Members of the Viola genus play important roles in traditional Asian herbal medicine. This study investigates the ability of Viola odorata L. extracts to inhibit Na+,K+-ATPase, an essential animal enzyme responsible for membrane potential maintenance. The root extract of V. odorata strongly inhibited Na+,K+-ATPase, while leaf and seeds extracts were basically inactive. A UHPLC-QTOF-MS/MS metabolomic approach was used to identify the chemical principle of the root extract's activity, resulting in the detection of 35,292 features. Candidate active compounds were selected by correlating feature area with inhibitory activity in 14 isolated fractions. This yielded a set of 15 candidate compounds, of which 14 were preliminarily identified as procyanidins. Commercially available procyanidins (B1, B2, B3 and C1) were therefore purchased and their ability to inhibit Na+,K+-ATPase was investigated. Dimeric procyanidins B1, B2 and B3 were found to be inactive, but the trimeric procyanidin C1 strongly inhibited Na+,K+-ATPase with an IC50 of 4.5 µM. This newly discovered inhibitor was docked into crystal structures mimicking the Na3E1∼P·ADP and K2E2·Pi states to identify potential interaction sites within Na+,K+-ATPase. Possible binding mechanisms and the principle responsible for the observed root extract activity are discussed.

Department of Chemical Biology Faculty of Science Palacky University Olomouc Czech Republic

Department of Experimental Biology Faculty of Science Palacky University Olomouc Czech Republic

Department of Experimental Physics Faculty of Science Palacky University Olomouc Czech Republic

Laboratory of Growth Regulators Institute of Experimental Botany of the Czech Academy of Sciences Palacky University Olomouc Czech Republic

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Kaplan JH. Biochemistry of Na, K-ATPase. Annu. Rev. Biochem. 2002;71:511–535. doi: 10.1146/annurev.biochem.71.102201.141218. PubMed DOI

Zdravkovic I, Zhao C, Lev B, Cuervo JE, Noskov SY. Atomistic models of ion and solute transport by the sodium-dependent secondary active transporters. Biochim. Biophys. Acta Biomembr. 2012;1818:337–347. doi: 10.1016/j.bbamem.2011.10.031. PubMed DOI

Adams KF, et al. A perspective on re-evaluating digoxin’s role in the current management of patients with chronic systolic heart failure: Targeting serum concentration to reduce hospitalization and improve safety profile. Eur. J. Heart Fail. 2014;16:483–493. doi: 10.1002/ejhf.64. PubMed DOI

De Carvalho Aguiar P, et al. Mutations in the Na+/K+-ATPase α3 gene ATP1A3 are associated with rapid-onset dystonia parkinsonism. Neuron. 2004;43:169–175. doi: 10.1016/j.neuron.2004.06.028. PubMed DOI

Segall L, et al. Alterations in the α2 isoform of Na, K-ATPase associated with familial hemiplegic migraine type 2. Proc. Natl. Acad. Sci. U. S. A. 2005;102:11106–11111. doi: 10.1073/pnas.0504323102. PubMed DOI PMC

Li Z, et al. Dihydroouabain, a novel radiosensitizer for cervical cancer identified by automated high-throughput screening. Radiother. Oncol. 2020;148:21–29. doi: 10.1016/j.radonc.2020.03.047. PubMed DOI

Wha Jun D, et al. Ouabain, a cardiac glycoside, inhibits the fanconi anemia/BRCA pathway activated by DNA interstrand cross-linking agents. PLoS ONE. 2013;8:e75905. doi: 10.1371/journal.pone.0075905. PubMed DOI PMC

Nilubol N, et al. Four clinically utilized drugs were identified and validated for treatment of adrenocortical cancer using quantitative high-throughput screening. J. Transl. Med. 2012;10:198. doi: 10.1186/1479-5876-10-198. PubMed DOI PMC

Guo J, et al. Screening of natural extracts for inhibitors against Japanese encephalitis virus infection. Antimicrob. Agents Chemother. 2020 doi: 10.1128/AAC.02373-19. PubMed DOI PMC

Prassas I, Paliouras M, Datti A, Diamandis EP. High-throughput screening identifies cardiac glycosides as potent inhibitors of human tissue kallikrein expression: Implications for cancer therapies. Clin. Cancer Res. 2008;14:5778–5784. doi: 10.1158/1078-0432.CCR-08-0706. PubMed DOI

Rupaimoole R, Yoon B, Zhang WC, Adams BD, Slack FJ. A high-throughput small molecule screen identifies ouabain as synergistic with mir-34a in killing lung cancer cells. Iscience. 2020;23(2):100878. doi: 10.1016/j.isci.2020.100878. PubMed DOI PMC

Zhang L, et al. Quantitative high-throughput drug screening identifies novel classes of drugs with anticancer activity in thyroid cancer cells: Opportunities for repurposing. J. Clin. Endocrinol. Metab. 2012;97:E319–E328. doi: 10.1210/jc.2011-2671. PubMed DOI PMC

Cayo MA, et al. A drug screen using human iPSC-derived hepatocyte-like cells reveals cardiac glycosides as a potential treatment for hypercholesterolemia. Cell Stem Cell. 2017;20:478–489.e5. doi: 10.1016/j.stem.2017.01.011. PubMed DOI PMC

Song Y, et al. Inhibitors of Na+/K+ ATPase exhibit antitumor effects on multicellular tumor spheroids of hepatocellular carcinoma. Sci. Rep. 2020;10:1–16. doi: 10.1038/s41598-019-56847-4. PubMed DOI PMC

Simpson CD, et al. Inhibition of the sodium potassium adenosine triphosphatase pump sensitizes cancer cells to anoikis and prevents distant tumor formation. Cancer Res. 2009;69:2739–2747. doi: 10.1158/0008-5472.CAN-08-2530. PubMed DOI

Blanco G, Mercer RW. Isozymes of the Na-K-ATPase: Heterogeneity in structure, diversity in function. Am. J. Physiol. Renal Physiol. 1998;275:633–650. doi: 10.1152/ajprenal.1998.275.5.F633. PubMed DOI

Toyoshima C, Kanai R, Cornelius F. First crystal structures of Na+, K+-ATPase: New light on the oldest ion pump. Structure. 2011;19:1732–1738. doi: 10.1016/j.str.2011.10.016. PubMed DOI

Clausen MV, Hilbers F, Poulsen H. The structure and function of the Na, K-ATPase isoforms in health and disease. Front. Physiol. 2017;8:371. doi: 10.3389/fphys.2017.00371. PubMed DOI PMC

Yap JQ, Seflova J, Sweazey R, Artigas P, Robia SL. FXYD proteins and sodium pump regulatory mechanisms. J. Gen. Physiol. 2021 doi: 10.1085/jgp.202012633. PubMed DOI PMC

Monti JLE, Montes MR, Rossi RC. Steady-state analysis of enzymes with non-Michaelis-Menten kinetics: The transport mechanism of Na+/K+-ATPase. J. Biol. Chem. 2018;293:1373–1385. doi: 10.1074/jbc.M117.799536. PubMed DOI PMC

Post RL, Kume S, Tobin T, Orcutt B, Sen AK. Flexibility of an active center in sodium-plus-potassium adenosine triphosphatase. J. Gen. Physiol. 1969;54:306–326. doi: 10.1085/jgp.54.1.306. PubMed DOI PMC

Albers RW. Biochemical aspects of active transport. Annu. Rev. Biochem. 1967;36:727–756. doi: 10.1146/annurev.bi.36.070167.003455. PubMed DOI

Kubala M. ATP-binding to P-type ATPases as revealed by biochemical, spectroscopic, and crystallographic experiments. Proteins Struct. Funct. Bioinform. 2006;64:1–12. doi: 10.1002/prot.20969. PubMed DOI

Clarke RJ, Catauro M, Rasmussen HH, Apell HJ. Quantitative calculation of the role of the Na+,K+-ATPase in thermogenesis. Biochim. Biophys. Acta Bioenerg. 2013;1827:1205–1212. doi: 10.1016/j.bbabio.2013.06.010. PubMed DOI

Páez O, et al. A Model for the homotypic interaction between Na+, K+-ATPase β1 subunits reveals the role of extracellular residues 221–229 in Its Ig-like domain. Int. J. Mol. Sci. 2019;20(18):4538. doi: 10.3390/ijms20184538. PubMed DOI PMC

Tokhtaeva E, et al. Epithelial junctions depend on intercellular trans-interactions between the Na, K-ATPase β1 subunits. J. Biol. Chem. 2011;286:25801–25812. doi: 10.1074/jbc.M111.252247. PubMed DOI PMC

Vagin O, Tokhtaeva E, Sachs G. The role of the β1 subunit of the Na, K-ATPase and its glycosylation in cell-cell adhesion. J. Biol. Chem. 2006;281:39573–39587. doi: 10.1074/jbc.M606507200. PubMed DOI

Kitamura N, et al. Mouse Na+/K+-ATPase β1-subunit has a K+-dependent cell adhesion activity for β-GlcNac-terminating glycans. Proc. Natl. Acad. Sci. U.S.A. 2005;102:2796–2801. doi: 10.1073/pnas.0409344102. PubMed DOI PMC

Rajasekaran SA, et al. Na, K-ATPase β-subunit is required for epithelial polarization, suppression of invasion, and cell motility. Mol. Biol. Cell. 2001;12:279–295. doi: 10.1091/mbc.12.2.279. PubMed DOI PMC

Laursen M, Yatime L, Nissen P, Fedosova NU. Crystal structure of the high-affinity Na+, K+-ATPase–ouabain complex with Mg2+ bound in the cation binding site. Proc. Natl. Acad. Sci. U.S.A. 2013;110:10958–10963. doi: 10.1073/pnas.1222308110. PubMed DOI PMC

Aperia A, Akkuratov EE, Fontana JM, Brismar H. Na+-K+-ATPase, a new class of plasma membrane receptors. Am. J. Physiol.-Cell Physiol. 2016;310(7):C491–C495. doi: 10.1152/ajpcell.00359.2015. PubMed DOI

Xie Z, Askari A. Na+/K+-ATPase as a signal transducer. Eur. J. Biochem. 2002;269:2434–2439. doi: 10.1046/j.1432-1033.2002.02910.x. PubMed DOI

Hamlyn JM, et al. Identification and characterization of a ouabain-like compound from human plasma. Proc. Natl. Acad. Sci. U.S.A. 1991;88:6259–6263. doi: 10.1073/pnas.88.14.6259. PubMed DOI PMC

Schoner W. Endogenous cardiac glycosides, a new class of steroid hormones. Eur. J. Biochem. 2002;269:2440–2448. doi: 10.1046/j.1432-1033.2002.02911.x. PubMed DOI

Bagrov AY, Shapiro JI, Fedorova OV. Endogenous cardiotonic steroids: Physiology, pharmacology, and novel therapeutic targets. Pharmacol. Rev. 2009;61:9–38. doi: 10.1124/pr.108.000711. PubMed DOI PMC

Lewis LK, et al. Ouabain is not detectable in human plasma. Hypertension. 1994;24:549–555. doi: 10.1161/01.HYP.24.5.549. PubMed DOI

Baecher S, Kroiss M, Fassnacht M, Vogeser M. No endogenous ouabain is detectable in human plasma by ultra-sensitive UPLC-MS/MS. Clin. Chim. Acta. 2014;431:87–92. doi: 10.1016/j.cca.2014.01.038. PubMed DOI

Ballard HE, Sytsma KJ, Kowal RR. Shrinking the violets: Phylogenetic relationships of infrageneric groups in Viola (Violaceae) based on internal transcribed spacer DNA sequences. Syst. Bot. 1999;23:439–458. doi: 10.2307/2419376. DOI

Rahman IU, et al. Contributions to the phytotherapies of digestive disorders: Traditional knowledge and cultural drivers of Manoor Valley, Northern Pakistan. J. Ethnopharmacol. 2016;192:30–52. doi: 10.1016/j.jep.2016.06.049. PubMed DOI

Bhatt VP, Negi GCS. Ethnomedicinal plant resources of Jaunsari tribe of Garhwal Himalaya, Uttaranchal. Indian J. Tradit. Knowl. 2006;5:331–335.

Verma G, Dua VK, Agarwal DD, Atul PK. Anti-malarial activity of Holarrhena antidysenterica and Viola canescens, plants traditionally used against malaria in the Garhwal region of north-west Himalaya. Malar. J. 2011;10:1–5. doi: 10.1186/1475-2875-10-20. PubMed DOI PMC

Chung IM, Kim MY, Park WH, Moon HI. Aldose reductase inhibitors from Viola hondoensis W. Becker et H Boss. Am. J. Chin. Med. 2008;36:799–803. doi: 10.1142/S0192415X08006247. PubMed DOI

Zhu Y, Zhao L, Wang X, Li P. Pharmacognostical and phytochemical studies of Viola tianschanica Maxim.—An Uyghur ethnomedicinal plant. J. Pharm. Pharmacogn. Res. 2016;4:95–106.

Wang H, Cong WL, Fu ZL, Chen DF, Wang Q. Anti-complementary constituents of Viola kunawarensis. Nat. Prod. Res. 2017;31:2312–2315. doi: 10.1080/14786419.2017.1297446. PubMed DOI

Feyzabadi Z, Ghorbani F, Vazani Y, Zarshenas MM. A critical review on phytochemistry, pharmacology of Viola odorata L. and related multipotential products in traditional Persian medicine. Phytother. Res. 2017;31:1669–1675. doi: 10.1002/ptr.5909. PubMed DOI

Cu JQ, Perineau F, Gaset A. Volatile components of violet leaves. Phytochemistry. 1992;31:571–573. doi: 10.1016/0031-9422(92)90040-W. DOI

Akhbari M, Batooli H, Kashi FJ. Composition of essential oil and biological activity of extracts of Viola odorata L. from central Iran. Nat. Prod. Res. 2012;26:802–809. doi: 10.1080/14786419.2011.558013. PubMed DOI

Parsley NC, et al. PepSAVI-MS reveals anticancer and antifungal cycloviolacins in Viola odorata. Phytochemistry. 2018;152:61–70. doi: 10.1016/j.phytochem.2018.04.014. PubMed DOI PMC

Narayani M, Chadha A, Srivastava S. Cyclotides from the indian medicinal plant Viola odorata (Banafsha): Identification and characterization. J. Nat. Prod. 2017;80:1972–1980. doi: 10.1021/acs.jnatprod.6b01004. PubMed DOI

Rizwan K, et al. A comprehensive review on chemical and pharmacological potential of Viola betonicifolia: A plant with multiple benefits. Molecules. 2019;24:3138. doi: 10.3390/molecules24173138. PubMed DOI PMC

Zubek S, Rola K, Szewczyk A, Majewska ML, Turnau K. Enhanced concentrations of elements and secondary metabolites in Viola tricolor L. induced by arbuscular mycorrhizal fungi. Plant Soil. 2015;390:129–142. doi: 10.1007/s11104-015-2388-6. DOI

Welling MT, Liu L, Rose TJ, Waters DLE, Benkendorff K. Arbuscular mycorrhizal fungi: effects on plant terpenoid accumulation. Plant Biol. 2016;18:552–562. doi: 10.1111/plb.12408. PubMed DOI

Harrison MJ. Molecular and cellular aspects of the arbuscular mycorrhizal symbiosis. Annu. Rev. Plant Biol. 1999;50(1):361–389. doi: 10.1146/annurev.arplant.50.1.361. PubMed DOI

Katoch M, Singh A, Singh G, Wazir P, Kumar R. Phylogeny, antimicrobial, antioxidant and enzyme-producing potential of fungal endophytes found in Viola odorata. Ann. Microbiol. 2017;67:529–540. doi: 10.1007/s13213-017-1283-1. DOI

Rauf A, et al. Proanthocyanidins: A comprehensive review. Biomed. Pharmacother. 2019;116:108999. doi: 10.1016/j.biopha.2019.108999. PubMed DOI

Rárová L, et al. Identification of narciclasine as an in vitro anti-inflammatory component of Cyrtanthus contractus by correlation-based metabolomics. J. Nat. Prod. 2019;82:1372–1376. doi: 10.1021/acs.jnatprod.8b00973. PubMed DOI

Enomoto H, Takahashi S, Takeda S, Hatta H. Distribution of flavan-3-ol species in ripe strawberry fruit revealed by matrix-assisted laser desorption/ionization-mass spectrometry imaging. Molecules. 2019;25:103. doi: 10.3390/molecules25010103. PubMed DOI PMC

Willer EA, et al. The vascular barrier-protecting hawthorn extract WS® 1442 raises endothelial calcium levels by inhibition of SERCA and activation of the IP3 pathway. J. Mol. Cell. Cardiol. 2012;53:567–577. doi: 10.1016/j.yjmcc.2012.07.002. PubMed DOI

Schwinger RHG, Pietsch M, Frank K, Brixius K. Crataegus special extract WS 1442 increases force of contraction in human myocardium cAMP-independently. J. Cardiovasc. Pharmacol. 2000;35:700–707. doi: 10.1097/00005344-200005000-00004. PubMed DOI

Koch E, Malek FA. Standardized extracts from hawthorn leaves and flowers in the treatment of cardiovascular disorders—preclinical and clinical studies. Planta Med. 2011;77:1123–1128. doi: 10.1055/s-0030-1270849. PubMed DOI

Yokomichi T, et al. Ursolic acid inhibits Na+/K+-ATPase activity and prevents TNF-α-induced gene expression by blocking amino acid transport and cellular protein synthesis. Biomolecules. 2011;1:32–47. doi: 10.3390/biom1010032. PubMed DOI PMC

Chen RJY, et al. Steroid-like compounds in Chinese medicines promote blood circulation via inhibition of Na+/K+-ATPase. Acta Pharmacol. Sin. 2010;31:696–702. doi: 10.1038/aps.2010.61. PubMed DOI PMC

Svedström U, et al. High-performance liquid chromatographic determination of oligomeric procyanidins from dimers up to the hexamer in hawthorn. J. Chromatogr. A. 2002;968:53–60. doi: 10.1016/S0021-9673(02)01000-2. PubMed DOI

Hellenbrand N, Sendker J, Lechtenberg M, Petereit F, Hensel A. Isolation and quantification of oligomeric and polymeric procyanidins in leaves and flowers of Hawthorn (Crataegus spp.) Fitoterapia. 2015;104:14–22. doi: 10.1016/j.fitote.2015.04.010. PubMed DOI

Svedström U, Vuorela H, Kostiainen R, Laakso I, Hiltunen R. Fractionation of polyphenols in hawthorn into polymeric procyanidins, phenolic acids and flavonoids prior to high-performance liquid chromatographic analysis. J. Chromatogr. A. 2006;1112:103–111. doi: 10.1016/j.chroma.2005.12.080. PubMed DOI

Cui T, et al. Quantification of the polyphenols and triterpene acids in Chinese hawthorn fruit by high-performance liquid chromatography. J. Agric. Food Chem. 2006;54:4574–4581. doi: 10.1021/jf060310m. PubMed DOI

Cui T, Nakamura K, Tian S, Kayahara H, Tian Y-L. Polyphenolic content and physiological activities of Chinese hawthorn extracts. Biosci. Biotechnol. Biochem. 2006;70:2948–2956. doi: 10.1271/bbb.60361. PubMed DOI

Yang B, Liu P. Composition and health effects of phenolic compounds in hawthorn (Crataegus spp.) of different origins. J. Sci. Food Agric. 2012;92:1578–1590. doi: 10.1002/jsfa.5671. PubMed DOI

Zumdick S, Petereit F, Luftmann H, Hensel A. Preparative isolation of oligomeric procyanidins from Hawthorn (Crataegus spp.) Pharmazie. 2009;64:286–288. PubMed

Souccar C, et al. Inhibition of gastric acid secretion by a standardized aqueous extract of Cecropia glaziovii Sneth and underlying mechanism. Phytomedicine. 2008;15:462–469. doi: 10.1016/j.phymed.2008.02.006. PubMed DOI

Mizukasi S, Tanabe Y, Noguchi M, Tamaki E. p-coumaroylputrescine, caffeoylputrescine and feruloylputrescine from callus tissue culture of Nicotiana tabacum. Phytochemistry. 1971;10:1347–1350. doi: 10.1016/S0031-9422(00)84339-3. DOI

Li Z, et al. Deep annotation of hydroxycinnamic acid amides in plants based on ultra-high-performance liquid chromatography-high-resolution mass spectrometry and its in silico database. Anal. Chem. 2018;90:14321–14330. doi: 10.1021/acs.analchem.8b03654. PubMed DOI

Ryabinin AA, Il’Ina EM. The alkaloid of Salsola subaphylla. Dok. Akad. Nauk SSSR. 1949;67:513.

Bardon C, et al. Biological denitrification inhibition (BDI) with procyanidins induces modification of root traits, growth and N status in Fallopia x bohemica. Soil Biol. Biochem. 2017;107:41–49. doi: 10.1016/j.soilbio.2016.12.009. DOI

Kraus TEC, Zasoski RJ, Dahlgren RA, Horwath WR, Preston CM. Carbon and nitrogen dynamics in a forest soil amended with purified tannins from different plant species. Soil Biol. Biochem. 2004;36:309–321. doi: 10.1016/j.soilbio.2003.10.006. DOI

Čechová P, Berka K, Kubala M. Ion Pathways in the Na+/K+-ATPase. J. Chem. Inf. Model. 2016;56:2434–2444. doi: 10.1021/acs.jcim.6b00353. PubMed DOI

Chebrolu S, Ma H, Artigas P. State-Dependent Movement between the First and Last External Loops of the Na/K Pump α Subunit. Biophys. J. 2014;106:582a. doi: 10.1016/j.bpj.2013.11.3227. DOI

Young VC, Artigas P. Displacement of the Na+/K+-pump’s transmembrane domains demonstrate conserved conformational changes in P-type 2 ATPases. Proc. Natl. Acad. Sci. U.S.A. 2021;118:e2019317118. doi: 10.1073/pnas.2019317118. PubMed DOI PMC

Kubala M, et al. Flavonolignans as a novel class of sodium pump inhibitors. Front. Physiol. 2016;7:115. doi: 10.3389/fphys.2016.00115. PubMed DOI PMC

Gu L, et al. Concentrations of proanthocyanidins in common foods and estimations of normal consumption. J. Nutr. 2004;134:613–617. doi: 10.1093/jn/134.3.613. PubMed DOI

Kosińska A, Andlauer W. Cocoa polyphenols are absorbed in Caco-2 cell model of intestinal epithelium. Food Chem. 2012;135:999–1005. doi: 10.1016/j.foodchem.2012.05.101. PubMed DOI

Mendoza-Wilson AM, et al. Absorption of dimers, trimers and tetramers of procyanidins present in apple skin by IEC-18 cell monolayers. J. Funct. Foods. 2016;27:386–391. doi: 10.1016/j.jff.2016.09.020. DOI

Deprez S, Mila I, Huneau JF, Tome D, Scalbert A. Transport of proanthocyanidin dimer, trimer, and polymer across monolayers of human intestinal epithelial Caco-2 cells. Antioxid. Redox Signal. 2001;3:957–967. doi: 10.1089/152308601317203503. PubMed DOI

Serra A, et al. Distribution of procyanidins and their metabolites in rat plasma and tissues in relation to ingestion of procyanidin-enriched or procyanidin-rich cocoa creams. Eur. J. Nutr. 2013;52:1029–1038. doi: 10.1007/s00394-012-0409-2. PubMed DOI

Prasain JK, et al. Liquid chromatography tandem mass spectrometry identification of proanthocyanidins in rat plasma after oral administration of grape seed extract. Phytomedicine. 2009;16:233–243. doi: 10.1016/j.phymed.2008.08.006. PubMed DOI PMC

Tsang C, et al. The absorption, metabolism and excretion of flavan-3-ols and procyanidins following the ingestion of a grape seed extract by rats. Br. J. Nutr. 2005;94:170–181. doi: 10.1079/BJN20051480. PubMed DOI

Serra A, et al. Determination of procyanidins and their metabolites in plasma samples by improved liquid chromatography–tandem mass spectrometry. J. Chromatogr. B. 2009;877:1169–1176. doi: 10.1016/j.jchromb.2009.03.005. PubMed DOI

Serra A, et al. Bioavailability of procyanidin dimers and trimers and matrix food effects in in vitro and in vivo models. Br. J. Nutr. 2010;103:944–952. doi: 10.1017/S0007114509992741. PubMed DOI

Shoji T, et al. Apple procyanidin oligomers absorption in rats after oral administration: Analysis of procyanidins in plasma using the porter method and high-performance liquid chromatography/tandem mass spectrometry. J. Agric. Food Chem. 2006;54:884–892. doi: 10.1021/jf052260b. PubMed DOI

Ottaviani JI, Kwik-Uribe C, Keen CL, Schroeter H. Intake of dietary procyanidins does not contribute to the pool of circulating flavanols in humans. Am. J. Clin. Nutr. 2012;95:851–858. doi: 10.3945/ajcn.111.028340. PubMed DOI

Rios LY, et al. Cocoa procyanidins are stable during gastric transit in humans. Am. J. Clin. Nutr. 2002;76:1106–1110. doi: 10.1093/ajcn/76.5.1106. PubMed DOI

Holt RR, et al. Procyanidin dimer B2 [epicatechin-(4β-8)-epicatechin] in human plasma after the consumption of a flavanol-rich cocoa. Am. J. Clin. Nutr. 2002;76:798–804. doi: 10.1093/ajcn/76.4.798. PubMed DOI

Sano A, et al. Procyanidin B1 is detected in human serum after intake of proanthocyanidin-rich grape seed extract. Biosci. Biotechnol. Biochem. 2003;67:1140–1143. doi: 10.1271/bbb.67.1140. PubMed DOI

Fedosova NU. Purification of Na, K-ATPase from pig kidney in Methods in Molecular Biology. New York: Humana Press Inc.; 2016. pp. 5–10. PubMed

Trott O, Olson AJ. AutoDock Vina: Improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J. Comput. Chem. 2009;31:455–461. PubMed PMC

Salentin S, Schreiber S, Haupt VJ, Adasme MF, Schroeder M. PLIP: Fully automated protein-ligand interaction profiler. Nucleic Acids Res. 2015;43:W443–W447. doi: 10.1093/nar/gkv315. PubMed DOI PMC