2.1 Drugs and Chemicals Diazepam was obtained from Square Pharmaceuticals Ltd. Lychee honey (1 kg) was collected from a cultivated hive in the lychee garden of Dinajpur district (25.63 ° N 88.65 ° E) of Bangladesh in the month of April 2018.
2.2 Preparation of the Test Samples Collected viscous Lychee honey (LCH) were kept in airtight glass jar at room temperature (250C) and passed through a sieve (0.5 mm mesh) to remove non soluble particles (bee particles, wax, pollen, egg) and other coarse material.
2.3 Physical Properties of Test Samples 2.3.1 Determination of Moisture Content and Total Soluble Solids Moisture content can be deduced from refractive index of honey [13]. A portable honey refractometer (Biobase BK-PRN3, China) with 58 – 92% Brix range, thermoregulated at 200C and calibrate with distilled water, was used for measuring the refractive index of honey. Total Soluble Solids were deduced from its Brix value and temperature correction was applied according to ISO 2173:2003. 2.3.2 Determination of pH The pH of the sample was measured using a pH meter (Biobase pH-10S, China), calibrated at pH 4.01 and 7.00. Honey sample was prepared as 10% (w/v) solution in distilled water and reading was taken in triplicate. 2.3.3 Determination of Optical density (OD) Optical density was determined from a 10% (w/v) honey solution in distilled water by using a UV-VIS Spectrophotometer (Biobase BK-UV1800, China). Absorbance was taken at 530 nm using distilled water as blank. The method was performed as described by Wakhle in 1997. Absorbance values obtained was compared with the standard set by United State Department of Agriculture (1985). 2.3.4 Determination of Honey Density 1 ml honey was drawn with a syringe, both empty and filled weight was measured using an Automatic Electronic Analytical Balance (Biobase BA2004N, China). The mass of the honey was determined from the difference of these two weights. Finally, Density was calculated as described by Kinoo et al. in 2012 using the below formula.
2.4 Acute Toxicity Test Before execution of the in-vivo experimental methods, acute toxicity was observed. Lychee Honey was orally administered to 20 experimental animals at the dose of 5g, 7.5g, 10g & 15g per kg of body weight. Rodents were then observed for next 72 hours for any number of deaths or any unusual symptoms or behavior.
2.5 Experimental Animal Female Albino mice, 45 days of age having 27-32 g body weight, were selected for the evaluation. Rodents were accustomed with a 12 h light/dark cycle, ambient temperature, air ventilation and ad libitum food and water at animal house of Institute for Pharmaceutical Skill Development and Research. Total six groups were formed which consisted five mice in each and orally challenged with respective agents. Group 1: Blank (No gavage), Group 2: Control (Distilled Water), Group 3: Diazepam (1mg/kg), Group 4: LCH-2 (2g/kg body weight, equivalent to 25% w/v in 0.15ml distilled water), Group 5: LCH- 4 (4g/kg body weight, equivalent to 50% w/v in 0.15ml distilled water), Group 6: LCH-6 (6g/kg body weight, equivalent to 75% w/v in 0.15ml distilled water).
2.6 Experimental Design A novelty-induced environmental challenge was designed by combining three apparatus in a continuous series. After oral gavage, mice were placed in Open Field, Hole Cross and Elevated Plus Maze (EPM) sequentially in a row and allowed to explore each for three minutes. For first interval, mice experienced 0-3rd min at open field, 4-6th min at hole cross (however for simplifying, the time denoted as 0 min for hole cross) and 7-9th min at EPM (the time denoted as 0 min for EPM). For each rodent, the cascade was repeated in 30, 60, 90 and 120 minute intervals accordingly. 2.6.1 Open Field Test Mice were placed in an open cubic box measuring a dimension of 60x60x60 cm having a tiled (5x5 cm) floor alternatively colored black and white. This method was performed as described by Billah et al. (2016) [19] to asses sedative-anxiolytic activities by observing parameters such as Number of Square crossed, Grooming and Rearing. 2.6.2 Hole Cross Test In this experiment mice were freely allowed to cross a 3 cm hole made on a partition at 7 cm floor height which divided a 30x20x14 cm box into two equal compartments. Number of holes crossed was counted during the observation as parameter of exploratory behavior. This method was performed as described by Nawrin et al. (2015). 2.6.3 Elevated Plus Maze Test In Elevated Plus Maze (EPM) apparatus, mice were allowed to move in any direction of a Plus shaped mirror-designed two open arms intersecting with two closed arms. Each arm had a length of 14 cm, width of 5 cm and the close arm had wall height of 14 cm. Open and close arm entry and duration were observed as parameter indicating anxiolytic potentials as described by Hawiset et al., (2011).
2.7 Statistical Analysis Statistical analysis of data was done by utilizing the method of one-way analysis of variance (ANOVA) followed by Dunnett’s t tests using SPSS 24 for windows. The results obtained were compared with the control group. P values < 0.05, 0.01 and 0.001 were considered statistically significant.