1 Introduction

Male sexual inadequacy is a problem facing many men throughout the world.  Aphrodisiacs and drugs that inhibit premature ejaculation are useful in certain forms of male sexual inadequacies.  In Sri Lanka, according to Ayurvedic system of medicine, several plants are claimed to possess aphrodisiac potential^{[1, 2]}. However, in most cases the validity of these claims has not been scientifically tested.  Kernels of seeds of Terminalia catappa Linn. (Family: Combretaceae, Kottamba in Sinhala and Amandi in Tamil) is one such plant^[2].

2 Materials and methods

2.1 Animals, reagents and instruments

Healthy, sexually experienced male (275-325 g) and female (180-200 g) crossbred albino rats were kept in well ventilated animal house conditions (temperature: 28-31℃; photoperiod: 12 h natural light and 12 h dark; humidity: 50-55%) with free access to pelleted food (Vet House Ltd., Colombo, Sri Lanka) and tap water. Methyl cellulose was purchased from Griffin and George Ltd (London, UK), oestradiol benzoate, progesterone and ether from Fluka (Buchs, Switzerland), and enzyme assay kits from Randox Laboratories Ltd. (Co. Antrim, UK).  The haemocytometer was purchased from Fison Scientific Equipments (Loughborough, UK), animal balance (MP 6000) from Chyo Balance Corporation (Tokyo Japan), hot plate (Model MK 350A) from  Muromachi kikai Co. Ltd. (Tokyo, Japan), Wifug Lab Centrifuge from Eltex of Sweden Ltd. (Bradford, UK), spectrophotometer (Jasco V500) from Jasco Corporation (Tokyo, Japan) and microscope from Olympus Optical Co. Ltd. (Tokyo, Japan).

2.2 Collection of seeds, preparation of seed suspension (SS) and phytochemical analysis  

Ripe and partially dried fruits (with gray coloured pericarp) of T. catappa were collected from the campus of University of Colombo, Sri Lanka, during March and April, 1999 and authenticated by Professor A. S. Seneviratne, Department of Botany, University of Colombo. The seeds were decoated using a pen knife and the white colored kernel  was exposed.  The kernels of the fruit were dried in an oven at 60℃ to a fixed weight (usually within 4 days).  These were then macerated in a porcelain mortar until a semisolid paste was obtained.  This material was stored at 4℃ until use.  The paste was suspended (SS) in 1% methyl cellulose (vehicle) 2-3 h prior to experimental use to obtain the desired concentrations (750, 1500, 3000 mg/kg ) in 1 mL.

The semisolid paste was subjected to standard chemical tests as described by Farnsworth^[4] to determine the presence (qualitatively) or absence of alkaloids, flavonoids, phenols, steroids and triterpenoids, oils, saponins, amino acids and peptides.

2.3 Effects on rat male sexual behaviour

A total of 36 male rats were randomly divided into three groups (n=12 per group) and each group was orally treated either with 1 mL of 1500 mg/kg (SS) or 3000 mg/kg SS or 1% methyl cellulose (vehicle).  These rats were individually placed in cages 3 h following the administration and were given a 10 min adaptation period.  A female that had been brought into oestrus (oestradiol benzoate 12 μg in olive oil injected subcutaneously 56 h prior to pairing plus progesterone 0.5 mg in olive oil injected subcutaneously 8 h prior to pairing) was placed in the cage.

The following parameters of sexual behaviour were monitored until ejaculation or 15 min after pairing; mount frequency, intromission frequency, mount latency (time from the introduction of the receptive female to the first mount), intromission latency (time from the introduction of the receptive female to the first intromission), ejaculatory latency (time from the introduction of the receptive female to ejaculation).

Using these measures the following parameters were computed: % mounted, % intromitted, % ejaculated, copulatory efficiency (number of intromissions/number of mounts) and intercopulatory interval (average time between intromissions).

In the fertility study, male rats were randomly assigned to two groups.  One group (n=6) was orally treated with 1 mL of 3000 mg/kg SS and the other group (n=6) 1 mL of vehicle (at 12:00) for 7 consecutive days.

2.4 Effects on male fertility

Libido, ejaculatory competence and fertility of these rats were assessed 7 days prior to treatment, and on days 1, 4 and 7 during treatment, and day 7 post-treatment.  Each male was paired overnight with a pro-oestrous female (at 16:30-17:00).  The pre-coital sexual behaviour (chasing, nosing, anogenital sniffing, genital grooming and attempted clasping and mounts) of the paired rats was observed 1-2 h later.  Successful mating was confirmed by the presence of sperm in the vaginal smear the following morning (08:00-08:30).  If spermatozoa were present, their numbers were estimated (in duplicate) using an improved Neubauer haemocytometer and gross morphology were noted by microscopic examination (×400). 

At day 14 post-coitum  the mated females were subjected to laparotomy under ether anaesthesia and the number of conceptus (both viable and dead) were counted. In addition, the number and the gross morphology of the corpora lutea in each ovary were recorded.

The following reproductive parameters were then computed: index of libido=(number mated/number paired)×100%; quantal pregnancy = (number pregnant/number mated)×100%; fertility index=(number pregnant/number paired)×100%; implantation index=(total number of implantations/number mated)×100%; pre-implantation loss=［(number of corpora lutea-number of implantations)/number of corpora lutea］×100%;  post-implantation loss=［(total number of implants-number of viable implants)/ total number of implants］×100%. 

2.5 Effects on morphology and wet weights of accessory sexual organs

To determine antiandrogenic effects of SS on wet weight of selected organs of the male reproductive system  randomly selected male rats were orally treated with either 1 mL of 3000 mg/kg of SS (n=6) or 1 mL vehicle daily for 7 consecutive days (between 13:00-14:00).  On day 1 post-treatment, these rats were killed with an overdose of ether and the animals were weighed.  The gross external morphology of the testes, excurrent ducts and sexual accessory glands was noted.  Weights were recorded for the paired seminal vesicles with coagulation glands (glandular sections were not removed), lateral prostates, testes, epididymides or vasa deferentia and were expressed as a percentage of body weight.  The rats were weighed before the commencement of treatment and on day 1 post-treatment.

2.6 Effects on haematology

On day 1 post-treatment blood was collected from the tail of the rats (used in 2.4) under aseptic conditions and red blood cell (RBC) counts, white blood cell (WBC) counts, packed cell volume (PCV), haemoglobin content and differential white cell counts (DC) were estimated as described by Cheesbrough et al^[5].

2.7 Evaluation of sedative potential

The SS was evaluated using the rat hole-board technique^[6].  Randomly selected male rats were treated orally either with 1 mL 750 (n=12), 1500 (n=12) or 3000 (n=12) mg/kg of SS or 1 mL of vehicle (n=12).  Three h later these rats were individually placed on the centre of rat hole-board and given a 7.5 min trial period. The number of head dips, rears, locomotory activity and the number of faecal boluses produced were recorded.  The time per head dip was then calculated. 

2.8 Evaluation of muscle strength and coordination

Randomly selected male rats were either orally treated with 1 mL of 3000 mg/kg SS (n=9) or 1 mL of vehicle (n=9).  Three hours post-treatment each of these rats was subjected to bar holding test (to evaluate muscle strength^[7])  and the time taken (in sec) for the rat to fall from the bar was determined.  Immediately following this test, these rats were subjected to Bridge test (to evaluate muscle coordination^[7])   and the latency to slide off (in sec) was recorded.

2.9 Evaluation of analgesic effect

Analgesic activity of the SS was evaluated by means of  tail flick^[8] and hot plate^[8] tests with a cut off time of 10 and 20 sec respectively, to prevent tissue damage.  Randomly selected male rats were orally treated with either 1 mL of 3000 mg/kg seed suspension (n=12) or 1 mL of vehicle (n=12).  Three-four hours before the treatment and 3 and 6 h post-treatment, the time taken (in sec) to flick the tail (reaction time) when immersed in a water bath (5-6 cm from the tip) at 55℃^[9] and the time taken (in sec) to lick either of the hind paws when placed on a hot plate were determined.

2.10 Evaluation of hypoglycaemic effect

Twelve male rats were deprived of food 14-16 h and randomly divided into two groups.  One group was orally treated with 1 mL of 3000 mg/kg SS (n=6) and the other with 1 mL of vehicle (n=6). Blood (3-5 drops) was collected from the tail of these rats using aseptic precautions under mild ether anaesthesia 20-30 min before treatment and 3 h post-treatment.  Blood was allowed to clot at room temperature (28-30℃) and serum was collected following centrifugation at 3200×g for 5 min.  The serum was stored at 4℃ until glucose levels were determined (within 12 h) using Randox glucose oxidase-peroxidase kit and a spectrophotometer.

2.11 Effects on liver function

The liver toxicity of the seed suspension was tested in randomly selected male rats orally treated with either 1 mL of 3000 or 1500 mg/kg of SS or vehicle (n=6 per group) for 7 consecutive days (between 13:00-14:00).  On day 1, post-treatment 2 mL of blood was collected from the tail using aseptic precautions under mild ether anaesthesia.

The blood was allowed to clot (25-30 min) at room temperature (28-30℃) and subjected to 15 min centrifugation using a Wifug Lab Centrifuge at 3200×g.  Serum was collected and activities of serum glutamic-oxaloacetic transaminase (EC 2.6.1.1, SGOT) and glutamic-pyruvate transaminase (EC 2.6.1.2, SGPT) were determined (within 1-2 h) using a Randox enzyme assay kit and a spectrophotometer.  All readings were taken within 10 min after incubation.

2.12 Adverse effects

All treated rats were observed at least once daily for any overt signs of toxicity (salivation, rhinorrhoea, lachrymation, ptosis, squinted eyes, writhing, convulsions, tremors, yellowing of fur, loss of hair) stress (erection of fur, and exophthalmia) and changes in behaviour (such as spontaneous movements in the cage, climbing, cleaning of face, nongenital self grooming).  In addition, food and water intake were noted.

2.13 Statistical analysis

Data are represented as mean±SEM.  Statistical analyses were made using Mann-Whitney U-test and G-test(in the case of proportional data).  Significance was inferred when P<0.05.

3 Results

3.1 Phytochemical analysis

Preliminary chemical testing showed the presence of alkaloids, oils, amino acids and peptides in the seed kernel. Detailed chemical isolation is ongoing.

3.2 Sexual behaviour

Table 1 summarises the data obtained with the sexual behaviour study. The only significant effect observed with the lower dose of SS was the prolongation of the time taken to mount, intromit or ejaculate.  In contrast, with the higher dose, all the behavioural parameters monitored were significantly altered (Table 1) except the frequencies of mounting and intromission, and copulatory efficiency.

Table 1. Effects of oral administration of different doses of seed kernel suspension (SS) of T. catappa on masculine sexual behaviour of rats (mean±SEM, n=12, ranges in parentheses).