Anti-apoptotic and anti-inflammatory activity of Gentiana lutea root extract

Materials

Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum were from Euroclone. Acridine orange, aluminium chloride, ascorbic acid, dithiotreitol (DTT), dimethyl sulfoxide (DMSO), DPPH (2,2-diphenyl-1-picrylhydrazyl), ethidium bromide, Folin–Ciocalteau’s reagent, glutathione reductase, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], NADPH, Triton X-100, tannic acid, quercetin, sulfosalicilic acid, reduced glutathione (GSH), 5,5′-dithiobis(2-nitrobenzoic acid), lipopolysaccharide (LPS) and tert-butyl hydroperoxide (t-BHP), HPLC-grade acetonitrile (≥ 99.9) and formic acid were purchased from Sigma-Aldrich. 2′,7′-dichlorofluorescein diacetate (DCFH2-DA) was purchased from Cayman Chemical. Fluorogenic caspase-3 substrate, acetyl-Asp-Glu-Val-aspaminomethylcoumarin (Ac-DEVD-AMC), doxorubicin and vinblastine were from Alexis Biochemicals. Polyvinylidene difluoride (PVDF) was from Bio-Rad. Anti-Bcl-2 (cat. no. sc-509), anti-Bax (cat. no. sc-493), anti-Sirt-1 (cat. no. sc-15404) and anti-actin (cat. no. sc-1616) antibodies were from Santa Cruz Biotechnology, Inc.

The analytical standards of loganic acid, swertiamarin, sweroside, gentiopicroside, amarogentin and isogentisin were purchased from PhytoLab (Germany). The purity of all standard compounds was ≥ 97% (determined by HPLC). All other chemicals were reagent grade. Deionized water (> 18-MΩ cm resistivity) was obtained from a Milli-Q SP Reagent Water System (Millipore, Bedford, MA, USA). Stock solutions of vinblastine and doxorubicin were prepared in DMSO and stored in the dark at − 20 °C. All solvents and solutions were filtered before HPLC analysis through 0.45-μm PTFE filters purchased from Phenomenex (Bologna, Italy).

Preparation of G. lutea extracts

Commercial dried roots of G. lutea, collected in French Alpes, were purchased from an herbal shop. A voucher specimen was retained in the Laboratory of Cellular Biochemistry of Department of Biotechnological and Applied Clinical Sciences, University of L’Aquila (FBGL-0004).

Roots were cut into small pieces and finely powered using a mechanical blender.

Aliquots of 10 g of obtained powder were extracted with methanol (3 × 50 ml, 24 h each, at room temperature) or with H2O (3 × 50 ml, 24 h each, at room temperature or 1 × 50 ml, for 10 min, at 95 °C). After filtration through Whatman No. 1 filter paper, the alcoholic extracts were collected and concentrated under vacuum by using a rotary evaporator, yielding brown residues. The aqueous extracts were collected, frozen and lyophilized. The extracts were stored in darkness at 4 °C until used.

The methanolic and aqueous extracts were reconstituted, at a concentration of 50 mg/ml, in DMSO and distilled water, respectively.

Determination of total phenolic content

Total phenolic content was determined using the Folin–Ciocalteau method with some modifications (Stratil et al. 2006). Briefly, the reaction mixture with 1 mg of extract, 100 µl of Folin–Ciocalteau’s reagent and 800 µl of 2.5% sodium carbonate was incubated at 25 °C for 1 h and then the absorbance was measured at 765 nm in a Perkin–Elmer Lambda 19 spectrophotometer. Tannic acid was used as a standard. The results were compared with the tannic acid calibration curve and expressed as milligrams of tannic acid equivalents (TAE)/g of sample.

Determination of total flavonoid content

The total flavonoid content of extracts was determined by the aluminium chloride (AlCl3) method (Ordoñez et al. 2006). The reaction mixture with aliquots of 500 µl of extract solution, containing 2 mg of extract, and an equal volume of 2% AlCl3 in ethanol was incubated for 1 h at 25 °C. The absorbance was recorded at 420 nm in a Perkin–Elmer Lambda 19 spectrophotometer.

Known concentrations of quercetin were used to generate a standard calibration plot. The concentrations of flavonoids in the samples were calculated from the calibration plot and expressed as milligrams of quercetin equivalent (QE)/g of sample.

Determination of total flavonol content

Flavonols were determined according to Kumaran and Karunakaran (2007), with some modifications. Aliquots of 200 µl of extract solution, containing 5 mg of extract, were incubated with an equal volume of 2% AlCl3 and 600 µl of 5% sodium acetate. The reaction mixture was incubated at 25 °C for 150 min and the absorbance was recorded at 440 nm in a Perkin–Elmer Lambda 19 spectrophotometer. Known concentrations of quercetin were used to generate a standard calibration plot. The concentrations of flavonol in the test samples were calculated from the calibration plot and expressed as milligrams of quercetin equivalent (QE)/g of sample.

DPPH free radical scavenging assay

DPPH quenching activity of extracts was evaluated by spectrophotometric assays. Increasing concentrations of samples in methanol were mixed with 100 µL of 1 mM DPPH solution in methanol. After 30 min of incubation at room temperature, absorbance was recorded at 517 nm on a Perkin-Elmer spectrophotometer Lambda 19. Ascorbic acid was used as a reference standard. Radical-scavenging ability was calculated according to the following formula:

$$\% \;of\;Inhibition = \, \left[ - A_ } \right)/A_ } \right] \, \times \, 100$$

where Acontrol is the absorbance of the DPPH solution without the samples, and Asample is the absorbance of the tested samples. The antioxidant activity was expressed as IC50 value, calculated as the minimum concentration of samples required to inhibit 50% of the DPPH radical.

HPLC–MS triple quadrupole

HPLC–MS studies were performed using an Agilent 1290 Infinity series and a Triple Quadrupole 6420 from Agilent Technology (Santa Clara, CA) equipped with an ESI source operating in negative ionisation mode. The separation was achieved on a Synergi Polar-RP C18 (4.6 mm × 150 mm, 4 µm) analytical column from Phenomenex (Chesire, UK). The mobile phases for HPLC–ESI–MS (triple quadrupole) analyses were water with 0.1% formic acid (80) and acetonitrile (20) at a flow rate of 0.6 mL/min in isocratic conditions. The solvent composition varied as follows: 0–7 min, 20% B; 7–15 min, 20–90% B; 15–18 min, 90% B; 18–25 min, 90–20% B. The column temperature was set at 30 °C and the injection volume was 10 μl. In HPLC-ESI–MS, ion source was operating in negative ionization (NI) mode. Optimization of the HPLC/ESI–MS conditions was carried out by flow injection analysis (FIA) of the analytes (1 μl of a 50 μg/ml individual standard solutions). The optimum ESI ion source conditions were as follows: gas temperature, 350 °C; nebulizer gas (nitrogen) pressure, 50 psi; drying gas (nitrogen) flow rate, 11 ml/min; capillary voltage, 4000 V. Quantifications were performed by using selected ion monitoring (SIM) mode, fragmentor 70 V, dwell time 150 and all the analytes monitored in two time segment, loganic acid, gentiopicroside swertiamarin and sweroside from 0 to 9 min, and amarogentin, gentiopicroside and isogentisin from 9 until the end of the run. The different ions were: the deprotonated molecular ions [M–H]− of loganic acid, amarogentin and isogentisin at m/z 375, 585 and 257, respectively and the [M + HCOOH–H]− ions of swertiamarin, sweroside and gentiopicroside at m/z 419, 403 and 401, respectively.

Cell culture

The human SH-SY5Y neuroblastoma cell line and the murine RAW 264.7 macrophage-like cell line, obtained from the American Type Culture Collection (ATCC) and the European Collection of Cell Cultures (ECACC), respectively, were grown in DMEM medium. The media were supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 2 mM glutamine. Cells were maintained at 37 °C in a humidified 5% CO2 atmosphere. Cell viability was determined by trypan blue exclusion test.

Cytotoxicity assay

The effects of G. lutea extract on cell viability were evaluated in vitro using the MTT colorimetric method (Mosmann 1983). Exponentially growing cells were seeded in 96-well plates and, after 24 h of growth, were treated with increasing concentrations of commercial G. lutea methanolic extract, from 50 to 800 µg/ml. The same amount of DMSO was added to negative controls. After treatment, MTT reagent, at a concentration of 0.5 mg/ml, was added to each well and the cells were incubated for additional 3 h at 37 °C. The absorbance at 570 nm was determined in a microplate reader (BioRad, Model 550) after solubilizing the formazan crystals by addition of 100 μl of acidified isopropanol (0.04 M HCl in isopropanol). Viability of untreated cells was considered 100%. Cell survival was determined comparing the absorbance of treated and untreated cells.

For evaluation of combined effects of the extract and some pro-apoptotic agents, SHSY-5Y cells were treated with increasing concentrations, from 50 to 800 µg/ml, of methanolic extract of G. lutea root, alone or in combination with concentrations from 0.05 to 0.8 µM of doxorubicin and vinblastine for 48 h. At the end of incubation, cell survival, compared with untreated controls, was evaluated using the MTT assay.

Apoptosis evaluation

Nuclear morphology was assessed by acridine orange/ethidium bromide double staining assay. After washing with PBS, cells were stained with a fluorescent solution containing 100 µg/ml ethidium bromide and 100 µg/ml acridine orange in PBS and immediately observed with a fluorescence microscope. Cells showing condensed and fragmented chromatin were considered apoptotic. A minimum of 400 cells were counted for each determination.

Caspase-3 activity

Cells were washed with PBS and then resuspended in a lysis buffer, containing 50 mM Tris–HCl, pH 7.4, 10 mM EGTA, 1 mM EDTA, 10 mM DTT, 1% (v/v) Triton X-100, for 30 min at 4 °C. After centrifugation at 15,000 g for 15 min at 4 °C, supernatants were collected and used for detection of caspase activity.

Cell lysate (60 µg of proteins) was mixed with 20 μM fluorogenic caspase-3 peptide substrate, Ac-DEVD-AMC, in the reaction buffer (50 mM Tris–HCl, pH 7.4, 10 mM EGTA, 1 mM EDTA, 10 mM DTT). The reaction mixture was incubated for 30 min, at 37 °C (Köhlex et al. 2002).

Fluorescence was measured on a Perkin-Elmer LS-50B spectrofluorometer, setting excitation at 380 nm and emission at 460 nm.

Detection of intracellular reactive oxygen species (ROS)

DCF fluorescence was used to detect the generation of cellular ROS (LeBel et al. 1992). Cells were exposed to 200 µg/ml of G. lutea root extract alone or in combination with 0.1 µM vinblastine for 12, 24 and 36 h at 37 °C and to 200 µM t-BHP for 2 h at 37 °C, as positive control. After treatments, cells were incubated with DCFH2-DA to a final concentration of 20 µM at 37 °C for 30 min. After washing with PBS, fluorescence intensity of cells was analyzed with a Perkin–Elmer LS-50B spectrofluorometer, setting excitation at 485 nm and emission at 530 nm.

Glutathione determination

Cells were washed with PBS and resuspended in 5 mM EDTA and 5% (w/v) sulfosalicilic acid. Total glutathione (GSH) concentrations was measured as previously reported (Rahman et al. 2006) with some modifications.

An aliquot (10 µl) of cell extract was added to 930 µl of 0.1 M potassium phosphate buffer (pH 7.5), containing 5 mM EDTA, 58 µM NADPH and 40 µg/ml 5,5′-dithiobis(2-nitrobenzoic acid). The assay was initiated by addition of 60 µl of glutathione reductase (3 U/ml). The rate of formation of 5-thio-2-nitrobenzoic acid, proportional to total glutathione concentration, was followed at 412 nm for 2 min at 25 °C in a Perkin–Elmer Lambda 19 spectrophotometer. GSH concentration values were calculated from a standard curve and expressed as nmol/106 cells.

Western blot analysis

Cells were washed with PBS and lysed in a buffer containing 10 mM Hepes, pH 7.2, 5 mM MgCl2, 142 mM KCl, 0.2% (v/v) Nonidet P-40, 1 mM EDTA and a suitable cocktail of protease inhibitors, at 4 °C for 30 min. Proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes. The blots were incubated with monoclonal anti-Bcl-2, anti-Bax and anti-Sirt-1 antibodies, and polyclonal anti-actin antibody, for 1 h at room temperature. The membranes were then probed with the appropriate peroxidase-conjugated secondary IgG antibodies. The blots were visualized using an enhanced chemiluminescent detection system (Thermo Scientific, Rockford, USA) and the intensity of each band was quantified by ImageJ software and normalized with actin levels.

TNF-α determination

RAW 264.7 cells were seeded at a density of 1.5 × 105 cells/well in 24-well plates for 24 h. The following treatments were performed: (1) cell stimulation with 200 ng/ml lipopolysaccharide (LPS) from Pseudomonas aeruginosa for 1 h, after pre-treatment with 200 µg/ml of G. lutea root methanolic extract for 7 h; (2) simultaneous exposure of cells to LPS and methanolic extract for 1 h.

Cell-free supernatants of treated and untreated cells were collected and TNF-α levels were quantified using an enzyme-linked immunoadsorbent assay (ELISA) kit (PeproTech, Rocky Hill, NJ) according to the manufacturer’s instructions.

Statistical analysis

Data are reported as mean ± SD. Statistical differences were calculated using the Student’s t test. Results were considered statistically significant at p value < 0.05.

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