Identification of five newly described bioactive chemical compounds in methanolic extract of Mentha viridis by using gas chromatography-mass spectrometry ( GC-MS )

The bioactive compounds were screened by gas chromatography-mass spectrometry (GC-MS) method. Twenty one bioactive phytochemical compounds were identified in the methanolic extract of Mentha viridis using GC-MS method. The identification of phytochemical compounds is based on the peak area, retention time, molecular weight and molecular formula. GC-MS analysis of M. viridis revealed the existence of the 3,6-Octadecadiynoic acid, methyl ester, 2,5-Dimethyl-4-hydroxy-3(2H)-furanone, 4Hpyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl, benzofuran, R-Limonene, 2-methoxy-4-vinylphenol, 2hydroxy-5-methylbenaldehyde, tetra-acetyl-d-xylonic nitrile, Ficusin, Phen-1,4-diol, 2,3-dimethyl-5trifluoromethyl, n-Hexadecanoic acid, 7-Methyl-Z-tetradecen-1-ol acetate, Ethyl 9,12,15octadecatrienoate, Methyl 19-methyl-eicosanoate, Ethyl iso-allocholate, and Tocopherol. Five new bioactive chemical compounds 3-(N,N-Dimethyllayrylammonio), 1b,4a-eboxy-2H-cyclopenta [3,4]cyclopropal[8,9] cycloundec, 5H-Cyclopropa[3,4]benz [1,2-e]azulene-5-one, 2,2,4-Trimethyl-3-(3,8), 12,16-tetramethyl-hepta deca, and 4H-Cyclopropa[5,6]benz [1,2:7,8] azulene[5,6-b] oxiren-4-one are described and may in future be suitable sources for phytotherapy purposes. Mentha viridis contain chemical constitutions which may be useful for various herbal formulation exhibiting cardiac tonic, analgesic, antiasthamatic, anti-inflammatory and antipyretic properties.

been grown in damp or wet places (Gulluce et al., 2007;Mkaddem et al., 2009;Ozturk et al., 2009).Lamiaceae family consists of 200 genera and more than 4000 species (Ramesh et al., 2007).The phytochemical investigations on the Mentha species revealed that they possessed flavonoids and their glycosides, phenolic compounds, triterpenoids, steroids, and lignans (Monte et al., 1998;Areias et al., 2001;Ali et al., 2002;Zheng et al., 2007).Many species within this family are medicinal plants that apply in food, row and cooked forms and in human disease therapy (Santos et al., 2012).
Mentha spp.has been used for liver complaints due to its anti-inflammatory and treatment of bronchitis, nausea, flatulence, anorexia, and ulcerative colitis (Zhang et al., 2006).Essential oil formation in the plants is highly dependent on climatic conditions, especially day length, irradiance, temperature, and water supply (Franz and Novak, 2010).The leaves, flowers and stems of the Mentha species have been used as carminative, antispasmodic, antiemetic, stimulant, analgesic, and emmenagogue in traditional medicine all around the world.Their leaves have also been consumed as herbal tea and spice (Iscan et al., 2002).
Mentha species usually contain the monoterpene menthol in their constitutions, food products, menthol, and cosmetic (Simões et al., 2007;Oliveira et al., 2014).The chemical composition of the essential oils is influenced by factors such as leaf development and the emergence of new organs, which may lead to lower concentrations of these metabolites caused by translocation, as well as by effects such as seasonality, rain levels, and the stress to which the plant is exposed.These effects can directly influence the quantity and quality of the constituents in the essential oil (Gobbo et al., 2007;Mkaddem et al., 2009).The objective of this research was to determine the phytochemical composition of methanolic extract of Mentha viridis.

Collection and preparation of plant material
Mentha viridis seeds were purchased from local market in Hilla city, middle of Iraq.After thorough cleaning and removal of foreign materials, the seeds were stored in airtight container to avoid the effect of humidity and then stored at room temperature until further use (Ameera et al., 2015;Huda et al., 2015a).

Preparation of sample
About seventeen grams of methanolic extract of Mentha viridis powdered were soaked in twenty five ml methanol for ten h in a rotatory shaker.Whatman No.1 filter paper was used to separate the extract of plant.The filtrates were used for further phytochemical analysis.It was again filtered through sodium sulphate in order to remove the traces of moisture (Huda et al., 2015b).

Gas chromatography -mass spectrum analysis
The GC-MS analysis of the plant extract was made in a QP 2010 Plus SHIMADZU instrument under computer control at 70 eV (Mohammed and Imad, 2013).About 1 μl of the methanol extract was injected into the GC-MS using a micro syringe and the scanning was done for 45 min.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 70 eV.The column employed here for the separation of components was Elite 1 (100% dimethyl poly siloxane) (Imad et al., 2014;Muhanned et al., 2015).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.

Conclusion
Mentha viridis is native plant of Iraq.It contains chemical constitutions which may be useful for various herbal formulation as anti-inflammatory, analgesic, antipyretic, cardiac tonic and antiasthmatic.

Table 1 .
Major phytochemical compounds identified in methanolic extract of Mentha viridis leaves.