Green synthesis of Nerium oleander-conjugated gold nanoparticles and study of its in vitro anticancer activity on MCF-7 cell lines and catalytic activity
© The Author(s) 2018
Received: 25 January 2018
Accepted: 26 March 2018
Published: 19 April 2018
Metal nanoparticles has been area of intense research interest in recent years because of their applications in diversified areas such as catalysis, cancer therapy, drug delivery, medicine, biotechnology, electronics, etc. [1–7]. Among various metal nanoparticles, the studies on gold nanoparticles (AuNPs) have been carried out most extensively due to its least toxicity to animal and microorganism cells compared to the other metal nanoparticles. AuNPs have different physicochemical properties compared to the bulk solids because of its large surface to volume ratio. Depending upon their shape, size, degree of aggregation and stabilizing ligands, AuNPs exhibit different colors [8–11]. AuNPs are widely applied in enzyme immobilization in biosensor for the detection of virus, bacteria and pathogen and in biomedical science such as the nanobiodiagnostics and controlled drug delivery [12–14]. It has also been applied in surface enhanced Raman spectroscopy, optical sensor and in biomedical application . Colloidal AuNPs stabilized with phytochemicals in aqueous medium are required for many of its applications. In a bottom-up synthetic strategy, plant and fungi extract mediated solution phase synthesis of AuNPs involving reduction of Au(III) to Au(0) has gained profound significance in recent years because of the renewable and biocompatible nature of the plant and fungi extracts, eco-friendly aqueous medium and mild reaction condition [16–18]. Additional advantage of this method is that the extract itself acts as a stabilizer and no additional stabilizers are required. The extracts of Aspergillus fischeri , Epicoccum nigrum , bark of Mimusops elengi , leaf of Chrysophyllum cainito (Star apple) , bark of Abroma augusta Linn,  Backhousia citriodora , Breynia rhamnoides , Piper betle , pear extract  etc. have been utilized for the synthesis of AuNPs.
Nerium oleander, commonly known as Karabi, is an evergreen beautiful flowering shrub that belongs to the family Apocynaceae. The flowers of Nerium oleander grow in clusters in terminal branches, each 2.5–5 cm, funnel shaped with five lobes and white or pink colors. A number of plant secondary metabolites such as steroids, terpenoids, flavonoids, cardenolides, cardiac glycosides, long chain esters have been reported in the bark extract of Nerium oleander [28–31]. Tremendous biological effects such as heart tonic [32–35], diuretic , cytotoxic , antibacterial, anti-platelet aggregation [38–42], anti-inflammatory, hepatoprotective [43–46], antitumor, anti hyperlipidemic, anti-ulcer, anti-depressant action in central nervous system [47–53] have been reported. During our investigations on the utilization of triterpenoids (C30 s) as renewable functional nano entities [54–58] it occurred to us that the medicinally important bark extract of Nerium oleander, rich in polyphenolic compounds, can be utilized for the synthesis AuNPs from HAuCl4. In this paper we report the green synthesis of gold-conjugated nanoparticles by the reaction of aqueous chloroauric acid with an ethanol extract of the stem bark of the Nerium oleander under very mild reaction conditions without any additional stabilizing or capping agents. Synthesis of AuNPs carried out with increasing concentration of the bark extract showed that the smaller sized AuNPs form flower-like bigger-sized AuNPs composed of smaller sized AuNPs. The stabilized gold nanoparticles have been characterized by surface plasmon resonance (SPR) spectroscopy, high resolution transmission electron microscopy (HRTEM) and X-ray diffraction studies. Anticancer activity of the synthesized AuNPs studied against MCF-7 breast cancer cell lines indicated selective apoptosis of the cancer cells compared to normal cells. Catalytic activity of the synthesized AuNPs has also been demonstrated for model chemical transformations in aqueous medium at room temperature.
2.1 Preparation of stem bark extract of Nerium oleander
Air dried stem bark of Nerium oleander (white flower variety) was finely powdered using a grinder. Finely powdered stem bark of Nerium oleander (7.5 g) was suspended in methanol (50 mL) and refluxed with magnetic stirring for 2 h, cooled at room temperature and then filtered via a sintered glass funnel. Volatiles of the filtrate were removed under reduced pressure to afford a sticky solid (1.00 g). The stem bark extract (0.100 g) was dissolved in distilled water (10 mL) and sonicated in an ultra sonicator bath for 10 min to get a semi-transparent solution (10 mg/mL).
2.2 Synthesis of gold nanoparticles
Aliquots of Au(III) solution (0.2 mL, 11.6 mM each) were added drop wise to the stem bark extract solution of Nerium oleander contained in a vial (4 mL) and the final volume was made up to 4 mL to prepare a series of stabilized AuNPs where concentration of the bark extract varied from 200, 400, 600, 800, 1200–4000 mg/L, keeping the concentration of Au(III) fixed (0.58 mM). UV–visible spectroscopic measurement of the solutions were carried out after 7 h of mixing of HAuCl4 and the stem bark extract of Nerium oleander.
2.3 Procedure of the catalytic reduction
To study the catalytic activity of the green synthesized colloidal AuNPs using the stem bark extract of Nerium oleander, two model reactions were carried out (a) the reduction of 3-nitrophenol to 3-aminophenol and (b) the reduction of 4-nitrophenol to 4-aminophenol, by sodium borohydride (16.38 mM) at room temperature in the presence of freshly synthesized colloidal AuNPs. Both the reactions were monitored by UV–visible spectroscopy.
2.4 DPPH assay
HRTEM images of AuNPs were recorded in JEOL JEM-2100 instrument. X-ray diffraction (XRD) patterns of the stabilized AuNPs were recorded Rigaku Miniflex II diffractometer with Cu-Kα radiation (λ = 1.54 Å). Mass spectra were recorded in Shimadzu GCMS QP 2100 Plus instrument. UV–visible spectrophotometry was carried out in Shimadzu 1601 spectrophotometer. For HRTEM analysis one drop of colloidal gold nanoparticles was placed over a carbon coated copper grid, allowed to dry in air and then under reduced pressure.
2.6 Cell culture and maintenance
MCF-7 breast cancer cell line was obtained from Jadavpur University, Kolkata, India. The cell line was cultured in DMEM complete media with 10% FBS (fetal bovine serum). l-glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 μg/mL) were required for the culture of the cell. Cultivated cells were incubated under 5% CO2 at 37 °C temperature in a CO2 incubator. Cells were grown in an exponential form until it reaches 1 × 106 cells/mL growth.
2.7 Selection of subjects for lymphocytes
Lymphocytes were separated from six different human samples belonging to the same geographical area and having the same environmental condition. The subjects were devoid of any kind of drug and anti-oxidant supplementation. Written consents were provided by these patients. Total process of lymphocytes separation was abided by Helsinki . Ethical committee of Vidyasagar University had approved the process.
2.8 Isolation of human lymphocytes
Blood samples (5 mL) were collected from six healthy persons by vein-puncture in a heparin coated vacutainers according to the method of Hudson and Hay . Blood was diluted with PBS (Phosphate Saline buffer, 1:1 v/v) and centrifuged using Histopaque 1077 (Sigma) at 1500 rpm for 40 min for separation of the layers. Then the separation of lymphocytes were carried out following the previously described method.
2.9 Intracellular reactive oxygen species generation
Intracellular ROS measurement was performed using H2DCFDA according to the procedure of Das et al. . The drug (100 μg/mL) was treated with 2 × 105 cells/mL for 24 h. After the treatment schedule, cells were washed with culture media followed by incubation with 1 μg/mL H2DCFDA for 30 min at 37 °C. Then the cells were washed three times with fresh culture media. DCF fluorescence was determined at 485 nm excitation and 520 nm emission using a Hitachi F-7000 Fluorescence Spectrophotometer. All the measurements were carried out in triplicate.
3 Results and discussion
3.1 Synthesis of AuNPs, UV–visible spectroscopy, HRTEM, DLS and XRD studies
3.2 Mechanism of the formation of stabilized AuNPs
3.3 Application of stabilized AuNPs
To demonstrate the usefulness of the stabilized AuNPs, in vitro anticancer activity and catalytic activity were studied which are discussed in the following sections.
3.3.1 Application of stabilized AuNPs as an anti-cancer drug
To find out whether AuNPs can act as a drug, dose dependent cytotoxicity assay of the freshly prepared AuNPs (0.5 mL, 1000 mg/L, synthesised with HAuCl4 (0.58 mM) and the stem bark extract (400 mg/L) was carried out on MCF-7 cell line. Non-radioactive colorimetric assay technique using tetrazolium salt, 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) was used for the cell viability studies.
The half inhibitory concentration (IC50) values for the AuNPs drug and the plant extract against the cancer cell MCF-7 were 74.04 and 130.87 μg/mL respectively (Additional file 1: Figure S4). Multiple linear regressions were used for comparison of data through statistica version 5.0 (Stat soft, India) software package. The anticancer activity of the plant extract, though less effective compared to the AuNPs drug (as obvious from the above mentioned IC50 values), might be due to the antioxidants present in the extract (as evident from the DPPH assay discussed in Sect. 3.4) .
The cytotoxicity assay was carried out against normal lymphocytes. The % of lymphocyte killing is more in case of the drug compared to the plant extract. 100 μg/mL dose of the drug can be used as a biologically safe dose because at this particular dose it killed the cancer cell massively and the cytotoxicity is minimum. From the point of cytotoxicity, the plant extract is better but the anti-cancer activity was insignificant compared to the drug. AuNPs are more efficient to kill the cancer cells compared to the plant extract.
3.3.2 Intracellular reactive oxygen species (ROS) measurement
3.4 Determination of antioxidant activity of the bark extract by DPPH assay
3.5 Catalytic activity
AuNPs with very high surface to volume ratio of have recently been utilized as a catalyst for various kinds of chemical transformations. To test whether the stem bark extract of Nerium oleander derived colloidal AuNPs can be utilized as a catalyst for photocatalytic reduction of toxic pollutants, we chose the sodium borohydride reduction of 3-nitrophenol to 3-aminophenol and 4-nitrophenol to 4-aminophenol as model reactions.
3.5.1 Reduction of 3-nitrophenol to 3-aminophenol by stabilized AuNPs
3.5.2 Reduction of 4-nitrophenol to 4-aminophenol by stabilized AuNPs
The phytochemicals present in the stem bark extract have been utilized for the synthesis of gold nanoparticles at room temperature under very mild conditions without any additional stabilizing agents. The antioxidant activity of the stem bark extract has been studied against the long lived 2,2-diphenylpicrylhydrazyl (DPPH) radical at room temperature. According to our knowledge, this is the first report of the study of antioxidant property of the stem bark extract of Nerium oleander and its utilization in the green synthesis of gold nanoparticles. A mechanism for the synthesis of the gold nanoparticles has also been proposed. The present study also demonstrated the in vitro anticancer activity of the stabilized AuNPs on MCF-7 cell lines significantly killing the cancer cells at 74 μg/mL. The normal lymphocytes were found to be non-toxic at this dose. The catalytic activities of the stabilized AuNPs have also been demonstrated for borohydride reduction of 3- and 4-nitrophenols.
All authors have contributed to the writing of the manuscript. All authors read and approved the final manuscript.
ACB thanks UGC, New Delhi and AD thanks DST-Inspire for research fellowships. BGB thanks Science and Engineering Research Board (SERB), India (ref. EMR/2016/001123), India Srilanka project (DST/INT/SL/P25/2016), UGC-MRPMAJOR-CHEM-2013-35629, UGC-SAP DRS II and DST-FIST New Delhi and Vidyasagar University for financial support and infrastructural facilities.
The authors declare that they have no competing interests.
Ethics approval and consent to participate
Ethical committe of Vidyasagar University had approved the process.
This study was supported by a Grant of the Science and Engineering Research Board (SERB), India (ref. EMR/2016/001123), India Srilanka project (DST/INT/SL/P25/2016), UGC-MRPMAJOR-CHEM-2013-35629, UGC-SAP DRS II and DST-FIST New Delhi.
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