Trace Element and Phytochemical Characterization of Pinus pinaster and Pinus halepensis Bark Extracts with Protective Effects Against Phenylhydrazine-induced Hemolytic Anemia in Rats.

Anemia, characterized by decreased hemoglobin levels and impaired oxygen-carrying capacity, remains a major global public health concern. Conventional therapies, such as blood transfusions and oral iron supplementation, are often associated with adverse effects and limited absorption, highlighting the need for safer and more effective alternatives. Natural products rich in polyphenols and essential minerals have attracted increasing attention due to their multifunctional therapeutic potential. In this context, the present study is the first to investigate the anti-anemic potential of Pinus pinaster Aiton and Pinus halepensis Mill. bark extracts through an integrated phytochemical, nutritional, in vivo, in vitro, and in silico approach. Phytochemical profiling by HPLC-MS/MS revealed several major phenolic compounds, including quercetin, catechin, protocatechuic acid, and taxifolin-3-O-glucoside. The extracts exhibited high total polyphenol and flavonoid contents and strong antioxidant activity, as demonstrated by DPPH, ABTS, PR, and TAC assays. Mineral analysis revealed significant levels of essential elements, including iron, magnesium, potassium, sodium, and zinc. In phenylhydrazine-induced anemic Wistar rats, 21-day treatment with the extracts significantly restored hematological parameters (RBC, Hb, and HCT), serum iron and ferritin levels, and improved renal and hepatic biomarkers compared with untreated anemic controls. In vitro assays further demonstrated anti-hemolytic activity and enhanced erythrocyte membrane stability. Molecular docking analysis supported these findings by revealing favorable interactions between the major phenolic compounds and key targets involved in erythropoiesis, iron metabolism, and oxidative stress, including the erythropoietin receptor, superoxide dismutase, and ferrochelatase. Collectively, these findings highlight the potent anti-anemic and protective effects of Pinus bark extracts and suggest their potential as promising natural therapeutic agents.