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1182 DOI 10.1002/mnfr.201200678 Mol. Nutr. Res. 2013, 57, 1182–1197

RESEARCH ARTICLE and from – fermented beverages inhibit markers of inflammation in macrophages and carbohydrate-utilizing enzymes in vitro

Michelle H. Johnson1, Elvira Gonzalez de Mejia1,2, Junfeng Fan3, Mary Ann Lila4 and Gad G. Yousef4

1 Division of Nutritional Sciences, University of Illinois, Urbana, IL, USA 2 Department of Food Science and Human Nutrition, University of Illinois, Urbana, IL, USA 3 College of Bioscience and Biotechnology, Beijing Forestry University, Beijing, P. R. China 4 Plants for Human Health Institute, NC Research Campus, North Carolina State University, Kannapolis, NC, USA

Scope: are an excellent source of dietary flavonoids which have several health benefits. Received: October 14, 2012 Methods and results: We evaluated well-characterized anthocyanins (ANCs) and proantho- Revised: January 14, 2013 (PACs) from fermented blueberry–blackberry beverages. were produced from Accepted: January 23, 2013 highbush and blackberries grown in Illinois and blended to create ratios rang- ing from 100% blueberry to 100% blackberry. Total ANCs of the were strongly corre- lated to total phenolics (r = 0.99, p < 0.05) and to antioxidant capacity (r = 0.77, p < 0.05). ANC- and PAC-enriched fractions were purified from each wine blend and a phenolic profile was generated. ANCs increased with more blackberries from 1114 to 1550 mg -3-O- glucoside (C3G) equivalents/L. Hydrolysable were identified in the PAC-enriched frac- tion. Both ANC- and PAC-enriched fractions inhibited starch-degrading enzyme ␣-glucosidase and dipeptidyl peptidase-IV activity. Computational docking demonstrated that - 3-arabinoside effectively inactivated dipeptidyl peptidase-IV by binding with the lowest inter- action energy (−3228 kcal/mol). ANC and PAC (100 ␮M C3G and epicatechin equivalents, respectively) from blueberry–blackberry blends reduced LPS-induced inflammatory response in mouse macrophages via the nuclear factor kappa B-mediated pathway. Conclusion: ANC- and PAC- (including hydrolysable tannins in blackberry) enriched fractions from blueberry and blackberry fermented beverages are beneficial sources of , inhibitors of carbohydrate-utilizing enzymes, and potential inhibitors of inflammation.

Keywords: / / Diabetes / Hydrolysable tannins / Inflammation / Proantho- cyanidin

1 Introduction

Correspondence: Professor Elvira Gonzalez de Mejia, Depart- Blueberries and blackberries are a rich source of anthocyanins ment of Food Science and Human Nutrition, University of Illi- (ANCs), the water-soluble pigments which give berries their nois at Urbana-Champaign, 228 ERML, 1201 W. Gregory Avenue, , blue, and coloring [1]. Berries also contain poly- Urbana, IL 61801, USA merized phenolic compounds including hydrolysable tannins E-mail: [email protected] (HTs), such as ellagitannins and gallotannins, and condensed Fax: +1-217-265-0925 tannins known as proanthocyanidins (PACs) [2]. The con- Abbreviations: ANC, anthocyanin; C3G, cyanidin-3-O-glucoside; sumption of ANCs is among the highest of all flavonoids COX-2, cyclooxygenase-2; DPP-IV, dipeptidyl peptidase-IV; EAE, ellagic acid equivalents; HT, hydrolysable ; iNOS, inducible nitric oxide synthase; MTS, 3-(4,5-dimethylthiazol-2-yl)- PAC, ; PES, phenazine ethosulfate; p-p65 NF␬B, 5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; phosphorylated p-65 subunit of nuclear factor kappa B; TE,trolox NO,nitricoxide;ORAC, oxygen radical absorbance capacity; equivalents

C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.mnf-journal.com Mol. Nutr. Food Res. 2013, 57, 1182–1197 1183 due to their wide distribution in [3]. Positive health tioxidant capacity, the ability to reduce carbohydrate-utilizing effects have been attributed to the high capacity enzymes activity, and reduce inflammation. of these . ANCs have been established as hav- ing a high antioxidant capacity by scavenging free radicals and reducing oxidative stress [4]. PACs may also reduce oxidative 2 Materials and methods stress and inflammation [5]. Increased phenolic intake has been seen to correlate with health benefits and is linked with 2.1 Materials decreased incidence of several diseases, including metabolic syndrome [6] and diabetes [4]. Wines used were produced from highbush blueberries Fermentation is known to increase the phenolic content of ( corymbosum) and blackberries ( spp.) grown berry juice products by increasing extraction from the skins, at Dixon Springs Agricultural Center in Simpson, Illinois. including ANCs [7], and therefore may increase the antioxi- Blueberry wine was fermented as previously described [14]. dant capacity [8] and potential for health benefits. Moderate Blueberry (V. corymbosum) Blue Chip, Bluecrop, wine consumption as part of a healthy diet may reduce the Blue Haven, Blue Jay, Blueray, Bluetta, Collins, Coville, Dar- risk of certain chronic diseases linked to inflammatory pro- row, Earliblue, Elliot, Jersey, Late Blue, and Spartan were cesses [6, 9]. The health benefits and sensory properties at- collected from Dixon Springs Agricultural Center during the tributed to fermented berry products may be due to the high ripening season of 2010. Blueberry juice from crushed frozen amounts of polyphenolic compounds present [10–12]. blueberries was fermented with Saccharomyces bayanus at Type 2 diabetes occurs when the body is unable to pro- 23ЊC until alcohol content reached 5–6%; finished wine was vide a proper insulin response to deal with glucose from the filtered through cheesecloth. Blackberry wine was prepared diet, leading to excessive blood glucose and insulin resistance. at the same time using a similar procedure [14]. Blackberries Several studies have been conducted to evaluate the reduction (Rubus fruticosus) cultivars A-1937, A-2215, A-2241 Natchez, of absorption of glucose by berries and berry compounds to A-2315, APF 27, APF 40, APF 41, and Prime Jan were col- inhibit ␣-glucosidase and ␣-amylase [13–15]. One potential lected from Dixon Springs Agricultural Center during the therapy for diabetics targets these carbohydrate-utilizing ripening season of 2010. Upon collection, the berries were enzymes; natural sources of inhibitors of these enzymes stored frozen at −18ЊC until use. Wine was prepared using would be beneficial to avoid undesirable side effects that oc- 90.7 kg blackberries, with roughly even mixtures of all culti- cur with drug therapy [16]. Dipeptidyl peptidase-IV (DPP-IV, vars listed above. To the blackberry lot, 180 mL SO2 solution EC.3.4.14.5) is another current pharmaceutical target for di- (50 ppm), 3 g pectic enzyme solution (dissolved in water), abetes treatment. Inhibitors of this enzyme are emerging as and 10 kg sucrose solution were added. Dry wine yeast Lavin antidiabetes therapy and allow for stimulation of insulin se- EC 1118 (S. bayanus) obtained from Presque Isle Wine Cel- cretion to consequently decrease blood glucose levels [17]. lars (North East, PA) was rehydrated at 40ЊC for 20 min with However, the inhibitory potential on DPP-IV by berry com- occasional gentle stirring to remove clumps prior to inocula- pounds needs to be further studied. tion of the juice. Per manufacturer’s recommendations, EC Chronic inflammation due to abnormal circulating lev- 1118 was inoculated at 40 or 50 mg dry yeast/100 mL, and els of pro-inflammatory cytokines is associated with the held at 21 ± 1ЊC during fermentation. Blends ranging from development of diabetes and other metabolic diseases. A 100% blueberry to 100% blackberry were made using room stimulated inflammatory cascade may be mediated by nu- temperature fermented wines. Blends are presented as a ra- clear factor kappa B (NF␬B) signaling pathway to upreg- tio of %blueberry : %blackberry. The ratios were 100:0, 70:30, ulate pro-inflammatory enzymes, such as inducible nitric 50:50, 30:70,and0:100 of blueberry:blackberry wine blends, oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Di- respectively. etary flavonoids have been linked to anti-inflammatory path- All solvents used for phenolic extraction were HPLC- ways [3], and many berries or berry compounds have been grade and were purchased from Fisher Scientific (Pittsburg, investigated for their anti-inflammatory properties and an- PA). Amberlite XAD-7 was purchased from Sigma-Aldrich tioxidant capacity or radical scavenging activity [4, 18–21]. (St. Louis, MO), Sephadex LH-20 was purchased from GE Life Fermented juices and wines from berries have been inves- Sciences (Buckinghamshire, UK). Murine macrophage RAW tigated [11, 22], and in several studies they were found to 264.7 cell line and DMEM with L-glutamine were purchased inhibit inflammatory production or expression more from American Type Culture Collection (ATCC) (Manassas, than their unfermented counterparts [12, 23–25]. VA). Fetal bovine serum was from Invitrogen (Grand Island, The objective of this study was to evaluate well- NY). CellTiter 96R AQueous One Solution Proliferation assay characterized ANCs-enriched fraction and PACs-enriched kit (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)- fraction present in blueberry–blackberry fermented bever- 2-(4-sulfophenyl)-2H-tetrazolium (MTS)/phenazine ethosul- ages for their effect on markers of inflammation in RAW fate (PES)) was purchased from Promega Corporation 264.7 macrophages and carbohydrate utilization in vitro. (Madison, WI). Mouse monoclonal antibodies (actin, iNOS, We hypothesized that blends of ANCs and PACs present COX-2) and rabbit polyclonal antibody (p-p65 NF␬B) were in blueberry–blackberry fermented beverages have high an- obtained from Santa Cruz Biotechnology (Santa Cruz, CA),

C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.mnf-journal.com 1184 M. H. Johnson et al. Mol. Nutr. Food Res. 2013, 57, 1182–1197 and anti-mouse and anti-rabbit IgG horseradish peroxidase for C3G; and 1000 as the conversion factor from gram to mil- conjugated secondary antibodies were from GE Health- ligram; and 0.45 as the conversion factor from the established care (Buckinghamshire, UK). Compounds used as stan- method [27] to the plate reader method. The AOAC Official dards and their purity were as follows: Trolox (≥97%), Method [28] has been standardized and validated and uses glucose (≥99.5%), gallic acid (≥98%), ellagic acid (≥95%), C3G to express ANC as equivalents as it is the most common (-)-epicatechin (≥99%), and cyanidin-3-O-glucoside (C3G) ANC pigment found in nature. Similarly, (-)-epicatechin was (≥99%). All chemicals and reagents were purchased from used to quantify PACs due to common use as a standard. Sigma (St. Louis, MO), unless otherwise specified. The pH differential method utilizes C3G to calculate total monomeric ANCs and referred them as C3G equivalents, and, therefore, this was used as the quantification standard 2.2 Total phenolic (TP) content for the HPLC peaks. TP content was quantified using the Folin–Ciocalteu method adapted to a microassay [26]. Briefly, to a 96-well flat bottom 2.5 Phenolic extraction and preparation of enriched plate, 50 ␮L of 1 N Folin–Ciocalteu’s phenol reagent and 50 ␮L fractions of either sample, standard or blank were added; this mixture was allowed to stand for 5 min before the addition of 100 ␮L Phenolic extraction was adapted from the method by Grace of 20% Na CO . The solution was then allowed to stand for 2 3 et al. [29]. To each blend, 0.1% TFA was added to stabilize 10 min before reading at 690 nm using a Synergy 2 multiwell the ANCs during isolation. A rotary evaporator with tem- plate reader (Biotek, Winooski, VT). Results were expressed perature not exceeding 40ЊC was used to remove the alco- as milligrams ellagic acid equivalents (EAEs)/L wine. hol completely and most of the water from 600 mL of each blend. The dealcoholized and concentrated beverage blends 2.3 Antioxidant capacity were mixed with amberlite XAD-7 resin preconditioned with acidified water (0.1% TFA), settled at room temperature for Antioxidant capacity was measured by the oxygen radical 20 min, and water was decanted. This process was repeated absorbance capacity assay as described by Prior et al. [27], three more times until the supernatant became clear where using 20 ␮L trolox standard, samples, or blank (75 mM , pectins, phenolic acids, and other impurities were re- buffer, pH 7.4), 120 ␮L of 116.9 nM fluorescein moved from samples. XAD-7 is a moderately polar resin and (final concentration 70 nm/well), 60 ␮Lof40mM␣,␣- is used to remove relatively polar compounds from nonaque- azodiisobutyramidine dihydrochloride (AAPH) per well. A ous solvents, and to remove nonaromatic compounds from black walled 96-well plate was read at 485 and 582 nm every polar solvents. Acidified water was used to precondition the 2 min at sensitivity 60 at 37ЊC using a Synergy 2 multiwell resin and was acidified to protect the ANCs from degrada- plate reader (Biotek). Results were expressed as millimoles tion. The sample with resin was loaded onto a column (30 × Trolox equivalents (TE)/L. 3 cm) and washed thoroughly with 1.5 L acidified methanol (0.1% TFA) to elute the polyphenolic mixture from resin. The polyphenolic mixture was concentrated to remove any 2.4 Total ANC content methanol on a rotary evaporator (not exceeding 40ЊC) to yield a crude extract which contained ANC and PAC phenolics Total ANCs from wine blends were determined using the present in wine. This was then partitioned with ethyl acetate AOAC Official Method adapted from Lee et al. [28] for mi- to remove any nonpolar compounds, and the polar eluate was croassay. Samples were diluted to a factor of 1:10 using then concentrated again on a rotary evaporator (not exceeding two buffers (pH 1.0, KCl buffer and pH 4.5, sodium acetate 40ЊC). Samples were then stored at −80ЊC and subsequently buffer). Diluted solutions at each pH were transferred to a lyophilized on a Labconco freeze-dryer (Kansas City, MO). 96-well plate and the absorbance was read at 520 and 700 To generate ANC- and PAC- enriched fractions, approx- nm on a Synergy 2 multiwell plate reader (Biotek). The total imately 4 g of the freeze dried material dissolved in 10 mL ANCs were expressed as C3G equivalents in milligram per of acidified water:methanol (80:20; 0.1% TFA) was loaded on liter using the equation below: a Sephadex LH-20 column (30 × 3 cm) preconditioned with   mg water:methanol (80:20; 0.1% TFA). With an isocratic elution Total monomeric anthocyanins L using the same solvent ratio, fractions were collected starting A × MW × D × 1000 when colored material began to elute to generate ANC frac- = × 0.45 tions 1–4, and then 50% methanol was used to remove any ␧ × PL remaining ANC as fraction 5. Next, 70% acetone was used where: A = (A520–A700)atpH1.0 − (A520–A700)atpH4.5; MW = to elute the PAC, and fractions were collected as PAC 1–4. 449.2 g/mol for C3G; D = dilution factor determined experi- The solvents were evaporated on a rotary evaporator (not ex- mentally with pH 1.0 buffer; PL is the constant at path length ceeding 40ЊC), and fractions were freeze dried, weighed, and 1cm;␧ is 26 900 L/mol cm, the molar extinction coefficient stored at −80ЊC until further use. Phenolic extraction and

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Figure 1. Phenolic extraction and fractionation procedure to prepare anthocyanin (ANC)- and proanthocyanidin (PAC)-enriched fractions. fractionation procedure to prepare ANC- and PAC-enriched 25%, 30%, 60%, and 10% of solvent B at 0, 5, 15, 20, 25, fractions, indicating the methods conducted are presented in 45, and 60 min, respectively. For PAC analyses, the same in- Fig. 1. strumentation was used but with mobile phase consisting of

ANC 1–5 and PAC 1–4 were collected from each blend. 95% H2O: 5% ACN with 0.1% formic acid (A) and of 95% The following enriched fractions were used for further anal- ACN: 5% H2O with 0.1% formic acid (B). The flow rate was ysis because they had the highest ANC or PAC concentra- held constant at 1 mL/min with a step gradient of 0%, 5%, tion based on HPLC analysis: ANC-3 for 100% blueberry 30%, 60%, 90%, and 0% of solvent B at 0, 5, 40, 45, 50, and also for 50:50%; ANC-2, for 100% blackberry; PAC-1, for and 55 min, respectively. ANC was detected at 520 nm and 100% blueberry; PAC-4 for 50:50%; PAC-2 for 100% black- PAC at 280 nm using a DAD. Chemstation software (Agilent berry. The other fractions were not studied beyond HPLC Technologies) was used for both sample analysis protocol and analysis. data processing. A reference blueberry extract (post-amberlite extract 22%) was always run as an internal standard, during HPLC analysis, to account for any variation in the reten- 2.6 ANC and PAC analysis tion time among runs. For qualitative peak identification of specific ANCs, we used pure standards that were already in- ANC analysis was conducted on a 1200 HPLC (Agilent Tech- corporated into our HPLC library, and then we did compar- nologies, Santa Clara, CA) using a Supelcosil LC-18 RP col- isons to references in the literature. MS was generated and umn (250 × 4.6 mm, 5 ␮M) (Supelco, Bellefonte, PA). Sam- identification of ANCs was performed; accurate determina- ples were prepared by dissolving 2 mg of each fraction in 1 tion was accomplished using our own reference library of mL of methanol and filtering through 0.2 ␮M PTFE syringe peaks generated from the phenolic analysis of many blue- filters (Fisher Scientific) before injection. The mobile phase berry cultivars on the same equipment and under exactly the consisted of 5% formic acid in H2O (A) and 100% methanol same analytical conditions. ANC were identified based on (B). The flow rate was held constant during the HPLC anal- comparison to standards as our previously published data ysis at 1 mL/min with a step gradient of 10%, 15%, 20%, indicates [20, 29].

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Using the peak areas as measured by HPLC at 520 nm, using the DPP-IV GloTM Protease Assay (Promega, Madi- total ANC were quantified from a standard curve gener- son, WI) following the manufacture’s protocol. Briefly, 50 ␮L ated from 0.125, 0.25, 0.5, and 1.0 mg/mL of C3G and are of DPP-IV GloTM reagent was added to a black-walled 96-well presented as C3G equivalents. In the same way, total PAC plate containing 50 ␮L of blank, positive control, or treatment. was calculated as epicatechin equivalents from the peak ar- The blank contained the vehicle control only while positive eas measured at 280 nm. Fractions containing the highest control contained the vehicle and purified DPP-IV enzyme total ANCs and PACs were considered ANC and PAC en- at a final concentration of 1 ng/mL. Treatments used were riched fractions, respectively. To further elucidate the compo- enriched ANC or known inhibitor, diprotin A, and the puri- sition of the PAC, samples were analyzed with HPLC–ESI– fied DPP-IV enzyme at a final concentration of 1 ng/mL. The MS using the same protocol above except at a flow rate of content of the wells was gently mixed using a plate shaker 0.4 mL/min and Eclipse XDB-C18 RP column (250 mm × at 400 rpm for 30 s. DPP-IV cleavage of the provided sub- 3mm× 5 ␮m, Agilent Technology, Inc., Wilmington, DE). strate generated a luminescent signal by luciferase reaction LC/MS Accurate–Mass 6220 model (Agilent Technology, with the amount of DPP-IV enzyme available to bind Gly-Pro- Inc.) was used for PAC analysis. Mass analyses conditions aminoluciferin proportional to relative light units produced. were as follows: gas temperature at 350ЊC, gas flow 10 L/min, This signal in relative light unit was measured after 30 min in nebulizer 45 psi, ion source dual ESI, positive mode, and in- an Ultra Microplate Reader (Biotek Instruments, Winooski, jection volume was 10 ␮L (2 mg/mL sample). MassHunter VT) and was compared to the inhibitory action of diprotin A. Workstation Software for data acquisition version 2.B.00 and Diprotin A linear standard curve (y =−50.41x + 86.35, R2 = qualitative analysis version B.03.01 (Agilent Technology, Inc.) 0.94) where y is the % maximum activity of the enzyme and was used to run samples and analyze the total ion spectrum x is the log10 of the concentration (␮M) of known inhibitor data at 150–3000 m/z range. (diprotin A) was used to calculate IC50 values. A computational docking study to predict the affin- ity of main ANC present in the blends as C3G (Pro- 2.7 Enzymatic studies with ␣-glucosidase tein Data Bank (PDB): 10099), -3-galactoside (PDB: and ␣-amylase 671499), malvidin-3-arabinoside, delphinidin-3-glucoside (PDB: 13563), and delphinidin-3-arabinoside and diprotin The methods for enzymatic inhibition assays were adapted A with DPP-IV enzyme (PDB: 1R9M) were conducted from Kwon et al. [16]. For the ␣-glucosidase assay, to a 96-well with the protein-ligand docking program SwissDock plate, 50 ␮L of sample (0.1 mg freeze dried product/mL) or (http://swissdock.vital-it.ch/, accessed July 2012). The PDB positive control (1 mM acarbose) were added to 100 ␮Lofa format files of ANC and diprotin A were drawn by chem- 1U/mL␣-glucosidase solution (in 0.1 M sodium phosphate ical drawing software Marvisketch (ver 5.10.3, ChemAxon). buffer pH 6.9) and incubated for 10 min. In total, 50 ␮L The OpenBabel GUI chemical expert system was used for of a 5 mM p-nitrophenyl-␣-D-glucopyranoside solution (in converting chemical file format (PDB file) of ANC (malvidin- 0.1 M sodium phosphate buffer pH 6.9) were added briefly 3-galactoside and delphinidin-3-arabinoside) and diprotin A to each well and incubated at 25ЊC for 5 min before reading to Mol2 files which can be read by the docking program. the absorbance at 405 nm. For the ␣-amylase assay, 500 ␮L Docking results predicting binding modes were visualized of sample or positive control (1 mM acarbose) were added with UCSF Chimera1.6.2, for displaying, animating, and an- to 500 ␮Lof13U/mL␣-amylase solution (Type VI-B from alyzing large biomolecular systems using 3-D graphics and porcine pancreas in 0.02 M sodium phosphate buffer pH 6.9) built-in scripting. At the end, the best conformation was se- and incubated in test tubes at 25ЊC for 10 min before 500 ␮L lected based on the lowest FullFitness [30]. The FullFitness of 1% soluble starch solution (dissolved in sodium phosphate is the sum of the total energy of the system and includes a buffer and boiled for 10–15 min) was added to each tube and solvation term: incubated for another 10 min. Finally, 1 mL of dinitrosalicylic acid color reagent was added and the tubes were placed in 100ЊC water for 5 min. The mixture was diluted with 10 mL ligand recept = E + E + E + ⌬G . + (␴ × SASA) of distilled water and absorbance was read at 520 nm. Results intra intra inter elec solv were presented as percent inhibition relative to the current diabetes drug acarbose as the positive control, which was set ligand recept at 100% inhibition. where Eintra and Eintra are the internal energy of ANC and DPP-IV, respectively; Einter is the interaction energy between the ANC and DPPI-IV, and is equal to the sum of the van der

2.8 DPP-IV inhibition and computational docking Waals and the electrostatic interaction energies; ⌬Gelec.solv is study electrostatic potential of solvent and calculated using the ana- lytical Generalized Born Molecular Volume (GB-MV2) model Measurements of the activity and potential inhibition of DPP- implemented in CHARMM; SASA is the solvent accessible IV (EC 3.4.14.5), a type II membrane glycoprotein, were done surface area; ␴ is 0.0072 kcal/(mol A˚ 2).

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2.9 Cell proliferation and treatment tein was loaded in 4–20% Tris–HCl gels (BioRad) for pro- tein separation. The resolved were transferred to a Murine macrophage cell line RAW 264.7 was cultured in polyvinylidene fluoride membrane (Millipore, Billerica, MA) DMEM supplemented with 1% penicillin/streptomycin, 1% and blocked with 3% milk in 0.1% Tris-buffered saline with sodium pyruvate, and 10% fetal bovine serum at 37ЊCin5% Tween 20 (TBST) for 1 h at room temperature. After block-

CO2/95% air using a CO2 Jacketed Incubator (NuAIRE DH ing, the membrane was washed with 0.1% TBST (five times, Autoflow, Plymouth, MN). The cell proliferation assay was 5 min each) and incubated with primary antibody (1:500) at conducted using the CellTiter 96R AQueous One Solution 4ЊC overnight. The membrane was washed again and incu- Proliferation assay kit containing a tetrazolium compound; bated with anti-mouse or anti-rabbit IgG horseradish perox- MTS; and an electron coupling reagent, PES (Promega, idase conjugate secondary antibody (1:2500) for 1 h at room Madison, WI). Briefly, 1 × 104 cells were seeded in a 96-well temperature. After incubation and repeated washing, the ex- plate and the total volume was adjusted to 200 ␮L with growth pression of proteins was visualized using chemiluminescent medium. After 24 h incubation, the cells were treated with 5, reagent (GE Healthcare, Pittsburgh, PA) following manufac- 50, or 100 ␮M C3G (for ANC) or epicatechin (for PAC) equiv- turer’s instructions. The membrane picture was taken with alents, and incubated for an additional 24 h. After this time, a GL 4000 Pro Imaging system (Carestream Health, Inc., the growth medium was replaced with 100 ␮L fresh growth Rochester, NY). medium and 20 ␮L MTS/PES were added to each well. The plate was incubated for 2 h at 37ЊCin5%CO2/95% air and the absorbance was read at 515 nm in an Ultra Microplate 2.12 Statistical analyses Reader (Biotek Instruments). The percentage of viable cells was calculated with respect to untreated cells in complete Data were expressed as means of independent duplicates with media. at least three replications. Statistical analysis was conducted For treatments, RAW 264.7 macrophages were seeded at using the proc GLM procedures of SAS version 9.3 (SAS 2 × 105 in a six-well plate, and the total volume was adjusted Institute, Inc., Cary, NC). Group mean comparisons were to 2 mL with growth medium and incubated for 48 h at conducted using LSD means and were considered to be sig-

37ЊCin5%CO2/95% air. After 48 h incubation, the cells nificant at p < 0.05 based on minimum significant differences were treated with 25, 50, or 100 ␮MC3G(forANC)orwith from one-way analysis of variance (ANOVA) with ␣ = 0.05. epicatechin (for PAC) equivalents, stimulated with 1 ␮g/mL Correlations were done using Pearson’s correlation values LPS and incubated for an additional 24 h. After 24 h, spent with p < 0.05. medium was collected for NO production measurement and whole cell lysates were collected for Western blot analysis of inflammatory markers, as follows. 3 Results

3.1 Blackberry increased total ANCs, total phenolics, 2.10 NO measurement in supernatant of RAW 264.7 and antioxidant capacity of wine blends cell culture Blueberry–blackberry wine blends had total ANCs ranging Nitrite measurement was performed using Griess reaction. from 76.8 (100% blueberry) to 137.2 mg C3G equivalents/L Briefly, 100 ␮L of the growth medium diluted 1:5 were plated (100% blackberry); total ranging from 2196.7 in 96-well plate and an equal amount of Griess reagent (100% blueberry) to 4029.0 mg EAE/L wine (100% black- (1% sulfanilamide and 0.1% N-1-(naphthyl)ethylenediamine- berry); and antioxidant capacity from 24.5 (70:30 blend) diHCl in 2.5% H3PO4) was added. The plate was incubated to 37.4 mmol TE/L (100% blackberry), as can be seen in for 5 min at room temperature and the absorbance was Table 1. Both total polyphenols and total ANCs increased measured at 550 nm in an Ultra Microplate Reader (Biotek consistently with a higher percent of blackberry in the blend, Instruments). The amount of NO was calculated using with the highest values for the 100% blackberry wines. An- sodium nitrite standard curve (y = 0.14x + 0.05, R2 = 0.997). tioxidant capacity increased with 70% blackberry in the blend, with the highest antioxidant capacity seen again for the 100% blackberry blend. 2.11 Western blot analysis Table 1 also shows that based on our previous study [14], and for comparative purposes, blueberry juice decreased 16.1 Treated cells were washed twice with ice-cold DMEM and brix, pH decreased by 2.5, and total polyphenols increased twice with ice-cold PBS and the cells were lysed with by 625.4 mg/L EAE from week 0 to fermented wine at 200 ␮L of Laemmli buffer (BioRad, Hercules, CA) con- room temperature. Blackberry juice decreased 17.5 brix, pH taining 5% ␤-mercaptoethanol. The cell lysates were son- increased only slightly (by 0.3), and total polyphenols in- icated for 30 s and then boiled for 5 min. Protein was creased 643.8 mg/L EAE from week 0 to fermented wine quantified using the RCDC Assay (BioRad), and 30 ␮gpro- at room temperature.

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Ta b l e 1 . Total anthocyanins (ANCs [TA]) and total phenolics (TPs) of wine blends and antioxidant capacity (AC) of wine anthocyanins (ANC) and proanthocyanidins (PAC)1)

Blend ratio Total ANCs Total phenolics Antioxidant capacity (%blueberry (mg C3G- (mg EAE2)/L wine) (mmol Trolox Eq./L) :%blackberry) equivalents/L wine) Wine ANC3) PAC3)

100:0 76.8 ± 2.6d 2196.7 ± 42.7e 25.3 ± 2.2b 407.3 ± 48.8bc 657.0 ± 63.7a 70:30 94.3 ± 6.2cd 2611.2 ± 76.6d 24.5 ± 0.82b 667.6 ± 58.2ab 540.5 ± 74.1a 50:50 105.8 ± 6.3bc 2932.3 ± 28.5c 25.3 ± 1.0b 758.8 ± 58.2a 646.6 ± 50.0a 30:70 121.7 ± 8.7ab 3403.4 ± 50.5b 26.5 ± 0.1b 545.0 ± 63.5abc 604.7 ± 38.6a 0:100 137.2 ± 9.6a 4029.0 ± 68.4a 37.4 ± 1.4a 757.2 ± 45.9a 579.2 ± 52.3a Correlations4) TA_wine 1.00 0.99 0.77 0.61 −0.35 TP_wine 1.00 0.84 0.59 −0.34 AC_wine 1.00 0.44 −0.26 AC_ANC 1.00 −0.41 AC_PAC 1.00 Total ANC5) 0.93 0.93 0.70 0.55 0.00 Total PAC 5) 0.78 0.72 0.31 0.82 0.52

1) Values are means ± SEM. Means with different letters in each column are significantly different (p < 0.05). 2) EAE: ellagic acid equivalents. 3) ANC and PAC at 0.1mg freeze dried product/mL. 4) Correlation values are Pearson’s r-values (p < 0.05). 5) Total ANC and PAC are based on HPLC data presented in Table 2. Note based on our previous results [14]: for blueberries: Wk0 (unfermented): pH, 6.3; Brix, 29.7; total polyphenols (mg/L EAE), 993.8 ± 6.9. For blackberries: Wk0 (unfermented): pH, 3.1; Brix, 29.0; total polyphenols (mg/L EAE), 2141.3 ± 101.1. Week 0 = no fermentation occurred; values are averages of independent duplicates.

3.2 Main ANCs and PACs in the blueberry 30:70, 50:50; blueberry:blackberry) contained in addition to and blackberry enriched fractions PAC, a complex chemical composition of HTs. Basic struc- tures for ANCs, PACs, and HTs are presented in Fig. 2. ANC identified for each blend are presented in Table 2. Chro- Figure 3 shows the composition of a mixture of blueberry: matographic analysis revealed up to 17 ANCs present in blue- blackberry, with a blend of 50% each. Based on the relative berry and blackberry blend fermented beverages. Malvidin- abundance of the ESI chromatogram, HTs are the predom- 3-galactoside was the main ANC present in 100% blueberry, inant polymerized compounds in the matrix obtained from while delphinidin-3-arabinoside was the predominant ANC mostly blackberry wine. PACs (condensed tannins) were poly- in the blends containing blackberry. Total ANC, using total meric forms of and/or epicatechin molecules (m/z peak areas at 520 nm as quantified with HPLC in compari- 291.0234 each [M + H]+), while HTs were more complex, son with the commercial standard C3G, increased with more diverse, and with larger molecular size [20]. Based on LC– blackberry in the blend and ranged from 1112.3 (70:30 blend) MS data across all PAC, blueberry contributed PACs, while to 1550.4 mg C3G equivalents/L (100% blackberry), confirm- blackberry contributed mostly HTs (Fig. 3). The HTs in black- ing the increase in total ANC seen by the wines before phe- berry included gallic acid (m/z: 170.2129) and ellagic acid nolic separation. The identification of ANCs and PACs were (m/z: 303.0307) which form gallotannins and ellagitannins, based on their m/z, UV absorption (520 nm for ANC, and 280 respectively, in diversified polymers ranging from 465.269 to nm for PAC), and our previously published data on blueberry 2486.2511 m/z [M + H]+ present as shown in Fig. 3. More and blackberry species [19,20,29]. Total PAC were quantified than 1000 HTs have been identified from simple glucogallin using peak areas at 280 nm and ranged from 560.3 (100% to pentameric ellagitannins with molecular weight ranging blueberry) to 1249.0 mg (-)-epicatechin equivalents/L (100% from 332 to 5000 [35]. LC–MS/MS fragmentation of HTs in blackberry; Table 2). Further HPLC-ESI-MS analyses for PAC blackberry samples led to the identification of several ma- revealed complex tannin composition with highly polymer- jor compounds based on their m/z, UV absorption at 280 ized compounds. In the fraction mostly composed of blue- nm, and published data such as ellagic acid pentoside (m/z: berry matrix (100:0, 70:30, 50:50; blueberry:blackberry), in ad- 435.2345), ellagic acid glucose (m/z: 465.0272), galloyl–HHP dition to monomers, PAC oligomers and polymers were the glucose (m/z: 633.0259), sanguiim H-6 (m/z: 1871.2658), nob- predominant compounds ranging from 2- to 14-units of cate- otanin A/malabathrin B (m/z: 1736.1203), and lambertianin chin/epicatechin. This was consistent with published data on isomers A, C, and D (m/z: 1871.1403.5335 and 1870.9546, complex PAC in blueberry and cacao extracts [31–34]. Frac- respectively). These identifications were in agreement with tions derived mostly from blackberry matrix/blend (0:100, previous publications [20,36–40]. A comprehensive structural

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Ta b l e 2 . Anthocyanins (ANC) identification and quantification by HPLC, and total proanthocyanidins (PACs) determined at maximum absorption of 520 and 280 nm, respectively

RT (min) ANC ID ANCs (mg C3G equivalents/L) per blend (%Blueberry : %Blackberry)

100:0 70:30 50:50 30:70 0:100

24.44 Delphinidin-3-galactoside 10.8 11.2 9.1 6.9 nd 25.98 Delphinidin-3-glucoside 26.8 51.0 34.7 12.3 8.8 27.33 Cyanidin-3-galactoside 6.5 6.3 nd 17.7 23.8 28.86 Delphinidin-3-arabinoside 11.9 nd 993.3 1079.3 1244.7 29.69 Cyanidin-3-O-glucoside 23.4 564.1 12.1 10.2 10.1 30.82 Cyanidin-3-arabinoside 140.2 16.7 8.0 nd 116.8 31.62 -3-glucoside 7.3 79.6 45.7 100.9 9.6 32.69 -3-glucoside 39.8 6.7 6.7 8.4 10.1 33.95 Petunidin-3-arabinoside 178.5 34.5 23.7 14.3 9.7 34.39 Malvidin-3-galactoside 292.1 32.7 11.9 38.4 6.7 35.19 Malvidin-3-glucoside 11.1 69.3 22.6 49.6 6.4 37.14 Malvidin-3-arabinoside 278.9 127.2 111.5 44.7 7.5 39.03 Delphinidin-6-acetyl-3-glucoside 7.3 12.6 10.3 nd nd 40.55 Cyanidin-6-acetyl-3-glucoside 7.8 nd 6.1 6.1 43.9 41.70 Malvidin-6-acetyl-galactoside 26.7 13.6 8.0 7.7 nd 42.25 Petunidin-6-acetyl-3- nd 11.9 10.9 nd nd 44.40 Malvidin-6-acetyl-3-glucoside 10.8 19.4 14.6 nd nd Total ANC 1113.7 1112.3 1403.1 1448.2 1550.4 PAC total [mg 560.3 1166.7 1244.7 1240.5 1249.0 (-)-epicatechin equivalents/L] Bold values indicate the concentration of the predominant ANC in 100% blueberry and 100% blackberry. nd, peak not detected.

elucidation of HTs in blackberry and other plant species was enzyme are shown in Table 3. There was a strong negative recently reviewed [41]. correlation (r =−0.97, p < 0.05) between the amount of ANC

Freeze-dried ANC had an antioxidant capacity ranging required to inhibit ␣-glucosidase by 50% (IC50) and the total from 407.3 (100% blueberry) to 758.8 mmol TE/L (50:50 ANC present as determined by HPLC. Similarly, there was blend), with an increase in all blends containing blackberry a strong negative correlation (r =−0.93, p < 0.05) between

compared to 100% blueberry (Table 1), however, there was no PAC needed to inhibit ␣-glucosidase by 50% (IC50) and the difference in the antioxidant capacity of PAC, having an av- total PAC present as determined by HPLC. erage antioxidant capacity of 605.6 mmol TE/L. Correlations Compared to a standard curve of diprotin A, a known in- between components of the wines and enriched-fractions are hibitor of DPP-IV containing an Ile-Pro-Ile sequence, ANC shown in Table 1. and PAC all had the ability to inhibit DPP-IV activity at con- centrations of 20 and 40 ␮M in C3G and epicatechin equiv- alents, respectively. ANC were more effective at inhibiting

DPP-IV activity than the PAC, as seen by IC50 values in 3.3 ANCs significantly inhibited activity Table 3. Overall, the 50:50 blend was the most effective at ␣ of -glucosidase and DPP-IV reducing the activity of DPP-IV with an IC50 value of 4.0 ␮M C3G, comparable to the known inhibitor’s IC50 value of 4.3 ± ␣ While -amylase was not inhibited by either the ANC or 1.6 mg/mL. Correlations between IC50 values needed to in- ␣ the PAC (data not shown), -glucosidase was significantly hibit the enzyme are shown in Table 3, as well as between IC50 inhibited (Table 3). The ANC concentration needed to inhibit values to inhibit both DPP-IV and ␣-glucosidase. A strong ␣ -glucosidase by 50% (IC50) in C3G equivalents ranged from negative correlation between amount of ANC (r =−0.90, ␮ ␮ 27.6 M for 100% blackberry to 36.2 M for 100% blueberry, p < 0.05) and IC50 values to inhibit DPP-IV indicated that with the 50:50 blend having an IC50 value in between the less of the compound was needed to inhibit the activity of the 100% blends. When compared to the IC50 values for PAC, enzyme. Conversely, there was a strong positive correlation ␮ which ranged from 54.3 M in epicatechin equivalents for between PAC and the IC50 value to inhibit DPP-IV, indicating 100% blackberry to 115.1 ␮M for 100% blueberry, it can be that higher PAC was required in the sample to inhibit the en- seen that less ANC was needed to reduce ␣-glucosidase than zyme activity. Results from the computational docking study PAC. Correlations between IC50 values needed to inhibit the showed that the FullFitness of the ANCs with the DPP-IV

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Figure 2. Basic chemical structures for bioactive phytochemicals found in blueberry and blackberry: an- thocyanins (ANCs; cyanidin, pe- onidin, delphinidin, petunidin and malvidin), proanthocyanidin (PAC; catechin, epicatechin), and hyrolyz- able tannins (gallic acid, ellagic acid). Additions of glycoside(s) from different types to anthocyani- dins results in formation of ANCs listed in Table 2. Addition of gluco- side(s) and/or polymerization of gal- lic/ellagic acids results in the forma- tion of hydrolysable tannins (HTs) shown in Fig. 3. Proanthocyanin polymers are multiunits of and/or epicatechins. had no obvious differences (from −3228 to −3052 kcal/mol) or 100% blackberry were cytotoxic to RAW 264.7 mouse (Table 4). Moreover, they were as low as those of diprotin A macrophages at concentrations up to 100 ␮M in C3G or (−3208 kcal/mol), which is a potent DPP-IV inhibitor. These epicatechin equivalents, respectively. None of the ANC or results suggest that ANCs contained in blueberry and black- PAC (100 ␮M C3G or epicatechin equivalents) increased berry have strong DPP-IV inhibiting effect. Among the four production of any of the inflammatory proteins tested when chains in DPP-IV, chain C bound each ANC with the lowest not stimulated with LPS. All three (100:0, 50:50, and 0:100) interaction energy. Malvidin-3-galactoside in blueberry and blueberry–blackberry blend ANC at 100 ␮M were able to lower delphinidin-3-arabinoside in blackberry, present in the high- total p-p65 NF␬B subunit following LPS-stimulation by 68.9, est concentrations, were presumed to be the major active 88.6, and 80.8%, respectively (Fig. 5A). ANC from the blends components of blueberry and blackberry. The interactions of containing 50% or 100% blackberry were able to significantly chain C with the two ANCs are shown in Fig. 4. These re- inhibit the activation of the p-p65 NF␬B subunit at 50 ␮Mby sults indicate that delphinidin-3-arabinoside and malvidin-3- 88.6 and 72.0%, respectively. PAC from the 100% blueberry galactoside could effectively inhibit the activity of the enzyme. and the 50:50 blends were also able to significantly decrease the p-p65 NF␬B subunit at 100 ␮M epicatechin equivalents by 55.7 and 40.0%, respectively; however, PAC from the 100% 3.4 Markers of inflammation were inhibited blackberry blend did not significantly decrease this inflamma- in the NF␬B-mediated pathway tory protein (Fig. 5A). This can be attributed to the fact that, unlike blueberry with more polymeric PACs or condensed Proliferation assays showed that none of the ANC or PAC tannins, blackberry contains HTs. COX-2 expression was de- from 100% blueberry, 50% blueberry : 50% blackberry, creased by all ANC at 100 ␮M C3G equivalents; 52.4% for

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Figure 3. HPLC–ESI–MS chromatograph for PAC from 50:50 blend of blueberry:blackberry. The large bold numbers refer to average degree of polymerization for proanthocyanidins (PACs), consisting of dimers (2 units of catechin/epicatechin) to 14-mers, with peaks contained two bold numbers indicated the presence of a doubly charged ion and the presence of both degrees of polymerization (i.e., 7 of 14 heptamer and 14-mers). Mass ions tagged with HT refer to polymerized hydrolysable tannins (HTs) contributed only by blackberry. HTs included gallic acid (m/z: 170.2129) and ellagic acid (m/z: 303.007) that formed gallotannins and ellagitannins in diversified polymers shown in LC–MS spectrum ranging from of 465.268 to 2486.2511 m/z [M + H]+.

100:0, 67.8% for 50:50, and 72.2% for 0:100 (Fig. 5B). PAC mouse macrophages following LPS-stimulation at 100 ␮M from 100% blackberry and the 50:50 blends were also able to epicatechin equivalents, with the 50:50 blend the most ef- decrease COX-2 expression significantly by 64.3 and 65.2%, fective to decrease iNOS expression by 57.4% compared to respectively. PAC at 100 ␮M epicatechin equivalents from the 53.0% inhibition by 100:0 and 42.3% by 0:100. iNOS protein 100% blueberry blend decreased COX-2 expression by 36.8%, produces the inflammatory marker NO when active, so NO however, this decrease was not significant. production was also measured. As seen in Fig. 6B, ANC at iNOS expression was decreased by ANC with the 100% 100 ␮M were able to decrease NO production with black- blueberry and 50:50 blends at 100 ␮M C3G equivalents, berry in the blend by 37.9% for 50:50 and 48.1% for with ANC from the 100% blueberry the most effective with 0:100; however, the 100% blueberry ANC was unable to 75.7% inhibition compared to 40.2% for 50:50 (Fig. 6A). PAC show a significant decrease in NO production. PAC from from all blends were able to decrease iNOS expression in 100:0, 50:50,and0:100 at 100 ␮M epicatechin equivalents

Ta b l e 3 . Determination of IC50 values (mean ± standard error) of anthocyanins (ANC) and proanthocyanidins (PAC) needed to inhibit enzymes involved in diabetes management.1)

Enriched 100% 50%:50% 100% Correlations2) fraction Blueberry Blackberry ␣-glu_ ␣-glu_ DPP-IV_ DPP-IV_ ANC PAC ANC PAC

b b b ␣-Glucosidase IC50 ANC- 36.2 ± 13.2 33.7 ± 9.8 27.6 ± 9.7 1.00 0.90 0.98 −0.86 values (␮M) PAC- 115.1 ± 28.5a 71.3 ± 28.9ab 54.3 ± 30.8ab 1.00 −0.99 −1.00 Dipeptidyl ANC- 15.9 ± 0.7a 4.2 ± 1.5b 5.5 ± 0.4b 1.00 −0.95 peptidase-IV PAC- 16.6 ± 1.1a 19.3 ± 1.1a 18.2 ± 3.2a 1.00 (DPP-IV) IC50 values (␮M)

1) IC50 values were determined from at least two independent duplicates done in triplicate. Values are means ± SEM. Means with different letters in each row are significantly different for each enzyme (p < 0.05). Positive control of inhibition for ␣-glucosidase was acarbose having an IC50 value of 500 ␮M. The positive control of inhibition for DPP-IV was diprotin A (Ile-Pro-Ile) at 0.1 mg/mL, and had an IC50 value of 4.3 ± 1.6 mg/mL. 2) Correlation values are Pearson’s r-values (p < 0.05).

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Ta b l e 4 . Interactions of chains of dipeptidyl peptidase-IV (DPP-IV) and anthocyanins (ANCs)

Chain of FullFitness SimpleFitnessa) H bond Distance of ⌬G (kcal) DPP-IV (kcal/mol) (kcal/mol) position H bond (A)˚

Cyanidin-3-O-glucoside A −3096.22 83.08 −−−7.34 B −3076.18 84.01 PHE95 HN−C3G O8 2.135 −7.68 C −3118.97 85.40 TRP62 NH−C3G O1 2.563 −7.26 D −3051.96 78.58 TRP62 O−C3G H7 2.032 −7.83 Malvidin-3-galactoside A −3079.03 124.70 −−−7.23 B −3063.75 115.45 ASN74 O−M3GA H11 2.622 −7.79 C −3106.89 115.08 PHE461 HN−M3GA O10 2.275 −7.87 ILE107 O−M3GA H 2.691 D −3033.99 116.42 TRP305 NH−M3GA O4 2.145 −7.59 Malvidin-3-arabinoside A −3094.00 116.12 GLY99 HN−M3A O7 2.151 −7.63 B −3075.47 110.77 −−−7.88 C −3118.97 113.62 ARG669 O−M3A H4 2.836 −7.73 D −3046.40 113.68 −−−7.45 Delphinidin-3-glucoside A −3102.27 78.94 −−−7.59 B −3081.15 80.25 −−−7.47 C −3125.95 82.11 HIS533 HN−D3G O9 2.168 −7.64 ASN506 O−D3G H 2.114 LYS41 HN−D3G O 2.385 D −3052.84 79.68 −−−7.48 Delphinidin-3-arabinoside A −3202.45 35.04 ASN74 O−D3A H8 2.043 −7.64 ILE102 HN−D3A O4 2.467 ASN92 O−D3A H14 2.150 B −3183.53 30.69 ASN74 O−D3A H8 1.763 −8.38 ILE102 HN−D3A O9 2.194 C −3227.73 36.22 ILE107 HN−D3A O10 2.223 −7.62 TRP62 O−D3A H14 1.990 ILE63 O−D3A H9 2.338 D −3155.24 39.17 −−−7.03 Diprotin A A −3183.60 −7.07 LYS71 HZ3−DA O4 2.618 −7.62 B −3161.06 −8.22 LEU90 O−DA H12 2.582 −8.25 PHE95 O−DA H22 2.643 HIS100 O−DA H23 2.428 ILE102 HN−DA O4 1.874 C −3208.29 −9.23 LYS122 O−DA H22 2.678 −7.72 GLU191 OE2−DA H12 1.875 D −3135.71 −11.14 −−−7.17

C3G, M3GA, M3A, D3G, D3A, and DA represent cyanidin-3-O-glucoside, malvidin-3-galactoside, malvidin-3-arabinoside, delphinidin-3- glucoside, delphinidin-3-arabinoside, and diprotin A, respectively. Other abbreviations indicate the three-letter amino acid code in the binding site. a) The SimpleFitness is equal to the total energy of the system calculated with the CHARMM22 molecular mechanics force field, and neglects the effect of solvent known to have an important contribution to the binding free energy. also decreased NO production by 16.4, 4.8, and 14.7%, re- els increased with more blackberry in the blend associ- spectively; however, these decreases were not significant ated with higher ANC concentration. To our knowledge, (p > 0.05). this is the first reported analysis of ANCs from fermented blueberry–blackberry blends; however, the results from blueberry and blackberry wines were comparable to previ- 4 Discussion ously reported values [24]. In this study, we show that the ANC profile from fermented berry beverages may be altered Fermented berry beverages have been established as hav- from profiles previously generated for blackberry [20] or ing an increased phenolic content and higher antioxidant even phenolic-rich blueberry extracts [29]. Malvidin contains capacity than their nonfermented counterparts [7–9,23]. Ben- two methoxy side chains on its B ring, while delphinidin eficial effects of berries in the diet have been attributed has two hydroxyl groups, which may contribute to the higher to their high phenolic content, especially their ANC con- antioxidant capacity seen for the blackberry blends. Delphini- tent [40]. Wines were tested for their total polyphenols, to- din (45.0–74.9 mg/100 g fresh weight, FW) and malvidin tal ANC content, and antioxidant capacity, and these lev- (37.1–62.2 mg/100 g FW) were the predominant ANCs in the

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Figure 4. Computational visualization of the most optimal docking conformation of (A) delphinidin-3-arabinoside, (B) malvidin-3- galactoside, and (C) diprotin A, with chain C of the DPP-IV enzyme.

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Figure 5. Effect of anthocyanin (ANC; cyanidin-3-O-glucoside equivalents) and proanthocyanidin (PAC; epicatechin equivalents) at 50 and 100 ␮MonNF␬B (A) and COX-2 expression (B). Western blots are shown above with p-p65 subunit of NF␬B detected at 65 kDa, COX-2 at 70–72 kDa, and actin at 43 kDa from whole cell lysates. Values are relative expression of protein of interest over actin; means ± SEM with different letters are significantly different (n = 4, p < 0.05). varieties tested in highbush and lowbush blueberries [42], or formed during the fermentation process or during wine similar to the findings from the fermented products. maturation. While the process of ANC transformation is not During wine processing from berries, phenolic com- clear, it is supported by the changes seen in wines during pounds may be effectively extracted into the wines; aging as native blackberry ANCs are transformed during and delphinidin-3-glucoside has been identified as a major com- after alcoholic fermentation. ponent in red wine and had the most variation among all Our results showed inhibition of ␣-glucosidase activ- ANCs [43]. Although C3G has been identified as the major ity; other studies on red and black currants and red and ANC in different blackberry species, cyanidin 3-rutinoside green gooseberries have shown strong inhibition of intestinal was the major component in the wines from Andean black- glucose absorption by inhibiting ␣-glucosidase activity [13]. berry (Rubus glaucus Benth.) [44]. These results contrast with ANCs present in blueberry fruits and their fermented prod- the major ANC identified in our study as delphinidin-3- ucts appear to be more potent at inhibiting ␣-glucosidase than arabinoside, though many unknown compounds in the pre- the PACs. While a previous study in our laboratory evaluated vious study were only tentatively identified. Authors agree blueberry wines and found strong inhibition of ␣-amylase that fermentation causes an absolute and relative change in in vitro [14], we believe that during the phenolic extraction ANC content, allowing for new pigments to be generated process, phenolic acids were removed indicating these may

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Figure 6. Effect of anthocyanin (ANC; cyanidin-3-O-glucoside equi- valents) and proanthocyanidin (PAC; epicatechin equivalents) at 50 and 100 ␮M on (A) iNOS expression. Western blots are shown above with iNOS detected at 130 kDa and actin at 43 kDa from whole cell lysates. Values are relative expression of protein of interest over actin. (B) NO assay presents data from treatments at 100 ␮M cyanidin-3-O-glucoside (C3G) equivalents (ANC) or epicat- echin equivalents (PAC) and was measured from spent medium. Means ± SEM with different letters are significantly different (n = 4, p < 0.05). be the bioactive compounds with ability to inhibit ␣-amylase the major active components. To confirm these results, the activity rather than the ANC or PAC. Grussu et al. [45] demon- SwissDock computational docking study demonstrated that strated that it is not the ANCs that are required to inhibit delphinidin-3-arabinoside and malvidin-3-galactoside were ␣-amylase because both red and yellow showed able to interact with DPP-IV through ligand interaction, and similar inhibitory capacity. therefore potentially inactivate the activity of the enzyme. A study of rowanberry PAC-rich fraction had a low The most optimal conformation happened in chain C of level of inhibition compared to the whole extract [46], sug- DPP-IV indicated by the lowest interaction energies of −3228 gesting that tannin and ellagitannin components are not kcal/mol (for delphinidin-3-arabinoside) and −3107 kcal/mol influential or are poor inhibitors of ␣-glucosidase. Our re- (for malvidin-3-galactoside), which are comparable to the in- sults indicate that there was some inhibition of the enzyme teraction energy of the known inhibitor diprotin A with the by the PAC-enriched fraction, but the ANC-enriched frac- enzyme of −3208 kcal/mol. Due to the lowest interaction en- tion was more potent. The efficacy of the ANCs to inhibit ergy of chain C, we concluded that the chain C was the major DPP-IV enzyme activity at a rate comparable to a known in- binding site of these ANCs. hibitor (diprotin A) indicated that ANCs may be able to act Our results agree with previous studies that reported the as naturally occurring DPP-IV inhibitors of DPP-IV. If DPP- ability of berries to reduce inflammation through decreased IV is inactivated by or natural compounds, the degra- expression of enzymes iNOS and COX-2 [19, 20]. The up- dation of glucagon-like peptide-1 and glucose-dependent in- stream mediator in the NF␬B-mediated pathway, the p-p65 sulinotropic polypeptide is prevented, and these hormones subunit of NF␬B that lead to the induction of these inflamma- are able to inhibit glucagon release and stimulate insulin tory markers was inhibited by ANC 50:50% blend and 100% release, respectively, thereby lowering blood glucose levels blackberry. The strong anti-inflammatory effect was consid- [17]. All of the ANCs tested showed similar Fullfitness ener- ered to be exerted by a combination of various gies from the computational modeling study; therefore, they compounds rather than any single compound. have no major difference in anti-DPP-IV activity. Due to the Berry extracts can inhibit ␣-glucosidase at concentrations higher amounts of malvidin-3-galactoside in blueberry and less than 50 ␮g GAE/mL, an amount that can be eas- delphinidin-3-arabinoside in blackberry, these ANC are likely ily reached in the after consumption

C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.mnf-journal.com 1196 M. H. Johnson et al. Mol. Nutr. Food Res. 2013, 57, 1182–1197 of berries or juices [47]. Additionally, a previous study re- [9] Walzen, R. L., Review: wine and health—state of proofs ports that a moderate drink of 140 mL of wine made from and research needs. Inflammopharmacology 2008, 16, highbush blueberry would correspond to an intake of 2.42 265–271. mmol of TE of Oxygen Radical Absorbance Capacity beta- [10] Vuong, T., Martin, L., Matar, C., Antioxidant activity of fer- /day [43]. mented berry juices and their effects on nitric oxide and tu- In conclusion, ANC and PAC at 100 ␮M C3G/epicatechin mor necrosis factor-alpha production in macrophages 264.7 equivalents obtained from blueberry–blackberry blends af- gamma NO(-) cell line. J. Food Biochem. 2006, 30, 294–268. fected LPS-induced inflammatory response in RAW 264.7 [11] Juan, C., Jianquan, K., Junni, T.,Zijian, C. et al., The profile in mouse macrophages. These ANC and PAC reduced the phos- polyphenols and volatile compounds in alcoholic beverages phorylation of the p65-subunit NF␬B, decreased iNOS and from different cultivars of mulberry. Food Chem. 2012, 77, COX-2 expression, and decreased NO production. Along with C430-C436. the high antioxidant capacity and phenolic content and abil- [12] Lim, J. W., Hwang, H. J., Shin, C. S., Polyphenol compounds ity to decrease enzyme activity related to improved glucose and anti-inflammatory activities of Korean black utilization, bioactive ANC and PAC compounds present in (Rubus coreanus Miquel) wines produced from juice sup- plemented with pulp and . J. Agric. 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