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Identification of tanshinone IIA metabolites in rat liver microsomes by liquid chromatography–tandem mass spectrometry
Journal of Chromatography A, 1104 (2006) 366–369
Short communication
Identification of tanshinone IIA metabolites in rat liver microsomes by liquid chromatography–tandem mass spectrometry Peng Li a , Guang-Ji Wang a,∗ , Jing Li a,b , Hai-Ping Hao a , Chao-Nan Zheng a a
Key Lab of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University, Nanjing 210038, China b Department of Pharmacology, School of Medical, Jiangsu University, Zhenjiang 212001, China Received 24 November 2005; received in revised form 7 December 2005; accepted 9 December 2005
Abstract Tanshinone IIA, the major component extracted from Radix salvia miltiorrhiza, has been observed to possess various kinds of pharmacological activities including antioxidant, prevention of angina pectoris and myocardial infarction and anticancer. Tanshinone IIA was incubated with rat liver microsomes and the resulting metabolites were identified by liquid chromatography/tandem mass spectrometry. The results showed the formation of three main hydroxyl metabolites. The three hydroxyl metabolites of tanshinone IIA were proved to be tanshinone IIB, hydroxytanshinone IIA and przewaquinone A by comparing the tandem mass spectra and the chromatographic retention time with that of the respective authentic compounds. Tanshinone IIB, hydroxytanshinone IIA and przewaquinone A are all the chemical components of total tanshinones. It was reasonable to presume that the three hydroxy metabolites of tanshinone IIA were pharmacologically active the same as tanshinone IIA and the total tanshinones. © 2005 Elsevier B.V. All rights reserved. Keywords: Tanshinone IIA; Metabolites identification; LC–MS–MS
1. Introduction Tanshinone IIA is the major component extracted from Radix salvia miltiorrhiza (RSM, the root of Salvia miltiorrhiza), which is a well-known traditional Chinese herbal medicine called “Danshen” and widely adopted in traditional Chinese medicinal preparations [1,2]. Tanshinone IIA has been observed to possess various kinds of pharmacological activities including antioxidant [3], prevention of angina pectoris and myocardial infarction [4] and anticancer [5,6]. As compared to the extensive research of the pharmacological activites of tanshinone IIA, few studies have dealt with its metabolism and pharmacokinetics. Recent research indicated that tanshinone IIA may possess complex inhibiting and inducting characters upon the CYP450 enzyme [7,8]. The metabolites identification and further metabolism study of tanshinone IIA is essential for explaining the mechanism of these characters.
∗
Corresponding author. Tel.: +86 25 85391035; fax: +86 25 85306750. E-mail address: [email protected] (G.-J. Wang).
0021-9673/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2005.12.047
In present study, we identified the metabolites of tanshinone IIA in rat microsomes with the existence of a NADPH generating system using a LC–MS–MS system and the molecular structure was elucidated and confirmed by comparing the chromatographic retention time and tandem mass spectra with that of the standard compounds. 2. Experimental 2.1. Chemicals and reagents Tanshinone IIA (mw = 294), Tanshinone IIB (mw = 310), Hydroxytanshinone IIA (mw = 310) and Przewaquinone A (mw = 310) were generously provided by Professor Houwei Luo (Department of Natural Medicinal Chemistry, China Pharmaceutical University). The purity of all chemicals was proved above 99% and their chemical structures are shown in Fig. 1. Glucose 6-phosphate, NADP and glucose 6-phosphate dehydrogenase were purchased from Sigma Chemical (St. Louis, MO, USA). HPLC grade acetonitrile was obtained from Fisher Scientific (Toronto, Canada). Deionized water was purified using a
P. Li et al. / J. Chromatogr. A 1104 (2006) 366–369
367
Fig. 1. Chemical structure of tanshinone IIA, tanshinone IIB, hydroxytanshinone IIA and przewaquinone A.
Milli-Q system (Millipore, Milford, MA, USA). Ethyl acetate and other chemicals and solvents used were all of analytical grade. 2.2. Liver microsome preparation and in vitro incubation Rat liver microsome was prepared according to the method of Abernathy et al. [9]. Microsomal protein concentration was determined by the method of Lowry et al. [10], using bovine serum albumin as the standard. For the in vitro metabolites identification, rat liver microsomal preparations were incubated with the existence of a NADPH-regenerating system including 0.5 mM NADP+ , 10 mM glucose 6-phosphate, 10 mM MgCl2 and 1.0 Unit/ml glucose-6-phosphate dehydrogenase at a final incubation volume of 200 l. After incubating at 37 ◦ C in a shaking water bath for 3 h, the reaction was terminated by the addition of 800 l ice-cold ethyl acetate. The mixture was shaken mechanically and centrifuged at 2000 × g for 5 min. Then, the upper organic layer was evaporated to dry in the Thermo Savant SPD 2010 SpeedVac System (Thermo Electron Corporation, USA) set at 40 ◦ C. The residues were then reconstituted in 200 l acetonitrile for LC–MS–MS analysis.
The mass detection was conducted using a Finnigan TSQ Quantum Discovery Max system (Thermo Electron, San Jose, CA, USA) equipped with an electrospray ionization source. Data acquisition was performed with Xcalibur 1.2 software (Thermo Finnigan, USA). A gradient with mobile phase composed of acetonitrile and 0.05% formic acid was used to obtain the baseline separation of all metabolites at a flow rate of 0.2 ml/min. The gradient start at 50% acetonitrile and 50% water for 20 min, followed by a linear gradient increase to 95% acetonitrile in only 1 min. This percentage was maintained for 10 min before a linear change back to 50% acetonitrile in 1 min. The gradient finished after the 50% acetonitrile maintained for 3 min. Spray voltage was set at 4 kV and capillary temperature was set at 350 ◦ C. Nitrogen was used both as sheath gas setting at 30 × 105 Pa and as auxiliary gas setting at 0.5 × 105 Pa. The mass spectrometer was operated in positive ion mode and scanned over the range m/z 100–800. Argon was used as collision gas for the tandem mass experiments. CID experiments were carried out using collision energy of 25 eV, following isolation of the precursor ion over a selected mass window of 1 Da. 3. Results and discussion
2.3. Instruments and analytical condition
3.1. Metabolic profiles in rat microsome
LC experiments were conducted using a Finnigan SurveyorTM HPLC system (Thermo Electron, San Jose, CA, USA). Separations of analytes were achieved using a 250 mm × 2.0 mm Shim-pack VP-ODS analytical column (Shimadzu, Kyoto,Japan.) protected by a Securityguard (Phenomenex Inc.).
The characterization of tanshinone IIA metabolites was performed using the rat liver microsomes incubation samples by LC–MS–MS. Three metabolites, M1, M2 and M3, were identified from the incubation samples. The resulting total ion chromatograms and the reconstructed ion chromatograms generated
368
P. Li et al. / J. Chromatogr. A 1104 (2006) 366–369
Fig. 2. Total ion chromatogram and extracted ion chromatograms for tanshinone IIA incubated with rat liver microsomes in the presence of a NADPH-generating system. Fig. 3. MS2 product ions spectra of the protonated molecules of tanshinone IIA.
by monitoring the detected ions of tanshinone IIA and its three metabolites are shown in Fig. 2. 3.2. Metabolites identification Tanshinone IIA is observed as its protonated molecule [M + H]+ at m/z 295 with a retention time of 30.4 min. The product ions spectra of the protonated molecules of tanshinone IIA are showed in Fig. 3. The major product ion of tanshinone IIA at m/z 277 is corresponding to H2 O loss from the parent ion m/z 295. Ji and Luo studied the MS fragmentation mechanism of tanshinones and proved hydrogen at C-1 and oxygen at C-11 to be the source of the H2 O dissociated [11]. Two additional fragment ions at m/z 280 and m/z 262 were formed, presumably, via the loss of the neutral species CH3 from the protonated ion and the m/z 277, respectively. A fragment ion at m/z 249 formed by the loss of CO from the m/z 277. The proposed fragmentation mechanism of tanshinone IIA is illustrated in Fig. 4.
M1 and M3 were all observed as protonated molecules [M + H]+ at m/z 311 with a retention time of 10.7 and 18.7 min, respectively. M2 is observed as its dehydrated protonated molecule [M + H − H2 O]+ at m/z 293 with a retention time of 17.9 min. The mass spectrums of M3 exhibited almost no protonated molecule [M + H]+ at m/z 311 detected under the Q1 MS fullscan mode. The fragment ion m/z 293 of M1, M3 and the dehydrated protonated parent ion m/z 293 of M2 were all extended lose of H2 O to form the fragment ion m/z 275. This indicated that they may be the hydroxyl metabolites of tanshinone IIA. The M1, M2 and M3 were unambiguously confirmed to be tanshinone IIB, hydroxytanshinone IIA and przewaquinone A, respectively, by comparison of their fragmentation upon CID and the chromatographic retention times with those of the standard compounds. Retention time, m/z of the parent ion and MS2 data of tanshinone IIA and its metabolites obtained from LC–MS–MS are summarized in Table 1. They all show the same
Fig. 4. Fragmentation mechanism of tanshinone IIA.
P. Li et al. / J. Chromatogr. A 1104 (2006) 366–369
369
Table 1 LC–MS–MS product ion spectra of tanshinone IIA and its three hydroxy metabolites Compound Tanshinone IIA Tanshinone IIB (M1) Hydroxytanshinone IIA (M2) Przewaquinone A (M3)
Retention time (min)
Parent ion
Product ion spectra (fragment ions)
30.4 10.7 17.9 18.7
[M + H]+
280; 277; 262; 249 293; 278; 275; 250; 247 275; 265; 247 293; 278; 275; 265; 247
fragmentation mechanism as tanshinone IIA. The product ions corresponding to H2 O loss from the parent ions with the same fragmentation mechanism as tanshinone IIA, despite the fact that they have the hydroxyl group in their molecules [11]. Tanshinone IIB, hydroxytanshinone IIA and przewaquinone A, the hydroxyl metabolites of tanshinone IIA, were also the chemical components of total tanshinones with pharmacological activities such as antioxidant, anti-inflammatory and prevention of angina pectoris and myocardial infarction [12–14]. It was then reasonable to presume that the three hydroxyl metabolites of tanshinone IIA were pharmacologically active the same as tanshinone IIA or the total tanshinones. 4. Conclusion In present study three hydroxyl metabolites of tanshinone IIA, which were tanshinone IIB, hydroxytanshinone IIA and przewaquinone A, were characterized in rat liver microsome. The structure of these metabolites was conformed by comparing the chromatography retention time and MS–MS collision chromatograms with that of the standard compounds. Acknowledgements The project was financially supported by National “863” Project (No. 2003AA2Z347A); program in International Cooperation (No. BZ2004042) and Key Lab of Drug Metabolism and Pharmacokinetics of Jiangsu province (No. BM2001201). We
295 [M + H]+ 311 [M + H − H2 O]+ 293 [M + H]+ 311
thank Prof. Sun Fenzhi for editorial assistance and professor Houwei Luo for his valuable chemical support. Technical assistances from Mr. Jianguo Sun, Hao Li, and Mrs. Yan Liang were highly appreciated. References [1] A.R. Lee, W.L. Wu, W.L. Chang, H.C. Lin, M.L. King, J. Nat. Prod. 50 (1987) 157. [2] U. Breyer-Pfaff, K. Nill, Xenobiotica 25 (1995) 1311. [3] E.H. Cao, X.Q. Liu, J.J. Wang, N.F. Xu, Free Radic. Biol. Med. 20 (1996) 801. [4] B.L. Zhao, W. Jiang, Y. Zhao, J.W. Hou, W.J. Xin, Biochem. Mol. Biol. Int. 38 (1996) 1171. [5] S.L. Yuan, X.J. Wang, Y.Q. Wei, Ai. Zheng. 22 (2003) 1363. [6] X. Wang, Y. Wei, S. Yuan, G. Liu, Y. Lu, J. Zhang, W. Wang, Int. J. Cancer 116 (2005) 799. [7] Y.F. Ueng, Y.H. Kuo, H.C. Peng, T.L. Chen, W.C. Jan, F. Peter Guengerich, Y.L. Lin, Xenobiotica 33 (2003) 603. [8] Y.F. Ueng, Y.H. Kuo, S.Y. Wang, Y.L. Lin, C.F. Chen, Life Sci. 74 (2004) 885. [9] C.O. Abernathy, E. Hodgson, F.E. Guthrie, Biochem. Pharmacol. 20 (1971) 2385. [10] O.H. Lowry, N.J. Rosebrough, AL. Farr, R.J. Randall, J. Biol. Chem. 193 (1951) 265. [11] J. Ji, H.W. Luo, J. Chin. Pharm. Univ. 19 (1988) 197. [12] S.Y. Ryu, M.H. Oak, K.M. Kim, Planta Med. 65 (1999) 654. [13] A.C. Goren, G. Topcu, S. Oksuz, G. Kokdil, W. Voelter, A. Ulubelen, Nat. Prod. Lett. 16 (2002) 47. [14] J.S. Ko, S.Y. Ryu, Y.S. Kim, M.Y. Chung, J.S. Kang, M.C. Rho, H.S. Lee, Y.K. Kim, Arch. Pharm. Res. 25 (2002) 446.
Short communication
Identification of tanshinone IIA metabolites in rat liver microsomes by liquid chromatography–tandem mass spectrometry Peng Li a , Guang-Ji Wang a,∗ , Jing Li a,b , Hai-Ping Hao a , Chao-Nan Zheng a a
Key Lab of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University, Nanjing 210038, China b Department of Pharmacology, School of Medical, Jiangsu University, Zhenjiang 212001, China Received 24 November 2005; received in revised form 7 December 2005; accepted 9 December 2005
Abstract Tanshinone IIA, the major component extracted from Radix salvia miltiorrhiza, has been observed to possess various kinds of pharmacological activities including antioxidant, prevention of angina pectoris and myocardial infarction and anticancer. Tanshinone IIA was incubated with rat liver microsomes and the resulting metabolites were identified by liquid chromatography/tandem mass spectrometry. The results showed the formation of three main hydroxyl metabolites. The three hydroxyl metabolites of tanshinone IIA were proved to be tanshinone IIB, hydroxytanshinone IIA and przewaquinone A by comparing the tandem mass spectra and the chromatographic retention time with that of the respective authentic compounds. Tanshinone IIB, hydroxytanshinone IIA and przewaquinone A are all the chemical components of total tanshinones. It was reasonable to presume that the three hydroxy metabolites of tanshinone IIA were pharmacologically active the same as tanshinone IIA and the total tanshinones. © 2005 Elsevier B.V. All rights reserved. Keywords: Tanshinone IIA; Metabolites identification; LC–MS–MS
1. Introduction Tanshinone IIA is the major component extracted from Radix salvia miltiorrhiza (RSM, the root of Salvia miltiorrhiza), which is a well-known traditional Chinese herbal medicine called “Danshen” and widely adopted in traditional Chinese medicinal preparations [1,2]. Tanshinone IIA has been observed to possess various kinds of pharmacological activities including antioxidant [3], prevention of angina pectoris and myocardial infarction [4] and anticancer [5,6]. As compared to the extensive research of the pharmacological activites of tanshinone IIA, few studies have dealt with its metabolism and pharmacokinetics. Recent research indicated that tanshinone IIA may possess complex inhibiting and inducting characters upon the CYP450 enzyme [7,8]. The metabolites identification and further metabolism study of tanshinone IIA is essential for explaining the mechanism of these characters.
∗
Corresponding author. Tel.: +86 25 85391035; fax: +86 25 85306750. E-mail address: [email protected] (G.-J. Wang).
0021-9673/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2005.12.047
In present study, we identified the metabolites of tanshinone IIA in rat microsomes with the existence of a NADPH generating system using a LC–MS–MS system and the molecular structure was elucidated and confirmed by comparing the chromatographic retention time and tandem mass spectra with that of the standard compounds. 2. Experimental 2.1. Chemicals and reagents Tanshinone IIA (mw = 294), Tanshinone IIB (mw = 310), Hydroxytanshinone IIA (mw = 310) and Przewaquinone A (mw = 310) were generously provided by Professor Houwei Luo (Department of Natural Medicinal Chemistry, China Pharmaceutical University). The purity of all chemicals was proved above 99% and their chemical structures are shown in Fig. 1. Glucose 6-phosphate, NADP and glucose 6-phosphate dehydrogenase were purchased from Sigma Chemical (St. Louis, MO, USA). HPLC grade acetonitrile was obtained from Fisher Scientific (Toronto, Canada). Deionized water was purified using a
P. Li et al. / J. Chromatogr. A 1104 (2006) 366–369
367
Fig. 1. Chemical structure of tanshinone IIA, tanshinone IIB, hydroxytanshinone IIA and przewaquinone A.
Milli-Q system (Millipore, Milford, MA, USA). Ethyl acetate and other chemicals and solvents used were all of analytical grade. 2.2. Liver microsome preparation and in vitro incubation Rat liver microsome was prepared according to the method of Abernathy et al. [9]. Microsomal protein concentration was determined by the method of Lowry et al. [10], using bovine serum albumin as the standard. For the in vitro metabolites identification, rat liver microsomal preparations were incubated with the existence of a NADPH-regenerating system including 0.5 mM NADP+ , 10 mM glucose 6-phosphate, 10 mM MgCl2 and 1.0 Unit/ml glucose-6-phosphate dehydrogenase at a final incubation volume of 200 l. After incubating at 37 ◦ C in a shaking water bath for 3 h, the reaction was terminated by the addition of 800 l ice-cold ethyl acetate. The mixture was shaken mechanically and centrifuged at 2000 × g for 5 min. Then, the upper organic layer was evaporated to dry in the Thermo Savant SPD 2010 SpeedVac System (Thermo Electron Corporation, USA) set at 40 ◦ C. The residues were then reconstituted in 200 l acetonitrile for LC–MS–MS analysis.
The mass detection was conducted using a Finnigan TSQ Quantum Discovery Max system (Thermo Electron, San Jose, CA, USA) equipped with an electrospray ionization source. Data acquisition was performed with Xcalibur 1.2 software (Thermo Finnigan, USA). A gradient with mobile phase composed of acetonitrile and 0.05% formic acid was used to obtain the baseline separation of all metabolites at a flow rate of 0.2 ml/min. The gradient start at 50% acetonitrile and 50% water for 20 min, followed by a linear gradient increase to 95% acetonitrile in only 1 min. This percentage was maintained for 10 min before a linear change back to 50% acetonitrile in 1 min. The gradient finished after the 50% acetonitrile maintained for 3 min. Spray voltage was set at 4 kV and capillary temperature was set at 350 ◦ C. Nitrogen was used both as sheath gas setting at 30 × 105 Pa and as auxiliary gas setting at 0.5 × 105 Pa. The mass spectrometer was operated in positive ion mode and scanned over the range m/z 100–800. Argon was used as collision gas for the tandem mass experiments. CID experiments were carried out using collision energy of 25 eV, following isolation of the precursor ion over a selected mass window of 1 Da. 3. Results and discussion
2.3. Instruments and analytical condition
3.1. Metabolic profiles in rat microsome
LC experiments were conducted using a Finnigan SurveyorTM HPLC system (Thermo Electron, San Jose, CA, USA). Separations of analytes were achieved using a 250 mm × 2.0 mm Shim-pack VP-ODS analytical column (Shimadzu, Kyoto,Japan.) protected by a Securityguard (Phenomenex Inc.).
The characterization of tanshinone IIA metabolites was performed using the rat liver microsomes incubation samples by LC–MS–MS. Three metabolites, M1, M2 and M3, were identified from the incubation samples. The resulting total ion chromatograms and the reconstructed ion chromatograms generated
368
P. Li et al. / J. Chromatogr. A 1104 (2006) 366–369
Fig. 2. Total ion chromatogram and extracted ion chromatograms for tanshinone IIA incubated with rat liver microsomes in the presence of a NADPH-generating system. Fig. 3. MS2 product ions spectra of the protonated molecules of tanshinone IIA.
by monitoring the detected ions of tanshinone IIA and its three metabolites are shown in Fig. 2. 3.2. Metabolites identification Tanshinone IIA is observed as its protonated molecule [M + H]+ at m/z 295 with a retention time of 30.4 min. The product ions spectra of the protonated molecules of tanshinone IIA are showed in Fig. 3. The major product ion of tanshinone IIA at m/z 277 is corresponding to H2 O loss from the parent ion m/z 295. Ji and Luo studied the MS fragmentation mechanism of tanshinones and proved hydrogen at C-1 and oxygen at C-11 to be the source of the H2 O dissociated [11]. Two additional fragment ions at m/z 280 and m/z 262 were formed, presumably, via the loss of the neutral species CH3 from the protonated ion and the m/z 277, respectively. A fragment ion at m/z 249 formed by the loss of CO from the m/z 277. The proposed fragmentation mechanism of tanshinone IIA is illustrated in Fig. 4.
M1 and M3 were all observed as protonated molecules [M + H]+ at m/z 311 with a retention time of 10.7 and 18.7 min, respectively. M2 is observed as its dehydrated protonated molecule [M + H − H2 O]+ at m/z 293 with a retention time of 17.9 min. The mass spectrums of M3 exhibited almost no protonated molecule [M + H]+ at m/z 311 detected under the Q1 MS fullscan mode. The fragment ion m/z 293 of M1, M3 and the dehydrated protonated parent ion m/z 293 of M2 were all extended lose of H2 O to form the fragment ion m/z 275. This indicated that they may be the hydroxyl metabolites of tanshinone IIA. The M1, M2 and M3 were unambiguously confirmed to be tanshinone IIB, hydroxytanshinone IIA and przewaquinone A, respectively, by comparison of their fragmentation upon CID and the chromatographic retention times with those of the standard compounds. Retention time, m/z of the parent ion and MS2 data of tanshinone IIA and its metabolites obtained from LC–MS–MS are summarized in Table 1. They all show the same
Fig. 4. Fragmentation mechanism of tanshinone IIA.
P. Li et al. / J. Chromatogr. A 1104 (2006) 366–369
369
Table 1 LC–MS–MS product ion spectra of tanshinone IIA and its three hydroxy metabolites Compound Tanshinone IIA Tanshinone IIB (M1) Hydroxytanshinone IIA (M2) Przewaquinone A (M3)
Retention time (min)
Parent ion
Product ion spectra (fragment ions)
30.4 10.7 17.9 18.7
[M + H]+
280; 277; 262; 249 293; 278; 275; 250; 247 275; 265; 247 293; 278; 275; 265; 247
fragmentation mechanism as tanshinone IIA. The product ions corresponding to H2 O loss from the parent ions with the same fragmentation mechanism as tanshinone IIA, despite the fact that they have the hydroxyl group in their molecules [11]. Tanshinone IIB, hydroxytanshinone IIA and przewaquinone A, the hydroxyl metabolites of tanshinone IIA, were also the chemical components of total tanshinones with pharmacological activities such as antioxidant, anti-inflammatory and prevention of angina pectoris and myocardial infarction [12–14]. It was then reasonable to presume that the three hydroxyl metabolites of tanshinone IIA were pharmacologically active the same as tanshinone IIA or the total tanshinones. 4. Conclusion In present study three hydroxyl metabolites of tanshinone IIA, which were tanshinone IIB, hydroxytanshinone IIA and przewaquinone A, were characterized in rat liver microsome. The structure of these metabolites was conformed by comparing the chromatography retention time and MS–MS collision chromatograms with that of the standard compounds. Acknowledgements The project was financially supported by National “863” Project (No. 2003AA2Z347A); program in International Cooperation (No. BZ2004042) and Key Lab of Drug Metabolism and Pharmacokinetics of Jiangsu province (No. BM2001201). We
295 [M + H]+ 311 [M + H − H2 O]+ 293 [M + H]+ 311
thank Prof. Sun Fenzhi for editorial assistance and professor Houwei Luo for his valuable chemical support. Technical assistances from Mr. Jianguo Sun, Hao Li, and Mrs. Yan Liang were highly appreciated. References [1] A.R. Lee, W.L. Wu, W.L. Chang, H.C. Lin, M.L. King, J. Nat. Prod. 50 (1987) 157. [2] U. Breyer-Pfaff, K. Nill, Xenobiotica 25 (1995) 1311. [3] E.H. Cao, X.Q. Liu, J.J. Wang, N.F. Xu, Free Radic. Biol. Med. 20 (1996) 801. [4] B.L. Zhao, W. Jiang, Y. Zhao, J.W. Hou, W.J. Xin, Biochem. Mol. Biol. Int. 38 (1996) 1171. [5] S.L. Yuan, X.J. Wang, Y.Q. Wei, Ai. Zheng. 22 (2003) 1363. [6] X. Wang, Y. Wei, S. Yuan, G. Liu, Y. Lu, J. Zhang, W. Wang, Int. J. Cancer 116 (2005) 799. [7] Y.F. Ueng, Y.H. Kuo, H.C. Peng, T.L. Chen, W.C. Jan, F. Peter Guengerich, Y.L. Lin, Xenobiotica 33 (2003) 603. [8] Y.F. Ueng, Y.H. Kuo, S.Y. Wang, Y.L. Lin, C.F. Chen, Life Sci. 74 (2004) 885. [9] C.O. Abernathy, E. Hodgson, F.E. Guthrie, Biochem. Pharmacol. 20 (1971) 2385. [10] O.H. Lowry, N.J. Rosebrough, AL. Farr, R.J. Randall, J. Biol. Chem. 193 (1951) 265. [11] J. Ji, H.W. Luo, J. Chin. Pharm. Univ. 19 (1988) 197. [12] S.Y. Ryu, M.H. Oak, K.M. Kim, Planta Med. 65 (1999) 654. [13] A.C. Goren, G. Topcu, S. Oksuz, G. Kokdil, W. Voelter, A. Ulubelen, Nat. Prod. Lett. 16 (2002) 47. [14] J.S. Ko, S.Y. Ryu, Y.S. Kim, M.Y. Chung, J.S. Kang, M.C. Rho, H.S. Lee, Y.K. Kim, Arch. Pharm. Res. 25 (2002) 446.