Drugs and Chemicals Jujube honey (1 kg) was collected from a cultivated hive in the jujube garden of Gazipur district (23.53°N 92.39°E) in the month of May 2019. This raw honey was stored in an airtight glass jar at room temperature (25?C). Diazepam was obtained from Square Pharmaceuticals Ltd. For the preparation of test samples, the viscous jujube honey was allowed to pass through a sieve (0.5 mm mesh) to remove nonsoluble particles (wax, bee particle, egg, pollen) and other coarse material.
Determination of Moisture and Total Soluble Solids The moisture content of honey can be deduced from its refractive index (12). To measure the refractive index of honey, a portable honey refractometer (Biobase BK-PRN3, China), having the Brix range of 58 – 92%, thermoregulated at 200C and calibrated with distilled water, was used. Temperature correction was applied according to ISO 2173:2003 (13). Total Soluble Solids were inferred from Brix value of honey.
Determination of pH A pH meter (Biobase pH-10S, China), calibrated at pH 4.01 and 7.00 was used to measure the pH of the sample. The pH measurement was done in triplet where the honey sample was prepared as 10% (w/v) solution in distilled water.
Determination of Optical Density (OD) Optical density was determined by using a UV-VIS Spectrophotometer (Biobase BK-UV1800, China) from a 10% (w/v) honey solution in distilled water. Absorbance was taken at 530 nm. Distilled water was used as blank. The method was performed according to the procedure described by El Sohaimy et al. 2015 (15). The obtained absorbance values were compared with standard set by United State Department of Agriculture (1985).
Determination of Honey Density At first, empty weight of a syringe was taken. After drawing 1 ml honey with the syringe, filled weight was taken. Automatic Electronic Analytical Balance (Biobase BA2004N, China) was used for weight measurement. From the difference of these two weights, mass of the honey was determined. Finally, Density of honey was calculated by using the formula given below as described by Kinoo et al. in 2012.
Assessment of Sedative-Anxiolytic potential Acute Toxicity Test Acute toxicity was evaluated before commencing the in-vivo experimental scheme. Jujube Honey (JH) was orally administered to 20 experimental rodents at the dose of 5g, 7.5g, 10g & 15g per kg of body weight (n=5). The animals were then monitored for the following 72 hours to observe any number of deaths or any unusual symptoms or behavior (18).
Experimental Animal Female Swiss Albino mice, having 27-32 g of body weight with 45 days of age were selected for the assessment. These animals were habituated with a 12 h light/dark cycle, air ventilation, ambient temperature and ad libitum food and water at animal house of Institute for Pharmaceutical Skill Development and Research. Mice were divided into six groups, each containing 5 animals for control, standard, and test samples, for every experiment to challenge them orally with respective agents. Group 1: Blank (No gavage), Group 2: Control (Distilled Water), Group 3: Diazepam (1mg/kg), Group 4: JH-2 (2g/kg body weight, in 0.15ml distilled water), Group 5: JH-4 (4g/kg body weight, in 0.15ml distilled water), Group 6: JH-6 (6g/kg body weight, in 0.15ml distilled water).
Experimental Design Three apparatus were arranged in a continuous series to design a cascade of novelty induced environmental challenge. This method was performed as described by Billah et al., 2019 (19). Mice were placed in Open Field, Hole Cross and Elevated Plus Maze (EPM) sequentially in a row immediately after oral gavage. Behavior of each mice was observed for three minutes in each field. In first interval, mice were placed at open field for the first 3 minutes which was continued with the shifting of mice at hole cross apparatus for the next 3 minutes (however for simplifying, the time denoted as 0 min for hole cross) and at EPM for the last 3 minutes (the time denoted as 0 min for EPM). For each animal, the workflow was repeated in 30, 60, 90 and 120-minute intervals accordingly.
Elevated Plus Maze Test In this experiment, mice were placed in the center of the maze and allowed to move in any direction. It has mirror-designed plus shaped two open arms intersecting with two closed arms each having a length of 14 cm and width of 5 cm. The close arm has wall height of 14 cm. Duration and entry in open and closed arms were observed as parameters to assess the anxiolytic potential. This test was executed as described by Hawiset et al., 2011.
Statistical Analysis Statistical analysis of data was performed by using the method of one-way analysis of variance (ANOVA), followed by Dunnett’s t-test by using SPSS 24 for windows. The obtained results were compared with the control group. P values < 0.05, 0.01 and 0.001 were accepted as statistically significant.