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\def\RunningAuthor{Anitha Roy et al.}
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\articleType{Original Article}
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\title{Controlling of oral pathogens using ginger oleoresin mediated silver nanoparticles}
\author{Karthikeyan~Gayathri,  
            Anitha~Roy\textsuperscript{*},  
            Thangavelu~Lakshmi, 
            Rajeshkumar~S~\\[5pt]{Department of Pharmacology\unskip, Saveetha Dental College, Saveetha Institute of Medical and Technical Sciences, Chennai, Tamil Nadu, India}}

\begin{abstract}
Silver nanoparticles have become a substance of great interest among researches in the present time due to its low cost and its wide range of applications in various fields. Moreover, recently, green nanotechnology has been gaining popularity because of its exclusion of damaging reagents and cost-effectiveness. In this study, ginger oleoresin mediated silver nanoparticles were prepared and confirmed with UV-Vis spectroscopy. The ginger oleoresin mediated silver nanoparticles were then tested for its antibacterial activity using agar well diffusion method against \textit{Staphylococcus aureus}, \textit{Streptococcus mutans} and \textit{Enterococcus faecalis}. The antimicrobial activity was found to be highest against \textit{Streotococcus mutans} among the test organisms. Silver nanoparticle synthesised using natural substances can be used in control of oral pathogens.
\end{abstract}\def\keywordstitle{Keywords}

\begin{keywords}Ginger Oleoresin,\newline Green synthesis,\newline oral pathogens,\newline silver nanoparticles
\end{keywords}
\twocolumn[ \maketitle {\printKwdAbsBox}]
\makeatletter{\raggedright\textsuperscript{*}Corresponding Author\par Name:\ Anitha~Roy~\\ Phone:\ 9840787458~\\  Email:\ anitharoy2015@gmail.com
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\section{Introduction}
Silver nanoparticles have become a substance of great interest and focus in researches in recent years due to its low cost and its wide range of applications like such as catalysis, electrical batteries, bio-sensing and biotechnology. In recent times metal nanoparticles have been manufactured from eco-friendly sources such as fruits, fungi, plant extracts and microorganisms. Studies report that the reduction of metal ions using plants are much faster as compared to microorganisms \unskip~\citep{582688:13495102,582688:13495103}.

 Recently green nanotechnology has been gaining popularity because of its exclusion of damaging reagents and cost-effectiveness. Several studies have shown that silver nanoparticles have antifungal and antibacterial activity \unskip~\citep{582688:13495102,582688:13495104,582688:13495106}. Green synthesis of silver nanoparticles using the various parts of the plant for example leaf extract, seed extract, plant latex, microorganisms and some biopolymers have been reported. Polymers such as polyethylene glycol, polyvinylpyrrolidone, polyacrylonitrile, poly(methyl methacrylate), polyaniline and poly(vinyl alcohol) were also used as reducing and stabilizing agents for the formation of well-dispersed AgNPs.\unskip~\citep{582688:13495098,582688:13495101}. \textit{Zingiber officinale is }an aromatic and pungent spice that has unique culinary, medicinal value. It is cultivated in tropical and subtropical countries. Ginger is effective against the growth of various microorganism, which cause many diseases \unskip~\citep{582688:13495100}. In this study, ginger oleoresin mediated silver nanoparticles were prepared and evaluated for its antimicrobial activity.
    
\section{Materials and Methods}
\textbf{Plant material}

Ginger oleoresin was obtained from Synthite Industries Ltd, Kerala, India as a gift sample. 

\textbf{Synthesis of nanoparticles}

 90 mL of 1 mM silver nitrate solution was mixed with 10 mL of ginger oleoresin extract (1 mL of ginger oleoresin in 9 mL of 50 \% ethanol) solution and was kept in magnetic stirrer for nanoparticle synthesis formation. The colour change was observed visually, and photographs were recorded. 

\textbf{Conformation of synthesis of NPs}

The synthesised NP's solution was preliminary confirmed by UV-Vis-spectroscopy. 3 mL of the solution was taken in cuvette and scanned in double beam UV-Vis-spectrophotometer from 300 nm to 700 nm wavelengths. The results were recorded for graphical analysis.

\textbf{Preparation of nanoparticles powder}

 The prepared nanoparticle solution was centrifuged using refrigerated lark centrifuge at 8000 rpm for 15 minutes, and the pellet were collected and washed twice with distilled water. The final purified pellets were collected and dried at 60\ensuremath{^{0}}C and stored in airtight Eppendorf tube \unskip~\citep{582688:13495095}.

\textbf{Antibacterial activity of nanoparticles against oral pathogens }

Agar well diffusion method was used to determine the antibacterial activity of synthesised ginger oleoresin mediated silver nanoparticles. Different concentrations of AgNP's were tested against \textit{Staphylococcus mutans}, \textit{Enterococcus faecalis},\textit{ Staphylococcus aureus}. The fresh bacterial culture was dispersed on the surface of Muller Hinton agar plates. Different concentrations of nanoparticles (20, 50, and 100 \ensuremath{\mu }L) were incorporated into the wells, and the plates were incubated at 37\ensuremath{^\circ}C for 24 hours. Amoxycillin was used as positive control and the inhibition zone was recorded in each plate.
    
\section{Results and Discussion}

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\caption{\boldmath {A \& B Visual observation of Ginger oleoresin mediated silver nanoparticles before and after formation}}
\label{f-ecb258db8dca}
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\caption{\boldmath {UV {\textendash}Vis Spectroscopy of Ginger oil mediated silver nanoparticles}}
\label{f-d71824d78a24}
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\caption{\boldmath {Antibacterial activity of Ginger oleoresin mediated AgNPs against test microorganism }}
\label{f-3594e8615c8a}
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\egroup
\textbf{Visual observation}

It is a well-known fact that AgNPs exhibit dark brown colour depending on the intensity and size of nanoparticles. The colour change occurs due to the excitation of the surface plasmon resonance (SPR) of the silver nanoparticles. When Ginger oleoresin was added to the silver nitrate solution, the colour of the solution gradually changed from a turbid whitish-yellow colour to dark brown. This colour change has been shown in Figure~\ref{f-ecb258db8dca} A and Figure~\ref{f-ecb258db8dca} B. This colour change was indicative of the formation of silver nanoparticles which was further confirmed by UV {\textendash} Vis spectrophotometry.

\textbf{UV-Vis spectroscopy }

The UV {\textendash} Vis spectra was recorded for the prepared ginger oleoresin mediated silver nanoparticles (Figure~\ref{f-d71824d78a24}). It was observed from the spectra that the AgNP's surface plasmon resonance peak occurs at 440 nm with high absorbance, which is indicative for silver nanoparticles. This confirms the formation of Ginger oleoresin mediated silver nanoparticles.

\textbf{Antibacterial activity}

The synthesised ginger oleoresin mediated AgNP's by green techniques were studied for antimicrobial activity against the pathogen such as\textit{Staphylococcus aureus}, \textit{Streptococcus mutans} and \textit{Enterococcus faecalis}. The diameter of the zone of inhibition (ZOI) was recorded in millimetre against each concentration of bacterial species. (Figure~\ref{f-3594e8615c8a}). The results were tabulated, and the graph was plotted (Figure~\ref{f-3594e8615c8a}). The zone of inhibition for \textit{Enterococcus faecalis} was noted as 9mm at 20 \ensuremath{\mu }L, 11mm at 50 \ensuremath{\mu }L and 13 mm at 100 \ensuremath{\mu }l. The zone of inhibition for \textit{S.aureus} was noted as 10mm at 20 \ensuremath{\mu }L, 12mm at 50 \ensuremath{\mu }L and 13 mm at 100 \ensuremath{\mu }L. The zone of inhibition for \textit{S.mutans} was noted as 10mm at 20 \ensuremath{\mu }L, 12mm at 50 \ensuremath{\mu }L, 16mm at 100 \ensuremath{\mu }L. The zone of inhibition was the highest for \textit{S. mutans}. The results obtained were comparable to the study conducted by \unskip~\citep{582688:13495099}. In the present study\textit{, }ginger oleoresin was used for the synthesis of silver nanoparticles. One of the main components of ginger oleoresin is ginger oil, which has numerous phytochemicals. The ginger oleoresin contains ginger essential oil and 6-gingerol and a structurally related vanilloid \unskip~\citep{582688:13495100}. This oil is shown to have a greater affinity towards the wall cell present in the gram-positive bacteria. The gram-positive bacteria have a cell wall composed of thick layers of peptidoglycans which the oil will attack and destroy. \unskip~\citep{582688:13495097}. Green synthesised nanoparticles have numerous benefits of being eco-friendly and compatible with pharmaceutical and other biomedical applications \unskip~\citep{582688:13495105,582688:13495095}. In recent years, the number of infections connected with antibiotic-resistant bacteria has amplified, so the use of green synthesis of antimicrobials will be useful in combating this problem. The main reason for the use of nanoparticles is for good bioavailability, and it easily enter the cell membrane to improve the activity \unskip~\citep{582688:13495096}.
    
\section{Conclusion}
The present study concludes that ginger oleoresin is an effective means to produce silver nanoparticles in an ecofriendly manner and that oral pathogens can be controlled by using ginger oleoresin mediated silver nanoparticles. Ginger oleoresin mediated silver nanoparticles was showing maximum activity against \textit{Streptococcus mutans}. Hence, it may be used to control infections caused by \textit{Streptococcus mutans}.
\section*{Acknowledgement} The authors thank Synthite Industries Pvt Limited, Kerala for providing ginger oleoresin as a gift sample. 
    

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