Study of chemical composition of Foeniculum vulgare using Fourier transform infrared spectrophotometer and gas chromatography-mass spectrometry

Medicinal plants are potential sources of natural compounds with biological activities and therefore attract the attention of researchers worldwide. The objective of this research was to determine the chemical composition of seeds extract from methanol. The phytochemical compound screened by gas chromatography mass spectrometry (GC-MS) method. Fifty six bioactive phytochemical compounds were identified in the methanolic extract of Foeniculum vulgare. The identification of phytochemical compounds is based on the peak area, retention time molecular weight, molecular formula, MS Fragmentions and Pharmacological actions. The Fourier transform infrared spectroscopy (FTIR) analysis of F. vulgare seeds proved the presence of alkenes, aliphatic fluoro compounds, alcohols, ethers, carboxlic acids, esters, nitro compounds, alkanes, hydrogen bonded alcohols and phenols.


INTRODUCTION
Bitter Fennel (Foeniculum vulgare Mill.) is one of the oldest herbs and possesses beneficial medicinal effects, belongs to the Apiaceae family and native to Mediterranean regions (Hornok, 1992).In botany the Umbllifererae (apiaceae) family is widespread and includes 300 genus and 3000 aromatic herbaceous species (Hay et al., 1993).F. vulgare is a well known aromatic medicinal plant which is used in traditional medicine as spice and substrate for different industrial purpose (Telci et al., 2009).Fennel is used for various purposes in the food, cosmetic, and medical industries.
Fennel essential oil has a valuable antioxidant, and has antibacterial, anticancer and antifungal activity (Lucinewton et al., 2005;El-Awadi and Esmat, 2010;Altameme et al., 2015a).It is cultivated and also widespread in many parts of Mediterranean and midlist countries such as Italy, Turkey and Iran (Marino et al., 2007;Altameme et al., 2015b).The increasing commercial value of fennel necessitates the need to identification, recognizing and conservation the existing diversity.The fruits of sweet fennel contain essential oil which is rich source of anethole, limonene, fenchone, estragole and camphene among them the anethole is the most important constituent with determinant role in quality of the essential oil of seeds (Gross et al., 2002;Hameed et al., 2015a).These depend upon internal and external factors affecting the plant such as genetic structures and ecological conditions (Telci et al., 2009).

Collection and preparation of plant material
The seeds 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 (Hameed et al., 2015b).

Preparation of sample
About four grams of the plant sample powdered were soaked in 50 ml methanol individually.It was left for two weeks so that alkaloids, flavonoids and other constituents if present will get dissolved (Hameed et al., 2015c).The methanol extract was filtered using Whatman No.1 filter paper and the residue was removed (Hamza et al., 2015).

Identification of component by gas chromatography -mass spectrum analysis
The physicochemical properties of F. vulgare are presented in Table 1.Interpretation of mass spectroscopy (GC-MS) was conducted using data base of National Institute Standard and Technology (NIST) having more than 62000 patterns.The spectrum of the unknown component was compared with the spectrum of the known component stored in the NIST library (Mohammed and Imad, 2013;Imad et al., 2014a).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.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.The greater the concentration in the sample, bigger was the signal obtained which was then processed by a computer (Imad et al., 2014b;Hameed et al., 2015d).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 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 Hussein et al. 61 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 minute (Imad et al., 2014c;Kareem et al., 2015).The column employed here for the separation of components was Elite 1(100% dimethyl poly siloxane).

Figure 3 .
Figure 3. Structure of L-Fenchone present in the methanolic seeds extract of F. vulgare using GC-MS analysis.

Figure 6 .
Figure 6.Structure of Estragole present in the methanolic seeds extract of F. vulgare using GC-MS analysis.

Figure 8 .
Figure 8. Structure of Benzaldehyde ,4-methoxy present in the methanolic seeds extract of Foeniculum vulgare using GC-MS analysis.

Figure 9 .
Figure 9. Structure of Anethole present in the methanolic seeds extract of F. vulgare using GC-MS analysis.

Figure 12 .
Figure 12.Structure of Ascaridole epoxide present in the methanolic seeds extract of F. vulgare using GC-MS analysis.

Figure 13 .
Figure 13.Structure of d-Mannose present in the methanolic seeds extract of F. vulgare using GC-MS analysis.

Figure 17 .
Figure 17.Structure of Pterin -6-carboxylic acid present in the methanolic seeds extract of F. vulgare using GC-MS analysis.

Figure 19 .
Figure 19.Structure of 4-Methoxybenzoic acid, allyl ester present in the methanolic seeds extract of F. vulgare using GC-MS analysis.

Figure 20 .
Figure 20.Structure of Arisaldehyde dimethyl acetal present in the methanolic seeds extract of F. vulgare using GC-MS analysis.

Figure 23 .
Figure 23.Structure of 1-Heptatriacotanol present in the methanolic seeds extract of F. vulgare using GC-MS analysis.

Figure 29 .
Figure 29.Structure of Corymbolone present in the methanolic seeds extract of F. vulgare using GC-MS analysis.

Figure 30 .
Figure 30.Structure of Apiol present in the methanolic seeds extract of F. vulgare using GC-MS analysis.

Figure 32 .
Figure 32.Structure of Fenretinide present in the methanolic seeds extract of F. vulgare using GC-MS analysis.

Figure 33 .
Figure 33.Structure of Dihydroxanthin present in the methanolic seeds extract of F. vulgare using GC-MS analysis.

Figure 38 .
Figure 38.Structure of Gibberellic acid present in the methanolic seeds extract of F. vulgare using GC-MS analysis.

Figure 43 .
Figure 43.Structure of Cis-Vaccenic acid present in the methanolic seeds extract of F.vulgare using GC-MS analysis.

Figure 46 .
Figure 46.Structure of 9-Octadecenamide, (Z) present in the methanolic seeds extract of F. vulgare using GC-MS analysis.

Figure 50 .
Figure 50.Structure of Phthalic acid, decyl oct-3-ylester present in the methanolic seeds extract of F. vulgare using GC-MS analysis.

Figure 53 .
Figure 53.Structure of Oxiraneoctanoic acid, 3-octyl-,methyl ester present in the methanolic seeds extract of F. vulgare using GC-MS analysis.

Figure 55 .
Figure 55.Structure of lngol 12-acetate present in the methanolic seeds extract of F. vulgare using GC-MS analysis.

Table 1 .
Major phytochemical compounds identified in methanolic extract of Foeniculum vulgare.