Griffonia simplicifolia negatively affects sexual behavior in female rats.
At present Griffonia simplicifolia is used in food supplement aimed to treat mood disorders as well as to reduce food intake and body weight. The plant has gained increasing interest for its high content in 5-hydroxy-L-tryptophan (5-HTP) particularly in the seed. The present study was designed to evaluate the influence of a seed extract of the plant, dosed at 25, 50 and 100 mg/kg, on the sexual behavior of ovariectomized hormone-primed rats after acute and subchronic treatment. The single administration of G. simplicifolia significantly reduced lordosis response and increased rejection behavior in female rats treated with the highest dose while it did not influence preceptive behaviors. On the other hand the subchronic administration of the extract significantly reduced proceptivity but not receptivity, and increased rejection behavior. All the tested dosages were able to markedly decrease food intake and body weight after a 9-day treatment. Taken together the present results, possibly ascribed to increased levels of 5-hydroxytryptamine (5-HT) in the brain, suggest a cautious administration of the plant extract owing to its negative influence on female sexual behavior.
[C] 2010 Elsevier GmbH. All rights reserved.
Keywords: Griffonia simplicifolia 5-hydroxy-L-tryptophan (5-HTP) Female rat Receptivity Proceptivity Body weight
Griffonia simplicifolia Baill. (fam. Caesalpinaceae) is a tropical non-climbing shrub native to West Africa (Ghana, Ivory Coast, Togo). It is common in the coastal plain as well as in secondary forest. It grows to a height of about 3 m and bears greenish flowers followed by black pods which mature in the period between December and February. The seeds, naturally released by ripe fruits, are harvested and dried (Irvine 1961).
Traditional uses of the plant include use of the stem and roots as chewing sticks, use of the leaves for wound healing and leaf juice to treat urinary disturbances. A decoction of stems and leaves provides relief from nausea. The seeds are also reputed to exert aphrodisiac effects as well as to have antibiotic properties (Ayensu 1978).
The great interest in the plant is due to the high concentration of 5-hydroxy-L-tryptophan (5-HTP) found in the mature seeds of the plant (Bell and Fellows 1966). The amino acid was particularly found in the seed (6-10% fresh weight) but also in the leaves (0.3-1.2% fresh weight) and in the pods (0.1-0.2% dry weight) (Fellows and Bell 1970). It is well-known that 5-HTP is metabolized to 5-hydroxytryptamine (5-HT) and then to 5-hydroxy-indole-3-acetic acid (5-HIAA) in all tissues containing 5-HTP decarboxylase. Consequently increased levels of 5-HTP in the blood can induce a corresponding increase in 5-HT levels in the brain with associated physiological effects.
The modern therapeutic applications of G. simplicifolia seed include the treatment of anxiety and depression, insomnia, migraine and headache besides the regulation of appetite leading to weight reduction in obese patients. These indications are due to the main role of serotonin as neurotransmitter in the brain, particularly in the pathways controlling mood, sleep and eating (Dubovsky and Thomas 1995).
It has been shown that 5-HT is able to influence the receptive behavior in female rats. Depending on the subtype of serotonin receptors which were activated, 5-HT receptor agonists may exert inhibitory or facilitatory effects on lordosis response (Mendelson 1992; Uphouse 2000). The 5-[HT.sub.1A] receptors located in the ventromedial nucleus of hypothalamus (VMN) mediate a lordosis inhibiting effect (Uphouse et al. 1996); on the other hand the activation of 5-[HT.sub.2A/2C] produces a facilitatory effect (Wolf et al. 1998). Drugs which elevate extracellular 5-HT as well as 5-HT reuptake inhibitors such as fluoxetine are known to inhibit sexual behavior in female rats (Mendelson 1992; Uphouse 2000). Recently a reduction in the lordosis response in female rats acutely administered with fluoxetine, and a reduction in proceptivity after both acute and subchronic treatment with the drug were demonstrated (Sarkar et al. 2008).
Taking in account the above mentioned findings, we hypothetized that G. simplicifolia administration could influence female sexual behavior. In the following experiments we examined the effects of acute and subchronic administration of G. simplicifolia seed extract on receptive and proceptive behaviors of ovariectomized hormone-primed rats. In addition we evaluated the effects of the extract administered at the same dosages on feeding behavior and body weight gain.
Materials and methods
Adult female Sprague-Dawley rats weighing 200-250g were obtained from Charles River Laboratories (Calco, Como, Italy). They were housed two per cage in polycarbonate cages in a colony room maintained in standard conditions at 22[degrees]C and 60% humidity with a 12:12 reversed light/dark cycle. Commercial rat pellets (Global Diet 2018, Mucedola S.r.l., Milan, Italy) and water were always available. After one week of adaptation, all rats were bilaterally ovariectomized under ketamine hydrochloride (Ketavet 100[R], Farmaceutici Gellini Spa, Italy) plus xylazine hydrochloride (Rompun[R], Bayer, Germany) anesthesia.
Sexually experienced Sprague-Dawley male rats, weighing approximately 350-400 g, were used as stimulus animals in mating tests.
Animal care, maintenance and surgery were conducted in accordance with Italian law(D.L. n. 116/1992) and European legislation (EEC n. 86/609). The experimental designs and procedures received the approval of the Bioethical Committee of the Italian Institute of Health.
The ovariectomized rats were induced into estrus by the subcutaneous (s.c.) injection of 1.5 [mu]g estradiol benzoate/rat (Estradiolo AMSA[R], AMSA) twice a week, specifically 52 and 28 h prior to test sexual receptivity and the s.c. injection of 500 [mu]g of progesterone (Prontogest[R], AMSA) 4 h before testing. Hormones were delivered in a peanut oil vehicle and administered in the volume of 0.3 ml/rat.
G. simpiicifolia seed dried extract (batch 0200849), standardized to 20% 5-HTP, was supplied by EPO S.r.l. (Milan, Italy). It was suspended in water by tragacanth gum and administered by oral gavage in a volume of 5 ml/kg at three dose levels: 25, 50 and 100mg/kg b.w. The treatments were performed both acutely and repetitively (daily for 9 days) using the same dosages. Control animals received an equal volume of vehicle solution (tragacanth gum and water). The behavioral tests were carried out 45 min after the single dose or 4h after the last dose when the extract was sub-chronically administered.
All behavioral tests were performed during the dark phase of the light/dark cycle under red light illumination in a clear plexiglass arena (70 cm x 35 cm x 40 cm high). The behavioral parameters were scored by observers unaware of the drug treatment. A single male rat was placed in the arena for 5 min and was permitted two intromissions with a stimulus female to ensure his sexual vigor. Following the 5-min period, an experimental female rat was placed in the arena with the male rat. Each female received 10 mounts with or without intromission. If the female received an ejaculation, she was placed in an adjacent arena with another sexually experienced male until testing was completed. The lordosis response was scored on a four-point scale (0-3) as described by Hardy and DeBold (1971). For each rata lordosis quotient (LQ) was calculated by dividing the number of lordosis scores of 2 or 3 by the total number of mounts x 100. A lordosis rating (LR) for each rat was calculated as the sum of 0, 1, 2, 3 response scores divided by the number of mounts. During the test also the number of proceptive (hops, darts and ear wiggles) and rejection (kicking, boxing, running away) behaviors exhibited per minute were recorded.
Four groups of six female rats each, maintained in individual cages, were used to assess the influence of three dose levels (25, 50, 100 mg/kg/day) of G. simplicifolia extract administered daily for 9 consecutive days. Every morning preweighed chow pellets (about 30 g) were introduced into the cage. The amounts of food consumed were determined after 24 h. At the end of the experiment, cumulative food intake (grams of food/100 g of rat body weight) was calculated.
Body weight of individually housed rats was measured at the beginning and the end of the treatments. The total change over the 9-day period was calculated.
The data expressed as mean [+ or -] SEM were obtained from groups of 6-8 rats each. One-way analysis of variance (ANOVA) with Dunnett's test for post-hoc comparisons between individual treatment groups and controls was performed using GraphPad Prism version 5.0 (GraphPad Software Inc, San Diego, CA, USA). The percentages of rats exhibiting rejection behavior in treated and control groups were compared using Fisher's test. In all cases, p < 0.05 was taken as the level of significance.
Female sexual behavior after acute treatment
The single administration of G. simplicifolia extract induced a dose dependent decrease in LQ values. However, a significant difference in comparison with vehicle group was observed only in the group of rats treated with the highest dose of the extract (100mg/kg) (Fig. 1). Similarly a significant reduction in LR values was found only after the administration of the highest dose (p < 0.05 vs vehicle group) (Fig. 1). Proceptive behaviors exhibited during the mating test were not significantly influenced by the acute treatment with the extract, even if a tendency to a decrease was observed (Table 1). A dose dependent increase in the rejective behaviors was observed in the females treated with the extract: the difference both in the number of the behaviors and in the percentage of rats exhibiting this effect was found statistically significant in comparison with vehicle group after the administration of the highest dose of the extract (p <0.05) (Table 1).
Table 1 Proceptive and rejective behaviors exhibited by female rats acutely treated with Griffonia simplicifolia extract during the sexual receptivity test. Treatment (mg/kg) Proceptive Rejective % of behaviors/min behaviors/min animals exhibiting rejective behaviors Vehicle 8.10 [+ or -] 1.22 0.06 [+ or -] 0.06 12.5 Extract 25 8.05 [+ or -] 0.92 0.10 [+ or -] 0.10 12.5 Extract 50 6.51 [+ or -] 0.67 0.41 [+ or -] 0.16 50.0 Extract 100 5.33 [+ or -] 0.49 0.70 [+ or -] 0.21 * 75.0 [dagger] Values are mean [+ or -] SEM, n = 8. One-way ANOVA followed by Dunnett's post-hoc test: *p < 0.05 vs vehicle group; Fisher's test:[dagger] p < 0.05 vs vehicle group.
[FIGURE 1 OMITTED]
Female sexual behavior after subchronic treatment
The daily administration of G. simplicifolia extract for 9 days did not affect female receptive behavior; consequently LQ. and LR values were approximately equal in all experimental groups (Fig. 2). On the other hand a dramatic decrease in the proceptive behaviors was detected in all treated groups: the more marked reduction was found in rats treated with the highest doses of the extract (p <0.001 vs vehicle treated rats) (Table 2). On the other hand the number of rejection behaviors were significantly increased by the administration of the extract dosed at 50 and 100 mg/kg (p <0.05 and p <0.01 vs vehicle group, respectively) (Table 2). Also the percentage of rats displaying rejective behaviors was significantly increased by the same dosages of the extract (p <0.05) (Table 2).
Table 2 Proceptive and rejective behaviors exhibited by female rats subchronically treated with Griffonia simplicifolia extract during the mating test. Treatment Proceptive Rejective % of (mg/kg) behaviors/min behaviors/min animals exhibiting rejective behaviors Vehicle 8.10 [+ or -] 1.22 0.06 [+ or -] 0.06 12.5 Extract 25 3.96 [+ or -] 0.47 ** 0.16 [+ or -] 0.07 50.0 Extract 50 3.41 [+ or -] 0.62 *** 0.46 [+ or -] 0.12 * 75.5 [dagger] Extract 100 2.98 [+ or -] 0.41 *** 0.63 [+ or -] 0.14 ** 87.5 [dagger] Values are mean [+ or -] SEM, n = 8. One-way ANOVA followed by Dunnett's post-hoc test: * p < 0.05, ** p <0.01, *** p < 0.001 vs vehicle group. Fisher's test: [dagger] p <0.05 vs vehicle group.
[FIGURE 2 OMITTED]
The daily administration of the plant extract at all tested dosages was able to significantly reduce the total amount of food intake/100g b.w. during the 9-day treatment (Fig. 3).
[FIGURE 3 OMITTED]
The repeated treatment with the plant extract at all tested dosages induced a significant decrease in body weight of rats in comparison with vehicle group (p <0.01) (Fig. 4).
[FIGURE 4 OMITTED]
It is well-known that sexual behavior in female rats is characterized by both appetitive and consummatory components, named proceptivity and receptivity (Beach 1976). The receptive behavior consists in a reflexive posture, called lordosis, which represents the female readiness to allow copulation (Beach 1976). The term proceptivity includes a number of soliciting behaviors such as hops and darts, approach and withdrawl from the male, ear-wigglings, representing the female's effort to arouse male's attention and to stimulate him to initiate mounting (Beach 1976; Madlafousek and Hlinak 1983). Gonadal steroids exert a crucial influence on the central control of female sexual behavior (Pfaff et al. 1994). Estrogen is required for the elicitation of lordosis response which can be facilitated by progesterone. This effect, observed only in estrogen-primed rats, allows to reduce the dose of estrogen required for lordosis to occur. On the other hand progesterone is not able to induce lordosis response in ovariectomized females not pretreated with estrogen. Even if the expression of the female receptive posture in rodents requires the coordinated activity of forebrain, midbrain, brainstem and spinal cord neurons, specific brain areas such as the hypothalamic ventromedial nucleus (VMN) and the medial preoptic area (mPOA) are crucial for the elicitation of lordosis response (Etgen et al. 1999: Pfaff and Modianos 1985). In these areas the steroid effect is modulated by the presence of neurotransmitters such as dopamine and serotonin. In particular. 5-HT seems to differently regulate female sexual behavior depending on the type of activated receptors. The inhibitory and facilitatory influence of 5-HT receptor agonists and antagonists on female lordosis reflex has been exhaustively reviewed by Uphouse (2000). From a clinical point of view, it is well-known that serotonin levels increase with the use of selective serotonin reuptake inhibitor antidepressants (SSRIs), and a higher incidence of sexual dysfunction is found in women treated with SSRI (Lane 1997; Montgomery et al. 2002).
In our study we focused our attention on the activity of G. simplicifolio seed extract which is rich in 5-HTP, a direct precursor in the biosynthesis of serotonin. Here we studied the influence of acute and repeated treatment with the extract on female sexual behavior. The acute treatment with the highest dose (100 mg/kg) was able to reduce sexual receptivity, as indicated by a significant decrease in LQ and LR values. When subchronically administered, the extract failed to influence both receptivity parameters. Therefore we can conclude that the supplementation with G. simplicifolia seed extract gives up a transient inhibitory influence on female receptivity which disappears after repeated treatments.
On the other hand we observed a different trend in the evaluation of proceptive behaviors which were markedly decreased after the subchronic treatment with the extract but not after the acute administration. Many researchers, according to Beach's (1976) formulation, view proceptivity as a manifestation of sexual motivation. Although sexual behavior greatly differs between rat and human, some aspects of neuroanatomical and neuroendocrine substrates of sexual responses have been conserved across evolution in different mammalian species, and the effect of different pharmacological agents are similar in rodents and in humans (Pfaus et al. 2003). Considering that desire in women is homologous to appetitive aspects of sexual activity in female rats, we can surmise that a repeated supplementation of G. simplicifolia leads to a decrease in sexual desire. The negative influence of the plant extract on female sexual behavior was confirmed by the finding of a higher incidence of rejection behaviors during the mating test following the acute (100 mg/kg) and subchronic (50 and 100 mg/kg) administration of the extract.
Taken together these results lead to an intriguing conclusion, taking into account the widespread utilization of herbal supplements containing G. simplicifolia in humans. However, it is always difficult to correlate the doses used in experimental animals with the doses recommended in clinical use.
Considering that one of the major claim of G. simplicifolia extract is the reduction of food intake and consequently of body weight, we evaluated the influence of our extract, at the same dosages used in the sexual behavior study, on feeding and body weight after repeated treatments. There is good evidence that brain serotonin is involved in the regulation of food intake (Meguid et al. 2000) and that increased 5-HT levels cause the suppression of carbohydrate intake (Mancilla-Diaz et al. 2005). Recently, it has been demonstrated that 5-HTP (3-200 mg/kg i.p.) acute administration is able to suppress food intake (Amer et al. 2004). But at our knowledge the anorectic effect of G. simplicifolia has not been demonstrated until now. We found that all tested dosages of the extract were able to significantly reduce both food intake and body weight.
Even if the experimental studies in laboratory rodents could be only indicative of pharmacological effects in humans and the transfer of the effective doses is always uncertain, our results suggest caution in the utilization of food supplements containing G. simplicifolia owing to potential side effects which were unknown so far.
The authors gratefully acknowledge to EPO S.r.1. for supplying Griffonia simplicifolia seed extract used for the experiments.
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G. Carnevale (a), V. Di Viesti (a), M. Zavatti (b), A. Benelli (b), P. Zanoli (a), *
(a) Department of Biomedical Science, University of Modena and Reggio Emilia, Via Campi 287, 41100 Modena, Italy
(b) Department of Anatomy and Histology, University of Modena end Reggio Emilla, via Del Pozzo 71, 41100 Modena, Italy
* Corresponding author at: Department of Biomedical Sciences, Section of Pharmacology, Via Campi 287, 1-41100 Modena, Italy. Tel.: +39 0592055165: fax: +39 0592055376.
E-mail address: firstname.lastname@example.org (P. Zanoli).
0944-7113/$ - see front matter [C] 2010 Elsevier GmbH. All rights reserved.
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|Author:||Carnevale, G.; Di Viesti, V.; Zavatti, M.; Benelli, A.; Zanoli, P.|
|Publication:||Phytomedicine: International Journal of Phytotherapy & Phytopharmacology|
|Date:||Oct 1, 2010|
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