The Lipophilic Extract of Hypericum perforatum Exerts Significant Cytotoxic Activity Against T24 and NBT-II Urinary Bladder Tumor Cells

Abstract

Hypericum perforatum L. (St. John’s wort) is a medicinal plant used for many pathologies, especially for the treatment of mild to moderate depression. In the present study we have investigated the cytotoxic activity of the locally collected (Epirus region) Hypericum perforatum L. against cultured T24 and NBT-II bladder cancer cell lines. The lipophilic extract of the herb, prepared using petroleum ether, induced apoptosis displaying LC₅₀ values at concentrations as low as 4 and 5 μg/mL. A fraction of this extract displayed 60 % cell growth inhibition at a concentration of 0.95 μg/mL. Evaluating the importance of various biologically active components of the extract, it was found that hypericins (hypericin, pseudohypericin, etc.) were identified only in the methanolic (lipophobic) extract of the herb, and not in the active lipophilic extract. In addition, hyperforin concentrations in the lipophilic extract and its most active fraction, were 0.94 μg/mL, and 0.17 μg/mL, respectively, while the active cytotoxic concentration of pure hyperforin appeared in the range of 1.8 μg/mL - 5.0 μg/mL. Therefore, pure hyperforin does not seem to contribute significantly to the cytotoxicity activity. Chlorophylls were identified in low, not significantly different, concentrations in all extracts and fractions and were not correlated to the biological activity.

Owing to the combination of significant cytotoxic activity, natural abundance and low toxicity, the lipophilic extract of Hypericum perforatum holds the promise of being an interesting, new, antiproliferative agent against bladder cancer that deserves further investigation.

Abbreviations

HP:Hypericum perforatum L.

AS:aqueous solution

MS:methanolic solution

ME:methanolic extract

NPMF:non polar methanolic fraction

PMF:polar methanolic fraction

PEE:petroleum ether (lipophilic) extract

PEE (1,2,3,4,5,6,7):petroleum ether extract's fractions

PBS:phosphate buffered saline

DMSO:dimethyl sulfoxide

Hypericum perforatum L. - St. John’s wort - hypericin - pseudohypericin - hyperforin - chlorophylls - bladder tumor

References

• 1 Landis S H, Murray T, Bolden S, Wingo P A. Cancer Statistics, 1998.  CA Cancer J Clin. 1998;  48 6-29
• 2 Koss L G. Tumors of bladder. In: Koss LG, editor Diagnostic Cytology of the Urinary Tract With Histopathology and Clinical Correlations. New York; Lippincott-Raven 1995: pp 71-80
• 3 Kurth K H, Boufoux C, Sylvester R, van der Meijden A PM, Oosterlinck W, Brausi M. Treatment of superficial bladder tumors: achievements and needs.  Eur Urol. 2000;  37 1-9
• 4 Nseyo U O, Lamn D L. Therapy of superficial bladder cancer.  Semin Oncol. 1996;  23 598-604
• 5 Stavropoulos N E, Hastazeris K, Filiadis I, Michailidis I, Ioachim E, Liamis Z. et al . Intravesical instillations of interferon gamma in the prophylaxis of high risk superficial bladder cancer - results of a controlled prospective study.  Scand J Urol Nephrol. 2002;  36 218-22
• 6 Prout GR J r, Barton B A, Grifin P P, Friedell G H. Treated history of non-invasive grade 1 transitional cell carcinoma.  J Urol. 1992;  148 1413-9
• 7 Nseyo U O, DeHaven J, Dougherty T J, Potter W R, Merill D L, Lundahl S L. et al . Photodynamic therapy (PDT) in the treatment of patients with resistant superficial bladder cancer: a long-term experience.  J Clin Laser Med Surg. 1998;  16 61-8
• 8 Blumenthal M, Busse W R, Goldberg A, Gruenwald J, Hall T, Riggins C W, Rister R S, editors . The complete German Commission E monographs: therapeutic guide to herbal medicines. Boston; American Botanical Council 1998
• 9 St John’s W ort. (Hypericum perforatum). In: Upton R editor American herbal pharmacopoeia (AHP). St. Cruz; 1997: pp 1-32
• 10 Malamas M, Marselos M. The tradition of medicinal plants in Zagori, Epirus (Northwestern Greece).  J Ethnopharmacol. 1992;  37 197-203
• 11 Brantner A, Della Loggia R, Sosa S, Kartnig T. Untersuchungen zur antiphlogistichen Wirkung von Hypericum perforatum L.  Sci Pharm. 1994;  62 7-8
• 12 Shakirova K K, Garagulya A D, Khazanovich R L. Antimicrobial properties of some species of St. John’s wort cultivated in Uzbekistan.  Mikrobiol Zh. 1970;  32 494-7
• 13 Yip L, Hudson J B, Gruszecka-Kowalik , Zalkow L H, Towers G H. Antiviral activity of a derivative of the photosensitive compound hypericin.  Phytomedicine III. 1996;  2 185-90
• 14 Harrer G, Sommer H. Treatment of mild/moderate depressions with Hypericum .  Phytomedicine. 1994;  1 3-8
• 15 Linde K, Murlow C J, Berner M, Egger M. St John’s wort for depression.  Cochrane Database Syst Rev. 2005;  18 CD000448
• 16 Bilia A R, Gallori S, Vincieri F F. St John’s wort and depression efficacy, safety and tolerability - an update.  Life Sci. 2002;  70 3077-96
• 17 Pietta P, Gardana C, Pietta A. Comparative evaluation of St. John’s wort from different Italian regions.  Il Farmaco. 2001;  56 491-6
• 18 Singer A, Wonneman M, Mueller W E. Hyperforin, a major antidepressant constituent of St. John’s wort inhibits serotonin uptake by elevating free intracellular Na^{+ 1} .  J Pharmacol Exp Ther. 1999;  290 1363-8
• 19 Butterweck V, Petereit F, Winterhoff H, Nahrstedt A. Solubilized-hypericin and pseudohypericin from Hypericum perforatum exert anti-depressant activity in the forced swimming test.  Planta Med. 1998;  64 291-4
• 20 Butterweck V, Jurgenliemk G, Nahrstedt A, Winterhoff H. Flavonoids from Hypericum perforatum show antidepressant activity in the forced swimming test.  Planta Med. 2000;  66 3-6
• 21 Skalkos D, Tatsis E, Gerothanasis I P, Troganis A. Towards a consensus structure of hypericin in solution: direct evidence for a single tautomer and different ionization states in protic and nonprotic solvents by the use of variable temperature gradient ¹H NMR.  Tetrahedron. 2002;  58 4925-9
• 22 United States P harmacopoeia. St. John’s wort monograph, 9^th Supplement. 1998: pp 4709-4710
• 23 AOAC method 9 42.04, Chlorophylls in p lants. In: Cunniff P, editor Official Methods of Analysis of AOAC International. Gaithersburg, MD; 1998
• 24 Fotsis T, Pepper M S, Aktas E, Breit S, Rasku S, Adlercreutz H. et al . Flavonoids, dietary-derived inhibitors of cell proliferation and in vitro angiogenesis.  Cancer Res. 1997;  57 2916-21
• 25 Hostanska K, Reichling J, Bommer S, Weber M, Saller R. Aqueous ethanolic extract of St. John’s wort (Hypericum perforatum L.) induces growth inhibition and apoptosis in human malignant cells in vitro .  Pharmazie. 2002;  57 323-31
• 26 Hostanska K, Reichling J, Bommer S, Weber M, Saller R. Hyperforin a constituent of St John’s wort (Hypericum perforatum L.) extract induces apoptosis by triggering activation of caspases and with hypericin synergistically exerts cytotoxicity towards human malignant cell lines.  Eur J Pharm Biopharm. 2003;  56 121-32
• 27 Schempp C M, Kirkin V, Simon-Haarhaus B, Kersten A, Kiss J, Termeer C C. et al . Inhibition of tumour cell growth by hyperforin, a novel anticancer drug from St. John’s wort that acts by induction of apoptosis.  Oncogene. 2002;  21 1242-5