Fatty acid standard

The fatty acid content was measured on an Agilent 7890A gas chromatograph with an FID detector on an Omegawax 250 column (30 m x 0.25 mm x 0.25 μm) using the program developed in our laboratory: injector temperature: 250 °C; split 100:1; gas pressure 17.81 psi; carrier gas: nitrogen; Owen program: 140 °C (hold 2 min) to 240 °C (4 °C/min; hold 10 min); FID detector temperature: 260 °C. Before the analysis, a series of fatty acid standards (Sigma-Aldrich) was run to determine the retention times for each compound. Each sample was esterified and measured in 3 replicates.

A gas chromatography (GC)/flame-ionization detection instrument (6890 N; Agilent Technologies, Santa Clara, CA, USA) with an autosampler (7683; Agilent Technologies, USA) was used to determine the fatty acid composition in the present study. Initially, finely ground samples (20 g) were extracted in duplicate according to Folch et al [14 (link)] with a chloroform-methanol (2:1 v/v) solution. Methylation to convert fatty acids into methyl esters was performed using 25% boron trifluoride in methanol at 80°C for 1 h. Fatty acid methyl esters were subsequently mixed with 1.5 mL hexane and 1 μL of the sample was injected into the GC port using an autosampler. The injector temperature was set at 250°C with a 100:1 split ratio. Fatty acid methyl esters were separated using a WCOT-fused silica capillary column (100 m×0.25 mm i.d., 0.20 μm film thickness; Varian Inc., Palo Alto, CA, USA) with a 1.0 mL/min helium flow. The detailed program of the oven was: 150°C/1 min, 150°C to 200°C at 7°C/min, 200°C/5 min, 200°C to 250°C at 5°C/min, and 250°C/10 min. The detector temperature was 275°C. Fatty acids were identified by comparing the identified peaks with the retention time of fatty acid standards (47,015-U; Sigma-Aldrich, St. Louis, MO, USA). The peak area of each identified fatty acid was used to calculate the proportion (%) of the total identified peak area.

The camellia seeds (Camellia oleifera Abel.) were provided by a local forest farm (Haikou country, Hainan province, China). The raw material (dry camellia seeds) contained 9.38% moisture content. The seeds were sealed in plastic containers and stored in a refrigerator at 4°C until extraction.
Fatty acid standards, gallic acid, Folin phenol reagent, α‐tocopherol, squalene, 5α‐cholestane, and 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) were purchased from Sigma‐Aldrich Co. (Steinheim, Germany). All other reagents and chemicals were of analytical grade and purchased from Guangzhou Chemical Reagents Co (Guangzhou China).

Chloroform, ethyl alcohol (96% w/w), methanol, n-hexane, formic acid (99% w/w) hydrochloric acid (37% w/w) and anhydrous sodium sulphate were of analytical grade and were purchased from Carlo Erba (Milan, Italy). Boron trifluoride (approximately 10% w/w in methanol for gas chromatography (GC) derivatization) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Fatty acid standards (C16:0, C16:1, C17:0, C18:0, C18:1, C18:2, C18:3) were also purchased from Sigma-Aldrich.

Quercetin (≥95%), resveratrol (≥95%), NADPH (99%), dimethyl sulfoxide (DMSO, 99.8%), and all fatty acid standards used in the GC–MS analysis were purchased from Sigma (Sigma-Aldrich, St. Louis, MI, USA). The chromatographically pure methyl tert-butyl ether (MTBE), acetonitrile and, methanol (MeOH), and n-hexane were obtained from Merck (Merck, Darmstadt, Germany), and bacterial culture media and all other analytically pure reagents were purchased from Sinopharm (Sinopharm, Shanghai, China). Ultrapure water was obtained using a PALL Lab Water Purification System (Port Washington, NY, USA).

Folin-Ciocalteu phenol reagent, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), ascorbic acid (AA), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), potassium persulfate, 14% boron trifluoride-methanol (BF3-methanol), anhydrous sodium sulfate, n-hexane, sodium hydroxide, fatty acid standards (palmitic, stearic, oleic, linoleic, and linolenic acids), chloroform, ethanol, and methanol were purchased from Sigma Aldrich (St. Louis, MO, USA). CS and FS were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All chemicals were of analytical grade and used without further purification.

Walnut (Juglans regia L.) nuts were purchased from the local market in Shandong Province and harvested in October 2019 from Nanshan District, Jinan City located in Shandong Province. Walnut oil was extracted from Shandong walnuts according to the cold-press method with minor modification [15 (link)]. The preparation process of PUFA is based on previous literature [16 (link)] and prepared by a professional following a standard protocol. PUFA is stored at − 20 °C to prevent oxidation and contamination. And free fatty acids were separated and determined by HPLC–UV equipped with Lichrosorb RP-18 column (particle size 5 µm; 150 × 4.6 mm, Merck). The HPLC conditions were as follows: The column temperature was maintained at 30 °C with a flow rate of 0.9 mL/min, the UV absorption at a wavelength of 192 nm and the mobile phase composition was water/acetonitrile (1:9) isocratic for 15 min. The free fatty acid peaks were identified by matching them with fatty acid standards (Sigma-Aldrich, USA). All other chemical reagents were purchased from Aladdin Reagent Co. (Shanghai, China).