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SCIENTIFIC RESEARCH GATE
International Journal of Biomedical Papers 2016; 1: 5–12

International
Journal of
Biomedical
Papers

www.scigatejournals.com/publications/index.php/ijbp

Phytochemical Analyses and Anti-Microbial Effects of Alstonia
boonei (De Wild), Bridelia ferrugenea (Benth), Eucalyptus
tereticornis (Dehnh), Terminalia schimperiana (Hochst) and
Polyalthia longifolia (Sonn) Thwaites.
Olanipekun Mary K 1, Adedeji Damilola E1, Kayode Joshua1, David O.Moses1, Adewuyi
Damilare1
1. Department, Plant Science and Biotechnology, Ekiti-State University, Ado-Ekiti, Nigeria

Abstract
Background and Objectives: This study examined the bioactive ingredients and anti-bacterial activities of five medicinal plants
– Alstonia boonei, Bridelia ferrugenea, Eucalyptus tereticornis, Terminalia schimperiana and Polyalthia longifolia – used
traditionally in the study area. This study aimed at validating, scientifically, the ethnobotanical utilization of these species.
Methods: The leaves and stem barks of these plants were collected, dried, made to powders. The powdered leaves and stems
were extracted using aqueous and ethanol solvent. The extracts were used for the preliminary phytochemical screening and
antimicrobial studies using standard methods.
Results: The plants were found to contain tannins, saponins, flavonoids, alkaloids, cardiac glycosides and terpenoids. Extracts
from these plants, at high concentration (100mg/ml), inhibited the growth of the bacteria studied. Salmonella typhi was the most
resistance organism (at 100mg/ml) while Staphlococcus spp strains were the most sensitive organisms to both aqueous and
ethanol extracts of all the plants at varying concentrations. The ethanol solvent extracted the phyto- chemicals in the plants better
than water extract.
Conclusion: The results obtained in this study demonstrated the potencies of these species on the bacterial isolates of
Staphlococcus aureus, Escherichia coli, Enterococcus faecalis and Salmonella typhi (at 50mg/ml and 100 mg/ml). Thus,
confirming their suitability in the traditional treatments of diseases, such as dysentery, diarrhea, malaria and typhoid fever.
Key words: Bioactive ingredients, anti-bacterial, medicinal plants, ethnobotany

1. Introduction
Medicinal plants have been used and identified throughout human history. Their use as sources of relief from
illnesses can be traced back to five millennia in the form of written document of the early civilization in China, India
and near East (1, 2). Estimate by the World Health Organization (WHO) reported that over 80% of the people in the
developing world relied on medicinal plants as components of health care (3).
Plants have the ability to synthesis a wide variety of chemical compounds that are used to perform important
biological functions and to defend against attack from disease-causing organisms such as insects, bacteria, virus,
fungi and herbivorous animals (4, 5. 6). The plants have the potential and some important active ingredients that


* Corresponding author: J. Kayode
Tel: +2348035063504
E-mail: Joshua.kayode@eksu.edu.ng

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Olanipekun Mary K et al.

International Journal of Biomedical Papers 2016; 1: 5–12

served as therapeutic properties. Almost all plants are medicinal and the application of medicinal plants especially in
traditional medicine is currently well acknowledged (4. 5, 7, 8). Therefore, the continued investigation into the
secondary metabolites or bioactive ingredients present in medicinal plants as an effective and alternate measure to
the treatment of diseases cannot be overemphasized (9).
The synthetic treatment of the emergence and spread of Salmonella typhi causing typhoid fever for instance is
expensive and currently found to be resistance to many common used standard antibiotics such as Amoxicillin,
Ampicillin, Chloramphenicol and Ciprofloxacin. Therefore, there has been gradual shift recently from the use of
synthetic drugs in the treatment of diseases to the use of plant products due to emergence of multi drugs resistant
phenomena. Also, it has been proven scientifically that some plants have many compounds that exhibited potent
effects on the diseased causing organisms. Similarly, various bioactive compounds used as starting materials in the
preparation of modern medicines were derived from plants (9-13). The plants are now known to have greater
bioactive ingredients than synthetic formulation.
Recent initiatives on the use of medicinal plants necessitated the need for the scientific validation of plants used
traditionally for medicines. Plants are natural, cheap, available, holistic, less toxic and easy to prepare especially to
the rural dwellers. The aim of this study is to determine the antibacterial potentials of some medicinal plants used
traditionally for treating diseases in the study area.
2. Materials and methods
2. 1 Collection of Plant Materials:
Fresh leaves and stem barks of Alstonia boonie, Bridelia ferruginea, Eucalyptus tereticornis, Terminalia
schimperiana and Polyalthia longifolia were collected from Ekiti State University Agricultural Farm. The plants
were scientifically identified and authenticated at the herbarium of the Department of Plant Science and
Biotechnology, Ekiti State University, Ado-Ekiti, Nigeria. The plants materials were air dried at room temperature,
ground and stored in air-tight containers to avoid volatilization of the active ingredients.
2.2 Extraction of the plant samples
The air dried plants materials were pulverized with an electric blender to powdered form, 10g of each of the
powdered samples was dissolved in 100 ml of distilled water, shaken regularly and left for three days to ensure deep
extraction of the bioactive ingredients present in the plant samples. The solutions were filtered through eight layers
of muslin cloth followed by Whatman No.1 filter paper; the filtrates were centrifuged for 10 min. The supernatant
was collected and further filtered till the volume was reduced to one-fourth of the original volume of the solvent
used (that was 100 ml) giving the concentration of 400mg/ml (14, 15).
Also 10g of each of the powdered samples was suspended in 100 ml of distilled ethanol in a conical flask, plugged
with cotton wool and then kept on a rotary shaker 190-220 rpm for 24 h. The supernatant was collected slowly and
evaporated in wide mouthed evaporating bowls at room temperature for 2-3 days until the final volume was reduce
to one fourth of the original volume of the solvent used (i.e. 100ml) giving the concentration of 400mg/ml (14) and
stored at 40C in airtight bottles.
2. 3 Phytochemical Screening of the Plant Samples
The phytochemical screening of the medicinal plants used was carried out in the Laboratory of the Department of
Chemistry, Afe Babalola University, Ado-Ekiti, Nigeria. The leaves and the barks of the plant samples were
screened for the presence of bioactive ingredients such as alkaloids (according to 14), tannins (according to 16),
glycosides, saponins and terpenoids (according to 17) and flavonoids (according to 18).
2. 4 Sources of Bacteria
Bacterial strains were obtained from the stock culture of the Department of Microbiology, Ekiti State University,
Ado-Ekiti. The bacteria included Staphlococcus aureus, Escherichia coli, Enterococcus faecalis, Salmonella typhi
.The strains were kept at 40C on agar slant and sub cultured and incubated at 37 0C for 24 h on nutrient agar (SigmaAldrich, Germany).
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Olanipekun Mary K et al.

International Journal of Biomedical Papers 2016; 1: 5–12

2. 5 Antimicrobial Assays
These were conducted at the Laboratory of the Department of Plant Science and Biotechnology, Ekiti State
University, Ado-Ekiti, Nigeria, between November 2015 and April 2016, to determine the in vitro anti-bacteria
effects of the administration of aqueous and ethanolic extracts of the above-stated plants. Micro-titer method was
used to screen for the anti-bacterial activity in accordance with the Clinical and Laboratory Standards Institute
(CLSI) guidelines (19).
The plant extracts were dissolved in 5% of Dimethylsulphate (DMSO) to achieve a test concentrations ranging from
100 mg/ml to 6.25 mg/ml. 100ml inoculums suspension of giving bacterial cells in a nutrient broth was added into
each well with 100ml of each plant extract which was incubated at 37°c for 24hours (i.e. the bacteria strain) and the
minimum inhibitory concentration (MIC) determined, the last well that is next to the well with visible bacterial
growth was taken to be the MIC.
2. 6 Determination of Minimum Inhibitory Concentration: Micro-broth dilution method was used for the
determination of minimum inhibitory concentration (MIC) of the extract (as described by 20). Blood broth was used
to prepare different concentrations ranging from 6.25mg/ml to100mg/ml by serial dilutions. Each prepared
concentration in tubes was inoculated with 100ul of each of the standardized culture of the test bacteria. Tube
containing blood broth without extract was used as negative control. The tubes were incubated aerobically at 37°C
for 24h. The first tube in the series shows no sign of visible growth; this was taken as the MIC.
2. 7 Determination of Minimum Bactericidal Concentration (MBC): The MBC is the lowest concentration of
the tested compound that produced a 99.9% reduction on a bacterial viable count on the agar plate. A loop-full of
culture from the first three broth tubes that showed no growth in the MIC tubes were inoculated on sterile Nutrient
Broth Agar plates and observed for growth after incubation at 37°C for 24hours. The MBC was taking as the least
concentration of the extracts that showed no growth.
2. 8 Determination of Minimum Inhibitory Concentration Index (MICI)
The minimum inhibitory concentration index (MICI), was calculated as the ratio of MBC and MIC (21) as follow:
When MBC/MIC ≤ 2.0, the extract was considered bactericidal,
When MBC/MIC >2 but <16, the extract was considered bacteriostatic, and,
When MBC/MIC ≥16.0, the extract was considered ineffective
3. Results
The preliminary phytochemical screening of the selected plants revealed the presence or absence of some bioactive
ingredients. The secondary metabolites such as tannins, saponins, flavonoids, alkaloids, cardiac glycosides and
terpenoids were found to be variously distributed in the plant samples as shown in Table 1 below. The aqueous and
ethanolic extracts showed the level of the availability of the bioactive ingredients which subsequently shows that
ethanol is a better extraction medium than the aqueous solvent. Similarly, the considerable availability of each of the
bioactive parameters revealed the level of antibacterial activities shown on the tested pathogens. Cardiac glycosides,
flavonoids, saponins and terpenoids were present in all the plant samples. Alkaloids were found present in
abundance in Terminalia schimperiana and in substantial quantities in Alstonia boonei, and Polyalthia longifolia.
Tannins were present in all the plants.
Tables 2 to 6 show the MIC, MBC and MICI (i.e. MBC/MIC) of the aqueous and ethanol extracts of Alstonia
boonie, Bridelia ferruginea, Eucalyptus tereticornis, Terminalia schimperiana and Polyalthia longifolia
respectively. The extracts of the plants were screened against bacterial strains of Escherichia coli, Salmonella typhi
A, B and C strains, Enterecoccus faecalis strains A and B and Staphylococcus aureus strains A and B. The plants
extract inhibited the growth of the tested organisms at varying concentrations ranging from 6.25mg/ml-100mg/ml.

Page | 7

Olanipekun Mary K et al.

International Journal of Biomedical Papers 2016; 1: 5–12

The ethanol extracts of stem barks of all the studied plants possessed high inhibitory effects on Staphylococcus
aureus, Escherichia coli, Enterococcus faecalis, Salmonella typhi strains than the aqueous extracts at the
concentration of 50 mg/ml and 100mg/ml respectively (Tables 2-6). However, aqueous extract of Terminalia
schimperiana stem bark moderately inhibited the growth of the resistant Salmonella typhi at the minimum inhibitory
concentration of 6.25%, minimum bactericidal concentration of 50% and minimum inhibitory concentration index of
4** (Table 5).
Table 1. Phytochemicals in the selected traditionally utilized medicinal plants examined
Constituents
A.b
T.s
B.f
W
E
W
E
W

E.t
E

W

+
+
+
+
+
+
Tannins
+
++
+
+
+
Saponins
+
++
+
+
+
+
+
Flavonoids
+
+
Alkaloids
++
+
++
+
++
+
+
Cardiac glycosides
+
+
++
+
Terpenoids
Legend: +++ = present in abundance, ++ = moderately present, + = present, - = absent A.b=Alstonia boonei , T.s =
E.c= Enantia chloranta, B.f= Bridelia ferruginea, C.c= Cymbopogon citratus, E.c= Eucalyptus tereticornis, P.L=
Aqueous, E=Ethanol.

P.i
E

W

E

+
+
+
+
+
+
++
+
+
++
+
+
+
Terminalia schimperiana,
Polyalthia longifolia. W=

Table 2. The antibacterial activity of the leaf and stem extracts of Alstonia boonei against bacterial pathogens.
Bacteria
Leaf
Stem
MIC
MBC
MICI
MIC
MBC
Aq
Eth
Aq
Eth
Aq
Eth
Aq
Eth
Aq
Eth
12.5
50
25
50
4**
1*
25
25
50
50
Staphlococus aureus A
6.25
25
50
50
8**
2*
50
25
100
50
Staphlococus aureus B
12.25
6.25
50
25
8**
4**
100
25
100
25
Escherichia coli
12.25
6.25
50
25
2*
4**
100
50
100
50
Salmonella Typhi A
6.25
25
50
25
4**
1*
100
25
100
50
Salmonella Typhi B
25
25
100
50
1
2*
25
25
50
50
Salmonella Typhi C
25
25
50
50
4**
2*
12.5
25
25
25
Enterococcus faecalis A
25
25
50
50
4**
2*
6.25
12.5
25
25
Enterococcus faecalis B
Legend: *=Bactericidal effect, **=Bacteriostatic effects, ***= ineffective
Table 3. The antibacterial activity of the leaf and stem extracts of Bridelia ferruginea against bacterial pathogens
Bacteria
Leaf
Stem
MIC
MBC
MICI
MIC
MBC
Aq
Eth
Aq
Eth
Aq
Eth
Aq
Eth
Aq
Eth
25
25
50
50
2*
2*
6.25
25
12.5
50
Staphlococus aureus A
50
25
100
50
2*
2*
12.5
50
25
100
Staphlococus aureus B
50
25
50
50
1*
2*
12.5
50
25
100
Escherichia coli
50
25
100
50
2*
2*
25
50
50
100
Salmonella Typhi A
50
25
100
50
2*
2*
12.5
50
50
100
Salmonella Typhi B
50
25
100
50
2*
2*
12.5
50
50
100
Salmonella Typhi C
25
25
50
50
2*
2*
12.5
50
25
50
Enterococcus faecalis A
6.25
25
25
50
4**
2*
12.5
25
25
50
Enterococcus faecalis B
Legend: *=Bactericidal effect, **=Bacteriostatic effects, ***= ineffective
Table 4. The antibacterial activity of the leaf and stem extracts of Eucalyptus tereticornis against bacterial pathogens
Bacteria
Leaf
Stem
MIC
MBC
MICI
MIC
MBC
Aq
Eth
Aq
Eth
Aq
Eth
Aq
Eth
Aq
Eth
25
25
25
100
1*
4**
12.5
25
100
50
Staphlococus aureus A
12.5
25
50
100
4**
4**
50
50
100
50
Staphlococus aureus B
25
25
100
100
4**
4**
25
50
100
100
Escherichia coli
25
12.5
100
25
4**
2
25
50
100
50
Salmonella Typhi A
50
25
100
100
2*
4**
12.5
25
100
50
Salmonella Typhi B
50
25
100
100
2*
4**
50
50
100
50
Salmonella Typhi C
25
25
50
50
2*
2
25
50
25
50
Enterococcus faecalis A
6.25
12.5
25
50
4**
4**
12.5
50
25
25
Enterococcus faecalis B
Legend: *=Bactericidal effect, **=Bacteriostatic effects, ***= ineffective

MICI
Aq
Eth
2*
2*
2*
2*
1*
1*
1*
1*
1*
2*
2*
2*
2*
1*
4**
2*

MICI
Aq
Eth
2*
2*
2*
2*
2*
2*
2*
2*
4**
2*
4**
2*
2*
1*
2*
2*

MICI
Aq
Eth
8**
2*
2*
1*
4**
2*
4**
1*
8**
2*
2*
1*
1*
1*
2*
1*

Page | 8

Olanipekun Mary K et al.

International Journal of Biomedical Papers 2016; 1: 5–12

Table 5. The antibacterial activity of the leaf and stem extracts of Terminalia schimperiana against bacterial pathogens.
Bacteria
Leaf
Stem
MIC
MBC
MICI
MIC
MBC
Aq
Eth
Aq
Eth
Aq
Eth
Aq
Eth
Aq
Eth
25
25
50
50
2*
2*
6.25
50
25
50
Staphlococus aureus A
12.5
25
25
50
2*
1*
6.25
25
25
50
Staphlococus aureus B
12.5
25
50
50
2*
2*
6.25
50
50
100
Escherichia coli
25
25
50
50
2*
2*
12.5
50
50
100
Salmonella Typhi A
12.5
25
50
50
4**
2*
6.25
50
25
100
Salmonella Typhi B
12.5
25
50
50
4**
2*
6.25
50
25
100
Salmonella Typhi C
12.5
25
25
50
2*
1*
6.25
50
25
100
Enterococcus faecalis A
12.5
12.5
25
25
2*
2*
6.25
50
25
100
Enterococcus faecalis B
Legend: *=Bactericidal effect, **=Bacteriostatic effects, ***= ineffective
Table 6. The antibacterial activity of the leaf and stem extracts of Polyalthia longifolia against bacterial pathogens
Bacteria
Leaf
Stem
MIC
MBC
MICI
MIC
MBC
Aq
Eth
Aq
Eth
Aq
Eth
Aq
Eth
Aq
6.25
50
50
100
4**
2*
25
50
25
Staphlococus aureus A
12.5
50
100
100
8**
2*
25
50
50
Staphlococus aureus B
12.5
50
100
100
8**
2*
12.5
50
100
Escherichia coli
50
50
100
100
2*
2*
25
25
50
Salmonella Typhi A
12.5
50
50
100
4**
2*
50
100
50
Salmonella Typhi B
50
50
50
100
1*
2*
25
100
25
Salmonella Typhi C
12.5
50
50
100
4**
2*
12.5
50
25
Enterococcus faecalis A
6.25
12.5
25
50
4**
4**
12.5
100
25
Enterococcus faecalis B
Legend: *=Bactericidal effect, **=Bacteriostatic effects, ***= ineffective

Eth
100
100
100
50
100
100
50
100

MICI
Aq
Eth
4**
1*
4**
2*
8**
2*
4**
2*
4**
2*
4**
2*
4**
2*
4**
2*

MICI
Aq
1*
2*
8**
2*
1*
1*
2*
2*

Eth
2*
2*
2*
2*
1*
1*
1*
1*

4. Discussion
The presence of bioactive ingredients explains the various therapeutic healing activities that these plants could exert
on various diseases causing organisms. Alkaloids are used as analgesic and as antibacterial (22, 23). The presence of
saponins helps in wounds healing and in arresting bleeding treatments (24). It also helps in protection against attack
by pathogens. Saponins, alkaloids and flavonoids have been documented to possess medicinal properties (25, 26).
Tannins have considerable beneficial effect on vascular health and help to draw out all irritants from the skin. They
are useful as anti-inflammatory against pathogens and in the treatments of burns and other wounds based on their
anti-microbial, anti-hemorrhagic and antiseptic potentials. Tannins are used as anti-helminthes, antioxidants,
antimicrobial, anti-viral and in cancer treatment (18).
The results of the MBC/MIC showed that ethanol extract exerted bactericidal effect on the pathogens. Hence, the
strong activities of these extracts on S. aureus, E. coli, Salmonella typhi and E. faecalis, suggested that it may be
used in the treatments of diseases caused by these organisms such as gastro-intestinal infection like diarrhea and
dysentery. They will also be useful for the treatments of wound infections as previously reported by (27). This study
also revealed the potential of the extracts of Terminalia schimperiana stem barks in the treatment of typhoid fever
caused by S. typhi. Salmonella typhi A, B and C bacteria were the most resistant organisms to all the plants extracts
except the water extract of Terminalia schimperiana stem bark. Staphlococcus spp strains are the most sensitive
organisms to all the plants extracts. Similar results were obtained with extracts from leaves of Kalanchoe pinnate
when tested against S.aureus, E. coli, B. subtilis, P. auruginosa and C. albicansas (28).
Antibacterial activity obtained in this study varied with the type of solvents used for the extraction of the plants
used. Medicinal and healing properties of the herbs are the results of the presence of biochemical ingredients present
in the plants. However, the abilities of these chemicals to carry out the healing activities depend on the solubility of
these compounds in the various solvents. The ethanol extracts of the samples demonstrated better antibacterial
activities when compared to the aqueous extracts. This might be attributed to the organic nature of ethanol and its
high capacity to dissolve more organic and active antimicrobial compounds (29). Similarly, the antimicrobial
activities of the aqueous extracts could be attributed to their anionic components such as thiocynate, nitrates,
chlorides, sulphate and other water soluble components which are naturally occurring in the plant materials (30).

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Olanipekun Mary K et al.

International Journal of Biomedical Papers 2016; 1: 5–12

These results confirmed the previous assertions that ethanol is a better solvent for extraction of antimicrobial
substances from medicinal plants compared to other solvents (31-36).
In conclusion, the use of plant extracts with known antimicrobial potentials can be of great significance in
therapeutic treatments. However, there are reports of various types of contaminants in herbal medicine which
include microorganisms and toxins produced by microorganisms, pesticides and heavy metals (37). Hence,
sterilization is needed especially for aqueous extract before use in order to get rid of these contaminations. The
results of this study suggested that Alstonia boonie, Bridelia ferruginea, Eucalyptus tereticornis, Terminalia
schimperiana and Polyalthia longifolia served as potential sources of bioactive healthy compounds in the diet and
their consumption could be useful in the prevention of diseases. These plants could be used in the development of
standardized herbal medicine to treat infectious diseases. The activities of the extracts from these plants against the
tested isolates justify their local use. However, further studies are required towards the isolation and identification of
active principles present in them, the toxins produced by the plants in case of pollution, pesticides and
microorganism’s attacks as well as the heavy metals present in the extracts. All these could possibly alter their
pharmeceutical efficacies.
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