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

Plant material and extract preparation

The inner bark from T. rosea (Bertol.) DC and T. chrysantha (JACQ) G. Nicholson were collected at Universidad Tecnológica de Pereira campus in April 2011. The plants were identified at the Colombian National Herbarium (Voucher No. COL 582577 for T. rosea and COL 587611 for T. chrysantha). The collection and processing of the material were covered by the collection permission number 1133/2014, issued by the National Environmental Licensing Authority (ANLA) of Colombia.

Extracts were obtained as previously described⁶. Plant material (inner bark from T. chrysantha and T. rosea) was dried and macerated in methanol analytical grade (14 L) for 48 h. This was followed by homogenization, filtration, and concentration under vacuum using a vacuum rotary evaporator (Heidolph, Laborota model) at 40 °C to obtain the crude extract. This procedure was repeated three times. Crude extracts were dissolved in 400 mL of distilled water, and underwent liquid–liquid extraction with increasing polarity solvents (solvent volume 1.6 L): n-hexane, chloroform (CHCl₃), ethyl acetate (EtOAc), and n-butanol (all solvents were analytical grade). Each extract was vacuum dried by vacuum rotary evaporator obtaining the following mass for each extract: n-hexane (0.3 g), chloroform (1.2 g), ethyl acetate (3.7 g), n-butanol (12.5 g) and aqueous (21.7 g). Endotoxin levels in the extracts were assayed using the Limulus Amebocyte Lysate Test, E-Toxate Kit (Sigma Chemical Co, Saint Louis, MO, USA, Cat No. ET0200-1KT). All samples were negative for the presence of endotoxins (detection limit 0.05–0.1 EU/mL). The extracts were kept refrigerated at 4 °C in amber tubes protected from light, heat, air and moisture. For each one of the biological assays, the extracts were dissolved in 0.1% DMSO (Dimethyl sulfoxide, Merck, Darmstadt, Germany, Cat No. 1029521000).

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. rosea⁶. The extracts were evaluated using normal and reverse phase thin layer chromatography (TLC). Chromatographic plates were revealed with aluminum chloride (AlCl₃, Sigma Chemical Co, Saint Louis, MO, USA, Cat No. 563919-25G) and ferric chloride (FeCl₃, Sigma Chemical Co, Saint Louis, MO, USA, Cat No. 157740-1KG) for detection of flavonoids, phenols and phenolic acids; potassium hydroxide (KOH, Merck, Darmstadt, Germany, Cat No. 1050331000) in analytical grade ethanol for detection of anthrones, quinones and coumarins; oleum (Sigma Chemical Co, Saint Louis, MO, USA, Cat No. 778990-500ML) for detection of sesquiterpenic lactones; and the Liebermann-Burchard reagent for detection of terpenes and steroids.

Total phenolic content

The total phenolic content of each extract was determined according to the Folin–Ciocalteu colorimetric method¹⁵, 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 Na₂CO₃ (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; R²=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 using the method described by Ou et al, with some modifications¹⁶. 2,2’-Azobis (2-amidinopropane) dihydrochloride (AAPH) and sodium fluorescein stock solutions were prepared in a 75 mM, pH 7.0 phosphate buffer solution. 31 μL of each sample were diluted in 187 μL of fluorescein (120 nM) and incubated at 37 °C for 10 min. The reaction was started by the addition of 31 μL of AAPH (143 mM) to reach a final volume of 249 μL per well. Extracts were evaluated at 10, 15, and 20 μg/mL. A Trolox® standard curve was prepared (10, 20, 40, and 60 μM). Changes in fluorescence were measured with a Varian Cary Eclipse 1.1 fluorescence spectrophotometer at 2 min intervals for 120 min with emission and excitation wavelengths of 515 and 493 nm, respectively. The excitation slit was 5 nm, as was the emission slit^17,18. The antioxidant capacity was calculated as the area under the curve (AUC)¹⁹ and expressed as μmol Trolox® equivalents per g of extract (μmol TE/g of extract).

Cell culture

Human hepatocarcinoma cell line (HepG2; ATCC; CRL-11997) was purchased from American Type Culture Collection (ATCC, Rockville, MD, USA) and cultured with Dulbecco’s Modified Eagle Medium (DMEM), supplemented with Glutamax (GIBCO/BRL, USA, Cat No. 10564-011) and 10% heat inactivated FBS (GIBCO, Cat No. 16140071), 200 μg/mL Penicillin, 200 μg/mL streptomycin, 400 μg/mL neomycin (GIBCO, Cat No. 15640-055), 5 μg/mL amphotericin, 0.05 mM 2-β-mercaptoethanol, and 1 mM sodium pyruvate (all from Sigma Chemical Co, Saint Louis, MO, USA, Cat No. A9528-50MG, M7522-100ML, S8636-100ML, respectively). Cells were maintained at 37 °C with 5% CO₂ atmosphere.

Cell viability test

To determine the effects of extracts on HepG2 cells, cell viability was tested using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) method²⁰. Cells (5 × 10⁴ 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% CO₂ 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 × 10⁵ 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: Alpha-lipoic acid (ALA, 50 μM, Sigma Chemical Co, Saint Louis, MO, USA, Cat No. T1395-1G), Curcumin (CUR, 15 μM, Sigma Chemical Co, Saint Louis, MO, USA, Cat No. C7727-500MG)^21–24, ethyl acetate extracts from T. chrysantha (0.5 μg/mL), ethyl acetate extract from T. rosea (1 μg/mL) and H₂O₂ (0.6 mM). H₂O₂ was used as an oxidative stress inductor. After exposure, cells were harvested and used for nuclear and cytosolic protein extraction simultaneously, following the specifications included in the Nuclear Extraction Kit (Cayman Chemical, Ann Arbor MI, USA, Item No 10009277). Protein fractions were quantified using the BCA Protein Quantification Kit (Abcam, Cambridge, UK, ab102536). Nrf2 was detected by using the Nrf2 transcription factor assay kit (Cayman Chemical, Ann Arbor MI, USA, Item No 600590), and following the manufacturer’s instructions. The absorbance of each well was measured at 450 nm in an ELx800 BioTek microplate reader.

qRT-PCR assays

HepG2 cells (3 × 10⁵ cells/well) were treated with ALA (50 μM), CUR (15 μM, ethyl acetate extract from T. chrysantha (0.5 μg/mL), ethyl acetate extract from T. rosea (1 μg/mL) and H₂O₂ (0.6 mM) for durations of 0, 6 and 12 hours. After treatment, mRNA extraction was performed using the RNeasy Plus Mini Kit (Qiagen, Maryland, USA, Cat No. 74134). The mRNA was quantified with a NanoDrop 2000c (Thermo Fisher Scientific, Waltham, MA). The expression of the NQO1 gene (an Nrf2-dependent gene containing ARE sequences in its promoter region^25,26 that is expressed in response to oxidative stress) was evaluated by qRT-PCR and quantified using the 2^-ΔΔCt method, using predesigned TaqMan Gene Expression Assays (code Hs00168547, Applied Biosystems, Foster City, CA, Cat No. 4331182) and the TaqMan® RNA-to-CT^TM 1-Step kit (Applied Biosystems, Foster City, CA, Cat No. 4392653). The run method was holding 48 °C and 15 min, 95 °C 10 min and cycling (40 cycles) 95 °C 15 sec, 60 °C 1 min. β-actin was used as an endogenous control.

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).

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. rosea⁶ (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, H₂O₂, 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, H₂O₂ and the extracts. However, significant differences were found only after exposure to ALA, CUR and, H₂O₂ (p<0.01).

Figure 1. Nrf2 levels in cytosol (C) and nucleus (N) after 0 (white bars) and 4 hours (black bars) exposure to 50 mM ALA, 15 mM CUR, ethyl acetate extracts from T. chrysantha (0.5 μg/mL) and T. rosea (1 µg/mL) and induction of oxidative stress with 0.6 mM H₂O₂.

Kruskal Wallis ** p<0.01, *** p<0.001. ALA: Alpha-lipoic acid; CUR: Curcumin.

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 H₂O₂, 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.

Figure 2. Relative NQO1 mRNA levels with 0, 6 and 12 hours post-exposure to 50 mM ALA, 15 mM CUR, ethyl acetate extracts from T. chrysantha (0.5 μg/mL) and T. rosea (1 µg/mL) and induction of oxidative stress with 0.6 mM H₂O₂.

Kruskal Wallis * p<0.05. ALA: Alpha-lipoic acid; CUR: Curcumin.