Protodioscin

RPMI 1640 medium and L-Glutamine were purchased from Lonza, Fetal Bovine Serum was from Gibco BRL (Cergy Pontoise, France), Bradford reagent was obtained from Bio-Rad (Hercules, CA, USA), and β-Nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt hydrate (NADPH) from Roche Diagnostics (Mannheim, Germany). ProtoDioscin and Dioscin natural compounds tested in this study were purchased from Sigma Aldrich, and their purity was ≥ 99%. Cepltatin (Cisplatine) was obtained from Pharmedic Laboratories Pvt (Ltd.) Pakistan. All other chemicals and biochemical reagents were purchased from Sigma-Aldrich. Two human breast adenocarcinomas cell lines came from the Curie Institute bank (Translational Research Department, Breast Cancer Biology Team, Paris, France). They were subsequently obtained from the parent cultures of the laboratory of Experimental Oncology and Natural Substances of the Faculty of Science and Technology of Sultan Moulay Slimane University, Beni Mellal.

Tribulus terrestris dry extract (origin China) was purchased from Xi'an Green Life Natural Products Co., Ltd. (China), in November 2013, batch number: 20121023, CAS number: 18642-44-9. Fruits were used to prepare the dry extract (alcohol/water extraction was used according to the manufacturer). The steroid saponin concentration has been determined to be a minimum of 40% of the dry matter. Cyclophosphamide, protodioscin, and all other chemicals and reagents were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Acetonitrile was purchased from Tedia (Fairfield, OH, USA). Acetic acid was obtained from Synth (São Paulo, Brazil). Purified water used on HPLC (high-performance liquid chromatography) analysis was prepared using Milli-Q Plus® (Millipore, Bedford, USA).

Fusarium proliferatum KF 3360 strain cultures on solid PDA medium were used to evaluate the efficiency of plant metabolites in reducing the fungal growth in vitro. Four arbitrary concentrations of plant metabolite standards, namely, quercetin-3-glucoside, kaempferol-3-rutinoside, isorhamnetin-3-O-rutinoside, ferulic acid, chlorogenic acid, neochlorogenic acid, protodioscin, DIMBOA (all supplied by Sigma Aldrich, Taufkirchen, Germany) were tested (1000, 100, 10, and 1 μg/mL). Sterile 90 mm Petri plates contained 15 mL of PDA medium and were inoculated with a single PDA plug (1 cm²) of mycelium. The solutions of plant metabolites were then applied onto the plug and then incubated for seven days at room temperature with a 12 h photoperiod. The experiment was conducted in triplicate, and colony diameter was measured daily. For protodioscin and DIMBOA, 100 μg/mL concentration was chosen for liquid cultures and 1 μg/mL for chlorogenic acid, while 10 μg/mL was used for the remaining compounds.