Catalytic, antioxidant and anticancer activities of gold nanoparticles synthesized by kaempferol glucoside from Lotus leguminosae

Mohamed Habib Oueslati; Lotfi Ben Tahar; Abdel Halim Harrath

doi:10.1016/j.arabjc.2018.09.003

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Original article

13 (

); 3112-3122

doi:

10.1016/j.arabjc.2018.09.003

Catalytic, antioxidant and anticancer activities of gold nanoparticles synthesized by kaempferol glucoside from Lotus leguminosae

Mohamed Habib Oueslati^{^a,^b,⁎}, Lotfi Ben Tahar^{^a,^c,^d}, Abdel Halim Harrath^{^e}

a Northern Border University, College of Science, P.O. Box 1231, Arar 91431, Saudi Arabia

b Carthage University, Department of Chemistry, Preparatory Institute for Scientific and Technical Studies, P.O. Box 51, La Marsa 2070, Tunisia

c Tunis El Manar University, Faculty of Science of Tunis, Campus Universitaire, 2092, Tunisia

d Carthage University, Faculty of Science of Bizerte, UR11ES30, Synthèse et Structures de Nanomatériaux, 7021 Jarzouna, Tunisia

e King Saud University, College of Science, Department of Zoology, P.O. Box 2455, 1145 Riyadh, Saudi Arabia

⁎Corresponding author at: Northern Border University, College of Science, P.O. Box 1231, Arar 91431, Saudi Arabia. oueshabib@yahoo.fr (Mohamed Habib Oueslati)

Received: 2018-2-26, Accepted: 2018-9-10, Published: 2018-01-01

Disclaimer:
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.

Peer review under responsibility of King Saud University.

Abstract

The aim of this study was to evaluate gold nanoparticles (AuNPs) for their anticancer activity against MCF-7 cancer cells, antioxidant activities and catalysis of the reduction of p-nitrophenol (p-NP). AuNPs were synthesized using kaempferol 3-O-β-D-apiofuranosyl-7-O-α-L-rhamnopyranoside (KG) from the plant Lotus leguminosae. The structure was determined using nuclear magnetic resonance (NMR) and electrospray (ES)-HRMS. The isolated compound was involved in the synthesis and stabilization of AuNPs. A number of parameters such as the pH and the mass ratio (HAuCl₄/KG) have been optimized to produce very stable colloids of an almost spherical shaped AuNPs with an average diameter of about 37 nm. UV–Vis spectrophotometry, infrared (IR) spectroscopy, transmission electron microscopy (TEM), and thermogravimetric analysis (TGA) studies were employed to characterize the produced nanoparticles. In vitro anticancer studies were performed to assess the chemotherapeutic potential of formulating NPs against a human breast carcinoma cell line (MCF-7). It was observed that the synthesized AuNPs have mild to low cytotoxicity in MCF-7 cells at higher concentrations. The microscopic observations showed no significant changes in the morphology of control cells or the treated cells. AuNPs also displayed strong DPPH radical scavenging compared to the flavonoid extract, with an IC₅₀ of 30.56 μg/mL. In addition, the biosynthesized AuNPs showed a highly improved catalytic activity for the reduction of p-nitrophenol (p-NP) to p-aminophenol (p-AP), indicative of its potential application in the chemical industry.

Keywords

Kaempferol glucoside

AuNPs

Anticancer

Antioxidant

Biocatalytic

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1 Introduction

Over the last few decades, the synthesis of gold nanoparticles (AuNPs) with controlled morphology and surface functionality has gained tremendous attention due to their potential application in various fields. The applications include medical diagnosis, therapy, drug delivery (Cui et al., 2017; Yang et al., 2017; Li et al., 2017, 2018; Lai et al., 2018 ), electronic conductors, and catalysis (Daniel and Astruc, 2004; Astruc, 2007; Tong et al., 2009 ). The remarkable physical (in particular, the optoelectronic properties), chemical and biological properties of AuNPs can be finely tuned by changing the size, shape, surface chemistry, and aggregation state. The microstructural parameters are in turn closely dependent on the elaboration route and the physiochemical conditions of the reaction medium. Although several chemical procedures were used for the synthesis of AuNPs, the release of toxic and hazardous by products restricts their use in biomedical applications (Rao et al., 2004; Shah et al., 2014). Furthermore, the use of toxic chemicals and solvents in these methods may prove to be problematic for downstream biological applications of AuNPs. As alternatives to the chemical routes, green chemistry routes, involving the utilization of plant extracts and microorganisms, have received growing attention in recent years. The microbial approach suffers from a number of drawbacks; it often requires complicated processes such as the purification and adherence of bacteria on the particle surface, which may increase the potential danger to public environmental hygiene and human health (Ahmed et al., 2016). In contrast, plant extract mediated synthesis routes should be the most suitable method of AuNPs preparation due to their simplicity, low toxicity, ease of biodegradability, reliability, energy efficiency, and environmentally friendly processing (Shah et al., 2014). In general, the synthesis of AuNPs using a plant extract involves the reduction of an Au³⁺ salt by at least one easily oxidizable molecule of the plant extract (Parida et al., 2011; Lakshmanan et al., 2016). Green synthesis of AuNPs employing flavonoid (luteolin, quercetin, rutin, curcumin) reducing agents of chloroauric acid (HAuCl₄) has been reported (Sindhu et al., 2014a,b; Nirmala et al., 2017 ). Flavonoids involved in the green chemistry synthesis of AuNPs have, in fact, multiple roles, as they possess functional groups that can reduce Au³⁺ species, catalyse their reduction and cap the produced AuNPs, resulting in very stable colloids (Sathishkumar et al., 2018). Recently, qualitative and quantitative studies have shown that the genus Lotus contains a large number of polyphenols, which are largely linked to its pharmacological activities, such as antioxidant, anti-obesity, anti-ischaemia, antiviral, and anticancer (Deng et al., 2013; Valls et al., 2009). In the present contribution, a kaempferol glucoside (Fig. 1) was obtained from the ethanolic extract of Lotus leguminosae, and its molecular structure was resolved using nuclear magnetic resonance (1D and 2D-NMR), electrospray (ES)-HRMS and comparison with literature data. The isolated compound was used for the synthesis of Au⁰ nanoparticles from HAuCl₄ at room temperature. To tune the size and/or morphology of the AuNPs, a number of synthetic parameters, including the pH, the mass ratio HAuCl₄/KG and the incubation time, were varied. The anticancer activity in MCF-7 cancer cells and antioxidant activity assessment of aqueous colloids of the synthesized AuNPs was reported. In addition, the potential of the AuNPs as catalysts for the chemical reduction of p-NP at room temperature was evaluated.

2 Materials and methods

2.1

2.1 Plant materials

The areal parts of lotus plants were collected in Arar, Saudi Arabia, in May 2016 and the parent plant was identified by Dr. Arabi Guetet (College of Science, Department of Biology, Northern Border University, Kingdom of Saudi Arabia). A voucher specimen was deposited in the herbarium of the same department.

2.2

2.2 Chemicals

Chloroauric acid (HAuCl₄·3H₂O, 99.99%), gallic acid (99%), sodium hydroxide (99%), 2,2-diphenyl-1-picrylhydrazyl (DPPH) (99%), p-nitrophenol (99%) and NaBH₄ (99%) were purchased from Sigma-Aldrich. Solvents (ethanol (99%), ethyl acetate (99%), pyridine (99%) and acetic anhydride (99%)) were purchased from Fisher Scientific UK. Silica gel high-purity grade (pore size 60 A, 70–230 mesh 63–200 mm) for column chromatography and TLC silica gel 60 F₂₅₄ were obtained from Sigma Aldrich. All of these chemicals were of analytical grade and were used without any further treatment.

2.3

2.3 Extraction and purification of KG

First, 250 g of areal parts of Lotus was extracted three times with ethanol (2.5 L) at room temperature. After evaporation of the solvent using a rotary evaporator (Buchi-R 210), the obtained ethanolic extract (17.46 g) was then fractionated by column chromatography (SiO₂; d,i = 4 cm; L = 80 cm) and eluted with increasing amount of ethanol in ethyl acetate (EtOAc) to yield four fractions (Fr.1-Fr.4) according to TLC plates analysis, revealed by sulfuric acid in ethanol 10%. Fr. 1 (3.85 g), Fr. 2 (4.82 mg), Fr. 3 (2.56 g) and Fr. 4 (3.28 g). The precipitation of fraction Fr.2 in EtOAc produced an impure solid (2.62 g), which was purified by silica gel flash column chromatography using EtOAc/MeOH (80:20) to obtain pure compound (2.62 g) noted KG (Fig. 1S).

2.4

2.4 Synthesis of gold nanoparticles

For the synthesis of AuNPs, a fixed volume of 25 mL of 1 mmol L⁻¹ aqueous solution of HAuCl₄·3H₂O was mixed with 100 mg of KG. The resulting solution was then gently stirred at room temperature for different times, between 2 and 72 hr. The formation of AuNPs can be observed during the first 15 min of reaction by a color change from yellow to wine-red, which is a characteristic feature of very small colloidal AuNPs. Note that the pH of the reaction medium was adjusted to the desired value by adding 0.1 M NaOH. The resulting colloidal solution was centrifuged at 16,000 rpm for approximately 20 min, resulting in a reddish precipitate. The precipitate was finally washed twice with Milli-Q water with intermittent centrifugation and stored in a clean and dry small bottle at 4 °C until further use. It is important to note that the obtained AuNPs are readily re-dispersed in water resulting in very a stable colloid over several months. To achieve the synthesis of pure nanocrystalline powders of Au⁰ with tuned size and morphology, three synthesis parameters were varied: the mass ratio of HAuCl₄/KG, the pH of the reaction medium and the reaction duration, t. The three parameters were varied in the range 4–10, 1/16–1/4 and 2–72 hr, respectively.

2.5

2.5 Characterization techniques

The proton (¹H, 500 MHz) and carbon 13 (¹³C, 125 MHz) nuclear magnetic resonance (NMR) spectra of the acetylated KG were recorded in deuterated chloroform (CD₃Cl) by using a JEOL JNM ECX 500 NMR spectrometer. The chemical shifts are given in parts per million (ppm) and the coupling constants in hertz. In each of the measurements, the residual solvents (CHCl₃) are used as the internal standard. IR characterization was carried out on a Thermo Scientific Nicolet iS5 Infrared Spectrometer using the KBr pellet method over the range 400–4000 cm⁻¹ with a resolution of 4 cm⁻¹. UV–Visible spectroscopy was recorded on a double-beam spectrophotometer (Jasco V-670) covering a wavelength range from 190 to 800 nm and equipped with 1 cm wide quartz cells. The particle morphology was observed by a JEM-1010 transmission electron microscope (TEM) operating at an accelerating voltage of 100 kV. For TEM analysis, a drop of the AuNPs colloid was deposited on a carbon-coated copper grid and allowed to dry at room temperature. Particle size distribution was plotted using the Image J analysis software by counting more than 200 particles selected from different TEM micrographs. Thermogravimetric analysis (TGA) (Mettler Toledo TGA/SDTA 851e) of the as-prepared powders was recorded from ambient to 800 °C in air with a flow rate of 30 mL min⁻¹ and a heating rate of 5 °C min⁻¹.

2.6

2.6 Cell culture and treatments

The MCF-7 human breast adenocarcinoma cell line was obtained from American Type Culture Collection (ATCC, Rockville, MD, USA). The cells were maintained in DMEM with 10% foetal bovine serum (FBS) and 1% penicillin/streptomycin in a completely humidified atmosphere with 95% air and 5% CO₂ at 37 °C. The viability of the cells was determined by staining with trypan blue. The cells were counted using a cell counter (Bio Rad TC20 automated cell counter) and diluted in medium at a density of 1 × 10⁵ cells/ml to be used in the experiments. A stock solution of AuNPs was prepared in Millipore water (w/v) and was then diluted in cell culture medium to obtain the desired concentrations for cell treatment.

2.7

2.7 Cytotoxicity assay

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay with modification was used to analyse the cytotoxic activity of AuNPs (Sindhu et al., 2014a,b). A Cell Titre 96® non-radioactive cell proliferation assay kit (Promega, Madison, WI, USA) was used following the manufacturer’s instructions. Briefly, the MCF-7 cells (1 × 10⁴ cells/well) were grown overnight in 96-well flat-bottom cell culture plates and were then exposed to six different concentrations of two-fold dilutions of 1000 µg/ml AuNPs (1000 µg/ml, 500 µg/ml, 250 µg/ml, 125 µg/ml, 62.5 µg/ml and 31.25 µg/ml) for 24 hr. A negative control (untreated) was also maintained for comparison. After the completion of the desired treatment, 15 µl of MTT reagent, provided in the kit, was added to each well and further incubated for 3 hr at 37 °C. Finally, the medium with MTT solution was removed, and 200 µl of solubilization solution was added to each well and further incubated for 30 min with occasional vortexing. The optical density (OD) of each well was measured at 550 nm using a Synergy microplate reader (BioTek, Winooski, VT, USA). The results were generated from three independent experiments, and each experiment was performed in triplicate. The percentage of cytotoxicity compared to the untreated cells was estimated. To examine if there were any morphological alterations induced by AuNPs in MCF-7 cells, images were taken under a phase contrast inverted microscope equipped with a digital camera (Olympus IX51, Tokyo, Japan) at 100× magnification before adding the MTT reagent.

2.8

2.8 Statistical analysis

The cytotoxicity assay (MTT) was carried out with three independent replicates, and the values are presented as the mean ± standard error of mean (SEM). The data were statistically analysed using Student’s t-test for comparison between the means with a significance level of P < 0.05.

2.9

2.9 Light microscopy

The morphological appearance of MCF-7 cancer cell lines and their structural changes after 24 h of treatment with the AuNPs were observed with an inverted phase contrast microscope and the images were captured by an MC 170 HD camera (Leica, Germany).

2.10

2.10 Antioxidant activities

The antioxidant potential of the AuNPs was determined by DPPH^• free radical scavenging assays. The DPPH free radical scavenging potential of synthesized AuNPs was examined according to a standard procedure (Brand-Williams et al., 1995). Briefly, aliquots (1.6 mL) of various concentrations of AuNPs (20–150 μg/mL) in Milli-Q water were added to 0.4 mL of 0.1 mM DPPH in methanol. The reaction mixture was mixed properly and incubated in the dark at 37 °C at selected durations up to 30 min while shaking at 100 rpm. Absorbance was read against a blank at 517 nm using a UV–vis spectrophotometer. The percentage inhibition of DPPH oxidation was calculated using the following equation: $% Inhibition = [(A_{blank} - A_{sample}) / A_{blank}] \times 100$ where A_blank is the absorbance value of the control and A_sample is the absorbance value of the test sample. Ascorbic acid was used as a reference, and tests were conducted in triplicate.

2.11

2.11 Catalytic reduction reaction of nitrophenol and nitrobenzaldehyde

The reduction of p-NP to p-AP by NaBH₄ using the as-produced AuNPs as catalyst was studied as a model reaction (Zeynizadeh et al., 2017). To study the reaction, 2.7 mL (2 · 10⁻⁵ M) p-NP was mixed with 0.2 mL of reducing agent NaBH₄ (2 · 10⁻² M). Then, 0.1 mL AuNPs (1 mg/mL) was introduced into a quartz cuvette along with the reaction mixture. The reaction time began when a catalyst AuNPs was added to the p-NP and NaBH₄ mixture. Immediately after the addition of catalyst AuNPs, UV–visible spectra of the sample were recorded against ultrapure water as a blank in the range of 200–700 nm.

3 Results and discussion

3.1

3.1 Structure of KG compound

The KG compound was obtained as a yellow amorphous powder, and its molecular formula (C₂₆H₂₈O₁₄) was determined using NMR (Table 1S), electrospray ES-HRMS, and infrared (IR) spectroscopic analyses. The IR spectrum (Fig. 2) exhibited the features of a hydroxyl group (3415 cm⁻¹), a carbonyl group (1650 cm⁻¹), and aromatic rings (1598 cm⁻¹, 1491 cm⁻¹). The UV spectrum (Fig. 3) showed absorption maxima at 265 and 348 nm, suggesting that KG contains the flavonol skeleton (Hussein et al., 2005). In addition, the ¹H NMR spectrum for acetylated KG (Table 1S) exhibited A and B ring signals typical of kaempferol glucoside (Shang et al., 2017). The spectrum showed two meta coupled doublets at δ_H 7.06 (J = 2.0 Hz) and δ_H 6.76 (J = 2.0 Hz) assigned to H-8 and H-6, respectively, of ring A and two pairs of doublets at δ_H 7.87 (J = 8.0 Hz) and δ 7.25 (J = 8.0 Hz) assigned to H-2′,6′ and H-3′,5′, respectively, of ring B. The ¹³C NMR spectrum (Table 1S) confirmed the presence of the kaempferol skeleton with 15 sp² carbons characteristic of the kaempferol skeleton (Shang et al., 2017). The ¹H NMR spectrum also showed signals of two anomeric proton resonances at δ_H 5.60 (br s, H- $1^{''}$ ) and δ_H 5.57 (br s, H- $1^{'''}$ ), indicating the presence of two monosaccharides. These results were deduced previously from the ¹H NMR and ¹³C NMR and by comparison with literature data (Spanou et al., 2008) and indicated that KG was kaempferol 3-O- α -D-apiofuranosyl-7-O-α-L-rhamnopyranoside (Fig. 1).

IR spectrum of the dried AuNPs. IR spectrum of the KG is also given for comparison.

3.2

3.2 Synthesis and characterization of AuNPs

To achieve the synthesis of pure nanocrystalline powders of Au⁰ with tuned size and morphology, three synthesis parameters were varied: the mass ratio HAuCl₄/KG (1/4, 1/8, and 1/16), the pH (4, 6, 8 and 10) of the reaction medium and the reaction duration (2–72 hr).

3.2.1

3.2.1 Effect of the mass ratio HAuCl₄/KG

Previous studies showed that in alkaline medium, Au³⁺ can undergo reduction to nanosized Au⁰ metal in contact with flavonoids (Irfan et al., 2017). Moreover, based on a study of selected flavonoids, it has been demonstrated that the reduction potential of the flavonoids is pH dependent, decreasing as the pH increases. Therefore, high alkalinity should render the oxidation of any flavonoids easier, thus leading to easier reduction of Au³⁺ ions to Au⁰. In our case, in a preliminary experiment to achieve the synthesis of AuNPs in the presence of flavonoids, we fixed the pH value at 10 by adding drops of 0.1 M NaOH solution to the chloroauric acid and the KG solution. The effect of the mass ratio of HAuCl₄/KG was studied by taking three different values: 1/4, 1/8 and 1/16. Photographs of the resulting colloids obtained after 2 hr of incubation along with the associated UV–vis spectra are given in Fig. 4. The color exhibited by the formed AuNPs is due to the excitation of surface plasmon resonance (SPR) vibration in the AuNPs. The KG reduced the gold ions, Au³⁺, to the metal, Au⁰. The mechanism for the formation of AuNPs is illustrated in Fig. 5. Au³⁺ ions make an intermediate complex with KG via its hydroxyl groups. Then, the OH groups undergo oxidation to keto forms which consequently lead to the reduction of gold ion (Au³⁺) to gold metal Au (0). The reduced seed particles undergo agglomeration and form the clusters, which act as nucleation centers and catalyze the reduction of remaining metal ions into AuNPs (Raghavan, et al., 2015; Nazeruddin et al., 2014). The formation of AuNPs was confirmed by the appearance of an intense absorption band at wavelengths of approximately 500–580 nm, which is typical of SPR of spherically shaped AuNPs (Chow and Zukoski, 1994). The SPR maxima were evident at different positions: 528, 534, 568, and 645 nm. Variation in the position of the SPR band in the range ∼ 530–570 is explained by the formation of AuNPs with variable size. The NPs obtained with the mass fraction 1/4 are expected to show the smallest spherically shaped mean size (528 nm) since the SPR band position exhibited at the lowest wavelengths (Toh et al., 2013). In addition, for the mass ratio of 1/4, the second SPR band at higher wavelengths is likely due to the existence of a fraction of AuNPs epitaxial NPs (Suvith and Philip, 2014). Indeed, we recorded the TEM image of the sample with the smallest mass ratio Au/KG (1/4) (Fig. 6). As can be clearly seen, it exist a small fraction of the AuNPs showing a shape anisotropy (indicated by arrows). Therefore, the appearance of the second SRP weak band at higher wavenumbers is due to the presence of the few elongated AuNPs with high epitaxy. The presence of the small fraction of AuNPs with shape anisotropy could be due to the lack of KG molecules compared to the amount of Au³⁺ ions initially present in the reaction medium that couldn’t ensure the complete transformation of the NPs to the spherical shape. With the increasing KG mass (molar ratios 1/8 and 1/16), the second SRP band vanishes indicating the transformation of all the AuNPs to the isotropic spherical shape. Further, as the quantity of GK is increased, the SRP band is red shifted and broadened indicating an increase of both NPs size and particle size distribution. It is noted that with further increasing KG mass (molar ratio less than 1/16), we have observed the gradual formation of precipitate with time indicating agglomeration of the produced NPs. From the above interpretations one would suggest that at the beginning of the growth step of the AuNPs, anisotropic NPs formed firstly, then they transform to the isotropic spherical ones.

The UV–vis spectra of AuNPs obtained with different HAuCl4/KG ratios. The inset shows photos of corresponding colloids taken after an incubation time of 2 hr.

The possible mechanism for the synthesis of gold nanoparticles in the presence of KG molecules.

A selected TEM image of AuNPs produced with the molar ratio HAuCl4/KG = 1/4. The arrows show few selected AuNPs exhibiting shape anisotropy.

3.2.2

3.2.2 Effect of pH

The formation and the colloidal stability of AuNPs in the presence of KG have been reported to be strongly dependent on the pH of the reaction medium (Pintoa et al., 2017; Toh et al., 2013). To study the effect of pH on the room temperature formation and stability of AuNPs using the KG, the pH was systematically varied between 4 and 10 while maintaining the mass ratio of KG at 1/8. Photographs of the resulting suspensions obtained after 2 hr of incubation along with the associated UV–vis spectra are shown in Fig. 7. As can be clearly seen in the inset of Fig. 7, the color of the obtained colloids changes from greenish to red-wine with the increase in pH from 4 to 10. The color change is traduced by a blueshift in a monotonic manner of the SPR band position from 580 to 535 nm. As outlined before, the change in SPR position can be correlated to a change in the Au NPs size (Guo et al., 2014; Sau and Rogach, 2010). Furthermore, the intensity of the SPR band increases with the increase in the alkalinity of the reaction medium, indicating an increasing reaction yield as a consequence of an easier reduction of Au³⁺ in alkaline medium (Qiua et al., 2018).

UV–vis spectra of AuNPs prepared at different pH. The inset shows photos of corresponding colloids taken after an incubation time of 2 hr.

3.2.3

3.2.3 Effect of incubation time

In this case, the intensity of the SPR band increases with the increase in incubation time without any appreciable change in band position (Fig. 8). The results indicate that the growth of the AuNPs is limited in time; it stopped after an incubation time of approximately 2 hr. Additionally, the increase in the SPR band intensity indicates an increased formation of AuNPs with time. Moreover, UV–visible measurements after three days of incubation do not reveal any noticeable change in the intensity of the SPR band, indicating that the formation of AuNPs the desirable size is limited in time. Furthermore, the observation shows that the AuNP colloid remains stable in water for a long incubation time. The produced NPs were further characterized by TEM, IR and TGA.

UV–vis spectra of AuNPs obtained at different incubation times under a constant pH (10.0) and a HAuCl4/KG ratio of (1/8).

A selected TEM image is shown in Fig. 9(a). The Au grains are of nanosized dimensions and are almost spherical in shape and well dispersed without any aggregation. A size distribution histogram (Fig. 9(b)) shows an average NPs size of ∼37 nm with an SD of ∼11 nm. FTIR analysis of the Au powder (Fig. 2) revealed the features of KG grafted on the AuNPs surface. For instance, 3431, 2923, 1655, 1636, 1559, 1457 and 1090 cm⁻¹ are assigned to the hydroxyl, the ketone (C⚌O), the aromatic rings, and the C—O stretching, respectively. Compared to the free KG, the shift in the band positions of OH (3431 vs. 3404 for KG) and C⚌O (1650 vs. 1656 for KG) (1090 vs. 1040 for KG) observed in the functionalized AuNPs suggests the contribution of flavonoid molecules to the formation and stabilization of the colloidal AuNPs. TG analysis of the produced AuNPs (Fig. 10) further confirms the capping of the flavonoid molecules onto the AuNPs surface. Indeed, the TG thermogram showed a two-stage weight loss. During the first stage (∼70–120 °C), the absorbed water departs, while during the second stage (150–490 °C), the organic entities are burned (Khalil et al., 2012). The mass per cent of KG molecules grafted onto the AuNPs is estimated to be approximately 29%.

A selected TEM image of AuNPs (a) with its associated particle size distribution histogram (b).

3.2.4

3.2.4 Antioxidant activity assays

To evaluate the reaction mechanism of the free radical scavenging reaction in the presence of the biofabricated nanoparticles AuNPs, UV–vis spectra were recorded in the spectra range 400–700 nm. As can be seen, the absorbance at the maximum wavelength (λ_max = 517 nm) of DPPH^• decreases with time traduced by a gradual color change of the DPPH solution from purple to pale yellow (Fig. 11). The results suggest that the biofabricated AuNPs transfer electron or hydrogen to the free radical at the atomic level DPPH^• and convert it to the pale yellow stable DPPH-H (Du et al., 2013).

Scavenging of DPPH• to DPPH-H by AuNPs: a. UV–Visible spectra; b. plausible mechanism.

The results for the effect of AuNPs on DPPH radical scavenging activity are shown in Fig. 12. The DPPH radical scavenging activity tends to increase as the concentration of AuNPs increases. AuNPs showed the highest free radical scavenging activity (IC₅₀ = 30.54 μg/mL) compared to KG (IC₅₀ = 48.9 µg/mL), which was lower than that of the gallic acid standard (IC₅₀ = 11.92 µg/mL). The increased activity of the AuNPs can be explained on the basis of the adsorption of KG on spherical nanoparticles having a larger surface area. The free radical scavenging activity of various synthesized nanoparticles is also reported recently because of the high surface area to volume ratio. (Balasubramani et al., 2015; Swamy et al., 2015).

Antioxidant activity (DPPH free radical scavenging assay) of ascorbic acid, KG and the AuNPs.

3.2.5

3.2.5 Cytotoxicity assay

To evaluate the cytotoxic activity of AuNPs, the MTT assay was performed. This assay is useful in measuring the IC₅₀ value, which is the concentration of the test compound that can inhibit or kill 50% of the total cells. Fig. 13 shows the per cent viability of cells exposed to different concentrations of green AuNPs. A concentration dependent decrease in cell viability was observed. The decrease was significant (p < 0.05) at higher concentrations only. The cell proliferation was inhibited by 46% at the highest concentration of 1000 µg/ml used in this study. Therefore, determination of the IC₅₀ value was not possible. At the lowest concentration, the cell growth was decreased up to only 6%. These data suggest that AuNPs have mild to low cytotoxicity in MCF-7 cells at higher concentrations.

Cytotoxic effects of AuNPs analysed by MTT assay on MCF-7 cells treated as indicated with two-fold dilutions for 24 hr. All data are expressed as mean ± SE for three independent experiments. * Significant (p < 0.05) compared with control.

The microscopic observation revealed no significant changes in morphology in the control cells (Fig. 14) or the treated cells. The control cells appeared to have a normal shape, were attached to the surface and reached approximately 95–100% confluence. The population of MCF-7 cells at higher concentrations was decreased between 20 and 40% (Fig. 14E and F). However, there were no dead or detached cells observed. It has been reported that AuNPs can be used in the destruction of cancer cells and act as potential therapeutic agents (Mata et al., 2016). Recently, tea flavonoids have been found to cause significant morphological changes in MCF-7 cells and also and inhibit cell growth after 48 h of treatment (Chen et al., 2014). The cytotoxic effect of AuNPs prepared using broccoli extract in breast cancer cell lines has been observed and the IC₅₀ after 24 h is reported to be 160 μg/mL (Khoobchandani et al., 2013). Our results showed no significant effect of the green gold nanoparticles synthesized from Lotus leguminosae on MCF-7 cells. An earlier study showed that the capability of nanoparticles to induce cytotoxicity is based on their shape and size and the nature of biomolecules grafted onto the surface of nanoparticles (Rajkuberan et al., 2015).

The MCF-7 cell morphology was examined by phase contrast inverted microscopy after treated with AuNPs for 24 hr. (A) control (B) 62.5 µg/ml (C) 125 µg/ml (D) 250 µg/ml (E) 500 µg/ml (F) 1000 µg/ml. Magnification: 100×.

3.2.6

3.2.6 Reduction of 4-nitrophenol

The toxicity of nitrophenols to biological systems has led to its classification as a priority pollutant by the US Environmental Protection Agency (EPA) (Emmanuel et al., 2014). Nonetheless, the class of chemicals and their derivatives are currently increasingly used in many fields, such as the pharmaceutical industry, petrochemicals, pesticides, plastics, explosives and dyes (Mori et al., 2007; Nemanashi and Meijboom, 2013). To face real situations of nitrophenol resource pollution, the adaptation of highly advanced materials and methods would be of crucial importance in limiting the environmental impact of the chemicals. The chemical reduction to more safe materials is a method of choice for the transformation of nitrophenols (Wanga and Wang, 2018; Akçay and Akçay, 2004). Owing to their high specific area, dispersion ability, surface modifiability, and biocompatibility, appropriate nanoparticles such as Au, Ag, and NiFe₂O₄@Cu have proven to be good agents for the chemical reduction of nitrophenols (Zeynizadeh et al., 2017; Emmanue et al., 2014; Pradhan et al., 2002 ). Here, we investigated the catalytic efficacy of the synthesized gold nanoparticles for the catalytic reduction of p-NP to p-AP by NaBH₄. The reduction reaction can be easily monitored by UV–Visible spectrophotometry. The p-NP solution without AuNPs in the presence of NaBH₄ shows a color change from light to bright yellow and the corresponding absorption band undergoes a redshift from 317 to 400 nm without any appreciable change in intensity/position over time (Fig. 2S). This is due to the formation of the p-nitrophenolate ion in alkaline conditions (Zeynizadeh et al., 2017). After the addition of AuNPs, the 4-nitrophenolate features disappear rapidly in less than 1 min. Simultaneously, two new bands appeared at 246 and 300 nm due to the formation of p-AP whose intensity increased with time, thus showing the high catalytic efficiency of AuNPs in the reduction reaction of p-NP. The complete reduction of p-NP to p-AP is supported by the color change from bright yellow to colourless (see the inset of Fig. 15 The reaction could be considered as a catalytic hydrogenation concept for the reduction of nitro group with NaBH₄ through activated hydride on the surface of gold nanoparticles. Therefore Nitrophenol also adsorbs onto the gold nanoparticle surface. Once the two substrates are adsorbed on the surface of the gold nanoparticles, hydrogen transfer from the gold hydride complex to nitrophenol occurs. The nitro phenol was reduced into the corresponding nitroso, hydroxylamine, and amino compound consecutively. Therefore, the plausible mechanism for reduction of p-NP with Au-NPs are given in Fig. 16.

UV–vis. absorption spectra recorded at selected reaction times showing the reduction of 4-NP by NaBH4 catalyzed by AuNPs.

Plausible catalytic hydrogenation mechanism for p-NP by Au-NPs system.

4 Conclusion

In conclusion, flavonoid functionalized gold nanoparticles were successfully synthesized by a feasible green method using kaempferol glucoside isolated from Lotus leguminosae. The synthesized gold nanoparticles were found to be almost spherical in shape and tending to possess ellipsoidal shape for low HAuCl₄/KG mass ratios. They displayed good free radical scavenging activities in vitro, with an IC₅₀ = 30.56 μg/mL. Further, they exhibited low cytotoxicity in MCF-7 cells at higher concentration effects against MCF-7 cancer cell lines. In addition, the biosynthesized nanoparticle AuNPs were highly catalytic for the reduction of p-nitrophenol (p-NP) to p- aminophenol (p-AP), indicative of a potential application in the chemical industries industry.

Acknowledgments

The authors would like to extend their sincere appreciation to the Deanship of Scientific Research at Northern Border University for its funding of this research through the research project No. 6969-SCI-2017-1-7-F.

References

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Appendix A

Supplementary material

Supplementary data to this article can be found online at https://doi.org/10.1016/j.arabjc.2018.09.003.

Appendix A

Supplementary material

The following are the Supplementary data to this article:

Supplementary data 1

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Show Sections

[1] Ahmed S., Annu Ikram S., Yudha S.S., . Biosynthesis of gold nanoparticles: a green approach. J. Photochem. Photobiol. B. 2016;161:141-153.
[Google Scholar]

[2] Akçay M., Akçay G., . The removal of phenolic compounds from aqueous solutions by organophilic bentonite. J. Hazard. Mater. 2004;113:189-193.
[Google Scholar]

[3] Astruc D., . Nanoparticles and Catalysis. Wiley-VCH; 2007.

[4] Balasubramani G., Ramkumar R., Krishnaveni N., Pazhanimuthu A., Natarajan T., Sowmiya R., Perumal P., . Structural characterization, antioxidant and anticancer properties of gold nanoparticles synthesized from leaf extract (decoction) of Antigonon leptopus Hook. & Arn. J. Trace Elem. Med. Biol.. 2015;30:83-89.
[Google Scholar]

[5] Brand-Williams W., Cuvelier M.E., Berset C., . Use of a free radical method to evaluate antioxidant activity. LWT − Food Sci. Technol.. 1995;28:25-30.
[Google Scholar]

[6] Chen X., Li Y., Lin Q., Wang Y., Sun H., Wang J., Cui G., Cai L., Dong X., . Tea polyphenols induced apoptosis of breast cancer cells by suppressing the expression of surviving. Sci. Rep.. 2014;4:4416.
[Google Scholar]

[7] Chow M.K., Zukoski C.F., . Gold sol formation mechanisms: role of colloidal stability. J. Colloid. Interface Sci.. 1994;165:97-109.
[Google Scholar]

[8] Cui H.D., Hu D.H., Zhang J.N., Gao G.H., Cai L.T., . Theranostic gold cluster nanoassembly for simultaneous enhanced cancer imaging and photodynamic therapy. Chin. Chem. Lett.. 2017;28(7):1391-1398.
[Google Scholar]

[9] Daniel M.C., Astruc D., . Gold nanoparticles: assembly, supra molecular chemistry, quantum-size-related properties, and applications toward biology, catalysis, and nanotechnology. Chem. Rev.. 2004;104:293-346.
[Google Scholar]

[10] Deng J., Chen S., Yin X., Wang K., Liu Y., Li S., Yang P., . Systematic qualitative and quantitative assessment of anthocyanins, flavones and flavonols in the petals of 108 lotus (Nelumbonucifera) cultivars. Food Chem.. 2013;139:307-312.
[Google Scholar]

[11] Du L., Suo S., Wang G., Jia H., Liu K.J., Zhao B., Liu Y., . Mechanism and cellular kinetic studies of the enhancement of antioxidant activity by using surface-functionalized gold nanoparticles. Chem. Eur. J.. 2013;l19:1281-1287.
[Google Scholar]

[12] Emmanuel R., Karuppiah C., Chen S.M., Palanisamy S., Padmavathy S., Prakash P., . Green synthesis of gold nanoparticles for trace level detection of a hazardous pollutant (nitrobenzene) causing Methemoglobinaemia. J. Hazard. Mater.. 2014;279:117-124.
[Google Scholar]

[13] Guo Q., Guo Q., Yuan J., Zeng J., . Biosynthesis of gold nanoparticles using a kind of flavonol: dihydromyricetin. Colloids Surf. A Physicochem. Eng. Asp.. 2014;441:127-132.
[Google Scholar]

[14] Hussein A.A., Barberena I., Correa M., Coley P.D., Solis P.N., Gupta M.P., . Cytotoxic flavonol glycosides from Triplaris cumingiana. J. Nat. Prod.. 2005;68:231-233.
[Google Scholar]

[15] Irfan M., Moniruzzaman M., Ahmad T., Mandal P.Ch., Bhattacharjee S., Abdullah B., . Ionic liquid based extraction of flavonoids from Elaeis guineensis leaves and their applications for gold nanoparticles synthesis. J. Mol. Liq.. 2017;241:270-278.
[Google Scholar]

[16] Khalil M.M.H., Ismail E.H., El-Magdoub F., . Biosynthesis of Au nanoparticles using olive leaf extract. Arab. J. Chem.. 2012;5:431-437.
[Google Scholar]

[17] Khoobchandani M., Zambre A., Katti K.B.S., Lin C.H., Katti K.V., . Green nanotechnology from brassicaceae development of broccoli phytochemicals-encapsulated gold nanoparticles and their applications in nanomedicine. Int. J. Green Nanotechnol.. 2013;5(1):1-15.
[Google Scholar]

[18] Lai Y., Deng Y., Yang G., Li S., Zhang Ch., Liu X., . Molecular imprinting polymers electrochemical sensor based on AuNPs/PTh modified GCE for highly sensitive detection of carcinomaembryonic antigen. J. Biomed. Nanotechnol.. 2018;14(10):1688-1694.
[Google Scholar]

[19] Lakshmanan A., Umamaheswari C., Nagarajan N.S., . A facile phyto-mediated synthesis of gold nanoparticles using aqueous extract of Momordica cochinchinensis rhizome and their biological activities. J. Nanosci. Technol.. 2016;2:76-80.
[Google Scholar]

[20] Li Y., Sun J., Chen Q., Chen Z., Zhu L., . Fast drug release of liposome-gold conjugation under light irradiation and the comparison with liposome-gold hybrid. Nanosci. Nanotechnol. Lett.. 2017;9:982-987.
[Google Scholar]

[21] Li T., Yi H., Liu Y., Wang Z., Liu Sh., He N., Liu H., Deng Y., . One-step synthesis of DNA templated water-soluble Au-Ag bimetallic nanoclusters for ratiometric fluorescence detection of DNA. J. Biomed. Nanotechnol.. 2018;14:150-160.
[Google Scholar]

[22] Mata R., Nakkala J.R., Sadras S.R., . Polyphenol stabilized colloidal gold nanoparticles from Abutilon indicum leaf extract induces apoptosis in HT-29 colon cancer cells. Colloids Surf. B. 2016;143:499-510.
[Google Scholar]

[23] Mori T., Watanuki T., Kashiwaguru T., . Diesel exhaust particles disturb gene expression in mouse testis. Environ. Toxicol.. 2007;22:58-63.
[Google Scholar]

[24] Nazeruddin G.M., Prasad N.R., Prasad S.R., Shaikh Y.I., Waghmare S.R., Adhyapak P., . Coriandrum sativum seed extract assisted in situ green synthesis of silver nanoparticle and its anti-microbial activity. Ind. Crops Prod.. 2014;60:212-216.
[Google Scholar]

[25] Nemanashi M., Meijboom R., . Synthesis and characterization of Cu, Ag and Au dendrimer-encapsulated nanoparticles and their application in the reduction of 4-nitrophenol to 4-aminophenol. J. Colloid Interf. Sci.. 2013;389:260-267.
[Google Scholar]

[26] Nirmala J.G., Akila S., Narendhirakannan R.T., Chatterjee S., . Vitisvinifera peel polyphenols stabilized gold nanoparticles induce cytotoxicity and apoptotic cell death in A431 skin cancer cell lines. Adv. Powder Technol.. 2017;28:1170-1184.
[Google Scholar]

[27] Parida U.K., Bindhani B.K., Nayak P.L., . Green synthesis and characterization of gold nanoparticles using onion (Allium cepa) extract. World J. Nano Sci. Eng.. 2011;1:93-98.
[Google Scholar]

[28] Pintoa R.J.B., Lucasa J.M.F., Moraisa M.P., Santosa S.A.O., Silvestrea A.J.D., Marques P.A.A.P., Freire C.S.R., . Demystifying the morphology and size control on the biosynthesis of gold nanoparticles using Eucalyptus globulus bark extract. Ind. Crops Prod.. 2017;105:83-92.
[Google Scholar]

[29] Pradhan N., Pal A., Pal T., . Silver nanoparticle catalyzed reduction of aromatic nitro compounds. Colloids Surf. A. 2002;196:247-257.
[Google Scholar]

[30] Qiua W.Y., Wang K., Wanga Y.Y., Dinga Z.C., Wua L.X., Cai W.D., Yana J.K., . pH dependent green synthesis of gold nanoparticles by completely C₆-carboxylated curdlan under high temperature and various pH conditions. Int. J. Biol. Macromol.. 2018;106:498-506.
[Google Scholar]

[31] Rajkuberan C., Susha K., Sathishkumar G., Sivaramakrishnan S., . Antibacterial and cytotoxic potential of silver nanoparticles synthesized using latex of Calotropis gigantea L. Spectrochim. Acta A. 2015;136:926-930.
[Google Scholar]

[32] Rao C.N.R., Muller A., Cheetham A.K., . The Chemistry of Nanomaterials: Synthesis, Properties and Applications. Wiley-VCH; 2004.

[33] Raghavan B.S., Kondath S., Anantanarayanan R., Rajaram R., . Kaempferol mediated synthesis of gold nanoparticles and their cytotoxic effects on MCF7 cancer cell line. Process Biochem.. 2015;50:1966-1976.
[Google Scholar]

[34] Shah M., Badwaik V., Kherde Y., Waghwani H.K., Modi T., Aguilar Z.P., Rodgers H., Hamilton W., Marutharaj T., Webb C., Lawrenz M.B., Dakshinamurthy R., . Gold nanoparticles: various methods of synthesis and antibacterial applications. Front Biosci.. 2014;19:1320-1344.
[Google Scholar]

[35] Sau T.K., Rogach A.L., . Nonspherical noble metal nanoparticles: colloid chemical synthesis and morphology control. Adv. Mater.. 2010;22:1781-1804.
[Google Scholar]

[36] Sathishkumar P., Gu F.L., Zhan Q., Palvannan T., Mohd Yusoff A.R., . Flavonoids mediated ‘Green’ nanomaterials: a novel nanomedicine system to treat various diseases – current trends and future perspective. Mater Lett.. 2018;210:26-30.
[Google Scholar]

[37] Shang Y.Y., Qin Q., Li M.S., Xu H.Y., Ge Y.B., . Flavonol glycosides from the leaves of Elaeagnus Pungens. Nat. Prod. Res.. 2017;31:1066-1072.
[Google Scholar]

[38] Sindhu K., Bhuvanasree S.R., Rajaram A., Rajaram R., . Synthesis and characterisation of morin reduced gold nanoparticles and its cytotoxicity in MCF-7 cells. Chem Biol Interact.. 2014;224:78-88.
[Google Scholar]

[39] Sindhu K., Rajaram A., Sreeram K.J., Rajaram R., . Curcumin conjugated gold nanoparticle synthesis and its biocompatibility. RSC Adv.. 2014;4:1808-1818.
[Google Scholar]

[40] Swamy M.K., Akhtar M.S., Mohanty S.K., Sinniah U.R., . Synthesis and characterization of silver nanoparticles using fruit extract of Momordica cymbalaria and assessment of their in vitro antimicrobial, antioxidant and cytotoxicity activities. Spectrochim. Acta A. 2015;151:939-944.
[Google Scholar]

[41] Spanou C., Bourou G., Dervishi A., Aligiannis N., Angelis A., Komiotis D., Skaltsounis A.L., Kouretas D., . Antioxidant and chemopreventive properties of polyphenolic compounds derived from Greek Legume plant extracts. J. Agric. Food Chem.. 2008;56:6967-6976.
[Google Scholar]

[42] Suvith V.S., Philip D., . Catalytic degradation of methylene blue using biosynthesized gold and silver nanoparticles. Spectrochim. Acta A. 2014;118:526-532.
[Google Scholar]

[43] Toh Y.R., Yu P., Wen X., Tang J., Hsieh T.S., . Induced pH-dependent shift by local surface plasmon resonance in functionalized gold nanorods. Nanoscale Res. Lett. 2013:103-109.
[Google Scholar]

[44] Tong L., Wei Q., Wei A., Cheng J.X., . Gold nanorods as contrast agents for biological imaging: optical properties, surface conjugation and photothermal effects. Photochem. Photobiol.. 2009;85:21-32.
[Google Scholar]

[45] Valls J., Millلn S., Martd M.P., Borràs E., Arola L., . Advanced separation methods of food anthocyanins, isoflavones and flavanols. J. Chromatogr. A. 2009;1216(43):7143-7172.
[Google Scholar]

[46] Wanga J., Wang S., . Activation of persulfate (PS) and peroxymonosulfate (PMS) and application for the degradation of emerging contaminants. Chem. Eng. J.. 2018;334:1502-1517.
[Google Scholar]

[47] Yang J., Nian F., Guo Z., Bai T., Peng G., . Effect of gold nanoparticles with different diameters on rat neural stem and progenitor cells. Nanosci. Nanotechnol. Lett.. 2017;9:1491-1496.
[Google Scholar]

[48] Zeynizadeh B., Mohammadzadeh I., Shokri Z., Hosseini S.A., . Synthesis and characterization of NiFe₂O₄@Cu nanoparticles as a magnetically recoverable catalyst for reduction of nitroarenes to arylamines with NaBH₄. J. Colloid Interface Sci.. 2017;500:285-293.
[Google Scholar]

Catalytic, antioxidant and anticancer activities of gold nanoparticles synthesized by kaempferol glucoside from Lotus leguminosae