Piper betle-mediated green synthesis of biocompatible gold nanoparticles
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Here, we report the novel use of the ethonolic leaf extract of Piper betle for gold nanoparticle (AuNP) synthesis. The successful formation of AuNPs was confirmed by UV-visible spectroscopy, and different parameters such as leaf extract concentration (2%), gold salt concentration (0.5 mM), and time (18 s) were optimized. The synthesized AuNPs were characterized with different biophysical techniques such as transmission electron microscopy (TEM), Fourier transform infrared (FT-IR) spectroscopy, X-ray diffraction (XRD), and energy-dispersive X-ray spectroscopy (EDX). TEM experiments showed that nanoparticles were of various shapes and sizes ranging from 10 to 35 nm. FT-IR spectroscopy revealed that AuNPs were functionalized with biomolecules that have primary amine group –NH2, carbonyl group, –OH groups, and other stabilizing functional groups. EDX showed the presence of the elements on the surface of the AuNPs. FT-IR and EDX together confirmed the presence of biomolecules bounded on the AuNPs. Cytotoxicity of the AuNPs was tested on HeLa and MCF-7 cancer cell lines, and they were found to be nontoxic, indicating their biocompatibility. Thus, synthesized AuNPs have potential for use in various biomedical applications.
KeywordsNanocrystalline materials Biomaterials Crystal growth Electron microscopy Fourier transform infrared spectroscopy Biosynthesis Nucleation
Piper betle leaf extract
transmission electron microscopy
Fourier transforms infrared spectroscopy
energy-dispersive X-ray spectroscopy.
Synthesis of gold nanoparticles (AuNPs) has gained immense significance during the last few years due to their catalytic, optical, and electrical properties . The existing chemical and physical methods are successful in producing well-defined AuNPs, but these processes usually require use of toxic chemicals. Therefore, the synthesized nanoparticles are not useful in medical and biological applications [2, 3, 4]. For this reason, the synthesis of AuNPs using an eco-friendly method is important to address the growing concerns on the overall toxicity of nanoparticles for medical and biotechnological applications. AuNPs have been considered important due to their unique and tunable surface plasmon resonance (SPR) property and their applications in biomedical science including drug delivery, tissue/tumor imaging, photo thermal therapy, immune chromatography, and identification of pathogens in clinical specimens . It is known that the physico-chemical properties of AuNPs are strongly dependent upon their interaction with capping agent molecules . Indeed, the surface chemistry of AuNPs can modify their interaction with external systems . This study describes an eco-friendly method for the synthesis of AuNPs using Piper betle as the reducing and stabilizing agent.
P. betle Linn (family: Piperaceae) leaves are widely used as a post-meal mouth freshener, and the crop is extensively grown in India, Sri Lanka, Malaysia, Thailand, Taiwan, and other Southeast Asian countries. The leaves of this plant are economically and medicinally important and have been traditionally used in India, China. In Thailand leaves are used to prevent oral malodor since it has an antibacterial activity against obligate oral anaerobes responsible for halitosis . Aqueous extracts of P. betle have also been shown to reduce the adherence of early dental plaque bacteria . The leaves of P. betle have a strong pungent and aromatic flavor and are used as a mouth freshener, in wound healing , as a digestive and pancreatic lipase stimulant , antioxidant [12, 13], antifungal, antibacterial [1, 14, 15, 16], anti-inflammatory, bioprotective , and antidiabetic  agent. In Chinese folk medicine, it is used for curing wind-cold cough, bronchial asthma, rheumatism, stomachalgia, and pregnancy edema . P. betel leaves contain a significant amount of antioxidants such as hydroxychavicol, eugenol, ascorbic acid, and β-carotene . We have successfully synthesized AuNPs by hypothesizing that the presence of strong antioxidants and flavonoids would assist in the reduction of gold ions to AuNPs. To aid this process, we applied microwave (MW) irradiation, which has the advantages of homogeneous heating that directly influence the nucleation process of AuNPs synthesis [21, 22]. Cytotoxicity tests were done to examine the effect of the synthesized AuNPs on the proliferation of two human cancer cell lines and revealed that the synthesized AuNPs were biocompatible. Therefore, AuNPs synthesized using this method can potentially be used in biomedical applications.
P. betel was collected from a local market in Guwahati, India. The reagents were of analytical grade obtained either from Merck (Mumbai, India) or Sisco Research Laboratories (Mumbai, India). 3,4,5-Dimethylthiazol-2-yl-2-5-diphenyltetrazolium bromide (MTT) was purchased from Hi Media (Bangalore, India). Cell lines were obtained from the National Centre for Cell Sciences (Pune, India). Cell culture-related plasticware were obtained from Sigma-Aldrich (Bangalore, India).
Preparation of leaf extract
P. betel leaves were washed with deionized water to remove adsorbed dirt. The leaves were chopped into small pieces (2 × 2 cm) and dried at room temperature (25°C) under shade. The dried leaves were powered in a mixer grinder (Bajaj Model GX 11, Mumbai, India). Five grams of powder was dissolved in 50 ml of ethanol and kept at 4°C for 1 week to get the leaf extract. The P. betle leaf extract (PLE) was filtered using a Whatman (50 mm; Sigma, Bangalore, India) filter paper, and the filtrate was stored at 4°C for various experiments.
Synthesis of AuNPs
The synthesis of AuNPs was carried out by varying the PLE concentration (0.5% to 4%) against 0.5 mM HAuCl4 in a total volume of 2 ml made up with double distilled water. The resulting mixtures were placed in a domestic microwave oven (900 W, 2.45 GHz, LG MO- MC-767 W/WS, LG Corp., Noida, India) and irradiated for 15 s. To obtain the optimum concentration of HAuCl4, the experiments were carried out by varying the gold solution concentration against 2% of PLE. The optimum time for synthesis was determined by incubating 2% of PLE with 0.5 mM by varying the MW irradiation time from 12 to 30 s with an interval of 2 s.
Characterization of AuNPs
All UV-visible (UV–vis) spectroscopic measurements of the synthesized AuNPs were carried out on a Cary 100 BIO UV–vis spectrophotometer (Varian, Palo Alto, CA, USA).
Transmission electron microscope
Colloidal solution (5 ml) of synthesized AuNPs was centrifuged twice at 20,000 rpm for 20 min to remove the non-covalently bounded molecules on the their surfaces. The resulting pellet was redispersed in 1 ml of distilled water, a few drops were placed over a carbon-coated copper grid, and the water was evaporated in a hot air oven (Daihan Labtech Co.Ltd. model LDO-150 F, New Delhi, India) at 60°C for 4 h. Transmission electron microscope (TEM) measurements were performed on a TEM instrument (JEOL model 2100, JEOL Ltd., Tokyo, Japan) operated at 190 V of 200 kV.
XRD, FT-IR, and EDX analyses
To obtain the X-ray diffraction (XRD) pattern, AuNP solution was placed on a microscope glass slide and allowed to dry in a hot air oven at 50°C, and the process was repeated to form a layer on the glass slide. The dried samples were analyzed with the help of an XRD instrument (Bruker Advance D8 XRD machine, Bruker, Madison, WI, USA) with a Cu source at 1.5406 Å wavelength in thin film mode.
AuNP colloidal solution (50 ml) was synthesized with optimum parameters (2% of PLE, 0.5 mM HAuCl, and 18 s) and centrifuged at 20,000 rpm for 20 min, and the process was repeated twice to remove non-covalently bounded molecules. The resulting pellet was resuspended in 5 ml of distilled water and lyophilized (Christ Gefriertrocknungsanlagen GmbH Model 1–4, Osterode, Germany) for 16 h. Infrared spectra were recorded using a Fourier transform infrared (FT-IR) spectroscope (Spectrum One, Perkin Elmer, Waltham, MA, USA) from 4,000/cm to 450/cm, with a resolution of 2 cm and five scans/sample by using 1 mg of finely powdered AuNPs prepared with 200 mg of KBr.
The elemental composition of the AuNPs was obtained by using energy-dispersive X-ray spectroscopy (EDX; LEO 1430 VP, Carl Zeiss AG, Oberkochen, Germany) at variable pressure and a scanning electron microscope equipped with INCA Oxford EDX facility at an acceleration voltage of 10 keV.
To maintain the HeLa (human cervical cancer) and MCF-7 (human breast cancer) cells, we used the minimal essential medium (MEM) containing 1.0 mM sodium pyruvate, 0.1 mM nonessential amino acids, 1.5 g/L sodium bicarbonate, 2 mM L-glutamine supplemented with 10% FBS (heat inactivated), and 1% antibiotic-antimycotic solution (1,000 U/mL penicillin G, 10 mg/mL streptomycin sulfate, 5 mg/mL gentamycin, and 25 μg/mL amphotericin B). The cells were cultured at 37°C in a humidified incubator (HF 160 W, Heal Force, Shanghai, China) supplemented with 5% CO2.
where Atreated and Acontrol are the absorbances of the treated and untreated cells, respectively.
Results and discussion
Effect of PLE and HAuCl4 on the AuNP synthesis
UV-visible spectral analysis of the reaction mixtures and their λ maxpeak positions
Number of runs
PLE extract (%)
MW irradiation time (s)
Observed λ maxpeak position
Time-dependent synthesis of AuNPs
Synthesized AuNPs were characterized with various biophysical techniques
Possibility of functional groups being involved in the synthesis of AuNPs
PLE provided a nontoxic coating on the surface of AuNPs
The method of AuNP synthesis described here has advantages such as rapidness, use of biologically benign biomaterial (PLE), and most importantly, resulting in biocompatible AuNPs. The synthesized AuNPs have insignificant toxicity at a maximum dose of 100 μM; thus, the particles could be very effective as a drug delivery tool and for other biomedical applications.
This is to declare that the present work is not published anywhere and submitted elsewhere for the publication. All authors materially participated in the present research work, and there will not be any conflicts among authors in the future. Therefore, I request you to consider the present article entitled ‘Piper betle-mediated green synthesis of biocompatible gold nanoparticles’ for publication in the peer-reviewed journal International Nano Letters. Utpal Bora.
We thank the Department of Science and Technology, Government of India, for funding this work (Project No. R & D/07/1479/ENV/P/UB/04). PJB thanks MHRD for providing research fellowship and PS thanks DBT Biotech Hub for the post doctoral trainee ship.
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