Acetonitrile

The samples of the worker bees and the brood were lyophilized using a Labconco Freezone 2.5 freeze dryer (Labconco, USA) (pressure: 0.024 mbar; temperature: 50 °C, time: 168 h).
Analytical portions of 5 g of the lyophilized animals or honey were shaken with 10 mL of acetonitrile (Chempur, Poland). Then, a mixture of salts containing 4 g of anhydrous magnesium sulfate (VI) (Chempur, Poland), 1 g of sodium chloride (Chempur, Poland), 1 g of trisodium citrate (Chempur, Poland), and 0.5 g of sesquihydrate disodium hydrogen citrate (Chempur, Poland) was added. The contents were shaken for 2 min and centrifuged for 5 min at 4500 rpm at 21 °C. Six millilitres of the acetonitrile phase was transferred to a polypropylene test tube that contained 150 mg of PSA (primary secondary amine) (Agilent, USA) and 900 mg of anhydrous sodium sulfate(VI) (Chempur, Poland). The extract was vigorously shaken for 2 min and centrifuged for 5 min as described above. Four millilitres of the obtained extract was taken and transferred to a glass tube, evaporated to dryness on a Heidolph Efficient Labware 4000 rotary evaporator (Heidolph, Germany), and then dissolved in 4 mL of petroleum ether (Chempur, Poland).

The sample preparation followed the previously described method [45 (link)]. The contents of the salvianolic acid and tanshinones were determined using a HPLC (high-performance liquid chromatography) system (Agilent Zorbax, Agilent, Santa Clara, CA, USA). The chromatographic separation was performed using a C18 column (Agilent Eclipse XDB-C18-USP L1, 4.6 mm × 250 mm, 5 μm particle size) at 30 °C and a sample injection volume of 20 μL. The detection at 280 nm, using a flow rate of 1.0 mL/min over a gradient of methanol containing 0.4% acetic acid (buffer A), triple distilled water containing 0.4% acetic acid (buffer B), and acetonitrile (Fisher Scientific) containing 0.4% acetic acid (buffer C). The gradient conditions are shown in Supplementary Material Table S3. The standards of rosmarinic acid (RA), salvianolic acid B (SAB), cryptotanshinone (CT), and tanshinone II A (T-II A) were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China).

Fructose concentration was measured by HPLC (12000 Series, Agilent Technologies, Palo Alto, CA, USA) using a Shodex Asahipak NH2P-50 4E column (4.6 mm ID × 250 mm; Shodex, Kanagawa, Japan). The fructose were detected with a refractive index detector (RID; 1200, Agilent Technologies, Palo Alto, CA, USA) at 30 °C with a flow rate 1 mL/min of a mixture of acetonitrile (J.T. Baker Chemical Co. Phillipsburg, NJ, USA) and water (H2O/CH3CN = 40/60) as a mobile phase.

The reducing sugar (xylose, arabinose, glucose, and fructose) contents were measured before and after heat treatment using a 1260 Infinity II preparative ultra-high performance liquid chromatography and reflective index detector (UHPLC-RID) system (Agilent, Santa Clara, CA, USA) with a modified protocol described by Chua et al. [21 (link)]. Before injection, 1.0 mL of each sample was filtered through a 0.2 μm Minisart RC 15 syringe filter (Sartorius, Goettingen, Germany), and then, 200 μL of each filtered sample was mixed with 200 μL of deionised (DI) water (arium^®, Sartorius Stedim Biotech, Goettingen, Germany) and 600 μL of pure acetonitrile (Tedia, Fairfield, OH, USA) and kept at 4 °C for more than 2 h to allow protein precipitation. Each sample was centrifuged at 10,000× g at 4 °C for 15 min to remove the precipitate, and the collected supernatant was injected to the UHPLC system for sugar analysis. The sugars were separated using a Zorbax Eclipse Plus C18 column (150 × 4.6 mm, Agilent, Santa Clara, CA, USA) using a mobile phase containing 80% acetonitrile and 20% DI water at a flow rate of 1.4 mL/min at 40 °C. A series of external xylose, arabinose, glucose, and fructose standards were used for identification and quantification.