Plant material
Plant material was collected from natural habitats in Kokrajhar, Assam, India and identified by a taxonomist. Herbarium was made and submitted to the Department of Botany herbarium museum and assigned a voucher number (NEHU-12098). A copy of the herbarium has been retained in the Parasitology and Ethnopharmacology Lab. The taxonomic status of plant was also verified with the database of Kew working list of all known plant species (www.theplantlist.org). The roots were washed in tap water, air-dried and powdered in a blender. The powdered plant material was extracted in methanol using Soxhlet apparatus. The yield of extract was 7.98 % (w/w).
Experimental animals
Albino rats of either sex (Wistar strain), weighing about 180 to 220 g, and Swiss mice of both either sex, weighing about 25 to 30 g were used to perform in vivo experiments. Animals were maintained in acrylic cages and had ad libitum access to food and water. S. obvelata infection was maintained in Swiss mice as described by Gogoi and Yadav (2016) [18]. Infection of H. diminuta was maintained in Wistar rats as described by Tangpu et al. (2006) [19].
All the experiments on laboratory animals were done after the approval from the Institutional Ethics Committee (Animal models), North-Eastern Hill University, Shillong, vide, Member Secretary, IEC, NEHU, letter dated December 4, 2014. All experiments on animals adhere to the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines, the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines and the EU Directive 2010/63/EU for animal experiments.
Ethnomedicinal survey
Firstly, data was collected from traditional healers, called as Ojhas, and local people, using a closed type semi-structured questionnaire, about different medicinal plants used against intestinal-helminthic infections in different villages of Kokrajhar district, Assam, India. The study involved 300 respondents, interviewed from April, 2014 to April, 2015. The questionnaire included queries such as, the local names of various plants that are used by the natives against worm infections, the form of their preparations, their dosage, perceived efficacy, and opinion about adverse effects.
Anthelmintic assays
In vitro study
H. diminuta and S. obvelata were collected from infections maintained in rats and mice respectively in the laboratory. The parasites were washed in phosphate-buffered saline (PBS) and exposed to 10, 20 and 30 mg/ml concentrations of extract in separate petridishes (n = 5) inside an incubator at 37 ± 1 °C. A group of worms (n = 5) were placed in PBS alone and served as negative control. In addition, another set (n = 5) was placed in reference drugs, praziquantel (PZQ) (1 mg/ml) and albendazole (ABZ) (5 mg/ml). Observations for parasites paralysis and mortality were made at regular time intervals [20].
In vivo study
For in vivo testings in H. diminuta, rats were orally fed with 5 cysticercoid larvae to induce infection. Animals were divided into 5 groups (n = 5). Group 1 served as negative control and was administered only the vehicle. Group 2 to Group 4 of animals were administered with 125 mg/kg, 250 mg/kg, and 500 mg/kg doses of extract to animals. Group 5 of animals served as positive control and were administered PZQ at 5 mg/kg concentration. Eggs per gram (EPG) counts were done counts were done 3 days prior to and after the dosings. Animals were sacrificed on the 29th day post-inoculation to calculate the worm burden.
For in vivo testings in S. obvelata, mice were kept in infected bed for 15 days. Establishment of infection was confirmed by cellophane tape test. Animals were divided into 5 groups, with 5 animals in each group. Group 1 served as negative control and was administered only the vehicle. Group 2 to 4 of animals were given 125 mg/kg, 250 mg/kg, and 500 mg/kg doses of extract. Group 5 of animals were administered ABZ (20 mg/kg). EPG count was done for 3 days prior to and after dosing. The animals were dosed for 5 days and they were sacrificed for worm recovery count on day 11. The percentage reductions in EPG and worm counts were done as described by Kozan et al. (2006) [21].
The prescribed dose administered by practitioners to their clients was taken as the median dose i.e., 250 mg/kg body weight (b.w.) and two doses, one exponentially lower (125 mg/kg b.w.) and the other higher (500 m g/kg b.w.) to the median dose were selected.
Statistical analysis
The experimental data are represented as mean ± standard error of mean (SEM). The in vitro data were analyzed by student’s t-test and the p value ≤ 0.05 was considered to be statistically significant between the values from control and treated groups. The data from in vivo tests was analyzed by one-way analysis of variance (ANOVA), followed by Tukey’s test. The p value ≤ 0.05 was considered to be statistically significant. Statistical calculations were done using OriginPro 8.