Materials and methods
HepG2 cell line was purchased from Pasteur institute-Iran. RPMI 1640, Fetal Bovine Serum (FBS), Penicillin–Streptomycin (Pen-Strep®), MTT solution, trichloroacetic acid (TCA), Arbutin (HPLC grade), and trypan blue were purchased from Sigma-Aldrich Chemical Co, UK. t-BHP was purchased from Merck Co, Germany.
Preparing Pyrus biossieriana Buhse leaf extract
The fresh leaves of P. biossieriana Buhse were collected from Babol city—Iran, washed, dried for 6 days, and then chopped. 400 g of powder was extracted with 2000 mL methanol (63%). The methanol portion was evaporated using a rotary evaporator. The extract was stored at − 70 °C [1, 12].
Determining the arbutin and flavonoid content of the extract
The arbutin content of the extract was measured by HPLC. The calibration curve for arbutin over the known concentration range was linear (r = 0.99) (Fig. 1a) We performed HPLC using a Knauer Smartline Liquid Chromatography System (Knauer, Germany). Similar to our previous investigation , we used a Prontosil #60-5, C18 H column that was 4.6 mm in diameter and 250 mm in length. The mobile phase consisted of a 50:50 mixture of methanol and water containing 1% acetonitrile. We set the flow rate at 0.7 mL/min . The percentage of arbutin recovered was determined by spiking a sample containing 7573 mg/L arbutin with an arbutin standard solution (2500 mg/L) to yield solutions with final concentrations of 7065, 6304, and 5036 mg/L. HPLC for standard arbutin (2500 mg/L) was performed using a Eurospher C-18 column (4.6 × 250 mm), mobile phase (methanol/water 50/50; flow rate: 0.7 mL/min) and was detected in 286 nm wavelength [1, 12, 13] (Fig. 1b) We then run an HPLC to detect arbutin in the extract and another HPLC for arbutin in extract and hydroquinone (as internal standard), in the same condition (Fig. 1c, d).
Determination of the total phenolic content
The total amount of phenolic compounds was determined by Folin-Ciocalteu reagent using the method described by Singleton and Rossi with some modifications . In brief, 1 mL of extract (diluted tenfold), was mixed with 5 mL Folin-Ciocalteu reagent (diluted 1:10 with ultrapure water). After 2 min, 4 mL of sodium carbonate solution (75 g/L) was added and kept at room temperature for 2 h. The absorbance was measured at 765 nm. Total phenolic content was expressed as gallic acid equivalent (GAE) in milligrams per gram  (Fig. 1e).
Determination of the total flavonoids content
The total amount of flavonoids was determined using aluminum chloride (quercetin was used as standard). Different concentrations of standards were prepared in 60% methanol. 1 mL sample/standard was incubated in the test tube, then 1 mL of 2% aluminum chloride was added to each tube. Finally, 6 ml potassium acetate was added and OD was measured after 40 min at 415 nm .
HepG2 cells were plated in 24 multi-well flat-bottom culture plates, at 1.7 × 105 cells per well and incubated (37 °C with 5% CO2 and 95% humidity) for 48 h. HepG2 cells were then divided into 16 groups each including 5 wells. The wells were exposed to different concentrations of extract (1.5 mg/mL, 2.0 mg/mL, and 2.5 mg/mL) prepared in complete media, and incubated for 24 h. After 24 h the media were removed and wells were washed twice with sterile distilled water, then different concentrations of t-BHP (0, 100 µM, 150 µM, 200 µM), were dissolved in complete media and were added to all groups, except control and incubated for 24 h .
Cell viability assay (MTT assay)
200 μL of MTT solution was added to each well and incubated at 37 °C in dark place for 4 h. Acid-isopropanol (as calibrator) (1 mL of 0.04 N HCl in isopropanol) was added to all wells and mixed thoroughly to dissolve dark blue crystals . Using a spectrophotometric method, all wells were read at 570 nm as test and 630 nm as reference wavelengths .
Total antioxidant capacity (TAC)
Using ferric-reducing antioxidant power (FRAP), the total antioxidant capacity (TAC) of samples was measured. . 1.5 mL of FRAP reagent was added to test tubes, incubated at 37°c for 5 min, then 50 µL of HepG2 cells media (test solution) was added to tubes, mixed thoroughly, and incubated in 37°c for 15 min. The absorbance was read at 593 .
The Griess reagent system is based on the chemical reaction which uses sulfanilamide (SA) and N-1-naphthyl ethylenediamine dihydrochloride (NED) under acidic (phosphoric acid) conditions. This system detects NO2 in a variety of biological samples. 50 µL of samples and 50 µL of sulfanilamide were mixed gently in a microtube and incubated in dark place for 5 min. 50 µL NED was also added to each microtube. Then 50 µL Vanadium chloride (VCL3) was added to all samples, incubated at 37 °C for 45- 60 min and the absorbance of each tube was read at 540 nm .
Lipid peroxidation was estimated by TBARS assay, a colorimetric test for determining lipid peroxidation, based on the reaction of 2-Thiobarbituric Acid (TBA) and MDA. 0.5 mL of cell media was added to the tube containing 2 mL of reagent, mixed thoroughly, and incubated in boiling water for 15 min, allowed to reach room temperature, then centrifuged in 1500 RPM for 5 min. The OD of samples were read at 593 nm .
All groups were containing 5 wells (N = 5), except for MTT that was performed in triplicate (N = 3) to check the reproducibility. Results are presented as the mean ± standard deviation (SD). Statistical analysis was performed using one-way analysis of variance (ANOVA) with subsequent post hoc comparisons by LSD (SPSS, Ver 21.0, IBM-USA). The criterion for statistical significance is expressed as p < 0.05. The normality of data was checked using the Kolmogorov–Smirnov test.
The arbutin content of the extract was measured by HPLC (12.6% of the dry weight of the leaves). The total flavonoid and phenolic contents in the extract were 119 ± 6.93 mg/g and 418 ± 10.07 mg/g respectively. All groups that received t-BHP and extract, showed significantly reduced cell viability when compared to the control group. Incubation of cells with the extract alone partially reduced their viability in a dose-dependent manner in which incubation of cells with the extract at different concentrations of 1.5, 2.0, and 2.5 mg/mL reduced the cell viability to 94.36 ± 2.56% (p < 0.016), 81.99 ± 2.07% (p < 0.001), and 73.25 ± 1.81% (p < 0.001) respectively. In the group treated with the extract concentration of 1.5 mg/mL and 200 μM t-BHP, the cell viability decreased to 72.52 ± 1.1% (p < 0.001). The minimum rate of cell viability was observed when t-BHP at the concentration of 200 µM was added to cells pretreated with 2.5 mg/mL of extract. In this case, the cell viability dropped to 43.48 ± 3.20% (p < 0.001). Indeed, preincubation of HepG2 cells with extract (1.5 mg/mL and 2.0 mg/mL) attenuated t-BHP toxicity when the groups exposed to all concentrations of t-BHP (p values < 0.001) (Table 1) Exposure to high levels of nitrite results in toxicity, which induces the production of reactive ROS and causes oxidative stress . Pretreatment of cells with the extract only, slightly increased the nitrite production [31 ± 3.33 µM in the group treated with 1.5 mg/mL concentration of extract (p = 0.09) and 31.8 ± 1.1 µM in the group treated with 2.0 mg/mL concentration of the extract when compared to the control group (p = 0.02)]. Exposure of the cells pretreated with 1.5 mg/mL of extract to different t-BHP doses (100 µM, 150 µM, 200 µM) increased the nitrite production to 40 ± 2.63, 41 ± 0.8, and 42.5 ± 1.94 µM respectively (p < 0.001). The results showed a decrease in nitrite production in t100/E1.5 treated group (40 ± 2.63 µM) and t100/E2.0 mg/mL treated group (43.7 ± 2.80 µM) (p < 0.001) (Table 2). Treatment of the cells with different concentrations of extract revealed that the extract increases the antioxidant capacity in a dose-dependent manner in which treatment of the cells with 1.5, 2.0, and 2.5 mg/mL concentrations of extract increased this parameter to 1814 ± 64.85, 2498 ± 37.83, and 3952 ± 37.83 µM (p values < 0.001). Exposure of t-BHP alone in different doses (100 µM, 150 µM, and 200 µM) reduced the antioxidant capacity in a dose-dependent manner to 140 ± 12.69 (p = 0.078), 96 ± 9.76 (p = 0.008), and 80 ± 8.8 µM (p = 0.001) respectively when compared to control group. Exposure of the cells treated with 2.0 mg/mL concentration of the extract to t-BHP concentrations (100 µM, 150 µM, 200 µM), showed a slightly decreasing trend in the antioxidant capacity (t100/E2.0, 2438 ± 32.23, p = 0.03, t150/E2.0, 2294 ± 30.81, p < 0.001, and t200/E2.0, 2234 ± 12.14, p < 0.001). A similar trend was shown when cells treated with 2.5 mg/mL concentration of the extract were exposed to 100 µM, 150 µM, and 200 µM concentrations of t-BHP in which the parameter dropped to 3832 ± 34.49, 3508 ± 23.93, and 3075 ± 97.91 µM respectively (p < 0.001). (Table 3) Lipids are the most susceptible biological molecules to the attack of ROS and RNS. Lipid peroxidation plays a role in the disturbance of fine structures, functional loss, and permeability of biomembranes and results in production of toxic products which are chemically reactive and covalently modify a variety of biomolecules including DNA bases . TBA assay (TBA test) is widely used to assess the products of lipid peroxidation. From a molecular point of view, the mechanism is based on the MDA (an end product of lipid peroxidation) reaction with TBA and production of a red adduct . The results of TBA test showed that t-BHP alone increased the parameter dose-dependently in which 100, 150, and 200 µM concentrations could increase TBA results to 3.17 ± 0.58, 3.86 ± 0.06, and 5.91 ± 0.18 µM (p < 0.001). Interestingly, all groups pretreated with 1.5, 2.0, and 2.5 mg/mL concentrations of the extract that were exposed to 200 µM concentration of t-BHP, showed the highest rates of lipid peroxidation [3.76 ± 0.09, 3.96 ± 0.14, and 6.23 ± 1.36 µM respectively when they were compared to corresponding groups with the same extract concentrations (p < 0.001)]. Our results showed that the extract suppresses the lipid peroxidation more effectively at 1.5 and 2.0 mg/mL concentrations (Table 4).
In the present study, it was shown that t-BHP induced strong inhibition on cell growth, and pretreatment with P. biossieriana Buhse leaf extract (1.5 mg/mL and 2.0 mg/mL), significantly protected the HepG2 cells against oxidative damage. In the previous studies, the extract showed significant anti-hyperglycemic and anti-hyperlipemic activity . Besides, it attenuates lipid peroxidation and NO production in t-BHP treated cells. Finally, our results showed that the groups treated with extract only but not t-BHP, had lower cell viability [E 1.5 mg/mL (p value = 0.016), E 2.0 mg/mL (p value ≤ 0.001), and E 2.5 mg/mL (P value ≤ 0.001)] when compared to the control group. We suggest investigating the effects of commercial arbutin, or other antioxidants reported in extract or the separated fractions of extract in further in vitro and in vivo investigations. The present study showed that P. biossieriana Buhse leaf extract attenuates t-BHP- induced cytotoxicity in HepG2 cells. Pretreatment with certain concentrations of extract protected HepG2 cells against alterations induced by t-BHP, probably through quenching radical species, reducing the rate of MDA formation, and NO production.
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