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INTERNATIONAL JOURNAL OF PURE AND APPLIED SCIENCES

FULL LENGTH RESEARCH PAPER

Determination of Activity, Time Survival and Pharmacokinetics of

Extracts From Momordica charantia on Some Bacterial Pathogens
*1
Abalaka, M. E., 2Olonitola, O.S., 3Onaolapo, J.A. and 2Inabo, H.I.
1
Department of Microbiology, Federal University of Technology, Minna.
2
Department of Microbiology, Ahmadu Bello University, Zaria.
3
Department of Pharmacognosy, Ahmadu Bello University, Zaria.

* Corresponding Author email: modorc2005@yahoo.com

Received: June 2009 Accepted: July 2009

__________________________________________________________________
ABSTRACT

The activity of crude ethanolic, petroleum ether and water extracts of Momordica
charantia was determined against four bacterial pathogens. The Minimum
Inhibitory Concentration (MIC) of 0.1, 1 and 10 mg/ml and Minimum Bactericidal
Concentration (MBC) of 10, 10 and 100 mg/ml of the three extracts respectively
were recorded against the test organisms. The Time Survival analysis indicates
there was a gradual inhibition of bacterial growth when cultures of the pathogens
were challenged with MBC of the extracts. The phytochemicals contained in the
crude extracts (Alkaloids, Tannins, Glycosides and Steroids) may have been
responsible for the pharmacokinetics observed in these analyses. It is
recommended that extracts from Momordica charantia be further tested for
toxicity in order for wide scale use to treat certain human ailments.

Keywords: Momordica charantia, Time Survival, Pharmacokinetics, toxicity

__________________________________________________________________

INTRODUCTION
Bitter melon grows in tropical areas, including
parts of the Amazon, east Africa, Asia, and the
Caribbean, and is cultivated throughout South
America for food and medicine. It's a slender,
climbing annual vine with long-stalked leaves
and yellow, solitary male and female flowers
borne in the leaf axils. The fruit looks like a
warty gourd, usually oblong and resembling a
small cucumber. The young fruit is emerald
green, turning to orange-yellow when ripe. At
maturity, the fruit splits into three irregular
valves that curl backwards and release
numerous reddish-brown or white seeds
encased in scarlet arils (Sofowora, 1993). The
Latin name Momordica means "to bite,"
referring to the jagged edges of the leaves,
which appear as if they have been bitten.
6
In the Amazon, local people and indigenous
tribes grow bitter melon in their gardens for
food and medicine. A leaf tea is used for
diabetes, to expel intestinal gas, to promote
menstruation, and as an antiviral for measles,
hepatitis, and feverish conditions.
In Brazilian herbal medicine, bitter melon is
used for tumors, wounds, rheumatism, malaria,
vaginal discharge, inflammation, menstrual
problems, diabetes, colic, fevers, and worms. It
is also used to induce abortions and as an
aphrodisiac. It is prepared into a topical
remedy for the skin to treat vaginitis,
hemorrhoids, scabies, itchy rashes, eczema,
leprosy and other skin problems (Gislene et
al., (2000). Many Researchers have studied
African plants and have found them active in
vitro against so many important clinical
Int. Jor. P. App. Scs., 3(3):6-13, 2009________________________________________www.ijpas.com
pathogens. Studies by Rotimi and Mosadomi
(1987) on nine (9) African chewing sticks
showed that they are effective against oral
anaerobes. Mann et al. (1997) studied the
antimicrobial properties of Calotropis procera
reported that it has activity on Pseudomonas
aeruginosa, Salmonella typhi, Clostridium
perfringens. Amba (1993) reported an active
principle from leaf extracts of neem plant used
in suppressing egg – hatch of root rot
nematodes. Rotimi et al (1987) examined the
aqueous extracts of roots of Fagara
zanthoxyloides (Pakoata) and reported that it
has significant anti-sickling effects on the red
blood cells of patient with sickle cell disease.
So many plants have been studied for activity
against pathogens with the aim of developing
active drugs against them in the face of the
development of spontaneous resistance by
pathogens to orthodox drugs. Our present work
was borne out of believe that the plant,
Momordica charantia, could be a useful
source of antibacterial drug with a very wide
spectrum of activity against the test pathogens
and probably lots more.
MATERIALS AND METHODS
Sample collection: Plant materials,
(Mormodica charantia) were collected from
Bida, Niger State. Identification was carried
out by local people and confirmed by a
Botanist and Taxonomist (Dr. O.A. Falusi,
Dept. of Biological Sciences, Federal
University of Technology, Minna, Niger
State).
Interviews were conducted among some
Herbalists to know the diseases this plant is
used to treat in their localities.
Extraction and preparation of plants’
materials: Ethanol, Petroleum ether and Water
were used as solvents for the extraction of the
plant materials. The method of Silva et al
(1997) was adopted. Twenty (20) grammes
each of ground samples was suspended in
distilled water, 95% ethanol and petroleum
ether (100ml each) for a period of about 120
hours. The extracts were decanted and filtered
and the filtrate evaporated in vacuo at 450C.
The residue was reconstituted in 95% ethanol
and reserved as stock concentration then
stored.
Test Organisms and Collection of Test
Organisms: A total of four bacteria
Staphylococcus aureus, Salmonella typhi,
Escherichia coli and Streptococcus pyogenes
were used for this analysis. The test organisms
were obtained from the culture bank of
Microbiology Department Federal University
of Technology, Minna, Niger State.
7
Sensitivity Testing: Each of the
microorganisms was subjected to the action of
the extracts using the agar cup plate technique
as described by Mitscher et al., (1972). Using
cork borer N04, three holes were bored on the
surface of the agar medium equidistant from
one another. The bottom of each hole was
sealed with molten agar to avoid seepage.
When solidified, each of the cups or holes
made was filled with known volume and
concentration of the pre-prepared extract
solution and allowed to fully diffuse. The
surface of the agar was streaked for confluent
growth with an 18 hour culture of the test
organism which has been previously
standardized to 106 and incubated at the
temperature of 37oC for 24 hours.
Minimum inhibitory concentration (MIC):
Using tube dilution method, the least
concentration of fractions from the plant
extract in which there was no turbidity was
taken as the minimum inhibitory concentration
(MIC) (Hugo and Rusell, 1983). The MIC of
the plant extracts was determined by serially
diluting extract from 101 to 1010. 1ml of each
of the dilutions representing a known
concentration of the extract was introduced
into 9ml of nutrient broth in the test tube. This
mixture was then inoculated with 0.1ml culture
of the test organism previously standardized to
106. This was then incubated at 37oC for 24
hours. The least concentration of plant extract
in the test tube with no turbidity was taken as
the Minimum Inhibitory Concentration (MIC).
Minimum bactericidal concentration
(MBC): This was an offshoot of the
previously determined MIC. The MBC of the
plant extracts was determined by first serially
diluting extract from 101 to 1010. 1ml of each
of the dilutions representing a known
concentration of the extract was introduced
into 9ml of nutrient broth in the test tube. This
mixture is then inoculated with 0.1ml culture
of the test organism previously standardized to
106. This was then incubated at 37oC for 24
hours. The least concentration of plant extract
in the test tube with no turbidity was taken as
the Minimum Inhibitory Concentration
(MIC).Subsequently, those tubes that showed
no turbidity were plated out on nutrient agar
plates and absence of growth on incubation for
24 hours was confirmatory for Minimum
Bactericidal Concentration (MBC).
Phytochemical analysis of Plant extracts for
Active Components:
Phytochemical screening of the extracts was
carried out according to the methods described
by Odebiyi and Sofowora (1978) and Trease
Int. Jor. P. App. Scs., 3(3):6-13, 2009________________________________________www.ijpas.com

and Evans (1989) for the detection of active indicative of the absence of
components like saponins, tannins, alkaloids, flavonoids.
phlobatanins, glycosides and e.t.c g. Steroids- Salkowski test: 5 drops of
3 concentrated H2SO4 was added to
a. Alkaloids- 1cm of 1%HCL was
1cm3 of the extract in a test tube. Red
added to 3 cm3 of the extract in a test
colouration was observed which is
tube. The mixture was then heated for
indicative for the presence of steroids.
20 minutes, cooled and filtered.
h. Phlobatanins- 1cm3 of the extract
About 2 drops of Mayer’s reagent
was added to 1%HCl. No red
to1cm3 of the extract. A creamy
precipitate observed which means
precipitate was an indication of the
negative result.
presence of alkaloids.
i. Triterpenes- 1cm3 of the extract was
b. Tannins- 1cm3 of freshly prepared
added to 5 drops of acetic anhydride
10%KOH was added to 1cm3 of the
and a drop of concentrated H2SO4
extract. A dirty white precipitate
added. The mixture was then steamed
showed the presence of tannins.
for 1 hour and neutralized with NaOH
c. Phenolics- Two drops of 5%FeCl3
followed by addition of chloroform.
was added to 1 cm3 of the extract in a
Absence of blue-green colour
test tube. Absence of greenish
indicates the absence of triterpenes.
precipitate indicates the absence of
phenolics.
d. Glycosides- 10cm3 of 50% H2SO4 Time survival Analysis: In this analysis the
was added to 1cm3 of the extract and Minimum Bactericidal Concentration (MBC)
the mixture heated in boiling water against each organism was used in order to
for about 15 minutes. 10cm3 of evaluate the rate of cell lyses as the organisms
Fehling’s solution was then added and were challenged with the extracts. The test was
the mixture boiled. A brick-red carried out by inoculating 0.1ml of 18 hour
precipitate was confirmatory for the culture of the test organism into nutrient broth
presence of glycosides. containing a known concentration of the
e. Saponins- (i) Frothing test: extract. About 0.1ml of the organism growing
2cm3 of the extract was vigorously in the broth was plated out on a nutrient
shaken in the test tube for 2 minutes. medium at the initial 0 hours then at 1 hour
No frothing was observed. intervals subsequently until the 7th hour. The
(ii) Emulsion test: 5 drops of olive oil plates were incubated for 24 hours at 37oC
was added to 3cm3 of the extract in after which the resultant growth was counted
the test tube and vigorously shaken. using the colony counter.
Absence of stable emulsion formed
indicates the absence of saponins.
f. Flavonoids- 1cm3 of 10% NaOH was
added 3cm3 of the extract. There was RESULTS
no yellow colouration which is
__________________________________________________________________________________

Table 1 Results of Sensitivity Analysis showing zones of inhibition (in mm) around crude extracts at
varying concentrations.

___________________________________________________________________________________
Conc. of extracts Pet. Ether Ethanol water
___________________________________________________________________________________
(mg/ml) S.a S.t E.c S.p S.a S.t E.c S.p S.a S.t E.c S.p
___________________________________________________________________________________
0.1 0 0 0 0 0 0 0 14±1 0 0 0 0
1 9±1 0 9±3 8±1 15±4 0 13±1 15±2 11±1 0 13±4 15±1
10 10±2 12±2 12±3 10±0 17±3 10±0 16±0 19±2 13±1 10±3 14±1 16±0
100 13±1 15±3 16±2 13±1 22±2 13±1 20±2 24±2 15±2 13±3 18±2 21±1
___________________________________________________________________________________
Key: S.a=Staphylococcus aureus, S.t=Salmonella typhi, E.c=Escherichia coli, S.p=Streptococcus
pyogenes.

___________________________________________________________________________________

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Int. Jor. P. App. Scs., 3(3):6-13, 2009________________________________________www.ijpas.com

From table 1 it is clear the ethanol extract was
active against S. pyogenes at the concentration
of 0.1mg/ml while the remaining organisms
were susceptible at 1mg/ml but S. typhi was
resistant. As the concentration of extracts
increased there was corresponding increase in
susceptibility of organisms and activity
extracts.

___________________________________________________________________________________

Table 2 Breakpoints used for antibiotic resistance testing (NCCLS 1996)

Breakpoints used Standards for Breakpoint µg/ml

Salmonella Breakpoints Susceptible Resistant
<= >
Tetracycline NCCLS 4 8
Chloramphenicol NCCLS 8 16
Florfenicol SVARM 2002 16 16
ß-Lactam
Ampicillin NCCLS 8 16
Cephalosporins
3rd gen. cephalosporin1 NCCLS 2 4
Fluoroquinolones
Ciprofloxacin 2
Enrofloxacin 2 NCCLS 0,25 1
Quinolones
Nalidixic acid NCCLS 16 16
Sulfonamides
Sulfonamide/TMP
Trimethoprim 3 NCCLS 8 8
Sulfonamide NCCLS 256 256
Aminoglycosides
Streptomycin SVARM 2002 32 32
Gentamicin NCCLS 4 8
Neomycin 2 SVARM 2002 16 16

Table 3: Results of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal

Concentration (MBC)
_____________________________________________________________________
Minimum Inhibitory Conc of Minimum Bactericidal Conc of
Extract Px Ex Wx Px Ex Wx
Organisms mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml
______________________________________________________________________
S. aureus 1±0 1±0 1±0 10±0 10±0 10±0
S. typhi 10±0 10±0 10±0 100±0 100±0 100±0
E. coli 1±0 1±0 1±0 100±0 10±0 10±0
S. pyogenes 1±0 0.1±0 1±0 10±0 1±0 10±0
______________________________________________________________________
Key:
Px – Petroleum ether extract
Ex – Ethanol extract
Wx - Water extract

9
Int. Jor. P. App. Scs., 3(3):6-13, 2009________________________________________www.ijpas.com

Table 4: Results of phytochemical screening of the extracts

Extracts Petroleum ether extract Ethanol extract Water extract
Plant Chemical
______________________________________________________________________
Alkaloids + + +
Tannins + + +
Phenolics - - -
Glycosides - + +
Saponins - - -
Flavonoids - - -
Steroids - + -
___________________________________________________________________________
Key:
+ = Present
- = Absence
The results of the phytochemical analysis show (b) Tannins
that the following organic components are (c) Glycosides
present in the plant extracts (d) Steroids
(a) Alkaloids

8
Log of Bacterial population

6 S. aureus
S. pyogenes
5
S. typhi
4 E. coli
3

0
ur
r

s
s

s
s
u

ur

ur

ur
ur

ur
ur
ho
ho

ho

ho

ho

ho
ho

ho
0

Time in hours

Fig 1 Time survival analysis with

ethanol extract

10
Int. Jor. P. App. Scs., 3(3):6-13, 2009________________________________________www.ijpas.com

7.2
Log of bacterial population
7
6.8 S. aureus
6.6 S. pyogenes
6.4 S. typhi
6.2 E. coli
6
5.8
5.6
5.4

our our urs urs urs urs urs urs

h h o o o o o o
0 1 2h 3h 4h 5h 6h 7h
Time in hours

Fig 2 Time survival analysis with petroleum ether

extract

6
Log of Bacterial population

5 S. aureus
S. pyogenes
4 S. typhi
E. coli
3

0
ur ur ur
s
ur
s
ur
s
ur
s
ur
s
ur
s
ho ho ho ho ho ho ho ho
0 1 2 3 4 5 6 7
Time in hours

Fig 3 Time survival analysis with Water extract

___________________________________________________________________________________

11
Int. Jor. P. App. Scs., 3(3):6-13, 2009________________________________________www.ijpas.com
DISCUSSION
Results of sensitivity analysis revealed
different level of activity with different
extracts. The activity of ethanolic extracts
against all test organisms is the highest
compared with water and petroleum ether
extracts. However, petroleum ether extract had
activity on Salmonella typhi more than the
other solvents. This was displayed by zones of
inhibition created round the extracts as the
organisms grew (table 1).
Preliminary results of activity of
antimicrobial agents such as plant active
components, antibiotics are usually expressed
in vitro as zones of inhibition around the
chemical. Gislene et al., (2000) observed
similar trend in their work on the antibacterial
activity of plant extracts and phytochemicals
on antibiotic resistant bacteria in Brazil.
According to them, any chemical that
demonstrates activity with zones of inhibition
of 7mm and above is acceptable as being
active. From these results, the least zone of
inhibition is 8mm. It can therefore, be said that
the extracts of this plant are active against
these organisms.
Antibiotic breakpoint: Antibiotic breakpoint
is a maximum MIC threshold for predicting
successful antibiotic therapy. The antibiotic
breakpoint for each antibiotic in tables 2 are
very much similar to the results obtained in
this work. This means the results of the
extracts compare favourably with those of
refined standards antibiotics.
According to Baker and Silverton, (1985) an
organism is said to be sensitive, moderately
sensitive or resistant to a chemical agent
(extract) when the zone of diameter of
inhibition is compared with zone of inhibition
produced by a known standard antibiotic. The
results of these experiments are in conformity
with this position.
The MIC and MBC are usually in vitro tests
conducted to verify the activity of chemicals
against selected test organisms. Minimum
Inhibitory Concentration is the least
concentration of the chemicals that disallow or
inhibit the growth an organism. The MIC of all
extracts against S. aureus, S. pyogenes and E.
coli was 1mg/ml and 10mg/ml against S. typhi
with the exception of ethanolic extract
demonstrating an MIC of 0.1mg/ml against S.
pyogenes. The Minimum Bactericidal
Concentration on the other hand was 10mg/ml
for both Staphylococcus aureus and
Streptococcus pyogenes while the MBC of 100
12
mg/ml were recorded for Salmonella typhi and
Escherichia coli. Based on the low MIC and
MBC obtained from the present results it is
evident that the chemical components from
extracts of Momordica charantia are active
against the test organisms and could be a good
source of drugs to cure ailments/diseases
caused by these organisms. According to
Mitscher et al., (1972) for any plant material to
be of clinical importance, it should be effective
in vitro at the concentration of 0.1 mg/ml or 1
mg/ml and should be considered an effective
therapeutic agent if it produces zones of
clearing or inhibition of between 15-22mm on
the target pathogenic organisms. The MIC of
0.1 mg/ml as well as the zones of inhibition of
between 15-22mm has been recorded in this
work.
From table 4 it is obvious that Momordica
charantia contains Alkaloids, Tannins,
Glycosides and Steroids. The presence of these
organic compounds in the plant extracts may
have been responsible for the plant’s activity
on microorganisms. Rotimi and Mosadomi,
(1987) explained that tannin-like substances
are present in the bark and pulp of plants and
that they inhibit bacterial growth and are
capable of protecting these plants against
bacterial infection.
The time survival analysis: In drug
administration (chemotherapy), disease
resolution and drug metabolism is of
paramount importance to the clinician. It is
very important to note that any useful
chemotherapeutic agent should lead to disease
resolution as quickly as possible and for such
agent to be removed from the system as soon
as its objectives are accomplished. The time
survival curves as shown in figures 1, 2 and 3
reveal the rate of cell lyses by each extract.
Furthermore, cell lyses was at the rate of 0.7
log of cell population on the average, showing
the kinetics of cell destruction by the extracts.
From these results it is clear that extracts of
Momordica charantia are well able to destroy
bacterial cells given the right concentrations.
CONCLUSION
Medicinal plants possess many potentially
valuable therapeutic agents which are effective
in the control of organisms that cause several
diseases in man and other living things.
Research into antimicrobial agents of plant
origin is very important therefore, in order that
we might discover such potentials and use
them against these pathogens that are fast
 Int. Jor. P. App. Scs., 3(3):6-13, 2009________________________________________www.ijpas.com

developing resistance against common Rotimi, V.O. and Mosadomi, H.A. (1987). The
antibiotics. effect of crude extracts of nine African
chewing sticks on oral anaerobes.
Journal of medical microbiology 23: 55-
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