Organic beetroot (Beta vulgaris L.) stalks and leaves, grown in May and April 2016, were obtained from a producer in the city of Limeira, São Paulo state, Brazil. All reagents used for analysis were of analytical grade. High-performance liquid chromatography (HPLC)-grade acetonitrile was obtained from J.T. Baker (Phillipsburg, NJ, USA), and phosphoric acid was obtained from Labsynth (Diadema, SP, Brazil). Vitexin-2-rhamnoside standard was purchased from Sigma-Aldrich (São Paulo, Brazil).
Preparation of the raw material used in the experimental diets
An overview of the preparation of the beet stalks and leaves and extract to be used for supplementation of the diets is presented in Fig. 1. Beet stalks and leaves were sanitized, and excess moisture was removed at room temperature. Stalks/leaves were divided into two groups; one group was placed on aluminum trays and taken to an oven to perform the dehydration process (Clarice Brand, Pinhalzinho, SC, Brazil) at 180 °C for 45 min (SL). The samples were homogenized and stored in hermetically sealed containers at − 80 °C. The other group was placed in aluminum trays, frozen and submitted to the lyophilization process (Ly) (Laboratório de Apoio Central – LAC, Faculty of Food Engineering, University of Campinas, SP, Brazil) (Fig. 1). The samples were stored in hermetically sealed containers at − 80 °C. The beet stalk and leaf extract that was used to supplement the mice diet (EX) was prepared only using dehydrated beet stalks and leaves (SL). These two extracts (lyophilized and dehydrated at 180 °C/45 min) were used to determine the total and individual phenolic compound concentration in the samples. The extract was prepared using 1.0 ± 0.1 g of oven-dried beet stalks and leaves. The raw material was placed in a conical centrifuge tube with 10 mL of ultrapure water and stirred on a tube shaker (Phoenix Luferco AP56) for 5 min, followed by centrifugation (5810 R Centrifuge, Germany) at 4,000 rpm for 15 min at room temperature (25 ± 1 °C). After centrifugation, the supernatant was filtered through Whatman no. 3 filter paper, 10 mL of 80% ethanol was added to the solid residue in the conical tube, and the process was repeated under the same conditions. The extracts obtained by the two sequential extractions of the same raw material were mixed, and the volume was completed to 20 mL with 80% ethanol in a volumetric flask. The extract was stored at − 80 °C in amber glass until used for HPLC analysis, and the extract (EX) obtained from dehydrated beet stalks and leaves (SL) was stored at − 80 °C until used to supplement the diet (EX group).
Sample preparation for the analysis of beet stalks and leaves by HPLC
Freeze-dried and oven-dried beet stalk and leaf samples were prepared using an extraction protocol similar to that used for preparation of the extract applied for supplementation of the experimental diets. The samples (1.0 g) were extracted by two sequential extractions. The first extraction was carried out using 10 mL of water and by stirring for 5 min. After centrifugation, the supernatant was collected, and the solid residue re-extracted with 10 mL of 80% ethanol by stirring for 5 min. Both extracts were combined, and the volume brought up to 20 mL in a volumetric flask. The extracts were filtered through syringe filters (nylon, 25 mm, 0.22 µm; Analytica, Barueri, São Paulo, Brazil) before chromatographic analysis.
Identification of the compounds present in the extracts by ultra-high performance liquid chromatography coupled with mass spectrometry (UHPLC–MS/MS)
The compounds present in the extract were identified using a UHPLC–MS/MS 8040 instrument (Shimadzu, Kyoto, Japan) consisting of a liquid chromatography system coupled to a triple quadrupole mass spectrometer, equipped with an electrospray ionization (ESI) source. Chromatographic separation was performed on a Kinetex C18 column (2.6 µm, 3.0 mm i.d., 100 mm; Phenomenex, California, USA) using a binary mobile phase. Solvent A was water, and solvent B was acidified acetonitrile (0.1% formic acid). The elution gradient used at 40 °C was as follows: 0 min, 98% A; 5 min, 98% A; 15 min, 85% A; 20 min, 80% A; 25 min, 65% A; 30 min, 20% A; 34 min, 20% A; 35 min, 98% A, at a flow rate of 0.3 mL/min. The autosampler temperature was maintained at 10 °C, and the injection volume was 10 µL. The ESI source parameters were as follows: capillary voltage, − 3.5 kV; heat block temperature, 500 °C; desolvation line temperature, 250 °C; drying gas flow (N2), 10 L/min; nebulizing gas flow (N2), 1.5 L/min; collision-induced dissociation gas pressure (Ar), 224 kPa. For each compound, ESI(−)–MS/MS data were first collected for the identification of deprotonated molecules [M − H], and two of the most selective product ions were chosen for the MRM transitions using a dwell time of 20 ms. Data were acquired and processed with Labsolution software (version 5.53 SP2, Shimadzu). The recorded masses were processed throughout the chromatogram during the time interval of the peaks present at different wavelengths of detection (260, 290, 335, 360 and 484 nm). The extract was then reinjected, the detected masses were examined for fragmentation using different capillary voltages, and the detected fragments were recorded. The main compound present in the extracts was identified as vitexin-2-rhamnoside based on its molecular weight (MW 578) and the presence of the fragments (m/z) 457, 274, 413 and 293. Most compounds present in the extracts were related to vitexin, an apigenin flavone glucoside, presenting the characteristic 293 ion in the MS/MS spectra.
HPLC analysis
The HPLC analysis of the samples was carried out in an EXTRACT-US analysis system (FAPESP 2013/04304-4, patent pending), consisting of an HPLC pump (PU2080, Jasco, Kyoto, Japan), an HPLC ternary gradient unit (LG 2080-2, Jasco, Japan), a three-line degasser (DG 2080-55, Jasco, Kyoto, Japan), a UV-Vis detector (UV-7075, Jasco) and five automatic two-position 10-port valves (Waters Corporation, Milford, MA, USA). The compounds were separated by an adaptation of the method developed by Rostagno et al. using a fused core-type column (Kinetex C18, 2.6 μm, 100 A, 100 × 4.6 mm; Phenomenex, Torrance, CA, USA) [28]. The column was maintained at room temperature. The mobile phase consisted of water with 1% v/v phosphoric acid (solvent A), and acetonitrile with 1% v/v phosphoric acid (solvent B). The gradient profile was: 2 min, 88% A; 4 min, 80% A; 6 min, 70% A; 8 min, 40% A; 10 min, 20% A; 13 min, 20% A; and 14 min, 95% A. The equilibration time between runs was 3 min. Flow rate was 1.2 mL/min, and injection volume was 5 μL. Peaks were recorded and integrated at 320 nm. The software for the control of the system was developed by Kalatec (Campinas, SP, Brazil). ChromNav software from Jasco was used for data acquisition and processing. The vitexin-2-O-rhamnoside compound was identified by comparing the retention times of the peak obtained in analysis of the extracts to the peak obtained in analysis of the authentic standard. The vitexin-2-O-rhamnoside standard solution (100 mg/L) was diluted in a mixture of methanol and water (90 : 10 v/v) to prepare the calibration curve (six points: 100, 50, 25, 2.5, 1 and 0.5 ppm). The calibration curve for the compound was prepared by plotting concentration versus area. All compounds present in the samples were expressed as vitexin-2-rhamnoside equivalents (VRE). The analysis was performed in duplicate.
Experimental protocol
The experiments involving animal procedures followed the Guide for the Care and Use of Laboratory Animals published by the National Institute of Health, and the guidelines of the Brazilian College for Animal Experimentation. Experiments were approved by the Ethics Committee on Animal Use – CEUA, UNICAMP, under protocol 4239-1. Forty Swiss male mice, 21 days old, were obtained from the UNICAMP Animal Centre. The animals were allowed to acclimate for 3 weeks before the beginning of the experiment and were then divided randomly into five groups with a similar mean weight and standard deviation: (CT) standard group (n = 8); (HF) high-fat diet group (n = 8); (HFEX) group fed high-fat diet supplemented with extract of dried beet stalks and leaves (n = 8); (HFSL) group fed high-fat diet supplemented with dried beet stalks and leaves (n = 8); and (HFLy) group fed high-fat diet supplemented with lyophilized beet stalks and leaves. Diet and water were given ad libitum throughout the experiment. The animals were kept in a temperature-controlled environment (25 ± 1 °C) with a 12-h light cycle. An analytical balance (Mark 500, Bel Engineering, Italy) was used to weigh the animals and to analyze the food intake and was checked once a week during the 8-week experiment. The experimental protocol of supplementation with beet stalks and leaves is presented in Figure 2; 0.5% of dehydrated or lyophilized beet stalks and leaves was mixed directly into the diet (HFSL and HFLy) (Figure 1). The beet stalk and leaf extract were added directly into the diet (HFEX), and the total volume (10 mL/100 g) was adjusted to enhance the total phenolic compounds expressed as VRE/100 g diet compared to the phenolic concentration in the HFSL diet (Figure 1). Therefore, the group supplemented with dehydrated (powder) beet stalks and leaves (HFSL) received 2.04 mg VRE/100 g of diet, the animals supplemented with beet stalk and leaf extract (HFEX) received 2.04 mg VRE/100 g of diet, and the group supplemented with lyophilized beet stalks and leaves received 4.10 mg VRE/100 g of diet. All groups were treated for 8 weeks. The composition and nutritional value of the control diet and the high-fat diet are shown in Table 1.
Table 1.
Composition and nutritional value of the diets.
Ingredients (g/100g)
|
Standard (g)
|
High-fat diet 60%
|
Standard (g)
|
High-fat diet (g)
|
Extract of stalks and leaves (g)
|
Dried stalks and leaves (g)
|
Lyophilized stalks and leaves (g)
|
(CT)
|
(HF)
|
(HFEX)
|
(HFSL)
|
(HFLy)
|
Corn starch
|
41.07
|
2.00
|
2.00
|
2.00
|
2.00
|
Casein
|
11.00
|
16.10
|
16.10
|
16.10
|
16.10
|
Wheat flour
|
20.00
|
24.00
|
24.00
|
24.00
|
24.00
|
Extract of beet stalks and leaves (mL)
|
--
|
--
|
10.00
|
--
|
--
|
Dehydrated beet stalks and leaves
|
--
|
--
|
--
|
0.50
|
0.50
|
Dextrinized corn starch
|
11.50
|
3.91
|
3.91
|
3.91
|
3.91
|
Sucrose
|
2.50
|
10.00
|
10.00
|
10.00
|
10.00
|
Soybean Oil
|
4.00
|
4.00
|
4.00
|
4.00
|
4.00
|
Lard
|
-
|
30.00
|
30.00
|
30.00
|
30.00
|
Cellulose microfiber (fiber)
|
5.00
|
5.00
|
5.00
|
4.50
|
4.50
|
Mineral mix
|
3.50
|
3.50
|
3.50
|
3.50
|
3.50
|
Vitaminmix
|
1.00
|
1.00
|
1.00
|
1.00
|
1.00
|
L-cistine
|
0.18
|
0.24
|
0.24
|
0.24
|
0.24
|
Choline bitartrate
|
0.25
|
0.25
|
0.25
|
0.25
|
0.25
|
Total
|
100
|
100
|
100
|
100
|
100
|
Energy value (kcal/g)
|
3.90
|
5.35
|
5.35
|
5.35
|
5.35
|
Composition of standard diet (CT), high-fat diet 60% (HF), high-fat diet 60% supplemented with extract of stalks and leaves (HFEX), high-fat diet 60% supplemented with dried stalks and leaves (HFSL) and high-fat diet 60% supplemented with lyophilized stalks and leaves (HFLy).
*CT diet contains 10% of dietary energy as lipids, and high-fat diets (HF, HFEX, HFSL and HFLy) contain 60% of dietary energy as lipids.
|
Intraperitoneal glucose tolerance test (ipGTT)
Basal blood glucose levels were measured after 4 h of fasting with an Accu-Check Performa glucometer (Roche). The test was done after an intraperitoneal injection of glucose solution (1.0 g glucose/kg body weight). Blood glucose concentration was measured in blood from tail-tip bleedings and were used to determine glucose levels at 0, 15, 30, 60 and 120 min. Area under the curve values were determined by the trapezoidal method.
Western blotting
At the end of the experiment, the animals were anesthetized with a solution of sodium ketamine (0.1 g/kg), diazepam (5 mg/kg) and xylazine (3 mg/kg), and decapitation was used to cull mice that had been starved for 12 h. The negative control liver samples were weighed, frozen in liquid nitrogen and stored at −80 °C for further analysis. To evaluate insulin signalling, a bolus injection of regular insulin (5 UI) was administered (Humulin, Eli Lilly and Company, USA) through the abdominal cava vein; subsequently, hepatic samples were extracted after 45 s, this tissue being the positive control for the stimulus. The samples were homogenized in freshly prepared ice-cold buffer [1% (v/v) Triton X-100, 0.1 mol/L Tris, pH 7.4, 0.1 mol/L sodium pyrophosphate, 0.1 mol/L sodium fluoride, 0.01 mol/L EDTA, 0.01 mol/L sodium vanadate, 0.002 mol/L PMSF and 0.01 mg/mL aprotinin] using a tissue homogenizer (Bead Ruptor 12 Homogenizer, Omni International, Kennesaw, GA, USA). Insoluble material was removed by centrifugation (10,000 g) for 30 min at 4 °C. The protein concentration of the supernatant was determined using the Biuret dye-binding method. The supernatant was suspended in Laemmli sample buffer (1 mmol sodium phosphate/L, pH 7.8, 0.1% bromophenol blue, 50% glycerol, 10% sodium dodecyl sulphate (SDS), 2% mercaptoethanol). Immunoblotting was performed by using protein extract (50 μg) from each mouse sample and polyacrylamide gels (SDS-PAGE) using a miniature gel apparatus (BioRad, Richmond, CA, USA). Electrotransfer of proteins from the gel to a nitrocellulose membrane was performed for 120 min at 120 V (constant) in a transfer buffer that contained methanol and SDS. Membranes were then blocked with a solution containing 5% fat-free milk in Tris-buffered saline (TBS)–Tween 20 (TTBS; 10 mmol Tris/L, 150 mmol NaCl/L, 0.5% Tween 20) for 2 h at room temperature. Nitrocellulose membranes were probed overnight at 4 °C with specific antibodies [pAKT (#4060), AKT (#4691) and β-actin (ab8227)]. The membranes were then incubated with a horseradish peroxidase-conjugated secondary antibody (diluted 1 : 5000 in TTBS containing 3% dry fat-free milk) at room temperature, which was followed with a 2-h incubation. Bands were detected by chemiluminescence (Thermo Scientific #34078) and quantified by densitometry (UN-Scan-it Gel 6.1, Silk Scientific Inc, Orem, UT, USA).
Haematoxylin and eosin staining
The hepatic tissues of the mice (n = 3 per group) were perfused with 4% paraformaldehyde solution during sacrifice. Then, liver sections of approximately 3 cm were fixed in 6% formaldehyde for approximately 6 h. The liver sections were then washed in running water for 40 min and processed in the following solutions: 70% alcohol solution; 80% alcohol solution; 90% alcohol solution; 99% alcohol solution; xylol I; xylol II; paraffin I; paraffin II, at 65 °C. After being embedded in paraffin, the slices were incorporated into a mould, cut into sections of 5 μm thickness using a microtome and finally stained with haematoxylin and eosin (H&E) for visualization of hepatocyte morphology. NAFLD activity score [29] was applied for semiquantitative analysis of the three defined criteria of NASH: steatosis (0–3), ballooning (0–3) and lobular inflammation (0–2) using a Leica DMI 4000 B (Switzerland) microscope.
Fatty acid profile
Hepatic total lipids were extracted from 200 mg of tissue following the method of Folch [30]. The samples were homogenized in 1 mL Folch solution (2 :1 v/v chloroform/methanol) and centrifuged at 1,000 rpm for 10 min. The supernatants were collected, and the lipid-containing fraction was dried. Lipids were transmethylated, and the resulting fatty acid methyl esters were analyzed by gas chromatography as previously described [31].
Data presentation and statistical analysis
The results are expressed as the mean ± SE of the indicated number of experiments. The Levene test for the homogeneity of variances was initially used to check the fit of data to the assumptions for parametric ANOVA. Data were analyzed by one-way ANOVA followed by LSD post hoc tests if there were significant interactions among CT, HF and HFEX; CT, HF and HFSL; or CT, HF and HFLy groups. The level of significance was set at P < 0.05.