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Biogenic gold nanoparticles synthesized using Crescentia cujete L. and evaluation of their different biological activities
Biocatalysis and Agricultural Biotechnology 11 (2017) 75–82
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Biogenic gold nanoparticles synthesized using Crescentia cujete L. and evaluation of their different biological activities
MARK
Prabukumar Seetharaman, Rajkuberan Chandrasekaran, Sathishkumar Gnanasekar, ⁎ Illaiyaraja Mani, Sivaramakrishnan Sivaperumal Department of Biotechnology, Bharathidasan University, Tiruchirappalli 620024, Tamil Nadu, India
A R T I C L E I N F O
A B S T R A C T
Keywords: Crescentia cujete Antibacterial Anticancer Gold nanoparticles
Crescentia cujete L. (Calabash tree) is a common tree distributed throughout the world. The tree and its fruit have high ethnobotanical value and practiced in Indian folk medicine. Nanotechnology is gaining more importance due to its myriad of application in physical, chemical and biological sciences. Herewith we present a comprehensive study on the benign synthesis of gold nanoparticles using an aqueous leaf extract of Crescentia cujete L. (CCAuNPs). In a shorter period of time, the reaction completed when an aqueous extract of Crescentia cujete was employed as a reducing agent to reduce Au3+ ions into nanoparticles. Generated CCAuNPs generates a shift in the reaction medium due to the excitation of surface Plasmon resonance (SPR) which produces an intense absorbance peak at 560 nm in UV–Vis spectroscopy. Fourier Transform Infrared spectroscopy (FTIR) reveals the functional group moieties involved in reduction and stabilization of CCAuNPs. Transmissions Electron Microscope (TEM) studies depict the anisotropic shape of CCAuNPs with mean size 32.89 mm. Additionally, XRay Diffraction (XRD) study depicts face centered cubic crystalline structure and Dynamic Light Scattering (DLS) revealed that synthesized CCAuNPs were stable with highly negatively charged. CCAuNPs exhibited extraordinary bactericidal activity against the tested pathogens. 3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2H tetrazolium bromide (MTT) assay determined that CCAuNPS conferred strong cytotoxicity against the HeLa cell line. Therefore, the present study shows the utility in synthesizing gold nanoparticles with Phytoconstituents and having a wide range of applications in medical science.
1. Introduction In the recent era, nanomaterials are a choice of interests among the research fraternity due to its plethora of applications in science and technology. Nanoparticles synthesis is a kind of bottom-up approach where atoms and molecules build up to form nanoclusters (Lee et al., 2015). Such kind of nanomaterials has prominent applications due to its insightful optical and spectral properties. Nanomaterials, in particular, noble metallic nanomaterials (Silver, gold, copper, zinc, palladium, selenium and iron oxide) are gaining more popularity and can be used for diversified applications (Islam et al., 2015). Among them, gold nanoparticles (AuNPs) are in the top niche due to its properties such as catalytic, optical, electronic, controllable size, dispersity, biocompatibility and strong adsorbing capacity (Lee et al., 2010). Gold nanoparticles exhibit versatility in surface modification, surface Plasmon resonance (SPR), non-toxicity, photothermal properties and have found major applications in cancer diagnosis and therapy (Cai et al., 2008; Ghosh et al., 2008).
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Corresponding author. E-mail address: srkbtge123@rediffmail.com (S. Sivaperumal).
http://dx.doi.org/10.1016/j.bcab.2017.06.004 Received 2 March 2017; Received in revised form 5 June 2017; Accepted 7 June 2017 Available online 08 June 2017 1878-8181/ © 2017 Elsevier Ltd. All rights reserved.
Conventionally AuNPs synthesized by microwave irradiation, electrochemical, ablation sonochemical, thermal decomposition and chemical reduction techniques involve expensive techniques; require high energy, usage of toxic chemicals and leaves hazardous by-products (Sathishkumar et al., 2016; Rajathi et al., 2014). Physio -chemical methods are robustly scaled up for the AuNPs production with uniform size and shape, but toxicity makes them unsuitable for biomedical applications (Narayanan and Sakthivel, 2011). Hence it is imperative to develop an environmentally friendly method for the synthesis of AuNPs to utilize its potential benefit maximum to the human welfare. Green synthesis of metallic nanoparticles can be achieved by using bioresources like bacteria, fungi, algae, and plants. But preferably, plant-based synthesis is more advantages over a microbial route which require elaborate time and high-cost downstream processing (Kuppusamy et al., 2016). Many earlier studies have reported the synthesis of metallic nanoparticles using plants, roots, seeds, flowers and explored its therapeutic potency as bactericidal, fungicidal, nematicidal, mosquitocidal and anticancer agent (Noruzi, 2015). In the
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Fig. 1. (a) Color change pattern, formation of purple red color indicates the synthesis of CCAuNPs (b) and (c) UV–Visible spectroscopy shows the effect of various reaction kinetics for efficient synthesis of CCAuNPs, in optimal conditions temperature and time. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Moreover, the size and shape of the biogenic nanoparticles can be well tuneable to achieve uniformity by altering the reaction kinetics, such as metal ion concentration, leaf extract concentration, temperature, pH, time and stoichiometric proportion (Shankar et al., 2004). Crescentia cujete is an important ethano-botanically plant possessing various curative properties used in traditional and folk medicine (Prabukumar et al., 2015). It is widely used for the treatment of broad spectrum diseases such as arthritis, meningitis, eye lesions, menstrual disorder, bronchitis, viral diseases, and bacterial infections. Moreover, virtually the plant posses’ high concentrations of manganese, iron, zinc and copper. Phytochemicals saponins, flavonoid, cardenolides, tannins phenol and hydrogen cyanide were present in the plant (Ejelonu et al., 2011). In this context, we have studied the fabrication of gold nanoparticles using an aqueous leaf extract of Crescentia cujete and evaluated their potency in antibacterial and anticancer studies.
Fig. 2. FTIR transmittance of aqueous leaf extract and synthesized CCAuNPs shows the stretches of major functional biomolecules in reduction and stabilization of CCAuNPs.
2. Materials and methods plant-based synthesis of nanoparticles, crude plant extract contains proteins, phenols, flavanoids, terpenoids, carbohydrates play a vital role in reducing metallic ions to nanosize and also act as capping/stabilizing agent in the nanoparticles formation (Noruzi et al., 2011).
2.1. Materials Chloroauric acid (HAuCl4), and 3-(4, 5-dimethylthiazol-2-yl)-2, 5–90, diphenyl-2H-tetrazolium bromide (MTT) was purchased from 76
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Fig. 3. (a) and (b) TEM micrograph at different magnification displays anisotrophic CCAuNPs (c) Particle size distribution graph of CCAuNPs, the mean size was calculated as 32.89 nm (d) X-ray diffractogram intensities were interpreted with JCPDS and depicts that synthesized CCAuNPs were cubic crystalline in nature. Fig. 3e DLS analysis shows the particle size distribution of CCAuNPs Fig. 3f Zeta potential value of CCAuNPs.
2.3. Synthesis of C. cujete L. Gold nanoparticles (CCAuNPs)
Sigma-Aldrich (St. Louis, MO, USA). All other analytical reagents used in this study were purchased from Merck, India.
For CCAuNPs synthesis, to get better efficiency, monodispersity, and yield of CCAuNPs, different parameters such as time, temperature, substrate and aqueous extract concentration, stoichiometric proportion and pH were optimized. Based on the initial test with UV-spectroscopy, the optimized parameters for CCAuNPs are fixed to be 1 mM HAuCl4; 10% C. cujete L. aqueous extract, 90 (HAuCl4) ml: 10 (aqueous extract) ml stoichiometric proportion, 60 °C, 25 min at neutral pH. After adding the reaction mixtures, the solution was observed for a color change from pale yellow to pinkish violet color, which indicates the formation of CCAuNPs. Before characterization, colloidal CCAuNPs was dialyzed by using a cellulose tube (MW cutoff 12 400 D) against 1000 ml of deionized water for 9 h at 30 °C to remove unreacted ions and
2.2. C. cujete L. aqueous extract preparation Fresh and disease symptomless leaves of C. cujete L. were collected from the University campus. After collection, the leaves were rinsed thoroughly in distilled water and eviscerated leaves shadowed dried for five days at room temperature. Using a kitchen blender, the dried leaves were crushed thoroughly for powder formation. To prepare the aqueous extract, 10 gm of powder was mixed with 100 ml of double distilled water and kept in an orbital shaker at 37 °C for 6 h. Then the aqueous mixture was filtered through What Mann Filter No: 1 and the filtered extract were stored in a refrigerator until further use. 77
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Fig. 4. (a) Antimicrobial activity of CCAuNPs against a. E.Coli, b. P.aeruginosa c. V.cholerae, d. S.typhi, e. S.flexneri f. B. subtilis Concentration range 1–25 µg, 2–50 µg, 3–75 µg, 4–100 µg. 5Positive control streptomycin 6-C. cujete L. aqueous extract.
the dimension of CCAuNPs, XRD analysis was performed. Prior analysis, CCAuNPs was lyophilized for powder formation and XRD spectra were recorded in XRD 600, Shimadzu, Japan instruments (voltage 40Kv; current 30 mA; CuKα radiation). Using Debye-Scherrer equation, the mean particle size (L) (PAN analytical X-pert PRO Model) of the CCAuNPs was calculated using the formula
molecules. Finally, the dialyzed CCAuNPs is resuspended in MILLI-Q water centrifuged and then used for further characterization. 2.4. Characterization of CCAuNPs The synthesized CCAuNPs was initially confirmed by UV–Vis spectroscopy using Cyber lab- 100 spectrophotometer operated at the wavelength of 300–700 nm. FTIR analysis was carried out to identify the reduced bio-compound present in CCAuNPs. To analyze CCAuNPs, the sample (CCAuNPs) was mixed with KBr powder to pelletize and dried. The spectra were recorded using JASCO 460 PLUS FTIR spectrometer in the diffuse reflectance (Wavelength range between 4000 cm −1to 400 cm −1). The topology features of CCAuNPs were analyzed using the microscopic images observed with JEOL 3010 transmission electron microscope. To perform TEM, the sample was resuspended thrice in MILLI-Q water, purified by centrifugation and allowed for sonication. For this, a drop of solution was used to make two thin films onto copper coated grid and allowed for drying under an infrared lamp. After drying, the micrographic images were taken using TEM with different magnification operated at an accelerating voltage of 100 KeV. To precisely orient
L = 0.9λ/β cos θ where λ – wavelength of the X- ray; θ - Bragg’s angle; β - full width and half maximum. Dynamic light scattering (DLS) and Zeta potential were performed to measure hydrodynamic distribution and surface charge of CCAuNPs. For DLS analysis, synthesized CCAuNPs were sonicated for 10–20 min to disperse the nanoparticles and analyzed using the Malvern Zetasizer, Nano-ZS90analyzer. 2.5. Antibacterial activity of CCAuNPs The bactericidal activity of CCAuNPs was evaluated against gram positive and negative pathogens using the agar well diffusion method (Rajkuberan et al., 2015). Test pathogens such as E. Coli (MTCC 1687), 78
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Fig. 5. Bactericidal effects against tested pathogens (Zone of inhibition) with triplicate.
P.aeruginosa (MTCC 1688), V.cholerae (MTCC 3906), S. typhi (MTCC 531), S.flexneri (MTCC 9543) and B. subtilis (MTCC 441) were purchased from Microbial Type Culture Collection (MTCC), Government of India. For the assay, test pathogens were subcultured individually in nutrient broth for 12 h at 37 °C. A 20 ml of Muller Hinton Agar (MHA) medium was poured into each petri plates and each bacterial strain was swabbed uniformly into plates using sterile cotton swabs. Wells of 6 mm diameter were made onto each bacterium inoculated agar plate using sterile gel borer. In the well, different concentrations of CCAuNPs (25 µg/ml, 50 µg/ml, 75 µg/ml and 100 µg/ml) along with positive control streptomycin antibiotic was added. Further, the plates were incubated at 37 °C for 24 h. After the incubation period, the plates were visually observed for the development of zone of inhibition and measured using a meter ruler in mm. 2.6. Anticancer activity of CCAuNPs 2.6.1. Cell culture The HeLa cell line was obtained from National Centre for Cell Science [NCCS], Pune and grown in Dulbecco’s modified Eagle's medium [DMEM] containing 10% fetal bovine serum [FBS]. All cells were maintained at 37° C, 5% CO2, 95% air, and 100% relative humidity. Maintenance cultures were passaged weekly, and the culture medium was changed twice a week. 2.6.2. In-vitro cytotoxicity assay The cell viability assay was measured using MTT assay (Rajkuberan et al., 2016). 100 µl of cell suspensions/ well were seeded into 96-well plates at a plating density of 10,000 cells/well and incubated to allow for cell attachment at 37 °C, 5% CO2, 95% air and 100% relative humidity. After 24 h, the medium replaced with fresh medium containing serially diluted CCAuNPs (62.5 µg, 125 µg, 250 µg, 500 µg and 1000 µg) and the cells were allowed to incubate for 48 h at 37 °C, 5% CO2, 95% air, and 100% relative humidity. After 48 h of incubation, 15 µl of MTT (5 mg/ml) was added to each well and incubated at 37 °C for 4 h. After the incubation time, the MTT solution was removed and 100 µl DMSO was added and further incubated for 30 min 37 °C. Finally, the absorbance was measured at 570 nm using a Bio- Rad micro plate reader. The % cell inhibition was determined using the following formula.
Fig. 6. (a) Images of biosynthesized CCAuNPs treated with HeLa cell line at different concentration. Control without CGAuNPs. Fig. 6 (b) Cytotoxicity effect of CCAuNPs on HeLa cell line: increased concentration of CGAuNPs (62.5–1000 µg/ml) (X-axis) inhibits the viability of cells (Y-axis) all the data were expressed in mean ± SD of three experiments.
and Log concentration and IC50 were determined using GraphPad Prism software. 3. Results and discussion Bioprospecting of plants is still in the limelight of research throughout the world due to its immeasurable treasures of phytochemical constituents and its wide therapeutic applications in cancer, HIV/AIDS, Alzheimer's, malaria, and tuberculosis (Cowan, 1999). In traditional folk, medicinal plants are used for treating various diseases.
%Cell Inhibition=A570 of test/A570 of control x100. Nonlinear regression graph was plotted between % Cell inhibition 79
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2013). As reported tannins and other phenolic moieties will oxidize in the presence of AuCl4- and thus hydroxyl groups will convert into carbonyl group. The π electron of C˭O groups by Red/Ox system transfer free orbital of metal ions and convert that to the metallic particles (Shabestariana et al., 2017). In CCAuNPs, the absorbance spectrum at 1743 cm−1 resembles the binding of carbonyl group with metallic nanoparticles. However, unassigned peaks may be attributed to the formation of new bonds between metallic nanoparticles CCAuNPs and the functional group present in the aqueous leaf extract. Fig. 3a – c TEM images depict the anisotropic (spherical, triangular, hexagonal) CCAuNPs with an average mean size of 32.89 nm. From the TEM image, an important feature that can be visualized is the particles are surrounded by a thin layer of matrix probably formed in the production process from aqueous extracts of C. cujete L. (Philip, 2010). Further, the matrix provides stability and prevent from the aggregation of CCAuNPs. With JCPDS intensities, the XRD pattern of CCAuNPs was determined. In the Fig. 3d, after reduction the diffraction peaks were noticed at 2θ = 38.03°, 46.76°, 64.55° and 77.58° were indexed as (111), (200), (220) and (311) implies that the CCAuNPs were facedcentered cubic (fcc) lattice (JCPDS, No. 04–0784). The strong reflection observed at (111) may be specified due to the development of nanocrystals. Our results were matched with those reported for the standard gold metal (Au°) (Singh et al., 2013). Using Debye–Scherrer's equation, the mean size of the CCAuNPs was calculated by determining the width of (111) peak and found to be 30 nm respectively which is similar to the TEM measurement. The hydrodynamic study and Zeta potential of CCAuNPs were evaluated through DLS. It confirms that the CCAuNPs were finely distributed within the 100 nm size range (Fig. 3e). Zeta potential corresponds that negative potential value (−26.4 mV) was suggesting the high stability of NPs. This phenomenon could be due to the presence of the capping agent, which generates repulsive forces between the NPs. (Fig. 3f) (Philip, 2009). Moreover, CCAuNPs warped with anionic compounds present in aqueous extract so that CCAuNPs were stable due to electrostatic repulsion (Namvar et al., 2015).
But scientific information is scarce on the exploration of active constituents in utilizing and developing as a drug. In this context, a new modern technology, nanotechnology has the possibility to develop plant compounds as a drug or drug precursor for disease management in pharmaceutics. The present study focused on fabricating gold nanoparticles from plant C. cujete L. for its improved biomedical applications. 3.1. Synthesis of CCAuNPs The reaction was prompt when an aqueous leaf extract of C. cujete L. was added in 1 mM HAuCl4, the reaction turned into straw yellow to purple red due to excitation of Surface Plasmonic Vibrations (SPR) (Aromal and Philip, 2012). The reaction initiated by 5 min and overall reaction completed within 25 min, which indicated the reduction of Au3+ to Au° ions and thus the formation of CCAuNPs was visually confirmed (Fig. 1a). UV–Vis spec measures the SPR of CCAuNPs which gives an intensely absorb spectrum at 560 nm (Toderas et al., 2009). This is because due to the collective oscillation of free conduction electrons of CCAuNPs in resonance induced by the interacting electromagnetic field (Sarwar et al., 2017). 3.2. Characterization of CCAuNPs The optical characteristics of gold nanoparticles are directly related to the SPR or inter band transition, particularly on size, shape, dielectric constant and surrounding environment highly influence the intensity range and bandwidth of SPR spectra (Toderas et al., 2009). In our study, UV–Vis spectroscopic analysis of synthesized CCAuNPs produces intensely absorb spectrum at 560 nm, also the synthesized CCAuNPs were stable at room temperature for more than a month. In the Fig. 1b, the range of wavelength throughout the incubation temperature was 540–590 with the continuous increase in intensity which reflects the presence of a considerable amount of monodispersed and stable AuNPs (Shankar et al., 2004). Temperature above 60 °C synthesis was achieved but the particles easily decomposed quickly in a short period of time. CCAuNPs synthesized at 60 °C showed a broad absorbance black shift due to the presence of mono and polydispersed CCAuNPs. Fig. 1c depicts the UV–Vis data of CCAuNPs analyzed at different time intervals, initially, reaction proceeds within 5 mins, but the intensity of SPR raised with respect to time intervals and sharpened at 25 °C; which resembles that synthesized CCAuNPs is a clear indication of the existence of the stable nanoparticles in colloidal form. Due to the dependence of Au binding with pH, the absorption mechanism of Au is ionic slightly than covalent. At acidic pH, gold tends present in solution in anionic form (AuCl4) and the functional groups such as hydroxyl groups on the biomass surface undergo protonation and become positively charged. The overall positively charged surface could promote the interaction between protonated functional groups and the negatively charged (AuCl4) through electrostatic attraction or electrovalent bond (Dubey et al., 2010). Finally, the optimized parameters to attain narrow size distribution and maximum yield of CCAuNPs were fixed as 1 mM HAuCl4; 10% C. cujete L. aqueous extract, 90 (HAuCl4) ml: 10 (aqueous extract) ml stoichiometric proportion, 60 °C, 25 min at neutral pH. The functional group of active biomolecules was ascertained using FTIR spectroscopy. In the Fig. 2 the CCAuNPs, major vibrations stretches were observed at 3365.89 cm−1, 2682.34 cm−1, 1335.78 cm−1 and 765.27 cm−1. The transmittance at 3365.89 cm−1 corresponds to O-H stretch or H-bonded of alcohols and phenol groups. Vibrations stretch at 2682.34 cm−1 attributes to O-H stretch of carboxylic acid groups. The band at 1335.78 cm−1 corresponds to a C-N stretch of aromatic amines and transmittance at 765.27 cm−1 attributes to N-H wag of 1° and 2° amines. The consistent vibrational bands such as O-H, C-N and N-H are derived from the compounds flavonoids, tannins, terpenoids, phenols and proteins (Zhou et al., 2010; El-Batal et al.,
3.3. Antibacterial activity of CCAuNPs The antibacterial activity of the CCAuNPs was evaluated against the human bacterial pathogens such as E.coli (MTCC 1687), P. aeruginosa (MTCC 1688), V. cholerae (MTCC 3906), S. typhi (MTCC 531), S. flexneri (MTCC 9543) and B. subtilis (MTCC 441) and the result of the inhibitory zone (mm) is represented in (Figs. 4a–f; 5). CCAuNPs gave the highest zone of inhibition against S. flexneri, whereas lower zone of inhibition was recorded against V. cholerae. The antibacterial effect of CCAuNPs at different concentrations was examined. At different doses of CCAuNPs, it provided a maximum zone of inhibition against each pathogen. However, control exhibited effective inhibitory than CCAuNPs against the tested pathogens. The inhibitory action of nanoparticles depends on sizes, surface area, dosage level, pathogens (Genus and Species) and type of phytoconstituents coated on the surface of AuNPs (Edison and Sethuraman, 2012). The antimicrobial action of CCAuNPs for Gram-positive and Gram-negative bacteria are different. The main difference is the structure of the membrane, i.e., the peptidoglycan layer’s thickness. In Gram-positive bacteria, peptidoglycan layers are very thick than Gramnegative bacteria (Shamaila et al., 2016). In the study, CCAuNPs exhibited effective inhibitory activity in Gram-negative pathogens E. coli, P. aeruginosa, V. cholerae, S. typhi, S. flexneri than gram-positive pathogen B. subtilis due to the difference in the membrane thickness. AuNPs exert the antibacterial effect in two steps. One is to change membrane potential and inhibit F-type ATP synthase, its activities to decrease the ATP level, indicating a general decline in metabolism. The other is to inhibit the subunit of ribosomal protein S10 (encoded by rpsJ) having a tRNA-binding function. AuNPs modifies 4, 6- diaminopyrimidine thiol as an analog of bacterial tRNA base has potential ability to inhibit the tRNA function for tRNA binding, indicating a 80
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collapse of the protein process (Umadevi et al., 2011). Moreover, AuNPs exhibit ROS-independent mechanism of action in pathogens suggests their low toxicity to mammalian cells (Cui et al., 2012). 3.4. Anticancer activity of CCAuNPs The cell viability assay is one of the important methods in toxicology analysis, in which the cell's response to a drug can be evaluated (Ramamurthy et al., 2013). In the study, CCAuNPs exhibited cytotoxicity to the HeLa cell line in a dose-response relationship. As the dosage of the CCAuNPs was increased, the percentage of growth inhibition was also increased. From the analysis, Inhibitory Concentration rate (IC50) of CCAuNPs in HeLa cell line was found to be 316 µg/ml. A similar study, also shown the dose-response relationship of nanoparticles synthesized from Cassia auriculata leaf extract (Rani et al., 2009), exhibited an IC50 value of 400 mg, A. leptopus derived AuNPs (IC50value: 257.8 µg/ml) (Prasanna et al., 2009). Observations of morphological changes in HeLa cell line, when exposed to CCAuNPs, was captured and recorded (Fig. 6a & b) using phase contrast microscopy. The changes include are cell burst, retardation of cell growth, cell clumping, loss of membrane stability (Balasubramani et al., 2015). The important factor of AuNPs in anticancer activity is surface properties. In general, AuNPs are positively charged, while cancer cell membranes are negatively charged materials containing lipids, phosphate groups; which allow AuNPs uptake and internalization. When NPs come into contact with the cells they are taken up by a variety of mechanisms such as clathrindependent endocytosis and macropinocytosis. These can lead to activation of cellular signaling processes producing ROS, which in turn activate DNA damage and activate Caspase-mediated cell cycle arrest in G2/M phase and induce apoptosis. (Patil and Kim, 2017). Moreover, an increase in the activities of caspase-9 and caspase-3/7 and a significant decrease in the level of ATP, along with a significant increase in protein concentration p53, Bax and downregulates Bcl-2 which results in activation of programmed cell death (Jeyaraj et al., 2015). 4. Conclusion To summarize, herewith we have synthesized gold nanoparticles from the aqueous leaf extract of C. cujete L. The synthesized CCAuNPs were characterized using UV–Vis, FTIR, TEM, DLS and XRD which implies that CCAuNPs are in nano regime and highly stable. Further, CCAuNPs were assessed for its biomedical applications. CCAuNPs perform an effective bactericidal activity against both gram positive and negative pathogens. Moreover, biogenic CCAuNPs exhibited prominent anticancer activity in HeLa cell line. CCAuNPs induce toxicity by triggering mitochondrial-driven apoptosis via activation of procaspase-3 and procaspase-9 and finally cell death. From the outcome of the study, it is perceptible that biosynthesized CCAuNPs is a clean, inexpensive, commercially valid drug to treat infectious diseases. Further, its potential should be corroborated in different types of cancer cells, elucidation of molecular mechanisms and in preclinical test assessment. Acknowledgements The authors declare no conflict of interests References Aromal, S.A., Philip, D., 2012. Benincasa hispida seed mediated green synthesis of gold nanoparticles and its optical nonlinearity. Phys. E 44, 1329–1334. Balasubramani, G., Ramkumar, R., Krishnaveni, N., Pazhanimuthu, A., Natarajan, T., Sowmiya, R., Perumal, P., 2015. Structural characterization, antioxidant and anticancer properties of gold nanoparticles synthesized from leaf extract (decoction) of Antigonon leptopus Hook & Arn. J. Trace Elem. Med. Biol. 30, 83–89. Cai, W., Gao, T., Hong, H., Sun, J., 2008. Applications of Au NPs in cancer nanotechnology. Nanotechnol. Sci. Appl. 1, 17–32. Cowan, M.M., 1999. Plant products as antimicrobial agents. Clin. Microbiol. Rev. 12, 564–582.
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Toderas, F., Iosin, M., Astilean, S., 2009. Luminescence properties of gold nanorods. Nucl. Instrum. Methods Phys. Res. B 267, 400–402. Umadevi, M., Rani, T., Balkrishnan, T., Ramanibai, R., 2011. Antimicrobial activity of silver nanoparticles under an ultrasonic field. Int J. Pharm. Sci. Nanotechnol. 4, 1491–1496. Zhou, Y., Lin, W., Huang, J., Wang, W., Gao, Y., Lin, L., Li, Q., Lin, L., Du, M., 2010. Biosynthesis of gold nanoparticles by Foliar Broths: roles of biocompounds and other attributes of the extracts. Nanoscale Res. Lett. 5, 1351–1359.
Nanomaterials 6 (4), 71. Shankar, S.S., Rai, A., Ahmad, A., Sastry, M., 2004. Rapid synthesis of Au, Ag, and bimetallic Au core—Ag shell nanoparticles using neem (Azadirachta indica) leaf broth. J. Colloid Interface Sci. 275, 496–502. Singh, D.K., Jagannathan, R., Khandelwal, P., Abraham, P.M., Poddar, P., 2013. In situ synthesis and surface functionalization of gold nanoparticles with curcumin and their antioxidant properties: an experimental and density functional theory investigation. Nanoscale 5, 1882–1893.
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Biogenic gold nanoparticles synthesized using Crescentia cujete L. and evaluation of their different biological activities
MARK
Prabukumar Seetharaman, Rajkuberan Chandrasekaran, Sathishkumar Gnanasekar, ⁎ Illaiyaraja Mani, Sivaramakrishnan Sivaperumal Department of Biotechnology, Bharathidasan University, Tiruchirappalli 620024, Tamil Nadu, India
A R T I C L E I N F O
A B S T R A C T
Keywords: Crescentia cujete Antibacterial Anticancer Gold nanoparticles
Crescentia cujete L. (Calabash tree) is a common tree distributed throughout the world. The tree and its fruit have high ethnobotanical value and practiced in Indian folk medicine. Nanotechnology is gaining more importance due to its myriad of application in physical, chemical and biological sciences. Herewith we present a comprehensive study on the benign synthesis of gold nanoparticles using an aqueous leaf extract of Crescentia cujete L. (CCAuNPs). In a shorter period of time, the reaction completed when an aqueous extract of Crescentia cujete was employed as a reducing agent to reduce Au3+ ions into nanoparticles. Generated CCAuNPs generates a shift in the reaction medium due to the excitation of surface Plasmon resonance (SPR) which produces an intense absorbance peak at 560 nm in UV–Vis spectroscopy. Fourier Transform Infrared spectroscopy (FTIR) reveals the functional group moieties involved in reduction and stabilization of CCAuNPs. Transmissions Electron Microscope (TEM) studies depict the anisotropic shape of CCAuNPs with mean size 32.89 mm. Additionally, XRay Diffraction (XRD) study depicts face centered cubic crystalline structure and Dynamic Light Scattering (DLS) revealed that synthesized CCAuNPs were stable with highly negatively charged. CCAuNPs exhibited extraordinary bactericidal activity against the tested pathogens. 3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2H tetrazolium bromide (MTT) assay determined that CCAuNPS conferred strong cytotoxicity against the HeLa cell line. Therefore, the present study shows the utility in synthesizing gold nanoparticles with Phytoconstituents and having a wide range of applications in medical science.
1. Introduction In the recent era, nanomaterials are a choice of interests among the research fraternity due to its plethora of applications in science and technology. Nanoparticles synthesis is a kind of bottom-up approach where atoms and molecules build up to form nanoclusters (Lee et al., 2015). Such kind of nanomaterials has prominent applications due to its insightful optical and spectral properties. Nanomaterials, in particular, noble metallic nanomaterials (Silver, gold, copper, zinc, palladium, selenium and iron oxide) are gaining more popularity and can be used for diversified applications (Islam et al., 2015). Among them, gold nanoparticles (AuNPs) are in the top niche due to its properties such as catalytic, optical, electronic, controllable size, dispersity, biocompatibility and strong adsorbing capacity (Lee et al., 2010). Gold nanoparticles exhibit versatility in surface modification, surface Plasmon resonance (SPR), non-toxicity, photothermal properties and have found major applications in cancer diagnosis and therapy (Cai et al., 2008; Ghosh et al., 2008).
⁎
Corresponding author. E-mail address: srkbtge123@rediffmail.com (S. Sivaperumal).
http://dx.doi.org/10.1016/j.bcab.2017.06.004 Received 2 March 2017; Received in revised form 5 June 2017; Accepted 7 June 2017 Available online 08 June 2017 1878-8181/ © 2017 Elsevier Ltd. All rights reserved.
Conventionally AuNPs synthesized by microwave irradiation, electrochemical, ablation sonochemical, thermal decomposition and chemical reduction techniques involve expensive techniques; require high energy, usage of toxic chemicals and leaves hazardous by-products (Sathishkumar et al., 2016; Rajathi et al., 2014). Physio -chemical methods are robustly scaled up for the AuNPs production with uniform size and shape, but toxicity makes them unsuitable for biomedical applications (Narayanan and Sakthivel, 2011). Hence it is imperative to develop an environmentally friendly method for the synthesis of AuNPs to utilize its potential benefit maximum to the human welfare. Green synthesis of metallic nanoparticles can be achieved by using bioresources like bacteria, fungi, algae, and plants. But preferably, plant-based synthesis is more advantages over a microbial route which require elaborate time and high-cost downstream processing (Kuppusamy et al., 2016). Many earlier studies have reported the synthesis of metallic nanoparticles using plants, roots, seeds, flowers and explored its therapeutic potency as bactericidal, fungicidal, nematicidal, mosquitocidal and anticancer agent (Noruzi, 2015). In the
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Fig. 1. (a) Color change pattern, formation of purple red color indicates the synthesis of CCAuNPs (b) and (c) UV–Visible spectroscopy shows the effect of various reaction kinetics for efficient synthesis of CCAuNPs, in optimal conditions temperature and time. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Moreover, the size and shape of the biogenic nanoparticles can be well tuneable to achieve uniformity by altering the reaction kinetics, such as metal ion concentration, leaf extract concentration, temperature, pH, time and stoichiometric proportion (Shankar et al., 2004). Crescentia cujete is an important ethano-botanically plant possessing various curative properties used in traditional and folk medicine (Prabukumar et al., 2015). It is widely used for the treatment of broad spectrum diseases such as arthritis, meningitis, eye lesions, menstrual disorder, bronchitis, viral diseases, and bacterial infections. Moreover, virtually the plant posses’ high concentrations of manganese, iron, zinc and copper. Phytochemicals saponins, flavonoid, cardenolides, tannins phenol and hydrogen cyanide were present in the plant (Ejelonu et al., 2011). In this context, we have studied the fabrication of gold nanoparticles using an aqueous leaf extract of Crescentia cujete and evaluated their potency in antibacterial and anticancer studies.
Fig. 2. FTIR transmittance of aqueous leaf extract and synthesized CCAuNPs shows the stretches of major functional biomolecules in reduction and stabilization of CCAuNPs.
2. Materials and methods plant-based synthesis of nanoparticles, crude plant extract contains proteins, phenols, flavanoids, terpenoids, carbohydrates play a vital role in reducing metallic ions to nanosize and also act as capping/stabilizing agent in the nanoparticles formation (Noruzi et al., 2011).
2.1. Materials Chloroauric acid (HAuCl4), and 3-(4, 5-dimethylthiazol-2-yl)-2, 5–90, diphenyl-2H-tetrazolium bromide (MTT) was purchased from 76
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Fig. 3. (a) and (b) TEM micrograph at different magnification displays anisotrophic CCAuNPs (c) Particle size distribution graph of CCAuNPs, the mean size was calculated as 32.89 nm (d) X-ray diffractogram intensities were interpreted with JCPDS and depicts that synthesized CCAuNPs were cubic crystalline in nature. Fig. 3e DLS analysis shows the particle size distribution of CCAuNPs Fig. 3f Zeta potential value of CCAuNPs.
2.3. Synthesis of C. cujete L. Gold nanoparticles (CCAuNPs)
Sigma-Aldrich (St. Louis, MO, USA). All other analytical reagents used in this study were purchased from Merck, India.
For CCAuNPs synthesis, to get better efficiency, monodispersity, and yield of CCAuNPs, different parameters such as time, temperature, substrate and aqueous extract concentration, stoichiometric proportion and pH were optimized. Based on the initial test with UV-spectroscopy, the optimized parameters for CCAuNPs are fixed to be 1 mM HAuCl4; 10% C. cujete L. aqueous extract, 90 (HAuCl4) ml: 10 (aqueous extract) ml stoichiometric proportion, 60 °C, 25 min at neutral pH. After adding the reaction mixtures, the solution was observed for a color change from pale yellow to pinkish violet color, which indicates the formation of CCAuNPs. Before characterization, colloidal CCAuNPs was dialyzed by using a cellulose tube (MW cutoff 12 400 D) against 1000 ml of deionized water for 9 h at 30 °C to remove unreacted ions and
2.2. C. cujete L. aqueous extract preparation Fresh and disease symptomless leaves of C. cujete L. were collected from the University campus. After collection, the leaves were rinsed thoroughly in distilled water and eviscerated leaves shadowed dried for five days at room temperature. Using a kitchen blender, the dried leaves were crushed thoroughly for powder formation. To prepare the aqueous extract, 10 gm of powder was mixed with 100 ml of double distilled water and kept in an orbital shaker at 37 °C for 6 h. Then the aqueous mixture was filtered through What Mann Filter No: 1 and the filtered extract were stored in a refrigerator until further use. 77
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Fig. 4. (a) Antimicrobial activity of CCAuNPs against a. E.Coli, b. P.aeruginosa c. V.cholerae, d. S.typhi, e. S.flexneri f. B. subtilis Concentration range 1–25 µg, 2–50 µg, 3–75 µg, 4–100 µg. 5Positive control streptomycin 6-C. cujete L. aqueous extract.
the dimension of CCAuNPs, XRD analysis was performed. Prior analysis, CCAuNPs was lyophilized for powder formation and XRD spectra were recorded in XRD 600, Shimadzu, Japan instruments (voltage 40Kv; current 30 mA; CuKα radiation). Using Debye-Scherrer equation, the mean particle size (L) (PAN analytical X-pert PRO Model) of the CCAuNPs was calculated using the formula
molecules. Finally, the dialyzed CCAuNPs is resuspended in MILLI-Q water centrifuged and then used for further characterization. 2.4. Characterization of CCAuNPs The synthesized CCAuNPs was initially confirmed by UV–Vis spectroscopy using Cyber lab- 100 spectrophotometer operated at the wavelength of 300–700 nm. FTIR analysis was carried out to identify the reduced bio-compound present in CCAuNPs. To analyze CCAuNPs, the sample (CCAuNPs) was mixed with KBr powder to pelletize and dried. The spectra were recorded using JASCO 460 PLUS FTIR spectrometer in the diffuse reflectance (Wavelength range between 4000 cm −1to 400 cm −1). The topology features of CCAuNPs were analyzed using the microscopic images observed with JEOL 3010 transmission electron microscope. To perform TEM, the sample was resuspended thrice in MILLI-Q water, purified by centrifugation and allowed for sonication. For this, a drop of solution was used to make two thin films onto copper coated grid and allowed for drying under an infrared lamp. After drying, the micrographic images were taken using TEM with different magnification operated at an accelerating voltage of 100 KeV. To precisely orient
L = 0.9λ/β cos θ where λ – wavelength of the X- ray; θ - Bragg’s angle; β - full width and half maximum. Dynamic light scattering (DLS) and Zeta potential were performed to measure hydrodynamic distribution and surface charge of CCAuNPs. For DLS analysis, synthesized CCAuNPs were sonicated for 10–20 min to disperse the nanoparticles and analyzed using the Malvern Zetasizer, Nano-ZS90analyzer. 2.5. Antibacterial activity of CCAuNPs The bactericidal activity of CCAuNPs was evaluated against gram positive and negative pathogens using the agar well diffusion method (Rajkuberan et al., 2015). Test pathogens such as E. Coli (MTCC 1687), 78
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Fig. 5. Bactericidal effects against tested pathogens (Zone of inhibition) with triplicate.
P.aeruginosa (MTCC 1688), V.cholerae (MTCC 3906), S. typhi (MTCC 531), S.flexneri (MTCC 9543) and B. subtilis (MTCC 441) were purchased from Microbial Type Culture Collection (MTCC), Government of India. For the assay, test pathogens were subcultured individually in nutrient broth for 12 h at 37 °C. A 20 ml of Muller Hinton Agar (MHA) medium was poured into each petri plates and each bacterial strain was swabbed uniformly into plates using sterile cotton swabs. Wells of 6 mm diameter were made onto each bacterium inoculated agar plate using sterile gel borer. In the well, different concentrations of CCAuNPs (25 µg/ml, 50 µg/ml, 75 µg/ml and 100 µg/ml) along with positive control streptomycin antibiotic was added. Further, the plates were incubated at 37 °C for 24 h. After the incubation period, the plates were visually observed for the development of zone of inhibition and measured using a meter ruler in mm. 2.6. Anticancer activity of CCAuNPs 2.6.1. Cell culture The HeLa cell line was obtained from National Centre for Cell Science [NCCS], Pune and grown in Dulbecco’s modified Eagle's medium [DMEM] containing 10% fetal bovine serum [FBS]. All cells were maintained at 37° C, 5% CO2, 95% air, and 100% relative humidity. Maintenance cultures were passaged weekly, and the culture medium was changed twice a week. 2.6.2. In-vitro cytotoxicity assay The cell viability assay was measured using MTT assay (Rajkuberan et al., 2016). 100 µl of cell suspensions/ well were seeded into 96-well plates at a plating density of 10,000 cells/well and incubated to allow for cell attachment at 37 °C, 5% CO2, 95% air and 100% relative humidity. After 24 h, the medium replaced with fresh medium containing serially diluted CCAuNPs (62.5 µg, 125 µg, 250 µg, 500 µg and 1000 µg) and the cells were allowed to incubate for 48 h at 37 °C, 5% CO2, 95% air, and 100% relative humidity. After 48 h of incubation, 15 µl of MTT (5 mg/ml) was added to each well and incubated at 37 °C for 4 h. After the incubation time, the MTT solution was removed and 100 µl DMSO was added and further incubated for 30 min 37 °C. Finally, the absorbance was measured at 570 nm using a Bio- Rad micro plate reader. The % cell inhibition was determined using the following formula.
Fig. 6. (a) Images of biosynthesized CCAuNPs treated with HeLa cell line at different concentration. Control without CGAuNPs. Fig. 6 (b) Cytotoxicity effect of CCAuNPs on HeLa cell line: increased concentration of CGAuNPs (62.5–1000 µg/ml) (X-axis) inhibits the viability of cells (Y-axis) all the data were expressed in mean ± SD of three experiments.
and Log concentration and IC50 were determined using GraphPad Prism software. 3. Results and discussion Bioprospecting of plants is still in the limelight of research throughout the world due to its immeasurable treasures of phytochemical constituents and its wide therapeutic applications in cancer, HIV/AIDS, Alzheimer's, malaria, and tuberculosis (Cowan, 1999). In traditional folk, medicinal plants are used for treating various diseases.
%Cell Inhibition=A570 of test/A570 of control x100. Nonlinear regression graph was plotted between % Cell inhibition 79
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2013). As reported tannins and other phenolic moieties will oxidize in the presence of AuCl4- and thus hydroxyl groups will convert into carbonyl group. The π electron of C˭O groups by Red/Ox system transfer free orbital of metal ions and convert that to the metallic particles (Shabestariana et al., 2017). In CCAuNPs, the absorbance spectrum at 1743 cm−1 resembles the binding of carbonyl group with metallic nanoparticles. However, unassigned peaks may be attributed to the formation of new bonds between metallic nanoparticles CCAuNPs and the functional group present in the aqueous leaf extract. Fig. 3a – c TEM images depict the anisotropic (spherical, triangular, hexagonal) CCAuNPs with an average mean size of 32.89 nm. From the TEM image, an important feature that can be visualized is the particles are surrounded by a thin layer of matrix probably formed in the production process from aqueous extracts of C. cujete L. (Philip, 2010). Further, the matrix provides stability and prevent from the aggregation of CCAuNPs. With JCPDS intensities, the XRD pattern of CCAuNPs was determined. In the Fig. 3d, after reduction the diffraction peaks were noticed at 2θ = 38.03°, 46.76°, 64.55° and 77.58° were indexed as (111), (200), (220) and (311) implies that the CCAuNPs were facedcentered cubic (fcc) lattice (JCPDS, No. 04–0784). The strong reflection observed at (111) may be specified due to the development of nanocrystals. Our results were matched with those reported for the standard gold metal (Au°) (Singh et al., 2013). Using Debye–Scherrer's equation, the mean size of the CCAuNPs was calculated by determining the width of (111) peak and found to be 30 nm respectively which is similar to the TEM measurement. The hydrodynamic study and Zeta potential of CCAuNPs were evaluated through DLS. It confirms that the CCAuNPs were finely distributed within the 100 nm size range (Fig. 3e). Zeta potential corresponds that negative potential value (−26.4 mV) was suggesting the high stability of NPs. This phenomenon could be due to the presence of the capping agent, which generates repulsive forces between the NPs. (Fig. 3f) (Philip, 2009). Moreover, CCAuNPs warped with anionic compounds present in aqueous extract so that CCAuNPs were stable due to electrostatic repulsion (Namvar et al., 2015).
But scientific information is scarce on the exploration of active constituents in utilizing and developing as a drug. In this context, a new modern technology, nanotechnology has the possibility to develop plant compounds as a drug or drug precursor for disease management in pharmaceutics. The present study focused on fabricating gold nanoparticles from plant C. cujete L. for its improved biomedical applications. 3.1. Synthesis of CCAuNPs The reaction was prompt when an aqueous leaf extract of C. cujete L. was added in 1 mM HAuCl4, the reaction turned into straw yellow to purple red due to excitation of Surface Plasmonic Vibrations (SPR) (Aromal and Philip, 2012). The reaction initiated by 5 min and overall reaction completed within 25 min, which indicated the reduction of Au3+ to Au° ions and thus the formation of CCAuNPs was visually confirmed (Fig. 1a). UV–Vis spec measures the SPR of CCAuNPs which gives an intensely absorb spectrum at 560 nm (Toderas et al., 2009). This is because due to the collective oscillation of free conduction electrons of CCAuNPs in resonance induced by the interacting electromagnetic field (Sarwar et al., 2017). 3.2. Characterization of CCAuNPs The optical characteristics of gold nanoparticles are directly related to the SPR or inter band transition, particularly on size, shape, dielectric constant and surrounding environment highly influence the intensity range and bandwidth of SPR spectra (Toderas et al., 2009). In our study, UV–Vis spectroscopic analysis of synthesized CCAuNPs produces intensely absorb spectrum at 560 nm, also the synthesized CCAuNPs were stable at room temperature for more than a month. In the Fig. 1b, the range of wavelength throughout the incubation temperature was 540–590 with the continuous increase in intensity which reflects the presence of a considerable amount of monodispersed and stable AuNPs (Shankar et al., 2004). Temperature above 60 °C synthesis was achieved but the particles easily decomposed quickly in a short period of time. CCAuNPs synthesized at 60 °C showed a broad absorbance black shift due to the presence of mono and polydispersed CCAuNPs. Fig. 1c depicts the UV–Vis data of CCAuNPs analyzed at different time intervals, initially, reaction proceeds within 5 mins, but the intensity of SPR raised with respect to time intervals and sharpened at 25 °C; which resembles that synthesized CCAuNPs is a clear indication of the existence of the stable nanoparticles in colloidal form. Due to the dependence of Au binding with pH, the absorption mechanism of Au is ionic slightly than covalent. At acidic pH, gold tends present in solution in anionic form (AuCl4) and the functional groups such as hydroxyl groups on the biomass surface undergo protonation and become positively charged. The overall positively charged surface could promote the interaction between protonated functional groups and the negatively charged (AuCl4) through electrostatic attraction or electrovalent bond (Dubey et al., 2010). Finally, the optimized parameters to attain narrow size distribution and maximum yield of CCAuNPs were fixed as 1 mM HAuCl4; 10% C. cujete L. aqueous extract, 90 (HAuCl4) ml: 10 (aqueous extract) ml stoichiometric proportion, 60 °C, 25 min at neutral pH. The functional group of active biomolecules was ascertained using FTIR spectroscopy. In the Fig. 2 the CCAuNPs, major vibrations stretches were observed at 3365.89 cm−1, 2682.34 cm−1, 1335.78 cm−1 and 765.27 cm−1. The transmittance at 3365.89 cm−1 corresponds to O-H stretch or H-bonded of alcohols and phenol groups. Vibrations stretch at 2682.34 cm−1 attributes to O-H stretch of carboxylic acid groups. The band at 1335.78 cm−1 corresponds to a C-N stretch of aromatic amines and transmittance at 765.27 cm−1 attributes to N-H wag of 1° and 2° amines. The consistent vibrational bands such as O-H, C-N and N-H are derived from the compounds flavonoids, tannins, terpenoids, phenols and proteins (Zhou et al., 2010; El-Batal et al.,
3.3. Antibacterial activity of CCAuNPs The antibacterial activity of the CCAuNPs was evaluated against the human bacterial pathogens such as E.coli (MTCC 1687), P. aeruginosa (MTCC 1688), V. cholerae (MTCC 3906), S. typhi (MTCC 531), S. flexneri (MTCC 9543) and B. subtilis (MTCC 441) and the result of the inhibitory zone (mm) is represented in (Figs. 4a–f; 5). CCAuNPs gave the highest zone of inhibition against S. flexneri, whereas lower zone of inhibition was recorded against V. cholerae. The antibacterial effect of CCAuNPs at different concentrations was examined. At different doses of CCAuNPs, it provided a maximum zone of inhibition against each pathogen. However, control exhibited effective inhibitory than CCAuNPs against the tested pathogens. The inhibitory action of nanoparticles depends on sizes, surface area, dosage level, pathogens (Genus and Species) and type of phytoconstituents coated on the surface of AuNPs (Edison and Sethuraman, 2012). The antimicrobial action of CCAuNPs for Gram-positive and Gram-negative bacteria are different. The main difference is the structure of the membrane, i.e., the peptidoglycan layer’s thickness. In Gram-positive bacteria, peptidoglycan layers are very thick than Gramnegative bacteria (Shamaila et al., 2016). In the study, CCAuNPs exhibited effective inhibitory activity in Gram-negative pathogens E. coli, P. aeruginosa, V. cholerae, S. typhi, S. flexneri than gram-positive pathogen B. subtilis due to the difference in the membrane thickness. AuNPs exert the antibacterial effect in two steps. One is to change membrane potential and inhibit F-type ATP synthase, its activities to decrease the ATP level, indicating a general decline in metabolism. The other is to inhibit the subunit of ribosomal protein S10 (encoded by rpsJ) having a tRNA-binding function. AuNPs modifies 4, 6- diaminopyrimidine thiol as an analog of bacterial tRNA base has potential ability to inhibit the tRNA function for tRNA binding, indicating a 80
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collapse of the protein process (Umadevi et al., 2011). Moreover, AuNPs exhibit ROS-independent mechanism of action in pathogens suggests their low toxicity to mammalian cells (Cui et al., 2012). 3.4. Anticancer activity of CCAuNPs The cell viability assay is one of the important methods in toxicology analysis, in which the cell's response to a drug can be evaluated (Ramamurthy et al., 2013). In the study, CCAuNPs exhibited cytotoxicity to the HeLa cell line in a dose-response relationship. As the dosage of the CCAuNPs was increased, the percentage of growth inhibition was also increased. From the analysis, Inhibitory Concentration rate (IC50) of CCAuNPs in HeLa cell line was found to be 316 µg/ml. A similar study, also shown the dose-response relationship of nanoparticles synthesized from Cassia auriculata leaf extract (Rani et al., 2009), exhibited an IC50 value of 400 mg, A. leptopus derived AuNPs (IC50value: 257.8 µg/ml) (Prasanna et al., 2009). Observations of morphological changes in HeLa cell line, when exposed to CCAuNPs, was captured and recorded (Fig. 6a & b) using phase contrast microscopy. The changes include are cell burst, retardation of cell growth, cell clumping, loss of membrane stability (Balasubramani et al., 2015). The important factor of AuNPs in anticancer activity is surface properties. In general, AuNPs are positively charged, while cancer cell membranes are negatively charged materials containing lipids, phosphate groups; which allow AuNPs uptake and internalization. When NPs come into contact with the cells they are taken up by a variety of mechanisms such as clathrindependent endocytosis and macropinocytosis. These can lead to activation of cellular signaling processes producing ROS, which in turn activate DNA damage and activate Caspase-mediated cell cycle arrest in G2/M phase and induce apoptosis. (Patil and Kim, 2017). Moreover, an increase in the activities of caspase-9 and caspase-3/7 and a significant decrease in the level of ATP, along with a significant increase in protein concentration p53, Bax and downregulates Bcl-2 which results in activation of programmed cell death (Jeyaraj et al., 2015). 4. Conclusion To summarize, herewith we have synthesized gold nanoparticles from the aqueous leaf extract of C. cujete L. The synthesized CCAuNPs were characterized using UV–Vis, FTIR, TEM, DLS and XRD which implies that CCAuNPs are in nano regime and highly stable. Further, CCAuNPs were assessed for its biomedical applications. CCAuNPs perform an effective bactericidal activity against both gram positive and negative pathogens. Moreover, biogenic CCAuNPs exhibited prominent anticancer activity in HeLa cell line. CCAuNPs induce toxicity by triggering mitochondrial-driven apoptosis via activation of procaspase-3 and procaspase-9 and finally cell death. From the outcome of the study, it is perceptible that biosynthesized CCAuNPs is a clean, inexpensive, commercially valid drug to treat infectious diseases. Further, its potential should be corroborated in different types of cancer cells, elucidation of molecular mechanisms and in preclinical test assessment. Acknowledgements The authors declare no conflict of interests References Aromal, S.A., Philip, D., 2012. Benincasa hispida seed mediated green synthesis of gold nanoparticles and its optical nonlinearity. Phys. E 44, 1329–1334. Balasubramani, G., Ramkumar, R., Krishnaveni, N., Pazhanimuthu, A., Natarajan, T., Sowmiya, R., Perumal, P., 2015. Structural characterization, antioxidant and anticancer properties of gold nanoparticles synthesized from leaf extract (decoction) of Antigonon leptopus Hook & Arn. J. Trace Elem. Med. Biol. 30, 83–89. Cai, W., Gao, T., Hong, H., Sun, J., 2008. Applications of Au NPs in cancer nanotechnology. Nanotechnol. Sci. Appl. 1, 17–32. Cowan, M.M., 1999. Plant products as antimicrobial agents. Clin. Microbiol. Rev. 12, 564–582.
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