M. Yusha’u et al.

International Journal of Biomedical and Health Sciences 0794-4748/2010 $12.00 + 0.00

Vol. 6, No. 3, September 30, 2010  2010 African Studies on Population and Health
Printed in Nigeria http://www.asopah.org

IJBHS 2010050/6304

Antibacterial activity of Adansonia digitata stem bark

extracts on some clinical bacterial isolates
M. Yusha’u*, M. M. Hamza, and N. Abdullahi
Department of Biological Sciences, Bayero University, P. M. B. 3011, Kano -Nigeria

(Received April 15, 2010; Accepted June 16, 2010)

ABSTRACT: Powdered stem bark of Adansonia digitata (L.) was extracted with ethanol and chloroform using
percolation method and Sohxlet extractor. The extracts were tested for antimicrobial activity against clinical bacterial
isolates of Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis and Staphylococcus specie using disc diffusion
and microbroth dilution technique. The extracts were further screened for the presence of secondary metabolites using
standard techniques. Results of sensitivity test using both procedures showed that ethanol extracts of the plant were
more active than chloroform extract on the isolates tested irrespective of the method of extraction employed. The
results of phytochemical screening indicated the presence of alkaloids, flavonoids, reducing sugars, steroids and tannins
in either or both extracts. This indicates that Adansonia digitata root bark has the potential for the production of drugs
against some clinical bacterial isolates.

Keywords: Antibacterial activity, Adansonia digitata, Extracts, Clinical isolates

Introduction
Adansonia digitata is a deciduous, massive majestic tree up to 25m high, which may live for hundreds
of years. It has thick, angular, wide spreading branches and a short, stout trunk which attains 10 – 14m or
more in girth and often becomes deeply fluted. The form of the trunk varies. In young trees it is conical, in
mature trees it may be cylindrical, bottle shaped, or tapering with branching near the base.
Villagers often plant baobabs within their own courtyards and nurture them until they are 2 – 3m tall,
before transplanting them along the edges of cultivated fields. It is used as a boundary marker to make the
dividing line between plots (Rocheleau et al., 1988).
The fruit pulp is probably the most important food stuff. It is dry and mealy and it is used in cool and
hot drinks. Pulp can be dissolved in water or milk and the liquid is used as a drink, as a sauce for food, as a
fermenting agent in local brewing or as a substitute for cream of tartar in baking. The energy value of pulp
is similar to that of baobab leaves (Becker, 1983).

*Correspondence author e-mail: mryushau@gmail.com

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The leaves of the baobab tree are a staple food source for rural populations in many parts of Africa,
especially the central region of the continent (Yazzie et al., 1994). They are eaten both fresh and as a dry
powder. In Nigeria, the leaves are locally known as “kuka” and are used to make “kuka soup”
(http://en.wikipedia.org/wiki/baobab). During the rainy season the baobab leaves are tender and people
harvest the leaves fresh. During the last month of the rainy season, leaves are harvested in great abundance
and are dried for domestic use and for marketing during the dry season. The leaves are typically sun-dried
and either stored as whole leaves or pounded and sieved into a fine powder. In markets, the powder is the
most common form (Sidebe et al., 1998b). The leaves of Adansonia digitata are important protein sources
in complementing the amino acid profile and thereby improving the protein quality of the diet (Nordeide et
al., 1996). Young leaves are commonly used as a vegetable in soups or cooked and eaten as spinach
(Venter and Venter, 1996). Dried green leaves are used throughout the year, mostly in soups served with
the staple dish of millet (Delisle et al., 1997). Flowers can be eaten raw or used as flavour in drinks.
The seeds are characterized as a potential source of protein and roasted seeds are used as coffee
substitute in some areas (Dirar, 1993). The seeds are mostly used as a thickener for soups, but may be also
be fermented into a seasoning, roasted for direct consumption, or pounded to extract vegetable oil.
(http://en.wikipedia.org/wiki/baobab).
The bark produces strong fibres used in making ropes, mats, bags and hats. The smooth fibers of the
inner side of the bark are more important than the outer bark for weaving (Igboeldi et al., 1997). The wood
is whitish, spongy and light (air – dried 320 kgm-3 and is used mainly for fuel (Venter and Venter, 1996).
Hollow trees provide reservoirs of fresh water which are used by nomads, particularly in the western part of
Sudan (Tothill, 1954). Water storage capacities range from 1000 to 9000 litres per tree (Craig, 1991).
The use of trees as cattle feed is extremely important in the savanna areas especially in the arid zones,
where animals obtain much of their feed in the form of pods and leaves. The pulp and seeds have a high
nutritional value and are recommended for feeding the herd late in the dry season when grazing is poor
(Venter and Venter, 1996).
In the folk medicine baobab pulp is used in the treatment of fevers, diarrhoea, malaria, haemoptysis and
scorbutic complaints (vitamin C deficiency) and dysentery. Pulp extract is applied as eye-drops in cases of
measles (FAO, 1988). In many medicinal uses, stem bark is used. When prepared it is made into a
decoction for internal use and functions due to its soluble and insoluble tannin, and gummy and
albuminious constituents beta-sitosterol has been studied and this occurs in the bark and also the seed oil
(Asolkar et al., 1992). Root bark is a also used in traditional medicine. This contains Beta-sitosterol and
two glycosides (Ramesh et al., 1992). The leaves form a component or herbal remedies and a mash
prepared from the dried powdered roots is given to malarial patients as a tonic. A semi-fluid gum obtained
from baobab bark is used to treat sores (FAO, 1988).

Materials and Methods

(a) Collection and Identification of plant materials

Stem bark of Adansonia digitata was scraped using sterile knife at Kirikasamma Local Government
Area of Jigawa state. The tree was identified using guide (Aliyu, 2006). The scrapings were air-dried and
ground into fine powder using mortar and pestle in the laboratory as described by Mukhtar and Tukur
(1999).

(b) Extraction

Fifteen grams of the powdered plant material was dispensed in 150ml of ethanol and chloroform in
separate conical flasks, kept for two weeks with shaking at regular intervals after which the content was
filtered and the filtrate was evaporated at 30oC. They were labeled EPE and CPE for ethanol and
chloroform percolation extracts respectively. Two batches of fifteen grams were extracted with the same
solvents using soxhlet extractor for one hour and labeled ESE and CSE for ethanol and chloroform soxhlet
extracts respectively. All the extracts were allowed to evaporate at room temperature (Fatope et al., 1993).

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(c) Phytochemical screening

(i) Test for alkaloids

To 0.1ml of the extract and fractions in a test tube, 2 – 3 drops of Dragendoff’s reagent was added.
Orange red precipitate with turbidity denoted the presence of alkaloids (Ciulci, 1994).

(ii) Test for flavonoids

To 4mg/ml of the extracts and fractions a piece of magnesium ribbon was added followed by drop-wise
addition of concentrated HCl. A colour change from orange to red indicated the presence of flavones; red to
crimson indicated the presence of flavonoids (Sofowora, 1993).

(iii) Test for reducing sugars

To 1ml of extract and fractions in separate test tubes, 2.0mls of distilled water were added followed by
addition of Fehling’s solution (A + B) and the mixtures were warmed at 40oC. Appearance of brick red
precipitate at the bottom of the test tube indicated the presence of reducing sugar (Brain and Turner, 1975).

(iv) Test for steroids

Two milliliters of the extracts were evaporated to dryness in separate test tubes and the residues
dissolved in acetic anhydride followed by addition of chloroform. Concentrated sulphuric acid was added
by means of a pipette via the side of the test tubes. Formation of brown ring at the interface of the two
liquids and violet colour in the supernatant layer denoted the presence of steroids (Ciulci, 1994).

(v) Test for tannins

Two milliliters of the extract/fraction was diluted with distilled water in separate test tubes, 2 – 3 drop
of 5% ferric chloride (FeCl3) solution was added. A green – black or blue colouration indicated tannin
(Ciulci, 1994).

(d) Bioassay studies

(i) Disc preparation

Sensitivity discs were punched from Whatman No. 1 filter paper, sterilized in Bijou bottles by
autoclaving at 121oC for 15mins. Sensitivity discs were prepared by weighing the appropriate amount of
the extract or fraction and serial doubling dilution in Dimethyl-sulfoxide (DMSO) followed by placing the
improvised paper discs in the solution such that each disc absorbed 0.01ml to make the disc potency of
15µg, 30µg and 60µg (Akinyemi et al., 2005; Vallekobia et al., 2001).

(ii) Test isolates

Identified clinical tract isolates were collected from the microbiology laboratory of Aminu Kano
Teaching Hospital (AKTH) and maintained on nutrient agar slants in the refrigerator (4oC) prior to use.

(iii) Inoculum standardization

A loopful of the test isolate was picked using a sterile wire loop and emulsified in 3 – 4mls of sterile
physiological saline. The turbidity of the suspension was matched with that of 0.5 McFarland Standard
(Cheesbrough, 2004).

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(iv) Sensitivity testing

Using sterile swab stick, standardized inocula of each isolate was swabbed onto the surface of Mueller
Hinton Agar in separate Petri dishes. Discs of the extracts and standard Tetracycline (TET 30µg) were
placed onto the surface of the inoculated media. The plates were inverted and allowed to stand for 30mins
for the extract to diffuse into the agar after which the plates were incubated aerobically at 350C for 18
hours. This was followed by measurement of zone of inhibition formed by the test organisms around each
of the extract and standard antibiotic discs (NCCLS, 1999).

(e) Micro-broth dilution technique

(i) Minimum inhibitory concentration (MIC)

Minimum inhibitory concentrations of the extract and fractions were prepared by serial doubling
dilution using distilled water to obtain concentrations of 2000µg/ml, 1000µg/ml and 500µg/ml. Equal
volume (2mls) of extract and Mueller – Hinton broth were mixed. Specifically 0.1ml of standardized
inocula (3.3 x 106 CFU/ml) was added to each of the test tubes above. The tubes were incubated aerobically
at 350C for 24 hours. Tubes containing broth and leaf extracts without inocula which served as positive
control while tubes containing broth and inocula served as negative control. The tubes were observed after
24 hours of incubation to determine minimum inhibitory concentration. That is the lowest concentration
that showed no evidence of growth (Akinyemi et al., 2005; Vallekobia et al., 2001).

(ii) Minimum Bactericidal Concentration (MBC)

Sterile Mueller-Hinton agar plates were separately inoculated with sample from each of the test tubes
that showed no evidence of growth. The plates were further incubated at 35oC for 24 hours and observed.
The highest dilution that yielded no bacterial growth was regarded as MBC (Akinyemi et al., 2005;
Vallekobia et al., 2001).

Results and Discussion

High yield of extract was obtained at the end of Sohxlet extraction using chloroform, with extract
having yellow colour and gummy texture. The high yield of this extract may be due to the high solubility of
the compounds present in low polar solvent coupled with the effect of temperature provided by the Sohxlet
extraction system. There exist differences in the colour of the extracts based on the extraction solvent
irrespective of the method with ethanol extracts being reddish brown and chloroform extracts being yellow
but except for ethanol percolation extract that has powdery texture, all the extracts had gummy texture.
Sensitivity of the test isolates to A. digitata root bark extracts using disc diffusion method was indicated
by observation and measurement of inhibition zones formed around discs prepared from various
concentrations of the extracts. Absence of turbidity in tube cultures indicates the activity of the extract
using micro-broth dilution technique, the least concentration amongst the tubes without evidence of
turbidity was considered the minimum inhibitory concentration (MIC). Microbroth dilution technique
employed in this research is important in determining whether the extract and fractions are capable of
inhibiting the growth or completely killing the test isolates.
The results of sensitivity tests using both procedures indicated that ethanol extracts of the plant were
more active than chloroform extract on the isolates tested irrespective of the method of extraction
employed. The activity exhibited by the extracts may be related to the presence of tannins in addition to
flavonoids that are reported to be responsible for antimicrobial properties of some ethno-medicinal plants
(Singh and Bhat, 2003).
The results of phytochemical screening of ethanol and chloroform extracts of A. digitata using
percolation and Sohxlet extraction method revealed the presence of flavonoids and Steroids in all extracts
irrespective of either the solvent used or extraction method employed. Alkaloids were present in Sohxlet
extracts while tannins were present in percolation extracts hence their presence is dependent upon method

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of extraction employed. Reducing sugars is present in all extract with the exception of ethanol sohxlet
extracts (Table 2). These metabolites have been reported to possess antimicrobial activity (Cowan, 1999).
In particular the flavonoids were reported to be responsible for antimicrobial activity associated with some
ethnomedicinal plants (Singh and Bhat, 2003).

Table 1: Physical properties of Adansonia digitata extracts.

Physical parameters EPE ESE CPE CSE

Weight extracted 15 15 15 15
Weight of extract 1.3 1.2 1.3 2.0
Percentage yield 8.67 8.67 8.67 13.33
Colour Reddish brown Reddish brown Yellow Yellow
Texture Powdery Gummy Gummy Gummy
Key: EPE – Ethanol Percolation Extracts, ESE – Ethanol Soxhlet Extract, CPE – Chloroform Percolation
Extract, CSE - Chloroform Soxhlet Extract

Table 2: Phytochemical properties of Adansonia digitata extracts.

Phytochemical Tests
Extracts Alkaloids Flavonoids Reducing sugars Steroids Tannins
EP - + + + +
ES + + - + -
CP - + + + +
CS + + + + -
Key: EP – Ethanol Percolation, ES – Ethanol Soxhlet, CP – Chloroform Percolation,
CS - Chloroform Soxhlet, + - Present, - - Absent

Table 3: Sensitivity of clinical isolates (mm) to Adansonia digitata extracts using Disc Diffusion
Method.

EP ES CP CS TET(S)
Isolates 15 30 60 15 30 60 15 30 60 15 30 60 30
E. coli 8 9 9 6 6 6 6 6 6 6 6 6 6
Klebsiella pneumoniae 6 8 8 6 6 6 6 6 6 6 6 6 6
Proteus mirabilis 6 6 6 6 6 6 6 6 6 6 6 10 9
Staphylococcus specie 6 6 6 6 6 9 6 6 6 6 6 6 (33)
Key: EP – Ethanol Percolation, ES – Ethanol Soxhlet, CP – Chloroform Percolation,
CS - Chloroform Soxhlet, TET – Tetracycline, (S) - Streptomycin
TET - Tetracycline, S – Streptomycin

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Table 4: Sensitivity of clinical isolates to Adansonia digitata extracts using Micro-broth Dilution
Technique.

EPE (µg/ml) ESE (µg /ml)

Isolates MIC MBC MIC MBC
Escherichia coli 1000 ** 2000 **
Klebsiella pneumoniae ** ** ** **
Proteus mirabilis ** ** ** **

Staphylococcus specie ** ** ** **

Key: EPE – Ethanol Percolation Extracts, ESE – Ethanol Soxhlet Extract,

CPE – Chloroform Percolation Extract, CSE - Chloroform Soxhlet Extract

Conclusion

From the results of this work, it can be concluded that Adansonia digitata has the potential for the
production of drug for the treatment of bacterial infections.

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