Article pubs.acs.org/JAFC
Inhibition of Xanthine Oxidase by Rhodiola crenulata Extracts and Their Phytochemicals Yung-Hung Chu,† Chao-Jung Chen,† Shih-Hsiung Wu,‡,§ and Jung-Feng Hsieh*,†,§ †
Department of Food Science, Fu Jen Catholic University, 510 Zhongzheng Road, Xinzhuang, Taipei 24205, Taiwan Institute of Biological Chemistry, Academia Sinica, 128 Academia Road, Section 2, Nankang, Taipei 11529, Taiwan
‡
ABSTRACT: Using a fractionation technique, four phytochemicals were isolated from Rhodiola crenulata extracts. These compounds were identified as 4′-hydroxyacetophenone (4-HAP), epicatechin-(4β,8)-epicatechin gallate (B2−3′-O-gallate), salidroside, and p-tyrosol using mass spectrometry and nuclear magnetic resonance spectroscopy. The inhibition of xanthine oxidase (XO) activity by these purified compounds was then evaluated and compared to that of a known XO inhibitor (allopurinol; IC50 = 12.21 ± 0.27 μM). Both 4-HAP and B2−3′-O-gallate showed an XO inhibitory effect, for which the half maximal inhibitory concentration (IC50) values were 15.62 ± 1.19 and 24.24 ± 1.80 μM, respectively. However, salidroside and p-tyrosol did not show significant inhibitory effects on XO at 30 μM. Furthermore, an inhibition kinetics study indicated that 4-HAP and B2−3′-O-gallate are mixed competitive inhibitors. The inhibition constants (Ki) of 4-HAP and B2−3′-O-gallate were 8.41 ± 1.03 and 6.16 ± 1.56 μM, respectively. These results suggest that 4-HAP and B2−3′-O-gallate are potent XO inhibitors. KEYWORDS: xanthine oxidase, inhibitor, hyperuricemia, Rhodiola crenulata
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INTRODUCTION Xanthine oxidase (XO, EC 1.2.3.2) can catalyze the oxidation of xanthine and hypoxanthine to yield uric acid. Uric acid is the final metabolite of purine compounds in humans, and most uric acid is excreted into the urine by the kidneys. The overproduction of uric acid by XO will lead to hyperuricemia, which is a key cause of gout.1 Gout is a metabolic disorder associated with abnormal amounts of uric acid in the body, which causes inflammation, gouty arthritis, and uric acid nephrolithiasis.2 Accordingly, the use of the XO inhibitors that block the synthesis of uric acid in the body could be one of the therapeutic approaches for the treatment of gout. Among the known XO inhibitors, allopurinol (1H-pyrazolo[3,4-d]pyrimidin-4-ol) has been used for the treatment of gout for many years. However, several studies have indicated that allopurinol may induce hypersensitivity syndrome and Stevens−Johnson syndrome in patients.3 Hence, the identification of novel, efficient, and less toxic XO inhibitors for nutraceutical and pharmaceutical applications is necessary. The search for novel XO inhibitors from medicinal plants would be beneficial to treat gout. Several medicinal plants have been evaluated for their XO inhibitory property. Among the 122 water extracts (WE) prepared from 122 traditional Chinese medicinal plants, 40 were shown to be inhibitory at concentrations of 100 μg/mL.4 XO inhibitors were also purified from medicinal plants. For example, oleuropein isolated from the leaves of Olea europaea showed a strong inhibitory effect on XO in a competitive mode.5 Cinnamaldehyde isolated from Cinnamomum cassia twigs has high XO inhibitory activity.6 Furthermore, Arimboor et al.7 reported that tetrahydroamentoflavone isolated from Semecarpus anacardium also has XO inhibitory activity (IC50 = 50 ± 3 μg/mL). Our preliminary results revealed that the WE of Rhodiola crenulata exhibited significant XO inhibitory activity (IC50 = 59.5 ± 1.7 μg/mL). R. crenulata is an important member of the © 2014 American Chemical Society
genus Rhodiola L., which is found mostly in the northwest region of China. The extracts of R. crenulata have been made into pharmaceutical preparations and functional foods.8 Previous studies have demonstrated that the roots of R. crenulata possess beneficial properties, including the scavenging of active-oxygen species,9 anti-Alzheimer’s disease effects,10 and blood-glucose-lowering activity.11 In the previous phytochemical studies, more than 100 compounds were isolated from R. crenulata, including phenols, phenylpropanoids, phenylethanoids, flavonoids, monoterpenoids, cyanogens, and their corresponding glycosides.12 Among the isolated compounds, salidroside and p-tyrosol were the major bioactive constituents and showed antioxidant, antifatigue, and antiinflammatory activities.13 Because of the important properties of R. crenulata, its study is a subject of great interest. However, the inhibition of XO by R. crenulata extracts and their bioactive constituents has never been reported. In this paper, we describe the isolation and characterization of the bioactive constituents, including 4′-hydroxyacetophenone (4-HAP), epicatechin-(4β,8)-epicatechin gallate (B2−3′O-gallate), p-tyrosol, and salidroside, from R. crenulata and their inhibitory activity against XO. The objective of this study was to investigate the XO inhibitory effects of R. crenulata extracts and the phytochemicals contained therein.
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MATERIALS AND METHODS
Preparation of the Crude Extract. The dried roots of R. crenulata were obtained from a traditional Chinese medicine pharmacy in Chiayi in south Taiwan. Their authenticity was confirmed by Dr. Hsiang-Wen Tseng (Industrial Technology Research Institute, Received: Revised: Accepted: Published: 3742
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Figure 1. Scheme employed for the extraction of WE and its fractions from R. crenulata. Identification of Purified Active Compounds. The chemical structures of the purified active compounds were identified by ultraviolet spectroscopy (UV, Hitachi, U-1900), Fourier transform infrared spectroscopy (FTIR, Bruker, Tensor27), polarity meter (PerkinElmer, 341), high-resolution electrospray ionization−TOF mass spectrometry (ESI−MS, BioTOF III; Bruker Daltonics, Inc., Billerica, MA), and nuclear magnetic resonance spectroscopy (NMR). NMR spectra, including 1H, 13C, distortionless enhancement by polarization transfer (DEPT), 1H−1H correlation spectroscopy (COSY), nuclear Overhauser effect spectrometry (NOESY), heteronuclear single-quantum coherence (HSQC), and heteronuclear multiple-bond correlation (HMBC), were recorded with a Bruker LC-SPE-NMR AVII 500 MHz spectrometer.14 Screening of Purified Active Compounds for XO Inhibitors. The XO inhibitory activity was assayed spectrophotometrically at 295 nm under aerobic conditions.14 Absorbance (A) at 295 nm was measured using a UV spectrophotometer (U-1900, Hitachi HighTechnologies Corporation, Minato-ku, Japan). The reaction mixture contained 200 mM sodium pyrophosphate buffer (pH 7.5), 100 μM xanthine, and 0.01 unit/mL of XO. The absorption rate at 295 nm indicates the formation of uric acid at 30 °C. The samples included WE and its five fractions (fractions 1−5), 4-HAP, B2−3′-O-gallate, salidroside, p-tyrosol, and allopurinol. Samples were dissolved directly in the buffer and incorporated into an enzyme assay to evaluate their inhibitory activities. Each sample was tested 3 times. The purified active compounds and allopurinol were tested for XO inhibitory activity at different concentrations (0−30 μM). The sample was dissolved directly in the buffer and incorporated into the enzyme assay to assess the inhibitory activity, and each sample was performed in triplicate. The inhibitory activity was determined by IC50, which was obtained from percent inhibition calculated by
Taiwan) using DNA technology and the internal transcribed spacer sequence database. A total of 70 g of R. crenulata roots were milled using a laboratory-scale milling machine and extracted 3 times with distilled water (700 mL) at 30 °C. After 15 min of centrifugation at 12000g, the supernatant of collected samples were freeze-dried to yield the WE. The specimen was stored in the refrigerator (−20 °C) for further solid-phase extraction (SPE). SPE Procedure. WE were redissolved in distilled water and underwent column chromatography on a vacuum manifold (Phenomenex, Torrance, CA) using SPE cartridges (6 mL/1000 mg, BAKERBOND octadecyl C18, J.T.Baker, Deventer, Netherlands). Each sample (50 mg) was chromatographed on a SPE cartridge. The sample was eluted with a stepwise method of 0, 10, 20, 30, and 40% methanol in distilled water. A total of five fractions, one for each methanol elution (20 mL), were collected. Samples were concentrated at 35 °C using a rotary evaporator and then freeze-dried. These five fractions (fractions 1−5) were then analyzed in the XO assay to assess the inhibitory activity. HPLC Analysis of the WE of R. crenulata and Its Fractions. WE of R. crenulata and its fractions were dissolved in distilled water and subjected to column chromatography on the high-performance liquid chromatography (HPLC) system composed of a pump (PU-980, JASCO), a detector (UV-970, JASCO), and a C18 packed column (4.6 × 250 mm, 5 μm spherical, Dikma Technologies, Inc.). The mobile phase was solvent A (100% acetonitrile) and solvent B (ultrapure water). Elution conditions were as follows: 2−50% of A to B for 0−55 min (linear gradient) at a flow rate of 1 mL/min. The effluent was monitored through an optical absorption study carried out at 280 nm. Isolation of Active Compounds from the WE of R. crenulata. WE and the fourth fraction (fraction 4) were redissolved in distilled water and subjected to column chromatography on the HPLC system composed of a pump (PU-980, JASCO), a detector (UV-970, JASCO) and a C18 packed column (4.6 × 250 mm, 5 μm spherical, Dikma Technologies, Inc.). The mobile phase was solvent A (100% acetonitrile) and solvent B (ultrapure water). Elution conditions for WE were as follows: 2−2% of A to B for 0−7 min and 2−30% A to B for 7−35 min (linear gradient). Elution conditions for fraction 4 were 14−14% A to B for 0−20 min and 14−18% A to B for 20−35 min (linear gradient). The elution began with solvent at a flow rate of 1 mL/min. The effluent was monitored through an optical absorption study carried out at 280 nm. The active compounds were then collected.
percent inhibition (%) = [(A without sample − A with sample)]× 100%/A without sample Lineweaver−Burk and Dixon Plots. To determine the mode of inhibition by 4-HAP and B2−3′-O-gallate, Lineweaver−Burk plot analysis was performed. This kinetics study was conducted in the absence and presence of inhibitors with varying concentrations of xanthine as the substrate. The initial velocity was expressed as the absorbance rate at 295 nm per 10 s in the assay. To determine the competitive inhibition constant (Ki) and uncompetitive inhibition constant (Ki′), Dixon plot analysis was performed. Ki is the 3743
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equilibrium constant for an inhibitor binding to XO, and Ki′ is the equilibrium constant for an inhibitor binding to the XO−xanthine complex. The Ki and Ki′ values of purified XO inhibitors were determined from the Dixon plot according to the method reported by Cornish-Bowden.15 This kinetics study was carried out with varying concentrations of inhibitors and xanthine. The initial velocity was expressed as the absorbance rate at 295 nm per 10 s in the assay. Statistical Analysis. Data were expressed as the mean ± standard deviation. The data were analyzed using the Statistical Package for the Social Sciences software (SPSS for Windows, version 10.0.7C, SPSS, Inc., Chicago, IL). Statistical significance among the treatments was determined by a one-way analysis of variation (ANOVA), followed by a Duncan’s multiple range test. Three determinations for each treatment were made, and the significance level was set at p < 0.05.
solvent at a flow rate of 1 mL/min, and a gradient elution from H2O/acetonitrile (98:2) to H2O/acetonitrile (50:50) over 55 min was performed to analyze these samples. The HPLC profile showed significant differences in the phytochemical patterns for each fraction. As shown in Figure 3, five major peaks were found in the WE at the elution times of 2−3, 5−7, 14−15, 21−22, and 23−24 min. Three peaks were found in fraction 1 at the elution times of 2−3, 5−7, and 14−15 min, while two peaks were found in fractions 2 and 3 at the elution times of 21−22 and 23−24 min. Furthermore, other minor peaks were found in fractions 3−5 at the elution time of 24−38 min. As previously mentioned, fractions 3−5 were used to inhibit XO at the concentration of 30 μg/mL, and the XO inhibitory activities were 17.2, 72.0, and 64.7%, respectively. Our results suggest that the peaks found in fraction 4 at the elution time of 24−38 min exhibit XO inhibitory properties. Isolation and Identification of XO Inhibitors from R. crenulata. The WE and fraction 4 were analyzed and purified by HPLC (Figure 4). As shown in Figure 4A, a total 100 mg of WE was subjected to HPLC. Two major pure compounds (arrows, compounds 1 and 2) were isolated, which were collected at the elution times of 21−22 and 23−24 min, respectively. The total yields of compounds 1 and 2 were 6.4 and 9.8 mg, respectively. Furthermore, a total of 157 mg of fraction 4 was then analyzed and purified by HPLC (Figure 4B). The individual peak was collected and used to inhibit XO. Consequently, two pure compounds (arrows, compounds 3 and 4) were isolated and had XO inhibitory properties. These two compounds were collected at the elution times of 16−17 and 19−20 min, and the total yields of compounds 3 and 4 were 3.7 and 6.4 mg, respectively. Four purified compounds were analyzed using ESI−MS and NMR and were identified as salidroside, p-tyrosol, B2−3′-O-gallate, and 4-HAP. The following sections will focus on the spectral data of these compounds obtained from FTIR, UV, ESI−MS, and NMR. Salidroside (Compound 1). Compound 1 was a white amorphous powder (Figure 4A), [α]20 D −28.30° (c 0.00883, MeOH). UV (MeOH) λmax, nm: 230, 273. IR νmax: 3363, 2925, 2886, 1614, 1597, 1516, 1449, 1369, 1237, 1160, 1076, 1024. ESI−MS m/z: 323.1106 [M + Na]+ (calculated for C14H20O7). 1 H NMR (CD3OD, 500 MHz) δ: 7.06 (2H, d, J = 8.4 Hz, H-2, 6), 6.69 (2H, d, J = 8.4 Hz, H-3, 5), 2.83 (1H, m, H-7), 4.03 (1H, m, H-8), 3.70 (1H, m, H-8), 4.29 (1H, d, J = 7.8 Hz, HGlu.-1), 3.17 (1H, m, H-Glu.-2), 3.27 (1H, m, H-Glu.-3), 3.24 (1H, m, H-Glu.-4), 3.35 (1H, m, H-Glu.-5), 3.86 (1H, dd, J = 11.9, 2.0 Hz, H-Glu.-6), 3.66 (1H, dd, J = 11.9, 5.2 Hz, H-Glu.-6). 13C NMR (CD3OD, 125 MHz) δ: 130.9 (C-1), 131.1 (C-2, 6), 116.3 (C-3, 5), 157.0 (C-4), 36.6 (C-7), 72.3 (C-8), 104.6 (C-Glu.-1), 75.3 (C-Glu.-2), 78.1 (C-Glu.-3), 71.8 (C-Glu.-4), 78.3 (C-Glu.-5), 63.0 (C-Glu.-6). Compound 1 was identified as salidroside by direct comparison to commercial salidroside (Sigma Chemical Co., St. Louis, MO). On the basis of the data described above and the verified reference,17 the structure of compound 1 was elucidated as salidroside. Yuan et al.18 reported that salidroside exhibits antioxidant activity and a protective effect against furan-induced hepatocyte damage in mice. p-Tyrosol (Compound 2). Compound 2 was a white amorphous powder (Figure 4A). UV (MeOH) λmax, nm: 223, 263. IR νmax: 3386, 3141, 3024, 1598, 1510, 1452, 1363, 1230, 1105, 1051, 1015. ESI−MS m/z: 138 [M] (calculated for C8H10O2). 1H NMR (CD3OD, 500 MHz) δ: 7.04 (2H, d, J = 8.6 Hz, H-2, 6), 6.71 (2H, d, J = 8.6 Hz, H-3, 5), 2.73 (2H, t,
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RESULTS AND DISCUSSION XO Inhibitory Activity of Fractionated Extracts of R. crenulata. The procedure for fractionation of the extracts of R. crenulata is shown in Figure 1. R. crenulata (70 g) was extracted with distilled water and freeze-dried to yield 14 g of WE, which displayed XO inhibitory activity (IC50 = 59.5 ± 1.7 μg/mL). The WE were then chromatographed on the SPE cartridges and eluted with varying concentrations of methanol. Finally, five fractions (fractions 1−5) were collected using a stepwise method of 0, 10, 20, 30, and 40% methanol in distilled water; the total yield of these fractions was 98.2%, and the yield of each fraction was 84.3, 9.4, 3.0, 1.1, and 0.4%, respectively. The IC50 values of WE and the associated fractions (fractions 1−5) were 59.5 ± 1.7, 862.2 ± 31.3, 524.2 ± 28.6, 94.7 ± 3.2, 16.1 ± 1.5, and 19.2 ± 1.8 μg/mL, respectively. Thus, we tested the inhibition of XO using WE and fractions at a final concentration of 0−60 μg/mL. Our results indicated that XO inhibition activity was 25.7% for WE and 2.8, 5.5, 17.2, 72.0, and 64.7% for fractions 1−5, respectively, at a concentration of 30 μg/mL (Figure 2). Among those fractions, the fourth
Figure 2. Inhibition of XO by the WE and its fractions at a final concentration of 30 μg/mL. Each value is represented as the mean ± standard deviation from triplicate measurements. WE, water extracts; Fr, fraction.
fraction (fraction 4) had the highest XO inhibitory activity (IC50 = 16.1 ± 1.5 μg/mL). Chen et al.16 reported that the crude extracts of Koelreuteria henryi, Prunus campanulata, and Rhodiola rosea possessed strong XO inhibitory activity, with IC50 values of 91.8 ± 1.7, 64.6 ± 5.8, and 56.0 ± 1.0 μg/mL, respectively. These values indicate that the WE and fraction 4 would be plentiful sources of XO inhibitors. HPLC Analysis of the WE and Its Fractions. To establish the fingerprint chromatogram of the WE and its five fractions, samples were analyzed by HPLC. The elution began with 3744
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Figure 3. HPLC chromatograms of the WE and its fractions. Flow rate, 1.0 mL/min; UV detection, 280 nm. WE, water extracts; Fr, fraction.
Table 1. Structure, Molecular Weight, Molecular Formula, and IC50 Values of XO Inhibitors
a
IC50 values are expressed as the mean ± standard deviation. 3745
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Figure 4. Isolation of active compounds from the (A) WE and (B) fraction 4. Arrow, active compounds; flow rate, 1.0 mL/min; UV detection, 280 nm.
J = 7.2 and 14.5 Hz, H-7), 3.70 (2H, t, J = 7.2 and 14.5 Hz, H-8). 13C NMR (CD3OD, 125 MHz) δ: 131.1 (C-1), 131.2 (C-2, 6), 116.3 (C-3, 5), 154.9 (C-4), 39.6 (C-7), 64.8 (C-8). Compound 2 was identified as p-tyrosol by direct comparison to commercial p-tyrosol (Sigma Chemical Co., St. Louis, MO). On the basis of the data described above and the verified reference,19 the structure of compound 2 was elucidated as p-tyrosol. Dewapriya et al.20 suggested that p-tyrosol has antioxidant activity and a protective effect against dopaminergic neuronal cell death in an in vitro model of Parkinson’s disease. B2−3′-O-Gallate (Compound 3). Compound 3 was a brown amorphous powder (Figure 4B), [α]20 D −57.00° (c 0.00667, MeOH). UV (MeOH) λmax, nm: 213, 228, 274. IR νmax: 3321, 1690, 1609, 1520, 1448, 1370, 1284, 1235, 1156, 1099, 1039. ESI−MS m/z: 731.1615 [M + H]+ (calculated for C37H30O16). 1H NMR (CD3COCD3, 500 MHz) δ: 5.26 (2H, s, H-2U, 2L), 4.06 (1H, s, H-3U), 4.89 (1H, s, H-4U), 6.03 (3H, m, H-6U, 8U, 6L), 6.77 (3H, m, H-2′U, 6′U, 5′L), 6.98 (1H, s, H-5′U), 5.62 (1H, s, H-3L), 2.98 (1H, d, J = 17.5 Hz, H-4L), 3.13 (1H, dd, J = 17.1 and 4.4 Hz, H-4L), 7.13 (3H, m, H-2′L, 2″L, 6″L), 7.05 (1H, s, H-6′L). 13C NMR (CD3COCD3, 125 MHz) δ: 78.0 (C-2U), 72.9 (C-3U), 36.7 (C-4U), 157.7 (C-5U), 97.2 (C-6U, 8L), 158.2 (C-7U), 96.3 (C-8U), 158.2 (C-9U), 107.9 (C-10U), 132.5 (C-1′U), 115.6 (C-2′U), 145.3 (C-3′U, 4′U, 3′L), 119.4 (C-5′U), 119.1 (C-6′U), 77.2 (C-2L), 69.1 (C-3L),
26.6 (C-4L), 155.5 (C-5L, 7L), 95.8 (C-6L), 156.0 (C-9L), 99.6 (C-10L), 131.2 (C-1′L), 115.5 (C-2′L), 145.2 (C-4′L), 115.3 (C-5′L), 114.7 (C-6′L), 122.0 (C-1″L), 110.2 (C-2″L, 6″L), 145.7 (C-3″L, 5″L), 138.7 (C-4″L), 166.2 (C-7″L). On the basis of the data described above and the verified reference,21 the structure of compound 3 was elucidated as B2−3′-O-gallate. Lourenço et al.22 reported that this compound has antioxidant activity and reduces the formation of UV-induced α-tocopheroxyl radicals. 4-HAP (Compound 4). Compound 4 was a white amorphous powder (Figure 4B). UV (MeOH) λmax, nm: 222. IR νmax: 3305, 1661, 1602, 1576, 1512, 1430, 1357, 1279, 1220, 1166, 1130, 1107, 1075, 1021. ESI−MS m/z: 136 [M] (calculated for C8H8O2). 1H NMR (CD3OD, 400 MHz) δ: 7.88 (2H, d, J = 8.9 Hz, H-2, 6), 6.84 (2H, d, 2, J = 8.8 Hz, H-3, 5), 2.52 (3H, s, H-8). 13C NMR (CD3OD, 100 MHz) δ: 130.4 (C-1), 132.3 (C-2, 6), 116.4 (C-3, 5), 164.2 (C-4), 199.7 (C-7), 26.5 (C-8). Compound 4 was identified as 4-HAP by direct comparison to commercial 4-HAP (Sigma Chemical Co., St. Louis, MO). On the basis of the data described above and the verified reference,23 the structure of compound 4 was elucidated as 4-HAP. Chen et al.24 reported that 4-HAP exhibits antiallergic activities, and against β-hexosaminidase, it could release a marker of degranulation in rat basophilic leukemia (RBL)-2H3 cells. 3746
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Modes of Inhibition of XO by B2−3′-O-gallate and 4-HAP. The 4-HAP and B2−3′-O-gallate were analyzed by Michaelis−Menten kinetic studies to determine their modes of inhibition of XO. In this study, the Michaelis constant (Km) of XO was 5.33 ± 0.21 mM, which was similar to that reported by Tung et al.27 They reported that the Km value of XO was 21 μM. As shown in Figure 6, Lineweaver−Burk plots of B2−
XO Inhibitory Activity of 4-HAP, B2−3′-O-Gallate, Salidroside, p-Tyrosol, and Allopurinol. In the previous XO inhibitor studies, quercetin-3-O-rhamnopyranoside and luteolin isolated from Acacia confusa showed a strong inhibitory effect on XO, with IC50 values of 37.7 and 11.6 μM, respectively.25 The inhibitory effects of 4-HAP, B2−3′-O-gallate, salidroside, p-tyrosol, and allopurinol on XO were tested at different concentrations. XO inhibition increased significantly with the addition of allopurinol (p < 0.05; Figure 5). When
Figure 5. Inhibitory effects of allopurinol, B2−3′-O-gallate, and 4-HAP on XO. Each value is represented as the mean ± standard deviation from triplicate measurements.
15 μM of the inhibitor was added, the XO inhibition by allopurinol was 60.8%. Similar trends were also observed in the results for 4-HAP and B2−3′-O-gallate; they showed inhibitory effects on XO. When examining the inhibition of XO by 4-HAP at 25 μM and B2−3′-O-gallate at 30 μM, the XO inhibitory activities were 70.3 and 57.5%, respectively. 4-HAP and B2−3′O-gallate showed remarkable inhibitory effects on XO (p < 0.05). However, we failed to observe any significant inhibitory effects on XO by salidroside or p-tyrosol at 30 μM (data not shown). As previously mentioned, five major peaks and one minor peak were found in the HPLC profile of WE (Figure 3). Salidroside and p-tyrosol indicated two major peaks in WE following elution times of 21−22 and 23−24 min, respectively. We observed that WE displayed XO inhibitory activity; however, the five major peaks in WE did not present significant inhibitory effects on XO, suggesting that the minor peak in WE at an elution time of 24−38 min exhibits XO. Nguyen et al.26 reported that allopurinol is a substrate and a specific potent inhibitor of XO, but oxypurinol (1H-pyrazolo[3,4-d]-pyrimidine-4,6-diol) is the basic functioning ingredient found in allopurinol. XO can catalyze the conversion of allopurinol into oxypurinol, and inhibition occurs mainly through direct substrate competition in the breakdown of purines. The XO inhibitory activities of purified salidroside, p-tyrosol, B2−3′-O-gallate, and 4-HAP from R. crenulata were evaluated and compared to that of allopurinol. The structure, molecular weight, molecular formula, and IC50 values of allopurinol, 4-HAP, B2−3′-O-gallate, p-tyrosol, and salidroside are shown in Table 1. Allopurinol, 4-HAP, and B2−3′-O-gallate showed strong inhibitory effects on XO, and the IC50 values of these compounds were 12.21 ± 0.27, 15.62 ± 1.19, and 24.24 ± 1.80 μM, respectively. This result indicated that 4-HAP and B2−3′-O-gallate have the potential to be XO inhibitors. However, the IC50 values of both salidroside and p-tyrosol were higher than 200 μM.
Figure 6. Lineweaver−Burk plots for the inhibition of XO by XO inhibitors with xanthine as the substrate: (A) B2−3′-O-gallate and (B) 4-HAP.
3′-O-gallate and 4-HAP were constructed. The Lineweaver− Burk plots of B2−3′-O-gallate (Figure 6A) and 4-HAP (Figure 6B) had no intersection on the y or x axis, indicating that the type of inhibition was a mixed-type mode. Therefore, the Lineweaver−Burk plots revealed that B2−3′-O-gallate and 4-HAP behaved as mixed-type inhibitors. Several mixed-type XO inhibitors have been reported. For example, a Lineweaver− Burk plot indicated that 3,4-dihydroxybenzoic acid isolated from the flowers of Chrysanthemum sinense appears to be a mixed-type inhibitor of XO.26 Furthermore, Day et al.28 reported that quercetin-3′-glucuronide showed mixed competitive inhibition of XO. Dixon plots of B2−3′-O-gallate and 4-HAP on XO were also evaluated (Figure 7). The Dixon plots indicated that B2−3′-Ogallate (Figure 7A) and 4-HAP (Figure 7B) were mixed-type inhibitors of XO, and the values of Ki were 7.26 ± 0.38 and 8.45 ± 0.34 μM, respectively. Jiao et al.29 reported that apigenin4′-O-(2″-O-p-coumaroyl)-β-D-glucopyranoside purified from Palhinhaea cernua inhibits XO with mixed-type inhibition, with Ki and Ki′ values of 14.35 and 93.68 μM, respectively. The Ki 3747
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AUTHOR INFORMATION
Corresponding Author
*Telephone: +886-2-29052516. Fax: +886-2-29053622. E-mail: [email protected]. Author Contributions §
Shih-Hsiung Wu and Jung-Feng Hsieh contributed equally to this work. Funding
The authors thank the National Science Council (NSC 992313-B-030-001-MY3) for financial support. Notes
The authors declare no competing financial interest.
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ABBREVIATIONS USED 4-HAP, 4′-hydroxyacetophenone; B2−3′-O-gallate, epicatechin-(4β,8)-epicatechin gallate; IC50, half maximal inhibitory concentration; Ki, inhibition constant; Ki′, uncompetitive inhibition constant; Km, Michaelis constant; NMR, nuclear magnetic resonance spectroscopy; SPE, solid-phase extraction; WE, water extracts; XO, xanthine oxidase
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REFERENCES
(1) Unno, T.; Sugimoto, A.; Kakuda, T. Xanthine oxidase inhibitors from the leaves of Lagerstroemia speciosa (L.) Pers. J. Ethnopharmacol. 2004, 93, 391−395. (2) Tung, Y. T.; Chang, S. T. Inhibition of xanthine oxidase by Acacia confusa extracts and their phytochemicals. J. Agric. Food Chem. 2010, 58, 781−786. (3) Hammer, B.; Link, A.; Wagner, A.; Böhm, M. Hypersensitivity syndrome during therapy with allopurinol in asymptomatic hyperuricemia with a fatal outcome. Dtsch. Med. Wochenschr. 2001, 26, 1331−1334. (4) Kong, L. D.; Cai, Y.; Huang, W. W.; Cheng, C. H.; Tan, R. X. Inhibition of xanthine oxidase by some Chinese medicinal plants used to treat gout. J. Ethnopharmacol. 2000, 73, 199−207. (5) Flemmig, J.; Kuchta, K.; Arnhold, J.; Rauwald, H. W. Olea europaea leaf (Ph.Eur.) extract as well as several of its isolated phenolics inhibit the gout-related enzyme xanthine oxidase. Phytomedicine 2011, 18, 561−566. (6) Ngoc, T. M.; Khoi, N. M.; Ha, D. T.; Nhiem, N. X.; Tai, B. H.; Don, D. V.; Luong, H. V.; Son, D. C.; Bae, K. Xanthine oxidase inhibitory activity of constituents of Cinnamomum cassia twigs. Bioorg. Med. Chem. Lett. 2012, 22, 4625−4628. (7) Arimboor, R.; Rangan, M.; Aravind, S. G.; Arumughan, C. Tetrahydroamentoflavone (THA) from Semecarpus anacardium as a potent inhibitor of xanthine oxidase. J. Ethnopharmacol. 2011, 133, 1117−1120. (8) Shen, C.; Jiang, L.; Gu, X.; Qiu, S.; Shen, X.; Yang, Q. Antioxidant compounds and antioxidant activity of Rhodiola drink. Acad. Period. Farm. Prod. Process. 2010, 12, 32−34. (9) Chen, D.; Fan, J.; Wang, P.; Zhu, L.; Jin, Y.; Peng, Y.; Du, S. Isolation, identification and antioxidative capacity of water-soluble phenylpropanoid compounds from Rhodiola crenulata. Food Chem. 2012, 134, 2126−2133. (10) Zhang, J.; Zhen, Y. F.; Ren, P. B. C.; Song, L. G.; Kong, W. N.; Shao, T. M.; Li, X.; Chai, X. Q. Salidroside attenuates β amyloidinduced cognitive deficits via modulating oxidative stress and inflammatory mediators in rat hippocampus. Behav. Brain Res. 2013, 244, 70−81. (11) Wang, J.; Rong, X.; Li, W.; Yang, Y.; Yamahara, J.; Li, Y. Rhodiola crenulata root ameliorates derangements of glucose and lipid metabolism in a rat model of the metabolic syndrome and type 2 diabetes. J. Ethnopharmacol. 2012, 142, 782−788. (12) Yang, Y. N.; Liu, Z. Z.; Feng, Z. M.; Jiang, J. S.; Zhang, P. C. Lignans from the root of Rhodiola crenulata. J. Agric. Food Chem. 2012, 60, 964−972.
Figure 7. Dixon plots for the inhibition of XO by XO inhibitors with xanthine as the substrate: (A) B2−3′-O-gallate and (B) 4-HAP.
value of 4-HAP was higher than that of B2−3′-O-gallate. This indicated that B2−3′-O-gallate showed a higher binding affinity than 4-HAP to XO. Furthermore, the Ki′ of B2−3′-O-gallate and 4-HAP were 24.43 ± 0.65 and 69.58 ± 1.13 μM, respectively. In addition, we noted that the values of Ki for B2−3′-O-gallate and 4-HAP were smaller than values of Ki′. As mentioned previously, Ki is the equilibrium constant for an inhibitor binding to XO, while Ki′ is the equilibrium constant for inhibitor binding to the XO−xanthine complex. Cortés et al.30 reported that, in the reversible mixed competitive inhibition, the values of Ki were smaller than the values of Ki′. This indicated that the binding affinity for the inhibitor−XO complex was higher than that for the inhibitor−XO−xanthine complex. Therefore, the inhibition kinetics study indicates that B2−3′-O-gallate and 4-HAP are mixed competitive inhibitors of XO. Although the binding sites and mechanisms of inhibition remain to be determined, the finding of mixed competitive inhibition of XO suggests that B2−3′-O-gallate and 4-HAP may bind to either XO or the XO−xanthine complex. In summary, the WE of R. crenulata displayed XO inhibitory activity, and four active compounds, salidroside, p-tyrosol, B2−3′-O-gallate, and 4-HAP, were isolated from R. crenulata. Among these compounds, B2−3′-O-gallate and 4-HAP showed excellent XO inhibitory activity with a mixed competitive mode. Thus, B2−3′-O-gallate and 4-HAP have great potential to prevent hyperuricemia caused by XO. Therefore, our results suggest that B2−3′-O-gallate and 4-HAP could be adopted as candidates to treat gout, and further evaluation should be conducted with in vivo studies. 3748
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(13) Sun, L.; Isaak, C. K.; Zhou, Y.; Petkau, J. C.; Karmin, O.; Liu, Y.; Siow, Y. L. Salidroside and tyrosol from Rhodiola protect H9c2 cells from ischemia/reperfusion-induced apoptosis. Life Sci. 2012, 91, 151− 158. (14) Hsieh, J. F.; Wu, S. H.; Yang, Y. L.; Choong, K. F.; Chen, S. T. The screening and characterization of 6-aminopurine-based xanthine oxidase inhibitors. Bioorg. Med. Chem. 2007, 15, 3450−3456. (15) Cornish-Bowden, A. A simple graphical method for determining the inhibition constants of mixed, uncompetitive and non-competitive inhibitors. Biochem. J. 1974, 137, 143−144. (16) Chen, C. H.; Chan, H. C.; Chu, Y. T.; Ho, H. Y.; Chen, P. Y.; Lee, T. H.; Lee, C. K. Antioxidant activity of some plant extracts towards xanthine oxidase, lipoxygenase and tyrosinase. Molecules 2009, 14, 2947−2958. (17) Akita, H.; Kurashima, K.; Nakamura, T.; Kato, K. Chemoenzymatic syntheses of naturally occurring β-glucosides. Tetrahedron: Asymmetry 1999, 10, 2429−2439. (18) Yuan, Y.; Wu, S. J.; Liu, X.; Zhang, L. L. Antioxidant effect of salidroside and its protective effect against furan-induced hepatocyte damage in mice. Food Funct. 2013, 4, 763−769. (19) Park, C. H.; Kim, K. H.; Lee, I. K.; Lee, S. Y.; Choi, S. U.; Lee, J. H.; Lee, K. R. Phenolic constituents of Acorus gramineus. Arch. Pharm. Res. 2011, 34, 1289−1296. (20) Dewapriya, P.; Himaya, S. W.; Li, Y. X.; Kim, S. K. Tyrosol exerts a protective effect against dopaminergic neuronal cell death in in vitro model of Parkinson’s disease. Food Chem. 2013, 141, 1147−1157. (21) Saito, A.; Mizushina, Y.; Ikawa, H.; Yoshida, H.; Doi, Y.; Tanaka, A.; Nakajima, N. Systematic synthesis of galloyl-substituted procyanidin B1 and B2, and their ability of DPPH radical scavenging activity and inhibitory activity of DNA polymerases. Bioorg. Med. Chem. 2005, 13, 2759−2771. (22) Lourenço, C. F.; Gago, B.; Barbosa, R. M.; de Freitas, V.; Laranjinha, J. LDL isolated from plasma-loaded red wine procyanidins resist lipid oxidation and tocopherol depletion. J. Agric. Food Chem. 2008, 56, 3798−3804. (23) Vogl, M.; Kratzer, R.; Nidetzky, B.; Brecker, L. Candida tenuis xylose reductase catalysed reduction of acetophenones: The effect of ring-substituents on catalytic efficiency. Org. Biomol. Chem. 2011, 9, 5863−5870. (24) Chen, H. J.; Shih, C. K.; Hsu, H. Y.; Chiang, W. Mast celldependent allergic responses are inhibited by ethanolic extract of adlay (Coix lachryma-jobi L. var. ma-yuen Stapf) testa. J. Agric. Food Chem. 2010, 58, 2596−2601. (25) Hsieh, C. Y.; Chang, S. T. Antioxidant activities and xanthine oxidase inhibitory effects of phenolic phytochemicals from Acacia confusa twigs and branches. J. Agric. Food Chem. 2010, 58, 1578−1583. (26) Nguyen, M. T.; Awale, S.; Tezuka, Y.; Ueda, J. Y.; Tran, Q. I.; Kadota, S. Xanthine oxidase inhibitors from the flowers of Chrysanthemum sinense. Planta Med. 2006, 72, 46−51. (27) Tung, Y. T.; Hsu, C. A.; Chen, C. S.; Yang, S. C.; Huang, C. C.; Chang, S. T. Phytochemicals from Acacia confusa heartwood extracts reduce serum uric acid levels in oxonate-induced mice: Their potential use as xanthine oxidase inhibitors. J. Agric. Food Chem. 2010, 58, 9936−9941. (28) Day, A. J.; Bao, Y.; Morgan, M. R.; Williamson, G. Conjugation position of quercetin glucuronides and effect on biological activity. Free Radical Biol. Med. 2000, 29, 1234−1243. (29) Jiao, R. H.; Ge, H. M.; Shi, D. H.; Tan, R. X. An apigeninderived xanthine oxidase inhibitor from Palhinhaea cernua. J. Nat. Prod. 2006, 69, 1089−1091. (30) Cortés, A.; Cascante, M.; Cárdenas, M. L.; Cornish-Bowden, A. Relationships between inhibition constants, inhibitor concentrations for 50% inhibition and types of inhibition: New ways of analysing data. Biochem. J. 2001, 357, 263−268.
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Inhibition of Xanthine Oxidase by Rhodiola crenulata Extracts and Their Phytochemicals Yung-Hung Chu,† Chao-Jung Chen,† Shih-Hsiung Wu,‡,§ and Jung-Feng Hsieh*,†,§ †
Department of Food Science, Fu Jen Catholic University, 510 Zhongzheng Road, Xinzhuang, Taipei 24205, Taiwan Institute of Biological Chemistry, Academia Sinica, 128 Academia Road, Section 2, Nankang, Taipei 11529, Taiwan
‡
ABSTRACT: Using a fractionation technique, four phytochemicals were isolated from Rhodiola crenulata extracts. These compounds were identified as 4′-hydroxyacetophenone (4-HAP), epicatechin-(4β,8)-epicatechin gallate (B2−3′-O-gallate), salidroside, and p-tyrosol using mass spectrometry and nuclear magnetic resonance spectroscopy. The inhibition of xanthine oxidase (XO) activity by these purified compounds was then evaluated and compared to that of a known XO inhibitor (allopurinol; IC50 = 12.21 ± 0.27 μM). Both 4-HAP and B2−3′-O-gallate showed an XO inhibitory effect, for which the half maximal inhibitory concentration (IC50) values were 15.62 ± 1.19 and 24.24 ± 1.80 μM, respectively. However, salidroside and p-tyrosol did not show significant inhibitory effects on XO at 30 μM. Furthermore, an inhibition kinetics study indicated that 4-HAP and B2−3′-O-gallate are mixed competitive inhibitors. The inhibition constants (Ki) of 4-HAP and B2−3′-O-gallate were 8.41 ± 1.03 and 6.16 ± 1.56 μM, respectively. These results suggest that 4-HAP and B2−3′-O-gallate are potent XO inhibitors. KEYWORDS: xanthine oxidase, inhibitor, hyperuricemia, Rhodiola crenulata
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INTRODUCTION Xanthine oxidase (XO, EC 1.2.3.2) can catalyze the oxidation of xanthine and hypoxanthine to yield uric acid. Uric acid is the final metabolite of purine compounds in humans, and most uric acid is excreted into the urine by the kidneys. The overproduction of uric acid by XO will lead to hyperuricemia, which is a key cause of gout.1 Gout is a metabolic disorder associated with abnormal amounts of uric acid in the body, which causes inflammation, gouty arthritis, and uric acid nephrolithiasis.2 Accordingly, the use of the XO inhibitors that block the synthesis of uric acid in the body could be one of the therapeutic approaches for the treatment of gout. Among the known XO inhibitors, allopurinol (1H-pyrazolo[3,4-d]pyrimidin-4-ol) has been used for the treatment of gout for many years. However, several studies have indicated that allopurinol may induce hypersensitivity syndrome and Stevens−Johnson syndrome in patients.3 Hence, the identification of novel, efficient, and less toxic XO inhibitors for nutraceutical and pharmaceutical applications is necessary. The search for novel XO inhibitors from medicinal plants would be beneficial to treat gout. Several medicinal plants have been evaluated for their XO inhibitory property. Among the 122 water extracts (WE) prepared from 122 traditional Chinese medicinal plants, 40 were shown to be inhibitory at concentrations of 100 μg/mL.4 XO inhibitors were also purified from medicinal plants. For example, oleuropein isolated from the leaves of Olea europaea showed a strong inhibitory effect on XO in a competitive mode.5 Cinnamaldehyde isolated from Cinnamomum cassia twigs has high XO inhibitory activity.6 Furthermore, Arimboor et al.7 reported that tetrahydroamentoflavone isolated from Semecarpus anacardium also has XO inhibitory activity (IC50 = 50 ± 3 μg/mL). Our preliminary results revealed that the WE of Rhodiola crenulata exhibited significant XO inhibitory activity (IC50 = 59.5 ± 1.7 μg/mL). R. crenulata is an important member of the © 2014 American Chemical Society
genus Rhodiola L., which is found mostly in the northwest region of China. The extracts of R. crenulata have been made into pharmaceutical preparations and functional foods.8 Previous studies have demonstrated that the roots of R. crenulata possess beneficial properties, including the scavenging of active-oxygen species,9 anti-Alzheimer’s disease effects,10 and blood-glucose-lowering activity.11 In the previous phytochemical studies, more than 100 compounds were isolated from R. crenulata, including phenols, phenylpropanoids, phenylethanoids, flavonoids, monoterpenoids, cyanogens, and their corresponding glycosides.12 Among the isolated compounds, salidroside and p-tyrosol were the major bioactive constituents and showed antioxidant, antifatigue, and antiinflammatory activities.13 Because of the important properties of R. crenulata, its study is a subject of great interest. However, the inhibition of XO by R. crenulata extracts and their bioactive constituents has never been reported. In this paper, we describe the isolation and characterization of the bioactive constituents, including 4′-hydroxyacetophenone (4-HAP), epicatechin-(4β,8)-epicatechin gallate (B2−3′O-gallate), p-tyrosol, and salidroside, from R. crenulata and their inhibitory activity against XO. The objective of this study was to investigate the XO inhibitory effects of R. crenulata extracts and the phytochemicals contained therein.
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MATERIALS AND METHODS
Preparation of the Crude Extract. The dried roots of R. crenulata were obtained from a traditional Chinese medicine pharmacy in Chiayi in south Taiwan. Their authenticity was confirmed by Dr. Hsiang-Wen Tseng (Industrial Technology Research Institute, Received: Revised: Accepted: Published: 3742
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Figure 1. Scheme employed for the extraction of WE and its fractions from R. crenulata. Identification of Purified Active Compounds. The chemical structures of the purified active compounds were identified by ultraviolet spectroscopy (UV, Hitachi, U-1900), Fourier transform infrared spectroscopy (FTIR, Bruker, Tensor27), polarity meter (PerkinElmer, 341), high-resolution electrospray ionization−TOF mass spectrometry (ESI−MS, BioTOF III; Bruker Daltonics, Inc., Billerica, MA), and nuclear magnetic resonance spectroscopy (NMR). NMR spectra, including 1H, 13C, distortionless enhancement by polarization transfer (DEPT), 1H−1H correlation spectroscopy (COSY), nuclear Overhauser effect spectrometry (NOESY), heteronuclear single-quantum coherence (HSQC), and heteronuclear multiple-bond correlation (HMBC), were recorded with a Bruker LC-SPE-NMR AVII 500 MHz spectrometer.14 Screening of Purified Active Compounds for XO Inhibitors. The XO inhibitory activity was assayed spectrophotometrically at 295 nm under aerobic conditions.14 Absorbance (A) at 295 nm was measured using a UV spectrophotometer (U-1900, Hitachi HighTechnologies Corporation, Minato-ku, Japan). The reaction mixture contained 200 mM sodium pyrophosphate buffer (pH 7.5), 100 μM xanthine, and 0.01 unit/mL of XO. The absorption rate at 295 nm indicates the formation of uric acid at 30 °C. The samples included WE and its five fractions (fractions 1−5), 4-HAP, B2−3′-O-gallate, salidroside, p-tyrosol, and allopurinol. Samples were dissolved directly in the buffer and incorporated into an enzyme assay to evaluate their inhibitory activities. Each sample was tested 3 times. The purified active compounds and allopurinol were tested for XO inhibitory activity at different concentrations (0−30 μM). The sample was dissolved directly in the buffer and incorporated into the enzyme assay to assess the inhibitory activity, and each sample was performed in triplicate. The inhibitory activity was determined by IC50, which was obtained from percent inhibition calculated by
Taiwan) using DNA technology and the internal transcribed spacer sequence database. A total of 70 g of R. crenulata roots were milled using a laboratory-scale milling machine and extracted 3 times with distilled water (700 mL) at 30 °C. After 15 min of centrifugation at 12000g, the supernatant of collected samples were freeze-dried to yield the WE. The specimen was stored in the refrigerator (−20 °C) for further solid-phase extraction (SPE). SPE Procedure. WE were redissolved in distilled water and underwent column chromatography on a vacuum manifold (Phenomenex, Torrance, CA) using SPE cartridges (6 mL/1000 mg, BAKERBOND octadecyl C18, J.T.Baker, Deventer, Netherlands). Each sample (50 mg) was chromatographed on a SPE cartridge. The sample was eluted with a stepwise method of 0, 10, 20, 30, and 40% methanol in distilled water. A total of five fractions, one for each methanol elution (20 mL), were collected. Samples were concentrated at 35 °C using a rotary evaporator and then freeze-dried. These five fractions (fractions 1−5) were then analyzed in the XO assay to assess the inhibitory activity. HPLC Analysis of the WE of R. crenulata and Its Fractions. WE of R. crenulata and its fractions were dissolved in distilled water and subjected to column chromatography on the high-performance liquid chromatography (HPLC) system composed of a pump (PU-980, JASCO), a detector (UV-970, JASCO), and a C18 packed column (4.6 × 250 mm, 5 μm spherical, Dikma Technologies, Inc.). The mobile phase was solvent A (100% acetonitrile) and solvent B (ultrapure water). Elution conditions were as follows: 2−50% of A to B for 0−55 min (linear gradient) at a flow rate of 1 mL/min. The effluent was monitored through an optical absorption study carried out at 280 nm. Isolation of Active Compounds from the WE of R. crenulata. WE and the fourth fraction (fraction 4) were redissolved in distilled water and subjected to column chromatography on the HPLC system composed of a pump (PU-980, JASCO), a detector (UV-970, JASCO) and a C18 packed column (4.6 × 250 mm, 5 μm spherical, Dikma Technologies, Inc.). The mobile phase was solvent A (100% acetonitrile) and solvent B (ultrapure water). Elution conditions for WE were as follows: 2−2% of A to B for 0−7 min and 2−30% A to B for 7−35 min (linear gradient). Elution conditions for fraction 4 were 14−14% A to B for 0−20 min and 14−18% A to B for 20−35 min (linear gradient). The elution began with solvent at a flow rate of 1 mL/min. The effluent was monitored through an optical absorption study carried out at 280 nm. The active compounds were then collected.
percent inhibition (%) = [(A without sample − A with sample)]× 100%/A without sample Lineweaver−Burk and Dixon Plots. To determine the mode of inhibition by 4-HAP and B2−3′-O-gallate, Lineweaver−Burk plot analysis was performed. This kinetics study was conducted in the absence and presence of inhibitors with varying concentrations of xanthine as the substrate. The initial velocity was expressed as the absorbance rate at 295 nm per 10 s in the assay. To determine the competitive inhibition constant (Ki) and uncompetitive inhibition constant (Ki′), Dixon plot analysis was performed. Ki is the 3743
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equilibrium constant for an inhibitor binding to XO, and Ki′ is the equilibrium constant for an inhibitor binding to the XO−xanthine complex. The Ki and Ki′ values of purified XO inhibitors were determined from the Dixon plot according to the method reported by Cornish-Bowden.15 This kinetics study was carried out with varying concentrations of inhibitors and xanthine. The initial velocity was expressed as the absorbance rate at 295 nm per 10 s in the assay. Statistical Analysis. Data were expressed as the mean ± standard deviation. The data were analyzed using the Statistical Package for the Social Sciences software (SPSS for Windows, version 10.0.7C, SPSS, Inc., Chicago, IL). Statistical significance among the treatments was determined by a one-way analysis of variation (ANOVA), followed by a Duncan’s multiple range test. Three determinations for each treatment were made, and the significance level was set at p < 0.05.
solvent at a flow rate of 1 mL/min, and a gradient elution from H2O/acetonitrile (98:2) to H2O/acetonitrile (50:50) over 55 min was performed to analyze these samples. The HPLC profile showed significant differences in the phytochemical patterns for each fraction. As shown in Figure 3, five major peaks were found in the WE at the elution times of 2−3, 5−7, 14−15, 21−22, and 23−24 min. Three peaks were found in fraction 1 at the elution times of 2−3, 5−7, and 14−15 min, while two peaks were found in fractions 2 and 3 at the elution times of 21−22 and 23−24 min. Furthermore, other minor peaks were found in fractions 3−5 at the elution time of 24−38 min. As previously mentioned, fractions 3−5 were used to inhibit XO at the concentration of 30 μg/mL, and the XO inhibitory activities were 17.2, 72.0, and 64.7%, respectively. Our results suggest that the peaks found in fraction 4 at the elution time of 24−38 min exhibit XO inhibitory properties. Isolation and Identification of XO Inhibitors from R. crenulata. The WE and fraction 4 were analyzed and purified by HPLC (Figure 4). As shown in Figure 4A, a total 100 mg of WE was subjected to HPLC. Two major pure compounds (arrows, compounds 1 and 2) were isolated, which were collected at the elution times of 21−22 and 23−24 min, respectively. The total yields of compounds 1 and 2 were 6.4 and 9.8 mg, respectively. Furthermore, a total of 157 mg of fraction 4 was then analyzed and purified by HPLC (Figure 4B). The individual peak was collected and used to inhibit XO. Consequently, two pure compounds (arrows, compounds 3 and 4) were isolated and had XO inhibitory properties. These two compounds were collected at the elution times of 16−17 and 19−20 min, and the total yields of compounds 3 and 4 were 3.7 and 6.4 mg, respectively. Four purified compounds were analyzed using ESI−MS and NMR and were identified as salidroside, p-tyrosol, B2−3′-O-gallate, and 4-HAP. The following sections will focus on the spectral data of these compounds obtained from FTIR, UV, ESI−MS, and NMR. Salidroside (Compound 1). Compound 1 was a white amorphous powder (Figure 4A), [α]20 D −28.30° (c 0.00883, MeOH). UV (MeOH) λmax, nm: 230, 273. IR νmax: 3363, 2925, 2886, 1614, 1597, 1516, 1449, 1369, 1237, 1160, 1076, 1024. ESI−MS m/z: 323.1106 [M + Na]+ (calculated for C14H20O7). 1 H NMR (CD3OD, 500 MHz) δ: 7.06 (2H, d, J = 8.4 Hz, H-2, 6), 6.69 (2H, d, J = 8.4 Hz, H-3, 5), 2.83 (1H, m, H-7), 4.03 (1H, m, H-8), 3.70 (1H, m, H-8), 4.29 (1H, d, J = 7.8 Hz, HGlu.-1), 3.17 (1H, m, H-Glu.-2), 3.27 (1H, m, H-Glu.-3), 3.24 (1H, m, H-Glu.-4), 3.35 (1H, m, H-Glu.-5), 3.86 (1H, dd, J = 11.9, 2.0 Hz, H-Glu.-6), 3.66 (1H, dd, J = 11.9, 5.2 Hz, H-Glu.-6). 13C NMR (CD3OD, 125 MHz) δ: 130.9 (C-1), 131.1 (C-2, 6), 116.3 (C-3, 5), 157.0 (C-4), 36.6 (C-7), 72.3 (C-8), 104.6 (C-Glu.-1), 75.3 (C-Glu.-2), 78.1 (C-Glu.-3), 71.8 (C-Glu.-4), 78.3 (C-Glu.-5), 63.0 (C-Glu.-6). Compound 1 was identified as salidroside by direct comparison to commercial salidroside (Sigma Chemical Co., St. Louis, MO). On the basis of the data described above and the verified reference,17 the structure of compound 1 was elucidated as salidroside. Yuan et al.18 reported that salidroside exhibits antioxidant activity and a protective effect against furan-induced hepatocyte damage in mice. p-Tyrosol (Compound 2). Compound 2 was a white amorphous powder (Figure 4A). UV (MeOH) λmax, nm: 223, 263. IR νmax: 3386, 3141, 3024, 1598, 1510, 1452, 1363, 1230, 1105, 1051, 1015. ESI−MS m/z: 138 [M] (calculated for C8H10O2). 1H NMR (CD3OD, 500 MHz) δ: 7.04 (2H, d, J = 8.6 Hz, H-2, 6), 6.71 (2H, d, J = 8.6 Hz, H-3, 5), 2.73 (2H, t,
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RESULTS AND DISCUSSION XO Inhibitory Activity of Fractionated Extracts of R. crenulata. The procedure for fractionation of the extracts of R. crenulata is shown in Figure 1. R. crenulata (70 g) was extracted with distilled water and freeze-dried to yield 14 g of WE, which displayed XO inhibitory activity (IC50 = 59.5 ± 1.7 μg/mL). The WE were then chromatographed on the SPE cartridges and eluted with varying concentrations of methanol. Finally, five fractions (fractions 1−5) were collected using a stepwise method of 0, 10, 20, 30, and 40% methanol in distilled water; the total yield of these fractions was 98.2%, and the yield of each fraction was 84.3, 9.4, 3.0, 1.1, and 0.4%, respectively. The IC50 values of WE and the associated fractions (fractions 1−5) were 59.5 ± 1.7, 862.2 ± 31.3, 524.2 ± 28.6, 94.7 ± 3.2, 16.1 ± 1.5, and 19.2 ± 1.8 μg/mL, respectively. Thus, we tested the inhibition of XO using WE and fractions at a final concentration of 0−60 μg/mL. Our results indicated that XO inhibition activity was 25.7% for WE and 2.8, 5.5, 17.2, 72.0, and 64.7% for fractions 1−5, respectively, at a concentration of 30 μg/mL (Figure 2). Among those fractions, the fourth
Figure 2. Inhibition of XO by the WE and its fractions at a final concentration of 30 μg/mL. Each value is represented as the mean ± standard deviation from triplicate measurements. WE, water extracts; Fr, fraction.
fraction (fraction 4) had the highest XO inhibitory activity (IC50 = 16.1 ± 1.5 μg/mL). Chen et al.16 reported that the crude extracts of Koelreuteria henryi, Prunus campanulata, and Rhodiola rosea possessed strong XO inhibitory activity, with IC50 values of 91.8 ± 1.7, 64.6 ± 5.8, and 56.0 ± 1.0 μg/mL, respectively. These values indicate that the WE and fraction 4 would be plentiful sources of XO inhibitors. HPLC Analysis of the WE and Its Fractions. To establish the fingerprint chromatogram of the WE and its five fractions, samples were analyzed by HPLC. The elution began with 3744
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Figure 3. HPLC chromatograms of the WE and its fractions. Flow rate, 1.0 mL/min; UV detection, 280 nm. WE, water extracts; Fr, fraction.
Table 1. Structure, Molecular Weight, Molecular Formula, and IC50 Values of XO Inhibitors
a
IC50 values are expressed as the mean ± standard deviation. 3745
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Figure 4. Isolation of active compounds from the (A) WE and (B) fraction 4. Arrow, active compounds; flow rate, 1.0 mL/min; UV detection, 280 nm.
J = 7.2 and 14.5 Hz, H-7), 3.70 (2H, t, J = 7.2 and 14.5 Hz, H-8). 13C NMR (CD3OD, 125 MHz) δ: 131.1 (C-1), 131.2 (C-2, 6), 116.3 (C-3, 5), 154.9 (C-4), 39.6 (C-7), 64.8 (C-8). Compound 2 was identified as p-tyrosol by direct comparison to commercial p-tyrosol (Sigma Chemical Co., St. Louis, MO). On the basis of the data described above and the verified reference,19 the structure of compound 2 was elucidated as p-tyrosol. Dewapriya et al.20 suggested that p-tyrosol has antioxidant activity and a protective effect against dopaminergic neuronal cell death in an in vitro model of Parkinson’s disease. B2−3′-O-Gallate (Compound 3). Compound 3 was a brown amorphous powder (Figure 4B), [α]20 D −57.00° (c 0.00667, MeOH). UV (MeOH) λmax, nm: 213, 228, 274. IR νmax: 3321, 1690, 1609, 1520, 1448, 1370, 1284, 1235, 1156, 1099, 1039. ESI−MS m/z: 731.1615 [M + H]+ (calculated for C37H30O16). 1H NMR (CD3COCD3, 500 MHz) δ: 5.26 (2H, s, H-2U, 2L), 4.06 (1H, s, H-3U), 4.89 (1H, s, H-4U), 6.03 (3H, m, H-6U, 8U, 6L), 6.77 (3H, m, H-2′U, 6′U, 5′L), 6.98 (1H, s, H-5′U), 5.62 (1H, s, H-3L), 2.98 (1H, d, J = 17.5 Hz, H-4L), 3.13 (1H, dd, J = 17.1 and 4.4 Hz, H-4L), 7.13 (3H, m, H-2′L, 2″L, 6″L), 7.05 (1H, s, H-6′L). 13C NMR (CD3COCD3, 125 MHz) δ: 78.0 (C-2U), 72.9 (C-3U), 36.7 (C-4U), 157.7 (C-5U), 97.2 (C-6U, 8L), 158.2 (C-7U), 96.3 (C-8U), 158.2 (C-9U), 107.9 (C-10U), 132.5 (C-1′U), 115.6 (C-2′U), 145.3 (C-3′U, 4′U, 3′L), 119.4 (C-5′U), 119.1 (C-6′U), 77.2 (C-2L), 69.1 (C-3L),
26.6 (C-4L), 155.5 (C-5L, 7L), 95.8 (C-6L), 156.0 (C-9L), 99.6 (C-10L), 131.2 (C-1′L), 115.5 (C-2′L), 145.2 (C-4′L), 115.3 (C-5′L), 114.7 (C-6′L), 122.0 (C-1″L), 110.2 (C-2″L, 6″L), 145.7 (C-3″L, 5″L), 138.7 (C-4″L), 166.2 (C-7″L). On the basis of the data described above and the verified reference,21 the structure of compound 3 was elucidated as B2−3′-O-gallate. Lourenço et al.22 reported that this compound has antioxidant activity and reduces the formation of UV-induced α-tocopheroxyl radicals. 4-HAP (Compound 4). Compound 4 was a white amorphous powder (Figure 4B). UV (MeOH) λmax, nm: 222. IR νmax: 3305, 1661, 1602, 1576, 1512, 1430, 1357, 1279, 1220, 1166, 1130, 1107, 1075, 1021. ESI−MS m/z: 136 [M] (calculated for C8H8O2). 1H NMR (CD3OD, 400 MHz) δ: 7.88 (2H, d, J = 8.9 Hz, H-2, 6), 6.84 (2H, d, 2, J = 8.8 Hz, H-3, 5), 2.52 (3H, s, H-8). 13C NMR (CD3OD, 100 MHz) δ: 130.4 (C-1), 132.3 (C-2, 6), 116.4 (C-3, 5), 164.2 (C-4), 199.7 (C-7), 26.5 (C-8). Compound 4 was identified as 4-HAP by direct comparison to commercial 4-HAP (Sigma Chemical Co., St. Louis, MO). On the basis of the data described above and the verified reference,23 the structure of compound 4 was elucidated as 4-HAP. Chen et al.24 reported that 4-HAP exhibits antiallergic activities, and against β-hexosaminidase, it could release a marker of degranulation in rat basophilic leukemia (RBL)-2H3 cells. 3746
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Modes of Inhibition of XO by B2−3′-O-gallate and 4-HAP. The 4-HAP and B2−3′-O-gallate were analyzed by Michaelis−Menten kinetic studies to determine their modes of inhibition of XO. In this study, the Michaelis constant (Km) of XO was 5.33 ± 0.21 mM, which was similar to that reported by Tung et al.27 They reported that the Km value of XO was 21 μM. As shown in Figure 6, Lineweaver−Burk plots of B2−
XO Inhibitory Activity of 4-HAP, B2−3′-O-Gallate, Salidroside, p-Tyrosol, and Allopurinol. In the previous XO inhibitor studies, quercetin-3-O-rhamnopyranoside and luteolin isolated from Acacia confusa showed a strong inhibitory effect on XO, with IC50 values of 37.7 and 11.6 μM, respectively.25 The inhibitory effects of 4-HAP, B2−3′-O-gallate, salidroside, p-tyrosol, and allopurinol on XO were tested at different concentrations. XO inhibition increased significantly with the addition of allopurinol (p < 0.05; Figure 5). When
Figure 5. Inhibitory effects of allopurinol, B2−3′-O-gallate, and 4-HAP on XO. Each value is represented as the mean ± standard deviation from triplicate measurements.
15 μM of the inhibitor was added, the XO inhibition by allopurinol was 60.8%. Similar trends were also observed in the results for 4-HAP and B2−3′-O-gallate; they showed inhibitory effects on XO. When examining the inhibition of XO by 4-HAP at 25 μM and B2−3′-O-gallate at 30 μM, the XO inhibitory activities were 70.3 and 57.5%, respectively. 4-HAP and B2−3′O-gallate showed remarkable inhibitory effects on XO (p < 0.05). However, we failed to observe any significant inhibitory effects on XO by salidroside or p-tyrosol at 30 μM (data not shown). As previously mentioned, five major peaks and one minor peak were found in the HPLC profile of WE (Figure 3). Salidroside and p-tyrosol indicated two major peaks in WE following elution times of 21−22 and 23−24 min, respectively. We observed that WE displayed XO inhibitory activity; however, the five major peaks in WE did not present significant inhibitory effects on XO, suggesting that the minor peak in WE at an elution time of 24−38 min exhibits XO. Nguyen et al.26 reported that allopurinol is a substrate and a specific potent inhibitor of XO, but oxypurinol (1H-pyrazolo[3,4-d]-pyrimidine-4,6-diol) is the basic functioning ingredient found in allopurinol. XO can catalyze the conversion of allopurinol into oxypurinol, and inhibition occurs mainly through direct substrate competition in the breakdown of purines. The XO inhibitory activities of purified salidroside, p-tyrosol, B2−3′-O-gallate, and 4-HAP from R. crenulata were evaluated and compared to that of allopurinol. The structure, molecular weight, molecular formula, and IC50 values of allopurinol, 4-HAP, B2−3′-O-gallate, p-tyrosol, and salidroside are shown in Table 1. Allopurinol, 4-HAP, and B2−3′-O-gallate showed strong inhibitory effects on XO, and the IC50 values of these compounds were 12.21 ± 0.27, 15.62 ± 1.19, and 24.24 ± 1.80 μM, respectively. This result indicated that 4-HAP and B2−3′-O-gallate have the potential to be XO inhibitors. However, the IC50 values of both salidroside and p-tyrosol were higher than 200 μM.
Figure 6. Lineweaver−Burk plots for the inhibition of XO by XO inhibitors with xanthine as the substrate: (A) B2−3′-O-gallate and (B) 4-HAP.
3′-O-gallate and 4-HAP were constructed. The Lineweaver− Burk plots of B2−3′-O-gallate (Figure 6A) and 4-HAP (Figure 6B) had no intersection on the y or x axis, indicating that the type of inhibition was a mixed-type mode. Therefore, the Lineweaver−Burk plots revealed that B2−3′-O-gallate and 4-HAP behaved as mixed-type inhibitors. Several mixed-type XO inhibitors have been reported. For example, a Lineweaver− Burk plot indicated that 3,4-dihydroxybenzoic acid isolated from the flowers of Chrysanthemum sinense appears to be a mixed-type inhibitor of XO.26 Furthermore, Day et al.28 reported that quercetin-3′-glucuronide showed mixed competitive inhibition of XO. Dixon plots of B2−3′-O-gallate and 4-HAP on XO were also evaluated (Figure 7). The Dixon plots indicated that B2−3′-Ogallate (Figure 7A) and 4-HAP (Figure 7B) were mixed-type inhibitors of XO, and the values of Ki were 7.26 ± 0.38 and 8.45 ± 0.34 μM, respectively. Jiao et al.29 reported that apigenin4′-O-(2″-O-p-coumaroyl)-β-D-glucopyranoside purified from Palhinhaea cernua inhibits XO with mixed-type inhibition, with Ki and Ki′ values of 14.35 and 93.68 μM, respectively. The Ki 3747
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AUTHOR INFORMATION
Corresponding Author
*Telephone: +886-2-29052516. Fax: +886-2-29053622. E-mail: [email protected]. Author Contributions §
Shih-Hsiung Wu and Jung-Feng Hsieh contributed equally to this work. Funding
The authors thank the National Science Council (NSC 992313-B-030-001-MY3) for financial support. Notes
The authors declare no competing financial interest.
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ABBREVIATIONS USED 4-HAP, 4′-hydroxyacetophenone; B2−3′-O-gallate, epicatechin-(4β,8)-epicatechin gallate; IC50, half maximal inhibitory concentration; Ki, inhibition constant; Ki′, uncompetitive inhibition constant; Km, Michaelis constant; NMR, nuclear magnetic resonance spectroscopy; SPE, solid-phase extraction; WE, water extracts; XO, xanthine oxidase
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REFERENCES
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Figure 7. Dixon plots for the inhibition of XO by XO inhibitors with xanthine as the substrate: (A) B2−3′-O-gallate and (B) 4-HAP.
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