Introduction

Sexual function is a multifaceted action comprising of a complex interplay between the endocrine, vascular, and nervous systems as well as other biostructures that are essential for satisfactory sexual arousal, intercourse, and performance. Despite the importance of satisfactory sexual function to the normal male species, demanding living conditions, certain prescriptive and several nonprescriptive medications, change in lifestyle, pollution, dietary deficiencies, and toxicants have negative effects on sexual life and result in sexual dysfunction (Chen et al. 2019; Kenneth 2001), with World Health Organization (WHO) listing sexual dysfunction as a major public health priority in both the rich and poor economic nations (WHO 2016). This has triggered research to synthesize drugs, such as Alprostadil, Buspirone, Cyproheptadine, and Sildenafil, which have been implicated to have serious side effects (Malviya et al. 2016). In Nigeria, a variety of natural products comprising crude extracts and aphrodisiac concoctions from plants are employed as alternatives to the synthesized drugs to mediate sexual motivation, prolonged duration, improved desire, and alertness with most of the crude extracts and aphrodisiac concoctions made from psychoactive plants, such as Cannabis sativa (Bonini et al. 2018).

Due to C. sativa been illicit in Nigeria, naturally available and licit psychoactive plants, Datura stramonium and Nicotiana tabacum are employed as alternatives for its traditional medicine and recreational usage. Their rates of prevalence are on the increase due to the different and new experiences they offer (Abdurahman et al. 2019; Dumbili 2020). Although, the use of crude extracts and aphrodisiac concoctions from these psychoactive plants for treatment and management of sexual dysfunction have been observed in folklore. There has been dearth of scientific validation for the traditional claims of the plants to improve or deplete sexual behavior and function during chronic exposure. Furthermore, the comparative effects between the illicit and licit substances to mediate sexual function or dysfunction in chronic studies are scarce.

Sexual behavior and function are initiated via sexual excitement, a process implicated to elevate nitric oxide (NO) release from endothelial cells and nervous terminations via nitric oxide synthase (NOs) action. Nitric oxide synthase (NOs) uses molecular oxygen and l-arginine as the substrate to trigger the release of NO, which then diffuses across the localized cell membrane into smooth muscles to trigger guanylyl cyclase enzymatic activation. This process in turn catalyzes guanosine-5′-triphosphate (GTP) biotransformation into 3′-5′-cyclic guanosine monophosphate (cGMP), a second messenger. The production of cGMP results in cGMP-dependent protein kinase (PKG) step activation, which in turn phosphorylates proteins that are crucial for relaxation. Consecutively, cGMP production also triggers intracellular calcium ion (Ca2+) concentrations reduction. Both post-cGMP production processes trigger arterial and trabecular smooth muscle relaxation, leading to arterial dilation, elevated blood flow to the penis, and a successive erection and stiffness of the penis (Masuku et al. 2020). However, inhibition feedback is induced by phosphodiesterase-5 (PDE-5) enzyme activity on the cGMP to induce penile detumescence and arteriolar vasoconstriction (Castela and Costa 2016), a process if overactivated may result in reduced cGMP levels and subsequently sexual dysfunction. Therefore, the ability of a drug to suppress PDE-5 enzyme activity will improve cGMP levels as well as sexual function (Odubanjo et al. 2018). More so, suppression of acetylcholinesterase (AChE) activity increases acetylcholine (ACh) concentrations at penis corpus cavernosum smooth muscle cells to induce relaxation at the muscle and increase penile tumescence by triggering endothelium NO production (Ojo et al. 2019). The renin-angiotensin system (RAS) has also been observed to play key roles in sexual function (Masuku et al. 2020). Meanwhile, reactive oxygen species (ROS) production has been implicated to oxidize tetrahydrobiopterin (BH4) (a substrate for NO synthesis) to dihydrobiopterin (BH2), resulting in uncoupling of NOs, which in turn results in superoxide production instead of NO production, thus leading to decreased NO levels (Zhao et al. 2015). Another essential toxicity indicator in sexual function/dysfunction is lactate dehydrogenase (LDH), an oxidoreductase (XOR) enzyme that is released into the bloodstream by cells after tissue damage (Akintunde et al. 2015). Several studies have evaluated the effects of the plants on sexual function but the present study will focus on comparing the abilities of the alkaloids-rich extracts from the plants to modulate sexual behavior and key enzymes relevant to sexual function in male Wister rats to justify their use as aphrodisiac agents.

Materials and methods

Sample collection and preparation

Cannabis sativa and Nicotiana tabacum leaves, and Datura stramonium seeds were obtained in towns of Akure South Local Government of Ondo State in Nigeria. The seeds of D. stramonium and leaves of C. sativa and N. tabacum were authenticated and identified at the CERAD (Centre for Research and Development) unit of the Federal University of Technology, Akure, Nigeria. The seeds and leaves were cleansed from debris, stalks, and other unwanted materials, and were afterward powdered with an electric blender.

Chemicals and reagents

Acetylthiocholine iodide, acetic acid, angiotensin-1 converting enzyme, methanol, estradiol benzoate, progesterone, hydrogen peroxide, l-arginine, Coomassie blue G, para-nitrophenyl phenylphosphonate (PNPPP), nitrate, and Ellman’s reagent was obtained from Sigma Chemical Co, St. Louis, MO, USA; Organon Limited, Kolkata, India; and Cadilla Healthcare Limited, Daman, India. All reagents used were of analytical grade and the water used was distilled.

Preparation of alkaloid-rich extracts

Alkaloid-rich extracts were prepared of Cannabis sativa and Nicotiana tabacum leaves and Datura stramonium seeds according to the methods of Ademiluyi et al. (2016).

Experimental protocol

Sixty-five (65) Wistar albino rats (male) weighing an average of 203 ± 10 g were purchased from the Biochemistry department animal house in the Federal University of Technology, Akure, Nigeria. Their handling and use were approved by the Animal ethical committee, Centre for Research and Development (CERAD) of the Federal University of Technology, Akure with the ethical number FUTA/ETH/2020/016. Stainless steel cages were used to house the Wistar albino rats in a room where 12-h light/dark cycle, relative humidity (60–70%), and room temperature (25–27 °C) were maintained all through the timing of the experiment. The rats were acclimatized for 2 weeks, after which they were grouped with each group consisting of five (5) rats randomly selected and housed in a cage. The animals were grouped and doses of extract selected according to the Organization for Economic Co-operation and Development (OECD) guideline (2001) as described by Patrick-Iwuanyanwu et al (2010) as follows:

  • Group 1 consisted of the normal control rats orally administered distilled water only.

  • Group 2 consisted of rats orally administered alkaloid-rich extracts of C. sativa once daily at doses of 5 mg/kg bwt.

  • Group 3 consisted of rats orally administered alkaloid-rich extracts of C. sativa once daily at doses of 50 mg/kg bwt.

  • Group 4 consisted of rats orally administered alkaloid-rich extracts of C. sativa once daily at doses of 500 mg/kg bwt.

  • Group 5 consisted of rats orally administered alkaloid-rich extracts of C. sativa once daily at doses of 2000 mg/kg bwt.

  • Group 6 consisted of rats orally administered alkaloid-rich extracts of D. stramonium once daily at doses of 5 mg/kg bwt.

  • Group 7 consisted of rats orally administered alkaloid-rich extracts of D. stramonium once daily at doses of 50 mg/kg bwt.

  • Group 8 consisted of rats orally administered alkaloid-rich extracts of D. stramonium once daily at doses of 500 mg/kg bwt.

  • Group 9 consisted of rats orally administered alkaloid-rich extracts of D. stramonium once daily at doses of 2000 mg/kg bwt.

  • Group 10 consisted of rats orally administered alkaloid-rich extracts of N. tabacum once daily at doses of 5 mg/kg bwt.

  • Group 11 consisted of rats orally administered alkaloid-rich extracts of N. tabacum once daily at doses of 50 mg/kg bwt.

  • Group 12 consisted of rats orally administered alkaloid-rich extracts of N. tabacum once daily at doses of 500 mg/kg bwt; and.

  • Group 13 consisted of rats orally administered alkaloid-rich extracts of N. tabacum once daily at doses of 2000 mg/kg bwt.

Experimental animals were administered the alkaloid-rich extracts through oral gavage within the hours of 09:00 to 11:00. They were allowed free access to rat chow and water ad libitum and the experiment lasted for 90 days after acclimatization.

Sexual behavioral assessment

On the 90th day of administration, alkaloid-treated male rats were each paired with estrous Wistar albino female rats to assess the sexual behavior of the alkaloid-treated male rats. The Female rats were induced to estrous by the administration of estradiol benzoate at a dose of 2 μg/kg body weight at 48 h and progesterone, 500 μg/kg body weight at 4 h respectively before the commencement of sexual behavioral studies. The sexual behavior was assessed using a plastic box (80 × 60 × 60 cm) that is clear and appropriate for digital video recordings in a different room from the administration room for 30 min per session. The behavioral study was carried out during the early hours starting at 6:00 a.m. Parameters assessed for sexual behavior were mounting number and latency and intromission number and latency (Ademosun et al. 2019; Guohua et al. 2009).

Tissue homogenate preparation

On the 91st day after commencement of experiment, alkaloid-treated male rats were anesthetized using mild diethyl ether. Alkaloid-treated male rats were sacrificed while the penile tissues were excised and washed quickly in a cold saline solution, after which they were homogenized in 0.1 M physiological buffer (pH 7.4) and centrifuged in a 10,000 g refrigerated centrifuge for 10 min. The supernatant was decanted and used for biochemical analyses.

Biochemical assays

The total protein content of penile tissue homogenate was measured using Coomassie blue method with bovine serum albumin serving as the standard (Bradford 1976). Nitric oxide (NO) level was measured using 2% VCl3 in 5% HCl, 0.1% N-(1-naphthyl)-ethylenediamine dihydrochloride, and 2% sulfanilamide (in 5% HCl) while the reduction of nitrate to nitrite by VCl3 was measured to determine the levels of NO at 540 nm (Miranda et al. 2001). The phosphodiesterase-5 activity was measured using p-nitrophenyl phenylphosphonate (5 mM) and 20 mM Tris buffer (pH 8.0), while the resultant p-nitrophenol produced was measured at 400 nm at intervals of 60, 120, 180, 240, and 300 s (Kelly and Butler 1977). Arginase activity was measured using 1.0 mM Tris–HCl buffer (pH) containing 1.0 mM MnCl2 and 0.1 M arginine solution. The reaction was terminated with 2.5 ml Ehrlich reagent (2.0 g of p-dimethylaminobenzaldehyde in 20 ml of absolute HCl) after 10 min of incubation at 37 °C and measured at 450 nm (Zhang et al. 2001); acetylcholinesterase activity was measured using 5,5′-dithiobis (2-nitrobenzoic) acid (3.3 mM) prepared by 0.1 M buffer (pH 7.0) constituting of NaHCO3. AChE iodide served as the substrate for the assay while absorbance was measured at 412-nm at intervals of 30, 60, 90, 120, 150, and 180 s (Perry et al. 2000). Angiotensin-1 converting enzyme (ACE) was measured using HHL (hippuryl-l-histidyl l-leucine) as substrate while the resultant hippuric acid (Bz-Gly) was measured to determine the activity of ACE at 228 nm (Cushman and Cheung 1971). Reactive oxygen species (ROS) level was measured using N–N-diethyl-para-phenylenediamine (DEPPD) (6 mg/ml) and ferrous sulfate (4.37 µM) prepared in 0.1 M sodium acetate (pH 4.8). H2O2 production was used to measure ROS level at 505 nm (Hayashi et al. 2007). Lactate dehydrogenase activity was measured using a commercially available kit (Randox Laboratories UK) (Weisshaar 1975).

Statistical analysis

All data were analyzed GraphPad Prism version 8.0 for windows using one-way analysis of variance (ANOVA) followed by Duncan multiple tests. Data were expressed as mean ± standard error of mean (SEM) of five (5) rats per group.

Results

Figure 1 revealed the effects of alkaloid extracts of Cannabis sativa, Datura stramonium, and Nicotiana tabacum at varying doses (5, 50, 500, and 2000 mg/kg bwt) on the mounting number (1a), mounting latency (1b), intromission number (1c), and intromission latency (1d) in comparison to the normal control group. Typically, the administration of C. sativa significantly increased (p ˂ 0.05) the mounting and intromission number at doses of 5 and 50 mg/kg bwt though a significant decline in mounting and intromission number was observed at higher doses of 500 and 2000 mg/kg bwt of C. sativa. Although similar trends were also observed with the alkaloid extracts of N. tabacum administration, the effects were not significantly different (p ˂ 0.05) compared to the normal control group. However, results revealed that alkaloid extracts of D. stramonium significantly reduced (p ˂ 0.05) the mounting and intromission number in a concentration-dependent manner compared to the normal control group. Furthermore, the effects of alkaloid extract of C. sativa and N. tabacum on mounting and intromission were observed to be biphasic while alkaloid extracts of D. stramonium were observed to significantly increase (p ˂ 0.05) the mounting and intromission latency in a concentration-dependent manner compared to the normal control group.

Fig. 1
figure 1

a Mounting number. C5—orally administered alkaloid extracts of C. sativa at 5 mg/kg bwt; C50—orally administered alkaloid extracts of C. sativa at 50 mg/kg bwt; C500—orally administered alkaloid extracts of C. sativa at 500 mg/kg bwt; C5000—orally administered alkaloid extracts of C. sativa at 5000 mg/kg bwt; D5—orally administered alkaloid extracts of D. stramonium at 5 mg/kg bwt; D50—orally administered alkaloid extracts of D. stramonium at 50 mg/kg bwt; D500—orally administered alkaloid extracts of D. stramonium at 500 mg/kg bwt; D5000—orally administered alkaloid extracts of D. stramonium at 5000 mg/kg bwt; T5—orally administered alkaloid extracts of N. tabacum at 5 mg/kg bwt; T50—orally administered alkaloid extracts of N. tabacum at 50 mg/kg bwt; T500—orally administered alkaloid extracts of N. tabacum at 500 mg/kg bwt; T5000—orally administered alkaloid extracts of N. tabacum at 5000 mg/kg bwt. Results are expressed as mean ± SEM (n = 5). Bars with superscript # are significantly (p < 0.05) from the normal control group. b C5—orally administered alkaloid extracts of C. sativa at 5 mg/kg bwt; C50—orally administered alkaloid extracts of C. sativa at 50 mg/kg bwt; C500—orally administered alkaloid extracts of C. sativa at 500 mg/kg bwt; C5000—orally administered alkaloid extracts of C. sativa at 5000 mg/kg bwt; D5—orally administered alkaloid extracts of D. stramonium at 5 mg/kg bwt; D50—orally administered alkaloid extracts of D. stramonium at 50 mg/kg bwt; D500—orally administered alkaloid extracts of D. stramonium at 500 mg/kg bwt; D5000—orally administered alkaloid extracts of D. stramonium at 5000 mg/kg bwt; T5—orally administered alkaloid extracts of N. tabacum at 5 mg/kg bwt; T50—orally administered alkaloid extracts of N. tabacum at 50 mg/kg bwt; T500—orally administered alkaloid extracts of N. tabacum at 500 mg/kg bwt; T5000—orally administered alkaloid extracts of N. tabacum at 5000 mg/kg bwt. Results are expressed as mean ± SEM (n = 5). Bars with superscript # are significantly (p < 0.05) from the normal control group. c C5—orally administered alkaloid extracts of C. sativa at 5 mg/kg bwt; C50—orally administered alkaloid extracts of C. sativa at 50 mg/kg bwt; C500—orally administered alkaloid extracts of C. sativa at 500 mg/kg bwt; C5000—orally administered alkaloid extracts of C. sativa at 5000 mg/kg bwt; D5—orally administered alkaloid extracts of D. stramonium at 5 mg/kg bwt; D50—orally administered alkaloid extracts of D. stramonium at 50 mg/kg bwt; D500—orally administered alkaloid extracts of D. stramonium at 500 mg/kg bwt; D5000—orally administered alkaloid extracts of D. stramonium at 5000 mg/kg bwt; T5—orally administered alkaloid extracts of N. tabacum at 5 mg/kg bwt; T50—orally administered alkaloid extracts of N. tabacum at 50 mg/kg bwt; T500—orally administered alkaloid extracts of N. tabacum at 500 mg/kg bwt; T5000—orally administered alkaloid extracts of N. tabacum at 5000 mg/kg bwt. Results are expressed as mean ± SEM (n = 5). Bars with superscript # are significantly (p < 0.05) from the normal control group. d C5—orally administered alkaloid extracts of C. sativa at 5 mg/kg bwt; C50—orally administered alkaloid extracts of C. sativa at 50 mg/kg bwt; C500—orally administered alkaloid extracts of C. sativa at 500 mg/kg bwt; C5000—orally administered alkaloid extracts of C. sativa at 5000 mg/kg bwt; D5—orally administered alkaloid extracts of D. stramonium at 5 mg/kg bwt; D50—orally administered alkaloid extracts of D. stramonium at 50 mg/kg bwt; D500—orally administered alkaloid extracts of D. stramonium at 500 mg/kg bwt; D5000—orally administered alkaloid extracts of D. stramonium at 5000 mg/kg bwt; T5—orally administered alkaloid extracts of N. tabacum at 5 mg/kg bwt; T50—orally administered alkaloid extracts of N. tabacum at 50 mg/kg bwt; T500—orally administered alkaloid extracts of N. tabacum at 500 mg/kg bwt; T5000—orally administered alkaloid extracts of N. tabacum at 5000 mg/kg bwt. Results are expressed as mean ± SEM (n = 5). Bars with superscript # are significantly (p < 0.05) from the normal control group

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The result of the effects of C. sativa, D. stramonium, and N. tabacum at varying doses (5, 50, 500, and 2000 mg/kg bwt) on nitric oxide (NO) levels in normal rats is presented in Fig. 2. Results revealed that administration of C. sativa alkaloids at 5, 50, and 500 mg/kg bwt significantly (p ˂ 0.05) elevated the NO level in comparison with the normal control group. The most prominent effect of C. sativa was observed at 500 mg/kg bwt while a decline in trend was observed at 2000 mg/kg bwt C. sativa alkaloid administration. Additionally, a similar trend was observed with N. tabacum alkaloid administration, with the effects of C. sativa alkaloids posing a prominent NO level than that observed with N. tabacum alkaloids. However, D. stramonium alkaloids administration significantly (p ˂ 0.05) depleted the level of NO in a concentration-dependent manner, when compared to the normal control group.

Fig. 2
figure 2

Nitric oxide level. C5—orally administered alkaloid extracts of C. sativa at 5 mg/kg bwt; C50—orally administered alkaloid extracts of C. sativa at 50 mg/kg bwt; C500—orally administered alkaloid extracts of C. sativa at 500 mg/kg bwt; C5000—orally administered alkaloid extracts of C. sativa at 5000 mg/kg bwt; D5—orally administered alkaloid extracts of D. stramonium at 5 mg/kg bwt; D50—orally administered alkaloid extracts of D. stramonium at 50 mg/kg bwt; D500—orally administered alkaloid extracts of D. stramonium at 500 mg/kg bwt; D5000—orally administered alkaloid extracts of D. stramonium at 5000 mg/kg bwt; T5—orally administered alkaloid extracts of N. tabacum at 5 mg/kg bwt; T50—orally administered alkaloid extracts of N. tabacum at 50 mg/kg bwt; T500—orally administered alkaloid extracts of N. tabacum at 500 mg/kg bwt; T5000—orally administered alkaloid extracts of N. tabacum at 5000 mg/kg bwt. Results are expressed as mean ± SEM (n = 5). Bars with superscript # are significantly (p < 0.05) from the normal control group

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Figure 3 revealed the effects of C. sativa, D. stramonium, and N. tabacum at varying doses (5, 50, 500, and 2000 mg/kg bwt) on the activity of phosphodiesterase-5 (PDE-5) in normal rats. C. sativa alkaloids significantly reduced (p ˂ 0.05) the activities of PDE-5 in normal rats when compared to the normal control group. Meanwhile, D. stramonium alkaloids significantly (p ˂ 0.05) increased the activities of PDE-5 in a concentration-dependent manner. However, reduction in PDE-5 activities was also observed with N. tabacum alkaloid administration while showing no significant level of difference (p ˂ 0.05) in comparison to the normal control group.

Fig. 3
figure 3

Phosphodiesterase-5 activity. C5—orally administered alkaloid extracts of C. sativa at 5 mg/kg bwt; C50—orally administered alkaloid extracts of C. sativa at 50 mg/kg bwt; C500—orally administered alkaloid extracts of C. sativa at 500 mg/kg bwt; C5000—orally administered alkaloid extracts of C. sativa at 5000 mg/kg bwt; D5—orally administered alkaloid extracts of D. stramonium at 5 mg/kg bwt; D50—orally administered alkaloid extracts of D. stramonium at 50 mg/kg bwt; D500—orally administered alkaloid extracts of D. stramonium at 500 mg/kg bwt; D5000—orally administered alkaloid extracts of D. stramonium at 5000 mg/kg bwt; T5—orally administered alkaloid extracts of N. tabacum at 5 mg/kg bwt; T50—orally administered alkaloid extracts of N. tabacum at 50 mg/kg bwt; T500—orally administered alkaloid extracts of N. tabacum at 500 mg/kg bwt; T5000—orally administered alkaloid extracts of N. tabacum at 5000 mg/kg bwt. Results are expressed as mean ± SEM (n = 5). Bars with superscript # are significantly (p < 0.05) from the normal control group

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C. sativa, D. stramonium, and N. tabacum at varying doses (5, 50, 500, and 2000 mg/kg bwt) also caused significant (p ˂ 0.05) effects on arginase activity in normal rats (Fig. 4). The activities of arginase were significantly reduced (p ˂ 0.05) in C. sativa and N. tabacum alkaloids treated groups in a concentration-dependent manner in comparison to the normal control group. Whereas, administration of D. stramonium alkaloid caused a significant (p ˂ 0.05) increase in the activities of arginase in a concentration-dependent manner in comparison to the normal control group.

Fig. 4
figure 4

Arginase activity. C5—orally administered alkaloid extracts of C. sativa at 5 mg/kg bwt; C50—orally administered alkaloid extracts of C. sativa at 50 mg/kg bwt; C500—orally administered alkaloid extracts of C. sativa at 500 mg/kg bwt; C5000—orally administered alkaloid extracts of C. sativa at 5000 mg/kg bwt; D5—orally administered alkaloid extracts of D. stramonium at 5 mg/kg bwt; D50—orally administered alkaloid extracts of D. stramonium at 50 mg/kg bwt; D500—orally administered alkaloid extracts of D. stramonium at 500 mg/kg bwt; D5000—orally administered alkaloid extracts of D. stramonium at 5000 mg/kg bwt; T5—orally administered alkaloid extracts of N. tabacum at 5 mg/kg bwt; T50—orally administered alkaloid extracts of N. tabacum at 50 mg/kg bwt; T500—orally administered alkaloid extracts of N. tabacum at 500 mg/kg bwt; T5000—orally administered alkaloid extracts of N. tabacum at 5000 mg/kg bwt. Results are expressed as mean ± SEM (n = 5). Bars with superscript # are significantly (p < 0.05) from the normal control group

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The result of C. sativa, D. stramonium, and N. tabacum administration at varying doses (5, 50, 500, and 2000 mg/kg bwt) on acetylcholinesterase (AChE) activity are presented in Fig. 5. The activity of AChE was significantly (p ˂ 0.05) reduced in normal rats at C. sativa, D. stramonium, and N. tabacum administration in a concentration-dependent manner, with D. stramonium alkaloid posing the most prominent effect when compared to the normal control group.

Fig. 5
figure 5

Acetylcholinesterase activity. C5—orally administered alkaloid extracts of C. sativa at 5 mg/kg bwt; C50—orally administered alkaloid extracts of C. sativa at 50 mg/kg bwt; C500—orally administered alkaloid extracts of C. sativa at 500 mg/kg bwt; C5000—orally administered alkaloid extracts of C. sativa at 5000 mg/kg bwt; D5—orally administered alkaloid extracts of D. stramonium at 5 mg/kg bwt; D50—orally administered alkaloid extracts of D. stramonium at 50 mg/kg bwt; D500—orally administered alkaloid extracts of D. stramonium at 500 mg/kg bwt; D5000—orally administered alkaloid extracts of D. stramonium at 5000 mg/kg bwt; T5—orally administered alkaloid extracts of N. tabacum at 5 mg/kg bwt; T50—orally administered alkaloid extracts of N. tabacum at 50 mg/kg bwt; T500—orally administered alkaloid extracts of N. tabacum at 500 mg/kg bwt; T5000—orally administered alkaloid extracts of N. tabacum at 5000 mg/kg bwt. Results are expressed as mean ± SEM (n = 5). Bars with superscript # are significantly (p < 0.05) from the normal control group

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Figure 6 showed that the activities of angiotensin-1 converting enzyme (ACE) were significantly (p ˂ 0.05) reduced in a concentration-dependent manner during chronic administration of C. sativa and N. tabacum at doses of 5, 50, and 500 mg/kg. However, the reduction pattern was absent at 2000 mg/kg bwt administration which showed a concomitant increase but lower than that observed in the normal control group. Interestingly, chronic administration of D. stramonium was observed to increase ACE activities in a concentration-dependent manner.

Fig. 6
figure 6

Angiotensin-1 converting enzyme activity. C5—orally administered alkaloid extracts of C. sativa at 5 mg/kg bwt; C50—orally administered alkaloid extracts of C. sativa at 50 mg/kg bwt; C500—orally administered alkaloid extracts of C. sativa at 500 mg/kg bwt; C5000—orally administered alkaloid extracts of C. sativa at 5000 mg/kg bwt; D5—orally administered alkaloid extracts of D. stramonium at 5 mg/kg bwt; D50—orally administered alkaloid extracts of D. stramonium at 50 mg/kg bwt; D500—orally administered alkaloid extracts of D. stramonium at 500 mg/kg bwt; D5000—orally administered alkaloid extracts of D. stramonium at 5000 mg/kg bwt; T5—orally administered alkaloid extracts of N. tabacum at 5 mg/kg bwt; T50—orally administered alkaloid extracts of N. tabacum at 50 mg/kg bwt; T500—orally administered alkaloid extracts of N. tabacum at 500 mg/kg bwt; T5000—orally administered alkaloid extracts of N. tabacum at 5000 mg/kg bwt. Results are expressed as mean ± SEM (n = 5). Bars with superscript # are significantly (p < 0.05) from the normal control group

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Figure 7 reveals the effects of C. sativa, D. stramonium, and N. tabacum at varying doses (5, 50, 500, and 2000 mg/kg bwt) on reactive oxygen species (ROS) levels in comparison with normal control rats. Administration of C. sativa alkaloid at 5 and 50 mg/kg bwt was observed to lower the level of ROS while higher doses of 500 and 2000 mg/kg bwt of the alkaloids significantly (p ˂ 0.05) elevated ROS level. However, D. stramonium and N. tabacum alkaloids administration significantly (p ˂ 0.05) increased ROS level in normal rats, with D. stramonium posing a higher level of ROS production.

Fig. 7
figure 7

Reactive oxygen species production level. C5—orally administered alkaloid extracts of C. sativa at 5 mg/kg bwt; C50—orally administered alkaloid extracts of C. sativa at 50 mg/kg bwt; C500—orally administered alkaloid extracts of C. sativa at 500 mg/kg bwt; C5000—orally administered alkaloid extracts of C. sativa at 5000 mg/kg bwt; D5—orally administered alkaloid extracts of D. stramonium at 5 mg/kg bwt; D50—orally administered alkaloid extracts of D. stramonium at 50 mg/kg bwt; D500—orally administered alkaloid extracts of D. stramonium at 500 mg/kg bwt; D5000—orally administered alkaloid extracts of D. stramonium at 5000 mg/kg bwt; T5—orally administered alkaloid extracts of N. tabacum at 5 mg/kg bwt; T50—orally administered alkaloid extracts of N. tabacum at 50 mg/kg bwt; T500—orally administered alkaloid extracts of N. tabacum at 500 mg/kg bwt; T5000—orally administered alkaloid extracts of N. tabacum at 5000 mg/kg bwt. Results are expressed as mean ± SEM (n = 5). Bars with superscript # are significantly (p < 0.05) from the normal control group

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The effects of C. sativa, D. stramonium, and N. tabacum at varying doses (5, 50, 500, and 2000 mg/kg bwt) on the activity of lactate dehydrogenase (LDH), a functional asthenospermia marker in rat testes, are shown in Fig. 8. Administration of C. sativa alkaloids improved the activity of LDH at 5 and 50 mg/kg bwt doses, though dosages of 500 and 2000 mg/kg bwt showed inhibitory effects on LDH activities. Effects of D. stramonium and N. tabacum were revealed to inhibit significantly (p ˂ 0.05) the activities of LDH. The figure further revealed that the potency of D. stramonium alkaloids to inhibit LDH activities was higher in comparison to that expressed by N. tabacum and alkaloids.

Fig. 8
figure 8

Lactate dehydrogenase activity. C5—orally administered alkaloid extracts of C. sativa at 5 mg/kg bwt; C50—orally administered alkaloid extracts of C. sativa at 50 mg/kg bwt; C500—orally administered alkaloid extracts of C. sativa at 500 mg/kg bwt; C5000—orally administered alkaloid extracts of C. sativa at 5000 mg/kg bwt; D5—orally administered alkaloid extracts of D. stramonium at 5 mg/kg bwt; D50—orally administered alkaloid extracts of D. stramonium at 50 mg/kg bwt; D500—orally administered alkaloid extracts of D. stramonium at 500 mg/kg bwt; D5000—orally administered alkaloid extracts of D. stramonium at 5000 mg/kg bwt; T5—orally administered alkaloid extracts of N. tabacum at 5 mg/kg bwt; T50—orally administered alkaloid extracts of N. tabacum at 50 mg/kg bwt; T500—orally administered alkaloid extracts of N. tabacum at 500 mg/kg bwt; T5000—orally administered alkaloid extracts of N. tabacum at 5000 mg/kg bwt. Results are expressed as mean ± SEM (n = 5). Bars with superscript # are significantly (p < 0.05) from the normal control group

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Discussions

As observed in the present study, mounting number, mounting latency, intromission number, and intromission latency were greatly altered by exposure to the different concentrations of C. sativa, D. stramonium, and N. tabacum alkaloids in comparison to the normal control group. Interestingly, the study revealed a curvilinear model of effect on sexual activities. This biphasic model of the effect of the alkaloids is consistent with other studies that observed that chronic administration of plant-based psychoactive substances improves sexual function at low doses while at high doses impairment of testosterone secretion and sperm production was observed (German et al. 2014; Johnson et al. 2020). At the lower doses of administration (5 and 50 mg/kg bwt), the alkaloid extracts tend to favorably influence sexual activities but at higher doses of 500 and 2000 mg/kg bwt, the sexual activities were adversely affected with C. sativa having the least deterioration. The decreased mounting and intromission latencies observed at lower doses may indicate the potential of C. sativa and N. tabacum to enhance the sex desire and sexual behavior in sexual dysfunctional individuals (Outhoff 2009). The trend of compromised sexual behavior at higher doses of C. sativa and N. tabacum and decreased sexual activities in D. stramonium groups as observed in the present study could result from elevated arginase and PDE-5 activities which have been established to result in lower copulation and sexual dysfunction (Adefegha et al. 2021).

Nitric oxide (NO) release is central to the initiation of activation of soluble guanylate cyclase, inhibition of vascular smooth muscle proliferation, smooth muscle cell relaxation, and maintenance of vascular endothelial cells, which leads to vasodilation and reduced intracellular calcium levels during sexual activities (Andersson 2011). Results from the present study showed that C. sativa and N. tabacum posed a significant curvilinear effect while D. stramonium showed a concentration-dependent reduction in NO level. The possible explanation may be that the C. sativa and N. tabacum alkaloids increased endothelial tissue NOs activity expression and/or inhibited NO elimination, thereby resulting in increased nitric oxide bioavailability (Sasatomi et al. 2008).

The first-line of drugs endorsed by the United States of Food and Drug Administration (FDA) in the management and treatment of sexual dysfunction are drugs that can prevent the PDE-5 enzyme from depleting cGMP levels and impeding arterial blood flow, a process that results in vasodilation and corpus cavernosum tissue relaxation as well as prolonged penile erection and improved sexual function (Hatzimouratidis et al. 2016; Sommer and Schulze 2005). Interestingly, C. sativa and N. tabacum alkaloids inhibited the activities of PDE-5 while D. stramonium alkaloids upregulated the activities of the enzyme. The ability of C. sativa and N. tabacum alkaloids to inhibit PDE-5 may be one of the mechanisms employed by the plant alkaloids to improve mounting and intromission numbers as observed in the present study, as the inhibition of PDE-5 will reduce rapid hydrolysis of cGMP by PDE-5 and thereby, prolonged penile erection and improved sexual functions. Meanwhile, the dose-dependent increase in PDE-5 activity observed with D. stramonium administration may be due to disorder in the NO/cGMP pathway, a major factor in sexual dysfunction (Lugnier 2006).

Another key mediator of reduced NO production during sexual activity is elevated arginase activity as observed in the present study. Whenever the activity of arginase is elevated, hydrolysis of l-arginine to urea and ornithine is increased via urea cycle, while the bioavailability and concentration of NO are depleted, due to NO synthase and arginase dependence on the same substrate (l-arginine) (Caldwell et al. 2018). The present study observed that C. sativa and N. tabacum both decreased arginase activity significantly while D. stramonium increased arginase activity significantly. This may be the possible explanation for the effect of the alkaloids on NO release, as NO release is linked with an agent inhibitory effect on arginase activity (Lorenzen et al. 2009; Adefegha et al. 2018). Although most sexual dysfunction management/treatment drugs are PDE-5 inhibitors and not arginase inhibitors, administration of PDE-5 inhibitors to manage/treat sexual dysfunction where arginase activity prevails is usually ineffective. Therefore, the inhibitory effect of both C. sativa and N. tabacum alkaloid extracts on arginase activity may be another mechanism employed by the plants to improve the mounting and intromission number observed in the present study and further indicate their potential to improve sexual function as observed in folklore.

Acetylcholine (ACh), a parasympathetic neurotransmitter, mediates sexual function by relaxing the corpus cavernosum smooth muscle of the penis and enhances penile tumescence by triggering endothelial NO production (Ojo et al. 2019). Therefore, the ability of a drug to inhibit the activity of AChE, the enzyme that hydrolysis ACh to choline and acetate, will increase ACh levels and enhance penile erection (Heidelbaugh 2010; Ojo et al. 2019). Interestingly, all the alkaloid extracts inhibited AChE activity significantly with D. stramonium showing the highest level of inhibition. This substantiates a previous study that implicated D. stramonium alkaloids (atropine, hyoscyamine, and scopolamine) as a potent cholinesterase inhibitor (Fasakin et al. 2021).

Another pathway that has also been implicated in sexual function/dysfunction pathophysiology is the renin-angiotensin system (RAS) pathway (Odubanjo et al. 2018). The enzyme angiotensin-1 converting enzyme (ACE) is the enzyme tasked with catalyzing angiotensin-1 conversion to the potent angiotensin-2, the main constituent of the renin-angiotensin system (RAS), which mediates stimulated contraction of the corpus cavernosum smooth muscles during sexual functions (Fraga-Silva et al. 2013; Oboh et al. 2017). Therefore, the high activities of ACE observed with D. stramonium administration may result in chronic high levels of angiotensin-2 which have been implicated to result in angiotensin type-1 receptor (AT1R) activation, leading to stimulation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and vasoconstriction, which results into excessive superoxide anions generation, depleted endothelial nitric oxide concentration, and sexual dysfunction (Ademiluyi et al. 2019; Jiménez et al. 2019; Passaglia et al. 2015; Rincón et al. 2015). However, the reduction in ACE activities observed during C. sativa and N. tabacum administration may suggest their ability to act as ACE inhibitors at the dosages.

Furthermore, the present study observed that the alkaloid extracts elevated ROS production in a concentration-dependent manner, with significant levels of increase observed mostly at 500 and 2000 mg/kg bwt. The study also observed that D. stramonium had the highest level of ROS production. The elevation of ROS level has been linked with angiotensin-2 production (Adefegha et al. 2018), a process that was also observed in the present study. Meanwhile, elevated ROS have been implicated to react with NO to produce peroxynitrite (ONOO–) which decreases the bioavailability and concentration of NO (Zhao et al. 2015). Furthermore, the resulting peroxynitrite was implicated to cause the generation of powerful oxidants such as nitrogen dioxide (NO2) and hydroxyl radicals which result in oxidative stress and consecutively, sexual dysfunction (Tabit et al. 2010).

LDH activity has been widely used in assessing cellular ATP levels during anaerobic conditions due to its stability (Akintunde et al. 2015). The present study observed that C. sativa had a curvilinear effect on the enzyme while D. stramonium and N. tabacum significantly reduced the enzyme activity. The inhibitory effect of D. stramonium and N. tabacum, and C. sativa only at high doses on LDH activity can indicate an alteration to the metabolic pathway responsible for energy (ATP) production. The finding agrees with previous studies that observed abnormal spermatogenesis and sexual dysfunction in link with depleted ATP levels (Klaassen and Watkins 2003; Akintunde et al. 2015), an indication that only C. sativa alkaloids at doses of 5 and 50 mg/kg bwt may have potential to ameliorate depleted ATP levels in sexual dysfunctional individuals.

Conclusions

Although, the relationship between psychoactive plant alkaloids and sexual function may be complex, the effect of the alkaloids on sexual behavior and key enzymes relevant to sexual function may proffer us mechanisms of action to justify their use in folklore as crude extracts or ingredients of aphrodisiac concoctions. According to our results, C. sativa and N. tabacum showed significant potency to improve sexual function (especially at 5 and 50 mg/kg bwt) while D. stramonium showed significant potency to facilitate sexual dysfunction across all doses during chronic exposure. This is an indication that the use of D. stramonium as ingredients of aphrodisiac concoctions may be to elevate the concoction’s psychoactive effects and not majorly to improve the aphrodisiac potentials of the concoction. However, the use of C. sativa and N. tabacum crude extracts and as aphrodisiac concoction ingredients in folklore may be justified but should be discouraged as its administration at high doses was observed to pose toxicological effects that may result in sexual dysfunction and even cause hazardous effects to users. Furthermore, molecular and clinical studies, as well as chemical identification and isolation of the bioactive compounds of these psychoactive plants, are recommended to evaluate their pharmacokinetic, pharmacodynamic, and toxicological mechanism of action to mediate the observed sexual function or dysfunction.