Chinese Journal of Natural Medicines 2014, 12(2): 01420147
Chinese Journal of Natural Medicines
Two new steroidal saponins from the rhizomes of Dioscorea zingiberensis ZHENG Lu1#, ZHOU Yuan1#, ZHANG Jia-Yu2, SONG Min1, YUAN Ye1, XIAO Yan-Jiao1, XIANG Ting1* 1
Research Institute of Traditional Chinese Medicine, Yangtze River Pharmaceutical Group Co., Ltd., Taizhou, 225321, Chinaχ School of Chinese Pharmacy, Beijing University of Chinese Medicine, Beijing 100029, China
2
Available online 20 Feb. 2014
[ABSTRACT] AIM: To investigate the chemical constituents of Dioscorea zingiberensis C. H. Wright. METHODS: The compounds were isolated by various chromatographic techniques, and the structures of the new steroidal saponins were elucidated by extensive 1D- and 2D-NMR, MS, and IR spectral analysis. RESULTS: The 70% EtOH extract of the rhizomes of Dioscorea zingiberensis afforded two new steroidal saponins, zingiberenosides A (1) and B (2), along with eight known analogues, 3, 26-dihydroxy-25(R)-furosta-Ƹ5, 20(22)-diene-3-O--L- rhamnopyra-
nosyl-(12)-O--D-glucopyranoside (3), methyl parvifloside (4), deltoside (5), methyl deltoside (6), zingiberensis new saponin (7), deltonin (8), progenin III (9) and diosgenin-diglucoside (10). CONCLUSION: Two new steroidal saponins were isolated from Dioscorea zingiberensis and their structures determined. [KEY WORDS] Dioscorea zingiberensis; Dioscoreaceae; Steroidal saponins; Zingiberenoside
[CLC Number] R284.1
[Document code] A
[Article ID] 2095-6975(2014)02-0142-06
Introduction Dioscorea zingiberensis C. H. Wright, common name ‘Huang Jiang’, is a perennial herb, and is distributed widely in Henan, Hubei, Sichuan, and the south of Shanxi provinces [1-2]. It is used to treat cough with lung heat, pyretic stranguria, anthracia, swelling, ulcers, and sprains. As an important species of the Dioscoreaceae, D. zingiberensis has attracted much attention for the high content of diosgenin and steroid saponins [3], as well as excellent pharmacologic action, such as reducing the content of cholesterol
[Received on] 10-Nov.-2012 [Research funding] This project was supported by the Science and Technology Achievement Transformation Project of Jiangsu Province (No. BA2010144); and the scientific and technological major special project for “Significant Creation of New Drugs” (No. 2012ZX09101231). [ Corresponding author] XIANG Ting: Dr., Senior Engineer, Tel: 86-10-80728999-6255, Fax: 86-10-80728999-6255. E-mail: tingxiang [email protected] # Co-first authors These authors have no conflict of interest to declare. Copyright © 2014, China Pharmaceutical University. Published by Elsevier B.V. All rights reserved
in blood [4], and decreasing stenocardia and regulating metabolism [5]. Furthermore, its total steroidal saponins could treat atherosclerosis, high blood fat, wheeze, inflammation. and tumors [6-7]. Previous phytochemical investigations of its rhizomes led to the isolation of several steroidal saponins [5, 8-15]. In this paper, the isolation and structural elucidation of two new steroidal saponins and eight known derivatives from the 70% EtOH extract of the dried rhizome of D. zingiberensis are reported.
Results and Discussion A 70% EtOH extract of the commercially available rhizomes of D. zingiberensis was fractionated by macroporous resin and silica gel, and then separated by ODS column chromatography and semi-preparative HPLC to afford two new steroidal saponins, zingiberenoside A (1) and zingiberenoside B (2), together with eight known analogues (Fig. 1). The structures of the new compounds were elucidated by extensive 1D- and 2D-NMR, MS, and IR spectral analyses. Compound 1 was obtained as a white amorphous powder, and was deduced to possess a furostanol group based on TLC using Ehrlich’s reagent [20]. The IR spectrum showed absorp-
– 142 –
ZHENG Lu, et al. / Chin J Nat Med, 2014, 12(2): 142147
Fig. 1 Structures of compounds 1 10 [M + Na]+ ion peak at m/z 1069.520 32 (Calcd. 1069.519 54). The 1H and 13C NMR (pyridine-d 5 , 500/125 MHz) data (Tables 1 and 2), assigned by 1 H- 1 H COSY, HSQC, and
tion bands of hydroxyl (3 421 cm1), methyl (2 930 cm1), and olefinic (1 644 cm1) groups. The molecular formula was assigned to be C 51 H 82 O 2 on the basis of positive HRESI-MS Table 1 No. 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 OCH 3
13
C NMR data for the aglycone of compounds 1 10 (in pyridine-d 5 , 125 MHz) 1 37.9 30.5 78.4 39.3 141.2 122.2 32.8 31.8 50.7 37.5 21.6 40.0 43.8 55.3 34.9 84.8 64.9 14.5 19.8 103.9 12.2 152.8 33.9 24.1 31.8 75.3 17.7
2 37.9 30.5 78.4 39.3 141.2 122.2 32.8 31.8 50.7 37.5 21.6 40.0 43.8 55.3 34.8 84.8 64.9 14.5 19.8 103.9 12.2 152.7 33.9 24.1 31.8 75.3 17.7
3 37.9 30.6 78.7 39.4 141.2 122.2 32.8 31.8 50.7 37.5 21.6 40.0 43.8 55.3 34.9 84.8 64.9 14.5 19.8 103.9 12.1 152.8 33.8 24.1 31.8 75.3 17.7
4 37.8 30.5 78.5 39.3 141.2 122.2 32.5 32.0 50.7 37.4 21.4 40.1 41.2 56.9 32.7 81.6 64.6 16.6 19.7 40.9 16.6 113.0 37.4 28.5 34.6 75.6 17.5 47.6
5 37.9 30.5 78.6 39.3 141.2 122.2 32.0 31.2 50.7 37.6 21.4 40.1 41.1 56.9 32.6 81.6 64.2 16.6 19.7 40.8 16.8 111.0 37.5 28.7 34.6 75.6 17.8
– 143 –
6 37.8 30.5 78.6 39.3 141.2 122.1 32.0 31.2 50.7 37.5 21.4 40.3 41.1 56.9 32.8 81.4 64.5 16.6 19.7 40.0 16.6 113.0 37.6 28.5 34.6 75.6 17.5 47.6
7 37.8 30.5 78.7 39.3 141.2 122.1 32.7 32.0 50.6 37.5 21.5 40.2 40.8 57.0 32.6 81.5 63.3 16.7 19.7 42.3 15.4 109.6 32.2 29.6 30.9 67.2 17.7
8 37.8 30.5 78.5 39.3 141.1 122.1 32.6 32.0 50.6 37.5 21.4 40.2 40.8 57.0 32.7 81.5 63.2 16.7 19.7 42.3 15.4 109.6 32.2 29.6 31.0 67.2 17.7
9 37.8 30.5 78.6 39.3 141.2 122.1 32.7 32.0 50.6 37.4 21.5 40.2 40.8 57.0 32.6 81.5 63.2 16.7 19.7 42.4 15.4 109.6 32.2 29.6 30.9 67.2 17.7
10 37.8 30.9 78.7 39.6 141.2 122.1 32.7 32.0 50.6 37.5 21.5 40.2 40.8 57.0 32.6 81.5 63.2 16.7 19.8 42.3 15.4 109.6 32.2 29.6 30.9 67.2 17.7
ZHENG Lu, et al. / Chin J Nat Med, 2014, 12(2): 142147
Table 2
13
C NMR data for the sugar moiety of compounds 1–10 (in pyridine-d 5 , 125 MHz)
No. inner 1
1
2
3
4
5
6
7
8
9
10
Glc
Glc
Glc
Glc
Glc
Glc
Glc
Glc
Glc
Glc
100.4
100.3
100.3
100.3
100.4
100.4
100.3
100.4
100.7
102.6
2
77.6
77.6
80.0
77.7
77.7
77.7
77.6
78.8
78.2
78.2
3
76.6
76.6
78.3
76.6
76.6
76.6
76.6
76.6
80.0
77.2
4
82.4
81.9
72.2
81.8
82.4
82.4
81.9
82.4
72.0
81.6
5
78.1
78.0
78.2
78.0
78.1
78.1
78.0
78.1
78.8
76.8
6
62.3
61.8
63.0
61.9
62.3
62.3
61.9
62.3
63.0
62.8
intermediate
Glc
Glc
Glc
1
104.9
104.9
104.9
2
74.1
74.1
74.1
3
88.7
88.6
88.7
4
69.8
69.8
69.8
5
78.4
78.4
78.4
6
62.1
62.1
62.1
terminal 1
Glc
Glc
Glc
Glc
Glc
Glc
Glc
Glc
105.3
106.3
106.3
105.3
105.3
106.3
105.6
105.3
2
75.3
75.9
75.9
75.3
75.3
75.9
75.3
75.1
3
79.0
79.1
79.1
78.5
78.5
78.5
78.6
78.3
4
71.6
72.0
72.0
71.6
71.6
72.0
71.6
71.9
5
78.9
79.0
79.0
78.8
78.8
79.1
77.7
78.6
6
62.4
62.9
62.9
62.4
62.4
62.9
62.5
terminal
Rha
Rha
Rha
Rha
Rha
Rha
Rha
Rha
Rha
62.5
1
102.2
102.1
102.4
102.2
102.2
102.2
102.2
102.2
102.4
2
72.8
72.8
73.0
72.8
72.8
72.8
72.8
72.8
72.9
3
73.1
73.1
73.2
73.1
73.1
73.1
73.1
73.1
73.2
4
74.5
74.5
74.5
74.5
74.5
74.5
74.5
74.5
74.5
5
69.8
69.7
69.9
69.7
69.8
69.8
69.7
69.8
69.8
6
19.0
19.0
19.0
19.0
19.0
19.0
19.0
19.0
19.0
26-O1
Glc
Glc
Glc
Glc
Glc
Glc
105.6
105.3
105.3
105.4
105.5
105.6
2
75.6
75.6
75.6
75.6
75.6
75.6
3
78.9
78.9
79.0
78.9
78.9
78.9
4
72.1
72.1
72.1
72.1
72.1
72.1
5
78.7
78.7
78.9
78.7
78.8
78.8
6
63.2
63.2
63.0
63.3
63.2
63.3
HMBC, revealed the presence of three singlets for tertiary
=^ ´ H 0.75 (H 3 -18, s), 1.07 (H 3 -19, s) and 1.66 (H 3 -21, s), two dou` ^ ´ H 1.03 (H 3 -27, d, J = 7.0 Hz) and 1.78 (H 3 -Rha-6, d, J = 6.0 Hz), and an olefinic proton at ´ H 5.49 (H-6, d, J = 4.0 Hz). Two pairs of olefinic carbon signals at ´ C 141.2, 122.2 and 103.9, 152.8 in the 13C NMR spectrum were assigned to C-5(6) and C-20(22) by comparison with the known compounds huangjiangsu A [8] and ceparoside C [21]. Comparison of the 1H and 13C NMR spectra data of 1 with those of huangjiangsu A and ceparoside C indicated it had the
same furostanol aglycone, 3, 26-dihydroxyfurosta-5, 20(22)-diene. The only difference among huangjiangsu A, ceparoside C and 1 was that one sugar unit of huangjiangsu A and ceparoside C was galactose, while it was glucose in compound 1. The R-orientation of C-25 was evident by the chemical shifts of ´ H-26a (3.89) and ´ H-26b (3.64) (¸´ H ˘ 0.48 ppm) [22]. HMBC correlations (Fig. 2) from ´ H-21 (1.66) to C-20, C-22, and C-17, from ´ H-19 (0.75) to C-5, C-10, and C-1, and from the olefinic proton ´ H-6 (5.31) to C-4, C-7, and C-8 confirmed the presence of two double bonds at C-5(6) and
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ZHENG Lu, et al. / Chin J Nat Med, 2014, 12(2): 142147
C-20(22). ROESY correlations (Fig. 2) of CH 3 -18/CH 3 -19, CH 3 -18/H-8, and CH 3 -19/H-8 suggested that CH 3 -18, CH 3 -19, and H-8 were on the same face of the molecule. ROESY correlations between H-14/H-16, H-14/H-17, and H-16/H-17 indicated they were also on the same face of 1.
relation from terminal-glc-H-? }´ H 5.29) to intermediate-glc-C-3 (´ C 88.7). Thus, 2 was elucidated as 26-O--D-glucopyranosyl- 3,26-dihydroxy-(25R)-furosta5,20(22)-dien-3-O-[-D-glucopyranosyl-(13)--D-glucop yranosyl-(1 4)]-[-L-rhamnopyranosyl-(1 2)]--D-glu copyranoside, named zingiberenoside B.
Experimental
Fig. 2
Key HMBC and ROESY correlations of 1
Acid hydrolysis of 1 with 10% HCl-dioxane (1 : 1, V/V) afforded the sugar components D-glucose and L-rhamnose identified by TLC and GC analysis. The monosaccharides were identified and their linkages were also determined by the analysis of 1H NMR, 13C NMR, COSY, HSQC, HMBC, and TOCSY spectra. The 13C NMR data for the sugar moieties indicated that all the monosaccharides were in pyranose forms. The ¤-anomeric configuration for rhamnose was determined by the chemical shift of C-5 (´ C 69.8) [23]. The anomeric configurations for the glucose units were determined from their large 3J 1, 2 coupling constants (J = 7.0, 8.0, 8.0 Hz respectively). HMBC correlations from inner-glc-H-1 (´ H 4.97) to C-3 (´ C 78.4), and from glc-H-1 (´ H 4.85) to C-26 (´ C 75.3) suggested that 1 had a 3-O-,26-O-disaccharide structure. The linkages of the other sugar moieties were defined by HMBC correlations from terminal-glc-H-1 (´ H 5.14) to inner-glc-C-4 (´ C 82.4), and from terminal-rha-H-1 (´H 6.27) to inner-glc-C-2 (´C 7 7 . 6 ) . Thus, 1 was unambiguously elucidated as 26-O-ª-Dglucopyranosyl-3,26-dihydroxy-(25R)-furosta-5,20(22)dien-3-O--D-glucopyranosyl-(14)-[-L-rhamnopyranosyl- (12)]--D-glucopyranoside, and named zingiberen oside A. Compound 2 was obtained as a white amorphous powder and was deduced to possess a furostanol group based on TLC using Ehrlich’s reagent. The IR spectrum showed absorption bands of hydroxyl (3 421 cm1), methyl (2 930 cm1), and olefinic (1 646 cm1) groups. The molecular formula was assigned to be C 57 H 92 O 27 on the basis of a positive H R E S I M S [M + Na]+ ion peak at m/z 1 231.564 11 (Calcd. 1 231.572 37). The 1H and 13C NMR data (Tables 1 and 2) indicated that compound 2 possessed the same aglycone as 1, except that 2 had one more sugar unit, supported by the HMBC cor-
General HRESI-MS were measured on a Bruker APEX IV FT-MS (7.0T) spectrometer (America) in positive ion mode. Optical rotations were measured on an Autopol-IV polarimeter (RudoPH Research Analytical, America). IR spectra were obtained using a NEXUS-470 FTIR (Nicolet, Waltham, America) spectrometer. 1D- and 2D-NMR spectra were recorded on Vnmrs-500 spectrometer (Palo Alto, America). Semi- preparative HPLC was performed on a Aglient model 1260 (Agilent ODS Rp-C 18 column, 250 mm × 10 mm i.d., 5 < # = ^
# _ (ELSD, Chicago, America). GC analysis was carried out on an Agilent 6890N gas chromatograph, with an HP-5 capillary column (28 m × 0.32 mm) and an FID detector operated at 260 °C (column temp. 180 ¹ ?' #1 N 2 as carrier gas. Macroporous resin (HPD100) were purchased from Hebei Bao-En Biotech Ltd. (Cangzhou, China) Column chromatography was performed using silica gel (200–300 mesh) (Qingdao Marine Chemical Ltd., Qingdao, China). Sephadex LH-20 (GE Healthcare, Fairfield, America) and ODS (50 < , YMC, JP). All the solvents were of analytical grade and were purchased from Beijing Chemical Company Ltd., Beijing, China. Plant material The rhizomes of Dioscorea zingiberensis C.H.Wright were purchased from Hebei Anguo Hongfa Chinese Herbal Medicine Co. Ltd., and were originally collected from Shanxi province, China (date of collection: 20100719). The plant material was identified by Prof. TU Peng-Fei. A voucher specimen (No. 20100905) was deposited in the Herbarium of the Modern Research Center for Traditional Chinese Medicine (TCM), Peking University, Beijing. Extraction and isolation Dried crude materials (10 kg) were ground and extracted twice with 70% EtOH at 80 °C for 1 h. After removing the ethanol, the residue was suspended in water (30 L) and subjected to column chromatography (CC) on macroporous resin with an EtOH-H 2 O gradient (0 : 100, 50 : 50, 5 : 5) to yield three fractions }^'?. Fr. 2 (75 g) was chromatographed on silica gel (1.0 kg, 100 cm × 10 cm) with CH 2 Cl 2 ¥ H 2 O (70 : 30 : 10, lower layer) elution to yield seven fractions (Frs. AG). Fr. G was applied to CC on Sephadex LH-20 with MeOH-H 2 O (70 : 30), then to ODS with ACN-H 2 O gradient (10 : 90, 20 : 80, 30 : 70, 40 : 60) elution, to yield compounds 7 (750 mg), 8 (800 mg), 9 (45 mg), 10 (55 mg), and Fr. Ga. Fr. Ga was separated by
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ZHENG Lu, et al. / Chin J Nat Med, 2014, 12(2): 142147
semi-preparative HPLC with ACN-H 2 O (31 : 69) to afford compounds 1 (60 mg), 2 (40 mg), 3 (10 mg), 4 (25 mg), 5 (16 mg), and 6 (14 mg). Acid hydrolysis and sugar analysis Compounds 1 (3 mg) and 2 (3 mg) were heated separately in 10% HCl-dioxane (3 mL, 1 : 1) at 90 °C for 4 h in a sealed ampoule. After the dioxane was removed, the aqueous solution was extracted with EtOAc (3 mL × 3) to yield the aglycone and sugar, respectively. The sugar components in the aqueous layer after acid hydrolysis were identified by comparison with standard sugars on silica gel TLC, using n-BuOH-HOAc-H 2 O (40 : 10 : 50, upper layer) as the solvent system, and phenylamine-diphenylamine as the chromogenic reagent. For the sugars of 1 and 2, the R f values of glucose and rhamnose on TLC were 0.26 and 0.48, respectively. Furthermore, the results were confirmed by GC analysis of the methyl sugar peracetates. The aqueous layer was evaporated and dissolved in anhydrous pyridine (100
Chinese Journal of Natural Medicines
Two new steroidal saponins from the rhizomes of Dioscorea zingiberensis ZHENG Lu1#, ZHOU Yuan1#, ZHANG Jia-Yu2, SONG Min1, YUAN Ye1, XIAO Yan-Jiao1, XIANG Ting1* 1
Research Institute of Traditional Chinese Medicine, Yangtze River Pharmaceutical Group Co., Ltd., Taizhou, 225321, Chinaχ School of Chinese Pharmacy, Beijing University of Chinese Medicine, Beijing 100029, China
2
Available online 20 Feb. 2014
[ABSTRACT] AIM: To investigate the chemical constituents of Dioscorea zingiberensis C. H. Wright. METHODS: The compounds were isolated by various chromatographic techniques, and the structures of the new steroidal saponins were elucidated by extensive 1D- and 2D-NMR, MS, and IR spectral analysis. RESULTS: The 70% EtOH extract of the rhizomes of Dioscorea zingiberensis afforded two new steroidal saponins, zingiberenosides A (1) and B (2), along with eight known analogues, 3, 26-dihydroxy-25(R)-furosta-Ƹ5, 20(22)-diene-3-O--L- rhamnopyra-
nosyl-(12)-O--D-glucopyranoside (3), methyl parvifloside (4), deltoside (5), methyl deltoside (6), zingiberensis new saponin (7), deltonin (8), progenin III (9) and diosgenin-diglucoside (10). CONCLUSION: Two new steroidal saponins were isolated from Dioscorea zingiberensis and their structures determined. [KEY WORDS] Dioscorea zingiberensis; Dioscoreaceae; Steroidal saponins; Zingiberenoside
[CLC Number] R284.1
[Document code] A
[Article ID] 2095-6975(2014)02-0142-06
Introduction Dioscorea zingiberensis C. H. Wright, common name ‘Huang Jiang’, is a perennial herb, and is distributed widely in Henan, Hubei, Sichuan, and the south of Shanxi provinces [1-2]. It is used to treat cough with lung heat, pyretic stranguria, anthracia, swelling, ulcers, and sprains. As an important species of the Dioscoreaceae, D. zingiberensis has attracted much attention for the high content of diosgenin and steroid saponins [3], as well as excellent pharmacologic action, such as reducing the content of cholesterol
[Received on] 10-Nov.-2012 [Research funding] This project was supported by the Science and Technology Achievement Transformation Project of Jiangsu Province (No. BA2010144); and the scientific and technological major special project for “Significant Creation of New Drugs” (No. 2012ZX09101231). [ Corresponding author] XIANG Ting: Dr., Senior Engineer, Tel: 86-10-80728999-6255, Fax: 86-10-80728999-6255. E-mail: tingxiang [email protected] # Co-first authors These authors have no conflict of interest to declare. Copyright © 2014, China Pharmaceutical University. Published by Elsevier B.V. All rights reserved
in blood [4], and decreasing stenocardia and regulating metabolism [5]. Furthermore, its total steroidal saponins could treat atherosclerosis, high blood fat, wheeze, inflammation. and tumors [6-7]. Previous phytochemical investigations of its rhizomes led to the isolation of several steroidal saponins [5, 8-15]. In this paper, the isolation and structural elucidation of two new steroidal saponins and eight known derivatives from the 70% EtOH extract of the dried rhizome of D. zingiberensis are reported.
Results and Discussion A 70% EtOH extract of the commercially available rhizomes of D. zingiberensis was fractionated by macroporous resin and silica gel, and then separated by ODS column chromatography and semi-preparative HPLC to afford two new steroidal saponins, zingiberenoside A (1) and zingiberenoside B (2), together with eight known analogues (Fig. 1). The structures of the new compounds were elucidated by extensive 1D- and 2D-NMR, MS, and IR spectral analyses. Compound 1 was obtained as a white amorphous powder, and was deduced to possess a furostanol group based on TLC using Ehrlich’s reagent [20]. The IR spectrum showed absorp-
– 142 –
ZHENG Lu, et al. / Chin J Nat Med, 2014, 12(2): 142147
Fig. 1 Structures of compounds 1 10 [M + Na]+ ion peak at m/z 1069.520 32 (Calcd. 1069.519 54). The 1H and 13C NMR (pyridine-d 5 , 500/125 MHz) data (Tables 1 and 2), assigned by 1 H- 1 H COSY, HSQC, and
tion bands of hydroxyl (3 421 cm1), methyl (2 930 cm1), and olefinic (1 644 cm1) groups. The molecular formula was assigned to be C 51 H 82 O 2 on the basis of positive HRESI-MS Table 1 No. 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 OCH 3
13
C NMR data for the aglycone of compounds 1 10 (in pyridine-d 5 , 125 MHz) 1 37.9 30.5 78.4 39.3 141.2 122.2 32.8 31.8 50.7 37.5 21.6 40.0 43.8 55.3 34.9 84.8 64.9 14.5 19.8 103.9 12.2 152.8 33.9 24.1 31.8 75.3 17.7
2 37.9 30.5 78.4 39.3 141.2 122.2 32.8 31.8 50.7 37.5 21.6 40.0 43.8 55.3 34.8 84.8 64.9 14.5 19.8 103.9 12.2 152.7 33.9 24.1 31.8 75.3 17.7
3 37.9 30.6 78.7 39.4 141.2 122.2 32.8 31.8 50.7 37.5 21.6 40.0 43.8 55.3 34.9 84.8 64.9 14.5 19.8 103.9 12.1 152.8 33.8 24.1 31.8 75.3 17.7
4 37.8 30.5 78.5 39.3 141.2 122.2 32.5 32.0 50.7 37.4 21.4 40.1 41.2 56.9 32.7 81.6 64.6 16.6 19.7 40.9 16.6 113.0 37.4 28.5 34.6 75.6 17.5 47.6
5 37.9 30.5 78.6 39.3 141.2 122.2 32.0 31.2 50.7 37.6 21.4 40.1 41.1 56.9 32.6 81.6 64.2 16.6 19.7 40.8 16.8 111.0 37.5 28.7 34.6 75.6 17.8
– 143 –
6 37.8 30.5 78.6 39.3 141.2 122.1 32.0 31.2 50.7 37.5 21.4 40.3 41.1 56.9 32.8 81.4 64.5 16.6 19.7 40.0 16.6 113.0 37.6 28.5 34.6 75.6 17.5 47.6
7 37.8 30.5 78.7 39.3 141.2 122.1 32.7 32.0 50.6 37.5 21.5 40.2 40.8 57.0 32.6 81.5 63.3 16.7 19.7 42.3 15.4 109.6 32.2 29.6 30.9 67.2 17.7
8 37.8 30.5 78.5 39.3 141.1 122.1 32.6 32.0 50.6 37.5 21.4 40.2 40.8 57.0 32.7 81.5 63.2 16.7 19.7 42.3 15.4 109.6 32.2 29.6 31.0 67.2 17.7
9 37.8 30.5 78.6 39.3 141.2 122.1 32.7 32.0 50.6 37.4 21.5 40.2 40.8 57.0 32.6 81.5 63.2 16.7 19.7 42.4 15.4 109.6 32.2 29.6 30.9 67.2 17.7
10 37.8 30.9 78.7 39.6 141.2 122.1 32.7 32.0 50.6 37.5 21.5 40.2 40.8 57.0 32.6 81.5 63.2 16.7 19.8 42.3 15.4 109.6 32.2 29.6 30.9 67.2 17.7
ZHENG Lu, et al. / Chin J Nat Med, 2014, 12(2): 142147
Table 2
13
C NMR data for the sugar moiety of compounds 1–10 (in pyridine-d 5 , 125 MHz)
No. inner 1
1
2
3
4
5
6
7
8
9
10
Glc
Glc
Glc
Glc
Glc
Glc
Glc
Glc
Glc
Glc
100.4
100.3
100.3
100.3
100.4
100.4
100.3
100.4
100.7
102.6
2
77.6
77.6
80.0
77.7
77.7
77.7
77.6
78.8
78.2
78.2
3
76.6
76.6
78.3
76.6
76.6
76.6
76.6
76.6
80.0
77.2
4
82.4
81.9
72.2
81.8
82.4
82.4
81.9
82.4
72.0
81.6
5
78.1
78.0
78.2
78.0
78.1
78.1
78.0
78.1
78.8
76.8
6
62.3
61.8
63.0
61.9
62.3
62.3
61.9
62.3
63.0
62.8
intermediate
Glc
Glc
Glc
1
104.9
104.9
104.9
2
74.1
74.1
74.1
3
88.7
88.6
88.7
4
69.8
69.8
69.8
5
78.4
78.4
78.4
6
62.1
62.1
62.1
terminal 1
Glc
Glc
Glc
Glc
Glc
Glc
Glc
Glc
105.3
106.3
106.3
105.3
105.3
106.3
105.6
105.3
2
75.3
75.9
75.9
75.3
75.3
75.9
75.3
75.1
3
79.0
79.1
79.1
78.5
78.5
78.5
78.6
78.3
4
71.6
72.0
72.0
71.6
71.6
72.0
71.6
71.9
5
78.9
79.0
79.0
78.8
78.8
79.1
77.7
78.6
6
62.4
62.9
62.9
62.4
62.4
62.9
62.5
terminal
Rha
Rha
Rha
Rha
Rha
Rha
Rha
Rha
Rha
62.5
1
102.2
102.1
102.4
102.2
102.2
102.2
102.2
102.2
102.4
2
72.8
72.8
73.0
72.8
72.8
72.8
72.8
72.8
72.9
3
73.1
73.1
73.2
73.1
73.1
73.1
73.1
73.1
73.2
4
74.5
74.5
74.5
74.5
74.5
74.5
74.5
74.5
74.5
5
69.8
69.7
69.9
69.7
69.8
69.8
69.7
69.8
69.8
6
19.0
19.0
19.0
19.0
19.0
19.0
19.0
19.0
19.0
26-O1
Glc
Glc
Glc
Glc
Glc
Glc
105.6
105.3
105.3
105.4
105.5
105.6
2
75.6
75.6
75.6
75.6
75.6
75.6
3
78.9
78.9
79.0
78.9
78.9
78.9
4
72.1
72.1
72.1
72.1
72.1
72.1
5
78.7
78.7
78.9
78.7
78.8
78.8
6
63.2
63.2
63.0
63.3
63.2
63.3
HMBC, revealed the presence of three singlets for tertiary
=^ ´ H 0.75 (H 3 -18, s), 1.07 (H 3 -19, s) and 1.66 (H 3 -21, s), two dou` ^ ´ H 1.03 (H 3 -27, d, J = 7.0 Hz) and 1.78 (H 3 -Rha-6, d, J = 6.0 Hz), and an olefinic proton at ´ H 5.49 (H-6, d, J = 4.0 Hz). Two pairs of olefinic carbon signals at ´ C 141.2, 122.2 and 103.9, 152.8 in the 13C NMR spectrum were assigned to C-5(6) and C-20(22) by comparison with the known compounds huangjiangsu A [8] and ceparoside C [21]. Comparison of the 1H and 13C NMR spectra data of 1 with those of huangjiangsu A and ceparoside C indicated it had the
same furostanol aglycone, 3, 26-dihydroxyfurosta-5, 20(22)-diene. The only difference among huangjiangsu A, ceparoside C and 1 was that one sugar unit of huangjiangsu A and ceparoside C was galactose, while it was glucose in compound 1. The R-orientation of C-25 was evident by the chemical shifts of ´ H-26a (3.89) and ´ H-26b (3.64) (¸´ H ˘ 0.48 ppm) [22]. HMBC correlations (Fig. 2) from ´ H-21 (1.66) to C-20, C-22, and C-17, from ´ H-19 (0.75) to C-5, C-10, and C-1, and from the olefinic proton ´ H-6 (5.31) to C-4, C-7, and C-8 confirmed the presence of two double bonds at C-5(6) and
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ZHENG Lu, et al. / Chin J Nat Med, 2014, 12(2): 142147
C-20(22). ROESY correlations (Fig. 2) of CH 3 -18/CH 3 -19, CH 3 -18/H-8, and CH 3 -19/H-8 suggested that CH 3 -18, CH 3 -19, and H-8 were on the same face of the molecule. ROESY correlations between H-14/H-16, H-14/H-17, and H-16/H-17 indicated they were also on the same face of 1.
relation from terminal-glc-H-? }´ H 5.29) to intermediate-glc-C-3 (´ C 88.7). Thus, 2 was elucidated as 26-O--D-glucopyranosyl- 3,26-dihydroxy-(25R)-furosta5,20(22)-dien-3-O-[-D-glucopyranosyl-(13)--D-glucop yranosyl-(1 4)]-[-L-rhamnopyranosyl-(1 2)]--D-glu copyranoside, named zingiberenoside B.
Experimental
Fig. 2
Key HMBC and ROESY correlations of 1
Acid hydrolysis of 1 with 10% HCl-dioxane (1 : 1, V/V) afforded the sugar components D-glucose and L-rhamnose identified by TLC and GC analysis. The monosaccharides were identified and their linkages were also determined by the analysis of 1H NMR, 13C NMR, COSY, HSQC, HMBC, and TOCSY spectra. The 13C NMR data for the sugar moieties indicated that all the monosaccharides were in pyranose forms. The ¤-anomeric configuration for rhamnose was determined by the chemical shift of C-5 (´ C 69.8) [23]. The anomeric configurations for the glucose units were determined from their large 3J 1, 2 coupling constants (J = 7.0, 8.0, 8.0 Hz respectively). HMBC correlations from inner-glc-H-1 (´ H 4.97) to C-3 (´ C 78.4), and from glc-H-1 (´ H 4.85) to C-26 (´ C 75.3) suggested that 1 had a 3-O-,26-O-disaccharide structure. The linkages of the other sugar moieties were defined by HMBC correlations from terminal-glc-H-1 (´ H 5.14) to inner-glc-C-4 (´ C 82.4), and from terminal-rha-H-1 (´H 6.27) to inner-glc-C-2 (´C 7 7 . 6 ) . Thus, 1 was unambiguously elucidated as 26-O-ª-Dglucopyranosyl-3,26-dihydroxy-(25R)-furosta-5,20(22)dien-3-O--D-glucopyranosyl-(14)-[-L-rhamnopyranosyl- (12)]--D-glucopyranoside, and named zingiberen oside A. Compound 2 was obtained as a white amorphous powder and was deduced to possess a furostanol group based on TLC using Ehrlich’s reagent. The IR spectrum showed absorption bands of hydroxyl (3 421 cm1), methyl (2 930 cm1), and olefinic (1 646 cm1) groups. The molecular formula was assigned to be C 57 H 92 O 27 on the basis of a positive H R E S I M S [M + Na]+ ion peak at m/z 1 231.564 11 (Calcd. 1 231.572 37). The 1H and 13C NMR data (Tables 1 and 2) indicated that compound 2 possessed the same aglycone as 1, except that 2 had one more sugar unit, supported by the HMBC cor-
General HRESI-MS were measured on a Bruker APEX IV FT-MS (7.0T) spectrometer (America) in positive ion mode. Optical rotations were measured on an Autopol-IV polarimeter (RudoPH Research Analytical, America). IR spectra were obtained using a NEXUS-470 FTIR (Nicolet, Waltham, America) spectrometer. 1D- and 2D-NMR spectra were recorded on Vnmrs-500 spectrometer (Palo Alto, America). Semi- preparative HPLC was performed on a Aglient model 1260 (Agilent ODS Rp-C 18 column, 250 mm × 10 mm i.d., 5 < # = ^
# _ (ELSD, Chicago, America). GC analysis was carried out on an Agilent 6890N gas chromatograph, with an HP-5 capillary column (28 m × 0.32 mm) and an FID detector operated at 260 °C (column temp. 180 ¹ ?' #1 N 2 as carrier gas. Macroporous resin (HPD100) were purchased from Hebei Bao-En Biotech Ltd. (Cangzhou, China) Column chromatography was performed using silica gel (200–300 mesh) (Qingdao Marine Chemical Ltd., Qingdao, China). Sephadex LH-20 (GE Healthcare, Fairfield, America) and ODS (50 < , YMC, JP). All the solvents were of analytical grade and were purchased from Beijing Chemical Company Ltd., Beijing, China. Plant material The rhizomes of Dioscorea zingiberensis C.H.Wright were purchased from Hebei Anguo Hongfa Chinese Herbal Medicine Co. Ltd., and were originally collected from Shanxi province, China (date of collection: 20100719). The plant material was identified by Prof. TU Peng-Fei. A voucher specimen (No. 20100905) was deposited in the Herbarium of the Modern Research Center for Traditional Chinese Medicine (TCM), Peking University, Beijing. Extraction and isolation Dried crude materials (10 kg) were ground and extracted twice with 70% EtOH at 80 °C for 1 h. After removing the ethanol, the residue was suspended in water (30 L) and subjected to column chromatography (CC) on macroporous resin with an EtOH-H 2 O gradient (0 : 100, 50 : 50, 5 : 5) to yield three fractions }^'?. Fr. 2 (75 g) was chromatographed on silica gel (1.0 kg, 100 cm × 10 cm) with CH 2 Cl 2 ¥ H 2 O (70 : 30 : 10, lower layer) elution to yield seven fractions (Frs. AG). Fr. G was applied to CC on Sephadex LH-20 with MeOH-H 2 O (70 : 30), then to ODS with ACN-H 2 O gradient (10 : 90, 20 : 80, 30 : 70, 40 : 60) elution, to yield compounds 7 (750 mg), 8 (800 mg), 9 (45 mg), 10 (55 mg), and Fr. Ga. Fr. Ga was separated by
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ZHENG Lu, et al. / Chin J Nat Med, 2014, 12(2): 142147
semi-preparative HPLC with ACN-H 2 O (31 : 69) to afford compounds 1 (60 mg), 2 (40 mg), 3 (10 mg), 4 (25 mg), 5 (16 mg), and 6 (14 mg). Acid hydrolysis and sugar analysis Compounds 1 (3 mg) and 2 (3 mg) were heated separately in 10% HCl-dioxane (3 mL, 1 : 1) at 90 °C for 4 h in a sealed ampoule. After the dioxane was removed, the aqueous solution was extracted with EtOAc (3 mL × 3) to yield the aglycone and sugar, respectively. The sugar components in the aqueous layer after acid hydrolysis were identified by comparison with standard sugars on silica gel TLC, using n-BuOH-HOAc-H 2 O (40 : 10 : 50, upper layer) as the solvent system, and phenylamine-diphenylamine as the chromogenic reagent. For the sugars of 1 and 2, the R f values of glucose and rhamnose on TLC were 0.26 and 0.48, respectively. Furthermore, the results were confirmed by GC analysis of the methyl sugar peracetates. The aqueous layer was evaporated and dissolved in anhydrous pyridine (100
