EVALUATION OF THE IN VIVO ANTI-HELMINTHIC ACTIVITY, OF BRIDELIA MICRANTHA, CHENOPODIUM AMBROSOIDES AND OCIMUM AMERICANUM EXTRACTS AGAINST SCHISTOSOMA MANSONI INFECTION IN MICE.

Schistosomiasis is a chronic debilitating global disease affecting approximately 600 million people in 74 developing countries, with 800 million, mostly children at risk. Chemotherapy is the only immediate recourse to minimize the prevalence and incidence of this disease worldwide. Presently, Praziquantel is the only drug of choice for the treatment of all forms of schistosomiasis, however it shows low efficacy against schistosomula and juvenile stages. This dependence on a single drug with the likely potential for development of resistance to Praziquantel has justified the search for new alternative chemotherapies. Medicinal plants are potential candidates as sources of new drug prototypes. This study provides findings on the schistosomicidal activity of Bridelia micrantha, Chenopodium ambrosoides and Ocimum americanum plant extracts against Schistosoma mansoni infection in mice. Seven week old BALB/c mice were infected with approximately 250 cercariae and treated on the third and fourth week post infection with five crude extracts from the 3 plants for respective efficacy studies. Praziquantel treated group and infected control group served as controls. Perfusion was performed for all groups on the sixth week after infection for worm recovery. Worm recovery analysis confirmed that the three extracts have antischistosomal properties. Furthermore, pathology showed resolving granulomas and immune profiling results confirmed the extracts had immunomodulatory activity which could potentially be important in inhibiting infection. International Journal of Advanced Research (IJAR) Vol.6 (1) pp. 1097-1108 (2018) See more at: http://www.journalijar.com/uploads/581_IJAR-21094.pdf

Schistosomiasis is a chronic debilitating global disease affecting approximately 600 million people in 74 developing countries, with 800 million, mostly children at risk. Chemotherapy is the only immediate recourse to minimize the prevalence and incidence of this disease worldwide. Presently, Praziquantel is the only drug of choice for the treatment of all forms of schistosomiasis, however it shows low efficacy against schistosomula and juvenile stages. This dependence on a single drug with the likely potential for development of resistance to Praziquantel has justified the search for new alternative chemotherapies. Medicinal plants are potential candidates as sources of new drug prototypes. This study provides findings on the schistosomicidal activity of Bridelia micrantha, Chenopodium ambrosoides and Ocimum americanum plant extracts against Schistosoma mansoni infection in mice. Seven week old BALB/c mice were infected with approximately 250 cercariae and treated on the third and fourth week post infection with five crude extracts from the 3 plants for respective efficacy studies. Praziquantel treated group and infected control group served as controls. Perfusion was performed for all groups on the sixth week after infection for worm recovery. Worm recovery analysis confirmed that the three extracts have antischistosomal properties. Furthermore, pathology showed resolving granulomas and immune profiling results confirmed the extracts had immunomodulatory activity which could potentially be important in inhibiting infection.
1098 2014). Although clinically insignificant, resistance has been reported in Egypt where some individuals could not be cured for schistosomiasis leading to identification of resistant isolates (Ismail et al. 1999). Studies have also shown that Praziquantel does not prevent re-infection and is not effective against schistosomula, which can result in low cure rates in areas where schistosomiasis is hyperendemic (Sabah et al. 1986). Praziquantel has further been reported to induce hemorrhage in the lung tissue of the host as well as abdominal pain and diarrhea (Flisser and McLaren 1989; Kabatereine et al. 2003). This has encouraged new studies in search of alternative therapies that could either replace or complement the use of Praziquantel in the treatment for schistosomiasis.
Natural products with therapeutic properties have been used for a long time. The main sources include mineral, plant and animal products (De Pasquale 1984). Plants have remained important sources contributing to 25% of prescribed drugs and 11% of drugs considered by WHO as essential (Rates 2001).
Bridelia micrantha, Ocimum americanum and Chenopodium ambrosoides have been used in traditional medicine to treat various ailments including as antihelminthics (Osebe et al. 2016). Their antischistosomal activity has also been reported in preliminary studies (Moilo et al. 2014; Waiganjo, Yole and Ochanda 2014). It will be interesting to determine if the extracts have immunomodulatory properties, a quality which has been observed for Praziquantel (Mutapi et al. 2005;Tweyongyere et al. 2011;Bourke et al. 2013). It will also be important to determine if the extracts have activity against 3 week old juvenile worms relative to Praziquantel.

Materials and Methods:-
Plant material, extraction and preparation of extracts Bridelia micrantha (bark), Chenopodium ambrosoides (leaves and fruits) and Ocimum americanum (whole plant) were collected from various parts of Kenya. They were dried under shade at room temperature with periodic turning to prevent moulding until they were completely dry, then ground to fine powder with sieving through 0.5mm mesh to standardize particle size.
To prepare water extracts, the ground powder was soaked in distilled water for 72 h then filtered through cotton wool and subsequently Whatman ® qualitative filter paper, grade 1 (Sigma-Aldrich, Taufkirchen Germany). The filtrates were then frozen to -10 o C for 12 h and resultant ice blocks placed in vacuum chamber of a freeze dryer which was adjusted to -100 o C and 1 torr one at a time. Freeze drying was allowed to run until only a solid extract remained in the vacuum chamber.
To prepare hexane and methanol extracts O. americanum powder was soaked in analytical grade hexane (Unilabs, Nairobi Kenya) and C. ambrosoides fruit powder in analytical grade methanol (Pancreac Quimica, Barcelona Spain) for 72 h. Filtration was done first through cotton wool then using Whatman ® qualitative filter paper grade 1. The organic solvents were evaporated using a rotor evaporator (Joh, Achelis and Johne, Bremen Germany) at 70 o C until pasty residues remained. The extracts were then transferred to small jars and further dried in fume chambers at room temperature for approximately 2 weeks.

1.2
Infection of BALB/c mice with S. mansoni cercariae S. mansoni eggs recovered from chronically infected olive baboon (Papio anubis) was used to infect naïve snails (Biomphalaria pfeifferi) which were maintained at Institute of Primate Research's (IPR) malacology lab. The cercariae shed from snails were quantified and 250 used to infect each mouse. 7 week old BALB/c mice were used for this study where each group was composed of 10 mice being 5 of either sex. Infection was by ring method as described by Smithers and Terry (1965). The animals were maintained in cages each with 5 mice at IPR's rodent house with food pellets (Unga Group LTD, Kenya) and water ad libitum. Temperature was maintained at 20 o C ± 3 o C and relative humidity at above 50% with natural day/night cycle.

1.3
Treatment and worm recovery from infected mice The two mice treatment groups were treated by administering the preparation orally on the third and fourth week respectively and a dose being administered two days after the initial treatment. Dosage for the plant extracts treatment groups was 150 mg/kg as recommended in earlier studies (Moilo et al. 2014; Waiganjo, Yole and Ochanda, 2014). Praziquantel groups' dosage was 900 mg/kg and they served as drug control group. An infected control group was also included where treatment was withheld. The administered high dose of 900 mg/kg has been shown to be highly effective in mice while at the same time being within toxicity limits (Frohberg .1984;Muchirah et al. 2012).

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Worm recovery was performed on the sixth week post infection by perfusion using a perfusion pump (Manostat®, division of Barant Company, England) (Smithers and Terry 1965). Recovered worms from were manually counted and numbers analyzed as follows; Test of significance was performed between the treatment groups using student's t-test for paired samples and ANOVA at 95% significance 1. 4 Gross pathology and histopathology analysis Fresh liver recovered from mice were observed for adhesion between liver lobes, general inflammation and presence of granuloma. The severity of granuloma presence per liver lobe was graded subjectively to None, Few (1-3), Moderate (4-10) and Severe (more than 10).
The livers were then fixed in 10% formaldehyde, processed as per routine and sectioned to 0.7µm thickness using a microtome before mounting on slides (Hopwood 1996;Carson 2007; Carson and Christa 2009). Staining was done using haematoxylin and eosin dyes. Under an ocular micrometer, 10 granulomas from each animal's liver were measured on their vertical and horizontal dimensions, the average of which was assumed to be the granuloma diameter (Farah et al. 2000).

1.5
Immune reaction of treatment groups 1.5. 1 ELISA and Flow cytometry reagents and kits ELISA 96 well plates (Nunc-Immuno TM plate marxi sorp TM ) and goat anti-mouse IgG were obtained from Sigma-Aldrich, USA while the substrate (SureBlue TM , TMB microwell peroxidase substrate -1 component) was procured from KPL, Gaithersburg USA.
All materials used in flow cytometry were obtained from BD Biosciences (Califonia, USA). BD cytometric bead array (CBA) mouse Th1/Th2 cytokine kit contained all the necessary reagents and capture antibodies specific for IL-2, IL-4, IL-5, IFN- and TNF proteins. BD FACSCalibur TM flow cytometer, BD CellQuest TM Pro version 5.2.1 and FCap Array software version 1 were used in the analysis of cytokine profiles. BD Falcon TM 12 x 75mm sample acquisition tubes for the flow cytometer and 15ml conical propylene tubes were also used.
Serum was prepared from blood that was recovered from mice during perfusion. The blood was initially left on the bench to clot at room temperature then kept at 4 o C overnight. The following morning, the blood was retrieved and centrifuged at 450 g at 4 o C for 15 minutes then supernatant recovered and stored in 1 ml microfuge tubes at -20 o C for subsequent studies.

1.5.2
Determination of specific IgG profiles of the different treatments using ELISA Soluble egg antigen (SEA), 0-3 h release protein (0-3 h) and soluble adult worm antigen preparation (SWAP) antigens were diluted in 1 X PBS at a concentration of 1mg/ml and 50µl of each added to ELISA plates wells. This was allowed to stand overnight at 4 o C then washed three times using wash buffer with ELISA washer system (Dynex Technologies LTD, Guernsey channel Great Britain).
After draining all wash buffer, 100µl blocking buffer was added to each well and allowed to stand at 37 o C for 1 h, then washed three times. 50 µl of antibody mixture prepared by mixing mice serum with blocking buffer at a concentration of 2.5µl/ml was added to each well in duplicate and allowed to stand at 37 o C for 2 h. This was washed six times and 50 µl conjugate goat anti-mouse IgG in a dilution of 1µl/2ml of 1% BSA added then incubated at 37 o C for 1.5 h. The plates were then washed six times and 50µl of substrate added. This was allowed to stand in a dark room for 30 min and reading done using ELISA reader (Dynatech Laboratories, USA) at OD 450. Test of significance of the mean OD readings was performed using ANOVA at 95% significance

1.5.3
Determination of cytokine profiles of the different treatments using flow cytometry 1.5.3. 1 Mixing of mouse Th1/Th2 cytokine capture beads and cytokine assay Reconstitution and serial dilution of standards was done according to manufacturer instructions. Mixing of mouse Th1/Th2 cytokine capture beads was also done as per manufacturer instructions. Briefly, each of the Capture Bead suspension was individually vortexed and 10µl aliquot of each pooled in a single tube labeled "Mixed Capture 1100 Beads" and vortexed thoroughly. 50 µl of the Th1/Th2 cytokine standard solutions were added to control tubes as indicated by manufacturer.
The test groups' serum samples were retrieved and 50µl of each added to an acquisition tube then 50µl of mouse Th1/Th2 PE Detection Reagent added. This mixture was then incubated in darkness for 2 h at room temperature. 1ml of wash buffer was added to each of the tubes and centrifuged at 200 g for 5 min then supernatant discarded. 300µl of wash buffer was then added to resuspend the bead pellet and suspension loaded on BD FACSCalibur TM flow cytometer. The data was acquired using BD CellQuest TM Pro version 5.2.1 and analyzed using FCap Array software version 1. Test of significance of mean cytokine concentrations was performed using ANOVA at 95% significance.

Results:-
Worm efficacy results are summarized in Tables 1 and 2 below. 1. 6 Effect of the plant extracts on the survival of 4 week and 3 week old schistosomes Other than O. americanum water extract, all treatments were significantly better than IC for the 4 week old worms efficacy study (Table 1). For the 3 week old worms efficacy study, all treatments were significantly better than IC except for B. micrantha water extract ( Table 2). Praziquantel was highly effective against both three and four weeks old worms

1.7
Four and week old worms treatment groups liver gross pathology analysis Results of liver gross pathology are shown in Tables 3 and 4. Adhesion between liver lobes was observed for all treatment groups. All the treatment groups in the 4 week old worms efficacy study except B. micrantha water extract and O. americanum hexane extract presented with few to no observable granulomas correlating with general inflammation observations. C. ambrosoides water extract, O. americanum water extract and Praziquantel treatment groups had no observable general inflammation.
All the treatment groups for the 3 week old worms efficacy study except C. ambrosoides water extract, O. americanum hexane extract and O. americanum water extract presented with moderate, few to no observable granulomas. B. micrantha water extract and Praziquantel had no observable inflammation which correlates with the few to no observable granulomas observed 1. 8 Histopathology of mice liver biopsies Using one way ANOVA (ά = 0.05) the means were significantly different although the post hoc Tukey HSD did not detect any differences between paired means. In decreasing order, the average granuloma sizes were infected control (240.9µm), O. americanum water extract (234.6µm), C. ambrosoides methanol extract (171.1µm), O. americanum hexane extract (170µm), Praziquantel (160µm), B. micrantha water extract (153.2µm) and finally C. ambrosoides water extract (70µm) (Figure 1).
Histopathology changes -determined mainly by extent of lymphocytic cellular infiltration-of liver from group treated with Praziquantel was mild with few areas of necrosis and granuloma. Other than for B. micrantha group where histopathology changes were moderate, the other groups varied ranging from mild to severe. Infected control group had highest number of granuloma per slide and none was observed to be resolving. The other treatment groups however had granuloma which were resolving being without a distinct central egg. The photomicrographs of the liver sections are shown in Figure 2.

1.9
IgG profiles of the different treatment groups using ELISA There was no statistical difference for IgG profiles using SWAP antigen in 4 week old worm treatment study. OD readings in decreasing order were C. ambrosoides methanol extract treatment (0.586), B. micrantha water extract

Cytokine profiles of the different treatment groups using Flow cytometry
Statistical difference for 4 week old efficacy study was observed for all cytokine concentration groups except for IL-4 which was only detectable for mice treated with B. micrantha water extract. Infected control and naïve cytokine concentrations were lower than in all treatment groups except for IL-2 where C. ambrosoides water extract and B. micrantha water extract treatment groups had lower concentrations. The cytokine concentrations for treatment groups is summarized in table 5.
Statistical difference for 3 week old worm efficacy study was observed for TNF and IL-5. IL4 was only detectable for mice treated with C. ambrosoides methanol extract. Infected control had IL-2 concentration of 4.91 pg/ml which was higher than for four treatment groups including Praziquantel. Naïve IL-2 concentration was lower than for all treatment groups except B. micrantha water extract. These differences were however not statistically significant. IFN-γ also lacked a statistical difference but concentration for infected control was relatively low and undetectable for naïve group. The cytokine concentrations for treatment groups is summarized in table 6.

Discussion:-
In recent years, a substantial interest in natural products to the treatment of Neglected Tropical Diseases, including schistosomiasis, has been growing. The interest has been exploited and stimulated as an effort to develop a new medicine as an alternative method to this parasitosis treatment. This study investigated the in vivo efficacy of Bridelia micrantha, Ocimum americanum and Chenopodium ambrosoides against 3 and 4 week old worms of S. mansoni.
Generally, all treatments were more effective than the untreated control group suggesting they contain phytochemicals that have activity against S. mansoni. Treatment with Praziquantel -which acted as drug control group-was as expected, observed to be more effective than the crude plant extracts. Crude plant extracts typically contain many different biomolecules and their concentration may be suboptimal relative to the pure active ingredient in Praziquantel.
It was interesting to observe that O. americanum water extract was more effective against 3 week old worms while B. micrantha water extract was more effective against 4 week old worms. By week three after infection, majority of the worms are already in the portal blood stream (Doenhoff et al. 1978), where they remain in adult stage. This means the worms were mostly exposed to the treatment in the same location. Although it is not known which antigens, it is suggested that tegumental antigens of schistosomes vary as the worms mature from cercaria to schistosomule to adult which is thought to be a protective mechanism (Sepulveda et al. 2010). This therefore suggests there are worm structural differences that may have led to the two extracts having more pronounced activity in either the 3 week or 4 week old worms.

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Our findings were however not consistent with studies performed by Sabah et al (1986), since Praziquantel was observed to be as effective against the juvenile worms. This may be due to differences in experimental design where we exposed the mice to 900mg/kg dose compared to 250mg/kg in Sabah's study. There may also be some differences between the Kenyan strain of S. mansoni when compared to the Puerto Rican one.
While the pathology for the plant extracts was not as remarkable as for the Praziquantel treated groups, the observation of resolving granulomas could be an indicator that the extracts have comparable properties noting that the eggs were produced by adult female worms in the days following treatment for both studies. Potentially at higher doses than the administered 150 mg/kg, the effect would be more pronounced. Alternatively, pathology of mice done serially over a few weeks would better show the disease progression. IgG responses were observed to be similar across the treatment groups, infected control and naïve despite variation in worm numbers suggesting insignificant IgG effect in early stage infection. Naïve OD readings were potentially due to non specific binding Interestingly, Praziquantel profile was not highest for any of the cytokines while infected control was low in all groups except for IL-2. While it is known that innate immunity can develop following prolonged exposure owing to responses against dead worms (Mitchell et  . Prominence of Th1 cytokines especially IFN has an effect of down regulating Th2 cytokines and consequently a reduction of granuloma size and fibrosis (Cheever. 1992;Cheever et al. 1998). IL-5 serves to recruit eosinophils but has no obvious effect on granuloma size, IL-4 leads to formation of IgE with varied effect on granuloma while IL-2 increases granuloma size and fibrosis (Cheever. 1992). Although TNF has not been widely studied, it is thought to have a role in periportal fibrosis (Cheever. 1992;Ramadan et al. 2013). This suggests the inflammation causing cytokines may be important in acting against the miracidia within eggs and thereafter, other cytokines resolve the granuloma implying the importance of the cytokine balance. This suggests the plant extracts elicited cytokine responses that compared well with Praziquantel and may potentially be protective.

Conclusion:-
According to the results the plant extracts used in this study could be developed to be important alternatives to Praziquantel offering antischistosomal activity, improve pathology upon treatment and elicit mechanisms for protection to reinfection. Therefore, this study opens up perspectives for future researches on the substance or the compound isolation, making possible potential new treatment alternatives against S. mansoni. While the toxicology of these plants extracts have been studied where B. micrantha water extract was determined to be relatively toxic, the drug candidates will also need to be assessed in terms of metabolism, chemistry and treatment regimen (Nwaka and       None -No granuloma observed per lobe; Few-1 to 3 granuloma per lobe; Moderate-4 to 10 granuloma per lobe; Severe-more than 10 granuloma per lobe Table 5:-Mean cytokine concentrations of mice treated against 4 week old worms. Serum was prepared from blood drawn from BALB/c mice treatment groups then analyzed using flow cytometry. Statistical difference was analyzed using ANOVA (ά = 0.05) and paired difference using students t-test (p>0.05) Cytokine concentration in pg/ml