Chemical profile and toxicogenetic safety assessment of Smallanthus sonchifolius (yacon) organic extracts

Abstract The interest in Smallanthus sonchifolius (yacon) has strongly resurfaced due to its multiple beneficial effects on human health. This study aimed at determining the toxicity and the chemical profile of an ethanol extract (EE) and a crude lactone mixture (CLM) of yacon leaves. Cytotoxicity and genotoxicity tests were performed by the MTT assay and the alkaline version of the comet assay respectively. The phytochemical analysis, performed by chromatographic and spectroscopy techniques, revealed the presence of nine sesquiterpene lactones (STLs) and two acyclic diterpene acids. In all cases, cell viability was inversely proportional to the extract concentration employed. The effects obtained with the highest dose of EE were significantly different from those obtained with the negative and solvent controls. Conversely, no significant differences were observed between the lowest doses of EE and controls. As for CLM, all tested doses showed statistically significant increases, as compared to negative and solvent controls. Graphical Abstract


Introduction
Smallanthus sonchifolius (Poepp.& Endl.) H. Robinson (Asteraceae), commonly known as yacon, is an edible tuberous shrub native to the Andean regions of South America that has been used for centuries by indigenous people.As a functional food, it can be used as a dietary supplement to prevent colon cancer, as immunomodulatory agent, and as regulator of glucose homeostasis and lipid metabolism (De Almeida et al. 2015;Caetano et al. 2016).Yacon is currently used as a traditional herbal remedy for its anti-diabetic properties, for the prevention of obesity and in the treatment of liver diseases.Ethanol extracts of yacon have shown anti-inflammatory and neuroprotective activity (Baek et al. 2018) and have proved to increase sperm counts and serum testosterone levels in rats (Park and Han 2013).Sesquiterpene lactones-rich yacon extracts have shown their potential to be used as topical anti-inflammatory agents (Oliveira et al. 2013).We have recently reported the genotoxic and cytotoxic effects of an aqueous extract of yacon prepared using a procedure that was like that commonly followed in popular medicine (tea bags) (Moreira Szokalo et al. 2020).Since studies on genotoxicity of S. sonchifolius organic extracts are lacking, this study aimed at investigating the in vitro toxicity of an ethanol extract (EE) and a crude lactone mixture (CLM), in terms of cell viability and genotoxic activity, and the chemical profile of EE and CLM.
The acyclic diterpenes smaditerpenic acid E (2) and smaditerpenic acid F (3) were identified, in both extracts, based on accurate mass comparisons and MS data (Mercado et al. 2010).The germacranolide STLs identified share a common melampolide backbone and have been previously reported in S. sonchifolius.

Cytotoxicity assay
In the present study, the CHO-K1 cell line was used to evaluate the potential toxicity of S. sonchifolius extracts (EE and CLM).In all cases, cell viability was inversely proportional to the extract concentration (Figure S3).The CC 50 values obtained were 40.3 mg/ mL for EE and 5.8 mg/mL for CLM.Extract concentrations allowing >80% viability in the MTT assay were chosen for evaluation in the comet assay.

Genotoxicity assay
Figure S4 shows DNA damage in CHO-K1 cells treated with different concentrations of EE (A) and CLM (B) of S. sonchifolius.The highest dose of EE (15 mg/mL) showed significant differences (p < 0.01) in DNA damage, as compared to the negative and solvent (DMSO) controls, but no differences were found between the lowest doses of EE (10 mg/mL) and controls.All tested doses of CLM showed statistically significant increases (p < 0.05) in DNA damage.The results obtained herein show that 5.8 mg/mL of CLM presented the same toxic effects as 40.3 mg/mL of EE.Previous studies of our group have shown that some STLs exhibit a strong cytotoxic effect in leukemia and pancreas cancer cells, while their cytotoxic effect on normal human cells was weak.This behavior is similar in terms of genotoxicity, since the CLM extract exerted toxic effects at much lower doses (0.5 mg/mL; 1 mg/mL and 2 mg/mL), while the genotoxic effect of EE was only detected at the highest dose (15 mg/mL).

Conclusions
The results obtained in this study indicate that STLs-rich yacon extracts present dosedependent cytotoxic and genotoxic effects.While the ethanol extract showed a genotoxic effect at the highest dose tested (15 mg/mL), a crude lactone mixture extract exerted the toxic effect at much lower doses (0.5 mg/mL).These results support our assumptions regarding the contribution of STLs to the genotoxicity of yacon leaves extracts.Based on these findings, we suggest that the STLs content and the risk/benefit ratio must be considered, and we propose to establish a maximum STLs content in yacon leaf extracts to ensure its safety when they are to be used as an herbal medicine.To the best of our knowledge, this is the first time that the toxicity of yacon organic extracts is reported.