Cytotoxicity and Coagulation Effects of Methanolic Extracts of Punica granatum, Quercus infectoria, and Achillea millefolium: an in vitro Study

Objective: Considering the historical use of three plant species Punica granatum, Quercus infectoria , and Achillea millefolium in traditional medicine for hemostatic purposes, along with the variation observed in some experimental research, the aim of this study was to comparatively evaluate the effect of methanol extracts derived from these three medicinal plants on blood clotting time in vitro. Methods: Methanolic (70%) extracts of P. granatum flower, Q. infectoria galls and A. millefolium aerial parts were prepared by maceration method. The total tannin and phenolic content were determined using a colorimetric method. The cytotoxic activity of extracts on NIH/3T3 cell lines was evaluated by the colorimetric MTT assay. Non-toxic concentrations were tested in vitro on blood coagulation profiles including prothrombin time (PT) and activated partial thromboplastin time (aPTT) of healthy human volunteers. Results: Q. infectoria had the highest phenolic content at 287.18 mg gal/g, followed by P. granatum and A. millefolium. P. granatum exhibited the highest tannin content (119.21 mg catechin/g DW), followed by Q. infectoria (109.30 mg catechin/g DW) and A. millefolium (54.35mg catechin/g DW). The highest total flavonoid content was found in P. granatum, A. millefolium , and Q. infectoria with 98, 85.5, 5.22 mg Rutin/g DW, respectively. Non-toxic concentrations were selected to perform coagulation experiments. Based on the plant-derived coagulation assays, pomegranate extract notably reduced the PT to 11.57 ± 0.37 s compared with the control value of 14.47 ± 0.41 s. Conversely, gall oak extract markedly increased the coagulation time to 17.07 ± 1.07 s. Similarly, pomegranate significantly decreased aPTT to 33.83 ± 0.79 s relative to the control (36.30 ± 2.04 s), whereas gall oak considerably prolonged it to 50.00 ± 2.04 s. Conclusion: Methanolic extracts of P. granatum show promise as procoagulants, while Q. infectoria acts as an anticoagulant, and A. millefolium remains neutral in this context. Keywords: Punica granatum , Quercus infectoria , Achillea millefolium , coagulation profile, cytotoxicity, in vitro assay