Blood–brain barrier transport and neuroprotective potential of blackberry-digested polyphenols: an in vitro study
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Epidemiological and intervention studies have attempted to link the health effects of a diet rich in fruits and vegetables with the consumption of polyphenols and their impact in neurodegenerative diseases. Studies have shown that polyphenols can cross the intestinal barrier and reach concentrations in the bloodstream able to exert effects in vivo. However, the effective uptake of polyphenols into the brain is still regarded with some reservations. Here we describe a combination of approaches to examine the putative transport of blackberry-digested polyphenols (BDP) across the blood–brain barrier (BBB) and ultimate evaluation of their neuroprotective effects.
BDP was obtained by in vitro digestion of blackberry extract and BDP major aglycones (hBDP) were obtained by enzymatic hydrolysis. Chemical characterization and BBB transport of extracts were evaluated by LC–MSn. BBB transport and cytoprotection of both extracts was assessed in HBMEC monolayers. Neuroprotective potential of BDP was assessed in NT2-derived 3D co-cultures of neurons and astrocytes and in primary mouse cerebellar granule cells. BDP-modulated genes were evaluated by microarray analysis.
Components from BDP and hBDP were shown to be transported across the BBB. Physiologically relevant concentrations of both extracts were cytoprotective at endothelial level and BDP was neuroprotective in primary neurons and in an advanced 3D cell model. The major canonical pathways involved in the neuroprotective effect of BDP were unveiled, including mTOR signaling and the unfolded protein response pathway. Genes such as ASNS and ATF5 emerged as novel BDP-modulated targets.
BBB transport of BDP and hBDP components reinforces the health benefits of a diet rich in polyphenols in neurodegenerative disorders. Our results suggest some novel pathways and genes that may be involved in the neuroprotective mechanism of the BDP polyphenol components.
KeywordsBlackberry In vitro digestion Neuronal cells Brain endothelial cells Microarrays
iNOVA4Health Research Unit (LISBOA-01-0145-FEDER-007344), which is cofunded by Fundação para a Ciência e Tecnologia / Ministério da Ciência e do Ensino Superior, through national funds, and by FEDER under the PT2020 Partnership Agreement, is acknowledged. Authors would like to acknowledge to COST FA1005–INFOGEST and to FCT for financial support of CNS (IF/01097/2013), IF (SFRH/BD/86584/2012), APT (PD/BD/52473/2014), and DB and MAB (Strategic Project to iMed.ULisboa, UID/DTP/04138/2013). CNS and DS acknowledge funding via BacHBerry (Project No. FP7-613793; http://www.bachberry.eu) and DS and GMcD acknowledge funding from the Scottish Government Rural and Environmental Sciences and Analytical Services (RESAS) Department. Funding from Tecnimede - Sociedade Técnico Medicinal S.A. (Abrunheira, Sintra, Portugal), the European Regional Development Fund (FEDER) and the System of Incentives for the Research and Technological Development (QREN) of the Portuguese Government is also acknowledged. Extensive revising of the English written work by GMcD is also acknowledged.
Compliance with ethical standards
Conflict of interest
AF is employee of Tecnimede-Sociedade Técnico Medicinal, S.A. The other authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
- 1.Williams RJ, Spencer JP (2011) Flavonoids, cognition, and dementia: actions, mechanisms, and potential therapeutic utility for Alzheimer disease. Free Radic Biol Med 52(1):35–45. https://doi.org/10.1016/j.freeradbiomed.2011.09.010 CrossRefGoogle Scholar
- 3.Garcia G, Nanni S, Figueira I, Ivanov I, McDougall GJ, Stewart D, Ferreira RB, Pinto P, Silva RF, Brites D, Santos CN (2017) Bioaccessible (poly)phenol metabolites from raspberry protect neural cells from oxidative stress and attenuate microglia activation. Food Chem 215:274–283. https://doi.org/10.1016/j.foodchem.2016.07.128 CrossRefGoogle Scholar
- 6.Virgili F, Marino M (2008) Regulation of cellular signals from nutritional molecules: a specific role for phytochemicals, beyond antioxidant activity. Free Radic Biol Med 45(9):1205–1216. https://doi.org/10.1016/j.freeradbiomed.2008.08.001 CrossRefGoogle Scholar
- 8.Gee JM, DuPont MS, Day AJ, Plumb GW, Williamson G, Johnson IT (2000) Intestinal transport of quercetin glycosides in rats involves both deglycosylation and interaction with the hexose transport pathway. J Nutr 130(11):2765–2771Google Scholar
- 9.Manach C, Scalbert A, Morand C, Remesy C, Jimenez L (2004) Polyphenols: food sources and bioavailability. Am J Clin Nutr 79(5):727–747Google Scholar
- 12.Ho L, Ferruzzi MG, Janle EM, Wang J, Gong B, Chen TY, Lobo J, Cooper B, Wu QL, Talcott ST, Percival SS, Simon JE, Pasinetti GM (2012) Identification of brain-targeted bioactive dietary quercetin-3-O-glucuronide as a novel intervention for Alzheimer’s disease. FASEB J 27(2):769–781. https://doi.org/10.1096/fj.12-212118 CrossRefGoogle Scholar
- 15.Fornasaro S, Ziberna L, Gasperotti M, Tramer F, Vrhovsek U, Mattivi F, Passamonti S (2016) Determination of cyanidin 3-glucoside in rat brain, liver and kidneys by UPLC/MS-MS and its application to a short-term pharmacokinetic study. Sci Rep 6:22815. https://doi.org/10.1038/srep22815 CrossRefGoogle Scholar
- 18.Macedo D, Tavares L, McDougall GJ, Vicente Miranda H, Stewart D, Ferreira RB, Tenreiro S, Outeiro TF, Santos CN (2015) Poly)phenols protect from alpha-synuclein toxicity by reducing oxidative stress and promoting autophagy. Hum Mol Genet 24(6):1717–1732. https://doi.org/10.1093/hmg/ddu585 CrossRefGoogle Scholar
- 20.Tavares L, Carrilho D, Tyagi M, Barata D, Serra AT, Duarte CM, Duarte RO, Feliciano RP, Bronze MR, Chicau P, Espirito-Santo MD, Ferreira RB, dos Santos CN (2010) Antioxidant capacity of Macaronesian traditional medicinal plants. Molecules 15(4):2576–2592. https://doi.org/10.3390/molecules15042576 CrossRefGoogle Scholar
- 22.Palmela I, Sasaki H, Cardoso FL, Kim KS, Brites D, Brito MA (2012) Time-dependent dual effects of high levels of unconjugated bilirubin on the human blood–brain barrier lining. Front Cell Neurosci 6. https://doi.org/10.3389/fncel.2012.00022
- 23.Tavares L, Figueira I, Macedo D, McDougall GJ, Leitão MC, Vieira HLA, Stewart D, Alves PM, Ferreira RB, Santos CN (2012) Neuroprotective effect of blackberry (Rubus sp.) polyphenols is potentiated after simulated gastrointestinal digestion. Food Chem 131(4):1443–1452. https://doi.org/10.1016/j.foodchem.2011.10.025 CrossRefGoogle Scholar
- 24.Gomes A, Pimpão RC, Fortalezas S, Figueira I, Miguel C, Aguiar C, Salgueiro L, Cavaleiro C, Gonçalves MJ, Clemente A, Costa C, Martins-Loução MA, Ferreira RB, Santos CN (2015) Chemical characterization and bioactivity of phytochemicals from Iberian endemic Santolina semidentata and strategies for ex situ propagation. Ind Crops Prod 74:505–513. https://doi.org/10.1016/j.indcrop.2015.04.037 CrossRefGoogle Scholar
- 25.Terrasso AP, Pinto C, Serra M, Filipe A, Almeida S, Ferreira AL, Pedroso P, Brito C, Alves PM (2015) Novel scalable 3D cell based model for in vitro neurotoxicity testing: combining human differentiated neurospheres with gene expression and functional endpoints. J Biotechnol 205:82–92. https://doi.org/10.1016/j.jbiotec.2014.12.011 CrossRefGoogle Scholar
- 29.Brito C, Simão D, Costa I, Malpique R, Pereira CI, Fernandes P, Serra M, Schwarz SC, Schwarz J, Kremer EJ, Alves PM (2012) Generation and genetic modification of 3D cultures of human dopaminergic neurons derived from neural progenitor cells. Methods 56(3):452–460. https://doi.org/10.1016/j.ymeth.2012.03.005 CrossRefGoogle Scholar
- 31.Palmela I, Cardoso FL, Bernas M, Correia L, Vaz AR, Silva RFM, Fernandes A, Kim KS, Brites D, Brito MA (2011) Elevated Levels of bilirubin and long-term exposure impair human brain microvascular endothelial cell integrity. Curr Neurovasc Res 8(2):153–169. https://doi.org/10.2174/156720211795495358 CrossRefGoogle Scholar
- 32.Eigenmann DE, Xue G, Kim KS, Moses AV, Hamburger M, Oufir M (2013) Comparative study of four immortalized human brain capillary endothelial cell lines, hCMEC/D3, hBMEC, TY10, and BB19, and optimization of culture conditions, for an in vitro blood–brain barrier model for drug permeability studies. Fluids Barriers CNS 10(1):1–17. https://doi.org/10.1186/2045-8118-10-33 CrossRefGoogle Scholar
- 34.Ishisaka A, Ichikawa S, Sakakibara H, Piskula MK, Nakamura T, Kato Y, Ito M, Miyamoto K-i, Tsuji A, Kawai Y, Terao J (2011) Accumulation of orally administered quercetin in brain tissue and its antioxidative effects in rats. Free Radic Biol Med 51(7):1329–1336. https://doi.org/10.1016/j.freeradbiomed.2011.06.017 CrossRefGoogle Scholar
- 35.Bohn T, McDougall GJ, Alegria A, Alminger M, Arrigoni E, Aura AM, Brito C, Cilla A, El SN, Karakaya S, Martinez-Cuesta MC, Santos CN (2015) Mind the gap-deficits in our knowledge of aspects impacting the bioavailability of phytochemicals and their metabolites—a position paper focusing on carotenoids and polyphenols. Mol Nutr Food Res 59(7):1307–1323. https://doi.org/10.1002/mnfr.201400745 CrossRefGoogle Scholar
- 36.Marques Peixoto F, Fernandes I, Gouvêa ACMS, Santiago MCPA, Galhardo Borguini R, Mateus N, Freitas V, Godoy RLO, Ferreira IMPLVO (2016) Simulation of in vitro digestion coupled to gastric and intestinal transport models to estimate absorption of anthocyanins from peel powder of jabuticaba, jamelão and jambo fruits. J Funct Foods 24:373–381. https://doi.org/10.1016/j.jff.2016.04.021 CrossRefGoogle Scholar
- 37.McDougall GJ, Conner S, Pereira-Caro G, Gonzalez-Barrio R, Brown EM, Verrall S, Stewart D, Moffet T, Ibars M, Lawther R, O’Connor G, Rowland I, Crozier A, Gill CI (2014) Tracking (poly)phenol components from raspberries in ileal fluid. J Agric Food Chem 62(30):7631–7641. https://doi.org/10.1021/jf502259j CrossRefGoogle Scholar
- 39.Figueira I, Garcia G, Pimpao RC, Terrasso AP, Costa I, Almeida AF, Tavares L, Pais TF, Pinto P, Ventura MR, Filipe A, McDougall GJ, Stewart D, Kim KS, Palmela I, Brites D, Brito MA, Brito C, Santos CN (2017) Polyphenols journey through blood–brain barrier towards neuronal protection. Sci Rep 7 (11456). https://doi.org/10.1038/s41598-017-11512-6
- 40.Ghersi-Egea JF, Leninger-Muller B, Suleman G, Siest G, Minn A (1994) Localization of drug-metabolizing enzyme activities to blood–brain interfaces and circumventricular organs. J Neurochem 62(3):1089–1096. https://doi.org/10.1046/j.1471-4159.1994.62031089.x CrossRefGoogle Scholar
- 41.Shawahna R, Uchida Y, Declèves X, Ohtsuki S, Yousif S, Dauchy S, Jacob A, Chassoux F, Daumas-Duport C, Couraud P-O, Terasaki T, Scherrmann J-M (2011) Transcriptomic and quantitative proteomic analysis of transporters and drug metabolizing enzymes in freshly isolated human brain microvessels. Mol Pharm 8(4):1332–1341. https://doi.org/10.1021/mp200129p CrossRefGoogle Scholar
- 44.Bieger J, Cermak R, Blank R, de Boer VC, Hollman PC, Kamphues J, Wolffram S (2008) Tissue distribution of quercetin in pigs after long-term dietary supplementation. J Nutr 138(8):1417–1420Google Scholar
- 45.Pogacnik L, Pirc K, Palmela I, Skrt M, Kim KS, Brites D, Brito MA, Ulrih NP, Silva RF (2016) Potential for brain accessibility and analysis of stability of selected flavonoids in relation to neuroprotection in vitro. Brain Res 1651:17–26. https://doi.org/10.1016/j.brainres.2016.09.020 CrossRefGoogle Scholar
- 47.Youdim KA, Qaiser MZ, Begley DJ, Rice-Evans CA, Abbott NJ (2004) Flavonoid permeability across an in situ model of the blood–brain barrier. Free Radic Biol Med 36(5):592–604. https://doi.org/10.1016/j.freeradbiomed.2003.11.023 CrossRefGoogle Scholar
- 58.DeCoster MA, Schabelman E, Tombran-Tink J, Bazan NG (1999) Neuroprotection by pigment epithelial-derived factor against glutamate toxicity in developing primary hippocampal neurons. J Neurosci Res 56(6):604–610. 10.1002/(SICI)1097-4547(19990615)56:6<604::AID-JNR6>3.0.CO;2-B CrossRefGoogle Scholar
- 59.Amano S, Yamagishi S, Inagaki Y, Nakamura K, Takeuchi M, Inoue H, Imaizumi T (2005) Pigment epithelium-derived factor inhibits oxidative stress-induced apoptosis and dysfunction of cultured retinal pericytes. Microvasc Res 69(1–2):45–55. https://doi.org/10.1016/j.mvr.2004.11.001 CrossRefGoogle Scholar
- 62.Adams LS, Phung S, Yee N, Seeram NP, Li L, Chen S (2010) Blueberry phytochemicals inhibit growth and metastatic potential of MDA-MB-231 breast cancer cells through modulation of the phosphatidylinositol 3-kinase pathway. Cancer Res 70(9):3594–3605. https://doi.org/10.1158/0008-5472.CAN-09-3565 CrossRefGoogle Scholar
- 64.Zhao W, Wang J, Bi W, Ferruzzi M, Yemul S, Freire D, Mazzola P, Ho L, Dubner L, Pasinetti GM (2015) Novel application of brain-targeting polyphenol compounds in sleep deprivation-induced cognitive dysfunction. Neurochem Int 89:191–197. https://doi.org/10.1016/j.neuint.2015.07.023 CrossRefGoogle Scholar
- 65.O’Neil BJ, McKeown TR, DeGracia DJ, Alousi SS, Rafols JA, White BC (1999) Cell death, calcium mobilization, and immunostaining for phosphorylated eukaryotic initiation factor 2-alpha (eIF2alpha) in neuronally differentiated NB-104 cells: arachidonate and radical-mediated injury mechanisms. Resuscitation 41(1):71–83CrossRefGoogle Scholar
- 68.Kampinga HH (1993) Thermotolerance in mammalian cells. Protein denaturation and aggregation, and stress proteins. J Cell Sci 104(Pt 1):11–17Google Scholar
- 71.Lin ST, Tu SH, Yang PS, Hsu SP, Lee WH, Ho CT, Wu CH, Lai YH, Chen MY, Chen LC (2016) Apple polyphenol phloretin inhibits colorectal cancer cell growth via inhibition of the type 2 glucose transporter and activation of p53-mediated signaling. J Agric Food Chem 64(36):6826–6837. https://doi.org/10.1021/acs.jafc.6b02861 CrossRefGoogle Scholar
- 72.Sakagami H, Hashimoto K, Suzuki F, Ogiwara T, Satoh K, Ito H, Hatano T, Takashi Y, Fujisawa S-i (2005) Molecular requirements of lignin–carbohydrate complexes for expression of unique biological activities. Phytochemistry 66(17):2108–2120. https://doi.org/10.1016/j.phytochem.2005.05.013 CrossRefGoogle Scholar
- 73.Ohkawa H, Sohma H, Sakai R, Kuroki Y, Hashimoto E, Murakami S, Saito T (2002) Ethanol-induced augmentation of annexin IV in cultured cells and the enhancement of cytotoxicity by overexpression of annexin IV by ethanol. BBA Mol Basis Dis 1588(3):217–225. https://doi.org/10.1016/S0925-4439(02)00168-0 CrossRefGoogle Scholar
- 76.Teske BF, Fusakio ME, Zhou D, Shan J, McClintick JN, Kilberg MS, Wek RC (2013) CHOP induces activating transcription factor 5 (ATF5) to trigger apoptosis in response to perturbations in protein homeostasis. Mol Biol Cell 24(15):2477–2490. https://doi.org/10.1091/mbc.E13-01-0067 CrossRefGoogle Scholar
- 82.Cavet ME, Harrington KL, Vollmer TR, Ward KW, Zhang JZ (2011) Anti-inflammatory and anti-oxidative effects of the green tea polyphenol epigallocatechin gallate in human corneal epithelial cells. Mol Vis 17:533–542Google Scholar
- 83.Catalan U, Fernandez-Castillejo S, Angles N, Morello JR, Yebras M, Sola R (2012) Inhibition of the transcription factor c-Jun by the MAPK family, and not the NF-kappaB pathway, suggests that peanut extract has anti-inflammatory properties. Mol Immunol 52(3–4):125–132. https://doi.org/10.1016/j.molimm.2012.05.007 CrossRefGoogle Scholar