Protective effects of stem bark of Harungana madgascariensis on the red blood cell membrane

Anemia is a condition that has multiple origins. One such origin is the destruction of red blood cells’ (RBCs) membrane induced by free radicals. Treatment of anemia could therefore be enhanced by the use of free radicals’ scavengers potentially found in some medicinal plants. In this study, the protective effect of Harungana madagascariensis on the RBCs’ membrane physiology was investigated in vitro and in vivo.

In vitro hemolytic anemia was induced by incubation of fresh human RBCs with carbontetrachloride (CCl₄) in Olive oil (Oo). Relaxation times of protons excited at 20 MHz (Carr-Purcell-Meiboom-Gill pulse sequence) in the absence or presence of paramagnetic Mn²⁺ ions (T _2i for “extracellular” water and T _2a for “intracellular” water, respectively) were determined at several temperatures (25–37°C) via Nuclear Magnetic Resonance (NMR) on a Bruker Minispec spectrometer. Water exchange times (T_e) were consequently calculated using the Conlon-Outhred equation: 1/T_e = (1/T_2a) – (1/T_2i). Morphological characteristics (mean cell volume, V, and cell surface area, A) were determined by photonic microscopy and the RBCs’ diffusional water permeability (P _d ) was calculated as P_d = (1/T_e)*(V_a/A), where V_a is the aqueous volume in the RBC and is about 0.7 of the cell volume (V). The activation energy of the diffusional process (E_a) for the respective temperature range was estimated using the Arrhenius modified equation k = A(T/T₀)ⁿ*e^-Ea/RT. Inhibition of the water diffusion induced by incubation with para-chloro-mercuribenzoic acid (PCMB) at 25, 30 and 37°C was calculated as I(%) = [(Pd control – Pd sample)/Pd control]*100.

To investigate the protective influence of the extract on the RBC membrane, inhibition of the water permeability was evaluated on membranes pre-incubated with the Harungana madagascariensis extract. Male rats were used in in vivo investigations. Malondialdehyde (MDA) and cholesterol in the RBC membrane were estimated by induction of lipid peroxidation while the antioxidant properties of catalase (CAT) and superoxide dismutase (SOD) on the membrane were evaluated in regard to their antioxidant properties on the membrane.

T _2a significantly decreased at each temperature. Te results were higher in both RBCs and RBCs + extract groups incubated with PCMB compared to non-incubated controls, but differences were not statistically significant. A high percentage (73.81 ± 7.22) of RBCs pre-incubated with the extract presented the regular biconcave shape. Inhibition by PCMB of the RBCs’ membrane water permeability was increased at 30°C and decreased in the presence of extract (25°C and 37°C), while Ea decreased from 30.52 ± 1.3 KJ/mol to 25.49 ± 1.84 KJ/mol. Presence of the Harungana madagascariensis extract normalized the SOD and CAT activities as well as the MDA and membrane cholesterol concentrations altered by the CCl₄-induced oxidative stress.