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\begin{document}


\def\RunningAuthor{Kousalya P and Doss VA}
\firstPage{2099}
\articleType{Original Article}
\receivedDate{18 Sep 2020}
\acceptedDate{19 Oct 2020}
\revisedDate{10 Oct 2020}
\journalVolume{2020, 11 (SPL4)}
\journalDoi{ijrps.v11iSPL4.4425}
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\title{Assessment of phytochemicals and quantification of primary and secondary metabolites of \textit{Artabotrys hexapetalus } (L.f.) Bhandari leaves}
\author{Kousalya~P\textsuperscript{*}\textsuperscript{1,2}, 
            Doss~VA\textsuperscript{1}~\\[5pt]{\textsuperscript{1}Department of Biochemistry, PSG college of Arts and Science, Coimbatore-641014, Tamilnadu, India}~\\{\textsuperscript{2}Department of Biochemistry, Bharathidasan College of Arts and Science, Erode - 638116, Tamilnadu, India}}

\begin{abstract}
The main goal of the research was to explore the existence of phytochemicals, quantification of primary and secondary metabolites of leaves extract of \textit{Artabotrys hexapetalus } (L.f.) Bhandari. The phytochemical activity of leaves of \textit{Artabotrys hexapetalus} was assessed using different solvent extracts like water, ethanol, acetone, chloroform and petroleum ether. Among the different solvent extracts, aqueous leaves extract revealed the high content of phytochemicals. So the aqueous leaves extract was used for further investigations. Aqueous leaves extract of \textit{Artabotrys hexapetalus} was subjected to quantitative analysis of primary metabolites like carbohydrates, proteins and amino acids. Quantitative analysis of secondary metabolites like flavonoids, tannins and phenols were performed using aqueous leaves extract of \textit{Artabotrys hexapetalus}. Qualitative screening of phytochemicals reported the existence of carbohydrates, amino acids, proteins, flavonoids, alkaloids, saponins, phenols, glycosides, tannins and diterpenes. Quantitative analysis showed the presence of carbohydrates (43.16\ensuremath{\pm}1.0 mg/g extract), proteins (60.4\ensuremath{\pm}0.88 mg/g extract), amino acids (19.33 \ensuremath{\pm} 1.30 mg/g extract), flavonoids (28.3 \ensuremath{\pm}0.91 mg/g extract), tannins (24.53\ensuremath{\pm}1.02 mg/g extract) and phenols (7.63\ensuremath{\pm}0.85 mg/g extract). The present study concluded that aqueous leaves extract of \textit{Artabotrys hexapetalus} as a potential source of phytochemicals, primary and secondary metabolites.
\end{abstract}\def\keywordstitle{Keywords}

\begin{keywords}Artabotrys hexapetalus,\newline Primary metabolites,\newline Secondary metabolites,\newline Flavonoids,\newline Phenols,\newline Phytochemicals,\newline Tannins
\end{keywords}
\twocolumn[ \maketitle {\printKwdAbsBox}]
\makeatletter{\raggedright\textsuperscript{*}Corresponding Author\par Name:\ Kousalya~P~\\ Phone:\ 9788291776~\\  Email:\ kousiviswa@gmail.com
        \par}\vspace*{-11pt}\hrulefill\par{\fontsize{12}{14}\selectfont ISSN: 0975-7538}\par% 
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\section{Introduction}
Plants contain natural bioactive compounds called phytochemicals. Phytochemicals are classified into two categories primary metabolites and secondary metabolites \unskip~\citep{911123:20571666}. Carbohydrates, amino acids, chlorophyll and proteins are primary metabolites. Secondary metabolites include alkaloids, flavonoids, terpenoids, tannins, phenols, glycosides, saponins \unskip~\citep{911123:20571665}. The medicinal property of a plant is due to secondary metabolites \unskip~\citep{911123:20571663}. Secondary metabolites are free radicals scavengers \unskip~\citep{911123:20571673}. Analysis of phytochemicals and quantification of primary and secondary metabolites will be helpful to identify bioactive compounds. These bioactive compounds have therapeutic value in treating diseases.

\textit{Artabotrys hexapetalus} (L.f.) Bhandari is a member of the Annonaceae family. It is located in India, South China and Srilanka. Within India, it is indigenous to south India and very commonly cultivated in gardens for its very fragrant flowers. \textit{A.hexpetalus} was used in traditional Chinese medicine to treat malaria \unskip~\citep{911123:20571667} and scrofula \unskip~\citep{911123:20571669}. This study aims to analyse the phytoconstituents, quantification of primary metabolites (carbohydrates, proteins and amino acids) and secondary metabolites (flavonoids, tannins and phenols) in aqueous leaves extract of \textit{Artabotrys hexapetalus}.
\begin{table*}[!htbp]
\caption{\boldmath {Qualitative phytochemical analysis} }
\label{tw-a6e346233f7a}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.15779999999999998\linewidth-2\tabcolsep}p{\dimexpr.5022\linewidth-2\tabcolsep}p{\dimexpr.34\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}S.No. & Phytochemicals & Name of the Test\\
\tblmidrule 
\multicolumn{1}{p{\dimexpr(.15779999999999998\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{1.}} &
  \multicolumn{1}{p{\dimexpr(.5022\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{Alkaloids }} &
  Dragendorff's \\
 &
   &
  Wagner's \\
 &
   &
  Mayer's  \mbox{}\protect\newline Hager's\\
\multicolumn{1}{p{\dimexpr(.15779999999999998\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{2.}} &
  \multicolumn{1}{p{\dimexpr(.5022\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{Flavonoids}} &
  Alkaline reagent \\
 &
   &
  Shinoda \\
 &
   &
  Lead acetate \\
\multicolumn{1}{p{\dimexpr(.15779999999999998\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{3.}} &
  \multicolumn{1}{p{\dimexpr(.5022\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{Saponins }} &
  Foam \\
 &
   &
  Froth \\
\multicolumn{1}{p{\dimexpr(.15779999999999998\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{4.}} &
  \multicolumn{1}{p{\dimexpr(.5022\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{Carbohydrates }} &
  Fehling's \\
 &
   &
  Benedict's \\
 &
   &
  Molisch \\
\multicolumn{1}{p{\dimexpr(.15779999999999998\linewidth-2\tabcolsep)}}{\multirow{4}{\linewidth}{5.}} &
  \multicolumn{1}{p{\dimexpr(.5022\linewidth-2\tabcolsep)}}{\multirow{4}{\linewidth}{Aminoacids and proteins}} &
  Ninhydrin \\
 &
   &
  Xanthoproteic \\
 &
   &
  Million's \\
 &
   &
  Biuret \\
6. &
  Phenols  &
  Ferric chloride \\
\multicolumn{1}{p{\dimexpr(.15779999999999998\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{7.}} &
  \multicolumn{1}{p{\dimexpr(.5022\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{Glycosides }} &
  Keller Killani \\
 &
   &
  Borntrager  \\
 &
   &
  Modified Borntrager's \\
\multicolumn{1}{p{\dimexpr(.15779999999999998\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{8.}} &
  \multicolumn{1}{p{\dimexpr(.5022\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{Tannins }} &
  Ferric chloride \\
 &
   &
  Lead acetate \\
\multicolumn{1}{p{\dimexpr(.15779999999999998\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{9.}} &
  \multicolumn{1}{p{\dimexpr(.5022\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{Sterols and Terpenes }} &
  Salkowski's (Sterols and triterpenes)\\
 &
   &
  Copper acetate (Diterpenes)\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Qualitative phytochemical analysis} }
\label{tw-c8aae4239207}
\centering 
\begin{threeparttable}

\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.14\linewidth-2\tabcolsep}p{\dimexpr.14\linewidth-2\tabcolsep}p{\dimexpr.1151\linewidth-2\tabcolsep}p{\dimexpr.16490000000000002\linewidth-2\tabcolsep}p{\dimexpr.14\linewidth-2\tabcolsep}p{\dimexpr.14\linewidth-2\tabcolsep}p{\dimexpr.16\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\multicolumn{2}{p{\dimexpr(.28\linewidth-2\tabcolsep)}}{Plant constituents } & Water  & Ethanol  & Acetone  & Chloroform  & Petroleum ether \\
\tblmidrule 
\multicolumn{7}{p{\dimexpr(1\linewidth-2\tabcolsep)}}{Alkaloids }\\
1. &
  Dragendorff's  &
  + &
  - &
  - &
  ++ &
  ++\\
2. &
  Wagner's  &
  ++ &
  + &
  + &
  ++ &
  -\\
3. &
  Mayer's  &
  - &
  - &
  - &
  - &
  -\\
4. &
  Hager's  &
  ++ &
  + &
  - &
  - &
  -\\
\multicolumn{7}{p{\dimexpr(1\linewidth-2\tabcolsep)}}{Flavonoids }\\
1. &
  Alkaline reagent  &
  ++ &
  - &
  - &
  - &
  +\\
2. &
  Shinoda  &
  + &
  - &
  - &
  + &
  -\\
3. &
  Lead acetate  &
  - &
  - &
  - &
  - &
  -\\
\multicolumn{7}{p{\dimexpr(1\linewidth-2\tabcolsep)}}{Saponins }\\
1. &
  Foam  &
  ++ &
  - &
  -- &
  - &
  -\\
2. &
  Froth  &
  - &
  ++ &
  ++ &
  - &
  -\\
\multicolumn{7}{p{\dimexpr(1\linewidth-2\tabcolsep)}}{Carbohydrates }\\
1. &
  Fehling's &
  + &
  + &
  - &
  ++ &
  -\\
2. &
  Benedict's  &
  ++ &
  ++ &
  + &
  - &
  -\\
3. &
  Molisch's  &
  ++ &
  - &
  - &
  - &
  +\\
\multicolumn{7}{p{\dimexpr(1\linewidth-2\tabcolsep)}}{Proteins and Aminoacids }\\
1. &
  Million's &
  ++ &
  - &
  - &
  - &
  -\\
2. &
  Biuret &
   &
  - &
  - &
  - &
  -\\
3. &
  Xanthoproteic &
  + &
  + &
  - &
  - &
  -\\
4. &
  Ninhydrin &
  ++ &
  - &
  - &
  - &
  -\\
\multicolumn{7}{p{\dimexpr(1\linewidth-2\tabcolsep)}}{Phenols }\\
1. &
  Ferric chloride &
  ++ &
  ++ &
  ++ &
  + &
  +\\
\multicolumn{7}{p{\dimexpr(1\linewidth-2\tabcolsep)}}{Glycosides }\\
1. &
  Keller Killani &
  ++ &
  - &
  - &
  - &
  +\\
2. &
  Borntrager &
  - &
  - &
  - &
  - &
  -\\
3. &
  Modified Borntrager's &
  - &
  - &
  - &
  - &
  -\\
\multicolumn{7}{p{\dimexpr(1\linewidth-2\tabcolsep)}}{Tannins }\\
1. &
  Ferric chloride &
  + &
  - &
  - &
  - &
  -\\
2. &
  Lead acetate &
  + &
  + &
  + &
  - &
  -\\
\multicolumn{7}{p{\dimexpr(1\linewidth-2\tabcolsep)}}{Sterols And Terpenes }\\
1. &
  Salkowski's  \mbox{}\protect\newline (Sterols and Trierpenes) &
  - &
  - &
  - &
  - &
  -\\
2. &
  Copper acetate (Diterpenes) &
  ++ &
  + &
  + &
  - &
  -\\
\tblbottomrule 
\end{tabulary}\par 
\begin{tablenotes}\footnotesize 
    
\item{+indicates the presence of phytochemicals and {\textendash} indicates the absence of phytochemicals}
\end{tablenotes}
\end{threeparttable}

\end{table*}

    
\section{Materials and Methods }
\textbf{Plant sample collection and authentication}

The leaves of \textit{Artabotrys hexapetalus} were gathered from Erode district, Tamilnadu, India. The plant was validated (BSIS/RC/5/23/2016/Tech/2101) by Botanical Survey of India Taxonomist, Southern Regional Centre, Coimbatore, Tamilnadu, India.

\textbf{Preparation of plant extract}

Fresh leaves of \textit{Artabotrys} \textit{ hexapetalus }were collected from Erode district, Tamilnadu, India. Collected fresh leaves were cleaned with sterile water and dried under shade. Dried leaves were subjected to mechanical grinding to obtain a coarse powder.

The coarse powder of \textit{A.hexapetalus} leaves (20 grams) was soaked with 200 ml of different solvents like water, ethanol (alcohol), acetone, chloroform and petroleum ether in the ratio of 1:10 for three days. Then the plant sample was extracted with a muslin cloth and used to analyse the phytoconstituents. 

\textbf{Preparation of aqueous leaves extract of \textit{Artabotrys} \textit{ hexapetalus}}

Large scale aqueous leaves extract, was prepared by soaking 40g leaves powder in 400ml distilled water for three days. Then the plant sample was extracted with a muslin cloth. The filterate obtained was evaporated to dryness in a microwave oven under controlled temperature. Finally, 8 gram of greenish-brown powdered crystals were obtained, and it was kept for later analysis in tightly sealed desiccators.

\textbf{Qualitative phytochemical analysis}

\textit{Artabotrys} \textit{ hexapetalus} leaves extracted with aqueous (water), ethanol (alcohol), acetone, chloroform and petroleum ether were analysed for qualitative phytochemicals (Table~\ref{tw-a6e346233f7a}) using standard procedures \unskip~\citep{911123:20571677,911123:20571680}. 

\textbf{Analysis of total carbohydrates }

Quantitative evaluation of carbohydrates in \textit{Artabotrys} \textit{ hexapetalus} leaves was performed with anthrone reagent using a standard protocol \unskip~\citep{911123:20571664}.

\textbf{Analysis of total proteins }

The protein content of \textit{Artabotrys} \textit{ hexapetalus} leaves was evaluated according to the Lowry method \unskip~\citep{911123:20571668}.

\textbf{Analysis of total amino acids}

Standard procedure was followed to determine the amino acid content of \textit{Artabotrys} \textit{ hexapetalus} leaves \unskip~\citep{911123:20571672} \textbf{.}

\textbf{Analysis of flavonoids}

Aluminium chloride method was used to assess the flavonoids of \textit{Artabotrys} \textit{ hexapetalus} leaves \unskip~\citep{911123:20571681}.

\textbf{Analysis of tannins by Folin- Denis method }

The tannin content of \textit{Artabotrys} \textit{ hexapetalus} leaves was analysed by Folin-Denis method \unskip~\citep{911123:20571662}.

\textbf{Analysis of total phenols }

Assessment of phenols in \textit{Artabotrys} \textit{ hexapetalus} leaves was done by standard procedure \unskip~\citep{911123:20571671}.

\textbf{Statistical analysis }

Quantitative estimation of primary and secondary metabolites were done in triplicates with standards. The results of the quantitative analysis were given as mean \ensuremath{\pm} standard deviation.
    
\section{Results and Discussion}
The result of phytochemical analysis of different solvent extracts of \textit{A.hexapetalus} leaves has been listed in Table~\ref{tw-c8aae4239207}. Among the various solvent extract of \textit{Artabotrys} \textit{ hexapetalus} leaves, aqueous extract revealed more phytoconstituents. Carbohydrates, amino acids, proteins, alkaloids, flavonoids, tannins, diterpenes, phenols, saponins, and glycosides were reported in the aqueous leaves extract of \textit{Artabotrys} \textit{ hexapetalus}. When compared to aqueous extract, other extracts showed fewer phytoconstituents. So the aqueous leaves extract of \textit{Artabotrys hexapetalus} was taken for further studies.

\textbf{Analysis of total carbohydrates}

The amount of carbohydrate in\textbf{\space }aqueous leaves extract of \textit{A.hexapetalus} was found to be 43.16\ensuremath{\pm} 1.0 mg/g extract (Figure~\ref{f-8e93dbf409fe}).


\bgroup
\fixFloatSize{images/d18ce553-3920-47b7-af53-3bace6f753d7-upicture1.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/d18ce553-3920-47b7-af53-3bace6f753d7-upicture1.png}{\includegraphics{images/d18ce553-3920-47b7-af53-3bace6f753d7-upicture1.png}}{}
\makeatother 
\caption{\boldmath {Quantification of Phytoconstituents in aqueous extract of \textit{A.hexapetalus} leaves}}
\label{f-8e93dbf409fe}
\end{figure}
\egroup
\textbf{Analysis} \textbf{ of total proteins}

The total protein content of\textbf{\space }aqueous leaves extract of \textit{A.hexapetalus} was found to be 60.4 \ensuremath{\pm} 0.88 mg/g extract.

\textbf{Analysis of total amino acids}

The amount of amino acid in\textbf{\space }aqueous leaves extract of \textit{A.hexapetalus} was found to be 19.33\ensuremath{\pm} 1.30 mg/g extract.

\textbf{Analysis} \textbf{ of flavonoids}

Flavonoid content of\textbf{\space }aqueous leaves extract of \textit{A.hexapetalus} was found to be 28.3 \ensuremath{\pm} 0.91 mg/g extract. Flavonoids are a potent radical scavenger and possess the metal chelating ability \unskip~\citep{911123:20571670,911123:20571679,911123:20571678}. Flavonoids possess anticancer activity, anti-inflammatory activity and lower the risk of heart disease \unskip~\citep{911123:20571676}.

\textbf{Analysis of tannins}

The tannin content of\textbf{\space }aqueous extract of \textit{A.hexapetalus} leaves was found to be 24.53 \ensuremath{\pm} 1.02 mg/g extract. Tannin plays a protective role against oxidative stress \unskip~\citep{911123:20571674}. Tannins are very effective against microorganisms and parasites. It is used in the treatment of inflammation in mouth and diarrhoea \unskip~\citep{911123:20571675}.

\textbf{Analysis of total phenols}

Phenol content of\textbf{\space }aqueous extract of \textit{A.hexapetalus} leaves was found to be 7.63 \ensuremath{\pm} 0.85 mg/g extract. Plant phenols possess radical scavenging ability and protection against UV radiation \unskip~\citep{911123:20571661}.
    
\section{Conclusions}
Phytochemical analysis of \textit{A.hexapetalus} leaves was performed using different solvent extracts like aqueous, ethanol, acetone, chloroform and petroleum ether. Among the different solvent extracts, an aqueous extract of \textit{A.hexapetalus} leaves revealed the presence of phytoconstituents like carbohydrates, amino acids, proteins, alkaloids, flavonoids, tannins, diterpenes, phenols, saponins and glycosides. Quantitative analysis of carbohydrates, proteins, amino acids, flavonoids and phenols were done using aqueous extract of\textit{ A.hexapetalus} leaves. This study concludes that aqueous extract of \textit{A.hexapetalus} leaves is a potent source of phytochemicals, primary metabolites and secondary metabolites. Secondary metabolites act as antioxidants and scavenge free radicals. \textit{A.hexapetalus} leaves can act as a natural antioxidant.

\textbf{Conflict of Interest}

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

\textbf{Funding Support}

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

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