Evaluation of the Effect of Epilobium angustifolium Aqueous Extract on LNCaP Cell Proliferation in In Vitro and In Vivo Models^[*]

Abstract

Epilobium sp. are commonly used in traditional medicine in the treatment of early stages of benign prostatic hyperplasia and inflammation. It is suggested that a dominating constituent, oenothein B, is responsible for the extracts therapeutic effects. Several bioactivities were established for extracts and oenothein B in various in vitro models, but due to the questionable bioavailability of this dimeric macrocyclic ellagitannin, their significance in the in vivo effects remains unresolved. We have thus focused our attention on a complex comparative investigation of the in vitro and in vivo activities of phytochemically characterized Epilobium angustifolium aqueous extract and oenothein B on prostate cancer cells proliferation.

Incubation of different cell lines with E. angustifolium aqueous extract resulted in a significant reduction of proliferation of PZ-HPV-7 and LNCaP cells, which was partly associated with antiandrogenic activity. These effects were fully congruent with oenothein B, examined in parallel. Oral supplementation of rats implanted with LNCaP cells with E. angustifolium aqueous extract 50–200 mg/kg b. w. resulted in a reduction of the occurrence of prostatic adenoma up to 13 %. Oenothein B was not detected in the urine and feces of the E. angustifolium aqueous extract-treated group, however, conjugates of nasutins gut microbiota metabolites of ellagitannins were detected in the urine, while in human volunteers supplemented with Epilobium tea, only urolithin conjugates were present.

Despite observing significant and consistent effects in vitro and in vivo, we were unable to point out unequivocally the factors contributing to the observed E. angustifolium aqueous extract activity, facing the problems of an unknown metabolic fate of oenothein B and interspecies differences in E. angustifolium aqueous extract gut microbiota metabolism.

Key words

Epilobium angustifolium - Onagraceae - oenothein B - prostate cancer - LNCaP - polyphenols metabolites

^* Dedicated to Professor Dr. Max Wichtl in recognition of his outstanding contribution to pharmacognosy research.

• References

• 1 Wichtl M. Herbal Drug and Phytopharmaceuticals, 3th ed. Stuttgart: Medpharm GmbH; 2004: 191-194
• 2 Granica S, Piwowarski JP, Czerwińska ME, Kiss AK. Phytochemistry, pharmacology and traditional uses of different species belonging to the genus of Epilobium (Onagraceae): a review. J Ethnopharmacol 2014; 156: 316-346
• 3 Lesuisse D, Berjonneau J, Ciot C, Devaux P, Doucet B, Gourvest JF, Khemis B, Lang C, Legrand R, Lowinski M, Maquin P, Parent A, Schoot B, Teutsch G, Chodounska H, Kasal A. Determination of oenothein B as the active 5-alpha-reductase-inhibiting principle of the folk medicine Epilobium parviflorum . J Nat Prod 1996; 59: 490-492
• 4 Ducrey B, Marston A, Gohring S, Hartmann RW, Hostettmann K. Inhibition of 5 alpha-reductase and aromatase by the ellagitannins oenothein A and oenothein B from Epilobium species. Planta Med 1997; 63: 111-114
• 5 Vitalone A, Guizzetti M, Costa LG, Tita B. Extracts of various species of Epilobium inhibit proliferation of human prostate cells. J Pharm Pharmacol 2003; 55: 683-690
• 6 Vitalone A, McColl J, Thome D, Costa LG, Tita B. Characterization of the effect of Epilobium extracts on human cell proliferation. Pharmacology 2003; 69: 79-87
• 7 Stolarczyk M, Piwowarski JP, Granica S, Stefańska J, Naruszewicz M, Kiss AK. Extracts from Epilobium sp. herbs, their components and gut microbiota metabolites of Epilobium ellagitannins-urolithins inhibit hormone-dependent prostate cancer cells (LNCaP) proliferation and PSA secretion. Phytother Res 2013; 27: 1842-1848
• 8 Kiss AK, Kowalski J, Melzig MF. Induction of neutral endopeptidase activity in PC-3 cells by an aqueous extract of Epilobium angustifolium L. and oenothein B. Phytomedicine 2006; 13: 284-289
• 9 Hiermann A, Bucar F. Studies of Epilobium angustifolium extracts on growth of accessory sexual organs in rats. J Ethnopharmacol 1997; 55: 179-183
• 10 Kujawski R, Mrozikiewicz PM, Bogacz A, Cichocka J, Mikołajczak PŁ, Czerny B, Bobkiewicz-Kozłowska T, Grześkowiak E. Influence of standardized extract of Epilobium angustifolium on estrogen receptor α and β expression in in vivo model. Ginekol Pol 2010; 81: 600-605
• 11 Kujawski R, Derebecka-Hołysz N, Mikołajczak PŁ, Mrozikiewicz PM, Bobkiewicz-Kozłowska T. Investigation of the molecular mechanisms of action of natural herbal drugs in benign prostate hyperplesia. Acta Biochim Pol 2007; 54: 145
• 12 Koziorowski M, Mrozikiewicz PM, Mścisz A, Otta H, Mikołajczak PŁ, Król D. Czy Epilobium angustifolium może wspomagać leczenie i zapobiegać hormonozależnym przerostom prostaty. Herba Pol 2006; 52: 74
• 13 Schepetkin IA, Ramstead AG, Kirpotina LN, Voyich JM, Jutila MA, Quinn MT. Therapeutic potential of polyphenols from Epilobium angustifolium (Fireweed). Phytother Res 2016; 30: 1287-1297
• 14 Piwowarski JP, Granica S, Stefańska J, Kiss AK. Differences in metabolism of ellagitannins by human gut microbiota ex vivo cultures. J Nat Prod 2016; 79: 3022-3030
• 15 Ducrey B, Wolfender JL, Marston A, Hostettmann K. Analysis of flavonol glycosides of thirteen Epilobium species (Onagraceae) by LC-UV and thermospray LC-MS. Phytochemistry 1995; 38: 129-137
• 16 Stolarczyk M, Naruszewicz M, Kiss AK. Extracts from Epilobium sp. herbs induce apoptosis in human hormone-dependent prostate cancer cells by activating mitochondrial pathway. J Pharm Pharmacol 2013; 65: 1044-1054
• 17 Kaškonienė V, Stankevičius M, Drevinskas T, Akuneca I, Kaškonas P, Bimbiraitė-Survilienė K, Maruška A, Ragažinskienė O, Kornyšova O, Briedis V, Ugenskienė R. Evaluation of phytochemical composition of fresh and dried raw material of introduced Chamerion angustifolium L. using chromatographic, spectrophotometric and chemometric techniques. Phytochemistry 2015; 115: 184-193
• 18 Monschein M, Jaindl K, Buzimkić S, Bucar F. Content of phenolic compounds in wild populations of Epilobium angustifolium growing at different altitudes. Pharm Biol 2015; 53: 1576-1582
• 19 Baert N, Kim J, Karonen M, Salminen JP. Inter-population and inter-organ distribution of the main polyphenolic compounds of Epilobium angustifolium . Phytochemistry 2017; 134: 54-63
• 20 Shikov AN, Poltanov EA, Dorman HJ, Makarov VG, Tikhonov VP, Hiltunen R. Chemical composition and in vitro antioxidant evaluation of commercial water-soluble willow herb (Epilobium angustifolium L.) extracts. J Agric Food Chem 2006; 54: 3617-3624
• 21 Kiss A, Krupa K, Galambosi B, Shikov AN. Comparison of fermented and non-fermented Epilobium angustifolium herb extracts on prostate cells proliferation. Planta Med 2014; 80: LP49
• 22 Vitali F, Pennisi G, Attaguile G, Savoca F, Tita B. Antiproliferative and cytotoxic activity of extracts from Cistus incanus L. and Cistus monspeliensis L. on human prostate cell lines. Nat Prod Res 2011; 25: 188-202
• 23 Weijerman PC, König JJ, Wong ST, Niesters HG, Peehl DM. Lipofection-mediated immortalization of human prostatic epithelial cells of normal and malignant origin using human papillomavirus type 18 DNA. Cancer Res 1994; 54: 5579-5583
• 24 Wang CC, Chen LG, Yang LL. Antitumor activity of four macrocyclic ellagitannins from Cuphea hyssopifolia . Cancer Lett 1999; 140: 195-200
• 25 Kim AR, Kim SN, Jung IK, Kim HH, Park YH, Park WS. The inhibitory effect of Scutellaria baicalensis extract and its active compound, baicalin, on the translocation of the androgen receptor with implications for preventing androgenetic alopecia. Planta Med 2014; 80: 153-158
• 26 Harada N, Atarashi K, Murata Y, Yamaji R, Nakano Y, Inui H. Inhibitory mechanisms of the transcriptional activity of androgen receptor by resveratrol: Implication of DNA binding and acetylation of the receptor. J Steroid Biochem Mol Biol 2011; 123: 65-70
• 27 Liu S, Yuan Y, Okumura Y, Shinkai N, Yamauchi H. Camptothecin disrupts androgen receptor signaling and suppresses prostate cancer cell growth. Biochem Biophys Res Commun 2010; 394: 297-302
• 28 Fu M, Wang C, Zhang X, Pestell RG. Acetylation of nuclear receptors in cellular growth and apoptosis. Biochem Pharmacol 2004; 68: 1199-1208
• 29 Link A, Balaguer F, Goel A. Cancer chemoprevention by dietary polyphenols: promising role for epigenetics. Biochem Pharmacol 2010; 80: 1771-1792
• 30 Lee YH, Kim YJ, Kim HI, Cho HY, Yoon HG. Potent HAT inhibitory effect of aqueous extract from Bellflower (Platycodon grandiflorum) roots on androgen receptor-mediated transcriptional regulation. Food Sci Biotechnol 2007; 16: 457-462
• 31 Lee YH, Jung MG, Kang HB, Choi KC, Haam S, Jun W, Kim YJ, Cho HY, Yoon HG. Effect of anti-histone acetyltransferase activity from Rosa rugosa Thunb. (Rosaceae) extracts on androgen receptor-mediated transcriptional regulation. J Ethnopharmacol 2008; 118: 412-417
• 32 Kim MJ, Seong AR, Lee YH, Kim YJ, Shiota M, Yokomizo A, Naito S, Lee J, Jun W, Yoon HG. Histone acetyltransferase inhibitory activity of Bokbunja (Rubus coreanus Miq.) ethanol extract on androgen receptor-dependent prostate cancer cell growth. Food Sci Biotechnol 2010; 19: 1503-1511
• 33 Sakagami H, Jiang Y, Kusama K, Atsumi T, Ueha T, Toguchi M, Iwakura I, Satoh K, Ito H, Hatano T, Yoshida T. Cytotoxic activity of hydrolyzable tannins against human oral tumor cell lines – a possible mechanism. Phytomedicine 2000; 7: 39-47
• 34 Kiss A, Kowalski J, Melzig MF. Effect of Epilobium angustifolium L. extracts and polyphenols on cell proliferation and neutral endopeptidase activity in selected cell lines. Pharmazie 2006; 61: 66-69
• 35 Miyamoto K, Nomura M, Murayama T, Furukawa T, Hatano T, Yoshida T, Koshiura R, Okuda T. Antitumor activities of ellagitannins against sarcoma-180 in mice. Biol Pharm Bull 1993; 16: 379-387
• 36 Okuda T, Yoshida T, Hatano T. Ellagitannins as active constituents of medicinal plants. Planta Med 1989; 55: 117-122
• 37 Moore BP. The chemistry of the nasutins. Aust J Chem 1964; 17: 901-907
• 38 González-Barrio R, Truchado P, Ito H, Espín JC, Tomás-Barberán FA. UV and MS identification of urolithins and nasutins, the bioavailable metabolites of ellagitannins and ellagic acid in different mammals. J Agric Food Chem 2011; 59: 1152-1162
• 39 Kosmala M, Zduńczyk Z, Juśkiewicz J, Jurgoński A, Karlińska E, Macierzyński J, Jańczak R, Rój E. Chemical composition of defatted strawberry and raspberry seeds and the effect of these dietary ingredients on polyphenol metabolites, intestinal function, and selected serum parameters in rats. J Agric Food Chem 2015; 63: 2989-2996
• 40 Seeram NP, Aronson WJ, Zhang Y, Henning SM, Moro A, Lee RP, Sartippour M, Harris DM, Rettig M, Suchard MA, Pantuck AJ, Belldegrun A, Heber D. Pomegranate ellagitannin-derived metabolites inhibit prostate cancer growth and localize to the mouse prostate gland. J Agric Food Chem 2007; 55: 7732-7737
• 41 Sánchez-González C, Ciudad CJ, Noé V, Izquierdo-Pulido M. Walnut polyphenol metabolites, urolithins A and B, inhibit the expression of the prostate-specific antigen and the androgen receptor in prostate cancer cells. Food Funct 2014; 5: 2922-2930
• 42 Granica S, Bazylko A, Kiss AK. Determination of macrocyclic ellagitannin-oenothein B in plant materials by HPLC-DAD-MS: method development and validation. Phytochem Anal 2012; 23: 582-587
• 43 Bialonska D, Kasimsetty SG, Khan SI, Ferreira D. Urolithins, intestinal microbial metabolites of pomegranate ellagitannins, exhibit potent antioxidant activity in a cell-based assay. J Agric Food Chem 2009; 57: 10181-10186
• 44 Grupillo M, Lakomy R, Geng X, Styche A, Rudert WA, Trucco M, Fan Y. An improved intracellular staining protocol for efficient detection of nuclear proteins in YFP-expressing cells. Biotechniques 2011; 51: 417-420
• 45 Perez OD, Krutzik PO, Nolan GP. Flow cytometric analysis of kinase signaling cascades. Methods Mol Biol 2004; 263: 67-94
• 46 Piwowarski JP, Granica S, Zwierzyńska M, Stefańska J, Schopohl P, Melzig MF, Kiss AK. Role of human gut microbiota metabolism in the anti-inflammatory effect of traditionally used ellagitannin-rich plant materials. J Ethnopharmacol 2014; 155: 801-809