ERECTOGENIC ACTIVITIES AND MECHANISM OF ACTION OF AQUEOUS EXTRACT OF Ocimum gratissimum LEAF AND THYMOL ON PENILE TISSUE IN WISTAR RATS

Abstract

Erectile dysfunction is an increasing social and health concern. Relaxation of Corpus Cavernosum Smooth Muscle (CCSM) is required for penile erection. Thymol and thymol-containing plant, Ocimum gratissimum (OG), have been shown to have relaxant activity on aortic smooth muscle. However, modulation of smooth muscle relaxation is different across various tissues. There is thus, dearth of information on the erectogenic activities of OG or thymol. This study was designed to investigate the penile erectogenic activities of OG and thymol, and their probable mechanism of action in Wistar rats. Fresh OG leaves were procured from Bodija market, authenticated at Forestry Research Institute of Nigeria (FHI:110026), air-dried and macerated in distilled water to obtain Aqueous extract of OG (AeOG) which was lyophilised. Thirty-two male rats (170-190g) were used for in-vivo and ex-vivo experiments. Twenty rats for in-vivo study were grouped into four (n=5) and administered 0.5 mL/kg distilled water (control), 0.5 mL/kg corn oil, 300 mg/kg AeOG and 7.5 mg/kg analytical grade thymol orally for 28 days. Mounting latency and frequency were assessed as indices of mating behaviour. Serum Luteinizing Hormone and penile calcium-ATPase activities, Nitric Oxide (NO), cyclic Guanosine Mono-Phosphate (cGMP) and calcium levels were assayed spectrophotometrically. Ex-vivo effects of AeOG and thymol were assessed in 24 CCSM obtained from 12 rats. Using organ bath set-up, CCSM (n=6) were incubated in Kreb’s solution (control) or Kreb’s solution containing 60 μg/mL AeOG, 1% v/v ethanol or 0.06 μg/mL thymol. The CCSM were pre-contracted with phenylephrine (10-7M) or potassium chloride (KCl, 60 mM) for Maximum Contraction Response (MCR), and then relaxed with acetylcholine (10-5M) for Maximum Relaxation Response (MRR). Erectogenic mechanisms were then assessed via co-incubation with pathway inhibitors including Methylene Blue (MB, 10-4M), nifedipine (10-4M) or 5-(1,4-diazepan-1- ylsulfonyl)isoquinoline (HA-1077, 10-3M) for cGMP, calcium channel or Rho-Kinase blockade, respectively. Data were analysed using descriptive and ANOVA at α0.05. Mounting latency reduced in AeOG group by 59.6%. Mounting frequency increased by 63.8% and 47.7% in AeOG and thymol groups, respectively. Luteinizing Hormone (2.4±0.5 vs 1.1±0.1 μIU/mL) and Calcium-ATPase (4.3±0.1 vs 3.8±0.1 nmol/mg protein/hr) increased in AeOG group compared with control. Calcium (AeOG=18.7±0.3 and Thymol=18.8±0.2 10-4M/mL), NO (AeOG=11.4±2.0 and Thymol=12.9±1.6 μM/mL) and cGMP (AeOG=32.3±1.3 and Thymol=33.2±1.1 pM/mL) levels were similar to control (18.8±0.2 10-4M/mL, 11.6±1.1 μM/mL, 33.1±0.6 pM/mL, respectively). The MCR to phenylephrine (77.5±2.2%) was reduced by AeOG (67.2±4.6%) and thymol (68.7±1.1%), while MRR was only increased by thymol (65.0±1.9%) compared with control (54.2±2.1%). The MRR in AeOG (52.6±3.74%) was promoted by MB (60.7±3.4%) and reduced by nifedipine (46.1±1.9%). None of the inhibitors reduced the MRR in thymol. During KCl pre-contraction, MCR was reduced by AeOG (51.5±3.1%) and thymol (57.6±1.5%) compared with control (63.1±2.9%). Both AeOG (64.4±2.6%) and thymol (54.7±2.6%) increased MRR compared with control (47.6±3.4%). The effect of AeOG and thymol on MRR were reduced by nifedipine (56.8±2.9% and 47.5±2.3%, respectively) and HA-1077 (52.2±1.6% and 45.5±2.4%, respectively). The aqueous extract of Ocimum gratissimum leaf and thymol promoted penile erection. This erectogenic activity involved reduction of penile smooth muscle contraction sensitivity to calcium via Rho-Kinase pathway.