Abstract

Introduction: The role of Tridax procumbens leaf extract in erectile dysfunction (ED) of chronic variable stress (CVS) etiology is unknown. This study investigates the potential of the ethanol fraction of Tridax procumbens leaf (EETP) in modulation of CVS-induced ED.

Methods: Twenty-five male Wistar rats were divided into five groups of five rats each. Groups 1 & 2 (without stress) were treated with normal saline (vehicle) and 100 mg/kg of EETP, respectively. Groups 3–5 were treated as stress groups, with Group 3 co-treated with 100 mg/kg of EETP, and group 4 co-treated with vitamin C (7 mg/kg). Treatments were administered by oral gavage once daily for seven weeks. Oxidative biomarkers, cortisol, testosterone, and sperm parameters were determined, as well as the contractile mechanism of the corpus cavernosa to cumulative doses of calcium chloride, potassium chloride, acetylcholine, and sodium nitroprusside. Furthermore, the contractile mechanism was also determined after incubation in acetovanillone, nicorandil, methyl blue, and glibenclamide.

Results: Serum cortisol was significantly reduced, while testosterone was significantly increased in the EETP supplemented groups when compared to the CVS-only exposed group. Furthermore, malonaldehyde activity was decreased while superoxide dismutase concentration was increased in the EETP- and vitamin C-supplemented groups when compared to the CVS-only exposed group. Contraction (%) responses to calcium chloride and potassium chloride were also significantly reduced in the CVS-only exposed group when compared to the EETP-supplemented groups. The relaxation responses (%) to acetylcholine and SNP were significantly increased in the CVS group supplemented with EETP and vitamin C when compared to the CVS-only exposed group. The incubation of the cavernosa tissues in acetovanillone and nicorandil resulted in increased relaxation (%) in the CVS-only group, while incubation in glibenclamide caused increased relaxation in the EETP-supplemented groups compared to CVSonly exposed group. Sperm motility (%) was significantly reduced while abnormal spermatozoa was increased in the CVS-only exposed group when compared to the groups supplemented with EETP and Vitamin C.

Conclusion: Variable stress-induced dysfunctions in erectile mechanism were attenuated through supplementation with EETP.

Introduction

For decades, increasing incidence of erectile dysfunction (ED) has been a problem for public health1. Currently, a prevalence of 52% is reported in the United States, with approximately 40% of men having it at 40 years old and almost 70% by the age of 70 years1, 2. Occurrence in Nigeria is also high. A cross-sectional descriptive study previously revealed a prevalence of 45.7%3. Other studies revealed a prevalence of 41.5% in the Niger Delta4, 66.4% in Abuja5, 46.9% in Ilorin6, and 58.9% in Ogbomosho, in the southwestern region7 of the country.

The development of ED is multifactorial and usually involves neurologic, cardiovascular, hormonal, psychological. and local anatomic systems1. Chronic variable stress resulting in oxidative stress is also a known cause of erectile dysfunction and male infertility8. Numerous orthodox treatments are in common use, as some herbs and supplements have been found to be effective in the treatment of ED9. Furthermore, complementary and alternative medicine is hugely popular in Africa due to cultural beliefs, affordability, and accessibility10.

Tridax procumbens is a natural plant of the Asteraceae family that occurs naturally in tropical Africa, Asia, Australia, and India. Bioactive compounds, such as hydroxycinnamates, flavonoids, alkaloids, phytosterols, vitamin C, linoleic acid, proteins, tannins, and carotenoids, have been reported to be present in the plant11.

There is evidence that extracts of Tridax procumbens have potentials for modulating ED caused by experimental hypertension12 as well as paroxetine-induced13 ED. Vitamin C, a synthetic antioxidant, has been reported to attenuate chronic variable stress-induced ED14. Several reports have shown that Tridax procumbens has potent antioxidant properties15, 16, 17, 18.

Tridax procumbens leaf extract has shown potential in attenuating erectile dysfunction in experimental hypertensive and paroxetine-induced rats. However, the function of Tridax procumbens leaf extract in erectile dysfunctions of chronic variable stress etiology is unknown. This study investigates the potential of EETP in modulating and/or attenuating CVS-induced ED and sperm impairments in male Wistar rats.

Methods Animals, acclimatization and ethical approval

Twenty-five healthy adult male Wistar rats (180–200 g) were purchased from the Lagos State University College of Medicine animal house. Animals underwent acclimatization for two weeks within temperatures of 21–26 0C and 30%–70 % relative humidity. They were allowed access to standard food (Ladokun feeds Nigeria Ltd.) in pellet form and drinking water ad libitum. The NIH guide procedures for laboratory animal safety and use was adhered to at all times throughout the study and the procedures used on the animals were certified by the Lagos State University College of Medicine Animal Ethics Committee (AREC/2021/025).

Collection and preparation of ethanol extract of Tridax procumbens leaves (EETP)

Leaves of Tridax procumbens were collected from Lagos State University, Ojo, Lagos state, Nigeria in May 2022. The collected plant samples were authenticated by a certified taxonomist, with voucher number FHI 1008876. The extraction was performed according to previous literature13. Leaves were air-dried and ground to produce 300 g of product in smooth, powdered form. This was placed in a clean, conical flask and mixed with 5 liters of 96% ethanol, creating a suspension that was left to stand for 48 hours. The mixture was then filtered and the filtrate was left to stand for another 48 hours. After 48 hours, the filtrate was decanted carefully and allowed to concentrate by evaporation in a rotary evaporator at 35–40 0C. The yield was 5.83% (17.5 g) of a light-brown powdery extract.

Drugs and chemicals for the study

Vitamin C produced by the Emzor pharmaceutical company, Lagos, Nigeria was used. The potassium chloride, glucose, sodium bicarbonate, potassium phosphate, magnesium sulphate, calcium chloride, and sodium chloride used for the physiological salt solution were manufactured by J.T Baker Chemical Company, USA. Drugs used to assess dose response of the tissues included phenylephrine (PHE) and acetylcholine (Ach) purchased from Tocris, UK. Sodium nitroprusside was manufactured by Suvidhinath Laboratories, India, while acetovanillone, nicorandil, and N-nitro-l-arginine methyl ester (L-NAME) were purchased from AK Scientific, Inc., CA, USA. Glibenclamide was manufactured by Emcure Pharmaceuticals Ltd., while methyl blue, calcium chloride, and potassium chloride was produced by J.T Baker Chemical Company, USA.

Study design and treatment

Twenty-five male Wistar rats were divided into five groups of five rats each. Groups 1 and 2 (non-stress groups) were treated with normal saline (vehicle) and 100 mg/kg of EETP, respectively12. Groups 3, 4, and 5 were stress groups, with groups 3 and 4 co-treated with 100 mg/kg of EETP and vitamin C (7 mg/kg)14, respectively, while Group 5 received no supplementation. These treatments were administered via oral gavage once daily for seven weeks.

Procedure for variable stress exposure

The stress model of Mueller and Bale19 was used with slight modifications, as reported by Salami et al.14. These included sleep deprivation initiated by saturating the beddings of the cage with tepid water through the night; immobilization by restraining each rat in a 50 cl cylindrical container of 3 cm diameter for 20 minutes; fear inducement by placing a predator (cat) in cages with rats separated by a wire mesh; noise stress created by exposing rats to 100 decibels of noise during light cycle for 4 hours; stress from multiple cage changes by changing rats’ cages every 20 minutes interval during a light cycle for a period of 2 hours; and finally, exposure to foreign objects by introducing rough marbles and luminous beads in the cages of the animals at night. A different stress model was used each day for the entire duration of the stress exposure.

Animal sacrifice and serum collection

Animals were anaesthetized with 30 mg/kg of pentobarbital and sacrificed by cervical dislocation. Blood samples were taken from heart ventricles using sterile 5 ml syringes and needles; samples were then placed in plain bottles and allowed to stand for 15 minutes at room temperature. They were then centrifuged using a cold centrifuge (Model SM112, Uniscope Laboratory Centrifuge, England) at 4000 rpm for 15 mins, yielding a supernatant serum, which was carefully aspirated into empty plain bottles and stored at –40C.

Collection and preparation of corpus cavernosa

The corpus cavernosa of the rats were surgically removed and placed in a petri dish that contained physiological salt solution (PSS).

The corpus cavernosa of each animal across the groups were later suspended by a thread in a 50 ml chamber of the organ bath with PSS. Each corpus cavernosum was anchored with a stainless-steel hook to an electronic transducer (7004 model, Ugo-Basile, Varese, Italy) connected to a data capsule (model 17400) for isometric contraction recordings12. The organ bath was bubbled with a gas mixture of 95% oxygen and 5% carbon dioxide at a pH of 7.35–7.40, with temperature maintained at 370C.

Experiments regarding the contractile activity of the cavernosa tissue

The corpus cavernosa were allowed to equilibrate in the PSS for a period of 90 minutes. During this period, cavernosa tissue was stimulated three times at 30-minute intervals with 10-7 M phenylephrine. After equilibration, contractile responses of the cavernosa tissue to cumulative doses of acetylcholine (10-9–10-5 M), sodium nitroprusside (10-9–10-5 M), and potassium chloride (10–60 mM) were determined and recorded. Furthermore, the following were also investigated:

Addition of cumulative doses of calcium (10–60 mM, Tocris Biotechne, UK) to calcium-free Tyrode solution in the tissue chamber was used to investigate the influence of extracellular calcium influx on the contractile activity of the cavernosa tissue of all groups. Ca-free Tyrode solution was prepared by making Tyrode solution without calcium but with EDTA (0.5 mM). The influence of NADPH oxidase inhibitor on the contractile activity of the cavernosa tissue from all the groups was examined by incubating the tissue in acetovanillone (10-4 M, AK Scientific, Inc., CA, USA) for 15 minutes. Contractile responses of the tissue to cumulative doses of acetylcholine (10-9–10-5 M) were then recorded. The nitric oxide activity in the cavernosa tissues across groups was investigated by incubating cavernosa tissue in N-nitro-l-arginine methyl ester (L-NAME (10-4M; AK Scientific, Inc. CA, USA) for 15 minutes. Contractile responses of the tissue to cumulative doses of ACh (10-9–10-5 M) were then determined and recorded. The activity of the ATP-sensitive K+ channel was investigated in cavernosa tissue across groups by incubating cavernosa tissue in glibenclamide (10-4 M, AK Scientific, Inc., CA, USA) for 15 minutes. Contractile responses of the tissue to cumulative doses of ACh (10-9–10-5 M) were then determined and recorded. Nitric oxide donor and ATP-sensitive potassium channel activity in the corpus cavernosum tissues across groups was investigated by incubating the tissues in nicorandil (300 µM) for 15 minutes. Subsequently, the contractile responses of the cavernosa tissue to the cumulative doses of Ach (10-9–10-5 M) were obtained and recorded.Incubation for 15 minutes in methylene blue (10 µM, AK Scientific, Inc., CA, USA) was used to investigate the activity of soluble guanyl cyclase activity in the cavernosa tissue across groups. Contractile responses of the tissue to cumulative doses of ACh (10-9–10-5 M) were then determined and recorded after incubation.

Contractile responses were allowed to occur in a steady state before the addition of further doses and tissues were washed thrice before the use of a separate drug.

Determination of serum superoxide dismutase (SOD) and MDA concentration

Serum SOD was determined as described by Sun and Zigman20, while the method of Buege and Aust21 was used to determine MDA (i.e., an index of lipids peroxidation).

Determination of serum cortisol and testosterone

Serum cortisol was determined using the cortisol ELISA kit (Calbiotech, CA 92020, USA), while and testosterone was determined using the ELISA kit (Accu-Bind ELISA Lake Forest, CA 92630, USA).

Analysis of sperm motility, and morphology

The cauda epididymis was macerated and then immersed in 10 ml of PSS in a sterile specimen bottle. Using a pipette, an aliquot of the suspension was placed on a slide. The percentage of motility was then evaluated based on five different fields with a microscope (Cetti reset microscope). Five different fields were checked and recorded for progressive sperm motility in percentages, while morphological abnormalities were determined using a portion of sperm suspension placed on a glass slide and smeared out with another slide and stained with Leishman’s stain for morphological examination. Abnormalities of the sperm cells noted included coiled tail, headless, rotated head, and microcephaly conditions.

Statistical analysis

Results are presented here as mean ± standard error of mean in percentages. Tension was expressed as a percentage of the initial contraction to phenylephrine. Prism GraphPad (version 8.0.2) was the statistical software used for data analysis. Data were analyzed using the ANOVA test, with p values of less than 0.05 considered statistically significant.

Table 1.

Serum hormones and oxidative biomarkers across groups

Control EETP EETP+CVS EETP+VIT C CVS Cortisol (ng/ml) 28.5 ± 0.4 27.9 ± 0.6 25.2 ± 0.5* 31.5 ± 1.0 34.9 ± 1.3 Testosterone (ng/ml) 3.8 ± 0.2 4.0 ± 0.2 5.7 ± 0.3* 5.2 ± 0.4* 3.6 ± 0.3 MDA (µm/ml) 0.5 ± 0.01 0.5 ± 0.1 0.6 ± 0.1 0.7 ± 0.1 2.6 ± 0.7* SOD (µm/ml) 1.64 ± 0.05 1.67 ± 0.03 1.73 ± 0.05 1.71 ± 0.04 1.44 ± 0.22

Table 2.

Sperm motility (%) and abnormal sperm morphology (%) across treatment groups

Control EETP EETP+CVS EETP+VIT C CVS Motility (%) 55.22 ± 3.39 58.10 ± 2.96 76.25 ± 4.71 68.06 ± 7.65 12.00 ± 3.39* Abnormal morphology (%) 5.40 ± 0.93 5.60 ± 1.29 4.80 ± 0.80 5.80 ± 1.77 8.00 ± 1.61*

Table 3.

The mean body weights on the first, fourth and seventh week of treatment across groups

Weeks Control EETP CVS+EETP CVS+VIT C CVS 1 st