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Inhibitory effect of Rhus verniciflua Stokes extract on human aromatase activity; butin is its major bioactive component
Bioorganic & Medicinal Chemistry Letters 24 (2014) 1730–1733
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Inhibitory effect of Rhus verniciflua Stokes extract on human aromatase activity; butin is its major bioactive component Myeong Hyeon Park a, , In Sook Kim a, , Sun-A Kim a, Chun-Soo Na b, Cheol Yi Hong b, Mi-Sook Dong c,⇑, Hye Hyun Yoo a,⇑ a b c
Institute of Pharmaceutical Science and Technology and College of Pharmacy, Hanyang University, Ansan, Gyeonggi-do 426-791, Republic of Korea Lifetree Biotech Co., Ltd, Suwon, Gyeonggi-do 441-350, Republic of Korea School of Life Sciences and Biotechnology, Korea University, Seoul 136-713, Republic of Korea
a r t i c l e
i n f o
Article history: Received 13 January 2014 Revised 4 February 2014 Accepted 14 February 2014 Available online 26 February 2014 Keywords: Rhus verniciflua Stokes Human aromatase CYP19 Butin
a b s t r a c t Rhus verniciflua Stokes has been used as a traditional herbal medicine in Asia. In this study, the effect of R. verniciflua extract on human aromatase (cytochrome P450 19, CYP19) activity was investigated to elucidate the mechanism for the effect of R. verniciflua extract on androgen hormone levels. Androstenedione was used as a substrate and incubated with R. verniciflua extract in cDNA-expressed CYP19 supersomes in the presence of NADPH, and estrone formation was measured using liquid chromatography–tandem mass spectrometry. R. verniciflua extract was assessed at concentrations of 10–1000 lg/mL. The resulting data showed that R. verniciflua extract inhibited CYP19-mediated estrone formation in a concentrationdependent manner with an IC50 value of 136 lg/mL. Subsequently, polyphenolic compounds from R. verniciflua extract were tested to identify the ingredients responsible for the aromatase inhibitory effects by R. verniciflua extract. As a result, butin showed aromatase inhibitory effect in a concentration-dependent manner with an IC50 value of 9.6 lM, whereas the inhibition by other compounds was negligible. These results suggest that R. verniciflua extract could modulate androgen hormone levels via the inhibition of CYP19 activity and butin is a major ingredient responsible for this activity. Ó 2014 Elsevier Ltd. All rights reserved.
The stem bark of Rhus verniciflua Stokes (Anacardiaceae) is a folk herbal medicine used in Asian countries such as Korea, Japan, and China as an invigorant and to treat gastritis, stomach cancer, paralysis, high blood pressure, menstrual cycle irregularities, poor blood circulation, and atherosclerosis.1–3 It has been reported to possess various pharmacological activities, including antioxidant,4–6 antiproliferative,7,8 anti-inflammatory,9 antitumor,10–12 antimutagenic,13 and antirheumatoid arthritis14 effects. With regard to R. verniciflua as a folk remedy invigorant, the effect of R. verniciflua extract on androgen levels has been studied.15,16 According to the report by Seong et al.15 when mouse Leydig cells were treated with R. verniciflua extract, androgen hormone levels were increased. In addition, when rats were orally administered with the flavonoid fraction of R. verniciflua extract, plasma testosterone levels were elevated and the spermatozoon motility and epididymal sperm concentration were significantly ⇑ Corresponding authors. Tel.: +82 31 400 5804; fax: +82 31 400 4783. E-mail addresses: [email protected] (M.-S. Dong), [email protected] (H.H. Yoo). M.H. Park and I.S. Kim contributed equally to this work. http://dx.doi.org/10.1016/j.bmcl.2014.02.039 0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
increased.16 These reports suggest that R. verniciflua extract could enhance the androgen-dependent male sexual function. To characterize the mechanism of such an androgen-stimulating effect by R. verniciflua, we investigated the effect of R. verniciflua extract on the pathways for androgen biosynthesis using an androgen receptor (AR) nuclear translocation assay and an AR binding assay. However, R. verniciflua extract showed minimal effects in those tests (data not shown). Subsequently, we tested the effects on aromatase activity. Aromatase (CYP19) is an enzyme responsible for the conversion of androgens into estrogens. The enzyme is distributed in many tissues including gonads, brain, adipose tissue, placenta, blood vessels, skin, bone, and endometrium.17 Aromatase plays an important role in the regulation of sexual differentiation and reproduction during adult life and is involved in other physiological and behavioral processes, including neuroplasticity, cell growth, migration, and neuroprotection.17 Androgens are converted to estrogens by the aromatase enzyme complex.18,19 Thus, the modulation of aromatase activity may affect the androgen hormone level. Inhibition of aromatase activity would be helpful for androgen level retention. In this study, we investigated the effect of R. verniciflua extract and its polyphenolic ingredients on human aromatase activity to
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M. H. Park et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1730–1733
understand the mechanism of elevation of the androgen hormone level and to identify the active compound responsible for this activity. R. verniciflua extract was prepared according to the previous report.16 For aromatase inhibition assay, the incubation mixture consisted of 0.05 mg/mL human CYP 19A1 supersomes (BD Biosciences, Woburn, MA, USA), various concentrations of the test compound, 1 lM androstenedione, 3.3 mM magnesium chloride, and an NADPH-generating system (0.1 M glucose-6-phosphate, 10 mg/mL b-NADP+, and 1 U/mL glucose-6-phosphate dehydrogenase) in a total volume of 1 mL potassium phosphate buffer (0.1 M, pH 7.4). The reaction mixture was incubated for 30 min at 37 °C. After incubation, the reaction was stopped by the addition of 1 mL of ethyl acetate and 10 lL of IS solution (4 lg/mL cortisone). The reaction sample was extracted by liquid–liquid extraction. For liquid–liquid extraction, ethylacetate (4 mL) was added to the reaction sample. The mixture was vortexed for 2 min and centrifuged for 5 min at 13200g. The upper layer (5 mL) was collected and evaporated under nitrogen flow at 40 °C. The residue was reconstituted in 100 lL of methanol and transferred to a HPLC vial for liquid chromatography-–tandem mass spectrometric (LC–MS/MS) analysis. Estrone formation was determined using LC–MS/MS analysis and the details are provided as a Supplementary data. To characterize a chemical profile of the R. verniciflua extract used in this study, the major polyphenolic ingredients of the extract were determined using LC–MS/MS analysis (Supplementary data). Various phenolic compounds such as 2,4-dihydroxybenzoic acid, protocatechuic acid, caffeic acid, chlorogenic acid, p-coumaric acid, phloretin-20 -O-glucoside, kaempferol-3-O-glucoside, quercetin, butein, kaempferol, gallic acid, 2,6,30 ,40 tetrahydroxy-2-benzylcoumaran-3-one, fustin, fisetin, and sulfuretin have been reported as ingredients of R. verniciflua extracts.4,10,20 Based on these
OH OH
O
OH
OH
O
OH
OH
OH
OH
OH
Garbanzol OH
OH
O
OH
O
Taxifolin OH
OH OH
O
OH
OH
OH
OH
OH
OH
O
Dehydrokaempferol
O
H
OH
O
Quercetin
Butin
O
O
Butein
OH
OH
O
OH
OH OH OH
OH
2,4-dihydroxybenzaldehyde
OH
2,4-dihydroxybenzoic acid
3,4-dihydroxybenzoic acid O
OH HO
O
O
Sulfuretin
OH
OH
HO H 3CO
2-methoxybenzene-1,4-diol
OH
OH
OH
HO
Resorcinol
OH OH
O
Fisetin OH
O
OH
OH
O
OH
O
O
Fustin
O
OH
OH
O
OH
literature data and our phytochemical study results, we determined the contents of 15 polyphenolic compounds (Fig. 1) in R. verniciflua extract. The mean contents of those compounds are presented in Table 1. The inhibitory effects of R. verniciflua extract on human aromatase activity were evaluated in cDNA-expressed CYP19 supersomes. In this study, androstenedione was used as a substrate and estrone formation was measured using LC–MS/MS. The assay system was tested with formestane, a well-known CYP19 selective inhibitor. Formestane selectively inhibited estrone formation in a concentration-dependent manner with an IC50 value of 0.47 lM (Fig. 2A). When R. verniciflua extract was tested in a concentration range of 10–1000 lg/mL, R. verniciflua extract exhibited concentrationdependent inhibitory effects on the estrone formation mediated by CYP19 with an IC50 value of 136.2 lg/mL (Fig. 2B). To investigate the ingredients responsible for the aromatase inhibitory effect of R. verniciflua extract, a total of 15 phenolic compounds contained in R. verniciflua extract (Fig. 1) were evaluated for their inhibitory potential against aromatase activity. All of the test compounds were assayed at 10 lM. The resulting data are assembled in Table 1. The positive control (formestane) inhibited estrone formation by 94.9% at 10 lM. Among the compounds tested, butin showed a significant inhibitory activity (by 50%) on estrone formation but other compounds exhibited minimal effects. When butin was tested at various concentrations, it inhibited estrone formation in a concentration-dependent manner with an IC50 value of 9.6 lM (Fig. 2C). In the previous study by Na et al. R. verniciflua extract elevated the plasma testosterone in male rats.16 In addition, in the recent clinical study with R. verniciflua extract, the extract has been found to increase the testosterone level in middle-aged men. When R. verniciflua extract was administered for 8 weeks, total testosterone and free testosterone levels were significantly increased in subjects
OH
OH
Garllic acid
Figure 1. Chemical structures of polyphenolic compounds from R. verniciflua extract.
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M. H. Park et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1730–1733
Table 1 Content and aromatase inhibitory activity of polyphenolic compounds in R. verniciflua extract
a
Compounds
Contents (%)
Estrone formationa (% of control)
Fustin Fisetin Gallic acid Butin Butein Sulfuretin Resorcinol Garbanzol Quercetin Taxifolin Dehydrokaempferol 2,4-Dihydroxybenzaldehyde 2,4-Dihydroxybenzoic acid 3,4-Dihydroxybenzoic acid 4-Hydroxybenzoic acid Formestane (positive control)
12.1 6.7 3.4 0.1 0.5 0.4 0.9 0.7 0.1 1.0 0.1 0.3 0.07 0.09 0.04 —
109.9 117.3 112.2 50.0 87.5 99.0 91.8 99.3 96.0 90.7 101.7 91.7 93.9 83.5 91.8 5.1
All compounds tested were assayed at 10 lM and the data shown represent the mean of duplicate experiments.
Estrone formation (%)
120
(A)
120
(B)
120
100
100
100
80
80
80
60
60
60
40
40
40
20
20
20
0
0
0.01
0.10
1.00
Formastane (μM)
10.00
(C)
0 10
100
1000
R. verniciflua extract (μg/mL)
1
10
100
Butin (μM)
Figure 2. Effects of R. verniciflua extract and butin on estrone formation in CYP19 supersomes. (A) Formestane (positive control); (B) R. verniciflua extract; (C) Butin.
(unpublished data). The present results may support the enhancing effects of R. verniciflua extract for androgen hormones and provide its possible mechanism. Thus, R. verniciflua may modulate androgen hormone level by inhibiting the conversion of androgens to estrogens via the inhibition of CYP19 activity. R. verniciflua extract showed a concentration-dependent inhibitory activity on aromatase but its potency was relatively weak. Our results indicated that butin is a major component responsible for its aromatase inhibitory activity and the weak activity of the extract may be due to low content of butin in the extract. According to previously reported papers, butin has a reactive oxygen species scavenging or antioxidant activities.21,22 As for hormone-related activities, butin has been reported to exhibit an uterotrophic effect as a weak estrogen.23 This is thought to be due to a structural similarity between butin and sex hormones. In this context, butin may react as a competitive inhibitor on human aromatase. Considering that the other flavonoid ingredients lack the inhibitory effect, the absence of a substituent at the C3 position is supposed to be important for an aromatase inhibitory activity. This is accordance with the previous reported data by Jeong et al.24; Among the 28 flavonoids tested, several flavonoids such as apigenin, chrysin, hesperetin, and naringin exhibited inhibitory effects on aromatase. All of these active flavonoids do not have a substituent at the C3 position. In conclusion, the present study demonstrates that R. verniciflua extract inhibited a conversion of androstenedione to estrone mediated by human aromatse and butin is a major ingredient responsible for this activity. This result suggests that R. verniciflua extract could modulate androgen hormone levels via the inhibition of CYP19 activity. In addition, butin could be a lead compound for development of aromatase inhibitors.
Acknowledgments This research was supported in part by Industrialization Support Program for Bio-technology of Agriculture and Forestry, Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea (810009-03-2-SB250) and in part by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2011-0012319). Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2014. 02.039. References and notes 1. Hong, D. H.; Han, S. B.; Lee, C. W.; Park, S. H.; Jeon, Y. J.; Kim, M.-J.; Kwak, S.-S.; Kim, H. M. Arch. Pharmacol. Res. 1999, 22, 638. 2. Kang, D. G.; Lee, A. S.; Mun, Y. J.; Woo, W. H.; Kim, Y. C.; Sohn, E. J.; Moon, M. K.; Lee, H. S. Biol. Pharm. Bull. 2004, 27, 366. 3. Kim, J. H.; Go, H. Y.; Jin, D. H.; Kim, H.-P.; Hong, M. H.; Chung, W.-Y.; Park, J.-H.; Jang, J. B.; Jung, H.; Shin, Y. C. Cancer Lett. 2008, 265, 197. 4. Lim, K.-T.; Hu, C.; Kitts, D. Food Chem. Toxicol. 2001, 39, 229. 5. Jung, C. H.; Jun, C.-Y.; Lee, S.; Park, C.-H.; Cho, K.; Ko, S.-G. Biol. Pharm. Bull. 2006, 29, 1603. 6. Kim, J.; Kim, H.; Jung, C.; Hong, M.; Hong, M.; Bae, H.; Lee, S.; Park, S.; Park, J.; Ko, S. Int. J. Mol. Med. 2006, 18, 201. 7. Lim, K.-T. J. Toxicol. Public Health 1999, 15, 169. 8. Kitts, D. D.; Lim, K.-T. J. Toxicol. Environ. Health, A 2001, 64, 357. 9. Lee, J.; Huh, J.; Jeon, G.; Yang, H.; Woo, H.; Choi, D.; Park, D. Int. Immunopharmacol. 2009, 9, 268. 10. Lee, J.-C.; Lim, K.-T.; Jang, Y.-S. Bba-Gen. Subj. 2002, 1570, 181.
M. H. Park et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1730–1733 11. Lee, J. C.; Lee, K. Y.; Kim, J.; Na, C. S.; Jung, N.; Chung, G. H.; Jang, Y. S. Food Chem. Toxicol. 2004, 42, 1383. 12. Jang, H.-S.; Kook, S.-H.; Son, Y.-O.; Kim, J.-G.; Jeon, Y.-M.; Jang, Y.-S.; Choi, K.-C.; Kim, J.; Han, S.-K.; Lee, K.-Y. Bba-Gen. Subj. 2005, 1726, 309. 13. Park, K.-Y.; Jung, G.-O.; Lee, K.-T.; Jongwon, C.; Choi, M.-Y.; Kim, G.-T.; Jung, H.J.; Park, H.-J. J. Ethnopharmacol. 2004, 90, 73. 14. Choi, J.; Yoon, B.-J.; Han, Y. N.; Lee, K.-T.; Ha, J.; Jung, H.-J.; Park, H.-J. Planta Med. 2003, 69, 899. 15. Seong, H.; Choi, S.; Jang, Y.; Min, K.; Woo, J.; Jang, W.; NC, J.; Na, C.; Jung, I. Korean J. Anim. Rep. 2001, 25, 125. 16. Na, C. S.; Choi, B. R.; Choo, D. W.; Choi, W. I.; Kim, J. B.; Kim, H. C.; Park, Y. I.; Dong, M. S. J. Toxicol. Public Health 2005, 21, 309. 17. Di Nardo, G.; Gilardi, G. Biotechnol. Appl. Biochem. 2013, 60, 92.
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18. Simpson, E. R.; Mahendroo, M. S.; Means, G. D.; Kilgore, M. W.; Hinshelwood, M. M.; Graham-Lorence, S.; Amarneh, B.; Ito, Y.; Fisher, C. R.; Michael, M. D. Endocr. Rev. 1994, 15, 342. 19. Kragie, L. Endocr. Res. 2002, 28, 121. 20. Kim, S.-A.; Kim, S. H.; Kim, I. S.; Lee, D.; Dong, M.-S.; Na, C.-S.; Nhiem, N. X.; Yoo, H. H. Food Chem. 2013, 141, 3813. 21. Zhang, R.; Chae, S.; Kang, K. A.; Piao, M. J.; Ko, D. O.; Wang, Z. H.; Park, D. B.; Park, J. W.; You, H. J.; Hyun, J. W. Mol. Cell. Biochem. 2008, 318, 33. 22. Zhang, R.; Kang, K. A.; Piao, M. J.; Chang, W. Y.; Maeng, Y. H.; Chae, S.; Lee, I. K.; Kim, B. J.; Hyun, J. W. Food Chem. Toxicol. 2010, 48, 922. 23. Bhargava, S. J. Ethnopharmacol. 1986, 18, 95. 24. Jeong, H. J.; Shin, Y. G.; Kim, I. H.; Pezzuto, J. M. Arch. Pharmacol. Res. 1999, 22, 309.
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Inhibitory effect of Rhus verniciflua Stokes extract on human aromatase activity; butin is its major bioactive component Myeong Hyeon Park a, , In Sook Kim a, , Sun-A Kim a, Chun-Soo Na b, Cheol Yi Hong b, Mi-Sook Dong c,⇑, Hye Hyun Yoo a,⇑ a b c
Institute of Pharmaceutical Science and Technology and College of Pharmacy, Hanyang University, Ansan, Gyeonggi-do 426-791, Republic of Korea Lifetree Biotech Co., Ltd, Suwon, Gyeonggi-do 441-350, Republic of Korea School of Life Sciences and Biotechnology, Korea University, Seoul 136-713, Republic of Korea
a r t i c l e
i n f o
Article history: Received 13 January 2014 Revised 4 February 2014 Accepted 14 February 2014 Available online 26 February 2014 Keywords: Rhus verniciflua Stokes Human aromatase CYP19 Butin
a b s t r a c t Rhus verniciflua Stokes has been used as a traditional herbal medicine in Asia. In this study, the effect of R. verniciflua extract on human aromatase (cytochrome P450 19, CYP19) activity was investigated to elucidate the mechanism for the effect of R. verniciflua extract on androgen hormone levels. Androstenedione was used as a substrate and incubated with R. verniciflua extract in cDNA-expressed CYP19 supersomes in the presence of NADPH, and estrone formation was measured using liquid chromatography–tandem mass spectrometry. R. verniciflua extract was assessed at concentrations of 10–1000 lg/mL. The resulting data showed that R. verniciflua extract inhibited CYP19-mediated estrone formation in a concentrationdependent manner with an IC50 value of 136 lg/mL. Subsequently, polyphenolic compounds from R. verniciflua extract were tested to identify the ingredients responsible for the aromatase inhibitory effects by R. verniciflua extract. As a result, butin showed aromatase inhibitory effect in a concentration-dependent manner with an IC50 value of 9.6 lM, whereas the inhibition by other compounds was negligible. These results suggest that R. verniciflua extract could modulate androgen hormone levels via the inhibition of CYP19 activity and butin is a major ingredient responsible for this activity. Ó 2014 Elsevier Ltd. All rights reserved.
The stem bark of Rhus verniciflua Stokes (Anacardiaceae) is a folk herbal medicine used in Asian countries such as Korea, Japan, and China as an invigorant and to treat gastritis, stomach cancer, paralysis, high blood pressure, menstrual cycle irregularities, poor blood circulation, and atherosclerosis.1–3 It has been reported to possess various pharmacological activities, including antioxidant,4–6 antiproliferative,7,8 anti-inflammatory,9 antitumor,10–12 antimutagenic,13 and antirheumatoid arthritis14 effects. With regard to R. verniciflua as a folk remedy invigorant, the effect of R. verniciflua extract on androgen levels has been studied.15,16 According to the report by Seong et al.15 when mouse Leydig cells were treated with R. verniciflua extract, androgen hormone levels were increased. In addition, when rats were orally administered with the flavonoid fraction of R. verniciflua extract, plasma testosterone levels were elevated and the spermatozoon motility and epididymal sperm concentration were significantly ⇑ Corresponding authors. Tel.: +82 31 400 5804; fax: +82 31 400 4783. E-mail addresses: [email protected] (M.-S. Dong), [email protected] (H.H. Yoo). M.H. Park and I.S. Kim contributed equally to this work. http://dx.doi.org/10.1016/j.bmcl.2014.02.039 0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
increased.16 These reports suggest that R. verniciflua extract could enhance the androgen-dependent male sexual function. To characterize the mechanism of such an androgen-stimulating effect by R. verniciflua, we investigated the effect of R. verniciflua extract on the pathways for androgen biosynthesis using an androgen receptor (AR) nuclear translocation assay and an AR binding assay. However, R. verniciflua extract showed minimal effects in those tests (data not shown). Subsequently, we tested the effects on aromatase activity. Aromatase (CYP19) is an enzyme responsible for the conversion of androgens into estrogens. The enzyme is distributed in many tissues including gonads, brain, adipose tissue, placenta, blood vessels, skin, bone, and endometrium.17 Aromatase plays an important role in the regulation of sexual differentiation and reproduction during adult life and is involved in other physiological and behavioral processes, including neuroplasticity, cell growth, migration, and neuroprotection.17 Androgens are converted to estrogens by the aromatase enzyme complex.18,19 Thus, the modulation of aromatase activity may affect the androgen hormone level. Inhibition of aromatase activity would be helpful for androgen level retention. In this study, we investigated the effect of R. verniciflua extract and its polyphenolic ingredients on human aromatase activity to
1731
M. H. Park et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1730–1733
understand the mechanism of elevation of the androgen hormone level and to identify the active compound responsible for this activity. R. verniciflua extract was prepared according to the previous report.16 For aromatase inhibition assay, the incubation mixture consisted of 0.05 mg/mL human CYP 19A1 supersomes (BD Biosciences, Woburn, MA, USA), various concentrations of the test compound, 1 lM androstenedione, 3.3 mM magnesium chloride, and an NADPH-generating system (0.1 M glucose-6-phosphate, 10 mg/mL b-NADP+, and 1 U/mL glucose-6-phosphate dehydrogenase) in a total volume of 1 mL potassium phosphate buffer (0.1 M, pH 7.4). The reaction mixture was incubated for 30 min at 37 °C. After incubation, the reaction was stopped by the addition of 1 mL of ethyl acetate and 10 lL of IS solution (4 lg/mL cortisone). The reaction sample was extracted by liquid–liquid extraction. For liquid–liquid extraction, ethylacetate (4 mL) was added to the reaction sample. The mixture was vortexed for 2 min and centrifuged for 5 min at 13200g. The upper layer (5 mL) was collected and evaporated under nitrogen flow at 40 °C. The residue was reconstituted in 100 lL of methanol and transferred to a HPLC vial for liquid chromatography-–tandem mass spectrometric (LC–MS/MS) analysis. Estrone formation was determined using LC–MS/MS analysis and the details are provided as a Supplementary data. To characterize a chemical profile of the R. verniciflua extract used in this study, the major polyphenolic ingredients of the extract were determined using LC–MS/MS analysis (Supplementary data). Various phenolic compounds such as 2,4-dihydroxybenzoic acid, protocatechuic acid, caffeic acid, chlorogenic acid, p-coumaric acid, phloretin-20 -O-glucoside, kaempferol-3-O-glucoside, quercetin, butein, kaempferol, gallic acid, 2,6,30 ,40 tetrahydroxy-2-benzylcoumaran-3-one, fustin, fisetin, and sulfuretin have been reported as ingredients of R. verniciflua extracts.4,10,20 Based on these
OH OH
O
OH
OH
O
OH
OH
OH
OH
OH
Garbanzol OH
OH
O
OH
O
Taxifolin OH
OH OH
O
OH
OH
OH
OH
OH
OH
O
Dehydrokaempferol
O
H
OH
O
Quercetin
Butin
O
O
Butein
OH
OH
O
OH
OH OH OH
OH
2,4-dihydroxybenzaldehyde
OH
2,4-dihydroxybenzoic acid
3,4-dihydroxybenzoic acid O
OH HO
O
O
Sulfuretin
OH
OH
HO H 3CO
2-methoxybenzene-1,4-diol
OH
OH
OH
HO
Resorcinol
OH OH
O
Fisetin OH
O
OH
OH
O
OH
O
O
Fustin
O
OH
OH
O
OH
literature data and our phytochemical study results, we determined the contents of 15 polyphenolic compounds (Fig. 1) in R. verniciflua extract. The mean contents of those compounds are presented in Table 1. The inhibitory effects of R. verniciflua extract on human aromatase activity were evaluated in cDNA-expressed CYP19 supersomes. In this study, androstenedione was used as a substrate and estrone formation was measured using LC–MS/MS. The assay system was tested with formestane, a well-known CYP19 selective inhibitor. Formestane selectively inhibited estrone formation in a concentration-dependent manner with an IC50 value of 0.47 lM (Fig. 2A). When R. verniciflua extract was tested in a concentration range of 10–1000 lg/mL, R. verniciflua extract exhibited concentrationdependent inhibitory effects on the estrone formation mediated by CYP19 with an IC50 value of 136.2 lg/mL (Fig. 2B). To investigate the ingredients responsible for the aromatase inhibitory effect of R. verniciflua extract, a total of 15 phenolic compounds contained in R. verniciflua extract (Fig. 1) were evaluated for their inhibitory potential against aromatase activity. All of the test compounds were assayed at 10 lM. The resulting data are assembled in Table 1. The positive control (formestane) inhibited estrone formation by 94.9% at 10 lM. Among the compounds tested, butin showed a significant inhibitory activity (by 50%) on estrone formation but other compounds exhibited minimal effects. When butin was tested at various concentrations, it inhibited estrone formation in a concentration-dependent manner with an IC50 value of 9.6 lM (Fig. 2C). In the previous study by Na et al. R. verniciflua extract elevated the plasma testosterone in male rats.16 In addition, in the recent clinical study with R. verniciflua extract, the extract has been found to increase the testosterone level in middle-aged men. When R. verniciflua extract was administered for 8 weeks, total testosterone and free testosterone levels were significantly increased in subjects
OH
OH
Garllic acid
Figure 1. Chemical structures of polyphenolic compounds from R. verniciflua extract.
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Table 1 Content and aromatase inhibitory activity of polyphenolic compounds in R. verniciflua extract
a
Compounds
Contents (%)
Estrone formationa (% of control)
Fustin Fisetin Gallic acid Butin Butein Sulfuretin Resorcinol Garbanzol Quercetin Taxifolin Dehydrokaempferol 2,4-Dihydroxybenzaldehyde 2,4-Dihydroxybenzoic acid 3,4-Dihydroxybenzoic acid 4-Hydroxybenzoic acid Formestane (positive control)
12.1 6.7 3.4 0.1 0.5 0.4 0.9 0.7 0.1 1.0 0.1 0.3 0.07 0.09 0.04 —
109.9 117.3 112.2 50.0 87.5 99.0 91.8 99.3 96.0 90.7 101.7 91.7 93.9 83.5 91.8 5.1
All compounds tested were assayed at 10 lM and the data shown represent the mean of duplicate experiments.
Estrone formation (%)
120
(A)
120
(B)
120
100
100
100
80
80
80
60
60
60
40
40
40
20
20
20
0
0
0.01
0.10
1.00
Formastane (μM)
10.00
(C)
0 10
100
1000
R. verniciflua extract (μg/mL)
1
10
100
Butin (μM)
Figure 2. Effects of R. verniciflua extract and butin on estrone formation in CYP19 supersomes. (A) Formestane (positive control); (B) R. verniciflua extract; (C) Butin.
(unpublished data). The present results may support the enhancing effects of R. verniciflua extract for androgen hormones and provide its possible mechanism. Thus, R. verniciflua may modulate androgen hormone level by inhibiting the conversion of androgens to estrogens via the inhibition of CYP19 activity. R. verniciflua extract showed a concentration-dependent inhibitory activity on aromatase but its potency was relatively weak. Our results indicated that butin is a major component responsible for its aromatase inhibitory activity and the weak activity of the extract may be due to low content of butin in the extract. According to previously reported papers, butin has a reactive oxygen species scavenging or antioxidant activities.21,22 As for hormone-related activities, butin has been reported to exhibit an uterotrophic effect as a weak estrogen.23 This is thought to be due to a structural similarity between butin and sex hormones. In this context, butin may react as a competitive inhibitor on human aromatase. Considering that the other flavonoid ingredients lack the inhibitory effect, the absence of a substituent at the C3 position is supposed to be important for an aromatase inhibitory activity. This is accordance with the previous reported data by Jeong et al.24; Among the 28 flavonoids tested, several flavonoids such as apigenin, chrysin, hesperetin, and naringin exhibited inhibitory effects on aromatase. All of these active flavonoids do not have a substituent at the C3 position. In conclusion, the present study demonstrates that R. verniciflua extract inhibited a conversion of androstenedione to estrone mediated by human aromatse and butin is a major ingredient responsible for this activity. This result suggests that R. verniciflua extract could modulate androgen hormone levels via the inhibition of CYP19 activity. In addition, butin could be a lead compound for development of aromatase inhibitors.
Acknowledgments This research was supported in part by Industrialization Support Program for Bio-technology of Agriculture and Forestry, Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea (810009-03-2-SB250) and in part by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2011-0012319). Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2014. 02.039. References and notes 1. Hong, D. H.; Han, S. B.; Lee, C. W.; Park, S. H.; Jeon, Y. J.; Kim, M.-J.; Kwak, S.-S.; Kim, H. M. Arch. Pharmacol. Res. 1999, 22, 638. 2. Kang, D. G.; Lee, A. S.; Mun, Y. J.; Woo, W. H.; Kim, Y. C.; Sohn, E. J.; Moon, M. K.; Lee, H. S. Biol. Pharm. Bull. 2004, 27, 366. 3. Kim, J. H.; Go, H. Y.; Jin, D. H.; Kim, H.-P.; Hong, M. H.; Chung, W.-Y.; Park, J.-H.; Jang, J. B.; Jung, H.; Shin, Y. C. Cancer Lett. 2008, 265, 197. 4. Lim, K.-T.; Hu, C.; Kitts, D. Food Chem. Toxicol. 2001, 39, 229. 5. Jung, C. H.; Jun, C.-Y.; Lee, S.; Park, C.-H.; Cho, K.; Ko, S.-G. Biol. Pharm. Bull. 2006, 29, 1603. 6. Kim, J.; Kim, H.; Jung, C.; Hong, M.; Hong, M.; Bae, H.; Lee, S.; Park, S.; Park, J.; Ko, S. Int. J. Mol. Med. 2006, 18, 201. 7. Lim, K.-T. J. Toxicol. Public Health 1999, 15, 169. 8. Kitts, D. D.; Lim, K.-T. J. Toxicol. Environ. Health, A 2001, 64, 357. 9. Lee, J.; Huh, J.; Jeon, G.; Yang, H.; Woo, H.; Choi, D.; Park, D. Int. Immunopharmacol. 2009, 9, 268. 10. Lee, J.-C.; Lim, K.-T.; Jang, Y.-S. Bba-Gen. Subj. 2002, 1570, 181.
M. H. Park et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1730–1733 11. Lee, J. C.; Lee, K. Y.; Kim, J.; Na, C. S.; Jung, N.; Chung, G. H.; Jang, Y. S. Food Chem. Toxicol. 2004, 42, 1383. 12. Jang, H.-S.; Kook, S.-H.; Son, Y.-O.; Kim, J.-G.; Jeon, Y.-M.; Jang, Y.-S.; Choi, K.-C.; Kim, J.; Han, S.-K.; Lee, K.-Y. Bba-Gen. Subj. 2005, 1726, 309. 13. Park, K.-Y.; Jung, G.-O.; Lee, K.-T.; Jongwon, C.; Choi, M.-Y.; Kim, G.-T.; Jung, H.J.; Park, H.-J. J. Ethnopharmacol. 2004, 90, 73. 14. Choi, J.; Yoon, B.-J.; Han, Y. N.; Lee, K.-T.; Ha, J.; Jung, H.-J.; Park, H.-J. Planta Med. 2003, 69, 899. 15. Seong, H.; Choi, S.; Jang, Y.; Min, K.; Woo, J.; Jang, W.; NC, J.; Na, C.; Jung, I. Korean J. Anim. Rep. 2001, 25, 125. 16. Na, C. S.; Choi, B. R.; Choo, D. W.; Choi, W. I.; Kim, J. B.; Kim, H. C.; Park, Y. I.; Dong, M. S. J. Toxicol. Public Health 2005, 21, 309. 17. Di Nardo, G.; Gilardi, G. Biotechnol. Appl. Biochem. 2013, 60, 92.
1733
18. Simpson, E. R.; Mahendroo, M. S.; Means, G. D.; Kilgore, M. W.; Hinshelwood, M. M.; Graham-Lorence, S.; Amarneh, B.; Ito, Y.; Fisher, C. R.; Michael, M. D. Endocr. Rev. 1994, 15, 342. 19. Kragie, L. Endocr. Res. 2002, 28, 121. 20. Kim, S.-A.; Kim, S. H.; Kim, I. S.; Lee, D.; Dong, M.-S.; Na, C.-S.; Nhiem, N. X.; Yoo, H. H. Food Chem. 2013, 141, 3813. 21. Zhang, R.; Chae, S.; Kang, K. A.; Piao, M. J.; Ko, D. O.; Wang, Z. H.; Park, D. B.; Park, J. W.; You, H. J.; Hyun, J. W. Mol. Cell. Biochem. 2008, 318, 33. 22. Zhang, R.; Kang, K. A.; Piao, M. J.; Chang, W. Y.; Maeng, Y. H.; Chae, S.; Lee, I. K.; Kim, B. J.; Hyun, J. W. Food Chem. Toxicol. 2010, 48, 922. 23. Bhargava, S. J. Ethnopharmacol. 1986, 18, 95. 24. Jeong, H. J.; Shin, Y. G.; Kim, I. H.; Pezzuto, J. M. Arch. Pharmacol. Res. 1999, 22, 309.