FRACTIONS OF Drymaria cordata (Linn) MODULATE MITOCHONDRIAL-MEDIATED APOPTOSIS IN NORMAL RAT LIVER AND MONOSODIUM GLUTAMATE-INDUCED UTERINE HYPERPLASIA

Abstract

The mitochondria have become an important component of apoptosis execution machinery. Mitochondrial Permeability Transition (mPT) pore opening constitutes the point of no return for apoptosis to take place when cytochrome C is released into the cytosol. Some dietary components of plant origin induce apoptosis in tumour cells via the induction of mPT pore opening. In folkloric medicine, Drymaria cordata (DC) is used to treat benign tumours in the liver and uterus. Therefore, this study was designed to investigate the effects of Methanol Extract of DC (MEDC) on mitochondrial-mediated apoptosis in rat liver and its ameliorative potential on Monosodium Glutamate (MSG)-induced uterine hyperplasia. The Drymaria cordata leaf was harvested, authenticated at the Department of Botany, University of Ibadan (UIH: 22555), air-dried, milled and extracted with methanol for 72 hours. The MEDC obtained was partitioned successively using chloroform, ethyl acetate and water to obtain CFDC, EFDC and AFDC fractions, respectively. Thirty female Wistar rats (100.0±8.0 g) were used for in vitro study using the fractions (10-90 µg/mL). Another set of 20 female Wistar rats (150.0±10.0 g) were divided into four groups (n=4) and orally treated for twenty-eight days as follows; control (1 mL/kg distilled water), 100 mg/kg CFDC, 200 mg/kg MSG and MSG+CFDC. Rats were sacrificed and the liver mitochondria isolated using differential centrifugation. The mPT pore, mitochondrial ATPase (mATPase), Cytochrome C release (CCR) and nuclear DNA (nDNA) fragmentation were determined by standard methods using a spectrophotometer. Caspases (Casp9 and Casp3) activities, estrogen, progesterone and total cholesterol levels were determined using ELISA, while the fibroblast cell count density was measured histomorphometrically using TS View CX Image® Software. The CFDC was subjected to GC-MS analysis to identify the compounds in the fraction. Data were analysed using descriptive statistics and ANOVA at α0.05.

The mPT pore opening was induced by MEDC (1.3, 2.7, 4.5, 10.2 and 13.3 folds), CFDC (3.6, 13.4, 15.3, 17.8 and 17.4 folds) at 10, 30, 50, 70 and 90 µg/mL, respectively, when compared with the control. The EFDC and AFDC did not induce pore opening at all concentrations tested. The CFDC enhanced mATPase activity (10.9±0.3 µmol Pi/mg protein/min) relative to control (3.2±0.2 µmol Pi/mg protein/min) and CCR (6.3±0.2 nmol/mg protein) relative to control (2.1±0.1 nmol/mg protein) maximally at 90 µg/mL. The CFDC (50, 100 and 200 mg/kg) increased hepatic nDNA fragmentation (30.1, 37.3 and 48.2%) relative to control (22.4%); increased activation of Casp9 (37.2±2.1, 51.0±3.2, 65.4±3.4 ng/mL) relative to control (29.2±2.3 ng/mL) and Casp3 (9.5±0.2, 12.3±0.2, 15.4±0.4 ng/mL) relative to control (8.5±0.2 ng/mL). The CFDC significantly reduced MSG-induced uterine hyperplasia from 0.009.±0.002 to 0.003±0.001 fibroblast count/µm2. In addition, the estrogen (142.2±8.3 pg/mL), progesterone (33.1±1.3 ng/mL) and total cholesterol (77.3±2.2 mg/dL) levels in the MSG-treated rats were significantly reduced to 86.4±6.3 pg/mL, 25.4±2.2 ng/mL and 40.6±2.3 mg/dL, respectively. The GC-MS analysis of CFDC revealed the presence of 3-(p-fluorobenzoyl)-propinoic acid (70.2%), bis(2-ethylhexyl) phthalate (12.0%) and n- hexadecanoic acid (7.5%). The chloroform fraction of methanol extract of Drymaria cordata induced mitochondrial-mediated apoptosis and protects against monosodium glutamate-induced uterine hyperplasia.