Nobiletin

SMD, used for gastrointestinal dysmotility in China (approval number: guo-yao-zhun-zi Z20025044; specification: 10 mL/division), was obtained from Hunan Hansen Pharmaceutical Company, Ltd. (Yi Yang, China). HSA was acquired from Sigma Chemical Co. (St. Louis, MO, USA), standards, including arecaidine, arecoline, norisoboldine, linderane, costunolide, dehydrocostus lactone, synephrine, rutin, limonin, eriocitrin, narirutin, naringin, hesperidin, heohesperidin, poncirin, naringenin, hesperetin, nobiletin, tangeretin, and sorbic acid with a purity of over 98%, were purchased from Yuan-ye Bio-Technology Co., Ltd. (Shanghai, China). The formic acid, acetonitrile, and methanol used for HPLC analysis were chromatographic grade and purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

We accurately weighed 2 mg of each of the following: narirutin, hesperidin, nobiletin, tangeretin, and 5-demethylnobiletin (Shanghai Yuanye Biological Technology Co., Ltd. China). Then, the standard substances were dissolved in methanol (HPLC grade) individually to afford standard solutions of different concentrations. Then, 5 mg of GPE was dissolved in methanol (1 ml). This sample solution was filtered through a microporous membrane (0.22 μm) to afford the test solution.
The chromatographic separation conditions were carried out as follows: a liquid chromatography system (UltiMate3000 HPLC System, Thermo Fisher Scientific, Waltham, MA, United States) with a Welch C18 column (4.6 × 250 mm long, 5 μm) was used, mobile phase A was acetonitrile (HPLC grade), and mobile phase B was 0.5% glacial acetic acid aqueous solution. The mobile phase gradient was as follows: 0 min, 12% A; 0–20 min 25% A; 20–35 min 45% A; 35–40 min, 100% A. The flow rate was set at 0.5 ml/min, and the column temperature was 35°C. The injection volume was 5 μl, and the detection wavelength was 285 nm. The sample solution and standard solutions of different concentrations were analysed under these conditions. The compounds in GPE were analysed according to the chromatographic peak area of the standard substance.

Eleven samples of Aurantii fructus (AF) samples from different habitats were collected and identified by Associate Professor Tang Qi. Reference standards included synephrine, eriocitrin, neoeriocitrin, rutin, narirutin, naringin, hesperidin, neohesperidin, limonin, xanthotol, poncirin, naringenin, hesperetin, nobiletin, tangeretin and auraptene with a purity of over 98% were purchased from Yuan-ye Bio-Technology Co., Ltd. (Shanghai, China). Chromatographic grade formic acid, methanol, ethanol and acetonitrile were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China) and used for extractive and analytical procedures.

Citri Reticulatae Pericarpium was supplied by Scientific Research Institute of Beijing Tongrentang Co., Ltd. Total 21 reference standards, including vicenin-2 (6), vicenin-3 (25), eriocitrin (40), isoquercitroside (45), troxerutin (49), rhoifolin (66), naringin (67), naringenin (68), naringenin-7-O-glucoside (71), hesperidin (78), neohesperidin (82), hesperetin (108), isosinensetin (122), sinensetin (133), quercetagetin-3,5,6,7,3′,4′-hexamethyl ether (144), nobiletin (151), 4´,5,6,7-tetramethoxyflavone (153), tangeretin (168), 5-demethylnobiletin (174), 8-hydroxy-3,5,6,7,3′,4′-hexamethoxyflavone (180), 5-hydroxy-3,7,3',4'-tetramethoxyflavone (181), were purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). The purity of all the reference standards was > 98%. The distilled water was obtained from Watsons, whereas LC–MS grade formic acid was obtained from Fisher-Scientific (Fair Lawn, USA). The LC–MS-grade acetonitrile and methanol were purchased from Merck (Germany).

The reference standards for naringenin, nobiletin, narirutin, naringin, hesperidin, neohesperidin, sulfamethoxazole (internal standard, IS1), and honokiol (internal standard, IS2) were purchased from the Shanghai Yuanye Bio-Technology Co., Ltd.(Shanghai, China). Meranzin hydrate (MH) was obtained from the Huaxi Medical University Medicine Factory (Chengdu, China). Formic acid, methanol and acetonitrile (HPLC grade) were purchased from Sigma (United States). Water was purified by a Milli-Q water purification system (Millipore, Burlington, MA, United States). ZQ was obtained from the pharmacy in Xiangya Hospital, Central South University and authenticated by Professor S. Y. Hu.