The activity of Mangifera indica L.

leaf extracts against

the tetanus causing bacterium, Clostridium tetani
Godfrey S. Bbosa1,*, Aloysius Lubega1, Nathan Musisi2, David B. Kyegombe1, Paul
Waako1, Jasper Ogwal-Okeng1 and Olwa Odyek1
1

Department of Pharmacology and Therapeutics, Faculty of Medicine, Makerere University, PO Box 7072, Kampala, Uganda and 2Department of
Veterinary Microbiology and Parasitology, Makerere University, PO Box 7062, Kampala, Uganda

Abstract
Mangifera indica L. is a common horticulture and medicinal
plant, which is used traditionally to treat various infections.
A previous study has shown its leaf extracts to have antibacterial activity against Staphylococcus aureus, Escherichia
coli and Pseudomonas aeruginosa. This study investigated the
activity of the leaf extracts against Clostridium tetani, which
causes many deaths around the world. Ether and ethanolic
leaf extracts were obtained by sequential extractions.
Qualitative studies were carried out to determine the different classes of compounds in the extracts. The chemical tests
showed that the ether extract had saponins, steroids and
triterpenoids, while the ethanol extract had alkaloids,
anthracenosides, coumarins, flavonones, reducing sugars,
catechol and gallic tannins, saponins, steroids and triterpenoids. The minimum inhibitory concentration (MIC) of
the extracts against the study organism was determined
using the gradient serial dilution method. Gentamycin and
distilled water were used as controls. Both the ethereal and
ethanolic fractions showed anti-clostridium tetani activity
with an MIC of 6.25 and 12.5 mg ml)1, respectively.
Key words: Clostridium tetani, minimum inhibitory
concentration

Introduction
Traditional medicinal plants have been used in the treatment of infectious diseases for a long time and this practice
is still widespread in developing countries. This is partly
because they are more accessible and affordable to the

*Correspondence: E-mail: godfossa@yahoo.com

All authors declare no conflicts of interest

54

communities than allopathic medicine (WHO, 1998a;

Marjorie, 1999; Kamatenesi, 2002). One of the plants
traditionally used in the treatment of fever, cough and
wounds is Mangifera indica L. (Anacardiaceae). It is a large
evergreen tree found all over the tropical regions of the
world where it is important as a horticultural and medicinal plant. The leaves contain saponins, glycosides,
unsaturated sterols, polyphenols, euxanthin acid, mangiferine, mangin and gallic tannins, which are reported to
have antibacterial activity on various microorganisms
(Dweck, 2001; Hirte, 2002).
Leaf extracts of this plant have been found to have
antibacterial activity against Staphylococcus aureus,
Escherichia coli and Pseudomonas aeruginosa (Bbosa et al.,
2007). Traditionally, the leaf extract of this plant is commonly applied to wounds but the exact therapeutic effect is
not documented. One possible benefit is its anti-clostridial
activity against Clostridium tetani, a bacterium that causes
tetanus.
Tetanus is a major problem around the world especially
in the rural communities of the developing countries
(Steinglass, 1993; WHO, 1999a). It manifests as neonatal
tetanus in the newborn, maternal tetanus in the mothers
and in adults who have sustained injuries. According to
the Centre for Disease Control and Prevention (CDC, 2003,
2004), the most common occurrence of tetanus infection
is in the yards, gardens, homes and farms. The infection is
common in individuals who are not immunized and those
who fail to clean all injuries and wounds sustained (AAP,
2003; Mylonakis, 2003).
Tetanus leads to both maternal and neonatal death.
Maternal tetanus is responsible for at least 5% of maternal
deaths, c. 30,000 deaths annually worldwide (Fauveau,
1993; Cook, 2003). According to WHO. (1999b), it is
estimated that about 90,000 women die annually from
puerperal infections caused by unclean delivery practices

 2007 African Journal of Ecology, Afr. J. Ecol., 45 (Suppl. 3), 5458

Activity of Mangifera indica L. against Clostridium tetani

with sepsis related to pregnancy and other complications

of unsafe abortions posing serious threats.
In Uganda, 3,433 cases are reported annually with
2,403 deaths, 316 neonatal tetanus cases and 2.2 live
births per 1,000 (UNICEF, 1999; WHO, UNICEF & UNFPA,
2000; WHO, 1998b, 2004; NaTHNaC, 2005). In newly
borne infants, the umbilical stump become infected with
spores from the dirty environment, while others are
infected through cultural practices when their wounds
come in contact with contaminated animal faeces (World
Health Organization (WHO), 1999b; Centre for Disease
Control and Prevention (CDC), 2003, 2004). The current
preventive measures that involve immunization with the
toxoids, cleaning of the wounds and use of sedatives and
antibacterial agents to infected people only cover 60% and
in some places, a steady decline and accessibility have been
reported (UNICEF, 2004; Semwanga & Ddembe, 2006). In
order to alleviate these problems, alternative sources of
medicines particularly from plants need to be investigated.

Materials and methods

Processing and extraction of plant materials
Mature leaves were collected from Mulago Hill in Kampala,
the capital city of Uganda (11001340 m above sea level).
The leaves were cleaned using distilled water and then
dried in the shade for 3 weeks. Dry leaves were then
pounded in a metallic mortar to a fine powder. Sequential
extraction was done using ether and ethanol, respectively.
A total of 100 g of the plant powder in Erlenmeyer flasks
was soaked in 500 ml of the solvent for 4 days with
occasional shaking to facilitate extraction of the active
compounds from the plant leaves. The mixture was decanted and filtered using Whatman No.1 filter paper in a
Buchner funnel using a suction pump. The residue was
dried in the shade for 3 days in preparation for the ethanol
extraction. All reagents were supplied by Biochemicals and
Reagents for Life Science Research, SigmaAldrich Co. Ltd
(Munich, Germany); 20022003. The recovery of the
ethereal and ethanol solvents from the filtrate was carried
out by a Heidolph model rotary evaporator to obtain the
dry ethereal and ethanolic leaf extracts.

Qualitative tests on the extracts

The extracts were tested for alkaloids, steroids and triterpenoids, tannins, anthracenosides, reducing sugars,

 2007 African Journal of Ecology, Afr. J. Ecol., 45 (Suppl. 3), 5458

55

flavones, saponins and coumarins using standard methods

(Ciulei, 1964).

The anti-clostridium tetani studies on the dry M. indica leaf

extracts
The process followed the established procedures for testing
antimicrobial agents (Case, 1998). Standard C. tetani
bacterial organisms from the American Type Culture
Collection (ATCC 10779) were obtained from the Department of Veterinary Microbiology and Parasitology, Makerere University, Uganda. The extracts were dissolved in a
few drops of dimethylsulfoxide (DMSO) and topped up with
distilled water to give a stock solution of concentration of
100 mg ml)1 of ethereal and ethanolic leaf extracts. The
DMSO and distilled water are the commonly used solvents
in preparing solutions for antimicrobial studies (Carter &
Cole, 1990). The stock solution was kept at 4C. The
organisms were isolated on cooked meat and then cultured
on Reinforced Clostridial Medium (Oxoid CM 149 or BD
218081) (Biomed Diagnostic Systems, BD; Loveton Circle
Sparks, Maryland, USA) at 37C under anaerobic conditions for 36 h. This formed the bacterial stock solutions for
use in the agar-well diffusion assays using MuellerHinton
agar (Biomed Diagnostic Systems, BD; Loveton Circle
Sparks, Maryland, USA).

Preparation of media
Direct sensitivity testing MuellerHinton agar was used.
The medium was prepared by adding 40 g of agar
powder to 1 l of distilled water and boiling to dissolve.
The solution was autoclaved at 121C for 15 min,
cooled to 50C in a water bath. It was then transferred
into sterile Petri dishes. It was allowed to cool and
solidify under sterile conditions. They were then incubated for 24 h at 37C to ensure that there was no
contamination by bacterial organisms. Wells of 6 mm
diameter and 5 mm depth were made in the solidified
agar using a sterile borer (Case, 1998).

Agar-well diffusion assay

Cultures of C. tetani were inoculated on the solidified
MullerHinton agar on each Petri dish by streaking using
a wire loop. About 100 mg ml)1 (100,000 lg ml)1) of the
test extracts and 10 lg ml)1 of gentamycin (positive
control) were dispensed into the wells made (Brunton et al.,

56

Godfrey S. Bbosa et al.

2006; Carter & Cole, 1990). The plates were incubated at

37C for 36 h under anaerobic conditions. The sensitivity
of the test organisms to the extracts was determined by
measuring the diameters of the zone of inhibition surrounding the wells. Distilled water was used as the negative control. The diameters of the zones of inhibition were
measured with a ruler.

Table 1 Different classes of compounds in each solvent leaf

extract of Mangifera indica
Solvent leaf extract

Class of compounds

Ether

Saponinsa
Steroids and triterpenoidsa
Alkaloidsa
Anthracenosides
Reducing sugars
Steroids and triterpenoids
Saponins
Coumarins
Catechol and gallic tanninsa
Flavonoids

Ethanol

Determination of the minimum inhibitory concentration by

serial dilution method
The agar-well diffusion assay was used. A tenfold dilution
of the stock extract solution of bacterial broth was carried
out. Ten test tubes were arranged in a row and serial
dilutions of the crude extract solution were carried out
with 100 mg ml)1 (100,000 lg ml)1) as the highest
concentration in tube-1 in order to determine the rough
values of the minimum inhibitory concentration (MIC).
The 10 lg ml)1 gentamycin solution as the highest
concentration was prepared in the same way as the stock
leaf extract solution. About 0.5 ml of distilled water was
put in each test tube. Then 0.5 ml of the stock solution
from tube-1 was put into tube-2. It was mixed carefully
and the process was repeated such as from tube-1 to
tube-2 up to the tenth, producing a tenfold dilution. The
cultures of C. tetani were inoculated again on Muller
Hinton agar and incubated at 37C for 36 h under
anaerobic conditions. The lowest concentration that
inhibited growth of microorganisms was the rough MIC
value, which was recorded. The process was repeated
three times in order to obtain average MIC values. Gentamycin, distilled water and DMSO were also tested on
C. tetani test organisms to ensure that they had no activity
on the organisms, which would lead to false results.

Results
Phytochemistry
Qualitative chemical tests on ethereal and ethanolic extracts indicated that the extracts contained saponins, steroids and triterpenoids, alkaloids, anthracenosides,
coumarins, flavonones, reducing sugars, catechol and
gallic tannins. The saponins, steroids and triterpenoids
were present in both the ethereal and ethanolic fractions
but much more concentrated in the ethereal fraction. The
ethanolic fraction had more compounds than the ethereal
fraction (Table 1).

High levels of compound in the solvent extract.

Table 2 Mean MIC(mg ml)1) for the different solvent extracts of

Mangifera indica
Solvent
extract

Mean diameter
of inhibition (mm)

Mean MIC
(mg ml)1)

Ether
Ethanol
Gentamycin
Distilled water
DMSO

19.0
16.0
21.0
0
0

6.25
12.5
0.1256
Growth
Growth

Anti-clostridium tetani activity

The ether extract showed the highest activity on C. tetani
with zones of inhibition of 19 mm and MIC of
6.25 mg ml)1. The ethanolic extract had zones of inhibition of 16 mm and MIC of 12.5 mg ml)1. Gentamycin,
used as positive control, had zones of inhibition of 21 mm
and MIC of 0.156 mg ml)1 (Table 2). Distilled water as
negative control and DMSO that was used as solvent in
making the solutions for the study showed no anti-clostridial tetani activity on the test organisms.

Discussion
The phytochemical study on the M. indica leaves showed
that it contains different compounds such as alkaloids,
anthracenosides, coumarins, flavonones, reducing sugars,
tannins, saponins, steroids and triterpenoids (Table 1).
These compounds are found in many plants and are
reported to have antimicrobial activity on a variety of

 2007 African Journal of Ecology, Afr. J. Ecol., 45 (Suppl. 3), 5458

Activity of Mangifera indica L. against Clostridium tetani

microorganisms such as the bacteria, fungi, viruses and

many others (Marjorie, 1999; Dweck, 2001; Hirte, 2002).
A previous study on M. indica leaf extract indicates that
these compounds had antibacterial activity on Staph.
aureus, E. coli and P. aeruginosa (Bbosa et al., 2007). The
anti-clostridial tetani activity tests on the M. indica leaf extract showed that ethereal fraction had the highest activity
(MIC of 6.25 mg ml)1). This could be due to high levels of
steroids and triterpenoids and saponins present in the
extract (Tables 1 and 2). The ethanolic fraction had a
slight activity on the study organisms (MIC of
12.5 mg ml)1) as compared with the ethereal fraction
(Tables 1 and 2). However, the M. indica leaf extracts
showed a lower anti-clostridial tetani activity compared
with the positive control gentamycin (MIC of
0.1256 mg ml)1; Table 2). This was because gentamycin
was a pure drug in comparison to the crude leaf
extracts, which were impure. Gentamycin is an antibiotic, which is commonly used in antibacterial activity
studies as well as in the treatment of various bacterial
infections in both humans and animals (Brunton et al.,
2006; Carter & Cole, 1990). Mangifera indica leaves
contain compounds with anti-clostridial tetani activity,
which could be isolated and purified. The compounds
could serve as an alternative source of antibacterial
agents that can be used in the treatment of various
bacterial infections such as C. tetani infection that causes
tetanus especially in the rural areas where modern
medicines are not easily accessible.

Acknowledgements
We appreciate the contribution of SIDA SAREC by sponsoring this research and Makerere University where study
was carried out. We also acknowledge the contribution of
the anonymous reviewers of this manuscript.

References
American Academy of Pediatrics (AAP). (2003) Tetanus. In: Pickering Report of the Committee on Infectious Diseases (Eds R. Poore
and S. V. Houten), 26th Edn. American Academy of Pediatrics,
IL.
Bbosa, G.S., Kyegombe, D.B., Ogwal-Okeng, J., Bukenya-Ziraba, R.,
Odyek, O. & Waako, P. (2007) Antibacterial activity of Mangifera indica (L.). Afr. J. Ecol. 45(Suppl.1), 1316.
Brunton, L.L., Lazo, J.S. & Parker, K.L. (2006) Goodman &
Gilmans The Pharmacological Basis of Therapeutics, 11th Edn.
McGraw-Hill Medical Publishing Division, New York.

 2007 African Journal of Ecology, Afr. J. Ecol., 45 (Suppl. 3), 5458

57

Carter, G.R. & Cole, J.R. Jr (1990) Diagnostic Procedures in Veterinary Bacteriology and Mycology. Academic Press, London and
New York.
Case, C.L. (1998) Discover a new antibiotic. Strategies for Success.
Skyline College, CA.
Centre for Disease Control and Prevention (CDC). (2003) Tetanus
surveillance. Available at: http://www.cdc.gov/nip/publications/
pink/tetanus.
Centre for Disease Control and Prevention (CDC). (2004) Tetanus
in National Immunisation Program. Available at: http://
www.cdc.gov/nip/publications/pink/tetanus.pdf.
Ciulei, I. (1964) Practical Manuals on the Industrial Utilisation
of Medicinal and Aromatic Plants. University of Bucharest,
Romania.
Cook, M.(2003) Federation Programme Action, Newsletter March
2003. Soroptimist International of Great Britain and Ireland.
Dweck, A.C.(2001) Ethnobotanical Plants from Africa. Black Medicare Ltd, Iltshire. Available at: http://www.dweckdata.co.uk
(accessed January 2007).
Fauveau, V. (1993) Maternal tetanus: magnitude, epidemiology
and potential control measures. Int. J. Gynecol. Obstet. 40(1),
312.
Hirte, M. (2002) Benefits of Mango for the Human Health. Available
at: http://www.preda.org/archives.
Kamatenesi, M.M. (2002) The Socio-cultural Aspects in Utilization of
Medicinal Plants in Reproductive Health Care in Western Uganda.
Unpublished Report, Department of Botany, Makerere University, Kampala, Uganda.
Marjorie, M.C. (1999) Plant products as antimicrobial agents.
Clin. Microbiol. Rev. 12, 564582.
Mylonakis, E. (2003) Division of Infectious Disease. VeriMed
Healthcare Network, Massachusetts General Hospital, Boston,
MA.
National Travel Health Network and Centre (NaTHNaC). (2005)
Tetanus. Travel Health Surveillance Section of the Health Protection
Agency for communicable Disease Surveillance Centre. Available at:
http://www.nathnac.org/pro/factsheets/documents/
Tetanus.pdf (accessed January 2005).
Semwanga, A.R. & Ddembe, W.W. (2006) An Evaluation of
Healthcare Policy in Immunisation Coverage in Uganda.
Information Systems Department, Faculty of Computing and
Information Technology, Makerere University. Available at:
http://www.albany.edu/cpr/sds/conf2006/proceed/papers/
RWASH189.pdf.
Steinglass, R. (1993) Tetanus, Disease Control Priorities in Developing Countries. Oxford University Press, Oxford.
UNICEF (1999) Programming for Safe Motherhood: Guidelines for
Maternal and Neonatal Survival. UNICEF, New York City.
UNICEF (2004) Global and Regional Trends in Immunization Coverage. Available at: http://www.childinfo.org/eddb/immuni/
trends.htm.
WHO, UNICEF & UNFPA (2000) Maternal and Neonatal Tetanus
Elimination by 2005. Available at: http://www.unicef.org/
immunization/MNTE_strategy_paper.pdf.

58

Godfrey S. Bbosa et al.

World Health Organization (WHO). (1998a) Quality Control

Methods for Medicinal Plant Materials. WHO, Geneva.
World Health Organization (WHO). (1998b) EPI Information
Systems: Global Summary, September 1998. WHO EPI GEN 98.10, Geneva.
World Health Organization (WHO). (1999a) Progress towards
the global elimination of neonatal tetanus, 19901998. Wkly
Epidemiol. Rec. 74(10), 7380.
World Health Organization (WHO). (1999b) The High-Risk
approach: the WHO recommended strategy to accelerate

elimination of neonatal tetanus. Wkly Epidemiol. Rec. 71(5),

3337.
World Health Organization (WHO). (2004) Neonatal tetanus
reported cases. Vaccines, Immunization and Biologicals, Geneva.
Available at: http://wwwnt.who.int/vaccines/globalsummary/
timeseries/tsincidenceneo.htm.

(Manuscript accepted 20 August 2007)

 2007 African Journal of Ecology, Afr. J. Ecol., 45 (Suppl. 3), 5458