Researcher
2010;2(10)
Phytochemical Analysis and Broad Spectrum Antimicrobial Activity
of Laggera pterodonta (DC.) Sch. Bip. (Aerial Part)
1*
Egharevba, Henry Omoregie; 2Abdullahi, Makailu Sabo; 3Okwute, Simon Koma; 1Okogun, Joseph Ibumeh
1
Department of Medicinal Plant Research and Traditional Medicine
National Institute for Pharmaceutical Research &Development (NIPRD), Abuja, Nigeria.
2
National Research Institute for Chemical Technology, Zaria, Nigeria.
3
Department of Chemistry, University of Abuja, Nigeria
*Corresponding author
E-mail: omoregieegharevba@yahoo.com
ABSTRACT: Laggera pterodonta (DC.) Sch. Bip. aerial part was extracted successively with hexane, ethyl acetate
and methanol. The extracts were screened in vitro for activity against standard strains microbes and clinical isolates.
The zones of inhibition, minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and
minimum fungicidal concentration (MFC) were determined. The in vitro antimicrobial screening revealed that the
extract exhibited varying activity against different microbes with zones of inhibition ranging from 14-32mm, MIC
ranging from 1.25 - 5mg/ml, and MBC/MFC of 2.5-10mg/ml. The highest activity was an MIC of 1.25 mg/ml and
MBC of 2.5mg/ml. The activities observed could be due to the presence of some of the secondary metabolites like,
alkaloids, tannins, sterols, glycosides, saponins, terpenes and flavonoids present in the plant. Isolation work to
determine compound(s) responsible for activities is ongoing.
[Egharevba, Henry Omoregie; Abdullahi, Makailu Sabo; Okwute, Simon Koma; Okogun, Joseph Ibumeh.
Phytochemical Analysis and Broad Spectrum Antimicrobial Activity of Laggera pterodonta (DC.) Sch. Bip. (Aerial
Part). Researcher. 2010;2(10):35-40]. (ISSN: 1553-9865).
Key words: Laggera pterodonta, phytoconstituents, antimicrobial, MIC, MBC, MFC
galleried forest. It grows in Senegal, Sierra Leone,
Nigeria and West Cameroons, and probably
occurring elsewhere in the region (Burkill, 1985;
Wu et al., 2006), It is widespread in other part of
tropical Africa. It is found growing as common
weeds or weed of cultivation in Nigeria around
December to March. Germination starts around
August and September and grows to maturity in
December/January. The matured herb could be up
to 1.7m high, viscid and strongly aromatic. In Lake
Province of Tangayika, it is reported to kill
tobacco plants if growing nearby (Burkill, 1985). It
has been reported for use in China as anti
inflammatory agent for treatment of hepatitis,
arthritis, bronchitis and nephritis (Wu et al., 2006
& 2007), in Cameroun for the preservation of
seeds against insects attack (Njan et al. 2007), and
in Nigeria for athlete foot, skin infections,
paediatric malaria and wounds. Some chemical
isolation and characterization work has been
reported for the Asian species growing in China.
Some of the isolated compounds include, 1β,9βDihydroxy-5,11(13)-dien-eudesman-13-oic acid
(1), 1β,3α-Dihydroxy-5,11(13)-dien-eudesman13-oic acid (2), 2β-Dihydroxy-ilicic acid (3),
INTRODUCTION
Current trends in drug development process
are focused on natural sources, especially sources of
plant origin due to some proven correlation between
the folkloric medicinal uses of some of these plants to
biological activity. In spite of the growing interests in
plant- based drugs, only a fraction of higher plants
have been chemically investigated, and even a
smaller percentage have been evaluated by biological
or pharmacological screening (Hamburger and
Hostettmann, 1991). Laggera pterodonta has been
used as traditional medicine in Northern Nigeria and
other parts of the tropics for centuries. The whole has
been employed in herbal medicine in china and some
tropical countries of the world, however very limited
work has been reported on its antimicrobial activities
has been reported, and activity related studies has
been reported for species found in Nigeria and West
Africa. This makes it a very interesting plant of study
as a source of anti-infective.
Laggera pterodonta belong to the family
Asteraceae (Compositae) and the genus Laggera
which consist of 20 species. The plant is spread
throughout the sub-Saharan Africa and the
tropical countries of Asia, especially Southeast
Asia, in open waste spaces and partially shaded
35
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Pterodonta A, which is 1β-hydroxy-pterodontic
acid-1-O-β-D-glucopyranoside (4), Pterodonta B,
which is 9β-hydroxy-pterodontic acid-9-O-β-Dglucopyranoside (5).
However, the folkloric use as an antiinfective has not been fully investigated especially
on Nigerian species of the plant. Reports of
chemical constituents of the oil from the species in
Benin and Cameroon showed variation in
composition (Asfaw et al., 1999 and 2000). We
have earlier reported the activity of the crude
methanol
extract
against
Mycobacterium
tuberculosis BCG strains (Egharevba et al., 2010).
This work is aimed at investigating the
antimicrobial potential of the Nigerian species
with a view towards isolating some of the
bioactive constituents in subsequent studies.
drugs were sparfloxacin (0.2mg/ml), erythromycin
(0.5mg/ml) and flouconazole (0.5mg/ml), all of
sigma chemicals UK obtained from Zayo-Sigma
Abuja Nigeria.
Antimicrobial Screening
Organism Source
The organisms used include standard strains,
Staphylococcus aureus NCTC 6571, Bacilluc subtilis
NCTC 8236, Eschericia coli NCTC 10418,
Pseudomonas aeruginosa NCTC 6750, Salmonella
typhimurium ATCC 9184, Klebsiella pneumonia
ATCC 10031 and Staphylococcuc aureus ATCC
13704, and clinical isolates, Staphylococcus aureus,
Methicilin
Resistant
Staphylococcus
aureus,
Streptococcus pyogenes, Streptococcus faecalis,
Corynebacterium ulcerans Listeria monocytogenes,
Bacillus subtilis, Bacillus cereus, Escherichia coli,
Klebsiella pneumonia, Klebsialla ozaenae, Proteus
mirabilis,
Proteus
vulgaris,
Pseudomonas
aeruginosa, Pseudomonas flourescense, Salmonella
typhimurium, Shigella dysenteriae, Aspergillus nigre,
candida albicans, Microsporum gypseum and
Trichophyton rubrum. The typed strains and clinical
isolates were obtained from the department of
medical Microbiology Ahmadu Bello University
Teaching Hospital (ABUTH) and department of
Pharmaceutical Microbiology, Ahmadu Bello
University (ABU) Zaria, Nigeria, respectively. All
the organisms were checked for purity and
maintained at 4oC in slants of nutrient agar and
sabouraud dextrose agar (SDA) for bacteria and fungi
respectively. Well diffusion method described by
Hugo and Russel (1992) was used to determine the
antimicrobial activities (zone of inhibition) of the
extracts against the organisms.
MATERIALS AND METHODS
All the solvents and reagent used in the
study were of Analar grade and, unless otherwise
stated, were sourced from Zayo-Sigma, Abuja,
Nigeria.
Collection and Extraction of Plant Material
The plant was collected on the on 15
November, 2009 from Chaza village, Suleja, Niger
State, Nigeria, and authenticated by the Taxonomist
in the Department of Medicinal Plant Research and
Traditional Medicine of the National Institute for
Pharmaceutical Research and Development (NIPRD)
Abuja, Nigeria. The plant was assigned a voucher
specimen number NIPRD/H/6403. The roots were
removed and the aerial part air-dried for two weeks,
and then crushed with a mechanical grinder. The
crushed plant was kept in an air-tight cellophane bag
until used. The crushed material was later macerated
successively for 24hrs in hexane, ethyl acetate and
98% methanol. The extracts were concentrated in
rotary evaporator and allowed to dry in the hood. The
solid extracts were used for phytochemical analysis
and antimicrobial screening.
Preparation of the Inoculums
A loopful of the test organism was taken
from their respective agar slants and sub-cultured into
test-tubes containing nutrient broth for bacteria and
sabouraud dextrose liquid for fungi. The test-tubes
were incubated for 24hrs at 37oC for bacteria and for
48hrs at 30oC for the fungi. The obtained
microorganisms in the broth were standardized using
normal saline to obtain a population density of
108cfu/ml for the bacteria. For the fungi, fungal
spores were harvested after 7 days old SDA slant
culture was washed with 10ml normal saline in 2%
Tween 80 with the aid of glass beads to help in
dispersing the spores. The spores suspension were
standardized to 105cfu/ml.
Phytochemical screening
The presence of some secondary metabolites
in the plant was determined using standard methods
(Sofowora, 2008; Evans, 2002).
Preparation of Extract Stock Concentration for
Antimicrobial screening
A test stock concentration of 10mg/ml for
methanol extracts was prepared by dissolving 0.1g of
extract in 10mls of distilled water in a test tube. For
the ethyl acetate and hexane extracts a concentration
of 10mg/ml was prepared by disolving 0.1g in 10mls
of dimethyl sofoxide (DMSO). The positive control
Preparation of Media
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The medium was prepared according to
manufacturer’s
instruction
(Oxoids
Limited
Basingstoke, Hampshire, England). 40g of Blood
Agar (52g of SDA) were weighed into a conical flask
1000ml of distilled water was added and capped with
a cotton wool. The media were boiled to dissolution
and then sterilized at 121oC for 15mins. The media
were allowed to cool to 45oC and 20ml of the
sterilized medium was poured into sterile petri-dishes
and allowed to cool and solidify. The plates were
labeled with the test microorganism (each plate with
a test microbe). The microbes were spread evenly
over the surface of the medium with the aid of a glass
spreader. The plates were dried at 37oC for 30mins
and divided into two sets to be used for the well
diffusion method and the disc diffusion method
respectively.
concentration which showed no turbidity in the test
tube was recorded as the MIC.
Minimum Bactericidal/Fungicidal Concentration Broth Dilution Method
Blood and sabouraud media were prepared,
sterilized at 121oC for 15mins and was poured into
sterile petri-dishes and left to cool and solidify. The
contents of the MIC in the serial dilution were then
sub-cultured onto the media and incubated at 37oC
for 24hrs and 30oC for 1-7days for bacteria and fungi
respectively, and observed for colony growth. The
MBC/MFC was the plate with the lowest
concentration of extract and without colony growth.
RESULTS
The results of phytochemical screening is
shown in tables 1 while that of microbial screening
are as shown in table 2 below.
Zone of Inhibition - Well Diffusion Method
A standard cork borer of 5mm in diameter
was used to cut well at the center of each inoculated
plate and the agar removed from the well. 0.1ml of
the text solution (extract) was then introduced into
the well created at the center for each plate. The
bacteria plates were incubated at 37oC for 24hrs
while the fungal plates were incubated at 30oC for 17days, and observed for the zone of inhibition of
growth. The zones were measured with a transparent
ruler and the result recorded in millimeters. The
screening was done in triplicates. Sterilized distilled
water and DMSO were used as negative control.
DISCUSSION
The methano extract was generally the most
active among the extract followed by the hexane
extract. However the ethyl acetate extract exhibited
good activity against three fungi speciess unlike the
other two extracts which were only active against
candida albicans. The extracts were differentially
active against food pathogens like the Staphylococcus
aureus and enteric organisms like Listeria
monocytogenes,
Streptococcus
faecalis
and
pyogenes, Bacillus subtilis and Bacillus cereus,
Proteus vulgaris, Salmonella typhimurium, Shigella.
The observed activity may be due to the presence of
some metabolites like alkaloid, saponins, flavonoids
and terpenes, which are known to possess various
biological activities in different organisms (Okhale
et al., 2010; Sadique et al., 1987; Chukwujekwu et
al., 2006; Sheeba et al., 2009).
Minimum Inhibitory Concentration - Broth
Dilution Method
MIC of the extracts was also carried out using broth
dilution method as described in Ibekwe et al, 2001.
The nutrient broth and sabouraud dextrose liquid
were prepared according to the manufacturer’s
instruction (10ml of each broth was dispensed into
separate test-tube and was sterilized at 121oC for
15mins and then allowed to cool. Two-fold serial
dilutions of the extracts in the broth were made from
the stock concentration of the extract to obtain 10, 5,
2.5, 1.25, 0.625 mg/ml for methanol, and 5, 2.5, 1.
25, 0.625, 0.3125 mg/ml for ethyl acetate and hexane
extracts. 0.1ml of the standardized inoculums of the
microbes was inoculated into the different
concentrations of the extracts in the broth. The test
tubes of the broth were incubated at 37 oC for 24hrs
and 30oC for 1-7days for bacteria and fungi
respectively and observed for turbidity. The lowest
CONCLUSION
The result supports the folkloric use of the
plant as antimicrobial in treatment of athlete’s feet,
wound and skin disease as well as in malaria. It also
shows that the crude plant may be useful for the
management of gastroenteritis and respiratory
diseases. Work is ongoing in our laboratory to
characterize and screen some compounds isolated
from the plant, which are thought to be responsible
for these activity.
Table 1: Phytochemical Analysis
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METABOLITE
RESULT
Carbohydrate
+
Tannins
+
Saponins
-
Terpenes
+
Sterols
+
Flavonoids
+
Alkaloids
+
Phenols
+
Volatile Oil
+
Resin
+
Balsam
+
Cardiac glycoside
+
Phlobatannins
+
+ = present, - = not detected
Table 2: Zone of Inhibition
S/N
TEST ORGANISM
1
Staphylococcus aureus
2
Bacillus subtilis
3
Escherichia coli
4
6
Pseudomonas
aeruginosa
Salmonella
typhimurium
Klebsiella pneumoniae
7
Staphylococcus aureus
8
Candida albicans
9
10
Staphylococcus aureus
Methicilin Resistant
Staph. aureua
Streptococcus pyogenes
Streptococcus faecalis
Corynebacterium
ulcerans
Listeria monocytogenes
5
11
12
13
14
STRAIN
S
ZONES OF
INHIBITION
MIC
MBC/MFC
m
27
e
20
h
27
m
2.5
e
1.25
h
1.25
m
5
e
5
h
2.50
25
20
26
2.5
1.25
1.25
5
5
2.50
27
0
28
2.5
-
1.25
5
-
2.50
30
0
27
1.25
-
1.25
5
-
5.00
24
24
24
2.5
1.25
1.25
5
5
5.00
25
27
27
2.5
1.25
1.25
5
2.5
2.50
27
22
27
2.5
1.25
1.25
10
5
5.00
24
0
29
2.5
-
1.25
5
-
2.50
30
32
26
25
22
0
1.25
1.25
1.25
1.25
1.25
-
2.5
2.5
2.5
2.5
5.00
-
Isolate
Isolate
Isolate
30
24
27
26
27
0
27
26
27
1.25
2.5
2.5
1.25
1.25
-
1.25
1.25
1.25
2.5
5
5
2.5
2.5
-
2.50
2.50
2.50
Isolate
29
23
25
2.5
1.25
1.25
5
5
2.50
NCTC
6571
NCTC
8236
NCTC
10418
NCTC
6750
ATCC
9184
ATCC
10031
ATCC
13704
ATCC
10231
Isolate
Isolate
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15
16
17
18
19
20
21
22
Bacillus subtilis
Isolate
29
27
20
Bacillus cereus
Isolate
30
24
22
Escherichia coli
Isolate
27
0
25
Klebsiella pneumoniae
Isolate
27
24
24
Klebsiella ozaenae
Isolate
24
27
Proteus mirabilis
Isolate
0
24
Proteus vulgaris
Isolate
27
24
25
Pseudomonas
Isolate
24
0
0
aeruginosa
23 Pseudomonas
Isolate
30
0
0
flourescenses
24 Salmonella
Isolate
22
27
24
typhimurium
25 Shigella dysenteriae
Isolate
27
28
27
26 Aspergillus flavus
Isolate
0
0
0
27 Aspergillus nigre
Isolate
0
0
0
28 Candida albicans
Isolate
17
24
25
29 Microsporum gypseum
Isolate
0
18
0
30 Trichophyton rubrum
Isolate
0
14
0
m= methanol extract; e= ethylacetate extract; h= hexane extract
2.5
1.25
2.5
2.5
2.5
2.5
2.5
1.25
1.25
1.25
1.25
-
1.25
1.25
1.25
1.25
1.25
1.25
1.25
-
5
2.5
5
5
5
5
5
2.5
5
5
5
-
5.00
5.00
5.00
5.00
2.50
5.00
2.50
-
1.25
-
-
2.5
-
-
2.5
1.25
1.25
5
2.5
2.50
2.5
5
-
1.25
1.25
-
1.25
1.25
-
5
10
-
2.5
5
-
2.50
2.50
-
SOME REPORTED COMPOUNDS FROM THE SPECIES IN CHINA
R1
14
1
9
2
3
R3
10
5
4
6
8
7
12
11
COOH
(1). R1=R3=OH, R2=H
(2). R1=R2=OH, R3=H
(4). R1=O-β-D-Glc, R2=R3=H
(5). R1=R2=H, R3= O-β-D-Glc
R2
15
13
14
HO
Compounds 1,2,4,5
1
9
2
3
10
5
6
4
HO
8
7
12
11
COOH
13
(3)
National Institute for Pharmaceutical Research and
Development (NIPRD)
Idu, PMB 21 Garki, Abuja, Nigeria
Tel. +234-803-645-3033
Email: omoregieegharevba@yahoo.com
ACKNOWLEDGEMENT
The authors are grateful to the management of the
National Institute of Pharmaceutical Research and
Development (NIPRD) and staff of the department of
Medicinal Plant Research and Traditional Medicine
for their supports.
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CORRESPONDENCE
Egharevba, Henry O.
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