INTRODUCTION
Medicinal plants are of great importance to the health of individuals and the
society. The medicinal value of these plants lies in some chemical substances
that produce a definite physiological action on the human body (Edeoga
and Gomina, 2000).
Plants as sources of remedy for many diseases date back to the early century
(Karou et al., 2005). In Africa, particularly
West Africa, new drugs are often beyond the reach of the poor. Hence, upto 80%
o the population use medicinal plants as remedy against infections and diseases
(Kirby, 1996; Hostettmann and Maston,
2002).
Treatment offered by traditional healers is primary health care that
has sustained the Nigerian community before and after colonization and
the medicinal plants used by African traditional healers are selected
not on the basis of their chemical constituent, but on their perceived
ability to restored patients disease condition to normal.
The use of plants as antibacterial agents is gradually attracting attention
probably due to the high cost, unavailability and resistance of the drugs.
Khaya grandifoliola is a medicinal plant endemic to Nigeria. It is a
tall, woody tree belonging to the family Meliaceae and commonly called African
Mahogany (Hutchinson and Dalziel, 1978). It is widely distributed
across West Africa from the Guinea coast to Cameron and extending eastward through
Congo Basin to Uganda and some parts of Sudan. It grows up to 40 m high and
5 m girth. The bark is grey in colour and yield a bitter gum when wounded.
Khaya species are valuable indigenous traditional medicine in West Africa.
Its bitter bark is mostly the part that is used to make concoctions to treat
some illness like fever, lumbago, cough, rheumatism, stomach ache, gastric pains
and as remedy against worm infestation. The anti malarial activity of the stem
bark was also reported (Agbedahunsi et al., 1998;
). The stem bark was also found to possess anti ulcer property (Njifutie
and Njikam, 2006), anti anaemic (Adeyemi and Gbilade,
2006), hypoglycaemic, hypoproteinaemic and hypocholesterolaemic effects
(Bumah et al., 2005). Some of the chemical constituents
reported include limonoids (Zhang et al., 2008).
The aqueous extract of K. grandifoliola is used in traditional
certain in Nigeria as remedy against cough, mycobacterium tuberculosis
and bacterial infections.
This present study was necessitated in order to justify the folkloric
usage of the plant. Herein, we report the effect of the aqueous extract
of the plant on methicillin resistant Staphylococcus aureus (MRSA)
implicated in many bacterial infections which are drug resistant.
MATERIALS AND METHODS
Collection and identification of plant material: The stem bark
of the tree plant Khaya grandifoliola was collected from
a local government area in Benue State, Nigeria around April, 2007. The
plant was identified by Mr. Sunny A of the Department of Pharmacognosy,
Faculty of Pharmacy, University of Benin, Benin City, Nigeria, where a
voucher specimen (218) was deposited.
Preparation and extraction of plant sample: The fresh stem bark
of K. grandifoliola was chopped into pieces and sun dried for a
period of two weeks, reduced to fine powder with the aid of a mechanical
grinder. The milled powder sample was collected and stored in glass jars,
tightly covered and kept for further studies.
Extraction of the plant material (800 g) with methanol (3 L) by maceration
for 48 h and filtration of the extract was carried out at room temperature
25°C. The reddish brown extract was concentrated to dryness using
a rotary evaporator at 30°C at reduced pressure. The dried extract
was stored in a refrigerator at -4°C until use.
Phytochemical screening: Phytochemical tests were carried out on the
powdered sample using standard procedures, to identify the constituents as described
by Sofowora (2008), Trease and Evans (2002)
and Harborne (1973).
Carbohydrate: 0.1 g of the powdered sample (Khaya grandifolila
powdered stem bark) was measured into a beaker and 20 mL of distilled
water was added. The beaker was heated in a water bath for over 5 min.
The mixture was filtered using a filter paper into another beaker to obtain
a filtrate, which was used to test for.
Molisch’s test for carbohydrates: Two milliliter of the
filtrate from above was measured into a test tube and 2 drops of alcoholic
solution of α-naphthol added, then the test tube was slanted and
concentrated sulphuric acid added down the side into the test tube without
mixing.
Saponins: Two milliliter of the filtrate from above was measured
into another test tube and 10 mL of distilled water was added it was shaken
vigorously for over a minutes.
Tannins: Powdered Khaya grandifoliola stem bark
(2.5 g) was weighed into a conical flask and mixed with 50 mL of water,
boiled in a water bath for 5 min. The mixture was filtered hot using a
filter paper and the filtrate collected in a beaker. Two milliliter of
the filtrate was mixed with 10 mL of distilled water and then a drop of
iron chloride was added.
Flavonoids: Five milliliter of dilute ammonia solution was added
to a portion of the aqueous filtrate followed by the addition of concentrated
sulphuric acid, (1 mL) to 2 mL of potassium hydroxide solution and allowed
to mix. Then into the acid base mixture, a small quantity of aqueous filtrate
of the sample was added and observed for colour change.
Anthraquinones: Powdered sample (2.5 g) was shaken with 5 mL of
benzene and then 2.5 mL of 10% ammonia solution was added and shaken.
Alkaloids: Extraction of 5 g of the powdered sample was carried
out by boiling in 50 mL of distilled water in a water bath for 30 min.
It was then filtered into a test tube and the filtrate collected. The
filtrate was tested with alkaloidal reagents (Dragendorfs, Wagner’s
and Mayer’s reagent) and results compared to blanks.
Tropane alkaloids: Vitali- Morin test; powdered sample (0.5 g)
was mixed with chloroform, boiled and allowed to extract. The mixture
was filtered using a filter paper into a beaker. Two milliliter of the
filtrate was measured into a small crucible and evaporated to dryness.
The residue was moistened with a few drops of concentrated nitric acid
and evaporated to dryness on a water bath. A few drops of 10% of potassium
hydroxide solution in alcohol was added and mixed.
Isoquinoline alkaloids: Emetine was used as standard. Powdered
sample (1.0 g) was added to 2.5 mL of water and 10 mL of HCl. The mixture
was allowed to stand for 5 min then filtered. Two milliliter of the filtrate
was taken and a few crystals of potassium chlorate were added.
Antimicrobial evaluation
Susceptibility testing: The agar-well diffusion method was used to determine
the antimicrobial activity of the extract (Barry and Thornsberry,
1995). The bacteria used for this study were obtained from the department
of microbiology, University of Benin Teaching Hospital, Nigeria.
The susceptibility of Methicillin Resistant Staphylococcus aureus
(MRSA) isolates using the E test strip was first carried out by the disk
diffusion method using mannitol salt agar. Each isolates was grown in
a nutrient broth for 18 h. One milliliter of the cultures of these organisms
were sub-cultured into 9 mL nutrient broth and shaken in a water bath
for 4 h at 80 throws min-1 and the dilutions made to obtain
105 cells mL-1. One milliliter of this final dilution
was used to flood an already prepared Mueller-Hinton agar and the excess
drained into disinfectant jar.
The extract was tested at 20 mg mL-1 concentration. This was
prepared by dissolving 2 g of the crude extract in 5 mL of sterile distilled
water. 0.005 mL (50μL) was then delivered into wells (5 mm in diameter)
bored into the surface of the already seeded Mueller-Hinton agar plates.
Standard antibiotics made from ciprofloxacin (0.010 g mL-1)
and gentamycin (0.010 g mL-1) were assayed along using the
agar-well diffusion technique. The plates were allowed to stand on the
bench for 30-40 min and then incubated at 37°C for 24 h.
Determination of Minimum Inhibitory Concentration (MIC): The modified
agar-well diffusion technique (Okeke et al., 2001)
was used to determine the MIC of the extract. A two fold serial dilution was
prepared by first reconstituting in sterile distilled water, then diluting to
achieve a decreasing concentration of 20.0, 16.0, 12.0, 8.0, 4.0, 2.0, 1.0,
0.8, 0.6, 0.4, 0.2 and 0.1 mg mL-1, respectively. Each dilution was
introduced into Mueller-Hinton agar plates already seeded with standardized
inoculums (approximately 105 cfu mL-1) of the test bacterial
isolates. All test plates were incubated at 37°C for 24 h. The least concentration
of extract showing a clear zone of inhibition was regarded as the MIC.
RESULTS AND DISCUSSION
The phytochemical components of K. grandifoliola is presented in Table
1. Results obtained from the qualitative phytochemical tests carried out
on the powdered sample revealed that the stem bark contained a wide array of
phytochemicals these include carbohydrate, saponins, tannins, flavonoids, anthraquinones,
alkaloids and specific alkaloids such as emetine (isoquinolic alkaloid) and
strychnine (indole alkaloids). The absence of tropane alkaloids and brucine
(indole alkaloid) was also observed. Knowing the phytochemical constituent can
help one to speculate on the medicinal value of the stem bark. Flavonoids have
been reported to have antibacterial and antimicrobial properties (Tsuchiya
et al., 1996). Tannins have antimicrobial (Ya et
al., 1988) and antioxidant properties. Crude saponin extract from Sorghum
bicolor has antimicrobial activity (Seotan et al.,
2006). Alkaloids have pronounced physiological effect particularly on the
nervous system (Sofowora, 2008; Levetin
and McMahon, 2003). The presence of these phytochemicals in the stem bark
suggests that the plant is pharmacologically active, supporting the claim by
traditional healers.
| Table 2: |
Zone of inhibition (mm) produced by extract and standard antimicrobial
agents |
|
| Cip: Ciprofloxacin, Gen: Gentamycin |
The result contradicted the reported phytochemical components indicating absence
of flavonoids in the stem bark of this same plant (Ibrahim
et al., 2006).
The results of the antimicrobial activity of the crude extract at 0.020
g mL-1 concentration, ciprofloxacin 10 mg L-1 and
gentamycin 0.010 g L-1 tested against the isolates is given
in Table 2.
The crude extract showed activity against all the test isolates having
a greater activity against the gram positives especially the MRSA isolates.
The mean zone diameter measured for the MRSA isolates was the highest,
ranging from 18.3- 21.0 mm (Fig. 1). This is of great
interest, because, bacterial resistance to antibiotics is a difficult
problem facing the world of medicine today. Substances that are able to
significantly control the activity of resistant strains of bacteria are
highly esteemed; the stem bark of Khaya grandifoliola is
proving to be one of such.
Similar concentrations were not used for the crude extract and the standards,
but the results are comparable because the crude extract is a mixture
of pharmacologically and non-pharmacologically active components while
the standards are purified antibiotics. The mean zone diameter measured
for the crude extract is within a reasonable range of that measured for
the standards, indicating that when further isolation and purification
of the extract is carried out, increased activity most assuredly would
be obtained.
The Minimum Inhibitory Concentration (MIC) for all test isolates was
0.4 mg mL-1 except for B. subtilis which was
0.2 mg mL-1 (Table 3). This concentration
is remarkable for a crude extract and further work should be carried out
to isolate, characterize and purify the active constituents of this indigenous
plant with view to determining its spectrum of activity as well as adding
it to already established antimicrobial agents especially those that are
active against resistant strains of bacteria.
|
| Fig. 1: |
Zones of inhibition produced by extracts at the tested concentration |
| Table 3: |
Minimum Inhibitory Concentration (MIC) of K. grandifoliola
stem bark extract |
|
| MRSA; Methicillin Resistant Staphylococcus aureus |
The antimicrobial activity exhibited by this plant could be attributed to the
phytochemicals that it contains. Of particular interest is its activity against
the Methicillin Resistant Staphylococcus aureus (MRSA) isolates. This
is probably due to the presence of flavonoids (Tsuchiya et
al., 1996). This is an indication that the stem bark if processed could
be a good first line drug for gram positive bacteria (especially the Staphylococcus
specie). This result proves that the use of the stem bark of Khaya grandifoliola
to cure several illnesses, especially those caused by microbes, is valid. It
is expected that the results from this study would serve as background knowledge
for further studies on this plant, which would result to discovering other medicinally
useful properties.
CONCLUSION
An aqueous solution of the methanolic extract of the powdered stem bark of
Khaya grandifoliola possesses antimicrobial potentials against both gram
positive and gram negative bacteria, especially on some resistant strains of
Staphylococcus. It is therefore confirmed as a useful antimicrobial agent.
The powdered stem bark is rich in phytochemicals and secondary metabolites such
as tannins, alkaloids, flavonoids, anthraquinones and saponins, which are probably
responsible for its medicinal properties.
ACKNOWLEDGMENTS
The researchers are greatful university of Benin for the technical assistance
provided fot the research work. We appreciate the assistance of Mr. Simon Peters
for the collection of the plant sample.
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