In vitro Antibacterial, Antifungal and Phytochemical Analysis of Methanolic Extract of Fruit Cassia

18 Pages·2016·2.22 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. (3):
Pg. 1329-1346
www.orientjchem.org
In vitro Antibacterial, Antifungal and Phytochemical
Analysis of Methanolic Extract of Fruit Cassia fistula
MOHANAD JAWAD KADHIM1, GHAIDAA JIHADI MOHAMMED2
and IMAD HADI HAMEED3*
1Department of Genetic Engineering,, Al-Qasim Green University, Iraq.
2College of Science, Al-Qadisyia University, Iraq.
3College of Nursing, Babylon University, Iraq.
*Corresponding author E-mail: [email protected]
http://dx.doi.org/10.13005/ojc/320307
(Received: April 02, 2016; Accepted: May 05, 2016)
ABSTRACT
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 analysis of Cassia fistula revealed the presence of the Oxacyclododecan-2-one, Imidazole
,2-amino-5-[(2-carboxy)vinyl], D-Glucose , 6-O-á-D-galactopyranosyl, 2-Nonanone, Eicosanoic
acid , phenylmethyl ester, Phenol , 4-(2-propenyl), Eugenol, Caryophyllene, ß-copaene,
Azulene,1,2,3,3a,4,5,6,7-octahydro-1,4-dimethyl-7-(1-methylethenyl), á-acorenol, Spiro[5.5]
undec-8-en-1-one, Isoaromadendrene epoxide, Tetraacetyl-d-xylonic nitrile , Benzyl Benzoate,
N-Isobutyl-(2E,4Z,8Z,10E)-dodecatetraenamide, Phenethylamine , 3-benzyloxy-2-fluoro-ß-hydroxy,
4a-Hydroxy-4-nitroperhydronaphthalen-1-one, Dasycarpidan -1-methanol, acetate (ester), Propanoic
acid , 2-(3-acetoxy-4,4,14-trimethylandrost-8-en-17-yl), Carda-4,20(22)-dienolide,3-[(6-deoxy-3-O-
methyl-á-L-mannopyranosyl, Cis-13-Eicosenoic acid , 16-Nitrobicyclo[10.4.0]hexadecane-1-ol-13-
one, Strychane ,1-acetyl-20á-hydroxy-16-methylene, 2,4,6-Decatrienoic acid , 1a,2,5,5a,6,9,10,10a-
octahydro-5,5a-dihydro, Vitamin E and Glycine ,N-[(3á,5ß,12á)-3,12-dihydroxy-24-oxocholan-24-
yl]. The FTIR analysis of Cassia fistula leaves proved the presence of Alkenes, Aliphatic fluoro
compounds, Alcohols, Ethers, Carboxlic acids, Esters, Nitro Compounds, Alkanes, Alcohols and
Phenol. Cassia fistula was highly active against Aspergillus terreus (6.99±0.29). Methanolic extract
of bioactive compounds of Cassia fistula was assayed for in vitro antibacterial activity against eleven
pathogenic bacteria by using the diffusion method in agar. The zone of inhibition were compared with
different standard antibiotics. The diameters of inhibition zones ranged from 1.00±0.05 to 6.02±0.23
mm for all treatments.
Keywords: GC/MS, Bioactive compounds, FT-IR, Cassia fistula.
INTRODUCTION medicines, dietary supplements and other useful
commercial products1. Also, has been reported
Cassia fistula plant have naturally occurring to contain anthraquinone the principal laxative
bioactive compounds and are mostly secondary constituent of many plants used as purgative2.
metabolites which are now a days being used as Plants have been an important source of medicine
1330 KADHIM et al., Orient. J. Chem., Vol. 32(3), 1329-1346 (2016)
with qualities for thousands of years3,4. Mainly on rpm and supernatant was injected in GC-MS for
traditional remedies such as herbs for their history, analysis20,21.
they have been used as popular folk medicines5,6.
Since thousands of years back, plants are used as a Gas chromatography-mass spectrometry (GC-
major source for medicine as they found to possess MS) analysis
a reservoir of bioactive compound7-9. Cassia fistula GC-MS of methanol extract was performed
contains alkaloids, tannins, flavonoids,terpenes, using Agilent 7890A. The run time was 30 minutes.
sugars, and glucosides. Tannins are naturally ionization of sample components were performed
occurring and water soluble phenolic compounds, on EI mode (70 eV). The carrier gas was helium at
which precipitate proteins from aqueous media10. 1.0ml/min flow rate. 0.5 ml of sample was injected in
Cassia fistula shows the presence of glycoside a split mode of 20:1.The mass spectrum scan range
natural product, which is used to enhance the cardiac was set at 29.0 to 500(m/z) 22.
contractile force in patient with congestive heart
failure11 glycoside also plays major role in the cancer Identification of compounds
therapy12. They also play major role in controlling Interpretation of mass spectrum of GC-
topical disease as eczema etc. Many metabolites MS was done using the database of National
have found to possess interesting biological activities Institute Standard and Technology (NIST). The mass
such as bactericidal, fungicidal, hepatoprotective and spectrum of phytochemicals was compared with the
muscle relaxant13. A fruit is cylindrical pod and seeds spectrum of known compounds stored in the NIST
many in black, sweet pulp separated by transverse library23.
partitions. The long pods which are green, when
unripe, turn black on ripening after flowers shed14. Determination of antibacterial activity of crude
Pulp is dark brown in colour, sticky, sweet and bioactive compounds of Cassia fistula
mucilaginous, odour characteristic, and somewhat The anti-bacterial activity was evaluated
disagreeable15. Each compartment contains one seed using Mueller-Hinton agar. The bacterial plates
which is flat, oval, reddish brown with a well-marked were incubated at 37 °C for 24 h. After incubation,
raphe. The seed contains a whitish endosperm in the diameter of the inhibition zone was measured
which the yellowish embryo is embedded16. Fruits to evaluate the antimicrobial activity. Each test was
are used in the treatment of diabetes, antipyretic, performed twice and the average of the results
abortifacient, demulcent, lessens inflammation and was calculated. The extraction solvents were used
heat of the body; useful in chest complaints, throat as negative control24,25. The test pathogens were
troubles, liver complaints, diseases of eye and swabbed in Muller Hinton agar plates. 60ìl of plant
gripping17. The aim of this research was study the extract was loaded on the bored wells. The wells
phytochemical composition of Cassia fistula and to were bored in 0.5 cm in diameter. The plates were
evaluate the isolates for possible in vitro antifungal incubated at 37C° for 24 h and examined. After the
and antibacterial activities. incubation the diameter of inhibition zones around
the discs was measured26.
MATERIALS AND METHODS
Determination of antifungal activity
Solvent extraction Five-millimeter diameter wells were cut
The shade dried powdered of Cassia fistula from the agar using a sterile cork-borer, and 50 ìl of
was extracted with methanol. The extract was filtered the samples solutions Cassia fistula was delivered
with Whatman’s filter paper. Filtrate was concentrated into the wells. Antimicrobial activity was evaluated
under reduced pressure and preserved at 5Cº in dark by measuring the zone of inhibition against the test
air tight bottles18,19. microorganisms. Methanol was used as solvent
control. Amphotericin B and fluconazole were used
Sample preparation for GC-MS analysis as reference antifungal agent. The tests were carried
50 µl of sample (Cassia fistula) was out in triplicate. The antifungal activity was evaluated
dissolved in 2ml of methanol and kept in ultrasonic by measuring the inhibition-zone diameter observed
bath for 25 min and centrifuged for 10 min. at 6000 after 48 h of incubation27,28.
1331 KADHIM et al., Orient. J. Chem., Vol. 32(3), 1329-1346 (2016)
et
Pharmacological actions Biological activities such as antitumor, anti- inflammatory and antiplatel Antimicrobial and anti- inflammatory activity Anti-inflammatory activities Anti-bacterial activity Anti-inflammatory activity Antioxidant, anti microbial and anti inflammatory
4
3
1
of Cassia fistula MS Fragment- ions 55,69,84,98 55,69,109,135 60,73,85,110, 126,182,212,261 58,67,71,85,99, 113,127,142 57,71,85,91,97, 108,126,147,167,207 51,65,77,91,107,115,
ct
a
xtr
e
c
oli
n
ha e
et ur
n m uct
d i str
fie al
nti mic
e
d e
s i Ch
d
n
u
o
p
m
o
c
or phytochemical olecular Exact WeightMass 184 184.146 153 153.054 342 342.116 142 142.136 402 402.35 134 134.073
aj M
M
Table 1: Formula CHO 11202 CHNO 6732 CHO 122211 CHO 918 CHO 27462 CHO 910
T (min) 3.367 3.613 3.751 4.878 5.833 7.344
R
Phytochemical compound Oxacyclododecan-2-one Imidazole ,2-amino-5- [(2-carboxy)vinyl]- D-Glucose , 6-O-±- D-galactopyranosyl- 2-Nonanone Eicosanoic acid , phenylmethyl ester Phenol , 4-(2-propenyl)-
S. No. 1 2 3 4 5 6
1332 KADHIM et al., Orient. J. Chem., Vol. 32(3), 1329-1346 (2016)
Anti-insect activity Anti-inflammatory activity Anti-inflammatory, antibiotic, antioxidant, anticarc inogenic and local anaesthetic Anti-Candida Antioxidant and anti-inflammatory activities Antibacterial agents, anticonvulsant agents and anti- tuberculosis Biological activities including antimicrobial, antioxidant and anti-inflammatory
5,65,77,91,103,115, 131,137,149,164 9,79,93,105,120,133, 161,175,189,204 7,79,91,105,119, 147,161,189,204 1,91,107,121,133, 175,189,204 1,93,105,119,147, 175,189,204,222 7,79,91,120,135,164 7,93,107,135,149, 220
1,5 21, 5,6 47, 5,6 33, 5,8 61, 9,8 61, 5,6 5,6 62,
5 1 5 1 5 1 5 1 5 1 5 5 1

164.084 204.188 204.188 204.188 222.198 164.12 220.183
4 4 4 4 2 4 0
6 0 0 0 2 6 2
1 2 2 2 2 1 2
CHO 10122 CH 1524 CH 1524 CH 1524 CHO 1526 CHO 1116 CHO 1524
8.769 9.644 10.606 10.411 10.331 11.183 12.253
e e
Eugenol Caryophyllene ß-copaene Azulene,1,2,3,3a,4,5,6,7- octahydro-1,4-dimethyl-7- (1-methylethenyl)- ±-acorenol Spiro[5.5]undec-8-en-1-on Isoaromadendrene epoxid
7 8 9 10 11 12 13
1333 KADHIM et al., Orient. J. Chem., Vol. 32(3), 1329-1346 (2016)
ory mor
nti-viral effects nti-inflammatory nalgesic, anti-inflammat nknown nknown nknown nti-microbial and anti-tu nti-inflammatory activity
A A A U U U A A
3,112,133,197,223, 281 5,77,91,105,167, 212 6,81,95,115, 167,247 7,91,105,15,166,243 7,98,125,149167,213 9,83,97,111,124,167, 222,256,284,326 9,121,159,187,213, 281,337,355,415 4,91,105,145,179,201, 245,275,321,336,354, 384,446
0,7 51, 1,6 94, 7,6 52, 5,7 5,6 0,6 80, 5,6 33, 6,7 31, 68,
6 2 5 1 5 1 6 5 6 1 5 2 5 2 3

343.09 212.084 247.194 261.117 213.1 326.199 430.308 532.304
3 2 7 1 3 6 0 2
4 1 4 6 1 2 3 3
3 2 2 2 2 3 4 5
CHNO 14199 CHO 14122 CHNO 1625 CHFNO 15162 CHNO 10154 CHO 27424 CHO 30448
12.797 13.346 14.228 14.416 15.468 15.87 16.745 18.473
Tetraacetyl-d-xylonic nitrile Benzyl Benzoate N-Isobutyl-(2E,4Z,8Z,10E) -dodecatetraenamide Phenethylamine , 3-benzyloxy-2-fluoro- ß-hydroxy- 4a-Hydroxy-4- nitroperhydronaphthalen -1-one Dasycarpidan -1- methanol, acetate (ester) Propanoic acid , 2- (3-acetoxy-4,4,14- trimethylandrost-8-en-17-yl)- Carda-4,20(22)-dienolide, 3-[(6-deoxy-3-O-methyl -±-L-mannopyranosyl
4 5 6 7 8 9 0 1
1 1 1 1 1 1 2 2
1334 KADHIM et al., Orient. J. Chem., Vol. 32(3), 1329-1346 (2016)
s
Anti-inflammatory activity Anti-inflammatory and antiviral agents New chemical compound Anti-Candida Anti-inflammatory activitie and their role in disease prevention and therapy Unknown
9,83,97,125,138,208, 292,310 9,81,98,126,158, 209,267,297 0,88,130,166,239, 338 9,91,122,149,284, 330,380,413,465 1,91,121,165,205, 275,303,344,372,430 7,81,117,147,238,255, 314,356,398,413,431,
5,6 49, 5,6 73, 7,7 81, 5,7 12, 7,7 46, 5,6 73, 47
5 2 5 1 5 2 5 3 5 2 5 2 4

7 4 9 2 1 4
8 9 9 8 8 1
0.2 7.1 8.1 6.2 0.3 9.3
1 9 3 9 3 4
3 2 3 4 4 4
0 7 8 6 0 9
1 9 3 9 3 4
3 2 3 4 4 4
CHO 20382 CHNO 16274 CHO 30406 CHO 29502 CHNO 26435
18.611 19.08 19.846 21.929 26.484 28.893
o
Cis-13-Eicosenoic acid 16-Nitrobicyclo[10.4.0] hexadecane-1-ol-13-one Strychane ,1-acetyl-20 ±-hydroxy-16-methylene- 2,4,6-Decatrienoic acid , 1a,2,5,5a,6,9,10, 10a-octahydro-5,5a-dihydr Vitamin E Glycine ,N-[(3±,5ß,12±) -3,12-dihydroxy-24 -oxocholan-24-yl]-
2 3 4 5 6 7
2 2 2 2 2 2
KADHIM et al., Orient. J. Chem., Vol. 32(3), 1329-1346 (2016) 1335
Statistical analysis RESULTS AND DISCUSSION
Results of the study were based on
analysis of variance (ANOVA) and differences were Identification of phytochemical compounds
considered significant at p < 0.05. Plants have been a common source of
medicinal property, either in the form of pure active
Fig. 1: GC-MS chromatogram of Cassia fistula
Table 2: FT-IR peak values of Cassia fistula
No. Peak (Wave Intensity Bond Functional group Group
number cm-1) assignment frequency

1 675.09 57.314 C-H Alkenes 675-995
2 904.61 72.826 C-H Alkenes 675-995
3 921.97 72.347 C-H Alkenes 675-995
4 1014.56 51.417 C-F stretch Aliphatic fluoro compounds 1000-10150
5 1072.42 57.43 C-F stretch Aliphatic fluoro compounds 1000-10150
6 1242.16 72.837 C-O Alcohols, Ethers, Carboxlic acids, Esters 1050-1300
7 1317.38 74.466 NO2 Nitro Compounds 1300-1370
8 1361.74 72.514 NO2 Nitro Compounds 1300-1370
9 1373.32 72.15 - Unknown -
10 1595.13 69.134 - Unknown -
11 1732.08 83760 - Unknown -
12 2752.42 90.502 - Unknown -
13 2848.86 77.274 - Unknown -
14 2918.3 72.553 C-H Alkanes 2850-2970
15 3277.06 76.261 O-H Hydrogen bonded Alcohols, Phenols 3200-3600
1336 KADHIM et al., Orient. J. Chem., Vol. 32(3), 1329-1346 (2016)
compound or as traditional preparations and it are detected was shown in Figure 1. Chromatogram
reasonable to use local plants29. Identifying on the GC-MS analysis of the methanol extract of Cassia
plant phytochemistry provides a fundamental use of fistula showed the presence of twenty seven major
plants as storage of chemical agents in the field of peaks and the components corresponding to the
medicine13. The phytochemical analyses of plant from peaks were determined as follows. The first set up
Cassia fistula was studied extensively. Identifying the peak were determined to be 1,7-Dioxaspiro[5,5]
importance of secondary metabolites in the field of undec-2-ene Figure 2. The second peak indicated
medicine, the presence of tannins, phlobatanins, to be 2,4-Dihydroxy-2,5-dimethyl-39(2H)-furan-3-
saponins, flavonoids, terpenoids, glycosides and one Figure 3. The next peaks considered to be
steroids was detected. Folklore medicine are widely á-D-Glucopyranoside , O-á-D-glucopyranosyl-(1.
used in our ancient period13. Gas chromatography fwdarw.3)-ß-D-fruc, d-Mannose, 5,7-Dodecadiyn
and mass spectroscopy analysis of compounds -1,12-diol, 3-Trifluoroacetoxypentadecane,
was carried out in methanolic leaves extract of 3-Trifluoroacetoxypentadecane, Pterin-6-carboxylic
Cassia fistula, shown in Table 1. The GC-MS acid, Imidazole-4-carboxylic acid ,2-fluoro-1-
chromatogram of the 27 peaks of the compounds methoxymethyl-,ethyl ester, D-Carvone, Pyrrolizin-
Table 3: Antimicrobial activity of Cassia fistula
Microbe Plant Cefotoxime Streptomycin Amphotericin B Fluconazol
Streptococcus pneumonia 4.19±0.15 1.00±0.07 2.00±0.11 - -
Pseudomonas eurogenosa 5.73±0.20 1.30±0.09 1.07±0.06 - -
Staphylococcus epidermidis 3.00±0.10 1.31±0.09 1.56±0.09 - -
Salmonella typhi 4.15±0.14 1.01±0.08 1.22±0.08 - -
Bacillus subtilis 4.00±0.12 1.49±0.10 1.00±0.04 - -
Escherichia coli 4.83±0.19 2.03±0.11 2.08±0.10 - -
Proteus mirabilis 6.02±0.23 1.90±0.09 1.00±0.05 - -
Streptococcus pyogenes 3.04±0.11 1.16±0.09 1.82±0.05 - -
Staphylococcus aureus 5.20±0.20 2.00±0.10 2.11±0.12 - -
Streptococcus faecalis 4.65±0.20 1.58±0.08 1.00±0.05 - -
Klebsiella pneumonia 5.23±0.23 1.27±0.07 1.95±0.09 - -

Fungi/ Yeast
Aspergillus niger 5.89±0.22 - - 1.94±0.08 2.11±0.19
Aspergillus terreus 6.99±0.29 - - 4.00±0.16 1.95±0.18
Aspergillus flavus 5.96±0.28 - - 2.00±0.20 3.00±0.22
Aspergillus fumigatus 6.00±0.29 - - 2.06±0.09 2.85±0.17
Candida albicans 5.25±0.20 - - 2.00±0.19 2.00.±0.11
Saccharomyces cerevisiae 3.92±0.18 - - 1.89±0.09 2.90±0.19
Fusarium sp. 5.01±0.24 - - 2.99±0.10 3.00±0.21
Microsporum canis 4.00±0.19 - - 3.31±0.19 1.97±0.10
Streptococcus faecalis 3.80±0.27 - - 3.73±0.14 2.60±0.20
Mucor sp. 4.00±0.19 - - 2.00±0.17 1.99±0.18
Penicillium expansum 4.12±0.19 - - 2.61±0.19 2.60±0.17
Trichoderma viride 5.08±0.23 1.99±0.15 2.30±0.18
Trichoderma horzianum 3.95±0.17 0.94±0.01 2.99±0.19
Trichophyton mentagrophytes 4.00±0.19 2.71±0.14 1.00±0.14
ª The values (average of triplicate) are diameter of zone of inhibition at 100 mg/mL crude extract, 30 µg/mL
of antibiotics (Streptomycin; Rifambin; Kanamycin; Cefotoxime and chloramphenicol).
KADHIM et al., Orient. J. Chem., Vol. 32(3), 1329-1346 (2016) 1337
Fig. 2: Structure of Oxacyclododecan-2-one Fig. 3: Structure of Imidazole ,2-amino-5-
with RT: 3.367 present in Cassia fistula [(2-carboxy)vinyl] with RT: 3.613 present in
Cassia fistula
Fig. 4: Structure of D-Glucose , 6-O-α-D- Fig. 5: Structure of 2-Nonanone with
galactopyranosyl with RT: 3.751 present in RT: 4.878 present in Cassia fistula
Cassia fistula
1338 KADHIM et al., Orient. J. Chem., Vol. 32(3), 1329-1346 (2016)
Fig. 6: Structure of Eicosanoic acid , Fig. 7: Structure of Phenol , 4-(2-propenyl)
phenylmethyl ester with RT: 5.833 present in with RT: 7.344 present in Cassia fistula
Cassia fistula
Fig. 9: Structure of Caryophyllene
Fig. 8: Structure of Eugenol with
with RT: 9.644 present in Cassia fistula
RT: 8.769 present in Cassia fistula