Toxicity and anti-prolific properties ofXysmalobium undulatumwater extract during short-term exposure to two-dimensional and three-dimensional spheroid cell cultures

Abstract

Xysmalobium undulatum (Uzara) is one of the most widely used indigenous traditional herbal remedies in Southern Africa. Commercially available Uzara plant material was used to prepare a crude aqueous extract, of which the toxicity potential was investigated in the hepatic HepG2/C3A cell line in both traditional two-dimensional (2D) and rotating three-dimensional (3D) spheroid cell cultures. These cultures were treated over a period of 4 days at concentrations of 200, 350, 500, and 750 mg/kg plant extract to protein content. Basic physiological parameters of the cell cultures were measured during exposure, including cell proliferation, glucose uptake, intracellular adenosine triphosphate levels, and adenylate kinase release. The results indicated that all physiological parameters monitored were affected in a dose dependent manner, with the highest concentration of Uzara crude water extract (750 mg/kg) resulting in toxicity. Anti-proliferating effects of Uzara crude water extract were observed in both the 2D and 3D cell cultures, with the most pronounced effects at concentrations of 350, 500, and 750 mg/kg. Discrepancies between results obtained from the 2D and 3D cell culture models may be attributed to the type of repair system that is initiated upon exposure, depending on where cells are within the cell cycle. DNA repair systems differ in cells within the G1 phase and non-diving cells, (i.e. cells found predominantly in in vitro 3D and the in vivo situation).

A sub-chronic Xysmalobium undulatum hepatotoxicity investigation in HepG2/C3A spheroid cultures compared to an in vivo model

“…A statistically significant decrease in the growth curve of the valproic acid compared to the untreated control group (p < 0.0001) was observed on day 21, as well as the crude X. undulatum aqueous extract at 200 mg/kg and 250 mg/kg relative to the untreated control (p < 0.0001). This growth inhibition corresponds well to previous studies with valproic acid and crude X. undulatum aqueous extract at various concentrations (Wrzesinski & Fey, 2012;Calitz et al, 2018). The crude X. undulatum aqueous extract contains the cardenolide uzarin, which has been shown to possess cytotoxic and anti-prolific effects (Krishna et al, 2015;Calitz et al, 2018).…”

“…We have previously reported (Calitz et al, 2018) the incidence of hepatotoxicity and anti-prolific activity associated with acute administration of a crude X. undulatum aqueous extract at concentrations 200, 350, 500 and 750 mg/kg in both two-dimensional (2D) and three-dimensional (3D) cell culture models. Furthermore, there has been reports of a toxic digitalis-like action on the heart when X. undulatum is administered in extremely high concentrations (Vermaak et al, 2014).…”

“…A crude X. undulatum aqueous extract was prepared and characterized, and the results previously published (Calitz et al, 2018). Briefly, commercially available dried and milled X. undulatum material was purchased from Afrinatural holdings (Prestige laboratory supplies, Mt Edgecombe, Johannesburg).…”

“…The chemical composition of the X undulatum aqueous extract was determined on a Waters Acquity Ultra Performance Liquid Chromatographic system with PDA detector (Waters, Milford, MA, USA), and the presence of the major active constituent, uzarin, was confirmed and quantified (226.4 µg/mg X. undulatum (n = 2)), as previously published (Calitz et al, 2018).…”

“…The test substances consisted of 200 and 250 mg/kg crude X. undulatum aqueous extract in water and 300 mg/kg valproic acid in phosphate buffered saline (PBS) as positive hepatotoxicity control, with the control group receiving only PBS. Treatment concentrations for X. undulatum were based on available literature and previously acute and sub-chronic in vitro studies (Vermaak et al, 2014;Calitz et al, 2018), while the valproic acid concentration was based on its EC 50 (485 mg/kg) and LD 50 (600 mg/kg) values in rodents (Lӧscher, 2007;Lee et al, 2008;Drugbank, 2017). Fresh stock solutions of the test substances were prepared daily and diluted according to the weight of each test subject prior to administration thereof.…”