In vitro Antibacterial Properties of Total Alkaloids Extract from Mitragyna Inermis (Willd.) O. Kuntze, a West African Traditional Medicinal Plant
Etienne-Francois O. Akomo,
Louis Clement Obame,
Alfred S. Traore
The antimicrobial activity of the total alkaloids from
the leaves of Mitragyna inermis (Willd.) O. Kuntze (Rubiaceae)
From Burkina Faso was evaluated using disc diffusion essay and broth microdilution
assay. The extract was used against ten (10) reference bacterial strains
and three (3) clinical isolates including Gram(+) and Gram(-) strains.
The alkaloids showed moderate activity against mcroorganisms tested. The
highest Diameter of Inhibition Zone (DIZ) was obtained with S. aureus
ATCC9244 (23 ± 1 mm). The lowest Minimum Inhibitory Concentration
(MIC) obtained was 0.625 mg mL-1 recorded with three (3) Gram(+)
bacteria (S. aureus ATCC 25293, S. aureus (clinical isolate)
and S. carmorum LMG 13567) and one Gram(-) strain (P.
mirabilis CIP104588. Results showed that Gram(+) bacteria are more
sensitive to alkaloids from M. inermis than Gram(-) bacteria. This
study confirmed the use of the plant in traditional medicine against some
to cite this article:
C. Zongo, Etienne-Francois O. Akomo, A. Savadogo, Louis Clement Obame, Jean Koudou and Alfred S. Traore, 2009. In vitro Antibacterial Properties of Total Alkaloids Extract from Mitragyna Inermis (Willd.) O. Kuntze, a West African Traditional Medicinal Plant. Asian Journal of Plant Sciences, 8: 172-177.
Indigenous medicinal plants are often the only means for the treatment of several
infections in Africa (Fennell et al., 2004; Taylor
et al., 2001). According to WHO (2003) 80%
of the population use traditional medicine for their primary health care.
Mitragyna inermis (Willd.) O. Kuntze (Rubiaceae) is a medicinal plant
widely known and used in folk medicine in West Africa. This plant is a shrub
growing on low alluvial plains and swampy savannah of many countries of West
Africa (Pillay, 1964; Shellard and
Wade, 1969; Kerharo Adam, 1974).
In traditional medicine, M. inermis is used to treat several diseases
such as fever, headache, diarrhoea, dysentery, cholera, malaria and other diseases.
(Nacoulma/Ouèdraogo, 1996; Ouedraogo
et al., 2007). According to the traditional knowledge, many scientific
studies have been carried out to confirm the activities the plant is assumed
to exert in vivo.
The in vitro antiplasmodial activity of the plant has been demonstrated
by Traore-Keita et al. (2000), Mustofa
et al. (2000), Kohler et al. (2002),
Azas et al. (2002) and Fiot
et al. (2005). The cardiovascular properties of the aqueous extract
have been also demonstrated (Ouedraogo et al., 2004).
Toxicity studies including cytotoxicity, genotoxicity, acute and chronic toxicity
have been carried out (Azas et al., 2002; Toure
et al., 1996; Traore et al., 2000;
Monjanel-Mouterde et al., 2006). Several compounds
including alkaloids and non-alkaloids have been purified from M. inermis
(Shellard and Sarpong, 1969, 1970;
Shellard et al., 1971; Cheng
et al., 2002; Fiot et al., 2005). The
chloroformic extract from the plant showed antibacterial activity against
Escherichia coli, Staphylococcus aureus and Streptococcus sp.
(Umeh et al., 2005). Some studies showed that
the pharmacological and biological properties were mostly due to alkaloids (Traore-Keita
et al., 2000). More recently, antimicrobial activity of different
extracts from M. inermis against bacteria and fungi has been demonstrated
and chemical analysis revealed the presence of alkaloids in the active extracts
(Asase et al., 2008).
However, there is a few scientific data on the antibacterial properties
of the plant. In the present study, in vitro antibacterial activity
of the total alkaloids of M. inermis was evaluated against clinical
and reference Gram-positive and Gram-negative bacteria.
MATERIALS AND METHODS
Plant material: The leaves of Mitragyna inermis were collected
in December 2007, at 45 km from Ouagadougou in Burkina Faso. The samples
were carefully dried in the laboratory under continuous ventilation, away
from sun light and dust. The leaves were then crushed to fine powder with
a mechanical crusher and the powder was kept in plastic bags and stored
away from light and moisture until required.
The plant was taxonomically authenticated at the Laboratory of Plant
Biology and Ecology of the University of Ouagadougou where a voucher specimen
Total alkaloids extraction: The dried powder of the leaves was
moistened with ammonia (28%) and extracted with chloroform at room temperature
for a total period of 24 h. The chloroform extract was then filtered and
a first liquid-liquid partition of the alkaloids was made with hydrochloric
acid (HCl) (5%). The aqueous layer from the first partition was made alkaline
again (pH 9-10) with ammonia and a second partition with chloroform was
made. Finally, chloroform was totally evaporated from the organic phase
to give a total alkaloids powder.
Microbial strains: The total alkaloids extract of leaves of M. inermis
was tested against a panel of microorganisms, including reference strains (Bacillus
cereus LMG 13569, Enterococcus faecalis CIP 103907, Escherichia
coli CIP 105182, Listeria innocua LMG 1135668, Salmonella enterica
CIP 105150, Shigella dysenteria CIP 5451, Staphylococcus aureus
ATCC 9244, Proteus mirabilis CIP 104588 Staphylococcus aureus
ATCC 25293 and Staphylococcus camorum LMG 13567) and Clinical strains
(Pseudomonas aeruginosa, Staphylococcus aureus and Streptococcus
pyogenes). These clinical strains were isolated at the Laboratoire de Biologie
Médicale Saint Camille in Ouagadougou. The identification of these clinical
strains was based on their biochemical profiles as recommended by the manual
Bactériologie Médicale (Le-Minor and Veron, 1984).
Antibiotics and media: Commercially available antibiotics discs,
ampicillin 33 μg and Tetracyclin 30 μg were purchased from Beckton
Dickinson and used as references for the test. All media used were from
Fluka BioChemica. Chloroform was analytical grade.
Disc diffusion method: The in vitro antibacterial activity of total
alkaloids extract of M. inermis was studied by the paper disc diffusion
method (Bauer et al., 1966; Pelczar
et al., 1993; Ayandele and Adebiyi, 2007)
using Mueller-Hinton agar plates. Briefly, to activate the microorganisms, they
were grown on nutrient broth at 37 °C for 18 h. The other night cultures
were suspended in saline solution (0.9% NaCl) and adjusted to a turbidity of
0.5 Mac Farland standards corresponding to 108 cfu mL-1.
Each bacterial suspension was used to inoculate 90 mm diameter Petri plates
with a sterile non toxic cotton swab. Six millimeter paper discs (Whatman No.
3) soaked with 10 μL of the total alkaloid extract dilution (100 mg mL-1
in dimethylsulphoxyde (DMSO) were placed on the agar. The quantity of extract
was then 1000 μg per disc. Paper discs soaked in DMSO without extract were
used as negative control and DMSO didnt show inhibition effects to mcroorganisms
growth. The plates were incubated aerobically at 37 °C for 18 to 24 h. All
tests were performed in duplicate and the antibacterial activity was expressed
as the mean of Diameters of Inhibition Zone (DIZ) produced.
Broth microdilution assay: A microdilution broth susceptibility assay
was used, as recommended by the National Committee for Clinical
Laboratory Standards (2006) for the determination of the Minimum Inhibitory
Concentration (MIC) and the minimum bactericidal concentration (MBC). Briefly,
the total alkaloids extract was properly prepared, sterilized by filtration
through 0.22 μm sterilizing Millipore express filter and transferred in
sterile 96 well-plates previously filed with sterile nutrient broth to obtain
a twofold serial dilutions ranging from 19,5312 to 2500 μg mL-1.
Then plates were inoculated with microbial suspensions diluted from the same
0.5 Mac Farland standards to have 5x105 cfu mL-1 in each
well. A number of wells were reserved in each plate for sterility control (no
inoculum added), inoculum viability (no extract added) and the DMSO inhibitory
effect. The final volumes in wells were 200 μL. After 24 h aerobically
incubation at 37 °C, bacterial growth was indicated by the presence of turbidity
and a pellet on the well bottom. MIC was defined as the lowest extract concentration
demonstrating no visible growth in the broth and the MBC was defined as the
lowest concentration of the extract at which 99.99% or more of the initial inoculum
was killed. To determine MBC values, 100 μL of bacterial suspension from
subculture demonstrating no visible growth were removed to spread onto Plate
Count Agar (PCA) medium plates. Plates were incubated at 37 °C for a total
period of 48 h.
Time-kill assay: In order to evaluate the efficiency of the alkaloids
as a function of the time, two bacteria (E. coli CIP 105182 and S.
enterica CIP 105150) were arbitrary chosen to perform a time-kill assay
according to NCCLS guidelines (National Committe for Clinical
Laboratory, 1999). For this test, other night bacteria cultures were adjusted
to the same 0.5 Mac Farland standard and then diluted in 50 mL of sterile nutrient
broth to have approximately 105 cfu mL-1. The alkaloids
were added at the concentration of 3 mg mL-1 for both bacteria. This
concentration chosen arbitrary was slightly greater than their MBC values. The
cultures were incubated aerobically at 37 °C in an incubator shaker (INNOVATM
4000). Each hour, an aliquot of 100 μL was removed from each culture and
diluted with 10 mL sterile isotonic water. Successive dilutions were made from
this initial dilution and the obtained suspension was used to inoculate 90 mm
diameter Petri plates previously filed with PCA (Plate Count Agar). After 48
h incubation at 37 °C, The number of viable bacteria was evaluated by Colonies
Forming Units (CFU) counting. The experiment was carried out twice.
RESULTS AND DISCUSSION
The aim of this research was to study the antibacterial activity of total alkaloids
of M. inermis. Present results exhibited moderate to significant antibacterial
activity against bacterial strains used for this test. The diameters of inhibition
zone (DIZ) indicated the susceptibility of all tested bacteria to alkaloids
excepted to P. aeruginosa. An antibacterial activity is recorded when
a DIZ more than 9 mm is observed around the paper disc (Kitzberger
et al., 2006). The largest DIZ (23 ± 1) mm were obtained with
S. aureus ATCC92449. It has frequently been reported that Gram-positive
bacteria are more sensitive to plant extract and their components than Gram-negative
bacteria (Kelmanson et al., 2000; Masika and Afolayane,
2002 ; Sahin et al., 2002; Karaman
et al., 2003; Karou et al., 2006; Masoodi
et al., 2008). The results of this study confirmed these observations.
Diameter of inhibition values obtained with gram-positive bacteria are larger
than those obtained with gram-negative bacteria (Table 1).
Earlier study with alkaloids of Sida acuta (Karou
et al., 2006) gave DIZ values greater than those obtained in this
study. However, it is difficult to make a comparison because alkaloids from
M. inermis are different from those from S. acuta. It is also
indicated that the DIZ value is determined by the initial population density
of the microorganisms, their growth rate and the rate of diffusion of the antimicrobial
agent (Hugo and Russell, 1998).
The lowest MIC (0.625 mg mL-1) was recorded with P. mirabilis
CIP104588, S. aureus (clinical isolate), S. carmorum LMG13567
and S. aureus ATCC 25293. The antibacterial activity was considered bactericide
when the ratio MBC/MIC is 1 or 2 and bacteriostatic when this ratio is 3 or
more. In this case, alkaloids of M. inermis can be considerate bactericide
to tested bacteria (Table 2). The mode of action of the alkaloids
of M. inermis which include indole and oxindole alkaloids (Shellard
and Sharpong, 1969) is not known in detail. They should have a particular
mechanism which can justified their actions against Gram negative and Gram positive
bacteria because the outer membrane of Gram-negative bacteria is known to present
a barrier to the penetration of numerous antibiotic molecules.
|| Diameters of inhibition zone by disc diffusion assay
|ALK: Alkaloids of M. inermis; AMP: Ampicillin;
||MIC and MBC values of total alkaloids of M. inermis
in the microdilution assay
|nd: Not determine
||Evolution of bacteria exposed to alkaloids (3 mg mL-1)
in the time-kill assay
It have been reported that alkaloids of the quinolone family act by targeting
bacterial DNA synthesis essential enzymes such as DNA gyrase and DNA topoisomerase
IV, that play important roles in DNA replication (Drica and
Zhao, 1997; Khodursky and Cozzarelli, 1998) or competitively
inhibit electron transport in the respiratory chain (Dekker
et al., 1998; Kunze et al., 1987).
Other studies have shown that bisindole monoterpenoid alkaloids act as DNA intercalating
agents or like topoisomerase inhibitors (Angenot et al.,
1991; Bonjean et al., 1998).
In the time-kill assay performed to follow the alkaloids on microorganisms
as a function of the time, both test bacteria (E. coli CIP 105182
and S. enterica CIP 105150) showed the same clearance time (Fig.
1). After 9 h exposition, any CFU was observed in the plates traducing
that there was no viable microorganism in the initial inoculums.
In conclusion, the study has shown that alkaloids from M. inermis
also have in vitro antimicrobial activities, which could support
the use of the plant by traditional healers to treat various infective
diseases Further studies could led to the most active alkaloids and could
led to a new antimicrobial agent.
This study was partially supported by CORUS 034. We gratefully think
Dr. Imaël H.N. Bassole for his helpful comments on the manuscript
and technical help and Mr. Dieudonné Kinda for his help during
plant material collection. We also think Laboratoire de Biologie Médicale
Saint Camille for providing clinical bacterial strains.
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