Inhibition of Trypanosoma brucei brucei by Extracts from Waltheria indica L. (Sleepy Morning)
The study was conducted to evaluate the inhibitory activity of polar and non-polar extracts from Waltheria indica against Trypanosoma brucei brucei, the causative parasite of trypanosomosis, for the first time. The parasites Trypanosoma brucei brucei were cultured using 96 well micro titer plate and maintained at 37°C. About 20-25 parasites per microscope field were dozed with 1, 2 and 4 mg mL-1 of the extracts of the plant. Two controls were set up; one is Berenil (the reference drug), which was used as positive control and a negative control group without extract/Berenil. Results showed that the ethanol extract was the most active with Inhibitory Concentration (IC50) value of 0.5-1 mg mL-1 while the petroleum ether extract was moderate against the trypanosome parasites with IC50 value of 3.8-4.0 mg mL-1. The results also showed that the parasites survived more than 4 h when no plant extract/berenil was present. Phytochemical screening revealed a range of compounds including, alkaloids, terpenes, flavonoids, tannins, glycosides, steroids, among others, which could be responsible for the trypanocidal activity of the extracts. Further studies including in vivo assays for the determination of efficacy and toxicity are planned and will be carried out.
June 28, 2010; Accepted: August 21, 2010;
Published: February 07, 2011
African trypanosomosis remains a disease with unsatisfactory medical control
(Maikai et al., 2008). The control of trypanosomosis
continues to rely principally on chemotherapy and chemoprophylaxis using the
salts of the three compounds, diminazene; an aromatic diamidine; homidium, a
phenanthridine and isometadium, phenanthridine-aromatic amidine (Anene
et al., 2001). However, the therapeutic and prophylatic use of trypanocides
is beset by numerous limitations, including toxicity and the development of
resistance by the parasites (Gutteridge, 1985). The
emergence of drug resistance trypanosome strains is considered a very serious
problem in trypanosomosis control, particularly for the resource-poor at risk
populations and farmers in Africa. Recent surveys in Eastern and Southern Africa
(Ndungu et al., 1999) and in West Africa (Mcdermott
et al., 2000; Maikai et al., 2007)
have shown that the prevalence of trypanocidal drug resistance might even be
higher than hitherto expected. The limited availability and affordability of
pharmaceutical medicines emphasizes the need for research into a more comprehensive,
formidable and cheaper sources of trypanocide. It is estimated that some 20,000
species of higher plants are used medicinally throughout the world (Tagboto
and Townson, 2001). Plants have provided the basis for traditional treatment
for different types of diseases and still offer an enormous potential source
of new chemotherapeutic agents. Plants present a spectrum of biological compounds
with activities against virus, cancer and parasites. Plants contain compounds
mainly secondary metabolites such as alkaloids, Glycosides, flavonoids, terpenes
and coumarins (Rates, 2001) which have been reported
to provide better and cheaper alternative drugs against parasites (Nwude
and Ibrahim, 1980; Freiburghaus et al., 1996;
ITDG and IIRR, 1996; Adewummi et
al., 2001; Nok, 2005).
Waltheria indica L. (sleepy morning) is a short-lived shrub that is
popular among traditional healers, here in Sokoto State, in the treatment of
skin diseases, malaria and typhoid fever, sickle cell anaemia and epilepsy.
In addition the aqueous extract of the plant was shown elsewhere (Bala
et al., 2009) to possess an in vitro trypanocidal activity.
However, no scientific data has been published to show the trypanocidal effects
of various extracts of the plant.
Therefore this study is designed to determine the inhibitory effects and phytochemical analysis of polar and non polar extracts of Waltheria indica on Trypanosoma brucei brucei parasites in vitro. This is the first report on the trypanocidal activity of the various extracts of the plant.
MATERIALS AND METHODS
Collection of the plant: The plant used in this study was collected
in October 2007 from Dabagi farm of the Usmanu Danfodiyo University, Sokoto
and transported to the Botany Unit of the Usmanu Danfodiyo University, Sokoto,
for identification with the help of a taxonomist, A.M. Umar. A voucher specimen
of the plant was deposited in the herbarium of the Department where it was identified.
Preparation of the plant extracts: Whole plant of Waltheria indica
was cut into pieces, air-dried (under shade to avoid destruction of the
active components) at room temperature and pulverized using mortar and pestle.
Various extracts of the plant part were prepared in a separating funnel using
activity guided fractionation method as described by Hostettamnn
et al. (1995). In this method, 40 g of the pulverized plant part
was dissolved in 50 mL of water and ethanol. It was then left to stand for 24
h after which it was filtered. The residue was discarded and to the water-ethanol
filtrate, 50 mL petroleum ether was added and shaken for 30 min in a separating
funnel. The petroleum ether extract was separated and evaporated in vacuo at
45°C and the residue was weighed and stored. To the water-ethanol filtrate,
50 mL ethyl acetate was added using the same procedure above and then followed
by n-Butanol. Finally, the water-ethanol filtrate was separated, dried, weighed
and stored until required.
Test organisms: Trypanosoma brucei brucei were obtained from stabilates maintained at the Nigerian Institute of Trypanosomiasis and Onchocerciasis Research (NITOR) Vom, Plateau State. The parasites were maintained in the laboratory by continuous passage in rats until required. Passage was considered necessary when parasitaemia reaches a range of 16-32 parasites per field (usually 3-5 days post infection). In passaging, 1x103 parasites in 0.1-0.2 mL blood/PBS solution was introduced intraperitoneally into clean rats acclimatized under laboratory condition for 1 week.
In vitro study of extracts of Waltheria indica: Assessment
of in vitro trypanocidal activity was performed in triplicates in 96
well micro titer plates (Atawodi et al., 2003),
but with slight modification. The 0.5 g of each extract was dissolved in 25
mL distilled water to obtain 20 mg mL-1 concentration. Using the
same process two other concentrations of 10 and 5 mg mL-1 were also
formed, giving three different concentrations for each extract. The 20, 10 and
5 mg mL-1 concentrations of each plant extract was prepared in triplicates.
Twenty microliter of blood containing about 20-25 parasites per field obtained as described above, was mixed with 5 μL of each extract concentration to produce effective test concentrations of 4, 2 and 1 mg mL-1, respectively. To ensure that the effect monitored was that of the extract alone, two sets of control were set up. First Diminazene aceturate (Berenil-a commercial trypanocide) was used as positive control and then blood suspended in normal saline was used as a second (negative) control. Berenil was prepared in the same concentration as the extracts (445 mg Diminazene aceturate + 555 mg Antipyrine, Eagle Chemical Company LTD, Ikeja, Nigeria).
Each of the test mixtures was incubated for 5 min in closed Eppendorf tubes maintained at 37°C. Two microliter of test mixture was placed on separate microscope slides and covered with a cover slip and the parasites observed for a total duration of 60 min.
The concentration at which 50% of the trypanosomes were cleared by each treatment was recorded and taken as the IC50 value of the respective extract.
Phytochemical analysis: Phytochemical screening of the ethanol and petroleum
ether extract of Waltheria indica was carried out to determine the presence
of secondary metabolites using standard procedures (El-Olemy
et al., 1994; Harborne, 1973). Flavinoids were
determined by acidifying 2 mL of each extract by adding equal volume of 0.1
MHCl. 0.5 mL of 0.5 M NaOH was added to the acidified extract. A yellow colour
indicates the presence of flavinoids (El-Olemy et al.,
1994; Harborne, 1973). The presence of saponins was
assessed by vigorously mixing 1 mL of each extract in a corked test tube. Persistent
froth, lasting for several minutes was indicative of the presence of saponin.
The presence of saponin was confirmed by shaking 2 mL of diluted fraction with
1 mL of olive oil for about half a minute. A thick white emulsion confirmed
the presence of saponin (El-Olemy et al., 1994).
Tannins were determined by using 5% ferric chloride solution which was added
drop wise to 3 mL of diluted extract. The presence of condensed tannins was
indicated by green-black colouration, while blue-black was indicative of hydrolysable
tannins (El-Olemy et al., 1994). Triterpenes and
Steroids were assessed by the Liebermann and Burchard Method as done by El-Olemy
et al. (1994). To about 1 mL of the fraction was added equal volume
of concentrated H2SO4 and acetic anhydride. Green-pink
to purple violet indicated the presence of triterpenes. The presence of sterols
is indicated by pink to brown/yellowish brown colour. Blue-green colour is indicative
of both sterols and triterpenes. The presence of Alkaloids was determined by
stirring 1 mL of each extract with 5 mL of 10% aqueous hydrochloric acid on
a steam bath for 20 min, which was allowed to cool and was later filtered. One
microliter of the filtrate was treated with drops of Mayers reagent. Appearance
of creamy precipitate indicated the presence of alkaloids. Glycosides were determined
by adding 10 cm3 of 50% H2SO4 to 1 cm3
of each extract in a test tube. The mixture was heated in boiling water for
15 min. The 10 cm3 of Fehlings solution was added and the mixture
was heated again in a boiling water bath. A brick-red precipitate indicated
the presence of glycosides (El-Olemy et al., 1994).
Statistical analysis: Percentage inhibition of parasite population caused
by each treatment was calculated with respect to the parasite-laden control
without treatment as number of parasites in the test well divided by the number
of parasites in the control multiplied by a hundred. Final percentage inhibition
for each concentration was obtained as mean of percentage inhibition for that
concentration. Data in Table 1 were evaluated statistically
with one-way ANOVA followed by Duncans Multiple Range test.
Inhibitory activity of polar and nonpolar extracts of Waltheria indica analyzed against Trypanosoma brucei brucei parasites in vitro are presented in Table 1.
The results showed that at 1.0 mg mL-1 only the ethanol extract of the plant had effect on the trypanosome parasites, producing 93.33% inhibition of trypanosome parasites and comparing favourably with the standard drug of choice Berenil which produces an inhibition of 98.4%.
At 2.0 mg mL-1, ethanol extract inhibited the trypanosomes parasites with a percentage of 96.2. The other extracts had no such effect on the parasites while Berenil 100% inhibited the trypanosome parasites at this concentration.
At 4.0 mg mL-1, 52.1 and 98.8% inhibition was found for petroleum ether and ethanol extracts respectively. Berenil was able to clear the parasites at this concentration with 100% inhibition.
The IC50 value for the ethanol extract was found to be between 0.5-1 mg mL-1 while that of the petroleum ether extract was between 3.8-4 mg mL-1.
Results also showed that after 5 min incubation in Eppendorf tubes maintained at 37°C, the trypanosome parasites survived for about 4 h when no extract/Berenil was present.
The results showed the ethanol extract to be the most effective against Trypanosoma brucei brucei parasite on all concentrations tested while the petroleum ether extract was moderate, active only at the highest concentration of 4 mg mL-1.
The result of the qualitative phytochemical screening of the ethanol extract of Waltheria indica showed an appreciable presence of tannins, terpenes, flavinoids, phenols and glycosides. Alkaloids and steroids were mildly present. In the petroleum ether extract glycosides and steroids were highly present while tannins, terpenes and flavonoids were mildly detected. Saponins and anthraquinones were not detected in both extracts (Table 2).
activity of various extracts of Waltheria indica against Tyrpanosoma
brucei brucei parasites in vitro
|Each value represents mean±SEM of 3 replicates. *p<0.05;
of qualitative phytochemical analysis of the ethanol and petroleum ether
extracts of Waltheria indica
|-: Absent; +: Mildly present; ++: Highly present
Natural products isolated from higher plants have provided novel clinically
active drugs which are one of the keys to the discovering of naturally occurring
therapeutic agents through bioassay-guided fractionation and purification procedures.
When fractionation or purification enhances activity, it often leads to the
discovery of new active molecules. On the other hand if it leads to reduction
in activity, the constituents are synergistic, as such drugs are to be formulated
at the stage of highest activity. In this study, W. indica was subjected
to fractionation and the bioactive constituents were found to be in the ethanol
extract, the polar portion. This had antitrypanosomal activity at all concentrations
tested. The trypanocidal activity of the polar portion of Waltheria indica
seen in this study is in agreement with reports from several researchers (Atindehou
et al., 2004; Ogbunugafor et al., 2007;
Abdel-Sattar et al., 2009). However, the results
were in disagreement with the findings of Hoet et al.
(2004), Agbedahunsi et al. (2006) and Ene
et al. (2009) who respectively found the ethyl acetate, chloroform
and methylene chloride extracts of some plant materials to have the most antitrypanosomal
The observation that different solvent extracts of plants have different trypanocidal
activities is in line with the report of Freiburghaus et
al. (1996), who clearly indicated that different solvent extracts of
the same plant may exhibit different trypanocidal activity, just as extracts
of different parts of the same plants. Therefore, different solvent extracts
of plants should be tested separately for medicinal purposes. The petroleum
ether extract of Waltheria indica was found to be moderately active against
the trypanosome parasites, an indication that it may also contain some active
constituents that have trypanocidal potential. Although the concentration of
the extracts used in this study was high (up to 20 mg mL-1), the
morphology of the red blood cells was maintained while that of the trypanosome
parasites was affected, when compared to the parasite laden control. This is
an indication that a higher trypanocidal activity may be obtained by increasing
the concentration of the extracts.
The trypanocidal potential of the extracts was further confirmed by phytochemical
analysis which revealed a range of compounds that could be responsible for the
activity. The phytochemical analysis revealed both extracts to contain tannins,
terpenes, flavonoids, steroids, phenols and glycosides. Terpenes, steroids and
alkaloids have been reported to have high antitrypanosomal effects (Hoet
et al., 2007; Ene et al., 2009). Anthrquinones
which was absent in both extracts was reported to have activity against T. congolense
(Nok et al., 1996). Some literatures have reported
that flavonoids had antitrypanosomal activity (Tarus et
al., 2002). Therefore this study has shed new light, as an added data
on our earlier findings (Bala et al., 2009),
on the trypanocidal activity of the ethanol and petroleum ether extracts of
The mechanism by which the extracts exhibit trypanocidal action is not immediately
known. However accumulated evidence (Sepulveda-Boza and
Cassels, 1996) suggest that many natural products exhibit their trypanocidal
activity by virtue of their interference with the redox balance of the parasites
acting either on the respiratory chain or on the cellular defences against oxidative
stress. This is because natural products possess structures capable of generating
radicals that may cause peroxidative damage to trypanothione reductase that
is very sensitive to alterations in redox balance. It is also known that some
agents act by binding with the kinetoplast DNA of the parasite. Many others
too act by static action affecting growth and multiplication of trypanosomes.
In conclusion, results have clearly shown that Waltheria indica possesses anti-trypanosomal activity in vitro. The trypanocidal property was found to be higher in the polar portion and moderate, at the highest test concentration, in the non polar part. The antitrypanosomal activity
of the extracts is not surprising because earlier, Bala
et al. (2009) has shown the aqueous extract to possess in vitro
trypanocidal activity. Phytochemical screening reveals a range of compounds,
alkaloids, terpenes, flavonoids, tannins, glycosides, steroids, among others,
which could be responsible for the trypanocidal activity of the extracts. Further
studies are in progress to identify the active constituents, toxicity and the
trypanocidal activity of the extracts in vivo using animal models.
The authors wish to thank Head and Technicians of Department of Biochemistry, Usmanu Danfodiyo University, Sokoto for creating such an enabling environment in their lab for this work to be conducted. We also wish to thank Professor N.S. Naqvi for dedicating his pension as postgraduate scholarship which provided financial support for this study.
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