Present study was approved by the ethical committee (Faculty of Science, University of Zagreb, Croatia). Male and female Swiss albino inbred mice 2 to 3 months old, weighing 20 to 25 g, obtained from Department of Animal Physiology, Faculty of Science, University of Zagreb, were used in this study. The animals were kept in individual cages during the experiment and at 12 hours of light per day. They were fed a standard laboratory diet (4 RF 21, Mucedola, Settimo Milanese, Italy) and tap water ad libitum. Maintenance and care of all experimental animals were carried out according to the guidelines in force in Republic of Croatia (Law on the Welfare of Animals, N.N. #19, 1999) and carried out in compliance with the Guide for the Care and Use of Laboratory Animals, DHHS Publ. # (NIH) 86–123.
Water-soluble derivative of propolis (WSDP)
Row Croatian propolis was collected by scraping it off from hive frames. The collected propolis samples were kept desiccated in the dark until analysis at room temperature. Water-soluble derivative of propolis (WSDP) was prepared by the method described in our previous paper. Briefly, Croatian propolis from beehives kept at the outskirts of Zagreb was extracted with 96% ethanol, which was filtered and evaporated to dryness in vacuum evaporator. The resultant resinous product was added to a stirred solution of 8% L-lysine (Sigma Chemie, Deisenhofen, Germany) and freeze-dried to yield the WSDP, a yellow-brown powder.
The chemical profile of propolis from the northern hemisphere, often named as “poplar-type” propolis can be characterized by the three analytical parameters: total flavonol and flavone content, total flavanone and dihydroflavonol content, and total polyphenolics content. According to Popova et al., spectrophotometric procedures for quantification of the three main groups of bioactive substances in propolis could be used for quality assessment of different propolis samples, and results of those analyses correlate with biological activity, especially in the “poplar-type” of propolis. The spectrophotometric assay based on the formation of aluminium chloride complex was applied for quantification of total flavones/flavonols and expressed as quercetin equivalent. For the quantification of flavanones and dihydroflavonols propolis, we used 2,4-dinitrophenylhydrazine method. Total polyphenolics content was measured by the Folin–Ciocalteu procedure. Total phenol content was expressed as gallic acid equivalents (mg/g), total flavonoid contents as quercetin equivalents (mg/g), while total flavanones and dihydroflavonols content was expressed as naringenin equivalents (mg/g) from calibration curves recorded for the standards.
WSDP contained: flavones and flavonols 2.13%, flavanones and dihydroflavonols 9.06%, total flavonoids 11.19%, total polyphenols 70.48%.
Ethanolic extract of propolis (EEP)
Ethanolic propolis extract (EEP) was prepared by the method described elsewhere[13, 36]. Briefly, propolis (10 g) was crushed into small pieces in a mortar and mixed vigorously with 34.85 ml 80% (V/V) ethanol during 48 h at 37 ± 1°C. After extraction, the ethanolic extract of propolis was filtered through Whatman N0. 4 paper and than the extract was lyophilized. Spectrophotometric analysis has shown that EEP contained: flavones and flavonols 1.6%, flavanones and dihydroflavonols 38.60%, total flavonoids 40.20%, total polyphenols 84.40%.
Antioxidant capacity of the extracts
β-Carotene−linoleic acid assay
The antioxidant activity of the extracts was evaluated using β-carotene−linoleic acid system according to Amarowicz et al.. In short, 1 mL of β-carotene solution in chloroform (0.2 mg mL-1) was pipetted into a round-bottom flask. To the solution, 20 mg of linoleic acid and 200 mg of Tween 40 were added. After removing chloroform in a rotary evaporator, 50 mL of aerated distilled water was added to the oily residue. Aliquots (5 mL) of thus obtained emulsion were transferred to a series of tubes containing 2 mg of extract or 0.5 mg of BHA (positive control). Emulsion without antioxidant served as control. After addition of the emulsion to the tubes, they were placed in a water bath at 50°C for 2 h. During that period, the absorbance of each sample was measured at 470 nm at 15 min intervals, starting immediately after sample preparation (t = 0 min) until the end of the experiment (t = 120 min). The rate of β-carotene bleaching (R) for the extracts, BHA and water, was calculated according to first-order kinetics. The percent of antioxidant activity (ANT) was calculated as described in Al-Saikhan et al., using the equation:
control and R
sample are average bleaching rates of water control and antioxidant (plant extract or BHA), respectively.
DPPH radical-scavenging activity
The scavenging effect for DPPH free radical was monitored as described in Zovko Končić at al. with minor modification. Briefly, 1.0 mL of 0.16 mM DPPH methanolic solution was added to 1.0 mL of either methanolic solution of extract (sample) or methanol (control). The mixtures were vortexed and then left to stand at room temperature in the dark. After 30 min absorbance was read at 517 nm. Radical-scavenging activity (RSA) for DPPH free radical was calculated using the following equation:
control is the absorbance of the methanol control and A
sample is the absorbance of the extract. Synthetic antioxidant, BHA, was used as positive control. DPPH radical-scavenging activity was calculated as the concentration that scavenges 50% of DPPH free radical and thus has RSA = 50% (EC50).
The reducing power of the extracts
The reducing power of extracts was determined according to the method of Yen and Chen. Briefly, extracts (0.2–1.0 mg) were dissolved in 1.0 mL of distilled water and mixed with 2.5 mL of 0.2 M phosphate buffer (pH 6.6) and 2.5 mL of 1% potassium ferricyanide. The mixtures were incubated at 50°C for 20 min. After incubation, 2.5 mL of a 10% trichloroacetic acid was added to the mixtures. Following that, samples were centrifuged for 10 min. Aliquots of 2.5 mL of the upper layer were combined with 2.5 mL of water and 0.5 mL of the 0.1% solution of ferric chloride. Absorbance of the reaction mixture was read spectrophotometrically at 700 nm. Ascorbic acid was used as positive control.
Chelating activity (ChA)
The chelation of iron (II) ions was studied as described by Decker and Welch. An aliquot of the extract in methanol (1.3 mL) was added to 100 μL of 2 mM FeCl2. After 5 min, the reaction was initiated by adding 200 µl of 5 mM ferrozine. Following 10 min incubation at room temperature, the absorbance at 562 nm was recorded. For preparation of control, 1.3 mL of methanol was used instead of extract solution. EDTA was used as a chelating standard. The Fe2+-chelating activity (ChA) was calculated using the equation below:
control is the absorbance of the negative control (solution to which no extract was added) and A
sample is the absorbance of the extract solution. Chelating activity was expressed as ChEC50, the concentration that chelates 50% of Fe2+ ions and thus has ChA = 50%.
Seventy mice were randomly divided into four groups, as follows:
Group (i): control animals (healthy, nondiabetic animals); received 0.5 mL distilled water intraperitonealy (i.p.) per day by injection for 7 days;
Group (ii): alloxan controls; injected i.v. with alloxan in a single dose of 75 mg kg-1 body weight; these served as the untreated diabetic group;
Group (iii): received WSDP i.p. in a daily dose of 50 mg kg-1 for 7 days starting 2 days after alloxan injection; these served as the WSDP-treated diabetic group.
Group (iv): received EEP i.p. in a daily dose of 50 mg kg-1 for 7 days starting 2 days after alloxan injection; these served as the EEP -treated diabetic group.
Five mice from each group were used on the 9th day after alloxan injection. After desinfection of the external abdominal region, each animal was inoculated with 3 mL of saline solution and after gentle agitation of the abdominal wall, the solution containing peritoneal cells was removed for cellular evaluation. The following variables were analyzed: toxicity analysis, animal weight loss, hematological, biochemical parameters (total cholesterol and triglyceride), determination of lipid peroxidation of liver and kidney cells and their histopathological analysis.
The remaining animals, i.e., 8–11 animals of each group were used for the survival analysis (increased lifespan).
Induction of experimental diabetes and determination of serum glucose level
Diabetes was induced in Swiss albino mice by a single intravenous injection of alloxan monohydrate (75 mg kg-1, i.v.) in total volume of 0.5 mL of freshly prepared saline solution. Blood glucose level was tested before alloxan injection and 48 h after treatment, to monitor the immediate diabetogenesis. After 48 h, the animals with blood glucose level above 11 mmol L-1 were selected for the study (diabetic mice) and then treated with WSDP or EEP. Blood glucose level was determined by test strips of blood glucose (Betachek Visual blood glucose test strips, Sydney, Australia).
Effect of WSDP or EEP on body weight in alloxan induced diabetic mice
During the study period of 50 days, the body weights of the mice were recorded every 4 days using an electronic balance. From these data, the mean change in body weight was calculated. The maximum percentage of animal weight loss, an indicator of toxicity, was calculated for individual animals as:
For the survival analysis Swiss albino mice were given test components i.p. at doses of 50 mg kg-1 for 7 days starting 2 days after the alloxan injection. The end point of the experiment was determined by the spontaneous death of animals. The results are expressed as percentage of mean survival time of the treated animals over the mean survival time of the control group with diabetes (treated vs. control, T/C%). The percentage of increased lifespan (ILS%) was calculated according the formula: where T represents mean survival time of treated animals and C represents mean survival time of the control group.
The haematological analysis was performed on blood obtained from the tail vein of experimental and control mice on day 9 after alloxan injection. Blood was collected into EDTA tubes. The measurement of the leukocyte, erythrocytes, haemoglobin, hematocrit, MCV, MCH, MCHC and platelets was made in an automatic cell counter (Cell-Dyn® 3200, Abbott, USA).
Serum samples and biochemical determinations
Animals were treated with test components, blood samples were collected and centrifuged at 2200 rpm for 10 minutes. Serum was used for the determination of total protein, glucose, urea, creatinine, bilirubin, alcaline phosphatase (ALP), aspartate and alanine aminotransferases (AST and ALT) and lactic dehydrogenase (LDH). Biochemical parameters were made using serum samples from both control and experimental groups in an automatic cell counter. Serum triglycerides and total cholesterol were determined by enzymatic methods according to the commercial kit’s instructions (Thermo Electron, Australia). The total concentration of triglycerides or total cholesterol was estimated by measuring the absorbance of sample and standard by spectrophotometer (Shimadzu, UV-160) at a wavelength of 500 nm.
Prepations of tissue homogenate and protein estimation
Samples of liver and kidneys (100 mg mL-1 buffer) were homogenized in 50 mM phosphate buffer (pH 7.0), and then centrifuged at 10,000 rpm for 15 min; the supernatant thus obtained was used for biochemical analysis. The protein concentration in each fraction was determined by the standard laboratory procedure, at NanoDrop 1000 Spectrophotometer V3.7. using crystalline bovine serum albumin as standard.
Determination of lipid peroxidation
The extent of lipid peroxidation was determined by the method of Ohkawa et al.. To a tube containing 0.2 mL of 8.1% sodium dodecyl sulfate, 1.5 mL of 20% acetic acid (pH 3.5), and 1.5 mL of 0.81% thiobarbituric acid aqueous solution were added. To this reaction mixture, 0.2 mL of each tissue homogenate was added. The mixture was then heated in boiling water for 60 min. After cooling to room temperature, 5 mL of butanol:pyridine (15:1, v/v) solutions were added. The mixture was then centrifuged at 5000 rpm for 15 min. The upper organic layer was separated, and the intensity of the resulting pink color was read at 532 nm. Tetramethoxypropane was used as an external standard. The level of lipid peroxides was expressed as nmoles of malondialdehyde (MDA) formed/mg protein.
For the histopathological changes, liver and kidney tissues from diabetic control mice treated with physiological and ethanolic solution and diabetic mice treated with WSDP and EEP were fixed in 10% neutral buffered formalin for 24 hours, dehydrated in a graded alcohol series and after chloroform treatment embedded in paraplast. Deparaplasted 5–6 μm thick sections were stained with hematoxylin and eosin (HE) following standard protocol. Stained slides were examined under a light microscope (Nikon Eclipse E600) at 100, 200, 400 and 1000x magnification. Liver sections were examined for vacuolization, lymphocyte infiltrations, necrosis and apoptosis. The percentage of apoptotic cells was determined by counting 200 cells in randomly selected microscopical fields of vision.
Kidney sections were examined for lymphocyte infiltrations, reduction of Bowman’s spaces and changes in renal tubules.
Photomicrographs were taken by digital camera (Nikon DMX1200) and Imaging Software Lucia G 4.80 (Laboratory Imaging Ltd., Prague, Czechoslovakia).
The experiments were performed in triplicate. The results were expressed as mean ± SD. Statistical comparisons were made using one-way ANOVA, followed by Dunnett’s post-hoc test for multiple comparisons with the control and Student’s t-test for comparison between samples. Statistical analyses were performed using the JMP V6 from SAS software (SAS Institute, Cary, NC, USA). A value of P < 0.05 was considered to indicate statistical significance.