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\def\RunningAuthor{Rajeshkumar S}
\firstPage{4152}
\articleType{Original Article}
\receivedDate{23 Feb 2020}
\acceptedDate{26 Mar 2020}
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\title{\textbf{\textit{Cynodon dactylon} Mediated Synthesis of Selenium Nanoparticles and Its Antimicrobial Activity Against Oral Pathogens}}
\author{Akansha~Kishen\textsuperscript{1},  
            Rajeshkumar~S\textsuperscript{*}\textsuperscript{2}, 
            Preejitha~V.~B.\textsuperscript{1}~\\[5pt]{\textsuperscript{1}Department of Pathology, Saveetha Dental College and Hospital, SIMATS, Chennai {\textendash} 600077, Tamilnadu, India}~\\{\textsuperscript{2}Nanobiomedicine Lab, Department of Pharmacology, Saveetha Dental College and Hospital, SIMATS, Chennai {\textendash} 600077, Tamilnadu, India}}

\begin{abstract}
Cynodon is a genus of plant in the grass family. It is found in the tropical regions and is also known as druva grass, Bermuda grass and Indian Doab. It is a herb extensively used in Indian Ayurvedic system of medicine. It has many health benefits and is useful in controlling hypertension, skin diseases and diabetes. Selenium nano particles have an excellent anti-cancer, biocidal, antioxidant, photo reactive property. The size of SeNPs ranges from 30nm to 200 nm. These particles can be used in arthritis, cancer, diabetes etc. To prepare the selenium nano particles using Cynodon dactylon, fresh \textit{Cynodon dactylon} was dried and made into a fine powder and powder mixed with distilled water. Selenium nanoparticles were synthesized using plant extract and sodium selenite. Anti microbial activity against oral pathogens tested using agar well diffusion method. The diameter of zone of inhibition was recorded on each plate. The colour change was indicative of the formation of SeNPs that was further confirmed by UV-vis spectrophotometer. The Cynodon dactylon mediated SeNPs showed high anti microbial activity against all the three micro-organisms especially Candida albicans and S.mutans. Therefore, diseases can be prevented as it is very effective in inhibiting the growth of the colonies.
\end{abstract}\def\keywordstitle{Keywords}

\begin{keywords}Cynodon dactylon,\newline selenium nano particles,\newline antimicrobial activity,\newline spectrophotometry
\end{keywords}
\twocolumn[ \maketitle {\printKwdAbsBox}]
\makeatletter{\raggedright\textsuperscript{*}Corresponding Author\par Name:\ Rajeshkumar~S~\\ Phone:\ 9629739263~\\  Email:\ ssrajeshkumar@hotmail.com
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\section{Introduction}
In many areas of treatment, namely, cancer, ceramics, drug delivery, catalysis and diagnosis, nano particles are being used. \unskip~\citep{775014:18431679} These nano particles have remarkable properties and potential applications which are due to their large surface area and their surface Plasmon resonance (SPR) effect. Selenium nano particles present a remarkable bactericidal effect and are also known to the most powerful antibiotics besides its usage in anti cancer and anti viral therapies. \unskip~\citep{775014:18431671} There are numerous methods that are used to obtain the high yield and good quality of the necessary products. Among many experiments, there are some that has ease of control over different particle size and re-distribution with numerous experimental aids. \unskip~\citep{775014:18431678} These are thermal decomposition method, laser ablation method, radiation method, micro emulsion method etc. Various nano structured and nano phase materials are worldwide used selectively in biological and pharmaceutical applications. \unskip~\citep{775014:18431672} The main aim of the usage of nano particles is also due to the current rise in the resistance of different bacteria to antibiotics to the overuse and exploitation has become a crucial area of concern. This has eventually led to renewed interests in investigation; the proper efficacy of selenium nano particles as a potent alternative. \unskip~\citep{775014:18431676} The antimicrobial activity of selenium materials are being used for many latest applications. This includes prevention of bacterial colonization in variety of medical devices like that of catheters, prosthesis and dental materials. \unskip~\citep{775014:18431680} A new research has been proposed that nanoparticles have the potent cytoprotective activity towards HIV infected cells. Selenium nano particles have an excellent anti cancer, biocidal, antioxidant, photo reactive property. The size of SeNPs ranges from 30nm to 200 nm.

Cynodon dactylon belongs to the genus of plant in the grass family. It is found in the tropical as well as subtropical regions. There are various regional names through which it is known as; these are druva grass, Bermuda grass and Indian Doab. It is a herb extensively used in Indian Ayurvedic system of medicine. \unskip~\citep{775014:18431674} Has many health benefits and useful in controlling hypertension, skin diseases and diabetes particularly. The phytochemical compounds extracted from these plants are widely used in various pharmaceutical companies. The main phytochemicals extracted includes glycosides, reducing sugar, carbohydrates, alkaloids, glycosides, flavonoids, alkaloids, terpenoids, triterpenoids, volatile oils and fixed oils. The juice extracted from this plant act as an astringent and is applied externally to fresh cuts and wounds. This plant is being used as a folk remedy for calculus, carbuncles, dysentery, snake bites etc. \unskip~\citep{775014:18431673}

The main objective of the present study to synthesise the selenium nano particle using the plant extract of Cynodon dactylon and test for its anti microbial activity. 
    
\section{Materials and Methods}
\textbf{Plant material }

Fresh Cynodon dactylon was obtained for the experiment.

\textbf{Preparation of Cynodon dactylon}

Fresh Cynodon dactylon was dried and made into a fine powder. 40mg of the powder was taken in a mixer and 60mL of distilled water measured using measuring jar and was added and mixed with a stirrer. The extract was boiled for 3 to 5 minutes and filtered using a filter paper. Figure~\ref{f-b9d37701caf9} demonstrate the boiling of the Cynodon dactylon and Figure~\ref{f-d05255d43b1e} demonstrates the filtration of the extract.

\textbf{Preparation of selenium nanoparticles}

0.519g of sodium selenite powder was added to 60mL of distilled water and to this previously prepared Cynodon dactylon extract was added. This is depicted in Figure~\ref{f-999ed79aa46b}. The mixture was kept in a magnetic stirrer and the readings were taken for analysing the synthesis of nano particles. \unskip~\citep{775014:18431681}  The colour was observed visually after every two hours \& proper pictures were taken of the beaker.

After 3 days of observation the mixture was taken in the graduated centrifuge pellet. 12mL of six pellets were being filled and was kept inside the centrifuge machine for 5 minutes. After this process the supernatant was being separated from the filtrate. The final supernatant from all the six pellets were taken into one graduated pellet and stored in a refrigerator.

\textbf{Antimicrobial activity of nano particles against oral pathogens}

To evaluate the antimicrobial activity of selenium nano particles the agar well diffusion method was being used. \textit{Streptococcus mutans}, \textit{Candida albicans} and \textit{Lactobacillus species} were used to test the Cynodon dactylon mediated selenium nano particles. Freshly prepared colloidal suspensions were being dispersed on the surface of Rose Bengal agar plate and Muller Hington agar plates. Different concentrations of nanoparticles i.e. 50, 100 and 150 \ensuremath{\mu}L were being used. Therefore, this different concentration of nanoparticles was incorporated into the wells and the plates were incubated for 24 hours at 37 degree Celsius. Amoxicillin and fluconazole were being used as antibiotics. \unskip~\citep{775014:18431682}  This antibiotic acted as a positive control. After incubation, the zone of inhibition was being recorded for each plate.
    
\section{Results and Discussion}
\textbf{Visual observation}

The selenium nano particles exhibit a dark green colour which basically depends on the intensity and the size of nanoparticles. The selenium nanoparticles have undergone colour change basically due to the surface Plasmon resonance (SPR). \unskip~\citep{775014:18431677} When Cynodon dactylon was being added to the sodium selenite solution, the colour of the solution transformed from a turbid light greenish colour to dark brown greenish colour. This colour change is being depicted in Figure~\ref{f-b9d37701caf9}. Thus, this colour change was a proper indication for the formation of the selenium nano particles which was further analysed and confirmed by the Ultraviolet-visible spectrophotometry.


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\caption{\boldmath {The \textit{Cynodon dactylon} extract preparation}}
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\caption{\boldmath {Filtered Plant extract }}
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\caption{\boldmath {Visual observation of selenium nanoparticles synthesis using Cynodon dactylon}}
\label{f-999ed79aa46b}
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\caption{\boldmath {Graph showing the absorbance of rosemary oleoresin mediated selenium nanoparticles by UV- Visible Spectroscopy}}
\label{f-a8886a833b76}
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\caption{\boldmath {Zone of inhibition for oral pathogens at 50, 100and 150\ensuremath{\mu }L depicted in the graph}}
\label{f-3d3cbca0d193}
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\caption{\boldmath {Antibacterial activity of Cynodon dactylon mediated selenium nano particles for \textit{Candida albicans}}}
\label{f-304bf9a0e3a1}
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\caption{\boldmath {Antibacterial activity of Cynodon dactylonmediated selenium nano particles for \textit{Lactobacillus species}}}
\label{f-0ac69232f05c}
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\caption{\boldmath {Antibacterial activity of Cynodon dactylon mediated selenium nano particles for \textit{Streptococcus mutans}}}
\label{f-74aeff2283ca}
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UV {\textendash}vis spectrophotometry involves the basic principle i.e. Beer- Lambert Law. The theory associated with this concept states that the energy difference between the higher energy state and the ground energy state is actually equal to the absorbed UV- radiation. This is a proper renowned technique, nowadays used to confirm the formation and stability of selenium nano particles. The UV- vis spectra graph is depicted in the Figure~\ref{f-a8886a833b76}  which was recorded for the \textit{Cynodon dactylon} mediated selenium nanoparticles.

\textbf{Antimicrobial activity}

The Cynodon dactylon mediated selenium nano particles were studied for antimicrobial activity against the oral pathogens, \textit{Candida albicans}, \textit{Streptococcus species }and \textit{S. mutans}. The diameter of the zone of inhibition (ZOI) was being recorded around various wells which had different concentrations level of Cynodon dactylon mediated selenium nano particles against the microorganism species. \unskip~\citep{775014:18431675} The result was tabulated and graph was being plotted which is depicted in Figure~\ref{f-3d3cbca0d193}.

The result formulated showed that the antimicrobial effect of the prepared selenium nano particles was almost similar against two microorganisms used in the study with a maximum zone of inhibition against \textit{Candida albicans } and \textit{S. mutans}. With the increase in the concentration there was a gradual increase in the zone of inhibition. 

The zone of inhibition for the prepared SeNP's against Lactobacillus at the concentration 50, 100, 150 \ensuremath{\mu }L was found to be 9mm, 9mm and 10mm respectively. The antibiotic for \textit{Lactobacillus species} showed the maximum zone of inhibition of 17 mm. Similarly, for \textit{Candida albicans} the zone of inhibition was found to be 9mm, 10mm and 11mm respectively. The antibiotic used here showed the least zone of inhibition as compared to other microorganisms' i.e.12 mm. For the case of \textit{Streptococcus mutans} the zone of inhibition at different concentration was 25mm, 30mm and 33mm respectively. The antibiotic used here showed a moderate zone of inhibition of 15mm. The ZOI for \textit{S. mutans} is shown in the Figure~\ref{f-74aeff2283ca}, for \textit{Lactobacillus species} it in depicted in Figure~\ref{f-0ac69232f05c} and for \textit{Candida albicans} it is shown in the Figure~\ref{f-304bf9a0e3a1}.

The antimicrobial activity of the freshly synthesised selenium nanoparticles was subjected to the selenium cations which were released from the mixture, that penetrates and lead to the changes in the membrane of the microorganism. Thus, this led to the increase of the membrane permeability of a particular microorganism which finally let to the cell death.
    
\section{Conclusion }
In the present study it is concluded that the Cynodon dactylon mediated selenium nano particles showed high antimicrobial activity against the entire three microorganisms especially Candida albicans and Streptococcus mutans'. Therefore, diseases caused by these microorganisms can be prevented as it is very effective in inhibiting the growth of colonies. On further studies, these nano particles can be used to cure diseases as it can be effective management on sensitive microorganisms. 

\textbf{Funding Support}

None.

\textbf{Conflict of Interest}

None.


    

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