Analysis of bioactive chemical components of two medicinal plants ( Coriandrum sativum and Melia azedarach ) leaves using gas chromatography-mass spectrometry (GC-MS)

The main objective of this study was to determine the phytochemical composition in the leaves of Coriandrum sativum , using methanolic extraction and report the main functional components by using IR technique. The phytochemical compounds in the extract were then screened by GC-MS method. Seven bioactive phytochemical compounds were identified in the methanolic extract of C. sativum : 1,6-octadien-3-ol, 3,7-dimethyl, 1,6-octadien-3-ol,3,7-dimethyl, 2-aminobenzoate, bicyclo[2.2.1]heptan-2-one,1,7,7-trimethyl., geranyl vinyl ether, 9,10-secocholesta-5,7,10(19)-triene-3,24,25-triol., ascorbic acid 2,6-dihexadecanoate and 7aH-cyclopenta[a] cyclopropa[f]cycloundecene. Thirteen bioactive phytochemical compounds were identified in the methanolic extract of Melia azedarach . In the present investigation, a variety of compounds have been detected in M. azedarach including trichloromethane, propanedioic acid, diethyl ester, 2-pyrrolidinyl-methylamine, butanedioic acid, diethyl ester, 2-piperidimethanamine, butanedioic acid, hydroxyl-, diethyl ester, 2,5-dimethylhexane-2,5-dihydroperoxide, dithiocarbamate, s-methyl-,n-(2-methyl-3-oxobutyl), triethyl citrate, y-sitosterol, ethyl 9,12,15-octadecatrienoate, hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl)ethyl ester, and octadecane, 3-ethyl-5-(2-ethylbutyl). It contains chemical constitutions which may be useful for various herbal formulation as anti-inflammatory, analgesic, antipyretic, cardiac tonic and antiasthamatic. C. sativum is highly active against Aspergillus terreus 6.01 ± 0.200. Bioactive compounds of C. sativum and M. azedarach were assayed for in vitro antibacterial activity against Staphylococcus aureus, Proteus mirabilis, Pseudomonas aerogenosa, Escherichia coli and Klebsiella pneumonia using the diffusion method in agar. The zone of inhibition was compared with different standard antibiotics. The diameters of inhibition zones ranged from 5.60 ± 0.320 to 1.96 ± 0.200 mm for all treatments.


INTRODUCTION
Coriander is an annual popular culinary medicinal plant with a distinctive pungent, fatty, and aldehydic aroma (Msaada et al., 2009).Coriander is recognized as one of the most important spices in the world and is of great significance in international trade (Msaada et al., 2007).The bioactive non-nutrient plant compounds in fruit, vegetables, grains, and other plant foods have been linked to reductions in the risk of major chronic diseases (Altameme et al., 2015).Coriander has been cultivated since ancient times and is originally from the Mediterranean and Middle Eastern region and grows extensively in India, Russia, Central Europe, Asia and Morocco.The Coriander essential oil is generally obtained by steam distillation of the dried fully ripe fruits (seeds) and oil has a characteristic odor of linalool and a mild, sweet, warm and aromatic flavor (Ramadan and Moersel, 2006).The seeds have medical uses and traditionally applied for curing digestive disorder, pain in joints and rheumatism.Stomachic, spasmolytic, carminative, diarrhoea and dyspepsia of various origin's coriander are also used in aromatherapy (Gil et al., 2002;Eikani et al., 2007;Grosso et al., 2008, Hussein et al., 2015;Imad et al., 2015).It is used to treat female diseases such as menoxenia, ovulation type dysfunctional uterine bleeding (Paarakh, 2009).It is used for treating leucorrhea; spermatorrhea.Coriander fruit possess stimulant and carminative properties (Khare, 2007).The fruits are used as astringent, anthelmintic, emollient, stomachic, antibilious, digestive, appetizer, constipating, diuretic, antipyretic, refrigerant, tonic, expectorant, anodyne, antidiabetic and dyspepsia (Paarakh, 2009;Hameed et al., 2015a).Melia azedarach, family Meliaceae is from west Asia (Sumathi, 2013).It is a moderate-sized deciduous tree 9 to 12 m in height dark grey and a cylindrical bole.M. azedarach is traditionally been used as anthelmintic, astringent and stomachic agent.It is widely distributed in Himalayan region.The leaves are bi-or trip innate, pinnate opposite or alternate, ovate orlanceolate, serrate, acuminate, glabrous on both surfaces (Bergsson et al., 2002;Dawson et al., 2002;Lee et al., 2002;Hameed et al., 2015b).M. azedarach is used for the treatment of inflammations, leprosy and cardiac disorders.Its fruits and leaves extracts possess antiviral, antifertility activity, ovicidal and larvicidal activity (Wandscheer et al., 2004;Corpinella et al., 2007;Mandal and Dhaliwal, 2007).The plant possesses antioxidant, antimalarial, antihepatotoxic, antibacterial, antiparasitic, and antiulcer properties (Dai et al., 1999;Devi et al., 2001;Bahuguna et al., 2009;Samudram et al., 2009;Nahak et al., 2010;Hameed et al., 2015c).The aim of the study was to investigate the presence of phytochemical compounds from the leaves of Coriandrum sativum and M. azedarach by using gas chromatography-mass spectrometry and evaluation antibacterial activity.

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

Preparation of sample
About 20 g of the plant sample powdered were soaked in 120 ml methanol for 18 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.

Gas chromatography -Mass spectrum analysis
GC-MS (Agilent 7890 A) was used in this study to identify the components present in the extract (Mohammed and Imad, 2013;Kareem et al., 2015).About 1 μL of the methanol extract was injected into the GC-MS using a micro syringe (Imad et al., 2015a).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 retention time 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 (Imad et al., 2015b).The mass/charge (M/Z) 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.The flow rate of helium was set to 1 ml per minute.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).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 (Muhanned et al., 2015;Imad et al., 2015c).

Determination of antibacterial activity of crude bioactive compounds of C. sativum and M. azedarach
Proteus mirabilis, Escherichia.coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Staphylococcus aureus were swabbed in Mueller Hinton agar plates.60 μL of plant extract was loaded on the bored wells.The wells were bored in 0.5 cm in diameter.The plates were incubated at 37°C for 24 h and examined.After the incubation the diameter of inhibition zones around the discs was measured.

Determination of antifungal activity
Five-millimeter diameter wells were cut from the agar using a sterile cork-borer, and 50 μl of the samples solutions (C.sativum and M. *Corresponding author.E-mail: imad_dna@yahoo.com.Tel: 009647716150716. Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License azedarach) was delivered into the wells.Antimicrobial activity was evaluated by measuring the zone of inhibition against the test microorganisms.Methanol was used as control.Amphotericin B and fluconazole were used as reference antifungal agents.The tests were carried out in triplicate.The antifungal activity was evaluated by measuring the inhibition-zone diameter observed after 48 h of incubation (Huda et al., 2015;Ameera et al., 2015).

Statistical analysis
Data were analyzed using analysis of variance (ANOVA) and differences among the means were determined for significance at p < 0.05 using Duncan's multiple range test using SPSS software) version 9.1.

RESULTS AND DISCUSSION
Gas chromatography and mass spectroscopy analysis of compounds was carried out in methanolic leaves extract of C. sativum and M. azedarach shown in Tables 1 to 2. The GC-MS chromatogram of the twenty peaks of the compounds detected is shown in Figures 1 and 2. Chromatogram GC-MS analysis of the methanol extract of C. sativum showed the presence of 20 major peaks and the components corresponding to the peaks were determined as follows.The first set up peak was determined to be 1,6-octadien-3-ol,3,7-dimethyl (Figure 2).The second peak was indicated to be 1,6-octadien-3ol,3,7-dimethyl, 2-aminobenzoate (Figure 3).The next peaks was considered to be bicyclo[2.2.1]heptan-2one,1,7,7-trimethyl, geranyl vinyl ether, 9,10secocholesta-5,7,10(19)-triene-3,24,25-triol, ascorbic acid 2,6-dihexadecanoate, 7ah-cyclopenta[a] cyclopropa[f]cycloundecene (Figures 4 to 9).Coriander oil may have future use as a free radical scavenger, preventing oxidative deterioration in foods.In a report by Ramadan and Moersel (2006) coriander oil was shown to have greater activity against the radical generating activity of 1,1-diphenyl-2-picrylhydrazyl in several oils.Recently, Coriander oil has been reported to possess many medicinal properties, including antimicrobial properties against selected pathogenic (Martins et al., 2003;Ishikawa et al., 2003) antioxidant (Quynh et al., 2009), antidiabetic (Pourmortazavi and Hajimirsadeghi, 2007), anticancer and antimutagenic activities (Mohammad et al., 2011).Coriandrum sativum can act as source for oleic acid as the percentage found was 38.55% and soxhlation method can be used to extract it from the fruit (Padmaa, 2014).Shahidi (2008) reported that their synergistic effects are rendered by a combination of phytochemicals present in source materials, and complementary nature of phytochemicals from different sources are important factors to consider in the formulation of functional foods and in the choice of a healthy diet.
Medicinal plants are used in traditional treatments to cure variety of diseases (Hariprasad and Ramakrishnan, 2011).Various scientific studies reported the analgesic, anticancer, antiviral, antimalarial, antibacterial, antifeedent and antifertility activity of this plant (Vishnukanta, 2008;Sen and Batra, 2011).Gas chromatography and mass spectroscopy analysis of compounds was carried out in methanolic leaves extract of M. azedarach as shown in Table 2. Chromatogram GC-MS analysis of the methanol extract of M. azedarach showed the presence of 13 major peaks and the components corresponding to the peaks were determined as follows.The first set up peak was determined to be Trichloromethane (Figure 10).The second peak indicated to be Propanedioic acid and diethyl ester (Figure 11).The next peaks considered to be 2-pyrrolidinyl-methylamine, butanedioic acid, diethyl ester, 2-piperidimethanamine, butanedioic acid, hydroxyl, diethyl ester, 2,5-dimethylhexane-

Antibacterial and antifungal activity
K. pneumoniae, P. aeroginosa, E. coli, S. aeureus and P. mirabilis were five clinical pathogens selected for THE antibacterial activity maximum zone formation against E. coli (Table 3).Methanolic extraction of plant showed notable antifungal activities against Aspergillus niger, Aspergillus terreus, Aspergillus flavus and Aspergillus fumigates (Table 4).C. sativum and M. azedarach was very highly active against A. terreus (6.01 ± 0.200).Aspergillus was found to be sensitive to all test medicinal plants and mostly comparable to the standard reference antifungal drug amphotericin B and fluconazole to some extent.

Conclusion
C. sativum and M. azedarach are native plant of Iraq.Thus, the GC-MS analysis of methanolic leaves extract of C. sativum and M. azedarach showed a highly complex profile containing approximately 20 components.This study may be useful to explore the pharmacological and biosynthetic activity of the plants further.,69,111,149,237,281,358,400,46 Immune enhancement and anti-microorganism.,74,84,98,112,134, 154,182,213,239,275, 270,299,330 antioxidant, antiinflammatory and anthelmintic activities  ,71,85,99,127,183, 197,225,281,295,337, 364 anti-oxidant and antiinflammatory effect

Figure 6 .
Figure 6.Structure of Geranyl vinyl ether present in the methanolic leaves extract of Coriandrum sativum by using GC-MS analysis.

Figure 8 .
Figure 8. Structure of Ascorbic acid 2,6-dihexadecanoate present in the methanolic leaves extract of Coriandrum sativum by using GC-MS analysis.

Table 1 .
Major phytochemical compounds identified in methanolic extract of Coriandrum sativum leaves.

Table 2 .
Major phytochemical compounds identified in methanolic extract of Melia azedarach leaves.

Table 3 .
Zone of inhibition (mm) of test bacterial strains to Coriandrum sativum and Melia azedarach bioactive compounds and standard antibiotics.

Table 4 .
Zone of inhibition (mm) of Aspergillus spp.test to Coriandrum sativum and Melia azedarach bioactive compounds and standard antibiotics.