Chapter 7
Species Identification of Black Cohosh by L C - M S for Quality Control Kan He, Bo Lin Zheng, Calvin Hyungchan Kim, Ling-ling Rogers, and Qun Yi Zheng* Department of Research and Development, Pure World Botanicals, Inc., 375 Huyler Street, South Hackensack, NJ 07606
A method to directly identify triterpene glycosides using reversed-phase liquid chromatography with positive atmospheric pressure c h e m i c a l ionization mass spectrometry (LC/(+)APCIMS) was developed. Based on the analysis of the molecular weight, fragment ions, and selected ion chromatograms, a number of triterpene glycosides, including actein (1), 27-deoxyactein (2), cimicifugoside M (3), and cimicifugoside (4), from Cimicifuga racemosa were studied. Cimicifugoside M was further isolated and was identified as a new triterpene glycoside, cimigenol 3-O-α-L-arabinopyranoside. Meanwhile, a chromone, cimifugin (5), was also isolated from C. foetida. Cimicifugoside M and cimifugin can serve as markers for species identification. The method can therefore be used to distinguish black cohosh products from different Cimicifuga species for quality control purposes. The four triterpene glycosides (1-4) were evaluated for the estrogen— receptor-binding activity and it was found that they do not bind to estrogenic receptors.
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© 2002 American Chemical Society
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Introduction Cimicifuga racemosa (L.) Nutt (Ranunculaceae), commonly known as black cohosh, is a Native American medicinal plant. Its roots and rhizomes have long been used to treat a variety of women's complaints, especially painful periods, menopausal disorders, premenstrual complaints, dysmenorrhea and uterine spasms in the 19 century. The modern preparation of black cohosh extract is the major component found in commercial European preparations for menopausal disorder and has been widely used in Europe for over 40 years in over 1.5 million cases (7). Numerous clinical studies using Remifemin for the black cohosh extract have confirmed its efficacy as an alternative to estrogen treatment. According to the German Commission Ε Monographs, black cohosh consists of dried rhizomes and roots of Cimicifuga racemosa (2). Currently, the most common commercially available preparation of black cohosh is a powdered extract which is standardized for its content of triterpene glycosides (calculated as 27-deoxyactein). Some products using Asian Cimicifuga species, such as C. foetida, C. simplex, or a combination of species are also found on the marketplace. A n H P L C analytical method with U V and E L S D detectors for analysis of triterpene glycosides has been developed. However, this system is still unable to identify triterpene glycosides using the above mentioned detectors without the availability of standards of each peak. This leads to difficulty of species identification due to the complicated H P L C chromatograms they presented. Therefore, it is necessary to find some unique marker compounds which can serve as specific indicators to distinguish different species in the genus. Not much chemical information on Cimicifuga racemosa can be found in early literature. Only actein (1), 27-deoxyactein (2), and cimicifugoside (4) were reported from black cohosh ( J- 7). We have isolated these three major triterpene glycosides during the analytical method development (Figure 1). In addition, the compound cimicifugoside M (3), was isolated and identified as a new triterpene glycoside based on N M R spectroscopic data. A number of triterpene structures have been recently elucidated by two groups (8-9), adding to 18, the total number of triterpene glycosides in the black cohosh. The presence of complicated matrix interferences in the black cohosh extract and low U V absorption due to the absence of a strong chromophore in the triterpene structures makes the identification of these compounds difficult. Using an atmospheric pressure chemical ionization (APCI) as an H P L C detector has become the method of choice for the analysis of triterpene glycosides in black cohosh. APCI is an ionization technique in which the analyzed molecules react with reagent ions at atmospheric pressure. The reagent ions are formed via solvent vaporization and subsequent interaction with high voltage, these creating a corona discharge. The formed ions are then transmitted through an electrostatic field and captured in the ion trap. APCI is a soft ionization technique which provides little or no fragmentation for thermally stable compounds. However, due to the high temperature needed for vaporization (450°C), both the molecular ion and th
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fragmentation ions of the thermally unstable triterpene glycosides will be detected by mass spectrometer. Cimicifuga species are known to contain highly oxygenated triterpene glycosides. The oxygen atoms provide the important fragment ions which are derived from the loss of a water, a sugar, or an acetyl group. Identification of the compound is based on a group of fragment ions. We have used this method to identify several triterpene glycosides and found that one triterpene, cimicifugoside M , which was only detected in C. racemosa, and a nontriterpene glycoside, cimifugin, which was only detected in C. foetida and C. simplex can specifically serve as markers for species differentiation. We have applied this method to check more than 30 commercially available black cohosh powder extracts. The results of our analyses are presented and discussed in the following sections.
Figure 1. Triterpene glycosides (1-4) isolated from C. racemosa. Cimifugin (5) isolated from C foetida.
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Identification of New Triterpene Glycoside The four pure triterpene glycoside standards used in the L C / M S experiment were actein (1), 27-deoxyactein (2), cimicifugoside M (3) and cimicifugoside (4). These standards were isolated from Cimicifuga racemosa and were compared by Ή and C N M R data with literature (10-13). Cimicifugoside M is a new compound which was first isolated from C. racemosa and the spectral data are listed as follows. H R - F A B M S : found: 621.3996; calcd. for C H 0 621.3927. I R v 3448,2942,1642,1455, 1383,1255,1167,1149,1070 cm . H-NMR(400MHz,pyridine-d ): δ = 1.24,1.57 (m, 2H, H-l), 1.94 (qd, 1H, J = 11.6, 3.6 Hz, H-2), 2.36 (m, 1H, H-2), 3.48 (dd, 1H, J = 11.6,4.4 Hz, H-3), 1.31 (dd, 1H, J = 12.4, 3.6 Hz, H-5), 0.71 (br. q, 1H, J =12.4 Hz, H-6), 1.51 (m, 1H, H-6), 1.19,2.06 (m, 2H, H-7), 1.70 (m, 1H, H-8), 1.08,2.05 (m,2H,H41), 1.54,1.68 (m,2H,H-12),4.24(d, 1H, J = 8.6 Hz, H - l 5), 1.50 (m, 1H, H-17), 1.14 (s, 3H, H-l8), 0.27, 0.51 (d, 2H, J = 4.0 Hz, H-19), 1.68 (m, 1H, H-20), 0.84 (d, 3H, J = 6.4 Hz, H-21), 1.04,2.25 (m, 2H, H-22), 4.74 (d, 1H, J = 9.2 Hz, H 23), 3.75 (s, 1H, H-24), 1.45 (s, 3H, H-26), 1.47 (s, 3H, H-27), 1.17 (s, 3H, H-28), 1.26 (s, 3H, H-29), 1.01 (s, 3H, H-30), 4.78 (d, 1H, J = 7.0 Hz, Η-Γ), 4.44 (dd, 1H, J =7.4, 7.0 Hz, H-2'), 4.15 (br. d, 1H, J = 7.6 Hz, H-3'), 4.31 (br. s, 1H, H-4 ), 3.78, 4.29 (dd, 2H, J = 10.4 Hz, H-5 ). C NMR(400 M H z , pyridine-d ): δ = 32.46 (C-l), 30.11 (C-2), 88.63 (C-3), 41.38 (C-4), 47.63 (C-5), 21.10 (C-6), 26.38 (C-7), 48.66 (C-8), 20.04 (C-9), 26.70 (C-10), 26.48 ( C - l l ) , 34.11 ( C-12), 41.88 (C-13), 47.32 (C-14), 80.25 (C-15), 112.00(C-16), 59.61 (C-17), 19.54(C-18),30.90(C-19),24.13 (C-20), 19.61 (C-21), 38.19 (C-22), 71.86 (C-23), 90.21 (C-24), 70.98 (G-25), 25.44 (C-26), 25.21 (C-27), 11.84 (C-28), 27.77 (C-29), 15.43 (C-30), 107.49 (C-Γ), 72.98 (C-2') 74.68 (C-3'), 69.55 (C-4'), 66.77 (C-5 ). A l l the protons and protonated carbons in 3 are assigned based on a combination of data obtained by DEPT, Ή - Ή COSY, TOCSY, H M Q C , HSQC, and R O S E Y N M R spectra. These assignments are supported by the close similarity of the Ή and C N M R spectroscopic data with those obtained for cimicifugoside (4). The partial structures deduced from different spin systems by the Ή - Ή correlation spectroscopy are connected by analysis of an H M B C spectrum. As shown in Figure 2, the methyl protons at δ 1.26 and 1.01 (CH -29 and CH -30) show long-range correlations with the carbons at δ 41.38 (C-4), 88.63 (C-3), and 47.63 (C-5). This indicates that C-4 is connected to C-29, C-30, C-3, and C-5. The methyl protons at δ 1.14 (CH -18) exhibit long-range correlations with the carbons at δ 41.88 (C-13), 34.11 (C-12), 47.32 (C-14), and 59.61 (C-17), suggesting that C-18, C-12, C-14, and C-17 are connected with C-13. Similarly, the correlation of the protons at CH -28 (δ 1.17) with C-14, C-13, and C-15 (δ 80.25) indicates that C-14 is connected to C-28, C-13, and C-15. Also, the methyl signal at δ 0.84 (CH -21) is correlated with the signals at δ 24.13 (C-20), 59.61 (C-17), and 38.19 (C-22), indicating that C-20 is connected to C-21, C-17, and C-22. Furthermore, methyl signals at δ 1.45 (CH -26) and 1.47 (CH -27) display long-range correlations with carbons at δ 70.98 (C-25) and 90.21 13
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94 (C-24) indicating that C-25 is connected with C-26, C-27,and C-24. correlations give further evidence that 3 possesses a cimigenol aglycone.
These
Figure 2. Partial structures of 3 and significant long-range correlations observed in the HMBC spectrum.
The relative stereochemistry of 3 is determined by analysis of the R O S E Y and N O E difference spectra as well as the coupling constants. A l l the N O E correlations indicate the structural configuration as shown in the Figure 3. Combining all the spectroscopic data, cimicifugoside M is determined to be cimigenol 3-0-a-Larabinopyranoside.
Figure 3. Structural configuration of 3 and some important NOE correlations observed in the ROESY and NOE difference spectra.
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LC/MS Analysis of the Triterpene Glycosides and Cimifugin Structurally, there are two major types of triterpene glycosides based on the literature report. Actein with acteol aglycone contains a xylosyl and cimicifugoside with cimigenol aglycone which contains a xylosyl or arabinosyl. LC/(+) APCI-MS experiments were initially carried out on four pure triterpene glycosides (1-4). Both actein and 27-deoxyactein contain an acetyl group at the 12-position; therefore, the loss of an acetyl group provides useful diagnostic information for the presence of these two compounds. In the mass spectrum of actein (Figure 4), the pseudo molecular ion [MH] was observed at m/z 677. The other major peaks observed at m/z 659 and 599 were derived from the loss of water (18 amu) from [MH] and the subsequent loss of acetic acid (60 amu), respectively. A peak at m/z 527 is derived from a loss of xylosyl from [MH] , which provides the evidence of the existence of acteol aglycone. Other fragment ions observed at m/z 467,449 and 432 were formed from [MH] by loss of an acetyl and xylosyl group followed by two successive losses of water. Some minor peaks, due to a loss of water or acetic acid from [MH] or other fragment ions, were also observed. The suggested pathway involves the protonation of an oxygen atom followed by a series of neutral molecule losses. The oxygen atom in the ring undergoes ring opening followed by water loss. In the spectrum of 27-deoxyactein, the major peak which is due to a loss of an acetyl group was observed at m/z 601. The loss of an additional xylosyl group followed by three successive losses of water results in the fragments observed at m/z 469,451,433 and 415, respectively. Since the difference in molecular weight between actein and 27deoxyactein is 16 amu, the loss of a water molecule (-18 amu) always produces a fragment with a m/z difference of 2 amu when compared to the corresponding fragment ions. This provides diagnostic information which is useful in distinguishing these two compounds. In the mass spectra of cimicifugoside M and cimicifugoside, the pseudo molecular ion [ M H ] is observed at m/z 621. Loss of water molecules from both the molecular ions and the fragment ions gives the characteristic fragmentation patterns of these two compounds. The structural difference between cimicifugoside M and cimicifugoside is due to the different sugar moieties, arabinosyl and xylosyl. Since the molecular weights of the sugars are the same, it is not possible to distinguish them based on the fragment ions, but they can be distinguished by the differences in retention time. The protonated molecular ion of actein [ M H ] observed at m/z 677 is weak, but an intense series of peaks is produced by a group of characteristic fragment ions generated by neutral molecule losses. These fragment ions which are used for identification purposes are observed at m/z 677,659, 617, 599, 581,527,467,449 and 431. Similarly, identification of 27-deoxyactein can be made based on the presence of the molecular ion and other fragment ions observed at m/z 661, 601, 583,469,451,433 and 415. Peaks observed at m/z 621, 603, 585, 512,489,471, 453, 435 and 417 indicated the presence of cimicifugoside M or cimicifugoside. +
+
+
+
+
+
+
96 A n alcoholic extract of C. racemosa was scanned under the (+)APCIMS mode and more than 20 peaks were resolved by the H P L C condition. Many of them could be positional, steric isomers, or structural difference, such as xylosyl and arabinosyl substitute, which have the same molecular weight and similar fragment patterns to those of actein, 27-deoxyactein, or cimicifugoside. A s discussed previously, these fragments were derived from the water, sugar, or acetyl loses and only those peaks which showed the same fragment patterns with standards were related to triterpene glycosides. In this way, we found that all of the major peaks seen in the total ion current, displayed black cohosh triterpene fragment patterns. Using the molecular ion and fragment ion information obtained from (+) A P C I / M S , most triterpene glycosides of C. racemosa can be classified. However, the absolute structural identification of each peak from L C still has to be based on the direct comparison of each standard.
467.5 449.6
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Figure 4. Total ion chromatogram and mass spectra of actein (1), 27deoxyactein (2), cimicifugoside M (3), and cimicifugoside (4).
97 Two compounds were eluted (t around 12 min and 13 min) in the alcoholic extract of C. foetida. The U V spectra of both compounds exhibits absorption maxima at the 233,251,256 and 298 nm. These two components were not related to triterpene glycosides since no characteristic fragments were found. The first peak shows the M H ion at m/z 469 and its aglycone M H at m/z 307 derived from losing a glucosyl while the second peak (5) only displays the molecular ion at m/z 307 in the mass spectra. We isolated 5 from C. foetida and its structure was elucidated as cimifugin based on the spectroscopic method, mainly N M R . Both these two compounds, cimifugin and cimifiigin 7-
