Phytochemical and Bioactivity Evaluation of Bee Pollen and Androecia of Castanea, Salix, and Quercus Species

Qualitative and quantitative differences in the chemical composition between bee pollen originated from <i>Castanea sativa</i> (Türkiye and Slovenia), <i>Salix</i> spp. (Türkiye and Slovenia), and <i>Quercus</i> spp. (Türkiye) and androecia of <i>Castanea sativa</i>, <i>Salix alba</i>, and <i>Quercus pubescens</i> (apetalous trees) were evaluated for the first time by new high-performance thin-layer chromatography (HPTLC) and ultra-performance liquid chromatography (UPLC) methods using marker compounds. <i>N</i>¹,<i>N</i>⁵,<i>N</i>¹⁰-tricaffeoylspermidine was isolated, and its structure was elucidated by nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HRMS). It was the main and the marker compound common to bee pollen (≈3-41 mg/g) and androecia (≈3-6 mg/g) samples. To the best of our knowledge, this is the first report of the identification of <i>N</i>¹,<i>N</i>⁵,<i>N</i>¹⁰-tricaffeoylspermidine in bee pollen originated from <i>Salix</i> spp. and androecia of <i>C. sativa</i>, <i>S. alba</i>, and <i>Q. pubescens</i>. The botanical origins of bee pollen were determined via phytochemical profiling using HPTLC-image analyses showing that bee pollen from the same botanical source had almost identical profiles regardless of collection location, geographical differences, and the bee race. <i>In vitro</i> tests and HPTLC-effect-directed analyses (EDAs) were performed to assess antioxidant and xanthine oxidase (XO) inhibitory activities of bee pollen, androecia, and <i>N</i>¹,<i>N</i>⁵,<i>N</i>¹⁰-tricaffeoylspermidine. HPTLC-EDA combined with image analyses was used for comparing the activities of bee pollen, androecia, <i>N</i>¹,<i>N</i>⁵,<i>N</i>¹⁰-tricaffeoylspermidine, and also other marker compounds (quercetin, myricitrin, hyperoside, quercitrin, isoquercitrin, and rutin). The remarkable bioactivity of <i>N</i>¹,<i>N</i>⁵,<i>N</i>¹⁰-tricaffeoylspermidine was for the first time evaluated by HPTLC-EDA and <i>in vitro</i> tests. This is the first study performing HPTLC-XO inhibitory activity analyses on the HPTLC NH₂ F_254S plates. Further bioactivity studies on botanically and chemically well-characterized bee pollen samples are needed to aid in the use of bee pollen-containing supplements in the prevention and treatment of diseases.