Component Analysis of Isoflavonoids in Pueraria montana and Pueraria lobata by High Performance Liquid Chromatography

Component Analysis of Isoflavonoids in Pueraria montana and Pueraria lobata 115 Component Analysis of Isoflavonoids in Pueraria montana and Pueraria lobata by High Performance Liquid Chromatography Chian-Ying Yang 1, Chien-Chih Chen 2, Su-Ju Lin 3, Chia-Chi Chen 3 and Horng-Liang Lay 3 * 1 Institute of Tropical Agriculture & International Cooperation, National Pingtung University of Science & Technology, Neigu, Pingtung Hsien 912, Taiwan (ROC) 2 National Research Institute of Chinese Medicine, Taichung 404, Taiwan (ROC) 3 Department of Plant Industry, National Pingtung University of Science & Technology, Pingtung Hsien 912, Taiwan (ROC) ABSTRACT Radix of Pueraria spp. is a popular traditional Chinese medicine. There are two major species of Pueraria in Taiwan, i.e., Pueraria montana and Pueraria lobata. The major constituents of Puerariae radix were isoflavonoids, which include puerarin, daidzin, genistin, genistein, daidzein and daidzein-4,7- diglucoside. This study developed simultaneous analysis of multi-component HPLC method to determine the six components of isoflavonoids in root, vine, leaf, and petiole parts of P. montana and P. lobata for establishing a standard quality control of Pueraria spp. Results showed that the highest level of isoflavonoids was observed in the root and vine of P. lobata, and the daidzein-4,7-diglucoside was only found in root and vine of P. lobata. Levels of isoflavones existed among species, as well as organs, were significantly different. Key words: Puerariae radix, P. Montana, P. lobata, Puerarin, Daidzin, Genistin, Genistein, Daidzein, Daidzein-4,7-diglucoside. v å ¼¾e¼ g g e * Ê Å, layhl@mail.npust.edu.tw i 2004 9 i 16 i ªi 2005 1 i 7 i Á vá 2:115-122 (2005) Crop, Environment & Bioinformatics 2: 115-122 (2005) 189 Chung-Cheng Rd., Wufeng, Taichung Hsien 41301, Taiwan (ROC) 1 Õ 2» 3 3 Ê 3 * 1 xô»ç ~ æ µ 2 x æ µ 3 xô»ç v î (Pueraria spp.) á v š eœ¹ Ð lîƒ (P. lobata)g (P. montana) g nù puerarin daidzin genistin genistein daidzein g daidzein-4,7-diglucoside tæ le g eáù e e} e¼ HPLC i¾ ˆ }Á g } ( g Ý) e¼ Ð þ µ» x g l (P. lobata) g á daidzein-4,7-diglucoside ¹ g } e } g 價 þ Ü j Ü j d d4,7

116 Crop, Environment & Bioinformatics, Vol. 2, June 2005 INTRODUCTION Radix of Pueraria spp., referred to as Puerariae radix in crude herb, is one of very important pharmaceutical source in the traditional Chinese medicine. The herb has been used to treat the common cold, influenza, shoulder or wrist stiffen, vascular hypertension (Luo and Chin 1995). The major constituents of Puerariae radix are isoflavonoids, which include puerarin, daidzin, genistin, genistein, daidzein, and daidzein-4,7-diglucoside. These isoflavonoids with many important biochemical and pharmacological activities have been reported recently (Yanagihara et al. 1993, Kim et al. 1998). Chinese herbal medicines are natural products, they are difficult to control the quality of raw materials, due to the influencing factors such as botanical origins, production, harvesting seasons, plant parts chosen for use, and storage conditions. In recent years, several analytical methods including thin layer chromatography (TLC) (Huang 1995), high-performance liquid chromatography (HPLC) (Hayakawa et al. 1984, Kitada et al. 1985, Oshima et al. 1988, Yasuda et al. 1993, Wen et al. 1993, Okamura et al. 1999, Yu et al. 2002), high-speed counter-current chromatography (Cao et al. 1999) and capillary zone electrophoresis (CZE) (Wang et al. 1998, Chen et al. 2001, Cao et al. 2002) have been employed for evaluating the quality of Puerariae radix. However, these analytical methods can only analyze two to five components in the crude herb or in the concentrated preparation. In this study, six major constituents puerarin, daidzin, genistin, genistein, daidzein and daidzein-4,7-diglucoside of Puerariae radix were used as marker substances and their structures were shown in Fig. 1. These compounds in the root, vine, leaf, and petiole of two major species of Pueraria in Taiwan, i.e., P. montana and P. lobata, and that in the commercial Puerariae radix were determined by HPLC. This method is precise, reliable and efficient for the individual and simultaneous determination of the six isoflavonoids components, and used to establish a standard for the quality control of Pueraria spp. MATERIALS AND METHODS 1. MATERIALS Different plant parts, which included root, vine, leaf, and petiole of P. montana and P. lobata were collected from the campus of National Pingtung University of Science and Technology (NPUST), Pingtung Hsien, Taiwan in March, 2002. These materials were verified by Professor Sin-Zen Yang, Department of Forestry, NPUST. Voucher specimens are maintained in the laboratory of corresponding author. Commercial Puerariae radix sample was obtained from local herbal market. All samples were dried by drying oven at 60 for 24 hours, and pulverized through a #8 mesh sieve (2.36 mm). R 2 O R 3 R 1 O O R 4 R 1 R 2 R 3 R 4 Puerarin H H Glu OH Daidzin H Glu H OH Genistin OH Glu H OH Genistein OH H H OH Daidzein H H H OH Daidzein-4-7-diglucoside H Glu H Glu Fig. 1. Structures of the marker substances in Pueraria spp. 2. CHEMICALS AND REAGENTS Reference standards, puerarin, genistin, genistein, and the internal standard methylparaben were purchased from Sigma Chemical Co. (St. Louis, Mo, USA); Daidzin and daidzein were purchased from Fluka Chemie AG (Switzerland). Daidzein-4, 7-diglucoside was isolated from the root of P. lobata in our laboratory; and mass spectrometry, NMR spectrometry and infrared spectrometry were applied to determine compound characteristics. Abovementioned analytical values obtained agreed with the previous investigations (Kobayashi and Ohta 1983, Kinjo et al. 1987). The 95% ethanol was purchased from Taiwan Tobacco and Wine Board. Acetonitrile and methanol (HPLC grade) were purchased from Mallinckrodt, Inc. (USA), and phosphoric acid from Kanto Chemical (Japan). Ultra-pure distilled water with a resistivity

Component Analysis of Isoflavonoids in Pueraria montana and Pueraria lobata 117 greater than 18 MΩ was prepared with a mini-q system (Millipore, Bedford, MA, USA). Samples for HPLC were filtered through a 0.45 µm membrane filter (Millipore, Bedford, MA, USA). All reagents were analytical grade otherwise described. 3. HPLC INSTRUMENTS AND CONDITIONS HPLC was conducted by a Hitachi system equipped with a degasser DG-2410, pump L-7100, UV/Vis detector L-7420, photodiode array detector L-4500 and autosampler L-7200. Peak areas were calculated with D-7000 HSM (HPLC system manager) software. A column of reverse phase Cosmosil 5C 18-AR (Nacalai, 4.6 mm I.D. 250 mm) was use. Column oven was set at 30. The mobile phase is the mixtures of 10%, 40% and 100% acetonitrile aqueous solutions used in the linear gradient elution are shown in Table 1. Detection wavelength was set at UV 245 nm. Flow rate was set at 1.0 ml min -1. 20 µl of each sample solution prepared as described above was injected into the HPLC column for analysis. The results were quantified by interpolating into the linear regression plot made from standard solution. 4. PREPARATION OF STANDARD AND INTERNAL STANDARD SOLUTION (1) Preparation of standard solutions All the standards were dissolved in 70% methanol to give sequential concentrations, puerarin 3,600.0 µg ml -1 and 2.5 µg ml -1, daidzin 400.0 µg ml -1 and 10.0 µg ml -1, genistin 70.0 µg ml -1, genistein 35.0 µg ml -1, Table 1. Gradient elution program using mobile phase A, B and C; flow rate l ml min -1. Time (min) A (%) B (%) C (%) 0 100 0 0 15 90 10 0 25 75 25 0 35 55 45 0 45 45 55 0 50 25 75 0 55 0 0 100 60 0 0 100 65 100 0 0 A: water (adjusted to ph 3.0 with phosphoric acid). B: 40% acetonitrile (adjusted to ph 3.0 with phosphoric acid). C: 100% acetonitrile (adjusted to ph 3.0 with phosphoric acid). daidzein 160.0 µg ml -1, and daidzein-4,7-diglucoside 300.0 µg ml -1 were prepared as the standard stock solution. (2) Preparation of internal standard solution Methylparaben at 44 µg ml -1 was prepared as the internal standard stock solution. 5. PREPARATION OF SAMPLE SOLUTION All samples (2 g) were accurately weighed and extracted with 100 ml ethanol by refluxing at 80 for 3 hours, each was filtered and concentrated by evaporator. Then, the solution was adjusted to 10 ml by adding 70% methanol, while internal standard solution was added to each solution to a concentration of 22.0 µg ml -1 at the same time. There were test solutions used for subsequent HPLC analysis after filtration through a 0.45 µm membrane filter. 6. CALIBRATION METHOD The standard stock solutions of each marker substance described above was diluted by 70% methanol to give sequential concentrations are: puerarin at 112.50, 225.00, 450.00, 900.00 and 1,800.00 µg ml -1 (high concentration range), and at 0.08, 0.16, 0.31, 0.62 and 1.25 µg ml -1 (low concentration range); daidzin at 12.50, 25.00, 50.00, 100.00 and 200.00 µg ml -1 (high concentration range), and at 0.31, 0.62, 1.25, 2.50 and 5.00 µg ml -1 (low concentration range); genistin at 2.19, 4.38, 8.75, 17.50 and 35.00 µg ml -1 ; genistein at 1.09, 2.19, 4.38, 8.75 and 17.50 µg ml -1 ; daidzein at 5.0, 10.0, 20.0, 40.0 and 80.0 µg ml -1 ; daidzein-4,7- diglucoside at 9.38, 18.75, 37.50, 75.00 and 150.00 µg ml -1. Each dilution contained the internal standard solution, methylparaben, at 22.0 µg ml -1. After filtering through a 0.45 µm membrane filter, 20 µl of each concentration was injected into the HPLC column for analysis. Linear regression model was used to describe the relationship between peak area and concentration of standard solution. 7. VALIDATION (1) Precision Standard stock solutions were diluted to three different concentrations with 80% methanol (Table 3). Both an intra-day test (injecting each concentration of standard solution three times within 24 hours), and an inter-day test (injecting each concentration of standard

118 Crop, Environment & Bioinformatics, Vol. 2, June 2005 solution four times over 7 days with each injection separated by at least 24 hours) were run to check the reproducibility of HPLC analysis. The standard deviation (S.D.) and relative standard deviation (R.S.D.) were calculated. (2) Accuracy Each of standard stock solutions of isoflavonoids at various concentrations were spiked into a sample solution of the vine of P. lobata, and internal standard solution was added into each solution to a concentration of 22.0 µg ml -1. Then the solution was filtered and subjected to HPLC analysis in triplicates. The recovery rate (%) was calculated by the equation of ((C3-C2)/C1) 100%, in which C1 represents the amount of each standard spiked, C2 represents the amount of each marker in ethanol solution of the vine of P. lobata, and C3 represents the total amount of each markers in the solution. Table 2. Calibration curves of marker substances. Compound Concentration range µg ml -1 Regression equation r Puerarin 112.50~1800.00 (high) 0.08~1.25 (low) y=0.063x-0.4992 y=0.1587x -0.008 0. 9998 0.9989 Daidzin 12.50~200.00 (high) 0.31~5.00 (low) y=0.0607x-0.1755 y=0.0308x-0.0225 0. 9996 0.9987 Genistin 1.09~70.00 y=0.034x-0.0013 0.9999 Daidein 0.63~80.00 y=0.1533x-0.1468 0.9986 Genistein 1.09~17.50 y=0.0838x+0.0238 0.9999 Daidzein-4',7-9.38~150.00 y=0.0322x-0.1759 0.9994 diglucoside n 5 Table 3. Relative standard deviations of intra-day and inter-day analysis of marker substances. Concentration Mean±S.D. (R.S.D. %) Compound (µg ml -1 ) Intra-day (n=5) Inter-day (n=4) 112.50 113.13±2.77 (2.45) 115.29±0.44 (0.38) Puerarin 450.00 458.12±2.78 (0.61) 460.31±3.39 (0.74) 1800.00 1819.10±7.03 (0.39) 1828.23±7.11 (0.39) 6.25 6.05±0.12 (1.98) 5.92±0.02 (0.33) Daidzin 25.00 25.54±0.04 (0.16) 25.91±0.03 (0.12) 200.00 203.93±0.53 (0.26) 198.91±0.39 (0.20) 2.19 2.23±0.03 (1.35) 2.28±0.07 (3.10) Genistin 8.75 9.06±0.04 (0.44) 9.09±0.01 (0.11) 35.00 34.97±0.12 (0.34) 35.70±0.37 (1.04) 1.09 1.14±0.03 (2.63) 1.11±0.04 (3.60) Genistein 4.38 4.55±0.11 (2.42) 4.64±0.09 (1.94) 17.50 17.46±0.11 (0.63) 17.92±0.24 (1.34) 1.25 1.29±0.03 (2.33) 1.33±0.03 (2.26) Daidzein 5.00 5.28±0.01 (0.19) 5.48±0.01 (0.18) 80.00 81.79±0.31 (0.38) 78.59±0.52 (0.66) Daidzein-4',7-9.38 10.00±0.01 (0.10) 10.28±0.21 (2.04) diglucoside 37.50 38.06±0.10 (0.26) 37.30±0.18 (0.48)! 150.00 152.33±0.52 (0.34) 150.62±1.77 (1.17)

Component Analysis of Isoflavonoids in Pueraria montana and Pueraria lobata 119 RESULTS AND DISCUSSION 1. SEPARATION OF MARKER SUBS- TANCES BY HPLC HPLC chromatograms of the ethanol extract in the vine of P. lobata (Fig. 2) indicated that the puerarin, daidzin, genistin, genistein, daidzein, daidzein-4,7- diglucoside, and the internal standard were well separated, with retention time of 15.86, 24.51, 27.86, 41.77, 47.61, 8.01, and 32.61 min, respectively. The peak purities of components in the vine of P. lobata were evaluated with a photodiode array detector where high peak purities were shown irrespective of each component (Fig. 3). Apparently, there was no interaction between components in this plant. Therefore, the above conditions can be used for quantification of the marker substances. Fig. 2. HPLC chromatogram of puerarin, daidzin, genistin, genistein, daidzein daidzein-4,7-diglucoside, and methylparaben in vine of P. lobata. IS: Internal standard. Fig. 3. Peak purity of puerarin, daidzin, genistin, genistein, daidzein daidzein-4,7-diglucoside, and methylparaben in standard solutions of vine of P. lobata by HPLC photodiode array detector. IS: Internal standard.

120 Crop, Environment & Bioinformatics, Vol. 2, June 2005 2. CALIBRATION CURVE The linear regression equations, correlation coefficients and concentration range of calibration lines for those marker substances were listed in Table 2. All calibration curves obtained linear correlation with correlation coefficient of 0.9986 ~0.9999. 3. PRECISION AND ACCURACY The relative standard deviations of the intra-day and inter-day ranged between 0.10~2.63% and 0.11~3.60%, suggesting that the method used had a high reproducibility (Table 3). The compound analyses were obtained (Table 4) with the recovery rates of greater than 91.08%. 4. ANALYSIS OF THE SAMPLE SOLUTION Amounts of puerarin, daidzin, genistin, genistein, daidzein, and daidzein-4,7- diglucoside in vines, leaves, and petioles of P. montana and P. lobata, and commercial crude drug sample were successfully determined within 65 minutes (Table 5). Significant differences in individual and total isoflavonoid contents were observed among various plant sections and species. Results showed that the root from P. lobata had the highest content of total isoflavonoid, followed by the vine, and puerarin was the major component of isoflavonids in this species. Puerarin was undetectable or had a low level in P. montana. Daidzein-4,7- diglucoside was only existed in root and vine of P. lobata. In addition, there were not significant differences among roots and vines of P. lobata, and commercial crude drug sample in total isoflavonoids. Table 4. Recovery rates of marker substances from Pueraria spp. Compound Concentration (µg ml -1 ) Recovery rate (%) Mean±S.D. (R.S.D. %)* 112.50 93.31±1.75 (1.88) Puerarin 450.00 93.54±1.80 (1.92) 1800.00 96.19±1.37 (1.42) 3.13 95.52±2.39 (2.50) Daidzin 25.00 94.38±3.30 (3.50) 200.00 101.04±2.63 (2.60) 2.19 99.08±0.18 (0.18) Genistin 8.75 91.08±1.28 (1.41) 35.00 96.40±0.14 (0.15) 1.09 93.49±2.62 (2.80) Genistein 4.38 112.06±1.79 (1.60) 17.50 97.67±3.13 (3.20) 0.63 115.20±0.92 (0.80) Daidzein 5.00 92.40±1.94 (2.10) 80.00 115.49±2.56 (2.22) Daidzein-4',7-9.38 102.61±1.21 (1.18) diglucoside 37.50 103.01±1.14 (1.11) 150.00 102.44±0.72 (0.70) * n 3. Table 5. Content of marker substances in various plant sections of both P. momtana and P. lobata, and in commercial sample.! Puerarin Daidzin Genistin Daidzein Genistein P. montana Daidzein-4',7 -diglucoside Root 0.62±0.02 104.09±0.20 124.06±3.28 11.09±1.57 18.29±0.17 -- 258.15 Vine 0.50±0.08 80.13±2.59 71.56±1.06 15.47±0.78 25.24±2.18 -- 192.90 Petiole -- 25.92±0.10 27.28±0.43 8.97±0.09 9.49±0.31 -- 71.66 Leaf -- 144.60±1.73 105.74±1.69 7.72±0.13 15.36±2.55 -- 273.42 P. lobata Root 4,037.06±0.29 730.47±0.59 108.52±0.27 13.83±0.43 135.89±0.09 208.98±1.82 5,025.77 Vine 3,516.53±0.57 815.26±0.31 172.07±3.17 21.71±1.16 171.07±0.27 218.94±0.49 4,696.64 Petiole 4.25±0.11 21.93±0.28 8.01±0.25 6.06±0.54 11.22±0.37 -- 51.47 Leaf 10.58±0.04 53.09±0.30 35.57±0.73 5.51±0.42 8.46±0.25 -- 113.21 Commercial sample 2907.75±0.23 745.87±2.41 435.89±3.66 23.76±4.35 483.70±4.28 208.47±3.35 4,805.44 Data represented as mean (µg g -1 ) ± R.S.D. (%), n 7. -- Not detected. Total

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