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\def\RunningAuthor{Vijey Aanandhi M and Ganesh S}
\firstPage{1396}
\articleType{Original Article}
\receivedDate{25 Feb 2021}
\acceptedDate{30 Mar 2021}
\revisedDate{28 Mar 2021}
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\title{In-vitro Cytotoxic activity of Papaverine compound isolated from Aqueous Ethanolic leaf extract of \textit{Erythrina subumbrans }(Hassk.) Merr.}
\author{Ganesh~S\textsuperscript{1}, 
            Vijey~Aanandhi~M\textsuperscript{*}\textsuperscript{2}~\\[5pt]{\textsuperscript{1}Research Scholar, Department of Pharmaceutical Chemistry and Analysis, School of Pharmaceutical Sciences, Vels Institute of Science, Technology and Advanced Studies (VISTAS), Pallavaram, Chennai {\textendash} 600 117, Tamil Nadu, India}~\\{\textsuperscript{2}Department of Pharmaceutical Chemistry and Analysis, School of Pharmaceutical Sciences, Vels Institute of Science, Technology and Advanced Studies (VISTAS), Pallavaram, Chennai {\textendash} 600 117, Tamil Nadu, India}}

\begin{abstract}
Natural compounds that occur either in pure form or as extract after standardization from medicinal plants, provide a limitless chance for new discovery leads because of consummate chemical diversity. The rising demand for compounds that occur naturally during screening studies, that has therapeutic action predominantly on the vegetative part of the plant has grown across the world. In this study, Papaverine compound was isolated from aqueous Ethanolic extract (80\%) from vegetative (leaf) part of herbal plant \textit{Erythrina subumbrans} (Hassk.)  Merr. Isolated compound was subjected for characterization studies like Thin layer chromatography (TLC), Mass spectrophotometry (MS), Fourier Transform Infra-Red (FTIR), \ensuremath{^{1}}H and \ensuremath{^{13}}C Nuclear magnetic resonance spectroscopy. Confirmatory study using the aqueous ethanolic crude extract by MS technique and mass library match confirms the presence of Papaverine compound. Further, in-vitro cytotoxic activity using Human Melanoma cell line (A375) was carried out using the isolated compound (papaverine), Aqueous ethanolic extract and standard Cisplatin. Six different Concentration ranging from 3.125 to 100 \ensuremath{\mu}g/ml were prepared. Control samples were prepared without standard and samples solution. Absorbance measured at 570 nm in UV-Vis spectrophotometer using a microplate reader and the \% of viability was calculated. IC\ensuremath{_{50}} value of Aqueous ethanolic extract of \textit{Erythrina subumbrans}, isolated compound-Papaverine and standard Cisplatin exhibits value of 82.14\ensuremath{\mu}g, 23.43 \ensuremath{\mu}g \& 4.02 \ensuremath{\mu}g respectively.  
\end{abstract}\def\keywordstitle{Keywords}

\begin{keywords}Erythrina subumbrans (Hassk.) Merr.,\newline Papaverine,\newline Human Melanoma Cell Line (A375),\newline Cisplatin
\end{keywords}
\twocolumn[ \maketitle {\printKwdAbsBox}]
\makeatletter{\raggedright\textsuperscript{*}Corresponding Author\par Name:\ Vijey~Aanandhi~M~\\ Phone:\ 9840956519~\\  Email:\ hodpchemistry@velsuniv.ac.in
        \par}\vspace*{-11pt}\hrulefill\par{\fontsize{12}{14}\selectfont ISSN: 0975-7538}\par% 
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\section{Introduction}
Plants are the vital source for the presence of numerous naturally occurring compounds, such as extract from plants either in pure from or as extract after standardization from medicinal plants, provide a limitless chance for new discovery leads because of consummate chemical diversity \unskip~\citep{1061695:21888933}. Per statement by WHO (World Health Organization), greater than 80 \% of the world population trusts on traditional medication system for the needs of primary healthcare system. The herbal plant selected for traditional purpose contains natural occurring substance which can be used to treat infectious disease as well as chronic disease. The dependency on Ethanopharamacognosy were grown significantly due to severe adverse reaction on usage of chemically synthesized drug and resistance by microorganism after prolonged usage of drug. Based on that, peoples found chemical constituents from plants are found to be safe and alternatively effective against broad spectrum of microorganism with less adverse reactions. Many biological activities that are beneficial were reported like analgesic, Antimicrobial, Antidiarrheal, Anthelmintic, wound healing activity and anticancer activity. Many cases were reported to believe good benefit on usage of herbal products. However, scientific Trials are required to describe the bioavailability, safety, efficacy and pharma kinetic mechanism of the herbal product. Clinical Trials are conducted to safeguard the participants health as well as evaluating the long term and immediate side effects and their outcomes are consistently measured before the drugs are distributed the wider group \unskip~\citep{1061695:21888925}. 

After cardiac disorder, cancer represents second position for the maximum number of deaths in the world \unskip~\citep{1061695:21888930}. About 6 \% of all death in India were due to cancer as reported by the International Agency for Research on Cancer in 2008, reported about 8 \% of the cancer deaths globally \unskip~\citep{1061695:21888927}. Regardless of current advancement on usage of cancer therapies includes radiation and chemotherapy, the mortality rate stood high in terms of cancer disease \unskip~\citep{1061695:21888926}. This is due to the adverse side effects associated with cancer therapies \unskip~\citep{1061695:21888936}. The search for Tumor cell therapy using the natural products were grown significantly, in which using the plant source are the greatest area of interest. Although, there were ethnobotanical bibliography that were published locally for the treatment of cancer, there were very few experiments were establish the presence of cytotoxic activity of the plants. The vital screening data for the possible antineoplastic principles of the selected plants species were assessed through cytotoxic screening studies \unskip~\citep{1061695:21888935}. This research experiment was carried out to evaluate the cytotoxic activity of aqueous ethanolic extract vegetative (leaf) part of \textit{Erythrina subumbrans (Hassk.)} Merr. along with the isolated compound and standard cisplatin for the traditional use in treatment of skin cancer. 
    
\section{Materials and Methods}
\textbf{Material}

Chemical such as Cisplatin, Dimethyl sulfoxide (DMSO), Ethanol, Methanol, Silica gel, Hexane, Ethyl acetate, chloroform and Iodine reagent were purchased from SISCO research laboratories Ltd, Maharashtra, India. Other chemicals procured from local manufactures (Analytical reagent grade).

\textbf{Plant material}

The leaves of Erythrina subumbrans (Hassk.) Merr were collected from Western Ghats of India and authenticated at Siddha central research institute (Ministry of AYUSH, Government of India), Chennai by Research Officer and Head of pharmacognosy department Dr K.N. Sunil Kumar and confirmed by Assistant Director in-charge Dr P. Sathiyarajeswaran (Authentication certificate 112.04011901 dated 04 Apr 2019). The harvested vegetative part was dried in a nominal condition and stored in container made of Kraft paper. 

\textbf{Preparation of plant Extract}

The plant material was washed with distilled water for several times and was subjected to air drying under the shade. After drying, they were ground by an electric mixer until they became a powder. Then the powdered samples were stored in a dark place and subjected to extraction method \unskip~\citep{1061695:21888932}. Extraction of powdered samples was done using aqueous ethanol (80\%) in 1000ml for 72 hours. Sample mixtures were filtered and concentrated under reduced pressure using a rotary evaporator and keep stored at refrigerator storage condition (2-8\ensuremath{^\circ} C). 

\textbf{Procedure for Isolation of compounds}

Eight (8) gram of the aqueous ethanolic extract was dissolved in methanol and adsorbed in silica gel 100 {\textendash} 200 mesh size. After evaporation of the solvent, it was made into a fine powder. For column packing, Dry packing method was followed. Powdered extract was loaded into silica gel column (100 {\textendash} 200 mesh size) (Figure~\ref{f-a2fe506c857d}). The column was prepared by keeping the cotton at the bottom followed by 2-4 gm of silica gel, followed by powdered extract then silica gel about 2-4 gm followed by cotton at the top. About 200-300mL of Hexane was added to wet the column and ensured for the absence of cracking of column bed. The column was eluted initially with 100\% hexane followed by gradually increasing polarity with Hexane: Ethyl acetate (90:10; 80:20; 70:30; 60:40; 50:50; 40:60; 30:70; 20:80) and finally with 100\% Ethyl acetate. The column was further eluted with Ethylacetate: Chloroform (90:10; 80:20; 70:30; 60:40; 50:50; 40:60; 30:70; 20:80) and finally with 100\% chloroform. The column was further eluted with Chloroform: Methanol (98:2; 96:4; 94:2; 92:8; 90:10; 85:15; 80:20). Total 124 Fractions were collected and monitored under TLC. 

\textbf{Cytotoxicity Assay by MTT Method}

The MTT system meant for measuring the activity of cells (i.e., live cells) via mitochondrial dehydrogenases. The key component is (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide) or MTT. Dissolved MTT is converted to an insoluble purple formazan by cleavage of the tetrazolium ring by mitochondrial dehydrogenase enzymes of viable cells. Using DMSO, insoluble formazan can be solubilized. The resulting colored solution (purple) is spectrophotometrically measured. Degree of cytotoxicity caused by the test material can be measured by the change in amount of formazan formed with can be differentiated by either decrease or increase in cell number \unskip~\citep{1061695:21888934}. 

Human melanoma cell line (A375) was procured from NCCS, stock cells were cultured in medium supplemented with 10\% inactivated Fetal Bovine Serum (FBS) in a humidified atmosphere of 5\% CO\ensuremath{_{2}} at 37\ensuremath{^\circ}C until confluent \unskip~\citep{1061695:21888924}. 

The monolayer cells were trypsinized with TPVG solution (0.2 \% trypsin, 0.02 \% EDTA, 0.05 \% glucose in PBS) and the cell count was adjusted to 1.0 x 105 cells/ml using respective media containing 10\% FBS. To each well of the 96 well microtiter plate, 100\ensuremath{\mu }l of the diluted cell suspension (50,000cells/well) was added \unskip~\citep{1061695:21888929}. After 24 h, when a partial monolayer was formed, the supernatant was flicked off, washed the monolayer once with medium and 100\ensuremath{\mu }l of different test concentrations of test drugs were added on to the partial monolayer in microtiter plates. The plate was then incubated at 37\ensuremath{^\circ}C for 24hrs in 5\% CO\ensuremath{_{2}} atmosphere. After incubation the test solutions in the wells were discarded and 100\ensuremath{\mu }l of MTT (5 mg/10 ml of MTT in PBS) was added to each well. The plate was incubated for 4h at 37\ensuremath{^\circ}C in 5\% CO\ensuremath{_{2}} atmosphere. The supernatant was removed and 100\ensuremath{\mu }l of DMSO was added and the plate was gently shaken to solubilize the formed formazan \unskip~\citep{1061695:21888931}. The absorbance was measured using a microplate reader at a wavelength of 570 nm \unskip~\citep{1061695:21888928}.
    
\section{Results}
\textbf{Plant Extract}

The percentage yield of the ethanolic extracts was calculated by using the following equation: yield (\%) = (weight of extract/weight of dried plant material) x 100 and the obtained yield was presented in Table~\ref{tw-6aa8309b0c8d}.


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\caption{\boldmath {Thin layer chromatography of isolated compound}}
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\caption{\boldmath {Isolated compound-Papaverine}}
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\caption{\boldmath {Cytotoxicity assay {\textendash} A375 cell line (Ethanolic extract)}}
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\fixFloatSize{images/0a9bacce-efbf-41e3-96bc-f18504168c3a-upicture6.png}
\begin{figure*}[!htbp]
\centering \makeatletter\IfFileExists{images/0a9bacce-efbf-41e3-96bc-f18504168c3a-upicture6.png}{\includegraphics{images/0a9bacce-efbf-41e3-96bc-f18504168c3a-upicture6.png}}{}
\makeatother 
\caption{\boldmath {Cytotoxicity assay {\textendash} A375 cell line (Standard-Cisplatin)}}
\label{f-d8eeffc8077e}
\end{figure*}
\egroup

\begin{table*}[!htbp]
\caption{\boldmath {Extract Yield values} }
\label{tw-6aa8309b0c8d}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.32260000000000005\linewidth-2\tabcolsep}p{\dimexpr.20439999999999998\linewidth-2\tabcolsep}p{\dimexpr.2168\linewidth-2\tabcolsep}p{\dimexpr.2562\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack Solvent system & \cAlignHack Powder weight (g)  & \cAlignHack Extract weight (g)  & \cAlignHack Extract Yield (\%) \\
\tblmidrule 
\cAlignHack Aqueous ethanol (80\%) &
  \cAlignHack 200 g &
  \cAlignHack 8.26 g &
  \cAlignHack 4.13 \%\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Thin layer chromatography-R\ensuremath{_{f}} value} }
\label{tw-151015c713b3}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.25210000000000004\linewidth-2\tabcolsep}p{\dimexpr.31639999999999993\linewidth-2\tabcolsep}p{\dimexpr.20639999999999997\linewidth-2\tabcolsep}p{\dimexpr.22509999999999998\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack Sample & \cAlignHack Solvent system & \cAlignHack Number of spots & \cAlignHack Rf value\\
\tblmidrule 
\cAlignHack Isolated compound &
  \cAlignHack Chloroform: Ethanol (90:10) &
  \cAlignHack 1 &
  \cAlignHack 0.52\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Cytotoxicity assay -Ethanolic extract} }
\label{tw-978700f3b80d}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.1294\linewidth-2\tabcolsep}p{\dimexpr.1081\linewidth-2\tabcolsep}p{\dimexpr.0925\linewidth-2\tabcolsep}p{\dimexpr.11\linewidth-2\tabcolsep}p{\dimexpr.11\linewidth-2\tabcolsep}p{\dimexpr.1165\linewidth-2\tabcolsep}p{\dimexpr.11459999999999999\linewidth-2\tabcolsep}p{\dimexpr.1081\linewidth-2\tabcolsep}p{\dimexpr.1108\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack Extract &
  \cAlignHack Blank &
  \cAlignHack Control &
  \multicolumn{6}{p{\dimexpr(.67\linewidth-2\tabcolsep)}}{\cAlignHack Concentration Unit: \ensuremath{\mu}g}\\
\rowcolor{kwdboxcolor}\cAlignHack  &
  \cAlignHack  &
  \cAlignHack  &
  \cAlignHack 3.12 &
  \cAlignHack 6.25 &
  \cAlignHack 12.5 &
  \cAlignHack 25 &
  \cAlignHack 50 &
  \cAlignHack 100\\
\tblmidrule 
\cAlignHack Reading 1 &
  \cAlignHack 0.025 &
  \cAlignHack 1.227  &
  \cAlignHack 1.220  &
  \cAlignHack 1.204 &
  \cAlignHack 1.163 &
  \cAlignHack 1.111 &
  \cAlignHack 0.881 &
  \cAlignHack 0.483\\
\cAlignHack Reading 2 &
  \cAlignHack 0.023 &
  \cAlignHack 1.223 &
  \cAlignHack 1.214 &
  \cAlignHack 1.202 &
  \cAlignHack 1.155 &
  \cAlignHack 1.106 &
  \cAlignHack 0.877 &
  \cAlignHack 0.478\\
\cAlignHack Reading 3 &
  \cAlignHack 0.023 &
  \cAlignHack 1.234 &
  \cAlignHack 1.217 &
  \cAlignHack 1.200 &
  \cAlignHack 1.159 &
  \cAlignHack 1.102 &
  \cAlignHack 0.877 &
  \cAlignHack 0.472\\
\cAlignHack Mean &
  \cAlignHack 0.024 &
  \cAlignHack 1.228 &
  \cAlignHack 1.217 &
  \cAlignHack 1.202 &
  \cAlignHack 1.159 &
  \cAlignHack 1.106 &
  \cAlignHack 0.878 &
  \cAlignHack 0.478\\
\multicolumn{2}{p{\dimexpr(.2375\linewidth-2\tabcolsep)}}{\cAlignHack Mean OD-Mean B} &
  \cAlignHack 1.204 &
  \cAlignHack 1.193 &
  \cAlignHack 1.178 &
  \cAlignHack 1.135 &
  \cAlignHack 1.083 &
  \cAlignHack 0.855 &
  \cAlignHack 0.454\\
\multicolumn{2}{p{\dimexpr(.2375\linewidth-2\tabcolsep)}}{\cAlignHack Standard Dev.} &
  \cAlignHack 0.006 &
  \cAlignHack 0.003 &
  \cAlignHack 0.002 &
  \cAlignHack 0.004 &
  \cAlignHack 0.005 &
  \cAlignHack 0.002 &
  \cAlignHack 0.006\\
\multicolumn{2}{p{\dimexpr(.2375\linewidth-2\tabcolsep)}}{\cAlignHack Viability in \%} &
  \cAlignHack 100.000 &
  \cAlignHack 99.087 &
  \cAlignHack 97.841 &
  \cAlignHack 94.271 &
  \cAlignHack 89.898 &
  \cAlignHack 70.966 &
  \cAlignHack 37.697\\
\multicolumn{9}{p{\dimexpr(1\linewidth-2\tabcolsep)}}{\rAlignHack IC\ensuremath{_{50}} value: 82.14}\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Cytotoxicity assay -Isolated compound (Papaverine)} }
\label{tw-5926648e67bb}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.21689999999999998\linewidth-2\tabcolsep}p{\dimexpr.0703\linewidth-2\tabcolsep}p{\dimexpr.09179999999999996\linewidth-2\tabcolsep}p{\dimexpr.09510000000000002\linewidth-2\tabcolsep}p{\dimexpr.1045\linewidth-2\tabcolsep}p{\dimexpr.0998\linewidth-2\tabcolsep}p{\dimexpr.1008\linewidth-2\tabcolsep}p{\dimexpr.1073\linewidth-2\tabcolsep}p{\dimexpr.1135\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack Isolated Compound & \cAlignHack Blank & \cAlignHack Control & \multicolumn{6}{p{\dimexpr(.621\linewidth-2\tabcolsep)}}{\cAlignHack Concentration Unit: \ensuremath{\mu}g}\\
\rowcolor{kwdboxcolor}\cAlignHack  & \cAlignHack  & \cAlignHack  & \cAlignHack 3.12 & \cAlignHack 6.25 & \cAlignHack 12.5 & \cAlignHack 25 & \cAlignHack 50 & \cAlignHack 100\\
\tblmidrule 
\cAlignHack Reading 1 &
  \cAlignHack 0.025 &
  \cAlignHack 1.227  &
  \cAlignHack 1.187 &
  \cAlignHack 1.123 &
  \cAlignHack 1.002 &
  \cAlignHack 0.691 &
  \cAlignHack 0.284 &
  \cAlignHack 0.116\\
\cAlignHack Reading 2 &
  \cAlignHack 0.023 &
  \cAlignHack 1.223 &
  \cAlignHack 1.188 &
  \cAlignHack 1.129 &
  \cAlignHack 1.017 &
  \cAlignHack 0.688 &
  \cAlignHack 0.278 &
  \cAlignHack 0.118\\
\cAlignHack Reading 3 &
  \cAlignHack 0.023 &
  \cAlignHack 1.234 &
  \cAlignHack 1.184 &
  \cAlignHack 1.128 &
  \cAlignHack 1.016 &
  \cAlignHack 0.684 &
  \cAlignHack 0.288 &
  \cAlignHack 0.112\\
\cAlignHack Mean &
  \cAlignHack 0.024 &
  \cAlignHack 1.228 &
  \cAlignHack 1.186 &
  \cAlignHack 1.127 &
  \cAlignHack 1.012 &
  \cAlignHack 0.688 &
  \cAlignHack 0.283 &
  \cAlignHack 0.115\\
\multicolumn{2}{p{\dimexpr(.2872\linewidth-2\tabcolsep)}}{\cAlignHack Mean OD-Mean B} &
  \cAlignHack 1.204 &
  \cAlignHack 1.163 &
  \cAlignHack 1.103 &
  \cAlignHack 0.988 &
  \cAlignHack 0.664 &
  \cAlignHack 0.260 &
  \cAlignHack 0.092\\
\multicolumn{2}{p{\dimexpr(.2872\linewidth-2\tabcolsep)}}{\cAlignHack Standard Dev.} &
  \cAlignHack 0.006 &
  \cAlignHack 0.002 &
  \cAlignHack 0.003 &
  \cAlignHack 0.008 &
  \cAlignHack 0.004 &
  \cAlignHack 0.005 &
  \cAlignHack 0.003\\
\multicolumn{2}{p{\dimexpr(.2872\linewidth-2\tabcolsep)}}{\cAlignHack Viability in \%} &
  \cAlignHack 100.000 &
  \cAlignHack 96.540 &
  \cAlignHack 91.586 &
  \cAlignHack 82.038 &
  \cAlignHack 55.134 &
  \cAlignHack 21.561 &
  \cAlignHack 7.611\\
\multicolumn{9}{p{\dimexpr(1\linewidth-2\tabcolsep)}}{\rAlignHack IC\ensuremath{_{50}} value: 24.43}\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Cytotoxicity assay- standard Cisplatin} }
\label{tw-197289a64d31}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.1322\linewidth-2\tabcolsep}p{\dimexpr.0878\linewidth-2\tabcolsep}p{\dimexpr.11\linewidth-2\tabcolsep}p{\dimexpr.11\linewidth-2\tabcolsep}p{\dimexpr.11\linewidth-2\tabcolsep}p{\dimexpr.11\linewidth-2\tabcolsep}p{\dimexpr.11\linewidth-2\tabcolsep}p{\dimexpr.11\linewidth-2\tabcolsep}p{\dimexpr.12\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack Cisplatin &
  \cAlignHack Blank &
  \cAlignHack Control &
  \multicolumn{6}{p{\dimexpr(.67\linewidth-2\tabcolsep)}}{\cAlignHack Concentration Unit: \ensuremath{\mu}g}\\
\rowcolor{kwdboxcolor} &
   &
   &
  \cAlignHack 3.12 &
  \cAlignHack 6.25 &
  \cAlignHack 12.5 &
  \cAlignHack 25 &
  \cAlignHack 50 &
  \cAlignHack 100\\
\tblmidrule 
\cAlignHack Reading 1 &
  \cAlignHack 0.025 &
  \cAlignHack 1.227  &
  \cAlignHack 0.811  &
  \cAlignHack 0.525 &
  \cAlignHack 0.247 &
  \cAlignHack 0.110 &
  \cAlignHack 0.081 &
  \cAlignHack 0.041\\
\cAlignHack Reading 2 &
  \cAlignHack 0.023 &
  \cAlignHack 1.223 &
  \cAlignHack 0.805 &
  \cAlignHack 0.525 &
  \cAlignHack 0.244 &
  \cAlignHack 0.108 &
  \cAlignHack 0.074 &
  \cAlignHack 0.040\\
\cAlignHack Reading 3 &
  \cAlignHack 0.023 &
  \cAlignHack 1.234 &
  \cAlignHack 0.818 &
  \cAlignHack 0.520 &
  \cAlignHack 0.240 &
  \cAlignHack 0.116 &
  \cAlignHack 0.083 &
  \cAlignHack 0.046\\
\cAlignHack Mean &
  \cAlignHack 0.024 &
  \cAlignHack 1.228 &
  \cAlignHack 0.811 &
  \cAlignHack 0.523 &
  \cAlignHack 0.244 &
  \cAlignHack 0.111 &
  \cAlignHack 0.079 &
  \cAlignHack 0.042\\
\multicolumn{2}{p{\dimexpr(.22\linewidth-2\tabcolsep)}}{\cAlignHack Mean OD-Mean B} &
  \cAlignHack 1.204 &
  \cAlignHack 0.788 &
  \cAlignHack 0.500 &
  \cAlignHack 0.220 &
  \cAlignHack 0.088 &
  \cAlignHack 0.056 &
  \cAlignHack 0.019\\
\multicolumn{2}{p{\dimexpr(.22\linewidth-2\tabcolsep)}}{\cAlignHack Standard Dev.} &
  \cAlignHack 0.006 &
  \cAlignHack 0.007 &
  \cAlignHack 0.003 &
  \cAlignHack 0.004 &
  \cAlignHack 0.004 &
  \cAlignHack 0.005 &
  \cAlignHack 0.003\\
\multicolumn{2}{p{\dimexpr(.22\linewidth-2\tabcolsep)}}{\cAlignHack Viability in \%} &
  \cAlignHack 100.000 &
  \cAlignHack 65.403 &
  \cAlignHack 41.489 &
  \cAlignHack 18.267 &
  \cAlignHack 7.279 &
  \cAlignHack 4.622 &
  \cAlignHack 1.550\\
\multicolumn{9}{p{\dimexpr(1\linewidth-2\tabcolsep)}}{\rAlignHack IC\ensuremath{_{50}} value: 4.02}\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}
\textbf{Isolation of Active constituents}

Isolated fractions were subjected to thin layer chromatography (TLC) as per conventional method using silica gel 60F\ensuremath{_{254}}, Size: 5x3 cm (Merck) were cut using TLC cutter. TLC Plate markings were made with soft pencil. The Solvent (Mobile phase) system: Chloroform: Ethanol (90:10). Pre-saturation with mobile phase was allowed for 30 minutes. Plates were kept inside the chamber and the elution was performed using solvent system. After completion of the elution the plate was dried and subjected to visualize under UV chamber and sprayed using different spray reagents. Rf values determined by using following formula (Rf = Distance travelled by the solute / Distance travelled by the solvent).

Similar identical spots were observed for fractions number 82-91. These fractions were combined and evaporated under reduced pressure. To remove unwanted elements like chlorophyll which present in the extract, resulting crude materials were further purified by using activated charcoal in hot ethanol and the fractions are kept for crystallization. Isolated compound with the yield of 32.4 mg and identification was made by TLC study. After completion of the elution the plate was dried and subjected to visualize under UV chamber and sprayed using different spray reagents (Iodine Reagents). Similar spots were observed (refer to Figure~\ref{f-aa653d67da0f}). The Rf value of the obtained spot is 0.52 (refer to Table~\ref{tw-151015c713b3}). The obtained solid was submitted for Mass spectra, FT-IR spectra, \ensuremath{^{13}}C-NMR and \ensuremath{^{1}}H-NMR analysis. 

\textbf{Structural interpretation}

Based on the data obtained from Electron spray mass spectrophotometry (EI-MS), FTIR, \ensuremath{^{1}}H NMR and \ensuremath{^{13}}C-NMR, the following impression were made. Description of the compound was found to be Colorless solid and the melting point is 149 - 152 \ensuremath{^\circ}C. The molecular mass of the compound (EI-MS) m/z: 340.26.In \ensuremath{^{1}}H-NMR: delta (\ensuremath{\delta }) 3.66 {\textendash} 3.67 (6H, d, -OCH\ensuremath{_{3}}); 3.87 (3H, s, -OCH\ensuremath{_{3}}); 3.89 (3H, s, -OCH\ensuremath{_{3}}); 4.43 {\textendash} 4.47 (2H, m, -CH\ensuremath{_{2}}); 6.77 {\textendash} 6.81 (2H, m, Ar-H); 7.00 {\textendash} 7.01 (1H, d, Ar-H); 7.31 (1H, s, Ar-H ); 7.51 {\textendash} 7.53 (2H, m, Ar-H); 8.24 {\textendash} 8.26 (1H, d, Ar-H) 

\ensuremath{^{13}}C-NMR: \ensuremath{\delta } 40.77 (s); 55.35 {\textendash} 55.65 (m); 67.19 {\textendash} 67.25 (d); 104.26 (s); 105.52 {\textendash} 105.62 (d); 111.88 (s); 112.58 {\textendash} 112.70 (m); 118.37 (s); 120.55 (s); 122.07 (s); 132.24 (s); 132.81 (s); 140.44 {\textendash} 140.60 (t); 147.11 (s); 148.53 (s); 149.55 (s); 152.14 (s); 157.89 (s) and FTIR (KBr cm-1): 3422 cm\ensuremath{^{-1 }} ; 3019 cm\ensuremath{^{-1}}; 3006 cm\ensuremath{^{-1}}; 2938 cm\ensuremath{^{-1}}; 2835 cm\ensuremath{^{-1}}; 1507 cm\ensuremath{^{-1}} ; 1416 cm\ensuremath{^{-1 }} ;\ensuremath{^{}} 1353 cm\ensuremath{^{-1}};\ensuremath{^{}} 909 cm\ensuremath{^{-1 }}

\textbf{Structural Impression}

The molecular weight of the isolated compound was determined by high-resolution mass spectrometry and the molecular ion peak appeared at m/z 340.26. The FTIR spectra showed the peaks at 3422 (-OCH\ensuremath{_{3}} group), 2938 (=CH stretch),  2835 ({\textendash}CH\ensuremath{_{3}} asymmetric stretch). The carbonyl group C=N stretching band is located at  1507 and =CH double bond showed  1416 cm\ensuremath{^{-1}}. In the fingerprint region, the {\textendash}CH\ensuremath{_{2}} bending vibration peaks are observed at 1353 and  909 cm\ensuremath{^{-1}}. The \ensuremath{^{1}}H-NMR spectrum of compound was showed the signals of the methoxy protons of at  3.66 {\textendash} 3.67 (6H, d, -OCH\ensuremath{_{3}}); 3.87 (3H, s, -OCH\ensuremath{_{3}}); 3.89 (3H, s, -OCH\ensuremath{_{3}}). The {\textendash}CH\ensuremath{_{2}} proton resonated as multiplet at  4.43 {\textendash} 4.47. The signals for the seven aromatic protons appeared at 6.77 to 8.26. The \ensuremath{^{13}}C-NMR spectrum Methoxy carbons appeared at  40.77 (s); 55.35 {\textendash} 55.65 (m); 67.19 {\textendash} 67.25 (d). The aromatic carbons were showed signals between 132.24 - 157.89. Based on the pattern of the spectrum was identical with that of compound 1-[(3,4-dimethoxyphenyl) methyl]-6,7-dimethoxyisoquinoline alkaloid (Papaverine) and molecule formula is C\ensuremath{_{20}}H\ensuremath{_{21}}NO\ensuremath{_{4\ }} and the structure  shown in Figure~\ref{f-dbef77453dad}. 

\textbf{Cytotoxicity Assay by MTT Method}

The absorbance value for the percentage of viability was calculated using the formula (\% of viability = Sample abs/Control abs x 100). The cell growth inhibition against Human Melanoma cell line (A375) for various concentration were shown in Tables~\ref{tw-978700f3b80d}, \ref{tw-5926648e67bb} and~\ref{tw-197289a64d31}  and in pictorial presentation from Figures~\ref{f-f41dd617c513}, \ref{f-496aa5bcd1e2} and~\ref{f-d8eeffc8077e}. Based on the rise in concentration of aqueous ethanolic extract, isolated compound-papaverine and standard cisplatin, there is rise in level of viability of cells for standard followed by isolated compound and crude extract. From the obtained data, IC\ensuremath{_{50}} value of Aqueous ethanolic extract of \textit{Erythrina subumbrans} (Hassk.)  Merr, isolated compound-Papaverin and standard Cisplatin exhibits value of 82.14\ensuremath{\mu}g, 24.43 \ensuremath{\mu}g \& 4.02 \ensuremath{\mu}g respectively which indicates significant cytotoxic activity exhibited by isolated compound Papaverin against standard Cisplatin and less or no activity observed in aqueous ethanolic extract.
    
\section{DISCUSSION}
The extraction activity was carried out on powdered leaf samples of \textit{Erythrina subumbrans }(Hassk.) Merr using aqueous ethanol (80 \%) as solvent for extraction. The activity was carried out for 72 hours and the final resulting solution was filtered, and the final yield is 4.13 \% (refer to Table~\ref{tw-6aa8309b0c8d}). The obtained aqueous ethanolic extract were further dissolved in methanol and adsorbed in silica gel 100-200 mesh size. Evaporated to dryness and powdered. Fine powdered were packed in column prepared in Hexane. Initial flow was with Hexane followed by gradually increasing the polarity to Ethyl acetate and then to Chloroform. Fractions were confirmed by TLC by traditional TLC system. Identical fractions were combined and evaporated under reduced pressure. Isolated compound with yield of 32.4mg and it was further confirmed by TLC study and the R\ensuremath{_{f}} value of obtained spot is 0.52 (refer to Table~\ref{tw-151015c713b3}).

Isolated compound was further subjected for FTIR, LC-MS, \ensuremath{^{1}}H and \ensuremath{^{13}}CNMR study. From the data obtained, the m/z is 340.26. \ensuremath{^{1}}H-NMR spectrum of compound was showed the signals of the methoxy protons of at 3.66 {\textendash} 3.67 (6H, d, -OCH\ensuremath{_{3}}); 3.87 (3H, s, -OCH\ensuremath{_{3}}); 3.89 (3H, s, -OCH\ensuremath{_{3}}). {\textendash}CH\ensuremath{_{2}} proton resonated as multiplet at 4.43 {\textendash} 4.47. The signals for the seven aromatic protons appeared at 6.77 to 8.26. \ensuremath{^{13}}C-NMR spectrum Methoxy carbons appeared at 40.77 (s); 55.35 {\textendash} 55.65 (m); 67.19 {\textendash} 67.25 (d). The aromatic carbons were showed signals between 132.24 - 157.89. The obtained pattern is identical to that of iso-quinoline alkaloid Papaverine (1-[(3,4-dimethoxyphenyl) methyl]-6,7-dimethoxyisoquinoline alkaloid.

Cytotoxic activity of aqueous ethanolic extract of \textit{Erythrina subumbrans} (Hassk.)  Merr, Isolated compound-Papaverine and standard cisplatin was carried out in Human Melanoma cancer cell (A375) cells. Concentration ranging from 3.12 \ensuremath{\mu}g/ml to 100\ensuremath{\mu}g/ml was prepared and treated in similarly (and evaluated for viability of cells (\%). IC\ensuremath{_{50}} value of Aqueous ethanolic extract of erythrina subumbrans (82.14), isolated compound-Papaverine (24.43) and standard Cisplatin (4.02) which indicates significant cytotoxic activity exhibited by isolated compound Papaverin against standard Cisplatin.
    
\section{Conclusion}
In this study, leaves of \textit{Erythrina subumbrans} (Hassk.) Merr. was collected from Western Ghats of India and authenticated at Siddha central research institute, Chennai. Extraction was carried out using aqueous Ethanol (80\%) and the obtained extract was subjected for isolation of compound. Isolated compound-papaverine was confirmed by FTIR, LC-MS, \ensuremath{^{1}}H \& \ensuremath{^{13}}CNMR study. Further, cytotoxic activity was carried for extract, isolated compound and standard using Human Melanoma (A375) cells. IC\ensuremath{_{50}} value of Aqueous ethanolic extract of \textit{Erythrina subumbrans (Hassk.) } Merr (82.14), isolated compound-Papaverine (24.43) and standard Cisplatin (4.02). From the data, it is concluded that significant activity was obtained from Papaverine compared to standard Cisplatin. And no/less activity was observed from crude extract. 
    
\section{Acknowledgement}
The authors are thankful to the School of Pharmaceutical Sciences, Vels Institute of Science, Technology, and Advanced Studies, and its management for providing research facilities and encouragement.

\textbf{Funding Support}

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

\textbf{Conflict of Interest}

The authors declare that there is no conflict of interest for this study.
    

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