Fitoterapia 82 (2011) Contents lists available at ScienceDirect. Fitoterapia. journal homepage:

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1 Fitoterapia 82 (2011) Contents lists available at ScienceDirect Fitoterapia journal homepage: Comparative analysis of the chemical constituents of two varieties of Pueraria candollei Gorawit Yusakul a, Waraporn Putalun a,, Orapin Udomsin a, Thaweesak Juengwatanatrakul b, Chaiyo Chaichantipyuth c a Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen, 40002, Thailand b Faculty of Pharmaceutical Sciences, Ubon Rajathanee University, Ubon Ratchathani 34190, Thailand c Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, 10330, Thailand article info abstract Article history: Received 28 April 2010 Received in revised form 8 September 2010 Accepted 14 September 2010 Available online 18 September 2010 Keywords: Pueraria candollei Isoflavonoid HPLC A high-performance liquid chromatography (HPLC) method was developed to determine the contents of miroestrol and deoxymiroestrol in the tubers of Pueraria candollei var. mirifica and P. candollei var. candollei. The linear detection ranges were μg/ml for miroestrol and μg/ml for deoxymiroestrol. The limit of detection (LOD) and limit of quantification (LOQ) were 0.2 and 0.78 μg/ml, respectively, for miroestrol and 0.78 and 1.56 μg/ml, respectively, for deoxymiroestrol. Our results suggest that both varieties of P. candollei can produce miroestrol and deoxymiroestrol and that the developed HPLC method can be applied for quality control of plants and their products Elsevier B.V. All rights reserved. 1. Introduction Pueraria candollei Wall. ex Benth. (Kwao Kruea Khao), which belongs to the family Leguminosae, has long been used in Thai traditional medicine for rejuvenation. There are two varieties of Kwao Kruea Khao found in Thailand, P. candollei var. candollei and P. candollei var. mirifica [1]. Due to the similarity of the botanical characteristics between the two varieties, the tuberous roots of both varieties have been used as sources of Kwao Kruea Khao for use in traditional Thai medicine [2]. The tuberous root of P. candollei exhibits dosedependent estrogenic effects on the reproductive system [3]. This result confirmed that P. candollei could be used as an alternative source of estrogenic compounds. P. candollei tuberous extract had also been shown to prevent bone loss [4] and to possess antioxidant activity [5]. The tubers were found to contain active constituents related to the plant's rejuvenating properties. The active compounds from the Corresponding author. Tel.: ; fax: address: waraporn@kku.ac.th (W. Putalun). tuberous roots of P. candollei are chromenes, such as miroestrol and deoxymiroestrol (Fig. 1), and isoflavonoids, such as puerarin, daidzin, genistin, daidzein and genistein [2,6 8]. and deoxymiroestrol are known to be potent estrogenic compounds [6,9]. Sugiyama et al. reported that these two compounds bind the estrogen receptor alpha stronger than other isoflavonoids found in this plant [10]. Moreover, isoflavonoids that include puerarin, daidzin, daidzein, genistin and genistein have been recognized as major constituents of P. candollei. The concentrations of phytoestrogens in the tuberous roots of P. candollei are variable and depend on the cultivation conditions [11]. To date, P. candollei has had a high market demand due to its high medicinal values. Analytical methods to determine the miroestrol and deoxymiroestrol contents of the plants and/or plant-derived products have become important for quality control. This paper is the first to report a method for the determination of the miroestrol and deoxymiroestrol contents of two varieties of P. candollei. In this study, we developed an HPLC method and completed a comparative study of the miroestrol and deoxymiroestrol contents of two X/$ see front matter 2010 Elsevier B.V. All rights reserved. doi: /j.fitote

2 204 G. Yusakul et al. / Fitoterapia 82 (2011) varieties of P. candollei. The method developed can be used for standardization and quality control of P. candollei. In addition, the total isoflavonoid content was evaluated and compared among plants of different varieties grown in different locations using anti-puerarin and anti-daidzin polyclonal antibodies (PAbs) [12]. 2. Experimental 2.1. Chemicals and deoxymiroestrol were isolated from the tuberous roots of P. candollei var. mirifica as described previously [6]. NMR analysis was performed to compare the isolated compounds with the authentic standards of miroestrol and deoxymiroestrol and was provided by Dr. C. Chaichantipyuth, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Thailand. Daidzin was purchased from Nacalai Tesque, Inc. (Tokyo, Japan). Puerarin was obtained from ChromaDex, Inc. (CA, USA). Peroxidase-labeled anti-rabbit IgG was obtained from MP Biomedicals (OH, USA). 2,2 -Azino-bis- (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) was purchased from Wako (Osaka, Japan). All other chemicals were standard commercial products of analytical grade Plant materials Tubers of P. candollei var. candollei and mirifica were obtained from Suranaree University of Technology, Nakhon Ratchasima province, and from Ubon Rajathanee University, Ubon Ratchathani province, Thailand. Reference specimens (NI-PSKKU ) were deposited at the Herbarium of the Faculty of Pharmaceutical Sciences, Khon Kaen University, Thailand Sample preparation for miroestrol, deoxymiroestrol and HPLC analyses Dried powder (1 g) was washed with 5 ml of hexane for 1 h with sonication and then extracted four times with 5 ml of ethyl acetate chloroform (3:1, v/v) with sonication for 1 h. The extracts were combined and evaporated at 60 C. The H H OH OH residual solid was dissolved in 1 ml of ethanol for HPLC analysis. The mobile phase consisted of 20% acetonitrile containing 1.5% acetic acid at a flow rate of 1.0 ml/min. The detection wavelength was 254 nm. HPLC was performed using a PerkinElmer Series 200 LC pump connected to a PerkinElmer 785A UV/VIS detector and a PE Nelson computer. An RP-18 column (LiChroCART, 125 mm 4 mm, 5 μm particle size, Merck, Germany) was used. A series of standard solutions was prepared by mixing miroestrol and deoxymiroestrol to obtain solutions with final concentrations of 25.00, 12.50, 6.25, 3.13, 1.56 and 0.78 μg/ml for each compound. Calibration curves for miroestrol and deoxymiroestrol were constructed. Triplicate injections were made for each of the six concentrations. The linearity of each standard curve was confirmed by plotting the peak height versus the concentration. The correlation coefficients and the regression equations are shown in Table Sample preparation and isoflavonoid analysis Dried samples (30 mg) were powdered and extracted four times with 0.5 ml ethanol with sonication. The extracts were combined, evaporated and then re-dissolved in 1 ml ethanol. The total isoflavonoid content including puerarin, daidzin, genistin, daidzein and genistein, of the extracted solutions were determined using an indirect competitive ELISA using anti-puerarin and anti-daidzin polyclonal antibodies (PAbs) as described previously [12], but with some modifications. Mixtures of puerarin ovalbumin and daidzin ovalbumin (100 μl; 0.5 μg/ml each in 50 mm carbonate buffer, ph 9.6) were adsorbed to the surfaces of the wells of a 96-well immunoplate, and the plate was incubated at 37 C for 1 h. The plate was washed three times with 0.05% Tween 20 in phosphate-buffered saline (TPBS). The plate was then treated with 300 μl of 0.2% gelatin in phosphate-buffered saline (GPBS) for 1 h and washed three times with TPBS. A 50-μL volume of various concentrations of puerarin and daidzin (ratio 1:1) or of samples dissolved in 20% ethanol was incubated with 50 μl of anti-puerarin and anti-daidzin PAbs (5 μg/ml and 2 μg/ml, respectively) for 1 h. The plate was washed three times with TPBS, and then the PAbs were combined with 100 μl of a 1000-fold diluted solution of peroxidase-conjugated goat anti-rabbit IgG for 1 h. After washing the plate three times with TPBS, 100 μl of a substrate solution (100 mm citrate buffer (ph 4.0) containing 0.003% H 2 O 2 and 0.3 mg/ml of ABTS) was added, and the plate was incubated for 15 min. The absorbance at 405 nm was measured using a microplate reader (Model 550 Microplate H R O HO O R =OH, miroestrol; R= H, deoxymiroestrol Fig. 1. The chemical structures of miroestrol and deoxymiroestrol. Table 1 Regression equations, correlation coefficients, limits of quantification (LOQ) and limits of detection (LOD) for miroestrol and deoxymiroestrol determined by HPLC. Compound Regression equation y =0.1289x y =0.077x Correlation coefficient (R 2 ) LOQ LOD

3 G. Yusakul et al. / Fitoterapia 82 (2011) Fig. 2. HPLC chromatograms of (A) miroestrol [1] and deoxymiroestrol [2] (B) the tuber cortex of P. candollei var. mirifica (Ubon Ratchathani province) (C) the tuber cortex of P. candollei var. mirifica (Nakhon Ratchasima province) (D) the tuber cortex of P. candollei var. candollei (Ubon Ratchathani province) and (E) the tuber cortex of P. candollei var. candollei (Nakhon Ratchasima province). Reader BioRad Laboratories, CA, USA). All reactions were carried out at 37 C. 3. Results and discussion In order to determine the contents of miroestrol and deoxymiroestrol in the tubers of Pueraria candollei var. mirifica and var. candollei, we developed a quantitative HPLC method. The optimum HPLC separation of both miroestrol and deoxymiroestrol was achieved using 20% acetonitrile containing 1.5% acetic acid. The HPLC chromatograms obtained are shown in Fig. 2(A). The retention times were 5.6 min for miroestrol and 11.9 min for deoxymiroestrol. Linearity was verified by construction of a calibration curve for each compound. For each curve, standard solutions were prepared at six concentrations, and chromatograms were recorded. The Table 2 Intra- and inter-day precision values for HPLC analysis of miroestrol and deoxymiroestrol at different concentrations (n=5). Concentration, RSD, RSD Intra-day Inter-day Intra-day Inter-day Table 3 Recovery of miroestrol and deoxymiroestrol by HPLC. Spiked level Measured amount RSD Recovery Measured amount RSD Recovery 19.76± ± ± ± ± ± ± ±

4 206 G. Yusakul et al. / Fitoterapia 82 (2011) Table 4, deoxymiroestrol and total isoflavonoid contents in different parts of tubers from two varieties of Pueraria candollei. Varieties/collection place Part Content (μg/g dry wt.) Total isoflavonoid Total chromene (mg/g dry wt.) P. candollei var. mirifica Ubon Rajathanee Tuber cortex 61.99± ± ± ±0.46 Whole tuber 34.04± ± ± ±0.12 Tuber without cortex 10.41± ± ± ±0.05 Nakhon Ratchasima Tuber cortex 69.37± ± ± ±0.21 Whole tuber 11.69±0.53 ND 11.69± ±0.01 Tuber without cortex ND ND ND 0.01±0.00 P. candollei var. candollei Ubon Rajathanee Tuber cortex ± ± ± ±0.17 Whole tuber 75.46± ± ± ±0.29 Tuber without cortex 26.85± ± ± ±0.16 Nakhon Ratchasima Tuber cortex ND 14.21± ± ±0.15 Whole tuber ND ND ND 1.74±0.16 Tuber without cortex ND ND ND 1.36±0.15 ND: not detected. correlation coefficients obtained for miroestrol and deoxymiroestrol were and , respectively (Table 1). The linear range was μg/ml for miroestrol and μg/ml for deoxymiroestrol. Intra-day and inter-day assay precision values were evaluated using the variation in the measurement of miroestrol and deoxymiroestrol expressed as the relative standard deviation (RSD). As shown in Table 2, the maximum intra-day and inter-day RSDs were 0.93% and 2.63%, respectively, for miroestrol and 3.24% and 5.17%, respectively, for deoxymiroestrol. The limits of detection for miroestrol and deoxymiroestrol were 0.20 and 0.78 μg/ml, respectively. The established HPLC analysis for chromenes used in this study, with a limit of quantification of 0.78 μg/ml for miroestrol and 1.56 μg/ml for deoxymiroestrol revealed a difference in the chromene contents among the plant samples. The accuracy of this method was confirmed by performing a recovery experiment. The accuracy values were determined by analyzing samples spiked with standard miroestrol and deoxymiroestrol at three different concentrations: 5, 10 and 20 μg/ml. P. candollei samples were spiked with known amounts of the standard compounds, extracted and analyzed using the developed HPLC method. The accuracy of the HPLC method was found to be N96% at all of the concentration levels for all samples (Table 3). These results demonstrate that the developed HPLC method can be used for the accurate determination of the miroestrol and deoxymiroestrol contents in P. candollei samples. For sample preparation, a mixture of ethyl acetate and chloroform (3:1, v/v) was found to be suitable for extraction of miroestrol and deoxymiroestrol, whereas ethanol and ethyl acetate were not suitable. Using ethyl acetate chloroform (3:1, v/v), high-quality HPLC chromatograms of miroestrol and deoxymiroestrol isolated from crude extract samples can be obtained. The HPLC profiles of P. candollei var. mirifica tuber cortex samples were determined (Fig. 2B and C) and compared with that of P. candollei var. candollei (Fig. 2D and E) obtained under the same conditions. Comparison of the HPLC profiles for plants of different varieties grown in different locations revealed differences in miroestrol and deoxymiroestrol contents (Fig. 2). Previously, we produced anti-daidzin and anti-puerarin PAbs. Anti-daidzin PAbs are cross-reactive with genistin, daidzein and genistein, other major isoflavonoids found in P. candollei, and anti-puerarin PAbs are cross-reactive only with daidzein. Based on these results, ELISA using these antipuerarin and anti-daidzin antibodies can detect five of the major isoflavonoids found in P. candollei [12]. The calibration range of the isoflavonoid was μg/ml. Intra-assay and inter-assay precision levels were determined. The maximum intra-assay RSD was 1.80%, and the maximum inter-assay RSD was 2.55%. A recovery experiment was performed to evaluate the accuracy of this method. For each sample, % of the spiked isoflavonoid was recovered, indicating that this ELISA system is accurate. Therefore, the combination assay system using anti-puerarin and antidaidzin PAbs for indirect competitive ELISA represents a useful methodology for determining the concentrations of major isoflavonoids in P. candollei samples. In order to evaluate the variability of the natural bioactive compounds found in the two varieties of P. candollei, a comparative analysis of the concentrations of miroestrol, deoxymiroestrol and isoflavonoids was undertaken. The miroestrol, deoxymiroestrol and isoflavonoid contents of different parts of the tuber were determined. As shown in Table 4, the results revealed variability in the chromene and total isoflavonoid contents between the samples. The tuber cortex contained higher levels of miroestrol, deoxymiroestrol and isoflavonoid than the other parts in the same tuber. These results suggest that a large proportion of the accumulated chromenes are found in the tuber cortex of P. candollei. The highest levels of miroestrol (182.18±8.25 μg/g dry wt.), deoxymiroestrol (154.34±5.70 μg/g dry wt.) and total isoflavonoid (5.45±0.17 mg/g dry wt.) were obtained in the tuber cortex of P. candollei var. candollei collected from the Ubon Ratchathani province. and deoxymiroestrol could not be detected in whole tuber or the tuber without the cortex of P. candollei var. candollei or in the tuber without the cortex of P. candollei var. mirifica collected from the Nakhon Ratchasima province (Table 4). Interestingly, both two varieties of P. candollei collected from the Ubon Ratchathani province had high contents of miroestrol, deoxymiroestrol and isoflavonoids. It has been reported that the miroestrol and deoxymiroestrol contents in the tubers of P. candollei var.

5 G. Yusakul et al. / Fitoterapia 82 (2011) mirifica were 20 μg/g and 20 μg/g dry wt., respectively [6]. As shown by the data presented in Table 4, the miroestrol and deoxymiroestrol contents (up to ± 8.25 μg/g and ±5.70 μg/g dry wt., respectively) found in our study were nine-fold higher than the contents reported previously. In addition, the total isoflavonoid contents in P. candollei tubers from our study (up to 5.45±0.04 mg/g dry wt.) were higher than that reported previously (1.98±0.04 mg/g dry wt.) [11]. In summary, our findings indicate that both two varieties of P. candollei can produce chromenes, miroestrol and deoxymiroestrol. This is the first report detailing an HPLC method developed for the determination of miroestrol and deoxymiroestrol concentrations and the first report of a comparative analysis of the two varieties of P. candollei. Moreover, total isoflavonoid contents were determined, and our results revealed a correlation between the chromene and total isoflavonoid contents in P. candollei. Due to the high market demand for P. candollei, the concentrations of phytoestrogen constituents in the tubers of P. candollei should be determined to evaluate the raw plant material and/or plant-derived products to ensure the quality of the plant and/or products. Our method and the information reported herein may be valuable for a quality assessment of P. candollei. Acknowledgments This work was supported by a grant from Khon Kaen University (542101) and the Thailand Research Fund (RSA ). References [1] Niyomdham C. Nord J Bot 1992;12:339. [2] Cain JC. Nature 1960;188:774. [3] Cherdshewasart W, Kitsamai Y, Malaivijitnond S. J Reprod Dev 2007;53:385. [4] Urasopan N, Hamada Y, Cherdshewasart W, Malaivijitnond S. Maturitas 2008;59:137. [5] Cherdshewasart W, Sutjit W. Phytomedicine 2008;15:38. [6] Chansakaow S, Ishikawa T, Seki H, Sekine K, Okada M, Higuchi Y, et al. J Nat Prod 2000;63:175. [7] Chansakaow S, Ishikawa T, Sekine K, Okeda M, Higuchi Y, Kudo M, et al. Planta Med 2000;66:572. [8] Ingham JL, Tahara S, Dziedzic. Z Naturforsch C 1986;41:403. [9] Jones HE, Pope GS. J Endocrinol 1961;22:303. [10] Sugiyama H, Kumamoto T, Suganami A, Nakanishi W, Sowa Y, Takiguchi M, et al. Biochem Biophys Res Commun 2009;379:139. [11] Cherdshewasart W, Subtang S, Dahlan W. J Pharm Biomed Anal 2007;43:428. [12] Pongkitwitoon B, Sakamoto S, Tanaka H, Tsuchihashi R, Kinjo J, Morimoto S, et al. Planta Med 2010;76:831.