Antioxidant effects of phenolic compounds, vitamin and mineral contents of Yakutboleti (Hortiboletus rubellus) mushroom extracts under in vitro conditions.

The aim of this study was to investigate the phenolic compounds, vitamin and mineral contents as well as antioxidant effects of Yakutboleti mushroom (Hortiboletus rubellus), which is rarely found in beech and oak forests in summer, under in vitro conditions. There are limited number of studies on this species in the literature. Mushroom samples collected from Istıranca Forests (Kırklareli) in August 2022 and stored at -80 °C were subjected to freeze-drying (lyophilisation). Extraction was performed using ethanol, methanol, dimethylsulfoxide (DMSO), phosphate buffer and ethyl acetate solvents. The extraction process included homogenisation (45 min), agitation (125 rpm, 24 h) and centrifugation (10,000 rpm, 20 min, 10 °C). Total phenolics (Folin-Ciocalteu, gallic acid equivalent - GAE), total flavonoids (Al³⁺ complexation, quercetin equivalent - QE) and free radical scavenging activity (DPPH radical scavenging, BHA/BHT standard) were analysed spectrophotometrically. Phenolic profile and vitamin content were characterised by LC-MS, elemental profile by ICP-MS and bioactive metabolites by QTOF-MS. Laccase activity was evaluated by kinetic measurements. Folin-Ciocalteu reaction was used for the determination of total phenolic matter. The highest total phenolic content (11.57 mg GAE/g) was determined in the extract. Total flavonoid content was analysed by spectrophotometric method based on the principle of flavonoid complexation with aluminium ion (Al³⁺) and the results were reported in quercetin viii equivalent (QE). The highest flavonoid content was found in the methanol extract with a value of 1.45 mg QE/g. In the determination of free radical scavenging activity, the method developed by Blois and based on DPPH radical scavenging was used. BHA (butylated hydroxyanisole) and BHT (butylated hydroxytoluene) were preferred as standard solutions. Methanol extract (39.64% inhibition, 1000 µg/mL) showed the highest free radical scavenging activity. Phenolic substance profile determination analysis by LC-MS revealed a remarkable phenolic profile in lyophilized Yakutboleti mushroom extracts. Indole-3-acetic acid compound was detected at a high concentration of 9489.16 μg/g in the non-hydrolyzed extract. After hydrolysis, the concentration of Protocatechic Acid reached 907.92 μg/g and increased 16 times. It was also determined that new bioactive compounds such as Jaseosidin (67.95 μg/g) and Isorhamnetin (61.51 μg/g) were released as a result of hydrolysis. QTOF-MS analysis revealed critical metabolites of Yakutboleti mushroom with pharmacological potential. In positive ion mode, Brefeldin A with anticancer activity and the neuroprotective antioxidant ergothioneine were detected. At the same time, waxenic acid, a trans fatty acid also found in milk and dairy products, was detected. In the negative ion mode, quinolin-8-ol compound showing neurodegenerative effect by metal chelation was determined. These findings prove the bioactive diversity of the fungus can be a source of therapeutic applications. The fat-soluble vitamin content of Yakutboleti mushroom was analyzed by LC-MS. Vitamin D₃ compound was detected at a concentration of 512.68 µg/kg (ppb). Vitamin B profile was analyzed by LC-MS. The highest concentration was determined in nicotinic acid (B3) with 2326,08 µg/kg (ppb). The elemental profile of the Yakutboleti mushroom was analyzed by ICP-MS (Inductively Coupled Plasma Mass Spectrometry). The highest concentrations were phosphorus (775,921.41 µg/kg) and potassium (1,005,692.17 µg/kg). Arsenic (601.34 µg/kg) was detected at a remarkable level, while the accumulation of essential minerals such as iron (12,046.98 µg/kg) and zinc (52,630.00 µg/kg) stood out. The low level of toxic metal cadmium (14.09 µg/kg) was recorded as a positive finding. ix Laccase activity of the extracts was evaluated by kinetic measurements. The highest enzyme activity was detected in ethanol extract with 0.22 U/L, which was significantly higher than methanol (0.12 U/L) and DMSO (0.11 U/L) extracts. No measurable laccase activity was detected in phosphate buffer and ethyl acetate extracts. In these comprehensive analyses, it is noteworthy that various bioactive phenolic compounds (Indole-3-Acetic Acid, Protocatechic Acid, Jaseosidin, Isorhamnetin), metabolites with pharmacological potential (Brefeldin A, ergothionine, quinolin-8-ol) were detected in Yakutboleti mushroom, especially the significant antioxidant capacity of methanol extract and laccase activity of ethanol extract indicate the potential of this rare mushroom species for functional food and biotechnological applications.