Chemicals/drugs
Lauric acid, Sodium nitroprusside (SNP), Acetylcholine (Ach), Phenylephrine (PE) and Streptozocin were obtained from Ak Scientific Inc. (USA) Sildenafil citrate was obtained from Pfizer pharmaceutical company (USA).
Animals
Thirty male Wistar rats (12 weeks old) weighing 150–200 g were used. The animals were kept in the animal house of Human Physiology Department, Ahmadu Bello University (ABU), Zaria. They were maintained under standard conditions and given food and water ad libitum. Ethical approval was obtained from the Ahmadu Bello University Animal care and use committee with the approval number, ABUCAUC/2017/019. Animals were handled in compliance with the World Medical Association Declaration of Helsinki regarding the ethical conduct of research involving animals [16].
The animals were divided into six groups (n = 5) as follows:
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Group 1 (NC) Normal Control animals
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Group 2 (DM-UT) Diabetic untreated animals
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Group 3 (DM + LA 90) Diabetic treated with lauric acid 90 mg/Kg orally
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Group 4 (DM + LA 180) Diabetic treated with lauric acid 180 mg/Kg orally [17]
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Group 5 (DM + LA 360) Diabetic treated with lauric acid 360 mg/Kg orally [17]
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Group 6 (DM + Sild) Diabetic rats treated with Sildenafil (20 mg/Kg) [18]
Diabetes was induced with diabetes by the administration of a single intraperitoneal injection of streptozotocin (65 mg/kg) in 0.1 M citric acid buffer at pH 4.5 [19]. The blood glucose level of the rats was checked 3 days after. Animals with fasting blood glucose levels greater than 200 mg/dL were marked as diabetic and placed in the diabetic groups (groups 2, 3 and 4 and 5). All treatments were for 4 weeks, and then the animals were sacrificed by cervical dislocation following chloroform inhalation.
Acute toxicity study
The acute toxicity was estimated following Lorke’s method [20], which involves two phases. For the first phase, nine male Wistar rats were divided into three groups of three rats each. Each group received 10, 100 and 1000 mg/kg of lauric acid, respectively. For the second phase, three male Wistar rats were divided into three groups of one rat each. Each group received 1600, 2900 and 5000 mg/kg of lauric acid, respectively. In both phases, the animals were observed for 24 h for any case of mortality or abnormal behaviour [21].
Measurement of blood glucose level
Blood glucose level was measured with the aid of the Accu-Chek glucose glucometer (Roche, USA), which works based on the glucose oxidase principle [22]. Blood was obtained by pricking the tip of the rat tail with a lancet.
Tissue preparation
After the rat has been sacrificed, the penis was cut out as a whole. The corpus cavernosum was then isolated from the surrounding penile tissues. Each cavernosal strip with both ends tied with a silk thread was placed in a 50 mL organ bath chamber filled with Krebs buffer solution with the following composition NaCl (119 mM), KCl (4.7 mM), NaHCO3 (15 mM), KH2PO4 (1.2 mM), MgSO4 (1.2 mM), CaCl2 (1.6 mM), and Glucose (11.5 mM). The Krebs solution was supplied with 95% O2 and 5% CO2 and maintained at a temperature of 37 °C. One end of the cavernosal strip was tied to a tissue holder and the other knotted to an Ugo Basile 7004 force transducer (Ugo Basile, Varese, Italy). The force transducer was connected to a 17,400 data capsule (Ugo Basile, Varese, Italy) which was then connected to a computer system. Thus, data relating to the contraction and relaxation response of the cavernosal tissues could be captured, displayed and then analysed. 2 g of tension was applied to each strip and allowed to equilibrate for 60–90 min. During the equilibration periods, the tissue was contracted with phenylephrine once every 30 min, and the organ bath solution was washed off and replaced 5 min after each injection of phenylephrine. Thereafter the contractile or relaxant responses of the tissue to the test drugs were evaluated [23].
Relaxation response to acetylcholine
Relaxation response of the corpus cavernosal strip to acetylcholine was used to evaluate endothelium-dependent relaxation. Following a contraction of the cavernosal strip with phenylephrine (10–7) or KCl (60 mM), the cumulative concentration of (10–9–10–3 M) was added to the organ bath. The concentration-relaxation response was then obtained [24]
Relaxation response to sodium nitroprusside
Relaxation response of the corpus cavernosal strip to sodium nitroprusside (SNP) was used to evaluate endothelium-independent relaxation. Cumulative doses of Sodium nitroprusside (SNP) (10–9–10–4 M) were injected into the organ bath after pre-contracting the tissues with phenylephrine (10–7) or KCl (60 mM) [24].
Statistical analysis
The contraction or relaxation responses were evaluated as percentage contraction or relaxation. The final results were presented as mean ± standard deviation (SD). Data were analysed using ANOVA and Bonferonni post-tests with the aid of Graph pad prism 5. Values with P < 0.05 were considered significant.
