Evaluation In Vitro of Toxicity of Hydroalcoholic Extract of Leaves and Roots from Yacon ( Smallanthus sonchifolius )

Abstract

Yacon is an Andean plant that has been used in folk medicine for its medicinal properties. The beneficial effects of this plant are possibly due to the high content of phenolic compounds present in its leaves and roots. This study evaluated the in vitro toxicity of the hydroalcoholic extract of leaves and roots from yacon (1, 10, 50, and 100 μg/mL) through cell viability tests, genotoxic and mutagenic activity in leukocytes culture cells; and cytotoxicity and apoptosis cell death (1, 10, 50, 100, and 500 μg/mL) in cell line originally established from the primary mouse embryonic fibroblast cells that were cultured by the designated protocol, so-called 3T3 protocol “3-day transfer, inoculum 3 × 10⁵ cells” (3T3 cell line). No mutagenic and cytotoxic activities were observed in leukocyte cultures. Cytotoxic activity was evidenced in the highest concentrations of yacon leaf extract (50 and 100 μg/mL), whereas all concentrations tested with yacon leaf extract there was induction for apoptosis in the 3T3 cells. Genotoxic potential was observed only at higher doses of leaf (50 and 100 μg/mL) and root (100 μg/mL) extract. These results suggest that yacon leaf at high concentrations may present toxic potential showing concentration-dependent behavior; however, in vivo studies should be performed to validate these results.

Introduction

The yacon is a plant originating from the Andean regions and has been used in folk medicine for its medicinal properties for the treatment of diabetes and cholesterol disorders.^1,2 This plant belongs to the family Asteraceae, also known as Compositae, and its scientific name is Smallanthus sonchifolius (Poepp. & Endl.) H. Robinson. The names Polymnia sonchifolia Poepp. & Endl. and Polymnia edulis Wedd are also used in the scientific literature.³

The yacon is a tuberous root containing fructooligosaccharides as carbohydrate reserves, substances that can benefit human health.⁴ It has been reported that the medicinal properties of yacon are due to the presence of phenolic compounds,⁵ which have a high concentration in the leaves and roots.⁶ Studies have shown that Yacon has hypoglycemic,^7
–9 antioxidant,^10,11 and hypolipidemic activities.¹²

Phytotherapy and the use of medicinal plants is an ancient practice with a great current trend in folk medicine. This increasing use of “natural products” without sufficient data on side effects, dose, and toxicity is becoming a major problem today.¹³ A study demonstrated that consumption of the yacon leaf may result in renal toxicity.¹⁴ Thus, the objective of this study was to investigate the cytotoxic, genotoxic, and mutagenic effects of the hydroalcoholic extract of leaves and roots from yacon in cell cultures of human leukocytes and cytotoxicity and apoptosis response in the 3T3 cell line.

Materials and Methods

Plant material and preparation of the extract

The yacon roots were provided by Emater/RS and cultivated in a property in Alegrete/RS, Brazil (geographical coordinates: 29° 48′ 88′′ S and 55° 40′ 22′′ W). The roots and leaves were duly transported fresh to Uruguaiana/RS, Brazil (geographical coordinates: 29° 44′ 58′′ S and 57° 5′ 18′′ W), where they were processed immediately.

The hydroalcoholic yacon extract was obtained from 70% ethanol:water (v/v). The roots were washed, peeled, and dried at 37°C for 72 h, and the leaves were washed and dried at 37°C for 5 h. Both roots and leaves were placed in contact with the solvent for 7 days in the dark, according to a slightly modified method previously described.¹⁵ After filtering in filter paper to remove the vegetal residue, the extract was concentrating under reduced pressure using a rotary evaporator at 40°C, the remaining water portion was lyophilized. The lyophilized product was stored at −70°C until further use.

Human blood samples and cell cultures of human leukocyte preparation

Peripheral blood was collected from a male human volunteer by venipuncture into sterile vials containing 68 I.U. of sodium heparin (BD Vacutainer^®; Becton, Dickinson and Company, NJ, USA) per milliliter of blood, and then whole-blood cultures were established. This study was approved by the Committee of Ethics in Research in Humans (CEP) of the Federal University of Pampa (UNIPAMPA), Uruguaiana, Rio Grande do Sul, Brazil (Authorization No. 27045614.0.0000.5323).

The leukocyte cultures were prepared with 1 mL of whole-blood sample and immediately transferred to 9 mL of Roswell Park Memorial Institute (RPMI) 1640 culture medium supplemented with 25% fetal bovine serum (FBS), 1% streptomycin/penicillin, and 2% phytohemagglutinin. ^16
–18 The cells were then placed in a microaerophilic environment with 5% carbon dioxide (CO₂) at 37°C for 72 h. The solutions analyzed included the extract of leaves and roots from yacon diluted in phosphate-buffered saline (PBS), pH 7.4, at concentrations of 1.0, 10, 50, and 100 μg/mL. PBS was used as negative control, and 100 μM hydrogen peroxide (H₂O₂) was used as positive control. Each group consisted of three culture flasks. Genetic parameters were analyzed after 72 h of growth, which included cellular viability, comet assay, and micronucleus test.

Cellular viability in cell cultures of human leukocyte

The viability was determined using trypan blue solution, which is assessed by the loss of membrane integrity. This test was performed by mixing 100 μL of each sample with 100 μL of 0.4% trypan blue solution and checking after 3 min. A total of 100 cells were counted for each survival determination. The test cytotoxic is based on the principle that living cells have intact cell membranes that exclude the dye, whereas the dead cells do not. Thus, a viable cell will have a clear cytoplasm, and an already nonviable cell will have a blue cytoplasm as it absorbs the dye. The cells were analyzed through microscopic observation, and the percentage of viable cells was determined.¹⁹

Comet assay and micronucleus test

The principle of this technique is DNA migration in an agarose matrix under electrophoretic conditions. After incubation, the samples were mixed with low-melting point agarose and placed on a microscope slide precoated with normal melting point agarose. The slides were immersed in a lysis solution, and electrophoresis was performed for 20 min at 300 mA and 25 V. Then, the slides were neutralized and left to dry overnight at room temperature. The dry slides were rehydrated and then fixed for 10 min and left to dry again. The last stage was coloring and the use of stop solution.²⁰ The slides were analyzed under blind conditions. DNA damage was defined as DNA damage index by counting 100 cells in the slides and visually scored according to tail length, with scores ranging from 0 (no migration, cells without morphological alterations) to 4 (maximal migration, fragmented DNA totally, comet tail format, and maximum injury). When observed under a microscope, the cells have the appearance of a comet, with a head (nuclear region) and a tail containing DNA fragments that migrated toward the positive.²¹

For the micronucleus, slides were stained by panoptic method and then analyzed under an optical microscope with immersion lens. For each slide, 100 cells were counted, and the micronucleus frequency was determined.²²

Cell line culture and treatments

The 3T3-Swiss albino cell line was obtained from American Type Culture Collection (ATCC^® CCL-92™). Cell treatment and all experimental procedures were performed at Federal University of Pampa (Uruguaiana, RS, Brazil). Cells were routinely maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FBS and 2 g/L HEPES buffer (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; pH 7.4). Cells were plated (5 × 10⁴cells/mL) in 24-well plates and cultured under 37°C and 5% CO₂ conditions for 24 h to reach 60–70% of confluence before treatment. Yacon leaf and root extract was diluted in culture medium to final concentrations of 1, 10, 50, 100, and 500 μg/mL just before use. The 3T3 cells were treated in the dark for 24 h.

Colorimetric 3-(4,5-dimethylthiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay

MTT (3-(4,5-dimethylthiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; Sigma, Inc., Saint Louis, MO, USA) is a yellow tetrazolium salt that is reduced to purple formazan crystals. The MTT assay is widely used for the assessment of cytotoxicity, cell viability, and proliferation studies in cell biology. Preconfluent 3T3 cells were incubated with different concentrations of yacon leaf and root extract for 24 h. Cells were then incubated with 1 mg/mL MTT for 2 h at 37°C. Purple crystals were dissolved in dimethylsulfoxide (Sigma, Inc.). The absorbance was measured using a spectrophotometric microplate reader (Spectra Max M5; Molecular Devices, Sunnyvale, CA, USA) at 570 and 630 nm. Percentages of cell viability were calculated relative to cell controls.

Cell apoptosis assay in yacon leaf extract

Induction of apoptosis was evaluated through caspase-3/7 activity was measured with the CellEventTM caspase-3/7 Green Detection Reagent (Life Technologies). On cleavage by activated caspase-3/7, the probe becomes fluorescent and free to bind to DNA. After treatment with the extracts the cells were incubated for 10 min according to kit instructions and the green fluorescence was evaluated using Floid Cell Imaging (Life Technologies).

Statistical analysis

Data were expressed as mean ± standard deviation. Comparisons between groups were performed using one-way analysis of variance, followed by post hoc Bonferroni test for multiple comparisons. Results were considered statistically significant when P < .05. These analyses were performed using the free software R version 3.1.1 (R Core Team, 2014).

Results

Cellular viability in cell cultures of human leukocyte

Result of the cellular viability assay is demonstrated in Figure 1A. All treatments resulted in significantly higher (P < .05) viability than that of positive control, being similar to negative control.

FIG. 1.

Effect of yacon in human leukocytes culture. In (A) cellular viability; (B) comet assay; (C) frequency of micronucleus. Averages followed by different letters differ by one-way ANOVA followed by Bonferroni's comparison post hoc test at P < .05. ANOVA, analysis of variance; L, yacon leaf extract; NC, negative control; PC, positive control; R, yacon root extract.

Comet assay

Figure 1B shows the results of comet assay. Leaf extract at both 10 and 1 μg/mL concentrations and root extract at 50, 10, and 1 μg/mL concentrations showed similar results to those of the negative control but were significantly smaller (P < .05) than that of the positive control. Leaf extract at 100 μg/mL concentration showed significantly higher results than those of the positive control. At 50 μg/mL concentration (leaf extract) and 100 μg/mL concentration (root extract), similar results to those of the positive control were obtained. Leaf extract (100 and 50 μg/mL) and root extract (100 μg/mL) showed genotoxic activity at higher concentrations tested, whereas the root extract showed lesser DNA damage than that with leaf extract.

Micronucleus test

The results of micronucleus frequency are demonstrated in Figure 1C. All treatments showed significantly smaller (P < .05) values than those of the positive control, but were similar to those of the negative control. These results demonstrated that there was no mutagenic activity at all the concentrations tested for both extracts.

Effect of yacon in 3T3 cell line

In the yacon root extract treatment, none of the test groups showed a decrease in cell viability compared with the control group, as well as the concentrations of 1 and 10 μg/mL of the leaf extract; however, the doses 50, 100, and 500 μg/mL, a percentage of reduced cell viability was found to be 72%, 46%, and 22%, respectively (Fig. 2A).

FIG. 2.

Effect of yacon in 3T3 cell line. In (A) cellular viability; (B) cell apoptosis assay. Averages followed by different letters differ by one-way ANOVA followed by Bonferroni's comparison post hoc test at P < .05.

Cell apoptosis assay

The Figure 2B showed monitoring apoptosis by fluorescent detection of caspase-3/7 in leaf extract yacon treatment. There was an increase in the activity of caspase-3/7 in all concentrations tested.

Discussion

Our findings demonstrate, for the first time the toxic effects of the hydroalcoholic extract of leaves and roots from yacon in cell cultures of human leukocytes and 3T3 cell line. The main results suggest that there was cytotoxic activity in the highest concentrations of leaf extract. Genotoxic potential was observed only at higher doses of leaf and root extract, whereas no mutagenic activity was observed at any dose tested.

Plants are important sources of natural antioxidants that play a fundamental role in protecting the human body against various diseases besides great potential in improving these processes.²³ Thus, many plants as yacon are being used indiscriminately without any data on the safety and tolerability of its compounds in humans. Therefore, cytotoxic, mutagenic, and genotoxic assays are needed to establish the safety of these compounds. Cell cultures, because of their ability to multiply in vitro, are an attractive alternative to assess the toxicity of natural products and thus estimate the initial doses for in vivo toxicity studies.²⁴

Our study demonstrated that leaf extract at the highest concentrations in 3T3 cell line (500, 100, and 50 μg/mL) showed a significant decrease in cell viability, indicating a concentration-dependent cytotoxic effect. The other concentrations of the leaf and root extracts did not alter the cellular viability, indicating no cytotoxic activity. Our findings corroborate those of Barcellona et al. ²⁴ that although using cell lines and different concentrations also observed cell viability decreased with increasing concentrations of the 10% decoction of yacon leaves. This study evaluated the potential toxicity of the 10% decoction (2, 10, 20, 100, and 200 μg of dry extract/mL) of yacon leaves in four different cell lines (Hep-G2, COS1, CHO-K1, and Vero cell lines). The concentration at which cell viability was inhibited to 50% of control values (IC50) was 100 μg/mL (Hep-G2 and CHO-K1 cell lines), 175 μg/mL (COS1 cell line), and 125 μg/mL (Vero cell line).

On the basis of cytotoxicity data of the leaf extract in 3T3 cell line we carry out additional studies on apoptosis, caspase-3/7 activation were examined. Apoptosis is a programmed and orderly form of cell death that eliminates undesirable cells without inflammatory consequence close to the tissue, is also important for the maintenance of tissue homeostasis or may contribute to the pathogenesis of diseases. Apoptosis is characterized by morphological changes (shrinkage, depolarization of mitochondria, condensation, and nuclear fragmentation), biochemical and molecular, and can be measured by several methods that evaluate these changes that occur in the cell during this process.^25,26 The most evident biochemical characteristic of apoptosis is the activation of caspases (cysteine-dependent aspartate-specific proteases), which are a large family of cysteine proteases that have the ability to recognize and cleave substrates on the carboxy-terminal side of specific aspartic acid residues. Caspases in relation to their function can be classified as initiators (caspases-1, -2, -4, -5, -8, -9, -10, -11, and -12) or executors (caspases-3, -6, and -7). All caspases are synthesized as inactive zymogens and activated by a series of cleavages or allosteric conformational changes. In general, the activity of caspase-3/7 is greatly enhanced in the apoptosis process and activity detection is considered a reliable marker for cells that are dying or apoptosed.^27
–29 In this study, we observed through intense fluorescence that there was induction to apoptosis at all concentrations tested of yacon leaf extract, including those that did not present differences in cell viability. These results suggest that possibly longer treatment with yacon leaf extract would result in cytotoxicity even at the lower doses.

There are several methods to evaluate genotoxic influence, and in this study, we used the comet assay and micronucleus frequency test. Genotoxic activity was determined through the comet assay involving electrophoresis of lysed cells embedded in agarose on a microscope slide. Undamaged DNA stays within the core, whereas fragmented DNA migrates from the core toward the anode, forming an image resembling a comet with a head and a tail, and the relative content of DNA in the tail indicates the frequency of breaks.^30,31

Genotoxicity is usually a result of hereditary mutation characteristics or breakage of DNA strands, leading to apoptosis, carcinogenesis, or alteration of phenotype.³² Our findings demonstrated that these extracts have genotoxic potential at higher concentrations.

Mutagenic activity was determined through the micronucleus frequency assay. Micronuclei are structures constituted of chromatin genetic material and can be whole chromosomes or acentric chromosome fragments that cannot incorporate into the nucleus during cell division, thereby forming a small independent nucleus, appearing around the nuclear envelope or the cytoplasm of cells exposed to toxic agents. Micronucleus frequency determination provides an estimate of the presence of chromosomal changes.³³ No mutagenic activity was observed in for any of the doses administered. These results corroborate with the findings of Sakuma et al. ³⁴ who evaluated the mutagenic potential of the extract from the leaf and stem of yacon by the bacterial reverse mutation test (Ames test) and the mouse bone marrow micronucleus test and did not observe gene mutations or clastogenic activity in vivo. Another study carried out by Cuyacot³⁵ evaluated the effect of the ethanolic extract of yacon (5, 15, 25, 35, and 50 mg/mL) on Allium cepa and no chromosomal aberration was found with the frequency of micronuclei and chromosome fragments being equal to that of the control.

In conclusion, Yacon can be considered as a plant with multiple functions, in addition to not exhibiting mutagenic activity. We observed that leaf extract induces concentration-dependent cytotoxicity and apoptosis in 3T3 cell line. Genotoxic potential was demonstrated only at higher concentrations of leaf and root extracts. These results suggest that yacon leaf at high concentrations may present toxic potential showing concentration-dependent behavior; however, in vivo studies should be performed to validate these results.

Footnotes

Acknowledgments

This study is based on Dr. Patrícia Martinez-Oliveira's doctoral thesis, a copy of which can be found online (Universidade Federal do Pampa [UNIPAMPA], http://dspace.unipampa.edu.br:8080/jspui/handle/riu/4084).

Author Disclosure Statement

No competing financial interests exist.

Funding Information

No funding was received.

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