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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
shaded 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

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

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compounds
include,
1,9-Dihydroxy5,11(13)-dien-eudesman-13-oic acid (1),
1,3-Dihydroxy-5,11(13)-dien-eudesman13-oic acid (2), 2-Dihydroxy-ilicic acid
(3), Pterodonta A, which is 1-hydroxypterodontic acid-1-O--D-glucopyranoside
(4), Pterodonta B, which is 9-hydroxypterodontic acid-9-O--D-glucopyranoside
(5).
However, the folkloric use as an
anti-infective
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.

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
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

Phytochemical screening
The presence of some secondary metabolites
in the plant was determined using standard methods
(Sofowora, 2008; Evans, 2002).

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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.

tubes of the broth were incubated at 37oC for 24hrs

and 30oC for 1-7days for bacteria and fungi
respectively and observed for turbidity. The lowest
concentration which showed no turbidity in the test
tube was recorded as the MIC.

Preparation of Media
The medium was prepared according to
manufacturers
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 45 oC 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.

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.

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 30 oC 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.

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).

RESULTS
The results of phytochemical screening is
shown in tables 1 while that of microbial screening
are as shown in table 2 below.
DISCUSSION

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 manufacturers
instruction (10ml of each broth was dispensed into
separate test-tube and was sterilized at 121 oC 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

CONCLUSION
The result supports the folkloric use of the
plant as antimicrobial in treatment of athletes 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.

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Table 1: Phytochemical Analysis

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

Staphylococcus aureus

Bacillus subtilis

Escherichia coli

Pseudomonas
aeruginosa
Salmonella
typhimurium
Klebsiella pneumoniae

Staphylococcus aureus

Candida albicans

9
10

Staphylococcus aureus
Methicilin Resistant
Staph. aureua
Streptococcus pyogenes
Streptococcus faecalis

11
12

STRAIN
S
NCTC
6571
NCTC
8236
NCTC
10418
NCTC
6750
ATCC
9184
ATCC
10031
ATCC
13704
ATCC
10231
Isolate
Isolate
Isolate
Isolate

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

2.50

27

28

2.5

1.25

2.50

30

27

1.25

1.25

5.00

24

24

24

2.5

1.25

1.25

5.00

25

27

27

2.5

1.25

1.25

2.5

2.50

27

22

27

2.5

1.25

1.25

10

5.00

24

29

2.5

1.25

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
-

30
24

26
27

27
26

1.25
2.5

1.25
1.25

1.25
1.25

2.5
5

2.5
2.5

2.50
2.50

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13

Corynebacterium
Isolate
27
0
27
ulcerans
14 Listeria monocytogenes
Isolate
29
23
25
15 Bacillus subtilis
Isolate
29
27
20
16 Bacillus cereus
Isolate
30
24
22
17 Escherichia coli
Isolate
27
0
25
18 Klebsiella pneumoniae
Isolate
27
24
24
19 Klebsiella ozaenae
Isolate
24
27
20 Proteus mirabilis
Isolate
0
24
21 Proteus vulgaris
Isolate
27
24
25
22 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

2.50

2.5
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
1.25
1.25
-

5
5
2.5
5
5
5
5
5

5
2.5
5
5
5
-

2.50
5.00
5.00
5.00
5.00
2.50
5.00
2.50
-

1.25

2.5

2.5

1.25

1.25

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
2
3

10
5

R3
9

8
7

12

11

COOH

(1). R1=R3=OH, R2=H

(2). R1=R2=OH, R3=H
(4). R1=O--D-Glc, R2=R3=H
(5). R1=R2=H, R3= O--D-Glc

R2
15

13

14

HO

Compounds 1,2,4,5

1
2
3

HO

10
4

15

8
7

12

COOH

11
13

(3)
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.

CORRESPONDENCE
Egharevba, Henry O.
National Institute for Pharmaceutical Research and
Development (NIPRD)
Idu, PMB 21 Garki, Abuja, Nigeria
Tel. +234-803-645-3033
Email: omoregieegharevba@yahoo.com

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Manuscript submission date: 20th September 2010.

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