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\def\RunningAuthor{Cynthia C. and Horne Iona Averal}
\firstPage{3164}
\articleType{Original Article}
\receivedDate{03 Apr 2020}
\acceptedDate{22 May 2020}
\revisedDate{10 May 2020}
\journalVolume{2020, 11}
\journalIssue{3}
\journalDoi{ijrps.v11i3.2430}
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\title{\textbf{Phytochemical characterization and cytotoxicity analysis of ayurvedic formulation of drug}}
\author{Cynthia~C.\textsuperscript{*}\textsuperscript{1}, 
            Horne~Iona~Averal\textsuperscript{2}~\\[5pt]{\textsuperscript{1}Department of Biotechnology, Bishop Heber college, Tiruchirapalli, Tamil Nadu- 620017, India}~\\{\textsuperscript{2}Department of Zoology, Holy Cross College, Trichirappalli, Tamil Nadu-620002, India}}

\begin{abstract}
The liver is a major endocrine organ which controls the significant metabolic activity in the human body. Treatment of several liver disorders with herbal based plant drugs is popularising nowadays due to their hepatoprotective and nontoxic effect. Livoral, a polyherbal Ayurvedic formulation is one such drug commonly recommended for liver disorders in India. The current work was aimed at studying the phytochemical characterisation and cytotoxic activity of livoral. Phytochemical analysis of extracts of livoral in methanol, hexane, acetone and petroleum ether was studied by TLC method. The cytotoxic activity of livoral was analysed by MTT assay in Vero cell lines at a concentration ranging from 10\ensuremath{\mu }g/ml to 500\ensuremath{\mu }g/ml. The phytochemical analysis revealed the presence of Phyto compound such as  tannins, phenols, steroids, alkaloids, terpenes and carbohydrates in all the four types of livoral extracts. Phytochemicals such as phenols, terpenes and alkaloids were already known for their hepatoprotective activity. MTT assay revealed the nontoxic effect of livoral up to 300 \ensuremath{\mu }g/ml concentrations. Thus, based on the results of the phytochemical and cytotoxic analysis, the livoral was proven to be one of the best herbal drugs for liver disorders with hepatoprotective constituents and non-cytotoxic effect. 
\end{abstract}\def\keywordstitle{Keywords}

\begin{keywords}Livoral,\newline phytochemical characterization,\newline cytotoxic analysis,\newline hepatoprotective
\end{keywords}
\twocolumn[ \maketitle {\printKwdAbsBox}]
\makeatletter{\raggedright\textsuperscript{*}Corresponding Author\par Name:\ Cynthia~C.~\\ Phone:\ +91- 8220821265~\\  Email:\ cynthiachainsam@gmail.com
        \par}\vspace*{-11pt}\hrulefill\par{\fontsize{12}{14}\selectfont ISSN: 0975-7538}\par% 
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\section{Introduction}
In the last decade, Ayurveda medicine has been extensively studied due to one of the world's oldest traditional medical systems originated in India and several countries more than three thousand years ago. In recent years, this area has seen significant advances in the country's modern health care systems. This is currently under consideration, for it promotes health by the use of herbal drugs, regular diets and other unique health-related practices. Over the past three decades, there has been a sustained research activity in herbal based medicinal products, and it has been supplemented tremendously with more than 75\% of people relying on them for some part of primary health care issues in worldwide \unskip~\citep{805818:19238667}. 

Herbal drugs have better cultural acceptability, better suitable activity with the human body and low-level side effects. However, in the last few years, their use has been increasing in developed countries. Many herbs and herbal drugs were used as medical prescription drugs in the European Union, and their sales were around more than 6 billion in 1991 and maybe over 20 billion at present. 

Over the years, this field has reached a mature stage in the USA, herbal drugs and compounds are currently sold in retail health food stores with a turnover of about more than 4 billion in 2000 which has been more anticipated to increase double by the turn of the entire centuries. 

Recently, this has caused considerable interest within the community due to illness in India the herbal based drug market is about more than 1 billion and overall export of herbal-based drugs are around the year 80 million \unskip~\citep{805818:19238666}. Herbal medicines and herbal drugs also find everywhere in a market as health care compounds whose current market is estimated at 80{\textendash}250 billion in India and also in rest of world \unskip~\citep{805818:19238676}.

The liver is a major organ of the human body that significance plays a crucial role in the metabolic elimination and biotransformation of a foreign toxic substance. Liver cirrhosis was one of the major medical issues of alcohol abuse. Modern medical practices followed have adverse effects over them. Hence, Ayurvedic formulations of herbal origin can be considered as a curative alternative with minimal no side effects \unskip~\citep{805818:19238674}.

Livoral is one such ancient Ayurvedic formulation of herbal origin produced by Dharma pharmacy, to balance Pitta (Fire) in the body. It is made up of 15 herbs which are believed to have a hepatoprotective effect and commonly used to detoxify, guard and rejuvenate liver from various infections by eliminating natural toxins, from the body and to treat liver disorders like hepatomegaly, fatty liver, liver cirrhosis due to alcohol. 

It has also been used to support digestion. However, there was no elaborate scientific study regarding their hepatoprotective activity. With this background, the present study has been formulated to study the phytochemical characterisation and the cytotoxicity effect of livoral. 
    
\section{Materials and Methods}
\textbf{Collection of sample}

Livoral syrup was purchased from Dharma pharmacy Pvt. ltd, Chennai. The composition of livoral syrup is given in Table~\ref{tw-55d87455db2f}. 

\textbf{Cell culture}

Kidney Cell lines (African Green Monkey) were purchased from NCCS, Pune and transfer and cultured the cell line by DEEM liquid medium contains Fetal Bovine serum (10\%), Penicillin (100 ug/ml) and Streptomycin (100 \ensuremath{\mu}g/ml) and inoculated medium was incubated for 5\% Co\ensuremath{_{2}} incubator at 37\ensuremath{^{o}}C. 

\textbf{Phytochemical characterisation }

The phytochemical tests were carried out for four extracts (methanol, hexane, acetone, petroleum ether) of livoral to identify the major components by using the TLC method.

\textbf{Flavonoids}

The sample was spotted on the TLC plate, and 1\% AICl\ensuremath{_{3}} reagent was sprayed. Yellow fluorescence under UV indicates the presence of flavonoids.

\textbf{Phenols}

The sample was spotted on the TLC plate, and Anisaldehyde- H\ensuremath{_{2}}SO\ensuremath{_{4}} was sprayed. Spot of violet colour on the plate it indicates phenols presence 

\textbf{Steroids}

The sample was spotted on the TLC plate, and Anisaldehyde H\ensuremath{_{2}}SO\ensuremath{_{4}} was sprayed. The appearance of red colour indicates steroids presence.

\textbf{Amino acids}

The sample was spotted on a TLC plate, and Ninhydrin reagent was sprayed. The appearance of reddish spot indicates the presence of amino acids. 

\textbf{Alkaloids}

The sample was spotted on the TLC plate, and Dragendorff's reagent was sprayed. The appearance of the spot by orange colour indicates the presence of the alkaloids. 

\textbf{Hydroquinone derivatives}

The sample was spotted on the TLC plate, and 10\% KOH reagent was sprayed. The appearance of red, yellow, blue colour indicates the presence of anthraquinone, anthrones, coumarins. 

\textbf{Test for tannins}

The sample was spotted on the TLC plate, and 10\% FeCl\ensuremath{_{3}} reagent was sprayed. The appearance of blue and green colour changes it mention the presence of tannins.


\begin{table*}[!htbp]
\caption{\boldmath {Ethnobotanical Information of drug} }
\label{tw-55d87455db2f}
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\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack S. No. & \cAlignHack Plant Name & \cAlignHack Quantity per 5 ml\\
\tblmidrule 
\cAlignHack 1. &
  \cAlignHack \textit{Phyllanthusamarus} &
  \cAlignHack 5 mg\\
\cAlignHack 2. &
  \cAlignHack \textit{Fumariavaillantii} &
  \cAlignHack 5 mg\\
\cAlignHack 3. &
  \cAlignHack \textit{Ionidiumsuffruticosum} &
  \cAlignHack 5 mg\\
\cAlignHack 4. &
  \cAlignHack \textit{Evolvulusalsinoides} &
  \cAlignHack 5 mg\\
\cAlignHack 5. &
  \cAlignHack \textit{Hibiscus rosainensis} &
  \cAlignHack 5 mg\\
\cAlignHack 6. &
  \cAlignHack \textit{Wedeliacalendulacea} &
  \cAlignHack 5 mg\\
\cAlignHack 7. &
  \cAlignHack \textit{Murrayakoengi} &
  \cAlignHack 5 mg\\
\cAlignHack 8. &
  \cAlignHack \textit{Hydrocotyleasiatica} &
  \cAlignHack 5 mg\\
\cAlignHack 9. &
  \cAlignHack \textit{Solanumnigrum} &
  \cAlignHack 1 mg\\
\cAlignHack 10. &
  \cAlignHack \textit{Andrographispaniculata} &
  \cAlignHack 1 mg\\
\cAlignHack 11. &
  \cAlignHack \textit{Cuminumcyminum} &
  \cAlignHack 1 mg\\
\cAlignHack 12. &
  \cAlignHack \textit{Foeniculumvulgare} &
  \cAlignHack 1 mg\\
\cAlignHack 13. &
  \cAlignHack \textit{Vitisvinifera} &
  \cAlignHack 1 mg\\
\cAlignHack 14. &
  \cAlignHack \textit{Emblicaofficinalis} &
  \cAlignHack 1 mg\\
\cAlignHack 15. &
  \cAlignHack \textit{Cyperusrotundus} &
  \cAlignHack 1 mg\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Herbal constituents of drug and their effect on Liver} }
\label{tw-44edaf0aa99f}
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\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack S. No. & \cAlignHack Plant Name & \cAlignHack Activity on liver\\
\tblmidrule 
\cAlignHack 1. &
  \cAlignHack \textit{Phyllanthusamarus} &
  \cAlignHack liver damage in rat\\
\cAlignHack 2. &
  \cAlignHack \textit{Fumariavaillantii} &
  \cAlignHack liver injury in rats\\
\cAlignHack 3. &
  \cAlignHack \textit{Ionidiumsuffruticosum} &
  \cAlignHack High fat diet in rat \\
\cAlignHack 4. &
  \cAlignHack \textit{Evolvulusalsinoides} &
  \cAlignHack liver damage in rat\\
\cAlignHack 5. &
  \cAlignHack \textit{Hibiscus rosasinensis} &
  \cAlignHack liver damage in rat\\
\cAlignHack 6. &
  \cAlignHack \textit{Wedeliacalendulacea} &
  \cAlignHack liver injury in rats\\
\cAlignHack 7. &
  \cAlignHack \textit{Murrayakoengi} &
  \cAlignHack antioxidant activity\\
\cAlignHack 8. &
  \cAlignHack \textit{Hydrocotyleasiatica} &
  \cAlignHack liver damage in rat \\
\cAlignHack 9. &
  \cAlignHack \textit{Solanumnigrum} &
  \cAlignHack liver damage in rat \\
\cAlignHack 10. &
  \cAlignHack \textit{Andrographispaniculata} &
  \cAlignHack Antihepatotoxicactivity \\
\cAlignHack 11. &
  \cAlignHack \textit{Cuminumcyminum} &
  \cAlignHack Hepatoprotective activity \\
\cAlignHack 12. &
  \cAlignHack \textit{Foeniculumvulgare} &
  \cAlignHack Hepatoprotective action \\
\cAlignHack 13. &
  \cAlignHack \textit{Vitisvinifera} &
  \cAlignHack liver damage in rat \\
\cAlignHack 14. &
  \cAlignHack \textit{Emblicaofficinalis} &
  \cAlignHack Inhibit the hepatotoxicity  \\
\cAlignHack 15. &
  \cAlignHack \textit{Cyperusrotundus} &
  \cAlignHack liver damage in rat \\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Phytochemical Screening of drug (Methanol Extract)} }
\label{tw-ef9655cd94c4}
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  \cAlignHack Yellow flouresense under the UV &
  \cAlignHack -\\
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  \cAlignHack Anisaldehyde H\ensuremath{_{2}}S0\ensuremath{_{4}} &
  \cAlignHack Phenols and Steroids &
  \cAlignHack Violet / Red &
  \cAlignHack ++\\
\cAlignHack 3 &
  \cAlignHack Ninhydrin &
  \cAlignHack Amino acids &
  \cAlignHack Red &
  \cAlignHack -\\
\cAlignHack 4 &
  \cAlignHack Dragon droff's &
  \cAlignHack Alkaloids &
  \cAlignHack Orange &
  \cAlignHack +\\
\cAlignHack 5 &
  \cAlignHack Pancal D &
  \cAlignHack Tannins and Terpens &
  \cAlignHack Blue /Green &
  \cAlignHack ++\\
\cAlignHack 6 &
  \cAlignHack Con. H\ensuremath{_{2}}S0\ensuremath{_{4}} &
  \cAlignHack Carbohydrates &
  \cAlignHack Gray/Brown &
  \cAlignHack +\\
\tblbottomrule 
\end{tabulary}\par 
\begin{tablenotes}\footnotesize 
    
\item{++= shows highest level  + = presence ofphytocompounds  - = absence of phytocompounds  }
\end{tablenotes}
\end{threeparttable}

\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Phytochemical Screening of Livoral (Petroleum Ether Extract)} }
\label{tw-b28257b26ca0}
\centering 
\begin{threeparttable}

\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.13350000000000001\linewidth-2\tabcolsep}p{\dimexpr.24849999999999998\linewidth-2\tabcolsep}p{\dimexpr.2138\linewidth-2\tabcolsep}p{\dimexpr.2389\linewidth-2\tabcolsep}p{\dimexpr.1653\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack S.No. & \cAlignHack Reagent & \cAlignHack Analysis & \cAlignHack Color Appearance & \cAlignHack Result\\
\tblmidrule 
\cAlignHack 1 &
  \cAlignHack 1\% Alcl\ensuremath{_{3}} in ethanol &
  \cAlignHack Flavonoids &
  \cAlignHack Yellow fluorescence under the UV &
  \cAlignHack -\\
\cAlignHack 2 &
  \cAlignHack Anisaldehyde H\ensuremath{_{2}}S0\ensuremath{_{4}} &
  \cAlignHack Phenols and Steroids &
  \cAlignHack Violet / Red &
  \cAlignHack ++\\
\cAlignHack 3 &
  \cAlignHack Ninhydrin &
  \cAlignHack Amino acids &
  \cAlignHack Red &
  \cAlignHack -\\
\cAlignHack 4 &
  \cAlignHack Dragon droff's &
  \cAlignHack Alkaloids &
  \cAlignHack Orange &
  \cAlignHack +\\
\cAlignHack 5 &
  \cAlignHack Pancal D &
  \cAlignHack Tannins and Terpenes &
  \cAlignHack Blue /Green &
  \cAlignHack ++\\
\cAlignHack 6 &
  \cAlignHack Con. H\ensuremath{_{2}}S0\ensuremath{_{4}} &
  \cAlignHack Carbohydrates &
  \cAlignHack Gray/Brown &
  \cAlignHack +\\
\tblbottomrule 
\end{tabulary}\par 
\begin{tablenotes}\footnotesize 
    
\item{++= shows highest level  + = presence of phytocompounds  - = absence of phytocompounds}
\end{tablenotes}
\end{threeparttable}

\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Phytochemical Screening of drug (Acetone Extract)} }
\label{tw-6c674b7a5572}
\centering 
\begin{threeparttable}

\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.1417\linewidth-2\tabcolsep}p{\dimexpr.2333\linewidth-2\tabcolsep}p{\dimexpr.2084\linewidth-2\tabcolsep}p{\dimexpr.26650000000000002\linewidth-2\tabcolsep}p{\dimexpr.1501\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack S.No. & \cAlignHack Reagent & \cAlignHack Analysis & \cAlignHack Color Appearance & \cAlignHack Result\\
\tblmidrule 
\cAlignHack 1 &
  \cAlignHack 1\% Alcl\ensuremath{_{3}} in ethanol &
  \cAlignHack Flavonoids &
  \cAlignHack Yellow fluorescence under the UV &
  \cAlignHack -\\
\cAlignHack 2 &
  \cAlignHack Anisaldehyde H\ensuremath{_{2}}S0\ensuremath{_{4}} &
  \cAlignHack Phenols and Steroids &
  \cAlignHack Violet / Red &
  \cAlignHack ++\\
\cAlignHack 3 &
  \cAlignHack Ninhydrin &
  \cAlignHack Amino acids &
  \cAlignHack Red &
  \cAlignHack -\\
\cAlignHack 4 &
  \cAlignHack Dragon droff's &
  \cAlignHack Alkaloids &
  \cAlignHack Orange &
  \cAlignHack +\\
\cAlignHack 5 &
  \cAlignHack Pancal D &
  \cAlignHack Tannins and Terpenes &
  \cAlignHack Blue /Green &
  \cAlignHack ++\\
\cAlignHack 6 &
  \cAlignHack Con. H\ensuremath{_{2}}S0\ensuremath{_{4}} &
  \cAlignHack Carbohydrates &
  \cAlignHack Gray/Brown &
  \cAlignHack +\\
\tblbottomrule 
\end{tabulary}\par 
\begin{tablenotes}\footnotesize 
    
\item{++= shows highest level  + = presence of phytocompounds  - = absence of phytocompounds}
\end{tablenotes}
\end{threeparttable}

\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Phytochemical Screening of Drug (Hexane Extract)} }
\label{tw-ef15abfec9c3}
\centering 
\begin{threeparttable}

\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.1114\linewidth-2\tabcolsep}p{\dimexpr.23869999999999997\linewidth-2\tabcolsep}p{\dimexpr.225\linewidth-2\tabcolsep}p{\dimexpr.22490000000000002\linewidth-2\tabcolsep}p{\dimexpr.20\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack S.No. & \cAlignHack Reagent & \cAlignHack Analysis & \cAlignHack Color Appearance & \cAlignHack Result\\
\tblmidrule 
\cAlignHack 1 &
  \cAlignHack 1\% Alcl\ensuremath{_{3}} in ethanol &
  \cAlignHack Flavonoids &
  \cAlignHack Yellow fluorescence under the UV &
  \cAlignHack -\\
\cAlignHack 2 &
  \cAlignHack Anisaldehyde H\ensuremath{_{2}}S0\ensuremath{_{4}} &
  \cAlignHack Phenols and Steroids &
  \cAlignHack Violet / Red &
  \cAlignHack ++\\
\cAlignHack 3 &
  \cAlignHack Ninhydrin &
  \cAlignHack Amino acids &
  \cAlignHack Red &
  \cAlignHack -\\
\cAlignHack 4 &
  \cAlignHack Dragon droff's &
  \cAlignHack Alkaloids &
  \cAlignHack Orange &
  \cAlignHack +\\
\cAlignHack 5 &
  \cAlignHack Pancal D &
  \cAlignHack Tannins and Terpenes &
  \cAlignHack Blue /Green &
  \cAlignHack ++\\
\cAlignHack 6 &
  \cAlignHack Con. H\ensuremath{_{2}}S0\ensuremath{_{4}} &
  \cAlignHack Carbohydrates &
  \cAlignHack Grey/Brown &
  \cAlignHack +\\
\tblbottomrule 
\end{tabulary}\par 
\begin{tablenotes}\footnotesize 
    
\item{++= shows highest level  + = presence of phytocompounds  - = absence of phytocompounds}
\end{tablenotes}
\end{threeparttable}

\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Cell Viability Percentage of Vero Cells incubated with drug} }
\label{tw-978e1575a4c0}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.16666666666666664\linewidth-2\tabcolsep}p{\dimexpr.20360000000000007\linewidth-2\tabcolsep}p{\dimexpr.1574333333333334\linewidth-2\tabcolsep}p{\dimexpr.1574333333333334\linewidth-2\tabcolsep}p{\dimexpr.1574333333333334\linewidth-2\tabcolsep}p{\dimexpr.1574333333333334\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack S. No. & \cAlignHack Livoral concentration (\ensuremath{\mu }g/ml) & \multicolumn{3}{p{\dimexpr(.4723000000000002\linewidth-2\tabcolsep)}}{\cAlignHack Cell viability (\%) \mbox{}\protect\newline (in triplicates)} & \cAlignHack Mean Value (\%) \\
\tblmidrule 
\cAlignHack 1. &
  \cAlignHack Control &
  \cAlignHack 100 &
  \cAlignHack 100 &
  \cAlignHack 100 &
  \cAlignHack 100\\
\cAlignHack 2. &
  \cAlignHack 10 \ensuremath{\mu }g/ml &
  \cAlignHack 105.72 &
  \cAlignHack 100.22 &
  \cAlignHack 108.69 &
  \cAlignHack 104.87\\
\cAlignHack 3. &
  \cAlignHack 20 \ensuremath{\mu }g/ml &
  \cAlignHack 103.89 &
  \cAlignHack 102.97 &
  \cAlignHack 101.37 &
  \cAlignHack 102.74\\
\cAlignHack 4. &
  \cAlignHack 40 \ensuremath{\mu }g/ml &
  \cAlignHack 97.71 &
  \cAlignHack 98.16 &
  \cAlignHack 99.54 &
  \cAlignHack 98.47\\
\cAlignHack 5. &
  \cAlignHack 60 \ensuremath{\mu }g/ml &
  \cAlignHack 91.76 &
  \cAlignHack 94.50 &
  \cAlignHack 97.48 &
  \cAlignHack 94.58\\
\cAlignHack 6. &
  \cAlignHack 80 \ensuremath{\mu }g/ml &
  \cAlignHack 90.61 &
  \cAlignHack 87.18 &
  \cAlignHack 87.64 &
  \cAlignHack 88.47\\
\cAlignHack 7. &
  \cAlignHack 100 \ensuremath{\mu }g/ml &
  \cAlignHack 79.63 &
  \cAlignHack 81.69 &
  \cAlignHack 77.57 &
  \cAlignHack 79.63\\
\cAlignHack 8. &
  \cAlignHack 200 \ensuremath{\mu }g/ml &
  \cAlignHack 69.79 &
  \cAlignHack 72.08 &
  \cAlignHack 67.50 &
  \cAlignHack 69.79\\
\cAlignHack 9. &
  \cAlignHack 300 \ensuremath{\mu }g/ml &
  \cAlignHack 62.01 &
  \cAlignHack 58.58 &
  \cAlignHack 64.30 &
  \cAlignHack 61.63\\
\cAlignHack 10. &
  \cAlignHack 400 \ensuremath{\mu }g/ml &
  \cAlignHack 45.08 &
  \cAlignHack 52.40 &
  \cAlignHack 48.97 &
  \cAlignHack 48.81\\
\cAlignHack 11. &
  \cAlignHack 500 \ensuremath{\mu }g/ml &
  \cAlignHack 21.51 &
  \cAlignHack 21.96 &
  \cAlignHack 16.93 &
  \cAlignHack 20.13\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\bgroup
\fixFloatSize{images/f84d18e1-d0ad-4b0d-a36b-09c96219256e-upicture1.png}
\begin{figure*}[!htbp]
\centering \makeatletter\IfFileExists{images/f84d18e1-d0ad-4b0d-a36b-09c96219256e-upicture1.png}{\includegraphics{images/f84d18e1-d0ad-4b0d-a36b-09c96219256e-upicture1.png}}{}
\makeatother 
\caption{\boldmath {Phytochemical screening of drug in various extracts (Methanol, Petroleum Ether, Acetone, Hexane)}}
\label{f-6798794a7f70}
\end{figure*}
\egroup

\bgroup
\fixFloatSize{images/6fc76a44-64ba-46cb-aadf-7f27c94895c3-upicture2.png}
\begin{figure*}[!htbp]
\centering \makeatletter\IfFileExists{images/6fc76a44-64ba-46cb-aadf-7f27c94895c3-upicture2.png}{\includegraphics{images/6fc76a44-64ba-46cb-aadf-7f27c94895c3-upicture2.png}}{}
\makeatother 
\caption{\boldmath {Cytoxicity effect of livoral by MTT assay on Vero cells}}
\label{f-09e87aad6dfd}
\end{figure*}
\egroup

\bgroup
\fixFloatSize{images/e71e1161-b80c-449b-9ff8-f56d63c3a339-upicture3.png}
\begin{figure*}[!htbp]
\centering \makeatletter\IfFileExists{images/e71e1161-b80c-449b-9ff8-f56d63c3a339-upicture3.png}{\includegraphics{images/e71e1161-b80c-449b-9ff8-f56d63c3a339-upicture3.png}}{}
\makeatother 
\caption{\boldmath {Control and livoral treated cells}}
\label{f-a3bbcddfdb3d}
\end{figure*}
\egroup
\textbf{Test for terpenes}

The sample was spotted on the TLC plate, and Pancal D reagent was sprayed. The appearance of grey, green colour indicates the presence of terpenes. 

\textbf{Carbohydrates}

The sample was spotted on the TLC plate, and concH\ensuremath{_{2}}SO\ensuremath{_{4\ }} reagent was sprayed. Its appear of grey/ brownish colour indicates the presence of carbohydrates.

\textbf{Cytotoxicity analysis of livoral by MTT assay}

\textit{In vitro} cytotoxicity experiments were performed by (MTT) assay, livoral syrup was tested by using Vero cells soaked on 3-(4,5-dimethylthiazol-2-yl )-2,5-diphenyltetrazolium bromide. Initially, trypsinisation was carried for collection of the cultured Vero cells then pooled the cells in a 15 ml tube with a cape. Then, collected cells (1\ensuremath{\times}10\ensuremath{^{5}} cells/ml) cells were plated at a density of (200 \ensuremath{\mu}L/well) into 96-well microtiter tissue culture plate with a supplement in liquid medium (DMEM) containing Fetal Bovine serum (10\%), Penicillin (100 ug/ml) and Streptomycin (100 \ensuremath{\mu}g/ml) and incubated 24-48h at 37\ensuremath{^{0}}C. After incubation 96 well plate was washed with sterile 1X Phosphate buffer solution and treated with different concentrations of the livoral syrup with serum-free liquid DMEM medium. 

All the samples were replicated three times, and the cells were incubated in 5\% CO\ensuremath{_{2}} incubator at 37\ensuremath{^\circ}C for 24 h. After the CO\ensuremath{_{2\ }} incubation period add the of 20 \ensuremath{\mu}L of MTT(5 mg/ml) into the wells and the cells were incubated 2-4 h again until it showed purple colour precipitates were observed under visible in the plate under an inverted microscope. Later, the medium added with 220 \ensuremath{\mu}L MTT, were aspirated off the entire wells and all the sample well rewashed with 1X PBS solution (200 \ensuremath{\mu}l). Similarly, to dissolve formazan crystals and add 100 \ensuremath{\mu}L DMSO, then the plate was shaken for 5 min in an orbital shaker. After shaking all sample were measured the absorbance at 570 nm and read out using a microplate reader. \textbf{\space }

\textbf{Statistical analysis}

Cell viability percentage of and values of IC50 were calculated using GraphPad Prism 6.0 software with triplicates.
    
\section{Results and Discussion}
As on date therapeutic treatment drugs like herbal-based has been popularised for the treatment of liver disorders by markets leading pharmaceutical industries worldwide, The results were directly compared with the previously reported findings on hepatoprotective agents \unskip~\citep{805818:19238673}.  Livoral, an Ayurvedic polyherbal formulation is one such hepatoprotective agent which is widely prescribed for liver disorders in India. The herbal constituents of livoral and their proven effect on the liver were listed in Table~\ref{tw-44edaf0aa99f}. From the table, it is evident that the livoral is a herbal formulation made up of 15 herbs which were known to have a protective effect on the liver \unskip~\citep{805818:19238676}.

The phytochemical studies revealed the presence of steroids, phenols, alkaloids, tannins, terpenes and carbohydrates in the methanol, petroleum ether, acetone and hexane  extracts of livoral syrup as shown in Table~\ref{tw-ef9655cd94c4}, Table~\ref{tw-b28257b26ca0}, Table~\ref{tw-6c674b7a5572}, and Table~\ref{tw-ef15abfec9c3} (Figure~\ref{f-6798794a7f70} ). The past decade has seen an increase in the use of herbal-based drugs was a rich source of active phytocompounds and have been extensively used for treatment reaction. Thus, phytochemical technique analyses play a significant role in search and to find novel drug molecules \unskip~\citep{805818:19238668}. Recently, investigators have reported that alkaloids, polyphenols, glycosides, carotenoids, coumarins and flavonoids have hepatoprotective activity \unskip~\citep{805818:19238669}. This evidence shows that many phyto compounds and drugs have more activity to sense, induce microsomal enzymes activity either by mobilising the hepatotoxin excretion or lipid peroxidation inhibition. Triterpenes and alkaloids have been well-resulted act as hepatoprotective agents. Polyphenols are reported to have an anti-oxidant property \unskip~\citep{805818:19238675}. Anti-oxidants molecules are synthetic or natural origin and have good ability to stabilise or deactivate scavenging free radicals. In this study, highly reactive\textbf{\space }free radicals are major causative agents for various cellular injuries, including liver damage. Anti-oxidant therapy is a promising strategy to improve liver injury. Thus, it is evident from the photochemical analysis that livoral has a hepatoprotective and anti-oxidant property and can be used for the treatment of liver disorders \unskip~\citep{805818:19238670,805818:19238671}.

Cytotoxic activity of livoral was tested against Vero cell line at different concentrations to determine the IC\ensuremath{_{50}} value (50\% growth inhibition) by MTT assay. Results of different levels of livoral, including 10 \ensuremath{\mu }g/ml to 500 \ensuremath{\mu }g/ml were shown in Table~\ref{tw-978e1575a4c0}  (Figure~\ref{f-09e87aad6dfd}). In Vero cells incubated with livoral sample at 10\ensuremath{\mu }g/ml, the amount of formazan was similar to control cells, the quantity of formazan produced varied between different concentrations of livoral treated cells (Figure~\ref{f-a3bbcddfdb3d}). The formation of formazan crystals decreases when the concentration of livoral increases in Vero cells. The IC50 value of livoral on Vero cell line was 217.2 \ensuremath{\mu }g /ml. Together, these results showed that the livoral possess no toxic effect on Vero cells. The livoral was found to have a nontoxic effect on Vero cells up to 300 \ensuremath{\mu }g/ml concentration. This might be due to the presence of phytochemicals such as phenols, steroids, alkaloids, tannins, terpenes and carbohydrates, which are already proven to have a hepatoprotective and anti-oxidant property which are essential for cell survival. Thus livoral syrup is shown to have no cytotoxic effect making it a nontoxic drug of choice for the treatment of liver disorders \unskip~\citep{805818:19238666,805818:19238677}. Taken together, and the current study suggests that livoral is a hepatoprotective and nontoxic drug for the treatment of liver disorders. \unskip~\citep{805818:19238672,805818:19238678}
    
\section{Conclusion}
It can be concluded four extracts like methanol, hexane, acetone, petroleum ether of that Livoral, and its secondary metabolites were found viz flavonoids, steroids, hydroquinone derivatives, phenols, alkaloids also proved to have highest Cytotoxic activity of livoral was tested against Vero cell line. Therefore, This formulation can be used as a potential source of the natural drug.
\section*{Acknowledgement}Authors are indebted to the management of Holy Cross College who have supported profoundly in performing this work. Authors are also grateful to Trichy Research Institute of Biotechnology for their research help and the software analysis.

\textbf{Conflicts of Interest}

Author's declare no conflicts of Interest

\textbf{Funding Support}

None
    

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