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A new compound from the leaves of Panax ginseng
Fitoterapia 78 (2007) 556 – 560 www.elsevier.com/locate/fitote
A new compound from the leaves of Panax ginseng Li-jun Wu a,⁎, Li-bo Wang a , Hui-yuan Gao a , Bin Wu a , Xiao-mei Song b , Zhi-shu Tang b a
School of Traditional Chinese Madica, Shenyang Pharmaceutical University, Shenyang 110016, China b School of Shanxi University of Chinese Medicine, Xianyang 712046, China Received 2 October 2006; accepted 19 June 2007 Available online 3 July 2007
Abstract Two compounds were isolated from the leaves of Panax ginseng. Their structures were identified as 3β,6α,12β-triol22,23,24,25,26,27-hexanordammaran-20-one, and dammar-20(22),24-diene-3β,6α,12β-triol by spectral and chemical methods. The complete signal assignments of the two compounds were carried out by means of 2D NMR spectral analysis. © 2007 Elsevier B.V. All rights reserved. Keywords: Panax ginseng; 3β,6α,12β-triol-22,23,24,25,26,27-Hexanordammaran-20-one; Dammar-20(22),24-diene-3β,6α,12β-triol
1. Introduction Panax ginseng, an ancient and famous herbal drug in traditional Chinese medicine was used to treat various diseases such as psychiatric neurologic diabetes mellitus [1]. A number of dammarane oligoglycosides constituents with anticancer, anti-arrhythmia and inhibitory activities on reducing side effects of steroid hormones were found and reported [2–10]. In order to find additional active agents from this plant, a further study on the chemical constituents was carried out, and a new compound and a novel natural product were obtained. This paper describes the isolation and structure elucidation of the new compound named 3β,6α,12β-triol-22,23,24,25,26,27-hexanordammaran-20-one (1). Moreover, the novel natural product, no previously isolated in any plant, was identified as dammar-20(22),24diene-3β,6α,12β-triol (2). 2. Experimental 2.1. Material Melting Point: X-4 micro melting point determination apparatus (uncorrected). ESI-MASS spectrum: LC-MSDTrap-SL. HR-FABMS: Bruker APEX. 1 H-NMR (600 MHz) and 13C-NMR (150 MHz):Bruker ARX-600. ⁎ Corresponding author. E-mail address: [email protected] (L. Wu). 0367-326X/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2007.06.002
L. Wu et al. / Fitoterapia 78 (2007) 556–560
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Table 1 H and 13C NMR data for 1 (600 and 150 MHz, DMSO-d6, J in Hertz and δ in ppm)
1
C
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 28 29 30 3-OH 6-OH 12-OH
HMQC
HMBC
δH
δC
0.93 and 1.53 1.84 2.92 – 0.75(1H, d, J 10.2) 3.87 (1H, m) 1.43 and 1.51 overlapped – 1.31(1H, m) – 1.62 and 1.66 overlapped 3.37(1H, m) 1.84(1H, m) – 1.07(2H,overlapped) 1.45 and 1.53(overlapped) 2.75(1H,m) 1.00(3H, m) 1.00(3H, m) – 2.13(3H, m) 1.21(3H, s) 0.83(3H, s) 0.82(3H, s) 4.20(1H, d, J 5.4.) 3.93(1H, d, J 6.6) 4.37(1H, d, J 6.0)
38.5 27.1 77.0 38.7 60.4 66.3 46.8 40.4 49.6 40.0 32.3 69.9 53.4 50.7 31.8 26.8 51.8 16.9 17.2 212.7 30.0 31.1 15.8 16.7 – – –
C-6, C-7
C-11, C-8 C-12 C-12, C-17, C-20 C-13 C-20 C-20 C-7, C-8, C-9, C-14 C-1, C-5, C-9, C-10 C-17, C-20 C-4, C-5,C-30 C-3, C-4, C-29 C-13, C-14, C-2, C-3, C-4 C-5, C-6 C-7 C-12, C-13
2.2. Plant P. ginseng (Araliaceae), leaves collected in Jilin province North-East district of China, were identified by Prof. Sun Qishi. A voucher was deposited in the Traditional Chinese medica of Shenyang Pharmaceutical University. 2.3. Extraction and isolation P. ginseng air-dried leaves extracted with 75% EtOH at 25 °C. The extract was subjected to a macro-reticular absorption resin (D101). The 37% EtOH fraction (45 g) was Si-gel CC eluting with CHCl3–MeOH mixture to give 400 fractions. Fractions No. 65–98, No. 99–102 were separated repeatedly on Si-gel CC eluting with CHCl3–MeOH mixtures to obtain compound 2 and compound 1, respectively. Compound 1. White amorphous powder. HR-FAB-MS m/z 415.2906 [M + Na]+ HR-FAB-MS (m/z). Calculated for C24H40O4Na (M + Na)+: 415.2912. 1H and 13C NMR: Table 1. Compound 2. White powder. 1H-NMR(600 MHz, pyr-d5) δ: 0.96, 1.00, 1.15, 1.45, 1.57, 1.61, 1.82, 1.99 (3H each, m, H-18, H-19, H-30, H-29, H-27, H-26, H-21, H-28), 2.77(2H, m, H-23), 3.55(1H, m, H-3), 3.94(1H, m, H-12), 4.42(1H, m, H-6), 5.22(1H, m, H-24), 5.49(1H, m, H-22). 13C-NMR data are given in Table 2. 3. Results and discussion Compound 1, white amorphous powder, mp. 252–254 °C, gave positive reaction to the Libermann–Burchard test. Its molecular formula was determined to be C24H40O4 based on the NMR and HR-FAB-MS. The 1H-NMR(600 MHz, DMSO-d6) spectrum showed six methyl signals and three OH as doublets proton at δ 4.37, 4.20 and 3.93. The 13C-NMR showed six methyl signals and one keto group at δ 212.7. In the HMBC spectrum, the long-range correlation between the
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L. Wu et al. / Fitoterapia 78 (2007) 556–560
Table 2 C-NMR data for compounds 2 and Rh4 (150 MHz, pyridine-d5)
13
C
2
Rh4 [11]
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 1′ 2′ 3′ 4′ 5′ 6′
39.6 28.2 78.4 40.4 61.8 67.7 47.7 41.5 50.5 40.4 32.7 72.6 50.7 50.9 32.3 28.9 50.4 17.7 17.1 140.1 13.2 123.2 27.5 123.5 131.3 25.7 17.5 32.0 16.5 17.7
38.9 27.3 79.5 39.8 60.9 78.0 44.7 40.8 50.0 39.2 31.1 71.2 49.8 50.3 32.0 26.9 50.1 17.2 17.2 139.5 12.5 122.9 29.4 124.7 130.7 25.1 16.8 31.1 15.8 16.2 105.4 74.8 79.0 72.0 77.5 62.5
methyl proton signal at δ 2.13 and the carbon signals at δ 212.7 and 51.8 could be identified. Moreover, the HMBC spectrum also showed the long-range correlation between the proton signal at δ1.84(1H, m, H-13) and the carbons at δ 16.7 (C-30), 31.8(C-15), 51.8(C-17), 69.9(C-12), and the long-range correlation between the proton signal at δ 2.75(1H, m, H-
Fig. 1. HMBC correlations of compound 1.
L. Wu et al. / Fitoterapia 78 (2007) 556–560
559
Fig. 2. Compound 1.
17) and δ 53.4 (C-13), 69.9(C-12), 212.7(C-20) also could be found. In addition, the long-range correlation between the proton signal at δ 0.82 (3H, m, H-30) and the carbons at δ 31.8 (C-15), 40.4 (C-8), δ 1.00 (3H, s, H-18) and δ 40.4 (C-8), 46.8(C-7), 49.6(C-9), 50.7(C-14) also could be identified in the HMBC correlations. Thus, the structure of piece A of compound 1 could be obtained (Fig. 1). Beside that, the long-range correlation between the proton signal at δ 2.92(1H, m, H-3) and the carbon signals at δ 15.8 (C-29), 31.1(C-28), δ 0.83(3H, m, H-29) and δ 31.1 (C-28), δ 0.83(3H, m, H-19) and δ 38.5 (C-1), 49.6(C-9), δ 0.75(1H, m, H-5) and 15.8(C-29), 17.2(C-19), 31.1(C-28), 38.7(C-4), 46.8(C-7), 66.3(C-6) also could be elucidated. Thus, the structure of piece B of compound 1 could be elucidated (Fig. 1). Segments A and B could be connected at the carbon signals at 46.8 (C-7) and 49.6 (C-9). Further more, the relative configuration of 1 was identified by the NOESY correlation between the proton signal at the δ 1.84 (1H, m, H-13) and δ 1.00 (3H, m, H-18), 2.75 (1H, m, H-17) and the proton signal at the δ 3.37 (1H, m, H-12) and the δ 0.82 (3H, m, H-30) were obtained and the NOESY spectrum also showed the correlation between the proton signal at the δ 1.21(3H, m, H-28) and δ 2.92(1H, m, H-3), 3.93(1H, m, 6OH),0.75(1H, m, H-5), and the signal at δ 0.83 (1H, m, H-29) and δ 4.20 (1H, m, 3-OH), 3.87(1H, m, H-6). The 13C-NMR signals of compound 1 were found to be similar to that of ginsengoside-Rh4 except for the data of C-6 and the side-chain part (C20–C27) [11]. Therefore, compound 1 was identified as 3β,6α,12β-triol-22,23,24,25,26,27-hexanordammaran20-one (Fig. 2). Compound 2, white amorphous powder, mp. 130–132 °C, gave positive reaction to the Libermann–Burchard test. Its molecular formula was determined to be C30H50O3 based on ESI-MS, m/z: 457[M–H]– and NMR (Table 2).
Fig. 3. Compound 2.
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L. Wu et al. / Fitoterapia 78 (2007) 556–560
The 1H-NMR spectrum showed eight methyl signals and two olefinic proton signals at δ 5.23 and 5.49. The 13C-NMR and DEPT spectra showed eight methyl signals and two double bonds signals at δ 123.2(C-22), 123.5 (C-24), 134.2(C-25), 140.1(C-20). Compound 2 could be confirmed to be the aglycone of ginsengoside Rh4 [dammara-20(22),24-dien20(22),24-diene] (Fig. 3) with a little difference of signal at C-6 (δ 67.7) by the comparison of the Rh4 carbon chemical shift reported [11– 13] (Table 2). Furthermore, based on analysis of HMQC and HMBC spectra, the long-range correlations between the proton signal at δ 1.22(H-5) and the carbons at δ 67.7(C-6), 47.7(C-7), δ 1.98(H-13) and δ 17.7(C-30),72.6(C-12) revealed the carbon signal at δ 67.7 should be assigned to the position of C-6 not C-12, Meanwhile, the signal at δ 72.6 should be assigned to the carbon of C-12. This result corrected an error found in the Ref. [11]. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13]
Chang HM, But PP. Pharmacology and application of Chinese materia medica. Singapore: World Scientific; 1996. p. 556. Chen YJ, Zhang BF, Zhang SL, Yao X, Dou DK. Bull Natl Nat Sci China 1995;3:34. Dou D, Wen Y, Pei Y, Chen Y. Chin Mater Med 1997;22:174. Kim HS, Lee JH, Goo YS, Na SY. Brain Res Bull 1998;46:245. Rudakewich M, Ba F, Benishin CG. Planta Med 2001;67:533. Lee TF, Shiao YJ, Chen CF, Wang CH. Planta Med 2001;67:634. Lee KY, Park JA, Chung E, Lee YH, Kim S, Lee SK. Cancer Lett 1996;110:193. Yun YS, Lee YS, Jo SK, Jung IS. Planta Med 1993;59:521. Zhu JH, Takeshita T, Kitagawa I, Morimoto K. Cancer Res 1995;55:1221. Bae EA, Han MJ, Baek NI, Dong DH. Arch Pharm Res 2001;24:297. Nam-In Baek, Dong Seon Kim, You Hui Lee. Planta Med 1996;62:86. Kim S, Park J, Ryu J, Lee Y, Kim T, Kim J, et al. Arch Pharm Res 1996;19:551. Chen Y, Xu S, Ma Q. Shenyang Coll Pharm 1987;4:282.
A new compound from the leaves of Panax ginseng Li-jun Wu a,⁎, Li-bo Wang a , Hui-yuan Gao a , Bin Wu a , Xiao-mei Song b , Zhi-shu Tang b a
School of Traditional Chinese Madica, Shenyang Pharmaceutical University, Shenyang 110016, China b School of Shanxi University of Chinese Medicine, Xianyang 712046, China Received 2 October 2006; accepted 19 June 2007 Available online 3 July 2007
Abstract Two compounds were isolated from the leaves of Panax ginseng. Their structures were identified as 3β,6α,12β-triol22,23,24,25,26,27-hexanordammaran-20-one, and dammar-20(22),24-diene-3β,6α,12β-triol by spectral and chemical methods. The complete signal assignments of the two compounds were carried out by means of 2D NMR spectral analysis. © 2007 Elsevier B.V. All rights reserved. Keywords: Panax ginseng; 3β,6α,12β-triol-22,23,24,25,26,27-Hexanordammaran-20-one; Dammar-20(22),24-diene-3β,6α,12β-triol
1. Introduction Panax ginseng, an ancient and famous herbal drug in traditional Chinese medicine was used to treat various diseases such as psychiatric neurologic diabetes mellitus [1]. A number of dammarane oligoglycosides constituents with anticancer, anti-arrhythmia and inhibitory activities on reducing side effects of steroid hormones were found and reported [2–10]. In order to find additional active agents from this plant, a further study on the chemical constituents was carried out, and a new compound and a novel natural product were obtained. This paper describes the isolation and structure elucidation of the new compound named 3β,6α,12β-triol-22,23,24,25,26,27-hexanordammaran-20-one (1). Moreover, the novel natural product, no previously isolated in any plant, was identified as dammar-20(22),24diene-3β,6α,12β-triol (2). 2. Experimental 2.1. Material Melting Point: X-4 micro melting point determination apparatus (uncorrected). ESI-MASS spectrum: LC-MSDTrap-SL. HR-FABMS: Bruker APEX. 1 H-NMR (600 MHz) and 13C-NMR (150 MHz):Bruker ARX-600. ⁎ Corresponding author. E-mail address: [email protected] (L. Wu). 0367-326X/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2007.06.002
L. Wu et al. / Fitoterapia 78 (2007) 556–560
557
Table 1 H and 13C NMR data for 1 (600 and 150 MHz, DMSO-d6, J in Hertz and δ in ppm)
1
C
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 28 29 30 3-OH 6-OH 12-OH
HMQC
HMBC
δH
δC
0.93 and 1.53 1.84 2.92 – 0.75(1H, d, J 10.2) 3.87 (1H, m) 1.43 and 1.51 overlapped – 1.31(1H, m) – 1.62 and 1.66 overlapped 3.37(1H, m) 1.84(1H, m) – 1.07(2H,overlapped) 1.45 and 1.53(overlapped) 2.75(1H,m) 1.00(3H, m) 1.00(3H, m) – 2.13(3H, m) 1.21(3H, s) 0.83(3H, s) 0.82(3H, s) 4.20(1H, d, J 5.4.) 3.93(1H, d, J 6.6) 4.37(1H, d, J 6.0)
38.5 27.1 77.0 38.7 60.4 66.3 46.8 40.4 49.6 40.0 32.3 69.9 53.4 50.7 31.8 26.8 51.8 16.9 17.2 212.7 30.0 31.1 15.8 16.7 – – –
C-6, C-7
C-11, C-8 C-12 C-12, C-17, C-20 C-13 C-20 C-20 C-7, C-8, C-9, C-14 C-1, C-5, C-9, C-10 C-17, C-20 C-4, C-5,C-30 C-3, C-4, C-29 C-13, C-14, C-2, C-3, C-4 C-5, C-6 C-7 C-12, C-13
2.2. Plant P. ginseng (Araliaceae), leaves collected in Jilin province North-East district of China, were identified by Prof. Sun Qishi. A voucher was deposited in the Traditional Chinese medica of Shenyang Pharmaceutical University. 2.3. Extraction and isolation P. ginseng air-dried leaves extracted with 75% EtOH at 25 °C. The extract was subjected to a macro-reticular absorption resin (D101). The 37% EtOH fraction (45 g) was Si-gel CC eluting with CHCl3–MeOH mixture to give 400 fractions. Fractions No. 65–98, No. 99–102 were separated repeatedly on Si-gel CC eluting with CHCl3–MeOH mixtures to obtain compound 2 and compound 1, respectively. Compound 1. White amorphous powder. HR-FAB-MS m/z 415.2906 [M + Na]+ HR-FAB-MS (m/z). Calculated for C24H40O4Na (M + Na)+: 415.2912. 1H and 13C NMR: Table 1. Compound 2. White powder. 1H-NMR(600 MHz, pyr-d5) δ: 0.96, 1.00, 1.15, 1.45, 1.57, 1.61, 1.82, 1.99 (3H each, m, H-18, H-19, H-30, H-29, H-27, H-26, H-21, H-28), 2.77(2H, m, H-23), 3.55(1H, m, H-3), 3.94(1H, m, H-12), 4.42(1H, m, H-6), 5.22(1H, m, H-24), 5.49(1H, m, H-22). 13C-NMR data are given in Table 2. 3. Results and discussion Compound 1, white amorphous powder, mp. 252–254 °C, gave positive reaction to the Libermann–Burchard test. Its molecular formula was determined to be C24H40O4 based on the NMR and HR-FAB-MS. The 1H-NMR(600 MHz, DMSO-d6) spectrum showed six methyl signals and three OH as doublets proton at δ 4.37, 4.20 and 3.93. The 13C-NMR showed six methyl signals and one keto group at δ 212.7. In the HMBC spectrum, the long-range correlation between the
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L. Wu et al. / Fitoterapia 78 (2007) 556–560
Table 2 C-NMR data for compounds 2 and Rh4 (150 MHz, pyridine-d5)
13
C
2
Rh4 [11]
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 1′ 2′ 3′ 4′ 5′ 6′
39.6 28.2 78.4 40.4 61.8 67.7 47.7 41.5 50.5 40.4 32.7 72.6 50.7 50.9 32.3 28.9 50.4 17.7 17.1 140.1 13.2 123.2 27.5 123.5 131.3 25.7 17.5 32.0 16.5 17.7
38.9 27.3 79.5 39.8 60.9 78.0 44.7 40.8 50.0 39.2 31.1 71.2 49.8 50.3 32.0 26.9 50.1 17.2 17.2 139.5 12.5 122.9 29.4 124.7 130.7 25.1 16.8 31.1 15.8 16.2 105.4 74.8 79.0 72.0 77.5 62.5
methyl proton signal at δ 2.13 and the carbon signals at δ 212.7 and 51.8 could be identified. Moreover, the HMBC spectrum also showed the long-range correlation between the proton signal at δ1.84(1H, m, H-13) and the carbons at δ 16.7 (C-30), 31.8(C-15), 51.8(C-17), 69.9(C-12), and the long-range correlation between the proton signal at δ 2.75(1H, m, H-
Fig. 1. HMBC correlations of compound 1.
L. Wu et al. / Fitoterapia 78 (2007) 556–560
559
Fig. 2. Compound 1.
17) and δ 53.4 (C-13), 69.9(C-12), 212.7(C-20) also could be found. In addition, the long-range correlation between the proton signal at δ 0.82 (3H, m, H-30) and the carbons at δ 31.8 (C-15), 40.4 (C-8), δ 1.00 (3H, s, H-18) and δ 40.4 (C-8), 46.8(C-7), 49.6(C-9), 50.7(C-14) also could be identified in the HMBC correlations. Thus, the structure of piece A of compound 1 could be obtained (Fig. 1). Beside that, the long-range correlation between the proton signal at δ 2.92(1H, m, H-3) and the carbon signals at δ 15.8 (C-29), 31.1(C-28), δ 0.83(3H, m, H-29) and δ 31.1 (C-28), δ 0.83(3H, m, H-19) and δ 38.5 (C-1), 49.6(C-9), δ 0.75(1H, m, H-5) and 15.8(C-29), 17.2(C-19), 31.1(C-28), 38.7(C-4), 46.8(C-7), 66.3(C-6) also could be elucidated. Thus, the structure of piece B of compound 1 could be elucidated (Fig. 1). Segments A and B could be connected at the carbon signals at 46.8 (C-7) and 49.6 (C-9). Further more, the relative configuration of 1 was identified by the NOESY correlation between the proton signal at the δ 1.84 (1H, m, H-13) and δ 1.00 (3H, m, H-18), 2.75 (1H, m, H-17) and the proton signal at the δ 3.37 (1H, m, H-12) and the δ 0.82 (3H, m, H-30) were obtained and the NOESY spectrum also showed the correlation between the proton signal at the δ 1.21(3H, m, H-28) and δ 2.92(1H, m, H-3), 3.93(1H, m, 6OH),0.75(1H, m, H-5), and the signal at δ 0.83 (1H, m, H-29) and δ 4.20 (1H, m, 3-OH), 3.87(1H, m, H-6). The 13C-NMR signals of compound 1 were found to be similar to that of ginsengoside-Rh4 except for the data of C-6 and the side-chain part (C20–C27) [11]. Therefore, compound 1 was identified as 3β,6α,12β-triol-22,23,24,25,26,27-hexanordammaran20-one (Fig. 2). Compound 2, white amorphous powder, mp. 130–132 °C, gave positive reaction to the Libermann–Burchard test. Its molecular formula was determined to be C30H50O3 based on ESI-MS, m/z: 457[M–H]– and NMR (Table 2).
Fig. 3. Compound 2.
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The 1H-NMR spectrum showed eight methyl signals and two olefinic proton signals at δ 5.23 and 5.49. The 13C-NMR and DEPT spectra showed eight methyl signals and two double bonds signals at δ 123.2(C-22), 123.5 (C-24), 134.2(C-25), 140.1(C-20). Compound 2 could be confirmed to be the aglycone of ginsengoside Rh4 [dammara-20(22),24-dien20(22),24-diene] (Fig. 3) with a little difference of signal at C-6 (δ 67.7) by the comparison of the Rh4 carbon chemical shift reported [11– 13] (Table 2). Furthermore, based on analysis of HMQC and HMBC spectra, the long-range correlations between the proton signal at δ 1.22(H-5) and the carbons at δ 67.7(C-6), 47.7(C-7), δ 1.98(H-13) and δ 17.7(C-30),72.6(C-12) revealed the carbon signal at δ 67.7 should be assigned to the position of C-6 not C-12, Meanwhile, the signal at δ 72.6 should be assigned to the carbon of C-12. This result corrected an error found in the Ref. [11]. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13]
Chang HM, But PP. Pharmacology and application of Chinese materia medica. Singapore: World Scientific; 1996. p. 556. Chen YJ, Zhang BF, Zhang SL, Yao X, Dou DK. Bull Natl Nat Sci China 1995;3:34. Dou D, Wen Y, Pei Y, Chen Y. Chin Mater Med 1997;22:174. Kim HS, Lee JH, Goo YS, Na SY. Brain Res Bull 1998;46:245. Rudakewich M, Ba F, Benishin CG. Planta Med 2001;67:533. Lee TF, Shiao YJ, Chen CF, Wang CH. Planta Med 2001;67:634. Lee KY, Park JA, Chung E, Lee YH, Kim S, Lee SK. Cancer Lett 1996;110:193. Yun YS, Lee YS, Jo SK, Jung IS. Planta Med 1993;59:521. Zhu JH, Takeshita T, Kitagawa I, Morimoto K. Cancer Res 1995;55:1221. Bae EA, Han MJ, Baek NI, Dong DH. Arch Pharm Res 2001;24:297. Nam-In Baek, Dong Seon Kim, You Hui Lee. Planta Med 1996;62:86. Kim S, Park J, Ryu J, Lee Y, Kim T, Kim J, et al. Arch Pharm Res 1996;19:551. Chen Y, Xu S, Ma Q. Shenyang Coll Pharm 1987;4:282.