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Pharmacological profiling of Argemone mexicana for its aphrodisiac potentials in male Wistar rats
110
Asian Pacific Journal of Reproduction 2014; 3(2): 110-115
Asian Pacific Journal of Reproduction Journal homepage: www.apjr.net
Document heading
doi: 10.1016/S2305-0500(14)60013-0
Pharmacological profiling of Argemone mexicana for its aphrodisiac potentials in male Wistar rats 1
2
2
2
3
4
Asuntha G , Prasanna Raju Y *, Harini Chowdary V , Vandana KR , Arun Rasheed , Prasad KVSRG Department of Pharmacy, S P W Polytechnic, Tirupati- 517502, India Pharmaceutics Division, Sree Vidyanikethan College of Pharmacy, A. Rangampet, Tirupati, 517102, India 3 Al-Shifa College of Pharmacy, Poonthavanam, 679325, India 4 Institute of Pharmaceutical Technology, Sri Padmavathi Viswavidyalayam, Tirupati- 51750, India 1 2
ARTICLE INFO
Keywords:
Sexual dysfunction Testosterone Protopine alkaloid Flavanoid Estrous cycle Sexual behavioral parameters
ABSTRACT Objective: To study the aphrodisiac potentials of ethanol extract of Argemone mexicana L. (A. mexicana) of Papaveraceae family in sexually sluggish male Wistar rats. Methods: The sexually inactive male rats were divided into two groups of 8 rats each. The test group animals were treated with ethanol extract of A. mexicana (EEAM) at 1 g/kg daily oral dose for 28 days. Other group animals were treated with sildenafil citrate at an oral dose of 5 mg/kg. The latencies of mount, intromission, ejaculation; post ejaculatory pause and frequencies of mount, intromission, and ejaculation were measured on 0, 7th, 14th, 21st and 28th days. Serum testosterone levels were estimated using ELISA. Results: The EEAM was nonlethal even at dose of 4.0 g/kg. The oral dosing of EEAM has significantly enhanced the orientation of males towards female by increase in ano-genital investigatory behavior, frequencies of mount, intromission, and ejaculation (P< 0.01). The latencies of mount, intromission and ejaculation were significantly decreased (P<0.05). The EEAM has produced marked variation in sexual behavior characteristics and was able to elevate the serum testosterone levels (P<0.01) on par to that of sildenafil citrate. Conclusion: The EEAM has elevated sexual dysfunctions in male rats. These potentials may be related to protopine alkaloids and flavanols by means of physiological stimulus for penile vasculature. Thus, results support the use of EEAM in enhancing sexual behavior in sluggish male rats.
1. Introduction H uman sexuality is a multi-faceted physiologic
phenomenon in touch with psychological, clinical, behavioral, moral and cultural insights. It has fascinated the people in all walks of life for ages. Sexual themes have been common in art and literature, and glorified in Hindu temples, praised poetically, condemned morally, and studied historically[1]. Human sexuality is well associated with socioeconomic development. As per WHO, a majority of the urban population in developed and developing *Corresponding author: Dr. Prasanna Raju Y, Professor, Sree Vidyanikethan College of Pharmacy, A.Rangampet, Tirupati, IN 517102. Tel: +91-9885729290 Fax: +91-8772248226 E-mail: [email protected]
countries is suffering with sexual disorders/dysfunctions.
Sexual dysfunction (SD), especially erectile dysfunction,
is a serious public health problem as reflected in epidemiological data[2]. This could be due to globalization, un-uniform urbanization and increased stress conditions. This may lead unhealthy social imbalance. Male SD affects not only sexual relationships, but also overall quality of life. Male SD is the most commonly prevailing among age group of 40–70 years worldwide, with a prevalence of more than 20%. In developed and urban areas, male SD is prevailing as high as 34.8%[3]. Male SD is characterized by erectile dysfunction and premature ejaculation. General medical illness, psychosocial factors, nonpsychiatric medication, psychiatric disorders and psychotropic medication would lead SD in male. The patients suffering with a primary
Asuntha G et al./ Asian Pacific Journal of Reproduction (2014)110-115
sexual disorder that predates their depression and in other cases, psychiatric illness (neurologic illness) and genitourinary trauma or infections cause SD. A classical drawing of mechanisms and hormonal involvement in SD is attributed as estrogen, testosterone and progesterone are physiological components in women sexual function and testosterone alone is significant in male[4]. Oxytocin is well related to orgasm and may influence feelings of desire. In addition to hormones, the neurotransmitters also have significant physiologic effect on sexual functioning. The effect that dopamine and norepinephrine appears to have on sexual functioning could explain the diminished feelings of desire and arousal experienced by patients with depression. Dopamine appears to be related to motivated behaviors which influences desire and may play a role in maintaining sexual focus. Norepinephrine provokes sexual arousal and vasocongestion. To avoid persistent arousal with nitric oxide (NO) release, serotonin (5-HT) systems are activated to suspend vasocongestion, thus turning off arousal. In addition, serotonin may diminish NO function and decrease genital sensation. Basic and clinical research advances in SD treatment have led to newer options such as therapeutic agents for intracavernous, intraurethral and oral use. Selective and reconstructive vascular surgery was codified in cases of arterial insufficiency, with no current indications for venous ligation procedures and resulted poor outcomes. Therefore, continuing research is required to explore safe and effective new drugs for male SD. Plants are reliable source and play a key role in the world of health. The use of plant materials is age old practice to treat SD in India and world as well. The medicinal plants and herbs have proven effective in sexual function in male animals. Aphrodisiacs are the substances, which can enhance sexual functions. This property has been attributed to many plants, animal products and synthetic compounds but none of these drugs are up to reputation. Hence, there is a need to search for potential and inexpensive drug candidates for the management of human sexuality problems. Argemone mexicana L. (A. Mexicana) of Papaveraceae family had antiinflammatory, expectorant, emetic, anthelmintic, antipyretic and sedative properties [5]. In our survey, it was observed that A. mexicana is used folklore of Chittoor district of Andhra Pradesh, India for aphrodisiac purpose. But, no systematic pharmacological studies were reported to support the aphrodisiac activity of A. mexicana. Hence, the present study explored the aphrodisiac efficiency of ethanol extract of A. mexicana in male Wistar rats.
2. Materials and methods
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2.1. Plant material The whole plant of A. mexicana was collected locally near S rinivasa M angapuram area, T irupati, C hittoor District, Andhra Pradesh, India in the month of April-June 2004 according to its seasonal availability. The plant was authenticated by Prof. T. Vedavathi (Retd.), Department of Botany, Sri Venkateswara University, Tirupati, India. A
voucher specimen was deposited at the herbarium of the Institute of Pharmaceutical Technology, Sri Padmavathi Mahila Viswavidyalayam, Tirupati, India (Ref: 01/AM/06IPT-SPMVV/TPT/2004). 2.2. Preparation of ethanol extract of A. mexicana (EEAM)
The dried, coarse powdered whole plant of A. mexicana was boiled in 1 000 mL of 95% ethanol under reflux by
(250 g)
heating over water bath and the process was continued with the same marc in three batches. The extracts were vacuum dried and the yield of the ethanol extract was 34.25% (w/w). The suspension of the ethanol extract was prepared with 2% Tween 80 before administration to animals. 2.3. Experimental animals Male albino rats of Wistar strain weighing 150-200 g were
used for the study. They were provided in polypropylene cages and standard laboratory conditions were maintained with a 12-12 h light-dark cycle along with free access to standard rat pellet diet (Lipton, India Ltd.) and drinking water. The animals were acclimatized to the laboratory conditions for ten days before commencing the experiment. The experimental protocol was endorsed by the institutional animal ethical committee (Ref. no: 1220/a/08/CPCSEA). 2.4. Acute toxicity studies Ethanol extract of A. mexicana (EEAM) used in this study was subjected to acute toxicity studies. The rats were fasted overnight with free access to drinking water and divided into six groups, each of six animals. Group 1 animals served as a control and received distilled water orally (2 mL/kg). Animals in groups 2 to 6 were administered with 0.25, 0.5, 1.0, 2.0 and 4.0 g/kg, respectively, of the EEAM extract by gastric intubation with a soft rubber catheter. The animals were kept under observation continuously for 2 h and then at onehourly interval until the twenty-fourth hour. Behavioral, neurological and autonomic profiles were recorded at these
112
Asuntha G et al./ Asian Pacific Journal of Reproduction (2014)110-115
points of time. The animals were observed for mortality up to the 48th h. 2.5. Experimental procedure Before the drug treatments, the male rats were given
training individually with normal adult female rat (in estrous cycle) for sexual experience. Then the male rats were divided into sexually active and sexually inactive groups, based on their copulatory behavior. A male was considered sexually active when it attempted to mount any female introduced into the cage. The average mountings in normal male rats were found to be 4-10 in 5 min. The animal with less than 4 mounts was considered inactive. Sexually inactive male rats were selected to conduct the study. The female sexual behavior is limited to the period of estrous, that coincides with ovulation and during which the animal is said to be in heat. The estrous female arouses sexual interest in male rat by physical changes in the genital region and the production of pheromones. These are sexual scents found in animals that produce dramatic sex-seeking behavior in animals[6]. The female rats respond to each mount with a lordosis response. This response occurs when the female is receptive to mounting male and consists of an arching of the back to a concave position with deviation laterally and the neck extended[7] . The female rat with estrous cycle was confirmed by vaginal smear method. A dropper with a drop of distilled water was introduced into the animal’s vagina and the secretions were collected and were tested under microscope. Estrous cycle was confirmed when 50% or more of the cells were cornified. The sexually inactive male rats were divided into two groups and each contains eight animals. One group was treated with sildenafil citrate (SC) (gift sample from Ranbaxy Laboratories, India) at a dose of 5 mg/kg (p.o.) and the second group was treated with EEAM at a dose of 1 g/kg (p.o.) daily for 28 days. The third group contains eight sexually active male rats was used as positive control. T he highly receptive female ( in estrous stage ) was introduced into the home cage of the male rats and the following male sexual behavioral parameters were recorded during a period of 30 min: Latency (time) of first mount, number of mounts, latency of first intromission, number of intromission, latency of ejaculation (time from intromission to ejaculation), number of ejaculations and post ejaculatory pause (time from ejaculation to next mount or intromission)[8, 9]. All the groups were tested for copulatory behavior on 0, 7th, 14th, 21st and th 28 days.
2.6. Estimation of serum testosterone Blood samples were collected from retro-orbital plexus before treatment, 30 min after administration of EEAM on th th st th 0 day, 7 , 14 , 21 , 28 days and 7 days after withdrawal of treatment. Serum was separated upon centrifugation at 3 0 0 0 rpm for 20 min [10, 11] . S erum testosterone concentrations were determined in triplicate by ELISA method using testosterone ELISA kit (Model ADI-901-065; ® Enzyo Life Sciences, India).
2.7. Statistical analysis The data were expressed as mean ± S.E.M. The results were statistically treated with one-way analysis of variance ( ANOVA ) followed by T ukey- K ramer test for multiple columns comparison. The statistically significance was set accordingly.
3. Results 3.1. Acute toxicity studies The oral administration of EEAM did not provoke any gross behavioral changes or manifestations of toxic symptoms such as increased or decreased motor activity, muscle relaxation, tremors, clonic convulsions, spasticity, loss of right reflex, tonic extensions, ataxia, lacrimation, salivation, weight loss, watery diarrhea, writhing and urination over a period of 48 h. Ethanol extract of A. mexicana was found to be non-lethal even at the maximum single dose of 4.0 g/kg. Hence, the dose of the EEAM selected for the study was 1 g/kg orally.
3.2. Sexual behavioral parameters Sexually active male rats were used as positive control to compare the effect of ethanol extract of A. mexicana (EEAM) in elevating the sexual behavior and characteristics in sluggish male rats. The results of EEAM were also compared with that of sildenafil citrate (SC), a well known aphrodisiac moiety. No negative control group was used, since two test group animals used were of sexually inactive. Overall, it was observed that the sexual behavior in sluggish male rats was elevated with EEAM treatment. Before start of the treatment, treated group animals have exhibited different mount and intromission latencies from control group animals (P<0.05). th At the end of the treatment period i.e. on 28 day, significant decrease in mount latency was noticed in both test groups compared to control group. Dissimilar reduction in mount latency was observed between two test groups soon after dosing as shown in Table 1. Treatment with EEAM and SC was resulted with delayed ejaculation latency in animals on th 7 day (P<0.05) as presented in Figure 1.
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Asuntha G et al./ Asian Pacific Journal of Reproduction (2014)110-115
Table 1 Effect of ethanol extract of A. mexicana (EEAM) on mount and intromission latencies. Treatment period
Latency Mount
Before treatment
Intromission Mount Intromission Mount Intromission Mount Intromission Mount Intromission Mount Intromission
0 day th
7 day th
14 day th
21 day st
28 day th
Control
Treatment group SC (5 mg/kg)
180.00依11.23
356.00依13.62
180.30依9.76
204.00依10.45
380.50依11.50
180.00依1.34
196.00依6.46
296.00依12.42
180.50依9.12
184.00依10.32
217.00依14.52
180.00依14.96
168.00依17.23
161.00依13.36
181.00依16.15
142.50依11.30
158.00依14.64
49.50依2.30
EEAM (1 g/kg) 70.72依8.70
71.83依5.50
*
49.10依10.96
380.00依19.36
53.66依6.66
49.50依2.34
62.50依3.50
50.50依3.76
49.16依9.36
49.00依9.73
39.66依6.32
49.00依8.82
43.62依8.19
35.33依16.65
49.00依8.08
36.66依4.61
28.10依 0.61
30.83依1.44
*
#
Values are expressed as mean 依 S.E.M., n=8, P<0.05 considered significant as compared to control, P<0.05 considered significant as compared to sildenafil citrate (SC). ##
Ejaculation latency(sec)
400
*
animals when compared to SC treated group animals and control group even after treatment on 7th day as depicted in Figure 2.
## *
300
100 Before treatment
0 day
7th day
14th day
21st day
28th day
Figure 1. Effect of ethanol extract of A. mexicana on delaying the
ejaculation latency.
Values are expressed as mean ± S.E.M. (n=8). *P<0.05 considered
significant as compared to control and ##P<0.01 considered significant as compared to SC.
The post ejaculatory pause was lasted for long time in both
test groups compared control group before the treatment. The pause was lasted for long time in EEAM treated group
Post ejaculatory pause(sec)
200
0
#
400 300
*
200
*
#
100 0
Before treatment 0 day
7th day
14th day
21st day
28th day
Figure 2. Effect of ethanol extract of A. mexicana on post ejaculatory
pause.
Values are expressed as mean ± S.E.M. (n=8). *P<0.05 considered
significant as compared to control and #P<0.05 considered significant as compared to SC.
Table 2
Effect of ethanol extract of A. mexicana (EEAM) on mount and intromission frequencies. Treatment period Before treatment 0 day th
7 day th
14 day th
21 day st
28 day th
Frequency Mount
Intromission Mount Intromission Mount Intromission Mount Intromission Mount Intromission Mount Intromission
Values are expressed as mean 依 S.E.M., n=8, P<0.05 and considered significant as compared to sildenafil citrate (SC). *
**
Control 13.00依1.60 10.00依0.24 12.57依2.90 9.66依0.526 13.00依3.52 10.90依0.45 12.50依2.90 10.10依0.56 12.80依1.81 10.50依1.03 13.00依0.83 10.30依0.34
Treatment group SC (5 mg/kg) 4.00依0.46 2.00依0.18 10.24依3.82 7.30依0.27 12.50依1.17 * 9.50依0.30 15.00依0.84 10.91依0.22 16.50依0.38 ** 13.90依0.52 23.40依4.31 15.10依0.32
EEAM (1 g/kg) 3.80依0.55 2.16依0.02 6.00依0.98 2.30依0.13 7.00依0.86 3.50依0.18 14.30依1.68 7.10依0.39 18.50依3.51 ## 10.00依0.32 21.50依3.52 # 12.60依0.42
P<0.01 considered significant as compared to control, #P<0.05 and ##P<0.01
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Asuntha G et al./ Asian Pacific Journal of Reproduction (2014)110-115
Before treatment, the sexually active group animals have
exhibited high mount frequency than sexually inactive group animals. The mount frequency was elevated in EEAM and SC treated sexually sluggish rats as shown in Table 2. Before starting and 0 day of the treatment, intromission frequencies were more in control group animals compared with that of test group animals. An increased intromission frequency was observed with SC treatment from 7th day onwards (P<0.05) and same time the intromission frequency was continued to be less in EEAM treated animals than that of with SC treatment even on 28th day as evidenced Table 2. During the study it was noticed that ejaculation frequencies in control and test group animals were found to be different before and after the treatment. 3.3. Serum testosterone levels T he serum testosterone levels in inactive rats were
significantly low compared positive control group active rats. Upon treatment with EEAM serum testosterone levels were continued to be lower on 0, 7th and 14th day than untreated animals. But, upon treatment with SC, serum testosterone levels were elevated. T he gradual elevation of serum testosterone levels was observed with EEAM treatment and the similar trend was lasted up to 7 days after withdrawal of the treatment. Figure 3 demonstrates that serum testosterone levels in both test group animals were significantly different on 14th day observations (P<0.01). The difference in serum testosterone levels of test group animals was gradually diminished and the similar effect was continued even after withdrawal of the treatment. Post ejaculatory pause(sec)
3
2.5
** ##
2
1.5 1
*
0.5
0 Before treatment 0 day
7th day
14th day
21st day
28th day
After withdrawal of treatment
Figure 3. Effect of ethanol extract of A. mexicana on serum testosterone levels. Values are expressed as mean ± S.E.M. (n=8). **P<0.01 considered significant as compared to control and ##P<0.01 considered significant as compared to SC.
4. Discussion Aphrodisiacs are substances that stimulate or increase sexual desire and performance. Sexual desire is controlled
and regulated by the central nervous system, which integrates olfactory, tactile, auditory, and mental stimuli. SD can occur even if sexual desire remains strong, because sexual performance depends on a neurovascular event via the hemodynamic mechanisms of penile erection. Sexual function is said to be a sequence of physiological responses to stimulation which involves increasing vasocongestion and myotonia tumescence, subsequent release of the vascular activity, muscle tone and experience four phases of sexual response cycle[12]. Latency for mount and intromissions are indicators of sexual motivation. Number of intromission, number of ejaculations is considered for sexual performance and facilitation[13, 14]. As per the statistically significant results emanated, the ethanol extract of A. mexicana decreased the latency for mount and intromission and significantly increased the number of intromissions and ejaculations as compared to control group. As per the earlier phytochemical studies, EEAM contains protopine alkaloids, flavanols, phenolics and fatty acids[15]. Flavanoids have been shown to be responsible for endothelium-dependent nitric oxide-mediated relaxation. Nitric oxide is a major physiological stimulus for penile vasculature and trabecular smooth muscles, all essential for penile erection. It is also assimilated that EEAM stimulate testosterone production and thereby maintain and improve reproductive health and quality of life. The present study shows for the first time that A. mexicana is able to significantly increase testosterone levels in male rats treated with the EEAM daily for 28 days, in parallel with the elicitation of an improved copulatory activity. Testosterone seems to be involved in the complex mechanism regulating the copulatory behavior by acting both peripherally and centrally in concert with other determinants. Certain plants were proven to have improved sexual function with increased levels of testosterone[16, 17]. Testosterone may facilitate male sexual behavior by increasing dopamine release and thereby potentiating neurotransmission[18]. Hence the ethanol extract of A. mexicana increases the sexual motivation, desire, potency, performance and facilitation to meet the cardinal phases of sexuality. Sexual arousal and functions are related to hormonal function. Testosterone in male and estrogen in female have the most marked effect on human sexuality. The present findings provide experimental evidence for aphrodisiac potentials of A. mexicana. The results of present work demonstrated that the ethanol extract of A. mexicana has significant ability in enhancing copulatory ability and sexual performance in sexually sluggish animals. Ethanol extract of A. mexicana has capability to increase testosterone levels
Asuntha G et al./ Asian Pacific Journal of Reproduction (2014)110-115
in sexually impaired male rats and it remained unaffected even after withdrawal of treatment. The ethanol extract of A. mexicana has similar potentials of SC with slow onset of action. Further studies are required to explore the chemical constituents responsible for the aphrodisiac activity and also to establish the mode of action. Conflict of interest statement The authors report no declarations of interest.
References [1] Devendra KS, Vikas S, Nagendra SC, Dixit VK. Effect of ethanolic extract of Paederia foetida Linn. leaves on sexual behavior and
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[8] C hauhan NS , S harma V , D ixit VK . E ffect of A steracantha longifolia seeds on sexual behaviour of male rats. Nat Prod Res
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[11]Sharma V, Thakur M, Dixit VK. A comparative study of ethanolic
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Asian Pacific Journal of Reproduction 2014; 3(2): 110-115
Asian Pacific Journal of Reproduction Journal homepage: www.apjr.net
Document heading
doi: 10.1016/S2305-0500(14)60013-0
Pharmacological profiling of Argemone mexicana for its aphrodisiac potentials in male Wistar rats 1
2
2
2
3
4
Asuntha G , Prasanna Raju Y *, Harini Chowdary V , Vandana KR , Arun Rasheed , Prasad KVSRG Department of Pharmacy, S P W Polytechnic, Tirupati- 517502, India Pharmaceutics Division, Sree Vidyanikethan College of Pharmacy, A. Rangampet, Tirupati, 517102, India 3 Al-Shifa College of Pharmacy, Poonthavanam, 679325, India 4 Institute of Pharmaceutical Technology, Sri Padmavathi Viswavidyalayam, Tirupati- 51750, India 1 2
ARTICLE INFO
Keywords:
Sexual dysfunction Testosterone Protopine alkaloid Flavanoid Estrous cycle Sexual behavioral parameters
ABSTRACT Objective: To study the aphrodisiac potentials of ethanol extract of Argemone mexicana L. (A. mexicana) of Papaveraceae family in sexually sluggish male Wistar rats. Methods: The sexually inactive male rats were divided into two groups of 8 rats each. The test group animals were treated with ethanol extract of A. mexicana (EEAM) at 1 g/kg daily oral dose for 28 days. Other group animals were treated with sildenafil citrate at an oral dose of 5 mg/kg. The latencies of mount, intromission, ejaculation; post ejaculatory pause and frequencies of mount, intromission, and ejaculation were measured on 0, 7th, 14th, 21st and 28th days. Serum testosterone levels were estimated using ELISA. Results: The EEAM was nonlethal even at dose of 4.0 g/kg. The oral dosing of EEAM has significantly enhanced the orientation of males towards female by increase in ano-genital investigatory behavior, frequencies of mount, intromission, and ejaculation (P< 0.01). The latencies of mount, intromission and ejaculation were significantly decreased (P<0.05). The EEAM has produced marked variation in sexual behavior characteristics and was able to elevate the serum testosterone levels (P<0.01) on par to that of sildenafil citrate. Conclusion: The EEAM has elevated sexual dysfunctions in male rats. These potentials may be related to protopine alkaloids and flavanols by means of physiological stimulus for penile vasculature. Thus, results support the use of EEAM in enhancing sexual behavior in sluggish male rats.
1. Introduction H uman sexuality is a multi-faceted physiologic
phenomenon in touch with psychological, clinical, behavioral, moral and cultural insights. It has fascinated the people in all walks of life for ages. Sexual themes have been common in art and literature, and glorified in Hindu temples, praised poetically, condemned morally, and studied historically[1]. Human sexuality is well associated with socioeconomic development. As per WHO, a majority of the urban population in developed and developing *Corresponding author: Dr. Prasanna Raju Y, Professor, Sree Vidyanikethan College of Pharmacy, A.Rangampet, Tirupati, IN 517102. Tel: +91-9885729290 Fax: +91-8772248226 E-mail: [email protected]
countries is suffering with sexual disorders/dysfunctions.
Sexual dysfunction (SD), especially erectile dysfunction,
is a serious public health problem as reflected in epidemiological data[2]. This could be due to globalization, un-uniform urbanization and increased stress conditions. This may lead unhealthy social imbalance. Male SD affects not only sexual relationships, but also overall quality of life. Male SD is the most commonly prevailing among age group of 40–70 years worldwide, with a prevalence of more than 20%. In developed and urban areas, male SD is prevailing as high as 34.8%[3]. Male SD is characterized by erectile dysfunction and premature ejaculation. General medical illness, psychosocial factors, nonpsychiatric medication, psychiatric disorders and psychotropic medication would lead SD in male. The patients suffering with a primary
Asuntha G et al./ Asian Pacific Journal of Reproduction (2014)110-115
sexual disorder that predates their depression and in other cases, psychiatric illness (neurologic illness) and genitourinary trauma or infections cause SD. A classical drawing of mechanisms and hormonal involvement in SD is attributed as estrogen, testosterone and progesterone are physiological components in women sexual function and testosterone alone is significant in male[4]. Oxytocin is well related to orgasm and may influence feelings of desire. In addition to hormones, the neurotransmitters also have significant physiologic effect on sexual functioning. The effect that dopamine and norepinephrine appears to have on sexual functioning could explain the diminished feelings of desire and arousal experienced by patients with depression. Dopamine appears to be related to motivated behaviors which influences desire and may play a role in maintaining sexual focus. Norepinephrine provokes sexual arousal and vasocongestion. To avoid persistent arousal with nitric oxide (NO) release, serotonin (5-HT) systems are activated to suspend vasocongestion, thus turning off arousal. In addition, serotonin may diminish NO function and decrease genital sensation. Basic and clinical research advances in SD treatment have led to newer options such as therapeutic agents for intracavernous, intraurethral and oral use. Selective and reconstructive vascular surgery was codified in cases of arterial insufficiency, with no current indications for venous ligation procedures and resulted poor outcomes. Therefore, continuing research is required to explore safe and effective new drugs for male SD. Plants are reliable source and play a key role in the world of health. The use of plant materials is age old practice to treat SD in India and world as well. The medicinal plants and herbs have proven effective in sexual function in male animals. Aphrodisiacs are the substances, which can enhance sexual functions. This property has been attributed to many plants, animal products and synthetic compounds but none of these drugs are up to reputation. Hence, there is a need to search for potential and inexpensive drug candidates for the management of human sexuality problems. Argemone mexicana L. (A. Mexicana) of Papaveraceae family had antiinflammatory, expectorant, emetic, anthelmintic, antipyretic and sedative properties [5]. In our survey, it was observed that A. mexicana is used folklore of Chittoor district of Andhra Pradesh, India for aphrodisiac purpose. But, no systematic pharmacological studies were reported to support the aphrodisiac activity of A. mexicana. Hence, the present study explored the aphrodisiac efficiency of ethanol extract of A. mexicana in male Wistar rats.
2. Materials and methods
111
2.1. Plant material The whole plant of A. mexicana was collected locally near S rinivasa M angapuram area, T irupati, C hittoor District, Andhra Pradesh, India in the month of April-June 2004 according to its seasonal availability. The plant was authenticated by Prof. T. Vedavathi (Retd.), Department of Botany, Sri Venkateswara University, Tirupati, India. A
voucher specimen was deposited at the herbarium of the Institute of Pharmaceutical Technology, Sri Padmavathi Mahila Viswavidyalayam, Tirupati, India (Ref: 01/AM/06IPT-SPMVV/TPT/2004). 2.2. Preparation of ethanol extract of A. mexicana (EEAM)
The dried, coarse powdered whole plant of A. mexicana was boiled in 1 000 mL of 95% ethanol under reflux by
(250 g)
heating over water bath and the process was continued with the same marc in three batches. The extracts were vacuum dried and the yield of the ethanol extract was 34.25% (w/w). The suspension of the ethanol extract was prepared with 2% Tween 80 before administration to animals. 2.3. Experimental animals Male albino rats of Wistar strain weighing 150-200 g were
used for the study. They were provided in polypropylene cages and standard laboratory conditions were maintained with a 12-12 h light-dark cycle along with free access to standard rat pellet diet (Lipton, India Ltd.) and drinking water. The animals were acclimatized to the laboratory conditions for ten days before commencing the experiment. The experimental protocol was endorsed by the institutional animal ethical committee (Ref. no: 1220/a/08/CPCSEA). 2.4. Acute toxicity studies Ethanol extract of A. mexicana (EEAM) used in this study was subjected to acute toxicity studies. The rats were fasted overnight with free access to drinking water and divided into six groups, each of six animals. Group 1 animals served as a control and received distilled water orally (2 mL/kg). Animals in groups 2 to 6 were administered with 0.25, 0.5, 1.0, 2.0 and 4.0 g/kg, respectively, of the EEAM extract by gastric intubation with a soft rubber catheter. The animals were kept under observation continuously for 2 h and then at onehourly interval until the twenty-fourth hour. Behavioral, neurological and autonomic profiles were recorded at these
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points of time. The animals were observed for mortality up to the 48th h. 2.5. Experimental procedure Before the drug treatments, the male rats were given
training individually with normal adult female rat (in estrous cycle) for sexual experience. Then the male rats were divided into sexually active and sexually inactive groups, based on their copulatory behavior. A male was considered sexually active when it attempted to mount any female introduced into the cage. The average mountings in normal male rats were found to be 4-10 in 5 min. The animal with less than 4 mounts was considered inactive. Sexually inactive male rats were selected to conduct the study. The female sexual behavior is limited to the period of estrous, that coincides with ovulation and during which the animal is said to be in heat. The estrous female arouses sexual interest in male rat by physical changes in the genital region and the production of pheromones. These are sexual scents found in animals that produce dramatic sex-seeking behavior in animals[6]. The female rats respond to each mount with a lordosis response. This response occurs when the female is receptive to mounting male and consists of an arching of the back to a concave position with deviation laterally and the neck extended[7] . The female rat with estrous cycle was confirmed by vaginal smear method. A dropper with a drop of distilled water was introduced into the animal’s vagina and the secretions were collected and were tested under microscope. Estrous cycle was confirmed when 50% or more of the cells were cornified. The sexually inactive male rats were divided into two groups and each contains eight animals. One group was treated with sildenafil citrate (SC) (gift sample from Ranbaxy Laboratories, India) at a dose of 5 mg/kg (p.o.) and the second group was treated with EEAM at a dose of 1 g/kg (p.o.) daily for 28 days. The third group contains eight sexually active male rats was used as positive control. T he highly receptive female ( in estrous stage ) was introduced into the home cage of the male rats and the following male sexual behavioral parameters were recorded during a period of 30 min: Latency (time) of first mount, number of mounts, latency of first intromission, number of intromission, latency of ejaculation (time from intromission to ejaculation), number of ejaculations and post ejaculatory pause (time from ejaculation to next mount or intromission)[8, 9]. All the groups were tested for copulatory behavior on 0, 7th, 14th, 21st and th 28 days.
2.6. Estimation of serum testosterone Blood samples were collected from retro-orbital plexus before treatment, 30 min after administration of EEAM on th th st th 0 day, 7 , 14 , 21 , 28 days and 7 days after withdrawal of treatment. Serum was separated upon centrifugation at 3 0 0 0 rpm for 20 min [10, 11] . S erum testosterone concentrations were determined in triplicate by ELISA method using testosterone ELISA kit (Model ADI-901-065; ® Enzyo Life Sciences, India).
2.7. Statistical analysis The data were expressed as mean ± S.E.M. The results were statistically treated with one-way analysis of variance ( ANOVA ) followed by T ukey- K ramer test for multiple columns comparison. The statistically significance was set accordingly.
3. Results 3.1. Acute toxicity studies The oral administration of EEAM did not provoke any gross behavioral changes or manifestations of toxic symptoms such as increased or decreased motor activity, muscle relaxation, tremors, clonic convulsions, spasticity, loss of right reflex, tonic extensions, ataxia, lacrimation, salivation, weight loss, watery diarrhea, writhing and urination over a period of 48 h. Ethanol extract of A. mexicana was found to be non-lethal even at the maximum single dose of 4.0 g/kg. Hence, the dose of the EEAM selected for the study was 1 g/kg orally.
3.2. Sexual behavioral parameters Sexually active male rats were used as positive control to compare the effect of ethanol extract of A. mexicana (EEAM) in elevating the sexual behavior and characteristics in sluggish male rats. The results of EEAM were also compared with that of sildenafil citrate (SC), a well known aphrodisiac moiety. No negative control group was used, since two test group animals used were of sexually inactive. Overall, it was observed that the sexual behavior in sluggish male rats was elevated with EEAM treatment. Before start of the treatment, treated group animals have exhibited different mount and intromission latencies from control group animals (P<0.05). th At the end of the treatment period i.e. on 28 day, significant decrease in mount latency was noticed in both test groups compared to control group. Dissimilar reduction in mount latency was observed between two test groups soon after dosing as shown in Table 1. Treatment with EEAM and SC was resulted with delayed ejaculation latency in animals on th 7 day (P<0.05) as presented in Figure 1.
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Table 1 Effect of ethanol extract of A. mexicana (EEAM) on mount and intromission latencies. Treatment period
Latency Mount
Before treatment
Intromission Mount Intromission Mount Intromission Mount Intromission Mount Intromission Mount Intromission
0 day th
7 day th
14 day th
21 day st
28 day th
Control
Treatment group SC (5 mg/kg)
180.00依11.23
356.00依13.62
180.30依9.76
204.00依10.45
380.50依11.50
180.00依1.34
196.00依6.46
296.00依12.42
180.50依9.12
184.00依10.32
217.00依14.52
180.00依14.96
168.00依17.23
161.00依13.36
181.00依16.15
142.50依11.30
158.00依14.64
49.50依2.30
EEAM (1 g/kg) 70.72依8.70
71.83依5.50
*
49.10依10.96
380.00依19.36
53.66依6.66
49.50依2.34
62.50依3.50
50.50依3.76
49.16依9.36
49.00依9.73
39.66依6.32
49.00依8.82
43.62依8.19
35.33依16.65
49.00依8.08
36.66依4.61
28.10依 0.61
30.83依1.44
*
#
Values are expressed as mean 依 S.E.M., n=8, P<0.05 considered significant as compared to control, P<0.05 considered significant as compared to sildenafil citrate (SC). ##
Ejaculation latency(sec)
400
*
animals when compared to SC treated group animals and control group even after treatment on 7th day as depicted in Figure 2.
## *
300
100 Before treatment
0 day
7th day
14th day
21st day
28th day
Figure 1. Effect of ethanol extract of A. mexicana on delaying the
ejaculation latency.
Values are expressed as mean ± S.E.M. (n=8). *P<0.05 considered
significant as compared to control and ##P<0.01 considered significant as compared to SC.
The post ejaculatory pause was lasted for long time in both
test groups compared control group before the treatment. The pause was lasted for long time in EEAM treated group
Post ejaculatory pause(sec)
200
0
#
400 300
*
200
*
#
100 0
Before treatment 0 day
7th day
14th day
21st day
28th day
Figure 2. Effect of ethanol extract of A. mexicana on post ejaculatory
pause.
Values are expressed as mean ± S.E.M. (n=8). *P<0.05 considered
significant as compared to control and #P<0.05 considered significant as compared to SC.
Table 2
Effect of ethanol extract of A. mexicana (EEAM) on mount and intromission frequencies. Treatment period Before treatment 0 day th
7 day th
14 day th
21 day st
28 day th
Frequency Mount
Intromission Mount Intromission Mount Intromission Mount Intromission Mount Intromission Mount Intromission
Values are expressed as mean 依 S.E.M., n=8, P<0.05 and considered significant as compared to sildenafil citrate (SC). *
**
Control 13.00依1.60 10.00依0.24 12.57依2.90 9.66依0.526 13.00依3.52 10.90依0.45 12.50依2.90 10.10依0.56 12.80依1.81 10.50依1.03 13.00依0.83 10.30依0.34
Treatment group SC (5 mg/kg) 4.00依0.46 2.00依0.18 10.24依3.82 7.30依0.27 12.50依1.17 * 9.50依0.30 15.00依0.84 10.91依0.22 16.50依0.38 ** 13.90依0.52 23.40依4.31 15.10依0.32
EEAM (1 g/kg) 3.80依0.55 2.16依0.02 6.00依0.98 2.30依0.13 7.00依0.86 3.50依0.18 14.30依1.68 7.10依0.39 18.50依3.51 ## 10.00依0.32 21.50依3.52 # 12.60依0.42
P<0.01 considered significant as compared to control, #P<0.05 and ##P<0.01
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Before treatment, the sexually active group animals have
exhibited high mount frequency than sexually inactive group animals. The mount frequency was elevated in EEAM and SC treated sexually sluggish rats as shown in Table 2. Before starting and 0 day of the treatment, intromission frequencies were more in control group animals compared with that of test group animals. An increased intromission frequency was observed with SC treatment from 7th day onwards (P<0.05) and same time the intromission frequency was continued to be less in EEAM treated animals than that of with SC treatment even on 28th day as evidenced Table 2. During the study it was noticed that ejaculation frequencies in control and test group animals were found to be different before and after the treatment. 3.3. Serum testosterone levels T he serum testosterone levels in inactive rats were
significantly low compared positive control group active rats. Upon treatment with EEAM serum testosterone levels were continued to be lower on 0, 7th and 14th day than untreated animals. But, upon treatment with SC, serum testosterone levels were elevated. T he gradual elevation of serum testosterone levels was observed with EEAM treatment and the similar trend was lasted up to 7 days after withdrawal of the treatment. Figure 3 demonstrates that serum testosterone levels in both test group animals were significantly different on 14th day observations (P<0.01). The difference in serum testosterone levels of test group animals was gradually diminished and the similar effect was continued even after withdrawal of the treatment. Post ejaculatory pause(sec)
3
2.5
** ##
2
1.5 1
*
0.5
0 Before treatment 0 day
7th day
14th day
21st day
28th day
After withdrawal of treatment
Figure 3. Effect of ethanol extract of A. mexicana on serum testosterone levels. Values are expressed as mean ± S.E.M. (n=8). **P<0.01 considered significant as compared to control and ##P<0.01 considered significant as compared to SC.
4. Discussion Aphrodisiacs are substances that stimulate or increase sexual desire and performance. Sexual desire is controlled
and regulated by the central nervous system, which integrates olfactory, tactile, auditory, and mental stimuli. SD can occur even if sexual desire remains strong, because sexual performance depends on a neurovascular event via the hemodynamic mechanisms of penile erection. Sexual function is said to be a sequence of physiological responses to stimulation which involves increasing vasocongestion and myotonia tumescence, subsequent release of the vascular activity, muscle tone and experience four phases of sexual response cycle[12]. Latency for mount and intromissions are indicators of sexual motivation. Number of intromission, number of ejaculations is considered for sexual performance and facilitation[13, 14]. As per the statistically significant results emanated, the ethanol extract of A. mexicana decreased the latency for mount and intromission and significantly increased the number of intromissions and ejaculations as compared to control group. As per the earlier phytochemical studies, EEAM contains protopine alkaloids, flavanols, phenolics and fatty acids[15]. Flavanoids have been shown to be responsible for endothelium-dependent nitric oxide-mediated relaxation. Nitric oxide is a major physiological stimulus for penile vasculature and trabecular smooth muscles, all essential for penile erection. It is also assimilated that EEAM stimulate testosterone production and thereby maintain and improve reproductive health and quality of life. The present study shows for the first time that A. mexicana is able to significantly increase testosterone levels in male rats treated with the EEAM daily for 28 days, in parallel with the elicitation of an improved copulatory activity. Testosterone seems to be involved in the complex mechanism regulating the copulatory behavior by acting both peripherally and centrally in concert with other determinants. Certain plants were proven to have improved sexual function with increased levels of testosterone[16, 17]. Testosterone may facilitate male sexual behavior by increasing dopamine release and thereby potentiating neurotransmission[18]. Hence the ethanol extract of A. mexicana increases the sexual motivation, desire, potency, performance and facilitation to meet the cardinal phases of sexuality. Sexual arousal and functions are related to hormonal function. Testosterone in male and estrogen in female have the most marked effect on human sexuality. The present findings provide experimental evidence for aphrodisiac potentials of A. mexicana. The results of present work demonstrated that the ethanol extract of A. mexicana has significant ability in enhancing copulatory ability and sexual performance in sexually sluggish animals. Ethanol extract of A. mexicana has capability to increase testosterone levels
Asuntha G et al./ Asian Pacific Journal of Reproduction (2014)110-115
in sexually impaired male rats and it remained unaffected even after withdrawal of treatment. The ethanol extract of A. mexicana has similar potentials of SC with slow onset of action. Further studies are required to explore the chemical constituents responsible for the aphrodisiac activity and also to establish the mode of action. Conflict of interest statement The authors report no declarations of interest.
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