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Planta Med 2003; 69(9): 856-858
DOI: 10.1055/s-2003-43222
Letter
© Georg Thieme Verlag Stuttgart · New York

Antiandrogenic Activity of the Phytoestrogens Naringenin, 6-(1,1-Dimethylallyl)naringenin and 8-Prenylnaringenin

Oliver Zierau1 , Colm Morrissey2 , R. William G. Watson2 , Pia Schwab3 , Susanne Kolba1 , Peter Metz3 , Günter Vollmer1
  • 1Institut für Zoologie, Technische Universität Dresden, Germany
  • 2Department of Surgery, Mater Misericordiae University Hospital, Conway Institute of Biomolecular and Biomedical Research, Dublin Molecular Medicine Centre, University College Dublin, Ireland
  • 3Institut für Organische Chemie, Technische Universität Dresden, Germany
Colm Morrissey was funded by an EU (E)UROESTROGEN(E)S grant contract no: QLK6-CT-2000-00565. This paper was supported by the Deutsche Forschungsgemeinschaft Vo410/6 - 1 to Günter Vollmer
Further Information

Dr. Oliver Zierau

Technische Universität Dresden

Institut für Zoologie

Zellescher Weg 20

01217 Dresden

Germany

Phone: +49-351-46 33 78 41

Fax: +49-351-46 3319 23

Email: Oliver.Zierau@mailbox.tu-dresden.de

Publication History

Received: February 19, 2003

Accepted: April 26, 2003

Publication Date:
04 November 2003 (online)

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Table of Contents #

Abstract

Naturally occurring naringenin derivatives, known for their estrogenic activity, were tested in two independent (anti-)androgen screening assays. Using a yeast-based androgen receptor assay relatively strong antiandrogen activities were demonstrated for 6-(1,1-dimethylallyl)naringenin and 8-prenylnaringenin, while the parent compound naringenin did not show recognizable antiandrogen activity. In an androgen receptor activity assay based on the analysis of prostate specific antigen (PSA) concentrations in the supernatants of treated PC3(AR)2 cells the antiandrogenic activity of 6-(1,1-dimethylallyl)naringenin was detected at concentrations of 10-5 M. 8-Prenylnaringenin or naringenin have no detectable antiandrogenic effect. In summary, for the first time we provide evidence of the antiandrogenic activity of 6-DMA-N in two independent model systems. In conclusion, we demonstrated the ability of prenylated naringenins not only to act via the estrogen receptor but also through the androgen receptor.

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Abbreviations

DHT:dihydrotestosterone

PSA:prostate-specific antigen

Bic:bicalutamide

6-DMA-N:6-(1,1-dimethylallyl)naringenin

8-P-N:8-prenylnaringenin

E2:17-β estradiol

Phytoestrogens are plant derived compounds with estrogenic activity that may exhibit their actions via binding to the estrogen receptor (ER) and have been implicated in protecting against heart disease and cancer progression [1]. A considerable amount of information is available on the estrogen-like actions of many phytoestrogens [2], in addition, there have been a number of reports on the interaction of phytoestrogens with other steroid receptors, particularly on those exhibiting (anti-) androgenic activities [3]. Recently, a number of xenoestrogens were described that have antiandrogenic potential [4].

A flavonoid recently isolated from hops (Humulus lupulus), 8-Prenylnaringenin (8-P-N) (Fig. [1] a), also described as 8-isopentenylnaringenin, derived from Anaxagorea luzonensis [5] has been characterized as an extremely potent phytoestrogen [5], [6], [7], [8]. Only some of the estrogenic activities of the structurally related 6-(1,1-dimethylallyl)naringenin (6-DMA-N) (Fig. [1] b) from the African tree Monotes engleri [9] have been characterized [10], [11], while the potential estrogenic effects of naringenin (Fig. [1] c) present in grapefruit (Citrus grandis) are controversial [11], [12], [13].

A new efficient chemical synthesis [10] for production of larger quantities of 8-prenylnaringenin and 6-(1,1-dimethylallyl)naringenin allowed us to study the estrogenicity and the (anti-)androgenicity of these compounds.

To study the (anti-)androgenic potential of these synthetic derivatives and compare the activity to the parental compound naringenin, two different bioassays were used: a yeast-based androgen receptor assay [4] and an androgen receptor activity assay based on the analysis of prostate specific antigen (PSA) concentrations in the supernatants of treated PC3(AR)2 cells [3]. In comparison to dihydrotestosterone (DHT) none of the three naringenins shows androgenic action in the yeast-based androgen receptor assay (Fig. [2]). Furthermore, naringenin does not inhibit DHT activity at any of the concentrations used (Fig. [3] c). On the other hand, 6-DMA-N and 8-P-N are able to inhibit the androgenic effects induced by 10-8 M DHT at concentrations above 5 × 10-6 M (Figs. [3] a + b). DHT binding to the androgen receptor is counteracted by simultaneous incubation with the substituted naringenins. In conclusion, the yeast based androgen receptor assay clearly demonstrates the anti-androgenic activities of 6-DMA-N and 8-P-N (Figs. [3] a + b).

To verify these results we measured the antiandrogenic potential of 6-DMA-N, 8-P-N and naringenin. PSA was used as a marker of androgenic control. PSA production was assessed in androgen receptor positive PC3(AR)2 cells treated with bicalutamide, 6-DMA-N, 8-P-N, naringenin and estradiol. PSA secretion and cell number were assessed in cells treated with and without DHT (10-8 M). Bicalutamide an antiandrogen used for prostrate cancer treatment and 6-DMA-N display antiandrogenic activity inhibiting PSA production relative to control. 8-P-N, naringenin and estradiol have no antiandrogenic effect (Fig. [4]).

6-DMA-N has antiandrogenic activity, however it was not as effective as the Bic control. Stimulating the PC-3(AR)2 cells with estradiol (E2) demonstrated that the existing antiandrogenic activity is not due to the described estrogenic potency of the compounds.

In summary, for the first time we provide evidence of the antiandrogenic activity of 6-DMA-N in two independent model systems. In conclusion, we demonstrated the ability of prenylated naringenins to act through the androgen receptor. This represents a further step towards the characterization of naringenin derived phytohormones, which may be useful in treatment or prevention of hormone related diseases.

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Fig. 1 Chemical structures of the naringenin derivatives.

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Fig. 2 Androgen inducible yeast receptor assay. Androgenic potentials of 8-prenylnaringenin, 6-(1,1-dimethylallyl)naringenin and naringenin were tested in a concentration-dependent manner. Experimental conditions and treatment procedures are described in Materials and Methods.

Zoom Image

Fig. 3 Androgen inducible yeast receptor assay. Antiandrogenic potentials of 6-(1,1-dimethylallyl)naringenin (a), 8-prenylnaringenin (b), and naringenin (c) were tested in a concentration-dependent manner in combination with 10-8 M dihydrotestosterone. Experimental conditions and treatment procedures are described in Materials and Methods.

Zoom Image

Fig. 4 PSA concentration in the supernatant of treated PC3(AR)2 cells. Antiandrogenic potentials of 6-(1,1-dimethylallyl)naringenin, 8-prenylnaringenin and naringenin were tested at concentrations of 10-5 M and compared to the effect of bicalutamide (10-5 M) and estradiol (10-8 M) with simultaneous dihydrotestosterone (10-6 M) treatment. Experimental conditions and treatment procedures are described in Materials and Methods.

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Materials and Methods

17β-Estradiol (E2) and dihydrotestosterone (DHT) were obtained from Sigma (Deissenhofen, Germany), bicalutamide (Bic) was obtained from E. Von Angerer (Institut für Pharmazie, Universität Regensburg, Germany), naringenin was purchased from several commercial sources, while 8-prenylnaringenin and 6-(1,1-dimethylallyl)naringenin were synthesized from naringenin as described previously [10]. The purity of the compounds was assessed to be > 99 % (gas chromatography and HPLC). Naringenin, 8-prenylnaringenin and 6-(1,1-dimethylallyl)naringenin were used as racemic mixtures. The yeast-based androgen receptor assay and the PC-3(AR)2 cells were cultured as previously described [4], [3]. For the assessment of the antiandrogenicity a concentration-dependent assay in the widely used androgen inducible yeast screen androgen receptor assay was performed [4]. The yeast strain contained both a stably transfected androgen receptor (AR) construct and an expression plasmid carrying androgen-responsive sequences controlling the reporter gene lac-Z encoding the enzyme β-galactosidase. (Anti-)Androgenic activity from the enzymatic hydrolysis of chlorophenol red β-D-galactopyranoside was read at 540 nm using a colorimetric assay.

In a concentration-dependent analysis of reporter gene activity, half maximal induction of β-galactosidase activity by dihydrotestosterone was antagonized by the test substances and therefore the antiandrogenic activity could be estimated.

To test the antiandrogenicity of the naringenins in a mammalian model system in vitro PC3(AR)2 cells [3] were grown in 24-well plates and treated for 6 days in charcoal stripped, phenol red free RPMI-1640 medium. PSA concentration of the supernatant measured using a total PSA test (Abbott Diagnostics. Chicago, IL) on an AXSYM automated immunoassay analyzer (Abbott Diagnostics). Cell number was determined using the crystal violet assay. PSA concentrations were normalized using cell number and expressed relative to control.

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Acknowledgements

Our special thanks to Prof. Terry Brown who supplied us with the PC3(AR)2 cell line. Our thanks to Dr. Peadair Mc Ging and Ann Touhy at the Mater Misericordiae hospital biochemistry laboratories at the University College Dublin.

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References

  • 1 Knight D C, Eden J A. A review of the clinical effects of phytoestrogens.  Obstet Gynecol. 1996;  87 897-904
    PubMedSearch in Google Scholar
  • 2 Whitten P L, Patisaul H B. Cross-species and interassay comparisons of phytoestrogen action.  Environ Health Perspect. 2001;  109 5-20
    PubMedSearch in Google Scholar
  • 3 Rosenberg Zand R S, Jenkins D J, Brown T J, Diamandis E P. Flavonoids can block PSA production by breast and prostate cancer cell lines.  Clin Chim Acta. 2002;  317 7-26
    PubMedSearch in Google Scholar
  • 4 Sohoni P, Sumpter J P. Several environmental oestrogens are also anti-androgens.  J Endocrinol. 1998;  158 327-39
    CrossrefPubMedSearch in Google Scholar
  • 5 Kitaoka M, Kadokawa H, Sugano M, Ichikawa K, Taki M, Takaishi S, Iijima Y, Tsutsumi S, Boriboon M, Akiyama T. Prenylflavonoids: a new class of non-steroidal phytoestrogen (Part 1). Isolation of 8-isopentenylnaringenin and an initial study on its structure-activity relationship.  Planta Med. 1998;  64 511-5
    PubMedSearch in Google Scholar
  • 6 Miyamoto M, Matsushita Y, Kiyokawa A, Fukuda C, Iijima Y, Sugano M, Akiyama T. Prenylflavonoids: a new class of non-steroidal phytoestrogen (Part 2). Estrogenic effects of 8-isopentenylnaringenin on bone metabolism.  Planta Med. 1998;  64 516-9
    PubMedSearch in Google Scholar
  • 7 Milligan S R, Kalita J C, Heyerick A, Rong H, De Cooman L, De Keukeleire D. Identification of a potent phytoestrogen in hops (Humulus lupulus L.) and beer.  J Clin Endocrinol Metab. 1999;  84 2249-52
    CrossrefPubMedSearch in Google Scholar
  • 8 Milligan S R, Kalita J C, Pocock V, Van De Kauter V Stevens J F, Deinzer M L, Rong H, De Keukeleire D. The endocrine activities of 8-prenylnaringenin and related hop (Humulus lupulus L.) flavonoids.  J Clin Endocrinol Metab. 2000;  85 4912-5
    CrossrefPubMedSearch in Google Scholar
  • 9 Seo E K, Silva G L, Chai H B, Chagwedera T E, Farnsworth N R, Cordell G A, Pezzuto J M, Kinghorn A D. Cytotoxic prenylated flavanones from Monotes engleri .  Phytochemistry. 1997;  45 509-15
    CrossrefPubMedSearch in Google Scholar
  • 10 Gester S, Metz P, Zierau O, Vollmer G. An efficient synthesis of the potent phytoestrogens 8-prenylnaringenin and 6-(1,1-dimethylallyl)naringenin by europium(III)-catalyzed Claisen rearrangement.  Tetrahedron. 2001;  57 1015-18
    CrossrefPubMedSearch in Google Scholar
  • 11 Zierau O, Gester S, Schwab P, Metz P, Kolba S, Wulf M, Vollmer G. Estrogenic activity of the phytoestrogens naringenin, 6-(1,1-dimethylallyl)naringenin and 8-prenylnaringenin.  Planta Medica. 2002;  68 449-51
    Thieme ConnectPubMedSearch in Google Scholar
  • 12 Ruh M F, Zacharewski T, Connor K, Howell J, Chen I, Safe S. Naringenin: a weakly estrogenic bioflavonoid that exhibits antiestrogenic activity.  Biochem Pharmacol. 1995;  50 1485-93
    CrossrefPubMedSearch in Google Scholar
  • 13 Zand R S, Jenkins D J, Diamandis E P. Steroid hormone activity of flavonoids and related compounds.  Breast Cancer Res Treat. 2000;  62 35-49
    CrossrefPubMedSearch in Google Scholar

Dr. Oliver Zierau

Technische Universität Dresden

Institut für Zoologie

Zellescher Weg 20

01217 Dresden

Germany

Phone: +49-351-46 33 78 41

Fax: +49-351-46 3319 23

Email: Oliver.Zierau@mailbox.tu-dresden.de

#

References

  • 1 Knight D C, Eden J A. A review of the clinical effects of phytoestrogens.  Obstet Gynecol. 1996;  87 897-904
    PubMedSearch in Google Scholar
  • 2 Whitten P L, Patisaul H B. Cross-species and interassay comparisons of phytoestrogen action.  Environ Health Perspect. 2001;  109 5-20
    PubMedSearch in Google Scholar
  • 3 Rosenberg Zand R S, Jenkins D J, Brown T J, Diamandis E P. Flavonoids can block PSA production by breast and prostate cancer cell lines.  Clin Chim Acta. 2002;  317 7-26
    PubMedSearch in Google Scholar
  • 4 Sohoni P, Sumpter J P. Several environmental oestrogens are also anti-androgens.  J Endocrinol. 1998;  158 327-39
    CrossrefPubMedSearch in Google Scholar
  • 5 Kitaoka M, Kadokawa H, Sugano M, Ichikawa K, Taki M, Takaishi S, Iijima Y, Tsutsumi S, Boriboon M, Akiyama T. Prenylflavonoids: a new class of non-steroidal phytoestrogen (Part 1). Isolation of 8-isopentenylnaringenin and an initial study on its structure-activity relationship.  Planta Med. 1998;  64 511-5
    PubMedSearch in Google Scholar
  • 6 Miyamoto M, Matsushita Y, Kiyokawa A, Fukuda C, Iijima Y, Sugano M, Akiyama T. Prenylflavonoids: a new class of non-steroidal phytoestrogen (Part 2). Estrogenic effects of 8-isopentenylnaringenin on bone metabolism.  Planta Med. 1998;  64 516-9
    PubMedSearch in Google Scholar
  • 7 Milligan S R, Kalita J C, Heyerick A, Rong H, De Cooman L, De Keukeleire D. Identification of a potent phytoestrogen in hops (Humulus lupulus L.) and beer.  J Clin Endocrinol Metab. 1999;  84 2249-52
    CrossrefPubMedSearch in Google Scholar
  • 8 Milligan S R, Kalita J C, Pocock V, Van De Kauter V Stevens J F, Deinzer M L, Rong H, De Keukeleire D. The endocrine activities of 8-prenylnaringenin and related hop (Humulus lupulus L.) flavonoids.  J Clin Endocrinol Metab. 2000;  85 4912-5
    CrossrefPubMedSearch in Google Scholar
  • 9 Seo E K, Silva G L, Chai H B, Chagwedera T E, Farnsworth N R, Cordell G A, Pezzuto J M, Kinghorn A D. Cytotoxic prenylated flavanones from Monotes engleri .  Phytochemistry. 1997;  45 509-15
    CrossrefPubMedSearch in Google Scholar
  • 10 Gester S, Metz P, Zierau O, Vollmer G. An efficient synthesis of the potent phytoestrogens 8-prenylnaringenin and 6-(1,1-dimethylallyl)naringenin by europium(III)-catalyzed Claisen rearrangement.  Tetrahedron. 2001;  57 1015-18
    CrossrefPubMedSearch in Google Scholar
  • 11 Zierau O, Gester S, Schwab P, Metz P, Kolba S, Wulf M, Vollmer G. Estrogenic activity of the phytoestrogens naringenin, 6-(1,1-dimethylallyl)naringenin and 8-prenylnaringenin.  Planta Medica. 2002;  68 449-51
    Thieme ConnectPubMedSearch in Google Scholar
  • 12 Ruh M F, Zacharewski T, Connor K, Howell J, Chen I, Safe S. Naringenin: a weakly estrogenic bioflavonoid that exhibits antiestrogenic activity.  Biochem Pharmacol. 1995;  50 1485-93
    CrossrefPubMedSearch in Google Scholar
  • 13 Zand R S, Jenkins D J, Diamandis E P. Steroid hormone activity of flavonoids and related compounds.  Breast Cancer Res Treat. 2000;  62 35-49
    CrossrefPubMedSearch in Google Scholar

Dr. Oliver Zierau

Technische Universität Dresden

Institut für Zoologie

Zellescher Weg 20

01217 Dresden

Germany

Phone: +49-351-46 33 78 41

Fax: +49-351-46 3319 23

Email: Oliver.Zierau@mailbox.tu-dresden.de

   Permissions and Reprints
Zoom Image

Fig. 1 Chemical structures of the naringenin derivatives.

Zoom Image

Fig. 2 Androgen inducible yeast receptor assay. Androgenic potentials of 8-prenylnaringenin, 6-(1,1-dimethylallyl)naringenin and naringenin were tested in a concentration-dependent manner. Experimental conditions and treatment procedures are described in Materials and Methods.

Zoom Image

Fig. 3 Androgen inducible yeast receptor assay. Antiandrogenic potentials of 6-(1,1-dimethylallyl)naringenin (a), 8-prenylnaringenin (b), and naringenin (c) were tested in a concentration-dependent manner in combination with 10-8 M dihydrotestosterone. Experimental conditions and treatment procedures are described in Materials and Methods.

Zoom Image

Fig. 4 PSA concentration in the supernatant of treated PC3(AR)2 cells. Antiandrogenic potentials of 6-(1,1-dimethylallyl)naringenin, 8-prenylnaringenin and naringenin were tested at concentrations of 10-5 M and compared to the effect of bicalutamide (10-5 M) and estradiol (10-8 M) with simultaneous dihydrotestosterone (10-6 M) treatment. Experimental conditions and treatment procedures are described in Materials and Methods.