Sexual health is important to overall good health and well-being (Mulhall et al., 2008). Recently, female sexual dysfunction has attracted much interest in the field of medical sciences (Aslan et al., 2008, Both et al., 2008a, Both et al., 2008b, Clayton, 2007, Derogatis and Burnett, 2008, Garcia et al., 2008, Harte and Meston, 2008, Hayes et al., 2008, Laan et al., 2008). Female sexual dysfunction, which consists of multiple disorders, are categorized as hypoactive sexual desire disorder, orgasmic disorder, sexual arousal disorder, and sexual pain disorder (Aslan et al., 2008, Beharry et al., 2008, Berman et al., 1999, Clayton, 2007, Derogatis and Burnett, 2008, Goldstein et al., 2000, Traish et al., 2004). Female sexual arousal disorder refers to a lack of responsiveness to sexual stimulation in women (Wincze and Carey, 2001). Physiologically, female sexual arousal disorder is characterized by the absence of vaginal lubrication and expansion (Both et al., 2008a, Clayton, 2007, Laan et al., 2008, Wincze and Carey, 2001).
Estradiol plays a significant role in regulating female sexual function (Clayton, 2007, Garcia et al., 2008). Studies on the effect of estrogen in the rat model suggested that estrogens increased libido, increased vaginal lubrication, and decreased pain during intercourse, indicating that estradiol is an important regulator of vaginal hemodynamics (Min et al., 2002, Min et al., 2003, Traish et al., 2004).
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The lack of established, clinically efficient treatments for sexual motivation disorders makes it difficult to evaluate if any model has predictive validity (Huh et al., 2008). However, the model proposed and validated in rats by Ågmo et al. (2004) may be analogous to the human condition. Ågmo (2002) suggested that sexual motivation and copulatory behavior in rats can be partly dissociated; while copulation does not occur in the absence of motivation, rats may approach a sexual incentive without engaging in copulatory behavior. In recent years, sexual motivation studies using Agmo's model in rats were reported by numerous researchers (Ågmo, 2002, Ågmo, 2003b, Ågmo, 2003a, Ågmo et al., 2004, Chu and Ågmo, 2008, De Gasperin-Estrada et al., 2008, Ellingsen and Ågmo, 2004, Hurtazo et al., 2008, Nocjar and Panksepp, 2007, Portillo and Paredes, 2004, Romero-Carbente et al., 2007, Spiteri and Ågmo, 2006, Viitamaa et al., 2006).
Many natural herbs purportedly enhance libido. One of such plants is an evergreen shrub or tree that grows in certain areas of East Africa and the Arabian Peninsula, commonly known as khat (Kalix, 1990). Khat (Catha edulis) belongs to the suborder Rosidae and family Celastraceae. Chewing its fresh leaves is a common practice during social gatherings in the local populations that often last for several hours (Al-Motarreb et al., 2002, Kassie et al., 2001). After the first few hours of consumption, users of khat demonstrate increased alertness, better self-esteem, augmented feeling of elation, enhanced imaginative ability, and improved capacity to relate ideas (Cox and Rampes, 2003). However, toward the end of the khat session, some users experience a depressive stage often manifested as irritability, anorexia, hyperthermia, insomnia, mydriasis, and endocrine disturbances (Al-Motarreb et al., 2002, Hassan et al., 2002).
Khat chewing has been reported to affect both male and female sexual behavior. Studies have shown that khat possessed aphrodisiac activities (Bentur et al., 2008, Giannini et al., 1992, Krikorian, 1984), and could be used to treat premature ejaculation (Luqman and Danowski, 1975). Likewise, it increased sexual desire in females (Elmi, 1983). On the other hand, loss of libido (Kervingant, 1959 , Krikorian, 1984), impairment of sexuality (Chanoit et al., 1980, Halbach, 1972), inability to sustain erection (Elmi, 1983), and spermatorrhea due to khat chewing have also been documented (Elmi, 1983, Granek et al., 1988, Halbach, 1972).
There are also contradicting reports on chronic uses of khat. Dalu (2000) reported that long-term and regular consumption of khat may lead to progressive and diminished sex performance and sexual impotence in male. El-Shoura et al. (1995) reported that khat chewing adversely affected semen volume, sperm count, sperm motility, motility index, and percentage of normal spermatozoa in humans and resulted in abnormal sperm morphology. In contrast, a more recent study using rabbits showed that khat stimulated spermatogenesis, and had no deleterious effect on the testis (Al-Mamary et al., 2002). Furthermore, Adeoya-Osiguwa and Fraser (2005) documented the effects of cathine and norephedrine on mouse and human sperms, and that they were capable of stimulating the final stage of sperm capacitation. In addition, they suggested that moderate levels of cathine and norephedrine, especially in the female reproductive tract, could have a positive effect on the natural fertility. It is also reported that countries where khat use has a deep-rooted cultural tradition have high population growth rates (Al-Hebshi and Skaug, 2005).
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In view of the prevalence of sexual dysfunction in female, this part of study aimed to investigate the effect of khat alkaloid on the sexual motivation vaginal secretions and estradiol levels of female rats, with special emphasis on the importance of the sustained release effect.
5.2 MATERIALS AND METHODS
Isolated alkaloid (cathine and norephedrine) of khat extract were prepared as previously described in chapter 2, section2.?.?. Khat extract microcapsules containing cathine and norephedrine (KE235), norpseudoephedrine-HCl microcapsules (NPE235), norephedrine-HCl microcapsules (NE112), and microcapsules containing both norpseudoephedrine-HCl and norephedrine-HCl at ratios of 1:1 (NPENE235-11), 2:1 (NPENE235-21) and 4:1 (NPENE235-41) were previously prepared in chapter 3.
The female rats (Sprague Dawley) used in this study were obtained from the animal house at the Universiti Sains Malaysia, Penang, Malaysia. The rats were housed in cages containing wood shavings as bedding and were fed with commercial pellets and water under a reversed light-dark cycle. Ambient temperature was maintained at 28 Â± 1Â°C, and relative humidity was 72 Â± 2%. All the animal studies were approved by the Animal Ethics Committee of the School of Pharmaceutical Sciences, Universiti Sains Malaysia.
5.2.3 SEXUAL MOTIVATION STUDY
5.2.3 (a) Determination of the estrous cycle of female rats
The vaginal secretions of 50 female rats (5-10 female rats per day) were collected every morning (7 am) and examined microscopically using a light microscope (Leica DMLB). The characteristics of the four phases (proestrus, estrus, metestrus, and diestrus) of the estrous cycle of the rats was determined by the proportion of the epithelial cells, cornified cells, and leukocytes in the vaginal smear (Marcondes et al., 2002). A female rat that had proestrus phase in the morning was marked and selected for the sexual motivation test at 2 pm of the same day. A total of 30 female rats of 4-6 months old, weighing 200-230 g, were selected for this part of study.
5.2.3 (b) Animal treatment groups
The selected female rats were randomly assigned to one of the following groups (N = 6 rats per group): Group I received 2 mL of 5% carboxymethylcellulose (CMC) suspension and served as the control; Groups II, III, and IV received 2 mL of 5% CMC suspension containing 10, 20, and 50 mg/kg khat extracts as low, medium, and high doses, respectively. Group V received 2 mL of 5% CMC suspension containing khat extract microcapsules (equivalent to 20 mg/kg khat extract). The various suspensions were given using metal oral feeding needles connected to 5-mL disposable plastic syringes.
5.2.3 (c) Arena cage
The method used in the sexual motivation test was a modified procedure of Ågmo (2003b). As shown in Figure 1, the testing arena prepared was oval in shape (100 cm length Ã- 50 cm width Ã- 45 cm high), with two chamber openings (25 cm length Ã- 15 cm width Ã- 25 cm high). The chamber front was made of wire mesh (1-mm wire, mesh size 12 mm Ã- 12 mm) that allowed experimental and incentive animals to see, smell, and hear each other. Although limited physical contact was possible through the mesh, copulatory interactions could not occur. A virtual area (30 Ã- 20 cm) adjacent to the incentive chamber was defined as the incentive zone. The wall and incentive chambers were made of 0.3-mm sheet metal zinc painted black on the inside wall. The upper surface of the removable arena woody floor was partitioned into squares of 5 cm2 each to facilitate ease of determination of the distance traveled by the female rats during the experiments. In addition, the arena floor also contained an incentive and nonincentive zones of 600 cm2 each. During the test, the arena was placed on the floor and an overhead video camera (Sony, DCR-TRV355E, Tokyo, Japan) was installed at a height of 2 m above the arena to capture all the activities of the rats.
Figure 5.1: A modified apparatus of Ågmo used in the sexual motivation test.
5.2.3 (d) Test procedures
During the test, one male rat was kept in the chamber that was fixed to the arena cage facing the incentive zone, while a female rat was kept in the other chamber facing the nonincentive zone. After receiving the test dose, each female rat under study was left in the arena for 10 minutes to acclimatize to the environment. Subsequently, the video camera was switched on for 60 minutes to record all the activities of the rat. After the completion of experiment, the recorded film was transferred to a computer. The time spent in the incentive and nonincentive zones and number of visits in incentive and nonincentive zones of the rats were recorded and calculated using the EthoLog software version 2.2, Behavioural Transcription Tool Sao Paulo, SP, Brazil (Ottoni, 2000).
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On the other hand, the total time and distance of movement of the rats recorded in the computer were calculated manually using Windows Media Player software and the removable arena floor. The total time of the distance moved by the rat on the removable arena floor was recorded by Windows Media Player software. The position of the rat and the distance traveled by the rat on removable arena floor was measured at every 5 seconds using a ruler.
5.2.3 (e) Measurements data
The behavioral measurements include preference score (time spent in the incentive zone/total time spent in the incentive and nonincentive zones), number of visits in incentive, and nonincentive zones, and the velocity (distance moved/time of moving). The behavioral coding was conducted by raters "blind" to the condition of the animals.
5.2.4 DETERMINATION OF ESTRADIOL AND VAGINAL SECRETIONS
5.2.4 (a) Determination of the estrous cycle of female rats
A separate group of 50 female rats were monitored for 1 month (before the experiments) to determine their natural estrous cycles microscopically as mentioned previously. The time and duration of the estrous cycle for each female rat were recorded. The female rats that had an estrous cycle in the last 10 days of the first month were selected (18 female rats, 7-10 months old, weighing between 190 and 250 g).
5.2.4 (b) Metabolism cages
In the second month, the selected female rats were housed separately in metabolism wire cages. Each cage was placed on top of a funnel device resting on a metal wire screen (mesh size <1 mm2) so that urine could be collected in a plastic vial while the fecal pellets were retained on the wire screen. Feeding and watering compartments of the metabolism cage were incorporated in such a way that food and water would not contaminate the urine and fecal matters. Each female rat under study was left in the metabolism cage for 24 hours (first day) to acclimatize to the environment.
5.2.4 (c) Animal treatment groups
The female rats were randomly divided to three groups. Group A received 2 mL of 0.5% CMC as the control, Group B received 2 mL of 0.5% CMC containing 20 mg/kg khat extract, and Group C received 2 mL of 0.5% CMC containing khat extract microcapsules (equivalent to 20 mg/kg khat extract). All the test samples were administered orally starting from the first day of the second month in the morning at 7 am for 30 days using metal feeding needles. The body weights of female rats were recorded every 4 days.
5.2.4 (d) Urine collection
During the last 10 days of the study, total urine samples from each rat were collected twice daily at 7 am and 7 pm. The urine samples were centrifuged at 3,500 rpm for 10 minutes to remove the precipitated residue. The volume and the pH of the supernatant portions were determined before storing at -20Â°C until use.
5.2.4 (e) Determination of vaginal secretion's volume and pH
Vaginal secretions of the female rats were examined twice daily at 7 am and 7 pm during the last 10 days of treatment. A preweighed sterilized cotton swab (W1) was used to swab the rat's vagina in a circular motion for 5 seconds, then removed and weighed (W2). Vaginal secretion was assumed to be proportional to the weight of the fluid absorbed by the cotton swab and expressed as the difference in weights of W2 and W1. Microscopic examinations of the vaginal cells were made twice daily. Finally, the resultant cotton swab was soaked in 2 mL of distilled water. After shaking for 5 minutes, the pH was recorded.
5.2.4 (f) Determination of estradiol levels
Estradiol levels in the urine samples were determined using an AxSYM Random Access Immunoassay Analyzer (Abbott Laboratories, Abbott Park, IL, USA) with a microparticle enzyme immunoassay reagent kit. This procedure was carried out at the LAM WAH EE Hospital, Penang, Malaysia. According to the information provided by the manufacturer, the antibody used for the assay of estradiol was highly specific for 17-Î² estradiol, with relatively low cross-reactivity to other naturally occurring steroids.
5.2.5 STATISTICAL ANALYSIS
Each of the results produced: preference score, time spent in incentive zone, time spent in non-incentive zone, number of visits in incentive zone, number of visits in non-incentive zone, distance moved, time of moving, velocity of movement, body weight, estradiol level, vaginal secretions weight, vaginal secretions pH, urine volume, and urine pH were statistically analyzed using one way analysis of variance (SPSS software, Version 13, SPSS Inc., Chicago, IL, USA). When there was a statistically significant difference, post hoc Tukey Honestly Significant Difference (Tukey-HSD) test was performed. A statistically significant difference was considered when P < 0.05.
5.3 RESULTS AND DISCUSSION
5.3.1 Sexual Motivation
In general, all the female rats under study showed preference scores larger than 0.5, stayed a significantly longer time, and made more visits in the incentive zone than the nonincentive zone. The preference score, time spent in incentive zone, and numbers of visits in incentive zone were affected by all the treatments, as shown in Table 5.1 and Figures 5.2-5.7. Post hoc Tukey-HSD test showed that the preference score, time spent in incentive zone, and number of visits in incentive zone of rats receiving the control and 10, 20, and 50 mg/kg khat extracts belonged to the same subset, while rats receiving khat extract microcapsules belonged to another subset. Rats receiving khat extract microcapsules also showed greater mean velocity of movements to reach their partners than the other groups (Table 5.1). In contrast, the time spent in nonincentive zone, number of visits in nonincentive zone, distance moved, and time of moving were unaffected by all the treatments (Table 5.1). Preference score is a good indicator of the intensity of sexual motivation in animal studies. Changes in preference score between one dose and another become meaningful only when combined with data on changes in time spent in the incentive and nonincentive zones (Ellingsen and Ågmo, 2004).
At the end of the 1-month test period, the body weight of female rats in Group A (control group) increased by 9%, but those in Group B (khat extract) and Group C (khat extract microcapsules) decreased by 12% and 7%, respectively. The mean body weight of the control group was significantly different from the khat extract and khat extract microcapsule groups (P < 0.001). This finding is in agreement with previous workers. They documented that chronic consumption of khat reduced body weight (10%) in human and baboon (Al-Dubai et al., 2006, Mwenda et al., 2006).
Female rats in the treatment and control groups produced similar amounts of urine that did not differ in pH (P > 0.05). However, the group given khat extract microcapsules did have an increase in the weight of vaginal secretions and decrease in pH during the estrous cycle compared with the other groups (Table 5.2 and Figures 5.8, 5.9 & 5.10). During the estrous cycle for all the female rats, estradiol levels in the urine were significantly higher at the proestrus phase than the other phases. Additionally, rats given khat extract microcapsules also showed statistically higher estradiol levels than rats given khat extract or control (Table 5.2 and Figures 5.8, 5.9 & 5.10).
The present study showed that the acute treatment with 50 mg/kg as high dose of khat extract increased the activity of female rats when compared with the lower doses (10 and 20 mg/kg) but did not increase sexual motivation-i.e., female rats given the lower doses of khat extract spent almost similar amount of time in both the incentive and nonincentive zones as the higher doses.
These results suggested that if any effect of repeated administration of lower doses of khat extract on sexual motivation existed, it could have been masked by increased side effects related to the general activity elevations (Banjaw and Schmidt, 2005). Most female rats receiving 50 mg/kg of khat extract experienced tremors, twitching of the mouth and nose regions. On the contrary, female rats given khat extract microcapsules exhibited lesser side effects but enhanced sexual motivation.
Numerous studies investigated the effects of khat in male rats and mice (Abdulwaheb et al., 2007, Banjaw et al., 2003, Connor et al., 2002, Nyongesa et al., 2007). Recently, Abdulwaheb et al. (2007) reported that low doses of khat extract (100-200 mg/kg/day) exerted enhanced sexual motivation/arousal, while high doses of the khat extract (400 mg/kg/day) inhibited sexual motivation/arousal and performance in male rats. However, the amount of fresh khat leaves normally consumed by an average habitual human user is about 100-200 g (equivalent to approximately 2-4 g of dried khat extract) (Kalix, 1990).
Taking the average adult male body weight of 70 kg, the amount of khat extract consumed would be approximately 30 to 60 mg/kg. The 10 to 50 mg/kg khat extract used in this study was closer to the human range and was lower than the low dose of 100 mg/kg given to the animals in previous studies. It is apparent that the effect of khat on sexual motivation is not directly correlated to the amount of khat consumed, but more importantly to the suitable amount of khat ingested.
The mode of khat administration influences the absorption and the severity of its side effects (Toennes et al., 2003). Typically, khat leaves are ingested in two ways. The first entails chewing several leaves in the buccal cavity and swallowing the juice slowly. The second involves slow consumption of leaves by masticating each thoroughly during the 3-4 hours of khat session (Al-Hebshi and Skaug, 2005, Kalix, 1990). In some African countries, such as Ethiopia and Somalia, the mode of consumption is slightly different but similarly aimed at obtaining a sustained release by completely chewing the soft stem with young leaves and then swallowing the juice (around 15 minutes); the stem and leaf residues are then ejected from the mouth and other new stem/leaves are chewed. Comparing with the normal mode of khat administration, bolus administration of khat extract to the animals does not simulate closely the preferred khat chewing performed by khat users. Additionally, bolus administration may induce too many negative side effects, which would cover up/interfere any potential pro sexual effects.
Studies on the pharmacokinetic of khat chewing reported that cathine/norephedrine has a slower onset of action, with a serum half-life in humans of about 3 hours. It is excreted unchanged in the urine within about 24 hours (Cox and Rampes, 2003, Toennes et al., 2003). This property may explain why the sustained release of khat alkaloids facilitates better absorption into the systemic circulation to exert its effects. The gastrointestinal transit time can be controlled by the gelatin coating in the microcapsules due to its mucoadhesive property (Wang et al., 2000). As the microcapsules move through the gastrointestinal tract, gelatin coatings of the microcapsules are able to retain the particles longer in the upper gastrointestinal tract due to their mucoadhesive property, leading to improved absorption profile. It is well-known that gelatin microcapsules are useful to achieve the sustained release of many drugs (Chang et al., 2006, Lu et al., 2007, Pamujula et al., 2004, Tsuyoshi et al., 1987). Gelatin not only displays mucoadhesive properties, but also acts as an absorption enhancer (Wang et al., 2002). As the microcapsules adhered onto the gastrointestinal mucosal membrane, the concentration of the khat alkaloids around the adhesion site was increased, thus led to a concentration gradient and better absorption potential. The increased amounts of vaginal secretions accompanied by decreased vaginal pH seen in the present study may also be contributed by khat extract microcapsules-induced elevation in estradiol.
Hormones play a significant role in regulating female sexual function. Estradiol levels affect cells in both the peripheral and central nervous system and influence nerve transmission. In animal models, a decreased circulating level of estrogen following bilateral ovariectomy decreased vaginal lubrication (Min et al., 2002, Min et al., 2003). Ovariectomy caused significant vaginal tissue atrophy with diminished vaginal fluid transudate secretion in the basal state. Conversely, estrogen administration to ovariectomized animals restored vaginal lubrication (Min et al., 2002). Moreover, estrogen relieves symptoms of vaginal dryness and burning when applied topically or locally (Ayton et al., 1996). Female genital sexual arousal is a physiological process modulated by psychosocial factors, hormonal milieu, and neurovascular input. It involves vascular and nonvascular smooth muscle relaxation, which affects vaginal lubrication and pH (Min et al., 2002). Vaginal atrophy can occur with any estrogen-deficient state. Low estrogen conditions can decrease normal flora (lactobacilli) and elevate vaginal pH (Lipsky et al., 2003).
Although the peripheral effects of estradiol may be important for sexual arousal disorder, the sexual motivation effect of khat alkaloid is more likely due to their effects on the central nervous system. It was documented that khat alkaloids may exert their effects based on two main neurochemical pathways, namely dopamine and noradrenalin (Cox and Rampes, 2003). Khat extract also appeared to be associated directly or indirectly with either dopamine or serotonin release or transporter function (Kalix, 1992). Kalix and Braenden (1985) suggested that khat alkaloids induce the release of dopamine from the central nervous system dopamine terminals, resulting in an increase in the activity of the dopaminergic pathways. In addition, these alkaloids increase the release of noradrenalin from its storage sites and facilitate noradrenalin transmission. Drake (1988) further proposed that the effects of khat alkaloids are caused by an inhibition of noradrenalin uptake. Other researchers attempted to identify a mechanism of action of khat alkaloids in somatic cells focused mainly on responses in the central nervous system. Khat alkaloids were shown to cause a blockage of noradrenaline transporter (Cleary et al., 2002, Cleary and Docherty, 2003).
The study from Abdulwaheb et al. (2007) revealed that low doses of khat extract exerted enhanced sexual motivation/arousal in sexually experienced animals, while high doses produced opposite effects on both sexual motivation/arousal and performance. They postulated that alteration of both dopamine (at low dose) and/or serotonin (at high dose) levels in the CNS could explain the biphasic sexual behavior of rats after khat administration. Our result further supported that low dose of khat alkaloids released in a sustained manner from the khat extract microcapsules were able to enhance sexual motivation in the female rats.
The sustained release of khat alkaloid from microcapsules enhanced sexual motivation, up-regulated estradiol, and increased vaginal secretions in female rats when compared with non-encapsulated bolus administration of khat extract. The administration of khat extract microcapsules mimics the way of khat use by chewing. In conclusion, the results of the present study indicate that the khat extract microcapsules prepared from dried khat alkaloid exhibited great potential in enhancing libido in females.