Evaluation of anti-bacterial activity and bioactive chemical analysis of Ocimum basilicum using Fourier transform infrared ( FTIR ) and gas chromatography-mass spectrometry ( GC-MS ) techniques

The objective of this research was to determine the chemical composition of aerial parts extract from methanol. The phytochemical compound screened by gas chromatography-mass spectrometry (GCMS) method. Thirty one bioactive phytochemical compounds were identified in the methanolic extract of Ocimum basilicum. The identification of phytochemical compounds is based on the peak area, retention time molecular weight, molecular formula, MS fragment-ions and pharmacological actions. GC-MS and Fourier transform infrared (FT-IR) analyses of O. basilicum revealed the existence of Paromomycin, Stevioside, Campesterol and Ascaridole epoxide, aliphatic fluoro compounds, alcohols, ethers, carboxlic acids, esters, nitro compounds, alkanes, H-bonded H-X group, hydrogen bonded alcohols and phenols. Methanolic extract of bioactive compounds of O. basilicum was assayed for in vitro antibacterial activity against Pseudomonas aerogenosa, Proteus mirabilis, Escherichia coli, Staphylococcus aureus and Klebsiella pneumonia by using the diffusion method in agar. The zone of inhibition was compared with different standard antibiotics. The diameters of inhibition zones ranged from 5.70±0.10 to 0.55±0.29 mm for all treatments.


INTRODUCTION
Plants secondary metabolites have recently been referred to as phytochemicals.Phytochemicals are naturally occurring and biologically active plant compounds that have potential disease inhibiting capabilities (Rukayadi et al., 2006;Yoshikawa et al., 2007;Hameed et al., 2015a;Al-Marzoqi et al., 2016).It is believed that phytochemicals may be effective in combating or preventing disease due to their antioxidant *Corresponding author.E-mail: imad_dna@yahoo.com.Tel: 009647716150716.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License effect (Halliwell and Gutteridge, 1992;Altameme et al., 2015a).Plant materials have played an important role in traditional methods of field crop and stored grain protection against insect pest infestation since time immemorial (Ogendo et al., 2003;Al-Marzoqi et al., 2015).Ocimum species contains a wide range of essential oils rich in phenolic compounds and a wide array of other natural products including polyphenols such as flavonoids and anthocyanins.The genus Ocimum comprises more than 150 species and is considered as one of the largest genera of the Lamiaceae family (Holm, 1996;Hameed et al., 2015b).Ocimum basilicum L. (sweet basil) is an annual herb which grows in several regions all over the world.The plant is widely used in food and oral care products.The essential oil of the plant is also used as perfumery (Bauer et al., 1997;Chiang, 2005).Kéita et al. (2000) reported O. basilicum and O. gratissimum to be potential insecticides.The leaves and seeds are rich in essential oils, which are repellent, toxic or growth inhibitory to many insects.A high degree of polymorphism in the genus Ocimum determines a large number of subspecies, different varieties and forms producing essential oils with varying chemical composition offering variable level of medicinal potential.Essential oils extracted from Ocimum plants have been reported to possess interesting biological properties.These volatile oils have been applied in perfumery, to inhibit growth of microorganisms, in food preservation and in aromatherapy.The potential uses of O. basilicum, Ocimum canum Ocimum gratissimum and Ocimum sanctum essential oils, particularly as antioxidant and antimicrobial agents have also been explored (Politeo et al., 2007;Koba et al., 2009;Zhang et al., 2009;Hameed et al., 2015c;Altameme et al., 2015b;Hussein et al., 2016a).O. basilicum has been traditionally used for the treatment of many ailments, such as headaches, coughs and diarrhea and it is generally recognized as safe and is a rich source of phenolic antioxidant compounds and flavonoids (Juliani and Simon, 2002).The aims of this study were to determine the phytochemical composition of aerial parts and evaluation of anti-bacterial activity.

Collection and preparation of plant
The aerial parts were dried at room temperature for ten days and when properly dried then powdered using clean pestle and mortar, and the powdered plant was size reduced with a sieve (Altameme et al., 2015c;Hameed et al., 2015d;Hussein et al., 2016b).The fine powder was then packed in airtight container to avoid the effect of humidity and then stored at room temperature.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 (Jasim et al., 2015;Hadi et al., 2016;Hussein et al., 2016c).The methanol extract was filtered using Whatman No.1 filter paper and the residue was removed.

Gas chromatography-mass spectrum (GC-MS) analysis
The GC-MS analysis of the plant extract (O.basilicum) 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.The time from when the injection was made (Initial time) to when elution occurred is referred to as the retention time (RT).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/min.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 (Yang et al., 2010).

Determination of antibacterial activity of crude bioactive compounds of O. basilicum
The test pathogens (Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, and Staphylococcus aureus) were swabbed in Muller 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 37C° for 24 h and examined.After the incubation the diameter of inhibition zones around the discs was measured.

RESULTS AND DISCUSSION
Medicinal herbs are known as sources of active compounds that are widely sought after worldwide for their natural properties.They have been used since ancient times as sources of flavorings and for their pharmaceutical properties (Bais et al., 2002).Phytochemicals may be effective in combating or preventing disease due to their antioxidant effect (Halliwell and Gutteridge, 1992).A great number of organizations and scientists turn their attention to traditional therapies in order to find and conserve important resources and up to 80% of the population relies on traditional medicines or folk remedies for primary health care needs (Smith et al., 2010).Gas chromatography and mass spectroscopy analysis of compounds was carried out in methanolic aerial parts extract of O. basilicum, shown in Table 1.The GC-MS chromatogram of the 31 peaks of the compounds detected was shown in Figure 1.Chromatogram GC-MS analysis of the methanol extract of O. basilicum showed the presence of thirty one major peaks and the components corresponding to the peaks were determined    as follows.The first set up peak were determined to be 1,2,4-Triazole (Figure 2).The second peak indicated to be 4-[N-(2-hydroxyethyl)-N-nitro]amino (Figure 3).The next peaks were considered to be, 9-Acetoxynonanal,                              The FTIR analysis of O. basilicum aerial parts proved the presence of aliphatic fluoro compounds, alcohols, ethers, carboxlic acids, esters, nitro compounds, alkanes, H-bonded H-X group, hydrogen bonded alcohols and phenols which shows major peaks at 1028.06, 1095.57, 1238.30, 1317.38, 1396.46, 1396.46, 1616.35, 3010.88 and 3275.13 (Table 2 and Figure 33).Baritaux et al. (1992) isolated and analyzed essential oil from basil (O.basilicum) by steam distillation, then it were recovered into a pentane/dichlorometane mixture containing known amounts of two internal standards, nonane and octadecane.Laakso et al. (1990) (Morales and Simon, 1996;Phippen and Simon, 1998).An important approach to discover new medicines is survey of natural products, such as medicinal plants or their secondary metabolites that modulate painful conditions (Baratta et al., 1998;Chiang et al., 2005).Previous studies (Bakkali et al., 2008;Quintans-Júnior et al., 2008;Oliveira et al., 2009;Li and Vederas, 2009;Venâncio et al., 2011) have demonstrated that several Ocimum species are used to treat central nervous system (CNS) disorders in various regions of the world, mainly in developing countries, and their analgesic profile is frequently reported.In this study, five clinical pathogens were selected for antibacterial activity, namely, S. aureus, K. pneumoniae, P. aeruginosa, E. coli and Proteus mirabilis.Maximum zone formation is against E. coli (5.70±0.10)(Table 3).Essential oils derived from several Ocimum spp.have been reported to be active against several Gram-positive and Gram-negative bacteria as well as against yeasts and fungi due to their terpenic constituents.Recently, essential oils and extracts of certain plants have been shown to have antimicrobial effects as well as imparting flavour to foods (Zhang et al., 2009;Shareef et al., 2016;Kadhim et al., 2016).Ocimum spp.contain a wide range of essential oils rich in phenolic compounds and a wide array of other natural products including polyphenols such as flavonoids and anthocyanins.Antiviral and antimicrobial activities of this plant have also been reported (Li and Vederas, 2009;Venâncio et al., 2011).

Conclusion
The results of this study provide data on phytochemical characteristics of O. basilicum.O. basilicum is native plant of Iraq.It contains chemical constitutions which may be useful for various herbal formulation as antiinflammatory, analgesic, antipyretic, cardiac tonic and antiasthamatic.

Table 1 .
Phytochemical compounds identified in methanolic extract of O. basilicum.

Table 2 .
FT-IR peak values of Ocimum basilicum methanolic aerial parts extract.

Table 3 .
Zone of inhibition (mm) of test bacterial strains to Ocimum basilicum bioactive compounds and standard antibiotics.