1 2 3 4 3 5 6 8 7 9 9 11 10 8 10 4 6 16 12 14 15 17 18 13 B A Method and Materials WBE from Wild Blueberry Association and cranberry from a local cranberry farm in Wisconsin were freeze dried and extracted using the method described by Tulio et al. [2]. Prior to treatment, fruit extracts were suspended in phosphate buffered saline (PBS; pH 7.4), vortexed, centrifuged and filtered through 0.22 μm nylon syringe filters. ORAC (Oxygen Radical Absorbance Capacity) Fruit extracts were analyzed for antioxidant capacity by ORAC assay using the method modified by Edirisinghe et al.[3]. ORAC values for WBE and RCE extract stock solution of 100 mg/mL are 587 ± 63.8 and 349 ± 22.4 TE/ml, respectively (n=6). Phenolic Profiling HR-MS (Exactive Orbitrap) An Accela HPLC system comprising an HPLC pump, a PDA detector and Exactive Orbitrap mass spectrometer fitted with ESI (Thermo Fisher Scientific) was used to identify the anthocyanins following the procedure described by Mullen et al. [4]. LC-MS (Quattro Micro) A Waters (Milford, MA) Alliance 2795 HPLC system with a 996 PDA, and Quattro Micro triple quadrupole mass spectrometer was used to separate and quantify anthocyanins in berry extracts. Analyses were carried out at 40 ºC using a Synergi RP-Max column, 250 x 2.0 mm i.d., 4 μm packing (Phenomenex) eluted with a 50 min gradient program of 2-20% acetonitrile (1% formic acid) in 1% aqueous formic acid at a flow rate of 0.4mL/min. PDA monitoring was set at 520 nm and the spectra were recorded from 200 to 600 nm with a resolution of 1.2 nm and acquisition of 1 spectra/sec. MS was set up in ESI+ mode, source temperature at 100 °C, cone at 30 V, and capillary voltage at 3000 V. In Vitro Assay Akt assay, cell migration assay and tube formation assay were performed using methods described by Chang et al. [5]. Phosphorylated Akt (p-AKT) levels were expressed as p-Akt/Total Akt. Effects of Wild Blueberry and Cranberry Fruit Extracts on Endothelial Cell Function Claire Wei-Ju Chang 1 , Artemio Z. Tulio, Jr. 1 , Joseph Jablonski 1 Archana Kangath 2 , Krishnankutty Sandhya 2 , Indika Edirisinghe 2 , Ravi K. Tadapaneni 2 , Kevin White 3 , Britt Burton-Freeman 2 , Lauren S. Jackson 1 1 U.S. Food and Drug Administration, Bedford Park, IL 2 Institute of Food Safety and Health, Illinois Institute of Technology, Bedford Park, IL 3 U.S. Food and Drug Administration, College Park, MD Figure 1: Phosphorylated Akt (ser 473) in response to wild blueberry (WBE) and cranberry extracts (RCE) . WBE and RCE increased p-Akt levels in vitro in HUVEC compared to PBS control (n=2). Introduction Polyphenolic antioxidants activate signaling molecules in phosphoinositide 3-kinase (PI3K)/ protein kinase B (Akt) pathway mediating endothelial function [1]. Activation of PI3K/Akt pathway phosphorylate endothelial nitric oxide synthase (eNOS) resulting in nitric oxide production and vascular smooth muscle relaxation. We hypothesized that polyphenolic antioxidants such as anthocyanins derived from fruits induce the redox sensitive PI3K/Akt pathway and activate downstream signals resulting in endothelial cell migration and tube formation. Objective To investigate the effects of polyphenolic antioxidants on the activation of p-Akt and endothelial cell function using anthocyanin- rich fruit extracts from wild blueberry (WBE) and cranberry (RCE). References 1. Edirisinghe I, Burton-Freeman B, Kappagoda T, (2008) The mechanism of the endothelium dependent relaxation evoked by a grape seed extract. Clin. Sci. (London), 114, 331–337. 2. Tulio, A. Jr. ; Edirisinghe, I.; Chang, C.; Jablonski, J.E.; Tadapaneni, K.; Burton-Freeman, B.; Jackson. L.S. In vitro estimation of antioxidant activities in red and white cranberry extracts. Poster at ACS Fall 2010 National Meeting & Exposition, Boston, MA. 3. Edirisinghe, I.; Banaszewski K.; Cappozzo, J.; Krishnankutty, S.; Ellis, C. L.; Tadapaneni, R.; Kappagoda, C. T. and Burton-Freeman, B. Strawberry anthocyanin and its association with postprandial inflammation and insulin. British Journal of Nutrition. 2011, (in press. 4. Mullen, W.; Larcombe, S.; Arnold, K.; Welchman, H. Crozier, A. 2010. Use of Accurate Mass Full Scan Mass Spectrometry for the Analysis of Anthocyanins in Berries and Berry-Fed Tissues. J. Agric. Food Chem. 58, 3910–3915. 5. Chang, C.W.; Edirisinghe, I.; Jablonski, J.E.; Tadapaneni, R.K.; Tulio, A.Z. Jr.; Burton-Freeman, B.; Jackson, L.S. Polyphenol-rich fruit modulate endothelial cell function via PI3 Kinase/Akt Pathway in Vitro in Human Umbilical Vein Endothelial Cell. Poster at Experimental Biology 2011, Washington, DC. Conclusion In summary, our study shows that malvidin-3-O-glucoside and peonidin-3-O-galactoside were the most abundant anthocyanin compounds in wild blueberry and cranberry fruits, respectively. There was diversity and distinct differences in the composition of individual anthocyanins in wild blueberry with respect to cranberry fruit extracts. However, the total anthocyanin contents between these fruits were very similar. Our findings with HUVECs suggested that the group of anthocyanins specific to each fruit may be responsible for the induction of redox-sensitive PI3K/Akt pathway and the activation of downstream signals resulting in endothelial cell migration and tube formation. Further studies are needed to verify the potential health benefits of the anthocyanins in wild blueberry and cranberry fruits. Figure 3: The effects of 0.2 mg/mL of wild blueberry (WBE) and cranberry extracts (RCE) on cell migration. Cell migration was improved when HUVECs were treated with WBE or RCE compared to PBS control. Cell migration activity attenuated when cells were treated with Wortmannin (W) , a PI3 kinase inhibitor alone with fruit extracts, suggesting the involvement of redox sensitive PI3K/Akt pathway. W WBE+W 0 h 5 h 24 h Control-PBS WBE RCE RCE+W Figure 4: The effects of 0.2 mg/mL of wild blueberry (WBE) and cranberry extracts (RCE) on tube formation. Tube formation microscopic images indicated that angiogenesis increased after treatment of HUVEC with different fruit extracts compared to control-PBS at 5h. Angiogenesis was abolished when cells were treated with Wortmannin (W), a PI3 kinase inhibitor with fruit extracts suggesting the involvement of the redox sensitive PI3K/Akt pathway. 5 h WBE WBE + W 24 h WBE 5 h PBS PBS + W 24 h PBS 0 h WBE 0 h PBS 5 h RCE RCE + W 24 h RCE 0 h RCE 0.14 0.02 0.04 0.06 0.08 0.1 0.12 0 Control WBE RCE p-Akt / Total Akt a Identification and quantification using HPLC and Quattro-Micro MS coupled with PDA at 520 nm. b Identified using Exactive Orbitrap high resolution-MS. c Actual mass from Quattro Micro MS. d nd, not detected. * Expressed as malvidin-3-O-glucoside equivalents. peak t R (min) anthocyanin chemical formula exact mass (m/z) b actual mass (m/z) c wild blueberry (mg/100 g FW) cranberry (mg/100 g FW) 1 16.96 delphinidin 3-O -galactoside C 21 H 21 O 12 465.103305 465 6.5 ± 2.8* nd d 2 18.50 delphinidin-3-O -glucoside C 21 H 21 O 12 465.103305 465 4.0 ± 2.7 nd 3 19.73 cyanidin-3-O -galactoside C 21 H 21 O 11 449.108390 449 9.5 ± 4.6 14.8 ± 3.0 4 21.43 cyanidin-3-O -glucoside C 21 H 21 O 11 449.108390 449 5.8 ± 2.3 trace 5 22.51 petunidin 3-O -galactoside C 22 H 23 O 12 479.118955 479 1.1 ± 0.8* nd 6 22.51 cyanidin-3-O -arabinoside C 20 H 19 O 10 419.097825 419 2.7 ± 0.7 23.4 ± 6.3 7 23.65 petunidin-3-O -glucoside C 22 H 23 O 12 479.118955 479 4.9 ± 2.2* nd 8 24.83 peonidin-3-O -galactoside C 22 H 23 O 11 463.124040 463 7.6 ± 3.3 29.9 ± 6.4 9 26.55 peonidin-3-O -glucoside C 22 H 23 O 11 463.124040 463 2.3 ± 5.6 0.8 ± 0.3 9 26.55 malvidin-3-O -galactoside C 23 H 25 O 12 493.134605 493 8.9 ± 2.4 nd 10 28.10 peonidin-3-O -arabinoside C 21 H 21 O 10 433.113475 433 nd 7.6 ± 1.6 10 28.10 malvidin-3-O -glucoside C 23 H 25 O 12 493.134605 493 13.1 ± 2.8 6.5 ± 1.5 11 29.43 malvidin-3-O -arabinoside C 20 H 19 O 11 463.124040 463 5.2 ± 2.4* nd 12 31.10 delphinidin-3-(6"-acetoyl)glucoside C 22 H 23 O 13 507.113870 507 1.4 ± 0.5* nd 13 32.49 petunidin-3-(6"-acetoyl)galactoside C 24 H 25 O 13 521.128967 521 trace nd 14 34.46 cyanidin-3-(6"-acetoyl)glucoside C 23 H 23 O 12 491.118955 491 1.1 ± 0.5* nd 15 35.90 petunidin-3-(6"-acetoyl)glucoside C 24 H 25 O 13 521.128967 521 0.8 ± 0.2* nd 16 36.85 malvidin-3-(6"-acetoyl)galactoside C 24 H 27 O 13 535.144617 535 trace nd 17 39.15 peonidin-3-(6"-acetoyl)glucoside C 24 H 25 O 12 505.134605 505 trace nd 18 40.25 malvidin-3-(6"-acetoyl)glucoside C 25 H 27 O 13 535.144617 535 7.6 ± 3.5* nd total anthocyanins 82.5 83.0 Table 1. Anthocyanins Detected from Wild Blueberry (WBE) and Cranberry Extracts (RCE) in Phosphate Buffer Saline Using Reversed-Phase HPLC-MS a Figure 2. Anthocyanin profiles of [A] cranberry (RCE) and [B] wild blueberry extracts (WBE) in phosphate buffer saline detected at 520 nm. Peak identities and quantities are shown in Table 1. Five major anthocyanin compounds were detected in RCE, whereas, 12 major anthocyanin compounds were detected in WBE. View publication stats View publication stats