Evaluation of Antifungal and Antibacterial Activity and Analysis of Bioactive Phytochemical ...

20 Pages·2016·2.9 MB·English

ISSN: 0970-020 X
ORIENTAL JOURNAL OF CHEMISTRY
CODEN: OJCHEG
An International Open Free Access, Peer Reviewed Research Journal
2016, Vol. 32, No. (4):
Pg. 1769-1788
www.orientjchem.org
Evaluation of Antifungal and Antibacterial Activity and
Analysis of Bioactive Phytochemical Compounds of
Cinnamomum zeylanicum (Cinnamon bark) using Gas
Chromatography-Mass Spectrometry
IMAd HAdI HAMEEd*1, HUdA JASIM ALTAMEME2 and GHAIdAA JIHAdI MOHAMMEd3
1College of Nursing, Babylon University, Iraq.
2Department of Biology, Babylon University, Iraq.
3College of Science, Al-Qadisiya University, Iraq.
*Corresponding author E-mail: [email protected]
http://dx.doi.org/10.13005/ojc/320406
(Received: May 31, 2016; Accepted: July 19, 2016)
ABSTRACT
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),
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, Á-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, 9-Octadecenamide,
17.alfa.-21ß-28,30-Bisnorhopane, 17.alfa.-21ß-28,30-Bisnorhopane, Androstan-3-one,cyclic 1,2-
ethanediyl mercaptole , (5á), (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-6á,11ß-
dihydroxy-16á,17á-propylmethylenediox, (+)-ã-Tocopherol,O-methyl and Stigmasterol. Cinnamomum
zeylanicum 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.
Keywords: Antifungal, Antibacterial, Cinnamomum zeylanicum,
Gas chromatography-mass spectrometry, Fourier-transform infrared spectroscopy.
HAMEED et al., Orient. J. Chem., Vol. 32(4), 1769-1788 (2016) 1770
INTROdUCTION for 45 minutes. As the compounds were separated,
they eluted from the column and entered a detector
Cinnamomum zeylanicum Blume which was capable of creating an electronic signal
(Lauraceae), is called true cinnamon. Cinnamon is an whenever a compound was detected23. The greater
evergreen of tropical area reachingabout nine meters the concentration in the sample, bigger was the
high and covered with a smooth, pale bark1-3. It is signal obtained which was then processed by a
considered to be the native of Sri Lanka and Malabar computer. The time from when the injection was
Coast of India4,5. Cinnamon mainly contains essential made (Initial time) to when elution occurred referred
oils and important compounds like cinnamaldehyde, to as the Retention time (RT) 24. While the instrument
eugenol, cinnamic acid and cinnamate. It has was run, the computer generated a graph from the
traditionally been used to treat toothache, fight bad signal called Chromatogram. Each of the peaks in the
breath and treatment common cold6-9. The bark chromatogram represented the signal created when
of tree consists of volatile oil, possesses many a compound eluted from the Gas chromatography
medicinal properties like antibacterial, anti-oxidant, column into the detector. The X-axis showed the RT
anti-ulcer, antidiabetic10-12 and antifungal (Bruneton and the Y-axis measured the intensity of the signal
et al., 1998). Cinnamaldehye is the most prevalent to quantify the component in the sample injected.
with concentration of 6,000 –30,000 ppm13,14. As individual compounds eluted from the Gas
The aims of this study were analysis of chemical chromatographic column, they entered the electron
compounds of Cinnamomum Zeylanicum (Cinnamon ionization (mass spectroscopy) detector, where they
bark) and evaluation of antifungal and antibacterial were bombarded with a stream of electrons causing
activity. them to break apart into fragments. The fragments
obtained were actually charged ions with a certain
MATERIALS ANd METHOdS mass25-27. The M/Z (mass/charge) ratio obtained was
calibrated from the graph obtained, which was called
Collection and preparation of plant material as the Mass spectrum graph which is the fingerprint
Cinnamomum zeylanicum (Cinnamon of a molecule. Before analyzing the extract using
bark) were purchased from local market in Hilla city, Gas Chromatography and Mass Spectroscopy, the
middle of Iraq. After thorough cleaning and removal temperature of the oven, the flow rate of the gas used
of foreign materials, the Cinnamon bark was stored and the electron gun were programmed initially. The
in airtight container to avoid the effect of humidity temperature of the oven was maintained at 100°C.
and then stored at room temperature until further Helium gas was used as a carrier as well as an
use15-17. eluent. The flow rate of helium was set to 1ml per
minute. The electron gun of mass detector liberated
Preparation of sample electrons having energy of about 70eV. The column
About eighteen grams of methanolic extract employed here for the separation of components was
of Cinnamomum zeylanicum powdered were soaked Elite 1 (100% dimethyl poly siloxane). The identity
in thirty three ml methanol for ten hours in a rotatory of the components in the extracts was assigned by
shaker. Whatman No.1 filter paper was used to the comparison of their retention indices and mass
separate the extract of plant18-22. The filtrates were spectra fragmentation patterns with those stored
used for further phytochemical analysis. It was again on the computer library and also with published
filtered through sodium sulphate in order to remove literatures. Compounds were identified by comparing
the traces of moisture. their spectra to those of the Wiley and NIST/EPA/
NIH mass spectral libraries28.
Gas chromatography – mass spectrum
analysis determination of antibacterial activity of
The GC-MS analysis of the plant extract crude bioactive compounds of Cinnamomum
was made in a (QP 2010 Plus SHIMADZU) zeylanicum
instrument under computer control at 70 eV. About The test pathogens (E. coli, Pseudomonas
1ìL of the methanol extract was injected into the GC- aeruginosa, Klebsiella pneumonia and
MS using a micro syringe and the scanning was done Staphylococcus aureus) were swabbed in Muller
1771 HAMEED et al., Orient. J. Chem., Vol. 32(4), 1769-1788 (2016)

Pharmacological actions New chemical compound Anti- convulsant activity; anti-microbial, anti-daibtic, and antiobesity New chemical compound New chemical compound Anti-cancer activity Antimutagenic and anti-malarial Anti-inflammatory Antineoplastic and anti- inflammatory
6 6
19 5, 5, 13
um ns 06 164. 2,10 1,10 05, 03, 117,
mum zeylanic MS Fragment- io 55,69,98 51,63,77,86,1 53.79.91.119. 51,59,65,77,9 118,136 51,59,65,77,9 121,136,151 51,65,78,91,1 115,134 51,63,77,91,1 115,131,159 51,77,91,105,
o
m
a
n
n
Ci
of
ct
a
olic extr emical ucture
han Ch str
et
m
n
d i
e
fi
nti
e
d
al compounds i cular Exact htMass 98.0368 106.042 196.146 136.052 152.084 134.073 160.052 136.089
hemic Mole Weig 98 106 196 136 152 134 160 136
c
e 1: Phyto Formula CHO 562 CHO 76 CHO 12202 CHO 882 CHO2 912 CHO 910 CHO 1082 CHO 912
Tabl T (min) 419 67 859 095 284 942 715 121
R 3. 3. 3. 5. 5. 5. 6. 7.
yl-
Phytochemical compound 6-Oxa-bicyclo[3.1.0] hexan-3-one Benzaldehyde Cyclohexene, 4-isopropenyl-1- methoxymethoxymeth Benzoic acid methyl ester Benzaldehyde dimethyl acetal Benzenepropanal Benzylidenema- lonaldehyde 3-Phenylpropanol
S No. 1 2 3 4 5 6 7 8
1772 HAMEED et al., Orient. J. Chem., Vol. 32(4), 1769-1788 (2016)
Antityrosinase activity; anti-inflammatory and antitermitic New chemical compound New chemical compound New chemical compound New chemical compound New chemical compound Anti-tyrosinase activities; anti- tyrosinase and anti- melanogenic activities New chemical compound
4
0
3,74,77,91,103, 131 3,78,92,105, 134 3,77,91,105, 133,148 7,91,103,119,131, 160,204,250,280 9,77,91,105,119, 147,161,175,189,2 7,79,107,119,133, 175,189,204 3,77,85,91,103, 131,147,161,178 9,77,93,107,119, 148,161,189,204
1,6 15, 1,6 15, 1,6 15, 1,7 47, 5,6 33, 5,6 47, 1,6 18, 5,6 33,
5 1 5 1 5 1 5 1 5 1 5 1 5 1 5 1

58 73 89 3 88 88 63 88
0 0 0 2 1 1 0 1
2. 4. 8. 6. 4. 4. 8. 4.
3 3 4 5 0 0 7 0
1 1 1 4 2 2 1 2
2 4 8 6 4 4 8 4
3 3 4 5 0 0 7 0
1 1 1 4 2 2 1 2
CHO 98 CHO 910 CHO 1012 CHO 30324 CH 1224 CH 1524 CHO 10103 CH 1524

9 2 9 4 7 5 1 5
1 8 1 1 9 3 8 5
6 0 2 4 7 9 1 4
7. 8. 8. 8. 8. 8. 9. 9.
Cinnamaldehyde, (E)- 2-Propen- 1- ol, 3-phenyl- 9-Methoxybicyclo [6.1.0]nona – 2, 4,6- triene 1,3-Bis(cinnamoy- loxymethyl)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)
9 10 11 12 13 14 15 16
1773 HAMEED et al., Orient. J. Chem., Vol. 32(4), 1769-1788 (2016)
y d d d d
Anti-inflammatory activit New chemical compoun New chemical compoun New chemical compoun New chemical compoun Antifungal and anti-HIV Anti-wood-decay fungal activity and moderate antimicrobial activity
85 4
1 0
5,75,91,103,118, 146,161,178 9,93,105,119,133, 161,175,189,204 7,81,91,105,119, 161,175,189,204 5,66,91,105,115, 142,157,183,200 1,115,128,143,157, 1,93,119,161,189,2 9,95,121,134,161, 204,222
1,6 31, 5,7 47, 5,6 33, 3,6 31, 1,9 9,8 5,7 89,
5 1 5 1 5 1 5 1 5 5 5 1

3 8 8 2 7 8 8
6 8 8 7 5 9 9
0 1 1 1 1 1 1
8. 4. 4. 2. 0. 2. 2.
7 0 0 0 0 2 2
1 2 2 2 2 2 2
8 4 4 2 0 2 2
7 0 0 0 0 2 2
1 2 2 2 2 2 2
HO 103 H 24 H 24 H 22 H0 2 HO 26 HO 26
C10 C15 C15 C15 C15 C15 C15

1 97 71 8 69 41 02
0 4 7 8 0 8 0
9.7 10. 10. 10. 11. 11. 12.
c- 5,
Trans-2-Hydroxy innamic acid , methyl ester y-Muurolene ß-Guaiene Cadala-1(10), 3,8-triene Isolongifolene,4, 9,10-dehydro- Cubenol Tau-Muurolol
7 8 0 1 2 3 4
1 1 2 2 2 2 2
1774 HAMEED et al., Orient. J. Chem., Vol. 32(4), 1769-1788 (2016)
e
Anti-fungal and as hepatoprotective and cytotoxic activities New chemical compound New chemical compound New chemical compound Good anti-inflammatory activity Anti-Feeding effect: Larva New chemical compound Anti-inflammatory activity and, antibacterial activity
9,95,121,137,161,204,222 5,81,91,105,123,145,161, 73,191,205,221,234,252 5,79,91,107,149,175, 18,236 1,65,77,103,131,160, 88,205,234 5,69,83,123,180, 22,264,282 ###### 7,67,79,95,109,135,155, 73,232,261,291,321,352 9,72,83,114,184, 12,264,281
7 5 1 5 2 5 1 5 2 # 5 1 5 2

8 3 8 9 6 5 1 2
9 7 7 8 5 2 6 7
1 1 1 0 2 2 2 2
2. 2. 6. 4. 2. 2. 2. 1.
2 5 3 3 8 0 5 8
2 2 2 2 2 4 3 2
2 2 6 4 2 2 2 1
2 5 3 3 8 0 5 8
2 2 2 2 2 4 3 2
O O 2 O 2 O 4 O 2 O 8 O 4
H26 H24 H24 H14 H34 H34 H36
C15 C15 C15 C13 C18 C20 C21

9 4 1 7 8 2 6 4
3 8 1 5 6 5 7 3
1 4 6 8 9 9 1 8
2. 3. 4. 4. 6. 7. 8. 8.
1 1 1 1 1 1 1 1
‘-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
5 6 7 8 9 0 1 2
2 2 2 2 2 3 3 3
1775 HAMEED et al., Orient. J. Chem., Vol. 32(4), 1769-1788 (2016)
A nd nd nd nd
S u u u u
R o o o o
1,95,109,149,163,177,191, Important for anti-M 17,246,313,369,384 activity 5,67,81,95,132,189, New chemical comp 19,241,257,289,350 ###### New chemical comp 3,69,83,109,124,149,193, New chemical comp 15,308,340,383,400,442 5,91,121,149,223,,279,297, New chemical comp 51,387,416,445,488 7,91,137,165,205,260, Anti-oxidant activity 02,344,486,430 5,69,83,133,213,255, Anti-platelet 00,351,369,412
8 2 5 2 # 5 2 5 3 5 3 5 3

6 6 9 1 1 1 1
7 7 8 3 4 8 7
3 3 1 2 2 3 3
4. 4. 5. 0. 8. 0. 2.
8 8 2 2 8 3 1
3 3 3 5 4 4 4
4 4 5 0 8 0 2
8 8 2 2 8 3 1
3 3 3 5 4 4 4
O 10 O 8 O 2 O
H 48 H36 H36 H50 H48
C28 C27 C27 C29 C29

7 7 2 1 1 7 1
8 4 6 3 3 2 4
2 4 5 8 0 4 8
0. 0. 0. 0. 5. 6. 8.
2 2 2 2 2 2 2
17.alfa.-21ß-28, 30-Bisnorhopane Androstan-3-one, cyclic 1,2-ethanediyl mercaptole , (5±) (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-6±, 11ß-dihydroxy-16±, 17±-propylmethylenediox (+)-³-Tocopherol,O-methyl- Stigmasterol
3 4 5 6 7 8 9
3 3 3 3 3 3 3
HAMEED et al., Orient. J. Chem., Vol. 32(4), 1769-1788 (2016) 1776
Table 2: Zone of inhibition (mm) of test bacterial strains to Cinnamomum
zeylanicum bioactive compounds and standard antibiotics
Plant Bacteria
Antibiotics Proteus Pseudomonas Escherichia Klebsiella Staphylococcus
mirabilis eurogenosa coli pneumonia 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
Table 3: Zone of inhibition (mm) of Aspergillus Spp. test to
Cinnamomum zeylanicum bioactive compounds and standard antibiotics
Plant Aspergillus Spp.
Antibiotics Aspergillus Aspergillus Aspergillus Aspergillus
niger terreus flavus fumigatus
Plant 3.00±0.120 4.71±0.52 6.16±0.42 5.19±0.02
Amphotericin B 2.61±0.270 4.28±0.610 3.95±0.5 4.00±0.820
Fluconazol 4.79±0.211 3.21±0.25 2.90±0.451 4.70±0.930
Control 0.00 0.00 0.00 0.00
Fig. 1: GC-MS chromatogram of methanolic extract of Cinnamomum zeylanicum
1777 HAMEED et al., Orient. J. Chem., Vol. 32(4), 1769-1788 (2016)
Hinton agar plates. 60ìl of plant extract was loaded determination of antifungal activity
on the bored wells. The wells were bored in 0.5cm Five-millimeter diameter wells were cut from
in diameter. The plates were incubated at 37C° the agar using a sterile cork-borer, and 50 ìl of the
for 24 hrs and examined. After the incubation the samples solutions (Cinnamomum zeylanicum) was
diameter of inhibition zones around the discs was delivered into the wells. Antimicrobial activity was
measured29. evaluated by measuring the zone of inhibition against
Fig. 2: Structure of 6-Oxa-bicyclo[3.1.0]hexan- Fig. 3: Structure of Benzaldehyde present in
3-one present in the methanolic extract of C. the methanolic extract of C. zeylanicum using
zeylanicum using GC-MS analysis GC-MS analysis
Fig. 4: Structure of Cyclohexene,4-isopropenyl- Fig. 5: Structure of Benzoic acid, methyl
1-methoxymethoxymethyl present in the ester present in the methanolic extract of C.
methanolic extract of C. zeylanicum using zeylanicum using GC-MS analysis
GC-MS analysis
HAMEED et al., Orient. J. Chem., Vol. 32(4), 1769-1788 (2016) 1778
the test microorganisms. Methanol was used as Statistical analysis
solvent control. Amphotericin B and fluconazole Data were analyzed using analysis of
were used as reference antifungal agent30,31. The variance (ANOVA) and differences among the means
tests were carried out in triplicate. The antifungal were determined for significance at P < 0.05 using
activity was evaluated by measuring the inhibition- Duncan’s multiple range test (by SPSS software)
zone diameter observed after 48 h of incubation. Version 9.1 .
Fig. 6: Structure of Benzaldehyde dimethyl
Fig. 7: Structure of Benzenepropanal present in
acetal present in the methanolic extract of C.
the methanolic extract of C. zeylanicum using
zeylanicum using GC-MS analysis
GC-MS analysis
Fig. 8: Structure of Benzylidenemalonaldehyde Fig. 9: Structure of 3-Phenylpropanol present
present in the methanolic extract of C. in the methanolic extract of C. zeylanicum
zeylanicum using GC-MS analysis using GC-MS analysis