2.1. Plant sampling and extracts preparation
Plant samples of different parts of Nitraria retusa were collected from a salt flat “sebkha of El Kelbia” located in the region of Kairouan, Tunisia with the GPS following coordinates N 35 48 44, E 10 09 06. After rinsing samples with distilled water and dried in shadow. The organs of N. retusa (stems and leaves) were ground separately using a ball mill type “Dangoumeau” and extracted with 70% of ethanol with a ratio of 1 g of plant powder in 10 ml of extraction solvent. Then the solution was kept in the dark for 2 weeks. During this period, the extract solution was manually shaked for 5-10 minutes every day. Finally, the extract was filtered through 0.22 𝜇m filter (MILLIPORE, U.S.A.), and stored for further experiments .
2.2. Chemical analyses of the general composition of Nitraria retusa extracts (stem extract and leaf extract) using reverse phase-high performance liquid chromatography (RP-HPLC)
Before injection into the HPLC system, N. retusa organ extracts were passed through a 0.45 μm nylon filter. The separation of selected phenolic compounds was carried out using HPLC system (consisting of a vacuum degasser, an autosampler, and a binary pump with a maximum pressure of 600 bar, Agilent 1260, Agilent technologies, Germany) equipped with a reversed phase C18 analytical column of 4.6 x100 mm and 3.5μm particle size (Zorbax Eclipse XDB C18). Column temperature was maintained at 25°C. The injected sample volume was 2 μL and the flow-rate of mobile phase was 0.4 mL/min. Mobile phase B was milli-Q water consisted of 0.1% formic acid and mobile phase A was methanol. The optimized chromatographic condition was revealed as follows: 10% A, 90% B (0 min), 20% A, 80% B (5 min), 30% A, 70% B (10 min), 50% A, 50% B (15 min), 70% A, 30% B (20 min), 90% A, 10% B (25 min), 50% A, 50% B (30 min), 10% A,90% B (35 min). UV-vis absorption spectra were recorded online during the HPLC analysis. The DAD detector was set to a scanning range of 200-400 nm. Peak identification was obtained comparing the retention time and the UV-vis spectra of Nitraria retusa phenolics chromatogram with those of available standards . Quantification was performed by reporting the measured integration area in the calibration equation of the corresponding standard.
2.3. Preadipocytes differentiation and Oil-Red-O staining procedures
3T3-L1 preadipocytes were seeded into 96-well plates at 1.0 x 104 cells/well and cultured for additional two days until full confluence. Two days later (Day 0), cells were incubated with a differentiation cocktail (MDI) containing 1/10 insulin solution, 1/10 dexamethasone solution and 1/10 3-isobutyl-1-methylxanthine solution in standard culture medium for 3 days followed by additional 48h with standard culture medium containing insulin alone. The differentiation-maintenance medium was changed every 2 days . To investigate the effect N. retusa stem and leaf extracts on adipogenesis in 3T3-L1, different doses (25, 50, 100, 200 and 400 μg/mL) were added to the differentiation-induction and differentiation-maintenance media. The same procedure was conducted to investigate the effect of the determined phenolic compounds of N. retusa (luteolin-7-O-glucoside, isorhamnetin-3-O-rutinoside and isorhamnetin) using the following concentrations: 5, 25, 50, 100 and 200 μM. The staining procedure was conducted according to the adipogenesis assay kit (Cayman chemical company). The absorbance was read at 490 nm with a 96-well plate reader. The lipid droplet content was reported as percentage of control cells.
2.4. Cell culture
Murine 3T3-L1 preadipocytes (Riken Tsukuba Japan) were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin (5000 μg/mL)-streptomycin (5000 IU/mL) in 75-cm2 tissue culture flasks. Medium was changed every 3 days and cell passage was carried out at 80% confluence at one on two ratio using 0.25% trypsin (1 mM EDTA). 3T3-L1 cells were cultured in a humidified incubator at 37°C and 5% CO2 .
2.5. Cell proliferation assay (MTT assay)
Cell proliferation was investigated by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. 3T3-L1 cells were seeded in 96-well plates at 1 × 105 cells/mL. After incubation for 7 days (adipocytes), leaf and stem extract samples diluted in medium were added at final concentrations of 25, 50, 100, 200, 400 μg/mL. The same procedure was conducted to investigate the effect of the determined phenolic compounds of N. retusa using 5, 25, 50, 100 and 200 μM concentrations for luteolin-7-O-glucoside, isorhamnetin-3-O-rutinoside and isorhamnetin. MTT was added after treatment for 7 days and the resulting formazan was completely dissolved by 100 μL of 10% sodium dodecyl sulfate (SDS) for 24h. The absorbance was determined at 570 nm in a multi-detection microplate reader (Power-scan HT, Dainippon Pharmaceutical, NJ, USA). Absorbance caused by the ability of the sample to reduce MTT or by its color, was corrected using plates as blanks, prepared in the same conditions in the absence of cells.