P hydroxybenzoic

Compounds were separated on a 250 × 4.6‐mm stainless steel column Hypersil XDB‐ C18 (Agilent, Santa Clara, CA, USA), packed with 5 μm particles using a stepwise mobile phase gradient prepared from 1% aqueous acetic acid (component A) and methanol (component B; v/v). The gradient was as follows: 0 min, 5% component B into A; 5 min 20% component B into A; 20 min 35% component B into A; 35 min 55% component B into A; and 45 min 75% component B into A. The mobile phase flow rate was 1 mL·min⁻¹, the sample injection volume was 10 μL and the elution was performed at 25 °C. Liquid chromatography pumps, autosampler, column oven and diode array detector were monitored and controlled by hp chem station rev.10.0 software (Agilent). Retention times were compared with standards using UV spectra (λ = 254 nm for benzoic acid derivatives: protocatechuic, p‐hydroxybenzoic, vanillic and syringic acids; 280 nm for gallic acid; 325 nm for cinnamic acid derivatives: p‐coumaric ferulic, caffeic and chlorogenic acids) as a comparative parameter. Following PhAs, standards were used: p‐hydroxybenzoic, m‐hydroxybenzoic, protocatechuic, gallic, vanillic, syringic, trans‐cinnamic, p‐coumaric, caffeic, ferulic, rosmarinic, chlorogenic, coumaric, o‐coumaric, isovanillic and gentisic acids (Sigma‐Aldrich).

All the used reagents and solvents were of analytical or liquid chromatography purity and were obtained from Merck (Darmstadt, Germany). Formic and phosphoric acids, anhydrous sodium carbonate, aluminium chloride, sodium acetate and 96% ethanol were analytical grade, while methanol and acetonitrile were for liquid chromatography use. The Folin–Ciocalteu phenol reagent (2 N), 2,2-Diphenyl-1-picrylhydrazyl and 6-hydroxy- 2,5,7,8-tetramethyl-2-carboxylic acid (Trolox) used for the determination of total polyphenols composition and antioxidant activity were purchased from Sigma-Aldrich (St. Louis, MO, USA). All phenolic standards (caffeic, gallic, ferulic, p-coumaric, p-hydroxybenzoic, 3,4-dihydroxybenzoic, t-cinnamic and chlorogenic acids, catechin, epicatechin, quercetin, rutin, t-resveratrol, ursolic and oleanolic acids) had purities corresponding to high performance liquid chromatography (HPLC) and were purchased from Sigma-Aldrich (Steinheim, Germany). Stock and working standard solutions were prepared in methanol. Deionised water, produced by a Milli-Q Millipore system (Bedford, MA, USA), was used for the preparation of aqueous solutions and mobile phases.

Ethanol, hexane, acetonitrile, sodium hydroxide, and hydrochloric acid were purchased from Fisher Scientific (Mississauga, ON, Canada). Suprapur formic acid (FA) was purchased from VWR (Mississauga, ON, Canada). 2,2-Diphenyl-1-picrylhydrazyl (DPPH), 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), fluorescein, 2,2′-Azobis (2-methylpropion-amidine) dihydrochloride (AAPH), Trolox, gallic, protocatechuic, p-hydroxybenzoic, gentisic, 3-hydroxy-benzoic, vanillic, caffeic, syringic, p-coumaric, ferulic, sinapinic, and o-coumaric acids were purchased from Sigma (Sigma-Aldrich Canada Ltd., Oakville, ON, Canada). All the chemicals and reagents used in the study are of analytical grade. The nano pure water was obtained from Milli-Q integral water purification system (Millipore (Canada) Ltd., Etobicoke, ON, Canada).

Neutral-detergent fibre (NDF) was determined using a solution package containing sodium lauryl sulphate, EDTA disodium, sodium borate and sodium phosphate dibasic together with glycol and α-amylase (all Ankom Technology, NewYork, NY, USA). To study digestibility, pepsin (0.7 FIG-U/mg) and a mixture of pancreatin enzymes (350 FIG-U/g protease, 6000 FIG-U/g lipase and 7500 FIG-U/g amylase) were used (all Merck, Darmstadt, Germany). To determine antioxidant activities, 2,2′-azinobis(3-ethylbenzo-thiazoline-6-sulfonic acid) diammonium salt (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH) and standard 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox) (Sigma-Aldrich, St. Louis, MO, USA) were used. PCL was measured and evaluated using ACL and ACW kits supplied by Analytik Jena AG (Jena, Germany). For the determination of phenolic profile, the standard compounds (epigallocatechin, catechin, epicatechin, rutin, quercetin, kaempferol, neochlorogenic, chlorogenic, gallic, protocatechuic, p-hydroxybenzoic, vanillic, caffeic, syringic, p-coumaric, ferulic, sinapic, ellagic, o-coumaric, cinnamic acids and protocatechuic acid ethyl ester) were purchased from Sigma-Aldrich, St. Louis, MO, USA. All standards and solvents were HPLC-grade (purity ≥ 98.5%).

The analytic phenolic components as followed: resveratrol, hydroxybenzoic acids (i.e., salicylic acid, p-hydroxybenzoic, syringic acid, gallic acid, vanillic acid, 3,4-dimethoxybenzoic acid, protocatechuic acid, and gentisic acid), hydroxycinnamic acids (i.e., p-coumaric acid, o-coumaric acid, m-coumaric acid, caffeic acid, ferulic acid, sinapinic acid, and chlorogenic acid), and flavonoids (i.e., naringenin, hesperidin, naringin, kaempferol, and rutin) were purchased from Sigma-Aldrich (Saint Louis, MO, USA). The compounds were dissolved in dimethyl sulfoxide (DMSO) and stored at −20 °C. Reagent chemicals used in the present study were of analytical grade.

Dehulled adlay (DA) was obtained from Xingren, Guizhou Province, China (N 25°18′, E 105°34′) and sealed in plastic bags and stored until use (4 °C). B.subtilis BJ3-2 was provided by Dr. Wu (College of life sciences, Guizhou University, Guizhou, China). Human glioma U251 cells, human hepatocellular carcinoma HepG-2 cells, human colonic cancer Hct-15 cells, human non-small cell lung cancer A549 cells, human pancreatic cancer Panc-1cells, human leukemia K562 cells, and human embryonic kidney 239T cells were obtained from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). DMEM medium, RPMI-1640 medium, fetal bovine serum (FBS), and streptomycin were obtained from Gibco (Grand Island, NY, USA). Phosphate buffer solution (PBS), MTT reagent, dimethyl sulfoxide (DMSO), and Hoechst 33258 were purchased from Solarbio Science and Technology Ltd. (Beijing, China). Specific primary antibodies against Bax (ab32503), Bcl (ab32124), Caspase-3 (ab32351), Caspase-8 (ab32397), Caspase-9 (ab32539), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (ab8245) were purchased from Abcam Technology, Inc. (Danvers, MA, USA). TMP, protocatechuic, 2,3,4-trihydroxybenzoic, caffeic, chlorogenic, p-hydroxybenzoic, trans-cinnamic and ferulic acids, and rutin were obtained from Sigma Chemical Co. (St. Louis, MO, USA).

Absolute ethanol (EtOH), sodium hydroxide (NaOH), formic acid ≥ 95%, and all the standards used in the present work (ferulic, p-coumaric, o-coumaric, m-coumaric, p-hydroxybenzoic, caffeic, syringic, citric, vanillic, and protocatechuic acids, syringaldehyde, vanillin, quercetin, and kaempferol) were obtained from Sigma-Aldrich, St. Louis, MO, USA. Acetonitrile HPLC Plus Gradient grade, hexane, and ethyl acetate (EtOAc) were from Carlo Erba, Val de Reuil, France. Phosphoric acid 85% p.a. was from Panreac, Barcelona, Spain. Water was purified by a Milli-Q water purification system from Millipore, Burlington, MA, USA.