Breast Cancer Research and Treatment 83: 221–231, 2004. © 2004 Kluwer Academic Publishers. Printed in the Netherlands. Report Growth inhibitory activity of extracts and purified components of black cohosh on human breast cancer cells Linda Saxe Einbond 1 , Masahito Shimizu 1 , Danhua Xiao 1 , Paiboon Nuntanakorn 2 , Jin T. E. Lim 1 , Masumi Suzui 1 , Colette Seter 1 , Thomas Pertel 1 , Edward J. Kennelly 2 , Fredi Kronenberg 1 , and I. Bernard Weinstein 1 1 Columbia University College of Physicians and Surgeons, New York, NY; 2 Lehman College in the Graduate Center, The City University of New York, Bronx, NY, USA Key words: Actaea racemosa, actein, Cimicifuga racemosa, cinnamic acid ester, cyclin D1, triterpene glycoside Summary The purpose of this study was to determine whether black cohosh contains constituents that inhibit the growth of human breast cancer cells, and therefore might eventually be useful in the prevention or treatment of breast cancer. Black cohosh rhizomes were extracted with methanol/water and fractionated by solvent–solvent partitioning to yield three fractions: hexane, ethyl acetate and water. The ethyl acetate fraction displayed the highest potency in two cell-based assays, growth inhibition and cell cycle analysis. This fraction inhibited growth of both the ER + MCF7 and ER - MDA-MB-453 human breast cancer cell lines with IC 50 values of about 20 and 10 μg/ml, respectively. It also induced cell cycle arrest at G1 when tested at 30 μg/ml and at G2/M at 60 μg/ml in MCF7 cells. This suggests that the extract contains a mixture of components with the more active (or more abundant) causing G1 arrest and the less active causing G2/M arrest. We then examined specific components in this extract. The triterpene glycoside fraction obtained by polyamide column chromatography, and the specific triterpene glycosides actein, 23-epi-26-deoxyactein and cimiracemoside A, inhibited growth of the MCF7 human breast cancer cells and induced cell cycle arrest at G1. The most potent compound, actein, decreased the level of cyclin D1, cdk4 and the hyperphosphorylated form of the pRb protein and increased the level of p21 cip1 in MCF7 cells, changes that may contribute to the arrest in G1. Further studies are in progress to identify the mechanisms by which actein and related compounds present in black cohosh inhibit growth of human breast cancer cells. Introduction There is growing interest in the use of herbs to aid in the maintenance of women’s health. Plants contain a wide variety of chemicals that have potent biological effects, including anticancer activity [1]. Identification of the active components, their mechanisms of action, and their possible interactions is important in order to assess their potential for clinical use and possible adverse side effects. Black cohosh (Actaea racemosa L. syn. Cimici- fuga racemosa {L.} Nutt) is a North American pe- rennial in the buttercup family (Ranunculaceae) that has been in use in Europe for the last 50 years as a natural alternative to hormone replacement therapy and for other gynecological conditions [2]. The plant grows in the understory of hardwood forests east of the Mississippi River, especially in Ohio and West Virginia [3]. Related Asian species have been em- ployed in Traditional Chinese Medicine to treat infec- tious diseases [4]. The rhizome has been in use for hundreds of years by Native Americans for a variety of purposes that include inflammatory conditions, stimu- lation of menstrual flow, dysmenorrhea, suppression of cough, treatment of diarrhea, and rheumatism [5]. American women are increasingly turning to black cohosh as a ‘more natural’ alternative to estrogen re- placement therapy, in the belief that it has the benefits,