Characterization of the XIAP-Inhibiting Proanthocyanidin Fraction of the Aerial Parts of Ephedra sinica

S. Schäfer; S. Salcher; M. Seiter; C. Ranninger; M. Möst; P. Obexer; C. G. Huber; M. J. Ausserlechner; S. Schwaiger; H. Stuppner

doi:10.1055/s-0042-107253

Planta Medica, Inhaltsverzeichnis

Planta Med 2016; 82(11/12): 973-985
DOI: 10.1055/s-0042-107253

Biological and Pharmacological Activity

Original Papers

Georg Thieme Verlag KG Stuttgart · New York

Characterization of the XIAP-Inhibiting Proanthocyanidin Fraction of the Aerial Parts of Ephedra sinica ^[*]

S. Schäfer

¹Institute of Pharmacy, Pharmacognosy and Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck, Innsbruck, Austria

,

S. Salcher

³Department of Pediatrics II, Medical University Innsbruck, Innsbruck, Austria

⁴Tyrolean Cancer Research Institute, Innsbruck, Austria

,

M. Seiter

²Department of Pediatrics I, Medical University Innsbruck, Innsbruck, Austria

⁴Tyrolean Cancer Research Institute, Innsbruck, Austria

,

C. Ranninger

⁵Department of Molecular Biology, Division of Chemistry and Bioanalytics, University of Salzburg, Salzburg, Austria

,

M. Möst

⁶Department of Zoology, University of Cambridge, Cambridge, United Kingdom

,

P. Obexer

³Department of Pediatrics II, Medical University Innsbruck, Innsbruck, Austria

⁴Tyrolean Cancer Research Institute, Innsbruck, Austria

,

C. G. Huber

⁵Department of Molecular Biology, Division of Chemistry and Bioanalytics, University of Salzburg, Salzburg, Austria

,

M. J. Ausserlechner

²Department of Pediatrics I, Medical University Innsbruck, Innsbruck, Austria

,

S. Schwaiger

¹Institute of Pharmacy, Pharmacognosy and Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck, Innsbruck, Austria

,

H. Stuppner

¹Institute of Pharmacy, Pharmacognosy and Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck, Innsbruck, Austria

› Institutsangaben

Abstract

The X-linked inhibitor of apoptosis protein is a cellular protein that inhibits the activity of mammalian caspases and promotes resistance to apoptosis. The ethanol extract of the aerial parts of Ephedra sinica has been identified to possess inhibitory activity of the X-linked inhibitor of apoptosis protein by an in vitro fluorescence polarization assay using the BIR3 domain of the X-linked inhibitor of apoptosis protein. Bioactivity-guided fractionation identified proanthocyanidin-enriched fractions as the active principles. The most active fraction showed an IC₅₀ value of 27.3 µg/mL (CI₉₅: 25.9–28.9 µg/mL) corresponding to 9.6 µM (CI₉₅: 9.1–10.1 µM) calculated by the use of the determined average molecular weight of 2853.5. Samples were analyzed by a thiolytic degradation/HPLC-MS assay, UHPLC-HRMS, and 1D NMR.

The thiolytic degradation/HPLC-MS assay revealed a mean degree of polymerization of 9.5 ± 0.2 units (calculated average MW 2853.5) for the active fraction and 11.4 ± 0.6 units (calculated average MW 3437.0) for the most related inactive fraction. Chemical characterization identified (epi)gallocatechin (76.6 ± 1.0 % active; 80.7 ± 2.7 % inactive sample) and (epi)catechin units as building blocks. Interestingly, the investigated proanthocyanidins turned out to be a complex mixture of double linked A-type (binding 2-O-7″, 4–6″) and single linked B-type units.

This study identified oligomeric proanthocyanidins as active principles of E. sinica in vitro by a fluorescence polarization assay and via protein fragment complementation analysis.

Key words

Ephedra sinica - Ephedraceae - proanthocyandin - XIAP - fluorescence polarization

Volltext

Referenzen

References
1 Lowe SW, Lin AW. Apoptosis in cancer. Carcinogenesis 2000; 21: 485-495
2 Höffeler F. Die Maschinerie der Apoptose: Chronik eines angekündigten Todes. Biol Unserer Zeit 2004; 34: 16-23
3 Hengartner MO. The biochemistry of apoptosis. Nature 2000; 407: 770-776
4 Tewari KM, Dhaneshwar SS. Inhibitors of apoptosis proteins (IAPS): clinical significance in cancer treatment research. J Can Res Updates 2012; 1: 212-220
5 Holcik M, Korneluk RG. XIAP, the guardian angel. Nat Rev Mol Cell Biol 2001; 2: 550-556
6 Kashkar H. X-linked inhibitor of apoptosis: a chemoresistance factor or a hollow promise. Clin Cancer Res 2010; 16: 4496-4502
7 Blumenthal M, Penny K. Ma huang: ancient herb, modern medicine, regulatory dilemma; a review of the botany, chemistry, medicinal uses, safety concerns, and legal status of ephedra and its alkaloids. HerbalGram 1995; 34: 22-26
8 Hurlbut JA, Carr JR, Singleton ER, Faul KC, Madson MR, Storey JM, Thomas TL. Solid-phase extraction cleanup and liquid chromatography with ultraviolet detection of ephedrine alkaloids in herbal products. J AOAC Int 1998; 81: 1121-1127
9 Nam NH, Kim HM, Bae KH, Ahn BZ. Inhibitory effects of Vietnamese medicinal plants on tube-like formation of human umbilical venous cells. Phytother Res 2003; 17: 107-111
10 Nam NH, Lee CW, Hong DH, Kim HM, Bae KH, Ahn BZ. Antiinvasive, antiangiogenic and antitumour activity of Ephedra sinica extract. Phytother Res 2003; 17: 70-76
11 Tao HM, Wang LS, Cui ZC, Zhao DQ, Liu YH. Dimeric proanthocyanidins from the roots of Ephedra sinica . Planta Med 2008; 74: 1823-1825
12 Reti L. Ephedra bases. In: Manske RHF, Holmes HL, editors The alkaloids: chemistry and physiology, Vol. 3. New York: Academic Press, Inc.; 1953: 339-362
13 Guyot S, Marnet N, Laraba D, Drilleau JF. Reversed-phase HPLC following thiolysis for quantitative estimation and characterization of the four main classes of phenolic compounds in different tissue zones of a french cider apple variety (Malus domestica var. kermerrien). J Agric Food Chem 1998; 46: 1698-1705
14 Matthews S, Mila I, Scalbert A, Pollet B, Lapierre C, Hervé du Penhoat CLM, Rolando C, Donnelly DMX. Method for estimation of proanthocyanidins based on their acid depolymerization in the presence of nucleophiles. J Agric Food Chem 1997; 45: 1195-1201
15 Gu L, Kelm MA, Hammerstone JF, Beecher G, Holden J, Haytowitz D, Prior RL. Screening of foods containing proanthocyanidins and their structural characterization using LC-MS/MS and thiolytic degradation. J Agric Food Chem 2003; 51: 7513-7521
16 Thompson RS, Jacques D, Haslam E, Tanner RJN. Plant proanthocyanidins. Part I. Introduction; the isolation, structure, and distribution in nature of plant procyanidins. J Chem Soc Perkin Trans 1 1972; 1387-1399
17 Li HJ, Deinzer ML. Tandem mass spectrometry for sequencing proanthocyanidins. Anal Chem 2007; 79: 1739-1748
18 Li HJ, Deinzer ML. The mass spectral analysis of isolated hops A-type proanthocyanidins by electrospray ionization tandem mass spectrometry. J Mass Spectrom 2008; 43: 1353-1363
19 Reed JD, Krueger CG, Vestling MM. MALDI-TOF mass spectrometry of oligomeric food polyphenols. Phytochemistry 2005; 66: 2248-2263
20 Mouls L, Mazauric JP, Sommerer N, Fulcrand H, Mazerolles G. Comprehensive study of condensed tannins by ESI mass spectrometry: average degree of polymerisation and polymer distribution determination from mass spectra. Anal Bioanal Chem 2011; 400: 613-623
21 Venter PB, Senekal ND, Kemp G, Amra-Jordaan M, Khan P, Bonnet SL, van der Westhuizen JH. Analysis of commercial proanthocyanidins. Part 3: The chemical composition of wattle (Acacia mearnsii) bark extract. Phytochemistry 2012; 83: 153-167
22 Czochanska Z, Foo LY, Newman RH, Porter LJ. Polymeric proanthocyanidins – stereochemistry, structural units, and molecular weight. J Chem Soc Perkin Trans 1 1980; 1980: 2278-2286
23 Seiter MA, Salcher S, Rupp M, Hagenbuchner J, Kiechl-Kohlendorfer U, Mortier J, Wolber G, Rollinger JM, Obexer P, Ausserlechner MJ. Discovery of Sanggenon G as a natural cell-permeable small-molecular weight inhibitor of X-linked inhibitor of apoptosis protein (XIAP). FEBS Open Bio 2014; 4: 659-671
24 Stefan E, Aquin S, Berger N, Landry CR, Nyfeler B, Bouvier M, Michnick SW. Quantification of dynamic protein complexes using Renilla luciferase fragment complementation applied to protein kinase A activities in vivo . Proc Natl Acad Sci U S A 2007; 104: 16916-16921
25 Nikolovska-Coleska Z, Xu L, Hu Z, Tomita Y, Li P, Roller PP, Wang R, Fang X, Guo R, Zhang M, Lippman ME, Yang D, Wang S. Discovery of embelin as a cell-permeable, small-molecular weight inhibitor of XIAP through structure-based computational screening of a traditional herbal medicine three-dimensional structure database. J Med Chem 2004; 47: 2430-2440
26 Schmidt BM, Howell AB, McEniry B, Knight CT, Seigler D, Erdman jr. JW, Lila MA. Effective separation of potent anti proliferation and antiadhesion components from wild blueberry (Vaccinium angustifolium ait.) fruits. J Agric Food Chem 2004; 52: 6433-6442
27 Mayer R, Stecher G, Wuerzner R, Silva RC, Sultana T, Trojer L, Feuerstein I, Krieg C, Abel G, Popp M, Bobleter O, Bonn GK. Proanthocyanidins: target compounds as antibacterial agents. J Agric Food Chem 2008; 56: 6959-6966
28 Ortega T, De La Hera E, Carretero ME, Gomez-Serranillos P, Naval MV, Villar AM, Prodanov M, Vacas V, Arroyo T, Hernandez T, Estrella I. Influence of grape variety and their phenolic composition on vasorelaxing activity of young red wines. Eur Food Res Technol 2008; 227: 1641-1650
29 Packer L, Rimbach G, Virgili F. Antioxidant activity and biologic properties of a procyanidin-rich extract from pine (pinus maritima) bark, pycnogenol. Free Radic Biol Med 1999; 27: 704-724
30 Pierini R, Kroon PA, Guyot S, Ivory K, Johnson IT, Belshaw NJ. Procyanidin effects on oesophageal adenocarcinoma cells strongly depend on flavan-3-ol degree of polymerization. Mol Nutr Food Res 2008; 52: 1399-1407
31 Zhang SG, Ding F, Luo AP, Chen AG, Yu ZC, Ren SH, Liu ZH, Zhang L. XIAP is highly expressed in esophageal cancer and its downregulation by RNAi sensitizes esophageal carcinoma cell lines to chemotherapeutics. Cancer Biol Ther 2007; 6: 973-979
32 Hümmer W, Schreier P. Analysis of proanthocyanidins. Mol Nutr Food Res 2008; 52: 1381-1398
33 Gea A, Stringano E, Brown RH, Mueller-Harvey I. In situ analysis and structural elucidation of sainfoin (Onobrychis viciifolia) tannins for high-throughput germplasm screening. J Agric Food Chem 2011; 59: 495-503
34 Saucier C, Mirabel M, Daviaud F, Longieras A, Glories Y. Rapid fractionation of grape seed proanthocyanidins. J Agric Food Chem 2001; 49: 5732-5735
35 Kawakami K, Aketa S, Nakanami M, Iizuka S, Hirayama M. Major water-soluble polyphenols, proanthocyanidins, in leaves of persimmon (Diospyros kaki) and their α-amylase inhibitory activity. Biosci Biotechnol Biochem 2010; 74: 1380-1385
36 Nonaka GI, Sakai R, Nishioka I. Hydrolysable tannins and proanthocyanidins from green tea. Phytochemistry 1984; 23: 1753-1755
37 Cai Y, Evans FJ, Roberts MF, Phillipson JD, Zenk MH, Gleba YY. Polyphenolic compounds from Croton lechleri . Phytochemistry 1991; 30: 2033-2040
38 Nonaka GI, Morimoto S, Kinjo JE, Nohara T, Nishioka I. Tannins and related compounds. L. Structures of proanthocyanidin A-1 and related compounds. Chem Pharm Bull (Tokyo) 1987; 35: 149-155
39 Kolodziej H, Sakar MK, Burger JFW, Engelshowe R, Ferreira D. A-type proanthocyanidins from Prunus spinosa . Phytochemistry 1991; 30: 2041-2047
40 Kamiya K, Watanabe C, Endang H, Umar M, Satake T. Studies on the constituents of bark of Parameria laevigata Moldenke. Chem Pharm Bull (Tokyo) 2001; 49: 551-557
41 Nonaka G, Morimoto S, Nishioka I. Tannins and related compounds. Part 13. Isolation and structures of trimeric, tetrameric, and pentameric proanthicyanidins from cinnamon. J Chem Soc Perkin Trans 1 1983; 1983: 2139-2145
42 Lou HX, Yamazaki Y, Sasaki T, Uchida M, Tanaka H, Oka S. A-type proanthocyanidins from peanut skins. Phytochemistry 1999; 51: 297-308
43 Cui CB, Tezuka Y, Kikuchi T, Nakano H, Tamaoki T, Park JH. Constituents of a fern, Davallia mariesii MOORE. II. Identification and 1H- and 13C-nuclear magnetic resonance spectra of procyanidin B-5, epicatechin-(4beta-8)-epicatechin-(4beta-6)-epicatechin, and epicatechin-(4beta-6)-epicatechin-(4beta-8)-epicatechin-(4beta-6)-epicatechin. Chem Pharm Bull (Tokyo) 1992; 40: 889-898
44 Porter LJ, Wong RY, Benson M, Chan BG, Viswanadhan VN, Gandour RD, Mattice WL. Conformational-analysis of flavans, ¹H NMR and molecular mechanical (MM2) studies of the benzopyran ring of 3′,4′,5,7-tetrahydroxyflavan-3-ols. The crystal and molecular structure of the procyanidin (2R,3S,4R)-3′,4′,5,7-tetramethoxy-4-(2,4,6-trimethoxyphenyl)-flavan-3-ol. J Chem Res (M) 1986; Issue 3: 830-880
45 Nonaka G, Nishioka I, Nagasawa T, Oura H. Tannins and related compounds. I. Rhubarb (1). Chem Pharm Bull (Tokyo) 1981; 29: 2862-2870
46 Venter PB, Senekal ND, Amra-Jordaan M, Bonnet SL, van der Westhuizen JH. Analysis of commercial proanthocyanidins. Part 2: An electrospray mass spectrometry investigation into the chemical composition of sulfited quebracho (Schinopsis lorentzii and Schinopsis balansae) heartwood extract. Phytochemistry 2012; 78: 156-169
47 Venter PB, Sisa M, van der Merwe MJ, Bonnet SL, van der Westhuizen JH. Analysis of commercial proanthocyanidins. Part 1: the chemical composition of quebracho (Schinopsis lorentzii and Schinopsis balansae) heartwood extract. Phytochemistry 2012; 73: 95-105
48 Antal DS, Schwaiger S, Ellmerer-Müller EP, Stuppner H. Cotinus coggygria wood: novel flavanone dimer and development of an HPLC/UV/MS method for the simultaneous determination of fourteen phenolic constituents. Planta Med 2010; 76: 1765-1772
49 The R Core Team. R: A language and environment for statistical computing. Version 3.3.0 (2016-05-03). Vienna, Austria:: R Foundation for Statistical Computing; 2016. Available at https://cran.r-project.org/doc/manuals/r-release/fullrefman.pdf Accessed May 10, 2016
50 Veisova D, Rezabkova L, Stepanek M, Novotna P, Herman P, Vecer J, Obsil T, Obsilova V. The C-terminal segment of yeast BMH proteins exhibits different structure compared to other 14-3-3 protein isoforms. Biochemistry 2010; 49: 3853-3861
51 Du Y, Khuri FR, Fu H. A homogenous luminescent proximity assay for 14-3-3 interactions with both phosphorylated and nonphosphorylated client peptides. Curr Chem Genomics 2008; 2: 40-47
52 Du Y, Masters SC, Khuri FR, Fu H. Monitoring 14-3-3 protein interactions with a homogeneous fluorescence polarization assay. J Biomol Screen 2006; 11: 269-276

Zusatzmaterial

Supporting Information

Characterization of the XIAP-Inhibiting Proanthocyanidin Fraction of the Aerial Parts of Ephedra sinica [*]

Characterization of the XIAP-Inhibiting Proanthocyanidin Fraction of the Aerial Parts of Ephedra sinica ^[*]