M hydroxydiphenyl

Cassia seeds were obtained from a market in Ya’an, China and dried at a temperature of 45 °C for two days. Subsequently, the dried Cassia seeds were ground to pass through a 60 mesh sieve and stored at −20 °C for further analysis.
Trifluoroacetic acid, rhamnose, glucuronic acid, galacturonic acid, mannose, glucose, galactose, xylose, arabinose, m-hydroxydiphenyl, griess reagent, sodium nitroprusside (SNP), 1-phenyl-3-methyl-5-pyrazolone (PMP), 2,2-diphenyl-1-(2,4,6-trinitrophenyl) hydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), hydrogen peroxide, vitamin C, and butylated hydroxytoluene (BHT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other reagents and chemicals used were of analytical grade.

Grape pomace was collected by processing two different Vitis vinifera varieties (Fetească Neagră and Rară Neagră) from a 2019 harvest, cultivated in the Bugeac area, Republic of Moldova. The grape pomace was dried in an oven with air circulation Zhicheng ZRD-A5055 (Zhicheng, Shanghai, China) at 50 °C until constant weight was achieved. Then, dried pomace was powdered using a food processor (KitchenAid, Benton Harbor, MN, USA) and separated it on the following particle size intervals: <125 µm, ≥125–<200 µm and ≥200–<300 µm using an analytical sieve shaker Retsch AS 200 Basic (Retsch GmbH, Haan, Germany).
Nitric acid, sulfuric acid, citric acid, sulfamic acid, hydrochloric acid, sodium hydroxide, m-hydroxydiphenyl, potassium hydroxide, sodium tetraborate, D-galacturonic acid, and ethyl alcohol were acquired from Sigma-Aldrich (Darmstadt, Germany).

The flower of snow chrysanthemum (C. tinctoria) was purchased from a local market in Ya’an, China. Samples were dried at the temperature of 45 °C for 2 days. Subsequently, the samples were ground to pass through a 60 mesh sieve and stored at −20 °C for further analysis.
Trifluoroacetic acid, rhamnose (Rha), mannose (Man), glucuronic acid (GlcA), galacturonic acid (GalA), glucose (Glc), galactose (Gal), xylose (Xyl), arabinose (Ara), 1-phenyl-3-methyl-5-pyrazolone (PMP), m-hydroxydiphenyl, griess reagent, sodium nitroprusside (SNP), 2,2-diphenyl-1-(2,4,6-trinitrophenyl) hydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), vitamin C, butylated hydroxytoluene (BHT), and 6-hydroxy-2,5,7,8-tetramethyl chroman-2-carboxylic acid (Trolox) were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other reagents and chemicals used were of analytical grade.

GAGs were extracted from ~2x10⁷ cells and highly-sulfated cell surface CS/DS were released by β-elimination and purified as described previously [23 (link)]. The HexUA content was determined using an m-hydroxydiphenyl reaction. Uronic acid was hydrolyzed in 80% sulfuric acid containing tetraborate at 80°C, incubated with m-hydroxydiphenyl (Sigma Aldrich) at room temperature and measured at 540nm using heparin as standard [5 (link)].
10 μg CS/DS were digested with 10 mU of chondroitin ABC for 2h. The unsaturated disaccharides were labeled with 5 μl of 0.1 M 2-Aminoacridon (AMAC) in 15% CH₃COOH/DMSO solution. After 10 min incubation at RT, 1 M NaBH₃CN was added and the mixture was incubated 16 h at 37°C followed by fluorophore assisted carbohydrate electrophoresis (FACE). AMAC-labeled disaccharides were separated on 30% Borate-polyacrylamid gel [35 (link)]. HS were analyzed as described before. In order to analyze HS composition, cell pellets of B16V cell lines were prepared as described previously. After enzymatic removal of CS/DS, the heparin lyase I-, II- and III- digested GAGs were fractioned by RPIP-HPLC. The peaks were identified by co-elution with standard HS disaccharides [5 (link)].

Fruiting body of Coriolus versicolor used in this study is cultivated in Changbai Mountain district and identified by Professor Yi Xin at Department of Biotechnology, Dalian Medical University, Dalian, P.R. China. TriFlouracetic Acid (TFA) CAS No: 76-05-1, m-hydroxydiphenyl, CAS No: 580-51-8, Sulfamic Acid CAS No: 5329-14-6, Sephacryl S-500 High Resolution, CAS No: GE17-0613-01, 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyl tetrazolium bromide (MTT) Cat No: 3511-096-K, Concanavalin A (ConA) CAS No: 11028-71-0, Neutral Red CAS No: 553-24-2, and Cyclophosphamide (CP) CAS No: 6055-19-2, were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Medium RPMI 1640 with improved nutrient solution was purchased from Gibco industry (Grand Island, NY, USA, Cat No: 11875119). CCK-8, Sarcoma 180 (S-180) were purchased from (KeyGENBioTCH, Cat No: KGA317) China. All other reagents and chemicals used were of analytical grade made in China.
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Dalian Medical University. All surgeries were performed under sodium pentobarbital anesthesia, and efforts were made to minimize suffering.

Different chrysanthemum teas, including C. tinctoria (snow chrysanthemum tea), C. indicum, C. morifolium ‘Huangju’, C. morifolium ‘Gongju’, and C. morifolium ‘Hangbaiju’, were purchased from a local market in Ya'an, China. Chrysanthemum teas were dried at the temperature of 45 °C for 2 days, and then the dried samples were ground to pass through a 60 mesh sieve, and stored at −20 °C for further analysis.
Trifluoroacetic acid, monosaccharide standards (rhamnose, mannose, glucuronic acid, galacturonic acid, glucose, galactose, xylose and arabinose), 1-phenyl-3-methyl-5-pyrazolone (PMP), hydrogen peroxide (H₂O₂), m-hydroxydiphenyl, griess reagent, vitamin C, sodium nitroprusside (SNP), 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), butylated hydroxytoluene (BHT), 2,2-diphenyl-1-(2,4,6-trinitrophenyl) hydrazyl (DPPH), sodium azide, bovine serum albumin (BSA), and aminoguanidine were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other reagents and chemicals used were of analytical grade.

All of the chemicals and reagents used in this study were of analytical grade. Ethanol, acetone, sodium carbonate (Na₂CO₃), and potassium sulfate (K₂SO₄) were purchased from Gram-mol doo (Zagreb, Croatia); sodium tetraborate, L-cystein, sulfamic acid, gelatin, potassium hydroxide, chloroform, 1-phenyl-3-methyl-5-pyrazolone (PMP), and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) from Acros Organics (Geel, Belgium); acetonitrile, trichloroacetic acid (TCA), silicone oil, and Folin–Ciocalteu reagent from Fisher Scientific (Leicestershire, UK); barium chlorid (BaCl₂) from abcr GmbH (Karlsruhe, Germany), fucoidan from Fucus vesiculosus, D-galacturonic acid, phenol, m-hydroxydiphenyl, 2,2-diphenyl-1-picrylhydrazyl (DPPH), arabinose, D-(+)-glucose, L-rhamnose, D-(−)-fructose, trimethylamine, 2,2′-Azobis(2-methylpropionamidine) dihydrochloride (AAPH), D-(+)-mannose, L-(−)-fucose, and ammonium acetate from Sigma-Aldrich (St. Louis, Missouri, USA); ethyl acetate, hexane, and absolute ethanol from Carlo Erba Reagents (Cornaredo, Italy); hydrochloric acid (HCl) from TKI Hrastnik (Hrastnik, Slovenia); sodium hydroxide from Lach-Ner (Zagreb, Croatia); sulfuric acid (H₂SO₄) from Scharlab S.L. (Barcelona, Spain); D-(−)-ribose from TCI (Portland, OR, USA); and fluorescein sodium salt from Honeywell Riedel-de-Haën (Bucharest, Romania).