Life Sciences 73 (2003) 1215 – 1229 www.elsevier.com/locate/lifescie
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Pharmacological effects of Cimicifuga racemosa F. Borrelli a,*, A.A. Izzo a, E. Ernst b a
Department of Experimental Pharmacology, University of Naples ‘‘Federico II’’, Via D. Montesano 49, 80131 Naples, Italy b Department of Complementary Medicine, School of Postgraduate Medicine and Health Studies, University of Exeter, EX2 4NT, UK Received 24 June 2002; accepted 12 February 2003
Abstract Cimicifuga racemosa is widely employed to relieve menopause symptoms for its hormonal-like action. However, recent experimental studies have not found an estrogenic action by this plant. The purpose of this systematic review is to analyse all experimental studies (in vivo and in vitro) performed on C. racemosa to elucidate its mechanism of action. Animal and in vitro experiments on C. racemosa were identified through a computerised literature searches performed on Medline (PubMed), Embase, Amed, CISCOM and Cochrane Library databases. In addition, bibliographies of the articles thus located were scanned for further relevant publications and manufactures of commercial C. racemosa preparations were asked to contribute published and unpublished material. No language restrictions were imposed. A total of 15 animal and 15 in vitro studies on C. racemosa have been found. Their results suggest that C. racemosa possesses a central activity instead of a hormonal effect. Further biological and chemical investigations are required to define its mechanism of action and to identify the compounds responsible of its actions. D 2003 Elsevier Science Inc. All rights reserved. Keywords: Herbal medicine; Cimicifuga racemosa; Black cohosh; Climacteric disorders; Mechanism of action; Menopausal symptoms; Sex hormones
Introduction Climacteric is the period of passage from reproductive stage of life of women to the non-productive state. It encompasses premenopause, perimenopause (menopause) and postmenopause phases. Climac-
* Corresponding author. Tel.: +39-081-678436; fax: +39-081-678403. E-mail address:
[email protected] (F. Borrelli). 0024-3205/03/$ - see front matter D 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S0024-3205(03)00378-3
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teric is characterised by important hormonal changes. In particular, the ovaries stop producing oestrogen and progesterone and there is an increased production of gonadotropin hormones such as luteinizing hormone (LH) and follicle stimulating hormone (FSH) (Fig. 1). These hormonal changes can cause a
Fig. 1. Anterior pituitary-releasing hormones and their action (above). Gonadotropin-releasing hormone (GnRH) produced by the hypothalamus, regulates the synthesis and release of gonadotropic hormones (LH = Luteinizing hormone; FSH = Folliclestimulating hormone) from the anterior pituitary which in turn stimulate the release of oestrogen and progesterone. Hormonal changes during climacteric period (below). Increase of LH and FSH with a concomitant dramatically stop production of oestrogen and progesterone by ovaries.
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Fig. 2. Cimicifuga racemosa.
wide variety of vasomotor, vaginal and psychological symptoms and diseases including hot flushes, tissue atrophy, sexual dysfunction, impaired sleep and emotional disturbances. Accelerated bone demineralisation leading to osteoporosis, acceleration of the rate of development of coronary heart disease and cardiovascular fatality can also occur. Currently, hormone replacement therapy (HRT) is commonly used to combat the symptoms and diseases associated with falling oestrogen and progesterone levels. However, HRT is associated with adverse effects and an increased risk of endometrial or breast cancer. Many women are therefore against HRT (Whitehead et al., 1981; Bergkvist and Persson, 1996) and choose to employ herbal remedies for menopausal symptoms. Cimicifuga racemosa or black cohosh (Ranunculaceae; Fig. 2), a herbaceous perennial plant native to North America, is widely employed to Table 1 Terms used for computer searches Cimicifuga racemosa (botanical name) Actaea racemosa (first botanical name) Black cohosh (common English name) Rattleweed (common English name)
Black snakeroot (common English Bugbane (common English Rattleroot (common English Rattletop (common English
name) name) name) name)
Traubensilberkerze (common German name) Wanzenkraut (common German name) Cimicifuga (common Italian name) Remifemin (tradename)
Actein (active ingredient) Cimicifugoside (active ingredient) Cimicifugic acid (active ingredient) Fukinolic acid (active ingredient)
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Table 2 Animal experiments to investigate the mechanism of action of Cimicifuga racemosa Author(s), year
Animals (length of treatment)
Extract(s), dosage, (administration)
Test(s) used
Main result
Comments
Mercier and Balansard, 1935
Dogs
Cardiac function
Rats of different ages, Immature mice
A glycoside fraction insoluble in water possessed cardiotoxic activity CR increased uterine and ovary weight
This fraction induced arterial hypotension, bradycardia and death
Gizicky, 1944
Foldes, 1959
Mice (4 days) Rats (3 days)
4 glycosidic fractions, 0.02 – 0.03 g/kg (iv) 50% water/alcohol 1 ml/rat 0.25 ml/mouse (sc) Alcoholic (RemifeminR) 0.2 ml/die/mouse 0.2 – 0.6 ml/die/rat (ND)
Siess and Immature mice Seybold, 1960 (52 days)
Alcoholic (RemifeminR) 0.05 ml/die/mouse (os or sc) Ovariectomized Dichloromethane Jarry and Harnischfeger, rats (3 – 3.5 days) and Ethanol fractions 108 1985 mg/rat (ip or os) Jarry et al., Ovariectomized Chloroform fraction 1985 rats (3 – 3.5 days) isolated from a methanol extract. 108 mg/rat (ip) Duker et al., Ovariectomized CHCl3 140 mg/rat 1991 rats (3 days) (i.p.) Einer Jensen Ovariectomized 50% water/ethanol et al., 1996 rats Mice (3 days) 6 – 600 mg/kg (os and sc) Eagon et al., Ovariectomized CR root. ND 1997 rats (3 weeks) (added to rat diet)
Eagon et al., 1999
Ovariectomized rats
CR, ND
Jarry et al., 1999
Ovariectomized rats (7 days)
Dichloromethane 60 mg/rat (sc)
Uterine weight, ovary weight
Effect was related to age of animals
Uterine weight, induction of oestrus, sedative effect, changes in ovaries and thyroid function Uterine weight, induction of oestrus
The extract increased No differences were observed between the the uterine weight and induced oestrus other parameters in control and treated groups
FSH levels, LH levels, prolactin levels
NC Dichloromethane fraction (ip) reduced LH levels
LH levels
CHCl3 fraction reduced LH levels
LH levels
CHCl3 reduced LH levels CR did not show estrogenic effects
Vaginal cornification, uterine weight Uterine weight, c-myc expression, serum CP levels and hepatic CP mRNA levels Uterine weight, LH levels LH levels, uterine weight and ERa,IgF1,C3, collagenI, osteocalcin expression
No effect was observed
CR root increased uterine weight and CP levels
CR increased uterine weight and decreased LH levels CR either reduced LH levels, Collagen1 and Osteocalcin expression or increased ERa expression
NC
Formononetin, a constituent of the extract, did not reduce LH levels NC Estradiobenzoate increased uterine weight in mice NC
No information reported about dose and type of extract Similar effects were observed with estradiol. Estradiol increased IgF1 and C3 expression
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Table 2 (continued ) Author(s), year
Animals (length of treatment)
Extract(s), dosage, (administration)
Test(s) used
Main result
Comments
Lohning et al., 1999
Mice (1 hour)
ND 25 – 100 mg/kg (os)
Body temperature, Ketamine-induced sleeping. [Central activity (D2)] Number and size of tumour, FSH, LH, prolactin levels, organ weights
CR decreased body temperature and prolongated sleeping time CR did not show oestrogen effects
Effects comparable to a D2 agonist. Effects inhibited by D2 antagonist Mestranol showed oestrogen effects
PYR and DPD levels Femoral density Days of oestrus Uterine and ovary weight
CR reduced PYR/DPD levels and bone loss CR increased the days of oestrus
Effects comparable to raloxifene
Freudenstein et al., 2000
Ovariectomized rats with ER-positive mammary gland tumour (7 weeks) Ovariectomized Nisslein and Freudenstein, rats (>5 weeks) 2000 Immature female Liu et al., 2001b mice (14 days)
Isopropanolic 1 – 100 human therapeutic dosage (os) Isopropanolic extract ND CR 2.5% 75 – 300 mg/kg (os)
Effect less than estradiol
CR, Cimicifuga racemosa; ER, oestrogen receptor; PR, progesterone receptor; LH, luteinising hormone; FSH, folliclestimulating hormone; PYR, pyridinoline; DPD, deoxypyridinoline; CP, ceruloplasmin; PS, prolactin secretion; ND, not described; NC, no comment.
alleviate perimenopausal and postmenopausal symptoms, including hot flushes, profuse sweating and sleep disturbances (Murray and Pizzorno, 1999; Kass-Annese, 2000). An ethanolic extract of the rhizome of this plant standardised to contain 1 mg of triterpenes calculated as 27-deoxyacteine per 20 mg tablet (trade name: REMIFEMINR) is commercially available. This plant, known alternatively as Actaea racemosa, black snakeroot, bugbane, rattleroot, rattletop and rattleweed, has also shown a diuretic, antidiarrhoeal, anti-inflammatory activity (Murray and Pizzorno, 1999; Foster, 1999; Mckenna et al., 2001). A recent systematic review of the clinical effectiveness of C. racemosa has produced unconvincing results (Borrelli and Ernst, 2002). The aim of this review is to summarise all experimental studies of C. racemosa in order to critically evaluate the existing data about the plant’s mechanism of action.
Methods Computerised literature searches were performed to identify all animals and in vitro experiments carried out with C. racemosa. Databases included Medline, Embase, Amed, Phytobase, PubMed, CISCOM (Research Council for Complementary Medicine, London) and Cochrane Library (all from their respective inception to December 2001). In addition, several (n = 3) manufacturers of C. racemosa preparations were asked to contribute published and unpublished material and our own files were hand searched. Bibliographies of the articles thus located were scanned for further relevant publications. No language restrictions were imposed. The search terms used are shown in Table 1. All animal and in vitro experiments performed with C. racemosa for any topics were included. Studies of this plant in combination with other substances were excluded. The data were validated and extracted according to predefined criteria and is reported in narrative and tabular form
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Table 3 In vitro experiments to investigate the mechanism of action of Cimicifuga racemosa Author(s), year
Cell line(s)
Jarry et al., 1985
Uterine and pituitary Chloroform fraction cells obtained from isolated from a ovariectomized rats methanol extract 0.02% Uterine cells CHCl3 Fractions obtained from of the CHCl3 ovariectomized rats extract ND
Duker et al., 1991
Extract(s), concentration(s)
Test(s) used
Result
Comments
ER binding assay
The fraction bound to ER
NC
ER binding assay
Whole extract did not bind to ER. Some fractions bound to ER CR inhibited cell proliferation at concentration >2.5 Ag/ml
NC
Nesselhut et al., 1993
435 E2 positive breast cancer cell line
ND 0.025 – 25 Ag/ml
Cell proliferation
Jarry et al., 1996
Porcine: granulosa (GC) and luteal (LC) cells Human: granulosa (GC) cells
Commercial extract of CR 0.01 – 1 mg
OPS
Harnischfeger and Cillien, 1996
Fibroblasts FTDE cells Carcinoma AN3 and MX1 cells
CHCl3 and ER binding assay, butanolic subfraction cell proliferation of an alcoholic extract 1 – 5 Ag/ml; >10 Ag/ml
Eagon et al., 1996
ER from lives of mature ovariestomized rats
Full-strength extract obtained from root of CR ND
ER binding assay
Zava et al., 1998
MCF7 and T47D
50% ethanol/H2O 100 Ag/ml
ER/PR binding assay, T47D cell proliferation
50% and 96% ethanolic extract 0.01 – 100 Ag/ml
MCF-7 cell proliferation
Lohning et al., MCF7 2000
The extract inhibited OPS in porcine and human GC. No effect on porcine LC Both the fractions bound to ER. The butanolic fraction increased the proliferation of AN3 at 1 – 5 Ag/ml and decreased it at >10 Ag/ml The extract did not bind to ER
CR did not bind to ER/PR and did not modify cell proliferation Both extracts increased cell proliferation to 0.1 – 10 Ag/ml. 100 Ag/ml were ineffective
The effect was reduced by estradiol and increased by an oestrogen antagonist The effect was partially inhibited by an oestrogen antagonist The effect was comparable to oestrogen and genistein (flavonoid)
An inhibitor of estradiol and a phytoestrogen bound to ER Estradiol and a phytoestrogen increased the cell proliferation Estradiol increased cell proliferation. A ER antagonist abolished the effect of the extract
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Table 3 (continued ) Author(s), year
Cell line(s)
Extract(s), concentration(s)
Test(s) used
Result
Comments
Eagon et al., 1999
ND
CR ND
ER binding assay
The extract bound ER
Lohning et al., Primary pituitary 1999 cells
ND 10 – 100 Ag/ml
Basal and TRH stimulated PS
Dixon Shanies and Shaikh, 1999
T47D
Extract obtained from root of CR 0.01 – 1% v/v
No information about material and method used The effect was reduced by a D2 antagonist NC
Jarry et al., 1999
ER from porcine uteri
Ethanolic extract (fractions) 35 Ag/ml
CR reduced both basal and TRH stimulated PS T47D cells The extract proliferation inhibited cell proliferation to 0.1 – 1% v/v CR bound to ER binding assay; Luciferase expression ER and activated the transcription in a MCF7-a- and of oestrogen h ER expressing regulated genes subclone MCF-7 cells CR inhibited proliferation cells proliferation
MCF7 cells Freudenstein and Bodinet, 1999
Isopropanolic/ aqueous extract 0.1 – 100 Ag/ml
Liu et al., 2001a
Methanolic extract 200 Ag/ml
ER a and h binding CR did not assay; In vitro assays show activity
CR 2.5% 4.75 Ag/l
MCF-7 cells proliferation; ER expression
Liu et al., 2001b
Pure human recombinant diluted ER a-h MCF7 cells
The effect of CR was similar to estradiol and was reduced by an antiestrogen Estradiol stimulated and tamoxifen inhibited cells proliferation Other plant tested shown activity
CR increased The effect was cells proliferation similar to and ER expression estradiol
CR, Cimicifuga racemosa; ER, oestrogen receptor; PR, progesterone receptor; TRH, thyreotropin-releasing hormone; OPS, ovarian-progesterone secretion; PS, prolactin secretion; ND, not described; NC, no comment.
below. It should be mentioned that the methodological quality of the extracted experimental data was not assessed.
Results A total of 15 animal and 15 in vitro studies on C. racemosa met our inclusion/exclusion criteria (Mercier, 1935; Gizicky, 1944; Foldes, 1959; Siess and Seybold, 1960; Jarry and Harnischfeger, 1985; Jarry et al., 1985, 1996, 1999; Duker et al., 1991; Nesselhut et al., 1993; Einer Jensen et al., 1996; Harnischfeger and Cillien, 1996; Eagon et al., 1996, 1997, 1999; Zava et al., 1998; Lohning et al., 1999, 2000; Dixon Shanies and Shaikh, 1999; Freudenstein and Bodinet, 1999; Freudenstein et al., 2000; Nisslein and Freudenstein, 2000; Liu et al., 2001a,b). Key data of all studies are summarised in Tables 2 and 3. Details are also reported below in narrative form according to type of experiment.
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Plasma hormone levels in ovariectomized rats Jarry and Harnischfeger (1985) and Jarry et al. (1985) studied the effect of a C. racemosa extract on the serum concentration of LH and FSH. The extract was found to depress plasma LH levels but not FSH levels. A successive fractionated extraction of C. racemosa extract led either to a dichloromethane and an ethanol fraction (Jarry and Harnischfeger, 1985) or to a chloroform fraction (Jarry et al., 1985). The LH-suppressive substances resided only in the dichloromethane extract, and the ethanolic fraction showed no effect on LH levels. Similarly, Duker et al. (1991) demonstrated that LH secretion was affected by a lipophilic but not a hydrophilic extract of C. racemosa (140 and 216 mg/rat, respectively, intraperitoneally). Subsequently, Eagon et al. (1999) reported that an extract of C. racemosa decreased the LH levels in ovariectomized rats. The authors did not provide any information on dose and type of extract used. Jarry et al. (1999) confirmed an estrogenic action of a C. racemosa extract (commercially available extract, BNO 1055) comparing its effect with that of estradiol on serum levels of LH and other parameters regulated by oestrogen receptors. Both the herbal extract as well as estradiol (60 mg/rat and 8 Ag/rat for seven days, respectively, s.c.) had oestrogen-like effects in the brain and reduced LH levels. By contrast, recently Freudenstein et al. (2000) did not find any oestrogen agonistic effects on plasma hormone levels (prolactin, FSH, LH) of ovariectomized rats treated with a isopropanolic extract of C. racemosa (RemifeminR). In this experiment, vehicle (negative control), C. racemosa extract (1–100 human therapeutic dosage, orally) and mestranol (positive control), were administered daily for seven weeks. Uterine weight and induction of oestrus Gizicky (1944) assessed the effect of a tincture of C. racemosa (50% water/alcohol) on uterine and ovary weight both in rats and mice. Chronic treatment with C. racemosa increased the uterine and ovary weight of animals, which showed a certain grade of maturity of follicles. The extract seemed to act by transforming larger follicles in corpus luteum. In addition, C. racemosa established menstrual cycles in immature and climacteric rats, which had already lost their cycle. Foldes (1959) studied the effect of a C. racemosa extract (RemifeminR) both on uterine growth in mice (three weeks old) and on induction of oestrus in ovariectomized rats. The extract induced in a dose-dependent manner the oestrus in ovariectomized rats (0.1–0.3 mg/day, twice daily for 3 days) and increased the uterine weight of the mice (0.2 ml/day for 4 days). Siess and Seybold (1960) reported that the alcoholic extract of C. racemosa (RemifeminR) administered for 52 days did not modify the uterine weight and did not induce oestrus in immature mice. Einer Jensen et al. (1996) investigated the oestrogen effects of C. racemosa on uterine growth in immature mice and on vaginal cornification in ovariectomized rats. Vehicle (negative control), estradiol-benzoate (positive control, 0.4 mg/kg) and a commercially available extract of C. racemosa (rhizome of C. racemosa extracted with a 50% water/ethanol mixture, 6–600 mg/kg) were administered for three days. Estradiol-benzoate increased significantly the average wet weight of the mouse uteri. By contrast, no signs of uterotrophic or vaginotrophic effects were found in the mice and rats treated with the extract. Eagon et al. (1997, 1999) found that an extract of C. racemosa obtained from the root (not rhizome) of the plant administered to ovariectomized rats for three weeks, increased the uterine growth. Jarry et al. (1999) and Freudenstein et al. (2000) observed that C. racemosa but not estradiol or mestranol did not modify the uterine weight of ovariectomized rats. In addition, no increase
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of the expression of IgF1 and C3 in the uterus of the animals treated with C. racemosa was observed suggesting no estrogenic action of the extract on the uterine tissue (Jarry et al., 1999). Finally, an extract of C. racemosa administered to immature female mice for 14 days, did not increase uterine or ovarian weight although it prolonged significantly the days of oestrus (Liu et al., 2001b). Estradiol (0.5 mg/kg), used as positive control, increased significantly the weight of both the uterus and ovary as well as the days of oestrus. Bone loss Jarry et al. (1999), in an experiment carried out on ovariectomized rats, showed that an extract of C. racemosa (BNO 1055) as well as estradiol (60 mg/rat and 8 Ag/rat for seven days, respectively) possessed an oestrogen agonist effect on bone (femur) increasing the expression of collagen I and osteocalcin. Similarly, Nisslein and Freudenstein (2000) reported that an isopropanolic extract of C. racemosa (administered for three weeks) reduced significantly both some urinary parameters of bone metabolism and the bone loss observed in control animals (not significantly). The effects induced by the extract was similar to those induced by the oestrogen, raloxifene. Oestrogen receptor competitive binding assay Jarry et al. (1985, 1999) and Duker et al. (1991) demonstrated the endocrine activity of C. racemosa using the in vitro oestrogen receptor-binding assay. They observed that a lipophilic extract of the plant (35 Ag/ml) was able to bind to oestrogen receptors of rat uteri. By chromatographic separation of the lipophilic extract, they isolated at least three different endocrine active compounds: (i) compounds which were not ligands for the oestrogen receptor but suppressed LH release after chronic treatment (perhaps acting on CNS such as an alpha-2 agonist), (ii) compounds binding to the oestrogen receptor and also suppressing LH release and (iii) compounds which are ligands for the oestrogen receptor but without an effect on LH release. One of these compounds was identified as the isoflavon formononetin, which has been shown to be a competitor in the oestrogen receptor assay, but failed to reduce the serum levels of luteinizing hormone in ovariectomized rats. Similarly, Harnischfeger and Cillien (1996) reported that a butanol-derived and a chloroform-derived subfraction of an alcoholic extract of C. racemosa were able to bind to oestrogen receptors as well as a flavonoid, genistein. Recent research conducted by Eagon et al. (1996), Zava et al. (1998) and Liu et al. (2001a) contradicted these data. Eagon et al. (1996) assessed the estrogenic activity of C. racemosa extract in an in vitro competitive oestrogen receptor binding assay in parallel with diethylstilbestrol (DES), a potent inhibitor of estradiol binding. The assay was conducted on cytosolic oestrogen receptors from livers of ovariectomized rats incubated with 5 nM of [3H]-estradiol. The results showed a significant inhibition of estradiol binding in the presence of DES but not with the extract. Similarly, Zava et al. (1998) tested the ability of C. racemosa extract (50% ethanol/water) to bind to oestrogen receptors. The authors reported that C. racemosa (0.1 mg) did not possess oestrogen/progesterone receptorsbinding activity on intact human breast cancer cell lines (MCF7 and T47D). In this experiment other plants (e.g. liquorice, thyme) showed oestrogen receptor binding activity on the same cell lines. Finally, Liu et al. (2001a) demonstrated that a methanolic extract of C. racemosa (200 Ag/ml) was not able to bind to human recombinant diluted oestrogen receptors (a and h). Other plants (e.g. hops, red clover) were able to bind to oestrogen receptors (a and h). In an abstract, Eagon et al. (1999), in
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contradiction with previous results (Eagon et al., 1996) reported that an extract of C. racemosa binds to oestrogen receptors. However, this abstract did not report any information on concentration or type of extract used. Cell proliferation Nesselhut et al. (1993), Dixon Shanies and Shaikh (1999) and Freudenstein and Bodinet (1999) assessed the estrogenic activity of C. racemosa on in vitro growth of human breast cancer cell lines (T47D and MCF-7) characterised by an oestrogen dependent cell proliferation. The authors found that C. racemosa significantly inhibited cell proliferation. In addition, the extract antagonised estrogenic activity of estradiol and augmented the anti-proliferative effect of oestrogen antagonists, such as Raponticin (Nesselhut et al., 1993) or Tamoxifen (Freudenstein and Bodinet, 1999). Harnischfeger and Cillien (1996) observed that a butanolic derived subfraction of an alcoholic extract of C. racemosa stimulated the proliferation of carcinoma AN3 (an oestrogen sensitive cell line) at concentrations between 1–5 Ag/ml and suppressed it at concentration above 10 Ag/ml. Oestrogen (10 9 –10 6 M) used as positive control increased the AN3 proliferation. Oestrogen and the butanolic fraction had no effects on the growth of FTDE and MX1 cells (not sensitive oestrogen cell lines). Lohning et al. (2000) and Liu et al. (2001b) observed a dose dependent increase on MCF-7 cell number induced by C. racemosa or estradiol. This effect was observed using an extract of C. racemosa in very low concentrations (see Table 3). Higher concentrations of the extract (100 Ag/ml) were ineffective. Furthermore, the proliferative effect of C. racemosa was completely abolished in the presence of an oestrogen receptor antagonist, ICI 182,789 (Lohning et al., 2000). Similarly, Zava et al. (1998) reported that a high concentration of C. racemosa (100 Ag/ml) did not influence the proliferation of T47D breast cancer cells. Hormonal secretion Jarry et al. (1996) investigated the effect of a commercially available extract of C. racemosa on in vitro ovarian steroid secretion. The extract (0.01–1 mg) inhibited progesterone-secretion from both porcine and human granulosa cells in a dose-dependent manner. Since this effect was only partially reversed by the oestrogen-antagonist Tamoxifen, the authors concluded that at least C. racemosa possesses endocrine active constituents, which partially act via the ovarian oestrogen receptor. Lohning et al. (1999) assessed the estrogenic activity of ethanolic extracts of C. racemosa on primary pituitary cells. The ethanolic extract (10 –100 Ag/ml) reduced significantly both basal and TRH stimulated prolactin secretion of primary pituitary cell cultures. As C. racemosa effects on prolactin secretion were reduced by the D2-antagonist haloperidol, a dopaminergic activity of the extract can be assumed. Oestrogen receptors expression Jarry et al. (1999) evaluated the effects of a hydrophilic and lipophilic extract of C. racemosa on luciferase expression in an MCF7-a-and h oestrogen receptor-expressing subclone. The effects of the extracts were compared with the effects of estradiol. The lipophil extract of C. racemosa (35 Ag/ml) as
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well as estradiol was able to activate the transcription of oestrogen regulated genes. This effect was significantly reduced by the specific antioestrogen ICI 182,780. The hydrophil extract of C. racemosa was inactive. More recently, Liu et al. (2001b) investigated the effect of C. racemosa on the oestrogen receptor level of human breast cancer MCF-7 cells. C. racemosa (4.75 Ag/l) as well as estradiol increased oestrogen receptor levels thus demonstrating an oestrogen-like action. Miscellaneous Foldes (1959) studied the effect of an alcoholic extract of C. racemosa on sedation in 60 mice treated with a lethal dose of amphetamine (20 mg/kg). The extract did not protect against the effect of amphetamine. Subsequently, the author assessed either histological changes of the ovaries or changes in thyroid function and hormone mobilisation on rats treated with C. racemosa. No differences were observed between control and treated groups. Mercier and Balansard (1935) assessed the effect of four glycosidic fractions obtained from the rhizome of C. racemosa on cardiac functions in dogs. Only the fraction insoluble in water (0.02–0.03 g/ kg, iv) induced strong arterial hypotension, decrease of cardiac contraction and definite bradycardia up to the point of death. Lohning et al. (1999) demonstrated that the activity of C. racemosa was mediated by central D2receptors. The authors studied the effect of the plant on body temperature and ketamine-induced sleeping time in mice. Bromocriptine (a D2-agonist) (5 mg/kg, intraperitoneally) was used as positive control. C. racemosa (25–100 mg/kg, orally) caused both a fall in body temperature in a dose-dependent manner and a significant prolongation of ketamine-induced sleeping time. These effects were inhibited by pretreatment with a D2-receptor antagonist thus demonstrating a central activity of the plant mediated by D2-receptors. Freudenstein et al. (2000) studied the effect of an isopropanolic extract of C. racemosa (Remifemin) on the number and size of oestrogen dependent mammarian gland tumours induced in ovariectomized rats. Vehicle and mestranol were used as negative and positive controls. C. racemosa (1–100 human therapeutic dosage, orally) administration daily for seven weeks did not increase the number or the size of tumours. By contrast, mestranol (450 Ag/kg/day for seven weeks) significantly increased the tumour size. Liu et al. (2001a) evaluated the estrogenic activity of C. racemosa on several in vitro assays (alkaline phosphatase induction, progesterone receptor and presenelin-2 mRNA expression). They found that a methanol extract of this plant (20 Ag/ml) did not show estrogenic activity in any assay.
Discussion Early animal studies on an extract of C. racemosa had postulated its capacity to possess «oestrogenlike» activity as evidenced by an increase in uterine weight and an induction of oestrus Gizicky, 1944; Foldes, 1959). Further studies (Jarry and Harnischfeger, 1985; Jarry et al., 1985, 1999; Duker et al., 1991) suggested that C. racemosa contained three synergistically acting compounds which were able to reduce serum LH levels and bind to oestrogen receptors (so as to increase the amount of oestrogen in the blood which decreases the menopause symptoms). Actein and cimicifugoside were believed to be partly
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responsible for the reduction in LH, while the isoflavone formononetin was thought to bind to oestrogen receptors. However, recent investigations imply that C. racemosa contain compounds that act by a mechanism that does not involve oestrogen receptors (Freudenstein et al., 2000; Zava et al., 1998; Liu et al., 2001b). A central activity mediated by dopaminergic-2 receptors rather than to an estrogenic activity (Lohning et al., 1999) has been also hypothesised. However, it should be taken into account that such hypothetical mechanism is based on indirect evidence and is not supported by binding studies. This theory is further supported by chemical studies reporting that ethanolic and isopropanolic extracts do not contain the isoflavone formononetin and the flavone kaempferol (Struck et al., 1997; Hagels et al., 2000). These substances are concentrated in the aerial parts of the plant. As some of the early experiments did not use standardised extracts, the detection of formononetin and the effect of C. racemosa on oestrogen receptors and LH levels observed in these investigations could be due to contaminants or variations in content of active compounds. Furthermore, the doses used in early experiments to study the effect of C. racemosa on LH levels were higher than the doses used in later animal experiments yielding positive results (Jarry and Harnischfeger, 1985; Duker et al., 1991; Lohning et al., 1999). It should be also mentioned that the concentration of the active ingredients of C. racemosa could vary depending on a number of factors, including growing season, harvesting, exsiccation procedure, preservation and storage of crude drugs. Lack of estrogenic activity was also demonstrated by a further studies carried out in vivo. The presence of an oestrogen-like substance in the extract of C. racemosa should increase the organ weights and the number and size of tumours; these effects have not been observed in ovariectomized rats treated daily for seven weeks with the extract (Freudenstein et al., 2000). All the investigations (with the exception of Eagon et al., 1997, 1999) which used an extract of root) agree on the lack of estrogenic effects of C. racemosa on uterine tissue [Einer Jensen et al., 1996; Jarry et al., 1999; Freudenstein et al., 2000; Liu et al., 2001b). By contrast, there are conflicting results on the effect of C. racemosa on cell proliferation. Some investigators have found that an extract of C. racemosa inhibited the proliferation of human oestrogen receptor-positive breast cancer cells (T47D and MCF-7) (Nesselhut et al., 1993; Dixon Shanies and Shaikh, 1999; Freudenstein and Bodinet, 1999). Since dopaminergic agonists also cause a significant decrease in proliferation of MCF-7 cells (Johnson et al., 1995), the effect of the extract could be due to the presence dopaminergic substances in the plant. Recently it has been reported that the cinnamic acid ester fukinolic acid, found in high concentration in the rhizome of C. racemosa (Struck et al., 1997; Hagels et al., 2000), increases the proliferation of oestrogen dependent MCF-7 cell systems (Kruse et al., 1999). Thus, the stimulant effect of C. racemosa observed with low concentrations (Harnischfeger and Cillien, 1996; Lohning et al., 2000; Liu et al., 2001b), could be due to a loss of dopaminergic activity and a predominance of the fukinolic acid effect. The effect of C. racemosa on bone loss could be linked with its capacity to reduce prolactin levels (effect demonstrated due to an action on D2-receptor). High prolactin levels have been associated with a premature loss of bone mass mainly due to concomitant hypogonadism (Koloszar et al., 1997; Sanfilippo, 1999). Treatment with a dopamine agonist, suppressing serum prolactin levels, prevents additional bone loss and increases slightly (not significantly) bone mineral density (Di Somma et al., 1998; Colao et al., 2000). Two recent experiments on the expression of oestrogen receptors reported an increase in receptor levels by C. racemosa (Jarry et al., 1999; Liu et al., 2001b). Since D1-like dopamine receptors mediate the in vitro transcriptional activation of oestrogen receptors by dopamine (Tsai and O’Malley, 1994;
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O’Malley et al., 1995), the possibility that the increase of oestrogen expression is due to the presence of a dopaminergic substance cannot be excluded. Conclusion In conclusion, the effects of C. racemosa are probably due to substances with dopaminergic activity rather than an oestrogen-like activity. Consistently, dopaminergic drugs reduce some symptoms (e.g. hot flashes) associated to menopause (Bider et al., 1989). Further studies are required to explain why C. racemosa is devoid of the typical side effects associated with dopaminergic drugs (Borrelli and Ernst, 2002; Webster, 1996) and to determinate the pharmacokinetic properties of this plant extract. Acknowledgements The authors wish to thank the SESIRCA–Regione Campania and the Enrico and Enrica Sovena Foundation (Rome, Italy) for their support. References Bergkvist, L., Persson, I., 1996. Hormone replacement therapy and breast cancer. A review of current knowledge. Drug Safety 15, 360 – 370. Bider, D., Mashiach, S., Serr, D.M., Ben-Rafael, Z., 1989. Endocrinological basis of hot flushes. Obstetrical and Gynecological Survery 44, 495 – 499. Borrelli, F., Ernst, E., 2002. Cimicifuga racemosa: A systematic review of its clinical and pharmacological effects. European of Journal Clinical Pharmacology (in press). Colao, A., Di Somma, C., Loche, S., Di Sarno, A., Klain, M., Pivonello, R., Pietrosante, M., Salvatore, M., Lombardi, G., 2000. Prolactinomas in adolescents: persistent bone loss after 2 years of prolactin normalization. Clinical Endocrinology 52, 319 – 327. Di Somma, C., Colao, A., Di Sarno, A., Klain, M., Landi, M.L., Facciolli, G., Pivonello, R., Panza, N., Salvatore, M., Lombardi, G., 1998. Bone marker and bone density responses to dopamine agonist therapy in hyperprolactinemic males. Journal of Clinical Endocrinology and Metabolism 83, 807 – 813. Dixon Shanies, D., Shaikh, N., 1999. Growth inhibition of human breast cancer cells by herbs and phytoestrogens. Oncology Rep. 6, 1383 – 1387. Duker, E.M., Kopanski, L., Jarry, H., Wuttke, W., 1991. Effects of extracts from Cimicifuga racemosa on gnadotropin release in menopausal women and ovariectomized rats. Planta Medica 57, 420 – 424. Eagon, C.L., Elm, M.S., Eagon, P.K., 1996. Estrogenicity of traditional Chinese and Western herbal remedies. Proceeding of the American Association for Cancer Research 37, 284. Eagon, C.L., Elm, M.S., Teepe, A.G., Eagon, P.K., 1997. Medicinal botanicals: Estrogenicity in rat uterus and liver. Proceeding of the American Association for Cancer Research 38, 293. Eagon, P.K., Tress, N.B., Ayer, H.A., Wiese, J.M., Henderson, T., Elm, M.S., Eagon, C.L., 1999. Medicinal botanicals with hormonal activity. Proceeding of the American Association for Cancer Research 40, 161 – 162. Einer Jensen, N., Zhao, J., Andersen, K.P., Kristoffersen, K., 1996. Cimicifuga and Melbrosia lack oestrogenic effects in mice and rats. Maturitas 25, 149 – 153. Foldes, J., 1959. The actions of an extract of Cimicifuga racemosa. Arzneimittelforschung 13, 623 – 624. Foster, S., 1999. Black cohosh: Cimicifuga racemosa. A literature review. Herbal Gram, 35 – 50. Freudenstein, J., Bodinet, C., 1999. Influence of an isopropanolic aqueous extract of Cimicifuga racemosa rhizoma on the proliferation of MCF-7 cells (Abstractband). 23rd Int LOF-Symposium on Phyto-Oestrogens, University of Gent, Belgium.
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