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

Introduction

Cinnamomum zeylanicum Blume (Lauraceae), is called true cinnamon. Cinnamon is an evergreen of tropical area reachingabout nine meters high and covered with a smooth, pale bark^1-3. 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^6-9. The bark of tree consists of volatile oil, possesses many medicinal properties like antibacterial, anti-oxidant, anti-ulcer, antidiabetic^10-12 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.

Materials and Methods

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^15-17.

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^18-22. 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 1μL 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 detected²³. 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^25-27. 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 libraries²⁸.

Determination of antibacterial activity of crude bioactive compounds of Cinnamomum zeylanicum.

The test pathogens (E. coli, Pseudomonas aeruginosa, Klebsiella pneumonia and   Staphylococcus aureus) were swabbed in Muller Hinton agar plates. 60μl 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 measured²⁹.

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 (Cinnamomum zeylanicum) was delivered into the wells. Antimicrobial activity was evaluated by measuring the zone of inhibition against the test microorganisms.   Methanol was used as solvent control. Amphotericin B and fluconazole were used as reference antifungal agent^30,31. The tests were carried out in triplicate. The antifungal activity was evaluated by measuring the inhibition-zone diameter observed after 48 h of incubation.

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 .

Results and Discusion

Analysis of phytochemical compounds of methanolic extract of Cinnamomum zeylanicum was carried out by gas chromatography-mass spectroscopy (Table1). The GC-MS chromatogram of the thirty nine peaks of the compounds detected are shown in Figure 1. Chromatogram GC-MS analysis of the methanolic extract of Cinnamomum zeylanicum showed the presence of thirty nine major peaks and the components corresponding to the peaks were determined as follows. The First set up peak were determined to be 6-Oxa-bicyclo[3.1.0]hexan-3-one Figure 2. The second peak indicated to be Benzaldehyde Figure 3. The next peaks considered to be Cyclohexene,4-isopropenyl-1-methoxymethoxymethyl, Benzoic acid , methyl ester, Benzaldehyde  dimethyl acetal,  -Oxa-bicyclo[3.1.0]hexan-3-one, 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), Cis – 2-Methoxycinnamic acid, Bicyclo[3.1.1]hept-2-ene,2,6- dimethyl-6-(4-methyl-3-pentenyl), Trans-2-Hydroxycinnamic acid , methyl ester, y-Muurolene, ß-Guaiene, Cadala-1(10),3,8-triene, Isolongifolene,4,5,9,10-dehydro,  Cubenol, Tau-Muurolol, Α-Cadinol , Spiro[tricyclo[4.4.0.0(5.9)]decane-10.2oxirane],1-methyl-4-isoprol,  6-Isopropenyl-4,8q-dimethyl-1,2,3,5,6,7,8,8a-octahydronaphthalen, Ethyl9,9-difomylnona-2,4,6,8-tetraenoate,  Trans-13-Octadecenoic acid, Tributyl acetylcitrate, 9,12,15-Octadecatrienoic acid ,2,3-dihydroxypropyl ester , (z,z,z), 9-Octadecenamide, 17.alfa.-21ß-28,30-Bisnorhopane, 17.alfa.-21ß-28,30-Bisnorhopane, Androstan-3-one,cyclic 1,2-ethanediyl mercaptole , (5α), (4H)4a,5,6,7,8,8a-Hexahydrobenzopyran-5-one-3-carboxamide,2, 4H-Cyclopropa[5´,6´]benz [1´,2´,7,8]azuleno[5,6]oxiren-4-one,8,8a, (22S)-21-Acetoxy-6α,11ß-dihydroxy-16α,17α-propylmethylenediox, (+)-γ-Tocopherol,O-methyl and Stigmasterol Figure 4-40. Five clinical pathogens were selected for antibacterial activity namely, (Klebsiella pneumoniae, Pseudomonas aeroginosa, E.coli, Staphylococcus aeureus and Proteus mirabilis. Maximum zone formation was against klebsiella pneumoniae, Table 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.

Table 1: Phytochemical compounds identified in methanolic extract of Cinnamomum zeylanicum.   Click here to View table

 

Table 2: Zone of inhibition (mm) of test bacterial strains to Cinnamomum zeylanicum bioactive compounds and standard antibiotics. 

/ Plant Antibiotics	Bacteria
	Proteus mirabilis	Pseudomonas eurogenosa	Escherichia coli	Klebsiella pneumonia	Staphylococcus aureus
Plant	4.92±0.22	3.99±0.31	5.39±0.22	6.12±0.52	5.19±0.02
Rifambin	0.70±0.3	0.96±0.11	1.00±0.13	0.90±0.20	0.93±0.50
Streptomycin	2.00±0.10	1.20±0.18	0.97±0.53	1.34±0.47	1.80±0.38
Kanamycin	0.39±0.17	0.60±0.33	1.00±0.19	0.98±0.40	0.50±0.12
Cefotoxime	0.89±0.6	1.40±0.26	1.36±0.40	0.96±0.39	1.90±0.36

 

Figure 1: GC-MS chromatogram of methanolic extract of Cinnamomum zeylanicum. Click here to View Figure

 

Figure 2: Structure of 6-Oxa-bicyclo[3.1.0]hexan-3-one present in the methanolic extract of C. zeylanicum using GC-MS analysis. Click here to View Figure

 

Figure 3: Structure of Benzaldehyde present in the methanolic extract of C. zeylanicum using GC-MS analysis. Click here to View Figure

 

Figure 4: Structure of Cyclohexene,4-isopropenyl-1-methoxymethoxymethyl present in the methanolic extract of C. zeylanicum using GC-MS analysis. Click here to View Figure

 

Figure 5: Structure of Benzoic acid, methyl ester present in the methanolic extract of C. zeylanicum using GC-MS analysis.  Click here to View Figure

 

Figure 6: Structure of Benzaldehyde  dimethyl acetal present in the methanolic extract of C. zeylanicum using GC-MS analysis. Click here to View Figure

 

Figure 7: Structure of Benzenepropanal present in the methanolic extract of C.zeylanicum using GC-MS analysis Click here to View Figure

 

Figure 8: Structure of Benzylidenemalonaldehyde present in the methanolic extract of C. zeylanicum using GC-MS analysis.  Click here to View Figure

 

Figure 9: Structure of 3-Phenylpropanol present in the methanolic extract of C. zeylanicum using GC-MS analysis.  Click here to View Figure

 

Figure 10: Structure of Cinnamaldehyde, (E) present in the methanolic extract of C. zeylanicum using GC-MS analysis. Click here to View Figure

 

Figure 11: Structure of 2-Propen- 1- ol,3-phenyl present in the methanolic extract of C. zeylanicum using GC-MS analysis. Click here to View Figure

 

Figure 12: Structure of 9-Methoxybicyclo[6.1.0]nona – 2,4,6- triene present in the methanolic extract of C. zeylanicum by using GC-MS analysis.  Click here to View Figure

 

Figure 13: Structure of 1,3-Bis(cinnamoyloxymethyl)adamantine present in the methanolic extract of C. zeylanicum using GC-MS analysis. Click here to View Figure

 

Figure 14: Structure of Alfa . – Copaene present in the methanolic extract of C. zeylanicum using GC-MS analysis. Click here to View Figure

 

Figure 15: Structure of Naphthalene , 1,2,3,5,6,7,8,8a-octahydro-1,8a-dimethyl-7-(1-methyl)present in the methanolic extract of C. zeylanicum using GC-MS analysis. Click here to View Figure

 

Figure 16: Structure of Cis – 2-Methoxycinnamic acid present in the methanolic extract of C. zeylanicum using GC-MS analysis.  Click here to View Figure

 

Figure 17: Structure of Bicyclo[3.1.1]hept-2-ene,2,6- dimethyl-6-(4-methyl-3-pentenyl) present in the methanolic extract of C. zeylanicum using GC-MS analysis. Click here to View Figure

 

Figure 18: Structure of Trans-2-Hydroxycinnamic acid , methyl ester present in the methanolic extract of C. zeylanicum by using GC-MS analysis. Click here to View Figure

 

Figure 19: Structure of y-Muurolene present in the methanolic extract of C. zeylanicum using GC-MS analysis.  Click here to View figure

 

Figure 20: Structure of Naphthalene present in the methanolic extract of C. zeylanicum by using GC-MS analysis.  Click here to View Figure

 

Figure 21: Structure of ß-Guaiene present in the methanolic extract of C. zeylanicum using GC-MS analysis.  Click here to View Figure

 

Figure 22: Structure of Cadala-1(10),3,8-triene present in the methanolic extract of C. zeylanicum using GC-MS analysis. Click here to View Figure

 

Figure 23: Structure of Isolongifolene,4,5,9,10-dehydro present in the methanolic extract of C. zeylanicum using GC-MS analysis. Click here to View Figure

 

Figure 24: Structure of Cubenol present in the methanolic extract of C. zeylanicum by using GC-MS analysis.  Click here to View Figure

 

Figure 25: Structure of Tau-Muurolol present in the methanolic extract of C. zeylanicum using GC-MS analysis.  Click here to View Figure

 

Figure 26: Structure of Α-Cadinol present in the methanolic extract of C. zeylanicum using GC-MS analysis. Click here to View Figure

 

Figure 27: Structure of Spiro[tricyclo[4.4.0.0(5.9)]decane-10.2oxirane] present in the methanolic extract of C. zeylanicum using GC-MS analysis. Click here to View Figure

 

Figure 28: Structure of 6-Isopropenyl-4,8q-dimethyl-1,2,3,5,6,7,8,8a-octahydronaphthalen  present in the methanolic extract of C. zeylanicum using GC-MS analysis. Click here to View Figure

 

Figure 29: Structure of Ethyl9,9-difomylnona-2,4,6,8-tetraenoate present in the methanolic extract of C. zeylanicum using GC-MS analysis. Click here to View Figure

 

Figure 30: Structure of Trans-13-Octadecenoic acid present in the methanolic extract of C. zeylanicum using GC-MS analysis. Click here to View Figure

 

Figure 31: Structure of Tributyl acetylcitrate present in the methanolic extract of C. zeylanicum using GC-MS analysis. Click here to View Figure

 

Figure 32: Structure of 9,12,15-Octadecatrienoic acid ,2,3-dihydroxypropyl ester present in the methanolic extract of C. zeylanicum using GC-MS analysis. Click here to View Figure

 

Figure 33: Structure of 9-Octadecenamide present in the methanolic seeds extract of C. zeylanicum using GC-MS analysis.  Click here to View Figure

 

Figure 34: Structure of 17.alfa.-21ß-28,30-Bisnorhopane present in the methanolic extract of C. zeylanicum using GC-MS analysis. Click here to View Figure

 

Figure 35: Structure of Androstan-3-one,cyclic 1,2-ethanediyl mercaptole , (5α) present in the methanolic extract of C. zeylanicum using GC-MS analysis. Click here to View Figure

 

Figure 36: Structure of (4H)4a,5,6,7,8,8a-Hexahydrobenzopyran-5-one-3-carboxamide,2 present in the methanolic extract of C. zeylanicum using GC-MS analysis. Click here to View Figure

 

Figure 37: Structure of 4H-Cyclopropa[5´,6´]benz[1´,2´,7,8]azuleno[5,6]oxiren-4-one,8,8a  present in the methanolic extract of C. zeylanicum using GC-MS analysis.  Click here to View Figure

 

Figure 38: Structure of (22S)-21-Acetoxy-6α,11ß-dihydroxy-16α,17α-propylmethylenediox  present in the methanolic extract of C. zeylanicum using GC-MS analysis.  Click here to View Figure

 

Figure 39: Structure of (+)-γ-Tocopherol, present in the methanolic extract of C. zeylanicum using GC-MS analysis. Click here to View Figure

 

Figure 40: Structure of Stigmasterol present in the methanolic extract of C. zeylanicum using GC-MS analysis. Click here to View Figure

 

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.

Acknowlgements

Special thanks to Prof. Abdul-Kareem, Babylon University, Faculty of science for women, for his special care.

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