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\def\RunningAuthor{Kesri Nandan Sharma and Nitu Bhatnagar}
\firstPage{254}
\articleType{Original Article}
\receivedDate{21 Jun 2020}
\acceptedDate{03 Oct 2020}
\revisedDate{26 Jul 2020}
\journalVolume{2021, 12}
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\journalDoi{ijrps.v12i1.4174}
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\title{\textbf{HPTLC } \textbf{study to determine the antioxidant activity of dried leaves of \textit{Portulaca oleracea L.}}}
\author{Kesri~Nandan~Sharma\textsuperscript{*}\textsuperscript{1}, 
            Nitu~Bhatnagar\textsuperscript{2}~\\[5pt]{\textsuperscript{1}Research Scholar, Department of Chemistry, Manipal University Jaipur {\textendash} 303 007, Rajasthan, India}~\\{\textsuperscript{2}Department of Chemistry, Manipal University Jaipur {\textendash} 303 007, Rajasthan, India}}

\begin{abstract}
This present study involves the assessment of the anti-oxidant activity study of the sample which was obtained from the methanolic extracts of dried leaves of \textit{Portulaca Oleracea L.}(common name Purslane). Purslane is a rich source of Vitamin A, Vitamin-C and some other B-complex vitamins like riboflavin, niacin, pyridoxine and carotenoids which are known powerful natural anti-oxidants.  Anti-oxidants are compounds that inhibit oxidation. This methanolic extract of leaves was evaluated for the determination of its anti-oxidant efficiency by using 1,1{\textendash}diphenyl-2-picryl-hydrazyl (DPPH) by using Silica TLC plates on Camag High-Performance Thin Layer Chromatography (HPTLC) system using visionCATS software. Densitograms and chromatographs obtained show the presence of anti-oxidant activity. It is a rapid, inexpensive and straightforward method to measure anti-oxidant properties of substances after separation by HPTLC. It involves the use of the free radical, 2, 2-Diphenyl-1- picrylhydrazyl (DPPH) which is widely used to test the ability of compounds to act as free radical scavengers or hydrogen donors and to evaluate anti-oxidant activity. When Anti-oxidants substances react with DPPH, which is a stable free radical becomes paired off in the presence of a hydrogen donor (e.g., a free radical scavenging anti-oxidant) and is reduced to the DPPHH. As a consequence, the absorbance's decreased from the DPPH.
\end{abstract}\def\keywordstitle{Keywords}

\begin{keywords}Densitogram,\newline Derivatization,\newline DPPH,\newline Nutrients,\newline Purslane,\newline visionCATS
\end{keywords}
\twocolumn[ \maketitle {\printKwdAbsBox}]
\makeatletter{\raggedright\textsuperscript{*}Corresponding Author\par Name:\ Kesri~Nandan~Sharma~\\ Phone:\ +91-9829014052~\\  Email:\ sharma\_kesri@rediffmail.com
        \par}\vspace*{-11pt}\hrulefill\par{\fontsize{12}{14}\selectfont ISSN: 0975-7538}\par% 
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        \vspace*{-2pt}{\fontsize{9.12}{10.12}\selectfont\textcopyright\ \@copyrightYear\ $|$ All rights reserved.}\par%
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\section{Introduction}
\textit{Portulaca oleracea} L.(Purslane) has wide cosmopolitan distribution around the globe as it can be seen in fields, gardens, vineyards, lawns, driveways, dunes and river banks. It is better known as wild plants but also an edible vegetable rich in many beneficial nutrients for human consumption \unskip~\citep{910209:20563689}. It contains more omega-3 fatty acids and alpha-linolenic acid (both are good anti-oxidants) in than any other leafy vegetable plant. \unskip~\citep{910209:20563690}, and is a nutritious food rich in omega-3 fatty acids and anti-oxidants \unskip~\citep{910209:20563686}. It possesses many anti-oxidant properties due to the excellent value contents of vitamins, minerals, essential fatty acids and other compounds and having rich medicinal properties \unskip~\citep{910209:20563682}, possess potent pharmacological activities as anti-oxidant \unskip~\citep{910209:20563684}.\textit{\space }

\textit{Portulaca oleracea L} extracts showed the highest amount of total phenolics and revealed significant anti-oxidant activities \unskip~\citep{910209:20563687} and are one of the richest green plant sources of omega-3 fatty acid \unskip~\citep{910209:20563691}. 


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\caption{\boldmath {Structure of DPPH}}
\label{f-9c1246bd3111}
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\caption{\boldmath {HPTLC Plate image under normal white light }}
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\caption{\boldmath {HPTLC Plate image under UV light at 254 nm}}
\label{f-813556b49f59}
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\caption{\boldmath {HPTLC Plate image under UV light at 366 nm}}
\label{f-256504dc0e5d}
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\caption{\boldmath {Densitogram at 254 nm}}
\label{f-579b921efd6e}
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\caption{\boldmath {Densitogram at 366 nm}}
\label{f-cd6d175feea1}
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\caption{\boldmath {Derivatized HPTLC Plate image in white light}}
\label{f-19bf3a8cffe8}
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\caption{\boldmath {Derivatized HPTLC Plate image at 366 nm}}
\label{f-6679e156fced}
\end{figure}
\egroup
Purslane is a nutritious vegetable used for human consumption \unskip~\citep{910209:20563688} and having anti oxidative property. Phytochemical investigations revealed that this plant has a wide range of secondary metabolites including alkaloids, terpenoids, flavonoids and organic acids \unskip~\citep{910209:20563685}. Anti-oxidants are the compounds that inhibit oxidation and oxidation is a chemical reaction that can produce free radicals \unskip~\citep{910209:20563681}, thereby leading to chain reactions that may damage the cells of organisms. Many anti-oxidants like thiols or ascorbic acid (vitamin C) terminate these chain reactions. To balance the oxidative stress, plants and animals maintain complex systems of overlapping anti-oxidants, such as glutathione and enzymes (e.g., catalase and superoxide dismutase), produced internally, or the dietary anti-oxidants vitamin C \& vitamin E \unskip~\citep{910209:20563683}.


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\caption{\boldmath {*Chromatograph A1, A2, A3, A4 and A5 obtained at 254 nm}}
\label{f-529405cd4179}
\end{figure*}
\egroup

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\caption{\boldmath {**Chromatograph B1, B2, B3, B4 and B5 obtained at 366 nm}}
\label{f-33035daa33c7}
\end{figure*}
\egroup

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\caption{\boldmath {Final result image of plate by TLC Visualizer}}
\label{f-ec4092e1aaf8}
\end{figure*}
\egroup
DPPH (2,2 diphenyl-1-picrylhydrazyl) is a free radical which produces a violet solution dissolved in Methanol \unskip~\citep{910209:20563693}. It is stable at room temperature and ambient environmental conditions. Anti-oxidants present in the extract reacts with the DPPH free radicals and breaks the chain reaction and further formation of free radicals \unskip~\citep{910209:20563692} this causes a colour change in DPPH to yellow. The formula of DPPH is C18H12N5O6 and its Molecular Weight is 394.32 g/mol. Structure of DPPH (Figure~\ref{f-9c1246bd3111}). High-Performance Thin Layer Chromatography (HPTLC) is a powerful and versatile chromatographic technique for the separation and analysis of natural products as compared to other techniques such as HPLC, spectrophotometry, titrimetric. HPTLC system of Camag brand consists of following components such as Plate Developing Chambers (Twin trough), TLC Applicator (Linomet 5), TLC plate heater, Derivatier Sprayer, Detection component (Scanner 4), Evaluation is done by software (visionCATS) and documentation by TLC Visualizer.


\begin{table*}[!htbp]
\caption{\boldmath {Evaluation result HPTLC Chromatograph are obtained at 254 nm} }
\label{tw-8e7a114c0798}
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\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.1137\linewidth-2\tabcolsep}p{\dimexpr.1325\linewidth-2\tabcolsep}p{\dimexpr.1075\linewidth-2\tabcolsep}p{\dimexpr.12010000000000002\linewidth-2\tabcolsep}p{\dimexpr.13129999999999997\linewidth-2\tabcolsep}p{\dimexpr.12520000000000001\linewidth-2\tabcolsep}p{\dimexpr.13360000000000001\linewidth-2\tabcolsep}p{\dimexpr.1361\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack * & \cAlignHack Volume & \cAlignHack Peak & \multicolumn{3}{p{\dimexpr(.37660000000000004\linewidth-2\tabcolsep)}}{\cAlignHack Max} & \multicolumn{2}{p{\dimexpr(.2697\linewidth-2\tabcolsep)}}{\cAlignHack Area}\\
\rowcolor{kwdboxcolor}\cAlignHack  & \cAlignHack  & \cAlignHack  & \cAlignHack Rf & \cAlignHack Height & \cAlignHack \% & \cAlignHack A & \cAlignHack \%\\
\tblmidrule 
\cAlignHack A1 &
  \cAlignHack 2.0 \ensuremath{\mu}l &
  \cAlignHack 1 &
  \cAlignHack 0.124 &
  \cAlignHack 0.0326 &
  \cAlignHack 22.66 &
  \cAlignHack 0.00103 &
  \cAlignHack 11.81\\
\cAlignHack A1 &
  \cAlignHack 2.0 \ensuremath{\mu}l &
  \cAlignHack 2 &
  \cAlignHack 0.876 &
  \cAlignHack 0.1112 &
  \cAlignHack 77.34 &
  \cAlignHack 0.00767 &
  \cAlignHack 88.19\\
\cAlignHack A2 &
  \cAlignHack 4.0 \ensuremath{\mu}l &
  \cAlignHack 1 &
  \cAlignHack 0.039 &
  \cAlignHack 0.0201 &
  \cAlignHack 7.50 &
  \cAlignHack 0.00036 &
  \cAlignHack 2.21\\
\cAlignHack A2 &
  \cAlignHack 4.0 \ensuremath{\mu}l &
  \cAlignHack 2 &
  \cAlignHack 0.124 &
  \cAlignHack 0.0503 &
  \cAlignHack 18.77 &
  \cAlignHack 0.00153 &
  \cAlignHack 9.45\\
\cAlignHack A2 &
  \cAlignHack 4.0 \ensuremath{\mu}l &
  \cAlignHack 3 &
  \cAlignHack 0.173 &
  \cAlignHack 0.0291 &
  \cAlignHack 10.85 &
  \cAlignHack 0.00125 &
  \cAlignHack 7.70\\
\cAlignHack A2 &
  \cAlignHack 4.0 \ensuremath{\mu}l &
  \cAlignHack 4 &
  \cAlignHack 0.381 &
  \cAlignHack 0.0153 &
  \cAlignHack 5.71 &
  \cAlignHack 0.00062 &
  \cAlignHack 3.82\\
\cAlignHack A2 &
  \cAlignHack 4.0 \ensuremath{\mu}l &
  \cAlignHack 5 &
  \cAlignHack 0.745 &
  \cAlignHack 0.0460 &
  \cAlignHack 17.15 &
  \cAlignHack 0.00277 &
  \cAlignHack 17.07\\
\cAlignHack A2 &
  \cAlignHack 4.0 \ensuremath{\mu}l &
  \cAlignHack 6 &
  \cAlignHack 0.887 &
  \cAlignHack 0.1073 &
  \cAlignHack 4.02 &
  \cAlignHack 0.00968 &
  \cAlignHack 5.75\\
\cAlignHack A3 &
  \cAlignHack 6.0 \ensuremath{\mu}l &
  \cAlignHack 1 &
  \cAlignHack 0.037 &
  \cAlignHack 0.0222 &
  \cAlignHack 6.76 &
  \cAlignHack 0.00045 &
  \cAlignHack 3.21\\
\cAlignHack A3 &
  \cAlignHack 6.0 \ensuremath{\mu}l &
  \cAlignHack 2 &
  \cAlignHack 0.126 &
  \cAlignHack 0.0699 &
  \cAlignHack 21.28 &
  \cAlignHack 0.00213 &
  \cAlignHack 15.11\\
\cAlignHack A3 &
  \cAlignHack 6.0 \ensuremath{\mu}l &
  \cAlignHack 3 &
  \cAlignHack 0.174 &
  \cAlignHack 0.0417 &
  \cAlignHack 12.70 &
  \cAlignHack 0.00173 &
  \cAlignHack 12.26\\
\cAlignHack A3 &
  \cAlignHack 6.0 \ensuremath{\mu}l &
  \cAlignHack 4 &
  \cAlignHack 0.385 &
  \cAlignHack 0.0229 &
  \cAlignHack 6.98 &
  \cAlignHack 0.00093 &
  \cAlignHack 6.59\\
\cAlignHack A3 &
  \cAlignHack 6.0 \ensuremath{\mu}l &
  \cAlignHack 5 &
  \cAlignHack 0.750 &
  \cAlignHack 0.0526 &
  \cAlignHack 16.02 &
  \cAlignHack 0.00321 &
  \cAlignHack 22.78\\
\cAlignHack A3 &
  \cAlignHack 6.0 \ensuremath{\mu}l &
  \cAlignHack 6 &
  \cAlignHack 0.935 &
  \cAlignHack 0.1190 &
  \cAlignHack 36.24 &
  \cAlignHack 0.00564 &
  \cAlignHack 40.05\\
\cAlignHack A4 &
  \cAlignHack 8.0 \ensuremath{\mu}l &
  \cAlignHack 1 &
  \cAlignHack 0.039 &
  \cAlignHack 0.0272 &
  \cAlignHack 7.13 &
  \cAlignHack 0.00053 &
  \cAlignHack 3.18\\
\cAlignHack A4 &
  \cAlignHack 8.0 \ensuremath{\mu}l &
  \cAlignHack 2 &
  \cAlignHack 0.126 &
  \cAlignHack 0.0832 &
  \cAlignHack 21.83 &
  \cAlignHack 0.00274 &
  \cAlignHack 16.52\\
\cAlignHack A4 &
  \cAlignHack 8.0 \ensuremath{\mu}l &
  \cAlignHack 3 &
  \cAlignHack 0.177 &
  \cAlignHack 0.0526 &
  \cAlignHack 13.81 &
  \cAlignHack 0.00217 &
  \cAlignHack 13.06\\
\cAlignHack A4 &
  \cAlignHack 8.0 \ensuremath{\mu}l &
  \cAlignHack 4 &
  \cAlignHack 0.384 &
  \cAlignHack 0.0284 &
  \cAlignHack 7.45 &
  \cAlignHack 0.00118 &
  \cAlignHack 7.09\\
\cAlignHack A4 &
  \cAlignHack 8.0 \ensuremath{\mu}l &
  \cAlignHack 5 &
  \cAlignHack 0.753 &
  \cAlignHack 0.0565 &
  \cAlignHack 14.82 &
  \cAlignHack 0.00318 &
  \cAlignHack 19.17\\
\cAlignHack A4 &
  \cAlignHack 8.0 \ensuremath{\mu}l &
  \cAlignHack 6 &
  \cAlignHack 0.939 &
  \cAlignHack 0.1332 &
  \cAlignHack 34.95 &
  \cAlignHack 0.00680 &
  \cAlignHack 40.97\\
\cAlignHack A5 &
  \cAlignHack 10.0 \ensuremath{\mu}l &
  \cAlignHack 1 &
  \cAlignHack 0.039 &
  \cAlignHack 0.0324 &
  \cAlignHack 7.38 &
  \cAlignHack 0.00070 &
  \cAlignHack 3.50\\
\cAlignHack A5 &
  \cAlignHack 10.0 \ensuremath{\mu}l &
  \cAlignHack 2 &
  \cAlignHack 0.129 &
  \cAlignHack 0.0937 &
  \cAlignHack 21.33 &
  \cAlignHack 0.00320 &
  \cAlignHack 16.08\\
\cAlignHack A5 &
  \cAlignHack 10.0 \ensuremath{\mu}l &
  \cAlignHack 3 &
  \cAlignHack 0.181 &
  \cAlignHack 0.0610 &
  \cAlignHack 13.89 &
  \cAlignHack 0.00242 &
  \cAlignHack 12.15\\
\cAlignHack A5 &
  \cAlignHack 10.0 \ensuremath{\mu}l &
  \cAlignHack 4 &
  \cAlignHack 0.389 &
  \cAlignHack 0.0338 &
  \cAlignHack 7.70 &
  \cAlignHack 0.00142 &
  \cAlignHack 7.15\\
\cAlignHack A5 &
  \cAlignHack 10.0 \ensuremath{\mu}l &
  \cAlignHack 5 &
  \cAlignHack 0.758 &
  \cAlignHack 0.0633 &
  \cAlignHack 14.42 &
  \cAlignHack 0.00368 &
  \cAlignHack 18.53\\
\cAlignHack A5 &
  \cAlignHack 10.0 \ensuremath{\mu}l &
  \cAlignHack 6 &
  \cAlignHack 0.944 &
  \cAlignHack 0.1549 &
  \cAlignHack 35.28 &
  \cAlignHack 0.00847 &
  \cAlignHack 42.61\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Evaluation result HPTLC Chromatographs are obtained at 366 nm} }
\label{tw-c3c5e1d99c46}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.125\linewidth-2\tabcolsep}p{\dimexpr.125\linewidth-2\tabcolsep}p{\dimexpr.125\linewidth-2\tabcolsep}p{\dimexpr.12500000000000004\linewidth-2\tabcolsep}p{\dimexpr.12190000000000005\linewidth-2\tabcolsep}p{\dimexpr.12085000000000003\linewidth-2\tabcolsep}p{\dimexpr.13330000000000007\linewidth-2\tabcolsep}p{\dimexpr.12395000000000005\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack ** & \cAlignHack Volume & \cAlignHack Peak & \multicolumn{3}{p{\dimexpr(.3677500000000001\linewidth-2\tabcolsep)}}{\cAlignHack Max} & \multicolumn{2}{p{\dimexpr(.25725000000000012\linewidth-2\tabcolsep)}}{\cAlignHack Area}\\
\rowcolor{kwdboxcolor}\cAlignHack  & \cAlignHack  & \cAlignHack  & \cAlignHack Rf & \cAlignHack Height & \cAlignHack \% & \cAlignHack A & \cAlignHack \%\\
\tblmidrule 
\cAlignHack B1 &
  \cAlignHack 2.0 \ensuremath{\mu}l &
  \cAlignHack 1 &
  \cAlignHack 0.827 &
  \cAlignHack 0.1072 &
  \cAlignHack 28.53 &
  \cAlignHack 0.00450 &
  \cAlignHack 30.49\\
\cAlignHack B1 &
  \cAlignHack 2.0 \ensuremath{\mu}l &
  \cAlignHack 2 &
  \cAlignHack 0.933 &
  \cAlignHack 0.2685 &
  \cAlignHack 71.47 &
  \cAlignHack 0.01025 &
  \cAlignHack 69.51\\
\cAlignHack B2 &
  \cAlignHack 4.0 \ensuremath{\mu}l &
  \cAlignHack 1 &
  \cAlignHack 0.831 &
  \cAlignHack 0.2239 &
  \cAlignHack 35.97 &
  \cAlignHack 0.01148 &
  \cAlignHack 40.74\\
\cAlignHack B2 &
  \cAlignHack 4.0 \ensuremath{\mu}l &
  \cAlignHack 2 &
  \cAlignHack 0.940 &
  \cAlignHack 0.3985 &
  \cAlignHack 64.03 &
  \cAlignHack 0.01670 &
  \cAlignHack 59.26\\
\cAlignHack B3 &
  \cAlignHack 6.0 \ensuremath{\mu}l &
  \cAlignHack 1 &
  \cAlignHack 0.064 &
  \cAlignHack 0.0175 &
  \cAlignHack 2.19 &
  \cAlignHack 0.00046 &
  \cAlignHack 1.22\\
\cAlignHack B3 &
  \cAlignHack 6.0 \ensuremath{\mu}l &
  \cAlignHack 2 &
  \cAlignHack 0.260 &
  \cAlignHack 0.0141 &
  \cAlignHack 1.77 &
  \cAlignHack 0.00039 &
  \cAlignHack 1.04\\
\cAlignHack B3 &
  \cAlignHack 6.0 \ensuremath{\mu}l &
  \cAlignHack 3 &
  \cAlignHack 0.836 &
  \cAlignHack 0.3062 &
  \cAlignHack 38.26 &
  \cAlignHack 0.01653 &
  \cAlignHack 44.23\\
\cAlignHack B3 &
  \cAlignHack 6.0 \ensuremath{\mu}l &
  \cAlignHack 4 &
  \cAlignHack 0.948 &
  \cAlignHack 0.4625 &
  \cAlignHack 57.79 &
  \cAlignHack 0.02000 &
  \cAlignHack 53.50\\
\cAlignHack B4 &
  \cAlignHack 8.0 \ensuremath{\mu}l &
  \cAlignHack 1 &
  \cAlignHack 0.067 &
  \cAlignHack 0.0191 &
  \cAlignHack 2.15 &
  \cAlignHack 0.00042 &
  \cAlignHack 0.97\\
\cAlignHack B4 &
  \cAlignHack 8.0 \ensuremath{\mu}l &
  \cAlignHack 2 &
  \cAlignHack 0.840 &
  \cAlignHack 0.3647 &
  \cAlignHack 41.03 &
  \cAlignHack 0.01978 &
  \cAlignHack 46.29\\
\cAlignHack B4 &
  \cAlignHack 8.0 \ensuremath{\mu}l &
  \cAlignHack 3 &
  \cAlignHack 0.953 &
  \cAlignHack 0.5050 &
  \cAlignHack 56.81 &
  \cAlignHack 0.02253 &
  \cAlignHack 52.74\\
\cAlignHack B5 &
  \cAlignHack 10.0 \ensuremath{\mu}l &
  \cAlignHack 1 &
  \cAlignHack 0.067 &
  \cAlignHack 0.0230 &
  \cAlignHack 2.31 &
  \cAlignHack 0.00056 &
  \cAlignHack 1.15\\
\cAlignHack B5 &
  \cAlignHack 10.0 \ensuremath{\mu}l &
  \cAlignHack 2 &
  \cAlignHack 0.265 &
  \cAlignHack 0.0199 &
  \cAlignHack 1.99 &
  \cAlignHack 0.00059 &
  \cAlignHack 1.23\\
\cAlignHack B5 &
  \cAlignHack 10.0 \ensuremath{\mu}l &
  \cAlignHack 3 &
  \cAlignHack 0.844 &
  \cAlignHack 0.4033 &
  \cAlignHack 40.48 &
  \cAlignHack 0.02209 &
  \cAlignHack 45.89\\
\cAlignHack B5 &
  \cAlignHack 10.0 \ensuremath{\mu}l &
  \cAlignHack 4 &
  \cAlignHack 0.958 &
  \cAlignHack 0.5501 &
  \cAlignHack 55.22 &
  \cAlignHack 0.02489 &
  \cAlignHack 51.72\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Result Analysis} }
\label{tw-54347036e6e9}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.3777\linewidth-2\tabcolsep}p{\dimexpr.3093\linewidth-2\tabcolsep}p{\dimexpr.313\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack Rf Value & \cAlignHack Colour  & \cAlignHack Intensity \\
\tblmidrule 
\cAlignHack 0.87 &
  \cAlignHack Yellow  &
  \cAlignHack High \\
\cAlignHack 0.78 &
  \cAlignHack Yellow  &
  \cAlignHack High \\
\cAlignHack 0.18 &
  \cAlignHack Yellow  &
  \cAlignHack Low \\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

    
\section{Materials and Methods}
The whole plant of \textit{Portulaca oleracea } along with leaves on which study is performed was authenticated by Raw Materials Herbarium \& Museum, Delhi (RHMD), National Institute of Science Communication \& Information Resources (CSIR-NISCAIR) New Delhi and requisite certificates are obtained.

DPPH reagent (2,2-Diphenyl-1picrylhydrazyl), Ethanol, Camag HP-TLC System comprising vision CATS- Software, Linomat 5, Twin Trough Chamber, Visualizer, Scanner4, Dipping Chamber, Centrifuge machine, Ultrasonic bath, Silica gel TLC plates, Ethyl Acetate, Methyl ethyl ketone, Formic Acid, Distilled Water

\textbf{Reagent Preparation}

Exactly 7.89 mg of DPPH reagent is weighed by analytical balance and diluted to 100 ml by 99.5 \% ethanol. The concentration of this solution is 0.2 mM. It is kept in an amber-coloured bottle which is then wrapped with aluminium foil as DPPH activity is light sensitive.

\textbf{Sample preparation}

One gram of fine powder made from dried leaves of \textit{Portulaca oleracea } (Purslane) is precisely weighed and dissolved in 10 ml of Methanol and then kept in Ultrasonic bath for 15 minutes after its supernatant is taken with the help of a pipette. After that, it is centrifuged at 3000 rpm for 10 minutes in a centrifuge machine, and the final extract is taken for the analysis. 
    
\section{\textbf{PROCEDURE}}
The study is performed in a dark environment only as the DPPH activity is light sensitive, DPPH reagent is dissolved in ethanol and dissolved. After application of the sample on stationary phase HPTLC plates silica gel 60 F 254 of size 10 cm x10 cm the and standard are run in the mobile phase which contains Ethyl Acetate: Methyl ethyl ketone: Formic Acid: Water in a ratio of 5:3:1:1 volume/volume. 

After the development of standard and sample, the plate is then derivatized with DPPH reagent. A working program is prepared in visionCATS software. 

A specific volume of sample 2\ensuremath{\mu}l, 4\ensuremath{\mu}l, 6\ensuremath{\mu}l, 8\ensuremath{\mu}l and 10\ensuremath{\mu}l is taken by using 100 \ensuremath{\mu}l syringe and applied through sample applicator. 

After application, the plate is air-dried for 10 minutes. For the development of chromatogram, a filter paper lined in the Twin Trough Chamber for uniform saturation chamber is used. 

Chamber saturation is done for 20 minutes (according to USP chapter 202). Then the applied plate is placed in a twin trough chamber layer facing towards the paper. The plate is a run-up to the marked position, i.e. 70 mm and then removed and air-dried for 5 minutes. 

The dip tank is used for derivatization with DPPH reagent. It is kept in the dark place for 10 min for development of proper intensity of colour. Then images are taken in visible light if anti-oxidant is present it will show the yellow colour spot. 
    
\section{Results and Discussion}
Images of HPTLC plate after application of sample of methanolic extract applied in a volume of by microsyringe in 2\ensuremath{\mu}l, 4\ensuremath{\mu}l, 6\ensuremath{\mu}l, 8\ensuremath{\mu}l and 10\ensuremath{\mu}l concentrations is obtained under normal white light (Figure~\ref{f-6a314163a39e}), under U.V. light at 254 nm (Figure~\ref{f-813556b49f59}) and when under U.V. light at 366 nm (Figure~\ref{f-256504dc0e5d}). 

First Densitogram is obtained by keeping the developed HPTLC plate in TLC Scanner and vision CATS software with settings at wavelength 254 nm and other as under:- 

Scanner type used as Single \ensuremath{\lambda }; Optimization - Resolution; Measurement mode {\textendash}absorption; 

Detector mode {\textendash} Automatic ; Scanning speed - 20 mm/s; Data resolution 100 \ensuremath{\mu }m/step ; Slit 6 x 0.45 mm, micro; lamp- Deuterium Lamp (Figure~\ref{f-579b921efd6e}).  

Second Densitogram is obtained by keeping the developed HPTLC plates in TLC Scanner and vision CATS software with settings at wavelength 366 nm and other as under, Scanner type used as Single \ensuremath{\lambda } ; Optimization - Resolution ; Filter {\textendash} K 400 ; Measurement mode{\textendash}Fluorescence; Detector mode {\textendash} Automatic ; Scanning speed - 20 mm/s; Data resolution 25 \ensuremath{\mu }m/step ; Slit 6 x 0.45 mm, micro; lamp- Mercury Lamp (Figure~\ref{f-cd6d175feea1}).  

The developed plate is then derivatized by using freshly prepared DPPH reagents and images are observed under white light (Figure~\ref{f-19bf3a8cffe8}) and at 366 nm (Figure~\ref{f-6679e156fced}). 

\textbf{Evaluation of results}

Area, Area \% and Rf values of the peaks detected during densitometry analysis of extract are given in Table~\ref{tw-8e7a114c0798} for five chromatographs are obtained at 254 nm on applying 2\ensuremath{\mu}l, 4\ensuremath{\mu}l, 6\ensuremath{\mu}l, 8\ensuremath{\mu}l and 10\ensuremath{\mu}l consequently. (Figure~\ref{f-529405cd4179}) and in Table~\ref{tw-c3c5e1d99c46} for the other five chromatographs are obtained at 366 nm on applying the same concentrations as above consequently (Figure~\ref{f-33035daa33c7}). TLC visualize used for imaging and documentation in which yellow colour bands can be seen at 0.18, 0.78 and 0.87 Rf values (Figure~\ref{f-ec4092e1aaf8}), and detailed results can be seen as in Table~\ref{tw-54347036e6e9}. 
    
\section{Conclusion }
This study concludes that when five tracks of different concentrations, i.e. 2\ensuremath{\mu}l,4 \ensuremath{\mu}l,6 \ensuremath{\mu}l, 8 \ensuremath{\mu}l and 10 \ensuremath{\mu}l were applied and studied at 254 nm and 366 nm and again studied at after derivatization by DPPH reagent, it is found that the yellow colour band present at Rf 0.18, Rf 0.78 and Rf 0.87 shows the presence of anti-oxidant activity present in the methanolic extract of \textit{Portulaca oleracea} dried leaves. 
\section*{Acknowledgement}Manipal University Jaipur for providing central lab facility and resources to conduct this study and M/S Anchrom Enterprises (India) Pvt Ltd, Mumbai for providing their Camag HPTLC application lab facilities and assistance to conduct this study. 

\textbf{Funding Support}

The authors declare that they have no funding support for this study.

\textbf{Conflict of Interest}

The authors declare that they have no conflict of interest for this study.
    

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