Introduction

The mechanism of penile erection involves a haemodynamic process in the cavernosal tissues and nervous system [1]. Erectile dysfunction (ED) results from the inability to attain and maintain erection needed for sexual satisfaction [1, 2]. It is associated with pathological and physiological changes in the corpus cavernosal tissues [2]. In addition, ED has been linked to several pathological conditions such as hypercholesterolemia, hypertension, cardiovascular diseases and diabetes [3, 4]. Studies have also shown that depression as well as the use of antidepressant drugs may lead to ED [5, 6]. However, drugs such as sildenafil or sildenafil citrate, vardenafil, and tadalafil, are currently in use for the treatment of ED [7, 8]. These drugs possess attendant side effects including dizziness, blurred vision, nasal congestion, hearing loss, dyspepsia, nasopharyngitis, myalgia [8, 9]. Sildenafil citrate, a PDE-5′ inhibitor, acts by inhibiting PDE-5, an enzyme that promotes degradation of CGMP thus enhancing the attainment of penile rigidity [9].

For effective management and treatment of ED, therapeutic options should exceed the use of PDE-5 inhibitors [10]. There are indications that some crucial enzymes such as arginase, acetylcholinesterase (AChE) and adenosine diaminase (ADA), linked to biomolecules such as cGMP, NO, ACh and adenosine involved in penile erection, could be therapeutic targets [10, 11]. Considering the vast range of bioactivity and broad spectrum of specificity of plants, bioactive compounds in plants may represent safe, natural, alternative and effective therapeutics or therapeutic sources for the treatment of erectile dysfunction [11, 12]. Holistic treatment of ED may actually involve elevation in nitric oxide (NO) production, repair of endothelial dysfunction, enhancement of antioxidant status, as well as increase in the levels of cyclic-guanosine monophosphate (cGMP), acetylcholine (ACh) and adenosine in the cavernosal tissue [12, 13].

Beta-caryophyllene (BCP) is a natural, volatile, bicyclic sesquiterpene lactone compound abundantly present as an essential oil component of many dietary and edible plants including spices [14]. Previous studies have also demonstrated the antioxidant, antimicrobial, anticancer, anti-inflammatory, anti-arthritic, cardio protective, anticholinesterase effects in vitro and in vivo [15,16,17,18,19,20]. There is dearth of information on the erectogenic effects of BCP, and the possible mechanisms of BCP in ED. Therefore, this study was convened to assess the effect of β-caryophyllene on sexual performance, lipid peroxidation and crucial enzymes linked to erectile function in penile tissue of paroxetine-induced rats.

Materials and methods

Chemicals, reagents and equipment

5,5-Dithio-bis-(2-nitrobenzoic acid), trichloroacetic acid, thiobarbituric acid (TBA), β-caryophyllene (purity 98.5%), tween 80, acetylthiocholine iodide, and adenosine were purchased from Sigma–Aldrich (St. Louis, MO, USA). Sodium dodecyl sulphate and acetic acid were sourced from BDH Chemicals Ltd, (Poole, England). Other chemicals and reagents used were of analytical grade and water was glass distilled. Kenxin refrigerated centrifuge Model KX3400C and UV–vis spectrophotometer (Model 6305; Jenway, Barloworld Scientific, Dunmow, United Kingdom) were used for centrifuging and the measurement of the absorbance respectively.

Bioassay

Animal ethics

All the animals received humane care according to the criteria outlined in the guide for the care and use of laboratory animals prepared by the National Academy of Science and published by the National Institute of Health (USA). The animal ethic committee of the Federal University of Technology, Akure, Nigeria gave an approval for the use of laboratory animals with reference number of FUTA/ETH/2019/012. The ethic regulations are in accordance with national and institutional guidelines for the protection of animals’ welfare during experiments.

Experimental design

50 Male and 50 female Wistar rats weighing 185–210 g were purchased from the Department of Veterinary Medicine, University of Ibadan, Nigeria. The rats were acclimatized for a period of 2 weeks. The animals were kept in wire mesh cages under controlled light cycle (12 h light/12 h dark). They were fed with commercial growers and water was administered ad libitum. After 2 weeks of acclimatization, the male animals were randomly divided into ten groups (n = 5 in each group);

Group I Normal control rats—was administered the vehicle (1% ethanol).

Group II β-caryophyllene (10 mg/kg).

Group III β-caryophyllene (20 mg/kg).

Group IV Sildenafil citrate (20 mg/kg/day).

Group V β-caryophyllene (20 mg/kg/day) + sildenafil citrate (20 mg/kg/day).

Group VI Paroxetine induced erectile dysfunction (PD) rat.

Group VII Paroxetine induced erectile dysfunction + β-caryophyllene (10 mg/kg).

Group VIII Paroxetine induced erectile dysfunction + β-caryophyllene (20 mg/kg).

Group IX Paroxetine induced erectile dysfunction + sildenafil citrate (20 mg/kg/day).

Group X Paroxetine induced erectile dysfunction + β-caryophyllene (20 mg/kg/day) + sildenafil citrate (20 mg/kg/day).

Paroxetine was administered orally for the first 7 days, after which the rats were treated with the vehicle (1% ethanol), β-caryophyllene, and sildenafil citrate orally for the next 7 days based on their respective groups. The doses of β-caryophyllene (10 and 20 mg/kg/day), paroxetine (20 mg/kg) and sildenafil (20 mg/kg) used were in accordance with previous investigation with slight modification [21, 22]. On the 7th and 14th day only, the female rats were introduced into the cages of the male rat for the sexual performance assays (male paired with female in a single cage) according to the modified method of Thawatchai et al. [23]. The animals were subjected to euthanasia, after been anaesthetized with isoflurane. The penile tissues were excised, rinsed in ice-cold normal saline, blotted and weighed for subsequent analysis.

Sexual performance study

The sexual performance study was carried out according to the modified method of Thawatchai et al. [23]. In order to access the sexual behaviours, estrous female rats was paired with male rats of each group. The female rats were induced with progesterone (1 mg/kg body weight) and estradiol benzoate (10 µg/mg body weight) at 4 and 24 h respectively, injected via intraperitoneal and subcutaneous route respectively) before copulation. Sexual performance was monitored inside a clear rectangular plastic chamber (60 cm length × 40 cm breath × 30 cm height) and recorded using digital video recording on the 7th and 14th day of the experiment, in a quiet room for 1 h. Mounting number, which is the number of mounts without intromission from the time of introduction of the female to the male; mounting latency, which is the time of introduction of the female to the first mount by the male; intromission number, which is the number of intromission form the introduction of the female until the end of the experiment); and intromission latency, which is the interval from the time of introduction of the female to the first intromission by the male, were subsequently determined [24].

Biochemical analysis

Acetylcholinesterase (AChE) inhibitory assay

A mixture of penile tissue homogenate (200 μL), 5,5′-dithio-bis-(2-nitrobenzoic) acid [3.3 mM, 100 μL) and 0.1 M phosphate buffer (pH 8.0) was incubated for 20 min at 25 °C, after which acetylthiocholine iodide was added. The absorbance of the mixture was measured at 412 nm immediately after the addition of the substrate [25]. AChE activity was calculated with respect to the total protein. The specific activity of AChE was expressed as µmol. AChE/h/mg protein.

Phosphodiesterase-5 (PDE-5) activity assay

The penile tissue was prepared with 0.1 M Tris–HCl buffer containing 1 mM CaCl2 and 50 mM NaCl (pH 8.0). The reaction mixture containing 5 mM of the substrate (p-nitrophenyl phenylphosphonate), penile homogenate, 20 mM Tris buffer (pH 8.0), was incubated at 37 °C for 10 min. The intensity of p-nitrophenol produced was measured as change in absorbance after 5 min at 400 nm [26]. The specific activity of PDE-5 was calculated and expressed as µmol./min/mg protein.

Arginase activity assay

In brief, the arginase activity in penile homogenate was firstly activated by adding Tris–HCl (75 μL, 50 mmol/l, pH 7.5) containing 10 mmol/l MnCl2, which was preincubated for 10 min at body temperature, followed by catalyzing the L-arginine by arginase by incubating the mixture containing activated arginase, L-arginine (50 μL, 0.5 mol/l, pH 9.7) 1 h. This was halted by adding 400 μL of the acid solution mixture [H2SO4/H3PO4/H2O at ratio 1:3:7 (v/v/v)]. For the development of urea concentration, α-isonitrosopropiophenone (25 μL 9% in absolute ethanol) was then added and the mixture was heated at 100 °C for 45 min [27]. The urea concentration was determined spectrophotometrically at 550 nm measured the amount of urea produced, after normalization with protein, was used as an index for arginase activity.

Adenosine deaminase (ADA) activity

Briefly, 50 μL of penile tissue homogenate reacted with 21 mmol/L of adenosine, pH 6.5, and was incubated at 37 °C for 60 min. The protein content used for the experiment was adjusted to between 0.7 and 0.9 mg/mL [28]. Results were expressed in units per liter (U/L). One unit (1 U) of ADA is defined as the amount of enzyme required to release 1 mmol of ammonia per minute from adenosine at standard assay conditions.

Angiotensin-I converting enzyme (ACE) assay

The ACE activity was carried out according to the method described by Cushman and Cheung [29] with slight modification. The penile tissue homogenate was pre-incubated at 37 °C for 15 min. The ACE substrate [150 μL, 8.33 mM hippuryl-l-histidyl-leucine in 125 mM of Tris − HCl buffer (pH 8.3)] was added to the mixture, which was incubated at 37 °C for 30 min. The reaction was halted with 250 μL of 1 M HCl. The enzymatic product [hippuric acid (Bz-Gly)] was extracted with 1.5 mL of ethyl acetate, and centrifuged to separate the ethyl acetate layer. Thereafter, 1 mL of ethyl acetate layer was transferred to a clean test tube and evaporated to dryness. Distilled water (1 mL) was added and its absorbance was measured at 228 nm. The specific activity of ACE was calculated with respect to the total protein and reported as μmol./min/ mg protein.

Lipid peroxidation assay

Lipid peroxidation was determined as the formation of thiobarbibutric acid-reactive substances (TBARS) during an acid heating reaction according to the method of Ohkawa et al. [30]. Briefly, the reaction mixture consisting of 200 μL of the penile homogenates or standard (0.03 mM malondialdehyde (MDA)), 200 μL of 8.1% sodium dodecyl sulfate (SDS), 500 μL of 0.8% TBA, and 500 μL of acetic acid solution (2.5 M HCl, pH 3.4) was heated at 95 °C for 1 h. The absorbance was measured at 532 nm, and the penile tissue lipid peroxidation levels were expressed as micromoles MDA per milligram of protein.

Statistical analysis

All data were expressed as the means ± standard error of mean (SEM). One-way ANOVA was used for the statistical analysis followed by the post hoc Tukey’s test; p < 0.05 was considered to represent a significant difference using Prism software (GraphPad, version 6.0, Carlsbad, CA, USA).

Results

As shown in Tables 1 and 2, the results of mounting and intromission latencies as well as mounting and intromission frequencies on the 7th day of experiment revealed that there was significant (p < 0.05) increase in mounting and intromission latencies as well as significant (p < 0.05) reduction in mounting and intromission frequencies of PD group when compared with NC group. In a similar manner, there was significant (p < 0.05) elevation in both mounting and intromission latencies and significant (p < 0.05) decrease in mounting and intromission frequencies in PD group as compared to the NC group on the 14th day (Table 1). However, mounting and intromission latencies were significantly (p < 0.05) reduced in PD rat groups administered BCP (10 and 20 mg/kg), SD (20 mg/kg) and combination of BCP (20 mg/kg) and SD (20 mg/kg) in comparison to PD control group (Table 1). Conversely, treatment of PD rats with BCP (10 and 20 mg/kg), SD (20 mg/kg) and combination of BCP (20 mg/kg) and SD (20 mg/kg) showed significant (p < 0.05) increase in mounting and intromission frequencies (Table 2).

Table 1 Effects of β-caryophyllene on mount and intromission latency in paroxetine-induced sexual dysfunction in rats
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Table 2 Effects of β-caryophyllene on mount and intromission frequency in paroxetine-induced sexual dysfunction in rats
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As presented in Fig. 1, the effects of BCP, SD as well as the combination of BCP and SD on AChE activity in penile tissues of NC and PAR-induced rats were determined. A significant (p < 0.001) increase in AChE activity of PD group in comparison to the NC group was observed. However, there was decrease in AChE activity in PD rat groups treated with BCP (10 mg/kg), BCP (20 mg/kg) and SD (20 mg/kg) when compared with PD group. Interestingly, treatment with the combination of BCP (20 mg/kg) and SD (20 mg/kg) in PD rats showed significant (p < 0.05) reduction in AChE activity as compared with PD group.

Fig. 1
figure 1

Influence of β-caryophyllene, sildenafil and their combination on acetylcholinesterase (AChE) of normal and paroxetine-induced sexual dysfunction in rats’ penile tissue. Bars represent mean ± standard deviation of five readings (n = 5). #p < 0.05; ##p < 0.01; ###p < 0.001; different from NC. *p < 0.05; **p < 0.01; ***p < 0.001; different from PD. NC normal control; SD sildenafil; PD paroxetine; BCP β-caryophyllene, BCP + SD combination of β-caryophyllene and sildenafil

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In this study, significant (p < 0.01) elevation in PDE-5 activity of PD rat group and significant (p < 0.05) decrease in PDE-5 activity of SD group in comparison with NC group were observed (Fig. 2). In addition, the administration of BCP (20 mg/kg) and SD (20 mg/kg) in PD rats caused significant (p < 0.05) reduction in PDE-5′ activity as compared to PD rat group (Fig. 2). Furthermore, the result depicts that the combined treatment of PD rats with BCP and SD significantly (p < 0.01) decreased PDE-5′ activity in comparison to the PD group (Fig. 2).

Fig. 2
figure 2

Influence of β-caryophyllene, sildenafil and their combination on phosphodiesterase 5 (PDE-5’) of normal and paroxetine-induced sexual dysfunction in rats’ penile tissue. Bars represent mean ± standard deviation of five readings (n = 5). #p < 0.05; ##p < 0.01; ###p < 0.001; different from NC. *p < 0.05; **p < 0.01; ***p < 0.001; different from PD. NC normal control; SD sildenafil; PD paroxetine; BCP β-caryophyllene, BCP + SD combination of β-caryophyllene and sildenafil

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The result of the effect of BCP on arginase activity in penile tissues of paroxetine induced erectile dysfunction in rats is presented in Fig. 3. Arginase activity was significantly (p < 0.01) higher in PD group when compared with NC group. However, there was significant reduction in arginase activity of PD rat groups administered BCP (20 mg/kg) and SD (20 mg/kg) (p < 0.05) and the combination of BCP (20 mg/kg) and SD (20 mg/kg) (p < 0.01) in comparison to PD control group (Fig. 3).

Fig. 3
figure 3

Influence of β-caryophyllene, sildenafil and their combination on arginase of normal and paroxetine-induced sexual dysfunction in rats’ penile tissue. Bars represent mean ± standard deviation of five readings (n = 5). #p < 0.05; ##p < 0.01; ###p < 0.001; different from NC. *p < 0.05; **p < 0.01; ***p < 0.001; different from PD. NC normal control; SD sildenafil; PD paroxetine; BCP β-caryophyllene, BCP + SD combination of β-caryophyllene and sildenafil

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As shown in Fig. 4, there was significant (p < 0.05) decrease in ADA activity of penile tissues in rats treated with SD and combination of BCP and SD when compared with NC group. ADA activity was significantly (p < 0.01) higher in PD group as compared to NC group. Significant reduction in ADA activity of PD rats treated with BCP (20 mg/kg) (p < 0.05), SD (20 mg/kg) (p < 0.01), and the combination of BCP (20 mg/kg) and SD (20 mg/kg) (p < 0.01) in comparison to PD rat group was observed.

Fig. 4
figure 4

Influence of β-caryophyllene, sildenafil and their combination on adenosine deaminase (ADA) of normal and paroxetine-induced sexual dysfunction in rats’ penile tissue. Bars represent mean ± standard deviation of five readings (n = 5). #p < 0.05; ##p < 0.01; ###p < 0.001; different from NC. *p < 0.05; **p < 0.01; ***p < 0.001; different from PD. NC normal control; SD sildenafil; PD paroxetine; BCP β-caryophyllene, BCP + SD combination of β-caryophyllene and sildenafil

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Figure 5 reveals the effect of SD, BCP and their combination on ACE activity in penile tissues of normal and paroxetine-induced rats. The activity of ACE was significantly (p < 0.01) elevated in PD rat group when compared with NC group. Furthermore, PD rat groups treated with BC (10 mg/kg) (p < 0.05), BCP (20 mg/kg) and SD (20 mg/kg) and combination of BCP (20 mg/kg) and SD (20 mg/kg) had significantly (p < 0.05) lower ACE activity in comparison to the PD group.

Fig. 5
figure 5

Influence of β-caryophyllene, sildenafil and their combination on angiotensin-I converting enzymes of normal and paroxetine-induced sexual dysfunction in rats’ penile tissue. Bars represent mean ± standard deviation of five readings (n = 5). #p < 0.05; ##p < 0.01; ###p < 0.001; different from NC. *p < 0.05; **p < 0.01; ***p < 0.001; different from PD. NC normal control; SD sildenafil; PD paroxetine; BCP β-caryophyllene, BCP + SD combination of β-caryophyllene and sildenafil

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Figure 6 depicts a significant (p < 0.01) increase in the level of lipid peroxidation (LPO) of PD rat group when compared with NC group. However, the administration of BCP (10 & 20 mg/kg), SD (20 mg/kg) and both BCP (20 mg/kg) and SD (20 mg/kg) in PD rats caused significant (p < 0.01) reduction in LPO level as compared to PD group (Fig. 6). Normal rats treated with SD and BCP + SD had significantly (p < 0.05) lower LPO in comparison to the NC group (Fig. 6).

Fig. 6
figure 6

Influence of β-caryophyllene, sildenafil and their combination on lipid peroxidation level of normal and paroxetine-induced sexual dysfunction in rats’ penile tissue. Bars represent mean ± standard deviation of five readings (n = 5). #p < 0.05; ##p < 0.01; ###p < 0.001; different from NC. *p < 0.05; **p < 0.01; ***p < 0.001; different from PD. NC normal control; SD sildenafil; PD paroxetine; BCP β-caryophyllene, BCP + SD combination of β-caryophyllene and sildenafil

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Figure 7 reveals the histological representative micrographs of rat penile tissue (corpus cavernosum). Control rats show intact penile erectile tissues with large vascular cavernous spaces as well as regularly arranged connective tissue fibres and smooth muscles. Numerous endothelial cells lines cavernous spaces were seen. Fatty (adipose) infiltration into erectile tissues indicated by numerous empty cellular structures are obvious in PD rat groups (PIED). Additionally, the connective tissue fibres and smooth muscles were loosely arranged in PD rat group. Furthermore, PD rat groups treated with BC, SD and the combination of BCP and SD, showed near normal erectile tissues (Table 3).

Fig. 7
figure 7

Histological representative micrographs of rat penile tissue (corpus cavernosum). Control rats show intact penile erectile tissues with large vascular cavernous spaces (arrows), and regularly arranged smooth muscles. Paroxetine-induced erectile dysfunction (PD) rats show obvious fatty (adipose) infiltration into erectile tissues (dashed arrows) and loosely arranged connective tissue fibres and smooth muscles (circled area). Other treatment groups show near normal erectile tissues

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Table 3 Histopathological Score of rats induced with paroxetine and treated with β-Caryophyllene
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Discussion

Endothelial dysfunction is a crucial underlying factor for ED and vascular disease [3, 22]. Release of NO is central to the induction of smooth muscle cell relaxation, maintenance of vascular endothelial cells, inhibition of vascular smooth muscle proliferation and activation of soluble guanylate cyclase, thus resulting in lower intracellular calcium levels and vasodilatation [31]. Paroxetine is a selective serotonin reuptake inhibitors (SSRIs), that is often used to treat a wide range of depressive, anxiety, and other psychiatric disorders, with good therapeutic results [32, 33]. However, paroxetine has been reported to cause sexual dysfunction [34]. Erectile dysfunction in human subjects and animals have been associated with impairment in erection, oxidative degradation of penile tissues [3, 31]. Hence, there is continuous search for safe, natural and effective therapeutic agents from several phytochemicals from plant sources [19]. Plant phytochemicals have been reported to modulate pathophysiological pathways linked to ED [19, 35]. Considering the various biological properties of BCP, it is regarded as a multifunctional and polypharmacological agent for therapeutic development in complex diseases [21, 36, 37]. The mechanisms of sexual behavior involve initiation (arousal component), an intromission count, hit rate and copulation. The observed increase in both mounting and intromission latencies (Table 1) of PD group suggest that certain form of sexual dysfunction has occurred [38]. The decreased mounting and intromission latencies observed in PD rat groups administered BCP (10 and 20 mg/kg), SD (20 mg/kg) and combination of BCP (20 mg/kg) and SD (20 mg/kg) indicates the restoration of the desire for sex thus enhancing sexual behavior [38]. Furthermore, reduction in mounting and intromission frequencies (Table 2) in PD group noticed in this study support previous assertions that paroxetine; an antidepressant drug could cause sexual dysfunction and lower copulation in male rats [22, 32,33,34]. The observed increase in mounting and intromission frequencies of PD rats treated with BCP (10 and 20 mg/kg), SD (20 mg/kg) and the combination of BCP (20 mg/kg) and SD (20 mg/kg) could be attributed to possible enhancement of copulation and erectogenic efficiency of β-caryophyllene, sildenafil and their combination [39, 40]. Reduction in mounting and intromission latencies as well as elevation of mounting and intromission frequencies in rats with erectile dysfunction by β-caryophyllene, sildenafil and their combination agree with previous investigation where prophylactics and/or plant phytochemicals were reported to enhance sexual function in male rats [41,42,43].

Modulation in the activities of critical enzymes such as AChE, PDE-5, arginase, ADA, ACE, and oxidative stress index (lipid peroxidation) in penile tissues are crucial therapeutic strategies towards the management/treatment of endothelial dysfunction and erectile dysfunction [10, 36, 37]. In this study, elevation in AChE activity observed in PD group may be connected to erectile dysfunction. Increased AChE activity depletes acetylcholine level in penile tissues thus causing impairment of penile or sexual function [22]. Acetylcholine; a neurotransmitter which is a key determinant in the regulation of erection and improvement of erectile function via its action on vascular endothelium, which induces the release of NO and elevates cGMP [10, 36]. The reduction in AChE activity of rats treated with the combination of β-Caryophyllene (20 mg/kg/day) and sildenafil (20 mg/kg/day) in PD-Induced rats in this study indicate decreased degradation of acetylcholine in the penile tissues by β-Caryophyllene and sildenafil. This suggests that β-caryophyllene and sildenafil are promising and effective therapeutic agents for the management of anti-depressant-induced erectile dysfunction [44]. The result is in agreement with earlier reports from our laboratory on the inhibitory effect of plant phytochemicals on penile and heart AChE in vivo [22, 37, 44]. The combination of BCP and SD in PD-induced ED showed better reductive effect on AChE activity as compared to treatment with BCP or SD. This could be attributed to the mechanism of action of SD as a strong PDE-5′ inhibitor and not a specific inhibitor of AChE [9, 22]. Hence, the use of SD may not effectively improve erectile function in ED cases related to the reduction of acetylcholine or high AChE activity [22].

The use of PDE-5′ inhibitors has been established as an effective strategy for the treatment of antidepressant-induced ED in men [38, 44]. In this study, the administration of PD led to a significant increase in PDE-5′ activity in rats’ penile tissues. The observed increase in the activity of PDE-5′ in PD group may be linked with impairment in erectile function [9]. Elevated PDE-5 activity has been demonstrated to cause rapid degradation of cGMP, which can hinder the processes involved in penile erection. Thus, cGMP plays a major role in the erectile process as it triggers penile erection [35]. It induces the activation of cGMP-dependent protein kinase, which reduces calcium levels and trigger the relaxation of smooth muscles, contraction of the penile veins and rigidity of penile erection [2]. High PDE-5′ activity may indicate low levels of cGMP in the corpus cavernosum [45]. In this study, SD (20 mg/kg), BCP (20 mg/kg) and the combination of BCP and SD significantly reduced PDE-5′ activity in penile tissues of PD-induced rats. The observed inhibitory effects suggest that BCP are potent inhibitors of PDE-5′ activity. This indicates that BCP and SD could prevent rapid degradation of cGMP levels in corpus cavernosum of penile tissues, hence inducing the relaxation of smooth muscles and improving erectile process in antidepressant-induced ED [20]. The result suggests that BCP may show effective potentials in improving erectile function in anti-depressant-induced sexual dysfunction. The combination of BCP and SD in PD induced ED showed better inhibitory effect on PDE-5′ activity in comparison with treatment with either BCP or SD. Furthermore, the side effects associated with the use of sildenafil limit its use hence BCP may represent a safe and effective therapy that could reduce the side effects associated with SD [46].

The L-arginine-nitric oxide-guanylyl cyclase-cyclic guanosine monophosphate pathway is very important in the mechanism of penile erection [47]. The availability of NO is important to the erectile process [47]. Moreover, in the erectile tissue, NO is produced from L-arginine and oxygen in a reaction regulated by NO synthase. In the normal erectile process, sexual arousal stimulates the release of NO which penetrates into the smooth muscle cells and binds to guanylyl cylase thereby changing its conformation to form cGMP—an intracellular trigger of penile erection [31]. However, in ED cases, arginase competes with NO synthase to reduce the availability of NO in the endothelial cells [37, 48]. Findings from this study revealed that the administration of PD increased arginase activity in rat penile tissues. This indicates that antidepressants caused elevation in the activity of arginase, an enzyme that hinder the relaxation of smooth muscle cavernosal tissues and reduction of NO levels. However, treatment with BCP and SD reduced arginase activity in penile tissues of PD-induced rats. Our findings showed that BCP effectively reduced arginase activity in penile tissues of PD-induced rats as much as SD. The combination of BCP and SD in PD induced ED showed better inhibitory effect on arginase activity than treating with either BCP or SD in PD induced ED. The implication of this result is that administration of BCP may reduce the availability of NO as much as that of SD. In addition, BCP are not selective only for PDE-5′ but exhibit multiple inhibitory effects on different enzymes linked to ED. However, our findings suggest that the combinatorial effects of both BCP and SD showed better therapeutic action on arginase activity.

The administration of PD increased the activity of ADA in PD-induced rats. The observed increase in ADA activity in PD-induced rats could be associated with the alteration of adenosine signaling. Alteration of adenosine signaling has been linked with impairment in erectile function [11]. Adenosine acts as a neuromodulator in the corpus cavernosum, hence causing smooth muscle relaxation of penile tissues [11, 49]. ADA regulates adenosine levels in corpus cavernosal tissues by breaking it down to inosine [50]. However, the increase in ADA activity may deplete adenosine levels in the corpus cavernosum and impair penile erection. Our findings revealed that treatment with SD and BCP (20 mg/kg) reduced ADA activity in PD-induced rats. The combinatorial effects of BCP and SD showed better inhibitory action against ADA activity when compared to the treatment with either BCP or SD in PD induced ED. The observed decrease in ADA activity could be attributed to the interaction of the flavonoids with the enzyme. Previous report has shown that compounds such as carotenoids, quercetin, myricetin, naringin and kaempferol are potent inhibitors of ADA [51]. Although, the possible mechanism of action involved in the inhibition of ADA has not been fully understood, however there are indications that the hydroxyl groups of some of these flavonoids may interact with the active site of the enzyme, hence reducing the activity of the enzyme [51]. This finding suggests that BCP may alleviate the impairment of erectile function in anti-depressant-induced ADA by improving adenosine signaling in penile tissues.

Evidence has shown that ACE is involved in mechanisms involving penile erection via the regulation of angiotensin II (Ang II) levels in corpus cavernosal tissues [52]. ACE converts Ang I to Ang II and degrades bradykinin in the renin-angiotensin system (RAS). Ang II is a vasoconstrictor produced at physiological levels in the endothelial cells of the human corpus cavernosal tissues [53]. However, high levels of Ang II have been associated with the pathogenesis of ED. In this study, the administration of PD led to an increase in ACE activity in PD induced rats, which correlates with rapid conversion of Ang I to Ang II with concomitant increase in the levels of Ang II. Experimental investigations have shown that Ang II may induce smooth muscle cell contraction [53, 54] and elicit harmful effects such as fibrosis, proliferation and oxidative damage to corpus cavernosal tissues by binding to angiotensin II receptor type I (AT1). However, our findings revealed that treatment with SD and BCP (10 and 20 mg/kg) led to a significant decrease in ACE activity in PD-induced rats. The significant reduction of ACE activity observed in PD-induced rats treated with BCP indicates reduction in vasoconstriction necessitated by AngII. Moreover, treatment with BCP showed significant reduction in ACE activity compared to SD. The combination of BCP and SD in PD induced ED showed better inhibitory effect on ACE activity than treating with either BCP or SD in PD induced ED. This finding suggests that the use of BCP may be an effective strategy for the treatment of erectile dysfunction associated with hypertension.

Oxidative stress has been implicated as one of the factors involved in the pathogenesis of ED [55]. Lipid peroxidation is an important oxidative stress biomarker that has been identified in cavernosal tissues of ED patients [1]. Malondialdehyde (MDA) is a veritable index of lipid peroxidation [56]. The observed elevation in MDA levels in rats’ penile tissues could be attributed to increased oxidative damage to the penile tissue. This implies that administration of PD may aggravate free radicals release that will accelerate the destruction of the penile tissues and membrane lipid peroxidation. However, the observed decrease in MDA level in PD rats treated with BCP, SD and the combination of BCP and SD, could be attributed to the antioxidant properties of BCP and SD, which include free radical scavenging ability, ferric reducing power, iron chelating ability and donation of hydrogen. The observed non-significant difference in MDA production between the PD rats treated with either BCP or SD or the combination of BCP and SD may indicate possible safety in the use of BCP, SD or the combination of BCP and SD.

As observed in the histological architectures of the penile tissues (Fig. 7), fatty infiltration into erectile tissues as well as the loosely arranged connective tissue fibres and smooth muscles are indicative of erectile dysfunction in PD rats. Paroxetine, being one of the commonest selective serotonin reuptake inhibitors (SSRIs) often used, has been associated with damage to penile tissues due to oxidative insults, thus affecting the penile cell structural components (mainly proteins and lipid). Hence, the observed adipose tissue infiltration seen in PD group. This corroborates the assertion that excessive intake of paroxetine may cause erectile dysfunction [34]. However, BCP attenuated the PD induced erectile dysfunction in rat as shown by the intact nature of the penile tissues in BCP and SD treated PD rats. This could be attributed to the possible enzyme modulation and antioxidant properties of BCP and/ or SD. From this study, β-Caryophyllene and sildenafil significantly modulate the activities of critical enzymes (phosphodiesterase-5′, arginase, acetylcholinesterase, adenosine deaminase and Angiotensin-I converting enzyme) and lipid peroxidation (malondialdehyde), in penile tissues of paroxetine-induced erectile dysfunction in rats. The combination of BCP and SD in PD induced ED showed better reductive effect on critical enzymes activity than treating with BCP or SD alone in PD induced ED. This could be possible mechanisms by which BCP and/or SD alone attenuate PD induced erectile dysfunction in rats. Further studies in animal models at the molecular level are required to substantiate these findings.