The effects of Black Cohosh ( Cimicifuga racemosa) on bone in ovarectomized rats

1. The effects of Black Cohosh (Cimicifuga racemosa) on bone in ovarectomized rats By: Mindi J. Littleton Introduction Materials and Methods Results Estrogen receptors are found throughout most of the body. Since these receptors are present in different tissues in the body, there are numerous problems that arise when an estrogen deficiency occurs during menopause and post- menopause in women, one of the more severe of which is osteoporosis. The reduction of estrogen in women causes a decrease in osteoblast activity. This decrease causes osteoclasts to be more predominately produced which causes loss of bone resulting in osteoporosis2. One of the main, short term preventive treatments for osteoporosis is hormone replacement therapy (HRT)3. However, HRT causes an increased risk of cardiovascular disease, breast cancer, and endometrial cancer4. HRT also can cause vaginal dryness, decreased sexual desires, and uterine bleeding4, which has caused women to look for alternative treatments, such as Cimicifuga racemosa. Many studies have demonstrated the beneficial effects of Cimicifuga racemosa on ovarectomized rats. They have shown that Cimicifuga racemosa has preventive effects, such as reversal of bone-loss, bone fracture resistance, and an increase in overall bone quality2. It has also been shown that Cimicifuga racemosa does not cause an increase in the uterine weight, suggesting that this treatment does not produce estrogenic effects1. One large problem with Cimicifuga racemosa is that it is not regulated by the Food and Drug Administration. This lack of regulation allows the herbal treatment to be used without any clinical trials being performed. This lack of testing gives reason for this experiment to be performed to show if Cimicifuga racemosa actually prevents osteoporosis. Twenty-six female Sprague-Dawley rats were obtained from the Zivic-Miller Laboratory at 3 weeks of age. The rats were housed in standard laboratory caging (49x31x17.5 cm) with food and water available ad libitum. The twenty-six rats were divided into four groups, all of the groups were ovarectomized after the rats reached an average weight of 200 grams. The ovarectomies were performed using 1mL of Ketamine for anesthesia. The rats were then allowed to heal for seven days before dosing. All of the rats were fed a phytoestrogen free diet (AIN-93G) obtained from Purina test diets after the ovarectomies were performed. Each rat was given 0.1mL of Ketamine before each dosage. The rats were then gavaged with either 3mL of physiological saline alone or 3mL of physiological saline with the equivalent daily dosage. Group 1 was the control group containing eight rats receiving physiological saline without the presence of Cimicifuga racemosa. The second group of six rats received a dosage equal to the equivalent of one recommended daily dosage of Cimicifuga racemosa based on the average mg/kg body weight. The third group of six rats received the equivalent of five dosages of Cimicifuga racemosa (mg/kg body weight), the fourth group of six rats received the equivalent of ten dosages of Cimicifuga racemosa (mg/kg body weight) with each gavage. Each rat was given the equivalent dosage every other day for four weeks using an 18 gage gavage needle. After the four week dosing period the rats were sacrificed and both femurs were removed. A random femur from each rat was dried and weighed. A water volume measurement was taken by placing a femur in a graduated cylinder filled with water and recording the difference in the water level. The weight and volume measurement were then used to calculate a density measurement. The femurs were then x-rayed and the length was measured using a ruler on the hard copy of the x-ray. A radiological density of the femurs was then measured in photoshop CS2 by taking a 12 by 12 pixel measurement in two places on the bone shaft. The density was recorded from the histogram in photoshop then the two measurements were averaged. The results were tested using an Analysis of Variance Test (ANOVA). All of the rats survived the ovarectomies. There were no observed differences between groups in external or internal physical appearance. There also were no observable behavioral differences between groups. The Anova test was run between groups for the weight to volume ratio, the density value, and the weight to length ratio. The weight to volume ratio showed no statistical difference with a significance value of 0.26 (see Table 1). There was no statistical difference in the density measurement with a significance value of 0.18 (see Table 1). No statistical significance was seen in the weight to length ratio with a significance value of 0.17 (see Table 1). The lack of significance can also be seen with the graphs to the left. Conclusion Based on these results the hypothesis that Black Cohosh (Cimicifuga racemosa) will prevent the development of osteoporosis was rejected. References 1. Kretzschmar G, Nisslein T, Zierau O, Vollmer G. 2005. No estrogen-like effects of an isopropanolic extract of Rhizoma Cimicifugae racemosae onuterus and vena cava of rats after 17 day treatment. Journal of Steroid Biochemistry and Molecular Biology. 97: 271-277. 2. NiBlein T, and Freudenstein J. 2003. Effects of an Isopropanolic extract of Cimicifuga racemosa on urinary crosslinks and other parameters of bone quality in an ovariectomized rat model of osteoporosis. Journal of Bone and Mineral Metabolism. 21: 370-376 3. Seidolva-Wuttke D, Jarry H, Becker T, Christoffel V, and Wuttke W. 2003b. Pharmacology of Cimicifuga racemosa extract BNO 1055 in rats: bone, fat and uterus. Maturitas. 1: 39-50. 4. Wuttke W, Seidlova-Wuttke D, Gorkow C. 2003. The Cimicifuga preparation BNO 1055 vs. conjugated estrogens in a double-blind placebo-controlled study: effects on menopause symptoms and bone markers. Maturitas. 1: 67-77. Hypothesis Black Cohosh (Cimicifuga racemosa) will prevent the development of osteoporosis in ovarectomized rats. Acknowledgements Marietta College Biology Department Dr. David Brown Dr. Peter Hogan Dr. Steven Spilatro Dr. David McShaffrey Marietta Psychology Department Dr. Jennifer McCabe Marietta Memorial Hospital: Radiology Department Amiee Phillips Special Thanks to: Rhonda Littleton Brandon Vrable Diana Rohlman