Isorhamnetin

All the Chinese herbs were provided by Tongde Hospital of Zhejiang Province (Hangzhou, China). All the chemical standards, including l (+)-arginine, proline, adenine, succinic acid, isoleucine, leucine, gallic acid, phenylalanine, protocatechuic acid, l-tryptophan, protocatechuic aldehyde, neo-mangiferin, chlorogenic acid, caffeic acid, amygdalin, mangiferin, rutin, liquiritin, luteoloside, naringin, berberrubine, baicalin, quercetin, epimedin A, epimedin B, epimedin C, icarrin, isorhamnetin, glycyrrhizic acid, limonin, aaikosaponin A, and 3-N-butyl-4,4-dihydrophthalide, were purchased from Shanghai Yuanye Bio-Technology Co. Ltd (Shanghai, China). Azoxymethane (AOM) was obtained from Sigma-Aldrich (St. Louis, MO, United States) and dextran sodium sulfate (DSS) from MP Biomedicals (Santa Ana, CA United States). TUNEL apoptosis assay kits, radioimmunoprecipitation assay (RIPA) buffer, BCA protein assay kits, and anti-MMP9 antibody (AF5234) were purchased from Beyotime (Beijing, China). Mouse IL-6 and IL-17A ELISA kits were from LiankeBio (Hangzhou, China), and antibodies to p-STAT3 (AF3293), STAT3 (AF6294), NF-κB-p65 (BF8005), Bcl-2 (BF9103), Lamin B (AF5161), and β-actin (T0022) were purchased from Affinity Biosciences (Changzhou, China). Antibodies to IL-17RA (sc-376374) and Bax (sc-20067) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, United States).

Epicatechin, kaempferol, quercetin, rutin, isoquercitrin, syringic acid, p-coumaric acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, ferulic acid and gallic acid were purchased from Adamas Reagent, Ltd. (Shanghai, China). Other standard reference materials including 4-methoxysalicylic acid, p-hydroxycinnamic acid, isoferulic acid, 1,3-dicaffeoylquinic acid, gentiopicrin, grosvenorine, diosmin, isorhamnetin and baicalein were obtained from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). 2,4,6-tri(pyridin-2-yl)-1,3,5-triazine (TPTZ), 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS), 4-nitrophenyl α-D-glucopyranoside (p-NPG) were all obtained from Adamas Reagent, Ltd. (Shanghai, China). Acetonitrile and formic acid (99.9%, HPLC grade) were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Cellulase (400 u/mg) from Trichoderma Vride G, hemiCellulase (20,000 u/g) and pectinase (500 u/mg) from Aspergillus niger were all supplied from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). All other reagents and chemicals used in this study are analytical grade.

Dried FSI (sun dried with the water content of 8.0 ± 0.5%) was purchased from Hebei Anguo Yao Yuan Trading Co., Ltd. (Baoding, China). Rutin (RU), quercetin (QU), hyperoside (HY), quercitrin (QI), kaempferol (KA), gentianoside, protocatechuic acid, isorhamnetin, kaempferol-3-O-Rutinoside, narcissoside, chlorogenic acid, 4-nitrophenyl β-D-glucopyranoside (PNPG), acarbose, α-glucosidase (Yeast) (100 U/mg) and all chemicals were obtained from Shanghai Yuan Ye Biological Technology Co., Ltd. (Shanghai, China).

The fresh fruits of sea buckthorn used in this experiment were provided and identified by Qinghai Nationalities University. They were stored in the −18 °C freezer until use.
Reference substances of rutin (purity ≥ 98%), quercetin (purity ≥ 98%), isorhamnetin (purity ≥ 98%) were purchased from Shanghai yuanye Bio-Technology Co., Ltd. (Shanghai, China). Ultrapure water was produced by Barnstead TII super Pure Water System (MA, USA). D101 macroporous resin was purchased from Tianjin Bohong Resin Technology Co., Ltd. (Tianjin, China). All other analytical grade chemicals used in this experiment were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).
High-fat feed was purchased from SPF (Beijing, China) Biotechnology Co., Ltd. (Beijing, China). All detection kits (total cholesterol test kit, triglyceride detection kit, protein carbonyl detection kit, high-density lipoprotein cholesterol detection kit, low-density lipoprotein cholesterol detection kit, aspartate aminotransferase detection kit, alanine transaminase detection kit and Bradford protein detection kit) were purchased from Nanjing Jiancheng Bioengineering Research Institute (Nanjing, Jiangsu, China).

The general viability of the cultured cells was determined through the reduction of 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to formazan [20 (link)]. Exponentially growing B16F10 cells were trypsinized, harvested and equal numbers of cells (2 × 10⁵ cells/ml) in 100 μl medium were plated in 96-well microplates. After overnight incubation, 100 μl of different concentrations (8, 16, 32 μM) of Isorhamnetin and Kaempferide (purity >98%; Shanghai yuanye Bio-Technology Co., Ltd., China) was added to the well for 24 h. The untreated controls were exposed to fresh medium. Following this, 50 μl 5 mg/ml MTT solution was added to each well and incubated for 4 h. After aspirating the culture medium, the resulting formazan was dissolved with 150 μl dimethylsulfoxide (Sigma, USA). The plates were then placed on a shaker for 5 min and measured at 570 nm by using a microplate reader (Thermo Varioskan Flash 3001, USA).