Plant material and extraction procedure
Helianthus tuberosus L. were planted in a sunny position, in the first half of March, every 30 cm in a row. The distance between rows was about 80 cm. The tubers were planted in a depth of 10–15 cm. The leaves and tubers of Helianthus tuberosus L. were collected from the region of Subcarpathian Voivodeship in Poland during the September 2017. The collected tubers and leaves were transported to the laboratory and prepared for further analysis. To remove the soil and other impurities, the plant material was cleaned by washing with deionized water. Then, samples of leaves and tubers were used for solvent extraction.
The fresh plant extracts were prepared by using ultrasound-assisted extraction method (UAE). UAE was performed according to the method described by Ying et al. [22] in ultrasonic bath (Digital Ultrasonic Cleaner) equipped with time controller. About 15 g of plant material was packed to the glass tubes and extracted with a 200 ml of ethanol in room temperature. The mixture was homogenized for 50 min (10 cycles for 5 min). Then, obtained extracts were collected and filtered through Whatman filter paper No. 10 and evaporated at 50 °C using a rotary evaporator. Tuber and leaves extracts were stored in the dark in 4 °C for further analysis.
Total phenolic content determination
The total phenolic content of leaves and tubers Helianthus tuberosus L. extracts were determined spectrophotometrically by the Folin-Ciocalteu method according to the procedure reported by Singleton et al. with some modifications [23]. The 300 μL of leaves extract solutions and 1500 μL of 1:10 Folin-Ciocalteau reagent were mixed and after 6 min in the dark, 1200 μL of sodium carbonate (7.5%) was added. After 2 h of incubation in the dark at room temperature, the absorbance at 740 nm was measured spectrophotometrically by AquamateHelion (Thermo Scientific). The total phenolic concentration was calculated from a gallic acid (GA) calibration curve (10–100 mg·mL− 1). Data were expressed as gallic acid equivalents (GA)·g− 1 of extract averaged from three measurements.
Total flavonoids content determination
The total flavonoid content of plant extracts were evaluated using aluminium nitrate nonahydrate according to the procedure reported by Woisky and Salatino with modifications [24]. The 600 μL of plant extracts solutions and 2400 μL of mixture (80% C2H5OH, 10% Al(NO3)3 × 9H2O and 1 M C2H3KO2) were mixed. After 40 min of incubation at room temperature, the absorbance at 415 nm was measured spectrophotometrically by AquamateHelion (Thermo Scientific). The total flavonoids concentration in extracts were calculated from a quercetin hydrate (Qu) calibration curve (10–100 mg·mL− 1) and expressed as quercetin equivalents (Qu)·g− 1 of extract averaged from three independent measurement.
DPPH radical scavenging assay
Antioxidant activity of plant extract was analysed using DPPH free radical scavenging assay, according to the method described by Brand-Williams et al. [25]. 167 μL of 4 mM ethanol solution of DPPH was mixed with 33 μL analysed samples in different concentrations (250 μg·ml− 1 – 5000 μg·ml− 1). The absorbance was measured at λ = 516 nm in every 5 min for 30 min using UV-Vis spectrophotometer Filter Max 5 (Thermo Scientific). DPPH solution mixed with equal volume of distilled water was served as a control. The percentage of the DPPH radical scavenging were calculated using the equation:
$$ \%\mathrm{DPPH}\bullet \mathrm{scavenging}=\left[\mathrm{Abs}\ \mathrm{control}-\mathrm{Abs}\ \mathrm{sample}\right]/\mathrm{Abs}\ \mathrm{control}\times \kern0.37em 100\% $$
Cell culture
HaCaT cells (normal human keratinocytes, ATCC®) and BJ cells (fibroblasts, ATCC®CRL-2522™) was obtained from the American Type Culture Collection (Manassas, VA 20108, USA). HaCaT cells were maintained in a DMEM (Dulbecco’s modified essential medium, Gibco) with L-glutamine, supplemented with 5% (vol/vol) FBS (fetal bovine serum, Gibco), and 1% (vol/vol) antibiotic (100 U·mL− 1Penicillin and 1000 μg·mL− 1 Streptomycin, Gibco). Fibroblast were maintained in a MEM (Minimum Essential Medim, Gibco) contains Earle’s salt and L-glutamine, supplemented with 5% (vol/vol) FBS (fetal bovine serum, Gibco), and 1% (vol/vol) antibiotic (100 U·mL− 1 Penicillin and 1000 μg·mL− 1 Streptomycin, Gibco). All cultured cells were kept at 37 °C in a humidified atmosphere of 95% air and 5% of carbon dioxide (CO2).When the cells reached confluence, the culture medium was removed from the flask (VWR) and cells were rinsed two times with sterile PBS (Phosphate-Buffered Saline, Gibco). The confluent layer was trypsinized using Trypsin/EDTA (Gibco) and then resuspended in fresh medium. Cells were treated with varying concentrations (25, 50, 100, 250, 500 μg·mL− 1) of ethanolic tuber and leaves topinambour extract suspended in DMSO and its value has not exceed 1%.
Cell ViabilityAssay
Cell growth was measured using the neutral red dye (Sigma Aldrich). This assay is based on the initial protocol described by Borenfreund et al. and determines the accumulation of the neutral red dye in the lysosomes of viable, uninjured cells [26].
Cells were placed in 96-well plates at a density of 1 × 104 cells per well with fresh medium. After 24 h of pre-culture, medium was aspirated and varying concentrations (25, 50, 100, 250, 500 μg·mL− 1) of extracts were added into each well and cultured for another 24 h. The control group were unexposed cells. Following exposure to leaves and tuber extracts, cells were incubated for 2 h with neutral red dye (40 μg·mL− 1) dissolved in serum free medium (DMEM or MEM for HaCaT and fibroblasts respectively). After this, cells were washed with Phosphate Buffered Saline (PBS), and then added 150 μL destain solution (EtOH/AcCOOH/H2O2, 50%/1%/49%) per well, followed by gentle shaking for 10 min, until the neutral red has been extracted from the cells and has formed a homogenous solution. Neutral red dye uptake was determined by measuring the optical density (OD) of the eluted dye at 540 nm in microtiter plate reader spectrophotometer FilterMax F5 (Thermo Fisher). The experiments were performed in triplicates for each extract concentration and presented as percentage of control values.
Detection of intracellular reactive oxygen species level
To assay the capacity of obtained plant extracts to generate intracellular level of reactive oxygen species in HaCaT and fibroblasts cells, the fluorogenic dye H2DCFDA was used. After passively diffusion into the cells, H2DCFDA was deacetylated by intracellular esterases into the nonfluorescent compound, that upon oxidation by ROS is converted to the highly fluorescent 2′,7′-dichlorofluorescein (DCF) [27].
Cells were seeded in 96-well plates at a density of 1 × 104cells per well and initially cultured before the experiment for 24 h. After this, culture medium was changed on 10 μM H2DCFDA (Sigma Aldrich) in serum-free medium (DMEM or MEM for HaCaT and fibroblasts respectively). Cells were incubated in H2DCFDA for 45 min before extracts treatment. Then HaCaT and fibroblast cells were exposed into different extract concentration (25, 50, 100, 250, 500 μg·mL− 1), cells treated with 1 mM hydrogen peroxide (H2O2) was used as a positive control. The DCF fluorescence was measured every 30 min for a total 90 min using a microplate reader FilterMax F5 (Thermo Fisher Scientific) at maximum excitation of 485 nm and emission spectra of 530 nm.
Real-time PCR for mRNA expression analyses
For the experiment, cell were seeded on 6-well plate at a density 5 × 105 cells per well. After 24 h of pre-incubation, cells were treated with obtained leaves and tuber extracts and incubated for 24 h. Then, samples were collected, and total RNA was extracted from HaCaT and fibroblast cells using a EURx Universal RNA Purification Kit according to the manufacturer’s protocol. The quality and quantity of the mRNA were determined spectrophotometrically at 260 and 280 nm (ND/1000 UV/VIS; ThermoFisher NanoDrop). The reverse transcription (RT) reaction was performed at a final volume 20 μL with 1 μg of RNA (as a cDNA template) using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystem™) according to the manufacturer’s protocol. The RT-PCR run was performed using the Bio Rad C1000 Touch™ Thermal Cycler (Bio Rad Laboratories). The products obtained from the RT reaction were amplified using TaqMan Gene Expression Master Mix (Life Technologies Applied Biosystems) kit with TaqMan probes as primes for the specific genes encoding Nox-4 (assay ID Hs00276431_m1), SOD-1 (assay ID Hs00166575_m1), GAPDH (assay ID Hs02786624_g1). Amplification was carried out in a total volume of 20 μL containing 1× TaqMan Gene Expression Master Mix and 1 μL of RT product, which was used as the PCR template. Standard qPCR procedures were performed as follows: 2 min at 50 °C and 10 min at 95 °C, followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C using an Bio Rad CFX Connect™ Real-Time System (Bio Rad Laboratories).The mRNA expression was calculated relative to a nontargeting control in each experiment. The experiments were repeated in triplicate. The expression level of the gene was calculated using the comparative threshold cycle (Ct) method [28] GADPH was used as the reference gene.
Statistical analysis
Each value is the mean of three replicates. Values of different parameters were expressed as the mean ± standard deviation (SD). The two-way analysis of variance (ANNOVA) and Bonferroni post-test between groups were performed at the level P value of < 0.05 to evaluate the significance of differences between values. Statistical analyses were performed using GraphPad Prism 5.0 (GraphPad Software, Inc., Sand Diego CA).