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Evaluación antibacteriana y perl cromatográco (CG-EM) de
fracciones de hojas y tallo de Calotropis procera (Linn)
Antibacterial screening and GC-MS prole of leave and stem fractions of
Calotropis procera (Linn)
Resumen
La medicina tradicional, también conocida como medicina ancestral o popular, comprende sistemas de conocimientos que se de-
sarrollaron a lo largo de generaciones dentro de varias sociedades antes de la era de la medicina moderna. La cromatografía en
columna fue utilizada para recolectar fracciones crudas; se utilizaron disolventes como éter de petróleo, cloroformo y metanol. Las
actividades antibacterianas de las fracciones crudas de Calotropis procera (hojas y tallo) se evaluaron en este estudio, utilizando
algunos microorganismos seleccionados como Escherichia coli, Shigella dysenteriae ATCC 24162, Salmonella typhi y Klebsiella
pneumoniae ATCC 34089; para tal n, la planta fue extraída usando acetona y metanol acuoso. El método de difusión en disco de
papel fue usado después de que la zona de inhibición alrededor de los discos fue estimada. Los resultados mostraron que en las
hojas de la planta, E. coli con la fracción acuoso-metanol y K. pneumoniae ATCC 34089 con la fracción del éter de petróleo-acetona,
tuvieron el rendimiento más alto de 30mm respectivamente; mientras que en el tallo, K. pneumoniae ATCC 34089 con la fracción
del metanol-metanol tuvo el rendimiento más alto de 25mm. La elucidación estructural de los compuestos bioactivos en los extrac-
tos fue evaluada usando CG-EM, identicándose compuestos químicos como fenol, palmitato metílico, ácido ftálico, ácido 9-octade-
cenoico y otros compuestos conocidos como antimicrobianos valiosos, con actividades biológicas y propiedades antioxidantes. Las
hojas y el tallo han presentado buenos compuestos químicos, que pueden ser responsables del efecto antimicrobiano observado.
Palabras Clave: análisis de hojas; Calotropis procera; fracciones brutas; cromatografía de gases; espectrometría de masas;
toquímica
Abstract
Traditional medicine also known as Indigenous or folk medicine comprises of knowledge systems that developed over generations
within various societies before the era of modern medicine. The column chromatography was used to collect the crude fractions;
solvents like petroleum ether, chloroform and methanol were used. The antibacterial activities of the crude fractions of Calotropis
procera (leaf and stem) were evaluated in this study using some selected microorganisms like Escherichia coli, Shigella
dysenteriae ATCC 24162, Salmonella typhi and Klebsiella pneumoniae ATCC 34089, whereby the plant was extracted using
(acetone, methanol and aqueous). The paper disc method was used after which the zone of inhibition around the discs was
estimated. The results showed that in the leaf of the plant; E. coli with aqueous-methanol fraction and K. pneumoniae ATCC 34089
with acetone-petroleum ether fraction had the highest yield of 30mm respectively while in stem; K. pneumoniae ATCC 34089 with
methanol-methanol fraction had the highest yield of 25mm. The structural elucidation of the bioactive compounds in the extracts
were evaluated using GC-MS which reveals the chemical compounds like phenol, methyl palmitate, phthalic acid, 9-octadecenoic
acid and other compounds known for valuable antimicrobial, biological activities and antioxidant properties. The leaves and the
stem are having good chemical compounds that can be responsible for the antimicrobial property observed.
Keywords: analysis leaves; Calotropis procera; crude fractions; gas chromatography; mass spectrometry; phytochemistry stem.
Oluwakemi-Sola Asoso
1
; Muftau-Kolawole Oladunmoye
2
; Ayodele Oluyemisi Ogundare
2
(Recibido: Febrero 2018, Aceptado: Mayo - 2018)
1
Department of Biological Sciences, Afe Babalola University, Ado-Ekiti, Nigeria, E-mail address:oyesolakemi@gmail.com
2
Department of Microbiology, School of Sciences, Federal University of Technology, Akure, Ondo State, Nigeria. E-mail:
chourlar@yahoo.com.
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INTRODUCTION
The World Health Organizaon (WHO) denes
tradional medicine as the sum of total of the
knowledge, skills, and pracces based on the
theories, beliefs and experiences indigenous to
dierent cultures, whether explicable or not,
used in the maintenance of health as well as
in the prevenon, diagnosis, improvement or
treatment of physical and mental illness (1).
The use of herbal medicine as alternave therapy
has prevalent throughout the world due to the
growing resistance of pathogens to convenonal
anbiocs (2). The need for more potent, safe
and aordable drugs has led to intensied
research into herbal drugs, the result of which
is the introducon of new herbal preparaon
for therapeuc uses (3).Medicinal plants are
frequently used as remedies for many infecous
diseases (4). The treatment and control of
diseases by the use of the available medicinal
plants in a locality have been helpful and of a
priority to majority of urban and rural dwellers in
healing various diseases because of the reliability
and stability in plant products for healing (5).
Calotropis procera (Apple of Sodom) is a shrub or
small tree, which has become a serious weed in
pastures and overgrazed rangelands. It is a nave
to West Africa as far as south as Angola, North and
East African, Madagascar, the Arabian Peninsula,
Southern Asia, India and China to Malaysia.
Calotropis was formerly placed in the family of
Asclepiadaceous (the milkweed family), which is
now considered a subfamily of the Apocynaceae
(6).
MATERIALS AND METHOD
Collecon of plant samples
Apparently healthy plant namely Calatropis
procera were collected from Ado-Eki, Eki State
Nigeria.
The plants parts leave and stem were air-dried
for 5 weeks at room temperature (25 + 2 oC) and
then ground to powder with a mechanical grinder
(Thomas Wiley machine, model 5 USA). Powders
(200gs) of each plant were extracted with 1litre
of sterile aqueous water, ethanol, methanol and
acetone separately at room temperature (25 + 2
oC). They were labeled as crude extracts.
Anbacterial Screening of the Crude Fracons
The evaluaon of anmicrobial acvity was
performed for all fracons by the paper disc
agar diusion method following the standard
rules of anmicrobial sensivity tests by the
Clinical and Laboratory Standards Instute (7).
The anbacterial eect of the fracons was
carried out whereby the discs were previously
impregnated with the plant crude fracons and
placed on the sterile prepared medium. The
plates were incubated at 37 oC for 24 - 48 hours.
The sensivity of the test organisms to each of
the extracts was indicated by clearing around
each disc. The diameter as an index of the degree
of sensivity was measured with a transparent
plasc ruler.
Column chromatography and fracon extracts
Glass wool was placed at the outlet of a column
aer which, one gram of plant extract powder
was weighed into it and subjected to column
chromatography (30 x 8 cm column) using
60 g of silica gel 60 F254 (Merck, 0.020 mm
thickness). The column was successively eluted
rst with petroleum ether (150 ml) and then with
chloroform (80 ml): methanol (2 ml) (40:1) and
nally with 100% methanol (150 ml). Each 100
ml eluent was collected into a round boom ask
(250 ml) capacity and dislled to obtain fracons.
The fracons collected were numbered. GC-MS
(Gas Chromatography-Mass Spectrophotometry)
analysis was carried out in GCMS-QP2010 PLUS
Shamadzu.
Anbacterial screening of crude fracons
The anbacterial screening of the crude fracons
of Calotropis procera leaf was examined; some
chemicals were used in the fraconalizaon of the
crude sample, which are methanol, chloroform
and petroleum ether. Methanol, aqueous and
acetone leaf fracons showed the highest zone of
inhibion were: 30, 20 and 20 mm respecvely.
The use of petroleum ether do not show any
signicant anbacterial eects against the
isolates while aqueous crude fracons shows an
eecve anbacterial acvity against S. aureus at
zone of inhibion of 15 mm (Table 1).
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Table 2 shows the antibacterial screening
of the stem crude fractions to indicate
the effectiveness of various bioactive or
metabolite fractions found in the crude stem
extracts of the plant (C. procera). During
ORGS
A
C
M
A
C
C
A
C
P
M
M
M
C
M
P
A
Q
M
A
Q
P
A
Q
C
E
C
E
P
E
M
E.coli - - - 4 20 2 30 - 20 - - -
S. aureus - - - 10 5 - - - 15 - - -
E.coli
ATCC 35218 - - - - 20 - - - - - - -
S. typhi 20 15 20 18 5 - - - - 10 5 7
S. dysenteriae 20 - - 14 - - - - - 4 14 -
S. dysenteriae
ATCC 24162 2 - - 22 14 2 - - - - - -
K. pneumonia
ATCC 34089 20 - 30 - - - - - - - - -
K.pneumoniae 24 2 - - - - - - - - - -
ACM- Acetone methanol, ACC- Acetone Chloroform, ACP-Acetone Pet. Ether, MM-methanol methanol, MC-Methanol Chloroform, MP-Methanol pet. Ether,
AQC-Aqueous Chloroform, AQP Aqueous Pet.ether, AQM- Aqueous Methanol, EM-Ethanol Methanol, EC- Ethanol Chloroform, EP- Ethanol Pet. Ether. (-):
did not show inhibition zone.
ACM- Acetone methanol, ACC- Acetone Chloroform, ACP-Acetone Pet. Ether, MM-methanol methanol, MC-Methanol Chloroform, MP-
Methanol pet. Ether, AQC-Aqueous Chloroform, AQP-Aqueous Pet.ether, AQM- Aqueous Methanol, EM-Ethanol Methanol, EC- Ethanol
Chloroform, EP- Ethanol Pet. Ether. (-): did not show inhibition zone.
this research, also methanol was found to
show a significant antibacterial effect on K.
pneumoniae at zone of inhibition of 25 mm.
Both methanol and aqueous crude fractions
have shown good antibacterial activities.
Tables 3-8 and Figures 1 -2, indicate the results
of GC-MS analysis with peaks of secondary
metabolites of the crude fractions. Those
shows diverse peaks which implies different
chemical compounds that are been found in
the fractions of C. procera.
The chemical compounds of the crude
ORGS
A
C
M
A
C
C
A
C
P
M
M
M
C
M
P
A
Q
C
A
Q
P
A
Q
M
E
M
E
C
E
P
E. coli - - - - - - - - 5 - - -
S. dysenteriae ATCC 24162 - - - - - - 10 3 3 6 - -
K. pneumonia ATCC 34089 - - - 25 - 9 - - - - - -
S. typhi - 5 10 2 - 7 - - 21 6 - -
Table 2. Screening of antibacterial crude fractions of Calotropis procera stem (mm)
Table 1: Antibacterial screening of crude fractions of Calotropis procera leaf (mm)
fractions were determined and listed out with
their retention time and the concentration
of the chemical compounds in percentage.
The secondary metabolites identified were
phenol, methyl palmitate, 9-octadecenoic
acid, phthalic acid, dimethyl sulfoxide, phytol
and among others.
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Figure 1: GCMS ANALYSIS WITH PEAKS OF SECONDARY METABOLITES OF
MMS (METHANOL METHANOL STEM FRACTION)
Table 3. Chemical compounds of methanol- methanol stem fraction of C.
procera, identified by GC-MS analysis.
S/N Chemical compounds
Retention
Time (mins)
Concentration
(%)
1 phenol 14.725 1.761
2 Methyl 38.401 7.053
3 9.12 octadecadienoic acid (z,z)- Methyl ester 39.625 2.814
4 9- octadecadienoic acid 39.666 4.602
5 octadecadienoic acid 40.184 34.65
6 Methyl 19,22- hepatadeca dienoate 40.225 8.98
7 9 – octadecadienoic acid 40.7033 31.16
8 Tetradecanoic acid 41.080 4.21
9 Phthalic acid 42.117 4.76
Identified by GC-MS analysis
Table 4. Chemical compounds of methanol- aqueous stem fraction of C. procera, identified by GC-MS analysis
S/N CHEMICAL COMPOUNDS RETENTION TIME (min) CONCENTRATION(%)
1 Hydrazine 7.797 0.92
2 Thietane 7.876 0.86
3 Dimethyl Sulfoxide 9.282 13.23
4 2(5H)-Furanone 11.905 13.53
5 Triacetin 23.798 1.22
6 4-methyl-5-methoxy-1,2,4-triazole-3-thione 24.018 1.33
7 Methyl palmitate 38.386 4.40
8 9-octadecenoic acid 39.650 0.69
9 Methyl palmitate 39.807 2.08
10 Benzenemethanamine 41.512 11.17
11 Phthalic acid 42.109 18.97
12 1H-Indole 42.219 22.26
13 2-methyl-7-phenylindole 42.345 7.24
14 (2,3-Diphenylcyclopropyl) methylphenyl sulfoxide 43.060 2.09
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Table 5. Chemical compounds of chloroform-aqueous leaf fraction of C. procera,
identified by GC-MS analysis
Figure 2: GCMS analysis with peaks of secondary metabolites of pal (pet-ether
acetone leaf fraction)
Table 6. Chemical compounds of pet-ether acetone leaf fraction of C. procera, identified by GC-MS analysis
S/N
CHEMICAL
COMPOUNDS
RETENTION TIME(min)
CONCENTRATION
(%)
1 Dodecane 15.802 1.67
2 Phenol 29.061 2.79
3 Cyclohexadecane 31.151 1.55
4 9-Octadecene 36.744 1.68
5 Dodecane 36.744 1.22
6 Phthalic acid 37.435 4.64
7 Phthalic acid 37.749 37.68
8 Methyl palmitate 38.401 12.72
9 9-octadecenoic acid 39.658 12.72
10 Methyl palmitate 39.823 8.60
11 Phthalic acid 42.109 6.94
S/N CHEMICAL COMPOUNDS RETENTION TIME (min) CONCENT RATION (%)
1 2-Hexadecene 37.380 1.29
2 11-tetradecyn-1-ol acetate 37.451 2.94
3 2-pentadecanone 37.521 10.40
4 Phthalic acid 37.741 24.54
5 3,7,11,15-tetramethyl-2-hexadecenol-ol 37.953 2.70
6 Methyl palmitate 38.393 7.66
7 Hexadecanoic acid 38.951 0.97
8 9,12-octadecadienoic acid(z,z)-methyl ester 39.611 1.80
9 7,10,13-hexadecatrienoic acid 39.650 5.53
10 Phytol 39.744 28.64
11 Methyl palmitate 39.815 2.75
12 Cyclopentanone 39.909 1.11
13 Bromacetic acid 40.577 3.88
14 4,8,12,16 tetramethylheptadecan-4-olide 40.993 3.88
15 Phthalic acid 42.101 2.66
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The table 6 shows the explanation of the Figure 1.
above, GCMS analysis with peaks of secondary
metabolites of pal (pet-ether acetone leaf fraction)
S/N
CHEMICAL
COMPOUNDS
RETENTION
TIME(min)
CONCENTRATION(%)
1 Dimethyl sulfoxide 9.400 91.44
2 3-acetoxy-3-
hydroxypropionic
acid
16.878 5.05
3 Methyl palmitate 38.401 3.51
Table 7. Chemical compounds of methanol-methanol leaf fraction of C.
procera, identified by GC-MS analysis.
Table 8. Chemical compounds of methanol- aqueous leaf fraction of C.
procera, identified by GC-MS analysis.
S/N
CHEMICAL
COMPOUNDS
RETENTION
TIME (min)
CONCENTRATION(%)
1 Butane 9.965 65.81
2 n- hexadecanoic acid 39.116 26.20
3 9-octadecenoic acid 40.074 2.20
4 Octadecanoic acid 40.208 5.79
DISCUSSION
Antibacterial activities of the crude fractions
showed that this plant can be used for curing many
diseases such as pneumonia, dysentery among
others. The results demonstrated that the crude
extracts were more efficient than the fractions,
this findings might suggest that Phytochemical
constituents in combination may be having synergy
in their efficacy, which is in agreement with many
other report that have shown higher antibacterial
potency of crude extracts as compared to the
fraction (8).
The difference of various plant extracts in the
antimicrobial activity is expected, as the activity
is based not only on the different structures of
microorganisms but also on their susceptibilities
(9). This inhibitory action could be attributed
to the phytochemical constituents, since these
constituents are well established as antimicrobial
agents (10). In the present study, in the agar
diffusion assay the methanol and chloroform
extracts showed the larger inhibition halo enabling
to observe the extraction potential of the solvent
employed.
Differences of biological activity between the
fractions can be partly explained by quantitative
and qualitative variations in the secondary
metabolites present in the fractions which are
in support with (11) and could be due to the use
of different parts of the plants and leading to
the extraction of different compounds and with
antimicrobial activity. GC-MS technique was used
to effect complete separation and identification of
the pure compounds in the combined fractions.
The presence or absence of functional groups in
an organic molecule determines the manner in
which that organic molecule will fragment. The
structure of the compounds can be deducing
by interpretation of various mass peaks in each
spectrum. The compounds identified are phthalic
acid, butane, methyl palmitate, n-hexadecanoic
acid, phytol among others.
CONCLUSIONS
The present study has investigated the efficacy
of Calotropis procera which can be considered in
folklore or traditional medicine, edible vegetable
and animal forage.
The overall results showed that the crude fractions
of this plant has an appreciable antibacterial
activity on the selected microorganisms.
The chemical compounds present in the plant
simplifies that C. procera extracts could maximally
serve as alternative to highly rated synthesized
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drugs, whose costs are unaffordable by the
common man.
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