Integrated in vitro and computational assessment of Syzygium aromaticum polyphenolic extract: bioactivities and toxicity profiling

Abstract

In this study, we aimed to identify the main polyphenolic compounds of Syzygium aromaticum polyphenolic extract (SAPE) and evaluate their antioxidant, antidiabetic, hemolytic activities and acute toxicity. HPLC-MS-MS was used to identify the polyphenolic compounds of SAPE. DPPH, FRAP and TAC methods were used to evaluate the antioxidant activity of SAPE. The antidiabetic activity of our extract was assessed by inhibiting α-amylase and α-glucosidase. The hemolytic activity of our extract was also evaluated by exposing rat red blood cells to varying doses to determine its toxicity to blood cells. The acute toxicity of SAPE was assessed by examining biochemical and hematological parameters, as well as through histological examination. An in silico study was carried out to evaluate the activity of the molecules identified in SAPE against biological targets. Statistical analyses were performed to assess the significance of the results. UHPLC-MS/MS analysis showed Chrologenic acid, 4-Caffeoylquinic acid, piperic acid, and other chemicals. The SAPE has demonstrated significant antioxidant activity (DPPH IC₅₀: 67.92 µg/mL, FRAP EC₅₀: 29.47 µg/mL, TAC: 644.90 µg EAA/mg) compared to BHT, making it a promising agent for applications as a natural antioxidant. The extract demonstrated the ability to inhibit α-amylase and α-glucosidase at concentrations of 0.388 ± 0.024 and 0.067 ± 0.003 mg/mL, respectively. The SAPE caused significant hemolysis at higher concentrations (33.33–100 mg/mL) but was non-toxic at lower levels (3.7–11.11 mg/mL). Acute toxicity assessment of SAPE (up to 5000 mg/kg) revealed no signs of toxicity. However, significant dose-dependent alterations were observed in relative liver weight and certain biochemical/hematological parameters, without histopathological changes in the organs. In silico analysis identified 4,5-O-dicaffeoylquinic acid, petunidine 3-O-glucoside, and catechin 7-O-beta-D-xyloside as the most active molecules of SAPE against NADPH oxidase, the antidiabetic enzymes α-amylase and α-glucosidase, and human TRPV4 ankyrin, validating the therapeutic potential of the extract. Statistical analysis validated concentration-dependent toxicity, with elevated doses exhibiting effects analogous to complete hemolysis.