Plant material and preparation of E. diffusum whole plant methanol extract (EDME)

The whole plant of Equisetum diffusum D. Don has been collected from the foothills of the northern part of West Bengal (Darjeeling hilly region). The plant specimen was identified by Dr. R. K. Gupta, Scientist ‘E’ of the Central National Herbarium, Botanical Survey of India (BSI), Howrah, India. A voucher specimen number NBU/SS-002 was deposited in Central National Herbarium, BSI, India. The whole plant methanol extract of the E. diffusum (EDME) was prepared following a previously established procedure [17]. The extract (EDME) was then stored in an airtight container at 4 °C for future experimental usage. For animal feeding, the extract was reconstituted in 0.5% carboxymethylcellulose (CMC).

In silico toxicity study ADME-toxicity study

From the previous GC–MS data [17], the canonical SMILES (Simplified Molecular Input Line Entry System) format and 2D SDF (Structure Data File) format of all the phyto-compounds were retrieved from the NCBI (National Center for Biotechnology Information) PubChem database [24]. Then, in the Maestro flatform, ligands were prepared in the LigPrep module of Maestro (LigPrep, Maestro Version 12.5.139, Schrödinger, LLC, New York) using the OPLS3 (Optimized Potentials for Liquid Simulations) force field. The assumption of the pharmacokinetics profile and toxicity prediction of all the potential compounds was conducted using the QikProp module of Maestro (QikProp, Maestro Version 12.5.139, Schrödinger, LLC, New York), Toxicity descriptor algorithm TOPKAT (Toxicity Prediction by Komputer Assisted Technology) in the BIOVIA Discovery Studio, and ADMETlab 2.0 server [25].

In vitro toxicity study Cytotoxicity assay

To determine the cytotoxicity of the plant extract, the spectrophotometric MTT (3-(4, 5-dimethyl thiazolyl-2)-2, 5-diphenyltetrazolium bromide) (HiMedia, India) test was conducted as per standard protocol with slight modifications [26]. Briefly, 5 × 103 HEK293 cells and 2 × 104 Huh7 cell line were cultured in DMEM (Dulbecco's Modified Eagle Medium) culture medium (HiMedia, India) with 10% FBS (Foetal Bovine Serum) and 1% antibiotic solution at 37 °C with 5% CO2 in a CO2 incubator for 24 h. After 24 h of incubation, each well was treated with different concentrations of EDME and incubated at 37 °C with 5% CO2 for 24 h. Next, MTT (5 mg/ml) dissolved in Phosphate buffer saline (PBS) was added in each well and incubated for 3 h to allow it to form purple-colored formazan crystals. Finally, Dimethyl sulfoxide (DMSO) (Sigma Aldrich, USA) was added to dissolve the formazan crystals and was incubated for 15 min at room temperature. After the incubation period, absorbance was recorded at 570 nm/590 nm in a multi-mode microplate reader (BioTek SynergyTM H1, Vermont, USA). All experiments were replicated thrice independently to calculate cell viability using the following equation: Cell viability (%) = (OD Treated/OD Control) × 100.

In vivo toxicity study Experimental animals

For the in vivo studies, Wistar albino rats of both sexes (8–12 weeks old; 120 ± 10 g) were used. Rats were purchased from an authorized animal dealer (M/s Chakraborty Enterprise, Kolkata, India; Regd. No. 1443/PO/Bt/s/11/CPCSEA). All the animals were kept in polypropylene cages (max. 4 rats per cage) and were maintained at a room temperature of 25 ± 3 °C. Rats were supplemented with standard feed and water ad libitum. The animals were kept in the animal house of the Department of Zoology, University of North Bengal, where they were acclimatized to laboratory conditions for 7 days before the commencement of experiments. The experimental procedures were approved by the Institutional Animal Ethical Committee (Approval number: IAEC/NBU/2018/03) (IAEC) of CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals) (now CCSEA or Committee for Control and Supervision of Experiments on Animals) of the University of North Bengal, West Bengal, India.

Acute toxicity studies

Acute toxicity test was performed as per the Organization for Economic Cooperation and Development guidelines (OECD) 423 [27]. In this test, Wistar albino rats (120 ± 10 g) of both sexes were categorized into 5 groups; each contained 6 rats; 3 males and 3 females. The first group contained normal animals receiving only normal water; the other four groups were considered as experimental dose groups. In the experimental dose groups, EDME was administrated orally by using gavage in single doses of 250 mg/kg, 500 mg/kg, 1000 mg/kg, and 2000 mg/kg of body weight on day one. All animals were then observed at 30 min, 1 h, 2 h, 4 h, 6 h, 12 h, and 24 h and, thereafter, for 14 days for any delayed toxicological effects. Animals were observed daily for any behavioral change, such as consumption rate of food and water, aggressiveness, sedation, diarrhea, rising of fur, lethargy, mortality, and morbidity till the end of the experimental tenure. The body weight of the rats from each experimental group was recorded at day 0 followed by each week till the end of the experimental schedule. Based on acute toxicity results, the doses for the sub-acute toxicity study were selected.

Sub-acute toxicity studies

Sub-acute toxicity test was performed according to the OECD guidelines 407 [28]. In this experimental set, Wistar albino rats (120 ± 10 g) of both sexes were categorized into 4 groups; each group contained 6 rats (n = 6); which were further subdivided into 3 males and 3 females separately. Group I represented the normal control group and were administered with normal water for 28 days orally. Group II was considered as a low-dose group and received 250 mg/kg body weight of EDME. Group III was considered as a high- dose group and the animals in this group received 500 mg/kg body weight of EDME. Group IV served as vehicle control and was administered with 0.5% CMC orally for 28 days. All the animals from both the dose groups (group II and group III) received the treatment orally once daily at a fixed time continually for 28 days. During the experimental schedule, all the rats were observed for any abnormal behavioural signs, food intake, fur irritation, lethargic behaviour, mortality, and morbidity till the completion of the experiment. At the end of the experimental period, all the rats were kept fasted overnight (on day 28) and were sacrificed by cervical dislocation after euthanizing with sodium pentobarbital (60 mg/kg; i.p.) on the 29th day following standard procedure [29].

Body weight and relative body organ weight

Body weight from each experimental group was recorded before commencing the experiment on day 0. After starting the experimental tenure, body weight from each experimental group was measured at the end of each week till the end of the experimental schedule. On the 29th day, before the sacrifice, the final body weights were recorded, and immediately after the sacrifice, body organs (liver, kidneys, and spleen) were dissected out, and measured separately to obtain their absolute organ weight. From the absolute organ weight value, the relative organ weight of each animal in all the experimental groups was calculated by this formula [30]: Relative organ weight (ROW) = Absolute organ weight (g) × 100/ Body weight of rat on day of sacrifice (g).

Blood sample collection

Blood samples were collected by cardiac puncture into EDTA-coated vials (for hematological parameters) as well as in non-EDTA-coated vials (for serum parameters) following standard protocol [30].

Hematological analyses

For hematological parameters, collected blood samples (in EDTA-coated vials) were analysed using an automated hematology analyser (Sysmex XN-1000, Mumbai, India) following standard protocol [29]. The following parameters were considered for hematological analyses: red blood cell count (RBC), white blood cell count (WBC), hemoglobin (Hb) content, platelets (Pt) count, neutrophils (NP), lymphocytes (LC), monocytes (MC), eosinophils (EP), basophils (BP), hematocrit (HCT), Mean Red Blood Cell Volume (MCV), Mean Corpuscular Hb (MCH), Mean Corpuscular Hb Concentration (MCHC), and Mean Platelet cell Volume (MPV).

Biochemical parameters

For serum analyses, blood samples collected into non-EDTA coated vials were centrifuged (5000 rpm for 10 min, 4ºC) and serum was collected. The collected serum samples were analysed for different biochemical parameters. To assess the liver function alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total protein content, and albumin content were measured. The measurement of urea and creatinine concentrations was done to evaluate kidney function. The amount of total cholesterol, triglyceride, HDL-C, and LDL-C were determined to evaluate the effect of the extract on the lipid profile. In addition, the amount of glucose and electrolytes (Sodium and Potassium) were also measured. All the biochemical parameters were measured using commercial Coral Kits (Coral clinical systems, India) following the manufacturers’ protocol using a spectrophotometer (UV-1900i VIS-spectrophotometer, Shimadzu, Japan).

Histopathological examinations

Organs such as liver, kidney, spleen, stomach, and intestine were collected immediately after sacrifice from the rats of each group, washed with chilled PBS (pH 7.4), and fixed in 4% formalin. After fixation, the tissues were dehydrated using serial dilutions of ethanol, followed by embedding in paraffin wax. Tissues were then cut at 5 μm thickness and stained with hematoxylin–eosin. The tissues were then examined under a light microscope (Nikon Eclipse E200, Nikon, Tokyo, Japan) with 10X and 40X objectives and a scale bar was attached (100 µm and 25 µm) respectively [29].

Statistical analyses

Quantitative data concerning the body weight, and organ weight data was expressed as mean ± standard error mean (SEM). For the remaining assays, all data were expressed as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) or two-way ANOVA, following the post hoc analysis with Dunnett’s multiple comparisons test, was performed. Values of p ≤ 0.05 were taken to indicate a statistical difference. All the statistical analyses were performed using GraphPad Prism Version 7.00 (San Diego, United States of America).