Chemicals
The following chemicals were procured from the respective companies to carry out this study. Colchicine (Sigma-Aldrich), disodium hydrogen phosphate (Na2HPO4) (Himedia), DPX (SDFCL), Giemsa stain (Sigma-Aldrich), glacial, acetic acid (Himedia), methanol (Rankem), nitric acid (HNO3) (Himedia), potassium chloride (KCl) (SDFCL), potassium dihydrogen phosphate (KH2PO4) (Rankem), sodium chloride (NaCl) (Sigma-Aldrich), and sulphuric acid (H2SO4) (Himedia).
Plant material
Fresh leaves of S. sesban var. bicolor and roots of C. compressus, and A. racemosus were collected from natural habitats in Assam with the help of traditional healers. Herbariums were made and identified by a plant taxonomist from the Department of Botany, North-Eastern Hill University (NEHU) and voucher specimen numbers were assigned. Plant materials were washed, shade dried and powdered in a kitchen mixer. The plant materials were then extracted in methanol using Soxhlet apparatus and stored at 4°C in vials. The powdered plant material and plant methanolic extracts were used to carry out further analysis.
Experimental animals
Laboratory inbred Wistar strain rats weighing about 150 to 200 g which were procured from the animal room of the Department of Zoology, NEHU, were used to perform genotoxicity studies. Animals were kept in separate cages with ad libitum access to water and food. Experiments in laboratory animals was performed after due approval from the Institutional Ethics Committee (IEC). All the animals were handled strictly according to the guidelines stated by the IEC and comply with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines.
Genotoxicity studies
Genotoxicity studies were conducted on Wistar rats following the protocol of Guo and Wu (2008) with minor modifications. The animals were dosed according to the dosage administered by traditional healers to their clients. The recommended dose was taken as the median dose and two doses, one exponentially lower and the other higher that the median doses were selected for the study. Accordingly, animals were dosed 100, 200 and 400 mg/kg b.w. of S. sesban var. bicolor leaf extract, 175, 350 and 700 mg/kg b.w. of C. compressus root extract and 125, 250 and 500 mg/kg b.w. of A. racemosus root extract.
Animals were divided into three groups, with five animals in each. Group 1 served as the control and received only the vehicle. Group 2 and 3 received highest and lowest dose respectively. All the animals were dosed for 14 days. At the end of the dosing period, the animals were subjected to mitotic arrest by injecting colchicine intraperitonially (4mg/kg). The animals were then sacrificed after 2 h, according to the prescribed guidelines of IEC (Animal Model) of NEHU.
The femurs were dissected out and flushed with pre warmed (37±1°C) 0.06 M KCl through a 40 μm cell strainer into a 50 ml centrifuge tube. The samples were kept in Carnoy’s fixative then centrifuged at 1500 rpm for 15 min (4 times) with KCl. After each centrifugation, the supernatant was discarded and the pellet was used to carry out further examinations. Finally, the cells were resuspended in 2 ml fixative and stored at -20° C for further use.
To make the slides, a few drops of the cell suspension was dropped on grease free, methanol cooled slides from a height of about 1 m. The slides were then warmed in flame for 2 seconds to allow the cells to spread following which they were allowed to dry, and then stained in 5% buffered Giemsa stain, (pH – 7) for 10 minutes. The slides were then washed in distilled water to remove excess stain and allowed to dry. The slides were mounted in DPX and observed in oil at 100 x for various chromosomal aberrations which included chromatid breaks (CB), isochromatid breaks (ICB), chromosomal fragments (CF), exchanges (E) and sister chromatid unions (SCU). To score various chromosomal aberrations, about 100 well spread metaphase plates were examined per animal (Nath et al., 2017).
Elemental analysis
To perform elemental analysis, the powdered plant material (1 g) was subjected to acid digestion using HCl and HNO3 in the ratio 2:1. The acid mixture was made to evaporate in a hot plate following which distilled water was added and then filtered. The filtrate was made to a final volume of 50 ml then subjected to elemental by inductively coupled plasma-optical emission spectrometry (ICP-OES) using Thermo Fischer iCAP 7600. Essential elements such as chromium (Cr), cobalt (Co), copper (Cu), iron (Fe), manganese (Mn) and nickel (Ni) were estimated. Toxic heavy metals arsenic (As), cadmium (Cd), and lead (Pb) were also evaluated. The values were compared with the WHO/FAO permissible limits (ppm) and inferences were drawn (WHO, 1998; FAO/WHO, 1999; WHO, 2005; Valadez-Vega et al., 2011; Shah et al., 2013; Dabanovic et al., 2016).
Statistical analysis
The results obtained in the study were analysed using OriginPro 8. All data are represented as mean ± standard error of mean (SEM). P≤0.05 was considered to be significantly different.