Superior protective effects of in vitro propagated green garlic against hydrogen peroxide-induced cytotoxicity in human hepatoma cells

Garlic was grown either by planting the cloves of autochthonous garlic cultivar Slavonian winter garlic (cv. Slavonski ozimi; collected in July 2016) directly in the field on 5 November 2016 (conventional garlic – CG) or by propagating garlic (PG) in vitro and planting the transplants in the field on 9 March 2017. Plant material was supplied by the Josip Juraj Strossmayer University of Osijek, Faculty of Agrobiotechnical Sciences Osijek, Osijek, Croatia. Field in-row spacing for planting CG was 10 cm and inter-row spacing 35 cm (10x35 cm). PG was planted at 5x20 cm plant spacing. In other words, plant density of CG was 28.5 and of PG 100 plants per square meter. Both CG and PG were grown on 4 m² parcels in quadruplicate following the randomised block design. At the end of the vegetation period on 11 July 2017, which corresponds to the harvest time, when 70 % of the aboveground part of CG is dry. We collected CG bulbs (cloves) and the leafy part of PG, as it did not form true bulbs with cloves and remained green. This was expected due to skipped vernalisation. During the harvest, we recorded the yield (weight of PG leafy part and CG bulbs) as well as the number of leaves per plant (Table 1).

In vitro propagation of garlic started in November 2016. Healthy and vital garlic cloves collected in July 2016 were peeled and washed under tap water for 15 min. Cleaned cloves were pre-sterilised as described previously (7, 8, 9) with some modifications. Briefly, cloves were immersed in 70 % ethanol for 2 min and then in 2 % sodium hypochlorite with 2 drops of liquid detergent for 20 min for pre-sterilisation. The cloves were then rinsed with sterile distilled water five times and germs explanted from cloves in a microbiological safety cabinet. To minimise potential contamination, germ surface was also sterilised by immersion in 70 % ethanol (Gram-mol, Zagreb, Croatia, CAS 64-17-5) for 1 min and then in a solution of 2 % sodium hypochlorite and two drops of liquid detergent for 10 min. Rinsed with distilled water, the germs were cut vertically in two to three pieces and placed on a Linsmaier & Skoog (LS) medium supplemented with 30 g/L sucrose, 0.1 g/L myo-inositol (HiMedia Laboratories, Mumbai, India), 8 g/L agar (Acros Organics, New Jersey, USA, CAS 9002-18-0), 1 mg/L 6-benzylaminopurine (Acros Organics, CAS 121439-7), and 0.1 mg/L indole butyric acid (Acros Organics, CAS 87-51-4) and cultured in a growth chamber. The explants were sub-cultured several times, each time after three weeks on the same medium, by separating new micro-shoots as new explants. After gaining about 1700 healthy and viable garlic explants, the explants were sub-cultured on rooting LS medium supplemented with 1 mg/L indole butyric acid and 0.1 mg/L 6-benzylaminopurine. Temperature (21 °C±1 °C) and photoperiod (16 hours of light) were the same throughout cultivation. Rooted garlic plantlets were transferred to polystyrene trays with 78 mL cells filled with sterile substrate (Potgrond H, Klasmann-Deilmann GmbH, Geeste, Germany) and placed in a growth chamber for acclimatisation. Ex vitro garlic plants were kept under the same light and temperature conditions as those in vitro, with relative air humidity maintained at 90 % for the first week and then gradually decreased to constant 60 % and temperature gradually decreased to 15 °C. After one month of acclimatisation, the transplants were put into soil on 9 March 2017 and were about the same size as conventional garlic (CG) planted in November 2016 but with fewer true leaves formed.

CG and PG garlic extracts were prepared after sample collection from the field in July 2017. Garlic powder was prepared as follows: 400 g of fresh and peeled CG garlic cloves and 400 g of fresh PG leafy part were submerged in liquid nitrogen and then lyophilised. The lyophilisate was then ground into fine powder and stored at -30 °C until analysis.

Extraction for the analysis of antioxidant activity, total phenolic content, and total flavonoid content was performed by adding 1 g of freeze-dried garlic powder to 10 mL of 80 % methanol (v/v) and using an ultrasonic bath at 25 °C for 30 min. Then the extracts were filtered through a 0.2 μm pore size nylon membrane filter (Whatman Inc., Piscataway, NJ, USA) and stored at 4 °C in sealed glass test tubes until analysis.

Extraction for high-performance liquid chromatographic (HPLC) determination of alliin was done by adding 1 g of garlic powder to 10 mL of acetonitrile in the Erlenmeyer flask. The flasks were closed, placed in a water bath shaker and shaken at 80 °C for 4 h. The samples were then centrifuged, and their supernatant filtered through a 0.2 μm pore size nylon membrane filter (Whatmann Inc.). Extracts were kept in sealed glass vials at 4 °C until analysis.

Total phenolic content was determined with the Folin-Ciocalteu method (10) as described by Chen et al. (11). Briefly, CG and PG extracts (100 μL) were diluted with 5.9 mL of distilled water and mixed. Next, 200 μL of Folin-Ciocalteu reagent was added to the mixture and 2 mL of sodium carbonate solution added 1 min later. The mixture was allowed to react at room temperature in the dark for 120 min. The absorbance was measured at 735 nm. Gallic acid was used as a standard, and the results were expressed as mg of gallic acid equivalents (GAE) per gram of garlic powder.

Total flavonoid content was determined as described by Molina-Quijada et al. (12). Briefly, a 0.05 mL of methanolic extract was mixed with 3.65 mL of distilled water and 0.15 mL of a 5 % (w/v) solution of NaNO₂. After 5 min, 0.15 mL of 10 % (w/v) AlCl₃ was added to the mixture, kept for another 1 min, and 1 mL of 1 mol/L NaOH was added. Then, the absorbance of samples was measured at 415 nm with a Cecil 9000 UV-visible spectrophotometer (Cecil Instrumentation Services, London UK). Total flavonoid content was expressed as quercetin equivalents, i.e. in milligrams of quercetin per gram of garlic powder. All determinations were carried out in triplicate.

The DPPH free radical (DPPH•) scavenging ability of garlic extracts was determined as described previously (13). Briefly, a 0.3 mmol/L DPPH• stock solution was prepared by dissolving 7.6 mg of DPPH in 100 mL of methanol. Properly diluted CG and PG extracts of alliin (0.1 mL) were mixed with 2 mL of 0.1 mmol/L of DPPH• solution. The mixture was left in the dark for 25 min and the absorbance measured at 517 nm using a Cecil 9000 UV-visible spectrophotometer (Cecil Instrumentation Services). Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) was used as a standard reference to convert the AOX of each extract solution to the Trolox equivalent antioxidant activity (TEAC). The results were expressed as millimol of TEAC per gram of garlic powder. All determinations were carried out in triplicate.

Alliin content in CG and PG samples was determined with a HPLC Varian ProStar system equipped with a 230SDM pump, 410 autosampler, and a 330 UV diode-array detector (Varian, Palo Alto, CA, USA). The chromatographic column was a 250x4 mm I.D., 5 μm particle size Gemmini C18 with a 4x3.0 mm I.D. 5 μm particle size guard column (Phenomenex, Torrence, USA). The mobile phase consisted of acetonitrile and water. The isocratic elution was carried out with 10 % acetonitrile and 90 % water in 7 min. The flow rate was 1 mL/min. The amount injected was 100 μL. The UV spectra were recorded from 200 to 400 nm. The working wavelength for quantitative analysis was 210 nm. Compounds in samples were identified by their retention time and UV spectra, which were compared to the known standards. Alliin content was expressed as milligrams of alliin per gram of garlic powder. All determinations were carried out in triplicate.

The human hepatoma cell line HepG2 was purchased from Sigma-Aldrich (St. Louis, MO, USA), cultivated in 1 % glucose Dulbecco’s Modified Eagle’s Medium supplemented with L-glutamine (2 mm), and 10 % foetal bovine serum. Cell cultures were routinely grown in 25 cm² flasks (TPP Techno Plastic Products AG, Trasadingen, Switzerland) in a humidified atmosphere with 5 % CO₂ at 37 °C until the growth reached the exponential stage. Growth medium was refreshed every three or four days.

To determine the antioxidant activity of garlic extracts, hepatoma cells were exposed to 200 μmol/L H₂O₂ (Kemika, Zagreb, Croatia) to induce oxidative stress (14, 15, 16, 17, 18, 19, 20). The protective effect of garlic extracts was evaluated by adding CG and PG extracts to a cell culture medium 20 min after cell exposure to H₂O₂. Exposure to garlic extract lasted 24 h at 37 °C. The effects of CG and PG extracts were compared with the antioxidant activity of commercial alliin (S-Allyl-L-cysteine sulphoxide, Sigma-Aldrich) alone. All extracts were dissolved, sterilised by filtering through Whatman 0.2 μm pore size nylon membrane filters, and then added to cells in concentrations below 1000 μg/mL for garlic or 100 μmol/L for commercial alliin, which turned out not be toxic to HepG2 cells in a preliminary 24-hour exposure test, while higher concentrations induced apoptosis (data not shown). These were 10, 100, and 1000 μg/mL for CG and PG extracts and 1, 10, and 100 μmol/L for commercial alliin, prepared by diluting garlic stock solutions with PBS buffer until they corresponded to the concentration of applied alliin alone. Cells exposed to H₂O₂ alone (positive control) and non-treated cells (negative control) were included in each experiment.

The metabolic activity and viability of HepG2 cells was assessed using the WST-8 colorimetric assay (CCK-8, Sigma). The cells were seeded in quadruplicate in 96-well tissue culture plates at the concentration of 1x10⁵ cells/mL. After a 48-hour incubation, the cells were treated with 200 μmol/L H₂O₂ and after 20 min with different concentrations of CG and PG extracts and alliin for 24 h. At the end of the treatment, the cells were washed with PBS, and 10 μL of WST-8 solution was added to each well (21). The cells were incubated in a CO₂ incubator at 37 °C for 4 h. Optical density inside the well was determined with a Victor3™ multilabel plate reader (Perkin Elmer, Waltham, MA, USA). Data are expressed as the percentage of absorbance of corresponding positive and negative control.

The level of intracellular reactive oxygen species (ROS) was measured with a Sigma fluorescent 2’,7’-dichlorodihydrofluorescin diacetate (DCFH-DA) probe. All of the measurements were performed in dark sided 96-well microplates. Following the treatment, the cells were washed with PBS and loaded (30 min, 37 °C in the dark) with 50 μmol/L DCFH-DA dye. Fluorescence intensity was detected by Victor3™ multilabel plate reader at an excitation wavelength of 485 nm and an emission wavelength of 535 nm (22). Data are expressed as a percentage of fluorescence of corresponding positive and negative control.

Cell glutathione (GSH) was quantified with a fluorescent monochlorobimane (mBCl) probe. The cells (1x10⁵ cells/mL) were seeded in black 96-well tissue culture plates for 48 h before the treatment. The cells were then exposed to 200 μmol/L H₂O₂ for 20 min and then to different concentrations of CG and PG extracts and alliin for 24 h. Followed washing with PBS and incubation with 50 μm mBCl at 37 °C in the dark for 20 min (23). The amount of GSH was analysed using a Victor3™ multilabel plate reader at an excitation wavelength of 355 nm and emission wavelength of 460 nm. Data are expressed as a percentage of fluorescence of the corresponding positive and negative control.

For factorial analysis of variance (ANOVA) and the Tukey HSD test of differences between treatments we used the SAS 9.1 (SAS Institute Inc., Carry, NC, USA) statistical package. The data are reported in figures as means with standard deviations in error bars. Statistical significance was set at P<0.05.