Echinatin

Total flavonoid contents were determined by sodium nitrite–aluminum nitrate colorimetric method using rutin as standard (Hao et al., 2018 (link)). The standards rutin, echinatin, and LCA were purchased from Biosynth Carbosynth, United States. The contents of echinatin and LCA were determined by LC-MS/MS.

To extract the metabolites, 10 mg of freeze-dried samples was subjected to ultrasonic-assisted extraction twice with 1 mL of methanol for 30 min. After centrifugation at 12,000× g for 10 min, the supernatants were dried and finally redissolved with 300 μL of methanol.
Total flavonoids content was measured using the sodium nitrite–aluminum nitrate colorimetric method using rutin as the standard [23 (link)].
The contents of specialized metabolites were analyzed using HPLC. The supernatant samples were filtered through 0.22 μm millipore filters. The samples were then analyzed using HPLC (LC-2030C, Shimadzu, Kyoto, Japan), using a reversed-phase C18 column (150 mm × 4.6 mm, 5 μm, Shimadzu, Kyoto, Japan) under the following chromatographic conditions: mobile phase, acetonitrile (A) and 0.1% formic acid (B); flow rate, 1 mL/min; sample injection volume, 10 µL; detection wavelengths, 254 nm and 370 nm; column temperature, 40 °C. The gradient elution approach was set as follows: 0~25 min, 30~55% A; 25~27 min, 55~95% A; 27~30 min, 95% A; 30~31 min, 95~30% A; 31~35 min, 30% A. The standards of rutin, echinatin, isoliquiritigenin, licochalcone C, and LCA were purchased from Biosynth Carbosynth, Staad, Switzerland.