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Cimicifuga extract BNO 1055: reduction of hot flushes and hints on antidepressant activity
Maturitas 44 Suppl. 1 (2003) S51 /S58 www.elsevier.com/locate/maturitas
Cimicifuga extract BNO 1055: reduction of hot flushes and hints on antidepressant activity Hilke Winterhoff a,*, Barbara Spengler b, Volker Christoffel b, Veronika Butterweck a, Anke Lo¨hning a a
Institute for Pharmacology and Toxicology, Central Clinic, University of Mu¨nster, Domagkstrasse 12, 48149 Mu¨nster, Germany b BIONORICA AG, Kerschensteinerstr 11-15, 92318 Neumarkt Opf., Germany Received 20 May 2002; accepted 4 September 2002
Abstract Ethanolic- and isopropanolic-aqueous extracts of Cimicifuga racemosa are used for the treatment of climacteric complaints. As hot flushes and psychic complaints seem to be special targets for Cimicifuga extracts in clinical studies, these parameters were studied in experimental animals. Hot flush equivalents were measured in castrated rats as a quick increase in peripheral temperature with the aid of a transmitter implanted subcutaneously on the ventral side. The hot flush equivalents proved to respond to estrogen and the antidopaminergic drug veralipride but they were also reduced very effectively by Cimicifuga extract BNO 1055 (which is contained in Klimadynon† /Menofem† ). In addition, an ethanolic-aqueous extract of C. racemosa was studied in the tail suspension test (TST), a behavioural test indicative for antidepressant activity. A significant decrease of the period of immobility was observed after treatment with 30 mg/kg body weight (bw) imipramine or with 50 or 100 mg/kg bw Cimicifuga extract. These findings in pharmacological tests */a reduction of the frequency of hot flush equivalents and hints on antidepressant activity of Cimicifuga extracts */are in good agreement with the therapeutical responses in climacteric women. # 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Cimicifuga racemosa ; Hot flush equivalents; Antidepressant activity
1. Introduction Cimicifuga racemosa was traditionally used by North American Indians to treat pain of the joints,
* Corresponding author. Tel.: /49-251-83-55-507; fax: /49251-83-55-501. E-mail address: [email protected] (H. Winterhoff).
neuralgia, rheumatism, pains during labour, gynecological disorders and climacteric complaints. The medicinal use of this plant was adopted by European immigrants in North America. In Europe, the plant is known and used since 17th century to treat catarrh, pain of the joints, neuralgia, rheumatism, pain in pregnancy and labour. Since the late 1950s, the efficiency of the drug in more specific gynecological indications like
0378-5122/02/$ - see front matter # 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0378-5122(02)00348-1
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H. Winterhoff et al. / Maturitas 44 (2003) S51 /S58
intermittent bleeding, disturbances of the menstrual cycle, PMS as well as in climacteric complaints was described (for an overview, see [1]). Corresponding to the indications of the monograph of the German Commission E ethanolicand isopropanolic-aqueous extracts of C. racemosa are used to improve premenstrual and dysmenorrhoic as well as neurovegetative climacteric complaints. The efficiency of such a treatment in climacteric women could be proven in clinical studies by a significant reduction of the modified Kuppermann index. Not all items of the Kuppermann index are influenced to the same degree by Cimicifuga extracts. Hot flushes as well as psychic complaints are described as specific targets for the drug, whereas other symptoms were improved only slightly or not at all. Thus, it was the aim of our study to investigate the efficacy of an ethanolic-aqueous extract of C. racemosa in pharmacological test systems with special stress on these central effects: the improvement in depressant situation and the reduction of the frequency and severity of hot flushes. The vasomotor flushes observed in climacteric or ovariectomized women are caused by the estrogen deficiency; they represent an inadequate activation of heat loss mechanisms with an increase in peripheral perfusion and temperature and a decrease in core body temperature. Immediately before the sensation of a hot flush the skin conductance is increased, the peripheral blood flow and the heart rate is increased, paralleled by an increase of serum LH concentrations and intense sweating. The augmented peripheral perfusion precedes the rise in peripheral temperature; a significant increase in epinephrine is observed 3 and 6 min after the onset of increased peripheral perfusion [2,3]. Hot flushes can be treated effectively with estrogens, but alpha-adrenergic agonists, dopaminergic as well as antidopaminergic drugs were shown to be effective too [4 /7]. Thus the anti-flush activity of Cimicifuga extracts observed in clinical studies must not necessarily indicate an estrogenic activity of those extracts but may be caused by compounds that elicit other central effects. To
measure flush equivalents in rats, a test system had to be established and checked for sensitivity. Three animal models were used in the past to study this vasomotor syndrome: the raise in tail flick temperature in morphine-dependent rats following naloxone injection [8 /11], the reduced drop in tail temperature of estrogen-deficient rats during transition from the resting to the active phase [12] or the raise in tail temperature following ovariectomy [13]. All these experimental models determine the effects of estrogen deprivation but the comparability to flush events seems questionable. Therefore, it was the aim of our study to develop a more reliable method to measure flush equivalents in ovariectomized rats.
2. Material and methods 2.1. C. racemosa extract BNO 1055 The test substance in the present tests was the C. racemosa extract BNO 1055 (dry extract prepared with ethanol 58%, v/v, DEV 6.1 /9.1). 2.2. Telemetric measurement of flush equivalents in rats As experimental animals female Wistar rats (Harlan-Winkelmann, Borchen, Germany) were used, ovariectomized and kept on an estrogenand phytoestrogen-free diet (ssniff EF, 1/0, 10 mm) for 3 weeks before the experiments were started. The rats were single housed with free access to food and tap water; a 12 h light/dark change and a room temperature of 259/1 8C was maintained. Thermosensors and transmitters (PDT-4000 EMitter, Vital View† , Mini Mitter Co., Inc., Sunpriver, OR) were implanted subcutaneously on the ventral side; the cages (Macrolon III) were placed upon the corresponding receivers. The data were transferred directly from the receiver to a computer to allow a continuous recording of the course of skin temperature. A hot flush was defined as a quick increase of peripheral temperature of more than 1 8C (corresponding to the definition of the rise of finger temperature in climacteric women acc. to [14]).
H. Winterhoff et al. / Maturitas 44 (2003) S51 /S58
S53
Fig. 1. Incidence of hot flush equivalents in intact (open bars) and castrated (filled bars) rats. Each bar represents the number of flush equivalents per day.
The number of such hot flush equivalents was cumulated over a period of 24 h. The recording of the hot flushes was started not earlier than 8 days after the implantation of the transmitter to avoid possible influences of the anaesthesia and surgery on the hot flush equivalents. The dry extract was suspended in water with sonification and kept in a homogeneous suspension by stirring during application. It was administered once daily for 5 days in the afternoon orally by gavage in a final volume of 10 ml/kg bw. Animals were observed for clinical symptoms and body weight was determined daily to detect possible toxic effects. 2.3. Tail suspension test (TST) in mice TST was introduced as a screening test for antidepressant activity by Steru et al. [15]. Mice suspended by their tails without any contact to the ground are used as test animals. During the observation period of 6 min, there is a change between searching behaviour with intense movements and waiting behaviour with a typical motionless position, in part accompanied by ptosis. This test is derived from the Porsolt test in rats, in which a reduction of the period of learned immobility indicates antidepressant activity
[16,17]. Correspondingly, the cumulative period of immobility in TST is the test parameter which is reduced by antidepressants. Unspecific effects caused by motor stimulation are detected by an open field test performed prior to the TST. The animals (female NMRI-mice, 18/27 g bw, Harlan-Winkelmann, Borchen, Germany) were housed in groups of 5 with free access to food (Altromin 1324) and tap water; a 12 h light/dark change and a room temperature of 259/1 8C was maintained. They were acclimatized for at least 5 days before use in experiments. Test solutions were prepared directly before use by suspending the extract in water with the aid of a sonifier and maintaining a homogeneous suspension by stirring. The animals were treated with the test preparations in a volume of 10 ml/kg bw by gavage. The mice were assigned randomly to the experimental groups. The test period was recorded on a videotape which was evaluated by a person unaware of the treatment. TST was performed 60 min after one single application of the test substances. A second assay was performed after a treatment period of 8 days with daily application of the Cimicifuga extract, the last application 1 h before TST. The results were compared with those of a control group treated with tap water and an imipraminetreated group.
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Fig. 2. Reduction of the number of hot flush equivalents per day by estradiolvalerat (2 mg/kg per day per os). Each bar represents the number of flush equivalents per day.
To study motility, an open field test was performed in the animals after the same treatment as for TST. Sixty minutes after gavage the mice were placed in the open field, during a 5 min observation period the number of line crossings was recorded [18,19]. 2.4. Statistics Each value represents the mean9/S.E.M. of 7/ 10 animals per group. Significance of differences to the control group were evaluated by the ANOVA test and are marked with an asterisk for values with P B/0.05.
3. Results 3.1. Telemetric measurement of flush equivalents in rats In the first step, the test parameter ‘‘flush equivalents in rats’’ had to be checked for its sensitivity. The frequency of short-term increases in skin temperature, which can be considered as a flush equivalent, was studied before and 2 weeks following castration in the same animals. As shown in Fig. 1, the number of hot flush equiva-
Fig. 3. Reduction of the number of hot flush equivalents by an ethanolic Cimicifuga extract (100 mg/kg per day). Each bar represents the number of flush equivalents per day.
lents clearly and significantly increased with the removal of the ovaries. Next, the reaction to treatments effective in patients with hot flushes had to be checked. As shown in Fig. 2, estradiolvalerate (2 mg/kg bw per day) reduced the number of hot flush equivalents. A comparable effect was seen after application of 100 mg/kg bw per day of the antidopaminergic drug veralipride (Agradil† , data not shown). These findings in rats confirm a comparable response and sensitivity of the hot flush equivalents to the hot flushes observed in women. Following oral administration of 100 mg/kg bw of the C. racemosa extract, the number of hot flush
H. Winterhoff et al. / Maturitas 44 (2003) S51 /S58
S55
Fig. 4. Skin temperature of a rat during the pretreatment period.
equivalents was clearly reduced, it returned to pretreatment values after cessation of the treatment (Fig. 3). In Fig. 4, the direct registration of skin temperature of a control period and following application of the Cimicifuga extract (Fig. 5) are shown. It is obvious that Cimicifuga extract causes not only a reduction of the incidence of hot flush equivalents but also a decrease of the variation of the course of temperature.
3.2. TST in mice Sixty minutes after gavage, both dosages of the Cimicifuga extract and the imipramine significantly reduced the period of immobility (Fig. 6). Central stimulation could be excluded because
Cimicifuga extract as well as imipramine even reduced motility in the open field test after acute application (Fig. 7). The same test was performed after an 8-day treatment period. Again the Cimicifuga extract as well as imipramine reduced the period of immobility significantly (Fig. 8), whereas the motility was no longer reduced significantly (number of line crossings for control group 919/11, for imipramine group 799/16, and for Cimicifuga extract group 769/11). As substances inhibiting the re-uptake of noradrenaline, dopamine or serotonine proved to be active in this test [20,21], the activity in this test system does not indicate the mode of antidepressant activity of Cimicifuga extracts. This has to be studied in further experiments.
Fig. 5. Skin temperature of a rat during treatment with 100 mg/kg per day of an ethanolic Cimicifuga extract.
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H. Winterhoff et al. / Maturitas 44 (2003) S51 /S58
Fig. 6. TST in female NMRI mice 1 h after gavage.
4. Discussion In our test system, hot flush equivalents can be measured in castrated female rats. They responded similarly to hot flushes in women, they were increased following castration and proved to be reduced by treatment with estrogen as well as by the antidopaminergic drug veralipride. In accor-
dance with the results in clinical reports, the ethanolic-aqueous Cimicifuga extract clearly and effectively reduced the hot flush incidence in rats. In a behavioural test system indicative for antidepressant activity, TST, Cimicifuga extract like antidepressant drugs significantly reduced the cumulative period of immobility. The effect was observed after acute and repeated treatment and
Fig. 7. Motility in the open field 1 h after gavage.
H. Winterhoff et al. / Maturitas 44 (2003) S51 /S58
S57
Fig. 8. TST in female NMRI mice after an 8-day pretreatment period.
seems to be specific, as an unspecific stimulation of locomotor activity could be excluded. Both activities of the ethanolic-aqueous Cimicifuga extract BNO 1055, anti-flush activity and hints on antidepressant activity, are in good accordance with the efficacy of the C. racemosa preparation BNO 1055 (Klimadynon† / † Menofem ) in the treatment of climacteric complaints.
References [1] Boblitz N, Liske E, Wu¨stenberg P. Traubensilberkerze DAZ 2000;140:107. [2] Mashchak CA, Kletzky OA, Artal R, Mishell DR, Jr.. The relation of physiological changes to subjective symptoms in postmenopausal women with and without hot flushes. Maturitas 1984;6(4):301 /8. [3] Kronenberg F, Cote LJ, Linkie DM, Dyrenfurth I, Downey JA. Menopausal hot flushes: thermoregulatory, cardiovascular, and circulating catecholamine and LH changes. Maturitas 1984;6(1):31 /43. [4] Vercellini P, Sacerdote P, Trespidi L. Veralipride for hot flushes induced by a gonadotropin-releasing hormone agonist: a controlled study. Fertil Steril 1994;62(5):938 /42. [5] Tulandi T, Kinch RA, Guyda H, Mazella L, Lal S. Effect of methyldopa on menopausal flushes, skin temperature, and luteinizing hormone secretion. Am J Obstet Gynecol 1984;150(6):709 /12.
[6] Clayden JW, Bell JW, Pollard P. Menopausal flushing: double-blind trial of a non-hormonal medication. Br Med J 1974;1(905):409 /12. [7] Melis GB, Cagnacci A, Gambacciani M, Paoletti AM, Moggi L, Fioretti P. Restoration of luteinizing hormone response to naloxone in postmenopausal women by chronic administration of the antidopaminergic drug veralipride. J Clin Endocrinol Metab 1988;66(5):964 /7. [8] Simpkins JW, Katovich MJ, Song IC. Similarities between morphine withdrawal in the rat and the menopausal hot flush. Life Sci 1983;32(17):1957 /66. [9] Simpkins JW. Spontaneous skin flushing episodes in the aging female rat. Maturitas 1984;6(3):269 /78. [10] Katovich MJ, Simpkins JW, O’Meara J. Effects of opioid antagonists and their quaternary analogs on temperature changes in morphine-dependent rats. Life Sci 1986;39(20):1845 /54. [11] Merchenthaler I, Funkhouser JM, Carver JM, Lundeen SG, Ghosh K, Winneker RC. The effect of estrogens and antiestrogens in a rat model for hot flush. Maturitas 1998;30(3):307 /16. [12] Berendsen HH, Weekers AH, Kloosterboer HJ. Effect of tibolone and raloxifene on the tail temperature of oestrogen-deficient rats. Eur J Pharmacol 2001;419(1):47 /54. [13] Pan Y, Anthony MS, Binns M, Clarkson TB. A comparison of oral micronized estradiol with soy phytoestrogen effects on tail skin temperatures of ovariectomized rats. Menopause 2001;8(3):171 /4. [14] Meldrum DR, Tataryn IV, Frumar AM, Erlik Y, Lu KH, Judd HL. Gonadotropins, estrogens, and adrenal steroids during menopausal hot flush. J Clin Endocrinol Metab 1980;50(4):685 /9. [15] Steru L, Chermat R, Thierry B, Simon P. The tail suspension test: a new method for screening antidepres-
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sants in mice. Psychopharmacology (Berlin) 1985;85(3):367 /70. [16] Porsolt RD, Le Pichon M, Jalfre M. Depression: a new animal model sensitive to antidepressant treatments. Nature 1977;266(5604):730 /2. [17] Porsolt RD, Anton G, Blavet N, Jalfre M. Behavioural despair in rats: a new model sensitive to antidepressant treatments. Eur J Pharmacol 1978;47(4):379 /91. [18] Barnett SA, Cowan PE. Activity, exploration, curiosity, and fear. Interdiscip Sci Rev 1978;1:43 /61.
[19] Open field test, In: Vogel HG, Vogel WH, editors. Drug Discovery and Evaluation. Berlin: Springer, 1997. p. 206 /7. [20] Perrault GH, More IE, Zivkovic B, Sanger DJ. Activity of litoxetine and other serotonin uptake inhibitors in the tail suspension test in mice. Pharmacol Biochem Behav 1992;42:45 /7. [21] Teste JF, Pelsy-Johann I, Decelle T, Boulu RG. Antiimmobility activity of different antidepressant drugs using the tail suspension test in normal or reserpinized mice. Fundam Clin Pharmacol 1993;7(5):219 /26.
Cimicifuga extract BNO 1055: reduction of hot flushes and hints on antidepressant activity Hilke Winterhoff a,*, Barbara Spengler b, Volker Christoffel b, Veronika Butterweck a, Anke Lo¨hning a a
Institute for Pharmacology and Toxicology, Central Clinic, University of Mu¨nster, Domagkstrasse 12, 48149 Mu¨nster, Germany b BIONORICA AG, Kerschensteinerstr 11-15, 92318 Neumarkt Opf., Germany Received 20 May 2002; accepted 4 September 2002
Abstract Ethanolic- and isopropanolic-aqueous extracts of Cimicifuga racemosa are used for the treatment of climacteric complaints. As hot flushes and psychic complaints seem to be special targets for Cimicifuga extracts in clinical studies, these parameters were studied in experimental animals. Hot flush equivalents were measured in castrated rats as a quick increase in peripheral temperature with the aid of a transmitter implanted subcutaneously on the ventral side. The hot flush equivalents proved to respond to estrogen and the antidopaminergic drug veralipride but they were also reduced very effectively by Cimicifuga extract BNO 1055 (which is contained in Klimadynon† /Menofem† ). In addition, an ethanolic-aqueous extract of C. racemosa was studied in the tail suspension test (TST), a behavioural test indicative for antidepressant activity. A significant decrease of the period of immobility was observed after treatment with 30 mg/kg body weight (bw) imipramine or with 50 or 100 mg/kg bw Cimicifuga extract. These findings in pharmacological tests */a reduction of the frequency of hot flush equivalents and hints on antidepressant activity of Cimicifuga extracts */are in good agreement with the therapeutical responses in climacteric women. # 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Cimicifuga racemosa ; Hot flush equivalents; Antidepressant activity
1. Introduction Cimicifuga racemosa was traditionally used by North American Indians to treat pain of the joints,
* Corresponding author. Tel.: /49-251-83-55-507; fax: /49251-83-55-501. E-mail address: [email protected] (H. Winterhoff).
neuralgia, rheumatism, pains during labour, gynecological disorders and climacteric complaints. The medicinal use of this plant was adopted by European immigrants in North America. In Europe, the plant is known and used since 17th century to treat catarrh, pain of the joints, neuralgia, rheumatism, pain in pregnancy and labour. Since the late 1950s, the efficiency of the drug in more specific gynecological indications like
0378-5122/02/$ - see front matter # 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0378-5122(02)00348-1
S52
H. Winterhoff et al. / Maturitas 44 (2003) S51 /S58
intermittent bleeding, disturbances of the menstrual cycle, PMS as well as in climacteric complaints was described (for an overview, see [1]). Corresponding to the indications of the monograph of the German Commission E ethanolicand isopropanolic-aqueous extracts of C. racemosa are used to improve premenstrual and dysmenorrhoic as well as neurovegetative climacteric complaints. The efficiency of such a treatment in climacteric women could be proven in clinical studies by a significant reduction of the modified Kuppermann index. Not all items of the Kuppermann index are influenced to the same degree by Cimicifuga extracts. Hot flushes as well as psychic complaints are described as specific targets for the drug, whereas other symptoms were improved only slightly or not at all. Thus, it was the aim of our study to investigate the efficacy of an ethanolic-aqueous extract of C. racemosa in pharmacological test systems with special stress on these central effects: the improvement in depressant situation and the reduction of the frequency and severity of hot flushes. The vasomotor flushes observed in climacteric or ovariectomized women are caused by the estrogen deficiency; they represent an inadequate activation of heat loss mechanisms with an increase in peripheral perfusion and temperature and a decrease in core body temperature. Immediately before the sensation of a hot flush the skin conductance is increased, the peripheral blood flow and the heart rate is increased, paralleled by an increase of serum LH concentrations and intense sweating. The augmented peripheral perfusion precedes the rise in peripheral temperature; a significant increase in epinephrine is observed 3 and 6 min after the onset of increased peripheral perfusion [2,3]. Hot flushes can be treated effectively with estrogens, but alpha-adrenergic agonists, dopaminergic as well as antidopaminergic drugs were shown to be effective too [4 /7]. Thus the anti-flush activity of Cimicifuga extracts observed in clinical studies must not necessarily indicate an estrogenic activity of those extracts but may be caused by compounds that elicit other central effects. To
measure flush equivalents in rats, a test system had to be established and checked for sensitivity. Three animal models were used in the past to study this vasomotor syndrome: the raise in tail flick temperature in morphine-dependent rats following naloxone injection [8 /11], the reduced drop in tail temperature of estrogen-deficient rats during transition from the resting to the active phase [12] or the raise in tail temperature following ovariectomy [13]. All these experimental models determine the effects of estrogen deprivation but the comparability to flush events seems questionable. Therefore, it was the aim of our study to develop a more reliable method to measure flush equivalents in ovariectomized rats.
2. Material and methods 2.1. C. racemosa extract BNO 1055 The test substance in the present tests was the C. racemosa extract BNO 1055 (dry extract prepared with ethanol 58%, v/v, DEV 6.1 /9.1). 2.2. Telemetric measurement of flush equivalents in rats As experimental animals female Wistar rats (Harlan-Winkelmann, Borchen, Germany) were used, ovariectomized and kept on an estrogenand phytoestrogen-free diet (ssniff EF, 1/0, 10 mm) for 3 weeks before the experiments were started. The rats were single housed with free access to food and tap water; a 12 h light/dark change and a room temperature of 259/1 8C was maintained. Thermosensors and transmitters (PDT-4000 EMitter, Vital View† , Mini Mitter Co., Inc., Sunpriver, OR) were implanted subcutaneously on the ventral side; the cages (Macrolon III) were placed upon the corresponding receivers. The data were transferred directly from the receiver to a computer to allow a continuous recording of the course of skin temperature. A hot flush was defined as a quick increase of peripheral temperature of more than 1 8C (corresponding to the definition of the rise of finger temperature in climacteric women acc. to [14]).
H. Winterhoff et al. / Maturitas 44 (2003) S51 /S58
S53
Fig. 1. Incidence of hot flush equivalents in intact (open bars) and castrated (filled bars) rats. Each bar represents the number of flush equivalents per day.
The number of such hot flush equivalents was cumulated over a period of 24 h. The recording of the hot flushes was started not earlier than 8 days after the implantation of the transmitter to avoid possible influences of the anaesthesia and surgery on the hot flush equivalents. The dry extract was suspended in water with sonification and kept in a homogeneous suspension by stirring during application. It was administered once daily for 5 days in the afternoon orally by gavage in a final volume of 10 ml/kg bw. Animals were observed for clinical symptoms and body weight was determined daily to detect possible toxic effects. 2.3. Tail suspension test (TST) in mice TST was introduced as a screening test for antidepressant activity by Steru et al. [15]. Mice suspended by their tails without any contact to the ground are used as test animals. During the observation period of 6 min, there is a change between searching behaviour with intense movements and waiting behaviour with a typical motionless position, in part accompanied by ptosis. This test is derived from the Porsolt test in rats, in which a reduction of the period of learned immobility indicates antidepressant activity
[16,17]. Correspondingly, the cumulative period of immobility in TST is the test parameter which is reduced by antidepressants. Unspecific effects caused by motor stimulation are detected by an open field test performed prior to the TST. The animals (female NMRI-mice, 18/27 g bw, Harlan-Winkelmann, Borchen, Germany) were housed in groups of 5 with free access to food (Altromin 1324) and tap water; a 12 h light/dark change and a room temperature of 259/1 8C was maintained. They were acclimatized for at least 5 days before use in experiments. Test solutions were prepared directly before use by suspending the extract in water with the aid of a sonifier and maintaining a homogeneous suspension by stirring. The animals were treated with the test preparations in a volume of 10 ml/kg bw by gavage. The mice were assigned randomly to the experimental groups. The test period was recorded on a videotape which was evaluated by a person unaware of the treatment. TST was performed 60 min after one single application of the test substances. A second assay was performed after a treatment period of 8 days with daily application of the Cimicifuga extract, the last application 1 h before TST. The results were compared with those of a control group treated with tap water and an imipraminetreated group.
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H. Winterhoff et al. / Maturitas 44 (2003) S51 /S58
Fig. 2. Reduction of the number of hot flush equivalents per day by estradiolvalerat (2 mg/kg per day per os). Each bar represents the number of flush equivalents per day.
To study motility, an open field test was performed in the animals after the same treatment as for TST. Sixty minutes after gavage the mice were placed in the open field, during a 5 min observation period the number of line crossings was recorded [18,19]. 2.4. Statistics Each value represents the mean9/S.E.M. of 7/ 10 animals per group. Significance of differences to the control group were evaluated by the ANOVA test and are marked with an asterisk for values with P B/0.05.
3. Results 3.1. Telemetric measurement of flush equivalents in rats In the first step, the test parameter ‘‘flush equivalents in rats’’ had to be checked for its sensitivity. The frequency of short-term increases in skin temperature, which can be considered as a flush equivalent, was studied before and 2 weeks following castration in the same animals. As shown in Fig. 1, the number of hot flush equiva-
Fig. 3. Reduction of the number of hot flush equivalents by an ethanolic Cimicifuga extract (100 mg/kg per day). Each bar represents the number of flush equivalents per day.
lents clearly and significantly increased with the removal of the ovaries. Next, the reaction to treatments effective in patients with hot flushes had to be checked. As shown in Fig. 2, estradiolvalerate (2 mg/kg bw per day) reduced the number of hot flush equivalents. A comparable effect was seen after application of 100 mg/kg bw per day of the antidopaminergic drug veralipride (Agradil† , data not shown). These findings in rats confirm a comparable response and sensitivity of the hot flush equivalents to the hot flushes observed in women. Following oral administration of 100 mg/kg bw of the C. racemosa extract, the number of hot flush
H. Winterhoff et al. / Maturitas 44 (2003) S51 /S58
S55
Fig. 4. Skin temperature of a rat during the pretreatment period.
equivalents was clearly reduced, it returned to pretreatment values after cessation of the treatment (Fig. 3). In Fig. 4, the direct registration of skin temperature of a control period and following application of the Cimicifuga extract (Fig. 5) are shown. It is obvious that Cimicifuga extract causes not only a reduction of the incidence of hot flush equivalents but also a decrease of the variation of the course of temperature.
3.2. TST in mice Sixty minutes after gavage, both dosages of the Cimicifuga extract and the imipramine significantly reduced the period of immobility (Fig. 6). Central stimulation could be excluded because
Cimicifuga extract as well as imipramine even reduced motility in the open field test after acute application (Fig. 7). The same test was performed after an 8-day treatment period. Again the Cimicifuga extract as well as imipramine reduced the period of immobility significantly (Fig. 8), whereas the motility was no longer reduced significantly (number of line crossings for control group 919/11, for imipramine group 799/16, and for Cimicifuga extract group 769/11). As substances inhibiting the re-uptake of noradrenaline, dopamine or serotonine proved to be active in this test [20,21], the activity in this test system does not indicate the mode of antidepressant activity of Cimicifuga extracts. This has to be studied in further experiments.
Fig. 5. Skin temperature of a rat during treatment with 100 mg/kg per day of an ethanolic Cimicifuga extract.
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H. Winterhoff et al. / Maturitas 44 (2003) S51 /S58
Fig. 6. TST in female NMRI mice 1 h after gavage.
4. Discussion In our test system, hot flush equivalents can be measured in castrated female rats. They responded similarly to hot flushes in women, they were increased following castration and proved to be reduced by treatment with estrogen as well as by the antidopaminergic drug veralipride. In accor-
dance with the results in clinical reports, the ethanolic-aqueous Cimicifuga extract clearly and effectively reduced the hot flush incidence in rats. In a behavioural test system indicative for antidepressant activity, TST, Cimicifuga extract like antidepressant drugs significantly reduced the cumulative period of immobility. The effect was observed after acute and repeated treatment and
Fig. 7. Motility in the open field 1 h after gavage.
H. Winterhoff et al. / Maturitas 44 (2003) S51 /S58
S57
Fig. 8. TST in female NMRI mice after an 8-day pretreatment period.
seems to be specific, as an unspecific stimulation of locomotor activity could be excluded. Both activities of the ethanolic-aqueous Cimicifuga extract BNO 1055, anti-flush activity and hints on antidepressant activity, are in good accordance with the efficacy of the C. racemosa preparation BNO 1055 (Klimadynon† / † Menofem ) in the treatment of climacteric complaints.
References [1] Boblitz N, Liske E, Wu¨stenberg P. Traubensilberkerze DAZ 2000;140:107. [2] Mashchak CA, Kletzky OA, Artal R, Mishell DR, Jr.. The relation of physiological changes to subjective symptoms in postmenopausal women with and without hot flushes. Maturitas 1984;6(4):301 /8. [3] Kronenberg F, Cote LJ, Linkie DM, Dyrenfurth I, Downey JA. Menopausal hot flushes: thermoregulatory, cardiovascular, and circulating catecholamine and LH changes. Maturitas 1984;6(1):31 /43. [4] Vercellini P, Sacerdote P, Trespidi L. Veralipride for hot flushes induced by a gonadotropin-releasing hormone agonist: a controlled study. Fertil Steril 1994;62(5):938 /42. [5] Tulandi T, Kinch RA, Guyda H, Mazella L, Lal S. Effect of methyldopa on menopausal flushes, skin temperature, and luteinizing hormone secretion. Am J Obstet Gynecol 1984;150(6):709 /12.
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