Antibacterial, anticancer, anti-diabetic and catalytic activity of bio-conjugated metal nanoparticles

All the experiments were performed in triple distilled water and glassware were cleaned with aqua regia and washed with triple distilled water. ${\rm HAuC}{{{\rm l}}_{4}}$, ${\rm AgN}{{{\rm O}}_{3}}$, Whatman filter paper disks, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and $\alpha {{\rm \text -}}$amylase were purchased from Sigma Aldrich. Luria-Bertani broth was purchased from Himedia, India.

Fresh leaves of Saraca asoca were collected from parking lot of Siksha O Anusandhan Deemed to be University, and thoroughly washed with triply distilled water. 10 g of Saraca asoca leaves were taken in 50 ml triply distilled water and boiled in microwave oven for 1 min. This process was repeated for 1 h interval up to 8 h and then allowed to cool at room temperature. This was then filtered and centrifuge at 14,000 rpm for 40 min. The supernatant was stored at $-20\,{}^\circ {\rm C}$ and used as stock solution.

To 10 ml, 0.25 mM aqueous ${\rm HAuC}{{{\rm l}}_{4}}$ solution varying amount of aqueous leaf extract of Saraca asoca ($100\,\mu {\rm l}$, $200\,\mu {\rm l}$ and $300\,\mu {\rm l}$ in 10 ml) were added with stirring. The formation of AuNP were confirmed by visual observation of ruby red color and the progress of the reaction was monitored by UV–vis spectrophotometer by measuring the characteristic surface plasmon resonance (SPR) band of AuNP in the wavelength range 200 nm to 800 nm. Similar synthesis procedure was followed for the preparation of AgNP. Varying amounts of leaf extract of Saraca asoca ($200\,\mu {\rm l}$, $300\,\mu {\rm l}$ and $400\,\mu {\rm l}$) were added to 10 ml, 0.25 mM aqueous ${\rm AgN}{{{\rm O}}_{3}}$ solution. The appearance of bright orange color indicated the formation of AgNP in solution. The bio-synthesized nanoparticle solutions were kept in refrigerator and used as stock solution for further use.

Surface plasmon resonance band of biosynthesized nanoparticles were measured using Perkin Elmer Lambda 35 spectrophotometer ranging from 200 nm to 800 nm. SEM analysis was performed by using Zeiss Merlin compact Microscope, Oxford instruments by using drop crusting the samples on carbon tape, and TEM images were taken by using JEOL JEM 2100 HR-transmission electron microscope by placing the samples on copper grid. The XRD analysis was carried out using Bruker D8 Advance Diffractometer (Bruker AXS, Germany) with ${\rm Cu{\text -}K}\alpha$ radiation $(\lambda =1.54\,{{\rm}\mathring{\rm A}})$ placing concentrated sample on glass slide and dried at $70\,{}^\circ {\rm C}$ for overnight. The FTIR analyses were performed using Bruker FTIR ALPHA by making KBr pellets.

Antibacterial activities of nanoparticles were done in E. coli MG1655 and B. subtilis 168 stains. In dried Luria-Bertani agar plate containing E.coli and B. subtilis strains, filter paper disks were placed. Then, $40\,\mu {\rm l}$ of AuNP, AgNP and leaves extract was spotted on paper disks and air-dried for 30 min. After that, plates were incubated at $37\,{}^\circ {\rm C}$ for 10 h, same concentration of ampicillin, kanamycin $\left(0.25\,{\rm mM} \right)$ were taken as controls. Clear zones were noted as the inhibitory effect of nanoparticles on growing lane of culture.

Prostate cancer (Du145) cell line (from the ATCC) was taken for studying the anticancer activity. Cells were grown in RPMI medium (Roswell Park Memorial Institute medium) with 10% bovine serum (FBS) supplement and antibiotics. The cell viability of the green synthesized AuNP and AgNP was measured by standard MTT (3-(4, 5-dimethylthiazol-2-yl)-2-5 diphenyl tetrazolium bromide) assay by following the procedure reported earlier [22].

For study the anti-diabetic activity α-amylase inhibition assay model was used following the method as described earlier [23]. In brief, sample was mixed with 0.5 ml of $\alpha {{\rm \text -}}$amylase solution ($0.5{\rm mg}\times {\rm m}{{{\rm l}}^{-1}}$ in 0.02 M sodium phosphate buffer, pH 6.9) and incubated at $37\, {}^\circ{\rm C}$ for 10 min followed by addition of 0.5 ml of starch solution (1%) and again incubated at $37\, {}^\circ{\rm C}$ for 10 min. Finally, the reaction was terminated using 1 ml of dinitrosalicylic acid and placed in a water bath ($100\, {}^\circ{\rm C}$) for 5 min. The mixture was then diluted with 10 ml of deionised water and measured the absorbance at 540 nm. During calculation absorbance intensity value of only nanoparticles should be subtracted from the test value as metal nanoparticles itself shows absorbance peak at visible region. The inhibition of $\alpha {{\rm \text -}}$amylase was calculated using the following equation:

$$\begin{align} \newcommand{\e}{{\rm e}} \displaystyle {\rm Inhibition}\,{\rm of}\,\alpha {{\rm \text -}{\rm amylase}}~ \left(\% \right){\rm =}\frac{{\rm Ab}{{{\rm s}}_{{\rm control}}}-{\rm Ab}{{{\rm s}}_{{\rm sample}}}}{{\rm Ab}{{{\rm s}}_{{\rm control}}}} \times{\rm 100,}\nonumber \end{align}$$

where ${\rm Ab}{{{\rm s}}_{{\rm control}}}$ and ${\rm Ab}{{{\rm s}}_{{\rm sample}}}$ are the absorbance of solution without and with sample, respectively.

Chick chorioallantoic membrane (CAM) assay was done to check the biocompatibility. In brief, fertilised chicken eggs were obtained from the local government poultry farm (CPDO, Bhubaneswar) at embryonic days 5. Eggs were placed horizontally and open the top of the egg shell to expose the CAM. Sterile filter paper discs were soaked with test sample (0.25 mM nanoparticles and raw extract), dried and placed on the exposed blood vessels for 5 h. Before and after 5 h of incubation, images were taken to compare the changes of blood vessels.

Catalytic activity study of green synthesised AuNP and AgNP were performed in 4-nitrophenol (4-NP) reduction model in presence of ${\rm NaB}{{{\rm H}}_{4}}$ by following the reported method. In brief, $100\,\mu {\rm l}$ of nanoparticles (final concentration is 0.025 mM) suspension and $100\,\mu {\rm l}$ of ${\rm NaB}{{{\rm H}}_{4}}$ solution (final concentration is 10 mM) with $700\,\mu {\rm l}$ of water was taken in a 1 ml quartz cuvette, which was place in the cell holder of the spectrophotometer for 30 s. Then absorbance was taken immediately after adding $100\,\mu {\rm l}$ of 4-NP solution (final concentration is 0.1 mM) and continued the time dependent spectra measurement to monitor the reaction process.

Aqueous extract of Saraca asoca leaves have been used for the synthesis of metal nanoparticles. Metal nanoparticles are successfully prepared by adding suitable ratios of metal precursors and leaf extract. It is observed that at room temperature extract acts as both reducing agent and stabilizing agent

It is known that the surface plasmon resonances (SPR) property of metal nanoparticles is unique with their type of metals, size and shape. After visual observation, SPR measurement using UV-visible spectrophotometer is carried out for monitoring the nanoparticles synthesis. The observed SPR bands at 428 nm and 532 nm in the UV-visible spectra are the signature for the formation of AgNP and AuNP, respectively (figure 1). To optimize the reaction parameters, the concentration of reagents and reaction time are varied. The optimum reaction conditions are found to be $200\,\mu {\rm l}$ leaf extract, $0.25{\rm mM}$ ${\rm HAuC}{{{\rm l}}_{4}}$ in 10 ml and 12 h reaction time for AuNP and $300\,\mu {\rm l}$ leaf extract, 0.25 mM ${\rm AgN}{{{\rm O}}_{3}}$ in 10 ml and reaction time 8 h for AgNP (figures S1 and S2 in supplementary (stacks.iop.org/ANSN/9/035001/mmedia)).

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The formation of nanoparticles and their morphology, shape and size have been authenticated by SEM (figure 2) and TEM (figure 3) analyses. Both the SEM and TEM results revealed that the green synthesized AuNP and AgNP are spherical in nature with average particle size 24 nm and 36 nm, respectively, with relatively good monodispersity.

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The crystalline nature and crystallite size of nanoparticles are identified by x-ray diffraction (XRD) analysis (figure 4). XRD pattern of AuNP and AgNP have shown diffraction peaks at $38.8{}^\circ$, $44.9{}^\circ$, $64.7{}^\circ$, $77.8{}^\circ$ and $82.3{}^\circ$ in the $2\theta$ range $30{}^\circ \!-\!90{}^\circ$ which are corresponds to the (111), (200), (220), (311) and (222) planes. Both data show that the biosynthesized nanoparticles are highly crystalline in nature with a crystallite size 11–18 nm for AuNP and 15–25 nm for AgNP. The crystallite size is calculated from the full width at half maxima (FWHM) of the high-intensity diffraction peak using the Debye-Sherrer formula:

$$\begin{align} \newcommand{\e}{{\rm e}} \displaystyle D=\frac{0.9\lambda}{\beta \cos \theta},\nonumber \end{align} \tag{ 1 }$$

where $D$ is the mean grain size, $\lambda$ is the x-ray wavelength for ${\rm Cu{\text -}K}\alpha$ $(\lambda =1.5406\,{{\rm}\mathring{\rm A}})$, $\beta$ is FWHM of diffraction peak of plane (111) and $\theta$ is the diffraction angle.

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To identify the probable biomolecules responsible for reduction and stabilization of nanoparticles present in the leaf extract, Fourier transform infrared (FTIR) analysis are performed. Changes of spectral peaks between pure plant extract and biosynthesized NPs give the information about the role of possible functional groups for the synthesis of nanoparticles. The dried extract have shown intense IR bands at $3400\,{\rm c}{{{\rm m}}^{-1}}$, $2927\,{\rm c}{{{\rm m}}^{-1}}$, $1612\,{\rm c}{{{\rm m}}^{-1}}$, $1400\,{\rm c}{{{\rm m}}^{-1}}$, $1280\,{\rm c}{{{\rm m}}^{-1}}$, $1112\,{\rm c}{{{\rm m}}^{-1}}$ and $1069\,{\rm c}{{{\rm m}}^{-1}}$ indicating the presence of (N–H or O–H), C–H, –C=O, aldehydic –C–H/C–O–H, aliphatic ester, alcoholic –C–O and –C–O–C–functional groups. The bands at 1280, 1112 and $1069\,{\rm c}{{{\rm m}}^{-1}}$ get vanished after the formation of nanoparticles, indicating that functional groups present in that region are responsible for reduction of metal ions and functional group corresponding to $3400\,{\rm c}{{{\rm m}}^{-1}}$, $2927\,{\rm c}{{{\rm m}}^{-1}}$, $1612\,{\rm c}{{{\rm m}}^{-1}}$ and $1400\,{\rm c}{{{\rm m}}^{-1}}$ that are N–H or O–H, responsible for capping and stabilization of nanoparticles (figure 5).

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Antibacterial activity of AgNP is well known but AuNP usually does not show antibacterial activity. Recent report discloses that sub-nanometer dimension AuNP displays good antibacterial activity [24]. The preliminary screening for antibacterial potency of bio-synthesized AuNP and AgNP are done by disc diffusion assay using gram negative E. coli and gram positive B. subtilis strains. 1 mM of AgNP displays very good antibacterial activity with zone of inhibition 9.7 mm and 11 mm whereas 2.5 mM of AuNP shows antibacterial activities at high concentration with zone of inhibition 9.6 mm and 7.7 mm for E. coli and B. subtilis respectively (figure 6(a) and table S1). These results are comparable with standard antibiotics kanamycin and ampicillin (figure S3). Previous literature also reports the good antibacterial activity of as-synthesized AgNP against E. Coli and B. subtilis with zone of inhibition 15.1 nm and 14.3 nm using concentration $39\,{\rm mg}\times {\rm m}{{{\rm l}}^{-1}}$ and $78\,{\rm mg}\times {\rm m}{{{\rm l}}^{-1}}$, respectively [9]. Screening of anticancer activity by MTT assay shows that AgNP significantly inhibits the growth of prostate cancer cell line DU145 (p53 mutant) with IC₅₀ of $50\,\mu {\rm M}$ whereas AuNP is inactive (figure 6(b) and table S1). Prasannaraj et al also reports the anticancer activity of green synthesized AgNP on prostate cancer cell line PC3 where p53 is null. This indicates the anticancer activity of AgNP may be p53 independent [15]. The antidiabetic activities of bio-synthesized nanoparticles are also tested by $\alpha {{\rm \text -}}$amylase inhibitory assay. Both nanoparticles, inhibit $\alpha {{\rm \text -}}$amylase activity with IC₅₀ value of $1.5\,{\rm mM}$ and 0.35 mM for AuNP and AgNP, respectively (table S1 and figure S4). The in vitro toxicity study of nanoparticles are done by chicken embryo assay (CAM assay), which confirm the biocompatibility of green synthesized nanoparticles (figure S5).

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It is known that nitro-aromatic compounds are the main organic hazard in environment, having mutagenic or carcinogenic activities. Therefore, to minimize the toxic nitro-phenol, conversion of nitro group to amine group is crucial. Metal nanoparticles acts as catalyst for this conversion. Here synthesized NPs exhibited excellent catalytic performance toward the reduction of 4-nitrophenol (4-NP) by ${\rm NaB}{{{\rm H}}_{4}}$ in a water-based system (figure 7).

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