Bajopas Volume 7 Number 1 June, 2014 http://dx.doi.org/10.4314/bajopas.v7i1.

20

Bayero Journal of Pure and Applied Sciences, 7(1): 105 – 115

Received: December 2011
Accepted: February 2014
ISSN 2006 – 6996

PHYTOCHEMICAL SCREENING, ANTIBACTERIAL AND TOXICOLOGICAL

ACTIVITIES OF ACACIA NILOTICA EXTRACTS
1
Okoro, S. O., 2Kawo, A. H. and 2Arzai, A. H.
1
Department of Physiology, Bayero University, P. M. B. 3011, Kano, Nigeria
2
Department of Microbiology, Bayero University, Kano, Nigeria
2
Department of Microbiology, Bayero University, Kano, Nigeria
ABSTRACT
The phytochemical screening, antibacterial and toxicological activities of extracts of the leaves,
stem bark and roots of Acacia nilotica were investigated. The phytochemical analyses according to
standard screening tests using conventional protocols revealed the presence of tannins and sterols
in the leaves stem barks and roots of the plant. Alkaloids were detected only in the leaves.
Glycosides, saponins, resins and flavonoids were not detected in the plant. In-vitro agar-diffusion
sensitivity tests of crude extract fractions of the plant extracts using ethanol, chloroform,
methanol, petroleum ether, water and ethyl acetate were investigated on nine bacterial isolates.
The extract fractions generally exhibited marked antibacterial activities on Klebsiella pneumoniae,
Pseudomonas aeruginosa, Proteus vulgaris, Salmonella typhi, Shigella dysenteriae Staphylococcus
aureus and Escherichia coli except on Streptococcus pneumoniae and Streptococcus pyogenes. All
the leaves extract fractions of the plant exhibited weak or no antibacterial activity on the bacterial
isolates tested but the stem bark and root extracts generally exhibited strong antibacterial
activities on them. The minimum inhibitory concentration and minimum bactericidal concentration
studies revealed that some bacterial isolates were inhibited at concentrations of about 12.5mg/ml
and 50mg/ml and killed at concentrations of about 100mg/ml and 400mg/ml. Toxicity studies of
the ethanol extracts revealed that they exhibited no significant toxicity (LD50 of 123.86µg/ml
and312.55µg/ml) against Artemia salina . These results suggest that the plant may not be toxic to
man and could be a potential source of novel antibacterial compound.
Keywords: Phytochemical Screening, Antibacterial Activity, Toxicological Activity Acacia nilotica,
Extracts
INTRODUCTION
Acacia nilotica (bagaruwa in Hausa) has been MATERIALS AND METHODS
designated and used as medicinal plants in parts of Collection and Identification of Plant Materials
Northern Nigeria, West Africa, North Africa and other The plant materials were collected from
parts of the world. The plant is used to treat infections Ungogo Local Government Area of Kano State. The
such as diarrhea, dysentery, leprosy, cancers, ulcer, plants were however identified at the Botany Unit of
and diabetes (Aliyu, 2006; the Department of Biological Sciences by Prof. B. S.
http//:en.wikipedia.org/wiki.acacia-nilotica, 2008). Aliyu and with the aid of botanical keys (Arber, 1972).
Antimicrobial drug resistance is not only on the The parts of the plants mentioned above were
increase, it is also a serious problem to the medical collected fresh, healthy and free from organic
profession. Moreover, the toxicity of some medicinal contaminants that may interfere with the substances
plants has been severally reported. For instance, of interest by washing them with clean water
Bryophyllum calycinum whole plant, Annona (Onoruvwe et al., 1998).
senegalensis root, Hymenocardia acida stem bark, Extraction and Fractionation of Plant Materials
Erythrophleum suaveollens leaves and Spondiatus The specimens were dried at room temperature
preussii extracts were toxic to brine shrimps and (300C), and kept away from sunlight to prevent
caused chromosomal damage in rats (Sowemimo et changes in the nature of the plants’ constituents. The
al; 2007). The vast number of chemicals used specimens were grounded to powder (fine texture)
industrially and pharmacologically provides an ever with mortar and pistol. One hundred grammes of
increasing hazard to the liver. These chemicals are powdered specimens were separately percolated in
thought to be responsible for one of the most one liter of 96% alcohol for seven days followed by
common type of liver diseases, such as chemical filtration.
hepatitis (Sule, 2006). The extracts were concentrated using a laboratory
Brine shrimps have been used as a benchtop rotary vacuum evaporator at a temperature of 400C.
bioassay for the discovery and purification of bioactive The crude extracts were weighed labeled and stored
natural products and they are excellent choice for in a refrigerator at a temperature of 40C. A fraction of
elementary toxicity investigations of consumer each extract was partitioned between water and
products (Lieberman, 2008). Thus, this research is chloroform mixture (300:300). This was shaken for
focused on the study of phytochemical screening, about one hour and allowed to settle for 24 hours in a
antibacterial activity and toxicity of the plant extracts. separating funnel.

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The water, chloroform and interface fractions were 25ml of 0 .1N iodine and 10ml of 4% NaOH. The
separated in glass beakers and labeled respectively. resulting mixture was kept in the dark for 15minutes.
These fractions were again concentrated using a Ten (10) ml of water was used to dilute the mixture
rotary vacuum evaporator, weighed, labeled and and acidified with 10ml 4% sulphuric acid. The
stored in a refrigerator at 40C respectively. Similarly, a mixture was titrated with 0.1N sodium thiosulphate
fraction of each of the chloroform soluble extract was solution and starch solution was used as indicator.
partitioned in a mixture of absolute methanol and Titration value corresponds to the sum of tannins and
petroleum ether (300:300). Again, the methanol and pseudo tannins concentration A. Another 25ml of each
petroleum ether fractions were concentrated using water extract was placed in a stoppered conical flask
rotary evaporator, weighed, labeled and stored as followed by 15ml of gelatin. This volume was made up
above. Finally, each of the water soluble fractions to 100ml with water and filtered. Aliquot of 20ml was
were partitioned between water and ethyl acetate placed in a volumetric flask, 25ml of 0.1N iodine and
(300:300). The water and ethyl acetate fractions were 10ml of 4% NaOH were added mixed and kept in the
concentrated using a rotary vacuum evaporator, dark for 15 minutes. The mixture was diluted with
weighed, labeled and stored as above (Fatope et al; 10ml of water and acidified with 10ml of 4% sulphuric
1993 and Adoum et al; 1997). acid. This was finally treated with 0.1N sodium
Qualitative Phytochemical Analysis of Plant thiosulphate using starch as indicator. The titration
Extracts value that was obtained corresponds only to the
The phytochemical analyses were carried out pseudo tannins concentration B. The tannins and
using standard screening tests and conventional pseudo tannins content of each sample was then
protocols for the presence of alkaloids (Sofowora, calculated using the formula below:
1993), tannins (Trease et al., 1989), glycosides A = (Blank - Exp. A) x 0.029 x 100g % / 5 (volume
(Ciulei, 1994), saponins (Turner and Brain, 1975), taken).
sterol (Sofowora, 1993) and resins (Sofowora, 1993). B = (Blank - Exp. B) x 0.029 x 100g% / 5 (volume
Extraction of Alkaloids for Quantitative taken).
Analysis Where A = % of tannins and pseudo tannins, B = %
Five, (5g) of the powdered leaf extract was of pseudo tannins only
extracted with 50ml methanol. From the extract, Therefore % of true tannins = A - B g% w/v. (El-
10ml was placed in 250ml separating funnel and 5ml Olemy et al., 1994).
of dilute sulphuric acid and distilled water was added. Preparation Sensitivity Discs
The extract was shaken twice with 10ml chloroform. Preparation of sensitivity discs were done in the
The combined chloroform-extract was transferred to a laboratory. Whatman,s No 1 filter paper were used.
second separating funnel containing 5ml of dilute These were obtained by punching the filter paper with
sulphuric acid and 10ml of distilled water. The a paper punch (6mm diameter). The disc were
chloroform layer was discarded after shaking and the sterilized by autoclaving at 1210c for 15 minutes and
aqueous acidic layer was transferred to the content of impregnated with the prepared extracts. The
the first separating funnel. The extract was basified impregnated discs were stored in a refrigerator for
with ammonia solution and was shaken for 30 future use. Various test solutions were prepared in
seconds. accordance with the dilution method used by Baker et
The alkaloids were completely extracted by al. (1993). Stock solutions of each fraction were
successive portions of chloroform. The combined prepared by dissolving 100mg of the extract in 10ml
chloroform extract was shaken with 5ml of water and of dimethyl sulphur oxide (DMSO). Each stock solution
was run through a plug of cotton wool previously thus has a concentration of 100,000µg/ml. A 1ml
moistened with chloroform. The content was covered concentrations of 1,000µg/ml, 5,000µg/ml,
with a little anhydrous sodium sulphate, which was 10,000µg/ml and 50,000µg/ml of each extract was
washed in 5ml of chloroform. The filtrate was then prepared, which was used to impregnate 100 filter
placed into 25ml conical flask after which the paper discs. The disc potency would therefore be 10,
chloroform was distilled completely followed by the 50, 100 and 500µg/disc. Another 1ml of 1: 1 ratio
addition of 5ml of neutral alcohol, which was combined forms from the above concentrations of the
evaporated on a boiling water bath. The residue was individual extract were used to impregnate other 100
further heated on a water bath for 15 minutes. The filter paper discs (Baker et al., 1993 and Mukhtar and
residue was dissolved in 2ml of chloroform and 20ml Okafor; 2002).
0.02N sulphuric acid. The content was warmed to Collection and Identification of Test Organisms
remove chloroform. The excess acid was titrated with The organisms tested with various extracts for
0.02N sodium hydroxide using methyl red as indicator, antibacterial activity were pure clinical isolates of
a colour change from pink to yellow was observed. Escherichia coli, Staphylococcus aureus Streptococcus
The available content of the sample was then pneumoniae, Klebsiella pneumoniae, Shigella
calculated using the formula dysenteriae, Salmonella typhi, Streptococcus
Alkaloid content = ( mls taken of 0.02N NaOH x pyogenes. Pseudomonas aeroginosa and Proteus
0.00578/10) = g% w/v (El- Olemy et al., 1994). vulgaris. The bacterial isolates were obtained from the
Quantitative Analysis of Tannins Using Aminu Kano Teaching Hospital, Kano. They were
Iodometric Method subsequently transported to the laboratory in nutrient
From water extract of each specimen 5ml agar (NA) slant culture medial bottles. Confirmatory
was placed into a stoppered conical flask followed by tests were carried on each of the isolates.

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Bioassay extract solid medium and incubated for further 24

Nutrient agar was used to subculture Escherichia coli, hours. The highest dilution that shows no single
Staphylococcus aureus, Klebsiella pneumoniae, bacterial colony was taken as the minimum
Shigella dysenteriae, Salmonella typh, Pseudomonas bactericidal concentration (MBC) as reported by
aeruginosa and Proteus vulgaris, while blood agar (Baker et al., 1993).
was used to subculture Streptococcus pneumoniae Brine Shrimp Lethality Bioassay
and Streptococcus pyogenes for 18 – 24 hours. Eggs of Artema salina (about 50mg) were
Sensitivity tests were done using diffusion method placed into a hatching chamber containing sea water
(Baker et al., 1993 and Mukhtar and Tukur 2000). The (instant sea water can be prepared by dissolving
organisms were inoculated by streaking method in 2.86g of NaCl in 75ml distilled water) and kept under
which the surface of nutrient agar and blood agar a fluorescent bulb for 24hours for the eggs to hatch
plates was streaked with sterile swabs containing each into shrimp larvae. In addition, 20mg of each plant
of the standard inoculum. The filter paper discs fraction was weighed into sterile vials, and dissolved
impregnated with the above concentrations of extracts in 2ml absolute methanol. 500, 50 and 5µl of each
was then placed on the surface of the inoculated these solutions was transferred into empty vials
nutrient agar and blood agar plates with the aid of corresponding to 1000, 100 and 10µg/ml
sterilized pair of forceps. Discs impregnated with concentrations respectively. Each of these dosages for
DMSO only were placed at the centre of some plates each fraction was prepared in triplicate. The vial used
to serve as negative controls while disc impregnated for the control experiment was stained with 1ml
with perfloxacin and recophin were placed at the methanol. All vials containing the dosages and the
centre of some plates to serve as positive controls. A control was left overnight for the methanol to
pre – diffusion time of 30 seconds was allowed for the vaporize, leaving only the plant extract as residue.
extracts to diffuse from the discs into the agar Methanol is a poison to the shrimp larva.
medium before incubation. The plates were inverted To each of the vials containing the plant
and incubated at 370C for 24 hours. The degree of fraction-residue (9-vials per fraction), 2 drops of
sensitivity of the organisms to the extracts was dimethyl sulphoxide (DMSO) were added to re-
determined by measuring diameter of visible zones of dissolve the dosages followed by 4ml of sea- water.
inhibition to the nearest millimeter with respect to Ten (10) larvae of Artema salina were introduced into
each isolate and extract concentration. The following each of the test vials using Pasteur’s pipette. The
keys were adopted: 0mm zone of inhibition – indicates volume of each vial was adjusted to 5ml with sea-
no effects.Less than 8mm zone of inhibition – water. Two drops of DMSO followed by 4ml of sea-
indicates low sensitivity.More than 8mm zone of water was added to the control vial, and 10 larvae of
inhibition – indicates high sensitivity (Mukhtar and Artema salina were introduced. The volume was
Okafor, 2002). adjusted to 5ml with sea- water. Twenty- four hours
after the inoculation, the number of surviving shrimp
Determination of Minimum Inhibitory larvae at each dosage was counted and recorded. LC50
Concentration (MIC) values were determined with 95% confidence intervals
Minimum inhibitory concentration, (MIC) of the by analyzing the data on Kintech AT-compatible
extracts was determined using the tube dilution computer loaded with Finney program (Guerrero and
method (Baker et al., 1993). Dilution of the plants Robledo, 1993; Meyer et al., 1982).
extracts was incorporated in nutrient broth in 1: 1
ratio Initial rough estimates of the MIC values of the RESULTS AND DISCUSSION
plant extracts against the test organisms were Tables 1 and 2 show the physical properties
estimated to determine the range of MIC values. of Acacia nilotica extract fractions recovered from leaf,
Consequently, the following concentrations were stem bark and root of both plants. The solvents used
prepared for each extract, using the dilution formula: include ethanol, chloroform, methanol, petroleum
400, 200, 100, 50, 25, 12.5, 6.25mg/ml. In addition, ether, water and ethyl acetates. Twenty- one extracts
0.1ml of standard suspension of the test organisms were obtained from the partition method of extraction
was added to each tube. The tubes were incubated at used namely ethanol, chloroform, chloroform/water
370c for 24 hours. A tube containing extract and interface, methanol, petroleum ether, water and ethyl
growth medium without inoculum would be included acetates. All the leaf extracts were gummy in texture
to serve as control. The presence of growth (turbid and dark green in colour. The other extracts were
solution) or absence of growth (clear solution) at the either gummy or granular in texture, dark brown or
end of incubation period was recorded. The lowest brown in colour. Most of the extracts were however
concentration of the extract showing no growth was granular in texture.
regarded as the minimal inhibitory concentration
(MIC).

Determination of Minimum Bactericidal

Concentration (MBC)
The minimum bactericidal concentration,
(MBC) was determined by sub culturing the last test
dilution that showed visible growth (turbidity) and all
others in which there was no growth on a fresh

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Table 3 shows the qualitative phytochemical screening of ethanolic extracts of the percentage weight of alkaloids in ethanol leaf extract was 7.8%. Alkaloids were not
A. nilotica, namely the leaf, stem bark and root. The results showed that glycosides detected in the stem bark and root extracts, and were therefore not determined
saponins, resins and flavonoids were not detected in the plant. Alkaloids were detected quantitatively. Tables 5 – 7 show the results of antibacterial activities of various leaf,
only in the leaves of the plant. Sterols and tannins were detected in the leaves, stem stem bark and root extract fractions of A. nilotica. Recophin and perfloxacin were used
bark and root of the plant. This is similar to report of Banso (2009), that ethanolic as positive controls for the sensitivity tests based on their levels of antibacterial activities
extract of Acacia nilotica stem contain active principles e.g. terpenoids, tannins, on the bacterial isolates tested. While filter paper discs soaked in the dimethyl sulphur
alkaloids, etc. Table 4 shows the percentage weight of tannins in water extracts of leaf, oxide (DMSO) were used as negative control against the bacterial isolates.
stem bark and root of Acacia nilotica were 2.64%, 6.09% and 5.26% respectively. While,

Table 1: Weights of Acacia nilotica Extract Fractions Recovered

Plant Ethanol Extract Chloroforn Chloroform/ Petroleum Methanol Extract Water Extract Ethyl Acetate
Parts Extract Water Interface Ether Extract Extract
Extract

WR WR WR WR WR WR WR

Initial Final % Initial Final % Initial Final % Initial Final % Initial Final % Initial Final % Initial Final %
(g) (g) (g) (g) (g) (g) (g) (g) (g) (g) (g) (g) (g)
(g)

LF 100.00 34 34.00 25.00 8.10 32.40 25.00 1.50 6.00 6.00 1.30 21.70 6.00 2.30 38.33 12.40 6.00 48.39 12.40 4.50 36.29
SB 100.00 30.5 30.5 25.00 9.50 38.00 25.00 3.40 13.60 9.00 4.50 50.00 9.00 4.30 47.78 10.80 3.20 29.63 10.80 4.00 37.03
RO 100.00 27.8 27.8 22.00 7.00 31.81 22.00 5.00 22.72 7.00 3.00 42.90 7.00 2.40 34.29 8.60 3.80 44.19 8.60 2.40 27.91
Key: LF = leaf, SB = stem bark, RO = root

Table 2: Texture and Colour of Acacia nilotica Extract Fractions

Plant Parts Ethanol Extract Chloroforn Chloroform/ Petroleum Methanol Extract Water Extract Ethyl Acetate
Extract Water Interface Ether Extract Extract
Extract
Texture Colour Texture Colour Texture Colour Texture Colour Texture Colour Texture Colour Texture Colour
Leaf Gummy Dark Gummy Dark Gummy Dark Gummy Dark Gummy Dark Gummy Dark Gummy Dark
green Green Green Green Green Green Green
Stem Bark Granular Dark Gummy Dark Granular Dark Granular Dark Granular Dark Granular Brown Granular Brown
Brown Brown Brown Brown Brown
Root Granular Dark Granular Dark Granular Dark Granular Dark Granular Dark Granular Brown Granular Brown
Brown Brown Brown Brown Brown

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Table 3: Qualitative Determination of Phytochemicals Present in the Plant

Phytochemicals Acacia nilotica

Leaf Stem Root
Bark
Glycosides - - -
Alkaloids + - -
Saponins - - -
Flavonoids - - -
Sterols + + +
Resins - - -
Tannins + + +

Key: + Phytochemicals detected, - Phytochemicals not detected

Table 4: Tannins and Alkaloids in the Leaf, Stem Bark and Root Extracts Acacia nilotica

Plant Extracts Percentage of Weight Percentage Weight of

Tannins/5ml of Alkaloids/Five
Extract Grammes of Extract
Leaf 2.64 7.8
Stem Bark 6.09 _
Root 5.26 _

Perfloxacin was used on Staphylococcus Banso (2009) reported that ethanol extract
aureus, Klebsiella pneumoniae, Pseudomonas exhibited antimicrobial activity against Streptococcus
aeroginosa, Proteus vulgaris and Salmonella typhi. viridans, Staphylococcus aureus, Escherichia coli,
Recophin was used against Escherischia coli, Bacillus subtilis and Shigella sonnei. Abeer and Sanaa
Streptococcus pneumoniae, Streptococcus pyogenes (2007); reported that A. nilotica ethanol and
and Shigella dysenteriae. chloroform fruit extracts showed varying degrees of
Ethanol and chloroform/water extracts of activity against Gram- negative bacteria (Escherichia
Acacia nilotica stem bark appear to have the highest coli, Proteus vulgaris, Klebsiella pneumoniae and
antibacterial activities on the bacterial isolates tested, Pseudomonas aeruginosa) and Gram-positive bacteria
followed by methanol and ethyl acetate extracts, in (Staphylococcus aureus). Olaleye (2007) reported that
that order. While all the isolates tested were generally methanol extracts of alkaloids and saponins from
sensitive to the various extract fractions, S. Hibiscus sabdariffa had some pharmacologic actions
pneumoniae and S. pyogenes were resistant to them on bacterial isolates like E. coli, K. pneumoniae, S.
at the concentrations used. Chloroform and petroleum aureus etc. Philips (2010) reported that tannins and
ether extracts however, exhibited no antibacterial alkaloids are natural products that have medicinal
activeities on any of the isolates at the concentrations properties. He also said that some remedial values of
used. Similarly, ethanol and chloroform/water tannins include application on burn to heal injury and
interface extracts appears to have the highest cuts to stop bleeding. Moreover, it stop infections on
antibacterial activities on the bacterial isolates tested the skin surface, internally tannins continue to heal
at the concentrations used. This is in line with the the wound. In the case of third degree burns using
report of Abeer and Sanaa (2007), that ethanolic bark strong tannins sources will not only prevent
extract of A. nilotica exhibited higher antibacterial septicemia, but also helps to save life. Alkaloids often
activities than chloroform extract on some bacterial have pharmacological effects and are used as
isolates. Moreover, this may be due to the ability of medications. Examples are cocaine, caffeine, nicotine,
ethanol to extract a wide range of chemical morphine and quinine (Philips, 2010). Therefore,
constituents of the plant while the chloroform might antibacterial activity showed in this present work may
have extracted less number of the ingredients (Abeer be due to tannins and alkaloids. The results of the
and Sanaa, 2007). Furthermore, ethanol extract was study also revealed that the extracts of the stem bark
the first solvent used for extraction of the plant and extracts of the root of the plant should be
constituents before portions of the extracts were preferred for the treatment of bacterial infections as
partitioned between other solvents in this present the stem bark and root extracts had better
study. There is also evidence that some of the extract antibacterial activity on the organisms tested.
fractions that showed no antibacterial activity or weak
antibacterial activity will show profound antibacterial
activities at higher concentrations from the pilot
studies conducted.

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Table 5: Antibacterial Activity of Stem Bark Extracts of Acacia nilotica

Bacterial Diameter of Zones of Inhibition (mm)/ Extracts Concentration (µg/disc)

Isolates
Petroleum Chloroform Ethyl Positive Negative
Ethanol Methanol Chloroform Ether Water Water Acetate Control Control
Interface (µg) (DMSO)

10 50 100 500 10 50 100 500 10 50 100 500 10 50 100 500 10 50 100 500 10 50 100 500 10 50 100 500

Staphylococcus 0 0 0 0 0 0 0 0 0 0 0 10 0 0 0 8 0 0 0 0 0 0 0 0 0 0 0 0 30 (PER) 0
aureus
Escherichia coli 0 0 8 8 0 0 0 0 0 0 0 10 0 0 0 8 0 0 0 0 0 0 0 0 0 0 0 8 20 (REC) 0

Klebsiella 0 0 8 10 0 0 0 0 0 0 0 10 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 30 (PER) 0
pneumoniae
Streptococcus 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 28 (REC) 0
pneumoniae

Streptococcus 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 15 (REC) 0
pyogenes
Pseudomonas 0 0 8 10 0 0 0 0 0 0 0 10 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 8 27 (PER) 0
aeruginosa
Proteus 0 0 10 12 0 0 0 0 0 0 0 8 0 0 0 8 0 0 0 8 0 0 0 0 0 0 8 8 25 (PER) 0
vulgaris
Salmonella 0 0 8 10 0 0 0 0 0 0 0 10 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 8 25 (PER) 0
typhi
Shigella 0 0 0 14 0 0 0 8 0 0 0 0 0 0 0 0 0 0 0 8 0 0 8 10 0 0 8 8 23(REC) 0
dysenteriae

Key: PER = Perfloxacin, REC = Recophin, 0 = No Activity

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Table 6: Antibacterial Activity of Root Extracts of Acacia nilotica

Bacterial Diameter of Zones of Inhibition (mm)/ Extracts Concentration (µg/disc)

Isolates
Petroleum Chloroform Ethyl Positive Negative
Ethanol Methanol Chloroform Ether Water Water Acetate Control Control
Interface (µg) (DMSO)

10 50 100 500 10 50 100 500 10 50 100 500 10 50 100 500 10 50 100 500 10 50 100 500 10 50 100 500

Staphylococcus aureus 0 0 0 0 0 0 0 0 0 0 0 10 0 0 0 8 0 0 0 0 0 0 0 0 0 0 0 0 30 (PER) 0

Escherichia coli 0 0 8 8 0 0 0 0 0 0 0 10 0 0 0 8 0 0 0 0 0 0 0 0 0 0 0 8 20 (REC) 0

Klebsiella 0 0 8 10 0 0 0 0 0 0 0 10 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 30 (PER) 0
pneumoniae
Streptococcus 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 28 (REC) 0
pneumoniae

Streptococcus 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 15 (REC) 0
pyogenes
Pseudomonas 0 0 8 10 0 0 0 0 0 0 0 10 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 8 27 (PER) 0
aeruginosa
Proteus 0 0 10 12 0 0 0 0 0 0 0 8 0 0 0 8 0 0 0 8 0 0 0 0 0 0 8 8 25 (PER) 0
vulgaris
Salmonella 0 0 8 10 0 0 0 0 0 0 0 10 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 8 25 (PER) 0
typhi
Shigella 0 0 0 14 0 0 0 8 0 0 0 0 0 0 0 8 0 0 0 8 0 0 8 10 0 0 8 8 23(REC) 0
dysenteriae

Key: PER = Perfloxacin, REC = Recophin, 0 = No Activity

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Table 7: Antibacterial Activity of Leaf Extracts of Acacia nilotica

Bacterial Diameter of Zones of Inhibition (mm)/ Extracts Concentration (µg/disc)

Isolates
Petroleum Chloroform Ethyl Positive Negative
Ethanol Methanol Chloroform Ether Water Water Acetate Control Control
Interface (µg) (DMSO)

10 50 100 500 10 50 100 500 10 50 100 500 10 50 100 500 10 50 100 500 10 50 100 500 10 50 100 500

Staphylococcus aureus 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 30 (PER) 0

Escherichia coli 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 20 (REC) 0

Klebsiella 0 0 8 12 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 30 (PER) 0
pneumoniae
Streptococcus 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 28 (REC) 0
pneumoniae

Streptococcus 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 15 (REC) 0
pyogenes
Pseudomonas 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 27 (PER) 0
aeruginosa
Proteus 0 0 0 8 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 25 (PER0 0
vulgaris
Salmonella 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 25 (PER) 0
typhi
Shigella 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 23 (REC) 0
dysenteriae

Key: PER = Perfloxacin, REC = Recophin, 0 = No Activity

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The results of the MIC and MBC conducted (tables 8 – who investigated the antibacterial activity of extracts
10) showed that the growth of most of the bacterial of guava, jambolan, pomegranate (high contents of
isolates tested (except S. pneumoniae and S. tannins) and other plants against some antibiotic
pyogenes) were inhibited at concentrations ranging resistant bacteria. In that, study the MIC values of
from 12.5mg/ml to 50mg/ml and that they were killed the plant extracts were between 10mg/ml and
at concentrations ranging from 50mg/ml to 400mg/ml. 400mg/ml.
This agrees with the reports of Gislene et al., (2000),

Table 8: Minimum Inhibitory and Minimum Bactericidal Concentrations (mg/ml) of Acacia nilotica
of Stem Bark Extracts
Ethanol Methanol Chloroform Petroleum Chloroform Water Ethyl
Ether Water Acetate
Bacterial Interface
Isolates
MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC
Staphylococcus
aureus - - 25 200 - - - - 50 200 50 200 50 400
Escherichia coli
- - 50 400 - - - - 25 200 50 400 50 400
Klebsiella
pneumoniae 12.5 100 50 400 - - - - 25 200 - - 50 400
Streptococcus
pneumoniae - - - - - - - - - - - - - -
Streptococcus
pyogenes - - - - - - - - - - - - -
Pseudomonas
aeruginosa 12.5 200 50 400 - - - - 12.5 100 - - 25 200
Proteus
vulgaris 12.5 200 - - - - - 25 200 - - 25 200
-
Salmonella
typhi 12.5 100 - - - - - - 12.5 100 - - - -
Shigella
dysenteriae 12.5 200 - - - - 25 200 50 200 25 200
12.5 200
Key: - = No Activity

Table 9: Minimum Inhibitory and Minimum Bactericidal Concentrations (mg/ml) of Acacia nilotica
Root Extracts
Ethanol Methanol Chloroform Petroleum Chloroform Water Ethyl
Ether Water Acetate
Bacterial Interface
Isolates
MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC
Staphylococcus
aureus 25 200 -
-
Escherichia coli
50 400 25 200 - 50 400
Klebsiella
pneumoniae 12.5 200 25 200 - -
Streptococcus
pneumoniae - - - -
- - -
Streptococcus
pyogenes - - -
- - - -
Pseudomonas
aeruginosa 12.5 100 25 200 - 50 400
-
Proteus
vulgaris 12.5 200 - 50 400 50 400 50 400 50 400
-
Salmonella
typhi 12.5 200 25 200 - 50 400
-
Shigella
dysenteriae 12.5 100 50 100 - - - 50 400 25 200 50 400

Key: - = No Activity

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Table 10: Minimum Inhibitory and Minimum Bactericidal Concentrations (mg/ml) of Acacia nilotica

Bacterial Ethanol Methanol Chloroform Petroleum Chloroform Water Ethyl

Isolates Ether Water Acetate
Interface
MIC MBC MIC MIC MBC MIC MBC

MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC
Staphylococcus
aureus - - - - - - - - -
Escherichia coli
- - - - - - - - -
Klebsiella
Pneumoniae 12.5 200 - - - - - - -
Streptococcus
Pneumoniae - - - - - - - - -
Streptococcus
Pyogenes - - - - - - - - -
Pseudomonas
aeruginosa - - - - - - - - -
Proteus
Vulgaris 50 400 - - - - - - -
Salmonella
Typhi - - - - - - - - -
Shigella
Dysenteriae - - - - - - - - -
Leaf Extracts

Key: - = No Activity

Table 11 shows the toxicity study of ethanolic extracts using brine shrimps lethality test. The LC50 for
leaf and stem back extracts of A. nilotica are 253.27µg/ml, 312.55µg/ml and 123.86 respectively. However, the
recommended cut off point for detecting cytotoxic activity using brine shrimp lethality test is 20µg/ml (Geran et
al; 1972; Massele et al., 1995). It therefore follows that A. nilotia extracts may not be toxic to humans. Brine
shrimps lethality test is a general bioassay, which is indicative of cytotoxicity, antibacterial activities, pesticide
effects and pharmacologic actions of plant extracts (Olaleye, 2007).

Table 11: Brine Shrimp Lethality Assay of Acacia nilotica Ethanolic Extracts
Plant Extracts Acacia nilotica
LC50 (µg/ml)
Leaf 253.27
Stem Bark 312.55
Root 123.86
CONCLUSION were generally sensitive to the extracts, S.
From the results of the phytochemical screening, it pneumoniae and S. pyogenes were resistant to them.
was discovered that the leaves, stem bark and roots The study also revealed that, the stem bark and the
of A. nilotica contain tannins and sterols. Glycosides root extracts of the plant should be preferred for the
saponins, resins and flavonoids were not detected in treatment of bacterial infections. The study also
the plant. Alkaloids were present only in the leaves. showed that A. nilotica extracts may not be toxic to
While S. aureus, E. coli, K. pneumoniae, P. humans. Therefore, the claims of literatures that A.
aeruginosa, P. vulgaris, S. typhi and S. dysenteriae nilotica has antibacterial activities is hereby verified.

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