الفهرس 
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Abstract
Type 2 diabetes mellitus (T2DM) is the most common type of diabetes mellitus (DM) and is regarded as a chronic metabolic disorder characterized by inadequate insulin secretion or action in insulin- sensitive tissues, resulting in an elevated blood glucose concentration (hyperglycemia). Several complications arise due to DM, such as hypoglycemia, cardiovascular and neurological diseases, kidney failure, limb amputation and vision problems. According to current estimates, it is widely accepted that the number of diabetic patients (most with T2DM) can reach 366 million by the year 2030, promoted by a dramatic increase in the incidence of obesity and a sedentary lifestyle (Kolluru et al., 2012).
The economic burden resulting from the total cost of care of T2DM patients and its complications is very high. Thus, much interest has been focused on the development of alternative medicinal approaches, like natural bioactive compounds with the ability to improve glycemic control and lower the risk of T2DM complications (Tan et al., 2019).
Propolis is a resinous material produced by bees using the collected exudates and buds of plants in combination with beeswax and enzymes. Propolis is well known for its biological properties, including antioxidant, antiviral, antibacterial, antifungal, antiatherogenic, and antiproliferative activities, and has been used for folk medicine in many countries (Karimian et al., 2019).
Aim of the Study:
The current study aimed to evaluate and improve the efficiency of propolis harvested from the western honey bee workers against type-2 diabetes mellitus in vivo, using a chitosan-based nanocarrier.
Experimental Methodology and Results of the Study:
Collection of Propolis Samples:
Carniolan hybrid Egyptian forager honey bee workers (Apis mellifera carnica) in the age group of 20-45 days were employed in the current study. The used bee colonies were reared under the special breeding program at the apiaries of the Honey bee Research Department, Plant Protection Institute, Agricultural Research Center, Giza, Egypt, during the period from April (2018) to July (2018).
The collection of propolis was accomplished through using PVC ”propolis traps” with a series of 2-3 mm-wide grooved slits over the sheets’ entire surface. The plastic sheets were arranged contiguous to each other. The propolis deposition on the trap sheets took around 3 weeks.
Preparation of the Ethanolic Extract of Propolis:
The ethanolic extract of propolis (EEP) was produced by macerating the collected propolis at room temperature in the ratio of 10 g of propolis to 100 mL of 80% (v/v) ethanol. Afterwards, the ethanolic extracts were sieved by Whatman (No.1) filter paper and incubated at room temperature the extract gained a viscous honey-like consistency.
Chemical characterization of the Collected Propolis:
Estimation of Total Phenolic Components:
Total phenolic content was determined using the Folin-Ciocalteau colorimetric modified method with gallic acid as a standard. The total phenolic content was expressed as micrograms of gallic acid equivalent (GAE) per mL of sample extract (mg of GAE/ mL of extract).
The Folin-Ciocalteau colorimetric method illustrated that the total phenolics present in the EEP sample were 61.9 ± 3.09 mg/mL GAE/mL.
Estimation of Total Flavones, Flavonols, and Flavanones:
The total flavonoid content of extracted EEP was investigated using the aluminum chloride colorimetry method. The total flavonoid content was expressed as milligrams of quercetin equivalent per gram of extract (mg of QCE/g of extract).
The aluminum chloride colorimetry method revealed that the total flavonoids present in the EEP sample were 127.4 ± 2.7 mg/mL QCE/g.
Biochemical characterization of Propolis by Gas chromatography-Mass Spectrometry (GC-MS):
The GC-MS analysis of the harvested EEP revealed the presence of 23 major spectrophotometric peaks, attributed to several phenolic and flavonoid compounds.
Formulation of the Nanogel Carrier Particles:
Due to their favorable characteristics like being biodegradable, biocompatible and non-toxic, polyacrylic acid (PAA) and chitosan (CS) were regarded as ideal candidates, as a polymer and a respectively, for the formulation of the nanogel carrier for the EEP in the present study.
Preparation of Chitosan:
The CS was prepared from the cuticle of the oriental hornet due to its convenient large size. The crushed exoskeleton samples were treated by boiling in NaOH for 1 h to dissolve the sugars and proteins, thus isolating the crude chitin. Afterwards, the ground exoskeletons were then demineralized with 1% HCl overnight and deproteinized in 2% NaOH solution for 1 h. The residual chitin was then washed with deionized water.
The process of deacetylation was performed by adding 50% NaOH to the chitin samples and then boiling them at 100°C for 2 h.
The degree of deacetylation, XD, was estimated using CHN elemental analysis and substituting in the following equation:
X_D=100 .(4-0.583093×W_(C⁄N))
where WC/N is the mass ratio between carbon and nitrogen present in the CS sample.
The CHN analysis revealed the percentages of N, C and H, in the prepared CS sample to be 7.6, 40.4 and 7.42%, respectively. Subsequently, when substituting in the formula, it was revealed that the prepared CS showed a 90.04% deacetylation degree, which was considered to be an acceptable percentage.
Preparation of the Chitosan-Polyacrylic Acid Hydrogel Polymer:
The polyacrylic acid - chitosan (PAA-CS) hydrogel composite was prepared while maintaining a temperature not exceeding 70oC to prevent the denaturation of the EEP.
Purified CS solution was introduced to AA aqueous solution in a 250-ml standard three-necked flask. At this temperature, a solution of 0.66 mM K2S2O8 was added dropwise into the CS-AA mixture, to initiate the polymerization reaction. During the preparation of EEP-loaded hydrogel, a saturated EEP sample was introduced with the CS-AA mix before initiating polymerization with K2S2O8.
Afterwards, the polymer solution was poured into 15 cm Petri dishes and oven-incubated for 48 h at 60oC for dehydration to produce polymer ring films. Each of the resulting polymer films (blank or EEP-loaded) was rod-milled thoroughly using a home-made rod mill, then sonicated and centrifuged to remove insoluble materials.
Nano-formulation and Purification of the Hydrogel Polymer:
The nano-formulation and encapsulation of the EEP into the polymer matrix was accomplished through nano-emulsification and solvent exchange procedures. This emulsification was aided by exposure to a high-energy ultrasonic device.
For further purification, the resultant nanoparticles were dialyzed using cellophane-based dialysis membrane for 24 h against distilled water to remove any unreacted molecules.
Characterization and Drug-polymer Biocompatibility:
The utilized characterization techniques served to confirm the major structural components of the loaded EEP, in addition to thoroughly perceiving the interactive, physical and chemical properties of the carrier nanoparticles with the loaded EEP.
Determination of the Surface Chemistry:
A sample of each prepared nanogel (blank chitosan-polyacrylic acid nanoparticles: BNPs; and EEP-encapsulated nanoparticles: PNPs) was suspended in ethyl acetate and sonicated for 30 min (42 KHz) prior to being loaded on a TEM sample grid. Afterwards, the TEM grids were left to dry in air, and no stains were applied.
Transmission Electron Microscopy (TEM):
The results provided by the transmission electron microscopy (TEM) revealed that the produced BNPs were regularly spherical with an outer shell-like edge. Regarding the size range, it was revealed that the BNPs were 17 to 29 nm in diameter, while the PNPs were 24 to 32 nm in diameter.
Dark Field Electron Diffraction (DFED):
Based on the DFED ring patterns, the crystalline nature of the BNPs and PNPs was elaborated by the appearance and disappearance of diffraction dots. Both the BNPs and PNPs showed an amorphous nature.
Determination of Propolis-polymer Interaction and Biocompatibility:
Fourier Transform Infrared (FT-IR) Spectroscopy:
The FT-IR technique (Thermo Nicolet 6700, UK) was employed to interpret the chemical composition and functional groups of the NPs, revealing any surface interactions, either chemical or physical.
The comparative FT-IR data obtained from the PAA, CS and CS-PAA samples showed that the chemical composition of the CS-PAA copolymer was found to be nearly identical to that of PAA, indicating that CS was most-likely involved within the inner core structure of the BNPs.
When comparing the FT-IR spectra of BNPs and PNPs, the characteristic peaks of EEP were almost completely masked in the FT-IR spectra of the PNPs formulation, which showed near-identical spectrograms to those of BNPs.
X-ray Diffraction (XRD):
The used X-ray Diffractometer was equipped with Cu-Kα as a radiation source (λ = 1.54Å) at 40 Kv, 35 mA, 25oC and scanning speed of 0.02o/sec. The diffraction peaks were recorded between 2o and 60o 2 θ.
The comparative XRD analysis between BNPs and PNPs indicated that the BNPs’ spectroscopic fingerprint was present also in the PNPs. Moreover, the PNPs showed additional peaks with higher intensity, which were most probably attributed to the loaded EEP.
Studying the Anti-diabetic Effect of Propolis and Nano-propolis in Wistar Rats:
Preparation of the Study Sample:
Male albino Wistar rats (Rattus norvegicus) were obtained from the animal house at the medical research center (MRC), Ain Shams University, and were employed as animal models in the present research. All the conducted animal studies were approved and were following the Ethical Principles in Animal Research adopted by the Faculty of Science, Ain Shams University.
The housing conditions of the studied rats involved a standard pellet diet and water, ad libitum; temperature (24 ± 2oC); RH (60-70%); photoperiod (12L:12D); a noise level <50 dB.
Chemical Induction of Type-2 Diabetes in Wistar Rats:
Type 2 diabetes was achieved in the experimental group using a mixture of high-fat diet (HFD) for two weeks, and a single intraperitoneal streptozotocin (STZ; 35 mg/kg) dose, a diabetogenic agent that intoxicates the pancreatic beta cells, resulting in a partial or complete interruption in the secretion of insulin.
After 72 h, all the rats were fasted for 16 h and their blood glucose levels were monitored from the tip of the tail vein using a glucose kit and an auto-analyzer (ACCU-CHEK Active, Roche diagnostics). The rats used in the experiments were considered diabetic when their fasting blood glucose levels were above 200 mg/dL. Afterwards, the treatment was introduced to the diabetic rats through an oral gavage, daily for 4 weeks.
Experimental Design:
Thirty male Wistar albino rats were used in the current research, and were categorized into 6 groups (5 animals/ group) as follows:
group I: Included non-diabetic rats that received only distilled water (1 ml/kg) and were labeled as the negative control.
group II: Included diabetic rats that did not receive any treatment and were labeled as the positive control.
group III: Included diabetic rats treated with EEP at a dose of 300 mg/kg.
group IV: Included diabetic rats treated with BNPs at a dose of 300 mg/kg.
group V: Included diabetic rats treated with PNPs at a dose of 300 mg/kg.
group VI: Included diabetic rats treated with Metformin at a dose of 100 mg/kg.
Evaluating the antihyperglycemic effects of the applied agents (free and nano-encapsulated EEP and Metformin) on the body weight, using a digital scale (Weston), and blood glucose, using a glucose kit and an auto-analyzer (ACCU-CHEK Active, Roche diagnostics), was recorded weekly.
The experimental data revealed that there was not a marked increase in the total body weight of the diabetic rat groups when compared with the normal control group.
Moreover, the blood glucose level in diabetic rats was significantly higher than normal ones (P < 0.05), confirming the development of insulin resistance and hyperglycemia due to T2DM.
Blood Sampling and Analysis:
At the termination of the experiment (after 4 weeks), rats were abstained 16 h after their last feeding, and then blood samples were collected from the orbital vein of the rats under ether-induced anesthesia, into Eppendorf tubes (1.5 mL). The collected blood samples permitted to clot at room temperature, then centrifuged at 3000 ×g for 20 min at 4∘C. The obtained serum was divided and rapidly put in storage at -80°C for further biochemical assessments.
During the treatment period, the diabetic rats which did not receive any treatment showed increasing hyperglycemia from 1st till 4th week, while the treated diabetic rat groups showed an improvement in the blood glucose levels, except for the group treated with BNPs. The most significant improvement in blood glucose levels was detected in the group treated with PNPs (more than four folds), followed by those treated with EEP and those treated with Metformin, respectively.
This indicated a strong potential exhibited by propolis in establishing a possible glycemic control in the diabetic animal model, in addition to a possible efficiency of the CS-PAA carrier particles in improving such glycemic control potential when compared to the EEP.
Biochemical Assays:
Determination of Serum Insulin Level (SIL):
The serum insulin levels (SIL) were detected employing a rat insulin ELISA kit (EMD Millipore, rat/mouse insulin ELISA kit, USA), which quantifies insulin using Sandwich ELISA method based. The enzyme activity is measured spectrophotometrically (Bio-Tek EL800 plate reader) by the increased absorbency at 450 nm, corrected from the absorbency at 590nm. The intensity of the color generated was directly proportional to the amount of insulin present in the sample.
After the treatment period, the untreated diabetic positive control group showed SILs significantly lower than the negative control group (P < 0.05). On the other hand, both the groups treated with EEP and PNPs showed improved SILs, respectively, compared with the positive control group. However, those treated with the BNPs or Metformin did not show considerable improvement in SILs.
Determination of Lipid Peroxidation:
Lipid peroxide was determined by estimation of malondialdehyde (MDA) in the serum using colorimetric kits (Bio-diagnostic Chemical Company) based on the principle that thiobarbituric acid (TBA) reacts with MDA in acidic medium at 95°C for 30 min to form pink-colored TBA reactive product, which was read at 532 nm and expressed as ”n” moles of malondialdehyde (MDA) produced/ml. The amount of pigment produced from MDA-TBA condensation reveals the level of lipid peroxidation.
The positive control group showed a significantly high MDA levels when compared with the negative control (P < 0.05). Moreover, the experimental group treated with BNPs also showed elevated MDA levels, compared to the negative control group, while those treated with PNPs showed significantly reduced MDA levels (P < 0.05), followed by those treated with EEP and those treated with Metformin, respectively.
Determination of Total Antioxidant Capacity (TAC):
The estimation of the antioxidative capacity was achieved by the reaction of the antioxidants in the serum sample with a pre-specified quantity of exogenously offered hydrogen peroxide (H2O2). The remaining H2O2 is estimated by a colorimetric enzymatic reaction.
The TAC levels were significantly lower in the positive control group than in the negative control (P < 0.05). Concerning the efficiency of the applied therapy in improving TAC levels, the experimental group treated with PNPs showed the highest TAC levels, followed by those treated with EEP, BNPs, and Metformin, respectively.
Determination of Alanine Aminotransferase (ALT):
Serum ALT was spectrophotometrically determined using commercial kits (Bio-diagnostic Chemical Company) at 340 nm according to the supplier’s guidelines, by measuring the change in NADH concentration, which directly correlates with the concentration of ALT.
The serum ALT levels were significantly higher in the positive control group when compared to the negative one (P < 0.05), these levels were reduced in the rest of the experimental group, with the lowest levels detected in the experimental group treated with PNPs, followed by those treated with EEP, and those treated with Metformin. On the other hand, the experimental group treated with BNPs showed high ALT levels.
Histopathological Examination:
Sections of pancreatic tissues were fixed in 10% formalin for 7 days and processed by paraffin embedding and dehydration in a graded alcohol series. After fixation, the tissues were sliced and stained with hematoxylin and eosin (HE). The tissue sections were analyzed under a trinocular research microscope (Olympus BX 43) fitted with a camera (Olympus DP 27) linked to Cellsens dimensions software (Olympus).
The histopathological observation of the pancreatic tissue of the studied rats provided insight into the antidiabetic potential of the used propolis-based therapy in STZ-induced diabetic rats. The stimulated T2DM created obvious pancreatic damage culminating in a reduction in the pancreatic islets overall possibly due to a decline in the number of β‑cells. The diabetic positive control group showed severe atrophy of the exocrine acini with necrobiotic changes accompanied by β‑cells vacuolation.
Conclusion:
The findings of the present study concluded that propolis showed promising anti-diabetic effects as an oral therapeutic, such effects were further improved after being loaded on a chitosan-based carrier.