Acacetin

Acacetin (≥97% purity by HPLC), can be isolated from S. tridactyla, S. involucrata or other Asteraceae family (Chik et al., 2015 (link); Li et al., 2008 (link)) was purchased from Sigma-Aldrich (St. Louis, MO, United States). All experimental animal care and housing protocols were approved by the Laboratory Animal Care Committee of Chang Gung University of Science and Technology (IACUC approval number: 2013-007). Male C57BL/6 mice (4 weeks old) were purchased from the National Laboratory Animal Center in Taiwan. Mice were fed a standard chow diet or a HFD with water, and all mice were housed in temperature-controlled environment. The 4-week-old mice were randomly subdivided into 4 groups of 8 mice who were treated as follows for 16 weeks. In the N group, the mice were fed a normal diet (11.4% fat) and received DMSO by intraperitoneal injection. In the HFD group, the mice were fed an HFD diet (60% fat) and received DMSO by intraperitoneal injection. In the AC5 group, the mice were fed an HFD (60% fat) and received 5 mg/kg Acacetin dissolved in DMSO by intraperitoneal injection. Finally, in the AC10 group, the mice were fed an HFD (60% fat) and received 10 mg/kg Acacetin dissolved in DMSO by intraperitoneal injection. The intraperitoneal injections were performed twice a week for 10 weeks (from age 11 weeks to age 20 weeks).

For the HPLC–DAD analysis, a Hewlett—Packard HP series 1100 (Hewlett—Packard, Wilmington, DE, USA) instrument operated with ChemStation software v. A.09.03 and a Discovery C18 column (250 × 4.6 mm, particle size of 5 µm) was used. The sample was eluted with an isocratic mixture of methanol/acetonitrile/water (25:25:50) with a flow rate of 1 mL/min and measured at a wavelength of 260 nm with a complete screen from 200 to 400 nm. Thirty microlitres of a stock solution of 3 mg/mL of extract in HPLC—grade MeOH was injected into the instrument. Additionally, 30 µL of 15 flavonoid standards (vanillin, quercetin, catechin, luteolin, pinocembrin, chrysin, baicalein, myricetin, naringenin, naringin, gallic acid, catechol, apigenin, acacetin and kaempferol; Sigma—Aldrich, St. Louis, MO, USA) were injected at a concentration of 1 mg/mL.

Methoxylated flavonoids were quantified in the hydroalcoholic propolis extract using a previously reported LC-MS method [35 (link)]. Six standards were used: jaceosidin, eupatilin (ALB Technology, Hong Kong, China), casticin, acacetin, eupatorin and hispidulin (Sigma, Neustadt, Germany). Calibration curves in the 0.02–6 μg/mL range with good linearity (R² > 0.99) were used to determine the concentration of methoxylated flavones.

The specific compounds, were: Caffeic acid, CAPE, chrysin, luteolin, daidzein, suberic acid, apigenin, (Alfa Aesar), pinocembrin, isorhamnetin, isosakuranetin, vitexin, orientin, rosmarinic acid, myricetin, vanillin, ursolic acid, hydroxytyrosol, tangeretin, chrysoeriol, betulinic acid, eriodictyol, sakuranetin, naringenin, t-cinnamic acid, genistein, diosmetin, resveratrol, galangin, pinocembrin 7-methyl ether, techtochrysin, (Extrasynthese) rutin, isoferulic acid, ellagic acid, kaempferol, quercetin, corosolic acid, acacetin, diosmin, protocatechuic acid ethyl ester, hesperetin, phloridzin, chlorogenic acid, p-coumaric acid, (±)catechin, maslinic acid, (Sigma Aldrich), rhamnetin, syringic acid, protocatechuic acid, ferulic acid, kaempferide, adipic acid, pinostrobin, gallic acid, pinobanksin (Fluka), naringin, hesperidin, (Acros Organics) pinobanksin-3O-acetate (Interchim Inc), cinnamylidenacetic acid, artepillin C (Wako Chemicals). o-Orselllinaldehyde was purchased from Santa Cruz Biotechnology (USA).
Water, acetonitrile, mEthanol and formic acid were purchased from Fisher Scientific, UK and they were of LC-MS grade. Ethanol was purchased from Merck, Germany. PTFE filters (0.45 μm) were obtained from Macherey-Nagel, Germany.

Cell lysates were obtained from 3T3-L1 or HepG2 cells. Cells were scraped and lysed with M-PER lysis buffer. After centrifugation for 15 min at 16,000×g, the supernatant was obtained, and protein content was quantified using Bradford reagent. Before drug treatment, the samples were diluted to achieve a protein concentration of 1 mg/mL. Samples were treated with the Kaem or DMSO for 2 h at 25 °C and then incubated with pronase (5, 10, and 20 µg/mL) or distilled water for 10 min at 25 °C. After the reaction, SDS was added to the sample and the samples were heated at 100 °C. A portion of each sample was used for LC–MS/MS analysis. Sample preparation and proteome analysis were conducted as indicated in the previous publication⁶⁹ . For western blot analysis, VDAC1 or Na⁺K⁺ ATPase was used as an internal control. For the structure–activity-relationship (SAR) analysis, kaempferol (Sigma-Aldrich, 60010), Acacetin (Sigma-Aldrich, 00017), isosakuranetin (Sigma-Aldrich, PHL82569), Biochanin A (Sigma-Aldrich, D2016), (−)Epicatechin (Sigma-Aldrich, E4018), Genistein (Sigma-Aldrich, G6649) were used.

Propolis sample was obtained from Taishan fir in the autumn. Adenosine diphosphate (ADP) was purchased from Arkray (Aggrepack, Japan). Collagen was purchased from NYCOMED (Kollagenreagens Horm, Austria). Thrombin receptor activator peptide (TRAP) was purchased from Bachem (Germany). Caffeic acid phenethyl ester (CAPE), galangin, acacetin, and pinostrobin were purchased from Sigma Chemical Co. (USA).

Pepsin, pancreatin, α-amylase, amyloglucosidase, standards of gallocatechin, catechin, epicatechin, procyanidin B2, quercetin, quercetin 3-O glucoside, naringenin, naringin, luteolin, apigenin, acacetin, rutin, neohesperidin, taxifolin, phloretin, kaempferol, kaempferol-3-O-glucoside, quinic acid, protocatechuic acid, 2,5-dihydroxybenzoic acid, 4-hydroxybenzoic acid, 2,4,6-trihydroxybenzaldehyde, syringic acid, chlorogenic acid, 4-O-caffeoylquinic acid, caffeic acid, coumaric acid, formic acid, and shikimic acid were obtained from Sigma Chemical (St. Louis, MO, USA). Acetone, methanol and acetonitrile were LC-MS grade from J.T. Baker Inc. 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (TROLOX, Sigma-Aldrich, St. Louis, MO, USA), 2,2′ -azo-bis (2-amidino-propane) dihydrochloride (AAPH, Sigma-Aldrich, USA), 2,2-diphenyl-1-picrylhydrazyl (DPPH), COX-2 kit no. 560,131 from Cayman Chemical Co., Ann Arbor, MI, USA.