**2. Subjects and methods**

### **2.1 Animals and maintenance**

In bred adult (12–15 weeks of age), female Parkes strain mice were used for this study. Mice were maintained under hygienic conditions in a well-ventilated room with 12-h photoperiod (8 AM to 8 PM, light) with 50 20% relative humidity, <sup>25</sup> 2° C temperature and were fed pelleted food (Mona Laboratory Animal Feeds, Varanasi, India); drinking water was available *ad libitum*. Five mice in each group were housed in polypropylene cages (430 mm 270 mm 300 mm), with dry rice husk as the bedding material. General health condition and body weight of the animals were monitored regularly during the entire tenure of the experiment. All experiments were conducted in accordance with principles and procedures approved by Departmental Research Committee under supervision of Committee for the Purpose of Control and Supervision of Experiments on Animals, (CPCSEA), Govt. of India (2007).

Preparations of different doses of *Cannabis* extracts:

Leaves and flowers of fresh *Cannabis* plant (100 g cannabis plant) were extensively ground in mortar and pestle with 1 ml autoclaved double distilled water. From the 1 g/ml paste, 12 mg was weighed and further dissolved in 1 ml autoclaved double-distilled water to make a stock solution of 12 mg/ml. This solution was filtered to get a clear solution. Finally, the mice were gavaged *Cannabis* by means of a 100 μl micro-pipette using the 12 mg/ml stock.

#### **2.2 Purity assessment of** *Cannabis* **preparations**

The dry-weight ratio of D9-tetrahydrocannabinol (THC) to cannabidiol (CBD) and the percent CBD and THC in the cannabis variant found in this region of the world has been previously reported [21]. The proportion of high THC/CBD chemotype plants in most accessions assigned to *C. sativa* was of 25% (Hillig and Mahlberg) [21].
