Bioactive Compounds, Antioxidant, Anti-Inflammatory, Anti-Cancer, and Toxicity Assessment of Tribulus terrestris-In Vitro and In Vivo Studies

Abstract

Tribulus terrestris L. belongs to the family Zygophyllaceae and integral part of various ancient medicinal systems including Chinese, Indian, and European to combat various health ailments. The aim of the present study was to assess the phytochemical constituents, in vitro antioxidant activity using DPPH, FRAP, and H₂O₂ assays, in vitro anticancer activity using MTT assay, and in vitro and in vivo anti-inflammatory properties of T. terrestris. The acute and sub-acute toxicity of extracts exhibiting most biological potential was examined using murine models. Liquid-liquid partitioning followed by RP-HPLC sub-fraction of crude extract was performed. After that, ESI-MS/MS analysis was done for the timid identification of bioactive metabolites responsible for bioactivities of sub-fractions and HPLC analysis to quantify the compounds using external standards. Among all extracts, T. terrestris methanol extract was noted to hold maximum phenolic (341.3 mg GAE/g) and flavonoid (209 mg QE/g) contents, antioxidant activity in DPPH (IC₅₀ 71.4 µg/mL), FRAP (35.3 mmol/g), and H₂O₂ (65.3% inhibition) assays, anti-inflammatory activities in vitro at 400 µg/mL (heat-induced hemolysis, % inhibition 68.5; egg albumin denaturation, % inhibition 75.6%; serum albumin denaturation, % inhibition 80.2), and in vivo at 200 mg/kg (carrageenan-induced paw edema, % inhibition 69.3%; formaldehyde-induced paw edema, % inhibition 71.3%) and anticancer activity against breast cancer cell (MCF-7) proliferation (IC₅₀ 74.1 µg/mL). Acute and sub-acute toxicity studies recorded with no change in body weight, behavior, hematological, serum, and histopathological parameters in treated rats with T. terrestris methanol extracts when compared to control group. Fraction B obtained through liquid-liquid partitioning resulted in more bioactive potential as compared to the parent methanol extract. RP-HPLC analysis of fraction B resulted with four sub-fractions (TBTMF1-TBTMF4), wherein TBTMF3 delineated notable bioactive capabilities as compared to other fractions and parent methanol extract. ESI-MS/MS analysis of TBTMF3 resulted with tentative identification of myricetin, rutin, liquitrigenin, physcion, and protodioscin. It can be stated that T. terrestris is a potential bearing herb and findings of current study further verify the claims made in ancient medicinal systems. However, after investigation of each identified compound, it must be considered for drug discovery.

Keywords: ESI-MS/MS; RP–HPLC; antioxidant; bioassay-guided fractionation; cancer; inflammation; liquitrigenin; myricetin; phytochemicals; rutin; toxicity.

Figure 1

(A) Carrageenan-induced and (B) formaldehyde-induced paw edema inhibition. Values are mean ± SEM of three readings. The standard drug (Indomethacin) exhibited potent inhibition of 86.3% (p < 0.0001) and 89.3% (p < 0.0001) against carrageenan and formaldehyde intoxicated paw edema at 100 mg/kg, respectively when compared to control (normal saline = 0% inhibition). On the other hand, dichloromethane and 70% aqueous methanol extracts induced moderate to non-substantial inhibition in both assays.

Figure 2

Histopathology results of acute and subacute toxicity of T. terrestris methanol extract.

Figure 3

Overlay of HPLC-chromatograms of standards (blue) and the TBTMF3 fraction (red) recorded at 280 nm by HPLC-DAD.