Deoxicholate and BBR were purchased from Sigma. Ketamine hydrochloride and xylazine were from Alfasan (Holland). The chemical used including NaoH, KH2Po4, NaCL, HCL and other materials and solvents were analytical grade. Equipments were used including rotary (Buchi, Switzerland), centrifuge, filter amicon 3000 KD, and spectrophotometer UV-Vis (SPEKOL 1300; Analytik Jena, Germany).
Preparation of BBR-loaded micelle solution
Thin film hydration method was used for preparation of BBR-loaded micelle solution [23]. Briefly, in a round-bottom flask BBR (1% w/w) and deoxicholate (49% w/w) as surfactant, was mixed with minimum amount of methanol as organic solvent. The rotary evaporator was applied for more than 2 h to evaporate solvent under reduced pressure and to deposit thin film. Then, in the hydration phase, deionized water (50% w/w) added to it, and bain-marie was applied to 50 °C, while stirring was done for a 20 min at the same temperature until a clear solution was obtained that was light yellow.
Characterization
Dynamic light scattering was reported size and size distribution, PDI, Zeta potential of nano micelles (Malvern instruments, UK). The transmission electron microscope (TEM) was applied to show the size of nanomicellar formulations (PHILIPSCM 300, Netherlands).
For determination of encapsulation efficiency, an indirect method was used. Briefly, after centrifugation of micelles at 4,000 rpm for 30 minutes, the concentration of BBR in the samples was determined by spectroscopy UV-Vis. The drug encapsulation efficacy was calculated by the following equations [24]:
Drug release study
The Release behavior of BBR from nano micelles was investigated in SGF and SIF. To prepare the SGF, 246 µL HCl and 200 mg NaCl was added to 60 ml deionized water (DW), and pH was adjusted at 2, then the final volume was filled to 100 mL with DW. Also SIF was prepared by dissolving 680 mg of KH2PO4 and 61.6 mg of NaOH in 60 mL DW, and pH was adjusted at 6.5. Then the final volume was filled to 100 mL with DW. Samples were diluted in a ratio of 1:10 in SIF and SGF, and incubated at 37 oC. Then sampling carried out at time points 0, 30 min, 1, 2, 4, 8, 24 h [25]. Then the determination of BBR was accomplished by spectroscopy UV-Vis.
Animal Groups and Ethical considerations
96 adult male wistar-rats, weighting 200-220 g (aged 4-5 weeks) were purchased from Pharmacology School of Tehran University of Medical Sciences. All of them were kept in an animal house under standard laboratory condition at temperature of 22±1 oC and humidity of 80%, with a typical 12 h light/dark cycle and were allowed to have free access to food and water. All phases of the experiment were approved by the ethics committee of Tehran University of Medical Sciences for the maintenance and application of laboratory animals(IR.TUMS.MEDICINE.REC.1398.593). All attempts were made to minimize the animal suffering. After completing the experiments, the animals were euthanized by carbon dioxide gas. To collect brain tissue samples, the animals underwent spinal cord in complete anesthesia. Animal carcasses were disposed of with hospital waste.
The rats were randomly divided into 8 groups (12 rats in each group) as follow:
The control group
The stroke group
The nano micelle group (without drug)
The pretreated group with BBR (100 mg/kg)
The pretreated group with different doses of nano BBR (100, 75, 50, 25 mg/kg)
The control group hasn’t received any drug and no induction has been made on it. The stroke group Induction has been performed but has not received drug. Also, groups 4-8 were treated orally with BBR and nano-BBR for 14 days with concentrations of 25, 50, 75, 100 mg/kg. For oral administration of berberine hydrochloride, it (approximately 1% of body weight) was dissolved in water [26].
Stroke inducing by BCCAO model
For the inducing of C.I, rats were anesthetized by an intraperitoneal (i.p) injection of 50 mg/kg ketamine and 2-8 mg/kg xylazine. Rats were placed on its back. The animal’s tail and paws were fixed using adhesive tape. A sagittal ventral midline incision (~ 1cm length) was performed, then the bilateral common carotid arteries revealed, and both carotid arteries were carefully separated from the vagal nervous. Also, it is crucial to avoid any manipulations of the vagal nervous. A 5-0 silk suture loop was made around each carotid artery. Both carotid arteries were occluded for 30 minutes. Then a five minutes reperfusion period was initiated. After the reperfusion the wounds were sutured and with an anti-septic solution were disinfected. Thereafter, rats were returned to their animal cages [27-29].
Biochemical assays
Rats were decapitated under deep anesthesia, the brains were removed, and all tissues were stored at −80°C for later biochemical assays. The animal’s brains were homogenized in buffer (TRIS HCL, SDS, DTT, NP40, and glycerol), and centrifuged for 5 min [30], then the levels of IL-1β and TNF-α in serum were detected with ELISA kits, also the activity of MDA (as marker for lipid peroxidation) in serum was measured using the commercial kit according to the manufacturer’s instructions.
Data analysis
Data analysis was carried out using GraphPad prism version 7 software. All data were analyzed using one-way analysis of variance (ANOVA) and Tukey post-hoc. Data were presented as Mean ± SEM. Statistically, differences with p<0.05 were recognized as significant.