Plant material
Leaves of G. tinctoria Mol. (Nalca) were collected in April 2012 in the Araucanía Region of Chile. Leaves were air-dried, knife grounded and sieved up to a particle size of 500 µm. The voucher material was deposited at Herbarium of Pharmacy School, Universidad Andres Bello (Collection number HPS-UNAB/14024).
Preparation of the methanolic extract from G. tinctoria leaves
The methanolic extract was obtained by successive maceration of 100 g of dried plant material (average particle size < 500 µm) with hexane, dichloromethane, ethyl acetate, and methanol, at room temperature for 48 h. The solid residue/solvent ratio was 1/10 (w/v). The dispersion was continually stirred at 800 rpm. Finally, the crude extracts were concentrated under reduced pressure to dryness.
Polyphenol content
The quantification of polyphenols was done according to the Folin–Ciocalteau method with modifications [21]. The phenolic content was expressed as mg gallic acid equivalents/g dry methanolic extract (mg GAE/g extract). The measurements were made in triplicate.
Fractionation of G. tinctoria extract by pH-zone refinement using centrifugal partition chromatography (CPC)
The extract of G. tinctoria was fractionated using a Spot-CPC-250-B Bio-Extractor (CPC, Armen, France). The system had a four-way switching valve that allowed operation in either descending or ascending modes. The CPC unit was connected to a SPOT.PREP II system (Armen, France), with integrated UV detector and fraction collector. CPC separation was performed using the pH zone refinement method described by Ref. [22] with slight modifications. The two-phase solvent system was composed of methyl tert-butyl ether/1-butanol/acetonitrile/water with a volume ratio of 4:2:3:8. The solvent mixture was equilibrated in a separation funnel and divided in an upper and lower phase, where 10 mM trifluoroacetic acid (TFA) was added as a retainer to the upper organic phase and 10 mM NH3 was added as a displacer to the lower phase. The CPC rotor was first filled with 1.5 column volumes using the upper phase at 30 mL/min and 500 rpm rotation. The lower phase was pumped into the system (descending mode) at a flow rate of 12 mL/min and rotation was increased from 500 to 2000 rpm. The samples (1 g of G. tinctoria extract) were dissolved in 10 mL 1:1 mixture of upper (with 10 mM TFA) and lower (without NH3) layers and injected into the CPC system at the same time the lower phase was introduced. Elution was monitored using a scan in the range of 200–600 nm; and at 254 and 360 nm wavelengths, by collecting fractions in 32 mL tubes. Fractions with similar concentrations were combined according to on-line UV spectra and thin layer chromatography results.
Liquid chromatography parameters and MS parameters
Fast preliminary analysis of the G. tinctoria extract was performed using a RP-HPLC–UV with a monolithic RP-18e column Chromolith 100–10 mm (Merck, Germany). The mobile phases employed were (A) water with 0.1% TFA and (B) acetonitrile while the gradient program was: (0.00 min, 0% B); (10.00 min, 20% B); (12.00 min, 20% B); (14.00 min, 0% B) and 2 min for column equilibration before injections. The flow rate employed was 5.00 mL/min, and the injection volume was 50 μL. Liquid chromatography-mass spectrometry was performed using an Acclaim UHPLC C18 column (Acclaim, 150 mm × 4.6 mm internal diameter, 2.5 μm, Thermo Fisher Scientific, Bremen, Germany) set at 25 °C. The wavelengths used for detection were 354, 254, 280, and 330 nm, and DAD was acquired from 200 to 800 nm for full characterization of peaks. Mobile phases employed were acetonitrile (B) and 1% aqueous formic solution (A) while the gradient program was: (0.00 min, 7% B); (5.00 min, 7% B); (10.00 min, 25% B); (15.00 min, 33% B); (20.00 min, 85% B); (25.00 min, 90% B); (35.00 min, 7% B) and 15 min for column equilibration before injections. The flow rate employed was 1.00 mL/min, and the injection volume was 10 μL. Standards and the extract dissolved in methanol were maintained at 10 °C during storage in the auto-sampler. The HESI II and Orbitrap spectrometer parameters were set as informed previously [23]. Peak identification was done analyzing the mass spectra, fragmentation patterns and by comparison with pure standards, accordingly.
In vitro antioxidant activity of G. tinctoria extract
Ferric reducing antioxidant power (FRAP)
In vitro antioxidant activity of G. tinctoria extract was determined using the FRAP assay with modifications [24]. The FRAP reagent was prepared mixing 1020 µL of sodium acetate 300 mM at pH 3.6, 100 µL of 2,4,6-tripyridyl-s-triazine 10 mM, and 100 µL of FeCl3·6H2O 20 mM. Then, 10 µL of extract dissolution was added to the FRAP reagent. The calibration curve was prepared with FeSO4 solution in concentrations ranging from 15 to 35 µM and using the FRAP reagent as blank. The absorbance was measured at 593 nm in a Jenway 6405 UV–Vis spectrophometer, after 4 and 60 min of heating at 40 °C. The average of three measurements were expressed as µmol Fe2+/g dry methanolic extract. Quercetin was used as reference compound. Ferric reducing antioxidant power values of G. tinctoria extract and quercetin at 4 and 60 min were reported.
2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity
The method is based on the decrease of absorbance of DPPH dissolved in methanol when mixed with an antioxidant compound. The decrease in absorbance at 515 nm is inversely proportional to the antioxidant activity. An aliquot of 100 µL of G. tinctoria extract were added to 3.9 mL of DPPH methanolic solution (25 mg/L). The absorbance at 515 nm was recorded at the beginning (Absorbanceonset) and in steady state (Absorbanceend) at room temperature, and the remnant DPPH was calculated as follows:
$$\% DPPH_{rem} = \frac{{Absorbance_{end} }}{{Absorbance_{onset} }}*100$$
The antiradical efficiency (AE) was expressed as [25]:
$$AE = \frac{1}{{EC_{50} *T_{EC50} }}$$
where EC50 is the concentration of antioxidant required to decrease the initial DPPH radical concentration by 50% and is obtained from a curve of remnant DPPH % versus extract concentration. TEC50 is defined as the time required to reach steady state at EC50. Gallic acid was used as reference compound. The average of three measurements were reported as L/(min × mg of extract).
Isolation and culture of human umbilical vein endothelial cells
This investigation conforms to the principles outlined in the Declaration of Helsinki and counts with approval from the Ethics Committee of Universidad de Concepción and the Ethics and Scientific Committee of Chilean National Health System, Concepción division (CEC 19-05-3, 25-06-2019). Patient written informed consents were obtained. HUVECs were isolated by collagenase digestion (0.25 mg/mL collagenase Type I from Clostridium histolyticum, Invitrogen, Carlsbad, CA, USA) as previously described [26]. After isolation, cells were cultured (37 °C, 5% CO2) in M199 (Gibco Life Technologies, Grand Island, NY, USA) containing 5 mM d-glucose, 10% newborn calf serum, 10% fetal calf serum, 3.2 mM l-glutamine, and 100 U/mL penicillin–streptomycin (primary culture medium). Cells were used up to passage 2 and experiments were performed on cells depleted of serum (12 h) and later incubated as reported in each experiment.
Cell viability assay
To determine the toxicity of extracts, HUVECs were seeded in 96-well plate dishes and incubated (24 h) in M199 (control) in the absence or presence of H2O2 (100 µM), and/or G. tinctoria extract (50, 100, 200 and 300 μg/L) or vehicle of extract (dimethyl sulfoxide, DMSO). The sample labels were chosen according to minority components added to the culture medium, thus cells incubated only with M199 were labeled as control, those incubated in presence of H2O2 as H2O2, those incubated in presence of DMSO (1% v/v) as vehicle and those incubated in presence of G. tinctoria extract as G. tinctoria. After the treatments, cytoxicity was evaluated by Alamar Blue (resazurin) assay (Thermo Fisher Scientific, Waltham, MA, USA) incubated at 10% v/v by 2 h (37 °C). Fluorescence was measured at 590 nm emission using Synergy 2 (Biotek, Winooski, VT, USA) microplate reader after 570 nm excitation. Data were expressed as a percentage of change in relative fluorescence units (RFU) compared to the average of the control.
Live cell imaging
HUVECs were seeded in 6-well plate dishes and incubated (24 h) in M199 in the absence (labeled as control) or presence of 25 mM d-glucose (high d-glucose), H2O2 (100 µM) and/or G. tinctoria extract (200 μg/L) or vehicle (DMSO, 1% v/v). The sample labels were also chosen according to minority components added to the culture medium, thus those incubated in presence of 25 mM d-glucose were labeled as HG, those incubated in presence of 25 mM d-glucose and of G. tinctoria extract as HG+G. tinctoria, and those in presence of hydrogen peroxide and of G. tinctoria extract as H2O2+G. tinctoria. Imaging was performed using a Nikon Eclipse Ti-U coupled to a Nikon DS-QiMc camera (Nikon Instruments Inc., Melville, USA). Images were created by capturing phase contrast with a 40× lens. The apoptotic-like cells in each field were quantified and the rate of apoptotic-like cells was expressed in relation to the total number of cells in each field.
Gene expression of apoptotic gene Noxa
To determine the expression of phorbol-12-myristate-13-acetate -induced protein 1 (Noxa), total RNA was isolated by chomczynski-fenol method [17] from HUVECs incubated in absence (control) or presence of high d-glucose (25 mmol/L, 24 h), H2O2 (100 µM) and/or G. tinctoria extract (200 µg/L) or vehicle (DMSO, 1% v/v). Non-quantitative polymerase chain reaction (PCR) was performed using Swift Max Pro thermal cycler (Esco technologies, Horsham, USA) in a reaction mixture containing 0.5 μmol/L primers, deoxynucleotide triphosphates, thermostable deoxyribonucleic acid polymerase and a reaction buffer (SapphireAmp Fast PCR Master Mix, Clontech laboratories, Mountain View, USA). The oligonucleotide primers for Noxa were gently provided by Dr. Roxana Pincheira (University of Concepción) and 28S was used as housekeeping. The amplicon images (PCR bands) in agarose gel were captured under ultraviolet (UV) light, documented and analyzed using ImageJ software (Java-based imaging processing program, National Institute of Health, USA) [17].
Intracellular ROS levels
HUVECs were grown in 24-well plates and incubated in absence (control) or presence of high d-glucose (25 mmol/L, 24 h), H2O2 (100 µM) and/or G. tinctoria extract (200 μg/L) or vehicle (DMSO, 1% v/v). After treatments, cells were exposed (45 min, 37 °C) to 10 μmol/L of CM-H2DCFDA (Molecular Probes, Leiden, The Netherlands) in phosphate buffered saline (37 °C, pH 7.4). The fluorescence of CM-H2DCFDA (λexc/λem: 495/510 nm) was determined in a Synergy 2 (Biotek, Winooski, VT, USA) microplate reader [17].
Intracellular NO levels
HUVECs were grown on microscope coverslips and incubated with M199 (labeled as Control), G. tinctoria extract (200 μg/L) (labeled as G. tinctoria), ellagic acid-enriched fraction (labeled as F3) or DMSO (labeled as vehicle, 1% v/v). To determine intracellular NO, cells were loaded (45 min, 37 °C) with 10 μM of 4-amino-5-methylamino-2′,7′-difluorofluorescein (DAF-FM) (Molecular Probes, Leiden, The Netherlands). The fluorescence was determined in fixed cells and the signal density was analyzed by ImageJ software (Java-based imaging processing program, National Institute of Health, USA) [25]. Histamine (10 μM, 5 min) was used as positive control [27].
Wire myography in placental veins
Human placental veins of 2–3 mm external diameter were isolated, freed of adhering connective tissue, and cut into 2-mm ring segments. Two stainless steel wires were inserted through the vessel ring, one connected to a Grass FT03 force displacement transducer for isometric recording of the contractions on a Grass recorder (Model 79 D), and the other was used to adjust the resting tension vessels to 1.0 g. Vessels were bathed in modified Krebs–Ringer solution [mM: 119 NaCl, 4.6 KCl, 15 NaHCO3, 1.5 CaCl2, 1.2 MgCl2, 1.2 NaH2PO4, 5 d-glucose (pH 7.4, 37 °C, 95% O2/5% CO2)]. After the optimal resting tension was obtained, the tissues were allowed to equilibrate for 1 h. At the beginning and end of each experiment, the maximal contractile response to 90 mM KCl solution was recorded. Only if the contractile responses were similar in magnitude (with variation less than 10%), the data from this particular experiment were included in the analysis [28]. After maximal response to KCl, vessels were washed and equilibrated for 30–40 min. Vessels were incubated with U46619 (0.1 µM, thromboxane A2 analog) and after 5 min 200 μg/L of G. tinctoria extract were added in the experimental group. Changes in isometric tension were registered for 15 min following incubation with the extract.
Statistical analysis
The data of FRAP assay were analyzed with a one-way analysis of variance at the 95% confidence level. Differences among the mean values of antioxidant power at 4 and 60 min were determined with Duncan’s multiple-range test.
In endothelial cell and vascular reactivity assays, the values are mean ± S.E.M., where n indicates the number of different biological samples in triplicated. Comparisons between two groups were performed by means of Student’s unpaired t-test or Mann–Whitney test for parametric or non-parametric data, respectively. The statistical software GraphPad Prism 6.0 (GraphPad Software Inc., San Diego, CA, USA) was used for data analysis; p < 0.05 was considered statistically significant.