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\begin{document}


\def\RunningAuthor{Bheemreddy Thrinitha et al.}
\firstPage{185}
\articleType{Original Article}
\receivedDate{06.07.2019}
\acceptedDate{30.10.2019}
\revisedDate{21.10.2019}
\journalVolume{2020, 11}
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\def\authorCount{3}
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\title{\textbf{Evaluation of in vitro antioxidant activity of different extracts of entire plant of \textit{Ipomoea pestigridis Linn}}}
\author{Bheemreddy~Thrinitha\textsuperscript{*}\textsuperscript{1},  
            Murali~R\textsuperscript{1}, 
            Manichandrika~P\textsuperscript{2}~\\[5pt]{\textsuperscript{1}Bojjam Narasimhulu Pharmacy college for woman, Vinay Nagar,Champapet, Hyderabad, India}~\\{\textsuperscript{2}Department of Pharmacy, Annamalai University, Annamalai Nagar-608 002, Tamil Nadu, India}}

\begin{abstract}
\textit{Ipomoea pestigridis} (Linn) (family Convolvulaceae) is commonly known as ``Tiger Foot Morning Glory'' in English and locally known as `Pulichuvadi' or `Pulichuvadu' in Malayalam.\textit{\space } The current study, aerial parts of different concentrates(Pet.ether, ethyl acetate, and methanol) of \textit{I.pestigridis}, was evaluated for its \textit{in-vitro } antioxidant potential by nitric oxide activity, total antioxidant activity, iron chelating activity taking ascorbate \& Ethylenediamine tetraacetate as the standard correspondingly. An IC\ensuremath{_{50}} value was originated that EA concentrates of \textit{I.pestigridis}  more efficient in nitric oxide activity, total antioxidant activity, Iron chelating capacity compared methanolic \& PE concentrates. The ethyl acetate concentrates of \textit{I.pestigridis}  \& ascorbic acid exhibited antioxidant potential possessing IC\ensuremath{_{50\ }}226\ensuremath{\mu}g/ml \& 66\ensuremath{\mu}g/ml (Nitric oxide). 185\ensuremath{\mu}g/ml \& 60\ensuremath{\mu}g/ml (total antioxidant) , 287\ensuremath{\mu}g/ml \& 65\ensuremath{\mu}g/ml (iron-chelating Activity) respectively. The difference in the scavenging potential of the extracts can be due to variation in the percentage of bioactive compounds present in different solvents.  Invitro antioxidant studies obviously show EA concentrates of \textit{I.pestigridis} have better antioxidant activity. These results indicate that aerial parts of methanolic concentrate \textit{I.pestigridis}  could serve as a natural antioxidant, which may be useful in preventing free radical-induced diseases. 
\end{abstract}\def\keywordstitle{Keywords}

\begin{keywords}I.pestigridis,\newline Nitric oxide radical scavenging,\newline total antioxidant,\newline Iron chelating activity
\end{keywords}
\twocolumn[ \maketitle {\printKwdAbsBox}]
\makeatletter{\raggedright\textsuperscript{*}Corresponding Author\par Name:\ Bheemreddy~Thrinitha~\\ Phone:\ 9704245374~\\  Email:\ Thrinitha.bheemreddy@gmail.com
        \par}\vspace*{-11pt}\hrulefill\par{\fontsize{12}{14}\selectfont ISSN: 0975-7538}\par% 
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\section{Introduction}
Free radicals might be defined as one or more unpaired electrons present in the molecules as it's outmost orbital and are able of autonomous existence. It is regularly represented as hydrogen radical (H\ensuremath{^{\ensuremath{\cdot}}}), which contains one proton and one electron (unpaired) is the simplest free radical. A free radical reaction might be finished by a reaction among two free radicals or by neutralization by substances such as the antioxidants \unskip~\citep{630001:14741809}. Free radicals may be generated both \textit{in vivo} and \textit{in vitro } through one of the subsequent mechanisms. Loss of a single electron from a regular molecule. Addition of an electron to regular molecule \unskip~\citep{630001:14741811}. Ethnomedical literature contains a huge amount of herbs that may be used for the various diseases, in which ROS and free radical participate vital responsibility. A huge numerical herbs are used for strong antioxidant activity \unskip~\citep{630001:14741804}. Current reports revealed that there is a converse connection among the intake of antioxidant-rich foods and the occurrence of human diseases \unskip~\citep{630001:14741811}.


\begin{table*}[!htbp]
\caption{\boldmath {Nitric oxide scavenging activity of \textit{I. pestigridis} PE Extract } }
\label{tw-f3eccc8dfd0d}
\centering 
\begin{threeparttable}

\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.1389\linewidth-2\tabcolsep}p{\dimexpr.31159999999999997\linewidth-2\tabcolsep}p{\dimexpr.23200000000000003\linewidth-2\tabcolsep}p{\dimexpr.3175\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor} S.no &  Extract (\ensuremath{\mu}g/ml) & \multicolumn{2}{p{\dimexpr(.5495\linewidth-2\tabcolsep)}}{\cAlignHack \% of activity(\ensuremath{\pm}SEM)*}\\
\rowcolor{kwdboxcolor} &  &  PE extract   & Ascorbic acid  \\
\tblmidrule 
1 &
  125 &
  34.65\ensuremath{\pm}0.025 &
  48.54 \ensuremath{\pm}0.046\\
2 &
  250 &
  40.34\ensuremath{\pm}0.042 &
  57.31 \ensuremath{\pm}0.034\\
3 &
  500 &
  45.34\ensuremath{\pm}0.052 &
  68.24 \ensuremath{\pm}0.045\\
4 &
  1000 &
  55.02\ensuremath{\pm}0.021 &
  76.34 \ensuremath{\pm}0.012\\
\multicolumn{2}{p{\dimexpr(.4505\linewidth-2\tabcolsep)}}{} &
  IC\ensuremath{_{50}}  = 750 \ensuremath{\mu}g/ml &
  IC\ensuremath{_{50}}  = 135 \ensuremath{\mu}g/ml\\
\tblbottomrule 
\end{tabulary}\par 
\begin{tablenotes}\footnotesize 
    
\item{* Every value was articulated as mean \ensuremath{\pm} SEM for 3 experimentation}
\end{tablenotes}
\end{threeparttable}

\end{table*}
\textit{Ipomoea pestigridis} (Linn) (family Convolvulaceae) is commonly known as ``Tiger Foot Morning Glory'' in English and locally known as `Pulichuvadi' or `Pulichuvadu' in Malayalam \unskip~\citep{630001:14741819,630001:14741816}. \textit{I.pestigridis} was used for the treatment of wound healing \unskip~\citep{630001:14741806,630001:14741807}. \textit{I.pestigridis} was used for different diseases like headaches, swellings, poisonous stings, snake bites. \textit{I.pestigridis} was used for analgesic, antimicrobial, thrombocytic, cytotoxic activity \unskip~\citep{630001:14741817}. Still, no literature are available on the antioxidant activity of the entire plant of \textit{I.pestigridis}. Thus, the present study to assess the antioxidant activities of the entire plant of \textit{I.pestigridis.}
    
\section{Materials and Methods}
\textbf{Collection and Identification of Plant }

The entire plant of \textit{I.pestigridis} (family Convolvulaceae ) were gathered form kalakad , Tirunelveli District of Tamilnadu India. Plant identification was made from Botanical investigation of India, Palayamkottai The \textit{I.pestigridis} were desiccated under shadowy, segregate, crushed through a grinder \unskip~\citep{630001:14741820}.


\begin{table*}[!htbp]
\caption{\boldmath {Nitric oxide scavenging activity of \textit{I. pestigridis} EA Extract } }
\label{tw-b7e40abc555e}
\centering 
\begin{threeparttable}

\def\arraystretch{1.1}
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\begin{tabulary}{\linewidth}{p{\dimexpr.13109999999999998\linewidth-2\tabcolsep}p{\dimexpr.29279999999999998\linewidth-2\tabcolsep}p{\dimexpr.3261\linewidth-2\tabcolsep}p{\dimexpr.25\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor} S.no &  Extract (\ensuremath{\mu}g/ml) & \multicolumn{2}{p{\dimexpr(.5761\linewidth-2\tabcolsep)}}{\cAlignHack \% of activity(\ensuremath{\pm}SEM)*}\\
\rowcolor{kwdboxcolor} &  &  EA extract  & Ascorbic acid \\
\tblmidrule 
1 &
  125 &
  43.54 \ensuremath{\pm}0.022 &
  48.54 \ensuremath{\pm}0.046\\
2 &
  250 &
  55.32 \ensuremath{\pm}0.043 &
  57.31 \ensuremath{\pm}0.034\\
3 &
  500 &
  64.56 \ensuremath{\pm}0.065 &
  68.24 \ensuremath{\pm}0.045\\
4 &
  1000 &
  69.34 \ensuremath{\pm}0.054 &
  76.34 \ensuremath{\pm}0.024\\
\multicolumn{2}{p{\dimexpr(.4238999999999999\linewidth-2\tabcolsep)}}{} &
  IC\ensuremath{_{50}}  = 198 \ensuremath{\mu}g/ml &
  IC\ensuremath{_{50}}  = 135 \ensuremath{\mu}g/ml\\
\tblbottomrule 
\end{tabulary}\par 
\begin{tablenotes}\footnotesize 
    
\item{  * Every value was articulated as mean \ensuremath{\pm} SEM for 3 experimentation}
\end{tablenotes}
\end{threeparttable}

\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Nitricoxide scavenging activity of \textit{I. pestigridis} methanol Extract} }
\label{tw-8301e02283dc}
\centering 
\begin{threeparttable}

\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.104\linewidth-2\tabcolsep}p{\dimexpr.27519999999999996\linewidth-2\tabcolsep}p{\dimexpr.32019999999999996\linewidth-2\tabcolsep}p{\dimexpr.3006\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}S.no & Extract(\ensuremath{\mu}g/ml) & \multicolumn{2}{p{\dimexpr(.6208\linewidth-2\tabcolsep)}}{\cAlignHack \% of activity(\ensuremath{\pm}SEM)*}\\
\rowcolor{kwdboxcolor} &  & Methanolic extract  & Ascorbate \\
\tblmidrule 
1 &
  125 &
  32.34 \ensuremath{\pm}0.025 &
  48.54 \ensuremath{\pm}0.046\\
2 &
  250 &
  44.84 \ensuremath{\pm}0.054 &
  57.31 \ensuremath{\pm}0.034\\
3 &
  500 &
  52.38 \ensuremath{\pm}0.062 &
  68.24 \ensuremath{\pm}0.045\\
4 &
  1000 &
  66.22 \ensuremath{\pm}0.038 &
  76.34 \ensuremath{\pm}0.024\\
\multicolumn{2}{p{\dimexpr(.37919999999999995\linewidth-2\tabcolsep)}}{} &
  IC\ensuremath{_{50}}= 435 mg/ml  &
  IC\ensuremath{_{50}}= 135 mg/ml\\
\tblbottomrule 
\end{tabulary}\par 
\begin{tablenotes}\footnotesize 
    
\item{*Every value was articulated as mean \ensuremath{\pm} SEM for 3 experimentation}
\end{tablenotes}
\end{threeparttable}

\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Total antioxidant activity of \textit{I. pestigridis} PE Extract } }
\label{tw-376fbb3f7a41}
\centering 
\begin{threeparttable}

\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.0822\linewidth-2\tabcolsep}p{\dimexpr.3129\linewidth-2\tabcolsep}p{\dimexpr.28290000000000006\linewidth-2\tabcolsep}p{\dimexpr.32199999999999996\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}S.no &  Extract  (\ensuremath{\mu}g/ml) & \multicolumn{2}{p{\dimexpr(.6049\linewidth-2\tabcolsep)}}{\cAlignHack \% inhibition(\ensuremath{\pm}SEM)*}\\
\rowcolor{kwdboxcolor} &  & PE extract  & Ascorbate\\
\tblmidrule 
1 &
  50 &
  12.23 \ensuremath{\pm}0.043 &
  50.76 \ensuremath{\pm}0.024\\
2 &
  100 &
  21.32 \ensuremath{\pm}0.012 &
  61.68 \ensuremath{\pm}0.035\\
3 &
  200 &
  29.22 \ensuremath{\pm}0.076 &
  74.64 \ensuremath{\pm}0.048\\
4 &
  300 &
  39.55 \ensuremath{\pm}0.046 &
  98.12 \ensuremath{\pm}0.021\\
\multicolumn{2}{p{\dimexpr(.3951\linewidth-2\tabcolsep)}}{} &
  IC\ensuremath{_{50\ }} = 460 \ensuremath{\mu}g/ml &
  IC\ensuremath{_{50\ }} = 57 \ensuremath{\mu}g/ml\\
\tblbottomrule 
\end{tabulary}\par 
\begin{tablenotes}\footnotesize 
    
\item{* Every value was articulated as mean \ensuremath{\pm} SEM for 3 experimentation}
\end{tablenotes}
\end{threeparttable}

\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Total antioxidant activity of \textit{I. pestigridis} EA Extract } }
\label{tw-2a577c5c9413}
\centering 
\begin{threeparttable}

\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.0929\linewidth-2\tabcolsep}p{\dimexpr.2752\linewidth-2\tabcolsep}p{\dimexpr.28949999999999996\linewidth-2\tabcolsep}p{\dimexpr.34239999999999995\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}S.no &  Extract  (\ensuremath{\mu}g/ml) & \multicolumn{2}{p{\dimexpr(.6318999999999999\linewidth-2\tabcolsep)}}{\cAlignHack \% inhibition(\ensuremath{\pm}SEM)*}\\
\rowcolor{kwdboxcolor} &  & EA extract  & Ascorbate\\
\tblmidrule 
1 &
  50 &
  33.45 \ensuremath{\pm}0.043 &
  50.76 \ensuremath{\pm}0.024\\
2 &
  100 &
  40.65 \ensuremath{\pm}0.054 &
  61.68 \ensuremath{\pm}0.035\\
3 &
  200 &
  50.85 \ensuremath{\pm}0.062 &
  74.64 \ensuremath{\pm}0.048\\
4 &
  300 &
  59.46 \ensuremath{\pm}0.014 &
  98.12 \ensuremath{\pm}0.021\\
\multicolumn{2}{p{\dimexpr(.3681\linewidth-2\tabcolsep)}}{} &
  IC\ensuremath{_{50}}  = 190 \ensuremath{\mu}g/ml &
  IC\ensuremath{_{50}}  = 57 \ensuremath{\mu}g/ml\\
\tblbottomrule 
\end{tabulary}\par 
\begin{tablenotes}\footnotesize 
    
\item{* Every value was articulated as mean \ensuremath{\pm} SEM for 3 experimentation}
\end{tablenotes}
\end{threeparttable}

\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Total antioxidant activity of \textit{I. pestigridis} methanol Extract } }
\label{tw-96340a124a87}
\centering 
\begin{threeparttable}

\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.10740000000000002\linewidth-2\tabcolsep}p{\dimexpr.30389999999999993\linewidth-2\tabcolsep}p{\dimexpr.28330000000000002\linewidth-2\tabcolsep}p{\dimexpr.3054\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}S.no &  Extract  (\ensuremath{\mu}g/ml) & \multicolumn{2}{p{\dimexpr(.58870000000000005\linewidth-2\tabcolsep)}}{\cAlignHack \% inhibition(\ensuremath{\pm}SEM)*}\\
\rowcolor{kwdboxcolor} &  & Methanolic extract  & Ascorbate\\
\tblmidrule 
1 &
  50 &
  17.82 \ensuremath{\pm}0.035 &
  50.76 \ensuremath{\pm}0.024\\
2 &
  100 &
  26.54 \ensuremath{\pm}0.042 &
  61.68 \ensuremath{\pm}0.035\\
3 &
  200 &
  37.22 \ensuremath{\pm}0.033 &
  74.64 \ensuremath{\pm}0.048\\
4 &
  300 &
  44.32 \ensuremath{\pm}0.012 &
  98.12 \ensuremath{\pm}0.021\\
\multicolumn{2}{p{\dimexpr(.41129999999999995\linewidth-2\tabcolsep)}}{} &
  IC\ensuremath{_{50}}  = 340 \ensuremath{\mu}g/ml &
  IC\ensuremath{_{50}}  = 57 \ensuremath{\mu}g/ml\\
\tblbottomrule 
\end{tabulary}\par 
\begin{tablenotes}\footnotesize 
    
\item{* Every value was articulated as mean\ensuremath{\pm} SEM for 3 experimentation}
\end{tablenotes}
\end{threeparttable}

\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Iron-binding potential of \textit{I.pestigridis} PE concentrates} }
\label{tw-c39ec0531536}
\centering 
\begin{threeparttable}

\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.10190000000000001\linewidth-2\tabcolsep}p{\dimexpr.26899999999999995\linewidth-2\tabcolsep}p{\dimexpr.2610000000000001\linewidth-2\tabcolsep}p{\dimexpr.3681\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}S.no &  Extract  (\ensuremath{\mu}g/ml) & \multicolumn{2}{p{\dimexpr(.6291000000000001\linewidth-2\tabcolsep)}}{\cAlignHack \% inhibition (\ensuremath{\pm}SEM)*}\\
\rowcolor{kwdboxcolor} &  &  PE extract   & Ethylenediamine tetraacetate \\
\tblmidrule 
1 &
  125 &
  29.56 \ensuremath{\pm}0.018 &
  57.52 \ensuremath{\pm}0.014\\
2 &
  250 &
  37.48 \ensuremath{\pm}0.038 &
  64.76 \ensuremath{\pm}0.022\\
3 &
  500 &
  45.23 \ensuremath{\pm}0.042 &
  82.12 \ensuremath{\pm}0.045\\
4 &
  1000 &
  55.48 \ensuremath{\pm}0.023 &
  96.34 \ensuremath{\pm}0.034\\
\multicolumn{2}{p{\dimexpr(.37089999999999996\linewidth-2\tabcolsep)}}{} &
  IC\ensuremath{_{50}}  = 760 \ensuremath{\mu}g/ml &
  IC\ensuremath{_{50}}  = 65 \ensuremath{\mu}g/ml\\
\tblbottomrule 
\end{tabulary}\par 
\begin{tablenotes}\footnotesize 
    
\item{* Every value was articulated as mean \ensuremath{\pm} SEM for 3 experimentation}
\end{tablenotes}
\end{threeparttable}

\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Iron-binding potential of \textit{I.pestigridis} of EA concentrates} }
\label{tw-fb94bd5c144d}
\centering 
\begin{threeparttable}

\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.11439999999999998\linewidth-2\tabcolsep}p{\dimexpr.2682\linewidth-2\tabcolsep}p{\dimexpr.27620000000000005\linewidth-2\tabcolsep}p{\dimexpr.3412\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor} S.no &  Extract  (\ensuremath{\mu}g/ml) & \multicolumn{2}{p{\dimexpr(.6174\linewidth-2\tabcolsep)}}{\cAlignHack \% inhibition (\ensuremath{\pm}SEM)*}\\
\rowcolor{kwdboxcolor} &  &  EA extract   & Ethylenediamine tetraacetate \\
\tblmidrule 
1 &
  125 &
  38.12 \ensuremath{\pm}0.010 &
  57.52 \ensuremath{\pm}0.014\\
2 &
  250 &
  53.92 \ensuremath{\pm}0.024 &
  64.76 \ensuremath{\pm}0.022\\
3 &
  500 &
  61.43 \ensuremath{\pm}0.036 &
  82.12 \ensuremath{\pm}0.045\\
4 &
  1000 &
  69.87 \ensuremath{\pm}0.065 &
  96.34 \ensuremath{\pm}0.034\\
\multicolumn{2}{p{\dimexpr(.3826\linewidth-2\tabcolsep)}}{} &
  IC\ensuremath{_{50}}  = 217 \ensuremath{\mu}g/ml &
  IC\ensuremath{_{50}}  = 65\ensuremath{\mu}g/ml\\
\tblbottomrule 
\end{tabulary}\par 
\begin{tablenotes}\footnotesize 
    
\item{* Every value was articulated as mean \ensuremath{\pm} SEM for 3 experimentation}
\end{tablenotes}
\end{threeparttable}

\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Iron-binding potential of \textit{I.pestigridis} Methanolic concentrates } }
\label{tw-fcf0a0583d93}
\centering 
\begin{threeparttable}

\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.1055\linewidth-2\tabcolsep}p{\dimexpr.26099999999999998\linewidth-2\tabcolsep}p{\dimexpr.31509999999999998\linewidth-2\tabcolsep}p{\dimexpr.3184\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}S.no &  Extract  (\ensuremath{\mu}g/ml) & \multicolumn{2}{p{\dimexpr(.63349999999999994\linewidth-2\tabcolsep)}}{\cAlignHack \% inhibition(\ensuremath{\pm}SEM)*}\\
\rowcolor{kwdboxcolor} &  & Methanol extract   & EthyleneDiamine tetraacetate  \\
\tblmidrule 
1 &
  125 &
  30.84 \ensuremath{\pm}0.045 &
  57.52 \ensuremath{\pm}0.014\\
2 &
  250 &
  43.22 \ensuremath{\pm}0.063 &
  64.76 \ensuremath{\pm}0.022\\
3 &
  500 &
  55.84 \ensuremath{\pm}0.072 &
  82.12 \ensuremath{\pm}0.045\\
4 &
  1000 &
  67.32 \ensuremath{\pm}0.028 &
  96.34 \ensuremath{\pm}0.034\\
\multicolumn{2}{p{\dimexpr(.3665\linewidth-2\tabcolsep)}}{} &
   IC\ensuremath{_{50}}  = 385 \ensuremath{\mu}g/ml &
  IC\ensuremath{_{50}}  = 65\ensuremath{\mu}g/ml\\
\tblbottomrule 
\end{tabulary}\par 
\begin{tablenotes}\footnotesize 
    
\item{* Every value was articulated as mean \ensuremath{\pm} SEM for 3 experimentation}
\end{tablenotes}
\end{threeparttable}

\end{table*}
\textbf{Preparation of Concentrates }

The pulverized materials were progressively concentrated with PE (40-60\ensuremath{^{0}}C) through hot constant percolation  method in Soxhlet equipment \unskip~\citep{630001:14741813} for twenty-four hours. At that moment, the marc was subjected to EA (76-78\ensuremath{^{0}}C) for 24 hrs \& then mark was subjected to methanol for 24 hrs. The concentrates were concentrated through the rotational evaporator and subjected to solidify drying in a lyophilizer till dry powder was acquired. \unskip~\citep{630001:14741803}.

\textbf{Assessment of Antioxidant potential through invitro methods}

The variety of concentrates of \textit{I.pestigridis} \textit{}were used assessment of antioxidant\textit{\space }activity by \unskip~\cite{630001:14741810} method was adopted for NO radical assay \& \unskip~\cite{630001:14741818} method described for total antioxidant activity, and \unskip~\cite{630001:14741805} method was adopted to determine the Iron chelating activity. 
    
\section{Results and Discussion}
\textbf{Nitric oxide scavenging activity}

Nitric oxide scavenging activity of PE concentrates of \textit{I. pestigridis} appeared in Table~\ref{tw-f3eccc8dfd0d}. The PE concentrates of \textit{I. pestigridis} exhibit a more Nitric oxide scavenging activity of 55.02\% at 1000 \ensuremath{\mu}g/ml \& ascorbate was recorded 76.34\% at 1000 \ensuremath{\mu}g/ml. The IC\ensuremath{_{50}} of the PE concentrates of \textit{I. pestigridis} \& ascorbic acid were recorded 750\ensuremath{\mu}g/ml \& 135\ensuremath{\mu}g/ml correspondingly.

Nitric oxide scavenging activity of the EA concentrates of \textit{I. pestigridis} appeared in Table~\ref{tw-b7e40abc555e}. The EA concentrates of \textit{I. pestigridis} exhibit a more Nitric oxide scavenging activity of 69.34\% at 1000 \ensuremath{\mu}g/ml \& ascorbic acid was recorded at 76.34\% at 1000 \ensuremath{\mu}g/ml. The IC\ensuremath{_{50}} of the EA concentrates of \textit{I. pestigridis} \& ascorbic acid were recorded 198\ensuremath{\mu}g/ml \& 135\ensuremath{\mu}g/ml correspondingly.\textbf{\space }

Nitric oxide scavenging activity of methanol concentrates of \textit{I. pestigridis} appeared in Table~\ref{tw-8301e02283dc}. The methanol concentrates of \textit{I. pestigridis} exhibit a more Nitric oxide scavenging activity of 66.22\% at 1000 \ensuremath{\mu}g/ml \& ascorbic acid was recorded at 76.34\% at 1000 \ensuremath{\mu}g/ml. The IC\ensuremath{_{50}} of methanol concentrates of \textit{I. pestigridis} \& ascorbic acid were recorded 435\ensuremath{\mu}g/ml \& 135\ensuremath{\mu}g/ml correspondingly.\textbf{\space }

IC\ensuremath{_{50}} values \& Nitric oxide scavenging potential revealed that ethyl acetate concentrates of \textit{I. pestigridis} is better activity in scavenging Nitric oxide scavenging activity when compared methanol \& PE extracts.

\textbf{Phosphomolybdic acid method}

Total antioxidant activity of PE concentrates of \textit{I. pestigridis} appeared in Table~\ref{tw-376fbb3f7a41}. The PE concentrates of \textit{I. pestigridis} exhibit a more total antioxidant activity of 39.55\% at 300 \ensuremath{\mu}g/ml \& ascorbic acid was recorded at 98.12\% at 300 \ensuremath{\mu}g/ml. The IC\ensuremath{_{50}} of the PE concentrates of \textit{I. pestigridis} \& ascorbic acid were recorded 460\ensuremath{\mu}g/ml \& 57\ensuremath{\mu}g/ml correspondingly.

The total antioxidant activity of the EA concentrates of \textit{I. pestigridis} appeared in Table~\ref{tw-2a577c5c9413}. The EA concentrates of \textit{I. pestigridis} exhibit a more total antioxidant activity of 59.46\% at 300 \ensuremath{\mu}g/ml \& ascorbic acid was recorded at 98.12\% at 300 \ensuremath{\mu}g/ml. The IC\ensuremath{_{50}} of the EA concentrates of \textit{I. pestigridis} \& ascorbic acid were recorded 190\ensuremath{\mu}g/ml \& 57\ensuremath{\mu}g/ml correspondingly.

The total antioxidant activity of methanol concentrates of \textit{I. pestigridis} appeared in Table~\ref{tw-96340a124a87}. The methanol concentrates of \textit{I. pestigridis} exhibit a more total antioxidant activity of 44.32\% in both table (Table~\ref{tw-96340a124a87}) and text. The IC\ensuremath{_{50}} of the methanol concentrates of \textit{I. pestigridis} \& ascorbic acid were recorded 460\ensuremath{\mu}g/ml \& 57\ensuremath{\mu}g/ml correspondingly.

IC\ensuremath{_{50}} values \& total antioxidant potential revealed that ethyl acetate concentrates of \textit{I. pestigridis} is better activity in scavenging total antioxidant potential when compared methanol \& PE extracts

\textbf{Iron chelating potential}

The iron complex potential of PE concentrates \textit{I.pestigridis} \textbf{\textit{\space }}\textit{\space }\& Ethylenediamine tetraacetate were appeared in Table~\ref{tw-c39ec0531536}. The more iron-binding potential of PE concentrates \& Ethylenediamine tetraacetate  1000 \ensuremath{\mu}g/ml were recorded, 55.48\% \& 96.34 \%. The IC\ensuremath{_{50}} of PE concentrates of \textit{I.pestigridis} \textit{\textbf{\space }} \textit{\space }\& Ethylenediamine tetraacetate were found as 760\ensuremath{\mu}g/ml \& 65\ensuremath{\mu}g/ml correspondingly.

The iron complex potential of EA concentrates of \textit{I.pestigridis}\textit{\textbf{\space }}\textit{\space }\& Ethylenediamine tetraacetate was were presented in Table~\ref{tw-fb94bd5c144d}. The more iron-binding capacity of EA concentrates \& Ethylenediamine tetraacetate 1000 \ensuremath{\mu}g/ml was recorded at 69.87\% \& 96.34 \%.  The IC\ensuremath{_{50}} value of ethyl acetate concentrates of \textit{I.pestigridis} \textit{\textbf{\space }} \textit{\space }\& Ethylenediamine tetraacetate were found 217\ensuremath{\mu}g/ml \& 65\ensuremath{\mu}g/ml correspondingly.

The iron complex potential of methanolic concentrates of \textit{I.pestigridis}\textit{\textbf{\space }} \textit{\space }\& Ethylenediamine tetraacetate were presented in Table~\ref{tw-fcf0a0583d93}. The more iron-binding potential of methanolic concentrates \& Ethylenediamine tetraacetate 1000 \ensuremath{\mu}g/ml were recorded, 67.32\% \& 96.34 \%. The IC\ensuremath{_{50}} value of methanol concentrates of \textit{I.pestigridis} \textit{\textbf{\space }}\& Ethylenediamine tetraacetate was recorded as 385\ensuremath{\mu}g/ml \& 65\ensuremath{\mu}g/ml correspondingly.

IC\ensuremath{_{50}} values \& iron-binding potential revealed that ethyl acetate concentrates of \textit{I.pestigridis}\textit{\textbf{\space }}\textit{\space }is a huge activity in iron-chelating potential when compared methanol \& petroleum ether concentrates. But when compared to all the three concentrates, the ethyl acetate concentrates of the \textit{I.pestigridis}\textit{\textbf{\space }} \textit{\space }showed the better result.

Assessment of antioxidant activity, so many \textit{in vitro } methods have been used a variety of concentrates of \textit{I.pestigridis}. The results of antioxidant activity by NO radical activity, total antioxidant activity \& iron-chelating potential were expressed in terms of \% inhibition of generated free radicals, respectively, with respect to various concentrations. NO is produced from L-arginine through vascular endothelial cells, phagocytes, and certain cells of the brain. NO is a free radical since its unpaired electron and displays significant reactivity with confident types of proteins and other free radicals. The toxicity of Nitric oxide becomes a side effect when it reacts with superoxide radical, forming a highly reactive peroxynitrite anion (ONOO\ensuremath{-}) \unskip~\citep{630001:14741814}. NO produced from sodium nitroprusside reacts with oxygen to form nitrite. The nitrite ions diazotize with sulphanilamide acid and couple with naphthyl ethylenediamine, producing pink coloured, which absorbs at 546 nm \unskip~\citep{630001:14741815}. Among the three different plant concentrates tested, interestingly, in the NO radical activity of the EA extract of\textit{ I.pestigridis }exhibited more NO radical potential comparable with that of ascorbic acid. 

The iron-chelating potential of all the concentrates was measured by Fe-ferrozine complex formation. Ferrozine-Fe complex is producing red coloured, which absorbs at 562nm \unskip~\citep{630001:14741821}. It was revealed that Ethylenediamine tetraacetate, which forms \ensuremath{\sigma  } bond with iron, are efficient as secondary antioxidants, for the basis that they decrease the redox potential, thereby stabilizing the oxidized form of the iron ion \unskip~\citep{630001:14741808}. The iron-chelating potential of ethyl acetate extract of \textit{I.pestigridis }exhibited a higher ability in scavenging compared to standard Ethylenediamine tetraacetate. 
    
\section{Conclusions}
The current trends, antioxidative activity of the herbs having more interest due to their possible use as natural additives to substitute synthetic ones. Among the three various extract ethyl acetate extracts of \textit{I.pestigridis }exhibited higher potency of antioxidant activity. These results indicate that ethyl acetate extract of \textit{I.pestigridis }might serve as a natural antioxidant, which may be useful in preventing free radical-induced diseases.  
    

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