In silico, in vitro, and in vivo acute and sub-acute toxicity profiling of whole plant methanol extract of Equisetum diffusum D. Don from the sub-Himalayan West Bengal, India, having ethnobotanical uses

Abstract

Background: Equisetum diffusum D. Don commonly known as 'Himalayan horsetail', has been traditionally used in the treatment of back pain, bone fracture and dislocation, and arthritis by various tribal communities of India. Our previous study confirmed the anti-inflammatory efficacy of the plant through in silico, in vitro, and in vivo model studies. Therefore, the current research is focused on safety dose evaluation for the first-time of the whole-plant methanol extract (EDME) of E. diffusum through appropriate in silico, in vitro, and in vivo approaches.

Method: The whole plant, along with its rhizomes, was collected, and the methanol extract was prepared. The in silico ADMET study was performed to predict the pharmacokinetics profile and toxicity of all the identified phyto-compounds of EDME previously screened by GC-MS study. In vitro cytotoxicity study of EDME was performed using two cell lines: kidney (HEK293) and liver (Huh7) cell lines. The in vivo toxicity study of EDME was validated by the acute toxicity (OECD 423, 2002) and sub-acute toxicity assays (OECD 407, 2008) in the Wistar Albino rat model.

Results: The in silico ADMET study of all 47 bioactives predicted good pharmacokinetic and low toxicity profiles. In vitro cytotoxicity showed higher IC₅₀ values of EDME viz., 672 ± 15.7 μg/mL and 1698 ± 6.54 μg/mL for both kidney (HEK293) and liver (Huh7) cell lines, respectively, which were considered as low-toxic. Based on acute oral toxicity, the LD₅₀ value of the extract was considered "non-toxic" up to a feeding range of 2000 mg/kg of body weight. The regular consumption of the extract for an extended period (28 days) was also qualified as safe based on the body and organ weight, hematological, biochemical, and histoarchitecture results in the sub-acute toxicity assay.

Conclusion: The detailed in silico, in vitro, in vivo (acute and sub-acute oral toxicity) studies gave us a new insight to the safety dose evaluation of Equisetum diffusum, which may serve as a reliable documentation for undertaking the experimental validation of the ethnobotanical uses of the plant which would help in the field of drug development for the treatment of inflammation related complications.

Keywords: Equisetum diffusum D. Don; ADME-Toxicity; Acute toxicity; HEK293; Huh7; Sub-acute toxicity.

Fig. 1

GC–MS chromatogram of Equisetum diffusum D. Don whole plant methanol extract

Fig. 2

Cytotoxicity of E. diffusum whole plant methanol extract (EDME) against two cell lines. A HEK293, kidney cell line; B Huh7, liver cell line

Fig. 3

Body weight of rat of both sexes during acute toxicity study with different doses of EDME (A-Male; B-Female)

Fig. 4

Histological sections of major organs of different experimental groups. ‘Li’ denotes Liver (A-D); ‘Ki’ denotes Kidney (E–H); ‘Sp’ denotes Spleen (I-L); ‘St’ denotes Stomach (M-P) and ‘In’ denotes Intestine (Q-T). The first column represents normal control group, followed by vehicle control group, low dose group, and high dose group. The main images represent sections observed under 10X magnification; insets represent 40X magnified images. Scale bars are 100 µm in 10X and 25 µm in 40X. Following yellow markings are the labelling of histological structures: Hepatocytes (H); Hepatic artery (Ha); Portal vein (PV); Sinusoids (S); Kupffer cell (K); Central vein (CV); vascularised Glomeruli (G); Urinary space (Us); Bowman’s capsule (Bc); White pulp (Wp); Red pulp (Rp); Gastric gland (Gg); Mucosal layer (M); Sub-mucosal layer (SM); Intestinal villi (Iv); Duodenal/Brunner’s gland (Dg)