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\def\RunningAuthor{Heni Setyowati ER et al.}
\firstPage{3755}
\articleType{Original Article}
\receivedDate{06.07.2019}
\acceptedDate{29.10.2019}
\revisedDate{13.10.2019}
\journalVolume{10}
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\journalDoi{ijrps.v10i4.1765}
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\def\journalTitle{International Journal of Research in Pharmaceutical Sciences}

\title{GC-MS analysis of phytochemical components in the ethanolic extract of Acanthus illicifolius from mangrove forest Purworejo Indonesia}
\author{Heni~Setyowati~Esti~Rahayu\textsuperscript{*}\textsuperscript{1},  
            Nasruddin\textsuperscript{2},  
            Kartika~Wijayanti\textsuperscript{1},  
            Puspita~Septie~Dianita\textsuperscript{3}, 
            Prasojo~Pribadi\textsuperscript{3}~\\[5pt]{\textsuperscript{1}Department of Maternity Nursing, Faculty of Health Science, Universitas Muhammadiyah Magelang, Indonesia}~\\{\textsuperscript{2}Department of Medical Laboratory Science, Faculty of Nursing and Health Sciences, Universitas Muhammadiyah Semarang, Indonesia}~\\{\textsuperscript{3}Department of Pharmacy, Faculty of Health Science, Universitas Muhammadiyah Magelang, Indonesia}}

\begin{abstract}
Jeruju has the potential as a source of bioactive components. Steroids are commonly found in mangroves, especially in the mangrove leaves (Acanthus Illicifolius) or better known as jeruju plants. However, the use of mangroves in Indonesia as a medicinal plant is still very limited. Therefore, it is necessary to explore the bioactive components of mangrove plants. The purpose of this study was to determine the active content of jeruju leaves (Acanthus Illicifolius) as an analgesic with the Gas Cromatographic Mass Spectrometry (GC-MS) analysis method. The design of this study was experiments with the stages are sampel preparation, extraction and fractionation and GC-MS analysis. The results of the Acanthus Illicifolius study contain protein, carbohydrate, saponin, sterooid, triterpenoids, and phenol. Thin Layer Chromatography (TLC) results showed that the compound separation was evidenced by the presence of spots as a result of elution with the detection of ferric chloride spray at UV 254 nm and 365 nm. Blotches eluted fluorescently on UV 365 nm with spot colors of phenolic compounds in visible black gray with phenol Rf detected 0.85. At the retention time of 40.22 to 43.89 minutes, there were 3 peaks of a large chromatogram and indicated the presence of topiramate, budesonide, and spingosine. 
\end{abstract}\def\keywordstitle{Keywords}

\begin{keywords}Acanthus Illicifolius,\newline Phytochemical,\newline GC-MS,\newline Mangrove
\end{keywords}
\twocolumn[ \maketitle {\printKwdAbsBox}]
\makeatletter{\raggedright\textsuperscript{*}Corresponding Author\par Name:\ Heni~Setyowati~Esti~Rahayu~\\ Phone:\ ~\\  Email:\ henisetyowati@ummgl.ac.id
        \par}\vspace*{-11pt}\hrulefill\par{\fontsize{12}{14}\selectfont ISSN: 0975-7538}\par% 
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\section{Introduction}
One of the substances contained in plants that can reduce or eliminate pain, namely steroids. Steroids are important compounds in the pharmaceutical field and are widely used in medicine such as anti-bacterial, anti-inflammatory, and as a pain reliever \unskip~\citep{634378:14858189}. Steroids are found in mangroves, especially in the mangrove leaves (Acanthus Illicifolius) or better known as jeruju plants. Jeruju (Acanthus Illicifolius) is one of the mangrove species. Jeruju is naturally found in wetland areas in the river mouth and is classified as emergent aquatic plants\unskip~\citep{634378:14858191}. Mangroves in Indonesia are the largest in the world, both in terms of an area quantity (\ensuremath{\pm}42,550 km\ensuremath{^{2}}) and a number of species (\ensuremath{\pm}45 species) \unskip~\citep{634378:14858200}. However, the medicinal value of mangrove plants has not been studied in several countries, one of which is in Indonesia. Moreover, the use of mangroves in Indonesia as a medicinal plant is still very limited. 

Jeruju has the potential as a source of bioactive components. Jeruju has metabolite content, with the chemical composition of alkaloids, flavonoids, fatty acids, steroids, lignans, and phenol components, terpenoids\unskip~\citep{634378:14858192,634378:14858201} and megastigmane glycosides \unskip~\citep{634378:14858202}. Several studies have reported that Acanthus Illicifolius has pharmacological activity including hepatoprotective \unskip~\citep{634378:14858182} , anticancer \unskip~\citep{634378:14858183,634378:14858187}, antiulcer \unskip~\citep{634378:14858185}, antibacterial \unskip~\citep{634378:14858196} and antileismania \unskip~\citep{634378:14858195}. The wide spectrum of activities, active components, and diversity of mangrove species can make it an endless source of active ingredients to be explored. The magnitude of this potential can be used as a comparative superior product of state revenue \unskip~\citep{634378:14970187}. 

Exploration and research on the potential of mangroves as medicinal raw materials needs to be continued. Therefore,  it is necessary to explore the bioactive components of mangrove plants in each species. This study tries to increase the medicinal value of mangroves by providing evidence-based in the form of information on the active compounds of Jeruju plants (Acanthus Illicifolius).  The purpose of this study was to determine the active content of Jeruju (Acanthus Illicifolius) with Gas Chromatography-Mass Spectrometry analysis method. 
    
\section{Materials and Methods}
\textbf{Sample preparation}

The samples used in this study were mangrove leaf samples (Acanthus Illicifolius) obtained from Demang Gedi Mangrove Forest, Purworejo Regency. The samples that have been obtained are then washed thoroughly, dried, then milled to form powder.

\textbf{Extraction and Fractionation}

Mangrove leaf powder (Acanthus Illicifolius) was macerated using 95\% ethanol for 5x24 hours, then filtered and evaporated with a rotary evaporator to obtain a concentrated extract, solvent-free and brown in color. Ethanol extract of mangrove leaves (Acanthus Illicifolius) was then fractionated until the hexane fraction was obtained.

\textbf{GC-MS Analysis}

The hexane fraction from the ethanol extract of mangrove leaves (Acanthus Ilicifolius) was analyzed using GC-MS (Mass Gas Spectroscopy). GC-MS analysis plays a key role in the analysis of unknown plant components \unskip~\citep{634378:14858198}. As much as 1 \ensuremath{\mu}l of hexane fraction from the ethanol extract of mangrove leaves (Acanthus Illicifolius) was used in GC-MS for analysis of different compounds. Chromatographic instruments and conditions were carried out on the GC-MS system. Samples of 1 \ensuremath{\mu}l were injected into GC-MS, then the columns used were capillary model agilent number 19091S-433 HP-5MS 5\% Phenyl Methyl Siloxane with 30 m length, 250 \ensuremath{\mu}m diameter and 0.25 \ensuremath{\mu}m thickness. 

The oven temperature used is between 100-220\ensuremath{^{\rm O}}C. The rate of increase in temperature is 15\ensuremath{^{\rm O}}C / minute, and the flow rate is 1.0 ml/minute. The carrier gas is 10.5 psi pressurized helium, and the total rate is 140 ml/minute, and the split ratio is 1:50. Components evaluated will be detected in the mass detector. The known spectrum components of compounds will be stored in the NIST library and determine in the name of the compound, molecular weight, and included in the class of compounds such as triterpenoids, alkaloids, flavonoids, lermic acid, phenols, and others which are useful compounds for GC-MS analysis \unskip~\citep{634378:14858188}.
    
\section{Results and Discussion}
To determine the content of the compounds present in the hexane fraction of the mangrove ethanol extract (Acanthus Illicifolius), it was necessary to analyze it using the GC-MS analysis method. The results of the analysis using Gas Chromatographic Mass Spectrometry (GC-MS) can be seen in Table~\ref{tw-1ad1db7a69c1}.

\textbf{Phytochemical Screening}

The phytochemical analysis results were given in Table~\ref{tw-6f5a3d52907c}, which shows that this plant Acanthus Illicifolius contains protein, carbohydrate, saponin, sterooid, triterpenoidsdanfenol. To determine the separation of compounds, Thin Layer Chromatography (TLC) identification tests were carried out with the results, as shown in Figure~\ref{f-5361794188a9} .


\begin{table*}[!htbp]
\caption{\boldmath {Screening of Phytochemical Analysis of Ethanolic Extract Acanthus ilicifolius} }
\label{tw-6f5a3d52907c}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.564\linewidth-2\tabcolsep}p{\dimexpr.436\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Test & Result\\
\tblmidrule 
Protein {\textendash} xanthoprotein &
  +\\
Carbohydrate {\textendash} molish &
  +\\
Saponin &
  +\\
Sterol {\textendash} libermanbauchard &
  -\\
Triterpenoid &
  +\\
Fenol &
  +\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {GC-MS Analysis of ethanolic extract Acanthus ilicifolius} }
\label{tw-1ad1db7a69c1}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.0809\linewidth-2\tabcolsep}p{\dimexpr.1529\linewidth-2\tabcolsep}p{\dimexpr.36620000000000005\linewidth-2\tabcolsep}p{\dimexpr.2485\linewidth-2\tabcolsep}p{\dimexpr.1515\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}No.  & Ret.Time \mbox{}\protect\newline min & Hit \mbox{}\protect\newline  & Area  \mbox{}\protect\newline counts*min & Rel. Area  \mbox{}\protect\newline \textbf{\%}\\
\tblmidrule 
1  &
  1,68 &
  D-Glucosyl-{\ss}1-1'-D-erythro-sphingosine &
  156129,185 &
  0,95\\
2  &
  33,38 &
  D-Glucosyl-{\ss}1-1'-D-erythro-sphingosine &
  85719,422 &
  0,52\\
3  &
  36,75 &
  D-Glucosyl-{\ss}1-1'-D-erythro-sphingosine &
  125711,878 &
  0,77\\
4  &
  39,47 &
  D-Glucosyl-{\ss}1-1'-D-erythro-sphingosine &
  174208,756 &
  1,06\\
5  &
  39,89 &
  9S,15S-Dihydroxy-11-oxothromboxa-5Z,13E,17Z-trienoic acid &
  316697,981 &
  1,93\\
6  &
  40,22 &
  Topiramate &
  3067696,597 &
  18,68\\
7  &
  40,41 &
  9-Oxo-15S-hydroxy-5Z,8(12),13E,17Z-prostatetraenoic acid &
  388786,063 &
  2,37\\
8  &
  42,45 &
  Budesonide &
  410972,511 &
  2,50\\
9  &
  42,82 &
  D-Glucosyl-{\ss}1-1'-D-erythro-sphingosine &
  148678,921 &
  0,91\\
10  &
  43,89 &
  D-Lactosyl-{\ss}1-1'-D-erythro-sphingosine &
  6622679,105 &
  40,34\\
11  &
  44,04 &
  D-Glucosyl-{\ss}1-1'-D-erythro-sphingosine &
  1217324,578 &
  7,41\\
12  &
  44,19 &
  9-Oxo-15S-hydroxy-5Z,8(12),13E,17Z-prostatetraenoic acid &
  2562621,816 &
  15,61\\
13  &
  44,53 &
  D-erythro-C18-sphingosine &
  1006239,954 &
  6,13\\
14  &
  44,90 &
  9S,15S-Dihydroxy-11-oxothromboxa-5Z,13E,17Z-trienoic acid &
  134898,453 &
  0,82\\
\multicolumn{3}{p{\dimexpr(.60\linewidth-2\tabcolsep)}}{Total:} &
  16418365,221  &
  100,00 \\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

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\caption{\boldmath {TLC Ethanolic Extract of Acanthus ilicifolius Leaves }}
\label{f-5361794188a9}
\end{figure}
\egroup

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\makeatother 
\caption{\boldmath {GC-MS chromatogram for defatted ethanolic extract of Acanthus ilicifolius}}
\label{f-696e8a9dd753}
\end{figure}
\egroup
Based on the results of Thin Layer Chromatography (TLC), as shown in Figure~\ref{f-5361794188a9}, it was seen that the separation of compounds was evidenced by the presence of spots as a result of elution with the detection of ferric chloride spray at 254 nm and UV 365 nm UV. Blotches eluted fluorescently on UV 365 nm with spot colors of phenolic compounds in visible black gray with phenol Rf detected 0.85.

\textbf{GC-MS} \textbf{\space }

Besides the characterization using phytochemical screening analysis, the ethanol extract obtained was also characterized using GC-MS. Based on the analysis using GC-MS, it shows the identification of compounds in the form of budesonide. Based on Figure~\ref{f-696e8a9dd753} above, it can be seen that at the first 1.5 minutes, no chromatogram peak has been identified, which identifies the presence of certain compounds. At retention times between 1.68 to 39.89 minutes, the presence of a chromatogram peak representing the presence of spingosin and trienoic acid. At retention time of 40.22 to 43.89 minutes, there were 3 large peaks of a chromatogram and indicated the presence of topiramate, budesonide, and spingosine based on mass spectrometry and molecular structure images of compounds that appeared in the GC-MS database. Budesonide is a group of alkaloids. While the retention time between 44.04 to 44.90 shows the presence of a chromatogram that represents the presence of prostatetraenoic acid, trienoic acid, and spingosin. As for the results of gas chromatography analysis in ethanol extract in full as in Table~\ref{tw-1ad1db7a69c1}. 

The results of GC-MS analysis of ethanol samples removed from this study indicate the presence of D-Glucosyl-{\ss}1-1'-D-erythro-sphingosine; 9S, 15S-Dihydroxy-11-oxothromboxa-5Z, 13E, 17Z-trienoic acid; Topiramate; 9-Oxo-15S-hydroxy-5Z, 8 (12), 13E, 17Z-prostatetraenoic acid; Budesonide; D-Lactosyl-{\ss}1-1'-D-erythro-sphingosine; D-erythro-C18-Sphingosine. The presence of budesonide was observed at RT: 42.45 minutes, less concentration caused the peak area to be largely undetectable depending on the chemical and physical properties of the system they added, and an analytical protocol adopted to monitor lipids. Oxidation may not be enough to make a valid judgment. 

The results of the phytochemical screening analysis showed that this plant contained protein, carbohydrate, saponin, sterooid, triterpenoids, and phenol. In the thin layer chromatography test showed that Acanthus Illicifolius ethanol extract contained phenol. These results are supported by research conducted by \unskip~\citep{634378:14858184}, which shows that Ailicifolius is rich in long-chain alcoholic sources, terpenoids, steroids, and triterpenoidalsaponins. Several phytochemical studies revealed the presence of 2-benzoxazolinone, glucosidelignans, benzoxazinoideglucoside, flavone glycosides, and phenylhanoid glycosides in this plant \unskip~\citep{634378:14858193}. Ethanol extract of leaves containing methylapigenin 7-o-\ensuremath{\beta }-D-glucuronate, flavone glycosides \unskip~\citep{634378:14858199}. Phytochemical analysis Ailicifolius shows the presence of saponins, tannins, cardiac glycosides, terpenoids, flavanoids, anthraquionones, and alkaloids \unskip~\citep{634378:14858197}. Another study conducted by \unskip~\cite{634378:14858190} showed that methanol extract produced the maximum number of phytoconstituents for the leaves of this plant. Tannin, glycosides, flavonoids, proteins, carbohydrates, terpenoids, phenols, steroids, and positive saponins are found in methanol extract, while alkaloids are only found in water and ethanol extracts. \unskip~\cite{634378:14858186} conducted a phytochemical analysis of 7 types of mangroves from studies showing plants. These contain triterpenoids, cholesterol, phytol, squalene, and phytosterol from six mangrove leaves, and triterpenoids are the largest constituents for A. Ilicifolius. 

GC-MS analysis of this study shows the presence of D-Glucosyl-{\ss}1-1'-D-erythro-sphingosine; 9S, 15S-Dihydroxy-11-oxothromboxa-5Z, 13E, 17Z-trienoic acid; Topiramate; 9-Oxo-15S-hydroxy-5Z, 8 (12), 13E, 17Z-prostatetraenoic acid; Budesonide; D-Lactosyl-{\ss}1-1'-D-erythro-sphingosine; D-erythro-C18-Sphingosine. Previous studies showed different results, in a study conducted by \unskip~\citet{634378:14858197} which showed the presence of 1,2-Benzisoxazol-3-(2H)-one, 2-chloroethyl linoleate, Tricyclo (7,16)] triacontane, 1 (2 ..., 2H Benzocyclohepten-2-one, decanhydro-9-, Retinoyl-beta-6', 3'-lactone. Seven lignanglucosides, two phenylethanoid glycosides, and five benzoxazinoidglucosides has been isolated from the ilicifolius A plant \unskip~\citep{634378:14858194,634378:14858193}. A new acanthicifoline alkaloid has also been reported to be successfully isolated from this plant \unskip~\citep{634378:14858184}. The difference in content between this study and previous research can be due to differences in the use of chemical constituents used for isolation of compounds from the Acanthus ilicifolius plant: Use of different parts of plants such as roots, leaves, air parts, stems, pods will produce different chemical groups\ensuremath{^{16}}. Suspected as a biomarker compound in this study budesonide had an effect of decreasing in vitro production from LTB4 (\ensuremath{-}27.9\%, \ensuremath{-}23\%), PGE2 (\ensuremath{-}75.5\%, \ensuremath{-}44\%), and IL-1\ensuremath{\beta } (\ensuremath{-}84.2\%, \ensuremath{-}52\% ) and inhibits the production of polyclonal stimulated cytokines by unselected lymphocytes and monocytes stimulated by LPS. Previous research stated that large amounts of stigmasterol and steroids were found in ilicifoliusA plants, which have a hypercholesterolemic effect\ensuremath{^{4}}. 2-Benzoxazoline is a synthetic compound widely used as a central nervous system depressant, also shows antipyretic activity, hypnosis and muscle relaxants have been isolated from this plant \unskip~\citep{634378:14858184,634378:14858202}. A detailed study of A. Ilicifolius and its phenolic fraction relating to anti-inflammatory activities required. Further research developed towards phytochemical characterization of the A. Ilicifolius fraction and isolation-identification of responsible bioactive compounds.
    
\section{Conclusion}
Acanthus Illicifolius contains protein, carbohydrate, saponin, sterooid, triterpenoids, and phenol. Thin Layer Chromatography (TLC) results showed that the compound separation was evidenced by the presence of spots as a result of elution with the detection of ferric chloride spray at 254 nm and UV 365 nm UV. Blotches eluted fluorescently on UV 365 nm with spot colors of phenolic compounds in visible black gray with phenol Rf detected 0.85. At the retention time of 40.22 to 43.89 minutes, there were 3 peaks of a large chromatogram and indicated the presence of topiramate, budesonide, and spingosine. 
\section*{Acknowledgement}The Director of Research and Community Service, the Ministry of Research and Technology of Higher Education who has provided research funding. The Institute for Research, Development, and Community Service of Universitas Muhammadiyah Magelang has facilitated this research activity.
    

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