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eCAM Advance Access originally published online on April 20, 2005
eCAM 2005 2(2):223-226; doi:10.1093/ecam/neh086
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Brief Communication

Isolation of CYP3A4 Inhibitors from the Black Cohosh (Cimicifuga racemosa)

Sachiko Tsukamoto, Maki Aburatani and Tomihisa Ohta*

Graduate School of Natural Science and Technology, Kanazawa University Kakuma-machi, Kanazawa 920-1192, Japan

*For reprints and all correspondence: Tomihisa Ohta, Graduate School of Natural Science and Technology, Kanazawa University, Kakuma-machi, Kanazawa, 920-1192 Japan. Tel: +81-76-234-4468; Fax: +81-76-264-6241; E-mail: ohta{at}p.kanazawa-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Recent investigation on drug interaction has shown that some foods and herbal medicines increase the oral availability of a variety of CYP3A4 substrates, which is caused by the reduction of CYP3A4 in intestinal epithelium. During the course of our investigation on CYP3A4 interaction, we found that the commercially available dietary supplement made from black cohosh (Cimicifuga racemosa) showed CYP3A4 inhibition. Black cohosh has been used for the treatment of menopausal and post-menopausal symptoms as a dietary supplement. Bioassay-guided isolation from the supplement afforded six active principles, which were identified as cycloartanoid triterpene glycosides.

Keywords: black cohosh – Cimicifuga racemosa – CYP3A4 – cytochrome P450 – inhibition – triterpene glycoside


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
The rhizomes of Cimicifuga species (Ranunculaceae) are used as Traditional Chinese Medicines. Among Cimicifuga species, C. heracleifolia, C. dahurica and C. foetida are listed in the Chinese Pharmacopoeia and used as an anti-inflammatory, antipyretic and analgesic remedy (1). Cimicifuga racemosa, commonly known as black cohosh, is a herb used among Native Americans to treat a variety of ailments, including diarrhea, sore throat and rheumatism. It is best known for its health benefit in treating menopausal disorders (2). It is used therapeutically to reduce the frequency and intensity of hot flushes (3), and in the USA the extract of C. racemosa is available for sale as a dietary supplement used for the treatment of menopausal and post-menopausal symptoms (4). Cytochrome P450 (CYP) enzymes belong to heme-containing monooxygenases and constitute three families, CYP1, CYP2 and CYP3 (5), and these enzymes are recognized to be responsible for drug metabolism, carcinogenesis and degradation of xenobiotics. Among a family of CYP enzymes, CYP3A4 is the most abundant enzyme in human liver microsomes and intestinal epithelium; ~30% of the total CYP was suggested to be CYP3A4 (6), and >50% of clinically used drugs are oxidized by CYP3A4 (7,8). It is widely known that concomitant oral administration of several foods and herbs affects drug metabolism in humans by inhibiting CYP3A4 activity. Among them, the inhibition by grapefruit juice has been well studied and it is reported that concomitant intake of the juice alters the pharmacokinetics of various drugs, including cyclosporin (9,10), midazolam (11), dihydropyridine-type calcium channel blockers (12) and triazolam (13). In the course of our study on CYP inhibitors from foods, we have reported the isolation and structural elucidation of CYP inhibitors from grapefruit (Citrus paradisii) juice (14–16), white pepper, Piper nigrum (17,18) and the strawberry fruit, Fragaria ananassa (19). Recently, we found that the commercially available black cohosh, C. racemosa Nutt, showed CYP3A4 inhibition. Here we report the isolation, structural identification and CYP inhibitory activity of constituents from black cohosh.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
General Methods

Nuclear magnetic resonance (NMR) spectra were recorded on a JEOL GSX-500 NMR spectrometer in pyridine-d5. Mass spectra were measured on a JEOL SX-102 mass spectrometer.

Isolation of Triterpene Glycosides 1–6 from C. racemosa

Commercially available black cohosh, C. racemosa Nutt, was generously provided by from Tokiwa Phytochemical Co., Ltd. (Japan). The powder of black cohosh (20 g) was suspended in water and extracted with ethyl acetate (EtOAc). The organic layer was then partitioned between hexane and 90% methanol (MeOH)-H2O. The polar fraction (6.1 g), which exhibited significant CYP3A4 inhibition, 44% at 5 mg/ml, was subjected to silica gel chromatography with EtOAc/MeOH followed by octadecyl silica gel (ODS) chromatography with MeOH/H2O and then acetonitrile (CH3CN)/H2O to afford a potent CYP3A4 inhibitory fraction. The purification by ODS high-performance liquid chromatography (HPLC) with CH3CN/H2O gave six triterpene glycosides 1–6. On the basis of 1H NMR spectra in C5D5N and fast atom bombardment mass spectrometry (FABMS), the six triterpene glycosides were identified as cimiracemoside H (1, 2.0 mg, 0.01% yield) (19), 26-deoxyactein (2, 1.1 mg, 0.0055%) (20,21), 23-O-acetylshengmanol 3-O-ß-D-xylopyranoside (3, 1.9 mg, 0.0095%) (22), actaeaepoxide 3-O-ß-D-xylopyranoside (4, 1.1 mg, 0.0055%) (23), 25-O-acetylcimigenol 3-O-{alpha}-L-arabinopyranoside (5, 5.9 mg, 0.029%) (24) and 25-O-acetylcimigenol 3-O-ß-D-xylopyranoside (6, 4.2 mg, 0.021%) (25,26) (Figure 1).



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  		Figure 1 Structures of CYP inhibitors isolated from C. racemosa

 
Assay of CYP3A4 Inhibition

CYP3A4 activity was based on nifedipine oxidation. Various amounts (0–10 µM, final concentration) of samples in 1 ml of dimethylsulfoxide (DMSO) were added to 192 µl of solution containing 100 mM phosphate buffer (pH 7.4) containing 50 µM nifedipine (Wako Pure Chemical Industries, Ltd, Osaka, Japan), 5 mM glucose-6-phosphate (Oriental Yeast Co., Ltd, Tokyo, Japan), 0.5 mM b-NADP+ (Oriental Yeast Co., Ltd), 0.5 mM MgCl2 and 4.3 µg/ml glucose-6-phosphate dehydrogenase (Oriental Yeast Co., Ltd) and incubated at 37°C for 5 min. CYP3A4 (Gentest Co., Woburn, MA) was also pre-incubated in 7 µl of the buffer at 37°C for 5 min and added to the sample solution. After the incubation at 37°C for 1 h, the reaction was quenched by the addition of 100 ml of MeOH. After adding 3.7 µg of 6-methoxycarbonyl-5-methyl-7-(2-nitrophenyl)-4,7-dihydrofuro[3,4-b]pyridin-1-(3H)-one in 1 µl of DMSO as an internal standard, the reaction mixture was extracted with 1 ml of ether, and the ether layer was evaporated. The residue was dissolved in 100 µl of MeOH, and an aliquot (20 µl) was analyzed by reverse-phase HPLC (column, TSK-gel ODS-120T, 4.6 mm i.d. x 150 mm; mobile phase, 64% MeOH-H2O; flow rate, 1.0 ml/min; detection, UV 254 nm); retention times: 2.9 min for the internal standard, 4.0 min for the nifedipine metabolite (nifedipine pyridine) and 5.5 min for nifedipine. The IC50 value, the concentration required for 50% inhibition of CYP3A4 activity, was calculated from the data of duplicate measurements.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Bioassay-guided Isolation of Triterpene Glycosides 1–6 from C. racemosa

The CYP inhibition was tested using human CYP3A4 as shown in Methods. Commercially available supplement of C. racemosa was extracted with EtOAc, and the organic layer was then partitioned between hexane and 90% MeOH-H2O. The polar fraction exhibited significant CYP inhibition. Forty-four percent inhibition at 5 mg/ml was as potent as that by ketoconazole, 58% at 5 mg/ml. Bioassay-guided fractionation afforded six triterpene glycosides 1–6. On the basis of their 1H NMR spectra in C5D5N and FABMS, they were identified as cimiracemoside H (1, 0.01% yield) (20), 26-deoxyactein (2, 0.0055%) (21,22), 23-O-acetylshengmanol 3-O-b-d-xylopyranoside (3, 0.095%) (23), actaeaepoxide 3-O-ß-D-xylopyranoside (4, 0.0055%) (24), 25-O-acetylcimigenol 3-O-{alpha}-L-arabinopyranoside (5, 0.0029%) (25) and 25-O-acetylcimigenol 3-O-ß-D-xylopyranoside (6, 0.0021%) (26,27). The CYP inhibitory activities of 1–6 are shown in Table 1, which indicates moderate IC50 values.


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  		Table 1 CYP3A4 inhibition of C. racemosa metabolites

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
Bailey et al. demonstrated that the blood concentration of anti-hypertensive drugs such as nifedipine was kept at a high level with grapefruit juice co-administered to mask the taste of alcohol. Since that time there has been a great deal of interest in food–drug interactions because of the clinical risks associated with changes in the bioavailability or metabolic rate of clinically administered drugs. In spite of the increasing use of herbal remedies and nutraceuticals, information on the drug interaction that occurs with them including foodstuffs, is still insufficient to understand the clinical risks.

We have studied drug interactions with foodstuffs such as grapefruit juice and pepper, isolating several potent CYP3A4 inhibitors in each material. The fact that a foodstuff contains more than one inhibitor suggests that the clinical effect of drug interaction with herbs and foodstuffs could be better understood by studying the mixture of inhibitors and/or an extract of these.

During our study on CYP3A4 inhibitors contained in food, we found that the supplement of C. racemosa exhibited potent inhibition. The polar fraction from the extract showed 44% inhibition at 5 mg/ml, which was as potent as the inhibition produced by ketoconazole, 58% at 5 µg/ml. We clarified the main constituents, cognate triterpene glycosides, as the CYP3A4 inhibitory principles. To date, highly potent CYP3A4 inhibitors in food have been reported, e.g. paradisins A and B (IC50, 0.07 and 0.07 µM) from grapefruit juice (15), dipiperamide A (IC50, 0.18 µM) from white pepper (17) and gomisin C (IC50, 0.254 µM) from Schisandra fruit (29). Although the IC50 value of each isolate was moderate, the high content of a series of cognates in the supplement could result in the exhibition of significant CYP inhibition. Care should be taken to avoid concomitant intake of black cohosh with any kind of medication because of the possibility of increasing the bioavailable concentration of drugs in the blood by the downregulation suppression of CYP3A4. Medical practitioners therefore should pay more attention in order to avoid any trouble with diet containing black cohosh.

The IC50, 0.027 mg/ml, of the extract of black cohosh was very low in spite of the weak activities of the isolated compounds. The IC50 value indicates that 40 mg of the black cohosh extract could be estimated to inhibit the metabolism of roughly half a dose of nifedipine for 1 day.

Evaluation of the IC50 of both constituents and the extract may be useful for clinical study of drug interactions of herbs and foodstuffs predicted to be a clinical risk, especially for the study of the mechanism of action and the pharmacokinetics.


   Acknowledgments
 
The authors are grateful to Tokiwa Phytochemical Co., Ltd (Japan) for the generous gift of black cohosh.


   References
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. New Medicinal College of Jiangsu (ed.). Dictionary of Chinese Materia Medica 1977;Shanghai Shanghai Scientific and Technological Press 451
  2. Lieberman S. A review of the effectiveness of Cimicifuga racemosa (black cohosh) for the symptoms of menopause J Women's Health 1998; 7: 525–29[Web of Science][Medline]
  3. McKenna D, Jones K, Humphrey S, Hughes K. Black cohosh: efficacy, safety, and use in clinical and preclinical applications Altern Ther 2001; 7: 93–100
  4. Borrelli F, Izzo AA, Ernst E. Pharmacological effects of Cimicifuga racemosa Life Sci 2003; 73: 1215–29[CrossRef][Web of Science][Medline]
  5. Nelson DR, Koymans L, Kamataki T, Stegeman JJ, Feyereisen R, Waxman DJ, Waterman MR, Gotoh O, Coon MJ, Estabrook RW, Gunsalus IC, Nebert DW. P450 superfamily: update on new sequences, gene mapping, accession numbers and nomenclature Pharmacogenetics 1996; 6: 1–42[Web of Science][Medline]
  6. Shimada T, Yamazaki H, Mimura M, Inui Y, Guengerish FP. Interindividual variations in human liver cytochrome P-450 enzymes involved in the oxidation of drugs, carcinogens and toxic chemicals: studies with liver microsomes of 30 Japanese and 30 Caucasians J Pharmacol Exp Ther 1994; 270: 414–23[Abstract/Free Full Text]
  7. Guengerish FP. Role of cytochrome P450 enzymes in drug–drug interactions Adv Pharmacol 1997; 43: 7–35
  8. Rendic S and DiCarlo FJ. Human cytochrom P450 enzymes: a status report summarizing their reactions, substrates, inducers, and inhibitors Drug Metab. Rev. 1997; 29: 413–580[Web of Science][Medline]
  9. Ducharme MP, Warbasse LH, Edwards DJ. Disposition of intravenous and oral cyclosporine after administration with grapefruit juice Clin Pharmacol Ther 1995; 57: 485–91[CrossRef][Web of Science][Medline]
  10. Yee GC, Stanley DL, Pessa LJ, Dalla CT, Beltz SE, Ruiz J. Effect of grapefruit juice on blood cyclosporin concentration Lancet 1995; 345: 955–56[CrossRef][Web of Science][Medline]
  11. Kupferschmidt HH, Ha HR, Ziegler WH, Meier PJ, Krahenbuhl S. Interaction between grapefruit juice and midazolam in humans Clin Pharmacol Ther 1995; 58: 20–8[CrossRef][Web of Science][Medline]
  12. Bailey DG, Arnold JM, Spence JD. Grapefruit juice and drugs. How significant is the interaction? Clin Pharmacokinet 1994; 26: 91–8[Web of Science][Medline]
  13. Hukkinen SK, Varhe A, Olkkola KT, Neuvonen PJ. Plasma concentrations of triazolam are increased by concomitant ingestion of grapefruit juice Clin Pharmacol Ther 1995; 58: 127–31[CrossRef][Web of Science][Medline]
  14. Ohta T, Nagahashi M, Hosoi S, Tsukamoto S. Dihydroxybergamottin caproate as a potent and stable CYP3A4 inhibitor Bioorg Med Chem 2002; 10: 969–73[CrossRef][Medline]
  15. Ohta T, Maruyama T, Nagahashi K, Miyamoto Y, Hosoi S, Kiuchi F, Yamazoe Y, Tsukamoto S. Paradisin C: a new CYP3A4 inhibitor from grapefruit Juice Tetrahedron 2002; 58: 6631–35[CrossRef]
  16. Ohta T, Miyamoto Y, Maruyama T, Kiuchi F, Tsukamoto S. Localization and contents of paradisins, the most potent CYP3A4 inhibitors, in a grapefruit Citrus paradisii and grapefruit Juice Nat Med 2002; 56: 264–7
  17. Tsukamoto S, Cha B-C, Ohta T. Dipiperamides A, B, and C: bisalkaloids from the white pepper Piper nigrum inhibiting CYP3A4 activity Tetrahedron 2002; 58: 1667–71[CrossRef]
  18. Tsukamoto S, Tomise K, Miyakawa K, Cha B-C, Abe T, Hamada Y, Hirota H, Ohta T. CYP3A4 inhibitory activity of new bisalkaloids dipiperamides D and E and cognate alkaloids from the white pepper Bioorg Med Chem 2002; 10: 2981–85[CrossRef][Medline]
  19. Tsukamoto S, Tomise K, Aburatani M, Onuki H, Hirota H, Ishiharajima E, Ohta T. Isolation of cytochrome P450 inhibitors from strawberry fruit, Fragaria ananassa Duch. cv. Tochiotome J Nat Prod 2004; 67: pp. 1839–41[CrossRef][Medline]
  20. Shao Y, Harris A, Wang M, Zhang H, Cordell GA, Bowman M, Lemmo E. Triterpene glucosides form Cimicifuga racemosa J Nat Prod 2000; 63: 905–10[CrossRef][Medline]
  21. Koeda M, Aoki Y, Sakurai N, Nagai M. Studies on the Chinese crude drug ‘Shoma.’ IX. Three novel cycloanostanol xylosides, cimicifugosides H-1, H-2 and H-5, form Cimicifuga Rhizome Chem Pharm Bull 1995; 43: 771–6
  22. Chen S-N, Li W, Fabricant DS, Santarsiero BD, Mesecar A, Fitzloff JF, Fong HHS, Farnsworth NR. Isolation, structure elucidation, and absolute configuration of 26-deoxyactein from Cimicifuga racemosa and clarification of nomenclature associated with 27-deoxyactein J Nat Prod 2002; 65: 601–5[CrossRef][Medline]
  23. Kusano A, Shibano M, Kitagawa S, Kusano G, Nozoe S, Fushiya S. Studies on the constituents of Cimicifuga species. XV. Two new diglycosides from the aerial parts of Cimicifuga simplex WORMSK Chem. Pharm. Bull. 1994; 42: 1940–43
  24. Wende K, Mugge C, Thurow K, Schopke T, Lindequist U. Actaeaepoxide 3-O-b-d-xylopyranoside, a new cycloartane glycoside from the rhizomes of Actaea racemosa (Cimicifuga racemosa) J Nat Prod 2001; 64: 986–9[CrossRef][Medline]
  25. Ye W, Zhang J, Che C-T, Ye T, Zhao S. New cycloartane glycosides from Cimicifuga dahurica Planta Med 1999; 65: 770–1[Medline]
  26. Kadota S, Li JX, Tanaka K, Namba T. Constituents of Cimicifugae rhizoma II. Isolation and Strucures of new cycloartenol triterpenoids and related compounds from Cimicifuga foetida L. Tetrahedron 1995; 51: 1143–66[CrossRef]
  27. Takemoto T, Kusano G, Kawahara M. Studies on the constituents of Cimicifuga spp. VI. Structures of 25-O-acetylcimigenoside and 25-O-methylcimigenoside Yakugaku Zasshi 1970; 90: 64–7[Medline]
  28. ‘The Pharmacopoeia of Chinese People's Republic’ vol. I 1990;Beijing The People's Health Publishing House & The Chemical Industry Publishing House 59
  29. Iwata H, Tezuka Y, Kadota S, Hiratsuka A, Watabe T. Identification and characterization of potent CYP3A4 inhibitors in Schisandra fruit extract Drug Metab Dispos 2004; 32: 1351–8[Abstract/Free Full Text]
Received September 30, 2004; Revision received January 18, 2005. accepted March 28, 2005


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No characterisation of the extract and questionable in-vivo correlation.
Beat Meier, et al.
Evidence-based Complementary and Alternative Medicine, 12 Aug 2005 [Full text]
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