Amelioration of Trypanosome-infection-induced Alterations in Serum Cholesterol, Triglycerides and Proteins by Hydro-ethanolic Extract of Waltheria indica in Rats
A study was conducted to investigate the amelioration potential of trypanosome infection induced alterations in rat serum cholesterol, triglycerides and proteins by hydro-ethanolic extract of Waltheria indica L. The results from the study showed that infection with Trypanosoma brucei brucei resulted in significant (p<0.05) increases in the concentrations of serum triglycerides (hyperglyceridemia), total protein (hyperproteinaemia) and globulin (hyperglobulinaemia) while the levels of cholesterol and albumin were significantly reduced leading to hypocholesterolaemia and hypoalbuminaemia, respectively. However, treatment with the hydro-ethanolic extract of W. indica significantly (p<0.05) ameliorates these infection induced biochemical changes in fashion comparable to the standard trypanocide Berenil®. The corrective action of the plant may revolve around reduction in parasite load coupled with some hepato-protective properties that need further research to ascertain.
September 11, 2011; Accepted: November 24, 2011;
Published: January 04, 2012
Trypanosomiasis is a disease caused by infection with trypanosome parasites
that live in the blood of the host, commonly transmitted by tsetse flies. It
remains a significant health problem in sub-saharan Africa. It affects most
species of domestic animals and man, causing alterations in serum biochemistry
of the hosts including decrease in blood albumin, increase in globulin levels,
hypoglycaemia and increase in triglycerides, have been reported in donkeys,
horses, rats and rabbits (Rouzer and Cerami, 1980; Marques
et al., 2000; Bala et al., 2011b).
Increased serum lipids in trypanosome infected animals has led to the suggestion
that lipolysis is the major mechanism for supplying the high energy demanded
by the fever following trypanosome infection (Akinbamijo
et al., 1994). Adamu et al. (2009)
also reported a significant reduction in serum levels of cholesterol, triglycerides
and high density lipoprotein in Trypanosma brucei brucei infected pigs
while Rouzer and Cerami (1980) found a significant increase
in triglycerides in T. brucei brucei infected rabbits. Because of the
aforementioned and many others (Orhue et al., 2005;
Orhue and Nwanze, 2006), it could logically be put forward
that trypanosomes can cause serious alterations in hosts serum biochemistry.
Waltheria indica L. (sleepy morning) is a popular medicinal plant here
in Sokoto State, Nigeria. Phytochemical screening of the plant revealed that
it is rich in alkaloids, tannins, flavonoids terpenes, among others and the
pharmacological actions of W. indica are believed to be due to the presence
of these phytochemicals (Bala et al., 2011a).
The present study is designed to assess the amelioration potential of the hydroethanolic
extract of W. indica on trypanosome induced changes in serum cholesterol,
triglycerides and proteins in albino rats. This is because the ethanol extract
of the plant was shown elsewhere (Bala et al., 2011b)
to significantly reduced parasitaemia and also improved the hypoglycaemic condition
of trypanosome infected and treated rats.
MATERIALS AND METHODS
Collection of the plant: The plant used in this study was collected
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 voucher specimen of the plant was deposited
in the herbarium of the Department where it was identified.
Preparation of plant material: The whole plant was cut into pieces,
air-dried (in open air in the laboratory to avoid destruction of the active
components) at room temperature and pulverized using mortar and pestle. Forty
grams of the coarse powder of the plant part were dissolved in 50 mL of water
and ethanol (50% v/v). It was then left to stand for 72 h (3 days) at room temperature
after which it was sieved first with cotton wool and then with number 1 Whatmann
filter paper. It was then evaporated to dryness under reduced pressure and stored
until required (Hostettamnn et al., 1995).
Parasites and animals: T. brucei brucei were obtained from stabilities 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 one week. Thirty adult Wistar albino rats of the same age group, weighing between 200-260 g, were obtained from the Animal House of the Department of Biological Sciences, Usmanu Danfodiyo University Sokoto. They were housed in metal cages in the Parasitology Laboratory of the same Department. They were allowed to acclimatize for two weeks and were fed growers mash (Pfizer Nigeria PLC) and given drinking water ad libitum. The rats were sorted and grouped into 5 groups of 6 rats each as follows:
||Group A (n = 6) Uninfected untreated rats as positive
||Group B (n = 6) Infected but untreated rats as negative control
||Group C (n = 6) Infected treated with 300 mg kg-1 body
weight concentration of plant extract for 5 days
||Group D (n = 6) Infected and treated with 300 mg kg-1
b. wt. plant extract for 10 days
||Group E (n = 6) Infected and treated with full dosage of Berenil®
(3.5 mg kg-1) once intraperitoneally.
The animals were subjected to the same physical conditions. A clean environment was maintained throughout the course of the experiment. They were fed on growers mash (Pfizer Nigeria PLC) and water given ad libitum, throughout the duration of the study.
Animal inoculation: At the end of the acclimatization period, the experimental
animals were inoculated with T. brucei brucei parasites, using needle
and syringe (Onyeyili et al. (1994). One millilitre
(1 mL) of infected blood was taken from the donor rat with fulminating parasitaemia
and was diluted with 9 mL phosphate buffered saline (pH 8.0). The trypanosomes
were counted and thereafter 0.5 mL of the diluted blood containing approximately
2.0x106 trypanosomes per mL of blood was inoculated into the rats
in groups B, C, D and E. Inoculation was done intraperitoneally as described
by Onyeyili et al. (1994) and was preceded by cleaning the area to be
inoculated with cotton wool soaked in 70% alcohol. The same process (cleaning)
was repeated after the injection, to prevent secondary infection by microorganism.
Preparation and administration of the treatments: Twenty percent concentration of the extract was prepared. The rats in groups C and D received 300 mg kg-1 body weight for 5 and 10 days, respectively as indicated above. Administration was done orally with the aid of oral cannular. The rats in group E received full dosage of Berenil®. The indicated dose of Berenil® is 3.5 mg kg-1 body weight as recommended by the manufacturer. Administration was carried out intramuscularly with the help of a syringe and needle. Administration of all treatments began day 5 after inoculation (peak of parasitaemia).
Serum collection (separation of plasma from blood): Three milliliters (3 mL) of blood was collected from the rats in plain sample bottles. Plasma was obtained from blood by centrifugation at 2000 rpm for 10 min. Blood samples were analyzed within a few hours of sample collection by enzymatic colorimetric methods using the appropriate commercial kits according to established techniques.
Biochemical analysis: Determination of biochemical parameters were carried
out using previously described protocols. The total cholesterol was determined
by the Enzymatic Method of Allain et al. (1974).
Triglycerides were determined by Enzymatic Hydrolysis with Lipases as described
by Tietz (1990). Serum total protein and albumin were
analyzed using the Biuret and Bromocresol Green methods, respectively. Serum
globulin was determined as the difference between serum total protein and albumin
concentrations (Harshmohan, 2002). In both cases, commercially
available test kits, products of Randox Laboratories, U.K. were used and with
the manufacturers instructions strictly adhered to.
Statistical analysis: Results were expressed as mean + standard error of the mean of 6 animals. The efficacy of treatments between treated and control groups and between treated groups was compared using one way ANOVA and correlation analysis. Post test analysis was done using the Duncans multiple range test. Values of p<0.05 were considered as statistically significant. In all cases data was entered into computer and analyzed using SPSS (Version 11.0) statistical package.
Results from this study showed that at the peak of parasitaemia all the infected
animals developed hypocholesterolaemia. The level of cholesterol of the untreated
rats continued to decline culminating in the death of animals in that group.
||Changes in serum cholesterol in Trypanosoma brucei brucei
infected rats before and after treatment
||Triglycerides concentration from control and experimental
groups post inoculation and treatment
However, the cholesterol level increased in the treated animals. Percentage
restoration of cholesterol was 29.03, 53.16 and 30.85% for groups C, D and E,
respectively. There was no statistical difference (p>0.05) between the standard
drug Berenil® and W. indica in their normocholesterol
restoration potential in the treated animals (Fig. 1).
On day 5 post inoculation (peak of parasitaemia) the infected animals showed an elevated concentration of triglycerides which is almost 3-fold its initial value. The triglycerides concentration continued to increase in the untreated rats, resulting in the death of animals in that group. However, following treatment, the triglycerides concentration dropped significantly (p<0.05). Percentage drop in triglycerides concentration was in the order 52.99, 56.45 and 57.91% for groups C, D and E, respectively (Fig. 2). There was no significant difference (p>0.05) between Berenil® and W. indica in their amelioration effects on triglycerides levels in the treated animals.
||Changes in protein concentration in Trypanosoma brucei
brucei infected rats before and after treatment
|| Serum Albumin levels of test and experimental groups post
inoculation and treatment
The results of protein analysis indicated an increase in total protein concentration
(hyperproteinaemia) in the infected animals when compared to the normal control.
The protein concentration of the untreated animals continued to increase resulting
in the death of animals in that group. Treatment with Berenil®/W.
indica resulted in reduction in the concentration of the proteins. Percentage
restoration being 8.51, 15.34 and 16.64% for animals in groups C, D and E, respectively
(Fig. 3). There was no significant difference (p>0.05)
between the Berenil® and W. indica in their restoration
effects on total protein levels of the treated animals. Evaluation of serum
albumin showed that there was a significant (p<0.05) decrease in values obtained
for infected animals relative to the uninfected control. In the untreated animals,
the infection-associated hypoalbuminaemia continued to manifest. Following treatment
with Berenil® and ethanol extract of W. indica, there
was a significant (p<0.05) amelioration of the infection-associated hypoalbuminaemia
in the treated animals. Percentage amelioration of the albumin levels in the
treated animals was in the order 24.39, 33.33 and 33.11% for groups C, D and
E, respectively (Fig. 4).
||Levels of plasma globulin concentration in trypanosome infected
rats before and after treatment
There was no significant difference (p>0.05) between Berenil®
and the ethanol fraction of W. indica on their effect on correcting hypoalbuminaemia
of the treated animals.
The levels of plasma globulin showed a 2-fold increase above the pre- infection values, leading to hyperglobulinaemia. This increase was significant (p<0.05) when compared with the uninfected control. The hyperglobulinaemic condition of the untreated animals continued to manifest, culminating in death of the animals in that group. However, treatment with Berenil®W. indica resulted in significant reduction in the parasite-induced hyperglobulinaemia. Percentage restoration being in the order 27.67, 42.25 and 46.77% for groups C, D and E, respectively (Fig. 5). No significant difference (p>0.05) was found between Berenil® and ethanol extract of W. indica in reducing the parasite associated hyperglobulinaemia.
Biochemical evaluation of the body fluids gives an indication of the functional
state of the various body organs and biochemical changes in body fluids that
result from infections depend on the species of the parasite and its virulence
(Anosa, 1988). It has been observed that protozoan parasites
including trypanosomes depend on their hosts for energy and nutrients required
for their growth, motility and reproduction. There is evidence (Gillett
and Owen, 1992) suggesting that parasites can take up the lipids and cholesterol
they need from lipoproteins present in the host body which may lead to alterations
in serum concentrations of such metabolites. The role of lipids in pathogenesis
of trypanosomiasis had been reported (Tizard et al.,
1978). Hypocholesterolaemia during trypanosome infection has been shown
to occur in sheep (Katunguka-Rwakishava et al., 1992),
pigs (Adamu et al., 2009) and in humans (Awobode,
2006), as also recorded in this study.
The 3-fold increase in triglycerides seen in this study contrasted the work
of Awobode (2006) who reported a reduced triglycerides
concentration in humans infected with Trypanosoma brucei gambiense. However,
the work of McGowan et al. (1983) who showed that
the level of parasitaemia is directly proportional to the triglyceride concentration
in Trypanosoma cruzi infected murine animals (Calomys callosus).
The increase in the serum triglycerides concentrations observed in this study
may be due to reduced serum concentration of albumin. Albumin (reduced plasma
concentration of which lead to reduced total serum proteins) is required to
bind to neutral fats (triglycerides and cholesterol) for these lipids to be
transported in the plasma (because the lipids are hydrophobic in nature and
therefore, require some forms of hydrophilic adaptation in the form of lipoproteins).
However, infection can lead to a fall in serum albumin due to decreased synthesis
of albumin and or increased catabolism of albumin, consequently causes reduction
in the binding capacity and leading to increased plasma free concentration of
these analytes. Treatment with the ethanol extract of W. indica significantly
improved the levels of serum cholesterol and triglycerides of the treated animals
possibly by increasing the synthesis of albumin which now binds and transports
the lipids. The plant extract may also have helped the treated animals to degrade
and remove the triglycerides efficiently, as Rouzer and
Cerami (1980) attributed trypanosome-induced hyperglyceridemia in rabbits
to be due to defective triglyceride removal by the host. The amelioration of
the cholesterol and triglycerides levels of the treated animals by W. indica
was comparable to that of the standard trypanocide Berenil.
Hyperproteinaemia, hyperglobulinaemia coupled with hypoalbuminaemia in the
infected animals, as evident in this study, is similar to observations found
in other mammalian hosts parasitized by trypanosome species (Singh
et al., 1988). Hyperproteinaemia due to trypanosome infection may
be due to lysis of the parasites by the host immune system which leads to accumulation
of the parasites proteins in the body of the host. Hypoalbuminaemia during infections
could be due to malnutrition, hepatic damage, gastrointestinal malabsorption
and or increased protein need following infection while the increase in globulin
levels in the infected animals may be caused by damage to the liver by trypanosomes
and or antibody production by the host against the trypanosomes. In this study,
there was a statistically significant increase in total protein and globulin
and a drop in serum albumin 5 days post infection. Worthy of note however, is
the ability of W. indica to effectively control or resist these changes.
This is because treatment with the plant resulted in significant reduction in
parasite-induced hyperproteinaemia and hyperglobulinaemia as well as a significant
amelioration of infection associated hypoalbuminaemia which favourably compared
to the standard trypanocide.
The mechanism by which W. indica achieves its action is not immediately
apparent, it is logical to speculate that the mechanism may revolve around the
reduction in parasite load in the treated animals or the plant may have phytochemicals
that have hepato-protective activity. As put forward by Pagana
and Pagana (2010), albumin is used to test how well the liver and kidneys
are working. Damaged liver cells lose their ability to make proteins. The livers
ability to make proteins may be used to predict the course of certain liver
disease. Therefore, the fact that the plant extract was able to ameliorate the
hyperproteinaemia and correct the hypoalbuminaemia is suggesting some hepato-protective
potential of the extract but this needs further studies on liver enzymes, as
well as the effect of the extract on the excretory and synthetic functions of
It could be concluded that the trypanosome infection induces serious alterations in the host biochemistry and treatment with the ethanol extract of W. indica has significant amelioration effect comparable to the standard trypanocide, Berenil, coupled with suggestive hepato-protective property which needs further studies to ascertain. This is part of an ongoing research.
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