Drugs and preparation of extracts
The authenticated sample of dried Nutmeg of Myristica fragrans Houtt and buds (Clove) of Syzygium aromaticum Merr. & Perry. were procured from the market (Delhi, India), and the standard reference drug Penegra (Sildenafil citrate) was obtained from Zydus Cadila (Ahmadabad, India).
To prepare 50% ethanolic extracts of Nutmeg and Clove, 50 g. powder of each drug was extracted with 200 ml of distilled water and 200 ml of absolute alcohol (v/v) by a soxhlet apparatus. The extracts were filtered, and the filtrates were evaporated to dryness at low temperature under reduced pressure. The yield of 50% ethanolic extracts thus obtained was approximately 7.6 g. for Nutmeg and 5.4 g. for Clove.
The test drugs were administered to the animals and the doses of 500 mg /kg; p.o. were selected according to Dhawan [24] multiplying the Unani clinical dose with the conversion factor of 12. The dose of the referent drug (5 mg/kg; p.o.) was also calculated in the same manner.
Animals
Adult Swiss mice (25–35 g.) were used for the study. The animals were housed under standard laboratory conditions (relative humidity 65 ± 2%, temperature 23 ± 2°C and 12 h light: dark cycle.) They were fed with standard rodent pellet diet (Gold Mohar, Lipton-India, Ltd.) and tap water ad libitum. The study was approved by the departmental ethical committee for animal care and use.
Mounting behaviour test
Mount is operationally defined as the male assuming the copulatory position but failing to achieve intromission. To quantify mounting behaviour, non-oestrous female mice were paired with males treated with single dose of the drugs (500 mg/kg; p.o.). Animals were observed for 3 hrs and their behaviours were scored as described [25]. Males were placed individually in a glass cage. After 15 minutes of acclimatization, a non-oestrous female was introduced into the arena. The number of mounts were recorded during a 15 minutes observation period at the start of 1st hr. Then the female was separated for 105 minutes. Again the female was introduced and the number of mounts was observed for 15 minutes as before at 3rd hr. All the experiments were performed between 09.00 to 12.00 hrs during day time at room temperature 26–27°C.
To determine the effects of Nutmeg, Clove and Penegra on mounting four groups of six animals each were taken for the study. All drugs were dissolved in distilled water just before the administration. The first group received distilled water (10 ml/kg; p.o.) and served as control. Groups II and III were given the extracts of Nutmeg (500 mg/kg; p.o.) and Clove (500 mg/kg; p.o.) respectively, while the group IV received Penegra (5 mg/kg; p.o.) and served as standard.
Assessment of mating performance
Male mice divided into 4 groups of six each were used in the study. Group I served as control and received distilled water (10 ml/kg;p.o.). Group II and III were administered extract of Nutmeg (500 mg/kg; p.o.) and Clove (500 mg/kg; p.o.) while group IV received Penegra (5 mg/kg; p.o.) and served as standard. The drugs were administered in the evening (17.00 – 18.00 h.) and each male was placed in a separate cage. After 1 hr, five oestrous female were admitted into each cage and they were cohabitated overnight. The stage of the oestrous cycle was determined according to the criteria laid down by Ecksterin et. al. [26]. The vaginal smear of each female mouse was examined under a microscope for the presence of sperm. The number of sperm positive female was recorded in each group.
Toxicity study
To determine general short term toxicity, the animals were divided into 3 groups, each containing 6 mice. Group I animals served as control and received distilled water in an identical manner. The groups II & III were given extract of Nutmeg (500 mg/kg; p.o.) and Clove (500 mg/kg; p.o.) respectively. The animals were observed continuously for 1 hr for any gross behavioural changes or death, if any, and intermittently for the next 6 hrs and then again at 24 hrs after drug administration. The behavioural parameters like convulsions, hyper activity, sedation, grooming, loss of righting reflex and increased respiration were observed.
Statistical analysis
The statistical comparison between control, standard and treated groups was done by using the analysis of variance method (ANOVA) with the confidence level of 95% (P < 0.05)
