Nrf2-Mediated Antioxidant Activity of the inner bark extracts obtained from Tabebuia rosea (Bertol) DC and Tabebuia chrysantha (JACQ) G. Nicholson.

Background: Several ethnobotanical and ethnopharmacological studies have shown the therapeutic potential of plants from the genus Tabebuia, which have long been used in traditional medicine in rural areas of South America, for the treatment of several human diseases. This study aimed to evaluate the Nrf2-mediated antioxidant activity of the inner bark extracts obtained from Tabebuia rosea and Tabebuia chrysantha. Methods: The antioxidant activity of extracts obtained from the inner bark of T. rosea and T. chrysantha was evaluated using the Oxygen radical absorbance capacity (ORAC) technique. The effect of extracts on the viability of HepG2 cells was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method. The translocation of Nrf2 to the nucleus after exposure of HepG2 cells to the extracts and controls (α-lipoic acid, curcumin and hydrogen peroxide) was evaluated using the Nrf2 transcription factor kit. Induction of the Nrf2-mediated antioxidant response gene ( NQO1) was evaluated by real-time PCR. Results: The ethyl acetate extract obtained from both species displayed the highest ORAC activity (12,523 and 6,325 µmoles Eq Trolox/g extract). In addition, the extracts had the ability to activate and to translocate Nrf2 to the nucleus, as well as to induce the expression of NQO1. Conclusion: These results indicate that the ethyl acetate extracts obtained from the inner bark of T. chrysantha and T. rosea have an important antioxidant effect mediated by Nrf2 activation, and could be used as a new source of natural antioxidants.


Abstract
Several ethnobotanical and ethnopharmacological studies have Background: shown the therapeutic potential of plants from the genus , which have Tabebuia long been used in traditional medicine in rural areas of South America, for the treatment of several human diseases. This study aimed to evaluate the Nrf2-mediated antioxidant activity of the inner bark extracts obtained from and .

Tabebuia rosea
Tabebuia chrysantha The antioxidant activity of extracts obtained from the inner bark of Methods: T. and was evaluated using the Oxygen radical absorbance rosea T. chrysantha capacity (ORAC) technique. The effect of extracts on the viability of HepG2 cells was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method. The translocation of Nrf2 to the nucleus after exposure of HepG2 cells to the extracts and controls (α-lipoic acid, curcumin and hydrogen peroxide) was evaluated using the Nrf2 transcription factor kit. Induction of the Nrf2-mediated antioxidant response gene ( ) NQO1 was evaluated by real-time PCR.
The ethyl acetate extract obtained from both species displayed the Results: highest ORAC activity (12,523 and 6,325 µmoles Eq Trolox/g extract). In addition, the extracts had the ability to activate and to translocate Nrf2 to the nucleus, as well as to induce the expression of .

NQO1
These results indicate that the ethyl acetate extracts obtained Conclusion: from the inner bark of and have an important antioxidant T. chrysantha T. rosea effect mediated by Nrf2 activation, and could be used as a new source of natural antioxidants.

Amendments from Version 1
We include in the methods that H 2 O 2 was used as and oxidative stress inductor.
In Table 1, we change Chloroform and Ethyl acetate with CHCl 3 and EtOAc in order to homogenize the text in Table 1 and Table 2.
In Table 1, we include the statistically significant differences found. Also, these differences are included in the text.
In the results section, we also clarify that both T. chrysantha and T. rosea decreased the Nrf2 levels in the cytoplasm after 4 hours of exposure, although the differences are not significant.
In the discussion section, we also clarify that only the ethyl acetate extract from T. chrysantha significantly increased the expression of NQO1 in HepG2 cells after six 6 hours of exposure compared to ALA and CUR.

Introduction
Nature's compounds reveal a great diversity of chemical structures and physicochemical properties, as well as biological ones. Over the years, plants have been used for the treatment of various diseases, including those of inflammatory origin, such as arthritis, obesity, and cancer. Plants of the genus Tabebuia belong to the Bignoniaceae family, which is composed of about 120 genera with 827 species, and is considered the second most diverse family of species of neotropical woody plants in dry forests 1 . There are reports on the presence of chemical compounds-including quinones and phenols, among others-in this family 2,3 . The Tabebuia genus comprises about 100 species of trees and shrubs, mainly distributed from Mexico to several regions of Central and South America, which have been used in traditional medicine. Plants of this genus are an important source of bioactive molecules such as: naphthoquinones; quinones; phenols; and molecules with anti-inflammatory, antioxidant, anti-microbial and anti-proliferative activity 4-8 .
A large number of chemical compounds exert their antioxidant effects through the activation of key transcriptional regulation mechanisms, such as the transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2) 9 . Under normal physiological conditions, this factor is in the cytoplasm inhibited by Keap1 (Kelch ECH associating protein 1), which leads to its degradation 10 . In cells exposed to oxidative stress, Nrf2 is not targeted for ubiquitin-dependent degradation. Instead, it is released and translocated to the nucleus, where it activates its antioxidant response through binding to Antioxidant Response Elements (ARE sites), allowing the coordinated expression of more than 200 detoxifying enzymes and antioxidants, such as NAD(P)H quinone oxidoreductase (NQO1) and heme oxygenase 1 (HO-1), among others [11][12][13][14] . The aim of this study was to evaluate the Nrf2mediated antioxidant activity of extracts obtained from the inner bark of Tabebuia rosea and Tabebuia chrysantha in HepG2 cells.

Preliminary phytochemical screening
The preliminary phytochemical screening was performed using selective derivatization reactions for the characterization of secondary metabolites present in the n-hexane, chloroform, ethyl acetate and n-butanol extracts obtained from inner bark, as previously reported for T. rosea 6 . The extracts were evaluated using normal and reverse phase thin layer chromatography (TLC

Total phenolic content
The total phenolic content of each extract was determined according to the Folin-Ciocalteu colorimetric method 15 , using gallic acid as standard. Briefly, Folin-Ciocalteu's reactive (Merck, Darmstadt, Germany, Cat No. 1090010100) was diluted 10-fold with distilled water. 25 µL of the samples (1 mg/mL) were added to the Folin-Ciocalteu's reactive. After the addition of Na 2 CO 3 (20%), the reaction was maintained at room temperature (RT) in the dark for 30 min, and absorbance was measured at 760 nm in a Shimadzu UV-1700 spectrophotometer. Gallic acid (0.25-5 mg/mL) was used to generate a standard curve (y=0.101x+0.086; R 2 =0.996). Results are presented as mg gallic acid equivalents per g of extract (mg GAE/g extract). All experiments were performed in triplicate.
Oxygen radical absorbance capacity (ORAC) Oxygen radical absorbance capacity was determined

Cell viability test
To determine the effects of extracts on HepG2 cells, cell viability was tested using the MTT (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide) method 20 . Cells (5 × 10 4 cell/well) were treated with a concentration of 2 µg/mL of the extracts (the n-butanol extract from T. chrysantha was used at 1 µg/mL) diluted in DMSO (final concentration 0.1%), and incubated for 24 hours. After treatment, the medium was discarded, 200 µL of DMEM medium containing 0.5 mg/mL MTT (Sigma Chemical Co, Saint Louis, MO, USA, Cat No. M2128-500MG) was then added to each well. The plates were incubated for 4 hours at 37°C in a 5% CO 2 atmosphere. The medium was discarded and 100 µL of DMSO was then added to solubilize the formazan crystals. Absorbance was measured in an ELISA microplate reader at 492 nm (ELx800; BioTek Instruments Inc., USA). Viability percentage was calculated based on the non-treated control. Three independent assays were performed, each one in triplicate.

Nrf2 nuclear activation
The HepG2 cell line (3 × 10 5 cell/well) was cultured in DMEM medium using a T25 flask. The medium was discarded, and the cells were exposed at two time points (0 and 4 hours) to:

Statistical analysis
Each experiment was performed at least in duplicate. Results were expressed as the mean ± SD of at least three independent experiments. Statistical analysis was performed using the Kruskal-Wallis test and a p-value less than 0.05 was considered statistically significant. The statistical tests were applied using GraphPad Prism, version 5.02 (GraphPad Software, San Diego, CA, USA).

Results
Preliminary phytochemical screening, total phenolic content, ORAC and cell viability The preliminary phytochemical screening of the inner bark extracts obtained from T. rosea and T. chrysantha did show the presence of anthrones, quinones and coumarins, as previously   reported for T. rosea 6 ( Table 1). Sesquiterpenic lactones were present in the n-hexane, chloroform and ethyl acetate extracts of T. rosea, but were absent from the n-hexane extract from T. chrysantha. Steroids were identified in chloroform and ethyl acetate extracts of both species. The presence of flavonoids and phenolic acids was observed only in the ethyl acetate extract from T. rosea. The total phenolic content in the extracts was determined by the Folin Ciocalteu colorimetric method. The ethyl acetate extracts obtained from T. rosea and T. chrysantha exhibited the highest total phenolic content (2.18 ± 0.29 and 2.08 ± 0.72 mg GAE/g extract, respectively), whereas the chloroform and aqueous extracts displayed the lowest phenolic content (0.63 ± 0.11, 1.55 ± 0.78, -0.668 ± 0.23 and 0.07 ± 0.03 mg GAE/g extract, respectively). The total phenolic content of the ethyl acetate extract from T. rosea was significantly higher (p <0.05) than its chloroform extract. The order of total phenolic content in both species is: ethyl acetate> n-butanol> chloroform> aqueous (Table 2). The ORAC results indicated that the ethyl acetate extracts from T. rosea, at a concentration of 10 µg/mL, have the best antioxidant activity (12,523.41 ± 840.46 µmol TE/g extract) and even this activity was superior to that obtained with the controls, showing a significant difference (p<0.001) compared to the antioxidant activity of quercetin (Table 2). Both the chloroform and n-butanol extracts from T. rosea also showed important activity. Among the T. chrysantha extracts, the ethyl acetate extract displayed the best antioxidant activity (6,325.74 ± 1,057.14 µmol TE/g extract); however, this activity did not exceed that obtained with luteolin and quercetin ( Table 2). The MTT assay revealed that neither the extracts from the inner bark of T. rosea nor those from T. chrysantha affected the viability of the cells studied, since viability was greater than 80% after 24 hours of exposure ( Table 2).

Effect of T. rosea and T. chrysantha extracts on activation and nuclear translocation of Nrf2
The Nrf2 detection test allowed for the evaluation of the ability of the extracts to activate and translocate Nrf2 to the nucleus. Nrf2 detection enabled comparisons of the basal Nrf2 status in both the cytosol and the nucleus. It also allowed for comparison of their associated changes after the exposure of HepG2 cells to the ethyl acetate extracts from T. chrysantha (0.5 µg/mL) and T. rosea (1 µg/mL), which displayed the best antioxidant activity in the ORAC assay. As shown in Figure 1, the exposure of HepG2 cells to ALA, CUR, H 2 O 2 , and the ethyl acetate extract from both T. chrysantha and T. rosea, decreased the Nrf2 levels in the cytoplasm after 4 hours of exposure, although the differences are not significant. This decrease was measured in relation to their basal level (non-exposed cells). As expected, an increase in Nrf2 levels in the nucleus was observed after exposure to ALA, CUR, H 2 O 2 and the extracts. However, significant differences were found only after exposure to ALA, CUR and, H 2 O 2 (p<0.01).

Effect of extracts on the expression of NQO1
Transcriptional regulation of antioxidant response genes against oxidative stress represents a defense against cell damage. In this study, we evaluated the expression of the gene coding for the antioxidant enzyme NQO1, which is involved in protection against oxidative stress. The level of expression of the NQO1 gene was evaluated (qRT-PCR) and quantified using the 2 -ΔΔCt method.
The results indicate that the ethyl acetate extract from both T. chrysantha and T. rosea, as well as the culture of HepG2 cells in the presence of H 2 O 2 , significantly increased the expression levels of NQO1 after six hours of exposure (p<0.05), compared to the controls ALA and CUR (Figure 2). The relative expression levels of NQO1 gene decreased significantly after 12 hours post-exposure.

Discussion
Oxidative stress is important because of its relation with a wide variety of disorders associated with an increase in the levels of oxidative markers and damaged cellular components, such as Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis 21 , premature aging, inflammatory diseases and cancer 27 .
Plants are widely used as sources of antioxidants due to their phenolic compounds and ability to scavenge ROS and free radicals, which makes them among the most potent and therapeutically useful biocompounds 28 . Some studies have evaluated the antioxidant activity in extracts obtained from T. chrysantha and T. rosea 6,29,30 , demonstrating the potential of these plants in the search for new molecules with significant biological effects. Previous studies have also shown the potential anti-inflammatory activity of T. chrysantha and T. rosea 6,30 . Such activity can also be associated with and effect on Nrf2, a molecule that not only regulates oxidative/xenobiotic stress response, but also represses inflammation by opposing transcriptional upregulation of a number of pro-inflammatory cytokine genes 31 . It is due to this that the antioxidant activity of the inner bark extracts obtained from T. chrysantha and T. rosea and its association with the activation-dependent translocation of Nrf2 to the nucleus and the induction in the expression of NQO1 gene was evaluated.  The ethyl acetate extracts from both T. chrysantha and T. rosea displayed strong antioxidant activities due to their oxygen radical absorbance capacity, which could be related to the high total phenol content found with the Folin Ciocalteu method. This capacity could also be related to phenols previously reported in T. rosea, like gentisic acid 32 or phenols found in the same genus, such as α-tocopherol and γ-tocopherol 33 . The results of the present study are in agreement with those previously reported for T. rosea 6 . This is the first study involving the ethyl acetate extract from T. chrysantha. An evaluation of the scavenging hydroxyl radical capacity of the methanolic and aqueous extracts from T. chrysantha revealed significant scavenging of the hydroxyl radical (80 and 83%, respectively), and reductions in the production of the peroxyl radical 30 .
Given that ethyl acetate extracts were the most active of those produced, and that they did not affect the viability of HepG2 cells, these extracts were used to evaluate effects on Nrf2 translocation and expression of antioxidant response genes. We compared basal Nrf2 levels in both the cytosol and the nucleus, as well as the changes associated with exposure to the extracts. As expected, after 4 hours of exposure of HepG2 cells to the extracts, Nrf2 translocate from the cytoplasm to the nucleus; however, this effect was more pronounced after exposure to ALA, CUR, and H 2 O 2 . Flavonoids found in the ethyl acetate extract of T. rosea 6 could be related to its ability to induce the activation and translocation of Nrf2, as they possibly have the same action mechanism as resveratrol, whose ability to activate Nrf2 translocation to the nucleus in astrocytes after 2.5 hours of exposure has been demonstrated previously 34 . ALA may induce Nfr2 translocation to the nucleus after 1 hour of treatment 35 . A recent study did show that pau d'arco (an extract from the inner bark of T. impetiginosa) has the capacity to activate and translocate Nrf2 to the nucleus via a MEK (MAPK/ERK kinase)independent mechanism 36 . The results show that the ethyl acetate extracts obtained from T. chrysantha and T. rosea did induce the nuclear translocation of Nrf2 in HepG2 cells. Therefore, the upregulated expression of the NQO1 gene by the ethyl acetate extracts is due to the stabilization and nuclear accumulation of Nrf2.
Antioxidant activity through upregulated expression of NQO1 gene has been reported for β-lapachone 37 , a quinone that has previously been isolated from T. chrysantha 38 . It is also possible that steroids found within ethyl acetate extracts of T rosea and T. chrysantha during the Lieberman-Burchard test could be responsible for the overexpression of NQO1 gene, as that has been reported for steroids like 17β-estradiol on CCD841CoN cell line 39 . The Nrf2 pathway is considered the most important in the cell for protection against oxidative stress, which is generated by an accumulation of ROS and/or electrophiles, leading to the oxidation of biomolecules, membrane damage, DNA adduct formation, and mutagenicity. All these changes lead to degeneration of tissues, premature aging, apoptotic cell death and the development of cancer 13 .
Nrf2 is a major activator of the phase II antioxidant genes such as SOD, CAT, GST, HO-1 and NQO1 40 . Our results demonstrated that the ethyl acetate extracts from T. chrysantha and T. rosea increased the expression of NQO1 in HepG2 cells after 6 hours of exposure compared to ALA and CUR, although only a significant difference was found for T. chrysantha. It has been shown that overexpression of Nrf2, by Nrf2-cDNA, upregulates the expression and induction of the NQO1 gene in response to antioxidants and xenobiotics 41 . In addition, Nrf2-null mice exhibited a marked decrease in NQO1 expression and induction, indicating that Nrf2 plays an essential role in the in vivo regulation of NQO1 in response to oxidative stress 13 . NQO1 overexpression is also considered a cytoprotective mechanism after exposure to toxic metals 42 . Tabebuia is the largest genus of the Bignoniaceae family and several bioactive compounds such as: naphthoquinones; quinones and phenols have been extracted so far, due to the traditional use of numerous species of this genus. Very few studies have evaluated the anti-oxidant activity of extracts obtained from both and and only one study has evaluated the effect of T. rosea T. chrysantha T. extracts on Nrf-2 activation and translocation. impetiginosa The study from Garzón-Castaño describes for the first time the effect of the inner bark extracts from et al. and on the Nrf-2-mediated antioxidant activity. The best antioxidant activity T. rosea T. chrysantha (evaluated using the ORAC Technique) was displayed by the ethyl acetate extracts from both species. This activity was clearly associated with the activation and translocation of Nrf-2 to the nucleus and the induction in the expression of the gene in HepG2 cells. This study contributes to the knowledge on NQO1 the biological activity of plants from the genus that have not been studied extensively and Tabebuia highlight its importance on the search of new molecules with antioxidant activity.
Finally, the research paper is very clear and presents results of great expectation.

Is the work clearly and accurately presented and does it cite the current literature? Yes
Is the study design appropriate and is the work technically sound? The benefits of publishing with F1000Research: Your article is published within days, with no editorial bias You can publish traditional articles, null/negative results, case reports, data notes and more The peer review process is transparent and collaborative Your article is indexed in PubMed after passing peer review Dedicated customer support at every stage For pre-submission enquiries, contact research@f1000.com