Current Research Journal of Biological Sciences 2(2): 143-146, 2010
ISSN: 2041-0778
© M axwell Scientific Organization, 2010
Submitted Date: January 26, 2010
Accepted Date: February 08, 2010
Published Date: March 10, 2010
Antibacterial Activity and Phytochemical Screening of Ethanolic Extracts
Obtained from Selected Sudanese Medicinal Plants
1
Hatil Hashim EL-Kamali and 2 Mohamm ed Y agoub E L-amir
Department of Botany, Faculty of Science and Technology,
2
Departm ent of Microbiolog y, Faculty of Medical Laboratories,
Omd urman Islamic University, P.O. Box 382, Omdurman, Sudan
1
Abstract: Selected plants (8 species) having a history of use in Sudanese traditional medicine for the treatment
of infectious diseases were investigated for antibacterial activity in vitro. Phytochemical screening of these
plants was performed for constituents: alkaloids, flavonoids, tannins, anthraquinone s, saponins and volatile oils.
Moisture, ash, crude fibres and soluble ethanol extractive contents have been carried out. The antibacterial
screening of the ethanol extracts of the selected plants was performed by the agar well diffusion method against
clinical isolates Gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis) and Gram-negative
bacteria (Escherichia co li and Pseudom onas aeruginosa). All the eight ethano lic extracts show ed go od ac tivity
against four tested bacteria. The activity of the Cymbopogon schoenathus spp. proximus aerial parts,
Cymbopogon nervatus inflorescence and Cassia oc ciden talis seed extracts w ere more pronounced. T he results
of the antibacterial activity screening support the ethno medical uses of these plants. Further studies on the
isolation and characterization of the Cymbopogon schoenathus spp. proximus and Cymbopogon nervatus,
partially o r totally responsible for th e observed antibacterial pro perties in progress.
Key w ords: Antibacterial activity, ethanolic extracts, medicinal plants, phytochemical screening, Sudan
INTRODUCTION
The use of medicinal plants to treat human diseases
has its roots in p re-historical times. M edicinal plants are
used by 80 % o f the w orld po pulation as the only availab le
medicines especially in developing countries. In Sudan,
the percentage of p eople dependant on med icinal plants
for health care is estimated as over 90%. These plants and
derived products play an imp ortant role in the primary
health care of Sudan (U NID O, 199 6).
The phytochemistry of Cassia species, Am brosia
maritima, Geigeria alata, Cymbopogon nervatus and
Cymbopogon schoenathus spp. proximus have been
received considerab le interest (Modawi et al., 1984; Rizk,
1986; Franz, 1993; Farnsworth, 1995; Ross et al., 1997).
In previo us pu blication we reported the screen ing of a
series of selected medicinal plants from central Sudan
(EL-Kamali et al., 1998).
In this study we examined the phytochemical
screening and antibacterial effects o f ethanolic extracts of
selected medicinal plants in central Sudan.
MATERIALS AND METHODS
Plant ma terial: Eight plants (Table 1) commonly used in
Sudanese traditiona l medicine w ere collected from the
various parts of C entral Sudan, nam ely: Ed-Dam er,
Er-Rahad, Um m R uwaba, E L-O beid and G edare f, in
March - Octo ber (19 99-2001). The taxon omic identity of
the plants was confirmed according to the studies of ELAmin, (1990) and Broun and Massey (1929). Voucher
herbarium specimens have been deposited at the
herbarium of the Department of Botany, Omdurman
Islamic University, Sudan. The plant material was shadedried and then ground in a Wiley grinder with a 2mm
diameter mesh.
Used bacteria: Clinical isolates Gra m-positive b acteria
(Staphylococcus aureus and Bacillus subtilis) and Gramnegative bacteria (Escherichia co li and Pseudomonas
aeruginosa) obtained from Khartoum hospital were grown
in nutrient broth medium an d incubated at 37ºC for 48 h
followed by frequen t sub culturing (every 48 h) to refresh
medium. Bacterial strains w ere maintained on M ullerHinton Agar (composition: Beef, infusion form, 300 g;
casein hydrolysate, 17.5 g; starch, 1.5 g; Agar No.1,
10 g; distilled w ater, 1000 m l; pH 7 .4).
Studied activity: W ell diffusion method (Collins
et al., 1995) for assessing the antibacterial activity was
applied. Wells were made by flamed and then cooled
cork-borer (9 mm) filled (0.01 ml) with the test solution
Corresponding Author: Dr. Hatil Hashim EL-Kamali, Department of Botany, Faculty of Science and Technology,
Omdurman Islamic University, P.O. Box 382, Omdurman, Sudan
143
Curr. Res. J. Biol. Sci., 2(2): 143-146, 2010
Table 1: Traditional uses of selected medicinal plants of central Sudan
Botanic name/ (Family)
Local name/(Locality)
Part uses
A m b ro si a m a ri ti m a
Dam sisa (Ed-D amer)
Leaves
L. (Asteraceae)
Ge igeria a lata
Gud-gad (Er-Rahad)
Ae rial parts
(DC.) Benth. & H ook (Asteraceae)
Cas sia acc identa lis
L. (Caesalpiniaceae)
Soreib (Umm Row aba)
Seeds
Cassia senna L. (Caesalpiniaceae)
San asan a (Ed-D amer)
Pods
Cassia tora L. (Caesalpiniaceae)
Kaw al(Umm Row aba)
Fermented leaves
R h yn ch o si a m i ni m a (L.)DC . Var.
me mn onia (Del.) Cooke (Fabaceae)
Cymbopogon nervatus (Hoch st.)
Chiov. (Poaceae)
Cymbopogon schoenanthus (L.)
Sp reng . spp . proximus (H och st.
E x A.R ic h.) M air e &
Weiller (Poaceae)
Traditional uses
To treat backache, stomach pains, malaria and for gastro-intestinal disturbances.
To treat cough, intestinal complaints and diabetes.
Irg el Dam (Geda ref)
Roo ts
To relieve backache and to treat hypertension.
The aqueous extracts of senna is known to be laxative and stomachic.
Kawal sprinkled over popular food in West Central Sudan as an appetizer and
is also popularly considered to treat diabetes.
To treat diabetes, tonsillitis, backache and diarrhea.
Nal (G edaref)
Inflorescence
To treat kidney pains and for bladder infections.
Ma hareib (Ed -Dam er)
Ae rial parts
To treat constipation, intestinal complaints, carminative, stomachic and as an
appetizer.
Table 2: Antibacterial activity of the ethanolic extracts of selected medicinal plants from central Sudan
Na lidixic
Bac teria
A.m G.a C.o C.sn C.t
R.m C.n
C.s
Ge ntam icin
Tetracycline
acid
Am picillin Strep tom ycin Colistin Nitro furan toin
Staphylococcus
12 16
14 12
12
16
15
20
15
7
0
0
9
0
12
aureus
Bac illus sub tilis
15 22
17 13
17
15
20
24
16
18
13
0
16
0
19
Esch erichia coli
15 13
15 15
14
13
14
14
0
15
13
0
10
13
9
Pseudomonas
15 13
16 0
13
14
16
15
15
0
0
0
10
13
0
aeruginosa
A.m = A m b ro si a m a ri ti m a; G.a = Ge igeria a lata; C.o = Cas sia occ identa lis; C.sn = Cassia senna; C.t = Cassia tora; R.m = R h yn ch o si a m i ni m a var. me mn onia ;
C.n = Cymbopogon nervatus; C.s = Cymbopogon schoenanthus ssp. proximus. Values are inhibition zon e (IZ ) dia me ters/m m; 0 = N o inh ibitio n.; Ethanol alone did not show
any activities
Table 3: Properties and phytochemical screening of selected medicinal plants from central Sudan
Plants
Plant part
Et OH extract
Phytochemical
M o is tu re (% )
% yield
screening
A m b ro si a m a ri ti m a
Leaves
1.5
Alkaloids, Flavonoids,
20.0
Vo latile oil
Ge igeria a lata
Ae rial parts
1.8
Alkaloids, Flavonoids,
20.0
Tan nins, V olatile oil
Cas sia occ identa lis
Seeds
1.3
Anthraquinones,
15.0
Tannins
Cassia senna
Pods
2.5
Anthraquinones,
15.0
Tannins
Cassia tora
Fermented
2.6
Anthraquinones,
25.0
leaves
Tannins
R h yn ch o si a m i ni m a
Roo ts
5.0
Saponins,
5.0
var. me mn onia
Anthraquinones
Cymbopogon nervatus Inflorescence
2.0
Flavonoids, Tannins,
25.0
Vo latile oil
Cymbopogon
Ae rial parts
1.3
Flavonoids, Tannins,
20.0
schoenanthus ssp.
Vo latile oil
proximus
and the standa rd reference antibiotics (Gentamicin-25 mg,
Tetracycline – 25 m g, Nalidixic acid – 30 mg , Am picillin
– 25 mg, Streptomycin – 25 mg, Colistin – 25 mg and
Nitrofurantoin – 25 mg) were used.
T ota l a sh (% )
A cid in so lu ble as h ( %)
S ulf ate d a sh (% )
20.0
10.0
2.5
25.0
10.0
1.5
5.0
1.5
3.5
2.5
1.5
2.1
20.0
7.0
3.3
15.0
4.5
2.4
5.0
7.0
4.4
10.0
8.0
4.8
incubated in the upright position at 37 ºC for 24h. The
plates were checked for bacterial growth after the
incubation period and the resultant zon es of growth
inhibition were accurately measured an d exp ressed in
mm.
Antibacterial testing: The inoculum size of each test
bacteria was adjusted to a 1 ml of bacterial suspension.
Using a flamed and then cooled 9mm. cork-borer, five
cups were cut out of each of the ag ar plates wh ich were
previously inoculated with the test organism. T he cut
discs of agar were removed and decontaminated. By
means of standard pipettes, 0.1ml of each of the different
test suspensions was added to the appropriate cups.
Concentration of 25 mg / ml was tested for each plant
extract. Each test was replicated twice. In each plate one
well at the middle was saturated only with the solvent
(ethan ol) and used as a negative control. After filling the
reservoirs with the appropriate dilutions, the plates w ere
Properties of selected sudanese med icinal plants: The
moisture, ash, acid-insoluble ash, sulfated ash, crude fiber
and alcohol-soluble extractive of selected Sudanese
medicinal plants (A. maritima, C.alata, C. occidentalis,
C. senna, C. tora, R. minima var. memnonia,
Cymbopogon nervatus and C. schoenanthus spp.
proximus) were determined a ccording to stan dard
procedu res (AO AC , 1980; Lo u, 1980).
Phytochemical tests: Phytochemical screening was
carried out acco rding to Farnsworth (19 66).
144
Curr. Res. J. Biol. Sci., 2(2): 143-146, 2010
Extraction procedures: Dried and powdered plant
materials (100 g) were extracted with 80% ethanol (500
ml) in Soxhlet Apparatus for 6 h. The solvent was
removed in vaccum (R otava por R E) to give concentrated
extract and the residue kept in the refrigerator until use.
obtained by hydrodistillation for 4 hours in a C levengertype apparatus (BP, 1980).
RESULTS AND DISCUSSION
In the present work medicinal uses and the yields of
ethan olic extracts of candidate plant sp. were
phytochemically screened and tested for their antibacterial
activity. The results are shown in Tables 1-3. The
ethanolic extracts of all the examined plants showed
stronger grow th inhibition against Gram-positive bacteria.
Study on antibacterial activity of 8 p lants brought to
light some very interesting results. C. nervatus ssp.
proximus, C. nervatus and Cassia oc ciden talis appeared
to be the most active against tested bacteria. For
Cymbopogon species this effect might be due to the high
content of terpenoids w hereas for C. occidentalis the
activity might be related to the presence of tannins.
A glance at Table 2 reveals that various plant
ethan olic extracts may be bro adly d ivided into two
categories (1), those having a relatively higher propensity
to act on Gram-positive bacteria (2), those equally or
nearly equally effective against both types of bacteria.
Ethanolic extracts of G. alata, C. schoenanthus ssp.
proximus showed relatively higher propensity to act on
Gram- positive bacteria. EtOH extracts of C. oc ciden talis,
C. tora, C. senna, R. minima var. memnonia, A. maritima
and C. nervatus showed equal or nearly equal
antibacterial activity both against Gram-p ositive and
Gram-negative bacteria. Compared to the standard,
C. occidentalis, C. nervatus, C. schoenanthus ssp.
proximus ethanolic extracts exhibited a broader spectrum
of antibacterial activity.
EL-Magboul et al. (1985) stated that chloroformic
extracts of C. schoenanthus ssp. proximus was foun d to
be weak activity against S. aureus, B. sub tilis, E. coli and
P. aeruginosa except methano lic extrac t against S.
aureus. However, in the present stud y ethanolic extract of
this plant was highly effective against all tested bacteria.
The obtained results may provide a support to some uses
of the plants in traditional medicine.
Tests:
Alkaloids: The ethanolic extract (30 ml) was evaporated
to dryness in an eva porating dish on w ater bath. Five ml
of 2NHC l were adde d and stirred w hile heating on the
water bath for 10 min., cooled, filtered and the filtrate was
treated with a few drops of Mayer reagent. The samples
were then observed for the presence of turbidity or
precipitation.
Flavonoids: The alcohol extract (75 m l) of plant samp le
were evaporated to dryness on a water bath, cooled and
the residue was defatted by washing several times w ith
petroleum ether. The defatted residue was dissolved in 30
ml 80% ethanol and filtered. The filtrate wa s treated with
a few drops of concentrated HCl and magnesium turnings
(0.5 g). The prese nce of flavonoids was ind icative if a
pink or magenta-red colour developed within 3 min.
Tannins: The alcoholic extract (25 ml) was evaporated to
dryness on a water bath. The residue was extracted
several times with n-hexane and filtered, the insoluble
residue was stirred with 10 ml of hot saline solution, the
mixture was cooled, filtered and the volume of filtrate
was adjusted to 10 ml with more saline solution. To 5 ml
of this solution, few drops of ferric chloride test reagent
were added. An intense green, purple, blue or black
colour was taken as an evidence for the presence of
tannins.
Saponins: One gram of ethanol extract was dissolved in
10 ml of distilled water in a test tube and shaked
vigorously for 1-2 min. The presence of saponins was
indicated by ch aracteristic hon eyco mb fro th at least 1 cm
in height, which persisted for 30 min.
Anthraquinone glycosides: To 1 g of the powdered plant
material, 10 ml of N/2 potassium hydroxide containing 1
ml of 3% hydrogen peroxide solution was added. The
suspension was boiled for 3-5 min. then cooled, filtered
and 5 ml of the filtrate was acidified with 10 drops of
glacial acetic acid. This acidified mixture was extracted
by shaking with 10 ml of benzene. A 5 ml aliquot of the
benzene solution was shaken with 3 ml of 10%
ammonium hydroxide solution an d the two layers were
allowed to separate. A pink to red colouration of the
alkaline layer indicated the presence of anthraquinone.
CONCLUSION
Compared to reference antibiotics, the spectrum of
antibacterial activity of most investgated plants was found
to be clearly superior. The demonstration of broad
spectrum of C. schoenanthus ssp. proximus, C. nervatus
and C. occid entalis may help to discover new chemical
classes of antibiotic substances that could serve as
selective agents for infectious disease chemotherapy and
control. The effect of these plants on more patho genic
organisms, and toxicological investigations and further
purification, however, need to be carried out.
Essential oils: The oil o f A. maritima, G. alata, C.
nervatus and C. schoenanthus spp. proximaus was
145
Curr. Res. J. Biol. Sci., 2(2): 143-146, 2010
Farnsworth, N.R., 1966. Biology and Phytochemical
screening of plants. Pharm. Sci., 55: 225-276.
Farnsworth, N.R., 1995. NAPRALERT Database
Chicago, Unive rsity of Illinois at Chicago.IL
Mrch1995 production.
Franz, G., 1993. The senna drug and its chemistry.
Pharmacology, 47(Suppl 1): 2-6.
Mod awi, B.M ., H.R.Y. M agar, A .M. Satti and
R.J.H. Duprey, 1984. Chemistry of Sudanese Flora:
Cymbopogon nervatus. Nat. Prod. J., 47(1): 167-169.
Rizk, A.M., 1986. The phytochemistry of the flora of
qatar. Published by king print of richmond on behaf
of scientific and applied research center.
Unive rsity of Qatar.
Ross, S.A., K.A. EL-Sayed, M.A. EL-Sohly, M.T.
Hamann, O.R. Abdel-H alim and M.M . Ahmed, 1997.
Phytochemical analysis of Geigeria alata and
Francocuria crispa essen tial oils. Plan ta. M ed.,
63(5): 479-482.
Unido, 1996. Project for the Government of Sudan
prepared by unido industrial utilization of medicinal
and aromatic plants in Sudan (Draft) Sudan.
Lou, 1980. General control methods for vegetable drugs.
W HO /Pharm, 80 (502): 26-32 .
REFERENCES
EL-Amin, H.M ., 1990 . Trees and shrubs of the Sudan.
Publisher: Ithaca Press, Exeter, UK.
AL-Magboul, A .Z ., A . F aro uk , A .K . B ash ir a nd A.M .
Salih, 1985. Antibacterial activity of Sudanese plants
used in folkloric medicine (III). Fitoterapia, 56:
195-200.
AOAC, 1980. Official M ethod s of A nalysis. 11th E dn.,
Washington D.C.
British Pharmacopoea (BP), 1980. Her Majesty s
Stationery O ffice. Lo ndon.
Broun, A.F. and R.E. Massey, 1929. Flora of the Sudan.
Thomas Murby and Co. 1. Fleet Lane, London,
E.C.4.
Collins, C.H ., P.M . Lyne and J.M . Gran ge, 1995.
Microbiological Methods. 7th Edn., Oxford:
Butterworth-Heine-man, pp: 178-179.
EL-Kamali, H.H ., A .H . A hm ed , A .S . M o hame d, A .A .M .
Yahia, I.H. EL-Tayeb and A.A. Ali, 1998.
Antibacterial properties of essential oils from Nige lla
sativa seeds, Cymbopogon citratus leaves and
Pulicaria undu lata aerial parts. Fitoterapia, 69:
77-78.
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