Evaluation of Antifungal and Antibacterial Activity and Analysis of Bioactive Phytochemical Compounds of Cinnamomum zeylanicum (Cinnamon bark) using Gas Chromatography-Mass Spectrometry

Phytochemicals are chemical compounds often referred to as secondary metabolites. Thirty nine bioactive phytochemical compounds were identified in the methanolic extract of Cinnamon bark. The identification of phytochemical compounds is based on the peak area, retention time molecular weight and molecular formula. GC-MS analysis of Cinnamomum zeylanicum revealed the existence of the 6 -Oxa-bicyclo[3.1.0]hexan-3-one, Benzaldehyde, Cyclohexene,4- isopropenyl-1-methoxymethoxymethyl, Benzoic acid- methyl ester, Benzaldehyde dimethyl acetal, Benzenepropanal, Benzylidenemalonaldehyde, 3-Phenylpropanol, Cinnamaldehyde, (E), 2-Propen- 1- ol,3-phenyl, 9-Methoxybicyclo[6.1.0]nona – 2,4,6- triene , 1,3-Bis(cinnamoyloxymethyl) adamantine, Alfa.– Copaene, Naphthalene , 1,2,3,5,6,7,8,8a-octahydro-1,8a-dimethyl-7-(1-methyl), contain chemical constitutions which may be useful for various herbal formulation as anti-inflammatory, analgesic, antipyretic, cardiac tonic and antiasthamatic. Cinnamomum zeylanicum was highly active against Aspergillus flavus (6.16±0.42). Methanolic extract of bioactive compounds of Cinnamomum zeylanicum was assayed for in vitro antibacterial activity against Pseudomonas aerogenosa, Escherichia coli , Proteus mirabilis, Staphylococcus aureus and Klebsiella pneumonia by using the diffusion method in agar. The zone of inhibition were compared with different standard antibiotics. The diameters of inhibition zones ranged from 6.12±0.52 to 0.39±0.17 mm for all treatments.


INTROdUCTION
C i n n a m o m u m zey l a n i c u m B l u m e (Lauraceae), is called true cinnamon.Cinnamon is an evergreen of tropical area reachingabout nine meters high and covered with a smooth, pale bark [1][2][3] .It is considered to be the native of Sri Lanka and Malabar Coast of India 4,5 .Cinnamon mainly contains essential oils and important compounds like cinnamaldehyde, eugenol, cinnamic acid and cinnamate.It has traditionally been used to treat toothache, fight bad breath and treatment common cold [6][7][8][9] .The bark of tree consists of volatile oil, possesses many medicinal properties like antibacterial, anti-oxidant, anti-ulcer, antidiabetic [10][11][12] and antifungal (Bruneton  et al., 1998).Cinnamaldehye is the most prevalent with concentration of 6,000 -30,000 ppm 13,14 .The aims of this study were analysis of chemical compounds of Cinnamomum Zeylanicum (Cinnamon bark) and evaluation of antifungal and antibacterial activity.

Collection and preparation of plant material
Cinnamomum zeylanicum (Cinnamon bark) were purchased from local market in Hilla city, middle of Iraq.After thorough cleaning and removal of foreign materials, the Cinnamon bark was stored in airtight container to avoid the effect of humidity and then stored at room temperature until further use [15][16][17] .

Preparation of sample
About eighteen grams of methanolic extract of Cinnamomum zeylanicum powdered were soaked in thirty three ml methanol for ten hours in a rotatory shaker.Whatman No.1 filter paper was used to separate the extract of plant [18][19][20][21][22] .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
The GC-MS analysis of the plant extract was made in a (QP 2010 Plus SHIMADZU) instrument under computer control at 70 eV.About 1ìL of the methanol extract was injected into the GC-MS using a micro syringe and the scanning was done for 45 minutes.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 23 .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 referred to as the Retention time (RT) 24 .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 [25][26][27] .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.Compounds were identified by comparing their spectra to those of the Wiley and NIST/EPA/ NIH mass spectral libraries 28 .

determination of antibacterial activity of crude bioactive compounds of Cinnamomum zeylanicum
The test pathogens (E.coli, Pseudomonas a e r u g i n o s a , K l e b s i e l l a p n e u m o n i a a n d Staphylococcus aureus) were swabbed in Muller

zeylanicum using GC-MS analysis
Hinton agar plates.60ìl of plant extract was loaded on the bored wells.The wells were bored in 0.5cm in diameter.The plates were incubated at 37C° for 24 hrs and examined.After the incubation the diameter of inhibition zones around the discs was measured 29 .

determination of antifungal activity
Five-millimeter diameter wells were cut from the agar using a sterile cork-borer, and 50 ìl of the samples solutions (Cinnamomum zeylanicum) was delivered into the wells.Antimicrobial activity was evaluated by measuring the zone of inhibition against

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 (by SPSS software) Version 9.1 .2. Methanolic extraction of plant showed notable antifungal activities against Aspergillus niger, Asp.terreus, Asp.flavus, and Asp.fumigatus Table 3. Cinnamomum zeylanicum was very highly active against Aspergillus flavus (6.16±0.42).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
From the results obtained in this study, it could be concluded that Cinnamomum zeylanicum acts possesses remarkable antimicrobial activity, which is mainly due to (E)-cinnamaldehyde.According to these findings, it could be said that the methanolic extract of Cinnamomum zeylanicum acts as antifungal and antibacterial agents.