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Stability of black cohosh triterpene glycosides and polyphenols: potential clinical relevance.

ARTICLE INFO

Keywords: Black cohosh

Actaea racemosa

Cimicifuga racemosa

Stability

Shelf life

Analysis

Clinical

ABSTRACT

Concurrent to a clinical trial of black cohosh for menopausal hot flashes, the long-term stability of the black cohosh, over the duration of the clinical trial, was examined. Analytical results showed that the major constituents, both triterpene glycosides and polyphenols, were stable over the 3-year period of testing. These results indicate that a black cohosh product stored for several years in a controlled environment does not undergo significant changes in its major constituents. These results have implications not only for clinical research in natural products, but for basic science, as well as the dietary supplements industry.

[c] 2013 Published by Elsevier GmbH.

Introduction

Black cohosh, Actaea racemosa L (syn. Cimicifuga racemosa (L) Nutt), a plant native to Eastern North America, has a long history of medicinal use. Native Americans as well as early colonists in North America used the root of this species as a treatment for conditions such as general malaise, malaria, rheumatism, abnormalities in kidney function, sore throat, menstrual irregularities, and during childbirth (Barton 1798; Rafinesque 1828; Low Dog et al. 2003). However, in the recent past few decades black cohosh has become known as an herbal therapy for menopausal symptoms, particularly for hot flashes (Borrelli et al. 2003). Despite this widespread use and its sale as a common herbal supplement, the mechanism of action of this plant for hot flashes is still not known. In the past two decades, more and better designed clinical studies have been conducted but most are of short duration and relatively small, and results have been mixed with respect to outcome (Stoll 1987; Jacobson et al. 2001; Pockaj et al. 2004; Frei-Kleiner et al. 2005; Newton et al. 2006; Pockaj et al. 2006; Wuttke et al. 2006; Geller et al. 2009).

For better evaluation of the varied results from clinical trials, it is necessary to examine the long-term stability of black cohosh and whether this might change over the duration of a clinical trial, which typically takes multiple years to accrue the full complement of subjects and to run all to completion. As a parallel study to a clinical trial of black cohosh for menopausal hot flashes that was conducted at Columbia University between 2001 and 2006, we evaluated the stability of the black cohosh capsules used in the study. The major triterpene glycosides and polyphenolic constituents (Fig. 1) were analyzed during the clinical trial (from 2003 to 2005). The triterpene glycosides were chosen since most commercial products use these as marker compounds for standardization.

Our previous research on the stability of black cohosh constituents showed that triterpene glycosides in black cohosh were stable, but polyphenols changed over time (Jiang et al. 2008). Study of an 85-year-old black cohosh sample showed a similar result (Jiang et al. 2005). In that case a museum specimen of ground black cohosh root collected in 1919 was carefully examined, providing these surprising conclusions. The specimen was not carefully curated over the years, and for decades stored in a laboratory basement subject to occasional flooding. We had expected to find that this specimen had degraded significantly, but this was far from the case. In the current research we undertook a controlled study of stability over a shorter period that would reflect the time line expected for a clinical study--from sourcing of the raw materials and its chemical evaluation through the clinical trial. Our current study indicated that when sealed and maintained in a controlled room temperature of 20-25 [degrees]C (68-77 [degrees]F), a black cohosh product can be stored for several years without significant change in the polyphenols and triterpene glycoside constituents. In this paper, we report the results of this stability study.

Materials and methods

Black cohosh capsule and storage

The black cohosh capsules were maintained throughout the clinical trial by the Columbia Presbyterian Medical Center Research Pharmacy and were provided for analysis at 6-month intervals for a 3-year period. The capsules were a combined effort of Pure-World Botanicals Inc. (now, Naturex, South Hackensack, NJ) which provided the extract, and Pharmavite which encapsulated the powdered black cohosh in 2001. The capsules were stored at 20-25 C (68-77 F), in sealed bottles protected from light.

Chemicals and reagents

HPLC grade acetonitrile (J.T. Baker, Phillipsburg, NJ) and methanol (E. Merck, Darmstadt, Germany) were used for sample preparation and HPLC analysis. Reagent grade Chloroform (J.T. Baker, Phillipsburg. NJ), Dimethyl sulfoxicle (DMSO) (EMD Chemical Inc., Germany). sodium hydroxide (Fisher Scientific, NJ), and formic acid (purity >98%, E. Merck, Darmstadt, Germany) were used in this study.

Standards

23-Epi-26-deoxyactein (1) (formerly known as 27-deoxyactein, 60.74% purity) were purchased from ChromaDex (Santa Ana, USA). Caffeic acid (3) and ferulic acid (4) were purchased from Sigma Chemical Co. (St. Louis, MO). Isoferulic acid (5)(97.37% purity), fukinolic acid (6) (95.90% purity), cimicifugic acid A (7) (96.95% purity), and cimicifugic acid B (8) (93.74% purity) were isolated from the black cohosh extract (Fig. 1). The method to isolate the compounds 5-8 were described in our previous publications (Jiang et al. 2005: Jiang et al. 2006).

HPLC

The samples and standards were analyzed using HPLC with a Waters 2695 separations module (Milford, MA) equipped with a 996 photodiode array detector (PDA), and operated with Empower software. Separations were carried out on a 125 mm 4.0 mm i.d. Hypersil ODS column (Agilent, Santa Clara, CA) for triterpene glycosides and a 250 mm x 4.6 mm i.d., 5 [micro]m Aqua C18 column (Phenomenex, Torrance, CA) for polyphenolic constituents. Both analyses were performed at 25 [degrees]C with a flow rate of 1.0 ml/min. The sample volume injected was 10 [micro]l, and data was analyzed at 203 nm for triterpene glycosides, and 320 nm for polyphenols. The mobile phase for the analysis of triterpene glycosides consisted of a step gradient starting with 5% (v/v) acetonitrile (solvent A) in water (B) and increasing to 100% acetonitrile over 55 min. The gradient profile was: 0-18 min: 5-28% A; 18-36 min: 28-35% A: 36-45 min: 35-55% A; 45-55 min: 55-100% A. The solvent system for the analysis of polyphenols was composed of acetonitrile (A) and 10% aqueous formic acid (B) using a step gradient elution of 5-15% A at 0-15 min. 15% A at 15-20 min, 15-50% A at 20-50 min, and 50-100% A at 50-55 min. The UV-vis spectra were recorded from 200 to 500 nm.

LC-MS

Mass spectra were recorded on a LCQ Mass Spectrometer (ThermoFinnigan. San Jose, USA) equipped with an atmospheric pressure chemical ionization (APCI) source. APCI was performed to analyze trite rpene glycosides with the discharge current at 5 [micro]A. The vaporizer and capillary temperatures were set to 450.0 and 150.0 [degrees]C, respectively. The sheath gas and auxiliary gas, both nitrogen, had flow rates of 80 and 10 units, respectively. A mass range of 50-1000 amu was scanned.

Sample preparation for analysis

For triterpene glycosides

The content from 12 black cohosh capsules (5.3-5.6g) was dissolved with 50ml 0.5% NaOH water solution. The solution was sonicated for 1 h, and then it was transferred into a separatory funnel. The solution was partitioned with [CHCl.sub.3] (50 ml) by shaking it vigorously for 2 min, centrifuged, and the chloroform layer was transferred into a flask. The NaOH solution was partitioned 3 more times with chloroform (50 ml). All chloroform fractions were combined and evaporated, and the residue (70-90 mg) was dissolved in DMSO (10 ml) and filtered with a 0.45 [micro]m membrane filter for the further analysis.

For polyphenols

The powder from 5 black cohosh capsules (2.2-2.4g) was dissolved with 70% Me0H water solution (25 ml). After the bottle was sealed, the solution was sonicated for 5 min and then filtered into HPLC vials through a 0.45 [micro]m membrane filter.

Sample analysis

The black cohosh capsule content was analyzed with HPLC-PDA and LC-MS every six months. The capsules were studied at six time points (TP) between 2003 and 2005 and the results compared with the original PureWorld HPLC. For the quantitative analysis the amounts of four major triterpene glycosides, 1.2, and two unknown triterpene glycosides C and D (Fig. 2), were studied and calculated based on one standard compound, 23-epi-26-deoxyactein (1). Six major polyphenols (3-8) were measured based on the standard constituents 3-8 (Fig. 3).

Validation of HPLC method

Validation of this assay was in compliance with the AOAC Guidelines for Single Laboratory Validation of Chemical Methods for Dietary Supplements and Botanicals. The method was validated with respect to linearity, recovery. and sensitivity, which was reported in our previous publication (Jiang et al. 2008).

Results and discussion

The HLPC and LC-MS spectra of triterpene glycosides of the capsule are shown in Fig. 2. The amounts of four selected major triterpene glycosides are listed in Table 1. Triterpene glycosides in black cohosh could be divided into two types. One type was the saturated triterpene glycosides, such as 23-epi-26-deoxyactein (1) and actein (2). Their UV spectra often showed weak end absorption. Another type was the unsaturated triterpene glycosides with 1-3 isolated double-bonds; the typical compounds included cimicifugoside H-1 and cimiracemosicles F. The UV spectra of these compounds displayed a strong UV absorption peak at 208-225 nm. If the amounts of cimicifugoside H-1 and cimirace-moside F were determined with a standard compound such as 23-epi-26-deoxyactein (1), there would be a large error due to a significant difference between their UV absorption properties. Therefore, when one standard compound is used to quantify the total amount of triterpene glycosides in black cohosh, as is the case in certain commercial products, this calculation is likely to be inaccurate.

Table 1 Four major triterpene glycosides in the
capsule tested at six time points (TP).

Compound    Amount
           (%) (a)

          1 TP (b)  2 TP (b)   3TP (b)      4 TP      5 TP      6 TP

1         1.476 [+  1.666 [+  1.435 [+  0.671 [+  0.722 [+  0.723 [+
             or -]     or -]     or -]     or -]     or -]     or -]
             0.085     0.042     0.026     0.031     0.054     0.060

2                                       0.683 [+  0.430 [+  0.528 [+
                                           or -]     or -]     or -]
                                           0.030     0.007     0.021

C         0.948 [+  D.927 [+  0.854 [+  1.012 [+  1.045 [+  1.158 [+
             or -]     or -]     or -]     or -]     or -]     or -]
             0.008     0.050     0.079     0.015     0.027     0,025

D         1.417 [+  1.266 [+  1.135 [+  1.365 [+  1.405 [+  1.413 [+
             or -]     or -]     or -]     or -]     or -]     or -]
             0.133     0.073     0.032     0.033     0.051     0.095

Total        3.840     3.860     3.424     3.731     3.601     3.823

(a.) The percentage was calculated based on 40 mg of
extract in each capsule. Data are the mean
[+ or -] SD. and n = 3 - 7.

(b.) Compounds 1 and 2 were overlapped.


Since the standard used for the analysis of triterpene glycosides in this study was the saturated compound (23-epi-26-deoxyactein), constituents that could be included for the quantitative analyses were these major triterpene glycosides of similar structures only. From HPLC UV (or PDA) chromatograms in Fig. 2, compound A (peak A) appeared to be the major triterpene glycoside in the capsule. However, the peak area of compound A decreased significantly between HPLC and LC-MS analysis. According to the UV spectra of peak A, this compound is likely an unsaturated triterpene glycoside, and verified later to be cimirace-moside F. Therefore, it was not used for the calculation of the total amount of triterpene glycosides based on the standard compound 1.

As seen in Table 1, the amount of four selected (major) triter-pene glycosides was in the range 3.42-3.86%. Results from the six time points tested showed that these four compounds did not decrease significantly. Therefore, these triterpene glycosides were stable over the testing period. This is further confirmed by our other studies on the stability of black cohosh compounds (Jiang et al. 2005; Jiang et al. 2008). Over the course of the study, three different HPLC columns with the same stationary phase were used, accounting for small variation in the retention times. Furthermore, when the HPLC column used in this study was new, 23-epi-26-deoxyactein (1) and actein (2) could be separated. However, as the column efficiency decreased, these two compounds merged into one peak on the HPLC chromatogram of the capsule (Fig. 2).

The HPLC-PDA chromatogram for the polyphenols is shown in Fig. 3. The six major polyphenolic constituents were identified by comparing the UV spectra and a spiking experiment. The total amounts of the six major polyphenolic constituents 3-8 from the six time point tests were similar (Table 2), indicating that those compounds in the capsule were stable during the testing period.

Table 2 Polyphenols in the capsule tested in the six time points (TP).

Compound    Amount
           (%) (a)

              1 TP      2 TP      3 TP      4 TP       5TP      6 TP

3         0.034 [+  0.034 [+  0.036 [+  0.038 [+  0.031 [+  0.030 [+
             or -]     or -]     or -]     or -]     or -]     or -]
             0.002     0.002     0.002     0.000     0.001     0.000

4         0.034 [+  0.034 [+  0.033 [+  0.033 [+     0.032  0.028 [+
             or -]     or -]     or -]     or -]   1-0.000     or -]
             0.000     0.000     0.002     0.001               0.000

5         0.292 [+  0.293 [+  0.295 [+  0.293 [+  0.292 [+  0.294 [+
             or -]     or -]     or -]     or -]     or -]     or -]
             0.009     0.011     0.008     0.003     0.000     0.002

G         0.238 [+  0.234 [+  0.247 [+  0.258 [+  0.231 [+  0.257 [+
             or -]     or -]     or -]     or -]     or -]     or -]
             0.024     0.024     0.027     0.001     0.002     0.003

7         0.215 [+  0.211 [+  0.260 [+  0.257 [+  0.200 [+  0.211 [+
             or -]     or -]     or -]     or -]     or -]     or -]
             0.018     0.019     0.027     0.002     0.004     0.004

8         0.3O2 [+  0.292 [+  0.310 [+  0.311 [+  0.291 [+  0.309 [+
             or -]     or -]     or -]     or -]     or -]     or -]
             O.O15     0.020     0.023     0.002     0.005     0.003

Total        1.115     1.098     1.181     1.190     1.077     1.129

a The percentage was calculated based on 40 mg of
extract in each capsule. Data are the mean
[+ or -] SD, and n = 3 - 9.


As a comparative study, we also examined phytochemical profile of the polyphenols from the black cohosh extract from Pure-World Botanicals, Inc (now Naturex) which was used to produce the capsule. HPLC chromatograms of the powdered extract and native ethanolic extract (without excipient) are shown in Fig. 3C and D. The HPLC analysis indicated that there were high ratios of compounds 6-8 in the native ethanolic extract (Fig. 3D). However, the same ratios significantly decreased in the powdered extract, especially in the capsule (Fig. 3C and B), suggesting that some major polyphenols decomposed during the production of the black cohosh capsule. This result was also in agreement with our previous study (Jiang et al. 2008). As for the differences observed in the HPLC chromatograms for the powered extract and the capsule extract (Fig. 3B and C), this was caused by the excipients in powdered extract put into the capsule. According to our previous study, the excipient tricalcium phosphate, could selectively absorb fukiic acid ester derivatives, thus preventing these compounds from being extracted with aqueous alcohol (Jiang et al. 2008). Therefore, the HPLC chromatogram for the polyphenols of the extract with excipient in the capsule was different from that of the extract alone, without excipient.

Our previous study indicated that polyphenolic constituents from black cohosh were usually unstable. These compounds tended to be oxidized or hydrolyzed depended on the storage conditions (Jiang et al. 2005; Jiang et at. 2008). When in open-air, the major polyphenols decomposed significantly, especially in a high temperature and high moisture environment. However, such decomposition could be reduced if samples were stored in a sealed container (Jiang et at. 2008). The black cohosh capsule used in the clinical study was stored in constant temperature, and unsealed just before stability test, and therefore, most constituents were stable.

The method for the quantitative analysis of polyphenols by HPLC-PDA was validated with respect to linearity, recovery, and sensitivity. Detailed results are available in our previous paper (Jiang et at. 2008).

Black cohosh triterpene glycosides and polyphenols were stable over the 3-year period of the clinical trial. This indicates a black cohosh product can be stored for several years in a controlled environment without significant change in these compounds. These data also suggest that the differing results of the clinical trials with respect to efficacy for treating hot flashes are most likely not due to the instability of the compounds tested in this current study. These compounds were chosen to include triterpene glycosides used in the standardization of commercial products. However, for products marketed for hot flashes, these are marker compounds for plant identity. We do not know which compounds are responsible for the biological activity (in this case--on hot flashes). Since we still do not know the mechanism of hot flashes, we cannot test any particular substances for biological activity without a relevant target.

These results have implications for research on natural products, and for commercial products. As Chen et al. (2008) point out in their report on the steps taken for FDA approval of the first new drug application (NDA) of a botanical in contemporary times, the tea plant (Camellia sinensis (L.) Kuntze) as a topical treatment for perianal and genital condyloma, there are many aspects of a plant's biology and provenance that need to be documented in order to assure batch to batch consistency during the trial and into the market.

Acknowledgement

This project was supported by grant #P50-AT00090 from the National Institutes of Health-National Center for Complementary and Alternative Medicine (NIH-NCCAM). Its conclusions are solely those of the authors and do not necessarily reflect the official views of NIH-NCCAM.

* Corresponding author. Tel.: +1 718 960 1105: fax: +1 718 960 8236.

E-mail addresses: edward.kennelly@lehman.cuny.edu, kennelly@lehman.cuny.edu (EJ. Kennelly).

1 Current affiliation: Dali University, College of Pharmacy, Dali 671000. Yunnan, China.

2 Current affiliation: Stanford University School of Medicine, Stanford, CA, USA.

0944-7113/$ - see front matter [c] 2013 Published by Elsevier GmbH. http://dx.doi.org/10.1016/j.phymed.2012.12.011

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Bei Jiang (a), (b), (1) Fredi Kronenberg (a), (2), Michael J. Balick (c), Edward J. Kennelly (b), *

(a) The Richard and Hinda Rosenthal Center for Complementary & Alternative Medicine. College of Physicians &Surgeons, Columbia University. New York, NY 10032, USA

(b) Department of Biological Science, Lehman College and The Graduate Center, City University of New York. Bronx, NY 10468. USA

(c) The New York Botanical Garden. Bronx. NY 10458, USA
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Author:Jiang, Bei; Kronenberg, Fredi; Balick, Michael J.; Kennelly, Edward J.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:1USA
Date:Apr 15, 2013
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