immunoassays, Chem. Asian J. 12 (2017) 2780–2789. [2] J. Shu, Z. Qiu, S. Lv, K. Zhang, D. Tang, Plasmonic enhancement coupling with defect-engineered TiO2-x: a mode for sensitive photoelectrochemical biosensing, Anal. Chem. 90 (2018) 2425–2429. [3] D. Jain, H.K. Daima, S. Kachhwaha, S.L. Kothari, Synthesis of plant-mediated silver nanoparticles using papaya fruit extract and evaluation of their anti-microbial activities, Digest J. Nanomater. Biostruct. 4 (2009) 723–727. [4] S. Vijayakumar, S. Ganesan, Gold nanoparticles as an HIV entry inhibitor, Curr. HIV Res. 10 (2012) 643–646. [5] O.Y. Damoah, R. Wang, J. Zhou, Y. Ding, Versatile nanosystem-based cancer theranostics: design inspiration and predetermined routing, Theranostics 6 (2016) 986–1003, https:// doi.org/10.7150/thno.14860. [6] L. Mocan, C.T. Matea, D. Bartos, O. Mosteanu, T. Pop, T. Mocan, C. Iancu, Advances in cancer research using gold nanoparticles mediated photothermal ablation, Clujul Med. 89 (2016) 199–202, https://doi.org/10.15386/cjmed-573. [7] N.S. Abadeer, C.J. Murphy, Recent progress in cancer thermal therapy using gold nanoparticles, J. Phys. Chem. C 120 (2016) 4691–4716, https://doi.org/10.1021/acs.jpcc.5b11232. [8] K. Shameli, M.B. Ahmad, S.D. Jazayeri, S. Sedaghat, P. Shabanzadeh, H. Jahangirian, Y. Abdollahi, Synthesis and characterization of polyethylene glycol mediated silver nanoparticles by the green method, Int. J. Mol. Sci. 13 (6) (2012) 6639–6650. [9] K. Xin Lee, K. Shameli, M. Miyake, N. Kuwano, N.B.B. Ahmad Khairudin, S.E.B. Mohamad, Y.P. Yew, Green synthesis of gold nanoparticles using aqueous extract of Garcinia mangostana fruit peels, J. Nanomater. (2016) 1–9, https://doi.org/10.1155/2016/8489094. [10] S. Vijayakumar, S. Ganesan, Preparation and characterization of gold nanoparticles with different capping agents, Int. J. Green Nanotechnol. 3 (1) (2011) 47–55.
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Synthesis of gold nanoparticles using fruit extracts [28] C.C. Liaw, F.R. Change, C.Y. Lin, C.J. Chon, H.F. Chiu, M.J. Wu, Y.C. Wu, New cytotoxic monotetrahydrofuran annonaceous acetogenins from Soursop, J. Nat. Prod. 65 (2002) 470–475. [29] R.D. Gibbs, Comparative chemistry and phylogeny of flowering plants, Tans. R. Soc. Cancer (1974) 48. [30] J.B. Harborne, Phytochemical Methods. A Guide to Modern Techniques of Plant Analysis, 1st ed., Chapman and Hall, London, 1984. [31] G.E. Trease, W.C. Evans, Textbook of Pharmacognosy, 14th ed., Sanders, London, W.B., 1989. [32] T. Mosmann, Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays, J. Immunol. Methods 65 (1983) 55–63. [33] Z. Wang, Q. Zhang, D. Kuehner, A. Ivaska, L. Niu, Green synthesis of 1–2 nm gold nanoparticles stabilized by amineterminated ionic liquid and their electrocatalytic activity in oxygen reduction, Green Chem. 10 (2008) 907–909. [34] C. Umamaheswari, A. Lakshmanan, N.S. Nagarajan, Green synthesis, characterization and catalytic degradation studies of gold nanoparticles against congo red and methyl orange, J. Photochem. Photobiol., B: Biol. 178 (2018) 33–39. [35] A. Ghasemzadeh, N. Ghasemzadeh, Flavonoids and phenolic acids: role and biochemical activity in plants and human, J. Med. Plants Res. 5 (2011) 6697–6703. [36] J. Conde, J.T. Dias, V. Grazu´, M. Moros, P.V. Baptista, J.M. delaFuente, Revisiting 30 years of biofunctionalization and surface chemistry of inorganic nanoparticles for nanomedicine, Front. Chem. (2014), https://doi.org/10.3389/fchem.2014.00048. [37] E.C. Dreaden, L.A. Austin, M.A. Mackey, M.A. El-Sayed, Size matters: gold nanoparticles in targeted cancer drug delivery, Ther. Deliv. 3 (2012) 457–478.
761 [38] A. Mocanu, O. Horovitz, C.P. Racz, M. Tomoaia-Cotisel, Green synthesis and characterization of gold and silver nanoparticles, Rev. Roum. Chim. 60 (2015) 721–726. [39] B. Mondal, N. Kamatham, S.R. Samanta, P. Jagadesan, J. He, V. Ramamurthy, Synthesis, characterization, guest inclusion, and photophysical studies of gold nanoparticles stabilized with carboxylic acid groups of organic cavitands, Langmuir 29 (2013) 12703–12709. [40] L. Ning, W. De-Ning, Y. Sheng-Kang, Hydrogen-bonding properties of segmented polyether poly (urethane urea) copolymer, Macromolecules 30 (1997) 4405–4409. [41] V. Lin, N.B. Colthup, W.G. Fateley, J.G. Grasselli, The Handbook of Infrared and Raman Characteristic Frequencies of Organic Molecules, 1st ed., Academic Press, Boston, 1991. [42] A. Vedpriya, Living systems: eco-friendly nanofactories, Dig. J. Nanomater. Bios. 5 (2010) 9–21. [43] S. Vijayakumar, S. Ganesan, Size-dependent in vitro cytotoxicity assay of gold nanoparticles, Toxicol. Environ. Chem. 95 (2013) 277–287, https://doi.org/10.1080/ 02772248.2013.770858. [44] S. Vijayakumar, S. Ganesan, In vitro cytotoxicity assay on gold nanoparticles with different stabilizing agents, J. Nanomater. (2012) 1–9. [45] G. Arun, M. Eyini, P. Gunasekaran, Green synthesis of silver nanoparticles using the mushroom fungus Schizophyllum commune and its biomedical applications, Biotechnol. Bioprocess Eng. 19 (2014) 1083–1090. [46] M.R. Bindhu, M. Umadevi, Antibacterial activities of green synthesized gold nanoparticles, Mater. Lett. 120 (2014) 122–125. [47] M. Koperuncholan, Bioreduction of chloroauric acid (HAuCl4) for the synthesis of gold nanoparticles (GNPs): a special empathies of pharmacological activity, Int. J. Phytopharm. 5 (4) (2015) 72–80.
King Saud University
Journal of Saudi Chemical Society www.ksu.edu.sa www.sciencedirect.com
ORIGINAL ARTICLE
Eco-friendly synthesis of gold nanoparticles using fruit extracts and in vitro anticancer studies S. Vijayakumar Department of Science and Humanities, Sri Ramakrishna Institute of Technology, Coimbatore, India Received 12 November 2018; revised 17 December 2018; accepted 20 December 2018 Available online 30 December 2018
KEYWORDS Gold nanoparticles; Aegle marmelos; Eugenia jambolana; Soursop; Anticancer activity
Abstract Gold nanoparticles are biocompatible and are having several applications in biomedical Sciences and Engineering. Integration of nanoscience in medicine leads to the development of biomedical products that helps the Society in a faster and safer manner. In the present research work, bioreduction and biofunctionalization of gold nanoparticles are performed with fruit extracts of Aegle marmelos, Eugenia jambolana and soursop. The nanoparticles are characterized using UV– Vis spectroscopy, Transmission Electron Microscopy, Fourier Transform Infrared Spectroscopy and Zeta potentiometer. The qualitative phytochemical analysis of the fruit extracts shows the presence of alkaloids, amino acid, flavonoids, phenol, proteins, tannin, reducing sugars and total Sugars. The in vitro anticancer activity was confirmed by MTT assay on the human breast cancer cell line MCF-7 at different concentrations. The flavonoids present in the fruit extracts are potential reducing agent which is responsible for the formation of gold nanoparticles. Stabilization of gold nanoparticles are performed by the carboxylate group present in the proteins. Also, the nanoparticles are held apart from each other by the electrostatic repulsions that exist due to the presence of like charges surrounding the gold nanoparticles. This study proves that the fruit extracts can be used for the synthesis and stabilization of gold nanoparticles. Further, the engineered nanoparticles capped with bioactive compounds are potential anticancer agents against breast cancer cell line MCF-7. Ó 2019 King Saud University. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction Nanotechnology is an interdisciplinary domain of technological pool containing Physics, Chemistry, Biology, E-mail address: [email protected] Peer review under responsibility of King Saud University.
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Medicine and Material Science having applications ranging from material science to personal care products [1], Quantum dot-based photoelectric conversion for immunoassays [2] and development of an improved photoelectrochemical sensing platform [3]. The smaller size and unique properties of the nanoparticles have substantially improved their application in different areas such as anti HIV agent [4] and anticancer agent as well as therapy [5–7]. Green synthesis of gold nanoparticles are possible and has better stability over the conventional methods of preparation [8–10]. Different parts of the fruits, plants and biological entities are used for the
https://doi.org/10.1016/j.jscs.2018.12.002 1319-6103 Ó 2019 King Saud University. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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Fig. 1 The schematic illustration of the development of fruit extracts using soxhlet apparatus and the formation of gold nanoparticles using aegle marmelos, eugenia jambolana and soursop fruit extracts.
preparation of nanoparticles [11]. The nanosize and distinctive properties of nanoparticle have considerably improved in the recent years [12]. There is a variety of recent articles highlighting the advantages of nanoscale materials used for the investigation into biomedical applications [13–17]. During the past twenty years, technology shows a convergence of nano-engineering and nanomedicine, giving rise to many new technologies and ideas that can be achieved in medicine [18–21]. Nature has perfected with the art of biology at nanoscale and many of the inner workings cells naturally occur at the nanoscale. Nanoscale materials have a larger surface area to volume ratio than bulk materials. The larger surface area of nanoparticles enhances the chemical reactivity which helps to create better catalytic applications. The preparation of gold nanoparticles using plant extracts providesan efficient and environmentally friendly method to produce well defined size of nanoparticles [22]. Eugenia jambolana has immense health benefits. It is believed to be of a special use in treatment of diabetes and diarrhea. Several studies provide evidence that it has hypoglycemic activity by stimulating insulin secretion with up to 30 per cent reduction in blood sugar [23]. Literature reveals that the Aegle marmelos commonly has a fraction of ethyl acetate suppressed TNF – alpha-mediated, translocation of NF kappa B, ERK phosphorylation and inhibited AKT in tumor xenografts [24]. Soursop fruit extracts has anticancer activities that kill cancer cells and leaving healthy cells untouched. The benefits of soursop fruits are that they alleviate cough, back pain, stress, migraine headache and high blood pressure. Also, it showed cardio depressant activities in animals and anti-
cancer activities and is a more effective agent against multidrug resistant cancer cell lines [25–28]. In the present research work, chloroauric acid is reduced by the flavonoids of fruit extracts. The carboxylate group present in the protein of the fruit extract stabilizes the particles through electrostatic repulsion. Also it proves the possibilities of green synthesis of gold nanoparticles (AuNps) capped with Aegle marmelos, Eugenia jambolana and soursop extracts and in vitro anticancer activity. The schematic methodology for the preparation of gold nanoparticles using fruit extract is shown in Fig. 1. 2. Materials and methods 2.1. Chemicals and reagents The chemicals used for the synthesis of AuNps are hydrogen tetrachloro aurate (HAuCl4H2O) which was purchased from Hi-media, Coimbatore, India. Doubly distilled water was used for the preparation of Aegle marmelos, Eugenia jambolana and soursop extracts. In vitro assay reagents such as Phosphate buffered saline (PBS), Dimethyl sulphoxide (DMSO), trypsin-ethylenediaminetetraacetic acid (EDTA) and Fetal bovine serum (FBS) reagents were purchased from Sigma Aldrich, India. Eugenia jambolana is a very popular and seasonal fruit being sold everywhere in India and as a roadside avenue tree. It was obtained from the local garden near Coimbatore, Aegle marmelos was obtained from the Perur Patteeswarar temple. Soursop was purchased from Kutralam
Synthesis of gold nanoparticles using fruit extracts
Fig. 2
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Schematic visualization of reduction and stabilization of gold nanoparticles using different fruit extracts.
forest base, Thirnelveli District, Tamilnadu. All the chemicals and solvents were used as received. The illustration of reduction and stabilization of nanoparticles by the phytochemical is shown in Fig. 2. 2.2. Preparation of fruit extracts The fruits are washed with deionized water several times and chopped into small pieces. Then, 100 mg of fruit is mixed with 100 ml of deionized water and put inside a glass bowl of soxhlet apparatus. The mixture is boiled continuously for 12 h. The evaporated gases are condensed and recirculated for boiling. The entire system was allowed to cool down to room temperature. The fruit extracts were stored at 4 °C after filtration with Whatman No. 1 filter paper and used within a week. 2.3. Phytochemical qualitative analysis Bioactive compounds like alkaloids, amino acid, flavonoids, phenol, proteins, tannin, reducing sugars and total Sugars present in Aegle marmelos, Eugenia jombolana and soursop are qualitatively analyzed [29–31]. The extracts were treated with various chemical reagents and their color reactions were observed to identify the presence of different phytoconstituents. 2.4. Preparation of gold nanoparticles using Aegle marmelos, Eugenia jambolana and soursop extracts The Aegle marmelos, Eugenia jambolana and soursop fruit extracts were used to obtain phytochemical derived reducing and stabilizing agents for the generation of AuNps. About 1 ml of 5 mM tetra chloroauric acid (HAuCl4) was diluted by adding 50 ml of deionized water and heated until it boils. Then 10 ml of Aegle marmelos, Eugenia jambolana and soursop extracts was added in a separate beaker containing gold chloride solution. The reaction mixture was stirred well continuously using a magnetic stirrer. The color of the solution turned wine red from pale yellow within 5 min of the addition of fruit extracts, which indicates the formation of AuNps. The reaction mixture was allowed to cool after 10 min. AuNps stabilized with Aegle marmelos, Eugenia jambolana and soursop extracts were labeled as S1, S2 and S3.
2.5. Cytotoxicity studies MCF-7 cells are widely studied model for hormone-dependent human breast cancer. They are derived from a pleural effusion of a malignant breast cancer and having estrogen and progesterone receptors. The human breast cancer cell line, Michigan Cancer Foundation-7 (MCF-7) was received from National Centre for Cell Science at Pune. The cell lines are grown in 10% fetal bovine serum (FBS) contained in Eagle minimum essential medium. The cells were maintained in an incubator with 95% air and 100% relative humidity at 37 °C in 5% CO2. The viable cells were counted using a hemocytometer and diluted with medium containing 5% FBS to give a final density of 1 105 cells/mL. The cytotoxicity evaluation of Aegle marmelos, Eugenia jambolana and soursop extract stabilized AuNps was performed using tetrasolium dye MTT assay as described by Mossman [32]. About 1 105 mL 1 cells of MCF-7 in their experimental growth phase was seeded in a flat bottomed 96 well polystyrene coated plate and incubated for cell attachment. The cells were treated with serial concentrations (0, 20, 40, 60, 80, 100, 120) mgm/mL of the test samples after 24 h. About 100 ml of these different sample dilutions was added to the appropriate wells already containing 100 ml of the medium. Further, the plates were incubated for 48 h. Each well was added with phosphate buffered saline (PBS) containing 15 ml of MTT (5 mg/mL) reagent and incubated at 37 °C about 4 h. The medium without samples served as control. The medium with MTT was then flicked off and the formosan crystals were solubilized in 100 ml of DMSO. The absorbance was measured at 570 nm using a micro plate reader. The percentage of cell viability was then calculated with respect to control. 3. Characterization Characterization is a collection of tools to create knowledge on synthesis, properties and applications of nanoparticles. AuNps synthesized using Aegle marmelos, Eugenia jambolana and soursop fruit extracts were characterized by Ultraspec-3000 series UV–Visible spectrophotometer (Department of Nanoscience and Nanotechnology, Sri Ramakrishna Engineering College, Coimbatore, India) to study the peak absorption band. About 1 ml solution of AuNps in water was used.
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The formation of AuNps was confirmed by UV–visible spectroscopy, which is based on localized SPR band exhibited at a particular wavelength. High Resolution Transmission Electron Microscopy, JEOL JEM 2100 operating at an accelerating Voltage of 200 KV with a lattice resolution of 0.14 nm and point to point resolution of 0.19 nm (PSG Institute of Advanced Studies, Coimbatore, India) was used to confirm the formation of nanoparticles, particle distribution and size range. The crystalline nature of nanoparticles can be analyzed with the help of SAED pattern. The AuNps images were formed from the interaction of electron transmitted through the thin film of specimen. The Zeta potentiometer quantifies the effective electric charges on the surface of nanoparticles. The stability of the particles are directly proportional to the magnitude of the Zeta potential. The electrostatic repulsion increases with the increase in zeta potential and stability as well. Zeta potential measurements were performed using a Malvern Instruments Zeta sizer 1000 Hs operating with a variable power of (5–50 mW) He-Ne laser at 633 nm (Sri Ramakrishna Engineering College, Coimbatore). SHIMADZU FTIR spectrometer was used to identify the FTIR spectrum. The FTIR spectra of the sample were recorded in KBr phase in the frequency region of 10,000–400 cm 1. The cytotoxicity assay on AuNps capped with Aegle marmelos, Eugenia jambolana and soursop on breast cancer cell lines were analyzed by MTT assay and was performed in KMCH Pharmacy College, Coimbatore. 4. Results The curative properties of medicinal plants are possibly due to the presence alkaloids, flavonoids, phenols, alkaloids, tannins and proteins. The ethanolic extract of whole fruits was found to have more constituents of phytochemicals. The results of phytochemical analysis of fruit extracts are shown in Table 1. Further it shows that the qualitative phytochemicals present in soursop is more than that of Aegle marmelos and Eugenia jambolana. The reduction of hydrogen tetra chloroaurate into AuNps during exposure to the fruit extracts could be followed by color change in the solution. AuNps exhibit dark pinkish and wine red color in aqueous solution due to the SPR phenomenon. When metal absorbs light of a resonant wavelength, it causes the electron cloud to vibrate, thus dissipating the
Table 1
energy. This process takes place on the surface of nanoparticles and therefore called Surface Plasmon Resonance (SPR). The collective oscillations of electrons of a AuNps upon interaction with light of suitable energy cause the nanoparticles to obtain a specific color. The frequency of oscillation is strongly dependent on the particle size, shape, and aggregation. The SPR were found to be 519 nm, 523 nm and 526 nm for Aegle marmelos, Eugenia jambolana and soursop fruit extracts stabilized AuNps. The SPR peak increases with increase in particle size, which is shown in Fig. 3. The TEM image of gold nanoparticle is shown in Fig. 4 (a), (b) and (c) and SAED patterns are shown in Fig. 4 (d), (e) and (f) respectively. TEM images show the spacing between the AuNps. Several particles with a diameter greater than the specified sizes were also present, but it was suspected that these particles arose from the overlapping of two or more small particles [33]. The phytochemicals present in fruit extracts act as a reducing as well as a stabilizing agent. It helps the nanoparticles to stay apart and thus provides stability to nanoparticles. The diffraction patterns obtained for AuNps are (1 1 1), (2 0 0), (2 2 0) and (3 1 1) reflections which are face-centered-cubic (FCC) structure of nanoparticles. The corresponding d-spacing are calculated as 2.40 A˚, 2.05 A˚, 1.467 A˚ and 1.22 A˚. The SAED shows that the samples have crystalline AuNps as deciphered from diffraction pattern using X-rays. The average size of the AuNps was found to be 18 nm, 28 nm and 16 nm, respectively for Aegle marmelos, Eugenia jambolana and soursop fruit extracts stabilized AuNps. Another characterization is Zeta potential which is a measure of the effective electric charge on the nanoparticle surface and quantifies the charges. This was already in the literature stating that the prepared AuNps are stable without any aggregation [34]. The zeta potential gives information about particle stability. The higher the magnitude of potential exhibits increased electrostatic repulsion and stability. The particle size distribution and zeta potential graph are shown in Fig. 5 (a), (b), (c) and (d), (e), (f) respectively. EDAX is an attachment to electron microscopy instruments and is one of an X-ray technique. The spectra show the peaks relevant to the composition of elements present in the sample. The qualitative analysis can be performed using the instrumental technique. The EDAX of bio functionalized AuNps showed strong signals for gold atoms and the weaker
Result of qualitative analysis of phyto constituents in fruit extracts.
Chemical test
Result
Phytochemical identified
Aegle marmelos
Annona muricata
Eugenia jambolana
Dragendroff’s Ninhydrin Alkaline reagent Ferric chloride Bromine Fehling Ninhydrin Anthrone reagent
Reddish brown precipitate Blue color Intense yellow coloration & disappeared on the addition of dilute HCl Bluish black color
Alkaloids Amino acid Flavonoids
++ + ++
+ + ++
++ + +
Phenol
++
++
+
Decoloration of Bromine Brick red color Violet color Blue-green color
Tannin Reducing sugars Proteins Total Sugars
++ + + +
++ ++ ++ ++
+ ++ ++ ++
Note: + lower, ++ higher.
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Fig. 3 The Surface Plasmon Resonance of gold nanoparticles stabilized with aegle marmelos, eugenia jambolana and soursop fruit extracts.
signals for carbon, oxygen and chloride which is shown in Fig. 6. FTIR analysis was used to identify the chemical components responsible for the reduction of Au+ ions and stabilization. This measures infrared intensity versus wavelength (wavenumber) of light. It is used to determine the nature of associated molecules of plants or their extracts with nanoparticles. The FTIR of the bio-functionalized AuNps using the aqueous extracts of Aegle marmelos, Eugenia jambolana and soursop are shown in Fig. 7. The reduction of chloroauric acid may be due to the presence of flavonoids in the fruit extract [35]. The electrostatic repulsion among the nanoparticles are due to the presence of
757 carbohydrate group. It is found that the aqueous medium of fruit extract has the ability to perform dual functions of reduction and stabilization [26–39]. As noted above, the functional groups include primary amino group, ester moiety, mono substituted benzene ring and carboxylic group. The sharp peaks at 1639.49 cm 1 and at 1546 cm 1 were assigned to the C‚O stretching vibration (amide I) and a mixed vibration of NH deformation and CN stretch (amide II) in amides respectively. In addition, the IR bands at approximately 3371.59 cm 1 could also be observed, which may be assigned to O–H stretching vibration in the carboxyl moiety [40]. By contrast, the absorption band corresponding to amide II on the IR spectrum of AuNPs was broadened and blue shifted to 1560 cm 1, whereas the band corresponding to amide was also broadened but red shifted to 1620 cm 1. Generally, such broadening and shifting of the vibrational spectra have been ascribed to the formation of hydrogenbonded molecular complexes. Another feature of the gold particles was the presence of a strong and broad band centered at 3440 cm 1. On the basis of literature values [41], this band was attributed to be the various O–H stretching vibrations. The association of O–H O hydrogen bonding interaction with the ester moiety stretching was identified. Most importantly the peak at 1380 cm 1 was assigned to the bending of methyl group, 1226 cm 1 was assigned to C–O stretching of ester group and 1260 cm 1 was assigned to C–O stretching of a methoxyl group. Further IR clusters from 2800 cm 1 to 3200 cm 1 are attributed to aromatic CH of monosubstituted benzene. Also the intense band of C–O stretches in the ester moiety were identified at 1735 cm 1 and 1732 cm 1. It was thus concluded that chemical constituents of fruit extracts could serve as a reducing and stabilizing agent.
Fig. 4 (a), (b), (c) are Transmission electron micrograph and (d), (e), (f) are SAED pattern of AuNps stabilized with aegle marmelos, eugenia jambolana and soursop fruit extracts.
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Fig. 5 The Size distribution (a), (b), (c) and Zeta potentials (d), (e), (f) of gold nanoparticles stabilized with aegle marmelos, eugenia jambolana and soursop fruit extracts.
MTT cytotoxicity assay reveals that the sample were capable of metabolizing a dye (3-(4,5-dimethylthiozol-2-yl)-2,5-di phenyl tetrazolium bromide) efficiently. Spectrophotometric analysis was performed for the precipitate dissolved in detergent solution. After 24 h of treatment, it was found that the cell viability depends on the concentration of gold nanoparticles. The results demonstrate that the phytochemicals capping the nanoparticles acted upon the cancer cell lines and destroy them by influencing the chemical reactions. The concentration dependent toxicity of AuNps provides new opportunities for the safe application of nanoparticles in molecular therapy. Soursop capped gold nanoparticles treated MCF-7 cells exhibited cell death higher than the nanoparticles capped with Aegle marmelos and Eugenia jambolana. At higher concentration this shows more cell death which is about 30% more than others. The cell viability analysis is shown in Fig. 8. The result of this study suggests that the cytotoxicity of greener synthesized gold nanoparticles stabilized with soursop was increased with the increasing concentration. Also as per the experimental studies and literature survey the soursop is a fruit which has better anticancer effects. The IC50 values of AuNPs stabilized with Aegle marmelos, Eugenia jambolana and soursop fruit extracts are tabulated in Table 2.
5. Discussions Interestingly gold nanoparticles were formed within 30 min due to the rapid reduction of chloroaurate ions by the fruit extracts. It is clear from these studies that the plant extract mediated biosynthesis is very simple, fast, economic, ecofriendly and safe for human therapeutic use. Thus, the greener method of nanoparticles synthesis has a much reduced impact to the environment and has recently emerged as a viable alternative to conventional physical, chemical and even microbial methods. Research analysis proves that the biomoities present in the fruit extracts are responsible for the reduction and capping of nanoparticles [42–46]. The cytotoxicity of gold nanoparticles was increased with the increasing concentration of nanoparticles. Recent studies shows that the cytotoxicity depends on the stabilizing agent and size of the AuNps [47]. In the current work, MCF-7, human breast cancer cells showed 60% cell death at 120 mg/ ml concentrations of AuNps. In biomedicine, a lower concentration of drug has been used for treatment. Due to the presence of microvasculature in cancerous tumor, the AuNp based targeted drug delivery is possible to destroy the cancer cells by photothermal therapy. Therefore, it might be worth-
Synthesis of gold nanoparticles using fruit extracts
759
Fig. 7 FTIR analyses of gold nanoparticles stabilized with aegle marmelos, eugenia jambolana and soursop fruit extracts.
Fig. 6 Elemental analyses of gold nanoparticles stabilized with (a) aegle marmelos, (b) eugenia jambolana and (c) soursop fruit extracts.
while to explore the biosynthesized nanoparticles as a possible source of novel anticancer drugs. 6. Conclusion Simple, economic and non-toxic AuNps were synthesized using Aegle marmelos, Eugenia jambolana and soursop fruit extracts and in vitro anticancer activity was studied. Qualitative tests confirmed the presence of bioactive phytoconstituents in these fruit extracts. FTIR spectra revealed the presence of reducing and stabilizing groups in the extract responsible for AuNp synthesis. The synthesized AuNps were near spherical. The in-vitro anticancer activity was confirmed by MTT assay on MCF-7 cell lines that showed IC50 values of soursop stabilized AuNps as 98 ± 4 lg/mL which is much better than AuNps stabilized with Aegle marmelos and Eugenia jambolana. This study suggests that green synthesized AuNps might be a potential agent in cancer therapy. These particles are easily sol-
Fig. 8 Cell lines in the exponential growth phase were exposed to different concentrations of AuNps stabilized with aegle marmelos, eugenia jambolana and soursop fruit extracts. Cell viability was determined by the MTT assay as described in the experimental section. Each result represents the mean viability ± standard deviation (SD) of three independent experiments and each of these was performed in triplicate. Cell viability was calculated as the percentage of viable cells compared to untreated controls.
Table 2 The summary of physical and chemical properties of gold nanoparticles. Sample
SPR absorption
Particle size
IC50 value (mg mL 1)
S1 S2 S3
519 nm 523 nm 526 nm
18 nm 16 nm 28 nm
172 ± 4 163 ± 4 98 ± 4
760 uble in water and stable for more than 5 months. The toxicity due to the neglected dimensions must be a major concern since AuNps have been widely used in biomedical applications. Further these green synthesized AuNps may be used in developing catalysts, sensors, nanomedicine, target delivery and biomarker applications. Funding This work was supported by the Tamil Nadu State Council for Science and Technology, Government of Tamil Nadu [grant numbers TNSCST/S&T Projects/AR/PHY/2011-2012/2124, 2012]. Acknowledgements This work was funded by the Tamilnadu State Council for Science and Technology, Government of Tamilnadu, India. The author acknowledges the Management, SNR Sons Charitable Trust, Dr. M. Paulraj, Principal, Sri Ramakrishna Institute of Technology; sincere thanks to Dr. S. Harihara Sivakumar, KMCH Pharmacy College, Coimbatore for providing facilities and support to execute the research work successfully. References [1] J. Shu, D. Tang, Current advances in quantum-dots-based photoelectrochemical immunoassays, Chem. Asian J. 12 (2017) 2780–2789. [2] J. Shu, Z. Qiu, S. Lv, K. Zhang, D. Tang, Plasmonic enhancement coupling with defect-engineered TiO2-x: a mode for sensitive photoelectrochemical biosensing, Anal. Chem. 90 (2018) 2425–2429. [3] D. Jain, H.K. Daima, S. Kachhwaha, S.L. Kothari, Synthesis of plant-mediated silver nanoparticles using papaya fruit extract and evaluation of their anti-microbial activities, Digest J. Nanomater. Biostruct. 4 (2009) 723–727. [4] S. Vijayakumar, S. Ganesan, Gold nanoparticles as an HIV entry inhibitor, Curr. HIV Res. 10 (2012) 643–646. [5] O.Y. Damoah, R. Wang, J. Zhou, Y. Ding, Versatile nanosystem-based cancer theranostics: design inspiration and predetermined routing, Theranostics 6 (2016) 986–1003, https:// doi.org/10.7150/thno.14860. [6] L. Mocan, C.T. Matea, D. Bartos, O. Mosteanu, T. Pop, T. Mocan, C. Iancu, Advances in cancer research using gold nanoparticles mediated photothermal ablation, Clujul Med. 89 (2016) 199–202, https://doi.org/10.15386/cjmed-573. [7] N.S. Abadeer, C.J. Murphy, Recent progress in cancer thermal therapy using gold nanoparticles, J. Phys. Chem. C 120 (2016) 4691–4716, https://doi.org/10.1021/acs.jpcc.5b11232. [8] K. Shameli, M.B. Ahmad, S.D. Jazayeri, S. Sedaghat, P. Shabanzadeh, H. Jahangirian, Y. Abdollahi, Synthesis and characterization of polyethylene glycol mediated silver nanoparticles by the green method, Int. J. Mol. Sci. 13 (6) (2012) 6639–6650. [9] K. Xin Lee, K. Shameli, M. Miyake, N. Kuwano, N.B.B. Ahmad Khairudin, S.E.B. Mohamad, Y.P. Yew, Green synthesis of gold nanoparticles using aqueous extract of Garcinia mangostana fruit peels, J. Nanomater. (2016) 1–9, https://doi.org/10.1155/2016/8489094. [10] S. Vijayakumar, S. Ganesan, Preparation and characterization of gold nanoparticles with different capping agents, Int. J. Green Nanotechnol. 3 (1) (2011) 47–55.
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