Antischistosomal Effects Of Solanum Incanum And Carica Papaya Crude Extracts On The Parasite Schistosoma Mansoni In Vivo And In Vitro
S Muchika, H Kutima, R Maranga, D Yole
antischistosomal, granuloma, igg, plant extacts, worm count
S Muchika, H Kutima, R Maranga, D Yole. Antischistosomal Effects Of Solanum Incanum And Carica Papaya Crude Extracts On The Parasite Schistosoma Mansoni In Vivo And In Vitro. The Internet Journal of Tropical Medicine. 2010 Volume 7 Number 2.
In schistosomiasis infection, the disease is managed by exposing the definitive host to a dose of Praziquantel. However, Praziquantelis still not reaching the majority of those who most need it due to its high cost and there is possibility of drug resistance, hence need for alternatives. Antischistosomal effects of crude
Schistosomiasis is a major disease of public health in humans, occurring in over 74 countries of the tropics and sub-tropics (WHO, 2010). It affects an estimated 207 million or more individuals and cause an estimated 500,000 deaths every year. Its current increased prevalence in many areas has numerous causes, including increased irrigation in areas with inadequate waste disposal and breakdown in public health infrastructure. The most common significant clinical effects of infection are intestinal and hepatic manifestations, which can result in serious illness or death (David
The possible existence of
Plants are frequently discussed as possible sources of novel drugs, and in recent years they have been investigated as potential sources of antiparasitic agents including novel antischistosomal agents (Sher, 2001; Hagan
Methanol was added to the other samples in the bottles until the samples were slightly submerged, soaked for 36 h and the content was filtered. The filtrate was placed in a round bottomed flask and fixed on a clean rotary vacuum evaporator (RE-100 Bibby, made in Japan). Temperature was set at 70○C, the mouth of the machine closed and power switched on. The machine was switched off when methanol stopped running from the distiller. The sample was then removed from the machine and further dried on a water bath until there was no evaporation of methanol (methanol extract).
BALB/c mice acquired from IPR Animal Resources Department were housed in cages, in groups of five per cage. They were maintained on a commercial diet and water
Mice were divided into six categories of 15 mice each, representing treatment and infected control groups infected with
Sampling time points
Blood was obtained via heart puncture as follows; 5 naive mice (uninfected control) were sampled at wk 0; and 5 mice from each infected/ treated groups at wk 6. The blood was used to prepare serum for IgG ELISA. Five mice from all experimental groups and controls (PZQ and infected control) were perfused for worm recovery at week 6 and their livers were observed for gross pathology.
Schistosome Specific IgG enzyme Linked Immunosorbent Assay (IgG-ELISA)
Blood obtained from heart puncture was allowed to stand at room temperature for 3 h and incubated overnight at 4 ○C. The clotted blood was centrifuged in a Microfuge (Sorvall RT 6000D made in Japan) at 2000 rpm for 20 minutes and sera retrieved. Nunc-ImmunoTM plates (MaxiSorp TM Surface) ELISA plates were coated overnight with 50 μl of 10 μg/ml of soluble worm antigen preparation (SWAP) or 18 hr schistosomule soluble antigen (SSP) diluted in bicarbonate buffer, pH 9.6 and incubated overnight at 4 o C. The antigen was dispensed off on a blotting paper. The plates were washed six times using the washing buffer (0.05 % Tween 20 in PBS). This was followed by blocking of the non-specific binding sites with 100 μl 3% BSA in PBS and incubating at 37 o C for 1 h. The plates were washed off unbound BSA six times with washing buffer. Diluted (1:200) serum samples (50 μl) was dispensed into each well in duplicates and incubated overnight at 4 o C, and then washed as above. After washing the unbound serum, 50 μl of 1:2000 peroxidase conjugated rabbit anti-mouse IgG was dispensed into the wells and incubated for 1 h at 37oC. The unbound conjugate was washed off as before and
50 μl TMB micro well peroxidase substrate (Sure Blue TM TMB) was added. The plates were incubated at 37 o C in the dark for 30 minutes and optical density was read at 630 nm in an ELISA micro plate reader.
At week 6 post-infection (2 weeks post-treatment), five mice from each group were perfused according to a modified method of Smithers and Terry (1965). Adult worm recovery was done according to the method described by Yole
Gross Pathology Examination
At week 6, gross pathology of the liver was observed in all the groups. The indices of comparison were; inflammation, adhesions and presence or absence of granulomas on the liver. The granulomas were categorized into none (no granuloma), few (1-3 granulomas), moderate (4-10 granulomas) and severe (>10 granulomas) per lobe.
Two millilitre of each of plant extract (5 ug/ml, 15 ug/ml and 30/ml) was dispensed in a well of 24 cell culture plate containing an aliquote of 20 cercariae. Two replicates for each concentration was made. Each preparation was observed under a dissecting microscope for cercariae motility at the following time points: 5, 10, 20, 30, 45 and 60 minutes. Immobile cercariae were noted at every point; when all cercariae were immobile before 1 h, the experiment was terminated. At the end of each experiment, iodine was added for clarity in counting of the total number of cercariae as a confirmation of accuracy of the counting procedure.
Analysis of worm recovery and immunological data was performed using Student’s t-test using computer excel programme and significance difference was defined as p<0.05. Gross pathology was noted by visual observation of liver tissues and cercaricidal assay was by enumerating the larvae under a microscope in percentages.
The maturation level of the Kibwezi isolate of
The mean number of
Worms recovered from different groups were subjected to Student’s t test to determine their significant difference in comparison with each other. Infected-untreated control was significantly different from PZQ control at p<0.001. All the four treatment groups were significantly different from infected-untreated control;
There was a significant difference between three treatment groups and PZQ:
Fig 1: Percentage Worm Recovery in Treatments
In all mice in the six groups, all liver were inflamed and had adhesions. In infected-untreated control group, most of the mice had moderate granuloma while two mice had severe granuloma. Praziquantel group had only one mouse with few granulomas and the rest had no granuloma. In
Immunoglobulin G Response to SWAP and SSP-specific Antigen
IgG responses were analyzed in seru collected from naive group, infected-untreated control and from treatment groups week-2 post-treatment (6 weeks post-infection). The results are shown in Feig 2.
IG response to SWAP antigen in infected-untreated control at week 6 was not very high (O.D 0.319). Praziquantel had a high IgG response in week 6 (O.D 0.352) which was significantly higher than the response in infected-untreated control (p<0.05).
In assay for SSP-specific IgG responses, infected-untreated control group had higher IgG responses (O.D 232) than Praziquantel treatment (O.D 0.162). This difference was significant (p<0.05).
IgG responses to SSP antigen were lower in relation to IgG responses to SWAP antigens in all the treatments and controls
Wk = week
SWAP = soluble worm antigen preparation
SSP =18 hr schistosomule soluble antigen
The cercaricidal effect of the plant extracts is shown in Table 1. The results are the means of duplicate observations of lowest (5 µg/ml), moderate (15 µg/ml) and maximum (30 µg/ml) concentrations of the plant extracts at different time intervals.
In both aqueous and methanol
At a concentration of 30 µg/ml of
*_ cercariae were dead and lysed
_ Observation stopped
This study compared the efficacy of
The infected-untreated group had the highest worm burden (57±1.3) while Praziquantel had the lowest worm burden (25±2).
Gross pathological observations revealed that the livers of all the mice were inflamed and had adhesions, a manifestation of an infection. Granuloma formation in the liver was the worst in infected-untreated control group while Praziquantel had the lowest granuloma formation level. Although Praziquantel is the most efficacious drug (Utzinger and Keiser, 2004), some mice under this treatment had granulomas. This can be linked to the fact that some parasites may have delayed to mature thus escaped the effect of Praziquantel at the time of its administration. This is because Praziquantel drug has a short half-life of 1–1.5 hours (Ross
Infected-untreated control had high IgG responses to both SWAP and SSP antigens. The elevated levels of IgG responses in infected-untreated control can be associated with a high worm burden leading to a high level of circulating parasite antigens many of which are not related to protection (Njoroge
The IgG responses in Praziquantel for both antigens were relatively high, and in this case, unlike the untreated control, it had the lowest worm burden and the lowest pathology. Praziquantel kills the worms directly and also, induces schistosome-specific immune response which reduces the worm burden further. This results in reduced pathology, as lower number of worms translates to lower egg production, and hence fewer granulomas.
Generally, in this assay, IgG responses to SWAP in all the 6 treatments were higher, compared to responses in SSP. The low IgG responses to SSP antigens could be due to reduced schistosomule antigens as the worms matured.
The two plants extracts demonstrated efficacy to
This work has been supported by Jomo Kenyatta University of Agriculture and Technology Research Grant. We acknowledge technical assistance offered by Kiio Kithome, Sammy Kisara and Simon Kiarie.