Determination of Phytocomponents in Methanolic Extract of Annona muricata Leaf Using GC-MS Technique

The aim of this study was to carry out for identification of bioactive compounds from the methanolic extract of Annona muricata leaves by Gas chromatography and Mass spectroscopy (GC-MS). GCMS analysis of methanolic extract was done by standard protocol using the equipment Perkin-Elmer Gas Chromatography–Mass Spectrometry, while the mass spectra of the compounds found in the extract was matched with the National Institute of Standards and Technology (NIST) library. The GC-MS analysis revealed the presence of various compounds like 4H-Pyran-4-one, 2,3-dihydro-3,5dihydroxy-6, Tetradecanoic acid, 3,7,11,15-Tetramethyl-2-hexadecen-1-ol, Hexadecanoic acid methyl ester, nHexadecanoic acid, Phytol and Octadecanoic acid in the methanolic extract of Annona muricata. These findings support the traditional use of Annona muricata in various disorders. Keyword: Gas chromatography and Mass spectroscopy, Annona muricata, Phytochemistry INTRODUCTION Phytochemistry or plant chemistry has developed in recent years as a distinct discipline, somewhere in between natural product organic chemistry and plant biochemistry and is closely related to both. It is concerned with the enormous variety of organic substances that are elaborated with and accumulated by plants and deals with the chemical structures of these substances, their biosynthesis, turn over and metabolism, their natural distribution and their biological function. It has been shown that in vitro screening methods could provide the needed preliminary observations necessary to elect crude plant extracts with potentially useful properties for further chemical and pharmacological investigations. Plants have been an important source of medicine with qualities for thousands of years. Plants are used medicinally in different countries, and they are the source of many potent and powerful drugs. Mainly on traditional remedies such as herbs for their history, they have been used as popular folk medicines Plant produces many chemicals to protect itself but recent research demonstrates that emphasizes the plant source of most of these protective, disease-preventing compounds. Phytochemicals are the chemicals extracted from plants. These organic chemicals are classified as primary or secondary constituents, depending on their role in plant metabolism. Primary constituents include the common sugars, aminoacids, proteins, purines and pyrimidines of nucleic acids, chlrophyll’s etc. Secondary constituents are the remaining plant chemicals such as alkaloids (derived from aminoacids), terpenes (a group of lipids) and phenolics (derived from carbohydrates) A true nutritional role for phytochemicals is becoming more probable every day as research uncovers more of their remarkable benefits. Within a decade, there were a number of dramatic advances in analytical techniques including TLC, UV, NMR and GC-MS that were powerful tools for separation, identification and structural determination of phytochemicals. The chosen medicinal plant namely as Annona muricata. Annona muricata L. belongs to the family of Annonaceae has a widespread pantropical distribution and has been pridely known as corossol. It is a widespread small tree and has its native in Central America. The fruit of Annona muricata Linn. is found to be edible in Yunnan province of China9 and their fruits is used commercially for the production of juice, candy and sherbets. Intensive chemical investigations of the leaves and seeds of this species have resulted in the isolation of a great number of acetogenins. The isolated compounds display some of the interesting biological or the pharmacological activities, such as antitumoral, cytotoxicity, antiparasitic and pesticidal properties. Roots of these species are used in traditional medicine due to their antiparasitical and pesticidal properties . The aim of this study is to determine the organic compounds present in the Annona muricata leaf extract with the aid of GC-MS Technique, which may provide an insight in its use in tradition medicine. MATERIAL AND METHODS Plant materials The fully mature Annona muricata leaves were collected in April 2013 from Tamil University, Thanjavur District, Shibula et al. / Determination of Phytocomponents... IJPPR, Volume 7, Issue 6 : December 2015 Page 1252 Tamil Nadu, India from a single herb. The leaves were identified and authenticated by Dr.S.John Britto, The Director, the Rapiant Herbarium and centre for molecular systematics, St. Joseph’s college Trichy-Tamil Nadu. India. A Voucher specimen has been deposited at the Rabinat Herbarium, St. Josephs College, Thiruchirappalli, Tamil nadu, India. Preparation of extracts The collected Annona muricata leaves were washed several times with distilled water to remove the traces of impurities from the leaves. The leaves were dried at room temperature and coarsely powdered. The powder was extracted with 70% methanol for 48 hours. A semi solid extract was obtained after complete elimination of alcohol under reduced pressure. The extract was stored in desiccator until used. The extract contained both polar and non-polar phytocomponents of the plant material used. GC –MS analysis GC-MS analysis was carried out on a GC clarus 500 Perkin Elmer system comprising a AOC-20i autosampler and gas chromatograph interfaced to a mass spectrometer instrument employing the following conditions: column Elite-1 fused silica capillary column (30 x 0.25mm ID x 1μMdf, composed of 100% Dimethyl polydiloxane), operating in electron impact mode at 70eV; Helium gas (99.999%) was used as carrier gas at a constant flow of 1 ml /min and an injection volume of 0.5 μI was employed (split ratio of 10:1) injector temperature 250 oC; ionsource temperature 280 oC. The oven temperature was programmed from 110 oC (isothermal for 2 min), with an increase of 10 oC/min, to 200oC, then 5oC/min to 280oC, ending with a 9min isothermal at 280oC. Mass spectra were taken at 70eV; a scan interval of 0.5 seconds and fragments from 40 to 450 Da. Total GC running time is 36min. min. The relative percentage amount of each component was calculated by comparing its average peak area to the total areas. Software adopted to handle mass spectra and chromatograms was a TurboMass Ver 5.2.0 RESULTS AND DISCUSSION Gas chromatography–mass spectrometry (GC-MS) is an analytical method that combines the features of gaschromatography and mass spectrometry to identify different substances within a test sample. Applications of GC-MS include drug detection, fire investigation, environmental analysis, explosives investigation, and identification of unknown samples. Additionally, it can identify trace in materials that were previously thought to have disintegrated beyond identification. GC-MS has been widely heralded as a “gold standard” for forensic substance identification because it is used to perform a specific test. A specific test positively identifies the actual presence of a particular substance in a given sample 13,14. Plants have an almost limitless ability to synthesize aromatic substances, most of which are phenols or their oxygen substituted derivatives. Most are secondary metabolites, of which at least 12,000 have been isolated, a number estimated to be less than 10% of the total. These substances serve as plant defense mechanisms against, insects and herbivores. Flavonoids exhibit several Table 1: Shows the components identified in methanolic extract of Annona muricata (GC MS study) Peak R.Time Area % Height % Molecular Formula Name of the compounds 1 4.620 0.65 0.78 C6H14N2 1-Pyrrolidineethanamine 2 5.120 1.27 1.55 C6H8O4 2,4-Dihydroxy-2,5-dimethyl-3(2H)-furan-3-one 3 7.744 4.32 3.78 C6H8O4 4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6methyl 4 8.595 1.73 1.65 C9H17N 1-(3-Methyl-3-butenyl) Pyrrolidin 5 8.833 2.27 1.65 C8H8O 1-(3-Methyl-3-butenyl)pyrrolidine 6 12.344 1.51 1.16 C15H24 Cyclohexane, 1-ethenyl-1-methyl-2,4-bis(1methylethenyl)-, [1S-(1.alpha.,2.beta.,4.beta.)]7 13.285 2.15 1.67 C15H24 Bicyclo[7.2.0]undec-4-ene, 4,11,11-trimethyl-8methylene-, [1R-(1R*,4E,9S*)]8 6.034 0.73 0.85 C15H24 Naphthalene, 1,2,3,5,6,8A-hexahydro-4,7-dimethyl1-(1-methylethyl)-, (1S-cis)$$ 9 20.208 1.03 1.00 C15H24 Tetradecanoic acid 10 20.400 4.08 2.67 C5H6N2O 2-Amino-3-hydroxypyridine 11 21.320 22.65 24.96 C20H38 2,6,10-Trimethyl,14-ethylene-14-pentadecne 12 21.406 2.49 2.28 C20H40O (2E)-3,7,11,15-tetramethyl-2-hexadecene 13 21.649 3.50 3.94 C20H40O 3,7,11,15-Tetramethyl-2-hexadecen-1-ol 14 21.891 6.16 7.14 C20H40O 3,7,11,15-Tetramethyl-2-hexadecen-1-ol 15 22.438 0.68 0.80 C17H34O2 Hexadecanoic acid, methyl ester 16 22.864 20.86 19.87 C16H32O2 n-Hexadecanoic acid 17 24.597 9.11 10.12 C22H42O2 Phytol 18 24.845 5.42 5.73 C17H32O2 cis-10-Heptadecenoic acid 19 24.892 6.06 5.03 C18H30O2 9,12,15-Octadecatrienoic acid, (Z,Z,Z)20 25.050 3.29 3.37 C18H36O2 Octadecanoic acid 100.00 100.00 Shibula et al. / Determination of Phytocomponents... IJPPR, Volume 7, Issue 6 : December 2015 Page 1253 biological effects such as anti-inflammatory, anti-fungal, anti-hepatotoxic and anti-ulcer actions. Identification of components Interpretation on mass spectrum GC-MS was conducted using the database of National Institute Standard and Technology (NIST) having more than 62,000 patterns. The spectrum of the unknown component was compared with the spectrum of the known components stored in the NIST library. The name, molecular weight and structure of the components of the test materials were ascertained. The biological activities listed are based on Dr. Duke’s Phytochemical and Ethnobotanical Databases by Dr. Jim Duke of the Agricultural Research Service/USDA. GC-MS Analysis Twenty compounds were identified in Annona muricata by GC-MS analysis. The active principles with their retention time (RT), molecular formula, molecular weight (MW) and concentration (%) are presented in (Table 1 and Fig 1). The prevailing compounds were 4H-Pyran-4one, 2,3-dihydro-3,5-dihydroxy-6, Tetradecanoic acid, 3,7,11,15-Tetramet