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Advances in Microbiology, 2016, 6, 691-698
Published Online August 2016 in SciRes. http://www.scirp.org/journal/aim
http://dx.doi.org/10.4236/aim.2016.69068
How to cite this paper: Abdllha, H.B., Mohamed, A.I., Almoniem, K.A., Adam, N.I., Alhaadi, W., Elshikh, A.A., Ali, A.J., Ma-
kuar, I.G., Elnazeer, A.M., Elrofaei, N.A., Abdoelftah, S.F. and Hemidan, M.N. (2016) Evolution of Antimicrobial, Antioxidant
Potentials and Phytochemical Studies of Three Solvent Extracts of Five Species from Acacia Used in Sudanese Ethnomedi-
cine. Advances in Microbiology, 6, 691-698. http://dx.doi.org/10.4236/aim.2016.69068
Evolution of Antimicrobial, Antioxidant
Potentials and Phytochemical Studies of
Three Solvent Extracts of Five Species from
Acacia Used in Sudanese Ethnomedicine
Hajir B. Abdllha1, Alaa I. Mohamed1, Khansa A. Almoniem1, Naga I. Adam1, Wdeea Alhaadi1,
Ahmed A. Elshikh2, Ahmed J. Ali1, Ismail G. Makuar2, Anas M. Elnazeer2, Nagat A. Elrofaei3,
Samir F. Abdoelftah4, Monier N. Hemidan2
1Microbiology Department, Faculty of Medicinal Laboratory Science, Omdurman Islamic University, Omdurman,
Sudan
2Microbiology Department, Faculty of Pure and Applied Sciences, International University of Africa, Khartoum,
Sudan
3Biotechnology Department, Faculty of Science and Technology, Omdurman Islamic University, Omdurman,
Sudan
4Zology Department, Faculty of Science and Technology, Omdurman Islamic University, Omdurman, Sudan
Received 28 March 2016; accepted 8 August 2016; published 11 August 2016
Copyright © 2016 by authors and Scientific Research Publishing Inc.
This work is licensed under the Creative Commons Attribution International License (CC BY).
http://creativecommons.org/licenses/by/4.0/
Abstract
The ethanol, chloroform and acetone extracts of five species from Acacia (Acacia albidia stems,
Acacia mellifera aerial parts, Acacia nubica aerial parts, Acacia seyal var. seyal stems and Acacia
tortilis aerial parts) were investigated for their antimicrobial activity against two standard bac-
terial strains of Gram +ve bacteria (Staphylococcus aureus (ATCC 25923)), Gram −ve bacteria
(Pseudomonas aeruginosa (ATCC 27853)) and standard fungi Candida albicans (ATCC 90028) using
the agar-plate well diffusion method. The chloroform extract was inactive compared to ethanol
and acetone extracts. But ethanol extracts showed the maximum antimicrobial activity against the
test organism. Amongst the plant species screened, ethanol extract of Acacia seyal stems showed
maximum inhibitory activity (38 mm) and (37 mm) against Staphylococcus aureus and Candida al-
bicans, respectively. The ethanol, chloroform and acetone extracts of Acacia mellifera (aerial parts)
did not show any activity against the test organisms. Cholorophorm and acetone extracts via DPPH,
the radical scavenging activities were found to be 91 ± 0.03, 88 ± 0.01 and 85 ± 0.04, respectively.
The results of phytochemical screening showed that all extracts of studied plant contain flavono-
ids, saponins, terpenoids, steroids, alkaloids, phenols and tannins.
H. B. Abdllha et al.
692
Keywords
Antimicrobial, Antioxidant, Phytochemical, Sudanese Ethnomedicine Acacia Species
1. Introduction
According to World Health Organization medicinal plants would be the best source to obtain a variety of drugs.
About 80% of individuals from developed countries use traditional medicine, which has compounds derived
from medicinal plants. Therefore, such plants should be investigated to better understand their properties, safety
and efficiency. For a long period of time, plants have been a valuable source of natural products for maintaining
human health, especially in the last decade, with more intensive studies for natural therapies [1].
Some antibiotics have become almost obsolete because of drug resistance and consequently new drugs must
be sought for. Herbal treatment is one possible way to treat diseases caused by multidrug resistant bacteria. The
use of plant extracts and phytochemicals, with known antibacterial properties, may be of immense importance in
therapeutic treatments. In the past few years, a number of studies have been conducted in different countries to
prove such efficiency [2].
Acacia is the most significant genus of family: Leguminosae, first of all described by Linnaeus in 1773. It is
estimated that there are roughly 1380 species of Acacia worldwide, about two-thirds of them native to Australia
and rest of spread around tropical and subtropical regions of the world [3] [4]. The aim of this study is to inves-
tigate the antimicrobial, antioxidant potentials and phytochemical studies of five species from Acacia to ascer-
tain the rationale for its use in traditional medicine.
2. Material and Method
2.1. Plant Material
The plants used in this study were collected from Khartoum university-faculty of agriculture, Acacia seyal was
collected from local market. This plant was identified in the Botany department, Faculty of science and tech-
nology, Omdurman Islamic University by Prof. Hatil Hashim Al-Kamali and by comparison with herbarium of
the department. The plant was spread and dried in the shade for three weeks and then pulverized with mechani-
cal grinder.
2.2. Preparation of Plant Extracts
Each of the coarsely powdered plant material (200 g) was exhaustively extracted for 24 hours with 1 liter of
ethanol in conical flasks. The ethanolic extract was filtered and evaporated. The extracted plant material was
then air-dried, repacked in conical flask and exhaustively extracted with 800 ml of chloroform extract was fil-
tered and evaporated. The extracted plant material was then air-dried and repacked in conical flask and exhaus-
tively extracted with 600 ml of acetone. Each residue was weighed and determined by sensitive balance.
2.3. Preparation of Bacterial Suspensions
One ml aliquots of a 24 hours broth culture of the test organisms were aseptically distributed onto nutrient agar
slopes and incubated at 37˚C for 24 hours. The bacterial growth was harvested and washed off with 100 ml ste-
rile normal saline, to produce a suspension containing about 108 - 109 C.F.U./ml. The suspension was stored in a
refrigerator at 4˚C till used.
The average number of viable organisms per ml of the stock suspension was determined by means of the sur-
face viable counting technique [5]. Serial dilutions of the stock suspension were made in sterile normal saline
solution and 0.02 ml volumes on drop of the appropriate dilutions were transferred by micro pipette onto the
surface of dried nutrient agar plates. The plates were allowed to stand for two hours at room temperature for the
drops to dry and then incubated at 37˚C for 24 hours. After incubation, the number of developed colonies in
each drop was counted. The average number of colonies per drop (0.02 ml) was multiplied by 50 and by the di-
lution factor to give the viable count of the stock suspension, expressed as the number of colony forming units
H. B. Abdllha et al.
693
per ml suspension (C.F.U./ml).
Each time a fresh stock suspension was prepared. All the above experimental conditions were maintained
constant so that suspensions with very close viable counts would be obtained.
2.4. Preparation of Fungal Suspension
The fungal cultures was maintained on Sabouraud dextrose agar, incubated at 25˚C for 4 days. The fungal
growth was harvested and washed with sterile normal saline and finally suspension in 100 ml of sterile normal
saline, and the suspension was stored in the refrigerator until used.
2.5. In Vitro Testing of Extracts for Antimicrobial Activity
2.5.1. Testing for Antibacterial Activity
The cup-plate agar diffusion method was adopted according to [6] with some minor modifications to assess the
antibacterial activity of the prepared extracts.
One ml of the standardized bacterial stock suspension 108 - 109 C.F.U/ml were thoroughly mixed with 100 ml
of sterile molten nutrient agar which was maintained at 45˚C. 20 ml aliquots of the inoculated nutrient agar were
distributed into sterile Petri-dishes.
The agar were left to dry and in each of these plates 4 cups (10 mm in diameter) were cut using a sterile cork
borer (No. 4) and agar discs were removed.
Alternate cups were filled with 0.1 ml sample of each extracts using automatic Microlitre-pipette, and allowed
to diffuse at room temperature for two hours. The plates were then incubated in the upright position at 37˚C for
18 hours.
Two replicates were carried out for each extracts against each of the test organisms. Simultaneously addition
of the respective solvents instead of extracts was carried out as controls. After incubation, the diameters of the
resultants and growth inhibition zones were measured, averaged and the mean values were tabulated.
2.5.2. Testing for Antifungal Activity
The same method as for bacteria was adopted. Instead of nutrient agar, Sabouraud dextrose agar was used. The
inoculated medium was incubated at 25˚C for two days for the Candida albicans.
2.6. Antioxidant Activity
The antioxidant activity has been carried out using two assays:
2.6.1. DPPH Free Radical Scavenging Activity
The DPPH radical scavenging was determined according to the method of [7], with some modification. The test
samples were allowed to react with 2.2 di(4-tretoctylphenyl)-1-picryl-hydrazyl stable free radical (DPPH) for
half an hour at 37˚C in 96-wells plate. The concentration of DPPH was kept at (300 μM). The test sample was
dissolved in DMSO while DPPH was prepared in ethanol. After incubation decrease in absorbance was meas-
ured at 517 nm using multiplate reader spectrophotometer. Percentage of radical scavenging activity of the sam-
ple was determined in comparison with a DMSO treated control. All tests were conducted triplicate.
2.6.2. Iron Chelating Activity Assay
The iron chelating ability was determined according to the modified method of [8], in which the Fe2+ was moni-
tored by measuring the formation of ferrous ion-ferrozine complex. The experiment was carried out in 96
micrometer plate. The plant extract was mixed with FeSO4. And the reaction was initiated by adding 5 mM fer-
rozine. The mixture was shaken, left at 25˚C for 10 min, and finally the absorbance was measured at 562 nm,
using multi-plate spectrophotometer. EDTA was used as positive control, and DMSO as control. All tests were
done in triplicate.
2.7. Phytochemical Screening of Extracts
The ethanol, chloroform and acetone extracts were used for the detection of the following tests according to
standard methods. Described [9] [10].
H. B. Abdllha et al.
694
2.7.1. Phenols
Two ml of extract was added to one ml of distilled water and warmed at 45˚C - 50˚C. Then 2 ml of 3% FeCl3
was added. Appearance of green or blue color indicate the presence of phenols.
2.7.2. Flavonoids
One ml of extract was added to one ml of 10% KOH. It was gently shaken. Appearance of yellow color indi-
cated the presence of flavonoids.
2.7.3. Tannins
One ml of extract was added to one ml of 3% FeCl3. A greenish black precipitate indicated the presence of tan-
nins.
2.7.4. Alkaloids
One ml of Dragendorff reagent was added to 1 ml of filtrate. The formation of cloudy orange was observed.
2.7.5. Terpenoids and Steroids
Five ml of extract was mixed in two ml of chloroform. Then 3 ml concentrated sulphuric acid was carefully
added to observe a reddish brown coloration between upper and lower layer was observed.
2.7.6. Saponins
Approximately 0.2 ml of extract was mixed with 5 ml of distilled water. Mixture was shaken vigorously for 5
min. Persistence of foams indicated the presence of saponins.
3. Results and Discussion
The mean of diameters of the growth inhibition zone produced by extracts of five species of Acacia on the stan-
dard strains is shown in Table 1. The results of antimicrobial tests were interpreted as active (>18 mm), mod-
erately active (14 - 18 mm) and inactive (<14 mm) [11] [12]. The ethanol extract of A. seyal stems showed the
highest inhibitory activity (38 mm) and (37 mm) against Staphylococcus aureus and Candida albicans respec-
tively and had the highest antioxidant activity with 91 ± 0.03 using DPPH assay, but other extracts had less ac-
tivity (Table 2), followed by acetone extract which showed the good activity (30 mm) against Pseudomonas
aeruginosa and Staphylococcus aureus. These might be due to the presence of alkaloids, flavonoids, steroids
and trepenoids in ethanol extracts, and alkaloids, flavonoids, saponins, tannins, steroids, trepenoids and phenols
in acetone extracts (Table 3).
Table 1. Antimicrobial activities of Acacia species against standard organisms.
Plants Extracts Concentration (%)
Test organism
MIDZ (mm)
P.a. S.a. C.a.
A. seyal (stems)
Ethanol
10%
5%
2.5%
1.25
Control
35
30
29
25
-
38
30
28
20
-
37
35
30
24
-
Chloroform
10%
5%
2.5%
1.25
Control
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
Acetone
10%
5%
2.5%
1.25%
Control
30
28
22
19
-
30
29
23
20
-
29
28
25
20
-
H. B. Abdllha et al.
695
Continued
A. albida (aerial parts)
Ethanol
10%
5%
2.5%
1.25%
Control
20
18
13
-
-
-
-
-
-
-
20
19
17
15
-
Chloroform
10%
5%
2.5%
1.25%
Control
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
Acetone
10%
5%
2.5%
1.25%
Control
20
18
15
-
-
-
-
-
-
-
-
-
-
-
-
A. tortilis (aerial parts)
Ethanol
10%
5%
2.5%
1.25%
Control
20
17
-
-
-
20
19
18
-
-
23
20
17
-
-
Chloroform
10%
5%
2.5%
1.25%
Control
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
Acetone
10%
5%
2.5%
1.25%
Control
23
20
15
-
-
18
-
-
-
-
25
23
19
9
-
A. nubica (aerial parts)
Ethanol
10%
5%
2.5%
1.25%
Control
31
28
22
19
-
25
22
19
-
-
27
23
20
16
-
Chloroform
10%
5%
2.5%
1.25%
Control
25
23
20
-
-
-
-
-
-
-
-
-
-
-
-
Acetone
10%
5%
2.5%
1.25%
Control
29
25
22
15
-
23
20
19
-
-
25
20
19
18
-
A. mellifera (aerial parts)
Ethanol
10%
5%
2.5%
1.25%
Control
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
Chloroform
10%
5%
2.5%
1.25%
Control
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
Acetone
10%
5%
2.5%
1.25%
Control
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
MDIZ = Mean Diameter of Inhibition Zone = average of two replicates in millimeters, Tested conc of extract (0.1 ml/well). S.a. = Staphylococcus
aurous, P.s. = Pseudomonas aeruginosa, C.a. = Candida albicans.
H. B. Abdllha et al.
696
Table 2. Antioxidant activity of ethanol, chloroform and acetone extracts of Acacia species.
Plants species Solvents Results
A. seyal (stems)
Ethanol 91 ± 0.03
Chloroform 88 ± 0.01
Acetone 85 ± 0.04
A. nubica (aerial parts)
Ethanol 83 ± 0.02
Chloroform 28 ± 0.20
Acetone 79 ± 0.03
A. tortilis (aerial parts)
Ethanol 83 ± 0.02
Chloroform 42 ± 0.7
Acetone 82 ± 0.04
A. albida (aerial parts)
Ethanol 89 ± 0.05
Chloroform 12 ± 0.04
Acetone 81 ± 0.04
A. mellifera (aerial parts)
Ethanol 37 ± 0.08
Chloroform 9 ± 0.10
Acetone 49 ± 0.01
Table 3. Phytochemical studies of ethanol, chloroform and acetone extracts of Acacia species.
Plants species Solvents Class of chemical compounds
Alkaloids Flavonoids Saponins Tannins Steroids Terpenoids Phenols
A. albida
Ethanol − + − + + + +
Chloroform + − + − + + +
Acetone + + − − + + −
A. mellifera
Ethanol + − − − + + −
Chloroform + − − − + + −
Acetone + + + − + + −
A. nubica
Ethanol + + + + + + +
Chloroform + − + − + + −
Acetone + + + + + + +
A. seyal Ethanol + + − − + + −
Acetone + + + + + + +
A. tortilis Ethanol + + − − + + −
Chloroform + + + − + + −
+ve = Present, −ve = Absent.
H. B. Abdllha et al.
697
[13]-[16] found that some compounds isolated from different plants such as flavonoids, tannin, quinines and
coumarins had been extracted and found to be of inhibitory effect on numerous bacteria strains as well as fungi
and yeast. Alkaloids had been demonstrates to have high antibacterial activity against Gram-negative and Gram
positive bacteria [17]. Among the extracts tested acetone extract showed antimicrobial activity against at least
one strains, while chloroform extract was not active against any of the test organisms. This means that the plant
part used and the type of extraction might have resulted in nil activity in the test performed in this study. Some
of these plants were also screened previously against other test strains [18] [19] and were found to exhibit simi-
lar results to those obtained in this study with varying degree in potency. The differences in potency may be due
to locality of the plant species, time of collection of the plant sample, storage condition different sensitivity of
the test strains and method of extraction [20].
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