Biochemical analysis of Origanum vulgare seeds by fourier-transform infrared (FT-IR) spectroscopy and gas chromatography-mass spectrometry (GC-MS)

Medicinal plants are potential sources of natural compounds with biological activities, and therefore attract the attention of researchers worldwide. The objective of this research is to determine the chemical composition of methanolic seed extract. The phytochemical compound screened by spectroscopy and gas chromatography-mass spectrometry (GC-MS) method. Sixteen bioactive phytochemical compounds were identified in the methanolic extract of Origanum vulgare. The identification of phytochemical compounds is based on the peak area, retention time molecular weight, molecular formula, MS Fragmentions and pharmacological actions. GC-MS analysis of O. vulgare revealed the existence of the 1,7-Dioxaspiro[5,5]undec-2-ene, 2,4-Dihydroxy-2,5-dimethyl-39(2H)-furan3-one, 2,4-Difurobenzene, 1-benzyloxy, α-D-Glucopyranoside, O-αGlucopyranosyl, 4-Hexenal, 6hydroxy-4-methyl,dimethyl acetal, acetate, 4H-pyran-4-one,2,3,-dihydro-3,5-dihydroxy-6-methyl, Benzofuran, 4-Amino-1,5-pentandioic acid, 2-Methoxy-4-vinylphenol, d-Mannose, 7-Isopropyl-10-methyl1-oxo-1,5-dithia-spiro[5,5]undecane-2-carboxy, Phytol, Cis-Vaccenic acid, N-MethylNbenzyltetradecanamine, 3,8,8-Trimethoxy-3-piperidyl-2,2-binaphthalene-1,1,4,4-tetrone, and 17-(1,5Dimethylhexyl)10,13-dimethyl. The FTIR analysis of O. vulgare seeds proved the presence of alkenes, aliphatic fluoro compounds, alcohols, ethers, carboxlic acids, esters and hydrogen bonded alcohols, phenols.


INTRODUCTION
Origanum vulgare L. is a perennial aromatic herb belonging to the family Lamiaceae (Skoula and Harborne, 2002) used for thousands of years as spices and as local medicines in traditional medicine (Altameme et al., 2015). O. vulgare L. is the most wide spread among all the species within the genus. It is distributed all over Europe, West and Central Asia up to Taiwan (Ietswaart, 1980;. Aerial flowering parts of O. vulgare subsp. viride are used in Iranian traditional medicine as diuretic, stomachic, antineuralgic, antitussive and expectorant (Afsharypuor et al. 1997). O. vulgare plays a primary role *Corresponding author. E-mail: imad_dna@yahoo.com. Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License among culinary herbs in world trade (Oliver, 1997). Taxonomic studies on the basis of morphological characters have led to the discrimination of several subspecies. Ietswaart (1980), distinguished six subspecies of O. vulgare, that is, hirtum, vulgare, virens, viride, gracile and glandulosum. Only O. vulgare L. subspecies hirtum has the leaf anatomy which corresponds to that of commercially marketed European oregano (Skoula and Harborne, 2002).
Oregano is used worldwide both as spice and crude drug, which is mainly provided by species of Origanum genus (Franz and Novak, 2002). Oregano was found to be strong antimicrobial agent and had a significant spasmolytic effect on smooth muscle . The fumigant toxicity and insecticidal effect of oregano essential oils for storeroom insects has also been proved (Marn et al., 1999;Imad et al., 2015). Many herbs are commonly used in home-type cure therapies, complementary medicine and modern medicine because of their perceived antioxidant, antimicrobial, anticancer, etc. properties. Origanum species are counted among these herbs, since they show high activities according to their assessment for the above biological properties (Mechergui et al., 2010;Hussein et al., 2015). A remarkable phytochemical polymorphism with several chemotypes is also reported by several studies on this species that shows marked spatial segregation in nature (D'antuono et al., 2000;Radušiene et al., 2005;Hameed et al., 2015c).
Furthermore, O. vulgare has an antioxidant property and is applied in human health. Cervato et al. (2000), prove that the antioxidant activities of extracts of oregano's leaves (both aqueous and methanolic extracts) can inhibit all phases of lipid peroxidative process (Sahin et al., 2004;Singh, 2007;Hussein et al., 2015). The biological activity of essential oils and herb extracts cause a high pharmaceutical and industrial interest in O. vulgare, since antimicrobial, antifungal, insecticidal and antioxidative effects have been reported (Kulisic et al. 2004;Bakkali et al. 2008;. Origanum tea is a treatment for indigestion, coughs, and to stimulate menstruation. The oil of Origanum is used for toothache, and in some cosmetics. Its leaves and flowering stems are natural antiseptics because of high thymol content (Bhat et al., 2002;Singh, 2003;Dalpe, 2004;Bilalis et al., 2011).
The study, aim to study the analysis of chemical compounds of Origanum vulgare seeds by fouriertransform infrared (FT-IR) spectroscopy and gas chromatography-mass spectrometry (GC-MS).

Collection and preparation of plant material
The seeds were dried at room temperature for two weeks, and when properly dried then powdered using clean pestle and mortar, and the powdered plant was size reduced with a sieve. The fine powder was then packed in airtight container to avoid the effect of humidity and then stored at room temperature. About thirteen grams of the plant sample powdered were soaked in 100 ml methanol individually. It was left for 96 h so that alkaloids, flavonoids and other constituents if present will get dissolved. The methanol extract was filtered using Whatman No.1 filter paper and the residue was removed .

Gas chromatography -mass spectrum analysis
The GC-MS analysis of the plant extract was made in a (Agilent 7890 A) instrument under computer control at 70 eV. About 1μL of the methanol extract was injected into the GC-MS using a micro syringe and the scanning was done for 45 min (Imad et al., 2014a). As the compounds were separated, they eluted from the column and entered a detector which was capable of creating an electronic signal whenever a compound was detected. The greater the concentration in the sample, bigger was the signal obtained which was then processed by a computer. The time from when the injection was made (Initial time) to when elution occurred is referred to as the Retention time (RT). While the instrument was run, the computer generated a graph from the signal called Chromatogram. Each of the peaks in the chromatogram represented the signal created when a compound eluted from the Gas chromatography column into the detector. The X-axis showed the RT and the Y-axis measured the intensity of the signal to quantify the component in the sample injected. As individual compounds eluted from the gas chromatographic column, they entered the electron ionization (mass spectroscopy) detector, where they were bombarded with a stream of electrons causing them to break apart into fragments (Mohammed and Imad, 2013;Imad et al 2014b;Kareem et al., 2015).
The fragments obtained were actually charged ions with a certain mass .The M/Z (Mass / Charge ) ratio obtained was calibrated from the graph obtained, which was called as the Mass spectrum graph which is the fingerprint of a molecule. Before analyzing the extract using gas chromatography and mass spectroscopy, the temperature of the oven, the flow rate of the gas used and the electron gun were programmed initially. The temperature of the oven was maintained at 100°C. Helium gas was used as a carrier as well as an eluent. The flow rate of helium was set to 1 ml per min. The electron gun of mass detector liberated electrons having energy of about 70eV. The column employed here for the separation of components was Elite 1(100% dimethyl poly siloxane). The identity of the components in the extracts was assigned by the comparison of their retention indices and mass spectra fragmentation patterns with those stored on the computer library and also with published literatures. Compounds were identified by comparing their spectra to those of the Wiley and NIST/EPA/NIH mass spectral libraries (Imad et al., 2014c;Muhanned et al., 2015).

CONCLUSION
O. vulgare is native plant of Iraq. It contain chemical constitutions which may be useful for various herbal formulation as anti-inflammatory, analgesic, antipyretic, cardiac tonic and antiasthamatic.