Plant material
Fresh roots of C. compressus were collected from fields in Kokrajhar, Assam, India. They were brought to the laboratory, washed with tap water, shade dried, powdered, and extracted with methanol. Herbarium of plant material was made and identified by a plant taxonomist from the Department of Botany in the same institute. It was identified by comparing with existing specimen and an accession number was assigned. The herbarium specimen has been deposited in the herbarium museum of the Department of Botany and a copy of the herbarium specimen has also been retained in the laboratory.
Experimental animals
Swiss albino mice of either sex (25–30 g) were used for in vivo studies against Syphacia obvelata and Wistar rats of either sex (150–190 g) of about 7 to 8 weeks were used for in vivo studies against Hymenolepis diminuta. All the animals used were procured from the laboratory of the Department of Zoology. They were inbred and maintained in separate cages in adequate laboratory conditions at 22 °C ± 3 °C. They had ad libitum access to food and water and cages were cleaned once daily. Adequate lighting to maintain 12 h day and 12 h night cycle was provided [17]. Approval and permission to use the animals was obtained from the Institutional Ethics Committee (IEC) (animal models). All experiments on laboratory animals were performed strictly according to the IEC and ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines.
Phytochemical analysis
The plant material was subjected to various qualitative tests to detect the presence of phytochemicals using standard protocols as described by Trease and Evans [18] and Sofowora [19].
In vitro anthelmintic study
Test parasites were collected from freshly necropsied rodents. Parasites (n = 5) were placed in three different concentrations of the extract, namely 10, 20, and 30 mg/ml in triplicates. Another set of parasites were placed in reference drug praziquantel (PZQ, 1 mg/ml) in case of H. diminuta and albendazole (ABZ, 5 mg/ml) in case of S. obvelata to compare the efficacy and a final set of worms were placed in phosphate-buffered saline (PBS) which served as control. Parasites were monitored for paralysis and mortality at regular intervals and the time taken for these events to occur were recorded and compared with that of the reference drug [4].
In vivo anthelmintic study
Adult Wistar rats pre-infected with cysticercoids (n = 5) were used for the in vivo study. Infection was maintained in the laboratory by feeding the intermediate host, Tribolium confusum with gravid segments. Prior to inoculation, T. confusum were starved for 48 h and restored to their diet after 72 h. After 28 days, they were dissected to retrieve the cysticercoids which were then inoculated to the rats. Establishment of infection was confirmed by the presence of gravid segments in the fecal matter after 18–20 days [4]. Against S. obvelata, infection was maintained in laboratory Swiss albino mice, by placing fecal matter containing eggs in the cages of uninfected mice. After ingestion, adults develop after a period of 18–20 days [8]. Animals in which parasite infection was established were used to carry out the study.
Animals were administered three different doses of the plant extract. Three doses were selected on the basis of the dose administered by traditional practitioners to their clients to compare the effects [8]. Accordingly, the prescribed dose of 350 mg/kg b.w. was taken as the median dose and two other doses, one exponentially lower (175 mg/kg b.w.) and one higher were selected (700 mg/kg b.w.) to compare the effects. One group of animals received reference drug, PZQ (5 mg/kg b.w.) in case of cestodes and ABZ (20 mg/kg b.w.) in case of nematodes, whereas the final group received only the vehicle (phosphate-buffered saline) and served as negative control. Each of the 5 groups comprised of 5 infected animals (n = 5) and a total of 25 animals were used for each in vivo study. Animals were dosed once daily in the morning for 5 days [17] and prior to sacrifice, they were euthanized using chloroform. After the study, the remains of the deceased animals were burnt in an incinerator. Animals were maintained and handled as per Organization for Economic Co-operation and Development (OECD) guidelines [17] and IEC.
Statistical analysis
Data obtained from the experiments were processed using OriginPro 8. All the experimental data are represented as mean ± standard error of mean (SEM). In vitro tests data were analyzed by Students’ t test and data from in vivo anthelmintic tests were analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test for multiple comparisons. p value ≤ 0.05 was considered to be statistically significant.