Sinensetin

Nobiletin (>98% purity) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Tangeretin, sinensetin, (>95% purity), and resveratrol (>99% purity) were obtained from Sigma-Aldrich (St. Louis, MO, USA). BACE1 inhibition was determined by enzymatic assay using the fluorescent resonance energy transfer (FRET)-based BACE1 kit from Invitrogen (Pan Vera, Madison, WI, USA). α-Secretase (tumor necrosis factor-α converting enzyme, TACE) and substrate were obtained from R&D Systems (Minneapolis, MN, USA). Trypsin, chymoTrypsin, elastase, and their substrates were obtained from Sigma-Aldrich (St. Louis, MO, USA). The measurement of fluorescence and optical density was performed with a BioTEK ELISA microplate fluorescence reader FLx 800 and BioTEK ELx 808 (Winooski, VT, USA), respectively.

The powdered form of OS was purchased from a local supplier (Ethno Resources, Sungai Buloh, Selangor Malaysia) with a particle size of 40 meshes and stored at room temperature in a dark environment. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin strep (PS), and trypsin were purchased from Gibco, Life Technologies (Rockville, MD, USA). Ethanol, acetonitrile, methanol, formic acid, and dimethyl sulfoxide (DMSO) were purchased from QRec Asia (Selangor, Malaysia). 2,2-Diphenyl-1-picrylhydrazyl (DPPH), 2,2’-Azino-bis(3-Ethylbenzothiazoline-6-sulphonic acid) (ABTS), standard rosmarinic acid, eupatorin, sinensetin, and 3′-hydroxy-5,6,7,4′-tetramethoxyflavone were acquired from Sigma-Aldrich, Taufkirchen, Germany. Trolox and ascorbic acid were purchased from Merck, Darmstadt Germany.

Naringenin, eriodictyol, apigenin, luteolin, liquiritigenin, butin, sakuranetin, kaempferol, quercetin, dihydrokaempferol, dihydroquercetin, eriocitrin, pinocembrin, narirutin, naringin, chrysin, genkwanin, baicalein, scutellarein, norwogonin, wogonin, and neoeriocitrin were purchased from Shanghai Yuanye Bio-Technology Co., Ltd (Shanghai, China). Hesperidin, neoHesperidin, sinensetin, isosinensetin, and nobiletin were obtained from Sigma-Aldrich (St Louis, MO, USA). 7-O-methyleriodictyol, isosakuranetin, and 5-HPMF were purchased from BioBioPha Co., Ltd (Kunming, China).

Solvents of analytical grade were obtained from VWR International s.r.l. (Milan, Italy).
Tween 20, ascorbic acid, Folin–Ciocalteu reagent, sodium carbonate, butylated hydroxytoluene (BHT), propyl gallate, 2,2-diphenyl-1-picrylhydrazyl (DPPH), tripyridyltriazine (TPTZ), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic) acid (ABTS) solution, β-carotene, linoleic acid, Orlistat, Trizma base, 4-nitrophenyl octanoate (NPC), maltose, α-amylase from porcine pancreas, α-glucosidase from Saccharomyces cerevisiae, o-dianisidine dihydrochloride, and peroxidase/glucose oxidase (PGO) were purchased from Sigma–Aldrich S.p.a. (Milan, Italy).
Acarbose from Actinoplanes sp. was obtained from Serva (Heidelberg, Germany). Caffeic acid, protocactechuic acid, p-coumaric acid, chlorogenic acid, vanillic acid, eriocitrin, gallic acid, apigenin, didymin, quercetin, hesperidin, neohesperidin, neoeriocitrin, naringin, narirutin, sinensetin, tangeretin, rutin, quercetin-O-glucoside, genistin, poncirin, luteolin, kaempferol, hesperetin, rhamnetin, umbelliferone, isopimpinellin, and bergapten were purchased from Sigma–Aldrich Chem. Co. (Milwaukee, WI, USA). Acetonitrile, formic acid, methanol, and water were HPLC-grade solvents, obtained from Carlo Erba Reagents (Milano, Italia).

Samples of Ganpu tea and the corresponding raw materials (including Pu-erh tea and GCP) were provided by Xinhui Hele Tea Art Co. Ltd. (Jiangmen, China). The reference standards of gallic acid, caffeine, hesperidin, naringin, neohesperidin, rutin, rhoifolin, and synephrine were obtained from the National Institute for Control of Biological and Pharmaceutical Products of China (Beijing, China). Naringenin, poncirin, nobiletin, sinensetin, tangeretin, and mass spectrometry (MS) grade formic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). Hesperetin and N-methyltyramine were acquired from Sinova (Shenzhen, China). MS-grade acetonitrile was purchased from Fisher Scientific (Pittsburgh, PA, USA). Water used in the experiment was distilled and further purified with a Milli-Q system (Millipore, Milford, MA, USA). All other reagents used were of analytical grade.

HPLC-grade mEthanol and formic acid were purchased from Tedia Company, Inc. (Fairfleld, OH, USA). Ethanol and mEthanol (analytical grade) were purchased from Sinopharm Chemical Reagent Co. (Shanghai, China). Water was purchased from Hangzhou Wahaha Group Co., Ltd. (Hangzhou, China). Eriocitrin, narirutin, neohesperidin, quercitrin, eridictyol, didymin, quercetin, naringenin, luteolin, sinensetin, nobiletin, and tangeretin were purchased from Sigma (St. Louis, MO, USA). Naringin and hesperidin were obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). The purity of all of the standards was beyond 95%.

Six independent plants were used as biological replicates, and about five leaves were sampled from each plant in secondary metabolic profiling. The secondary metabolic profiling was performed by LC-QTOF-MS using a modified method according to Yun et al. [37 (link)]. 100 mg freeze-dried powder was extracted with 80% methanol over night at 4°C. The mixture was centrifuged and filtered. Then, the metabolic profiling were performed using a QTOF 6520 mass spectrometer (Agilent Technologies, Palo Alto, CA, USA) coupled to a 1200 series Rapid Resolution HPLC system.
The raw data was processed by Agilent Mass Hunter Qualitative Analysis (version B. 04.00, Aglient Technologies) and Mass Profiler Software (version B.02.02, Aglient Technologies). Then PCA analysis was performed using the filtered and normalized data. Metabolite identification was carried out by comparing mass spectra and retention time with those of authentic standards. Nine authentic standards, namely, narirutin, naringin, hesperidin, neohesperidin, didymin, sinensetin, limonin, nomilin, and nobiletin were obtained from Sigma Chemical Co. (St. Louis, MO).