Rutin

Total flavonoid content (TFC) was measured by sodium nitrite aluminum nitrate colorimetry following the method described in Do Q et al. (Do et al., 2014 (link)), but with some slight modifications. An extract sample with a 1% volume fraction of hydrochloric acid ethanol solution was placed in an ultrasonic water bath (30° C) for 30 min and then centrifuged for 30 min. After that, rutin was used as standard (Sangon Biotech, Shanghai, China) for quantification under 510 nm wavelength (y = 1.498x + 0.0079, R = 0.9994). The results were displayed as mg/g dry weight (DW).

Total flavone content in the two samples was measured using the aluminum nitrate colorimetric method (30 (link)) with minor modifications. Sample extract (1.0 mL), 0.72 M NaNO₂ (0.15 mL), 0.27 M Al₂ (NO₃)₃ (0.15 mL), 1 M NaOH (2.0 mL) and 60% (v/v) ethanol (1.7 mL) were sequentially added into a 5 mL tube. The mixture was incubated for 15 min at 25^°C. Absorbance of the mixture was measured at 517 nm using a UV-2100 spectrophotometer (Shanghai, China). Each sample was measured three times. A standard solution was prepared using Rutin and a calibration curve was obtained with a range of concentrations from 0.75 to 75 μg/mL. Total flavone content was expressed as mg Rutin equivalent (RE)/g of sample. Rutin was purchased from Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China).

L. christinae Hance herbs (Harvested in October 2014 from Sichuan, China, subsequently dry and stored under the condition of dark) were purchased from Kang Jin Pharmaceutical (Xianning, China). Butylated hydroxytoluene (BHT), H₂O₂, and acetylcholine (ACh) were purchased from Sigma-Aldrich (St Louis, MO, USA). Ascorbic acid (Vitamin C) and Trolox were purchased from Richu Biosciences (Shanghai, China). Diphenylpicrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6 -sulphonic acid) (ABTS) were obtained from Aladdin Reagent (Shanghai, China). Rutin was purchased from Sangon Biotech (Shanghai, China). Dihydroethidium (DHE) was obtained from Thermo Fisher Scientific (MA, USA). 4′,6-diamidino-2-phenylindole (DAPI) was obtained from Calbiochem (Madrid, Spain). All other reagents used were of analytical grade. Double distilled water was used throughout the experiments.