Electronic Supplementary Material (ESM)

Title: Phenolic acids of the two major blueberry species in the US market and their antioxidant and anti-inflammatory activities

MATERIALS AND METHODS

HPLC-MS/MS analysis

The HPLC-MS/MS analyses were carried out using an Agilent 1100 HPLC system including an autosampler, a binary pump and a diode array detector (Agilent Technologies, Palo Alto, CA), which coupled to the 4000 Q TRAPTM mass spectrometer (Applied Biosystems, Forest City, CA). Separation was carried on a Phenominex Synergi Max-RP column (150×3.00 mm, 4 μm) using a flow rate of 0.4 mL/min. The solvent consisted of (A) 0.2% (v/v) of formic acid in water and (B) methanol. The 34 min gradient was as follows: 15-25 % B (0-10 min), 25-30 % B (10-22 min), 30-60 % B (22-25 min),60-60 % B (25-33 min), 60-15 % B (33-34 min). Multi-reaction monitoring (MRM) mode scan was used for quantitation. The mass spectrometer equipped with an ESI-Turbo V source was operated in the negative ion mode. Major parameters were auto-optimized by the instrument as follows: ion spray voltage, - 4.5 kV; 50 for curtain gas (CUR), 400 ºC for source temperature, 30 and 50 for nebulizing (GS1) and turbo spray gas (GS2). The method was validated by using the authentic standards. For quantification, the standard curves of eight phenolic acids were generated (50-2000 ng/mL),with ther2 all above 0.99.

ORAC assay

The eightPA mixtures (3,4-dihydroxybenzoic acid, vanillic acid, caffeic acid, p-coumaric acid, ferulic acid, chlorogenic acid, salicylic acid and gallic acid)representingthose in the two species were made using authentic standards according to their relevant profile and quantity(Table 1).PA mixture was dissolved in MeOHand diluted properly with phosphate buffer (0.75 M, pH7.0).The assaywas carried out on a FLUOstar Galaxy microplate reader equipped withfluorescence filters for an excitation wavelength of 485 nm andan emission wavelength of 520 nm. The temperature of the incubatorwas set to 37 C. Fluorescein wasused as fluorescence probe; AAPH was used as peroxyl generator and Trolox as standard. The results were expressed as μmol Troloxequivalent (TE) per gram.

CAP-e assay

Healthy human volunteers between the ages of 20 and 50 yearsold served as blood donors after informed consent, as approved by theSky Lakes Medical Center Institutional Review Board (KlamathFalls, OR). Peripheral venous blood samples were drawn into sodiumheparin and layered onto a double-gradient of Histopaque1119 and 1077. The vials were centrifuged at 2400 rpm for25 min. The red blood cells (RBC) were harvested using steriletransfer pipettes. RBC fraction was washedtwice in PBS without calcium or magnesium. Then the core of the packed RBC was transferred intonew vials and again washed twice in PBS without calcium or magnesium. RBC aliquots were stored at 4 Cuntil use.

RBC cell suspension was prepared for the CAP-e assay by addingpacked RBC (0.1 mL) into PBS (10 mL). The cell suspension was distributedin a V-bottom 96-well microtiter plate. Twelve wells werenot treated with any source of anti-oxidants, and served as negativecontrols (six wells) and positive controls (six wells) for minimumversus maximum oxidative damage. The remaining wells were treated with the testcompounds. RBC were incubatedwith PA mixture for 20 min. The cells were then lysed and the precursor dye was added tothe wells. Incubation was performed at room temperature for15 min, followed by two washes. Oxidation was carried out usingthe peroxyl free radical generator AAPH for 1 h. The green fluorescenceintensity, as a measure of oxidative damage, was measuredat 488 nm using a microplate reader. The inhibition of oxidative damage was calculated as thereduced fluorescence intensity of product-treated cells, comparedto cells treated only with the oxidizing agent. The CAP-e value was expressed as the IC50 dose (g/L) of the test products.

Secreted Alkaline Phosphatase(SEAP) reporter assay

Secreted Alkaline Phosphatase (SEAP) reporter assay was performed based on a recent report from our group [17].Placental alkaline phosphatase (PLAP) is one of the most stable isoenzymes, only existing in the placenta of higher primates. These characteristics make placental alkaline phosphatase suitable to use as a reporter gene for the analysis of promoter activity and gene expression in cell culture and animal serum. The natural form of PLAP is membrane anchored. The recombinant form of placental alkaline phosphatase (secreted alkaline phosphatase, SEAP) is used for reporter gene function. SEAP is designed by inserting a translational terminator after amino acid 489.

RAW-Blue cells (Invitrogen, San Diego, USA) are derived fromRAW264.7 macrophages with chromosomal integration of a SEAPreporter construct inducible by NF-B. RAW-Blue cells were cultured in DMEM supplemented with 10% (v/v) FBS (Hyclone, Logan, UT) and zeocineosin (200 µg/mL).The cells (1×105 cells/well) were treated with various concentrations of PA mixturesfor 24 h. After 7 hr LPS (100 ng/mL) stimulation, the supernatants were then collected for SEAP reporter assay. QUANTI-Blue powder was dissolved in endotoxin-free water and sterile filtered (0.22 μm). RAW-Blue cell supernatant (40 µL/well) was added to QUANTI-Blue solution (160 µL/well) and incubated at 37 °C for 0.5 - 1 hr. Absorbance was measured at 620 nm in a PolarStar microplate reader (BMG Labtech, Durham, NC)

TNF- and IL-6 ELISA

RAW 264.7 cells (5106cells/well) werepretreated with various concentrations of the PA mixtures for 1 hbefore LPS stimulation. After 18 h of LPS stimulation, supernatant wascollected; TNF-α and IL-6 in the supernatant was determined by ELISAusing Duoset ELISA kits (R&D, Minneapolis, MN) according to themanufacturer’s instructions. The optical density was determined using aBMG PolarStar microplate reader (BMG Labtech, Durham, NC) at450 nm. A mean value of triplicate samples for each experiment and twoseparate experiments were used for analysis.

1

Table 1. Retention time (RT), characteristic MS transitions, and contents of PA in highbush and lowbush BB by HPLC/MS2

Peak / Compounds / MW / MRM
transitions / RT
(min) / lowbush BB
(mg/g FW) / % in total PA / Highbush BB
(mg/g FW) / % in total PA
1 / gallic acid (1) / 170 / 169/125 / 6.1 / 2.3 ×10-4±5.0×10-6 / 0.05 / 2.8 ×10-3±2.5×10-5 / 2.15
2 / 3,4-dihydroxybenzoic acid (2) / 154 / 153/109 / 11.8 / 8.2 ×10-5±3.2×10-6 / 0.02 / 2.6 ×10-2±5.0×10-4 / 19.34
3 / chlorogenic acid (3) / 354 / 353/191 / 21.5 / 4.4 ×10-1±1.7×10-3 / 99.71 / 1.0 ×10-1±3.9×10-3 / 74.38
4 / vanillic acid (4) / 168 / 167/152 / 23.4 / 8.2 ×10-5±2.4×10-7 / 0.02 / 4.8 ×10-4±1.5×10-6 / 0.36
5 / caffeic acid (5) / 180 / 179/135 / 24.3 / 4.9 ×10-4±4.6×10-6 / 0.11 / 1.0 ×10-3±2.0×10-6 / 0.74
6 / p-coumaric acid (6) / 164 / 163/119 / 29.6 / 5.9 ×10-5±3.1×10-7 / 0.01 / 9.6 ×10-4±1.1×10-7 / 0.71
7 / ferulic acid (7) / 194 / 193/178 / 30.0 / 1.6 ×10-4±1.1×10-6 / 0.04 / 2.0 ×10-4±1.3×10-6 / 0.15
8 / salicylic acid (8) / 138 / 137/93 / 33.3 / 1.8 ×10-4±2.1×10-6 / 0.04 / 3.0 ×10-3±2.1×10-6 / 2.23
Total PA / 4.4×10-1 / 1.3×10-1

1

Figure 1. The PA chromatograms of lowbush (A) and highbush (B) BB analyzed by HPLC-MS/MS.

A.

B.

1