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\def\RunningAuthor{Anusha Anish et al.}
\firstPage{2990}
\articleType{Original Article}
\receivedDate{02 Nov 2020}
\acceptedDate{12 Dec 2020}
\revisedDate{05 Dec 2020}
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\title{Quantification of phytochemical constituents in sixteen commonly consumed green leafy vegetables}
\author{Anusha~Anish\textsuperscript{1},  
            Sherena~P~A\textsuperscript{2},  
            Annamala~P~T\textsuperscript{*}\textsuperscript{3}, 
            Mukkadan~J~K\textsuperscript{4}~\\[5pt]{\textsuperscript{1}Department of Pathology, PK DAS Institute of Medical Sciences, Hospital and Medical College, Palakkad, Kerala, India}~\\{\textsuperscript{2}Department of Biochemistry, Little Flower Medical Research Centre, Angamaly, Kerala, India}~\\{\textsuperscript{3}Department of Biochemistry, Jubilee Mission Medical College, Thrissur, Kerala, India}~\\{\textsuperscript{4}Little Flower Medical Research Centre, Angamaly, Kerala, India}}

\begin{abstract}
Sixteen green leafy vegetables - curry leaves (M\textit{urraya koenigii}), fenugreek (methi) leaves \textit{(Trigonella foenum-graecum}), coriander leaves (C\textit{oriandrum sativum}), broccoli leaves \textit{(Brassica oleracea}), bay leaves \textit{(Laurus nobilis}), oregano leaves (O\textit{riganum vulgare}), marjoram leaves (O\textit{riganum majorana}), basil leaves (O\textit{cimum basilicum}), pandan leaves (P\textit{andanus amaryllifolius}), sage leaves (S\textit{alvia officinalis}), thyme leaves (T\textit{hymus vulgaris}), lemon grass leaves (C\textit{ymbopogon citratus}), kaffirlime leaves \textit{(Citrus hystrix)} and parsley leaves (P\textit{etroselinum crispum}) were collected and analysed for the determination of major phytochemicals (polyphenols, flavonoids, chlorophills and xanthophylls). Basil leaves, sage leaves and oregano leaves were found to be rich in total polyphenols., basil leaves, mint leaves and thyme leaves were rich in flavonoids., coriander leaves, mint leaves and spinach leaves were rich in chlorophills., and coriander leaves, curry leaves and fenugreek leaves were found to be rich xanthophylls (carotenoids). This information will pave the way for selecting specific vegetables for the isolation and characterisation of individual phytochemical and its application in studying antimutagenic, anti-inflammatory and anticancer activities. 
\end{abstract}\def\keywordstitle{Keywords}

\begin{keywords}Chlorophylls,\newline Flavonoids,\newline Green leafy vegetables,\newline Polyphenols,\newline Xanthophylls
\end{keywords}
\twocolumn[ \maketitle {\printKwdAbsBox}]
\makeatletter{\raggedright\textsuperscript{*}Corresponding Author\par Name:\ Annamala~P~T~\\ Phone:\ +91 94470 86367~\\  Email:\ drannamala@gmail.com
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\section{Introduction}
Fruits, vegetables, and tea beverages contain active ingredients having anticancer and chemopreventive activities. Evidence supports the health benefits of phytochemicals, but, their efficacy and potentiality as medicine is to be addressed further. Phytochemicals are bioactive nutrient chemicals found in fruits, vegetables, grains and other plant foods that provide health benefits and reduce the risk of major chronic diseases \unskip~\citep{985333:21241632}. The phytochemicals derived from green leafy vegetables possess several disease preventing activities such as anti-inflammatory, antidiabetic, antiaging, antimicrobial, antiparasitic, antidepressant, anticancer, antioxidant and wound healing \unskip~\citep{985333:21241612,985333:21241608}. The knowledge of the content of phytochemicals such as carotenoids (xanthophylls), polyphenols, chlorophylls and flavonoids present in vegetables can help in their appropriate selection as per physiological needs. Fruits and vegetables contain polyphenols and flavonoids having antioxidant activity \unskip~\citep{985333:21241616}. Flavonoids present in vegetables, fruits, nuts, seeds, roots, and tea beverages have antioxidant function due to chelating and scavenging of free radicals and inhibition of oxidases \unskip~\citep{985333:21241615}. Many fruits and vegetables contain anticarcinogenic flavonoids \unskip~\citep{985333:21241626}. \unskip~\cite{985333:21241618} showed that anthocyanin flavonoids have antioxidant activities in in vitro studies of cell culture systems such as colon, liver, breast, leukemic cell, and keratinocytes. 

Chlorophylls are reported to have natural cancer prevention and anticarcinogenic effects, protect DNA from damage \unskip~\citep{985333:21241597}, promote weight loss by controlling appetite hormones, improve obesity-related risk factors, and potent anti-inflammatory activities \unskip~\citep{985333:21241594}. Xanthophyll pigments taken through diet are normal constituents in human blood and tissues. \unskip~\cite{985333:21241606} reported that high intake of fruits and vegetables have protective effect against cancer and other chronic diseases. The objective of the study is to quantitatively estimate the contents of major phytochemical constituents (polyphenols, flavonoids, chlorophylls and xanthophylls) in 16 different green leafy vegetables which are commonly consumed by people.
    
\section{Materials and Methods}
\textbf{Sample collection}

Leaves of 16 green vegetables - curry (M\textit{urraya koenigii}),  fenugreek (methi) (T\textit{rigonella foenum-graecum}), coriander (C\textit{oriandrum sativum}), broccoli (B\textit{rassica oleracea}),  bay (L\textit{aurus nobilis}),  oregano (O\textit{riganum vulgare}), marjoram (O\textit{riganum majorana}), basil (O\textit{cimum basilicum}), pandan (P\textit{andanus amaryllifolius}), sage (S\textit{alvia officinalis}),  thyme (T\textit{hymus vulgaris}),  lemon grass (C\textit{ymbopogon citratus}), kaffirlime (C\textit{itrus hystrix}) and parsley (P\textit{etroselinum crispum}) were collected from different regions.

\textbf{Solvents}

Methanol, ethyl alcohol, acetone, hexane, and toluene were purchased from Merck.

\textbf{Reagents and Standards}

Folin and Ciocalteu reagent, sodium nitrite, aluminium chloride, potassium hydroxide, sodium carbonate and sodium sulphate were also purchased from Merck. Pure gallic acid and quercetin were purchased from Sigma.

\textbf{Methods}

All the 16 leaves were washed, dried and ground before analysis and the content of phytochemical constituents measured spectrophotometrically.

\textbf{Total polyphenol content}

Folin-Ciocalteu reagent reacts with polyphenols in plant extracts forming a blue colored complex \unskip~\citep{985333:21241601}. The maximum absorption of the blue complex depends on the alkaline medium and the concentration of phenolic compounds. The reaction is found to be accurate and specific for polyphenolic compounds \unskip~\citep{985333:21241635}. Using this principle, content of total polyphenols in dried leaves was determined by spectrophotometric method \unskip~\citep{985333:21241636}. 0.5 g of the powdered leafy material was refluxed twice with 5 ml methanol and 50 ml water for  1 hr. at 80\ensuremath{^{0}}C. Cooled and filtered quantitatively to a 100 ml volumetric flask and diluted with  water which was used as test solution. 5 ml of the aliquot was pipetted into 25 ml volumetric flask and diluted with water. 50 mg of gallic acid was weighed into 100 ml volumetric flask, dissolved in 70\% methanol and diluted to mark with 70\% methanol and mixed well. 5 ml aliquot was pipetted into a 25 ml volumetric flask, diluted to the mark with deionized water and mixed well. This was used as standard.

A series of 10 ml test tubes containing 1.0 ml water was set up and to each test tube, 1.0 ml each of 70\% methanol (blank), standard solution and sample solution were added respectively followed by 1 ml of Folin - Ciocalteu reagent. To each test tube, 1.0 ml 70\% methanol was added, mixed well and allowed to stand at room temp for 10 min. 3.0 ml of saturated sodium carbonate solution was added to each of the tubes and mixed well. The test tubes were placed in a heated water bath set at 40\ensuremath{^{o}} C for 45 min and after the  reaction, the tubes were quickly cooled in an ice bath to room temperature. This solution was stable for 4 hrs. The absorbance of the two solutions (standard solution and sample solution) was noted at 750 nm against methanol blank. Total polyphenol content was expressed as gallic acid equivalents.

$\begin{array}{l}\mathrm{Total}\;\mathrm{polyphenols}\;\mathrm{in}\;\mathrm g/100\mathrm g=\\\frac{\mathrm{Abs}.\;\mathrm{of}\;\mathrm{sample}\times\mathrm{std}\;\mathrm{weight}\times\mathrm{dilution}\;\mathrm{of}\;\mathrm{sample}\times\mathrm{potency}\;\mathrm{of}\;\mathrm{std}}{\mathrm{Abs}.\;\mathrm{of}\;\mathrm{std}\times\mathrm{dilution}\;\mathrm{of}\;\mathrm{std}\times\mathrm{sample}\;\mathrm{weight}}\end{array} $

\textbf{Total flavonoid content}

Flavonoids form a complex with sodium nitrite in alkaline medium based on the nitration of aromatic ring bearing a catechol group \unskip~\citep{985333:21241598}. On addition of aluminium Al (III), a yellow complex was formed, which then turned red on addition of sodium hydroxide and the absorbance is measured at 510 nm \unskip~\citep{985333:21241629}. Aliquot of the samples for the estimation of flavonoids was prepared by the same procedure as employed for polyphenols. 50 mg of quercetin weighed into 100 ml volumetric flask, dissolved in 70\% methanol and diluted to mark with 70\% methanol and mixed well and 10 ml was pipetted into a 25 ml volumetric flask and diluted to the mark with deionized water and mixed well (standard solution).

A series of 10 ml test tubes containing 4 ml water was set up and to each test tube, 1.0 ml each of 70\% methanol (blank), standard solution and sample solution were added respectively and then 0.3 ml 5\% sodium nitrite was added. After 5 minutes 0.3 ml 10\% aluminium chloride was added to each tube. Again after five minutes 2 ml of 1M sodium hydroxide was added. Mixed well and the absorbance of the two solutions (standard and sample) was measured at 510 nm against methanol blank. Based on the measured absorbance the content of flavonoids was calculated and expressed as quercetin equivalent.

$\begin{array}{l}\mathrm{Total}\;\mathrm{flavonoids}\;\mathrm{in}\;\mathrm g/100\mathrm g=\\\frac{\mathrm{Abs}\;\mathrm{of}\;\mathrm{sample}\times\mathrm{std}\;\mathrm{wt}.\times\mathrm{dilution}\;\mathrm{of}\;\mathrm{sample}\times\mathrm{potency}\;\mathrm{of}\;\mathrm{std}}{\mathrm{Abs}.\;\mathrm{of}\;\mathrm{std}\times\mathrm{dilution}\;\mathrm{of}\;\mathrm{std}\times\mathrm{sample}\;\mathrm{volume}}\end{array} $

\textbf{Total chlorophyll content}

Chlorophyll pigments from the samples were  extracted with methyl alcohol. The content of chlorophylls a and b were estimated by  employing two wave lengths (642.5 nm and 660 nm) which transmit wave-lengths which are  absorbed in different degrees by chlorophylls a and b. 3.0 g of powdered leafy material was sonicated 4-5 times with 50 ml methanol each time for 20 minutes and filtered into a 250 ml standard flask and made up to the mark using methanol. 5 ml of the aliquot was further  diluted to 50 ml with acetone. The absorbance was noted at 642.5 nm and 660 nm.

$\begin{array}{l}\mathrm{Total}\;\mathrm{Chlorophyll}\;\mathrm{content}\%=\\\frac{\left(\mathrm{Abs}.\;\mathrm{at}\;660\times7.12+\mathrm{Absorbance}\;\mathrm{at}\;642.5\times16.8\right)\times250\times50}{\mathrm{Weight}\;\mathrm{of}\;\mathrm{sample}\;\mathrm{taken}\times10000\times5}\end{array} $

\textbf{Total xanthophyll content}

Xanthophylls have specific absorbace at 474 nm. This property is utilised in the estimation of xanthophylls. 3.0 g of representative sample of the ground leafy sample was refluxed with 10 ml hexane, 6 ml ethanol, 6 ml acetone, 7 ml toluene and 2 ml of 40\% methanolic potassium hydroxide in a water bath kept at 56\ensuremath{^{o}}C +/- 0.5\ensuremath{^{o}}C for 20 minutes.

The solution was cooled and 30 ml hexane was added and diluted to the mark with 10\% sodium sulphate solution and allowed to stand in the dark for 1 hour for phase separation. 1 ml of aliquot was pipetted from the upper phase into 50 ml volumetric flask and diluted to the mark with hexane: acetone: methanol (8:1:1) as solvent. The absorbance of the solution was measured at 474 nm for assessing the content of total xanthophylls.

$\mathrm{Total}\;\mathrm{xanthophylls}\;\left(\mathrm g/100\mathrm g\right)=\frac{\mathrm{Absorbance}\;474\times10.602}{\mathrm{Weight}\;\mathrm{of}\;\mathrm{sample}\;\mathrm{in}\;\mathrm{gram}} $

\textbf{Statistical analysis}

Data analysis was done using Microsoft excel 2010. All the data are presented as mean and standard deviation.
    
\section{Results and Discussion}
The results of the analysis of phytochemical constituents: polyphenols, flavonoids, chlorophylls and xanthophylls content are depicted in Table~\ref{tw-83a8fe44e9c1} and represented graphically in Figure~\ref{f-35f429b40435}.


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\caption{\boldmath {Graphical representation of the values of phytochemical analysis of the selected dehydrated green leafy vegetables}}
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\end{figure}
\egroup
The content of polyphenols was high in basil (7.28\ensuremath{\pm}0.02 g/100g), sage (6.60\ensuremath{\pm}0.60 g/100g) and oregano leaves (6.40\ensuremath{\pm}0.38 g/100g). Total flavonoids content was found to be high in  basil (23.72\ensuremath{\pm}0.02 g/100g), mint (22.72\ensuremath{\pm}0.18 g/100g) and thyme leaves (21.95+0.77  g/100g). The total chlorophylls content was high in coriander (4.77\ensuremath{\pm}0.17 g/100g), mint (4.77\ensuremath{\pm}0.18 g/100g), and spinach leaves (4.46\ensuremath{\pm}0.31 g/100g). Total xanthophylls content was found to be high in coriander (1.65\ensuremath{\pm}0.14 g/100g), fenugreek (1.45\ensuremath{\pm}0.19 g/100g) and curry leaves (1.33\ensuremath{\pm}0.20 g/100g). 

Green leafy vegetables in different forms are consumed by people depending on the geographical conditions and availability. Green leafy vegetable are identified as natural sources of antioxidants such as polyphenols, flavonoids, vitamins and carotenoids. The oxycarotenoids such as lutein and zeaxanthin present in green leafy vegetables have been proved to have many disease preventive properties \unskip~\citep{985333:21241620}.

\unskip~\cite{985333:21241631} reported that orange-fleshed sweet potato contained the highest level of total polyphenols. \unskip~\cite{985333:21241605} observed that flavonoids content was high in green leafy vegetables \textit{S androgynus} L\textit{\space } and \textit{P pinnata} of Indonesia.


\begin{table*}[!htbp]
\caption{\boldmath {Values of phytochemical constituents in g/100g - total polyphenols, total flavonoids, total chlorophylls and total xanthophylls contents in dehydrated green leafy vegetables (Values are expressed as mean \ensuremath{\pm}SD)} }
\label{tw-83a8fe44e9c1}
\centering 
\begin{threeparttable}

\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.20520000000000007\linewidth-2\tabcolsep}p{\dimexpr.2022\linewidth-2\tabcolsep}p{\dimexpr.19139999999999997\linewidth-2\tabcolsep}p{\dimexpr.1971\linewidth-2\tabcolsep}p{\dimexpr.20409999999999997\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Sample name  & Total Polyphenol Content (g/100g) & Total Flavonoids Content (g/100g) & Total Chlorophylls Content (g/100g) & Total Xanthophylls Content (g/100g)\\
\tblmidrule 
Basil leaves &
  \cAlignHack 7.28\ensuremath{\pm}0.02 &
  \cAlignHack 23.72\ensuremath{\pm}0.02 &
  \cAlignHack 0.46\ensuremath{\pm}0.02 &
  \cAlignHack 0.45\ensuremath{\pm}0.02\\
Bay leaves &
  \cAlignHack 2.88\ensuremath{\pm}0.40 &
  \cAlignHack 6.83\ensuremath{\pm}0.38 &
  \cAlignHack 0.68\ensuremath{\pm}0.06 &
  \cAlignHack 0.18\ensuremath{\pm}0.06\\
Broccoli leaves &
  \cAlignHack 1.06\ensuremath{\pm}0.18 &
  \cAlignHack 2.20\ensuremath{\pm}0.18 &
  \cAlignHack 3.88\ensuremath{\pm}0.19 &
  \cAlignHack 0.69\ensuremath{\pm}0.19\\
Coriander leaves &
  \cAlignHack 0.79\ensuremath{\pm}0.17 &
  \cAlignHack 2.64\ensuremath{\pm}0.17 &
  \cAlignHack 4.77\ensuremath{\pm}0.17 &
  \cAlignHack 1.65\ensuremath{\pm}0.14\\
Curry leaves &
  \cAlignHack 4.25\ensuremath{\pm}0.20 &
  \cAlignHack 6.42\ensuremath{\pm}0.20 &
  \cAlignHack 0.95\ensuremath{\pm}0.20 &
  \cAlignHack 1.33\ensuremath{\pm}0.20\\
Kaffir lime leaves &
  \cAlignHack 1.50\ensuremath{\pm}0.14 &
  \cAlignHack 2.64\ensuremath{\pm}0.14 &
  \cAlignHack 1.42\ensuremath{\pm}0.15 &
  \cAlignHack 0.53\ensuremath{\pm}0.14\\
Lemon grass &
  \cAlignHack 0.91\ensuremath{\pm}0.34 &
  \cAlignHack 1.66\ensuremath{\pm}0.29 &
  \cAlignHack 0.89\ensuremath{\pm}0.29 &
  \cAlignHack 0.13\ensuremath{\pm}0.04\\
Marjoram leaves &
  \cAlignHack 5.50\ensuremath{\pm}0.24 &
  \cAlignHack 19.18\ensuremath{\pm}0.27 &
  \cAlignHack 1.08\ensuremath{\pm}0.27 &
  \cAlignHack 0.16\ensuremath{\pm}0.03\\
Fenugreek leaves &
  \cAlignHack 1.84\ensuremath{\pm}0.24 &
  \cAlignHack 4.44\ensuremath{\pm}0.24 &
  \cAlignHack 3.94\ensuremath{\pm}0.25 &
  \cAlignHack 1.45\ensuremath{\pm}0.19\\
Mint leaves &
  \cAlignHack 7.01\ensuremath{\pm}0.18 &
  \cAlignHack 22.50\ensuremath{\pm}0.18 &
  \cAlignHack 4.77\ensuremath{\pm}0.18 &
  \cAlignHack 0.29\ensuremath{\pm}0.17\\
Oregano leaves &
  \cAlignHack 6.40\ensuremath{\pm}0.38 &
  \cAlignHack 11.02\ensuremath{\pm}0.38 &
  \cAlignHack 0.48\ensuremath{\pm}0.08 &
  \cAlignHack 0.15\ensuremath{\pm}0.09\\
Pandan leaves &
  \cAlignHack 1.04\ensuremath{\pm}0.27 &
  \cAlignHack 1.05\ensuremath{\pm}0.28 &
  \cAlignHack 1.95\ensuremath{\pm}0.28 &
  \cAlignHack 0.57\ensuremath{\pm}0.26\\
Parsley leaves &
  \cAlignHack 1.04\ensuremath{\pm}0.22 &
  \cAlignHack 0.92\ensuremath{\pm}0.22 &
  \cAlignHack 1.63\ensuremath{\pm}0.22 &
  \cAlignHack 0.22\ensuremath{\pm}0.08\\
Sage leaves &
  \cAlignHack 6.60\ensuremath{\pm}0.60 &
  \cAlignHack 11.63\ensuremath{\pm}0.60 &
  \cAlignHack 0.98\ensuremath{\pm}0.05 &
  \cAlignHack 0.44\ensuremath{\pm}0.06\\
Spinach leaves &
  \cAlignHack 0.84\ensuremath{\pm}0.31 &
  \cAlignHack 2.08\ensuremath{\pm}0.31 &
  \cAlignHack 4.46\ensuremath{\pm}0.31 &
  \cAlignHack 1.20\ensuremath{\pm}0.31\\
Thyme leaves &
  \cAlignHack 4.99\ensuremath{\pm}0.57 &
  \cAlignHack 21.95\ensuremath{\pm}0.77 &
  \cAlignHack 1.32\ensuremath{\pm}0.77 &
  \cAlignHack 0.40\ensuremath{\pm}0.14\\
\tblbottomrule 
\end{tabulary}\par 
\begin{tablenotes}\footnotesize 
    
\item{No of analysis conducted: 3; Method adopted for analysis: Spectrophotometry}
\end{tablenotes}
\end{threeparttable}

\end{table*}
In China, green-leafy vegetable varieties: lettuce (\textit{Lactuca sativa} L), pakchoi (Brassica chinensis L) and spinach (\textit{Spinacia oleracea} L) were analysed to determine chlorophyll content \unskip~\citep{985333:21241589}. \unskip~\cite{985333:21241600} quantified xanthophylls in selected vegetables commonly consumed in the Philippines. Very few reports are available in literature on the comprehensive assessment of flavonoids, polyphenols, chlorophills and xanthophylls in a variety of commonly consumed green leafy vegetables.

Phytochemicals possess biological activities such as antioxidant, antimicrobial, anticancer, modulation of detoxification enzymes, stimulation of immune system, decrease of platelet aggregation and modification of hormone metabolism \unskip~\citep{985333:21241603}. Studies indicate that high intake of polyphenols can decrease the risk of cardiovascular diseases, some forms of cancer and neurodegenerative diseases \unskip~\citep{985333:21241592}.

The mechanisms by which polyphenols exert anticarcinogenic activity may be by the removal of  carcinogenic agents \unskip~\citep{985333:21241613}, modulation of cancer cell signaling \unskip~\citep{985333:21241625}, cell cycle progression \unskip~\citep{985333:21241630}, promotion of apoptosis \unskip~\citep{985333:21241624} and modulation of enzyme activities \unskip~\citep{985333:21241614}. Polyphenols can enhance the activities of glutathione peroxidase, catalase, NADPH-quinone oxidoreductase, glutathione S-transferase and cytochrome P450 enzymes which may lead to the detoxification of carcinogenic agents \unskip~\citep{985333:21241619}. In addition, polyphenols may regulate signaling pathways (MAPK kinase and PI3 Kinase) involved in proliferation of cancer cells \unskip~\citep{985333:21241611,985333:21241596}. 

Flavonoids are hydroxylated phenolic compounds synthesized by plants having a benzo-\ensuremath{\gamma }-pyrone structure \unskip~\citep{985333:21241610}. Flavonoids are found to have potential role in promoting health and preventing chronic degenerative diseases \unskip~\citep{985333:21241616}. \unskip~\cite{985333:21241618} reported that flavonoids have biochemical and antioxidant effects associated with cancer, Alzheimer's disease, and atherosclerosis. Flavonoids have anti-oxidant, anti-inflammatory, anti-mutagenic, antimicrobial, anti-carcinogenic, and vascular activities.

Flavonoids are also known to be potent inhibitors of aldose reductase, Ca\ensuremath{^{2+}}-ATPase, xanthine oxidase, cyclo-oxygenase (COX), lipoxygenase and  phosphoinositide 3-kinase \unskip~\citep{985333:21241628,985333:21241604}. Flavonoids may act by exerting direct modulation of protein and lipid kinase signaling pathways \unskip~\citep{985333:21241607}, via the inhibition of MAPK signaling cascades, such as p38 or ERK1/2 \unskip~\citep{985333:21241622}.

Chlorophylls and their derivatives have wound healing \unskip~\citep{985333:21241595}, and anti-inflammatory activities \unskip~\citep{985333:21241633}. But, recent research is more focused on its role as potent anti-mutagen and anti-carcinogen \unskip~\citep{985333:21241617}. The actions of chlorophylls and their derivatives can be attributed to various mechanisms including antioxidant activity, modifier of genotoxic effect, inhibition of cytochrome P450 enzymes, induction of phase II enzymes, increased level of glutathione S-transferase, cell differentiation, cell arrest and apoptosis. Chlorophills inhibit lipid peroxidation \unskip~\citep{985333:21241623}, protein oxidation, DNA damage, and membrane damage \unskip~\citep{985333:21241593}.

Xanthophyll pigments mainly include lutein, zeaxanthin, \ensuremath{B }-cryptoxanthin, capsanthin, astaxanthin, and fucoxanthin \unskip~\citep{985333:21241599}. Administration of astaxanthin showed significant neuroprotective effects in an ischemic model of stroke in mice \unskip~\citep{985333:21241637}. This effect of astaxanthin may be due to the antioxidant activity by regulating glutathione peroxidase and catalase activities, decreased malondialdehyde and reactive oxygen species formations in focal cerebral ischemia in rats \unskip~\citep{985333:21241609}. It is also shown that astaxanthin protect glutamate-induced cytotoxicity in HT22 cells of Alzheimer's disease model by decreasing caspase activation and affecting the Akt/GSK-3B signaling pathway \unskip~\citep{985333:21241591}. Fucoxanthin, a xanthophyll, showed  neuroprotective effects by activating the Nrf2/HO-1 signaling and inducing Nrf2 nuclear translocation against cerebral ischemic/reperfusion injury \unskip~\citep{985333:21241627}.

Lutein, another xanthophyll, is also reported to have neuroprotective and antidepressant effects by  ameliorating the involvement of oxidative stress \unskip~\citep{985333:21241590}. Lutein pigment is distributed in the regions of human brain that controls cognition \unskip~\citep{985333:21241621}. Lutein and zeaxanthin are found to influence progression and minimize the effects of age-related macular degeneration, a multi factor disease mainly caused by inflammation and oxidative stress \unskip~\citep{985333:21241634}. Astaxanthin when applied on the human adenocarcinoma cell lines such as AGS, SNU-1, MKN-45, and KATO-III was found to suppress the proliferation of KATO-III and SNU-1 cells, but, did not affect the cell cycle of AGS and MKN-45 cell lines \unskip~\citep{985333:21241602}. 

The phytochemicals present in green leafy vegetables have various health benefits. Quantification of phytochemicals in commonly consumed green leafy vegetables can help in selecting specific leaves for the isolation and quantification of these constituents and to study their pharmaceutical actions and will also enable the appropriate selection of vegetables as per physiological needs.
    
\section{Conclusions}
Prevention and cure of diseases by using phytochemicals are well known. Fruits and vegetables are natural sources of phytochemicals that exhibit many biological properties which are beneficial to human health. Phytochemicals neutralize the harmful effects of free radicals and thus help in the prevention of many diseases. Phytochemicals are reported to have anti-oxidant, free-radical scavenging, anti-inflammatory, antibacterial, antiviral, anti-aging and anti-carcinogenic activities. This study highlights the dietary sources of various phytochemicals. This information will help in selecting specific vegetables for the isolation and quantification of individual phytochemicals and its application in studying their antimutagenic, anti-inflammatory and anticancer activities.
    
\section{Acknowledgement}
The authors acknowledge the support of Little Flower Medical Research Centre, Kerala and Care Kerala Limited, Kerala.

\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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