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Spirostanol steroids from the roots of Allium tuberosum
Steroids 100 (2015) 1–4
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Spirostanol steroids from the roots of Allium tuberosum Yun-Shan Fang a,b,1, Le Cai a,1, Ying Li a, Jia-Peng Wang a, Huai Xiao a, Zhong-Tao Ding a,⇑ a b
Key Laboratory of Medicinal Chemistry for Natural Resource, Ministry of Education, School of Chemical Science and Technology, Yunnan University, Kunming 650091, China School of Chemical Science and Technology, Kunming University, Kunming 650214, China
a r t i c l e
i n f o
Article history: Received 19 January 2015 Received in revised form 12 March 2015 Accepted 22 March 2015 Available online 30 March 2015 Keywords: Allium tuberosum Spirostanol saponins Tuberosines A–C Antibacterial activities
a b s t r a c t Three new spirostanol saponins named tuberosines A–C (1–3), together with three known ones tuberoside O (4), 25(S)-Schidigera-saponin D5 (5), and shatavarin IV (6) were isolated from the roots of Allium tuberosum. Their structures were established on the basis of extensive spectroscopic analyses. Whereas compounds 5 and 6 exhibited potent antibacterial activities against Bacillus subtilis (32 lg/mL) and Escherichia coli (16 lg/mL), the new saponin 2 showed only moderate antibacterial activities against these pathogens. The relationship between the antibacterial activities and the structures of these saponins are described. Ó 2015 Elsevier Inc. All rights reserved.
1. Introduction Plants of the genus Allium (Liliaceae), well known for their culinary uses, contain abundant sulfur compounds [1] and steroidal saponins [2–4]. Chinese chive (Allium tuberosum) grows naturally in central and northern parts of Asia and is cultured in China, Japan, Korea, India, Nepal, Thailand, and Philippines. It is a perennial plant whose stems, leaves and inflorescence are edible and have been used as herbal medicine for treatment of abdominal pain, diarrhea, hematemesis, snakebite, and asthma [5]. Its seeds have been reputedly used as a traditional Chinese medicine for treating both impotence and nocturnal emissions [6]. There is a popular belief in Chinese folk that A. tuberosum roots are effective in resisting gastric ulcer and treating dyspepsia [7]. Previous research revealed that A. tuberosum seeds contain amounts of steroidal saponins [8–11]. The steroidal saponins are naturally occurring glycosides that possess properties such as froth formation, hemolytic activity, toxicity to fish and complex formation with cholesterin [12]. During recent years, steroidal glycosides have attracted a growing interest owing to the wide range of their biological actions on living organisms, including antidiabetic [13], antitumor [14], antitussive actions, and as platelet aggregation inhibitors [15]. To find more bioactive steroidal saponins, we carried out a detailed phytochemical investigation on the roots of A. tuberosum, whose secondary metabolites has never been explored previously. Our experiments leaded to the discovery of ⇑ Corresponding author. Fax: +86 0871 65033910. 1
E-mail address: [email protected] (Z.-T. Ding). These authors contributed equally to this paper.
http://dx.doi.org/10.1016/j.steroids.2015.03.015 0039-128X/Ó 2015 Elsevier Inc. All rights reserved.
three new steroidal saponins, tuberosines A–C (1–3) (Fig. 1), together with three known ones tuberoside O (4) [15], 25(S)Schidigera-saponin D5 (5) [16], and shatavarin IV (6) [17]. The antibacterial activities of the isolated compounds have also been investigated. This paper deals with the isolation, structural elucidation, and antibacterial activities of the steroidal saponins from A. tuberosum roots. 2. Results and discussion Compound 1 was obtained as an amorphous powder, with the molecular formula C33H54O9, which was deduced from the HRESIMS data showing a [M-H] ion at m/z 593.3693. The IR spectrum of 1 showed the characteristic absorption of hydroxy groups at 3428 cm1. Of the 33 carbons, 27 were assigned to the aglycon part and 6 to the oligosaccharide moiety. The 1H NMR spectrum of the aglycon part of 1 showed two angular methyl signals at dH 0.75 and 0.97 (each 3H, s), two secondary methyl signal at dH 0.95 (3H, d, J = 6.6 Hz) and 1.05 (3H, d, J = 7.2 Hz) (Table 1). The 13 C NMR spectrum of 1 showed two signals at lower field than 100 ppm (Table 1); the signal at dC 103.8 was due to the anomeric carbon, and the signal at dC 111.1 was assignable to the C-22 carbon of the spirostan skeleton [18]. The above data were consistent with a (25S)-spirostanol monosaccharide. The chemical shift and coupling constant of H-27 (dH 1.05 d, J = 7.2 Hz) in 1 further confirmed the b-orientation of methyl-27 [19]. Comparison of the signals from the aglycon moiety in the 13C NMR spectra with those from markogenin [(25S)-5b-spirostane-2b,3b-diol] [20] showed that the aglycone moiety of 1 was markogenin and the sugar was attached to its C-3 position, which was further supported by the
2
Y.-S. Fang et al. / Steroids 100 (2015) 1–4 27
21
OH O
5'
HO
3'
19 11
R1 2 1
6'
R5 O 4'
12
R3
1'
3
O
2'
5
10
4 R 2
18 20 22 13
17
9 8 14 15 7 6
16
O 26
25
23 24
O
OR4 1 R1 =OH, R 2=R 3=R 4=R 5=H 2 R1 =R3 =OH, R 2=R 4=R 5=H 3 R1 =OH, R 2=R 3=R 4=H, R 5=α-L-Rha 4 R1 =R2 =OH, R 3=R 4=R 5=H 5 R1 =R2 =R3 =R5 =H, R4 =β-D-Glc 6 R1 =R2 =R3 =H, R4 =β-D-Glc, R 5=α-L-Rha
Fig. 1. Structures of compounds 1–6.
HMBC correlations of anomeric proton H-10 (dH 4.27 d, J = 7.8 Hz) with C-3 (dC 80.3) and H-3 (dH 3.99 m) with anomeric carbon C10 (dC 103.8) (Fig. 2). The sugar was assigned as glucopyranosyl moiety on the basis of a set of characteristic six carbon signals at dC 103.8, 78.9, 78.8, 75.6, 72.5, 63.5 in the 13C NMR spectrum. Table 1 NMR spectral data of 1 (1H: 600 MHz; 100 MHz) in CD3OD. Position
13
C: 150 MHz) and 2 (1H: 400 MHz;
1
13
2
dC
dH (J in Hz)
dC
dH (J in Hz)
1
40.8 68.8 80.3 32.4
5 6
43.3 27.6
7
27.8
8 9 10 11
37.6 37.9 38.6 23.0
m m m m m m m m m m m m m
33.3
2 3 4
12
42.1
13 14 15
42.6 58.3 33.5
16 17 18 19
83.3 64.5 17.8 24.9
1.65 1.41 3.58 3.99 1.77 1.61 1.30 1.98 1.39 1.31 1.24 1.57 1.77 – 1.49 1.31 1.72 1.17 – 1.17 1.92 1.20 4.35 1.71 0.75 0.97
20 21 22 23
44.3 15.6 111.1 28.5
m d (6.6)
24
27.8
25 26
29.4 66.9
27 10 20 30 40 50 60
17.2 103.8 75.6 78.8 72.5 78.9 63.5
1.81 0.95 – 1.42 1.04 1.87 1.39 1.63 3.88 3.23 1.05 4.27 3.20 3.23 3.23 3.31 3.81 3.60
1.80 m 1.53 m 3.63 m 4.02 m 1.94 m 1.21 m 1.38 m 1.81 m 1.24 m 1.43 m 1.05 m 1.59 m 2.14 m – 1.55 m 1.34 m 1.72 m 1.14 m – 1.14 m 1.86 m 1.66 m 4.36 m 1.71 m 0.74 s 3.87 d (11.6) 3.40 d (11.6) 1.82 m 0.95 d (7.2) – Ha1.90 m; Hb1.34 m
m m m m m m m m m s s
m m m m m dd (10.8, 2.4) (9.6, 7.8) d (7.2) d (7.8) m m m m m m
68.0 78.5 32.4 41.9 26.4 27.2 36.5 29.2 41.9 22.0 41.4 41.6 57.8 30.9 82.4 63.4 16.9 65.2 43.4 14.7 111.2 26.9 26.6 28.3 66.1 16.4 102.4 74.6 77.6 71.6 77.8 62.6
1.93 1.34 1.67 3.87 3.24 1.04 4.28 3.24 3.35 3.26 3.26 3.81 3.63
m m m dd (10.2, 2.4) dd (10.2, 7.2) d (6.8) d (8.0) m m m m m m
C:
The anomeric proton coupling constant of 7.8 Hz indicated the b-configuration of the glucopyranosyl [21], whose absolute configuration was confirmed to be D on the basis of the acid hydrolysis of 1 with 1 M HCl and the GC analysis of their trimethylsilyl L-cysteine derivatives. Therefore, the structure of 1 was determined as (25S)-5b-spirostan-2b,3b-diol 3-O-b-D-glucopyranoside and named tuberosine A. Compound 2, a white amorphous powder, exhibited the pseudomolecular ion peak at m/z 609.3641 [M-H] in the HRESIMS data, corresponding to the molecular formula C33H54O10. A detailed comparison of the 1H, 13C NMR chemical shift (Table 1) of 1 and 2, revealed that 2 was also a spirostanol steroidal saponin with a b-glucopyranosyl group at C-3. The main difference between them was that compound 2 possessed only one angular methyl group which was one less than 1. Meanwhile, compound 2 exhibited the signals of an additional oxygenated CH2 group (dH 3.40 d, J = 11.6 Hz; 3.87 d, J = 11.6 Hz; dC 65.2 t) in its NMR spectra. Combining with its molecular formula, there might be a hydroxy group substituted at C-18 or C-19 in compound 2. The absence of the angular methyl group at about 24 ppm indicated that the hydroxy group should be located at C-19, which was further supported by the HMBC correlations from H-19 to C-1 (dC 33.3 t), C-5 (dC 41.9 d), C-9 (dC 29.2 d), and C-10 (dC 41.9 s) (Fig. 2). On acid hydrolysis, 2 afforded D-glucose, which was identified by GC analysis of its trimethylsilyl L-cysteine derivative. Thus, compound 2 was established as (25S)-5b-spirostan-2b,3b,19-triol 3-O-b-Dglucopyranoside and named tuberosine B. Compound 3, a white amorphous powder, gives a molecular formula of C39H64O13 by HRESIMS (negative ion mode) at m/z 739.4271 [M-H]. Its spectral features and physicochemical properties suggested 3 to be a spirostanol saponin. Compound 3 containing 39 carbons, including 27 of the aglycon part and 12 of the oligosaccharide moiety. Comparison of the NMR data of 3 with those of 1 (Table 2) revealed that they shared the same skeleton and same glycosidic position at C-3. The molecular weight of 3 was 146 mass units greater than that of 1 indicating that 3 had an additional deoxyhexose group, which was identified as a rhamnose by its NMR data (dH 4.80 brs, 1.23 d, J = 6.0 Hz; dC 102.9 d, 73.7 d, 72.4 d, 72.2 d, 70.6 d, 17.8 d) [19]. The HMBC correlation between H-100 (dH 4.80 brs) and C-40 (dC 79.6) (Fig. 2) proved the (1 ? 4) linkage between glucose and rhamnose. The evaluation of chemical shifts and spin–spin couplings of two anomeric protons allowed the identification of one b-glucopyranose and one a-rhamnopyranose [2]. The absolute configuration of glucose was determined as D and that of rhamnose as L, as described above. Therefore, the structure of 3 was determined as (25S)-5b-spirostan-2b,3b-diol 3-O-a-L-rhamnopyranoyl-(1 ? 4)O-b-D-glucopyranoside and named tuberosine C. It’s worthy to note that all the three new spirostanol steroidal saponins (1–3) possess an unusual b-hydroxy substitution at C-2, which is normally a-orientation in this kind of compounds. The antimicrobial activities of the isolated compounds against Escherichia coli, Bacillus subtilis have also been investigated (Table 3). The results showed that 5 and 6 exhibited potent antibacterial activities against B. subtilis (32 lg/mL) and E. coli (16 lg/mL) (positive control, kanamycin: 2 lg/mL). New saponin 2 showed moderate antibacterial activities against B. subtilis (64 lg/mL) and E. coli (64 lg/mL). Compounds 1, 3, and 4 showed no or only very weak growth inhibition against above two microbes. Those saponins having a saccharide moiety at C-3 without any oxygen functionalities at C-2 (5 and 6) exhibited potent antibacterial activities, indicating 2-OH was negative to antibacterial activities. Compound 2, possessing a 2-OH, exhibited moderate antibacterial activities, probably because of the existence of 19OH. 4 showed no antibacterial activities indicating 5-OH has no positive effect on antibacterial activities.
3
Y.-S. Fang et al. / Steroids 100 (2015) 1–4
O
O OH
O
HO
O
HO
O
O HO
HO
O
HO
HO
OH
1
OH
O
HO
2
OH 21
HO 4"
HO 3"
2"
HO
6"
5"
O 1''
OH
6' 5'
O 4'
HO 2 1
O
3 1' O
HO
3'
2'
4
19
11
13 9 14
10 5
18
12
6
8 7
17
27
26 25 20 O 22 23 24
16
O
15
OH 3
Fig. 2. Key 1H–1H COSY (
Table 2 NMR spectral data of 3 in CD3OD (1H: 600 MHz;
13
) and HMBC (
) correlations of compounds 1–3.
Table 3 Antibacterial activities of compounds 1–6 (MIC: lg/mL).
C: 150 MHz).
Position
dC
dH (J in Hz)
Position
dC
dH (J in Hz)
Compounds
B. subtilis
E. coil
1
40.0
14.7
0.95 d (7.2)
67.9 79.4
m m m brs
21
2 3
1.66 1.41 3.58 3.99
22 23
111.1 27.7
4
31.5
24
26.9
256 64 256 128 32 16 2
42.5 26.7
25 26
29.4 66.1
7
26.9
27
16.4
8 9 10 11
36.8 37.0 37.8 22.1
m m m m m m m m m
m m m m m dd (10.2, 2.4) dd (10.2, 7.2) d (7.2)
256 64 256 256 16 16 2
5 6
10 20 30 40
102.6 74.9 76.7 79.6
4.29 3.24 3.43 3.48
d (7.8) m m m
12
41.3
1.77 1.61 1.31 1.98 1.39 1.31 1.24 1.58 1.77 – 1.52 1.31 1.72 1.18 –
– 1.42 1.04 1.87 1.39 1.64 3.88 3.24 1.05
1 2 3 4 5 6 Kanamycin
50
76.8
3.31 m
0
61.9
00
1 200
102.9 72.2
3.76 3.61 4.80 3.60
m m brs m
300 400 500 600
72.4 73.7 70.6 17.8
3.80 3.37 3.93 1.23
m m m d (6.0)
13
41.7
14 15
57.4 32.6
16 17 18 19 20
82.4 63.6 16.9 24.0 43.4
1.17 1.92 1.20 4.35 1.71 0.75 0.97 1.82
m m m m
6 m m m m m s s m
3. Experimental section 3.1. General experimental procedures Melting points were determined on a XRC-1 Melting Point Apparatus and uncorrected. Optical rotations were measured with a Jasco P-1020 digital polarimeter. A Nicolet Magna-IR 550 spectrometer was used for scanning IR spectroscopy with KBr pellets. NMR spectra were acquired with either a Bruker AM-400 spectrometer (400 MHz for 1H NMR and 100 MHz for 13C NMR) or a Bruker DRX-600 (600 MHz for 1H NMR and 150 MHz for 13C NMR) spectrometer. ESI-MS analyses were recorded with Agilent G3250AA and Auto Spec Premier P776 spectrometer. Silica gel (200–300 mesh or 300–400 mesh) was used for column chromatography. Sephadex LH-20 was purchased from Amersham Biosciences.
3.2. Plant material The roots of A. tuberosum were collected from Guandu District, Kunming City in Yunnan Province of China, in June 2011, and identified by professor Shu-Gang Lu from School of life Sciences, Yunnan University. The voucher specimen (2011-JCG-1) has been deposited in the school of Chemical Science and Technology of Yunnan University. 3.3. Extraction and isolation Air-dried and powdered roots (14.0 kg) of A. tuberosum were extracted three times with methanol at 60 °C. The extracts were combined, and evaporated under reduced pressure. The crude residue (1.4 kg) was suspended in H2O, and partitioned with petroleum ether, EtOAc, and 1-butanol successively. The 1-butanol fraction (200 g) was subjected to silica gel column eluted gradually with CHCl3–MeOH (200:1–1:1) to yield seven fractions (FrA1–A7). FrA4 (8.3 g) was further subjected to silica gel column chromatography using a EtOAc–MeOH gradient (20:1–2:1) to yield eight fractions (FrA4-1–FrA4-8). Fr4-4 (0.4 g) was separated by repeated column chromatography of silica gel, Sephadex LH-20 and yielded compounds 1 (7 mg), 2 (48 mg), and 4 (41 mg), respectively. Fr4-5 (0.3 g) was passed through a Sephadex LH-20 column and further purified by silica gel chromatography with CHCl3– MeOH (7:1) as the eluent to yield 3 (105 mg) and 5 (75 mg). FrA4-7 was separated by repeated silica column chromatography and Sephadex LH-20 to yield 6 (86 mg). Tuberosine A (1): white amorphous powder, m.p. 162–164 °C, 1 13 [a]20 C NMR data: see Table 1; IR D 60.0 (c 1.0, CH3OH); H and (KBr) v: 3428, 2933, 2353, 1678, 1531, 1082 cm1; HRESIMS m/z: 593.3693 [M-H] (calcd for C33H53O9 [M-H], 593.3690). Tuberosine B (2): white amorphous powder, m.p. 180–182 °C, 1 13 [a]20 C NMR data: see Table 1; IR D 56.1 (c 1.0, CH3OH); H and
4
Y.-S. Fang et al. / Steroids 100 (2015) 1–4
(KBr) v: 3427, 2931, 2353, 1633, 1454, 1044 cm1; HRESIMS m/z: 609.3641 [M-H] (calcd for C33H53O10 [M-H], 609.3639). Tuberosine C (3): white amorphous powder, m.p. 204–206 °C, 1 13 [a]20 C NMR data: see Table 1; IR D 77.9 (c 1.0, CH3OH); H and (KBr) v: 3428, 2933, 2353, 1678, 1531, 1081 cm1; HRESIMS m/z: 739.4271 [M-H] (calcd for C39H63O13 [M-H], 739.4269).
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.steroids.2015.03. 015. References
3.4. Acid hydrolysis and sugar analysis Saponin (4 mg) was dissolved in 1 M HCl (dioxane/H2O, 1:1, 2 mL) and stirred at 90 °C for 2 h. After cooling, the reaction mixture was neutralized with silver carbonate, and the solvent was evaporated. Then, the reaction mixture was extracted with CHCl3, and the aqueous layer was evaporated to give a mixture of monosaccharides. The residue was further dissolved in anhydrous pyridine (0.2 mL) followed by the addition of L-cysteine methyl ester hydrochloride (dry pyridine, 0.06 M, 0.2 mL). After heating at 60 °C for 2 h, and trimethylsilyl thiazolidine (0.2 mL) was added. Next, the mixture was heated at 60 °C for another 2 h and partitioned with n-hexane and water. The organic layer was analyzed by gas chromatography (GC) under the following conditions: capillary column, HP-5 (30 m 0.32 mm 0.25 lm, Dikma); FID detector at 300 °C; injection temperature, 280 °C; column temperature, 210 °C, maintained for 20 min; carrier gas, N2. The standard sugars were subjected to the same reaction and GC conditions. The retention times of thiazolidine derivatives D-rhamnose, L-rhamnose, D-glucose
and L-glucose were found to be 7.05 min, 7.53 min, 5.78 min and 6.39 min, respectively. 3.5. Antibacterial activities
Antibacterial activity assays were performed in sterilized 96well microplates using a microdilution method described previously [22]. The 18-h-old bacterial cultures from B. subtilis (ATCC 6633), and E. coli (ATCC 25922) were added to LB broth medium (1 L water, 10 g tryptone, 5 g yeast extract, and 10 g NaCl) to reach 1 105 CFU/mL. The samples were dissolved in dimethylsulfoxide (DMSO), and their final concentrations ranged from 0.5 to 512 lg/mL as determined by using a 2-fold serial dilution method. The wells containing strains and diluted samples were incubated at 37 °C for 18 h. Wells containing a culture suspension and DMSO were run as negative controls. Kanamycin was introduced in the experiments as a positive control. All experiments were repeated twice. The minimal inhibitory concentration (MIC) was defined as the lowest antibiotic concentration that produced complete growth inhibition of the microorganisms. Acknowledgements This project was financially supported by a Natural Science Foundation of China (No. 81460648) and a grant from the Program for Changjiang Scholars and Innovative Research Team in University (No. IRT13095).
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Contents lists available at ScienceDirect
Steroids journal homepage: www.elsevier.com/locate/steroids
Spirostanol steroids from the roots of Allium tuberosum Yun-Shan Fang a,b,1, Le Cai a,1, Ying Li a, Jia-Peng Wang a, Huai Xiao a, Zhong-Tao Ding a,⇑ a b
Key Laboratory of Medicinal Chemistry for Natural Resource, Ministry of Education, School of Chemical Science and Technology, Yunnan University, Kunming 650091, China School of Chemical Science and Technology, Kunming University, Kunming 650214, China
a r t i c l e
i n f o
Article history: Received 19 January 2015 Received in revised form 12 March 2015 Accepted 22 March 2015 Available online 30 March 2015 Keywords: Allium tuberosum Spirostanol saponins Tuberosines A–C Antibacterial activities
a b s t r a c t Three new spirostanol saponins named tuberosines A–C (1–3), together with three known ones tuberoside O (4), 25(S)-Schidigera-saponin D5 (5), and shatavarin IV (6) were isolated from the roots of Allium tuberosum. Their structures were established on the basis of extensive spectroscopic analyses. Whereas compounds 5 and 6 exhibited potent antibacterial activities against Bacillus subtilis (32 lg/mL) and Escherichia coli (16 lg/mL), the new saponin 2 showed only moderate antibacterial activities against these pathogens. The relationship between the antibacterial activities and the structures of these saponins are described. Ó 2015 Elsevier Inc. All rights reserved.
1. Introduction Plants of the genus Allium (Liliaceae), well known for their culinary uses, contain abundant sulfur compounds [1] and steroidal saponins [2–4]. Chinese chive (Allium tuberosum) grows naturally in central and northern parts of Asia and is cultured in China, Japan, Korea, India, Nepal, Thailand, and Philippines. It is a perennial plant whose stems, leaves and inflorescence are edible and have been used as herbal medicine for treatment of abdominal pain, diarrhea, hematemesis, snakebite, and asthma [5]. Its seeds have been reputedly used as a traditional Chinese medicine for treating both impotence and nocturnal emissions [6]. There is a popular belief in Chinese folk that A. tuberosum roots are effective in resisting gastric ulcer and treating dyspepsia [7]. Previous research revealed that A. tuberosum seeds contain amounts of steroidal saponins [8–11]. The steroidal saponins are naturally occurring glycosides that possess properties such as froth formation, hemolytic activity, toxicity to fish and complex formation with cholesterin [12]. During recent years, steroidal glycosides have attracted a growing interest owing to the wide range of their biological actions on living organisms, including antidiabetic [13], antitumor [14], antitussive actions, and as platelet aggregation inhibitors [15]. To find more bioactive steroidal saponins, we carried out a detailed phytochemical investigation on the roots of A. tuberosum, whose secondary metabolites has never been explored previously. Our experiments leaded to the discovery of ⇑ Corresponding author. Fax: +86 0871 65033910. 1
E-mail address: [email protected] (Z.-T. Ding). These authors contributed equally to this paper.
http://dx.doi.org/10.1016/j.steroids.2015.03.015 0039-128X/Ó 2015 Elsevier Inc. All rights reserved.
three new steroidal saponins, tuberosines A–C (1–3) (Fig. 1), together with three known ones tuberoside O (4) [15], 25(S)Schidigera-saponin D5 (5) [16], and shatavarin IV (6) [17]. The antibacterial activities of the isolated compounds have also been investigated. This paper deals with the isolation, structural elucidation, and antibacterial activities of the steroidal saponins from A. tuberosum roots. 2. Results and discussion Compound 1 was obtained as an amorphous powder, with the molecular formula C33H54O9, which was deduced from the HRESIMS data showing a [M-H] ion at m/z 593.3693. The IR spectrum of 1 showed the characteristic absorption of hydroxy groups at 3428 cm1. Of the 33 carbons, 27 were assigned to the aglycon part and 6 to the oligosaccharide moiety. The 1H NMR spectrum of the aglycon part of 1 showed two angular methyl signals at dH 0.75 and 0.97 (each 3H, s), two secondary methyl signal at dH 0.95 (3H, d, J = 6.6 Hz) and 1.05 (3H, d, J = 7.2 Hz) (Table 1). The 13 C NMR spectrum of 1 showed two signals at lower field than 100 ppm (Table 1); the signal at dC 103.8 was due to the anomeric carbon, and the signal at dC 111.1 was assignable to the C-22 carbon of the spirostan skeleton [18]. The above data were consistent with a (25S)-spirostanol monosaccharide. The chemical shift and coupling constant of H-27 (dH 1.05 d, J = 7.2 Hz) in 1 further confirmed the b-orientation of methyl-27 [19]. Comparison of the signals from the aglycon moiety in the 13C NMR spectra with those from markogenin [(25S)-5b-spirostane-2b,3b-diol] [20] showed that the aglycone moiety of 1 was markogenin and the sugar was attached to its C-3 position, which was further supported by the
2
Y.-S. Fang et al. / Steroids 100 (2015) 1–4 27
21
OH O
5'
HO
3'
19 11
R1 2 1
6'
R5 O 4'
12
R3
1'
3
O
2'
5
10
4 R 2
18 20 22 13
17
9 8 14 15 7 6
16
O 26
25
23 24
O
OR4 1 R1 =OH, R 2=R 3=R 4=R 5=H 2 R1 =R3 =OH, R 2=R 4=R 5=H 3 R1 =OH, R 2=R 3=R 4=H, R 5=α-L-Rha 4 R1 =R2 =OH, R 3=R 4=R 5=H 5 R1 =R2 =R3 =R5 =H, R4 =β-D-Glc 6 R1 =R2 =R3 =H, R4 =β-D-Glc, R 5=α-L-Rha
Fig. 1. Structures of compounds 1–6.
HMBC correlations of anomeric proton H-10 (dH 4.27 d, J = 7.8 Hz) with C-3 (dC 80.3) and H-3 (dH 3.99 m) with anomeric carbon C10 (dC 103.8) (Fig. 2). The sugar was assigned as glucopyranosyl moiety on the basis of a set of characteristic six carbon signals at dC 103.8, 78.9, 78.8, 75.6, 72.5, 63.5 in the 13C NMR spectrum. Table 1 NMR spectral data of 1 (1H: 600 MHz; 100 MHz) in CD3OD. Position
13
C: 150 MHz) and 2 (1H: 400 MHz;
1
13
2
dC
dH (J in Hz)
dC
dH (J in Hz)
1
40.8 68.8 80.3 32.4
5 6
43.3 27.6
7
27.8
8 9 10 11
37.6 37.9 38.6 23.0
m m m m m m m m m m m m m
33.3
2 3 4
12
42.1
13 14 15
42.6 58.3 33.5
16 17 18 19
83.3 64.5 17.8 24.9
1.65 1.41 3.58 3.99 1.77 1.61 1.30 1.98 1.39 1.31 1.24 1.57 1.77 – 1.49 1.31 1.72 1.17 – 1.17 1.92 1.20 4.35 1.71 0.75 0.97
20 21 22 23
44.3 15.6 111.1 28.5
m d (6.6)
24
27.8
25 26
29.4 66.9
27 10 20 30 40 50 60
17.2 103.8 75.6 78.8 72.5 78.9 63.5
1.81 0.95 – 1.42 1.04 1.87 1.39 1.63 3.88 3.23 1.05 4.27 3.20 3.23 3.23 3.31 3.81 3.60
1.80 m 1.53 m 3.63 m 4.02 m 1.94 m 1.21 m 1.38 m 1.81 m 1.24 m 1.43 m 1.05 m 1.59 m 2.14 m – 1.55 m 1.34 m 1.72 m 1.14 m – 1.14 m 1.86 m 1.66 m 4.36 m 1.71 m 0.74 s 3.87 d (11.6) 3.40 d (11.6) 1.82 m 0.95 d (7.2) – Ha1.90 m; Hb1.34 m
m m m m m m m m m s s
m m m m m dd (10.8, 2.4) (9.6, 7.8) d (7.2) d (7.8) m m m m m m
68.0 78.5 32.4 41.9 26.4 27.2 36.5 29.2 41.9 22.0 41.4 41.6 57.8 30.9 82.4 63.4 16.9 65.2 43.4 14.7 111.2 26.9 26.6 28.3 66.1 16.4 102.4 74.6 77.6 71.6 77.8 62.6
1.93 1.34 1.67 3.87 3.24 1.04 4.28 3.24 3.35 3.26 3.26 3.81 3.63
m m m dd (10.2, 2.4) dd (10.2, 7.2) d (6.8) d (8.0) m m m m m m
C:
The anomeric proton coupling constant of 7.8 Hz indicated the b-configuration of the glucopyranosyl [21], whose absolute configuration was confirmed to be D on the basis of the acid hydrolysis of 1 with 1 M HCl and the GC analysis of their trimethylsilyl L-cysteine derivatives. Therefore, the structure of 1 was determined as (25S)-5b-spirostan-2b,3b-diol 3-O-b-D-glucopyranoside and named tuberosine A. Compound 2, a white amorphous powder, exhibited the pseudomolecular ion peak at m/z 609.3641 [M-H] in the HRESIMS data, corresponding to the molecular formula C33H54O10. A detailed comparison of the 1H, 13C NMR chemical shift (Table 1) of 1 and 2, revealed that 2 was also a spirostanol steroidal saponin with a b-glucopyranosyl group at C-3. The main difference between them was that compound 2 possessed only one angular methyl group which was one less than 1. Meanwhile, compound 2 exhibited the signals of an additional oxygenated CH2 group (dH 3.40 d, J = 11.6 Hz; 3.87 d, J = 11.6 Hz; dC 65.2 t) in its NMR spectra. Combining with its molecular formula, there might be a hydroxy group substituted at C-18 or C-19 in compound 2. The absence of the angular methyl group at about 24 ppm indicated that the hydroxy group should be located at C-19, which was further supported by the HMBC correlations from H-19 to C-1 (dC 33.3 t), C-5 (dC 41.9 d), C-9 (dC 29.2 d), and C-10 (dC 41.9 s) (Fig. 2). On acid hydrolysis, 2 afforded D-glucose, which was identified by GC analysis of its trimethylsilyl L-cysteine derivative. Thus, compound 2 was established as (25S)-5b-spirostan-2b,3b,19-triol 3-O-b-Dglucopyranoside and named tuberosine B. Compound 3, a white amorphous powder, gives a molecular formula of C39H64O13 by HRESIMS (negative ion mode) at m/z 739.4271 [M-H]. Its spectral features and physicochemical properties suggested 3 to be a spirostanol saponin. Compound 3 containing 39 carbons, including 27 of the aglycon part and 12 of the oligosaccharide moiety. Comparison of the NMR data of 3 with those of 1 (Table 2) revealed that they shared the same skeleton and same glycosidic position at C-3. The molecular weight of 3 was 146 mass units greater than that of 1 indicating that 3 had an additional deoxyhexose group, which was identified as a rhamnose by its NMR data (dH 4.80 brs, 1.23 d, J = 6.0 Hz; dC 102.9 d, 73.7 d, 72.4 d, 72.2 d, 70.6 d, 17.8 d) [19]. The HMBC correlation between H-100 (dH 4.80 brs) and C-40 (dC 79.6) (Fig. 2) proved the (1 ? 4) linkage between glucose and rhamnose. The evaluation of chemical shifts and spin–spin couplings of two anomeric protons allowed the identification of one b-glucopyranose and one a-rhamnopyranose [2]. The absolute configuration of glucose was determined as D and that of rhamnose as L, as described above. Therefore, the structure of 3 was determined as (25S)-5b-spirostan-2b,3b-diol 3-O-a-L-rhamnopyranoyl-(1 ? 4)O-b-D-glucopyranoside and named tuberosine C. It’s worthy to note that all the three new spirostanol steroidal saponins (1–3) possess an unusual b-hydroxy substitution at C-2, which is normally a-orientation in this kind of compounds. The antimicrobial activities of the isolated compounds against Escherichia coli, Bacillus subtilis have also been investigated (Table 3). The results showed that 5 and 6 exhibited potent antibacterial activities against B. subtilis (32 lg/mL) and E. coli (16 lg/mL) (positive control, kanamycin: 2 lg/mL). New saponin 2 showed moderate antibacterial activities against B. subtilis (64 lg/mL) and E. coli (64 lg/mL). Compounds 1, 3, and 4 showed no or only very weak growth inhibition against above two microbes. Those saponins having a saccharide moiety at C-3 without any oxygen functionalities at C-2 (5 and 6) exhibited potent antibacterial activities, indicating 2-OH was negative to antibacterial activities. Compound 2, possessing a 2-OH, exhibited moderate antibacterial activities, probably because of the existence of 19OH. 4 showed no antibacterial activities indicating 5-OH has no positive effect on antibacterial activities.
3
Y.-S. Fang et al. / Steroids 100 (2015) 1–4
O
O OH
O
HO
O
HO
O
O HO
HO
O
HO
HO
OH
1
OH
O
HO
2
OH 21
HO 4"
HO 3"
2"
HO
6"
5"
O 1''
OH
6' 5'
O 4'
HO 2 1
O
3 1' O
HO
3'
2'
4
19
11
13 9 14
10 5
18
12
6
8 7
17
27
26 25 20 O 22 23 24
16
O
15
OH 3
Fig. 2. Key 1H–1H COSY (
Table 2 NMR spectral data of 3 in CD3OD (1H: 600 MHz;
13
) and HMBC (
) correlations of compounds 1–3.
Table 3 Antibacterial activities of compounds 1–6 (MIC: lg/mL).
C: 150 MHz).
Position
dC
dH (J in Hz)
Position
dC
dH (J in Hz)
Compounds
B. subtilis
E. coil
1
40.0
14.7
0.95 d (7.2)
67.9 79.4
m m m brs
21
2 3
1.66 1.41 3.58 3.99
22 23
111.1 27.7
4
31.5
24
26.9
256 64 256 128 32 16 2
42.5 26.7
25 26
29.4 66.1
7
26.9
27
16.4
8 9 10 11
36.8 37.0 37.8 22.1
m m m m m m m m m
m m m m m dd (10.2, 2.4) dd (10.2, 7.2) d (7.2)
256 64 256 256 16 16 2
5 6
10 20 30 40
102.6 74.9 76.7 79.6
4.29 3.24 3.43 3.48
d (7.8) m m m
12
41.3
1.77 1.61 1.31 1.98 1.39 1.31 1.24 1.58 1.77 – 1.52 1.31 1.72 1.18 –
– 1.42 1.04 1.87 1.39 1.64 3.88 3.24 1.05
1 2 3 4 5 6 Kanamycin
50
76.8
3.31 m
0
61.9
00
1 200
102.9 72.2
3.76 3.61 4.80 3.60
m m brs m
300 400 500 600
72.4 73.7 70.6 17.8
3.80 3.37 3.93 1.23
m m m d (6.0)
13
41.7
14 15
57.4 32.6
16 17 18 19 20
82.4 63.6 16.9 24.0 43.4
1.17 1.92 1.20 4.35 1.71 0.75 0.97 1.82
m m m m
6 m m m m m s s m
3. Experimental section 3.1. General experimental procedures Melting points were determined on a XRC-1 Melting Point Apparatus and uncorrected. Optical rotations were measured with a Jasco P-1020 digital polarimeter. A Nicolet Magna-IR 550 spectrometer was used for scanning IR spectroscopy with KBr pellets. NMR spectra were acquired with either a Bruker AM-400 spectrometer (400 MHz for 1H NMR and 100 MHz for 13C NMR) or a Bruker DRX-600 (600 MHz for 1H NMR and 150 MHz for 13C NMR) spectrometer. ESI-MS analyses were recorded with Agilent G3250AA and Auto Spec Premier P776 spectrometer. Silica gel (200–300 mesh or 300–400 mesh) was used for column chromatography. Sephadex LH-20 was purchased from Amersham Biosciences.
3.2. Plant material The roots of A. tuberosum were collected from Guandu District, Kunming City in Yunnan Province of China, in June 2011, and identified by professor Shu-Gang Lu from School of life Sciences, Yunnan University. The voucher specimen (2011-JCG-1) has been deposited in the school of Chemical Science and Technology of Yunnan University. 3.3. Extraction and isolation Air-dried and powdered roots (14.0 kg) of A. tuberosum were extracted three times with methanol at 60 °C. The extracts were combined, and evaporated under reduced pressure. The crude residue (1.4 kg) was suspended in H2O, and partitioned with petroleum ether, EtOAc, and 1-butanol successively. The 1-butanol fraction (200 g) was subjected to silica gel column eluted gradually with CHCl3–MeOH (200:1–1:1) to yield seven fractions (FrA1–A7). FrA4 (8.3 g) was further subjected to silica gel column chromatography using a EtOAc–MeOH gradient (20:1–2:1) to yield eight fractions (FrA4-1–FrA4-8). Fr4-4 (0.4 g) was separated by repeated column chromatography of silica gel, Sephadex LH-20 and yielded compounds 1 (7 mg), 2 (48 mg), and 4 (41 mg), respectively. Fr4-5 (0.3 g) was passed through a Sephadex LH-20 column and further purified by silica gel chromatography with CHCl3– MeOH (7:1) as the eluent to yield 3 (105 mg) and 5 (75 mg). FrA4-7 was separated by repeated silica column chromatography and Sephadex LH-20 to yield 6 (86 mg). Tuberosine A (1): white amorphous powder, m.p. 162–164 °C, 1 13 [a]20 C NMR data: see Table 1; IR D 60.0 (c 1.0, CH3OH); H and (KBr) v: 3428, 2933, 2353, 1678, 1531, 1082 cm1; HRESIMS m/z: 593.3693 [M-H] (calcd for C33H53O9 [M-H], 593.3690). Tuberosine B (2): white amorphous powder, m.p. 180–182 °C, 1 13 [a]20 C NMR data: see Table 1; IR D 56.1 (c 1.0, CH3OH); H and
4
Y.-S. Fang et al. / Steroids 100 (2015) 1–4
(KBr) v: 3427, 2931, 2353, 1633, 1454, 1044 cm1; HRESIMS m/z: 609.3641 [M-H] (calcd for C33H53O10 [M-H], 609.3639). Tuberosine C (3): white amorphous powder, m.p. 204–206 °C, 1 13 [a]20 C NMR data: see Table 1; IR D 77.9 (c 1.0, CH3OH); H and (KBr) v: 3428, 2933, 2353, 1678, 1531, 1081 cm1; HRESIMS m/z: 739.4271 [M-H] (calcd for C39H63O13 [M-H], 739.4269).
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.steroids.2015.03. 015. References
3.4. Acid hydrolysis and sugar analysis Saponin (4 mg) was dissolved in 1 M HCl (dioxane/H2O, 1:1, 2 mL) and stirred at 90 °C for 2 h. After cooling, the reaction mixture was neutralized with silver carbonate, and the solvent was evaporated. Then, the reaction mixture was extracted with CHCl3, and the aqueous layer was evaporated to give a mixture of monosaccharides. The residue was further dissolved in anhydrous pyridine (0.2 mL) followed by the addition of L-cysteine methyl ester hydrochloride (dry pyridine, 0.06 M, 0.2 mL). After heating at 60 °C for 2 h, and trimethylsilyl thiazolidine (0.2 mL) was added. Next, the mixture was heated at 60 °C for another 2 h and partitioned with n-hexane and water. The organic layer was analyzed by gas chromatography (GC) under the following conditions: capillary column, HP-5 (30 m 0.32 mm 0.25 lm, Dikma); FID detector at 300 °C; injection temperature, 280 °C; column temperature, 210 °C, maintained for 20 min; carrier gas, N2. The standard sugars were subjected to the same reaction and GC conditions. The retention times of thiazolidine derivatives D-rhamnose, L-rhamnose, D-glucose
and L-glucose were found to be 7.05 min, 7.53 min, 5.78 min and 6.39 min, respectively. 3.5. Antibacterial activities
Antibacterial activity assays were performed in sterilized 96well microplates using a microdilution method described previously [22]. The 18-h-old bacterial cultures from B. subtilis (ATCC 6633), and E. coli (ATCC 25922) were added to LB broth medium (1 L water, 10 g tryptone, 5 g yeast extract, and 10 g NaCl) to reach 1 105 CFU/mL. The samples were dissolved in dimethylsulfoxide (DMSO), and their final concentrations ranged from 0.5 to 512 lg/mL as determined by using a 2-fold serial dilution method. The wells containing strains and diluted samples were incubated at 37 °C for 18 h. Wells containing a culture suspension and DMSO were run as negative controls. Kanamycin was introduced in the experiments as a positive control. All experiments were repeated twice. The minimal inhibitory concentration (MIC) was defined as the lowest antibiotic concentration that produced complete growth inhibition of the microorganisms. Acknowledgements This project was financially supported by a Natural Science Foundation of China (No. 81460648) and a grant from the Program for Changjiang Scholars and Innovative Research Team in University (No. IRT13095).
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