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\def\RunningAuthor{Renugadevi Karthikeyan and Julius Amaldas}
\firstPage{1840}
\articleType{Original Article}
\receivedDate{10 Jan 2020}
\acceptedDate{25 Feb 2020}
\revisedDate{11 Feb 2020}
\journalVolume{2020, 11}
\journalIssue{2}
\journalDoi{ijrps.v11i2.2089}
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\title{GC-MS Analysis of Bioactive Compounds of \textit{Artemesia annua} and Assessment of its anti-proliferative activity against Human Cancer Cell Lines}
\author{Renugadevi~Karthikeyan\textsuperscript{*}\textsuperscript{1}, 
            Julius~Amaldas\textsuperscript{2}~\\[5pt]{\textsuperscript{1}Department of Biochemistry, Prince Shri Venkateshwara Arts and Science College, Gowrivakkam, Chennai -600073, Tamilnadu, India}~\\{\textsuperscript{2}Department of Biochemistry, Sree Balaji Dental College \& Hospital, Chennai - 600 100, Tamilnadu, India}}

\begin{abstract}
 Plant essential oil is found to possess many beneficial effects in the field of medicine. Especially essential oil derived from different species of plants is reported to have specific activity against cancer. \textit{Artemesiaannua} is an annual herb belonging to Asteraceae family has been traditionally used for treating several ailments. It is a conventional medicinal plant used in the treatment of chloroquine-resistant and cerebral malaria. The major aim of this study was to evaluate the anticancer potential of this plant against cancer cell line. HydrodistilledHydro distilled volatile oil obtained from aerial parts of \textit{A.annua} was analysed by GCMS. The majority of the compounds derived from essential oil of \textit{Artemesiaannua} are monoterpenes and sesquiterpenes. Artemisinin, the active component of Artemesiaannua is a sesquiterpenoid lactone containing unusual endoperoxide bridge that shows remarkable activity against numerous tumour growth and metastases. The pleiotropic nature of artemisinin induces oxidative stress, anti-angiogenetic effect and apoptosis triggering on cancer cells. The anti-proliferative effect of essential oil derived from \textit{Artemesiaannua} on human cancer cell lines like MCF-7, HT29 and AGS was done by MTT assay. Results shows the essential oil of artemisia annua have cytotoxic potential by inhibiting cell growth in a dose dependent manner. However, further investigations in isolating active cytotoxic components and understanding its molecular mechanisms will help in therapeutic management of cancer.
\end{abstract}\def\keywordstitle{Keywords}

\begin{keywords}Artemesiaannua,\newline Monoterpenes,\newline Sesquiterpenes,\newline Antiproliferative
\end{keywords}
\twocolumn[ \maketitle {\printKwdAbsBox}]
\makeatletter{\raggedright\textsuperscript{*}Corresponding Author\par Name:\ Renugadevi~Karthikeyan~\\ Phone:\ ~\\  Email:\ krenu2007@gmail.com
        \par}\vspace*{-11pt}\hrulefill\par{\fontsize{12}{14}\selectfont ISSN: 0975-7538}\par% 
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\section{Introduction}
\textit{Artemesia annua} belongs to the plant family of Astraceae is an annual short day plant with a strong fragnancefragrance origin to Asia and Eastern European countries. This aromatic species have many potent phytochmicals has been proven to possess anti-inflammatory, anti- microbial and antioxidant activities \unskip~\citep{708756:16803486}. This odorous herb is the natural source of Artemisinin, a sesquiterpene lactone with an ethnoproxide bridge responsible for its antimalarial activity \unskip~\citep{708756:16803476}, especially in cerebral malaria for centuries. Essential oil of \textit{A.annua} shows characteristic sweet aromas has been describes as fresh, grassy and bitter with acamphoraceous nuance that brings more commercial value \unskip~\citep{708756:16803477}.

 The terpenoids detected in the aerial parts of Artemesia include monoterpenes such as \ensuremath{\alpha }-phene, \ensuremath{\alpha }-terpinene and camphene. Sesquiterpens such as \ensuremath{\beta }-caryophyllene, germacrene, \ensuremath{\alpha }- copaeaeand \ensuremath{\alpha } -cerbebene and oxygenated terpenes such as Eucalypt, Camphor \unskip~\citep{708756:16803485}. Most of these compounds are regarded as aroma active and show characteristic odour. The volatile oils extracted from this compositae plant have high utilisation value. Here we report the extraction and identification of sesquiterpene from the leaves of \textit{A. annua} extract and studies for their antiproliferative effect against to human cancer cell lines namely, MCF-7, HT29 and AGS.

\textbf{Materials and Methods}

Leaves of \textit{Artemesia annua} were collected from Bangaluru, Karnataka. It was authenticated and voucher Specimen (No: PARC2017/3596) was retained for further reference. Leaves were cleant shade dried and the dry mass of the plant was hydrodistilledhydro distilled in a Clevenger type apparatus for 3 hours and the essential oil was isolated. The oil was separated from the aqueous phase with Dichloromethane and stored at 4\ensuremath{^{0}}C until further use. The yield was 0.39\% (V/M) on fresh weight bases.

\textbf{GCMS Analysis}

 GC was performed on Perkin Element auto XL system using 50m\ensuremath{\times}0.32mm film thickness as stationary phase and helium as carrier gas at a flow rate 2m /min. 0.1\ensuremath{\mu}l specimen was injected with an injector temperature of 200\ensuremath{^{0}} C. Initially temperature was programmed from 100\ensuremath{^{0}} C to 280\ensuremath{^{0}} C at 3 C/ min. Detector used was FID and MS was done at 70ev. Computer matched spectra with standard library were used to predict the compounds \unskip~\citep{708756:16803488}.

\textbf{Cell Culture}

 Cell lines MCF -7, HCT116, AGS were obtained from NCCS, cell repository, Pune, India. Cells were routinely cultured in DMEH with 1\% Penicillin- Streptomycin and maintained in a CO\ensuremath{_{2}} incubator at 37\ensuremath{^{0}} C.

\textbf{Cell Viability Assay}

 Cell Viability was assessed by seeding cells in 96 well plates at 1x10 densitydensities and allowed to attach overnight. The essential oil of \textit{A. annua} was dissolved in cell culture grade DMSO and dilutions made with cell culture media. Control cells had a maximum 0.1\% of DMSO at any concentration. Cells were treated with an increasing range of essential oil in various doses, (initially at logarithmic concentrations and then at various increasing doses) and were treated for 24 hrs and 48 hrs. 20\ensuremath{\mu}l of MTT in 5mg/ ml in culture media was added to the wells and incubated in dark for 3 hrs. Then so formed formazon crystal are dissolved in 15uml of DMSO and read at 540nm. The OD obtained at 540 nm was used to calculate the percentage of cell viability.


\begin{table*}[!htbp]
\caption{\boldmath {Shows the qualitative analysis of phytochemicals present in essential oil of A.annua characterised using GC-MS analysis} }
\label{tw-05a5e3f09a8d}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.08090000000000002\linewidth-2\tabcolsep}p{\dimexpr.28719999999999995\linewidth-2\tabcolsep}p{\dimexpr.15709999999999999\linewidth-2\tabcolsep}p{\dimexpr.17919999999999998\linewidth-2\tabcolsep}p{\dimexpr.29560000000000002\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}S.No & constitutent  &  RI & Molecular formula  & Biological activity\\
\tblmidrule 
1. &
  \ensuremath{\alpha }-pinene &
  928 &
   C\ensuremath{_{10}}H\ensuremath{_{16}} &
  Bactericidal activity\\
2. &
  Camphene &
  939 &
   C\ensuremath{_{10}}H\ensuremath{_{16\ }} &
  Hypolipedimic activity\\
3. &
  \ensuremath{\beta }-pinene &
  964 &
   C\ensuremath{_{10}}H16  &
  Bactericidal activity\\
4. &
  Myrcene &
  992 &
   C\ensuremath{_{10}}H\ensuremath{_{16}} &
  Antibacterial activity\\
5. &
  \ensuremath{\gamma }-Terpinene &
  1001 &
   C\ensuremath{_{10}}H\ensuremath{_{16}} &
  Antiproliferative activity\\
6. &
  Linalool &
  1006 &
   C\ensuremath{_{10}}H\ensuremath{_{18}}O  &
  Anti-inflammatory activity\\
7. &
  1,8-Cineole &
  1016 &
   C\ensuremath{_{10}}H\ensuremath{_{18}}O  &
  Antimicrobial activity\\
8. &
  Pinicamphone &
  1142 &
  C\ensuremath{_{10}}H\ensuremath{_{16}}O  &
  Antimicrobial activity\\
9. &
  \ensuremath{\alpha }-Terpineole &
  1181 &
  C\ensuremath{_{10}}H\ensuremath{_{18}}O  &
  Anticonvulsant activity\\
10. &
  Artemesia ketone &
  1192 &
  C\ensuremath{_{10}}H\ensuremath{_{16}}O  &
  Antimalarial activity\\
11. &
  Carvone &
  1223 &
  C\ensuremath{_{10}}H\ensuremath{_{14}}O  &
  Antimicrobial activity\\
12. &
  Ascaridol &
  1276 &
  C\ensuremath{_{10}}H\ensuremath{_{16}}O\ensuremath{_{2}} &
  Antitumor activity\\
13. &
  Undeane &
  1390 &
  C\ensuremath{_{11}}H\ensuremath{_{24}} &
  Antiviral activity\\
14. &
  3,5-Cycloheptadieny1-one &
  1403 &
  C\ensuremath{_{7}}H\ensuremath{_{6}}O\ensuremath{_{2}} &
  Antihypertensive activity\\
15. &
  \ensuremath{\alpha }-Guainene &
  1419 &
  C\ensuremath{_{15}}H\ensuremath{_{24}} &
  Antimicrobial activity \\
16. &
  Z-\ensuremath{\alpha }-Bisabolene &
  1490 &
  C\ensuremath{_{15}}H\ensuremath{_{24}} &
  Antioxidant activity\\
18. &
  \ensuremath{\beta }-Selinene &
  1531 &
  C\ensuremath{_{15}}H\ensuremath{_{24\ }} &
  Antioxidant activity\\
19. &
  Junipene &
  1598 &
  C\ensuremath{_{15}}H\ensuremath{_{26}}O  &
  Antibacterial activity\\
20.  &
  \ensuremath{\gamma }-Elemene &
  1626 &
  C\ensuremath{_{15}}H\ensuremath{_{24\ }} &
  Antimicrobial activity\\
21. &
  Germacrene B &
  1636 &
  C\ensuremath{_{15}}H\ensuremath{_{24}} &
  Antibacterial activity\\
22. &
  Humulene isomer &
  1640 &
  C\ensuremath{_{15}}H\ensuremath{_{24\ }} &
  Antineoplastic activity\\
23. &
  Caryophyllene oxide &
  1675 &
  C\ensuremath{_{15}}H\ensuremath{_{24}}O  &
  Antimicrobial activity\\
24. &
  \ensuremath{\alpha }-Cadinol &
  1681 &
  C15H\ensuremath{_{26}}O  &
  Anti-fungal activity\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\bgroup
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\makeatother 
\caption{\boldmath {MTT Assay of essential oil of \textit{A. annua} treated cancer cell lines}}
\label{f-8495bfa46d58}
\end{figure*}
\egroup
Figure~\ref{f-8495bfa46d58} Shows the cell viability analysis using MTT test. Cell viability was tested using essential oil of \textit{A. annua }plant extract and was tested using cell lines HT-29, MCF-7 and AGS respectively.  
    
\section{Results and Discussion}
\textbf{Constituents of the essential oil isolated from\textit{ Artemesia annua } leaves\textit{}}

Retention indices of most constituents identified by GC were compared with authentic standards available in the laboratory or with retention indices in close agreement with reference \unskip~\citep{708756:16803488,708756:16803487}. Further identification was achieved by GCMS. Mass spectra fragmentation patterns were compared with Wiley L. built in libraries and with those published in literature \unskip~\citep{708756:16803488,708756:16803484,708756:16803480,708756:16803481,708756:16803479,708756:16803482}. Greenish yellow oil with a pleasant aroma in 0.39\% (V/M) obtained from hydrodistillationhydro distillation of \textit{A.annua} shows different constituents which are completely volatile. Monoterpenes are found to be major (65.7\%) constituents of \textit{A. annua} followed by \ensuremath{\alpha } terpineole (14\%) Carvone (12.0\%) and \ensuremath{\alpha } terpenine (2.6\%). Sesquiterpenes (27.3\%) as monocyclic or bicyclic and in oxygenated forms occur as hydrocarbons (23.1\%)and alcohols (2.5\%), \ensuremath{\gamma }- Eumene and its derivative accounted for about 10 \% of total volatile followed by Z~\ensuremath{\alpha } {\textendash} bisabocene(5.4\%)(Table~\ref{tw-05a5e3f09a8d}).

\textbf{Essential oil of Artemesiaannua induced cytotoxicity in cancer cell lines}

Essential oil obtained from \textit{Artemesia annua} showed cytotoxicity against the cancer cell lines MCF-7 HT29 and AGS. Cytotoxicity of \textit{Artemesia annua} was evaluated by MTT assay, a simple and reliable experiment which measures cell viability and cytotoxicity for screening cytotoxic agents \unskip~\citep{708756:16803483}. Finding of this experiment demonstrated that essential oil of \textit{A.annua} was strongly cytotoxic on MCF-7, HT29, and AGS in a dose dependent manner. Here, we found that essential oil of \textit{A.annua} showed cytotoxic potential at doses ranging from (0-160 \ensuremath{\mu}g/ml) in the Figure~\ref{f-8495bfa46d58}, it implies that the  IC\ensuremath{_{50}} value of plant oil in HT 29 cell line is 40 \ensuremath{\mu}g/ml, in MCF-7 cells showed cytotoxic potential at doses ranging from (0-140 \ensuremath{\mu}g/ml) with an  IC\ensuremath{_{50}} value of 35 \ensuremath{\mu}g/ml, in AGS cell line with cytotoxic potential at doses ranging from (0-100 \ensuremath{\mu}g/ml) with an IC\ensuremath{_{50}} value of 38 \ensuremath{\mu}g/ml respectively. Previous reports show that the anti-tumour mechanism of \textit{Artemesia annua} is due to cleavage of ethnoperoide ring which is characteristic of sesquiterpenes by iron in cancer cells and formation of free radicals. Free radicals induce oxidative stress that cause cellular alterations like modulator of nuclear receptor responsiveness, DNA damage, apoptosis, tumortumour invasion and metastasis \unskip~\citep{708756:16803478}. However, our study implies about cytotoxic potential of \textit{Artemesia annua, } further investigations are needed to isolate bioactive constituents from essential oil of \textit{A.annua} for investigation of the molecular mechanisms as well as understanding the effects of \textit{A.annua} on other various tumor cell line models to explore its anticancer potential. 
    
\section{Conclusions}
 The molecular docking studies are one of the most influential techniques to find out new ligand for known protein and facilitates in treating dreadful diseases. In this present work, we have carried out molecular docking to analyze the binding properties of the mediator called 3HNG with Hexadecane, Hexadecanoic acid methyl ester, Quinoline, 1,2-dihydro-2,2,4-trimethyl) reported from Dictyotabartayresiana and 5-fluorouracil used as standard. The wet analysis carried out by us showed the best result with 3HNG and proven anti-colon cancer property. Among the various compound, of Hexadecanoic acid methyl ester and Quinoline, 1,2-dihydro-2,2,4-trimethyl) has higher binding energy than standard. So the present study may strongly conclude that anti-colon cancer property of the seaweed extract.
    

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