Antihemolytic activity of hydroalcoholic leaves and bark extracts from Rhamnus alaternus against AAPH induced hemolysis on human erythrocytes

Abstract

Rhamnus alaternus is a Mediterranean shrub that has been used in traditional medicine to treat various diseases. This study aimed to determine the phenolic composition, as well as antioxidant and antihemolytic activities of R. alaternus leaves (LRA) and bark (BRA), extracts against AAPH-induced hemolysis. The extraction yields were 19.8% and 18.2% for leaves and bark. Total polyphenols (88.1 ± 1.83 mg GAE/g) and condensed tannins (36.24 ± 5.44 mg CE/g) were higher in BRA extract than in LRA extract (80.22 ± 1.4 mg GAE/g and 23.48 ± 0.25 mg CE/g, respectively). However, LRA extract was found to be richer in total flavonoids (64.6 ± 2.6 mg QE/g) and flavones/flavonol (18.34 ± 1.65 mg QE/g) than BRA extract (39.87 ±0.58 mg QE/g and 10.08 ± 0.35 mg QE/g), respectively. The IC50 of DPPH and ABTS radical scavenging activity were 86.59 ± 2 µg/ml and 12.49 ± 0.29 µg/ml for LRA extract and 69.23 ± 2.14 µg/ml and 12.83 ± 0.13 µg/ml for BRA extract, respectively. Also, both extracts showed good reducing power with 157.09 ± 5.53 mg Asc E/g for LRA extract and 194.97 ± 1.46 mg Asc E/g for BRA extract. The hemolytic effect was tested on human erythrocytes, and both extracts did not have cytotoxic effects at low doses. To induce hemolysis, AAPH was used at a concentration of 200 mM with an incubation time of 4h. The antihemolytic activity of the two extracts showed that pretreatment of human erythrocytes with various doses significantly reduced AAPH-induced hemolysis in a dose-dependent manner. Indeed, at 200 µg/ml, the percentages of hemolysis inhibition were 99.41 ± 1.17% and 76.26 ± 12.03% for BRA and LRA extracts, respectively. BRA extract was more effective (IC50 = 106.70 ± 1.48 µg/ml) compared to LRA extract (IC50 = 148.64 ± 7.04 µg/ml). Our results demonstrate for the first time that R. alaternus attenuates AAPH-induced hemolysis and can be used to prevent and treat hemolytic anemias.