AU2021101319A4 - A process for synthesizing silver nanoparticles using piper nigrum concoction for anticancer activity - Google Patents

A process for synthesizing silver nanoparticles using piper nigrum concoction for anticancer activity Download PDF

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AU2021101319A4
AU2021101319A4 AU2021101319A AU2021101319A AU2021101319A4 AU 2021101319 A4 AU2021101319 A4 AU 2021101319A4 AU 2021101319 A AU2021101319 A AU 2021101319A AU 2021101319 A AU2021101319 A AU 2021101319A AU 2021101319 A4 AU2021101319 A4 AU 2021101319A4
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K. Thanigai Arul
G. Kavitha
K. Kavitha
E. Manikandan
K. Venugopal
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Abstract

The present disclosure relates to a process for synthesizing silver nanoparticles using Piper nigrum concoction for anticancer activity against. The process reports emphasis on silver nanoparticles are synthesized using Piper nigrum extract for in vitro cytotoxicity efficacy against MCF-7 and HEP-2 cells. The silver nanoparticles (Ag-NPs) are formed within 20 minutes and preliminarily confirmed by UV-Visible spectroscopy. Further, it is characterized by FT-IR and HR-TEM. The strong SPR broad peak observed at ~441 nm which confirms the formation of silver nanoparticles and further, the same sample are taken into the additional supporting characterization analysis of FT-IR, HRTEM-EDX, cytotoxicity cancer cell lines at room temperature. TEM images of biosynthesized Ag-NPs predominantly spherical shape with particle size in the range 5 nm to 40 nm. At various concentrations, biosynthesized silver nanoparticles showed a significant anticancer activity against both MCF-7 and Hep-2 cells compared to Piper nigrum extract. 18 100 grinding Pipernigrum(fuit) into fine powder using mortar and pestleandthereby dissolving 2ozoffiper nigrumpowderin,100 nil ofMillipore waterto formand mixture boiling the mixture at 80 degree Celsius for 10 min and thereafterfiltering the mixturethrough a syringe filter of 0.A5 micronto prepare Piper nigrum concoction a dding the Piper nigrumconcoction(10 ml) to 90 nl of 1 mMilver nitratesolution for the reduction ofAg* 10 ions and maintaining various temperatures such as RT, 40, 60, and 8O 0 C using water bath to optimize the synthesis stirring solutionat1000rpm for10 minutesandobservingcolor modulationatvarioustemperaturestoensure 108 theformationofsilvernanoparticles removingexcesssilver ionsbycentnfuging thesilver colloids at10,000rpmfor 15 minutesandthereafter 110 washing three times with Millipore water obtaining dried powder ofsilver nanoparticles by freeze-drying in Alpha Christ 2.0 lyophiliser for further 112 characterization Figure1 Figure2

Description

grinding Pipernigrum(fuit)into fine powder using mortar and pestleandthereby dissolving 2ozoffiper nigrumpowderin,100 nil ofMillipore waterto formand mixture
boiling the mixture at 80 degree Celsius for 10 min and thereafterfiltering the mixturethrough a syringe filter of 0.A5 micronto prepare Pipernigrumconcoction
a dding the Pipernigrumconcoction(10ml) to 90 nl of 1 mMilver nitratesolution for the reduction 0 ofAg* 10 ions and maintaining various temperatures such as RT, 40, 60, and 8O C using water bath to optimize the synthesis
stirring solutionat1000rpm for10 minutesandobservingcolor modulationatvarioustemperaturestoensure 108 theformationofsilvernanoparticles
removingexcesssilver ionsbycentnfuging thesilver colloids at10,000rpmfor 15 minutesandthereafter 110 washing three times with Millipore water
obtaining dried powder ofsilver nanoparticles by freeze-drying in Alpha Christ 2.0 lyophiliser for further 112 characterization
Figure1
Figure2
A PROCESS FOR SYNTHESIZING SILVER NANOPARTICLES USING PIPER NIGRUM CONCOCTION FOR ANTICANCER ACTIVITY FIELD OF THE INVENTION
The present disclosure relates to a process for concoction of Piper nigrum to synthesize the silver nanoparticles characterized by Fourier transform infrared spectroscopy (FTIR), UV-Visible spectroscopy, High resolution transmission electron microscopy (HR TEM) carried out for the first time for anticancer activity against MCF-7 and Hep-2 cell lines.
BACKGROUND OF THE INVENTION
Recently, metals nanoparticles have received considerable attention of researchers due to their wide unique properties as compared to bulk and possess immense applications in the fields of diagnostics, cell labeling, antimicrobial agents, drug delivery and cancer therapy. Silver nanoparticles have established substantial consideration for different reasons such as valuable antimicrobial agent, reveals low toxicity etc. Plant mediated synthesis is gaining cost effective, economic, eco-friendly and aiding to scale up synthesis of nanoparticles. Biosynthetic processes are an effective way to prepare silver nanoparticles using plants or their extract in a restricted approach due to their dispersion, size and shape. The plant-based nanoparticles has enhanced the opportunity of using beneficial nanoparticles in the diagnosis and treatment of human cancers. Noble metal silver (Ag), is a potential application in medicine traditionally due to its unique properties. There are various syntheses methods for Ag-NPs preparation, for example; sol-gel process, chemical precipitation, reverse micelle method, hydrothermal method, microwave, chemical vapor deposition and biological methods, etc. However; biological methods are preferred for being eco-friendly, cost effective, and don't involve the use of toxic chemicals.
Piper nigrum (black pepper) is a spicy plant in which the whole plant is medicinally high in nutritional and therapeutic compounds. It possesses many medicinal properties such as antipyretic, anti-inflammatory, analgesic, and antimicrobial properties.
However, there are various syntheses methods for Ag-NPs preparation, for example; sol-gel process, chemical precipitation, reverse micelle method, hydrothermal method, microwave, chemical vapor deposition and biological methods, etc. In order to overcome the aforementioned drawbacks, there exists a need to develop a process for synthesizing silver nanoparticles using Pipernigrum concoction for anticancer activity.
SUMMARY OF THE INVENTION
The present disclosure seeks to provide a process for synthesizing silver nanoparticles using Pipernigrum concoction for anticancer activity against MCF-7 and Hep-2 Cell Lines.
In an embodiment, a process for synthesizing silver nanoparticles using Piper nigrum concoction for anticancer activity against. The process comprises:
grinding Piper nigrum (fruit) into fine powder using mortar and pestle and thereby dissolving 20g of Pipernigrum powder in 100 ml of Millipore water to form and mixture; boiling the mixture at 80degree Celsius for 10 min and thereafter filtering the mixture through a syringe filter of 0.45 micron to prepare Piper nigrum concoction; adding the Piper nigrum concoction (10 ml) to 90 ml of1 mM silver nitrate solution for the reduction of Ag+ ions and maintaining various temperatures such as RT, 40, 60, and °C using water bath to optimize the synthesis; stirring solution at 1000 rpm for 10 minutes and observing color modulation at various temperatures to ensure the formation of silver nanoparticles; removing excess silver ions by centrifuging the silver colloids at 10,000 rpm for 15 minutes and thereafter washing three times with Millipore water; obtaining dried powder of silver nanoparticles by freeze-drying in Alpha Christ 2.0 lyophiliser for further characterization;
In an embodiment, the Piper nigrum seed concoction employed as a reducing and stabilizing agent for 1mM of silver nitrate. In an embodiment, the preliminary characterization of silver nanoparticles is carried out using UV-visible spectroscopy and UV Vis spectroscopy analysis is done using nanodrop 2000r in a scanning range of 200 nm to 800 nm.
In an embodiment, Millipore water is used as a blank, wherein the interactions between protein-silver nanoparticles are analyzed by Fourier transform infrared spectroscopy
(FTIR) in the range of 4000 to 400 cm-1 and transmission electron microscope (TEM) images of biosynthesized Ag NPs are obtained for size and shape determination using libra 200 HR TEM (m/s Carl Zeiss, Germany) operated at an accelerating voltage 120 kV and 200kV.
In an embodiment, the Ag-NPs sonicated for 5 minutes and a drop of diluted sample placed onto the carbon-coated copper grid and liquid fraction is allowed to evaporate at room temperature.
In an embodiment, the breast cancer cells (MCF-7), and Human Larynx Carcinoma cancer (Hep-2) cell lines are cultured in Dulbecco's modified Eagle's medium and supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin, wherein the cell lines are maintained at 5% CO 2 in CO 2 incubator and cultures are examined using an inverted microscope to evaluate the quality of confluency and confirming the absence of bacterial and fungal contaminants.
In an embodiment, a method for determining cytotoxic effect of silver nanoparticles and Piper nigrum extract and performing cell viability study with the MTT reduction assay comprises: seeding MCF-7 and Hep-2 cells in a 96-well plate at the density of 5x103 cells /well; allowing cells to attach and grow in 96-well plate for 24 h, in 200 g of EMEM with % FBS; removing the media and replacing with the suspension of various concentrations of silver nanoparticles 10 to100 mg/ml (minimum 4 wells are seeded with each concentration); incubating the cells for 48 h inside an incubator and thereafter adding 10 ml, 5 mg/ml MTT; incubating the cells are at 37 0 Celsius for another 4 h and adding 200 pl of DMSO to each well upon removing the incubator; and evaluating optical density (OD) of the formazan product at 620 nm using multi well spectrophotometer.
In an embodiment, a method for straining Acridine Orange/ Ethidium Bromide (AO/EtBr) for detection of apoptotic cells comprises: mixing acridine orange (AO) and ethidium bromide (EtBr) staining approximately 5 gl of dye mixture (100 mg.ml-1 acridine orange (AO) and 100 mg.ml-1 ethidium bromide
(EtBr)) with 9 ml of cell suspension (1 x 105 cells.ml-1 ) on a clean microscopic cover slip; and incubating the mixture for 2-3 min and visualizing the cells under fluorescence microscope at 40x magnification with excitation filter at 510-590 nm.
In an embodiment, a method for DAPI staining comprises: treating HEP-2 cells with the above methods for 48 h, and then fixed with methanol: acetic acid (3:1, v/v) prior to washing with PBS; staining the washed cells with 1 1 mg.ml- DAPI (4',6-diamidino-2-phenylindole, dihydrochloride) for 20 min in the dark; and recording the stained images with fluorescent microscope with appropriate excitation filter.
In an embodiment, the grouped data are statistically evaluated using GraphPad Prism 6 and Origin8.5 user interface.
An objective of the present disclosure is to characterize silver nanoparticles extracted from Piper nigrum by Fourier transform infrared spectroscopy (FTIR), UV-Visible spectroscopy, High resolution transmission electron microscopy (HR-TEM).
Another object of the present disclosure is to evaluate anticancer activity against MCF-7 and Hep-2 Cell Lines.
Yet another object of the present invention is to deliver an expeditious and cost effective process for synthesizing silver nanoparticles using Piper nigrum concoction for anticancer activity against.
To further clarify advantages and features of the present disclosure, a more particular description of the invention will be rendered by reference to specific embodiments thereof, which is illustrated in the appended drawings. It is appreciated that these drawings depict only typical embodiments of the invention and are therefore not to be considered limiting of its scope. The invention will be described and explained with additional specificity and detail with the accompanying drawings.
BRIEF DESCRIPTION OF FIGURES
These and other features, aspects, and advantages of the present disclosure will become better understood when the following detailed description is read with reference to the accompanying drawings in which like characters represent like parts throughout the drawings, wherein:
Figure 1 illustrates a flow chart of a process for synthesizing silver nanoparticles using Piper nigrum concoction for anticancer activity against in accordance with an embodiment of the present disclosure; Figures 2A and 2B illustrate visual observation of silver nitrate solution and biosynthesized silver nanoparticles in accordance with an embodiment of the present disclosure; Figure 3 illustrates an UV-visible spectrum of synthesized Ag-NPs from Piper nigrum extract at different temperature conditions in accordance with an embodiment of the present disclosure; Figures 4A and 4B illustrate FTIR spectrum of Piper nigrum Ag NPs biosynthesized and as synthesized Pipernigrum in accordance with an embodiment of the present disclosure; Figures 5A and 5B illustrate HRTEM image of Ag-NPs and magnified portion of Ag-NPs in accordance with an embodiment of the present disclosure; Figure 6 illustrates an elemental compositional analysis of Ag NPs by TEM image with EDAX in accordance with an embodiment of the present disclosure; Figure 7 illustrates a cytotoxicity of the green synthesized silver nanoparticles against the MCF-7 and the Hep-2 cell line in accordance with an embodiment of the present disclosure; Figures 8A and 8B illustrate efficacy of Piper nigrum and biosynthesized Ag-NPs against MCF-7 cells at different concentration and efficacy of Piper nigrum and biosynthesized Ag-NPs against Hep-2 cells at different concentration in accordance with an embodiment of the present disclosure; and Figures 9A, 9B, 9C and 9D illustrate apoptotic effects of fluorescence microscopy study of AO/EtBr stain respective, control cells appear in live cells in green colour, orange colour apoptotic cells and necrotic cells appearing in red colour, DAPI nuclear stain of control cells, and DAPI stain of Ag-NPs treated cells exhibited condensed form of nuclear materials in apoptotic cells in accordance with an embodiment of the present disclosure.
Further, skilled artisans will appreciate that elements in the drawings are illustrated for simplicity and may not have been necessarily been drawn to scale. For example, the flow charts illustrate the method in terms of the most prominent steps involved to help to improve understanding of aspects of the present disclosure. Furthermore, in terms of the construction of the device, one or more components of the device may have been represented in the drawings by conventional symbols, and the drawings may show only those specific details that are pertinent to understanding the embodiments of the present disclosure so as not to obscure the drawings with details that will be readily apparent to those of ordinary skill in the art having benefit of the description herein.
DETAILED DESCRIPTION
For the purpose of promoting an understanding of the principles of the invention, reference will now be made to the embodiment illustrated in the drawings and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, such alterations and further modifications in the illustrated system, and such further applications of the principles of the invention as illustrated therein being contemplated as would normally occur to one skilled in the art to which the invention relates.
It will be understood by those skilled in the art that the foregoing general description and the following detailed description are exemplary and explanatory of the invention and are not intended to be restrictive thereof.
Reference throughout this specification to "an aspect", "another aspect" or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Thus, appearances of the phrase "in an embodiment", "in another embodiment" and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment.
The terms "comprises", "comprising", or any other variations thereof, are intended to cover a non-exclusive inclusion, such that a process or method that comprises a list of steps does not include only those steps but may include other steps not expressly listed or inherent to such process or method. Similarly, one or more devices or sub-systems or elements or structures or components proceeded by "comprises...a" does not, without more constraints, preclude the existence of other devices or other sub-systems or other elements or other structures or other components or additional devices or additional sub-systems or additional elements or additional structures or additional components.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The system, methods, and examples provided herein are illustrative only and not intended to be limiting.
Embodiments of the present disclosure will be described below in detail with reference to the accompanying drawings.
Referring to Figure 1, a flow chart of a process for synthesizing silver nanoparticles using Pipernigrum concoction for anticancer activity against is illustrated in accordance with an embodiment of the present disclosure. At step 102, the process 100 includes grinding Piper nigrum (fruit) into fine powder using mortar and pestle and thereby dissolving 20g of Piper nigrum powder in 100 ml of Millipore water to form and mixture. The Piper nigrum (fruit) are collected from the local market and authenticated. The color modulation was observed at various temperatures to ensure the formation of silver nanoparticles.
At step 104, the process 100 includes boiling the mixture at 80degree Celsius for 10 min and thereafter filtering the mixture through a syringe filter of 0.45 micron to prepare Pipernigrum concoction.
At step 106, the process 100 includes adding the Pipernigrum concoction (10 ml) to ml of1 mM silver nitrate solution for the reduction of Ag+ ions and maintaining various temperatures such as RT, 40, 60, and 80degree Celsius using water bath to optimize the synthesis. Silver nitrate (AgNO 3) and MTT are purchased from Hi Media Laboratories Pvt. Ltd. India. The MCF-7 and Hep-2 cancer cell line is purchased from King Institute of Preventive Medicine and Research, Chennai-600025, India.
At step 108, the process 100 includes stirring solution at 1000 rpm for 10 minutes and observing color modulation at various temperatures to ensure the formation of silver nanoparticles.
At step 110, the process 100 includes removing excess silver ions by centrifuging the silver colloids at 10,000 rpm for 15 minutes and thereafter washing three times with Millipore water.
At step 112, the process 100 includes obtaining dried powder of silver nanoparticles by freeze-drying in Alpha Christ 2.0 lyophiliser for further characterization;
In an embodiment, the Piper nigrum seed concoction employed as a reducing and stabilizing agent for 1mM of silver nitrate. In an embodiment, the preliminary characterization of silver nanoparticles is carried out using UV-visible spectroscopy and UV Vis spectroscopy analysis is done using nanodrop 2000r in a scanning range of 200 nm to 800 nm.
In an embodiment, Millipore water is used as a blank, wherein the interactions between protein-silver nanoparticles are analyzed by Fourier transform infrared spectroscopy (FTIR) in the range of 4000 to 400 cm-1 and TEM images of biosynthesized Ag NPs are obtained for size and shape determination using libra 200 HR-TEM (m/s Carl Zeiss, Germany) operated at an accelerating voltage 120 kV and 200 kV.
In an embodiment, the Ag-NPs sonicated for 5 minutes and a drop of diluted sample placed onto the carbon-coated copper grid and liquid fraction is allowed to evaporate at room temperature.
In an embodiment, the breast cancer cells (MCF-7), and Human Larynx Carcinoma cancer (Hep-2) cell lines are cultured in Dulbecco's modified Eagle's medium and supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin, wherein the cell lines are maintained at 5% CO 2 in CO 2 incubator and cultures are examined using an inverted microscope to evaluate the quality of confluency and confirming the absence of bacterial and fungal contaminants. Breast cancer cells (MCF-7), and Human Larynx Carcinoma cancer
(Hep-2) cell lines are purchased from King Institute of Preventive Medicine and Research, ICMR, Chennai, India.
In an embodiment, a method for determining cytotoxic effect of silver nanoparticles and Piper nigrum extract and performing cell viability study with the MTT reduction assay includes seeding MCF-7 and Hep-2 cells in a 96-well plate at the density of 5x103 cells /well. The method includes allowing cells to attach and grow in 96-well plate for 24 h, in 200 gl of EMEM with 10% FBS. The method includes removing the media and replacing with the suspension of various concentrations of silver nanoparticles 10 tol00 mg/ml (minimum 4 wells are seeded with each concentration). The method includes incubating the cells for 48 h inside an incubator and thereafter adding 10 ml, 5 mg/ml MTT. The method includes incubating the cells are at 37degree Celsius for another 4 h and adding 200 pl of DMSO to each well upon removing the incubator. The method further includes evaluating optical density (OD) of the formazan product at 620 nm using multi well spectrophotometer. The results are given as mean of four independent experiments. The OD value is subjected to sort out percentage of viability by using the following formula,
% Cell Viability = OD Sample x 100% OD Control
In an embodiment, a method for straining Acridine Orange/ Ethidium Bromide (AO/EtBr) for detection of apoptotic cells comprises mixing acridine orange (AO) and ethidium bromide (EtBr) staining approximately 5 pl of dye mixture (100 mg.ml 1 acridine orange (AO) and 100 mg.ml-1 ethidium bromide (EtBr)) with 9 ml of cell suspension (1 x 105 cells.ml- 1) on a clean microscopic cover slip. The method further comprises incubating the mixture for 2-3 min and visualizing the cells under fluorescence microscope at 40x magnification with excitation filter at 510-590 nm.
In an embodiment, a method for DAPI staining comprises treating HEP-2 cells with the above methods for 48 h, and then fixed with methanol: acetic acid (3:1, v/v) prior to washing with PBS. The method comprises staining the washed cells with 1 mg.ml-1 DAPI (4',6-diamidino-2-phenylindole, dihydrochloride) for 20 min in the dark. The method further comprises recording the stained images with fluorescent microscope with appropriate excitation filter.
In an embodiment, the grouped data are statistically evaluated using GraphPad Prism 6 and Origin8.5 user interface. Values are presented as the mean SD of the four replicates of each experiment.
Figures 2A and 2B illustrate visual observation of silver nitrate solution and biosynthesized silver nanoparticles in accordance with an embodiment of the present disclosure. Silver nitrate solution (Figure 2A) is colourless and after adding Piper nigrum plant extract to silver nitrate solution, the colourless silver nitrate solution became dark red in colour (Figure 2B). This confirms that the silver nitrate is reduced and transformed into silver nanoparticles.
Figure 3 illustrates an UV-visible spectrum of synthesized Ag-NPs from Piger nigrum extract at different temperature conditions in accordance with an embodiment of the present disclosure. The UV-Vis spectra of synthesized Ag-NPs from Piper nigrum extract at different temperature conditions are as shown in the Figure 3. The colour change depicts the existence of the formation of silver nanoparticles in the Piper nigrum extract. It shows that the UV-Vis spectra of silver nanoparticle formation at different temperature using Piger nigrum nanoparticle extract (i) Room temperature (RT), (ii) 40 °C, (iii) 60 °C, (iv) 80 °C in aqueous medium. The surface Plasmon resonance (SPR) bands of colloidal silver for different temperatures are observed in the range 420 to 446 nm. At higher temperature (80 °C), intense SPR band observed compared other samples. The strong SPR broad peak observed at -441 nm which confirms the formation of silver nanoparticles and further, the same sample are taken into the additional supporting characterization analysis of FT-IR, HRTEM-EDX, cytotoxicity cancer cell lines at room temperature.
Figures 4A and 4B illustrate FTIR spectrum of Piper nigrum Ag NPs biosynthesized and as synthesized Pipernigrum in accordance with an embodiment of the present disclosure. FTIR spectrum of Ag-NPs Piper nigrum is as shown in the Figure 4A. Addition of silver nitrate, still, have the band at 3421 cm-1 which corresponds to "polymeric" OH stretching mode. An intense peak observed at 1645 cm-1 attributed to the C=O stretching mode of ketone (Figure 4B). The peaks at 1230 cm-1 and 1026 cm-1 which are corresponding to the amine C-N stretching, and the C-C stretching and the aliphatic fluoro compounds C-F stretching respectively present in the Ag-NPs and the peaks suppressed in comparison with Piper nigrum. The stretching vibration of nitro group of leaf (1388 cm-1), iodo compounds
OH group out of plane bending (521 cm-1, 565 cm-1) are completely suppressed due to the addition of silver nitrate, which means the complete reduction and stabilization of the silver nanoparticles. Here, it is confirmed that the modulated transmittance percentage of ketone, fluoro compounds and amine groups play a vital role for the bio reduction of silver nitrate to silver nanoparticles.
FTIR finger print spectrum of as synthesized Piper nigrum is depicted in Figure 4B inset. The extracted materials of vibrational bands assignments of Piper nigrum observed at 3421 cm- 1, 2933 cm- 1, 2388 cm- 1, 1632 cm- 1, 1388 cm- 1, 1221 cm-1, 1021 cm-1 565 cm-1 and 521 cm-1. The molecular vibrational bands at 3421 cm-1 which is corresponds to normal "polymeric" OH stretching mode. The peak at 2933 cm-1 associated to the methylene C-H asymmetric and symmetric stretching mode. The peak at 2388 cm-1 indicates the symmetric stretching of alkanes and the peak at 1632 cm-1 attributed to the C=O stretching mode of ketones. The peak at 1388 cm-1 corresponds to the N=O stretching of nitro groups of leaf extract. The 1021 cm-1 peak corresponds to the C-C stretch and the aliphatic fluoro compounds C- F stretch. The peaks at 521 cm-1 and 565 cm-1 which are corresponds the aliphatic iodo compounds, C-I stretch, alcohol and OH out-of-plane bending.
Figures 5A and 5B illustrate HRTEM image of Ag-NPs and magnified portion of Ag-NPs in accordance with an embodiment of the present disclosure. The surface morphology of green synthesized Ag NPs investigated by HRTEM and their surface crystalline, particle shape/size and compositional properties at the atomic-scale level. HRTEM image of Ag-NPs is shown the Figure 5A observed with particle size ~ 5 nm Ag nanoparticles shows uniform with well-crystallized particles. The overall particle size of Ag NPs range of 5 to 40 nm. The nanoparticles are embedded in a dense matrix which may be the organic stabilizing components of Pipernigrum extract. The shape of silver nanoparticles is spherical with core-shell structure indicated in the round red-circle of the Figure 5B.
Figure 6 illustrates an elemental compositional analysis of Ag NPs by TEM image with EDAX in accordance with an embodiment of the present disclosure. The elemental composition observer by silicon surface barrier detector determined using the selected area electron diffraction (SAED) (IH-300X) analysis shows (Figure 6) is performed at several points in the HRTEM system respectively. The identified elements are indexed in the EDAX spectrum as follows C, Cu, Ag and their Ka, Kp X-ray lines. The preeminent peaks of silver (Ag) concentrations from the extracted P. nigrum, C and Cu from the sample gird.
Figure 7 illustrates a cytotoxicity of the green synthesized silver nanoparticles against the MCF-7 and the Hep-2 cell line in accordance with an embodiment of the present disclosure. This study shows that the minimum dose shows enhanced anticancer activity. Respectively the Inhibitory concentration (IC 5 )0 value of the Phyto mediated Ag-NPs are observed at 43tg.ml- 1 against Hep-2 cells. The bar diagram indicates the efficacy of Piper nigrum and biosynthesized Ag-NPs against MCF-7 cells at different concentrations Figures 7(a-b). The bar diagram shows the efficacy of Piper nigrum and biosynthesized Ag-NPs against Hep-2 cells at different concentrations are shown in the Figures 7(c-d). In fact, silver nanoparticles may stimulate reactive oxygen species and effect in damage cellular components which lead to cell death.
Figures 8A and 8B illustrate efficacy of Piper nigrum and biosynthesized Ag-NPs against MCF-7 cells at different concentration and efficacy of Piper nigrum and biosynthesized Ag-NPs against Hep-2 cells at different concentration in accordance with an embodiment of the present disclosure. The cytotoxicity of the silver nanoparticle (Ag NPs) and Pipernigrum extract is studied against the MCF-7 (Figures 6a-b) and Hep-2 cell line by MTT assay (Figures 6c-d). The cytotoxicity effect of cancer cell is studied at different concentration (10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 pg.ml-1). The inhibitory concentration (IC 5 0) value of the Phyto mediated Ag-NPs are observed at the concentration of 52 pg.ml-lagainst MCF-7 cells and Piper nigrum plant extract are observed as 74tg.ml' show in the bar diagramFigure 8.
Figures 9A, 9B, 9C and 9D illustrate apoptotic effects of fluorescence microscopy study of AO/EtBr stain respective, control cells appear in live cells in green colour, orange colour apoptotic cells and necrotic cells appearing in red colour, DAPI nuclear stain of control cells, and DAPI stain of Ag-NPs treated cells exhibited condensed form of nuclear materials in apoptotic cells in accordance with an embodiment of the present disclosure. The induction of apoptosis, after the treatment with IC5 0 concentration of Ag-NPs is assessed by fluorescence microscopy after staining with acridine orange/ethidium bromide (AO/EtBr). Because AO can penetrate the normal cell membrane, the cells are observed as green fluorescence, while in apoptotic cells and apoptotic bodies are formed as a result of nuclear shrinkage, blebbing and are observed as orange-coloured bodies whereas, necrotic cells are observed as red colour fluorescence due to their loss of membrane integrity when viewed under fluorescence microscope Figures 9(a-b).
DAPI is a popular nuclear counter stain and the Ag-NPs induced nuclear fragmentation is observed by DAPI staining. The untreated cells showed normal nuclei (smooth nuclear) whereas after treatment of MCF-7 cells with Ag-NPs, the apoptotic nuclei (condensed or fragmented chromatin) are observed as shown in Figures 9 (c-d). Nuclear morphology analysis showed characteristic apoptotic changes, such as chromatin condensation, fragmentation of the nucleus, and formation of apoptotic bodies in the MCF-7 cells. Interestingly, some studies have reported that Ag-NPs can also induce DNA damage and apoptosis in cancer cells. With an increase the concentration of Ag-NPs, number of apoptotic cells are increased, which suggested that Ag-NPs could induce cell apoptosis. Respectively, UV-visible spectrum higher wavelength shift and TEM surface morphology particle crystalline size confirmation expose the similar observation.
In an embodiment, a simple, facile, inexpensive, eco-friendly and green synthesis of silver nanoparticles from the Piper nigrum in aqueous medium without employing manmade chemicals is reported. The UV-Vis spectroscopy and FT-IR analysis is confirmed the preliminary confirmation of the formation of silver nanoparticles. TEM image showed spherical shape with an average particle size of 20-40 nm. The biosynthesized silver nanoparticles and Piper nigrum extract showed promising anticancer activity against breast cancer cells (MCF-7) and human pharynx cancer cell line (Hep-2). From the study, it can be concluded that the silver nanoparticles synthesized using plant possess high anticancer activity against cell lines which further suggested the potential therapeutic use of these nanoparticles.
In the present innovative report emphasis on silver nanoparticles are synthesized using Piper nigrum extract for in vitro cytotoxicity efficacy against MCF-7 and HEP-2 cells. The silver nanoparticles (Ag-NPs) are formed within 20 minutes and preliminarily confirmed by UV-Visible spectroscopy. Further, it is characterized by FT-IR and HR-TEM. The strong SPR broad peak observed at ~441 nm which confirms the formation of silver nanoparticles and further, the same sample are taken into the additional supporting characterization analysis of FT-IR, HRTEM-EDX, cytotoxicity cancer cell lines at room temperature. TEM images of biosynthesized Ag-NPs predominantly spherical shape with particle size in the range 5 nm to nm. The compositional analysis are observed by EDAX. MTT assays for cytotoxicity are carried out using various concentrations of biosynthesized silver nanoparticles and Piper nigrum extract ranging from 10 to 100 pg are performed. At various concentrations, biosynthesized silver nanoparticles showed a significant anticancer activity against both MCF-7 and Hep-2 cells compared to Piper nigrum extract. Therefore, the results reveal excellent applications of green synthesis of silver nanoparticles using Piper nigrum. From the study, it can be concluded that the silver nanoparticles synthesized using plant possess high anticancer activity against cell lines which further suggested the potential therapeutic use of these nanoparticles.
The drawings and the forgoing description give examples of embodiments. Those skilled in the art will appreciate that one or more of the described elements may well be combined into a single functional element. Alternatively, certain elements may be split into multiple functional elements. Elements from one embodiment may be added to another embodiment. For example, orders of processes described herein may be changed and are not limited to the manner described herein. Moreover, the actions of any flow diagram need not be implemented in the order shown; nor do all of the acts necessarily need to be performed. Also, those acts that are not dependent on other acts may be performed in parallel with the other acts. The scope of embodiments is by no means limited by these specific examples. Numerous variations, whether explicitly given in the specification or not, such as differences in structure, dimension, and use of material, are possible. The scope of embodiments is at least as broad as given by the following claims.
Benefits, other advantages, and solutions to problems have been described above with regard to specific embodiments. However, the benefits, advantages, solutions to problems, and any component(s) that may cause any benefit, advantage, or solution to occur or become more pronounced are not to be construed as a critical, required, or essential feature or component of any or all the claims.

Claims (10)

WE CLAIM
1. A process for synthesizing silver nanoparticles (Ag-NPs) using Piper nigrum concoction for anticancer activity against, the process comprises:
grinding Piper nigrum (fruit) into fine powder using mortar and pestle and thereby dissolving 20 g of Piper nigrum powder in 100 ml of Millipore water to form and mixture; boiling the mixture at 80degree Celsius for 10 min and thereafter filtering the mixture through a syringe filter of 0.45 micron to prepare Pipernigrum concoction; adding the Piper nigrum concoction (10 ml) to 90 ml of 1 mM silver nitrate solution for the reduction of Ag+ ions and maintaining various temperatures such as RT, 40, 60, and 80 °C using water bath to optimize the synthesis; stirring solution at 1000 rpm for 10 minutes and observing color modulation at various temperatures to ensure the formation of silver nanoparticles; removing excess silver ions by centrifuging the silver colloids at 10,000 rpm for 15 minutes and thereafter washing three times with Millipore water; obtaining dried powder of silver nanoparticles by freeze-drying in Alpha Christ 2.0 lyophiliser for further characterization;
2. The process as claimed in claim 1, wherein the Piper nigrum seed concoction employed as a reducing and stabilizing agent for 1mM of silver nitrate.
3. The process as claimed in claim 1, wherein the preliminary characterization of silver nanoparticles is carried out using UV-visible spectroscopy and UV-Vis spectroscopy analysis is done using nanodrop 2000r in a scanning range of 200 nm to 800 nm.
4. The process as claimed in claim 3, wherein Millipore water is used as a blank, wherein the interactions between protein-silver nanoparticles are analysed by Fourier transform infrared spectroscopy (FTIR) in the range of 4000 to 400 cm-1 and TEM images of biosynthesized Ag NPs are obtained for size and shape determination using libra 200 HR-TEM (m/s Carl Zeiss, Germany) operated at an accelerating voltage 120 kV and 200 kV.
5. The process as claimed in claim 1, wherein the Ag-NPs sonicated for 5 minutes and a drop of diluted sample placed onto the carbon-coated copper grid and liquid fraction is allowed to evaporate at room temperature.
6. The process as claimed in claim 1, wherein the breast cancer cells (MCF-7), and Human Larynx Carcinoma cancer (Hep-2) cell lines are cultured in Dulbecco's modified Eagle's medium and supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin, wherein the cell lines are maintained at 5% CO2 in CO 2 incubator and cultures are examined using an inverted microscope to evaluate the quality of confluency and confirming the absence of bacterial and fungal contaminants.
7. The process as claimed in claim 1, wherein a method for determining cytotoxic effect of silver nanoparticles and Piper nigrum extract and performing cell viability study with the MTT reduction assay comprises:
seeding MCF-7 and Hep-2 cells in a 96-well plate at the density of 5x103 cells /well; allowing cells to attach and grow in 96-well plate for 24 h, in 200 gl of EMEM with 10% FBS; removing the media and replacing with the suspension of various concentrations of silver nanoparticles 10 tol00 mg/ml (minimum 4 wells are seeded with each concentration); incubating the cells for 48 h inside an incubator and thereafter adding 10 ml, 5 mg/ml MTT; incubating the cells are at 37degree Celsius for another 4 h and adding 200 gl of DMSO to each well upon removing the incubator; and evaluating optical density (OD) of the formazan product at 620 nm using multi well spectrophotometer.
8. The process as claimed in claim 1, wherein a method for straining Acridine Orange/ Ethidium Bromide (AO/EtBr) for detection of apoptotic cells comprises:
mixing acridine orange (AO) and ethidium bromide (EtBr) staining approximately 5 pl of dye mixture (100 mg.ml-1 acridine orange (AO) and 100 mg.ml-1 ethidium bromide (EtBr)) with 9 ml of cell suspension (1 x 105 cells.ml- 1) on a clean microscopic cover slip; and incubating the mixture for 2-3 min and visualizing the cells under fluorescence microscope at 40x magnification with excitation filter at 510-590 nm.
9. The process as claimed in claim 1, wherein a method for DAPI staining comprises:
treating HEP-2 cells with the above methods for 48 h, and then fixed with methanol: acetic acid (3:1, v/v) prior to washing with PBS; staining the washed cells with 1 mg.ml-1 DAPI (4',6-diamidino-2 phenylindole, dihydrochloride) for 20 min in the dark; and recording the stained images with fluorescent microscope with appropriate excitation filter.
10. The process as claimed in claim 1, wherein the grouped data are statistically evaluated using GraphPad Prism 6 and Origin 8.5 user interface.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114226712A (en) * 2021-12-14 2022-03-25 英特派铂业股份有限公司 Efficient fusion coating process for silver powder surface

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