Nobiletin

Dulbecco's modified Eagle's medium (DMEM) was purchased from Gibco (Logan, UT, USA). Fetal bovine serum (FBS) was purchased from Yeasen Biotechnology Co., Ltd. Cell counting kit 8 (CCK8) was purchased from DOJINDO (Japan). Antibodies against cleaved‐caspase 3, p‐AMPK, p‐S6, p‐P70S6K, and SIRT1 were purchased from Cell Signaling Technology (USA); anti‐AMPK and β‐actin were purchased from Santa Cruz Biotech (USA). Antibody against PARP‐2 was purchased from Abcam (UK). Nobiletin was purchased from Chengdu Must Bio‐Technology Co., Ltd (Sichuan, China) and dissolved in dimethyl sulfoxide and stored at −20°C until diluted upon use. Recombinant adenovirus vectors expressing green fluorescent protein (Ad‐Flag) and Flag‐tagged PARP‐2 (Ad‐PARP‐2) were purchased from Genechem Co., Ltd. (Shanghai, China). The viruses were extended in HEK293A cells and purified by virus purification kit (Biomiga, USA), then dialyzed in dilution buffer and stored at −80°C.

The positive control drug, dextromethorphan hydrobromide (DH) tablets, for the antitussive activity assay was purchased from Baiyunshan Guanghua (China). The ambroxol hydrochloride (AH) tablets, used for the expectorant activity assay, were obtained from Boehringer Ingelheim (Shanghai, China). The solvents for HPLC and LC/MS-IT-TOF analysis, HPLC-grade acetonitrile and purified water, used after filtration through membranes, were procured from Honeywell (America) and Yibao (China), respectively. Hesperidin (Chengdu Must Bio-Technology, Chengdu, China), nobiletin (Chengdu Must Bio-Technology, Chengdu, China), 3,5,6,7,8,3′,4′-heptamethoxyflavone (Weikeqi, Sichuan, China), tangeretin (Chengdu Must Bio-Technology, Chengdu, China), and 5-hydroxy-6,7,8,3′,4′-pentamethoxyflavone (Spring & Autumn, Nanjing, China) were all of reference standard grade, with the high purity above 98%. All other reagents were of analytical grade and pure.

Pericarpium Citri Reticulatae was purchased from a local herbal market. It was authenticated by Professor Xin-feng Gao. A voucher specimen was deposited in Herbaruim of Chengdu Institute of Biology, No. CIBI0064257.
Nobiletin and Tangeretin were purchased from Chengdu Must Bio-technology Co., LTD (China) and indentified in our laboratory for qualitative and quantitative analysis. β-naphthoflavone, polydiallyldimethylammonium chloride (PDDA), β-nicotinamide adenine dinucleotide phosphate hydrate (NADP), glucose-6-phosphate, yeast glucose-6-phosphate dehydrogenase, 4-nitrophenol (PNP) and 4-nitrocatechol (PNC) were purchased from Sigma (MO, USA). HPLC grade acetonitrile was purchased from Fisher Scientific (Fisher, Fair Lawn, USA). Deionized water was purified by a Milli-Q water system (Millipore Corp., Bedford, MA, USA). Tetraethyl orthosilicate (TEOS) was purchased from TCI (Tokyo, Japan). Other chemicals and solvents were of analytical reagent grade and were obtained from Chengdu Chemical Factory (Chengdu, China).