4.6.1. Preparation of Standard Solutions

In order to validate the changes of flavonoids during different growth stages after symbiotic culture, several monomer flavonoids from DAMs were tested. The standards of nobiletin (478-01-3), narcissin (604-80-8), isorhamnetin-3-O-beta-d-Glucoside (5041-82-7), tangeretin (481-53-8), rutin (153-18-4), quercetin (117-39-5), isorhamnetin (480-19-3), quercetin-7-O-glucoside (491-50-9), and kaempferol-3-O-glucoside (480-10-4) were purchased from the National Institutes for Food and Drug Control (Beijing, China) and the purity of all of them was over 98%. Each standard sample was dissolved in methanol to obtain 1 μg/mL of standard working solutions and used within 1 month at −20 ◦C.

### 4.6.2. Preparation of Sample Solutions

The preparation of the sample (approximately 20 mg) followed the same process as the above mentioned extract. In order to enrich the flavonoids, the extract was processed through C18-SPE solid phase extraction column (Waters Technology Co., Ltd., USA) and the column was rinsed with 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, and 90% absolute alcohol. The rinse solutions of 70%, 80%, 90%, and absolute alcohol, that were verified to contain flavonoids, were collected and freeze dried using a vacuum freeze-drying machine LGJ-18 (Beijing Songyuanhuaxing Technology Develop Co., Ltd, Beijing, China). The extract was re-dissolved with 2 mL methanol, then the sample solution was passed through a 0.22 μm syringe nylon filter and stored at −20 ◦C prior to analysis.
