Propolis and honey samples and extract preparations
Propolis and organic multi-floral honey samples were collected from hives installed in the Sefrou region, Morocco. The propolis sample was frozen at -20ºC and the organic honey was stored at 3 ºC throughout the experiment period. The raw propolis sample was macerated in 30 ml of ethanol (70%, v/v) under mechanical stirring for one week. The final extracts were filtered (Whatman, nº1), and the filtrate was concentrated in a rotary evaporator. Distilled water was added to prepare the chosen concentration (200 mg/kg b.wt and 100 mg/kg b.wt) [39]. Honey was dissolved in distilled water in order to prepare (1 g/kg b.wt and 2 g/kg b.wt ) for animals ‘feeding.
Chemical Analysis Of Honey And Propolis Extracts
Total carbohydrates
Total carbohydrate content was determined using the phenol-sulfuric acid method described by Ferrira-Santos et al. [40]. Honey or propolis extract (50 µL) was mixed with 150 µL of sulfuric acid (96–98% v/v). Then, 30 µL of phenol reagent (5%) was added and the final solution was heated for 5 min at 90 ◦C. The absorbance was measured at 490 nm by microplate reader after cooling down at room temperature for 5 min. Glucose (10–600 mg/L) was used as a standard to achieve the calibration curve (R2 = 0.992). The total carbohydrate content was expressed as a milligram of glucose equivalents (GlcE) per gram of extract (mg GLcE/g).
Soluble Protein Content
The soluble protein content was analyzed using the Bradford assay with some modifications [41]. A sample of 20 µL of honey or propolis extract was mixed with 230 µL of Bradford dye reagent. The microplate was placed in the dark for 5 min and the absorbance was measured at a wavelength of 595 nm by a UV/V spectrophotometer (Synergy HT, BioTek Instruments, Inc., U.S.A.). Bovine albumin serum was used to perform the standard curve (33–1000 mg/L, R2 = 0.989) and the results were expressed as milligram of BSA equivalents per gram of extracts (mg BSA/g).
Total Protein Content
The total protein content of honey and propolis was estimated by quantification of total nitrogen after sample acid digestion using a Kjeldahl digestor (Tecator, FOSS, Denmark), applying the nitrogen conversion factor (N × 6.25) [41].
Total Phenolic Content
The total phenolic content was determined by the method of Folin-Ciocalteu [42]. Briefly, 60 µL of Folin − Ciocalteu reagent and 15 µL of sodium carbonate solution (75 g/L) were added to 5 µL of honey or propolis hydro-ethanolic extract. The concentration of the produced coloration was measured at 700 nm by a UV/V spectrophotometer (Synergy HT, BioTek Instruments, Inc., U.S.A.) after incubating the mixture for 5 min at 60 ◦C. Gallic acid (0–500 mg/L) was used as a standard to achieve the calibration curve (R2 = 0.996) and the results were expressed in mg gallic acid equivalent (GAE) per gram of extracts (mg GAE/g).
Total Flavonoids Content
Total flavonoid content was determined [43]. One hundred microliters of honey or ethanolic extract of propolis were mixed with sodium nitrite (5%) and 150 µL of AlCl3 solution (10%). After 6 min, 200 µL of NaOH solution (1%) was added and the mixture was properly mixed and allowed to stand in the dark for 60 min. The absorbance was measured at 510 nm. Quercetin (2.6–142 mg/L) was used to perform the standard curve (R2 = 0.997) and the results were expressed in milligram of quercetin equivalent (QE) per gram of extracts (mg QE/g).
Identification And Quantification Of Polyphenols Compounds By Uhplc-dad
Honey and propolis samples were analyzed using a Shi-matzu Nexpera X2 UPLC chromatograph equipped with Diode Array Detector (DAD) (Shimadzu, SPD-M20A) following the method described by (Ferreira-Santos et al., 2019). Separation was performed on a reversed-phase Acquity UPLC BEH C18 column (2.1 mm × 100 mm, 1.7 µm particle size; from Waters) and a pre-column of the same material at 40 ◦C. The flow rate was 0.4 mL/min. HPLC grade solvents water/formic acid 0.1% (A) and acetonitrile (B) were used. The elution gradient for solvent B was as follows: from 0.0 to 5.5 min eluent B at 5%, from 5.5 to 17 min linearly increasing from 5 to 60%, from 17.0 to 18.5 min a linearly increasing from 60 to 100%; the column was equilibrated at 5% from 18.5 to 30.0 min. Phenolic compounds were identified by comparing their UV spectra and retention times with that of corresponding standards. Quantification was carried out using calibration curves for each analyzed compound using concentrations between 250 and 2.5 mg/L. In all cases, the coefficient of linear correlation was R2 > 0.99. Compounds were quantified and identified at different wavelengths (209–370 nm). The values of individual phenolic compounds were expressed in milligrams per kilogram of samples (mg/Kg). All analyses were made in triplicate.
Antioxidant Activity Of Honey And Propolis Extracts
Total antioxidant activity
The total antioxidant activity of honey or propolis samples was evaluated by the phosphomolybdenum method according to the method described by Prieto et al. [44] as follows: 1 ml of reagent solution (6 M sulfuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate) was added to 25 µL of ethanolic extract of propolis or honey and the mixture was incubated for 90 min in a water bath at 95°C. The absorbance was read at 695 nm and ascorbic acid was used as the standard calibration (0.171 to 0.872 mg/mL, R2 = 0.999). The results were expressed in milligrams of ascorbic acid equivalent (AAE) per gram of the sample (mg AAE/g).
Free radical scavenging activity (DPPH assay).
Two hundred and seventy µL of 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) solution (150 µM, prepared in methanol with an absorbance of 0.700 ± 0.01 at 515 nm) was added to 30 µl of different dilutions of honey or propolis extracts [44]. Then, the mixture reactions were incubated in the dark for 1 h at room temperature. The absorbance was measured at 515 nm and the antiradical activity (% inhibition) was calculated using Eq. (1). DPPH inhibition concentration at 50% (IC50) was determined using six different dilutions of each sample, considering that the percent inhibition had to be between 20% and 80%, and the results were expressed in micrograms of extracts per mL (mg/mL).
$$\text{%} \text{i}\text{n}\text{h}\text{i}\text{b}\text{i}\text{t}\text{i}\text{o}\text{n}=\frac{\text{A}\text{b}\text{s} \text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}-\text{A}\text{b}\text{s} \text{s}\text{a}\text{m}\text{p}\text{l}\text{e}}{\text{A}\text{b}\text{s} \text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}} \times 100 \left(1\right)$$
Alpha-amylase Inhibitory Assay
Five hundred µL of alpha-amylase solution (0.5 mg/mL) was incubated with 500 µL of different concentrations of honey or propolis extracts at 37 ◦C for 15 min. Afterward, 500 µL of starch solution (1%) was added and the mixture was incubated for 15 min at 37 ◦C. Immediately, 1 mL of dinitrosalicylic acid color reagent was added to the reaction and placed for 10 min in a boiling water bath. The final mixture was diluted 10 times and the absorbance of each dilution was read at 540 nm.
Alpha-glucosidase Inhibitory Assay
A mixture of different honey or propolis concentrations and p-nitrophenyl-R-d-glucopyranoside (pNPG, 3 mM) was added to the α-glucosidase solution (10U/mL). The mixture was incubated for 15 min at 37 ◦C, and the reaction was stopped by adding Na2CO3 solution (1 M). The intensity of p-nitrophenol coloration produced was measured at 400 nm. Alpha-amylase and alpha-glucosidase inhibition assays were determined as described previously by Laaroussi et al. [45].
Experimental Design
Twenty-five male Wistar rats weighing 150.12 ± 5.1 grams, obtained from the Animal Housing Breeding Center, Department of Biology, Faculty of Sciences Dhar El Mahraz, University Sidi Mohamed Ben Abdallah, Fez, Morocco, were used for the experiments. Rats were kept in a ventilated room and lived in standard environmental conditions (22 ± 3°C, 55 ± 5% humidity, 12 h light/dark cycles). The present work was approved by IRB/ethics committee of Sidi Mohamed Ben Abdellah University, Fez ( USMBA-SNAMOPEQ 2017-03). The manipulation of animals respected the EU Directive 2010/63/EU for animal experiments to avoid and minimize animal suffering and the number of animals experimented. All methods were performed in accordance with the relevant guidelines and regulations. The study is reported in accordance with ARRIVE guidelines (https://arriveguidelines.org).
For the first 7 weeks, groups 2, 3,4, and 5 received only D-glucose and had free access to tap water and normal rats’ chow diet. On the last day of the 7 weeks, rats in groups 3, 4, and 5 with fasting blood glucose greater than 9.4 mmol/L were treated with propolis, honey, and a mixture of honey and propolis respectively for the following 3 weeks.
Rats were randomly allocated into 5 groups, 5 rats in each group. The treatments and applied procedures are as follows:
Group 1 (control): received distal water (10 ml/kg b.wt) for 10 weeks.
Group 2 (diabetic): received daily by gavage D-glucose (10 g/kg b.wt) for the first 7 weeks,
Group 3 (diabetic + Propolis): received daily by gavage D-glucose (10 g/kg b.wt) for the first 7 weeks and treated with propolis extract (200mg/kg b.wt) for the following 3 weeks,
Group 4 (diabetic + honey): received daily by gavage D-glucose (10 g/kg b.wt) for the first 7 weeks and treated with honey (2g/kg b.wt) for the following 3 weeks,
Group 5 (diabetic, propolis, and honey): received daily by gavage D-glucose (10 g/kg b.wt) for the first 7 weeks and treated daily with 100mg/kg b.wt propolis extract + 1g/kg b.wt honey for the following 3 weeks.
Blood samples, urine samples including 24-hour collection, and histopathological specimens were prepared at the end of 10 weeks.
The treatment duration and honey and propolis extract doses were selected according to other studies [46–48].
Biochemical analysis
Aspartate aminotransferases (AST), alanine aminotransferases (ALT), lactate dehydrogenase (LDH), alkaline phosphatase (ALP), triglycerides (TG), total cholesterol (TC), low-density lipoprotein (LDL-C), high-density lipoprotein (HDL-C), serum uric acid, serum creatinine, blood urea, total bilirubin, serum albumin, and serum protein were measured by enzymatic methods, using specific commercial reagent kits. All the kits were purchased from Bio-Maghreb Casablanca, Morocco.
Urine and serum sodium, potassium, chloride, and phosphorus were analyzed using the ion-selective potentiometry method (Architect c8000i biochemistry analyzer), and calcium was determined based on its reaction with Arsenazo III (2,2′ -[1,8-Dihydroxy-3,6-disulphonaphthylene-2,7-bisazo]- bisbenzenear-sonic acid). The intensity of produced color was measured chromatically at 660/700 nm (Architect c8000i biochemistry analyzer).
Blood glucose and plasma insulin levels were determined by the radioimmunoassay method (Rat insulin RIA kit, Millipore, St Charles, MO, USA). Model homeostasis evaluation (HOMA-IR) (Eq. (1)), and homeostatic model-β (HOMA-β) were calculated following the formula described by Mattews et al. [49]. The quantitative insulin sensitivity check index (QUICKI) was determined according to Katz et al.[50].
$$HOMA-IR=\frac{Insulin \left(\frac{U}{L}\right) \times Glucose \left(\frac{mmol}{L}\right)}{22.5}$$
1
$$\text{H}\text{O}\text{M}\text{A}-{\beta }=\frac{20 \times \text{I}\text{n}\text{s}\text{u}\text{l}\text{i}\text{n} \left(\frac{\text{U}}{\text{L}}\right)}{\text{G}\text{l}\text{u}\text{c}\text{o}\text{s}\text{e} \left(\frac{\text{m}\text{m}\text{o}\text{l}}{\text{L}}\right)}- 3.5 \left(2\right)$$
$$\text{Q}\text{U}\text{I}\text{C}\text{K}\text{I}=\frac{1}{(\text{log}\text{f}\text{a}\text{s}\text{t}\text{i}\text{n}\text{g} \text{i}\text{n}\text{s}\text{u}\text{l}\text{i}\text{n} \text{l}\text{e}\text{v}\text{e}\text{l} \left(\frac{{\mu }\text{U}}{\text{m}\text{l}}\right)+\text{log}\text{f}\text{a}\text{s}\text{t}\text{i}\text{n}\text{g} \text{b}\text{l}\text{o}\text{o}\text{d} \text{g}\text{l}\text{u}\text{c}\text{o}\text{s}\text{e} \left(\frac{\text{m}\text{g}}{\text{d}\text{l}}\right))}$$
3
Liver, pancreas, and kidney antioxidant enzymes activities
At the end of the experiment (10 weeks), the kidney, liver, and pancreas were quickly removed, placed in ice-cold saline solution and trimmed off adipose tissue then homogenized in cold phosphate-buffered saline (0.1 M; pH 7.4) and centrifuged (-4°C). The supernatant was collected and stored at -20°C for analysis of the oxidative parameters.
Catalase (CAT) activity was calculated according to the method of Aebi [51]. A decrease in absorbance due to H2O2 degradation was monitored spectrophotometrically at 240 nm for 1 min and the activity was expressed as µmolH2O2/min/mg protein. Glutathione peroxidase (GPx) activity was estimated according to the method of Wa [52]. The activity was expressed as moles of GSH oxidized/min/mg protein.
Reduced glutathione (GSH) levels were measured following the protocol described by Ellman [53]. Briefly, 3 mL of sulfosalicylic acid (4%) was added to 500 mL of homogenate tissues. The mixture was centrifuged at 2,500 g for 15 min and then prepared Ellman’s reagent was added to 500 mL of supernatant. The absorbance was measured at 412 nm after 10 min. Total GSH content was expressed as µg/gram of tissue.
The formation of products of lipid peroxidation was quantified in liver and kidney tissues using the thiobarbituric acid-reactive substances (TBARS) method, as reported previously by Kassan et al. [54], The absorbance was measured at 532 nm. Results were expressed as malondialehyde (MDA) concentration (nmol/g tissue).
Statistical analysis
All data are presented as mean ± SD (standard deviation). Statistical comparisons between the groups were performed with one-way analysis of variance (ANOVA) followed by the Tukey test using GraphPad Prism® software (version 5.0; GraphPad Software, Inc., San Diego, USA). t-test was used to compare two means. Significance was accepted at p < 0.05.