Development of a Fast and Convenient Method for the Isolation of Triterpene Saponins from Actaea racemosa by High-speed Countercurrent Chromatography Coupled with Evaporative Light Scattering Detection

Abstract

In the present work, a fast and simple method for the separation and purification of triterpene saponins from Actaea racemosa was successfully established. Accelerated solvent extraction was used for defatting and extracting of the subaerial parts, giving a triterpene enriched crude extract. Size exclusion chromatography was used to separate actein and 23‐epi-26‐deoxyactein from other triterpenoids, which were collected in a third fraction. This most complex third fraction was applied to high-speed countercurrent chromatography, a well-established technique for the separation of saponins. Separation parameters were first optimized on an analytical level, using a hyphenated HSCCC-ELSD setup, before the system was scaled up to preparative size. The resulting two-phase solvent system, consisting of n‐hexane-acetone-ethyl acetate-2‐propanol-ethanol-water (3.5 : 1 : 2 : 1 : 0.5 : 2, v/v/v/v/v/v), enabled the isolation of 23-O-acetylshengmanol-3‐O-β-D-xylopyranoside (17.4 mg), cimiracemoside D (19.5 mg), 25-O-acetylcimigenol-3-O-β-D-xylopyranoside (7.1 mg) and the aglycone cimigenol (5.9 mg). Purity of the isolated substances was 96.8 %, 96.2 %, 97.9 %, and 98.4 %, respectively. The same method was suitable for the purification of actein and 23-epi-26-deoxyactein, with purities of 97.0 % and 98.3 %.

Key words

Actaea racemosa - Ranunculaceae - triterpene saponins - countercurrent chromatography - evaporative light scattering detection

References

• 1 Morelli V, Naquin C. Alternative therapies for traditional disease states: menopause.  Am Fam Physician. 2002;  66 129-134
• 2 Stammwitz U. Heilpflanze für das Klimakterium.  Pharm Ztg. 1993;  34 36-38
• 3 Wade C, Kronenberg F, Kelly A, Murphy P A. Hormone-modulating herbs: implications for women's health.  J Am Med Womens Assoc. 1999;  54 181-183
• 4 Borelli F, Ernst E. Cimicifuga racemosa: a systematic review of its clinical efficacy.  Pharmacol Res. 2008;  58 8-14
• 5 Palacio C, Masri G, Mooradian A D. Black cohosh for the management of menopausal symptoms: a systematic review of clinical trials.  Drugs Aging. 2009;  26 23-36
• 6 McKenna D J, Jones K, Humphrey S, Hughes K. Black cohosh: efficacy, safety, and use in preclinical applications.  Altern Ther Health Med. 2001;  7 93-100
• 7 Düker E M, Kopanski L, Jarry H, Wuttke W. Effects of extracts from Cimicifuga racemosa on gonadotropin release.  Planta Med. 1991;  57 420-424
• 8 Chen S N, Li W, Fabricant D S, Santarsiero B D, Mesecar A, Fitzloff J F, Fong H H S, Farnsworth N R. Isolation, structure elucidation, and absolute configuration of 26-deoxyactein from Cimicifuga racemosa and clarification of nomenclature associated with 27-deoxyactein.  J Nat Prod. 2002;  65 601-605
• 9 Chen S N, Fabricant D S, Lu Z Z, Fong H H S, Farnsworth N R. Cimiracemosides I–P, new 9,19-cyclolanostane triterpene glycosides from Cimicifuga racemosa.  J Nat Prod. 2002;  65 1391-1397
• 10 Shao Y, Harris A, Wang M, Zhang H, Cordell G A, Bowman M, Lemmo E. Triterpene glycosides from Cimicifuga racemosa.  J Nat Prod. 2000;  63 905-910
• 11 Wende K, Mügge C, Thurow K, Schöpke T, Lindequist U. Actaeaepoxide 3-O-β-D-xylopyranoside, a new cycloartane glycoside from the rhizomes of Actaea racemosa (Cimicifuga racemosa).  J Nat Prod. 2001;  64 986-989
• 12 Hamburger M, Wegner C, Bentin B. Cycloartane glycosides from Cimicifuga racemosa.  Pharm Pharmacol Lett. 2001;  2 98-100
• 13 Bedir E, Khan I. Cimiciracemosid A: a new cyclostanol xyloside from the rhizome of Cimicifuga racemosa.  Chem Pharm Bull. 2000;  48 425-427
• 14 Watanabe K, Mimaki Y, Sakagami H, Sashida Y. Cyloartane glycosides from the rhizomes of Cimicifuga racemosa and their cytotoxic activities.  Chem Pharm Bull. 2002;  50 121-125
• 15 Kusano A, Takahira M, Shibano M, In Y, Ishida T, Miyase T, Kusano G. Studies on the constituents of Cimicifuga species. XX. Absolute stereostructures of cimicifugoside and actein from Cimicifuga simplex WORMSK.  Chem Pharm Bull. 1998;  46 467-472
• 16 Shi S, Jiang D, Zhao M, Tu P. Preparative isolation and purification of triterpene saponins from Clematis mandshurica by high-speed counter-current chromatography coupled with evaporative light scattering detection.  J Chromatogr B. 2007;  852 679-683