Pinocembrin

HPLC-grade methanol was purchased from Merck (Merck & Co., Inc., Billerica, MA, USA), and analytical grade acetic acid and absolute ethanol were purchased from Chemical Reagent Factory of Zhejiang University (Hangzhou, Zhejiang, China). Absolute alcohol and acetic acid were purchased from Shanghai Chemical Reagent Company of Chinese Medical Group (Shanghai, China). Ultra-Pure water was purified by the Yjd-upws Ultra-Pure water system (Hangzhou, Zhejiang, China).
DPPH, vanillic acid, caffeic acid, ferulic acid, isoferulic acid, p-coumaric acid, cinnamic acid, 3,4-dimethoxycinnamic acid, CAPE, myricetin, apigenin, galangin, chrysin, pinocembrin, quercetin, kaempferol, luteolin and naringenin were purchased from Sigma–Aldrich (St. Louis, MI, USA), pinobanksin and 3-O-acetylpinobanksin were purchased from Ningbo Haishu Apexocean Biochemicals Co., Ltd (Ningbo, Zhejiang, China), and benzyl p-coumarate was purchased from Kunming BioBioPha Co., Ltd (Kunming, Yunnan, China).

Quercetin, rutin, ferulic acid, coumaric acid, pinocembrin, DPPH (2,2-diphenyl-1-picrylhydrazyl), ABTS (2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfphonic acid), TPTZ (2,4,6-tris(2-pyridyl)-s-triazine), Folin–Ciocalteu phenol reagents, Trolox, β-carotene/linoleic acid, 1,2-Bis(dimethylamino)ethane (TEMED), ethylenediamine tetraacetic acid (EDTA) and streptozotocin (STZ) were obtained from Sigma-Aldrich (St. Louis, MO, USA).

Gallic acid (3,4,5-trihydroxybenzoic acid), Caffeic acid, p-Coumaric acid, Ferulic acid, Luteolin, Quercetin, t-Cinnamic acid, Apigenin, Hesperidin, Rhamnetin, Chrysin, Pinocembrin, Protocatechuic acid, Chlorogenic acid, p-hydroxybenzoic acid, Epicatechin, Syringic acid, m-hydroxybenzoic acid, Ellagic acid, Myricetin, Resveratrol, Daidzein, Curcumin, Caffeic acid phenethyl ester (CAPE), 2,2-diphenyl-1-picrylhydrazyl (DPPH), Ascorbic acid were obtained from Sigma-Aldrich. Butylated hydroxytoluene (BHT), 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), were purchased from Fluka, USA. Potassium persulfate, Sodium carbonate, Folin–Ciocalteu solution, Aluminium chloride, Ethanol 96%, Methanol, were purchased from POCH, Poland.

The human breast cancer cell line MDA-MB-231 was maintained in complete DMEM supplemented with 10% fetal bovine serum (FBS) (Hyclone-GE Healthcare life Sciences, Pittsburg, USA), as previously described [22 (link)]. The human NSCLC cell line A549 was maintained in RPMI 1640 (Hyclone) supplemented with antibiotics (penicillin 50 U/mL; streptomycin 50 µg/mL) and 10% FBS. In all experiments, cell viability was higher than 99% using trypan blue dye exclusion. MH (UMF^® 20+) was purchased from ApiHealth (Auckland, New Zealand). As a control for MH, we used a sugar solution (designated Sugar Control or SC) containing equivalent concentrations of the three major sugars in honey (38.2% fructose, 31.3% Glucose, and 1.3% Sucrose), as described [23 (link)]. The MH flavonoid compounds (luteolin, quercetin, galangin, chrysin, and pinocembrin) were purchased from Sigma (St. Louis, MO, USA). For all reagents, appropriate dilutions to the desired concentrations were freshly made before addition to the cell cultures. Flavonoid stocks were made in DMSO and diluted to required concentrations in 5% DMEM for cell culture or in PBS for use in ELISA binding assay. Similarly-diluted DMSO solution in 5% DMEM or PBS was used as a control for the flavonoids.

Standards of phenolic acids and flavonoids (apigenin, 2,3-dihydroxybenzoic acid, caffeic acid, catechin, chlorogenic acid, chrysin, ferulic acid, galangin, gallic acid, hesperidin, m-hydroxybenzoic acid, p-hydroxybenzoic acid, 5-hydroxyferulic acid, kaempferol, methyl p-coumarate, morin, myricetin, naringenin, naringin, p-coumaric acid, pinocembrin, quercetin, quercitrin, rosmarinic acid, rutin, salicylic acid, salicylic acid glucoside, sinapic acid, syringic acid, trans-cinnamic acid, vanillic acid, p-coumaric acid-d₆, and salicylic acid-d₄) were purchased from Sigma-Aldrich (Steinheim, Germany).
The sodium molybdate dihydrate, sodium nitrite, sodium hydroxide, sodium carbonate, hydrogen peroxide, sodium ascorbate, dimethyl sulfoxide (DMSO), and bovine hemoglobin were also purchased from Sigma-Aldrich (Steinheim, Germany). The LC (liquid chromatography) grade methanol, analytical grade orthophosphoric acid, hydrochloric acid, aluminum chloride, sodium acetate, ethanol, and Folin–Ciocalteu reagent were purchased from Merck (Darmstadt, Germany). The DPPH (2,2-diphenyl-1-picrylhydrazyl) was obtained from Alfa-Aesar (Karlsruhe, Germany). All spectrophotometric data were acquired using a Jasco V-530 UV-Vis spectrophotometer (Jasco International Co., Ltd., Tokyo, Japan).

The proximal composition in waxes, resins, impurities, and ashes, as well as minerals and humidity, were determined previously according to the methods recommended by Bankova et al. [23 (link)]. The equipment used for the different determinations and analyses were Soxtec System HT 1043 (Tecator, DK-3400, Hilleroed, Denmark) and Muffle Oven Mod. 10PR/300 Serial 88 (Hobersal, Barcelona, Spain). Furthermore, Polyphenols Total Content (PTC), Flavonoids Total Content (FTC), and Flavanones, Flavones, and Flavonols were also determined, as was explained in earlier works [24 (link)]. The extraction assays used ethanol from Panreac and methanol (Labkem, Barcelona, Spain), and a Spectrophotometer DU 7400 (Beckman, Brea, CA, USA) was used for all the assays. The preparation of the calibration graph involved the following products from different trading houses: Pinocembrin, Med Chem Express, and Galangin, Med Chem Express, both from Sweden and Quercetin, Sigma Aldrich (St. Louis, MO, USA). The antioxidant scavenging profile was also performed. Using different homologated Kits: ABTS Antioxidant Capacity Batch 10022604 and DPPH Antioxidant Capacity Batch 10072004. BQC Redox Technologies (Asturias, Spain) allowed us to test the EPP quickly and efficiently [25 ,26 (link)].