Phytochemical analysis of Urtica dioica leaves by fourier-transform infrared spectroscopy and gas chromatography-mass spectrometry

Phytochemicals are chemical compounds formed during the plants normal metabolic processes. These chemicals are often referred to as secondary metabolites. The objective of this research was to determine the chemical composition of leaves extract from methanol. The phytochemical compound screened by gas chromatography-mass spectrometry (GC-MS) method. Fifteen bioactive phytochemical compounds were identified in the methanolic extract of Urtica dioica. The identification of phytochemical compounds is based on the peak area, retention time molecular weight, molecular formula, mass spectrometry (MS) fragment-ions and pharmacological actions. GC-MS analysis of U. dioica revealed the existence of the Oximemethoxy-phenyl, 2, 6,-Nonadienal, 3, 7-dimethyl, 1, 2, 3Butanetriol, Silane, triethyl(2-phenylethoxy), Benzofuran, 2,3,-dihydro, 2,5,5,8a-Tetramethyl-1,2,3,5,6,7,8, 8a-octahydronaphthalen-1-ol, 2H-Indeno[1,2-b]furan-2-one, 3,3a, 4,5,6,7,8, 8b-octahydro-8,8-dimet, 1Dodecanamine, N, N-dimethyl, 2(3H)-Naphthalenone, 4, 4a,5,6,7,8-hexahydro-1-methoxy, D-Fructose, diethyl mercaptal, pentaacetate, [1,1-Bicyclopropyl-2-octanoic acid 2hexyl-methyl ester, Estra-1,3,5(10)trien-17B-ol, Cyclopropaneoctanoic acid, 2-[2-pentylcycloproyl)methyl]-methyl, 1-Hydroxy-2-(2,3,4,6tetra-O-acetyl-beta-d-glucopyranosyl)-9H-xanthe and Ethyl isoallochlate. The FTIR analysis of U. dioica leaves proved the presence of aromatic rings, alkenes, aliphatic fluoro, alcohols, ethers, carboxlic acids, esters, nitro compounds, hydrogen bonded alcohols and phenols. It contain chemical constitutions which may be useful for various herbal formulation as anti-inflammatory, analgesic, antipyretic, cardiac tonic and antiasthamatic.


INTRODUCTION
Phytochemicals are defined as bioactive non-nutrient plant compounds in fruits, vegetables, grains, and other plant foods that have been linked to reducing the risk of major chronic diseases (Hai, 2004;Magee and Rowland, 2004;Altameme et al., 2015;Hameed et al., 2015a).
General description of Urtica dioica erect perennial, 50 to 300 cm tall with 4-sided stems, armed with stinging hairs, opposite leaves, 7 to 15 cm long, the stalks from about 1/10 as long to nearly 1/2 as long as the blades, depending on variety. The stipules prominent, mostly 10 to 15 mm long. Fruits are achenes, lens-shaped, flattened, about 1.5 mm long, enclosed by the 2 inner sepals. U. dioica has many hollow stinging hairs called trichomes on its leaves and stems, which act like hypodermic needles that inject histamine and other chemicals that produce the stinging sensation when contacted by humans and other animals (Kavalali, 2003;Petlevski et al., 2003;Gulcin, 2004).
This study aims to analyze the chemical compounds of U. dioica leaves by fourier-transform infrared (FT-IR) spectroscopy and gas chromatography-mass spectrometry (GC-MS).

Collection and preparation of plant material
The leaves were dried at room temperature for seven days 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 .

Preparation of sample
About 9 g of the plant sample powdered were soaked in 100 ml methanol individually. It was left for 72 h so that alkaloids, flavonoids and other constituents if present will get dissolved. The methanol extract was filtered using Whatman's 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 1 μL of the methanol extract was injected into the GC-MS using a micro syringe and the scanning was done for 45 min. 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 (Mohammed and Imad, 2013;Kareem et al., 2015;Imad et al., 2014). 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. The  fragments obtained were actually charged ions with a certain mass .The mass/charge (M/Z) ratio obtained was calibrated from the graph obtained, which was called the Mass spectrum graph which is the fingerprint of a molecule (Imad et al., 2014). Before analyzing the extract using GC-MS, 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) (Imad et al., 2014). 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.
New chemical compound 55,81,149,192,224,251,291,322 322 Adeyemo, 2006). Among those identified, phytocompounds have the property of antioxidant and antimicrobial activities (Silva et al., 2004;Sein et al., 2008). Plant based antimicrobials have enormous therapeutic potential as they can serve the purpose with lesser side effects. Continued  further exploration of plant derived antimicrobials is needed today.

Conclusion
U. dioica 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 antiasthamatic properties.

ACKNOWLGEMENT
The authors wish to express their deepest gratitude to Prof. Dr. Adul-Kareem for his valuable contributions and support throughout this study. They would also like to express their gratitude to Dr. Ali for his valuable suggestions