In vitro Antibacterial Activity of Five Sudanese Medicinal Plants Against Salmonella species

21 Pages·2014·0.81 MB·English

University of Africa Journal of Sciences

In vitro Antibacterial Activity of Five Sudanese
Medicinal Plants Against Salmonella species

Rashid. A. Hassouna*1, Amna. S. Khalid2, Hassan. S. Khalid3 and
Muddathir. S. Alhassan3

1 National Centre for Curriculum and Educational Research. Bakht El–Ruda
2 Faculty of Science and Technology. Al Neelain University. Khartoum. Sudan.
3 Medicinal and Aromatic Plants Research Institute. National Centre for Research .
Khartoum. Sudan.

*Corresponding author [email protected]

Keywords: Sudanese plants Tamarindus indica L. S. typhe, S. paratyphe.

Abstract:
Petroleum-ether and methanol extracts of Khaya senegalensis “bark &
leaves”, Senna alexandrina leaves, Solenostemna argel leaves,
Tamarindus indica L. fruits and Trigonella foenum graecum seeds,
were tested for their antimicrobial activity against Salmonella typhi
and S.paratyphi by the cup-plate agar diffusion technique. The
screening revealed that only petroleum ether extract of T.foenum
graecum possess MIC 100 mg / ml while the MIC of the methanolic
extracts against standard S.typhi (ATCC) were 50 mg / ml
K.senegalensis (Bark) , 50mg / ml K.senegalensis (Leaves),, 100 mg /
ml S.alexandrina (Leaves), 100 mg / ml S.argel (Leaves), 25 mg / ml
T.indica L (Fruits ) and 100 mg / ml T.foenum (seeds).

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The positive results obtained by the plants’ extracts were compared
with the in-vitro activity of five drugs of choice used for the treatment
of typhoid, namely Ciprofloxacin, Tetracycline, Chlorophenicol,
Streptomycin and Co - trimexazole. The results indicated that the most
effective extract was the methanolic extract of T.indica L (Aradaib),
which has MIC 25 mg / ml.
Introduction
Infectious diseases are caused by pathogenic microorganisms, such as
bacteria, fungi, viruses, or parasites. Diseases can spread, directly or
indirectly, from one person to another. These diseases are included in
the list of the ten leading causes of death worldwide (WHO 2011).
It has been estimated that in developed countries such as United States,
plant drugs constitute as much as 25% of the total drugs, while in fast
developing countries such as China and India, the contribution is as
much as 80% (Joy et. al 1998)
The use of traditional medicine are expanding globally, it continues to
be used not only for primary health care in developing countries, but
also in countries where conventional medicine predominates in the
national health care (WHO 2000).
Plants have been used to treat different disease since times
immemorial, it seems reasonable to view plant kingdom as striving to
slow down and weaken the continuous attacks of micro-organisms
(bacteria, fungi, viruses and other parasites) as well as Arthropods and
Herbivores (Abdallah 2009). The present study was designed to search
for newer, safer and more potent antimicrobial components which may

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accomplish our present need to treat and prevent the bacterial disease,
typhoid fever, caused by S. typhi and S. paratyphi

Materials and Methods
Collection and Authentication of Plants:
The plants under study namely K. senegalensis bark and leaves , S.
alexandrina leaves , S. argel leaves, T. indica L. fruits and T. foenum
graecum seeds where authenticated by Dr. Haider Abdelgadir at
Medicinal and Aromatic Plants Research Institute (MAPRI) , National
Centre for Research. Voucher Herbarium samples were deposited
there for further references with codes as: K.sen. Mell (2), S.alex. Caes
(2), S.arg. Ascl, (2), T. ind. Legum (2) and T.foe. Gr Papi (2).
Preparation of Plants’ Extracts:
The selected parts of five plants namely K. senegalensis bark , S.
alexandrina leaves , S. argel leaves, T. indica L. fruits and T. foenum
graecum seeds were dried in the shade and then ground to powder
with a mechanical grinder, passing through sieve 40 and stored in a
tight container.
50 g of each dried powdered material were successively macerated in
petroleum ether for three days and methanol for 5 days. Filtered throug
cotton wool. The total filtrate was concentrated by dryness, to give
methanol residue.
This methanol extract was then suspended in water and successively
partitioned with ethyl acetate (EtOAc) and Chloroform (CHCl ) to
3

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fraction, along with the residue present in the aqueous phase. Solvents
were evaporated under reduced pressure using rotary evaporator
(Sukhdev et al 2008). Extracts were placed in clean tubes till complete
dryness and stored in a refrigerator till use.
Preliminary antibacterial studies were conducted using dried plants‘
extracts on Rv. type of Salmonella typhe (ATCC19430) and
Salmonella paratyphyphe -A (ATCC 9150 / SARb42), supplied by
National Public Health Laboratory, Khartoum, Sudan. (N.P.H.L.K.S.).
As well as clinical isolates of S. typhi and S. paratyphe issued by
(Department of Microbiology, Faculty of Medical Laboratory
Sciences, Al Neelain University , Khartoum, Sudan) at concentrations
of 100 µl by applying the dilution method. The significant antibacterial
activity was determined by measuring the diameter of zone of
inhibition (Kavanagh 1972) and compared with those determined by
the standard drugs (Tables 1 & 6).
Preparation of suspension:
One ml aliquots of 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, washed off with
sterile normal saline, and finally suspended in 100 ml of normal saline
to produce a suspension containing about (108 – 109) colony forming
units per ml. (Miles and Misra 1938). The suspension was stored in
the refrigerator at 4◦ C till use.
Screening the antibacterial activity of the extracts:
Screening tests were performed by the cup – plate – agar diffusion
method ( Kavanagh 1972) . Three(3) ml of each of the 4 bacterial

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stock suspensions were thoroughly mixed with 300 ml of sterile
melted nutrient agar which was maintained at 45◦ C. 20 ml of each of
the inoculated nutrient agar were distributed into 6 sterile Petri-dishes.
The agar was left to set and in each of these plates, which were divided
into two halves, two cups in each half (10 mm in diameter) were cut
using sterile cork borer (No. 40,) each one of the 24 cups was
designed for one of the 12 extracts.
The agar discs were removed, alternate cups were filed with 0.1 ml of
each sample extract using adjustable volume automatic micro-titre
pipette, and allowed to diffuse at room temperature for 2 hours. The
plates were then incubated in the upright position at 37◦C for 18 hours.
After incubation the diameters of the inhibition zones were measured
(Table 1).
MIC of the active extracts:
The MIC of the active extracts was done by cup-plate-agar diffusion
method. The plants’ active extracts were prepared in series of
decreasing concentrations in the following order, 20%, 10%, 5%, 2.5%
and 1.25% (i.e. g per 100ml) (Table 3).
Another replicate experiment was carried out using the active extract
of T. indica concentrations, 10 %, 5 %, 2.5 %, 1.25 % and 0.625 % (i.e
g per 100 ml). After incubation, the diameters of the inhibition zones
were measured (Table 4).
The Sensitivity of Antibiotics:
Five antibacterial reference drugs (Ciprofloxacin, Tetracycline,
Chlormophenicol, Streptomycin and Co- trimexazole ), were prepared
in suspensions of 5 different concentrations (100 %, 40 %, 20 %, 10 %

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and 5 % (i.e g per100 ml), and by cup-diffusion method (Kavanagh,
1972), their activity against the four test organisms had been evaluated
(Table 6).
The clinical isolates were obtained from the faecal specimens. They
were purified by streaking on plates containing the appropriate
selective and differential culture media.
For the primary isolation of Salmonella and Shigella, Xylose Lysine
Deoxycholate agar medium (XLDA), Deoxycholate Citrate agar
(DCA) and Hektoen Enteric Agar (HEA) were used because they are
media of high selectivity that inhibit the growth of Gram-positive and
commensal organisms and differentiate between Lactose fermenters
and non-Lactose fermenters. (Cheesbrough 2006).
Differentiation between Salmonella and Shigellae had been done by
using Kligler’s ion agar medium (KIA) and Triple sugar ion agar
medium (TSIA), because they are helpful media in differentiating
Salmonellae and Shigellae. (Cheesbrough 2006).
Statistical analysis
All data were expressed as mean ± standard Error mean. Analysis of
variance was performed by ANOVA using the SPSS software
(version 10.0 for windows, 1999). Significant differences between
means were determined by Least Significant Difference (L.S.D). A
significant difference was considered at the level of P < 0.05. Tables (2
& 4 & 7).

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Results
The screening results showed that the inhibition zones of the
methanolic extracts against standard S.typhi (ATCC) were 15.0 mm K
.senegalensis (Bark) , 12.5 mm K. senegalensis (Leaves),, 12.0 mm
Senna alexandrina (Leaves), 16.0 mm S. argel (Leaves), 17.5 mm
T. indica L. (Fruits ) and 15.0 mm T.foenum. graecum (seeds) . While
The the inhibition zone of petroleum ether extract for T. foenum.
graecum (seeds) was 12.0 mm, the other petroleum ether plants'
extracts had weak activity (table 1)..
The inhibition zones of antibiotics using concentration 20 % against
S.typhe (ATCC) were 35.5 mm for Ciprofloxacin, 13.5 mm for
Tetracycline, 12.5 mm for Chloromphenicol ,15.5 mm for
Streptomycin and 00.0 mm for Co-trimexazole, (Table 6)..
Discussion
The preliminary screening generally showed that methanolic extracts
are more active than the petroleum ether extracts i.e. the activity is
displayed by the polar components of the plants towards the gram
negative bacteria namely S. typhe and S.paratyphe except the extract
of T. foenum graecum . As shown by the statistical analysis which
revealed a significant differences between the methanolic values and
petroleum ether values (table 1). Also the preliminary screening
revealed that the plant T. indica L. contains the most active
components compared to the other plants investigated, which confined
by the statistical analysis that showed a significant different of T.

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indical L. methanolic value with respect to the other five values (table
2). The higher antibacterial activity of methanol as an extracting
solvent is substantiated by the work of Abd Elhady et al (1994),
Muthu et al (2005), Cyrus et al (2008), Abdulahi et al (2010),
Mohamed et al (2010), and Shital et al (2010), who studied the
antibacterial activity of methanolic extracts of different plants beside
the plant T. indica L. against more than 20 species of bacteria and
showed that the methanolic extracts displayed broad spectrum activity
against gram negative bacteria.
The fact that T. indica L contains 32 fatty carboxylic acids saturated
and un saturated, β sitosterol and cycloatranol (Kanzada 2008), as an
antibacterial bio- active agents compared with the results shown in this
study, agrees with Mathu et, al (2005) , who found that methanolic
extract of T. indica L has anti – B. pseudomallei inhibitory potentials
under in vitro conditions, Abdel Gadier et, al (2007), who revealed
that methanolic extract of T. indica L has antibacterial activity on S.
typhi,
Abukakar et, al (2008), who stated that Phytochemical screening of T.
indica L indicated ten different compounds and the aqueous extract
has a positive activity against S. typhi, and Shital et, al (2010), who
found that the methanolic extract of T. indica L inhibited the growth of
S. typhimurium (NCIN – 2501).
Statistical analysis showed that T. indica L methanolic extract was
more active than Streptomycin, Tetracycline and Chloromphenicol
with respect of the significant difference of its value 15.0±0.20a

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compared to there values 12.25±0.25b, 0.0b, and 0.0b respectively
table (7). So we can suggest that T. indica L can be used in treatment
of typhoid in combination with the conventional antibiotics e.g.
Ciprofloxacin.

Acknowledgement
We are grateful to Medicinal & Aromatic Plants Research Institute,

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