Neohesperidin

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).

All the six herbal medicines (HMR: RM130121-08; DH: 130524; BS: RM130128-11; KXR: RM121026-04; HP: RM121026-05; ZS: RM121210-11) contained in MZRW were supported and authenticated by PuraPharm Company, Nanning department (Nanning, China). The reference standards of gallic acid, amygdalin, paeoniflorin, hesperidin, neohesperidin, naringin, magnolol, honokiol, aloe emodin, physcion, chrysophanol were purchased from Shanghai Yuan-Ye Bio-technology company (Shanghai, China). Emodin, rhein, albiflorin were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). The purity of all these reference standards was ≥98.0% (HPLC). HPLC grade of acetonitrile, methanol and formic acid were purchased from Merck (Darmstadt, Germany). Deionized water was purified by the Millipore water purification system (Millipore, Milford, MA, United States). All other reagents used were of analytical grade. Twelve male Sprague-Dawley rats (220–250 g) were kindly provided by the Experimental Animal Center of Chinese University of Hong Kong (Hong Kong). The rats were bred in an environmentally controlled room (22 ± 2°C, relative humidity 50 ± 20%) with a natural light-dark cycle for 7 days before the experiment carried out. The animal study was carried out in accordance with the Guideline for Animal Experimentation of Hong Kong.

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.

Naringenin, hesperetin, narirutin, naringin, hesperidin, neohesperidin, nobiletin, neohesperidin dihydrochalcone, and tert-butylhydroquinone were purchased from Yuanye (Shanghai, China). Dimethyl sulfoxide (DMSO), 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and bovine serum albumin (BSA) were purchased from Fluka (Sigma-Aldrich, Shanghai, China). Fetal bovine serum (FBS) and Dulbecco’s modified Eagle medium (DMEM) were provided by Gibco (Grand Island, NY, USA). Lipofectamine 2000 transfection reagent was obtained from Thermo Fisher Science (San Jose, CA, USA). All common reagents used in this study were of analytical grade.