100 A. PANOSSIAN ET AL. Copyright © 2004 John Wiley & Sons, Ltd. Phytochem. Anal. 15: 100–108 (2004) PHYTOCHEMICAL ANALYSIS Phytochem. Anal. 15, 100–108 (2004) Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002.pca.742 Copyright © 2004 John Wiley & Sons, Ltd. Received 21 February 2003 Revised 28 April 2003 Accepted 21 May 2003 Methods of Phytochemical Standardisation of Rhizoma Cimicifugae racemosae A. Panossian, 1 * A. Danielyan, 1 G. Mamikonyan 1 and G. Wikman 2 1 ‘ExLab’ Expert Analytical Laboratory of Armenian Drug Agency, 49/4 Komitas Str., Yerevan, 375051 Armenia 2 Scandinavian Herbal Institute, Gothenburg, Sweden The methanolic extract of roots of Cimicifuga racemosa and its methanolysis products have been analysed by GC-MS. 2-Hexylcyclopropaneoctanoic acid (9,10-methylenehexadecanoic acid) was found to be the marker most specific for the identification for this herb. For the phytochemical standardisation of Rhizoma Cimicifugae racemosae, validated methods for the quantitative analysis of formononetin (by TLC-fluorometry), of isoferulic acid (by GC-MS of the methyl ester), and of total triterpene glycosides (transformed to coloured complexes and measured photometrically) in roots of C. racemosa have been developed. The contents of formononetin, isoferulic acid and total triterpene glycosides (measured as actein) in the herb ranged from 0.0031 to 0.0035, from 1.22 to 1.35 and from 20.09 to 22.06 mg/g dry weight, respectively. Copyright © 2004 John Wiley & Sons, Ltd. Keywords: TLC-fluorometry; GC-MS; 2-hexylcyclopropaneoctanoic acid; 9,10-methylenehexadecanoic acid; formononetin; isoferulic acid; actein; Rhizoma Cimicifugae racemosae; Cimicifuga racemosa. * Correspondence to: A. Panossian, ‘ExLab’ Analytical Laboratory of Armenian Drug Agency, 49/4 Komitas Str., Yerevan 375051, Armenia. Email: phanos@sci.am 2002). The purpose of the current study was to develop sensitive quantitative assay methods with which to determine the amounts of triterpenes, formononetin and isoferulic acid in roots of C. racemosa. EXPERIMENTAL General procedures All solvents used for extraction and chromatography were of HPLC grade. Ethyl acetate, glacial acetic acid, chloroform and benzene were from Carl Roth (Karlsruhe, Germany), and methanol, toluene and propyl p -hydroxybenzoic acid were from Aldrich (Munich, Ger- many). Water used in HPLC mobile phases was distilled and subsequently filtered through a 0.45 μm membrane (Millipore, Bedford, MA, USA). Reference standards of formononetin (3) and isoferulic acid (2) were purchased from Extrasynthese (Genay, France). Actein (1), used for the determination of the standard curve, was purified from root extracts of C. racemosa and authenticated by NMR in comparison with authentic material (Wehrli and Nishida, 1979; Tables 1 and 2). The 1 H-NMR (at 330.077 MHz), HH-COSY, CH-correlated 2D-NMR (HMQC), 13 C-NMR and DEPT (at 75.46 MHz) spectra of 1 were measured in pyridine-d 5 or acetone-d 6 using a Varian (Palo Alto, CA, USA) Mercury 300 spectrometer. Superclean LC-18 3 mL tubes were obtained from Supelco (Bellefonte, PA, USA; lot number SP2030F). Plant materials Rhizoma Cimicifugae Racemosae (HE 10127) was pur- chased from Heinrich Klenk (Schwebheim, Germany; batch number 6721). Voucher specimens have been deposited in the herbarium of the Swedish Herbal Institute, Gothenburg. INTRODUCTION Rhizoma Cimicifugae Racemosae, black cohosh root, refers to the rhizome of Cimicifuga racemosa L. Nutt. (syn. Actaea racemosa L.; Ranunculaceae). This herb is a promising natural alternative to hormone replacement therapy for the relief of a variety of dysmenorrhoeal or climacteric (menopausal) symptoms (Jarry and Harnischgeger, 1985; Jarry et al., 1985; Stoll, 1987; Lehmann-Willenbrock and Riedel, 1988). Clinically ob- served activities (Stoll, 1987; Lehmann-Willenbrock and Riedel, 1988) of standardised black cohosh extracts (for example, Remifemin™, Klimadynon ® and CimiPure ® ) are often ascribed to the isoflavonoids formononetin (3) and genistein 4′ methyl ester (Jarry and Harnischgeger, 1985; Jarry et al., 1985) and/or to pentacyclic triterpene glycosides, namely actein (1) and the cimiracemosides A, B, C, E, F, G and H (Bradley, 1992; Bruchhausen et al., 1999; Dictionary of Natural Products on CD ROM, 2002). A related species, C. dahurica Maxim, has been used in traditional medicine to induce diaphoresis and promote skin eruption (Huang, 1993), and this plant also exhibited hypoglycaemic and anti-inflammatory activities in animals (Leung and Foster, 1996). Further chemical study of the phenolic compounds of C. dahurica has led to the isolation of a hypoglycaemic constituent isoferulic acid (2) (Liu et al., 1999). Since their initial isolation, no analytical assay has been published for the precise quantification of com- pounds 1–3 in samples of C. racemosa and related species. However, the concentration ranges of these com- pounds in plant preparations need to be established in order to prepare monographs on the medicinal plants. Furthermore, the actual presence of 3 in species of Cimicifuga has been questioned recently (Kennelly et al.,