Rutin (169050, J&K Scientific, Beijing, China) standard was of 98% purity. For NaR preparation, the rutin was dissolved in 0.1 M NaOH water solution to a final concentration of 10 mg/ml. The pH of NaR stock solution was adjusted to approximately 9.0 with 1 M HCl (low pH value would lead to rutin separating out). To identify NaR physicochemical properties, NaR was first purified using an MCI GEL CHP20P to remove the remaining NaOH in stock solution. The NaR attached on the MCI GEL was washed with double-distilled water (ddH2O) and eluted with methanol. After evaporation, dark brown solid NaR was obtained and used for further analysis.

HPLC analysis was performed with an Ultimate XB-C18 column (4.6 × 250 mm, 5 μm, Welch, Shanghai, China). The column temperature was set at 30°C, and the injection volume was 20 μl (0.1 mg/ml). The mobile phase was acetonitrile and 0.3% phosphoric acid (20:80, v/v), and the flow rate was 1 ml/min. Detection was performed at 254 nm.

TLC analysis was carried out on silica gel plates using ethyl acetate/methanol/FA/water (7:3:0.5:0.5, v/v) as mobile phase. The spots were visualized under an ultraviolet lamp (254 nm) with the help of sulfuric acid–vanillin.

For chemical structure identification of NaR, the 13C-NMR spectra were measured in CD3OD and recorded at 125 MHz. The 1H-NMR spectra were measured in CD3OD and recorded at 400/600 MHz. Chemical shifts were given in δ value.

13C-NMR (CD3OD, 125 MHz) of rutin: δ 179.4 (C-4), 166.0 (C-7), 163.0 (C-5), 159.3 (C-9), 158.5 (C-2), 149.8 (C-4′), 145.8 (C-3′), 135.6 (C-3), 123.6 (C-1′), 123.1 (C-6′), 117.7 (C-2′), 116.1 (C-5′), 105.6 (C-10), 104.7 (C-1″), 102.4 (C-1′′′), 100.0 (C-6), 94.9 (C-8), 78.2 (C-3″), 77.2 (C-5″), 75.7 (C-2″), 73.9 (C-4′′′), 72.2 (C-3′′′), 72.1 (C-2′′′), 71.4 (C-4″), 69.7 (C-5′′′), 68.6 (C-6″), and 17.9 (C-6′′′).

13C-NMR (CD3OD, 125 MHz) of NaR: δ 178.5 (C-4), 172.7 (C-7, invisible in the 13C broadband decoupling spectrum, assigned based on heteronuclear multiple bond coherence cross-peak), 162.5 (C-5), 159.0 (C-2), 158.5 (C-9), 150.6 (C-4′), 146.1 (C-3′), 135.4 (C-3), 123.5 (C-6′), 122.8 (C-1′), 117.3 (C-2′), 116.1 (C-5′), 105.6 (C-1″), 103.5 (C-10), 102.5 (C-6), 102.3 (C-1′′′), 96.5 (C-8), 78.3 (C-3″), 77.2 (C-5″), 75.7 (C-2″), 74.0 (C-4′′′), 72.2 (C-3′′′), 72.1 (C-2′′′), 71.4 (C-4″), 69.7 (C-5′′′), 68.7 (C-6″), and 17.9 (C-6′′′).

1H-NMR (CD3OD, 400 MHz) of rutin: δ 7.66 (1H, d, J = 1.9 Hz, H-2′), 7.62 (1H, d, J = 8.5, 1.9 Hz, H-6′), 6.86 (1H, d, J = 8.5 Hz, H-5′), 6.38 (1H, J = d, 1.9 Hz, H-8), 6.19 (1H, d, J = 1.9 Hz, H-6), 5.10 (1H, d, J = 7.5 Hz, H-1′′), 4.51 (1H, br s, H-1′′′), 3.82 to 3.24 (10H, m), and 1.12 (3H, d, J = 6.2 Hz, H-6′′′).

1H-NMR (CD3OD, 600 MHz) of NaR: δ 7.66 (1H, d, J = 1.9 Hz, H-2′), 7.63 (1H, d, J = 8.5, 1.9 Hz, H-6′), 6.84 (1H, d, J = 8.5 Hz, H-5′), 6.23 (1H, br s, H-8), 6.08 (1H, br s, H-6), 4.97 (1H, d, J = 7.8 Hz, H-1′′), 4.52 (1H, br s, H-1′′′), 3.82 to 3.24 (10H, m), and 1.15 (3H, d, J = 6.2 Hz, H-6′′′).

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