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Objective: To explore the phytocomponents in the ethanol extracts of Ixora coccinea Linn leaves using GC-MS analysis.
Methods: About 5 g of dried fine powder of I. coccinea leaves were extracted with ethanol at room temperature for overnight, condensed and analysed by GC-MS.
Results: The GC-MS analysis of the ethanol extracts of Ixora coccinea leaves resulted in the identification of 7 compounds. The major compounds identified from ethanol extract were Pentadecanoic acid, 14-methyl-methyl ester, 16 Octadecenoic acid Methyl ester, Heptadecanoic acid 16-methyl methyl ether.
Conclusion: The presence of 16 Octadecenoic acid methyl ester, an eukaryotic DNA polymerase inhibitor in the ethanol leaf extract concludes the plant possesses antitumour and anticancer activity.
Key words: Ixora coccinea, GC-MS analysis, phytochemicals, Rubiaceae.
Medicinal plants have been of age long remedies for human diseases because they contain components of therapeutic value. Plants are used in modern medicine where they occupy a very significant place as raw material for important drugs. However, over the millennia human beings used plants as the source of natural drugs.
Ixora coccinea Linn. (Rubiaceae) commonly known as jungle of geranium and red Ixora, is an evergreen shrub distributed allover India and Sri Lanka. About 400 species were recorded in the genus Ixora, whereas a few are commonly cultivated. The plant is evergreen multi branched shrub of varying heights (1.2-3.6m) .
In Ayurveda and other folklore medicines, various plants like flower, root, leaves and stem are used as the source of drug. Pharmacological studies suggest that the plant possesses antioxidant activity , anti-inflammatory , antidiarrheal, hypoglycaemea, hepatoprotective, wound healing, cytotoxic and antitumour, antiulcer have been reported. Also, a study showed that equivalent concentrations of methanolic extract of flower were more efficient in some cases, while in other leaf extracts were more efficient . This encouraging literature provides a niche for this investigation.
There are no published reports worldwide, regarding the chemical components present in the ethanol extract of Ixora coccinea, hence the present investigation are carried out. Since ethanol it is less toxic than methanol may be a good alternative and may be equally effective in terms of extraction for bioactive compounds. Hydro ethanolic combinations may reduce use of volatile/ toxic solvents in extraction hereby adopting a green chemistry approach.
So, the present study was aimed to investigate the possible chemical components by first preparing the ethanolic extract, separation and identification of compounds by subjecting it to GC-MS analysis. This results in identifying new novel metabolites that can be used as lead molecule for new drug discovery.
MATERIALS AND METHODS
Ixora coccinea was collected from college campus, Ramakrishna Mission Vivekananda College, Chennai (13.0839° N, 80.2700° E) Tamil Nadu, India and identified by Botany Department of the college. Herbarium was deposited in the departmental herbarium collection centre.
Preparation of extract
The leaf samples were air dried and powdered in a mechanical grinder. About 5 g of the leaf powder was weighted, transferred to flask, treated with 10 ml of ethanol until the powder was fully immersed. Incubate for overnight and filtered through a Whatmann No. 1 filterpaper. The filtrate is concentrated to 1 ml solution which contains both polar and non-polar components of the materials. About 2µL of the sample solution was employed in GC-MS for analysis of different compounds after filtration.
Gas chromatography-mass spectrometry (GC-MS) analysis
For GC – MS analysis, the ethanol extract were injected in a JEOL GCMATE II GC-MS (Agilent Technologies) with Data system is a high resolution, double focusing instrument. Chromatographic conditions were as follows: helium as carrier gas, flow rate of 1 mL/min; and the injector was operated at 110ºC and column oven temperature was programmed at 280°C at a rate of 5°C per min injection mode. MS conditions were as follows: Ionization voltage of 70eV; ion source temperature 250°C; mass range was 45-450 mass units .
Identification of phytocompounds
Interpretation on GC-MS was carried out using National Institute of Standard and Technology (NIST) database which are having more 62,000 patterns. The components of the test materials were ascertained by using these libraries . The spectrum of the test material can be identified by searching the libraries, tentatively by comparision with compound found in the database.
GC-MS is one of the modern techniques to identify the bioactive constituents like volatile matter, long chain, branched chain hydrocarbons, esters, acids, alcohols etc. The ethanol leaf extract of Ixora coccinea showed seven peaks indicating the presence of seven compounds. The active principles with their retention time (RT) molecular formula, molecular weight (MW) are presented in Table 1. The GC-MS spectrum confirmed the presence of various components with different retention times as illustrated in (Figure 1). The mass spectrometer analyzes the active compounds eluted at different times to identify the nature and structure of the compounds. These mass spectra are fingerprint of that compound which can be identified from the data library.
The major compounds of the leaf extract are Penadecanoic acid, 14 methyl, methyl ester (RT-22.27); 1,2 Benzenedicarboxylic acid, butyl cyclohexyl ester (RT-22.72); 16 Octadecenoic acid, methyl ester (RT-23.99); Heptadecanoic acid, 16 methyl methyl ester (RT-24.22).
The presence of 16-Octadecenoic acid methyl ester (Figure-2) showed the molecular ion peak at m/z 296.49, corresponding to the molecular formula C12H36O2 indicating two degrees of unsaturation in the molecule. 16 octadecenoic acid methyl ester is a monounsaturated fatty acid and it is a positional isomer of oleic acid. The isolated bioactive compounds pave way for further drug development.
The qualitative analysis of bioactive compound with precise information can be obtained by gas-chromatography coupled with mass spectrometry (GC-MS). As herbal preparations have chemical complexity it is very difficult to identify all of their constituents. Using these methods their quality and stability can be easily assessed.
The fingerprinting analysis of ethanol leaf extract of Ixora coccinea revealed the presence of seven compounds. Whereas Sumathy et al.,  reported twenty four compounds in the methanol extract of I. coccinea flower by GC-MS analysis. This is because most of these secondary metabolites being organic in nature are soluble in ethanol which is an organic solvent and not in water, a more polar solvent. Hence successful extraction of phytochemical compounds from plant material is largely dependent on the type of solvent used in the extraction procedure . Whereas the traditional healers make use of water primarily as a solvent but this study has shown that the ethanol extract contain more pharmacologically active metabolites.
Many of the bioactive compounds that are identified showed that they may possess anticancer activity. Lee Seong Wei et al., showed that the presences of Octadecatrienoic acid, Pentadecanoic acid are responsible to the antimicrobial property of Andrigraphis paniculata extract. Whereas Yoshiyuki et al.,  also reported that 16-Octadecenoic acid methyl ester selectively inhibit eukaryotic DNA polymerase activities in vitro conditions. In the present study revealed the presence of the same molecules in ethanol leaf extract, which is having anticancer activity. This is on par with our previous report that the occurrence of camptothecin an anticancer drug in I. coccinea . Hence it is concluded that the plant possesses anticancer activity.
Further study on in vitro apotopsis test should be carried out in the near future, before this plant can come to a commercial sense in cancer treatment.
In the present study various bio-active compounds have been identified from ethanol extract of the Ixora coccinea by Gas Chromatogram Mass spectrometry (GC-MS) analysis which justifies the use of whole plant for various ailments by traditional practitioners. Based on the results obtained, it could be concluded that Ixora coccinea contain various bioactive compounds that are having anticancer activity. Therefore, it is recommended as a plant of phytopharmaceutical importance.
The Authors are thankful to the Secretary, Ramakrishna Mission Vivekananda College, Mylapore, Chennai for providing all the facilities and support to carry out the work and are also thankful to Director, SAIF-IITM, for carrying out GC-MS analysis. The authors also thank Minor Research Project Scheme UGC, New Delhi for their financial support throughout the project.