Biological Validation of Antioxidant-Rich Extracts of Fragaria × ananassa, Pterocarpus marsupium, and Moringa oleifera for Erythroid Differentiation and Fetal Hemoglobin Induction in K-562 Cells: A Continuation of Phytochemical Characterization toward Sup

Abstract

Background: β-thalassemia is an extremely prevalent inherited anemia associated with impaired β-globin chain synthesis with ineffective erythropoiesis and also causes chronic anemia, iron overload, and oxidative stress. Conventional treatment options such as blood transfusion and iron chelation therapies exhibit clinical as well as economical limitations. A number of recent scientific studies proposed that naturally occurring botanical polyphenols might be exploited as an adjunctive therapy to counteract oxidative stress and promote erythropoiesis and fetal hemoglobin (HbF) production. In our previous study, Fragaria × ananassa, Pterocarpus marsupium and Moringa oleifera showed appreciable amount of biologically active phytochemical contents along with noteworthy antioxidant potentials; hence, the present investigation was planned to evaluate their efficacy on cellular level.
Objective: The current experimental study aimed at evaluating the effect of antioxidant rich botanical extracts and major phytoconstituents on K-562 human erythroleukemia cells. The Experimental design comprises the cytotoxicity, survival rates, erythroid differentiation, total hemoglobin (THb), fetal hemoglobin (HbF), protein expressions and gene regulation test to estimated their therapeutic applicability.
Materials and Methods: Our experiment comprises ethanolic extracts of Fragaria × ananassa, Pterocarpus marsupium, Moringa oleifera and purified compounds such as myricetin, fisetin and pterostilbene were employed at varying concentrations of 0.25, 0.5 and 1 mg/mL against K-562 cell line. The percent cell viability was accessed by MTT and Trypan blue dye exclusion assays. Experiment uses the Colorimetric estimation and enzyme-linked immunosorbent assay (ELISA) for THb and HbF quantification. The benzidine spot test was used for erythroid differentiation and the protein expressions and gene regulations were analyzed by immunocytochemistry and reverse transcriptase polymerase chain reaction (RT-PCR), respectively. Hydroxyurea was taken as a standard reference compound and one-way analysis of variance (ANOVA) followed by Student’s t-test were performed to obtain the statistical significance between the treatment groups, while p < 0.05 was considered as statistically significant.
Results: The crude extracts of all the three medicinal plants revealed concentration-dependent cytotoxic effect against K-562 cells. The maximum decrease in percent cell viability was observed in the treatment group of Moringa oleifera followed by Pterocarpus marsupium and Fragaria × ananassa at 1 mg/mL concentration with 29.16, 34.64 and 40.51% inhibition, respectively. However, in the case of purified compounds, no considerable reduction in cell viability was observed with myricetin recorded as the most effective compound with 71.23% survival rate at 1 mg/mL dose. An eminent increment in percent THb and HbF was recorded in all the treatment groups as compared to the control with hydroxyurea treatment found to be the most effective one with 206.94 and 212.61% changes, respectively. Myricetin and Moringa oleifera exerted significant effect on erythroid differentiation of erythroblasts which was supported by the results of protein expressions and gene regulations.
Conclusion: The current study provided experimental evidence in support of the antioxidant potential of medicinal plants traditionally used in the management of β-thalassemia as described in ethnopharmacological literature. The present findings demonstrated the effective role of natural polyphenols and flavonoids in maintaining cellular viability at lower concentrations and promoting erythropoiesis and HbF production. The results of this study highlight the need of exploring phytochemicals for the management of β-thalassemia.