In this study, we aimed to assess the effect and possible underlying mechanism of
Stress is regarded as one of the important factors of sexual dysfunction and infertility. It has been reported that stress can induce male sexual disturbances expressed as erectile dysfunction, ejaculatory disorders, loss of libido and a decrease in the frequency of intercourse [
Repeated exposure to immobilization/restraint stress has been recognized as the one of most commonly used stress inducer models in rodents and can produce the deleterious effects on numerous systems including sexual dysfunction as those observed in humans [
The current medication therapeutic strategies still focus only on the improvement of HPG-axis. In addition, most of them are expensive and produce serious side effects [
The fresh leaves of
Healthy, sexually mature, male Wistar rats weighing 250-350 g and female rats weighing 200-250 g were obtained from National Laboratory Animal Center, Salaya, Nakhon Pathom province, Thailand. They were housed in group of 6 per cage in standard metal cages at 24±2°C on 12:12 h light-dark cycle. All animals were given access to food and water
Total 42 male rats (n=6/group) were randomly divided into various groups as described below. Group I: Naïve control (nonstress group): rats were not given any substance. Group II: Vehicle plus stress: vehicle (distilled water) was administered to all rats at 45 minutes before the exposure to 12-hour-restraint stress exposure. Group III: Sildenafil: this group was served as positive control based on the sexual enhancing effect of Sildenafil citrate. Sildenafil citrate at dose of 5 mg/ kg was administered to the stress exposed rats at 45 minutes prior to the copulatory study. Group IV: Tianeptine: animals in this group were also used as positive control based on its antistress effect. All stress exposed rats were given Tianeptine at dose of 15 mg/ kg at 45 minutes prior to the copulatory study. Groups V-VII:
In this study, all assigned substances were orally given and stress exposure was performed by restraining rat for 12 hours (Based on the pilot data which demonstrated that this duration induced sexual dysfunction together with the decreased testosterone level and spermatogenesis). The treatments and the stress exposure were carried out once daily at a period of 14 days. Following stress exposure, the animals were subjected to 3-hour-refreshment period before subjecting to the sexual behavior evaluation. The sexual behaviors assessments were performed by an experienced observer blind to treatment at room temperature between 9.00 p.m. to 12.00 a.m. after the single intervention and 7 and 14 days of treatment.
In order to assess the sexual behaviors, estrous female rats were paired with male treated with the assigned substances. Female rats were induced to estrous by sequential administration of estradiol benzoate (Sigma, St. Louis, MO) at dose of 2
Mounting frequency: the frequency of mounts without intromission from the time of introduction of the female until ejaculation.
Intromission frequency: the frequency of intromissions from the time of introduction of the female until ejaculation.
Mount latency: the time interval between the introductions of the female to the first mount by the male.
Intromission latency: the interval from the time of introduction of the female to the first intromission by the male.
Ejaculation frequency: the frequency of ejaculation which characterized by longer, deeper pelvic thrusting, and slow dismount followed by a period of inactivity.
Ejaculation latency: the time interval between the first intromission and ejaculation [
A 12-hour-stress exposure was carried out between 6.00 a.m. to 6.00 p.m. for 14 days. In brief, all animals were placed in a transparent perforated plastic tube, 20 cm long, and 7 cm in diameter and the tubes were closed with Plexiglas lids. The animals fit tightly into the restrainers so it was not possible for them to turn around. The induction of stress was performed at the same period every day throughout the study period. Naïve control rats were maintained at room temperature with free access to food and water and were not subjected to any procedure until the experiment.
The determination of PDE-5 was carried out by using PDE-Glo™Phosphodiesterase Assay kit (Promega). The penis was isolated and prepared as homogenate by using lysate RIPA buffer. Then, the homogenate was subjected to a 14,000
At the end of experiment, all rats were killed to determine monoamine oxidase type B activity in medial preoptic area (MPOA) and nucleus accumbens (NAc) by using the peroxidase-linked photometric assay [
The venous blood of each animal was collected from tail vein and kept on ice. Then, it was subjected to a 2000 g-centrifugation at 4°C for 15 minutes. The obtained serum was kept at −80°C until used. Testosterone levels were measured using a radioimmunoassay (RIA) kit (TESTO-CT2, Cisbio International, France) whereas corticosterone levels were measured using Corticosterone Double Antibody Radioimmunoassay Kit (MP Biomedicals) for the quantitative determination of corticosterone in rat and mice serum. The amount of labeled testosterone bound to the antibody was inversely related to the amount of unlabeled testosterone present in the sample. The remaining radioactivity bound to the tube was measured with Gamma scintillation counter which calibrated for Iodine-125. The results were showed as ng/ml.
The brains were removed and fixed with 4% paraformaldehyde overnight and then postfixed with cryoprotective solution (30% sucrose in 0.1 M PBS) until they were sunk. Brain coronal sections containing nucleus accumbens and ventral tegmental area (VTA) at 10
The expression of endothelial nitric oxide synthase (eNOS) in penis was assessed via western blot assessment. In brief, homogenate of penis was prepared by using RIPA buffer with protease inhibitors. Then, the homogenate was subjected to a 14,000 g-centrifugation at 4°C for 20 minutes. The supernatant was collected and measured the level of protein by using NANO drop Spectrophotometers. Equal amounts of protein (100
The testes were removed, fixed with 4% paraformaldehyde for 24 hr, and postfixed with 30% sucrose. They were prepared as a 10
All data were expressed as mean ± SEM value. The significant difference among various groups was evaluated by ANOVA and followed by LSD test by using the SPSS software package for Windows. The statistical difference was regarded at p-value < 0.05.
The effects of
The effect of hydroalcoholic extracts of
The effect of hydroalcoholic extracts of
The effect of hydroalcoholic extracts of
After the single administration, the statistically significant differences between groups in mount and intromission latencies [(
When the treatment was prolonged to 14 days of treatment, data obtained from ANOVA analysis showed the significant differences between groups in mount latency (
The effect of
The effect of hydroalcoholic extracts of
The effect of
The effect of hydroalcoholic extracts of
Based on the crucial role of PDE-5 on penile erection [
The effect of hydroalcoholic extracts of
Since dopaminergic system plays an essential role on male sexual behaviors, the effect of
The effect of hydroalcoholic extracts of
Figure
The effect of hydroalcoholic extracts of
Based on the increased dopaminergic function in NAc in
The effect of hydroalcoholic extracts of
The effect of extract on TH-positive neuronal cell density in ventral tegmental area (VTA) was also assessed due to its critical role on the regulation of male sexual behaviors [
The effect of hydroalcoholic extracts of
Figure
The effect of hydroalcoholic extracts of
The current data is the first study which demonstrated the modulation effect of
Sexual arousal also contributes an important role on male sexual behavior especially in precopulatory behavior. The mesocorticolimbic DA tract which ascends from the ventral tegmental area (VTA) to the nucleus accumbens (NAc) and prefrontal cortex is reported to play a critical role on reinforcement and appetitive behaviors. However, various subregions of NAc exert different roles. The nucleus accumbens core (NAcC) is involved in the cognitive processing of cognitive processing of motor function related to reward and reinforcement whereas the nucleus accumbens shell (NAcS) is involved sexual performance, reward-related processing, and the inhibition of sexual behavior after ejaculation [
Copulatory behavior is also under the influence of testosterone. Testosterone especially metabolites of testosterone are essential for the dopaminergic function especially in MPOA. The low level of DA appears to exert disinhibition of genital reflex via the increase in parasympathetic nervous system leading to penile erection whereas the high dose of DA increase seminal emission and ejaculation [
Under normal circumstance, dopaminergic system plays the crucial role on the regulation of male sexual behavior [
This study failed to show the dose dependent manner because all observed parameters were associated with the multifactor and the masking effect of inactive ingredients could also contribute the role. Based on the pivotal roles of polyphenol on the suppression of MAO-B [
Taken all data together, sexual enhancing effect of
Schematic diagram showed the possible underlying mechanism of
The data used to support the findings of this study are available from the corresponding author upon request.
All authors declare no conflicts of interest.
This study was supported by the National Research Council of Thailand (NRCT), Invitation Research Grant of Faculty of Medicine, Khon Kaen University, and Integrative Complementary Alternative Medicine Research and Development Center, Khon Kaen University. Moreover, the authors also would like to express their sincere thanks to Associate Professor Panee Sirisa-ard for her authentication.