Drugs and chemicals

Ethanol extract of fruiting bodies and cracked spores Ganoderma lucidum in capsule form branded as ReishiMaxGLpTM, was purchased from Pharmanex Inc. (United States of America).

Animals

Male Swiss albino mice (Mus musculus) weighing about 22–25 g were utilized in this study. Thirty mice divided in five groups (n = 6) were used in this study. The animals were procured from Liaquat National Medical College, Karachi. The temperature of the area was kept 23 ± 0.5 °C. Water ad libitum was provided to the mice. Mice were acclimatized in laboratory environment before the start of the study.

Animal handling

The guidelines of National Institute of Health (NIH, 8th edition) were followed throughout the performance of research experiments. Handling of the animals was done according to the requirements mentioned in “Guidelines for the care and use of laboratory animals 8th edition” [18].

Dose selection

The selection of doses was made as per research literature, that is, 0.4 mg/kg for scopolamine, 0.1 mg/kg for physostigmine were used [19]. Ganoderma lucidum extract was given in two doses that is, 150 mg/kg and 300 mg/kg [20].

Animal groups and drug administration

The animals were divided in the following five groups:

Group I: Vehicle control group (administered distilled water 10 ml/kg, p.o.)

Group II: Negative control group (administered scopolamine 0.4 mg/kg, i.p.)

Group III: Treatment group (administered physostigmine 0.1 mg/kg i.p. + scopolamine 0.4 mg/kg i.p.)

Group IV: Treatment group (administered extract 150 mg/kg, p.o. + scopolamine 0.4 mg/kg, i.p.)

Group V: Treatment group (administered extract 300 mg/kg, p.o. + scopolamine 0.4 mg/kg, i.p.)

Elevated plus maze

Elevated plus maze (EPM) is considered as main model to evaluate learning and cognition in rodents. It is comprised of two open arms (16 × 5 cm) and two enclosed arms of same size as of open arms, having walls of about 5 cm. This instrument is protracted from a center (5 × 5 cm) and the instrument is elevated to the height of 25 cm. On the initial day of the experiment, (10th day of dosing), every animal was settled at the edge of any of the open arms. Initial Transfer Latency (ITL) was noted down on the initial trials for each animal. Initial transfer latency is defined as the time in which the animal moves from one of the open arms into any of the two closed arms with its all limbs. When the animal did not enter any closed arms within 90 s, the mouse was smoothly directed into one of the two closed arms and the ITL was assigned 90 s. The animal was then allowed to explore the maze for 120 s and then placed back into its home cages [21]. The remembrance of this memory was then evaluated later twenty-four (24) hours after first training; this is termed as Retention Transfer Latency or RTL [22, 23].

Morris water maze

Morris water maze (MWM) is considered as accurate model for evaluating learning of task and evaluating ongoing memory in rodents. Here MWM was utilized which was consisted of round tank 60 cm wide and 25 cm tall. This round tank was filled with water and temperature of water was kept 25 °C. The water was turned cloudy with nontoxic white colored dye. Tank was divided into four equal quadrants i.e. North, South, East and West, which were made on the tank by using two threads which were crossed in the middle perpendicularly to each other and were firmly attached on the top of the tank. In target quadrant of tank (Q4), a platform (6 × 6 cm) was placed. The water level was 1 cm above the platform which made it opaque. Every mouse was subjected to search the submersed platform and to stay on platform for 20 s. Every mouse was smoothly put in any quadrant (except Q4) during the trial session. The mouse was placed in such a way that it faced the wall of tank. After every trial the location of mouse was changed, and 120 s were assigned to every mouse to search submersed platform. The platform was kept untouched and the position was remained unaltered during the training session [24].

When the mouse was unable to find the platform in the prescribed period i-e 120 s, was smoothly guided towards the platform and allowed mouse to stay on platform for 20 s [25].

Escape latency (EL) recorded from 6th till 9th day of administration of drug for each animal. Trials for four consecutive days were done on every mouse. EL is defined as the period in which animal start moving from starting quadrant in search of hidden platform in the target quadrant. Training session for four consecutive trial days were done according to following pattern.

1st Day: Q1, Q2, Q3 and Q4.

2nd Day: Q2, Q3 Q4 and Q1.

3rd Day: Q3, Q4, Q1 and Q2.

4th Day: Q4, Q1, Q2 and Q3.

* “Q” is denoted for Quadrant.

After Escape latency session and testing, TSTQ (time spent in target quadrant) was noted. The platform was removed from the tank and each mouse could swim freely for 300 s. The average time spent in each quadrant was noted. The average time in target quadrant (Q4) was also recorded in which the mouse could find the removed platform. This finding of platform is considered as a sign of memory retrieval. The observer stood in the same position throughout the training and test days [24, 25].

Open field test

To rule out the effect of the extract on motor activity, we examined ambulatory behavior of the animals in open field test. An open-field is a wooden box (45 × 45 × 40 cm) with the floor partitioned into 16 squares. Every mouse was settled at the central square of the field and then ambulation (number of squares crossed) was noted for 10 min. This ambulation will represent the locomotor activity of the mice [26, 27].

Biochemical estimations of cholinergic status in mouse brain

At the end of the study, the mice were sacrificed. The whole brain was collected and subjected to homogenization in a glass homogenizer containing 10 ml of 0.9% of NaCl solution and homogenized in an ice bath. The homogenate was then placed in the centrifugation machine set at 3000 rpm for 10 min. The centrifugation caused the presence of cloudy supernatant liquid which was then used for detection of cholinesterase level in the brain [28].

To determine cholinesterase levels in brain of mouse the Ellman method was adopted. The method involves the acetylcholine iodide from and dithiobisnitrobenzoate. The reaction between dithiobisnitrobenzoate and acetylcholine iodide resulted in formation of thiocholine which formed yellow colour appearance and this yellow coloured precipitate formation in the reaction was considered as an end point of the method. The spectrophotometric analysis was done to determine rate of thiocholine formation from acetylcholine iodide in the presence of tissue cholinesterase and 5,5′-Dithiobis-(2-Nitrobenzoic Acid) (DTNB) was treated with the brain sample and the optical density (OD) of yellow colour compounds formed during the reaction was measured in spectrophotometer at the wavelength of 412 nm every minute [29].

Statistical analysis

The data were presented as mean ± standard error of mean (SEM). The data were analyzed by one-way ANOVA followed by Tukey’s post hoc test. A probability level of 0.05 or less was accepted as significant.