Gas chromatography mass spectrum and fourier-transform infrared spectroscopy analysis of methanolic extract of Rosmarinus oficinalis leaves

The aims of the study were to investigate the presence of phytochemical compounds from the leaves of Rosmarinus oficinalis, using methanolic extraction and report the main functional components by using FT-IR. The phytochemical compound screened by GC-MS method. Seventeen bioactive phytochemical compounds were identified in the methanolic extract of R. oficinalis. The identification of phytochemical compounds is based on the peak area, retention time molecular weight, molecular formula, chemical structure, MS Fragment ions and pharmacological actions. GC-MS analysis of Rosmarinus oficinalis revealed the existence of the α-pinene, Camphene, Eucalyptol, 2-Methoxy-4vinylohenol, 1-Oxaspiro[4,5]deca-3,6-diene,2,6,10,10-tetramethyl, 1-Oxaspiro[4,5] deca-3,6diene,2,6,10,10-tetramethyl, Neocurdione, Isoaromadendrene epoxide, 1b,4a-Epoxy-2Hcyclopenta[3,4]cyclopropa[8,9] cycloundec., Cis-Vaccenic acid, Phenanthrenol,4b,5,6,7,8,8a,9,10octahydro-4b,8,8-trimethyl-1, Galanthamine, Dibenz[a,c]cyclohexane,2,4,7-trimethoxy, 2,4a,7Trihydroxy-1-methyl-8-methyleneqibb-3-ene. 1,10-carboxylic acid, Retinoic acid, 7,8,12-Tri-O-acetyl-3desoxy-ingol-3-one and 4,6-Androstadien-3β-ol-17-one,acetate. The FTIR analysis of Rosmarinus oficinalis leaves proved the presence of Alkenes, Aliphatic fluoro compounds, Alcohols, Ethers, Carboxlic acids, Esters, Nitro compounds, Alkanes, Aldehydes, Ketones compounds. It contain chemical constitutions which may be useful for various herbal formulation as anti-inflammatory, analgesic, antipyretic, cardiac tonic and antiasthamatic.


INTRODUCTION
Rosmarinus officinalis , related to the lamiaceae family of plants.Rosemary is used as an antispasmodic in real colic and dysmenorrhoea, in relieving respiratory disorders and to stimulate growth of hair.Rosemary (Rosmarinus officinalis),which grows wild around Mediterranean area, is a species from Lamiaceae family.
It grows as a shrub or herbaceous plant with about 0.8 to 2m height (Atik bekkara et al, 2007).This plant likes dry and arid regions, hills and low mountains, calcareous, shale, clay and rocky substrates.The potent antioxidant properties of rosemary extracts have been attributed to its phenolic compounds, mainly rosmarinic acid and diterpenes carnosic acid and carnosol (Sergi and Leonor, 2002;Troncoso et al., 2005;Ameera et al., 2015).Rosemary extract relaxes smooth muscles and has choleretic, hepatoprotective and antitumerogenic activity (Al-Sereiti et al., 1999).Recent research shows that rosemary extracts possess strong anticancer properties.In the last few years gas chromatography mass spectrometry has become firmly established as a key technological platform for metabolite profiling in plant (Fiehn et al., 2003;Sumner et al., 2003;Fernie et al., 2004;Kell et al., 2005;Robertson et al., 2005).GC-MS based metabolome analysis has profound applications in discovering the mode of action of drugs or herbicides and helps unravel the effect of altered gene expression on metabolism and organism performance in biotechnological applications.Infrared spectroscopy provides a useful method for herbal analysis and elucidate the compounds structures as well as for quantitative analysis of drugs Bunaciu et al., 2010;Cheng et al., 2010;Bunaciu et al., 2010;Huda et al., 2015).Fourier transform infrared spectrometry is a physicochemical analytical technique and one of the most widely used methods to identify the structure of unknown composition or its chemical group, and the intensity of the absorption spectra associated with molecular composition or content of the chemical group (Surewicz et al., 1993;Imad et al., 2015).The present study involves an assessment using GC-MS and FT-IR spectroscopic techniques to investigate the authenticity of commercial sample of the herbal drug by analyzing their fingerprints.

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

Preparation of sample
About 20 g of the plant sample powdered were soaked in 100 ml methanol for 16 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 (Imad et al., 2015a).It was again filtered through sodium sulphate in order to remove the traces of moisture.

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 (Imad et al., 2014;Imad et al., 2015b andMuhanned et al., 2015).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).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 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 1ml per minute.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).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 (Imad et al., 2015c;Mohammed et al., 2015).

Fourier transform infrared spectrophotometer (FTIR)
The powdered sample of the plant specimen was treated for FTIR spectroscopy (Shimadzu, IR Affinity 1, Japan).The sample was run at infrared region between 400 and 4000 nm (Ameera et al., 2015 andHuda et al., 2015).

Figure 2 .
Figure 2. Structure of α-pinene present in the leaf extract of Rosmarinus oficinalis using GC-MS analysis.

Figure 3 .
Figure 3. Structure of camphene present in the leaf extract of Rosmarinus oficinalis using GC-MS analysis.

Figure 4 .
Figure 4. Structure of eucalyptol present in the leaf extract of Rosmarinus oficinalis using GC-MS analysis.

Figure 9 .
Figure 9. Structure of Neocurdione present in the leaf extract of Rosmarinus oficinalis using GC-MS analysis.

Figure 10 .
Figure 10.Structure of Isoaromadendrene epoxide present in the leaf extract of Rosmarinus oficinalis using GC-MS analysis.

Figure 12 .
Figure 12.Structure of Cis-Vaccenic acid present in the leaf extract of Rosmarinus oficinalis using GC-MS analysis.

Figure 14 .Figure 15 .
Figure 14.Structure of 2 Galanthamine present in the leaf extract of Rosmarinus oficinalis using GC-MS analysis.

Figure 17 .
Figure 17.Structure of Retinoic acid present in the leaf extract of Rosmarinus oficinalis using GC-MS analysis.

Table 1 .
Major phytochemical compounds identified in methanolic extract of Rosmarinus oficinalis leaves.