PHYTOFABRICATION OF SILVER NANO PARTICLES USING OCIMUM SANCTUM LEAF EXTRACT AND THEIR ANTIBACTERIAL AND ANTICANCER ACTIVITY THROUGH OXIDATIVE DAMAGE

Silver nanoparticles (AgNP) have found prominence in different fields such as medicine, catalysis, nanoelectronics, textile field, pollution and water treatment due to their unique attributes. Applications of AgNP are increasing rapidly in the medical purpose including drug delivery, treatment, diagnosis, medical device coating. Various chemical and physical methods are used to synthesize the AgNP conventionally. However, these processes are expensive and also have side effects. To solve these problems by modification in synthesis process for safer and more efficiency, synthesis of biogenic AgNP from plant extract, known as Green Nanotechnology, have come to play a very crucial role, In this study, we have reported the green synthesis of AgNP using Ocimum sanctum (Tulsi) leaf extract, which act as reducing agent as well as capping agent. Synthesized AgNPs were characterized and their antibacterial and anticancer activities were observed. The development of brown color by the addition of Tulsi signifies the formation of silver nanoparticles. UV-Vis absorption spectroscopy, XRD and zeta potential were applied to estimate the quantitative formation of silver nanoparticles. FTIR analysis was used to reveal that the AgNPs were stabilized by eugenols, terpenes, and other aromatic compounds present in the leaf extract. The antimicrobial and anticancer properties of AgNPs were assessed by various in vitro cellular assays. Our present study confirms that AgNP can be used as a dual therapeutic option for combating pathogenic microbial strains as well as hepatocellular cancer.


Introduction
Nanotechnology is the field for development of more consistent process for the synthesis of nanomaterials more than a range of size (with good monodispersity) and chemical composition [1]. Metallic nanoparticles have been gaining a lot of significance in the past few years due to their applicability in the field of physics, chemistry, medicine, biology and material science [2]. Metallic nanoparticles due to their high specific surface area and surface atoms have outstanding physicochemical characteristics, including optical, catalytic, electronic, magnetic and antibacterial properties. Synthesized metallic nanoparticles are enormous due to their influential applicability in various fields such as electronics, chemistry, energy, and medicine development [3]. Metallic nanoparticles, particularly noble metals are influential mainly because of their dominant optical absorption in the visible region caused by the exciting group of the free electron gas [4]. The silver nanoparticles have a wide range of interest as they have a larger number of apposite: nonlinear optics, spectrally selective coating for solar energy absorption, good electrical conductors, bio-labeling, antibacterial materials, intercalation materials for electrical batteries as optical receptors, chemically stable materials and catalyst in chemical reactions [5,6] In the medical field, silver and silver nanoparticles have broad applications, especially in skin ointments and creams to avoid infection of burns and open wounds [10]. Silver possess an inhibitory action toward many bacterial strains and microorganisms commonly present in medical and industrial processes [7]. The general method of synthesizing silver nanoparticles in chemical reduction is as colloidal dispersions in water or organic solvents [8]. The green synthesis method is an imperative http://sciforum.net/conference/mol2net-06 technique, utilizes nontoxic chemicals, eco-friendly solvent and renewable materials [9] and has capability to reduce metal by specific metabolic pathway. Many biological approaches of green synthesis have been reported till date using plant leaf extracts from different plants [9,10,11]. These biosynthesized nanoparticles act as an antibacterial agent [12][13][14][15][16][17][18].
Here we have observed the effect of these biogenic AgNPs on the prokaryotic and eukaryotic cell. The phytofabricated AgNPs had a positive effect against Gram-positive (S. aureus, B. subtilis) and Gram-negative (E. coli, P. aeruginosa) pathogenic bacterial species. We have also studied the mechanism behind the bacterial growth inhibition. Our present experiments with biogenic AgNP, destruction of bacterial cell wall might have caused due to one or multiple steps of signaling cascades resulting in defective cell wall synthesis or impaired cross-linking of polymer units, which have occurred due to the ROS generation in both Gram-positive and Gram-negative bacterial strains [28,29]. Silver ions have a great impact on altered cellular metabolism inhibiting proliferation and metastasis of cancer cells. Reactive oxygen species (ROS) generation plays a crucial role in the maintenance of the redox balance in most cancer cells and an elevated ROS level may promote oxidative damage leading to cellular abnormalities or death [30,31]. In this study, we investigated (AgNPs) and their antimicrobial as well as anticancer activities against different pathogenic bacterial strains and hepatocellular cancer cell lines. Thus, the development of these phytofabricated, non hazardous, eco-friendly AgNPs has the potential to emerge as an effective dual therapeutic agent.

Materials
AR-grade silver nitrate (AgNO3) was purchased from Sigma-Aldrich Chemicals and fresh Ocimum sanctum leaves were collected from local area Kolkata, India. All the chemicals and reagents required for bacterial culture media, MTT reagent, glutaraldehyde, fluorescence stains, different chemicals and reagents used for biological purposes were pruchased from Merck Ltd, and SRL Pvt. Ltd, Mumbai, India at the highest grade available. All the reagents were used without further purification. Deionised (Millipore) water was used throughout the experiment with resistivity at least 18 MΩ-cm. All the glasswares used in our experiments were cleaned with aqua regia solution followed by rinsing with ultrapure water.

Bacterial strain and Cell line
Gram-positive (Staphylococcus aureus 740 and Bacillus subtilis 441) and Gram-negative (Escherichia coli 443 and Pseudomona aeruginosa 1688) used for the bacterial experiment were procured from Microbial Type Culture Collection (MTCC), IMTECH, Chandigarh, India. Hep G2 are used for determine the cytotoxicity assay was obtained from Central Cell repository of National Center for Cell Science (NCCS), Pune, India.

Preparation of plant leaf extract
MOL2NET, 2020, 6, ISSN: 2624-5078 4 http://sciforum.net/conference/mol2net-06 The leaves of Ocimum sanctum were washed by diionized water to remove the dust and dirt particles. The leaves are then dried at room temperature. About 2 grams of the dried powder leaves was taken in to the 100 ml distilled water. The leaves were boiled for 5 minutes and then it was allowed to cool. The solution was filtered and then stored at 4° C. All synthesis was performed within a week after preparation of leaf extract.

Preparation of Silver Nitrate Solution
1.0 mM AgNO3 added in to distilled water and stirred continuously at 2 to 4 hours.

Synthesis of Silver Nanoparticles
150 ml of prepared silver nitrate stock solution was taken in a 250 ml beaker. After that, 10ml of leaf extract was added. At 90 o C the mixture was heated in a water bath for 1hr.

Purification of Synthesized Particles
After heating, precipitation was obtained by centrifugation at 9000 rpm for 25 minutes. Then the precipitation was washed by centrifuged repeatedly (3 times). Thus purified Ag Nps was obtained.

Physical Characterisation of Silver Nanoparticle
The absorbance spectra were measured using Ultraviolet-visible spectrophotometer (Bio-tek) at a wavelength of 250-800 nm. Silver nano particles were synthesized by reducing silver metal ions solution with Tulsi leaf extract were initially characterized using UV-Visible Spectrophotometer. The XRD (X Ray Diffractometer) patterns of the silvernano particle samples were recorded by X-ray powder Diffractometer model D8, Bruker AXS, Winconsin, USA, using Cu-Kα target employing wavelength of 1.5418 Å and operating at 35 kV with scan speed of 2s/step. Particle size and its distribution were assessed with field emission Scanning Electron Microscope (FESEM) using ZEISS. Electron interacts with the electrons in the sample, producing various signals that can be detected and that contain information about surface topography and composition of the samples. The Fourier transform infrared spectroscopy (FTIR) study was done using FTIR-8400S, Shimadzu in the wavenumber range from 400 cm −1 to 4000 cm −1 . FTIR Spectrometer to detect the possible functional groups in biomolecules present in the plant extract. The particle size distribution and stability were measured by DLS (Dynamic light Scattering) using Zetasizer (NANO ZS90, Malvern Instruments Ltd., UK). The surface charges of the nanoparticles were also measured by the Zetasizer.

Antibacterial activity determination (a) Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)
According to our previously reported protocol [28,29], MIC and MBC were evaluated by microdilution method in Luria broth. Different concentrations of AgNPs were added to the bacterial media containing the inoculums and incubated for 24 hrs. After incubation, the MIC values were obtained by checking the turbidity of the bacterial growth with UV absorption. The MBC values were determined by spreading the MIC dilutions of growth i.e. 10 μl of bacterial strain containing 2.5 × 10 5 CFU ml −1 bacteria were separately added to the several 1 ml nutrient broths (NBs) onto agar plates and incubating them for 24 hrs at 37° C. The lowest concentration of AgNPs, at which the bacterial strains were completely inhibited, was noted as the MBC value. This MBC value of the particles denotes the minimum http://sciforum.net/conference/mol2net-06 concentration of particles required for 100% bacterial killing compared to the positive control (no treatment). All assays were performed in a laminar air flow.

(b) Tolerance level
The tolerance levels of each bacterial strain against AgNP were determined by using the following formula [28,30].
The susceptibility of pathogenic bacteria to AgNP was examined according to a previously reported protocol by the Agar well diffusion method. The pathogenic strains were grown on LB Broth at 37°C overnight till a turbidity of 0.5 Mac Farland standards (10 8 CFU per ml). About 50μl of this suspension was used to inoculate 90mm diameter petridish filled with 30ml of Mueller Hinton Agar. Wells (diameter 2 = 0.563cm 2 ) were punched in the agar plates and treated with AgNPs at their MBC concentrations. The zone of inhibition diameter in the bacterial growth surrounding the disc (including the disc) was measured [28,30].

(d) Bacterial cell-viability assay
The viability of bacterial cells was analysed with help of 3-(4, 5-dimethylthiazol-2-yl) -2, 5diphenyltetrazolium bromide (MTT) following a standard protocol [28]. Cell viability was determined after exposure to different concentrations of AgNPs for 24 hrs at 37° C. After incubation, the bacterial cells were collected and centrifuged at 1400 rpm for 10 mins at 4° C then washed three times with sterile phosphate buffered saline (PBS, pH 7.4). Subsequently, the medium was replaced with fresh culture media containing 0.5 mg.ml −1 of MTT reagent and incubated for 3 hrs at 37° C. Then, HCl-isopropanol solution was added and after 15 mins of incubation at room temperature, absorbance of solubilized MTT formazan product was measured spectrophotometrically at 570 nm in a Shimadzu UV-Vis 1800 spectrophotometer.

(e) ROS generation in Bacterial cell
The bacterial intracellular ROS generation was measured by using 2, 7-dichlorofluorescein diacetate (DCFH2-DA) according to our previously reported protocol [29]. The DCFH2-DA passively enters the cells, reacts with the generated ROS and oxidizes as well as forming a highly fluorescent compound: 2, 7-dichlorofluorescein (DCF). After the exposure of bacteria cells with AgNPs, they were then cultured overnight and washed with PBS solutions. Then, the cells were incubated with the required amount of DCFH2-DA at 37° C for 30 mins. Finally, the bacteria cells were visualized under fluorescence microscope.

(b) Intracellular ROS generations were checked by DCFDA method in Hep G2 cells
Normally, the DCFDA enters the cell and reacts with the reactive oxygen to give a green fluorescent color compound dichlorofluorescein (DCF). Briefly, a stock solution of DCFDA (10mM) was prepared in methanol and was further diluted with PBS to a working concentration of 100μM. Hep G2 cells were treated with AgNP at LD50 for 12hrs at 37⁰C, and washed with ice-cold 1× PBS followed by an incubation with 100μM of DCFDA for 30mins in the dark at 37⁰C [32,33]. The fluorescence intensity was measured both spectroscopically (Hitachi, Japan) and under a fluorescence microscope in Hep G2 cells (Leica, Japan) at excitation and emission wavelengths of 485 nm and 520 nm respectively.

X-ray diffraction (XRD)
The crystalline character of these sythesized nanoparticles was confirmed by X-ray crystallography. The XRD pattern of AgNPs is given in Fig 1a. The pattern clearly shows the main peaks at (2θ) 38. 19 and 44.37 corresponding to the (111) and (200) planes, respectively. By comparing with JCPDS (file no: 89-3722), the typical pattern of green-synthesized AgNPs is found to possess a prominent structure. The average crystalline size of the silver nanoparticles was estimated using (Eq. 2), the Debye-Scherrer's equation D=0.9λ/β cosθ………………………….. (2) By calculating the width of (111) Bragg's reflection, we evaluate average size of the particle is 14 nm. In addition, three unassigned peaks appeared at 27.78 ⁰ , 32.34⁰ and 46.29⁰. These peaks were weaker than those of silver. This may be due to the bioorganic compounds occurring on the surface of the AgNPs.

Fourier Transform Infrared Spectroscopy (FTIR)
FTIR experiment was carried out in order to identify the presence of various functional groups in biomolecules responsible for the bioreduction of Ag and capping/stabilization silver nanoparticles. The obtained intense bands were compared with standard values to identify the functional groups as in Figure  2b. The bands at 3422 cm -1 in the spectra corresponds to O-H stretching vibration indicating the presence of alcohol and phenol group. Bands at 2921 cm -1 region arising from C-H stretching of aromatic compound were observed. The band at 1631 cm -1 assigned to C-N and C-C stretching indicating the presence of proteins. The band at 1450 cm -1 was assigned for N-H stretch vibration present in the amide linkages of the proteins. These functional groups have role in stability/capping of AgNP as reported in other studies. The bands at 1450 cm -1 were corresponds to N-H and C-N (amines) stretch vibration of the proteins respectively. The band at 1377 cm -1 exemplifies the N-O symmetry stretching typical of the nitro group.
The strong bands at 1074 cm -1 are corresponds to ether linkages and suggest the presence of flavanones adsorbed on the surface of synthesized metal nanoparticles. The immediate reduction and capping of silver ion into silver nanoparticles in this analysis might be due to flavanoids and proteins. The flavonoids present in the leaf extract are powerful reducing agents which may be actively involved and responsible for the reduction of Ag + to Ag 0 . http://sciforum.net/conference/mol2net-06

UV-Visible absorption studies
The synthesized AgNPs in aqueous solution was monitored to obtain the absorption spectra at a wavelength range of 250-800 nm as shown in Figure.2a. It was observed that solution of silver nitrate turned dark brown on addition of leaves extract; it indicated the formation of AgNPs, while no color change was observed in the absence of plant extract. In the UV Vis spectrum; a single, strong and broad Surface plasmon resonance (SPR) peak was observed at 450 nm that assign the synthesis of AgNPs. Particle size distribution and surface charge measurement

Size Determination of Silver Nanoparticle
Size distribution of the AgNPs was determined by DLS ( Figure.2b). Particle size distribution curve reveals that AgNPs obtained are polydispersed in nature having a P.D.I (Polydispersive Index) of 0.231 with average diameter ∼100 nm. The low P.D.I value indicates that the solution is homogenous in nature which is ideal for biological applications. The silver nanoparticles had quite high negative zeta potential which indicates the stability of the particles in aqueous solution. The details are given in the table below.

Field Emission Scanning Electron Microscope (FESEM) analysis
The FESEM images of the silver nanoparticles are shown in Figure.2c. The surface morphology of silver nanoparticles showed rod AgNP structure. In the present study, the particle size ranges from 25 to 100 nm.

Biosynthesized AgNP nanoparticles exhibits significant antibacterial activity against pathogenic bacterial strains (a) Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)
To evaluate the bactericidal activity of the synthesized AgNPs have been treated separately against Gram negative E.coli, P.aeruginosa and Gram positive S.aureus, B.subtilis. In the case of E.coli, P.aeruginosa strains the MIC values of AgNP was 38.46 ± 1.2 µg/ml, 44.65 ± 2.2 µg/ml and that in case of S.aureus, MOL2NET, 2020, 6, ISSN: 2624-5078 9 http://sciforum.net/conference/mol2net-06 B.subtilis the MIC value was 35.54 ± 1.43µg/ml, 41.3 ± 2.83 µg/ml respectively as shown in Figure.3. All bacterial strains were grown with the MIC dilutions on the sterile agar plates to obtain the MBC [28,29]. The MBC concentrations are noted where complete bacteria growth was inhibited on the agar plate after treatment with AgNPs. The MBC values of AgNP were at 135 ± 3.2 µg/ml, 143 ± 4.2 µg/ml, 132 ± 1.8 µg/ml, 140 ± 3.7µg/ml in case of E. coli, P. aeruginosa, S. aureus, B. subtilis bacterial strains respectively. Bacterial growth decreases with an increase in the concentration of AgNP. In the microdilution method, these may be inhibited due to the easy penetration of AgNP into the cells followed by bacteriostatic in different bacterial strains.

(b) Phytofabricated AgNPs show excellent bactericidal activity
The tolerance level of pathogenic strains against AgNP was evaluted from the respective MIC and MBC values. From these result, it is measured that the tolerance level to AgNP was 3.5, 3.2 in case of E. coli, P. aeruginosa and 3.7, 3.4 in case of S. aureus, B. subtilis respectively. The implication of the tolerance level is the difference between bactericidal agents from bacteriostatic agents. Bactericidal agents always kill microbes, but bacteriostatic agents inhibit the growth. When the MBC/MIC ratio is greater than or equal to 16 for particular bacteria, the antibacterial agents are considered as bacteriostatic type and when this ratio is less than or equal to 4, then the particles are considered as a bactericidal agent [28].Thus, the MBC/MIC ratio is an important parameter which reflects the bactericidal capacity of the AgNP. In this study, AgNP exhibits significant bactericidal activity against E. coli, S. aureus, P. aeruginosa, B. subtilis pathogenic strains.

(c) Selective bactericidal activity of AgNP confirmed by Disc diffusion method
This experiment was performed to observe the comparison of antibacterial activity of different AgNP samples. The zone of inhibitions of Gram-positive and Gram negative are given in Figure 4. Zone of inhibition having a diameter is significant when strain is treated with AgNP at their MBC concentration. So, from this study, it has become clear that the AgNP has really proved to be beneficial and needs to be administered for pathogenic E.coli, S.aureus, B.subtilis, P.aeruginosa growth inhibition. http://sciforum.net/conference/mol2net-06

(d) Treatment of E.coli, S.aureus, B.subtilis, P.aeruginosa with AgNP significantly diminishes the bacterial cell viability
AgNP is responsible for diminishing the bacterial cell survivability at their respective MBC conc. as depicted in Figure.5. This may be due to the ingression of AgNPs into the bacterial cells that can hinder the growth of the bacteria and acts as an ideal bactericidal agent.

(e) Mechanisms behind bactericidal activity of functionalized AgNPs
The phytofabricated AgNPs had the capability to induce intracellular ROS generation which in turn culminates in the bacterial cell death. In order to envisage the mechanisms behind the bactericidal activities of AgNPs, the intracellular ROS generation in the bacterial cells was determined by employing DCFH-DA as an intracellular ROS indicator which is depicted in Figure.6. The results indicate that the AgNP treated pathogenic bacterial cells augment the generation of ROS which is responsible for bacterial cell death. The enhanced ROS production is correlated with bacterial cell death. The intracellular ROS production contributes to the bacterial cell membrane damage, disruption in the electronic transport chain and the genetic material detonation [29,30].

Anticancer activity of AgNPs (a) Cytotoxicity of AgNPs
The in vitro cytotoxicity of the AgNPs is evaluated for determining the cytotoxic effects on HepG2 cell line. The cells were exposed to different concentrations of AgNPs (0-120 µg/ml) for a time span of 24hrs and was afterwards followed by MTT assay. It was observed that the AgNPs depicted a dose dependent decrease in the cell survivability. The LD50 of AgNP was calculated to be 46 ± 3.2 µM. Thus we can conclude that the synthesized phytofabricated AgNPs could exert encouraging anticancer activity as exhibited in Figure.7.

(b) Intracellular Reactive oxygen species generation induced by treatment of AgNP
The Reactive oxygen species (ROS) is analysed with the the dual aid of fluorescence microscopy and Spectro fluorometer by employing 2′, 7′ dichlorofluorscence in diacetate (DCF-DA) as a probe. The generation of reactive oxygen species is evaluated in case of hepatocellular carcinoma cells, Hep G2 with AgNPs at concentrations of 80 µM and 100 µM for 12hrs. The fluorescence microscopic images http://sciforum.net/conference/mol2net-06 clearly depicts that the enhancement of green color fluorescent intensity occurred in case of treated HepG2 cell lines after 12hrs ( Figure. 8a and 8b).

Conclusions
The synthesis of phytofabricated silver nanoparticle is inexpensive, non toxic and ecofriendly. The AgNPs were thoroughly charecterised with the UV-Vis, FTIR, XRD, DLS and FESEM. The results from UV-Vis spectral studies testified the presence of surface plasmon resonance of these biogenic silver nanoparticles. The biomolecules predominant in the O. sanctum leaves were primarily responsible for reducing and capping of AgNPs which were then analysed by FTIR measurements. Particle size and stabilization were determined with the aid of DLS and zeta potential techniques. FESEM studies depicted the formation of rod and uniform shaped silver nanoparticles with a size range of 25-100 nm. The XRD pattern additionally confirmed the development of the silver nanoparticles via the eco-friendly green synthetic route.
Although there has been significant progress in cancer diagnosis and treatment, cancer is still posing a worldwide grave threat. On the other hand, the global dissemination of pathogenic bacterial strains has emerged as a serious cotemporary challenges with respect to public health. Our results clearly depicts that the biogenic AgNPs are responsible for initiating the oxidative damage through augmented ROS generations both in case of prokaryotic and eukaryotic cells. The generated reactive oxygen or superoxide has the potential to directly interact with the cell metabolism in order to generate the hydroxyl radicals, which is responsible for damaging the DNA, lipid and proteins. Our results clearly suggest that these phytofabricated AgNPs can be considered as potential antibacterial agent against Gram-positive (S. aureus, B. subtilis) and Gram-negative (E. coli, P. aeruginosa) pathogenic bacterial strains. http://sciforum.net/conference/mol2net-06 In the present study with AgNPs, the probable reason behind the bacterial cell wall destruction might be the disruption in the steps of signaling cascades that leads to the defective cell wall synthesis.This phenomenon takes place due to the enhanced ROS generation in both Gram-positive and Gram-negative bacterial strains. Additionally, the phytofabricated leads to the death of human liver cancer due to enhanced intracellular ROS. In conclusion, our studies suggest that our phytofabricated AgNPs can emerge as potential anticancer and antibacterial agents.