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 g 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 luteaݡ100%;  post-implantation loss=(total number of implants-number of viable implants)/ total number of implantsݡ100%. 

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 3200g 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 3200g.  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 meanSEM.  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 (meanSEM, n=12, ranges in parentheses).

4 Discussion

At the doses tested, SS of T. catappa had no effect on orgasm or immediate fertility but caused marked changes in the male sexual behaviour.  The nature and severity of these demasculinisation effects, however, differed according to the dose used;  the lower dose impaired sexual arousability whilst the higher dose in addition, decreased sexual performance, sexual interest and libido.  Irrespective of the dose these inhibitory effects on sexual behaviour had a rapid onset and recovery.

Collectively, these data suggest a receptor-mediated action in the brain^[10].  Alkaloids and aminoacids are present in the SS of T. catappa and these can inhibit male sexual behaviour of rats^[10,11] in a similar fashion, as was evident in this study, through cholinergic and serotonergic receptors.  Subchronic treatment of high dose of SS caused moderate elevations in SGOT and SGPT activity indicating liver toxicity. This is a matter for concern. However, liver toxicity was not elicited with the lower dose. 

However, other than this effect on the liver, there were no overt signs of general toxicity, haemotoxicity, motor deficiencies, motor incoordination, general lethargy, behavioural abnormalities, stress or a lowering of body weight and suppression of food intake.

Thus, the antimasculine effects of SS can not be a consequence of generalised toxic effects but is due to a selective action.  With the lower dose only the time required for treated rats to mount, intromit or ejaculate was greatly prolonged whilst the other sexual parameters remained unchanged.  Taken together, the prolongation of these three latencies suggest a reduction in sexual arousability/motivation: as an inverse relationship exists between these three parameters of male sexual behaviour and sexual arousal/motivation^[12].

On the other hand, the prolongation of the ejaculatory latency by itself suggests an aphrodisiac action.  All the treated rats mounted and intromitted without any inhibition of mount-and-intromission frequencies or copulatory efficiency or intercopulatory interval.  This suggests that libido, sexual vigour and sexual performance were unimpaired during the aphrodisiac action.

A similar effect on copulatory behaviour of rats is reported with a water extract of Piper betel leaves^[13].  Further, the nonimpairment in the number of rats attempting intromission and in the number of intromissions made strongly suggests that the lower dose does not disrupt penile erectile function: in the rat, anteroflexions or flips are essential for penile insertion during copulation^[14].  The inability of the SS to prolong the reaction times in the tail flick and hot plate techniques suggest that the aphrodisiac action is not mediated via an impairment of penile sensitivity resulting from analgesia.  Nevertheless, SS induced elevation in threshold of cutaneous penile receptors cannot be ruled out.

The higher dose of SS, on the other hand, impaired all the parameters of sexual behaviour other than mounting-and-intromission frequency and copulatory efficiency indicating that sexual vigour remained uninhibited.  However, the anti-libido effect, at this dose, would reduce the aphrodisiac value of T. catappa seeds.  The rapid onset and reversibility of the sexual behavioural changes as well as  the lack of a significant change in either the absolute or relative weights of sexual accessory organs and unimpaired index of libido strongly suggest that the anti-libido effect is not mediated through a change in the blood testosterone level.

Hyperprolactenaemia in males causes deterioration of their sexual behaviour and libido^[15].  However, this mechanism seems unlikely to be operative in this study because it is generally accepted that only long-term hyperprolactenaemia can induce deficient sexual behaviour^[16].  Here, sexual behaviour was inhibited as early as 3 h of treatment.

Sedatives inhibit libido and sexual performance^[17].  The SS showed a sedative activity in a dose related manner as judged by the inhibition of three of the four parameters in the rat hole-board technique.  Thus sedative activity of the SS can account for the observed impairments of sexual behaviour and libido.  The failure of the SS to alter latencies of bar holding and Bridge tests provides further support to this notion: as false positive results are evident in rat hole-board technique by muscle deficiencies and their incoordinations.  The SS had no hypoglycaemic activity, thus, the sedation is unlikely to be mediated through a reduction in blood glucose level.

In conclusion, the present findings show that seeds of T. catappa possess potent aphrodisiac activity and provides scientific evidence in favour of  the claims made in Ayurvedic medicine in Sri Lanka regarding this action.  The results also suggest that moderate consumption of kernel of seed of T. catappa could be useful in the treatment of men with sexual dysfunctions resulting primarily from premature ejaculation.

References

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