Evaluation of Extraction Methods for Isolation and Detection of Formononetin in Black Cohosh (Actaea racemosa L.)

Black cohosh (Actaea racemosa L.) is a medicinal plant from which extracts of the roots and rhizomes (commonly known as rhizomes) have become a popular remedy for the relief of menopausal symptoms. While the chemical constituents responsible for the medicinal properties of the plant are unknown, extracts of the rhizomes are known to contain cycloartane triterpene glycosides, phenolics, and flavonoids. The possible presence of the phytoestrogenic isoflavone formononetin (7-hydroxy-4’-metoxy-isoflavone), however, could indicate that using black cohosh by women could enhance the risk of breast cancer similar to the risks associated with estrogen. Analyses for formononetin in black cohosh over the past several years, however, has been conflicting with some laboratories reporting the constituent in the rhizomes and others not. To determine whether these differences could be due to differences in the extraction and analysis methodology, extractions of the rhizomes were made with methanol, ethanol, and isopropanol and purified using a SPE C18 and DEAE Sephadex A-25 column. LC-MS-MS was used to detect the presence of formononetin using a Waters 2690 Alliance HPLC coupled with a Micromass Quattro 11 triple-quadruple mass spectrometer equipped with an electrospray source operated in the positive/negative ionization mode. The mass spectra of black cohosh constituents were obtained by infusing standard solutions into the mass spectrometer or by LC-MS, LC-MS-MS with product ion scanning. Formononetin at a concentration of <125 ng g -1 was identified in methanol extracts using LC-MS-MS with a microbore C18 column and with the ion pairs of m/z 269-253, 269-225, and 269-197. Introduction In the United States, some 25 to 40 percent of menopausal women have traditionally used some form of hormone replacement therapy (HRT) during or following menopause (Handa et al., 1996; Keating, et al., 1999; Stehman-Breen, et al., 1999). Reports during the past few years of adverse effects from HRT (Bush and Whitemen, 1999; Grodstein, et al., 1996), such as increased risk of breast and uterine cancer, have caused nationwide concern (Beckner, 2002). As result of the warnings about HRT, many women have been using botanical dietary supplements as an alternative to HRT with one of the more popular of these supplements being the roots and rhizomes (commonly referred to as rhizomes) of black cohosh (Actaea racemosa L.), a perennial plant native to the eastern woodlands of North America that was used by Native Americans as a therapeutic agent (Wood, 2000). Formononetin, an isoflavone, was initially proposed to be responsible for the pharmacological activity of black cohosh due to the potential phytoestrogenic activity of the compound (Kennely, et al., 2002). Such estrogenic activity, however, could Al-Amier et al.: Evaluation of Extraction Methods for Isolation and Detection of F