The effects of rhoicissus tridentate dichloromethane extract and its bioactive compounds on uterotonic and glucose lowering activity in-vitro.
Mvelase, Zinhle Priscilla.
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Poor myometrial contractility during labour is associated with an extended partuition process that can affect the neonate’s health status. Chronic use of conventional treatments for diabetes mellitus is sometimes associated with undesirable effects. In an effort to overcome these challenges, we explored the ability of a crude medicinal plant extract of Rhoicissus tridentate (RT) and its bioactive compounds, beta-sitesterol (BS) and arjunolic acid (AA) to act as a multi-target agent to manage both poor myometrium contractility and diabetes mellitus. We thus investigated the uterotonic effects of RT and its bioactive ingredients BS and AA using uterine strips in-vitro. Further we studied the glucose lowering effects and possible mechanisms of action of RT and its bioactive compounds using muscle (C2C12) and liver (Chang) cell lines.For uterotonic studies, diethylstilboestrol-treated female Sprague-Dawley rats were sacrificed by decapitation and 2-3 cm of the uterine muscle was isolated and suspended in an organ bath containing aerated de Jalon’s buffer maintained at 32 ºC. Each muscle strip was treated with graded concentrations of RT (0.24-62.08 mg/mL) or BS (0.09-57.10 μmol/mL) or AA (0.37-22.80 μmol/mL). Rhythmic contractions in myometrial tissue incubated in the absence of plant extract or oxytocin served as the absolute control. To determine the synergistic effect of RT and its bioactive ingredients, the uterine strips were pre-treated with oxytocin (1.11 nmol/mL) before being treated with various concentrations of RT, BS or AA. Afterwards, the force and rate of contractions were recorded. Thereafter, the uterine strips were harvested for prostaglandin F2 alpha (PGF2α) and oxytocin-receptor concentration measurements. In experiment 2, we assessed the effects of RT/BS/AA on glucose metabolism. Liver (Chang) and muscle (C2C12) cell lines were cultured in Eagle’s Minimal Essential Medium and Dulbecco’s Modified Eagle’s Medium, respectively. To initiate glucose utilization experiments, separate preparations of muscle and liver cell lines were incubated at 37 ºC in a humidified incubator with 5% CO2 with the following concentrations of each compound, 12.5, 25 and 50 μg/ml. Both the liver and muscle cell lines were challenged with 19 mmol and 29 mmol of glucose respectively. Cells incubated in DMSO (0.1%), insulin (40 μg/mL) or metformin (16 μg/ml) served as untreated and treated positive controls. Media glucose concentration and cell viability were measured at 0, 12, 24 and 48 hour post incubation. Thereafter, cells were assessed for glycogen, GLUT 4 and glycogen synthase while the media harvested was assessed for Alanine amino transferase (ALT) and aspartate amino transferase (AST) activity. Our results showed an increase in the force and rate of uterine muscle contraction following treatment with RT, BS and AA (p<0.05). While co-treatment of RT/BS with oxytocin had a synergistic effect on force and rate of myometrium contractility, RT treatment resulted in decreased PGF2α and oxytocin receptor concentration. Treatment with BS/AA resulted in an increase in PGF2α receptor concentration, (p<0.05) but a decrease in oxytocin receptors. RT/BS and oxytocin resulted in increased PGF2α and oxytocin receptor concentration. The administration of RT/BS/AA did not result in cell toxicity in both cell lines. The individual treatments of RT and BS resulted in decreased ``media glucose concentration with concomitant increases in glycogen concentration after 48 hours in both cell lines. The increased GLUT4 concentration in muscle cell lines following treatment RT or BS supported this. Furthermore, RT administration resulted in increased glycogen synthase concentration in both cell lines. The observations suggest that RT promotes uterine muscle contractility and glucose disposal in the liver and skeletal muscle cell lines. This may suggest that RT and its associated bioactive compounds may be of benefit in alleviating poor myometrium contractility and diabetes-associated hyperglycaemia with BS playing a more active role than AA.