2Kenya Medical Research Institute, Kenya
Fetal bovine serum (FBS) (SIGMA), RPMI 1640 media (SIGMA), analytical grade methanol, antibiotics (Penicillin & streptomycin) (SIGMA), sodium pentobarbital, distilled water and citrate saline (Sodium chloride and Sodium citrate).
Lannea schimperi and Searsia longipes stem barks were collected from Endasaki ward in Manyara region with geographical coordinate of 4° 25' 0" S, 35° 31' 0" E. Prior to sample collection, the aforementioned plants were identified by botanists from TPRI and the voucher specimens were prepared and thereafter preserved in NHT (National Herbarium of Tanzania) with voucher specimen number NM 01 and NM 02 for Lannea schimperi and Searsia longipes respectively.
Collected stem bark samples of Lannea schimperi and Searsia longipes were pulverized at Nelson Mandela African Institution of Science and Technology natural product laboratory and thereafter dried under shade for two weeks. Maceration method was used for extraction as described by Azwanida, (2015) [ 15]. Briefly, the dried plant materials were grinded by laboratory mill to get fine powder. Approximately, 1 kg of Lannea schimperi andSearsia longipes powder were measured by using weighing balance and soaked in 2.5 litters of methanol each and maintained for 48 hours. Afterward, extracts were filtered and concentrated under vacuum by using rotary evaporator. Following extraction process, 35 g and 45 g of Searsia longipes and Lannea schimperi crude extracts were obtained respectively. Moreover, in tradition settings people uses water for preparation of decoction from the aforementioned plants, therefore methanol were used as the extraction solvent in the present study since it has polarity close to water.
Snails were collected from two spots namely Dunga and Car wash, located along the shores of Lake Victoria in Kisumu, Kenya (Figure 1(B)). Briefly, snail’s collection was done by using standard scoop and about 699 snails of different species were obtained . Following collection, Biomphalaria pfeifferi snails were identified based on guideline described by PAHO, (1968)  and thereafter screened for cercariae shedding (Figure 1 (A)). Screening was done under inverted microscope (Olympus) after exposing snails plated on 24 well plates filled with snail’s water to the direct sun light for 1 hour. Positive snails which were shading Schistosoma mansoni cercariae were selected for use in bioassay .
Shedding of cercariae was done by exposing Biomphalaria pfeifferi snails to light for one hour at KEMRI schistosome laboratory. Thereafter, portion of the obtained cercariae were transformed to schistosomula and the remaining portion were used for plants extract bioassay against cercariae stage. In a nutshell, transformation was done by using slightly modified mechanical method prescribed by Ramalho-Pinto., et al. (1974) . Whereby, the obtained cercariaewere chilled on ice for 45 minutes, afterward cercariae suspension was centrifuged for 5 minutes at 1500 RPM. Supernatant was discarded and the pellet was suspended in RPMI 1640 (SIGMA) media. Following resumption, cercariae were mechanically transformed through vigorous agitation for two minutes by using vortex machine. After transformation, the obtained schistosomula were purified by using simple swilling method . In this method of purification, transformed mixture was poured in the petri dish and the latter was gently swirled for about 15 rounds. Following gentle swirling, schistosomula accumulated at the center of the petri dish. Thereafter, schistosomula were transferred to a graduated eppendorf tube by using micropipette. This process was repeated three times to ensure no schistosomula has remained. The obtained schistosomula were enumerated and afterward maintained in RPMI 1640 (SIGMA) media supplemented with 10% FBS (SIGMA) and 1% penicillin and streptomycin (SIGMA) ready for bioassay.
Adult worms used in the present study were obtained from Swiss albino mice pre infected with Schistosoma mansoni cercariae through abdominal percutaneous exposure . Briefly, 20 female Swiss albino mice weighing 18 ± 2g were anaesthetized with 0.2 ml of 5 mg/ml Sodium phenobarbital and shaved at the abdominal part. Thereafter, mice were infected with about 200 Schistosoma mansoni cercariae each. Infection was done by pouring the suspension containing the aforementioned number of the cercariae in special metal rings placed on shaved abdominal part of the mice as displayed in Figure 2. The suspension was maintained for 1 hour to allow penetration of the cercariae into the mice body. Following infection, mice were maintained for 8 weeks to allow the development of the schistosomula to an adult schistosome worms.
In vitro plant extracts bioassay against cercariae stage
Bioassay was conducted on 24 multi well plates to validate the activity of the extracts against cercariae stage of the Schistosoma mansoni by using methods described by Aziz., et al. (2011)  and Simões., et al. (2015)  with modifications. In this case, cercariae were subjected to different concentrations of the methanolic plants extracts namely Lannea schimperi and Searsia longipes. Briefly, 4 mg/ml stock solutions of the aforementioned extracts were prepared by dissolving an appropriate amount of the extract in distilled water, and the dissolubility was facilitated by heating the suspension at 370C for 15 minutes.
Following transformation of the cercariae stage of Schistosoma mansoni to schistosomula stage, bioassays were thereafter performed on 24 multi well plate to validate the activity of the plants extract against the latter stage. In this case, schistosomula were subjected to different concentrations of the methanolic plants extracts as previously described in cercariae bioassay. Briefly, 4 mg/ml stock solutions of the plant extracts were prepared after dissolving 20 mg of the extract powder in approximately 5 ml of the RPMI 1640 (SIGMA) media.
This test was performed to evaluate the activity of Lannea schimperi and Searsia longipes methanolic extracts against adult stage of the Schistosoma mansoni. In this case, previously perfused adult worms of both sexes were subject to different concentrations of the Lannea schimperi and Searsia longipes methanolic extracts. In a nutshell, for each extract 8 mg/ml stock solution was prepared in a graduated eppendorf tube. Afterward, from the stock solutions 6 serial concentrations of 2 mg/ml, 1 mg/ml, 0.5 mg/ml, 0.25 mg/ml, 0.05 mg/ml and 0.025 mg/ml were prepared in the 6 well plates in duplicate. RPMI 1640 (SIGMA) media supplemented with 10% FBS, 1% Penicillin and streptomycin was used for dilution as well as culture media. Additionally, wells containing only RPMI 1640 and 0.01 mg/ml praziquantel were used as negative and positive control respectively. Thereafter, 10 adult worms (5 females and 5 males) were placed in each well and each well contained a final volume of 5 milliliter. Monitoring was done from 0 to 48 hour in 24 hours interval. Henceforth, activity of the plant extracts was assessed under dissection microscope based on loss motility, mortality and structural changes .
Data were organized by Microsoft excel and analyzed by using graph pad prism software, whereby overall statistically significant different between treatments mean were determined by using one way ANOVA at p value of < 0.05. Further, multiple comparison between treatments and control were done by using Tukey Kramer test at p value of < 0.05.
Following In vitro bioassay on schistosomula stage of the Schistosoma mansoni, both plants exhibited significant anti-schistosomal activity at all tested concentrations. Additionally, both extracts were able to exhibit 100% mortality at concentration range of 0.25 mg/ml up to 2 mg/ml, whereby completely loss of motility was observed following 6 hours of schistosomula exposition. Regarding statistical analysis done using one way ANOVA at p value of < 0.05, overall significant difference between tested concentrations were observed. Further, statistically significant differences were also divulged when multiple comparison of the viability mean of each treatment were tested against that of the control by using Tukey Kramer test at p value of < 0.05. Moreover, time and concentration based reduction of schistosomula viability was also observed as depicted in Figure 3 and 4.
For in vitro bioassay against cercariae stage, both extracts exhibited significant activity manifested through reduction of the cercariae viability. Whereby, overall significant different between viability means were observed following statistical analysis by using one way ANOVA at p value < 0.05. Further, multiple comparison between treatments and control were done by using Tukey Kramer test at p value < 0.05 and statistically significant differences on viability means were observed up to the lower concentration of 0.05 mg/ml for Searsia longipes extract. Whilst for Lannea schimperi extract, significant differences in viability means were observed up the lowest concentration used. Furthermore, in all tested plants extracts concentration based reduction of cercariae viability were observed, particularly after 6 hours of exposition as depicted in Figure 5 and 6. Meanwhile, concentration and time dependent cercariae motility reduction were also manifested as shown on Table 1. Nevertheless, at the concentrations less than 0.5 mg/ml Lannea schimperi was able to induce tail detachment.
|Concentration||0 Hrs||1 Hrs||3 Hrs||6 Hrs|
Table 1: Motility of the live cercariae following exposure to different concentrations of Lannea schimperi and Searsia longipes methanolic extracts.
Searsia longipes methanolic extract (SLM) has divulged concentration based reduction of worm viability, where the viability reduction was observed to be high as concentration increased. At the three highest concentrations of 2 mg/ml, 1 mg/ml, and 0.5 mg/ml, worm viability was reduced to 0% after 48 hours of exposition as depicted in Table 3. Despite the effect on the viability of the worm, the extract has also demonstrated the concentration dependent reduction of worm motility especially at the concentrations distal to the lethal concentration. Meanwhile, Lannea schimperi methanolic extract (LSM)has exhibited 100% anti-schistosomal activity of all worms at two highest concentrations of 2 mg/ml and 1 mg/ml after 48 hours of exposition (Table 2). However at the concentration lower than 1 mg/ml the extract was observed to have no effect on the motility of the worms, hence the worms were highly motile even after 48 hours of exposure. Moreover, males worm were observed to be more susceptible to all tested concentrations of the extracts than female, whereby 100% mortality were observes at the concentration range of 0.025 to 2 mg/ml and 0.05 to 2 mg/ml for Searsia longipes and Lannea schimperi respectively. Additionally, dead male’s worm were also demonstrated fragmentation and tightly coiled characteristic as depicted on Figure 7. The viability of the control group subjected to RPMI 1640 only was 100% up to the end of the observation time.
|Concentration||0 Hrs||24 Hrs||48 Hrs|
bPositive control (0.01 mg/ml of Praziquantel)
Table 2: Percentage viability of the adult schistosome worms from 0 to 48 hours of exposure to different concentrations of Lannea schimperi methanolic extract.
|Concentration||0 Hrs||24 Hrs||48 Hrs|
bPositive control (0.01 mg/ml of Praziquantel)
Table 3: Percentage viability of the adult schistosome worms from 0 to 48 hours of exposure to different concentrations of Searsia longipes methanolic extract.
This study piggy back rode on a Kenya Medical Research Institute approved protocol, KEMRI/SERU/CBRD/PROP164/3406.
The authors declare to have no conflict of interest.
This research was funded by Nelson Mandela African Institution of Science and Technology through African Development Bank scholarship project. Moreover, heartfelt thanks are extended to KEMRI for the provision of the laboratory space, consumables and all necessary technical assistance.
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