Immunomodulatory and Antioxidant Activities of Pinus sylvestris L. Xylem Sap: Modulation of NK-92 Cell Receptors and Cytotoxic Function

This study investigated the phytochemical profile, antioxidant capacity, and immunomodulatory properties of Pinus sylvestris xylem sap harvested from the Kastamonu region of Türkiye during the early spring active sap-flow period. Quantitative screening demonstrated significant polyphenolic density, with total phenolic (39.65 ± 2.12 mg GAE/g) and flavonoid contents (21.48 ± 3.11 mg Catechin/g) reflecting a high relative accumulation of mobile, hydrophilic flavonoids. Targeted LC-MS/MS analysis further revealed the presence of several phenolic and flavonoid-related compounds, with catechin as the predominant compound in the directly extracted sample, while epicatechin and epigallocatechin were among the major compounds detected following acid hydrolysis. The sap exhibited consistent, robust antioxidant activity across DPPH (43.52 ± 2.76 mg TE/g), ABTS (41.64 ± 2.17 mg TE/g), and FRAP (39.21 ± 1.95 mg TE/g) assays, indicating multi-pathway radical scavenging and electron-donating capacities in cell-free chemical models. While direct exposure yielded concentration-dependent cytotoxicity against tumor lines (IC50 values: 7.6–12.5 µg/mL), subtoxic conditioning (1 µg/mL) and substantially enhanced NK-92-mediated lysis of target cancer cells, most prominently against MCF-7 breast carcinoma. Flow cytometric analysis revealed that xylem sap treatment selectively upregulated surface expression of CD16 (from 0.00% to 15.93%) and CD57 on NK-92 cells without altering baseline CD56. Furthermore, sap stimulation triggered a concentration-dependent surge in IFN-γ production and elevated cytotoxic responses in lymphocytes isolated from both healthy controls (n = 5) and breast cancer patients (n = 5). These findings indicate that conifer xylem sap represents in vitro immunomodulatory properties and enhances NK-92 cell-mediated cytotoxic responses in cell culture models.